Ⅰ. INTRODUCTION

More than one in every four South Koreans received treatment with periodontal disease, making it the most common oral disease [1]. In its early stage, called gingivitis, visual signs of redness, swelling, or bleeding may be present. In its advanced stage, called periodontitis, the symptoms are gingival recession, bone loss, or loose teeth [2]. Periodontitis is generally caused by bacteria in the mouth which infect the tissue around the teeth and cause inflammation [3]. Complex microorganisms, such as Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Treponema denticola, and Actinomycetes are the most important pathogens that cause periodontal disease [4]. When the bacteria stay on the teeth and aggregate, they form a biofilm called plaque, which hardens to calculus. Accumulation of calculus can spread below the gingival line, and when a dentist removes the calculus the perio- dontal disease process can be stopped [3].

The treatment of periodontitis mainly aims to eliminate infection and to regenerate destroyed bone and tissue. Elimination of infection is achieved by therapeutic procedures that include mechanical debridement (SRP) with antibiotics and surgical techniques [5, 6]. SRP is the most common procedure used for removing plaque and calculus followed by smoothing the surface of the tooth, but there are some limitations. The limitations include incomplete reduction or elimination of the anaerobic bacteria at the base of the pocket and difficulty in debriding inaccessible areas (furcation, within the gingival tissues, and in sites with deep probing depths). As for antibiotics they can reduce the number of pathogenic organisms and alter microorganisms to reduce their pathogenic potential. However, frequent use of antibiotics may lead to bacterial drug resistance [7, 8]. Therefore, a combination of SRP with adjuvant therapies has been proposed in the treatment of periodontitis.

Adjunctive therapies for periodontal disease treatment include mouth rinses, probiotics, laser, subgingival irrigation with hydrogen peroxide (H₂O₂) and antimicrobial photodynamic therapy (aPDT) [9]. Among these options, aPDT stands out for its wide spectrum of antimicrobial action that is independent of the antibiotic resistance pattern, minimal damage to host tissue, minimal risk of selection of photo-resistant cells, lack of mutagenicity to human cells, and being minimally invasive and low-cost [10]. Due to these advantages and its ability to overcome limitations of SRP, aPDT has been proposed as a highly effective adjunctive therapy for achieving optimal treatment outcomes in periodontal disease.

PDT mainly requires three components: a photosensitizer (PS), light, and oxygen. In general, PSs are active when they are exposed to specific light and the photodynamic activity is dependent on the presence of oxygen [11]. In the presence of oxygen, the light activated PS generates reactive oxygen species (ROS), which kill microorganisms that cause periodontal disease [12].

The type of PS used in PDT is a major determinant regarding outcome. Synthetic dyes (MB and toluidine blue), natural PSs (curcumin and hypericin), and tetra-pyrrole structures (phthalocyanines and porphyrins) have been studied. Methylene blue (MB) was the first synthetic drug in medicine, applied by Paul Guttmann and Paul Ehrlich against malaria in 1891 [13]. Numerous microscopic discoveries including the identification of Mycobacterium tuberculosis by Robert Koch were based on the biochemical properties of MB [14]. Staining with MB was also the beginning of modern drug research [15]. Presently, it is used clinically in medications to treat methemoglobinemia, urinary tract infections, cancer chemotherapy, and Alzheimer’s disease [16]. MB is also used as a PS in PDT in the treatment of periodontitis [17]. MB, with an absorption band located at 660 nm, is an amphipathic (one that has both polar and non-polar moieties) low-mass molecule. In view of its charge, it can bind to the lipopolysaccharides of the outer membrane of Gram-negative bacteria, also to the teichuronic acid residues of the outer membrane of Gram-positive bacteria [18].

The most investigated and effective PS for PDT in dentistry was MB from 2010 to 2020; indeed, it was applied in a total of 29 out of the 38 studies included in the review evaluated by Mylona et al [17]. In vitro and animal studies have proved that PDT using MB effectively inhibits the pathogen of periodontal disease [19, 20]. However, different results have been reported in several clinical trials comparing the efficacy of SRP and SRP + MB-PDT [21, 22, 23]. Thus, the purpose of this paper is to review MB-PDT from clinical trials of periodontitis which may serve to improve the treatment of periodontal diseases.

Ⅱ. MATERIALS AND METHODS

1. Search Strategy

An electronic search was conducted in regard to PDT applications in all fields of dentistry from July 2009 until June 2022. Database used was PubMed, with the following keywords and their combinations: (1) PDT; (2) periodontitis; (3) MB.

After applying the additional filters (published within the last 13 years, only clinical trials in humans, and only English language reports), the preliminary number of 17378 articles was reduced to 21.

Exclusion criteria:

After application and implementation of the eligibility criteria, 12 studies remained [24-35]. Details of the selection criteria are presented in Fig. 1.

