Effects of Sodium Hypochlorite Concentration and Application Time on Bacteria in an Ex Vivo Polymicrobial Biofilm Model.

INTRODUCTION: In endodontic treatment, it is important to remove or inactivate biofilms in the root canal system. We investigated the effects of different concentrations and application times of sodium hypochlorite (NaOCl) on the viability of bacteria in ex vivo polymicrobial biofilms of different maturation levels. METHODS: Polymicrobial biofilms were prepared from dental plaque samples and grown for 1, 2, and 3 weeks under anaerobic conditions on collagen-coated hydroxyapatite discs as an ex vivo biofilm model. The biofilms were then exposed to NaOCl at concentrations ranging from 0.1% to 2% for 1 or 3 minutes. The control group was exposed to sterile distilled water. Viability staining was performed and examined by confocal laser scanning microscopy to determine the percentage of biofilm bacteria killed by NaOCl. Scanning electron microscopy was also performed to visually examine the biofilms. RESULTS: Application of NaOCl at 0.5%-2% for both 1 and 3 min killed significantly more bacteria when compared to the controls (P < .05). Cell viability tended to be lower after the application of NaOCl for 3 minutes than that for 1 minute. CONCLUSIONS: Our experiments using an ex vivo model showed that within the range of 0.1%-2% of NaOCl, higher NaOCl concentrations and longer application times were more effective in killing biofilm bacteria, and that mature biofilms were more resistant to NaOCl than younger biofilms.

Chlorhexidine-Containing Electrospun Polymeric Nanofibers for Dental Applications: An In Vitro Study.

Chlorhexidine is the most commonly used anti-infective drug in dentistry. To treat infected void areas, a drug-loaded material that swells to fill the void and releases the drug slowly is needed. This study investigated the encapsulation and release of chlorhexidine from cellulose acetate nanofibers for use as an antibacterial treatment for dental bacterial infections by oral bacteria Streptococcus mutans and Enterococcus faecalis. This study used a commercial electrospinning machine to finely control the manufacture of thin, flexible, chlorhexidine-loaded cellulose acetate nanofiber mats with very-small-diameter fibers (measured using SEM). Water absorption was measured gravimetrically, drug release was analyzed by absorbance at 254 nm, and antibiotic effects were measured by halo analysis in agar. Slow electrospinning at lower voltage (14 kV), short target distance (14 cm), slow traverse and rotation, and syringe injection speeds with controlled humidity and temperature allowed for the manufacture of strong, thin films with evenly cross-meshed, uniform low-diameter nanofibers (640 nm) that were flexible and absorbed over 600% in water. Chlorhexidine was encapsulated efficiently and released in a controlled manner. All formulations killed both bacteria and may be used to fill infected voids by swelling for intimate contact with surfaces and hold the drug in the swollen matrix for effective bacterial killing in dental settings.

Morphological Analyses of Effects of Endodontic Irrigant Solutions Using a Root Canal Model and an Immersion Model.

Objective: This study aimed to compare an experimental model simulating clinical root canal irrigation (root canal model) with a conventional experimental model immersing dentin sample to irrigants (immersion model) to evaluate removal of the smear layer and decalcification of the root canal dentin using sodium hypochlorite (NaOCl) and two different concentrations of ethylenediaminetetraacetic acid (EDTA) solution. Materials and Methods: Forty-five single-rooted extracted human teeth were prepared using a Ni-Ti rotary file. EDTA, NaOCl, and citric acid were used in the root canal models and the immersion models. After the irrigation protocol, root canal surfaces were observed under scanning electron microscopy. Residual smear and decalcification of the root canal dentin were evaluated objectively by measuring the percentage of the area occupied by visible dentin tubules, the number of visible dentin tubules, and the mean area of a visible single dentin tubule. Results: Root canal and immersion models with the same irrigation protocol showed significantly different results for smear residues and decalcification of root canal dentin. In the root canal model, neither different EDTA concentrations nor the order of EDTA and NaOCl applications significantly impacted smear residues or decalcification of root canal dentin. Furthermore, no erosion of the root canal dentin surface was observed in any experimental groups in the root canal model using EDTA and NaOCl compared to intact dentin. Conclusions: Experimental design affected results for residual smear layer and decalcification of root canal dentin. The order of EDTA and NaOCl use and the concentration of EDTA did not affect results. EDTA and NaOCl irrigation did not cause erosion in the root canal model in this study.

