Schwann-endothelial crosstalk in neurovascularized cell sheets induced host CGRP release to drive mandibular bone regeneration.

BACKGROUND: Mandibular defects resulting from trauma, tumors, and infections present a considerable clinical challenge, profoundly affecting patients' psychological and physical well-being. Native bone tissue relies on tightly coordinated neurovascular crosstalk to regulate bone development, remodeling, and regeneration. Vascularization is well-studied in bone tissue engineering, whereas neural integration remains relatively unexplored. Consequently, the development of neurovascularized bone tissue engineering scaffolds represents a promising strategy for enhancing the outcomes of bone tissue regeneration. METHODS: Neurovascularized cell sheets were constructed through the co-culturing of dental pulp stem cells (DPSCs), endothelial cells (ECs), and Schwann cells (SCs). The angiogenic capacity of the sheets was evaluated using tube formation assays, along with the analysis of angiogenic markers through qRT-PCR and western blot. The neurogenic potential was assessed by examining the maturity of SCs, the expression of neurotrophic factors, and quantifying axon extension using trigeminal ganglion neurons (TGN) co-culture models. Subsequently, the bone regenerative efficacy was tested in a rat critical-sized mandibular defect model. Additionally, a Calcitonin gene-related peptide (CGRP) receptor antagonist was utilized to investigate the underlying molecular mechanism. RESULTS: SCs significantly enhanced ECs migration by 4.5-fold (P < 0.05) and tube formation while upregulating angiogenic marker expression. Reciprocally, ECs promoted SCs maturation and neurotrophic factor expression, accompanied by facilitating axonal elongation of neurons by 2.9-fold (P < 0.001). In vivo experiments demonstrated that neurovascularized cell sheets exhibited superior bone regeneration capacity compared to vascularized sheets by 2.1-fold (P < 0.001) or neurogenic sheets by 1.7-fold (P < 0.001). Mechanistically, CGRP secreted from host reinnervated nerve fibers activated the RAMP (receptor activity-modifying protein)-mediated PKA (protein kinase A)-CREB (cAMP-responsive element binding protein 1) signaling axis to amplify the osteogenic differentiation of DPSCs. CONCLUSION: Based on the physiological structure of bones, we developed a neurovascularized cell sheet demonstrating marked therapeutic efficacy in critical mandibular defect repair. Our findings improve the understanding of nerve and vessel interactions in skeletal repair and offer a promising strategy to address the challenges of critical bone defect healing. THE TRANSLATIONAL POTENTIAL OF THIS ARTICLE: This study establishes neurovascularized cell sheets as a promising therapeutic strategy for critical-sized bone defect reconstruction and identifies CGRP as a key target orchestrating bone regeneration. These findings provide a direct foundation for developing neurovascularized bio-grafts and related pharmaceutical interventions for bone repair applications.