iridoids and genipin

Natural cross-linkers are extensively employed due to their low toxicity and biocompatibility benefits. Genipin acts as a precursor for producing blue colorants. The formation of these colorants involves the cross-linking reaction between genipin and primary amines present in amino acids, peptides, and proteins. Genipin is extracted from Gardenia jasminoides and Genipa americana. This article explains the cross-linking mechanism of genipin with proteins/polysaccharides to provide an overall understanding of its properties. Furthermore, it explores new sources of genipin and innovative methodologies to make the genipin recovery process efficient. Genipin increases food products' texture, gel strength, stability, and shelf life. The antibacterial, anti-inflammatory, and antioxidant properties of chitosan, gelatin, alginate, and hyaluronic acid increased after genipin cross-linking. Lastly, drawbacks, toxicity, and directions regarding the genipin cross-linking have also been addressed. The review article covers how to recover and cross-link genipin with biopolymers for industrial applications.

Topics: Cross-Linking Reagents; Gardenia; Iridoids; Proteins; Rubiaceae

Genipin is an important monoterpene iridoid compound isolated from

Topics: Animals; Antineoplastic Agents; Color; Food Additives; Fruit; Humans; Iridoids; Pigments, Biological; Plant Extracts; Protective Agents

Topics: Antineoplastic Agents; Apoptosis; Humans; Iridoids; Neoplasms; Reactive Oxygen Species; Uncoupling Protein 2

Utility of plant-based materials in tissue engineering has exponentially increased over the years. Recent efforts in this area have been focused on substituting synthetic cross-linkers with natural ones derived from biological sources. These cross-linkers are essentially derived from the vegetative components of plants therefore suitably categorised as 'green' and renewable materials. Utilization of plant based cross-linkers in scaffolds and hydrogels offers several advantages compared to the synthetic ones. Natural compounds, like ferulic acid and genipin, when incorporated into scaffolds can promote cellular proliferation and growth, by regulation of growth factors. They participate in crucial activities, thus providing impetus for cell growth, function, differentiation and angiogenesis. Several natural compounds inherently possess anti-microbial, antioxidant and anti-inflammatory effects, which enhance the inherent characteristics of the scaffolds. Versatility of natural cross-linkers can be exploited for diverse applications. Integrating such potent molecules can enable the scaffold to display relevant characteristics for each function. This review article focuses on the recent developments with plant based cross-linkers that are employed for scaffold synthesis and their applications, which may be explored to synthesize scaffolds suitable for diverse biomedical applications.

Topics: Biocompatible Materials; Cell Differentiation; Cell Proliferation; Humans; Hydrogels; Iridoids; Materials Testing; Tissue Engineering; Tissue Scaffolds

Genipin is a naturally occurring nontoxic cross-linker, which has been widely used for drug delivery due to its excellent biocompatibility, admirable biodegradability and stable cross-linked attributes. These advantages led to its extensive application in the fabrication of hydrogels for drug delivery. This review describes the physicochemical characteristics and pharmacological activities of genipin and attempts to elucidate the detailed mechanisms of the cross-linking reaction between genipin and biomaterials. The current article entails a general review of the different biomaterials cross-linked by genipin: chitosan and its derivatives, collagen, gelatin, etc. The genipin-cross-linked hydrogels for various pharmaceutical applications, including ocular drug delivery, buccal drug delivery, oral drug delivery, anti-inflammatory drug delivery, and antibiotic and antifungal drug delivery, are reported. Finally, the future research directions and challenges of genipin-cross-linked hydrogels for pharmaceutical applications are also discussed in this review.

Topics: Biocompatible Materials; Chitosan; Cross-Linking Reagents; Hydrogels; Iridoids

Due to their strong biomimetic potential, silk fibroin (SF) hydrogels are impressive candidates for tissue engineering, due to their tunable mechanical properties, biocompatibility, low immunotoxicity, controllable biodegradability, and a remarkable capacity for biomaterial modification and the realization of a specific molecular structure. The fundamental chemical and physical structure of SF allows its structure to be altered using various crosslinking strategies. The established crosslinking methods enable the formation of three-dimensional (3D) networks under physiological conditions. There are different chemical and physical crosslinking mechanisms available for the generation of SF hydrogels (SFHs). These methods, either chemical or physical, change the structure of SF and improve its mechanical stability, although each method has its advantages and disadvantages. While chemical crosslinking agents guarantee the mechanical strength of SFH through the generation of covalent bonds, they could cause some toxicity, and their usage is not compatible with a cell-friendly technology. On the other hand, physical crosslinking approaches have been implemented in the absence of chemical solvents by the induction of β-sheet conformation in the SF structure. Unfortunately, it is not easy to control the shape and properties of SFHs when using this method. The current review discusses the different crosslinking mechanisms of SFH in detail, in order to support the development of engineered SFHs for biomedical applications.

Topics: Animals; Biocompatible Materials; Bombyx; Carbon Dioxide; Chemical Phenomena; Cross-Linking Reagents; Crystallography, X-Ray; Fibroins; Glutaral; Humans; Hydrogels; Hydrogen-Ion Concentration; Iridoids; Materials Testing; Models, Theoretical; Osmosis; Polymers; Silk; Stress, Mechanical; Surface-Active Agents; Temperature; Tissue Engineering

Genipin is an aglycone derived from the geniposide, the most abundant iridoid glucoside constituent of Gardenia jasminoides Ellis. For decades, genipin is the focus of studies as a versatile compound in the treatment of various pathogenic conditions. In particularly, Gardenia jasminoides Ellis has long been used in traditional Chinese medicine for the prevention and treatment of liver disease. Mounting experimental data has proved genipin possesses therapeutic potential for cholestatic, septic, ischemia/reperfusion-triggered acute liver injury, fulminant hepatitis and NAFLD. This critical review is a reflection on the valuable lessons from decades of research regarding pharmacological activities of genipin. Of note, genipin represents choleretic effect by potentiating bilirubin disposal and enhancement of genes in charge of the efflux of a number of organic anions. The anti-inflammatory capability of genipin is mediated by suppression of the production and function of pro-inflammatory cytokines and inflammasome. Moreover, genipin modulates various transcription factor and signal transduction pathway. Genipin appears to trigger the upregulation of several key genes encoding antioxidant and xenobiotic-metabolizing enzymes. Furthermore, the medicinal impact of genipin extends to modulation of regulated cell death, including autophagic cell death, apoptosis, necroptosis and pyroptosis, and modulation of quality of cellular organelle. Another crucial effect of genipin appears to be linked to dual role in targeting uncoupling protein 2 (UCP2). As a typical UCP2-inhibiting compound, genipin could inhibit AMP-activated protein kinase or NF-κB in circumstance. On the contrary, reactive oxygen species production and cellular lipid deposits mediated by genipin through the upregulation of UCP2 is observed in liver steatosis, suggesting the precise role of genipin is disease-specific. Collectively, we comprehensively summarize the mechanisms and pathways associated with the hepatoprotective activity of genipin and discuss potential toxic impact. Notably, our focus is the direct medicinal effect of genipin itself, whereas its utility as a crosslinking agent in tissue engineering is out of scope for the current review. Further studies are therefore required to disentangle these complicated pharmacological properties to confer this natural agent a far greater potency.

Topics: Animals; Anti-Inflammatory Agents; Antioxidants; Cell Death; Chemical and Drug Induced Liver Injury; Cholagogues and Choleretics; Humans; Iridoids; Liver; Massive Hepatic Necrosis; Mitochondria, Liver; Non-alcoholic Fatty Liver Disease; Uncoupling Protein 2

This review provides a report on cross-linking methods used for collagen modifications. Collagen materials have attracted significant academic interest due to its biological properties in native state. However, in many cases the mechanical properties and degradation rate should be tailored to especial biomedical and cosmetic applications. In the proposed review paper, the structure, preparation, and properties of several collagen based materials have been discussed in general, and detailed examples of collagen cross-linking methods have been drawn from scientific literature and practical work. Both, physical and chemical methods of improvement of collagenous materials have been reviewed. In the review paper the cross-linking with glutaraldehyde, genipin, EDC-NHS, dialdehyde starch, chitosan, temperature, UV light and enzyme has been discussed. A critical comparison of currently available cross-linking methods has been shown.

Topics: Animals; Chitosan; Collagen; Glutaral; Humans; Iridoids; Starch; Succinimides

Bone tissue scaffolds made from either natural or synthetic polymers are employed to promote bone healing. However, lack of sufficient or poor mechanical properties such as low integrity and stability reduces their medical applications. Crosslinking, defined as induction of chemical or physical links among polymer chains, is a simple method generally used to modify mechanical, biological and degradation properties of hydrogels. Although crosslinking through chemical reactions improves the mechanical properties of bone substitutes, most of the reagents used for this aim demonstrate undesirable effects and may exert toxic reactions. Glutaraldehyde is a widely-used chemical crosslinker with unique ability to crosslink a wide variety of biomaterials; however, many contradictory views have been recently raised on its cytotoxic effects. By keeping this limit in mind, green chemicals or natural crosslinking agents have been shown to provide desired improvements in mechanical properties of bone scaffolds. Therefore, developing more efficient crosslinking materials and methods are desirable to obtain crosslinked scaffolds with perfect properties in bone tissue engineering from different biopolymers such as collagen, gelatin, cellulose, chitosan, alginate, etc. In this review, we focused on developed or developing modalities used to improve mechanical properties of various bone scaffolds and matrices based on common crosslinking reagents.

Topics: Alginates; Biocompatible Materials; Bone and Bones; Chitosan; Citric Acid; Collagen; Cross-Linking Reagents; Gelatin; Glucuronic Acid; Glutaral; Glyoxal; Green Chemistry Technology; Hexuronic Acids; Humans; Iridoids; Tissue Engineering; Tissue Scaffolds

Platelet lysates (PLs) are a natural source of growth factors (GFs) known for its stimulatory role on stem cells which can be obtained after activation of platelets from blood plasma. The possibility to use PLs as growth factor source for tissue healing and regeneration has been pursued following different strategies. Platelet lysates are an enriched pool of growth factors which can be used as either a GFs source or as a three-dimensional (3D) hydrogel. However, most of current PLs-based hydrogels lack stability, exhibiting significant shrinking behavior. This chapter focuses on the application of supercritical fluid technology to develop three-dimensional architectures of PL constructs, crosslinked with genipin. The proposed technology allows in a single step operation the development of mechanically stable porous structures, through chemical crosslinking of the growth factors present in the PL pool, followed by supercritical drying of the samples. Furthermore gradient structures of PL-based structures with bioactive glass are also presented and are described as an interesting approach to the treatment of osteochondral defects.

Topics: Animals; Blood Platelets; Bone and Bones; Cartilage; Cross-Linking Reagents; Humans; Hydrogels; Intercellular Signaling Peptides and Proteins; Iridoids; Porosity

Genipin, an aglycone derived from the iridoid glycoside, geniposide, is isolated and characterized from the extract of Gardenia jasminoides Ellis fruit (family Rubiaceae). It has long been used in traditional oriental medicine for the prevention and treatment of several inflammation driven diseases, including cancer. Genipin has been shown to have hepatoprotective activity acting as a potent antioxidant and inhibitor of mitochondrial uncoupling protein 2 (UCP2), and also reported to exert significant anticancer effects. It is an excellent crosslinking agent that helps to make novel sustained or delayed release nanoparticle formulations. In this review, we present the latest developments of genipin as an anticancer agent and briefly describe its diverse mechanism(s) of action. Several lines of evidence suggest that genipin is a potent inhibitor of UCP2, which functions as a tumor promoter in a variety of cancers, attenuates generation of reactive oxygen species and the expression of matrix metalloproteinase 2, as well as induces caspase-dependent apoptosis in vitro and in in vivo models. These finding suggests that genipin can serve as both a prominent anticancer agent as well as a potent crosslinking drug that may find useful application in several novel pharmaceutical formulations.

Topics: Animals; Antineoplastic Agents; Cross-Linking Reagents; Humans; Iridoids; Nanoparticles; Neoplasms

Genipin is a fully assessed non-cytotoxic crosslinking compound. The chitosan|genipin physical properties such as morphology, roughness, porosity, hydrophilicity, ζ-potential, surface area and surface energy exert control over cell adhesion, migration, phenotype maintenance and intracellular signaling in vitro, and cell recruitment at the tissue-scaffold interface in vivo. For example a therapy using fucose|chitosan|genipin nanoparticles encapsulating amoxicillin, based on the recognition of fucose by H. pylori, leads to sharply improved clinical results. A bioactive scaffold sensitive to environmental stimuli provides an alternative approach for inducing adipose stem cell chondrogenesis: the expression of specific genes, the accumulation of cartilage-related macromolecules and the mechanical properties are comparable to the original cartilage-derived matrix (CDM), thus making the CDM|genipin a contraction-free biomaterial suitable for cartilage tissue engineering. For the regeneration of the cartilage, chitosan|genipin permits to modulate matrix synthesis and proliferation of chondrocytes by dynamic compression; chondrocytes cultured on the composite substrate produce much more collagen-II and sulfated GAG. The main advantages gained in the bone regeneration area with chitosan|genipin are: acceleration of mineral deposition; enhancement of adhesion, proliferation and differentiation of osteoblasts; promotion of the expression of osteogenic differentiation markers; greatly improved viability of human adipose stem cells.

Topics: Animals; Cell Differentiation; Cells, Cultured; Chitosan; Humans; Iridoids; Osteoblasts; Osteogenesis; Regeneration; Regenerative Medicine; Stem Cell Transplantation; Stem Cells; Tissue Engineering

Central neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), are one of the biggest health problems worldwide. Currently, there is no cure for these diseases. The Gardenia jasminoides fruit is a common herbal medicine in traditional Chinese medicine (TCM), and a variety of preparations are used as treatments for central nervous system (CNS) diseases. Pharmacokinetic studies suggest genipin is one of the main effective ingredients of G. jasminoides fruit extract (GFE). Accumulated research data show that genipin possesses a range of key pharmacological properties, such as anti-inflammatory, neuroprotective, neurogenic, antidiabetic, and antidepressant effects. Thus, genipin shows therapeutic potential for central neurodegenerative diseases. We review the pharmacological actions of genipin for the treatment of neurodegenerative diseases of the CNS. We also describe the potential mechanisms underlying these effects.

Topics: Animals; Anti-Inflammatory Agents; Humans; Hypoglycemic Agents; Iridoids; Nerve Growth Factors; Neurodegenerative Diseases; Neuroprotective Agents

The present review article intends to direct attention to the technological advances made since 2009 in the area of genipin-crosslinked chitosan (GEN-chitosan) hydrogels. After a concise introduction on the well recognized characteristics of medical grade chitosan and food grade genipin, the properties of GEN-chitosan obtained with a safe, spontaneous and irreversible chemical reaction, and the quality assessment of the gels are reviewed. The antibacterial activity of GEN-chitosan has been well assessed in the treatment of gastric infections supported by Helicobacter pylori. Therapies based on chitosan alginate crosslinked with genipin include stem cell transplantation, and development of contraction free biomaterials suitable for cartilage engineering. Collagen, gelatin and other proteins have been associated to said hydrogels in view of the regeneration of the cartilage. Viability and proliferation of fibroblasts were impressively enhanced upon addition of poly-l-lysine. The modulation of the osteocytes has been achieved in various ways by applying advanced technologies such as 3D-plotting and electrospinning of biomimetic scaffolds, with optional addition of nano hydroxyapatite to the formulations. A wealth of biotechnological advances and know-how has permitted reaching outstanding results in crucial areas such as cranio-facial surgery, orthopedics and dentistry. It is mandatory to use scaffolds fully characterized in terms of porosity, pore size, swelling, wettability, compressive strength, and degree of acetylation, if the osteogenic differentiation of human mesenchymal stem cells is sought: in fact, the novel characteristics imparted by GEN-chitosan must be simultaneously of physico-chemical and cytological nature. Owing to their high standard, the scientific publications dated 2010-2015 have met the expectations of an interdisciplinary audience.

Topics: Animals; Biocompatible Materials; Bone and Bones; Cartilage; Chitosan; Humans; Hydrogels; Iridoids; Osteogenesis; Regeneration; Tissue Engineering; Tissue Scaffolds

One of the popular approaches in controlling drug delivery from the polymeric carriers is suitably achieved by the inclusion of crosslinking agents into the formulations at different concentrations. Nevertheless, addition of the chemical crosslinkers such as glutaraldehyde, formaldehyde etc, used in the drug delivery systems causes very serious cytotoxic reactions. These chemical crosslinking agents did not offer any significant advantageous effects when compared to the natural crosslinking agents for instance genipin, which is quite less toxic, biocompatible and offers very stable crosslinked products. Based on the earlier reports the safety of this particular natural crosslinker is very well established, since it has been widely used as a Chinese traditional medicine for long-time, isolated from fruits of the plant Gardenia jasminoides Ellis. This concise article largely portrayed the value of this unique natural crosslinker, utilized in controlling the drug delivery from the various formulations.

Topics: Chemistry, Pharmaceutical; Cross-Linking Reagents; Delayed-Action Preparations; Drug Carriers; Drug Delivery Systems; Iridoids; Polymers; Solubility; Solvents; Technology, Pharmaceutical; Water

To review the application of genipin for the modification of natural biomaterials as a crosslinking agent and progress in research.. Domestic and foreign literature on application of genipin for the modification of natural biomaterials as a crosslinking agent was thoroughly reviewed. Results Genipin is an effective natural crosslinking agent with a very low level of cytotoxicity compared with conventional synthetic crosslinking agents. Tissues fixed with genipin can maintain a high level of stability as well as resistance to enzymatic degradation.. Genipin is a promising substitute for conventional synthetic crosslinking agents, which has offered an alternative for modification of natural biomaterials for tissue engineering.

Topics: Animals; Biocompatible Materials; Cross-Linking Reagents; Humans; Iridoids; Tissue Engineering; Tissue Fixation; Tissue Scaffolds; Toxicity Tests

The introduction of ninhydrin treatment as a chemical technique for the visualisation of latent fingermarks on porous surfaces revolutionised approaches to forensic fingermark examination. Since then, a range of amino acid sensitive reagents has been developed and such compounds are in widespread use by law enforcement agencies worldwide. This paper reviews the development and use of these reagents for the detection of latent fingermarks on porous surfaces. A brief overview is provided, including an historical background, forensic significance, and a general approach to the development of latent fingermarks on porous surfaces. This is followed by a discussion of specific amino acid sensitive treatments.

Topics: Amino Acids; Aza Compounds; Forensic Sciences; Indans; Iridoid Glycosides; Iridoids; Naphthoquinones; Ninhydrin; Surface Properties

Today, the blood vessel substitutes are in large demand for coronary and peripheral bypass procedures, and the demand cannot be met by conventional sources. This problem will be solved by applying tissue-engineered blood vessel in clinics. The prefabrication of vascular scaffold will be involved in engineering a blood vessel substitute. Biological tissues are important biomaterials fabricating vascular scaffold which can offer better constructs for adhesion and growth of cells onto synthetic materials. Because of immediate degradation of biological tissues obtained from the abattoir, cadaver or patient and the presence of antigenicity in allogenic or xenogenic tissues, the fresh biological tissues can not directly be preserved and applied. The use and preservation of these natural biomaterials have typically required pre-treatment aimed at (1) reducing the antigenicity of the materials, (2) enhancing the resistance of the materials to enzymatic degradation, (3) stabilizing the structure of the tissues and maintaining their mechanical properties. Physical and chemical methods for the pre-treatment of biological tissues are available. The predominant chemical agents that have been investigated for the pre-treatment of biological tissues for vascular scaffold are glutaraldehyde, polyepoxy compound, carbodiimide, genipin and proanthocyanidin. Typical and particularly promising physical pre-treatment of biological tissues for vascular scaffold is dye-mediated photooxidation. The crosslinking mechanisms of all classes of pre-treatments and the effects of pre-treatments on antigenicity, biostability, mechanical properties, cytoxicity and calcification of treated tissues are described in this article. The advantages and disadvantages of all pre-treatments are also reviewed. The trend of pre-treatment of biological tissues is to investigate and exploit the naturally occurring crosslinking reagent with less cytoxicity. Meanwhile, dye-mediated photooxidation crosslink is also a promising pre-treatment which should be widely applied in vascular scaffold.

Topics: Biocompatible Materials; Bioprosthesis; Blood Vessel Prosthesis; Cross-Linking Reagents; Ethyldimethylaminopropyl Carbodiimide; Glutaral; Humans; Iridoid Glycosides; Iridoids; Materials Testing; Pyrans; Stents; Tissue Engineering

Na(+)-dependent and -independent transport sites were elucidated for glycine and L-leucine, respectively, in Chang liver cells, a human culture cell line. Findings of acceleration of the L-leucine uptake by the cells in the acidic medium and synchronized acidification within the cell membrane vesicles with the uptake by them all suggested contransport of L-leucine and proton and the uptake of L-leucine dependent on the inward proton gradient in Chang liver cells. Cotransport of L-leucine and proton was also demonstrated in human peripheral lymphocytes and accelerated by the addition of concanavalin A, probably accompanied by membrane hyperpolarization. It was shown that the Na(+)-gradient-dependent uptake of glycine can be regulated by insulin and 17 beta-estradiol in the rat uterus and by Ca(2+)-calmodulin and membrane potential in Chang liver cells. D-Aspartate uptake as a model of glutamate transport was characterized in rat hippocampal slices and found to consist of Na(+)-dependent (higher-affinity) and -independent (lower-affinity) components. The vulnerability of hippocampal neurons to the Alzheimer beta-amyloid protein was confirmed in vitro with primary cultured rat hippocampal neurons in the presence of the amyloid protein beta 1-42 or its core fragments. The toxicity of the amyloid protein could be blocked by the addition of insulin and several other growth factors to the medium. The addition of genipin, a plant-derived iridoid, was demonstrated to prevent the toxicity of a synthetic fragment of beta 1-42, beta 25-35. Genipin had a neuritogenic activity in PC12h cells, a rat pheochromocytoma cell line, an activity extremely sensitive to inhibitors of the nitrogen oxide (NO) synthase and soluble guanylate cyclase and an NO scavenger. It was also demonstrated in PC12h cells that the activation of the MAP kinase cascade was essential for the neuritogenesis of genipin. These properties of genipin are very comparable to those of nerve growth factor in the cells. It is considered likely that various useful, neurotrophic substances and their extracts will be found in plants in future.

Topics: Amino Acids; Amyloid beta-Peptides; Animals; Biological Transport; Cell Membrane; Cells, Cultured; Dendrites; Hippocampus; Humans; Hydrocortisone; Iridoid Glycosides; Iridoids; Nerve Degeneration; Neurons; Plants, Medicinal; Pyrans

The suture-tendon interface often constitutes the point of failure in tendon suture repair. In the present study, we investigated the mechanical benefit of coating the suture with a cross-linking agent to strengthen the nearby tissue after suture placement in human tendons and we assessed the biological implications regarding tendon cell survival in-vitro.. Freshly harvested human biceps long head tendons were randomly allocated to control (n = 17) or intervention (n = 19) group. According to the assigned group, either an untreated or a genipin-coated suture was inserted into the tendon. 24 h after suturing, mechanical testing composed of cyclic and ramp-to-failure loading was performed. Additionally, 11 freshly harvested tendons were used for short-term in vitro cell viability assessment in response to genipin-loaded suture placement. These specimens were analyzed in a paired-sample setting as stained histological sections using combined fluorescent/light microscopy.. Tendons stitched with a genipin-coated suture sustained higher forces to failure. Cyclic and ultimate displacement of the tendon-suture construct remained unaltered by the local tissue crosslinking. Tissue crosslinking resulted in significant cytotoxicity in the direct vicinity of the suture (<3 mm). At larger distances from the suture, however, no difference in cell viability between the test and the control group was discernable.. The repair strength of a tendon-suture construct can be augmented by loading the suture with genipin. At this mechanically relevant dosage, crosslinking-induced cell death is confined to a radius of <3 mm from the suture in the short-term in-vitro setting. These promising results warrant further examination in-vivo.

Topics: Biomechanical Phenomena; Cell Survival; Humans; Iridoids; Suture Techniques; Sutures; Tendons; Tensile Strength

Inchinkoto (ICKT) is a popular choleretic and hepatoprotective herbal medicine that is widely used in Japan. Geniposide, a major ingredient of ICKT, is metabolized to genipin by gut microbiota, which exerts a choleretic effect. This study investigates the relationship between stool genipin-producing activity and diversity of the clinical effect of ICKT in patients with malignant obstructive jaundice. Fifty-two patients with malignant obstructive jaundice who underwent external biliary drainage were included. ICKT was administered as three packets per day (7.5 g/day) for three days and 2.5 g on the morning of the fourth day. Stool samples were collected before ICKT administration and bile flow was monitored on a daily basis. The microbiome, genipin-producing activity, and organic acids in stools were analyzed. The Shannon-Wiener (SW) index was calculated to evaluate gut microbiome diversity. The stool genipin-producing activity showed a significant positive correlation with the SW index. Stool genipin-producing activity positively correlated with the order Clostridia (obligate anaerobes), but negatively correlated with the order Lactobacillales (facultative anaerobes). Moreover, stool genipin-producing activity was positively correlated to the concentration valeric acid, but negatively correlated to the concentration of lactic acid and succinic acid. The change of bile flow at 2 and 3 days after ICKT administration showed significant positive correlation with genipin-producing activity (correlation coefficient, 0.40 and 0.29, respectively, P < 0.05). An analysis of stool profile, including stool genipin-producing activity, may predict the efficacy of ICKT. Modification of the microbiome may be a target to enhance the therapeutic effect of ICKT.

Topics: Adult; Aged; Aged, 80 and over; Bile; Carboxylic Acids; Cholagogues and Choleretics; Clostridium; Drugs, Chinese Herbal; Feces; Female; Gastrointestinal Microbiome; Humans; Iridoids; Jaundice, Obstructive; Lactobacillales; Male; Middle Aged; Neoplasms; Treatment Outcome

Ketorolac tromethamine gel (KT gel) and ketorolac tromethamine gel containing genipin (KTG gel) were prepared and their therapeutic effects on periodontitis were evaluated. The skin permeation rate of ketorolac from the KT gel and KTG gel was 5.75+/-0.53 and 5.82 +/- 0.74 microg/cm2/ h, respectively. The skin permeation rate of genipin from the KTG gel was 10.13 +/- 1.47 microg/ cm2/h. The tensile strength of the KTG gel was larger than the KT gel. After 4 weeks, the periodontal pocket depth of the KTG gel group (3.22 +/- 0.20 mm) significantly decreased compared with the non-treated group (4.50 +/- 0.25 mm) and the KT group (3.84 +/- 00.26 mm). The KTG gel did not induce separation of the stratum corneum and subcutaneous tissue, and the collagen layers of the corium were closer, more fibrous, and showed longer connections than in the other groups. The KTG gel appears to be effective against gingivitis in the periodontal pocket through its increased anti-inflammatory activity and the crosslinking of genipin with the biological tissue.

Topics: Adult; Animals; Chromatography, High Pressure Liquid; Double-Blind Method; Drug Combinations; Drug Compounding; Female; Gels; Humans; In Vitro Techniques; Iridoid Glycosides; Iridoids; Ketorolac Tromethamine; Male; Mice; Mice, Hairless; Middle Aged; Periodontal Diseases; Skin Absorption; Tensile Strength; Tissue Adhesives

The present study investigated the effects of genipin cross-linking on the gelling properties of ginkgo seed protein isolate (GSPI). Cross-linking of GSPI was achieved with different concentrations (0, 0.05, 0.1, 0.2, 0.4, 0.6% w/v) of genipin at pH 6.0. Compared to pure GSPI, genipin treatment led to lower solubility, surface hydrophobicity, and fluorescence intensity, while promoted protein aggregation. Cross-linked GSPI gels exhibited markedly improved gelling properties and water holding capacity (WHC), with up to 2.1-fold increases in gel hardness and 1.3-fold increases in WHC over non-treated GSPI gel. Electrophoresis and Fourier-transform infrared spectroscopy confirmed the cross-linking. Moreover, microstructural examination showed that cross-linking with genipin resulted in protein aggregation and more porous gel matrix. Overall, genipin cross-linking demonstrated great potential for the enhancement of gelling properties of ginkgo seed protein. The current research may expand the utilization of ginkgo seeds in food applications.

Topics: Cross-Linking Reagents; Gels; Ginkgo biloba; Iridoids; Protein Aggregates; Seeds

While current clinical utilization of large vascular grafts for vascular transplantation is encouraging, tissue engineering of small grafts still faces numerous challenges. This study aims to investigate the feasibility of constructing a small vascular graft from decellularized amniotic membranes (DAMs). DAMs were rolled around a catheter and each of the resulting grafts was crosslinked with (a) 0.1% glutaraldehyde; (b) 1-ethyl-3-(3-dimethylaminopropyl) crbodiimidehydro-chloride (20 mM)-N-hydroxy-succinimide (10 mM); (c) 0.5% genipin; and (d) no-crosslinking, respectively. Our results demonstrated the feasibility of using a rolling technique followed by lyophilization to transform DAM into a vessel-like structure. The genipin-crosslinked DAM graft showed an improved integrated structure, prolonged stability, proper mechanical property, and superior biocompatibility. After transplantation in rat abdominal aorta, the genipin-crosslinked DAM graft remained patent up to 16 months, with both endothelial and smooth muscle cell regeneration, which suggests that the genipin-crosslinked DAM graft has great potential to be

Topics: Amnion; Animals; Blood Vessel Prosthesis; Humans; Iridoids; Models, Animal; Rats; Tissue Engineering

Ferritin proteins are promising nano-carriers for bioactive compound delivery. However, the disassembly properties under acidic/alkaline conditions pose risks of cargo leakage. Herein, genipin-mediated chemical crosslinking method was provided as an alternative and effective strategy to construct robust ferritin nanocarrier through controlled-intramolecular conjugation. As indicated by SDS-/Native- PAGE, the crosslinking degree gradually increased with incubating time prolonging. CD results showed that the cross-linking would decrease α-helix content from 78.4 % to 52.7 % upon 6 h incubation. However, TEM images showed that the genipin-modification has subtle influence on its shell-like structure. Remarkably, the cross-linking can be well controlled by intramolecular subunit-subunit conjugation rather than intermolecular conjugation, giving an excellent monodispersity. Importantly, the covalent cross-linking can tight neighboring subunits and inhibit its disassociation, finally inhibiting the leakage of encapsulated-cargos from ferritin cavity under acidic environments. Such findings suggested that the genipin-mediated cross-linking strategy can fabricate robust nano-carriers for bioactive compound delivery.

Topics: Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Ferritins; Iridoids

Glottic insufficiency is one of the voice disorders affecting all demographics. Due to the incomplete closure of the vocal fold, there is a risk of aspiration and ineffective phonation. Current treatments for glottic insufficiency include nerve repair, reinnervation, implantation and injection laryngoplasty. Injection laryngoplasty is favored among these techniques due to its cost-effectiveness and efficiency. However, research into developing an effective injectable for the treatment of glottic insufficiency is currently lacking. Therefore, this study aims to develop an injectable gelatin (G) hydrogel crosslinked with either 1-ethyl-3-(3-dimethylaminpropyl)carbodiimide hydrochloride) (EDC) or genipin (gn). The gelation time, biodegradability and swelling ratio of hydrogels with varying concentrations of gelatin (6-10% G) and genipin (0.1-0.5% gn) were investigated. Some selected formulations were proceeded with rheology, pore size, chemical analysis and in vitro cellular activity of Wharton's Jelly Mesenchymal Stem Cells (WJMSCs), to determine the safety application of the selected hydrogels, for future cell delivery prospect. 6G 0.4gn and 8G 0.4gn were the only hydrogel groups capable of achieving complete gelation within 20 min, exhibiting an elastic modulus between 2 and 10 kPa and a pore size between 100 and 400 μm. Moreover, these hydrogels were biodegradable and biocompatible with WJMSCs, as > 70% viability were observed after 7 days of in vitro culture. Our results suggested 6G 0.4gn and 8G 0.4gn hydrogels as potential cell encapsulation injectates. In light of these findings, future research should focus on characterizing their encapsulation efficiency and exploring the possibility of using these hydrogels as a drug delivery system for vocal fold treatment.

Topics: Gelatin; Hydrogels; Iridoids; Vocal Cords

In recent years, there has been an increased interest in injectable, in situ crosslinking hydrogels due to their minimally invasive application and ability to conform to their environment. Current in situ crosslinking chitosan hydrogels are either mechanically robust with poor biocompatibility and limited biodegradation due to toxic crosslinking agents or the hydrogels are mechanically weak and undergo biodegradation too rapidly due to insufficient crosslinking. Herein, the authors developed and characterized a thermally-driven, injectable chitosan-genipin hydrogel capable of in situ crosslinking at 37 °C that is mechanically robust, biodegradable, and maintain high biocompatibility. The natural crosslinker genipin is utilized as a thermally-driven, non-toxic crosslinking agent. The chitosan-genipin hydrogel's crosslinking kinetics, injectability, viscoelasticity, swelling and pH response, and biocompatibility against human keratinocyte cells are characterized. The developed chitosan-genipin hydrogels are successfully crosslinked at 37 °C, demonstrating temperature sensitivity. The hydrogels maintained a high percentage of swelling over several weeks before degrading in biologically relevant environments, demonstrating mechanical stability while remaining biodegradable. Long-term cell viability studies demonstrated that chitosan-genipin hydrogels have excellent biocompatibility over 7 days, including during the hydrogel crosslinking phase. Overall, these findings support the development of an injectable, in situ crosslinking chitosan-genipin hydrogel for minimally invasive biomedical applications.

Topics: Chitosan; Humans; Hydrogels; Iridoids; Kinetics

Parkinson's Disease (PD) is a common neurodegenerative disorder affecting millions of people worldwide for which there are only symptomatic therapies. Small molecules able to target key pathological processes in PD have emerged as interesting options for modifying disease progression. We have previously shown that a (poly)phenol-enriched fraction (PEF) of Corema album L. leaf extract modulates central events in PD pathogenesis, namely α-synuclein (αSyn) toxicity, aggregation and clearance. PEF was now subjected to a bio-guided fractionation with the aim of identifying the critical bioactive compound. We identified genipin, an iridoid, which relieves αSyn toxicity and aggregation. Furthermore, genipin promotes metabolic alterations and modulates lipid storage and endocytosis. Importantly, genipin was able to prevent the motor deficits caused by the overexpression of αSyn in a Drosophila melanogaster model of PD. These findings widens the possibility for the exploitation of genipin for PD therapeutics.

Topics: alpha-Synuclein; Animals; Drosophila melanogaster; Iridoids; Lipids; Parkinson Disease; Phenols

Osteosarcoma is a highly invasive and early metastatic tumor. At present, the toxic and side effects of chemotherapy affect the quality of life of cancer patients to varying degrees. Genipin is an extract of the natural medicine gardenia with various pharmacological activities.. The purpose of this study was to investigate the effect of Genipin on osteosarcoma and its potential mechanism of action.. Crystal violet staining, MTT assay and colony formation assay were used to detect the effect of genipin on the proliferation of osteosarcoma. The effects of vitexin on migration and invasion of osteosarcoma were detected by scratch healing assay and transwell assay. Hoechst staining and flow cytometry were used to detect the effect of genipin on apoptosis of osteosarcoma cells. The expression of related proteins was detected by Western blot. An orthotopic tumorigenic animal model was used to verify the effect of genipin on osteosarcoma in vivo.. The results of crystal violet staining, MTT method and colony formation method proved that genipin significantly inhibited the proliferation of osteosarcoma cells. The results of the scratch healing assay and transwell assay showed that gen significantly inhibited the migration and invasion of osteosarcoma cells. The results of Hoechst staining and flow cytometry showed that genipin significantly promoted the apoptosis of osteosarcoma cells. The results of animal experiments show that genipin has the same anti-tumor effect in vivo. Genipin may inhibit the growth of osteosarcoma through PI3K/AKT signaling.. Genipin can inhibit the growth of human osteosarcoma cells, and its mechanism may be related to the regulation of PI3K/AKT signaling pathway.

Topics: Animals; Apoptosis; Bone Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Humans; Iridoids; Osteosarcoma; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Quality of Life; Signal Transduction

Polysaccharide-polypeptide nanocomplexes are promising colloidal Pickering stabilizers. The resulting Pickering emulsions, however, are susceptible to pH and ionic strength changes. This phenomenon was also observed in our recently developed Pickering emulsions stabilized by the chitosan (CS)-caseinophosphopeptides (CPPs) nanocomplexes. To improve the stability of these Pickering emulsions, we herein crosslinked the CS-CPPs nanocomplexes with a natural crosslinker genipin. The genipin-crosslinked CS-CPPs nanocomplexes (GCNs) were used to prepare Pickering emulsions. The impacts of genipin concentration, crosslinking temperature, and duration on the characteristics of GCNs and the GCNs-stabilized Pickering emulsions (GPEs) were systemically investigated. GCNs showed crosslinking strength-dependent variations in their physical properties. Crosslinking at a weak or strong condition weakened the emulsification ability of GCNs at low concentrations. A strong crosslinking condition also compromised the capacity of GCNs to stabilize a high fraction of oil. GPEs were oil-in-water type and gel-like. GCNs crosslinked at a lower temperature and for a shorter crosslinking duration stabilized stronger gel-like GPEs. Moreover, GPEs had high pH and ionic strength stabilities. This work provided a feasible way to enhance the stability and regulate the physical properties of Pickering emulsions stabilized by polysaccharide-polypeptide nanocomplexes.

Topics: Chitosan; Emulsions; Iridoids; Particle Size; Polysaccharides

Genipin is a natural crosslinker that improves the functional properties of proteins by modifying its structures. This study aimed to investigate the effects of sonication on the emulsifying properties of different genipin concentration-induced myofibrillar protein (MP) cross-linking. The structural characteristics, solubility, emulsifying properties, and rheological properties of genipin-induced MP crosslinking without sonication (Native), sonication before crosslinking (UMP), and sonication after crosslinking (MPU) treatments were determined, and the interaction between genipin and MP were estimated by molecular docking. The results demonstrated that hydrogen bond might be the main forces for genipin binding to the MP, and 0.5 μM/mg genipin was a desirable concentration for protein cross-linking to improve MP emulsion stability. Ultrasound treatment before and after crosslinking were better than Native treatment to improve the emulsifying stability index (ESI) of MP. Among the three treatment groups at the 0.5 μM/mg genipin treatment, the MPU treatment group showed the smallest size, most uniform protein particle distribution, and the highest ESI (59.89%). Additionally, the highest α-helix (41.96%) in the MPU + G5 group may be conducive to the formation of a stable and multilayer oil-water interface. Furthermore, the free groups, solubility, and protein exposure extent of the MPU groups were higher than those of UMP and Native groups. Therefore, this work suggests that the treatment of cross-linking followed by ultrasound (MPU) could be a desirable approach for improving the emulsifying stability of MP.

Topics: Emulsions; Iridoids; Molecular Docking Simulation; Proteins

A food dye from a South American fruit has become a test case for the ethical development of natural resources.

Topics: Food Coloring Agents; Fruit; Iridoids; Natural Resources; Rubiaceae

Topics: Animals; Antioxidants; Chemical and Drug Induced Liver Injury; Cholestasis; Glutathione; Iridoids; Liver; Mice

Glutamate-induced oxidative stress is well-known to play a crucial role in the development of neurodegenerative diseases, such as stroke. Genipin, a natural iridoid compound, has demonstrated potential neuroprotective properties but is unstable in physiological conditions. The present study aimed to develop new derivatives of genipin that exhibit improved stability and activity for the treatment of stroke. Nineteen new derivatives were thus designed and synthesized. Their neuroprotective effect against glutamate-induced injury was evaluated in HT22 cells. Among the newly synthesized derivatives, 3e demonstrated significantly greater neuroprotection and improved stability compared to genipin. Specifically, 0.01 μM of 3e was found to effectively attenuate glutamate-induced oxidative damage by inhibiting ROS over-accumulation, reducing MDA content, and restoring the endogenous antioxidative system. Further investigation revealed that 3e inhibited oxidative stress by downregulating the phosphorylation levels of p38 MAPK and activating Nrf2 and HO-1 proteins. These results suggested that 3e has the potential to serve as a promising candidate for the treatment of stroke by protecting against glutamate-induced oxidative stress.

Topics: Cell Line; Glutamic Acid; Iridoids; Molecular Structure; Neuroprotective Agents; NF-E2-Related Factor 2; Oxidative Stress; Reactive Oxygen Species

Topics: Animals; Guinea Pigs; Intraocular Pressure; Iridoids; Myopia; Sclera

Genipin, an aglycone of geniposide, is a rich iridoid component in the fruit of

Topics: Animals; Chromatography, High Pressure Liquid; Iridoids; Mass Spectrometry; Rats; Rats, Sprague-Dawley

Immobilizing microorganisms inside 3D printed semi-permeable substrates can be desirable for biotechnological processes since it simplifies product separation and purification, reducing costs, and processing time. To this end, we developed a strategy for synthesizing a feedstock suitable for 3D bioprinting of mechanically rigid and insoluble materials with embedded living bacteria. The processing route is based on a highly particle-filled alumina/chitosan nanocomposite gel which is reinforced by (a) electrostatic interactions with alginate and (b) covalent binding between the chitosan molecules with the mild gelation agent genipin. To analyze network formation and material properties, we characterized the rheological properties and printability of the feedstock gel. Stability measurements showed that the genipin-crosslinked chitosan/alginate/alumina gels did not dissolve in PBS, NaOH, or HCl after 60 days of incubation. Alginate-containing gels also showed less swelling in water than gels without alginate. Furthermore, E. coli bacteria were embedded in the nanocomposites and we analyzed the influence of the individual bioink components as well as of the printing process on bacterial viability. Here, the addition of alginate was necessary to maintain the effective viability of the embedded bacteria, while samples without alginate showed no bacterial viability. The experimental results demonstrate the potential of this approach for producing macroscopic bioactive materials with complex 3D geometries as a platform for novel applications in bioprocessing.

Topics: Alginates; Aluminum Oxide; Chitosan; Gels; Iridoids; Nanocomposites; Printing, Three-Dimensional

Tissue cross-linking represents an important and often used technique to enhance the mechanical properties of biomaterials. For the first time, we investigated biochemical and structural properties of genipin (GE) cross-linked equine pericardium (EP) using optical imaging techniques in tandem with quantitative atomic force microscopy (AFM). EP was cross-linked with GE at 37 °C, and its biochemical and biomechanical properties were observed at various time points up to 24 h. GE cross-linked EP was monitored by the normalized ratio between its second-harmonic generation (SHG) and two-photon autofluorescence emissions and remained unchanged for untreated EP; however, a decreasing ratio due to depleted SHG and elevated autofluorescence and a fluorescence band at 625 nm were found for GE cross-linked EP. The mean autofluorescence lifetime of GE cross-linked EP also decreased. The biochemical signature of GE cross-linker and shift in collagen bands were detected and quantified using shifted excitation Raman difference spectroscopy as an innovative approach for tackling artifacts with high fluorescence backgrounds. AFM images indicated a higher and increasing Young's modulus correlated with cross-linking, as well as collagen structural changes in GE cross-linked EP, qualitatively explaining the observed decrease in the second-harmonic signal. In conclusion, we obtained detailed information about the biochemical, structural, and biomechanical effects of GE cross-linked EP using a unique combination of optical and force microscopy techniques in a nondestructive and label-free manner.

Topics: Animals; Collagen; Elastic Modulus; Horses; Iridoids; Pericardium

Developing strategies to regulate the immune response poses significant challenges with respect to the clinical translation of tissue-engineered scaffolds. Prominent advancements have been made relating to macrophage-based therapies and biomaterials. Macrophages exhibit the potential to influence healing trajectory, and predominance of particular subtypes during early onset of healing influences repair outcomes. This study evaluated short- and long-term healing response and postoperative mechanical properties of genipin-cross-linked, electrochemically aligned collagen biotextiles with comparative administration of M0, M1, and M2 subtypes. Irrespective of macrophage subtype seeded, all the groups demonstrated existence of M2 macrophages at both time points as typified by arginase and Ym-1 expressions, and distinct absence of M1 macrophages, as indicated by lack of inducible nitric oxide synthase (iNOS) and interleukin-1β expression in all the groups for both time points. M2 macrophage-seeded collagen biotextiles revealed promising host tissue responses, such as reduced fibrous capsule thickness and minimal granulation tissue formation. Furthermore, the M2-seeded group displayed more abundant interstitial collagen deposition following degradation of the collagen threads. M2 macrophage supplementation improved structural and mechanical properties at the tissue and cellular level as indicated by increased modulus and stiffness. This study demonstrates improved biomechanical and histological outcomes following incorporation of M2 macrophages into genipin-cross-linked collagen biotextiles for tissue repair and offers future strategies focused on connective tissue regeneration. Impact statement Macrophages exhibit significant plasticity with complex phenotypes ranging from proinflammatory (M1) to proregenerative (M2). They release cytokines and chemokines governing immunological stability, inflammation resolution, and tissue healing and regeneration. However, utilization of macrophages as therapeutic tools for tissue engineering remains limited. In this study, genipin-cross-linked collagen biotextiles were employed to deliver M0, M1, and M2 macrophages and evaluate tissue responses and postsurgical mechanical properties

Topics: Collagen; Iridoids; Macrophages; Tissue Engineering; Tissue Scaffolds

Chitosan (CHI) based hydrogels promote wound healing and relieve inflammations and chronic infections. However, in hardly healable ulcers with excessively painful inflammations, anti-inflammatory activity of hydrogels can be enhanced by the sustained release of non-steroidal anti-inflammatory drugs or combining them with antibiotics. Thus, CHI was crosslinked with genipin (GP) to obtain biocompatible hydrogels. Moreover, their antibacterial activity was confirmed against Staphylococcus aureus and Escherichia coli with an almost 100% bacteria reduction and a potential antibacterial efficacy (R > 2). Furthermore, hydrogels effective healing of ulcerated wounds was corroborated by a significant improvement in metabolic activity (95.58 ± 4.40%), collagen and elastin quantities (1.48 ± 0.07 μg collagen and 5.82 ± 0.73 μg elastin per mg dermal tissue) and histological analysis. Finally, the sustained release of acetylsalicylic acid (ASA), cefuroxime (CFX), tetracycline (TCN) and amoxicillin (AMX) were studied, as well as their anti-inflammatory activity. Results confirm the synergistic anti-inflammatory activity by the significant reduction in the amount of pro-inflammatory cytokines when ASA was combined with CFX (5.39 ± 0.81 ng·mL

Topics: Anti-Bacterial Agents; Anti-Inflammatory Agents; Chitosan; Hydrogels; Iridoids; Wound Healing

Articular cartilage degeneration is still an unsolved issue owing to its weak repairing capabilities, which usually result in fibrocartilage tissue formation. This fibrous tissue lacks of structural and bio-mechanical properties, degrading over time. Currently, arthroscopic techniques and autologous transplantation are the most used clinical procedures. However, rather than restoring cartilage integrity, these methods only postpone further cartilage deterioration. Therefore, tissue engineering strategies aimed at selecting scaffolds that remarkably support the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) could represent a promising solution, but they are still challenging for researchers. In this study, the influence of two different genipin (Gp) crosslinking routes on collagen (Coll)-based scaffolds in terms of hMSCs chondrogenic differentiation and biomechanical performances was investigated. Three-dimensional (3D) porous Coll scaffolds were fabricated by freeze-drying techniques and were crosslinked with Gp following a "two-step" and an in "bulk" procedure, in order to increase the physico-mechanical stability of the structure. Chondrogenic differentiation efficacy of hMSCs and biomechanical behavior under compression forces through unconfined stress-strain tests were assessed. Coll/Gp scaffolds revealed an isotropic and highly homogeneous pore distribution along with an increase in the stiffness, also supported by the increase in the Coll denaturation temperature (T

Topics: Cartilage, Articular; Cell Differentiation; Cells, Cultured; Chondrogenesis; Collagen; Humans; Iridoids; Mesenchymal Stem Cells; Tissue Engineering; Tissue Scaffolds

Bone tissue repair is difficult due to the dense structure of the extracellular matrix. To solve this problem, a porous chitosan nanofiber scaffold (CSNFS) with an extracellular matrix-like structure was prepared via a facile cross-linked reaction of root-like chitosan nanofiber (CSNF) and collagen (Col) by using genipin (Gen) as the cross-linker. The optimal preparation conditions of CSNFS is weight ratio of CSNF:Col:Gen =1:1:0.1, crosslinked 48 h under 37 °C. CSNFS shows high porosity with adequate micro-scale pores, and its BET data shows that there are a large number of nano-scale pores. The CSNFS mechanical strength is higher than that of the chitosan scaffold both in dry and wet state. MC3T3 cells grow well on CSNFS, can overgrow the scaffold in three-dimensional space, adhere and differentiate well within those nanofiber structure. The cross-linked CSNFS has good biocompatibility and can be used as a repair material for bone tissue engineering.

Topics: Biocompatible Materials; Chitosan; Collagen Type I; Iridoids; Nanofibers; Osteoblasts; Porosity; Tissue Engineering; Tissue Scaffolds

Scleral crosslinking using genipin has been identified as a promising treatment approach for myopia control. The efficacy of genipin to alter biomechanical properties of the sclera has been shown in several animal models of myopia but its safety profile remains unclear. In this safety study, we aim to investigate the effect of scleral crosslinking using retrobulbar injections of genipin on retinal structure and function at genipin doses that were shown to be effective in slowing myopia progression in juvenile tree shrews. To this end, three or five retrobulbar injections of genipin at 0 mM (sham), 10 mM, or 20 mM were performed in one eye every other day. Form deprivation myopia was induced in the injected eye. We evaluated retinal function using full-field electroretinography and retinal structure using in vivo optical coherence tomography imaging and ex vivo histology. The optical coherence tomography results revealed significant thinning of the peripapillary retinal nerve fiber layer in all genipin treated groups including the lowest dose group, which showed no significant treatment effect in slowing myopia progression. In contrast, inducing form deprivation myopia alone and in combination with sham injections caused no obvious thinning of the retinal nerve fiber layer. Electroretinography results showed a significant desensitizing shift of the b-wave semi-saturation constant in the sham group and the second highest genipin dose group, and a significant reduction in b-wave maxima in the two highest genipin dose groups. The ex vivo histology revealed noticeable degeneration of photoreceptors and retinal pigment epithelium in one of two investigated eyes of the highest genipin dose group. While scleral crosslinking using genipin may still be a feasible treatment option for myopia control, our results suggest that repeated retrobulbar injections of genipin at 10 mM or higher are not safe in tree shrews. An adequate and sustained delivery strategy of genipin at lower concentrations will be needed to achieve a safe and effective scleral crosslinking treatment for myopia control in tree shrews. Caution should be taken if the proposed treatment approach is translated to humans.

Topics: Animals; Iridoids; Myopia; Sclera; Tupaiidae

Chitosan microgels are of significant interest in tissue engineering due to their wide range of applications, low cost, and immunogenicity. However, chitosan microgels are commonly fabricated using emulsion methods that require organic solvent rinses, which are toxic and harmful to the environment. The present protocol presents a rapid, non-cytotoxic, non-emulsion-based method for fabricating chitosan-genipin microgels without the need for organic solvent rinses. The microgels described herein can be fabricated with precise size control. They exhibit sustained release of biomolecules, making them highly relevant for tissue engineering, biomaterials, and regenerative medicine. Chitosan is crosslinked with genipin to form a hydrogel network, then passed through a syringe filter to produce the microgels. The microgels can be filtered to create a range of sizes, and they show pH-dependent swelling and degrade over time enzymatically. These microgels have been employed in a rat growth plate injury model and were demonstrated to promote increased cartilage tissue repair and to show complete degradation at 28 days in vivo. Due to their low cost, high convenience, and ease of fabrication with cytocompatible materials, these chitosan microgels present an exciting and unique technology in tissue engineering.

Topics: Animals; Chitosan; Emulsions; Iridoids; Microgels; Rats; Solvents; Tissue Engineering

Tympanic membrane perforation (TMP), a common disease, often needs a scaffold as the patch to support surgery. Due to the environment of auditory meatus, the patch can be infected by bacteria that results in failure; therefore, the ideal scaffold may combine biomimetic and antibacterial features. In this work, gelatin was used as the electrospinning framework, genipin as the crosslinking agent, and levofloxacin as an antibacterial in order to prepare the scaffold for TMP. Different contents of levofloxacin have been added to gelatin/genipin. It was found that, with the addition of levofloxacin, the gelatin/genipin membranes exhibit improved hydrophilia and enhanced tensile strength. The antibacterial and cell-cultured experiments showed that the prepared antibacterial membranes had excellent antibacterial properties and good biocompatibility, respectively. In summary, levofloxacin is a good group for the gelatin/genipin scaffold because it improves the physical properties and antibacterial action. Compared with different amounts of levofloxacin, a gelatin/genipin membrane with 1% levofloxacin is more suitable for a TM.

Topics: Anti-Bacterial Agents; Gelatin; Iridoids; Levofloxacin; Nanofibers; Tissue Scaffolds; Tympanic Membrane

A series of genipin derivatives included tricyclic cyclopentaimidazopyridine, cyclopentapyridopyrimidine, octahydrocyclopentapyridodiazepine, and tetracyclic decahydrobenzoimidazocyclopentapyridine were synthesized and developed as anti-inflammatory agents. All of them were tested against NO production in LPS-induced RAW264.7 cells. Based on IC

Topics: Animals; Anti-Inflammatory Agents; Cyclooxygenase 2; Indomethacin; Iridoids; Lipopolysaccharides; Mice; Nitric Oxide; RAW 264.7 Cells

Chemical cross-linking is an important step to grant satisfying properties to collagen-based materials. However, there are few comparative studies on crossing-linking of collagen-based fibrillar gels which are preferred biomaterials for similar properties to native tissues with different cross-linking agents. In this study, a fibrillar gel was fabricated with tilapia collagen and hyaluronic acid, and cross-linking conditions with EDC/NHS and genipin were discussed. Genipin gave gels much higher equilibrium cross-linking degree than EDC/NHS. ATR-FTIR and XPS showed EDC/NHS offered short-range cross-linking formed by amino and carboxyl groups in fibrils, while genipin induced long-range cross-linking by nucleophilic reaction through attack of amino groups in fibrils on carbon atoms at C-3 as well as ester groups in genipin, besides improved hydrogen bonds. XRD and SEM revealed the structural integrity of gels was strengthened after cross-linking, whereas fibril bundles disaggregated into thin fibrils. Consequently, swelling capacity and anti-degraded property were enhanced significantly, while thermal stability weakened. The fibrillar gels had good biocompatibility, but interestingly the appearance and migration of L929 fibroblasts were influenced by cross-linking degree. These results demonstrated that aquatic collagen-based fibrillar gel cross-linked by genipin had greater potential in biomaterials than EDC/NHS, whereas the cross-linking degree should be controlled.

Topics: Animals; Biocompatible Materials; Collagen; Cross-Linking Reagents; Gels; Hyaluronic Acid; Iridoids; Materials Testing; State Medicine; Tilapia

As a material for films used to keep fruits fresh, chitosan has attracted extensive interest because of its advantages of degradability, environmental friendliness, and biocompatibility. In this study, two amphiphilic chitosan derivative films were prepared by crosslinking N-2-hydroxypropyl-3-butyl ether-O-carboxymethyl chitosan (HBCC) and N-2-hydroxypropyl-3-(2-ethylhexyl glycidyl ether)-O-carboxymethyl chitosan (H2ECC)) with genipin, an excellent natural cross-linking agent. The microstructures, mechanical properties, water vapor permeability, swelling ratios, light transmittance, wettability, thermal stability, antibacterial properties, and biocompatibility of the crosslinked films were characterized. The results showed that the crosslinked films had compact structures, low moisture permeability, strong water resistance, strong ultraviolet resistance, unaffected visible light transmittance, and good hydrophilicity. Compared with the uncrosslinked films, the tensile strength of the genipin-crosslinked ones was increased by 328.33 % (HBCC) and 397.83 % (H2ECC). More importantly, the crosslinked films had strong antibacterial activity against Staphylococcus aureus and Escherichia coli and were non-toxic to endothelial cells. The crosslinked films could effectively prolong the preservation time of strawberries, inhibit the decay of strawberries, and inhibit the reduction of vitamin C in strawberries. In conclusion, genipin-crosslinked HBCC and H2ECC films are potential fruit preservation materials.

Topics: Anti-Bacterial Agents; Chitosan; Cross-Linking Reagents; Endothelial Cells; Escherichia coli; Fragaria; Iridoids

In this work, we studied the development of a biocomposite formulated with alginate and gelatin, crosslinked with genipin for application as support for β-galactosidase immobilization. Also, the biocomposites with the immobilized enzyme were characterized by thermal analyses and SAXS (size, density, and interconnectivity of alginate rods) for a detailed analysis of the microstructure, as well as the thermal and operational stabilities of the enzyme. The structural modifications of the biocomposite determined by SAXS demonstrate that the addition of both genipin and enzyme produced a significant reduction in size and density of the Ca(II)-alginate rods. Immobilized β-galactosidase could be stored for 175 days under refrigeration maintaining 80% of its initial activity. Moreover, 90% of its relative activity was kept after 11 reuses in a batch process of lactose hydrolysis. Thus, the biocomposite proved to be effective as support for enzyme immobilization.

Topics: Alginates; Aspergillus oryzae; beta-Galactosidase; Enzyme Stability; Enzymes, Immobilized; Gelatin; Hydrolysis; Iridoids; Lactose; Scattering, Small Angle; X-Ray Diffraction

The main goal of the study was to produce chitosan-collagen hydrogel composite scaffolds consisting of 3D printed poly(lactic acid) (PLA) strut and nanofibrous cellulose for meniscus cartilage tissue engineering. For this purpose, first PLA strut containing microchannels was incorporated into cellulose nanofibers and then they were embedded into chitosan-collagen matrix to obtain micro- and nano-sized topographical features for better cellular activities as well as mechanical properties. All the hydrogel composite scaffolds produced by using three different concentrations of genipin (0.1, 0.3, and 0.5%) had an interconnected microporous structure with a swelling ratio of about 400% and water content values between 77 and 83% which is similar to native cartilage extracellular matrix. The compressive strength of all the hydrogel composite scaffolds was found to be similar (∼32 kPa) and suitable for cartilage tissue engineering applications. Besides, the hydrogel composite scaffold comprising 0.3% (w/v) genipin had the highest tan δ value (0.044) at a frequency of 1 Hz which is around the walking frequency of a person. According to the in vitro analysis, this hydrogel composite scaffold did not show any cytotoxic effect on the rabbit mesenchymal stem cells and enabled cells to attach, proliferate and also migrate through the inner area of the scaffold. In conclusion, the produced hydrogel composite scaffold holds great promise for meniscus tissue engineering.

Topics: Animals; Cellulose; Chitosan; Collagen; Hydrogels; Iridoids; Meniscus; Polyesters; Printing, Three-Dimensional; Rabbits; Tissue Engineering; Tissue Scaffolds; Water

Currently, one of the biggest challenges of the colorant industry is to obtain natural blue colorants. Among the different options, a blue pigment can be formed by a crosslinking reaction between genipin and primary amine groups. However, at the industrial level, obtaining an ingredient from pure compounds, such as amino acids, is economically unfeasible. The present work aimed to study the reaction and kinetics of the blue color formation, starting the study with pure compounds (genipin and amino acids) to more complex and cheaper natural sources, such as Genipa americana L. fruits and milk. The reaction kinetics of the monomers/dimers for different amino acids reacting with genipin was evaluated, as well as the preferential amino acid, genipin:amino acid ratio and pH, to obtain the most rapid and intense blue color. Finally, the blue pigment formed using milk and its proteins was characterized by SDS-PAGE. The results suggest that the reaction kinetic is influenced by the type and concentration of the amino acid used and the pH of the medium, which could facilitate the further standardization of the industrial process. We also suggested milk as an excellent reaction medium to obtain the colorant from genipin as it presents an ideal pH and favorable amino acid composition to facilitate the reaction.

Topics: Amines; Amino Acids; Animals; Iridoids; Milk

Although three-dimensional (3D) co-culture of gingival keratinocytes and fibroblasts-populated collagen gel can mimic 3D structure of in vivo tissue, the uncontrolled contraction of collagen gel restricts its application in clinical and experimental practices. We here established a stable 3D gingival tissue equivalent (GTE) using hTERT-immortalized gingival fibroblasts (hGFBs)-populated collagen gel directly crosslinked with genipin/cytochalasin D and seeding hTERT-immortalized gingival keratinocytes (TIGKs) on the upper surface for a 2-week air-liquid interface co-culture. MTT assay was used to measure the cell viability of GTEs. GTE size was monitored following culture period, and the contraction was analyzed. Immunohistochemical assay was used to analyze GTE structure. qRT-PCR was conducted to examine the mRNA expression of keratinocyte-specific genes. Fifty µM genipin (G50) or combination (G + C) of G50 and 100 nM cytochalasin D significantly inhibited GTE contraction. Additionally, a higher cell viability appeared in GTEs crosslinked with G50 or G + C. GTEs crosslinked with genipin/cytochalasin D showed a distinct multilayered stratified epithelium that expressed keratinocyte-specific genes similar to native gingiva. Collagen directly crosslinked with G50 or G + C significantly reduced GTE contraction without damaging the epithelium. In summary, the TIGKs and hGFBs can successfully form organotypic multilayered cultures, which can be a valuable tool in the research regarding periodontal disease as well as oral mucosa disease. We conclude that genipin is a promising crosslinker with the ability to reduce collagen contraction while maintaining normal cell function in collagen-based oral tissue engineering.

Topics: Cells, Cultured; Collagen; Cytochalasin D; Fibroblasts; Gingiva; Humans; Iridoids; Keratinocytes; Tissue Engineering

Natural, high-performance fibers generally have hierarchically organized nanosized building blocks. Inspired by this, whey protein nanofibrils (PNFs) are assembled into microfibers, using flow-focusing. By adding genipin as a nontoxic cross-linker to the PNF suspension before spinning, significantly improved mechanical properties of the final fiber are obtained. For curved PNFs, with a low content of cross-linker (2%) the fiber is almost 3 times stronger and 4 times stiffer than the fiber without a cross-linker. At higher content of genipin (10%), the elongation at break increases by a factor of 2 and the energy at break increases by a factor of 5. The cross-linking also enables the spinning of microfibers from long straight PNFs, which has not been achieved before. These microfibers have higher stiffness and strength but lower ductility and toughness than those made from curved PNFs. The fibers spun from the two classes of nanofibrils show clear morphological differences. The study demonstrates the production of protein-based microfibers with mechanical properties similar to natural protein-based fibers and provides insights about the role of the nanostructure in the assembly process.

Topics: Iridoids; Nanostructures; Proteins; Tensile Strength

In this work, genipin crosslinked chitin nanofibril hydrogels/cryogels were successfully prepared. Due to the acidic disperse nature of positively charged chitin fibrils, the pH nature for the chemical crosslinking between chitin nanofibrils and genipin was restricted under 5.0. The pH nature and crosslinking temperature were found greatly influence the efficient chemical crosslinking between chitin nanofibril and genipin. Further ice templated crosslinking was found effective enhancing the mechanical properties of resulting chitin nanofibril hydrogels/cryogels. The cryogel prepared by genipin-crosslinked DEChNs showed interconnected pores, presenting a state of stacking layers. In addition, the deposition of hydroxyapatite (HA) on the surface of chitin nanofibril scaffolds was found effective for improving chondrocyte attachments and growths. The cells viability indicated the good cell compatibility of the cryogel. All the results indicated that the cryogel prepared by genipin-crosslinked DEChNs showed a potential for tissue engineering applications.

Topics: Chitin; Cross-Linking Reagents; Cryogels; Ice; Iridoids; Tissue Engineering; Tissue Scaffolds

Many surgical treatments require a suitable tissue adhesive that maintains its performance in wet conditions and can be applied simultaneously for hard and soft tissues. In the present study, a dual cross-linked tissue adhesive was synthesized by mixing the gelatin methacryloyl (Gel-MA) and gelatin-dopamine conjugate (Gel-Dopa). The setting reaction was based on a photopolymerization process in the presence of a combination of riboflavin and triethanolamine and a chemical cross-linking process attributed to the genipin as a natural cross-linker. Modified gelatin macromolecules were characterized and the best wavelength for free radical generation in the presence of riboflavin was obtained. Tissue adhesives were prepared with 30% hydrogels of Gel-MA and Gel-Dopa with different ratios in distilled water. The gelation occurred in a short time after light irradiation. The chemical, mechanical, physical, and cytotoxicity properties of the tissue adhesives were evaluated. The results showed that despite photopolymerization, chemical crosslinking with genipin played a more critical role in the setting process. Water uptake, degradation behavior, cytotoxicity, and adhesion properties of the adhesives were correlated with the ratio of the components. The SEM images showed a porous structure that could ensure the entry of cells and nutrients into the surgical area. While acceptable properties in most experiments were observed, all features were improved as the Gel-Dopa ratio increased. Also, the obtained hydrogels revealed excellent adhesive properties, particularly with bone even after wet incubation, and it was attributed to the amount of gelatin-dopamine conjugate. From the obtained results, it was concluded that a dual adhesive hydrogel based on gelatin macromolecules could be a good candidate as a tissue adhesive in wet condition.

Topics: Adhesives; Dihydroxyphenylalanine; Dopamine; Gelatin; Hydrogels; Iridoids; Methacrylates; Riboflavin; Tissue Adhesives; Water

Herein, a strong and stable gelatin-based composite hydrogel was fabricated by incorporation of amino-functionalized microfibrillated cellulose (AMFC) into gelatin matrix along with genipin crosslinking. The hydrogel consists of chemical and physical crosslinks among gelatin chains and AMFC fibrils. The morphology, swelling behavior and compressive properties of the composite hydrogels were investigated. The results show that the mechanical properties and structural stability of the gelatin hydrogels were improved remarkably by the addition of AMFC due to the formation of a hybrid network structure. The composite hydrogel has a compressive strength up to 1.52 MPa at a strain of 80 %, which is 41.2 and 1.8 times higher than that of the conventional physical and genipin-crosslinked gelatin hydrogels, respectively. Moreover, the developed gelatin-based composite hydrogels reinforced with AMFC exhibit good enzymatic stability, high surface hydrophobicity, tunable swelling property and excellent biocompatibility, which are expected to have potential applications in biomedical and pharmaceutical fields.

Topics: Cellulose; Gelatin; Hydrogels; Iridoids

Background and aims: Genipin, an iridoid derived from geniposide by β-glucosidase hydrolysis, has shown potential benefit in the treatment of heart function insufficiency despite its unclear therapeutic mechanism. This study aimed to investigate the primary cardioprotective mechanism of genipin. We hypothesized that genipin demonstrated the antiapoptosis and anti-inflammation for cardiac protection by inhibiting the cyclooxidase 2 (COX2)-prostaglandin D2 (PGD2) and murine double minute 2 (MDM2)-p53 pathways. Methods: The normal Sprague-Dawley rats were made into myocardial infarction models by conventional methods. Animals were treated with genipin for 5 weeks after myocardial infarction (MI). Morphometric and hemodynamic measurements were performed 5 weeks post-MI. Biological and molecular experiments were performed after the termination. Results: Both morphometry and hemodynamics in systole and diastole were significantly impaired in the model group but restored close to basal level after treatment with genipin. Genipin also restored the post-MI upregulated expressions of cytochrome c, p53, COX2, and PGD2 and downregulated expression of MDM2 to the approximate baseline. Genipin inhibited apoptotic and inflammatory pathways to prevent post-MI structure-function remodeling. Conclusions: This study showed the cardioprotective mechanism of genipin and implied its potential clinical application for the treatment of ischemic heart failure.

Topics: Animals; Cyclooxygenase 2; Heart Failure; Iridoids; Myocardial Infarction; Prostaglandin D2; Rats; Rats, Sprague-Dawley; Signal Transduction; Tumor Suppressor Protein p53

The kidney tubule consists of a single layer of epithelial cells supported by the tubular basement membrane (TBM), a thin layer of specialized extracellular matrix (ECM). The mechanical properties of the ECM are important for regulating a wide range of cell functions including proliferation, differentiation and cell survival. Increased ECM stiffness plays a role in promoting multiple pathological conditions including cancer, fibrosis and heart disease. How changes in TBM mechanics regulate tubular epithelial cell behavior is not fully understood. Here we introduce a cell culture system that utilizes in vivo-derived TBM to investigate cell-matrix interactions in kidney proximal tubule cells. Basement membrane mechanics was controlled using genipin, a biocompatibility crosslinker. Genipin modification resulted in a dose-dependent increase in matrix stiffness. Crosslinking had a marginal but statistically significant impact on the diffusive molecular transport properties of the TBM, likely due to a reduction in pore size. Both native and genipin-modified TBM substrates supported tubular epithelial cell growth. Cells were able to attach and proliferate to form confluent monolayers. Tubular epithelial cells polarized and assembled organized cell-cell junctions. Genipin modification had minimal impact on cell viability and proliferation. Genipin stiffened TBM increased gene expression of pro-fibrotic cytokines and altered gene expression for N-cadherin, a proximal tubular epithelial specific cell-cell junction marker. This work introduces a new cell culture model for cell-basement membrane mechanobiology studies that utilizes in vivo-derived basement membrane. We also demonstrate that TBM stiffening affects tubular epithelial cell function through altered gene expression of cell-specific differentiation markers and induced increased expression of pro-fibrotic growth factors.

Topics: Basement Membrane; Epithelial Cells; Iridoids; Kidney Tubules; Kidney Tubules, Proximal

To study the long-term safety of genipin treatment using a vacuum device with or without epithelial cells at different crosslinking times.. Twenty-five healthy New Zealand white rabbits were separated into five treatment groups: 0.25% genipin with epithelial cells for 5 minutes (G1), 0.25% genipin without epithelial cells for 5 minutes (G2), 0.25% genipin without epithelial cells for 10 minutes (G3), ultraviolet A-riboflavin collagen crosslinking (UVA), and controls (C). Before and 2, 4, 6, and 8 weeks after crosslinking treatment, anterior segment optical coherence tomography (ASOCT), in vivo confocal microscopy (IVCM), and the Pentacam system were used to evaluate the right eyes.. A demarcation line (DL) was observed in the corneal stroma in the G2, G3, and UVA groups. The DL depths in the G2 and G3 groups were stable but decreased in the UVA group over time. The density of keratocytes in these groups increased. Endothelial cell density was decreased in the UVA group. There were no differences in the endothelium before and after treatment in the G1, G2, G3, and C groups. The densitometry, as determined using the Pentacam system, significantly increased in the G2, G3, and UVA groups and was positively correlated with keratocyte densities.. A vacuum ring assisting local genipin immersion crosslinking without corneal epithelium can activate the keratocytes in the corneal stroma and was safe enough for the thin cornea.. Genipin can not only crosslink the collagen fibers but also activate the keratocytes and even may promote collagen fiber secretion.

Topics: Animals; Cornea; Corneal Stroma; Cross-Linking Reagents; Endothelium; Iridoids; Rabbits; Ultraviolet Rays

The minimally invasive treatment of intracranial aneurysms by endovascular coiling is attractive yet faces challenges related to the degradation of fibrin clots in the aneurysm sac over time. Fibrin gels cross-linked with genipin exhibit enhanced mechanical and chemical stability, but there are many unknowns related to best practices for delivery from endovascular devices and subsequent integration of cross-linkers with the nascent clot. Here, we describe the

Topics: Cross-Linking Reagents; Humans; Intracranial Aneurysm; Iridoids; Kinetics

In this study, we chemically modified poly(L-lactic acid) (PLLA) with functional amine groups and fabricated a PLLA membrane crosslinked with genipin as a biomembrane for inducing guided bone regeneration (GBR). The mechanical strength of the PLLA-amine membrane was improved by crosslinking with genipin compared to pure PLLA membrane. The surface of the PLLA-amine membrane crosslinked with genipin had many more uniform pores. Attachment and proliferation of MC3T3-E1 cells were increased and improved on the PLLA-amine membrane crosslinked with genipin. In an in vitro osteogenesis study, MC3T3-E1 cells on the PLLA membrane showed higher alkaline phosphatase (ALP) activity and calcification ability evaluated by alizarin red S staining than those on the pure PLLA membrane. When a skull defect hole of a rat was covered with the PLLA-amine membrane crosslinked with genipin, vigorous new bone regeneration determined by computed tomography at 8 weeks post operation was superior to that when the skull defect was covered with the pure PLLA membrane. Taken together, these results demonstrate that the PLLA-amine membrane crosslinked with genipin has a promising therapeutic application to GBR as a barrier membrane for covering the defect site.

Topics: Animals; Bone Regeneration; Calcification, Physiologic; Cell Proliferation; Guided Tissue Regeneration; Iridoids; Lactic Acid; Male; Mice; Osteogenesis; Polyesters; Rats; Rats, Sprague-Dawley; Skull

Surgical adhesives have partly replaced traditional sutures to seal and reattach tissues due to their superiorities in preventing liquids leakage and avoiding secondary damage in the surrounding wound area. Most of the existing adhesives are committed to promoting wound healing and functional recovery. A therapeutic adhesive that assists in clearing the residual tumors in the surgical area is undoubtedly meaningful to obtain a better clinical outcome. Herein, enlightened by commercial BioGlue (albumin/glutaraldehyde sealant), a biocompatible therapeutic albumin/genipin bioglue is designed for postoperative wound adhesion and tumor ablation. The albumin/genipin bioglue is formed by simple mixing of bovine serum albumin (BSA) and genipin (GP) under a 35 °C water bath for 24 h without further purification. The obtained dark-blue fluorescent adhesive exhibits a significant temperature increase accompanied by heating-induced curing once irradiated with an 808-nm laser. This unique characteristic allows BSA-GP a therapeutic adhesive for postoperative wound adhesion and photothermal elimination of residual tumors under laser irradiation. Moreover, its easy injectability and impressive photothermal efficacy also make it feasible for in situ tumor photothermal ablation. The ultrasimple synthetic strategy by mimicking BioGlue endows BSA-GP adhesive with large-scale production capacity and clinical transformation potential, which is a successful paradigm for reforming existing clinical products.

Topics: Humans; Iridoids; Neoplasm, Residual; Proteins; Serum Albumin, Bovine; Tissue Adhesives

Adipose-derived stem cells (ASCs) are an abundant and easily accessible multipotent stem cell source with potential application in smooth muscle regeneration strategies. In 3D collagen hydrogels, we investigated whether sustained release of growth factors (GF) PDGF-AB and TGF-β1 from GF-loaded microspheres could induce a smooth muscle cell (SMC) phenotype in ASCs, and if the addition of uniaxial cyclic stretch could enhance the differentiation level. This study demonstrated that the combination of cyclic stretch and GF release over time from loaded microspheres potentiated the differentiation of ASCs, as quantified by protein expression of early to late SMC differentiation markers (SMA, TGLN and smooth muscle MHC). The delivery of GFs via microspheres produced large ASCs with a spindle-shaped, elongated SMC-like morphology. Cyclic strain produced the largest, longest, and most spindle-shaped cells regardless of the presence or absence of growth factors or the growth factor delivery method. Protein expression and cell morphology data confirmed that the sustained release of GFs from GF-loaded microspheres can be used to promote the differentiation of ASCs into SMCs and that the addition of uniaxial cyclic stretch significantly enhances the differentiation level, as quantified by intermediate and late SMC markers and a SMC-like elongated cell morphology.

Topics: Adipose Tissue; Adult; Biomarkers; Bioreactors; Cell Differentiation; Cell Shape; Cells, Cultured; Cross-Linking Reagents; Gelatin; Gels; Humans; Intercellular Signaling Peptides and Proteins; Iridoids; Microspheres; Myocytes, Smooth Muscle; Phenotype; Stem Cells; Stress, Mechanical

Mounting evidence has shown that single-targeted therapy might be inadequate to achieve satisfactory effects. Thus, drug combinations are gaining attention as they can regulate multiple targets to obtain more beneficial effects. Heat shock protein 90 (HSP90) is a molecular chaperone that assists the protein assembly and folding of client proteins and maintains their stability. Interfering with the interaction between HSP90 and its client proteins by inhibiting the latter's activity may offer a new approach toward combination therapy. The HSP90 client protein AKT plays an important role in the inflammatory response syndrome caused by infections. In this study, the dietary flavone baicalein was identified as a novel inhibitor of HSP90 that targeted the N-terminal ATP binding pocket of HSP90 and hindered the chaperone cycle, resulting in AKT degradation. Combining baicalein with genipin, which was extracted from

Topics: Animals; Antioxidants; Cholagogues and Choleretics; Diet; Drug Delivery Systems; Drug Therapy, Combination; Flavanones; Gene Expression Regulation; HSP90 Heat-Shock Proteins; Inflammation; Iridoids; Lipopolysaccharides; Male; Mice; Phosphorylation; Proto-Oncogene Proteins c-akt; Pseudomonas aeruginosa; Pseudomonas Infections; Random Allocation; RAW 264.7 Cells

To evaluate the antileishmanial efficacy of genipin, which specifically inhibits uncoupling protein 2 (UCP2) that is induced in leishmaniasis to neutralize reactive oxygen species (ROS).. The effect of genipin was assessed against intracellular parasites in cultured macrophages and in suppressing spleen and liver parasite burdens in a BALB/c mouse model of visceral leishmaniasis by microscopic evaluation of intracellular amastigotes stained with Giemsa. ROS and mitochondrial membrane potential were measured by H2DCFDA- and JC-1-based fluorometric analysis. ELISA was performed for various Th1 and Th2 cytokines in both in vitro and in vivo infected conditions to evaluate the type of immunological responses. The role of UCP2 was assessed by lipofectamine-mediated transfection and overexpression in macrophages and short hairpin RNA-mediated knockdown of UCP2 in infected animals.. Genipin reduced the infection-induced UCP2 levels in macrophages, with optimum effect at 100 μM. Genipin reversed parasite-induced ROS suppression and mitochondrial membrane potential disruption. It has no inhibitory effect on promastigote or axenic amastigote forms, but markedly suppressed amastigote multiplication within macrophages, which was reversed by the ROS scavenger N-acetyl cysteine. Genipin administration (30 mg/kg/day) in infected mice showed significant suppression of liver and spleen parasite burdens with an enhanced host-favourable cytokine balance in a ROS-p38 mitogen-activated protein kinase-dependent manner. Co-treatment with genipin plus a sublethal dose of sodium antimony gluconate (SAG50) showed almost a curative reduction in spleen and liver parasite burden.. These results suggest the effectiveness of genipin as a synergistic agent for the front-line antileishmanial drug SAG in circumventing the resistance and toxicity problems associated with its high curative dose.

Topics: Animals; Immunologic Factors; Iridoids; Leishmaniasis, Visceral; Mice; Mice, Inbred BALB C; Pharmaceutical Preparations; Reactive Oxygen Species; Uncoupling Protein 2

Punicalagin (PA) not only binds type II collagen, but also blocks its MMP-13-mediated degradation, and genipin (GNP) is a collagen cross-linking agent. We hypothesized that these drugs could mitigate the loss of cartilage if administered in the early phase of osteoarthritis, and experiments were designed to provide proof-of-concept. Porcine cartilage was exposed to both drugs in a manner designed to simulate intra-articular (IA) injection. Based on penetration of PA into cartilage, the rate of drug diffusion was conservatively estimated at 2 μm per minute. GNP caused a measurable degree of cross-linking, increased compressive resistance and coefficient of friction, and substantially inhibited degradation by collagenase, but not by hyaluronidase. Pre-incubation of GNP with collagenase had no effect on enzymatic activity. PA did not cross-link collagen nor affect the mechanical properties of cartilage. It did, however, increase resistance to degradation by collagenase and hyaluronidase. Furthermore, it reacted with collagenase in solution and inhibited its subsequent enzymatic activity. Effects of PA and GNP were not additive. The chondroprotective effect of semi-weekly IA injections was investigated in the monoiodoacetate-induced model of OA in rats. Quantitative histology suggested that injection of PA decreased the amount of cartilage lost compared to saline-injected controls, and the addition of GNP made no difference. This study supports the notion that IA delivery of PA could mitigate OA-induced cartilage erosion.

Topics: Animals; Cartilage, Articular; Hydrolyzable Tannins; Injections, Intra-Articular; Iridoids; Male; Osteoarthritis; Rats; Swine

An emerging approach toward repair of connective tissues applies exogenous crosslinkers to mechanically augment injured structures in vivo. One crosslinker that has been explored for this purpose is the plant-derived small molecule genipin. However, genipin's high reactivity to primary amines in proteins, small size, and high diffusion coefficient necessitate localizing and controlling its delivery to avoid off-target or adverse effects. In this study, genipin-loaded polymers were evaluated for sustained local administration. Insoluble polymers comprising subunits of α-, β-, or γ-cyclodextrin, cyclic oligosaccharides possessing increasing cavity sizes, were compared to polymers comprising subunits of the non-cyclic polysaccharide dextran. Polymers made from β-cyclodextrin showed prolonged genipin release for over ten times longer than polymers made from α- or γ-cyclodextrins or dextran, indicating that genipin possesses molecular affinity for the β-cyclodextrin cavity. Modeling of complexation between genipin and cyclodextrin hosts supported this finding. Genipin released from all polymers was confirmed to be functional by exogenous collagen crosslinking through fluorometric and mechanical readouts. Co-incubation of genipin-loaded polymers with bovine tendon explants showed genipin crosslink-mediated coloration that was confined to the sites of exposure. Altogether, results indicate that host-guest interactions within a polymeric delivery vehicle can help to control and confine genipin release.

Topics: Animals; Cattle; Connective Tissue; Delayed-Action Preparations; Iridoids; Polymers

Topics: Animals; Anti-Inflammatory Agents; Cyclooxygenase 2; Dipeptides; Down-Regulation; Drug Design; Iridoids; Lipopolysaccharides; Macrophages; Mice; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; RAW 264.7 Cells; Signal Transduction; Tumor Necrosis Factor-alpha

Genipin, an iridoid substance, is mainly derived from Gardenia jasminoides Ellis of the traditional Chinese medicine and is widely used in raw materials for the food additive gardenia blue and biological materials. The developmental toxicity of genipin has not been investigated, and its underlying mechanism is unclear. Therefore, in this study we attempt to investigate the potential developmental toxicity of genipin in zebrafish embryos/larvae. The results showed zebrafish embryos treated with 50 μg/ml dose of genipin display inhibited hatching rates and body length. The pericardial edema was observed. It was also found that genipin could induce cardio-toxicity, hepatotoxicity and nephrotoxicity in zebrafish larvae. After genipin treatment, the suppression of antioxidant capacity and increase of oxidative stress were showed for the triggered generation of ROS and MDA, and decreased activity of SOD. Compared with the 0.5% DMSO group, a number of apoptotic cells in zebrafish were increased after genipin exposure. By measuring marker gene expression with the using of qRT-PCR, we proposed that developmental toxicity after genipin treatment might be associated with oxidative stress and apoptosis increase. Our research offers a better understanding for developmental toxicity of genipin.

Topics: Animals; Apoptosis; Biomarkers; Cholagogues and Choleretics; Embryo, Nonmammalian; Gene Expression Regulation, Developmental; Iridoids; Larva; Malondialdehyde; Oxidative Stress; Superoxide Dismutase; Zebrafish

Additive manufacturing holds promise for the fabrication of three-dimensional scaffolds with precise geometry, to serve as substrates for the guided regeneration of natural tissue. In this work, a bioinspired approach is adopted for the synthesis of hybrid hydroxyapatite hydrogels, which were subsequently printed to form 3D scaffolds for bone tissue engineering applications. These hydrogels consist of hydroxyapatite nanocrystals, biomimetically synthesized in the presence of both chitosan and l-arginine. To improve their mechanical properties, chemical crosslinking was performed using a natural crosslinking agent (genipin), and their rheology was modified by employing an acetic acid/gelatin solution. Regarding the 3D printing process, several parameters (flow, infill and perimeter speed) were studied in order to accurately produce scaffolds with predesigned geometry and micro-architecture, while also applying low printing temperature (15 °C). Following the printing procedure, the 3D scaffolds were freeze dried in order to remove the entrapped solvents and therefore, obtain a porous interconnected network. Evaluation of porosity was performed using micro-computed tomography and nanomechanical properties were assessed through nanoindentation. Results of both characterization techniques, showed that the scaffolds' porosity as well as their modulus values, fall within the corresponding range of the respective values of cancellous bone. The biocompatibility of the 3D printed scaffolds was assessed using MG63 human osteosarcoma cells for 7 days of culturing. Cell viability was evaluated by MTT assay as well as double staining and visualized under fluorescence microscopy, while cell morphology was analyzed through scanning electron microscopy. Biocompatibility tests, revealed that the scaffolds constitute a cell-friendly environment, allowed them to adhere on the scaffolds' surface, increase their population and maintain high levels of viability.

Topics: Chitosan; Durapatite; Humans; Iridoids; Porosity; Printing, Three-Dimensional; Tissue Engineering; Tissue Scaffolds; X-Ray Microtomography

Seven tacrine/CHR21 conjugates have been designed and synthesized. Compound 8-7 was confirmed as the most active AChE inhibitor with IC

Topics: Acetylcholinesterase; Alzheimer Disease; Autophagy; Drug Design; Humans; Iridoids; Molecular Structure; Structure-Activity Relationship; Tacrine

The small amount of proteins in starch-rich food industry byproducts can be an advantage to crosslink with genipin and tailor the performance of biobased films. In this work, genipin was combined with non- purified starch recovered from industrial potato washing slurries and used for films production. Starch recovered from potato washing slurries contained 0.75% protein, 2 times higher than starch directly obtained from potato and 6 times higher than the commercial one. Starch protein-genipin networks were formed with 0.05% and 0.10% genipin, gelatinized at 75 °C and 95 °C in presence of 30% glycerol. Bluish colored films were obtained in all conditions, with the higher surface roughness (Ra, 1.22 μm), stretchability (elongation, 31%), and hydrophobicity (water contact angle, 127°) for 0.10% genipin and starch gelatinized at 75 °C. Therefore, starch-rich byproducts, when combined with genipin, are promising for surpassing the starch-based films hydrophilicity and mechanical fragilities while providing light barrier properties.

Topics: Biofuels; Cross-Linking Reagents; Food Industry; Iridoids; Mechanical Phenomena; Microscopy, Atomic Force; Plant Proteins; Rheology; Solanum tuberosum; Solubility; Starch; Surface Properties; Suspensions

Genipin is a nontoxic natural cross-linker that was successfully used to prepare cross-linked enzyme aggregates (CLEAs) of Trametes versicolor laccase. The recovered activity of CLEAs was influenced by the co-solvent type, genipin concentration, cross-linking time, preparation pH, and bovine serum albumin (BSA; amino group feeder) concentration. The characteristics of CLEAs prepared using genipin under optimal conditions (genipin-BSA-CLEAs) were compared with those of typical CLEAs prepared using glutaraldehyde or dextran polyaldehyde. Genipin-BSA-CLEAs were nano-sized (average diameter, approximately 700 nm), had a ball-like shape, showed a narrow size distribution, and exhibited the highest substrate affinity among the prepared CLEAs. The thermal stability of genipin-BSA-CLEAs was 6.8-fold higher than that of free laccase, and their pH stability was also much higher than that of free laccase in the tested range. Additionally, genipin-BSA-CLEAs retained 85% of their initial activity after 10 cycles of reuse. Particularly, genipin-BSA-CLEAs showed higher thermal and pH stability than CLEAs that were cross-linked using glutaraldehyde. Therefore, genipin represents an alternative to toxic compounds such as glutaraldehyde during cross-linking to prepare CLEAs.

Topics: Cross-Linking Reagents; Enzyme Stability; Enzymes, Immobilized; Glutaral; Hydrogen-Ion Concentration; Iridoids; Kinetics; Laccase; Polyporaceae; Serum Albumin, Bovine; Temperature; Trametes

This study examined the release of vitamin B6 from a hydrogel made of whey protein isolate (WPI). Work was carried out at ambient temperature without preheating the whey protein. Native-state macromolecules were crosslinked with a nontoxic compound, genipin. Experimentation included a ninhydrin assay with UV-vis absorbance, FTIR,

Topics: Compressive Strength; Diffusion; Drug Carriers; Drug Liberation; Hydrogels; Iridoids; Vitamin B 6; Whey Proteins

Acellular nerve allografts (ANGs) represent a promising alternative in nerve repair. Our aim is to improve the structural and biomechanical properties of biocompatible Sondell (SD) and Roosens (RS) based ANGs using genipin (GP) as a crosslinker agent ex vivo. The impact of two concentrations of GP (0.10% and 0.25%) on Wistar rat sciatic nerve-derived ANGs was assessed at the histological, biomechanical, and biocompatibility levels. Histology confirmed the differences between SD and RS procedures, but not remarkable changes were induced by GP, which helped to preserve the nerve histological pattern. Tensile test revealed that GP enhanced the biomechanical properties of SD and RS ANGs, being the crosslinked RS ANGs more comparable to the native nerves used as control. The evaluation of the ANGs biocompatibility conducted with adipose-derived mesenchymal stem cells cultured within the ANGs confirmed a high degree of biocompatibility in all ANGs, especially in RS and RS-GP 0.10% ANGs. Finally, this study demonstrates that the use of GP could be an efficient alternative to improve the biomechanical properties of ANGs with a slight impact on the biocompatibility and histological pattern. For these reasons, we hypothesize that our novel crosslinked ANGs could be a suitable alternative for future in vivo preclinical studies.

Topics: Biocompatible Materials; Biomechanical Phenomena; Cross-Linking Reagents; Extracellular Matrix; Histocytochemistry; Iridoids; Nerve Regeneration; Nerve Tissue; Tissue Engineering; Tissue Scaffolds

Type 2 diabetes mellitus (T2DM) has a harmful effect on the stability and osseointegration of dental implants. T2DM induces mitochondrial damage by inhibiting AMPK signaling, resulting in oxidative stress and poor osteogenesis in the peri-implant bone area. Genipin is a major component of gardenia fruits with strong antioxidant, anti-inflammation, and antidiabetic actions, and it also can activate mitochondrial quality control via the AMPK pathway. The purpose of this study was to investigate the effects of genipin and insulin treatment on implant osseointegration in T2DM rats and explore the underlying mechanisms.. Streptozotocin-induced diabetic rats received implant surgery in their femurs and were then assigned to five groups that were subjected to different treatments for three months: control group, T2DM group, insulin-treated T2DM group (10 IU/kg), genipin-treated T2DM group (50 mg/kg), and the genipin and insulin combination-treated T2DM group. Then, we regularly assessed the weight and glucose levels of the animals. Rats were euthanized at 3 months after the implantation procedure, and the femora were harvested for microscopic computerized tomography analysis, biomechanical tests, and different histomorphometric assessment.. The results indicated that the highest blood glucose and oxidative stress levels were measured for the T2DM group, resulting in the poorest osseointegration. The combination-treated T2DM group mitigated hyperglycemia and normalized, reactivated AMPK signaling, and alleviated oxidative stress as well as reversed the negative effect of osseointegration. There were beneficial changes observed in the T2DM-genipin and T2DM-insulin groups, but these were less in comparison to the combination treatment group.. Our study suggests that treatment with genipin in combination with insulin could be an effective method for promoting implant osseointegration in T2DM rats, which may be related to AMPK signaling.

Topics: AMP-Activated Protein Kinases; Animals; Anti-Inflammatory Agents; Antioxidants; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Disease Models, Animal; Drug Therapy, Combination; Femur; Hypoglycemic Agents; Insulin; Iridoids; Male; Osseointegration; Oxidative Stress; Phytotherapy; Prostheses and Implants; Rats, Sprague-Dawley; Signal Transduction; Streptozocin

The objective of this research was to evaluate the immobilization of the enzyme β-galactosidase in a genipin-activated chitosan support. The influence of the number of spheres and substrate concentration on immobilization yield (IY) and enzyme activity (EA) was analyzed using experimental design. Thermal, operational and storage stabilities were assessed, and the enzymatic derivatives were characterized by thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). The TGA showed that the enzymatic derivatives kept their thermal behavior, and the SEM images revealed smooth surfaces in all the spheres. The optimized conditions for the immobilization process were 4.57 mg·mL

Topics: Animals; beta-Galactosidase; Chitosan; Enzyme Stability; Enzymes, Immobilized; Hydrolysis; Iridoids; Kluyveromyces; Lactose; Milk

The growth plate is a cartilage tissue near the ends of children's long bones and is responsible for bone growth. Injury to the growth plate can result in the formation of a 'bony bar' which can span the growth plate and result in bone growth abnormalities in children. Biomaterials such as chitosan microgels could be a potential treatment for growth plate injuries due to their chondrogenic properties, which can be enhanced through loading with biologics. They are commonly fabricated via an emulsion method, which involves solvent rinses that are cytotoxic. Here, we present a high throughput, non-cytotoxic, non-emulsion-based method to fabricate chitosan-genipin microgels. Chitosan was crosslinked with genipin to form a hydrogel network, and then pressed through a syringe filter using mesh with various pore sizes to produce a range of microgel particle sizes. The microgels were then loaded with chemokines and growth factors and their release was studied in vitro. To assess the applicability of the microgels for growth plate cartilage regeneration, they were injected into a rat growth plate injury. They led to increased cartilage repair tissue and were fully degraded by 28 days in vivo. This work demonstrates that chitosan microgels can be fabricated without solvent rinses and demonstrates their potential for the treatment of growth plate injuries.

Topics: Animals; Biocompatible Materials; Cartilage; Chitosan; Disease Models, Animal; Emulsions; Iridoids; Male; Microgels; Rats; Rats, Sprague-Dawley; Regeneration; Salter-Harris Fractures

Towards optimizing the growth of extracellular matrix to produce repair cartilage for healing articular cartilage (AC) defects in joints, scaffold-based tissue engineering approaches have recently become a focus of clinical research. Scaffold-based approaches by electrospinning aim to support the differentiation of chondrocytes by providing an ultrastructure similar to the fibrillar meshwork in native cartilage. In a first step, we demonstrate how the blending of chitosan with poly(ethylene oxide) (PEO) allows concentrated chitosan solution to become electrospinnable. The chitosan-based scaffolds share the chemical structure and characteristics of glycosaminoglycans, which are important structural components of the cartilage extracellular matrix. Electrospinning produced nanofibrils of ∼100 nm thickness that are closely mimicking the size of collagen fibrils in human AC. The polymer scaffolds were stabilized in physiological conditions and their stiffness was tuned by introducing the biocompatible natural crosslinker genipin. We produced scaffolds that were crosslinked with 1.0% genipin to obtain values of stiffness that were in between the stiffness of the superficial zone human AC of 600 ± 150 kPa and deep zone AC of 1854 ± 483 kPa, whereas the stiffness of 1.5% genipin crosslinked scaffold was similar to the stiffness of deep zone AC. The scaffolds were degradable, which was indicated by changes in the fibril structure and a decrease in the scaffold stiffness after seven months. Histological and immunohistochemical analysis after three weeks of culture with human articular chondrocytes (HACs) showed a cell viability of over 90% on the scaffolds and new extracellular matrix deposited on the scaffolds.

Topics: Biocompatible Materials; Cartilage, Articular; Cells, Cultured; Chitosan; Chondrocytes; Cross-Linking Reagents; Humans; Iridoids; Nanofibers; Polyethylene Glycols; Regeneration; Tissue Scaffolds

Genipin was reacted with benzylamine and several amino acids to prepare gardenia blue (GB). The time-course of GB formation with benzylamine was monitored by high-performance liquid chromatography (HPLC), liquid chromatography time-of-flight mass spectrometry (LC-TOFMS), and

Topics: Amino Acids; Benzylamines; Gardenia; Iridoids; Molecular Structure

PEG-interpenetrated dual-sensitive hydrogels that load nano lipid carrier (NLC) were researched and developed for topical drug administration. Natural antioxidant α-lipoic acid (ALA) was selected as our model drug. The α-lipoic acid (ALA) nano lipid carrier was successfully prepared by hot melt emulsification and ultrasonic dispersion method, and the physicochemical properties of the nano lipid carrier were investigated, including morphology, particle distribution, polydispersity coefficient, zeta potential and encapsulation efficiency. Carboxymethyl chitosan and poloxamer 407 contributed to pH- and temperature-sensitive properties in the hydrogel, respectively. Natural non-toxic cross-linking agent genipin reacted with carboxymethyl chitosan to form the hydrogel. Poly ethylene glycol (PEG), a polymer compound with good water solubility and biocompatibility, interpenetrated the hydrogel and influenced the mechanical strength and drug release behaviour. FI-IR test verified the successful synthesis of the hydrogel. The rheological parameters indicated that the mechanical strength of the hydrogel was positively correlated with the amount of PEG, and the

Topics: Administration, Topical; Drug Carriers; Drug Compounding; Hydrogels; Iridoids; Lipids; Nanostructures; Polyethylene Glycols

Genipin-1-β-d-gentiobioside (GG) is a kind of compound extracted from Gardenia jasminoides Ellis. The chemical structure of GG is similar to that of geniposide and has antidiabetic effects. We aimed to investigate the efficacy of GG on diabetic nephropathy (DN) in vivo and in vitro experiments and explore its potential mechanism.. For high-fat diet/streptozotocin-induced DN mice used in our study, the general features of mice were analysed after GG treatment. Oxidative stress parameters and inflammatory factors were also measured by commercial kits. Kidney damage was assessed using hematoxylin and eosin (H&E), periodic acid-Schiff (PAS) and Masson staining, respectively. In vitro, podocyte injury was assessed by TUNEL and flow cytometric analyses. AMP-activated protein kinase/silencing information regulator related enzyme 1 (AMPK/SIRT1)/nuclear factor-κB (NF-κB) pathway-related proteins were detected by AMPK-siRNA intervention and western blotting.. Treatment of GG could increase cell survival and attenuated kidney damage. Despite the presence of inflammatory and oxidative stress, when GG retained the expression of AMPK/SIRT1, it could be observed that the downstream NLRP3 inflammatory-related proteins were inhibited.. Results showed that the protective efficacy of GG on DN works together with hypoglycemia and suppressing oxidative stress and inflammation, which at least partly involved in APMK/SIRT1/NF-κB-dependent pathway.

Topics: AMP-Activated Protein Kinases; Animals; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Gardenia; Inflammation; Iridoids; Kidney; Male; Mice, Inbred C57BL; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Oxidative Stress; Phytotherapy; Plant Extracts; Podocytes; Sirtuin 1

The demand for biodegradable sustained release carriers with minimally invasive and less frequent administration properties for therapeutic proteins and peptides has increased over the years. The purpose of achieving sustained minimally invasive and site-specific delivery of macromolecules led to the investigation of a photo-responsive delivery system. This research explored a biodegradable prolamin, zein, modified with an azo dye (DHAB) to synthesize photo-responsive azoprolamin (AZP) nanospheres loaded with Immunoglobulin G (IgG). AZP nanospheres were incorporated in a hyaluronic acid (HA) hydrogel to develop a novel injectable photo-responsive nanosystem (HA-NSP) as a potential approach for the treatment of chorio-retinal diseases such as age-related macular degeneration (AMD) and diabetic retinopathy. AZP nanospheres were prepared via coacervation technique, dispersed in HA hydrogel and characterised via infrared spectroscopy (FTIR), X-ray diffraction (XRD) and thermogravimetric analysis (TGA). Size and morphology were studied via scanning electron microscopy (SEM) and dynamic light scattering (DLS), UV spectroscopy for photo-responsiveness. Rheological properties and injectability were investigated, as well as cytotoxicity effect on HRPE cell lines. Particle size obtained was <200 nm and photo-responsiveness to UV = 365 nm by decreasing particle diameter to 94 nm was confirmed by DLS. Encapsulation efficiency of the optimised nanospheres was 85% and IgG was released over 32 days up to 60%. Injectability of HA-NSP was confirmed with maximum force 10 N required and shear-thinning behaviour observed in rheology studies. In vitro cell cytotoxicity effect of both NSPs and HA-NSP showed non-cytotoxicity with relative cell viability of ≥80%. A biocompatible, biodegradable injectable photo-responsive nanosystem for sustained release of macromolecular IgG was successfully developed.

Topics: Azo Compounds; Drug Carriers; Drug Delivery Systems; Humans; Hyaluronic Acid; Hydrogels; Immunoglobulin G; Injections; Iridoids; Light; Macromolecular Substances; Nanomedicine; Nanospheres; Particle Size; Phototherapy; Prolamins; Rheology; Temperature; X-Ray Diffraction

In this work, a non-toxic chitosan-based carrier was constructed via genipin activation and applied for the immobilization of tannase. The immobilization carriers and immobilized tannase were characterized using Fourier transform infrared spectroscopy and thermogravimetric analysis. Activation conditions (genipin concentration, activation temperature, activation pH and activation time) and immobilizations conditions (enzyme amount, immobilization time, immobilization temperature, immobilization pH, and shaking speed) were optimized. The activity and activity recovery rate of the immobilized tannase prepared using optimal activation and immobilization conditions reached 29.2 U/g and 53.6%, respectively. The immobilized tannase exhibited better environmental adaptability and stability. The immobilized tannase retained 20.1% of the initial activity after 12 cycles and retained 81.12% of residual activity after 30 days storage. The catechins composition analysis of tea extract indicated that the concentration of non-ester-type catechins, EGC and EC, were increased by 1758% and 807% after enzymatic treatment. Biological activity studies of tea extract revealed that tea extract treated with the immobilized tannase possessed higher antioxidant activity, higher inhibitory effect on α-amylase, and lower inhibitory effect on α-glucosidase. Our results demonstrate that chitosan activated with genipin could be an effective non-toxic carrier for tannase immobilization and enhancing biological activities of tea extract.

Topics: alpha-Amylases; Antioxidants; Camellia sinensis; Carboxylic Ester Hydrolases; Chitosan; Drug Carriers; Drug Compounding; Enzyme Stability; Glycoside Hydrolase Inhibitors; Iridoids; Plant Extracts; Temperature; Time Factors

Wound dressing composed of polyelectrolyte complexes (PECs), based on chitosan/alginate/hyaluronic acid (CS/ALG/HYA) crosslinked by genipin, was prepared by freeze-dried molding. Genipin as excellent natural biological crosslinker was chose for high biocompatibility and improving mechanical properties of materials. The CS/ALG/HYA sponges (CAHSs) were characterized by FTIR, XRD, DSC and SEM. Porosity, swelling behavior and mechanical properties and in vitro degradation of CAHSs were investigated. The cytotoxicity assay was carried out on HUVEC cells in vitro and the result proves the good biocompatibility of CAHSs. Hemolysis tests indicated that the prepared CAHSs were non-hemolytic material (hemolysis ratio < 5%, no cytotoxicity). PT and aPPT coagulation tests demonstrated that CAHS2 and CAHS3 could both activate the extrinsic and intrinsic coagulation pathway and thus accelerated blood coagulation. Further, in a rat full-thickness wounds model, the CAHS2 sponge significantly facilitates wound closure compared to other groups. CAHSs exhibited adjustable physical, mechanical and biological properties. Thus, the chitosan-based polyelectrolyte composite sponges exhibit great potential as promising wound dressings.

Topics: Alginates; Animals; Biological Dressings; Chitosan; Cross-Linking Reagents; Female; Human Umbilical Vein Endothelial Cells; Humans; Hyaluronic Acid; Iridoids; Nanocomposites; Polyelectrolytes; Rabbits; Rats; Rats, Wistar

It is well known that cells can generate endogenous forces onto the extracellular matrix, but to what extent the mechanical properties of the matrix influences these endogenous cellular forces remains unclear. We therefore sought to quantify the influence of matrix rigidity on cell-matrix interactions by inducing cross-links using increasing concentrations of genipin (0.01-1 mM) or by blocking cross-link formation using beta-aminopropionitrile (BAPN) in engineered human tendon tissue constructs. The cell-matrix mechanics of the tendon constructs were evaluated as cell-generated tissue re-tensioning and stress-relaxation responses using a novel custom-made force monitor, which can apply and detect tensional forces in real-time in addition to mechanical failure testing. Genipin treatment had no influence on the biochemical profile (hydroxyproline, glycosaminoglycan and DNA content) of the constructs and cell viability was comparable between genipin-treated and control constructs, except at the highest genipin concentration. Endogenous re-tension after unloading was significantly decreased with increasing genipin concentrations compared to controls. Mechanical failure testing of tendon constructs showed increased (56%) peak stress at the highest genipin concentration but decreased (72%) with BAPN treatment when compared to controls. Tendon construct stiffness increased with high genipin concentrations (0.1 and 1 mM) and decreased by 70% in BAPN-treated constructs, relative to the controls. These data demonstrate that human tendon fibroblasts regulate their force exertion inversely proportional to increased cross-link capacity but did so independently of matrix stiffness. Overall, these findings support the notion of an interaction between cell force generation and cross-linking, and thus a role for this interplay in mechanical homeostasis of the tissue.

Topics: Collagen; Cross-Linking Reagents; Humans; Iridoids; Tendons; Tissue Engineering

An ideal scaffold for endodontic regeneration should allow the predictableness of the new tissue organization and limit the negative impact of residual bacteria. Therefore, composition and functionalization of the scaffold play an important role in tissue bioengineering. The objective of this study was to assess the morphological, physicochemical, biological and antimicrobial properties of a new solid chitosan-based scaffold associated with gelatin, microparticulate dentin and genipin.. Scaffolds based on chitosan (Ch); chitosan associated with gelatin and genipin (ChGG); and chitosan associated with gelatin, microparticulate dentin and genipin (ChGDG) were prepared by using the freeze-drying method. The morphology of the scaffolds was analyzed by scanning electron microscopy (SEM). The physicochemical properties were assessed for biodegradation, swelling and total released proteins. The biological aspects of the scaffolds were assessed using human cells from the apical papilla (hCAPs). Cell morphology and adhesion to the scaffolds were evaluated by SEM, cytotoxicity and cell proliferation by MTT reduction-assay. Cell differentiation in scaffolds was assessed by using alizarin red assay. The antimicrobial effect of the scaffolds was evaluated by using the bacterial culture method, and bacterial adhesion to the scaffolds was observed by SEM.. All the scaffolds presented porous structures. The ChCDG had more protein release, adhesion, proliferation and differentiation of hCAPs, and bacteriostatic effect on Enterococcus faecalis than Ch and ChGG (p < 0.05).. The chitosan associated with gelatin, microparticulate dentin and genipin has morphological, physicochemical, biological and antibacterial characteristics suitable for their potential use as scaffold in regenerative endodontics.

Topics: Chitosan; Dentin; Gelatin; Humans; Iridoids; Porosity; Regeneration; Tissue Engineering; Tissue Scaffolds

Physically crosslinked hydrogels with thixotropic properties attract considerable attention in the biomedical research field because their self-healing nature is useful in cell encapsulation, as injectable gels, and as bioinks for three-dimensional (3D) bioprinting. Here, we report the formation of thixotropic hydrogels containing nanofibers of double-hydrophobic elastin-like polypeptides (ELPs). The hydrogels are obtained with the double-hydrophobic ELPs at 0.5 wt%, the concentration of which is an order of magnitude lower than those for previously reported ELP hydrogels. Although the kinetics of hydrogel formation is slower for the double-hydrophobic ELP with a cell-binding sequence, the storage moduli G' of mature hydrogels are similar regardless of the presence of a cell-binding sequence. Reversible gel-sol transitions are demonstrated in step-strain rheological measurements. The degree of recovery of the storage modulus G' after the removal of high shear stress is improved by chemical crosslinking of nanofibers when intermolecular crosslinking is successful. This work would provide deeper insight into the structure-property relationships of the self-assembling polypeptides and a better design strategy for hydrogels with desired viscoelastic properties.

Topics: Amino Acid Sequence; Cross-Linking Reagents; Elastic Modulus; Elastin; Hydrogels; Hydrophobic and Hydrophilic Interactions; Iridoids; Nanofibers; Peptides; Rheology

Alzheimer's disease (AD) is a devastating brain disorder characterized by neurofibrillary tangles and amyloid plaques. Inhibiting Tau protein and amyloid-beta (Aβ) production or removing these molecules is considered potential therapeutic strategies for AD. Genipin is an aglycone and is isolated from the extract of Gardenia jasminoides Ellis fruit. In this study, the effect and molecular mechanisms of genipin on the inhibition of Tau aggregation and Aβ generation were investigated. The results showed that genipin bound to Tau and protected against heparin-induced Tau fibril formation. Moreover, genipin suppressed Tau phosphorylation probably by downregulating the expression of CDK5 and GSK-3β, and activated mTOR-dependent autophagy via the SIRT1/LKB1/AMPK signaling pathway in Tau-overexpressing cells. In addition, genipin decreased Aβ production by inhibiting BACE1 expression through the PERK/eIF2α signaling pathway in N2a/SweAPP cells. These data indicated that genipin could effectively lead to a significant reduction of phosphorylated Tau level and Aβ generation in vitro, suggesting that genipin might be developed into an effective therapeutic complement or a potential nutraceutical for preventing AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Cell Line, Tumor; Dose-Response Relationship, Drug; HEK293 Cells; Humans; Iridoids; Mice; Mice, Transgenic; Phosphorylation; Protein Structure, Tertiary; tau Proteins

The aim of the present study was to fabricate and characterise chitosan scaffolds from animal and fungal sources, with or without gelatine as a co-polymer, and cross-linked to 3-glycidyloxyproply trimethoxysilane (GPTMS) or genipin for application in dental root tissue engineering.\ Chitosan-based scaffolds were prepared by the emulsion freeze-drying technique. Scanning electron microscopy (SEM) and nano-focus computed tomography (nano-CT) were used to characterise scaffold microstructure. Chemical composition and cross-linking were evaluated by Fourier transform infrared-attenuated total reflectance spectroscopy. Compression tests were performed to evaluate scaffold mechanical properties. Scaffold degradation was evaluated by gravimetric method and SEM. Scaffold bioactivity immersed in simulated body fluid was evaluated by SEM, with associated electron dispersive X-ray spectroscopy, and apatite formation was examined by X-ray diffraction. Finally, human dental pulp stem cells (hDPSCs) viability was evaluated.\ The fabrication method used was successful in producing scaffolds with organised porosity. Chitosan source (animal vs. fungal), co-polymerisation with gelatine and cross-linking using GPTMS or genipin had a significant effect on scaffold properties and hDPSCs response. Chitosan-genipin (CS-GEN) scaffolds had the largest pore diameter, while the chitosan-gelatine-GPTMS (CS-GEL-GPTMS) scaffolds had the smallest. Animal chitosan-gelatine co-polymerisation increased scaffold compressive strength, while fungal chitosan scaffolds (fCS-GEL-GPTMS) had the fastest degradation rate, losing 80 % of their weight by day 21. Gelatine co-polymerisation and GPTMS cross-linking enhanced chitosan scaffolds bioactivity through the formation of an apatite layer as well as improved hDPSCs attachment and viability.\ Tailored chitosan scaffolds with tuned properties and favourable hDPSCs response can be obtained for regenerative dentistry applications.

Topics: Adolescent; Adult; Biocompatible Materials; Cells, Cultured; Chitosan; Compressive Strength; Dental Pulp; Female; Humans; Iridoids; Male; Materials Testing; Porosity; Printing, Three-Dimensional; Silanes; Stem Cells; Tissue Engineering; Tissue Scaffolds; Young Adult

Full thickness cutaneous wound therapy and regeneration remains a critical challenge in clinical therapeutics. Recent reports have suggested that mesenchymal stem cells exosomes therapy is a promising technology with great potential to efficiently promote tissue regeneration. Multifunctional hydrogel composed of both synthetic materials and natural materials is an effective carrier for exosomes loading. Herein, we constructed a biodegradable, dual-sensitive hydrogel encapsulated human umbilical cord-mesenchymal stem cells (hUCMSCs) derived exosomes to facilitate wound healing and skin regeneration process. The materials characterization, exosomes identification, and

Topics: Animals; Cell Movement; Collagen; Drug Liberation; Exosomes; Female; Hydrogels; Hydrogen-Ion Concentration; Iridoids; Mesenchymal Stem Cells; Particle Size; Rats; Rats, Sprague-Dawley; Skin; Wound Healing

Gardeniae Fructus (Zhizi in Chinese, ZZ in brief), a commonly used herbal medicine, has aroused wide concern for hepatotoxicity, but the mechanism remains to be investigated. This study was aimed at investigating the mechanism of ZZ-induced liver injury in vivo and in vitro based on metabolomics and evaluating the hepatotoxicity prediction ability of the in vitro model. SD rats were administered with extracted ZZ and HepG2 cells were treated with genipin, the major hepatotoxic metabolite of ZZ. Liver, plasma, intracellular and extracellular samples were obtained for metabolomics analysis. As a result, ZZ caused plasma biochemical and liver histopathological alterations in rats, and induced purine and amino acid metabolism disorder in the liver and pyrimidine, primary bile acids, amino acid metabolism and pantothenate and CoA biosynthesis disorder in the plasma. Pyrimidine, purine, amino acid metabolism and pantothenate and CoA biosynthesis were also found to be disturbed in the genipin-treated HepG2 cells, which exhibited similarity with the result in vivo. This study comprehensively illustrates the underlying mechanism involved in ZZ-related hepatotoxicity from the aspect of metabolome, and provides evidence that identifying hepatotoxicity can be achieved in cells, representing a non-animal alternative for systemic toxicology.

Topics: Animals; Cell Survival; Chemical and Drug Induced Liver Injury; Fruit; Gardenia; Hep G2 Cells; Humans; Iridoids; Plant Extracts; Rats; Rats, Sprague-Dawley

Bio-adhesives are essential for wound healing because of their convenience and safety. Although widely used as biomaterials, silk fibroin's (SF's) further application as bio-adhesive is hindered due to its weak stickiness with tissue and slow gelation speed. Here, a dopamine-modified SF-based bio-adhesive is fabricated by using genipin as the chemical cross-linking agent. Furthermore, metal ions have been used to adjust the adhesion property of the bio-adhesive. The experimental results shows that the dopamine-modified SF-based composite holds a better stickiness except slow gelation speed. The doping of Cu

Topics: 3T3 Cells; Adhesives; Animals; Biocompatible Materials; Cell Movement; Copper; Dopamine; Fibroins; Iridoids; Iron; Mice; Skin; Swine

Loading propolis by a simple process using genipin as a crosslinking agent and fabrication of a novel PVA/Chitosan-Propolis membrane scaffolds were reported for wound dressing applications. The research is focused on the effects of propolis on characterization properties of membrane such as chemical structure, surface morphology, degradation ratio, crystallinity, hydrophilicity, water uptake capacity, water vapour transmission rate and mechanical aspect. It was noticed that water uptake capacity and hydrophilicity properties of membrane considerably affected by the propolis. By addition of (0.50, % v/v) propolis, the contact angle of the PVA/Chitosan membrane was remarkably decreased from 86.29° ± 3 to 45 ± 2°. 3-(4,5-dimethylthiazoyl-2-yl)-2,5-diphenylte-trazolium (MTT) bromide test and SEM were used to analyse the cytocompatibility of the membranes and morphology of cells on membrane. The propolis incorporated membrane showed cell proliferation rate 176 ± 13%, 775 ± 1%, and 853 ± 23%, at 24 h, 27 h and 120 h, respectively. SEM images also supported the cell behaviour on membrane. DNA fragmentation was also investigated with genotoxicity test. The studies on the interactions between membranes and MEF cells revealed that the incorporation of propolis into membrane promoted cell proliferation. These overall results presented that propolis incorporated membranes could have potentially appealing application as scaffolds for wound healing applications.

Topics: Biocompatible Materials; Cell Proliferation; Chitosan; DNA Fragmentation; Humans; Iridoids; Membranes, Artificial; Mutagenicity Tests; Propolis; Wound Healing

To evaluate the effect of scleral crosslinking (SXL) on slowing experimental progressive myopia in tree shrew eyes using sub-Tenon's injections of genipin (GEN) at different concentrations and number of injections.. Three or five sub-Tenon's injections of GEN at 0 mM (sham), 10 mM, or 20 mM were performed in one eye every other day starting at 18 days of visual experience. Form deprivation (FD) myopia was induced in the injected eye between 24 and 35 days of visual experience; the fellow eye served as control. Tree shrews were randomly assigned to five experimental groups: FD (n = 8); FD + 5 × sham injections (n = 6); FD + 3 × GEN injections at 10 mM (n = 6) and 20 mM (n = 6); and FD + 5 × GEN injections at 20 mM (n = 6). Refractive state and ocular dimensions were measured daily.. Compared with the FD group, the sham-injected group showed a transient effect on slowing vitreous chamber elongation. With increasing GEN dose, SXL had an increasing treatment effect on slowing vitreous chamber elongation and myopia progression. In addition, SXL led to a dose-dependent shortening of the aqueous chamber depth and corneal thickening. Lens thickening was observed in the group with the highest concentration.. We have shown that SXL using GEN can slow axial elongation and myopia progression in tree shrews. The extent of this treatment effect was dose dependent. Several unexpected effects were observed (corneal thickening, decrease of the anterior chamber depth, and lens thickening), which require further optimization of the GEN delivery approach before clinical consideration.. The results of this preclinical study suggest that scleral crosslinking using genipin can slow myopia progression.

Topics: Animals; Iridoids; Myopia, Degenerative; Refraction, Ocular; Sclera; Tupaiidae

Delivering hydrophobic molecules through the intestine can be challenging due to limited cargo solubility and the harsh biochemical environment of the stomach. Here, we show that a protein-based nanocarrier system based on the abundant protein histone and the natural cross-linker genipin can deliver hydrophobic cargos, such as dyes and therapeutic molecules, through the gastrointestinal tract. Using hydrophobic near-infrared dyes as model cargos, a panel of potential protein carriers was screened, and histone was identified as the one with the best loading capability. The resulting nanoparticles had a positive ζ potential and were mucoadhesive. Cross-linking of the amine-rich nanocarrier with genipin was particularly effective relative to other proteins and increased the stability of the system during incubation with pepsin. Cross-linking was required for successful delivery of a hydrophobic dye to the colon of mice after oral gavage. To assess the platform for therapeutic delivery, another hydrophobic model compound, curcumin, was delivered using cross-linked histone nanoparticles in a murine colitis model and significantly alleviated the disease. Taken together, these results demonstrate that histone is a cationic, mucoadhesive, and cross-linkable protein nanocarrier that can be considered for oral delivery.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Colitis; Cross-Linking Reagents; Curcumin; Drug Carriers; Female; Gastrointestinal Tract; Histones; Hydrophobic and Hydrophilic Interactions; Iridoids; Mice; Mice, Inbred ICR; Nanoparticles

Decellularized nerve extracellular matrix (NECM) composited with chitosan are moldable materials suitable for spinal cord repair. But the rapid biodegradation of the materials may interrupt neural tissue reconstruction in vivo. To improve the stability of the materials, the materials produced by NECM and chitosan hydrogels were crosslinked by genipine, glutaraldehyde or ultraviolet ray. Physicochemical property, degradation and biocompatibility of materials crosslinked by genipin, glutaraldehyde or ultraviolet ray were evaluated. The scaffold crosslinked by genipin possessed a porous structure, and the porosity ratio was 89.07 + 4.90%, the average diameter of pore was 85.32 + 5.34 μm. The crosslinked degree of the scaffold crosslinked by genipin and glutaraldehyde was 75.13 ± 4.87%, 71.25 ± 5.06% respectively; Uncrosslinked scaffold disintegrated when immerged in distilled water while the scaffold crosslinked by genipin and glutaraldehyde group retained their integrity. The scaffold crosslinked by genipin has better water absorption, water retention and anti-enzymatic hydrolysis ability than the other three groups. Cell cytotoxicity showed that the cytotoxicity of scaffold crosslinked by genipin was lower than that crosslinked by glutaraldehyde. The histocompatibility of scaffold crosslinked by genipin was also better than glutaraldehyde group. More cells grew well in the scaffold crosslinked by genipin when co-cultured with L929 cells. The decellularized nerve extracellular matrix/chitosan scaffold crosslinked by the genipin has good mechanical properties, micro structure and biocompatibility, which is an ideal scaffold for the spinal cord tissue engineering.

Topics: Acrylic Resins; Biocompatible Materials; Chitosan; Cross-Linking Reagents; Extracellular Matrix; Iridoids; Tissue Engineering; Tissue Scaffolds

This study aimed to investigate the possibility of UCP-2 inhibitor in reducing acquired resistance of trastuzumab to improve the outcome of patients receiving trastuzumab therapy by exploring the relationship between UCP-2 expression and HER2 signaling pathway and examining whether UCP-2 expression was modulated by trastuzumab treatment.. 32 women diagnosed with primary HER2-positive breast cancer were recruited in this study. Needle biopsy was obtained from patients before they received at least four cycles neoadjuvant therapy containing trastuzumab in combination with chemotherapy. Surgical tumor biopsy was obtained during surgical procedure after the neoadjuvant therapy. Levels of HER2 phosphorylation and UCP-2 expression were detected by immunohistochemistry (IHC) and compared between tumor needle biopsy tissue and surgical tumor samples of these patients, as well as in BT474 breast cancer cells before and after trastuzumab treatment. HER2-selective phosphorylation/kinase activity inhibitor ONT-380 was used to identify the correlation between HER2 phosphorylation level and UCP-2 expression. UCP-2 inhibitor Genipin was then used to evaluate the apoptosis index in BT474 cells treated with trastuzumab.. UCP-2 expression was significantly elevated in surgical tumor samples from breast cancer patients receiving trastuzumab in a neoadjuvant setting. We further confirmed our findings in HER2-positive BT474 cell line and found that trastuzumab treatment induced phosphorylation of HER2 and the overexpression of UCP-2, and the latter can be reversed by HER2 selective kinase inhibitor ONT-380. Moreover, UCP-2 inhibitor Genipin significantly enhanced the proliferation suppression effects of trastuzumab and markedly promoted apoptosis.. Taken together, our study identified UCP-2 as a novel therapeutic target for HER2 positive breast cancer and UCP-2 inhibitor may have great potential to enhance the response rate and efficacy of trastuzumab therapy.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Drug Resistance, Neoplasm; Female; Humans; Iridoids; Neoadjuvant Therapy; Oxazoles; Pyridines; Quinazolines; Receptor, ErbB-2; Trastuzumab; Uncoupling Protein 2

Mucoadhesive buccal patch is a promising dosage form for a successful oral drug delivery, which provides unique advantages for various applications such as treatment of periodontal disease and postdental surgery disorders. The aim of this study is to synthesize a novel multifunctional mucoadhesive buccal patch in a multilayer reservoir design for therapeutic applications. The patches were fabricated through simultaneous electrospinning of chitosan/poly(vinylalcohol) (PVA)/ibuprofen and electrospraying of phenylalanine amino acid nanotubes (PhNTs) containing metronidazole into the electrospun mats through a layer-by-layer process. An electrospun poly(caprolactone) (PCL) was used as an impermeable backing layer to protect the mucoadhesive component from tongue movement and drug loss. Buccal patches were characterized using scanning electron microscopy (SEM) and field emission scanning electron microscopy (FESEM) and also evaluated in terms of physicomechanical parameters such as pH, weight, thickness, tensile strength, folding endurance, and mucoadhesive properties. The swelling index of the patches was examined with respect to the PVA/chitosan ratio. The effect of genipin addition to the electrospinning solution was also studied on mucoadhesive and swelling properties. The cell viability of buccal patches was assessed by methylthiazolydiphenyl-tetrazolium bromide test on L929 fibroblast cell line. The patch with an optimal amount of mucoadhesive polymers (PVA/chitosan 80:20) and crosslinking agent (0.05 g) indicated an ideal hemostatic activity along with antibacterial properties against Streptococcus mutans bacteria. The synthesized multifunctional mucoadhesive patch with a novel composition and design has a great potential for oral therapeutic applications.

Topics: Administration, Buccal; Animals; Cheek; Chitosan; Cross-Linking Reagents; Drug Delivery Systems; Ibuprofen; Iridoids; Metronidazole; Mice; Microscopy, Electron, Scanning; Nanotubes; Phenylalanine; Polyvinyl Alcohol; Streptococcus mutans; Tensile Strength; Tissue Adhesives

Decellularized extracellular matrix (dECM) derived from natural ECM is receiving considerable interest as a promising component of tissue adhesives because of its high biocompatibility and tissue regenerative ability. However, the availability of dECM as a tissue adhesive is limited because of the lack of a gelator that can crosslink low concentrations of dECM to form hydrogels. Here, we report dECM-based tissue adhesives using a genipin gelator. Based on the pH-dependent reactivity of genipin, genipin-terminated 4 arm-poly(ethylene glycol) (GeniPEG) was synthesized. dECM-based hydrogels were formed within a few seconds of mixing GeniPEG and dECM at an optimum pH through crosslinking of dECM and self-crosslinking between GeniPEG molecules. The hydrogels crosslinked with GeniPEG exhibited greater tissue adhesive strength to porcine-derived aorta tissue than those crosslinked with genipin. Moreover, GeniPEG can be applied to various dECMs, including those from the urinary bladder, heart, liver, pancreas, and small intestine. In vivo implantation experiments demonstrated biocompatibility and biodegradability of the dECM-GeniPEG hydrogels. Therefore, this dECM-based hydrogel may extend the possibility and availability of dECM as an organ-specific tissue adhesive and contribute to successful minimally invasive surgery. STATEMENT OF SIGNIFICANCE: There is a strong need to develop highly functional tissue adhesives with high biocompatibility, tissue adhesive strength, and tissue regenerative ability. In this report, dECM-based tissue adhesives were reported using a pH-driven genipin-gelator. Focusing on the pH-dependent reactivity of genipin, genipin-based gelators were synthesized to form dECM-based hydrogels in response to pH changes. The crosslinking reaction proceeded within a few seconds to form hydrogels. The hydrogels obtained had greater tissue adhesion to aorta tissue than that of the free genipin crosslinker. This gelator can be applied to various types of dECMs. This dECM-based hydrogel had high biocompatibility and tissue adhesive properties and is useful for sealing wounds and preventing postoperative complications.

Topics: Adhesives; Animals; Extracellular Matrix; Hydrogels; Hydrogen-Ion Concentration; Iridoids; Swine; Tissue Adhesives; Tissue Engineering

M2 macrophages are associated with deposition of interstitial collagen and other extracellular matrix proteins during the course wound healing and also inflammatory response to biomaterials. Developing advanced biomaterials to promote the M2 subtype may be an effective way to improve tissue reinforcement surgery outcomes. In this study, the effect of genipin, a naturally derived crosslinking agent, on M0 → M2-polarization was investigated. Genipin was introduced either indirectly by seeding cells on aligned collagen biotextiles that are crosslinked by the agent or in soluble form by direct addition to the culture medium. Cellular elongation effects on macrophage polarization induced by the collagen biotextile were also investigated as a potential inducer of macrophage polarization. M0 and M2 macrophages demonstrated significant elongation on the surface of aligned collagen threads, while cells of the M1 subtype-maintained a round phenotype. M0 → M2 polarization, as reflected by arginase and Ym-1 production, was observed on collagen threads only when the threads were crosslinked by genipin, implicating genipin as a more potent inducer of the regenerative phenotype compared to cytoskeletal elongation. The addition of genipin to the culture medium directly also drove the emergence of pro-regenerative phenotype as measured by the markers (arginase and Ym-1) and through the activation of the pSTAT6-PPAR-gamma pathway. This study indicates that genipin-crosslinked collagen biotextiles can be used as a delivery platform to promote regenerative response after biomaterial implantation. STATEMENT OF SIGNIFICANCE: The immune response is one of the key determinants of tissue repair and regeneration rate, and outcome. The M2 macrophage subtype is known to resolve the inflammatory response and support tissue repair by producing pro-regenerative factors. Therefore, a biomaterial that promotes M2 sub-type can be a viable strategy to enhance tissue regeneration. In this study, we investigated genipin-crosslinked electrochemically aligned collagen biotextiles for their capacity to induce pro-regenerative polarization of M0 macrophages. The results demonstrated that genipin, rather than matrix-induced cellular elongation, was responsible for M0 → M2 polarization in the absence of other bioinductive factors and maintaining the M2 polarized status of macrophages. Furthermore, we identified that genipin polarizes the M2 macrophage phenotype via activation of the pSTAT6-PPAR-ga

Topics: Iridoids; Macrophage Activation; Macrophages; Peroxisome Proliferator-Activated Receptors

Inulin-type fructans with different degrees of polymerization (DPs) were used as wall materials for the blue colorant produced from the crosslinking between genipin and milk proteins. The impact of using fructooligosaccharides (FOS) with DP = 5 and inulins with DP ≥ 10 (GR-In) and DP ≥ 23 (HP-In) on the physical (microstructure, size, water activity, wettability, solubility, water adsorption, glass transition temperature, and color), chemical (free genipin retention and moisture), and technological (colorant power, pH stability, and thermal stability) properties of the powdered blue colorant was examined. Inulins were more efficient carriers as seen from the physical characteristics of the microparticles. FOS and GR-In promoted higher retention of free genipin than HP-In. Additionally, their lower DP influenced the rehydration proprieties as well as the color intensity and colorant power. The DP did not affect the physical stability of the colorant at different pH conditions or at high temperature. Our findings demonstrated that the DP of the fructan exhibited a strong impact on the blue intensity of the samples and also their rehydration capacity.

Topics: Chemical Phenomena; Coloring Agents; Fructans; Humans; Inulin; Iridoids; Milk Proteins; Oligosaccharides; Particle Size; Polymerization; Powders; Solubility; Temperature; Water; Wettability

Gelatin has emerged as a biocompatible polymer with high printability in scaffold-based tissue engineering. The aim of the current study was to investigate the potential of genipin-crosslinked 3D printed gelatin scaffolds for temporomandibular joint (TMJ) cartilage regeneration. Crosslinking with genipin increased the stability and mechanical properties, without any cytotoxic effects. Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSC) on the scaffolds were compared to cell pellets and spheres. Although hBMSC seeded scaffolds showed a lower expression of chondrogenesis-related genes compared to cell pellets and spheres, they demonstrated a significantly reduced expression of collagen (COL) 10, suggesting a decreased hypertrophic tendency. After 21 days, staining with Alcian blue and immunofluorescence for SOX9 and COL1 confirmed the chondrogenic differentiation of hBMSC on genipin-crosslinked gelatin scaffolds. In summary, 3D printed gelatin-genipin scaffolds supported the viability, attachment and chondrogenic differentiation of hBMSC, thus, demonstrating potential for TMJ cartilage regeneration applications.

Topics: Cartilage; Gelatin; Humans; Iridoids; Printing, Three-Dimensional; Temporomandibular Joint; Tissue Scaffolds

Polyvinyl alcohol (PVA) hydrogel has gained interest in cartilage repair because of its highly swollen, porosity, and viscoelastic properties. However, PVA has some deficiencies, such as its poor biocompatibility and microstructure. This research aimed to design novel hydroxyapatite (HA)-collagen (COL)-PVA hydrogels. COL was added to improve cell biocompatibility, and the microstructure of the hydrogels was controlled by fused deposition modeling (FDM). The feasibility of the COL-HA-PVA hydrogels in cartilage repair was evaluated by in vitro and in vivo experiments. The scanning electron microscopy results showed that the hybrid hydrogels had interconnected macropore structures that contained a COL reticular scaffold. The diameter of the macropore was 1.08-1.85 mm, which corresponds to the diameter of the denatured PVA column. The chondrocytes were then seeded in hydrogels to assess the cell viability and formation of the cartilage matrix. The in vitro results revealed excellent cellular biocompatibility. Osteochondral defects (8 mm in diameter and 8 mm in depth) were created in the femoral trochlear of goats, and the defects were implanted with cell-seeded hydrogels, cell-free hydrogels, or a blank control. The in vivo results showed that the COL-HA-PVA hydrogels effectively repaired cartilage defects, especially the conditions inoculated with chondrocyte in advance. This research suggests that the COL-HA-PVA hydrogels have promising application in cartilage repair.

Topics: Animals; Biocompatible Materials; Cartilage; Cell Survival; Chondrocytes; Collagen; Cross-Linking Reagents; Elasticity; Goats; Hydrogels; In Vitro Techniques; Iridoids; Polyvinyl Alcohol; Porosity; Tissue Engineering; Tissue Scaffolds; Viscosity; Wound Healing

Novel eco-friendly and green dimethyldiallylammonium chloride (DMDAAC) grafted chitosan/genipin/cellulose hydrogel beads (CCBG-g-PDMDAAC), were fabricated as selective adsorbents for anionic dyes. The physical and chemical structural changes of the prepared hydrogels were evaluated by FTIR, XRD, SEM and TG-DSC analysis. Results showed CCBG-g-PDMDAAC efficiently and selectively adsorb anionic dyes (Reactive Red 195-RR195 and Methyl orange-MO) from mixture of dye solutions. Endowed chitosan-based hydrogels the advantage of acid insolubility and good adsorption. RR195 and MO adsorption process were described better with pseudo-second-order kinetic model and Langmuir isotherm model with a maximum adsorption capacity of 1333.52 and 190.48 mg/g, respectively, indicating that monolayer chemisorption controlled the sorption process. Moreover, the hydrogels exhibited nice reusability and against S. aureus and E. coli. The hydrogels are promising for the potential application in wastewater treatment and antibacterial simultaneously.

Topics: Adsorption; Anions; Anti-Bacterial Agents; Cellulose; Chitosan; Coloring Agents; Cross-Linking Reagents; Escherichia coli; Hydrogels; Hydrogen-Ion Concentration; Iridoids; Kinetics; Microbial Sensitivity Tests; Microspheres; Nitrogen; Polyethylenes; Quaternary Ammonium Compounds; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared; Staphylococcus aureus; Temperature; X-Ray Diffraction

To evaluate the use of genipin in delaying enzymatic digestion of corneal stroma.. Human corneal stromal tissue was treated with genipin, a known chemical crosslinker, and then along with control tissue was subjected to enzymatic digestion with collagenase. The effects of genipin treatment in retarding stromal digestion were analyzed with phase contrast microscopy, a protein quantification assay, second harmonic generation imaging, and transmission electron microscopy.. Genipin increased stromal resistance to enzymatic digestion when compared with untreated stroma. A morphologic analysis and protein quantification showed increased stromal resistance to enzymatic digestion once stromal tissue was treated with genipin. Second harmonic generation imaging revealed persistent fibrillar collagen signaling in genipin-treated tissue in contrast with untreated tissue suggesting that genipin retards collagenolysis.. Genipin increases stromal resistance to enzymatic digestion in controlled experiments as demonstrated by protein quantification studies and through morphologic imaging.. This study explores the novel use of genipin in delaying enzymatic stromal digestion. Delaying stromal melting in the setting of corneal infectious or autoimmune keratitis can potentially decrease clinical morbidity.

Topics: Collagen; Corneal Stroma; Cross-Linking Reagents; Digestion; Humans; Iridoids

To determine whether genipin (a natural crosslinker) could reduce the colonization and proliferation of bacteria and fungi in an ex vivo model of corneal infection.. This study, using an ex vivo model of bacterial and fungal keratitis, investigated the antimicrobial efficacy of genipin crosslinking. Excised corneoscleral buttons were wounded by scalpel incision and subsequently infected with Staphylococcus aureus, Pseudomonas aeruginosa, or Candida albicans. After inoculation, corneas were treated with genipin for 24 hours at 37°C. Histologic examinations were carried out, and the number of viable colony-forming units (CFU)/cornea was determined.. Genipin exerts bactericidal action against S. aureus and P. aeruginosa, as well as fungicidal action against C. albicans and significantly reduced the CFU compared to contralateral eyes that received saline treatment (P < 0.05).. These data identify genipin as a novel ocular antimicrobial agent that has the potential to be incorporated into the therapeutic armamentarium against microbial keratitis.. This study provided evidence for the antimicrobial and antifungal properties of genipin as an alternative crosslinker that could be used in the management of infectious keratitis.

Topics: Bacteria; Colony Count, Microbial; Cornea; Eye Infections, Bacterial; Fungi; Humans; In Vitro Techniques; Iridoids; Keratitis; Staphylococcus aureus

Suitable material for scaffolds that support cell attachment, proliferation, vascularization and contraction has always been a challenge in myocardial tissue engineering. Much research effort has been focused on natural polymers including collagen, gelatin, chitosan, fibrin, alginate, etc. Among them, a collagen/chitosan composite scaffold was widely used for myocardial tissue engineering. Due to the non-proliferative and contractile characteristics of cardiomyocytes, the biocompatibility and mechanical properties of the scaffolds are extremely important for supporting intercellular connection and tissue function for myocardial tissue engineering. To the best of our knowledge, the three crosslinking agents (glutaraldehyde (GTA), genipin (GP), tripolyphosphate (TPP)) have not yet been comparatively studied in myocardial tissue engineering. Thus, the aim of this study is to compare and identify the crosslinking effect of GTA, GP and TPP for myocardial tissue engineering. The collagen/chitosan scaffolds prepared with various crosslinking agents were fabricated and evaluated for physical characteristics, biocompatibility and contractile performance. All the groups of scaffolds exhibited high porosity (>65%) and good swelling ratio suitable for myocardial tissue engineering. TPP crosslinked scaffolds showed excellent mechanical properties, with their elastic modulus (81.0 ± 8.1 kPa) in the physiological range of native myocardium (20∼100 kPa). Moreover, GP and TPP crosslinked scaffolds exhibited better biocompatibility than GTA crosslinked scaffolds, as demonstrated by the live/dead staining and proliferation assay. In addition, cardiomyocytes within TPP crosslinked scaffolds showed the highest expression of cardiac-specific marker protein and the best contractile performance. To conclude, of the three crosslinking agents, TPP was recommended as the most suitable crosslinking agent for collagen/chitosan scaffold in myocardial tissue engineering.

Topics: Animals; Biocompatible Materials; Cell Proliferation; Cell Survival; Chitosan; Collagen; Cross-Linking Reagents; Elastic Modulus; Gelatin; Humans; Iridoids; Mice; Myocardial Contraction; Myocardium; NIH 3T3 Cells; Porosity; Pressure; Spectroscopy, Fourier Transform Infrared; Stress, Mechanical; Tensile Strength; Tissue Engineering; Tissue Scaffolds

Genipin is one of the major component in Gardenis fruit, which has long been used in the treatment of many chronic diseases, such as colitis. In the present study, we investigated the protective effects and mechanism of genipin on dextran sodium sulfate (DSS)-induced colitis in mice. Colitis was induced by giving 2.5% (wt/vol) DSS for 7 days. As the results show, DSS-induced body weight loss and colonic histological changes were inhibited by the treatment of genipin. DSS-induced MPO activity, MDA level, TNF-α, and IL-1β production in colonic tissues were also suppressed by genipin. To investigate the mechanism of genipin on DSS-induced colitis, the NF-κB and Nrf2 signaling pathways were detected. The results showed genipin significantly attenuated DSS-induced NF-κB activation and increased the expression of Nrf2 and HO-1 in a dose-dependent manner. The results of the present study indicated that genipin protected mice against colitis through inhibiting inflammatory and oxidative effects.

Topics: Animals; Colitis; Colon; Cytokines; Dextran Sulfate; Inflammation; Inflammation Mediators; Interleukin-1beta; Iridoids; Male; Mice; Mice, Inbred C57BL; NF-E2-Related Factor 2; NF-kappa B; Oxidative Stress; Signal Transduction; Tumor Necrosis Factor-alpha

Genipin can improve weak mechanical properties and control high degradation rate of gelatin, as a cross-linker of gelatin which is widely used in tissue engineering. In this study, genipin cross-linked gelatin biodegradable porous scaffolds with different weight percentages of gelatin and genipin were prepared for tissue regeneration and measurement of their various properties including morphological characteristics, mechanical properties, swelling, degree of crosslinking and degradation rate. Results indicated that the sample containing the highest amount of gelatin and genipin had the highest degree of crosslinking and increasing the percentage of genipin from 0.125% to 0.5% enhances ultimate tensile strength (UTS) up to 113% and 92%, for samples with 2.5% and 10% gelatin, respectively. For these samples, increasing the percentage of genipin, reduce their degradation rate significantly with an average value of 124%. Furthermore, experimental data are used to develop a machine learning model, which compares artificial neural networks (ANN) and kernel ridge regression (KRR) to predict degradation rate of genipin-cross-linked gelatin scaffolds as a property of interest. The predicted degradation rate demonstrates that the ANN, with mean squared error (MSE) of 2.68%, outperforms the KRR with MSE = 4.78% in terms of accuracy. These results suggest that machine learning models offer an excellent prediction accuracy to estimate the degradation rate which will significantly help reducing experimental costs needed to carry out scaffold design.

Topics: Cross-Linking Reagents; Gelatin; Iridoids; Machine Learning; Materials Testing; Microscopy, Electron, Scanning; Models, Theoretical; Neural Networks, Computer; Regression Analysis; Tissue Scaffolds

Bioinspired nonantibiotics can prove to be a better and an efficient tool to fight against antimicrobial resistance. In our study, biomaterial composed of zinc-carboxymethyl chitosan (CMC)-genipin was investigated for this purpose. Briefly, CMC was synthesized and transformed to porous scaffolds using the freeze drying method. The scaffolds were cross-linked and stabilized with genipin and zinc (2 M zinc acetate), respectively. FTIR spectroscopic data testified Zn complex formation and pointed out the absence of water molecule like that of zinc motif containing proteins. Hence, the complex may be termed as biomimetic. Genipin (0.5%) cross-linking appeared to contribute additively to the wet compressive strength of the zinc-CMC scaffolds. Biodegradation data revealed better stability of CMC-genipin-zinc scaffolds in enzymatic and nonenzymatic conditions than their redundant controls. The scaffolds seem to support adhesion and proliferation of human dental pulp stem cells and were hemocompatible to human red blood corpuscles, as revealed by scanning electron microscopy. The scaffolds were found to be antibacterial and mildly antibiofilm when tested against biofilm-forming bacteria, that is,

Topics: Adhesives; Biocompatible Materials; Biomimetic Materials; Biomimetics; Cells, Cultured; Chitosan; Dental Pulp; Erythrocytes; Humans; Iridoids; Materials Testing; Microbial Sensitivity Tests; Tissue Scaffolds; Zinc

Genipin has been shown to exert anti-inflammatory effects, but its mechanism in protecting the ethanol-induced acute gastric injuries remains largely unclear. The present study aimed to investigate the effects of genipin on ethanol-induced acute gastric injuries in mice. After intragastrical administration of genipin for 7 consecutive days, acute gastric injuries were induced in the mice by ethanol treatment for 1 h. The expression levels of MDA, MPO, SOD, CAT, and NO in gastric tissues, and the levels of IL-6, TNF-α, MTL, SP, VIP and SS in serum samples were measured by ELISA. In addition, Western blotting was used to determine the expression levels of proteins involved in NLRP3 signaling pathway. The findings revealed that oral administration of genipin significantly ameliorated the pathological injury of gastric mucosa induced by ethanol, decreased the oxidative stress induced by ethanol and suppressed the expression levels of in-flammatory cytokines in gastric tissues and serum samples. In addition, it was observed that oral administration of genipin could remarkably inhibit the expression levels of related proteins in the NLRP3 signaling pathway. In conclusion, these results suggest that genipin may exhibit protective roles in ethanol-induced gastric mucosal injuries by activating antioxidant system and attenuating inflammatory reaction.

Topics: Acute Disease; Animals; Anti-Inflammatory Agents; Disease Models, Animal; Ethanol; Gastric Mucosa; Humans; Inflammasomes; Iridoids; Male; Mice; NLR Family, Pyrin Domain-Containing 3 Protein; Oxidative Stress; Signal Transduction; Stomach Ulcer

Uncoupling protein 2 (UCP2) is a mitochondrial anion carrier which plays a key role in energy homeostasis. UCP2 is deregulated in several human cancers and has been suggested to regulate cancer metabolism. However, the role of UCP2 in gallbladder cancer has not been defined. Using clinical samples, we found highly expressed UCP2 in gallbladder cancer tissues, and higher expression levels of UCP2 correlated with worse clinical characteristics. To study whether UCP2 promotes gallbladder cancer growth, UCP2 stable knockdown cells were generated, and cell proliferation was suppressed in these knockdown cells. Further studies demonstrated that glycolysis was inhibited and IKKβ, as well as the downstream signaling molecules NF-κB/FAK/β-catenin, were downregulated in UCP2 knockdown cells. More importantly, gallbladder cancer cells became sensitive to gemcitabine treatments when UCP2 was inhibited. UCP2 knockdown suppressed the activation of the NF-κB/β-catenin axis and promoted the increases in mitochondrial ROS in gallbladder cancer cells exposed to gemcitabine treatments. The UCP2 inhibitor genipin suppressed xenograft tumor growth and sensitized grafted tumors to gemcitabine treatments. These results suggest targeting UCP2 as a novel therapeutic strategy for the treatment of gallbladder cancer.

