Morehead State University
Characterization of Osteoblastic Properties of 7F2 Cultures after Acclimation to Reduced Levels of Fetal Bovine Serum
Institution
Morehead State University
Faculty Advisor/ Mentor
Michael Fultz; Darrin Demoss; David Peyton
Abstract
Estrogen plays an important role in skeletal physiology by maintaining a remodeling balance between the activity of osteoblasts and osteoclasts. In an attempt to decipher the mechanism through which estrogen elicits its action on osteoblasts, experimentation necessitated a culturing environment reduced in estrogenic compounds. The selected media (OPTI-MEM) is enriched to sustain cultures under reduced fetal bovine serum (FBS) conditions; this media is devoid of the pH indicator phenol red, (a suspected estrogenic agent). This protocol reduced the concentration of FBS to 0% through successive, 24-hour incubations with diminishing amounts of total FBS (1%, 0.1%, and 0%). The protocol does not appear to alter the viability, cell morphology, or osteoblast phenotype of the cell lines utilized (7F2) when compared to control cells grown in various concentrations of FBS. Though the rate of mitotic divisions apparently declined, the utilization of colorimetric assays, immunohistochemical techniques, and RT-PCR have verified that the cell line still express osteoblast cell-specific markers. Experimental results indicate that 7F2 cells remain viable at low and high doses (0.1nM to 1.9mM) of estrogen following a 24-hour incubation, but that prolonged exposure (48 hrs) to high concentrations results in cell death. These experimental findings suggest the culture protocol developed has not altered the osteoblast nature of the cell lines and provides a model system to study estrogen’s antiresorptive role on skeletal turnover and the potential impact of calcium channel antagonists on estrogen’s mechanism of action.
Characterization of Osteoblastic Properties of 7F2 Cultures after Acclimation to Reduced Levels of Fetal Bovine Serum
Estrogen plays an important role in skeletal physiology by maintaining a remodeling balance between the activity of osteoblasts and osteoclasts. In an attempt to decipher the mechanism through which estrogen elicits its action on osteoblasts, experimentation necessitated a culturing environment reduced in estrogenic compounds. The selected media (OPTI-MEM) is enriched to sustain cultures under reduced fetal bovine serum (FBS) conditions; this media is devoid of the pH indicator phenol red, (a suspected estrogenic agent). This protocol reduced the concentration of FBS to 0% through successive, 24-hour incubations with diminishing amounts of total FBS (1%, 0.1%, and 0%). The protocol does not appear to alter the viability, cell morphology, or osteoblast phenotype of the cell lines utilized (7F2) when compared to control cells grown in various concentrations of FBS. Though the rate of mitotic divisions apparently declined, the utilization of colorimetric assays, immunohistochemical techniques, and RT-PCR have verified that the cell line still express osteoblast cell-specific markers. Experimental results indicate that 7F2 cells remain viable at low and high doses (0.1nM to 1.9mM) of estrogen following a 24-hour incubation, but that prolonged exposure (48 hrs) to high concentrations results in cell death. These experimental findings suggest the culture protocol developed has not altered the osteoblast nature of the cell lines and provides a model system to study estrogen’s antiresorptive role on skeletal turnover and the potential impact of calcium channel antagonists on estrogen’s mechanism of action.