Time Dependent Effects of Contemporary Irrigants on a Polymicrobial Biofilm

Abstract

INTRODUCTION: Removal of contaminated canal contents and biofilms by mechanical preparation alone are limited. Irregular cross sections, lateral canals, and apical deltas are mostly inaccessible to mechanical preparation (Siqueira et al., 1997). Therefore, the use of chemical dissolution and disinfection of these regions is necessary. Many studies have demonstrated the efficacy of sodium hypochlorite in providing these functions (Hand et al., 1978; Rosenfeld et al., 1978). However, adverse cytotoxic outcomes have been reported with its use (Joffe et al., 1991), as well as reports of allergic hypersensitivity (Kaufman et al., 1989). The ideal irrigant has attributes of; broad spectrum antimicrobial activity, ability to dissolve pulp tissue remnants, inactivates endotoxin, removes the smear layer components, and is systemically nontoxic to periodontal tissues. Such an irrigant does not yet exist (Zehnder, 2006). MTAD, a final irrigation solution containing 3% doxycycline, 4.25% citric acid, and 0.5% Tween-80 has been shown to remove smear layer on extracted human teeth and provides broad spectrum antimicrobial activity (Torbinejad et al., 2003). QMiX, a final irrigation solution containing; bisbiguanide, calcium chelating agent, cetrimide surfactant, and saline has demonstrated efficacy at removing smear layer and disinfecting dentin tubules (Wang et al., 2013). SmearClear, a smear layer removing irrigant containing EDTA, cetrimide and water, has been shown to remove smear layer efficiently in an ex-vivo, split-tooth model (Andrabi et al., 2013). The aims of this study are to; demonstrate the use of a novel polymicrobial biofilm model to test endodontic irrigants, measure the susceptibility of a biofilm to individual irrigant treatment groups, and to characterize the responses visually through scanning electron microscopy (SEM) and laser confocal microscopy. METHODS: Clinical endodontic microbial samples were collected from six adult patients that presented to the graduate endodontics clinic at the University of Minnesota School of Dentistry with a diagnosis of pulpal necrosis. For each of the 3 experimental runs a pool of one to three patients were combined to ensure a collection of heterogeneous microbial species. A series of baffled 500 mL Erlenmeyer culture flasks were used as a “bioreactor” system to facilitate biofilm formation on hydroxyapatite discs. A total of six discs were contained per flask (2 discs per cell strainer basket). The flasks were covered with a sterile, vented flask cap and placed within the anaerobic chamber for incubation at 37°C and 20 RPM. This allowed for biofilm formation on the hydroxyapatite discs under shear force conditions. The flasks were allowed to incubate according to the conditions described above for approximately 48 total hours. Following incubation, discs were randomly selected for placement into a sterile, flat-bottomed, 12-well polystyrene culture plate (Sigma-Aldrich) for treatment according to the irrigant protocol.Four irrigation solutions were independently used for testing on the 48 hour biofilms. Sterile phosphate buffered saline (PBS) was used as a negative control treatment group. MTAD (DENTSPLY, Int.), QMiX (DENTSPLY, Int.), and Smearclear (SybronEndo, Orange, CA) were used as challenge treatment groups. Groups included; 1X PBS at 1 minute exposure, MTAD at 1 minute exposure,MTAD at 20 minutes exposure, QMiX at 1 minute exposure,QMiX at 20 minutes exposure,SmearClear at 1 minute exposure, and SmearClear at 20 minutes exposure. Evaluation of biofilm disruption was determined by; cell viability staining, crystal violet biomass staining, SEM, and confocal laser microscopy. Microbial speciation was performed by the Forsyth Institute (Cambridge, MA) by HOMIM 16sRNA analysis. RESULTS: The flask bioreactor, as previously described, can be utilized for growing a polymicrobial biofilm suitable for testing the efficacy of antimicrobial and biofilm removing endodontic irrigants. When compared as a group, the endodontic irrigants in this study achieved a statistically significant reduction in cell viability as time of exposure increased. In a pairwise comparison between irrigants, the mean cell viability was lowest for QMiX. No statistically significant difference in the reduction of biomass between endodontic irrigants was observed. Comparing endodontic irrigants individually by time did not result in a statistically significant difference in reducing cell viability or biomass. None of the endodontic irrigants completely removed the biofilm, as observed by SEM and confocal laser microscopy. 6 of 12 microbial species recovered were found to be in common between the patient pools when analyzed by HOMIM 16sRNA identification. This study presents a simple model for growing endodontic biofilms under anaerobic conditions. CONCLUSIONS: Findings from this study support the inefficient removal of biofilm by the irrigants included in the study. It was determined that QMiX achieved the lowest cell viability over the 20 minute treatment exposure. As a group, the irrigants were statistically significant in reducing cell viability and reducing biomass. However, it could be concluded that they should not be utilized as a single irrigant to disinfect or remove biofilm.