Evaluation of the Effect of Two Chlorhexidine Preparations on Biofilm Bacteria In Vitro: A Three-Dimensional Quantitative Analysis

IntroductionMicroorganisms are essential in the development of periradicular diseases and are the major causative factors associated with endodontic treatment failures. Microbial biofilms are communities of bacteria that attach to surfaces and form heterogeneous three-dimensional structures. The purpose of this study was to develop a biofilm model that closely mimicked in vivo biofilm and to determine its susceptibility to endodontic antimicrobial irrigants by three-dimensional quantitative analysis.MethodsCollagen-coated hydroxyapatite (C-HA) and uncoated hydroxyapatite (HA) disks were inoculated with dispersed subgingival plaque for 3 weeks. Thick biofilms rich in spirochetes were formed on both substrates. Biofilms were subjected to 1-, 3-, and 10-minute exposures to CHX-Plus (Vista Dental Products, Racine, WI) and 2% chlorhexidine (CHX). After treatment, the volume ratio of dead bacteria to all bacteria in biofilms, indicated by the ratio of red and (red + green) fluorescence, was analyzed by confocal laser scanning microscopy for each medicament.ResultsThe proportion of killed bacteria was depended on the type of irrigant and the time of exposure in both C-HA and HA biofilm models (p = 0.00). CHX-Plus showed higher levels of bactericidal activity at all exposure times than 2% CHX (p < 0.001). The C-HA biofilm was thicker than the HA biofilm. Less bacteria were killed in C-HA biofilm than in the HA model.ConclusionsThis multispecies biofilm model and quantitative analysis methodology may be useful for the evaluation of the antimicrobial effectiveness of endodontic disinfecting agents.