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Prepubertal rabbit epiphyseal chondrocytes were grown in high density primary culture for 3 d. They were then incubated for 3 additional d in serum-free culture medium to which bFGF (1-50 ng/ml) was added. During the last 24 h incubation period, either IGF1 (1-80 ng/ml) or Insulin (1-5 micrograms/ml) was added to the culture medium. Chondrocyte DNA was significantly augmented with the increasing concentration of bFGF used, thus confirming its mitogenic effect on chondrocytes. On the other hand, bFGF was also shown to modulate the phenotypic expression of the chondrocytes. The 35S-sulfate incorporation into newly synthesized proteoglycans by the cultured cells decreased in a dose-dependent manner with bFGF concentration used. In addition, chondrocyte collagen gene expression was also shown to be modulated by bFGF. Total RNA extracted from the cultured cells was analyzed by dot blot and Northern blot with cDNA probes encoding for alpha 1 II and alpha 1 I procollagen chains. A significant lower level of type II collagen mRNA, the marker of chondrocytic phenotype, was observed when cells were grown in the presence of bFGF while the level of type I mRNA remained unchanged. When IGF1 or a high concentration of insulin was added to the cells during the last 24 h of incubation with bFGF, sulfated proteoglycan synthesis, as well as collagen type II mRNA level, were significantly stimulated when compared with chondrocytes incubated with bFGF alone. In conclusion, in the present experimental conditions, bFGF appears to be a growth promoting agent for chondrocytes in vitro with dedifferentiating action on chondrocyte phenotype. IGF1 or insulin used at a high concentration can prevent the dedifferentiating effect of bFGF without inhibiting its stimulating effect on chondrocyte DNA synthesis.

This paper reports data on the in vitro effects of insulin-like growth factor I (IGF-I) and basic fibroblast growth factor (bFGF) on the phenotypic expression of epiphyseal chondrocytes grown in serum-free (SF) culture medium. bFGF mostly stimulates chondrocyte DNA and inhibits sulfated proteoglycan synthesis and type II collagen mRNA. On the contrary, IGF-I is poorly mitogenic but strongly stimulates protein synthesis and type II collagen mRNA. In addition, IGF-I prevents the expression of type I collagen gene. Lastly, chondrocytes cultured in SF medium are able to locally produce IGF-I peptides. In conclusion, IGF-I and bFGF have opposite effects on the phenotypic expression of chondrocytes in vitro: bFGF is mostly mitogenic and IGF-I appears to be a differentiating factor.

This study concerns the immunoreactive somatomedin C secretion by prepubertal rabbit epiphyseal chondrocytes cultured in a defined serum-free medium. In such culture conditions, chondrocytes mainly synthesized Type II collagen (80% of total collagen) during 10 days. A small amount of Type I collagen was also found with a significant (p less than 0.05) higher level during the period of cell multiplication (6.4 +/- 1.5%) than when cells reached confluency (0.9 +/- 0.2%). During the 10 days of culture without serum and without hormone added, a Sm-C/IGF1 activity was measured by RIA at a mean level of 30 +/- 5 mU/ml/10 micrograms DNA. This value was significantly higher (p less than 0.001) than in the medium not incubated with the cells (1.7 +/- 0.9 mU/ml). When hGH was added to the culture medium during the period of cell division, the level of Sm-C/IGF1 activity was significantly elevated at 39 +/- 4 mU/ml/10 micrograms DNA (p less than 0.05) and at 55 +/- 3 mU/ml/10 micrograms DNA (p less than 0.001) with 50 ng/ml and 100 ng/ml hGH concentrations respectively. On the contrary, no difference was observed at confluency in treated and non treated cells.

To determine whether growth hormone has a direct effect on skeletal tissues not mediated by somatomedins, and to better define the role of somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) in skeletal development, bovine growth plate, and rabbit articular and growth plate chondrocytes in primary culture were evaluated under a variety of experimental conditions designed to elicit growth hormone and Sm-C/IGF-I stimulation. Under none of these conditions did bovine growth plate chondrocytes respond to either homologous bovine growth hormone or heterologous hGH. Under the same conditions, these cells were highly responsive to human Sm-C/IGF-I with respect to both [3H]thymidine and [35S]sulfate incorporation, indices of mitotic and differentiated cell functions, respectively. Similarly, both rabbit articular and growth plate chondrocytes showed enhanced incorporation of [3H] thymidine and [35S]sulfate in the presence of Sm-C/IGF-I, but did not respond to either native or recombinant hGH. Cells at different stages of maturation within the bovine growth plate differed in their reaction to Sm-C/IGF-I with proliferative zone cells manifesting a greater response to the peptide than cells of the reserve zone. These results suggest that the action of Sm-C/IGF-I on growth plate and articular chondrocytes is direct and that the effect of GH on these cells is indirect. The data further suggest that within the growth plate, the transition from reserve to proliferative status is associated with an increased Sm-C/IGF-I responsiveness, a change which may contribute to the functional differences in these cells.