Effects of 17-α ethynyl estradiol on proliferation, differentiation & mineralization of osteoprecursor cells

The steroidal estrogen 17α-ethynyl estradiol (EE) is an orally bio-active estrogen used in almost all modern formulations of estrogen-progestin combination preparations of oral contraceptives. Contrasting effects of treatment with combined oral contraceptives on bone mineral density of pre-, peri-, and post-menopausal women have been reported, and it has been suggested that the estrogen dose and the type of progestogen may be the main contributing factors for these contrasting results. The objective of this study was to evaluate the effects of EE on osteoprecursor cells.

The effects of single component of oral contraceptive, EE, were tested to see the relationship between EE and osteoblast proliferation, differentiation and mineralization. Tests used included a cell viability test, alkaline phosphatase (ALP) test, alizarin red-S staining, and a Western blot analysis. The effect on cell viability was determined by MTT assay. Differentiation and mineralization were examined using an ALP test and alizarin red-S staining. Protein expressions related to bone formation, such as estrogen receptor-alpha (ER-α), estrogen receptor-beta (ER-β), bone morphogenetic protein-2 (BMP-2), osteocalcin (OCN), and osteopontin (OPN) were evaluated by using a Western blot analysis.

Cultures growing in the absence of EE presented the lowest value for the MTT value. However, there were no significant changes in viability/proliferation when EE was added in the medium. Cultures growing in the absence of EE presented the highest value for the ALP activity, and the additional presence of EE resulted in dose-dependent decrease concerning ALP activity.

Our finding showed that EE in tested dosage within MC3T3-E1 cells seem to affect the proliferation and differentiation; however, significant differences are achieved in ALP activity in early differentiation phase and further studies are needed to elucidate the mechanisms of EE on bone.