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.