| Objective In the Endodontics Department clinical work,there are still a certain number of root canal treatment failure cases.This is mainly due to the continuous infection caused by the incomplete elimination of microorganisms in the root canal and extraapical.Enterococcus faecalis(E.faecalis)is a common microorganism causing infection.This study was designed to investigate the effects of D-leucine combined with MTAD on E.faecalis and its biofilm’s inhibition and dispersion,in order to find a more thorough root canal irrigation scheme to remove Enterococcus faecalis biofilm,to find a new root canal re-treatment method and reduce the failure rate of root canal therapy.Method E.faecalis was cultured to logarithmic growth stage.The suspension was diluted in BHI medium with a volume ratio of 1:10.The treatment group was added 0.2% chlorhexidine,MTAD,50 mmol D-leucine,50 mmol D-leucine +MTAD,while the non-treatment group was only added BHI for 10 hours.The optical density(OD600)of bacterial suspension at 600 nm was measured by enzyme label every 2 hours,draw the growth curve of single bacterial suspension.The suspension of logarithmic growth phase was added to 96-well plate,the treatment group was added to the above four groups of antimicrobial agents,the non-treatment group was added to the culture medium,and then cultured for 48 hours.The optical density(OD570)of biofilm at 570 nm was determined by crystal violet staining.E.faecalis 48 h biofilm model was constructed in 96-well plate.The four groups of antimicrobial agents were added to the treatment group,and the culture medium was added to the non-treatment group.The optical density(OD570)of the biofilm at 570 nm was determined by crystal violet staining.E.faecalis 48 h biofilm model was constructed in 48-well plates containing dentin and cementum.The treatment group was added to the said four groups of antimicrobial agents,and the non-treatment group was added to the culture medium,and then cultured for 2 hours.After irrigating,stain with AO/EB dye.Confocal laser scanning microscope was used to observe the remaining E.faecalis biofilm samples on dentin and cementum slices.Image J was used to measure green fluorescence area and red fluorescence area of biofilm,and the total biomassof bacterial biofilm was taken as the sum of red and green fluorescence area.Using SPSS21.0 software,Oneway-Anova variance analysis and SNK method,statistical analysis of data was conducted.When P < 0.05 was set,the difference was statistically significant.Result 1.The effect of D-leucine combined with MTAD on inhibiting the growth of suspended E.faecalis was better than that of D-leucine alone,but similar to that of MTAD alone.0.2% chlorhexidine had the best effect on inhibiting the growth of suspended E.faecalis.2.The effect of D-leucine combined with MTAD on inhibiting E.faecalis biofilm was better than that of D-leucine alone(P < 0.05).Compared with 0.2%chlorhexidine and MTAD alone,there was no significant difference(P > 0.05).3.Crystal violet staining showed that the effect of D-leucine combined with MTAD on the dispersing 48-hour E.faecalis biofilm was better than that of 0.2%chlorhexidine,MTAD alone and D-leucine alone(P < 0.05).4.Confocal laser scanning microscope showed that the remaining 48 hours E.faecalis biofilm on dentin and cementum slices in the D-leucine combined MTAD group was the least,which was significantly different from that in the 0.2%chlorhexidine group,MTAD group and D-leucine group(P < 0.05).Conclusion 1.D-leucine combined with MTAD could inhibit the growth of suspended E.faecalis,but there is no significant improvement in the inhibition effect of MTAD on suspended E.faecalis.2.D-leucine combined with MTAD could inhibit the formation of E.faecalis biofilm,but there is no significant improvement in the inhibition of E.faecalis biofilm formation by MTAD.3.D-leucine combined with MTAD can significantly improve the effect of MTAD on dispersing 48 hE.faecalis biofilm.The dispersed effect was better than0.2% chlorhexidine in root canal irrigation.4.D-leucine combined with MTAD could significantly improve the effect of MTAD to disperse 48 hE.faecalis biofilm on the dentin and cementum slice.The disperse effect was better than 0.2% chlorhexidine in root canal irrigation. |
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