Hypertrophic chondrocytes undergo further differentiation in culture

Conditions have been defined for promoting growth and differentiation of hypertrophic chondrocytes obtained in culture starting from chick embryo tibiae. Hypertrophic chondrocytes, grown in suspension culture as described (Castagnola P., G. Moro, F. Descalzi Cancedda, and R. Cancedda. 1986. J. Cell Biol. 102:2310-2317), when they reached the stage of single cells, were transferred to substrate-dependent culture conditions in the presence of ascorbic acid. Cells showed a change in morphology, became more elongated and flattened, expressed alkaline phosphatase, and eventually mineralized. Type II and X collagen synthesis was halted and replaced by type I collagen synthesis. In addition the cells started to produce and to secrete in large amount a protein with an apparent molecular mass of 82 KD in reducing conditions and 63 KD in unreducing conditions. This protein is soluble in acidic solutions, does not contain collagenous domains, and is glycosylated. The Ch21 protein, a marker of hypertrophic chondrocytes and bone cells, was synthesized throughout the culture. We have defined this additional differentiation stage as an osteoblast-like stage. Calcium deposition in the extracellular matrix occurred regardless of the addition of beta glycerophosphate to the culture medium. Comparable results were obtained both when the cells were plated at low density and when they were already at confluence and maintained in culture without passaging up to 50 d. When retinoic acid was added to the hypertrophic chondrocyte culture between day 1 and day 5 the maturation of the cells to the osteoblast-like stage was highly accelerated. The switch in the collagen secretion was already observed after 2 d and the production of the 63-kD protein after 3 d. Mineralization was observed after 15-20 d.