Dynamics and Inflammatory Potential of Enterococcus faecalis Biofilm on Root-Canal

Introduction: The persistence of bacteria in the root canal system can evoke immune response which ultimately manifests as apical periodontitis. Previous clinical observations have revealed biofilm mode of bacterial growth in root canal systems. Objective: The present investigation was to study the dynamics of Enterococcus faecalis mediated biofilm formation and their monocyte binding potential under different environmental conditions. Method: Stage-1: Intact non-carious human maxillary molars teeth (n=20), after sterilization was inoculated with E. faecalis (ATCC 29212) under nutrient-rich and nutrient-deprived conditions. Both groups were incubated anaerobically for a period of four months. Control specimens were also prepared by the same protocol except for bacterial inoculation. Scanning Electron Microscopy and Laser Confocal Scanning Microscopy (LCSM) were used to study the ultrastructure of the biofilm. LIVE/DEAD BacLight bacterial viability staining was conducted to observe the distribution of viable cells inside the biofilm. Energy-Dispersive-X-ray and Fourier-Transfer-Infra-Red-Spectroscopic analyses were conducted to study the physico-chemical changes in biofilm. Stage-2: The monocyte binding assay was carried out using fluorescent tagged c-AMP activated U937 (promonocytic cell line) cells under defined flow conditions and number of bound cells examined by LCSM. Result: It was found that E. faecalis produced a honey comb like biofilm structure on the root canal wall. Viable cells were also observed inside the biofilm structures. Advanced material characterization techniques highlighted biomineralization of the biofilm structure under optimal conditions. The U937 cell binding assay displayed distinct inflammatory potential of biofilm structures. Conclusion: This in vitro study highlighted development of E. faecalis mediated mineralized biofilm structures on root canals and their potential in eliciting an inflammatory response. This work was supported by National University of Singapore, ARF Grant number FY 03 R-224-000-021-101 and R-224-000-020-112.