Type X collagen synthesis during in vitro development of chick embryo tibial chondrocytes.

Abstract

Abstract. 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. 107:375-381). 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 der...

In vitro development of hypertrophic chondrocytes starting from selected clones of dedifferentiated cells.

“…Primary cultures, anchorage-dependent cultures, suspension cultures, and in vitro re.constitution of hypertrophic cartilage were performed as previously described (7,28). Briefly, Hamburger and Hamilton stage 28-30 (17) chick embryo tibiae were removed, cleaned, washed in Ca 2+-and Mg2+-free PBS, pH 7.2, and digested for 15 rain at 37°C with 400 U/ml collagenase I (CooperBiomedicai, Inc., Malvern, PA) and 0.25% trypsin (Gibco Ltd.).…”

“…These cells, as well as dedifferentiated chondrocytes from another source (4), maintain the dedifferentiated phenotype as long as the conditions for the cell attachment to the plastic are kept. When the same cells are transferred into suspension culture, they resume the chondrocytic phenotype and mature to hypertrophic chondrocytes through a transient stage of cell aggregation (7). This process is accompanied by an increase in the length of the cell cycle and in the number of quiescent and degenerating cells (16), as well as by a modulation in the transcriptional activity of the collagen genes (9).…”

Developmentally regulated synthesis of a low molecular weight protein (Ch 21) by differentiating chondrocytes.

“…Blenis and Hawkes have suggested that increased synthesis and deposition in the extracellular matrix of the 21K protein are general characteristics of the early stages of fibroblast transformation (3); we have found that the Ch 21 protein is synthesized in a large amount by normal chondrocytes. Although the real significance of this observation has to be further investigated, it is interesting to note that once more normal chondrocytes seem to share a property believed to be characteristic of transformed cells, other properties being: anchorage independent growth (8,13,28), capacity to form colonies in soft agar (13), presence on the cell surface of glycoproteins observed in transformed but not in normal fibroblasts (9). 4, 6, and 8).…”

“…Freshly dissociated chondrocytes from tibiae of stage 28-30 (11) embryos assume a fibroblast-like morphology and switch from the synthesis of type II to the synthesis of type I collagen when they are cultured as adherent cells on plastic dishes. When these dedifferentiated cells are transferred to agarose-coated dishes (suspension culture) they resume the chondrocyte phenotype and continue their maturation to single, isolated hypertrophic chondrocytes producing type X collagen (8). This process results in a large increase of the duration of all cell cycle phases and of the number of quiescent and degenerating cells (10).…”

Phylogeny and regulation of four lipocalin genes clustered in the chicken genome: evidence of a functional diversification after gene duplication

“…Methods for chondrocyte isolation and culture have been extensively described elsewhere (Castagnola et al, 1986 …”