Effects of coumestrol on neonatal and adult mice osteoblasts activities

Estrogen replacement therapy has been shown to reduce postmenopausal osteoporosis. In the present study, we examined the effects of the phytoestrogen coumestrol on neonatal and adult osteoblasts metabolism. Two different sources of osteoblast cells (neonatal mice calvaria and adult mice long bone) cultures were used in this study. The effects of coumestrol on the cellular activities were analyzed by the mitochondrial tetrazolium (MTT) assay, secretion of alkaline phosphatase (ALP), intracellular calcium content (Ca), and the gene expression of bone matrix protein, estrogen receptors (ER-α, ER-β), and osteoprotegerin (OPG) and osteoprotegerin ligand (OPGL). The results showed that the proliferation of neonatal mice osteoblast cells was enhanced by treatment of coumestrol. In the presence of 10^-9M coumestrol, the osteoblast proliferation attained 139.5% of the control and that the coumestrol can increase the intracellular calcium contents. Type I collagen gene expression was upregulated 167% at the 1st day's culture; ALP gene expression was upregulated 360% at the 7th day's culture; while the osteocalcin gene expression was upregulated 222% at the 14th day's culture. When adult mice osteoblasts were cultured in the presence of 10^-M coumestrol, the osteoblasts population increased significantly earlier and attained its maximal effect at the 21st day's culture with 207.4% of control group. The content of ER-β and osteoprotegerin secretion by neonatal mice control cells gradually increased during osteoblasts differentiation, whereas the ER-α and OPGL content were decreased in this study. The cellular responses to the estradiol and counmestrol were quite different in the osteoblasts derived from different age.