Immunocytochemical Evidence on the Effects of Glucocorticoids on Type I Collagen Synthesis in Human Osteoblastic Cells

The effects of glucocorticoids on DNA synthesis and cellular function were assessed in cultures of human osteoblastic cells by using indirect immunoperoxidase staining with a type I antiprocollagen antibody and by measuring procollagen type I N and C propeptides (PINP, PICP) in the culture medium by radiometric methods. Likewise, we analyzed the correlation between intracellular immunostaining and procollagen propeptides released into the culture medium, as well as the correlation between PINP and PICP. Human osteoblasts were cultured with and without addition of dexamethasone (DEX) at two supraphysiological concentrations, 10⁻⁶ M and 10⁻⁷ M, for 24 and 48 h. Treatment with DEX at 10⁻⁶ M was associated with a significant decrease in the percentage of cells showing intracellular type I procollagen immunoreactivity at 24 and 48 h (P < 0.05). Similar effects were observed with 10⁻⁷ M DEX. Dexamethasone 10⁻⁶ M and 10⁻⁷ M also induced significant decreases in PINP and PICP values after 24 and 48 h of treatment (P < 0.05). The decrease in intracellular procollagen immunoreactivity and propeptide secretion was not associated with a reduction in DNA synthesis. A highly significant correlation was observed between the values of PINP and PICP in the culture medium as well as between the values of intracellular immunostaining and PINP and PICP (P < 0.001). In conclusion, our results suggest that supraphysiological doses of glucocorticoids produce a direct inhibition on osteoblastic function through their effect on type I procollagen synthesis. Immunoperoxidase detection of type I intracellular procollagen as well as the quantification of PINP and PICP in the culture medium are reliable methods of assessing osteoblast function.