Reparative Dentin Formation by Dentin Matrix Proteins and Small Extracellular Vesicles

Dental pulp tissue, as the only vascularized connective tissue in the teeth, plays a critical role in maintaining teeth stability and health. Damaged pulp tissue can significantly affect normal dental physiological functioning and survival1. Direct and indirect dental pulp therapy induces tertiary mineralized dentin secretion to prevent dental pulp necrosis, preserve the integrity, and prolong the lifetime of the tooth during the early stages of pulp infection in young patients2. Recently, pulp-capping materials such as calcium silicate-based cement and calcium hydroxide have been used as natural tissue substitutes to Introduction: Vital pulp therapy aims at preserving pulp vitality and regenerating dentin. Therefore, the purpose of this study was to explore the effects of a combination of treated dentin matrix (TDM) proteins and dental pulp cell (DPC)-derived small extracellular vesicles (sEVs) on pulp-dentin complex repair. Methods: We prepared TDM by chemical demineralization and mechanical disruption of teeth to a powder preparation. The sEVs were isolated from culture supernatants of DPCs and identified by nanoparticle tracking analysis, Western blotting, and transmission electron microscopy. The effect of a combination of TDM proteins and DPC-derived sEVs on DPC proliferation, migration, and odontogenic differentiation was evaluated in vitro. A minipig model of pulp injury was used to compare the clinical outcomes and tissue responses attributed to 4 materials including TDM, sEV-TDM, sEVs, and mineral trioxide aggregate. Results: The sEV isolated from the cell supernatant promoted DPC proliferation and migration. The combination of TDM extracts and sEV synergistically promoted the migration of DPCs but suppressed their proliferation. Real-time polymerase chain reaction and Western blot revealed that sEV-TDM enhanced the odontoblast-related protein expressions in DPCs. In in vivo studies, TDM and sEV-TDM promoted the formation of continuous reparative dentin. Furthermore, odontoblastlike high columnar cells were observed on the pulp side of the dentin bridge. Conclusions: The sEV-TDM complex exhibits intrinsic biological activities, which has potential applications as a bioactive pulp capping material. (J Endod 2021;47:253?262.)