The Antimicrobial Activity Of Photodynamic Therapy Against Enterococcus Faecalis Using Two Different Formulations Of Methylene Blue: A Comparative Study

Objective:Enterococcus faecalis (E. faecalis) is known to be one of the predominantbacteria in teeth which root canal therapy fails and appears to be highly resistant tomedicament during treatment. It is hard to remove this pathogens completely bychemomechanical preparation and intracanal medication. Searching for an effectiveprocedures has become the inevitable trend to improve the success rate of endodontictreatment. Nowadays, a great deal of alternative methods against E. faecalis havebeen explored. New antimicrobial strategy including photodynaminc therapy (PDT)which has proved effective in helping fight systemic infections such as orthopaedicinfection, skin infection, and so on. In order to investigate PDT in the treatment of E.faecalis infection, this study aimed to assess the cytotoxicity of PDT on humanperiodontal ligament fibroblasts (hPDLFs) firstly, then assesed the antibacterialactivity of PDT against E. faecalis in an aqueous suspension and in an infected toothmodel with the technology of counting colony-forming units (CFU) and scanningelectronic microscope (SEM). The ultimate goal of this research was to explore thesafety and feasibility of PDT in endodontic treatment. In addition, we also compared the effectiveness of PDT in reducing E. faecalis using two different formulations ofmethylene blue (MB) and two different light delivery systems to analyze theinfluential factors of PDT further.Part Ⅰ: Cytotoxicity of PDT with two different formulations of MB on hPDLFs invitroObjective: To investigate the photodynamic effect of two different formulations ofMB on the killing ability to hPDLFs in vitro. Methods: Cultured hPDLFs wereincubated in a medium containing two different formulations of MB with5differentconcentration (0.1,1.0,10,25,50μmol/L) for10min, and irradiated5min using asemiconductor laser with a wavelength670nm. The power outputs were set at40mWand the total energy dose was12J. After treatment, the cells continue to cultivate24h,the cytotoxicity was investigated by light microscope and MTT assay. Results: Therewas not significant difference in the cytotoxicity of the two different formulations ofMB on hPDLFs (P>0.05). When the concentration of the photosensitizers was lessor epual to25μmol/L, the cells were normal in appearance and cytotoxicity score wasat1-2grade. While the concentration was more or epual to50μmol/L, the cellsshrinkaged and turned round, and cytotoxicity score was grade3. Conclusion: Thecytotoxicity of the two different formulations of MB on hPDLFs was effected by theconcentration of the photosensitizers, when using appropriate drug concentration, thecells had a lower toxicity.Part Ⅱ: Photodynamic effect of two different formulations of MB on E. faecalisin vitroObjective: To examine the photodynamic effect of two different formulations of MBon E. faecalis in planktonic cultures. Methods: The E. faecalis in planktonic cultureswere used to investigate the antibacterial activity of PDT using two differentformulations of MB. After10min pre-irradiated time, the cultured bacterium wereirradiated5min using a semiconductor laser with a wavelength670nm. The poweroutputs were set at50mW and the total energy dose was15J. Immediately after thetesting procedures, the bacteria groups were counted to measure the rate ofantibacterial introduced by PDT with two different formulations of MB. Results:There was not significant difference in the photodynamic effect of two differentformulations of MB on E. faecalis in planktonic cultures (P>0.05). The antibacterialrate were99.976%(MB dissolved in water) and99.982%(MB dissolved in a mixture), respectively. It was also observed that light alone reduced bacterial viabilityby67.5%while MB alone reduced bacterial viability by71.69%(MB dissolved inwater) and73.67%(MB dissolved in a mixture) respectively, which produced lesseffective than the PDT groups (P <0.05). Conclusions: As a new antimicrobialstrategy, PDT was efficient at killing E. faecalis in planktonic cultures. Only therequirement of both photosensitizer and light, PDT can play a greater therapeuticeffect.Part Ⅲ: Susceptibility of E. faecalis to PDT with two different light transmissionin a bovine tooth modelObjective: To evaluate the PDT effectiveness in reducing E. faecalis in infected rootcanl with two different light delivery systems. Methods: The model of bovine rootcanals infected with E. faecalis was established. Eighteen infected canals wererandomly divided into two groups with9samples in each. After10min pre-irradiatedtime, the specimens were irradiated5min with or without an intracanal optical fiberusing a semiconductor laser with a wavelength670nm. The power outputs were set at50mW and the total energy dose was15J. Samples were collected before and aftertesting procedures and CFU/mL was determined. Results: The antibacterial rate ofirradiation with an intracanal optical fiber was95.89%, while the other group was95.23%, and there was no significant statistical difference between the two groups (P>0.05). Conclusions: While the parameters of the illumination are suitable, PDTwould be effective against E. faecalis, regardless of the use of an intracanal opticalfiber.Part Ⅳ: Photodynamic effect of two different formulations of MB on E. faecalisin a bovine tooth modelObjective: To investigate the antibacterial activity of PDT against E. faecalis ininfected root canals using two different formulations of MB. Methods: The model ofbovine root canals infected with E. faecalis was established. Forty-five infected canalswere randomly divided into five groups with9samples in each. Five different rootcanal disinfection measures were applied for these groups respectively. The controlgroup were treated by ultrasonic irrigation with2.5%NaOCl, while the other four testgroups were treated by PDT alone (two different formulation of MB) or ultrasonicirrigation combined with PDT. All the specimens in the PDT groups and combinationgroups were irradiated5min using a semiconductor laser with a wavelength670nm.The power outputs were set at50mW and the total energy dose was15J. Samples were collected from root canals immediately before and after disinfection to quantifythe bacteria. After disinfection, three species per group were selected randomly andsplit longitudinally to examine the morphology of the residual bacteria inside thedentinal tubule with SEM. Results: Ultrasonic irrigation reduced bacterial viability by99.46%while PDT alone reduced bacterial viability by95.89%(dissolved in water)and98.3%(dissolved in a mixture), respectively. However, there were not statisticallysignificant among these three groups (P>0.05). The best result in this study wereformed by the combination groups which achieved99.96%(MB dissolved in water)and99.92%(MB dissolved in a mixture) reduction of E. faecalis activity respectively.The combination groups produced more significant bacterial killing than the PDTgroup (MB dissolved in water)(P <0.05) while there were not statistically significantamong other groups (P>0.05). Beyond that, the morphological change observedunder SEM was also in accordance with the antibacterial rate, with the most obviouschange in combination groups. Conclusions: As an effective adjunctive method forroot canal treatment, ultrasonic irrigation combined with PDT may improve theefficacy of root canal disinfection.