Introduction

Endodontics is a dental specialty concerned with the etiology, prevention, diagnosis, and management of pathological conditions affecting the dental pulp and their consequences in the periapical region. One of the fundamental principles is the prevention of infection and its control when present. When dental pathology is established, involving the periapical tissues and causing inflammation, it is classified as apical periodontitis. At this stage, the inflammatory response triggers the degradation of periapical tissues due to pathogens associated with the pulp infection. A fistula generally arises from an injury, frequently due to an infection. A dental fistula is a narrow tract that forms from the infected region of the tooth, typically at the root apex, to the external surface of the oral mucosa. This tract acts as a conduit for microorganisms and their byproducts. As the tract becomes filled with exudate, a localized edema may occur on the mucosal surface [1].

Antimicrobial photodynamic therapy—aPDT is a non-invasive treatment modality and ca be used as an adjunct to conventional periodontitis treatment. The objective of the therapy is to eliminate microbial cells through the significant production of reactive oxygen species. This technique targets both bacteria and fungi. The therapy operates through the interaction of a specific light wavelength with a photosensitizer (PS) in the presence of oxygen [2, 3].

The mechanism of action can be described by the Jablonski diagram. The photosensitizer (PS) absorbs visible light, promoting it from the ground singlet state to the singlet excited state, which has a brief lifetime. In this state, the PS can lose energy through non-radiative processes such as fluorescence or undergo intersystem crossing to the triplet state, which has a longer lifetime. From the triplet state, the PS may lose energy via phosphorescence or participate in the generation of reactive oxygen species (ROS) through two distinct mechanisms: Type I reaction, which involves electron transfer to produce free radicals, or Type II reaction, where energy is transferred to molecular oxygen to form singlet oxygen. The occurrence of Type I and Type II reactions is influenced by various factors, including the physicochemical properties of the photosensitizer (PS), such as its quantum yield and absorption spectrum, as well as the characteristics of the microenvironment, including the availability of molecular oxygen and the presence of solvents or other reactive species. [4].

Therefore, aPDT is utilized as an adjunctive treatment in endodontics. This non-invasive approach involves the application of a photosensitizer, which, when activated by a specific wavelength of light, generates reactive oxygen species. These reactive species effectively target and eradicate bacterial cells, including those within biofilms, enhancing the overall effectiveness of endodontic therapy [2, 3, 5, 6].

Phenothiazines are planar amphipathic tricyclic molecules featuring a quaternary intrinsic nitrogen atom and exhibit phototoxic activity against bacteria. In dentistry, phenothiazines are frequently employed as photosensitizers, with methylene blue (MB) and toluidine blue (TB) being key examples. These compounds are heterocyclic dyes comprising two benzene rings conjugated to a nitrogen atom and a sulfur atom. They exhibit significant absorption when exposed to visible light in the 600–660 nm range, which enhances their efficacy in generating reactive oxygen species within biological tissues [4, 7, 8].

The use of methylene blue in antimicrobial photodynamic therapy has demonstrated reliable outcomes. The hydrophilic nature of MB, combined with its low molecular weight and positive charge, facilitates its penetration through the cell walls of microorganisms [9].

It is essential to acknowledge that the antimicrobial efficacy of antimicrobial photodynamic therapy is contingent upon the match between the absorption spectrum of the photosensitizer and the emission spectrum of the light source. The effectiveness is influenced by factors such as the wavelength of the light, irradiance, exposure duration, and the absorption characteristics of the PS [10].

The aggregation of the photosensitizer is another factor influencing the efficacy of aPDT. This phenomenon, known as dimerization, can be controlled using surfactants. Sodium dodecyl sulfate (SDS) has been demonstrated to effectively reduce this aggregation [11]. Some researchers have demonstrated that methylene blue exhibits greater antimicrobial efficacy when dissolved in ethanol or urea solutions compared to water-based solutions. The addition of surfactants, such as SDS, has been shown to mitigate MB aggregation. This results in an increased concentration of monomeric MB and a reduction in aggregate formation, due to enhanced electrostatic and hydrophobic interactions among MB molecules [9, 12].

The use of methylene blue in a SDS solution at a 0.25% resulted in more effective microbial reduction during aPDT against Candida albicans compared to MB alone. The study found that MB, when in its monomeric form, was a more potent photosensitizer, with Type II photochemical reactions—involving singlet oxygen—being the most effective mechanism for inactivating C. albicans [11].

Following the disruption of the biofilm during endodontic therapy, residual bacterial populations may persist within the periodontal pockets, necessitating adjunctive antimicrobial treatment. Successful endodontic treatment requires precise canal instrumentation, effective irrigation, and appropriate intracanal medication. This process encompasses both manual and mechanized techniques, the application of irrigants, intracanal medicaments, and root canal sealers, all of which involve considerable time and resources. Antimicrobial photodynamic therapy (aPDT) may serve as an adjunctive treatment modality for periodontal disease, potentially reducing reliance on systemic antimicrobials and decreasing the risk of developing bacterial resistance [9].

