Extracellular Vesicles-Induced Cell Homing and Odontogenesis via microRNA Signaling for Dentin Regeneration

Reparative tertiary dentinogenesis requires the recruitment and odontogenic differentiation of dental pulp stem cells (DPSCs). Extracellular vesicles (EVs) as bioactive molecules have gained attention in regenerative medicine for their ability to mediate tissue repair through intercellular communication, influencing cell recruitment, proliferation, and differentiation. This study aimed to evaluate the effects of EVs on DPSC homing and odontogenic differentiation for dentin regeneration. DPSC-derived EVs were cultured in either growth (EV-G) or odontogenic differentiation (EV-O) conditions and isolated using a modified precipitation method. EVs were characterized by nanoparticle tracking analysis, scanning electron microscopy, antibody array, and cellular uptake assay. Treatment with 5 × 10⁸ EVs/mL significantly enhanced DPSC chemotaxis and proliferation compared with a no-treatment control and a lower dosage of EV (5 × 10⁷ EVs/mL). Gene expression and biochemical analyses revealed that EV-O up-regulated odontogenic markers including collagen type 1A1 (COL1A1), runt-related transcription factor 2 (RUNX2), and alkaline phosphatase (ALP). EV-O enhanced dentin regeneration by approximately 55% over vehicle controls in a rabbit partial dentinotomy/pulpotomy model. We identified key microRNAs (miR-21-5p, miR-221-3p, and miR-708-3p) in EV-O involved in cell homing and odontogenesis. In conclusion, our EV-based cell homing and odontogenic differentiation strategy has significant therapeutic potential for dentin regeneration.