M2c Macrophages Mediate <scp>YAP1</scp> to Promote Vascularized Bone Regeneration in Distraction Osteogenesis

Distraction osteogenesis (DO) is an efficient approach to addressing craniofacial deformities and bone defects. M2c macrophages play a crucial role in the formation of bone tissue; however, the underlying mechanism during the immunoregulation related to angiogenic osteogenesis remains unclear. In this study, we first isolate the cell culture medium from M2c macrophages to induce HUVECs and confirm the angiogenic ability. Then RNA-sequencing data identified key genes in M2c macrophages, verified by PCR and immunofluorescence (IF). Furthermore, siRNA inhibited YAP1 in HUVECs, after which the angiogenic activity of these cells and their effect on the vascular osteogenesis of BMSCs were evaluated through proliferation, migration, tube formation, and osteo-differentiation assays. In vivo, the rat mandibular DO model was established, YAP1 inhibitors were injected, and osteogenesis was investigated using micro-CT, H&E, Masson, and IHC staining. The M2c polarization in HUVECs enhanced their proliferation, migration, and tube formation abilities, which were reduced by the downregulated expression levels of YAP1. PCR and WB analyses revealed that inhibiting YAP1 expression in M2c could disrupt the expression levels of VEGF, FGF, and ANG1 in HUVECs, consequently diminishing their angiogenic capabilities. Moreover, HUVEC^CM significantly facilitated osteogenesis, while this process was hindered by inhibiting the expression of YAP1. Furthermore, inhibiting YAP1 in the context of DO resulted in the weakening of bone trabeculae formation and decreasing the expression levels of OCN and OPN. In conclusion, the current study indicated that M2c polarization plays a crucial role in DO, and YAP1-mediated regulation affects these interactions among immune responses, angiogenesis, and osteogenesis, laying a robust element for clinical and translational progress in bone tissue engineering.