Intracanal Antimicrobial Effects on SCAP Viability And Differentiation In Vitro

Objectives: Regenerative endodontic therapies have recently emerged as a viable strategy to restore tooth development by inducing stem cells from the apical papilla (SCAP) to migrate into the root canal and undergo odontogenic differentiation. Previous studies have indicated that intracanal antimicrobials used to disinfect the root canal in regenerative endodontic therapies may be cytotoxic to SCAP, leading to inconsistent treatment outcomes that limit the clinical use of this procedure. However, the effects of intracanal antimicrobial agents on the odontogenic differentiation capacity of SCAP at sub-lethal concentrations have not been examined. We aim to determine the effects of intracanal antimicrobials on SCAP viability and odontogenic differentiation capacity using a clinically-relevant concentration range (0.1-0.8 mg/mL).
Methods: Immature human third molars were collected from 71 patients and the apical papillae were mechanically harvested, enzymatically digested and filtrated to form single-cell suspensions. SCAP were positively selected using anti-STRO-1 immunomagnetic beads. The cytotoxic effects of intracanal antimicrobials were assessed using alamarBlue and Live/Dead assays after exposing STRO-1⁺ SCAP to four antimicrobial treatment groups for 7 days at 0.1-0.8 mg/mL. The odontogenic differentiation capacity of STRO-1⁺ SCAP was evaluated using immunocytochemistry to detect dentin matrix protein 1 expression.
Results: All concentrations of the triple antibiotic paste (TAP) significantly reduced STRO-1⁺ SCAP viability, while no double antibiotic paste (DAP) concentrations were significantly cytotoxic. Ca(OH)₂ and modified triple antibiotic paste (MTAP) concentrations below 0.4 mg/mL and 0.2 mg/mL, respectively, did not significantly reduce viability. Odontogenic differentiation was markedly reduced by all TAP concentrations, not impacted by Ca(OH)₂ or MTAP and DAP concentrations of 0.8 and 0.4 mg/mL, and enhanced by MTAP and DAP at concentrations of 0.2 and 0.1 mg/mL.
Conclusions: The varying effects of intracanal antimicrobials on STRO-1⁺ SCAP in vitro suggest that amendments to the current root canal disinfection protocol may ultimately improve the predictability and success of regenerative endodontic therapies.