Topics: Animals; Antineoplastic Agents; beta Catenin; Cell Line, Tumor; Cisplatin; Deoxycytidine; Drug Resistance, Neoplasm; Gallbladder Neoplasms; Gemcitabine; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Humans; Iridoids; Male; Mice; Mice, Nude; Mitochondria; Neoplasms, Experimental; NF-kappa B; Reactive Oxygen Species; Uncoupling Protein 2; Up-Regulation

Autologous and allogenic human pericardia used as biomaterials for cardiovascular surgery are traditionally crosslinked with glutaraldehyde. In this work, we have evaluated the resistivity to collagenase digestion and the cytotoxicity of human pericardium crosslinked with various concentrations of glutaraldehyde in comparison with pericardium crosslinked by genipin, nordihydroguaiaretic acid, tannic acid, and in comparison with unmodified pericardium. Crosslinking retained the wavy-like morphology of native pericardium visualized by second harmonic generation microscopy. The collagenase digestion products were analyzed using SDS-PAGE, capillary electrophoresis, and a hydroxyproline assay. Glutaraldehyde and genipin crosslinking protected the native pericardium efficiently against digestion with collagenase III. Only low protection was provided by the other crosslinking agents. The cytotoxicity of crosslinked pericardium was evaluated using xCELLigence by monitoring the viability of porcine valve interstitial cells cultured in eluates from crosslinked pericardium. The highest cell index, reflecting both the number and the shape of the monitored cells was observed in eluates from genipin. Crosslinking pericardium grafts with genipin therefore seems to be a promising alternative procedure to the traditional crosslinking with glutaraldehyde, because it provides similarly high protection against degradation with collagenase, without cytotoxic effects.

Topics: Biocompatible Materials; Cross-Linking Reagents; Glutaral; Humans; Iridoids; Masoprocol; Pericardium; Tannins; Transplants

Bovine serum albumin (BSA)-coated haemoglobin (Hb)-microcapsules prepared by co-precipitation of Hb and MnCO

Topics: Animals; Capsules; Cell Line; Cross-Linking Reagents; Hemoglobins; Humans; Iridoids; Mice; Serum Albumin, Bovine

Topics: Emulsions; Fixatives; Gelatin; Iridoids; Microscopy, Atomic Force; Molecular Structure; Nanoparticles; Particle Size; Tannins; Water

Bone allografts are the preferred method for bone augmentation in over 500,000 orthopedic surgical procedures in the US. Sterilization by ionizing radiation is the most effective method of minimizing the bioburden of bone allografts; however, radiation causes chain scission of collagen, resulting in the reduction of the allografts' mechanical strength. In this study, we doped bone allografts with vitamin E as radioprotectant using a novel two-step process to protect the collagen architecture against radiation damage and to preserve the mechanical strength of the construct. In addition, combining the radioprotectant with a cross-linking agent further minimized collagen degradation and further preserved the mechanical strength of the allografts. Both vitamin E and combined vitamin E/genipin-treated allograft were less cytotoxic to both osteoblasts and osteoclasts when compared to irradiated-only allografts. Host bone-allograft unionization was faster in a rat calvaria defect model with vitamin E-treated and combined vitamin E and genipin-treated allograft when compare to irradiated-only allografts. This method can enable the efficient and uniform radioprotective treatment of bone allograft of desired shapes for sterilization with improved mechanical strength and biointegration.

Topics: Allografts; Animals; Bone and Bones; Bone Transplantation; Collagen; Cross-Linking Reagents; Disease Models, Animal; Humans; Iridoids; Mechanical Phenomena; Osteoblasts; Osteoclasts; Radiation-Protective Agents; Skull; Sterilization; Vitamin E

We examined the morphology of a network made with native BSA molecules being crosslinked with genipin at ambient temperature. Ninhydrin assay, FTIR, WAXD, SEM and mechanical tests documented successful crosslinking that enhanced the structural properties of the three dimensional structure. Its hydrophilic nature allows swelling with water absorption, which can be monitored with the modified Flory-Rehner theory to predict the molecular weight between adjacent crosslinks, network mesh size and crosslinking density as a function of crosslinker addition. Characterisation studies were carried out with a view to developing a delivery vehicle for the controlled release of vitamin B6 over a prolonged period of observation. Moving boundaries associated with swelling of the protein matrix resulted in vitamin transport that could be described with the interplay of diffusional and relaxational kinetics via the Peppas-Sahlin equation. Combination of diffusion and swelling equilibrium theories unveils a measurable effect of network characteristics on vitamin B6 release.

Topics: Animals; Cattle; Delayed-Action Preparations; Diffusion; Iridoids; Kinetics; Molecular Weight; Serum Albumin, Bovine; Vitamin B 6

The present study focuses on the synthesis and evaluation of neomycin-loaded hydrogels as potential substrate for wound healing application. Herein, genipin crosslinked gelatin interpenetrated diosgenin-modified nanocellulose (DGN-NC) hydrogels were synthesized. The hydrogels' chemical structures as well as surface morphology, mechanical property, and thermal behavior were characterized. Swelling analysis and gelation kinetics of the hydrogels were studied and the results obtained showed good swelling capacity as well as high gel yield. In addition, the prepared loaded hydrogels were evaluated for antibacterial activity against human pathogenic E. coli and S. aureus bacteria with inhibition capacity determined in the range of 50-88%. In vitro cytocompatibility and drug release studies were also explored under simulated physiological conditions achieving high cell viability and release percentage >80% and >90% after 24 h, respectively. In effect, the design hydrogels in the present study possess adequate incorporated antibacterial properties with significant potentials towards wound dressing and healing applications.

Topics: Anti-Bacterial Agents; Bandages; Biocompatible Materials; Cell Line; Cellulose; Diosgenin; Drug Liberation; Escherichia coli; Fibroblasts; Gelatin; Hydrogels; Iridoids; Kinetics; Microbial Sensitivity Tests; Nanoparticles; Staphylococcus aureus; Wound Healing

Biomaterial-based therapy that can restore annulus fibrosus (AF) function in early stage and promote endogenous repair of AF tissues is a promising approach for AF tissue repair. In this study, we established a genipin-crosslinked decellularized AF hydrogels (g-DAF-G) that are injectable and could manifest better in situ formability than noncrosslinked decellularized AF hydrogel, while preserving the capacity of directing differentiation of human bone mesenchymal stem cells (hBMSCs) towards AF cells. Hematoxylin and eosin staining, 4',6-diamidino-2-phenylindole staining, and so forth showed that the majority of cellular components were removed, whereas extracellular matrix and microstructure were largely preserved. The storage modulus increased from 465.5 ± 9.4 Pa to 3.29 ± 0.24 MPa after 0.02% genipin crosslinking of decellularized AF hydrogels (DAF-G) to form g-DAF-G. AF-specific genes (COL1A1, COL5A1, TNMD, IBSP, FBLN1) were significantly higher in DAF-G and g-DAF-G groups than that in control group after 21 days of culturing. g-DAF-G significantly restored nucleus pulposus water content and preserved intervertebral structure in vivo. Summarily, we produced a novel AF regeneration biomaterial, g-DAF-G, which exhibited well biocompatibility, great bioactivity, and much higher mechanical strength than DAF-G. This study will provide an easy and fast therapeutic alternative to repair AF injuries or tears.

Topics: Animals; Annulus Fibrosus; Bone Marrow Cells; Cattle; Cell Differentiation; Cross-Linking Reagents; Humans; Hydrogels; Intervertebral Disc Degeneration; Iridoids; Mesenchymal Stem Cells

Three-dimensional (3D) biomimetic cell culture platforms offer more realistic microenvironments that cells naturally experience in vivo. We developed a tunable hyaluronan-based hydrogels that could easily be modified to mimic healthy or malignant extracellular matrices (ECMs). For that, we pre-functionalized our hydrogels with an adhesive polypeptide (poly-l-lysine, PLL) or ECM proteins (type III and type IV collagens), naturally present in tumorous tissues, and next, we tuned their stiffness by crosslinking with gradual concentrations of genipin (GnP). Then, we thoroughly characterized our substrates before testing them with glioblastoma and breast cancer cells, and thereafter with endothelial cells. Overall, our hydrogels exhibited (a) increasing stiffness with GnP concentration for every pre-functionalization and (b) efficient enzyme resistance with PLL treatment, and also with type IV collagen but to a lesser extent. While PLL-treated hydrogels were not favorable to the culture of any glioblastoma cell lines, they enhanced the proliferation of breast cancer cells in a stiffness-dependent manner. Contrary to type III collagen, type IV collagen pre-treated hydrogels supported the proliferation of glioblastoma cells. The as-desired HA-based 3D tumor-like models we developed may provide a useful platform for the study of various cancer cells by simply tuning their biochemical composition and their mechanical properties.

Topics: Biomechanical Phenomena; Biomimetic Materials; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cross-Linking Reagents; Extracellular Matrix; Female; Glioblastoma; Humans; Hyaluronic Acid; Hydrogels; Iridoids; Neoplasms; Tumor Microenvironment

Oral squamous cell carcinoma (OSCC) is a common malignant tumor of the head and neck, and it accounts for more than 90% of oral cancer. Due to high mortality, limitations of traditional treatment and many complications, new treatment methods are urgently needed. This study aimed to look into the effect of new potential anti-tumor drug, genipin, on OSCC treatment.. In vitro, CCK-8, colony formation, and flow cytometry were used to detect the effect of genipin on SCC-9 and SCC-15 cell lines. Immunofluorescence, real-time PCR, and Western blotting were used to investigate its mechanism. Xenograft tumor model was used to explore the role of genipin in vivo.. We found that genipin suppressed cell growth and induced apoptosis in vitro. In addition, the expression of p62 was down-regulated while Beclin1 and LC3II were up-regulated in SCC-25 and SCC-9 cells. 3-methyladenine (3-MA) significantly decreased LC3 (LC3II). Conclusively, this study implicated that genipin suppresses cell proliferation and stimulated apoptosis, and is the first exploration showing that genipin induces OSCC cell autophagy via PI3K/AKT/mTOR pathway inhibition.

Topics: Antineoplastic Agents; Apoptosis; Autophagy; Carcinoma, Squamous Cell; Cell Proliferation; Cells, Cultured; Drug Screening Assays, Antitumor; Humans; Iridoids; Mouth Neoplasms; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; TOR Serine-Threonine Kinases

The cardiac expression of the mitochondrial uncoupling protein (UCP)-2 is increased in patients with heart failure. However, the underlying causes as well as the possible consequences of these alterations during the transition from hypertrophy to heart failure are still unclear. To investigate the role of UCP-2 mechanistically, expression of UCP-2 was silenced by small interfering RNA in adult rat ventricular cardiomyocytes. We demonstrate that a downregulation of UCP-2 by siRNA in cardiomyocytes preserves contractile function in the presence of angiotensin II. Furthermore, silencing of UCP-2 was associated with an upregulation of glucose transporter type (Glut)-4, increased glucose uptake, and reduced intracellular lactate levels, indicating improvement of the oxidative glucose metabolism. To study this adaptation in vivo, spontaneously hypertensive rats served as a model for cardiac hypertrophy due to pressure overload. During compensatory hypertrophy, we found low UCP-2 levels with an upregulation of Glut-4, while the decompensatory state with impaired function was associated with an increase of UCP-2 and reduced Glut-4 expression. By blocking the aldosterone receptor with spironolactone, both cardiac function as well as UCP-2 and Glut-4 expression levels of the compensated phase could be preserved. Furthermore, we were able to confirm this by left ventricular (LV) biopsies of patients with end-stage heart failure. The results of this study show that UCP-2 seems to impact the cardiac glucose metabolism during the transition from hypertrophy to failure by affecting glucose uptake through Glut-4. We suggest that the failing heart could benefit from low UCP-2 levels by improving the efficiency of glucose oxidation. For this reason, UCP-2 inhibition might be a promising therapeutic strategy to prevent the development of heart failure.

Topics: Animals; Blood Pressure; Cardiomegaly; Cell Survival; Chronic Disease; Female; Glucose; Glucose Transporter Type 4; Heart Failure; Heart Ventricles; Humans; Hypertension; Iridoids; Male; Mitochondria, Heart; Models, Cardiovascular; Myocytes, Cardiac; Rats, Wistar; Spironolactone; Uncoupling Protein 2

High myopia can lead to blindness. Genipin is a collagen cross-linking agent that may be used to treat myopia. However, the mechanism of action of genipin for the treatment of myopia is unclear. This study investigated the effect of genipin on the scleral expression of the miR-29 cluster, matrix metalloproteinase 2 (MMP2), and collagen alpha1 chain of type I (COL1A1) in a guinea pig model of myopia.. The model of myopia was established by treating guinea pigs with a - 8D lens on both eyes for 21 days, and eyes with a refractive error of - 6D or greater were included. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and western blot were used to examine the mRNA and protein expression, respectively. A dual-luciferase assay was used to determine the direct targeting of the miR-29 cluster on the 3'-untranslated region (UTR) of the COL1A1 gene.. The scleral expression of miR-29a, miR-29b, and miR-29c as well as MMP2 was significantly increased, and the scleral expression of COL1A1 was significantly decreased in the myopia group. Genipin treatment reversed these effects in myopic eyes. The dual-luciferase assay showed that the luciferase activities were significantly decreased in human embryonic kidney (HEK) cells transfected with miR-29a and miR-29b, but not miR-29c, compared with those transfected with control miRNAs.. Genipin inhibits the scleral expression of the miR-29 cluster and MMP2 and promotes COL1A1 expression in a guinea pig model of myopia. Thus, genipin may promote COL1A1 expression by reducing the expression of the miR-29 cluster.

Topics: Animals; Blotting, Western; Cells, Cultured; Cholagogues and Choleretics; Collagen Type I; Collagen Type I, alpha 1 Chain; Disease Models, Animal; Female; Gene Expression Regulation; Guinea Pigs; HEK293 Cells; Humans; Iridoids; Male; Matrix Metalloproteinase 2; MicroRNAs; Myopia; Real-Time Polymerase Chain Reaction; RNA, Messenger; Sclera

Multifunctional carbon dots (CDs) present enormous potential in numerous applications and have attracted widespread attention for various applications in the biomedical field. Bacterial infection is a common health issue; the development of antibacterial materials with low toxicity and good biocompatibility is becoming more important. In this work, we synthesized a new type of nitrogen co-doped carbon dots-genipin covalent conjugate (N-CDs-GP) via hydrothermal methods. The microstructure and chemical composition of the N-CDs-GP were characterized. The biocompatibility, stability, antibacterial activity, and fluorescence performance of the N-CDs-GP were assessed. The results revealed that N-CDs-GP possessed high biocompatibility, high light stability, and broad antibacterial activity. Additionally, selective Gram-positive bacterial imaging by N-CDs-GP provided a more rapid method of bacterial detection. The N-CDs-GP have the potential to be applied as bioimaging and antibacterial agents and for bacterial discrimination.

Topics: Anti-Bacterial Agents; Biocompatible Materials; Breast Neoplasms; Carbon; Cell Line, Tumor; Cross-Linking Reagents; Escherichia coli; Female; Humans; Iridoids; Molecular Structure; Optical Imaging; Particle Size; Quantum Dots; Staphylococcus aureus; Surface Properties

In this work, coated liposome as a novel delivery system assembled from genipin-crosslinked with whey protein isolate (WPI) and sodium alginate (SA) as the second layer on WPI-coated liposomal was developed in order to increase its bioavailability and prolong release for drug and nutraceuticals. Therefore, the influence of GP and SA on physico-chemical properties, long term stability and in vitro release behavior of WPI coated nanoliposomes (NLs) for encapsulation of flaxseed oil were investigated successively. Compared with WPI-NLs and uncoated NLs, GP-WPI-NLs and SA-WPI-NLs displayed more significant changes in average diameter, zeta potential, entrapment efficiency, morphology, FT-IR, thermal properties, with lower n-3 fatty acids released. In terms of physical and oxidative stability during storage at 45 °C for 30 days, the GP was remarkably more effective than the SA. The results suggested that the coating with SA and cross-linking with GP altered the surface properties, and provided more chemical stability for the flaxseed oil as core material.

Topics: Alginates; Bioreactors; Calorimetry, Differential Scanning; Iridoids; Linseed Oil; Liposomes; Microscopy, Electron, Transmission; Spectroscopy, Fourier Transform Infrared; Surface Properties; Whey Proteins

It is of great clinical significance to design wound dressing materials with combined excellent wound healing properties and superior capability to suppress hypertrophic scar formation. This study aimed to examine if and how the cationicity of chitosan would affect the hypertrophic scar-related outcomes, through preparing carboxymethyl chitosan hydrogels with different genipin concentrations (2.5%, 5%, 10% and 15%, respectively). An optimum window of chitosan cationicity (5% in our case) demonstrated potential to mitigate hypertrophic scar in wound healing by suppressing the expression of a-smooth muscle actin (a-SMA) and promoting secretion of type I matrix metalloproteinases (MMP-1). In vivo, the CMCS-5% hydrogel again showed smaller, thinner and smoother wound appearance. Moreover, the CMCS-5% sample with additional incorporation of 2% (V/V) Aloe vera gel exhibited further improved performance in scar inhibition. Overall, such findings might have important implications in chitosan-based wound dressing design for high-quality wound repair and effective scar inhibition.

Topics: Animals; Bandages; Cations; Cells, Cultured; Chitosan; Cicatrix, Hypertrophic; Female; Humans; Hydrogels; Iridoids; Plant Preparations; Rats; Rats, Sprague-Dawley; Wound Healing

Ocular hypertension has been attributed to increased resistance to aqueous outflow often as a result of changes in trabecular meshwork (TM) extracellular matrix (ECM) using in vivo animal models (for example, by genetic manipulation) and ex vivo anterior segment perfusion organ cultures. These are, however, complex and difficult in dissecting molecular mechanisms and interactions. In vitro approaches to mimic the underlying substrate exist by manipulating either ECM topography, mechanics, or chemistry. These models best investigate the role of individual ECM protein(s) and/or substrate property, and thus do not recapitulate the multifactorial extracellular microenvironment; hence, mitigating its physiological relevance for mechanistic studies. Cell-derived matrices (CDMs), however, are capable of presenting a 3D-microenvironment rich in topography, chemistry, and whose mechanics can be tuned to better represent the network of native ECM constituents in vivo. Critically, the composition of CDMs may also be fine-tuned by addition of small molecules or relevant bioactive factors to mimic homeostasis or pathology. Here, we first provide a streamlined protocol for generating CDMs from TM cell cultures from normal or glaucomatous donor tissues. Second, we document how TM cells can be pharmacologically manipulated to obtain glucocorticoid-induced CDMs and how generated pristine CDMs can be manipulated with reagents like genipin. Finally, we summarize how CDMs may be used in mechanistic studies and discuss their probable application in future TM regenerative studies.

Topics: Cell Culture Techniques; Cells, Cultured; Cross-Linking Reagents; Extracellular Matrix; Glucocorticoids; Humans; Iridoids; Trabecular Meshwork

Prolonged healing is a severe problem for elderly and diabetic patients. Impaired angiogenesis, stem cell differentiation, and migration have been shown to delay wound healing. The chemokine stromal cell-derived factor-1 (SDF-1) plays an essential role in recruiting cells to wound sites and is suggested to be a candidate for tissue engineering. In this study, chitosan (CHI) scaffolds were crosslinked with nontoxic genipin (Gp) and further heparinized for SDF-1 immobilization. Then, the structures were evaluated for their physicochemical properties (porosity, swelling ratio, and water vapor transmission rate (WVTR)). The interaction between SDF-1 and heparin could sustain SDF-1 release, which has been shown to enhance human umbilical vein endothelial cell (HUVEC) 2D/3D migration. The investigation of the wound-healing activity of the SDF-1-loaded CHI scaffolds revealed a better wound recovery rate in vivo in healthy and streptozotocin-induced diabetic Sprague-Dawley (SD) rats. The histological analysis illustrated that the local of SDF-1 treatment scaffold at the wound site enhanced neovascularization. The wounds treated with SDF-1 scaffolds also exhibited higher vascular endothelial growth factor (VEGF) and transforming growth factor-beta (TGF-β) expression in Western blot assays. Based on the wound-healing activity and beneficial characteristics, the SDF-1-loaded CHI scaffold demonstrates potential as a material for treating skin wounds.

Topics: Animals; Blotting, Western; Cell Movement; Chemokine CXCL12; Chitosan; Collagen; Human Umbilical Vein Endothelial Cells; Humans; Iridoids; Male; Porosity; Rats; Rats, Sprague-Dawley; Streptozocin; Tissue Scaffolds; Transforming Growth Factor beta; Vascular Endothelial Growth Factor A; Wound Healing

A number of natural polymer biomaterial-based nerve guidance conduits (NGCs) are developed to facilitate repair of peripheral nerve injuries. Cross-linking ensures mechanical integrity and desired degradation properties of the NGCs; however, common methods such as formaldehyde are associated with cellular toxicity. Hence, there is an unmet clinical need for alternative nontoxic cross-linking agents. In this study, collagen-based NGCs with a collagen/chondroitin sulfate luminal filler are used to study the effect of cross-linking on mechanical and structural properties, degradation, biocompatibility, and immunological response. A simplified manufacturing method of genipin cross-linking is developed, by incorporating genipin into solution prior to freeze-drying the NGCs. This leads to successful cross-linking as demonstrated by higher cross-linking degree and similar tensile strength of genipin cross-linked conduits compared to formaldehyde cross-linked conduits. Genipin cross-linking also preserves NGC macro and microstructure as observed through scanning electron microscopy and spectral analysis. Most importantly, in vitro cell studies show that genipin, unlike the formaldehyde cross-linked conduits, supports the viability of Schwann cells. Moreover, genipin cross-linked conduits direct macrophages away from a pro-inflammatory and toward a pro-repair state. Overall, genipin is demonstrated to be an effective, safe, biocompatible, and anti-inflammatory alternative to formaldehyde for cross-linking clinical grade NGCs.

Topics: Animals; Anti-Inflammatory Agents; Axon Guidance; Biocompatible Materials; Cell Line; Cross-Linking Reagents; Fibroblasts; Humans; Iridoids; Rats; Schwann Cells; Tissue Engineering; Tissue Scaffolds

Tendinopathy is a common musculoskeletal disorder and current treatment options show limited success. Genipin is an effective collagen crosslinker with low cytotoxicity and a promising therapeutic strategy for stabilizing an intratendinous lesion.. This study examined the mechanical effect and delivery of intratendinous genipin injection in healthy and degenerated tendons.. Controlled laboratory study.. Bovine superficial digital flexor tendons were randomized into four groups: Healthy control (N = 25), healthy genipin (N = 25), degenerated control (N = 45) and degenerated genipin (N = 45). Degeneration was induced by Collagenase D injection. After 24h, degenerated tendons were subsequently injected with either 0.2ml of 80mM genipin or buffer only. 24h post-treatment, samples were cyclically loaded for 500 cycles and then ramp loaded to failure. Fluorescence and absorption assays were performed to analyze genipin crosslink distribution and estimate tissue concentration after injection.. Compared to controls, genipin treatment increased ultimate force by 19% in degenerated tendons (median control 530 N vs. 633 N; p = 0.0078). No significant differences in mechanical properties were observed in healthy tendons, while degenerated tendons showed a significant difference in ultimate stress (+23%, p = 0.049), stiffness (+27%, p = 0.037), work to failure (+42%, p = 0.009), and relative stress relaxation (-11%, p < 0.001) after genipin injection. Fluorescence and absorption were significantly higher in genipin treated tendons compared to control groups. A higher degree of crosslinking (+45%, p < 0.001) and a more localized distribution were observed in the treated healthy compared to degenerated tendons, with higher genipin tissue concentrations in healthy (7.9 mM) than in degenerated tissue (2.3 mM).. Using an ex-vivo tendinopathy model, intratendinous genipin injections recovered mechanical strength to the level of healthy tendons. Measured by genipin tissue distribution, injection is an effective method for local delivery.. This study provides a proof of concept for the use of intratendinous genipin injection in the treatment of tendinopathy. The results demonstrate that a degenerated tendon can be mechanically augmented by a clinically viable method of local genipin delivery. This warrants further in vivo studies towards the development of a clinically applicable treatment based on genipin.

Topics: Adhesives; Animals; Cattle; Collagen; Humans; Injections, Intralesional; Iridoids; Tendinopathy; Tendons; Tensile Strength

Biohydrogels, composed of naturally occurring biopolymers are typically preferred over their synthetic analogues in bioapplications thanks to their biocompatibility, bioactivity, mechanical or degradation properties. Shaping biohydrogels on the single-cell length scales (micrometers) is a key ability needed to create bioequivalent artificial cell/tissue constructs and cannot be achieved with current methods. This work introduces a method for photolithographic synthesis of arbitrarily shaped microgels composed purely of a biopolymer of choice. The biopolymer is mixed with a sacrificial photocrosslinkable polymer, and the mixture is photocrosslinked in a lithographic process, yielding anisotropic microgels with the biopolymer entrapped in the network. Subsequent ionic or covalent biopolymer crosslinking followed by template cleavage yields a microgel composed purely of a biopolymer with the 3D shape dictated by the photocrosslinking process. Method feasibility is demonstrated with two model polysaccharide biopolymers (alginate, chitosan) using suitable crosslinking methods. Next, alginate microgels were used as microtaggants on a pharmaceutical oral solid dose formulation to prevent its counterfeiting. Since the alginate is approved as an additive in the food and pharmaceutical industries, the presented tagging system can be implemented in practical use much easier than systems comprising synthetic polymers.

Topics: Alginates; Biopolymers; Dextrans; Fluorescence; Hydrogels; Iridoids; Methacrylates; Microgels

Back pain is a leading cause of global disability associated with intervertebral disc (IVD) pathologies. Discectomy alleviates disabling pain caused by IVD herniation without repairing annulus fibrosus (AF) defects, which can cause accelerated degeneration and recurrent pain. Biological therapies show promise for IVD repair but developing high-modulus biomaterials capable of providing biomechanical stabilisation and delivering biologics remains an unmet challenge. The present study identified critical factors and developed an optimal formulation to enhance the delivery of AF cells and transforming growth beta-3 (TGFβ-3) in genipin-crosslinked fibrin (FibGen) hydrogels. Part 1 showed that AF cells encapsulated in TGFβ-3-supplemented high-modulus FibGen synthesised little extracellular matrix (ECM) but could release TGFβ-3 at physiologically relevant levels. Part 2 showed that AF cells underwent apoptosis when encapsulated in FibGen, even after reducing fibrin concentration from 70 to 5 mg/mL. Mechanistic experiments, modifying genipin concentration and integrin binding site presence demonstrated that genipin crosslinking caused AF cell apoptosis by inhibiting cell-biomaterial binding. Adding integrin binding sites with fibronectin partially rescued apoptosis, indicating genipin also caused acute cytotoxicity. Part 3 showed that FibGen formulations with 1 mg/mL genipin had enhanced ECM synthesis when supplemented with fibronectin and TGFβ-3. In conclusion, FibGen could be used for delivering biologically active compounds and AF cells, provided that formulations supplied additional sites for cell-biomaterial binding and genipin concentrations were low. Results also highlighted a need for developing strategies that protect cells against acute crosslinker cytotoxicity to overcome challenges of engineering high-modulus cell carriers for musculoskeletal tissues that experience high mechanical demands.

Topics: Animals; Annulus Fibrosus; Apoptosis; Cattle; Cell-Matrix Junctions; Cross-Linking Reagents; Extracellular Matrix; Fibrin; Fibronectins; Humans; Hydrogels; Iridoids; Kinetics; Transforming Growth Factor beta3

The adipose stem cell is a potential candidate for the autologous chondrocytes repairing approach because of the abundance of fat in the animal body and its versatile differentiation capability. In this study, rat adipose stem cells (rASCs) were seeded into anti-oxidative N-acetylcysteine (NAC) grafted polyurethane (PU) scaffold and then combined with short dynamic compressive stimulation (24 h) to induce rASCs chondrogenesis differentiation in vitro. The inner pore surface of the PU scaffold was first modified via alginate and type I collagen to promote rASCs adherence. The modified layers crosslinked by genipin showed outstanding stability after ultrasonic treatment, indicating the modified layers were stable and can keep the cells adhesion well during dynamic compressive stimulation. After inner pore surface modification and 10 mM NAC grafting, the PU scaffold-A-C-G (graft 10 mM NAC) has shown the best proliferation efficiency with homogeneous cell distribution after 72hr static culture. After short term dynamic compressive stimulation, significant gene expression in chondrogenic markers, Sox-9, and Aggrecan, were noted in both PU scaffold-A-C-G and PU scaffold-A-C-G (graft 10 mM NAC). Considering the cell proliferation efficiency and gene expression, the anti-oxidative NAC grafted PU scaffold combined with short term dynamic compressive stimulation could be useful for cell culturing in stem cell therapy.

Topics: Acetylcysteine; Adipose Tissue; Aggrecans; Animals; Biocompatible Materials; Cell Adhesion; Cell Culture Techniques; Cell Differentiation; Cell Line; Cell Proliferation; Chondrocytes; Chondrogenesis; Gene Expression; Iridoids; Polyurethanes; Rats; SOX9 Transcription Factor; Stem Cells

Skin damaged during sea battles is vulnerable to seawater immersion and bacterial infection. Scaffolds with effective biological function are highly desired for treatment of naval combat wound injuries. Herein, we prepared composite scaffolds of CS/GEL/GMs-CIP. The chitosan (CS) and gelatin (GEL) were cross-linked by genipin as matrix, and then gelatin microspheres loading ciprofloxacin hydrochloride (GMs-CIP) were add. From in vitro characterization results, CS/GEL/GMs-CIP had high water absorption ability, proper porosity, satisfactory fracture resistance, and flexibility. Furthermore, CS/GEL/GMs-CIP composite scaffold had excellent biocompatibility. Antibacterial experiments confirmed that CS/GEL/GMs-CIP had a significant inhibitory effect on E. coli, S. aureus and P. aeruginosa. The in vivo wound healing was evaluated using animal wound infection model of seawater immersion, and it was observed that the prepared composite scaffolds accelerated wound healing, reepithelialization, collagen deposition. Further analysis of wound tissue indicated that the expression of anti-inflammatory factor (TGF-β1) was up-regulated, but the serum endotoxin levels and expression of pro-inflammatory factor (TNF-a, IL-6, and IL-1β) were down-regulated. In summary, we believe that CS/GEL/GMs-CIP composite scaffold may serve as a promising multifunctional dressing for healing with open trauma wound infections and wound with seawater immersion.

Topics: Animals; Anti-Bacterial Agents; Bacterial Infections; Cell Line, Tumor; Chitosan; Ciprofloxacin; Collagen; Cross-Linking Reagents; Cytokines; Gelatin; Iridoids; Male; Mice; Microspheres; Rats; Rats, Sprague-Dawley; Seawater; Transforming Growth Factor beta; Wound Healing

Neuropathic pain caused by traumatic neuromas is an extremely intractable clinical problem. Disorderly scar tissue accumulation and irregular and immature axon regeneration around the injury site mainly contribute to traumatic painful neuroma formation. Therefore, successfully preventing traumatic painful neuroma formation requires the effective inhibition of irregular axon regeneration and disorderly accumulation of scar tissue. Considering that chondroitin sulfate proteoglycans (CSPGs) can act on the growth cone and effectively inhibit axon regeneration, the authors designed and manufactured a CSPG-gelatin blocker to regulate the CSPGs' spatial distribution artificially and applied it in a rat model after sciatic nerve neurectomy to evaluate its effects in preventing traumatic painful neuroma formation.. Sixty female Sprague Dawley rats were randomly divided into three groups (positive group: no covering; blank group: covering with gelatin blocker; and CSPG group: covering with the CSPG-gelatin blocker). Pain-related factors were evaluated 2 and 8 weeks postoperatively (n = 30). Neuroma growth, autotomy behavior, and histological features of the neuromas were assessed 8 weeks postoperatively (n = 30).. Eight weeks postoperatively, typical bulb-shaped neuromas did not form in the CSPG group, and autotomy behavior was obviously better in the CSPG group (p < 0.01) than in the other two groups. Also, in the CSPG group the regenerated axons showed a lower density and more regular and improved myelination (p < 0.01). Additionally, the distribution and density of collagenous fibers and the expression of α-smooth muscle actin were significantly lower in the CSPG group than in the positive group (p < 0.01). Regarding pain-related factors, c-fos, substance P, interleukin (IL)-17, and IL-1β levels were significantly lower in the CSPG group than those in the positive and blank groups 2 weeks postoperatively (p < 0.05), while substance P and IL-17 remained lower in the CSPG group 8 weeks postoperatively (p < 0.05).. The authors found that CSPGs loaded in a gelatin blocker can prevent traumatic neuroma formation and effectively relieve pain symptoms after sciatic nerve neurotomy by blocking irregular axon regeneration and disorderly collagenous fiber accumulation in the proximal nerve stump. These results indicate that covering the proximal nerve stump with CSPGs may be a new and promising strategy to prevent traumatic painful neuroma formation in the clinical setting.

Topics: Administration, Topical; Animals; Axons; Behavior, Animal; Chondroitin Sulfate Proteoglycans; Cicatrix; Female; Ganglia, Spinal; Gelatin; Growth Cones; Interleukin-17; Interleukin-1beta; Iridoids; Nerve Regeneration; Neuralgia; Neuroma; Peripheral Nervous System Neoplasms; Random Allocation; Rats; Rats, Sprague-Dawley; rho GTP-Binding Proteins; Sciatic Neuropathy; Sciatica; Single-Blind Method

Due to its ubiquity and versatility in the human body, collagen is an ideal base material for tissue-engineering constructs. Chemical crosslinking treatments allow precise control of the biochemical and mechanical properties through macromolecular modifications to the structure of collagen. In this work, three key facets regarding the collagen crosslinking process are explored. Firstly, a comparison is drawn between the carbodiimide-succinimide (EDC-NHS) system and two emerging crosslinkers utilising alternate chemistries: genipin and tissue transglutaminase (TG2). By characterising the chemical changes upon treatment, the effect of EDC-NHS, genipin and TG2 crosslinking mechanisms on the chemical structure of collagen, and thus the mechanical properties conferred to the substrate is explored. Secondly, the relative importance of mechanical and biochemical cues on cellular phenomena are investigated, including cell viability, integrin-specific attachment, spreading and proliferation. Here, we observe that for human dermal fibroblasts, long-term, stable proliferation is preconditioned by the availability of suitable binding sites, irrespective of the substrate modulus post-crosslinking. Finally, as seen in the graphical abstract we show that by choosing the appropriate crosslinker chemistries, a materials selection map can be drawn for collagen films, encompassing both a range of tensile modulus and fibroblast proliferation which can be modified independently. Thus, in addition to a range of parameters that can be modified in collagen constructs, we demonstrate a route to obtaining tunable bioactivity and mechanics in collagen constructs is uncovered, that is exclusively driven by the crosslinking process.

Topics: Collagen; Cross-Linking Reagents; Human Body; Humans; Iridoids; Succinimides; Tissue Engineering

A layer-by-layer (LbL) coating was designed using ionic polysaccharides (chitosan, sodium alginate, sodium hyaluronate) and genipin (crosslinker), to sustain the release of diclofenac sodium salt (DCF) from soft contact lens (SCL) materials. The coating was hydrophilic, biocompatible, non-toxic, reduced bacterial growth and had minor effects on the physical properties of the material, such as wettability, ionic permeability, refractive index and transmittance, which remained within the recommended values for SCLs. The coating was applied on a silicone-based hydrogel and on commercial SofLens and Purevision SCLs. The coating attenuated the initial drug burst and extended the therapeutic period for, at least, two weeks. Relevantly, the problems of sterilizing drug loaded SCLs coated with biopolymers, using classic methods that involve high temperature or radiation, were successfully solved through high hydrostatic pressure (HHP) sterilization.

Topics: Alginates; Anti-Bacterial Agents; Bacteriological Techniques; Cell Line; Chitosan; Contact Lenses, Hydrophilic; Delayed-Action Preparations; Diclofenac; Drug Liberation; Hyaluronic Acid; Hydrogels; Iridoids; Polyhydroxyethyl Methacrylate; Technology, Pharmaceutical; Wettability

Curcumin (CUR) is a promising edible phytochemical compound with ideal ulcerative colitis (UC) treatment activity; however, it is characteristically instable in the digestive tract and has a short retention time in colon. Therefore, we designed and fabricated an oral food-grade nanocarrier composed of tannic acid (TA)-coated, Genipin (Gnp)-crosslinked human serum albumin (HSA) to encapsulate CUR (TA/CUR-NPs). The resulting CUR nanoparticles (NPs) were about 220 nm and -28.8 mV. With the assistance of TA layer and Gnp-crosslinking, the entire nano-scaled system effectively delayed CUR release in simulated gastric fluid, prolonged its colon adhesion and increased its uptake in Caco-2 cells. As expected, TA/CUR-NPs oral administration significantly alleviated colitis symptoms in DSS-treated mice when compared with controls by inhibiting the TLR4-linked NF-κB signaling pathway. Collectively, this study indicates that we have developed a convenient, eco-friendly, nano-scaled vehicle for oral delivery of CUR with anti-UC benefit.

Topics: Administration, Oral; Animals; Caco-2 Cells; Colitis, Ulcerative; Curcumin; Drug Delivery Systems; Humans; Iridoids; Male; Mice; Mice, Inbred BALB C; Nanoparticles; Serum Albumin, Human; Tannins

Crosslinking with genipin increases the acidic stability of chitosan-based materials, opening an opportunity to explore new applications. In this work, the viability of using chitosan-genipin solutions on cellulose-based materials coating was studied. Non-calendered paper and cardboard were used as raw materials. Different number of chitosan-genipin coating layers (1, 3, 6, 20, and 30) were applied and their influence on the materials mechanical, physicochemical, and barrier properties was studied. The small thickness and basis weight of non-calendered paper resulted in an inefficient adhesion of chitosan-genipin coating to the cellulose fibers. However, in cardboard, chitosan-genipin created a dense layer onto the cellulosic-fibers surface without impairing their mechanical properties. It conferred a greenish color, whose intensity increased with the layers number. The chitosan-genipin coating decreased the cardboard air and water vapor permeability up to 71 % and 52 %, respectively, and acted as a physical barrier for cardboard compounds leaching, being suitable for covering cellulose-based materials.

Topics: Antioxidants; Benzothiazoles; Cellulose; Chitosan; Coated Materials, Biocompatible; Cross-Linking Reagents; Iridoids; Materials Testing; Particle Size; Sulfonic Acids; Surface Properties

An important problem for researchers working in the field of dermatology is the preparation of the human skin equivalent (HSE). Here, we describe a simple and reliable protocol for preparing a skin model from the commercially available cell lines: keratinocytes, fibroblasts, and melanocytes. Importantly, in our 3D model, the keratinocytes are diverse that brings this model closer to the natural skin. For the production of HSE, we used available primary PCS-200-010, PCS-201-010, PCS-200-013, and immortalized CRL-4048 and CRL-4001 cell lines. We used genipin, which is necessary for collagen cross-linking and studied its cytotoxicity for keratinocytes and fibroblasts. The addition of 20 μM genipin reduced the shrinkage of the collagen in the constructs from 59% to 24% on day 12 of the culture of the construct. A higher concentration (80-200 µM) of genipin reduced shrinkage by 14% on average. Genipin in concentration 10 μM and below was not cytotoxic to the keratinocytes, and 150 μM and below to the fibroblasts. Hematoxylin and eosin staining showed that the morphology of HSEs was identical to that of native human skin. The immunohistochemical staining of the constructs showed the presence of vimentin-positive fibroblasts in the skin layer, while the melanocytes were in the epidermis and in the basal layer. We observed that the longer differentiation of constructs led to the higher secretion of GM-CSF, IL-10, IL-15, IL-1α, IL-6, IL-7, IL-8, and MCP-1. We also observed that the longer time of differentiation led to a more stable secretion of all analytes, which was reflected in the coefficient of variation. We described here a simple, reliable, and cost-effective production of the full-thickness human skin equivalents that can be used in the research and industry. With the global trend to decrease animal use for the research and testing, our HSE could be a useful testing tool and an alternative research model.

Topics: Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Collagen; Dermis; Epidermal Cells; Epidermis; Extracellular Matrix; Fibroblasts; Humans; Iridoids; Keratinocytes; Melanocytes; Models, Biological; Skin; Skin, Artificial

The cell-derived extracellular matrix (ECM) is associated with a lower risk of pathogen transfer, and it possesses an ideal niche with growth factors and complex fibrillar proteins for cell attachment and growth. However, the cell-derived ECM is found to have poor biomechanical properties, and processing of cell-derived ECM into gels is scarcely studied. The gel provides platforms for three-dimensional cell culture, as well as injectable biomaterials, which could be delivered via a minimally invasive procedure. Thus, in this study, an adipose-derived stem cell (ADSC)-derived ECM gel was developed and cross-linked by genipin to address the aforementioned issue. The genipin cross-linked ADSC ECM gel was fabricated via several steps, including rabbit ADSC culture, cell sheets, decellularization, freeze-thawing, enzymatic digestion, neutralization of pH, and cross-linking. The physicochemical characteristics and cytocompatibility of the gel were evaluated. The results demonstrated that the genipin cross-linking could significantly enhance the mechanical properties of the ADSC ECM gel. Furthermore, the ADSC ECM was found to contain collagen, fibronectin, biglycan, and transforming growth factor (TGF)-β1, which could substantially maintain ADSC, skin, and ligament fibroblast cell proliferation. This cell-derived natural material could be suitable for future regenerative medicine and tissue engineering application.

Topics: Adipocytes; Adipose Tissue; Animals; Cell Proliferation; Cells, Cultured; Extracellular Matrix; Gels; Iridoids; Male; Mice; Rabbits; Stem Cells; Tissue Engineering; Tissue Scaffolds

The signal transducer and activator of transcription-3 (STAT-3) can facilitate cancer progression and metastasis by being constitutively active via various signaling. Abundant evidence has indicated that STAT-3 may be a promising molecular target for cancer treatment.. In this study, a dual-luciferase assay-based screening of 537 compounds for STAT-3 inhibitors of hepatocellular carcinoma (HCC) cells was conducted, leading to the identification of genipin. Effects of genipin on HCC were assessed in a patient-derived xenograft nude mice model. Western blotting assay, chromatin immunoprecipitation (ChIP) assay, molecular docking study, tube formation assay, three-dimensional top culture assay, histological examination, and immunofluorescence were utilized to evaluate the regulatory signaling pathway.. Our research demonstrated that genipin suppresses STAT-3 phosphorylation and nuclear translocation, which may be attributed to the binding capacity of this compound to the Src homology-2 (SH2) domain of STAT-3. In addition, the therapeutic effects of genipin in a patient-derived HCC xenograft nude mice model were also demonstrated.. In conclusion, genipin showed therapeutic potential for HCC treatment by interacting with the SH2-STAT-3 domain and suppressing the activity of STAT-3. In the future, further research is planned to explore the potential role of genipin in combination with chemotherapy or radiotherapy for HCC.

Topics: Animals; Apoptosis; Biomarkers, Tumor; Carcinoma, Hepatocellular; Cell Movement; Cell Proliferation; Cholagogues and Choleretics; Epithelial-Mesenchymal Transition; Female; Gene Expression Regulation, Neoplastic; Humans; Iridoids; Liver Neoplasms; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Invasiveness; Neovascularization, Pathologic; Phosphorylation; Signal Transduction; STAT3 Transcription Factor; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

The purpose of this study was to determine whether genipin-induced crosslinked cell-derived matrix (XCDM) precipitates fibrotic phenotypes in human trabecular meshwork (hTM) cells by dysregulating β-catenin and Yes-associated protein (YAP)/ transcriptional coactivator with PDZ-binding motif (TAZ) signaling pathways.. Cell-derived matrices were treated with control or genipin for 5 hours to obtain respective uncrosslinked (CDM) and XCDMs and characterized. hTM cells were seeded on these matrices with/without Wnt pathway modulators in serum-free media for 24 hours. Elastic modulus, gene, and protein (whole cell and subcellular fractions) expressions of signaling mediators and targets of Wnt/β-catenin and YAP/TAZ pathways were determined.. At the highest genipin concentration (10% XCDM), XCDM had increased immunostaining of N-ε(γ-glutamyl)-lysine crosslinks, appeared morphologically fused, and was stiffer (5.3-fold, P < 0.001). On 10% XCDM, hTM cells were 7.8-fold (P < 0.001) stiffer, total β-catenin was unchanged, pβ-catenin was elevated, and pGSK3β was suppressed. Although 10% XCDM had no effect on cytoplasmic β-catenin levels, it reduced nuclear β-catenin, cadherin 11, and key Wnt target genes/proteins. The 10% XCDM increased total TAZ, decreased pTAZ, and increased cytoplasmic TAZ levels in hTM cells. The 10% XCDM increased total YAP, reduced nuclear YAP levels, and critical YAP/TAZ target genes/proteins. Wnt activation rescued hTM cells from 10% XCDM-induced stiffening associated with increased nuclear β-catenin.. Increased cytoplasmic TAZ may inhibit β-catenin from its nuclear shuttling or regulating cadherin 11 important for aqueous homeostasis. Elevated cytoplasmic TAZ may inhibit YAP's probable homeostatic function in the nucleus. Together, TAZ's cytoplasmic localization may be an important downstream event of how increased TM extracellular matrix (ECM) crosslinking may cause increased stiffness and ocular hypertension in vivo. However, Wnt pathway activation may ameliorate ocular hypertensive phenotypes induced by crosslinked ECM.

Topics: Adaptor Proteins, Signal Transducing; Aged; beta Catenin; Blotting, Western; Cells, Cultured; Cross-Linking Reagents; Extracellular Matrix; Female; Humans; Iridoids; Male; Microscopy, Atomic Force; Microscopy, Electron, Scanning; Middle Aged; Real-Time Polymerase Chain Reaction; Signal Transduction; Trabecular Meshwork; Transcription Factors; Wnt Signaling Pathway; YAP-Signaling Proteins

Topics: Calorimetry, Differential Scanning; Chitosan; Iridoids; Molecular Weight; Phase Transition; Rheology; Spectroscopy, Fourier Transform Infrared; Surface Properties; Vitrification; X-Ray Diffraction

Biomaterials used in tissue engineering and regenerative medicine applications benefit from longitudinal monitoring in a non-destructive manner. Label-free imaging based on fluorescence lifetime imaging (FLIm) and Raman spectroscopy were used to monitor the degree of genipin (GE) cross-linking of antigen-removed bovine pericardium (ARBP) at three incubation time points (0.5, 1.0, and 2.5 h). Fluorescence lifetime decreased and the emission spectrum redshifted compared to that of uncross-linked ARBP. The Raman signature of GE-ARBP was resonance-enhanced due to the GE cross-linker that generated new Raman bands at 1165, 1326, 1350, 1380, 1402, 1470, 1506, 1535, 1574, 1630, 1728, and 1741 cm

Topics: Animals; Cattle; Cross-Linking Reagents; Extracellular Matrix; Iridoids; Optical Imaging; Pericardium; Spectrum Analysis, Raman

Thermosensitive hydrogels based on polysaccharides are suitable candidates for the design of biodegradable and biocompatible injectable drug delivery systems. Thus, the combination of chitosan (CHI) and β-glycerol phosphate disodium salt (β-GP) has been intensively investigated to develop thermo-induced physical gels. With the aim of exploring the possibilities of optimization of these hydrogels, in this work, chitosan, β-GP and naturally extracted crosslinking agent, genipin (GEN), have been successfully combined, obtaining co-crosslinked hydrogels with both in situ physical and covalent crosslinking. A wide range of β-GP concentrations have been selected in order to analyze its influence on a variety of properties, including gelation time, pore size, water uptake ability, in vitro hydrolytic and enzymatic degradation, mucoadhesion and mechanical and rheological properties. Furthermore, the potential application of the developed systems for the administration and controlled release of an anti-inflammatory anionic drug, such as diclofenac, has been successfully demonstrated.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Chitosan; Diclofenac; Drug Delivery Systems; Drug Liberation; Glycerophosphates; Hydrogels; Iridoids; Rheology; Spectroscopy, Fourier Transform Infrared; Static Electricity; Temperature

The present manuscript deals with the elucidation of the mechanism of genipin binding by primary amines at neutral pH. UV-VIS and CD measurements both in the presence of oxygen and in oxygen-depleted conditions, combined with computational analyses, led to propose a novel mechanism for the formation of genipin derivatives. The indications collected with chiral and achiral primary amines allowed interpreting the genipin binding to a lactose-modified chitosan (CTL or Chitlac), which is soluble at all pH values. Two types of reaction and their kinetics were found in the presence of oxygen: (i) an interchain reticulation, which involves two genipin molecules and two polysaccharide chains, and (ii) a binding of one genipin molecule to the polymer chain without chain-chain reticulation. The latter evolves in additional interchain cross-links, leading to the formation of the well-known blue iridoid-derivatives.

Topics: Amines; Biocompatible Materials; Chitosan; Circular Dichroism; Computational Chemistry; Cross-Linking Reagents; Hydrogen-Ion Concentration; Iridoids; Kinetics; Lactose; Ligands; Magnetic Resonance Spectroscopy; Oxygen; Polysaccharides; Spectrophotometry, Ultraviolet

Genipin has been proposed as a possible neuroprotective therapy in myopia and glaucoma. Here, we aim to determine the effects of prolonged genipin-induced scleral stiffening on visual function.. Eyes from Brown Norway rats were treated in vivo with either a single 15 mM genipin retrobulbar injection or sham retrobulbar injection and were compared to naïve eyes. Intraocular pressure, optomotor response, and electroretinograms were repeatedly measured over 4 weeks following retrobulbar injections to determine visual and retinal function. At 4 weeks, we quantified retinal ganglion cell axon counts. Finally, molecular changes in gene and protein expression were analyzed via real-time polymerase chain reaction (RT-PCR) and proteomics.. Retrobulbar injection of genipin did not affect intraocular pressure (IOP) or retinal function, nor have a sustained impact on visual function. Although genipin-treated eyes had a small decrease in retinal ganglion cell axon counts compared to contralateral sham-treated eyes (-8,558 ± 18,646; mean ± SD), this was not statistically significant (. Posterior scleral stiffening with a single retrobulbar injection of 15 mM genipin causes no sustained deficits in visual or retinal function or at the molecular level in the retina and sclera. Retinal ganglion cell axon morphology appeared normal.. These results support future in vivo studies to determine the efficacy of genipin-induced posterior scleral stiffening to help treat ocular diseases, like myopia and glaucoma.

Topics: Animals; Glaucoma; Iridoids; Rats; Retina; Sclera

Mounting evidences have recognized that dual cross-link and double-network gels can promisingly recapitulate the complex living tissue architecture and overcome mechanical limitations of conventional scaffolds used hitherto in regenerative medicine. Here, dual cross-link gels formed of a bioactive lactose-modified chitosan reticulated via both temporary (boric acid-based) and permanent (genipin-based) cross-linkers are reported. While boric acid rapidly binds to lactitol flanking diols increasing the overall viscosity, a slow temperature-driven genipin binding process takes place allowing for network strengthening. Combination of frequency and stress sweep experiments in the linear stress-strain region shows that ultimate gel strength, toughness, and viscoelasticity depend on polymer-to-genipin molar ratio. Notably, herewith it is demonstrated that linear stretching correlates with strain energy dissipation through boric acid binding/unbinding dynamics. Strain-hardening effect in the nonlinear regime, along with good biocompatibility in vitro, points at an interesting role of present system as biological extracellular matrix substitute.

Topics: Biocompatible Materials; Boric Acids; Chitosan; Gels; Humans; Iridoids; Lactose; Regenerative Medicine; Stress, Mechanical; Viscosity

Rotavirus is the leading cause of acute gastroenteritis among young children worldwide. However, agents specifically designed to treat rotavirus infection have not been developed yet. In this study, the anti-rotavirus and anti-inflammatory effects of genipin, a chemical compound found in the fruit of Gardenia jasminoides, were evaluated. Genipin had an antiviral effect against the human rotavirus Wa and SA-11 strains in vitro, and it inhibited two distinct stages of the viral replication cycle: attachment and penetration (early stage) in pre-treatment and assembly and release (late stage) in post-treatment. Additionally, genipin downregulated nitric oxide synthase and pro-inflammatory cytokines in lipopolysaccharide-stimulated RAW264.7 cells and rotavirus-infected Caco-2 cells. Oral administration of genipin before and after viral infection with the murine rotavirus epidemic diarrhea of infant mice strain led to a reduced duration of diarrhea and faecal viral shedding and to decreased destruction of the enteric epithelium. Genipin could have potential as a natural compound with preventive and therapeutic effects against infection and colitis caused by rotavirus.

Topics: Animals; Animals, Newborn; Anti-Inflammatory Agents; Antiviral Agents; Caco-2 Cells; Cytokines; Diarrhea; Disease Models, Animal; Humans; Inflammation; Iridoids; Mice; Nitric Oxide Synthase; RAW 264.7 Cells; Rotavirus; Rotavirus Infections; Virus Replication; Virus Shedding

The genipin-crosslinked chitosan (GCS) nanocarrier has received a lot of attention due to its unique biological and chemical properties as an effective drug delivery system. GCS was modeled by considering two chitosan (CS) polymer sequences with six monomer units that are crosslinked by genipin. To investigate the characteristics of this model, we considered it as a nanocarrier of the anti-cancer drug cladribine (2CdA). Seven configurations of GCS and 2CdA (GCS/2CdA1-7) were optimized at M06-2X/6-31G(d,p) in aqueous solution. The average binding energy above 100 kJ mol

Topics: Chitosan; Drug Carriers; Drug Delivery Systems; Humans; Hydrogels; Hydrogen Bonding; Iridoids; Models, Chemical; Nanocomposites; Polymers

Microdiscectomy is the current standard surgical treatment for intervertebral disc (IVD) herniation, however annulus fibrosus (AF) defects remain unrepaired which can alter IVD biomechanical properties and lead to reherniation, IVD degeneration and recurrent back pain. Genipin-crosslinked fibrin (FibGen) hydrogel is an injectable AF sealant previously shown to partially restore IVD motion segment biomechanical properties. A small animal model of herniation and repair is needed to evaluate repair potential for early-stage screening of IVD repair strategies prior to more costly large animal and eventual human studies. This study developed an ex-vivo rat caudal IVD herniation model and characterized torsional, axial tension-compression and stress relaxation biomechanical properties before and after herniation injury with or without repair using FibGen. Injury group involved an annular defect followed by removal of nucleus pulposus tissue to simulate a severe herniation while Repaired group involved FibGen injection. Injury significantly altered axial range of motion, neutral zone, torsional stiffness, torque range and stress-relaxation biomechanical parameters compared to Intact. FibGen repair restored the stress-relaxation parameters including effective hydraulic permeability indicating it effectively sealed the IVD defect, and there was a trend for improved tensile stiffness and axial neutral zone length. This study demonstrated a model for studying IVD herniation injury and repair strategies using rat caudal IVDs ex-vivo and demonstrated FibGen sealed IVDs to restore water retention and IVD pressurization. This ex-vivo small animal model may be modified for future in-vivo studies to screen IVD repair strategies using FibGen and other IVD repair biomaterials as an augment to additional large animal and human IVD testing.

Topics: Animals; Biomechanical Phenomena; Fibrin Tissue Adhesive; Hydrogels; Intervertebral Disc; Intervertebral Disc Degeneration; Iridoids; Rats

Circadian rhythm disruption impacts a wide range of physiological processes, including fertility. However, the effect of circadian disruption on male spermatogenesis and fertility, and treatments for these effects have been largely unexplored at the molecular level.. In this study, we examined the effects of genipin on improving the reproductive health problems caused by circadian disruption. Three groups of animals were fed under different conditions: control group (normal T cycle with saline), group of shortened T cycles (Light/Dark = 4 hours/4 hours) with saline, and a group of shortened T cycles with genipin by oral gavage. The male fertility was evaluated by fertility study and pups parameters analysis after successful sexual behavior and mating with female mice. We sacrificed the treated animals after 5 or 10 weeks and collected the testis, sperm and serum for histological analysis, sperm motility assay, and serum hormone detection, respectively. Furthermore, the effect of genipin was assessed by detection of progesterone secretion and steroidogenic key proteins expression, including StAR and CYP11A1, in mouse Leydig tumor MLTC-1 cells.. Male mice exposed to shortened light-dark cycles, much shorter than 24 hours, had reduced fertility with decreased sperm concentrations and sperm motility. Male mice under circadian disruption have reduced testis size and abnormal morphology, leading to lower fertility rates, reduced litter size and pup body weight. Treatment with exogenous genipin, a natural plant-derived compound, alleviated circadian disruption-induced damage to fertility and spermatogenesis and normalized testosterone, dihydrotestosterone (DHT), and androstenedione (ASD) levels in the male mice. The levels of key proteins involved in steroidogenesis, StAR and CYP11A1, were reduced in mouse testes after the circadian disruption, but genipin treatment restored the reduction. The mRNA expression of SRD5A1, which encodes an androgen synthesis enzyme, was also upregulated by genipin treatment. Furthermore, genipin treatment showed a positive effect on steroidogenesis in MLTC-1 cells, resulting in an increase in hormone secretion and the upregulation of StAR and CYP11A1.. Our results showed an association between circadian disruption and reproductive health problems in male mice and indicated that treatments with genipin have positive effects on the reproductive health of male mice with circadian rhythm disorders.

Topics: 3-Oxo-5-alpha-Steroid 4-Dehydrogenase; Androstenedione; Animals; Cell Line, Tumor; Cholesterol Side-Chain Cleavage Enzyme; Circadian Rhythm; Dihydrotestosterone; Female; Fertility; Gene Expression; Iridoids; Male; Membrane Proteins; Mice, Inbred ICR; Reproduction; Spermatogenesis; Testis; Testosterone

We developed a hybrid nanostructured lipid carrier/dual pH- and thermo-sensitive hydrogel (NLC-Gel) for ocular delivery of quercetin (QN). The hydrogel consisted of carboxymethyl chitosan and poloxamer 407 (P407) was cross-linked using a naturally occurring cross-linker genipin (GP), while the hydrogel cross-linked using glutaraldehyde was used as a control. NLC loaded with quercetin/hydrogel cross-linked by a genipin hybrid drug delivery system (QN-NLC-Gel-GP) exhibited better cytocompatibility and lower ocular irritation than its glutaraldehyde counterpart. The levels of cellular uptake in NLC and NLC-Gel groups were improved. The results of fluorescence imaging and ex vivo transcorneal study indicated that NLC could facilitate transcorneal penetration of lipophilic molecules and incorporation into the hydrogel could increase the precorneal residence time of NLC. The area under the curve of quercetin in the NLC-Gel group was 4.4-fold that in the Eye drops group because of a longer precorneal retention time. In summary, the developed drug delivery system has enormous potential for ophthalmic applications.

Topics: Drug Carriers; Drug Delivery Systems; Hydrogels; Iridoids; Lipids

Topics: Adult; Coloring Agents; Dermatitis, Allergic Contact; Female; Humans; Iridoids; Plant Extracts; Rubiaceae; Tattooing

The protective effects of genipin against lipopolysaccharide (LPS)-induced acute lung injury (ALI) have been reported; however, the mechanism is unclear. Genipin performs its pharmacological effects via activation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway. In the present study, we aimed to determine whether the PI3K/AKT pathway is involved in the protective effects of genipin against mitochondrial-dependent apoptosis, endoplasmic reticulum stress (ERS), and inflammation in ALI. We constructed in vivo and in vitro models of LPS-induced ALI. PI3K/AKT signaling was inhibited using LY294002. Pretreatment with genipin increased AKT phosphorylation, indicating that PI3K/AKT signaling was upregulated. Genipin pretreatment prevented LPS-induced histopathological deterioration, increased pulmonary edema, and decreased oxygenation index, all of which were inhibited using LY294002. In addition, genipin pretreatment attenuated LPS-mediated mitochondrial apoptosis, as indicated by improved mitochondrial dysfunction, downregulation of BAX (BCL2 associated X, apoptosis regulator), upregulation of BCL2 (BCL2 apoptosis regulator), inhibited the release of cytochrome c, activation of caspase-3, and cell apoptosis. Genipin pretreatment inhibited the LPS-induced upregulation of AF4/FMR2 family member 4 (CHOP), glucose-regulated protein, 78 kDa (GRP78), and X-box binding protein 1 (XBP1) levels, indicating ERS suppression. Moreover, genipin pretreatment alleviated LPS-induced inflammation, indicating by blockade of nuclear factor kappa b (NF-κB) signaling activation and reduced tumor necrosis factor alpha (TNF-α), interleukin (IL)-1β, and IL-6 levels in the lung and bronchoalveolar lavage fluid. LY294002 could inhibit these genipin-induced protective effects against apoptosis, ERS, and inflammation. Thus, genipin significantly activates PI3K/AKT signaling to ameliorate mitochondria-dependent apoptosis, ERS, and inflammation in LPS-induced ALI.

Topics: A549 Cells; Acute Lung Injury; Animals; Anti-Inflammatory Agents; Apoptosis; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Humans; Iridoids; Lipopolysaccharides; Lung; Male; Mitochondria; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; Rats, Sprague-Dawley; Signal Transduction

Structural relaxation and glass transition were examined in the swelling behaviour of a high-solid biopolymer matrix; genipin-crosslinked gelatin. Degree of swelling was quantified by the Flory-Rehner theory that furnishes estimates of average molecular weight between crosslinks and network mesh size. Fourier transform infrared spectroscopy and wide angle X-ray diffraction described intermolecular interactions and the extent of amorphicity in the crosslinked matrix. Micro differential scanning calorimetry provided evidence of the changing thermodynamic characteristics of the gelatin network in the presence of the crosslinker. Modulated differential scanning calorimetry and small deformation oscillatory rheology unveiled the vitrification properties of the system. Experimental measurements were treated with the time-temperature superposition principle to unveil an extensive master curve through the rubbery, glass transition and glassy states. Viscoelastic behaviour was modelled with the combined predictions of the modified Arrhenius and William-Landel-Ferry theories to pinpoint the mechanical glass transition temperature that was compared with predictions from calorimetry. Comprehensive understanding of polymeric behaviour during swelling affords greater control in the design of targeted delivery matrices for drugs and other bioactive compounds.

Topics: Algorithms; Biopolymers; Chemical Phenomena; Cross-Linking Reagents; Gelatin; Glass; Iridoids; Mechanical Phenomena; Models, Theoretical; Molecular Structure; Molecular Weight; Phase Transition; Spectrum Analysis

Hydrogel membranes are often used as physical barriers in oral tissue reconstruction and facial surgery to isolate connective and epithelial tissues and form a closed space for undisturbed bone healing. In this study, gelatin and hyaluronic acid were crosslinked with genipin and loaded with a hinokitiol additive as a bacteriostatic agent for potential applications as regeneration membranes. This bifunctional membrane had biocompatibility and antibacterial activities on each membrane side for proper biodegradation. Different membrane groups of gelatin/hyaluronic acid were obtained via a solution casting technique and were genipin crosslinked. The membrane groups were further loaded with adequate hinokitiol at a loading concentration of up to 0.16 g/L (hinokitiol to phosphate buffered saline). Fourier transform infrared spectroscopy showed that gelatin and hyaluronic acid were crosslinked with genipin through cross-linking amide bond (CONH) formation with a cross-linking degree of over 84%. The groups with hinokitiol showed substantial antibacterial activity. Meanwhile, the addition of hinokitiol on hydrogel membranes did not significantly affect the tensile strength. However, it decreased the solubility of the membranes by slowing down the relaxation and degradation of their molecular junctions as hinokitiol is a hydrophobic compound with low permeability. Consequently, the degradation of hydrogel membranes with hinokitiol was delayed. In vitro cytocompatibility indicated that the cell viability of the groups with hinokitiol increased with incubation time, demonstrating that cell viability and proliferation were not affected by cell culture testing.

Topics: Animals; Anti-Bacterial Agents; Cross-Linking Reagents; Drug Evaluation, Preclinical; Gelatin; Hyaluronic Acid; Hydrogels; Iridoids; Materials Testing; Membranes, Artificial; Mice; Monoterpenes; NIH 3T3 Cells; Tropolone

Therapeutic proteins have been widely used in the treatment of various diseases, and effective carriers are highly required for achieving protein delivery to obtain favorable treatment potency.. A protein-polymer hybrid system was constructed through the genipin-mediated crosslinking of polyethyleneimine with a weight-average molecular weight of 25,000 g/mol (PEI25K) and ribonuclease A (RNase A), namely RGP.. The RGP nanoparticles were observed to be easily internationalized in HeLa cells owing to the introduction of positively charged PEI25K, thereby triggering the antiproliferative effects by cleaving RNA molecules in the tumor cells. Moreover, red fluorescence could be obviously visualized in the tumor cells after RGP delivery, which was attributed to the intrinsic characteristics of genipin.. The protein-polymer hybrid system prepared via the genipin-mediated crosslinking has exhibited potential to be used as a theranostic platform for both in vivo imaging and delivering diverse therapeutic proteins.

Topics: Apoptosis; Cell Proliferation; Cross-Linking Reagents; Drug Delivery Systems; Endosomes; HeLa Cells; Humans; Intracellular Space; Iridoids; Nanoparticles; Polyethyleneimine; Ribonuclease, Pancreatic; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared

The iridoid compound genipin (GNP) is a geniposide hydrolysate of β-glucosidase. GNP has many pharmacological effects, including antioxidant, anti-apoptotic, and anti-inflammation effects. However, its exact target and mechanism of action remain poorly understood. In this study, the binding of GNP to AKT protein was demonstrated via a GNP-modified magnetic microspheres (GNP-MMs) capture and immunofluorescence co-localization test. GNP-MMs fishing coupled with competitive testing and AKT plasma transport experiments indicate that GNP may act on the PH domain of AKT, and affect AKT plasma transport. The specific binding directly inhibits phosphorylation of AKT, affecting the downstream activation, and reducing inflammatory responses. The results indicate that GNP targets the PH domain region of AKT, inhibits the phosphorylation of AKT, and attenuates the transduction of AKT based inflammation signal pathway.

Topics: Cholagogues and Choleretics; Dose-Response Relationship, Drug; Drug Delivery Systems; HEK293 Cells; Humans; Inflammation; Inflammation Mediators; Iridoids; Protein Structure, Secondary; Protein Structure, Tertiary; Proto-Oncogene Proteins c-akt; Signal Transduction

Scleral stiffening has been proposed as a therapy for glaucoma and myopia. Previous

Topics: Animals; Glaucoma; Iridoids; Male; Myopia; Rats; Sclera

Decellularized human pericardium is under study as an allogenic material for cardiovascular applications. The effects of crosslinking on the mechanical properties of decellularized pericardium were determined with a uniaxial tensile test, and the effects of crosslinking on the collagen structure of decellularized pericardium were determined by multiphoton microscopy. The viability of human umbilical vein endothelial cells seeded on decellularized human pericardium and on pericardium strongly and weakly crosslinked with glutaraldehyde and with genipin was evaluated by means of an MTS assay. The viability of the cells, measured by their metabolic activity, decreased considerably when the pericardium was crosslinked with glutaraldehyde. Conversely, the cell viability increased when the pericardium was crosslinked with genipin. Coating both non-modified pericardium and crosslinked pericardium with a fibrin mesh or with a mesh containing attached heparin and/or fibronectin led to a significant increase in cell viability. The highest degree of viability was attained for samples that were weakly crosslinked with genipin and modified by means of a fibrin and fibronectin coating. The results indicate a method by which in vivo endothelialization of human cardiac allografts or xenografts could potentially be encouraged.

Topics: Allografts; Animals; Biocompatible Materials; Biomechanical Phenomena; Cell Survival; Collagen; Cross-Linking Reagents; Fibrin; Fibronectins; Glutaral; Heterografts; Human Umbilical Vein Endothelial Cells; Humans; Iridoids; Materials Testing; Microscopy, Fluorescence, Multiphoton; Pericardium; Surface Plasmon Resonance; Tensile Strength

Topics: Animals; Apoptosis; Cell Cycle; Cell Line; Cell Survival; Chromatography, High Pressure Liquid; Gardenia; Inflammation; Iridoids; Liver; Molecular Docking Simulation; Oxidative Stress; Phytochemicals; Plant Extracts; Rats; Receptors, Tumor Necrosis Factor, Type I; Reference Standards

Aim to assess the short-term effect of genipin collagen crosslinking (G-CXL) on corneal structure and biomechanical properties compared with ultraviolet A/riboflavin collagen crosslinking (UVA-CXL) in rabbit corneas.. Right eyes of 40 healthy rabbits were divided into the 0.20% G-CXL group, 0.25% G-CXL group, UVA-CXL group, and control group. Anterior segment optical coherence tomography (ASOCT) and in vivo confocal microscopy (IVCM) were performed before, 7 days after, and 14 days after the CXL treatment. Corneal strips were harvested for tensile strain measurements 7 and 14 days after the CXL treatment.. ASOCT showed the demarcation line (DL) in the UVA-CXL group was deeper than in the 0.20% G-CXL group and the 0.25% G-CXL group on day 7 (p=0.014) and day 14 (p=0.012). Nerve and keratocyte density in all CXL groups decreased, but was more obvious in the UVA-CXL group (p<0.001). Endothelial cell loss in the 0.20% G-CXL group, 0.25% G-CXL group, UVA-CXL group, and control group was 11.7%, 6.8%, 32.8%, and 2.0% 14 days after CXL, respectively. Young's modulus and stress in the 0.25% G-CXL group and the UVA-CXL group were statistically significantly higher than in the control group (p<0.05) 7 and 14 days after CXL. No statistically significant differences were observed between the 0.25% G-CXL group and the UVA-CXL group (p>0.05). The DL depth was positively correlated with Young's modulus (r=0.426, p=0.042) and stress (r=0.469, p=0.024).. The administration of 0.25% genipin enhances corneal biomechanical properties as long as 14 days after the CXL treatment with low toxicity. The DL exists in CXL-treated corneas, and the depth is related to the biomechanical properties.

Topics: Animals; Biomechanical Phenomena; Cell Count; Collagen; Cornea; Cross-Linking Reagents; Elastic Modulus; Endothelial Cells; Image Processing, Computer-Assisted; Iridoids; Keratinocytes; Rabbits; Tomography, Optical Coherence

Genipin, a natural electrophilic cross-linker, was applied (5, 10, 20, and 30 mM) to modify hempseed protein isolate (HPI). Genipin treatments resulted in general losses of total sulfhydryls (up to 2.9 nmol/mg) and free amines (up to 77.3 nmol/mg). Surface hydrophobicity decreased by nearly 90% with 30 mM genipin, corresponding to similar tryptophan fluorescence quenching. The genipin treatment converted HPI into highly cross-linked polymers. Hydrogels formed with such polymers when also incorporated with hemp oil emulsions exhibited substantially enhanced gelling ability: up to 3.3- and 2.6-fold increases, respectively, in gel strength and gel elasticity over genipin-untreated protein. The genipin-modified composite gels also exhibited superior water-holding capacity. Microstructural analysis revealed a compact gel network filled with protein-coated oil globules that interacted intimately with the protein matrix when treated with genipin. Such gels remained readily digestible. Hence, genipin-treated hemp protein hydrogels show promise as functional food components.

Topics: Cannabis; Cross-Linking Reagents; Emulsions; Hydrogels; Iridoids; Plant Proteins; Rheology; Seeds

Genipin, a natural compound from Gardenia jasminoides, is a well-known compound in Chinese medicine that is used for the treatment of cancer, inflammation, and diabetes. The use of genipin in classical medicine is hindered because of its unknown molecular mechanisms of action apart from its strong cross-linking ability. Genipin is increasingly applied as a specific inhibitor of proton transport mediated by mitochondrial uncoupling protein 2 (UCP2). However, its specificity for UCP2 is questionable, and the underlying mechanism behind its action is unknown. Here, we investigated the effect of genipin in different systems, including neuroblastoma cells, isolated mitochondria, isolated mitochondrial proteins, and planar lipid bilayer membranes reconstituted with recombinant proteins. We revealed that genipin activated dicarboxylate carrier and decreased the activity of UCP1, UCP3, and complex III of the respiratory chain alongside with UCP2 inhibition. Based on competitive inhibition experiments, the use of amino acid blockers, and site-directed mutagenesis of UCP1, we propose a mechanism of genipin's action on UCPs. At low concentrations, genipin binds to arginine residues located in the UCP funnel, which leads to a decrease in UCP's proton transporting function in the presence of long chain fatty acids. At concentrations above 200 μM, the inhibitory action of genipin on UCPs is overlaid by increased nonspecific membrane conductance due to the formation of protein-genipin aggregates. Understanding the concentration-dependent mechanism of genipin action in cells will allow its targeted application as a drug in the above-mentioned diseases.

Topics: Amino Acids; Animals; Cell Line, Tumor; Electron Transport Complex III; Humans; Ion Transport; Ions; Iridoids; Membrane Potential, Mitochondrial; Mice; Mitochondrial Proteins; Protons; Uncoupling Protein 1; Uncoupling Protein 2

Deposition of calcium phosphate minerals on the elastin-rich medial layers of arteries can cause severe cardiovascular complications. There are no available treatments for medial calcification, and the mechanism of mineral formation on elastin layers is still unknown. We recently developed an in vitro model of medial calcification using cross-linked elastin-like polypeptide (ELP) membranes immersed in simulated body fluid (SBF). While mineral phase evolution matched that observed in a mouse model of medial calcification, the long incubation required was a practical limitation of this model. Using higher SBF ion concentrations could be a solution to speed up mineral deposition, but its effect on the mineralization process is still not well understood. Here we analyze mineral formation and phase transformation on ELP membranes immersed in high concentration SBF. We show that while mineral deposition is significantly accelerated in these conditions, the chemistry and morphology of the minerals deposited on the ELP membranes and the overall mineralization process are strongly affected. Overall, this work suggests that while the use of low concentration SBF in this in vitro model is more appropriate to study medial calcification associated with the loss of calcification inhibitors, higher SBF ion concentration may be more relevant to study medial calcification in patients with life-threatening diseases such as chronic kidney disease.

Topics: Apatites; Biomimetic Materials; Calcium; Crystallization; Elastin; Escherichia coli; Iridoids; Membranes, Artificial; Peptides; Sodium

Meniscal injuries, particularly in the avascular zone, have a low propensity for healing and are associated with the development of osteoarthritis. Current meniscal repair techniques are limited to specific tear types and have significant risk for failure. In previous work, we demonstrated the ability of meniscus-derived matrix (MDM) scaffolds to augment the integration and repair of an in vitro meniscus defect. The objective of this study was to determine the effects of percent composition and dehydrothermal (DHT) or genipin cross-linking of MDM bioscaffolds on primary meniscus cellular responses and integrative meniscus repair. In all scaffolds, the porous microenvironment allowed for exogenous cell infiltration and proliferation, as well as endogenous meniscus cell migration. The genipin cross-linked scaffolds promoted extracellular matrix (ECM) deposition and/or retention. The shear strength of integrative meniscus repair was improved with increasing percentages of MDM and genipin cross-linking. Overall, the 16% genipin cross-linked scaffolds were most effective at enhancing integrative meniscus repair. The ability of the genipin cross-linked scaffolds to attract endogenous meniscus cells, promote glycosaminoglycan and collagen deposition, and enhance integrative meniscus repair reveals that these MDM scaffolds are promising tools to augment meniscus healing.

Topics: Animals; Cell Proliferation; Cells, Cultured; Extracellular Matrix; Female; Iridoids; Meniscus; Shear Strength; Swine; Tissue Engineering; Tissue Scaffolds

Genipin, as the most effective ingredient of various traditional medications, encompasses antioxidative, anti-inflammatory, and antibacterial capacities. More recently, it is suggested that genipin protects against septic liver damage by restoring autophagy. The purpose of the current study was to explore the protective effect of genipin against carbon tetrachloride- (CCl

Topics: Animals; Autophagy; Carbon Tetrachloride; Chemical and Drug Induced Liver Injury; Disease Models, Animal; Inflammation; Iridoids; Liver; Male; Medicine, Traditional; Mice; Rubiaceae

To investigate the effects of bio-crosslinker genipin pretreatment on type Ⅰ collagen mineralization.. Type Ⅰ collagen gels were prepared and pretreated with 0.5wt%genipin (experimental group) and deionized water (control group) for 2 h, respectively. The pretreated products were subjected to Fourier transform infrared spectroscopy (FT-IR). Reconstituted collagen fibrils were pretreated with genipin or deionized water for 2 h and were mineralized for 4 h. The collagen density and mineralization degree were examined with transmission electron microscopy (TEM) and analyzed with ImageJ software. Then scanning electron microscopy (SEM) and TEM were used to observe the mineralization of cross-linked demineralized dentin collagen.. FT-IR spectrum showed that the genipin was crosslinked with collagen. TEM observation and ImageJ results showed that after 4 h mineralization, the mineralization effect of 0.5wt% genipin group was significantly better than that of the control group[(73.3±5.3)%vs.(7.4±3.5)%,. The study demonstrates that 0.5 wt% concentration of genipin can significantly promote the mineralization of type Ⅰ collagen.

Topics: Collagen Type I; Iridoids; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Spectroscopy, Fourier Transform Infrared

Topics: Acetylcholinesterase; Alzheimer Disease; Amyloid beta-Peptides; Animals; Binding Sites; Cell Survival; Drug Design; Inhibitory Concentration 50; Iridoids; Molecular Docking Simulation; Neuroprotective Agents; PC12 Cells; Peptide Fragments; Protein Structure, Tertiary; Rats; Structure-Activity Relationship

Catechol-functionalized polymers are of particular interest because of their strong water-resistant adhesive properties. Hydroxymethyl chitosan (HECTS) has been used as an implantable biomaterial having good water solubility, biodegradability and biocompatibility. Here, hydrocaffeic acid (HCA) grafted HECTS (HCA-g-HECTS) was prepared through carbodiimide coupling and the tethered catechol underwent periodate (

Topics: Animals; Caffeic Acids; Catechols; Cell Line; Chitosan; Cross-Linking Reagents; Female; Hydrogels; Iridoids; Materials Testing; Mice; Rats; Rats, Sprague-Dawley; Tissue Adhesives

Imaging of implanted hydrogel-based biosystems usually requires indirect labeling of the vehicle or cargo, adding complexity and potential risk of altering functionality. Here, for the first time, it is reported that incorporation of genipin into the design of immunoisolation devices can be harnessed for in vivo imaging. Using cell-compatible in situ cross-linking reactions, a fast, efficient and noncytotoxic procedure is shown to maximize fluorescence of microcapsules. Moreover, genipin is validated as a quantitative imaging probe by injecting increasing doses of microcapsules in the subcutaneous space of mice, obtaining strong, stable fluorescence with good linearity of signal to microcapsule dose over several weeks. This allows immediate assessment of the actual injected dose and monitoring of its position over time, thereby significantly enhancing the efficacy and biosafety of the therapy. These outcomes may facilitate clinical translation and optimize medical applications of multiple hydrogel-based biotechnologies.

Topics: Animals; Fluorescence; Iridoids; Mice; Optical Imaging; Prostheses and Implants

Bronchopulmonary dysplasia is the most common chronic lung disease of infancy and is associated with pulmonary hypertension (PH). Inhibition of glycogen synthase kinase (GSK)-3 β has been shown to attenuate lung injury and PH in hyperoxia-exposed newborn rats. Genipin has been widely used for the treatment of inflammatory diseases. The aim of this study was to show that genipin decreased the expression of GSK-3 β in lung tissues of hyperoxia-exposed rat pups.. We established models of hyperoxia-exposed rat pups, evaluated lung injury and pulmonary hypertension and detected the mRNA and protein expression of key molecules.. Hyperoxia resulted in the reduction of survival rate and histologic injury of lung tissues; an increase of the messenger RNA (mRNA) expression of transforming growth factor-β1, extracellular matrix proteins collagen-I and fibronectin, and α-smooth muscle actin; an increase of right ventricular (RV) systolic pressure and the weight ratio of RV to left ventriclar (LV) plus septum (S) (RV/LV + S) were inhibited by genipin. Genipin also decreased the levels of tumor necrosis factor-α, interleukin-1 β, and interleukin-6 in both bronchoalveolar lavage fluid and lung tissues after hyperoxia exposure. In addition, genipin inhibited p65 nuclear factor-κB nuclear translocation and matrix metalloproteinase-2 and -9 expression. Moreover, hyperoxia resulted in an increase of methane dicarboxylic aldehyde content and a decrease of superoxide dismutase activity, catalytic subunit of glutamate-cysteine ligase, modified subunit of glutamate-cysteine ligase, and nuclear factor erythroid 2-related factor 2 expression were inhibited by genipin. All these effects induced by genipin were blocked by upregulation of GSK-3 β. Genipin downregulated GSK-3 β expression, decreased nuclear factor-κB translocation, increased nuclear factor erythroid 2-related factor 2 expression, attenuated inflammation and oxidative stress, leading to amelioration of lung injury and PH in hyperoxia-exposed rat pups.. Overall, genipin may provide a novel therapeutic option for preventing and treating infants with bronchopulmonary dysplasia.

Topics: Animals; Animals, Newborn; Anti-Inflammatory Agents; Antioxidants; Bronchopulmonary Dysplasia; Disease Models, Animal; Gardenia; Glycogen Synthase Kinase 3 beta; Humans; Hyperoxia; Hypertension, Pulmonary; Infant, Newborn; Interleukins; Iridoids; Lung; Lung Injury; NF-E2-Related Factor 2; NF-kappa B; Oxygen; Phytotherapy; Plant Extracts; Rats, Sprague-Dawley; Tumor Necrosis Factor-alpha

Mesenchymal stem cells (MSCs) are potential therapeutic candidates, owing to their ability to differentiate into several cell types. However, the gap between availability and demand of MSCs requires alternative expansion methods from 2D culture flasks. Microcarriers are a promising approach for MSC expansion due to their high surface area-to-volume ratio. However, current commercial microcarriers do not provide the highest cell yield due to low cell attachment efficiencies and difficulty in cell detachment. This study developed a hydrogel-based microcarrier from genipin cross-linked alginate-chitosan beads. Alginate beads were produced by electrospraying before being coated with chitosan and cross-linked in genipin. The degree of cross-linking was determined through fluorescence reading of the genipin-chitosan conjugates. MSCs cultured on these microcarriers had a 26% higher cell attachment and twice the proliferation rate compared to the commercial microcarrier Cytodex 1. Cells easily detached from the hydrogel beads and did not require extended incubation periods or intense agitation during cell harvest. There was no significant difference in gene expression between the two microcarriers for the positive MSC surface markers CD-90, CD-105, and CD-73 as well as showing either low or no signal for negative MSC surface markers CD-45 and CD-34. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 122-133, 2019.

Topics: Alginates; Cell Culture Techniques; Cell Line, Transformed; Chitosan; Humans; Iridoids; Mesenchymal Stem Cells; Microspheres

The natural liver extracellular matrix (ECM) achieved by decellularization holds great potential in the fields of tissue engineering and regenerative medicine. Additionally, the use of crosslinking agents on the ECM to stabilize its ultrastructure and enhance scaffold durability is gaining interest in tissue engineering. The objective of this study was to compare the scaffold properties of porcine liver ECM crosslinked with different agents (glutaraldehyde, genipin, and quercetin) to find the best strategy for producing a decellularized matrix with optimal and stable characteristics for transplantation and regeneration. The properties examined include mechanical properties, material stability, immunogenicity, and angiogenic capacity. Scaffolds were implanted into the greater omentum of rats, and their abilities to induce immune cell subpopulation invasion and neovascularization were evaluated. The results show that genipin crosslinking of decellularized liver matrices increased the mechanical and proangiogenic properties and reduced the inflammatory response in vivo.

Topics: Animals; Extracellular Matrix; Glutaral; Iridoids; Liver; Male; Rats; Swine; Tissue Engineering; Tissue Scaffolds; Transplantation, Heterologous

Enzymes are gradually increasingly preferred over chemical processes, but commercial enzyme applications remain limited due to their low stability and low product recovery, so the application of an immobilization technique is required for repeated use. The aims of this work were to produce stable enzyme complexes of cross-linked xylanase on magnetic chitosan, to describe some characteristics of these complexes, and to evaluate the thermal stability of the immobilized enzyme and its reusability. A xylanase was cross-linked to magnetite particles prepared by in situ co-precipitation of iron salts in a chitosan template. The effect of temperature, pH, kinetic parameters, and reusability on free and immobilized xylanase was evaluated. Magnetization, morphology, size, structural change, and thermal behavior of immobilized enzyme were described. 1.0 ± 0.1 μg of xylanase was immobilized per milligram of superparamagnetic chitosan nanoparticles via covalent bonds formed with genipin. Immobilized xylanase showed thermal, pH, and catalytic velocity improvement compared to the free enzyme and can be reused three times. Heterogeneous aggregates of 254 nm were obtained after enzyme immobilization. The immobilization protocol used in this work was successful in retaining enzyme thermal stability and could be important in using natural compounds such as Fe

Topics: Ascomycota; Biotechnology; Chitosan; Cross-Linking Reagents; Endo-1,4-beta Xylanases; Enzyme Stability; Enzymes, Immobilized; Fungal Proteins; Hydrogen-Ion Concentration; Iridoids; Kinetics; Magnetite Nanoparticles; Microscopy, Electron, Scanning; Temperature

Oxidative stress serves a vital function in the pathogenesis of age‑related macular degeneration (AMD); genipin (GP) possesses antioxidative properties. The present study aimed to investigate the effects of GP on retinal pigment epithelial (RPE) cells induced by H2O2 and the underlying mechanism. ARPE‑19 cells were subjected to H2O2 treatment to induce oxidative damage. Cell viability was determined via an MTT assay. Reactive oxygen species (ROS) levels and cell apoptosis were detected by flow cytometry. Nuclear factor‑erythroid 2‑related factor‑2 (Nrf2) signaling‑associated and the expression of apoptosis‑associated factors were measured using reverse transcription‑quantitative polymerase chain reaction assay and western blotting. The results revealed that 200 µM H2O2 and 30 µM GP were determined to be the optimal concentrations for subsequent experimentation. GP reversed the inhibitory effects of H2O2 by promoting cell viability, attenuating ROS accumulation and cell apoptosis, and increased the expression of Nrf2, heme oxygenase‑1 (HO‑1) and NAD(P)H: Quinine oxidoreductase 1 (NQO1); Nrf2 silencing inhibited HO‑1 and NQO1 expression. In addition, Nrf2 silencing enhanced the effects of H2O2 by promoting ROS production and cell apoptosis. Compared with H2O2, Nrf2 silencing further decreased the expression levels of B‑cell lymphoma‑2 (Bcl‑2), but increased that of Bcl‑2‑associated X protein and cleaved‑caspase‑3. The results of the present study revealed that Nrf2 silencing attenuated the protective effects of GP on H2O2‑induced injury in ARPE‑19 cells by promoting apoptosis and oxidation. Collectively, GP attenuated oxidative damage induced by H2O2 in ARPE‑19 cells. Furthermore, the molecular mechanism may be associated with the Nrf2 signaling pathway. The findings of the present study nay provide insight into a potential therapeutic agent for the treatment of AMD.

Topics: Apoptosis; Cell Line; Cell Survival; Cytoprotection; Epithelial Cells; Gene Silencing; Humans; Hydrogen Peroxide; Iridoids; NF-E2-Related Factor 2; Oxidation-Reduction; Oxidative Stress; Reactive Oxygen Species; Retinal Pigment Epithelium; Signal Transduction

Topics: Animals; Apoptosis; Energy Metabolism; Iridoids; Male; Mice; Mice, Inbred C57BL; Mitochondria; Neuroprotective Agents; Oxidative Stress; Reactive Oxygen Species; Reperfusion Injury; Signal Transduction; Sirtuin 3; Stroke; Uncoupling Protein 2

Injectable hydrogels and biodegradable nanoparticles are using in tissue engineering applications and drug delivery systems. To improve physiochemical properties of biomaterials and to develop their applications, hybrid systems consist of hydrogels, and biodegradable nanoparticles were synthesized. In this study, hybrid systems based on double crosslinked hyaluronic acid and PLGA/Dexamethasone sodium phosphate (PLGADEX) nanoparticles are designed and synthesized in several steps. At the first step, poly(l-lactide-co-glycolide) (PLGA) in a ratio of LLA:GA = 85:15 mol% was synthesized via ring-opening polymerization. Then, PLGADEX nanoparticles were synthesized in different ratios using the partially modified emulsification-diffusion method and fully characterized, and desirable nanoparticle was selected (PLGADEX20). At the second step, a double cross-linked hyaluronic acid (XHA) was prepared by mixing various ratios of amino-hyaluronic acid and aldehyde-hyaluronic acid in the presence of genipin. Finally, by mixing of various ratios of PLGADEX20 and Dexamethasone sodium phosphate (DEX) with different ratios of XHA, hybrid systems were prepared. Based on the characterization of hybrid samples and the release studies, hydrogels containing nanoparticles showed a controlled drug release, while the best sample with 3% of optimized nanoparticle was chosen. According to physiochemical and biological properties, these hybrid systems can be good candidates for anti-adhesion barriers, wound dressings, and novel drug delivery systems.

Topics: Calibration; Chemical Phenomena; Chromatography, Gel; Color; Cross-Linking Reagents; Dexamethasone; Drug Liberation; Fibroblasts; Humans; Hyaluronic Acid; Hydrogels; Iridoids; Nanoparticles; Particle Size; Polylactic Acid-Polyglycolic Acid Copolymer; Proton Magnetic Resonance Spectroscopy; Regression Analysis; Rheology; Spectroscopy, Fourier Transform Infrared; Temperature; Time Factors

Hydroxypropyl chitosan (HPCS) has recently attracted increasing attention in biomedical applications because it has enhanced water solubility, excellent biocompatibility, and better antioxidant and antibacterial activities compared with chitosan. However, HPCS doesn't meet the mechanical strength requirement in bone tissue engineering and is not suitable for cell adhesion and growth because of its hydrophilic nature and low crystallinity. In this study, nano-scaled hydroxyapatite (n-HA) and HPCS were synthesized, respectively, and then n-HA/HPCS nanocomposite scaffolds were developed by incorporating n-HA into HPCS matrix accompanied with crosslinking of HPCS by a naturally occurring compound, genipin (GP), which in turn greatly altered the hydrophilicity and mechanical properties. The nanocomposite scaffolds showed an open structure with interconnected pores and a rough morphology with n-HA inserted in the GP-crosslinked HPCS matrix. The porosity, swelling capacity, compressive strength, fluorescence emission and degradation rate can be regulated by varying GP concentrations and n-HA contents. An osteoconductive and osteogenic marine algae polysaccharide, fucoidan, was further adsorbed to the composite scaffolds via electrostatic interactions. Incorporation of n-HA and adsorption of FD into the composite scaffolds increased ALP activity in 7F2 osteoblast cells and promoted their mineralization. The FD-adsorbed n-HA/HPCS composite scaffolds can be a potential biomaterial for BTE applications.

Topics: Adsorption; Biocompatible Materials; Bone and Bones; Bone Regeneration; Cell Adhesion; Cell Line; Chitosan; Compressive Strength; Drug Discovery; Durapatite; Humans; Iridoids; Nanocomposites; Osteoblasts; Osteogenesis; Polysaccharides; Porosity; Surface Properties; Tissue Engineering; Tissue Scaffolds

Topics: Caffeic Acids; Chromatography, High Pressure Liquid; Discriminant Analysis; Drugs, Chinese Herbal; Iridoids; Mass Spectrometry; Multivariate Analysis

Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Bcl-2-Like Protein 11; Colorectal Neoplasms; Drug Resistance, Neoplasm; Drug Synergism; Endoplasmic Reticulum Stress; Female; Gardenia; Gene Knockdown Techniques; HCT116 Cells; Humans; Iridoids; Mice; Mice, Inbred BALB C; Mice, Nude; Oxaliplatin; Plant Extracts; Reactive Oxygen Species; Superoxide Dismutase; Transfection; Tumor Burden; Xenograft Model Antitumor Assays

Recently, various additive manufacturing (3D printing) approaches have been employed to fabricate dressings such as film scaffolds that possess well defined architecture and orientation at the micro level. In this study, crosslinked chitosan (CH) based film matrices were prepared using 3D printing with genipin (GE) as a crosslinker, with glycerol (GLY) and poly ethylene glycol (PEG) as plasticizer. The 3D printed films were functionally characterized using (tensile, fluid handling, mucoadhesion, drug dissolution, morphological properties and cell viability as well physico-chemical characterization using scanning electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction. CH-GE-PEG600 3D printed films having the ratio of 1:1 polymer: plasticizer was selected due to their appropriate flexibility. Fourier transform infrared results showed intermolecular interaction between CH, GE and PEG which was confirmed by X-ray diffraction showing amorphous matrix structure. In vitro mucoadhesion studies of CH-GE-PEG600 films showed the capability of the 3D printed film to adhere to the epithelial surface. Scanning electron microscopy images showed that the surface of the plasticised films were smooth indicating content uniformity of CH, GE and PEG whilst micro cracks in unplasticised films confirmed their brittle nature. Plasticised films also showed high swelling capacity which enhanced water absorption. Cytotoxicity (MTT) assay using human skin fibroblast cell lines demonstrated that more than 90% of cells were viable after 48 h confirming non-toxic nature of the 3D printed CH-GE-PEG600 films and therefore promising dressing for chronic wound healing applications.

Topics: Adhesiveness; Cell Line; Cell Survival; Chitosan; Cross-Linking Reagents; Drug Liberation; Excipients; Fibroblasts; Glycerol; Humans; Iridoids; Microscopy, Electron, Scanning; Plasticizers; Polyethylene Glycols; Printing, Three-Dimensional; Spectroscopy, Fourier Transform Infrared; Wound Healing; X-Ray Diffraction

Biopolymer based hydrogel materials are attractive options for a variety of medical applications, including drug delivery and tissue regeneration because of their innate biomimetic material properties. While biopolymers are typically selected for a specific application based off of their chemical properties; the overall material structure of the resulting hydrogel ultimately relates to its ability to function for its intended application. In view of this, it is imperative that the impact of commonly used drying procedures on hydrogel physical properties is well understood. Herein, the effects of post-synthesis drying techniques: air-drying and lyophilization, on genipin crosslinked chitosan hydrogel physical structure were studied. Chitosan-genipin hydrogels synthesized out of either 15 kDa MW or 50-190 kDa MW chitosan were either air-dried (AD), flash-frozen (FF) and then lyophilized, or step-down frozen (SD) and then lyophilized. Environmental scanning electron microscopy (ESEM) was employed to evaluate the resulting hydrogels physical structure as a function of chitosan molecular weight and drying condition. ESEM images revealed the presence of larger microscale pores within the SD samples compared to FF samples, but both treatments yielded the induction of micropore with sizes ranging between 9-400 μm in diameter into the hydrogels. Traditional hydrogel swelling studies were performed to assess the resulting hydrogels swelling profile as a function of chitosan molecular weight and drying treatment. Lyophilized hydrogels showed a five-fold increase in swelling ratio compared to AD hydrogels indicating a change in morphology due to the drying process. The results demonstrate that regardless of polymer molecular weight, post-processing technique had a strong correlation with hydrogel porosity.

Topics: Biocompatible Materials; Chitosan; Cross-Linking Reagents; Drug Delivery Systems; Hydrogels; Hydrogen-Ion Concentration; Iridoids; Microscopy, Electron, Scanning

Decellularized bovine pericardium (DBP)-based biomeshes are the gold standard in reconstructive surgery. In order to prolong their stability after the transplantation, various chemical cross-linking strategies are employed. However, structural and functional properties of the biomeshes differ in dependence on the cross-linker used. Here, we performed a bottom-up study of structural and functional alterations of DBP-based biomeshes following cross-linking with hexamethylene diisocyanate (HMDC), ethylene glycol diglycidyl ether (EGDE), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and genipin. The in vitro cytotoxicity tests supported their clinical applicability. Their structural differences (eg roughness, fibre thickness, pore morphology) were evaluated using the two-photon confocal laser scanning, atomic force, scanning electron and polarized light microscopies. HMDC and EDC samples appeared to be the roughest. Complex mechanical trials indicated the tendency to reduced Young's Modulus and mechanical anisotropy values of DBP upon cross-linking. The lowest mechanical anisotropy was found in EDC and genipin sample groups. In vitro collagenase susceptibility was the highest for EDC samples and the lowest for EGDE samples. The comparative analysis of the results allowed us to recognize the strengths and weaknesses of each cross-linker in relation to a particular clinical application.

Topics: Animals; Cattle; Cross-Linking Reagents; Iridoids; Materials Testing; Pericardium; Tissue Engineering; Transplantation, Heterologous

Nasal reconstruction is limited by the availability of autologous cartilage. The aim is to investigate an adhesive biomaterial for tissue engineering of nasal cartilage by evaluating mechanical properties of hydrogels made of fibrin crosslinked with genipin as compared to native tissue.. Hydrogels of fibrin, fibrin-genipin, and fibrin-genipin with extracellular matrix (ECM) particles were created and evaluated with mechanical testing to determine compression, tensile, and shear properties. Rabbit nasal septal cartilage was harvested and tested in these modalities for comparison. Transmission electron microscopy characterized hydrogel structure.. Fibrin-genipin gels had higher compressive, tensile, and shear moduli compared to fibrin alone or fibrin-genipin with ECM. However, all hydrogel formulations had lower moduli than the rabbit nasal septal cartilage. Electron microscopy showed genipin crosslinking increased structural density of the hydrogel and that cartilage ECM created larger structural features with lower crosslinking density.. The addition of genipin significantly improved mechanical properties of fibrin hydrogels by increasing the compressive, tensile, and shear moduli. The addition of cartilage ECM, which can add native structure and composition, resulted in decreased moduli values. Fibrin-genipin is a bioactive and biomechanically stable hydrogel that may offer promise as a scaffold for cartilage tissue engineering in nasal reconstruction, yet further augmentation is required to match material properties of native nasal cartilage.

Topics: Animals; Compressive Strength; Extracellular Matrix; Fibrin; Hydrogel, Polyethylene Glycol Dimethacrylate; Iridoids; Materials Testing; Microscopy, Electron, Transmission; Nasal Cartilages; Nasal Septum; Rabbits; Rhinoplasty; Shear Strength; Tensile Strength; Tissue Engineering; Tissue Scaffolds

A facile one-pot spray-drying process was developed for fabrication and in situ crosslinking of chitosan microspheres to improve the adsorption capacity by microscopic design. A fully biobased nature was achieved by utilizing genipin (GP) as a crosslinking agent and chitosan-derived nanographene oxide (nGO) as a property tuner. The produced chitosan microspheres were further proven as powerful adsorbents for common wastewater contaminants such as anionic dyes and pharmaceutical contaminants, here modeled by methyl orange (MO) and diclofenac sodium (DCF). By regulating the amount of GP and nGO, as well as by controlling the process parameters including the spray-drying inlet temperature and postheat treatment, the surface morphology, size, zeta potential, and adsorption efficiency of the microspheres could be tuned accordingly. The adsorption efficiency for MO and DCF reached 98.9 and 100%, respectively. The microspheres retained high DCF adsorption efficiency after six adsorption and desorption cycles, and the recyclability was improved by the incorporated nGO. The fabricated microspheres, thus, have great potential as reusable and eco-friendly adsorbents.

Topics: Adsorption; Azo Compounds; Chitosan; Diclofenac; Graphite; Iridoids; Microspheres; Wastewater; Water Purification; Wettability

The aim of this work is to design a wound dressing able to release chlorhexidine (CHX) as antiseptic agent, ensuring long-lasting antibacterial efficacy during the healing. The textile nonwoven (polyethylene terephthalate) (PET) of the dressing was first modified by chitosan (CHT) crosslinked with genipin (Gpn). Parameters such as the concentration of reagents (Gpn and CHT) but also the crosslinking time and the working temperature were optimized to reach the maximal positive charges surface density. This support was then treated by the layer-by-layer (LbL) deposition of a multilayer system composed of methyl-beta-cyclodextrin polymer (PCD) (anionic) and CHT (cationic). After a thermal treatment to stabilize the LbL film, the textiles were loaded with CHX as antiseptic agent. The influence of the thermal treatment i) on the cytocompatibility, ii) on the degradation of the multilayer system, iii) on CHX sorption and release profiles and iv) on the antibacterial activity of the loaded textiles was studied.

Topics: Adsorption; Anti-Bacterial Agents; Bandages; Cell Line; Cell Survival; Chlorhexidine; Coated Materials, Biocompatible; Cross-Linking Reagents; Drug Liberation; Hot Temperature; Humans; Iridoids; Microbial Sensitivity Tests; Polyelectrolytes; Polyethylene Terephthalates; Solubility; Staphylococcus aureus; Textiles; Time Factors; Wound Healing

Fluoxetine (FXT), a selective serotonin reuptake inhibitor (SSRI), is one of the most common psychiatric medications clinically prescribed; while over-produced serotonin may suppress neurite development. The role of major iridoids like geniposide (GPS) and genipin (GNP) from Gardenia jasminoides Ellis fruit (family Rubiaceae) in ameliorating the anti-neurite outgrowth effect of FXT is poorly understood. In this study, the effects of these iridoids on FXT-suppressed neurite outgrowth in Neuro2a neuroblastoma cells were investigated.. Neuro2a cells were treated with FXT and GPS. The effect of GPS-FXT co-treatment on neurite outgrowth was observed using inverted phase-contrast microscope imaging system, while neurite outgrowth markers - microtubule-associated protein-2 (MAP2) and growth-associated protein 43 (GAP43) were analyzed using RT-PCR, Western blot and immunofluorescence staining. The transcription factor-cAMP response element binding (CREB), and signaling pathways - mitogen-activated protein kinase (MAPK) and protein kinase B/mammalian target of rapamycin (AKT/mTOR) were also analyzed with the help of Western blot.. The results showed that FXT decreased the neurite outgrowth in Neuro2a cells and also downregulated gene and protein expression of MAP2 and GAP43. It also downregulated the protein expression of phosphorylated-CREB, MAPK, and AKT/mTOR signaling pathways. In contrast, GPS counteracted the effects of FXT. GPS-FXT co-treatment increased the percentage of neurite-bearing cells by 3.6-fold at 200 μM as compared to FXT treatment only.. This study has provided the possible molecular mechanism showing how FXT exerted its detrimental side-effects on the neurite differentiation, and via the same mechanism how GPS attenuated these side effects.

Topics: Cell Differentiation; Cell Line, Tumor; Cell Survival; Cyclic AMP Response Element-Binding Protein; Fluoxetine; GAP-43 Protein; Humans; Iridoids; MAP Kinase Signaling System; Microtubule-Associated Proteins; Mitogen-Activated Protein Kinases; Neural Stem Cells; Neurites; Neuroblastoma; Neurogenesis; Neuronal Outgrowth; Phosphorylation; Proto-Oncogene Proteins c-akt; Signal Transduction; TOR Serine-Threonine Kinases

Although the protective effects of genipin against acute lung injury (ALI) have been described previously, the associated mechanism remains unclear. We have previously reported that genipin exerts its pharmacological effects by regulating autophagy. Here, we hypothesized that the up-regulation of autophagy may contribute to the protective effects exhibited by genipin against ALI. In the present study, ALI was induced by intratracheal LPS administration in rats. Genipin treatment significantly reduced LPS-induced lung injury as evidenced by improved histopathology, decreased lung edema, total cells, and protein concentration in the bronchoalveolar lavage fluid (BALF). This protection was inhibited by 3-methyladenine (3-MA), an inhibitor of autophagy. Genipin treatment reduced the expression of P62 and increased the expression of Beclin-1 and LC3II, indicating increased autophagy. Genipin treatment also alleviated LPS-induced cell apoptosis (down-regulation of Bax, up-regulation of Bcl-2, and decreased number of terminal deoxynucleotidyl transferase dUTP nick end label-positive cells) and oxidative stress (increased SOD and decreased MDA content) in the lung. Furthermore, genipin attenuated LPS-induced production of TNF-α, IL-1β, and IL-6 in the lung and BALF. These protective effects induced by genipin were reversed by 3-MA treatment, indicating that autophagy was involved in the protective effects exerted by genipin against inflammation and apoptosis in ALI. In A549 cells incubated with LPS for 6 h, genipin treatment increased the number of GFP-LC3 punctae. 3-MA prevented the protective effects of genipin against mitochondrial dysfunction and cell death. These findings suggest that genipin protects against apoptosis and inflammation in LPS-induced ALI by promoting autophagy.

Topics: A549 Cells; Acute Lung Injury; Animals; Autophagy; Bronchoalveolar Lavage Fluid; Cell Survival; Cytokines; Humans; Iridoids; Lipopolysaccharides; Lung; Male; Protective Agents; Rats, Sprague-Dawley

To establish the procedures of genipin-linked scaffold for in situ tracheal reconstruction in a rabbit model, and to demonstrate whether stem cells can be further differentiated in the bioreactor in vivo. It will further provide an experimental and theoretical foundation for clinical application. Previously, in vitro evaluation proved the detergent–enzymatic method effectively removed stromal epithelial cells, and the number of nuclei was reduced significantly (p < 0.05). The content of type II collagen was not statistically reduced (p > 0.05). Plasmids with green fluorescence protein were transfected into 293T cells, and these cells subsequently synthesized lentivirus with green fluorescence protein that could infect other cells. After in vivo experiments, macroscopic specimen observation and hematoxylin and eosin staining comparison showed that the genipin cross-linked decellularized scaffold had low immunological rejection. Blood routine proved the progenitor cells (such as mononuclear cells) can be mobilized from the bone marrow by the growth factors, to allow their circulation into the peripheral blood. The immunohistochemistry of Type II collagen after surgery showed the expression level of bone marrow mesenchymal stem cells transplantated group was statistically higher than the autologous transplantated group (p < 0.05). The fluorescences of Bone marrow mononuclear cells (BMNCs) were traced after the specimens harvested. It successfully demonstrated that the procedures combining stem cells with the genipin cross-linked decellularized scaffold could apply to in situ airway construction. Compared to bone marrow mesenchymal stem cells, BMNCs can also be used to achieve chondrocyte differentiation; this procedure will avoid in vitro cell culture, shortening the time and economic costs.

Topics: Animals; Cell Differentiation; Collagen Type II; Cross-Linking Reagents; Cytokines; Escherichia coli; Green Fluorescent Proteins; HEK293 Cells; Humans; Iridoids; Lentivirus; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Rabbits; Regeneration; Tissue Engineering; Tissue Scaffolds; Trachea; Transfection

The aim of this study was to investigate the protective effects of a sustained release form of dexamethasone (dex) loaded chitosan-based genipin-cross-linked hydrogel (CBGCH) in a guinea pig model of cisplatin (CP) induced hearing loss.. Implantation of CBGCH was made by intratympanic (IT) injection. Ototoxicity was produced by intraperitoneal (IP) single dose of 14 mg/kg CP. Animals were randomly divided into four groups with 6 guinea pigs in each. Group 1 received only IP CP; group 2 received only IT dex-loaded CBGCH injections. Group 3 and group 4 received IP CP, plus IT nondrug CBGCH and IT dex-loaded CBGCH respectively 24 h prior to IP CP injections. Distortion product otoacoustic emissions (DPOAEs) and auditory brainstem response (ABR) measurements were obtained before the treatments and solely ABR measurements were done after 3 and 10 days. The ultrastructural effects were investigated by scanning electron microscopy (SEM) analysis.. The postCP ABR thresholds at 4, 8, 12, 16, 32 kHz frequencies were significantly better in group 4 than groups 1 and 3 (p < 0.05). The comparison of time effective ABR thresholds between groups 1 and 4 and between groups 3 and 4 showed significantly lower ABR thresholds in group 4 (p < 0.05). The SEM analysis showed that stereocilia of inner and outer hair cells were preserved in group 4, almost like group 2, whereas cytotoxic degenerations were noted in groups 1 and 3.. Intratympanic administration of dex-loaded CBGCH has been shown to provide functional and structural protection against CP-induced ototoxicity.

Topics: Animals; Anti-Inflammatory Agents; Antineoplastic Agents; Auditory Threshold; Biocompatible Materials; Chitosan; Cholagogues and Choleretics; Cisplatin; Delayed-Action Preparations; Dexamethasone; Drug Combinations; Evoked Potentials, Auditory, Brain Stem; Guinea Pigs; Hair Cells, Auditory, Inner; Hair Cells, Auditory, Outer; Hearing Loss; Hydrogels; Iridoids; Male; Microscopy, Electron, Scanning; Otoacoustic Emissions, Spontaneous; Random Allocation; Stereocilia

A promising vascular scaffold must possess satisfying mechanical properties, great hemocompatibility, and favorable tissue regeneration. Combining natural with synthetic materials is a popular method of creating/enhancing such scaffolds. However, the effect of additional modification on the materials requires further exploration.. We selected polycaprolactone (PCL), which has excellent mechanical properties and biocompatibility and can be combined with collagen. Electrospun fibers created using a PCL/collagen solution were used to fashion mixed nanofibers, while separate syringes of PCL and collagen were used to create separated nanofibers, resulting in different pore sizes. Mixed and separated nanofibers were cross-linked with glutaraldehyde (GA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and genipin; hence, we named them as mixed GA, mixed EDC (ME), mixed genipin (MG), separated GA, separated EDC (SE), and separated genipin (SG).. Fourier transform infrared (FTIR) spectroscopy and X-ray diffraction showed that cross-linking did not affect the main functional groups of fibers in all groups. ME, MG, SE, and SG met the requisite mechanical properties, and they also resisted collagenase degradation. In hemocompatibility assays, only ME and MG demonstrated ideal safety. Furthermore, ME and MG presented the greatest cytocompatibility. For vascular scaffolds, rapid endothelialization helps to prevent thrombosis. According to human umbilical vein endothelial cell migration on different nanofibers, ME and MG are also successful in promoting cell migration.. ME and MG may be promising candidates for vascular tissue engineering. The study suggests that collagen cross-linked by EDC/

Topics: Animals; Biocompatible Materials; Carbodiimides; Cell Movement; Cells, Cultured; Collagen; Cross-Linking Reagents; Endothelial Cells; Human Umbilical Vein Endothelial Cells; Humans; Iridoids; Materials Testing; Nanofibers; Platelet Adhesiveness; Polyesters; Rabbits; Rats; Regeneration; Succinimides; Tissue Engineering; Tissue Scaffolds

We address the hypothesis that uncoupling protein 2 (UCP2), a cellular glucose regulator, delays physiologic retinal vascular development (PRVD) by interfering with glucose uptake through glucose transporter 1 (Glut1).. In the rat 50/10 oxygen-induced retinopathy (OIR) model, retinal Glut1 and UCP2 were measured and compared to room air (RA)-raised pups at postnatal day 14 (p14). Pups in OIR and RA received intraperitoneal genipin, an UCP2 inhibitor, or control every other day from p3 until p13. Analyses at p14 included avascular/total retinal area (AVA), Western blots of retinal UCP2 and Glut1, and immunostaining of Glut1 in retinal cryosections. Intravitreal neovascular/total retinal area (IVNV) was analyzed at p18, and electroretinograms were performed at p26. Glut1 and phosphorylated VEGFR2 (p-VEGFR2), glucose uptake, adenosine triphosphate (ATP) production, and cell proliferation were measured in human retinal microvascular endothelial cells (hRMVECs) pretreated with genipin or transfected with UCP2siRNA, Glut1siRNA, or control siRNA when incubated with VEGF or PBS.. At p14, OIR pups had increased AVA with decreased Glut1 and increased UCP2 in the retina compared to RA retinas. Intraperitoneal genipin increased retinal Glut1 and reduced AVA. Compared to control, treatment with genipin or knockdown of UCP2 significantly increased Glut1, glucose uptake, ATP production, VEGF-induced p-VEGFR2 and cell proliferation in hRMVECs. Knockdown of Glut1 inhibited VEGF-induced p-VEGFR2. Genipin-treated OIR pups with decreased AVA at p14 had reduced IVNV at p18 and increased amplitudes in a- and b-waves at p26.. Extending PRVD by increasing retinal endothelial glucose uptake may represent a strategy to prevent severe retinopathy of prematurity and vision loss.

Topics: Adenosine Triphosphate; Animals; Animals, Newborn; Blotting, Western; Cholagogues and Choleretics; Diabetic Retinopathy; Disease Models, Animal; Electroretinography; Endothelium, Vascular; Female; Gene Silencing; Glucose; Glucose Transporter Type 1; Glycolysis; Injections, Intraperitoneal; Iridoids; Male; Oxygen; Pregnancy; Rats; Rats, Sprague-Dawley; Retinal Vessels; Transfection; Uncoupling Protein 2; Vascular Endothelial Growth Factor Receptor-2

Platelet-rich plasma (PRP) is rich in growth factors and has commonly been utilized in the repair and regeneration of damaged articular cartilage. However, the major drawbacks of direct PRP injection are unstable biological fixation and fast or burst release of growth factors. Fucoidan is a heparinoid compound that can bind growth factors to control their release rate. Furthermore, fucoidan can reduce arthritis through suppressing inflammatory responses and thus it has been reported to prevent the progression of osteoarthritis, promote bone regeneration and accelerate healing of cartilage injury. Injectable hydrogels can be used to deliver cells and growth factors for an alternative, less invasive treatment of cartilage defects. In this study, hyaluronic acid (HA) and fucoidan (FD) was blended with gelatin (GLT) and the GLT/HA/FD hybrid was further cross-linked with genipin (GP) to prepare injectable GP-GLT/HA/FD hydrogels. The gelation rate was affected by the GP, GLT, HA and FD concentrations, as well as the pH values. The addition of HA and FD to GLT networks improved the mechanical strength of the hydrogels and facilitated the sustained release of PRP growth factors. The GP-GLT/HA/FD hydrogel showed adequate injectability, shape-persistent property and strong adhesive ability, and was more resistant to enzymatic degradation. The PRP-loaded GP-GLT/HA/FD hydrogel promoted cartilage regeneration in rabbits, which may lead to an advanced PRP therapy for enhancing cartilage repair.

Topics: Animals; Cartilage; Cell Survival; Hyaluronic Acid; Hydrogels; Iridoids; Lipopolysaccharides; Mice; Platelet-Rich Plasma; Polysaccharides; Rabbits; RAW 264.7 Cells

Uncoupling protein 2 (UCP2) is upregulated in several human cancers which contributes to tumorigenesis. However, whether UCP2 expression is amplified in cholangiocarcinoma and whether UCP2 promotes cholangiocarcinoma progression are not known. Our results found that in human cholangiocarcinoma tissues, UCP2 was highly expressed in tumors and its levels were negatively associated with prognosis. Importantly, lymph node invasion of cholangiocarcinoma was associated with higher UCP2 expression. In cholangiocarcinoma cells, cell proliferation and migration were suppressed when UCP2 expression was inhibited via gene knockdown. In UCP2 knockdown cells, glycolysis was inhibited, the mesenchymal markers were downregulated whereas AMPK was activated. The increased mitochondrial ROS and AMP/ATP ratio might be responsible for this activation. When the UCP2 inhibitor genipin was applied, tumor cell migration and 3D growth were suppressed via enhancing the mesenchymal-epithelial transition of cholangiocarcinoma cells. Furthermore, cholangiocarcinoma cells became sensitive to cisplatin and gemcitabine treatments when genipin was applied. In conclusion, our results demonstrate that the amplified expression of UCP2 contributes to the progression of cholangiocarcinoma through a glycolysis-mediated mechanism.

Topics: AMP-Activated Protein Kinases; Bile Duct Neoplasms; Cell Line, Tumor; Cell Plasticity; Cholangiocarcinoma; Glycolysis; HEK293 Cells; Humans; Iridoids; Membrane Potential, Mitochondrial; Mitochondria; Signal Transduction; Uncoupling Protein 2

Genipin (GP) is a safe method for corneal crosslinking, even for very thin corneas. However, there have been no reports on the optimal GP concentration range to use in vivo for corneal crosslinking.. To investigate the safety of corneal crosslinking after a 24-h incubation with different concentrations of GP.. Twenty New Zealand white rabbits were divided into a phosphate-buffered saline (PBS) group, 0.2% GP crosslinking (GP-CXL) group, 0.25% GP-CXL group, and 0.3% GP-CXL group. Before and after surgery, the operated eyes of each group were characterized by confocal microscopy, and corneal buttons were excised for endothelium staining and electron microscopy.. The keratocyte structures in each GP group appeared to be similar to those in the PBS group. Through the confocal microscopy, the changes in corneal endothelial cell density also did not significantly differ among groups. There was a significant difference in apoptosis between the 0.3% GP-CXL and PBS groups (p < 0.05) and between the 0.3% GP-CXL and 0.25% GP-CXL groups (p < 0.05), but there were no significant differences between the 0.2 and 0.25% GP-CXL groups compared to the PBS group. Transmission electron microscopy showed endothelial cell damage in the 0.3% GP-CXL group, with minimal endothelial cell damage in the other groups.. Treatment of rabbit corneas with ≤0.25% GP resulted in minimal toxicity to keratocytes and endothelial cells, suggesting that it is a safe crosslinking agent at those concentrations.

Topics: Animals; Cornea; Corneal Keratocytes; Cross-Linking Reagents; Endothelial Cells; Iridoids; Photochemotherapy; Rabbits

Intron retention, one of the major types of alternative splicing in plants and animals, has also been reported existing in filamentous fungi's glycoside hydrolases. In this study, an intron-retained β-glucosidase gene transcript (bgl1B) from A. niger B2 strain was obtained. Compared with the normally spliced transcript bgl1A, bgl1B had an extra 51bp insertion, which was confirmed to be the sixth (the last) intron of this β-glucosidase gene. The bgl1A and bgl1B were expressed in Pichia pastoris and the purified enzymes were used to compare their catalytic properties. The results showed that the intron retention didn't impair the catalytic function. Instead, the intron-retained enzyme BGL1B had a better thermostability with a higher optimal temperature and a longer half-life under 50 °C. Also it exhibited a little higher k

Topics: Aspergillus niger; beta-Glucosidase; Cloning, Molecular; Genes, Fungal; Introns; Iridoids; Pichia

Back pain commonly arises from intervertebral disc (IVD) damage including annulus fibrosus (AF) defects and nucleus pulposus (NP) loss. Poor IVD healing motivates developing tissue engineering repair strategies. This study evaluated a composite injectable IVD biomaterial repair strategy using carboxymethylcellulose-methylcellulose (CMC-MC) and genipin-crosslinked fibrin (FibGen) that mimic NP and AF properties, respectively. Bovine ex vivo caudal IVDs were evaluated in cyclic compression-tension, torsion, and compression-to-failure tests to determine IVD biomechanical properties, height loss, and herniation risk following experimentally-induced severe herniation injury and discectomy (4 mm biopsy defect with 20% NP removed). FibGen with and without CMC-MC had failure strength similar to discectomy injury suggesting no increased risk compared to surgical procedures, yet no biomaterials improved axial or torsional biomechanical properties suggesting they were incapable of adequately restoring AF tension. FibGen had the largest failure strength and was further evaluated in additional discectomy injury models with varying AF defect types (2 mm biopsy, 4 mm cruciate, 4 mm biopsy) and NP removal volume (0%, 20%). All simulated discectomy defects significantly compromised failure strength and biomechanical properties. The 0% NP removal group had mean values of axial biomechanical properties closer to intact levels than defects with 20% NP removed but they were not statistically different and 0% NP removal also decreased failure strength. FibGen with and without CMC-MC failed at super-physiological stress levels above simulated discectomy suggesting repair with these tissue engineered biomaterials may perform better than discectomy alone, although restored biomechanical function may require additional healing with the potential application of these biomaterials as sealants and cell/drug delivery carriers.

Topics: Animals; Annulus Fibrosus; Biocompatible Materials; Biomechanical Phenomena; Carboxymethylcellulose Sodium; Cattle; Cross-Linking Reagents; Disease Models, Animal; Diskectomy; Fibrin; Hydrogels; In Vitro Techniques; Injections, Spinal; Intervertebral Disc Displacement; Iridoids; Materials Testing; Methylcellulose; Nucleus Pulposus

Geniposide is a natural hepatotoxic iridoid glycoside. Its hydrolysate of intestinal microbiota, genipin, is thought to be responsible for the hepatotoxicity. However, the underlying mechanism that genipin contributes to the hepatotoxicity of geniposide is not well understood. In this study, we found that genipin spontaneously converted into a reactive dialdehyde intermediate and covalently bond to the primary amine group of free amino acids in both of the phosphate buffers and geniposide-treated rats. Furthermore, genipin dialdehyde can form the covalent linkage to the primary amino group (ε) of lysine side chains of the hepatic proteins in geniposide-treated rats. Pretreatment with β-glucosidase or antibiotics significantly modulated the genipin dialdehyde formation and protein modification, revealing the essential role of microbial glycosidases. The levels of protein adduct were that mapped onto the hepatotoxicity of geniposide. In summary, this study demonstrates that the intestinal microbiota mediated covalent modification of the hepatic protein by genipin dialdehyde may play a crucial role in the liver injury of geniposide. The study is also helpful for understanding the contribution of intestinal microbiota to the metabolic activation of xenobiotics.

Topics: Aldehydes; Amino Acids; Animals; Anti-Bacterial Agents; beta-Glucosidase; Bile; Chemical and Drug Induced Liver Injury; Gastrointestinal Microbiome; Glutathione; Glycoside Hydrolases; Iridoids; Liver; Lysine; Male; Rats; Rats, Sprague-Dawley

Paclitaxel (PTX)-loaded genipin-crosslinked gelatin microspheres (GP-MS) are a prolonged IP delivery system under development for the treatment of peritoneal minimal residual disease (pMRD). Here, we show the use of a pharmacokinetic-pharmacodynamic (PKPD) modelling approach to inform the formulation development of PTX-GP-MS in a mice pMRD model.. PTX blood concentrations and survival data were obtained in Balb/c Nu mice receiving different single IP doses (7.5 and/or 35 mg/kg) of PTX-ethanolic loaded GP-MS (PTX. The model predicts that the dose range of 7.5-15 mg/kg of PTX

Topics: Albumin-Bound Paclitaxel; Animals; Antineoplastic Agents, Phytogenic; Cell Line, Tumor; Cross-Linking Reagents; Drug Carriers; Gelatin; Humans; Iridoids; Mice, Inbred BALB C; Mice, Nude; Microspheres; Models, Biological; Peritoneal Neoplasms; Xenograft Model Antitumor Assays

To investigate the effects of genipin on promoting brown adipose tissue activation and white adipose tissue browning.. The male C57BL/6J mice were divided into three groups: normal control group, genipin group and cold-stimulus group.Genipin group were treated consecutively with genipin at a dose of 15 mg/kg once a day for 9 days, normal control group were treated with the saline.The mice with cold-stimulus were exposed to 4℃ environment for 5 days.Daily food amount and body weight were measured.Morphological changes were observed in the subscapular region, inguinal region and epididymis around the adipose tissue.The expression of uncoupling protein 1 (UCP1) was determined by real-time PCR and Western blot respectively.. The wet weight of white fat in genipin-treated mice was decreased by 16% , and 28% in that of cold-stimulus mice, compared with the normal control group (P＜0.05).After treatments of genipin and cold-stimulus, the color of white adipose tissues was darker, and the size of lipid droplets in adipocytes was smaller, whereas the number was increased.Compared with the normal control group, UCP1 expression was increased obviously in fat tissues, including the subcutaneous and visceral white adipose tissues, and brown adipose tissue after treated with genipin and cold-stimulus (P＜0.05).. Genipin promoted activation of brown adipose tissue and browning of white adipose tissue by upregulating UCP1 expression, which could contribute to the loss of body weight against obesity.

Topics: Adipose Tissue, Brown; Adipose Tissue, White; Animals; Cholagogues and Choleretics; Iridoids; Male; Mice; Mice, Inbred C57BL; Obesity; Uncoupling Protein 1; Up-Regulation

Collagen, chitosan and hyaluronic acid based multicomponent injectable and in situ gellating biomimetic hybrid materials for bone tissue engineering applications were prepared in one-step procedure. The bioactive phase in the form of surface-modified silica particles was introduced to the solutions of biopolymers and simultaneously crosslinked with genipin both the biopolymer matrix and dispersed particles at 37 °C. The novel approach presented here involved the use of silica particles which surfaces were priory functionalized with amino groups. That modification makes possible the covalent attachment of silica particles to the polymeric hydrogel network on crosslinking with genipin. That methodology is especially important as it makes possible to obtain the hybrid materials (biopolymer-silica particles) in which the problems related to the potential phase separation of mineral particles, hindering their in vivo application can be eliminated. The hybrids of various compositions were obtained and their physicochemical and biological properties were determined. The in vitro experiments performed under simulated body fluid conditions revealed that the amino-functionalized silica particles covalently attached to the biopolymeric network are still bioactive. Finally, the in vitro cell culture studies shown that the materials developed are biocompatible as they supported MG-63 cells adhesion, proliferation as well as Alkaline phosphatase (ALP) expression.

Topics: Alkaline Phosphatase; Animals; Biocompatible Materials; Cell Adhesion; Cell Line; Cell Proliferation; Chitosan; Collagen; Hyaluronic Acid; Hydrogels; Injections; Iridoids; Minerals; Osteoblasts; Rats; Rheology; Silicon Dioxide; Wettability

Gardenia blue pigments derived from genipin reacting with amino acids have been used as natural food colorants for nearly 30 years in East Asia. However, their pharmacological effects, especially antidepressant-like effects, have not been reported so far. In this study, one of the gardenia blue pigments, was obtained from the reaction of genipin with tyrosine (genipin-tyrosine derivant (GTD)), and its antidepressant-like effects were investigated in lipopolysaccharide (LPS) or chronic unpredictable mild stress (CUMS) models. The results showed that GTD could attenuate depressive-like behaviors in both animal models. GTD reversed the LPS-induced cytokine increase of TNF-α, IL-6, and corticosterone (CORT) in mice plasma and hippocampus. In CUMS rats, GTD treatment significantly reduced hypothalamic-pituitary-adrenal (HPA) axis-related stress hormone levels in plasma including those of CORT, adrenocorticotropic hormone (ACTH), and corticotropin-releasing hormone (CRH). Besides, GTD increased plasma testosterone and hippocampal brain-derived neurotrophic factor (BDNF) levels in CUMS rats. GTD increased serotonin (5-HT), dopamine (DA), and norepinephrine (NE) in rat hippocampus and corpus striatum. Consistently, hippocampal metabolomic analysis demonstrated that GTD restored monoamine neurotransmitter metabolism, mitochondrial oxidative function, and membrane structural integrity. Our data suggested that GTD produced antidepressant-like activity through the restoration of the HPA axis hormone balance and the regulation of neurotransmitter release.

Topics: Animals; Antidepressive Agents; Brain-Derived Neurotrophic Factor; Corticosterone; Depression; Gardenia; Hippocampus; Humans; Interleukin-6; Iridoids; Male; Mice; Mice, Inbred C57BL; Pigments, Biological; Pituitary-Adrenal System; Plant Extracts; Rats; Rats, Sprague-Dawley; Serotonin; Tyrosine

The purpose of the present study was to identify the potential targets and markers for diagnosis, therapy and prognosis in patients with prolactinoma at the molecular level and to determine the therapeutic effects of genipin in prolactinoma. The gene expression profiles of GSE2175, GSE26966 and GSE36314 were obtained from the Gene Expression Omnibus database. The differentially expressed genes (DEGs) were identified after comparing between gene expression profiles of the prolactinoma tissues and normal tissues. Then, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and protein‑protein interaction (PPI) network analysis were conducted. In addition, in vitro, scratch assay, colony‑forming assay, Cell Counting Kit 8 (CCK8) assay and flow cytometry were performed to verify the functional effects of genipin. An aggregate of 12,695, 3,847 and 5,310 DEGs were identified from GSE2175, GSE26966 and GSE36314, respectively. The results of GO and KEGG analysis showed that the DEGs significant and important for prolactinoma were mostly involved with 'spindle pole' and 'oocyte meiosis'. A total of 20 genes were selected as hub genes with high degrees after PPI network analysis, including mitogen‑activated protein kinase 1 (MAPK1), MYC, early growth response 1 (EGR1), Bcl2 and calmodulin 1 (CALM1). CCK8 assay, colony‑forming assay and scratch assay were performed to verify the anti‑prolactinoma effect of genipin. The results of flow cytometry showed that apoptosis was increased by genipin. MAPK1, MYC, EGR1, Bcl2 and CALM1 were screened as main hub genes. Genipin upregulated the expression level of EGR1 and p21 (downstream mediator of EGR1) and EGR1, inhibited the proliferation and migration of prolactinoma cells. Genipin is a promising drug for treatment of patients with prolactinoma.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Humans; Iridoids; Mice; Pituitary Neoplasms; Prolactinoma; Protein Interaction Maps; Rats; Transcriptome

Collagen has been widely used for cartilage repair, but its low stiffness and rapid degradation disfavor chondrogenesis. Here we conjugated biocompatible carbon dot nanoparticles (CD NPs) onto collagen through a natural product crosslinker (genipin) to prepare an injectable hydrogel (termed collagen-genipin-CD nanoparticles, CGN). The CGN hydrogel showed increased stiffness due to the cross-linking effect of genipin and the presence of CD NPs, and could produce a moderate amount of reactive oxygen species (ROS) by photodynamic therapy (PDT). Both the stiffness enhancement and ROS generation resulted in improved chondrogenic differentiation of bone marrow-derived stem cells (BMSCs) and the subsequent enhanced cartilage regeneration for cartilage defect repair. Specifically, the CGN hydrogel presented a 21-fold higher compression modulus and a 39.3% lower degradation rate than the pure collagen hydrogel. A combination of both PDT and CGN hydrogel increased the BMSCs proliferation by 50.3%, upregulated their expression of cartilage-specific genes by multiple folds, and enhanced GAG secretion by 205.1% on day 21. This combination also accelerated the cartilage regeneration within as short as 8 weeks. The stiffness enhancement and ROS generation synergistically contributed to chondrogenic differentiation by regulating the TGF-β/SMAD and mTOR signaling pathway, respectively. The combination of CD-modified hydrogel injection and PDT treatment represents a new strategy for minimally invasive repair of cartilage defects.

Topics: Animals; Blotting, Western; Carbon; Cell Survival; Chondrogenesis; Collagen; Female; Hydrogels; Immunohistochemistry; Iridoids; Mice; Mice, Nude; Microscopy, Electron, Transmission; Nanocomposites; Photochemotherapy; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Singlet Oxygen; Spectroscopy, Fourier Transform Infrared

Extracellular matrix (ECM) hydrogels, produced by tissue decellularization are natural injectable materials suitable for neural tissue repair. However, the rapid biodegradation of these materials may disrupt neural tissue reconstruction in vivo. The aim of this study was to improve the stability of the previously described ECM hydrogel derived from human umbilical cord using genipin and N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), crosslinking at concentration of 0.5-10 mM. The hydrogels, crosslinked by genipin (ECM/G) or EDC (ECM/D), were evaluated in vitro in terms of their mechanical properties, degradation stability and biocompatibility. ECM/G, unlike ECM/D, crosslinked hydrogels revealed improved rheological properties when compared to uncrosslinked ECM. Both ECM/G and ECM/D slowed down the gelation time and increased the resistance against in vitro enzymatic degradation, while genipin crosslinking was more effective than EDC. Crosslinkers concentration of 1 mM enhanced the in vitro bio-stability of both ECM/G and ECM/D without affecting mesenchymal stem cell proliferation, axonal sprouting or neural stem cell growth and differentiation. Moreover, when injected into cortical photochemical lesion, genipin allowed in situ gelation and improved the retention of ECM for up to 2 weeks without any adverse tissue response or enhanced inflammatory reaction. In summary, we demonstrated that genipin, rather than EDC, improved the bio-stability of injectable ECM hydrogel in biocompatible concentration, and that ECM/G has potential as a scaffold for neural tissue application.

Topics: Carbodiimides; Cell Proliferation; Extracellular Matrix; Humans; Hydrogels; Iridoids; Mesenchymal Stem Cells; Nerve Regeneration; Tissue Engineering; Tissue Scaffolds; Umbilical Cord

Genipin is a compound derived from gardenia fruit extract. Although Genipin has anti-tumor effects in various cancers, its effect and mechanism in gastric cancer remain unclear. Here, we investigated the relationship between the anticancer effect of Genipin and signal transducer and activator of transcription (Stat3)/myeloid cell leukemia-1 (Mcl-1) in human gastric cancers.. MTT assays were performed to determine the cell viability of gastric cancer and gastric epithelial cell lines (AGS, MKN45, SNU638, MKN74, HFE-145). A TUNEL assay and Western blotting were carried out to investigate apoptosis. Stat3 activity was measured by proteome profiler phospho kinase array, immunofluorescence and immunoblotting. Mitochondria function was monitored with an XF24 analyzer and by flow cytometry, confocal microscopy using fluorescent probes for general mitochondrial membrane potential (MMP).. Genipin induced apoptosis in gastric cancer cells, including AGS and MKN45 cells. Genipin also reduced Mcl-1 mRNA and protein levels. Furthermore, we found that phosphorylation of Stat3 is regulated by Genipin. Additionally, the protein level of phospho Janus kinase 2 (JAK2) was decreased by Genipin treatment, indicating that the Stat3/JAK2/Mcl-1 pathway is suppressed by Genipin treatment in gastric cancer cells. Mcl-1 is closely related to mitochondrial function. These findings suggest that Genipin contributes to the collapse of mitochondrial functions like MMP.. Genipin induced apoptosis by suppressing the Stat3/Mcl-1 pathway and led to mitochondrial dysfunction. Our results reveal a novel mechanism for the anti-cancer effect of Genipin in gastric cancer.

Topics: Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Down-Regulation; Gene Knockdown Techniques; Humans; Iridoids; Janus Kinase 2; Membrane Potential, Mitochondrial; Mitochondria; Myeloid Cell Leukemia Sequence 1 Protein; Phosphorylation; Signal Transduction; STAT3 Transcription Factor; Stomach Neoplasms; Transfection

The piezoelectricity of collagen is purported to be linked to many biological processes including bone formation and wound healing. Although the piezoelectricity of tissue-derived collagen has been documented across the length scales, little work has been undertaken to characterise the local electromechanical properties of processed collagen, which is used as a base for tissue-engineering implants. In this work, three chemically distinct treatments used to form structurally and mechanically stable scaffolds-EDC-NHS, genipin and tissue transglutaminase-are investigated for their effect on collagen piezolectricity. Crosslinking with EDC-NHS is noted to produce a distinct self-assembly of the fibres into bundles roughly 300 nm in width regardless of the collagen origin. These fibre bundles also show a localised piezoelectric response, with enhanced vertical piezoelectricity of collagen. Such topographical features are not observed with the other two chemical treatments, although the shear piezoelectric response is significantly enhanced upon crosslinking. These observations are reconciled by a proposed effect of the crosslinking mechanisms on the molecular and nanostructure of collagen. These results highlight the ability to modify the electromechanical properties of collagen using chemical crosslinking methods.

Topics: Collagen; Cross-Linking Reagents; Elastic Modulus; GTP-Binding Proteins; Iridoids; Microscopy, Atomic Force; Nanostructures; Protein Glutamine gamma Glutamyltransferase 2; Succinimides; Tissue Engineering; Transglutaminases

The development of biodegradable scaffolds able to support cell growth has recently become of great importance. Therefore, the main objective of this work was the development of hybrid scaffolds made from the mixture of two biopolymers (collagen and chitosan) and the comparison of the effect of glutaraldehyde as crosslinking agent with three different crosslinking methods (chemical: genipin; physical: temperature and enzymatic: transglutaminase) in order to look for a promising candidate to substitute it. To achieve this purpose, the mechanical properties, structure, porosity, degree of crosslinking and swelling of the different scaffolds were assessed. The best ratio of biopolymers (collagen:chitosan) to form hybrid scaffolds was 1:1, which improve their mechanical and morphological properties compared to unitary scaffolds (only collagen or chitosan). In addition, the incorporation of 10% w/w transglutaminase (crosslinking agent) with respect to the mass of biopolymers made these scaffolds a good structure for the growth and proliferation of cells.

Topics: Biocompatible Materials; Biopolymers; Chitosan; Collagen; Cross-Linking Reagents; Iridoids; Microscopy, Electron, Scanning; Rheology; Tissue Engineering; Tissue Scaffolds

White spot syndrome virus (WSSV) is a serious epidemic pathogen of crustaceans and cause severe economic losses to aquaculture. However, no commercial drugs presently available to control WSSV infection. Genipin (GN) is a bioactive compound extracted from the fruit of Gardenia jasminoides and exhibits potential antiviral activity. In the study, the antiviral activity of GN against WSSV was investigated in crayfish Procambarus clarkii and in shrimp Litopenaeus vannamei. In vitro antiviral test showed that GN could inhibit WSSV replication in crayfish and in shrimp, and the highest inhibition on WSSV was over 99% when treatment with 50 mg/kg of GN for 24 h. In vivo antiviral test proved that GN could be used to treat and prevent WSSV infection. GN could also effectively protect crayfish from WSSV infection by reducing the mortality rate of WSSV-infected crayfish. Moreover, GN attenuated the WSSV-induced oxidative stress and inflammatory by upregulation the expression of antioxidant-related genes and downregulation the expression of inflammatory-related genes, respectively. Mechanically, GN inhibited WSSV replication at least via decreasing STAT (signal transducer and activator of transcription) gene expression to block WSSV immediate-early gene ie1 transcription. Additionally, the inhibition of BI-1 (Bax inhibitor-1) gene expression also played an important role in the suppression of WSSV infection. In conclusion, GN represented a potential therapeutic and preventive agent to block WSSV infection.

Topics: Animals; Antiviral Agents; Astacoidea; Dose-Response Relationship, Drug; Iridoids; Penaeidae; Random Allocation; White spot syndrome virus 1

Several strategies have been used to promote bone repair, with many failing due to the lack of osteoinduction. This report describes an approach for promoting bone healing that attempts to overcome prior shortcomings. First, the role was compared of different concentrations of gelatine (Gel), nanostructured-hydroxyapatite (nHA), simvastatin (Sim) and nHA-Sim particles on healing of small femoral bone defects in rabbits. The effective concentration of each was studied, and then a three-dimensional porous scaffold was designed using Gel, nHA and Sim, which was then cross-linked with genipin. Morphology, degradation profile and Sim delivery properties of the scaffolds were evaluated in vitro. Then, the scaffolds were subcutaneously tested in vivo to determine their biocompatibility, biodegradability and osteogenic properties. Finally, the scaffolds were implanted in a large radial bone defect model in rabbits and their effect on bone regeneration was investigated. The Gel, nHA and Sim with concentrations of 1, 1 and 5 mg/femoral hole were effective during bone healing respectively, and the Sim showed the most osteoinduction and osteoconduction when compared to controls. The Gel-Sim and Gel-nHA-Sim scaffolds continuously and homogenously released Sim into the simulated body fluid in vitro. Subcutaneously, the scaffolds were biocompatible, biodegradable and able to produce ectopic bone after 30 days. Thirty and 60 days after implantation of the scaffolds in radial bone defects, they were completely degraded and replaced with the new bone that had significantly superior morphology, mineral density, bioelectrical, biophysical and micromechanical properties compared with controls. Such bioactive grafts may be a suitable option for bone reconstruction, healing and repair.

Topics: Animals; Bone Regeneration; Bone Substitutes; Durapatite; Femur; Gelatin; Iridoids; Materials Testing; Porosity; Rabbits; Radius; Random Allocation; Simvastatin; Tissue Engineering; Tissue Scaffolds

Topics: Absorbable Implants; Animals; Biocompatible Materials; Cell Survival; Chitosan; Cross-Linking Reagents; Drug Delivery Systems; Hydrogels; Inflammation; Interleukin-4; Iridoids; Macrophages; Metal Nanoparticles; Mice; Osteoblasts; Phase Transition; Phenotype; Rats; Rats, Sprague-Dawley; RAW 264.7 Cells; Surface Properties; Titanium; Wound Healing

The demand of new strategies for the induction of bone regeneration is continuously increasing. Biomimetic porous gelatin-nanocrystalline hydroxyapatite scaffolds with tailored properties were previously developed, showing a positive response in terms of cell adhesion, proliferation, and differentiation. In the present paper, we focused on their osteoinductive properties. The effect of scaffolds on osteogenic differentiation of human mesenchymal stromal cells (hMSCs) was investigated in vitro. hMSCs were seeded on GEL (type A gelatin) and GEL containing 10 wt% hydroxyapatite (GEL-HA) and cultured in osteogenic medium. Results showed that GEL and GEL-HA10 sustained hMSC differentiation, with an increased ALP activity and a higher expression of bone specific genes. The osteoinductive ability of these scaffolds was then studied in vivo in a heterotopic bone formation model in nude mice. The influence of hMSCs within the implants was examined as well. Both GEL and GEL-HA10 scaffolds mineralized when implanted without hMSCs. On the contrary, the presence of hMSC abolished or reduced mineralization of GEL and GEL-HA10 scaffolds. However, we could observe a species-specific response to the presence of HA, which stimulated osteogenic differentiation of human cells only. In conclusion, the scaffolds showed promising osteoinductive properties and may be suitable for use in confined critical defects. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 914-923, 2018.

Topics: Animals; Biomarkers; Cell Differentiation; Cell Proliferation; Choristoma; Cross-Linking Reagents; Durapatite; Gelatin; Gene Expression Regulation; Humans; Iridoids; Male; Mesenchymal Stem Cells; Mice, Nude; Nanostructures; Osseointegration; Osteogenesis; X-Ray Microtomography

Complexes of Genipin and different water-soluble adjuvant polysaccharides, such as arabinogalactane, hydroxyethyl starch, fibergum, and oligosaccharides β-CD and HP-β-CD, were synthesized as drug delivery system using mechanochemical technology.. We have investigated physicochemical properties, stability, and hepatotoxicity of the synthesized complexes in solid state and aqueous solution. The formation of the complexes was evidenced by different physical and spectroscopy assays, and the stability constants of our synthesized Genipin-based complexes were also calculated.. The HP-β-CD inclusion complex showed the highest characteristics. We have found that the molecule of Genipin was completely included in the cyclodextrin cavity of the HP-β-CD. This complex of Genipin has shown a 6.14-fold increase of solubility compared with the original Genipin, and more stable in solvent and solid states.. The hepatotoxicity assays showed that our investigated complexes of Genipin are much safer than the original Genipin. These results suggest that new Genipin-based preparations can be synthesized with advantageous of higher stability and safety.

Topics: beta-Cyclodextrins; Cell Survival; Cholagogues and Choleretics; Drug Compounding; Drug Delivery Systems; Drug Liberation; Drug Stability; Hep G2 Cells; Humans; Hydroxyethyl Starch Derivatives; Iridoids; Solubility

The development of versatile tubular structures is a subject of broad interest in tissue engineering applications. Herein, we demonstrate the production of tubular structures based on chitosan through a combination of dipping, freeze-drying and supercritical technology approaches. The combination of these techniques yields versatile tubes with a perfectly defined hollow imprint, which upon chemical cross-linking with genipin acquire enhanced mechanical properties (Young Modulus ( E) and ultimate tensile stress (σ

Topics: Animals; Biocompatible Materials; Cell Line; Chitosan; Cross-Linking Reagents; Elastic Modulus; Fibroblasts; Freeze Drying; Iridoids; Mice; Tissue Engineering; Tissue Scaffolds

Schwann cell-seeded nerve guidance channels are designed to assist post-traumatic nerve regeneration in the PNS. Chitosan is a natural polymer well suited for tissue engineering as it is biocompatible, non-immunogenic, and biodegradable. Electrospun chitosan nanofibers utilized in nerve guidance channels have the capacity for guiding axonal growth within the channel lumen yet are limited in their capacity to maintain structural integrity within physiological environments. To address this, we attempted genipin crosslinking of chitosan nanofibers. Compared to neat chitosan nanofibers, genipin-treated nanofibers exhibited increased stiffness, resistance to swelling and lysozymal degradation. Furthermore, alignment and proliferation of purified Schwann cell cultures upon genipin-treated substratum was enhanced. When dorsal root ganglion explants were utilized as an in vitro model of peripheral nerve regeneration, emigrating neurons and Schwann cells assumed the uniaxial pattern of aligned electrospun chitosan nanofibers. Neurite growth along the nanofibers led, reaching a frontier more than twice that of the pursuant Schwann cells. Critically, neurite growth rate upon genipin-treated nanofibers demonstrated a 100% increase. Altogether, genipin treatment improves upon the physical and biological properties of chitosan nanofibers towards their utility in nerve guidance channel design.

