Photodynamic Therapy With Resveratrol and an Nd:YAG Laser for Enterococcus faecalis Elimination

Abstract

Background/Aim

Enterococcus faecalis is the leading cause of endodontic treatment failures. Despite various conventional disinfection approaches, microorganisms often persist in root canals. Photodynamic therapy (PDT) is an adjunct antimicrobial strategy employing a nontoxic photosensitizer (PS) and light source. This study evaluated the antimicrobial effect of PDT using an Nd:YAG laser and resveratrol (RSV) with or without pigment, and confirmed that RSV is nontoxic as a PS.

Materials and Methods

We employed laser irradiation at a 3W output power, using RSV and red pigment as the PS, on an E. faecalis bacterial solution. Subsequently, colony-forming units were quantified. The impact of RSV on osteoblasts was measured using an MTT assay.

Results

E. faecalis counts declined after laser irradiation. The combined application of laser irradiation with RSV, red pigment, or both showed a reduction compared to no irradiation and control groups without RSV and red pigment. The 50% cytotoxic concentration against osteoblast cells from mice incubated with RSV for 48 h was 162 μM. The value with RSV and laser was 201 μM and that with RSV and red pigment was 199 μM. The value with RSV, laser and red pigment was 357 μM.

Conclusion

The combination of Nd:YAG laser irradiation and RSV as the PS with pigment was efficacious for E. faecalis elimination without inducing any toxic effects on osteoblasts. This combination holds potential as a root canal irrigation strategy.

Enterococcus faecalis is a Gram-positive facultative anaerobic coccoid that plays a significant role in persistent infections and post-treatment endodontic diseases (1). Due to its unique morphological and genetic characteristics, E. faecalis can withstand intracanal procedures and systemic antibiotics, even under stressful ecological conditions (2). Consequently, it is a major contributor to endodontic treatment failures (3). The complex anatomy of root canals, including isthmuses, accessory canals, and dentinal tubules, allows bacteria such as E. faecalis to survive conventional cleaning methods (4). E. faecalis can persist under favorable nutritional conditions as a single microorganism and can form biofilms within dentinal tubules (5,6). These bacteria are capable of penetrating dentinal tubules up to a depth of 1,250 μm (7). Despite using various mechanical cleaning methods and chemical irrigants, root canals remain harbors for microorganisms (4). Sodium hypochlorite, the most commonly used irrigant, can only penetrate dentinal tubules up to 130 μm (8). Therefore, supplementary techniques, including photodynamic therapy (PDT), have been developed to enhance disinfection (9). In endodontics, PDT has been employed as an additional step for root canal treatment (10). High-power lasers have also recently been used for endodontic treatment (11). It has been demonstrated that an 810 nm diode laser is capable of obstructing dentinal tubules and decreasing the bacterial counts of Escherichia coli and E. faecalis (12).

PDT is an adjunctive method for bacterial inactivation (8). It involves using a nontoxic photosensitizer (PS) and light to create reactive oxygen species that can eliminate microorganisms (13,14). In endodontics, PDT using an 810 nm diode laser and indocyanine green has shown promise in reducing bacteria within root canals (15-17).

We previously reported the use of PDT with a diode laser and two types of PS (18,19). In one study, pyoktanin blue (PB) was used to stain a cyst during cystic brain tumor resection (20); in another, alkaline extract of the leaves of Sasa senanensis Rehder was used to obtain a polyphenol compound (21).

Resveratrol (RSV) is a polyphenolic antioxidant found in grapes, peanuts, and several other plants. It is involved in the prevention of inflammation, atherosclerosis, and carcinogenesis (22). Several studies have reported that RSV stimulates osteoblast differentiation (23,24).

In this study, we explored the effectiveness of PDT using a 1,064 nm neodymium:yttrium-aluminum-garnet (Nd:YAG) laser (ND Compact; Incisive Co., Ltd., Tokyo, Japan) with RSV as the PS. The Nd:YAG laser has a wavelength absorbed by black and red colors, such as hemoglobin and melanin. Since the RSV solution was colorless, red pigment (Caries Checker; Cimedical Co., Ltd., Hakusan, Japan) was used in combination with RSV. The study aimed to assess the antimicrobial impact of PDT with the Nd:YAG laser and RSV, both with and without a red pigment, to evaluate its efficacy against E. faecalis, while ensuring safety for osteoblasts.