2. Data Extraction

From the selected articles, the following data were extracted:

Ⅲ. RESULTS & DISCUSSION

Of those twelve qualifying articles, eleven compared SRP + PDT with SRP and one compared laser irradiation time and the level of MB dimerization (Table 1). In the articles that compared SRP + PDT with SRP five articles of SRP + PDT showed better results (Pocket Depth (PD) reduction, CAL gain, etc.) and the other six SRP + PDT showed nonsignificant results [24-35]. In the study of SRP with ultrasonic instrument (UPD) + aPDT and UPD showed no significant difference, suggesting that UPD rather than conventional SRP could have changed the result [25]. Studies with MB concentration of 0.05 mg/mL were performed in both parallel- group studies and split-mouth studies [26, 31, 32]. In parallel- group studies SRP + PDT had better results compared to SRP while in split-mouth studies there was no significant difference in results between SRP + PDT and SRP [26, 31, 32]. Studies with MB concentration of 0.1 mg/mL was performed with two studies showing better results and the other two studies showing nonsignificant results between groups SRP + PDT and SRP [27, 30, 34, 35]. Future research may resolve these inconsistent results with standardized protocols. Studies with MB concentration of 10 mg/mL was performed with only one study showing no significant difference between SRP + PDT and SRP due to the use of unsuitable laser wavelength [28, 29, 33]. In the one article that compared laser irradiation time and the level of MB dimerization, longer laser irradiation time and a lower level of MB dimerization by MB used in a sodium dodecyl sulfate solution showed better results (survival fraction reduction of microorganisms, change in the growth pattern of the colonies) compared to MB in aqueous solution [24].

The following parameters were important in description of PDT protocols: concentration, dimerization, and incubation time of the photosensitizer; irradiation time, fluence, total energy delivered, and wavelength of the laser. The ideal parameters of PDT are indicated in Table 2.

1. Photosensitizers

1) Concentration

It has been demonstrated that aPDT with higher MB concentration effectively killed the bacteria as compared to that with lower concentration [36]. Likewise, of the articles utilizing MB concentration of 0.05 to 10 mg/mL, when a higher concentration of MB (10 mg/mL) was used, PDT + SRP had higher improvements of PD reduction and CAL gain than SRP alone in controlled studies on chronic periodontitis [29, 33] (Fig. 2).

2) Dimerization

It is widely known that ionic dyes tend to aggregate in diluted solutions, leading to the formation of dimers [37]. However, MB has been approved as a PS for periodontal diseases because of its relatively low toxicity and high singlet oxygen generation of which its dimers produce less [24, 38]. Highly cytotoxic singlet oxygen can then lead to the photooxidation of certain amino acids, pyrimidine and purine bases of DNA/RNA, and unsaturated lipids, leading to DNA damage and/or damage to the cytoplasmic membrane allowing leakage of cellular contents or inactivation of membrane transport systems [39]. The use of MB in the surfactant vehicle (0.25% sodium dodecyl sulfate solution) showed better photodynamic action which could be explained by less dimerization, producing more singlet oxygen [24]. Through the visible absorption spectrum, the presence of dimers or monomers is shown, with the monomer showing absorption at 660 nm and the dimer at 610 nm, while in the surfactant vehicle (MBS) only one absorption peak was observed at 660 nm, referring to the monomer (Fig. 3).

2. Light Sources

PDT has been performed with various light sources, including lasers, lamps, laser-emitting diodes, and light-emitting diodes (LEDs) [40, 41]. Conventional lamps can be used with optical fibers to specify the light wavelength for tissue irradiation. However, they may have thermal effects, which must be avoided in PDT. Laser light sources are usually expensive and require the use of an optical system to expand the light beam for irradiation of a larger tissue area. Unlike the large and inefficient pumped dye lasers, diode lasers are small, cost-effective, simple to install, and have automated dosimetry [42]. Such lasers are now widely used for PDT in many articles [24-35]. Finally, with the recent developments in high-power LEDs they have been used as an alternative light source for PDT. Compared to other light sources, they are less expensive, less hazardous, thermally non-destructive, and easily available in flexible arrays [41, 43].

Clinical use of PDT largely depends on the method development of complex dosimetry: total light dose, light exposure time, and light delivery mode (single vs. fractionated or even metronomic). The fluence rate also affects PDT response despite the fact that three articles consulted in this review lacked fluence values. Furthermore, even with the same PS and for the same field of application, MB for the treatment of periodontitis, the protocols differed significantly between many articles in this review. Consequently, safe recommendations on aPDT protocols are yet to be determined for clinical use. Future studies should be performed with clear, validated protocols, so that standardization in dental applications could be achieved.

1) Wavelength

The rationale for using methylene blue as a photoactivatable agent in PDT is based on the generation of ROS with potent anti-bacterial effects, when excited with light at appropriate wavelength [50]. However, as shown in Fig. 4, methylene blue has two absorption peaks at 635- and 670-nm wavelengths and its absorption spectrum ranges from 609- to 690-nm wavelengths [51]. For that reason, the diode laser at 980 nm wavelength used by Malgikar et al. was unsuitable to achieve methylene blue photoactivation and effective antibacterial PDT [28].

3. Types of studies

In dental clinical trials, clinicians may randomize treatments over individuals (parallel-group design) or over segments in the mouth (split-mouth design) within each individual [52]. At low MB concentration (0.05 mg/mL) with mostly similar parameter conditions, results differed in parallel- group study and split-mouth study. Vohra et al. (parallel-group) reported significant reduction in PD for PDT + SRP group as compared to SRP group, while Muller et al. and Balata et al. (both split-mouth) reported no difference between groups regarding PD [26, 31, 32]. Since a potential for carry-across effect (bacterial contamination of one site to another) should be considered future studies are needed to definitively elucidate the benefit of PDT with lower MB concentration (0.05 mg/mL).

Ⅳ. CONCLUSIONS

Although the clinical evidence is yet limited, recent studies demonstrate that the adjuvant therapy, MB-PDT combined with SRP in the treatment of periodontitis show great potential as a treatment of periodontal diseases in comparison to SRP alone.