Topics: Animals; Biocompatible Materials; Chitosan; Cross-Linking Reagents; Ganglia, Spinal; Iridoids; Nanofibers; Nerve Regeneration; Neuronal Outgrowth; Neurons; Primary Cell Culture; Rats; Rats, Sprague-Dawley; Schwann Cells; Sciatic Nerve; Tissue Culture Techniques; Tissue Engineering; Tissue Scaffolds

Hyperlipidemia is a chronic disorder which plays an important role in the development of cardiovascular diseases, type 2 diabetes, atherosclerosis, hypertension, and nonalcoholic fatty liver disease. Genipin (GNP) is a metabolite from genipioside, which is an active component of the traditional Chinese medicine Gardenia jasminoides Ellis, and has been recognized as a beneficial compound against metabolic disorders. However, whether it can correct overnutrition-induced dyslipidemia is still unknown. In this study, the effects of GNP on attenuating hyperlipidemia and hepatic lipid accumulation were investigated using normal and obese mice induced with a high-fat diet (HFD) and primary hepatocytes treated with free fatty acids. We also sought to identify potential targets of GNP to mediate its effects in the liver. We found that obese mice treated with GNP showed a decrease in the body weight, serum lipid levels, as well as hepatic lipid accumulation. Besides, GNP regulated hepatic expression levels of lipid metabolic genes, which are important in maintaining systemic lipid homeostasis. At the molecular level, GNP increased the expression levels of miR-142a-5p, which bound to 3' untranslated region of Srebp-1c, an important regulator of lipogenesis, which thus led to the inhibition of lipogenesis. Collectively, our data demonstrated that GNP effectively antagonized HFD-induced hyperlipidemia and hepatic lipid accumulation in mice. Such effects were achieved by regulating miR-142a-5p/SREBP-1c axis.

Topics: Animals; Anti-Obesity Agents; Cells, Cultured; Computational Biology; Diet, High-Fat; Dose-Response Relationship, Drug; Fatty Acids, Nonesterified; Gene Expression Regulation; Genes, Reporter; Hyperlipidemias; Insulin Resistance; Iridoids; Lipid Metabolism; Lipotropic Agents; Liver; Male; Mice, Inbred C57BL; MicroRNAs; Non-alcoholic Fatty Liver Disease; Obesity; Random Allocation; Sterol Regulatory Element Binding Protein 1

Defects in the annulus fibrosus (AF) of intervertebral discs allow nucleus pulposus tissue to herniate causing painful disability. Microdiscectomy procedures remove herniated tissue fragments, but unrepaired defects remain allowing reherniation or progressive degeneration. Cell therapies show promise to enhance repair, but methods are undeveloped and carriers are required to prevent cell leakage. To address this challenge, this study developed and evaluated genipin-crosslinked fibrin (FibGen) as an adhesive cell carrier optimized for AF repair that can deliver cells, match AF material properties, and have low risk of extrusion during loading. Part 1 determined that feasibility of bovine AF cells encapsulated in high concentration FibGen (F140G6: 140 mg/mL fibrinogen; 6 mg/mL genipin) for 7 weeks could maintain high viability, but had little proliferation or matrix deposition. Part 2 screened tissue mechanics and in situ failure testing of nine FibGen formulations (fibrin: 35-140 mg/mL; genipin: 1-6 mg/mL). F140G6 formulation matched AF shear and compressive properties and significantly improved failure strength in situ. Formulations with reduced genipin also exhibited satisfactory material properties and failure behaviors warranting further biological screening. Part 3 screened AF cells encapsulated in four FibGen formulations for 1 week and found that reduced genipin concentrations increased cell viability and glycosaminoglycan production. F70G1 (70 mg/mL fibrinogen; 1 mg/mL genipin) demonstrated balanced biological and biomechanical performance warranting further testing. We conclude that FibGen has potential to serve as an adhesive cell carrier to repair AF defects with formulations that can be tuned to enhance biomechanical and biological performance; future studies are required to develop strategies to enhance matrix production.

Topics: Animals; Annulus Fibrosus; Biocompatible Materials; Cattle; Cell Proliferation; Cell Survival; Cells, Cultured; Fibrin; Glycosaminoglycans; Hydrogel, Polyethylene Glycol Dimethacrylate; Intervertebral Disc; Iridoids

Decellularized arteries have been considered as promising scaffolds for small-diameter vascular substitutes. However, weakened mechanical properties, immunological rejection and rapid degradation after transplantation still exist after decellularization. Previous studies indicated that genipin cross-linking can solve these problems. Therefore, genipin was selected as the cross-linking agent for the pre-treatment of decellularized arteries in our study. Histological analysis, scanning electron microscopy, mechanical properties analysis and subcutaneous embedding experiment were adopted to investigate the efficiency of decellularization and the effect of genipin cross-linking on improving mechanical, structural and biological properties of decellularized arteries. Decellularization protocols based on three trypsin concentrations were used to prepare decellularized arteries, after decellularization, arteries were cross-linked with genipin. Results showed that 0.5% trypsin was the most efficient concentration to remove cellular components and preserve ECM. However, mechanical properties of 0.5% trypsin decellularized arteries weakened significantly, while genipin cross-linking improved mechanical properties of decellularized arteries to the same level as fresh arteries. After 4 weeks subcutaneous embedding, cross-linked arteries caused the mildest inflammatory response. In conclusion, genipin could be employed as an ideal cross-linking agent to strengthen mechanical properties, enhance the resistance to degradation and reduce the antigenicity of decellularized arteries for small-diameter blood vessel tissue engineering applications.

Topics: Animals; Biomechanical Phenomena; Carotid Arteries; Cross-Linking Reagents; Dogs; Extracellular Matrix; Inflammation; Iridoids; Swine; Tissue Engineering; Tissue Scaffolds

In this paper we developed an innovative, effective and rapid one-step approach to crosslink mucoadhesive gelatin films for buccal drug delivery. The method, which involves the application of non-equilibrium pressure plasma for 3 or 5 minutes/side, was compared with a classical approach based on the use of a chemical crosslinking agent, namely genipin. Econazole nitrate (ECN), an imidazole antifungal agent used for the treatment of skin infections and mucosal candidiasis, was selected as model drug. X-Ray Diffraction characterization performed on the drug-containing gelatin films revealed that ECN undergoes to a topotactic transformation into Econazole (EC) immediately after mixing with gelatin suggesting the occurrence of an acid-base reaction between drug and gelatin during film processing. Plasma treatment, as well as genipin crosslinking, did not provoke any further variation of EC structure. However, plasma exposure significantly improved films adhesiveness and allowed to reach mucoadhesive strength values more than double with respect to those obtained with genipin, ascribable to the presence of polar and hydrophilic groups on the plasma treated film's surface. A residence time of at least 48 h was obtained by properly selecting the plasma exposure times. These results, together with the in-vitro data showing retention of antifungal efficacy against a strain of Candida albicans, demonstrated that plasma treatment was a valid and rapid alternative, easy to scale-up, to chemical crosslinking methods for the production of highly mucoadhesive gelatin-based films.

Topics: Adhesiveness; Administration, Buccal; Animals; Antifungal Agents; Atmospheric Pressure; Candida albicans; Cross-Linking Reagents; Drug Carriers; Drug Compounding; Drug Liberation; Econazole; Gelatin; Iridoids; Kinetics; Mouth Mucosa; Plasma Gases; Swine; Tensile Strength

Enzyme-assisted extraction in liquid-liquid two-phase aqueous system was applied for the first time in order to extract genipin from genipap. The effect of different commercial enzymes, their concentrations, and extraction parameters were investigated. Moreover, chitosan gels were prepared, crosslinked with glutaraldehyde or genipin and characterized by their textural and rheological properties. The crosslinked chitosan was used as support for the immobilization of model β-galactosidases. Among the different commercial enzymes tested for extraction, Celluclast 10% (36 °C and pH 3.7) provided an extraction of 196 mg.g

Topics: beta-Galactosidase; Chitosan; Cross-Linking Reagents; Enzymes, Immobilized; Gardenia; Gels; Glutaral; Iridoids; Rheology

Adipose-derived stem cells (ADSCs)-based tissue engineering was a promising method to treat intervertebral disc degeneration. Type II collagen is a native component in the nucleus pulposus (NP), and has the ability to promote ADSCs to differentiate into NP-like cells. In this article, we aimed to establish a genipin-cross-linked three-dimensional (3D) type II collagen scaffold, and determine the biological effects of the scaffold on ADSCs differentiating into a NP-like phenotype. Different concentrations of genipin were used to cross-link the 3D type II collagen scaffold. Microstructure, surface topography, mechanical strength, porosity, swelling property, and biological stability of the scaffolds were detected to evaluate the scaffold properties. Cell proliferation, gene and protein expression were measured to access the biological effects of the scaffolds on ADSCs, and the related molecular mechanism was investigated. Cross-linking by genipin increased the stability of the type II collagen scaffolds, but deformed the configuration of scaffolds and changed the intrinsic properties of type II collagen. scaffold cross-linked with 0.1% genipin improved the biostability on the basis of maintaining the configuration of scaffold. In addition, the 0.1% genipin-cross-linked scaffold promoted ADSCs proliferation and differentiation into NP-like cells, along with the increasing gene and protein expressions of Sonic Hedgehog (Shh). All these results suggested that 0.1% genipin was the optimal concentration to establish a bio-stable 3D type II collagen scaffold, which inducing ADSC proliferation and differentiation toward a NP-like phenotype through the activation of Shh signaling pathway. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1258-1268, 2018.

Topics: Adipose Tissue; Animals; Cell Death; Cell Differentiation; Cell Proliferation; Cell Shape; Cells, Cultured; Chickens; Collagen Type II; Cross-Linking Reagents; Gene Expression Regulation; Hedgehog Proteins; Iridoids; L-Lactate Dehydrogenase; Materials Testing; Nucleus Pulposus; Porosity; Rats, Sprague-Dawley; Signal Transduction; Stem Cells; Tissue Scaffolds

Genipin is the major active component of Gardeniae fructus and has been shown to ameliorate diabetes and insulin resistance in rat models. In this study, we first investigated the effect of genipin on obesity and the related lipid metabolism mechanisms in diet-induced obese rats. Our results showed that genipin reduced body weight, food intake, and visceral fat mass; ameliorated dyslipidemia, glucose intolerance, insulin intolerance, adipocyte hypertrophy, and hepatic steatosis; and reduced serum tumor necrosis factor-α level in diet-induced obese rats. Quantitative real-time reverse-transcription polymerase chain reaction results further illustrated that genipin promoted lipolysis and β-oxidation of fatty acid by upregulating gene expressions of hormone-sensitive lipase and adipose triglyceride lipase in white adipose tissue (WAT) and peroxisome proliferator-activated receptor-α and carnitine palmitoyltransferase 1α in hepatic tissue. Moreover, genipin promoted browning of WAT by upregulating the mRNA and protein levels of uncoupling protein 1 and PRD1-BF1-RIZ1 homologous domain containing 16 in WAT. Additionally, genipin inhibited gene expressions of activin receptor-like kinase 7, tumor necrosis factor-α, and interlukin-6 in WAT. These results indicated that genipin had a potential therapeutic role in obesity, in which regulation of lipid mobilization and browning of WAT were involved.

Topics: Adipose Tissue, White; Animals; Diet; Disease Models, Animal; Iridoids; Lipid Mobilization; Male; Obesity; Rats

A series of novel genipin glycoside derivatives incorporating 11 glycosidic moieties at either the 1 or 10 position of genipin were designed and synthesized. These compounds exhibited moderate to excellent inhibitory activities against tobacco mosaic virus. Especially, the in vitro and in vivo activities of compounds 6e, 7c, 7d, 7f, 7h, and 7i were comparable to that of ribavirin. In particular, compound 7c, the mannosyl derivative of genipin at the 10 position, showed the best activity. The series of genipin glycosyl derivatives also displayed fungicidal activities against 14 kinds of phytopathogenic fungi, especially for Rhizoctonia cerealis and Sclerotinia sclerotiorum. Moreover, compound 6h exhibited good insecticidal activity against diamondback moth; compounds 7b, 7c, and 7g exhibited moderate insecticidal activity against three kinds of Lepidoptera pests (oriental armyworm, cotton bollworm, and corn borer); and compound 7e showed excellent larvacidal activities against mosquito.

Topics: Animals; Antiviral Agents; Culicidae; Drug Design; Fungi; Fungicides, Industrial; Glycosylation; Insecticides; Iridoids; Molecular Structure; Moths; Structure-Activity Relationship; Tobacco Mosaic Virus

Understanding the effect of impact loading on the mechanical response of the intervertebral disc (IVD) is valuable for investigating injury mechanisms and devising effective therapeutic modalities. This study used 24 porcine thoracic motion segments to characterize the mechanical response of intact (N = 8), degenerated (Trypsin-denatured, N = 8), and repaired (Genepin-treated, N = 8) IVDs subject to impact loading. A meta-model analysis of poroelastic finite element simulations was used in combination with ex-vivo creep and impact tests to extract the material properties. Forward analyses using updated specimen-specific FE models were performed to evaluate the effect of impact duration. The maximum axial stress in the IVDs, Von-Mises stress in the endplates, and intradiscal pore pressure (IDP) were calculated, under a 400 N preload, subject to a sequence of impact loads for 10 impact durations (10-100 ms). The results were in good agreement with both creep and impact experiments (error < 10%). A significant difference was found in the maximum axial stress between the intact and degenerated disc groups. The IDP was also significantly lower in the degenerated disc group. The Von Mises stress in the adjacent endplates significantly increased with degeneration. It is concluded that the disc time-dependent response significantly changes with disc degeneration. Cross-linker Genipin has the potential to recover the hydraulic permeability and can potentially change the time dependent response, particularly in the IDP. This is the first study, to our best knowledge, which explores the effect of impact loading on the healthy, degenerated and repaired IVD using both creep and impact validation tests.

Topics: Animals; Biomechanical Phenomena; Computer Simulation; Finite Element Analysis; Intervertebral Disc; Intervertebral Disc Degeneration; Iridoids; Models, Biological; Pressure; Stress, Mechanical; Swine; Weight-Bearing

Skeletal muscle engineering aims at tissue reconstruction to replace muscle loss following traumatic injury or in congenital muscle defects. Skeletal muscle can be engineered by using biodegradable and biocompatible scaffolds that favor myogenic cell adhesion and subsequent tissue organization. In this study, we characterized scaffolds made of gelatin cross-linked with genipin, a natural derived cross-linking agent with low cytotoxicity and high biocompatibility, for tissue engineering of skeletal muscle. We generated gelatin-genipin hydrogels with a stiffness of 13 kPa to reproduce the mechanical properties characteristic of skeletal muscle and we show that their surface can be topographically patterned through soft lithography to drive myogenic cells differentiation and unidirectional orientation. Furthermore, we demonstrate that these biomaterials can be successfully implanted in vivo under dorsal mouse skin, showing good biocompatibility and slow biodegradation rate. Moreover, the grafting of this biomaterial in partially ablated tibialis anterior muscle does not impair muscle regeneration, supporting future applications of gelatin-genipin biomaterials in the field of skeletal muscle tissue repair. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2763-2777, 2018.

Topics: Animals; Cell Differentiation; Cell Line; Gelatin; Hydrogels; Iridoids; Mice; Muscle Development; Muscle, Skeletal; Tissue Engineering

Nanoparticles formed by the assembly of protein and polysaccharides are of great interest for the delivery of hydrophobic molecules. Herein, the formation of genipin-crosslinked nanoparticles from caseinate (CS) and chitosan (CH) is reported for the delivery of curcumin, a polyphenolic compound from turmeric, to cells. Genipin-crosslinked CS-CH nanoparticles (G-CCNPs) having a diameter of ∼250 nm and a low polydispersity index showed excellent stability over a wide pH range, as indicated by dynamic light scattering and transmission electron microscopic measurements. Cellular uptake of curcumin loaded into G-CCNPs by HeLa cells was improved, as measured by confocal laser scanning microscopy (CLSM) and fluorescence-activated cell-sorting analysis. Cell proliferation assays indicated that G-CCNPs were nontoxic and that curcumin's anticancer activity in vitro was also improved by G-CCNPs. Stability of curcumin at neutral pH was enhanced by G-CCNPs. CLSM study revealed that G-CCNPs were poorly internalized by HeLa cells, possibly because of strong cell membrane interactions and a negative zeta potential. Overall, our results suggested that the enhanced curcumin cytotoxicity might be associated with the enhanced stability of curcumin by G-CCNPs and free curcumin released from G-CCNPs into the cell. These biocompatible NPs might be suitable carriers for enhancing curcumin's therapeutic potential.

Topics: Antineoplastic Agents; Caseins; Cell Death; Chitosan; Curcumin; Drug Liberation; Fluorescence; HeLa Cells; Humans; Iridoids; Nanoparticles; Particle Size

The generation of biomimetic and biocompatible artificial tissues is the basic research objective for tissue engineering (TE). In this sense, the biofabrication of scaffolds that resemble the tissues' extracellular matrix is an essential aim in this field. Uncompressed and nanostructured fibrin-agarose hydrogels (FAH and NFAH, respectively) have emerged as promising scaffolds in TE, but their structure and biomechanical properties must be improved in order to broaden their TE applications. Here, we generated and characterized novel membrane-like models with increased structural and biomechanical properties based on the chemical cross-linking of FAH and NFAH with genipin (GP at 0.1%, 0.25%, 0.5% and 0.75%). Furthermore, the scaffolds were subjected to rheological (G, G', G″ modulus), ultrastructural and ex vivo biocompatibility analyses. Results showed that all GP concentrations increased the stiffness (G) and especially the elasticity (G') of FAH and NFAH. Ultrastructural analyses demonstrated that GP and nanostructuration of FAH allowed us to control the porosity of FAH. In addition, biological studies revealed that higher concentration of GP (0.75%) started to compromise the cell function and viability. Finally, this study demonstrated the possibility to generate natural and biocompatible FAH and NFAH with improved structural and biomechanical properties by using 0.1%-0.5% of GP. However, further in vivo studies are needed in order to demonstrate the biocompatibility, biodegradability and regeneration capability of these cross-linked scaffolds.

Topics: Biocompatible Materials; Biomechanical Phenomena; Colorimetry; Elasticity; Extracellular Matrix; Fibrin; Fibroblasts; Humans; Hydrogels; Iridoids; Materials Testing; Microscopy, Electron, Scanning; Porosity; Rheology; Sepharose; Stress, Mechanical; Tissue Engineering; Tissue Scaffolds; Viscosity

Accurate and efficient antigen delivery is crucial for inducing a strong and long-term immune response. A visible protein nanovaccine made from antigen could provide a novel and promising technology for secure and efficient delivery of the antigen with imaging visualization. In this study, a functional nanovaccine based on genipin crosslinked ovalbumin (OVA) fluorescent nanoparticles with chitosan (CS-OVA-NPs) was developed. The nanovaccine can carry abundant antigens by self-crosslinking without additional carriers. The fluorescence imaging technique was applied to monitor and reveal the process of antigen delivery in vivo based on the fluorescence of genipin with a non-invasive and real-time manner. This functional OVA nanovaccine can enhance the uptake of OVA in Dendritic Cells (DCs) and further promote DCs to maturate in vitro. In vivo study further indicated CS-OVA-NPs could trigger antigen-specific immune responses, which demonstrated that this fluorescent nanovaccine provided a novel design approach for accurate and efficient vaccine delivery.

Topics: Animals; Cell Survival; Cells, Cultured; Chitosan; Dendritic Cells; Drug Delivery Systems; Iridoids; Male; Mice; Mice, Inbred C57BL; Nanoparticles; Ovalbumin; Vaccines

To investigate the surgical outcomes of posterior scleral reinforcement (PSR) using genipin-cross-linked sclera to treat macular hole retinal detachment (MHRD) in highly myopic eyes.. Nineteen patients with high myopia (19 eyes) with MHRD were treated sequentially with genipin-cross-linked PSR and were followed at least for 1 year after the surgery. The best corrected visual acuity (BCVA), axial length (AL), optical coherence tomography (OCT) outcomes and the complications were evaluated.. Macular hole was closed in 73.7% of the eyes, foveal reattachment rate was 100%. The mean logMAR BCVA improved from 1.27±0.55 preoperatively to 0.88±0.55 postoperatively. The preoperative AL (29.88±1.97 mm) was decreased (27.73±1.84 mm) after the operation (p<0.001).. For at least a 1-year period of follow-up, PSR with genipin-cross-linked sclera should be considered as a preferred surgical approach to treat MHRD in highly myopic eyes, especially when foveal retinoschisis is also documented.

Topics: Adult; Aged; Axial Length, Eye; Cholagogues and Choleretics; Cross-Linking Reagents; Female; Follow-Up Studies; Humans; Iridoids; Male; Middle Aged; Myopia, Degenerative; Retinal Detachment; Retinal Perforations; Retrospective Studies; Sclera; Tomography, Optical Coherence; Visual Acuity

Synthetic antidepressants in current use for the complex etiopathogeneses of depression have slow response and remission as well as various unpleasant side effects. As a result, it is imperative to develop new antidepressants with more effectiveness and less severe side effects. Recent studies demonstrated that genipin, the aglycon of geniposide, extracted from Gardenia jasminoides Ellis has antidepressive effects. However, knowledge regarding the molecular mechanisms of its antidepressant effects remains limited. Employing a depression-like mouse model, we confirmed that genipin is capable of correcting depressions-like behaviors induced by prenatal stress in offspring from prenatally stressed dams (defined as PRS mice). In further experiments, we found that the effect of genipin on PRS mice occurs through DNA demethylation by inhibiting DNA methyltransferase 1 (DNMT1), normalizing the expression of reduced brain-derived neurotrophic factor (BDNF) in the hippocampus.

Topics: Animals; Antidepressive Agents; Brain-Derived Neurotrophic Factor; Depression; Disease Models, Animal; DNA (Cytosine-5-)-Methyltransferase 1; DNA Methylation; Female; Gardenia; Gene Expression Regulation; Hippocampus; Humans; Iridoids; Mice; Pregnancy; Prenatal Exposure Delayed Effects; Stress, Psychological

Excessive immune responses following the use of implantable, biomaterial-based medical devices represent a substantial challenge for treatment efficacy and patient well-being. Specifically, after implantation, pro-inflammatory M1 macrophages are activated by cytokines such as interferon-γ (IFN-γ) followed by anti-inflammatory M2 macrophages polarized by cytokines including interleukin-4 (IL-4), leading to healing and long-term stability of implants. Here, we report the loading of an immunomodulatory cytokine,IL-4, into TiO

Topics: Animals; Biomarkers; Bone and Bones; Cell Polarity; Coated Materials, Biocompatible; Cytokines; Hydrogels; Inflammation Mediators; Iridoids; Macrophages; Nanotubes; Prostheses and Implants; Rats, Sprague-Dawley; Spectroscopy, Fourier Transform Infrared; Titanium

Chitosan as a natural cationic polysaccharide has drawn wide interests as surface modification materials in orthopedic applications, with the potential to achieve combined osteogenic, antimicrobial and haemostatic functions. The cationicity of chitosan has been reported to play an important role in modifying the osteoblastic cell responses and the antibacterial activities, while its effect on the haemostatic properties has been rarely studied. To this aim, we prepared carboxymethyl chitosan hydrogels with different cationicity through crosslinking with different concentrations of genipin (1%, 2.5%, 5% and 10%). The genipin concentration strongly influenced both mesenchymal stem cell (MSC) responses and blood coagulation activity for chitosan-hydroxyapatite samples. Increasing genipin concentration overall enhanced the osteogenic and haemostatic potentials, and an optimum window of chitosan cationicity (5% genipin in our case) led to both the best MSC response and coagulant activities. In particular, the cationicity had demonstrated a profound modulation effect on the haemostatic activities of chitosan samples, through influencing three different aspects of the coagulation processes, including intrinsic coagulation pathway, aggregation and activation of platelet, and activation of erythrocyte. Tuning the crosslinking degree thus provides a simple and effective approach to achieving combined osteogenic and haemostatic functions, which has great potential in surface modification of surgical implants.

Topics: Chitosan; Erythrocytes; Humans; Hydrogels; Iridoids; Mesenchymal Stem Cells; Osteogenesis

The suture-tendon interface is often the weakest link in tendon-to-tendon or tendon-to-bone repair. Genipin is an exogenous collagen crosslink agent derived from the gardenia fruit that can enhance suture force to failure of the tendon-suture interface. Viable methods for intraoperative clinical delivery of genipin could be of clinical utility, but to our knowledge have not yet been extensively studied.. The purposes of this study were (1) to evaluate whether sutures precoated with genipin can augment the suture-tendon interface to improve force to failure, stiffness, and work to failure in healthy and degenerated tendons; and (2) to determine the effect of genipin on the extent and distribution of crosslinking.. Single-stitch suture pullout tests were performed ex vivo on 25 bovine superficial digital flexor tendons. To assess effects on native tissue, one group of 12 tendons was cut in proximal and distal halves and randomized to treatment (n = 12) and control groups (n = 12) in a matched-pair design. One simple stitch with a loop with either a normal suture or genipin-coated suture was applied to tendons in both groups. To simulate a degenerative tendon condition, a second group of 13 tendons was cut in proximal and distal halves, injected with 0.2 mL of collagenase D (8 mg/mL) and incubated for 24 hours before suturing with either a genipin-coated suture (n = 13) or their matched controls (n = 13). Sutures from all groups then were loaded to failure on a universal materials testing machine 24 hours after suturing. Suture pullout force, stiffness, and work to failure were calculated from force-displacement data and compared between the groups. Additionally, fluorescence was measured to determine the degree of crosslinking quantitatively and a qualitative analysis of the distribution pattern was performed by microscopy.. In healthy tendon pairs, the median maximum pullout force was greater with genipin-coated sutures than with control sutures (median, 42 N [range, 24-73 N] versus 29 N [range, 13-48 N]; difference of medians, 13 N; p = 0.003) with corresponding increases in the required work to failure (median, 275 mJ [range, 48-369 mJ] versus 148 mJ [range, 83-369 mJ]; difference of medians, 127 mJ; p = 0.025) but not stiffness (median, 4.1 N/mm [range, 2.3-8.1 N/mm] versus 3.3 N/mm [range, 1.1-9.6 N/mm]; difference of medians, 0.8 N/mm; p = 0.052). In degenerated tendons, median maximum pullout force was greater with genipin-coated sutures than with control sutures (median, 16 N [range, 9-36 N] versus 13 N [range, 5-28 N]; difference of medians, 3 N; p = 0.034) with no differences in work to failure (median, 75 mJ [range, 11-249 mJ] versus 53 mJ [range, 14-143 mJ]; difference of medians, 22 mJ; p = 0.636) or stiffness (median, 1.9 N/mm [range, 0.7-13.4 N/mm] versus 1.6 N/mm [range, 0.5-5.6 N/mm]; difference of medians, 0.3 N/mm; p = 0.285). Fluorescence was higher in tendons treated with genipin-coated sutures compared with the control group, whereas higher fluorescence was observed in the treated healthy compared with the degenerated tendons (difference of means -3.16; standard error 1.08; 95% confidence interval [CI], 0.97-5.34; p = 0.006/healthy genipin: mean 13.04; standard error 0.78; 95% CI, 11.47-14.62; p < 0.001/degenerated genipin: mean 9.88; SD 0.75; 95% CI, 8.34-11.40; p < 0.001).. Genipin-coated sutures improved force to failure of a simple stitch at the tendon-suture interface in healthy and degenerated tendons in an ex vivo animal model. Fluorescence was higher in tendons treated with genipin-coated sutures compared with the control group.. A genipin-coated suture represents a potential delivery vehicle for exogenous crosslink agents to augment suture retention properties. In vivo animal studies are the next logical step to assess safety and efficacy of the approach.

Topics: Animals; Cattle; Coated Materials, Biocompatible; Collagen; Cross-Linking Reagents; Equipment Design; Equipment Failure; In Vitro Techniques; Iridoids; Materials Testing; Suture Techniques; Sutures; Tendons

Tissue engineered nerve grafts (TENGs) are considered a promising alternative to autologous nerve grafting, which is considered the "gold standard" clinical strategy for peripheral nerve repair. Here, we immobilized tumor necrosis factor-α (TNF-α) inhibitors onto a nerve conduit, which was introduced into a chitosan (CS) matrix scaffold utilizing genipin (GP) as the crosslinking agent, to fabricate CS-GP-TNF-α inhibitor nerve conduits. The in vitro release kinetics of TNF-α inhibitors from the CS-GP-TNF-α inhibitor nerve conduits were investigated using high-performance liquid chromatography. The in vivo continuous release profile of the TNF-α inhibitors released from the CS-GP-TNF-α inhibitor nerve conduits was measured using an enzyme-linked immunosorbent assay over 14 days. We found that the amount of TNF-α inhibitors released decreased with time after the bridging of the sciatic nerve defects in rats. Moreover, 4 and 12 weeks after surgery, histological analyses and functional evaluations were carried out to assess the influence of the TENG on regeneration. Immunochemistry performed 4 weeks after grafting to assess early regeneration outcomes revealed that the TENG strikingly promoted axonal outgrowth. Twelve weeks after grafting, the TENG accelerated myelin sheath formation, as well as functional restoration. In general, the regenerative outcomes following TENG more closely paralleled findings observed with autologous grafting than the use of the CS matrix scaffold. Collectively, our data indicate that the CS-GP-TNF-α inhibitor nerve conduits comprised an elaborate system for sustained release of TNF-α inhibitors in vitro, while studies in vivo demonstrated that the TENG could accelerate regenerating axonal outgrowth and functional restoration. The introduction of CS-GP-TNF-α-inhibitor nerve conduits into a scaffold may contribute to an efficient and adaptive immune microenvironment that can be used to facilitate peripheral nerve repair.

Topics: Animals; Chitosan; Cross-Linking Reagents; Iridoids; Nerve Regeneration; Rats; Rats, Sprague-Dawley; Sciatic Nerve; Tissue Engineering; Tumor Necrosis Factor-alpha

In this study, central composite factorial design was used for the preparation and optimization of chitosan/Na-alginate hydrogel films containing metformin via solvent evaporation technique.. Low and high molecular weight (MW) chitosan was used as a polymer in different concentrations while genipin was used as a crosslinking agent. Drug release studies were performed in simulated gastric and intestinal fluids at pH 1.2 and 7.4 of formulated hydrogels.. For low MW chitosan hydrogel, the highest drug release at pH 1.2 was observed i.e., 9.82% for 2 hrs while at pH 7.4, 95.52% drug release was observed after 12 hrs. In case of high MW chitosan hydrogel 9.67% drug release at ph1.2 for 2 hrs and 90.63% drug release at pH 7.4 after 12 hrs was observed. The highest T50% of low MW and high MW chitosan hydrogel was observed as 22.72 and 33.34 hrs, respectively, while the highest dynamic swelling was observed as 8.21 and 7.9, respectively.. It was found that by changing the ratios of polymers as well as crosslinking agent, the release rate of metformin can be modified. Low MW chitosan hydrogel showed an increased release rate than high MW chitosan hydrogel and by increasing the concentration of crosslinking agent, the release rate was found to be decreased and vice versa.

Topics: Alginates; Chitosan; Cross-Linking Reagents; Drug Liberation; Gastric Juice; Glucuronic Acid; Hexuronic Acids; Hydrogels; Hypoglycemic Agents; Intestinal Secretions; Iridoids; Metformin

Hypoxia-inducible factor-1 (HIF-1) and vascular endothelial growth factor (VEGF) play important roles in cancer progression in various cancer cell lines. Although genipin, a constituent of Gardenia fruit, has been shown to have anti-tumor activity, its role in the suppression of HIF-1 and its downstream target genes is not well understood. We examined the effect of genipin on the intracellular level of HIF-1α and extracellular level of VEGF using the colon cancer cell line HCT116. We observed that genipin suppressed the accumulation of HIF-1α under hypoxia in various cancer cell lines, including HCT116, via the modulation of protein degradation. Genipin also suppressed the expression of VEGF and the invasion of colon cancer cells by blocking the extracellular signal-regulated kinase signaling pathway. Taken together, our results provide new insights into the potential role of genipin in suppressing colon cancer progression.

Topics: Cell Hypoxia; Colonic Neoplasms; Disease Progression; Extracellular Signal-Regulated MAP Kinases; HCT116 Cells; HT29 Cells; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Iridoids; Neoplasm Invasiveness; Neoplasm Metastasis; Proteolysis; Proto-Oncogene Proteins c-akt; Vascular Endothelial Growth Factor A

In this study, central composite design was utilized for the preparation and optimization of chitosan-gelatin hydrogel films containing metformin using different concentrations of genipin as crosslinking agent. Solvent evaporation technique was used to develop hydrogel films. The formulated hydrogels were subjected to study the drug release in the simulated gastric pH1.2 for 3h. Initial burst release was observed from hydrogel during the first 2h of dissolution in gastric simulated environment. Different polymer ratios in formulation showed significant influence on T

Topics: Administration, Oral; Animals; Chitosan; Delayed-Action Preparations; Gelatin; Hydrogen-Ion Concentration; Iridoids; Male; Metformin; Rabbits

Myopia progression is thought to involve biomechanical weakening of the sclera, which leads to irreversible deformations and axial elongation of the eye. Scleral crosslinking has been proposed as a potential treatment option for myopia control by strengthening the mechanically weakened sclera. The biomechanical mechanism by which the sclera weakens during myopia and strengthens after crosslinking is not fully understood. Here, we assess the effect of lens-induced myopia and exogenous crosslinking using genipin on the inelastic mechanical properties of the tree shrew sclera measured by cyclic tensile tests.. Cyclic tensile tests were performed on 2-mm wide scleral strips at physiological loading conditions (50 cycles, 0-3.3 g, 30 s cycle. -5D lens treatment significantly increased the cyclic softening response of the sclera when compared to contralateral control eyes (0.10% ± 0.029%, mean ± standard error, P = 0.037). Exogenous crosslinking of the lens treated sclera significantly decreased the cyclic softening response (-0.12% ± 0.014%, P = 2.2 × 10. Results indicated that cyclic tensile loading leads to an inelastic, cyclic softening of the juvenile tree shrew sclera. The softening rate increased during lens-induced myopia and was diminished after genipin crosslinking. This finding suggests that axial elongation in myopia may involve a biomechanical weakening mechanism that increased the cyclic softening response of the sclera, which was inhibited by scleral crosslinking using genipin.

Topics: Adhesives; Animals; Biomechanical Phenomena; Cross-Linking Reagents; Disease Models, Animal; Disease Progression; Iridoids; Myopia; Refraction, Ocular; Sclera; Tensile Strength; Tupaiidae

Diabetic nephropathy is one of the various complications of diabetes mellitus, affecting patients for lifetime. Earlier studies have revealed that genipin can not only improve diabetes, but also induce cytotoxicity. Therefore, it is not clear which effect of genipin on kidneys occurs, when it is used in the treatment of diabetes. In the present study, we performed nuclear magnetic resonance (NMR)-based metabolomics analysis of urine and kidney tissue samples obtained from diabetic rats to explore the change of endogenous metabolites associated with diabetes and concomitant kidney disease. Nine significant differential metabolites that were closely related to renal function were screened. They were mainly related to three metabolic pathways: synthesis and degradation of ketone bodies, glycine, serine and threonine metabolism, and butanoate metabolism, which are involved in methylamine metabolism, energy metabolism and amino acid metabolism. In addition, after the intervention of genipin, the metabolic levels of all the metabolites tended to be normal, indicating a protective effect of genipin on kidneys. Our results may be helpful for understanding the antidiabetic effect of genipin.

Topics: Amino Acids; Animals; Diabetes Mellitus, Experimental; Energy Metabolism; Hypoglycemic Agents; Iridoids; Kidney; Male; Metabolic Networks and Pathways; Metabolome; Metabolomics; Methylamines; Proton Magnetic Resonance Spectroscopy; Rats; Rats, Sprague-Dawley

Rapidly separating genepin-crosslinked gelatin (RS-GC) microneedles (MNs) mounted on the polyvinyl alcohol (PVA)-coated polylactic acid (PLA) MNs (RS-PGC-MNs) are fabricated, in which GC-MNs deliver insulin within the skin and the PLA supporting array is easily separated by the dissolution of the PVA layer. The release of insulin is controlled by utilizing the virtue of genipin as a crosslinking agent for producing biocompatible GC-MNs. The degree of crosslinking enhances the mechanical strength as well as humidity resistance. The in vitro and in vivo insulin release tests show significant changes in the release rates in the RS-PGC-MNs with different crosslinking degree. The hypoglycemic effect in diabetic mice demonstrate that the higher crosslinking GC-MNs result in characteristic controlled insulin release compared with other treatments and prolonged effectiveness of the RS-PGC-MNs. The proposed RS-PGC-MNs is a promising device for effective use as a noninvasive and painless controlled insulin delivery system.

Topics: Animals; Biocompatible Materials; Diabetes Mellitus, Experimental; Drug Delivery Systems; Gelatin; Humans; Humidity; Insulin; Iridoids; Mice; Needles; Polyesters; Stress, Mechanical

Topics: Antineoplastic Agents, Phytogenic; Biomarkers, Tumor; Carcinoma, Squamous Cell; Caspases; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation; Humans; Iridoids; Lacticaseibacillus casei; Mouth Neoplasms

Iridoid blue-based pigments (IBBP) extract of Genipa americana L. represents a natural alternative additive for food applications and also exerts desirable biological effects on human health. In this study the iridoids present in the extract were identified, the influence of pH and temperature on color difference (ΔE) of IBBP was evaluated using a central composite design (CCD) and finally the antioxidant capacity was monitored before and after its in vitro digestion. Ten glucoside iridoids were detected and the main compounds identified were genipin, genipin 1-β-gentiobioside and geniposide. It was also observed an increase of 17-18% of antioxidant capacity after the in vitro digestion, respectively. Among the conditions tested, the color of extract was more stable at 12-20 °C and low pH (3.0-4.0), suggesting that it is compatible for coloring acidic foods. Finally, the in vitro digestion also increased the antioxidant capacity (ORAC assay) by 39%.

Topics: Antioxidants; Chromatography, High Pressure Liquid; Humans; Hydrogen-Ion Concentration; Iridoids; Pigments, Biological; Plant Extracts; Rubiaceae; Spectrometry, Mass, Electrospray Ionization; Temperature

Studying genipin variable concentrations, treatment durations, and delivery methods as a substance to increase corneal stiffness by inducing corneal collagen cross-linking (CXL).. 100 bovine corneas treated with different genipin concentrations (0.1, 0.5, and 1%) and treatment durations (15 min, 40 min, 2 h, and 3 days) through different delivery methods compared to 10 controls treated with riboflavin/UV. Histology examination, enzymatic digestion with collagenase and thermal differential scanning calorimetry were performed on the different samples.. Bovine corneas soaked in 0.5% genipin morphologically showed 4.7% CXL in comparison to 5.6% in controls (p < 0.05). Corneas treated with topical 0.5% genipin, by a 140-µL drop applied hourly for 2 h, showed 7% corneal CXL. Corneas treated with topical genipin 0.5% for 30 min, 1 and 2 h showed 54 ± 6, 40 ± 7, and 39 ± 9% enzymatic degradation, respectively, in comparison to controls (74%). Corneas treated with 0.5% genipin for 1, 2, and 8 h showed higher thermal denaturation resistance (Td values of 64.9 ± 0.3, 64.7 ± 0.0 and 67.3 ± 0.9), respectively, in comparison to the control group (64.6 ± 0.5) (p < 0.05).. Genipin 0.5%, in a 140-µL drop applied hourly for 2 h, showed better potential to enhance corneal stiffness and stability through inducing CXL.

Topics: Animals; Biomechanical Phenomena; Cattle; Collagen; Cornea; Cross-Linking Reagents; Iridoids; Models, Animal; Time Factors; Ultraviolet Rays

Topics: Acetylation; beta-Glucosidase; Biotransformation; Cells, Immobilized; Chromatography, High Pressure Liquid; Crystallography, X-Ray; Fermentation; Iridoids; Isomerism; Models, Molecular; Protein Conformation; Proton Magnetic Resonance Spectroscopy; Temperature; Trichoderma

BACKGROUND Revascularization is a successful therapeutic strategy for myocardial infarction. However, restoring coronary blood flow can lead to ischemia-reperfusion (I/R) injury. Low-dose 4-hydroxy-2-nonenal (HNE) therapy appears to play a key role in myocardial tolerance to I/R injury. We hypothesized that the positive effects of HNE on myocardial I/R injury may be UCP3-dependent. MATERIAL AND METHODS Adult male wild-type (WT) or UCP3 knockout (UCP3-/-) mice were pre-treated with the UCP inhibitor genipin or saline 1 h before ischemia and underwent 30-min coronary artery ligation followed by 24-h reperfusion. Mice were treated with intravenous HNE (4 mg/kg) or saline 5 min before reperfusion. Echocardiography was conducted to measure left ventricular end-diastolic posterior wall thickness (LVPWd), end-diastolic diameter (LVEDD), and fractional shortening (FS). Infarct size was measured by TTC staining. qRT-PCR and Western blotting were used to assess the expression of UCP3, UCP2, and the apoptosis markers cytochrome C and cleaved caspase-3. RESULTS HNE improved survival at 24 h post-MI in wild-type mice (p<0.05) but not in UCP3-/- mice. HNE preserved LVEDD and FS in WT mice (p<0.05) but not in UCP3-/- mice. HNE reduced infarct size in WT mice (p<0.05) but not in UCP3-/- mice. HNE upregulated UCP3 expression (p<0.05) but did not affect UCP2 expression. HNE reduced apoptosis marker expression in WT mice (p<0.05) but not in UCP3-/- mice. HNE's positive effects were abrogated by genipin in an UCP3-dependent manner. CONCLUSIONS Low-dose HNE reperfusion therapy attenuates murine myocardial I/R injury in an UCP3-dependent manner. These effects are abrogated by genipin in an UCP3-dependent manner.

Topics: Aldehydes; Animals; Apoptosis; Coronary Vessels; Heart; Iridoids; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Myocardial Infarction; Myocardial Reperfusion; Myocardial Reperfusion Injury; Myocardium; Reperfusion Injury; Uncoupling Protein 3

In this study, central composite design was utilized for the optimization of genipin cross-linked chitosan/Eudragit®-L 100 interpenetrating hydrogel network films fabricated through solvent evaporation technique.. Hydrogel formulations were studied using response surface methodology; regression analysis and the surface plots were used to evaluate the effect of variables on T50% (the time for 50% of drug release) and dynamic swelling with optimum formulation selection. Initial burst release of drug was observed from the formulated hydrogels during the first 2 hours of dissolution at simulated gastric pH 1.2 and then slow release during the next 10 hours in the simulated intestinal fluid at pH 7.4. Different polymer ratios in formulation showed significant influence on T50% and dynamic swelling of hydrogel. The highest T50% was observed at 9.89 hour and dynamic swelling at 7.86 h.. It was observed that by changing the polymer ratio with cross-linker, release rate of metformin could be modified. Cross-linker also affects drug release rate, i.e. the release rate is decreased with the increase in its concentration. The physical state of hydrogel was investigated by scanning electron microscope.. It indicated the uniform distribution of drug in hydrogel matrix system. Moreover, the presence of hydrogen and ionic bonds between polymers and crosslinking agent formed interpenetrating hydrogel network, likely responsible for increased value of T50%, as confirmed by FTIR. Acute oral toxicity study was performed to investigate the toxic effect of crosslinking agent and polymer used in formulations.

Topics: Acrylates; Animals; Chitosan; Cross-Linking Reagents; Drug Liberation; Hydrogel, Polyethylene Glycol Dimethacrylate; Hydrogen-Ion Concentration; Iridoids; Male; Metformin; Particle Size; Polymers; Rabbits; Regression Analysis; Surface Properties

Genipin is a natural plant-derived compound that covalently cross-links biopolymers into lattice networks with good biocompatibility, controllable swelling, and mechanical properties. This protocol describes the genipin cross-linking of elastic proteins, including tropoelastin and elastin-based polypeptides, through steps of elastin phase-separation upon addition of salt and heat, centrifugation to rapidly concentrate the dense protein phase, and incubation. This method is applicable for the fabrication of elastic materials suitable for use as scaffolds for biomedical applications.

Topics: Cross-Linking Reagents; Elastin; Iridoids; Molecular Structure; Peptides

Gardenia blue is widely used in Eastern Asia as a natural food colorant. To evaluate the genotoxic potential of gardenia blue, as well as genipin, the natural starting material from which it is produced, a GLP-compliant test battery was conducted according to OECD guidelines. No evidence of mutagenicity of gardenia blue was detected in a 5-strain bacterial reverse mutation assay, with or without metabolic activation; an equivocal response for genipin occurred in S. typhimurium TA97a without metabolic activation. In in vitro micronucleus and chromosome aberration assays, genipin tested positive under some test conditions; however, gardenia blue tested negative in both assays. In combined micronucleus/comet assays conducted in male and female B6C3F1 mice, exposure to genipin at doses reaching maximal toxicity (74 and 222 mg/kg bw/day for males and females, respectively) or gardenia blue tested up to the limit dose (2000 mg/kg bw/day) did not induce micronuclei in peripheral blood or DNA damage in several examined tissues. Modified ("reverse") comet assays showed no evidence of DNA crosslinking potential of either genipin, known to form crosslinks with other macromolecules, or gardenia blue. Our results indicate that consumption of gardenia blue in food products does not pose a significant genotoxic concern for humans.

Topics: Animals; Chromosome Aberrations; Cisplatin; Comet Assay; Dose-Response Relationship, Drug; Female; Glucosides; Iridoids; Liver; Male; Mice; Micronucleus Tests; Mutagens; Salmonella typhimurium

The goal of this study was to improve the chemical stability of menhaden oil and control the lipolysis in emulsions with whey protein during in vitro digestion through EGCG conjugation and genipin-mediated interfacial cross-linking (CL). WPI-EGCG conjugate was successfully synthesized, confirmed by SDS-PAGE, ESI-MS, and phenolic group quantifications (125.3 mg/g), and characterized with far UV CD and ATR-FTIR. Emulsion particle diameter with WPI-EGCG is lower than with WPI. Compared to the native emulsion, WPI CL increased particle diameter and physical stability. Higher oxidative stability was observed for emulsions stabilized with WPI-EGCG conjugate than that with interfacial cross-linking due to the great antioxidant activity. Whereas, WPI CL is more effective than WPI-EGCG conjugate in hindering the rate and extent of lipolysis. The combination of EGCG conjugation and interfacial CL showed both the highest protection of menhaden oil against degradation and highest inhibition on the rate and extent of lipolysis of menhaden oil.

Topics: Antioxidants; Catechin; Circular Dichroism; Digestion; Electrophoresis, Polyacrylamide Gel; Emulsions; Fish Oils; Iridoids; Lipolysis; Oxidation-Reduction; Spectrometry, Mass, Electrospray Ionization; Spectroscopy, Fourier Transform Infrared; Whey Proteins

Studies on synthesis, physico-chemical and biological properties of novel biomimetic materials, potentially useful as injectable hydrogels are presented. These materials are in situ prepared chemically crosslinked collagen/chitosan/hyaluronic acid-based hydrogels exhibiting potential for tissue regeneration. Optimization of hydrogels involved testing the effect of various concentration of crosslinking agent (genipin) as well as different ratios of biopolymers used on their properties. The changes in the content of hyaluronic acid and in the genipin concentration used have been shown to be crucial. Employing the highest concentration of crosslinking agent studied (20 mM) the hydrogels of compact structure, characterized by good mechanical properties and prolonged degradation profile can be obtained. Changing the HA content in sol mixture the hydrogel of various wettability; more or less hydrophilic when compared to pure collagen/chitosan hydrogels can be fabricated. The in vitro cell culture study has shown that the surface of the prepared materials ensures suitable biocompatibility. These hydrogels can support the proliferation and adhesion of MG-63 cell line as it was demonstrated using Alamar Blue assay and SEM observations. It is believed that the collagen/chitosan/hyaluronic acid hydrogels crosslinked with genipin are particularly promising materials for bone regeneration procedures, especially attractive for regeneration of small bone losses. This is the first paper in the litearature presenting results of studies on that type of biopolymeric injectable hydrogels chemically crosslinked with genipin.

Topics: Cell Adhesion; Cell Proliferation; Cell Survival; Chitosan; Collagen; Cross-Linking Reagents; Dose-Response Relationship, Drug; Humans; Hyaluronic Acid; Hydrogels; Iridoids; Microscopy, Electron, Scanning; Particle Size; Structure-Activity Relationship; Surface Properties; Tissue Engineering; Tumor Cells, Cultured

Chinese giant salamander (CGS) has high medicinal value and long history of clinical use in ancient China. In this study, CGS skin (CGSS) collagen was extracted and purified to prepare collagen sponge by freeze-drying. TEMPO oxidized microfibrillated cellulose (TEMPO-MFC) and genipin were adopted to improve the mechanical properties of collagen sponge. The hygroscopicity, porosity, mechanical properties, hemostatic performance, morphology, and biodegradability of the resultant sponges were investigated in detail. The results indicated that CGSS collagen was type I collagen with intact triple-helical structure, and the prepared sponge had porous structure and excellent hemostatic performance with procoagulant ratio of 53.28%. However, the CGSS collagen sponge showed low tensile strength (TS) of 98.80 KPa, compression strength (CS) of 1.48 KPa, and elongation at break (E) of 4.72%. Incorporating 2.5% TEMPO-MFC into the native CGSS collagen sponge resulted in an increase of 188.26% in TS to 284.80 KPa, 166.89% in CS to 3.95 KPa, and 73.52% in E to 8.19%. The improvements were attributed to the physical filling of TEMPO-MFC in cavity and cavity wall of collagen sponge and the stable chemical linkage between carboxyl of TEMPO-MFC and amino group of collagen which effectively improved the toughening of sponge and formed good interface bonding, respectively. Subsequent 0.3% genipin treatment further improved the TS to 605.00 KPa and the CS to 8.66 KPa as a result of crosslinking reaction. Moreover, the composite reinforcing also improved the anti-degradation ability and procoagulant ratio of collagen sponge. All results suggested that the TEMPO-MFC toughened and genipin crosslinked CGSS composite collagen sponge is a promising rapid hemostatic material with high-strength and can be applicated in biomedical field.

Topics: Animals; Biocompatible Materials; Collagen Type I; Cross-Linking Reagents; Hemostatics; Humans; Iridoids; Porosity; Skin; Surface Properties; Urodela

The recently developed 3D bioprinting technology has greatly improved the ability to generate biomimetic tissues that are structurally and functionally relevant to their human counterparts. The selection of proper biomaterials as the bioinks is a key step toward successful bioprinting. For example, viscosity of a bioink is an important rheological parameter to determine the flexibility in deposition of free-standing structures and the maintenance of architectural integrity following bioprinting. This requirement, however, has greatly limited the selection of bioinks, especially for those naturally derived due to their commonly low mechanical properties. Here the generalization of a mechanism for extrusion bioprinting of bio-macromolecular components, mainly focusing on collagen and its derivatives including gelatin and gelatin methacryloyl, is reported. Specifically, a templating strategy is adopted using a composite bioink containing both the desired bio-macromolecular component and a polysaccharide alginate. The physically crosslinkable alginate component serves as the temporal structural support to stabilize the shape of the construct during bioprinting; upon subsequent chemical or physical crosslinking of the bio-macromolecular component, alginate can be selectively removed to leave only the desired bio-macromolecule. It is anticipated that this strategy is general, and can be readily expanded for use of a wide variety of other bio-macromolecular bioinks.

Topics: Alginates; Biocompatible Materials; Bioprinting; Breast Neoplasms; Cell Line, Tumor; Collagen Type I; Cross-Linking Reagents; Female; Humans; Iridoids; Lab-On-A-Chip Devices; Macromolecular Substances; Viscosity

Cross-linked gelatin microgels were formed in gelatin-in-maltodextrin water-in-water (W/W) emulsions and evaluated as carriers of the enzyme β-galactosidase (β-Gal). The phase behavior of aqueous gelatin/maltodextrin mixtures was studied in detail, focusing on the multiphase region of the phase diagram that is constituted by three equilibrium phases: two immiscible aqueous phases plus one solid phase. The solid phase was analyzed by Raman spectroscopy, and water-in-water emulsions were formed within the multiphase region. Gelation of the dispersed gelatin droplets was induced by cooling and cross-linking with genipin, which is a natural cross-linking reagent of low toxicity, leading to the formation of gelatin microgel particles. These microgels were studied as delivery vehicles for the enzyme lactase, used as a model active component. Various incorporation methods of the enzyme were tested, to achieve highest encapsulation yield and activity recovery. Microgel particles, loaded with the enzyme, can be freeze-dried, and the enzyme remained active after a complete cycle of freeze-drying and rehydration. The stability of the enzyme at 37 °C under gastric and neutral pH conditions was tested and led to the conclusion that the cross-linked microgels could be suitable for use in food-industry, where β-Gal carriers are of interest for hydrolyzing lactose in milk products.

Topics: beta-Galactosidase; Emulsions; Freeze Drying; Gelatin; Gels; Hydrogen-Ion Concentration; Iridoids; Particle Size; Phase Transition; Polysaccharides; Water

Continuous contraction of 3D skin equivalents in construction and use restricts their applications in clinical and pharmaceutical practices. So far, no effective method has been developed to inhibit such contraction. Hence, low cytotoxic cross-linkers, 1-ethyl-3-3-dimethylaminopropylcarbodiimide hydrochloride (EDC) and genipin, are investigated to reduce the contraction in this study. As found, both genipin and EDC at 0.2 and 0.4 mmol/L are nontoxic to collagen-entrapped fibroblasts and upregulate the extracellular matrix expression of fibroblasts in cross-linked collagen. Particularly, collagen cross-linking by intermediate concentrations of genipin, specifically 0.4 mmol/L, greatly reduces the contraction of 3D skin equivalents from 87% to 28% (n = 9, P < 0.05), while the collagen after EDC cross-linking at 0.4 mmol/L still presented severe contraction of 64% over a 21-day follow-up period. The inhibited contraction might relate to the increased gel stiffness and slowed collagen degradation. Moreover, the genipin cross-linking does not impair the formation of epidermal layers and improves the epidermal-dermal junction of skin equivalents as well. In this regard, genipin cross-linking might facilitate the applications of 3D skin equivalents in clinical practices and pharmacology testing.

Topics: Biocompatible Materials; Cell Survival; Cells, Cultured; Collagen; Cross-Linking Reagents; Fibroblasts; Gels; Humans; Imides; Iridoids; Keratinocytes; Propylamines; Skin, Artificial; Tissue Engineering

Organic photothermal sensitizers, such as indocyanine green (ICG), have been widely explored in photothermal therapy as a good substitute for inorganic materials owing to their advantageous biosafety and strong absorption in the near-infrared region. However, their intrinsic low stability and rapid clearance from the body requires further modification for efficient therapeutic application. In this work, we employed a covalent assembly strategy by covalently cross-linking genipin and a functional bola-amino acid to fabricate stable and degradable nanoparticles capable of loading ICG. The covalent assembly introduced strong covalent interactions in the assembly system together with functional linkers, which led to both enhanced stability and extended functionalities. This is distinguished from the conventional supramolecular strategy that relies only on weak noncovalent interactions. The functional building unit, consisting of phenylalanine and the disulfide bond, enables both good assembly and controllable degradation owing to the disulfide bond that responds to glutathione. The assembled nanoparticles show high stability, negligible toxicity, and considerable biodegradability. After loading ICG, the ICG-loaded nanoparticles possessed high photothermal conversion efficiency, and showed an enhanced photothermal effect in the near-infrared region. This covalent assembly strategy could be extended to various biomolecules containing a primary amino group for the fabrication of efficient and multifunctional nanomaterials used in biomedical applications.

Topics: Amino Acids; Apoptosis; Drug Carriers; Glutathione; Humans; Indocyanine Green; Iridoids; Light; MCF-7 Cells; Microscopy, Electron, Transmission; Nanoparticles; Neoplasms; Photosensitizing Agents; Phototherapy; Spectrometry, Fluorescence; Temperature

Amniotic membrane has the potential to be used as scaffold in various tissue engineering applications. However, increasing its biostability at the same time maintaining its biocompatibility is important to enhance its usage as a scaffold. This studied characteristics genipin-crosslinked amniotic membrane as a bioscaffold. Redundant human amniotic membranes (HAM) divided into native (nAM), decellularized (dAM) and genipin-crosslinked (clAM) groups. The dAM and clAM group were decellularized using thermolysin (TL) and sodium hydroxide (NaOH) solution. Next, clAM group was crosslinked with 0.5% and 1.0% (w/v) genipin. The HAM was then studied for in vitro degradation, percentage of swelling, optical clarity, ultrastructure and mechanical strength. Meanwhile, fibroblasts isolated from nasal turbinates were then seeded onto nAM, dAM and clAM for biocompatibility studies. clAM had the slowest degradation rate and were still morphologically intact after 30 days of incubation in 0.01% collagenase type 1 solution. The dAM had a significantly highest percentage of swelling than other groups (p < 0.05). Besides, the dAM retained the collagen content at similar level of nAM. Although the dAM had highest mechanical strength compared to the rest of the groups, the differences were statistically insignificant. Cell attachment on dAM and 0.5% clAM was higher compared to that on nAM and 1.0% clAM. In conclusion, clAM have better biostability and biocompatibility compared to the nAM and dAM. Together with other suitable characteristics of the clAM such as percentage of swelling, structural integrity and ECM content, clAM is suitable as scaffold for various tissue engineering applications.

Topics: Amnion; Biocompatible Materials; Cells, Cultured; Collagen; Collagenases; Cross-Linking Reagents; Extracellular Matrix Proteins; Female; Fibroblasts; Humans; Iridoids; Pregnancy; Tissue Engineering; Tissue Scaffolds; Turbinates

Lung-targeted genipin-crosslinked deacetylated chitosan (GEN-CS)/isoniazid (INH)/rifampin (RMP) nanogel particles (NGPs) were prepared as a treatment for tuberculosis caused by multidrug-resistant Mycobacterium tuberculosis (MTB) to surmount the undesirable side effects and decrease the cytotoxicity of INH and RMP when being against MTB. The size, morphology, in vitro release property, long-term antibacterial performance, stability, in vitro cytotoxicity, in vivo toxicity, and in vivo release property of GEN-CS/INH/RMP NGPs inhalation powder were investigated. The results showed that the GEN-CS/INH/RMP NGPs inhalation powder exhibited extended antibacterial activity because of its long-term release of INH and RMP. A simplex GEN-CS/INH/RMP NGPs pulmonary dose led to the therapeutic drug concentration of 40%-60% in lung and other organs (＜5%) for 24 h. Furthermore, this GEN-CS/INH/RMP NGPs lyophilized inhalation powder displayed lung-targeted property and lower in vivo toxicity. These results suggested that this GEN-CS/INH/RMP NGPs inhalation powder would be a more useful dosage form than separate dose of INH or RMP for MTB.

Topics: Anti-Bacterial Agents; Chitosan; Cross-Linking Reagents; Drug Delivery Systems; Drug Resistance, Multiple, Bacterial; Gels; Iridoids; Isoniazid; Lung; Microbial Sensitivity Tests; Mycobacterium tuberculosis; Nanoparticles; Rifampin

A novel chitosan-based nanoparticle (G-C-Q NPs), which simultaneously loaded catechin and quercetin, was synthesized through ionic gelation reaction between chitosan and sodium tripolyphosphate and subsequent modification by genipin. Chemical structure, size distribution, morphology, entrapment efficiency, in vitro release behavior, antioxidant and antibacterial effects of G-C-Q NPs were investigated. Results showed that G-C-Q NPs were dispersed as an ellipsoidal shape with an average diameter of 180.4 nm and a zeta potential of 31.79 ± 1.28 mV. Entrapment efficiency of catechin and quercetin reached 76.35 ± 1.37% and 52.23 ± 2.45%, respectively. In vitro release study showed that both catechin and quercetin had a sustainable release from the G-C-Q NPs. G-C-Q NPs had higher or comparable potency in scavenging DPPH, ABTS

Topics: Anti-Bacterial Agents; Antioxidants; Bacillus subtilis; Catechin; Chelating Agents; Chitosan; Escherichia coli; Iridoids; Nanoparticles; Quercetin; Staphylococcus aureus

Myopia has become a global public health problem, particularly in East Asia where myopic retinopathy has become one of the leading causes of blindness and visual impairment in the elderly population. The purpose of this study was to evaluate the efficacy of posterior scleral contraction/reinforcement (PSCR) surgery on controlling the progressive elongation of axial length of highly myopic eyes in young patients.. This is a prospective self-controlled interventional case series. Forty young patients (<18-years old) with progressive high myopia received PSCR with a genipin-crosslinked donor scleral strip for one eye and the fellow eye served as concurrent control without surgery. The main outcome measurement was the change of axial length over 2 to 3 years of follow-up.. Immediately after the surgery, axial length was shortened and subsequently increased by 0.32 mm over the follow-up period. In contrast, axial length of the fellow eyes increased by 0.82 mm over the same period (P < 0.001, paired t-test). PSCR delayed axial elongation in eyes with or without staphyloma. No significant change of visual acuity, cornea refractive power, or retina thickness was noted between the surgery and fellow eyes. None of the patients lost visual acuity compared with the baseline. The procedure was well tolerated with only temporary corneal refractive axis shifts that recovered by the 6-month postsurgical visit.. PSCR with genipin-crosslinked sclera is safe and effective to restrain eye globe elongation in young patients within a 2- to 3-year follow-up period.

Topics: Adolescent; Aged; Axial Length, Eye; Child; Child, Preschool; Cholagogues and Choleretics; Cross-Linking Reagents; Female; Humans; Intraocular Pressure; Iridoids; Male; Myopia, Degenerative; Ophthalmologic Surgical Procedures; Prospective Studies; Retinal Detachment; Sclera; Scleral Diseases; Tensile Strength; Tissue Donors; Tomography, Optical Coherence; Visual Acuity

A new approach for ionic liquid based enzyme-assisted extraction coupled with in-situ hydrolysis (ILEIH) of geniposide from Eucommia ulmoides Olive barks is presented, in which enzymatic hydrolysis is used in an ionic liquid aqueous medium to prepare genipin. The method relied on the use of single cellulase to the extract and hydrolyze geniposide, which is performed continuously in the same system; genipin is easy in preparation with exempting the isolation and purification of geniposide. The mechanism of ILEIH procedure was discussed in detail to illustrate the advantage of ILEIH in the extraction process. 0.5 mol/L [C6mim]Cl aqueous solution was selected as extraction solvent. The optimum conditions of 140 min treatment time, 19.81 mL/g liquid-solid ratio, 5.15 mg/mL enzyme concentration and 5.0 pH value for the ILEIH process were obtained after investigating by single factor experiments and Box-Benhnken design in terms of the genipin increment. And the first-order kinetic model based on β-glucosidase in the three reaction medium were established to study their impacts on the reaction rate. The proposed ILEIH method was validated by stability, repeatability, and recovery experiments and shows reliable data in the extraction and hydrolysis process. Therefore, this proposed approach is promising for the in-situ production of genipin and should be potentially applied to the obtaining of other active aglycons.

Topics: beta-Glucosidase; Cellulase; Chemistry Techniques, Analytical; Eucommiaceae; Hydrolysis; Ionic Liquids; Iridoids

With the rapid development of biomimetic polymers for cell-based assays and tissue engineering, crosslinking electrospun nanofibrous biopolymer constructs is of great importance for achieving sustainable and efficient three-dimensional scaffold constructs. Uncrosslinked electrospun gelatin nanofibrous constructs immediately and completely dissolved in aqueous solutions due to their aqueous solubility and poor storage stability. Here, a novel and versatile approach for the fabrication and crosslinking of electrospun gelatin construct with tunable porosity and high aspect ratio nanofibers is presented. Uncrosslinked electrospun gelatin/genipin nanofibrous and pure gelatin nanofibrous constructs exhibited smooth surfaces that were well-defined, with a diameter in the range of 448 ± 364 nm and 257 ± 57 nm, respectively. Dehydrothermal, genipin-EDC/Sulfo-NHS, and EDC/Sulfo-NHS crosslinking approaches were examined to achieve insoluble gelatin nanofibrous constructs that were suitable for cell-based assays. Mechanical characterization demonstrated that the pure gelatin nanofibrous construct crosslinked via EDC/Sulfo-NHS exhibited an increased mechanical strength and stiffness and showed no dissolution in aqueous solutions and retained its fiber morphology. An excellent 1 month storage stability was demonstrated at 22, 4, -20, and -80°C (dehydrated) and at 4°C (hydrated). The as-crosslinked gelatin nanofibrous construct was highly biocompatible (90% cell viability), as demonstrated by the promoted proliferation of PC12 cells.

Topics: Animals; Biocompatible Materials; Cell Proliferation; Cell Survival; Cross-Linking Reagents; Drug Stability; Drug Storage; Gelatin; Iridoids; Materials Testing; Nanofibers; Particle Size; PC12 Cells; Porosity; Rats; Succinimides; Surface Properties; Tensile Strength; Tissue Engineering; Tissue Scaffolds

Liver damage is a typical manifestation of nonalcoholic fatty liver disease (NAFLD). It originates from excessive fat accumulation, leading to hepatocyte death, inflammation, and fibrosis. Nonalcoholic steatohepatitis (NASH) is a type of NAFLD with a prevalence of 49% in morbidly obese patients. Pyroptosis plays an important role in the development of NASH; thus, it is important to elucidate the effect of lipid accumulation on pyroptosis. Genipin (GNP), a natural water-soluble cross-linking agent, has hepatoprotective effects and decreases lipid accumulation in the liver; however, the mechanisms underlying these effects are unknown.. In this study, qPCR and Western blot were used to examine pyroptotic gene expression in high-fat diet (HFD) induced obese mice and free fatty acids (FFAs) treated hepatocytes. At the same time, relative lactate dehydrogenase (LDH) release and Hoechst & propidium iodide (PI) staining were done to verify cell death. To explore the molecular mechanism, cell transfection were constructed with siRNA or plasmid to obtain knockdown or overexpression hepatocytes.. We found that HFD-fed mice and FFAs-treated hepatocytes had obvious pyroptosis, and addition of GNP reversed liver damage and inhibited pyroptosis both in vitro and in vivo. Besides, UCP2 knockdown cells showed suppressed FFAs-mediated pyroptosis, as determined by decreased pyroptotic gene expression, reduced lactate dehydrogenase (LDH) release, and reduced cell death. Consistent with this, cells transfected with UCP2 had upregulated pyroptotic gene expression, increased LDH release, and increased cell death.. GNP reverses HFD-induced liver damage and inhibits UCP2-mediated pyroptosis. Thus, GNP may serve as a potential therapeutic candidate for NAFLD.

Topics: Animals; Aspartate Aminotransferases; Cell Line; Cholagogues and Choleretics; Diet, High-Fat; Fatty Acids, Nonesterified; Hepatocytes; Iridoids; L-Lactate Dehydrogenase; Liver; Male; Mice; Mice, Inbred C57BL; Pyroptosis; RNA Interference; RNA, Small Interfering; Uncoupling Protein 2

Here, we have reported the influence of MMT and genipin in releasing curcumin from the Genipin crosslinked Chitosan/MMT nanoparticles, prepared by ionic gelation method. The nanoparticles were characterised using Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Diffractometry (XRD), Scanning Electron Microscopy (SEM), and Transmission Electron Microscopy (TEM). Zeta potential and average diameter of the nanoparticles were found in the range 32-47 mV and 430-560 nm. Swelling and release of curcumin from the nanoparticles increased with the decrease in pH of the medium, MMT, and genipin content. Curcumin released from the nanoparticles reduced the viability of MCF-7 and Hep G2 cells as compared to untreated cells. The nanoparticles increased the level of reduced glutathione (GSH), superoxide dismutase (SOD), and catalase level in human PBMCs and decreased the level of Lipid peroxidation suggesting an enhanced protection against cellular damage. Lower pH and higher MMT concentration in the nanoparticles improved the mucoadhesive properties.

Topics: Antineoplastic Agents; Bentonite; Cell Survival; Chitosan; Cross-Linking Reagents; Curcumin; Delayed-Action Preparations; Drug Liberation; Hep G2 Cells; Humans; Iridoids; MCF-7 Cells; Neoplasms

The wound healing for tympanic membrane (TM) perforations is one of the most common clinical treatment in otomicrosurgery. Recently, tissue-engineered composites and grafts, also new designs of biomaterials are applied to the management of TM perforation. In this work, a series of gelatin/genipin nanofibrous films were prepared as a patch for repairing TM perforation. Gelatin, a type of biomaterial with excellent electrospinning performance, has been used for preparing the nanofibers. The genipin, as a crosslinking agent, has been blended into the gelatin nanofibers. The reaction between gelatin and genipin engender suitable tensile strength and water-resistance for TM patch. The survival rate of human umbilical vein endothelial cells and fibroblasts demonstrated that the gelatin/genipin nanofiber scaffolds had good biocompatibility, which indicated the genipin was a kind of effective and nontoxic crosslinking agent for improving the mechanical property and water-resistance of gelatin films. In short, our work provides a novel macromolecular material with good mechanical properties, water-tolerance and excellent biocompatibility which could be used as a potential patch for TM repair.

Topics: Biocompatible Materials; Cell Survival; Cross-Linking Reagents; Gelatin; Human Umbilical Vein Endothelial Cells; Humans; Iridoids; Mechanical Phenomena; Nanofibers; Tissue Scaffolds; Tympanic Membrane; Tympanic Membrane Perforation

To demonstrate the function of uncoupling protein 2 (UCP2) in the regulation of human spermatozoa motility.. Semen samples were collected from donors with either normal spermatozoa motility (normospermia [NS]) or poor spermatozoa motility (asthenospermia [AS]). UCP2 protein in spermatozoawas quantified by Western blotting. The level of mitochondrial reactive oxygen species (mROS) was evaluated by MitoSOX Red. The activity of mitochondrial membrane potential (MMP) in spermatozoa was evaluated by a JC-1 assay and the ATP level was monitored by a luciferin-luciferase assay.. UCP2 was expressed in both NS and AS groups, with the former exhibiting a higher level than the latter. Immunofluorescence analysis shows that UCP2 is mainly located at the mid-region of human spermatozoa. The inhibition of UCP2 by a highly selective inhibitor, Genipin, results in not only impaired spermatozoa mobility (P<.05) but also an elevated level of mROS (P<.05), suggesting that UCP2 is involved in the maintenance of the spermatozoa mobility, which probably is achieved by promoting mROS elimination. Furthermore, H2O2 treatment of spermatozoa increases the mROS level coupled with the loss of spermatozoa mobility. Unexpectedly, this treatment also has a positive impact on the expression of UCP2 within a certain range of supplemental H2O2, indicating the moderate mROS level possibly serves as a feedback signal to stimulate the expression of UCP2. Finally, the treatment of spermatozoa by an ROS scavenger, N-acetyl-l-cysteine (NAC),decreases the level of mROS and increases the curvilinear velocity (VCL) of spermatozoa, but the UCP2 level is not affected.. These results suggest an UCP2-mROS-motility regulatory system exists for maintaining spermatozoa mobility in humans. In such a system, UCP2 fulfills its function by promoting mROS elimination, and slightly over-produced mROS in turn serves as a signal to stimulates the expression of UCP2. This regulatory system represents a new potential target for the discovery of novel pharmaceuticals for the treatment of patients with low spermatozoa motility.

Topics: Acetylcysteine; Humans; Hydrogen Peroxide; Iridoids; Male; Membrane Potential, Mitochondrial; Mitochondria; Reactive Oxygen Species; Sperm Motility; Spermatozoa; Uncoupling Protein 2

Self-assembling peptides (SAPs) are synthetic bioinspired biomaterials that can be feasibly multi-functionalized for applications in surgery, drug delivery, optics and tissue engineering (TE). Despite their promising biocompatibility and biomimetic properties, they have never been considered real competitors of polymers and/or cross-linked extracellular matrix (ECM) natural proteins. Indeed, synthetic SAP-made hydrogels usually feature modest mechanical properties, limiting their potential applications, due to the transient non-covalent interactions involved in the self-assembling phenomenon. Cross-linked SAP-hydrogels have been recently introduced to bridge this gap, but several questions remain open. New strategies leading to stiffer gels of SAPs may allow for a full exploitation of the SAP technology in TE and beyond. We have developed and characterized a genipin cross-linking strategy significantly increasing the stiffness and resiliency of FAQ(LDLK)3, a functionalized SAP already used for nervous cell cultures. We characterized different protocols of cross-linking, analyzing their dose and time-dependent efficiency, influencing stiffness, bioabsorption time and molecular arrangements. We choose the best developed protocol to electrospin into nanofibers, for the first time, self-standing, water-stable and flexible fibrous mats and micro-channels entirely made of SAPs. This work may open the door to the development and tailoring of bioprostheses entirely made of SAPs for different TE applications.

Topics: Biocompatible Materials; Cell Differentiation; Cell Line; Cell Proliferation; Cross-Linking Reagents; Humans; Hydrogels; Iridoids; Models, Molecular; Nanofibers; Neural Stem Cells; Peptides; Tissue Engineering; Tissue Scaffolds

Metastasis of hepatocellular carcinoma (HCC) is usually unrecognized before any pathological examination, resulting in time-taking treatment and poor prognosis. As a consequence, HCC patients usually show symptoms of depression. In order to suppress such psychiatric disorders and to facilitate better treatment outcome, antidepressants are prescribed. Up to present, information about the effect of antidepressants on HCC is still lacking. Therefore, we chose fluoxetine (FXT), one of the top five psychiatric prescriptions in the United States, together with the HepG2 cell model to explore its effect on HCC. Our study found that FXT (5 µM) increased the migratory distance of HepG2 cells by a factor of nearly 1.7 compared to control. In addition, our study also investigated the effect of genipin (GNP), which is an active compound from Gardenia jasminoides Ellis fruit (family Rubiaceae), on the FXT-induced HepG2 cells. Our study found that 30 and 60 µM GNP reduced the migratory distance by 42% and 74% respectively, compared to FXT treatment alone. Furthermore, we also found that FXT upregulated matrix metalloproteinases (MMPs) genes, increased the protein expression of MMPs, urokinase-type plasminogen activator (uPA), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), activator protein 1 (AP-1), phosphorylated mitogen-activated protein kinase (P-p38), phosphorylated protein kinase B (P-Akt), downregulated tissue inhibitor metalloproteinases (TIMPs) genes and decreased the TIMPs proteins expression whereas, GNP fully counteracted the action of FXT. Conclusively, this study has provided valuable information regarding the possible molecular mechanisms through which FXT affects the metastatic invasiveness of HepG2 cells and evidences to support that GNP counteracts such effect via the same molecular mechanisms.

Topics: Carcinoma, Hepatocellular; Fluoxetine; Hep G2 Cells; Humans; Iridoids; Liver Neoplasms; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Transcription Factor AP-1

As biocompatible matrices, porcine dermal scaffolds have limited application in tissue engineering due to rapid degradation following implantation. This study compared the physical, chemical and biomechanical changes that occurred when genipin and quercetin were used to crosslink dermal scaffolds and to determine whether quercetin could be used as an alternative to genipin. Physicochemical changes in the collagen were assessed using spectroscopic methods [X-ray diffraction analysis (XRD) and nuclear magnetic resonance (NMR) analysis]. The crosslinking reaction was evaluated by quantification of amino acids and the degree of this reaction by ninhydrin assay. Because the mechanical behaviour of the collagen matrices is highly influenced by crosslinking, the tensile strength of both sets of scaffolds was evaluated. The highest mechanical strength, stiffness, degree of crosslinking and changes in the packing features of collagen (measured by XRD) were achieved using genipin. Some of the results found in the quercetin-crosslinked scaffolds were possibly due to hydration and dehydration effects elicited by the solvents (phosphate-buffered saline or ethanol), as seen in the NMR results. In the quercetin-ethanol-crosslinked scaffolds, possible reorientation of the amino groups of the collagen molecule may have taken place. Therefore, depending on their proximity to the crosslinking reagent, different types and numbers of interactions may have occurred, inducing a higher crosslinking degree (as evidenced by the ninhydrin assay) and reduction in the free amino acids after reaction. Both crosslinking agents and solvents interfere in the physicochemical properties of collagen thereby inducing variations in the matrix structure. Quercetin-crosslinked scaffolds may have broader clinical application where a lower degree of crosslinking and stiffness is required. Copyright © 2016 John Wiley & Sons, Ltd.

Topics: Animals; Cross-Linking Reagents; Iridoids; Prosthesis Implantation; Quercetin; Stress, Mechanical; Swine; Tissue Scaffolds

The concept of scleral stiffening therapies has emerged as a novel theoretical approach for treating the ocular disorders glaucoma and myopia. Deformation of specific regions of the posterior eye is innately involved in the pathophysiology of these diseases, and thus targeted scleral stiffening could resist these changes and slow or prevent progression of these diseases. Here, we present the first systematic screen and direct comparison of the stiffening effect of small molecule collagen cross-linking agents in the posterior globe, namely using glyceraldehyde, genipin and methylglyoxal (also called pyruvaldehyde). To establish a dose-response relationship, we used inflation testing to simulate the effects of increasing intraocular pressure in freshly harvested rat eyes stiffened with multiple concentrations of each agent. We used digital image correlation to compute the mechanical strain in the tissue as a metric of stiffness, using a novel treatment paradigm for screening relative stiffening by incubating half of each eye in cross-linker and using the opposite half as an internal control. We identified the doses necessary to increase stiffness by approximately 100%, namely 30 mM for glyceraldehyde, 1 mM for genipin and 7 mM for methylglyoxal, and we also identified the range of stiffening it was possible to achieve with such agents. Such findings will inform development of

Topics: Animals; Collagen; Cross-Linking Reagents; Dose-Response Relationship, Drug; Glyceraldehyde; Intraocular Pressure; Iridoids; Pyruvaldehyde; Rats; Sclera

Microspheres can be regarded as a suitable platform for the development of ad hoc drug delivery systems, since the targeted release of a therapeutic agent can effectively contribute to support and improve a pharmacological protocol. However, several crucial factors related to the selection of materials, drugs and fabrication techniques should be critically analyzed in order to enhance the expected performance. Dealing with highly compatible materials, e.g. naturally-derived polymers and "green" reagents, can be a valid approach. For this aim, gelatin, chitosan and blend microspheres were produced by emulsion technique simply using distilled water and olive oil. Necessarily, due to the intrinsic instability of the selected materials in aqueous environment, microspheres were cross-linked with genipin, an extremely low cytotoxic agent, at three different concentration (i.e., 0.1, 0.5, 1% w/v). Collected microspheres were then loaded with methylene blue (MB), as drug model. Morphological analysis revealed homogeneous microspheres characterized by an average diameter comprised in the range 42-54μm. In vitro MB temporal delivery was assessed until complete release, which occurred in about 3days for gelatin and 30days for chitosan microspheres. Nanoindentation analysis was performed to evaluate how polymers and genipin influenced the mechanical properties of microspheres. Finally, the effect of released MB was investigated by means of chicken embryo chorioallantoic membrane assay, highlighting anti-angiogenic properties for gelatin differently from chitosan and blend microspheres.

Topics: Animals; Chick Embryo; Chickens; Chitosan; Drug Delivery Systems; Gelatin; Iridoids; Methylene Blue; Microspheres

Collagen cross-linking is an attractive therapeutic route aimed at supplementing natural collagen stabilisation. In this study the toxicity of the cross-linker genipin (GP) was examined in avascular (tendon) and vascular (dermis) tissue.. High doses of GP were injected intratendinously into three yearling horses and evaluated at various time points up to 30 days. A second group of three yearlings were injected into the dermis and evaluated at various time points up to 1 year. Metrics used included lameness, circumferential swelling, ultrasound evaluation, microscopic morphology, collagen production and systemic effect on blood parameters.. The tendon injection sites exhibited mild lameness and swelling with no apparent systemic toxicity or stabilisation defects. Treated tendons exhibited increased linear collagen microscopically. Dermal injections showed similar results, with mild swelling at the injection site. Microscopic morphology resulted in a decrease in dermal collagen at 30 days post-injection. Dermis injected at the high dose of 355 mmol/L examined 1 year post-treatment appeared similar to the untreated biopsies; however, there was an increase in mature collagen.. GP injection appeared to be well tolerated, with transient lameness and mild circumferential swelling when injected into the tendon and local tissue swelling when injected into the dermis. No systemic hypersensitivities or toxicities were observed. Microscopically, GP resulted in increased linear collagen in tendons at 30 days post-injection and overall increased collagen in dermal tissue when evaluated 1 year post-injection.

Topics: Animals; Collagen; Dermis; Horses; Injections, Intradermal; Iridoids; Lameness, Animal; Male; Pilot Projects; Tendon Injuries; Tendons; Wound Healing

Annulus fibrosus (AF) defects from intervertebral disk (IVD) herniation and degeneration are commonly associated with back pain. Genipin-crosslinked fibrin hydrogel (FibGen) is an injectable, space-filling AF sealant that was optimized to match AF shear properties and partially restored IVD biomechanics. This study aimed to enhance mechanical behaviors of FibGen to more closely match AF compressive, tensile, and shear properties by adjusting genipin crosslink density and by creating a composite formulation by adding Poly(D,L-lactide-co-glycolide) (PDLGA). This study also evaluated effects of thrombin concentration and injection technique on gelation kinetics and adhesive strength. Increasing FibGen genipin concentration from 1 to 36 mg/mL significantly increased adhesive strength (∼5 to 35 kPa), shear moduli (∼10 to 110 kPa), and compressive moduli (∼25 to 150 kPa) with concentration-dependent effects, and spanning native AF properties. Adding PDLGA to FibGen altered the material microstructure on electron microscopy and nearly tripled adhesive strength, but did not increase tensile moduli, which remained nearly 5× below native AF, and had a small increase in shear moduli and significantly decreased compressive moduli. Increased thrombin concentration decreased gelation rate to < 5 min and injection methods providing a structural FibGen cap increased pushout strength by ∼40%. We conclude that FibGen is highly modifiable with tunable mechanical properties that can be formulated to be compatible with human AF compressive and shear properties and gelation kinetics and injection techniques compatible with clinical discectomy procedures. However, further innovations, perhaps with more efficient fiber reinforcement, will be required to enable FibGen to match AF tensile properties.

Topics: Adhesiveness; Annulus Fibrosus; Biocompatible Materials; Fibrin; Iridoids; Materials Testing; Mechanical Phenomena; Polyglactin 910

Bone regeneration is a complicated process and includes a number of distinct and sequential stages of coordinated cellular actions under the regulation of multiple growth factors. Therefore, bone grafting materials in which growth factors can be incorporated and released in a programmed order in line with the bone tissue healing process may lead to desirable clinical outcomes. In the present study, a double-layered chitosan/gelatin/genipin (d-CSG/G) nanosphere coating is developed by using layer-by-layer electrophoretic deposition and genipin crosslinking. The surface morphology, physicochemical and mechanical properties of the coatings are explored. Cytochrome C is used as a therapeutic model protein and is successfully loaded on the inner and outer layers of the coating. The protein release can be controlled by the loading position, genipin concentration and thickness of the outer layer. Furthermore, the cell response to the coatings was evaluated. Real-time polymerase chain reactions, immunofluorescence staining and extracellular matrix mineralization assay confirmed that the functions of the loaded growth factor are fully preserved after fabrication. Overall, the d-CSG/G nanosphere coating could be a promising growth factor delivery system to promote bone tissue regeneration.

Topics: Animals; Biomimetics; Bone Morphogenetic Protein 2; Calcification, Physiologic; Cattle; Chitosan; Coated Materials, Biocompatible; Cross-Linking Reagents; Cytochromes c; Delayed-Action Preparations; Extracellular Matrix; Fluorescent Antibody Technique; Gelatin; Iridoids; Mesenchymal Stem Cells; Nanospheres; Osteocalcin; Rats; Real-Time Polymerase Chain Reaction; Recombinant Proteins; Solutions; Spectroscopy, Fourier Transform Infrared; Surface Properties; Transforming Growth Factor beta

Hepatic ischemia and reperfusion (IR) injury is closely linked to oxidative mitochondrial damage. Since mitochondrial quality control (QC) plays a pivotal role in the recovery of impaired mitochondrial function, mitochondrial QC has emerged as a potential therapeutic target. Genipin, an iridoid compound from Gardenia jasminoides, has been showed antioxidant and anti-inflammatory properties. In this study, we investigated the hepatoprotective mechanism of genipin against IR-induced hepatic injury, particularly focusing on mitochondrial QC. Male C57BL/6 mice underwent liver ischemia for 60min, followed by reperfusion for 6h. Genipin (100mg/kg, i.p.) or vehicle (10% Tween 80 in saline) was administrated to mice 1h before ischemia. Liver and blood samples were collected 6h after reperfusion. Hepatic IR increased hepatocellular oxidative damage and induced mitochondrial dysfunction. These phenomena were ameliorated by genipin. Hepatic IR also increased the level of mitochondrial fission, such as dynamin-related protein 1 and the level of PINK1 protein expression. In contrast, hepatic IR decreased the levels of mitochondrial biogenesis related proteins (e.g., peroxisome proliferator-activated receptor gamma coactivator 1α, nuclear respiratory factor 1, and mitochondrial transcription factor A), mitophagy related proteins (e.g., Parkin), and fusion related protein (e.g., mitofusin 2). Furthermore, hepatic IR decreased the levels of sirtuin1 protein and phosphorylation of AMP-activated protein kinase. Genipin alleviated these IR-induced changes. These data indicate that genipin protects against IR-induced hepatic injury via regulating mitochondrial QC. (225/250).

Topics: AMP-Activated Protein Kinases; Animals; Cytokines; Gardenia; Iridoids; Lipid Peroxidation; Liver Diseases; Male; Mice; Mice, Inbred C57BL; Mitochondria, Liver; Mitochondrial Swelling; Oxidative Stress; Protective Agents; Quality Control; Reperfusion Injury; Signal Transduction; Sirtuin 1

Stem cells have been assumed to demonstrate a reliance on anaerobic energy generation, suited to their hypoxic in vivo environment. However, we found that human mesenchymal stem cells (hMSCs) have an active oxidative metabolism with a range of substrates. More ATP was consistently produced from substrate oxidation than glycolysis by cultured hMSCs. Strong substrate preferences were shown with the ketone body, acetoacetate, being oxidised at up to 35 times the rate of glucose. ROS-generation was 45-fold lower during acetoacetate oxidation compared with glucose and substrate preference may be an adaptation to reduce oxidative stress. The UCP2 inhibitor, genipin, increased ROS production with either acetoacetate or glucose by 2-fold, indicating a role for UCP2 in suppressing ROS production. Addition of pyruvate stimulated acetoacetate oxidation and this combination increased ATP production 27-fold, compared with glucose alone, which has implications for growth medium composition. Oxygen tension during culture affected metabolism by hMSCs. Between passages 2 and 5, rates of both glycolysis and substrate-oxidation increased at least 2-fold for normoxic (20% O

Topics: Acetoacetates; Adenosine Triphosphate; Adipocytes; Aerobiosis; Anaerobiosis; Biomarkers; Cell Differentiation; Chondrocytes; Energy Metabolism; Glucose; Glutamine; Glycolysis; Humans; Iridoids; Mesenchymal Stem Cells; Osteocytes; Oxidation-Reduction; Oxidative Stress; Pyruvic Acid; Reactive Oxygen Species; Uncoupling Protein 2

Genipin, an aglycon of geniposide, has been reported to have anti-inflammatory effect. However, the anti-inflammatory activity of genipin on LPS-stimulated BV2 microglial cells has not been reported. In this study, we investigated the molecular mechanisms responsible for the anti-inflammatory activity of genipin both in vivo and in vitro. The levels of TNF-α, IL-1β, NO and PGE

Topics: Animals; Anti-Inflammatory Agents; Cell Survival; Cells, Cultured; Inflammation; Iridoids; Lipopolysaccharides; Mice; Microglia; NF-kappa B; Nitric Oxide

The ability to guide molecular self-assembly at the nanoscale into complex macroscopic structures could enable the development of functional synthetic materials that exhibit properties of natural tissues such as hierarchy, adaptability, and self-healing. However, the stability and structural integrity of these kinds of materials remains a challenge for many practical applications. We have recently developed a dynamic biopolymer-peptide co-assembly system with the capacity to grow and undergo morphogenesis into complex shapes. Here we explored the potential of different synthetic (succinimidyl carboxymethyl ester, poly (ethylene glycol) ether tetrasuccinimidyl glutarate and glutaraldehyde) and natural (genipin) cross-linking agents to stabilize membranes made from these biopolymer-peptide co-assemblies. We investigated the cross-linking efficiency, resistance to enzymatic degradation, and mechanical properties of the different cross-linked membranes. We also compared their biocompatibility by assessing the metabolic activity and morphology of adipose-derived stem cells (ADSC) cultured on the different membranes. While all cross-linkers successfully stabilized the system under physiological conditions, membranes cross-linked with genipin exhibited better resistance in physiological environments, improved stability under enzymatic degradation, and a higher degree of in vitro cytocompatibility compared to the other cross-linking agents. The results demonstrated that genipin is an attractive candidate to provide functional structural stability to complex self-assembling structures for potential tissue engineering or in vitro model applications.. Molecular self-assembly is widely used for the fabrication of complex functional biomaterials to replace and/or repair any tissue or organ in the body. However, maintaining the stability and corresponding functionality of these kinds of materials in physiological conditions remains a challenge. Chemical cross-linking strategies (natural or synthetic) have been used in an effort to improve their structural integrity. Here we investigate key performance parameters of different cross-linking strategies for stabilising self-assembled materials with potential biomedical applications using a novel protein-peptide co-assembling membrane as proof-of-concept. From the different cross-linkers tested, the natural cross-linker genipin exhibited the best performance. This cross-linker successfully enhanced the mechanical properties of the system enabling the maintenance of the structure in physiological conditions without compromising its bioactivity and biocompatibility. Altogether, we provide a systematic characterization of cross-linking alternatives for self-assembling materials focused on biocompatibility and stability and demonstrate that genipin is a promising alternative for the cross-linking of such materials with a wide variety of potential applications such as in tissue engineering and drug delivery.

Topics: Adipose Tissue; Cell Line; Cell Membrane; Cross-Linking Reagents; Humans; Iridoids; Peptides; Stem Cells

Herein, we show that a molecular assembly offers tremendous opportunities of affording existing building units with new physicochemical properties, holding promise in wide applications. Herein, we develop a facile covalent assembly using a natural occurring linker, genipin, to efficiently transform a traditional chemo drug, doxorubicin, into a nanophotomedicine. A possible mechanism is proposed, in which doxorubicin reacts with genipin through covalent bonding to produce poorly soluble units, which further form nuclei and mediate the interfacial assembly to generate uniform nanoparticles. Such assembled nanophotomedicine shows remarkably enhanced singlet oxygen generation ability (about 1000 folds), leading to a much higher photodynamic activity. Moreover, this self-carried nanodrug exhibits adjustable size, excellent colloidal stability, high capacity and preferable endocytosis. These favorable features lead to greatly improved anticancer efficiency under light at the same dosage, compared to that of pure doxorubin. We believe this study brings a new dimension to develop advanced drug delivery systems by molecular assembly.

Topics: Antineoplastic Agents; Doxorubicin; Drug Carriers; Iridoids; Nanoparticles; Photochemistry

One of the most interesting functions of albumin is the ability to interact with bioactive compounds. This study describes preparation of protein-based nanoparticles (NPs) for the preparation of solid dispersion of curcumin (CN). Fabrication of hydrosol system of dispersed CN in bovine serum albumin (BSA) was approached, followed by cross-linking with glutaraldehyde (Gta). Response surface methodology (RSM) was used to investigate the influence of input factors (pH, CN content and organic phase ratio (r)), on the particle size and CN entrapment efficiency (EE). Particle size, EE and CN loading efficiency (LE) at optimum condition (pH 7, r 10% and 3.4mg of CN content), were found to be in the range of 153-184.4nm, 72.54%, and 14.508μg/mg, respectively. In the optimum formulation, genipin (Gnp) was used at three different levels (0.1-0.2 and 0.3% w/w of BSA), as a safe, natural cross-linker instead of toxic Gta, to address the limitation of oral delivery purpose. AFM and SEM analysis revealed the spherical and smooth surface of Nps. Ninhydrin (NHD) assay and FT-IR analysis confirmed the cross-linking between BSA and Gnp. In vitro release studies ensure the efficiency of the formulation for sustained release of soluble CN.

Topics: Administration, Oral; Animals; Cattle; Curcumin; Drug Carriers; Drug Liberation; Glutaral; Iridoids; Particle Size; Serum Albumin, Bovine; Solubility; Water

Temporary tattoos made with an extract of the jagua fruit (Genipa americana L.) are becoming increasingly popular. It is claimed that it is 'dermatologically tested' and does not contain p-phenylenediamine. Extracts of jagua and gardenia fruits have been used by indigenous people in South America, as well as in traditional Chinese medicine, for centuries. Genipin is currently used for its cross-linking effect in the manufacture of polysaccharides, and is being investigated for its anti-inflammatory and other properties.. To report the presence of the allergenic substance genipin in a self-administered temporary tattoo dye made from the fruit juice of jagua (Genipa americana L.).. A 39-year-old female who repeatedly applied 'completely natural and 100% safe' Earth Jagua® tattoo, obtained via the internet, to her left hand developed allergic contact dermatitis within 6 weeks. Analysis of the dye showed the presence of geniposide and genipin.. Patch tests with the dye and with its main components, including genipin, gave strong positive reactions to the latter. There was no sensitization to other ingredients or p-amino compounds.. We report an extensively evaluated case of allergic contact dermatitis caused by a temporary Earth Jagua® tattoo. The allergen identified is genipin, a substance that is increasingly used for tattoos and as a therapeutic agent in medicine. This could result in an increase in the number of allergic reactions in the future.

Topics: Adult; Coloring Agents; Dermatitis, Allergic Contact; Dermatitis, Atopic; Female; Humans; Iridoids; Tattooing

Controlled release of growth factors allows the efficient, localized, and temporally-optimized delivery of bioactive molecules to potentiate natural physiological processes. This concept has been applied to treatments for pathological states, including chronic degeneration, wound healing, and tissue regeneration. Peptide microspheres are particularly suited for this application because of their low cost, ease of manufacture, and interaction with natural remodeling processes active during healing. The present study characterizes gelatin microspheres for the entrapment and delivery of growth factors, with a focus on tailored protein affinity, loading capacity, and degradation-mediated release. Genipin crosslinking in PBS and CHES buffers produced average microsphere sizes ranging from 15 to 30 microns with population distributions ranging from about 15 to 60 microns. Microsphere formulations were chosen based on properties important for controlled transient and spatial delivery, including size, consistency, and stability. The microsphere charge affinity was found to be dependent on gelatin type, with type A (GelA) carriers consistently having a lower negative charge than equivalent type B (GelB) carriers. A higher degree of crosslinking, representative of primary amine consumption, resulted in a greater negative net charge. Gelatin type was found to be the strongest determinant of degradation, with GelA carriers degrading at higher rates versus similarly crosslinked GelB carriers. Growth factor release was shown to depend upon microsphere degradation by proteolytic enzymes, while microspheres in inert buffers showed long-term retention of growth factors. These studies illuminate fabrication and processing parameters that can be used to control spatial and temporal release of growth factors from gelatin-based microspheres.

Topics: Animals; Drug Carriers; Drug Liberation; Gelatin; Intercellular Signaling Peptides and Proteins; Iridoids; Matrix Metalloproteinase 8; Microspheres; Swine

Acute liver failure (ALF) is a life-threatening syndrome resulting from massive inflammation and hepatocyte death. Necroptosis, a programmed cell death controlled by receptor-interacting protein kinase (RIP) 1 and RIP3, has been shown to play an important role in regulating inflammation via crosstalk between other intracellular signaling. The inflammasome is a major intracellular multiprotein that induces inflammatory responses by mediating immune cell infiltration, thus potentiating injury. Genipin, a major active compound of the gardenia fruit, exhibits anti-inflammatory, antioxidant, and anti-apoptotic properties. This study investigated the hepatoprotective mechanisms of genipin on d-galactosamine (GalN) and lipopolysaccharide (LPS)-induced ALF, particularly focusing on interaction between necroptosis and inflammasome. Mice were given an intraperitoneal injection of genipin (25, 50, and 100mg/kg) or necrostatin-1 (Nec-1, a necroptosis inhibitor; 1.8mg/kg) 1h prior to GalN (800mg/kg)/LPS (40μg/kg) injection and were killed 3h after GalN/LPS injection. Genipin improved the survival rate and attenuated increases in serum aminotransferase activities and inflammatory cytokines after GalN/LPS injection. Genipin reduced GalN/LPS-induced increases in RIP3, phosphorylated RIP1 and RIP3 protein expression, and RIP1/RIP3 necrosome complex, similar to the effects of Nec-1. GalN/LPS significantly increased serum levels of high-mobility group box 1 and interleukin (IL)-33, which were attenuated by genipin and Nec-1. Moreover, similar to Nec-1, genipin attenuated GalN/LPS-induced increases in the protein expression levels of NLRP3, ASC, and caspase-1, inflammasome components, and levels of liver and serum IL-1β. Taken together, our findings suggest that genipin ameliorates GalN/LPS-induced hepatocellular damage by suppressing necroptosis-mediated inflammasome signaling.

Topics: Animals; Apoptosis; Chemical and Drug Induced Liver Injury; Galactosamine; Inflammasomes; Iridoids; Lipopolysaccharides; Male; Mice; Mice, Inbred ICR; Necrosis; NLR Family, Pyrin Domain-Containing 3 Protein; Receptors, Pattern Recognition; Signal Transduction

Cisplatin (CP) is a potent and widely used chemotherapeutic agent. However, the clinical benefits of CP are compromised because it elicits nephrotoxicity and ototoxicity. In this study, we investigated the nephroprotective effects of the phytochemical genipin (GP) isolated from the gardenia (Gardenia jasminoides) fruit, using a murine model of CP-induced nephropathy. GP pretreatment attenuated the CP-induced renal tissue injury by diminishing the serum blood urea nitrogen, creatinine, and cystatin C levels, as well as those of kidney injury molecule-1. In addition, GP attenuated the CP-induced oxidative/nitrative stress by suppressing the activation of NADPH oxidase, augmenting the endogenous antioxidant defense system, and diminishing the accumulation of 4-hydroxynonenal and 3-nitrotyrosine in renal tissues. Furthermore, reduced levels of proinflammatory cytokines such as tumor necrosis factor-alpha and interleukin-1 beta indicated that CP-induced renal inflammation was mitigated upon the treatment with GP. GP also attenuated the CP-induced activation of mitogen-activated protein kinases, excessive activities of caspase-3/7 and poly(ADP-ribose) polymerase, DNA fragmentation, and apoptosis. When administered 12h after the onset of kidney injury, GP showed a therapeutic effect by ameliorating CP-induced nephrotoxicity. Moreover, GP synergistically enhanced the CP-induced cell death of T24 human bladder cancer cells. Collectively, our data indicate that GP attenuated the CP-induced renal tissue injury by abrogating oxidative/nitrative stress and inflammation and by blocking cell death pathways, thereby improving the renal function. Thus, our results suggest that the use of GP may be a promising new protective strategy against cisplatin-induced nephrotoxicity.

Topics: Aldehydes; Animals; Antioxidants; Apoptosis; Blood Urea Nitrogen; Caspase 3; Caspase 7; Cell Line, Tumor; Cisplatin; Creatinine; Cystatin C; Cytokines; Hepatitis A Virus Cellular Receptor 1; Humans; Inflammation; Iridoids; Kidney; Kidney Diseases; Male; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; Oxidative Stress; Poly(ADP-ribose) Polymerases; Tyrosine

Recent work demonstrated that 3D fibrin scaffolds function as an effective substrate for engineering tissues from pluripotent stem cells. However, the rapid degradation rate of fibrin remains a major limitation when differentiating human pluripotent stem cells for tissue engineering applications. The addition of crosslinking agents, such as genipin, during the polymerization process increases scaffold stability while decreasing the degradation rate of fibrin. Genipin crosslinking alters the physical characteristics of the fibrin scaffolds, which influences the behaviour of the differentiating cells seeded inside. It also possesses neuritogenic and neuroprotective properties, making it particularly attractive for engineering neural tissue from pluripotent stem cells. Here we show that genipin enhances neuronal differentiation of neural progenitors derived from human induced pluripotent stem cells (hiPSCs) in 2D culture and genipin concentration influences the morphological and mechanical properties of 3D fibrin scaffolds. These mechanically stable genipin-crosslinked fibrin scaffolds support hiPSC-derived neural aggregates and induce neurite outgrowth while remaining intact for 2 weeks as opposed to 5 days for unmodified fibrin scaffolds.

Topics: Cell Culture Techniques; Cell Differentiation; Cross-Linking Reagents; Fibrin; Humans; Induced Pluripotent Stem Cells; Iridoids; Nerve Tissue; Neuronal Outgrowth; Tissue Engineering; Tissue Scaffolds

To investigate, after 24 h, the safety of genipin or ultraviolet A (UVA)-riboflavin crosslinking of keratocytes and endothelial cells.. Fifteen New Zealand white rabbits were selected and divided into a PBS group (five rabbits), a 0.2% genipin crosslinking (GP-CXL) group (five rabbits), and a UVA-riboflavin crosslinking (UVA-CXL) group (five rabbits). In the GP-CXL and PBS groups, 0.2% genipin or PBS was applied to the corneal surface of the right eyes. In the UVA-CXL group, a clinical crosslinking procedure was used. Before and after surgery, the operated eyes of each group were characterized with confocal microscopy, and the corneal buttons were excised for endothelium staining and electron microscopy.. The corneal endothelial cell density of the GP-CXL, UVA-CLX, and PBS groups changed. There was a statistically significant difference in thickness and changes in corneal endothelial cell density between the UVA-CXL group and the PBS group (p<0.05), and between the UVA-CXL group and the GP-CXL group (p<0.05), but no statistically significant difference between the GP-CXL group and the PBS group. Confocal microscopy, transmission electron microscopy, and hematoxylin and eosin staining showed that there was keratocyte apoptosis in the anterior and middle stroma and endothelial cell damage in the UVA-CXL group. In the GP-CXL group, only active keratocytes were found and minimal endothelial cell damage.. Treatment of rabbit corneas with 0.2% genipin showed minimal toxicity toward keratocytes and endothelial cells. Genipin is safer than UVA-CXL for crosslinking of thin corneas.

Topics: Animals; Cell Count; Cholagogues and Choleretics; Collagen; Corneal Keratocytes; Corneal Stroma; Cross-Linking Reagents; Endothelium, Corneal; Female; Iridoids; Microscopy, Confocal; Microscopy, Electron, Scanning; Photochemotherapy; Photosensitizing Agents; Rabbits; Riboflavin; Ultraviolet Rays

Nile tilapia skin collagen sponges were fabricated by freeze-drying technology and modified with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in the presence of N-hydroxysuccinimide (EDC/NHS), genipin+PBS, genipin+ethanol, tea polyphenol (TP), nordihydroguaiaretic acid (NDGA) and diphenyl phosphoryl azide (DPPA). Physicochemical and biological properties, micromorphology and compatibility before and after modification were investigated to evaluate collagen sponge as a hemostatic biomedical material. The mechanical property of collagen sponges strengthened after cross-linking. The elongation at break of cross-linked collagen sponges decreased except for EDC/NHS, which was close to that of non-crosslinked. The collagen sponge cross-linked with EDC/NHS exhibited the highest hygroscopicity in comparison with other cross-linkers. The resistance to collagenase biodegradation of collagen sponges after cross-linking strengthened significantly except for NDGA. Collagen sponges cross-linked with EDC/NHS, TP and NDGA maintained high porosity (97-98%), similar to non-crosslinked (98.42%). Collagen sponges could shorten the blood coagulation time. From the variations of the FTIR spectrum pattern and SEM, DPPA could change the secondary structure of collagen and destroy the spongy structure of collagen sponge, which was not suitable for the cross-linking of collagen sponge. Whereas, EDC/NHS was recognized as a perfect cross-linker owing to its excellent properties and porous microstructure. All fabricated collagen sponges were recognized to be biocompatible by the hemolysis assay in vitro. Therefore, collagen sponge modified with EDC/NHS could be used as a perfect biomedical hemostatic material.

Topics: Animals; Azides; Biocompatible Materials; Cichlids; Collagen; Cross-Linking Reagents; Iridoids; Masoprocol; Materials Testing; Skin; Tissue Engineering

Several studies indicate that mitochondrial uncoupling protein 2 (UCP2) plays a pivotal role in cancer development by decreasing reactive oxygen species (ROS) produced by mitochondrial metabolism and by sustaining chemoresistance to a plethora of anticancer drugs. Here, we demonstrate that inhibition of UCP2 triggers Akt/mTOR pathway in a ROS-dependent mechanism in pancreatic adenocarcinoma cells. This event reduces the antiproliferative outcome of UCP2 inhibition by genipin, creating the conditions for the synergistic counteraction of cancer cell growth with the mTOR inhibitor everolimus. Inhibition of pancreatic adenocarcinoma cell growth and induction of apoptosis by genipin and everolimus treatment are functionally related to nuclear translocation of the cytosolic glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The synthetic compound (S)-benzyl-2-amino-2-(S)-3-bromo-4,5-dihydroisoxazol-5-yl-acetate (AXP3009), which binds GAPDH at its redox-sensitive Cys152, restores cell viability affected by the combined treatment with genipin and everolimus, suggesting a role for ROS production in the nuclear translocation of GAPDH. Caspase-mediated apoptosis by genipin and everolimus is further potentiated by the autophagy inhibitor 3-methyladenine revealing a protective role for Beclin1-mediated autophagy induced by the treatment. Mice xenograft of pancreatic adenocarcinoma further confirmed the antiproliferative outcome of drug combination without toxic effects for animals. Tumor masses from mice injected with UCP2 and mTOR inhibitors revealed a strong reduction in tumor volume and number of mitosis associated with a marked GAPDH nuclear positivity. Altogether, these results reveal novel mechanisms through which UCP2 promotes cancer cell proliferation and support the combined inhibition of UCP2 and of Akt/mTOR pathway as a novel therapeutic strategy in the treatment of pancreatic adenocarcinoma.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Pancreatic Ductal; Cell Line, Tumor; Cell Proliferation; Everolimus; Female; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Iridoids; Male; Pancreatic Neoplasms; Protein Transport; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction; TOR Serine-Threonine Kinases; Uncoupling Protein 2; Xenograft Model Antitumor Assays

Oenothein B (OeB), a dimeric macrocyclic ellagitannin isolated from eucalyptus leaves has been demonstrated as a promising natural bioactive compound for its remarkable antitumor, antioxidant, anti-inflammatory and immunomodulating effects. Unfortunately, early study indicates that OeB has quite low bioaccessibility for oral consumption due to their susceptibility to decomposition both in vitro and in vivo. Herein, we report the design and synthesis of food-grade polyelectrolyte complex coacervate using caseinophosphopeptides (CPPs) and chitosan (CS) to encapsulate OeB for enhanced protection through gastrointestinal (GI) tract. Turbidimetric titration, dynamic light scattering (DLS), ζ-potential and scanning electric microscopy (SEM), as well as theoretical calculations based on principle of charge neutralization, were conducted to provide tentative and quantitative description for phase behavior in formation and disassociation of the complex. The optimum fabrication conditions were found to be at pH 5.5, with CPP : CS at 1 : 1, using CS of high molecular weight (980 kDa). The genipin cross-linking protected the system from disassembling in harsh acidic environments. The best cross-linking conditions were found to be 0.6 mg ml

Topics: Chitosan; Drug Carriers; Drug Compounding; Drug Delivery Systems; Drugs, Chinese Herbal; Hydrolyzable Tannins; Iridoids; Nanoparticles; Particle Size; Polyelectrolytes

Genipin is a natural compound isolated from the fruit of Gardenia jasminoides with various pharmacological effects. In this study, we investigated whether genipin effectively alleviates allergic responses in a murine model of ovalbumin (OVA)-induced asthma. The mice were administered an intraperitoneal injection of OVA on day 0 and 14 to boost the immune response; genipin was then administered from day 18 to 23 by oral gavage. On days 21 to 23, mice were OVA-challenged using am ultrasonic nebulizer, and airway hyperresponsiveness (AHR) was determined on day 24 by plethysmography. Genipin significantly reduced the inflammatory cell count in bronchoalveolar lavage fluids (BALF) and AHR, which were accompanied by lower interleukin-5 (IL-5), IL-13 and OVA-specific immunoglobulin (Ig) E levels in the BALF or serum from OVA-induced asthmatic mice. In histology, genipin significantly decreased airway inflammation and mucus hypersecretion in OVA-induced asthmatic mice. Additionally, genipin inhibited OVA-induced increases in the expression of inducible nitric oxide synthase and cyclooxygenase-2 proteins. Further, genipin reduced the activity and protein levels of matrix metalloproteinase-9 in lung tissue from OVA induced asthmatic mice. Overall, genipin effectively alleviated the asthmatic inflammatory response in an OVA-induced asthmatic model. Therefore, our results suggest that genipin has therapeutic potential for treating asthma.

Topics: Animals; Anti-Inflammatory Agents; Asthma; Bronchial Hyperreactivity; Bronchoalveolar Lavage Fluid; Disease Models, Animal; Female; Gardenia; Hypersensitivity; Immunoglobulin E; Inflammation; Interleukin-13; Interleukin-5; Iridoids; Lung; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Nitric Oxide Synthase Type II; Ovalbumin

Polycystic ovary syndrome (PCOS) is a common endocrine disorder in women, and it is usually characterized by chronic inflammation, oxidative stress, and altered microRNA expression. The aim of this study is to investigate how the effects of iridoids with genipin stem nucleus inhibit PCOS complications. The interactions between iridoids were investigated, as well.. The chronic inflammation cell model was induced using lipopolysaccharide (LPS) in the RAW 264.7 and KGN cell lines. Levels of mRNA and protein expression were quantified using real time-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. The target of the iridoids was identified using the drug affinity responsive target stability (DARTS) method. The ability to scavenge free radicals was evaluated using the DPPH radical scavenging method and the ultra oxygen anion (O2-) radical scavenging method.. The cells recovered from the inflammatory conditions and showed significantly decreased levels of interleukins after treatment with iridoids. The iridoids were demonstrated to target NF-κB, inhibit the phosphorylation and degradation of IκB, inhibit the nuclear entry of NF-κB, and inhibit the expression of inflammatory factors. Though only genipin showed an efficient ability to scavenge O2-, the iridoids, IκB inhibitor (BAY 11-7085), and NF-κB inhibitor (PDTC) could inhibit LPS-induced oxidative stress on the cells, indicating that the iridoids exert their anti-oxidant effects via the NF-κB pathway. The expression levels of microRNAs (miRNAs) were also altered by LPS, but the iridoids could scarcely rescue the abnormal condition.. Chronic inflammation may be an important incentive for oxidative stress and abnormal microRNA expression in PCOS, and iridoids can protect patients from inflammatory damage by regulating the NF-κB pathway.

Topics: Animals; Blotting, Western; Cell Line, Tumor; Female; Fluorescent Antibody Technique; Humans; Inflammation; Iridoids; Lipopolysaccharides; Mice; NF-kappa B; Nitriles; Oxidative Stress; Phosphorylation; Polycystic Ovary Syndrome; RAW 264.7 Cells; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Sulfones

Geniposide and genipin have been found in Gardenia jasminoides Ellis, a traditional Chinese medicine that exhibits multiple biological functions. However, no report showing the effects of geniposide and genipin on gastric protection in Helicobacter pylori infections in vitro and in vivo has been done. In this study, we clarified how geniposide and genipin suppress H. pylori-mediated inflammation in gastric AGS cells and C57BL/6 mice. Our results demonstrated that genipin shows a strong ability to inhibit H. pylori growth and is able to reduce vacA and cagA gene expression of H. pylori in infected AGS cells. Genipin also attenuates the abilities of adhesion and invasion of H. pylori to AGS cells. An attenuation of interleukin (IL)-8 and IFN-γ production caused by genipin was observed to inhibit cell inflammatory responses. In the in vivo experiments, geniposide and genipin both showed suppressive effects on the vacA gene expression in mice after H. pylori infection. The serum levels of IFN-γ, IL-1β, immunoglobulin A, and Immunoglobulin M were decreased by geniposide and genipin in infected mice. The inflammatory maker COX2 was downregulated in H. pylori-infected mice after exposure to geniposide and genipin. Together, geniposide and genipin effectively exert a healthy promotion to reduce H. pylori infections in vivo by interfering with the growth and virulence of H. pylori as well as attenuating the gastric inflammation caused by an H. pylori infection.. Geniposide and genipin have a healthy promotion to eradicate H. pylori infections by interfering with the growth and virulence of H. pylori and to attenuate the gastric inflammation caused by an H. pylori infection.

Topics: Animals; Drugs, Chinese Herbal; Gardenia; Gastric Mucosa; Helicobacter Infections; Helicobacter pylori; Humans; Inflammation Mediators; Iridoids; Male; Mice; Mice, Inbred C57BL

The search for compounds with functional properties from natural sources has grown in recent years as people have developed healthier habits. Therefore, the aim of this study was to evaluate the extraction of bioactive compounds from various parts of unripe genipap fruit (Genipa americana L.) by using pressurized ethanol to verify which part of the fruit provides the greatest recovery of the iridoids genipin and geniposide. Two process variables were studied: temperature (50 and 80°C) and pressure (2, 12 and 20 bar). The whole fruit and the peel, mesocarp, endocarp, endocarp+seeds and seeds of the fruit were studied. The endocarp presented with the highest recovery of genipin (48.6±0.6mg/g raw material) and the extraction from the mesocarp allowed a greater recovery of geniposide (59±1mg/g raw material). The highest values of total phenolic content were obtained with mesocarp extracts. The endocarp and mesocarp extracts presented the highest antioxidant activity as measured by FRAP and DPPH. These results are promising and support the use of unripe genipap fruit as a source of iridoids and natural antioxidants.

Topics: Antioxidants; Ethanol; Food Handling; Fruit; Iridoids; Phenols; Plant Extracts; Rubiaceae; Seeds

Injectable hydrogels are becoming of increasing interest in the field of tissue engineering thanks to their versatile properties and to the possibility of being injected into tissues or devices during surgery. In peripheral nerve tissue engineering, injectable hydrogels having shear-thinning properties are advantageous as filler of nerve guidance channels (NGCs) to improve the regeneration process. In the present work, gelatin-based hydrogels were developed and specifically designed for the insertion into the lumen of hollow NGCs through a syringe during surgery. Injectable hydrogels were obtained using an agar-gelatin 20:80 weight ratio, (wt/wt) blend crosslinked by the addition of genipin (A/GL_GP). The physicochemical properties of the A/GL_GP hydrogels were analysed, including their injectability, rheological, swelling and dissolution behaviour, and their mechanical properties under compression. The hydrogel developed showed shear-thinning properties and was applied as filler of NGCs. The A/GL_GP hydrogel was tested in vitro using different cell lines, among them Schwann cells which have been used because they have an important role in peripheral nerve regeneration. Viability assays demonstrated the lack of cytotoxicity. In vitro experiments showed that the hydrogel is able to promote cell adhesion and proliferation. Two- and three-dimensional migration assays confirmed the capability of the cells to migrate both on the surface and within the internal framework of the hydrogel. These data show that A/GL_GP hydrogel has characteristics that make it a promising scaffold material for tissue engineering and nerve regeneration. Copyright © 2014 John Wiley & Sons, Ltd.

Topics: Agar; Alginates; Animals; Apoptosis; Cell Adhesion; Cell Movement; Cell Proliferation; Cell Survival; Compressive Strength; Gelatin; Hydrogels; Hydrogen-Ion Concentration; Iridoids; Materials Testing; Mice; Nerve Regeneration; Neurons; NIH 3T3 Cells; Rats; Regeneration; Rheology; Schwann Cells; Stress, Mechanical; Tissue Engineering; Tissue Scaffolds

Genipin is a natural low-toxic cross-linker for molecules with primary amino groups, and its use with collagen and gelatin has shown a great potential in tissue engineering applications. The fabrication of scaffolds with a well-organized micro and macro topology using additive manufacturing systems requires an accurate control of working parameters, such as reaction rate, gelling time, and diffusion constant. A polymeric system of 5% w/v gelatin in PBS with 2 mg/mL collagen solutions in a 1:1 weight ratio was used as template to perform measurements varying genipin concentration in a range of 0.1-1.5% w/w with respect to gelatin. In the first part of this work, the reaction rate of the polymeric system was estimated using a new colorimetric analysis of the reaction. Then its workability time, closely related to the gelling time, was evaluated thanks to rheological analysis: finally, the quantification of static and dynamic diffusion constants of genipin across nonreacting and reacting membranes, made respectively by agarose and gelatin, was performed. It was shown that the colorimetric analysis is a good indicator of the reaction progress. The gelling time depends on the genipin concentration, but a workability window of 40 min guaranteed up to 0.5% w/w genipin. The dynamic diffusion constant of genipin in the proposed polymeric system is in the order of magnitude of 10

Topics: Gelatin; Iridoids; Models, Chemical; Tissue Engineering

The synthesis and optimization of peptide-based hydrogel materials have gained growing interest in the last years, thanks to their properties, that make them appealing for diverse biotechnological applications, with a particular focus in the field of biomedicine. The self-assembling abilities of low molecular weight peptides make them ideal for designing advanced materials using mild reaction conditions. In this work, a biocatalytic approach has been used for the synthesis of an Fmoc-tripeptide that is able to self-assemble in water affording a self-supporting hydrogel. The mechanical properties of this material have been enhanced through chemical crosslinking by using a natural compound, genipin, that allows to minimize cytotoxic effects. Moreover, we have tested the potential of the prepared materials to be employed as drug delivery systems using naproxen as an anti-inflammatory model drug, and studying its release kinetics in aqueous medium. The cytotoxicity of the hydrogels has been evaluated, and their mechanical and morphological properties have been studied by rheology and SEM microscopy.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Biocatalysis; Biocompatible Materials; Biotechnology; Cell Survival; Cross-Linking Reagents; Drug Delivery Systems; Hydrogels; Iridoids; Materials Testing; Mice; Microscopy, Electron, Scanning; Naproxen; NIH 3T3 Cells; Oligopeptides; Rheology

The most common mode of surgical repair of ruptured tendons and ligaments involves the use of sutures for reattachment. However, there is a high incidence of rerupture and repair failure due to pulling out of the suture material from the damaged connective tissue. The main goal of this research was to achieve a localized delivery of crosslinking agent genipin (GP) from rapid-release biodegradable coatings on sutures, for strengthening the repair of ruptured connective tissue. Our hypothesis is that GP released from the suture coating will lead to exogenous crosslinking of native connective tissue resulting in beneficial effects on clinically relevant mechanical parameters such as tear resistance, tissue strength, and energy required to rupture the tissue (toughness). Sutures were successfully coated with a biodegradable polymer layer loaded with the crosslinking agent genipin, without compromising the mechanical properties of the suture. The rapid-release of genipin was achieved under both in vitro and ex vivo conditions. Exogenous crosslinking using these genipin releasing sutures was demonstrated using equine tendons. The tendons treated with genipin releasing sutures showed significant improvement in failure load, energy required for pull-out failure, and stiffness. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2199-2205, 2017.

Topics: Animals; Coated Materials, Biocompatible; Cross-Linking Reagents; Iridoids; Sutures; Tendon Injuries; Tendons

The therapeutic effect on HCl/ethanol induced gastric injury of Gardenia jasminoides (JXGJ-1 and JXGJ-2) were determined by a animal model. JXGJ-2 group reduced area of its gastric injury as compared to the control group, JXGJ-2 also helped in decreasing the gastric secretion volume results raised in pH value. The NO contents in serum, heart, liver, kidney and stomach of JXGJ-2 group were more than JXGJ-1 and control groups. JXGJ-2 reduce cytokine levels as compared to JXGJ-1 and control group. The serum and gastric tissue SOD, GSH-Px, GSH levels in JXGJ-2 treated mice were higher than JXGJ-1 treated and control mice, but the MDA, PC levels showed the crosscurrents, these levels were close to normal mice. Gardenia jasminoides could increase the occludin, EGF, EGFR, VEGF, IκB-α, nNOS, eNOS, Cu/Zn-SOD, Mn-SOD, CAT, GSH-Px (GSH1) mRNA and protein expressions and decrease the p38MAPK (p38), NF-κB, Bcl-2, COX-2, iNOS expressions in gastric tissues unlike to the control mice, JXGJ-2 had much better effect than JXGJ-1. JXGJ-1contained the higher genipin gentiobioside and gardenoside, they might be the key components of gastric injury inhibition. Gardenia jasminoides had a remarkable effect on gastric injury, and they were derived from two important components of genipin gentiobioside and gardenoside.

Topics: Animals; Antioxidants; Disease Models, Animal; Drugs, Chinese Herbal; Ethanol; Gardenia; Hydrochloric Acid; Iridoids; Male; Mice; Stomach; Stomach Diseases

High energy trauma to cartilage causes surface fissures and microstructural damage, but the degree to which this damage renders the tissue more susceptible to wear and contributes to the progression of post-traumatic osteoarthritis (PTOA) is unknown. Additionally, no treatments are currently available to strengthen cartilage after joint trauma and to protect the tissue from subsequent degradation and wear. The purposes of this study were to investigate the role of mechanical damage in the degradation and wear of cartilage, to evaluate the effects of impact and subsequent genipin crosslinking on the changes in the viscoelastic parameters of articular cartilage, and to test the hypothesis that genipin crosslinking is an effective treatment to enhance the resistance to biochemical degradation and mechanical wear. Results demonstrate that cartilage stiffness decreases after impact loading, likely due to the formation of fissures and microarchitectural damage, and is partially or fully restored by crosslinking. The wear resistance of impacted articular cartilage was diminished compared to undamaged cartilage, suggesting that mechanical damage that is directly induced by the impact may contribute to the progression of PTOA. However, the decrease in wear resistance was completely reversed by the crosslinking treatments. Additionally, the crosslinking treatments improved the resistance to collagenase digestion at the impact-damaged articular surface. These results highlight the potential therapeutic value of collagen crosslinking via genipin in the prevention of cartilage degeneration after traumatic injury. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:558-565, 2017.

Topics: Animals; Cartilage; Cattle; Collagenases; Elasticity; Friction; Iridoids; Stress, Mechanical

In this work, glucose oxidase (GOD) and catalase (CAT) were used simultaneously to produce gluconic acid from glucose. In order to reduce the distance between the two enzymes, and therefore improve efficiency, GOD and CAT were cross-linked together using genipin. Improvements in gluconic acid production were due to quick removal of harmful intermediate hydrogen peroxide by CAT. GOD activity was significantly affected by the proportion of CAT in the system, with GOD activity in the cross-linked multi-enzyme (CLME) being 10 times higher than that in an un-cross-linked GOD/CAT mixture. The glucose conversion rate after 15 h using 15 % glucose was also 10 % higher using the CLME than was measured using a GOD/CAT mixture.

Topics: Aspergillus niger; Catalase; Cross-Linking Reagents; Enzyme Activation; Gluconates; Glucose; Glucose Oxidase; Iridoids; Multienzyme Complexes

Genipin is a component of Japanese traditional herbal medicine (Kampo), inchinkoto, and is used for the treatment of various liver injuries. However, there are few scientific evidence for its anti-inflammatory effects and mechanisms. In inflamed liver, proinflammatory cytokines including tumor necrosis factor (TNF)-α and interleukin (IL)-1β stimulate liver cells, followed by the expression of inducible nitric oxide synthase (iNOS). Excessive levels of NO produced by iNOS have been implicated as one of the factors in liver injury. Thus it is essential to inhibit iNOS induction for the prevention of liver injury. In this study, we examined IL-1β-stimulated hepatocytes as a simple "in vitro liver injury model" to investigate liver protective effects of genipin.. Primary cultured rat hepatocytes were treated with IL-1β in the presence or absence of genipin. The induction of NO production and iNOS, and its signaling pathway were analyzed.. In IL-1β-stimulated hepatocytes, genipin inhibited the production of NO dose- and timedependently, and reduced the levels of iNOS protein and its mRNA expression. Genipin also reduced mRNA expressions of TNF-α and IL-6. Genipin inhibited two essential signaling pathways for iNOS induction, IκB degradation/NF-κB activation and type I IL-1 receptor upregulation. Transfection experiments revealed that genipin decreased the expression of iNOS mRNA through both inhibitions of the promoter activation and mRNA stabilization. Delayed administration of genipin after IL-1β addition also inhibited iNOS induction.. Genipin influenced the induction of inflammatory mediators, iNOS and TNF-α, in part through the inhibition of NF-κB activation in hepatocytes. Genipin may have therapeutic potential for organ injuries including liver.

Topics: Animals; Cells, Cultured; Drugs, Chinese Herbal; Hepatocytes; Humans; Interleukin-1beta; Interleukin-6; Iridoids; Liver; Liver Failure, Acute; Medicine, Kampo; NF-kappa B; Nitric Oxide; Nitric Oxide Synthase Type II; Plant Extracts; Primary Cell Culture; Protective Agents; Rats; Rats, Wistar; RNA, Messenger; Up-Regulation

The present study aims to investigate the physical-chemical and biological features exhibited by porous scaffolds for regeneration of cartilaginous tissues obtained through stabilization of 3D gelatin hydrogels by physical (DHT), chemical (BDDGE) and natural (Genipin) cross-linking approaches. The study aimed at comparatively assessing the porous microstructure and the long-term resistance of the scaffolds upon degradation in wet physiological conditions (37°C, pH=7.4). The degree of cross-linking increases as function of incorporation of cross-linkers which was maximum up to 73% for BDDGE. The infrared spectroscopy and thermal analysis confirmed the gelatin structure was preserved during the cross-linking treatments. Mechanical properties of the scaffolds were analysed by static and dynamic compression test, which showed different viscoelastic behaviour upon various cross-linking strategies. The biological performance of the scaffolds investigated using human chondrocytes showed good cell adhesion, viability and proliferation, as well as extensive 3D scaffold colonization. Besides, the analysis of gene expression related to the formation of new chondral tissue reported increasing ability with time in the formation of new extra-cellular matrix. In conclusion, out of three different cross-linking methods, the gelatin scaffolds subjected to dehydrothermal treatment (DHT) represented to be the most favourable 3D scaffold for cartilage regeneration.

Topics: Animals; Butylene Glycols; Cell Adhesion; Cell Line; Cell Proliferation; Cell Survival; Chondrocytes; Cross-Linking Reagents; Elasticity; Gelatin; Hot Temperature; Humans; Hydrogen-Ion Concentration; Iridoids; Porosity; Skin; Swine; Tissue Engineering; Tissue Scaffolds; Viscosity

We speculate that an acellular osteochondral xenograft may be a good alternative to allografts for repair of focal articular cartilage lesions. In order to make a xenograft resistant to enzymatic degradation and to prevent a chronic immune response it may be beneficial to stabilize it through crosslinking. The concept is analogous to treatment of porcine bioprosthetic heart valves with glutaraldehyde. The purpose of this study was to evaluate genipin, a natural crosslinking agent with low cytotoxicity, for stabilization of decellularized cartilage. Porcine articular cartilage discs were decellularized in SDS and nucleases and then crosslinked in genipin. The utility of genipin was determined from its effects on degree of crosslinking, mechanical properties, dimensional stability, enzymatic resistance, and in vitro biocompatibility. Degree of crosslinking, compressive moduli, and collagenase resistance varied over a wide range depending on genipin concentration. The equilibrium compressive modulus could be increased from approximately 50% to more than 120% that of native cartilage, and the time to complete degradation by collagenase could be extended from less than 12 h to more than 15 days. Radial shrinkage of approximately 4% was observed at a genipin concentration of 0.1% wt/vol, and cartilage coefficient of friction against glass increased in a concentration-dependent manner. Autologous chondrocytes displayed little difference in viability or their ability to attach and spread over the surface of genipin-fixed cartilage compared to non-crosslinked cartilage during 6 weeks of culture. These results indicate that genipin may be efficacious for stabilization of a decellularized porcine osteochondral xenograft. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1949-1957, 2017.

Topics: Animals; Cartilage, Articular; Collagenases; Compressive Strength; Cross-Linking Reagents; Iridoids; Materials Testing; Swine; Tissue Scaffolds

Injectable scaffolds for cardiac tissue regeneration are a promising therapeutic approach for progressive heart failure following myocardial infarction (MI). Their major advantage lies in their delivery modality that is considered minimally invasive due to their direct injection into the myocardium. Biomaterials comprising such scaffolds should mimic the cardiac tissue in terms of composition, structure, mechanical support, and most importantly, bioactivity. Nonetheless, natural biomaterial-based gels may suffer from limited mechanical strength, which often fail to provide the long-term support required by the heart for contraction and relaxation. Here we present newly-developed injectable scaffolds, which are based on solubilized decellularized porcine cardiac extracellular matrix (pcECM) cross-linked with genipin alone or engineered with different amounts of chitosan to better control the gel's mechanical properties while still leveraging the ECM biological activity. We demonstrate that these new biohybrid materials are naturally remodeled by mesenchymal stem cells, while supporting high viabilities and affecting cell morphology and organization. They exhibit neither in vitro nor in vivo immunogenicity. Most importantly, their application in treating acute and long term chronic MI in rat models clearly demonstrates the significant therapeutic potential of these gels in the long-term (12weeks post MI). The pcECM-based gels enable not only preservation, but also improvement in cardiac function eight weeks post treatment, as measured using echocardiography as well as hemodynamics. Infiltration of progenitor cells into the gels highlights the possible biological remodeling properties of the ECM-based platform.. This work describes the development of new injectable scaffolds for cardiac tissue regeneration that are based on solubilized porcine cardiac extracellular matrix (ECM), combined with natural biomaterials: genipin, and chitosan. The design of such scaffolds aims at leveraging the natural bioactivity and unique structure of cardiac ECM, while overcoming its limited mechanical strength, which may fail to provide the long-term support required for heart contraction and relaxation. Here, we present a biocompatible gel-platform with custom-tailored mechanical properties that significantly improve cardiac function when injected into rat hearts following acute and chronic myocardial infarction. We clearly demonstrate the substantial therapeutic potential of these scaffolds, which not only preserved heart functions but also alleviated MI damage, even after the formation of a mature scar tissue.

Topics: Animals; Cell Line; Chitosan; Extracellular Matrix; Humans; Hydrogels; Iridoids; Male; Mesenchymal Stem Cells; Mice; Myocardial Infarction; Myocardium; Rats; Rats, Wistar; Tissue Scaffolds

A kind of self-fluorescent, biocompatible, and low-toxic Genipin crosslinked Globin-PEI nanoparticle (Gb-G-PEI NP) with high enzymolysis-stability and photo-stability was synthesized successfully. The properties of the Gb-G-PEI NP were characterized, including its particle size, surface zeta potential, morphology, paclitaxel (PTX) loading capacity and release. The Gb-G-PEI NPs as imaging probe were investigated by Confocal Laser Scanning Microscope (CLSM) in vitro and by fluorescence imaging system in vivo. Cell imaging results showed that the tumor cell line (HepG-2) had the faster cell uptake rate and metabolism rate than the normal cell line (L-O2), this difference showed its tumor selectivity. MTT assay revealed that the PTX-loaded Gb-G-PEI NPs showed almost the equal potence to tumor cell HepG-2 as the free PTX at the same PTX concentration, while a lower cytotoxicity to normal cell L-O2, suggesting its promising utilization as a drug delivery system. The imaging on mice demonstrated the possibility of the self-fluorescent Gb-G-PEI NPs as probe in vivo. So Gb-G-PEI NPs can be potentially utilized as both tracking marker and tumor cell selective drug delivery system in the biomaterial field.

Topics: Animals; Drug Delivery Systems; Globins; Hep G2 Cells; Humans; Iridoids; Male; Mice; Mice, Inbred ICR; Microscopy, Confocal; Nanoparticles; Paclitaxel; Polyethyleneimine

Genipin-1-o-glucuronic acid and genipin-monosulfate are two major metabolites from geniposide and genipin. Based on diabetic rat model, we developed a simultaneous quantification method to investigate their comparative pharmacokinetics by online mircrodialysis-ultra performance liquid chromatography-mass spectrometry (MD-UPLC-MS/MS) without their standard compounds. Online microdialysis sampling could avoid unexpected contamination or degradation of the analytes during the storage and transfer steps. Combined with good sensitivity, selectivity and selectivity of UPLC-MS/MS, online MD-UPLC-MS/MS method could real-timely monitor metabolites in rat blood for quantitative analysis. Our research found that AUC

Topics: Animals; Chromatography, High Pressure Liquid; Diabetes Mellitus, Experimental; Iridoids; Linear Models; Male; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Sensitivity and Specificity; Tandem Mass Spectrometry

Degenerative disc disease, associated with discrete structural changes in the peripheral annulus and vertebral endplate, is one of the most common pathological triggers of acute and chronic low back pain, significantly depreciating an individual's quality of life and instigating huge socioeconomic costs. Novel emerging therapeutic techniques are hence of great interest to both research and clinical communities alike. Exogenous crosslinking, such as Genipin, and platelet-rich plasma therapies have been recently demonstrated encouraging results for the repair and regeneration of degenerated discs, but there remains a knowledge gap regarding the quantitative degree of effectiveness and particular influence on the mechanical properties of the disc. This study aimed to investigate and quantify the material properties of intact (N = 8), trypsin-denatured (N = 8), Genipin-treated (N = 8), and platelet-rich plasma-treated (N = 8) discs in 32 porcine thoracic motion segments. A poroelastic finite element model was used to describe the mechanical properties during different treatments, while a meta-model analytical approach was used in combination with ex vivo experiments to extract the poroelastic material properties. The results revealed that both Genipin and platelet-rich plasma are able to recover the mechanical properties of denatured discs, thereby affording promising therapeutic modalities. However, platelet-rich plasma-treated discs fared slightly, but not significantly, better than Genipin in terms of recovering the glycosaminoglycans content, an essential building block for healthy discs. In addition to investigating these particular degenerative disc disease therapies, this study provides a systematic methodology for quantifying the detailed poroelastic mechanical properties of intervertebral disc.

Topics: Animals; Biomechanical Phenomena; Computer Simulation; Cross-Linking Reagents; Disease Models, Animal; Elasticity; Finite Element Analysis; Humans; In Vitro Techniques; Intervertebral Disc Degeneration; Iridoids; Models, Biological; Platelet-Rich Plasma; Regenerative Medicine; Sus scrofa

To explore the roles of mitochondrial Uncoupling Protein 2 (UCP2) in cumulus cells (CCs), human CCs were cultured in vitro, and the UCP2 was inhibited by treatment with Genipin, a special UCP inhibitor, or by RNA interference targeting UCP2. No significant differences in adenosine triphosphate levels and the ratio of ADP/ATP were observed after UCP2 inhibition. UCP2 inhibition caused a significant increase in cellular oxidative damage, which was reflected in alterations to several key parameters, including reactive oxygen species (ROS) and lipid peroxidation levels and the ratio of reduced GSH to GSSG. UCP2 blocking resulted in an obvious increase in active Caspase-3, accompanied by the decline of proactive Caspase-3 and a significant increase in the LC3-II/LC3-I ratio, suggesting that UCP2 inhibition triggered cellular apoptosis and autophagy. The mRNA and protein expression of connexin 43 (Cx43), a gap junction channel protein, were significantly reduced after treatment with Genipin or siRNA. The progesterone level in the culture medium was also significantly decreased after UCP2 inhibition. Our data indicated that UCP2 plays highly important roles in mediating ROS production and regulating apoptosis and autophagy, as well as maintaining gap junction integrity and progesterone synthesis, which suggests that UCP2 is involved in the regulation of follicle development and early embryo implantation and implies that it might serve as a potential biomarker for oocyte quality and competency.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Apoptosis; Autophagy; Cumulus Cells; Female; Gap Junctions; Gene Knockdown Techniques; Humans; Iridoids; Oocytes; Oxidation-Reduction; Oxidative Stress; Progesterone; RNA, Small Interfering; Uncoupling Protein 2

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 °C and can be used immediately following neutralization or stored at 4 °C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

Topics: Animals; Cell Culture Techniques; Extracellular Matrix; Hydrogels; Iridoids; Lung; Mice; Rats; Swine; Temperature

Geniposide is a key iridoid glycoside from Gardenia jasminoides fructus widely used in traditional Chinese herbal medicine. However, detection of this small molecule represents a significant challenge mostly due to the lack of specific molecular recognition elements. In this study, we have performed in vitro selection experiments to isolate DNA aptamers that can specifically bind geniposide. Using a stringent selection procedure, we have isolated DNA aptamers that can distinguish geniposide from genipin and glucose, two structural analogs of geniposide. Two top aptamers exhibit low micromolar binding affinity towards geniposide, but show significantly reduced affinity to genipin and glucose. These aptamers have the potential to be further developed into analytical tools for the detection of geniposide.

Topics: Aptamers, Nucleotide; Gardenia; Glucose; Iridoids; Medicine, Chinese Traditional; Plant Extracts; SELEX Aptamer Technique

1. Geniposide (genipin 1-O-glucose), one of the major bioactive constituents isolated from Fructus Gardeniae, possesses many biological activities. In this study, an efficient strategy was developed using ultra-high-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometer (UPLC-LTQ-Orbitrap) to profile the in vitro and in vivo metabolic patterns of geniposide in rat liver microsomes (RLMs), plasma, urine, and various tissues. And post-acquisition data-mining methods including extracted ion chromatogram (EIC), multiple mass defect filters (MMDF), fragment ion searching (FISh), and isotope pattern filtering (IPF) were adopted to characterize the known and unknown metabolites. 2. A total of 33 metabolites were detected and interpreted according to accurate mass measurement, diagnostic fragment ions, relevant drug biotransformation knowledge, and bibliography data. Among them, 17 metabolites were detected in the plasma, 31 metabolites were identified in the urine, six metabolites could be found in rat heart, 12 in liver, three in spleen, six in lung, 12 in kidney, six in brain, and four in RLMs. 3. A series of corresponding reactions such as hydrolysis, hydroxylation, taurine conjugation, hydrogenation, decarboxylation, demethylation, sulfate conjugation, cysteine S-conjugation, glucosylation, and their composite reactions were all discovered. 4. The results could provide comprehensive insights and guidance for elucidation of side effect mechanism and safety monitoring as well as for rational formulation design in drug delivery system. The newly discovered geniposide metabolites could be targets for future metabolism studies on the important chemical constituents from herbal medicines.

Topics: Animals; Chromatography, High Pressure Liquid; Iridoids; Male; Mass Spectrometry; Metabolome; Rats, Sprague-Dawley; Spectrometry, Mass, Electrospray Ionization

Roles of dietary phytochemicals in cancer chemoprevention via induction of nuclear factor-erythroid-2-related factor 2 (Nrf2)-mediated antioxidant enzymes have been well established in a number of studies. In this study, FACS analysis was used to reveal that the intracellular reactive oxygen species level decreased at 0-25 μM of genipin treatment. Furthermore, immunofluorescence analysis and Western blotting were used to demonstrate that genipin treatment resulted in the upregulation and nuclear translocation of Nrf2, as well as upregulation of gastrointestinal glutathione peroxidase. Finally, we found that C-Jun-NH2-kinase (JNK) was also dose-dependently activated, where depleting JNK by using a biochemical inhibitor indicated that JNK was upstream of Nrf2. Interestingly, the antioxidant effects were limited to the treatment in the lower dosage of genipin, where higher dosage of genipin treatment resulted in the increased reactive oxygen species level and cytotoxicity. Thus, this study demonstrates for the first time that lower dosage of genipin results in the induction of JNK/Nrf2/ARE signaling pathway and protection from cell death.

Topics: Adenocarcinoma; Anticarcinogenic Agents; Cell Line, Tumor; Humans; Iridoids; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; NF-E2-Related Factor 2; Oxidative Stress; Reactive Oxygen Species; Response Elements; Stomach Neoplasms

Genipin gel dosimeters are hydrogels infused with a radiation-sensitive material which yield dosimetric information in three dimensions (3D). The effect of inorganic salts and glucose on the visible absorption dose-response, melting points and mass density of genipin gel dosimeters has been experimentally evaluated using 6-MV LINAC photons. As a result, the addition of glucose with optimum concentration of 10% (w/w) was found to improve the thermal stability of the genipin gel and increase its melting point (Tm) by 6 °C accompanied by a slight decrease of dose-response. Furthermore, glucose helps to adjust the gel mass density to obtain the desired tissue-equivalent properties. A drop of Tm was observed when salts were used as additives. As the salt concentration increased, gel Tm decreased. The mass density and melting point of the genipin gel could be adjusted using different amounts of glucose that improved the genipin gel suitability for 3D dose measurements without introducing additional toxicity to the final gel.

Topics: Dose-Response Relationship, Radiation; Gels; Glucose; Iridoids; Phantoms, Imaging; Photons; Radiation Dosage; Radiometry; Salts; Spectrophotometry; Temperature; Water

Optic atrophy 1 (Opa1) is a critical factor that regulates fusion and other important functions of mitochondria. In mitochondrion, the N-terminal mitochondrial targeting sequence of Opa1 precursors is removed to generate Opa1 long isoforms (L-Opa1), which are further cleaved into short isoforms (S-Opa1). In the present study, we found that retinal ischemia-reperfusion (I/R) injury and intravitreal injection of carbonylcyanide m-chlorophenyl hydrazone (CCCP) both dramatically induced Opa1 cleavage and caused loss of L-Opa1. In cultured neuronal cells under hypoxia-reoxygenation (H/R) injury, similar changes for Opa1 were also observed. In contrast, restoration of L-Opa1 level by overexpression of S1 cleavage site deletion Opa1 splice 1 (Opa1-ΔS1) not only normalized the H/R-induced mitochondrial morphology changes, but also inhibited the H/R-induced apoptosis, necrosis, and the intracellular ATP loss. Furthermore, recovering L-Opa1 level in the I/R-injured retina by intravitreal injection of genipin or overexpression of Opa1-ΔS1 inhibited apoptosis, necrosis, cell loss in the ganglion cell layer and retinal thickness reduction. Together, our data demonstrated the loss of L-Opa1 is involved in the development of retinal I/R injury, indicating restoring L-Opa1 level may be considered as a therapeutic target for I/R injury-related diseases, at least for the retina. Key messages: Retinal ischemia-reperfusion (I/R) or hypoxia-reoxygenation (H/R) injury induces L-Opa1 loss. Opa1-ΔS1 overexpression inhibits H/R-induced L-Opa1 loss. Opa1-ΔS1 overexpression inhibits H/R-induced mitochondria morphology change. Opa1-ΔS1 and genipin inhibit retinal I/R injury-induced necroptosis. Opa1-ΔS1 and genipin inhibit retinal I/R injury-induced neurodegeneration.

Topics: Animals; Apoptosis; Cell Death; Cell Line; GTP Phosphohydrolases; Humans; Iridoids; Male; Mitochondria; Protein Isoforms; Rats, Wistar; Reperfusion Injury; Retina; Retinal Neurons

At various developmental stages, pluripotent stem cells (PSCs) and their progeny secrete a large amount of extracellular matrices (ECMs) which could interact with regulatory growth factors to modulate stem cell lineage commitment. ECMs derived from PSC can be used as unique scaffolds that provide broad signaling capacities to mediate cellular differentiation. However, the rapid degradation of ECMs can impact their applications as the scaffolds for in vitro cell expansion and in vivo transplantation. To address this issue, this study investigated the effects of crosslinking on the ECMs derived from embryonic stem cells (ESCs) and the regulatory capacity of the crosslinked ECMs on the proliferation and differentiation of reseeded ESC-derived neural progenitor cells (NPCs). To create different biological cues, undifferentiated aggregates, spontaneous embryoid bodies, and ESC-derived NPC aggregates were decellularized. The derived ECMs were crosslinked using genipin or glutaraldehyde to enhance the scaffold stability. ESC-derived NPC aggregates were reseeded on different ECM scaffolds and differential cellular compositions of neural progenitors, neurons, and glial cells were observed. The results indicate that ESC-derived ECM scaffolds affect neural differentiation through intrinsic biological cues and biophysical properties. These scaffolds have potential for in vitro cell culture and in vivo tissue regeneration study.. Dynamic interactions of acellular extracellular matrices and stem cells are critical for lineage-specific commitment and tissue regeneration. Understanding the synergistic effects of biochemical, biological, and biophysical properties of acellular matrices would facilitate scaffold design and the functional regulation of stem cells. The present study assessed the influence of crosslinked embryonic stem cell-derived extracellular matrix on neural differentiation and revealed the synergistic interactions of various matrix properties. While embryonic stem cell-derived matrices have been assessed as tissue engineering scaffolds, the impact of crosslinking on the embryonic stem cell-derived matrices to modulate neural differentiation has not been studied. The results from this study provide novel knowledge on the interface of embryonic stem cell-derived extracellular matrix and neural aggregates. The findings reported in this manuscript are significant for stem cell differentiation toward the applications in stem cell-based drug screening, disease modeling, and cell therapies.

