ascorbate-2-phosphate and beta-glycerophosphoric-acid

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

A variety of different growth factors, most notably bone morphogenetic proteins (BMPs), have been shown to stimulate the osteogenic differentiation of mesenchymal stromal cells (MSCs) in vitro. Yet, due to the lack of comparative studies it remains unclear which protocol is the most effective in the induction of osteogenesis in MSC cultures. The aim of this study was to compare the most potent growth factors in regard to their osteoinductive potential. Human MSCs were cultured for 10 days in the presence of BMP-2, BMP-6, BMP-9 + IGF-2 and BMP-2, -6, -9 (day 1 + 2: 50 ng/ml; days 3-6: 100 ng/ml; days 7-10: 200 ng/ml). The formation of the osteoblast phenotype was assessed by quantification of osteoblast-related marker genes using reverse transcription polymerase chain reaction (RT-PCR) and alkaline phosphatase (ALP) staining. Matrix mineralization was assessed by alizarin red S and von Kossa staining. Statistical analysis was carried out using the one-way analysis of variance (ANOVA) followed by Scheffe's post hoc procedure. Among the tested growth factors the combination of BMP-2 + BMP-6 + BMP-9 most effectively induced the upregulation of collagen type I, collagen type V, osteocalcin, alkaline phosphatase, RUNX2, BMP-2, osteonectin and DLX5 (p < 0.01) and resulted in a consistent matrix mineralization. The findings suggest the combined addition of BMP-2, BMP-6 and BMP-9 to the osteoinductive culture medium containing dexamethasone, β-glycerophosphate and ascorbate-2-phosphate produces more potent osteoblast differentiation of human MSCs in vitro.

Topics: Alkaline Phosphatase; Ascorbic Acid; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 6; Calcification, Physiologic; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Collagen Type I; Collagen Type V; Core Binding Factor Alpha 1 Subunit; Culture Media; Dexamethasone; Glycerophosphates; Growth Differentiation Factor 2; Growth Differentiation Factors; Homeodomain Proteins; Humans; Insulin-Like Growth Factor II; Intercellular Signaling Peptides and Proteins; Mesenchymal Stem Cells; Osteoblasts; Osteocalcin; Osteogenesis; Osteonectin; Phenotype; Transcription Factors

The primary goal of periodontal treatment is regeneration of the periodontium. Current theories suggest that the periodontal ligament (PDL) cells have the capacity to participate in restoring connective and mineralized tissues, when appropriately triggered. We evaluated whether human PDL cell sheets could reconstruct periodontal tissue.. To obtain the cell sheet, human PDL cells were cultured on temperature-responsive culture dishes with or without osteogenic differentiation medium. The cell sheets were transplanted on periodontal fenestration defects of immunodeficient rats. Forty rats were divided in two groups: in one group, cell sheets cultured with control medium were transplanted and in the other, cell sheets cultured with osteogenic differentiation medium were transplanted. The defects were analysed histologically and histomorphologically after healing.. Most of the experimental group exhibited a new cementum-like layer and new attachment of collagen fibres to the layer. Histomorphological analyses indicated significant periodontal regeneration. The control group revealed dense extracellular matrix and fibre formation, but an obvious cementum layer was not observed.. Transplanted PDL cell sheets cultured with osteogenic differentiation medium induced periodontal regeneration containing an obvious cementum layer and Sharpey's fibres. Thus, the method could be feasible as a new therapeutic approach for periodontal regeneration.

Topics: Actins; Animals; Ascorbic Acid; Cell Culture Techniques; Cell Differentiation; Cells, Cultured; Cementogenesis; Culture Media, Conditioned; Dexamethasone; Glycerophosphates; Humans; Integrin-Binding Sialoprotein; Osteoblasts; Osteopontin; Periodontal Ligament; Rats; Rats, Nude; Regeneration; Sialoglycoproteins; Tooth Root

Human mesenchymal stem cells (hMSCs) isolated from bone marrow aspirates were cultured on silk scaffolds in rotating bioreactors for three weeks with either chondrogenic or osteogenic medium supplements to engineer cartilage- or bone-like tissue constructs. Osteochondral composites formed from these cartilage and bone constructs were cultured for an additional three weeks in culture medium that was supplemented with chondrogenic factors, supplemented with osteogenic factors or unsupplemented. Progression of cartilage and bone formation and the integration between the two regions were assessed by medical imaging (magnetic resonance imaging and micro-computerized tomography imaging), and by biochemical, histological and mechanical assays. During composite culture (three to six weeks), bone-like tissue formation progressed in all three media to a markedly larger extent than cartilage-like tissue formation. The integration of the constructs was most enhanced in composites cultured in chondrogenic medium. The results suggest that tissue composites with well-mineralized regions and substantially less developed cartilage regions can be generated in vitro by culturing hMSCs on silk scaffolds in bioreactors, that hMSCs have markedly higher capacity for producing engineered bone than engineered cartilage, and that chondrogenic factors play major roles at early stages of bone formation by hMSCs and in the integration of the two tissue constructs into a tissue composite.

Topics: Analysis of Variance; Ascorbic Acid; Bioreactors; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Culture Techniques; Chondrogenesis; Dexamethasone; Glycerophosphates; Humans; Immunohistochemistry; Insulin; Magnetic Resonance Imaging; Mesenchymal Stem Cells; Osteogenesis; Silk; Tissue Engineering; Transforming Growth Factor beta; Transforming Growth Factor beta1

Using a culture system that facilitates osteogenic differentiation of bone marrow-derived human mesenchymal stem cells, we analyzed gene-expression profiles during the mineralization process by means of a cDNA microarray system consisting of 23,040 genes. We compared expression profiles of the cells at days 3, 15, and 27 of incubation in media containing either a combination of 0.1 microM dexamethasone, 0.05 mM ascorbic acid-2-phosphate, and 10 mM beta-glycerophosphate, dexamethasone only, ascorbic acid-2-phosphate plus beta-glycerophosphate, or medium without any of these osteogenic supplements. Histochemical analysis revealed osteogenic differentiation of cells incubated in the presence of all three agents, but not in the other cultures. Comparison of the expression profiles disclosed transcriptional stimulation of 55 genes and repression of 82 genes among more than 20,000 examined. A set of differentially expressed genes we report here should contribute to a better understanding of the process of mineralization in the matrix surrounding human mesenchymal stem cells.

Topics: Antineoplastic Agents; Antineoplastic Agents, Hormonal; Ascorbic Acid; Cell Differentiation; Dexamethasone; DNA, Complementary; Down-Regulation; Glycerophosphates; Humans; Mesoderm; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; Time Factors; Transcription, Genetic; Up-Regulation