PubMed1996
A serum-free culture system has been developed to examine the biologic factors involved in the regulation of cellular maturation, extracellular matrix assembly, and calcification in the physis of the bovine fetal growth plate. Isolated prehypertrophic chondrocytes in high density culture undergo a process of cellular maturation whereby full expression of the hypertrophic phenotype is characterized first by type X collagen synthesis followed by matrix calcification. Using this culture system, we compared the capacity of tri-iodothyronine (T3) with thyroxine (T4) to stimulate expression of the hypertrophic phenotype and matrix calcification in three (B, C, and D) maturationally distinct prehypertrophic chondrocyte subpopulations. The B cell subpopulation was the most mature followed by C and D subpopulations in order of decreasing maturity. Comparisons were made to cultures in fetal calf serum (FCS). In Dulbecco's modified Eagle's medium supplemented with insulin, transferrin, and selenium, both hormones (T3/T4) separately induced, in a dose-dependent manner, chondrocyte maturation to the hypertrophic phenotype characterized by increased type X collagen mRNA and induction of protein synthesis of this molecule, together with increased alkaline phosphatase activity, and eventually calcification of the extracellular matrix. Such cellular maturation to the hypertrophic phenotype was not observed in the absence of T3 or T4 with subpopulations C and D. Only in older fetuses (> 210 days) was this observed and then only in the B subpopulation. Furthermore, T3 was at least 50-fold more potent than T4. The effects of T3 were most pronounced with the most immature cells (subpopulations C and D) where, in the case of the subpopulation C, in contrast to 0.5 nM T3 50 nM T4 was unable to induce expression of the hypertrophic phenotype. Alkaline phosphatase activity was also increased in the C cell subpopulation treated with 1 nM T3 (35.5 U/micrograms of DNA) over that supplemented with 50 nM T4 (7.8 U/micrograms of DNA). Furthermore, matrix calcification, measured by the incorporation of 45Ca2+ into the cell layer, always occurred earlier in cells cultured with T3 compared with T4. Cellular maturation to the hypertrophic phenotype was not accompanied by significant changes in DNA content; this ordinarily increases during culture in the presence of serum. Compared with cells cultured in the presence of serum, either thyroid hormone more potently induced cellular maturation. This study demonstrates that the most immature chondrocytes at the prehypertrophic stage are direct targets for T3 and T4 and, to a much a lesser degree, that either hormone is able to induce full chondrocyte hypertrophy from an early maturational stage leading to matrix calcification. But T3 is much more potent than T4. These studies also offer a new serum-free chemically defined medium containing T3 or T4 for the culture of defined prehypertrophic chondrocytes that supports matrix assembly, hypertrophic expression, followed by matrix calcification.