Materials and Methods

Bacterial inoculation. The E. faecalis standard strain (American Type Culture Collection BAA-2128™) was cultivated in 5 ml brain heart infusion (BHI) broth (33 g/l; Sigma-Aldrich, St. Louis, MO, USA) and incubated overnight at 37˚C. The turbidity of the prepared media was adjusted to 0.2 McFarland standard using a filter colorimeter. Next, 10 μl cultured medium, containing approximately 2×10⁸ E. faecalis cells, was combined with 200 μl BHI broth containing a 10% RSV solution (10 mM; Wako Pure Chemical Industries, Ltd., Osaka, Japan) in a 1.5 ml microtube (Watson Co., Ltd., Tokyo, Japan). For comparison, a control sample was prepared by adding 180 μl BHI broth and 20 μl dimethyl sulphoxide (DMSO) instead of the RSV solution to the 1.5 ml microtube containing 10 μl E. faecalis cultured medium.

To create a stock RSV solution (100 mM), the powder was initially dissolved in DMSO (Wako Pure Chemical Industries, Ltd., Osaka, Japan). Subsequently, a dilution was prepared by adding BHI broth for subsequent microbiological procedures.

Laser irradiation. We utilized an Nd:YAG laser, which emits light at a 1,064 nm wavelength and features a flexible fiber delivery system (Φ=0.4 mm). Laser irradiation was performed using a focused beam, with a pulse energy of 140 mJ and an output power of 3.5 W, operating at 25 Hz. Irradiation was performed at a distance of 6 mm from the bottom of the 1.5 ml microtube for 60 s. Laser irradiation was not performed for controls. Figure 1 shows the experimental setup, including the laser’s fiber tip and the 1.5 ml microtube containing the RSV solution.

Figure 1. Schematic of the experimental setup including the fiber tip of the Nd:YAG laser. Laser irradiation was performed at a distance of 6 mm from the bottom of the 1.5-ml microtube, which contained the resveratrol (RSV) solution.

Microbiological procedures.

RSV antibacterial assay. The antibacterial efficacy of RSV was assessed by establishing its minimum inhibitory concentrations (MICs) using the microdilution technique. Initially, RSV was adjusted to a concentration of 2,280 μg/ml in BHI medium, and subsequent two-fold serial dilutions (0, 8, 17, 35, 71, 142, 285, 570, 1,140, and 2,280 μg/ml) were prepared in U-shaped 96-well microplates (Watson, Tokyo, Japan). Overnight bacterial cultures, adjusted to an OD600 of 1.0 (equivalent to 10⁹ cells/ml), were further diluted to 1:100 with 100 μl BHI (yielding 10⁶ cells/ml). Subsequently, 10 μl bacterial cultures were added to each well, resulting in 100 μl total culture volume. The MICs of RSV were determined at 10 mM concentration after 24 h anaerobic incubation at 37˚C.

Control group experiments were conducted without RSV, using DMSO alone or combined with laser irradiation. For the experimental bacterial solution, the MIC of RSV was used.

Bacterial counts were determined through the following steps. Initially, 10 μl bacterial sample from a 1.5 ml microtube containing 200 μl bacterial solution was mixed with 90 μl BHI broth, and subsequent 10-fold serial dilutions were performed four times. Then, 50 μl from the resultant 100 μl bacterial solution was plated onto BHI agar plates (52 g/l distilled water) and incubated at 37˚C for 24 h. The resulting colony-forming units (CFUs) were counted. The experimental groups were treated with or without red pigment (5%).

RSV cytotoxicity assay. Osteoblast cells from mice (MC3T3-E1; American Type Culture Collection, Manassas, VA, USA) were seeded in 96-well plates at a density of 2.42×10³ cells per well (0.3 ml/well). The plates contained alpha-minimum essential medium (MEM, Sigma, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal calf serum. Following plating, the cells were incubated for 96 h. Immediately, the culture medium was substituted with 0.3 ml fresh medium containing different concentrations of RSV. Then, the cells were further incubated for 48 h. Subsequently, the medium was removed, and the cells were rinsed twice with fresh culture medium. Then, they were subjected to further incubation in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (FBS) for 48 h, enabling the viable cell count determination using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (25).

