Intracellular Bacteria in Stem Cells of the Apical Papilla: A "Trojan Horse" for Regenerative Endodontic Failure.

Abstract

Introduction: Regenerative endodontic procedures (REPs) show unpredictable outcomes in infected teeth due to persistent infection. While current disinfection protocols reduce but do not eliminate root canal bacteria, the fate of residual microorganisms during stem cell recruitment remains unclear. This study tested the hypothesis that endodontic pathogens invade and survive within stem cells of the apical papilla (SCAP), diminishing its biomineralization potential.

Methods: Internalization of SCAP by Gram-positive (Enterococcus faecalis, Actinomyces naeslundii) and Gram-negative (Porphyromonas gingivalis, Fusobacterium nucleatum) bacteria was characterized using colony-forming unit assays, confocal microscopy and flow cytometry. Intracellular survival of E. faecalis was monitored for 14 days in both planktonic and biofilm phases. The impact of intracellular E. faecalis on SCAP biomineralization and odontogenic differentiation was assessed using alkaline phosphatase activity, alizarin red staining, and quantitative real-time PCR. Statistical analyses were performed using one-way ANOVA with Dunnett's multiple comparison test or two-way ANOVA with Tukey's multiple comparison test. P<.05 was considered statistically significant.

Results: All tested bacteria invaded SCAP with differential efficiencies. E. faecalis and F. nucleatum demonstrated robust internalization (∼5-log CFU), while P. gingivalis and A. naeslundii showed reduced invasion (∼2.5 log CFU). E. faecalis persisted intracellularly for up to 14 days, with biofilm-phase bacteria showing enhanced survival in monolayer SCAP cultures. Intracellular E. faecalis abrogated SCAP mineralization while maintaining cell viability, accompanied by significant downregulation of all tested odontogenic genes (P<0.05).

Conclusions: Bacteria invade and establish persistent intracellular infections within SCAP, creating protected reservoirs. These intracellular bacteria severely compromise odontogenic differentiation without killing the host cells, representing a potentially unrecognized mechanism of REP failure. This "Trojan horse" strategy may explain the unpredictable outcomes of regenerative endodontics in infected teeth.