Estrogen added intermittently, but not continuously, stimulates differentiation and bone formation in SaOS-2 cells

Although it is well established that estrogen inhibits bone resorption, its effects on bone formation remain controversial. We studied the effects of intermittent and continuous treatment with estrogen on bone formation in vitro using long term cultures of SaOS-2 cells under conditions that permit mineralization. SaOS-2 cells cultured in dexamethasone, ascorbic acid and beta-glycerophosphate for up to 17 d formed mineralized bone nodules as visualized by von Kossa staining. Electron microscopic analysis of ultrathin sections of representative mineralized nodules showed the presence of mineral deposits, collagen fibrils and osteocytes. Both the mineralized nodule numbers and areas increased exponentially with time of culture after addition of beta-glycerophophate at day 8. Intermittent addition of 17beta-estradiol (E(2)) for 6 h or 24 h of every 48 h starting at day 3 or day 8 to the end of culture period resulted in a specific time- and dose-dependent stimulation of mineralized bone nodule number and area, and alkaline phosphatase activity which were accompanied with increase in cell numbers. On the other hand, continuous treatment with E(2) added every 48 h had no effect. The estrogen receptor alpha (ERalpha) mRNA expression was stimulated after 6 or 24-h (intermittent), but not after 48-h (continuous) treatment with E(2). The stimulatory effect of E(2), when added intermittently, but not continuously, on differentiation and bone formation in human osteoblasts in culture may be relevant to previous reports of stimulatory effects of E(2) on bone formation in vivo.