Thus, the aim of this protocol is to evaluate the photodynamic effect of methylene blue in 0.25% SDS for the treatment of apical periodontitis in presence of fistula. The hypothesis to be tested is that both interventions will exhibit equivalent efficacy, with no statistically significant difference. The evaluation will focus on the resolution of the fistula and the improvement of radiographic indicators, assessed at 15 and 30 days post-endodontic treatment for apical periodontitis [13].

Materials and methods

Interventions

All participants will be treated by the same experienced endodontist, who will oversee both data collection and procedural management. Each participant will receive conventional treatment.

A baseline periapical radiograph will be required for all participants, as well as follow-up radiographs at 15- and 30-days post-treatment. These radiographs will be obtained by a different operator, who will analyze them. This operator will be blinded to the intervention received by each participant.

Conventional treatment will be performed using the Protaper rotary system (Dentsply Maillefer rotary instruments). The canal irrigation will be with 5% sodium hypochlorite, 17% ethylenediaminetetracetic acid, and 2% chlorhexidine [14]. Subsequently, a methylene blue solution in a surfactant medium will be applied using a size 30 paper point cone (Dentsply), with a pre-irradiation time of 1 minute. The study group will receive a 3-minute irradiation period, while the control group will be administered the photosensitizer along with a simulated laser application. This simulation will be achieved by placing an aluminum foil barrier at the tip of the laser equipment to block the light. During these procedures, both the operator and participants will wear protective eyewear to ensure eye safety and concealment. Calcium hydroxide will be applied to the root canals as a long-term intracanal dressing for endodontic treatment.

A diode laser device (Therapy EC, DMC, São Carlos, Brazil) with a wavelength of 660 nm and a power output of 100 mW will be used, according to the parameters detailed in Table 1. The pre-irradiation and irradiation durations will be identical for both groups.

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Table 1. Dosimetric parameters.

https://doi.org/10.1371/journal.pone.0315169.t001

The photosensitizer used will be methylene blue at a concentration of 0.005%, dissolved in 0.25% sodium dodecyl sulfate.

Ethics and dissemination

The study will be conducted in accordance with Decree 158/19 of the Executive Branch, which regulates research involving human subjects in Uruguay. Additionally, the study will comply with Law 18,331 (Law on the Protection of Personal Data and the Action of "Habeas Data").

The Research Ethics Committee of Universidad Católica del Uruguay approved the protocol (211103b). Additionally, the protocol was registered under number NCT06413836 in the Clinical Trial Protocol Registration and Results System. The results from this trial will be presented at conferences and published upon completion of the study.

The study is anticipated to last up to 20 months, beginning from the recruitment date. The protocol was developed in accordance with the SPIRIT guidelines (Standard Protocol Items: Recommendations for Interventional Trials) (Fig 1), and the SPIRIT checklist is provided as an additional file. Upon completion of the study, the CONSORT guidelines (Consolidated Standards of Reporting Trials) will be adhered to for reporting.

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Fig 1. SPIRIT figure recommended by 2013 SPIRIT statement.

https://doi.org/10.1371/journal.pone.0315169.g001

Recruitment will commence following the acceptance of the protocol for publication.

Participants will be informed that they may withdraw from the study at any time and for any reason if they choose to do so. Researchers may also remove participants from the study if deemed necessary.

Outcome variables

Discussion

aPDT is considered a complementary approach in endodontic treatment. A primary limitation of this technique is the formation of compounds that can reduce the efficacy of the therapy. This group has been studying these compounds for several years [12, 17, 18].

On the other hand, SDS demonstrated the ability to mitigate the effect of dimer formation [19]. Therefore, this study aims to evaluate the effectiveness of methylene blue combined with 0.25% SDS in photodynamic therapy for treating apical periodontitis with fistulas. The objective is to assess how effectively this combination eliminates persistent microorganisms in root canals [8].

This study addresses the challenge of treating teeth with fistulas, which often require several clinical sessions. The main difficulty is effectively eliminating the bacteria that reside within the root canals of these teeth [4].

aPDT is a non-invasive therapeutic modality that can be employed as an adjunctive treatment for apical periodontal diseases. It reduces the reliance on systemic antimicrobials, thereby mitigating the risk of antimicrobial resistance associated with these medications.

Unlike antibiotics, aPDT exhibits low toxicity to humans and is not associated with the development of bacterial resistance [20]. Given the issue of antibiotics and their indiscriminate use by patients, it is essential to seek alternative and adjunctive methods for combating microorganisms. This is particularly pertinent in countries where, despite legislation regulating antibiotic sales, patients can still obtain these medications easily without prior consultation or authorization from a healthcare professional.

Supporting information

Acknowledgments

The authors would like to thank Universidade Nove de Julho (UNINOVE), Sao Paulo, Brazil, for training and Universidad Catolica del Uruguay for providing the equipment and services for participants that will be involved in the trial.