Confocal Fluorescence Imaging To Evaluate The Effect Of Antimicrobial Photodynamic Therapy Depth On P. Gingivalis And T. Denticola Biofilms.

BACKGROUND: Porphyromonas gingivalis and Treponema denticola are both principally implicated in the incidence of both periodontal disease and peri-implantitis. Recent studies have demonstrated that these bacteria exhibit symbiotic growth in vitro and a synergistic virulence in co-infection of animal models. Found at varying depths throughout the biofilm, these bacteria present a significant challenge to traditional antimicrobial treatment modalities. Antimicrobial photodynamic therapy ( aPDT ) has yielded high success against bacterial biofilms, namely those found in the oral cavity. Data on the use of aPDT against these particular periodontal pathogens is, however, scarce. Here, we studied the qualitative killing efficacy and depth of drug and laser penetration into defined P. gingivalis and T. denticola biofilms.

METHODS: P. gingivalis and T. denticola were incubated under anaerobic (10%CO2 , 10%H2 , 80%N2 ) conditions for two days in diluted TSB with PBS (TYGVS for T. denticola maintenance) to elicit biofilm growth on coverslip-modified polystyrene dishes. Treated biofilms were exposed to a purpurin-based sensitizer (25 µg/mL in DMSO) for 30 minutes, and then aPDT was carried out using a diode laser at 664 nm. Light doses of 15 and 45 J/cm2 were used. All biofilms were then exposed to Filmtracer™ LIVE/DEAD® Biofilm Viability Kit (Cat No. L10316). Qualitative analysis was performed using a Zeiss LSM 510 Meta NLO Confocal Microscope with attached Zeiss Axioimager Z1 and Axiovert 200 M for visual data collection, and images were processed using the ZEN Digital Imaging for Light Microscopy software suite. Analysis was performed in 2 × 3 stacks to assess the entire depth of both the biofilm and presumed drug/laser penetration.

RESULTS: Initial planktonic studies confirmed that the bacteria in question were present in the grown cultures and susceptible to aPDT exposure. Biofilm control groups were found to have significant levels of surviving bacterial colonies. Both treatment groups featured complete bacterial kill throughout the entirety of the biofilm (average: 23.17 µm; range: 18.13 - 27.20 µm).

CONCLUSIONS: The efficacy of the purpurin-based PS and aPDT is demonstrated to be effective at both high and low light doses. Bacterial kill was fully efficacious at each visualized biofilm layer (1.01 µm/z-level). This study serves as a proof of concept for future studies that must consider appropriate treatment parameters, including the amount of applied PS, and laser dose. These findings indicate that aPDT is a method that can be used to eliminate microorganisms associated with biofilms implicated in the etiology of peri-implantitis and periodontitis at large.