Effect Of MB-PDT On Enterococcus Faecalis Biofilm In Dentinal Tubules

ObjectivesIn this study,an in vitro model of Enterococcus faecalis biofilm-infected dentinal tubules was prepared on isolated teeth.After sterilization with conventional root canal irrigants and methylene blue-mediated photodynamic therapy(MB-PDT),the control of Enterococcus faecalis biofilm in dentinal canal was evaluated by confocal laser scanning microscopy(CLSM)and plate colony counting.To provide a new method for clinical prevention and treatment of pulp and periapical diseases.MethodsEnterococcus faecalis suspension was inoculated into the dentin tubules of isolated dentin lumps and single root canal teeth,and cultured in 37 ? incubator.Establishment of a dental tubule model infected by Enterococcus faecalis cultured in vitro for 1 day and 3 weeks,and sterilized with 5% NaOCl,2% CHX and MB-PDT.Each group was divided into 3 minutes,12 minutes and 30 minutes.MB-PDT combined with 5% NaOCl and MB-PDT combined with 2% CHX was used for 12 minutes.Then,the dentinal tubule model infected by Enterococcus faecalis after sterilization was stained with bacterial reactive fluorescent dyes.Laser scanning confocal microscopy(CLSM)was used to observe and scan immediately.Imaris software was used to reconstruct the three-dimensional images after three-dimensional reconstruction to calculate the proportion of dead bacteria in the biofilm of Enterococcus faecalis treated by different methods,and then the bactericidal efficacy of MB-PDT was evaluated.After 3 weeks of culture at 37?,5% NaOCl,2% CHX,MB-PDT and sterile saline were used to treat the isolated single root canal teeth for 12 minutes.The surface of root canal wall and dentin debris with different depths(100,200 and 300 microns)were collected before and after each group for colony culture and counting,and the bacterial clearance rate was calculated.Results1.The dentin tubule model of Enterococcus faecalis infection was successfully established.Under CLSM,it was observed that the bacteria arranged in a chain and Enterococcus faecalis colonized successfully.2.After three-dimensional reconstruction,bacterial biofilm images of infected dentinal tubules showed that 5% NaOCl,2% CHX and MB-PDT could effectively kill most of the bacteria in Enterococcus faecalis biofilm,but the sterilization effect of MB-PDT and 2% CHX was inferior to 5% NaOCl(P < 0.05);prolonging the action time,the proportion of bacteria in biofilm increased(P < 0.05);The resistance of bacterial biofilm to sterilization method is stronger than that of young bacterial biofilm,and it is not easy to be removed(P < 0.05).3.The biofilm images of Enterococcus faecalis infected dentinal tubules after three-dimensional reconstruction showed that MB-PDT combined with 5% NaOCl killed the most bacteria in Enterococcus faecalis biofilm,and the effect was better than that of 5% NaOCl,2% CHX,MB-PDT and MB-PDT combined with 2% CHX group(P < 0.05).4.The bacterial reduction rates of 5% NaOCl,2% CHX and MB-PDT groups at different root canal wall depths were significantly higher than those of saline group(P < 0.05).The bacterial reduction rates of 5% NaOCl group at the root canal wall surface and 100 and 200 ?m depth of dentinal canaliculus were the best,which were better than those of 2% CHX and MB-PDT(P < 0.05);the bacterial clearance rates of MB-PDT group at the depth of each dentinal canaliculus had no significant difference(P > 0.05).Conclusion:MB-PDT has good sterilization effect on Enterococcus faecalis biofilm in vitro.In the treatment of pulp and periapical diseases,MB-PDT can be used as a new auxiliary method to control Enterococcus faecalis infection and improve the success rate of root canal therapy.