Topics: Animals; Cell Differentiation; Cell Line; Cross-Linking Reagents; Extracellular Matrix; Glutaral; Iridoids; Mice; Mouse Embryonic Stem Cells; Neural Stem Cells; Tissue Scaffolds

An acid urease from Providencia rettgeri JN-B815 was purified via ultrasonication, ethanol precipitation, and DEAE ion-exchange column chromatography. It was found that the enzyme exhibits not only urease activity, but also urethanase activity, which made it possible to reduce EC already existed or would produce and its precursor urea at the same time. Then, crosslinked enzyme aggregates of P. rettgeri urease (PRU-CLEAs) were prepared using genipin as crosslinking agent. The purification process of acid urease, the effects of genipin concentration, and crosslinking time on PRU-CLEAs activity were investigated. The crosslinking was performed at pH 4.5 for 2.5 h, using 0.3% genipin as crosslinking agent, and 0.3 g · L(-1) bovine serum albumin as protein feeder. Using the obtained PRU-CLEAs, the removal rate of urea was up to 9.31 mg · L(-1) · h(-1). The removal rate of urea was still up to 7.56 mg · L(-1) · h(-1) after PRU-CLEAs was re-used for 6 times. When PRU-CLEAs were applied in a batch stirred and membrane reactor, the removal rate of urea in rice wine reached 5.16 mg · L(-1) · h(-1) and the removal rate of EC was 9.21 μg · L(-1) · h(-1). Furthermore, the treatment with PRU-CLEAs revealed no significant change of volatile flavor substances in Chinese rice wine. Thus PRU-CLEAs have great potential in the elimination of EC in Chinese rice wine.

Topics: Amidohydrolases; Cross-Linking Reagents; Enterobacteriaceae; Enzyme Activation; Ethanol; Iridoids; Oryza; Urea; Urease; Wine

Osteochondral xenografts are potentially inexpensive, widely available alternatives to fresh allografts. However, antigen removal from xenogenic cartilage may damage the extracellular matrix and reduce compressive stiffness. Non-crosslinked xenogenic cartilage may also undergo rapid enzymatic degradation in vivo. We hypothesized that natural crosslinking agents could be used in place of glutaraldehyde to improve the mechanical properties and enzymatic resistance of decellularized cartilage. This study compared the effects of genipin (GNP), proanthocyanidin (PA), and epigallocatechin gallate (EGCG), on the physical and mechanical properties of decellularized porcine cartilage. Glutaraldehyde (GA) served as a positive control. Porcine articular cartilage discs were decellularized in 2% sodium dodecyl sulfate and DNase I followed by fixation in 0.25% GNP, 0.25% PA, 0.25% EGCG, or 2.5% GA. Decellularization decreased DNA by 15% and GAG by 35%. For natural crosslinkers, the average degree of crosslinking ranged from approximately 50% (EGCG) to 78% (GNP), as compared to 83% for the GA control. Among the natural crosslinkers, only GNP significantly affected the disc diameter, and shrinkage was under 2%. GA fixation had no significant effect on disc diameter. Decellularization decreased aggregate modulus; GA and GNP, but not EGCG and PA, were able to restore it to its original level. GNP, PA, and GA conferred a similar, almost complete resistance to collagenase degradation. EGCG also conferred substantial resistance but to a lesser degree. Overall, the data support our hypothesis and suggest that natural crosslinkers may be suitable alternatives to glutaraldehyde for stabilization of decellularized cartilage. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1037-1046, 2016.

Topics: Animals; Cartilage; Cartilage, Articular; Catechin; Collagenases; Cross-Linking Reagents; Drug Evaluation, Preclinical; Grape Seed Extract; Heterografts; Iridoids; Proanthocyanidins; Swine

Autophagy is an essential cytoprotective system that is rapidly activated in response to various stimuli including inflammation and microbial infection. Genipin, an aglycon of geniposide found in gardenia fruit, is well known to have anti-inflammatory, antibacterial and antioxidative properties. This study examined the protective mechanisms of genipin against sepsis, with particular focus on the autophagic signalling pathway.. Mice were subjected to sepsis by caecal ligation and puncture (CLP). Genipin (1, 2.5 and 5 mg·kg(-1) ) or vehicle (saline) was injected i.v. immediately (0 h) after CLP, and chloroquine (60 mg·kg(-1) ), an autophagy inhibitor, was injected i.p. 1 h before CLP. Blood and liver tissues were isolated 6 h after CLP.. Genipin improved survival rate and decreased serum levels of aminotransferases and pro-inflammatory cytokines after CLP; effects abolished by chloroquine. The liver expression of autophagy-related protein (Atg)12-Atg5 conjugate increased after CLP, and this increase was enhanced by genipin. CLP decreased Atg3 protein liver expression, and genipin attenuated this decrease. CLP impaired autophagic flux, as indicated by increased liver expression of microtubule-associated protein-1 light chain 3-II and sequestosome-1/p62 protein; this impaired autophagic flux was restored by genipin, and chloroquine abolished this effect. Genipin also attenuated the decreased expression of lysosome-associated membrane protein-2 and Rab7 protein and increased expression of calpain 1 protein induced by CLP in the liver.. Our findings suggest that genipin protects against septic injury by restoring impaired autophagic flux. Therefore, genipin might be a potential therapeutic agent for the treatment of sepsis.

Topics: Alanine Transaminase; Animals; Aspartate Aminotransferases; Autophagy; Autophagy-Related Proteins; Chloroquine; Interleukin-6; Iridoids; Liver; Liver Diseases; Male; Mice; Mice, Inbred ICR; Microscopy, Electron, Transmission; Protective Agents; Sepsis; Tumor Necrosis Factor-alpha

In order to develop safer processes for the food industry, we prepared a chitosan support with the naturally occurring crosslinking reagent, genipin, for enzyme. As application model, it was tested for the immobilization of β-D-galactosidase from Aspergillus oryzae. Chitosan particles were obtained by precipitation followed by adsorption of the enzyme and crosslinking with genipin. The particles were characterized by Fourier transform infrared (FTIR) spectroscopy and thermogravimetric analysis (TGA). The immobilization of the enzyme by crosslinking with genipin provided biocatalysts with satisfactory activity retention and thermal stability, comparable with the ones obtained with the traditional methodology of immobilization using glutaraldehyde. β-D-Galactosidase-chitosan-genipin particles were applied to galactooligosaccharides synthesis, evaluating the initial lactose concentration, pH and temperature, and yields of 30% were achieved. Moreover, excellent operational stability was obtained, since the immobilized enzyme maintained 100% of its initial activity after 25 batches of lactose hydrolysis. Thus, the food grade chitosan-genipin particles seem to be a good alternative for application in food process.

Topics: beta-Galactosidase; Catalysis; Chitosan; Enzymes, Immobilized; Glutaral; Iridoids

Genipin (GNP) effectively inhibits uncoupling protein 2 (UCP2), which regulates the leakage of protons across the inner mitochondrial membrane. UCP2 inhibition may induce pancreatic adenocarcinoma cell death by increasing reactive oxygen species (ROS) levels. In this study, the hydroxyls at positions C10 (10-OH) and C1 (1-OH) of GNP were hypothesized to be the active groups that cause these inhibitory effects. Four GNP derivatives in which the hydroxyl at position C10 or C1 was replaced with other chemical groups were synthesized and isolated. Differences in the inhibitory effects of GNP and its four derivatives on pancreatic carcinoma cell (Panc-1) proliferation were assessed. The effects of GNP and its derivatives on apoptosis, UCP2 inhibition and ROS production were also studied to explore the relationship between GNP's activity and its structure. The derivatives with 1-OH substitutions, geniposide (1-GNP1) and 1-ethyl-genipin (1-GNP2) lacked cytotoxic effects, while the other derivatives that retained 1-OH, 10-piv-genipin (10-GNP1) and 10-acetic acid-genipin (10-GNP2) exerted biological effects similar to those of GNP, even in the absence of 10-OH. Thus, 1-OH is the key functional group in the structure of GNP that is responsible for GNP's apoptotic effects. These cytotoxic effects involve the induction of Panc-1 cell apoptosis through UCP2 inhibition and subsequent ROS production.

Topics: Apoptosis; Cell Line, Tumor; Cell Survival; Hep G2 Cells; Humans; Ion Channels; Iridoids; Mitochondrial Proteins; Reactive Oxygen Species; Structure-Activity Relationship; Uncoupling Protein 2

CHR20 and CHR21 are a pair of stable diastereoisomers derived from genipin. These stereoisomers are activators of neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS). In the rat retinal ganglion (RGC-5) cell model these compounds are non-toxic. Treatment of RGC-5 with 750 μM of sodium nitroprusside (SNP) produces nitrosative stress. Both genipin derivatives, however, protect these cells against SNP-induced apoptic cell death, although CHR21 is significantly more potent than CHR20 in this regard. With Western blotting we showed that the observed neuroprotection is primarily due to the activation of protein kinase B (Akt)/eNOS and extracellular signal-regulated kinase (ERK1/2) signaling pathways. Therefore, LY294002 (a phosphatidylinositol 3-kinase (PI3K) inhibitor) or PD98059 (a MAPK-activating enzyme inhibitor) abrogated the protective effects of CHR20 and CHR21. Altogether, our results show that in our experimental setup neuroprotection by the diasteromeric pair is mediated through the PI3K/Akt/eNOS and ERK1/2 signaling pathways. Further studies are needed to establish the potential of these compounds to prevent ntric oxide (NO)-induced toxicity commonly seen in many neurodegenerative diseases.

Topics: Animals; Apoptosis; Cell Line; Cell Survival; Chromones; Flavonoids; Gene Expression Regulation; Iridoids; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Morpholines; Neuroprotective Agents; Nitric Oxide; Nitric Oxide Donors; Nitroprusside; Oxidative Stress; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Proto-Oncogene Proteins c-akt; Rats; Retinal Ganglion Cells; Signal Transduction

The aim of the present investigation was to design biocompatible gelatin nanoparticles, capable of releasing the cytarabine drug in a controllable way by regulating the extent of swelling of nanoparticles. In order to achieve the proposed objectives, gelatin (Type A, derived from acid cured tissue) was modified by crosslinking with genipin and nanoparticles of crosslinked gelatin were prepared using single water in oil (W/O) emulsion technique. The nanoparticles were characterized by techniques like FTIR, SEM, TEM, particles size analysis, and surface potential measurements. The nanoparticle chemical architecture was found to influence drug-releasing capacity. The influence of experimental conditions such as pH and simulated physiological fluids as the release medium was also investigated on the release profiles of cytarabine. It is possible to fabricate high-performance materials, by designing of controlled size gelatin nanoparticles with good biocompatible properties along with desired drug release profiles.

Topics: Antimetabolites, Antineoplastic; Cytarabine; Drug Carriers; Drug Liberation; Gelatin; Hydrogen-Ion Concentration; Iridoids; Nanoparticles; Particle Size; Spectroscopy, Fourier Transform Infrared

It has been a great challenge to develop aldehyde-free tissue adhesives that can function rapidly and controllably on wet internal tissues with fine adhesion strength, sound biocompatibility and degradability. To this end, we have devised a mussel-inspired easy-to-use double-crosslink tissue adhesive (DCTA) comprising a dopamine-conjugated gelatin macromer, a rapid crosslinker (namely, Fe(3+)), and a long-term acting crosslinker (namely, genipin). As a mussel-inspired gluing macromer, dopamine is grafted onto gelatin backbone via an one-step reaction, the catechol groups of which are capable of performing strong wet adhesion on tissue surfaces. By addition of genipin and Fe(3+), the formation of catechol-Fe(3+) complexation and accompanying spontaneous curing of genipin-primed covalent crosslinking of gluing macromers in one pot endows DCTA with the double-crosslink adhesion mechanism. Namely, the reversible catechol-Fe(3+) crosslinking executes an controllable and instant adhesive curing; while genipin-induced stable covalent crosslinking promises it with long-term effectiveness. This novel DCTA exhibits significantly higher wet tissue adhesion capability than the commercially available fibrin glue when applied on wet porcine skin and cartilage. In addition, this DCTA also demonstrates fine elasticity, sound biodegradability, and biocompatibility when contacting in vitro cultured cells and blood. In vivo biocompatibility and biodegradability are checked and confirmed via trials of subcutaneous implantation in nude mice model. This newly developed DCTA may be a highly promising product as a biological glue for internal medical use including internal tissue adhesion, sealing, and hemostasis.. There is a great demand for ideal tissue adhesives that can be widely used in gluing wet internal tissues. Here, we have devised a mussel-inspired easy-to-use double-crosslink tissue adhesive (DCTA) that meets the conditions as an ideal tissue adhesive. It is composed of gelatin-dopamine conjugates - a gluing macromer, Fe(3+) - a rapid crosslinker, and genipin - a long-term acting crosslinker. This DCTA is constructed with a novel complexation-covalent double-crosslinking principle in one pot, in which the catechol-Fe(3+) crosslinking executes a controllable and instant adhesive curing, at the same time, genipin-induced covalent crosslinking promises it with long-term effectiveness in physiology conditions. This novel DCTA, with excellent wet tissue adhesion capability, fine elasticity, sound biodegradability, and biocompatibility, is a promising biological glue for internal medical use in surgical operations.

Topics: Animals; Biocompatible Materials; Bivalvia; Cell Proliferation; Cross-Linking Reagents; Dermis; Dopamine; Fibroblasts; Gelatin; Hemolysis; Humans; Implants, Experimental; Iridoids; Kinetics; Materials Testing; Mice, Nude; Organ Specificity; Sus scrofa; Tissue Adhesives

To evaluate the efficacy and safety of posterior scleral reinforcement (PSR) using genipin cross-linked sclera as the material to treat macular detachment and retinoschisis, both without macular hole, in highly myopic eyes.. Twenty-one patients with highly myopic eyes (24 eyes) with macular detachment and retinoschisis were treated sequentially with genipin cross-linked PSR and were followed for at least 1 year after surgery. The best-corrected visual acuity (BCVA), spherical equivalent (SE) power, axial length (AL), optical coherence tomography, and the complications were evaluated.. The mean SE decreased from -13.81±4.67 D preoperatively to -9.64±4.86 D postoperatively, while the improvement in the logMAR BCVA values was from 1.24±0.57 before surgery to 1.03±0.57 after surgery. The preoperative AL (29.73±2.31 mm) was decreased (28.08±2.08 mm) after the operation. The retina in 21 eyes (87.5%) was successful reattached and the macular detachment was significantly decreased in two eyes; a macular hole occurred in one eye.. For at least a 1 year period of follow-up, PSR with genipin cross-linked sclera was safe and effective to treat macular detachment and retinoschisis in high myopia when a macular hole was not present. The reinforcement effect tended to be stabilised and maintained for 6 months after treatment.

Topics: Adhesives; Adult; Aged; Female; Follow-Up Studies; Humans; Iridoids; Male; Middle Aged; Myopia, Degenerative; Ophthalmoscopy; Refraction, Ocular; Retinal Detachment; Retinoschisis; Retrospective Studies; Sclera; Scleral Buckling; Time Factors; Tomography, Optical Coherence; Treatment Outcome; Visual Acuity

In homeotherms, the hypothalamus controls thermoregulatory and adaptive mechanisms in energy balance, sleep-wake and locomotor activity to maintain optimal body temperature. Orexin neurons may be involved in these functions as they promote thermogenesis, food intake and behavioral arousal, and are sensitive to temperature and metabolic status. How thermal and energy balance signals are integrated in these neurons is unknown. Thus, we investigated the cellular mechanisms of thermosensing in orexin neurons and their response to a change in energy status using whole-cell patch clamp on rat brain slices. We found that warming induced an increase in miniature excitatory postsynaptic current (EPSC) frequency, which was blocked by the transient receptor potential vanilloid-1 (TRPV1) receptor antagonist AMG9810 and mimicked by its agonist capsaicin, suggesting that the synaptic effect is mediated by heat-sensitive TRPV1 channels. Furthermore, warming inhibits orexin neurons by activating ATP-sensitive potassium (KATP) channels, an effect regulated by uncoupling protein 2 (UCP2), as the UCP2 inhibitor genipin abolished this response. These properties are unique to orexin neurons in the lateral hypothalamus, as neighboring melanin-concentrating hormone neurons showed no response to warming within the physiological temperature range. Interestingly, in rats fed with western diet for 1 or 11weeks, orexin neurons had impaired synaptic and KATP response to warming. In summary, this study reveals several mechanisms underlying thermosensing in orexin neurons and their attenuation by western diet. Overeating induced by western diet may in part be due to impaired orexin thermosensing, as post-prandial thermogenesis may promote satiety and lethargy by inhibiting orexin neurons.

Topics: Acrylamides; Animals; Bridged Bicyclo Compounds, Heterocyclic; Capsaicin; Diet, High-Fat; Excitatory Postsynaptic Potentials; Hot Temperature; Hypothalamic Area, Lateral; Hypothalamic Hormones; Iridoids; KATP Channels; Male; Melanins; Miniature Postsynaptic Potentials; Neurons; Neurotransmitter Agents; Orexins; Patch-Clamp Techniques; Pituitary Hormones; Rats, Sprague-Dawley; Thermosensing; Tissue Culture Techniques; TRPV Cation Channels; Uncoupling Protein 2

Crosslinking of gelatin nanofibers maintaining a fibrous morphology after exposure to an aqueous solution is still a challenge. In this work, we developed an innovative method based on the use of an ad hoc designed co-axial needle to fabricate gelatin mats crosslinked with a very small amount of genipin and still able to retain their morphology when immersed in aqueous solution. Genipin-containing gelatin nanofibers are obtained by allowing mixing of the two solutions just within the needle. Genipin content of the electrospun mats can be modulated by varying feeding rates of the inner and outer solutions and their relative concentration. A subsequent thermal treatment of the mats, performed at 55 °C or 37 °C for 1 or 3 days and followed by rapid rinsing in ethanol and then in PB, allows one to obtain highly crosslinked gelatin nanofibers that perfectly maintain their morphology after immersion in an aqueous solution, display improved mechanical properties and enhanced stability. This new approach allows us to achieve gelatin mat stabilization using a very small amount of genipin with respect to other methods and to avoid post-treatment of the mats with the crosslinking agent, with a consequent significant reduction of the final cost of the materials. Moreover, in vitro tests demonstrate that the crosslinked mats support normal human primary chondrocyte culture, promoting their differentiation.

Topics: Biocompatible Materials; Cell Differentiation; Cell Survival; Cells, Cultured; Chondrocytes; Cross-Linking Reagents; Gelatin; Humans; Iridoids; L-Lactate Dehydrogenase; Materials Testing; Microscopy, Electron, Scanning; Nanofibers; Nanotechnology; Tissue Engineering; Tissue Scaffolds

Development of biomimetic scaffolds represents a promising direction in bone tissue engineering. In this study, we designed a two-step process to prepare a type of biomimetic hybrid hydrogels that were composed of collagen, hydroxyapatite (HAP) and alendronate (ALN), an anti-osteoporosis drug. First, water-soluble ALN-conjugated HAP (HAP-ALN) containing 4.0wt.% of ALN was synthesized by treating HAP particles with ALN. Hydrogels were then formed from HAP-ALN conjugate and collagen under physiological conditions using genipin (GNP) as the crosslinker. Depending on the ALN/collagen molar ratio and GNP concentration, the gelation time of hydrogels ranged from 5 to 37min. Notably, these hybrid hydrogels exhibited markedly improved mechanical property (storage modulus G'=38-187kPa), higher gel contents, and lower swelling ratios compared to the hydrogels prepared from collagen alone under similar conditions. Moreover, they showed tunable degradation behaviors against collagenase. The collagen/HAP-ALN hybrid hydrogels supported the adhesion and growth of murine MC3T3-E1 osteoblastic cells well. Such tough yet enzymatically degradable hybrid hydrogels hold potential as scaffolds for bone tissue engineering.

Topics: Alendronate; Animals; Biomimetic Materials; Bone and Bones; Bone Density Conservation Agents; Cell Adhesion; Cell Line; Cell Proliferation; Cell Survival; Collagen; Collagenases; Cross-Linking Reagents; Durapatite; Hydrogels; Hydrolysis; Iridoids; Mice; Osteoblasts; Phase Transition; Tissue Engineering; Tissue Scaffolds

In liver tissue engineering, scaffolds with porous structure desgined to supply nutrient and oxygen exchange for three-dimensional (3-D) cells culture, and maintain liver functions. Meanwhile, genipin, as a natural crosslinker, is widely used to crosslink biomaterials in tissue engineering, with lower cytotoxicity and better biocompatibility. In present study, chitosan/gelatin 3-D scaffolds crosslinked by genipin, glutaraldehyde or 1-(3-dimethylaminopropyl)-3-ethyl-carbodimide hydrochloride (EDC) were prepared and characterized by Fourier-transform infrared (FT-IR) and scanning electron microscopy (SEM). The biocompatibility of chitosan/gelatin scaffolds corsslinked with different crosslinkers was investigated by cell viability, morphology and liver specific functions. The result showed that the 1% and 2% genipin crosslinked chitosan/gelatin scaffolds possess ideal porosity. The genipin crosslinked 3-D scaffolds possessed the best biocompatibility than that of the others, and maintained liver specific functions when HepG2 cells seeded on scaffolds. The cellular morphology of HepG2 cells seeded on scaffolds showed that cells could penetrate into the scaffolds and proliferate significantly. Therefore, genipin crosslinked chitosan/gelatin scaffolds could be a promising biomaterial used in liver tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1863-1870, 2016.

Topics: Animals; Cattle; Cell Movement; Cell Proliferation; Cell Shape; Chitosan; Cross-Linking Reagents; Gelatin; Hep G2 Cells; Humans; Imaging, Three-Dimensional; Iridoids; Liver; Spectroscopy, Fourier Transform Infrared; Tissue Engineering; Tissue Scaffolds

In this study porous gelatin scaffolds were prepared using in-situ gas foaming, and four crosslinking agents were used to determine a biocompatible and effective crosslinker that is suitable for such a method. Crosslinkers used in this study included: hexamethylene diisocyanate (HMDI), poly(ethylene glycol) diglycidyl ether (epoxy), glutaraldehyde (GTA), and genipin. The prepared porous structures were analyzed using Fourier Transform Infrared Spectroscopy (FT-IR), thermal and mechanical analysis as well as water absorption analysis. The microstructures of the prepared samples were analyzed using Scanning Electron Microscopy (SEM). The effects of the crosslinking agents were studied on the cytotoxicity of the porous structure indirectly using MTT analysis. The affinity of L929 mouse fibroblast cells for attachment on the scaffold surfaces was investigated by direct cell seeding and DAPI-staining technique. It was shown that while all of the studied crosslinking agents were capable of stabilizing prepared gelatin scaffolds, there are noticeable differences among physical and mechanical properties of samples based on the crosslinker type. Epoxy-crosslinked scaffolds showed a higher capacity for water absorption and more uniform microstructures than the rest of crosslinked samples, whereas genipin and GTA-crosslinked scaffolds demonstrated higher mechanical strength. Cytotoxicity analysis showed the superior biocompatibility of the naturally occurring genipin in comparison with other synthetic crosslinking agents, in particular relative to GTA-crosslinked samples.

Topics: Animals; Biocompatible Materials; Cell Line; Cell Survival; Cross-Linking Reagents; Epoxy Resins; Gelatin; Glutaral; Iridoids; Isocyanates; Mice; Microscopy, Electron, Scanning; Porosity; Spectroscopy, Fourier Transform Infrared; Tensile Strength; Tissue Engineering; Tissue Scaffolds; Transition Temperature; Water

Decellularization techniques have been widely used as an alternative strategy for organ reconstruction. This study investigated the mechanical, pro-angiogenic and in vivo biocompatibility properties of decellularized airway matrices cross-linked with genipin. New Zealand rabbit tracheae were decellularized and cross-linked with genipin, a naturally derived agent. The results demonstrated that, a significant (p < 0.05) increase in the secant modulus was computed for the cross-linked tracheae, compared to the decellularized samples. Angiogenic assays demonstrated that decellularized tracheal scaffolds and cross-linked tracheae treated with 1% genipin induce strong in vivo angiogenic responses (CAM analysis). Seven, 15 and 30 days after implantation, decreased (p < 0.01) inflammatory reactions were observed in the xenograft models for the genipin cross-linked tracheae matrices compared with control tracheae, and no increase in the IgM or IgG content was observed in rats. In conclusion, treatment with genipin improves the mechanical properties of decellularized airway matrices without altering the pro-angiogenic properties or eliciting an in vivo inflammatory response.

Topics: Animals; Biocompatible Materials; Biomechanical Phenomena; Chick Embryo; Extracellular Matrix; Heterografts; Iridoids; Male; Materials Testing; Neovascularization, Physiologic; Rabbits; Rats; Tissue Engineering; Tissue Scaffolds; Trachea

Highly porous genipin cross-linked membranes of carboxymethylchitosan exhibiting different crosslinking degree (3%

Topics: Animals; Chitosan; Cross-Linking Reagents; Decapodiformes; Elastic Modulus; Iridoids; Membranes, Artificial; Muramidase; Porosity; Proton Magnetic Resonance Spectroscopy; Spectroscopy, Fourier Transform Infrared; Tensile Strength; Viscosity

The suture-tendon interface is often the weakest link in tendon to bone repair of massive rotator cuff tears. Genipin is a low-toxicity collagen crosslinker derived from the gardenia fruit that has been shown to augment collagen tissue strength and mechanically arrest tendon-tear progression.. The purpose of the current study was to evaluate whether genipin crosslinking can sufficiently augment the suture-tendon interface to improve suture pullout strength using simple single-loop sutures and the modified Mason-Allen technique. The study also aimed to assess whether time of genipin treatment is a relevant factor in efficacy.. In an ex vivo (cadaveric) sheep rotator cuff tendon model, a total of 142 suture pullout tests were performed on 32 infraspinatus tendons. Each tendon was prepared with three single-loop stitches. Two groups were pretreated by incubation in genipin solution for either 4 hours or 24 hours. Two corresponding control groups were incubated in phosphate buffered saline for the same periods. The same test protocol was applied to tendons using modified Mason-Allen technique stitch patterns. Each suture was loaded to failure on a universal materials testing machine. Suture pullout force, stiffness, and work to failure were calculated from force-displacement data, and then compared among the groups.. Median single-loop pullout force on tendons incubated for 24 hours in genipin yielded an approximately 30% increase in maximum pullout force for single-loop stitches with a median of 73 N (range, 56-114 N) compared with 56 N (range, 40-69 N; difference of medians = 17 N; p = 0.028), with corresponding increases in the required work to failure but not stiffness. Genipin treatment for 4 hours showed no added benefit for suture-pullout behavior (46 N, [range, 35-95 N] versus 45 N, [range, 28-63 N]; difference of medians, 1 N; p = 1). No tested genipin crosslinking conditions indicated benefit for tendons grasped using the modified Mason-Allen technique after 4 hours (162 N, [range, 143-193 N] versus 140 N, [range, 129-151 N]; difference of medians, 22 N; p = 0.114) or after 24 hours of crosslinking (172 N, [range, 42-183 N] versus 164 N [range, 151-180 N]; difference of medians, 8 N; p = 0.886).. Exogenous collagen crosslinking in genipin can markedly improve resistance to pullout at the tendon-suture interface for simple stitch patterns while the modified Mason-Allen stitch showed no benefit in an ex vivo animal model.. Tendon strength augmentation by genipin pretreatment offers the potential to improve suture retention properties. Future studies are warranted for the development of clinically viable intraoperative delivery strategies and in vivo testing for safety and efficacy.

Topics: Animals; Biomechanical Phenomena; Collagen; Cross-Linking Reagents; Disease Models, Animal; Equipment Failure; Iridoids; Materials Testing; Orthopedic Procedures; Rotator Cuff Injuries; Sheep; Stress, Mechanical; Suture Techniques; Sutures

Development of a reliable and selective anti-inflammatory agent of cyclooxygenase-2 (COX-2), induced or up-regulated by inflammatory/injury stimulus such as IL-1β, TNF-α and LPS in the various types of organs, tissues and cells, with low side effects is a long-standing medicinal chemistry problem with significant social implications.. To target druggable enzymome COX-2 by exploiting NSAIDs and genipin (GEP) in anti-inflammatory infection.. AGE pharmacophore reported herein may be an effective strategy to develop a novel anti-inflammatory agent and potential inhibitor of COX-2.

Topics: Animals; Anti-Inflammatory Agents; Arthritis, Experimental; Aspirin; Cyclooxygenase 2 Inhibitors; Drug Design; Iridoids; Male; Molecular Docking Simulation; Rats; Rats, Sprague-Dawley; Structure-Activity Relationship

Decellularized xenogeneic whole-liver matrices are plausible biomedical materials for the bioengineering of liver transplantation. A common method to reduce the inflammatory potential of xenogeneic matrices is crosslinking. Nevertheless, a comprehensive analysis of the immunogenic features of cross-linked decellularized tissue is still lacking. We aimed to reduce the immunogenicity of decellularized porcine whole-liver matrix through crosslinking with glutaraldehyde or genipin, a new natural agent, and investigated the mechanism of the immune-mediated responses. The histologic assessment of the host's immune reaction activated in response to these scaffolds, as well as the M1/M2 phenotypic polarization profile of macrophages, was studied in vivo. The genipin-fixed scaffold elicited a predominantly M2 phenotype response, while the glutaraldehyde-fixed scaffold resulted in disrupted host tissue remodeling and a mixed macrophage polarization profile. The specific subsets of immune cells involved in the responses to the scaffolds were identified in vitro. Crosslinking alleviated the host response by reducing the proliferation of lymphocytes and their subsets, accompanied by a decreased release of both Th1 and Th2 cytokines. Therefore, we conclude that the natural genipin crosslinking could lower the immunogenic potential of xenogeneic decellularized whole-liver scaffolds.

Topics: Allografts; Animals; Biocompatible Materials; Cell Proliferation; Coculture Techniques; Cytokines; Extracellular Matrix; Heterografts; Humans; Immune System; Iridoids; Leukocytes, Mononuclear; Liver; Liver Transplantation; Macrophages; Swine; T-Lymphocytes, Helper-Inducer; Tissue Engineering; Tissue Scaffolds

Peritoneal adhesions are a common complication after abdominal surgery. They cause small bowel obstruction, female infertility and chronic abdominal pain. Peritoneal adhesions also hamper uniform drug distribution in the peritoneal cavity, thereby reducing the efficacy of intraperitoneal chemotherapy after cytoreductive surgery.. The goal of this study was to develop a formulation that prevents peritoneal adhesions, evenly distributes in the abdominal cavity, and simultaneously extends residence time and improves local drug concentration. This report describes the formulation and characterization of genipin-crosslinked gelatin microspheres (GP-MS).. Spheroid gelatin microspheres were prepared by an emulsification solvent extraction method. A higher degree of crosslinking was obtained by increasing genipin concentration and crosslinking time. The degree of crosslinking allowed to tailor the degradation rate of GP-MS, hence their residence time. GP-MS did not affect cell viability. In vivo experiments showed excellent GP-MS biocompatibility and degradation characteristics. GP-MS were distributed evenly throughout the abdominal cavity. Adhesions were induced in Balb/c mice by application of an abraded peritoneal wall-cecum model. GP-MS-treated mice developed significantly less postsurgical adhesions compared to saline and Hyalobarrier(®) group. Histopathological examination showed a decrease of peritoneal inflammation over time in GP-MS-treated mice with complete recovery of peritoneal wounds post-operative day 14.. GP-MS are a promising strategy to prevent postoperative peritoneal adhesions and improve efficacy of postoperative intraperitoneal chemotherapy.

Topics: Abdominal Cavity; Animals; Cell Line, Tumor; Cell Survival; Cross-Linking Reagents; Disease Models, Animal; Emulsions; Female; Gelatin; Humans; Iridoids; Mice, Inbred BALB C; Microspheres; Peritoneum; Postoperative Complications; Surface-Active Agents; Tissue Adhesions

We previously obtained evidence in rainbow trout peripheral tissues such as liver and Brockmann bodies (BB) for the presence and response to changes in circulating levels of glucose (induced by intraperitoneal hypoglycaemic and hyperglycaemic treatments) of glucosensing mechanisms others than that mediated by glucokinase (GK). There were based on mitochondrial production of reactive oxygen species (ROS) leading to increased expression of uncoupling protein 2 (UCP2), and sweet taste receptor in liver and BB, and on liver X receptor (LXR) and sodium/glucose co-transporter 1 (SGLT-1) in BB. We aimed in the present study to obtain further in vitro evidence for the presence and functioning of these systems. In a first experiment, pools of sliced liver and BB were incubated for 6h at 15°C in modified Hanks' medium containing 2, 4, or 8mM d-glucose, and we assessed the response of parameters related to these glucosensing mechanisms. In a second experiment, pools of sliced liver and BB were incubated for 6h at 15°C in modified Hanks' medium with 8mM d-glucose alone (control) or containing 1mM phloridzin (SGLT-1 antagonist), 20μM genipin (UCP2 inhibitor), 1μM trolox (ROS scavenger), 100μM bezafibrate (T1R3 inhibitor), and 50μM geranyl-geranyl pyrophosphate (LXR inhibitor). The results obtained in both experiments support the presence and functioning of glucosensor mechanisms in liver based on sweet taste receptor whereas in BB the evidence support those based on LXR, mitochondrial activity and sweet taste receptor.

Topics: Animals; Bezafibrate; Chromans; Dose-Response Relationship, Drug; Endocrine System; Glucose; Iridoids; Liver; Liver X Receptors; Mitochondria; Oncorhynchus mykiss; Phlorhizin; Polyisoprenyl Phosphates

We previously developed a biomaterial scaffold that could effectively provide seed cells to a lesion cavity resulting from traumatic brain injury. However, we subsequently found that few transplanted human umbilical cord mesenchymal stem cells (hUC-MSCs) are able to migrate from the scaffold to the lesion boundary. Stromal derived-cell factor-1α and its receptor chemokine (C-X-C motif) receptor (CXCR)4 are chemotactic factors that control cell migration and stem cell recruitment to target areas. Given the low expression level of CXCR4 on the hUC-MSC membrane, lentiviral vectors were used to generate hUC-MSCs stably expressing CXCR4 fused to green fluorescent protein (GFP) (hUC-MSCs(CXCR4/GFP)). We constructed a scaffold in which recombinant human brain-derived neurotrophic factor (BDNF) was linked to chitosan scaffolds with the crosslinking agent genipin (CGB scaffold). The scaffold containing hUC-MSCs(CXCR4/GFP) was transplanted into the lesion cavity of a rat brain, providing exogenous hUC-MSCs to both lesion boundary and cavity. These results demonstrate a novel strategy for inducing tissue regeneration after traumatic brain injury.

Topics: Animals; Brain; Brain Injuries, Traumatic; Brain-Derived Neurotrophic Factor; Cell Adhesion; Cell Differentiation; Cell Movement; Cell Proliferation; Chitosan; Gene Expression Profiling; Green Fluorescent Proteins; Humans; Iridoids; Male; Rats; Rats, Sprague-Dawley; Receptors, CXCR4; Tissue Engineering; Tissue Scaffolds

Freestanding multilayered films were obtained using layer-by-layer (LbL) technology from the assembly of natural polyelectrolytes, namely chitosan (CHT) and chondroitin sulfate (CS). The morphology and the transparency of the membranes were evaluated. The influence of genipin (1 and 2 mg ml(-1)), a naturally-derived crosslinker agent, was also investigated in the control of the mechanical properties of the CHT/CS membranes. The water uptake ability can be tailored by changing the crosslinker concentration that also controls the Young's modulus and ultimate tensile strength. The maximum extension tends to decrease upon crosslinking with the highest genipin concentration, compromising the elastic properties of CHT/CS membranes: nevertheless, when using a lower genipin concentration, the ultimate tensile stress is similar to the non-crosslinked one, but exhibits a significantly higher modulus. Moreover, the crosslinked multilayer membranes exhibited shape memory properties, through a simple hydration action. The in vitro biological assays showed better L929 cell adhesion and proliferation when using the crosslinked membranes and confirmed the non-cytotoxicity of the developed CHT/CS membranes. Within this research work, we were able to construct freestanding biomimetic multilayer structures with tailored swelling, mechanical and biological properties that could find applicability in a variety of biomedical applications.

Topics: Adsorption; Animals; Biocompatible Materials; Cell Adhesion; Cell Line; Cell Survival; Chitosan; Chondroitin Sulfates; Cross-Linking Reagents; Elastic Modulus; Iridoids; Membranes, Artificial; Mice; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Spectroscopy, Fourier Transform Infrared; Stress, Mechanical; Tensile Strength

The possibility of genipin-crosslinked O-carboxymethyl chitosan-gum Arabic coacervate as a pH-sensitive delivery vehicle was investigated. O-carboxymethyl chitosan-gum Arabic coacervates separated in pH 3.0, 4.5, and 6.0 were crosslinked by genipin for different durations and the crosslinked products were subjected to crosslinking degree, swelling behavior, bovine serum albumin release profile, and microstructure characterization. Genipin-crosslinking greatly improved the stability of the coacervates against the simulated gastric solution and created certain pH-sensitivity. The coacervates displayed higher swelling ratios in the simulated gastric solution than in the simulated intestine and colon solutions; meanwhile, the coacervates prepared in pH 4.5 and 6.0 swelled more severely than the complex separated in pH 3.0. Nevertheless, the bovine serum albumin release in the simulated gastric solution from the microcapsules prepared in pH 6.0 was much lower than those prepared in pH 4.5 and 3.0, whose cumulative release percentages in the three simulated solutions were 17.14%, 55.23%, and 79.79%, respectively, in crosslinking duration 2 h. X-ray diffraction, scanning electron microscopy, and transmission electron microscopy analysis revealed that genipin-crosslinking improved the regularity and compactness of coacervate structure, whereas confocal laser scanning microscopy observation indicated that O-carboxymethyl chitosan content was possibly the major reason for the different swelling and bovine serum albumin release behavior of the coacervates. It was concluded that the genipin-crosslinked O-carboxymethyl chitosan-gum Arabic coacervate was a potential intestine-targeted delivery system and its delivery performance could be tailored by varying the crosslinking degree and coacervation acidity.

Topics: Capsules; Chitosan; Cross-Linking Reagents; Drug Delivery Systems; Drug Liberation; Gastric Juice; Gum Arabic; Hydrogels; Hydrogen-Ion Concentration; Intestinal Secretions; Iridoids; Serum Albumin, Bovine

Acute myocardial infarction (MI) leads to morbidity and mortality due to cardiac dysfunction. Here we identify sericin, a silk-derived protein, as an injectable therapeutic biomaterial for the minimally invasive MI repair. For the first time, sericin prepared in the form of an injectable hydrogel has been utilized for cardiac tissue engineering and its therapeutical outcomes evaluated in a mouse MI model. The injection of this sericin hydrogel into MI area reduces scar formation and infarct size, increases wall thickness and neovascularization, and inhibits the MI-induced inflammatory responses and apoptosis, thereby leading to a significant functional improvement. The potential therapeutical mechanisms have been further analyzed in vitro. Our results indicate that sericin downregulates pro-inflammatory cytokines (TNF-α and IL-18) and chemokine (CCL2) and reduces TNF-α expression by suppressing the TLR4-MAPK/NF-κB pathways. Moreover, sericin exhibits angiogenic activity by promoting migration and tubular formation of human umbilical vessel endothelial cells (HUVECs). Also, sericin stimulates VEGFa expression via activating ERK phosphorylation. Further, sericin protects endothelial cells and cardiomyocytes from apoptosis by inhibiting the activation of caspase 3. Together, these diverse biochemical activities of sericin protein lead to a significant recovery of cardiac function. This work represents the first study reporting sericin as an effective therapeutic biomaterial for ischemic myocardial repair in vivo.. Intramyocardial biomaterial injection is thought to be a potential therapeutic approach to improve cardiac performance after ischemic myocardial infarction. In this study, we report the successful fabrication and in vivo application of an injectable sericin hydrogel for ischemic heart disease. We for the first time show that the injection of in situ forming crosslinked sericin hydrogel promotes heart functional recovery accompanied with reduced inflammatory responses, attenuated apoptosis and increased microvessel density in the infarcted hearts. Further, we reveal that the improvement in those aspects is ascribed to sericin protein's functional bioactivities that are comprehensively uncovered in this study. Thus, we identify sericin, a natural protein, as a biomaterial suitable for myocardial repair and demonstrate that the in vivo application of this injectable sericin hydrogel can be an effective strategy for treating MI.

Topics: Animals; Apoptosis; Cell Movement; Cross-Linking Reagents; Cytokines; Cytoprotection; Fibrosis; Human Umbilical Vein Endothelial Cells; Hydrogel, Polyethylene Glycol Dimethacrylate; Inflammation; Injections; Iridoids; Male; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Myocardial Infarction; Myocytes, Cardiac; Neovascularization, Physiologic; NIH 3T3 Cells; Rats; Recovery of Function; Sericins; Transcription, Genetic; Vascular Endothelial Growth Factor A

Genipin has been reported to have anti-inflammatory effect. However, its role on lipopolysaccharide (LPS)-induced acute lung injury (ALI) has not been explored. This study aimed to evaluate the effect of genipin on murine model of acute lung injury induced by LPS. The mice were treated with genipin 1h before LPS administration. 12h later, the myeloperoxidase (MPO) in lung tissues and lung wet/dry ratio were detected. The levels of TNF-α, IL-1β and IL-6 in bronchoalveolar lavage fluid (BALF) were measured by ELISA. Apart from this, we use western blot to detect the protein expression in the NF-κB and NLRP3 signaling pathways. The results showed that the treatment of genipin markedly attenuated the lung wet/dry ratio and the MPO activity. Moreover, it also inhibited the levels of TNF-α, IL-1β, IL-6 in the BALF. In addition, genipin significantly inhibited LPS-induced NF-κB and NLRP3 activation. In conclusion, these results demonstrate that genipin protected against LPS-induced ALI through inhibiting NF-κB and NLRP3 signaling pathways.

Topics: Acute Lung Injury; Animals; Anti-Inflammatory Agents; Cytokines; Inflammation Mediators; Iridoids; Lipopolysaccharides; Lung; Male; Mice; Mice, Inbred BALB C; NF-kappa B; NLR Family, Pyrin Domain-Containing 3 Protein; Peroxidase; Signal Transduction

Accumulating evidences postulated the influential roles of macrophages in mediating hepatocellular carcinoma (HCC) initiation and progression. In this study, we demonstrate that a small molecule, genipin reduced HCC growth through suppressing IRE1α-mediated infiltration and priming of tumour associated macrophages (TAMs). Oral administration of genipin (30mg/kg/2days) suppressed orthotopic HCC tumour growth without challenging the viability and proliferation of HCC cells. Genipin reduced infiltration of inflammatory monocytes into liver and tumour thereby suppressed TAMs presence in HCC microenvironment. Suppression of HCC growth was diminished in HCC-implanted mice with depletion of TAMs by liposome clodronate. Genipin inhibited the TAMs migration, and reduced expression of TAMs-derived inflammatory cytokines that favors HCC proliferation. This is revealed by the in vivo deletion of IRE1α on TAMs in genipin-treated HCC-implanted mice. Diminishing IRE1α neutralised the inhibitory effect of genipin on TAMs. Silencing the expression of IRE1α greatly reduced TAMs migration and expression of inflammatory cytokines that prime HCC proliferation. Suppression of IRE1α led to reduced XBP-1 splicing and NF-κB activation. The reduced association of IRE1α with TRAF2 and IKK complex may be responsible for the genipin-mediated inactivation of NF-κB. The findings show the important role of TAMs in inhibitory effect of genipin on HCC, and TAMs-expressing IRE1α as a promising target for disrupting the tumour environment that favor of HCC development.

Topics: Animals; Carcinoma, Hepatocellular; Cell Proliferation; Endoribonucleases; Iridoids; Liver Neoplasms; Macrophages; Mice; Protein Serine-Threonine Kinases; Tumor Microenvironment

To examine the metabolism of genipin-1-β-d-gentiobioside (GG), its distribution and biotransformation in vivo and in vitro were investigated. Urine, plasma, feces, and various organs were collected after oral administration of GG to normal rats and pseudo-germ-free rats to evaluate GG metabolism in vivo. GG was incubated with intestinal flora and primary hepatocytes in vitro to investigate microbial and hepatic metabolism. Using HPLC-Q-TOF-LC/MS, 11 metabolites of GG were absolutely or tentatively identified in terms possible elemental compositions, retention times, and characteristics of fragmentation patterns corresponding to eight biotransformations: deglycosylation, hydroxylation, sulfate conjugation, glucuronidation, hydrogenation, demethylation, glycosylation, and dehydration. Fewer metabolites were detected in pseudo-germ-free rats than in conventional rats. Moreover, geniposide and genipin were generated by the deglycoslation of intestinal bacteria. Geniposidic acid was detected in rat primary-hepatocyte incubation. This study first explores the metabolism of GG in vivo and in vitro. The results can aid the elucidation of PK profiles and clinical usage of gardenia fruit.

Topics: Animals; Chromatography, High Pressure Liquid; Glycosylation; Hepatocytes; Hydroxylation; Iridoids; Male; Mass Spectrometry; Molecular Structure; Rats; Rats, Sprague-Dawley

Genipin is an ideal cross-linking agent in biomedical applications, which can undergo ring-opening polymerization in alkaline condition. The polygenipin can create short-range and long-range intermolecular cross-linking between protein chains. In this article, the polygenipin with different degree of polymerization was successfully prepared and used to fix gelatin composite materials. The short-range and long-range cross-linking effects of polygenipin were systematically studied. The results show that the composite materials present porous structure with tunable pore sizes in the gel state, which can be easily controlled by adjusting the degree of polymerization of polygenipin. Long-range cross-linking can increase the pore size of the gel. However, during the drying process, the composite films cross-linked by polygenipin with higher degree of polymerization shrank to smaller size to create more compact structure, resulting in the improvement of water resistance properties, thermal stability, tensile strength, and darker color for the composite films. It is interesting that the composite films can partly swell to the original gel structure when in contact with water and saturated water vapor. All the composite films have excellent barrier properties against UV light. However, the compatibility of gelatin and polygenipin is reduced when the degree of polymerization of polygenipin increases to a certain extent, which will result in the formation of phase separation structure. The obtained composite films are ideal candidates for food and pharmaceutical packaging materials. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2712-2722, 2016.

Topics: Biocompatible Materials; Cross-Linking Reagents; Gelatin; Iridoids; Polymerization; Porosity; Steam; Tensile Strength; Water

Genipin is a major active component of Fructus Gardenia, which has been widely used in Traditional Chinese Medicine for the treatment of various cardiovascular diseases. The aim of this study was to investigate the potential effects of genipin on hypertension and the related nephropathy and elucidate the underlying mechanisms of action. We first examined the effects of genipin on blood pressure and renal functions in the Spontaneously Hypertensive (SHR) rats. In the subsequent experiments with human mesangial cells (HMCs), the effects of genipin on angiotensin II (Ang II)-induced HMC proliferation, reactive oxygen species (ROS) generation, and cytokine prodution were examined using the MTT method, 2',7'-dichlorohydrofluorescein (DCFH-DA) staining, and the corresponding enzyme-linked immunosorbent assay (ELISA) kits, respectively. The effects of genipin on Ang II-induced activation of the MAPK pathway and up-regulation of TLR2, TLR4, and MyD88 were detected by real-time PCR and Western blot and further validated in MyD88 siRNA-transfected HMCs. Genipin not only significantly lowered blood pressure in SHR rats after an 8-week treatment, but effectively improved renal functions, evidenced by decreased serum creatinine and blood urea nitrogen (BUN), as well as urinary microalbumin (m-ALB) and N-acetyl-beta-d-glucosaminidase (NAG) upon administration with genipin. Mechanistic studies conducted in Ang II-treated HMCs showed that genipin was able to counteract Ang II-induced cell proliferation, ROS generation, and pro-inflammatory responses. These effects may be mediated through the TLR/MyD88/MAPK signaling pathway. These findings provide new insights into the molecular mechanisms of genipin in the treatment of renal damage in hypertension, which merits a further investigation.

Topics: Angiotensin II; Animals; Antihypertensive Agents; Blood Pressure; Cell Proliferation; Cytokines; Humans; Hypertension; Iridoids; Kidney; Male; MAP Kinase Signaling System; Mesangial Cells; Myeloid Differentiation Factor 88; Rats, Inbred SHR; Reactive Oxygen Species; Toll-Like Receptor 2; Toll-Like Receptor 4

Osteoblast and osteoclast communication (i.e. osteocoupling) is an intricate process, in which the biophysical profile of bone ECM is an aggregate product of their activities. While the effect of microenvironmental cues on osteoblast and osteoclast maturation has been resolved into individual variables (e.g. stiffness or topography), a single cue can be limited with regards to reflecting the full biophysical scope of natural bone ECM. Additionally, the natural modulation of bone ECM, which involves collagenous fibril and elastin crosslinking via lysyl oxidase, has yet to be reflected in current synthetic platforms. Here, we move beyond traditional substrates and use cell-derived ECM to examine individual and coupled osteoblast and osteoclast behavior on a physiological platform. Specifically, preosteoblast-derived ECM is crosslinked with genipin, a biocompatible crosslinker, to emulate physiological lysyl oxidase-mediated ECM crosslinking. We demonstrate that different concentrations of genipin yield changes to ECM density, stiffness, and roughness while retaining biocompatibility. By approximating various bone ECM profiles, we examine how individual and coupled osteoblast and osteoclast behavior are affected. Ultimately, we demonstrate an increase in osteoblast and osteoclast differentiation on compact and loose ECM, respectively, and identify ECM crosslinking density as an underlying force in osteocoupling behavior.

Topics: 3T3 Cells; Animals; Cell Communication; Cell Differentiation; Cross-Linking Reagents; Extracellular Matrix; Extracellular Matrix Proteins; Iridoids; Materials Testing; Mechanotransduction, Cellular; Mice; Osteoblasts; Osteoclasts; Osteogenesis; Protein-Lysine 6-Oxidase

Diabetes mellitus is a typical heterogeneous metabolic disorder characterized by abnormal metabolism of carbohydrates, lipids and proteins. Genipin possesses a wide spectrum of biological activities including ameliorating effects on diabetes, but the definite mechanism of this effect remains unknown. To investigate the antidiabetic activities of genipin and explore the biochemical changes of serum endogenous metabolites on diabetic rats induced by alloxan, (1)H NMR spectroscopy coupled with multivariate data analysis was used to. All rats were randomly divided into six groups including negative control (NC) group, diabetic mellitus (DM) group, metformin hydrochloride group, high dose group of genipin, middle dose group of genipin and low dose group of genipin. Diabetes was induced by a single intraperitoneal injection of 120mg/kg body weight of alloxan. Serum samples were collected for the (1)H NMR-based metabolomics and clinical biochemical analysis. Daily oral administration of genipin (25, 50 and 100mg/kg body weight) and metformin hydrochloride (125mg/kg) for two weeks showed beneficial effects on blood glucose level (P<0.01). Significant differences in the metabolic profile as well as the result of biochemical parameters between the diabetic group and the control group were observed. The PLS-DA scores and corresponding loading plots demonstrated that genipin significantly restored the abnormal metabolic state. Detailed analysis of the altered metabolite levels indicated that genipin significantly ameliorated the disturbance in glucose metabolism, tricarboxylic acid cycle, lipid metabolism and amino acid metabolism. Genipin showed the best anti-diabetic effects at a dose of 100mg/kg in rats. This finding indicates that chemical and metabolomic approaches could be powerful tools for the investigation of the biochemical changes in pathological conditions or drug treatment.

Topics: Animals; Blood Glucose; Diabetes Mellitus, Experimental; Hypoglycemic Agents; Iridoids; Magnetic Resonance Spectroscopy; Male; Metabolome; Metabolomics; Random Allocation; Rats; Rats, Sprague-Dawley

To investigate the potentiation effect of Genipin to Cisplatin induced cell senescence in HCT-116 colon cancer cells in vitro.. Cell viability was estimated by Propidium iodide and Hoechst 3342, reactive oxygen species (ROS) with DHE, mitochondrial membrane potential (MMP) with JC-1 MMP assay Kit and electron current production with microbial fuel cells (MFC).. Genipin inhibited the UCP2 mediated anti-oxidative proton leak significantly promoted the Cisplatin induced ROS and subsequent cell death, which was similar to that of UCP2-siRNA. Cells treated with Cisplatin alone or combined with Genipin, ROS negatively, while MMP positively correlated with cell viability. Cisplatin induced ROS was significantly decreased by detouring electrons to MFC, or increased by Genipin combined treatment. Compensatory effects of UCP2 up-regulation with time against Genipin treatment were suggested. Shorter the Genipin treatment before Cisplatin better promoted the Cisplatin induced ROS and subsequent cell death.. The interaction of leaked electron with Cisplatin was important during ROS generation. Inhibition of UCP2-mediated proton leak with Genipin potentiated the cytotoxicity of Cisplatin. Owing to the compensatory effects against Genipin, shorter Genipin treatment before Cisplatin was recommended in order to achieve better potentiation effect.

Topics: Apoptosis; Bioelectric Energy Sources; Cell Proliferation; Cellular Senescence; Cisplatin; Colonic Neoplasms; Drug Synergism; HCT116 Cells; Humans; Iridoids; Membrane Potential, Mitochondrial; Oxidative Stress; Reactive Oxygen Species

Geniposide (GE) is the main bioactive component of Gardeniae Fructus. The hepatotoxicity of geniposide limited clinical application. In order to get a new geniposide derivative that has less hepatotoxicity and still possesses the antidepressant activity, a new C-1 hydroxyl methylation derivative named methyl genipin (MG) was synthesized from geniposide. In the present study, we demonstrated that MG did not increase the liver index, alanine aminotransferase (ALT) and aspirate aminotransferase (AST). Histopathological examination suggested that no toxic damages were observed in rats treated orally with MG (0.72 mmol/kg). More importantly, a 7-day treatment with MG at 0.13, 0.26, and 0.52 mmol/kg/day could reduce the duration of immobility. It showed that the antidepressant-like effects of MG were similar to GE in the tail suspension test and the forced swim test. Furthermore, we found MG could be detected in the brain homogenate of mice treated orally with MG 0.52 mmol/kg/day for 1 day by HPLC. The area under the curve (AUC) of MG in the brain homogenate was enhanced to 21.7 times that of GE. The brain amount and distribution speed of MG were improved significantly after oral administration. This study demonstrated that MG possessed the antidepressant effects and could cross the blood-brain barrier, but had less hepatotoxicity.

Topics: Animals; Antidepressive Agents; Blood-Brain Barrier; Body Weight; Brain; Chromatography, High Pressure Liquid; Iridoids; Liver; Molecular Structure; Permeability; Rats; Tissue Distribution

Compounds that modulate cancer cell glucose metabolism could open new opportunities for antitumor therapy and for monitoring response using (18)F-FDG PET. Genipin, a natural dietary compound that blocks uncoupling protein 2 (UCP2)-mediated mitochondrial proton leakage, is a potential anticancer agent. We investigated the effect of genipin on glucose metabolism and the mitochondrial function of cancer cells.. Breast and colon cancer cells were assessed for effects of genipin on (18)F-FDG uptake. T47D breast cancer cells were further evaluated for time-dependent and dose-dependent effects on (18)F-FDG uptake, lactate release, oxygen consumption rate (OCR), reactive oxygen species (ROS) production, and mitochondrial membrane potential. The effects of UCP2 knockdown were evaluated using specific siRNA.. Cancer cells displayed significant reductions in (18)F-FDG uptake by genipin. T47D cells showed the greatest reduction to 32.6±1.0% of controls by 250μM genipin. The effect occurred rapidly, reaching a plateau by 1h that lasted up to 24h. The effect was dose-dependent with a half-inhibitory concentration of 60.8μM. An accompanying decrease in lactate release was consistent with reduced glycolytic flux. OCR was significantly decreased by genipin to 82.2±11.4% of controls, and ROS generation was increased to 156.7±16.0%. These effects were largely reproduced by UCP2 knockdown with specific siRNA.. Genipin decreased cancer cell (18)F-FDG uptake by reducing both glycolytic flux and mitochondrial oxidative respiration. This effect appeared to occur by blocking the ability of UCP2 to dissipate energy and restrict ROS production through proton leakage.

Topics: Biological Transport; Breast Neoplasms; Cell Line, Tumor; Colonic Neoplasms; Dose-Response Relationship, Drug; Fluorodeoxyglucose F18; Gene Expression Regulation, Neoplastic; Gene Knockdown Techniques; Glucose; Humans; Iridoids; Lactic Acid; Matrix Metalloproteinases; Reactive Oxygen Species; RNA, Small Interfering; Signal Transduction; Time Factors; Uncoupling Protein 2

The trabecular meshwork (TM) tissue controls drainage of aqueous humor from the anterior chamber of the eye primarily by regulating extracellular matrix (ECM) remodeling by matrix metalloproteinases (MMPs). Glaucomatous TM tissue is stiffer than age-matched controls, which may be due to alterations in ECM cross-linking. In this study, we used genipin or beta-aminopropionitrile (BAPN) agents to induce or inhibit matrix cross-linking, respectively, to investigate the effects on outflow resistance and ECM remodeling. Treatment with BAPN increased outflow rates in perfused human and porcine anterior segments, whereas genipin reduced outflow. Using a fluorogenic peptide assay, MMP activity was increased with BAPN treatment, but reduced with genipin treatment. In genipin-treated TM cells, Western immunoblotting showed a reduction of active MMP2 and MMP14 species and the presence of TIMP2-MMP14 higher molecular weight complexes. BAPN treatment increased collagen type I mRNA and protein levels, but genipin reduced the levels of collagen type I, tenascin C, elastin and versican. CD44 and fibronectin levels were unaffected by either treatment. Collectively, our results show that matrix cross-linking has profound effects on outflow resistance and ECM composition and are consistent with the emerging paradigm that the stiffer the ECM, the lower the aqueous outflow facility through the TM.

Topics: ADAMTS4 Protein; Adolescent; Adult; Aminopropionitrile; Animals; Aqueous Humor; Cells, Cultured; Collagen Type I; Collagen Type I, alpha 1 Chain; Cross-Linking Reagents; Extracellular Matrix; Extracellular Matrix Proteins; Humans; Iridoids; Matrix Metalloproteinases; Organ Culture Techniques; Swine; Trabecular Meshwork

The objective of this work was to correlate the physical and chemical properties of chitosan/poly(vinyl alcohol)/genipin (CS/PVA/GEN) and chitosan/poly(vinyl alcohol)/glutaraldehyde (CS/PVA/GA) hydrogels with their structural and mechanical responses. In addition, their molecular structures were determined and confirmed using FTIR spectroscopy. The results indicated that the hybrid hydrogels crosslinked with genipin showed similar crystallinity, thermal properties, elongation ratio and structural parameters as those crosslinked with glutaraldehyde. However, it was found that the elastic moduli of the two hybrid hydrogels were slightly different: 2.82±0.33MPa and 2.08±0.11MPa for GA and GEN, respectively. Although the hybrid hydrogels crosslinked with GEN presented a lower elastic modulus, the main advantage is that GEN is five to ten thousand times less cytotoxic than GA. This means that the structural and mechanical properties of hybrid hydrogels crosslinked with GEN can easily be tuned and could have potential applications in the tissue engineering, regenerative medicine, food, agriculture and environmental industries.

Topics: Biocompatible Materials; Biological Products; Chitosan; Glutaral; Hydrogels; Iridoids; Mechanical Phenomena; Polyvinyl Alcohol; Temperature; Tensile Strength

To evaluate the effect of genipin, a natural crosslinking agent, in rabbit eyes.. Department of Ophthalmology, Universidad Nacional de Colombia Centro de Tecnologia Oftalmica, Bogotá, Colombia.. Experimental study.. Ex vivo rabbit eyes (16; 8 rabbits) were treated with genipin 1.00%, 0.50%, and 0.25% for 5 minutes with a vacuum device to increase corneal permeability. Penetration was evaluated using Scheimpflug pachymetry (Pentacam). In the in vivo model (20 rabbits; 1 eye treated, 1 eye with vehicle), corneas were crosslinked with genipin as described. Corneal curvature, corneal pachymetry, and intraocular pressure (IOP) assessments as well as slitlamp examinations were performed 0, 7, 30, and 60 days after treatment.. In the ex vivo model, Scheimpflug pachymetry showed deep penetration in the rabbit corneas with an increase in corneal density and a dose-dependent relationship. Corneal flattening was observed in treated eyes (mean 4.4 diopters ± 0.5 [SD]) compared with the control eyes. Pachymetry and IOP were stable in all evaluations. No eye showed toxicity in the anterior chamber or in the lens.. Corneal crosslinking induced by genipin produced significant flattening of the cornea with no toxicity in rabbit eyes. This crosslinking could be useful in the treatment of corneal ectasia and in the modification of corneal curvature.. None of the authors has a financial or proprietary interest in any material or method mentioned.

Topics: Animals; Cornea; Corneal Pachymetry; Corneal Topography; Cross-Linking Reagents; Iridoids; Rabbits; Tonometry, Ocular

Extracellular matrix (ECM), comprised of multiple cues (chemical, physiomechanical), provides a niche for cell attachment, migration, and differentiation. Given that different cells give rise to distinct physiological milieus, the role of such microenvironmental cues on various cells has been well-studied. Particularly, the effect of various physiomechanical factors on stem cell lineage has been resolved into individual variables via ECM protein-coated polymeric systems. Such platforms, while providing a reductionist approach as a means to remove any confounding factors, unfortunately fall short of capturing the full biophysical scope of the natural microenvironment. Herein, the use of a cell-derived ECM platform is reported in which its crosslinking density is tunable; varying concentrations (0, 0.5, 1, 2% w/v) of genipin (GN), a naturally derived crosslinker with low toxicity, are used to form inter- and intrafibril crosslinks. ECM crosslinking produces GN concentration-dependent changes in ECM stiffness (<0.1-9.4 kPa), roughness (96-280 nm), and chemical composition (100-60% amine content). The effect of the various crosslinked ECM profiles on human mesenchymal stem cell differentiation, vascular morphogenesis, and cardiomyogenesis are then evaluated. Taken together, this study demonstrates that tunable crosslinked cell-derived ECM platform is capable of providing a comprehensive physiological platform, and envisions its use in future tissue engineering applications.

Topics: Cell Differentiation; Cellular Microenvironment; Coated Materials, Biocompatible; Extracellular Matrix; Human Umbilical Vein Endothelial Cells; Humans; Iridoids; Materials Testing; Mesenchymal Stem Cells; Myoblasts, Cardiac; Tissue Engineering

A delayed release bio-polymeric Dual-Biotic system has been extensively evaluated in this study to overcome the therapeutic issue of probiotic killing due to incorrect administration with the antibiotic.. In vitro and ex vivo release and characterization studies have been undertaken on the Dual-Biotic system. In vivo analyses utilizing a Large White pig model were also performed with commercial products used as a comparison. Intestinal fluid for probiotic quantification was aspirated using a surgically implanted intestinal cannula with Lactobacillus acidophilus cell counts determined through luminescence and inoculation onto Lactobacilli-specific agar. Plasma amoxicillin concentrations were determined through Ultra-Performance Liquid Chromatography. The reactional profile and crosslinking mechanism of ovalbumin and genipin was elucidated using molecular mechanic energy relationships in a vacuum system by exploring the spatial disposition of different concentrations of genipin with respect to ovalbumin with ovalbumin/genipin ratios of 1:1, 1:5 and 1:10.. In vivo evaluation of the Dual-Biotic system detailed maximum Lactobacillus viability (~455% baseline viability) 6 h after oral administration. Concurrent administration of the commercial products revealed a 75% decrease in bacterial viability when compared to the controls analyzed. A level A in vitro-in vivo correlation was also established with 96.9% predictability of amoxicillin release ascertained. The computational results achieved corroborated well with the experimental findings and physicochemical data.. Evaluation and correlation of the Dual-Biotic system has detailed the success of the formulation for the concurrent delivery of an antibiotic and probiotic.

Topics: Administration, Oral; Amoxicillin; Animals; Anti-Bacterial Agents; Chromatography, High Pressure Liquid; Computer Simulation; Cross-Linking Reagents; Drug Delivery Systems; Drug Liberation; Intestinal Mucosa; Iridoids; Lactobacillus acidophilus; Microbial Viability; Molecular Docking Simulation; Ovalbumin; Probiotics; Sus scrofa

Currently available clinical therapies are not capable to regenerate tissues that are lost by periodontitis. Tissue engineering can be applied as a strategy to regenerate reliably the tissues and function of damaged periodontium. A prerequisite for this regeneration is the colonization of the defect with the adequate cell populations. In this study, we proposed a bilayered system composed of (1) a platelet lysate (PL)-based construct produced by crosslinking of PL proteins with genipin (gPL) for the delivery of rat periodontal ligament cells (rat-PDLCs); combined with (2) an injectable composite consisting of calcium phosphate cement incorporated with PL-loaded poly(d, l-lactic-co-glycolic acid) microspheres. This system was expected to promote periodontal regeneration by the delivery of adequate progenitor cells and providing a stable system enriched with adequate cytokines and growth factors for the orchestration of tissue regrowth in periodontal defects. The bilayered system was tested in a three-wall intrabony defect in rats and the healing of periodontal tissue was assessed 6 weeks after surgery. Results showed that the bilayered system was able to promote the regrowth of functional periodontal tissues, both with (cells + gPL) and without the loading of PDLCs (gPL). Significant connective tissue attachment (45.0 ± 15.0% and 64.0 ± 15.0% for gPL and cells + gPL group, respectively) and new bone area (33.8 ± 21% and 21.3 ± 3% for gPL and cells + gPL group, respectively) were observed. Nevertheless, rat PDLCs delivered with gPL construct in the defect area were hardly visible 6 weeks after surgery and did not contribute for the regeneration of new periodontal tissue. Overall, our findings show that the bilayered system promotes the stabilization of PL proteins on the root surface and has a positive effect in the repair of periodontal tissues both in quality and in quantity.

Topics: Animals; Blood Platelets; Cytokines; Iridoids; Periodontal Ligament; Rats; Rats, Sprague-Dawley; Rats, Transgenic; Regeneration

The objective of this study was to demonstrate the therapeutic as well as biopolymer like characteristics of naturally occurring sericin protein for development of nanoparticulate system of atorvastatin (Atr) to improve therapeutic effect and to reduce toxicity. The sericin encapsulated atorvastatin nanoparticles (Seri-Atr NPs) were prepared by desolvation method utilizing genipin (Gn) as a natural and nontoxic crosslinker. The optimized NPs exhibited small particle size (166±0.30nm), high entrapment efficiency (91±0.69%) and uniform spherical shape with sustained release profile. Moreover, the results of pharmacokinetic studies indicated an increase in AUC0-∞ of NPs (1189.74±52.3hng/ml) compared with Atr (501.84±66hng/ml). The cellular uptake of NPs suggested an interaction of negatively charged particles with the cell surface and considerable reduction in systemic toxicity. Histopathology studies also demonstrated the therapeutic potential of sericin and cytocompatibility. Hence, genipin crosslinked sericin based nanoparticles represents a promising nanoplatform for improved therapeutic efficiency of Atr.

Topics: Animals; Atorvastatin; Bombyx; Cell Line; Cell Survival; Drug Liberation; Endocytosis; Hydroxymethylglutaryl CoA Reductases; Hypolipidemic Agents; Iridoids; Lipids; Liver; Macrophages; Mice; Nanoparticles; Particle Size; Sericins; Spectroscopy, Fourier Transform Infrared; Static Electricity; X-Ray Diffraction

In the present study genipin crosslinked chitosan (CHI) hydrogels, which had been constructed and reported in our previous studies (Gao et al., 2014 [22]), were further evaluated for their advantage as a carrier for silver sulfadiazine (AgSD) nanocrystal systems. Firstly, AgSD nanocrystals with a mean particle size of 289nm were prepared by wet milling method and encapsulated into genipin crosslinked CHI hydrogels. AgSD nanocrystals displayed a uniform distribution and very good physical stability in the hydrogel network. Swelling-dependent release pattern was found for AgSD nanocrystals from hydrogels and the release profile could be well fitted with Peppas equation. When AgSD nanocrystals were encapsulated in hydrogels their fibroblast cytotoxicity decreased markedly, and their antibacterial effects against Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa were still comparable to unencapsulated AgSD nanocrystals. In vivo evaluation in excision and burn cutaneous wound models in mice showed that AgSD nanocrystal hydrogels markedly decreased the expression of inflammatory cytokine IL-6, but increased the levels of growth factors VEGF-A and TGF-β1. Histopathologically, the wounds treated by hydrogels containing AgSD nanocrystals showed the best healing state compared with commercial AgSD cream, hydrogels containing AgSD bulk powders and blank hydrogels. The wounds treated by AgSD nanocrystal hydrogels were dominated by marked fibroblast proliferation, new blood vessels and thick regenerated epithelial layer. Sirius Red staining assay indicated that AgSD nanocrystal hydrogels resulted in more collagen deposition characterized by a large proportion of type I fibers. Our study suggested that genipin-crosslinked CHI hydrogel was a potential carrier for local antibacterial nanomedicines.

Topics: Chitosan; Drug Carriers; Hydrogels; Iridoids; Nanoparticles; Silver Sulfadiazine

Loss of vision due to corneal disease is a significant problem worldwide. Transplantation of donor corneas is a viable treatment option but limitations such as short supply and immune-related complications call for alternative options for the treatment of corneal disease. A tissue engineering-based approach using a collagen scaffold is a promising alternative to develop a bioengineered cornea that mimics the functionality of native cornea. In this study, an electrochemical compaction method was employed to synthesize highly dense and transparent collagen matrices. We hypothesized that chemical crosslinking of electrochemically compacted collagen (ECC) matrices will maintain transparency, improve stability, and enhance the mechanical properties of the matrices to the level of native cornea. Further, we hypothesized that keratocyte cell viability and proliferation will be maintained on crosslinked ECC matrices. The results indicated that uncrosslinked and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide (EDC-NHS) crosslinked ECC matrices were highly transparent with light transmission measurements comparable to native cornea. Stability tests showed that while the uncrosslinked ECC matrices degraded within 6 h when treated with collagenase, EDC-NHS or genipin crosslinking significantly improved the stability of ECC matrices (192 h for EDC-NHS and 256 h for genipin). Results from the mechanical tests showed that both EDC-NHS and genipin crosslinking significantly improved the strength and modulus of ECC matrices. Cell culture studies showed that keratocyte cell viability and proliferation are maintained on EDC-NHS crosslinked ECC matrices. Overall, results from this study suggest that ECC matrices have the potential to be developed as a functional biomaterial for corneal repair and regeneration.

Topics: Biocompatible Materials; Bioengineering; Cell Proliferation; Cell Survival; Collagen; Collagenases; Cornea; Cross-Linking Reagents; Electrochemistry; Humans; Iridoids; Keratinocytes; Materials Testing; Regeneration; Stress, Mechanical; Succinimides; Tissue Engineering

Kaposi's sarcoma-associated herpesvirus (KSHV) is a Gammaherpesvirus that causes acute infection and establishes life-long latency. KSHV causes several human cancers, including Kaposi's sarcoma, an acquired immune deficiency syndrome (AIDS)-related form of non-Hodgkin lymphoma. Genipin, an aglycone derived from geniposide found in Gardenia jasminoides, is known to be an excellent natural cross-linker, strong apoptosis inducer, and antiviral agent. Although evidence suggests antiviral activity of genipin in several in vitro viral infection systems, no inhibitory effect of genipin on KSHV infection has been reported. Thus, our aim was to determine, using the iSLK-BAC16 KSHV infection system, whether genipin has inhibitory effects on KSHV infection. For this purpose, we evaluated biological effects of genipin on KSHV infection and finally determined the underlying mechanisms responsible for the bioactive effects of genipin. A cytotoxicity assay revealed that genipin caused 50% cytotoxicity at 49.5 μM in iSLK-puro (KSHV-negative) cells and at 72.5 μM in iSLK-BAC16 (KSHV-positive) cells. Caspase 3/7 activities were slightly suppressed by genipin treatment in iSLK-BAC16 cells while significantly induced in iSLK-puro cells. Production of the KSHV latency-associated nuclear antigen (LANA), but not that of the R-transactivator (RTA) protein, was significantly induced by genipin treatment at lower concentration. Consistent with the LANA upregulation, KSHV LANA transcripts, but not RTA transcripts, were expressed at a higher level. Furthermore, KSHV intracellular copy numbers were slightly increased at lower concentration of genipin, while KSHV extracellular copy numbers were significantly increased at higher concentration of genipin. Interestingly, genipin treatment at a lower concentration did induce the expression of DNA (cytosine-5)-methyltransferase 1 (DNMT1); however, a co-immunoprecipitation assay showed that the DNMT1 and LANA induced by genipin did not co-precipitate from iSLK-BAC16 cells. Moreover, a chromatin immunoprecipitation assay demonstrated that genipin treatment enhanced the binding of CCCTC-binding factor (CTCF) to the CTCF-binding site in the KSHV latency control region but suppressed the binding of structural maintenance of chromosomes protein 3 (SMC3) to this site. Genipin treatment also led to the recruitment of additional RNA polymerase to the majority of binding sites of some interesting proteins in the KSHV latency control region, which mi

Topics: Antigens, Viral; Antiviral Agents; Apoptosis; Caspases; CCCTC-Binding Factor; Cell Cycle Checkpoints; Cell Line; Cell Survival; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; Gardenia; Gene Expression Regulation, Viral; Herpesvirus 8, Human; Humans; Iridoids; Nuclear Proteins; Protein Interaction Maps; Repressor Proteins; Sarcoma, Kaposi; Virus Latency

Coronary angioplasty is the most widely used technique for removing atherosclerotic plaques in blood vessels. The regeneration of the damaged intima layer after this treatment is still one of the major challenges in the field of cardiovascular tissue engineering. Different polymers have been used in scaffold manufacturing in order to improve tissue regeneration. Elastin-mimetic polymers are a new class of molecules that have been synthesized and used to obtain small diameter fibers with specific morphological characteristics. Elastin-like polymers produced by recombinant techniques and called elastin-like recombinamers (ELRs) are particularly promising due to their high degree of functionalization. Generally speaking, ELRs can show more complex molecular designs and a tighter control of their sequence than other chemically synthetized polymers Rodriguez Cabello et al (2009 Polymer 50 5159-69, 2011 Nanomedicine 6 111-22). For the fabrication of small diameter fibers, different ELRs were dissolved in 2,2,2-fluoroethanol (TFE). Dynamic light scattering was used to identify the transition temperature and get a deep characterization of the transition behavior of the recombinamers. In this work, we describe the use of electrospinning technique for the manufacturing of an elastic fibrous scaffold; the obtained fibers were characterized and their cytocompatibility was tested in vitro. A thorough study of the influence of voltage, flow rate and distance was carried out in order to determine the appropriate parameters to obtain fibrous mats without beads and defects. Moreover, using a rotating mandrel, we fabricated a tubular scaffold in which ELRs containing different cell adhesion sequences (mainly REDV and RGD) were collected. The stability of the scaffold was improved by using genipin as a crosslinking agent. Genipin-ELRs crosslinked scaffolds  show a good stability and fiber morphology. Human umbilical vein endothelial cells  were used to assess the in vitro bioactivity of the cell adhesion domains within the backbone of the ELRs.

Topics: Amino Acid Sequence; Atherosclerosis; Biocompatible Materials; Cell Adhesion; Cell Survival; Cross-Linking Reagents; Dynamic Light Scattering; Elastin; Human Umbilical Vein Endothelial Cells; Humans; Iridoids; Microscopy, Electron, Scanning; Nanofibers; Polymers; Tissue Engineering; Tissue Scaffolds

Uncoupling protein 2 (UCP2) has been suggested to inhibit mitochondrial production of reactive oxygen species (ROS) by decreasing the mitochondrial membrane potential. Experimental acute pancreatitis is associated with increased UCP2 expression, whereas UCP2 deficiency retards regeneration of aged mice from acute pancreatitis. Here, we have addressed biological and molecular functions of UCP2 in pancreatic stellate cells (PSCs), which are involved in pancreatic wound repair and fibrogenesis.. PSCs were isolated from 12 months old (aged) UCP2-/- mice and animals of the wild-type (WT) strain C57BL/6. Proliferation and cell death were assessed by employing trypan blue staining and a 5-bromo-2'-deoxyuridine incorporation assay. Intracellular fat droplets were visualized by oil red O staining. Levels of mRNA were determined by RT-PCR, while protein expression was analyzed by immunoblotting and immunofluorescence analysis. Intracellular ROS levels were measured with 2', 7'-dichlorofluorescin diacetate. Expression of senescence-associated beta-galactosidase (SA beta-Gal) was used as a surrogate marker of cellular senescence.. PSCs derived from UCP2-/- mice proliferated at a lower rate than cells from WT mice. In agreement with this observation, the UCP2 inhibitor genipin displayed dose-dependent inhibitory effects on WT PSC growth. Interestingly, ROS levels in PSCs did not differ between the two strains, and PSCs derived from UCP2-/- mice did not senesce faster than those from corresponding WT cells. PSCs from UCP2-/- mice and WT animals were also indistinguishable with respect to the activation-dependent loss of intracellular fat droplets, expression of the activation marker alpha-smooth muscle actin, type I collagen and the autocrine/paracrine mediators interleukin-6 and transforming growth factor-beta1.. A reduced proliferative capacity of PSC from aged UCP2-/- mice may contribute to the retarded regeneration after acute pancreatitis. Apart from their slower growth, PSC of UCP2-/- mice displayed no functional abnormalities. The antifibrotic potential of UCP2 inhibitors deserves further attention.

Topics: Animals; Biomarkers; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Gene Expression Profiling; Gene Expression Regulation; Genotype; Iridoids; Mice, Inbred C57BL; Mice, Knockout; Pancreatic Stellate Cells; Phenotype; Reactive Oxygen Species; Signal Transduction; Uncoupling Protein 2

Recently, a three-dimensional (3D) bioprinting process for obtaining a cell-laden structure has been widely applied because of its ability to fabricate biomimetic complex structures embedded with and without cells. To successfully obtain a cell-laden porous block, the cell-delivering vehicle, bioink, is one of the significant factors. Until now, various biocompatible hydrogels (synthetic and natural biopolymers) have been utilized in the cell-printing process, but a bioink satisfying both biocompatibility and print-ability requirements to achieve a porous structure with reasonable mechanical strength has not been issued. Here, we propose a printing strategy with optimal conditions including a safe cross-linking procedure for obtaining a 3D porous cell block composed of a biocompatible collagen-bioink and genipin, a cross-linking agent. To obtain the optimal processing conditions, we modified the 3D printing machine and selected an optimal cross-linking condition (∼1 mM and 1 h) of genipin solution. To show the feasibility of the process, 3D pore-interconnected cell-laden constructs were manufactured using osteoblast-like cells (MG63) and human adipose stem cells (hASCs). Under these processing conditions, a macroscale 3D collagen-based cell block of 21 × 21 × 12 mm

Topics: Bioprinting; Collagen; Humans; Iridoids; Porosity; Printing, Three-Dimensional; Tissue Engineering; Tissue Scaffolds

Several evidence indicate that metabolic alterations play a pivotal role in cancer development. Here, we report that the mitochondrial uncoupling protein 2 (UCP2) sustains the metabolic shift from mitochondrial oxidative phosphorylation (mtOXPHOS) to glycolysis in pancreas cancer cells. Indeed, we show that UCP2 sensitizes pancreas cancer cells to the treatment with the glycolytic inhibitor 2-deoxy-D-glucose. Through a bidimensional electrophoresis analysis, we identify 19 protein species differentially expressed after treatment with the UCP2 inhibitor genipin and, by bioinformatic analyses, we show that these proteins are mainly involved in metabolic processes. In particular, we demonstrate that the antioxidant UCP2 induces the expression of hnRNPA2/B1, which is involved in the regulation of both GLUT1 and PKM2 mRNAs, and of lactate dehydrogenase (LDH) increasing the secretion of L-lactic acid. We further demonstrate that the radical scavenger N-acetyl-L-cysteine reverts hnRNPA2/B1 and PKM2 inhibition by genipin indicating a role for reactive oxygen species in the metabolic reprogramming of cancer cells mediated by UCP2. We also observe an UCP2-dependent decrease in mtOXPHOS complex I (NADH dehydrogenase), complex IV (cytochrome c oxidase), complex V (ATPase) and in mitochondrial oxygen consumption, suggesting a role for UCP2 in the counteraction of pancreatic cancer cellular respiration. All these results reveal novel mechanisms through which UCP2 promotes cancer cell proliferation with the concomitant metabolic shift from mtOXPHOS to the glycolytic pathway.

Topics: Acetylcysteine; Carrier Proteins; Cell Line, Tumor; Cell Proliferation; Deoxyglucose; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Glucose Transporter Type 1; Glycolysis; Heterogeneous-Nuclear Ribonucleoprotein Group A-B; Humans; Insulin-Secreting Cells; Iridoids; L-Lactate Dehydrogenase; Membrane Proteins; Mitochondria; Oxidative Phosphorylation; Reactive Oxygen Species; RNA, Messenger; RNA, Small Interfering; Signal Transduction; Thyroid Hormone-Binding Proteins; Thyroid Hormones; Uncoupling Protein 2

The material surface plays an important role in the case of biomaterials used as tissue engineering scaffolds. On exposure to a biological environment, extra cellular matrix (ECM) proteins are adsorbed non-specifically onto the surface and cells interact indirectly with the surface through the adsorbed proteins. Most synthetic polymeric biomaterials lack the desirable surface properties for cells as well as have poor cellular adhesion due to their hydrophobic nature. The main objective of this study was to harness surface functionalization technologies to fabricate scaffolds that would be biocompatible and support the adhesion and proliferation of fibroblast cells. The collagen was immobilized on the surface of functionalized PLA via a novel natural cross-linking molecule genipin which resulted in improved cell proliferation of human dermal fibroblasts as compared to the PLA surface coated with collagen without genipin. It is believed that genipin helps reduce steric problems between the functional groups and large protein molecules, and enables immobilized peptide to move more freely in a biological environment.

Topics: Cells, Cultured; Fibroblasts; Humans; Iridoids; Lactic Acid; Materials Testing; Polyesters; Polymers; Tissue Engineering; Tissue Scaffolds

Our long-term goal is to develop smart biomaterials that can facilitate regeneration of critical-size craniofacial lesions. In this study, we tested the hypothesis that biomimetic scaffolds electrospun from chitosan (CTS) will promote tissue repair and regeneration in a critical size calvarial defect. To test this hypothesis, we first compared in vitro ability of electrospun CTS scaffolds crosslinked with genipin (CTS-GP) to those of mineralized CTS-GP scaffolds containing hydroxyapatite (CTS-HA-GP), by assessing proliferation/metabolic activity and alkaline phosphatase (ALP) levels of murine mesenchymal stem cells (mMSCs). The cells' metabolic activity exhibited a biphasic behavior, indicative of initial proliferation followed by subsequent differentiation for all scaffolds. ALP activity of mMSCs, a surrogate measure of osteogenic differentiation, increased over time in culture. After 3 weeks in maintenance medium, ALP activity of mMSCs seeded onto CTS-HA-GP scaffolds was approximately two times higher than that of cells cultured on CTS-GP scaffolds. The mineralized CTS-HA-GP scaffolds were also osseointegrative in vivo, as inferred from the enhanced bone regeneration in a murine model of critical size calvarial defects. Tissue regeneration was evaluated over a 3 month period by microCT and histology (Hematoxylin and Eosin and Masson's Trichrome). Treatment of the lesions with CTS-HA-GP scaffolds induced a 38% increase in the area of de novo generated mineralized tissue area after 3 months, whereas CTS-GP scaffolds only led to a 10% increase. Preseeding with mMSCs significantly enhanced the regenerative capacity of CTS-GP scaffolds (by ∼3-fold), to 35% increase in mineralized tissue area after 3 months. CTS-HA-GP scaffolds preseeded with mMSCs yielded 45% new mineralized tissue formation in the defects. We conclude that the presence of HA in the CTS-GP scaffolds significantly enhances their osseointegrative capacity and that mineralized chitosan-based scaffolds crosslinked with genipin may represent a unique biomaterial with possible clinical relevance for the repair of critical calvarial bone defects.

Topics: Alkaline Phosphatase; Animals; Bone Regeneration; Cell Differentiation; Cell Proliferation; Cells, Cultured; Chitosan; Durapatite; Female; Fluorescence; Iridoids; Mesenchymal Stem Cells; Mice; Nanofibers; Osseointegration; Radiographic Image Enhancement; Skull; Tissue Engineering; Tissue Scaffolds; Wound Healing; X-Ray Microtomography

Polyelectrolyte complex (PEC) is formed when polymers with opposite charges are combined in solution. PECs are recently gaining attention as carriers for controlled release of drugs and proteins. Herein, bone morphogenetic protein-2 (BMP-2) was immobilized in a PEC of natural polymers, chitosan and hyaluronic acid. Charge-to-charge stoichiometry of the formed PEC was estimated based on turbidity of combined chitosan and hyaluronic acid solutions. Free amino groups in chitosan were crosslinked with different amounts of genipin. The degree of crosslinking, consequently its effects in vitro in terms of swelling, degradation and cytocompatibility were analyzed. Immobilization of three different amount of BMP-2 in chitosan-hyaluronic acid PEC scaffold resulted sustained release of the growth factor for more than 30 days. Immobilization efficacies varied from 61% to 76% depending on the amount of BMP-2. Finally effects in osteogenic differentiation of the PEC with BMP-2 to MC3T3-E1 cells were determined by reverse transcriptase PCR.

Topics: Animals; Bone Morphogenetic Protein 2; Cell Differentiation; Cell Proliferation; Cells, Cultured; Chitosan; Cross-Linking Reagents; Delayed-Action Preparations; Electrolytes; Hyaluronic Acid; Immobilized Proteins; Iridoids; Mice; Osteoblasts; Polymers

Drug administration via buccal mucosa is an attractive drug delivery strategy due to good patient compliance, prolonged localized drug effect, and avoidance of gastrointestinal drug metabolism and first-pass elimination. Buccal drug delivery systems need to maintain an intimate contact with the mucosa lining in the wet conditions of the oral cavity for long enough to allow drug release and absorption. For decades, mucoadhesive polymers such as chitosan (CS) and its derivatives have been explored to achieve this. In this study, inspired by the excellent wet adhesion of marine mussel adhesive protein, we developed a buccal drug delivery system using a novel catechol-functionalized CS (Cat-CS) hydrogel. We covalently bonded catechol functional groups to the backbone of CS, and crosslinked the polymer with a non-toxic crosslinker genipin (GP). We achieved two degrees of catechol conjugation (9% and 19%), forming Cat9-CS/GP and Cat19-CS/GP hydrogels, respectively. We confirmed covalent bond formation during the catechol functionalization and GP crosslinking during the gel formation. The gelation time and the mechanical properties of Cat-CS hydrogels are similar to those of CS only hydrogels. Catechol groups significantly enhanced mucoadhesion in vitro (7 out of the 10 Cat19-CS hydrogels were still in contact with porcine mucosal membrane after 6 h, whereas all of the CS hydrogels lost contact after 1.5 h). The new hydrogel systems sustained the release of lidocaine for about 3 h. In-vivo, we compared buccal patches made of Cat19-CS/GP and CS/GP adhered to rabbit buccal mucosa. We were able to detect lidocaine in the rabbit's serum at concentration about 1 ng/ml only from the Cat19-CS patch, most likely due to the intimate contact provided by mucoadhesive Cat19-CS/GP systems. No inflammation was observed on the buccal tissue in contact with any of the patches tested. These results show that the proposed catechol-modified CS hydrogel is a promising mucoadhesive and biocompatible hydrogel system for buccal drug delivery.

Topics: Animals; Carbon-13 Magnetic Resonance Spectroscopy; Catechols; Chitosan; Cross-Linking Reagents; Diffusion; Drug Delivery Systems; Humans; Hydrogels; Iridoids; Kaplan-Meier Estimate; Male; Microscopy, Electron, Scanning; Mouth Mucosa; Rabbits; Rheology; Spectroscopy, Fourier Transform Infrared; Sus scrofa

Gardenamide A (GA) is a stable genipin derivative with neuroprotective properties. It rescued pheochromocytoma cell (PC12) sympathetic cultures and retinal neuronal cells from apoptosis insult induced by serum deprivation. GA attenuated the accumulation of intracellular reactive oxygen species (ROS) and the loss of mitochondrial membrane potential. Western blotting with specific phospho-antibodies indicated that GA increased the phosphorylation of both the protein kinase B (Akt) and the extracellular signal-regulated kinase (ERK1/2) in PC12 cells. The GA neuroprotective effect was inhibited by either the specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002 or the mitogen-activated protein kinase (MAPK) pathway inhibitor PD98059. These results propose that the neuroprotective effect of GA on PC12 neuronal cell cultures was mediated through both the PI3K/Akt and ERK1/2 signaling pathways. Therefore, GA may serve as a pharmacological tool to investigate neuroprotective mechanisms of neurons afflicted by different insults.

Topics: Animals; Apoptosis; Cell Survival; Cells, Cultured; Iridoids; MAP Kinase Signaling System; Neurons; Neuroprotective Agents; PC12 Cells; Phosphatidylinositol 3-Kinases; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Retinal Ganglion Cells; Signal Transduction

This paper utilized a quantitative (1)H nuclear magnetic resonance (qHNMR) method for assessing the purity of iridoids and secoiridoids. The method was fully validated, including specificity, linearity, accuracy, precision, reproducibility, and robustness. For optimization of experimental conditions, several experimental parameters were investigated, including relaxation delay (D1), scan numbers (NS) and power length (PL1). The quantification was based on the area ratios of H-3 from analytes relative to aromatic protons from 1,4-dinitrobenzene (internal standard) with methanol-d4 as solvent. Five iridoids and secoiridoids (sweroside, swertiamarin, gentiopicroside, geniposide, genipin) were analyzed. Furthermore, the results were validated by the high performance liquid chromatography coupled with ultraviolet detection (HPLC-UV) method. It can be concluded that the qHNMR method was simple, rapid, and accurate, providing a reliable and superior method for assessing the purity of iridoids and secoiridoids.

Topics: Chromatography, High Pressure Liquid; Iridoid Glucosides; Iridoids; Magnetic Resonance Spectroscopy; Molecular Structure; Pyrones; Reproducibility of Results; Sensitivity and Specificity

The natural crosslinking agent genipin has been applied widely in biomedicines and foods nowadays. Because of the special hemiacetal ring structure in its molecule, it can only be prepared by hydrolysis of geniposide according to biocatalysis. In this research, strategies including aqueous-organic biphasic catalysis and substrate fed-batch mode were adopted to improve the biocatalysis process of genipin. A 10 L ethyl acetate-aqueous biphasic system with geniposide fed-batch led to a satisfying genipin yield. With Fusarium solani ACCC 36223, 15.7 g/l genipin in the ethyl acetate phase was obtained, corresponding to space-time yields of 0.654 g l(-1) h(-1).

Topics: Batch Cell Culture Techniques; Cross-Linking Reagents; Culture Media; Fusarium; Hydrolysis; Iridoids

Injectable hydrogels are extensively used in drug delivery and tissue engineering to administer drugs, genes, growth factors and live cells. We report a method to produce tough, in-situ thermogelling, non-toxic, injectable hydrogels made of chitosan and hyaluronic acid co-crosslinked with β-glycerophophate and genipin. The gels are highly homogeneous and form within 32 min, i.e., faster than gels crosslinked with either genipin or β-glycerophophate. The shear strength of co-crosslinked hydrogels is 3.5 kPa, higher than any chitosan-based gel reported. Chondrocytes and nucleus pulposus cells thrive inside the gels and produce large amounts of collagen II. Injection in rats shows that the gels form in-vivo within a short time and remain well localized for more than one week while the rats remain healthy and active. The excellent mechanical properties, fast in-situ gelation, good biocompatibility and the ability to encapsulate live cells at physiological conditions make these hydrogels ideal for tissue engineering, especially cartilage regeneration.

Topics: Animals; Cartilage; Chitosan; Chondrocytes; Collagen; Drug Delivery Systems; Hyaluronic Acid; Hydrogels; Iridoids; Rats; Regeneration; Tissue Engineering

To modify a chitosan membrane (CM) by cross-linking the chitosan with genipin, a naturally occurring cross-linker extracted from Gardenia jasminoides fructus, with the aim of developing a new cell culture support and to observe the phenotypes of cultured human corneal epithelial cells (HCECs) on genipin-cross-linked chitosan membrane (GCM).. We tested the cross-linking characteristics and mechanical strength of the GCM. CMs modified by cross-linking with different concentrations of genipin were prepared to investigate the rate of membrane degradation. The biocompatibility of the GCMs was investigated by determining the viability of HCECs cultured on them in vitro. The morphology of the HCECs cultured on CM or GCM was analyzed by confocal microscopy and scanning electron microscopy (SEM). Immunocytochemical staining was conducted to determine the phenotypes of the cultured cells.. The fixation index of the GCM was 31 ± 3% after treatment of CM with 0.5mM genipin. A stress-strain test showed that the GCM could tolerate three times the mechanical force of noncross-linked CM. The biodegradation rate of GCM was much slower than for CM. A 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay showed that cell viability was not affected by cross-linking with 5.0mM genipin. SEM showed that the cultured HCECs adhered to and grew well on the surface of the GCM. Immunocytochemical staining showed keratin 3 (K3) and connexin 43 (Cx-43) immunoreactive HCECs on the GCM and their proliferative ability was not significantly affected by strong immunoreactivity of Ki-67 and p63 markers.. GCM has potential as a scaffold for corneal epithelium in ocular surface surgery and greater mechanical strength and slower degradation than unmodified CM.

Topics: Cells, Cultured; Chitosan; Cross-Linking Reagents; Epithelium, Corneal; Humans; Iridoids

Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that causes acute infection and establishes life-long latency. EBV causes several human cancers, including Burkitt's lymphoma, nasopharyngeal and gastric carcinoma. Antiviral agents can be categorized as virucides, antiviral chemotherapeutic agents, and immunomodulators. Most antiviral agents affect actively replicating viruses, but not their latent forms. Novel antiviral agents must be active on both the replicating and the latent forms of the virus. Gardenia jasminoides is an evergreen flowering plant belonging to the Rubiaceae family and is most commonly found growing wild in Vietnam, Southern China, Taiwan, Japan, Myanmar, and India. Genipin is an aglycone derived from an iridoid glycoside called geniposide, which is present in large quantities in the fruit of G. jasminoides. In this study, genipin was evaluated for its role as an antitumor and antiviral agent that produces inhibitory effects against EBV and EBV associated gastric carcinoma (EBVaGC). In SNU719 cells, one of EBVaGCs, genipin caused significant cytotoxicity (70 μM), induced methylation on EBV C promoter and tumor suppressor gene BCL7A, arrested cell-cycle progress (S phases), upregulated EBV latent/lytic genes in a dose-dependent manner, stimulated EBV progeny production, activated EBV F promoter for EBV lytic activation, and suppressed EBV infection. These results indicated that genipin could be a promising candidate for antiviral and antitumor agents against EBV and EBVaGC.

Topics: Antineoplastic Agents; Antiviral Agents; Apoptosis; Cell Cycle; Cell Line, Tumor; DNA Methylation; Gene Expression Regulation, Viral; HEK293 Cells; Herpesvirus 4, Human; Humans; Iridoids; Microfilament Proteins; Oncogene Proteins; Promoter Regions, Genetic; S Phase; Stomach Neoplasms; Viral Proteins; Virus Latency; Virus Replication

Sterilization through γ-irradiation has been reported to affect collagen mechanical properties, but its possible effects on gelatin based materials have not been investigated up to now. Herein we report the results of a mechanical, chemical and thermal study performed on gelatin films before and after γ-irradiation. The investigation was performed on uncrosslinked films as well as on crosslinked films. To this aim, two common crosslinking agents, glutaraldehyde and genipin, at different concentration (0.15, 0.30 and 0.67%) were used. The results indicate that sterilization significantly affects the mechanical properties of uncrosslinked films, whereas it displays a modest effect on gelatin swelling, release in solution, thermal stability and molecular structure. Both glutaraldehyde and genipin enhance the mechanical properties and stability in solution of the gelatin films. In particular, the values of Young modulus increase as a function of crosslinker concentration up to about 10 and 18 MPa for genipin and glutaraldehyde treated samples respectively. The results of in vitro study demonstrate that the films crosslinked with genipin do not display any cytotoxic reaction, whereas glutaraldehyde crosslinking provokes an acute and dose dependent cytotoxic effect.

Topics: Cross-Linking Reagents; Elastic Modulus; Gamma Rays; Gelatin; Glutaral; Iridoids; Materials Testing; Membranes, Artificial; Radiation Dosage; Sterilization; Stress, Mechanical; Tensile Strength

The aim of this study was to investigate the effects of genipin, a natural collagen cross-linking agent, on odontogenic differentiation of human dental pulp cells (hDPCs) because the mechanical properties of collagen allow it to serve as a scaffold for engineering of pulp-dentin complex. Furthermore, the role of extracellular signal-regulated kinase (ERK) was investigated as a mediator of the differentiation.. The odontogenic differentiation was analyzed by alkaline phosphatase activity, real time-polymerase chain reaction, Western blotting, and alizarin red S staining. The morphologic features of hDPCs cultured in genipin-treated collagen were evaluated by scanning electron microscopy. For the assessment of mechanical properties of collagen treated with genipin, the surface roughness and compressive strength were measured.. Alkaline phosphatase activity, the expression of odontogenic markers, and mineralized nodule formation increased in the genipin-treated group. Genipin also activated ERK, and treatment with ERK inhibitor blocked the expression of the markers. The cells cultured in genipin-treated collagen spread across the substrate and attached in close proximity to one another. The proliferation and differentiation of hDPCs cultured in genipin-treated collagen were facilitated. The mechanical properties of collagen, such as surface roughness and compressive strength, were increased by treatment with genipin.. Our results show that genipin promotes odontogenic differentiation of hDPCs via the ERK signaling pathway. Furthermore, the enhanced mechanical properties of the collagen scaffold induced by genipin may play important roles in cell fate. Consequently, the application of genipin might be a new strategy for dentin-pulp complex regeneration.

Topics: Cell Survival; Cells, Cultured; Collagen; Cross-Linking Reagents; Dental Pulp; Extracellular Signal-Regulated MAP Kinases; Humans; Iridoids; Odontogenesis; Plant Extracts; Rubiaceae

The present study provided a new approach to enhance the stability of protein-emulsified nanoemulsions and to control the lipase digestibility of lipid droplets through spontaneous cross-linking of the interfacial layer with genipin, a functional ingredient isolated from the fruit of Gardenia jasminoides E. Cross-linking casein-emulsified nanoemulsions under different genipin/casein mass ratios (1:20, 1:10, 1:5) significantly (p < 0.05) or very significantly (p < 0.01) enhanced their stability under harsh gastric pH environments and prevented nanoemulsion flocculation. As observed by transmission electron microscope (TEM), under the pH 1.2 condition, the genipin cross-linked nanoemulsion showed more compact microstructure with clear and defined contour as well as "core-shell" structure caused by the swelling of the surface protein film. Interestingly, the intestinal digestibility of lipid droplets was delayed very significantly (p < 0.01) after cross-linking the interfacial casein layer with genipin, which was enhanced by the increase in genipin/casein mass ratio and cross-linking time.

Topics: Caseins; Cross-Linking Reagents; Digestion; Fats; Gardenia; Humans; Hydrogen-Ion Concentration; Intestinal Mucosa; Iridoids; Lipase; Models, Biological; Plant Extracts; Protein Stability

In this study, pH responsive genipin-cross-linked Pluronic F127-chitosan nanoparticles (GNPs) was synthesized to encapsulate trehalose for intracellular delivery to cryopreserve primary human adipose-derived stem cells (hADSCs). Trehalose is a disaccharide of glucose used by lower organisms to survive extreme cold in nature and has been used to cryopreserve various biomacromolecules. However, it does not enter mammalian cells because of its highly hydrophilic nature, and has only been used in combination with other cell-penetrating cryoprotectants (such as dimethyl sulfoxide, DMSO) to cryopreserve mammalian cells. Our data show that trehalose can be efficiently encapsulated in our GNPs for intracellular delivery, which enables cryopreservation of primary hADSCs using the nontoxic sugar as the sole cryoprotectant. This capability is important because the conventional approach of cryopreserving mammalian cells using highly toxic (at body temperature) cell-penetrating cryoprotectants requires multistep washing of the cryopreserved cells to remove the toxic cryoprotectant for further use, which is time-consuming and associated with significant cell loss (∼10% during each washing step). By contrast, the trehalose-cryopreserved cells can be used without washing, which should greatly facilitate the wide application of the burgeoning cell-based medicine.

Topics: Adipose Tissue; Cell Survival; Cells, Cultured; Cryopreservation; Cryoprotective Agents; Humans; Hyaluronan Receptors; Iridoids; Nanoparticles; Platelet Endothelial Cell Adhesion Molecule-1; Poloxamer; Stem Cells; Transcription Factors; Trehalose

To solve the problem of synthesized magnetic nanoparticles in cancer therapy, a new drug delivery system synthesized from bacteria was used to load cytosine arabinoside (Ara-C). Genipin (GP) and poly-l-glutamic acid (PLGA) were selected as dual cross-linkers. The preparation and characterization of Ara-C-loaded GP-PLGA-modified bacterial magnetosomes (BMs) (ABMs-P), as well as their in vitro antitumor effects, were all investigated. Transmission electron micrographs (TEM) and Fourier transform infrared (FTIR) spectroscopy suggested that Ara-C could be bound to the membrane of BMs modified by GP-PLGA. The diameters of the BMs and ABMs-P were 42.0±8.6 nm and 74.9±8.2 nm, respectively. The zeta potential revealed that the nanoparticles were stable. Moreover, this system exhibited optimal drug-loading properties and long-term release behavior. The optimal encapsulation efficiency and drug-loading were 64.1%±6.6% and 38.9%±2.4%, respectively, and ABMs-P could effectively release 90% Ara-C within 40 days, without the release of an initial burst. In addition, in vitro antitumor experiments elucidated that ABMs-P is cytotoxic to HL-60 cell lines, with an inhibition rate of 95%. The method of coupling drugs on BMs using dual cross-linkers is effective, and our results reveal that this new system has potential applications for drug delivery in the future.

Topics: Antineoplastic Agents; Cell Survival; Cytarabine; Drug Carriers; Glutamic Acid; HL-60 Cells; Humans; Iridoids; Magnetosomes

The present study discusses about the preparation and characterization (thermal, mechanical, and electrical) of the genipin-crosslinked gelatin emulgels. Emulgels have gained importance in recent years due to their improved stability than emulsions and ability to control the drug release. Mustard oil was used as the representative oil. A decrease in the enthalpy and entropy of the formulations was observed with the increase in the oil fraction. The mechanical studies suggested formation of softer emulgels as the oil fraction was increased. As the proportion of the oil fraction was increased in the emulgels, there was a corresponding increase in the impedance. The drug release properties from the emulgels were also studied. Ciprofloxacin was used as the model antimicrobial drug. The drug release was higher from the emulgels whose electrical conductivity was higher.

Topics: Anti-Infective Agents; Chemistry, Pharmaceutical; Ciprofloxacin; Drug Liberation; Electricity; Emulsions; Gelatin; Gels; Iridoids

3D highly porous (93% total porosity) gelatin scaffolds were prepared according to a novel, simple method, which implies gelatin foaming, gelification, soaking into ethanol and successive freeze-drying. Reinforcement of the as-prepared scaffolds (GEL) was performed through immersion in aqueous solutions at different gelatin concentrations. Reinforcement solutions with and without genipin addition allowed to prepare two series of samples:cross-linked and uncross-linked samples, respectively. The amount of gelatin adsorbed onto the reinforced samples increases as a function of gelatin concentration in solution and provokes a drastic improvement of the compressive modulus and collapse strength up to values of about 30 and 4 MPa, respectively. The open and interconnected porosity, although slightly reduced, is still of the order of 80% in the samples reinforced with the highest concentration of gelatin. Water uptake ability evaluated after immersion in PBS for 20 s decreases with gelatin reinforcement. The presence of genipin in cross-linked samples reduces gelatin release and stabilizes the scaffolds in solution. Chondrocytes from human articular cartilage adhere, proliferate, and penetrate into the scaffolds. The evaluation of differentiation markers both on the supernatants of cell culture and by means of quantitative polymerase chain reaction (qPCR) indicates a dose-dependent promotion of cell differentiation.

Topics: Cartilage, Articular; Gelatin; Humans; Iridoids; Porosity; Regeneration; Tissue Scaffolds

In this study, we present gelatin-based thermoresponsive colloidal microgels that enable the controlled release of drugs by volume phase transition. The microgel was fabricated by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within three-dimensional gelatin networks crosslinked by genipin. We demonstrate that our gelatin-based thermoresponsive microgel exhibits a tunable deswelling to temperature increase, which positively correlated to the release of bovine serum albumin (BSA) as a function of poly(N-isopropylacrylamide-co-acrylamide) concentration. The microgel was enzymatically degradable by collagenase treatment. The extent of BSA release and biodegradability were tuned by controlling the crosslinking degree of the gelatin matrix. Meeting a great need for design and synthesis of auto-degenerating smart microgels that enable the controlled release of therapeutic proteins in responsive to external stimuli, our gelatin-based microgels that satisfy both thermoresponsivity and biodegradability have a great potential in tissue engineering applications as a soft microdevice element for drug delivery.

Topics: Acrylamides; Animals; Cattle; Colloids; Delayed-Action Preparations; Gelatin; Iridoids; Phase Transition; Polymers; Serum Albumin, Bovine

Renal damage precedes occurrence of stroke in high-sodium/low-potassium-fed stroke-prone spontaneously hypertensive rat (SHRSP). We previously reported a marked suppression of uncoupling protein-2 (UCP2) upon high-salt Japanese-style diet in SHRSP kidneys. Vegetable compounds are known to exert protective effects in cardiovascular diseases. We aimed at evaluating the impact of Brassica oleracea sprouts juice toward renal damage in Japanese diet-fed SHRSP and exploring the role of 5'-adenosine monophosphate-activated protein kinase (AMPK)/NAD-dependent deacetylase sirtuin-1 (SIRT1)/peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α)/peroxisome proliferator-activated receptor-α (PPARα)/UCP2 axis.. SHRSP received Japanese diet for 4 weeks. A group of SHRSP received Japanese diet and B. oleracea. A third group received Japanese diet, B. oleracea, and PPARα inhibitor (GW6471). A group of SHRSP fed with regular diet served as control.. Japanese diet induced marked increases of oxidative stress, inflammation, and proteinuria, along with glomerular and tubular damage, as compared with regular diet. A significant suppression of AMPK/UCP2 pathway was observed. Despite Japanese diet feeding, concomitant administration of B. oleracea prevented oxidative stress accumulation, inflammation, renal damage, and proteinuria. All components of the UCP2 regulatory pathway were significantly increased by B. oleracea. Superoxide dismutase 2 and phosphoendothelial nitric oxide synthase were also stimulated. Addition of PPARα inhibitor to B. oleracea and Japanese diet significantly reduced the B. oleracea beneficial effects. SBP levels were comparable among the different groups of rats.In vitro, UCP2 inhibition by genipin offset the antioxidant effect of B. oleracea in renal mesangial and proximal tubular cells.. B. oleracea administration prevented renal damage in salt-loaded SHRSP, independently from SBP, with parallel stimulation of AMPK/SIRT1/PGC1α/PPARα/UCP2 axis. Stimulation of the latter mechanism may provide relevant renal protective effect and play a therapeutic role in target organ damage progression in hypertension.

Topics: AMP-Activated Protein Kinases; Animals; Antioxidants; Blood Pressure; Brassica; Diet; Glomerular Mesangium; Hypertension; Ion Channels; Iridoids; Kidney Diseases; Kidney Glomerulus; Kidney Tubules, Proximal; Mitochondrial Proteins; Oxidative Stress; Plant Extracts; PPAR alpha; Proteinuria; Rats; Rats, Inbred SHR; Seedlings; Sodium Chloride, Dietary; Stroke; Uncoupling Protein 2

Extracellular matrices (ECM) obtained from in vitro-cultured cells have been given much attention, but its application in cardiac tissue engineering is still limited. This study investigates cardiomyogenic potential of fibroblast-derived matrix (FDM) as a novel ECM platform over gelatin or fibronectin, in generating cardiac cell lineages derived from H9c2 cardiomyoblasts. As characterized through SEM and AFM, FDM exhibits unique surface texture and biomechanical property. Immunofluorescence also found fibronectin, collagen, and laminin in the FDM. Cells on FDM showed a more circular shape and slightly less proliferation in a growth medium. After being cultured in a differentiation medium for 7 days, H9c2 cells on FDM differentiated into cardiomyocytes, as identified by stronger positive markers, such as α-actinin and cTnT, along with more elevated gene expression of Myl2 and Tnnt compared to the cells on gelatin and fibronectin. The gap junction protein connexin 43 was also significantly upregulated for the cells differentiated on FDM. A successive work enabled matrix stiffness tunable; FDM crosslinked by 2wt% genipin increased the stiffness up to 8.5 kPa, 100 times harder than that of natural FDM. The gene expression of integrin subunit α5 was significantly more upregulated on FDM than on crosslinked FDM (X-FDM), whereas no difference was observed for β1 expression. Interestingly, X-FDM showed a much greater effect on the cardiomyoblast differentiation into cardiomyocytes over natural one. This study strongly indicates that FDM can be a favorable ECM microenvironment for cardiomyogenesis of H9c2 and that tunable mechanical compliance induced by crosslinking further provides a valuable insight into the role of matrix stiffness on cardiomyogenesis.

Topics: 3T3 Cells; Animals; Cell Line; Cell Lineage; Cell Shape; Cellular Microenvironment; Cross-Linking Reagents; Culture Media; Cytoskeletal Proteins; Extracellular Matrix; Fibronectins; Gene Expression Profiling; Heart Ventricles; Iridoids; Mice; Muscle Development; Muscle Proteins; Myoblasts; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; Tissue Engineering; Tissue Scaffolds

We investigated the modulation of innate and adaptive immune cell activation by Eucommia ulmoides Oliver extract (EUE) and its ingredient genipin. As an innate immunity indicator, the phagocytic activity of macrophages was determined by measuring engulfed, fluorescently labeled Escherichia coli. As a surrogate marker for the respective activation of cellular and humoral adaptive immunity, concanavalin A (Con A) and lipopolysaccharide (LPS) induction of primary splenocyte proliferation was assayed in in vitro and ex vivo systems. EUE and genipin suppressed the proliferation of primary splenic lymphocytes induced by Con A or LPS, but not macrophage phagocytosis. Oral administration of EUE and genipin to mice decreased splenic lymphocyte proliferation induced by Con A or LPS. These results revealed that E. ulmoides and genipin suppressed cellular and humoral adaptive immunity, and they suggest that E. ulmoides and genipin are promising candidates for immunosuppressive drugs that target diseases that involve excessive activation of adaptive immunity.

Topics: Adaptive Immunity; Administration, Oral; Animals; Cell Proliferation; Concanavalin A; Eucommiaceae; Immunosuppressive Agents; Iridoids; Lipopolysaccharides; Lymphocytes; Macrophages; Male; Mice, Inbred BALB C; Phagocytosis; Plant Extracts; Spleen

Intervertebral discs (IVDs) are attractive targets for local drug delivery because they are avascular structures with limited transport. Painful IVDs are in a chronic inflammatory state. Although anti-inflammatories show poor performance in clinical trials, their efficacy treating IVD cells suggests that sustained, local drug delivery directly to painful IVDs may be beneficial.. The purpose of this study was to determine if genipin cross-linked fibrin (FibGen) with collagen Type I hollow spheres (CHS) can serve as a drug-delivery carrier for infliximab, the anti-tumor necrosis factor α (TNFα) drug. Infliximab was chosen as a model drug because of the known role of TNFα in increasing downstream production of several pro-inflammatory cytokines and pain mediators. Genipin cross-linked fibrin was used as drug carrier because it is adhesive, injectable, and slowly degrading hydrogel with the potential to seal annulus fibrosus (AF) defects. CHS allow simple and nondamaging drug loading and could act as a drug reservoir to improve sustained delivery.. This is a study of biomaterials and human AF cell culture to determine drug release kinetics and efficacy.. Infliximab was delivered at low and high concentrations using FibGen with and without CHS. Gels were analyzed for structure, drug release kinetics, and efficacy treating human AF cells after release.. Fibrin showed rapid infliximab drug release but degraded quickly. CHS alone showed a sustained release profile, but the small spheres may not remain in a degenerated IVD with fissures. Genipin cross-linked fibrin showed steady and low levels of infliximab release that was increased when loaded with higher drug concentrations. Infliximab was bound in CHS when delivered within FibGen and was only released after enzymatic degradation. The infliximab released over 20 days retained its bioactivity as confirmed by the sustained reduction of interleukin (IL)-1β, IL-6, IL-8, and TNFα concentrations produced by AF cells.. Direct mixing of infliximab into FibGen was the simplest drug-loading protocol capable of sustained release. Results show feasibility of using drug-loaded FibGen for delivery of infliximab and, in the context with the literature, show potential to seal AF defects and partially restore IVD biomechanics. Future investigations are required to determine if drug-loaded FibGen can effectively deliver drugs, seal AF defects, and promote IVD repair or prevent further IVD degeneration in vivo.

Topics: Antirheumatic Agents; Cells, Cultured; Drug Carriers; Fibrin Tissue Adhesive; Humans; Infliximab; Intervertebral Disc; Iridoids

Sepsis, a systemic inflammatory response to infection, initiates a complex immune response consisting of an early hyperinflammatory response and a subsequent hypoinflammatory response that impairs the removal of infectious organisms. The importance of sepsis-induced immunosuppression and its contribution to mortality has recently emerged. Apoptotic depletion of T lymphocytes is a critical cause of immunosuppression in the late phase of sepsis. Genipin is a major active compound of gardenia fruit that has anti-apoptotic and anti-microbial properties. This study investigated the mechanisms of action of genipin on immunosuppression in the late phase of sepsis. Mice received genipin (1, 2.5 and 5mg/kg, i.v.) at 0 (immediately) and 24h after cecal ligation and puncture (CLP). Twenty-six hours after CLP, the spleen and blood were collected. Genipin improved the survival rate compared to controls. CLP increased the levels of FADD, caspase-8 and caspase-3 protein expression, which were attenuated by genipin. Genipin increased the level of anti-apoptotic B-cell lymphoma-2 protein expression, while it decreased the level of pro-apoptotic phosphorylated-Bim protein expression in CLP. CLP decreased the CD4(+) and CD8(+) T cell population, while it increased the regulatory T cell (Treg) population and the level of cytotoxic T lymphocyte-associated antigen 4 protein expression on Treg. These changes were attenuated by genipin. The splenic levels of interferon-γ and interleukin (IL)-2 were reduced, while the levels of IL-4 and IL-10 increased after CLP. Genipin attenuated these alterations. These findings suggest that genipin reduces immunosuppression by inhibiting T lymphocyte apoptosis in the late phase of sepsis.

Topics: Adjuvants, Immunologic; Animals; Apoptosis; Caspase 3; Cecum; Cytokines; Disease Models, Animal; Fas-Associated Death Domain Protein; Gardenia; Humans; Immunosuppression Therapy; Iridoids; Male; Mice; Mice, Inbred ICR; Proto-Oncogene Proteins c-bcl-2; Sepsis; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Th1-Th2 Balance

In this study, a fucoidan-shelled chitosan bead was developed with the purpose of oral delivery of berberine to inhibit the growth of bacteria. The cross-linking level and swelling property of the beads were affected by the pH value and the composition of the genipin/fucoidan combined gelling agent. The drug release of the berberine-loaded beads was faster in simulated gastric fluid (pH 1.2) than those in simulated intestinal fluid (pH 7.4). Furthermore, a nanoparticles/beads complex system was developed by incorporation of berberine-loaded chitosan/fucoidan nanoparticles in the fucoidan-shelled chitosan beads. The nanoparticles/beads complex served as a drug carrier to delay the berberine release in simulated gastric fluid, with an estimated lag time of 2 h. Our results showed that the berberine-loaded beads and nanoparticles/beads complex could effectively inhibit the growth inhibition of common clinical pathogens, such as Staphylococcus aureus and Escherichia coli, and have the advantage of continually releasing berberine to inhibit the growth of the bacteria over 24 h.

Topics: Administration, Oral; Anti-Bacterial Agents; Berberine; Chitosan; Cross-Linking Reagents; Delayed-Action Preparations; Escherichia coli; Escherichia coli Infections; Humans; Iridoids; Polysaccharides; Staphylococcal Infections; Staphylococcus aureus

Bone cell activities are very important in bone remodeling. This study investigates the effects of lumbrokinase on bone cell activities in cultures. Moreover, a biodegradable composite (GGT) containing genipin-crosslinked gelatin and β-tricalcium phosphate was prepared to carry lumbrokinase (GGTLK). Rat calvarial bone defects were filled with GGT and GGTLK composites. Bone healing was monitored in vivo by bioluminescence imaging and micro-CT. Lumbrokinase was found to have a dose-dependent effect on bone cell activities. Low concentrations (<1μg/ml) of lumbrokinase increased the viability, total alkaline phosphatase activity and mobility of osteoblasts, the number of total calcified nodules and the expression of osteopontin and osteocalcin; however, they considerably reduced the total tartrate-resistant acid phosphatase activity of osteoclasts. IVIS images revealed a stronger fluorescent signal in GGTLK-treated animals than in GGT-treated animals. Micro-CT analysis revealed that GGTLK induced more new bone formation than did GGT. These observations suggest that lumbrokinase released from GGTLK composite can enhance bone tissue regeneration.

Topics: Animals; Biocompatible Materials; Bone Regeneration; Bone Substitutes; Calcium Phosphates; Cell Line; Dose-Response Relationship, Drug; Drug Carriers; Endopeptidases; Gelatin; Humans; Iridoids; Mice; Osteoblasts; Osteoclasts; Porosity; Rats; Rats, Sprague-Dawley; RAW 264.7 Cells; Skull; Wound Healing; X-Ray Microtomography

Collagen crosslinking enhances many beneficial properties of articular cartilage, including resistance to chemical degradation and mechanical wear, but many crosslinking agents are cytotoxic. The purpose of this study was to evaluate the effectiveness of genipin, a crosslinking agent with favorable biocompatibility and cytotoxicity, as a potential treatment to prevent the degradation and wear of articular cartilage. First, the impact of genipin concentration and treatment duration on the viscoelastic properties of bovine articular cartilage was quantified. Next, two short-term (15 min) genipin crosslinking treatments were chosen, and the change in collagenase digestion, cartilage wear, and the friction coefficient of the tissue with these treatments was measured. Finally, chondrocyte viability after exposure to these genipin treatments was assessed. Genipin treatment increased the stiffness of healthy, intact cartilage in a dose-dependent manner. The 15-min crosslinking treatments improved cartilage's resistance to both chemical degradation, particularly at the articular surface, and to damage due to mechanical wear. These enhancements were achieved without sacrificing the low coefficient of friction of the tissue and at a genipin dose that preserved chondrocyte viability. The results of this study suggest that collagen crosslinking via genipin may be a promising preventative treatment to slow the degradation of cartilage.

Topics: Animals; Cartilage; Cattle; Cell Survival; Chondrocytes; Collagenases; Cross-Linking Reagents; Drug Evaluation, Preclinical; Elasticity; Friction; Gardenia; Iridoids; Plant Extracts; Stress, Mechanical

We report on intriguing new phenomena related to the creation of chitosan hydrogels crosslinked with genipin. We found that the reaction between chitosan and genipin is very slow, sometimes requiring more than four days until completed. Further, we discovered that altering the pH within the small range of 4.00-5.50 dramatically affects the reaction, leading to hydrogels differing both in appearance and in properties. Increasing the pH by 1.5 units led to an almost fourfold decrease in the gelation time and more than tenfold equilibrium swelling. A correlation between the percentage of unreacted genipin molecules and the hydrogel properties was identified. The strong pH dependency was attributed to the degree of chitosan protonation and to the inability of protonated chitosan to react with genipin. This research shows, for the first time, that minor changes in the pH can lead to substantially different hydrogels.

Topics: Chitosan; Cross-Linking Reagents; Hydrogels; Hydrogen-Ion Concentration; Iridoids; Particle Size; Surface Properties

A 3D plotting system was used to make chitosan-based tissue scaffolds with interconnected pores using pure chitosan (C) and chitosan cross-linked with pectin (CP) and genipin (CG). A freeze-dried chitosan scaffold (CF/D) was made to compare with C, to observe the effects of structural differences. The fiber size, pore size, porosity, compression strength, swelling ratio, drug release efficacy, and cumulative weight loss of the scaffolds were measured. Osteoblasts were cultured on the scaffolds and their proliferation, type I collagen production, alkaline phosphatase activity, calcium deposition, and morphology were observed. C had a lower swelling ratio, degradation, porosity and drug release efficacy and a higher compressional stiffness and cell proliferation compared to CF/D (p < 0.05). Of the 3D-plotted samples, cells on CP exhibited the highest degree of mineralization after 21 d (p < 0.05). CP also had the highest swelling ratio and fastest drug release, followed by C and CG (p < 0.05). Both CP and CG were stiffer and degraded more slowly in saline solution than C (p < 0.05). In summary, 3D-plotted scaffolds were stronger, less likely to degrade and better promoted osteoblast cell proliferation in vitro compared to the freeze-dried scaffolds. C, CP and CG were structurally similar, and the different crosslinking caused significant changes in their physical and biological performances.

Topics: Alkaline Phosphatase; Animals; Biocompatible Materials; Bone Substitutes; Calcification, Physiologic; Cell Differentiation; Cell Line; Cell Proliferation; Chitosan; Collagen Type I; Hydrogels; Iridoids; Materials Testing; Mice; Microscopy, Electron, Scanning; Osteoblasts; Pectins; Porosity; Printing, Three-Dimensional; Tissue Engineering; Tissue Scaffolds

A model for the process impact temperature non-uniformity during high pressure processing (HPP) of genipap fruit purees was found during genipin recovery. Purees were subjected to HPP (130-530 MPa) under quasi-isobaric non-isothermal conditions (15 min; 0, 4.6 and 9.3mg pectinases/g fruit). Genipin and protein concentration was determined, and pH was measured. Polygalacturonase activity was quantified indirectly by protein content (mg/g fruit). First order kinetics described temperature changes (0-4 min). Polygalacturonase was activated at 130 MPa, inactivated reversibly at 330 MPa and activated again at 530 MPa. Enzyme reaction rate constant (k) was placed in the 0-4 min model and temperature from 2 to 15 min was described. Protein content and pH characterization in terms of decimal reduction time improved highly the 2-15 min model. Since temperature changes were modeled, more insight of its behavior in an HPP reactor was obtained, avoiding uniformity assumptions, making easier the industrial scale HPP implementation.

Topics: Aspergillus; Food Handling; Fruit; Gardenia; Iridoids; Kinetics; Models, Theoretical; Polygalacturonase; Pressure; Temperature

Catering the hydrogel manufacturing process toward defined viscoelastic properties for intended biomedical use is important to hydrogel scaffolding function and cell differentiation. Silk fibroin hydrogels may undergo "physical" cross-linking through β-sheet crystallization during high pressure carbon dioxide treatment, or covalent "chemical" cross-linking by genipin. We demonstrate here that time-dependent mechanical properties are tunable in silk fibroin hydrogels by altering the chronological order of genipin cross-linking with β-sheet formation. Genipin cross-linking before β-sheet formation affects gelation mechanics through increased molecular weight, affecting gel morphology, and decreasing stiffness response. Alternately, genipin cross-linking after gelation anchored amorphous regions of the protein chain, and increasing stiffness. These differences are highlighted and validated through large amplitude oscillatory strain near physiologic levels, after incorporation of material characterization at molecular and micron length scales.

Topics: Carbon Dioxide; Cell Differentiation; Cell Engineering; Cross-Linking Reagents; Fibroins; Humans; Hydrogels; Hydrogen-Ion Concentration; Iridoids; Protein Structure, Secondary; Silk

We report the effects of distinct concentrations of genipin and silk fibroin (SF):chitosan (CS) ratios on the formation of SF-CS composite microspheres. We selected microspheres featuring an SF:CS ratio of 1:1, encapsulated various concentrations of bovine serum albumin (BSA), and then compared their encapsulation efficiency and sustained-release rate with those of pure CS microspheres. We determined that the following five groups of microspheres were highly spherical and featured particle sizes ranging from 70 μm to 147 μm: mass ratio of CS:SF =1:0.5, 0.1 g or 0.5 g genipin; CS:SF =1:1, 0.05 g or 1 g genipin; and CS:SF =1:2, 0.5 g genipin. The microspheres prepared using 1:1 CS:SF ratio and 0.05 g genipin in the presence of 10 mg, 20 mg, and 50 mg of BSA exhibited encapsulation efficiencies of 50.16%±4.32%, 56.58%±3.58%, and 42.19%±7.47%, respectively. Fourier-transform infrared spectroscopy (FTIR) results showed that SF and CS were cross-linked and that the α-helices and random coils of SF were converted into β-sheets. BSA did not chemically react with CS or SF. Moreover, thermal gravimetric analysis (TGA) results showed that the melting point of BSA did not change, which confirmed the FTIR results, and X-ray diffraction results showed that BSA was entrapped in microspheres in a noncrystalline form, which further verified the TGA and FTIR data. The sustained-release microspheres prepared in the presence of 10 mg, 20 mg, and 50 mg of BSA burst release 30.79%±3.43%, 34.41%±4.46%, and 41.75%±0.96% of the entrapped BSA on the 1st day and cumulatively released 75.20%±2.52%, 79.16%±4.31%, and 89.04%±4.68% in 21 days, respectively. The pure CS microspheres prepared in the presence of 10 mg of BSA burst release 39.53%±1.76% of BSA on the 1st day and cumulatively released 83.57%±2.33% of the total encapsulated BSA in 21 days. The SF-CS composite microspheres exhibited higher sustained release than did the pure CS microspheres, and thus these composite microspheres might function as a superior drug carrier.

Topics: Chitosan; Cross-Linking Reagents; Delayed-Action Preparations; Drug Compounding; Fibroins; Iridoids; Microspheres; Particle Size; Serum Albumin, Bovine; Silk; Solubility; Surface Properties; Thermogravimetry; Water; X-Ray Diffraction

The objective of this work was to investigate material properties and osteogenic differentiation of human mesenchymal stem cells (hMSCs) in genipin (GN) crosslinked chitosan/nano β-tricalcium phosphate (CS/nano β-TCP) scaffolds, and compare the results with tripolyphosphate (TPP) crosslinked scaffolds. Porous crosslinked CS/nano β-TCP scaffolds were produced by freeze-gelation using GN (CBG scaffold) and TPP (CBT scaffold) as crosslinkers. The prepared CBT and CBG scaffolds were characterized with respect to porosity, pore size, water content, wettability, compressive strength, mass loss, and osteogenic differentiation of hMSCs. All scaffolds displayed interconnected honeycomb-like microstructures. There was a significant difference between the average pore size, porosity, contact angle, and percent swelling of CBT and CBG scaffolds. The average pore size of CBG scaffolds was higher than CBT, the porosity of CBG was lower than CBT, the water contact angle of CBG was higher than CBT, and the percent swelling of CBG was lower than CBT. At a given crosslinker concentration, there was not a significant difference in compressive modulus and mass loss of CBG and CBT scaffolds. Metabolic activity of hMSCs seeded in CBG scaffolds was slightly higher than CBT. Furthermore, CBG scaffolds displayed slightly higher extent of mineralization after 21 days of incubation in osteogenic medium compared to CBT.

Topics: Alkaline Phosphatase; Biocompatible Materials; Calcium Phosphates; Cell Differentiation; Cell Survival; Cells, Cultured; Chitosan; Compressive Strength; Gels; Humans; Iridoids; Materials Testing; Mesenchymal Stem Cells; Nanostructures; Osteogenesis; Polyphosphates; Porosity; Spectroscopy, Fourier Transform Infrared; Tissue Scaffolds; X-Ray Diffraction

Nanocarriers play an important role in improving the photo- and thermal-stability of photosensitizers to gain better pharmacokinetics behavior in tumor photothermal therapy. Herein, PEGylated chitosan (CG-PEG; PEG: polyethylene glycol) nanoparticles with ultrasmall size (∼5 nm) were prepared through a water-in-oil reverse microemulsion method using genipin as a cross-linker. Particle size and zeta-potential can be tuned by varying the molar ratio between chitosan amino groups and genipin. CG-PEG-ICG (ICG: indocyanine green) nanoparticles were fabricated by adding ICG to CG-PEG aqueous solution through a self-assembly method via electrostatic interaction. The resultant CG-PEG-ICG nanoparticles exhibited improved photo- and thermal-stability, good biocompatibility, and low toxicity. When irradiated with a laser, the cells incubated with CG-PEG-ICG nanoparticles showed very low cell viability (15%), indicating the CG-PEG-ICG nanoparticles possess high in vitro photothermal toxicity. Moreover, the CG-PEG nanocarriers can significantly alter the biodistribution and prolong the retention time of ICG in the mice body after intravenous injection. In vivo photothermal study of tumors injected with CG-PEG-ICG nanoparticles containing ICG at a concentration greater than 100 μg·mL(-1) (100 μL) induced irreversible tissue damage. The growth of U87 tumors was dramatically inhibited by CG-PEG-ICG nanoparticles, demonstrating that the CG-PEG nanoparticles may act as potential ICG nanocarriers for effective in vivo tumor photothermal therapy.

Topics: Animals; Cell Line, Tumor; Cell Proliferation; Chitosan; Drug Carriers; Iridoids; Low-Level Light Therapy; Mice; Nanoparticles; Neoplasms, Experimental; Particle Size; Photosensitizing Agents

To improve durability in wet conditions, electrospun chitosan (CTS) nanofibers were submersed into PBS (pH 7.4) solutions containing varied amounts of genipin (GP 0.1, 0.5, and 1% w/v) for crosslinking treatment. GP-crosslinking allowed the electrospun CTS nanofibers to maintain their fibrous morphology in wet state. Maximum tensile strength, 84.2% of the dry state strength, was attained when crosslinking was performed in GP 0.5% solution. GP-crosslinking also endowed the CTS nanofibers with enhanced resistances to swelling and enzymatic degradation. GP-crosslinked CTS nanofibers were found to significantly promote the adhesion and growth of the L929 fibroblasts, with the most suitable sample was the one crosslinked in the GP 0.5% solution as well. Our results suggest that crosslinking with the 0.5% GP in PBS could yield CTS nanofibers with improved wet stability in nanofiber structure and optimized mechanical and biological performances.

Topics: Animals; Biocompatible Materials; Cell Culture Techniques; Cell Line; Cell Proliferation; Cell Survival; Chitosan; Cross-Linking Reagents; Iridoids; Materials Testing; Mice; Muramidase; Nanofibers; Spectroscopy, Fourier Transform Infrared; Tensile Strength

In this study, spray-dried alfuzosin hydrochloride (ALF)-loaded casein (CAS) nanoparticles were successfully used for the preparation of a swellable floating matrix via direct compression. The developed NIR calibration model was able to assess ALF and CAS levels in five different batches of drug-loaded nanoparticles. The calibration and prediction plots exhibited good linearity with correlation coefficients of more than 0.9. The standard error of calibration and cross-validation was less than 5% of the measured values, confirming the accuracy of the model. A linear relationship was obtained correlating the actual drug entrapped and the predicted values obtained from the NIR partial least squares regression model. The un-crosslinked tablet demonstrated a substantial weight gain (317% after 2h) and completely disintegrated after 3-4h whereas both 10 and 40% w/w genipin-crosslinked tablets showed lower weight gain (114 and 42% after 2h, respectively). A rapid floating of the tablets within 5-15min (compared to 45min for the marketed tablet) was observed, with maintained floating for 24h. Marketed and prepared tablets succeeded to prolong ALF release for 24h. The development of drug-loaded CAS nanoparticles using spray-drying represents a new alternative for the preparation of swellable floating tablets for prolonged drug release.

Topics: Calibration; Caseins; Chemistry, Pharmaceutical; Drug Compounding; Iridoids; Nanoparticles; Spectroscopy, Near-Infrared; Tablets

To prepare pravastatin sodium-loaded chitosan microspheres to allow sustained drug release.. The drug-loaded chitosan microspheres were prepared by using genipin as the cross-linker. The influences of molecular weight of chitosan, volume ratio of oil and water, reaction temperature, and stirring speed on the formation of chitosan microspheres were investigated. The morphology of the microspheres was observed using scanning electron microscopy. The encapsulation efficiency, swelling ratio under different pH conditions, and in vitro drug release were measured.. The in vitro release of pravastatin sodium could last for at least 31 days. The drug release rate varied with the reaction condition. The drug entrapment efficiency of the microsphere was 54.7%. The optimal processing conditions were as follows: chitosan viscosity of 200-400 mPa·s, oil-water proportion of 10:1, stirring speed of 850 r/min, and reaction temperature at 40 degrees celsius;.. The pravastatin sodium-loaded microspheres show good sustained drug release property, and the drug release rate can be modified by controlling the cross-linking time.

Topics: Chitosan; Cross-Linking Reagents; Delayed-Action Preparations; Iridoids; Microscopy, Electron, Scanning; Microspheres; Pravastatin

Incomplete clearance of apoptotic cells and reactive oxygen species (ROS) release are known to trigger inflammasome activation causing severe inflammation in acute lung injury and various metabolic and autoimmune diseases. Moreover, it has been reported that apoptotic cell clearance and ROS-mediated apoptosis critically depend on mitochondrial uncoupling protein-2 (UCP2). However, the relationship between UCP2 and inflammasome activation has not been studied. This report investigates the role of UCP2 in the expression and activation of NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in human macrophages. We found that UCP2 overexpression significantly enhanced the expression levels of NLRP3. The NLRP3 expression levels were significantly suppressed in THP1 cells treated with genipin, a UCP2 inhibitor, compared to controls. In addition, genipin altered adenosine triphosphate (ATP)- and hydrogen peroxide (H2O2)-mediated interleukin-1 beta (IL-1β) secretion and significantly suppressed caspase-1 activity in inflammasome-activated human macrophages. Taken together, our results suggest that genipin modulates NLRP3 inflammasome activation and ATP- or H2O2-mediated IL-1β release.

Topics: Apoptosis; Carrier Proteins; Caspase 1; Cells, Cultured; Enzyme Activation; Gene Expression Regulation; Humans; Inflammasomes; Inflammation; Interleukin-1beta; Ion Channels; Iridoids; Macrophages; Mitochondrial Proteins; NLR Family, Pyrin Domain-Containing 3 Protein; Reactive Oxygen Species; Uncoupling Protein 2

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular behaviors on disease-relevant time scales. Previously, microcontact printing has been used to fabricate two-dimensional functional arterial mimics through patterning of extracellular matrix protein as guidance cues for tissue organization. Vascular muscular thin films utilized these mimics to assess functional contractility. However, the microcontact printing fabrication technique used typically incorporates hydrophobic PDMS substrates. As the tissue turns over the underlying extracellular matrix, new proteins must undergo a conformational change or denaturing in order to expose hydrophobic amino acid residues to the hydrophobic PDMS surfaces for attachment, resulting in altered matrix protein bioactivity, delamination, and death of the tissues. Here, we present a microfluidic deposition technique for patterning of the crosslinker compound genipin. Genipin serves as an intermediary between patterned tissues and PDMS substrates, allowing cells to deposit newly-synthesized extracellular matrix protein onto a more hydrophilic surface and remain attached to the PDMS substrates. We also show that extracellular matrix proteins can be patterned directly onto deposited genipin, allowing dictation of engineered tissue structure. Tissues fabricated with this technique show high fidelity in both structural alignment and contractile function of vascular smooth muscle tissue in a vascular muscular thin film model. This technique can be extended using other cell types and provides the framework for future study of chronic tissue- and organ-level functionality.

Topics: Cell Culture Techniques; Dimethylpolysiloxanes; Fibronectins; Humans; Iridoids; Microfluidic Analytical Techniques; Muscle, Smooth, Vascular

Genipin, a hydrolyzed metabolite of geniposide extracted from the fruit of Gardenia jasminoides Ellis, has shown promise in alleviating depressive symptoms, however, the antidepressant mechanism of genipin remains unclear and incomprehensive. In this study, the metabolic profiles of aqueous and lipophilic extracts in liver of the chronic unpredictable mild stress (CUMS)-induced rat with genipin treatment were investigated using proton nuclear magnetic resonance ((1)H NMR) spectroscopy coupled with multivariate data analysis. Significant differences in the metabolic profiles of rats in the CUMS model group (MS) and the control group (NS) were observed with metabolic effects including decreasing in choline, glycerol and glycogen, increasing in lactate, alanine and succinate, and a disordered lipid metabolism, while the moderate dose (50mg/kg) of genipin could significantly regulate the concentrations of glycerol, lactate, alanine, succinate and the lipid to their normal levels. These biomakers were involved in metabolism pathways such as glycolysis/gluconeogensis, tricarboxylic acid (TCA) cycle and lipid metabolism, which may be helpful for understanding of antidepressant mechanism of genipin.

Topics: Animals; Antidepressive Agents; Behavior, Animal; Body Weight; Depression; Disease Models, Animal; Iridoids; Liver; Magnetic Resonance Spectroscopy; Male; Metabolome; Metabolomics; Rats, Sprague-Dawley; Stress, Psychological

Scleral cross-linking (CXL) is a novel attempt to slow down the axial elongation process in animal eyes. As a natural CXL reagent, genipin would be also effective for the prevention of myopia process. Thus, the present study was designed to evaluate the effects of scleral cross-linking using genipin on the form-deprivation (FD) myopia process of guinea pigs.. Twenty-seven 3-week-old pigmented guinea pigs were randomly divided into three groups. Group A (n = 8) is the untreated control group. Group B (n = 8) is the FD control group, where all eyes were induced with monocular FD for 21 days. In Group C (n = 11), a sub-Tenon injection of 0.10 mL 0.50 % genipin was performed on FD eyes at day 0, 7 and 14 during the 21-day monocular FD. The ocular refraction, axial length, biomechanical test and light and electron microscopy were measured on all eyes to check the efficacy and safety of this scleral CXL technique.. Compared with Group A, significant increases in myopic refractive errors, axial elongation and reductions of scleral fibril diameter and density were observed in the 21-day FD eyes of Group B (P < 0.05). In Group C, the scleral CXL resulted in less myopia and axial elongation as compared with Group B (P < 0.05); a significant thickening of scleral fibrils was found after sub-Tenon injections of genipin; no histological damage on the retina or choroid was observed in Group C at the end of this study.. The FD myopia in guinea pig eyes was effectively blocked by the scleral CXL using sub-Tenon injections of genipin. No histological damage was found on the retina or choroid of these treated eyes. Further studies are needed to examine the long-term efficacy and safety of this CXL technique.

Topics: Animals; Axial Length, Eye; Cholagogues and Choleretics; Cross-Linking Reagents; Disease Models, Animal; Elasticity; Guinea Pigs; Injections, Intraocular; Iridoids; Myopia; Refraction, Ocular; Sclera; Sensory Deprivation

In this contribution, superhydrophilic chitosan-based scaffolds with ultrafast spreading property were fabricated and used to improve the trapped efficiency of cells. The ultrafast spreading property allowed cells to be trapped into the internal 3D porous structures of the prepared scaffolds more quickly and effectively. Cell adhesion, growth, and proliferation were also improved, which could be attributed to the combination of UV irradiation and ultrafast spreading property. The construction of ultrafast spreading property on the scaffold surface will offer a novel way to design more effective scaffold in tissue engineering that could largely shorten the therapeutic time for patients.

Topics: Animals; Biocompatible Materials; Cell Adhesion; Cell Line; Cell Proliferation; Cell Survival; Chitosan; Hydrophobic and Hydrophilic Interactions; Iridoids; Mice; Microscopy, Electron, Scanning; Photoelectron Spectroscopy; Porosity; Surface Properties; Tissue Engineering; Tissue Scaffolds; Ultraviolet Rays

Novel chitosan-polyvinyl pyrrolidone/45S5 Bioglass® (CS-PVP/BG) scaffolds were prepared via foam replication and chemical cross-linking techniques. The pristine BG, CS-PVP coated BG and genipin cross-linked CS-PVP/BG (G-CS-PVP/BG) scaffolds were synthesized and characterized in terms of chemical composition, physical structure and morphology respectively. Resistance to enzymatic degradation of the scaffold is improved significantly with the use of genipin cross-linked CS-PVP. The bio-effects of scaffolds on MC3T3-E1 osteoblast-like cells were evaluated by studying cell viability, adhesion and proliferation. The CCK-8 assay shows that cell viability on the resulting G-CS-PVP/BG scaffold is improved obviously after cross-linking of genipin. Cell skeleton images exhibit that well-stretched F-actin bundles are obtained on the G-CS-PVP/BG scaffold. SEM results present significant improvement on the cell adhesion and proliferation for cells cultured on the G-CS-PVP/BG scaffold. The drug release performance on the as-synthesized scaffold was studied in a phosphate buffered saline (PBS) solution. Vancomycin is found to be released in burst fashion within 24h from the pristine BG scaffold, however, the release period from the G-CS-PVP/BG scaffold is enhanced to 7days, indicating improved drug release properties of the G-CS-PVP/BG scaffold. Our results suggest that the G-CS-PVP/BG scaffolds possess promising physicochemical properties, sustained drug release capability and good biocompatibility for MC3T3-E1 cells' proliferation and adhesion, suggesting their potential applications in areas such as MC3T3-E1 cell stimulation and bone tissue engineering.

Topics: Animals; Cell Adhesion; Cell Line; Cell Proliferation; Ceramics; Chitosan; Delayed-Action Preparations; Glass; Iridoids; Mice; Osteoblasts; Povidone

One kind of adsorbent based on chitosan and gelatin with interpenetrating polymer networks (IPN) and porous dual structures was prepared using genipin as the cross-linker. These dual structures were demonstrated by means of Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TGA) and scanning electron microscopy (SEM). Adsorptions of acid orange II dye from aqueous solution were carried out at different genipin contents, adsorption times and pH values. The results showed that this material was put up the largest adsorption capacity when the genipin content is 0.25 mmol/L, meanwhile, the lower the solution pH value the greater the adsorption capacity. The chitosan/gelatin interpenetrating polymer networks porous material displayed pH-sensitive and rapidly response in adsorption and desorption to pH altered. It is indicated that the cross-linked chitosan/gelatin interpenetrating polymer networks porous material could be used as a recyclable adsorbent in removal or separation of anionic dyes as environmental pH condition changed.

Topics: Adsorption; Azo Compounds; Chitosan; Coloring Agents; Cross-Linking Reagents; Gelatin; Iridoids; Microscopy, Electron, Scanning; Naphthalenes; Porosity; Spectroscopy, Fourier Transform Infrared; Thermogravimetry; Water Pollutants, Chemical

Using live eukaryotic cells, including cancer cells, MCF-7 and HCT-116, normal hepatocytes and red blood cells in anode and potassium ferricyanide in cathode of MFC could generate bio-based electric current. Electrons and protons generated from the metabolic reaction in both cytosol and mitochondria contributing to the leaking would mediate the generation of electric current. Both resveratrol (RVT) and 2,4-dinitrophenol (DNP) used to induce proton leak in mitochondria were found to promote electric current production in all cells except red blood cells without mitochondria. Proton leak might be important for electric current production by bringing the charge balance in cells to enhance the further electron leak. The induced electric current by RVT can be blocked by Genipin, an inhibitor of UCP2-mediated proton leak, while that induced by DNP cannot. RVT could reduce reactive oxygen species (ROS) level in cells better than that of DNP. In addition, RVT increased mitochondrial membrane potential (MMP), while DNP decreased it. Results highly suggested the existence of at least two types of electric current that showed different properties. They included UCP2-mediated and non-UCP2-mediated electric current. UCP2-mediated electric current exhibited higher reactive oxygen species (ROS) reduction effect per unit electric current production than that of non-UCP2-mediated electric current. Higher UCP2-mediated electric current observed in cancer cells might contribute to the mechanism of drug resistence. Correlation could not be established between electric current production with either ROS and MMP without distinguishing the types of electric current.

