In Vitro Study On Removal Of Enterococcus Faecalis And Its Biofilm By Iron-Gallic Acid Nanase (Fe-GA)

Objective:Chronic periapical periodontitis is a common and frequently occurring disease in oral diseases.Although root canal therapy can solve most of the problems in periapical periodontitis,some intractable bacteria are difficult to be completely removed and develop into refractory periapical periodontitis with an incidence of about 10%-20%,which has always been a clinical problem.The detection rate of Enterococcus faecalis in the root canals of refractory periapical patients was more than 70%,mainly in the form of biofilm on the root canals and apical surfaces of affected teeth,and its resistance in the biofilm was 1000times that in the floating state,and it was difficult to be completely removed.Although traditional root canal washes,such as sodium hypochlorite and chlorhexidine,have been widely used in current root canal treatments,they can accidentally penetrate into oral tissue and produce toxicity and adverse reactions.Peroxidase can use physiological concentration of hydrogen peroxide to activate its enzyme-like reaction and locally produce a large number of bactericidal reactive oxygen species（ROS）,which can avoid denaturation or protease hydrolysis and maintain lasting bactericidal activity.It has been reported that nanomaterials with peroxidase activity have certain inhibitory effects on Enterococcus faecalis in root canals,but relevant studies are still few.The ferro-gallic acid nanomaterial（Fe-GA）synthesized successfully in laboratory has good peroxidase activity,good bactericidal effect and high biocompatibility.In this study,iron-gallic acid nanomaterial（Fe-GA）was used as the research object to explore its inhibitory effect on Enterococcus faecalis and biofilm,and establish a root canal model of Enterococcus faecalis infection in vitro.Fe-GA was used as the root canal washing solution,and compared with 3%sodium hypochlorite（Na OCl）,3%hydrogen peroxide（H₂O₂）and normal saline.The removal effect of Enterococcus faecalis on infected root canal was analyzed,which could be used as a reference for clinical discovery of a root canal flushing solution with high biological safety and good bactericidal effect.Methods:Ⅰ.Detection of bacteriostatic activity of Fe-GA against Enterococcus faecalis:Plate colony counting method was used to detect the bacteriostatic activity of Fe-GA against Enterococcus faecalis.Reactive oxygen species（ROS）level in bacterial cells was measured by reactive oxygen detection kit.The morphology of bacteria was observed by transmission electron microscope.The scavenging ability of Fe-GA on Enterococcus faecalis biofilm was studied by Confocal laserscanning microscopy（CLSM）and crystal violet biofilm staining.Ⅱ.Detection of cytotoxicity of Fe-GA:The toxic effect of Fe-GA on human gingival fibroblasts was further studied by CCK-8 method.Ⅲ.Detection of Fe-GA on the removal effect of Enterococcus faecalis and biofilm in root canals:The root canal model of bovine teeth infected with Enterococcus faecalis was established,and the sample teeth were randomly divided into 4 groups with 5 samples in each group,as follows:Group A（Fe-GA+H₂O₂）,Group B（3%Na OCl）,group C（3%H₂O₂）,and group D（normal saline）.Three samples were randomly selected from each group to calculate the reduction of the number of Enterococcus faecalis in the root canal after drug irrigation,and the remaining two samples were examined by scanning electron microscopy to observe the removal effect of Enterococcus faecalis and biofilm in the root canal.t test and one-way analysis of variance were used to analyze the comparative data.Results:Ⅰ.Detection of bacteriostatic activity of Fe-GA against Enterococcus faecalis:Under darkness or visible light,Fe-GA groups at all concentrations and 1m M H₂O₂showed no antibacterial effect;After being exposed to visible light for 10min and incubated in a 37℃water bath for 30 min,the inhibition rate of 1m M H₂O₂catalyzed by 200μg/ml and 400μg/ml Fe-GA on Enterococcus faecae was more than 90%,and there was no significant difference in the effect.No antibacterial effect was observed under dark conditions.Under darkness or visible light,low concentration（100μg/ml）Fe-GA catalyzed H₂O₂had no antibacterial effect.The antibacterial efficiency of Fe-GA is closely related to its concentration,whether it is irradiated by visible light and whether it is combined with hydrogen peroxide.The ROS test showed that Fe-GA combined with H₂O₂could induce ROS production in Enterococcus faecalis cells and promote bacterial lysis and death.Fe-GA was observed by transmission electron microscopy（TEM）to destroy bacterial cell wall and enhance the permeability of bacterial cell membrane.CLSM and crystal violet staining showed that Fe-GA+H₂O₂group could destroy the structure of Enterococcus faecalis biofilm and kill the bacteria in it.Ⅱ.Detection of cytotoxicity of Fe-GA:Human gingival fibroblasts（HGFs）were used as test cells,and the cytotoxicity of Fe-GA was detected by CCK-8 method.After 12h co-culture,the cell viability of 100μg and 200μg groups remained high regardless of whether they were combined with H₂O₂,while the cell viability of 400μg material group and1m M H₂O₂concentration group decreased.It also stayed above 50 percent.Ⅲ.Detection of the clearance effect of Fe-GA on Enterococcus faecalis and biofilm in root canal:In the infected root canal model,the clearance effect of Fe-GA group on Enterococcus faecalis and biofilm was equivalent to 3%Na OCl,and better than 3%H₂O₂and normal saline.Conclusion:Ⅰ.Fe-GA has good anti-Enterococcus faecalis and its biofilm.Ⅱ.the cytotoxicity test showed that Fe-GA had good cytocompatibility.Ⅲ.In vitro root canal experiment of bovine tooth infection,Fe-GA can clear Enterococcus faecalis and its biofilm in root canal.Therefore,Fe-GA can remove enterococcus faecalis and its biofilm safely and efficiently,providing a basis for clinical selection of irrigation drugs to control bacterial infection in root canals.