Background
Antimicrobial photodynamic therapy (aPDT) is a procedure in which oxygen-dependent activation of a photosensitizer by light (mainly lasers) leads to the generation of cytotoxic reactive oxygen species (ROS). Recently, this procedure was introduced in the medical [
1,
2] and dental [
3‐
6] fields. Besides confirmation of its antimicrobial potential
in vitro[
7,
8], prior clinical studies have evaluated the application of aPDT for the treatment of acne vulgaris in dermatology [
9], and of periodontal [
10,
11], endodontic [
12,
13], and peri-implant [
14] disease in dentistry. Further, since it uses light-generated compounds, aPDT may be able to eradicate multidrug-resistant bacteria without influencing the emergence of further resistance in those bacteria [
15].
Increasing evidence exists that oral care is critical for systemic health, especially in compromised patients. Previously, Yoneyama
et al. reported that good oral care lowers the risk of pneumonia and the rate of mortality in elderly institutionalized individuals [
16,
17]. Abe
et al. also suggested that oral hygiene was effective in the prevention of influenza [
18]. The US National Institutes of Health (NIH) has recommended that “all cancer patients should have an oral examination before the initiation of cancer therapy, and the treatment of preexisting or concomitant oral disease is essential in minimizing oral complications in all cancer patients” [
19]. Conventionally, mechanical tools such as a toothbrush, dental floss, or sponge brush accomplish the removal of dental bacterial plaque. However, mechanical plaque control is technically demanding, and can be physically stressful for subjects with a decreased range of arm motion, or a medical condition that impedes them from maintaining good oral hygiene. Therefore, the application of a non-mechanical means for controlling dental plaque formation could be of wide interest.
In 1993, Wilson
et al. [
20] proposed aPDT as an alternative to pharmaceutical and mechanical means of eliminating dental bacterial plaque. However, there are no clinical reports on the use of aPDT as a preventive oral care method for the control of dental plaque formation. If aPDT could be used as a novel method to control dental plaque formation, patients may be able to receive efficient oral care without the unpleasant stresses that accompany conventional mechanical tooth cleaning, such as bleeding or pain.
Accordingly, we aimed to investigate the inhibitory effects of aPDT in the oral cavity of healthy volunteers as a pilot study, prior to the clinical trial involving actual patients. We focused on toluidine blue O (TBO), which is a classic photosensitizer of phenothiazinium salt, because its bactericidal effects were already clarified in previous
in vitro studies [
8,
21‐
23], as well as in the treatment of periodontitis [
4]. Further, the bactericidal effects of TBO-mediated aPDT using high-power red light-emitting diode (LED) on two typical periodontopathic bacteria,
Porphyromonas gingivalis and
Aggregatibacter actinomycetemcomitans, have also been demonstrated
in vitro[
3].
Before the pilot study in healthy volunteers, the antimicrobial effects of aPDT on Streptococcus oralis (S. oralis), one of the typical facultative anaerobic bacterium in human dental plaque, and the cytotoxic effect of aPDT on fibroblasts, were examined in vitro. Further, the characterization of ROS generated during aPDT treatment was investigated by electron spin resonance (ESR).
Discussion
To our knowledge, this is the first clinical trial to demonstrate the effectiveness of aPDT in inhibiting dental plaque formation on teeth without inducing harmful effects to host tissues. Further, this study confirmed that TBO with red LED effectively reduced the bacteria
S. oralis, which is known as an initial colonizer in dental plaque formation, and is often detected in the blood of patients who suffer from infectious endocarditis [
25,
26]. The use of TBO with a high-power LED device and a 20 s irradiation time, led to a significant dose-dependent reduction in CFUs
in vitro. However, the reduction was less when 1000 μg/ml TBO (final concentration of 500 μg/ml) was used than when 500 μg/ml (final concentration of 250 μg/ml) was applied. A possible explanation was the fact that the blue-colored 1000 μg/ml TBO mixed bacterial solution was too dark for the red light to penetrate through the solution to the bottom of the 96-well plate, and thus the sensitization of TBO by LED irradiation was potentially blocked. Nonetheless, 1000 μg/ml TBO was used because in clinical situations, the immediate dilution of TBO with saliva and its superficial spread on the tooth surface could lead to the more efficient light sensitization of TBO than witnessed
in vitro. In the present study, the focus was only on
S. oralis; however, the formation of dental biofilm consists of different primary colonizers and complex inter-microbial interactions. Therefore, several other plaque forming bacteria including
Actinomyces viscosus and
Streptococcus sanguis should be investigated in future studies.
With respect to the safety of the procedure, TBO alone negatively influenced the viability of fibroblasts in a concentration-dependent manner, and the application of LED enhanced the affect. However, the
in vitro reduction in cell viability under the present aPDT conditions (1000 μg/ml TBO with 20 s LED irradiation) did not exceed that observed with other antiseptics, which indicated that the cytotoxicity of aPDT was within the conventional levels. Additionally, although prior reports have indicated the resistance of bacteria to antiseptics such as benzalkonium chloride, which is a quaternary ammonium cationic surfactant that interrupts the lipid membrane of cells [
27,
28], the lack of bacterial resistance following application of aPDT would be another benefit in clinical application [
29]. In the present study, however, we only observed acute cytotoxicity at 2 h after single application of aPDT, and thus further studies are required to investigate the long-term influence of aPDT on cell proliferation, as well as the cumulative action following repeated applications.
