Effects Of Antimicrobial Activity Of Different Irrigating Agents Residual In Dentine On Enterococcus Faecalis Biofilm

BackgroundE. faecalis has a high detected rate in root canal treatment failure and refractory periodical case. The anatomical structure of root canal system is complex and there are many places without mechanical preparation reach. Therefore, in order to prevent the occurrence of root canal infection and inhibit bacteria biofilm reformed in the root canal or maintain the number of bacteria in the root canal system below pathogenic is especially important. Nowad, most studies focus on the antimicrobial properties of the irrigating solutions, involving both forms of bacterial growth, plankton and biofilm. However, few studies look into the residual antibacterial activity of irrigating solutions. Most of the studies with bacterial invasion model have been based on cultural methods but many studies shown that only few dentinal tubules are invaded by bacteria even after prolonged time of incubation. More recent studies made bacterial invasion model by centrifugation methods. It can make the study of antimicrobial properties of irrigating solutions more reliable. Study the antibacterial activity of irrigating solutions combined with the new infected model can be more reliable. ObjectivesTo study the effects of antimicrobial activity of different irrigating agents residual in dentine surface on E. faecalis biofilm in24h, observed by confocal laser scanning microscopy. A new noninvasive E. faecalis infected dentinal tubules model was established by centrifugation methods to study the effectiveness of dentin disinfection in0d and7d by using confocal laser scanning microscopy. To evaluate the residual antibacterial effectiveness of root canal irrigating agents by dentin surface and dentinal tubules, and provide experimental evidence for choosing a clinic drugs to control E. faecalis infection.Materials and methods1. Effects of antimicrobial Activity of five irrigating agents Residual in dentine on E. faecalis BiofilmE. faecalis was cultured on brain-Heart infusion (BHI) agar plates for24h under anaerobic conditions at37℃. Identify the characteristic of E. faecalis and determine the growth curve of it in24h.60dentin slices specimens which4mm×4mm×0.5mm (width×lengthxheight) were sterilized. One was randomly incubated in BHI for24hours at37℃to ensure no bacterial contamination.60dentin slices were divided into6groups (n=10), and treated with0.9%physiological saline,2.5%and5.25%sodium hypochlorite solution (NaOCl),17%Ethylenediaminetetraacetic acid (EDTA),10%citric acid and2%chlorhexidine (CHX), respectively. After that, each group was incubated with400μl E. faecalis suspension for16h. All specimens were observed under confocal laser scanning microscopy (CLSM), after stained by fluorescent LIVE/DEAD BacLight Bacterial Viability stain. The scanned images were analyzed by using Bioimage-L software. The inhibition percentage of biofilm formation was calculated, compared with negative control. To compare the efficacies of the different protocols when the reduction percentage related to the control the Student one-way ANOVA, SNK (Student-Neuman-Keuls) was used.2. Patterns of E. faecalis infected dentin made by centrifugation:an in vitro studySeventeen human healthy premolars extracted for orthodontic reasons were collected. A root dentin block with a length of4mm was horizontally sectioned from each tooth below the cement-enamel junction and the root canal inside the blocks were enlarged to a Gates Glidden drill. Each cylindrical dentin block was splited into two semicylindrical halves. A total of33semicylindrical halves dentin specimens were made. The outer surfaces of the semicylindrical halves were ground by600-grit silicon carbide paper to achieve a standard thickness of2mm and to remove the root surface cement. The dentin specimens were then shaped by a water-cooled low-speed handpiece with a fine carbide bur to make the specimen fit the inner wall of a filter tube with0.45mm pore size. The size of the refined specimen was about4mm×4mm×2mm (width×length×height). The smear layer on both sides of the specimen was removed by immersion in5.25%NaOCl and10%citric acid each for4min in an ultrasonic bath specimens were rinsed in sterile water for1minute after smear layer removal. Each prepared dentin specimen was placed in a filter tube with the canal (pulpal) side up. Any gaps between the dentin