Effects of TGF-beta1 and triiodothyronine on cartilage maturation: in vitro analysis using long-term high-density micromass cultures of chick embryonic limb mesenchymal cells.

Endochondral ossification is initiated by differentiation of mesenchymal cells into chondrocytes, which produce a cartilaginous matrix, proliferate, mature, and undergo hypertrophy, followed by matrix calcification, and substitution of cartilage by bone. A number of hormones and growth factors have been implicated in this process. Using in vitro, long-term, high-density, micromass cultures of chick embryonic mesenchyme, that recapitulate the process of chondrogenesis, chondrocyte maturation, and hypertrophy, we have investigated the importance of a balance between proliferation and apoptosis in cartilage maturation, focusing specifically on the effects of transforming growth factor-beta1 (TGF-beta1) and the thyroid hormone, triiodothyronine (T3). Our results showed that TGF-beta1 stimulates proliferation, by week 2 of culture, and T3 inhibits proliferation by week 3. Cell size increases in cultures treated with T3. Collagen type X is expressed in all culture, and delay in matrix deposition is seen only in the cultures treated with TGF-beta1. T3 stimulates alkaline phosphatase activity, but not calcification. T3 enhances apoptosis, as seen by TUNEL staining, and internucleosomal DNA fragmentation. The results support the roles of T3 and TGF-beta in cartilage maturation, i.e., TGF-beta stimulates proliferation and suppresses hypertrophy, while T3 stimulates hypertrophy and apoptosis.