Spatiotemporal blood vessel specification at the osteogenesis and angiogenesis interface of biomimetic nanofiber-enabled bone tissue engineering.

While extensive research has demonstrated an interdependent role of osteogenesis and angiogenesis in bone tissue engineering, little is known about how functional blood vessel networks are organized to initiate and facilitate bone tissue regeneration. Building upon the success of a biomimetic composite nanofibrous construct capable of supporting donor progenitor cell-dependent regeneration, we examined the angiogenic response and spatiotemporal blood vessel specification at the osteogenesis and angiogenesis interface of cranial bone defect repair utilizing high resolution multiphoton laser scanning microscopy (MPLSM) in conjunction with intravital imaging. We demonstrate here that the regenerative vasculature can be specified as arterial and venous capillary vessels based upon endothelial surface markers of CD31 and Endomucin (EMCN), with CD31+EMCN- vessels exhibiting higher flowrate and higher oxygen tension (pO2) than CD31+EMCN+ vessels. The donor osteoblast clusters are uniquely coupled to the sprouting CD31+EMCN+ vessels connecting to CD31+EMCN- vessels. Further analyses reveal differential vascular response and vessel type distribution in healing and non-healing defects, associated with changes of gene sets that control sprouting and morphogenesis of blood vessels. Collectively, our study highlights the key role of spatiotemporal vessel type distribution in bone tissue engineering, offering new insights for devising more effective vascularization strategies for bone tissue engineering.