RAPID CHONDROCYTE MATURATION BY SERUM-FREE CULTURE WITH BMP-2 AND ASCORBIC-ACID

In serum-containing medium, ascorbic acid induces maturation of prehyp ertrophic chick embryo sternal chondrocytes. Recently, cultured chondr ocytes have also been reported to undergo maturation in the presence o f bone morphogenetic proteins or in serum-free medium supplemented wit h thyroxine. In the present study, we have examined the combined effec t of ascorbic acid, BMP-2, and serum-free conditions on the induction of alkaline phosphatase and type X collagen in chick sternal chondrocy tes. Addition of either ascorbate or rhBMP-2 to nonconfluent cephalic sternal chondrocytes produced elevated alkaline phosphatase levels wit hin 24-72 h, and simultaneous exposure to both ascorbate and BMP yield ed enzyme levels at least threefold those of either inducer alone. The effects of ascorbate and EMP were markedly potentiated by culture in serum-free medium, and alkaline phosphatase levels of preconfluent ser um-free cultures treated for 48 h With BMP + ascorbate were equivalent to those reached in serum-containing medium only after confluence. Wh ile ascorbate addition was required for maximal alkaline phosphatase a ctivity, it did not induce a rapid increase in type X collagen mRNA. I n contrast, BMP added to serum-free medium induced a three- to fourfol d increase in type X collagen mRNA within 24 h even in the presence of cyclohexamide, indicating that new protein synthesis was not required . Addition of thyroid hormone to serum-free medium was required for ma ximal ascorbate effects but not for BMP stimulation. Neither ascorbate nor BMP induced alkaline phosphatase activity in caudal sternal chond rocytes, which do not undergo hypertrophy during embryonic development . These results indicate that ascorbate + BMP in serum-free culture in duces rapid chondrocyte maturation of prehypertrophic chondrocytes. Th e mechanisms for ascorbate and BMP action appear to be distinct, while BMP and thyroid hormone may share a similar mechanism for induction. (C) 1997 Wiley-Liss, Inc.