Four hours after adding MTT reagent, the intracellular formazan that formed was dissolved using DMSO. The absorbance at 540 nm was gauged using a microplate reader (BioTek Japan, Tokyo, Japan). An additional absorbance measurement was taken at 630 nm for reference.

The control cells were treated with the same amounts of DMSO and cell damage induced by DMSO was subtracted from that induced by RSV. The concentration of RSV that reduced the viable cell number by 50% (CC₅₀) was determined from the dose-response curve. The mean CC₅₀ value was calculated from triplicate assays.

Statistical analysis. The groups were statistically compared using the Mann-Whitney U-test. All statistical analysis was performed using R with Stats package (Ver. 4. 2. 1) (The R Foundation for Statistical Computing, Vienna, Austria).

Results

Reductions in E. faecalis counts were observed following laser irradiation, RSV application, red pigment introduction, or their combination compared to the control group (untreated with laser, RSV, and red pigment) (Figure 2, Table I).

Figure 2. E. faecalis counts (colony-forming units).

Table I. E. faecalis colony-forming units.

Group A, RSV (–) Laser (–) Red pigment (–); group B, RSV (–) Laser (–) Red pigment (+); group C, RSV (–) Laser (+) Red pigment (–); group D, RSV (–) Laser (+) Red pigment (+); group E, RSV (+) Laser (–) Red pigment (–); group F, RSV (+) Laser (–) Red pigment (+); group G, RSV (+) Laser (+) Red pigment (–); group H, RSV (+) Laser (+) Red pigment (+). RSV: Resveratrol.

Significantly greater reductions were noted in the irradiation group compared to the non-irradiation group. Furthermore, the reduction in E. faecalis count was more pronounced in samples that received RSV with irradiation than in those that received RSV without irradiation. Some reduction in E. faecalis counts was also seen with the inclusion of red pigment compared to the group without pigment. Remarkably, with RSV after laser irradiation, almost no colonies were observed, and with RSV and red pigment after laser irradiation, colonies were completely absent. The differences between all group comparisons were statistically significant except for Group B×E (Mann-Whitney U-test; Table II).

Table II. Comparisons between groups according to E. faecalis colonyforming units.

*Mann-Whitney U-test. ^†No statistically significant difference.

The results of the cytotoxicity assay are presented in Figure 3. Osteoblasts were exposed to different concentrations of RSV, and cytotoxicity was evaluated using the MTT assay after 48 h of incubation. Each value represents the mean±standard deviation (n=8). Viable cell number was affected by the RSV concentration, with lower concentrations resulting in higher viable cell counts. The viable cell count in 1% DMSO-containing medium showed a13% reduction, and in a medium containing only 0.5% red pigment, there was a 49% reduction compared to the MEM medium. Figure 3A presents the results of the cytotoxicity assay with RSV solution and the CC₅₀ value of RSV was 162 μM. Figure 3B shows the results with RSV solution and laser irradiation and the CC₅₀ value of RSV was 201 μM. Figure 3C shows the cytotoxicity assay with RSV solution, and red pigment and the CC₅₀ value of RSV was 199 μM. Figure 3D shows the results with RSV solution, laser irradiation, and red pigment and the CC₅₀ value of RSV was 357 μM.

Figure 3. Cytotoxic activity of resveratrol (RSV). Confluent osteoblast cells (2.42×103/cm2) were inoculated into the 96-microwell plate and incubated for three days. Cells were exposed for 48 h to the indicated concentrations of RSV. After washing twice with culture medium, cells were further incubated for 48 h with fresh culture medium to determine the viable cell number by the MTT method. Data represent the mean+standard deviation (n=8). (A), RSV (+) Laser (–) Red pigment (–); (B), RSV (+) Laser (+) Red pigment (–); (C), RSV (+) Laser (–) Red pigment (+); (D), RSV (+) Laser (+) Red pigment (+).

Cell viability with RSV was reduced compared to that combined with laser irradiation. Cell viability with RSV and red pigment was reduced compared to that combined with laser irradiation.

Discussion

Numerous researchers have explored strategies for eradicating E. faecalis from root canals, resulting in the development of several irrigation techniques and devices to enhance irrigant efficiency and distribution (26). Lasers have emerged as a potential alternative to conventional cleaning and disinfection methods (27). PDT involves the interaction of light with a PS, a light-responsive chemical compound, in the presence of oxygen. The PS accumulates within target cells, including bacteria and tumor cells, which are then exposed to light of a specific wavelength. This process triggers the generation of reactive oxygen species, oxidatively damaging the target cells (28).