An extensive description of the microbiological effects of silver diamine fluoride on dental biofilms using an oral in situ model.

Silver diamine fluoride (SDF) has been long studied in laboratories, and its clinical effectiveness in the treatment and prevention of root caries has been reported. In the present study, we assessed the microbiological effects of SDF on dental biofilms grown on demineralized dentin in situ. Specifically, demineralized bovine root dentin slabs used as biofilm substrates were treated with 38% SDF, and the biofilms formed after this treatment were analyzed via real-time PCR, DEAD/LIVE cell staining, and SEM. Next, the viable cell count was determined, and microbial profiles were compared using 16S rRNA gene sequencing. Untreated slabs were used as controls. We observed significant decreases in viable cell counts (p < 0.05), number of biofilm-forming cells (p < 0.01), biofilm thickness (p < 0.01), and high proportion of dead cells with SDF treatment (p < 0.01). The microcolonies in the SDF-treated biofilms showed less complexity, and only a limited number of genera were differentially abundant between the groups. Microbial diversity index comparisons showed no significant differences between the groups with respect to treatments days (p = 0.362). Thus, SDF negatively influenced dental biofilm growth on demineralized root dentin in situ; however, its antimicrobial action did not target a specific oral taxon.

Assessment of the functional efficacy of root canal treatment with high-frequency waves in rats.

The purpose of this study was to develop a high-frequency wave therapy model in rats and to investigate the influence of high-frequency waves on root canal treatment, which may provide a novel strategy for treating apical periodontitis. Root canal treatments with and without high-frequency wave irradiation were performed on the mandibular first molars of 10-week-old male Wistar rats. The mesial roots were evaluated radiologically, bacteriologically, and immunohistochemically. At 3 weeks after root canal treatment, lesion volume had decreased significantly more in the irradiated group than in the non-irradiated group, indicating successful development of the high-frequency therapy model. The use of high-frequency waves provided no additional bactericidal effect after root canal treatment. However, high-frequency wave irradiation was found to promote healing of periapical lesions on the host side through increased expression of fibroblast growth factor 2 and transforming growth factor-ß1 and could therefore be useful as an adjuvant nonsurgical treatment for apical periodontitis.

Doxycycline release and antibacterial activity from PMMA/PEO electrospun fiber mats.

OBJECTIVE: To investigate the use of polymethyl methacrylate (PMMA) electrospun fiber mats containing different amounts of polyethylene oxide (PEO) as a doxycycline delivery system and to test antibacterial activity against an oral pathogen. METHODOLOGY: PMMA powders or PEO (mol wt 200 Kd) (10,20,30% w/w/) were dissolved in N, N-dimethylformamide (DMF) to obtain a final polymer concentration of 15% in DMF (w/v). 2% Doxycycline monohydrate was added to the solutions and submitted to vortex mixing. The solution was transferred to a plastic syringe and fit into a nanofiber electrospinning unit. The parameters applied were: voltage at 17.2 kV; distance of 20 cm between the needle tip and the collector plate; target speed at 2 m/min; and transverse speed at 1cm/min. Syringe pump speed was 0.15 mm/min. The drug release analysis was performed by removing aliquots of the drug-containing solution (in PBS) at specific periods. Doxycycline release was quantified using RP-HPLC. Fiber mats from all groups had their antibacterial action tested against S. mutans based on inhibition halos formed around the specimens. The experiments were performed in triplicate. Gravimetric analysis at specific periods was performed to determine any polymer loss. Morphological characterization of the electrospun fibers was completed under an optical microscope followed by SEM analysis. RESULTS: The addition of PEO to the PMMA fibers did not affect the appearance and diameter of fibers. However, increasing the %PEO caused higher doxycycline release in the first 24 h. Fibers containing 30% PEO showed statistically significant higher release when compared with the other groups. Doxycycline released from the fibers containing 20% or 30% of PEO showed effective against S. mutans. CONCLUSION: The incorporation of PEO at 20% and 30% into PMMA fiber mat resulted in effective drug release systems, with detected antibacterial activity against S. mutans.

