Study on the Mechanism of the Bone Formation Process by 17β‐estradiol during Rat Bone Marrow Stromal Cell Culture

Abstract

The aim of this study is to clarify the mechanism of bone formation by using 17β‐estradiol (E2) during rat bone marrow stromal cell culture. Stromal cells were separated from female rat femora (seven weeks old), and were cultured in an Eagle Minimal Essential Medium (MEM) containing 15% fetal calf serum (FCS). After confluence, the cells were subcultured in a MEM containing 15% FCS (sex steroids removed by charcoal treatment) with dexamethasone, Na betaglycerophosphate and ascorbic acid phosphate for 18 days. They were then cultured in medium containing E2 or insulin‐like growth factor‐I (IGF‐I) alone or with anti‐IGF‐I antibody. On the 14th day, formation of white colored nodules showing positive ALP activity in ALP staining and showing calcium deposition in alizarin red S staining was observed in each culture system, indicating the beginning of calcification. The addition of E2 (10−7M or 10−8M), or IGF‐I (10 and 100 ng/ml) enhanced ALP activity and calcium content significantly, but 17α‐estradiol did not. Anti‐IGF‐I antibody significantly inhibited enhancing effects of E2 and IGF‐I on ALP activity and calcium deposition. Incubating with E2 alone (10–8M), the medium IGF‐I concentration and IGF‐Im RNA expression in the cells were significantly increased on days 10~14, but the expression of IGF‐I receptor mRNA had not changed.These results suggest that E2 participates in bone formation by promoting IGF‐I production in the bone marrow stromal cells just before calcification starts. ACTA OBST GYNAEC JPN Vol. 54, No. 10, pp1457–1466, 2002