Methylene blue and hydrogen peroxide for photodynamic inactivation in root canal - A new protocol for use in endodontics

Objective: Antimicrobial photodynamic therapy (aPDT) is a controversial approach for endodontic disinfection. The objective of this study was to test the photosensitiser (PS) concentration and assess the optical shielding phenomenon, the use of hydrogen peroxide (H₂O₂) and minimal energy irradiation to optimise endodontic aPDT for suggesting a protocol for clinical use. Methods: Different parameters for aPDT were tested. Aqueous solutions of methylene blue (MB) at 50, 100, 150 and 300 µM were tested in vitro for optical shield and reactive oxygen species (ROS) production by the reduction of N,N-dimethyl-4-notrosoaniline (RNO) at 440 nm absorbance when irradiated using a diode laser (660 nm). Ten single-rooted teeth were inoculated with bioluminescent bacteria Pseudomonas aeruginosa for 72 hours to form biofilms. Bioluminescence imaging was used to serially evaluate the minimum energy necessary during endodontic aPDT using MB and a diode laser coupled to an optical fibre for intracanal microbial reduction. In addition, teeth (n=21) infected with Enterococcus faecalis were treated with sequential combinations of endodontic aPDT and H₂O₂ and the colony-forming unit (CFU) was determined. Results: ROS production was inversely proportional to the MB concentration in the solution due to quenching of MB. Optical shielding limited light penetration at high MB concentrations. The use of H₂O₂ before aPDT achieved higher disinfection compared to conventional aPDT or when MB was irradiated in an H₂O₂ solution. Energy irradiation of 9.6 J achieved a significant reduction and further light delivery did not produce further reduction. Conclusion: PS concentration of about 50 µM, biofilm pre-treatment with H₂O₂ for 1 min and energy irradiation around 10 J appear to be an effective protocol for endodontic aPDT.