Doxycycline release and antibacterial activity from PMMA/PEO electrospun fiber mats

Abstract Objective: To investigate the use of polymethyl methacrylate (PMMA) electrospun fiber mats containing different amounts of polyethylene oxide (PEO) as a doxycycline delivery system and to test antibacterial activity against an oral pathogen. Methodology: PMMA powders or PEO (mol wt 200 Kd) (10,20,30% w/w/) were dissolved in N, N-dimethylformamide (DMF) to obtain a final polymer concentration of 15% in DMF (w/v). 2% Doxycycline monohydrate was added to the solutions and submitted to vortex mixing. The solution was transferred to a plastic syringe and fit into a nanofiber electrospinning unit. The parameters applied were: voltage at 17.2 kV; distance of 20 cm between the needle tip and the collector plate; target speed at 2 m/min; and transverse speed at 1cm/min. Syringe pump speed was 0.15 mm/min. The drug release analysis was performed by removing aliquots of the drug-containing solution (in PBS) at specific periods. Doxycycline release was quantified using RP-HPLC. Fiber mats from all groups had their antibacterial action tested against S. mutans based on inhibition halos formed around the specimens. The experiments were performed in triplicate. Gravimetric analysis at specific periods was performed to determine any polymer loss. Morphological characterization of the electrospun fibers was completed under an optical microscope followed by SEM analysis. Results: The addition of PEO to the PMMA fibers did not affect the appearance and diameter of fibers. However, increasing the %PEO caused higher doxycycline release in the first 24 h. Fibers containing 30% PEO showed statistically significant higher release when compared with the other groups. Doxycycline released from the fibers containing 20% or 30% of PEO showed effective against S. mutans. Conclusion: The incorporation of PEO at 20% and 30% into PMMA fiber mat resulted in effective drug release systems, with detected antibacterial activity against S. mutans.

Development of a root canal treatment model in the rat.

Root canal treatment is performed to treat apical periodontitis, and various procedures and techniques are currently used. Although animal models have been used in the developmental research of root canal treatment, little of this research has used small animals such as rats, because of their small size. In this study, root canal treatment was performed on the rat mandibular first molar, which had four root canals, using a microscope, and the therapeutic effect was evaluated bacteriologically, radiologically and histopathologically. By performing root canal treatment, the level of bacteria in the mesial root of the treated teeth was reduced by 75% compared with the control. Additionally, the volume of the periapical lesions of the treated teeth as measured by micro-computed tomography decreased significantly 2 weeks after the root canal treatment when compared with the control. Histological evidence of healing was observed in the treatment group 8 weeks after root canal treatment. These results suggest that a root canal treatment model using rats can be used in developmental research for novel methods of root canal treatment.

Evaluation of Root Canal Dentin Erosion after Different Irrigation Methods Using Energy-dispersive X-ray Spectroscopy.

