Title

Maturation Specific Sensitivity of Osteocytes to Calcium and Phosphate Ion Pair-Induced Apoptosis

Date of Award

7-2012

Degree Type

Thesis

Degree Name

Master of Science in Biomedical Sciences

First Advisor

Christopher Adams, PhD, Thesis Advisor

Second Advisor

Susan Hingley, PhD

Third Advisor

Marina D'Angelo, PhD

Fourth Advisor

Marcus Bell, PhD

Abstract

During bone remodeling osteoblasts readily undergo apoptosis and previous work demonstrated the sensitivity of bone cells to microenvironmental apoptogens. As the lifespan of an osteocyte can extend into decades, osteocytic terminal differentiation must necessarily remove such sensitivity. Therefore, the goal of this study was to probe the relationship between osteocyte differentiation state and sensitivity to apoptosis. Two clones of murine long bone-derived osteocytes MLO-A5 and MLO-Y 4 cell lines, representative of early and late stage osteocytes, respectively, were seeded onto tissue culture wells, allowed to grow for 2, 7 or 14 days and subsequently exposed to increased levels of calcium and phosphate ion pairs for 24 hours. Following exposure, methyl thiazol tetrazolium (MTT) assays were performed to quantify cell death. Apoptosis was confirmed through caspase-3 staining. Osteocytic phenotypes were confirmed by alkaline phosphatase staining. Results indicated that, when treated with increased levels of the calcium and phosphate ion pair, both cell lines exhibited a time-in-culture dependent loss of sensitivity to apoptosis. However, at 7 days, the MLO-Y 4 cell line, representative of late osteocytes, exhibited greater sensitivity to apoptosis than the MLO-A5 cell line, representative of early osteocytes. These data suggest that, as opposed to a differentiation state dependent change in sensitivity to calcium and phosphate induced apoptosis, there are differences in the adaptability of each cell line to resistance to apoptosis. Thus, the cell line representative of late osteocytes is slower to become refractory to apoptosis. This work was supported by a grant from the Center for Chronic Disorders of Aging at the Philadelphia College of Osteopathic Medicine.

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