Graduation Date

5-2014

Document Type

Master's Thesis

Degree Name

Master of Science

Department or Program

Biological Sciences

Department or Program Chair

Mary B. Sevigny, Ph.D.

First Reader

Simon Melov, Ph.D.

Second Reader

Dorn Carranza, Ph.D.

Abstract

The mineralized matrix of bone makes it difficult to examine specific populations of cells which are integral to the tissue using traditional molecular methods. For this study we examined single cell cortical osteoblasts derived from the femurs of C57BL/6J mice. After isolating single cells from bone, we were able to individually analyze their gene expression profiles using the quantitative polymerase chain reaction (qPCR). The mice used for the study were divided into 4 treatment groups, including ovariectomized mice (OVX) and sham surgical controls (SHAM), treated with or without the anti-resorptive bisphosphonate drug Alendronate, an effective FDA approved therapeutic for slowing bone loss in association with osteoporosis. To administer Alendronate, mice were treated with 100 μg/kg Alendronate weekly via intraperitoneal (IP) injections. After a 16- week period post-surgery, mice were euthanized and bone tissue, including spines and femurs, were preserved for analysis (n= 10 mice per treatment per time point). Single cell cortical osteoblasts were obtained from preserved femur through serial collagenase digestion. Osteoblasts cells were collected by selecting for CD31-/CD45-/Alkaline Phosphatase+ using fluorescence-activated cell sorting (FACS). Over 100 osteoblast cells per treatment group were analyzed using qPCR for the detection of 96 specific transcripts. We confirmed the effects of bone loss in the mice due to OVX surgery via CT scans of the collected spines and femurs at a resolution of 9μm. We identified unique expression patterns in each treatment subset of osteoblast cells. The single cell gene expression analysis revealed that the cell populations that were undergoing bone loss had genetic signatures with distinctive gene expression profiles. Analysis of cells at a single cell level may lead to a better understanding of the effects of anti-resorptive agents on the cell populations found within bone.

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