Effects of calcium deficiency on chondrocyte hypertrophy and type X collagen expression in chick embryonic sternum

Maintenance of chick embryos in long-term culture without their calcareous egg- shell is a useful method for studying the relation- ship between calcium homeostasis and cell differ- entiation during skeletogenesis. Previously, we have shown that in shell-less (SL) embryos, cal- cium deficiency induces a cartilage-like pheno- type in osteogenic tissues, such as calvaria (Jacenko and Tuan (19861 Dev. Biol. 115215). In this investigation, we have studied the relation- ship between cartilage calcification and hypertro- phy, and the expression of type X collagen, a spe- cific product of hypertrophic chondrocytes. For this study, the cephalic (calcifying) and caudal (permanently cartilaginous) regions of sterna from day 18 and day 20 normal (NL) and SL em- bryos were metabolically labeled with (l4C1-pro- line. Analysis of the biosynthetic products re- vealed significant differences in type X collagen expression in the cephalic region of sternal carti- lage. In NL tissues, type X collagen production in- creased from 13.1% of total collagen at day 18 to 43.7% at day 20. In contrast, in SL embryos, type X collagen was not detectable until day 20, when it represented only 1% of total collagen. Comparison of the NL and SL embryos with respect to their serum calcium level and sternal calcium content and histology revealed a direct relationship be- tween low systemic calcium and limited cartilage hypertrophy, undermineralization, and decreased type X collagen production in the sternal cephalic cartilage. Supplementation of CaCO, to SL em- bryos increased their serum and sternal calcium, and restored cartilage hypertrophy, mineraliza- tion, and type X collagen synthesis in the cephalic portion of the sterna. These findings confirm that a critical relationship exists between calcium ho- meostasis, chondrocyte hypertrophy, mineraliza- tion, and type X collagen synthesis in the cephalic region of sternal cartilage. These results further demonstrate the importance of calcium in the morphogenetic events of endochondral ossifica- tion, in particular the transition from hyaline car- tilage to hypertrophic cartilage, and eventually to