Intravital microscopic studies of angiogenesis during bone defect healing in mice calvaria

Abstract Purpose Due to the great availability of specific antibodies, gene-targeted animals and knockout strains, mouse models came into the focus of musculoskeletal research. Herein, we introduce a calvarian defect model in mice that allows the repetitive analysis of blood vessel formation during bone repair by intravital microscopy. Methods The right parietal calvaria of 20 adult CD-1 mice were exposed by skin excision. Under continuous irrigation, a circular defect (O0.75 mm) was drilled into the calvarium without penetrating the inner cortical shell. A circular glass (O12 mm; thickness 0.15 mm) was fixed by two microscrews (M1; length 2 mm) to cover the bone defect. Angiogenesis was analysed by intravital microscopy at days 0, 3, 6, 9, 12, 15, 18 and 21. In addition, bone repair was evaluated by histomorphometry at days 3, 6, 9 and 15. Immunohistochemical stainings for the angiogenic growth factor vascular endothelial growth factor (VEGF) and the cell proliferation marker proliferating cell nuclear antigen (PCNA) were performed to assess angiogenic and proliferative activity during healing of the calvarian defect. Results Histomorphometry showed a typical pattern of intramembranous bone repair. Osseous bridging of the defect was observed at day 9. This was associated with the formation of a neo-periosteum, which covered the new woven bone and contained a dense network of newly formed blood vessels. At day 9, particularly cells of the neo-periosteum showed intense staining for VEGF, whilst PCNA-positive staining was found mainly in osteoblasts. At day 15, the major fraction of fibrous tissue was replaced by bone undergoing extensive remodelling. Intravital microscopy revealed an increase of vascular density between days 3 and 15. Blood vessel diameters showed an increase between days 3 and 9 and a subsequent decrease between days 9 and 21. Conclusions The present calvarian defect model provides a powerful tool to evaluate the process of angiogenesis during intramembranous bone repair in mice.