INTRODUCTION: The purpose of this study was to examine the level of erosion in root dentin caused by different irrigation methods and protocols. METHODS: Thirty-five extracted upper molar teeth were instrumented and divided into 7 groups to undergo treatment by different methods: negative control, GentleWave System (Sonendo Inc, Laguna Hills, CA), and syringe needle irrigation following different protocols. The teeth were instrumented to size #25/.08 or #30/.09 for needle irrigation groups and to ProTaper size S1 for the GentleWave group under 5% sodium hypochlorite (NaOCl). The needle irrigation groups were subjected to final rinses of 2 minutes of 3% NaOCl + 2 minutes of 8% EDTA (3% N2 + 8% E2), 2 minutes of 3% NaOCl + 2 minutes of 8% EDTA + 1 minute of 3% NaOCl (3% N2 + 8% E2 + 3% N1), 2 minutes of 5% NaOCl + 2 minutes of 17% EDTA (5% N2 + 17% E2), 2 minutes of 5% NaOCl + 2 minutes of 17% EDTA + 1 minute of 5% NaOCl (5% N2 + 17% E2 + 5% N1), and 5 minutes of 5% NaOCl + 5 minutes of 17% EDTA + 5 minutes of 5% NaOCl (5% N5 + 17% E5 + 5% N5), respectively. The root canal surface was observed by scanning electron microscopy, and the dentin composition was analyzed by continuous line scanning for 300 µm into dentin using energy-dispersive X-ray spectroscopy. RESULTS: A slight but statistically significant decrease of calcium and an increase of carbon was measured in the 5% N2 + 17% E2 group in comparison with the control; no significant difference was found among GentleWave, 3% N2 + 8% E2, and 5% N2 + 17% E2 (P > .05). A final 1-minute rinse with 3% or 5% NaOCl reduced calcium and phosphorus to a significantly lower level than in groups without a 1-minute final rinse (P < .05). Final irrigation with 5% NaOCl for 5 minutes removed almost all calcium and phosphorus. Scanning electron microscopy showed canal wall erosion when an additional final irrigation with NaOCl was done. CONCLUSIONS: NaOCl followed by final EDTA irrigation performed either by syringe needle or the GentleWave System caused minimal dentin erosion. Erosion was measured as increased loss of calcium and phosphorus in samples in which additional final irrigation was performed using NaOCl after EDTA.

Evaluation of the Susceptibility of Multispecies Biofilms in Dentinal Tubules to Disinfecting Solutions.

INTRODUCTION: The present study evaluated the effect of the source of biofilm bacteria on their susceptibility in dentinal tubules to disinfecting solutions using an infected dentin model. METHODS: Infected dentin blocks were prepared. Enterococcus faecalis strains VP3-181 and Gel 31 were introduced into dentinal tubules by centrifugation to form monospecies biofilms, whereas 3 specimens of pooled plaque bacteria collected from different donors were used to grow multispecies biofilms in dentin. After 1 and 3 weeks of incubation, the samples were subjected to sterile water, 2% chlorhexidine (CHX), and 2% sodium hypochlorite (NaOCl). After the 3-minute exposure, the proportions of killed bacteria in dentin canals were assessed by viability staining and confocal laser scanning microscopy. RESULTS: The proportion of killed bacteria in mature (3 weeks) mono- and multispecies biofilms was lower than in young biofilms (1 week) after treatment (P < .05). E. faecalis Gel 31 biofilms and multispecies biofilms were more resistant than VP3-181 biofilms. No differences in the susceptibilities to the disinfecting agents of the 3 multispecies biofilms were detected; 2% NaOCl was more effective against multispecies biofilms in dentin than 2% CHX (P < .05), whereas no significant difference was detected between 2% CHX and 2% NaOCl against the E. faecalis strains. CONCLUSIONS: Mature mono- and multispecies biofilms in dentinal tubules are more resistant to disinfectants than corresponding young biofilms. The susceptibility of the monospecies E. faecalis dentin biofilm showed strain-related differences, whereas the multispecies biofilms from different donors showed similar susceptibility.

Fatigue Resistance of a 3-dimensional Conforming Nickel-Titanium Rotary Instrument in Double Curvatures.

INTRODUCTION: Fatigue resistance of ProFile, Vortex Blue, and TRUShape files in artificial single curvature and in 2 different artificial double curvature canals was evaluated. METHODS: Three files (ProFile, Vortex Blue [size 20/.06], and TRUShape [size 20/.06v]) were subjected to fatigue tests in a single curvature (group 1: 60° curvature) and 2 different double curvatures (group 2: 60° and 30° curvatures; group 3: two 60° curvatures). The time to fracture and the total number of cycles to failure were recorded. The fracture surfaces of the fragments were examined with a scanning electron microscope. RESULTS: All files had significantly higher fatigue resistance in a single curvature canal than in the double curvature canals. In a single curvature group, the time to fracture of TRUShape and ProFile was longer than in Vortex Blue files. In both double curvature groups, TRUShape had the longest time to fracture among all files. The fatigue resistance (the time to fracture and number of cycles to failure) of ProFile and Vortex Blue was lower in group 3 than in group 2 (P < .05). However, there was no significant difference in fatigue resistance of TRUShape in the double curvature groups. The length of the fragment of TRUShape was longer than in Vortex Blue and ProFile files in group 3 (P < .05). CONCLUSIONS: The fatigue performance of TRUShape is different in double curvature canals, compared with conventional nickel-titanium rotary files. The fatigue resistance of TRUShape was superior to ProFile and Vortex Blue in double curvature canals.

