Coordinated development of embryonic long bone on chorioallantoic membrane in ovo prevents perichondrium-derived suppressive signals against cartilage growth.

Perichondrium has been shown to elicit signals to suppress differentiation and proliferation of chondrocytes during endochondral bone formation based on in vitro organ culture. However, these in vitro organ cultures did not allow the growth of bone collar, and thus the effect of perichondrium in a normal environment where development of adjacent embryonic tissues, including bone collar, is taking place has not yet been fully understood. Therefore, we examined the effect of perichondrium on cartilage development using chicken long bone organ cultures on chorioallantoic membrane in ovo, which supported bone collar development. In contrast to previous observations in in vitro organ cultures, in ovo organ cultures prevented overgrowth of epiphyseal cartilage due to the removal of perichondrium. This prevention was associated with the suppression of aggrecan gene expression in the absence of perichondrium in ovo. These results indicated that the perichondrium-derived activity that was observed in vitro to suppress cartilage development could be counterbalanced in ovo, where culture conditions are closer to those in in vivo. TUNEL assay indicated enhanced apoptosis in the presence of perichondrium in vitro, and removal of the perichondrium suppressed apoptosis. No major apoptosis was observed in ovo regardless of the presence or the absence of perichondrium. Thus, chondrogenesis in long bone could be coordinately regulated through modulation of apoptosis by perichondrium and adjacent embryonic tissues, including bone collar, as revealed in in ovo assay.