Effect Of MB-PDT Against Enterococcus Faecalis Biofilms In Vitro

Objectives:To establish an in vitro model of Enterococcus faecalis biofilm on bovine dentin blocks,and then scanned via scanning electron microscopy to observe the growth of Enterococcus faecalis at different stages in bovine dentin tubules.After sterilization,the activity of bacteria in biofilm was quantitatively analyzed by confocal laser scanning microscopy and Imaris software,in order to clarify the role of MB-PDT in controlling the Enterococcus faecalis biofilm and provide new ideas for endodontic therapies.Methods:Enterococcus faecalis was inoculated into sterile bovine dentin blocks and incubated at 37°C for one week or three weeks,to establish the endodontic bacterial biofilms formed at different time points.The structure of biofilms was observed by scanning electron microscopy.The 1 week sample was taken as a young biofilm model and the 3 weeks sample as a old biofilm model.5% NaOCl(group A),MB-PDT(group B),and sterile saline(group C)were used for sterilization.Each group was divided into three subgroups which were treated for 3,12,and 30 minutes,respectively.Subsequently,the samples were stained with fluorescent viability dyes and examined by confocal laser scanning microscopy.A 3D model was reconstructed and the proportion of dead cell volume was analyzed by using the Imaris software to evaluate the antibacterial effect of MB-PDT.1w biofilm samples were selected and treated with MB-PDT for 12 min.The morphology changes of biofilms were observed under scanning electron microscope to further clarify the bactericidal effect of MB-PDT.The aseptic bovine dentin block were subjected to the MB-PDT for 12 mins and 30 mins respectively,and then scanned via scanning electron microscopy to observe the microstructural changes of pulp chamber walls,so as to analyze the influence of MB-PDT on morphology of tooth tissue.Results:1.An in vitro model of Enterococcus faecalis biofilm at different time points was established successfully.The colonization of Enterococcus faecalis could be observed on the bovine dentine block under scanning electron microscope.2.The 3-dimentional reconstruction CLSM images showed that the majority of bacterial biofilm were killed by MB-PDT,but the sterilization effect was significantly lower than 5% NaClO(P<0.05).The proportions of dead cells subjected to the disinfecting approaches increased significantly with the increasing exposure.Remarkably more bacteria were killed in young endodontic biofilms than that in old biofilms(P<0.05).3.There were no microstructural changes in tooth structure after it had been subjected to MB-PDT for a certain treating time.Conclusion:MB-PDT has an effective bactericidal effect on E.faecalis biofilms,and there were no microstructural changes in tooth structure,but the sterilization effect of MB-PDT alone is not as good as that of 5% NaOCl.MB-PDT shows promise as an additional approach in infection control during endodontic therapies which can improve the sterilization rate.Laser scanning confocal microscopy can perform tomographic scanning of specimens with certain thickness.It is an effective way to observe the activity of bacteria in dentin tubules.