The presence of microorganisms in the root canal system is reported to be the main cause for failure of endodontic treatment due to its metabolic products as well as the resulting formation of a focus of infection. Moreover, the existence of resistant microorganisms inside the root canal system – especially in cases of re-treatment whenever the first treatment was not successful - makes it so that even when endodontic therapy is well performed, using strict measures for controlling infection, it is not effective in ensuring successful treatment.

Thus, new resources against intra and extra-canal (apical biofilm) infection are being pursued through tests with irrigation solutions, intra-canal medications, laser radiation and others. As a resource to be tested photodynamic therapy has the advantage of not inducing microbial resistance, which is currently very promising for use in areas where there are resistant microorganisms that may lead to unfavorable prognosis [1-6].

Enterococcus faecalis is a resistant microorganism that is frequently detected in cases of secondary infections in Endodontics [7]. However, its occurrence has also been reported in primary infections with techniques such as the PCR, in teeth as well as in periapical lesions [3,4,8] Furthermore, this microorganism is able to co-aggregate with Fusobacterium nucleatum, which the authors believe plays an important role in periapical lesions [8].

Several studies have shown that chemical-surgical preparation is able to achieve high levels of disinfection, but is unable to completely reduce the microbiota of the root canal system [9-12].

Photodynamic therapy (PDT) is a reaction between photosensitizers and light, producing a cytotoxic effect, usually via oxidative reactions. PDT, which is widely used in the disinfection of blood products, is efficient in the inactivation of viruses, resistant bacteria and yeast [13-17].

In Dentistry, photodynamic therapy has proven its effectiveness in reducing infection in peri-implantitis, both in in vitro and in vivo studies [18-20]. In endodontic therapy, PDT reduced the root canal infection in vitro significantly [21-24]. In in vivo studies, there were also an increase in the level of disinfection achieved using photodynamic therapy as an aid to endodontic treatment [6,25]

George and Kishen [26] tested methylene blue as a sensitizer, dissolved in water and in a mixture of glycerol, ethanol and water (MIX) in an E. faecalis suspension. The authors found that the sensitizer dissolved in the MIX solution was more effective in reducing the concentration of the microorganism. This is thought to be due to a higher interaction between the methylene blue and the cell acquired by this MIX.

The aim of this study was to test the effectiveness of methylene blue at two different concentrations diluted in water and also dissolved in a glycerol, ethanol and water solution in the disinfection of extracted bovine teeth infected with E. faecalis.

The crowns were cut off of twenty extracted bovine teeth and their root canals were emptied. They were then immersed in 1% sodium hypochlorite and agitated in an ultrasonic basin in order to remove pulp residue.

Following these procedures, the apical third of these teeth was enlarged by a #60 K-file (Dentsply-Maillefer, Ballaigues, Switzerland) in order to standardize the specimen. The roots were then externally waterproofed using cyanoacrylate (Super Bonder^® - Loctite Henkel, Itapevi, SP, Brazil) and dried at room temperature for 24 hours. Next, the roots were placed in 1.5 mL polypropylene tubes and the set was sterilized in an autoclave at 134°C for 15 minutes (Figure 1).

Figure 1: Picture of roots in polypropylene tubes for sterilization in an autoclave.

All specimens were checked for a lack of contamination by inoculating with sterile TSB, before the experiment.

Preparation of inoculation and contamination of the specimen

A suspension of 50 μL of the E. faecalis ATCC 29212 was incubated in 5 mL of Tryptic Soy Broth (TSB - Difco- Becton Drive, NJ, USA) at 37°C for 24 hours. The concentration of the inoculation was adjusted using the optic density at 600nm, corresponding to 3×10⁸ CFU/mL.

Next, the roots of the specimen were filled with the inoculation and were incubated over 2 weeks at 37°C with fresh culture media complemented every day. All the procedures were done in a flow chamber.