Evolution of resistance to cationic biocides in Streptococcus mutans and Enterococcus faecalis.

OBJECTIVES: The aim of this study was to investigate whether Streptococcus mutans and Enterococcus faecalis develop resistance to the cationic biocides chlorhexidine (CHX), cetylpyridinium chloride (CPC), and 12-methacryloyloxydodecylpyridinium bromide (MDPB). METHODS: The minimum inhibitory concentrations (MICs) of CHX, CPC, and MDPB were assessed after repeated exposure of S. mutans and E. faecalis to these biocides. Cell-surface hydrophobicity and protein expression profiles of bacterial cells were examined to elucidate possible resistance mechanisms. RESULTS: The MIC of CHX against E. faecalis showed constant increases up to 10 passages. No changes in the MICs of CPC and MDPB against E. faecalis were observed. The MICs of CHX, CPC, and MDPB against S. mutans did not increase. The surface hydrophobicity of E. faecalis significantly increased with increasing exposure to CHX and CPC. However, changes in protein expression profiles were only found in CHX-adapted E. faecalis, as evidenced by the emergence of a novel, approximately 19-kDa band following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. CONCLUSIONS: While E. faecalis and S. mutans did not exhibit increased resistance to CPC or MDPB, repeated exposure of E. faecalis to CHX led to resistance. It is likely that the acquisition of resistance is related to an altered protein composition. CLINICAL SIGNIFICANCE: Alkyl pyridinium compounds, such as CPC and MDPB, could have a lower risk to cause adaptation of E. faecalis, which is advantageous compared with CHX.

Inhibition of polysaccharide synthesis by the sinR orthologue PGN_0088 is indirectly associated with the penetration of Porphyromonas gingivalis biofilms by macrolide antibiotics.

Microbes commonly adhere to surfaces, aggregate in self-produced extracellular polymeric substances (EPS) and live in biofilms. Periodontitis is a serious oral infection that is initiated by the formation of biofilms by Porphyromonas gingivalis. EPS act as a barrier that protects biofilm-forming cells against sources of stress, including those induced by host immune cells and antimicrobial agents. Therefore, drugs intended to kill such micro-organisms cannot be used for the treatment of biofilm infections. Our previous studies revealed that subminimal inhibitory concentrations (subMIC) of two macrolide antibiotics (azithromycin, AZM and erythromycin, ERY) reduced P. gingivalis biofilms. Furthermore, we demonstrated that the Bacillus subtilis sinR orthologue (PGN_0088) inhibits the synthesis of carbohydrates that are components of EPS in P. gingivalis biofilms. Here, we constructed a novel sinR mutant from P. gingivalis ATCC 33277 and reveal that the increased abundance of carbohydrate in EPS of the mutant led to a reduced infiltration rate of AZM and ERY through EPS, and consequently elevated biofilm resistance to these macrolides. Detailed elucidation of the interaction between the product of the sinR gene and EPS will assist in the development of novel approaches that target EPS to prevent and inhibit the formation of biofilms.

Promotion of endodontic lesions in rats by a novel extraradicular biofilm model using obturation materials.

