tretinoin and beta-glycerophosphoric-acid

To investigate two potential strategies aimed at targeting the inflammatory pathogenesis of COPD: a small molecule, all trans retinoic acid (atRA) and human mesenchymal stem cells (hMSCs).. atRA was formulated into solid lipid nanoparticles (SLNs) via the emulsification-ultrasonication method, and these SLNs were characterised physicochemically. Assessment of the immunomodulatory effects of atRA-SLNs on A549 cells in vitro was determined using ELISA. hMSCs were suspended in a previously developed methylcellulose, collagen and beta-glycerophosphate hydrogel prior to investigating their immunomodulatory effects in vitro.. SLNs provided significant encapsulation of atRA and also sustained its release over 72 h. A549 cells were viable following the addition of atRA SLNs and showed a reduction in IL-6 and IL-8 levels. A549 cells also remained viable following addition of the hMSC/hydrogel formulation - however, this formulation resulted in increased levels of IL-6 and IL-8, indicating a potentially pro-inflammatory effect.. Both atRA SLNs and hMSCs show potential for modulating the environment in inflammatory disease, though through different mechanisms and leading to different outcomes - despite both being explored as strategies for use in inflammatory disease. atRA shows promise by acting in a directly anti-inflammatory manner, whereas further research into the exact mechanisms and behaviours of hMSCs in inflammatory diseases is required.

Topics: A549 Cells; Anti-Inflammatory Agents; Cell Survival; Collagen; Drug Carriers; Glycerophosphates; Humans; Hydrogels; Immunologic Factors; Immunomodulation; Interleukins; Lipids; Mesenchymal Stem Cell Transplantation; Methylcellulose; Nanoparticles; Pulmonary Disease, Chronic Obstructive; Signal Transduction; Tretinoin

Activin A is a growth factor released by mature osteoblasts that has a critical effect on bone formation. We investigated the effect of bone morphogenetic protein (BMP)-4 on activin A gene expression during in vitro osteogenic differentiation of mouse embryonic stem (ES) cells. Embryoid bodies were cultured in retinoic acid (RA) for three days and then without RA for two days. Seeded cells received osteogenic medium with β-glycerophosphate, L-ascorbic acid 2-phosphate and dexamethasone during 19 days, with or without BMP-4. Six independent experiments were carried out. Real-time PCR was used to detect gene expression of activin A, Oct-4, Nanog, osteocalcin, RUNX2 and bone alkaline phosphatase. Immunofluorescence was used to co-localize activin A with the undifferentiation marker stage-specific embryonic antigen 1. Cells treated with BMP-4 had an increased gene expression of activin A, osteocalcin and bone alkaline phosphatase (p < 0.05). In conclusion, BMP-4 increases activin A gene expression during mouse ES cell differentiation into bone precursors.

Topics: Activins; Animals; Ascorbic Acid; Bone Morphogenetic Protein 4; Cell Differentiation; Culture Media; Dexamethasone; DNA Primers; Fibroblasts; Gene Expression Regulation, Developmental; Glycerophosphates; Mice; Microscopy, Fluorescence; Mouse Embryonic Stem Cells; Osteogenesis; Real-Time Polymerase Chain Reaction; RNA, Messenger; Tretinoin

During mammalian embryogenesis, sclerotome-derived chondrocytes in the limb bud are arranged into a complicated bone shape with specific areas undergoing hypertrophy and calcification, creating a region-specific mineralized pattern in the cartilage. To follow chondrogenesis progression in vitro, we isolated limb cartilage from mice on embryonic day 13 (E13) and cultured it at the air-liquid interface after microsurgical removal of the ectoderm/epidermis. Explants underwent proper morphogenesis, giving rise to complete templates for limb bones in vitro. We found that region-specific calcification patterns resembling limbs of prepartum mature embryos could be induced in explants using culture medium containing high concentrations of CaCl2 (Ca), ascorbic acid (AA), and β-glycerophosphoric acid (BGP). In this culture system, excess amounts of all-trans retinoic acid (RA) severely disrupted morphogenesis and calcification patterns in limb cartilage. These effects were more pronounced in forearms than in phalanges. Although dissociated, the nascent chondrocytes in culture did not give rise to cartilage units even though augmented calcification was induced in these cell aggregates in the presence of RA. Taken together, our newly established culture system revealed that RA independently regulates three-dimensional morphogenesis and calcification.

