A novel cell culture model of chondrocyte differentiation during mammalianendochondral ossification

Endochondral ossification (EO) occurs in the growth plate where chondrocyte s pass through discrete stages of proliferation, maturation, hypertrophy, a nd calcification. We have developed and characterized a novel bovine cell c ulture model of EO that mirrors these events and will facilitate in vitro s tudies on factors controlling chondrocyte differentiation. Chondrocytes der ived from the epiphyses of long bones of fetal calves were treated with 5-a zacytidine (aza-C) for 48 h. Cultures were maintained subsequently without aza-C and harvested at selected time points for analyses of growth and diff erentiation status. A chondrocytic phenotype associated with an extensive e xtracellular matrix rich in proteoglycans and collagen types II and VI was observed in aza-C-treated and -untreated cultures. aza-C-treated cultures w ere characterized by studying the expression of several markers of chondroc yte differentiation. Parathyroid hormone-related protein (PTHrP) and its re ceptor, both markers of maturation, were expressed at days 5-9. Type X coll agen, which is restricted to the stage of hypertrophy, was expressed from d ay 11 onward. Hypertrophy was confirmed by a 14-fold increase in cell size by day 15 and an increased synthesis of alkaline phosphatase during the hyp ertrophic period (days 14-28). The addition of PTHrP to aza-C-treated cultu res at day 14 led to the down-regulation of type X collagen by 6-fold, show ing type X collagen expression is under the control of PTHrP as in vivo. Th ese findings show that aza-C can induce fetal bovine epiphyseal chondrocyte s to differentiate in culture in a manner consistent with that which occurs during the EO process in vivo.