Comparison of the effects of different dental pulp stem cell preconditioning protocols on the odontogenic potential of their derived small extracellular vesicles.

Regenerative endodontic therapy that is based on the use of stem cells or their derivatives to induce dentin-pulp complex regeneration has recently gained substantial interest. This study aimed to compare the effect of preconditioning through odontogenesis (OD) and/or low-level laser therapy photobiostimulation (LLLT) of human dental pulp stem cells (hDPSCs) on the regenerative potentials of their derived small extracellular vesicles (sEVs) aiming to develop a novel pulp capping therapy. In the current study, hDPSCs were subjected to OD, LLLT, or dual preconditioning protocols before the isolation of sEVs, then the derived sEVs were co-cultured with naïve hDPSCs. Cell viability and wound healing capacity were evaluated using MTT and scratch assays, respectively. RT-qPCR was performed for odontogenic gene expression (DSPP, DMP1, and COL1A1), followed by immunocytochemical staining. Matrix mineralization was also assessed, and relevant statistical analyses were performed. All sEVs-treated groups revealed enhanced cellular and odontogenic activities. OD-sEVs enhanced wound healing and mineralization, while LLLT-sEVs showed relatively higher viability, odontogenic gene and protein expression levels. However, no significant differences were detected between both groups. The novel dual preconditioning protocol surpassed either treatment alone representing a promising approach for odontogenic induction of mesenchymal stem cells. The results highlighted the regenerative potential of preconditioned sEVs as a biological, promising pulp capping material that could open new avenues in the field of regenerative endodontic treatment after being tested in-vivo in experimental animals.