Apoptotic Sensitivity in Osteoblasts is Maturation Specific

Date of Award

7-2013

Degree Type

Thesis

Degree Name

Master of Science (MS)

First Advisor

Christopher Adams, PhD

Second Advisor

Denah M Appelt, PhD

Third Advisor

Marina D'Angelo, PhD

Fourth Advisor

Marcus Bell, PhD

Abstract

Past research has shown that osteoblasts exhibit sensitivity to apoptogens present within their microenviromnent. Previous work from our laboratory observed small differences in apoptotic sensitivity with maturation in osteoblasts. Thus, our goal in this study has been to explore the mechanism by which maturing osteoblasts become more sensitive to apoptosis. For this study, the Human Fetal Osteoblast (hFOB) cell line was used. These cells show an early progenitor phenotype when cultured at 34°C and mature at temperatures above 37°C. Cells were cultured for 1, 2, 3, and 7 d at 34°C and 39°C and were then exposed to elevated levels of the phosphate and calcium ion pair. Cell viability was measured using the MTT assay and expressed as percentages of control cell numbers. Apoptosis was confirmed using Caspase-3/7 assay and Phi Phi Lux staining. Changes in mitochondrial membrane permeability were evaluated using the Mitochondrial ToxGlo and Cytochrome c assays, while the mitochondrial membrane potential (MMP) was measured using the confocal fluorochrome, MitoTracker Red. Results showed that hFOB cells show an increased sensitivity towards to apoptosis as they mature. Immature cells show a possible hyperpolarization of the MMP, while matured cells did not. Mature osteoblasts demonstrate an earlier release of cytochrome c compared to immature cells. Apoptosis was confirmed in both cell populations, with an increase in caspase-3 activity within 4 h of exposure of the ion pair. These results suggest that osteoblasts have increased sensitivity towards apoptosis as they mature. Furthermore, it seems as if matured osteoblasts show a more rapid response to the Pi/Ca2+ ion pair than do the immature cells. This study was supported by a grant from the Center for Chronic Disorders of Aging at the Philadelphia College of Osteopathic Medicine.

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