Effect Of Various Lasers On Smear Layer Removal From The Root Canal Wall And On The Enterococcus Faecalis Biofilms In Dentinal Tubules In Vitro

High-quality completion of root canal preparation and root canal disinfection are the prerequisite for the effective control of infection in the root canal therapy.However,during the root canal instrumention,due to the complexity of the root canal system and the limitations of instrumention.Most of the areas are not treated,especially in the apical third,because there are lateral accessory canal,fins,intercanal anastomoses,lateral branch of root canal,apical ramification,apical furcation.Debris and bacterias easily accumulated in these structures and formed smear layer,in addition the conventional irrigation and disinfection technology is superficial,so the smear layer in apical third is difficult to remove.In recent years,lasers as a novel adjuncting irrigation tool are used clinically,so the first purpose of this study is to evaluate the effect of laser-activated irrigations(LAI)using Er:YAG laser and the Er,Cr:YSGG laser on removing smear layer from the root canal wall.It has been well demonstrated that bacteria and their products contribute greatly to the development of pulpal and periapical diseases.The fundamental aim of endodontic therapy is the disinfection of root canals and prevention of reinfection.However,the success rate of endodontic therapy was reported about 85% and 90%.One of the main reasons of root canal therapy failure is the presence of microorganisms such as Enterococcus faecalis.Enterococcus faecalis is often detected from the teeth of root canal treatment failure and refractory periapical periodontits,which is a gram-positive facultative anaerobic bacteria.They can adhere to each other and invade the dentin tubules to form Enterococcus faecalis Biofilm,and they have the ability to withstand prolonged periods of nutrient limination and can resist the effects of irrigats and intracanal medicament.At present,Na OCl is the most commonly used canal irrigant,but it can only penetrate to a depth of 130?m into the dentinal tubules,microorganisms in the deeper layers of dentin still cannot be affected effectively.Currently,a variety of lasers were introduced into endodontic treatment for its bactericidal effect,such as Er,Cr: YSGG laser,Er: YAG laser,Nd: YAG laser,Diode laser,a PDT and etc.It's reported that they can directly or indirectly kill bacteria,yet little is known about their relative bactericidal effectiveness in the root canal environment,so the second purpose of this study is to compare the bactericidal effect of the various lasers.The experiment is divided into the following two parts:Part 1 Effect of different lasers on smear layer removal from the root canal wall Experiment one : The establishment of smear layer on the root canal wallIn this study,we collected human single teeth in vitro,by simulating the clinical root canal preparation protocol,they were then prepared with sequential M3 nickel-titanium rotary instruments(#19/.02,#20/.04,#25/.04,#25/.06,and #35/.04)to the working length using a crown-down technique.The 0.5% Na OCl(5 m L)irrigation was adopted during the instrumentation using a syringe with a 27-G side-vented needle,and then they were observed under scanning electron microscopy.Results showed that the smear layer was successfully generated within the root canals.Experiment Two: Effect of Laser-activated Irrigations on Smear Layer Removal from the Root Canal WallOn the basis of the established smear layer model,we selected 90 human single roots,and they were divided into 9 groups according to the final irrigation,and each group individually received one treatment as follows:(A)Na OCl,5.25% sodium hypochlorite(Na OCl)for 60 s,(B)Er:YAG+Na OCl,(C)Er,Cr:YSGG+Na OCl,(D)EDTA,17% ethylenediamine tetraacetic acid(EDTA)for 60 s,(E)Er:YAG+EDTA,(F)Er,Cr:YSGG+EDTA,(G)5.25%Na OCl+17%EDTA,Na OCl and EDTA for 30 s each,(H)Er:YAG+Na OCl+EDTA,(I)Er,Cr:YSGG+Na OCl+EDTA.For groups B?C?E?F,LAI with Na OCl or EDTA respectively for 60 s.For groups H?I,LAI with Na OCl and EDTA for 30 seconds each.Efficacies of SL removal were evaluated using a 5-grade scoring system under scanning electron microscopy.Results: The laser activations significantly enhanced the effect of the Na OCl,EDTA,and Na OCl + EDTA irrigations in removing SL(P < 0.05).The efficacies of SL removal were presented in the descending order,as follows: 1)LAI + Na OCl + EDTA,2)LAI + EDTA,3)LAI + Na OCl,4)Na OCl + EDTA,5)EDTA,and 6)Na OCl.Conclusions: Among the treatments,the LAI + Na OCl + EDTA was the most effective protocol in removing SL from the entire root canal wall,which may be effective for root canal treatment.Part 2: Effectiveness of various laser irradiation systems on Enterococcus faecalis biofilms in dentinal tubules in vitroExperiment one: Establishment of the E.faecalis biofilms in dentinal tubulesIn this study,a cylindrical root dentin block was horizontally sectioned from each single-rooted tooth at 1 mm below the cementoenamel junction,and then we received 4 × 2 × 3mm3 dentin specimens,and the samples were rinsed with 5.25% Na OCl and 17% EDTA in an ultrasonic bath for 4 min to remove the smear layer.After specimens were sterilized by autoclaving,and confirm that smear layer and microorganisms had been removed by SEM.Then dentinal tubules in semi-cylindrical dentin blocks were filled with E.faecalis by centrifugation and incubated to form 3-week-old biofilms,later they were examined by SEM and CLSM.Results showed that a heavy invasion by Enterococcus faecalis was detected by both SEM and CLSM throughout the dentinal tubules.Experiment two: Effect of Na OCl on dentin disinfection by using Confocal Laser Scanning MicroscopyOn the basis of the infectious Enterococcus faecalis model,the samples were randomly divided into four groups as followed: 5.25% Na OCl for 1,3,5 and 10 min respectively.After treatments,the proportion of bacteria killed was analyzed by CLSM by using LIVE/DEAD bacterial viability stain.Result: As the prolongation of the time of Na OCl,the bactericidal effect is enhanced,the difference is statistically significant(P <0.05).Conclusion: The killing of bacteria in infected dentin by Na OCl is time-dependent.When the exposure time is 10 min,Na OCl achieved the maximal bactericidal effect.Experiment three: Effect of Various Lasers Irradiation systems on E.faecalis biofilms in Dentinal tubulars by using Confocal Laser Scanning MicroscopyAfter 3 weeks of incubation,the infected dentin specimens were randomly subjected to 1 and 3 minutes of exposure to the following group,A:5.25% Na OCl,B :Nd: YAG laser,C: Diode laser,D: Er,Cr: YSGG / Na OCl,E: Er: YAG / Na OCl,F: Nd: YAP / Na OCl.The specimens were then stained for bacterial viability and examined by CLSM for 3-dimentional reconstruction analysis.The proportion of dead and live bacteria inside dentinal tubules were calculated by using Image-Pro-Plus software.Results showed that Er,Cr: YSGG,Er: YAG and Nd: YAP laser could significantly enhance the bactericidal effect of Na OCl(P <0.05).No significant difference in bacterial killing was detected between Er,Cr: YSGG / Na OCl and Er: YAG and between Nd: YAG laser and Diode laser (P> 0.05).The bactericidal effects against E.faecalis of laser activated groups were more effective than that of laser irradiation groups(P <0.05).The efficacies of killing bacteria were presented in the descending order,as follows:Er: YAG / Na OCl,Er,Cr: YSGG / Na OCl> Nd: YAP / Na OCl> Nd: YAG,Diode> Na OCl.Conclusion: Er: YAG and Er,Cr: YSGG laser can significantly enhance the bactericidal effect of Na OCl,especially the Er: YAG laser combined with NaOCl.