Killing effect of antibacterial photodynamic therapy - aPDT with long-term exposure against young and old Enterococcus faecalis biofilms in dentin

Background: Antibacterial photodynamic therapy - aPDT is a medical method that utilizes the activation of a nontoxic photoactive agent or photosensitizer by exposure to visible light of a specific wave-length in the presence of oxygen. The present study aimed to evaluate the killing in vitro effect of aPDT with 0.01% methylene blue (MB) against young and old Enterococcus faecalis ( E. faecalis ) biofilms in bovine dentin with the long-term exposure using confocal laser scanning microscopy (CLSM). Methods: Semicylindrical bovine dentin blocks were inoculated with E. faecalis and incubated in air to form 1- and 3-week-old biofilms. The biofilms in dentin were subjected to aPDT with 0.01% MB, 5% NaOCl and saline with the exposure of 3, 12 and 30 minutes. The dead portions of bacterial cells in E. faecalis biofilms were analyzed with using LIVE/DEAD bacteria viability staining and CLSM. Results: The visible changes in dentin structure caused by aPDT were verified with scanning electron microscopy. Significantly more bacteria were dead when aPDT with MB and 5% NaOCl were used with the long exposure time (12 and 30 minutes) than with 3 minutes (P < 0.05). The speed of killing was fastest during the first 3 minutes, and few more bacterial cells were killed after 12 minutes in the disinfection groups. Five percent NaOCl exhibited the highest effectiveness of bacterial killing in dentin at each time point than aPDT with MB groups (P < 0.05). The proportion of killed bacteria was higher in young biofilms than in mature biofilms in aPDT with MB and NaOCl groups (P < 0.05). Moreover, there were no clearly visible changes in structure of dentin surfaces subjected to aPDT with MB for 30 minutes. Conclusion: aPDT with 0.01% MB has the capability to kill bacterial cells in E. faecalis biofilms on bovine dentin, and does not result in visible changes of dentin structures. The antibacterial effect was time-dependent, but little additional killing was obtained after the first 12 minutes of exposure.