Apoptosis of terminal hypertrophic chondrocytes in an in vitro model of endochondral ossification.

It is widely accepted that growth plate chondrocytes undergo apoptosis when they reach the terminal hypertrophic stage of their differentiation during the process of endochondral ossification in vivo. In this report, an established chondrocyte cell culture model of mammalian endochondral ossification was utilized to investigate the fate of chondrocytes after they had entered hypertrophy in vitro. Fetal bovine epiphyseal chondrocytes were treated with the demethylating agent, 5-azacytidine, for 48 h and then cultured under azacytidine-depleted conditions. There was evidence for apoptosis in azacytidine-treated cells, as demonstrated by nuclear condensation and fragmentation (days 27 and 35) using transmission electron microscopy, and the detection of exposed phosphatidylserine on the plasma membrane surface of apoptotic chondrocytes (day 27) using fluorescence-labelled annexin V. Treated cultures on days 10 and 20 and untreated cultures at all corresponding time-points showed no morphological characteristics of apoptosis. In situ hybridization studies of treated cultures revealed that expression of the apoptotic suppressor, bcl-2, remained consistently high throughout the culture period, whilst the apoptotic inducer, bax, was not expressed until day 23. Quantification of these data showed a gradual shift in the ratio of the expression level of bcl-2 and bax in favour of bax with time in culture, particularly from day 23 onwards. Taken together, the results indicate that azacytidine-treated epiphyseal chondrocytes entered terminal hypertrophy from day 23 onwards in culture and died by apoptosis. This study confirms this culture system as a successful recapitulation of the entire mammalian chondrocyte differentiation pathway, including apoptosis. The culture model will prove valuable for studies of the apoptotic fate of terminally differentiated chondrocytes in the growth plate with a view to providing a better understanding of the underlying mechanisms of skeletal malformations and other pathological disorders such as osteoarthritis.