muramidase and genipin

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

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

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

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

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

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

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