Intravital imaging of neovascularization by two-photon laser scanning microscopy in tibial bone defects

Neovascularization plays a critical role in bone regeneration and skeletal development. Our understanding of weight-bearing bone healing has been hindered by the lack of a reliable method that allows tracking neovascularization at a high spatiotemporal resolution in living model. Thus, we employed two-photon laser scanning microscopy (TPLSM) for longitudinal analysis of angiogenesis of tibial bone defects in mice. In this study, we established an effective model for long-term visualization and longitudinal analyses of angiogenesis in tibial bone defect healing. The vessel structural can be imaged and analyzed in healthy and tibial bone defects mice for over 3 weeks. Blood flow could be tracked for 21 days post-surgery. During this tibial bone healing process imaging, we found the blood flow start at 12–14 days after surgery and the velocity reach 0.6205 mm/sed and 0.9784 mm/sed. After 21 days recovery, the vessel structural and functional recovered to normal with velocity of 3.7644 mm/s which corresponding to baseline. The establishment of a in vivo imaging platform provides a unique tool to better understand angiogenesis in tibial bone defects repair, enabling further investigation of structure and function of vascularization during weight-bearing bone healing.