PDT has been demonstrated to effectively lower E. faecalis counts in infected root canals compared to traditional endodontic approaches involving instrumentation and irrigation (3). A recent review by Plotino et al. investigated PDT in endodontics, highlighting that light sources emitting wavelengths primarily between 630 and 800 nm are commonly employed in PDT (1). In addition, Costa Lima et al. observed that root canal irrigation using the Nd:YAG laser with a wavelength of 1,064 nm exhibited superior smear layer removal compared to alternative methods, such as ultrasound, canal brushes, and the Protaper Universal System (27). The Nd:YAG laser has proven valuable in endodontic treatment; in this study, we used it in combination with RSV with or without a red pigment.

Our findings indicate that PDT involving RSV substantially reduced E. faecalis counts and the inclusion of red pigment in PDT further enhanced its efficacy. The combination of the pigment with RSV demonstrated greater reduction than RSV alone. Notably, the bacterial count was significantly diminished when RSV treatment was combined with 60 s of laser irradiation compared to RSV treatment alone. The results of this study suggest that optimizing laser power can enhance both bacterial and cellular activity, while excessive power may cause damage to both (29-31). The biostimulatory effects of lasers align with the Arndt-Schulz law of biology, where mild stimuli boost physiological activity, whereas strong stimuli impede it (32). Previous investigations have indicated potential drawbacks of laser irradiation, including temperature elevation in periapical tissues and stimulation of mammalian cell proliferation (33,34). Hence, the reduction in E. faecalis cell viability probably stemmed from laser irradiation itself, not just the heat generated by it. The presence of RSV was pivotal for the antimicrobial effectiveness of PDT. Therefore, meticulous selection of the appropriate PS in PDT is crucial for optimal antimicrobial outcomes.

RSV has been shown to enhance osteoblast differentiation and counteract the inhibitory effects of lipopolysaccharides on osteoblast differentiation in vitro (23,24). Consequently, in the context of an infected root canal, supplemental irrigation with PDT with RSV could promote osteogenesis in periapical lesions.

In this study, incubation of cells with RSV for 48 h resulted in a significant loss of viable cells with a CC₅₀ value of 162 μM. When cells were incubated with RSV and subjected to laser irradiation for 48 h, the cytotoxicity was slightly reduced (CC₅₀ value=201 μM). The addition of red pigment to the incubation of cells with RSV the cytotoxicity was also slightly reduced (CC₅₀ value=199 μM). Combining red pigment with RSV and laser irradiation further alleviated the cytotoxicity (CC₅₀ value=357 μM). Kaekehabadi demonstrated that, in an MTT assay, the number of viable human periodontal ligament cells in a 5.25% NaOCl solution decreased by 37.84% and in a 17% Ethylenediaminetetraacetic acid (EDTA) solution decreased 16.34% after 1 min incubation (35). Notably, a 5.25% NaOCl solution and 17% EDTA are commonly used for conventional root canal irrigation. In a clinical context, root canal irrigation is completed in less than 1 min, often involving lower laser irradiation power. Our findings suggest that an RSV concentration of approximately 2.3 mg/ml (10 mM) and a 1 min irrigation duration could be optimal, as RSV did not exhibit cytotoxicity to osteoblasts under these conditions. Wang et al. indicated that treating MC3T3-E1 cells with 30 μM RSV significantly boosted cell proliferation (36). Our study also shows the same results. Further investigations are warranted to explore varying RSV concentrations for different applications, such as an irrigant and medicament for root canal treatments.

Conclusion

Nd:YAG laser irradiation in combination with RSV as a PS in combination with a pigment is efficacious for E. faecalis elimination without causing toxicity to osteoblasts. This treatment shows promise as a root canal irrigant, but requires further investigation for clinical use.

Conflicts of Interest

The Authors have no competing financial interests to disclose in relation to this study.

Authors’ Contributions

Masaki Morikawa: Formal analysis, Data curation, Investigation, Writing; Shunsuke Uehara: Methodology, Supervision; Akihiro Yoshida: Methodology, Supervision; Hiroshi Sakagami: Validation, Writing-editing; Yoshiko Masuda: Supervision, Validation, Writing-editing, original draft.