specimen and the inner wall of tube were carefully sealed by composite light-cured for20s.10specimens were picked randomly, crystal violet was added to the specimens. Five of them were centrifuged at1400g,2000g,3600g, and5600g in a sequence twice each for5min. A fresh solution was added between every centrifugation, and the solution that had penetrated through the dentin piece was discarded. The other5were keeping in standing for24h.2specimens were picked randomly, E. Faecalis suspension was added to each dentin specimen before centrifugation, and observed by fluorescence microscope after stain with fluorescent acridine orange. All specimens remained were divided into two groups of10specimens each. In group A, E. Faecalis suspension was added to each dentin specimen before centrifugation. In group B (negative control) brain-Heart infusion (BHI) broth was added before centrifugation. In those two group halve dentin specimens were observed by scanning electron microscope (SEM) and the other halve specimens were observed by confocal laser scanning microscopy (CLSM) after stain with fluorescent LIVE/DEAD BacLight Bacterial Viability stain. 3. Effects of antimicrobial Activity of irrigating agents Residual in dentinal tubules on E. faecalis BiofilmSix human healthy premolars extracted for orthodontic reasons were collected. Specimens were prepared as described above, a total of12case of4mm×4mm×2mm (widthxlengthxheight) samples were prepared. The smear layer on both sides of the specimen was removed. Each prepared dentin specimen was placed in a filter tube with the canal (pulpal) side up. Any gaps between the dentin specimen and the inner wall of tube were carefully sealed by composite light-cured for20seconds. E. Faecalis suspension was added to each dentin specimen and centrifugated by the methods described above. The infected dentin specimens were divided into four groups,5.25%NaOC10d and7d group,2%CHX0d and7d group. The infected specimens were treated with5.25%NaOCl and2%CHX solution, respectively. The samples of0d group was observed by laser confocal scanning microscope after stained with fluorescent LIVE/DEAD BacLight Bacterial Viability stain, immediately after treaded with irrigating agents. The rest of samples were placed in the anaerobic incubator and keep the environment moist. The samples were took out of the anaerobic incubator after7d and observed by laser confocal scanning microscope after stained with fluorescent LIVE/DEAD BacLight Bacterial Viability stain. Images were analyzed by using Bioimage-L software.Results1. E. faecalis enter stable phase at16h. NaOCl groups shows the lowest residual activity (13.3%,18.8%); similar residual activity were found in17%EDTA and10%citric acid groups with inhibition of E. Faecalis biofilm formation43.8%and45.3%respectively;2%CHX group showed greatest residual activity (99.9%).2. Crystal violet dye penetrated into the cementum side of the dentin in the centrifugal group. Meanwhile, crystal violet only dye the root canal side of the dentin, no any dye the deep dentin tissue in the immersion group. Under fluorescence microscope a mound of green fluorescent were observed. Under CLSM, E. Faecalis penetrated into dentinal tubules in group A. It is a severe infection model. A large number of viable bacteria which were stained by green fluorescene showed in the dentin blocks. No bacteria were observed in control group, only a small amount of scattered light green fluorescence observed. Under SEM, E. Faecalis with normal Morphology were observed. There is a large number of E. faecalis in the dentinal tubules openings. Most bacteria in the dentinal tubules were normal at Morphology but irregular shape bacteria still can be seen occasionally. There were no bacteria observed in control group, collagen fibers interwoven into a network and embedded into the dentinal tubules wall.3. The results showed that viable biovolume of5.25%sodium hypochlorite7d group more than5.25%sodium hypochlorite0d group (P<0.05). The viable biovolume of2%chlorhexidine0d group compared with2%chlorhexidine7d group have no significant difference (P>0.05), indicating that the residual liquid of2%chlorhexidine can inhibit the E. faecalis within7d.Conclusion2%CHX was shown to be the most effective in inhibition of E. Faecalis biofilm formation compared with other five different irrigating agents.2%CHX was shown to be effective in inhibition of E. Faecalis in dentinal tubules at long-term.