Topics: 2,4-Dinitrophenol; Animals; Electricity; Electrodes; Electron Transport; Erythrocytes; Eukaryotic Cells; HCT116 Cells; Hepatocytes; Humans; Ion Channels; Iridoids; MCF-7 Cells; Membrane Potential, Mitochondrial; Mice; Mitochondrial Proteins; Protons; Rabbits; Reactive Oxygen Species; Resveratrol; Stilbenes; Uncoupling Protein 2

Natural compounds are becoming important candidates in cancer therapy due to their cytotoxic effects on cancer cells by inducing various types of programmed cell deaths. In this study, we investigated whether genipin induces programmed cell deaths and mediates in Egr1/p21 signaling pathways in gastric cancer cells. Effects of genipin in AGS cancer cell lines were observed via evaluation of cell viability, ROS generation, cell cycle arrest, and protein and RNA levels of p21, Egr1, as well as apoptotic marker genes. The cell viability of AGS cells reduced by genipin treatment via induction of the caspase 3-dependent apoptosis. Cell cycle arrest was observed at the G2/M phase along with induction of p21 and p21-dependent cyclins. As an upstream mediator of p21, the transcription factor early growth response-1 (Egr1) upregulated p21 through nuclear translocation and binding to the p21 promoter site. Silencing Egr1 expression inhibited the expression of p21 and downstream molecules involved in apoptosis. We demonstrated that genipin treatment in AGS human gastric cancer cell line induces apoptosis via p53-independent Egr1/p21 signaling pathway in a dose-dependent manner.

Topics: Antineoplastic Agents; Apoptosis; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cyclin-Dependent Kinase Inhibitor p21; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Iridoids; Molecular Structure; Repressor Proteins; Signal Transduction; Stomach Neoplasms; Structure-Activity Relationship

UCP2 plays a physiological role by regulating mitochondrial biogenesis, maintaining energy balance, ROS elimination, and regulating cellular autophagy in numerous tissues. But the exact roles of UCP2 in cumulus cells are still not clear. Genipin, a special UCP2 inhibitor, was added into the cultural medium to explore the roles of UCP2 in human cumulus cells. There were no significant differences in ATP and mitochondrial membrane potential levels in cumulus cells from UCP2 inhibiting groups as compared with the control. The levels of ROS and Mn-SOD were markedly elevated after UCP2 inhibited Genipin. However, the ratio of reduced GSH to GSSG significantly declined after treatment with Genipin. UCP2 inhibition by Genipin also resulted in obvious increase in the active caspase-3, which accompanied the decline of caspase-3 mRNA. The level of progesterone in culture medium declined obviously after Genipin treatment. But there was no significant difference in estradiol concentrations. This study indicated that UCP2 is expressed in human cumulus cells and plays important roles on mediate ROS production, apoptotic process, and steroidogenesis, suggesting UCP2 may be involved in regulation of follicle development and oocyte maturation and quality.

Topics: Apoptosis; Autophagy; Caspase 3; Cell Line; Cumulus Cells; Female; Glutathione; Humans; Ion Channels; Iridoids; Membrane Potential, Mitochondrial; Mitochondrial Proteins; Oxidative Stress; Reactive Oxygen Species; Superoxide Dismutase; Uncoupling Protein 2

Herb pair serves as the basic building block of a traditional Chinese medicine (TCM) formula. The rhubarb-gardenia herb pair (RGHP), composed of rhubarb and gardenia, has meaningful clinical effects to cure cholestasis diseases. This study was designed to confirm the expected synergistic effects of RGHP at pharmacodynamic and pharmacokinetic levels.. Thirty male Sprague-Dawley rats were divided into control, model and drug-treated groups. After intragastrically administrated with α-naphthylisothiocyanate (ANIT) to induce cholestasis, rats were treated with rhubarb, gardenia or RGHP. For pharmacodynamic study, biochemical and histopathological tests were performed to assess the hepatoprotective effects. While for pharmacokinetic study, a LC-MS method was developed for determination of five main chemical markers, namely genipin, rhein, aloe emodin, emodin and chrysophanol in rat plasma.. The biochemical and histopathological tests suggested that RGHP exerted enhanced hepatoprotective effects against the ANIT-induced cholestasis compared with single herbs. The pharmacokinetic study indicated RGHP could significantly elevate systemic exposure level and prolong retention time of five markers in comparison with rhubarb or gardenia alone.. The present study demonstrated the synergistic effects of RGHP in ANIT-induced cholestatic rats at pharmacodynamic and pharmacokinetic levels, and has significant enlightenments for the rational use of the related TCM formulas containing RGHP.

Topics: 1-Naphthylisothiocyanate; Alanine Transaminase; Alkaline Phosphatase; Animals; Anthraquinones; Aspartate Aminotransferases; Bilirubin; Cholestasis; Drug Synergism; Emodin; Fruit; Gardenia; Iridoids; Liver; Male; Plant Extracts; Protective Agents; Rats, Sprague-Dawley; Rheum; Rhizome

The aim of this study was to modulate the cationicity of chitosan to influence the mesenchymal stem cell (MSC) responses in terms of cell adhesion, proliferation, and differentiation. The authors prepared water-soluble carboxymethyl chitosan hydrogels using genipin as the crosslinking agent. The chitosan cationicity was modulated by varying the genipin content from 0.5 to 10 wt. %. The results indicated that the cationicity exerted a striking modulation effect on various MSC responses. The increase of the genipin content, i.e., decrease of the free amino group content (cationicity), overall promoted the MSC adhesion, cytoskeleton organization, proliferation, and differentiation into the osteogenic lineage. A surprising cell alignment effect was also observed on chitosan samples with high genipin concentrations (>2.5%). The chitosan sample with the highest genipin concentrations (10%) exhibited the best MSC proliferation and highest protein expression levels toward osteogenic lineages. The genipin content also showed a strong modulation effect on MSC condensation, and cell-cell and cell-matrix interactions, as suggested by the expressions of the sry related HMG box9 (Sox9), intercellular adhesion molecule 1, and N-Cadherin. Overall, the authors have demonstrated that modulation of cationicity (amino content) of chitosan is an effective and simple approach to tuning various MSC responses, including adhesion, proliferation, differentiation, as well as cell-cell interactions. Such findings might have important implications in biomaterial design for various biomedical applications.

Topics: Animals; Cations; Cell Adhesion; Cell Differentiation; Cell Proliferation; Chitosan; Hydrogels; Iridoids; Mesenchymal Stem Cells; Rats

Genipin, a natural and non-toxic cross linker, was used to prepare cross linked floating kappa carrageenan/sodium carboxymethyl cellulose hydrogels and the effect of genipin on hydrogels characterization was investigated. Calcium carbonates were employed as gas forming agents. Ranitidine hydrochloride was used as drug. Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and thermogravimetric analysis (TGA) were carried out to study the changes in the characteristics of hydrogels. Furthermore, scanning electron microscope (SEM) was performed to study microstructure of hydrogels. The result showed that all formulated hydrogels had excellent floating behavior. It was discovered that the cross linking reaction showed significant effect on gel strength, porosity and swelling ratio compared to non-cross linked hydrogels. It was found that the drug release was slower and lesser after being cross linked. Microstructure study shows that cross linked hydrogels exhibited hard and rough surface. Therefore, genipin can be an interesting cross linking agent for controlled drug delivery in gastrointestinal tract.

Topics: Carrageenan; Cross-Linking Reagents; Drug Carriers; Drug Delivery Systems; Drug Evaluation, Preclinical; Iridoids; Porosity; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction

Ischemic stroke causes extensive cellular loss that impairs brain functions, resulting in severe disabilities. No effective treatments are currently available for brain tissue regeneration. The need to develop effective therapeutic approaches for treating stroke is compelling. A tissue engineering approach employing a hydrogel carrying both cells and neurotrophic cytokines to damaged regions is an encouraging alternative for neuronal repair. However, this approach is often challenged by low in vivo cell survival rate, and low encapsulation efficiency and loss of cytokines. To address these limitations, we propose to develop a biomaterial that can form a matrix capable of improving in vivo survival of transplanted cells and reducing in vivo loss of cytokines. Here, we report that using sericin, a natural protein from silk, we have fabricated a genipin-cross-linked sericin hydrogel (GSH) with porous structure and mild swelling ratio. The GSH supports the effective attachment and growth of neurons in vitro. Strikingly, our data reveal that sericin protein is intrinsically neurotrophic and neuroprotective, promoting axon extension and branching as well as preventing primary neurons from hypoxia-induced cell death. Notably, these functions are inherited by the GSH's degradation products, which might spare a need of incorporating costly cytokines. We further demonstrate that this neurotrophic effect is dependent on the Lkb1-Nuak1 pathway, while the neuroprotective effect is realized through regulating the Bcl-2/Bax protein ratio. Importantly, when transplanted in vivo, the GSH gives a high cell survival rate and allows the cells to continuously proliferate. Together, this work unmasks the neurotrophic and neuroprotective functions for sericin and provides strong evidence justifying the GSH's suitability as a potential neuronal cell delivery vehicle for ischemic stroke repair.

Topics: Animals; Biocompatible Materials; Bombyx; Brain Ischemia; Cell Hypoxia; Cell Line; Cell Proliferation; Cell Survival; Cross-Linking Reagents; Cytokines; Glutathione Transferase; Green Fluorescent Proteins; Humans; Hydrogels; Iridoids; Mice; Mice, Inbred C57BL; Mice, Nude; Neurons; Neuroprotective Agents; Porosity; Regeneration; Sericins; Silk; Sincalide; Spectroscopy, Fourier Transform Infrared; Stroke; Tissue Engineering

A continuous process based on the combination of ultrasounds and/or microwaves pretreatments followed by enzymatic hydrolysis and simultaneous extraction (EHSE) has been proposed to recover genipin from Eucommia ulmoides bark. At first, in the pretreatment step, the mixture of 1.0 g dried bark powder and 10 mL deionized water were irradiated by microwave under 500 W for 10 min. Then, in hydrolysis step, the optimal conditions were as follows: 0.5 mg/mL of cellulase concentration, 4.0 pH of enzyme solution, 24 h of incubation time and 40 °C of incubation temperature. After incubation, 10 mL ethanol was added to extract genipin for 30 min by ultrasound. After EHSE treatment, the yield of genipin could reach 1.71 μmol/g. Moreover, scanning electron micrographs illustrated that severe structural disruption of plant was obtained by EHSE. The results indicated that the EHSE method provided a good alternative for the preparation of genipin from Eucommia ulmoides bark as well as other herbs.

Topics: Cellulase; Dose-Response Relationship, Radiation; Ethanol; Eucommiaceae; Hydrogen-Ion Concentration; Hydrolysis; Iridoids; Liquid Phase Microextraction; Microwaves; Plant Bark; Sonication; Temperature

Inflammasomes are cytoplasmic, multiprotein complexes that trigger caspase-1 activation and IL-1β maturation in response to diverse stimuli. Although inflammasomes play important roles in host defense against microbial infection, overactive inflammasomes are deleterious and lead to various autoinflammatory diseases. In the current study, we demonstrated that genipin inhibits the induction of IL-1β production and caspase-1 activation by NLRP3 and NLRC4 inflammasomes. Furthermore, genipin specifically prevented NLRP3-mediated, but not NLRC4-mediated, ASC oligomerization. Notably, genipin inhibited autophagy, leading to NLRP3 and NLRC4 inflammasome inhibition. UCP2-ROS signaling may be involved in inflammasome suppression by genipin. In vivo, we showed that genipin inhibited NLRP3-dependent IL-1β production and neutrophil flux in LPS- and alum-induced murine peritonitis. Additionally, genipin provided protection against flagellin-induced lung inflammation by reducing IL-1β production and neutrophil recruitment. Collectively, our results revealed a novel role in inhibition of inflammatory diseases for genipin that has been used as therapeutics for centuries in herb medicine.

Topics: Animals; Apoptosis Regulatory Proteins; Autophagy; Calcium-Binding Proteins; CARD Signaling Adaptor Proteins; Carrier Proteins; Caspase 1; Chemokines; Cytokines; Disease Models, Animal; Flagellin; Inflammasomes; Ion Channels; Iridoids; Lipopolysaccharides; Macrophage Activation; Macrophages; Mice; Mice, Knockout; Mitochondrial Proteins; NLR Family, Pyrin Domain-Containing 3 Protein; Peritonitis; Pneumonia; Protein Multimerization; Reactive Oxygen Species; Uncoupling Protein 2

In vivo examination of intradiscal pressure by quantitative discomanometry (QD).. To determine whether an injectable, exogenous crosslinking could acutely restore intradiscal pressure of stab-injured discs in vivo by short-term treatment.. Disc biomechanical performance depends on its integrity associated with the intradiscal pressure and mechanical properties. Genipin crosslink augmentation has demonstrated the in vitro biomechanical capability to improve intervertebral joint stability and increase mechanical properties of the annulus fibrosus.. 4 lumbar discs on each of 8 swine were randomly assigned to 4 groups: intact, injured, untreated, and crosslinked. A 16G needle was stabbed into the annulus fibrosus to create the disc injury model. An injection of 0.33% genipin solution was delivered into the annulus to treat the injury. QD technique was performed to examine the intradiscal pressure for the intact and injured discs at the time of surgery, while untreated and crosslinked discs were measured 1-week postsurgery. 4 QD parameters were analyzed and compared across the 4 groups: leakage pressure and volume, and saturation pressure and volume.. The leakage and saturation pressures of the injured group were significantly lower than those of the intact group (P = 0.004 and P = 0.01, respectively). The leakage and saturation pressures of untreated discs were statistically equivalent to the injured levels, but with a 2-times higher saturation volume. Relative to the untreated group, the leakage pressure and saturation pressure of genipin-crosslinked discs had a 617% (P = 0.008) and a 473% increase (P = 0.007), respectively.. A large disc injury produced by annular puncture immediately lowered intradiscal pressure when left untreated. Genipin crosslinking can restore intradiscal pressure acutely in vivo without any obvious morbidity associated with the injection.

Topics: Animals; Biomechanical Phenomena; Intervertebral Disc; Iridoids; Lumbar Vertebrae; Manometry; Spinal Injuries; Swine

To explore whether the inhibitory effect of Genipin on uncoupling protein-2 (UCP-2) in mitochondria is involved in energy metabolism of androgen-independent PC3 prostate cancer cells.. PC3 prostate cancer cells were cultured and treated with Genipin at the concentrations of 40, 80, and 160 μmol/L for 48 hours. Then the proliferation of the cells was detected by MTT assay, the expression of UCP-2 mRNA determined by RT-PCR, and the content of intracellular pyruvic acid (PA) and the activity of succinate dehydrogenase (SDH) in the mitochondria measured by visible spectrophotometry.. With the increased concentration of Genipin, the proliferative activity of the PC-3 cells, the expression level of UCP-2 mRNA, the content of intracellular PA and the activity of SDH in the cells were all decreased, namely, with the enhanced inhibitory effect of Genipin on UCP-2, a trend of reduction was observed in the proliferation of the cells, intracellular PA content, and SDH activity in the mitochondria.. Genipin is involved in the energy metabolism of androgen-independent PC3 prostate cancer cells by reducing the content of intracellular PA and the activity of SDH in the mitochondria, which may be associated with its inhibitory effect on UCP-2.

Topics: Cell Line, Tumor; Energy Metabolism; Humans; Ion Channels; Iridoids; Male; Mitochondria; Mitochondrial Proteins; Prostatic Neoplasms; Pyruvic Acid; RNA, Messenger; Succinate Dehydrogenase; Uncoupling Protein 2

To investigate the effects of modification of acellular bovine pericardium with 1-ethyl-3-(3- dinethylami-nopropyl) carbodimide (EDC)/N-hydroxysuccininide (NHS) or genipin and find out the best crosslinking reagent.. The cellular components of the bovine pericardiums were removed. The effects of decellularization were tested by HE staining. The acellular bovine pericardiums were crosslinked with EDC/NHS (EDC/NHS group) or genipin (genipin group). The properties of the crosslinked acellular matrix were evaluated by scanning electron microscope (SEM), matrix thickness, crosslinking index, mechanical property, denaturation temperature, enzymatic degradation, and cytotoxicity test before and after the crosslinking. Acellular bovine pericardium (ABP group) or normal bovine pericardium (control group) were harvested as controls.. SEM showed that collagen fibers were reticulated in bovine pericardial tissues after crosslinked by EDC/NHS or genipin, and relative aperture of the collagen fiber was from 10 to 20 μm. The thickness and denaturation temperature of the scaffolds were increased significantly after crosslinking with EDC/NHS or genipin (P < 0.05), while there was no significant difference between EDC/NHS group and genipin group (P > 0.05). The difference had no statistical significance in crosslinking index between EDC/NHS group and genipin group (t = 0.205, P = 0.218). The degradation rate in EDC/NHS group and genipin group was significantly lower than that in ABP group and control group (P < 0.05). Elastic modulus and fracture stress in EDC/NHS group and genipin group were significantly lower than those in ABP group (P < 0.05), but there was no significant difference among EDC/NHS group, genipin group, and control group (P > 0.05). The break elongation in EDC/NHS group and genipin group were significantly increased than those in ABP group and control group (P < 0.05). The difference had no statistical significance in stability and mechanical properties between EDC/NHS group and genipin group (P > 0.05). Cytotoxicity of genipin crosslinked tissue (grade 1) were much lower than that of EDC/NHS (grade 2) at 5 days.. Acellular bovine pericardium crosslinked with genipin has better biocompatibility than EDC/NHS.

Topics: Animals; Biocompatible Materials; Cattle; Cross-Linking Reagents; Culture Techniques; Extracellular Matrix; Iridoids; Pericardium; Temperature; Tissue Engineering

Guided bone regeneration (GBR) barrier membranes are used to prevent soft tissue infiltration into the graft space during dental procedures that involve bone grafting. Chitosan materials have shown promise as GBR barrier membranes, due to their biocompatibility and predictable biodegradability, but degradation rates may still be too high for clinical applications. In this study, chitosan GBR membranes were electrospun using chitosan (70% deacetylated, 312 kDa, 5.5 w/v%), with or without the addition of 5 or 10 mm genipin, a natural crosslinking agent, in order to extend the degradation to meet the clinical target time frame of 4-6 months. Membranes were evaluated for fibre diameter, tensile strength, biodegradation rate, bond structure and cytocompatibility. Genipin addition, at 5 or 10 mm, resulted in median fibre diameters 184, 144 and 154 nm for uncrosslinked, 5 mm and 10 mm crosslinked, respectively. Crosslinking, examined by Fourier transform infrared spectroscopy, showed a decrease in N-H stretch as genipin levels were increased. Genipin-crosslinked mats exhibited only 22% degradation based on mass loss, as compared to 34% for uncrosslinked mats at 16 weeks in vitro. The ultimate tensile strength of the mats was increased by 165% to 32 MPa with 10 mm crosslinking as compared to the uncrosslinked mats. Finally, genipin-crosslinked mats supported the proliferation of SAOS-2 cells in a 5 day growth study, similar to uncrosslinked mats. These results suggest that electrospun chitosan mats may benefit from genipin crosslinking and have the potential to meet clinical degradation time frames for GBR applications.

Topics: Biocompatible Materials; Bone and Bones; Bone Regeneration; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chitosan; Cross-Linking Reagents; Glutaral; Humans; Iridoids; Materials Testing; Microscopy, Electron, Scanning; Pressure; Spectroscopy, Fourier Transform Infrared; Stress, Mechanical; Tensile Strength; Tissue Engineering

To overcome release of silk sericin (SS) from semi-interpenetrating polymer network (semi-IPN) SS/poly(N-isopropylacrylamide) (PNIPAm) hydrogels, natural biocompatible genipin (GNP) was adopted as cross-linking agent of SS. The GNP/SS/PNIPAm hydrogels with various GNP contents were prepared by radical polymerization. Depending on GNP content, the resultant hydrogels present white, yellow, or dark blue. Required time of color change for GNP/SS mixture solution shortened with increasing GNP ratio. The GNP/SS/PNIPAm hydrogels present good oscillatory shrinking-swelling behavior between 20 and 37°C. The behaviors of L929 cell proliferation, desorption, and transshipment on the surface of hydrogels and tissue culture polystyrene were investigated by 3-(4,5-dimethy thioazol-2-yl)-2,5-di-phenytetrazoliumromide and scanning electron microscopy method. In comparison with pure SS/PNIPAm hydrogels, the introduction of certain GNP can accelerate cell adhesion and proliferation. Due to reversible change between hydrophobicity and hydrophilicity, by lowering temperature to 4°C from 37°C, L929 cells could spontaneously detach from the surface of hydrogels without the need for trypsin or ethylenediaminetetraacetic acid. The detached cells could subsequently be recultured. The prepared hydrogel and detached cells have potential applications in biomedical fields, such as organs or tissue regeneration and cancer or disease therapy.

Topics: Acrylic Resins; Animals; Cell Adhesion; Cell Line; Cell Proliferation; Hydrogels; Iridoids; Mice; Sericins

The application of porous hydroxyapatite (HAp)-collagen as a bone tissue engineering scaffold represents a new trend of mimicking the specific bone extracellular matrix (ECM). The use of HAp in reconstructive surgery has shown that it is slowly invaded by host tissue. Therefore, implant compatibility may be augmented by seeding cells before implantation. Human primary osteoblasts were seeded onto innovative collagen-gelatin-genipin (GP)-HAp scaffolds containing respectively 10%, 20%, and 30% HAp. Cellular adhesion, proliferation, alkaline phosphatase (ALP) activity, osteopontin (OPN), and osteocalcin (OC) expressions were evaluated after 3, 7, 15, and 21 days. The three types of scaffolds showed increased cellular proliferation over time in culture (maximum at 21 days) but the highest was recorded in 10% HAp scaffolds. ALP activity was the highest in 10% HAp scaffolds in all the times of evaluation. OC and OPN resulted in higher concentration in 10% HAp scaffolds compared to 20% and 30% HAp (maximum at 21 days). Finally, scanning electron microscopy analysis showed progressive scaffolds adhesion and colonization from the surface to the inside from day 3 to day 21. In vitro attachment, proliferation, and colonization of human primary osteoblasts on collagen-GP-HAp scaffolds with different percentages of HAp (10%, 20%, and 30%) all increased over time in culture, but comparing different percentages of HAp, they seem to increase with decreasing of HAp component. Therefore, the mechanical properties (such as the stiffness due to the HAp%) coupled with a good biomimetic component (collagen) are the parameters to set up in composite scaffolds design for bone tissue engineering.

Topics: Aged; Alkaline Phosphatase; Biomechanical Phenomena; Cell Proliferation; Cells, Cultured; Collagen; Compressive Strength; Durapatite; Elastic Modulus; Enzyme-Linked Immunosorbent Assay; Female; Gelatin; Humans; Iridoids; Male; Osteoblasts; Osteocalcin; Osteopontin; Tissue Engineering; Tissue Scaffolds

An efficient cold-mechanical/sonic-assisted extraction technique was developed for extraction of genipin from genipap (Genipa americana) peel. Ultrasound assisted extraction (285 W, 24 kHz) was performed at 5, 10 and 15 °C for 5, 10 and 15 min. After cold-extraction, genipin was separated from pectin and proteins by aid of fungal pectinesterase. The maximum yield of non-cross-linked genipin was 7.85±0.33 mg/g, at 10 °C for 15 min by means of ultrasound extraction. The protein amount in extracts decreased in all samples. If mechanical process is combined with ultrasound assisted extraction the yield is increased by 8 times after the pectinesterase-assisted polyelectrolyte complex formation between pectic polysaccharides and proteins, avoiding the typical cross-linking of genipin. This novel process is viable to obtain non-cross-linked genipin, to be used as a natural colorant and cross-linker in the food and biotechnological industries.

Topics: Aspergillus; Carboxylic Ester Hydrolases; Chemical Fractionation; Cold Temperature; Green Chemistry Technology; Iridoids; Mechanical Phenomena; Rubiaceae

The design and production of structures with nanometer-sized polymer films based on layer-by-layer (LbL) are of particular interest for tissue engineering since they allow the precise control of physical and biochemical cues of implantable devices. In this work, a method is developed for the preparation of nanostructured hollow multilayers tubes combining LbL and template leaching. The aim is to produce hollow tubes based on polyelectrolyte multilayer films with tuned physical-chemical properties and study their effects on cell behavior. The final tubular structures are characterized by differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), microscopy, swelling, and mechanical tests, including dynamic mechanical analysis (DMA) in physiological simulated conditions. It is found that more robust films could be produced upon chemical cross-linking with genipin. In particular, the mechanical properties confirms the viscoelastic properties and a storage and young modulus about two times higher. The water uptake decreases from about 390% to 110% after the cross-linking. The biological performance is assessed in terms of cell adhesion, viability, and proliferation. The results obtained with the cross-linked tubes demonstrate that these are more suitable structures for cell adhesion and spreading. The results suggest the potential of these structures to boost the development of innovative tubular structures for tissue engineering approaches.

Topics: Alginates; Animals; Biocompatible Materials; Calorimetry, Differential Scanning; Cell Adhesion; Cell Survival; Cells, Cultured; Chemical Phenomena; Chitosan; Glucuronic Acid; Hexuronic Acids; Iridoids; Mice; Microscopy, Electron, Scanning; Nanostructures; Polymers; Spectroscopy, Fourier Transform Infrared; Tissue Engineering

Recombinant elastin-like protein polymers are increasingly being investigated as component materials of a variety of implantable medical devices. This is chiefly a result of their favorable biological properties and the ability to tailor their physical and mechanical properties. In this report, we explore the potential of modulating the water content, mechanical properties, and drug release profiles of protein films through the selection of different crosslinking schemes and processing strategies. We find that the selection of crosslinking scheme and processing strategy has a significant influence on all aspects of protein polymer films. Significantly, utilization of a confined, fixed volume, as well as vapor-phase crosslinking strategies, decreased protein polymer equilibrium water content. Specifically, as compared to uncrosslinked protein gels, water content was reduced for genipin (15.5%), glutaraldehyde (GTA, 24.5%), GTA vapor crosslinking (31.6%), disulfide (SS, 18.2%) and SS vapor crosslinking (25.5%) (P<0.05). Distinct crosslinking strategies modulated protein polymer stiffness, strain at failure and ultimate tensile strength (UTS). In all cases, vapor-phase crosslinking produced the stiffest films with the highest UTS. Moreover, both confined, fixed volume and vapor-phase approaches influenced drug delivery rates, resulting in decreased initial drug burst and release rates as compared to solution phase crosslinking. Tailored crosslinking strategies provide an important option for modulating the physical, mechanical and drug delivery properties of protein polymers.

Topics: Cross-Linking Reagents; Disulfides; Drug Delivery Systems; Elastin; Fibronectins; Glutaral; Human Umbilical Vein Endothelial Cells; Humans; Iridoids; Mechanical Phenomena; Sirolimus; Water

A sensitive and specific method was developed and validated for the quantitation of one major metabolite of genipin in rats plasma. The major metabolite was isolated from rat bile via semi-preparative HPLC technology and its chemical structure was identified as genipin-1-o-glucuronic acid (GNP-GLU), which was for the first time used as a standard compound for quantitative analysis in rat plasma after administration of genipin. The application of high-performance liquid chromatography-tandem mass spectrometry in negative mode in multiple reaction monitoring mode was investigated. Chromatographic separation was achieved on an Eclipse XDB-C18 column using a mobile phase consisting of water with 0.1% formic acid (A)-acetonitrile (B). The limit of detecation was 0.214 ng/mL and the lower limit of quantification was 0.706 ng/mL. The calibration curve was linear from 1.27 to 3810 ng/mL for plasma samples, with a correlation coefficient of 0.9924. The intra- and inter-day precisions and accuracy were all within 15%. The recoveries of GNP-GLU and puerarin were above 90.0 and 76.2%, respectively. The highly sensitive method was successfully applied to estimate pharmacokinetic parameters of GNP-GLU following oral and intravenous administration of genipin to rats.

Topics: Animals; Chromatography, High Pressure Liquid; Drug Stability; Glucuronic Acid; Iridoids; Limit of Detection; Linear Models; Male; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Tandem Mass Spectrometry

A novel flavour microcapsule containing vanilla oil (VO) was developed using complex coacervation approach, aimed to control release of VO and enhance its thermostability for spice application in food industry. Viscosity of chitosan (CS) and VO/CS ratio were optimised for fabrication of microcapsules. The flavour microcapsules were evaluated by scanning electron micrograph (SEM), laser confocal microscopy (LSCM), particle size analyser, infrared spectrometer (FT-IR), thermal analysis and controlled-release analysis. The microcapsules were in spherical with good dispersibility when moderate viscosity CS was used. 94.2% of encapsulation efficiency was achieved in VO/CS ratio of 2:1. The FT-IR study proved chemical cross-linking reaction occurred between genipin and chitosan, but a physical interaction between CS and VO. A core-shell structure of microcapsule was confirmed by LSCM, which was beneficial to improve the thermostability of VO in microcapsule. Moreover, VO could be remained about 60% in the microcapsules after release for 30 days, which demonstrated the flavour microcapsules had good potential to serve as a high quality food spice with long residual action and high thermostability.

Topics: Capsules; Chitosan; Flavoring Agents; Food Handling; Iridoids; Microscopy, Confocal; Microscopy, Electron, Scanning; Plant Oils; Spectroscopy, Fourier Transform Infrared; Vanilla

Microbial bioburden in bone allografts can be reduced by gamma radiation; however, the radiation compromises collagen and increases the risk of graft failure. Genipin is an agent that may reduce bioburden by chemical crosslinking without causing mechanical detriment or cytotoxicity.. To evaluate genipin's ability to penetrate cortical bone while maintaining sporicidal activity, Bacillus subtilis spore strips were isolated between slices of bovine femoral cortical bone and immersed in genipin solutions for up to one week; spore viability was assessed with media-based assays. The mechanical effects of genipin treatment were assessed by performing three-point bending tests on genipin-treated cortical beams. Cytotoxicity studies were conducted by evaluating the adhesion and proliferation of murine MC3T3-E1 (P21) preosteoblasts on cortical bone slices which were treated with genipin and rinsed to different extents.. Genipin successfully penetrated cortical bone slices and sterilized B. subtilis populations after 48 hours (p>0.05) and one week (p<0.05). Genipin-treated cortical beams demonstrated dose-dependent increases in yield strain (p=0.02) and resilience (p<0.01), whereas other mechanical properties were not affected by genipin treatment. Seeding cells onto inadequately rinsed genipin-treated bones proved cytotoxic. However, with adequate post-treatment rinsing of the residual genipin, cell adhesion and proliferation was comparable to phosphate-buffered saline-treated controls (no genipin).. Genipin solutions can sterilize bacterial spore populations entrapped within the continuum of bone tissue while preserving mechanical properties of bone and allowing cell adhesion and proliferation. Provided that antimicrobial effects seen with bacterial spores extend broadly to all microbial and viral species, genipin holds strong potential for bone allograft sterilization.

Topics: 3T3 Cells; Allografts; Animals; Bone Transplantation; Cattle; Iridoids; Mice; Spores, Bacterial

The low stiffness of reconstituted collagen hydrogels has limited their use as scaffolds for engineering implantable tissues. Although chemical crosslinking has been used to stiffen collagen and protect it against enzymatic degradation in vivo, it remains unclear how crosslinking alters the vascularization of collagen hydrogels. In this study, we examine how the crosslinking agents genipin and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide alter vascular stability and function in microfluidic type I collagen gels in vitro. Under moderate perfusion (∼10 dyn/cm(2) shear stress), tubes of blood endothelial cells (ECs) exhibited indistinguishable stability and barrier function in untreated and crosslinked scaffolds. Surprisingly, under low perfusion (∼5 dyn/cm(2) shear stress) or nearly zero transmural pressure, microvessels in crosslinked scaffolds remained stable, while those in untreated gels rapidly delaminated and became poorly perfused. Similarly, tubes of lymphatic ECs under intermittent flow were more stable in crosslinked gels than in untreated ones. These effects correlated well with the degree of mechanical stiffening, as predicted by analysis of fracture energies at the cell-scaffold interface. This work demonstrates that crosslinking of collagen scaffolds does not hinder normal EC physiology; instead, crosslinked scaffolds promote vascular stability. Thus, routine crosslinking of scaffolds may assist in vascularization of engineered tissues.

Topics: Biocompatible Materials; Bioprosthesis; Cell Line; Collagen; Cross-Linking Reagents; Endothelial Cells; Ethyldimethylaminopropyl Carbodiimide; Humans; Iridoids; Materials Testing; Stress, Mechanical; Tissue Scaffolds

The fabrication of an instructive bioabsorbable scaffold is one of the main goals for tissue engineering applications. In this regard, genipin cross-linked gelatin scaffolds, produced by electrospinning, were tested as a platform to include decellularized rat brain extracellular matrix as an active agent to provide fundamental biochemical cues to the seeded cells. This approach is expected to furnish a suitable natural-based polymeric scaffold with sufficient temporal stability to support cell attachment and spreading, also providing tissue-specific signals that can contribute to the expression of the requested cellular phenotype. We first demonstrated the effectiveness of the proposed decellularization protocol and the cytocompatibility of the resulting brain matrix. Then, the in vitro biological assays of the conditioned electrospun scaffolds, using rat allogeneic mesenchymal stromal cells, confirmed their biocompatibility and showed a differentiative potential in presence of just 1% w/w decellularized rat brain extracellular matrix.

Topics: Animals; Brain; Cells, Cultured; Extracellular Matrix; Gelatin; Iridoids; Male; Mesenchymal Stem Cells; Rats; Tissue Engineering; Tissue Scaffolds

The interpenetrating polymer networks (IPN) hydrogels based on chitosan and gelatin using genipin as the cross-linker were prepared and characterized. The IPN formation of the genipin-cross-linked chitosan/gelatin hydrogel was confirmed by means of the instrinsic viscosity measurement, Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and the ninhydrin assays. The instrinsic viscosity measurement, FT-IR and SEM suggested that chitosan and gelatin were miscible in the molecular level. The miscibility leads to the formation of IPN after cross-linking. FT-IR also examined the cross-linking mechanism of genipin with primary amino groups. The degree of cross-linking increased with increase genipin concentration. Swelling results revealed that the IPN hydrogels are pH-sensitive, exhibiting reversibility and rather rapidly response in swelling to pH changes. It is expected this IPN hydrogel has potential as controlled drug delivery system or as alternative sorbents for biomedical and environmental use as pH altered.

Topics: Chitosan; Cross-Linking Reagents; Delayed-Action Preparations; Gelatin; Hydrogels; Iridoids; Microscopy, Electron, Scanning; Ninhydrin; Polymerization; Spectroscopy, Fourier Transform Infrared; Viscosity; Water

Genipin, the multipotent ingredient in Gardenia jasmenoides fruit extract (GFE), may be an effective candidate for treatment following stroke or traumatic brain injury (TBI). Secondary injury includes damage mediated by reactive oxygen species (ROS) and reactive nitrogen species (RNS), which can alter the biological function of key cellular structures and eventually lead to cell death. In this work, we studied the neuroprotective potential of genipin against damage stemming from ROS and RNS production in organotypic hippocampal slice cultures (OHSC), as well as its potential as a direct free radical scavenger. A 50 µM dose of genipin provided significant protection against tert-butyl hydroperoxide (tBHP), a damaging organic peroxide. This dosage of genipin significantly reduced cell death at 48 h compared to vehicle control (0.1% DMSO) when administered 0, 1, 6, and 24 h after addition of tBHP. Similarly, genipin significantly reduced cell death at 48 h when administered 0, 1, 2, and 6h after addition of rotenone, which generates reactive oxygen species via a more physiologically relevant mechanism. Furthermore, genipin significantly reduced both cell death and nitrite levels at 24 h caused by S-nitroso-N-acetylpenicillamine (SNAP), a direct nitric oxide (NO) donor, and successfully quenched 1,1-Diphenyl-2-picryl-hydrazyl (DPPH), a stable free radical, suggesting that genipin may act as a direct free radical scavenger. Our encouraging findings suggest that genipin should be tested in animal models of CNS injury with a significant component of ROS- and RNS-mediated damage, such as TBI and stroke, to assess its in vivo efficacy.

Topics: Analysis of Variance; Animals; Animals, Newborn; Ascorbic Acid; Biphenyl Compounds; Cell Death; Dose-Response Relationship, Drug; Hippocampus; Insecticides; Iridoids; Neuroprotective Agents; Nitric Oxide Donors; Organ Culture Techniques; Picrates; Reactive Nitrogen Species; Reactive Oxygen Species; Rotenone; S-Nitroso-N-Acetylpenicillamine; Time Factors

Crosslinking soft tissue has become more common in tissue engineering applications, and recent studies have demonstrated that soft tissue mechanical behavior can be directly altered through crosslinking. Using a recently reported test method that shears adjacent disc lamella, the effect of genipin crosslinking on interlamellar shear resistance was studied using in vitro bovine disc annulus. Specimens of adjacent lamella were dissected from four discs taken from three fresh frozen bovine tails. These specimens were paired and soaked in either 50 mM EPPS Phosphate (ph9) with 20 mM genipin at 37 °C for 4 h or in 50 mM EPPS Phosphate (ph9) of which twelve specimens (6 per treatment) were successfully tested and analyzed. Crosslinked specimens were noted to have significantly higher yield force per width (59%), peak force per width (70%), and resilience (69%) compared to sham treated controls, supporting the hypothesis that genipin crosslinking increases the resistance to interlamellar shear of the annulus interface. Additionally, a possible dependency may exist between the interlamellar shear strength and neighboring lamella because of the bridging fiber network previously described by other investigators.

Topics: Animals; Biomechanical Phenomena; Cattle; Cross-Linking Reagents; Intervertebral Disc; Iridoids; Shear Strength; Stress, Mechanical; Tissue Engineering

Genipin is a compound found in gardenia fruit extract with diverse pharmacological activities. However, the mechanism underlying genipin-induced cyclooxygenase-2 (COX-2) expression remains unknown. In this study, we investigated the effects of genipin on COX-2 expression and determined that exposure to genipin dose-dependently enhanced the production of prostaglandin E2 (PGE2), a major COX-2 metabolite, in RAW 264.7 cells. These effects were mediated by genipin-induced activation of the COX-2 promoter, as well as AP-1 and NF-κB luciferase constructs. Phosphatidylinositol-3-kinase/Akt and MAPKs were also significantly activated by genipin, and Akt and MAPKs inhibitors (PD98059, SB20358, SP600125, and LY294002) inhibited genipin-induced COX-2 expression. Moreover, genipin increased production of the ROS and the ROS-producing NAPDH-oxidase (NOX) family oxidases, NOX2 and NOX3. Inhibition of NADPH with diphenyleneiodonium attenuated ROS production, COX-2 expression and NF-κB and AP-1 activation. These results suggest that the molecular mechanism mediating ROS-dependent COX-2 up-regulation and PGE2 production by genipin involves activation of Akt, MAPKs and AP-1/NF-κB.

Topics: Animals; Cell Line; Cyclooxygenase 2; Iridoids; Macrophages; Mice; Mitogen-Activated Protein Kinases; NADPH Oxidases; NF-kappa B; Signal Transduction; Transcription Factor AP-1

Folate-chitosan conjugates were prepared by a concurrent functionalization and crosslinking reaction with the natural crosslinker genipin. Genipin molecule was employed simultaneously as crosslinker agent and spacer molecule in order to allow the functionalization with folic acid for active tumor targeting. The reaction was carried out in reverse microemulsion which provided colloidal size and monodisperse particle size distribution. The water solubility of the obtained folate-genipin-chitosan nanogels was studied as function of the pH of the medium and all nanoparticles were totally dispersible at physiological pH. The enzymatic degradability of the nanogels in a lysozyme solution was evaluated at acidic and physiological pH. QELS analyses of the swelling behavior of the nanogels with the pH did not show a clear pH-sensitivity. However, the study on the loading and release capacity of 5-fluorouracil revealed an interesting pH-responsive behavior of the nanogels that makes them promising as nanodevices for targeted anticancer drug delivery.

Topics: Chitosan; Drug Carriers; Folic Acid; Hydrogen-Ion Concentration; Iridoids; Nanostructures; Solubility; Water

Chitosan, a natural polysaccharide, has demonstrated potential as a degradable biocompatible guided bone regeneration membrane. This study aimed to evaluate the in vivo biocompatibility and degradation of chitosan nanofiber membranes, with and without genipin crosslinking as compared with a commercial collagen membrane in rat model. Chitosan nanofiber membranes, with and without genipin crosslinking, and collagen membrane (control) were implanted subcutaneously in the backs of 30 rats. The membranes were analyzed histologically at 2, 4, 8, 12, 16, and 20 weeks. Sections were viewed and graded by a blinded pathologist using a 4-point scoring system (0 = absent, 1 = mild, 2 = moderate, and 3 = severe) to determine the tissue reaction to the membranes and to observe membrane degradation. There was no statistically significant difference in histological scores among chitosan and collagen membranes at different time points. Absence or minimal inflammation was observed in 57-74% of the membranes across all groups. Most chitosan membranes persisted for 16-20 weeks, whereas most collagen membranes disappeared by resorption at 12-16 weeks. The general tissue response to chitosan nanofiber membranes with and without genipin crosslinking, was similar to that of control commercial collagen membrane. However, the chitosan membranes exhibited slower degradation rates than collagen membranes.

Topics: Animals; Chitosan; Iridoids; Male; Materials Testing; Membranes, Artificial; Nanofibers; Rats; Rats, Sprague-Dawley

The crosslinking agent genipin is increasingly invoked for the mechanical augmentation of collagen tissues and implants, and has previously been demonstrated to arrest mechanical damage accumulation in various tissues. This study established an in vitro dose-response baseline for the effects of genipin treatment on tendon cells and their matrix, with a view to in vivo application to the repair of partial tendon tears. Regression models based on a broad range of experimental data were used to delineate the range of concentrations that are likely to achieve functionally effective crosslinking, and predict the corresponding degree of cell loss and diminished metabolic activity that can be expected. On these data, it was concluded that rapid mechanical augmentation of tissue properties can only be achieved by accepting some degree of cytotoxicity, yet that post-treatment cell survival may be adequate to eventually repopulate and stabilize the tissue. On this basis, development of delivery strategies and subsequent in vivo study seems warranted.

Topics: Animals; Calorimetry, Differential Scanning; Cell Movement; Cell Survival; Cross-Linking Reagents; Elastic Modulus; Gene Expression Regulation; Horses; Iridoids; Mechanical Phenomena; Protein Denaturation; Reference Standards; Regression Analysis; Tendons; Time Factors; Wound Healing

The purpose of this paper is to analyze the properties of fabricating rat tail type I collagen scaffolds cross-linked with genipin under different conditions. The porous genipin cross-linked scaffolds are obtained through a two step freeze-drying process. To find out the optimal cross-link condition, we used different genipin concentrations and various cross-linked temperatures to prepare the scaffolds in this study. The morphologies of the scaffolds were characterized by scanning electron microscope, and the mechanical properties of the scaffolds were evaluated under dynamic compression. Additionally, the cross-linking degree was assessed by ninhydrin assay. To investigate the swelling ratio and the in vitro degradation of the collagen scaffold, the tests were also carried out by immersion of the scaffolds in a PBS solution or digestion in a type I collagenase respectively. The morphologies of the non-cross-linked scaffolds presented a lattice-like structure while the cross-linked ones displayed a sheet-like framework. The morphology of the genipin cross-linked scaffolds could be significantly changed by either increasing genipin concentration or the temperature. The swelling ratio of each cross-linked scaffold was much lower than that of the control (non-cross-linked).The ninhydrin assay demonstrated that the higher temperature and genipin concentration could obviously increase the cross-linking efficiency. The in vitro degradation studies indicated that genipin cross-linking can effectively elevate the biostability of the scaffolds. The biocompatibility and cytotoxicity of the scaffolds was evaluated by culturing rat chondrocytes on the scaffold in vitro and by MTT. The results of MTT and the fact that the chondrocytes adhered well to the scaffolds demonstrated that genipin cross-linked scaffolds possessed an excellent biocompatibility and low cytotoxicity. Based on these results, 0.3 % genipin concentrations and 37 °C cross-linked temperatures are recommended.

Topics: Animals; Cell Adhesion; Cells, Cultured; Chondrocytes; Collagen Type I; Cross-Linking Reagents; Dose-Response Relationship, Drug; Freeze Drying; In Vitro Techniques; Iridoids; Materials Testing; Microscopy, Electron, Scanning; Models, Animal; Rats; Rats, Sprague-Dawley; Stress, Mechanical; Temperature; Tissue Scaffolds

Novel porous scaffolds composed of chitosan, collagen and gelatin were prepared by the multistep procedure involving final freeze-drying and characterized. To eliminate the need for residual acid removal from the material after drying, carbon dioxide saturation process was used for chitosan blend formulation. The use of CO2 for chitosan dissolution made the scaffold preparation process more reproducible and economically sustainable. Genipin was applied to stabilize the structure of the scaffolds and those crosslinked at a level of 7.3% exhibited a homogenous porous structure (33.1%), high swelling capacity (27.6g/g for wound exudate like medium; 62.5 g/g for water), and were stable under cyclic compression. The values of other investigated parameters: dissolution degree (30%), lysozyme-induced degradation (5% after 168 h), good antioxidant properties (DPPH, ABTS, Fe(2+) assays) and especially very low in vitro cytotoxicity against fibroblasts (103%, MTT assay), were highly advantageous for possible biomedical applications of the novel materials.

Topics: Animals; Antioxidants; Biocompatible Materials; Carbon Dioxide; Chitosan; Collagen; Cross-Linking Reagents; Gelatin; Iridoids; Mice; Muramidase; NIH 3T3 Cells; Porosity; Solubility

Glucose-responsive systems are significant for self-regulated insulin delivery. The aim of this study was to assess the potential of genipin-crosslinked concanavalin A/GEA-chitosan microgels as a glucose-responsive insulin delivery system. A chitosan derivative (GEA-chitosan) was designed in this study as the polymer ligand of concanavalin A (Con A), which not only exhibits a strong affinity to Con A, but also could be directly crosslinked with Con A by genipin, thus avoiding the modification of Con A during an immobilization process. Glucose responsive microgels were fabricated by the reversed-phase emulsion crosslinking method. The in vitro release of insulin indicated that the insulin release was influenced by glucose concentrations, and a desired pulsatile release behavior was detected in response to stepwise glucose challenges for more than eight cycles. The release data were fitted well to an exponential model, without any significant influence of the surface effect. The released insulin was proved to remain active without destruction of the tertiary structure. The analysis of L929 cells viability suggested that these microgels possessed no in vitro cytotoxicity. The obtained genipin crosslinked Con A/GEA-chitosan microgels might be a potential candidate for self-regulated insulin delivery.

Topics: Animals; Cell Line; Cell Survival; Chitosan; Concanavalin A; Cross-Linking Reagents; Drug Delivery Systems; Fibroblasts; Gels; Glucose; Insulin; Iridoids; Mice

Genipin, the aglycon of geniposide found in gardenia fruit has long been considered for treatment of various diseases in traditional oriental medicine. Genipin has been used as a blue colorant in food industry. Genipin has recently been reported to have some pharmacological functions, such as antimicrobial, antitumor, and anti-inflammatory effects. The aim of this study was to examine whether genipin could modify CCL20 and IL-6, which are related to bone resorption in periodontal disease, expression in human periodontal ligament cells (HPDLCs).. CCL20 and IL-6 productions from HPDLCs were determined by ELISA. Western blot analysis was used for the detection of signal transduction molecules expressions in HPDLCs.. Genipin prevented IL-1β-mediated CCL20 and IL-6 production in HPDLCs. Moreover, genipin could suppress nuclear factor kappa B (NF-κB) p65, extracellular signalregulated kinase (ERK) and MAPK/ERK kinase (MEK) phosphorylations in IL-1β-stimulated HPDLCs. NF-κB inhibitor and ERK inhibitor significantly inhibited IL-6 and CCL20 productions from IL-1β-stimulated HPDLCs.. These data provide a novel mechanism through which genipin could be used to provide direct benefits in periodontal disease to inhibit IL-6 and CCL20 productions in periodontal lesions.

Topics: Cells, Cultured; Chemokine CCL20; Cholagogues and Choleretics; Humans; Interleukin-1beta; Interleukin-6; Iridoids; Periodontal Diseases; Periodontal Ligament

Uncoupling protein-2 (UCP2) may regulate glucose-stimulated insulin secretion. The current study investigated the effects of berberine, an alkaloid found in many medicinal plants, on oxidative stress and insulin secretion through restoration of UCP2 expression in high glucose (HG)-treated INS-1E cells and rat islets or in db/db mouse islets.. Mouse and rat pancreatic islets were isolated. Nitrotyrosine, superoxide dismutase (SOD)-1 and UCP2 expression and AMPK phosphorylation were examined by Western blotting. Insulin secretion was measured by ELISA. Mitochondrial reactive oxygen species (ROS) production was detected by confocal microscopy.. Incubation of INS-1E cells and rat islets with HG (30 mmol·L(-1); 8 h) elevated nitrotyrosine level, reduced SOD-1 and UCP2 expression and AMPK phosphorylation, and inhibited glucose-stimulated insulin secretion. HG also increased mitochondrial ROS in INS-1E cells. Co-treatment with berberine inhibited such effects. The AMPK inhibitor compound C, the UCP2 inhibitor genipin and adenovirus ucp2 shRNA inhibited these protective effects of berberine. Furthermore, compound C normalized berberine-stimulated UCP2 expression but genipin did not affect AMPK phosphorylation. Islets from db/db mice exhibited elevated nitrotyrosine levels, reduced expression of SOD-1 and UCP2 and AMPK phosphorylation, and decreased insulin secretion compared with those from db/m(+) mice. Berberine also improved these defects in diabetic islets and genipin blocked the effects of berberine.. Berberine inhibited oxidative stress and restored insulin secretion in HG-treated INS-IE cells and diabetic mouse islets by activating AMPK and UCP2. UCP2 is an important signalling molecule in mediating anti-diabetic effects of berberine.

Topics: AMP-Activated Protein Kinases; Animals; Berberine; Diabetes Mellitus, Experimental; Glucose; Insulin; Insulin Secretion; Insulinoma; Ion Channels; Iridoids; Islets of Langerhans; Male; Mice; Microscopy, Confocal; Mitochondria; Mitochondrial Proteins; Oxidative Stress; Pancreatic Neoplasms; Phosphorylation; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Uncoupling Protein 2

An extracellular β-glucosidase from Aspergillus niger Au0847 was purified to homogeneity by precipitation with ammonium sulfate, anion exchange, and gel filtration. The purified protein was composed of two subunits with molecular masses of 110 and 120 kDa. Au0847 β-glucosidase exhibited relatively high thermostability and pH stability, and its highest activity was obtained at 65°C and pH 4.6, respectively. As a potential metalloprotein, its enzymatic activity was potently stimulated by manganese ion and DTT. The β-glucosidase displayed avid affinity and high catalytic efficiency for geniposide. Au0847 β-glucosidase has potential value as an industrial enzyme for the hydrolysis of geniposide to genipin.

Topics: Aspergillus niger; Biotransformation; Enzyme Stability; Fungal Proteins; Glucosidases; Iridoids; Kinetics

People over the age of 50 are at risk of osteoporotic fracture, which may lead to increased morbidity and mortality. Osteoclasts are responsible for bone resorption in bone-related disorders. Genipin is a well-known geniposide aglycon derived from Gardenia jasminoides, which has long been used in oriental medicine for controlling diverse conditions such as inflammation and infection. We aimed to evaluate the effects of genipin on RANKL-induced osteoclast differentiation and its mechanism of action. Genipin dose-dependently inhibited early stage RANKL-induced osteoclast differentiation in bone marrow macrophages (BMMs) during culture. Genipin inhibited RANKL-induced IκB degradation and suppressed the mRNA expression of osteoclastic markers such as NFATc1, TRAP, and OSCAR in RANKL-treated BMMs, but did not affect c-Fos mRNA expression. Interestingly, genipin markedly inhibited c-Fos protein expression in BMMs, which was reversed in the presence of the proteosome inhibitor MG-132. Furthermore, genipin inhibited RANKL-mediated osteoclast differentiation, which was also rescued by overexpression of c-Fos and NFATc1 in BMMs. Taken together, our findings indicate that genipin down-regulated RANKL-induced osteoclast differentiation through inhibition of c-Fos protein proteolysis as well as inhibition of IκB degradation. Our findings indicate that genipin could be a useful drug candidate that lacks toxic side effects for the treatment of osteoporosis.

Topics: Animals; Cell Differentiation; Cells, Cultured; Depression, Chemical; Dose-Response Relationship, Drug; Gardenia; Iridoids; Male; Mice; Mice, Inbred ICR; Molecular Targeted Therapy; NF-kappa B; Osteoclasts; Osteoporosis; Phytotherapy; Proteasome Endopeptidase Complex; Proteolysis; Proto-Oncogene Proteins c-fos; RANK Ligand

The design of biomimetic coatings capable of improving the osseointegration of bone biomaterials is a current challenge in the field of bone repair. Toward this end, layer-by-layer (LbL) films composed of natural components are suitable candidates. Chondroitin sulfate A (CSA), a natural glycosaminoglycan (GAG), was used as the polyanionic component because it promotes osteoblast maturation in vivo. In their native state, GAG-containing LbL films are generally cytophobic because of their low stiffness. To stiffen our CSA-based LbL films, genipin (GnP) was used as a natural cross-linking agent, which is much less cytotoxic than conventional chemical cross-linkers. GnP-cross-linked films display an original combination of microscale topography and tunable mechanical properties. Structural characterization was partly based on a novel donor/acceptor Förster resonance energy transfer (FRET) couple, namely, FITC/GnP, which is a promising approach for further inspection of any GnP-cross-linked system. GnP-cross-linked films significantly promote adhesion, proliferation, and early and late differentiation of preosteoblasts.

Topics: Biocompatible Materials; Cell Adhesion; Cell Differentiation; Cell Proliferation; Cell Survival; Cells, Cultured; Chondroitin Sulfates; Cross-Linking Reagents; Fluorescence Resonance Energy Transfer; Humans; Iridoids; Microscopy, Atomic Force; Osteoblasts; Osteogenesis; Quartz Crystal Microbalance Techniques; Spectroscopy, Fourier Transform Infrared

The aim of the present study was to investigate the effects of genipin (Gp) cross-linking of chitosan (CHI) hydrogels on the cell adhesion and viability.. Series of Gp crosslinked CHI hydrogels were prepared by incubation of solutions containing a mixture of Gp and CHI in different ratios. The resulting hydrogels were characterized by scanning electron microscopy (SEM), parallel plate rheometer, contact angle and swelling ratio measurement. The in vitro cytocompatibility of hydrogels was evaluated with L929 fibroblasts by MTT method. The cell adhesion morphology on gel surface was characterized by SEM, and the cell viability was assessed through cell count and flow cytometry analysis.. It was found that macroporous structure of the CHI hydrogels could be tailored by varying Gp or CHI amount. Gp cross-linking of hydrogels enhanced their storage modulus significantly, and also altered their hydrophilicity and swell properties. The MTT results revealed that the cross-linked hydrogels did not induce cytotoxic effects. Cell count and flow cytometry analysis demonstrated that denser surface milieu of hydrogels could facilitate better cell adhesion and viability.. It could be concluded that increased cross-linking density significantly improved the cell adhesion and viability on hydrogel surface. This research provides prospective biocompatible approaches by making gel stiffness modifications to hydrogel scaffolds for the purpose of different tissue engineering.

Topics: Animals; Biocompatible Materials; Cell Adhesion; Cell Proliferation; Cell Survival; Chitosan; Cross-Linking Reagents; Fibroblasts; Hydrogels; Iridoids; Mice; Rheology

There is an unmet clinical need for a biomaterial sealant capable of repairing small annulus fibrosus (AF) defects. Causes of these defects include painful intervertebral disc herniations, microdiscectomy procedures, morbidity associated with needle puncture injury from discography, and future nucleus replacement procedures. This study describes the enhancements of a fibrin gel through genipin crosslinking (FibGen) and the addition of the cell adhesion molecules (CAMs), fibronectin and collagen. The gel's performance as a potential AF sealant is assessed using a series of in vitro tests. FibGen gels with CAMs had equivalent adhesive strength, gene expression, cytomorphology, and cell proliferation as fibrin alone. However, FibGen gels had enhanced material behaviors that were tunable to higher shear stiffness values and approximated human annulus tissue as compared with fibrin alone, were more dimensionally stable, and had a slower in vitro degradation rate. Cytomorphology of human AF cells cultured on FibGen gels exhibited increased elongation compared with fibrin alone, and the addition of CAMs to FibGen did not significantly affect elongation. This FibGen gel offers the promise of being used as a sealant material to repair small AF defects or to be used in combination with other biomaterials as an adhesive for larger defects.

Topics: Adhesiveness; Animals; Apoptosis; Biomimetic Materials; Cattle; Cell Adhesion Molecules; Cell Survival; Cells, Cultured; Fibrin Tissue Adhesive; Hardness; Intervertebral Disc; Iridoids; Materials Testing; Shear Strength

A facile 6-aminoagarose (AA) mediated synthesis of new fluorogenic amides of agarose with nicotinic (AA-NA) and picolinic acids (AA-PA) employing carbodiimide chemistry have been described. 6-Amino agarose (AA) was synthesized in a facile Mitsunobu-inspired microwave mediated method involving the reaction of agarose with phthalimide in presence of diisopropyl azodicarboxylate and triphenylphosphene (DIAD/TPP) followed by hydrazinolysis. All compounds were characterized by GPC, UV spectrophotometry, fluorescence spectroscopy, FT-IR, (1)H and (13)C NMR spectra. The fluorescence emissions (λmax 430 and 412 nm) of 1 × 10(-3)M solutions of AA-NA and AA-PA in water were significantly higher (ca. 82% and ca. 90%) than those of the molar equivalents (0.2mg) of NA and PA present in the 1 × 10(-3)M solutions of the amides, respectively. These fluorogenic pyridine carboxylic acid amides of agarose may find applications as sensors in biomedical and pharmaceutical industries.

Topics: Amides; Amino Sugars; Azo Compounds; Carbohydrate Conformation; Carboxylic Acids; Cross-Linking Reagents; Fluorescent Dyes; Hydrogels; Iridoids; Microwaves; Niacin; Nuclear Magnetic Resonance, Biomolecular; Organophosphorus Compounds; Phthalimides; Picolinic Acids; Pyridines; Sepharose; Solutions; Spectrophotometry; Spectroscopy, Fourier Transform Infrared

Chitosan blends have been widely investigated to create biomaterials with desirable physicochemical and biological properties for tissue engineering applications. A recurring difficulty, however, has been to maintain their stability in an aqueous environment. The rationale behind this study was to demonstrate that genipin crosslinking can improve and maintain the stability of chitosan/poly-l-lysine (PLL) blends. Four gel formulations were prepared by varying the weight ratios of chitosan and PLL. Electron microscopy revealed that genipin crosslinking provided a more homogenous gel surface compared to uncrosslinked gels. Moreover, it was discovered that 3h was sufficient to stabilize the gels. In vitro studies using fibroblasts demonstrated that genipin-crosslinked gels enhanced fibroblasts' attachment as compared to uncrosslinked gels. Moreover, cell viability was significantly improved by 1.6 times on 60:40 gels, and 6.5 times on 50:50 gels after crosslinking. Finally, proliferation was enhanced up to 5 times on 60:40 gels.

Topics: Biocompatible Materials; Cell Adhesion; Cell Proliferation; Chitosan; Fibroblasts; Gels; Iridoids; Microscopy, Electron; Polylysine; Regenerative Medicine

The purpose of this study is to explore depression metabolic markers in rat hippocampus and to investigate the anti-depressant effect of genipin and its mechanisms using nuclear magnetic resonance (NMR) metabonomics. Chronic unpredictable mild stress (CUMS) procedure was conducted to establish the depressive rat model. At the beginning of the third week, genipin low dose (25 mg x kg(-1)), middle dose (50 mg x kg(-1)), high dose (100 mg x kg(-1)), and venlafaxine (50 mg x kg(-1)) were given to the CUMS rats separately once daily for two weeks except control and model groups. Rat hippocampus was analyzed by 1H NMR based metabonomics after drug administration for 2 weeks. Significant differences in the metabolic profile of rat hippocampus of the CUMS treated group and the control group were observed with metabolic effects of CUMS including decreasing in glycine and N-acetylaspartate, increasing in inositol, glutamate, lactate, glutamine, taurine and alanine. Genipin showed ideal antidepressive effects at a dose of 50 mg x kg(-1) in rats, decrease of inositol, glutamate, lactate, alanine were observed, while glycine and N-acetylaspartate were increased. Important influence has been found on normal nervous system function of these significant changed metabolites, which suggests that the antidepressant effect of genipin may be played by enhancing the activity of neurons in hippocampus, repairing and improving the function of the neuron. The metabonomics approach is an effective tool for the investigation of the anti-depressant effect and pharmacologic mechanisms of genipin.

Topics: Alanine; Animals; Antidepressive Agents; Aspartic Acid; Behavior, Animal; Chronic Disease; Depression; Gardenia; Glutamic Acid; Glycine; Hippocampus; Inositol; Iridoids; Lactic Acid; Magnetic Resonance Spectroscopy; Male; Metabolomics; Plants, Medicinal; Rats; Rats, Sprague-Dawley

Application of biomodification techniques to dentin can improve its biochemical and biomechanical properties. Several collagen cross-linking agents have been reported to strengthen the mechanical properties of dentin. However, the characteristics of collagen that has undergone agent-induced biomodification are not well understood. The objective of this study was to analyze the effects of a natural cross-linking agent, genipin (GE), on dentin discoloration, collagen stability, and changes in amino acid composition and lysyl oxidase mediated natural collagen cross-links. Dentin collagen obtained from extracted bovine teeth was treated with three different concentrations of GE (0.01%, 0.1%, and 0.5%) for several treatment times (0-24 h). Changes in biochemical properties of NaB(3)H4-reduced collagen were characterized by amino acid and cross-link analyses. The treatment of dentin collagen with GE resulted in a concentration- and time-dependent pigmentation and stability against bacterial collagenase. The lysyl oxidase-mediated trivalent mature cross-link, pyridinoline, showed no difference among all groups while the major divalent immature cross-link, dehydro-dihydroxylysinonorleucine/its ketoamine in collagen treated with 0.5% GE for 24 h, significantly decreased compared to control (P < 0.05). The newly formed GE-induced cross-links most likely involve lysine and hydroxylysine residues of collagen in a concentration-dependent manner. Some of these cross-links appear to be reducible and stabilized with NaB(3)H4.

Topics: Animals; Cattle; Collagen Type I; Dentin; Hydroxylysine; Incisor; Iridoids; Lysine; Protein Stability; Tooth Discoloration

Decellularized tissues and organs represent a suitable option for tissue engineering when specific scaffolds are needed. However, the optimal conditions to completely remove all the cellular components and minimally affect the biochemical and structural properties of the extracellular matrix are still to be found. For this aim, bioreactors could be an alternative means to dynamically treat the biological samples, automatically controlling all the variables involved in the process and speeding up the entire procedure in order to deal with a suitable scaffold within a limited time period. This paper presents the characterization of rat tracheae decellularized in dynamic conditions, implementing a detergent-enzymatic method, previously considered. Only 6 cycles were enough to generate a tracheal matrix that was histologically and structurally similar to the native one. The network of collagen, reticular and elastic fibers was well preserved, such as the epithelial cilia, the luminal basement membrane and the main matrix components. The elastin content decreased, even if not significantly, after the decellularization protocol. Mechanical properties of the treated tissues were slightly affected by the procedure, and were partially recovered after crosslinking with genipin, a naturally-derived agent. The use of bioreactors could enhance the decellularization procedure of tissues/organs, but a careful selection of the processing parameters is needed in order to prevent large modifications compared to the native condition.

Topics: Animals; Biocompatible Materials; Biomechanical Phenomena; Cross-Linking Reagents; Elastin; Extracellular Matrix; Humans; Iridoids; Male; Materials Testing; Mesenchymal Stem Cells; Rats, Inbred BN; Trachea

Silver nanoparticles (AgNPs)-loaded alginate beads embedded in gelatin scaffolds were successfully prepared. The AgNPs-loaded calcium alginate beads were prepared by electrospraying method. The effect of alginate concentration and applied voltage on shape and diameter of beads was studied. The diameter of dry AgNPs-loaded calcium alignate beads at various concentrations of AgNO3 ranged between 154 and 171 μm. The AgNPs-loaded calcium alginate beads embedded in gelatin scaffolds were fabricated by freeze-drying method. The water swelling and weight loss behaviors of the AgNPs-loaded alginate beads embedded in gelatin scaffolds increased with an increase in the submersion time. Moreover, the genipin-cross-linked gelatin scaffolds were proven to be nontoxic to normal human dermal fibroblasts, suggesting their potential uses as wound dressings.

Topics: Alginates; Cell Survival; Cross-Linking Reagents; Excipients; Freeze Drying; Gelatin; Glucuronic Acid; Hexuronic Acids; Iridoids; Metal Nanoparticles; Particle Size; Silver

Selective isolation of Kunitz trypsin inhibitor (KTI) and lectin from soybean whey solutions by different types of chitosan beads was investigated. The chitosan beads were co-crosslinked with tripolyphosphate/genipin in solutions at pH 5, 7 or 9 (CB5, CB7, CB9). The maximum adsorption ratios of chitosan beads to KTI and lectin were observed at pH 4.4 and 5.4, respectively; highly selective separation was also demonstrated at these pHs. The adsorption ratios increased with temperature, rising between 5 and 25 °C. CB9 produced the best adsorption ratio, followed by CB7 then CB5. The critical interaction governing absorption of chitosan beads to KTI and lectin could be hydrogen bonding. At pH 9, KTI and lectin desorbed efficiently from CB7 with desorption ratios of 80.9% and 81.4%, respectively. The desorption was most likely caused predominantly by electrostatic repulsion. KTI and lectin can effectively be selectively isolated from soybean whey using this novel separation technique.

Topics: Adsorption; Chitosan; Cross-Linking Reagents; Glycine max; Hydrogen-Ion Concentration; Iridoids; Plant Lectins; Plant Proteins; Polyphosphates; Solutions; Trypsin Inhibitors

Renal tubular epithelial cell injury is a major pathological event that contributes to the development of diabetic kidney disease (DKD). Uncoupling protein-2 (UCP2), a mitochondrial membrane protein, has been reported to participate in the regulation of reactive oxygen species (ROS) generation, which contributes to tubular cell apoptosis induced by hyperglycemia. In this study, we found that genipin, a UCP2 inhibitor, dramatically boosted oxidative stress, attenuated antioxidative capacity, and exacerbated cell apoptosis accompanied with caspase-3 activation in rat renal proximal tubular cells (NRK-52E) incubated with high glucose. The present study results suggest that manipulation of UCP2 could be important in the prevention of oxidative stress damage in renal tubular epithelial cells induced by hyperglycemia in vitro.

Topics: Animals; Apoptosis; Cells, Cultured; Diabetic Nephropathies; Down-Regulation; Ion Channels; Iridoids; Kidney Tubules, Proximal; Mitochondrial Proteins; Rats; Uncoupling Protein 2

To investigate the property of genipin-crosslinked silk fibroin(SF)/chitosan(CS) microspheres for slow releasing of bovine serum albumin (BSA).. BSA-loaded genipin-crosslinked SF/CS microspheres were prepared by emulsion cross-linking technique. The micropheres were observed for surface morphology and size distribution under scanning electron microscope (SEM), and X-ray diffractometry (XRD) and fourier transform infrared spectroscopy (FTIR) were used to analyze their structural characteristics. BCA method was used for determining the drug entrapment, loading rate and cumulative drug release in 21 days.. The microspheres were spherical and showed a smooth surface with an average diameter of 7.84∓0.97 µm. The drug entrapment efficiency of the microspheres was (50.16∓4.32)% with a drug loading ratio of (1.25∓0.11)% and a cumulative release of the total drug of (75.2∓2.53)% in 21 days.. Genipin-crosslinked SF/CS microspheres have a high drug entrapment efficiency and possess good capacity of sustained drug release.

Topics: Chitosan; Delayed-Action Preparations; Emulsions; Fibroins; Iridoids; Microscopy, Electron, Scanning; Microspheres; Particle Size; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction; X-Rays

Genipin, an important bioactive component from Gardenia jasminoides Eills, was demonstrated to possess antidepressant-like effects in a previous study. However, the molecular mechanism of antidepressant-like effects on genipin was not clear. The present study aimed to investigate the possible mechanism of antidepressant-like effects on genipin with a chronic unpredictable mild stress (CUMS)-induced depression model in rats. In CUMS-induced depressive rats, bodyweight and 1% sucrose consumption decreased significantly compared with the normal control group. Furthermore, these changes could be significantly reversed by genipin application. The levels of 5-hydroxytryptamine (5-HT), norepinephrine (NE) in the hippocampus decreased and the level of 5-hydroxyindole acetic acid (5-HIAA) increased in the CUMS-induced depressive rats. However, pre-treatments with genipin significantly increased the levels of 5-HT, NE and decreased the level of 5-HIAA in the hippocampus. The concentration of cAMP in the hippocampus was increased by genipin compared to the CUMS-exposed model group. The mRNA expressions of 5-hydroxytryptamine 1A receptor (5-HT1AR), cAMP response element binding protein (CREB) and brain-derived neurotrophic factor (BDNF) in rats were decreased exposed to CUMS, which were reversed by genipin-treated rats exposed to CUMS. Compared to the CUMS-exposed model group, the mRNA expression of 5-hydroxytryptamine 2A receptor (5-HT(2A)R) was decreased significantly by genipin-treated rats. The mRNA and protein expression of CREB, BDNF were increased in genipin-treated rats compared to the CUMS-exposed model group. Moreover, the levels of corticosterone in serum were decreased by genipin-treated compared to the CUMS-exposed model group. These results suggest that the possible mechanism of antidepressant-like effects on genipin, at least in one part, resulted from monoaminergic neurotransmitter system and the potential dysfunctional regulation of the post-receptor signaling pathway, which particularly affected the 5-HT(1A)R, 5-HT(2A)R and BDNF levels in the hippocampus.

Topics: Animals; Antidepressive Agents; Brain-Derived Neurotrophic Factor; Depression; Disease Models, Animal; Hippocampus; Iridoids; Male; Phytotherapy; Rats; Rats, Sprague-Dawley; Real-Time Polymerase Chain Reaction; Receptors, Serotonin; Serotonin; Synaptic Transmission

This study focused on the synthesis and characterization of a natural polymeric system employing the interpenetrating polymer network (IPN) comprising curcumin as a bioactive. Biopolymers and actives such as chitosan, hypromellose, citric acid, genipin, and curcumin were used to develop an effective, biodegradable, and biocompatible film employed therapeutically as a wound healing platform. The semi-IPN films were investigated for their physicochemical, physicomechanical, and biological properties by quantification by FTIR, DSC, and Young's modulus. Following characterization, an optimum candidate formulation was produced whereby further in vitro and ex vivo studies were performed. Results revealed a burst release occurring at the first hour with 1.1 mg bioactive released when in contact with the dissolution medium and 2.23 mg due to bioactive permeation through the skin, thus suggesting that the lipophilic nature of skin greatly impacted the bioactive release rate. Furthermore, chemical and mechanical characterization and tensile strength analysis revealed that the degree of crosslinking and concentration of polymeric material used significantly influenced the properties of the film.

Topics: Administration, Cutaneous; Animals; Calorimetry, Differential Scanning; Chemistry, Pharmaceutical; Chitosan; Cross-Linking Reagents; Curcumin; Elastic Modulus; Iridoids; Kinetics; Permeability; Rats, Sprague-Dawley; Skin; Skin Absorption; Solubility; Spectroscopy, Fourier Transform Infrared; Technology, Pharmaceutical; Temperature; Tensile Strength; Water

Annulus fibrosus (AF) defects from annular tears, herniation, and discectomy procedures are associated with painful conditions and accelerated intervertebral disc (IVD) degeneration. Currently, no effective treatments exist to repair AF damage, restore IVD biomechanics and promote tissue regeneration. An injectable fibrin-genipin adhesive hydrogel (Fib-Gen) was evaluated for its performance repairing large AF defects in a bovine caudal IVD model using ex vivo organ culture and biomechanical testing of motion segments, and for its in vivo longevity and biocompatibility in a rat model by subcutaneous implantation. Fib-Gen sealed AF defects, prevented IVD height loss, and remained well-integrated with native AF tissue following approximately 14,000 cycles of compression in 6-day organ culture experiments. Fib-Gen repair also retained high viability of native AF cells near the repair site, reduced nitric oxide released to the media, and showed evidence of AF cell migration into the gel. Biomechanically, Fib-Gen fully restored compressive stiffness to intact levels validating organ culture findings. However, only partial restoration of tensile and torsional stiffness was obtained, suggesting opportunities to enhance this formulation. Subcutaneous implantation results, when compared with the literature, suggested Fib-Gen exhibited similar biocompatibility behaviour to fibrin alone but degraded much more slowly. We conclude that injectable Fib-Gen successfully sealed large AF defects, promoted functional restoration with improved motion segment biomechanics, and served as a biocompatible adhesive biomaterial that had greatly enhanced in vivo longevity compared to fibrin. Fib-Gen offers promise for AF repairs that may prevent painful conditions and accelerated degeneration of the IVD, and warrants further material development and evaluation.

Topics: Animals; Bioreactors; Cattle; Chondrogenesis; Compressive Strength; Fibrin Tissue Adhesive; Hydrogels; Intervertebral Disc; Intervertebral Disc Degeneration; Iridoids; Nitric Oxide; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Regeneration; Stress, Mechanical; Tensile Strength; Torque

Chitosan-silica porous hybrids were prepared by a novel strategy in order to improve the mechanical properties of chitosan (CHT) in the hydrogel state. The inorganic silica phase was introduced by sol-gel reactions in acidic medium inside the pores of already prepared porous scaffolds. In order to make the scaffolds insoluble in acidic media chitosan was cross-linked by genipin (GEN) with an optimum GEN concentration of 3.2 wt.%. Sol-gel reactions took place with Tetraethylorthosilicate (TEOS) and 3-glycidoxypropyltrimethoxysilane (GPTMS) acting as silica precursors. GPTMS served also as a coupling agent between the free amino groups of chitosan and the silica network. The morphology study of the composite revealed that the silica phase appears as a layer covering the chitosan membrane pore walls. The mechanical properties of the hybrids were characterized by means of compressive stress-strain measurements. By immersion in water the hybrids exhibit an increase in elastic modulus up to two orders of magnitude.

Topics: Chitosan; Compressive Strength; Cross-Linking Reagents; Elastic Modulus; Hydrogel, Polyethylene Glycol Dimethacrylate; Iridoids; Materials Testing; Membranes, Artificial; Phase Transition; Porosity; Silicon Dioxide; Solubility; Temperature; Tissue Scaffolds

Neuroinflammation and pro-inflammatory mediators play key roles in the pathogenesis of neurodegenerative diseases including stroke, which account for a significant burden of morbidity and mortality worldwide. Recently, the unsatisfactory pharmacotherapy and side effects of the drugs led to the development of alternative medicine for treating these diseases. Du Zhong (DZ), Eucommia ulmoides Oliver leaves, is a commonly used herb in the therapy of stroke in China. We hypothesize that the components from DZ inhibit neuroinflammation. In this study, DZ was extracted and the bioactive fractions with inhibitory effect on lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production in BV-2 microglial cells were further separated using chromatography. Two purified bioactive compounds, genipin (compound C) and 4-(1,2-dimethoxyethyl)benzene-1,2-diol (compound F), were isolated and identified after spectroscopic analysis. The results showed that they inhibited LPS-stimulated NO and tumor necrosis factor-alpha (TNF-α) production. Genipin exerted its anti-inflammatory effects through PI3K/Akt signaling pathway, whereas compound F inhibited phosphorylation of p38 mitogen-activated protein kinase (MAPK). In conclusion, genipin and compound F have potential for developing into new drugs for treating neurodegenerative diseases.

Topics: Animals; Anti-Inflammatory Agents; Catechols; Cell Line, Transformed; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Eucommiaceae; Gene Expression Regulation; Iridoids; Lipopolysaccharides; Magnetic Resonance Imaging; Mice; Microglia; Nitric Oxide Synthase Type II; Nitrites; Phosphatidylinositol 3-Kinases; Plant Preparations; RNA, Messenger; Signal Transduction

Genipin is obtained from the fruit of Gardenia jasminoides Ellis and acts as an herbal medicine or functional food in East Asia. In addition to produce natural colorant, it possesses widely antiinflammatory, antithrombotic, antidepressive and anticarcinogenic activities. However, little research focuses on the potential of genipin for drug-drug interactions. In this study, effects of genipin on mRNA and protein expression of cytochrome P450 (CYP) 2C19, CYP2D6 and CYP3A4 were detected by real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) and Western blot, respectively, in human hepatoma HepG2 cells. Enzyme activities of which were detected by luminogenic CYP assay in vitro. Moreover, effect of genipin on P-glycoprotein expression was analyzed by Western blot. Results showed that genipin possessed a significant induction on CYP2D6 and a remarkable inhibition on CYP2C19 and CYP3A4 not only from the expression of mRNA and protein (P<0.05 or P<0.01), but the level of enzyme activity. Moreover, a concentration-dependent induction of genipin on P-glycoprotein expression was observed. In conclusion, caution should be exercised with respect to the induction or inhibition of genipin on CYP isoenzymes and the strong induction on P-glycoprotein.

Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Cytochrome P-450 CYP2C19; Cytochrome P-450 CYP2C19 Inhibitors; Cytochrome P-450 CYP2D6; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inhibitors; Drug Interactions; Gardenia; Hep G2 Cells; Humans; Iridoids

Sickness behavior is a series of behavioral and psychological changes that develop in inflammatory disease, including infections and cancers. Administration of the bacterial endotoxin lipopolysaccharide (LPS) induces sickness behavior in rodents. Genipin, an aglycon derived from an iridoid glycoside geniposide extracted from the fruit of Gardenia jasminoides, has anti-inflammatory and antidepressant activities. However, the effects of genipin on inflammation-induced changes in emotional behaviors are unknown. In this study, we examined the effects of genipin on LPS-induced inflammation in BV-2 cells and sickness behavior in mice. Pretreatment with genipin inhibited LPS-induced increases in NO production and reduced the mRNA levels of inflammation-related genes (iNOS, COX-2, IL-1β and IL-6) in BV-2 cells. Oral administration of genipin ameliorated LPS-induced depressive-like behavior in the forced swim test and social behavior deficits 24h after LPS administration in mice. LPS-induced expression of mRNAs for inflammation-related genes and the number of c-fos immunopositive cells decreased in the paraventricular nucleus (PVN) of the hypothalamus and the central nucleus of the amygdala (CeA), suggesting that genipin attenuates LPS-induced changes of emotional behaviors through inhibition of neural activation and inflammatory responses in the PVN and CeA. These novel pharmacological effects of genipin may be useful for treatment of patients with sickness behavior.

Topics: Animals; Cell Line; Cell Survival; Central Amygdaloid Nucleus; Emotions; Illness Behavior; Iridoids; Lipopolysaccharides; Male; Mice; Paraventricular Hypothalamic Nucleus; Rubiaceae

Topics: Biocompatible Materials; Chitosan; Collagen; Humans; Iridoids; Nanocomposites; Regenerative Medicine; Tissue Scaffolds

Chitosan (CS) is a versatile biopolymer whose morphological and chemico-physical properties can be designed for a variety of biomedical applications. Taking advantage of its electrolytic nature, cathodic polarization allows CS deposition on electrically conductive substrates, resulting in thin porous structures with tunable morphology. Here we propose an easy method to obtain CS membranes with highly oriented micro-channels for tissue engineering applications, relying on simple control of process parameters and cathodic substrate geometry. Cathodic deposition was performed on two different aluminum grids in galvanostatic conditions at 6.25 mA cm(-2) from CS solution [1g L(-1)] in acetic acid (pH 3.5). Self-standing thin scaffolds were cross linked either with genipin or epichlorohydrin, weighted, and observed by optical and electron microscopy. Swelling properties at pH 5 and pH 7.4 have been also investigated and tensile tests performed on swollen samples at room temperature. Finally, direct and indirect assays have been performed to evaluate the cytotoxicity at 24 and 72 h. Thin scaffolds with two different oriented porosities (1000 µm and 500 µm) have been successfully fabricated by electrochemical techniques. Both cross-linking agents did not affected the mechanical properties and cytocompatibility of the resulting structures. Depending on the pH, these structures show interesting swelling properties that can be exploited for drug delivery systems. Moreover, thanks to the possibility of controlling the porosity and the micro-channel orientation, they should be used for the regeneration of tissues requiring a preferential cells orientation, e.g., cardiac patches or ligament regeneration.

Topics: Animals; Biocompatible Materials; Chemical Phenomena; Chitosan; Electrochemical Techniques; Endothelial Cells; Epichlorohydrin; Hydrogen-Ion Concentration; Iridoids; Mice; Microscopy, Electron, Scanning; Porosity; Regeneration; Tissue Engineering; Tissue Scaffolds

Human adipose derived stem cells have shown chondrogenic differentiation potential in cartilage tissue engineering in combination with biomimetic materials. In this study, the chondrogenic potential of a porous gelatin based scaffold genipin (GNP) crosslinked was investigated in human mesenchymal stem cells obtained from adipose tissue. Cells were cultured up to 4 weeks on the scaffold and on monolayer, MTT assay was performed to evaluate cell viability, light, and transmission electron microscopy were carried out to demonstrate cell proliferation, scaffold adhesion, and cell colonization inside the porous architecture of the biomaterial. The expression of chondrogenic markers such as SOX9, collagen type II, aggregan, and versican was investigated by Real Time PCR. Results showed an high cell viability, adhesion, and colonization of the scaffold. Real Time PCR data demonstrated an upregulation of all the chondrogenic markers analyzed. In conclusion, 3D gelatin GNP crosslinked porous scaffold provides an improved environment for chondrogenic differentiation of stem cells compared with cell monolayer culture system.

Topics: Adipose Tissue; Aggrecans; Biomimetic Materials; Cell Differentiation; Chondrogenesis; Collagen Type II; Gelatin; Humans; Iridoids; Mesenchymal Stem Cells; Microscopy; Microscopy, Electron, Transmission; Real-Time Polymerase Chain Reaction; SOX9 Transcription Factor; Tissue Scaffolds; Versicans

Casein micelles are porous colloidal particles, constituted of casein molecules, water, and minerals. The vulnerability of the supramolecular structure of casein micelles face to changes in the environmental conditions restrains their applications in other domains besides food. Thus, redesigning casein micelles is a challenge to create new functionalities for these biosourced particles. The objective of this work was to create stable casein microgels from casein micelles using a natural cross-linker, named genipin. Suspensions of purified casein micelles (25 g L(-1)) were mixed with genipin solutions to have final concentrations of 5, 10, and 20 mM genipin. Covalently linked casein microgels were formed via cross-linking of lysyl and arginyl residues of casein molecules. The reacted products exhibited blue color. The cross-linking reaction induced gradual changes on the colloidal properties of the particles. The casein microgels were smaller and more negatively charged and presented smoother surfaces than casein micelles. These results were explained based on the cross-linking of free NH2 present in an external layer of κ-casein. Light scattering and rheological measurements showed that the reaction between genipin and casein molecules was intramicellar, as one single population of particles was observed and the values of viscosity (and, consequently, the volume fraction of the particles) were reduced. Contrary to the casein micelles, the casein microgels were resistant to the presence of dissociating agents, e.g., citrate (calcium chelating) and urea, but swelled as a consequence of internal electrostatic repulsion and the disruption of hydrophobic interactions between protein chains. The casein microgels did not dissociate at the air-solution interface and formed solid-like interfaces rather than a viscoelastic gel. The potential use of casein microgels as adaptable nanocarriers is proposed in the article.

Topics: Caseins; Chromatography, High Pressure Liquid; Gels; Iridoids; Micelles; Microscopy, Electron, Scanning; Spectrophotometry, Ultraviolet; Surface Tension

Cellulose nanocrystal (CNC) reinforced chitosan based antimicrobial films were prepared by immobilizing nisin on the surface of the films. Nanocomposite films containing 18.65 μg/cm(2) of nisin reduced the count of L. monocytogenes by 6.73 log CFU/g, compared to the control meat samples (8.54 log CFU/g) during storage at 4 °C in a Ready-To-Eat (RTE) meat system. Film formulations containing 9.33 μg/cm(2) of nisin increased the lag phase of L. monocytogenes on meat by more than 21 days, whereas formulations with 18.65 μg/cm(2) completely inhibited the growth of L. monocytogenes during storage. Genipin was used to cross-link and protect the activity of nisin during storage. Nanocomposite films cross-linked with 0.05% w/v genipin exhibited the highest bioactivity (10.89 μg/cm(2)) during the storage experiment, as compared to that of the un-cross-linked films (7.23 μg/cm(2)). Genipin cross-linked films were able to reduce the growth rate of L. monocytogenes on ham samples by 21% as compared to the un-cross-linked films. Spectroscopic analysis confirmed the formation of genipin-nisin-chitosan heterocyclic cross-linked network. Genipin cross-linked films also improved the swelling, water solubility, and mechanical properties of the nanocomposite films.

Topics: Anti-Infective Agents; Chitosan; Cross-Linking Reagents; Iridoids; Listeria monocytogenes; Mechanical Phenomena; Microbial Sensitivity Tests; Nanocomposites; Nisin; Solubility; Spectroscopy, Fourier Transform Infrared; Surface Properties; Water

The surface characteristics of biomaterials, especially regarding the sustained delivery of bone morphogenetic protein-2 (BMP-2), can possibly provide a novel and effective drug delivery system that can enhance osteogenesis. In this study, we evaluated the BMP-2 adsorption and release ability of the surface biomimetic hydroxyapatite (HAp) nanostructure on a new HAp-coated genipin-chitosan conjugation scaffold (HGCCS), and the resulting osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. HGCCS exhibited a loading efficiency of 65% (1.30 μg), which is significantly higher than 28% (0.56 μg, p < 0.01) for the genipin cross-linked chitosan framework, as quantified by an enzyme-linked immunosorbent assay. More importantly, we found that the release of BMP-2 from HGGCS sustained for at least 14 days in simulated body fluid in vitro, which is much better than the burst release within 3 days for CGF. Moreover, the BMP-2 release from HGCCS induced an increase in alkaline phosphatase activity as an indicator of osteogenic differentiation of seeded BMSCs for 14 days in vitro. HGCCS also stimulated a high mRNA expression of osteogenic differentiation makers, runt-related transcription factor 2 for 14 days, osteopontin for 3 days, and osteocalcin for 14 days. The results of this study suggest that the surface biomimetic HAp nanostructure of HGCCS used as a delivery system for BMP-2 is capable of promoting osteogenic differentiation in vitro. These findings demonstrated that HAp nanostructure assembled on organic porous scaffold could work as both calcium source and absorption/release platform, which opened a new research avenue for cell growth factor release, and provided a promising strategy for design and preparation of bioactive scaffold for bone tissue engineering.

Topics: Adsorption; Alkaline Phosphatase; Animals; Biocompatible Materials; Bone Morphogenetic Protein 2; Bone Substitutes; Cells, Cultured; Chitosan; Delayed-Action Preparations; Durapatite; Femur; Growth Substances; Iridoids; Male; Mesenchymal Stem Cells; Nanostructures; Osteogenesis; Rats, Wistar; RNA, Messenger; Tibia; Tissue Scaffolds

Engineering metabolically demanding tissues requires the supply of nutrients, oxygen, and removal of metabolic byproducts, as well as adequate mechanical properties. In this work, we propose the development of chitosan (CHIT)/alginate (ALG) freestanding membranes fabricated by layer-by-layer (LbL) assembly. CHIT/ALG membranes were cross-linked with genipin at a concentration of 1 mg·mL(-1) or 5 mg·mL(-1). Mass transport properties of glucose and oxygen were evaluated on the freestanding membranes. The diffusion of glucose and oxygen decreases with increasing cross-linking concentration. Mechanical properties were also evaluated in physiological-simulated conditions. Increasing cross-linking density leads to an increase of storage modulus, Young modulus, and ultimate tensile strength, but to a decrease in the maximum hydrostatic pressure. The in vitro biological performance demonstrates that cross-linked films are more favorable for cell adhesion. This work demonstrates the versatility and feasibility of LbL assembly to generate nanostructured constructs with tunable permeability, mechanical, and biological properties.

Topics: Alginates; Animals; Biocompatible Materials; Cell Adhesion; Cells, Cultured; Chitosan; Cross-Linking Reagents; Fibroblasts; Glucuronic Acid; Hexuronic Acids; Iridoids; Membranes; Membranes, Artificial; Mice; Nanostructures; Permeability; Tensile Strength

Vascular disease is a leading cause of death globally and typically manifests chronically due to long-term maladaptive arterial growth and remodeling. To date, there is no in vitro technique for studying vascular function over relevant disease time courses that both mimics in vivo-like tissue structure and provides a simple readout of tissue stress. We aimed to extend tissue viability in our muscular thin film contractility assay by modifying the polydimethylsiloxane (PDMS) substrate with micropatterned genipin, allowing extracellular matrix turnover without cell loss. To achieve this, we developed a microfluidic delivery system to pattern genipin and extracellular matrix proteins on PDMS prior to cell seeding. Tissues constructed using this method showed improved viability and maintenance of in vivo-like lamellar structure. Functional contractility of tissues fabricated on genipin-modified substrates remained consistent throughout two weeks in culture. These results suggest that muscular thin films with genipin-modified PDMS substrates are a viable method for conducting functional studies of arterial growth and remodeling in vascular diseases.

Topics: Bioprinting; Cell Survival; Cells, Cultured; Dimethylpolysiloxanes; Fibronectins; Humans; Iridoids; Microfluidic Analytical Techniques; Models, Cardiovascular; Muscle, Smooth, Vascular; Tissue Engineering; Umbilical Arteries

In this study, an environmentally friendly method for preparation of chitosan crosslinked membranes with different genipin concentrations (0, 125, 250, and 500 mg/L) was developed. Genipin-crosslinked chitosan membranes were used for the immobilization of fungal pectinesterase (PE). PE was efficiently immobilized in chitosan membranes and used for modification of high-methoxylated pectins into low-methoxylated pectins. The charge density (ζ-potential), infrared spectroscopy (FTIR), and ion-exchange chromatography (IEC) revealed the modification of pectin for immobilized PE in genipin-crosslinked chitosan membrane. The ζ-potential results indicated a decrease of -5.2 mV in the pectin control solution, whereas using the PE immobilized in chitosan and genipin-chitosan membranes, the obtained values were -30.45 mV and -36.38 mV, respectively. Genipin-crosslinked chitosan membrane jointly with the immobilized PE represents an eco-friendly support to prepare tailor-made low-methoxylated lime pectin.

Topics: Carboxylic Ester Hydrolases; Chitosan; Enzymes, Immobilized; Fungal Proteins; Iridoids; Membranes, Artificial; Pectins

Topics: Alginates; Chitosan; Drug Carriers; Glucuronic Acid; Hexuronic Acids; History, 21st Century; Hydrogels; Hydrogen-Ion Concentration; Iridoids; Nanoparticles; Proteins

We report the versatility of polyion complex (PIC) micelles for the preparation of shell and core cross-linked (SCL and CCL) micelles with their surface properties determined by the constituent polymer composition and cross-linking agent. The negatively and positively charged PIC micelles with their molecular structure and properties depending on the mixing weight percentage and polymer molecular weight were first prepared by mixing the negatively and positively charged polyions, poly(acrylic acid) (PAA) and poly(L-lysine) (PLL). The feasibility of preparing SCL micelles was demonstrated by cross-linking the shell of the negatively and positively charged micelles using cystamine and genipin, respectively. The core of the micelles can be cross-linked by silica deposition to stabilize the assemblies. The shell and/or core cross-linked micelles exhibited excellent colloid stability upon changing solution pH. The drug release from the drug-loaded SCL micelles revealed that the controllable permeability of the SCL micelles can be achieved by tuning the cross-linking degree and the SCL micelles exhibited noticeable pH-responsive behavior with accelerated release under acidic conditions. With the versatility of cross-linking strategies, it is possible to prepare a variety of SCL and CCL micelles from PIC micelles.

Topics: Acrylic Resins; Antibiotics, Antineoplastic; Cross-Linking Reagents; Cystamine; Doxorubicin; Drug Carriers; Drug Compounding; Drug Stability; Feasibility Studies; Hydrogen-Ion Concentration; Iridoids; Kinetics; Materials Testing; Micelles; Models, Chemical; Molecular Weight; Permeability; Polylysine; Solubility; Surface Properties

Genipin, the aglycon of geniposide found in gardenia fruit has long been considered for treatment of inflammatory diseases in traditional oriental medicine. Genipin has recently been reported to have some pharmacological functions, such as antimicrobial, antitumor, and anti-inflammatory effects. The aim of this study was to examine whether genipin could modify matrix metalloproteinase (MMP)-1 and MMP-3, which are related to the destruction of periodontal tissues in periodontal lesion, expression in tumor necrosis factor (TNF)-α-stimulated human periodontal ligament cells (HPDLCs). Genipin prevented TNF-α-mediated MMP-1 and MMP-3 productions in HPDLCs. Moreover, genipin could suppress not only extracellular signal-regulated kinase (ERK) and Jun-N-terminal kinase (JNK) phosphorylations but also AMP-activated protein kinase (AMPK) phosphorylation in TNF-α-stimulated HPDLCs. Inhibitors of ERK and AMPK could inhibit both MMP-1 and MMP-3 productions. Moreover, we revealed the ERK inhibitor suppressed AMPK phosphorylation in TNF-α-stimulated HPDLCs. These data provide a new mechanism through which genipin could be used for the treatment of periodontal disease to prevent MMPs expression in periodontal lesion.

Topics: AMP-Activated Protein Kinases; Cells, Cultured; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Flavonoids; Humans; Iridoids; Matrix Metalloproteinase 1; Matrix Metalloproteinase 3; Periodontal Ligament; Protein Kinase Inhibitors; Tumor Necrosis Factor-alpha

Collagen (Coll), as the basic material of matrix scaffolds for cell growth, has been widely used in the field of tissue engineering and regenerative medicine. In this study, collagen protein was modified by L-lysine (Lys), and cross-linked by genipin (GN) to prepare the L-lysine-modified collagen (Lys-Coll-GN) scaffolds. Microstructure, pore size, porosity, stability and biocompatibility of Lys-Coll-GN scaffolds were observed. The results showed that the bond between L-lysine and collagen protein molecule was formed by generating amide linkage, and mouse embryo fibroblasts proliferation was not inhibited in the Lys-Coll-GN scaffolds. In the multiple comparisons of Coll-scaf- folds, Coll-GN scaffolds and Lys-Coll-GN scaffolds, Coll-scaffolds was the worst in mechanical characteristics while the highest in biodegradation rate. Compared to Coll-GN scaffolds, Lys-Coll-GN scaffolds had more fiber structure, higher interval porosity (P<0. 01). Although the tensile stress of Lys-Coll-GN scaffolds reduced significantly, its e- longation length extended when the scaffolds was fractured (P<0. 01). The percentage of Lys-Coll-GN scaffolds residual weight was lower than that of Coll-GN scaffolds after all the scaffolds were treated by collagenase for 5 days (P<0. 01). This study suggested that Lys-Coll-GN scaffold had good biocompatibility, and it improved the mechanical property and degradation velocity for collagen-based scaffold. This study gave a new predominant type of tissue engineering scaffold for the regenerative medicine.

Topics: Animals; Biocompatible Materials; Cell Proliferation; Collagen; Cross-Linking Reagents; Fibroblasts; Iridoids; Lysine; Mice; Porosity; Tissue Engineering; Tissue Scaffolds

Genipin-cross-linked caseinophosphopeptide (CPP)-chitosan (CS) nanoparticles (smaller than 300 nm) showed significantly improved stability and adjustable release profile in the gastrointestinal (GI) tract. Optimal purification of the nanoparticles was established by centrifugation to terminate the cross-linking reaction, which was further confirmed and characterized by FT-IR. Results from transmission electron microscopy (TEM), dynamic light scattering (DLS), and electrophoretic mobility (ζ-potential) measurements revealed that genipin cross-linking significantly prevented the bursting of the CPP-CS nanoparticles in simulated stomach acid and their precipitation under neutral intestinal environment. Pepsin showed little impact on the nanoparticle colloid stability; however, trypsin induced their aggregations. Genipin cross-linking slowed the burst release of (-)-epigallocatechin-3-gallate (EGCG) from the nanoparticles. The EGCG-loaded nanoparticles showed strong cytotoxicity against cancer cells; meanwhile, the net nanoparticles demonstrated high biocompatibility. The findings in the present work provide fundamental information for the rational design of biopolymer nanoparticles as an effective delivery systems for polyphenols.

Topics: Caseins; Catechin; Cell Line, Tumor; Cell Survival; Chitosan; Drug Carriers; Drug Delivery Systems; Gastrointestinal Tract; Humans; Iridoids; Nanoparticles; Peptide Fragments; Polyphenols; Proteoglycans

Mitochondrial uncoupling protein 2 (UCP2) is suggested to have a role in the development of nonalcoholic steatohepatitis (NASH). However, the mechanism remains unclear. Autophagy is an important mediator of many pathological responses. This study aims to investigate the relationship between UCP2 and hepatoma cells autophagy in palmitic acid- (PA-) induced lipotoxicity. H4IIE cells were treated with palmitic acid (PA), and cell autophagy and apoptosis were examined. UCP2 expression, in association with LC3-II and caspase-3, which are indicators of cell autophagy and apoptosis, respectively,was measured. Results demonstrated that UCP2 was associated with autophagy during PA-induced hepatic carcinoma cells injury. Tests on reactive oxygen species (ROS) showed that UCP2 overexpression strongly decreases PA-induced ROS production and apoptosis. Conversely, UCP2 inhibition by genipin or UCP2 mRNA silencing enhances PA-induced ROS production and apoptosis. Autophagy partially participates in this progress. Moreover, UCP2 was associated with ATP synthesis during PA-induced autophagy. In conclusion, increasing UCP2 expression in hepatoma cells may contribute to cell autophagy and antiapoptotic as result of fatty acid injury. Our results may bring new insights for potential NASH therapies.

Topics: Apoptosis; Autophagy; Carcinoma, Hepatocellular; Caspase 3; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Ion Channels; Iridoids; Liver Neoplasms; Microtubule-Associated Proteins; Mitochondrial Proteins; Non-alcoholic Fatty Liver Disease; Palmitic Acid; Reactive Oxygen Species; RNA, Messenger; Uncoupling Protein 2

Polysaccharides are frequently incorporated into scaffolds for tissue engineering applications to improve mechanical and biological properties. We evaluated the influence of a Ficoll® scaffold on collagen films, a scaffold that is extensively used for soft and hard tissue repair. To avoid cytotoxicity issues associated with chemical reagents, the influence of genipin, a naturally occurring crosslinking agent, was assessed. Ultra-structural level collagen films formed with and without Ficoll showed a fine fibrillar structure whereas genipin crosslinked films showed a coarse fibrillar and partially nodular structure. In contrast, glutaraldehyde crosslinked films lost their fibrillar pattern. Crosslinking significantly increased denaturation temperature (p < 0.001), stress (p < 0.0001) and force (p < 0.0001) at break. Collagen/Ficoll and collagen/Ficoll/genipin films showed the highest WI38 fibroblast attachment than any other scaffold (p < 0.003) and significantly greater WI38 fibroblast metabolic activity than other scaffolds (p < 0.001). By day 6. collagen/Ficoll/genipin films also induced higher and more aligned fibronectin matrix deposition than other scaffolds. Overall, this study indicates the suitability of collagen/Ficoll/genipin for tissue engineering applications.

Topics: Achilles Tendon; Animals; Biocompatible Materials; Cell Adhesion; Cell Line; Collagen; Cross-Linking Reagents; Fibroblasts; Ficoll; Glutaral; Humans; Immunohistochemistry; Iridoids; Microscopy, Phase-Contrast; Stress, Mechanical; Swine; Tissue Engineering; Tissue Scaffolds

For scaffolds in cartilage tissue engineering, it is the principle to design the materials with both favorable mechanical and biological property.. In this article, collagen hydrogels modified by two ways to improve mechanical strength were applied for in vivo cartilage reconstruction: one is collagen-alginate hydrogel (CAH) representative of mixture, the other is collagen hydrogel crosslinked by genipin (CGH). To investigate the biological activities of the two materials, it was designed as: scaffolds loaded with allogenous chondrocytes were encased in diffusion chamber, and then implanted subcutaneously in SD rats for 8 weeks.. Histologic, immunohistochemical, and RT-PCR results showed that collagen type Ⅱ and GAG, indicator of cartilage extracellular matrix (ECM) was highly expressed in constructs of chondrocyte-CAH. Significantly lower cell density and expression of cartilage specific protein were shown in constructs of chondrocyte-CGH than that in chondrocyte-CAH. This demonstrated that CAH may provide a more favorable environment for cartilage reconstruction. In addition, the model with diffusion chamber technique was viable for evaluation of scaffolds in vivo cartilage engineering in immunocompetent host. Instead, directly reconstruction of ectopic cartilage without diffusion chamber suffered from damaged tissue and less neo-cartilage matrix formed.. In conclusion, CAH is realistic as scaffold for in vivo cartilage tissue engineering with both satisfactory mechanical properties and biomimetic activity. And the model with diffusion chamber to reconstruct ectopic cartilage in immunocompetent animals is promising for evaluation of scaffolds. This study provided a new insight for in vivo cartilage tissue engineering.

Topics: Animals; Chondrocytes; Collagen Type II; Hydrogel, Polyethylene Glycol Dimethacrylate; Iridoids; Materials Testing; Rats; Rats, Sprague-Dawley; Tissue Engineering; Tissue Scaffolds

Novel casein (CAS)-based micelles loaded with the poorly soluble anti-cancer drug, flutamide (FLT), were successfully developed in a powdered form via spray-drying technique. Genipin (GNP) was used to crosslink CAS micelles as demonstrated by color variation of the micelles. Drug solubilization was enhanced by incorporation within the hydrophobic micellar core which was confirmed by solubility study and UV spectra. Spherical core-shell micelles were obtained with a particle size below 100 nm and zeta potential around -30 mV. At low drug loading, FLT was totally incorporated within micellar core as revealed by thermal analysis. However, at higher loading, excess non-incorporated drug at micelle surface caused a significant reduction in the surface charge density. Turbidity measurements demonstrated the high physical stability of micelles for 2 weeks dependent on GNP-crosslinking degree. In a dry powdered form, the micelles were stable for 6 months with no significant changes in drug content or particle size. A sustained drug release from CAS micelles up to 5 days was observed. After i.v. administration into rats, CAS micelles exhibited a prolonged plasma circulation of FLT compared to drug solution. Furthermore, a more prolonged drug systemic circulation was observed for GNP-crosslinked micelles. Overall, this study reports the application of spray-dried natural protein-based micelles for i.v. delivery of hydrophobic anti-cancer drugs such as FLT.

Topics: Animals; Antineoplastic Agents; Calorimetry, Differential Scanning; Caseins; Chemistry, Pharmaceutical; Chromatography, High Pressure Liquid; Cross-Linking Reagents; Drug Delivery Systems; Flutamide; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Iridoids; Male; Micelles; Particle Size; Rats; Rats, Sprague-Dawley; Spectroscopy, Fourier Transform Infrared; Time Factors; Ultraviolet Rays

Astrocytes can play dual roles in the response to spinal cord injury (SCI) acting as both an inhibitory barrier and a trophic support for growth axons. Therefore, migration of these cells into the defect as opposed to forming a scar at the periphery, may promote axon regeneration through the lesion. However, infiltration requires the conformal filling of the cyst-like lesion, which often forms after SCI, with a biomaterial scaffold encouraging of astrocyte migration. For this application, we investigated injectable collagen-based hydrogels covalently cross-linked with genipin and incorporating fibroblast growth factor-2 (FGF-2) either freely or encapsulated within lipid microtubules (LMTs). An outgrowth assay was used to evaluate in vitro the number of primary rat astrocytes infiltrating into the collagen gels and the distance to which they infiltrated. The presence of FGF-2 within the encapsulating gel significantly increased the number of astrocytes within the gel, their penetration distance into the gel, and caused them to move out in a chain-like pattern, compared to control gels without FGF-2. Genipin cross-linking of the collagen gel decreased the number of infiltrating astrocytes, compared to the non-cross-linked control gel; however, incorporation of FGF-2-containing LMTs within genipin-cross-linked gels restored the astrocyte infiltration to levels approaching non-cross-linked gels incorporating FGF-2. Overall, injectable collagen-genipin hydrogels containing FGF-2-containing LMTs are a promising candidate for the treatment for SCI through the attraction of astrocytes into the graft.

Topics: Animals; Astrocytes; Cell Survival; Cells, Cultured; Collagen; Fibroblast Growth Factor 2; Hydrogels; Iridoids; Nerve Regeneration; Rats; Rats, Sprague-Dawley; Spinal Cord Injuries; Tissue Engineering

As life spans increased, neurodegenerative disorders that affect aging populations have also increased. Progressive neuronal loss in specific brain regions is the most common cause of neurodegenerative disease; however, key determinants mediating neuron loss are not fully understood. Using a model of mitochondrial membrane potential (ΔΨm) loss, we found only 25% cell loss in SH-SY5Y (SH) neuronal mono-cultures, but interestingly, 85% neuronal loss occurred when neurons were co-cultured with BV2 microglia. SH neurons overexpressing uncoupling protein 2 exhibited an increase in neuron-microglia interactions, which represent an early step in microglial phagocytosis of neurons. This result indicates that ΔΨm loss in SH neurons is an important contributor to recruitment of BV2 microglia. Notably, we show that ΔΨm loss in BV2 microglia plays a crucial role in microglial activation and phagocytosis of damaged SH neurons. Thus, our study demonstrates that ΔΨm loss in both neurons and microglia is a critical determinant of neuron loss. These findings also offer new insights into neuroimmunological and bioenergetical aspects of neurodegenerative disease.

Topics: Animals; CA1 Region, Hippocampal; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cells, Cultured; Coculture Techniques; Cytophagocytosis; Dopamine; Dose-Response Relationship, Drug; Flow Cytometry; Green Fluorescent Proteins; High-Temperature Requirement A Serine Peptidase 2; Humans; Interleukin-2; Iridoids; L-Lactate Dehydrogenase; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Mice, Transgenic; Microglia; Mitochondrial Proteins; Neuroblastoma; Neurons; Polysaccharides; Proto-Oncogene Proteins; Proton Ionophores; Serine Endopeptidases; Time Factors; Trans-Activators; Transfection; Tubulin

The dielectric behavior of the gelatine-GAGs based blend systems has been studied to understand the dynamic behavior of the water at the protein-GAGs interfaces which are relevant for tissue engineering application. Impedance (Z) and phase have been measured as a function of frequencies from 0.01 Hz to 100 kHz. GAGs tunes the ionic charge drift which initiates polarization mechanisms through charge accumulation at structural interfaces and creates conduction currents. The admittance results showed that at high frequency, the conductivity increases with increasing GAGs concentration indicating changes in hydration shell of the gelatine by the GAGs.

Topics: Animals; Cattle; Electric Impedance; Electricity; Gelatin; Glycosaminoglycans; Iridoids; Materials Testing

In the engineering of soft tissues, designing the scaffolds with adequate strength and controllable biodegradation properties are essential. To fulfill such design criteria gelatin/carbohydrate chitosan (G/CC) scaffolds have gained much attention in engineering replacement of soft tissues. In this research, the influence of different cross-linking methods and parameters with genipin on the physico-chemical properties of G/CC scaffolds was investigated in detail. The scaffolds with different concentrations of G and CC were prepared and cross-linked trough two different methods, and the impact of cross-linker concentration beside of cross-linking time and temperature were fully analyzed. The obtained results suggested that the 1% genipin-cross-linked G60/CC40 scaffolds, prepared at room temperature for 24h through scaffold cross-linking method, could be a promising replacement in missing segments of load-bearing soft tissues. This article describes a systematic study of genipin cross-linking effects on G/CC scaffolds.

Topics: Biocompatible Materials; Chitosan; Cross-Linking Reagents; Elastic Modulus; Gelatin; Iridoids; Materials Testing; Microscopy, Electron, Scanning; Porosity; Tissue Engineering; Tissue Scaffolds; Water; Weight-Bearing

Helicobacter pylori is a significant human pathogen that recognizes specific carbohydrate receptors, such as the fucose receptor, and produces the vacuolating cytotoxin, which induces inflammatory responses and modulates the cell-cell junction integrity of the gastric epithelium. The clinical applicability of topical antimicrobial agents was needed to complete the eradication of H. pylori in the infected fundal area. In the present study, we combined fucose-conjugated chitosan and genipin-cross-linking technologies in preparing multifunctional genipin-cross-linked fucose-chitosan/heparin nanoparticles to encapsulate amoxicillin of targeting and directly make contact with the region of microorganism on the gastric epithelium. The results show that the nanoparticles effectively reduced drug release at gastric acids and then released amoxicillin in an H. pylori survival situation to inhibit H. pylori growth and reduce disruption of the cell-cell junction protein in areas of H. pylori infection. Furthermore, with amoxicillin-loaded nanoparticles, a more complete H. pylori clearance effect was observed, and H. pylori-associated gastric inflammation in an infected animal model was effectively reduced.

Topics: Amoxicillin; Animals; Cell Line; Cell Survival; Chitosan; Coculture Techniques; Cross-Linking Reagents; Fluorescent Antibody Technique; Fucose; Helicobacter Infections; Helicobacter pylori; Heparin; Humans; Hydrogen-Ion Concentration; Iridoids; Male; Mice; Mice, Inbred C57BL; Microbial Sensitivity Tests; Nanoparticles; Particle Size; Spectroscopy, Fourier Transform Infrared; Static Electricity

Mitochondria dysfunction has been reported in various kidney diseases but how it leads to kidney fibrosis and how this is regulated is unknown. Here we found that mitochondrial uncoupling protein 2 (UCP2) was induced in kidney tubular epithelial cells after unilateral ureteral obstruction in mice and that mice with ablated UCP2 resisted obstruction-induced kidney fibrosis. We tested this association further in cultured NRK-52E cells and found that TGF-β1 remarkably induced UCP2 expression. Knockdown of UCP2 largely abolished the effect of TGF-β1, whereas overexpression of UCP2 promoted tubular cell phenotype changes. Analysis using a UCP2 mRNA-3'-untranslated region luciferase construct showed that UCP2 mRNA is a direct target of miR-30e. MiR-30e was downregulated in tubular cells from fibrotic kidneys and TGF-β1-treated NRK-52E cells. A miR-30e mimic significantly inhibited TGF-β1-induced tubular-cell epithelial-mesenchymal transition, whereas a miR-30e inhibitor imitated TGF-β1 effects. Finally, genipin, an aglycone UCP2 inhibitor, significantly ameliorated kidney fibrosis in mice. Thus, the miR-30e/UCP2 axis has an important role in mediating TGF-β1-induced epithelial-mesenchymal transition and kidney fibrosis. Targeting this pathway may shed new light for the future of fibrotic kidney disease therapy.

Topics: Animals; Cell Line; Disease Models, Animal; Epithelial Cells; Epithelial-Mesenchymal Transition; Extracellular Matrix; Fibrosis; Humans; Ion Channels; Iridoids; Kidney Diseases; Kidney Tubules; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; MicroRNAs; Mitochondrial Proteins; Rats; Recombinant Proteins; RNA Interference; Signal Transduction; Time Factors; Transfection; Transforming Growth Factor beta1; Uncoupling Protein 2; Ureteral Obstruction

Chitosan nanoparticles crosslinked with genipin were prepared by reverse microemulsion that allowed to obtain highly monodisperse (3-20 nm by TEM) nanogels. The incorporation of genipin into chitosan was confirmed and quantitatively evaluated by UV-vis and (1)H NMR. Loosely crosslinked chitosan networks showed higher water solubility at neutral pHs than pure chitosan. The hydrodynamic diameter of the genipin-chitosan nanogels ranged from 270 to 390 nm and no remarkable differences were found when the crosslinking degree was varied. The hydrodynamic diameters of the nanoparticles increased slightly at acidic pH and the protonation of ionizable amino groups with the pH was confirmed by the zeta potential measurements. The biocompatible and biodegradable nature, as well as the colloidal and monodisperse particle size of the prepared nanogels, make them attractive candidates for a large variety of biomedical applications.

Topics: Biocompatible Materials; Chitosan; Hydrodynamics; Hydrogen-Ion Concentration; Iridoids; Nanogels; Particle Size; Polyethylene Glycols; Polyethyleneimine; Solubility; Spectrophotometry, Ultraviolet

Dynamic compression is an important physical stimulus for the physiology of chondrocyte and articular cartilage tissue engineering. In this study, modulation of chondrocyte behaviors in chitosan/collagen scaffolds with different mechanical properties under free-swelling or dynamic compression conditions was investigated. Rabbit chondrocytes were seeded in chitosan/collagen scaffolds crosslinked by genipin (GP) with different concentrations, and then cultured for 3 days prior to cyclic compression of 40% strain, 0.1 Hz, and 30 min/day for 2 weeks. The results showed that the cell proliferation was increased with increasing genipin concentrations and dynamic compression. On the other hand, although total glycosaminoglycans (GAGs) deposition was enhanced by dynamic compression under certain conditions, e.g. the GP0.5 chitosan/collagen scaffolds for 1 week of compression culture, normalized GAGs deposition per cell was decreased by dynamic compression. Our results suggest that while several studies suggest that dynamic compression benefits articular cartilage tissue engineering, many factors including scaffold types and compression conditions determine the outcome of dynamic compression culture.

Topics: Animals; Cartilage, Articular; Cell Culture Techniques; Cell Proliferation; Cells, Cultured; Chitosan; Chondrocytes; Collagen; Cross-Linking Reagents; Equipment Design; Glycosaminoglycans; Iridoids; Pressure; Rabbits; Stress, Mechanical; Tissue Engineering; Tissue Scaffolds

Genipin, an active constituent of Gardenia fruit, has been reported to show an anti-tumor effect in several cancer cell systems. Here, we demonstrate how genipin exhibits a strong apoptotic cell death effect in human non-small-cell lung cancer H1299 cells. Genipin-mediated decrease in cell viability was observed through apoptosis as demonstrated by induction of a sub-G1 peak through flow cytometry, DNA fragmentation measured by TUNEL assay, and cleavage of poly ADP-ribose-polymerase. During genipin-induced apoptosis, the mitochondrial execution pathway was activated by caspase-9 and -3 activation as examined by a kinetic study, cytochrome c release, and a dose-dependent increase in Bax/Bcl-2 ratio. A search for the downstream pathway reveals that genipin-induced apoptosis was mediated by an increase in phosphorylated p38MAPK expression, which further activated downstream signaling by phosphorylating ATF-2. SB203580, a p38MAPK inhibitor, markedly blocked the formation of TUNEL-positive apoptotic cells in genipin-treated cells. Besides, the interference of p38MAPK inhibited Bax expression and cytochrome c release. Altogether, our observations imply that genipin causes increased levels of Bax in response to p38MAPK signaling, which results in the initiation of mitochondrial death cascade, and therefore it holds promise as a potential chemotherapeutic agent for the treatment of H1299 cells.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Carcinoma, Non-Small-Cell Lung; Cell Survival; Cytochromes c; Dose-Response Relationship, Drug; Humans; Iridoids; Mitochondria; Molecular Targeted Therapy; p38 Mitogen-Activated Protein Kinases; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; Tumor Cells, Cultured

The progress in tissue regeneration is strongly dependent on the development of biocompatible materials with properties resembling those of a native tissue. Also, the application of noninvasive methods of delivering the scaffold into the tissue defect is of great importance. In this study we present a group of biopolymer-based materials as potential injectable scaffolds. In contrast to other studies involving collagen neutralization or additional incubation of gel in genipin solution, we propose collagen and collagen-chitosan gels crosslinked in situ with genipin. Since some parameters of the cells should be considered in the microscale, the steady-state fluorescence anisotropy was applied to study the microenvironment of the gels. To our knowledge we are the first to report on microrheological properties, such as gel time and microviscosity, for this group of hydrogels. Rapid gelation at physiological temperatures found makes these materials of special interest in applications requiring gel injectability. Physico-chemical investigation showed the influence of the crosslinking agent concentration and chitosan addition on the crosslinking degree, swelling ratio, gel microviscosity, and the degradation rate. Strong correlation was revealed between the surface wettability and the viability of cultured mesenchymal stem cells. Cytotoxicity studies indicated that the collagen-chitosan hydrogels showed the best biocompatibility.

Topics: Biopolymers; Cell Survival; Chitosan; Collagen; Cross-Linking Reagents; Fluorescence Polarization; Humans; Hydrogels; Injections; Iridoids; Materials Testing; Mesenchymal Stem Cells; Microscopy, Electron, Scanning; Regeneration; Tissue Engineering; Tissue Scaffolds; Viscosity

The immune response due to Galα1,3-Galβ1-4GlcNAc-R(α-Gal) epitopes is an important factor in bioprosthetic heart valve failure. The aim of this study was to evaluate the immune reaction and anticalcification effect of α-galactosidase and decellularization for glutaraldehyde (GA)/genipin fixed bovine pericardium using α1,3-galactosyltransferase knockout(α-Gal KO) mouse(C57BL/6). Bovine pericardial tissues were decellularized and treated with α-galactosidase before fixation with 0.25% GA/0.4% genipin in organic solvent (75% ethanol and 5% octanol) and treatment with glycine. The removal of α-gal epitope from the bovine pericardium was analyzed by 3,3'-Diaminobenzidine staining intensity. The bovine pericardial tissues were subcutaneously implanted into wild type mice (n=19) and α-Gal KO mice (n=66), which had been presensitized with rabbit red blood cells to maximize immunologic response or not, and anti α-Gal antibodies were measured at various time intervals. Calcium contents of the explanted tissues (n=104) were measured 3 months after implantation. The treatment of α-galactosidase effectively removed the α-gal epitopes expressed on bovine pericardial tissues. In both GA and genipin groups, titers for both anti α-Gal IgM and IgG of α-Gal KO mice increased according to the duration of implantation, and were lower in the groups with decellularization than without decellularization, and were lower in the groups with α-galactosidase+decellularization than with decellularization. The calcium contents of GA/genipin fixed tissues were lower in the groups with decellularization than without decellularization, and were lower in the groups with α-galactosidase+decellularization than with decellularization. Treatment of α-galactosidase with decellularization is useful for removal of the immunogenicity, and reduced calcification in both GA and genipin fixed bovine pericardia, supporting the hypothesis that the immune reaction may cause the calcification. Treatment of α-galactosidase has possible promise to enhance durability of bioprosthetic heart valve. To our knowledge, this is the first report that demonstrates the in vivo efficacy of α-galactosidase using presensitized α-Gal KO mouse to mimic the human immunologic environment.

Topics: alpha-Galactosidase; Animals; Bioprosthesis; Calcium; Cattle; Enzyme-Linked Immunosorbent Assay; Galactosyltransferases; Heart Valves; In Vitro Techniques; Iridoids; Mice; Mice, Inbred C57BL; Mice, Knockout; Pericardium

The feasibility of rat acellular spinal cord scaffolds for tissue engineering applications was investigated. Fresh rat spinal cords were decellularized and crosslinked with genipin (GP) to improve their structural stability and mechanical properties. The GP-crosslinked spinal cord scaffolds possessed a porous structure with an average pore diameter of 31.1 μm and a porosity of 81.5%. The resultant scaffolds exhibited a water uptake ratio of 229%, and moderate in vitro degradation rates of less than 5% in phosphate-buffered saline (PBS) and slightly more than 20% in trypsin-containing buffer, within 14 days. The ultimate tensile strength and elastic modulus of GP-crosslinked spinal cord scaffolds were determined to be 0.193±0.064 MPa and 1.541±0.082 MPa, respectively. Compared with glutaraldehyde (GA)-crosslinked acellular spinal cord scaffolds, GP-crosslinked scaffolds demonstrated similar microstructure and mechanical properties but superior biocompatibility as indicated by cytotoxicity evaluation and rat mesenchymal stem cell (MSC) adhesion behavior. Cells were able to penetrate throughout the crosslinked scaffold due to the presence of an interconnected porous structure. The low cytotoxicity of GP facilitated cell proliferation and extracellular matrix (ECM) secretion in vitro on the crosslinked scaffolds over 7 days. Thus, these GP-crosslinked spinal cord scaffolds show great promise for tissue engineering applications.

Topics: Animals; Biocompatible Materials; Cell Adhesion; Cell Survival; Cells, Cultured; Elastic Modulus; Glutaral; Iridoids; Mesenchymal Stem Cells; Porosity; Rats; Rats, Sprague-Dawley; Spinal Cord; Tissue Engineering; Tissue Scaffolds

To evaluate the effect of collagen cross-linking induced by genipin in porcine sclera.. Porcine cadaver eyes were treated with genipin at concentrations (by w/v) of 0.01%, 0.03%, 0.1%, 0.3%, 1.0% for 15 and 30 min. Riboflavin/ultraviolet A(UVA)-treated and untreated samples were used as controls. After treatment, scleral strips of 4.0 × 10.0 mm were cut. Twenty-four hours later, the stress-strain parameters of the strips were measured using a biomaterial microtester. The stress and Young's modulus at 8% strain were evaluated.. Compared with untreated groups, after treatment with genipin for 15 min, the stress was increased by 66-246%, depending on the concentration of genipin. As for the 30-min groups, the stress was 171-444% higher than that of the control. The difference of the Young's modulus between genipin 15-min groups, except the 0.01% groups (p = 0.095), also had statistical significance (p < 0.05). The Young's modulus had significant difference between the untreated group and the genipin 30-min groups (all p < 0.05). Of 0.3% genipin for 15 min or 0.01% genipin for 30 min had a similar stress-strain curve with those of eyes treated with the riboflavin/UVA group. The sclera exhibited a bluish colour which became deeper with increase concentration and cross-linking time.. Collagen cross-linking induced by genipin could increase the biomechanical strength in porcine sclera. The effect depends on the concentration and treatment time of genipin.

Topics: Adhesives; Animals; Collagen; Cross-Linking Reagents; Disease Models, Animal; Elasticity; Iridoids; Myopia; Sclera; Swine

Quantify changes in the flexion--extension neutral zone of the intervertebral disc with injections of increasing genipin concentration.. Bovine motion segments were treated with varying concentrations of genipin using bilateral injections of constant volume. After overnight static compression loading of the treated segments, anterior-posterior offset loading was used to simulate flexion-extension motion. Range of motion, neutral zone length, neutral zone stiffness, and an instability score were measured.. Injection of the disc annulus with increasing concentrations of genipin resulted in corresponding changes in flexion-extension neutral zone. A minimum concentration of 40 mM was needed to observe a significant change. The largest changes were observed with the 400 mM injection. Netural zone stability was the most sensitive of the metrics with a percent change of 48% at 40 mM and over 200% at 400 mM.. This study establishes the efficacy of using injection delivery to affect disc joint mechanics and quantifies the dose response between injected genipin and the flexion-extension stability of the disc.

Topics: Animals; Biomechanical Phenomena; Cattle; Dose-Response Relationship, Drug; Injections; Intervertebral Disc; Iridoids; Lumbar Vertebrae; Materials Testing; Pliability; Range of Motion, Articular

A direct one-pot production of genipin, an iridoid aglycone, from crude gardenia fruit was developed. The method relied on the use of single cellulase to disrupt plant cells and to cleave off sugar molecules simultaneously, thereby enhancing the release of intracellular iridoids and converting geniposide to genipin. During the biocatalysis, eco-friendly ethyl acetate was used to extract the product, providing the partial purification and the minimization of genipin degradation. By using 10 mg/mL cellulase and 24 h-incubation at 50 °C, pH 4, combined with in situ extraction, genipin with good purity was yielded at 58.83 mg/g, which increased 12.38 and 1.72 times compared with those obtained from the procedures without either the aid of enzyme or in situ extraction, respectively. Therefore, this integrated approach is promising for the production of genipin and should be potentially applied to the preparation of other plant aglycones.

Topics: Cellulase; Gardenia; Iridoid Glycosides; Iridoids; Plant Cells; Plant Extracts; Technology, Pharmaceutical

Chitosan microspheres have been explored for pharmaceutical applications, namely as a drug delivery systems for Helicobacter pylori gastric infection treatment, due to their mucoadhesive capacity. In this study, a different application of chitosan microspheres is proposed aiming the creation of an H. pylori-binding system where, after oral administration, microspheres will capture and remove these bacteria from infected patients, taking advantage of their muco/bacterial adhesive process. However, mucoadhesion is influenced by the degree of crosslinking necessary to avoid microspheres dissolution in the acidic gastric environment. During this work, the effect of genipin crosslinking on the stability, size, charge and mucoadhesive properties of chitosan microspheres under acidic pH was studied. Chitosan microspheres with ∼170 μm were produced by ionotropic gelation and subsequently covalently crosslinked with genipin in different degrees. The crosslinking reaction was followed by infrared spectroscopy and time-lapse fluorescence microscopy, since we have demonstrated that the fluorescence intensity of chitosan microspheres increases with genipin chemical bonding to chitosan. Results showed that both the zeta potential and the swelling capacity of chitosan microspheres decrease with increasing crosslinking. When immersed in simulated gastric fluid (SGF) with pepsin for 7 days, chitosan microspheres crosslinked with 10mM of genipin for 1h did not dissolve and doubled their size to approximately 345 μm. Furthermore, they maintained their in vitro mucoadhesion to soluble gastric mucins at both pH tested (3.6 and 6.5) and presented an in vivo retention time of around 2h in the stomach of C57BL/6 mice.

Topics: Adhesiveness; Administration, Oral; Animals; Anti-Bacterial Agents; Chemistry, Pharmaceutical; Chitosan; Cross-Linking Reagents; Drug Stability; Gastric Juice; Gastric Mucins; Helicobacter pylori; Hydrogen-Ion Concentration; Iridoids; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Microspheres; Particle Size; Pepsin A; Solubility; Spectroscopy, Fourier Transform Infrared; Stomach; Technology, Pharmaceutical; Time Factors; Time-Lapse Imaging

Rapid and controlled vascularization of engineered tissues remains one of the key limitations in tissue engineering applications. This study investigates the possible use of natural extracellular matrix-like scaffolds made of gelatin loaded with human vascular endothelial growth factor (VEGF), as a bioresorbable platform for long-term release and consequent angiogenic boosting. For this aim, gelatin was firstly electrospun and then cross-linked at two different concentrations (0.1% and 0.5% w/v) by using genipin, a low toxic agent, in order to fabricate a suitable substrate to be loaded with VEGF. Collected fibers were homogeneous and free of beads, the fibrous structure was retained after cross-linking. Mechanical properties were deeply affected by the chemical treatment showing a different behavior, depending on the testing conditions (i.e., dry or wet state). VEGF release was assessed by means of ELISA assay: a cumulative release of about 90% (0.1% w/v) and 60% (0.5% w/v) at 28 days was measured. Both VEGF loaded mats induced cell viability, endothelial differentiation and showed chemoattractive properties when tested on human mesenchymal stromal cells (hMSCs). In vitro and in vivo angiogenic assays demonstrated that the VEGF loaded mats induced an angiogenic potential in stimulating new vessel formation similar, if not superior, to fresh VEGF. VEGF retains bioactive and pro-angiogenic potential for up to 14 days. The results demonstrated that genipin cross-linked electrospun gelatin mats loaded with VEGF could be part of a useful strategy to stimulate and induce angiogenesis in tissue engineered applications.

Topics: Cells, Cultured; Gelatin; Humans; Iridoids; Mesenchymal Stem Cells; Microscopy, Electron, Scanning; Neovascularization, Physiologic; Tissue Engineering; Tissue Scaffolds; Vascular Endothelial Growth Factor A

Genipin-cross-linked hydrogels composed of biodegradable and pH-sensitive cationic poly(L-lysine) (PLL), poly(L-lysine)-block-poly(L-alanine) (PLL-b-PLAla), and poly(L-lysine)-block-polyglycine (PLL-b-PGly) polypeptides were synthesized, characterized, and used as carriers for drug delivery. These polypeptide hydrogels can respond to pH-stimulus and their gelling and mechanical properties, degradation rate, and drug release behavior can be tuned by varying polypeptide composition and cross-linking degree. Comparing with natural polymers, the synthetic polypeptides with well-defined chain length and composition can warrant the preparation of the hydrogels with tunable properties to meet the criteria for specific biomedical applications. These hydrogels composed of natural building blocks exhibited good cell compatibility and enzyme degradability and can support cell attachment/proliferation. The evaluation of these hydrogels for in vitro drug release revealed that the controlled release profile was a biphasic pattern with a mild burst release and a moderate release rate thereafter, suggesting the drug molecules were encapsulated inside the gel matrix. With the versatility of polymer chemistry and conjugation of functional moieties, it is expected these hydrogels can be useful for biomedical applications such as polymer therapeutics and tissue engineering.

Topics: 3T3 Cells; Animals; Bromelains; Compressive Strength; Cross-Linking Reagents; Doxorubicin; Drug Compounding; Fibroblasts; Freeze Drying; Hydrogels; Hydrogen-Ion Concentration; Iridoids; Mice; Microscopy, Electron, Scanning; Peptides; Polylysine; Spectroscopy, Fourier Transform Infrared; Time Factors

An in situ study using whole-organ culture system.. To study the effect of disc degeneration at different stages on its rheological and dynamic properties and to investigate the efficacy of exogenous cross-linking therapy.. Disc degeneration can involve protein denaturation or microdefects to the disc's collagen fiber network. A disc degeneration model using whole-organ culture technique can be effectively used for the screening of treatments of degenerated discs. Exogenous cross-linking therapy has been shown to enhance the mechanical properties of the disc by cross-linking collagen. However, the efficacy of this therapy on the degenerated disc is unclear.. A total of 40 porcine thoracic discs were assigned to 5 groups: intact discs, moderately degenerated discs, moderately degenerated discs with cross-linker augmentation, severely degenerated discs, and severely degenerated discs with cross-linker augmentation. The disc degeneration was simulated by trypsin digestion and mechanical fatigue loading. Rheological properties, dynamic properties, water content, and histological analysis were conducted after a 7-day incubation.. The mechanical properties of moderate degenerated discs significantly decrease both in rheological and dynamic properties, and laminate structure disorganization was observed. Mechanical defects of severely degenerated discs resulted in disc height loss, an increase in the aggregate modulus and stiffness modulus, and a decrease in the damping coefficient, hydraulic permeability, and water content. Cross-linker augmentation significantly recovered mechanical properties of moderately degenerated discs and restored the water content compared with the intact disc. However, the augmentation did not fully repair the severely degenerated discs.. Trypsin-induced extracellular matrix damage resulted in a change of the disc's biomechanics. Cross-linker augmentation recovers the rheological and dynamic properties of moderately degenerated discs but not of the severely degenerated discs. The genipin cross-linker may be able to improve the proteoglycan depletion effect in the nucleus pulposus but may not be effective to restore the structural damage in the collagen molecule of the anulus fibrosus.

Topics: Animals; Biomechanical Phenomena; Collagen; Computer Simulation; Cross-Linking Reagents; Intervertebral Disc; Intervertebral Disc Degeneration; Iridoids; Models, Biological; Organ Culture Techniques; Orthopedic Procedures; Proteoglycans; Rheology; Severity of Illness Index; Stress, Mechanical; Swine; Thoracic Vertebrae; Treatment Outcome; Trypsin

Solid wastes generated from the seafood industry represent an important environmental pollutant; therefore, utilization of those wastes for the development of processing biochemical tools could be an attractive and clean solution for the seafood industry. This study reports the immobilization of semi-purified acidic proteases from Monterey sardine stomachs onto chitin and chitosan materials extracted from shrimp head waste. Several supports (chitosan beads, chitosan flakes, and partially deacetylated flakes) were activated either with genipin or Na-tripolyphosphate and evaluated as a mean to immobilize acidic proteases. The protein load varied within the 67-91% range on different supports. The immobilization systems based on chitosan beads achieved the highest protein loads but showed the lowest retained catalytic activities. The best catalytic behavior was obtained using partially deacetylated chitin flakes activated either with genipin or Na-tripolyphosphate. According to results, the immobilization matrix structure, as well as acetylation degree of chitin-chitosan used, has considerable influence on the catalytic behavior of immobilized proteases. Partially deacetylated chitin flakes represent a suitable option as support for enzyme immobilization because its preparation requires fewer steps than other supports. Two abundant seafood by-products were used to obtain a catalytic system with enough proteolytic activity to be considered for biotechnological applications in diverse fields.

Topics: Animals; Biotechnology; Chitin; Chitosan; Enzymes, Immobilized; Fishes; Industrial Waste; Iridoids; Penaeidae; Peptide Hydrolases; Polyphosphates

Helicobacter pylori (H. pylori) colonizes the gastric mucosa of over 50% of the world population, causing several pathologies, such as gastric ulcers and gastric cancer. Since current antibiotic treatments are inefficient in 20% of cases alternative therapies are needed. This work reports the ability of chitosan microspheres to adhere to H. pylori and prevent/remove H. pylori colonization. Adhesion of H. pylori strains with different functional adhesins (BabA and/or SabA) to chitosan microspheres (diameter 167 ± 27 μm) occurs at both pH 2.6 and 6.0, but is higher at pH 6.0. Bacterial adhesion to a gastric cell line expressing sialylated carbohydrates (SabA receptors) was performed at the same pH values using H. pylori strains with and without SabA. At both pH values addition of microspheres to gastric cells before and after pre-incubation with H. pylori decreased bacterial adhesion to cells. Furthermore, the chitosan microspheres were non-cytotoxic. These findings reveal the potential of chitosan microspheres as an alternative or complementary treatment for H. pylori gastric eradication or prevention of H. pylori colonization.

Topics: Adhesins, Bacterial; Animals; Bacterial Adhesion; Cell Line, Tumor; Cell Survival; Chitosan; Cross-Linking Reagents; Fluorescein-5-isothiocyanate; Gastric Mucosa; Helicobacter Infections; Helicobacter pylori; Humans; Hydrogen-Ion Concentration; Iridoids; Microscopy, Electron, Scanning; Microspheres; Particle Size

The abilities of two natural products, genipin and lawsone, to enhance blood contaminated fingermarks on papers of various porosities and colour were investigated and compared to the routinely used amino acid reagents, ninhydrin and 1,8-diazafluoren-9-one (DFO). Fingermarks in blood were deposited as a split depletion series on various paper types and colours for ageing periods of 6 weeks, 4 weeks, 2 weeks and 1 week before enhancement. The developed marks were observed under different lighting conditions, recorded and graded by way of attributing quantitative data to each series. Results indicated that while genipin showed some potential as a reagent for the enhancement of latent fingermarks, it displayed no suitability for the enhancement of fingermarks in blood on paper. Lawsone also failed to successfully enhance either type of fingermark. Upon comparison of the results with those of ninhydrin and DFO it was found that ninhydrin displayed the highest success rate of development of these marks.

Topics: Aza Compounds; Blood; Coloring Agents; Dermatoglyphics; Humans; Indicators and Reagents; Iridoids; Naphthoquinones; Ninhydrin; Paper

Like other CNS neurons, mature retinal ganglion cells (RGCs) are unable to regenerate their axons after nerve injury due to a diminished intrinsic regenerative capacity. One of the reasons why they lose the capacity for axon regeneration seems to be associated with a dramatic shift in RGCs' program of gene expression by epigenetic modulation. We recently reported that (1R)-isoPropyloxygenipin (IPRG001), a genipin derivative, has both neuroprotective and neurite outgrowth activities in murine RGC-5 retinal precursor cells. These effects were both mediated by nitric oxide (NO)/S-nitrosylation signaling. Neuritogenic activity was mediated by S-nitrosylation of histone deacetylase-2 (HDAC2), which subsequently induced retinoic acid receptor β (RARβ) expression via chromatin remodeling in vitro. RARβ plays important roles of neural growth and differentiation in development. However, the role of RARβ expression during adult rat optic nerve regeneration is not clear. In the present study, we extended this hypothesis to examine optic nerve regeneration by IPRG001 in adult rat RGCs in vivo. We found a correlation between RARβ expression and neurite outgrowth with age in the developing rat retina. Moreover, we found that IPRG001 significantly induced RARβ expression in adult rat RGCs through the S-nitrosylation of HDAC2 processing mechanism. Concomitant with RARβ expression, adult rat RGCs displayed a regenerative capacity for optic axons in vivo by IPRG001 treatment. These neuritogenic effects of IPRG001 were specifically suppressed by siRNA for RARβ. Thus, the dual neuroprotective and neuritogenic actions of genipin via S-nitrosylation might offer a powerful therapeutic tool for the treatment of RGC degenerative disorders.

Topics: Animals; Cell Survival; Gene Expression; Iridoids; Male; Nerve Regeneration; Nitric Oxide Synthase Type I; Optic Nerve; Rats; Rats, Sprague-Dawley; Receptors, Retinoic Acid; Retina; Retinal Ganglion Cells; RNA, Small Interfering

Vascular smooth muscle cell (VSMC) proliferation and migration triggered by inflammatory stimuli contributes importantly to the pathogenesis of atherosclerosis and restenosis. On the other hand, genipin, an aglycon of geniposide, exhibits diverse pharmacological functions such as antitumor and anti-inflammatory effects. The protective effects of genipin on the cardiovascular system have also been reported. However, the molecular mechanism involved remains unknown. This study aimed to elucidate the precise function of genipin in VSMCs, focusing particularly on the role of heme oxygenase-1 (HO-1), a potent anti-inflammatory enzyme. We found that pretreatment of genipin induced HO-1 mRNA and protein levels, as well as its activity in VSMCs. Genipin inhibited TNF-α-induced VSMC proliferation and migration in a dose-dependent manner. At the molecular level, genipin prevented ERK/MAPK and Akt phosphorylation while left p38 MAPK and JNK unchanged. Genipin also blocked the increase of ROS generation induced by TNF-α. More importantly, the specific HO-1 siRNA partially abolished the beneficial effects of genipin on VSMCs. These results suggest that genipin may serve as a novel drug in the treatment of these pathologies by inducing HO-1 expression/activity and subsequently decreasing VSMC proliferation and migration.

Topics: Animals; Cell Movement; Cell Proliferation; Cholagogues and Choleretics; Enzyme Induction; Extracellular Signal-Regulated MAP Kinases; Heme Oxygenase-1; Iridoids; Male; Muscle, Smooth, Vascular; Myocytes, Smooth Muscle; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Tumor Necrosis Factor-alpha

The possibility to fabricate marine collagen porous structures crosslinked with genipin under high pressure carbon dioxide is investigated. Collagen from shark skin is used to prepare pre-scaffolds by freeze-drying. The poor stability of the structures and low mechanical properties require crosslinking of the structures. Under dense CO2 atmosphere, crosslinking of collagen pre-scaffolds is allowed for 16 h. Additionally, the hydrogels are foamed and the scaffolds obtained present a highly porous structure. In vitro cell culture tests performed with a chondrocyte-like cell line show good cell adherence and proliferation, which is a strong indication of the potential of these scaffolds to be used in tissue cartilage tissue engineering.

Topics: Animals; Carbon Dioxide; Cell Adhesion; Cell Line; Cell Proliferation; Chondrocytes; Collagen; Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Freeze Drying; Hydrogels; Iridoids; Materials Testing; Mice; Porosity; Sharks; Skin; Tissue Engineering; Tissue Scaffolds

A genipin-cross-linked chitosan/graphene oxide (GCS/GO) composite film was prepared using a solution casting method. Fourier transform infrared (FTIR) and ultraviolet-visible (UV-Vis) spectroscopy of the composite films showed that the interactions between the CS and oxygen-containing groups of GO resulted in good dispersion of the GO sheets in the CS network. The addition of GO decreased the expansion ratio of the composite films in physiological conditions and increased the resistance to degradation by lysozymes in vitro. As well, the tensile strength values of the GCS/GO films were significantly increased with the increasing load of GO. Moreover, the GCS/GO composite film also maintained the intrinsic fluorescence of GCS. The in vitro cell study results revealed that the composite films were suitable for the proliferation and adhesion of mouse preosteoblast (MC3T3-E1) cells. The GCS/GO biocomposite films might have a potential use in tissue engineering, bioimaging, and drug delivery.

Topics: 3T3 Cells; Absorbable Implants; Animals; Biocompatible Materials; Cell Survival; Fluorescent Dyes; Graphite; Iridoids; Membranes, Artificial; Mice; Oxides

Insulin resistance (IR) increases with age and plays a key role in the pathogenesis of type 2 diabetes mellitus. Oxidative stress and mitochondrial dysfunction are supposed to be major factors leading to age-related IR. Genipin, an extract from Gardenia jasminoides Ellis fruit, has been reported to stimulate insulin secretion in pancreatic islet cells by regulating mitochondrial function. In this study, we first investigated the effects of genipin on insulin sensitivity and the potential mitochondrial mechanisms in the liver of aging rats. The rats were randomly assigned to receive intraperitoneal injections of either 25mg/kg genipin or vehicle once daily for 12days. The aging rats showed hyperinsulinemia and hyperlipidemia, and insulin resistance as examined by the decreased glucose decay constant rate during insulin tolerance test (kITT). The hepatic tissues showed steatosis and reduced glycogen content. Hepatic malondialdehyde level and mitochondrial reactive oxygen species (ROS) were higher, and levels of mitochondrial membrane potential (MMP) and ATP were lower as compared with the normal control rats. Administration of genipin ameliorated systemic and hepatic insulin resistance, alleviated hyperinsulinemia, hyperglyceridemia and hepatic steatosis, relieved hepatic oxidative stress and mitochondrial dysfunction in aging rats. Furthermore, genipin not only improved insulin sensitivity by promoting insulin-stimulated glucose consumption and glycogen synthesis, inhibited cellular ROS overproduction and alleviated the reduction of levels of MMP and ATP, but also reversed oxidative stress-associated JNK hyperactivation and reduced Akt phosphorylation in palmitate-treated L02 hepatocytes. In conclusion, genipin ameliorates age-related insulin resistance through inhibiting hepatic oxidative stress and mitochondrial dysfunction.

Topics: Adenosine Triphosphate; Age Factors; Aging; Animals; Antioxidants; Cell Line; Dose-Response Relationship, Drug; Enzyme Activation; Fatty Liver; Hepatocytes; Hyperinsulinism; Hyperlipidemias; Hypoglycemic Agents; Insulin Resistance; Iridoids; JNK Mitogen-Activated Protein Kinases; Liver; Male; Malondialdehyde; Membrane Potential, Mitochondrial; Mitochondria, Liver; Oxidative Stress; Palmitic Acid; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species

The purpose of this work was to investigate the anti-depressant effect of genipin and its mechanisms using (1)H-NMR spectroscopy and multivariate data analysis on a chronic unpredictable mild stress (CUMS) rat model. Rat serum and urine were analyzed by nuclear magnetic resonance (NMR)-based metabonomics after oral administration of either genipin or saline for 2 weeks. Significant differences in the metabolic profile of the CUMS-treated group and the control group were observed, which were consistent with the results of behavioral tests. Metabolic effects of CUMS included decreases in serum trimetlylamine oxide (TMAO) and β-hydroxybutyric acid (β-HB), and increases in lipid, lactate, alanine and N-acetyl-glycoproteins. In urine, decreases in creatinine and betaine were observed, while citrate, trimethylamine (TMA) and dimethylamine were increased. These changes suggest that depression may be associated with gut microbes, energy metabolism and glycometabolism. Genipin showed the best anti-depressive effects at a dose of 100 mg/kg in rats. These results indicate that metabonomic approaches could be powerful tools for the investigation of the biochemical changes in pathological conditions or drug treatment.

Topics: Animals; Antidepressive Agents; Cholagogues and Choleretics; Chronic Disease; Dose-Response Relationship, Drug; Iridoids; Magnetic Resonance Spectroscopy; Male; Metabolome; Metabolomics; Rats; Rats, Sprague-Dawley; Stress, Psychological

Meniscus lesions are frequently occurring injuries with poor ability to heal. Typical treatment procedure includes removal of damaged regions, which can lead to sub-optimal knee biomechanics and early onset of osteoarthritis. Some of the drawbacks of current treatment approach present an opportunity for a tissue engineering solution. In this study, gelatin (G)/chitosan (Cs) scaffolds were synthesized via gel casting method and cross-linked with naturally derived cross-linker, genipin, through scaffold cross-linking method. Based on the characteristics of native meniscus tissue microstructure and function, three different layers were chosen to design the macroporous multilayered scaffolds. The multi-layered scaffolds were investigated for their ability to support human-derived meniscus cells by evaluating their morphology and proliferation using MTT assay at various time points. Based on structural, mechanical and cell compatibility considerations, laminated scaffolds composed of G60/Cs40, G80/Cs20 and G40/Cs60 samples, for the first, second and third layers, respectively, could be an appropriate combination for meniscus tissue engineering applications.