Topics: Animals; Ascorbic Acid; Calcification, Physiologic; Cartilage; Cell Differentiation; Cell Line; Extremities; Glycerophosphates; Mice; Morphogenesis; Tissue Culture Techniques; Tretinoin

Primordial germ cells (PGCs) are useful for producing transgenic chickens and preserving genetic material in avian species. In this study, we investigated the in vitro differentiation potential of chicken PGCs into different cell types. For differentiation into adipocytes, chicken PGCs were cultured for 21 days in induction media containing dexamethasone, insulin and/or 3-isobutyl-1-methylxanthine (IBMX), and differentiation rates ranging from 74% to 91% were identified by oil red-O and alkaline phosphatase (ALP) staining. For differentiation into neuron-like cells, chicken PGCs were cultured for 3 or 7 days in the induction media containing retinoic acid (RA) and IBMX, and differentiation rates ranging from 71% to 87% were identified by toluidine blue staining and immunohistochemical staining. For differentiation into osteoblasts, chicken PGCs were cultured for 15 or 21 days in the induction media containing desamethasone, beta-glycerol phosphate and/or vitamin C, and differentiation rates ranging from 47% to 79% were confirmed by Von Kossa, cytochemical and immunohistochemical staining. These data suggest that, like mammalian PGCs, chicken PGCs can differentiate into different cell types in vitro.

Topics: 1-Methyl-3-isobutylxanthine; Adipocytes; Adipogenesis; Alkaline Phosphatase; Animals; Ascorbic Acid; Cell Culture Techniques; Cell Differentiation; Chickens; Dexamethasone; Embryonic Stem Cells; Germ Cells; Glycerophosphates; Insulin; Neurogenesis; Neurons; Osteoblasts; Osteogenesis; Tretinoin

Retinoic acid (RA) exerts a wide variety of effects on development, cellular differentiation and homeostasis in various tissues. However, little is known about the effects of RA on the differentiation of periodontal ligament cells. In this study, we investigated whether RA can affect the dexamethasone-induced differentiation of periodontal ligament cells.. Human periodontal ligament cells were differentiated via culturing in the presence of dexamethasone, ascorbic acid, and beta-glycerophosphate for mineralized nodule formation, as characterized by von Kossa staining. Continuous treatment with all-trans-RA inhibited the mineralization in a dose-dependent manner, with complete inhibition over 1 microm RA. Other RA analogs, 9-cis-RA and 13-cis-RA, were also effective. Furthermore, addition of RA for just the first 4 days completely inhibited the mineralization; however, as RA was added at later stages of culture, the inhibitory effect was diminished, suggesting that RA had a phase-dependent inhibition of mineralization. RA receptor (RAR)-alpha agonist (AM-580), but not retinoid X receptor agonist (methoprene acid), inhibited the mineralization, and reverse transcription-polymerase chain reaction analysis revealed that RAR-alpha was expressed on the cells, suggesting that RAR-alpha was involved in the inhibitory mechanism. This inhibition was accompanied by inhibition of alkaline phosphatase activity; however, neither expression of platelet-derived growth factor (PDGF) receptor-alpha, PDGF receptor-beta, or epidermal growth factor (EGF) receptor, nor phosphorylation of extracellular signal-regulated kinases triggered by PDGF-ascorbic acid or PDGF-BB was changed, as assessed by flow cytometry or western blot analyses.. These findings suggest that RA is a potential negative regulator for differentiation of human periodontal ligament cells.

Topics: Alitretinoin; Alkaline Phosphatase; Ascorbic Acid; Benzoates; Cell Differentiation; Cells, Cultured; Cementogenesis; Dexamethasone; Enzyme Inhibitors; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Glycerophosphates; Humans; Isotretinoin; Periodontal Ligament; Receptor, Platelet-Derived Growth Factor alpha; Receptor, Platelet-Derived Growth Factor beta; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Tetrahydronaphthalenes; Time Factors; Transcription Factors; Tretinoin

This study examines the capability of NIH3T3 fibroblasts to express osteoblastic markers following stimulation with a number of hormones and growth factors in vitro. Of the agents tested, 1alpha,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) dose-dependently induced alkaline phosphatase (ALP) activity in NIH3T3 cells, and this effect was enhanced by the addition of dexamethasone (Dex), which when administered alone caused no detectable ALP expression. The combined use of 1,25(OH)(2)D(3) and Dex also stimulated the synthesis of osteocalcin, and osteopontin. Furthermore, cells treated with the both hormones, in the presence of beta-glycerophosphate and l-ascorbic acid, formed mineralized plaques, indicating an osteoblast (OB) phenotype. By contrast, the differentiation induced by 1,25(OH)(2)D(3) or 1,25(OH)(2)D(3) plus Dex was significantly antagonized by transforming growth factor-beta1 and all trans-retinoic acid. These data indicate that NIH3T3 cells have the potential to adopt an OB-like phenotype and may prove to be a convenient model for studying the early events of osteogenic differentiation and the specific interactions of 1,25(OH)(2)D(3) with glucocorticoids in controlling this process in vitro.

