Department of Periodontics, Seoul St Mary's Hospital, College of Medicine, The Catholic University of Korea, Seoul, Republic of Korea.
Indian J Med Res. 2012 Sep;136(3):466-70.
BACKGROUND & OBJECTIVES: 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.
INTERPRETATION & CONCLUSIONS: 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.
甾体雌激素 17α-乙炔基雌二醇（EE）是一种口服生物活性雌激素，几乎用于所有现代雌孕激素复方口服避孕药制剂。已有报道称，联合口服避孕药治疗对绝经前、围绝经期和绝经后妇女的骨密度有不同的影响，并且雌激素剂量和孕激素类型可能是导致这些结果不同的主要因素。本研究的目的是评估 EE 对成骨前体细胞的影响。
测试了口服避孕药的单一成分 EE，以观察 EE 与成骨细胞增殖、分化和矿化之间的关系。使用的测试包括细胞活力测试、碱性磷酸酶（ALP）测试、茜素红 S 染色和 Western blot 分析。通过 MTT 测定来确定细胞活力的影响。通过 ALP 测试和茜素红 S 染色来检测分化和矿化。通过 Western blot 分析评估与骨形成相关的蛋白表达，如雌激素受体-α（ER-α）、雌激素受体-β（ER-β）、骨形态发生蛋白-2（BMP-2）、骨钙素（OCN）和骨桥蛋白（OPN）。
在没有 EE 的情况下培养的细胞的 MTT 值最低。然而，当在培养基中添加 EE 时，细胞活力/增殖没有明显变化。在没有 EE 的情况下培养的细胞的 ALP 活性最高，而额外添加 EE 会导致 ALP 活性呈剂量依赖性下降。
我们的研究结果表明，在 MC3T3-E1 细胞中，测试剂量的 EE 似乎会影响增殖和分化；然而，在早期分化阶段，ALP 活性会有显著差异，需要进一步研究来阐明 EE 对骨骼的作用机制。