Topics: Adult; Biocompatible Materials; Cell Proliferation; Cell Survival; Cells, Cultured; Chitosan; Gelatin; Humans; Hydrogel, Polyethylene Glycol Dimethacrylate; Iridoids; Male; Menisci, Tibial; Models, Anatomic; Tissue Engineering; Tissue Scaffolds

The genipin-cross-linked silk sericin/poly(vinyl alcohol) (PVA) films were developed aiming to be applied as two-dimensional wound dressings for the treatment of superficial wounds. The effects of genipin cross-linking concentration on the physical and biological properties of the films were investigated. The genipin-cross-linked silk sericin/PVA films showed the increased surface density, tensile strength, and percentage of elongation, but decreased percentage of light transmission, water vapor transmission rate, and water swelling, compared to the non-cross-linked films. This explained that the cross-linking bonds between genipin and silk sericin would reduce the mobility of molecular chains within the films, resulting in the more rigid molecular structure. Silk sericin was released from the genipin-cross-linked films in a sustained manner. In addition, either L929 mouse fibroblast or HaCat keratinocyte cells showed high percentage of viability when cultured on the silk sericin/PVA films cross-linked with 0.075 and 0.1% w/v genipin. The in vivo safety test performed according to ISO 10993-6 confirmed that the genipin-cross-linked silk sericin/PVA films were safe for the medical usages. The efficacy of the films for the treatment of superficial skin wounds will be further investigated in vivo and clinically. The genipin-cross-linked silk sericin/PVA films would be promising choices of two-dimensional wound dressings for the treatment of superficial wounds.

Topics: Animals; Bandages; Biocompatible Materials; Iridoids; Mice; Polyvinyl Alcohol; Sericins; Silk; Wound Healing

The macroscopic viscoelastic behavior of collagen gel was studied through relaxation time distribution spectrum obtained from stress relaxation tests and viscoelastic constitutive modeling. Biaxial stress relaxation tests were performed to characterize the viscoelastic behavior of collagen gel crosslinked with Genipin solution. Relaxation time distribution spectrum was obtained from the stress relaxation data by inverse Laplace transform. Peaks at the short (0.3 s-1 s), medium (3 s-90 s), and long relaxation time (>200 s) were observed in the continuous spectrum, which likely correspond to relaxation mechanisms involve fiber, inter-fibril, and fibril sliding. The intensity of the long-term peaks increases with higher initial stress levels indicating the engagement of collagen fibrils at higher levels of tissue strain. We have shown that the stress relaxation behavior can be well simulated using a viscoelastic model with viscous material parameters obtained directly from the relaxation time spectrum. Results from the current study suggest that the relaxation time distribution spectrum is useful in connecting the macro-level viscoelastic behavior of collagen matrices with micro-level structure changes.

Topics: Collagen Type I; Elasticity; Finite Element Analysis; Gels; Iridoids; Stress, Mechanical; Viscosity

Bone TE uses a scaffold either to induce bone formation from surrounding tissue or to act as a carrier or template for implanted bone cells or other agents. We prepared different bone tissue constructs based on collagen, gelatin and hydroxyapatite using genipin as cross-linking agent. The fabricated construct did not present a release neither of collagen neither of genipin over its toxic level in the surrounding aqueous environment. Each scaffold has been mechanically characterized with compression, swelling and creep tests, and their respective viscoelastic mechanical models were derived. Mechanical characterization showed a practically elastic behavior of all samples and that compressive elastic modulus basically increases as content of HA increases, and it is strongly dependent on porosity and water content. Moreover, by considering that gradients in cellular and extracellular architecture as well as in mechanical properties are readily apparent in native tissues, we developed discrete functionally graded scaffolds (discrete FGSs) in order to mimic the graded structure of bone tissue. These new structures were mechanically characterized showing a marked anisotropy as the native bone tissue. Results obtained have shown FGSs could represent valid bone substitutes.

Topics: Bone and Bones; Cell Line, Tumor; Cell Survival; Collagen; Compressive Strength; Durapatite; Elastic Modulus; Humans; Iridoids; Materials Testing; Mechanical Phenomena; Microscopy, Electron, Scanning; Porosity; Spectrometry, X-Ray Emission; Tissue Scaffolds

Porous scaffolds for dermal tissue engineering were fabricated by freeze-drying a mixture of chitosan and gelatin (CG) solutions. Different crosslinking agents including glutaraldehyde, 1-(3-dimethylaminopropyl)-3-ethyl-carbodimide hydrochloride (EDC), and genipin were used to crosslink the scaffolds and improve their biostability. The porous structure and mechanical properties were determined for the scaffolds. The proliferation of human fibroblasts in the scaffolds was analyzed. It was found that EDC crosslinked scaffolds had the greatest amount of cells after four days. EDC crosslinked CG scaffolds had tensile modulus in a dry state and compressive modulus in a wet state similar to commercial collagen wound dressing. They also showed appropriate pore size, high water absorption, and good dimensional stability during cell culture. When human fibroblasts were seeded on acellular porcine dermis (APD), acellular human dermis (AHD), and CG scaffolds for 3D cell culture, they were well-distributed in the centre of the CG scaffolds but stayed only on the superficial layer of APD or AHD after seven days. A gelatin-based bioglue was applied to the CG scaffolds where the keratinocytes were seeded to mimic epidermal structure. After 14 days, the bioglue degraded and keratinocytes grew to form monolayers on the scaffolds. This study showed that CG scaffolds crosslinked by EDC and seeded with human fibroblasts could serve as dermal constructs, while the bioglue coating seeded with keratinocytes could serve as an epidermal construct. Such a combination could help regenerate skin with integrated dermal and epidermal layers and a have potential use in tissue-engineered skin.

Topics: Absorption; Carbodiimides; Cell Culture Techniques; Chitosan; Cross-Linking Reagents; Dermis; Dimethylamines; Fibroblasts; Freeze Drying; Gelatin; Glutaral; Humans; Iridoids; Keratinocytes; Stress, Mechanical; Tissue Engineering; Tissue Scaffolds; Water

Gamma irradiation is a proven sterilization method, but is not widely used on allografts for anterior cruciate ligament reconstruction (e.g., patella tendon) due to radiation-induced decreases in mechanical strength. Addressing this drawback would improve the safety and supply of allografts to meet current and future demand. It was hypothesized that genipin-induced collagen cross-linking would increase the tensile modulus of patella tendon tissue such that 5 MRad gamma irradiation would not reduce the tissue mechanical strength below the original untreated values. Optimized genipin treatment increased the tensile modulus of bovine tendons by ~2.4-fold. After irradiation, genipin treated tissue did not significantly differ from native tissue, proving the hypothesis. Optimized genipin treatment of human tendons increased the tensile modulus by ~1.3-fold. After irradiation, both control and genipin-treated tissues possessed ~50-60% of their native tendon modulus, disproving the hypothesis. These results highlight possible age- and species- dependent effects of genipin cross-linking on tendon tissue. Cross-linking of human allografts may be beneficial only in younger donor tissues. Future research is warranted to better understand the mechanisms and applications of collagen cross-linking for clinical use.

Topics: Animals; Cattle; Cell Death; Cross-Linking Reagents; Female; Humans; Iridoids; Male; Middle Aged; Patellar Ligament; Radiation-Protective Agents; Tensile Strength; Time Factors

Polymeric genipin macromers, prepared by ring-opening polymerization at various pH values, are used as crosslinking agents to fix collagen hydrogels. The results indicate that as the dark color of polymeric genipin itself and the networks formed by long-range intermolecular crosslinking, the genipin-fixed collagen hydrogels displace darker color. The polymeric genipin prepared at higher pH value needs longer time to fully crosslink with collagen molecules. Moreover, polymerization of genipin reduces the yield of genipin-fixed collagen hydrogels due to low extent of crosslinking. Specially, the microscope photographs present the porous networks structures of genipin-fixed collagen hydrogels. The pore size increases with the increase in polymerization degree of genipin. The data of FTIR indicate the likely transition of -NH(2) groups in collagen chains into C=N. Owning to much more number of hydrophilic groups and more porous networks, collagen hydrogels fixed by genipin with higher polymerization degree have higher water absorption capacity. The equilibrium swelling of genipin-fixed collagen hydrogels is pH-responsive, which show "M" type changes with the pH values. The results obtained in the study suggest that the polymeric genipin prepared at various pH values lead to significant influence to the crosslinking characteristics and properties of collagen hydrogels.

Topics: Animals; Cattle; Collagen; Cross-Linking Reagents; Hydrogel, Polyethylene Glycol Dimethacrylate; Iridoids; Polymerization; Solutions; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared; Surface Plasmon Resonance; Time Factors

The aim of this study was to develop a biocompatible monolayer substrate based on fibrin and chitosan for in vitro culture of chondrocytes. Fibrin-chitosan composite substrates combined the proved cell adhesion properties of fibrin with the hydrophilicity and poor adhesion capacity of chitosan. Chitosan microspheres were produced by coacervation method, agglomerated within a fibrin network and subsequently crosslinked with genipin. The composite substrate was stable for 28 days of culture due to the high crosslinking density. Human chondrocytes cultured on the composite substrate were viable during the culture period. At the end of culture time (28 days) the composite substrate showed low cellular proliferation, 41% more collagen type II and 13% more production of sulfated glycosaminoglycans with respect to the amounts found at 14 days. The study revealed that dedifferentiated chondrocytes cultured in monolayer on the composite substrate can re-acquire characteristics of differentiated cells without using three-dimensional substrates or chondrogenic media.

Topics: Cell Culture Techniques; Cell Shape; Cell Survival; Cells, Cultured; Chitosan; Chondrocytes; Cross-Linking Reagents; DNA; Extracellular Matrix; Fibrin; Humans; Iridoids

Terminal sterilization of musculoskeletal allografts by gamma radiation minimizes the risk of disease transmission but impairs allograft mechanical properties. Commonly employed crosslinking agents can sterilize tissues without affecting mechanical properties adversely; however, these agents are toxic. Genipin is reported to be a benign crosslinking agent that strengthens mechanical properties of tissues; however, the antimicrobial capacity of genipin is largely unknown. The present study's aims were: (1) to assess the sporicidal potential of genipin, (2) to improve antimicrobial capacity by changing chemical and physical treatment conditions. To establish genipin's sterilization potential Bacillus subtilis var. niger spore strips were treated with 0-10% genipin in PBS or in 1:1 DMSO:PBS up to 72 h at room temperature (RT). Sterilizing doses and concentrations of genipin were used to treat B. pumilus and Geobacillus stearothermophilus spores to assess broader spectrum sporicidal activity of genipin. Scanning electron microscopy (SEM) was performed to evaluate gross morphological changes after genipin treatment. Optimal sterilization conditions were determined by evaluating the effects of temperature (RT-50 °C), DMSO:PBS ratio (0:100-100:0), and treatment duration (24-72 h) on B. subtilis. Genipin penetration of full thickness bovine patellar tendon and cortical bone specimens was observed to assess the feasibility of the agent for treating grafts. Initial studies showed that after 72 h of treatment at RT with 0.63-10% genipin/DMSO:PBS B. subtilis spore strips were sterilized; 0.63% genipin/PBS did not sterilize spore strips at 72 h at RT. Genipin doses and concentrations that sterilized B. subtilis spore strips sterilized B. pumilus and G. stearothermophilus spore strips. SEM revealed no gross morphological differences between untreated and treated spores. Treatment optimization resulted in sterilization within 24 h with 100% PBS, and DMSO facilitated sporicidal activity. Genipin penetrated full thickness patellar tendon specimens and 3.72 ± 0.58 mm in cortical bone specimens. Genipin sterilizes B. subtilis, B. pumilus, and G. stearothermophilus spore strips. It penetrates soft and hard tissues at doses previously shown to be non-toxic and to improve mechanical strength in collagen-rich soft tissues. Further studies are indicated to assess genipin's effects on the mechanical properties of genipin-sterilized grafts, the ability of genipin to eradicate infe

Topics: Allografts; Animals; Bacillus subtilis; Biocompatible Materials; Cattle; Geobacillus stearothermophilus; Iridoids; Spores, Bacterial; Sterilization; Tendons

The aim of our work is to utilize the crosstalk between the vascular and the neuronal system to enhance directed neuritogenesis in uniaxial guidance scaffolds for the repair of spinal cord injury. In this study, we describe a method for angioneural regenerative engineering, i.e., for generating biodegradable scaffolds, produced by a combination of controlled freezing (freeze-casting) and lyophilization, which contain longitudinally oriented channels, and provide uniaxial directionality to support and guide neuritogenesis from neuronal cells in the presence of endothelial cells. The optimized scaffolds, composed of 2.5 % gelatin and 1 % genipin crosslinked, were characterized by an elastic modulus of ~51 kPa and longitudinal channels of ~50 μm diameter. The scaffolds support the growth of endothelial cells, undifferentiated or NGF-differentiated PC12 cells, and primary cultures of fetal chick forebrain neurons. The angioneural crosstalk, as generated by first forming endothelial cell monolayers in the scaffolds followed by injection of neuronal cells, leads to the outgrowth of long aligned neurites in the PC12/endothelial cell co-cultures also in the absence of exogenously added nerve growth factor. Neuritogenesis was not observed in the scaffolds in the absence of the endothelial cells. This methodology is a promising approach for neural tissue engineering and may be applicable for regenerative spinal cord injury repair.

Topics: Animals; Chick Embryo; Elastic Modulus; Endothelial Cells; Freeze Drying; Gelatin; Iridoids; Nerve Growth Factor; Neural Stem Cells; Neurogenesis; Neurons; PC12 Cells; Rats; Spinal Cord Injuries; Tissue Engineering; Tissue Scaffolds

The roles of uncoupling protein-2 (UCP2) on the androgen synthesis of granulosa cells derived from patients with polycystic ovary syndrome (PCOS) and normal subjects were explored. Primary human granulosa cells from 18 patients who received in vitro fertilization (IVF) were examined; nine patients had PCOS with hyperandrogenism. Primary cultures were treated with genipin, a proton leak inhibitor, guanosine diphosphate (GDP), an UCP inhibitor, and triiodothyronine (T3), an inducer of UCP gene expression. Mitochondrial membrane potential was determined using the JC-1 assay. T3 induced P450scc and UCP2 expressions and testosterone synthesis in both normal and PCOS granulosa cells. Their expressions in response to T3 treatments were correlated in the PCOS group. Differences in testosterone synthesis were observed between normal and PCOS cells in response to genipin. Increased mitochondrial membrane potential was observed in response to genipin and GDP; while T3 decreased it. Increased ovarian UCP2 expression in response to T3 treatment in PCOS may alter pregnenolone synthesis by influencing P450scc expression, thus altering testosterone production. Further in vivo studies are necessary to fully elucidate the role of UCP2 in the hyperandrogenism commonly observed in PCOS.

Topics: Adult; Aromatase; Cells, Cultured; Female; Granulosa Cells; Guanosine Diphosphate; Humans; Hyperandrogenism; Ion Channels; Iridoids; Membrane Potential, Mitochondrial; Mitochondrial Proteins; Polycystic Ovary Syndrome; Testosterone; Triiodothyronine; Uncoupling Protein 2

The use of chitosan films has been limited due to their high degradability in aqueous acidic media. In order to produce chitosan films with high antioxidant activity and insoluble in acid solutions caffeic acid was grafted to chitosan by a radical mechanism using ammonium cerium (IV) nitrate (60 mM). Genipin was used as cross-linker. This methodology originated films with 80% higher antioxidant activity than the pristine film. Also, these films only lost 11% of their mass upon seven days immersion into an aqueous solution at pH 3.5 under stirring. The films surface wettability (contact angle 105°), mechanical properties (68 MPa of tensile strength and 4% of elongation at break), and thermal stability for temperatures lower than 300 °C were not significantly influenced by the covalent linkage of caffeic acid and genipin to chitosan. Due to their characteristics, mainly higher antioxidant activity and lower solubility, these are promising materials to be used as active films.

Topics: Antioxidants; Caffeic Acids; Chitosan; Drug Stability; Glucosamine; Hydrogen-Ion Concentration; Iridoids; Mechanical Phenomena; Solubility; Surface Properties; Temperature

Autologous cell-based tissue engineering using three-dimensional scaffolds holds much promise for the repair of cartilage defects. Previously, we reported on the development of a porous scaffold derived solely from native articular cartilage, which can induce human adipose-derived stem cells (ASCs) to differentiate into a chondrogenic phenotype without exogenous growth factors. However, this ASC-seeded cartilage-derived matrix (CDM) contracts over time in culture, which may limit certain clinical applications. The present study aimed to investigate the ability of chemical crosslinking using a natural biologic crosslinker, genipin, to prevent scaffold contraction while preserving the chondrogenic potential of CDM. CDM scaffolds were crosslinked in various genipin concentrations, seeded with ASCs, and then cultured for 4 weeks to evaluate the influence of chemical crosslinking on scaffold contraction and ASC chondrogenesis. At the highest crosslinking degree of 89%, most cells failed to attach to the scaffolds and resulted in poor formation of a new extracellular matrix. Scaffolds with a low crosslinking density of 4% experienced cell-mediated contraction similar to our original report on noncrosslinked CDM. Using a 0.05% genipin solution, a crosslinking degree of 50% was achieved, and the ASC-seeded constructs exhibited no significant contraction during the culture period. Moreover, expression of cartilage-specific genes, synthesis, and accumulation of cartilage-related macromolecules and the development of mechanical properties were comparable to the original CDM. These findings support the potential use of a moderately (i.e., approximately one-half of the available lysine or hydroxylysine residues being crosslinked) crosslinked CDM as a contraction-free biomaterial for cartilage tissue engineering.

Topics: Adipocytes; Cells, Cultured; Chondrogenesis; Cross-Linking Reagents; Drug Implants; Equipment Design; Equipment Failure Analysis; Extracellular Matrix; Humans; Iridoids; Nanomedicine; Stem Cells; Tissue Scaffolds

Gardenia jasminoides J. Ellis (Rubiaceae) is a shrub tree species distributed all over the world. Now its pharmacological activities such as anti-atherosclerosis have been extensively studied.. To offer pharmacological proof for its further clinical application in cardiovascular diseases, the antithrombotic activities of the aqueous extract of G. jasminoides (GJ-ext) were studied in mouse and rat models.. GJ-ext was administrated orally to detect the effects on the models of carrageenan-induced tail thrombosis and arteriovenous shunt thrombosis. The effects of GJ-ext and geniposide (p.o.) on antiplatelet aggregation were examined. Geniposide and genipin were studied on venous thrombosis by oral administration.. GJ-ext (67, 133 and 266 mg/kg) and aspirin (50 mg/kg), respectively, decreased the length of tail thrombus with average thrombus inhibition rate of 21.9, 55.7, 65.8 and 57.6% at 48 h and 19.0, 54.5, 69.3 and 56.9% at 72 h after carrageenan injection and, meanwhile, improved thrombosis induced by arteriovenous shunt (silk thread) with 36.3, 45.5, 86.4 and 63.7% inhibition rate of thrombus respectively, and the ED(50) of GJ-ext was 160.8 mg/kg. Furthermore, GJ-ext (67 mg/kg) and geniposide (20 mg/kg) significantly inhibited platelet aggregation induced by thrombin/collagen with 45.1%/19.3% and 52.8%/26.2% aggregation rate. Geniposide (10-40 mg/kg) and genipin (5-20 mg/kg) inhibited venous thrombosis induced by tight ligation of the inferior vena cava, their ED(50) values were 18.4 and 8.6 mg/kg, respectively.. This study indicated that GJ-ext and geniposide demonstrated remarkable antithrombotic activities and supported their therapeutic uses for thrombotic diseases.

Topics: Administration, Oral; Animals; Arteriovenous Shunt, Surgical; Aspirin; Carrageenan; Disease Models, Animal; Dose-Response Relationship, Drug; Fibrinolytic Agents; Fruit; Gardenia; Iridoids; Male; Mice; Mice, Inbred ICR; Plant Extracts; Plants, Medicinal; Platelet Aggregation; Platelet Aggregation Inhibitors; Rats; Rats, Sprague-Dawley; Solvents; Time Factors; Venous Thrombosis; Water

Mitochondrial uncoupling protein 2 (UCP2) can moderate oxidative stress by favoring the influx of protons into the mitochondrial matrix, thus reducing electron leakage from respiratory chain and mitochondrial superoxide production. Here, we demonstrate that UCP2 inhibition by genipin or UCP2 siRNA strongly increases reactive oxygen species (ROS) production inhibiting pancreatic adenocarcinoma cell growth. We also show that UCP2 inhibition triggers ROS-dependent nuclear translocation of the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH), formation of autophagosomes, and the expression of the autophagy marker LC3-II. Consistently, UCP2 over-expression significantly reduces basal autophagy confirming the anti-autophagic role of UCP2. Furthermore, we demonstrate that autophagy induced by UCP2 inhibition determines a ROS-dependent cell death, as indicated by the apoptosis decrease in the presence of the autophagy inhibitors chloroquine (CQ) or 3-methyladenine (3-MA), or the radical scavenger NAC. Intriguingly, the autophagy induced by genipin is able to potentiate the autophagic cell death triggered by gemcitabine, the standard chemotherapeutic drug for pancreatic adenocarcinoma, supporting the development of an anti-cancer therapy based on UCP2 inhibition associated to standard chemotherapy. Our results demonstrate for the first time that UCP2 plays a role in autophagy regulation bringing new insights into mitochondrial uncoupling protein field.

Topics: Adenocarcinoma; Apoptosis; Autophagy; Blotting, Western; Cell Line, Tumor; Cell Proliferation; Cholagogues and Choleretics; Fluorescent Antibody Technique; Glyceraldehyde-3-Phosphate Dehydrogenases; Humans; Ion Channels; Iridoids; Mitochondrial Proteins; Oxidative Stress; Pancreatic Neoplasms; Protein Transport; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; RNA, Small Interfering; Uncoupling Protein 2

To propose a simple method for the development of genipin-crosslinked casein micelles as a new delivery platform for prolonged release of alfuzosin hydrochloride.. Crosslinked casein micelles entrapping alfuzosin were transformed into solid redispersible nanoparticles via spray-drying technique with no need for drying adjuvants based on the stabilizing effect of casein.. The nanoparticles displayed high production yields (86.99-94.63% w/w) with a reasonable drug incorporation efficiency ranged from 92.86 to 97.75%. The nanoparticles were readily reconstituted in aqueous solution with a particle size range of 122.1-260.0 nm and a zeta potential range of -21.6 to -36.6 mV indicating a good colloidal stability. No drug crystals were detectable in the scanning electron micrographs revealing successful encapsulation of alfuzosin into casein nanoparticles which was confirmed by differential scanning calorimetry. The nanoparticles succeeded in prolonging the drug release that could be controlled by modulating the genipin crosslinking degree. The release data showed a good fit into Higuchi release kinetics with non-Fickian type of drug diffusion.. These results demonstrated that genipin-crosslinking combined with spray-drying technique could be used as a promising tool to develop solid redispersible casein nanoparticles with sustained drug release properties.

Topics: Adrenergic alpha-1 Receptor Antagonists; Caseins; Cross-Linking Reagents; Delayed-Action Preparations; Desiccation; Iridoids; Nanoparticles; Quinazolines

DIABECELL® capsules comprise an inner core of alginate (Alg) coated with a polycationic polymer, poly-L-ornithine (PLO), designed as a stabilizing agent for strengthening the capsule wall, which is masked by an outer layer of biocompatible Alg. These polymeric microcapsules have demonstrated excellent mechanical properties and a reduction in hypoglycemia after tranplantation in human clinical trials; however, degradation of the outer Alg layer leaves the underlying layers of PLO exposed, which ultimately leads to reduced biocompatibility in vivo. Here we aim to improve capsule biocompatibility and to increase the hydrophilic properties of the capsule surface through chemical crosslinking/modification of the PLO layer using genipin. Fluorescence microscopy established crosslinking was limited to the layers of PLO. In vitro experiments confirmed islet viability and insulin release within chemically modified capsules over the course of a month and in vivo investigations demonstrated improved biocompatibility when comparing standard Alg/PLO/Alg capsules with genipin modified capsules.

Topics: Alginates; Animals; Animals, Newborn; Biocompatible Materials; Capsules; Coated Materials, Biocompatible; Cross-Linking Reagents; Diffusion Chambers, Culture; Drug Stability; Glucuronic Acid; Hexuronic Acids; Humans; Hydrophobic and Hydrophilic Interactions; Iridoids; Islets of Langerhans Transplantation; Materials Testing; Mice; Peptides; Sus scrofa; Transplantation, Heterologous

Biological tissues must be chemically fixed before they can be implanted in humans, due to the immediate degradation and presence of antigenicity of naturally derived tissues. To provide a crosslinking reagent which is cytocompatible and may prepare biocompatible fixed tissues, a novel crosslinking agent, alginate dialdehyde (ADA), was employed to fix biological tissues by our group. The study was to evaluate the cytocompatibility of ADA for biological tissue fixation. Glutaraldehyde and genipin counterparts were used as controls. The result suggested that the cytotoxicity of ADA was significantly lower than that of glutaraldehyde and genipin. Additionally, in the evaluation of cytotoxicity of fixed tissue itself and the residues, as well as the cell adhesion property, ADA-fixed tissue was significantly superior to its glutaraldehyde counterpart and comparable to its genipin counterpart. The results obtained in this study demonstrate that ADA is a cytocompatible crosslinking reagent for biological tissue fixation.

Topics: Alginates; Biocompatible Materials; Cellulose; Cross-Linking Reagents; Glucuronic Acid; Glutaral; Hexuronic Acids; Humans; Iridoids; Tissue Fixation

Bio-based fibrous nanocomposites of cellulose nanofibres and non-crosslinked/crosslinked collagen were prepared by in situ pH-induced fibrillation of collagen phase and sterilized using gamma rays at 25 KGy. Collagen phase is crosslinked using genipin, a bio-based crosslinker that introduces flexible crosslinks. Microscopy studies of the prepared materials showed nanostructured fibrous collagen and cellulose dispersed in collagen matrix. Mechanical performance of the sterilized nanocomposites was close to that of natural ligament and tendon, in simulated body conditions. Cytocompatibility studies indicated that these nanocomposites allowed human ligament cell and human endothelial cell adhesion, growth, and differentiation; which is eminently favourable to ligament tissue engineering.

Topics: Animals; Biocompatible Materials; Cell Adhesion; Cell Differentiation; Cellulose; Collagen; Cross-Linking Reagents; Gamma Rays; Hydrogen-Ion Concentration; Iridoids; Ligaments; Materials Testing; Mice; Microscopy, Electron, Scanning; Nanofibers; NIH 3T3 Cells; Tendons; Tissue Engineering; Tissue Scaffolds

As a biocompatible and bioactive natural tissue engineering scaffold, porcine acellular dermal matrix (PADM) has limitations for the application in tissue regeneration due to its low strength and rapid biodegradation. Here, purified PADM was modified by a nontoxic cross-linker (genipin) to enhance its mechanical properties and improve its resistance to enzymatic degradation. In vitro testing results demonstrated that the stiffness of the genipin cross-linked PADM was improved and biodegradation rate was decreased. Results of cell proliferation assays showed that the cross-linking reaction by genipin did not undermine the cytocompatibility of PADM. Furthermore, genipin cross-linking imparted an observable fluorescence allowing visualization of the scaffold's three-dimensional (3D) porous structure and cell distribution by confocal laser scanning microscopy (CLSM). Immunostaining of the cell nuclei and cytoskeleton indicated that MC3T3-E1 preosteoblasts were tightly adhered to and uniformly distributed onto the cross-linked PADM scaffold. Results of this study suggest that the 3D porous genipin cross-linked PADM with intrinsic fluorescence may have broader applications for tissue engineering scaffolds where higher mechanical stiffness is needed.

Topics: Acellular Dermis; Animals; Biocompatible Materials; Biomechanical Phenomena; Cell Proliferation; Cross-Linking Reagents; Fluorescence; Iridoids; Mice; Osteoblasts; Porosity; Swine; Tissue Engineering; Tissue Scaffolds

Oral and parenteral formulations are challenging to produce therapeutic concentration of flurbiprofen in the joints. This encourages for the development of formulation for long term drug retention in the joint through intra-articular (i. a.) administration. In this study, genipin cross-linked gelatin microspheres of flurbiprofen were prepared for i. a. delivery. The microspheres were prepared using emulsification-homogenization-cross-linking method by changing the experimental variables such as concentration of cross-linker, cross-linking time and cross-linking temperature. The microspheres showed drug entrapment up to 76.19% with a mean particle size range of 5.91-8.19 µm. The degree of cross-linking and water-soluble fraction were 8.27-59.33% and 12.29-81.23%, respectively. SEM confirmed smooth surface and spherical shape of the microspheres. FTIR and (13)C-NMR confirmed cross-linking of gelatin by genipin. No chemical change in encapsulated drug was observed by FTIR and TGA. DSC and XRD indicated the molecular dispersion of drug within microspheres. Optimized microspheres could prolong the drug release for more than 108 h with anomalous transport. Histopathology confirmed the biocompatibility of microspheres in the rat (Wistar) knee joint. After 96 h of i. a. injection, significant higher amount (42.56%) of administered drug in cross-linked microspheres was recovered than uncross-linked microspheres (8.27%) confirming better drug retention efficiency (p < 0.01).

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arthritis, Experimental; Biocompatible Materials; Chemistry, Pharmaceutical; Cross-Linking Reagents; Drug Carriers; Drug Compounding; Flurbiprofen; Injections, Intra-Articular; Iridoids; Male; Microscopy, Electron, Scanning; Particle Size; Rats; Rats, Wistar; Solubility; Spectroscopy, Fourier Transform Infrared; Surface Properties; Thermogravimetry; X-Ray Diffraction

Genipin, a compound derived from Gardenia jasminoides Ellis fruits, has been used over the years in traditional Chinese medicine to treat symptoms of type 2 diabetes. However, the molecular basis for its antidiabetic effect has not been fully revealed. In this study, we investigated the effects of genipin on glucose uptake and signaling pathways in C(2)C(12) myotubes. Our study demonstrates that genipin stimulated glucose uptake in a time- and dose-dependent manner. The maximal effect was achieved at 2 h with a concentration of 10 μM. In myotubes, genipin promoted glucose transporter 4 (GLUT4) translocation to the cell surface, which was observed by analyzing their distribution in subcellular membrane fraction, and increased the phosphorylation of insulin receptor substrate-1 (IRS-1), AKT, and GSK3β. Meanwhile, genipin increased ATP levels, closed K(ATP) channels, and then increased the concentration of calcium in the cytoplasm in C(2)C(12) myotubes. Genipin-stimulated glucose uptake could be blocked by both the PI3-K inhibitor wortmannin and calcium chelator EGTA. Moreover, genipin increases the level of reactive oxygen species and ATP in C(2)C(12) myotubes. These results suggest that genipin activates IRS-1, PI3-K, and downstream signaling pathway and increases concentrations of calcium, resulting in GLUT4 translocation and glucose uptake increase in C(2)C(12) myotubes.

Topics: Animals; Biological Transport; Calcium; Cell Line; Dose-Response Relationship, Drug; Glucose; Glucose Transporter Type 4; Insulin; Insulin Receptor Substrate Proteins; Iridoids; Mice; Muscle Fibers, Skeletal; Phosphorylation; Proto-Oncogene Proteins c-akt; Reactive Oxygen Species; Signal Transduction

Topics: Amyotrophic Lateral Sclerosis; Electron Transport; Humans; Iridoids; Mitochondria, Muscle; Models, Theoretical; Plant Extracts

Genipin-crosslinked complex microspheres made of the combination of two polymers, ethyl cellulose and chitosan, were prepared by spray drying method. Rifabutin, an anti-tuberculosis agent was used as a model drug. Effects of various specifications of ethyl cellulose and chitosan, different drug/polymers ratios and crosslinking effect of genipin on complex microspheres and drug release characteristics were compared to obtain optimized manufacturing parameters. The complex microspheres showed a significant different shape as compared to chitosan microspheres. Biphasic release features were observed in the in vitro and in vivo release study, consisting of an initial quick release phase and an extended sustained release phase. Furthermore, pulmonary drug concentrations of rats after administering the complex microspheres were maintained on a therapeutic level for at least 24 days.

Topics: Animals; Antitubercular Agents; Calorimetry, Differential Scanning; Cell Death; Cell Line, Tumor; Cellulose; Chemistry, Pharmaceutical; Chitosan; Cross-Linking Reagents; Drug Delivery Systems; Humans; Iridoids; Microscopy, Electron, Scanning; Microspheres; Particle Size; Rats; Rats, Sprague-Dawley; Rifabutin; Static Electricity; Viscosity

Silk sericin has recently been studied for its advantageous biological properties, including its ability to promote wound healing. This study developed a delivery system to accelerate the healing of full-thickness wounds. Three-dimensional scaffolds were fabricated from poly(vinyl alcohol) (PVA), glycerin (as a plasticizer) and genipin (as a crosslinking agent), with or without sericin. The physical and biological properties of the genipin-crosslinked sericin/PVA scaffolds were investigated and compared with those of scaffolds without sericin. The genipin-crosslinked sericin/PVA scaffolds exhibited a higher compressive modulus and greater swelling in water than the scaffolds without sericin. Sericin also exhibited controlled release from the scaffolds. The genipin-crosslinked sericin/PVA scaffolds promoted the attachment and proliferation of L929 mouse fibroblasts. After application to full-thickness rat wounds, the wounds treated with genipin-crosslinked sericin/PVA scaffolds showed a significantly greater reduction in wound size, collagen formation and epithelialization compared with the control scaffolds without sericin but lower numbers of macrophages and multinucleated giant cells. These results indicate that the delivery of sericin from the novel genipin-crosslinked scaffolds efficiently healed the wound. Therefore, these genipin-crosslinked sericin/PVA scaffolds represent a promising candidate for the accelerated healing of full-thickness wounds.

Topics: Animals; Bombyx; Iridoids; Male; Mice; Microscopy, Electron, Scanning; Rats; Rats, Sprague-Dawley; Sericins; Tissue Scaffolds; Wound Healing

Few studies exist on the mechanical performance of crosslinked electrospun chitosan (CS) fibre mats. In this study, we show that the mat structure and mechanical performance depend on the different crosslinking agents genipin, epichlorohydrin (ECH), and hexamethylene-1,6-diaminocarboxysulphonate (HDACS), as well as the post-electrospinning heat and base activation treatments. The mat structure was imaged by field emission scanning electron microscopy and the mechanical performance was tested in tension. The elastic modulus, tensile strength, strain at failure and work to failure were found to range from 52 to 592 MPa, 2 to 30 MPa, 2 to 31 per cent and 0.041 to 3.26 MJ m(-3), respectively. In general, neat CS mats were found to be the stiffest and the strongest, though least ductile, while CS-ECH mats were the least stiff, weakest, but the most ductile, and CS-HDACS fibre mats exhibited intermediary mechanical properties. The mechanical performance of the mats is shown to reflect differences in the fibre diameter, number of fibre-fibre contacts formed within the mat, as well as varying intermolecular bonding and moisture content. The findings reported here complement the chemical properties of the mats, described in part I of this study.

Topics: Biomechanical Phenomena; Chitosan; Cross-Linking Reagents; Electrochemistry; Epichlorohydrin; Iridoids; Microscopy, Electron, Scanning; Tissue Engineering

Carboxymethylchitosan (CMCS) microspheres were prepared by the carboxymethylation of chitosan (CS) beads using monochloroacetic acid. The CMCS microspheres were crosslinked using two different methods: the amine-amine crosslinker genipin and carbodiimide chemistry, yielding Gen-X CMCS and X-CMCS beads, respectively. The Gen-X CMCS beads were found to have poor degradation and drug release profiles. The X-CMCS microspheres displayed good potential for use in tissue engineering applications in which degradation and local drug delivery are desired. The X-CMCS beads displayed enzymatic degradation of 82.7 ± 1.2% in 100 μg/mL lysozyme after 1 month. An extended release of rhBMP-2 for at least 45 days was also observed with the X-CMCS microspheres. Scaffolds were formed by fusing beads together, and the X-CMCS beads were successfully incorporated into composite X-CMCS/CS scaffolds. The composite scaffolds had increased degradation of 14.5 ± 6.6% compared to 0.5 ± 0.4% for CS-only scaffolds, and the X-CMCS/CS scaffolds released more rhBMP-2 at all timepoints. The composite scaffolds also supported the attachment and proliferation of SAOS-2 cells. The addition of X-CMCS beads resulted in fabrication of scaffolds with improved properties for use in bone tissue engineering.

Topics: Biocompatible Materials; Bone and Bones; Bone Morphogenetic Protein 2; Bone Regeneration; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Chitosan; Cross-Linking Reagents; Durapatite; Humans; Iridoids; Microscopy, Electron, Scanning; Microspheres; Muramidase; Ninhydrin; Recombinant Proteins; Spectroscopy, Fourier Transform Infrared; Tissue Engineering; Tissue Scaffolds; Transforming Growth Factor beta

Stable surface modifications of polydimethylsiloxane (PDMS) are of crucial importance for the exploitation of the versatile physical properties of silicone in many biological applications. Surface hydrophobic recovery in fact poses severe time limitations to the observation of biological events and, in particular, to cell culturing. A novel method of stable modification of PDMS surface chemistry was therefore elaborated, relying on the use of genipin as a natural low-toxicity cross-linker, and involving free amine moieties. Its effectiveness to long-term cultures was studied by preparation of thin PDMS films with different stiffness. After assessment of surface chemistry and substrate stiffness, H9c2 muscle cells were cultured on the modified films, and differentiating myoblasts were observed for a period of four weeks since differentiation induction. A lower PDMS stiffness increased myotube width and supported a higher actin and myosin colocalization within myotubes, suggesting the achievement of myotube functional maturity. These results provide evidence of the effectiveness of the proposed procedures to PDMS surface chemistry modification. Furthermore, modified PDMS membranes prove to be suitable to several long-term studies of cell behaviour in vitro, including muscle cell contractility investigations.

Topics: Actins; Animals; Cell Differentiation; Cell Membrane; Cells, Cultured; Collagen; Dimethylpolysiloxanes; Iridoids; Myoblasts, Cardiac; Myosins; Rats; Surface Properties

Novel nanocomposites consisting of genipin-crosslinked chitosan (GC), poly(ethylene glycol) (PEG), zinc oxide (ZnO), and silver (Ag) nanoparticles were prepared for biomedical applications as the wound-healing materials. Various amounts of ZnO and Ag nanoparticles were dispersed in the GC/PEG hydrogel matrix without severe aggregation. The effects of composition and ZnO nanoparticles on the physico-chemical properties of nanocomposite samples were evaluated by infrared analysis, X-ray diffraction, and scanning electron microscopy. GC/PEG/ZnO/Ag nanocomposite showed the pH-sensitive swelling behavior and the improved mechanical properties. The antibacterial activities of nanocomposite films were tested toward the bacterial species Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis. GC/PEG/ZnO/Ag composite films had higher antibacterial activities than GC/PEG and GC/PEG/ZnO nanocomposite films. GC/PEG/ZnO/Ag composite films have potential application as wound and burn dressings.

Topics: Anti-Bacterial Agents; Bacteria; Bandages; Chitosan; Iridoids; Microscopy, Electron, Scanning; Nanocomposites; Polyethylene Glycols; Silver; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction; Zinc Oxide

In this article, silver and magnetite nanofillers were synthesized in modified κ-carrageenan hydrogels using the in situ method. The effect of metallic nanoparticles in gastro-intestinal tract (GIT) release of a model drug (methylene blue) has been investigated. The effect of nanoparticles loading and genipin cross-linking on GIT release of nanocomposite is also studied to finally provide the most suitable drug carrier system. In vitro release studies revealed that using metallic nanocomposites hydrogels in GIT studies can improve the drug release in intestine and minimize it in the stomach. It was found that cross-linking and nanofiller loading can significantly improve the targeted release. Therefore, applying metallic nanoparticles seems to be a promising strategy to develop GIT controlled drug delivery.

Topics: Carrageenan; Cross-Linking Reagents; Drug Carriers; Drug Liberation; Hydrogels; Hydrogen-Ion Concentration; Intestines; Iridoids; Metal Nanoparticles; Methylene Blue; Nanocomposites; Silver; Stomach

Emulsion gels are now emerging as a new class of biomaterials for controlled-release applications. Novel food-grade emulsion gels consisting of indomethacin-loaded vegetable oil droplets dispersed within genipin-cross-linked gelatin-based hydrogels were characterized for their physical and drug-release properties. Varying the weight ratio of the aqueous and oil phases between 5:1 and 5:5 was used to modulate construct swelling and drug release. The dispersed oil droplets generally became larger, more polydispersed and aggregated with an increase in oil fraction. Cross-linking with genipin increased the puncture strength of the gels vs. their uncross-linked counterparts and was necessary to prevent breakdown. Swelling of the emulsion gels demonstrated Fickian behaviour at all gelatin: oil ratios. Indomethacin release followed Fickian diffusion at higher oil fractions only, demonstrating coupled Fickian and super-Case-II transport at lower oil ratios (5:1, 5:2 and 5:3). Overall, the introduction of a dispersed oil phase within a hydrogel was exploited for the release of hydrophobic bioactive compounds, with tailoring of composition used to significantly alter release kinetics.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Delayed-Action Preparations; Diffusion; Drug Carriers; Emulsions; Gastric Mucosa; Gelatin; Gels; Indomethacin; Iridoids; Mechanical Phenomena; Plant Oils; Solubility; Swine; Water

Calcification plays a major role in the failure of bioprosthetic and other tissue heart valve substitutes. The objective of this study was to evaluate the anticalcification effect of decellularization and detoxification in glutaraldehyde (GA)/genipin- and solvent-fixed bovine pericardium using a rabbit intramuscular model which is effective for assessing calcification in bioprosthetic tissue.. Bovine pericardial tissues were fixed with 0.5% GA/0.4% genipin in organic solvent (75% ethanol + 5% octanol, 75% ethanol + 5% octanediol, or 70% ethanol + 10% isopropanol) and post-treated with glycine, glutamic acid, or sodium bisulfite. Decellularization was performed with 0.25% sodium dodecylsulfate prior to fixation. The material characteristics of the treated tissues were assessed by thermal stability test, uniaxial mechanical test, and light microscopy. Stability of the treated tissue was measured by the resistance to enzymatic degradation using protease. The tissues were intramuscularly implanted into 4-week-old rabbits for 8 weeks, and the anti-α-Gal(Gal[alpha]1,3-Gal[beta]1,4GlcNAc-R) antibodies (immunoglobulin G) were measured at various time intervals after implantation. Explanted tissues were examined by light microscopy and calcium contents of the explanted tissues were measured.. Differently treated tissues resulted in no significant alterations in material characteristics and morphology. GA groups are superior to genipin groups in tissue cross-linking without difference according to addition of decellularization, organic solvent treatment, and detoxification by resistance toward pronase degradation. The titer of anti-α-Gal antibodies gradually increased after implantation in all study groups. The titer of anti-α-Gal antibodies increased less in genipin groups than in GA groups, and less with decellularization than without decellularization. The calcium contents of genipin groups (n = 75) were significantly lower than those of GA groups (n = 118) (28.55 ± 11.22 μg mg(-1) vs 37.16 ± 7.75 μg mg(-1), p < 0.001). Calcium contents decreased with decellularization, organic solvent treatment, and detoxification, irrespective of the type of organic solvent and amino acids. Inorganic phosphorus contents of genipin groups (n = 66) were significantly lower than those of GA groups (n = 103) (55.36 ± 26.98 μg mg(-1) vs 75.67 ± 23.44 μg mg(-1), p = 0.000).. Genipin fixation is a novel alternative to conventional GA fixation in vitro material assessment and in vivo anticalcification effect. The addition of decellularization, organic solvent treatment, and detoxification prevented calcification of GA/genipin-fixed bovine pericardium in the rabbit intramuscular implantation model, irrespective of the type of organic solvent and amino acids.

Topics: Animals; Bioprosthesis; Calcinosis; Calcium; Cattle; Enzyme-Linked Immunosorbent Assay; Fixatives; Glutaral; Hot Temperature; Iridoids; Male; Materials Testing; Pericardium; Pronase; Rabbits; Solvents; Stress, Mechanical; Tissue Fixation; Transplantation, Heterologous

Genipin blue is a pigment prepared from the reaction of genipin with amino acid. We describe herein a new method used to prepare genipin blue, water-soluble blue pigments, through the reaction of hen egg protein with genipin. The effects of reaction time, reaction temperature, the pH value of the solution and the mass ratio of the reactants on the preparation are studied. One part of genipin reacted with eight parts of hen egg protein (w/w) in water (pH value of reaction system 7.5) at 60°C for 96 h and gave blue pigments with the maximum colour value of 146.2. The blue pigments showed identical absorption activity in UV spectroscopy (λ(max )= 584 nm) to that of gardenia blue pigments, which were prepared from the reaction of genipin with amino acid.

Topics: Color; Coloring Agents; Egg Proteins; Hydrogen-Ion Concentration; Iridoids; Temperature; Time Factors

This study proposes a biodegradable nerve conduit containing genipin-cross-linked gelatin annexed with tricalcium phosphate ceramic particles (genipin-gelatin-tricalcium phosphate, GGT) in peripheral nerve regeneration. Firstly, cytotoxicity tests revealed that the GGT-extracts were not toxic, and promoted the proliferation and neuronal differentiation of adipose tissue-derived stem cells (ADSCs). Secondly, the GGT composite film effectively supported ADSCs attachment and growth. Additionally, the GGT substrate was biocompatible with the neonatal rat sciatic nerve and produced a beneficial effect on peripheral nerve repair through in vitro tissue culture. Finally, the experiments in this study confirmed the effectiveness of a GGT/ADSCs nerve conduit as a guidance channel for repairing a 10-mm gap in a rat sciatic nerve. Eight weeks after implantation, the mean recovery index of compound muscle action potentials (CMAPs) was significantly different between the GGT/ADSCs and autografts groups (p < 0.05), both of which were significantly superior to the GGT group (p < 0.05). Furthermore, walking track analysis also showed a significantly higher sciatic function index (SFI) score (p < 0.05) and better toe spreading development in the GGT/ADSCs group than in the autograft group. Histological observations and immunohistochemistry revealed that the morphology and distribution patterns of nerve fibers in the GGT/ADSCs nerve conduits were similar to those of the autografts. The GGT nerve conduit offers a better scaffold for the incorporation of seeding undifferentiated ADSCs, and opens a new avenue to replace autologous nerve grafts for the rapid regeneration of damaged peripheral nerve tissues and an improved approach to patient care.

Topics: Adipose Tissue; Animals; Biocompatible Materials; Biodegradation, Environmental; Calcium Phosphates; Cell Differentiation; Cell Proliferation; Cell Shape; Cells, Cultured; Coculture Techniques; Electrophysiological Phenomena; Gelatin; Iridoids; Male; Microscopy, Electron, Scanning; Rats; Rats, Sprague-Dawley; Recovery of Function; Sciatic Nerve; Stem Cell Transplantation; Stem Cells; Sus scrofa; Tissue Scaffolds; Wound Healing

Genipin, a constituent of Gardenia jasminoides Ellis, is used in the treatment of hepatic disorders and inflammatory diseases in traditional medicine. Although mounting evidence suggests an anti-tumor activity of genipin in several cancer cell systems, the inhibitory effect of genipin on the growth of breast cancer cells has not been reported yet. The present study aimed to investigate the anti-proliferative activity of genipin in MDA-MB-231 human breast cancer cells. Herein, we showed that genipin efficiently induced apoptosis in MDA-MB-231 cells by the down-regulation of Bcl-2, up-regulation of Bax and proteolytic activation of caspase-3. Activation of JNK and p38 MAPK also increased by genipin. Importantly, genipin significantly inhibited invasive and migratory phenotypes of MDA-MB-231 cells. Taken together, this study demonstrates that genipin induces apoptosis and inhibits invasive/migratory abilities of highly invasive MDA-MB-231 human breast cancer cells, suggesting a potential application of genipin as a chemopreventive agent that may prevent or alleviate metastatic breast cancer.

Topics: Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Breast Neoplasms; Cell Line, Tumor; Cell Movement; Cell Proliferation; Female; Gardenia; Humans; Iridoids; MAP Kinase Signaling System

In this study, the obtainment and characterization of decellularized rat tracheal grafts are described. The detergent-enzymatic method, already used to develop bioengineered pig and human trachea scaffolds, has been applied to rat tracheae in order to obtain airway grafts suitable to be used to improve our knowledge on the process of tissue-engineered airway transplantation and regeneration. The results demonstrated that, after 9 detergent-enzymatic cycles, almost complete decellularized tracheae, retaining the hierarchical and mechanical properties of the native tissues with strong in vivo angiogenic characteristics, could be obtained. Moreover, to improve the mechanical properties of decellularized tracheae, genipin is here considered as a naturally derived cross-linking agent. The results demonstrated that the treatment increased mechanical properties, in term of secant modulus, without neither altering the pro-angiogenic properties of decellularized airway matrices or eliciting an in vivo inflammatory response.

Topics: Animals; Biomechanical Phenomena; Iridoids; Male; Mice; Microscopy, Electron, Scanning; Rats; Tissue Engineering; Tissue Scaffolds; Trachea

We investigated the hypothesis that exogenous collagen cross-linking can augment intact regions of tendon to mitigate mechanical propagation of partial tears. We first screened the low toxicity collagen cross-linkers genipin, methylglyoxal and ultra-violet (UV) light for their ability to augment tendon stiffness and failure load in rat tail tendon fascicles (RTTF). We then investigated cross-linking effects in load bearing equine superficial digital flexor tendons (SDFT). Data indicated that all three cross-linking agents augmented RTTF mechanical properties but reduced native viscoelasticity. In contrast to effects observed in fascicles, methylglyoxal treatment of SDFT detrimentally affected tendon mechanical integrity, and in the case of UV did not alter tendon mechanics. As in the RTTF experiments, genipin cross-linking of SDFT resulted in increased stiffness, higher failure loads and reduced viscoelasticity. Based on this result we assessed the efficacy of genipin in arresting tendon tear propagation in cyclic loading to failure. Genipin cross-linking secondary to a mid-substance biopsy-punch significantly reduced tissue strains, increased elastic modulus and increased resistance to fatigue failure. We conclude that genipin cross-linking of injured tendons holds potential for arresting tendon tear progression, and that implications of the treatment on matrix remodeling in living tendons should now be investigated.

Topics: Animals; Collagen; Cross-Linking Reagents; Disease Models, Animal; Elasticity; Horses; Iridoids; Lacerations; Pyruvaldehyde; Rats; Recovery of Function; Tendon Injuries; Tendons; Ultraviolet Therapy; Wound Healing

A sensitive high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of genipin in rat plasma after hydrolysis with sulfatase. Genipin could not be detected directly as it could be transformed into other forms such as conjugated-genipin immediately after administration. The conjugated genipin could be hydrolyzed by sulfatase to genipin. The conditions of hydrolysis were investigated. Genipin and the internal standard, peoniflorin (IS), were separated on a reversed-phase column by gradient elution and detected using an electrospray ion source on a 4000 QTrap triple-quadrupole mass spectrometer. The quantification was performed using multiple reaction monitoring with selected precursor-product ion pairs of the transitions m/z 225.0 → 122.7 and m/z 479.1 → 449.1 for genipin and peoniflorin. The assay was linear over the concentration range of 1.368-1368 ng/mL, with correlation coefficients of 0.9989. Intra- and inter-day precisions and accuracy were all within 15%. The lower limit of quantification was 1.368 ng/mL. The recoveries of genipin and peoniflorin were more than 53.3 and 51.2%. The highly sensitive method was successfully applied to estimated pharmacokinetic parameters of genipin following oral and intravenous administration to rats. The absolute bioavailability of genipin was 80.2% in rat, which is the first report.

Topics: Administration, Oral; Animals; Benzoates; Bridged-Ring Compounds; Chromatography, High Pressure Liquid; Drug Stability; Glucosides; Hydrolysis; Injections, Intravenous; Iridoids; Male; Monoterpenes; Rats; Rats, Sprague-Dawley; Reproducibility of Results; Sensitivity and Specificity; Sulfatases; Tandem Mass Spectrometry

Drug delivery is a promising technique to enhance the therapeutic efficacy of the drug. However, properties of carrier materials require intense improvement for effective transport of drug molecules. In the current study, attempts have been made to develop freeze dried gelatin matrices cross linked with genipin at various temperatures (5°C, 15°C and 25°C) prior to freeze-drying (-80°C). The freeze dried matrices thus obtained at the said temperatures are characterized for crosslinking density, compression strength, swelling behaviors. The matrix crosslinked at 25°C showed highest Flory-Rehner crosslinking density (467 ± 46) (p<0.05), highest compressive strength (12.36 ± 0.12) (p<0.05) and lowest equilibrium water content. In this context, scanning electron microscopy (SEM) was performed to study the surface morphology (size and shape of pores) of the crosslinked matrices. These images were further processed for quantitative analysis of morphological features, viz., areas, radius, ferret diameter, length of major and minor axis and eccentricity using MATLAB toolboxes. These quantitative analyses correlate transport and the release kinetics of model anti-inflammatory drug (indomethacin) from crosslinked matrices in vitro to tune as a controllable delivery system. The diffusional exponent (n) for all constructs ranging from 0.61 to 0.69 (p<0.05) (0.45

Topics: Anti-Inflammatory Agents; Drug Carriers; Drug Delivery Systems; Freeze Drying; Gelatin; Image Processing, Computer-Assisted; Indomethacin; Iridoids; Kinetics; Mechanical Phenomena; Microscopy, Electron, Scanning; Temperature

Eucommia ulmoides Oliver is a perennial woody plant distributed widely in China. To characterize some major compounds in E. ulmoides bark extract, six compounds were identified via high-performance liquid chromatography qualitative analysis. E. ulmoides bark extract protects against cadmium-induced oxidative damage in rat kidneys. Two compounds of E. ulmoides bark extract, geniposide and genipin, which were identified both in serum and in kidney tissue, showed inhibitory effects on nitric oxide production. This study provides biological evidence supporting the usefulness of E. ulmoides bark against cadmium-induced toxic oxidative stress in rat kidney tissue.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cadmium Poisoning; Drug Discovery; Drugs, Chinese Herbal; Eucommiaceae; Iridoids; Kidney; Macrophage Activation; Male; Mice; Mice, Inbred Strains; Nitric Oxide; Oxidative Stress; Plant Bark; Protective Agents; Rats; Rats, Sprague-Dawley; Renal Insufficiency

Intestinal microflora (IM) is able to produce toxic and carcinogenic metabolites and induce more potent cytotoxicity against cells than non-metabolites. This study was performed to investigate the cytotoxic responses of geniposide (GS) and its metabolite and to determine the role of metabolism by IM in GS-induced cytotoxicity. Genipin (GP), a GS metabolite, increased cytotoxic effects in cells, but GS did not. Following GS incubation with IM for metabolic activation, increased cytotoxicity was detected compared to GS. Western blot analysis revealed that the activated GS inhibited Bcl-2 expression with a subsequent increase in Bax expression. Likewise, GS activation by IM stimulated caspase-3 and the production of reactive oxygen species (ROS). In addition, activated GS-induced apoptosis was confirmed by apoptosis and ROS assays; N-acetyl-l-cysteine (NAC) suppressed ROS production and apoptotic cell death. Activated GS induced sustained JNK phosphorylation. Moreover, activated GS-induced cell death was reversed by SP600125. Taken together, these findings suggest that human IM is able to metabolize GS into GP, and the related biological activities induce apoptosis through ROS/JNK signaling.

Topics: Apoptosis; bcl-2-Associated X Protein; Biotransformation; Blotting, Western; Caspase 3; Cell Survival; Feces; Female; Hep G2 Cells; Humans; In Situ Nick-End Labeling; Intestines; Iridoids; Male; MAP Kinase Signaling System; Molecular Structure; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction

The pathogenesis of sepsis is characterized by overwhelming inflammatory responses that lead to tissue damage and organ failure. Toll-like receptor (TLR) signaling is crucial for induction of hyperinflammatory responses and tissue injury during sepsis. Genipin, an aglycon of geniposide, has antiinflammatory and antimicrobial activities. The purpose of this study was to test the hypothesis that genipin reduces multiple organ dysfunction and mortality during sepsis through inhibition of TLR signaling. Male ICR were subjected to sepsis by cecal ligation and puncture (CLP) or endotoxemia by lipopolysaccharide (LPS). Various doses of genipin (1, 2.5 and 5 mg/kg) or a vehicle were administered intravenously immediately after CLP or intraperitoneally after LPS treatment. In another set of survival tests, mice were treated with 2.5 mg/kg of genipin 0 and 24 h after CLP. Genipin was found to improve survival and to attenuate multiple organ dysfunction. Genipin attenuated production of proinflammatory cytokines and release of high-mobility group box 1 (HMGB1). Genipin prevented TLR2 and TLR4, myeloid differentiation factor 88 and the Toll/interleukin-1 receptor domain-containing adaptor protein, inducing interferon-β overexpression. Phosphorylation of mitogen-activated protein kinases and interferon regulatory factor 3 and translocation of nuclear factor (NF)-κB were prevented by genipin. Moreover, genipin attenuated increases in serum tumor necrosis factor-α and HMGB1 in LPS-induced endotoxemia. Pam3CSK4- and LPS-mediated production of nitrites and proinflammatory cytokines was suppressed by genipin in RAW264.7 cells. Genipin attenuated mortality and organ injuries during sepsis through interference with TLR signaling. Therefore, genipin might be useful as a potential therapeutic agent for treatment of sepsis.

Topics: Adaptor Proteins, Vesicular Transport; Animals; Anti-Inflammatory Agents; Cell Line; Cell Survival; Cytokines; HMGB1 Protein; Iridoids; Liver; Lung; Male; Mice; Mice, Inbred ICR; Mitogen-Activated Protein Kinases; Myeloid Differentiation Factor 88; Nitrites; Sepsis; Toll-Like Receptor 2; Toll-Like Receptor 4

It is known that endogenous levels of the incretin hormone glucagon-like peptide 1 (GLP1) can be enhanced by various secretagogues, but the mechanism underlying GLP1 secretion is still not fully understood. We assessed the possible effect of uncoupling protein 2 (UCP2) on GLP1 secretion in mouse intestinal tract and NCI-H716 cells, a well-characterized human enteroendocrine L cell model. Localization of UCP2 and GLP1 in the gastrointestinal tract was assessed by immunofluorescence staining. Ucp2 mRNA levels in gut were analyzed by quantitative RT-PCR. Human NCI-H716 cells were transiently transfected with siRNAs targeting UCP2. The plasma and ileum tissue levels of GLP1 (7-36) amide were measured using an ELISA kit. UCP2 was primarily expressed in the mucosal layer and colocalized with GLP1 in gastrointestinal mucosa. L cells secreting GLP1 also expressed UCP2. After glucose administration, UCP2-deficient mice showed increased glucose-induced GLP1 secretion compared with wild-type littermates. GLP1 secretion increased after NCI-H716 cells were transfected with siRNAs targeting UCP2. UCP2 was markedly upregulated in ileum tissue from ob/ob mice, and GLP1 secretion decreased compared with normal mice. Furthermore, GLP1 secretion increased after administration of genipin by oral gavage. Taken together, these results reveal an inhibitory role of UCP2 in glucose-induced GLP1 secretion.

Topics: Animals; Cell Line; Cholagogues and Choleretics; Enteroendocrine Cells; Glucagon-Like Peptide 1; Glucose; Humans; Ileum; Intestinal Mucosa; Ion Channels; Iridoids; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Obese; Mitochondrial Proteins; RNA, Small Interfering; Uncoupling Protein 2

We studied a model system of controlled drug release using beta-carotene and κ-carrageenan/NaCMC hydrogel as a drug and a device, respectively. Different concentrations of genipin were added to crosslink the beta-carotene loaded beads by using the dripping method. Results have shown that the cross-linked beads possess lower swelling ability in all pH conditions (pH 1.2 and 7.4), and swelling ratio decreases with increasing genipin concentration. Microstructure study shows that cross-linking has enhanced the stability and structure of the beads network. Determination of diffusion coefficient for the release of encapsulated beta-carotene indicates less diffusivity when beads are cross-linked. Swelling models using adaptive neuro fuzzy show that using genipin as a cross-linker in the kC/NaCMC hydrogels affects the transport mechanism. The model shows very good agreement with the experimental data that indicates that applying ANFIS modelling is an accurate, rapid and simple way to model in such a case for controlled release applications.

Topics: beta Carotene; Carboxymethylcellulose Sodium; Carrageenan; Cross-Linking Reagents; Diffusion; Drug Carriers; Drug Compounding; Drug Delivery Systems; Fuzzy Logic; Hydrogels; Iridoids; Microscopy, Electron, Scanning; Neural Networks, Computer

Bioengineered tissues created for transplant will be expected to survive and contribute to function over the lifetime of the individual. To evaluate potential intrinsic changes and degradation of the extracellular matrix of decellularized human tissue scaffolds, human decellularized tracheas were evaluated over a one year period in vitro. Human tracheas were decellularized and stored for one year in phosphate-buffered saline at 4 °C in the presence of antibiotics and anti-mycotics, and their structural, mechanical, and angiogenic properties compared to baseline values. Results showed that stored human decellularized tracheas were increasingly degraded resulting in a loss of extracellular matrix architecture - in particular of collagenous and elastic fiber structure -and decreased mechanical and angiogenic properties. The mechanical alterations of the extracellular matrix but not the deterioration and microstructure were not improved by using a natural cross-linking agent. These findings demonstrate that human decellularized tracheas, stored for one year in phosphate-buffered saline solution at 4 °C, would not meet the demands for a tissue engineering matrix and likely would not yield a suitable graft for lifelong implantation. The degradation phenomenon observed in vitro may be further enhanced in vivo, having clinical relevance for tissues that will be transplanted long-term and this should be carefully evaluated in pre-clinical settings.

Topics: Adult; Biomechanical Phenomena; Cross-Linking Reagents; Elastin; Extracellular Matrix; Humans; Iridoids; Male; Materials Testing; Mesenchymal Stem Cells; Microscopy, Electron, Scanning; Middle Aged; Neovascularization, Physiologic; Time Factors; Tissue Donors; Tissue Engineering; Tissue Preservation; Tissue Scaffolds; Trachea

In this paper, we present the first detailed study of the reaction kinetics and the characterization of the products from the endothermic reactions between β-lactoglobulin and genipin. The effects of the concentration, temperature, and pH were investigated. In the temperature range studied, the reaction was approximately a pseudo-first-order with respect to genipin and 0.22-order and -0.24-order with respect to β-lactoglobulin for pH 6.75 and 10.5 with corresponding activation energy (E(a)) estimated to be 66.2 ± 3.8 and 9.40 ± 0.36 kJ/mol, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies, validated by matrix-assisted laser desorption ionization-time of flight mass spectrometry, showed the presence of oligomeric, i.e., di-, tri-, quadri-, and pentameric, forms of cross-linked β-lactoglobulin by genipin at neutral but not alkaline pH; however, an extensive cross-linked network was not observed, consistent with the atomic force microscopy images. It was demonstrated that the reaction temperature and the concentration of genipin but not that of β-lactoglobulin positively affected the extent of the cross-linking reactions.

Topics: Calorimetry, Differential Scanning; Cross-Linking Reagents; Hydrogen-Ion Concentration; Iridoids; Kinetics; Lactoglobulins; Microscopy, Atomic Force; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Temperature; Thermodynamics

The edible blue pigments produced by gardenia fruits have been used as value-added colorants for foods in East Asia for 20 years. However, the biological activity of the blue pigments derived from genipin has not been reported.. The anti-inflammatory effect of blue pigments was studied in lipopolysaccharide (LPS) stimulated RAW 264.7 macrophage in vitro. The secretions of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) were inhibited in concentration-dependent manner by blue pigments. Real-time reverse-transcription polymerase chain reaction (Real-time RT-PCR) analyses demonstrated that the mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-6, and tumor necrosis factor alpha (TNF-α) was inhibited, moreover, ELISA results showed that the productions of IL-6 and TNF-α were inhibited. Cell-based ELISA revealed the COX-2 protein expression was inhibited. The proteome profiler array showed that 12 cytokines and chemokines involved in the inflammatory process were down-regulated by blue pigments. Blue pigments inhibited the nuclear transcription factor kappa-B (NF-κB) activation induced by LPS, and this was associated with decreasing the DNA-binding activity of p65 and p50. Furthermore, blue pigments suppressed the degradation of inhibitor of κB (IκB) α, Inhibitor of NF-κB Kinase (IKK) α, IKK-β, and phosphorylation of IκB-α. The anti-inflammatory effect of blue pigments in vivo was studied in carrageenan-induced paw edema and LPS-injecting ICR mice. Finally, blue pigments significantly inhibited paw swelling and reduced plasma TNF-α and IL-6 production in vivo.. These results suggest that the anti-inflammatory properties of blue pigments might be the results from the inhibition of iNOS, COX-2, IL-6, IL-1β, and TNF-α expression through the down-regulation of NF-κB activation, which will provide strong scientific evidence for the edible blue pigments to be developed as a new health-enhancing nutritional food for the prevention and treatment of inflammatory diseases.

Topics: Animals; Cell Line; Cyclooxygenase 2; Cytokines; Dinoprostone; Gene Expression Regulation, Enzymologic; Inflammation; Iridoids; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Inbred ICR; Models, Chemical; NF-kappa B; Nitric Oxide Synthase Type II; Nitrites; Pigmentation

Most infants with long-gap esophageal atresia receive an esophageal replacement with tissue from stomach or colon, because the native esophagus is too short for true primary repair. Tissue-engineered esophageal conducts could present an attractive alternative. In this paper, circular decellularized porcine esophageal scaffold tissues were implanted subcutaneously into Sprague-Dawley rats. Depending on scaffold cross-linking with genipin, glutaraldehyde, and carbodiimide (untreated scaffolds : positive control; bovine pericardium : gold standard), the number of infiltrating fibroblasts, lymphocytes, macrophages, giant cells, and capillaries was determined to quantify the host response after 1, 9, and 30 days. Decellularized esophagus scaffolds were shown to maintain native matrix morphology and extracellular matrix composition. Typical inflammatory reactions were observed in all implants; however, the cellular infiltration was reduced in the genipin group. We conclude that genipin is the most efficient and best tolerated cross-linking agent to attenuate inflammation and to improve the integration of esophageal scaffolds into its surrounding tissue after implantation.

Topics: Analysis of Variance; Animals; Antigens, CD; Cross-Linking Reagents; DNA; Esophagus; Graft Rejection; Immunohistochemistry; Inflammation; Iridoids; Leukocytes; Prostheses and Implants; Rats; Rats, Sprague-Dawley; Statistics, Nonparametric; Swine; Tissue Scaffolds; Transplantation, Heterologous

Stem cell transplantation has been widely acknowledged for their immense potential in regenerative medicine. In these procedures, the implanted cells need to maintain both their viability and functional properties for effective therapeutic outcomes. This has long been a subject of major concern and intensive studies. Microencapsulation of stem cells within polymeric microcapsules can be an efficient approach to achieve this goal, particularly for heart diseases. This study reports the use of biocompatible, fluorogenic genipin-cross-linked alginate chitosan (GCAC) microcapsules in delivery of human adipose stem cells (hASCs) with an aim to increase the implant retention in the infarcted myocardium for maximum clinical benefits. In vitro results show, under hypoxic conditions, the microencapsulated cells overexpressed significantly higher amount of biologically active vascular endothelial growth factor (VEGF). We investigated on the in vivo potential using immunocompetent female rats after induction of myocardial infarction. For this, animal groups (n = 8) received empty control microcapsules, 1.5 × 10(6) free male hASCs, or 1.5 × 10(6) microencapsulated male hASCs. Results show significant retention (3.5 times higher) of microencapsulated hASCs compared to free hASCs after 10 weeks of transplantation. Microencapsulated hASCs showed significantly attenuated infarct size compared to free hASCs and empty microcapsule group (21.6% ± 1.1% vs. 27.2% ± 3.1% vs. 33.3% ± 3.2%; p < 0.05), enhanced vasculogenesis, and improved cardiac function (fractional shortening: 24.2% ± 2.1% vs. 19.1% ± 0.5% vs. 12.0% ± 4.0%; p < 0.05). These data suggest that microencapsulated hASCs can contribute significantly to the improvement in cardiac functions. Their greater retentions exhibit reduced fibrosis and cardiac dysfunction in experimental animals. However, further research is needed to fully comprehend the underlying biological and immunological effects of microencapsulated hASCs, which jointly play important roles in cardiac repair.

Topics: Adipocytes; Alginates; Animals; Cell Differentiation; Cells, Cultured; Chitosan; Coronary Vessels; Disease Models, Animal; Female; Glucuronic Acid; Hexuronic Acids; Humans; Iridoids; Male; Myocardial Infarction; Myocardium; Neovascularization, Pathologic; Rats; Rats, Inbred Lew; Regenerative Medicine; Stem Cell Transplantation; Stem Cells; Vascular Endothelial Growth Factor A

Possible role of metabolism by the intestinal bacteria in geniposide-induced cytotoxicity was investigated in human hepatoma HepG2 cells. Initially, toxic effects of geniposide and its metabolite genipin were compared. Genipin, a deglycosylated form of geniposide, was cytotoxic at the concentrations that geniposide was not. As metabolic activation systems for geniposide, human intestinal bacterial cultures, fecal preparation (fecalase) and intestinal microbial enzyme mix were employed in the present study. When geniposide was incubated with human intestinal bacteria, either Bifidobacterium longum HY8001 or Bacteroides fragilis, for 24 h, the cultured media caused cytotoxicity in HepG2 cells. Fecalase and intestinal enzyme mix were also effective to metabolically activate geniposide to its cytotoxic metabolite. The present results indicated that genipin, a metabolite of geniposide, might be more toxic than geniposide, and that intestinal bacteria might have a role in biotransformation of geniposide to its toxic metabolite. In addition, among three activation systems tested, intestinal microbial enzyme mix would be convenient to use in detecting toxicants requiring metabolic activation by intestinal bacteria.

Topics: Bacteroides fragilis; Bifidobacterium; Biotransformation; Cell Survival; Dose-Response Relationship, Drug; Feces; Hep G2 Cells; Humans; Intestines; Iridoids

Novel polymeric hydrogel scaffolds for corneal epithelium cell culturing based on blends of chitosan with some other biopolymers such as hydroxypropylcellulose, collagen and elastin crosslinked with genipin, a natural substance, were prepared. Physicochemical and biomechanical properties of these materials were determined. The in vitro cell culture experiments with corneal epithelium cells have indicated that a membrane prepared from chitosan-collagen blend (Ch-Col) provided the regular stratified growth of the epithelium cells, good surface covering and increased number of the cell layers. Ch-Col membranes are therefore the most promising material among those studied. The performance of Ch-Col membranes is comparable with that of the amniotic membrane which is currently recommended for clinical applications.

Topics: Cells, Cultured; Chitosan; Cross-Linking Reagents; Epithelium, Corneal; Equipment Design; Equipment Failure Analysis; Humans; Hydrogels; Iridoids; Membranes, Artificial; Tissue Engineering; Tissue Scaffolds

In this study, genipin cross-linked chitosan microspheres of flurbiprofen for intra-articular (i.a.) delivery were prepared. Emulsion-cross-linking method was used to prepare the microspheres using different concentrations of genipin and drug-to-polymer ratios. The mean particle size was found to be in the range of 5.18-9.74 μm with good % drug entrapment up to 80.97%. SEM indicated the spherical shape with smooth surface of drug-loaded cross-linked microspheres. FTIR confirmed cross-linking of genipin with chitosan and the absence of chemical interactions between drug, polymer, and cross-linker, which was further confirmed by TGA showing unaltered melting point of entrapped drug. DSC and XRD revealed the molecular dispersion of drug within microspheres. The optimized microspheres were able to release the drug for more than 108 h. The biocompatibility of the microspheres in the rat (Sprague-Dawley) knee joints was confirmed by histopathology. The results of pharmacokinetic study, decreased K(a) and T(max), lower C(max) and AUC((0-24)) and delayed MRT, suggested the significant extended release of flurbiprofen from microspheres in comparison with its solution at P<0.05. The recovery of flurbiprofen as the percent of administered dose followed by 24h after i.a. injection of microspheres was found to be 8.7 folds higher than its solution.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Area Under Curve; Chitosan; Cross-Linking Reagents; Emulsions; Flurbiprofen; Injections, Intra-Articular; Iridoids; Knee Joint; Male; Microscopy, Electron, Scanning; Microspheres; Particle Size; Rats; Rats, Sprague-Dawley; Spectroscopy, Fourier Transform Infrared; Transition Temperature

To investigate the efficacy and safety of Genipin and UV-riboflavin crosslinking (UV-CLX) in corneal crosslinking.. Porcine eyes were separated in groups for each crosslinker, genipin 0.25% UV-CLX (clinical crosslinking procedure), glutaraldehyde 0.1% (gold standard crosslinker), and control eyes. Intraocular pressure (IOP) was continuously monitored by a pressure sensor cannulated to the anterior chamber and the volume was changed. The changes in ocular pressure as a function of change of the ocular volume were evaluated. Ocular rigidity was calculated as the exponential of polynomial quadratic fit. Endothelial damage was evaluated in a viability assay with alizarin red staining as the changes in cell counts.. Significant changes in IOP were observed in the globes were the cornea was stiffened with genipin and UV-CLX treatment (volume 200 μl: Genipin 19.4 mmHg, UVCRX 18.8 mmHg, glutaraldehide 23.9 mmHg, versus control 14.7 mmHg, and 400 μl genipin 31.5 mmHg, UV-CLX 26.0 mmHg, glutaraldehide 37.3 mmHg versus control 18.7 mmHg). The mean ocular ridigity coefficient was genipin 0.0078 mmHg/μl, UV-CLX 0.0065 mmHg/μl, glutaraldehide 0.0092 mmHg/μl, and 0.0046 mmHg/μl for control eyes. Endothelial cell damage was 5.9±1.8% (control), 10.3±1.7% (UV-CLX), 9.4±1.5% (Genipin 0.25%), and 40.1±6.2% (glutaraldehide). Some granules were observed in the UV-CLX group. Reduction of keratocites was observed in the UV CRX crosslinking.. Corneal crosslinking was similar between UV-CLX and genipin with minimal toxicity to endothelial cells. Stiffened corneas by any method induced substancially higher IOP elevation when ocular volume is increased.

Topics: Animals; Cell Survival; Cornea; Corneal Keratocytes; Cross-Linking Reagents; Endothelial Cells; Glutaral; Intraocular Pressure; Iridoids; Organ Culture Techniques; Riboflavin; Swine; Tonometry, Ocular; Ultraviolet Rays

Chitosan (CS), the deacetylated form of chitin, the second most abundant, natural polysaccharide, is attractive for applications in the biomedical field because of its biocompatibility and resorption rates, which are higher than chitin. Crosslinking improves chemical and mechanical stability of CS. Here, we report the successful utilization of a new set of crosslinkers for electrospun CS. Genipin, hexamethylene-1,6-diaminocarboxysulphonate (HDACS) and epichlorohydrin (ECH) have not been previously explored for crosslinking of electrospun CS. In this first part of a two-part publication, we report the morphology, determined by field emission scanning electron microscopy (FESEM), and chemical interactions, determined by Fourier transform infrared microscopy, respectively. FESEM revealed that CS could successfully be electrospun from trifluoroacetic acid with genipin, HDACS and ECH added to the solution. Diameters were 267 ± 199 nm, 644 ± 359 nm and 896 ± 435 nm for CS-genipin, CS-HDACS and CS-ECH, respectively. Short- (15 min) and long-term (72 h) dissolution tests (T(600)) were performed in acidic, neutral and basic pHs (3, 7 and 12). Post-spinning activation by heat and base to enhance crosslinking of CS-HDACS and CS-ECH decreased the fibre diameters and improved the stability. In the second part of this publication, we report the mechanical properties of the fibres.

Topics: Biocompatible Materials; Chitosan; Cross-Linking Reagents; Electric Conductivity; Hydrogen-Ion Concentration; Iridoids; Microscopy, Electron, Scanning; Nanofibers; Solubility; Spectroscopy, Fourier Transform Infrared; Thermogravimetry; Viscosity

Surface functionalization plays an important role in the design of biomedical implants, especially when layer forming cells, such as endothelial or epithelial cells, are needed. In this study, we define a novel nanoscale surface coating composed of collagen/alginate polyelectrolyte multilayers and cross-linked for stability with genipin. This buildup follows an exponential growth regime versus the number of deposition cycles with a distinct nanofibrillar structure that is not damaged by the cross-linking step. Stability and cell compatibility of the cross-linked coatings were studied with human umbilical vein endothelial cells. The surface coating can be covered by a monolayer of vascular endothelial cells within 5 days. Genipin cross-linking renders the surface more suitable for cell attachment and proliferation compared to glutaraldehyde (more conventional cross-linker) cross-linked surfaces, where cell clumps in dispersed areas were observed. In summary, it is possible with the defined system to build fibrillar structures with a nanoscale control of film thickness, which would be useful for in vivo applications such as inner lining of lumens for vascular and tracheal implants.

Topics: Alginates; Biocompatible Materials; Cell Adhesion; Cell Proliferation; Cell Shape; Cells, Cultured; Cross-Linking Reagents; Fibrillar Collagens; Glucuronic Acid; Glutaral; Hexuronic Acids; Human Umbilical Vein Endothelial Cells; Humans; Hydrogen-Ion Concentration; Iridoids; Microscopy, Atomic Force; Nanofibers; Spectroscopy, Fourier Transform Infrared; Surface Properties; Tissue Scaffolds

Genipin is a natural blue colorant in food industry. Inflammation is correlated with human disorders, and nuclear factor-κB (NF-κB) is the critical molecule involved in inflammation. In this study, the anti-inflammatory effect of genipin on the lipopolysaccharide (LPS)-induced acute systemic inflammation in mice was evaluated by NF-κB bioluminescence-guided transcriptomic analysis. Transgenic mice carrying the NF-κB-driven luciferase genes were administered intraperitoneally with LPS and various amounts of genipin. Bioluminescent imaging showed that genipin significantly suppressed LPS-induced NF-κB-dependent luminescence in vivo. The suppression of LPS-induced acute inflammation by genipin was further evidenced by the reductions of cytokine levels in sera and organs. Microarray analysis of these organs showed that the transcripts of 79 genes were differentially expressed in both LPS and LPS/genipin groups, and one third of these genes belonged to chemokine ligand, chemokine receptor, and interferon (IFN)-induced protein genes. Moreover, network analysis showed that NF-κB played a critical role in the regulation of genipin-affected gene expression. In conclusion, we newly identified that genipin exhibited anti-inflammatory effects in a model of LPS-induced acute systemic inflammation via downregulation of chemokine ligand, chemokine receptor, and IFN-induced protein productions.

Topics: Animals; Cell Line; Enzyme-Linked Immunosorbent Assay; Gene Expression Profiling; Humans; Immunohistochemistry; Inflammation; Iridoids; Lipopolysaccharides; Luminescence; Mice; NF-kappa B; Real-Time Polymerase Chain Reaction; Transcriptome

Guided tissue regeneration (GTR) is a surgical technique used to direct the formation of bone in the graft space by protecting it with a barrier membrane used to exclude soft tissues during healing. Chitosan has been advocated for GTR applications because of its biocompatibility, degradability, wound healing, and osteogenic properties. In this study, electrospun chitosan membranes, crosslinked with 5 mM or 10 mM geinipin, a natural crosslinker extracted from the gardenia plant, were evaluated for suture pullout strength, crystallinity, and cytocompatibility with normal human dermal fibroblast and TIB 71™ RAW 264.7 monocyte cells. Ultimate suture pullout strength was significantly lower (51-67%) than that of commercially available collagen membranes. Crystallinity of the electrospun chitosan mats decreased upon crosslinking by 14-17% (p = 0.013). The molecular weight of the chitosan polymer was decreased by 75% during the electrospinning process. Uncrosslinked and genipin-crosslinked chitosan mats were cytocompatible and supported fibroblast cell proliferation for 9 days. Uncrosslinked and genipin-crosslinked membranes did not activate monocytes to produce nitric oxide (NO) in vitro in the absence of lipopolysaccharide (LPS). Finally, chitosan membranes inhibited LPS-induced NO production of RAW 264.7 cells by 59-67% as compared to tissue culture plastic and collagen membrane. Improvements are needed in the tear strength of electrospun chitosan membranes for clinical application.

Topics: Animals; Biocompatible Materials; Cell Line; Cell Proliferation; Chitosan; Cross-Linking Reagents; Fibroblasts; Gardenia; Guided Tissue Regeneration; Humans; Iridoids; Mice; Monocytes; Nanofibers

Cancer cells exhibit an endogenous constitutive oxidative stress higher than that of normal cells, which renders tumours vulnerable to further reactive oxygen species (ROS) production. Mitochondrial uncoupling protein 2 (UCP2) can mitigate oxidative stress by increasing the influx of protons into the mitochondrial matrix and reducing electron leakage and mitochondrial superoxide generation. Here, we demonstrate that chemical uncouplers or UCP2 over-expression strongly decrease mitochondrial superoxide induction by the anticancer drug gemcitabine (GEM) and protect cancer cells from GEM-induced apoptosis. Moreover, we show that GEM IC(50) values well correlate with the endogenous level of UCP2 mRNA, suggesting a critical role for mitochondrial uncoupling in GEM resistance. Interestingly, GEM treatment stimulates UCP2 mRNA expression suggesting that mitochondrial uncoupling could have a role also in the acquired resistance to GEM. Conversely, UCP2 inhibition by genipin or UCP2 mRNA silencing strongly enhances GEM-induced mitochondrial superoxide generation and apoptosis, synergistically inhibiting cancer cell proliferation. These events are significantly reduced by the addition of the radical scavenger N-acetyl-l-cysteine or MnSOD over-expression, demonstrating a critical role of the oxidative stress. Normal primary fibroblasts are much less sensitive to GEM/genipin combination. Our results demonstrate for the first time that UCP2 has a role in cancer cell resistance to GEM supporting the development of an anti-cancer therapy based on UCP2 inhibition associated to GEM treatment.

Topics: Acetylcysteine; Apoptosis; Cell Line, Tumor; Cell Proliferation; Deoxycytidine; Drug Resistance, Neoplasm; Gemcitabine; Gene Expression Regulation, Neoplastic; Humans; Ion Channels; Iridoids; Mitochondrial Proteins; Neoplasms; Poly(ADP-ribose) Polymerases; RNA, Messenger; Superoxide Dismutase; Superoxides; Uncoupling Agents; Uncoupling Protein 2

Networks of discrete, genipin-crosslinked gelatin microfibers enveloping pancreatic islets were incorporated within barium alginate microcapsules. This novel technique enabled encapsulation of cellular aggregates in a spherical fibrous matrix <300 μm in diameter. Microfibers were produced by vortex-drawn extrusion within an alginate support matrix. Optimization culminated in a hydrated fiber diameter of 22.3 ± 0.4 μm, a significant reduction relative to that available through current gelatin microfiber spinning techniques, while making the process more reliable and less labor intensive. Microfibers were encapsulated at 40 vol % within 294 ± 4 μm 1.6% barium alginate microparticles by electrostatic-mediated dropwise extrusion. Pancreatic islets extracted from Sprague Dawley rats were encapsulated within the microparticles and analyzed over 21 days. Acridine orange and propidium iodide fluorescent viability staining and light microscopy indicated a significant increase in viability for islets within the fiber-embedded particles relative to fiber-free controls at days 7, 14, and 21. The fiber-embedded system also promoted cellular aggregate cohesion, reducing the incidence of dispersed islet morphologies within the capsules from 31 to 8% at day 21. Further enquiry into benefits of islet encapsulation within a protein fiber network will be the subject of future investigation.

Topics: Adhesiveness; Animals; Capsules; Cross-Linking Reagents; Gelatin; Iridoids; Islets of Langerhans; Male; Microscopy, Electron, Scanning; Rats; Rats, Sprague-Dawley; Static Electricity; Tissue Survival

Acellular porcine corneal stroma (APCS) has been proven to maintain the matrix microenvironment and is therefore an ideal biomaterial for the repair and reconstruction of corneal stroma. This study aims to develop a method to prepare cosmetic corneal lens implants for leukoma using genipin-crosslinked APCS (Gc-APCS). The Gc-APCS was prepared from APCS immersed in 1.0% genipin aqueous solution (pH 5.5) for 4 h at 37 °C, followed by lyophilization at -10 °C. The color of the Gc-APCS gradually deepened to dark-blue. The degree of crosslinking was 45.7 ± 4.6%, measured by the decrease of basic and hydroxy amino acids. The porous structure and ultrastructure of collagenous lamellae were maintained, and the porosity and BET SSA were 72.7 ± 4.6% and 23.01 ± 3.45 m(2)/g, respectively. The Gc-APCS rehydrated to the physiological water content within 5 min and was highly resistant to collagenase digestion. There were no significant differences in the areal modulus and curvature variation between Gc-APCS and nature porcine cornea. The dark-blue pigments were stable to pH, light and implantation in vivo. Gc-APCS extracts had no inhibitory effects on the proliferation of keratocytes. Corneal neovascularization, graft degradation and corneal rejection were not observed within 6 months.

Topics: Amino Acids; Animals; Biomechanical Phenomena; Cell Proliferation; Collagenases; Corneal Keratocytes; Corneal Stroma; Cosmetics; Cross-Linking Reagents; Female; Freeze Drying; Implants, Experimental; Iridoids; Lens, Crystalline; Male; Materials Testing; Porosity; Rabbits; Sus scrofa; Tissue Scaffolds

Modifications at C1, C7, C8, and C10 of genipin were conducted, and the neurotrophic effects of all derivatives were studied. Genipin derivatives 1-4 were obtained in mild to high yield. Compounds 1 and 4 are more stable than genipin if exposed to nucleophiles. All the derivatives display higher neurotrophic activities than genipin. Compound 4 is the most active, with the least optimal dose. Both genipin and 4 up-regulated the activity of nNOS in PC12 cells. The effect of 4 is inhibited not only by 7-NI, a specific inhibitor of nNOS, but also by L-NIO, a specific inhibitor of eNOS; in the case of genipin, its effect is only inhibited by 7-NI. All the results indicate that 4 is a promising lead compound for the development of new drugs in the treatment of neurodegenerative diseases with the ability to address multiple drug targets.

Topics: Animals; Cell Survival; Gardenia; Iridoids; Neurons; Neuroprotective Agents; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type III; PC12 Cells; Rats

Diabetic nephropathy (DN) is one of the most common causes of end stage renal disease (ESRD) in China, which requires renal replacement therapy. Recent investigations have suggested an essential role of podocyte injury in the initial stage of DN. This study investigated the potential therapeutic role of genipin, an active extract from a traditional Chinese medicine, on progression of DN in diabetic mice induced by intraperitoneally injection of streptozocin (STZ). In diabetic mice, orally administration of genipin postponed the progression of DN, as demonstrated by ameliorating body weight loss and urine albumin leakage, attenuating glomerular basement membrane thickness, restoring the podocyte expression of podocin and WT1 in diabetic mice. The protective role of genipin on DN is probably through suppressing the up-regulation of mitochondrial uncoupling protein 2 (UCP2) in diabetic kidneys. Meanwhile, through inhibiting the up-regulation of UCP2, genipin restores podocin and WT1 expression in cultured podocytes and attenuates glucose-induced albumin leakage through podocytes monolayer. Therefore, these results revealed that genipin inhibited UCP2 expression and ameliorated podocyte injury in DN mice.

Topics: Administration, Oral; Albuminuria; Animals; Cholagogues and Choleretics; Diabetes Mellitus, Experimental; Diabetic Nephropathies; Endopeptidases; Gene Expression Regulation; Iridoids; Male; Medicine, Chinese Traditional; Mice; Podocytes; Ubiquitin-Specific Proteases

Exogenous collagen cross-linking has been investigated as method of reinforcing scleral biomechanics, with the goal of counteracting scleral weakening that occurs at the onset of myopia. This study uses whole globe inflation testing to investigate the biomechanical effect of treating posterior sclera with the collagen cross-linking agents methylglyoxal and genipin. Pairs of porcine eyes were treated in four ways. Three groups involved 1% methylglyoxal: two-hour (Group I) or thirty-minute (Group II) incubation of the whole globe, and thirty-minute incubation of only the posterior sclera of the intact eye (Group III). Group IV consisted of a thirty-minute incubation of the posterior sclera in 1% genipin. Following treatment, each eye was subjected to inflation testing under physiological pressure levels (0-150 mmHg); four strain markers on the posterior pole were tracked, providing displacement measurements in two directions. Results were used to derive load versus deformation behavior and to calculate stiffness at 0.25% strain (toe stiffness) and at peak strain (peak stiffness). Toe stiffness of Group I was 4.8 and 1.3 times greater than controls (sagittal and transverse directions, respectively: 5.23 ± 0.39 vs. 0.90 ± 0.08 mHg, P < 0.001; and 3.41 ± 0.19 vs. 1.51 ± 0.22 mHg, P < 0.01; values in mean ± SE). Group II was 7.4 and 4.3 times stiffer than controls (sagittal and transverse directions, respectively: 5.26 ± 0.49 vs. 0.63 ± 0.10 mHg, P < 0.02; and 3.44 ± 0.44 vs. 0.65 ± 0.07 mHg, P < 0.003). Group III was 3.6 and 3.4 times stiffer than controls (sagittal and transverse directions, respectively: 5.21 ± 0.39 vs. 1.13 ± 0.31 mHg, P < 0.01; and 4.94 ± 1.48 vs. 1.13 ± 0.25, P < 0.01), while Group IV was 8.2 and 2.8 times stiffer than controls (sagittal and transverse: 12.36 ± 1.96 vs. 1.35 ± 0.14 mHg, P < 0.01; and 12.45 ± 1.34 vs. 3.27 ± 0.50 mHg, P < 0.05). In all groups, there was no significant difference in peak stiffness after scleral cross-linking (SXL). At low strain, the posterior sclera was stiffer in both measured directions following methylglyoxal and genipin treatments, however at peak strain the treated sclera was not stiffer. Additionally, the saturation level of scleral stiffening by methylglyoxal can be reached within thirty minutes of treatment.

Topics: Animals; Biomechanical Phenomena; Collagen; Cross-Linking Reagents; Elasticity; Iridoids; Ocular Physiological Phenomena; Pyruvaldehyde; Sclera; Swine

In this study, the effect of different crosslinkers including glutaraldehyde (GTA), genipin (GIP) and procyanidine (PA) on the properties of the electrospun gelatin membranes was compared. The water-resistant ability of the membranes could be significantly improved after being crosslinked with PA at T > 40 °C. In contrast with GTA and GIP, the PA-crosslinking process did not apparently affect the fibrous structure, and induced the lowest shrinkage of the membranes. At the concentration of 5% of PA, the ultimate tensile strength and elongation of the hydrated membrane were 0.87 MPa and 148%, respectively, which were higher than those of the GIP-crosslinked counterparts. In addition, the PA-crosslinked membranes displayed the highest resistance to pepsin degradation, and fibroblast cells could migrate deeper into the interior of the membranes due to the good preservation of the fibrous structure during the cell culture process.

Topics: Biflavonoids; Catechin; Cell Proliferation; Cells, Cultured; Cross-Linking Reagents; Fibroblasts; Gelatin; Glutaral; Humans; Iridoids; Membranes, Artificial; Pepsin A; Proanthocyanidins; Temperature

A transparent nanofibril suspension could be readily obtained by treating purified squid pen powder in water with ultrasonic irradiation. The obtained suspension is consisted of β-chitin nanofibrils (CNF) with 3-10 nm in width and several micrometers in length. The degree of acetylation (DA) of CNF was found to be 84% which is about 10% lower than that of untreated sample. The CNF suspension could be transformed into a durable 3-D hydrogels (CH) by simply heating to 180 °C for 1-4 h in an autoclave. Hydrophobic interaction between CNF was believed to play the major role for CNF self-assembling into hydrogels, since the as-prepared chitin hydrogels readily dissolved in a typical chaotropic solution (8 M urea) under room temperature. The hydrothermal duration and CNF concentration (0.3-2% (w/v)) strongly affected the physical properties of CH. The suspension of 1% (w/v) CNF treated with 4 h, 180 °C hydrothermal heating generated a CH with 99.3% water content, CNF with 87% crystallinity and an mechanical strength of 0.7 N breaking force.

Topics: Adhesives; Animals; Chitin; Cross-Linking Reagents; Decapodiformes; Hydrogels; Hydrophobic and Hydrophilic Interactions; Iridoids; Nanostructures; Sonication; Temperature; Water

A novel colorimetric assay for gabapentin in bulk drug and capsules has been developed via a safety-and-sustainability concerning concept. The method relied on the reaction of primary amino group of drug with non-toxic and eco-friendly genipin in totally aqueous medium to form the blue product which was subsequently measured by visible spectrophotometry at 590 nm. Under the optimized conditions, Beer's law was obeyed in the concentration range of 0.15-0.50 mM (r(2)=0.9998). It was accurate, precise and insensitive to the interferences from all related compounds specified in the United States Pharmacopeia as well as commonly used excipients. Furthermore, it gave the assay results in agreement with the pharmacopeial chromatographic method. Owing to the environmental concern and responsibility, a fast and facile method was also proposed for the treatment of waste generated from the assay based on the decoloration by using gypsum as a cheap and commonly available adsorbent. After the treatment, more than 95% of the initial blue product was removed from the waste solution and the treated waste was proven to be safe for aquatic organisms, as studied in brine shrimp and guppy fishes. Therefore, this work not only reports for the first time the application of naturally derived genipin to drug analysis, but also presents a new and contemporary paradigm that illustrates the fully benign-by-design development of the analytical methodologies in the era of Green Chemistry, starting from the safe source of reagents toward the safe sink when waste is released into the environment.

Topics: Amines; Biological Products; Capsules; Color; Colorimetry; Cyclohexanecarboxylic Acids; Gabapentin; gamma-Aminobutyric Acid; Hot Temperature; Hydrogen-Ion Concentration; Indicators and Reagents; Iridoids; Laboratories; Reproducibility of Results; Safety; Time Factors; Waste Management

Reconstruction of large bone defects remains problematic in orthopedic and craniofacial clinical practice. Autografts are limited in supply and are associated with donor site morbidity while other materials show poor integration with the host's own bone. This lack of integration is often due to the absence of periosteum, the outer layer of bone that contains osteoprogenitor cells and is critical for the growth and remodeling of bone tissue. In this study we developed a one-step platform to electrospin nanofibrous scaffolds from chitosan, which also contain hydroxyapatite nanoparticles and are crosslinked with genipin. We hypothesized that the resulting composite scaffolds represent a microenvironment that emulates the physical, mineralized structure and mechanical properties of non-weight bearing bone extracellular matrix while promoting osteoblast differentiation and maturation similar to the periosteum. The ultrastructure and physicochemical properties of the scaffolds were studied using scanning electron microscopy and spectroscopic techniques. The average fiber diameters of the electrospun scaffolds were 227 ± 154 nm as spun, and increased to 335 ± 119 nm after crosslinking with genipin. Analysis by X-ray diffraction, Fourier transformed infrared spectroscopy and energy dispersive spectroscopy confirmed the presence of characteristic features of hydroxyapatite in the composite chitosan fibers. The Young's modulus of the composite fibrous scaffolds was 142 ± 13 MPa, which is similar to that of the natural periosteum. Both pure chitosan scaffolds and composite hydroxyapatite-containing chitosan scaffolds supported adhesion, proliferation and osteogenic differentiation of mouse 7F2 osteoblast-like cells. Expression and enzymatic activity of alkaline phosphatase, an early osteogenic marker, were higher in cells cultured on the composite scaffolds as compared to pure chitosan scaffolds, reaching a significant, 2.4 fold, difference by day 14 (p < 0.05). Similarly, cells cultured on hydroxyapatite-containing scaffolds had the highest rate of osteonectin mRNA expression over 2 weeks, indicating enhanced osteoinductivity of the composite scaffolds. Our results suggest that crosslinking electrospun hydroxyapatite-containing chitosan with genipin yields bio-composite scaffolds, which combine non-weight-bearing bone mechanical properties with a periosteum-like environment. Such scaffolds will facilitate the proliferation, differentiation and maturation of osteobl

Topics: Animals; Bone and Bones; Cell Line; Cell Proliferation; Cell Survival; Chitosan; Durapatite; Iridoids; Mice; Microscopy, Electron, Scanning; Nanofibers; Osteoblasts; Spectroscopy, Fourier Transform Infrared; Tissue Engineering; X-Ray Diffraction

Controlled release carriers are often made into microspheres or tablets. Systematic and quantitative characterization of porous tissue engineered scaffolds as release carriers have not been done. Chitosan and chitosan crosslinked with various concentrations of genipin were made into porous tissue engineered scaffolds. Their thermal and enzymatic stabilities, hydrophobicities, porous structures, swelling and release properties, and compressional moduli were measured. The effects of scaffolds loaded with pentoxifylline (PTX) in suppressing inflammatory reactions in vitro were quantified.Fourier Transform Infrared spectra showed new bond formation after crosslinking chitosan with genipin. As genipin increased from 0.01% to 0.1%, the crosslinked chitosan scaffolds swelled 0.5% to 1.8% less, had 1.9-5% decrease in PTX release efficiencies, became less wettable, were less favorable for initial cell attachment, had 4-20% increase in Young's modulus and were more resistant to enzymatic degradation. In vitro tests showed that when PTX was released more slowly from crosslinked scaffolds, PTX became more effective in suppressing macrophage cells from releasing IL-6 and TNF-α.

Topics: Animals; Anti-Inflammatory Agents; Cell Line; Chitosan; Cross-Linking Reagents; Delayed-Action Preparations; Iridoids; Macrophages; Mice; Microscopy, Electron, Scanning; Pentoxifylline; Spectroscopy, Fourier Transform Infrared; Tissue Scaffolds

Hepatocellular carcinoma is one of the most malignant human cancers with high metastatic potential. The aim of this study is to investigate the anti-metastatic effect of genipin and its underlying mechanism.. The anti-metastatic potential of genipin was evaluated by both cell and animal model. Wound healing and invasion chamber assays were introduced to examine the anti-migration and anti-invasion action of genipin in human hepatocellular carcinoma cell HepG2 and MHCC97L; orthotopical implantation model was used for in vivo evaluation. Gelatin Zymography, Immunoblotting, quantitative real-time polymerase chain reaction and ELISA assays were used to study the mechanisms underlying genipin's anti-metastatic effect.. Genipin suppresses the motility and invasiveness of HepG2 and MHCC97L at non-toxic doses, which may be correlated to the inhibition of genipin on MMP-2 activities in the cells. No significant reduced expression of MMP-2 was observed either at mRNA or at protein level. Furthermore, genipin could specifically up-regulate the expression of TIMP-1, the endogenous inhibitor of MMP-2 activities. Silencing of TIMP-1 by RNA interference abolishes genipin's anti-metastaic effect. Activation of p38 MAPK signaling was observed in genipin-treated cells, which is responsible for the TIMP-1 overexpression and MMP-2 inhibition. Presence of SB202190, the p38 MAPK inhibitor, attenuates the anti-metastatic potential of genipin in hepatocellular carcinoma. Orthotopical implantation model showed that genipin could suppress the intrahepatic metastatic as well as tumor expansion in liver without exhibiting potent toxicity.. Our findings demonstrated the potential of genipin in suppressing hepatocellular carcinoma metastasis, and p38/TIMP-1/MMP-2 pathway may be involved as the key mechanism of its anti-metastasis effect.

Topics: Animals; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cholagogues and Choleretics; Down-Regulation; Gene Silencing; Humans; Imidazoles; Iridoids; Liver Neoplasms; Matrix Metalloproteinase 2; Mice; Mice, Nude; Neoplasm Transplantation; Neoplasms, Second Primary; p38 Mitogen-Activated Protein Kinases; Protein Kinase Inhibitors; Pyridines; RNA, Small Interfering; Signal Transduction; Tissue Inhibitor of Metalloproteinase-1; Up-Regulation

Collagen-based tissue mimics are important in clinical research because collagen is the main structural element in tendons. The current study aimed to improve the mechanical strength of Electronically Aligned Collagen (ELAC) threads by optimizing several crosslinking parameters. The results indicated that elevating the concentration of genipin to 2% and the solvent to 90% ethanol significantly enhanced the wet ultimate tensile stress of ELAC threads to 109 MPa with a crosslinking degree of 65%. Furthermore, significantly higher adhesion and proliferation of hMSCs was observed in ELAC threads crosslinked with 2% genipin in 90% ethanol compared to 0.625% genipin in 1X PBS. In conclusion, ELAC threads with mechanical strength on par with native tendon have significant potential to be used as scaffolds in tendon tissue engineering applications.

Topics: Biomechanical Phenomena; Biomimetic Materials; Cell Adhesion; Cell Proliferation; Collagen; Electrochemistry; Ethanol; Humans; Iridoids; Mechanical Phenomena; Mesenchymal Stem Cells; Tendons; Time Factors

Chitosan is a naturally occurring cationic polysaccharide and has attracted much attention in the past decade as an important ophthalmic biomaterial. We recently demonstrated that the genipin (GP) cross-linked chitosan is compatible with human retinal pigment epithelial cells. The present work aims to further investigate the in vivo biocompatibility of GP-treated chitosan (GP-chi group) by adopting the anterior chamber of a rabbit eye model. The glutaraldehyde (GTA) cross-linked samples (GTA-chi group) were used for comparison. The 7-mm-diameter membrane implants made from either non-cross-linked chitosan or chemically modified materials with a cross-linking degree of around 80% were inserted in the ocular anterior chamber for 24 weeks and characterized by slit-lamp and specular microscopic examinations, intraocular pressure measurements, and corneal thickness measurements. The interleukin-6 expressions at mRNA level were also detected by quantitative real-time reverse transcription polymerase chain reaction. Results of clinical observations showed that the overall ocular scores in the GTA-chi groups were relatively high. In contrast, the rabbits bearing GP-chi implants in the anterior chamber of the eye exhibited no signs of ocular inflammation. As compared to the non-cross-linked counterparts, the GP-chi samples improved the preservation of corneal endothelial cell density and possessed better anti-inflammatory activities, indicating the benefit action of the GP cross-linker. In summary, the intracameral tissue response to the chemically modified chitosan materials strongly depends on the selection of cross-linking agents.

Topics: Animals; Anterior Chamber; Biocompatible Materials; Chitosan; Cornea; Cross-Linking Reagents; Gene Expression Regulation; Glutaral; Inflammation; Interleukin-6; Intraocular Pressure; Iridoids; Prostheses and Implants; Rabbits

Primary hepatocyte is probably the preferred cell for cell therapy in liver regeneration. However, its non-ideal proliferation capacity and rapid loss of phenotype during 2D culture compromises the quality and quantity of the transplanted hepatocytes, resulting in variable success rates of this treatment. Many studies have shown that the formation of 3D hepatocellular spheroids aids in the maintenance of liver-specific functions in hepatocytes. However, many of the methodologies employed require a sophisticated set-up or specialized equipment which makes it uneconomical to scale up for clinical applications. In this study, we have developed dual-functioning genipin crosslinked gelatin microspheres that serve as cell carriers as well as porogens for delivering the model cells and also for creating cavities. The cells were first seeded onto genipin crosslinked gelatin microspheres for attachment, followed by encapsulation in alginate hydrogel. Collagenase, MMP-9, was introduced either in the culture media or mixed with alginate precursor solution to allow microsphere degradation for creating cavities within the gel bulk. Accordingly, the cells proliferate within the cavities, forming hepatocellular aggregates while the alginate hydrogel serves as a confinement, restricting the size and the shape of the aggregates to the size of the cavities. In addition, the final hepatocellular aggregates could be harvested from the system by removing the alginate hydrogel via citrate treatment. Therefore, this versatile platform not only has the advantage of injectability and simplicity, the cellular aggregates generated are in a controlled size and shape and can be extracted from the system.

Topics: Albumins; Alginates; Animals; Cell Culture Techniques; Cell Survival; Collagenases; Cross-Linking Reagents; Cytochrome P-450 CYP1A1; Gelatin; Hep G2 Cells; Hepatocytes; Humans; Hydrogel, Polyethylene Glycol Dimethacrylate; Hydrogels; Iridoids; Liver Regeneration; Male; Matrix Metalloproteinase 9; Mice; Mice, Inbred BALB C; Mice, Nude; Microspheres; Time Factors

A simple and versatile approach is proposed to use cross-linked polypeptide hydrogels as templates for silica mineralization, allowing the synthesis of polypeptide-silica hybrid hydrogels and mesoporous silica (meso-SiO(2)) by subsequent calcination. The experimental data revealed that the cross-linked polypeptide hydrogels comprised of interconnected, membranous network served as templates for the high-fidelity transcription of silica replicas spanning from nanoscale to microscale, resulting in hybrid network comprised of interpenetrated polypeptide nanodomains and silica. The mechanical properties of these as-prepared polypeptide-silica hybrid hydrogels were found to vary with polypeptide chain length and composition. The synergy between cross-link, hydrophobic interaction, and silica deposition can lead to the enhancement of their mechanical properties. The polypeptide-silica hybrid hydrogel with polypeptide and silica content as low as 1.1 wt% can achieve 114 kN/m(2) of compressive strength. By removing the polypeptide nanodomains, mesoporous silicas with average pore sizes ranged between 2 nm and 6 nm can be obtained, depending on polypeptide chain length and composition. The polypeptide-silica hybrid hydrogels demonstrated good cell compatibility and can support cell attachment/proliferation. With the versatility of polymer chemistry and feasibility of amine-catalyzed sol-gel chemistry, the present method is facile for the synthesis of green nanocomposites and biomaterials.

Topics: 3T3 Cells; Animals; Chromatography, Gel; Hydrogels; Iridoids; Mice; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Oxides; Peptides; Silicates; Spectroscopy, Fourier Transform Infrared

Uncoupling protein 2 (UCP2) was reported to be involved in lipid metabolism through regulating the production of superoxide anion. However, the role of UCP2 in hepatocytes steatosis has not been determined. We hypothesized that UCP2 might regulate hepatic steatosis via suppressing oxidative stress.. We tested this hypothesis in an in vitro model of hepatocytic steatosis in HepG2 cell lines induced by palmitic acid (PA). We found that treatment with PA induced an obvious lipid accumulation in HepG2 cells and a significant increase in intracellular triglyceride content. Moreover, the specific inhibition of UCP2 by genipin remarkably exacerbated PA-induced hepatocytes steatosis. Interestingly, the PA-induced superoxide overproduction can also be enhanced by incubation with genipin. In addition, administration with the antioxidant tempol abolished genipin-induced increase in intracellular lipid deposition. We further found that genipin significantly increased the protein expression of fatty acid translocase (FAT)/CD36.. These findings suggest that UCP2 plays a protective role in PA-induced hepatocytic steatosis through ameliorating oxidative stress.

Topics: CD36 Antigens; Fatty Liver; Hep G2 Cells; Humans; Ion Channels; Iridoids; Lipid Metabolism; Mitochondrial Proteins; Models, Biological; Oxidative Stress; Palmitic Acid; Triglycerides; Uncoupling Protein 2

To investigate the influence of cross-linking agents genipin and glutaraldehyde on the composite bio-sponge.. The composite bio-sponge was prepared with the technology of lyophilization. The degree of cross linking was determined using absorptiometry of trinitrobenzenesulfonic acid; the cytotoxicity was tested by MTT assay; the degradation rate in vitro was valuated by lysozyme degradation.. (1) The degree of cross linking of composite bio-sponge crosslinked using genipin and glutaraldehyde increased with the crosslinking time, and reached 26.43% and 54.63% respectively after crosslinking 3 d. (2) The water absorption rate of composite bio-sponge crosslinked using genipin was better than that of crosslinked using glutaraldehyde. (3) In the initial stage of cells incubation, all extracts of composite bio-sponges crosslinked using genipin and glutaraldehyde inhibited the growth of the cells, and the inhibition decreased with the incubation time; but the cytotoxicity of composite bio-sponge crosslinked using glutaraldehyde was higher than that of crosslinked using genipin. (4) After soaking in saline for 4 weeks, the degradation rate of composite bio-sponge crosslinked using genipin or glutaraldehyde was 32.1%, 28.4%, respectively; however, after soaking in saline containing lysozyme for 40 h, the degradation rate of composite bio-sponge was 36.7%, 31.2%, respectively.. Compared with the composite bio-sponge crosslinked using glutaraldehyde, the degree of cross linking and the cytotoxicity of the composite bio-sponge crosslinked using genipin decreased; however, the water absorption rate and the degradation rate increased.

Topics: Biocompatible Materials; Cross-Linking Reagents; Glutaral; Iridoids; Materials Testing

To optimize the process for preparing genipin with enzymolyzed geniposide by an orthogonal experiment.. The optimal enzymolysis process was selected by an orthogonal experiment, with the concentration of geniposide as the index as well as enzyme quantity, pH of enzymolysis solution, enzymolysis time and enzymolysis temperature as considerations.. The optimal hydrolysis conditions were as follow: rough genipin samples at the concentration of 40 g x L(-1) were selected and shook on a table concentrator at a speed of 100 r x min(-1), added with beta-glucosidase-geniposide 1 : 1 (weight proportion), with pH of enzymolysis solution at 4.5, hydrolyzation temperature at 50 degrees C, the conversion rate of genipin could reach 85.8%.. The process is so stable and feasible that it can provide theoretical basis for the preparation of genipin with enzymolyzed geniposide.

Topics: Hydrolysis; Iridoids; Technology, Pharmaceutical

The difficulty of eliminating Enterococcus faecalis and other bacteria infecting dental root canals makes it desirable to develop formulations capable of sustained release of antibiotics within the canal. With this function in view, in this work we compared the mechanical, drug release and biocompatibility properties of amoxicillin-loaded collagen (CL) and CL complexed with poly[(methyl vinyl ether)-co-(maleic anhydride)] (PVMMA), with or without glutaraldehyde (GTA) or the natural product genipin (GN) as cross-linker. Collagen was not denatured by complexation with PVMMA. Only CL-PVMMA-GN sponges did not disintegrate during 7 days exposure to cell culture medium (un-cross-linked CL disintegrated within 24 h and un-cross-linked CL-PVMMA within 4 days), and CL-PVMMA-GN sponges also exhibited the most appropriate combination of mechanical properties (hardness, modulus of deformability and plasticity). CL-PVMMA-GN sponges absorbed aqueous medium faster than other cross-linked formulations, but their maximum uptake was less; and drug release from CL-PVMMA-GN sponges tended to be faster than from any other, except un-cross-linked CL-PVMMA, maximum release taking about 4 days. No formulation significantly altered the viability of L929 fibroblast-like mouse connective tissue cells, but cells growing on sponges showed signs of non-adherence. It is concluded that genipin-cross-linked CL-PVMMA sponges merit further investigation as antibiotics vehicles and aids to tissue regeneration in the dental root canal.

Topics: Amoxicillin; Animals; Anti-Bacterial Agents; Biocompatible Materials; Cell Adhesion; Cell Line; Cell Survival; Collagen; Culture Media; Delayed-Action Preparations; Drug Liberation; Fibroblasts; Glutaral; Iridoids; Maleates; Materials Testing; Mice; Polyethylenes; Root Canal Filling Materials; Water

The gelatin plasma substitute is often polydisperse and heterogenous, making it difficult to determine the elimination rate and half-life in the body. In this study, one method was developed based on quantitative determination of hydroxyproline derivatives. Two plasma substitutes were prepared by succinylation and genipin-crosslinking, respectively. After transfusion, the blood samples were hydrolyzed and derivatized, and then analyzed by HPLC. A two-phase exponential association equation was used for fitting the time-concentration curves. The results indicated that this method could be used for quantitative determination of gelatin in blood, and the pharmacokinetic parameters such as elimination rate and half-life.

Topics: Animals; Blood Vessels; Blood Volume; Cattle; Cross-Linking Reagents; Dinitrofluorobenzene; Gelatin; Half-Life; Hemorrhage; Hydrolysis; Hydroxyproline; Iridoid Glycosides; Iridoids; Male; Plasma Substitutes; Rabbits; Succinic Anhydrides

Biochemical studies aimed at optimization of protein crosslinking formulations for the treatment of degenerative disc disease and subsequent biomechanical testing of tissues treated with these formulations.. To optimize protein crosslinking formulations for treatment of degenerating spinal discs.. Nonsurgical exogenous crosslinking therapy is a potential new, noninvasive technology for the treatment of degenerative disc disease. The technology is based on the injection of protein crosslinking reagents into the pathologic disc to restore its mechanical properties and also to potentially increase the permeability of the tissue and so facilitate the exchange of waste products and nutrients.. Diffusion of genipin (GP) was monitored following injection into spinal discs and the effects of surfactants on diffusion studied. Formulations for GP and methylglyoxal (MG) were biochemically optimized and used to treat bovine spinal discs. Their effects on bovine anulus tissue were evaluated using a circumferential tensile test, while the GP formulation was also tested with respect to its ability to reduce disc bulge under load.. GP exhibited a distinct time-dependent diffusion and sodium-dodecyl-sulfate, but not Tween-20, enhanced diffusion by 30%. Two crosslinkers, GP and MG, were inhibited by amines but enhanced by phosphate ions. Both formulations could enhance a number of physical parameters of bovine anulus tissue, while the GP formulation could reduce disc bulge following injections into spinal discs.. Formulations lacking amines and containing phosphate ions appear to be promising candidates for clinical use of the crosslinkers GP and MG.

Topics: Animals; Biomechanical Phenomena; Cattle; Cross-Linking Reagents; Diffusion; In Vitro Techniques; Injections, Spinal; Intervertebral Disc; Intervertebral Disc Degeneration; Iridoid Glycosides; Iridoids; Lumbar Vertebrae; Polysorbates; Pyruvaldehyde; Sodium Dodecyl Sulfate; Surface-Active Agents; Tensile Strength; Time Factors

This study evaluates the feasibility of poly(ε-caprolactone) as a tracheal replacement. To improve biocompatibility, the lumen was modified by gelatin hydrogel crosslinked with two different reagents, EDC and genipin. It was found that the choice of crosslinking agents could significantly affect human lung carcinoma cell proliferation. Genipin-crosslinked gelatin hydrogel had significantly better cell proliferation than EDC-crosslinked hydrogel. The study further investigated the performance of the PCL tube modified by genipin-crosslinked gelatin, using a rabbit tracheal implantation model with implants harvested and histologically examined. In vivo results showed that the PCL tube possessed suitable mechanical properties for resisting collapse during implantation. Additionally, PCL modified by genipin-crosslinked gelatin was found to suppress granulation tissue growth and prolong animal survival time in comparison with the original PCL tube. Genipin could be an effective treatment to reduce granulation tissue formation at the tracheal anastomoses.

Topics: Animals; Bioprosthesis; Cell Line, Tumor; Cell Proliferation; Gelatin; Humans; Hydrogels; Iridoid Glycosides; Iridoids; Polyesters; Porosity; Rabbits; Tissue Scaffolds; Trachea

A cross-linking reagent is required to improve mechanical strength and degradation properties of biopolymers for tissue engineering. To find the optimal preparative method, we prepared diverse genipin-cross-linked chitosan/collagen scaffolds using different genipin concentrations and various cross-linking temperatures and cross-linking times. The compressive strength increased with the increasing of genipin concentration from 0.1 to 1.0%, but when concentration exceeded 1.0%, the compressive strength decreased. Similarly, the compressive strength increased with the increasing of temperature from 4 to 20°C, but when temperature reached 37°C, the compressive strength decreased. Showing a different trend from the above two factors, the effect of cross-linking time on the compressive strength had a single increasing tendency. The other results also demonstrated that the pore size, degradation rate and swelling ratio changed significantly with different cross-linking conditions. Based on our study, 1.0% genipin concentration, 20°C cross-linking temperature and longer cross-linking time are recommended.

Topics: Animals; Biocompatible Materials; Biomechanical Phenomena; Cartilage; Cells, Cultured; Chitosan; Collagen; Compressive Strength; Cross-Linking Reagents; Dose-Response Relationship, Drug; Female; Iridoid Glycosides; Iridoids; Materials Testing; Porosity; Rabbits; Temperature; Time Factors; Tissue Engineering; Tissue Scaffolds

The fabrication of a fibrous collagen scaffold using electrospinning is desirable for tissue-engineering applications. Previously, electrospun collagen fibers were shown to be unstable in aqueous environments and, therefore, cross-linking is essential to stabilize these fibers. In this study genipin, a significantly less cytotoxic cross-linking agent compared to glutaraldehyde, was used to cross-link electrospun collagen fibers. The significance of this research lies in the use of four alcohol/water solvent systems to carry out the crosslinking reaction to maintain fibrous morphology during cross-linking. The four cross-linking conditions established were: (1) ethanol, 5% water and 3 days, (2) ethanol, 3% water and 5 days, (3) ethanol, 5% water and 5 days, and (4) isopropanol, 5% water and 5 days at a genipin concentration of 0.03 M. Results illustrated that genipin-cross-linking was effective in maintaining collagen fiber integrity in aqueous and cell culture media environments for up to 7 days. In addition, it was shown that fiber swelling could be controlled by using different cross-linking conditions. Swelling of cross-linked fibers immersed in Dulbecco's modified eagle medium for 7 days ranged from 0 to 59 ± 4%. The cross-linked fibers were analyzed using scanning electron microscopy, Fourier transform infrared spectroscopy and ninhydrin assay. Finally, studies using primary human fibroblasts indicated good cell adhesion to these scaffolds. Overall, our data suggest that these stabilized fibrous collagen scaffolds provide a promising environment for tissue-regeneration applications.

Topics: 2-Propanol; Cell Adhesion; Cell Count; Collagen; Ethanol; Fibroblasts; Glutaral; Humans; Iridoids; Materials Testing; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Ninhydrin; Spectroscopy, Fourier Transform Infrared; Time Factors; Tissue Engineering; Tissue Scaffolds; Water

The development of suitable biomimetic three-dimensional scaffolds is a fundamental requirement of tissue engineering. This paper presents the first successful attempt to obtain electrospun gelatin nanofibers cross-linked with a low toxicity agent, genipin, and able to retain the original nanofiber morphology after water exposure. The optimized procedure involves an electrospinning solution containing 30 wt.% gelatin in 60/40 acetic acid/water (v/v) and a small amount of genipin, followed by further cross-linking of the as-electrospun mats in 5% genipin solution for 7 days, rinsing in phosphate-buffered saline and then air drying at 37°C. The results of scanning electron microscopy investigations indicated that the cross-linked nanofibers were defect free and very regular and they also maintained the original morphology after exposure to water. Genipin addition to the electrospinning solution dramatically reduced the extensibility of the as-electrospun mats, which displayed further remarkable improvements in elastic modulus and stress at break after successive cross-linking up to values of about 990 and 21 MPa, respectively. The results of the preliminary in vitro tests carried out using vascular wall mesenchymal stem cells indicated good cell viability and adhesion to the gelatin scaffolds.

Topics: Animals; Cell Proliferation; Cross-Linking Reagents; Gelatin; Humans; Iridoid Glycosides; Iridoids; Materials Testing; Mechanical Phenomena; Mesenchymal Stem Cells; Nanofibers; Particle Size; Solutions; Spectroscopy, Fourier Transform Infrared; Sus scrofa; Tissue Engineering; Water; X-Ray Diffraction

In engineered regenerative medicine, various types of scaffolds have been customized to pursue the optimal environment for different types of therapeutic cells. In liver therapeutic research, hepatocytes require attachment to solid anchors for survival and proliferation before they could grow into cellular aggregates with enhanced functionalities. Among the various biomaterials scaffolds and vehicles, microspherical cell carriers are suited to these requirements. Individual spheres may provide two-dimensional (2D) cell-affinitive surfaces for cell adhesion and spreading; whereas multiple microcarriers may form three-dimensional (3D) matrices with inter-spherical space for cell expansion and multicellular aggregation. In this study, we culture human liver carcinoma cell line (HepG2) cells on genipin-crosslinked gelatin microspheres of two different sizes. Results suggest that both microcarriers support cell adhesion, proliferation, and spontaneous formation of hepatocellular aggregates, among which the spheres with bigger size (200-300 μm) seem more favorable than the smaller ones in terms of aggregate formation and liver specific functionalities. These findings suggest that the genipin-crosslinked microcarrier is a competent vehicle for liver cell delivery.

Topics: Cell Line, Tumor; Cell Proliferation; Cholagogues and Choleretics; Cross-Linking Reagents; Gelatin; Hepatocytes; Humans; Iridoid Glycosides; Iridoids; Microspheres; Molecular Structure; Tissue Scaffolds

Hydrogels are extensively studied as carrier matrices for the controlled release of bioactive molecules. The aim of this study was to design gelatin-based hydrogels crosslinked with genipin and study the impact of crosslinking temperature (5, 15 or 25°C) on gel strength, microstructure, cytocompatibility, swelling and drug release. Gels crosslinked at 25°C exhibited the highest Flory-Rehner crosslink density, lowest swelling ratio and the slowest release of indomethacin (Idn, model anti-inflammatory drug). Diffusional exponents (n) indicated non-Fickian swelling kinetics while drug transport was anomalous. Hydrogel biocompatibility, in vitro cell viability, cell cycle experiments with AH-927 and HaCaT cell lines indicated normal cell proliferation without any effect on cell cycle. Overall, these results substantiated the use of genipin-crosslinked hydrogels as a viable carrier matrix for drug release applications.

Topics: Adsorption; Animals; Anti-Inflammatory Agents; Biocompatible Materials; Cats; Cells, Cultured; Cross-Linking Reagents; Drug Carriers; Gelatin; Humans; Indomethacin; Iridoid Glycosides; Iridoids; Kinetics; Metabolome; Swine; Water

The ability of mesenchymal stem cells to self-renew and differentiate into specialized cell lineages makes them promising tools for regenerative medicine. Local injection and use of scaffolds had been employed earlier to deliver these cells; yet, an optimal delivery system remains to be identified. Here, using genipin, which is a non-toxic natural cross linker for proteins, we prepared alginate-chitosan polymeric microcapsules (GCAC) to develop an efficient stem cell delivery system. We investigated the properties of this membrane along with the encapsulated adipose tissue-derived stem cells (ASCs) and compared that with the widely used alginate poly-lysine (APA) membranes. The GCAC membrane was able to support cell viability, augment cell growth, and showed better results under external rotational and osmotic pressures with about 30% of the ruptured capsules in comparison to 60% ruptured APA capsules. The membrane also provided immune-protection to the entrapped cells as demonstrated by the lymphocyte proliferation assay. The capsule also has potential for long-term storage. The encapsulated four million ASCs also showed steady secretion of approximately 4600 pg vascular endothelial growth factor (VEGF) over 15-day time period comparable to that of free cells. Furthermore, the encapsulated ASCs showed around 3.8-fold increase in VEGF secretion after 72 h hypoxic conditions in comparison to normoxic conditions. This increased VEGF expression resulted in improved angiogenic potential of the bioactive capsules as noted by enhanced endothelial cell growth. GCAC encapsulation also did not show any effect on their differentiation ability. Thus, because of these biocompatible and bioactive attributes, genipin cross-linked polymeric microcapsules can emerge as a potentially important tool for improved stem cell-based therapy and cell delivery applications.

Topics: Adipose Tissue; Alginates; Animals; Capsules; Cell Proliferation; Cells, Cultured; Chitosan; Endothelial Growth Factors; Enzyme-Linked Immunosorbent Assay; Glucuronic Acid; Hexuronic Acids; Humans; Iridoid Glycosides; Iridoids; Lymphocytes; Polymers; Rats; Stem Cells; Transduction, Genetic

The aim of this study was to investigate the feasibility of fabricating porous crosslinked chitosan hydrogels in an aqueous phase using dense gas CO(2) as a foaming agent. Highly porous chitosan hydrogels were formed by using glutaraldehyde and genipin as crosslinkers. The method developed here eliminates the formation of a skin layer, and does not require the use of surfactants or other toxic reagents to generate porosity. The chitosan hydrogel scaffolds had an average pore diameter of 30-40 μm. The operating pressure had a negligible effect on the pore characteristics of chitosan hydrogels. Temperature, reaction period, type of biopolymer and crosslinker had a significant impact on the pore size and characteristics of the hydrogel produced by dense gas CO(2). Scanning electron microscopy and histological analysis confirmed that the resulting porous structures allowed fibroblasts seeded on these scaffolds to proliferate into the three-dimensional (3-D) structure of these chitosan hydrogels. Live/dead staining and MTS analysis demonstrated that fibroblast cells proliferated over 7 days. The fabricated hydrogels exhibited comparable mechanical strength and swelling ratio and are potentially useful for soft tissue engineering applications such as skin and cartilage regeneration.

Topics: Atmosphere; Carbon Dioxide; Cell Proliferation; Chitosan; Cross-Linking Reagents; Fibroblasts; Glutaral; Humans; Iridoid Glycosides; Iridoids; Materials Testing; Microscopy, Confocal; Porosity; Pressure; Solubility; Spectroscopy, Fourier Transform Infrared; Temperature; Tissue Engineering; Tissue Scaffolds; Water

This study proposes a biodegradable GGT composite nerve guide conduit containing genipin-cross-linked gelatin and tricalcium phosphate (TCP) ceramic particles in peripheral nerve regeneration. The proposed genipin-cross-linked gelatin annexed with TCP ceramic particles (GGT) conduit was dark bluish and round with a rough and compact surface. Water uptake and swelling tests indicated that the hydrated GGT conduit exhibited increased stability with not collapsing or stenosis. The GGT conduit had higher mechanical properties than the genipin-cross-linked gelatin without TCP ceramic particles (GG) conduit and served as a better nerve guide conduit. Cytotoxicity tests revealed that the GGT conduit was not toxic and that it promoted the viability and growth of neural stem cells. The experiments in this study confirmed the effectiveness of the GGT conduit as a guidance channel for repairing a 10-mm gap in rat sciatic nerve. Walking track analysis showed a significantly higher sciatic function index score and better toe spreading development in the GGT group than in the silicone group 8 weeks after implantation. Gross examination revealed that the diameter of the intratubular newly formed nerve fibers in GGT conduits exceeded those in silicone tubes after the implantation period. Histological observations revealed that the morphology and distribution patterns of nerve fibers in the GGT conduits at 8 weeks after implantation were similar to those of normal nerves. The quantitative results indicated the superiority of the conduits over the silicone tubes. Motor functional and histomorphometric assessments demonstrate that the proposed GGT conduit is a suitable candidate for peripheral nerve repair.

Topics: Animals; Biological Assay; Calcium Phosphates; Cell Adhesion; Cell Count; Cell Death; Cell Proliferation; Cross-Linking Reagents; Gelatin; Guided Tissue Regeneration; Iridoid Glycosides; Iridoids; Mechanical Phenomena; Nerve Regeneration; Neural Stem Cells; Rats; Rats, Sprague-Dawley; Rats, Wistar; Recovery of Function; Sciatic Nerve; Tissue Scaffolds; Walking; Wound Healing

Genipin, a natural cross-linking agent, was used for the immobilization of lipase from Candida sp. 99-125 by cross-linking to two kinds of mesoporous resins. Under optimum conditions, the activity recovery of immobilized lipase on resin NKA-9 could reach up to 96.99% when the genipin concentration was 0.5%, and it could reach up to 86.18% for S-8 with a genipin concentration of 0.25%. Compared with using glutaraldehyde as a cross-linking agent, the immobilized lipase using genipin showed better pH and thermal stability, storage stability, and reusability. The residual activity of immobilized lipase using genipin as cross-linker remained more than 60% of its initial activity after six hydrolytic cycles, whereas only about 35% activity remained by using glutaraldehyde as cross-linker.

Topics: Adsorption; Candida; Cross-Linking Reagents; Enzyme Stability; Enzymes, Immobilized; Glutaral; Hydrogen-Ion Concentration; Hydrolysis; Iridoid Glycosides; Iridoids; Lipase; Olive Oil; Plant Oils; Porosity; Resins, Synthetic; Temperature

Increasing evidence has revealed that the surface characteristics of biomaterials, such as chemical composition, stiffness, and topography, especially nanotopography, significantly influence cell growth and differentiation. In this study, we examined the effect of surface biomimetic apatite nanostructure of a new hydroxyapatite-coated genipin-chitosan conjugation scaffold (HGCCS) on cell shape, cytoskeleton organization, and osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells in vitro. Cell shape and cytoskeleton organization showed significant differences between cells cultured on genipin-cross-linked chitosan framework and those cultured on HGCCS with surface apatite network-like nanostructure after 7 days of incubation in the osteogenic medium. The result of specific alkaline phosphatase activity as an indicator of osteogenic differentiation showed that the alkaline phosphatase activity of rat bone marrow-derived mesenchymal stem cells was higher on HGCCS. Based on quantitative real-time polymerase chain reaction, HGCCS induced highest mRNA expression of osteogenic differentiation makers, runt-related transcription factor 2 by 7 days, osteopontin by 7 days, and osteocalcin by 14 days, respectively. The enhanced ability of cells on HGCCS to produce mineralized extracellular matrix and nodules was also assessed on day 14 with Alizarin red staining. The results of this study suggest that the surface biomimetic apatite nanostructure of HGCCS is a critical signal cue to promoting osteogenic differentiation in vitro. These findings open a new research avenue to controlling stem cell lineage commitment and provide a promising scaffold for bone tissue engineering.

Topics: Animals; Antigens, Differentiation; Biomimetic Materials; Bone Marrow; Calcification, Physiologic; Cell Differentiation; Cells, Cultured; Chitosan; Cytoskeleton; Durapatite; Iridoid Glycosides; Iridoids; Male; Mesenchymal Stem Cells; Nanostructures; Osteogenesis; Rats; Rats, Wistar; Tissue Engineering; Tissue Scaffolds

The vascular media, a layer of the blood vessel wall containing smooth muscle cells (SMCs), are often the target functional tissue in the construction of artificial vessel. It contributes to mechanical properties and biological functions of vessels. The present study aimed to study effects of crosslinking and biomolecule conditions in the development of mechanically strong and stable, biologically functional constructs with potential for vascular media regeneration. Genipin was used to crosslink collagen-chitosan-elastin (CCE) constructs. Results revealed that mechanical strength, stiffness, and stability of CCE constructs significantly increased with genipin concentration, but crosslinking significantly inhibited SMC contraction of and invasion in gel constructs. No contraction or invasion was observed in those crosslinked with genipin at 5 mM or above. attenuated total reflectance Fourier transform infrared results showed crosslinking changed functional groups on CCE depending on genipin concentration. To enhance biological activities on crosslinked constructs, soluble molecule factors were incorporated, and their effects on SMC activities were evaluated. These conditions include heparin, platelet-derived transforming growth factor (PDGF), high-concentrated fetal bovine serum (h-FBS), a mixture of heparin and PDGF, and a mixture of h-FBS and PDGF. The h-FBS and PDGF mixture was found to stimulate a 3.2-fold increase in SMC contraction of the crosslinked gels. It was also found that PDGF and h-FBS, separately and in combination, induced SMC invasion in the crosslinked gels, while heparin attenuated PDGF-induced SMC invasion. Our study suggests that designing high-performance acellular constructs to encourage tissue regeneration should use a combination of crosslinking condition and biomolecule factor, striking a balance between mechanical properties and biological functions.

Topics: Animals; Biopolymers; Cattle; Cell Survival; Collagen; Cross-Linking Reagents; Humans; Iridoids; Myocytes, Smooth Muscle; Rats; Regeneration; Solubility; Spectroscopy, Fourier Transform Infrared; Tensile Strength; Time Factors; Tunica Media

A facile microwave-induced method was developed for synthesizing water-soluble fluorescent derivatives of alginic acid (ALG) with four different diamines, hydrazine (HY), ethylenediamine (EDA), 1,6-hexanediamine (HDA), and 1,4-cyclohexanediamine (CHDA), followed by a cross-linking reaction with a natural cross linker genipin. The ethylenediamine derivative of alginic acid (ALG-EDA) exhibited good fluorescent activity, which upon cross linking was enhanced threefold. The other amide derivatives, for example, ALG-HY, ALG-HDA, and ALG-CHDA, were not fluorescent, but their respective crosslinked products exhibited excellent fluorescent activity. The fluorescence intensity had an inverse correlation with the number of carbon atoms present in the amine, which in turn was a function of degree of substitution (DS). These fluorescent polysaccharide derivatives are of potential utility in the domain of sensor applications.

Topics: Alginates; Carbohydrate Sequence; Glucuronic Acid; Hexuronic Acids; Iridoid Glycosides; Iridoids; Microwaves; Molecular Structure; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet

It has drawn a lot of attention to target signal transducer and activator of transcription 3 (STAT3) as a potential strategy for cancer therapeutics. Using several myelogenous cell lines, the effect of genipin (an active compound of Gardenia fruit) on the STAT3 pathway and apoptosis was investigated. Genipin suppressed the constitutive STAT3 activation in U266 and U937 cells and stimulated Src homology 2 domain-containing phosphatase 1 (SHP-1), which dephosphorylates and inactivates STAT3. Specifically, genipin blocked STAT3 activation via repressing the activation of c-Src, but not Janus kinase 1 (JAK1). Genipin also downregulated the expression of STAT3 target genes including Bcl-2, Bcl-x(L) , Survivin, Cyclin D1, and VEGF. Conversely, protein tyrosine phosphatase inhibitor pervanadate blocked genipin induced STAT3 inactivation. Using DNA fragmentation or TUNEL assays, we demonstrated the apoptotic effect of genipin on U266, MM.1S, and U937 cells. Furthermore, genipin effectively potentiated the cytotoxic effect of chemotherapeutic agents, such as bortezomib, thalidomide, and paclitaxel in U266 cells. Our data suggest that through regulation of Src and SHP-1, genipin antagonizes STAT3 for the induction of apoptosis in myeloma cells.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Boronic Acids; Bortezomib; Cell Line, Tumor; Electrophoretic Mobility Shift Assay; Humans; In Situ Nick-End Labeling; Iridoid Glycosides; Iridoids; Multiple Myeloma; Pyrazines; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; STAT3 Transcription Factor

Genipin has been widely used as a natural crosslinker to substitute chemical crosslinkers such as glutaraldehyde to crosslink various biomaterials like gelatin, collagen, and chitosan. However, there are contradicting views on the cytotoxicity and safety of genipin in tissue engineering. Therefore in this study, we aimed to evaluate the toxicity of genipin on skeletal tissues cells-osteoblasts and chondrocytes as they are also representatives of typical anchorage-dependent cells (ADCs) and nontypical ADCs. Results suggest that genipin toxicity is dose dependent and acute but not time dependent on both osteoblasts and chondrocytes. In particular, chondrocytes exhibit substantial alterations in the gene expression when exposed to Maximum nontoxic concentration (MaxNC) of genipin but there were no significant changes in the genes tested in osteoblasts. Since osteoblasts are typical ADCs, cellular focal adhesion assessment was carried out with F-actin being more contracted and unorganized when exposed to minimum toxic concentration (MinTC) of genipin. The mechanisms involved in cell deaths in both cell types are believed to be similar and hence using osteoblast as the model, cells were stained positive for Annexin-V and Reactive oxygen species (ROS) level were elevated at MinTC of genipin. Collectively, genipin induced cell apoptosis via ROS production, and apparently, gene expressions could also be altered at MaxNC. For this reason, we recommend the dose of genipin to be controlled within 0.5 mM.

Topics: Animals; Annexin A5; Cell Line; Cholagogues and Choleretics; Chondrocytes; Cross-Linking Reagents; Dose-Response Relationship, Drug; Focal Adhesions; Gene Expression Regulation; Humans; Iridoid Glycosides; Iridoids; Materials Testing; Models, Biological; Osteoblasts; Reactive Oxygen Species; Swine; Tissue Engineering

To investigate the effect of genipin on apoptosis in human leukemia K562 cells in vitro and elucidate the underlying mechanisms.. The effect of genipin on K562 cell viability was measured using trypan blue dye exclusion and cell counting. Morphological changes were detected using phase-contrast microscopy. Apoptosis was analyzed using DNA ladder, propidium iodide (PI)-labeled flow cytometry (FCM) and Hoechst 33258 staining. The influence of genipin on cell cycle distribution was determined using PI staining. Caspase 3 activity was analyzed to detect apoptosis at different time points. Protein levels of phospho-c-Jun, phosphor-c-Jun N-terminal kinase (p-JNK), phosphor-p38, Fas-L, p63, and Bax and the release of cytochrome c were detected using Western blot analysis.. Genipin reduced the viability of K562 cells with an IC(50) value of approximately 250 μmol/L. Genipin 200-400 μmol/L induced formation of typical apoptotic bodies and DNA fragmentation. Additionally, genipin 400 μmol/L significantly increased the caspase 3 activity from 8-24 h and arrested the cells in the G₂/M phase. After stimulation with genipin 500 μmol/L, the levels of p-JNK, p-c-Jun, Fas-L, Bax, and cytochrome c were remarkably upregulated, but there were no obvious changes of p-p38. Genipin 200-500 μmol/L significantly upregulated the Fas-L expression and downregulated p63 expression. Dicoumarol 100 μmol/L, a JNK1/2 inhibitor, markedly suppressed the formation of apoptotic bodies and JNK activation induced by genipin 400 μmol/L.. These results suggest that genipin inhibits the proliferation of K562 cells and induces apoptosis through the activation of JNK and induction of the Fas ligand.

Topics: Apoptosis; Cell Division; G2 Phase; Humans; Iridoid Glycosides; Iridoids; JNK Mitogen-Activated Protein Kinases; K562 Cells; Leukemia

Treatment of damaged intervertebral discs is a significant clinical problem and, despite advances in the repair and replacement of the nucleus pulposus, there are few effective strategies to restore defects in the annulus fibrosus. An annular repair material should meet three specifications: have a modulus similar to the native annulus tissue, support the growth of disc cells, and maintain adhesion to tissue under physiological strain levels. We hypothesized that a genipin crosslinked fibrin gel could meet these requirements. Our mechanical results showed that genipin crosslinked fibrin gels could be created with a modulus in the range of native annular tissue. We also demonstrated that this material is compatible with the in vitro growth of human disc cells, when genipin:fibrin ratios were 0.25:1 or less, although cell proliferation was slower and cell morphology more rounded than for fibrin alone. Finally, lap tests were performed to evaluate adhesion between fibrin gels and pieces of annular tissue. Specimens created without genipin had poor handling properties and readily delaminated, while genipin crosslinked fibrin gels remained adhered to the tissue pieces at strains exceeding physiological levels and failed at 15-30%. This study demonstrated that genipin crosslinked fibrin gels show promise as a gap-filling adhesive biomaterial with tunable material properties, yet the slow cell proliferation suggests this biomaterial may be best suited as a sealant for small annulus fibrosus defects or as an adhesive to augment large annulus repairs. Future studies will evaluate degradation rate, fatigue behaviors, and long-term biocompatibility.

Topics: Animals; Cattle; Cell Survival; Cells, Cultured; Cholagogues and Choleretics; Dose-Response Relationship, Drug; Fibrin; Humans; Hydrogels; Intervertebral Disc; Iridoid Glycosides; Iridoids; Time Factors; Tissue Adhesives

Recent advances in nerve repair technology have focused on finding more biocompatible, non-toxic materials to imitate natural peripheral nerve components. In this study, casein protein cross-linked with naturally occurring genipin (genipin-cross-linked casein (GCC)) was used for the first time to make a biodegradable conduit for peripheral nerve repair. The GCC conduit was dark blue in appearance with a concentric and round lumen. Water uptake, contact angle and mechanical tests indicated that the conduit had a high stability in water and did not collapse and cramped with a sufficiently high level of mechanical properties. Cytotoxic testing and terminal deoxynucleotidyl transferase dUTP nick-end labelling assay showed that the GCC was non-toxic and non-apoptotic, which could maintain the survival and outgrowth of Schwann cells. Non-invasive real-time nuclear factor-κB bioluminescence imaging accompanied by histochemical assessment showed that the GCC was highly biocompatible after subcutaneous implantation in transgenic mice. Effectiveness of the GCC conduit as a guidance channel was examined as it was used to repair a 10 mm gap in the rat sciatic nerve. Electrophysiology, labelling of calcitonin gene-related peptide in the lumbar spinal cord, and histology analysis all showed a rapid morphological and functional recovery for the disrupted nerves. Therefore, we conclude that the GCC can offer great nerve regeneration characteristics and can be a promising material for the successful repair of peripheral nerve defects.

Topics: Absorbable Implants; Animals; Caseins; Electrophysiology; Guided Tissue Regeneration; Histological Techniques; Image Processing, Computer-Assisted; Immunohistochemistry; In Situ Nick-End Labeling; Iridoid Glycosides; Iridoids; Mice; Mice, Transgenic; Nerve Regeneration; Peripheral Nerve Injuries; Rats; Schwann Cells; Sciatic Nerve

Gelatine is one of the most valuable natural polymers used for drug delivery applications. Gelatine-GAGs based composite system has been shown to act as good scaffolds for tissue engineering. The objective of the present study is to investigate the calorimetric properties of microporous gelatine-GAGs based blend, which were modified by co-crosslinking with a naturally occurring crosslinking agent genipin. The melting temperature (T(m)), enthalpy change (ΔH(m)) and heat capacity change (ΔC(p)) were systematically calculated over the experimentally observed systems using differential scanning calorimeter (DSC). The thermoporometry results suggest that the concentration of the glycosaminoglycans plays an important role in the pore size distribution of the blend matrices. The circular dichroism (CD) spectroscopy study, scanning electron microscopy (SEM) studies provide the valuable information about the structural features of the biodegradable blend that can be utilized for various biomedical applications. The results provide new insights into the thermal stability of blend and suggest potential strategies for its manipulation.

Topics: Calorimetry; Drug Carriers; Drug Design; Gelatin; Glycosaminoglycans; Iridoid Glycosides; Iridoids; Porosity; Thermodynamics; Transition Temperature; Viscosity

Genipin is the bioactive compound of geniposide and a natural cross-linking agent. In order to improve the preparation process of genipin, the hydrolysis of geniposide to genipin by immobilized β-glucosidase in an aqueous-organic two-phase system was studied. β-glucosidase was immobilized by the crosslinking-embedding method using sodium alginate as the carrier. The optimum reaction temperature, pH value and time were 55 °C, 4.5 and 2.5 h, respectively. To reduce genipin hydrolysis and byproduct production the reaction was carried out in an aqueous-organic two-phase system comprising ethyl acetate and sodium acetate buffer. The product was analyzed by HPLC, UV, IR, and NMR. The yield of genipin was 47.81% and its purity was over 98% (HPLC).

Topics: beta-Glucosidase; Enzyme Stability; Hydrogen-Ion Concentration; Hydrolysis; Iridoid Glycosides; Iridoids; Organic Chemistry Phenomena; Temperature; Time Factors

Genipin, a natural cross-linking reagent extracted from the fruits of Gardenia jasminoides, can be effectively employed in tissue engineering applications due to its low cytotoxicity and high biocompatibility. The cross-linking of collagen hydrogels with genipin was followed with one-photon fluorescence spectroscopy, second harmonic generation, fluorescence and transmission electron microscopy. The incubation with genipin induced strong auto-fluorescence within the collagen hydrogels. The fluorescence emission maximum of the fluorescent adducts formed by genipin exhibit a strong dependence on the excitation wavelength. The emission maximum is at 630 nm when we excite the cross-linked samples with 590 nm light and shifts to 462 nm when we use 400 nm light instead. The fluorescence imaging studies show that genipin induces formation of long aggregated fluorescent strands throughout the depth of samples. The second harmonic generation (SHG) imaging studies suggest that genipin partially disaggregates 10 μm "fiberlike" collagen structures because of the formation of these fluorescent cross-links. Transmission electron microscopy (TEM) studies reveal that genipin largely eliminates collagen's characteristic native fibrillar striations. Our study is the first one to nondestructively follow and identify the structure within collagen hydrogels in situ and to sample structures formed on both micro- and nanoscales. Our findings suggest that genipin cross-linking of collagen follows a complex mechanism and this compound modifies the structure within the collagen hydrogels in both micro- and nanoscale.