Although extraradicular biofilm formation is related to refractory periapical periodontitis, the mechanism of extraradicular biofilm development, as well as its effect on periapical lesions, is unknown. Therefore, we aimed to develop an in vivo extraradicular biofilm model in rats and to identify and quantify extraradicular biofilm-forming bacteria while investigating the effect of extraradicular biofilms on periapical lesions. Periapical lesions were induced by exposing the pulpal tissue of the mandibular first molars of male Wistar rats to their oral environment. Four weeks later, gutta-percha points were excessively inserted into the mesial root canals of the right first molars (experimental sites) but not the left first molars (control sites). After 6 and 8 weeks of pulp exposure, the presence of extraradicular biofilms was confirmed histomorphologically, and biofilm-forming bacteria were identified by using classical culture methods. The biofilms were observed in the extraradicular area of the experimental sites. Similar species were detected both inside and outside the root canals. The bacterial count, quantified by real-time PCR assays, in the extraradicular area gradually increased in the experimental sites until 20 weeks after pulp exposure. After 8 weeks of pulp exposure, the periapical lesion volume that was measured by micro-computed tomography was significantly larger in the experimental sites than in the control sites (P < 0.05 by Welch's t test). These results suggest that we developed an extraradicular biofilm model in rats and that extraradicular biofilms affect developing periapical lesions.

Porphyromonas gingivalis biofilms persist after chlorhexidine treatment.

Chlorhexidine (CHX) gluconate effectively reduces the viability of biofilm-forming bacteria, such as Porphyromonas gingivalis. However, it is impossible to completely remove biofilms. The goal of the present study was to assess the potential pathogenicity of residual P. gingivalis biofilms in vitro after treatment with CHX gluconate. Scanning and transmission electron microscopy and confocal laser imaging revealed that treatment with CHX gluconate disrupted individual biofilm-forming P. gingivalis cells but did not destroy the biofilms. The volumes of the protein and carbohydrate constituents in the residual biofilms were not significantly different from those of the controls. The physical resistance of the residual biofilms to ultrasonication was significantly higher than that of controls. The volume of P. gingivalis adherent to the residual biofilms was higher than that to saliva-coated wells. These findings suggest that although CHX gluconate caused disruption of biofilm-forming cells, the constituents derived from disrupted cells were maintained in the biofilms, which sustained their external structures. Moreover, the residual biofilms could serve as a scaffold for the formation of new biofilms.

The sinR ortholog PGN_0088 encodes a transcriptional regulator that inhibits polysaccharide synthesis in Porphyromonas gingivalis ATCC 33277 biofilms.

Biofilm-forming cells are distinct from well characterized planktonic cells and aggregate in the extracellular matrix, the so-called extracellular polymeric substances (EPS). The sinR gene of Bacillus subtilis encodes a transcriptional regulator that is known to be involved in the biosynthesis of EPS in biofilms. Porphyromonas gingivalis inhabits the subgingival and extraradicular biofilm of humans and is one of the primary pathogens that cause progressive marginal and refractory apical periodontitis. Furthermore, P. gingivalis possesses PGN_0088, which encodes a putative ortholog of B. subtilis sinR. Here, we investigated the role of PGN_0088 (sinR) on biofilm formation. P. gingivalis strains formed biofilms on saliva-coated glass surfaces in phosphate buffered saline. Quantitative analysis indicated that the biofilm of the sinR null mutant consisted of dense exopolysaccharide. Microscopic observations showed that the increased levels of exopolysaccharide produced by the sinR mutant changed the morphology of the EPS to a mesh-liked structure. Furthermore, physical analyses suggested that the enrichment of exopolysaccharide in the EPS enhanced the resistance of the biofilm to hydrodynamic shear force. The results presented here demonstrate sinR plays important roles in the ability of P. gingivalis strain ATCC 33277 to act as a negative mediator of exopolysaccharide accumulation and is indirectly associated with the structure of the EPS and the force of its adhesion to surfaces.

Time course of gene expression during Porphyromonas gingivalis strain ATCC 33277 biofilm formation.

Chronological gene expression patterns of biofilm-forming cells are important to understand bioactivity and pathogenicity of biofilms. For Porphyromonas gingivalis ATCC 33277 biofilm formation, the number of genes differentially regulated by more than 1.5-fold was highest during the growth stage (312/2,090 genes), and some pathogen-associated genes were time-dependently controlled.