Date of Award

1-1-2018

Embargo Period

1-1-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pathology and Laboratory Medicine

College

College of Graduate Studies

First Advisor

Amanda C. LaRue

Second Advisor

James J. Cray

Third Advisor

Victoria J. Findlay

Fourth Advisor

Lee R. Leddy

Fifth Advisor

Mary Ann McCrackin

Sixth Advisor

Meenal Mehrotra

Abstract

Bone remodeling requires a continuous supply of osteoblasts and osteoclasts from bone marrow (BM) stem cells. The traditional paradigm suggests mesenchymal stromal cells (MSC) generate osteoblasts, while hematopoietic stem cells (HSC) generate osteoclasts. However, our lab has shown, through murine clonal cell transplantation, HSCs are able to differentiate into osteoblasts, osteocytes, and hypertrophic chondrocytes during non-stabilized fracture repair. Based on these studies, we hypothesized that murine HSC-derived cells differentiate into functional osteoblasts that can promote fracture repair. HSC-derived CD45+ adherent BM cells were shown to differentiate into osteoblasts in vitro, based on flow cytometry, qRT-PCR, and histochemical staining. Use of the VavR double transgenic mouse model confirmed HSC origin. IGF-2 and, to a lesser extent, BMP-9 were found to enhance differentiation. Hematopoietic circulating cells that expressed CD34 (hematopoietic/endothelial marker) and osteocalcin (OCN; osteoblast marker) were found in murine peripheral blood (PB) at levels similar to those observed in human studies. Following non-stabilized tibial fracture, circulating CD34+OCN+ cells peaked three weeks post-fracture, suggesting involvement in cartilage callus transformation to bone and early mineralization based on micro-CT analysis. Immunofluorescent staining demonstrated presence of CD34+OCN+ cells within fracture callus. For potential therapeutic application, CD34+OCN+ cells were found elevated in PB after three days of AMD3100 treatment, a CXCR4 antagonist. However, short-term AMD3100 administration did not promote non-stabilized fracture repair. Lastly, we developed a murine femoral critical-sized defect atrophic nonunion model. Acellular skin graft was seeded with CD45+ adherent cells, IGF-2, and BMP-9 and tested for ability to prevent atrophic nonunion. The graft was shown to retain cells, secrete growth factor for up to 14 days, and appeared mineralized in treatment groups compared to controls. Although there was no evidence of enhanced repair based on bone morphometric analyses, we mainly attribute this result to technical difficulties in graft placement. Future studies will aim to expand our current findings and test ability of HSC-derived osteoblasts, in combination with scaffolds and growth factors, to prevent atrophic nonunion formation. Long-term, these studies have potential to demonstrate HSC-derived cells as a novel therapeutic source of osteoprogenitors with sustained engraftment that could overcome limitations associated with MSC clinical use.

Rights

All rights reserved. Copyright is held by the author.

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