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The Role of Angiogenesis in Osteogenic Mechanical Loading
Date of Award
Doctor of Philosophy (PhD)
Osteogenesis, the process of new bone tissue formation, is critically linked to angiogenesis, the process by which new vessels extend from the existing vascular system. However, the role of angiogenesis in osteogenic mechanical loading, a robust bone formation stimulus, is unknown. To study the interaction between angiogenesis and osteogenesis, one of two mechanical loading protocols designed to induce new bone formation at the midshaft of the ulna was applied to the right forelimb of each animal. The damaging loading protocol creates fatigue damage at the mid-diaphysis of the ulna that stimulates an abundant woven bone response with immediate decreases in stiffness and strength, accurately simulating a stress fracture. The non-damaging loading protocol stimulates lamellar bone formation at the same location without creating damage or decreasing bone strength, similar to physiological skeletal loading. The first aim was to determine the time course of changes in blood flow rate and vascularity using in vivo imaging and histological techniques following osteogenic mechanical loading. Using PET imaging, blood flow rate was increased as early as 4 hours following damaging mechanical loading, but there were no changes in blood flow rate following non-damaging mechanical loading. Investigation of this early response to damaging mechanical loading revealed inflammation-mediated vasodilation that was characterized by increased blood flow rates, NO signaling, and expanded vessels near the site of bone formation, and could be blocked using the NOS inhibitor, L-NAME. In the second aim, angiogenesis was inhibited following damaging and non-damaging loading by targeted nanoparticles in rats or gene knockout in mice. In rats, there were no changes in vascularity 3 days after damaging mechanical loading, but fewer angiogenic (CD105 positive) blood vessels were present at the site of bone formation in animals treated with anti-angiogenic nanoparticles. Seven days after loading, treatment was associated with less vasculature and less woven bone, demonstrating that vascular expansion by angiogenesis begins about 3 days after a stress fracture and is required for a full osteogenic response. Additionally, anti-angiogenic treatment resulted in significant decreases in the mechanical properties of the skeletal repair, particularly manifested in decreased post-yield energy dissipation. Finally, mice with HIF-1α selectively deleted from osteocalcin-expressing cells (osteoblasts and osteocytes) were subjected to damaging and non-damaging mechanical loading. As expected, ΔHIF-1α mice produced significantly less woven bone and had decreased vascularity following damaging mechanical loading. In addition, ΔHIF-1α mice had increased lamellar bone formation following non-damaging mechanical loading, due to non-angiogenic effects of the knockout. In total, the results from these studies have clarified the role of angiogenesis in osteogenic mechanical loading and may have implications for inducing rapid repair of stress fractures as well as increasing formation of lamellar bone.
Matthew J Silva
Fanxin Long, Larry A Taber, Stavros Thomopoulos