ESTROGEN ENHANCES DIFFERENTIATION OF OSTEOBLASTS IN MOUSE BONE-MARROWCULTURE

The effects of estrogen on bone are possibly mediated by several cell types. In the present study, the effect of 17 beta-estradiol (E-2) on osteoblast-like cells was investigated by using mouse bone marrow cult ures. Bone marrow cells were harvested from the shafts of femurs of 10 -week-old NMRI mice and cultured. On day 6, confluent primary cultures were trypsinized and subcultured. Under the conditions used (Keila, S ., Pitaru, S., Grosskopf, A., and Wernreb, M. Bone marrow from mechani cally unloaded rat bones expresses reduced osteogenic capacity in vitr o. J Bone Miner Res 9:321-327; 1994), the bone marrow cultures showed differentiation towards the osteoblastic phenotype. This was demonstra ted by the appearance of osteoblastic markers such as alpha 1(I) colla gen (COL1), alkaline phosphatase (ALP), osteocalcin (OCN), osteopontin (OF), and transforming growth factor-beta 1 (TGF-beta 1), which were detected by using reverse transcriptase polymerase chain reaction (RT- PCR). Bone nodule formation, including deposition of collagen fibers a nd matrix mineralization, was also studied at several time points of t he 3-week culture period. The effect of E-2 on the appearance of osteo blastic markers was studied by incubating cultures in the presence or absence of the hormone. The messenger ribonucleic acid (mRNA) for the estrogen receptor (ER) was found to be expressed at all time points as demonstrated by RT-PCR. When grown with E-2, the rate of cell prolife ration was increased in the early phase of cultures, but not after day 6. The addition of E-2 in subcultures resulted in an increase of leve ls of mRNA for COLI, ALP, OCN, OF, and TGF-beta 1. ALP activity was al so increased. Bone nodule formation, as well as calcium contents, were significantly increased in the cultures grown in the presence of E-2. All E-2 concentrations used (0.01-10 nmol/L) were effective but the m aximum response was obtained with 0.1 nmol/L E-2. Addition of the anti estrogen ICI 182,780 abolished the E-2-induced stimulation of prolifer ation and later an increase in ALP activity. Addition of ICI 182,780 w ithout the hormone did not cause any changes when compared to control cultures. In conclusion, our results demonstrate that E-2 stimulates s equential differentiation of osteoblasts and increases deposition and mineralization of matrix in mouse bone marrow cultures in an estrogen receptor-dependent manner. (C) 1998 by Elsevier Science Inc. All right s reserved.