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The influence of various culture conditions was studied on the effect of GH and insulin-like growth factor-I (IGF-I) on DNA and matrix synthesis in epiphyseal rat chondrocytes in monolayer culture.
Chondrocytes from enzymatically digested rat tibia epiphyseal growth plates were seeded in 48-well culture plates and precultured for 10 days in Ham's F-12 medium supplemented with 1% (v/v) newborn calf serum and 1% (v/v) of a serum substitute. After preculture, the medium was changed to Ham's F-12 medium supplemented with 1% serum from hypophysectomized rats, and the effect of GH and IGF-I on DNA synthesis ([3H]thymidine incorporation) and matrix production ([35S]sulphate uptake) was studied during an additional 96-h culture period. Isotopes were present during the last 24 h of culture.
Both hGH and IGF-I stimulated DNA synthesis in a dose-dependent manner. A maximal effect of GH was seen at a concentration of 25 µg/l (60 ± 11% stimulation over control) and for IGF-I at 10 µg/l (162 ± 12%). The stimulatory effects of the same concentrations of human GH (hGH) and IGF-I on [35S]sulphate uptake were 135 ± 25 and 320 ± 42% respectively. In-vitro pulse labelling revealed that GH did not produce a response during the first 3 days of culture (after addition of GH) but was effective during days 4 and 5 of culture. In contrast, IGF-I was effective throughout the culture period. Pretreatment of cells with GH or IGF-I for 2·5 days showed that GH but not IGF-I produced a sustained effect on [3H]thymidine uptake.
In order to study the influence of cell density on the effect of GH and IGF-I on DNA synthesis, the effect of added peptides was evaluated after different preculture periods (5–15 days). A maximal stimulatory effect of hGH was seen at a cell density of 150 000–300 000 cells/cm2. GH had no significant effect at a low (< 100 000 cells/cm2) or a high (>400 000 cells/cm2) cell density. The magnitude of the stimulatory effect of IGF-I was the same at densities between 10 000 and 250 000 cells/cm2, but was reduced at higher cell densities (over 250 000 cells/cm2).
Chondrogenic properties of cells that had been cultured for 15 days were verified in vitro by positive alcian blue staining and identification of type II collagen, and in vivo by development of cartilage nodules in nude mice.
The results from the present study clearly show that GH and IGF-I both stimulate DNA synthesis and matrix production in epiphyseal chondrocytes in monolayer culture. The results also demonstrate that expression of the effect of GH is highly dependent upon the culture conditions.
Journal of Endocrinology (1992) 133, 291–300
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