Effect of extracellular vesicles derived from either blood or dental pulp stem cells on human dental pulp stem cells: an in vitro study

OBJECTIVE: This study aimed to determine and compare the possible proliferative and differentiative effects of extracellular vesicles (EVs) derived from human dental pulp stem cells (hDPSCs-EVs) and human blood plasma (hBP-EVs) on hDPSCs as an in-vitro assessment for their ability on endogenous hDPSCs for osteo-odontogenic differentiation MATERIALS AND METHODS: hDPSCs were isolated from impacted third molars of healthy donors and characterized by flow cytometry. EVs were isolated from hDPSCs-conditioned media and human plasma using differential ultracentrifugation and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), bicinchoninic acid assay (BCA), and Western blotting (WB). hDPSCs were cultured in standard medium (control) or supplemented with 50 µg/mL hDPSCs-EVs or hBP-EVs. Proliferation was assessed by MTT assay. Mineralization was evaluated by Alizarin Red staining. Odontogenic differentiation was analyzed by quantitative real-time PCR for dentin sialophosphoprotein (DSPP) expression and immunocytochemical detection of osteocalcin (OCN). RESULTS: By flow cytometry, hDPSCs showed positive expression for CD90 and CD105 and negative expression for CD34 and CD45. Both EVs types demonstrated characteristic nanoscale morphology and EVs marker expression. hDPSCs-EVs significantly enhanced proliferation compared with hBP-EVs and control (P < 0.001). Additionally, mineral deposition, DSPP expression, and OCN-positive area were markedly higher in the hDPSCs-EVs group than in the hBP-EVs and control groups (P < 0.001). CONCLUSION: Although hBP-EVs exhibited measurable regenerative activity, tissue-specific hDPSCs- EVs produced significantly greater proliferative and osteo-odontogenic effects. These findings suggest that EVs bioactivity is strongly influenced by cellular origin and highlight the importance of lineage-specific vesicles in optimizing cell-free regenerative strategies for endodontic applications.