Experimental groups

All Groups: The canals of the teeth were filled with 0.5 mL of photosensitizer, which was left for 2 minutes (prior to irradiation). Irradiation was performed using a diode laser (Thera Lase^® - DMC Equipamentos Ltda, Sao Carlos, SP, Brazil), 660 nm wavelength, 100 mW power for 4 minutes and energy density of 850 J/cm² (Figure 2). After the canals were irradiated, they were irrigated and aspirated with 10 mL of sterile saline solution to eliminate the photosensitizer.

Figure 2: To illustrate how the irradiation was performed, showing the light spreading all over the surface dimensionally.

Group 1: photosensitizer was methylene blue at a 10 mM concentration diluted in distilled water.

Group 2: photosensitizer was methylene blue at a 10 mM concentration diluted in a glycerol: ethanol: water (30:20:50) solution.

Group 3: photosensitizer was methylene blue at a 100 mM concentration diluted in distilled water.

Afterwards, the canal was filled with peptonated water solution and the walls were filed with a #60 K-file; the content was later aspirated with micropipettes and those contents were transferred to 1mL of peptonated water solution and were vortexed (Genie 2™- Fisher Scientific, Bohemia, NY, USA), to get the samples homogenized. Serial dilutions of 10^-1 to 10^-4 were done.

Twenty-five μL was plated on the surface of mEnterococcus Agar^® (Difco - Becton Drive, NJ, USA) in triplicate, a selective media for E. faecalis–– in order to count the colonies of E. faecalis. The cultures were incubated at 37°C for 48 h.

The means obtained were: Group 1=513×10³, Group 2=1431×10³ and Group 3=2.96×10³ CFU/mL (Table 1). Statistical analysis was performed (Kruskall-Wallis and Student-Newman-Keuls tests) and it was detected higher disinfection achieved by G3 when compared with groups G1 and G2, and no significant difference between the groups G1 and G2 (P>0.05) (Table 1).

Table 1: CFU/mL means in the experimental groups.

Photodynamic therapy has long been used in Medicine, especially in Oncology therapy. In Dentistry, and more specifically in Endodontics, it is used because of the antimicrobial properties. Photodynamic therapy is safe and efficient as an aid to endodontic treatment in terms of an increase in intracanal disinfection. Some studies have shown the efficacy of this therapy against E. faecalis [17,22-26]. These studies present different options of sensitizers and different concentrations as well as different light wavelengths and application protocols, the most widely used sensitizer, however, is methylene blue, a phenothiazinic dye that is well known in Dentistry. In this study it was tested two different solutions (G1 and G2) for the same concentration (10 mM) of the dye, aiming at comparing the influence of solution in the antimicrobial effect of the PDT. These results did not show statistical difference between them which is contrary to the findings of George and Kishen [26] using methylene blue dissolved in a solution with glycerol:ethanol:water, perhaps because the protocol performed in this study was different of them. The parameters chosen were set by clinical conditions and the time of irradiation was similar to Garcez et al. [25] in their studies because the lack of consensus among authors towards optimal time-set and their good results in their clinical studies. When comparing the concentrations of the sensitizer the higher one (Group 3) was able to reduce a larger number of microorganisms, which implies that the concentration of the sensitizer is a factor that has influence in the antimicrobial effect of PDT and these findings are in accordance with other studies [18-20]. In the present study an increase of ten times in the concentration decreased the microbial load more than a hundred times. These results show a promising effect of the PDT in root canal disinfection. Besides the concentration of the dye or the type of solution that is made, variables such as prior irradiation time and irradiation time also can interfere in the efficacy of photodynamic therapy and there is not an established protocol followed by all researchers. Further studies must be carried out in order to get the most effective protocol of PDT towards endodontic infection. Moreover it is known that the endodontic microbiota can vary highly among the patients and even in different teeth of the same individual, thus it is crucial to develop more in vivo studies to achieve the benefits of the photodynamic therapy.