Physiological Concentrations of Glucocorticoids Stimulate Formation of Bone Nodules from Isolated Rat Calvaria Cellsin Vitro

Abstract

Isolated rat calvaria cells plated at low density in medium supplemented with ascorbic acid and organic phosphate form discrete three-dimensional mineralized nodules having the characteristics of bone. We have studied the effects of glucocorticoids on the formation of bone nodules by these cell populations. Cells isolated from 21-day-old fetal rat calvaria were maintained in vitro for up to 27 days. Dexamethasone (Dex) induced a dose-related increase in the number of nodules formed, with a peak at 10 nM and a half-maximal response at about 1 nM. Dex (10 nM) also significantly increased the size of bone nodules formed (P < 0.002). High concentrations of Dex (1 .mu.M) did not increase nodule number. In cells in primary culture maintained in medium containing 10 nM Dex, the increase in nodule number was 50-100% over the control value. The effect of Dex was much greater in first subculture cells, where the number of nodules was 600-800% higher than the control value. Dishes collected and quantitated from 12-27 days showed that nodule formation ceased between 15 and 18 days in cultures without Dex, whereas in the presence of Dex the number of nodules increased up to 27 days. Addition of 10 nM Dex only during specific periods resulted in significantly more nodules than in control cultures, but significantly fewer nodules than in cultures constantly exposed to Dex. Cell population doubling times during log phase growth were unaltered, but a significant increase in saturation density (P < 0.001) was observed with 10 nM Dex. Hydrocortisone also caused an increase in the number of nodules formed, with a maximal effect of 50 nM and a half-maximal response at 8 nM. The results indicate that physiological levels of glucocorticoids stimulate bone nodule formation in long term cell culture by increasing the number of cells forming bone nodules and that maximization of the stimulatory effect of glucocorticoids on bone formation may require constant exposure to low levels of the hormone.