Topics: Animals; Collagen; Hydrogels; Iridoid Glycosides; Iridoids; Microscopy, Confocal; Microscopy, Electron, Transmission; Rats; Spectrometry, Fluorescence

Genipin, an aglycon of geniposide, has been reported to exhibit diverse pharmacological functions such as antitumor and anti-inflammatory effects. This study aimed to elucidate the anti-inflammatory mechanism of genipin, focusing particularly on the role of heme oxygenase-1 (HO-1), a potent anti-inflammatory enzyme. In RAW264.7 cells, genipin increased HO-1 expression and its enzyme activity via a NF-E2-related factor 2 (Nrf2)-antioxidant response element (ARE) pathway. These effects were significantly inhibited by exposure to the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, or by expression of a dominant negative mutant of PI 3-kinase. Additional experiments showed that the activation of c-Jun NH(2)-terminal kinase 1/2 (JNK1/2) is required for genipin-induced phosphorylation and nuclear translocation of Nrf2 and antioxidant response element (ARE)-driven induction of HO-1, and acts as a downstream effector of PI 3-kinase. Furthermore, functional significance of HO-1 induction was revealed by genipin-mediated inhibition of lipopolysaccharide-stimulated inducible nitric oxide synthase expression or cyclooxygenase-2 promoter activity, the response was reversed by the blocking of HO-1 protein synthesis or HO-1 enzyme activity. Therefore, identification of PI 3-kinase-JNK1/2-Nrf2-linked signaling cascade in genipin-mediated HO-1 expression defines the signaling event that could participate in genipin-mediated anti-inflammatory response.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Line; Cyclooxygenase 2; Heme Oxygenase-1; Iridoid Glycosides; Iridoids; Macrophages; Mice; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinase 9; NF-E2-Related Factor 2; Nitric Oxide Synthase Type II; Phosphatidylinositol 3-Kinases; Signal Transduction; Up-Regulation

The genipin radiochromic gel offers enormous potential as a three-dimensional dosimeter in advanced radiotherapy techniques. We have used several methods (including Monte Carlo simulation), to investigate the water equivalency of genipin gel by characterizing its radiological properties, including mass and electron densities, photon interaction cross sections, mass energy absorption coefficient, effective atomic number, collisional, radiative and total mass stopping powers and electron mass scattering power. Depth doses were also calculated for clinical kilovoltage and megavoltage x-ray beams as well as megavoltage electron beams. The mass density, electron density and effective atomic number of genipin were found to differ from water by less than 2%. For energies below 150 keV, photoelectric absorption cross sections are more than 3% higher than water due to the strong dependence on atomic number. Compton scattering and pair production interaction cross sections for genipin gel differ from water by less than 1%. The mass energy absorption coefficient is approximately 3% higher than water for energies <60 keV due to the dominance of photoelectric absorption in this energy range. The electron mass stopping power and mass scattering power differ from water by approximately 0.3%. X-ray depth dose curves for genipin gel agree to within 1% with those for water. Our results demonstrate that genipin gel can be considered water equivalent for kilovoltage and megavoltage x-ray beam dosimetry. For megavoltage electron beam dosimetry, however, our results suggest that a correction factor may be needed to convert measured dose in genipin gel to that of water, since differences in some radiological properties of up to 3% compared to water are observed. Our results indicate that genipin gel exhibits greater water equivalency than polymer gels and PRESAGE formulations.

Topics: Absorption; Electrons; Gels; Iridoid Glycosides; Iridoids; Monte Carlo Method; Radiation Dosage; Radiometry; Radiotherapy; Scattering, Radiation; Water; X-Rays

Incorporation of nerve growth factor (NGF) into a nerve conduit can improve peripheral nerve regeneration. Here, genipin, a natural and low toxic agent, was used to crosslink chitosan, a natural polysaccharide, and concurrently to immobilize NGF onto modified chitosan, followed by fabrication of chitosan (CS)-genipin (GP)-NGF nerve conduits. MTT test showed that the cell viability of Schwann cells cultured in the conduit extract was not significantly different from that in plain medium. The neurite outgrowth measurement and immunocytochemistry with anti-growth-associated protein-43 and anti-neurofilament indicated that NGF released from CS-GP-NGF nerve conduits retained the bioactivity of stimulating neuronal differentiation of PC12 cells. Fracture strength measurements and vitamin B12 release analysis confirmed that CS-GP-NGF nerve conduits possessed good mechanical properties and adequate permeability. We also investigated the in vitro release kinetics of NGF from CS-GP-NGF nerve conduits by ELISA. The continuous release profile of NGF, within a 60-day time span, consisted of an initial burst that was controlled by a concentration gradient-driven diffusion, followed by a zero-order release that was controlled by a degradation of chitosan matrix. Collectively, CS-GP-NGF nerve conduits had an integrated system for continuous release of NGF, thus holding promise for peripheral nerve repair applications.

Topics: Animals; Animals, Newborn; Biocompatible Materials; Cell Culture Techniques; Cell Proliferation; Cell Survival; Chitosan; Cross-Linking Reagents; Iridoids; Microscopy, Electron, Scanning; Nerve Growth Factor; Nerve Regeneration; Neurites; PC12 Cells; Peripheral Nerves; Rats; Rats, Sprague-Dawley; Schwann Cells

This study focuses on a microgel-based functionalization method applicable to polyester textiles for improving their hydrophilicity and/or moisture-management properties, eventually enhancing wear comfort. The method proposed aims at achieving pH-/temperature-controlled wettability of polyester within a physiological pH/temperature range. First, primary amine groups are created on polyester surfaces using ethylenediamine; second, biopolymer-based polyelectrolyte microgels are incorporated using the natural cross-linker genipin. The microgels consist of the pH-responsive natural polysaccharide chitosan and pH/thermoresponsive poly(N-isopropylacrylamide-co-acrylic acid) microparticles. Scanning electron microscopy confirmed the microgel presence on polyester surfaces. X-ray photoelectron spectroscopy revealed nitrogen concentration, supporting increased microscopy results. Electrokinetic analysis showed that functionalized polyester surfaces have a zero-charge point at pH 6.5, close to the microgel isoelectric point. Dynamic wetting measurements revealed that functionalized polyester has shorter total water absorption time than the reference. This absorption time is also pH dependent, based on dynamic contact angle and micro-roughness measurements, which indicated microgel swelling at different pH values. Furthermore, at 40 °C functionalized polyester has higher vapor transmission rates than the reference, even at high relative humidity. This was attributed to the microgel thermoresponsiveness, which was confirmed through the almost 50% decrease in microparticle size between 20 and 40 °C, as determined by dynamic light scattering measurements.

Topics: Absorption; Acrylamides; Amination; Chitosan; Gels; Hydrogen-Ion Concentration; Iridoid Glycosides; Iridoids; Light; Microscopy, Electron, Scanning; Microspheres; Polyesters; Polyethylene Glycols; Polyethylene Terephthalates; Polymers; Scattering, Radiation; Temperature; Textiles; Wettability

Scaffolds based on gelatin (G) are considered promising for tissue engineering, able to mimic the natural extracellular matrix. G drawback is its poor structural consistency in wet conditions. Therefore, crosslinking is necessary to fabricate stable G scaffolds. In this work, a comparative study between the performance of two different crosslinkers, genipin (GP) and γ-glycidoxypropyltrimethoxysilane (GPTMS), is presented. Flat membranes by solvent casting and porous crosslinked scaffolds by freeze-drying were prepared. Infrared spectroscopy and thermal analysis were applied to confirm G chain crosslinking. Moreover, GP and GPTMS increased the stability of G in aqueous media and improved the mechanical properties. Crosslinking reduced the wettability, especially in the case of G_GPTMS samples, due to the introduction of hydrophobic siloxane chains. Both G_GP and G_GPTMS scaffolds supported MG-63 osteoblast-like cell adhesion and proliferation.

Topics: Animals; Calorimetry, Differential Scanning; Cell Count; Cell Line; Cross-Linking Reagents; Elastic Modulus; Gelatin; Humans; Iridoid Glycosides; Iridoids; Materials Testing; Porosity; Silanes; Solubility; Spectroscopy, Fourier Transform Infrared; Sus scrofa; Tissue Scaffolds; Wettability

In situ forming chitosan hydrogels have been prepared via coupled ionic and covalent cross-linking. Thus, different amounts of genipin (0.05, 0.10, 0.15, and 0.20% (w/w)), used as a chemical cross-linker, were added to a solution of chitosan that was previously neutralized with a glycerol-phosphate complex (ionic cross-linker). In this way, it was possible to overcome the pH barrier of the chitosan solution, to preserve its thermosensitive character, and to enhance the extent of cross-linking in the matrix simultaneously. To investigate the contributions of the ionic cross-linking and the chemical cross-linking, separately, we prepared the hydrogels without the addition of either genipin or the glycerol-phosphate complex. The addition of genipin to the neutralized solution disturbs the ionic cross-linking process and the chemical cross-linking becomes the dominant process. Moreover, the genipin concentration was used to modulate the network structure and performance. The more promising formulations were fully characterized, in a hydrated state, with respect to any equilibrium swelling, the development of internal structure, the occurrence of in vitro degradability and cytotoxicity, and the creation of in vivo injectability. Each of the hydrogel systems exhibited a notably high equilibrium water content, arising from the fact that their internal structure (examined by conventional SEM, and environmental SEM) was highly porous with interconnecting pores. The porosity and the pore size distribution were quantified by mercury intrusion porosimetry. Although all gels became degraded in the presence of lysozyme, their degradation rate greatly depended on the genipin load. Through in vitro viability tests, the hydrogel-based formulations were shown to be nontoxic. The in vivo injection of a co-cross-linking formulation revealed that the gel was rapidly formed and localized at the injection site, remaining in position for at least 1 week.

Topics: Animals; Biocompatible Materials; Biodegradation, Environmental; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Chitosan; Cross-Linking Reagents; Drug Carriers; Elastic Modulus; Female; Glycerol; Hydrogels; Hydrogen-Ion Concentration; Ions; Iridoid Glycosides; Iridoids; Male; Mice; Microscopy, Electron, Scanning; Muramidase; Porosity; Rats; Rats, Wistar; Spectroscopy, Fourier Transform Infrared

Collagen sponges loaded with polyphenols from Hamamelis virginiana were investigated as active materials for chronic wound dressings, evaluating in vitro the inhibition of two major enzymes that impair the wound healing process - myeloperoxidase (MPO) and collagenase. Prior to polyphenols loading, collagen was cross-linked with genipin to improve its biostability. The effect of genipin cross-linking and polyphenol concentration in the development of mechanically and enzymatically stable sponges was studied. The tensile strength of the cross-linked collagen increased with the increase of the cross-linking degree, coupled to decrease in the elongation and the swelling capacity of the sponges. The stability of the sponges to collagenase digestion reached maximum when 1 mM genipin was used. However, the biostability decreased more than 10-fold after loading the sponges with polyphenols (0.5 mg/mL), nevertheless, this effect was partially overcome using higher concentration of polyphenols (1 and 2 mg/mL) to inhibit collagenase. Moreover, the polyphenols released from the sponges were sufficient for complete inhibition of MPO activity. No considerable cytotoxicity of the genipin cross-linked collagen loaded with polyphenols was observed evaluating the NIH 3T3 fibroblasts viability.

Topics: Animals; Bandages; Biocompatible Materials; Chronic Disease; Collagen Type I; Cross-Linking Reagents; Drug Stability; Hamamelis; Iridoid Glycosides; Iridoids; Matrix Metalloproteinase Inhibitors; Mice; Microscopy, Electron, Scanning; NIH 3T3 Cells; Particle Size; Peroxidase; Plant Extracts; Plant Stems; Polyphenols; Wound Healing

Chitosan covalent nanogels cross-linked with genipin were prepared by template chemical cross-linking of chitosan in polyion complex micelle (PIC) nanoreactors. By using this method, we were able to prepare chitosan nanogels using only biocompatible materials without organic solvents. PIC were prepared by interaction between chitosan (X(n) = 23, 44, and 130) and block copolymer poly(ethylene oxide)-block-poly[sodium 2-(acrylamido)-2-methylpropanesulfonate] (PEO-b-PAMPS) synthesized by single-electron transfer-living radical polymerization (SET-LRP). PIC with small size (diameter about 50 nm) and low polydispersity were obtained up to 5 mg/mL. After cross-linking of chitosan with genipin, the nanoreactors were dissociated by adding NaCl. The dissociation of the nanoreactors and the formation of the nanogels were confirmed by (1)H NMR, DLS, and TEM. The size of the smallest nanogels was about 50 nm in the swollen state and 20 nm in the dry state. The amount of genipin used during reticulation was an important parameter to modulate the size of the nanogels in solution.

Topics: Acrylamides; Biocompatible Materials; Bioreactors; Chitosan; Cross-Linking Reagents; Ions; Iridoid Glycosides; Iridoids; Magnetic Resonance Spectroscopy; Micelles; Nanostructures; Nanotechnology; Particle Size; Polyethylene Glycols; Polymerization; Sodium Chloride

The purpose of this work is to investigate the effect of the genipin/hydroxypropyl-β-cyclodextrin (HP-β-CD) inclusion complex on the intestinal absorption of genipin and identify its mechanism of action. The phase solubility profile was classified as A(L) type, indicating the formulation of a 1:1 stoichiometry inclusion complex. Fourier transform infrared spectroscopy, Differential scanning calorimetry, X-ray powder diffractometry, and (1)H nuclear magnetic resonance (NMR) and two-dimensional (2D) (1)H rotating-frame Overhauser enhancement (ROESY) NMR spectroscopies further confirmed the formulation of the inclusion complex with superior dissolution properties than the drug alone. The results of single-pass intestinal perfusion showed that the intestinal absorption of genipin was affected by P-glycoprotein (Pgp). The absorption rate and permeability value of the inclusion complex were significantly higher than the free drug, suggesting that its enhancing effect was involved in its solubilizing effect and Pgp inhibitory effect. The mechanisms of HP-β-CD on Pgp inhibition were demonstrated by restraining the Pgp ATPase activity rather than changing the fluidity of the cell membrane.

Topics: 2-Hydroxypropyl-beta-cyclodextrin; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 1; beta-Cyclodextrins; Chemistry, Pharmaceutical; Cholagogues and Choleretics; Intestinal Absorption; Iridoid Glycosides; Iridoids; Magnetic Resonance Spectroscopy; Male; Rats; Rats, Sprague-Dawley; Solubility; Spectroscopy, Fourier Transform Infrared

The Warburg Effect is characterized by an irreversible injury to mitochondrial oxidative phosphorylation (OXPHOS) and an increased rate of aerobic glycolysis. In this study, we utilized a breast epithelial cell line lacking mitochondrial DNA (rho(0)) that exhibits the Warburg Effect associated with breast cancer. We developed a MitoExpress array for rapid analysis of all known nuclear genes encoding the mitochondrial proteome. The gene-expression pattern was compared among a normal breast epithelial cell line, its rho(0) derivative, breast cancer cell lines and primary breast tumors. Among several genes, our study revealed that over-expression of mitochondrial uncoupling protein UCP2 in rho(0) breast epithelial cells reflects gene expression changes in breast cancer cell lines and in primary breast tumors. Furthermore, over-expression of UCP2 was also found in leukemia, ovarian, bladder, esophagus, testicular, colorectal, kidney, pancreatic, lung and prostate tumors. Ectopic expression of UCP2 in MCF7 breast cancer cells led to a decreased mitochondrial membrane potential and increased tumorigenic properties as measured by cell migration, in vitro invasion and anchorage independent growth. Consistent with in vitro studies, we demonstrate that UCP2 over-expression leads to development of tumors in vivo in an orthotopic model of breast cancer. Genipin, a plant derived small molecule, suppressed the UCP2 led tumorigenic properties, which were mediated by decreased reactive oxygen species and down-regulation of UCP2. However, UCP1, 3, 4 and 5 gene expression was unaffected. UCP2 transcription was controlled by SMAD4. Together, these studies suggest a tumor-promoting function of UCP2 in breast cancer. In summary, our studies demonstrate that i) the Warburg Effect is mediated by UCP2; ii) UCP2 is over-expressed in breast and many other cancers; iii) UCP2 promotes tumorigenic properties in vitro and in vivo and iv) genipin suppresses the tumor promoting function of UCP2.

Topics: Adenosine Triphosphate; Blotting, Western; Breast Neoplasms; Cell Line; Cell Line, Tumor; Female; Humans; Immunohistochemistry; Ion Channels; Iridoid Glycosides; Iridoids; Membrane Potential, Mitochondrial; Mitochondrial Proteins; Reactive Oxygen Species; Smad4 Protein; Uncoupling Protein 2

For surface modification and nerve regeneration, chitosan, followed by nerve growth factor (NGF), was immobilized onto the interior surface of poly (lactic acit-co-glycolic) conduits, using EDC/NHS/MES system (EDCs) and genipin (GP). Four new conduits were, therefore, obtained and named by immobilizing order-EDCs/EDCs, GP/EDCs, EDCs/GP, and GP/GP groups. The immobilized methods used were evaluated and compared, respectively. The researchers found that the EDCs- and GP-cross-linked chitosan displayed higher hydrophilic than pure poly (DL-lactic acid-co-glycolic acid) (PLGA) in water contact angle experiment, which meant the cell compatibility was improved by the modification. Scanning electron microscopic observations revealed that the GP-cross-linking of chitosan greatly improved cell compatibility while cultured rat PC12 cells were flatter and more spindle-shaped than EDCs-cross-linked chitosan. The results concerning the GP-cross-linked chitosan revealed significant proliferation of the seeded cells relative to pure PLGA films, as determined by counting cells and MTT assay. The NGF was released from the modified conduits in two separate periods--an initial burst in 5 days and then slow release from day 10 to day 40. The GP/EDCs group had the highest NGF value among all groups after the 5th day. Finally, the controlled-release conduits were used to bridge a 10 mm rat sciatic nerve defect. Six weeks following implantation, morphological analysis revealed the highest numbers of myelinated axons in the midconduit and distal regenerated nerve in GP/EDCs group. Therefore, the results confirm that GP/EDCs groups with good cell compatibility and effective release of NGF can considerably improve peripheral nerve regeneration.

Topics: Alkanesulfonic Acids; Animals; Biocompatible Materials; Ethyldimethylaminopropyl Carbodiimide; Guided Tissue Regeneration; Implants, Experimental; Iridoid Glycosides; Iridoids; Lactic Acid; Male; Materials Testing; Morpholines; Nerve Growth Factor; Nerve Regeneration; PC12 Cells; Peripheral Nerves; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Rats; Rats, Sprague-Dawley; Succinimides; Surface Properties

Genipin, a herbal iridoid, is known to have both neuroprotective and neuritogenic activity in neuronal cell lines. As it is structurally similar to tetrahydrobiopterin, its activity is believed to be nitric oxide (NO)-dependent. We previously proposed a novel neuroprotective activity of a genipin derivative, (1R)-isoPropyloxygenipin (IPRG001), whereby it reduces oxidative stress in RGC-5, a neuronal precursor cell line of retinal origin through protein S-nitrosylation. In the present study, we investigated another neuritogenic property of IPRG001 in RGC-5 cells and retinal explant culture where in we focused on the NO-cGMP-dependent and protein S-nitrosylation pathways. IPRG001 stimulated neurite outgrowth in RGC-5 cells and retinal explant culture through NO-dependent signaling, but not NO-dependent cGMP signaling. Neurite outgrowth with IPRG001 requires retinoic acid receptor β (RARβ) expression, which is suppressed by an RAR blocking agent and siRNA inhibition. Thereby, we hypothesized that RARβ expression is mediated by protein S-nitrosylation. S-nitrosylation of histone deacetylase 2 is a key mechanism in chromatin remodeling leading to transcriptional gene activation. We found a parallelism between S-nitrosylation of histone diacetylase 2 and the induction of RARβ expression with IPRG001 treatment. The both neuroprotective and neuritogenic activities of genipin could be a new target for the regeneration of retinal ganglion cells after glaucomatous conditions.

Topics: Analysis of Variance; Animals; Cell Line, Transformed; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Enzyme Inhibitors; Histone Deacetylase 2; Humans; Iridoid Glycosides; Iridoids; Male; Neurites; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Synthase; Organ Culture Techniques; Rats; Rats, Sprague-Dawley; Receptors, Retinoic Acid; Retina; Retinal Ganglion Cells; RNA, Small Interfering; Signal Transduction

Various biodegradable hydrogels have been employed as injectable scaffolds for tissue engineering and drug delivery. We report a double-crosslinking strategy of biocompatible and biodegradable hydrogels derived from aminated and oxidized hyaluronic acid (HA) with genipin (GP), a compound naturally derived from the gardenia fruit. Fast gelation is attributed to the Schiff-base reaction between amino and aldehyde groups of polysaccharide derivatives, and the subsequent crosslinking with GP results in ideal biodegradability and mechanical properties. The gelation time, morphology, equilibrium swelling, compressive modulus and degradation of double-crosslinked hydrogels were examined. The double crosslinked hydrogels were examined in vivo via subcutaneous injection into a mouse model. Histological results indicated favourable biocompatility, as revealed by an absence of neutrophils and macrophages. These studies demonstrate that double-crosslinked HA hydrogels are potentially useful as injectable, biodegradable hydrogels in tissue-engineering applications.

Topics: Absorbable Implants; Animals; Drug Delivery Systems; Hyaluronic Acid; Hydrogels; Injections, Subcutaneous; Iridoid Glycosides; Iridoids; Male; Materials Testing; Mice; Mice, Inbred BALB C; Schiff Bases; Tissue Engineering; Tissue Scaffolds

Topics: Cell Proliferation; Cells, Cultured; Cross-Linking Reagents; Delayed-Action Preparations; Drug Carriers; Enzyme-Linked Immunosorbent Assay; Fibroblast Growth Factor 2; Gelatin; Heparin; Humans; Iridoids; Mesenchymal Stem Cells; Microscopy, Electron, Scanning; Polyesters; Polymers; Time Factors; Tissue Scaffolds

Topics: Calorimetry, Differential Scanning; Chemistry, Pharmaceutical; Cross-Linking Reagents; Delayed-Action Preparations; Drug Carriers; Drug Compounding; Gelatin; Iridoids; Microscopy, Electron, Scanning; Nanofibers; Serum Albumin, Bovine; Spectrophotometry, Ultraviolet; Stress, Mechanical; Technology, Pharmaceutical; Temperature; Time Factors; Tissue Engineering; Tissue Scaffolds

Topics: Alginates; Capsules; Chitosan; Cross-Linking Reagents; Drug Carriers; Glucuronic Acid; Hexuronic Acids; Iridoids; Microscopy, Confocal; Microscopy, Fluorescence; Microspheres; Particle Size; Technology, Pharmaceutical

The functional significance of uncoupling proteins UCP2 and UCP3 in the cell and the organism remains unknown. There have been reports about their involvement in cellular protection mechanisms underlying the phenomenon of ischemic preconditioning. The purpose of this study was to elucidate the role of uncoupling proteins UCP2 and UCP3 in the formation of cardioprotective effect of ischemic preconditioning. In experiments on isolated rat hearts we show here an increase in the level of UCP2 and UCP3 gene expression in the heart tissue under the influence of ischemic preconditioning with three episodes of 5 min stopping the flow perfusion. Similar effects were induced by a prolonged ischemia-reperfusion of myocardium. The blockade of the UCP2 activity by genipin (Wako Inc., USA, 10(-5) mol/L, isolated heart perfusion for 15 minutes) abolished the protective effect of adaptation to ischemia. It was concluded that uncoupling proteins take part in the cardioprotective effect ofischemic preconditioning.

Topics: Adaptation, Physiological; Animals; In Vitro Techniques; Ion Channels; Iridoids; Ischemic Preconditioning, Myocardial; Male; Mitochondrial Proteins; Myocardial Reperfusion Injury; Rats; Rats, Wistar; Uncoupling Protein 2; Uncoupling Protein 3

Microspheres of gelatin-A and κ-carrageenan were prepared by using genipin, a naturally occurring crosslinker, and sunflower oil as reaction media.. The variations in the size of the microspheres formed by varying the amount of surfactant (0.33-1.0 g/g of polymer), polymer (1.5-3.0 g), and crosslinker (0.2-0.8 mmol) were studied by scanning electron microscopy. The encapsulation of isoniazid was carried out by absorption. The isoniazid content in the prepared microspheres was determined. The release characteristic of isoniazid was also studied at pH values 1.2 and 7.4 by using UV-spectrophotometer.. Characterization of the isoniazid-loaded microspheres was carried out by using Fourier transform infrared spectrophotometry, differential scanning calorimetry, and X-ray diffractometery.

Topics: Antitubercular Agents; Calorimetry, Differential Scanning; Carrageenan; Cross-Linking Reagents; Drug Carriers; Electrolytes; Gelatin; Hydrogen-Ion Concentration; Iridoids; Isoniazid; Microspheres; Particle Size; Plant Oils; Spectroscopy, Fourier Transform Infrared; Sunflower Oil; Surface-Active Agents; X-Ray Diffraction

Composite nanofibers of poly(caprolactone) (PCL) and gelatin crosslinked with genipin are prepared. The contact angles and mechanical properties of crosslinked PCL-gelatin nanofibers decrease as the gelatin content increases. The proliferation of myoblasts is higher in the crosslinked PCL-gelatin nanofibers than in the PCL nanofibers, and the formation of myotubes is only observed on the crosslinked PCL-gelatin nanofibers. The expression level of myogenin, myosin heavy chain, and troponin T genes is increased as the gelatin content is increased. The results suggest that PCL-gelatin nanofibers crosslinked with genipin can be used as a substrate to modulate proliferation and differentiation of myoblasts, presenting potential applications in muscle tissue engineering.

Topics: Blotting, Western; Cross-Linking Reagents; DNA Primers; Fluorescent Antibody Technique; Gelatin; Iridoid Glycosides; Iridoids; Muscle Development; Myogenin; Myosin Heavy Chains; Nanofibers; Polyesters; Reverse Transcriptase Polymerase Chain Reaction; Tissue Engineering; Troponin T

Collagen and hydrazide-functionalized hyaluronic acid derivatives were hybridized by gelating and genipin crosslinking to form composite hydrogel. The study contributed to the understanding of the effects of adipic dihydrazide modification on the physicochemical and biological properties of the collagen/hyaluronic acid scaffold. The investigation included morphology observation, mechanical measurement, swelling evaluation, and collagenase degradation. The results revealed that the stability of composites was increased through adipic dihydrazide modification and genipin crosslinking. The improved biocompatibility and retention of hyaluronic acid made the composite material more favorable to chondrocytes growing, suggesting the prepared scaffold might be high potential for chondrogenesis.

Topics: Absorbable Implants; Adipates; Cell Adhesion; Cell Proliferation; Chondrocytes; Collagen Type I; Cross-Linking Reagents; Enzymes; Humans; Hyaluronic Acid; Immunohistochemistry; Iridoid Glycosides; Iridoids; Materials Testing; Microscopy, Electron, Scanning; Porosity; Spectrophotometry, Infrared; Spectroscopy, Fourier Transform Infrared; Stress, Mechanical; Tensile Strength; Tissue Scaffolds; Water

Carboxymethyl-hexanoyl chitosan (CHC) is an amphiphilic chitosan derivative with excellent swelling ability and water solubility under natural conditions. In this work, the influence of the degree of carboxymethyl and hexanoyl substitution on the pH-sensitive swelling behavior, drug release behavior, and antiadhesion behavior of CHC hydrogels (cross-linked with genipin) were studied. It was found that the pH sensitivity was more pronounced in CHC than in N,O-carboxymethyl chitosan because the hexanoyl group altered the state of water in CHC by inhibiting intermolecular hydrogen bonding. In addition, greater pH sensitivity was observed in samples bearing longer hydrophobic chains (carboxymethyl-palmityl chitosan). Interestingly, when used with ibuprofen (a poorly water-soluble therapeutic agent used here as a model drug), the bursting release of the drug was less prominent in the CHC samples having a high degree of carboxymethyl substitution. The CHC hydrogel also demonstrated good cell compatibility and its antiadhesive ability after grafting was altered by changes in the degree of hexanoyl substitution.

Topics: Animals; Calorimetry, Differential Scanning; Cell Adhesion; Cell Death; Cell Shape; Chitosan; Fibroblasts; Hydrogel, Polyethylene Glycol Dimethacrylate; Hydrogen-Ion Concentration; Ibuprofen; Iridoid Glycosides; Iridoids; Kinetics; Magnetic Resonance Spectroscopy; Mice; Solubility; Solutions; Spectroscopy, Fourier Transform Infrared; Temperature; Water

To improve survival during exposure to adverse conditions, probiotic Bifidobacterium adolescentis 15703T cells were encapsulated in novel mono-core and multi-core phase-separated gelatine-maltodextrin (GMD) microspheres where the gelatine (G) phase was cross-linked with genipin (GP). Microscopy showed that encapsulated cells were exclusively associated with maltodextrin (MD) core(s). Small (average diameter 37 microm) and large (70 microm) GMD and G microspheres were produced by modulating factors (e.g. mixing speed, surfactant, GP and G concentrations) affecting the size, structural stability and phase-separation. In vitro sequential gastro-intestinal (GI) juice challenge experiments revealed increased survival of cells encapsulated in GMD ( approximately 10(6-7) cfu mL(-1)) and G (approximately 10(5) cfu mL(-1)) microspheres as compared to free cells (approximately 10(4) cfu mL(-1)). In GMD microspheres, the bacteria derive energy from MD to survive during exposure to acid and bile salts. In conclusion, the novel food grade GMD microencapsulation formulation was shown to protect probiotic bifidobacteria from adverse conditions.

Topics: Bifidobacterium; Cells, Immobilized; Cross-Linking Reagents; Drug Compounding; Gastric Juice; Gastrointestinal Tract; Gelatin; Iridoid Glycosides; Iridoids; Phase Transition; Polysaccharides; Probiotics

To overcome the cytotoxicity of the chemical reagents used to fix bioprostheses, genipin, a naturally occurring crosslinking agent, was used to fix biological tissues in present study. We prepared the biological vascular scaffolds through cell extraction and fixing the porcine thoracic arteries with 1% (by w/v) genipin solution for 3 days, and then examined their mechanical properties and microstructures; glutaraldehyde- and epoxy-fixed counterparts were used as controls. HUVECs were seeded on the type I collagen-coated surface of different modified acellular vascular tissues (fixed with different crosslinking agents), and the growths of HUVECs on the specimens were demonstrated by means of MTT test, the secretion of PGI2 and vWF by HUVECs on the various specimens was also measured. Finally, HUVECs were seeded on the luminal surface of acellular biological vascular scaffolds (<6 mm internal diameter) which were, respectively, treated in the same manner described above, and then cultured for 9 days. On the ninth day, the HUVECs on the luminal surface of these vascular scaffolds were examined morphologically and by immunohistochemistry. Genipin-fixation can markedly diminish antigenicity of the vascular tissues through partially getting rid of cell or reducing the level of free amino groups in the vascular tissues. Genipin-fixed acellular vascular tissues mimicked the natural vessels due to the maintenance of the integrity of total structure and the large preservation of the microstructures of collagen fibers and elastic fibers; therefore, it appeared suitable to fabricate vascular scaffolds in mechanical properties. Compared to controls, the genipin-fixed acellular vascular tissues were characterized by low cytotoxicity and good cytocompatibility. The HUVECs can not only proliferate well on the genipin-fixed acellular vascular tissues, but also preserve the activities and function of endothelial cells, and easily make it endothelialized in vitro. The results showed that the genipin-fixed acellular porcine vascular scaffolds should be promising materials for fabricating vascular grafts or the scaffolds of tissue-engineered blood vessels.

Topics: Biocompatible Materials; Blood Vessels; Cells, Cultured; Cross-Linking Reagents; Crystallization; Endothelial Cells; Endothelium, Vascular; Humans; Iridoid Glycosides; Iridoids; Materials Testing; Tissue Engineering; Tissue Scaffolds

The traditional Japanese (kampo) medicine inchinkoto (ICKT) is used in Eastern Asia as a choleretic and hepatoprotective agent. Previously, we reported that ICKT ameliorates murine concanavalin A (con A)-induced hepatitis via suppression of interferon (IFN)-gamma and interleukin (IL)-12 production. In the present study, we investigated the active ingredients of ICKT.. ICKT and extracts of its component herbs were fractionated, and their effects on liver injury and cytokine production in vivo (biochemical markers of liver injury and cytokine levels in serum) and in vitro (cytokine and nitrite production in the cultures of splenocytes and peritoneal macrophages).. Decoctions of component herbs, Artemisiae Capillari Spica (Artemisia capillaris Thunberg: 'Inchinko' in Japanese), Gardeniae Fructus (Gardenia jasminoides Ellis: 'Sanshishi') and Rhei Rhizoma (Rheum palmatum Linné: 'Daio') were administered orally. Inchinko and Sanshishi decreased serum transaminases and IFN-gamma concentrations. Examination of fractions of component herbs suggested that capillarisin, a component of Inchinko, has potent hepatoprotective activity in vivo. In in vitro studies, capillarisin and genipin, an intestinal metabolite of geniposide that is contained in Sanshishi, were examined. IFN-gamma production was significantly suppressed by capillarisin and genipin in con A-stimulated splenocyte culture. Genipin also suppressed IL-1beta, IL-6, and IL-12p70 synthesis. Capillarisin and genipin decreased nitrite release from IFN-gamma-stimulated macrophages.. These results suggested that both Inchinko and Sanshishi may contribute to the protective effects of ICKT against con A hepatitis. Capillarisin was found to be potently hepatoprotective, and genipin may also contribute, especially via modulation of cytokine production.

Topics: Animals; Artemisia; Chemical and Drug Induced Liver Injury; Chromones; Concanavalin A; Cytokines; Disease Models, Animal; Gardenia; Hepatitis; Interferon-gamma; Iridoid Glycosides; Iridoids; Liver; Macrophages; Magnoliopsida; Male; Medicine, Kampo; Mice; Mice, Inbred BALB C; Nitrites; Phytotherapy; Plant Extracts; Rheum; Transaminases

Polypeptides based on the alternating hydrophobic and cross-linking domain structure of human elastin are capable of undergoing self-assembly to produce polymeric matrices with unique biological and mechanical properties. Here, we test the initial feasibility of using a genipin cross-linked elastin-based material as an acellular plug in the treatment of an osteochondral defect in the rabbit knee. Full-thickness defects in the weight-bearing surface of the medial femoral condyle in 18 New Zealand White rabbits were surgically produced and press fitted with cylindrical pads composed of genipin cross-linked elastin-like polypeptides, with identical wounds in the opposite knee left untreated as controls. The biocompatibility of the material, overall wound healing and regeneration of subchondral tissue was assessed at 2, 4 and 6weeks by histological evaluation, synovial fluid analysis and microcomputerized tomography scanning. Histological analysis revealed the regeneration of subchondral bone at the periphery of the material, with evidence of hyaline-like overgrowth across the apical surface in 11/16 cases. Pads developed tight contacts with host tissue and appeared completely biocompatible, with no evidence of localized immune response or increased inflammation compared to controls. The material was stable to 6weeks, with an aggregate elastic modulus calculated at approximately 470kPa when tested under confined compression. Further studies are required to assess material degradation over time and long-term replacement with repair tissue.

Topics: Animals; Biocompatible Materials; Cross-Linking Reagents; Drug Implants; Elastic Modulus; Elastin; Hardness; Iridoid Glycosides; Iridoids; Materials Testing; Osteochondritis; Peptides; Rabbits; Surface Properties; Treatment Outcome

The present study deals with the preparation of porous chitosan scaffolds by using a greener technique i.e., supercritical carbon dioxide (sc.CO2). 0.2 M chitosan (CS) solution in aqueous acetic acid was treated with 4% (w/v) genipin solution; the resulting hydrogels were subjected to solvent exchange prior to the final treatment procedures. Their morphology, pore structure, and physical properties were characterized by thermal analysis, X-ray diffractogram (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and the specific surface areas and porosimetry of scaffolds were determined by using N2 adsorption. The biological activity of scaffolds was investigated by immersing them into 1.5 x simulated body fluid (SBF) and cellular attachment study was assessed using Alamar Blue assay by seeding MG63 osteoblastic cells onto the scaffolds. The sc.CO2 assisted chitosan scaffold prepared by using green chemistry approach is highly pure and bioactive and can be served as a potential material for tissue engineering applications.

Topics: Calorimetry, Differential Scanning; Carbon Dioxide; Cell Line; Cell Proliferation; Chitosan; Cross-Linking Reagents; Humans; Iridoid Glycosides; Iridoids; Microscopy, Electron, Scanning; Osteoblasts; Porosity; Spectrometry, X-Ray Emission; Thermogravimetry; Tissue Engineering; Tissue Scaffolds; X-Ray Diffraction

Uncoupling protein (UCP)2 is a mitochondrial inner membrane protein that is expressed in mammalian myocardium under normal conditions and upregulated in pathological states such as heart failure. UCP2 is thought to protect cardiomyocytes against oxidative stress by dissipating the mitochondrial proton gradient and mitochondrial membrane potential (DeltaPsi(m)), thereby reducing mitochondrial reactive oxygen species generation. However, in apparent conflict with its uncoupling role, UCP2 has also been proposed to be essential for mitochondrial Ca(2+) uptake, which could have a protective action by stimulating mitochondrial ATP production.. The goal of this study was to better understand the role of myocardial UCP2 by examining the effects of UCP2 on bioenergetics, Ca(2+) homeostasis, and excitation-contraction coupling in neonatal cardiomyocytes.. Adenoviral-mediated expression of UCP2 caused a mild depression of DeltaPsi(m) and increased the basal rate of oxygen consumption but did not affect total cellular ATP levels. Mitochondrial Ca(2+) uptake was examined in permeabilized cells loaded with the mitochondria-selective Ca(2+) probe, rhod-2. UCP2 overexpression markedly inhibited mitochondrial Ca(2+) uptake. Pretreatment with the UCP2-specific inhibitor genipin largely reversed the effects UCP2 expression on mitochondrial Ca(2+) handling, bioenergetics, and oxygen utilization. Electrically evoked cytosolic Ca(2+) transients and spontaneous cytosolic Ca(2+) sparks were examined using fluo-based probes and confocal microscopy in line scan mode. UCP2 overexpression significantly prolonged the decay phase of [Ca(2+)](c) transients in electrically paced cells, increased [Ca(2+)](c) spark activity and increased the probability that Ca(2+) sparks propagated into Ca(2+) waves. This dysregulation results from a loss of the ability of mitochondria to suppress local Ca(2+)-induced Ca(2+) release activity of the sarcoplasmic reticulum.. Increases in UCP2 expression that lower DeltaPsi(m) and contribute to protection against oxidative stress, also have deleterious effects on beat-to-beat [Ca(2+)](c) handling and excitation-contraction coupling, which may contribute to the progression of heart disease.

Topics: 2,4-Dinitrophenol; Adenosine Triphosphate; Adenoviridae; Animals; Animals, Newborn; Calcium; Cells, Cultured; Electric Stimulation; Energy Metabolism; Excitation Contraction Coupling; Genetic Vectors; Homeostasis; Humans; Ion Channels; Iridoid Glycosides; Iridoids; Kinetics; Membrane Potential, Mitochondrial; Microscopy, Confocal; Mitochondria, Heart; Mitochondrial Proteins; Myocardial Contraction; Myocytes, Cardiac; Oxygen Consumption; Rats; Rats, Wistar; Transfection; Uncoupling Agents; Uncoupling Protein 2

We have characterized the relative efficacies of a number of protein crosslinking agents that have the potential for use in the crosslinking of proteinaceous matrices both in vitro and in vivo. The crosslinkers tested were; L: -threose (LT), Genipin (GP), Methylglyoxal (MG), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), proanthrocyanidin (PA) and glutaraldehyde (GA). The relative effectiveness of the crosslinkers with regard to their saturating concentrations was: GA > PA > EDC > MG = GP >> LT. Most of the crosslinkers displayed a pH dependence and were more effective at more alkaline pH. At optimal pH and saturating conditions, the relative reaction rates of the crosslinkers were: PA = GA > EDC > GP > MG >> LT.

Topics: Animals; Carbodiimides; Cattle; Cross-Linking Reagents; Extracellular Matrix Proteins; Glutaral; Hydrogen-Ion Concentration; Iridoid Glycosides; Iridoids; Kinetics; Osmolar Concentration; Proanthocyanidins; Protein Processing, Post-Translational; Proteins; Pyruvaldehyde; Solubility; Tetroses; Thermodynamics

We designed 1-alkyloxygenipins with the aim of improving the stability of genipins based on the structural and electronic properties of genipins, and prepared 1-alkyloxygenipins and examined their neuritogenic activities in PC12h cells. All genipin-derivatives exhibited electronic properties similar to those of genipin and induced significant neurite outgrowth. These compounds will be classified as nitric oxide synthase (NOS) activators (neuritogenic active compounds) since their lowest unoccupied molecular orbital (LUMO)-energies are similar to that of tetrahydrobiopterin (H4B). (1R)-isoPropyloxygenipin showed activity comparable to that of genipin, and unlike the parent compound genipin, it was found to be physiologically stable in rat liver homogenate.

Topics: Animals; Cell Line; Drug Stability; Iridoid Glycosides; Iridoids; Liver; Models, Molecular; Molecular Structure; Neurites; Neurons; Nitric Oxide Synthase; Protein Binding; Rats; Rats, Wistar

Microglia are the prime effectors in immune and inflammatory responses of the central nervous system (CNS). Under pathological conditions, the activation of these cells helps restore CNS homeostasis. However, chronic microglial activation endangers neuronal survival through the release of various proinflammatory and neurotoxic factors. Thus, negative regulators of microglial activation have been considered as potential therapeutic candidates to target neurodegeneration, such as that in Alzheimer's and Parkinson's diseases. Genipin, the aglycon of geniposide found in gardenia fruit has long been considered for treatment of various disorders in traditional oriental medicine. Genipin has recently been reported to have diverse pharmacological functions, such as antimicrobial, antitumor, and anti-inflammatory effects. The specific aim of this study was to examine whether genipin represses brain microglial activation. Genipin was effective at inhibiting LPS-induced nitric oxide (NO) release from cultured rat brain microglial cells. Genipin reduced the LPS-stimulated production of tumor necrosis factor-alpha, interleukin-1beta, prostaglandin E(2), intracellular reactive oxygen species, and NF-kappaB activation. In addition, genipin reduced NO release from microglia stimulated with interferon-gamma and amyloid-beta. Both pretreatment and post-treatment of genipin to LPS-stimulated microglia were effective at decreasing NO release. Furthermore, genipin effectively inhibited microglial activation in a mouse model of brain inflammation. These results suggest that genipin provide neuroprotection by reducing the production of various neurotoxic molecules from activated microglia.

Topics: Amyloid beta-Peptides; Animals; Anti-Inflammatory Agents; Blotting, Western; Brain; Cell Survival; Cells, Cultured; Cytokines; Immunohistochemistry; Indicators and Reagents; Inflammation; Interferon-gamma; Iridoid Glycosides; Iridoids; Lipopolysaccharides; Macrophage Activation; Male; Mice; Mice, Inbred C57BL; Microglia; NF-kappa B; Nitrites; Rats; Reactive Oxygen Species

Gelatin (Type B) nanoparticles were prepared by a single W/O emulsion technique and characterized by infrared (IR) spectra, transmission electron micrographs (TEM), surface potential measurements and magnetization studies. Whereas the IR spectra clearly confirmed the presence of gelatin, genipin and doxorubicin in the loaded nanoparticles, the transmission electron micrographs (TEM) image depicts smooth surface, spherical shape and non-uniform size of nanoparticles (up to 100 nm). The prepared nanoparticles were loaded with doxorubicin, a well known anticancer drug, and in vitro release dynamics of entrapped drug was investigated as a function of various experimental factors such as percent loading of the drug, chemical architecture of the nanocarriers, and pH, temperature, ionic strength and nature of the release medium in presence and absence of magnetic field. The nanoparticles were also studied for their water sorption capacity. The drug release process was analyzed kinetically using Ficks power law and a correlation was established between the quantity of released drug and swelling of the nanoparticles.

Topics: Doxorubicin; Gelatin; Iridoid Glycosides; Iridoids; Nanoparticles; Temperature

To investigate the beneficial effects of inchinkoto (ICKT) in the liver after 70% hepatectomy following ischemia reperfusion.. Wistar rats were divided into 3 groups: simple laparotomy and 70% hepatectomy (Hx), 70% hepatectomy following ischemia reperfusion (IR) with vehicle (IRHxV), 70% hepatectomy following IR with ICKT (1 or 2 g/kg of body weight; IRHxK). Vehicle or ICKT was administered for 3 days preoperatively. The hepatoduodenal ligament was clamped for 15 minutes before hepatectomy in the IRHx groups. Rats were killed 1 hours after hepatectomy. In other experiments, the hepatoduodenal ligament was clamped for 30 minutes, with or without ICKT treatment, to evaluate the effect of ICKT on IR injury-induced mortality. Serum transaminase levels and the gene expression of inflammatory cytokines and inducible nitric oxide synthase in the remnant liver were determined. Furthermore, the expression of antioxidant genes was evaluated by PCR array.. The elevation of serum transaminase levels, the upregulation of genes for inflammatory cytokines and inducible nitric oxide synthase, and the increased formation of nitrotyrosine observed in the remnant livers of the IRHxV group were all significantly attenuated by preoperative administration of ICKT in the IRHxK group. The expression of antioxidant genes was also higher in the IRHxK group compared with that of the IRHxV group. Moreover, administration of ICKT significantly reduced the mortality induced by IRHx after 30-minute ischemia.. Preoperative administration of ICKT provides beneficial effects through attenuating inflammatory responses and oxidative stress in the liver following IR and subsequent hepatectomy.

Topics: Animals; Antioxidants; Cytokines; Drugs, Chinese Herbal; Glutathione; Hepatectomy; Inflammation Mediators; Iridoid Glycosides; Iridoids; Liver; Male; Nitric Oxide Synthase; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Polymerase Chain Reaction; Rats; Rats, Wistar; Reactive Nitrogen Species; Reactive Oxygen Species; Reperfusion Injury; Survival Rate; Transaminases; Tyrosine

This study examined the effects of genipin, isolated from Gardenia jasminoides Ellis, on d-galactosamine (GalN) and lipopolysaccharide (LPS)-induced hepatic apoptosis and liver failure. Mice were given an intraperitoneal injection of genipin (25, 50, 100 and 200mg/kg) 1h before GalN (700mg/kg)/LPS (10microg/kg) administration. The survival rate of the genipin group was significantly higher than that of the control. Genipin markedly reduced the increases in serum aminotransferase activities and lipid peroxidation. The glutathione content decreased in GalN/LPS group, and this decrease was attenuated by genipin. Increases in serum tumor necrosis factor-alpha (TNF-alpha), which were observed in GalN/LPS-treated mice, were significantly reduced by genipin. Genipin attenuated the GalN/LPS-induced apoptosis of hepatocytes, as estimated by the caspase-3 and -8 activity assay, TNF-R1 associated death domain (TRADD) protein measurement and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method. Moreover, increased cytosolic cytochrome c protein was reduced by genipin. After 3h of GalN/LPS injection, nuclear phosphorylated c-Jun (p-c-Jun) level was significantly increased, whereas it was attenuated by genipin. Also, the increased nuclear level of nuclear factor-kappaB and the decreased cytosolic level of IkappaB-alpha protein were significantly attenuated by genipin. Our results suggest that genipin offers marked hepatoprotection against damage induced by GalN/LPS related with its antioxidative, anti-apoptotic activities, and inhibition of NF-kappaB nuclear translocation and nuclear p-c-Jun expression.

Topics: Animals; Apoptosis; Caspases; Cell Nucleus; Cytochromes c; Cytosol; Galactosamine; Gene Expression Regulation; Glutathione; I-kappa B Proteins; Iridoid Glycosides; Iridoids; Lipid Peroxidation; Lipopolysaccharides; Liver; Liver Failure, Acute; Male; Mice; Mice, Inbred ICR; NF-KappaB Inhibitor alpha; Oxidative Stress; Phosphorylation; Protein Transport; Proto-Oncogene Proteins c-jun; TNF Receptor-Associated Death Domain Protein; Transaminases; Transcription Factor RelA; Tumor Necrosis Factor-alpha

To evaluate the effect of genipin, a natural crosslinker, on porcine corneas.. Department of Ophthalmology, Universidad Nacional de Colombia, Bogota, Colombia.. Corneal strips (12.0 mm x 2.3 mm) were harvested from porcine eyes and treated by incubation with genipin at concentrations of 1.00%, 0.25%, and 0.10%. Parallel corneal strips from the same eye were used as untreated controls. After treatment at 20 degrees C for 40 minutes, tensile strain measurements were performed in a biomaterial tester. Porcine button corneas were treated with genipin 0.25% for 15 minutes and then digested by bacterial collagenase. Treated and untreated corneas were evaluated by light microscopy.. Young modulus and stiffness in treated corneas increased in a concentration-dependent manner. Genipin increased resistance to corneal collagenase 5-fold in comparison with the controls. A decrease in the interlamellar space in treated corneas was also observed.. Corneal collagen crosslinking induced with genipin produced a significant increase in biomechanical strength and resistance to bacterial collagenase. This crosslinker could be useful in treating corneal ectasia and corneal infectious and noninfectious diseases involving corneal melting.

Topics: Animals; Collagen; Collagenases; Corneal Stroma; Cross-Linking Reagents; Elasticity; Iridoid Glycosides; Iridoids; Swine; Tensile Strength

The capacities of urinary trefoil factor 3 (TFF3) and urinary albumin to detect acute renal tubular injury have never been evaluated with sufficient statistical rigor to permit their use in regulated drug development instead of the current preclinical biomarkers serum creatinine (SCr) and blood urea nitrogen (BUN). Working with rats, we found that urinary TFF3 protein levels were markedly reduced, and urinary albumin were markedly increased in response to renal tubular injury. Urinary TFF3 levels did not respond to nonrenal toxicants, and urinary albumin faithfully reflected alterations in renal function. In situ hybridization localized TFF3 expression in tubules of the outer stripe of the outer medulla. Albumin outperformed either SCr or BUN for detecting kidney tubule injury and TFF3 augmented the potential of BUN and SCr to detect kidney damage. Use of urinary TFF3 and albumin will enable more sensitive and robust diagnosis of acute renal tubular injury than traditional biomarkers.

Topics: Albuminuria; Animals; Biomarkers, Pharmacological; Carbapenems; Cisplatin; Gentamicins; Histocytochemistry; Iridoid Glycosides; Iridoids; Kidney Diseases; Kidney Tubules; Logistic Models; Neuropeptides; Rats; ROC Curve; Trefoil Factor-3

The present study aimed to investigate the antidepressant potential of genipin and its possible mechanisms. Mouse models of depression including the forced swimming test (FST) and the tail suspension test (TST) were used to evaluate the effects of genipin. A possible mechanism was explored in the test of antagonism of reserpine-induced ptosis and hypothermia in mice. The contents of monoamine neurotransmitters and their metabolites including epinephrine (NE), 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in mice hippocampi were determined by HPLC-ECD. The results showed that intra-gastric administration of genipin at 50, 100, 200mg/kg or fluoxetine at 7.5mg/kg for 7 days significantly reduced the duration of immobility in FST and TST, while it did not affect the locomotor activity in the open field test (OFT). However, the effect was not dose-dependent. When the mice were treated with genipin or fluoxetine for 7 days, both of them could antagonize reserpine-induced ptosis and hypothermia. The 5-HT and NE contents in mice hippocampi were decreased after the peritoneal injection of reserpine at 2.0mg/kg. The pre-treatment with genipin at 50, 100, 200mg/kg or fluoxetine at 7.5mg/kg for 7 days could elevate the contents of NE and 5-HT in mice hippocampi significantly. The results suggest that compared with fluoxetine, genipin exerts antidepressant-like effects significantly. A possible mechanism, at least in part, is the regulation of the 5-HT and NE levels in the hippocampus.

Topics: Animals; Antidepressive Agents; Behavior, Animal; Biogenic Monoamines; Blepharoptosis; Depression; Disease Models, Animal; Fluoxetine; Hindlimb Suspension; Hippocampus; Hypothermia; Iridoid Glycosides; Iridoids; Male; Mice; Mice, Inbred ICR; Motor Activity; Reserpine

The aim of this study was to first evaluate whether the chitosan hydrochloride-genipin crosslinking reaction is influenced by factors such as time, and polymer/genipin concentration, and second, to develop crosslinked drug loaded microspheres to improve the control over drug release. Once the crosslinking process was characterized as a function of the factors mentioned above, drug loaded hydrochloride chitosan microspheres with different degrees of crosslinking were obtained. Microspheres were characterized in terms of size, morphology, drug content, surface charge and capacity to control in vitro drug release. Clarithromycin, tramadol hydrochloride, and low molecular weight heparin (LMWH) were used as model drugs. The obtained particles were spherical, positively charged, with a diameter of 1-10 microm. X-Ray diffraction showed that there was an interaction of genipin and each drug with chitosan in the microspheres. In relation to the release profiles, a higher degree of crosslinking led to more control of drug release in the case of clarithromycin and tramadol. For these drugs, optimal release profiles were obtained for microspheres crosslinked with 1 mM genipin at 50 °C for 5 h and with 5 mM genipin at 50 °C for 5 h, respectively. In LMWH microspheres, the best release profile corresponded to 0.5 mM genipin, 50 °C, 5 h. In conclusion, genipin showed to be eligible as a chemical-crosslinking agent delaying the outflow of drugs from the microspheres. However, more studies in vitro and in vivo must be carried out to determine adequate crosslinking conditions for different drugs.

Topics: Analgesics, Opioid; Anti-Bacterial Agents; Anticoagulants; Chemistry, Pharmaceutical; Chitosan; Clarithromycin; Cross-Linking Reagents; Delayed-Action Preparations; Diffusion; Heparin, Low-Molecular-Weight; Iridoid Glycosides; Iridoids; Kinetics; Microscopy, Electron, Scanning; Microspheres; Osmolar Concentration; Particle Size; Surface Properties; Tramadol; X-Ray Diffraction

The concise synthesis of 5,6-dihydrovaltrate (2), the bioisostere of valtrate (1) showing anti-HIV activity by inhibition for nuclear export of Rev, has been achieved from the commercially available iridoid genipin (3). Analysis of steric influence of the substituents linked to the three hydroxyl groups was conducted by the synthesized three analogs (2a-2c). Consequently, attenuation of steric hindrance around the epoxy portion was revealed to enhance inhibitory potency for Rev-export. In addition to this finding, 1-acetoxy analog 2b was disclosed as the promising Rev-export inhibitor superior to 1.

Topics: Anti-HIV Agents; Cell Nucleus; HeLa Cells; HIV Infections; HIV-1; Humans; Iridoid Glycosides; Iridoids; Protein Transport; rev Gene Products, Human Immunodeficiency Virus

In this study, genipin-cross-linked collagen/chitosan biodegradable porous scaffolds were prepared for articular cartilage regeneration. The influence of chitosan amount and genipin concentration on the scaffolds physicochemical properties was evaluated. The morphologies of the scaffolds were characterized by scanning electron microscope (SEM) and cross-linking degree was investigated by ninhydrin assay. Additionally, the mechanical properties of the scaffolds were assessed under dynamic compression. To study the swelling ratio and the biostability of the collagen/chitosan scaffold, in vitro tests were also carried out by immersion of the scaffolds in PBS solution or digestion in collagenase, respectively. The results showed that the morphologies of the scaffolds underwent a fiber-like to a sheet-like structural transition by increasing chitosan amount. Genipin cross-linking remarkably changed the morphologies and pore sizes of the scaffolds when chitosan amount was less than 25%. Either by increasing the chitosan ratio or performing cross-linking treatment, the swelling ratio of the scaffolds can be tailored. The ninhydrin assay demonstrated that the addition of chitosan could obviously increase the cross-linking efficiency. The degradation studies indicated that genipin cross-linking can effectively enhance the biostability of the scaffolds. The biocompatibility of the scaffolds was evaluated by culturing rabbit chondrocytes in vitro. This study demonstrated that a good viability of the chondrocytes seeded on the scaffold was achieved. The SEM analysis has revealed that the chondrocytes adhered well to the surface of the scaffolds and contacted each other. These results suggest that the genipin-cross-linked collagen/chitosan matrix may be a promising formulation for articular cartilage scaffolding.

Topics: Animals; Biocompatible Materials; Cartilage, Articular; Cells, Cultured; Chitosan; Cholagogues and Choleretics; Chondrocytes; Collagen; Iridoid Glycosides; Iridoids; Materials Testing; Rabbits; Regeneration; Stress, Mechanical; Tissue Engineering; Tissue Scaffolds

Previously, we reported that genipin, a herbal iridoid, had neuritogenic and neuroprotective actions on PC12 cells. Although nitric oxide (NO)-activated signalings were proposed to be neuritogenic, the neuroprotective action of genipin remains to be elucidated. From the standpoint of NO activation, we tested a possible protective mechanism through the nitrosative Kelch-like ECH-associated protein (Keap1)/NF-E2-related factor 2 (Nrf2)-antioxidant response element pathway in rat retinal ganglion cells (RGC-5 cells) in culture, and in vivo, against hydrogen peroxide and optic nerve injury (ONI), respectively, using a long-acting (1R)-isoPropyloxygenipin (IPRG001). IPRG001 induced NO generation and the expressions of antioxidative enzymes, such as heme oxygenase-1 (HO-1), in RGC-5 cells. The protective action of IPRG001 depended on HO-1 and NO induction. We found that S-nitrosylation of Keap1 by IPRG001 may contribute to translocation of Nrf2 to the nucleus and triggered transcriptional activation of antioxidative enzymes. Furthermore, apoptotic cells were increased and 4-hydroxy-2-nonenal was accumulated in rat retina following ONI. Pre-treatment with IPRG001 almost completely suppressed apoptosis and accumulation of 4-hydroxy-2-nonenal in RGCs following ONI accompanied by HO-1 induction. These data demonstrate for the first time that IPRG001 exerts neuroprotective action in RGCs in vitro and in vivo, through the Nrf2/antioxidant response element pathway by S-nitrosylation against oxidative stress.

Topics: Aldehydes; Animals; Antioxidants; Blotting, Western; Cell Death; Cell Line; Chromatin; Coloring Agents; Cysteine Proteinase Inhibitors; Heme Oxygenase-1; Humans; Immunohistochemistry; Immunoprecipitation; In Situ Nick-End Labeling; Intracellular Signaling Peptides and Proteins; Iridoid Glycosides; Iridoids; Kelch-Like ECH-Associated Protein 1; NF-E2-Related Factor 2; Nitric Oxide; Oxidative Stress; Rats; Rats, Sprague-Dawley; Response Elements; Retinal Ganglion Cells; Reverse Transcriptase Polymerase Chain Reaction; RNA; RNA, Small Interfering; Signal Transduction; Tetrazolium Salts; Thiazoles

Genipin is a metabolite of geniposide isolated from an extract of Gardenia fructus. Some observations suggested that genipin could induce cell apoptosis in hepatoma cells and PC3 human prostate cancer cells. However, the effects of genipin on HeLa human cervical carcinoma cells are still unknown. In this study, we provided evidences that genipin induced the death of HeLa cells through apoptotic pathway in a dose-dependent manner. Genipin could remarkably induce cytotoxicity in HeLa cells and inhibit its proliferation. Induction of the apoptosis by genipin was confirmed by analysis of DNA fragmentation and induction of sub-G(1) peak through flow cytometry. The results also showed that genipin-treated HeLa cells cycle was arrested at G(1) phase. Western blot analysis revealed that the phosphorylated c-Jun NH(2)-terminal kinase (JNK) protein, phospho-Jun protein, p53 protein and bax protein significantly increased in a dose-dependent manner after treatment of genipin for 24 h, and to our knowledge, the activation of JNK maybe result in the increase of the p53 protein level, and the increase of the p53 protein led to the accumulation of bax protein, bax protein further induced cell apoptotic death eventually. Taken all these together, it is possible to develop genipin as an anti-cancer drug.

Topics: Antineoplastic Agents, Phytogenic; Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Cell Culture Techniques; Cell Cycle; Cell Proliferation; Cell Survival; DNA Fragmentation; Dose-Response Relationship, Drug; Enzyme Activation; Flow Cytometry; HeLa Cells; Humans; Iridoid Glycosides; Iridoids; JNK Mitogen-Activated Protein Kinases; Medicine, Chinese Traditional; Microscopy, Phase-Contrast; Phosphorylation; Tumor Suppressor Protein p53

To modulate the mechanical properties of nerve guide conduit for surgical manipulation, this study develops a biodegradable composite containing genipin cross-linked gelatin annexed with β-tricalcium phosphate ceramic particles as a nerve guide material. The conduit was dark bluish and round with a rough and compact outer surface compared to the genipin cross-linked gelatin conduit (without β-tricalcium phosphate). Water uptake and swelling tests indicate that the conduit noticeably increases the stability in water, and the hydrated conduit does not collapse and stenose. The conduit has a sufficiently high level of mechanical properties to serve as a nerve guide. After subcutaneous implantation on the dorsal side of a rat, the degraded conduit only evokes a mild tissue response, and the formation of a very thin fibrous capsule surrounds the conduit. This paper assesses the effectiveness of the conduit as a guidance channel when we use it to repair a 10 mm gap in the rat's sciatic nerve. The experimental results show no gross inflammatory reactions of the peripheral nerve tissues at the implantation site in either group. In overall gross examination, the diameter of the intratubular and newly formed nerve fibers in the conduits exceeds that of the silicone tubes during the implantation period. The quantitative results indicate the superiority of the conduits over the silicone tubes. This study microscopically observes the nerve regeneration in the tissue section at the middle region of all implanted conduits. Therefore, the histomorphometric assessment demonstrates that the conduit could be a candidate for peripheral nerve repair.

Topics: Absorbable Implants; Animals; Biocompatible Materials; Calcium Phosphates; Cross-Linking Reagents; Gelatin; Guided Tissue Regeneration; Implants, Experimental; Inflammation; Iridoid Glycosides; Iridoids; Nerve Regeneration; Peripheral Nerves; Rats; Sciatic Nerve

In the present research it is reported the development and characterization of novel polymer blends based on chitosan and poly(vinyl alcohol) (PVA). The hybrid polymeric network was synthesized and modified by chemical crosslinking using genipin for potential use in a variety of biomedical applications. The micro and nanostructures of the blended hydrogels and hybrids were characterized through Fourier Transform Infrared spectroscopy (FTIR) and swelling test analysis. Moreover, cytotoxicity and cell viability were also performed by MTT assay with cell culture. The chemical crosslinking was achieved via the relative intensity of the band at 1650 cm(-1) which is mostly associated with the reaction of genipin carboxymethyl group with the amino group of chitosan forming amides. It was found that by increasing the chitosan content relative to PVA the swelling index of the blend has decreased, reflecting the reduction on the mobility of polymer network and the hydrophilic behavior of the blend. The tested hydrogels have clearly presented adequate cell viability, non-toxicity and suitable properties which can be tailored for potential use in bioengineering applications.

Topics: Amides; Animals; Biocompatible Materials; Biotechnology; Cell Survival; Chitosan; Chlorocebus aethiops; Cross-Linking Reagents; Hydrogel, Polyethylene Glycol Dimethacrylate; Iridoid Glycosides; Iridoids; Materials Testing; Nanostructures; Polyvinyl Alcohol; Spectroscopy, Fourier Transform Infrared; Vero Cells

A porous-three-dimensional scaffold shows several advantages in terms of tissue engineering since it can provide a framework for cells to attach, proliferate and form an extracellular matrix. Sericin, a by-product from the silk industry, can form a three-dimensional scaffold with PVA after freeze-drying but has a fragile structure. Glycerin (as a plasticizer) and genipin (a crosslinking agent) are necessary to make a strong and stable matrix. Our objective was to investigate the properties of a three-dimensional silk sericin and PVA scaffold with and without glycerin and genipin at various concentrations. SEM showed that adding glycerin into scaffold gave better uniformity and porosity. Smaller pore sizes and better uniformity were found as the concentration of genipin in the scaffold increased. The results of FTIR indicated that glycerin retained a high moisture content and had a major effect at 3286 cm(-1), indicating the presence of water molecule in the matrix structure. Adding genipin into the scaffold resulted in a higher degree of crosslinking or fewer free ∈-amino groups, as shown by the decrease in the stretching (=C-H) peak and absorption peaks around 1370-1650 cm(-1), respectively. The sericin/PVA scaffold had a low water sorption capacity, but adding glycerin significantly increased this property. Genipin further enhanced the moisture absorption capacity of the scaffold and extended the time taken to reach equilibrium. After immersing the sericin/PVA scaffold into purified water, the scaffold completely dissolved within an hour, whereas the scaffolds containing glycerin or glycerin with 0.1% genipin swelled 8 and 11 times, respectively, compared with the initial stage after 6h of immersion. In terms of mechanical properties, the sericin/PVA/glycerin scaffold exhibited a similar compressive strength to the scaffold with a high genipin concentration, whereas a low concentration of genipin softened and reduced the compressive strength of the scaffold. A small amount of sericin was released from the scaffold and a higher concentration of genipin, resulting in less protein leaching compared to non-crosslinked sericin/PVA. The fraction of protein released from the sericin/PVA/glycerin scaffold was about 4%, with values of about 1 and 0.04% in the case of scaffolds with 0.01 and 0.1% genipin, respectively. All results indicated that the composition of the scaffolds had a significant effect on their physical properties, and that can easily be tuned to

Topics: Absorption; Amino Acid Sequence; Animals; Bombyx; Cross-Linking Reagents; Iridoid Glycosides; Iridoids; Mechanical Phenomena; Microscopy, Electron, Scanning; Molecular Weight; Polyvinyl Alcohol; Porosity; Sericins; Spectroscopy, Fourier Transform Infrared; Tissue Scaffolds

To improve water-resistant ability and mechanical properties of silk fibroin (SF)/hydroxybutyl chitosan (HBC) nanofibrous scaffolds for tissue-engineering applications, genipin, glutaraldehyde (GTA), and ethanol were used to crosslink electrospun nanofibers, respectively. The mechanical properties of nanofibrous scaffolds were obviously improved after 24 h of crosslinking with genipin and were superior to those crosslinked with GTA and ethanol for 24 h. SEM indicated that crosslinked nanofibers with genipin and GTA vapor had good water-resistant ability. Characterization of the microstructure (porosity and pore structure) demonstrated crosslinked nanofibrous scaffolds with genipin and GTA vapor had lager porosities and mean diameters than those with ethanol. Characterization of FTIR-ATR and (13)C NMR clarified both genipin and GTA acted as crosslinking agents for SF and HBC. Furthermore, genipin could induce SF conformation from random coil or α-helix to β-sheet. Although GTA could also successfully crosslink SF/HBC nanofibrous scaffolds, in long run, genipin maybe a better method due to lower cytotoxicity than GTA. Cell viability studies and wound-healing test in rats clarified that the genipin-crosslinked SF/HBC nanofibrous scaffolds had a good biocompatibility both in vitro and in vivo. These results suggested that genipin-crosslinked SF/HBC nanofibrous scaffolds might be potential candidates for wound dressing and tissue-engineering scaffolds.

Topics: Animals; Biocompatible Materials; Cells, Cultured; Chitosan; Cholagogues and Choleretics; Cross-Linking Reagents; Endothelial Cells; Fibroins; Iridoid Glycosides; Iridoids; Materials Testing; Molecular Structure; Nanofibers; Porosity; Protein Conformation; Rats; Swine; Tensile Strength; Tissue Engineering; Tissue Scaffolds; Wound Healing

A main challenge in tissue engineering and regenerative medicine is achieving local and efficient growth factor release to guide cell function. Gelatin is a denatured form of collagen that cells can bind to and degrade through enzymatic action. In this study, gelatin microspheres were used to release bone morphogenetic protein 2 (BMP2). Spherical microparticles with diameters in the range of 2-6 µm were created by an emulsification process and were stabilized by crosslinking with the small molecule genipin. The degree of crosslinking was varied by controlling the incubation time in genipin solution. Loading rate studies, using soy bean trypsin inhibitor as a model protein, showed rapid protein uptake over the first 24 h, followed by a levelling off and then a further increase after approximately 3 days, as the microspheres swelled. Growth factor release studies using microspheres crosslinked to 20%, 50% and 80% of saturation and then loaded with BMP2 showed that higher degrees of crosslinking resulted in higher loading efficiency and slower protein release. After 24 h, the concentration profiles produced by all microsphere formulations were steady and approximately equal. Microspheres incubated with adult human mesenchymal stem cells accumulated preferentially on the cell surface, and degraded over time in culture. BMP2-loaded microspheres caused a three- to eight-fold increase in expression of the bone sialoprotein gene after 14 days in culture, with more crosslinked beads producing a greater effect. These results demonstrate that genipin-crosslinked gelatin microspheres can be used to deliver growth factors locally to cells in order to direct their function.

Topics: Bone Morphogenetic Protein 2; Cells, Cultured; Drug Delivery Systems; Gelatin; Gene Expression Regulation; Humans; Integrin-Binding Sialoprotein; Iridoid Glycosides; Iridoids; Mesenchymal Stem Cells; Microspheres

Uncoupling protein-2 (UCP2) is known to suppress mitochondrial reactive oxygen species (ROS) production and is employed by drug-resistant cancer cells to mitigate oxidative stress. Using the drug-sensitive HL-60 cells and the drug-resistant MX2 subline as model systems, we show that genipin, a UCP2 inhibitor, sensitizes drug-resistant cells to cytotoxic agents. Increased MX2 cell death was observed upon co-treatment with genipin and different doses of menadione, doxorubicin, and epirubicin. DCFH-DA fluorimetry revealed that the increase in MX2 cell death was accompanied by enhanced cellular ROS levels. The drug-induced increase in ROS was linked to genipin-mediated inhibition of mitochondrial proton leak. State 4 and resting cellular respiratory rates were higher in the MX2 cells in comparison to the HL-60 cells, and the increased respiration was readily suppressed by genipin in the MX2 cells. UCP2 accounted for a remarkable 37% of the resting cellular oxygen consumption indicating that the MX2 cells are functionally reliant on this protein. Higher amounts of UCP2 protein were detected in the MX2 versus the HL-60 mitochondria. The observed effects of genipin were absent in the HL-60 cells pointing to the selectivity of this natural product for drug-resistant cells. The specificity of genipin for UCP2 was confirmed using CHO cells stably expressing UCP2 in which genipin induced an ∼22% decrease in state 4 respiration. These effects were absent in empty vector CHO cells expressing no UCP2. Thus, the chemical inhibition of UCP2 with genipin sensitizes multidrug-resistant cancer cells to cytotoxic agents.

Topics: Anthracyclines; Antineoplastic Agents; Cell Line, Tumor; Drug Resistance, Neoplasm; Drug Synergism; Fluorometry; Humans; Ion Channels; Iridoid Glycosides; Iridoids; Mitochondrial Proteins; Reactive Oxygen Species; Uncoupling Protein 2

In vitro study of genipin crosslinking effect on disc water content changes under compressive loading and unloading.. To investigate the influence of collagen crosslinking on hydration and fluid flow in different regions of intact discs, and to evaluate the nutritional implications.. Age-related reductions of nutrient supply and waste product removal are critically important factors in disc pathogenesis. Diffusion and fluid flow are blocked by subchondral bone thickening, cartilaginous endplate calcification, loss of hydrophilic proteoglycans, and clogging of anular pores by degraded matrix molecules. Previous studies demonstrated increased hydraulic permeability and macromolecular transport through crosslinked collagenous matrices. Genipin has also demonstrated the capability to increase retention of proteoglycans.. A total of 57 bovine lumbar motion segments were divided randomly into phosphate buffered saline and 0.33% genipin-soaked treatment groups. Water content changes were measured using a mass-loss technique in 3 intervertebral disc regions following successive stages of compressive loading and unloading (post-treatment, after 1 hour 750 N compression, and after a subsequent 24-hour period of nominal loading). Net flow of fluid into or out of a region was determined from the percentage change in mean water content from successive groups.. Fluid flow to and from the nucleus doubled with genipin crosslinking. Relative to the buffer-only controls, overall net fluid flow increased 103% in the nucleus pulposus, 36% in the inner anulus, and was 31% less in the outer anulus of genipin treated discs.. The effects of genipin crosslinking on matrix permeability and proteoglycan retention can alter hydration levels and fluid flow in the intervertebral disc. Resulting increases in fluid flow, including a doubling of flow to and from the nucleus, could lead to enhanced nutritional inflow and waste product outflow for the disc, and may have implications for emerging cell-based therapies.

Topics: Animals; Biomechanical Phenomena; Body Water; Cattle; Collagen; Compressive Strength; Cross-Linking Reagents; In Vitro Techniques; Intervertebral Disc; Iridoid Glycosides; Iridoids; Lumbar Vertebrae; Permeability; Proteoglycans; Weight-Bearing

Uncoupling protein (UCP) 2 is related to the dysfunction of beta cells induced by fatty acids. However, whether UCP2 has similar effects on alpha cell is still not clear. This study aimed to investigate the effects of UCP2 and its possible mechanisms in lipotoxicity-induced dysfunction of pancreatic alpha cells.. The alpha TC1-6 cells were used in this study to evaluate the effects of palmitate and/or UCP2 inhibit factors on the glucagon secretory function, glucagon content, the glucagon mRNA level and the nitrotyrosine level in the supernatant. Meantime, the expression levels of UCP2 and peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1 alpha) were measured by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Furthermore, the possible relationship between UCP2 and insulin signal transduction pathway was analyzed.. Palmitate stimulated alpha cell glucagon secretion and the expression of UCP2 and PGC-1 alpha, which could be partially decreased by the inhibition of UCP2. Palmitate increased nitrotyrosine level and suppressed insulin signal transduction pathway in alpha cells. Inhibition of UCP2 influenced the effects of free fatty acid on alpha cells and may relate to glucagon secretion.. UCP2 played an important role on alpha cell dysfunction induced by free fatty acid in vitro, which may be related to its effects on oxidative stress and insulin signal transduction pathway.

Topics: Animals; Cells, Cultured; Glucagon; Glucagon-Secreting Cells; Insulin; Insulin Receptor Substrate Proteins; Ion Channels; Iridoid Glycosides; Iridoids; Mice; Mitochondrial Proteins; Oxidative Stress; Palmitic Acid; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha; Phosphorylation; RNA, Messenger; Signal Transduction; Trans-Activators; Transcription Factors; Tyrosine; Uncoupling Protein 2

to investigate the ability of drug-loaded N,O-carboxymethyl chitosan (CMCS) hydrogels to modulate wound healing after glaucoma filtration surgery.. the drug-loaded CMCS hydrogels were in situ synthesized using genipin as the crosslinker in the presence of 5-fluorouracil (5FU) or bevacizumab. Their structures were characterized by FTIR, ultraviolet-visible (UV-vis) spectroscopy and scanning electron microscopy (SEM). In-vitro drug release experiments and in vivo evaluation in rabbits were performed.. the results of FTIR, UV-vis spectroscopy and SEM analyses indicated that 5FU was encapsulated into the CMCS hydrogels that were crosslinked by genipin. The in vitro drug release experiments showed that nearly 100% of 5FU was released from the drug-loaded hydrogels within 8 h, but less than 20% bevacizumab was released after 53 h. The in vivo evaluation in rabbits indicated that the drug-loaded CMCS hydrogels were nontoxic to the cornea and were gradually biodegraded in the eyes. Furthermore, the drug-loaded CMCS hydrogels effectively inhibited conjunctival scarring after glaucoma filtration surgery and controlled postoperative intraocular pressure (IOP).. the drug-loaded CMCS hydrogels provide a great opportunity to increase the therapeutic efficacy of glaucoma filtration surgery.

Topics: Administration, Topical; Animals; Antibodies, Monoclonal; Antibodies, Monoclonal, Humanized; Bevacizumab; Chitosan; Cicatrix; Conjunctiva; Cross-Linking Reagents; Delayed-Action Preparations; Drug Carriers; Female; Filtering Surgery; Fluorouracil; Glaucoma; Hydrogels; In Vitro Techniques; Intraocular Pressure; Iridoid Glycosides; Iridoids; Male; Microscopy, Electron, Scanning; Rabbits; Spectroscopy, Fourier Transform Infrared; Wound Healing

Parkinson's disease is a pervasive, ageing-related neurodegenerative disease the cardinal motor symptoms of which reflect the loss of a small group of neurons, the dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial oxidant stress is widely viewed as being responsible for this loss, but why these particular neurons should be stressed is a mystery. Here we show, using transgenic mice that expressed a redox-sensitive variant of green fluorescent protein targeted to the mitochondrial matrix, that the engagement of plasma membrane L-type calcium channels during normal autonomous pacemaking created an oxidant stress that was specific to vulnerable SNc dopaminergic neurons. The oxidant stress engaged defences that induced transient, mild mitochondrial depolarization or uncoupling. The mild uncoupling was not affected by deletion of cyclophilin D, which is a component of the permeability transition pore, but was attenuated by genipin and purine nucleotides, which are antagonists of cloned uncoupling proteins. Knocking out DJ-1 (also known as PARK7 in humans and Park7 in mice), which is a gene associated with an early-onset form of Parkinson's disease, downregulated the expression of two uncoupling proteins (UCP4 (SLC25A27) and UCP5 (SLC25A14)), compromised calcium-induced uncoupling and increased oxidation of matrix proteins specifically in SNc dopaminergic neurons. Because drugs approved for human use can antagonize calcium entry through L-type channels, these results point to a novel neuroprotective strategy for both idiopathic and familial forms of Parkinson's disease.

Topics: Animals; Biological Clocks; Brain; Calcium; Calcium Channel Blockers; Calcium Channels, L-Type; Calcium Signaling; Cyclophilins; Dihydropyridines; Dopamine; Gene Deletion; Ion Channels; Iridoid Glycosides; Iridoids; Male; Mice; Mice, Transgenic; Mitochondria; Mitochondrial Proteins; Neurons; Oncogene Proteins; Oxidative Stress; Parkinson Disease; Peptidyl-Prolyl Isomerase F; Peroxiredoxins; Protein Deglycase DJ-1; Purines; Superoxides; Uncoupling Protein 1

Degradation of genipin (GP), a low toxicity natural protein crosslinking agent, in aqueous solution was monitored by HPLC at various pH levels. Degradation of GP was consistent with a mechanism consisting of a first order reaction with a reversible first step. Formation of the intermediate was slowest at more neutral pHs while formation of the irreversible product was correlated to increasing alkalinity. Degradation at all pHs was enhanced by the presence of phosphate ions. Degradation of GP most likely proceeds via the reversible opening of the dihydropyran ring by water followed by irreversible polymerization of the intermediate. Degraded solutions containing no detectable GP or intermediate, however, are still capable of crosslinking proteins.

Topics: Cross-Linking Reagents; Drug Stability; Hydrogen-Ion Concentration; Iridoid Glycosides; Iridoids; Kinetics; Solutions

This study was aimed to detect the effect of genipin and Vitamin E (VitE) on non-alcoholic fatty liver disease. L02 cells were divided into five groups:control group, palmic acid treated group, VitE treated group, genipin treated group, and a combination group. All treatments were terminated at the end of 72 hours. Pathological changes of L02 cells were observed. Mitochondrial membrane potential changes were detected by flow cytometry. MDA, SOD, ALT, AST, GGT, TG in culture medium and expression of UCP2 mRNA and protein in L02 cells were detected. We also studied the effects of genipin and VitE on UCP2 and other related factors such as NF-kappaB and TNF-alpha on the L02 cell model of non-alcoholic fatty liver disease. In combination group, the degree of adipose degeneration of L02 cells mitigated significantly; mitochondrial membrane potential and the level of SOD activity increased; the level of MDA, ALT, AST, GGT, TG and the expression of UCP2, NF-kappaB,TNF-alpha in L02 cells decreased. The use of genipin in combination with VitE can increase mitochondrial membrane potential and markedly relieve the adipose degeneration of liver cells.

Topics: Cell Line; Drug Synergism; Fatty Liver; Humans; Ion Channels; Iridoid Glycosides; Iridoids; Liver; Membrane Potential, Mitochondrial; Mitochondrial Proteins; NF-kappa B; Non-alcoholic Fatty Liver Disease; Protective Agents; RNA, Messenger; Tumor Necrosis Factor-alpha; Uncoupling Protein 2; Vitamin E

The interaction between cells and biopolymers is the evaluation indicator of the biocompatibility of materials. The purpose of this work was to examine the responses of retinal pigment epithelial (RPE) cells to genipin (GP) or glutaraldehyde (GTA) cross-linked chitosan by means of cell viability assays, cytokine expression analyses, and apoptosis assays. Evaluations of non-cross-linked chitosan were conducted simultaneously for comparison. Both GP and GTA treated samples with the same extent of cross-linking (around 80%) were prepared by varying cross-linking time. Our results showed that GP cross-linking was carried out by either radical polymerization of the monomers or S(N)2 nucleophilic substitution reaction involving the replacement of the ester group on the monomer with a secondary amide linkage. On the other hand, GTA could react with free amino groups of chitosan, leading to the formation of either the Schiff bases or the Michael-type adducts with terminal aldehydes. The biocompatibility of non-cross-linked chitosan membranes was demonstrated by the absence of any signs of toxicity or inflammation reaction. The present study showed that the ARPE-19 cells exposed to GTA cross-linked chitosan membranes had significantly higher cytotoxicity, interleukin-6 levels, and number of TUNEL-positive nuclei than did those exposed to GP treated samples. In addition, the materials modified with GTA trigger apoptosis at an early stage and may induce toxicity in the RPE cells later. The findings suggest that while the chitosan molecules bridged by GP are satisfactorily cytocompatible, the counterparts treated by GTA do not seem to be tolerated. In terms of material safety, the GP cross-linked chitosan may be compatible with human RPE cells and may have a potential application as delivery carriers in the treatment of posterior segment diseases.

Topics: Cell Line, Transformed; Chitosan; Glutaral; Humans; Iridoids; Materials Testing; Membranes, Artificial; Retinal Pigment Epithelium

We have designed and developed a microfluidic system to study the response of cells to controlled gradients of mechanical stiffness in 3D collagen gels. An 'H'-shaped, source-sink network was filled with a type I collagen solution, which self-assembled into a fibrillar gel. A 1D gradient of genipin--a natural crosslinker that also causes collagen to fluoresce upon crosslinking--was generated in the cross-channel through the 3D collagen gel to create a gradient of crosslinks and stiffness. The gradient of stiffness was observed via fluorescence. A separate, underlying channel in the microfluidic construct allowed the introduction of cells into the gradient. Neurites from chick dorsal root ganglia explants grew significantly longer down the gradient of stiffness than up the gradient and than in control gels not treated with genipin. No changes in cell adhesion, collagen fiber size, or density were observed following crosslinking with genipin, indicating that the primary effect of genipin was on the mechanical properties of the gel. These results demonstrate that (1) the microfluidic system can be used to study durotactic behavior of cells and (2) neurite growth can be directed and enhanced by a gradient of mechanical properties, with the goal of incorporating mechanical gradients into nerve and spinal cord regenerative therapies.

Topics: Animals; Cell Adhesion; Chickens; Collagen; Cross-Linking Reagents; Elasticity; Ganglia, Spinal; Gels; Iridoid Glycosides; Iridoids; Microfluidic Analytical Techniques; Neurites; Neurogenesis; Tissue Engineering

Different lag-time of pulse-released nerve growth factor (NGF) from genipin-crosslinked gelatin within polycaprolactone (PCL) conduits was evaluated in large-gap peripheral nerve repair. In this study, 10% (w/v) gelatin was mixed with NGF, crosslinked with 0%, 0.1%, 0.5%, and 1% (w/v) genipin, and then sucked into the wall of PCL conduits. These controlled-release nerve conduits were named NCL (non-crosslink), LCL (low crosslink), MCL (medium crosslink), and HCL (high crosslink), respectively. The NGF releasing character showed four distinctive curves, including initial burst within 5 days, pulse releasing at 5-20 days, pulse releasing at 10-25 days, and steadily releasing. The bioactivity of the released NGF was shown by neurite outgrowth of PC12 cells after culturing in all groups. Finally, the controlled-release conduits were seeded with 9 x 10(3) Schwann cells. Conduits were used to bridge a 15-mm rat sciatic nerve defect, and the results were compared with the isografts (control group). Eight weeks after implantation, morphological analysis revealed that LCL, MCL, and HCL groups were similar to autograft treatment in the numbers and area of myelinated axons. The LCL group, although insignificant, showed a trend to have the highest myelinated axon counts of the conduit-treated groups. Thus, comparing the different NGF release characteristics among NCL, MCL, and LCL groups, we concluded that a high concentration of NGF at 5-10 days in LCL groups is needed in bridging a 15-mm peripheral nerve injury.

Topics: Animals; Axons; Blood Vessels; Cell Count; Cell Proliferation; Cell Shape; Cross-Linking Reagents; Gelatin; Iridoid Glycosides; Iridoids; Male; Myelin Sheath; Nerve Growth Factor; Nerve Regeneration; Neurites; PC12 Cells; Peripheral Nerves; Polyesters; Rats; Rats, Sprague-Dawley; Schwann Cells

The gelatin solution crosslinked by genipin (0, 0.1, 0.5, 1.0, and 1.5% w/w) was studied as a nerve growth factor (NGF) carrier (GGp0, GGp0.1, GGp0.5, GGp1.0, and GGp1.5) in a polycaprolactone conduit in large-gap nerve regeneration. The GGp0 and GGp0.1 displayed the highest activity of PC12 cells and inhibited the reduction of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl-tetrazolium bromide (MTT). No cytotoxicity was found in all groups by lactate dehydrogenase (LDH) release. The NGF-releasing characters were obtained by ELISA tests. A relatively fast release rate appeared during the first 10 days and then a subsequent slower release profile followed. NGF was higher in GGp0.1 than in GGp0 and GGp0.1 after 10 days. The bioactivity of the released NGF remains the same when measuring the neurite outgrowth of PC 12 cells. Finally, the controlled-release conduits were implanted into 12-mm long sciatic nerve gaps of rats. In addition, the best site of NGF carrier was determined either by filling carrier into the conduit lumen or by sucking carrier to the conduit wall. Four and 8 weeks after implantation, morphological analysis revealed that GGp0.1 conduits had markedly larger and more number of myelin axons in the midconduit and distal nerve. Further, sucking the carrier into the conduit wall was an efficient and convenient way to prevent the regeneration of axons and vessels from being impaired by the lumen's carrier. The genipin-crosslinked gelatin is a promising carrier in producing a high release concentration and a long release period of NGF to promote the regeneration over a large-gap nerve injury.

Topics: Animals; Cell Line; Cell Survival; Cross-Linking Reagents; Delayed-Action Preparations; Gelatin; Implants, Experimental; Iridoid Glycosides; Iridoids; L-Lactate Dehydrogenase; Male; Nerve Growth Factors; Nerve Regeneration; Neurites; Peripheral Nerves; Polyesters; Rats; Rats, Sprague-Dawley

Genipin is a naturally-derived biocompatible cross-linking agent commonly used to generate three dimensional tissue-engineered scaffolds or to fix biologically derived scaffolds prior to implantation. Here we propose a novel use for genipin as a long-term culture medium supplement to promote cross-linking of de novo cell products that are produced in engineered cartilage. We hypothesize that the application of genipin will stabilize the extracellular matrix components and increase the mechanical properties of developing engineered cartilage. Chondrocytes encapsulated in agarose hydrogel (a neutrally charged polysaccharide scaffold that is unaffected by genipin cross-linking) were cultured in a chemically-defined growth medium that was supplemented with varying concentrations of genipin (22 microM, 220 microM, 2200 microM) for various durations (continuous or intermittent). Tissues developed significantly higher mechanical properties (+28% dynamic modulus and +20% Young's modulus) by day 42 with genipin treatment compared to untreated controls. These increases were not immediate, but presented over culture time after genipin treatment. The genipin treated groups were also more resistant to cytokine-induced degradation with interleukin-1alpha; maintaining an E(Y) (+218%), G* (+390%) and glycosaminoglycan (GAG) content (+477%) over genipin-untreated constructs subjected to interleukin. We hypothesize two mechanisms through which the physical enhancement of tissue properties may be fostered: (1) by cross-link mediated reorganization and enhanced retention of cell-elaborated extracellular matrix components, and (2) through reduction of the loss of extracellular matrix components by increasing their resilience to catabolic degradation. These studies demonstrate a potential use of genipin as a medium supplement to develop enhanced engineered cartilage.

Topics: Animals; Biocompatible Materials; Cartilage; Cattle; Chondrocytes; Compressive Strength; Culture Media; Cytokines; Extracellular Matrix; Glycosaminoglycans; Inflammation; Interleukin-1alpha; Iridoid Glycosides; Iridoids; Materials Testing; Tissue Engineering

Macroporous calcium phosphate cements (CPCs) were developed using genipin-crosslinked gelatin microspheres (GMs) with two weight ratios (2.5 wt% and 5 wt%). The initial setting time of the composite was prolonged by GMs. After GMs/CPCs were soaked in phosphate-buffered saline (PBS) for several weeks, macropores appeared as a result of the degradation of GMs. The presence of GMs accelerated the setting reaction and improved the structure of the composite. The compressive strength increased up to 12 MPa (2.5 wt% GMs/CPCs) and 14 MPa (5 wt% GMs/CPCs) after one week of PBS soaking, then gradually decreased to 9 MPa (2.5 wt% GMs/CPCs) and 7 MPa (5 wt% GMs/CPCs) after three weeks of soaking, and further to 6 MPa (2.5 wt% GMs/CPCs) and 2 MPa (5 wt% GMs/CPCs) after five weeks of soaking. CPCs with 2.5 wt% GMs were the most favorable composite in the tested samples. Cell experiments showed that rat osteoblasts displayed normal morphologies when exposed to the 2.5 wt% GMs/CPCs, and proliferation of the cells was also enhanced. An in vivo study showed that new bone tissue was able to grow into the pores that resulted from GM degradation. This study suggests that the new composite could be a promising candidate for use as a bone substitute under non-compression-loaded circumstances.

Topics: Animals; Biocompatible Materials; Bone Cements; Bone Substitutes; Calcium Phosphates; Cells, Cultured; Gelatin; Iridoid Glycosides; Iridoids; Microscopy, Electron, Scanning; Microspheres; Rats; Rats, Sprague-Dawley

To compare the characteristics of gelatin microspheres crosslinked by glutaraldehyde (GA) or genipin (GP).. Gelatin microspheres, prepared by the improved emulsified cold-condensation method, were crosslinked by GP and GA, respectively. After being dispersed in PBS, two kinds of microspheres with 60% degree of cross linking were compared in terms of morphology, swelling and degrading properties. rhBMP-2 were loaded into the GP and GA microspheres, and the encapsulation rate, drug loading and releasing capacity were measured; 100%, 50% and 25% leaching liquid of GP and GA microspheres were respectively cultured with rat osteoblast (DMEM group as the control), and cell proliferation was measured by MTT method to grade the cell cytotoxicity.. GP and GA microspheres were both spherical with the diameters of (78 +/- 18) microm and (65 +/- 10) microm, and there were no difference between both microspheres in drug loading and encapsulation rate. But, GP microspheres, with long degrading period (28 days) compared to GA microspheres (21 days), had better dispersibility, and swelling rate (89.0% +/- 4.8%), the percentage of cumulative drug releasing at 10 days (78.80% +/- 4.96%) were both lower than GA microsphere (118.0% +/- 7.6%, 90.50% +/- 5.12%). The percentages of drug loading of GP and GA were (921 +/- 73) and (965 +/- 62) ng/g, and the encapsulation rates were 88.5% +/- 2.1% and 89.7% +/- 1.8%; showing no significant difference (P > 0.05). The cell cytotoxicity of 100%, 50% and 25% leaching liquid of GP microspheres was all at the level I, but leaching liquid of GA microspheres with corresponding concentration were at the levels of III, III and II.. GP crosslinked gelatin microspheres are superior to GA crosslinked gelatin microspheres and can be widely used in tissue engineering field.

Topics: Animals; Bone Substitutes; Cells, Cultured; Drug Delivery Systems; Gelatin; Glutaral; Iridoid Glycosides; Iridoids; Microspheres; Rats; Tissue Engineering

Owing to of the limited repair capacity of articular cartilage, it is essential to develop tissue-engineered cartilage for patients suffering from joint disease. Chondroitin sulfate (CS) and hyaluronan (HA) are the components of the cartilage extracellular matrix (ECM) and are known to influence the proliferation and differentiation of chondrocytes. Scaffolds composed of type-II collagen, CS, and HA may create an environment that can preserve the normal phenotype of cells to promote regeneration of cartilage-like constructs. In this investigation, we prepared and characterized 3-dimensional type-II collagen scaffolds both with and without HA and CS. Porous composite scaffolds fabricated by freeze-drying showed interconnected pores with mean diameters of 140+/-30 microm and porosities of 92-95% after cross-linking with genipin. After a 14-day in vitro culture, morphologically round chondrocytes were found to be uniformly distributed throughout the sponges. Expression of genes of aggrecan, type-II collagen and cartilage oligomeric matrix protein (COMP) was statistically and significantly increased on scaffolds with CS and HA than those without CS and HA. Furthermore, there was a markedly greater accumulation of proteoglycans (PGs) on the scaffolds with CS and HA.

Topics: Cartilage, Articular; Collagen Type II; Humans; Hyaluronic Acid; Iridoid Glycosides; Iridoids; Microscopy, Electron, Scanning; Tissue Engineering

Establishment of a comprehensive platform for the assessment of host-biomaterial interaction in vivo is an important issue. Nuclear factor-kappaB (NF-kappaB) is an inducible transcription factor that is activated by numerous stimuli. Therefore, NF-kappaB-dependent luminescent signal in transgenic mice carrying the luciferase genes was used as the guide to monitor the biomaterials-affected organs, and transcriptomic analysis was further applied to evaluate the complex host responses in affected organs in this study. In vivo imaging showed that genipin-cross-linked gelatin conduit (GGC) implantation evoked the strong NF-kappaB activity at 6h in the implanted region, and transcriptomic analysis showed that the expressions of interleukin-6 (IL-6), IL-24, and IL-1 family were up-regulated. A strong luminescent signal was observed in spleen on 14 d, suggesting that GGC implantation might elicit the biological events in spleen. Transcriptomic analysis of spleen showed that 13 Kyoto Encyclopedia of Genes and Genomes pathways belonging to cell cycles, immune responses, and metabolism were significantly altered by GGC implants. Connectivity Map analysis suggested that the gene signatures of GGC were similar to those of compounds that affect lipid or glucose metabolism. GeneSetTest analysis further showed that host responses to GGC implants might be related to diseases states, especially the metabolic and cardiovascular diseases. In conclusion, our data provided a concept of molecular imaging-guided transcriptomic platform for the evaluation and the prediction of host-biomaterial interaction in vivo.

Topics: Adhesives; Animals; Biocompatible Materials; Cross-Linking Reagents; Gelatin; Gene Expression Regulation; Genes, Reporter; Immunohistochemistry; Iridoid Glycosides; Iridoids; Luciferases; Luminescence; Luminescent Measurements; Materials Testing; Mice; Mice, Transgenic; NF-kappa B; Random Allocation; Transcription, Genetic

This paper demonstrates a proof-of-concept approach for producing genipin-gelatin microcapsules of precisely controlled and monodisperse size distributions by the microfluidic channels. We have demonstrated that one could control the size of emulsions from 130 microm to 580 microm in diameter (with a variation of less than 5%) by altering the relative sheath/sample flow rate ratio. In addition, Results show that the encapsulation and in vitro release of a model drug, 5-fluorouracil, to enhance the effect of controlled release. We demonstrated that the appropriate particle size for different release patterns is predictable, enabling better application of genipin-gelatin microcapsules as a drug carrier. The proposed microfluidic chip is capable of generating relatively uniform micro-droplets with well controllable diameter, and it has the added advantages of being a simple, low cost, and high throughput process.

Topics: Capsules; Drug Carriers; Drug Compounding; Emulsions; Fluorouracil; Gelatin; Iridoid Glycosides; Iridoids; Microfluidic Analytical Techniques; Microfluidics; Particle Size

Uncoupling protein 2 (UCP2) was reported to be involved in insulin-glucose homeostasis, based on well established event that inhibition of UCP2 stimulates insulin secretion in pancreatic beta-cells. However, the role of UCP2 on insulin-stimulated glucose uptake in adipose tissue, which is an indispensable process in insulin-glucose homeostasis, remains unknown. In this study, UCP2 was inhibited by genipin in 3T3-L1 adipocytes, which increased mitochondrial membrane potential, intracellular ATP level and production of reactive oxygen species (ROS). Importantly, insulin-stimulated glucose uptake in 3T3-L1 adipocytes was largely impaired in the presence of genipin, and recovered by CCCP, a mitochondrial uncoupler. Furthermore, genipin leaded to suppression of insulin signal transduction through hyperactivation of c-Jun N-terminal kinase (JNK) and subsequent serine phosphorylation of insulin receptor substrate-1 (IRS-1). These results suggest that mitochondrial uncoupling in adipocytes positively regulates insulin-stimulated glucose uptake in adipocytes, and UCP2 may play an important role in insulin resistance.

Topics: 3T3-L1 Cells; Adenosine Triphosphate; Adipocytes; Animals; Anthracenes; Biological Transport, Active; Glucose; Insulin; Insulin Resistance; Ion Channels; Iridoid Glycosides; Iridoids; JNK Mitogen-Activated Protein Kinases; Membrane Potential, Mitochondrial; Mice; Mitochondrial Proteins; Models, Biological; Protein Kinase Inhibitors; Reactive Oxygen Species; Signal Transduction; Uncoupling Protein 2

Our objective was to examine the potential of a genipin cross-linked human fibrin hydrogel system as a scaffold for articular cartilage tissue engineering. Human articular chondrocytes were incorporated into modified human fibrin gels and evaluated for mechanical properties, cell viability, gene expression, extracellular matrix production and subcutaneous biodegradation. Genipin, a naturally occurring compound used in the treatment of inflammation, was used as a cross-linker. Genipin cross-linking did not significantly affect cell viability, but significantly increased the dynamic compression and shear moduli of the hydrogel. The ratio of the change in collagen II versus collagen I expression increased more than 8-fold over 5 weeks as detected with real-time RT-PCR. Accumulation of collagen II and aggrecan in hydrogel extracellular matrix was observed after 5 weeks in cell culture. Overall, our results indicate that genipin appeared to inhibit the inflammatory reaction observed 3 weeks after subcutaneous implantation of the fibrin into rats. Therefore, genipin cross-linked fibrin hydrogels can be used as cell-compatible tissue engineering scaffolds for articular cartilage regeneration, for utility in autologous treatments that eliminate the risk of tissue rejection and viral infection.

Topics: Absorbable Implants; Animals; Cartilage, Articular; Cell Survival; Chondrocytes; Collagen; Compressive Strength; Cross-Linking Reagents; Fibrin; Humans; Hydrogels; Iridoid Glycosides; Iridoids; Rats; Rats, Sprague-Dawley; Regeneration; Shear Strength; Tissue Engineering

Exocytosis of endothelial Weibel-Palade bodies, which contain von Willebrand factor (VWF), P-selectin and other modulators, plays an important role in both inflammation and thrombosis. The present study investigates whether genipin, an aglycon of geniposide, inhibits endothelial exocytosis.. Human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords and cultured. The concentration of VWF in cell supernatants was measured using an ELISA Kit. P-selectin translocation on the cell surface was analyzed by cell surface ELISA. Cell viability was measured using a Cell Counting Kit-8. Mouse bleeding times were measured by amputating the tail tip. Western blot analysis was used to determine the amount of endothelial nitric oxide synthase (eNOS) and phospho-eNOS present. Nitric oxide (NO) was measured in the cell supernatants as nitrite using an NO Colorimetric Assay.. Genipin inhibited thrombin-induced VWF release and P-selectin translocation in HUVECs in a dose- and time-dependent manner. The drug had no cytotoxic effect on the cells at the same doses that were able to inhibit exocytosis. The functional study that demonstrated that genipin inhibited exocytosis in vivo also showed that genipin prolonged the mouse bleeding time. Furthermore, genipin activated eNOS phosphorylation, promoted enzyme activation and increased NO production. L-NAME, an inhibitor of NOS, reversed the inhibitory effects of genipin on endothelial exocytosis.. Genipin inhibits endothelial exocytosis in HUVECs. The mechanism by which this compound inhibits exocytosis may be related to its ability to stimulate eNOS activation and NO production. Our findings suggest a novel anti-inflammatory mechanism for genipin. This compound may represent a new treatment for inflammation and/or thrombosis in which excess endothelial exocytosis plays a pathophysiological role.

Topics: Animals; Bleeding Time; Cell Culture Techniques; Cell Survival; Endothelial Cells; Exocytosis; Female; Humans; Iridoid Glycosides; Iridoids; Mice; Nitric Oxide; P-Selectin; Pregnancy; Protein Transport; Umbilical Veins; von Willebrand Factor

In this study we investigated the effects of Gardenia jasminoides Ellis (GJE) extract and its constituents, such as ursolic acid and genipin, on gastritis in rats and the growth of human gastric cancer cells. The GJE extract, ursolic acid and genipin showed the acid-neutralizing capacities, the antioxidant activities, and the inhibitory effects on the growth of Helicobacter pylori (H. pylori), which are almost equivalent to positive control compounds. In addition, the GJE extract and ursolic acid had cytotoxic activity against AGS and SUN638 gastric cancer cells. The genipin and ursolic acid inhibited significant HCl/ethanol-induced gastric lesions. Taken together, GJE extract and its constituents might have antigastritic activities, associated with the antioxidant activities, acid-neutralizing capacities, and anti-H. pylori action. Also, we could suggest that genipin and ursolic acid may be useful for the treatment and/or protection of gastritis.

Topics: Animals; Anti-Ulcer Agents; Antineoplastic Agents, Phytogenic; Antioxidants; Cell Line, Tumor; Ethanol; Free Radical Scavengers; Gardenia; Gastritis; Helicobacter Infections; Helicobacter pylori; Indicators and Reagents; Iridoid Glycosides; Iridoids; Lipid Peroxidation; Male; Plant Extracts; Rats; Rats, Sprague-Dawley; Solvents; Stomach Neoplasms; Stomach Ulcer; Triterpenes; Ursolic Acid

Recent advances in hydrogel technology have focused on finding more biocompatible, non-toxic materials intended for pharmaceutical and biomedical applications. In this study, naturally occurring genipin was used for the first time to crosslink casein protein in aqueous system for the formation of novel hydrogel materials. For aqueous 8.0 wt% casein solution, its gelation in the presence of genipin was investigated by time sweep rheometric measurements. With the increase of genipin amount from 2.5 to 10.0 mmol/L, the gelation time decreased from 119.8. to 18.5 min when the reaction temperature was kept to be 35 degrees C. With the increase of the reaction temperature from 35 to 50 degrees C, the gelation time decreased from 44.7 to 27.6 min when genipin concentration was kept to be 5.0 mmol/L. The apparent activation energy was determined to be 28.6 kJ/mol according to the Arrhenius equation. Moreover, the mechanical strength of the crosslinked casein hydrogel could be tuned by the amount of genipin. (13)C NMR analyses confirmed the crosslinking reaction between casein and genipin. For the resultant casein hydrogels, their swelling characteristics and in vitro release profiles of bovine serum albumin (BSA) were studied in simulated gastrointestinal tract conditions (pH 1.2 and pH 7.4). At pH 1.2, the swelling ratio of the hydrogel and the release amount of the entrapped BSA were relatively low. However, high amounts of the swelling and BSA release could be observed at pH 7.4. The release behavior could be related to various crosslinking and swelling degrees of the hydrogel networks formed by various amounts of genipin. It is suggested that the genipin-crosslinked casein hydrogel might be a suitable polymeric carrier for protein drug delivery in the intestine.

Topics: Algorithms; Animals; Biological Availability; Caseins; Cattle; Cross-Linking Reagents; Delayed-Action Preparations; Elasticity; Hydrogels; Hydrogen-Ion Concentration; Iridoid Glycosides; Iridoids; Kinetics; Magnetic Resonance Spectroscopy; Mechanical Phenomena; Models, Chemical; Serum Albumin, Bovine; Temperature; Viscosity; Water

Genipin is an iridoid compound and an aglucon of geniposide isolated from Gardenia fructus. We have previously reported that genipin induces neurite outgrowth in PC12h and Neuro2a cells and protects against cytotoxicity induced by several conditions such as beta-amyloid peptide, serum deprivation, and oxidative stress in rat primary hippocampal neurons and Neuro2a cells. In this paper, we examined the protective effect of genipin on A23187 (a calcium ionophore)-induced cytotoxicity in Neuro2a cells. A23187 induced cytotoxicity in concentration- and time-dependent manners as assayed by measurements of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetazolium bromide (MTT) reduction activity and lactate dehydrogenase (LDH) release. The cytotoxicity was significantly suppressed by genipin in a concentration-dependent manner. A23187 also significantly activated caspase3/7, which is known to be the critical mediator of apoptosis, after 1 h, and the cytotoxicity was clearly blocked by an inhibitor of caspase 3/7. Furthermore, A23187 induced the expression of immunoglobulin-binding protein/glucose-regulated protein of 78 kDa (BiP/GRP78) protein, which is an endoplasmic reticulum (ER) stress marker protein, and the expression was suppressed by genipin. These results suggest that genipin protects Neuro2a cells from A23187-induced cytotoxicity mediated by caspase 3/7 and ER stress. Therefore, genipin may be effective in preventing neurodegeneration observed in Alzheimer's disease and Parkinson's disease involving ER stress.

Topics: Animals; Blotting, Western; Calcimycin; Caspase 3; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Ionophores; Iridoid Glycosides; Iridoids; Mice; Neurons; Neuroprotective Agents; Oxidative Stress; Time Factors

Type I collagen is a major component of the hybrid layer, and improvement of its mechanical properties may be advantageous during bonding procedures.. To investigate the effect of three different cross-linking agents (Glutaraldehyde [GD], Grape seed extract [GSE], and Genipin [GE]) on the tensile bond strength (TBS) of resin-dentin bonds.. Sixty-four sound human molars were collected and their occlusal surfaces were ground flat to expose dentin. Dentin surfaces were etched using a phosphoric acid and then teeth were randomly divided according to the dentin treatment: Control group (no treatment), 5% GD, 6.5% GSE, or 0.5% GE. Teeth were restored either with One Step Plus or Adper Single Bond Plus adhesive systems and resin composite. After 24 h, teeth were sectioned to produce a cross-sectional surface area of 1.0 mm(2) and tested for tensile bond strength. Data were statistically analyzed using ANOVA and Fisher's PLSD tests (p < 0.05). There was a statistically significant interaction between factors (treatment and adhesive p < 0.001). Treatment affected TBS (p < 0.0001), while no differences were observed between the adhesive systems (p = 0.6961).. Chemical modification to the dentin matrix promoted by GD and GSE, but not GE, resulted in increased bond strength. The application of selective collagen cross-linkers during adhesive restorative procedures may be a new approach to improve dentin bond strength properties.

Topics: Antioxidants; Collagen Type I; Cross-Linking Reagents; Dental Bonding; Dental Etching; Dentin; Dentin-Bonding Agents; Glutaral; Grape Seed Extract; Humans; Iridoid Glycosides; Iridoids; Materials Testing; Random Allocation; Surface Properties

Increased norepinephrine production by acute urine retention (AUR) induced sympathetic activation may contribute to acute liver injury (ALI) via the action of hepatic vasoconstriction and increased reactive oxygen species (ROS) production. We evaluated whether In-Chern-Hau-Tang, a hepatoprotective herb medicine, and its major ingredient genipin, may ameliorate norepinephrine-induced liver injury in the rat. We determined the effects of In-Chern-Hau-Tang and genipin on norepinephrine-induced oxidative stress in the Kupffer and endothelial cells and AUR-induced ALI in the rat via a chemiluminescence analyzer, physiologic and biochemical determination and western blot. The results of in vitro study showed that genipin with efficient H(2)O(2) and HOCl scavenging activities decreased norepinephrine-enhanced ROS production in the Kupffer cell and endothelial cell cultures. AUR activated hepatic sympathetic nervous activity lead to a hepatic hypoxia/hypoperfusion, and a reduction in bile flow. AUR increased intercellular adhesion molecular 1 (ICAM-1) protein expression, and hepatic ROS production from the activated leukocyte NADPH oxidase activity subsequently leading to plasma aspartate aminotransferase (AST) elevation. Hepatic sympathetic denervation, or oral pretreatment of In-Chern-Hau-Tang or genipin for 1 week ameliorated the level in AUR-induced hepatic hypoxia/hypoperfusion, and bile stasis. Hepatic denervation, In-Chern-Hau-Yang and genipin inhibited AUR-enhanced hepatic ICAM-1 expression, hepatic ROS production, leukocyte NADPH oxidase activity and plasma AST activity. In conclusion, In-Chern-Hau-Tang along with its active component, genipin, can ameliorate AUR-induced ALI via the alleviation of oxidative stress possibly by the inhibition of sympathetic induced hypoxia/hypoperfusion and leukocyte NADPH oxidase activity.

Topics: Animals; Drugs, Chinese Herbal; Free Radical Scavengers; Iridoid Glycosides; Iridoids; Liver; Luminescence; Oxidative Stress; Rats; Reactive Oxygen Species; Sympathetic Nervous System; Urinary Bladder

Use of polyelectrolyte multi-layers as biomaterials for cell attachment has been limited due to their gel-like characteristics. Herein, we attempt to improve the cellular adhesion properties of multi-layer films, reduce their gel-like nature and rigidify them through chemical cross-linking with genipin; a natural and non-cytotoxic compound. Chitosan (CH), hyaluronan (HA) and alginate (Alg) were used to assemble [CH-HA]n CH and [CH-Alg]n CH films, and the effects of genipin cross-linking on the cell adhesion properties of these multi-layers were investigated. Atomic force microscopy (AFM) confirmed that cross-linking affected each of the films differently. Quartz crystal microbalance with dissipation (QCM-D) revealed that [CH-HA]10 CH films were very viscoelastic, with thicknesses in the range 350-450 nm, while [CH-Alg]10 CH films only grew to thicknesses of approximately 100 nm. These differences were a result of the different growth regimes of these two polyelectrolyte systems. Cell adhesion studies using MC3T3 pre-osteoblasts and rat fibroblastic skin cells, carried out on both films demonstrated vast differences in cell adhesion. [CH-HA]n CH cross-linked films proved to be highly non-adhesive for pre-osteoblasts and fibroblastic skin cells. Conversely, cross-linking [CH-Alg]n CH films was shown to dramatically improve pre-osteoblast and rat fibroblastic skin cell adhesion, especially for high bi-layer numbers and using higher concentrations of cross-linker.

Topics: Animals; Cell Adhesion; Cross-Linking Reagents; Electrolytes; Fibroblasts; Iridoid Glycosides; Iridoids; Materials Testing; Mice; Osteoblasts; Polymers; Rats; Water

Accumulation of unfolding or misfolded proteins within the lumen of the endoplasmic reticulum (ER) triggers ER stress, and sustained ER stress ultimately leads to cell death. Both of these events are involved in the activation of glucose-regulated protein of 78 kDa (GRP78, also known as Bip), CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP, also known as growth arrest and DNA damage-inducible gene 153 (GADD153)), and caspase-12. ER stress has been shown to be involved in neurodegenerative disorders, such as Alzheimer, Parkinson, and polyglutamine diseases. We previously showed that genipin, a natural iridoid compound, has a protective effect against amyloid-beta (Abeta)-induced cytotoxicity. Here, we studied the protective effects of genipin on cytotoxicity induced in Neuro2a cells by the specific ER stress inducer tunicamycin (TM). TM treatment significantly reduced cell viability in a dose-dependent manner. Genipin dramatically rescued the cells against TM-induced cell death. In addition, genipin suppressed ER stress-induced upregulation of CHOP and GRP78. These data suggest that genipin is effective at protecting against neurodegenerative disorders.

Topics: Animals; Blotting, Western; Caspase 12; CCAAT-Enhancer-Binding Proteins; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Heat-Shock Proteins; Iridoid Glycosides; Iridoids; Mice; Neuroprotective Agents; Protein Folding; Reverse Transcriptase Polymerase Chain Reaction; Transcription Factor CHOP; Tunicamycin

A limitation with the use of polymers as stent matrices is their inherent mechanical weakness. In this study, a polymeric stent, made from chitosan-based films fixed by genipin which has a cyclic molecular structure, was developed (the genipin stent). The mechanical properties of the genipin stent were investigated; its counterpart fixed by a linear epoxy compound (the epoxy stent) and a commercially available metallic stent were used as controls. The results indicated that the cyclic crosslinking structures formed within the genipin stent matrix were beneficiary to the improvement of its mechanical property. Additionally, the tolerable compression load of the genipin stent was superior to that of the control metallic stent. The cytotoxicity of the genipin stent was significantly lower than the epoxy stent. The deployment of the genipin stent in rabbit infrarenal abdominal aortas was performed using a French sheath. At 3 months postoperatively, the retrieved arteries remained patent; no thrombosis was observed. A nearly intact layer of endothelial cells was seen on the stent-implanted vessel wall. To evaluate its possibility as a drug delivery vehicle, sirolimus (an anti-proliferative drug) was loaded in the genipin stent. It was found that the genipin stent with heparin coating exhibited a linear sustained-release profile and the released sirolimus still possessed its original activity in inhibiting smooth muscle cell proliferation. These findings suggest that the genipin stent with enhanced mechanical strength can be used as an attractive stent platform for local drug delivery.

Topics: Animals; Chitosan; Compressive Strength; Cross-Linking Reagents; Drug-Eluting Stents; Elastic Modulus; Equipment Design; Equipment Failure Analysis; Immunosuppressive Agents; Iridoid Glycosides; Iridoids; Polymers; Rabbits; Sirolimus

Human-like collagen (HLC) was cross-linked with hyaluronic acid by genipin in different ratio. The concentrations of hyaluronic acid in the mixture were 0, 0.01%, 0.05% and 0.1%. The blood vessel tubular grafts were then fabricated by freeze-drying. Microstructure, element composite, mechanical properties, cytotoxicity grade, and biocompatibility of different vascular scaffold groups were studied by scanning electron microscope (SEM), X-ray photoelectron spectroscopy (XPS), tensile test, burst pressure experiment, cytotoxicity experiment, endothelial cells planted in blood vessel scaffolds and hypodermic embedding of mice. The results showed that HLC-HA (0.05%) tubular scaffold exhibited interconnected well-distributed and porous structure and porosity of 94.38%; achieved the desirable mechanical property with stress of (1000.8 +/- 7.9) kPa and burst pressure of (1058.6 +/- 8.2) kPa, hypocytotoxicity, favourable cytocompatibility, hisocompatibility and disposition of degradation.

Topics: Adhesives; Animals; Biocompatible Materials; Biomimetic Materials; Blood Vessels; Collagen; Humans; Hyaluronic Acid; Iridoid Glycosides; Iridoids; Materials Testing; Mice; Tissue Engineering; Tissue Scaffolds

Here we have evaluated peripheral nerve regeneration with a porous biodegradable nerve conduit (PGGC), which was made from genipin-cross-linked gelatin. To examine the effect of pores, nonporous genipin-cross-linked gelatin conduit (GGC) was considered as the control. Both the PGGC and the GGC were dark blue in appearance with a concentric and round lumina. The PGGC featured an outer surface with pores of variable size homogeneously traversing, and a partially fenestrated inner surface connected by an open trabecular meshwork. The GGC had a rough outer surface whereas its inner lumen was smooth. Both PGGCs and GGCs had similar hydrophilicity on condition of the same material and cross-linking degree. The porosity of PGGCs and GGCs was 90.8 +/- 0.9% and 24.3 +/- 2.9%, respectively. The maximum tensile force of the GGCs (0.12 +/- 0.06 kN) exceeded that of the PGGCs (0.03 +/- 0.01 kN), but the PGGCs had a higher swelling ratio than GGCs at 0.5, 1, 3, 6, 12, 24, 48, 60, 72, and 84 h after soaking in deionized water. Cytotoxic testing revealed the soaking solutions of both of the tube composites would not produce cytotoxicity to cocultured Schwann cells. After subcutaneous implantation on the dorsal side of the rat, the PGGC was degraded completely after 12 weeks of implantation whereas a thin tissue capsule was formed encapsulating the partially degraded GGC. Biodegradability of both of the tube groups and their effectiveness as a guidance channel were examined as they were used to repair a 10 mm gap in the rat sciatic nerve. As a result, fragmentation of the GGC was still seen after 12 weeks of implantation, yet the PGGC had been completely degraded. Histological observation showed that numerous myelinated axons had crossed over the gap region in the PGGCs after 8 weeks of implantation despite only few myelinated axons and unmyelinated axons mostly surrounded by Schwann cells seen in the GGCs. In addition, the regenerated nerves in the PGGCs presented a significantly higher nerve conductive velocity than those in the GGCs (P < 0.05). Thus, the PGGCs can not only offer effective aids for regenerating nerves but also accelerate favorable nerve functional recovery compared with the GGCs.

Topics: Absorbable Implants; Animals; Biocompatible Materials; Cell Survival; Cross-Linking Reagents; Gelatin; Iridoid Glycosides; Iridoids; Nerve Regeneration; Peripheral Nerves; Permeability; Porosity; Rats; Rats, Sprague-Dawley; Schwann Cells

Gardeniae fructus (GF) has been used in traditional medicine for the treatment of inflammatory disease.. To evaluate the therapeutic effect of GF gel on the resolution of ecchymoses in rats.. Fifty hind limbs (in 25 Sprague-Dawley rats) were evaluated. The ecchymoses were produced by dropping a 100-g weight from a height of 20 cm on the posterior side of the hind limbs 25 times. Then, 0.5 g of hydrolyzed GF extracted gel was applied to the right hind limbs and 0.5 g of gel without GF extract powder was applied to the left hind limbs. The area of the ecchymosis was measured, and histological analysis was performed.. The area affected by the ecchymosis after 5 days was 15% in the control group and 2% in the GF gel group (p=.002). The mean duration for the ecchymosis was 5.8 days in the control group and 4.5 days in the GF gel group. The extravasated red blood cells and inflammation were less prominent in the GF gel group than in the control group.. The results of this study showed that hydrolyzed gel of GF extract, containing genipin, was effective for the treatment of ecchymoses in a rat model.

Topics: Animals; Disease Models, Animal; Ecchymosis; Gardenia; Hydrogels; Iridoid Glycosides; Iridoids; Phytotherapy; Plant Extracts; Plant Structures; Rats; Rats, Sprague-Dawley

Gardenia jasminoides E. (Rubiaceae) methanol extracts showed the highest level of antifungal activity against Pleurotus ostreatus, a wood-rotting fungus, compared to five other methanol plants extracts; [Thuja orientalis L. (Cupressaceae), Datura innoxia (Solanaceae), Ligustrum japonicum T. (Oleaceae), Juniperus chinensis var. procumbens (Cupressaceae) and Mallotus japonica M. (Euphorbiaceae)] and selected for further analysis. Two antifungal compounds were isolated from n-butanol and ethyl acetate solubles in the methanol extracts of Gardenia jasminoides leaves and stems by bioassay-guided fractionation, using Pleurotus ostreatus. The antifungal compounds found for the first time in Gardenia jasminoides against Pleurotus ostreatus were identified as genipin and geniposide based on instrumental analyses. Both also had potent inhibitory effects on two plant pathogenic fungi; Fusarium oxysporum and Corynespora cassiicola.

Topics: Antifungal Agents; Cholagogues and Choleretics; Fungi; Gardenia; Iridoid Glycosides; Iridoids; Microbial Sensitivity Tests; Molecular Structure; Plant Extracts

The aim of this study was to prepare the inclusion complex of genipin (GP) and beta-cyclodextrin (beta-CD) with improved stability, solubility, and bioavailability and to study the pharmacokinetics of beta-CD inclusion complex in mice.. Lyophilization was employed in the preparation of the inclusion complex of GP-beta-CD, whose formation was confirmed by infrared, ultraviolet, differential scanning calorimetry, X-ray diffraction, and phase solubility method. Comparative studies on the in vitro solubility and stability and in vivo evaluation of GP in mice were investigated. Liquid-liquid extraction was used for the isolation of GP in the assay of its concentration. After injection in the caudal vein at equal doses of the inclusion complex of free GP, the drug concentration in mice plasma at fixed time after administration was determined by high-performance liquid chromatography method.. The results demonstrated that GP-beta-CD solid powders showed improved stability and solubility in aqueous solution, when comparing with free GP. The results of the in vivo study showed that the inclusion complex of GP-beta-CD exhibited the dissimilar pharmacokinetics from that of free GP after intravenous administration. The inclusion complex of GP-beta-CD displayed longer MRT(0-infinity) and higher AUC(0-infinity) than free GP did.. The relative bioavailability of the inclusion complex of GP-beta-CD to free GP was 305.3%, which demonstrated that GP formulations containing beta-CD significantly increased the bioavailability.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; beta-Cyclodextrins; Biological Availability; Calorimetry, Differential Scanning; Drug Carriers; Drug Stability; Freeze Drying; Half-Life; Iridoid Glycosides; Iridoids; Male; Mice; Powder Diffraction; Random Allocation; Solubility; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared

To estimate the role of uncoupling proteins in aging rat heart recovery from prolonged ischemia we used genipin application during Langendorfpreparation. It was shown that genipin in dose-depended manner depressed coronary flow, heart rate and cardiac diastolic function. Such effect was similar to that observed during myocardial Ca2+ overload by gradually elevated CaCl2, in perfusion solution. Moreover, postischemic disturbances of cardiodynamic parameters, oxygen cost of myocardial work were much increased in genipin pretreated hearts that in control ones. Thus, genipin inhibition of UCP2 activity has cardiodepressive effects that imply UCPs in cardiac calcium regulation.

Topics: Aging; Animals; Calcium Chloride; Dose-Response Relationship, Drug; Heart Function Tests; In Vitro Techniques; Ion Channels; Iridoid Glycosides; Iridoids; Male; Mitochondrial Proteins; Myocardial Contraction; Myocardial Reperfusion; Myocardial Reperfusion Injury; Myocardium; Oxygen Consumption; Rats; Rats, Wistar; Uncoupling Protein 2

Genipin is aglycone of geniposide, one of the active compounds of Gardenia gasminoides Ellis. The gardenia fruit extract has been used in traditional Chinese medicine to relieve the symptoms of type 2 diabetes that is accompanied with extensive oxidative stress and endothelial dysfunction of NO production. Besides, genipin was shown to inhibit UCP-depended proton leak through the inner mitochondrial membrane that leads to increased membrane potential and ATP production. We studied the effects of genipin at ischemia/reperfusion-induced oxidative stress and activity of NOS isozymes using Langendorfperfused old rat heart model. Ischemia/reperfusion is well-known oxidative agent, and showed significant increasing of superoxide radical, hydrogen peroxide and hydroxyl radical. Genipin application in doze 10-5 mol/L for 15 min before prolonged ischemia exerted powerful antiradical and antilipoperoxidative effects. Heart ischemia/reperfusion was supported with peroxynitrite generation and nitrozative stress. We demonstrated the inhibitory property of genipin on INOS expression that possibly occurs via protein kinase A inhibition and stabilization of I-kappaB-NF-kappaB complex. Genipin stimulated cNOS activity seemingly activating PI3K/Akt signaling pathway. Although, post-ischemic recovery ofcardiodynamic parameters of old rat hearts were depressed due to "switching off" the NO production by inducible NOS which is important in early period of reperfusion. Thus, we conclude that genipin is powerfull antioxidant and posses insulin-like activity due to its property of managing the NO production at intracellular signal transduction cascade level.

Topics: Aging; Animals; In Vitro Techniques; Ion Channels; Iridoid Glycosides; Iridoids; Male; Mitochondrial Proteins; Myocardial Reperfusion Injury; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; Perfusion; Rats; Rats, Wistar; Reactive Oxygen Species; Uncoupling Protein 1

In this work, hybrid chitosan/hydroxyapatite composites material were developed and characterized. The polymer matrix was first dissolved in polylactic acid, and then hydroxyapatite (HA) was added as filler material. The effects of the added amounts of a crosslinking agent (genipin) and of the concentrations of lactic acid, and of the presence of HA powder on the evolution of rheological properties were evaluated. A significant decrease of gelation time with increasing amounts of crosslinking agent was observed, the effect being even more pronounced in the presence of HA. The chitosan matrix and the composites with a chitosan/HA weight ratio of 2/5 were characterized using microstructural analysis and in vitro tests. The formation of large pore sizes in the chitosan-based scaffolds was favored by low concentrations of lactic acid and genipin. The in vitro tests in synthetic body fluid revealed an extensive formation of an apatitic layer onto the surface of the chitosan/HA composite scaffolds crosslinked with genipin.

Topics: Biocompatible Materials; Body Fluids; Chitosan; Cross-Linking Reagents; Durapatite; Elasticity; Iridoid Glycosides; Iridoids; Lactic Acid; Materials Testing; Microscopy, Electron, Scanning; Polyesters; Polymers; Porosity; Rheology; Surface Properties; Tissue Scaffolds; Viscosity

To evaluate the effects of dentin collagen modifications induced by various cross-linkers on the stability of collagen matrix and the inhibition of root caries.. The following cross-linkers were tested: 5% glutaraldehyde (GA), 0.5% proanthocyanidin (PA), 0.625% genipin (GE). In the first experiment, cross-linker-treated demineralized human root dentin was digested with bacterial collagenase, centrifuged, and the supernatants were subjected to amino acid analysis to determine collagen content. The residues were analyzed by SDS-PAGE and hydroxyproline analysis. In the second experiment, bovine root surfaces were conditioned with phosphoric acid, treated with the cross-linkers, incubated with Streptococcus mutans and Lactobacillus acidophilus for 1 week and the root caries inhibition was evaluated with confocal microscopy. Lastly, the ability of the bacteria to colonize the root surface was evaluated. In this experiment slabs of bovine root were treated with the cross-linkers and incubated in a suspension of S. mutans and L. acidophilus. The slabs were washed, resuspended in water, glucose was added, and the pH measured.. While all collagen was digested with collagenase in the control groups, only a small proportion was solubilized in the GA-, PA-, and GE-treated groups. The root caries was significantly inhibited by treatment with PA or GA. Drops in pH in the cross-linker-treated groups were essentially the same as in the untreated group.. Naturally occurring cross-linkers, especially PA, could be used to modify root dentin collagen to efficiently stabilize collagen and to increase its resistance against caries.

Topics: Animals; Bacterial Adhesion; Cattle; Collagen; Colony Count, Microbial; Cross-Linking Reagents; Dentin; Glutaral; Humans; Iridoid Glycosides; Iridoids; Lactobacillus acidophilus; Proanthocyanidins; Root Caries; Streptococcus mutans; Tooth Root

Geniposide, an iridoid glucoside, is a major constituent in the fruits of Gardenia jasminoides (Gardenia fruits), a popular Chinese herb. Genipin, the aglycone of geniposide, is used to prepare blue colorants in food industry and also a crosslinking reagent for biological tissue fixation. In this study, we investigated the metabolism and pharmacokinetics of genipin and geniposide in rats. Blood samples were withdrawn via cardiopuncture and the plasma samples were assayed by HPLC method before and after hydrolysis with sulfatase and beta-glucuronidase. The results indicated that after oral administration of genipin or Gardenia fruit decoction, genipin sulfate was a major metabolite in the bloodstream, whereas the parent forms of genipin and geniposide were not detected. Importantly, oral administration of 200mg/kg of genipin resulted in a mortality of 78% (7/9) in rats.

Topics: Animals; Calibration; Chemical Phenomena; Chemistry, Physical; Cholagogues and Choleretics; Chromatography, High Pressure Liquid; Data Interpretation, Statistical; Gardenia; Hydrolysis; Injections, Intravenous; Iridoid Glycosides; Iridoids; Male; Plant Extracts; Rats; Rats, Sprague-Dawley; Reproducibility of Results

This study investigates the feasibility of a novel nanocomposite (GC/Ag) of a genipin-crosslinked chitosan (GC) film in which was embedded various amounts of Ag nanoparticles for wound-dressing applications. In situ UV-vis results revealed that adding chitosan solution did not affect the characteristics of Ag nanoparticles. The water uptake ratios and surface hydrophilicity of the GC/Ag nanocomposite were better and the degradation rates slightly lower than those of the pure GC film. The presence of Ag nanoparticles enhanced L929 cell attachment and growth. Its function as an anti-microbial agent in a GC/Ag nanocomposite was assessed for Ag contents of over 100 ppm. In conclusion, silver ions had dual functions--structural reinforcement and provision of antimicrobial properties to a biocompatible polymer.

Topics: Animals; Anti-Infective Agents; Cell Adhesion; Cell Count; Cell Death; Cell Line; Cell Proliferation; Chitosan; Cross-Linking Reagents; Escherichia coli; Fibroblasts; Iridoid Glycosides; Iridoids; Metal Nanoparticles; Mice; Microbial Sensitivity Tests; Muramidase; Nanocomposites; Silver; Spectrophotometry, Ultraviolet; Surface Properties; Water

Controlled release of a marker drug, 4,4'-bis(2-sulfostyryl) biphenyl (DSBP) from genipin crosslinked gelatin thin films, with application to drug delivery by transdermal patches is studied in this paper. A simple method for fabrication of nano-thin films, using basic lab equipment is introduced. This method consists of two steps: dipping of the substrate in a deposition solution, followed by centrifugation of the substrate. Also, swelling and drug release from thin films is modeled, using the Fick's second law of diffusion. The effect of genipin concentration on release of DSBP molecules from thin films is investigated, experimentally and numerically. The results show that controlled release of DSBP from the genipin-crosslinked gelatin thin films is achieved, using various concentrations of genipin in gelatin.

Topics: Absorption; Biphenyl Compounds; Delayed-Action Preparations; Diffusion; Drug Delivery Systems; Gelatin; Iridoid Glycosides; Iridoids; Kinetics; Microscopy, Atomic Force; Stilbenes; Surface Properties; Water

The positive interaction of materials with tissues is an important step in regenerative medicine strategies. Hydrogels that are obtained from polysaccharides and proteins are expected to mimic the natural cartilage environment and thus provide an optimum milleu for tissue growth and regeneration. In this work, novel hydrogels composed of blends of chitosan and Bombyx mori silk fibroin were cross-linked with genipin (G) and were freeze dried to obtain chitosan/silk (CSG) sponges. CSG sponges possess stable and ordered structures because of protein conformational changes from alpha-helix/random-coil to beta-sheet structure, distinct surface morphologies, and pH/swelling dependence at pH 3, 7.4, and 9. We investigated the cytotoxicity of CSG sponge extracts by using L929 fibroblast-like cells. Furthermore, we cultured ATDC5 cells onto the sponges to evaluate the CSG sponges' potential in cartilage repair strategies. These novel sponges promoted adhesion, proliferation, and matrix production of chondrocyte-like cells. Sponges' intrinsic properties and biological results suggest that CSG sponges may be potential candidates for cartilage tissue engineering (TE) strategies.

Topics: Animals; Biocompatible Materials; Bombyx; Cartilage; Chitosan; Hydrogel, Polyethylene Glycol Dimethacrylate; Hydrogels; Hydrogen-Ion Concentration; Iridoid Glycosides; Iridoids; Polysaccharides; Protein Conformation; Protein Structure, Secondary; Silk; Temperature; Tissue Engineering

The purpose of this study was to enhance the mechanical properties and slow the degradation of an electrospun fibrinogen scaffold, while maintaining the scaffold's high level of bioactivity. Three different cross-linkers were used to achieve this goal: glutaraldehyde vapour, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in ethanol and genipin in ethanol. Scaffolds with a fibrinogen concentration of 120 mg ml(-1) were electrospun and cross-linked with one of the aforementioned cross-linkers. Mechanical properties were determined through uniaxial tensile testing performed on scaffolds incubated under standard culture conditions for 1 day, 7 days and 14 days. Cross-linked scaffolds were seeded with human foreskin fibroblasts (BJ-GFP-hTERT) and cultured for 7, 14 and 21 days, with histology and scanning electron microscopy performed upon completion of the time course. Mechanical testing revealed significantly increased peak stress and modulus values for the EDC and genipin cross-linked scaffolds, with significantly slowed degradation. However, cross-linking with EDC and genipin was shown to have some negative effect on the bioactivity of the scaffolds as cell migration throughout the thickness of the scaffold was slowed.

Topics: Animals; Biocompatible Materials; Carbodiimides; Cattle; Cell Movement; Cross-Linking Reagents; Electrochemistry; Fibrinogen; Fibroblasts; Glutaral; Humans; Iridoid Glycosides; Iridoids; Microscopy, Electron, Scanning; Tensile Strength; Tissue Engineering

Chitosan hydrogel microspheres were obtained by cross-linking chitosan in its inverse emulsion using genipin as cross-linker. The genipin-cross-linked chitosan microspheres (ChGp) swell significantly in water at pH values below 6.5 and shrink to a smaller extent at pH values above 6.5. ChGp microspheres bind heparin in water. The kinetics of heparin binding was found to be pH dependent and was faster and more efficient at a lower pH. That can be also controlled by the weight of ChGp microspheres used. Rate and efficiency of heparin adsorption at pH 7.4, which is typical of blood, could be increased by quaternization of ChGp microspheres using glycidyltrimethylammonium chloride (GTMAC). The polymeric material obtained thus can be potentially useful for heparin removal in biomedical applications.

Topics: Adsorption; Chitosan; Cross-Linking Reagents; Heparin; Hydrogels; Hydrogen-Ion Concentration; Iridoid Glycosides; Iridoids; Kinetics; Microspheres

The conditions to determine geniposide and genipin using gradient liquid chromatography-tandem mass spectrometry (LC-MS/MS) via electrospray ionization were obtained using fractional factorial experimental design approaches, guided with Taguchi orthogonal arrays to enhance peak intensity. Geniposide, the major iridoid glycoside component of Gardenia herbs, which has been recognized to have choleretic effects, is transformed to genipin in animals. In this paper, the gradient establishment times, ionization source temperatures, and the concentrations of volatile additive ammonium acetate were investigated under the guidance of experimental designs to obtain LC-MS/MS signals of the highest peak intensity. Using geniposide and genipin standards, the methods are validated at the concentration ranges of 0.5-1000ng/mL and 10-5000ng/mL using ammonium adducts. The correlation coefficients of geniposide and genipin standard curves are greater than 0.999. Compared with the sensitivities of previously published LC-MS/MS methods, the methods developed in this work provide 6-fold sensitivity improvement. The lowest concentrations of geniposide and genipin, 0.19 and 2ng/mL, respectively, to generate detectable LC-MS/MS signal peaks are one order of magnitude lower than the repoered values in previous publications. The measurement accuracy and precision of geniposide are within 23% and 15%, respectively. The accuracy and precision of genipin are within 16% and 12.5%, respectively. When the validated calibration curves of geniposide and genipin are used to determine spiked control samples in rat blood dialysates, the geniposide determination errors are within 15% accuracy and within 5.8% precision, respectively, and the genipin determination errors are within 23% accuracy and within 3.6% precision, respectively.

Topics: Animals; Calibration; Chromatography, Liquid; Gardenia; Iridoid Glycosides; Iridoids; Rats; Reference Standards; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

One approach in tissue-engineering involves the implantation of decellularized, xenogenic scaffolds, with the expectation of repopulation in vivo. However, a major limitation of this method is the propensity to induce a strong immune host response. The study aim was to mitigate this immunogenicity by employing a crosslinking treatment with genipin.. Porcine matrices were prepared using a detergent-enzymatic treatment and fixed in 0.01% or 0.001% aqueous genipin. The mechanical properties of the matrices were monitored by tensile strength testing. The survival of chicken fibroblasts was used to determine cell-friendliness of the matrices. Non-fixed, decellularized biological scaffolds (n = 3) were implanted in a sheep model and compared to an equal number of genipin-fixed scaffolds (n = 6). Matrices implanted in the pulmonary position were explanted after six weeks and examined using light and transmission electron microscopy. The antibody reaction against porcine tissue in sheep serum was also determined.. Statistically significant differences were found between non-fixed leaflets, 0.001% genipin-and 0.6% glutaraldehyde (GA)-fixed leaflets for work to maximum load (non-fixed 0.00646 J; genipin-fixed 0.00509 J; GA-fixed 0.00543 J) and stiffness (non-fixed 9281 N/m; genipin-fixed 16214 N/m; GA-fixed 14401 N/m). Genipin-treated matrices were not cytotoxic. For all concentrations of genipin a high proportion of viable cells was present (79-100%). Low-dose GA (10 microg/ml) showed a distinct cytotoxicity (24.8% viability). At explant, an intense chronic inflammatory response was observed in non-fixed matrices, in contrast to genipin-fixed scaffolds. The sheep serum showed a marked decrease in IgG response in both 0.001% and 0.01% genipin-fixed matrices (IgG 30 and 20, respectively) when compared to non-fixed matrices (IgG 40).. Genipin crosslinking of the matrices attenuated, but did not eliminate, the inflammatory host reaction. Whether genipin treatment might extend the durability of xenogenic scaffolds remains to be investigated.

Topics: Animals; Bioprosthesis; Cross-Linking Reagents; Glutaral; Heart Valve Prosthesis; Immunoglobulin G; Inflammation; Iridoid Glycosides; Iridoids; Microscopy, Electron, Transmission; Pulmonary Valve; Sheep; Swine; Tensile Strength; Tissue Engineering; Tissue Scaffolds

Blends between chitosan (CS) and gelatin (G) with various compositions (CS/G 0/100 20/80, 40/60, 60/40 100/0 w/w) were produced, as candidate materials for biomedical applications. Different amounts of genipin (0.5 wt.% and 2.5 wt.%) were used to crosslink CS/G blends, promoting the formation of amide and tertiary amine bonds between the macromolecules and the crosslinker. The effects of composition and crosslinking on the physico-chemical properties of samples were evaluated by infrared analysis, thermogravimetry, contact angle measurements, dissolution and swelling tests. Mechanical properties of crosslinked samples were also determined through stress-strain and creep tests: samples stiffness increased with increasing the crosslinker amount and the CS content. Blend composition affected mouse fibroblasts adhesion and proliferation on substrates, depending on the crosslinker amount. Finally, crosslinked CS/G blends containing 80 wt.% G were found to support neuroblastoma cells adhesion and proliferation which made them promising candidates for uses in the field of nerve regeneration.

Topics: Animals; Biocompatible Materials; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Chitosan; Fibroblasts; Gelatin; Iridoid Glycosides; Iridoids; Mice; Neurons; NIH 3T3 Cells; Surface Properties; Tensile Strength

Topics: Adhesives; Animals; Biomechanical Phenomena; Cattle; Collagen; Compressive Strength; Cross-Linking Reagents; Glutaral; Glycation End Products, Advanced; Intervertebral Disc; Iridoid Glycosides; Iridoids; Tensile Strength

We have reported previously that genipin, a natural iridoid compound, induces neuritogenesis through a nitric oxide (NO)-cyclic GMP (cGMP)-cGMP-dependent protein kinase (PKG) signaling pathway in PC12h cells and that neuronal NO synthase (nNOS) is one of the target molecules of genipin in vitro. Recently, it has been suggested that the neurotrophic effects of NO are due to its direct activation of receptor-tyrosine kinase, especially TrkA. In this study, we investigated whether mouse neuroblastoma Neuro2a cells, which express nNOS but not TrkA, respond to genipin with neurite outgrowth through the mechanism observed in PC12h cells, to assess the involvement of TrkA in the mechanism. Neuro2a cells expressed all three types of NO synthase (NOS), and nNOS was detectable as the main component in Western blot analysis. Genipin significantly induced neurite outgrowth and activation of NADPH-diaphorase, which were significantly blocked by a non-selective NOS inhibitor. Both a soluble guanylate cyclase inhibitor and a PKG inhibitor also inhibited the genipin-induced neuritogenesis. Genipin induced sustained phosphorylation of mitogen-activated protein kinase (MAPK). In fact, the genipin-induced neurite outgrowth was completely inhibited by a specific MAPK kinase inhibitor. Moreover, a NOS inhibitor abolished MAPK phosphorylation as well as neurite outgrowth in genipin-treated cells. These results suggest that genipin induces neurite outgrowth through an NO-cGMP-PKG signaling pathway followed by MAPK phosphorylation without TrkA activation in Neuro2a cells and that PKG downstream to NOSs, which may be mainly nNOS, is very important for the signaling molecule to induce neuritogenesis by genipin.

Topics: Animals; Cell Line, Tumor; Cholagogues and Choleretics; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Iridoid Glycosides; Iridoids; Mice; Mitogen-Activated Protein Kinases; NADPH Dehydrogenase; Neurites; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Phosphorylation; Signal Transduction

Controlled crosslinking of collagen gels has important applications in cell and tissue mechanics as well as tissue engineering. Genipin is a natural plant extract that has been shown to crosslink biological tissues and to produce color and fluorescence changes upon crosslinking. We have characterized the effects of genipin concentration and incubation duration on the mechanical and fluorigenic properties of type I collagen gels. Gels were exposed to genipin (0, 1, 5, or 10 mM) for a defined duration (2, 4, 6, or 12 h). Mechanical properties were characterized using parallel plate rheometry, while fluorigenic properties were examined with a spectrofluorimetric plate reader and with a standard, inverted epifluorescent microscope. Additionally, Fourier transform infrared spectroscopy was used to characterize and track the crosslinking reaction in real-time. Genipin produced significant concentration- and incubation-dependent increases in the storage modulus, loss modulus, and fluorescence intensity. Storage modulus was strongly correlated to fluorescence exponentially. Minimal cytotoxicity was observed for exposure of L929 fibroblasts cultured within collagen gels to 1 mM genipin for 24 h, but significant cell death occurred for 5 and 10 mM genipin. We conclude that genipin can be used to stiffen collagen gels in a relatively short time frame, that low concentrations of genipin can be used to crosslink cell-populated collagen gels to affect cell behavior that is influenced by the mechanical properties of the tissue scaffold, and that the degree of crosslinking can be reliably assayed optically via simple fluorescence measurements.

Topics: Animals; Biocompatible Materials; Cells, Cultured; Collagen; Cross-Linking Reagents; Cytotoxins; Fibroblasts; Fluorescence; Gels; Iridoid Glycosides; Iridoids; Materials Testing; Mice; Molecular Structure; Spectrometry, Fluorescence; Spectroscopy, Fourier Transform Infrared; Stress, Mechanical; Tensile Strength; Tissue Engineering

When cultivated with Aspergillus niger, geniposide, an important drug, is transformed into genipin and genipinine. A simple and rapid HPLC method for simultaneous determination of geniposide and its two metabolites in broth of A. niger is described. The chromatographic separation was achieved on a C18 ODS column (250 x 4.6 mm) by gradient elution with 0.1% formic acid in water and 0.1% formic acid in acetonitrile as the gradient mixtures. The flow rate was 1 mL/min, the detection wavelength was 238 nm and the column temperature was kept at 28 degrees C. The retention times of geniposide, genipin and genipinine were 10.9, 13.8 and 21.5 min, respectively. The mean absolute recoveries of three analysts were over 98%. Quantification limits were 0.01 microg/mL for geniposide and 0.02 microg/mL for the two metabolites. The method was applied for the quantification of geniposide, genipin and genipinine during fermentation and the evaluation of the bioavailabilities of these three compounds in Caco-2 monolayer.

Topics: Aspergillus niger; Biological Availability; Caco-2 Cells; Chromatography, High Pressure Liquid; Humans; Iridoid Glycosides; Iridoids; Linear Models; Pyridines; Sensitivity and Specificity

The influence of mitochondrial uncoupling protein genipin on endothelium-dependent reaction of vessels, heart contractility and myocardial oxygen consumption was studied at streptozotocin-induced rat model of diabetes mellitus. The partial restoration of damaged at diabetes mellitus vascular reactions as well as decrease ofmyocardial oxygen consumption has been shown after intraperitoneal injection of genipin. For example, after the introduction of 10 mg/kg genipin were shown a partial restoration of endothelium-dependent dilatation of aorta and coronary vessels, increase of contractive strength-dependent responses of vascular smooth muscle, decrease of portal vein isolated strips stiffness, the improvement of contractive properties and decrease of myocardial stiffness. These data suggest a possible role for mitochondrial uncoupling protein in the development of changes of heart and vascular responses observed at experimental diabetes mellitus.

Topics: Animals; Coronary Vessels; Diabetes Mellitus, Experimental; Endothelium, Vascular; Ion Channels; Iridoid Glycosides; Iridoids; Male; Mitochondrial Proteins; Muscle Contraction; Muscle, Smooth, Vascular; Myocardium; Oxidation-Reduction; Oxygen Consumption; Phosphorylation; Rats; Rats, Wistar; Uncoupling Protein 1; Vasodilation

Nanometric silk-fibroin nets were fabricated by electrospinning from regenerated Bombyx mori silk-fibroin (SF)/formic acid solutions with the addition of genipin (GE), 2, 15 and 24 h after the solution preparation. After spinning, the pure fibroin nanofibers were water soluble and needed a further stabilization process, whereas the reaction of fibroin with genipin resulted in water-insoluble fibroin nets due to conformational changes induced in the fibroin by the genipin. SFGE nanofibers presented diameters ranging from 140 to 590 nm and were generally thinner than pure SF nanofibers. The secondary structure of SF into SFGE nanofibers showed the presence of a beta-sheet conformation together with beta-turn intermediates (turns and bends). The approach described in this paper provides an alternative method of designing SF nanofibers that are already water insoluble, without any stability post-treatment needed.

