Expression of the chondrogenic phenotype by mineralizing cultures of embryonic chick calvarial bone cells.
Cells released by sequential enzymatic digestion of 18-day chick calvariae were cultured over a 4-5 week period in Alpha modified Eagles medium. In some cultures the medium was supplemented with ascorbate and/or Na-beta-glycerophosphate. Microscopic examination of these cultures showed both polygonal and spindle-shaped cells. The biochemical nature of these cells was investigated by incubating the cultures with radiolabelled proline and subsequently analysing the medium and cell layer proteins by SDS/PAGE and fluorography. Osteoblast and chondrocyte-containing cultures were clearly distinguished in this way as the former cells secreted type I collagen while the latter secreted types II and X collagens as the major medium macromolecules. Type X collagen synthesis occurred after 14 days, but only in cultures supplemented with both ascorbate and Na-beta-glycerophosphate, and was maintained for the duration of the culture period. Unsupplemented cultures and those containing either ascorbate alone or Na-beta-glycerophosphate alone failed to synthesize type X collagen after 28 days. Isolated cells pulsed with radiolabelled proline at confluence and organ cultures of embryonic chick calvaria synthesized types I and V collagens only. These data demonstrate that the expression of phenotype by heterogeneous populations of bone cells could be modulated by a combination of culture conditions including the length of time in culture and conditions favourable for the formation of a mineralized matrix.
Department of Biochemistry and Molecular Biology, School of Biological Sciences, Manchester University Medical School, England.
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Shuttleworth
D000818:Animals; D001205:Ascorbic Acid / Q000494:pharmacology; D002356:Cartilage / Q000166:cytology / Q000187:drug effects / Q000196:embryology*; D002478:Cells, Cultured; D002642:Chick Embryo; D003094:Collagen / Q000096:biosynthesis / Q000302:isolation & purification; D046508:Culture Techniques / Q000379:methods; D005994:Glycerophosphates / Q000494:pharmacology; D006909:Hydroxyproline / Q000378:metabolism; D008970:Molecular Weight; D010012:Osteogenesis; D010449:Peptide Mapping; D010641:Phenotype; D011392:Proline / Q000378:metabolism; D014176:Protein Biosynthesis; D011506:Proteins / Q000302:isolation & purification
D005994:Glycerophosphates; D011506:Proteins; D003094:Collagen; D011392:Proline; C029620:alpha-glycerophosphoric acid; D001205:Ascorbic Acid; D006909:Hydroxyproline