Morehead State University

Development of an [i]in vintro[/i] Protocol to Study Estrogen-Mediated Osteoblast Activity in the Absence of Fetal Bovine Serum

Institution

Morehead State University

Abstract

Estrogen plays an important role in skeletal physiology by maintaining a remodeling balance between the activity of osteoblasts and osteoclasts. Experimental studies of estrogen-mediated osteoblast culture [i]in vitro[/i] show that estrogen directly acts on osteoblasts. In order to determine the mechanism by which estrogen elicits this action in osteosarcoma cells (7F2), experiments were carried out in the absence of estrogenic compounds. The culture medium (OPTI-MEM supplemented with 0.1% FBS) was changed to FBS-free medium 48 hrs before the start of an experiment to eliminate the estrogenic activity of FBS, and the use of OPTI-MEM medium eliminates phenol red, another potential estrogenic factor. This protocol development did not change the viability or the morphology of the cells as compared to control cells grown with FBS. Experimental results show that estrogen at both low and high doses (0.1nM to 1.9mM) stimulate cell proliferation of the 7F2 cells after 24 hours of incubation; demonstrating estrogen’s osteoblastic activity. Estrogen upregulates the transcription of osteoprotegerin (OPG), a decoy receptor, which antagonizes receptor activator of nuclear factor kB (RANK) preventing the differentiation/activation of osteoclasts. These experimental findings thus support the hypothesis of an antiresorptive role for estrogen in skeletal turnover.

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Development of an [i]in vintro[/i] Protocol to Study Estrogen-Mediated Osteoblast Activity in the Absence of Fetal Bovine Serum

Estrogen plays an important role in skeletal physiology by maintaining a remodeling balance between the activity of osteoblasts and osteoclasts. Experimental studies of estrogen-mediated osteoblast culture [i]in vitro[/i] show that estrogen directly acts on osteoblasts. In order to determine the mechanism by which estrogen elicits this action in osteosarcoma cells (7F2), experiments were carried out in the absence of estrogenic compounds. The culture medium (OPTI-MEM supplemented with 0.1% FBS) was changed to FBS-free medium 48 hrs before the start of an experiment to eliminate the estrogenic activity of FBS, and the use of OPTI-MEM medium eliminates phenol red, another potential estrogenic factor. This protocol development did not change the viability or the morphology of the cells as compared to control cells grown with FBS. Experimental results show that estrogen at both low and high doses (0.1nM to 1.9mM) stimulate cell proliferation of the 7F2 cells after 24 hours of incubation; demonstrating estrogen’s osteoblastic activity. Estrogen upregulates the transcription of osteoprotegerin (OPG), a decoy receptor, which antagonizes receptor activator of nuclear factor kB (RANK) preventing the differentiation/activation of osteoclasts. These experimental findings thus support the hypothesis of an antiresorptive role for estrogen in skeletal turnover.