Topics: Animals; Bombyx; Fibroins; Insect Proteins; Iridoid Glycosides; Iridoids; Nanostructures

Investigated the changes of crosslinking index, swelling ratio, degradation rate and cytotoxicity of genipin crosslinked gelatin accompany with crosslinking time. 1% genipin crosslinked gelatin were divided into 7 groups by crosslinking time: 10 min group, 30 min group, 1 h group, 2 h group, 12 h group, 24 h group, 72 h group. The results proved that genipin could crosslink gelatin effectively. Accompany with increasing of crosslinking time, crosslinking index increased, and swelling ratio, degradation rate decreased. In 10 min group, crosslinking index was low(26.7%), swelling ratio was high, (265%), completely degraded within 1 week. This indicated that biomaterials of 10 min group was instable and degraded easily. Compared with 10 min group, biomaterials of 30 min group changed significantly with crosslinking index(45.7%), swelling ratio (206%) and degration rate (completely degraded between 4 weeks and 8 weeks). This indicated that genipin could change the properties of gelatin within 30 min. Biomaterials after 30 min, crosslinking index increased, and swelling ratio, degradation rate decreased gradually accompanied with increasing of crosslinking time. Biomaterials of 72 h, crosslinking index was 73.1%, swelling ratio was 152%, and degradated 18.9% after 12 weeks. RGR (relative cell growth rate) of every group measured by MTT assay changed between 87.9% and 105.4%, indicated that the cytotoxicity of genipin crosslinked gelatin was very low.

Topics: Animals; Biodegradation, Environmental; Cell Survival; Cricetinae; Cricetulus; Cross-Linking Reagents; Gelatin; Iridoid Glycosides; Iridoids; Time Factors

Zanthoxylum limonella oil (ZLO) containing chitosan-gelatin complex microcapsules cross-linked with genipin, a cross-linker of natural origin, have been prepared by a complex coacervation process using the salting-out method. The effects of various parameters such as oil loading, degree of cross-linking, ratio of chitosan to gelatin, etc. on oil content, encapsulation efficiency and the release rate of ZLO have been studied. FT-IR spectroscopy has been used to understand the interaction between the polymers and oil. Scanning electron microscopy (SEM) has been employed to study the morphology of the prepared microcapsules.

Topics: Capsules; Chitosan; Cholagogues and Choleretics; Cross-Linking Reagents; Drug Compounding; Gelatin; Iridoid Glycosides; Iridoids; Microscopy, Electron, Scanning; Models, Chemical; Oils; Plant Extracts; Salts; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared; Zanthoxylum

Altered mechanical loading, secondary to biochemical changes in the nucleus pulposus, is a potential mechanism in disc degeneration. An understanding of the role of this altered mechanical loading is only possible by separating the mechanical and biological effects of early nucleus pulposus changes. The objective of this study was to quantify the mechanical effect of decreased glycosaminoglycans (GAG) and increased crosslinking in the nucleus pulposus using in vitro rat lumbar discs. Following initial mechanical testing the discs were injected according to the four treatment groups: PBS control, chondroitinase-ABC (ChABC) for GAG degradation, genipin (Gen) for crosslinking, or a combination of chondroitinase and genipin (ChABC+Gen). After treatment the discs were again mechanically tested, followed by histology or biochemistry. Neutral zone mechanical properties were changed by approximately 20% for PBS, ChABC, and ChABC+Gen treatments (significant only for PBS in a paired comparison). These trends were reversed with genipin crosslinking alone. With ChABC treatment the effective compressive modulus increased and the GAG content decreased; with the combination of ChABC+Gen the mechanics and GAG content were unchanged. Degradation of nucleus pulposus GAG alters disc axial mechanics, potentially contributing to the degenerative cascade. Crosslinking is unlikely to contribute to degeneration, but may be a potential avenue of treatment.

Topics: Animals; Biomechanical Phenomena; Chondroitin ABC Lyase; Cross-Linking Reagents; Glycosaminoglycans; Intervertebral Disc; Iridoid Glycosides; Iridoids; Lumbar Vertebrae; Male; Rats; Rats, Sprague-Dawley; Spinal Diseases; Weight-Bearing

Inchinkoto (ICKT), a herbal medicine, has been recognized in Japan and China as a "magic bullet" for jaundice. To explore potent therapeutic agents for cholestasis, the effects of ICKT or its ingredients on multidrug resistance-associated protein 2 (Mrp2/ MRP2)-mediated choleretic activity, as well as on antioxidative action, were investigated using rats and chimeric mice with livers that were almost completely repopulated with human hepatocytes. Biliary excretion of Mrp2 substrates and the protein mass, subcellular localization, and mRNA level of Mrp2 were assessed in rats after 1-wk oral administration of ICKT or genipin, a major ingredient of ICKT. Administration of ICKT or genipin to rats for 7 days increased bile flow and biliary excretion of bilirubin conjugates. Mrp2 protein and mRNA levels and Mrp2 membrane densities in the bile canaliculi and renal proximal tubules were significantly increased in ICKT- or genipin-treated rat livers and kidneys. ICKT and genipin, thereby, accelerated the disposal of intravenously infused bilirubin. The treatment also increased hepatic levels of heme oxygenase-1 and GSH by a nuclear factor-E2-related factor (Nrf2)-dependent mechanism. Similar effects of ICKT on MRP2 expression levels were observed in humanized livers of chimeric mice. In conclusion, these findings provide the rationale for therapeutic options of ICKT and its ingredients that should potentiate bilirubin disposal in vivo by enhancing Mrp2/MRP2-mediated secretory capacities in both livers and kidneys as well as Nrf2-mediated antioxidative actions in the treatment of cholestatic liver diseases associated with jaundice.

Topics: Animals; Antioxidants; ATP-Binding Cassette Transporters; Bile; Cells, Cultured; Cholagogues and Choleretics; Drugs, Chinese Herbal; Glutathione; Heme Oxygenase (Decyclizing); Iridoid Glycosides; Iridoids; Kidney; Liver; Male; Mice; NF-E2-Related Factor 2; Organic Anion Transporters; Rats; Rats, Sprague-Dawley

Elastin is an elastomeric, self-assembling extracellular matrix protein with potential for use in biomaterials applications. Here, we compare the microstructural and tensile properties of the elastin-based recombinant polypeptide (EP) EP20-244 crosslinked with either genipin (GP) or pyrroloquinoline quinone (PQQ). Recombinant EP-based sheets were produced via coacervation and subsequent crosslinking. The micron-scale topography of the GP-crosslinked sheets examined with atomic force microscopy revealed the presence of extensive mottling compared with that of the PQQ-crosslinked sheets, which were comparatively smoother. Confocal microscopy showed that the subsurface porosity in the GP-crosslinked sheets was much more open. GP-crosslinked EP-based sheets exhibited significantly greater tensile strength (P < or = 0.05). Mechanistically, GP appears to yield a higher crosslink density than PQQ, likely due to its capacity to form short-range and long-range crosslinks. In conclusion, GP is able to strongly modulate the microstructural and mechanical properties of elastin-based polypeptide biomaterials forming membranes with mechanical properties similar to native insoluble elastin.

Topics: Cross-Linking Reagents; Elastin; Iridoid Glycosides; Iridoids; Lysine; Microscopy, Atomic Force; Microscopy, Confocal; Peptides; PQQ Cofactor; Solvents; Tensile Strength; Water

Sutures are currently the gold standard for wound closure but they are still unable to seal tissue and may induce scarring or inflammation. Biocompatible glues, based on polysaccharides such as chitosan, are a possible alternative to conventional wound closure. In this study, the adhesion of laser-activated chitosan films is investigated in vitro and in vivo. In particular we examine the effect of varying the laser power, as well as adding a natural cross-linker (genipin) to the adhesive composition.. Flexible and insoluble strips of chitosan films (surface area approximately 34 mm(2), thickness approximately 20 microm) were bonded to sheep intestine using several laser powers (0, 80, 120, and 160 mW) at 808-nm wavelength. The strength of repaired tissue was tested by a calibrated tensiometer to select the best power. A natural cross-linker (genipin) was also added to the film and the tissue repair strength compared with the strength of plain films. The adhesive was also bonded in vivo to the sciatic nerve of rats and the thermal damage induced by the laser assessed 4 days post-operatively.. Chitosan adhesives successfully repaired intestine tissue, attaining a maximum repair strength of 14.7+/-4.3 kPa (n = 30) at the laser power of 120 mW. The chitosan-genipin films achieved lower repair strength (9.1+/-2.9 kPa). The laser caused partial demyelination of axons at the site of operation, but the myelinated axons retained a normal morphology proximally and distally.. The chitosan adhesive effectively bonded to tissue causing only localized thermal damage in vivo, when the appropriate laser parameters were selected.

Topics: Animals; Biocompatible Materials; Calorimetry, Differential Scanning; Chitosan; Hemostatics; Intestines; Iridoid Glycosides; Iridoids; Low-Level Light Therapy; Microscopy, Atomic Force; Microscopy, Electron, Scanning; Rats; Sciatic Nerve; Sheep; Spectrophotometry, Ultraviolet; Tensile Strength; Tissue Adhesives; Tissue Engineering

An improved technique for quantification of collagen immobilized on polymeric substrates is needed as tissue engineering evolves. Current immobilized protein quantification methods are indirect, time-consuming, and/or inaccurate. In this study, Sirius red colorimetric microassay was shown to be feasible for quantifying the density of collagen immobilized on aminolyzed poly(L-lactic acid) (PLLA) surfaces using the specific bonding of Sirius dye to collagen. It offers a number of advantages over traditional methods, including direct staining, high sensitivity, and high stability of the dye. The detection limit is approximately 0.1 microg/cm(2), and the dynamic range is greater than 50. Sirius red dye has not been used previously for quantification of protein immobilized on polymers. The collagen densities achieved with each of the two crosslinking reagents investigated, namely glutaraldehyde (GA) and genipin, were compared. The latter is an alternative crosslinker derived from a traditional Chinese medicine. The collagen densities immobilized by the two reagents were measured to be similar. This was confirmed by the similar behaviors of esophageal primary smooth muscle cells (ESMCs) on these two modified PLLA membranes; collagen grafted with either coupler was found to greatly promote, to a similar extent, cell attachment and both short-term (4 days) and long-term (12 days) proliferation compared with unmodified PLLA. Smooth muscle cells on both modified membranes were stained to display contractile alpha-actin protein filaments.

Topics: Actins; Animals; Biocompatible Materials; Cattle; Cell Survival; Cells, Cultured; Collagen; DNA; Esophagus; Extracellular Matrix; Glutaral; Iridoid Glycosides; Iridoids; Lactic Acid; Membranes, Artificial; Mitochondria; Myocytes, Smooth Muscle; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Polymers; Prostheses and Implants; Surface Properties; Swine; Tissue Engineering

We define "dual fingerprint reagents" as chemical formulations that produce with latent fingerprints in one stage impressions that are both colored and fluorescent. Solutions containing ninhydrin and group IIb metal salts appear to be true dual reagents. Application of these formulations to latent fingerprints on paper is as efficient as the two-step process beginning with ninhydrin and followed by treatment with metal salt. In the color mode, fingerprint detectability with the two ninhydrin-metal salt reagents (one with zinc chloride and the other with cadmium chloride) is comparable with that of ninhydrin itself, in spite of the difference in color. The sensitivity is significantly higher in the fluorescence mode. To view the latent impressions the exhibits are treated with ninhydrin-metal salt reagents and observed under white light illumination and under fluorescence conditions. Cooling to liquid nitrogen temperature enhances the fluorescence considerably. In the shorter wavelength domain, ninhydrin-metal salt reagents exhibit higher sensitivity than the recently reported dual reagent, genipin. The latter is advantageous, however, in the longer wavelength domain, on paper items with strong self-fluorescence, such as brown wrapping paper or paper printed with fluorescent ink. Upon reduction of the ninhydrin concentration 10-fold, ninhydrin-metal salt formulations become purely fluorogenic reagents; no color is noticed but the fluorescence is as intense as with concentrated solutions. Working at lower concentrations is an advantage from ecological and economical viewpoints.

Topics: Cadmium Chloride; Chlorides; Dermatoglyphics; Fluorescence; Humans; Indicators and Reagents; Iridoid Glycosides; Iridoids; Ninhydrin; Zinc Compounds

Genipin-fixed kappa-carrageenan was prepared in ambient conditions in aqueous solution using genipin, a naturally occurring crosslinker with kappa-carrageenan (kappaC). The crosslinked kappaC showed the greatest swelling capacity in acidic medium having pH 1.2 when compared to those in neutral and alkaline media. Enhanced stability of the crosslinked product with respect to the non-modified kappaC was confirmed by degradation studies in Ringer's solution, rheological and thermogravimetric measurements. Genipin fixation of kappaC was confirmed by measuring the bulk density, true density, pore volume, porosity, intrinsic viscosity, UV absorbance and optical rotation of the crosslinked kappaC, and as well as by optical microscopy, SEM, and MS/MS studies. The crosslinked product may be useful as super absorbent and sustained release formulation in biomedical applications.

Topics: Carrageenan; Cross-Linking Reagents; Drug Stability; Hydrogels; Hydrogen-Ion Concentration; Iridoid Glycosides; Iridoids; Macromolecular Substances; Microscopy, Electron, Scanning; Rheology; Spectrophotometry; Tandem Mass Spectrometry; Thermogravimetry; X-Ray Diffraction

The seeds and the pulp of Gardenia volkensii yielded one new iridiod, namely 4-(2N-gardenamide)n-butanoic acid, together with the known iridoids genipin and genipin gentiobioside, the pterocarpin medicarpin, coumarins, phenylpropanoids, benzenoids and triterpenes. The structures of these were determined on the basis of spectroscopic data and by comparison of obtained data with those from literature. The fruit extracts of this plant exhibited moderate radical scavenging activity against DPPH radical and moderate lethality against brine shrimps (Artemia salina).

Topics: Animals; Artemia; Biphenyl Compounds; Butyrates; Butyric Acid; Chromatography, Thin Layer; Dose-Response Relationship, Drug; Free Radical Scavengers; Gardenia; Hydrazines; Iridoid Glycosides; Iridoids; Magnetic Resonance Spectroscopy; Molecular Structure; Picrates; Plant Extracts; Pyridones; Seeds

This article describes the preparation and in vitro characterization of novel genipin cross-linked alginate-chitosan (GCAC) microcapsules that have potential for live cell therapy applications. This microcapsule system, consisting of an alginate core with a covalently cross-linked chitosan membrane, was formed via ionotropic gelation between calcium ions and alginate, followed by chitosan coating by polyelectrolyte complexation and covalent cross-linking of chitosan by naturally derived genipin. Results showed that, using this design concept and the three-step procedure, spherical GCAC microcapsules with improved membrane strength, suppressed capsular swelling, and suitable permeability can be prepared. The suitability of this novel membrane formulation for live cell encapsulation was evaluated, using bacterial Lactobacillus plantarum 80 (pCBH1) (LP80) and mammalian HepG2 as model cells. Results showed that capsular integrity and bacterial cell viability were sustained 6 mo postencapsulation, suggesting the feasibility of using this microcapsule formulation for live bacterial cell encapsulation. The metabolic activity of the encapsulated HepG2 was also investigated. Results suggested the potential capacity of this GCAC microcapsule in cell therapy and the control of cell signaling; however, further research is required.

Topics: Alginates; Capsules; Cell Line; Cell Proliferation; Chitosan; Drug Carriers; Glucuronic Acid; Hexuronic Acids; Humans; Iridoid Glycosides; Iridoids; Lactobacillus plantarum; Microscopy, Confocal; Plant Extracts; Polymers; Treatment Outcome; Trypan Blue

Gelatin microspheres cross-linked with genipin were developed to encapsulate the probiotic Bifidobacterium lactis Bb-12 The effects of different gelatin concentrations (10-19% w/v), bloom strengths (175 and 300), surfactants, stirring rates during emulsion formation and genipin concentrations (0-10 mM) on the microsphere sizes and viability of bacterial cells were investigated. Principal Component Analysis revealed microsphere size distribution differed depending on the presence or absence of surfactants as well as a trend of increasing micropshere size with increasing gelatin concentration and bloom strength. Lower stirring rates resulted in larger microspheres with higher encapsulation yields of bifidobacteria Microsphere size and cell viability were not significantly (p < 0.05) influenced by increasing genipin concentrations up to 10 mM whereas microsphere stability in simulated gastric juice increased with increasing genipin concentration. The encapsulation yields were higher in 175 bloom strength gelatin microspheres than in 300. Cold-stage scanning electron microscopy showed encapsulated bacteria distributed throughout the genipin cross-linked gelatin matrix.

Topics: Bifidobacterium; Cell Survival; Gelatin; Iridoid Glycosides; Iridoids; Microscopy, Electron, Scanning; Microspheres; Particle Size; Surface-Active Agents

Genipin, a reagent of plant origin was used for the immobilization of lipase by cross-linking to chitosan beads. The catalytic properties and operational and storage stabilities of the immobilized lipase were compared with the soluble lipase. Under optimum conditions, 198 microg protein was bound per g chitosan with a protein-coupling yield of 35%. The hydrolytic activity was 10.8 U/g chitosan and the relative specific activity was 108%. The immobilized lipase showed better thermal and pH stabilities compared to the soluble form. The immobilized enzyme exhibited mass transfer limitations as reflected by a higher apparent K(m) value and a lower energy of activation. The immobilized enzyme retained about 74% of its initial activity after five hydrolytic cycles.

Topics: Chitosan; Clinical Enzyme Tests; Cross-Linking Reagents; Enzyme Stability; Enzymes, Immobilized; Iridoid Glycosides; Iridoids; Lipase; Microspheres; Molecular Structure; Molecular Weight

Angiogenic therapies may need to select a stable agent to be delivered. In the study, a nonpeptide angiogenic agent, ginsenoside Rg(1) (Rg(1)), was encapsulated in the gelatin microspheres (MSs) crosslinked with genipin and intramuscularly injected into a rat model with infarcted myocardium. bFGF was used as a control. After swelling in an aqueous environment, the MSs without crosslinking became collapsed and stuck together. For those crosslinked, the swollen MSs appeared to be more stable with an increasing the degree of crosslinking. After it was released from MSs in vitro, the remaining activity of bFGF on HUVEC proliferation reduced significantly, while that of Rg(1) remained constant. An inspection of the retrieved hearts revealed a large aneurysmal left ventricle (LV) with a thinned myocardium and a significant myocardial fibrosis for that treated with the Empty MSs (without drug encapsulation). However, those receiving the MSs encapsulated with bFGF or Rg(1) attenuated the enlargement of the LV cavity and the development of myocardial fibrosis. The densities of microvessels found in the border zones of the infarct treated with the bFGF or Rg(1) MSs were significantly greater than that treated with the Empty MSs. These results indicated that Rg(1), a stable angiogenic agent, successfully enhanced the myocardial perfusion and preserved the infarcted LV function.

Topics: Angiogenesis Inducing Agents; Animals; Cell Proliferation; Cells, Cultured; Chemistry, Pharmaceutical; Coronary Circulation; Cross-Linking Reagents; Disease Models, Animal; Drug Carriers; Drug Compounding; Endothelial Cells; Feasibility Studies; Fibroblast Growth Factor 2; Fibrosis; Gelatin; Ginsenosides; Humans; Injections, Intramuscular; Iridoid Glycosides; Iridoids; Male; Microspheres; Myocardial Infarction; Neovascularization, Physiologic; Particle Size; Rats; Rats, Sprague-Dawley; Solubility; Time Factors; Ventricular Function, Left; Ventricular Remodeling

Recently there have been some developments in the preparation of controlled drug delivery systems for glaucoma. Many materials are being used in this area, namely gelatine and chitosan. Both of them present high levels of biocompatibility and biodegradability. In this paper, we wish to report the work we have been doing on the preparation and characterization of hydrogels based on gelatine and chitosan. The crosslinking agents used were 1-(3-(Dimethylamino)propyl)-3-Ethylcarbodiimide hydrocholide (CDI), 1,4-Butanodiol diglycidyl ether (epoxyde 1), Ethylene glycol diglycidyl ether (epoxyde 2) and genipin. The results obtained showed that all of the films were hydrogels. The surface and transversal cut showed a porous surface in all the films. The thermal analysis proved the modifications in the polymeric chains, with the stabilization of all of them by the crosslinking agents. The release pattern indicates that the gelatine films were the best since they release the adequate proportion of drug. Finally, the cytotoxicity showed that the gelatine films were all biocompatible, specially the ones crosslinked with one of the Epoxydes.

Topics: Animals; Biocompatible Materials; Cell Survival; Cells, Cultured; Chitosan; Chlorocebus aethiops; COS Cells; Cross-Linking Reagents; Drug Carriers; Drug Delivery Systems; Gelatin; Glaucoma; Humans; Iridoid Glycosides; Iridoids; Materials Testing; Models, Biological; Wettability

The inflammatory response is a key component in the biocompatibility of biomaterials. Among the factors that control the development of inflammation is a critical molecule nuclear factor-kappaB (NF-kappaB). Therefore, the aim of this study was to assess the feasibility of noninvasive whole-body real-time imaging for the evaluation of host-biomaterial interaction in the NF-kappaB transgenic mice. Transgenic mice, carrying the luciferase gene under the control of NF-kappaB, were constructed. In vivo bioluminescence imaging showed that the constitutive and induced NF-kappaB activities of transgenic mice were detected in most of the lymphoid tissues, demonstrating that NF-kappaB-driven luminescence reflected the inflammatory response in vivo. By the implantation of genipin-cross-linked gelatin conduit (GGC) and bacterial endotoxin-immersed GGC in the dorsal region, we detected a strong and specific luminescent signal from the tissue around the bacterial endotoxin-immersed GGC implant. Histological and immunohistochemical analysis also demonstrated that inflammation, characterized by the infiltration of immune cells, the accumulation of fluid, and the activation of NF-kappaB, was evoked around the same region. The correlation between the bioluminescence imaging and histological changes indicated that noninvasive imaging technique could be used to monitor the real-time inflammation in the implanted mice.

Topics: Animals; Biocompatible Materials; Diagnostic Imaging; Feasibility Studies; Gelatin; Gene Expression Regulation; Genes, Reporter; Immunohistochemistry; Inflammation; Iridoid Glycosides; Iridoids; Luciferases; Luminescent Measurements; Materials Testing; Mice; Mice, Transgenic; Microinjections; NF-kappa B; Prostheses and Implants

We evaluated peripheral nerve regeneration using biodegradable genipin-cross-linked gelatin nerve conduits (GGCs) with three different cross-linking degrees, 24, 36 and 51%. Biocompatibility and biodegradability of the GGC and its efficiency as a guidance channel were examined based on the repair process of a 10-mm gap in the rat sciatic nerve. From this pilot study we concluded that GGCs with a mean cross-linking degree of 36% can ensure nerve regeneration with a more mature structure, as demonstrated by better developed epineural and perineural organisation and axonal development, as well as better-recovered electrophysiology with a relatively positive sciatic functional index and a shorter latency of the muscle action potential curve. Regenerated nerves in the GGCs with mean cross-linking degrees of 24 and 51% were less favourable, due to irritation caused by degradation material and compression by the remaining tube walls, respectively.

Topics: Animals; Biocompatible Materials; Cross-Linking Reagents; Electrophysiology; Gelatin; Iridoid Glycosides; Iridoids; Materials Testing; Nerve Regeneration; Rats; Rats, Sprague-Dawley; Sciatic Nerve

It has been reported that genipin, the aglycone of geniposide, induces apoptotic cell death in human hepatoma cells via a NADPH oxidase-reactive oxygen species (ROS)-c-Jun NH(2)-terminal kinase (JNK)-dependent activation of mitochondrial pathway. This continuing work aimed to define that mixed lineage kinase 3 (MLK3) is a key mediator, which connect between ROS and JNK in genipin-induced cell death signaling. In PC3 human prostate cancer cells, genipin stimulated MLK3 activity in concentration- and time-dependent manner. The PC3 cells stably transfected with dominant-negative form of MLK3 was less susceptible to population of the sub-G1 apoptotic cells, activation of caspase, collapse of mitochondrial membrane potential, and release of cytochrome c triggered by genipin, suggesting a crucial role of MLK3 in genipin signaling to apoptotic cell death. Diphenyleneiodonium (DPI), a specific inhibitor of NADPH oxidase, markedly inhibited ROS generation and MLK3 phosphorylation in the genipin-treated cells. Pretreatment with SP0600125, a specific inhibitor of JNK but neither U0126, a specific inhibitor of MEK1/2 nor PD169316, a specific inhibitor of p38 suppressed genipin-induced apoptotic cell death. Notably, both the phosphorylation of JNK and induction of c-Jun induced by genipin were markedly inhibited in PC3-EGFP-MLK3 (K144R) cells expressing a dominant-negative MLK3 mutant. Taken together, our observations suggest genipin signaling to apoptosis of PC3 cells is mediated via activation of ROS-dependent MLK3, which leads to downstream activation of JNK.

Topics: Apoptosis; Blotting, Western; Butadienes; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Flow Cytometry; Green Fluorescent Proteins; Humans; Imidazoles; Iridoid Glycosides; Iridoids; JNK Mitogen-Activated Protein Kinases; Male; MAP Kinase Kinase Kinases; Membrane Potential, Mitochondrial; Mitochondria; Mitogen-Activated Protein Kinase Kinase Kinase 11; Nitriles; Prostatic Neoplasms; Reactive Oxygen Species; Recombinant Fusion Proteins; Signal Transduction; Transfection

A subset of neurons in the brain, known as 'glucose-excited' neurons, depolarize and increase their firing rate in response to increases in extracellular glucose. Similar to insulin secretion by pancreatic beta-cells, glucose excitation of neurons is driven by ATP-mediated closure of ATP-sensitive potassium (K(ATP)) channels. Although beta-cell-like glucose sensing in neurons is well established, its physiological relevance and contribution to disease states such as type 2 diabetes remain unknown. To address these issues, we disrupted glucose sensing in glucose-excited pro-opiomelanocortin (POMC) neurons via transgenic expression of a mutant Kir6.2 subunit (encoded by the Kcnj11 gene) that prevents ATP-mediated closure of K(ATP) channels. Here we show that this genetic manipulation impaired the whole-body response to a systemic glucose load, demonstrating a role for glucose sensing by POMC neurons in the overall physiological control of blood glucose. We also found that glucose sensing by POMC neurons became defective in obese mice on a high-fat diet, suggesting that loss of glucose sensing by neurons has a role in the development of type 2 diabetes. The mechanism for obesity-induced loss of glucose sensing in POMC neurons involves uncoupling protein 2 (UCP2), a mitochondrial protein that impairs glucose-stimulated ATP production. UCP2 negatively regulates glucose sensing in POMC neurons. We found that genetic deletion of Ucp2 prevents obesity-induced loss of glucose sensing, and that acute pharmacological inhibition of UCP2 reverses loss of glucose sensing. We conclude that obesity-induced, UCP2-mediated loss of glucose sensing in glucose-excited neurons might have a pathogenic role in the development of type 2 diabetes.

Topics: Adenosine Triphosphate; Animals; Diabetes Mellitus, Type 2; Dietary Fats; Glucose; Homeostasis; Humans; Ion Channels; Iridoid Glycosides; Iridoids; Mice; Mice, Obese; Mice, Transgenic; Mitochondrial Proteins; Neurons; Obesity; Potassium Channels, Inwardly Rectifying; Pro-Opiomelanocortin; Uncoupling Protein 2

Fibrin has been long used as an effective scaffolding material to grow a variety of cells and tissue constructs. It has been utilized mainly as a hydrogel in varying concentrations to provide an environment in which suspended cells work to rearrange the fibers and lay down their own extracellular matrix. For these fibrin hydrogels to be useful in many tissue-engineering applications, the gels must be cultured for long periods of time in order to increase their mechanical strength to the levels of native tissues. High concentrations of fibrinogen increase the mechanical strength of fibrin hydrogels, but at the same time reduce the ability of cells within the scaffold to spread and survive. We present a method to create a microporous, nanofibriliar fibrin scaffold that has controllable pore size, porosity, and microstructure for applications in tissue engineering. Fibrin has numerous advantages as a scaffolding material as it is normally used by the body as temporary scaffolding for tissue regeneration and healing, and can be autologously sourced. We present here a scaffolding process which enhances the mechanical properties of the fibrin hydrogel by forming it surrounding poly(methyl-methacrylate) beads, then removing the beads with acetone to form an interconnected microporous network. The acetone serves the dual purpose of precipitating and fixing the fibrinogen-based scaffolds as well as adding strength to the network during polymer bead removal. Effects of fibrinogen concentration and time in acetone were examined as well as polymerization with thrombin. A natural crosslinker, genipin, was also used to add strength to the scaffolds, producing a Young's modulus of up to 184+/-5 kPa after 36 h of reaction. Using these methods we were able to produce microporous fibrin scaffolds that support cell growth and have mechanical properties similar to many native tissues.

Topics: Animals; Biocompatible Materials; Cattle; Cell Proliferation; Fibrinogen; Iridoid Glycosides; Iridoids; Mice; Microscopy, Electron, Transmission; NIH 3T3 Cells; Porosity; Tissue Engineering; Tissue Scaffolds

A molecularly imprinted material was developed from hydrogels of chitosan (CS) cross-linked with genipin (GNP) using o-xylene as the template molecule. Gelling time, mechanical, and diffusion properties of CS-GNP hydrogels were initially investigated to establish optimal conditions to prepare molecularly imprinted hydrogels (MIHs). The elastic modulus was found to be directly proportional to the degree of cross-linking (R = moles of genipin/moles of glucosamine) while the diffusion of water, as monitored by magnetic resonance imaging, decreased with R. CS-GNP hydrogels of varying R were imprinted with o-xylene (MIH(o-xylene)). The adsorption capacity of o-xylene by MIH(o-xylene) was greater than the corresponding control hydrogels, particularly at R = 0.25. Freundlich isotherms yielded a better fitness than Langmuir ones and afforded n and Q(max)values of 2.55 and 103.3 mg/g, respectively. The imprinted hydrogel showed the highest adsorption capacity for o-xylene; however, the material was not highly selective as it also exhibited the capacity to adsorb m- and p-xylene isomers. In turn, the MIH(o-xylene) showed a low adsorption when 2-fluorotoluene was used in rebinding experiments, suggesting that molecular recognition by the binding sites is influenced by the electronic and steric properties of the analyte molecule, thus effectively confirming the imprinting effect within the MIH(o-xylene) network. This work opens the possibility to future development of materials with the capacity to adsorb o-xylene analogue molecules such as contaminants bearing chlorinated aromatic structures.

Topics: Animals; Chitosan; Cross-Linking Reagents; Crustacea; Hydrogels; Iridoid Glycosides; Iridoids; Kinetics; Microscopy, Electron, Scanning; Molecular Imprinting; Molecular Structure; Spectroscopy, Fourier Transform Infrared; Time Factors; Viscosity; Xylenes

Nine novel monoterpene alkaloid derivatives (3(a)-(c), 4(a)-(c), 5(a)-(c)) were prepared as a reaction of genipin 2 with L-amino acid methyl hydrochlorides utilized the reaction of reductive amination. Genipin was obtained by beta-glucosidase, catalyzed hydrolysis of the iridoid glucoside geniposide 1. The chemical structures were confirmed by 1D-, 2D-NMR and MS.

Topics: Alkaloids; Amino Acids; Fruit; Gardenia; Iridoid Glycosides; Iridoids; Magnetic Resonance Spectroscopy; Monoterpenes; Pyrans; Spectrometry, Mass, Electrospray Ionization

This paper reports the rheological behavior of chitosan solutions that have been cross-linked with different amounts of genipin, at body temperature and physiological pH. The effect of the cross-linker loading on the rheological properties of hydrogels has been evaluated. The oscillatory time sweep method was used to monitor the dynamic viscoelastic parameters during in situ (i.e., in the rheometer) gelation experiments, enabling the determination of the gelation time. The stress and frequency sweeps were employed to measure G' of the cured hydrogels. It was found that the solutions of chitosan cross-linked with genipin, under physiological conditions, could form relatively strong elastic gels when compared to those of pure chitosan. Moreover, the gelation time obtained from the crossover of G'' and G' was in excellent agreement with the value obtained from the Winter-Chambon criterion. A significant reduction on this parameter was achieved even at low genipin concentrations. This behavior suggests that these formulations are able to be produced in situ and thus constitute promising matrices for cells and bioactive molecule encapsulations.

Topics: Chitosan; Cross-Linking Reagents; Elasticity; Hydrogels; Iridoid Glycosides; Iridoids; Rheology

The suitability of chitosan-based hydrogels as scaffolds for the encapsulation of intervertebral disc (IVD) cells and the accumulation of a functional extracellular matrix mimicking that of the nucleus pulposus (NP) was investigated. The specific hypothesis under study was that the cationic chitosan would form an ideal environment in which large quantities of newly synthesized anionic proteoglycan could be entrapped. Indeed, all the formulations of cell-seeded chitosan hydrogels, studied under in vitro culture conditions, showed that the majority of proteoglycan produced by encapsulated NP cells was retained within the gel rather than released into the culture medium. This was not always the case when annulus fibrosus cells were encapsulated, as unlike the nucleus cells the annulus cells often did not survive when cultured in chitosan. The results support the concept that chitosan may be a suitable scaffold for cell-based supplementation to help restore the function of the NP during the early stages of IVD degeneration.

Topics: Animals; Cattle; Cell Culture Techniques; Cell Survival; Cell Transplantation; Cellulose; Chitosan; Chondrocytes; Coccyx; Gels; Glycerophosphates; Glycosaminoglycans; Intervertebral Disc; Iridoid Glycosides; Iridoids; Male; Proteoglycans; Pyrans; Transforming Growth Factor beta

Transforming growth factor-beta1 (TGF-beta1) is of great relevance to cartilage development and regeneration. A delivery system for controlled release of growth factors such as TGF-beta1 may be therapeutic for cartilage repair. We have encapsulated TGF-beta1 into poly(DL-lactide-co-glycolide) (PLGA) microspheres, and subsequently incorporated the microspheres into biodegradable hydrogels. The hydrogels are poly(ethylene glycol) based, and the degradation rate of the hydrogels is controlled by the non-toxic cross-linking reagent, genipin. Release kinetics of TGF-beta1 were assessed using ELISA and the bioactivity of the released TGF-beta1 was evaluated using a mink lung cell growth inhibition assay. The controlled release of TGF-beta1 encapsulated within microspheres embedded in scaffolds is better controlled when compared to delivery from microspheres alone. ELISA results indicated that TGF-beta1 was released over 21 days from the delivery system, and the burst release was decreased when the microspheres were embedded in the hydrogels. The concentration of TGF-beta1 released from the gels can be controlled by both the mass of microspheres embedded in the gel, and by the concentration of genipin. Additionally, the scaffold permits containment and conformation of the spheres to the defect shape. Based on these in vitro observations, we predict that we can develop a microsphere-loaded hydrogel for controlled release of TGF-beta1 to a cartilage wound site.

Topics: Absorbable Implants; Animals; Biocompatible Materials; Cartilage, Articular; Cells, Cultured; Delayed-Action Preparations; Drug Delivery Systems; Hydrogels; Iridoid Glycosides; Iridoids; Lung; Microscopy, Electron, Scanning; Microspheres; Mink; Pyrans; Tissue Engineering; Transforming Growth Factor beta; Transforming Growth Factor beta1

Gardenia fruit has been traditionally used as a folk medicine for centuries in Asian countries. Extraction with ethanol was used to obtain an extract (GFE) that contains two known constituents, geniposide and genipin, which were subsequently evaluated for anti-inflammatory activity. GFE, genipin, and geniposide showed acute anti-inflammatory activities in carrageenan-induced rat paw edema. In a dose-dependent manner, GFE also inhibited vascular permeability induced by acetic acid. Both genipin and geniposide inhibited production of exudate and nitric oxide (NO) in the rat air pouch edema model. However, genipin possessed stronger anti-inflammatory activity than geniposide, as demonstrated by the results with carrageenan-induced rat paw edema, carrageenan-induced air pouch formation, and measurement of NO content in the exudates. GFE caused a dose-dependent inhibition of acetic acid-induced abdominal writhing in mice. Collectively, genipin, rather than geniposide, is the major anti-inflammatory component of gardenia fruit.

Topics: Animals; Anti-Inflammatory Agents; Carrageenan; Dose-Response Relationship, Drug; Edema; Female; Fruit; Gardenia; Inflammation; Iridoid Glycosides; Iridoids; Male; Mice; Nitrates; Plant Extracts; Pyrans; Rats; Rats, Sprague-Dawley

We have demonstrated that a natural iridoid compound, genipin, induces neurite outgrowth through the nitric oxide (NO)-cGMP-protein kinase G signaling pathway in PC12h cells. PC12 cells, the parental cell line of PC12h cells, have been shown to carry out neurite extension that accompanies NO production in response to nerve growth factor (NGF). This neurite outgrowth was significantly inhibited by NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, in both PC12 and PC12h cells, suggesting that the neuritogenesis is NO-dependent in both cells. In this report, we investigated whether genipin also induces neurite outgrowth in PC12 cells in order to determine the NO-dependent neurotrophic action of genipin in more than just one cell type. Genipin induced marked neurite outgrowth in PC12h cells but not in PC12 cells. The genipin-induced neurite outgrowth was significantly inhibited by L-NAME in PC12h cells. An NO donor, NOR4, also significantly induced neurite outgrowth in a concentration-dependent manner in PC12h cells but not in PC12 cells. On the other hand, NGF-primed PC12 cells exhibited significant neurite extension, which was inhibited by L-NAME, in response to genipin. Interestingly, NGF-primed PC12 cells responded to NOR4 extending neurites and expressed detectable neuronal NO synthase protein which is not detected in naive PC12 cells. These results suggest that genipin exerts a neuritogenic action on neuronal cells which are responsive to NO itself. Furthermore, the results also suggest that PC12h cells are more suitable for the study of NO-dependent neuronal function than PC12 cells which were not responsive to NO.

Topics: Animals; Blotting, Western; Cell Differentiation; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Inhibitors; Gene Expression Regulation; Iridoid Glycosides; Iridoids; Nerve Growth Factor; Neurites; Neurons; NG-Nitroarginine Methyl Ester; Nitric Oxide; PC12 Cells; Pyrans; Pyridines; Rats

The physical properties and microstructure of gelatin-maltodextrin hydrogels fixed with genipin (GP) were investigated as a function of pH (3-7), maltodextrin (MD) (0-9%, w/w) and GP (0-10 mM levels), at a constant gelatin (G) concentration (10%, w/w). Network strength (elastic modulus, E) and swelling behavior were characterized by large deformation testing and by swelling index (SI). In general, network strength increased and swelling decreased at higher pH, MD and GP levels, except at pH 3, where E was independent of the GP concentration until approximately 7.5 mM, above which it declined. Confocal scanning laser microscopy (CLSM) images showed phase separation to be suppressed at pH 3, whereas at pH 7, separation into a self-similar dispersed phase was apparent. Overall, the judicious use of GP to crosslink G was an appropriate means of kinetically trapping MD within the gelatin network.

Topics: Animals; Chemical Phenomena; Chemistry, Physical; Gelatin; Hydrogels; In Vitro Techniques; Iridoid Glycosides; Iridoids; Microscopy, Confocal; Molecular Structure; Polysaccharides; Pyrans; Swine

Chitin and chitosan were hybridized in various weight percentages by genipin crosslinkage under various prefreezing temperatures to form tissue-engineering scaffolds via lyophilization. In addition, deposition of hydroxyapatite (HA) on the surface of the porous scaffolds was performed by precipitation method to achieve modified chemical compositions for chondrocyte attachments and growths. The experimental results revealed that a lower prefreezing temperature or a higher weight percentage of chitin in the chitin-chitosan scaffolds would yield a smaller pore diameter, a greater porosity, a larger specific surface area, a higher Young's modulus, and a lower extensibility. Moreover, a higher chitin percentage could also result in a higher content of amine groups after crosslink and a lower onset temperature for the phase transition after thermal treatment. A decrease in the prefreezing temperature from -4 degrees C to -80 degrees C, an increase in the chitin percentage from 20% to 50%, and an increase in the cycle number of alternate immersion for HA deposition from 1 to 5 generated positive effects on the cell number, the content of glycosaminoglycans, and the collagen level over 28-day cultivation of bovine knee chondrocytes.

Topics: Animals; Biocompatible Materials; Cartilage, Articular; Cattle; Cell Adhesion; Cell Culture Techniques; Cell Proliferation; Cell Survival; Cells, Cultured; Chitin; Chondrocytes; Cross-Linking Reagents; Durapatite; Elasticity; Extracellular Matrix; Iridoid Glycosides; Iridoids; Knee Joint; Materials Testing; Pyrans; Tissue Engineering

Uncoupling protein 2 (UCP2) negatively regulates insulin secretion. UCP2 deficiency (by means of gene knockout) improves obesity- and high glucose-induced beta cell dysfunction and consequently improves type 2 diabetes in mice. In the present study, we have discovered that the small molecule, genipin, rapidly inhibits UCP2-mediated proton leak. In isolated mitochondria, genipin inhibits UCP2-mediated proton leak. In pancreatic islet cells, genipin increases mitochondrial membrane potential, increases ATP levels, closes K(ATP) channels, and stimulates insulin secretion. These actions of genipin occur in a UCP2-dependent manner. Importantly, acute addition of genipin to isolated islets reverses high glucose- and obesity-induced beta cell dysfunction. Thus, genipin and/or chemically modified variants of genipin are useful research tools for studying biological processes thought to be controlled by UCP2. In addition, these agents represent lead compounds that comprise a starting point for the development of therapies aimed at treating beta cell dysfunction.

Topics: Adenosine Triphosphate; Aldehydes; Animals; Drugs, Chinese Herbal; Glucose; Heterocyclic Compounds, 3-Ring; Insulin; Insulin Secretion; Insulin-Secreting Cells; Ion Channels; Iridoid Glycosides; Iridoids; Islets of Langerhans; Male; Membrane Transport Proteins; Mice; Mice, Knockout; Mice, Obese; Mitochondria; Mitochondrial Membranes; Mitochondrial Proteins; Molecular Conformation; Obesity; Potassium Channels; Protons; Pyrans; Uncoupling Protein 2

The gelling properties (gel time (t(gel)) and gel strength) of a 10% (w/w) gelatin sol were investigated as a function of genipin (GP) concentration (0-15 mM) and temperature (25-55 degrees C) to discern mechanisms and optimal conditions for fixation. Gel time increased with increasing temperature, reached a maximum, and then declined as temperature was raised further. By contrast, network strength data followed the opposite trend. From the thermal behavior of t(gel) and network strength, it was inferred that gelation in the low-temperature regime was dominated by hydrogen bonding, while in the high-temperature regime it was dominated by covalent crosslinking. At higher temperatures, crosslinking was described by an Arrhenius rate law expression, with activation energies between 63.2 and 67.8 kJ/mol, depending on GP concentration. In the low temperature regime, an Arrhenius plot resulted in negative activation energies of -75.8 and -64.4 kJ/mol in the presence of 10 and 15 mM GP, respectively. With an increase in both GP concentration and temperature, the gelatin network gradually shifted from being dominated by hydrogen bonds (physical crosslinks) to covalent crosslinking (chemical crosslinks).

Topics: Animals; Cross-Linking Reagents; Gelatin; Hydrogels; In Vitro Techniques; Iridoid Glycosides; Iridoids; Kinetics; Swine; Thermodynamics

The mechanical stability of cross-linked and control spinal motion segments was evaluated using neutral zone, range of motion (ROM), and instability score metrics.. To determine if exogenous cross-linking could increase the stability of spinal motion segments.. The microstructure of the anulus fibrosus extracellular matrix can affect the stability of the intervertebral joint. Parallel testing in our laboratory has shown that exogenous cross-linking can improve the fatigue resistance of anulus fibrosus.. There were 3 separate experimental protocols conducted. The first study used calf lumbar intervertebral joints randomly divided into a genipin cross-linked group and phosphate buffered saline-soaked controls. After 2 days of soaking, flexion-extension ramp cycles were applied to the specimens. The second study repeated the test protocol using 22 moderately and severely degenerated human lumbar intervertebral joints. The third experiment compared the effect of cross-linking treatment on human discs with known degrees of preexisting mechanical instability. Each data set was used to assess joint instability by 3 calculations: ROM, neutral zone, and an instability score. Joint instability for each data set was evaluated using 3 calculations: ROM, neutral zone, and a novel instability score.. These results show that cross-link augmentation can effectively reduce instability of intervertebral discs. The stabilizing effect was observed to be higher in the more mechanically unstable discs. However, cross-linking did not appear to affect the total range of sagittal motion.. By reducing the neutral zone, exogenous cross-linking may help combat the progression of instability in degenerative disc disease.

Topics: Adhesives; Animals; Biomechanical Phenomena; Cattle; Cross-Linking Reagents; Humans; In Vitro Techniques; Intervertebral Disc; Intervertebral Disc Displacement; Iridoid Glycosides; Iridoids; Joint Instability; Joints; Lumbar Vertebrae; Pyrans; Range of Motion, Articular

The genipin cross-linked alginate-chitosan (GCAC) microcapsule, composed of an alginate core and a genipin cross-linked chitosan membrane, was recently proposed for live cell encapsulation and other delivery applications. This article for the first time describes the details of the microcapsule membrane characterization using a noninvasive and in situ method without any physical or chemical modifications on the samples. Results showed that the cross-linking reaction generated the fluorescent chitosan-genipin conjugates. The cross-linked chitosan membrane was clearly visualized by confocal laser scanning microscopy (CLSM). A straightforward assessment on the membrane thickness and relative intensity was successfully achieved. CLSM studies showed that the shell-like cross-linked chitosan membranes of approximately 37 microm in thickness were formed surrounding the microcapsule. The reaction variables, including cross-linking temperature and time significantly affected the fluorescence intensity of the membranes. Elevating the cross-linking temperature from 4 to 37 degrees C drastically intensified the membrane fluorescence, suggesting the attainment of a high degree of cross-linking on the chitosan membrane. Extended cross-linking time altered the cross-linked membranes in modulation. Although genipin concentration and cross-linking time had little effects on the membrane thickness, cross-linking at higher temperatures tended to form relatively thinner membranes.

Topics: Alginates; Capsules; Carbohydrate Conformation; Chitosan; Cross-Linking Reagents; Glucuronic Acid; Hexuronic Acids; Iridoid Glycosides; Iridoids; Microscopy, Atomic Force; Microscopy, Confocal; Pyrans

Numerous microcapsule systems have been developed for a wide range of applications, including the sustained release of drugs, cell transplantation for therapy, cell immobilization, and other biotechnological applications. Despite the fact that microcapsule membrane is a dominant factor governing overall microcapsule performance, its characterization is challenging. We report a new method for characterizing microcapsule membranes, using the most common alginate-poly-L-lysine-alginate (APA) microcapsule as an example. Our data demonstrate that genipin, a naturally derived reagent extracted from gardenia fruits, interacts with poly-L-lysine (PLL) and generates fluorescence. This fluorescence allows clear visualization and easy analysis of the PLL membrane in the APA microcapsules using confocal laser scanning microscopy. The results also show that PLL binding correlates to the reaction variables during PLL coating such as PLL concentration and coating time. In addition, five other different microcapsule formulations consisting of PLL and/or chitosan membranes were examined, and the results imply that this method can be extended to characterize a variety of microcapsule membranes. These findings suggest that genipin can serve as a fluorogenic marker for rapid characterization of microcapsule membranes, a superior method that would have important implications for microcapsule research and potential in many other applications.

Topics: Capsules; Iridoid Glycosides; Iridoids; Materials Testing; Membranes, Artificial; Microscopy, Confocal; Microscopy, Fluorescence; Polymers; Pyrans

Polyethylene glycol (PEG) hydrogels show promise as scaffolds for growth factor delivery to enhance cartilage repair. However, methods to control growth factor release in vivo are needed. We have recently shown that in vitro polymer degradation and in vitro growth factor release kinetics can be altered using PEG crosslinked with different concentrations of genipin. However, the degradation and behavior of PEG-genipin in vivo within the cartilage repair site are unknown. This study was conducted to test the hypotheses that the degradation of PEG-genipin can be altered in vivo within osteochondral defects by changing the concentration of genipin, and that PEG-genipin is biocompatible within the mammalian diarthrodial environment. PEG-genipin cylindrical polymers crosslinked using 8mM, 17.6 mM, or 35.2 mM of genipin were implanted into osteochondral defects made in the trochlea of 24 male Sprague- Dawley rats (48 knees). Rats were sacrificed at 5 weeks and gross, cross-sectional, and histologic assessments were performed. Altering the genipin concentration changed the in vivo degradation properties of the hydrogel ( p < 0.01). Consistent with in vitro findings, polymer degradation was inversely related to the concentration of genipin. Near-complete degradation was seen at 8 mM, intermediate degradation at 17.6 mM, and minimal degradation at 35.2 mM. The results of this study show the degradation of PEGgenipin can be altered in vivo within osteochondral defects by changing the concentration of genipin and that PEG-genipin is biocompatible within osteochondral defects. This new in vivo data support potential use of PEG-genipin polymer as an innovative delivery system to control in vivo release of growth factors for improving articular cartilage repair.

Topics: Absorbable Implants; Animals; Biocompatible Materials; Biodegradation, Environmental; Cholagogues and Choleretics; Drug Implants; Hydrogel, Polyethylene Glycol Dimethacrylate; Iridoid Glycosides; Iridoids; Knee Injuries; Male; Polyethylene Glycols; Pyrans; Rats; Rats, Sprague-Dawley

To determine in a lens epithelial cell line, alpha-TN4, whether genipin, an intestinal metabolite component of the herbal medicine inchin-ko-to, suppresses profibrogenic myofibroblast generation and upregulation of fibrogenic cytokines and to evaluate the potential benefit of the medicine in preventing posterior capsule opacification (PCO).. Department of Ophthalmology, Wakayama Medical University, Wakayama, Japan.. In this study, alpha-TN4 cell proliferation, migration, and expression of alpha-smooth muscle actin (alpha-SMA), the hallmark of myofibroblast generation, were assayed with a colorimetric assay, scratch wound assay, immunohistochemistry, and Western blot analysis. Gene expression of transforming growth factor-beta1 (TGF-beta1) and connective tissue growth factor (CTGF) was characterized with real-time reverse transcription-polymerase chain reaction. In addition, p38 mitogen-activated protein kinase (p 38 MAPK), extracellular signal-regulated kinase (ERK) limb, and Smad signalings were evaluated by Western blotting and immunohistochemistry. Cytotoxicity of genipin was evaluated using a commercial colorimetric assay kit for nuclear matrix protein 41/7 (NMP41/7) in culture medium.. Genipin suppressed cell proliferation and migration in association with inhibition of Smad and p38 MAPK phosphorylation, although ERK signaling was enhanced. Genipin suppressed mRNA expression of TGF-beta1 and CTGF. Cytoplasmic fiber formation declined based on less intense alpha-SMA immunocytochemical staining. However, alpha-SMA protein expression was actually not altered. This negative result suggests that genipin attenuated formation of alpha-SMA-containing cytoskeleton. Treatment of the cells with genipin for 48 hours did not increase the release of NMP41/7 to the medium, indicating this compound is not cytotoxic.. Because genipin suppressed alpha-TN4 lens cell fibrogenic behaviors, it may be of therapeutic value in preventing PCO.

Topics: Actins; Antigens, Nuclear; Blotting, Western; Cell Cycle Proteins; Cell Line; Cell Movement; Cell Proliferation; Connective Tissue Growth Factor; Drugs, Chinese Herbal; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Humans; Immediate-Early Proteins; Immunohistochemistry; Intercellular Signaling Peptides and Proteins; Iridoid Glycosides; Iridoids; Lens, Crystalline; Nuclear Matrix-Associated Proteins; p38 Mitogen-Activated Protein Kinases; Pyrans; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad Proteins; Transforming Growth Factor beta; Transforming Growth Factor beta1; Up-Regulation; Wound Healing

Inchin-ko-to is a herbal medicine which has therapeutic effects in ameliorating liver fibrosis or cholestatic liver diseases. Its main bioactive component is genipin, which is an intestinal bacterial metabolite of this medication. Accordingly, we determined whether or not Inchin-ko-to suppresses in a wound healing model subconjunctival fibroblast (SCF) migration proliferation and myofibroblast transdifferentiation since an inhibitory effect could be of value in improving trabeculotomy outcome.. Effects of genipin on SCF cell migration were examined subsequent to wounding confluent monolayer cultures. Alamar blue staining evaluated the effects of genipin (0-50 microg/ml) on fibroblast cell proliferation. Immunostaining determined alpha-smooth muscle actin (alphaSMA) expression. Western blotting evaluated (alphaSMA) expression and phospho-Smad2 formation. Real-time RT-PCR evaluated TGFbeta1 and collagen Ialpha2 mRNA expression. Enzyme-immunoassay determined culture medium collagen I content.. Genipin suppressed wound-induced cell migration and proliferation. It also decreased collagen type I TGFbeta1 and alphaSMA mRNA and protein expression. Smad2 signaling was inhibited by genipin in a dose-dependent manner.. Genipin suppresses injury-induced fibrogenic responses in SCFs. This result suggests that the herbal medicine Inchin-ko-to might have therapeutic value following trabeculotomy.

Topics: Actins; Blotting, Western; Cell Differentiation; Cell Movement; Cell Proliferation; Cells, Cultured; Collagen Type I; Conjunctiva; Drugs, Chinese Herbal; Fibroblasts; Humans; Iridoid Glycosides; Iridoids; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Smad2 Protein; Transforming Growth Factor beta1; Wound Healing

A biodegradable composite which was composed of genipin cross-linked gelatin mixing with tricalcium phosphate ceramic particles (GGT) was developed as a bone substitute. This study was evaluated by the biological response of rabbit calvarial bone to assess the potential of the GGT composite as a biodegradable and osteoconductive bone substitute. Eighteen New Zealand white rabbits were used for cranial implantation. Bone defects (15 x 15 mm) of nine rabbits were filled with the GGT composites, while the others were filled with the de-proteinized bovine bones as controls. Three rabbits were examined for each group in every time period at 4, 8 and 12 weeks post-surgery. The assessment included serial post-operative gross examinations, radiographic analyses and histological evaluations. This study demonstrated that this composite is: (1) malleable, with easily molded to the calvarial bone defect without fracture; (2) biocompatible, with no evidence of adverse tissue reaction; (3) osteoconductive, with progressive growth of new bone into the calvarial bone defect; (4) biodegradable, with progressive replacement of the composite by new bone. Additionally, results of both radiographic analyses and histological evaluations revealed obviously greater new bone ingrowth in the GGT composite compared with the de-proteinized bovine bone at the same implantation time. Therefore, the GGT composite could serve as a useful bone substitute for repairing bone defects.

Topics: Animals; Biocompatible Materials; Bone and Bones; Bone Regeneration; Bone Substitutes; Brain; Calcium Phosphates; Cross-Linking Reagents; Female; Gelatin; Iridoid Glycosides; Iridoids; Microscopy, Electron, Scanning; Osteoblasts; Pyrans; Rabbits; Radiography; Time Factors

We evaluated peripheral nerve regeneration using a biodegradable nerve conduit, which was made of genipin-cross-linked gelatin. The genipin-cross-linked gelatin conduit (GGC) was dark blue in appearance, which was concentric and round with a rough outer surface whereas its inner lumen was smooth. After subcutaneous implantation on the dorsal side of the rat, the GGC only evoked a mild tissue response, forming a thin tissue capsule surrounding the conduit. Biodegradability of the GGC and its effectiveness as a guidance channel were examined as it was used to repair a 10 mm gap in the rat sciatic nerve. As a result, tube fragmentation was not obvious until 6 weeks post-implantation and successful regeneration through the gap occurred in all the conduits at the three experimental periods of 4, 6, and 8 weeks. Histological observation showed that numerous regenerated nerve fibers, mostly unmyelinated and surrounded by Schwann cells, crossed through and beyond the gap region 6 weeks after operation. Peak amplitude and area under the muscle action potential curve both showed an increase as a function of the recovery period, indicating that the nerve had undergone adequate regeneration. Thus, the GGC can not only be an effective aids for regenerating nerves but can also lead to favorable nerve functional recovery.

Topics: Absorbable Implants; Animals; Biocompatible Materials; Cross-Linking Reagents; Gelatin; Guided Tissue Regeneration; Iridoid Glycosides; Iridoids; Materials Testing; Nerve Regeneration; Pyrans; Rats; Rats, Sprague-Dawley; Sciatic Neuropathy; Surface Properties; Treatment Outcome; Wound Healing

Thermosensitive polymer hydrogels that undergo a sol-to-gel transition in response to temperature changes are of great interest in therapeutic delivery and tissue engineering as injectable depot systems. A chitosan-based, injectable thermogel was prepared by grafting an appropriate amount of PEG onto the chitosan backbone and studied for drug release in vitro using bovine serum albumin (BSA) as a model protein. When more than approximately 40 wt.% of PEG was grafted to chitosan chains via covalent bonding, the aqueous solution of the resultant copolymer was an injectable liquid at low temperature and transformed to a semisolid hydrogel at body temperature. After an initial burst release in the first 5 h, a steady linear release of protein from the hydrogel was achieved for a period of approximately 70 h. Prolonged quasi-linear release of protein up to 40 days was achieved by crosslinking the hydrogel with genipin in situ, in a fashion suitable for protein encapsulation while maintaining the injectability of the hydrogel. The crosslinkage transformed the copolymer from a physical gel to an insoluble chemical gel and substantially reduced the initial burst release of protein. Both high performance liquid chromatography (HPLC) and gel electrophoresis indicated that the primary structure of BSA released from the hydrogels with or without genipin-crosslinking was generally conserved. The hydrogel can be prepared in solutions with a physiological pH, allowing the safe incorporation of bioactive molecules for a broad range of medical applications, particularly for sustained in vivo drug release and tissue engineering.

Topics: Chitosan; Chromatography, High Pressure Liquid; Cross-Linking Reagents; Delayed-Action Preparations; Electrophoresis, Polyacrylamide Gel; Excipients; Freeze Drying; Hot Temperature; Hydrogels; Iridoid Glycosides; Iridoids; Microscopy, Electron, Scanning; Polyethylene Glycols; Proteins; Pyrans; Serum Albumin, Bovine; Spectroscopy, Fourier Transform Infrared; Viscosity

The primary challenge for tissue engineering is to develop a vascular supply that can support the metabolic needs of the engineered tissues in an extracellular matrix. In this study, the feasibility of using a natural compound, ginsenoside Re, isolated from Panax ginseng in stimulating angiogenesis and for tissue regeneration was evaluated.. Effects of ginsenoside Re on the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) were examined in vitro. Additionally, angiogenesis and tissue regeneration in a genipin-fixed porous acellular bovine pericardium (extracellular matrix; ECM) incorporated with ginsenoside Re implanted subcutaneously in a rat model were investigated. Basic fibroblast growth factor (bFGF) was used as a control.. It was found that HUVEC proliferation, migration in a Transwell plate, and tube formation on Matrigel were all significantly enhanced in the presence of bFGF or ginsenoside Re. Additionally, effects of ginsenoside Re on HUVEC proliferation, migration, and tube formation were dose-dependent and reached a maximal level at a concentration of about 30 microg/ml. The in vivo results obtained at 1 week postoperatively showed that the density of neocapillaries and the tissue hemoglobin content in the ECMs were significantly enhanced by bFGF or ginsenoside Re. These results indicated that angiogenesis in the ECMs was significantly enhanced by loading with bFGF or ginsenoside Re. At 1 month postoperatively, vascularzied neo-connective-tissue fibrils were found to fill the pores in the ECMs loaded with bFGF or ginsenoside Re.. The aforementioned results indicated that like bFGF, ginsenoside Re-associated induction of angiogenesis enhanced tissue regeneration, supporting the concept of therapeutic angiogenesis in tissue-engineering strategies.

Topics: Angiogenesis Inducing Agents; Animals; Cattle; Cell Line; Cell Movement; Cell Proliferation; Extracellular Matrix; Fibroblast Growth Factor 2; Fixatives; Ginsenosides; Humans; Iridoid Glycosides; Iridoids; Male; Panax; Pericardium; Pyrans; Rats; Rats, Wistar; Staining and Labeling; Tissue Engineering

This study was to evaluate the feasibility of using an acellular bovine pericardium fixed with genipin (AGP) to repair an abdominal wall defect created in a rat model.. The glutaraldehyde-fixed acellular pericardium (AGA), the genipin-fixed cellular pericardium (GP), and a commercially available polypropylene mesh were used as controls.. Gross examination at 3-month post-operatively revealed that dense adhesions to the visceral organs were observed for the polypropylene mesh and the AGA patch, while a filmy to dense adhesion was seen for the GP patch. In contrast, no adhesion to the visceral organs was observed for the AGP patch. Histologically, inflammatory cells were found mainly surrounding the GP patch. In contrast, host cells (inflammatory cells, fibroblasts, and neo-capillaries) were able to infiltrate into the AGA and AGP patches. Unlike the AGA patch, the AGP patch retrieved at 1-month post-operatively became well integrated with the host tissue near the suture line. Additionally, there were some mesothelial cells, identified by the van Gieson stain, observed on the AGP patch. At 3-month post-operatively, a neo-peritoneum was observed on the AGP patch. The neo-peritoneum consisted of organized vascularized connective tissues covered by an intact layer of mesothelial cells. The calcium contents of the polypropylene mesh and the AGA patch increased significantly at 3-month post-operatively, while those of the GP and AGP patches stayed minimal throughout the entire course of the study.. The results obtained in the study revealed that the AGP patch effectively repaired abdominal wall defects in rats and successfully prevented the formation of post-surgical abdominal adhesions.

Topics: Abdomen; Abdominal Wall; Animals; Bioprosthesis; Calcium; Cattle; Cell-Free System; Cross-Linking Reagents; Feasibility Studies; Fixatives; Glutaral; Iridoid Glycosides; Iridoids; Male; Pericardium; Peritoneum; Polypropylenes; Pyrans; Rats; Rats, Wistar; Regeneration; Surgical Mesh; Tensile Strength; Tissue Adhesions; Tissue Fixation

Chronic rejection of arterial xenografts results in aneurysmal dilation, due to immune mediated processes. To minimize the immunologic degradation of the graft, a cell-extraction process employing sodium dodecyl sulfate (SDS) was used in the study to remove the cellular components in bovine carotid arteries. To further reduce their immunogenicity, the acellular arteries were fixed with glutaraldehyde (A-GA) or genipin (A-GP). The in vitro properties of all test samples were analyzed. Additionally, the in vivo performance of the heparinized A-GA and A-GP grafts (H-A-GA and H-A-GP) was evaluated in a canine model. It was found that the SDS treatment effectively removed cells from the arterial wall, but the main structures of the extracellular matrix were preserved with a portion of the water-soluble glycosaminoglycans removed. After cell extraction, the elastic lamellae in the media became straightened, and thus made the tissue less extensile. The heparinized tissues significantly reduced platelet adhesion. At retrieval, all implanted grafts were patent and not dilated. Chronic inflammatory response surrounding the implants was observed. However, fixation of acellular tissues by glutaraldehyde or genipin inhibited immune cell penetration into the media and limited tissue degradation, and therefore prevented the arterial wall from dilation. Nevertheless, the H-A-GP graft was superior to the H-A-GA graft in completeness of endothelialization on its luminal surface, and thus precluded thrombus formation.

Topics: Animals; Biomechanical Phenomena; Biotechnology; Blood Vessel Prosthesis; Cattle; Cross-Linking Reagents; Dogs; Fixatives; Glutaral; In Vitro Techniques; Iridoid Glycosides; Iridoids; Microscopy, Electron; Platelet Adhesiveness; Pyrans; Tissue Fixation; Transplantation, Heterologous

This study investigates the fluorogenic characteristics of the chitosan-genipin reaction for applications in microencapsulation research. Results showed that the chitosan-genipin reaction generated a colored and fluorescent product, with optimal excitation and emission wavelengths at 369 and 470 nm, respectively. Furthermore, it was found that reaction conditions affected the fluorescence intensity of the product. Mixture at the ratio of 4:1 (chitosan: genipin by weight) fluoresced the most. It also fluoresced stronger if the reaction occurred at higher temperature, with the intensity of 10.4 x 10(5) CPS at 37 degrees C, 5.9 x 10(5) CPS at 20 degrees C, and 2.5 x 10(5) CPS at 4 degrees C. As well, the fluorescence of the mixture developed gradually over time, attaining the emission maxima of 2.9 x 10(5), 7.6 x 10(5), and 10.0 x 10(5) CPS in 1, 6, and 18 h, respectively. Chitosan-coated alginate microcapsules were prepared without prior labeling, to which subsequent genipin treatment was applied in order to examine the potential of using genipin in microcapsule characterization. Chitosan bound to the alginate beads interacted with genipin, from which the resultant fluorescent signals allowed for clear visualization of the chitosan coating under confocal laser scanning microscopy. The relative fluorescence intensity across the chitosan membrane was found to be considerably higher than the controls (175 vs. 50). The membrane thickness measured was 29.2 +/- 7.3 microm. These findings demonstrate a convenient and effective way of characterizing chitosan-based microcapsules using genipin as a fluorogenic marker, a technique that will be useful in microcapsule research and other biomedical applications.

Topics: Capsules; Chitosan; Coated Materials, Biocompatible; Fluorescent Dyes; Iridoid Glycosides; Iridoids; Microscopy, Confocal; Pyrans; Spectrometry, Fluorescence; Spectrophotometry

Genipin, the aglycone of geniposide, exhibits anti-inflammatory and anti-angiogenic activities. Here we demonstrate that genipin induces apoptotic cell death in FaO rat hepatoma cells and human hepatocarcinoma Hep3B cells, detected by morphological cellular changes, caspase activation and release of cytochrome c. During genipin-induced apoptosis, reactive oxygen species (ROS) level was elevated, and N-acetyl-l-cysteine (NAC) and glutathione (GSH) suppressed activation of caspase-3, -7 and -9. Stress-activated protein kinase/c-Jun NH2-terminal kinase 1/2(SAPK/JNK1/2) but neither MEK1/2 nor p38 MAPK was activated in genipin-treated hepatoma cells. SP600125, an SAPK/JNK1/2 inhibitor, markedly suppressed apoptotic cell death in the genipin-treated cells. The FaO cells stably transfected with a dominant-negative c-Jun, TAM67, was less susceptible to apoptotic cell death triggered by genipin. Diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, inhibited ROS generation, apoptotic cell death, caspase-3 activation and JNK activation. Consistently, the stable expression of Nox1-C, a C-terminal region of Nox1 unable to generate ROS, blocked the formation of TUNEL-positive apoptotic cells, and activation of caspase-3 and JNK in FaO cells treated with genipin. Our observations imply that genipin signaling to apoptosis of hepatoma cells is mediated via NADPH oxidase-dependent generation of ROS, which leads to downstream of JNK.

Topics: Animals; Apoptosis; Carcinoma, Hepatocellular; Caspases; Cell Line; Humans; Iridoid Glycosides; Iridoids; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Mitochondria; Mitogen-Activated Protein Kinase 8; NADPH Oxidases; Pyrans; Rats; Reactive Oxygen Species

Nonliving synthetic materials have been widely used to repair myocardial defects; however, material-related failures do occur. To overcome these problems, an acellular bovine pericardium with a porous structure fixed with genipin (the AGP patch) was developed.. The AGP patch was used to repair a surgically created myocardial defect in the right ventricle of a rat model. A commercially available expanded polytetrafluoroethylene (e-PTFE) patch was used as a control. At retrieval, a computerized mapping system was used to acquire local epicardial electrograms of each implanted sample, and the appearance of each retrieved sample was grossly examined. The retrieved samples were then processed for histologic examination.. The amplitude of local electrograms on the AGP patch increased significantly with increasing implantation duration, whereas only low-amplitude electrograms were observed on the e-PTFE patch throughout the entire course of the study. No aneurysmal dilation of the implanted patches was seen for either studied group. Additionally, no tissue adhesion was observed on the outer (epicardial) surface of the AGP patch, whereas a moderate tissue adhesion was observed on the e-PTFE patch. On the inner (endocardial) surface, intimal thickening was observed for both studied groups; however, no thrombus formation was found. Intact layers of endothelial and mesothelial cells were identified on the inner and outer surfaces of the AGP patch, respectively. At 4 weeks postoperatively, smooth muscle cells, together with neomuscle fibers (with a few neocollagen fibrils), neoglycosaminoglycans, and neocapillaries, were observed to fill the pores in the AGP patch, an indication of tissue regeneration. These observations were more pronounced at 12 weeks postoperatively. In contrast, no apparent tissue regeneration was observed in the e-PTFE patch.. The present study indicated that the AGP patch holds promise to become a suitable patch for surgical repair of myocardial defects.

Topics: Animals; Bioprosthesis; Cattle; Electrocardiography; Heart Ventricles; Implants, Experimental; Iridoid Glycosides; Iridoids; Pericardium; Polytetrafluoroethylene; Porosity; Pyrans; Rats; Regeneration; Tissue Engineering

In the study, a novel drug-eluting stent for treating the coronary arterial stenosis was developed. Using a spray-coating method, aqueous bovine type I collagen and sirolimus were coated layer-by-layer alternatively onto the surface of a metallic stent and a topcoat of collagen was used as a barrier to control drug release. To prevent dissolution of the collagen matrices, the spray-coated collagen was further crosslinked by genipin, a naturally occurring crosslinking agent. The results obtained in the atomic force microscopy (AFM) examination suggested that the spray-coated collagen was tightly adhered to the surface of the stent. Additionally, the collagen coating was demonstrated by the scanning electron microscopy (SEM) to be sufficiently flexible to allow balloon expansion of the stent without cracking or peeling from the wire. The resistance against enzymatic degradation and the hemocompatibility of the collagen matrices increased significantly as their degree of crosslinking increased. All the studied sirolimus-loaded stents exhibited a nearly linear sustained-release profile (except at the end stage of release) with no significant burst releases. It was found that a topcoat of collagen on the collagen/sirolimus coated stent did slow down the release of sirolimus to some extent. Additionally, the number of layers of collagen/sirolimus coated significantly affected the duration of sirolimus released. Furthermore, the sustained-release duration of sirolimus was proportional to the actual amount of drug loaded on the stent. The aforementioned results indicated that the drug-eluting stent developed had a tightly adhered collagen coating and can be used as a drug reservoir to sustain release of sirolimus.

Topics: Animals; Coated Materials, Biocompatible; Collagen; Cross-Linking Reagents; Delayed-Action Preparations; Iridoid Glycosides; Iridoids; Materials Testing; Molecular Structure; Platelet Adhesiveness; Pyrans; Sirolimus; Solubility; Stents; Surface Properties

A two-component pH-sensitive hydrogel system composed of a water-soluble chitosan derivative (N,O-carboxymethyl chitosan, NOCC) and alginate cross-linked by genipin, glutaraldehyde or Ca2+ was investigated. Preparation and structures of these hydrogels and their swelling characteristics and release profiles of a model protein drug (bovine serum albumin, BSA) in simulated gastrointestinal media are reported. At pH 1.2, the swelling ratios of the hydrogels cross-linked by distinct methods were limited. Of note is that the lowest swelling ratios of test hydrogels were found at pH 4.0. At pH 7.4, the carboxylic acid groups on test hydrogels became progressively ionized and led to a significant swelling. There was barely any BSA released from the glutaraldehyde-cross-linked hydrogel throughout the entire course of the study. The amounts of BSA released at pH 1.2 from the genipin- and Ca(2+)-cross-linked hydrogels were relatively low (approx. 20%). At pH 4.0, there was still significant BSA release from the Ca(2+)-cross-linked hydrogel, while the cumulative BSA released from the genipin-cross-linked hydrogel was limited due to its shrinking behavior. At pH 7.4, the amount of BSA released from the genipin- and Ca(2+)-cross-linked hydrogels increased significantly (approx. 80%) because the swelling of both test hydrogels increased considerably. The aforementioned results indicated that the swelling behaviors and drug-release profiles of these test hydrogels are significantly different due to their distinct cross-linking structures.

Topics: Alginates; Animals; Biocompatible Materials; Calcium; Carboxylic Acids; Cattle; Chitosan; Cross-Linking Reagents; Drug Carriers; Drug Delivery Systems; Glucuronic Acid; Glutaral; Hexuronic Acids; Hydrogel, Polyethylene Glycol Dimethacrylate; Hydrogels; Hydrogen-Ion Concentration; Iridoid Glycosides; Iridoids; Models, Chemical; Pyrans; Serum Albumin, Bovine; Spectroscopy, Fourier Transform Infrared; Time Factors

Genipin, a hydrolytic product of geniposide extracted from gardenia fruit, was thoroughly studied as a potential fingerprint reagent, and optimal conditions for fingerprint development have been determined. Latent fingerprints on paper items that have been treated with a non-ink running formulation containing 0.17% of the reagent, showed up as both colored and fluorescent images. On brown wrapping paper and on papers with highly luminescent backgrounds, genipin developed more visible and clearer prints than did classical reagents such as ninhydrin or DFO. Another potential advantage of genipin is that it is totally harmless and an environmentally friendly reagent.

Topics: Aza Compounds; Dermatoglyphics; Humans; Humidity; Hydrogen-Ion Concentration; Indicators and Reagents; Iridoid Glycosides; Iridoids; Ninhydrin; Paper; Pyrans; Solvents; Spectrometry, Fluorescence; Temperature

Inchin-ko-to (ICKT), an herbal medicine, and its ingredients exert potent choleretic effects by a "bile acid-independent" mechanism. The current study was designed to determine whether ICKT or its ingredients potentiate multidrug resistance-associated protein 2 (Mrp2; Abcc2)-mediated choleresis in vivo. Biliary secretion of Mrp2 substrates and the protein mass, subcellular localization, and messenger RNA (mRNA) level of Mrp2 were assessed in rat liver after infusion of genipin, an intestinal bacterial metabolite of geniposide, a major ingredient of ICKT. The function of Mrp2 was also assessed by the adenosine triphosphate (ATP)-dependent uptake of Mrp2-specific substrates using canalicular membrane vesicles (CMVs) from the liver. Infusion of genipin increased bile flow by 230%. It also increased biliary secretion of bilirubin conjugates and reduced glutathione (GSH) by 513% and 336%, respectively, but did not increase bile acid secretion. The ATP-dependent uptake of estradiol 17-beta-D-glucuronide (E(2)17 beta G; by 265%), leukotriene C4 (LTC(4); by 161%), taurolithocholate-3-sulfate (TLC-3S; by 266%), and methotrexate (MTX; by 234%) was significantly stimulated in the CMVs from the liver. These effects were not observed in Mrp2-deficient rats. Under these conditions, genipin treatment increased the protein mass of Mrp2 in the CMVs but not the mRNA level. In immunoelectron microscopic studies, a marked increase in Mrp2 density in the canalicular membrane (CM) and microvilli was observed in the genipin-treated liver tissue sections when compared with the vehicle-treated liver tissue sections. In conclusion, genipin may enhance the bile acid-independent secretory capacity of hepatocytes, mainly by stimulation of exocytosis and insertion of Mrp2 in the bile canaliculi. ICKT may be a potent therapeutic agent for a number of cholestatic liver diseases.

Topics: Actin Cytoskeleton; Adenosine Triphosphate; Administration, Oral; Animals; Anions; ATP Binding Cassette Transporter, Subfamily B, Member 11; ATP-Binding Cassette Transporters; Bile; Cholagogues and Choleretics; Drugs, Chinese Herbal; Estradiol; Gene Expression; In Vitro Techniques; Iridoid Glycosides; Iridoids; Liver; Male; Membrane Transport Proteins; Multidrug Resistance-Associated Protein 2; Multidrug Resistance-Associated Proteins; Phalloidine; Pyrans; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tritium

It was reported that acellular biological tissues can provide a natural microenvironment for host cell migration and may be used as a scaffold for tissue regeneration. To reduce antigenicity, biological tissues have to be fixed with a crosslinking agent before implantation. As a tissue-engineering scaffold, it is speculated that the crosslinking degree of an acellular tissue may affect its tissue regeneration pattern. In the study, a cell extraction process was employed to remove the cellular components from bovine pericardia. The acellular tissues then were fixed with genipin at various known concentrations to obtain varying degrees of crosslinking. It was shown in the in vitro degradation study that after fixing with genipin, the resistance against enzymatic degradation of the acellular tissue increased significantly with increasing its crosslinking degree. In the in vivo subcutaneous study, it was found that cells (inflammatory cells, fibroblasts, endothelial cells, and red blood cells) were able to infiltrate into acellular tissues. Generally, the depth of cell infiltration into the acellular tissue decreased with increasing its crosslinking degree. Infiltration of inflammatory cells was accompanied by degradation of the acellular tissue. Due to early degradation, no tissue regeneration was observed within fresh (without crosslinking) and the 30%-degree-crosslinking acellular tissues. This is because the scaffolds provided by these two samples were already completely degraded before the infiltrated cells began to secrete their own extracellular matrix. In contrast, tissue regeneration (fibroblasts, neo-collagen fibrils, and neo-capillaries) was observed for the 60%- and 95%-degree-crosslinking acellular tissues by the histological examination, immunohistological staining, transmission electron microscopy, and denaturation temperature measurement. The 95%-degree-crosslinking acellular tissue was more resistant against enzymatic degradation than its 60%-degree-crosslinking counterpart. Consequently, tissue regeneration was limited in the outer layer of the 95%-degree-crosslinking acellular tissue throughout the entire course of the study (1-year postoperatively), while tissue regeneration was observed within the entire sample for the 60%-degree-crosslinking acellular tissue. In conclusion, the crosslinking degree determines the degradation rate of the acellular tissue and its tissue regeneration pattern.

Topics: Animals; Biocompatible Materials; Cattle; Cell Extracts; Cell-Free System; Connective Tissue; Cross-Linking Reagents; Extracellular Matrix; Foreign-Body Reaction; Iridoid Glycosides; Iridoids; Male; Materials Testing; Pericardium; Porosity; Prostheses and Implants; Pyrans; Rats; Rats, Wistar; Regeneration; Surface Properties; Tensile Strength; Tissue Engineering

Genipin, the hydrolytic product of geniposide, which is extracted from gardenia fruit, shows good potential as a fingerprint reagent. It develops latent fingerprints on paper as blue impressions with good contrast and resolution. Even very faint impressions that are barely visible in ambient light will fluoresce brightly upon illumination at ca. 590 nm and are best viewed with a barrier filter above 630 nm. Potential advantages of genipin are the combination of colorimetric and fluorogenic activity in one reagent as well as its being a safe and environmentally friendly natural product.

Topics: Dermatoglyphics; Fluorescent Dyes; Forensic Medicine; Humans; Indicators and Reagents; Iridoid Glycosides; Iridoids; Molecular Structure; Paper; Pyrans; Spectrometry, Fluorescence

We have demonstrated previously that a natural iridoid compound, genipin, induced neuritogenesis through activation of nitric oxide synthase (NOS) and mitogen-activated protein kinase (MAPK) in PC12h cells. In this paper, we investigated whether cyclic GMP (cGMP) and cGMP-dependent protein kinase (PKG) are involved in the neuritogenesis as a result of NOS activation. Furthermore, we also investigated the relationship between cGMP and MAPK activation in the signaling pathway. The genipin-induced neuritogenesis accompanied by induction of neurofilament was significantly inhibited by 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one (ODQ) and KT5823, inhibitors of soluble guanylate cyclase and PKG, respectively. Genipin-induced MAPK phosphorylation was also abolished by ODQ. These inhibitory effects of ODQ were similar to those observed for nerve growth factor (NGF)-induced neurite outgrowth and MAPK phosphorylation. The membrane-permeable cGMP analog, 8-Bromo-cGMP, had prominent neuritogenic activity, which was completely inhibited by a MAPK kinase inhibitor, PD98059. These results suggest that the soluble guanylate cyclase-PKG signaling pathway is important for MAPK activation by genipin as well as NGF during neuritogenesis in PC12h cells.

Topics: Animals; Blotting, Western; Cell Differentiation; Cyclic GMP; Cyclic GMP-Dependent Protein Kinases; Electrophoresis, Polyacrylamide Gel; Guanylate Cyclase; Iridoid Glycosides; Iridoids; Mitogen-Activated Protein Kinases; Nerve Growth Factors; Neurites; Neurofilament Proteins; Nitric Oxide; PC12 Cells; Phosphorylation; Pyrans; Rats; Signal Transduction

It was noted in our previous study that acellular tissues can provide a natural microenvironment for host cell migration and proliferation to accelerate tissue regeneration. The purpose of this study was to further investigate the tissue regeneration patterns in acellular bovine pericardia fixed with glutaraldehyde or genipin as a biological patch to repair a defect in the pulmonary trunk in a canine model. The implanted samples were retrieved at distinct durations postoperatively. The structural remodeling of retrieved samples was then examined. It was found that the degree of inflammatory reaction observed for the genipin-fixed acellular patch was significantly less than its glutaraldehyde-fixed counterpart. At 1 month postoperatively, intimal thickening was found on the inner surfaces of both studied groups. The intimal thickening observed on the glutaraldehyde-fixed acellular patch was significantly thicker than its genipin-fixed counterpart. An intact layer of endothelial cells was found on the intimal thickening of the genipin-fixed acellular patch, whereas endothelial cells did not universally and totally cover the entire surface of the glutaraldehyde-fixed acellular patch. Additionally, fibroblasts with neocollagen fibrils and myofibroblasts were observed in the acellular patches for both studied groups, an indication of tissue regeneration. This phenomenon was more prominent for the genipin-fixed acellular patch than its glutaraldehyde-fixed counterpart. At 6 months postoperatively, foci of chondroid and/or bony metaplasia were found in each retrieved sample for both studied groups. The observed adverse response of chondroid metaplasia may be attributed to a compliance mismatch at the implanted site of the canine pulmonary trunk after implantation or a lack of angiogenesis in the regenerated tissue observed at 1 month postoperatively. Bony metaplasia may then develop as in other chondroid tissues. It was reported that ischemia is a usual cause of metaplasia.

Topics: Animals; Calcium; Cattle; Dogs; Fixatives; Glutaral; Iridoid Glycosides; Iridoids; Pericardium; Pulmonary Artery; Pyrans; Regeneration; Temperature; Time Factors

A novel pH-sensitive hydrogel system composed of a water-soluble chitosan derivative (N,O-carboxymethyl chitosan, NOCC) and alginate blended with genipin was developed for controlling protein drug delivery. Genipin, a naturally occurring cross-linking agent, is significantly less cytotoxic than glutaraldehyde and may provide a less extent of cross-linking to form a semiinterpenetrating polymeric network (semi-IPN) within the developed hydrogel system. The drug-loading process used in the study was simple and mild. All procedures used were performed in aqueous medium at neutral environment. In the study, preparation of the NOCC/alginate-based hydrogels was reported. Swelling characteristics of these hydrogels as a function of pH values were investigated. Additionally, release profiles of a model protein drug (bovine serum albumin, BSA) from test hydrogels were studied in simulated gastric and intestinal media. The semi-IPN formation of the genipin-cross-linked NOCC/alginate hydrogel was confirmed by means of the scanning electron microscopy-energy dispersive X-ray spectrometer (SEM-EDS) and the ninhydrin assays. The percentage of decrease of free amino groups and cross-linking density for the NOCC/alginate hydrogel cross-linked with 0.75 mM genipin were 18% and 26 mol/m(3), respectively. At pH 1.2, the swelling ratio of the genipin-cross-linked NOCC/alginate hydrogel was limited (2.5) due to formation of hydrogen bonds between NOCC and alginate. At pH 7.4, the carboxylic acid groups on the genipin-cross-linked NOCC/alginate hydrogel became progressively ionized. In this case, the hydrogel swelled more significantly (6.5) due to a large swelling force created by the electrostatic repulsion between the ionized acid groups. The amount of BSA released at pH 1.2 was relatively low (20%), while that released at pH 7.4 increased significantly (80%). The results clearly suggested that the genipin-cross-linked NOCC/alginate hydrogel could be a suitable polymeric carrier for site-specific protein drug delivery in the intestine.

Topics: Alginates; Chitosan; Cross-Linking Reagents; Delayed-Action Preparations; Drug Carriers; Glucuronic Acid; Hexuronic Acids; Hydrogels; Hydrogen Bonding; Hydrogen-Ion Concentration; Iridoid Glycosides; Iridoids; Magnetic Resonance Spectroscopy; Microscopy, Electron, Scanning; Proteins; Pyrans; Serum Albumin, Bovine; Spectroscopy, Fourier Transform Infrared; Time Factors

A biodegradable composite (GGT) containing tricalcium phosphate ceramic particles and genipin crosslinked gelatin was developed for use as a bone substitute. The objective of this study was to assess the biocompatibility and the osteoconductivity of the GGT composite on new bone formation in vitro. Additionally, biodegradation and biocompatibility of the GGT composite in animals were investigated. Results of the GGT composites cocultured with osteoblasts showed that the concentration of genipin used as a crosslinking agent should be <0.5 wt % to avoid cytotoxicity. For in vivo degradation studies, we found that when the concentration of genipin in the composite <0.5 wt % was not enough to fully crosslink the gelatin, it results in a rapid degradation of the gelatin-genipin mixture. However, we also found that the foreign body capsule surrounding the GGT composite containing 1.0 wt % of the genipin was much thicker than that in the other three groups, that is, the composites containing 0.05, 0.1, and 0.5 wt % of the genipin. We therefore concluded that the ideal concentration of genipin used in the GGT was 0.5 wt %. Finally, we examined the organ culture units, which were maintained in cultured medium for 5 weeks. Morphology of tissue was observed and the quantitative evaluation of the regenerated bone was determined. We found that the GGT composites containing 0.5 wt % of the genipin had an excellent biocompatibility and could produce osteoconduction for the regenerating bone tissues.

Topics: Animals; Bone Substitutes; Calcium Phosphates; Cattle; Coculture Techniques; Gelatin; Iridoid Glycosides; Iridoids; Osteoblasts; Pyrans; Rats; Skull

Application of combining herbal medicine and biomedical material science to nerve regeneration is a new approach. In this study, we describe a novel use of purified genipin, which can be extracted from Gardenia jasminoides Ellis, fixing the gelatin to be an extracellular matrix for peripheral nerve regeneration. A 10-mm gap of rat sciatic nerve was created between the proximal and distal nerve stumps, which were sutured into silicone rubber tubes filled with either the genipin-fixed gelatin or collagen gel. Silicone rubber tubes filled with saline were used as controls. Six weeks after implantation, regeneration across the nerve gaps occurred in 80 and 90% of the animals from the groups of genipin-fixed gelatin and collagen, respectively, whereas only 30% in the control group. Large numbers of myelinated axons were also seen in the genipin-fixed gelatin (5104 +/- 3278) and the collagen groups (8063 +/- 1807). These findings indicated that the genipin-fixed gelatin could be an acceptable extracellular matrix for nerve regeneration.

Topics: Animals; Extracellular Matrix; Gelatin; Iridoid Glycosides; Iridoids; Male; Nerve Regeneration; Pyrans; Rats; Rats, Sprague-Dawley; Sciatic Nerve

Genipin, the aglycone of geniposide, is metabolically produced from the geniposide in body tissues. The purpose of this study is to clarify some pharmacological actions of genipin. Genipin showed concentration-dependent inhibition on lipid peroxidation induced by Fe++/ascorbate in rat brain homogenate. Genipin exhibited significant topical antiinflammatory effect shown as an inhibition of croton oil-induced ear edema in mice. Nitric oxide (NO) synthesis by inducible nitric oxide synthase (iNOS) is increased in inflammatory diseases and leads to cellular injury. Genipin concentration-dependently (50-300 microM) inhibited NO production and iNOS expression upon stimulation by lipopolysaccharide/interferon-gamma (IFN-gamma) in RAW 264.7, a murine macrophage cell line. Genipin markedly blocked lipopolysaccharide-evoked degradation of inhibitor-kappaB-beta (IkappaB-beta), indicating that it exhibits inhibitory effect on NO production through the inhibition of nuclear factor-kappaB (NF-kappaB) activation. It was also shown to contain potent antiangiogenic activity in a dose-dependent manner, which was detected by chick embryo chorioallantoic membrane assay. In summary, we demonstrate that genipin possesses antiinflammatory and is a specific hydroxyl radical scavenger. Its antiangiogenic and NO production-inhibitory properties are also presented.

Topics: Angiogenesis Inhibitors; Animals; Anti-Inflammatory Agents; Antioxidants; Brain; Cell Line; Cell Survival; Chorioallantoic Membrane; Dose-Response Relationship, Drug; Ear; Edema; Female; Gardenia; Immunoblotting; Interferon-gamma; Iridoid Glycosides; Iridoids; Lipid Peroxidation; Lipopolysaccharides; Macrophages; Male; Mice; Mice, Inbred ICR; Nitric Oxide; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Pyrans; Rats; Rats, Sprague-Dawley

A naturally occurring crosslinking agent, genipin, extracted from the fruits of Gardenia jasminoides ELLIS was used by our group to chemically modified biomolecules. Genipin and its related iridoid glucosides have been widely used as an antiphlogistic and cholagogue in herbal medicine. Our previous study showed that the cytotoxicity of genipin is significantly lower than glutaraldehyde. The study was to investigate the feasibility of using genipin to polymerize hemoglobin as a blood substitute. The results indicated that the rate of hemoglobin polymerization by glutaraldehyde was significantly faster than that by genipin and it readily produced polymers with molecular masses greater than 500,000 Da. It was found that the maximum degree of hemoglobin polymerization by genipin was approximately 40% if over-polymerization is to be prevented. With increasing the reaction temperature, hemoglobin concentration, and genipin-to-hemoglobin molar ratio, the duration taken to achieve the maximum degree of hemoglobin polymerization by genipin became significantly shorter. The P50 value of the unmodified hemoglobin was 9 mmHg, while that of the genipin-polymerized PLP-hemoglobin increased to 21 mmHg. It was found in a rat model that the genipin-polymerized PLP-hemoglobin resulted in a longer circulation time than the unmodified hemoglobin. In conclusion, the results of the study indicated that the genipin-polymerized hemoglobin solution has a lower oxygen affinity and a longer vascular retention time than the unmodified hemoglobin solution.

Topics: Animals; Biopolymers; Blood Substitutes; Chromatography, High Pressure Liquid; Cross-Linking Reagents; Exchange Transfusion, Whole Blood; Glycine; Hemoglobins; Iridoid Glycosides; Iridoids; Male; Methemoglobin; Molecular Structure; Pyrans; Pyridoxal Phosphate; Rats; Rats, Sprague-Dawley; Silicon Dioxide; Swine

Laser tissue soldering (LTS) is an alternative technique to suturing for tissue repair that avoids foreign body reaction and provides immediate sealing of the wound. One of the major drawbacks of LTS, however, is the weak tensile strength of the solder welds when compared to sutures. In this study, a crosslinking agent of low cytotoxicity was investigated for its ability to enhance the bond strength of albumin solders with sheep intestine.. Solder strips were welded onto rectangular sections of sheep small intestine using a diode laser. The laser delivered in continuous mode a power of 170 +/- 10 mW at lambda = 808 nm, through a multimode optical fiber (core size = 200 microm) to achieve a dose of 10.8 +/- 0.5 J/mg. The solder thickness and surface area were kept constant throughout the experiment (thickness = 0.15 +/- 0.01 mm, area = 12 +/- 1.2 mm2). The solder was composed of 62% bovine serum albumin (BSA), 0.38% genipin, 0.25% indocyanin green dye (IG), and water. Tissue welding was also performed with a BSA solder without genipin, as a control group. The repaired tissue was tested for tensile strength by a calibrated tensiometer. Murine fibroblasts were also cultured in extracted media from heat-denatured genipin solder to assess cell growth inhibition in a 48 hours period.. The tensile strength of the genipin solder was doubled that of the BSA solder (0.21 +/- 0.04 N and 0.11 +/- 0.04 N, respectively; P = 10(-15) unpaired t-test, N = 30). Media extracted from crosslinked genipin solder showed negligible toxicity to fibroblast cells under the culture conditions examined here.. Addition of a chemical crosslinking agent, such as genipin, significantly increased the tensile strength of adhesive-tissue bonds. A proposed mechanism for this enhanced bond strength is the synergistic action of mechanical adhesion with chemical crosslinking by genipin.

Topics: Adhesives; Albumins; Animals; Iridoid Glycosides; Iridoids; Laser Therapy; Pyrans; Sheep; Suture Techniques; Tensile Strength; Wound Healing

In this study amino-terminated poly(ethylene glycol) (PEG-diamine) hydrogels were crosslinked with genipin, a chemical naturally derived from the gardenia fruit. Dissolution, swelling, and PEG-genipin release properties were determined. The dissolution studies indicated that the hydrogels are water soluble, and that the dissolution rate was concentration, mass, and temperature dependent. The dissolution rates are easily tailored from 3 min to >100 days. The PEG-genipin release study indicated that the greatest release occurs within the first 24 h of immersion in water, and that incubation at 37 degrees C elicits a greater initial release than samples incubated at room temperature for all genipin concentrations. Through scanning electron microscopy it was observed that the hydrogels are porous, and surface morphology changes before and after swelling. Furthermore, smooth muscle cell (SMC) adhesion studies indicated that the PEG-genipin hydrogel is a suitable substrate for SMC seeding. Overall, the results of these studies indicate that PEG-genipin hydrogels may provide potential scaffolding for a variety of tissue engineering applications.

Topics: Adhesives; Animals; Aorta; Biocompatible Materials; Cell Adhesion; Hydrogel, Polyethylene Glycol Dimethacrylate; Iridoid Glycosides; Iridoids; Materials Testing; Microscopy, Electron, Scanning; Muscle, Smooth, Vascular; Polyethylene Glycols; Pyrans; Rats; Surface Properties; Tissue Engineering

Inchin-ko-to (TJ-135) is an herbal medicine used in Japan for treatment of icteric patients with cirrhosis. Its efficacy as an anti-fibrogenic drug was evaluated in relation to stellate cell activation.. Liver fibrosis was induced in rats by repeated injections of carbon tetrachloride (CCl4) or pig-serum. Oral administration of TJ-135 improved the mortality of rats given CCl4 with reduced extents of liver necrosis and fibrosis. Similar improvement of liver fibrosis was found in rats given pig-serum showing no liver necrosis. DNA synthesis of stellate cells activated in vitro after isolation from normal rat liver was decreased by culture with TJ-135 in a dose-related manner, accompanied by decreased smooth muscle alpha actin expression and contractility. Such attenuation was not found in the cells cultured with geniposide, an iridoid compound of TJ-135, but genipin, an aglycone of geniposide formed in the gut by action of bacterial flora, markedly decreased stellate cell activation without affecting synthesis of proteins other than collagen.. TJ-135 may be useful for treatment of liver fibrosis and portal hypertension through suppression of activated hepatic stellate cell function by genipin, an absorbed form of its component.

Topics: Animals; Carbon Tetrachloride; Cells, Cultured; Cholagogues and Choleretics; Drugs, Chinese Herbal; Iridoid Glycosides; Iridoids; Liver; Liver Cirrhosis, Experimental; Necrosis; Pyrans; Rats; Rats, Inbred F344; Survival Analysis; Swine

To evaluate the pharmacological actions of herbal medicines, metabolic activities of herbal medicine components, ginsenoside Rb1, glycyrrhizin, geniposide and baicalin to their bioactive compounds compound K, 18beta-glycyrrhetic acid, genipin and baicalein by fecal specimens were measured. Their metabolic activities were 646.1+/-591.4, 29.4+/-51.7, 926.3+/-569.6 and 3884.6+/-1400.1 micromol/h/g, respectively. The profiles of these metabolic activities of baicalin and ginsenoside Rb1 were not significantly different to those of water extracts of Scutellariae Radix and Ginseng Radix. None of the metabolic activities tested were different between males and females, or between ages. However, the difference in these metabolic activities in individuals was significant. These results suggest that the human intestinal microflora enzymes that convert herbal components to their bioactive compounds may be used as selection markers of responders to traditional medicines.

Topics: Adult; Chromatography, Thin Layer; Feces; Female; Flavanones; Flavonoids; Ginsenosides; Glycyrrhetinic Acid; Glycyrrhizic Acid; Humans; In Vitro Techniques; Iridoid Glycosides; Iridoids; Male; Panax; Plant Extracts; Pyrans; Scutellaria baicalensis; Stereoisomerism

The study was to investigate the crosslinking characteristics, mechanical properties, and resistance against enzymatic degradation of biological tissues after fixation with genipin (a naturally occurring crosslinking agent) and/or carbodiimide. Fresh tissue was used as a control. It was found that both genipin and carbodiimide are effective crosslinking agents for tissue fixation and genipin crosslinking is comparatively slower than carbodiimide crosslinking. Additionally, tissue fixation in genipin and/or carbodiimide may produce distinct crosslinking structures. Carbodiimide may form intrahelical and interhelical crosslinks within or between tropocollagen molecules, whereas genipin may further introduce intermicrofibrillar crosslinks between adjacent collagen microfibrils. The stability (denaturation temperature and resistance against enzymatic degradation) of the fixed tissue is mainly determined by its intrahelical and interhelical crosslinks. In contrast, intermicrofibrillar crosslinks significantly affect the mechanical properties (tissue shrinkage during fixation, tensile strength, strain at break, and ruptured pattern) of the fixed tissue. Moreover, the degree of enzymatic degradation of the fixed tissue may be influenced by three factors: the availability, to the enzyme, of recognizable cleavage sites, the degree of crosslinking, and the extent of helical integrity of tropocollagen molecules in tissue.

Topics: Animals; Carbodiimides; Collagen; Cross-Linking Reagents; Fixatives; Iridoid Glycosides; Iridoids; Molecular Structure; Pericardium; Protein Structure, Tertiary; Pyrans; Stress, Mechanical; Swine; Tensile Strength; Tissue Fixation

Gelatin microspheres have been widely evaluated as a drug carrier. Nevertheless, gelatin dissolves rather rapidly in aqueous environments, making the use of the polymer difficult for the production of long-term delivery systems. This adverse aspect requires the use of a crosslinking agent in forming nonsoluble networks in microspheres. However, the use of crosslinking agents such as formaldehyde and glutaraldehyde can lead to toxic side effects owing to residual crosslinkers. In an attempt to overcome this problem, a naturally occurring crosslinking agent (genipin) was used to crosslink gelatin microspheres as a biodegradable drug-delivery system for intramuscular administration. Glutaraldehyde was used as a control. In the in vitro study, the morphology, dynamic swelling, and antienzymatic degradation of test microspheres were evaluated. In the in vivo study, the biocompatibility and degradability of test microspheres were implanted in the skeletal muscle of a rat model via intramuscular injection. The results obtained in the study suggested that crosslinking of gelatin microspheres with glutaraldehyde or genipin may produce distinct crosslinking structures. The water transport mechanism in both the glutaraldehyde- and genipin-crosslinked gelatin microspheres exhibit anomalous behavior ranging from Fickian to Case-II extremes. The increase of the swelling diameter for the genipin-crosslinked microspheres was significantly less than that observed for the glutaraldehyde-crosslinked microspheres. In the animal study, it was found that the degree in inflammatory reaction for tissues implanted with the genipin-crosslinked microspheres was significantly less than that implanted with the glutaraldehyde-crosslinked microspheres. Additionally, the degradation rate of the genipin-crosslinked microspheres was significantly slower than their glutaraldehyde-crosslinked counterparts. These results indicated that the genipin-crosslinked gelatin microspheres may be used as a long-acting drug carrier for intramuscular administration.

Topics: Animals; Biocompatible Materials; Biodegradation, Environmental; Collagenases; Drug Carriers; Gelatin; Injections, Intramuscular; Iridoid Glycosides; Iridoids; Kinetics; Microscopy, Electron, Scanning; Microspheres; Muscle, Skeletal; Pyrans; Rats; Rats, Wistar; Spectrophotometry

A naturally occurring crosslinking agent (genipin) was used in this study to crosslink gelatin hydrogel to develop a wound-dressing membrane. The study was to investigate the in vitro characteristics of the genipin-crosslinked gelatin membrane. The glutaraldehyde-crosslinked counterpart, at a similar crosslinking degree, was used as control. Additionally, an in vivo experiment was undertaken to study the wound healings covered with the glutaraldehyde- and genipin-crosslinked dressings in a rat model. The in vitro results obtained suggested that crosslinking of gelatin membranes with glutaraldehyde or genipin may produce distinct crosslinking structures. The differences in crosslinking structure can significantly affect the mechanical property, water-vapor-transmission rate, swelling ratio, degradation against enzyme and cellular compatibility of the crosslinked membranes. In the in vivo study, it was found that the degree of inflammatory reaction for the wound treated with the genipin-crosslinked dressing was significantly less severe than that covered with the glutaraldehyde-crosslinked dressing throughout the entire course of the study. Additionally, the healing rate for the wound treated with the genipin-crosslinked dressing was notably faster than its glutaraldehyde-crosslinked counterpart.

Topics: Animals; Bandages; Biocompatible Materials; Cell Line; Cross-Linking Reagents; Fibroblasts; Gelatin; Glutaral; Humans; Inflammation; Iridoid Glycosides; Iridoids; Male; Membranes, Artificial; Microscopy, Electron, Scanning; Pyrans; Rats; Rats, Wistar; Tensile Strength; Water; Wound Healing; Wounds and Injuries

The purpose of this study was to prepare and evaluate in vitro the feasibility and cytocompatibility of a novel composite (GGT) as a large defect bone substitute. The composite is tricalcium phosphate ceramic particles combined with genipin crosslinked gelatin. After soaking the GGT composites in Ringer solutions at 37 degrees C for 7, 14, 28, 42, 56, and 84 days, the in vitro biologic degradation rate and biocompatibility were determined. Substances released from soaked GGT composites were analyzed with an ultraviolet visible light spectrophotometer. In addition, the solution soaking the GGT was co-cultured with osteoblasts to determine whether or not the released substances from GGT could facilitate the growth of bone cells. After they had been cultured for 2 days, the osteoblasts were tested for differentiation and proliferation by alkaline phosphatase (ALP) activity and a MTT assay. Results indicate that the concentration of the genipin solution is a critical factor in deciding the crosslinking degree of the GGT composite. Complete crosslinking reaction in the GGT composite occurred when 0.5 wt % of genipin had been added. Cytotoxic testing revealed that 80 ppm of the genipin in the culture medium served as the level over which cytotoxicity to osteoblasts could be produced. In addition, we found that gelatin and calcium continuously were released from the GGT composite in the soaking solution, which promoted differentiation and proliferation of the osteoblasts.

Topics: Alkaline Phosphatase; Animals; Animals, Newborn; Biocompatible Materials; Bone Substitutes; Calcium Phosphates; Cell Division; Cell Survival; Cells, Cultured; Gelatin; Iridoid Glycosides; Iridoids; Materials Testing; Molecular Structure; Osteoblasts; Pyrans; Rats; Rats, Wistar

Gardenia fruit (Gardenia jasminoides ELLIS) is widely used as a natural food colorant and as a traditional Chinese medicine for treatment of hepatic and inflammatory diseases. "Gardenia yellow" is a natural food colorant which is extracted by ethanol from gardenia fruit. The purpose of this study was to evaluate the genotoxicity of gardenia yellow. Genotoxicity of gardenia yellow and its components, crocetin, gentiobiose (a component of crocin), geniposide and genipin (formed by hydrolysis of geniposide), was studied by Ames test, rec-assay, and sister chromatid exchange (SCE) using V79 cells. Gardenia yellow and its components were found not to be mutagenic in the Salmonella reverse mutation assay. Gardenia yellow and genipin caused damage of DNA in rec-assay. Gardenia yellow induced a significant dose-dependent increase of SCE frequency (8.6 times at 1000 microg/ml as the value for the solvent control). Only genipin induced SCEs significantly among the components of gardenia yellow. Moreover, genipin induced a significant increase of tetraploids at all doses tested (95% at 8 microg/ml). Gardenia yellow preparation was analyzed by capillary electrophoresis (CE), and geniposide was detected. However, genipin was not observed. In conclusion, we have shown that genipin possesses genotoxicity. Furthermore, there were unidentified genotoxicants in gardenia yellow.

Topics: Bacillus subtilis; Carotenoids; Coloring Agents; Disaccharides; DNA Damage; Electrophoresis, Capillary; Food Coloring Agents; Gardenia; Iridoid Glycosides; Iridoids; Mutagenicity Tests; Plant Extracts; Pyrans; Sister Chromatid Exchange; Vitamin A

The possibility to stabilize gelatin films by crosslinking with genipin was investigated through a mechanical, chemical and thermal characterization of samples treated with genipin solutions at different concentrations. The extent of crosslinking, evaluated as difference between the number of free epsilon -amino groups before and after crosslinking, increases as a function of genipin concentration up to about 85%. Simultaneously, the deformability of the films decreases whereas the Young's modulus E, increases. Furthermore, crosslinking provokes a significant reduction of the swelling in physiological solution, and enhances the thermal stability of the samples, as indicated by the results of the d.s.c. investigation. The data obtained from the films treated with genipin at concentrations higher than 0.67% are quite similar, and indicative of a good stabilizing effect of genipin. In spite of the small gelatin release (2%) observed after 1 month of storage in buffer solution, the mechanical, thermal and swelling properties of the films are very close to those previously obtained for glutaraldehyde crosslinked gelatin, and suggest that genipin, which is by far less cytotoxic, can be considered a valid alternative for crosslinking gelatin biomaterials.

Topics: Biocompatible Materials; Calorimetry, Differential Scanning; Cholagogues and Choleretics; Cross-Linking Reagents; Gelatin; Iridoid Glycosides; Iridoids; Pyrans; Temperature; Time Factors

A cell extraction process was employed in the study to remove the cellular components from bovine pericardium, leaving a framework of largely insoluble collagen and elastin. It was hypothesized in the literature that this process may decrease the antigenic load (or increase the biocompatibility) within the material. Additionally, acellular tissues may provide a natural microenvironment for host-cell migration to regenerate the tissue. The study was to evaluate the biocompatibility of cellular and acellular bovine pericardia fixed with a naturally occurring crosslinking agent (genipin) implanted subcutaneously in a growing rat model. Additionally, the tissue regeneration rate in the genipin-fixed acellular tissue was investigated. The glutaraldehyde-fixed counterparts were used as controls. The results indicated that the degrees in inflammatory reaction for the genipin-fixed cellular and acellular tissues were significantly less than their glutaraldehyde-fixed counterparts. Additionally, it was noted that the inflammatory reactions for the glutaraldehyde-fixed cellular and acellular tissues lasted much longer than their genipin-fixed counterparts. The tissue regeneration rate for the genipin-fixed acellular tissue was significantly faster than its glutaraldehyde-fixed counterpart. The calcium content of each studied group, analyzed by atomic absorption. did not change significantly until at the 52nd week, postoperatively. The differences in calcium content between the cellular and acellular tissues were insignificant for both the glutaraldehyde- and genipin-fixed groups throughout the entire course of the study. In summary, the biocompatibility of the genipin-fixed cellular and acellular tissues was superior to their glutaraldehyde-fixed counterparts. The genipin-fixed acellular tissue provided a better microenvironment for tissue regeneration than its glutaraldehyde-fixed counterpart, due to its low cytotoxicity. These results suggested that the genipin-fixed acellular tissue might be used as a tissue-engineering matrix in the clinical applications.

Topics: Animals; Biocompatible Materials; Calcium; Cattle; CHO Cells; Cricetinae; Cross-Linking Reagents; Culture Techniques; Fixatives; Glutaral; Iridoid Glycosides; Iridoids; Male; Pericardium; Protein Denaturation; Pyrans; Rats; Rats, Wistar; Regeneration; Temperature; Tensile Strength

Heparinized biomaterials have been used to manufacture blood-contacting prostheses. The present study was intended to characterize the surface properties of a genipin-fixed biological tissue immobilized with heparin using the methods of ionic binding (the /h-i tissue) or covalent binding via multi-point attachment (the /h-m tissue) or end-point attachment (the /h-e tissue). The surface characteristics of test tissues evaluated were water contact angle, surface tension, protein adsorption, platelet adhesion, and cellular compatibility. Nonheparinized and the glutaraldehyde-fixed counterparts were used as controls. It was found that immobilization of heparin on the glutaraldehyde- and genipin-fixed tissues increased their hydrophilicity and surface tension and suppressed their mole ratio of adsorbed fibrinogen to adsorbed albumin and the amount of platelets adhered. Among the heparinized tissues, the /h-m tissue was more hydrophobic and had a higher mole ratio of adsorbed fibrinogen to adsorbed albumin and a greater amount of platelets adhered than the /h-i and /h-e tissues. In general, the surface characteristics of the /h-i tissue were comparable to the /h-e tissue. However, it is known that the ionically immobilized heparin may be displaced from the surface by an ion-exchange mechanism when exposed to blood. There were no significant differences in hydrophilicity, surface tension, the mole ratio of adsorbed fibrinogen to adsorbed albumin, and the amount of platelet adhesion between the glutaraldehyde- and genipin-fixed tissues in comparison with their respective counterparts. However, the cellular compatibility of the genipin-fixed tissues with or without heparinization was significantly superior to its glutaraldehyde-fixed counterparts.

Topics: Adsorption; Biocompatible Materials; Bioprosthesis; Blood Platelets; Blood Proteins; Cell Adhesion; Fixatives; Heparin; In Vitro Techniques; Iridoid Glycosides; Iridoids; Microscopy, Electron, Scanning; Pyrans; Surface Properties

The study was undertaken to investigate the stability of a biological tissue fixed with a naturally occurring crosslinking agent (genipin) at distinct elapsed storage durations. The glutaraldehyde-fixed counterpart was used as a control. Porcine pericardia procured from a slaughterhouse were used as raw materials. After fixation, the fixed tissues were sterilized in a graded series of ethanol solutions and thoroughly rinsed in phosphate buffered saline for 1 day, and then stored in a jar containing sterilized water. The samples were taken out and tested for their stability during the durations of 1day through 6 months after storage. The stability of each study group was tested by measuring its tensile strength, free-amino-group content, and denaturation temperature. Additionally, the cytotoxicity of each test sample and its corresponding storage solution were investigated in vitro using 3T3 fibroblasts. The results were examined using a microscope and 3-(4,5-dimethylthiazol-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. It was found that the stability of the genipin-fixed tissue during storage was superior to its glutaraldehyde-fixed counterpart. The differences in stability between the genipin- and glutaraldehyde-fixed tissues during storage may be caused by their differences in crosslinking structure. There was no apparent cytotoxicity for both the genipin-fixed tissue and its corresponding storage solution throughout the entire course of the study, whereas significant cytotoxicity was observed for both the glutaraldehyde-fixed tissue and its storage solution. However, the cytotoxicity of the glutaraldehyde-fixed tissue decreased with increasing elapsed storage duration, whereas that of its corresponding storage solution increased. This suggested that the toxic residues remaining in the glutaraldehyde-fixed tissue leached out slowly into its corresponding storage solution during the course of storage.

Topics: 3T3 Cells; Animals; Cross-Linking Reagents; Iridoid Glycosides; Iridoids; Mice; Pericardium; Pyrans; Swine; Tissue Fixation; Tissue Preservation

Prominent neurite outgrowth induced by genipin, a plant-derived iridoid, was substantially inhibited by addition of NG-nitro-L-arginine methyl ester (L-NAME), a nitric oxide (NO) synthase (NOS) inhibitor, and carboxy-PTIO, an NO scavenger, in PC12h cells. Increases of the NADPH-diaphorase activity and neuronal and inducible NOS proteins in cells preceded the neurite outgrowth after addition of genipin to medium. NO donors could induce the neurite outgrowth dose-dependently in the cells. On the other hand, an inhibitor of soluble guanylate cyclase (SGC), which is known to be a stimulatory target of NO, abolished greatly the genipin-induced neurite outgrowth. Addition of extracellular signal-regulated kinase (ERK) kinase inhibitors could almost completely abolish the neurite induction. L-NAME remarkably depressed genipin-stimulated phosphorylation of ERK-1 and -2. A neuritogenic effect of nerve growth factor (NGF) in PC12h cells was also remarkably inhibited by the NOS inhibitor, NO scavenger and SGC inhibitor. These findings suggest that induced NO production followed by cyclic GMP-mediated stimulation of the mitogen-activated protein kinase (MAPK) cascade is implicated in the neuritogenesis by genipin and NGF in PC12h cells.

Topics: Animals; Enzyme Activation; Enzyme Induction; Enzyme Inhibitors; Free Radical Scavengers; Guanylate Cyclase; Iridoid Glycosides; Iridoids; Mitogen-Activated Protein Kinases; Nerve Growth Factor; Neurites; NG-Nitroarginine Methyl Ester; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitric Oxide Synthase Type I; Nitric Oxide Synthase Type II; Nitric Oxide Synthase Type III; PC12 Cells; Phosphorylation; Pyrans; Rats

The study was to evaluate the characteristics of a chitosan membrane cross-linked with a naturally-occurring cross-linking reagent, genipin. This newly-developed genipin-cross-linked chitosan membrane may be used as an implantable drug-delivery system. The chitosan membrane without cross-linking (fresh) and the glutaraldehyde-cross-linked chitosan membrane were used as controls. The characteristics of test chitosan membranes evaluated were their cross-linking degree, swelling ratio, mechanical properties. antimicrobial activity, cytotoxicity, and degradability. It was found that cross-linking of chitosan membrane using genipin increased its ultimate tensile strength but significantly reduced its strain-at-fracture and swelling ratio. There was no significant difference in antimicrobial activity between the genipin-cross-linked chitosan membrane and its fresh counterpart. Additionally, the results showed that the genipin-cross-linked chitosan membrane had a significantly less cytotoxicity and a slower degradation rate compared to the glutaraldehyde-cross-linked membrane. These results suggested that the genipin-cross-linked chitosan membrane may be a promising carrier for fabricating an implantable drug-delivery system. The drug-release characteristics of the genipin-cross-linked chitosan membrane are currently under investigation.

Topics: Adhesives; Animals; Bacteria; Biocompatible Materials; Cell Survival; Chickens; Chitin; Chitosan; Colony-Forming Units Assay; Cross-Linking Reagents; Drug Carriers; Fibroblasts; Glutaral; Humans; Iridoid Glycosides; Iridoids; Membranes, Artificial; Muramidase; Pyrans

This study was designed to evaluate a newly developed biologic valved conduit fixed with genipin used to reconstruct the right ventricular outflow tract in a canine model.. Fresh bovine jugular veins with a retained native valve procured from a slaughterhouse were used as raw materials to fabricate the valved conduits. A naturally occurring crosslinking agent, genipin, was used to fix the procured jugular veins. The glutaraldehyde-fixed counterpart was used as a control. A canine model was used in the study.. Echocardiography revealed that the motion of the valvular leaflets in both the glutaraldehyde- and genipin-fixed conduits was satisfactory. The transvalvular pressure gradients of both studied groups were minimal. No endothelium-like cells were observed on the luminal surface of the conduit and the valvular leaflet for the glutaraldehyde-fixed group throughout the entire course of the study. In contrast, endothelium-like cells were observed on the entire surface of the genipin-fixed valved conduit retrieved at 6 months postoperatively in all the cases studied. There was no evidence of luminal fibrous peel in any the valved conduits studied. Degradation of valvular leaflet in one of the glutaraldehyde-fixed conduits was observed. In this particular case, thrombus formation was also observed on the surface of the valvular leaflet. On the other hand, no apparent degradation or thrombus formation was observed on the surfaces of the genipin-fixed valvular leaflet and conduit. A significantly more severe inflammatory reaction was observed for the glutaraldehyde-fixed conduit than for its genipin-fixed counterpart throughout the entire course of the study. The calcium contents of the samples before implantation and those retrieved at distinct implantation duration were minimal for both the glutaraldehyde- and genipin-fixed tissues.. Although further studies are necessary, the genipin-fixed valved conduit appears to have great potential in helping mitigate the complications observed in the commercially available conduits.

Topics: Adhesives; Animals; Blood Vessel Prosthesis Implantation; Cattle; Cross-Linking Reagents; Dogs; Echocardiography; Glutaral; Iridoid Glycosides; Iridoids; Jugular Veins; Pyrans; Ventricular Outflow Obstruction

Geniposide is one of the constituents of Gardenia fruit (Gardenia jasminoides Ellis, Rubiaceae), which has been used in traditional medicine. Although its anti-inflammatory and antithrombotic effects have been reported, the way it acts is still unclear. We have investigated the effects of geniposide and its metabolite genipin on thrombogenesis and platelet aggregation. In an in vivo model, geniposide and genipin significantly (P < 0.05) prolonged the time required for thrombotic occlusion induced by photochemical reaction in the mouse femoral artery. In an in vitro study, both geniposide and genipin inhibited collagen-induced, but did not inhibit arachidonate-induced, mouse platelet aggregation. However aspirin, a cyclooxygenase inhibitor, inhibited arachidonate-induced platelet aggregation but only partially inhibited the collagen-induced one. We also showed, by measuring PLA(2)-catalyzed arachidonic acid release, that geniposide inhibited phospholipase A(2) (PLA(2)) activity. We conclude that geniposide showed an antithrombotic effect in vivo due to the suppression of platelet aggregation. PLA(2) inhibition by geniposide is one possible anti-platelet mechanism.

Topics: Animals; Disease Models, Animal; Fibrinolytic Agents; Fruit; Iridoid Glycosides; Iridoids; Male; Mice; Phospholipases A; Phytotherapy; Plant Extracts; Platelet Aggregation; Pyrans; Rubiaceae; Thrombosis

Genipin, which was shown in our previous investigation to have prominent neuritogenic activity in paraneurons such as PC12h cells, was studied to determine whether it could prevent the toxicity of Alzheimer's amyloid beta protein (Abeta) in cultured hippocampal neurons. Increased release of lactate dehydrogenase from hippocampal neurons after 2 d of Abeta25-35 administration was prevented dose dependently by the addition of genipin 20-40 microm. Morphological observations and trypan blue staining of cells confirmed the protection of hippocampal neurons from Abeta toxicity by genipin. Geniposide had less effect in preventing Abeta toxicity.

Topics: Amyloid beta-Peptides; Animals; Cells, Cultured; Cholagogues and Choleretics; Dose-Response Relationship, Drug; Embryo, Mammalian; Hippocampus; Iridoid Glycosides; Iridoids; Nerve Growth Factor; Neurons; Pyrans; Rats

Topics: Administration, Oral; Animals; Chromatography, High Pressure Liquid; Drugs, Chinese Herbal; Humans; Iridoid Glycosides; Iridoids; Male; Mice; Mice, Inbred Strains; Plant Extracts; Pyrans; Rubiaceae

We showed previously that a Kampo (Chinese/Japanese herbal) medicine, Inchin-ko-to (ICKT), inhibits hepatocyte apoptosis induced by transforming growth factor beta1 in vitro. The present study investigated whether ICKT or its ingredients inhibit Fas-mediated liver apoptosis in vivo.. Acute liver injury was induced by an intravenous injection of anti-Fas antibody, Jo2. The effects of ICKT and its ingredients on lethality, histology, apoptotic index, serum transaminase levels, caspase activation, mitochondrial membrane potential (Deltapsi(m)), and mitochondrial permeability transition (MPT) were analyzed. Apoptosis in mouse hepatocytes in vitro was also evaluated.. Pretreatment with ICKT rescued 75% of Jo2-treated mice and markedly suppressed liver apoptosis/injury. Genipin, an intestinal bacterial metabolite of geniposide that is a major ingredient of ICKT, was found to be an active principle of ICKT. Genipin also suppressed in vitro Fas-mediated apoptosis in primary-cultured murine hepatocytes. Activation of caspase 3 and 8 in the liver homogenate and rapid reduction of triangle uppsi(m) of hepatocytes isolated from Jo2-treated mice were inhibited by genipin preadministration. The resistance to Ca(2+)-induced MPT was enhanced in liver mitochondria of genipin-treated mice.. These results suggest that the antiapoptotic activity of genipin via the interference with MPT is a possible mechanism for therapeutic effects of ICKT.

Topics: Alanine Transaminase; Animals; Apoptosis; Aspartate Aminotransferases; Cells, Cultured; Cholagogues and Choleretics; fas Receptor; Female; Glycyrrhizic Acid; Intracellular Membranes; Iridoid Glycosides; Iridoids; Liver; Membrane Potentials; Mice; Mice, Inbred BALB C; Mitochondria, Liver; Permeability; Plants, Medicinal; Pyrans; Ursodeoxycholic Acid

The study was designed to characterize the surface properties (including water contact angle, surface tension, protein adsorption, platelet adhesion, and cellular compatibility) of a biological patch fixed with genipin, a naturally occurring crosslinking agent. Fresh and glutaraldehyde-fixed counterparts were used as controls. It was found that both glutaraldehyde and genipin are effective crosslinking agents for biological tissue fixation. Fixation of biological tissue with glutaraldehyde or genipin significantly increased its hydrophilicity and surface tension and reduced its mol ratio of adsorbed fibrinogen to adsorbed albumin as well as the amount of adhered platelet. There were no significant differences in hydrophilicity, surface tension, the mole ratio of adsorbed fibrinogen to adsorbed albumin, and the amount of platelet adhesion between the glutaraldehyde- and genipin-fixed tissues. However, the cellular compatibilities of fresh and the genipin-fixed tissues were significantly superior to the glutaraldehyde-fixed tissue.

Topics: 3T3 Cells; Adsorption; Animals; Bioprosthesis; Chemical Phenomena; Chemistry, Physical; Cross-Linking Reagents; Fibrinogen; Glutaral; Iridoid Glycosides; Iridoids; Mice; Microscopy, Electron, Scanning; Pericardium; Platelet Adhesiveness; Pyrans; Serum Albumin; Surface Properties; Surface Tension; Swine; Tissue Fixation

The objective of the present study was to evaluate in vitro, using Chinese hamster ovary (CHO-K1) cells, the genotoxicity of genipin, a naturally occurring crosslinking agent. Glutaraldehyde, the most commonly used crosslinking agent for biologic tissue fixation, was employed as a reference chemical. The selected procedures for this evaluation were the micronucleus (MN) and sister chromatid exchange (SCE) assays with or without the addition of a metabolic activation system (S9 mix). Before starting the genotoxicity assays, the maximum noncytotoxic amounts of glutaraldehyde and genipin were determined using the MTT assay. The results obtained in the MTT assay revealed that the cytotoxicity of genipin was significantly lower than that of glutaraldehyde with or without S9 mix. The frequencies of MN observed in the cases drugged with varying concentrations of glutaraldehyde or genipin were not statistically different from those seen in the negative controls (blank) in the presence or absence of S9 mix. However, it was noted that glutaraldehyde significantly inhibited the cell-cycle progression while the cells drugged with genipin did not result in cell-cycle delay. In the SCE assay, the numbers of SCE per cell observed in the cases drugged with varying concentrations of glutaraldehyde were significantly greater than those found in the negative controls with or without S9 mix. Nevertheless, these numbers were still low compared to the numbers of SCE induced by the strong mutagens used as our positive control substances. This suggests that glutaraldehyde may produce a weakly clastogenic response in CHO-K1 cells. In contrast, the numbers of SCE per cell obtained in the cases drugged with genipin were comparable to those observed in the negative controls in those that were except drugged with the highest dose (50 ppm). This suggests that genipin does not cause clastogenic response in CHO-K1 cells provided its concentration is lower than 50 ppm. In conclusion, as far as cytotoxicity and genotoxicity are concerned, genipin is a promising crosslinking agent for biologic tissue fixation.

Topics: Animals; CHO Cells; Cholagogues and Choleretics; Cricetinae; Cross-Linking Reagents; Glutaral; Iridoid Glycosides; Iridoids; Micronucleus Tests; Pyrans; Sister Chromatid Exchange

In an attempt to overcome the cytotoxicity problem of the glutaraldehyde-fixed tissues, a naturally occurring crosslinking agent (genipin) was used by our group to fix biological tissues. The study was intended to investigate the rate of tissue fixation by genipin. Glutaraldehyde was used as a control. In addition, the degrees of tissue fixation by genipin at different pHs (pH 4.0, pH 7. 4, pH 8.5, or pH 10.5), temperatures (4 degrees C, 25 degrees C, 37 degrees C, or 45 degrees C), and initial fixative concentrations (0.250%, 0.625%, or 1.000%) were examined. The results obtained revealed that the rate of tissue fixation by glutaraldehyde was significantly faster than that by genipin. The degree of tissue fixation by genipin may be controlled by adjusting its fixation duration or fixation conditions. The order in degree of tissue fixation by genipin at different pHs, from high to low, was: at nearly neutral pH (pH 7.4 or pH 8.5) > at basic pH (pH 10.5) > at acidic pH (pH 4.0). The degrees of tissue fixation by genipin at different temperatures were about the same, except for that at 4 degrees C. In contrast, the initial fixative concentration did not seem to affect the degree of tissue fixation by genipin, if only the amount of genipin in the fixation solution was sufficient to complete tissue fixation. The concentrations of genipin in the aqueous solutions at different pHs, temperatures, and initial fixative concentrations tended to decrease with time with or without the occurrence of tissue fixation. This indicated that genipin was not stable in the aqueous solution. The instability of aqueous genipin was more remarkable with increasing pH or temperature. The results obtained in this study may be used to optimize the fixation process for developing bioprostheses fixed by genipin.

Topics: Animals; Cross-Linking Reagents; Glutaral; Hydrogen-Ion Concentration; Iridoid Glycosides; Iridoids; Pyrans; Swine; Temperature; Tissue Fixation

A recognized drawback of the currently available chemical cross-linking reagents used to fix bioprostheses is the potential toxic effects a recipient may be exposed to from the fixed tissues and/or the residues. It is, therefore, desirable to provide a cross-linking reagent which is of low cytotoxicity and may form stable and biocompatible cross-linked products. To achieve this goal, a naturally occurring cross-linking reagent -- genipin -- which has been used in herbal medicine and in the fabrication of food dyes, was used by our group to fix biological tissues. The study was to assess the cytotoxicity of genipin in vitro using 3T3 fibroblasts (BALB/3T3 C1A31-1-1). Glutaraldehyde, the most commonly used cross-linking reagent for tissue fixation, was used as a control. The cytotoxicity of the glutaraldehyde- and genipin-fixed tissues and their residues was also evaluated and compared. The observation in the light microscopic examination revealed that the cytotoxicity of genipin was significantly lower than that of glutaraldehyde. Additionally, the results obtained in the MTT assay implied that genipin was about 10000 times less cytotoxic than glutaraldehyde. Moreover, the colony forming assay suggested that the proliferative capacity of cells after exposure to genipin was approximately 5000 times greater than that after exposure to glutaraldehyde. It was noted that the cells seeded on the surface of the glutaraldehyde-fixed tissue were not able to survive. In contrast, the surface of the genipin-fixed tissue was found to be filled with 3T3 fibroblasts. Additionally, neocollagen fibrils made by these fibroblasts were observed on the genipin-fixed tissue. This fact suggested that the cellular compatibility of the genipin-fixed tissue was superior to its glutaraldehyde-fixed counterpart. Also, the residues from the glutaraldehyde-fixed tissue markedly reduced the population of the cultured cells, while those released from the genipin-fixed tissue had no toxic effect on the seeded cells. In conclusion, as far as cytotoxicity is concerned, genipin is a promising cross-linking reagent for biological tissue fixation.

Topics: 3T3 Cells; Animals; Cattle; Cholagogues and Choleretics; Cross-Linking Reagents; Fibroblasts; Formazans; Glutaral; Iridoid Glycosides; Iridoids; Mice; Mice, Inbred BALB C; Microscopy, Electron, Scanning; Pericardium; Prostheses and Implants; Pyrans; Tetrazolium Salts; Tissue Fixation

Bioadhesives are used for tissue adhesion and hemostasis in surgery. A gelatin-resorcinol mixture crosslinked with formaldehyde (GRF glue) and/or glutaraldehyde (GRG) is used for this purpose. Although the bonding strength of the GRF glue to tissue is satisfactory, concerns about the cytotoxicity of formaldehyde are reported in the literature. It was suggested that the cytotoxicity problem of the GRF glue may be overcome by changing its crosslinking method. The study was therefore undertaken to assess the feasibility of using an epoxy compound (GRE glue), a water-soluble carbodiimide (GAC glue), or genipin (GG glue) to crosslink with a gelatin hydrogel as new bioadhesives. GRF glue and GRG glue were used as controls. The results of our cytotoxicity study suggested that the cellular compatibility of the GAC and GG glues was superior to the GRF, GRG, and GRE glues. The gelation time for the GG glue was relatively longer than the GRF and GRG glues, while no gelation time could be determined for the GAC glue. Additionally, it took approximately 17 h for the GRE glue to become adhesive. The GRF and GRG glues had the greatest bonding strengths to tissue among all test adhesives, while the bonding strengths of the GAC and GG glues were comparable. In contrast, there was almost no bonding strength to tissue for the GRE glue. However, the GRF and GRG glues were less flexible than the GAC and GG glues. Subsequent to the bonding strength measurement, each test adhesive was found to adhere firmly to the tissue surface and underwent cohesive failure during the bond breaking. In conclusion, the GRF and GRG glues may be used as tissue adhesives when their ability to bind tissue rapidly and tightly is required; the GAC and GG glues are preferable when the adhesive action must be accompanied with minimal cytotoxicity and stiffness; and the GRE glue is not suitable for bioadhesion in clinical applications.

Topics: 3T3 Cells; Alginates; Animals; Carbodiimides; Cross-Linking Reagents; Drug Combinations; Formaldehyde; Gelatin; Glucuronic Acid; Hexuronic Acids; Hydrogel, Polyethylene Glycol Dimethacrylate; Iridoid Glycosides; Iridoids; Mice; Microscopy, Electron, Scanning; Molecular Structure; Pericardium; Pyrans; Resorcinols; Swine; Time Factors; Tissue Adhesives; Viscosity

Currently available crosslinking agents used in fixing bioprostheses are all highly (or relatively highly) cytotoxic, which may induce an adverse inflammatory reaction in vivo. It is therefore desirable to provide a crosslinking agent that is of low cytotoxicty and may form stable and biocompatible crosslinked products. To achieve this goal, a naturally occurring crosslinking agent-genipin-was used by our group to fix biological tissues. Genipin may be obtained from its parent compound, geniposide, which may be isolated from the fruits of Gardenia jasminoides Ellis. In our previous studies, it was found that the cytotoxicity of genipin is significantly lower than both glutaraldehyde and an epoxy compound. Also, it was shown that genipin can form stable and biocompatible crosslinked products. The present study further investigates the crosslinking characteristics and mechanical properties of a genipin-fixed bovine pericardium. Fresh and glutaraldehyde- and epoxy-fixed counterparts were used as controls. It was found that the denaturation temperatures of the glutaraldehyde- and genipin-fixed tissues were significantly greater than the epoxy-fixed tissue, although their fixation indices were comparable. The mechanical properties of fresh bovine pericardium are anisotropic. However, fixation tended to eliminate tissue anisotropy. The tendency in the elimination of tissue anisotropy for the genipin-fixed tissue was more remarkable than for the glutaraldehyde- and epoxy-fixed tissues. In addition, the genipin-fixed tissue had the greatest ultimate tensile strength and toughness among all the fixed tissues. Distinct patterns in rupture were observed in the study: The torn collagen fibers of the genipin- and glutaraldehyde-fixed tissues appeared to be bound together, while those of fresh and the epoxy-fixed tissues stayed loose. The results obtained in the study suggests that tissue fixation in glutaraldehyde, epoxy compound, and genipin may produce distinct crosslinking structures. The differences in crosslinking structure may affect the crosslinking characteristics and mechanical properties of the fixed tissues.

Topics: Animals; Biocompatible Materials; Bioprosthesis; Cattle; Cross-Linking Reagents; Iridoid Glycosides; Iridoids; Pericardium; Plants, Medicinal; Pyrans

The study investigates the mechanical properties of porcine aortic valve leaflets fixed with a naturally occurring crosslinking agent, genipin, at distinct pressure heads. Fresh and the glutaraldehyde-fixed counterparts were used as controls. Subsequent to fixation, the changes in leaflet collagen crimps and its surface morphology were investigated by light microscopy and scanning electron microscopy (SEM). Also, the crosslinking characteristics of each studied group were determined by measuring its fixation index and denaturation temperature. In the mechanical testing, tissue strips made from each studied group were examined in both the circumferential and radial directions. Histological and SEM comparisons between fresh porcine aortic valve leaflet and those fixed at medium or high pressure revealed that the following changes may occur: elimination of the natural collagen crimping, and extensive loss of the endothelial layer. The denaturation temperatures of the glutaraldehyde-fixed leaflets were significantly greater than the genipin-fixed leaflets; however, their fixation indices were comparable. Generally, fixation pressure did not affect the crosslinking characteristics of the genipin- and glutaraldehyde-fixed leaflets. It was found that fixation of porcine aortic valves in genipin or glutaraldehyde did not alter the mechanical anisotropy observed in fresh valve leaflets. This indicated that the intramolecular and intermolecular crosslinks introduced into the collagen fibrils during fixation is of secondary importance to the presence of structural and mechanical anisotropy in fresh leaflet. Tissue fixation in genipin or glutaraldehyde may produce distinct crosslinking structures. However, the difference in crosslinking structure between the genipin- and glutaraldehyde-fixed leaflets did not seem to cause any significant discrepancies in their mechanical properties when compared at the same fixation pressure. Nevertheless, regardless of the crosslinking agent used, changes in mechanical properties and ruptured patterns were observed when the valve leaflets were fixed at distinct pressures.

Topics: Animals; Aortic Valve; Bioprosthesis; Collagen; Cross-Linking Reagents; Glutaral; Heart Valve Prosthesis; Iridoid Glycosides; Iridoids; Microscopy, Electron, Scanning; Pyrans; Stress, Mechanical; Swine; Tensile Strength

Bioprostheses derived from biological tissues must be chemically modified and subsequently sterilized before they can be implanted in humans. Various crosslinking reagents, including formaldehyde, glutaraldehyde, dialdehyde starch, and epoxy compound, have been used to chemically modify biological tissues. However, these synthetic crosslinking reagents are all highly (or relatively highly) cytotoxic. It is therefore desirable to provide a crosslinking reagent suitable for use in biomedical applications that is of low cytotoxicity and that forms stable and biocompatible crosslinked products. This study evaluates the feasibility of using a naturally occurring crosslinking reagent--genipin--to chemically modify biological tissues. Genipin and its related iridoid compounds, extracted from gardenia fruits, have been used in traditional Chinese medicine for the treatments of jaundice and various inflammatory and hepatic diseases. In this feasibility study, the cytotoxicity of genipin and the crosslinking characteristics of genipin-fixed biological tissues were investigated. Fresh porcine pericardia procured from a slaughterhouse were used as raw materials. Glutaraldehyde and an epoxy compound (ethylene glycol diglycidyl ether), which has been used extensively in developing bioprostheses, were used as controls. It was found that the cytotoxicity of genipin was significantly lower than that of glutaraldehyde and the epoxy compound. The amino acid residues in the porcine pericardium that may react with genipin were lysine, hydroxylysine, and arginine. Additionally, the genipin-fixed tissue had a mechanical strength and resistance against enzymatic degradation comparable to the glutaraldehyde-fixed tissue. This suggests that genipin can form stable crosslinked products. The results of this in vitro study demonstrate that genipin is an effective crosslinking reagent for biological tissue fixation.

Topics: Animals; Bioprosthesis; Cell Survival; Cells, Cultured; Cross-Linking Reagents; Feasibility Studies; Fibroblasts; Glutaral; Humans; Infant, Newborn; Iridoid Glycosides; Iridoids; Male; Medicine, Chinese Traditional; Pericardium; Plant Extracts; Plants, Medicinal; Pyrans; Skin; Swine; Tissue Fixation

A recognized disadvantage of the currently available chemical reagents used to fix bioprostheses is the potential toxic effects a recipient may be exposed to from residues. It is therefore desirable to provide a crosslinking reagent that is of low cytotoxicity and can form stable and biocompatible crosslinked products. To achieve this goal, a naturally occurring crosslinking reagent-genipin-was used by our group to fix biological tissues. Genipin can be obtained from its parent compound geniposide, which can be isolated from the fruits of Gardenia jasminoides ELLIS. In our previous feasibility study, it was found that the cytotoxicity of genipin is significantly lower than both glutaraldehyde and an epoxy compound. Additionally, it was shown that genipin can form stable crosslinked products. The present study further investigates the biocompatibility of a genipin-fixed porcine pericardium implanted subcutaneously in a growing rat model. The fresh, glutaraldehyde-, and epoxy-fixed counterparts were used as controls. It was noted that the inflammatory reaction of the genipin-fixed tissue was significantly less than its glutaraldehyde- and epoxy-fixed counterparts. Also, the genipin-fixed tissue has tensile strength and resistance against in vivo degradation comparable to the glutaraldehyde-fixed tissue. Additionally, the calcium content of the genipin-fixed tissue measured throughout the entire course of the study was minimal. Nevertheless, further study in calcification for the genipin-fixed tissue should be conducted in a blood-contact environment. The results obtained in this subcutaneous study indicate that genipin is a promising crosslinking reagent for biological tissue fixation. However, further durability testing in vitro and in vivo are needed to determine the relative functional merits of this new crosslinker.

Topics: Animals; Biocompatible Materials; Calcium; Cross-Linking Reagents; Epoxy Compounds; Glutaral; Iridoid Glycosides; Iridoids; Male; Microscopy, Electron, Scanning; Pericardium; Plants, Medicinal; Pyrans; Rats; Rats, Wistar; Spectrophotometry, Atomic; Swine; Tensile Strength; Time Factors; Tissue Fixation; Transplantation, Heterologous

We examined effect of iridoid glucosides, aucubin, catalpol, geniposide and gardenoside, and their enzymic hydrolysates on neurite outgrowth of PC12h cells. Except for aucubin, these glucosides induced neurite outgrowth at 0.1 microgram/ml and above in medium after 3 d of treatment. Hydrolysates of the four glucosides all caused neuritogenesis. Geniposide hydrolysate enhanced responses of cells to carbachol and KCl-induced depolarization in terms of cytoplasmic free-calcium concentration. The aglucone of geniposide, genipin, also promoted neurite outgrowth in a dose-dependent manner (ED50 = 0.7 microM). The neuritogenic effect of genipin was partially or considerably inhibited in the presence of H-89 and genistein. All the results presented suggest that certain iridoid compounds can induce neuronal differentiation in PC12h cells through activation of components of the intracellular signal transduction pathway.

Topics: Animals; beta-Glucosidase; Calcium; Glucosides; Hydrolysis; Iridoid Glucosides; Iridoid Glycosides; Iridoids; Neurites; Neurons; PC12 Cells; Protein Kinase Inhibitors; Pyrans; Rats; Signal Transduction

Phosphatidylgenipin, synthesized via the transphosphatidylation reaction of 1,2-dipalmitoyl-3-sn-phosphatidylcholine to genipin by phospholipase D, was found to react with L-phenylalanine in chloroform and gave a clear blue solution. This blue solution was also formed in following organic solvents: ethanol, ethyl acetate, diethyl ether, benzene, and hexane. However, genipin and L-phenylalanine did not give any colored product under the same conditions. The blue pigment resulted from phosphatidylgenipin and L-phenylalanine showed lambda max at 615 nm in chloroform, and had a similar blue color to an aqueous solution of the natural blue pigment "gardenia blue." This is an example for the preparation of a hydrophobic pigment from a phosphatidyl derivative of a water-soluble compound.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Chloroform; Iridoid Glycosides; Iridoids; Phenylalanine; Phospholipase D; Pigments, Biological; Plants, Medicinal; Pyrans; Solubility; Solutions; Solvents; Spectrophotometry

The hepatotoxic effects of geniposide were investigated in rats. Increases in serum alanine aminotransferase and aspartate aminotransferase activities as a result of oral administration of 320 mg geniposide/kg body weight were suppressed when geniposide was administered ip or when the rats were pretreated with chloramphenicol. The non-protein sulphydryl content of the liver 4 hr after oral administration of geniposide decrease in a dose-dependent manner. Genipin, the aglycone of geniposide, had a marked reactivity with sulphydryl groups of glutathione and cysteine in vitro. The hepatotoxic effects of ip administration of genipin at a dose of 80 mg/kg body weight were comparable with those of oral administration of geniposide at a dose of 320 mg/kg. Buthionine sulphoximine pretreatment enhanced the toxicity of geniposide, while cysteine pretreatment completely suppressed it. These results suggest that the conversion of geniposide to genipin is causally related to the hepatotoxicity of geniposide and that hepatic non-protein sulphydryls are important in modulating the toxicity.

Topics: Administration, Oral; Alanine Transaminase; Animals; Aspartate Aminotransferases; Bilirubin; Buthionine Sulfoximine; Chloramphenicol; Cysteine; Injections, Intraperitoneal; Iridoid Glycosides; Iridoids; Liver; Male; Methionine Sulfoximine; Molecular Structure; Pyrans; Rats; Rats, Inbred Strains; Sulfhydryl Compounds

Topics: Animals; Cholagogues and Choleretics; Glucosides; Glycosides; Iridoid Glycosides; Iridoids; Lymphokines; Male; Pyrans; Rats; Rats, Inbred Strains

We made a study on choleretic property and mechanism of action of iridoid compounds as well as dehydrocholate (DHC), cholate (CA), and salicylate (SA), examining their effects on factors such as bile flow, bile acids, electrolytes (Na+, K+, Cl-, and HCO3-), and their metabolites. Each sample showed a characteristic property, respectively. Genipin and patrinoside decreased biliary concentrations of bile acids, Na+, Cl-, and HCO3-, corresponding to their rapid choleretic actions which were due to bile acids independent fraction. The choleretic action of DHC is approximately twice as potent as that of CA. Their actions were due to bile acids-dependent fraction. CA gave a marked increase in Na+ concentration but DHC did not. And both compounds gave a marked diminution in Cl- concentration and weakly decreased HCO3- concentration. SA showed a weak and durable choleretic action and also gave a marked increase in HCO3- concentration. The main metabolite detected from the bile given genipin was genipin-1-O-glucuronic acid (GGA). The periodical pattern of GGA level in bile was in agreement with that of genipin- induced choleretic action, and quantitatively cation, anion gap produced was nearly compensated by biliary concentration of GGA. From out various results, the choleretic mechanism of iridoid compounds is considered to be as follows: The hemiacetal moiety of them undergoes conjugation in the liver to give glucuronide. Glucuronide thus formed is secreted into the biliary tree being coupled mainly with Na+ and water is passively excreted.

Topics: Animals; Bicarbonates; Bile Acids and Salts; Chlorides; Cholagogues and Choleretics; Cholic Acids; Dehydrocholic Acid; Glucosides; Glycosides; Iridoid Glycosides; Iridoids; Male; Plant Extracts; Potassium; Pyrans; Rats; Rats, Inbred Strains; Sodium; Sodium Salicylate

In the experiment on continuous perfusion of rat stomach in vivo, genipin inhibited only the gastric acid secretion induced by carbachol, but not by tetragastrin, or histamine. In the experiment on isolated organs, genipin showed a weak competitive anti-acetylcholine action on the intestinal contraction. Based on these facts and results reported previously, it is conceivable that anti-cholinergic action at least partly contributes to the genipin-induced inhibitory effect on gastric functions. Erythritol clearance was increased with the increase in bile flow by administration of genipin. Genipin showed a significant choleretic action, and just then the concentration of biliary bile acid was decreased inversely. In the relationship between bile flow and biliary bile acid excretion rate, the slope of regression line obtained from genipin-treated group was not significantly different from that of control, and these lines were approximately parallel to each other. Genipin did not affect the concentration of sodium, potassium, chloride, or bicarbonate in the bile collected during the initial stage, in which bile flow was increased, after administration. It is concluded from these results that genipin-induced choleretic action proceeds by a mechanism wherein water is driven along osmotic gradient which originates in the transport of bile acid-independent fraction from hepatocytes into canaliculi, mainly through active Na+ transport.

Topics: Animals; Bile; Bile Acids and Salts; Carbachol; Cholagogues and Choleretics; Gastric Juice; Guinea Pigs; Histamine; In Vitro Techniques; Iridoid Glycosides; Iridoids; Male; Mice; Plant Extracts; Plants, Medicinal; Pyrans; Rats; Tetragastrin