Utilising high resolution imaging to interrogate blood vessel and bone cell interactions

Abstract

Fragility fractures represent a socio-economic burden, yet effective systematic treatments for the prevention of osteoporotic fractures are still lacking. Bone is a dynamic and highly vascularised tissue. Evidence suggests that with ageing, low bone mass and disruption of the microstructure and mechanical properties could be driven by reduced vascular supply and blood vessel attracting signals. However, it remains unclear whether alterations in the intracortical vasculature occur with age and if preservation of the cortical vasculature could prevent bone fractures. The main objective of this PhD was to develop and utilise high resolution micro-computed tomography (µCT) imaging ex-vivo of cortical bone microstructure to test the hypothesis that bone vascular networks are altered with age. The following aims have been addressed i) To develop an image processing and analysis framework that allows for systematic measurement of the 3D architecture of cortical porosity comprising vascular canal networks and osteocyte lacunae in murine cortical bone ii) Application and validation of developed methodology to assess the effects of age on vascular canal phenotype iii) Extension of methodology to a unique transgenic mouse strain were osteoblast-derived vascular endothelial growth factor (VEGF) has been deleted iv) Application of phase-contrast enhanced X-ray tomography imaging to visualise soft tissue within vascular canal networks. Using both desktop µCT (1.7 µm) and synchrotron X-ray tomography (0.65 μm), cortical bone microstructure was assessed at a sufficient spatial resolution to detect and extract cortical porosity. Extracted porosity measurements from synchrotron X-ray tomography were classified into vascular canals and osteocyte lacunae and 3D spatial relationships computed. First, the tibiofibular junction from 15-week and 10-month-old female C57BL/6J mice (n=6) was selected and vascular networks compared. It was found that the posterior region of the tibiofibular junction had a higher vascular canal volume than the anterior, lateral and medial regions (+127.62%, 693.08% and 659.64% respectively, p<0.05) at 15 weeks of age. By 10 months, bone cortices were thinner (-13.04%, p<0.01) and reduction in vascular density was evident in the posterior region (-46.54%, p<0.01) providing the first evidence for location of the intracortical vasculature impacting age related effects on bone porosity. To explore the effect of osteoblast-derived VEGF on the intracortical microstructure, VEGF was knocked out (KO) in mature osteocalcin (Ocn) expressing osteoblasts. Again, the tibiofibular junctions of aged (1 year) wildtype (WT) and transgenic (VEGF Ocn KO) female mice (n=5) were imaged, analysed and compared. Results revealed that the lack of osteoblast-derived VEGF increased total porosity (vascular networks and osteocyte lacunae combined) in the tibiofibular junction (+39.92%, p<0.01) with changes evident in the anterior and posterior compartments. Attempts to extend the use of the developed methodology to separate osteocyte lacunae from vascular canals was unsuccessful in the VEGF Ocn KO model due to low mineralised matrix in the VEGF Ocn KO bones. Finally, an approach that allows the 3D visualisation and assessment of the soft tissues in calcified bone using phase contrast-enhanced X-ray tomography has been reported. Using this technique, vascular structures were detected within 95.77% of the intracortical canals of the murine tibiofibular junction, supporting the theory that the intracortical network is the living space of the bone vasculature. This project has developed novel methodology which has allowed demonstration of an age-related reduction in the intracortical vasculature associated with reduced cortical bone thickness and deleterious changes in bone porosity due to the lack of VEGF, thus supporting further investigations into targeting the blood supply to treat age-related bone disease.

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