Type X Collagen Synthesis during In Vitro Development of Chick Embryo Tibial Chondrocytes

In the developing chick embryo tibia type X collagen is synthesized by chondrocytes from regions of hypertrophy and not by chondrocytes from other regions (Capasso, O., G. Tajana, and R. Cancedda, 1984, Mol. Cell. Biol. 4:1163-1168; Schmid, T. M., and T. F. Linsenmayer, 1985, Dev. Biol. 107:375381). To investigate further the relationship between differentiation of endochondral chondrocytes and type X collagen synthesis we have developed a novel culture system for chondrocytes from 29-31-stage chick embryo tibiae. At the beginning of the culture these chondrocytes are small and synthesize type II and not type X collagen, but when grown on agarose-coated dishes they further differentiate into hypertrophic chondrocytes that synthesize type X collagen. The synthesis of type X collagen has been monitored in cultured cells by analysis of labeled collagens and in vitro translation of mRNAs. When the freshly dissociated chondrocytes are plated in anchorage-permissive dishes, most of the cells attach and dedifferentiate, as revealed by their fibroblastic morphology. Dedifferentiated chondrocytes, after several passages, can still reexpress the differentiated phenotype and continue their development to hypertrophic, type X collagen-synthesizing chondrocytes. Hypertrophic chondrocytes, when plated in anchorage permissive dishes, attach, maintaining the differentiated phenotype, and continue the synthesis of type X collagen. T HE development and growth of long bones mainly occurs by endochondrai bone formation. During this process, chondrocytes in the bone growth region pass sequentially through a proliferative, a hypertrophic, and a degenerative stage (44). Differentiating chondrocytes synthesize collagens and other extracellular matrix macromolecules which are deposited in the cartilage. For several years type II collagen has been recognized as the only cartilage-specific collagen (29). More recently the existence of other minor collagens has been demonstrated, including 1 c~, 2a, 3a chains (6), and type IX (33, 34, 45) and X collagens. In particular, type X collagen, first detected in cultures of chick embryo tibial chondrocytes (8, 9, 37, 38) and in cultures of chick embryo sternal chondrocytes grown within collagen gels (16, 17) and subsequently in other species (35), becomes a major collagen in the regions where cartilage is removed and replaced by bone tissue. Its synthesis by hypertrophic chondrocytes and its association with endochondral development have been shown by metabolic labeling of proteins made by the cells (10, 20) and by immunofluorescent staining of cartilage with specific monoclonal antibodies (40). From the same regions of the tibiae the type X collagen deposited in the cartilage in vivo has been purified in milligram quantities (9, 32). After performing cultures of chondrocytes from sterna of chick embryos at different stages of development, Gibson and Flint (14) reported synthesis of type X collagen by chondrocytes derived from the presumptive calcification region but not by chondrocytes derived from the permanent cartilaginous region. In an attempt to establish a better correlation between differentiation of endochondral chondrocytes and synthesis of type X collagen, we developed a culture system in which tibial chondrocytes from 29-3 l-stage chick embryos (stage I chondrocytes) can continue their differentiation to endochondral hypertrophic chondrocytes synthesizing type X collagen (stage II chondrocytes) provided that their growth is anchorage independent. Under conditions of anchorage-dependent growth, most of the chondrocytes dedifferentiate, assume a fibroblastic morphology, and switch from the synthesis of type II to the synthesis of type I collagen; these cells express chondrogenic capability when retransferred to anchorageindependent growth conditions. In turn, stage II chondrocytes when plated on anchorage-permissive dishes maintain the differentiated phenotype and continue the synthesis of type X collagen. Mater ia ls and Me thods