Topics: 3T3 Cells; Alkaline Phosphatase; Animals; Biomarkers; Calcification, Physiologic; Calcitriol; Cell Differentiation; Cell Division; Collagen; Dexamethasone; Dose-Response Relationship, Drug; Fibroblasts; Glycerophosphates; Mice; Neoplasm Proteins; Osteoblasts; Osteocalcin; Osteopontin; Phenotype; Sialoglycoproteins; Transcription Factors; Transforming Growth Factor beta; Transforming Growth Factor beta1; Tretinoin

Pluripotent embryonic stem (ES) cells have the potential to differentiate to all fetal and adult cell types and might represent a useful cell source for tissue engineering and repair. Here we show that differentiation of ES cells toward the osteoblast lineage can be enhanced by supplementing serum-containing media with ascorbic acid, beta-glycerophosphate, and/or dexamethasone/retinoic acid or by co-culture with fetal murine osteoblasts. ES cell differentiation into osteoblasts was characterized by the formation of discrete mineralized bone nodules that consisted of 50-100 cells within an extracellular matrix of collagen-1 and osteocalcin. Dexamethasone in combination with ascorbic acid and beta-glycerophosphate induced the greatest number of bone nodules and was dependent on time of stimulation with a sevenfold increase when added to ES cultures after, but not before, 14 days. Co-culture with fetal osteoblasts also provided a potent stimulus for osteogenic differentiation inducing a fivefold increase in nodule number relative to ES cells cultured alone. These data demonstrate the application of a quantitative assay for the derivation of osteoblast lineage progenitors from pluripotent ES cells. This could be applied to obtain purified osteoblasts to analyze mechanisms of osteogenesis and for use of ES cells in skeletal tissue repair.

Topics: Animals; Antioxidants; Ascorbic Acid; Bone and Bones; Cell Differentiation; Cell Line; Cell Lineage; Cells, Cultured; Coculture Techniques; Dexamethasone; Embryo, Mammalian; Glucocorticoids; Glycerophosphates; Humans; Immunohistochemistry; Mice; Microscopy, Confocal; Osteoblasts; Osteogenesis; Stem Cells; Tretinoin

Embryonic stem (ES) cells have the capacity to differentiate into various cell types in vitro. In this study, we show that retinoic acid is important for the commitment of ES cells into osteoblasts. Culturing retinoic acid treated ES cells in the presence of the osteogenic supplements ascorbic acid and beta-glycerophosphate resulted in the expression of several osteoblast marker genes, osteocalcin, alkaline phosphatase, and osteopontin. However, there was only a slight amount of mineralized matrix secretion. Addition of bone morphogenic protein-2 or compactin, a drug of the statin family of HMG-CoA reductase inhibitors, resulted in a greatly enhanced formation of bone nodules. Compactin did not modify the expression of osteogenic markers, but at the late stage of differentiation promoted an increase in BMP-2 expression. These results establish ES-cell derived osteogenesis as an effective model system to study the molecular mechanisms by which the statin compactin promotes osteoblastic differentiation and bone nodule formation.

Topics: Alkaline Phosphatase; Animals; Antigens, Differentiation; Ascorbic Acid; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Cells, Cultured; Glycerophosphates; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin; Mice; Osteoblasts; Osteocalcin; Osteogenesis; Osteopontin; RNA, Messenger; Sialoglycoproteins; Stem Cells; Transforming Growth Factor beta; Tretinoin

Conditions have been defined for promoting growth and differentiation of hypertrophic chondrocytes obtained in culture starting from chick embryo tibiae. Hypertrophic chondrocytes, grown in suspension culture as described (Castagnola P., G. Moro, F. Descalzi Cancedda, and R. Cancedda. 1986. J. Cell Biol. 102:2310-2317), when they reached the stage of single cells, were transferred to substrate-dependent culture conditions in the presence of ascorbic acid. Cells showed a change in morphology, became more elongated and flattened, expressed alkaline phosphatase, and eventually mineralized. Type II and X collagen synthesis was halted and replaced by type I collagen synthesis. In addition the cells started to produce and to secrete in large amount a protein with an apparent molecular mass of 82 KD in reducing conditions and 63 KD in unreducing conditions. This protein is soluble in acidic solutions, does not contain collagenous domains, and is glycosylated. The Ch21 protein, a marker of hypertrophic chondrocytes and bone cells, was synthesized throughout the culture. We have defined this additional differentiation stage as an osteoblast-like stage. Calcium deposition in the extracellular matrix occurred regardless of the addition of beta glycerophosphate to the culture medium. Comparable results were obtained both when the cells were plated at low density and when they were already at confluence and maintained in culture without passaging up to 50 d. When retinoic acid was added to the hypertrophic chondrocyte culture between day 1 and day 5 the maturation of the cells to the osteoblast-like stage was highly accelerated. The switch in the collagen secretion was already observed after 2 d and the production of the 63-kD protein after 3 d. Mineralization was observed after 15-20 d.

Topics: Animals; Calcification, Physiologic; Calcium; Cartilage; Cell Differentiation; Cells, Cultured; Chick Embryo; Collagen; Culture Media; Extracellular Matrix; Glycerophosphates; Glycoproteins; Molecular Weight; Osteoblasts; Tretinoin