Dental Pulp Stem Cells Transfer Mitochondria via Tunneling Nanotubes to Drive Hypoxic Angiogenesis

The dental pulp resides within a rigid dentin chamber with a limited blood supply, creating a hypoxic environment that impedes tissue regeneration. While growth factor signaling in pulpal revascularization is well documented, the direct cellular mechanisms that protect endothelial cells (ECs) from hypoxia-induced apoptosis remain unclear. This study identifies intercellular mitochondrial transfer (MT) from dental pulp stem cells (DPSCs) to ECs as a critical survival and angiogenic mechanism under hypoxic stress. Using MitoTracker labeling and flow cytometry, we demonstrated that mitochondria are preferentially transferred from DPSCs to ECs, a process significantly upregulated by hypoxia. We found that contact-dependent mechanisms involving tunneling nanotube-like structures contribute to MT, as cytochalasin B treatment or Miro1 knockdown in DPSCs significantly reduced MT and impaired EC function. To assess the impact of this organelle exchange, we isolated mitochondria-recipient (Mito+) and nonrecipient (Mito-) ECs for analysis. Mito+ ECs exhibited enhanced mitochondrial membrane potential, improved energy metabolism, and yielded superior tube-forming capacity as compared with Mito- ECs. Furthermore, MT significantly reduced EC apoptosis under cobalt chloride-induced hypoxic stress. The in vivo Matrigel plug assay showed that inhibiting MT from DPSCs, genetically or by inhibiting mitochondrial respiration, markedly suppressed DPSC-supported angiogenesis and increased EC apoptosis. Mechanistically, RNA sequencing and Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that MT revives the EC transcriptome and activates the PI3K-AKT signaling pathway. Immunofluorescence confirmed upregulation of AKT signaling in recipient ECs and colocalization of P-AKT with transferred mitochondria. Furthermore, inhibition of AKT signaling with MK-2206 abolished the proangiogenic and prosurvival role associated with MT, suggesting a direct regulatory role. Collectively, these findings establish MT as a vital metabolic lifeline that prevents EC collapse and drives DPSC-supported angiogenesis in the hypoxic pulp during the vulnerable window of pulpal restoration, thereby emphasizing MT as a transformative regenerative endodontic target.