The analysis of ROS generated during TBO-mediated aPDT resulted in the novel finding that the hydroxyl radical was the primary product. Although the mechanism of aPDT [
30] is generally thought to take place by a Type I process, which produces a hydroxyl radical by electron transfer, or a Type II process, which yields singlet oxygen by energy transfer, no modality of cell death by TBO-mediated aPDT has been clarified. Singlet oxygen is regarded as the major damaging species in aPDT [
31], and the common photosensitizer methylene blue is a known producer of singlet oxygen. In our previous pilot study (data not shown), we confirmed the production of singlet oxygen in methylene blue-mediated aPDT. However, ESR analysis clearly revealed that the hydroxyl radical was the predominant product of TBO-mediated aPDT. Further, Type I machinery was speculated to play an important role in this aPDT procedure.
Following the results of the in vitro experiments, we attempted the clinical application of aPDT for the inhibition of dental plaque formation, and observed a significant suppression of plaque deposition on teeth treated with aPDT. The four day duration of the clinical trial was short, and thus a longer duration was desirable. However, the duration of this pilot study was limited to four days, due to consideration of the physical and mental stresses caused by prohibiting tooth brushing, and the concerns of volunteer dentists regarding the occurrence of tooth decay and gingival inflammation.
Regarding the mechanism of aPDT, a recent proteomic approach by Dosselli
et al. [
32] revealed that aPDT delays the growth of bacteria, and reduces the capacity of bacteria for glucose consumption. Combined, the bacterial killing effect and the retardation of bacterial growth with aPDT could reduce plaque deposition on teeth.
Nonetheless, the negative aspects of the clinical use of aPDT should be considered. Some reports have noted tooth staining with TBO [
33], however, residual staining of teeth and gingival tissue with TBO after the aPDT procedure was not visible, and did not present a problem in this clinical trial. Additionally, light energy has a biological effect on the activations of cells and tissues. Therefore, various positive and negative effects of the irradiation of teeth and gingival tissues, such as reducing gingival inflammation or inducing the calcification of dental pulp [
34], need to be clarified. In particular, although the performance of aPDT was only short term in this present study, more attention to the cumulative action of potential side effects should be paid in the long-term repeated usage for daily plaque control.
Additionally, in this clinical trial, the reduction rate in the plaque-deposition area was on average no more than approximately 56% following aPDT treatments, probably because the susceptibility of bacteria to aPDT was thought to be much lower in biofilms than in the planktonic condition [
35]. Hence, modification of the system for delivery of the photosensitizer into the biofilm should be examined for the effective enhancement of the inhibitory effects of aPDT on dental plaque formation. An example of a possible modification is the use of an antibody conjugated to photosensitizers [
36]. Similarly, the optimal power and time of LED irradiation, in combination with the frequency of aPDT procedure, should be clarified. In addition, the present pilot study was limited by the employment of healthy volunteer dentists as subjects, as well as by the small number of participants and the short duration of the clinical trial. Consequently, further investigations involving suitable conditions for clinical plaque control in a larger number of subjects and for a longer trial duration are required to confirm the safe and effective aPDT procedure compared with conventional treatments. In the future, aPDT could be used for plaque control at an office equipped with a specific appliance, or at home with the combination of tooth paste containing a photosensitizer and a light-emitting toothbrush [
5].
Acknowledgement
The authors are grateful to participating staff members from the Department of Periodontology, Tokyo Medical and Dental University (TMDU), and to Dr. Fumiko Kirikae from the Department of Infectious Diseases, Research Institute, National Center for Global Health and Medicine, for their enthusiastic support. We also thank Prof. Shinichi Arakawa (Lifetime Oral Health Care Sciences, TMDU), Ms. Masako Akiyama (University Research Administration Office, TMDU), Prof. Makoto Umeda (Department of Periodontology, Osaka Dental University), Dr. Norio Ueno (Tokyo Women’s Medical University), and Dr. Ayaka Yoshida (Department of Oral Science, Kanagawa Dental University Graduate School).
Funding
This work was supported by a Grant from the International Health Research (21–128), and a Grant from the National Center for Global Health and Medicine (24–116) to A. I-T.
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Competing interest
The authors declare that they have no competing interest.
Authors’ contributions
AI-T designed and performed all experiments and drafted the manuscript. AA designed and performed bacterial experiment and clinical trial, analyzed all experiments, and made manuscript revisions. YT designed and performed bacterial experiment and clinical trial, and contributed to manuscript preparation. TK designed and analysed cell experiment. TS made contributions to statistical analysis and interpretation of data. MCL designed and analysed ESR experiment. FY designed and assisted ESR experiment and analysed the data. YM made contribution during study protocol writing of clinical trial. TI assisted clinical trial and analysed the data. II conceived the conception of the study and assisted editing the manuscript. YI contributed to interpretation of results and revised the manuscript. All authors read and approved the final manuscript.