Functional vasculature is essential for maintaining skeletal integrity and preventing fracture. Microvessels within bone not only supply oxygen and nutrients directly to sites of active bone remodeling but also coordinate bone cell activity through vascular-bone tissue signaling. A number of clinical conditions that cause vascular dysfunction, including in limb vasculature, are associated with increased fracture incidence. However, the effects of these conditions on the health of vasculature within bone (osteovasculature), particularly on microvasculature, is not well understood. Current medical imaging techniques can measure macroscale changes to intraosseous perfusion in vivo in humans but cannot examine the structure or composition of the microvasculature. Further, even in animal models, osteovasculature is usually only measured at the end of the study using two-dimensional techniques, which do not accurately capture osteovascular structure or temporal changes to functional blood supply. Therefore, new tools are needed to characterize osteovasculature and assess whether osteovascular structure and function are altered in cases of clinical vascular dysfunction, such as obesity and ischemic stroke. Understanding how the microvasculature within bone is altered in these conditions may reveal potential roles for osteovascular involvement in skeletal fragility in these patients.

The goal of this dissertation was to develop and utilize techniques to assess intraosseous perfusion, osteovascular microstructure, and osteovascular composition in conditions associated with cardiovascular dysfunction. The first aim was to develop a technique for serial measurements of intraosseous perfusion in mice that would enable functional changes to blood supply to be assessed over time with disease progression or treatment. An existing laser Doppler flowmetry (LDF) technique was adapted and validated to quantify intraosseous perfusion weekly in the tibia without inducing inflammation or affecting future perfusion measurements. Next, the effect of obesity on osteovasculature was investigated in a mouse model of high fat diet-induced obesity. Intraosseous perfusion was measured with LDF and osteovascular microstructure was examined with immunofluoresence microscopy. Finally, the effect of ischemic stroke on osteovasculature was determined using a middle cerebral artery occlusion model in mice. Intraosseous perfusion was measured serially for four weeks during stroke recovery using our modified LDF technique, then osteovascular microstructure was quantified with contrast-enhanced micro-computed tomography, and endothelial cell composition was examined with immunofluoresence microscopy.

This work highlights the development of several techniques for quantifying the microvascular perfusion and structure within bone and demonstrates that osteovascular structure and function are altered in mouse models of obesity and stroke. These techniques can be used to inform future mechanistic studies of bone-vascular crosstalk, examine how osteovasculature is affected in other skeletal pathologies, and inform novel treatment strategies to mitigate skeletal fragility in these conditions.