Photodynamic treatment of endodontic polymicrobial infection in vitro

Abstract

We investigated the photodynamic effects of methylene blue on multispecies root canal biofilms comprising Actinomyces israelii, Fusobacterium nucleatum subspecies nucleatum, Porphyromonas gingivalis, and Prevotella intermedia in experimentally infected root canals of extracted human teeth in vitro. The 4 test microorganisms were detected in root canals by using DNA probes. Scanning electron microscopy showed the presence of biofilms in root canals before therapy. Root canal systems were incubated with methylene blue (25 microg/mL) for 10 minutes followed by exposure to red light at 665 nm with an energy fluence of 30 J/cm(2). Light was delivered from a diode laser via a 250-microm diameter polymethyl methacrylate optical fiber that uniformly distributed light over 360 degrees. Photodynamic therapy (PDT) achieved up to 80% reduction of colony-forming unit counts. We concluded that PDT can be an effective adjunct to standard endodontic antimicrobial treatment when the PDT parameters are optimized.

Figure 1

Optical fiber with a diameter of 250 µm that provided uniform illumination.

Figure 2

Multi-species biofilms on the root canal surface (A). Invasion of microorganisms in dentinal tubules (B).

Figure 3

Mean values of DNA probe counts for 4 species in root canal biofilms obtained from 5 experiments. Error bars denote the standard error of the mean.

Figure 4

Phototoxicity of multi-species root canal biofilms after incubation with 25 µg/ml MB dissolved in BHI broth (A) or in PBS (B) for 10 minutes followed by treatment with red light of 665 nm (30 J/cm²) and colony-forming assay. Each bar is the mean Log₁₀ CFU levels (± Standard Error). The combination of light and MB was significantly lower than control, light alone or MB alone (A). The combination of light and MB was significantly lower than either control or MB alone (B).

Figure 4

Phototoxicity of multi-species root canal biofilms after incubation with 25 µg/ml MB dissolved in BHI broth (A) or in PBS (B) for 10 minutes followed by treatment with red light of 665 nm (30 J/cm²) and colony-forming assay. Each bar is the mean Log₁₀ CFU levels (± Standard Error). The combination of light and MB was significantly lower than control, light alone or MB alone (A). The combination of light and MB was significantly lower than either control or MB alone (B).

Figure 5

Confocal scanning laser microscopy images (X–Z) obtained from the mid root area of canals (box) (a). Viable biofilms (green areas) were developed after infection with the mixture of 4 bacteria (b). The yellow areas represent reflected light (b). PDT induced destruction of root canal biofilms (red areas) (c). CSLM revealed the presence of foci of live bacteria following PDT (c, arrow). Scale bar = 100 µm.