Endothelial cell specification at the osteogenic and angiogenic interface in cranial bone tissue engineering

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Abstract Repair and reconstruction of bone loss due to tumor resection, trauma and infection remains a significant clinical challenge. Worldwide, autografts or allografts are used in approximately 3 million orthopaedic procedures annually, of which 6% are craniomaxillofacial in nature. Bone tissue engineering has been hailed as the ultimate solution for replacing bone autograft in repair of bone defects. However, the long-term success of bone tissue engineering is impeded by inadequate vascularization of the engineered construct. The current lack of progress in vascularization of tissue engineered scaffold is attributed to our incomplete understanding of angiogenesis and vascular beds in bone repair and regeneration. A functional blood vessel network consists of arteries, veins and a capillary interface that connects arterial and venous microvessels for proper vascular perfusion. While the specification of arterial and venous endothelium has been well studied during early embryonic development, the postnatal regulation of arterial and venous expansion and specification at capillary level during repair and regeneration is poorly understood. A series of recent studies have suggested that hypoxia affects the endothelial cell (EC) specification at the osteogenic and angiogenic interface in development and aging. Genetic manipulation of the hypoxia inducible factor 1 (HIF-1) pathway markedly affects the formation of specific subsets of capillary vessels, termed Type H (CD31highEmcnhigh) vessels that couple to OSX+ osteoblasts at the long bone metaphysis. To gain a better understanding of the critical role of hypoxia at the osteogenic and angiogenic interface in repair and regeneration, we established a series of novel imaging approaches that permit high resolution, quantitative, and functional analyses of capillary vessels that couple to Col (I) 2.3 GFP+ osteoblasts at a cranial bone defect site. Utilizing these novel imaging approaches in a layer-by-layer enabled, nanofiber-mediated cranial defect repair model, we demonstrate that osteogenesis- dependent angiogenesis consists of morphologically and functionally distinct CD31+Emcn+ and CD31+Emcn- vessels. Examination of blood vessel type distribution and bone regeneration demonstrates differential angiogenic responses and contrasting distributions of CD31+Emcn+ and CD31+Emcn- vessels associated with Col I (2.3) GFP+ osteoblasts, new bone and non-bone forming tissue, suggesting that EC specification at the capillary level is a key component of osteogenesis-dependent angiogenesis in bone repair and regeneration. Based on these findings, we propose to examine the effects of hypoxia on EC specification and the impact of dysregulation of EC specification on bone formation during cranial defect repair and regeneration. Three complementary Aims will combine imaging, genetic and engineering approaches to defining the osteogenesis- dependent EC specification and the role of hypoxia in repair and regeneration. The success of our study will provide novel insights into mechanisms of osteogenesis and angiogenesis in repair, potentially offering novel translational targets for bone regeneration.