Regulation of growth, protein synthesis, and maturation of fetal bovine epiphyseal chondrocytes grown in high-density culture in the presence of ascorbic acid, retinoic acid, and dihydrocytochalasin B

Phenotypic expression of chondrocytes can be modulated in vitro by changing the culture technique and by agents such vitamins and growth factors. We s tudied the effects of ascorbic acid, retinoic acid (0.5 and 10 mu M), and d ihydrocytochalasin B (3, 10, 20 mu M DHCB), separately or in combination (a scorbic acid + retinoic acid or ascorbic acid + DHCB), on the induction of maturation of fetal bovine epiphyseal chondrocytes grown for up to 4 weeks at high density in medium containing 10% fetal calf serum and the various a gents. In the absence of any agent or with retinoic acid or DHCB alone, the metabolic activity of the cells remained very low after day 6, with no ind uction of type I or X collagen synthesis nor increase in alkaline phosphata se activity. Chondrocytes treated with fresh ascorbic acid showed active pr otein synthesis associated with express ion of types I and X after 6 and 13 days, respectively. Th is maturation was not accompanied by obvious hypert rophy of the cells or high alkaline phosphatase activity. Addition of retin oic acid to the ascorbic acid-treated cultures decreased the level of type II collagen synthesis and delayed the induction of types I and X collagen, which were present only after 30 days. A striking increase in alkaline phos phatase activity (15-20-fold) was observed in the presence of both ascorbic acid and the highest dose of retinoic acid (10 mu M) DHCB was also a poten t inhibitor of the maturation induced by treatment with ascorbic acid, as t he chondrocytes maintained their rounded shape and synthesized type II coll agen without induction of type I or X collagen. The pattern of protein secr etion was compared under all culture conditions by two-dimensional gel elec trophoresis. The different regulations of chondrocyte differentiation by as corbic acid, retinoic acid, and DHCB were confirmed by the important qualit ative and quantitative changes in the pattern of secreted proteins observed by two-dimensional gel electrophoresis along the study. J. Cell. Biochem. 76:84-98, 1999. (C) 1999 Wiley-Liss, Inc.