Morehead State University
Characterization of Osteoblastic Properties of 7F2 and UMR-106 Cultures after Acclimation to Reduced Levels of Fetal Bovine Serum
Institution
Morehead State University
Faculty Advisor/ Mentor
Darrin DeMoss; Michael Fultz; 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 the development of a culturing environment reduced in estrogenic compounds. The selected media (OPTI-MEM) is enriched to sustain cultures under reduced fetal bovine serum (FBS) conditions and is devoid of the pH indicator phenol red. This protocol reduced the concentration of FBS supplementation to 0% through successive, 24-hour incubations with diminishing amounts of total FBS (1%, 0.1%, and 0%) and incorporated the use of charcoal filtered FBS. Although the rate of mitotic divisions declined, the protocol does not appear to alter the viability, cell morphology or osteoblast-like phenotype of 7F2 and UMR-106 cultures when compared to control cells grown in various concentrations of FBS. Utilizing a variety of techniques, cultures exposed to media devoid of FBS maintained the ability to express numerous osteoblast specific markers and some markers exhibited β-estradiol responsiveness. Hence, the cell culture protocol developed allowed cultures to proliferate while maintaining their osteoblastic phenotype and provides an alternative avenue to study the anti-resorptive role of estrogen on skeletal turnover. The use of calcium channel antagonists (diltiazem and nifedipine), to block voltage-regulated L-type calcium channels, were also investigated. This avenue of study was initially designed to observe the viability of osteoblast-like cells exposed to the antagonists and ultimately the flow of calcium across their membranes in a significantly reduced estrogen environment.
Characterization of Osteoblastic Properties of 7F2 and UMR-106 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 the development of a culturing environment reduced in estrogenic compounds. The selected media (OPTI-MEM) is enriched to sustain cultures under reduced fetal bovine serum (FBS) conditions and is devoid of the pH indicator phenol red. This protocol reduced the concentration of FBS supplementation to 0% through successive, 24-hour incubations with diminishing amounts of total FBS (1%, 0.1%, and 0%) and incorporated the use of charcoal filtered FBS. Although the rate of mitotic divisions declined, the protocol does not appear to alter the viability, cell morphology or osteoblast-like phenotype of 7F2 and UMR-106 cultures when compared to control cells grown in various concentrations of FBS. Utilizing a variety of techniques, cultures exposed to media devoid of FBS maintained the ability to express numerous osteoblast specific markers and some markers exhibited β-estradiol responsiveness. Hence, the cell culture protocol developed allowed cultures to proliferate while maintaining their osteoblastic phenotype and provides an alternative avenue to study the anti-resorptive role of estrogen on skeletal turnover. The use of calcium channel antagonists (diltiazem and nifedipine), to block voltage-regulated L-type calcium channels, were also investigated. This avenue of study was initially designed to observe the viability of osteoblast-like cells exposed to the antagonists and ultimately the flow of calcium across their membranes in a significantly reduced estrogen environment.