Abstract
<title>Abstract</title> <p>Background Irreversible pulpitis remains an unmet clinical challenge, as no existing therapy achieves physiological dentin–pulp complex regeneration. Dental pulp stem cell-derived extracellular vesicles (DPSC-EVs) hold regenerative promise, yet their therapeutic efficacy requires optimization. This study investigated whether hypoxic preconditioning enhances the pro-regenerative capacity of DPSC-EVs and sought to elucidate the underlying molecular mechanisms using integrated multi-omics analysis. Methods DPSCs were exposed to 1% O₂ for 48 h to obtain hypoxic EVs (Hypo-EVs); normoxic EVs (Nor-EVs) served as controls. EVs were isolated by ultracentrifugation and characterized. In vitro, the effects of Hypo-EVs on human umbilical vein endothelial cell (HUVEC) proliferation, migration, and tube formation, as well as on DPSC proliferation, migration, and mineralization, were evaluated. In vivo, a rat ectopic pulp regeneration model was established by co-transplanting DPSCs with Hypo-EVs or Nor-EVs. miRNA sequencing of Hypo-EVs and mRNA sequencing of Hypo-EV-treated DPSCs were performed, and candidate miRNAs identified through Venn analysis were validated by qPCR. Results Hypo-EVs exhibited higher protein content and a distinct microRNA profile compared with Nor-EVs. Functionally, Hypo-EVs significantly enhanced proliferation, migration, and tube formation in HUVECs, as well as proliferation, migration, and mineralization in DPSCs. In the rat model, co-transplantation of Hypo-EVs with DPSCs led to synergistic dentin–pulp complex regeneration, characterized by abundant neovascularization, a polarized odontoblast-like layer, mature collagen deposition, and de novo dentin formation. Multi-omics integration via Venn analysis of miRNA-seq and mRNA-seq data, confirmed by qPCR, identified hsa-miR-423-5p and hsa-miR-193b-3p as core candidate miRNAs regulating a tripartite program of osteo/odontogenic differentiation, angiogenesis, and hypoxia-adaptive autophagy–apoptosis homeostasis. Conclusions Hypoxic preconditioning is an effective bioengineering strategy to enhance the regenerative potency of DPSC-EVs. Hypo-EVs couple angiogenesis and odontogenesis to drive dentin–pulp complex regeneration through a distinct miRNA cargo that includes hsa-miR-423-5p and hsa-miR-193b-3p. These findings support the application of Hypo-EVs as a synergistic component in stem cell-based therapies and provide a foundation for future acellular regenerative strategies.</p>