Clinical efficacy of Methylene Blue mediated antimicrobial Photodynamic Therapy (PDT) using 680 nm Diode Laser in patients undergoing Open Flap Debridement: An Original Research

Abstract

Background

Inflammatory periodontal disease caused by dental plaque is characterised by the clinical signs of inflammation and loss of periodontal tissue support. The mechanical removal of this biofilm and adjunctive use of antibacterial disinfectants and antibiotics have been the conventional methods of periodontal therapy. The possibility of the development of resistance to antibiotics by the target organism has led to the development of a new antimicrobial concept with fewer complications. Photodynamic therapy (PDT) involves the use of low-power lasers with appropriate wavelengths to kill microorganisms treated with a photosensitiser drug. PDT could be a useful adjunct to mechanical as well as antibiotics in eliminating periodontal-pathogenic bacteria.

Methods

The patients selected were randomly divided into two groups. In the test group, Open Flap Debridement (OFD) with Methylene Blue-mediated Antimicrobial Photodynamic Therapy (PDT) using 680 nm Diode Laser application was done, and in the control group, OFD alone.

Results

The results of this study showed that diode laser irradiation (680 nm) combined with methylene blue dye reduced pocket probing depth and gingival bleeding by up to 95.85 % in the group treated with PDT with 5 min of irradiation.

Conclusions

The adjunctive use of photodynamic therapy with Diode 680 nm Laser and Methylene Blue Photosensitiser can be beneficial in reducing pocket probing depth, plaque index, and gingival bleeding index. Antimicrobial photodynamic therapy may hold promise as a substitute for currently available chemotherapy in the treatment of periodontal and peri-implant disease and in endodontic therapy.

Graphical abstract

1. Introduction

Periodontitis is an inflammatory reaction of the tissues surrounding a tooth, usually resulting from the extension of gingival inflammation induced by the bacteria residing in the plaque biofilms on the subgingival tooth surface.¹ Periodontal pockets may form in the typically healthy sulcus as a result of this inflammation causing the loss of lengthy junctional epithelium. Increased loss of connective tissue attachment, the development of intra-bony abnormalities, and eventually the potential loss of the tooth are probable outcomes.

The chronic nature, as well as the complexity and variety of the associated subgingival bacterial biofilms, are responsible for the numerous virulence factors and inflammatory markers characteristic of chronic periodontitis.

Mechanical removal of the biofilms has been the conventional approach to periodontitis therapy. Various local and systemic antibiotic regimens have been utilised in the treatment of periodontitis, but in most cases only slight improvements over mechanical debridement have been noted, along with concern about the development of increasing antibiotic resistance.²

Over the past decade, extensive investigation has been carried out into the antimicrobial action of photosensitising agents and light. When certain molecules absorb light, they may undergo an electronic transition to the singlet excited state (electron spins paired). The excited state can decay back to the ground state through a triplet (electron spins unpaired), and this triplet state can transfer energy directly to molecular oxygen (one of the few biological molecules naturally found in a triplet ground state). The triplet energy exchange can cause oxygen to undergo an electronic transition to its singlet state, and this highly reactive moiety can cause microbial cell death by several mechanisms, including lipid peroxidation, enzyme system inhibition, protein agglutination, and by reaction with other biological systems. Wilson first proposed the use of lethal photosensitization as a tool for the treatment of periodontal disease.³

Photodynamic therapy (PDT) has emerged in recent years as a non-invasive therapeutic modality for the treatment of various infections by bacteria, fungi, and viruses. This therapy is defined as an oxygen-dependent photochemical reaction that occurs upon light-mediated activation of a photosensitising compound leading to the generation of cytotoxic reactive oxygen species, predominantly singlet oxygen. PDT can be applied topically into a periodontal pocket, avoiding overdoses and side effects associated with the systemic antimicrobial agent administration. It also minimises the occurrence of bacterial resistance.⁴

Photodynamic therapy was discovered accidently at the beginning of the 20th century and was then applied in the medical field for the light-induced inactivation of cells, microorganisms, or molecules. Photodynamic therapy basically involves three nontoxic ingredients: visible harmless light, a nontoxic photosensitiser, and oxygen. It is based on the principle that a photosensitiser (i.e., a photoactivatable substance) binds to the target cells and can be activated by light of a suitable wavelength.⁵

In antimicrobial PDT, photosensitisers used are toluidine blue O and methylene blue. Both have similar chemical and physicochemical characteristics. Methylene blue is a redox indicator that is blue in an oxidising environment and becomes colourless upon reduction. Methylene blue combined with light has been reported to be beneficial in killing the influenza virus, Helicobacter pylori, and C. albicans. Methylene blue and toluidine blue O are very effective photosensitising agents for the inactivation of both gram-positive and gram-negative periodontopathic bacteria.⁶

The aim of this original research is a comparative evaluation of the clinical efficacy of Methylene Blue-mediated antimicrobial photodynamic therapy (PDT) using a 680 nm diode laser in patients undergoing open flap debridement to open flap debridement alone.

2. Materials and method

Patient Selection: Patients with clinical evidence of pocket depth≥5 mm (Stage III Grade A) were selected. Inclusion criteria included were: i) All patients having non-contributory medical history, ii) No history of antibiotic therapy in the last 6 months, iii) No history of periodontal therapy in the last 3 months, and iv) Patients showing optimum compliance in oral hygiene maintenance during the phase-1 (pre-surgical) therapy. Exclusion criteria included were: i) Patients having a compromised immune system, ii) Patients taking drugs known to cause gingival enlargement, iii) Pregnant and lactating mothers, and iv) Smokers.

The purpose of the investigation and the potential benefits and risks of the materials used and procedures to be performed were explained, and each patient signed a written consent form for their agreement to participate in the study. Pre-surgical (Phase-1) therapy was performed on all patients, which consisted of scaling and root planing, oral hygiene instructions, motivation and education, and occlusal adjustment when indicated.

Study Design: The patients selected were randomly divided into two groups. In the test group, OFD with methylene blue-mediated antimicrobial Photodynamic Therapy (PDT) using 680 nm diode laser application was done. In the control group, OFD alone was carried out. The patient's medical history, dental history, and personal history were taken, and clinical parameters, including Plaque Index (Silness and Loe, 1964),⁷ Pocket Probing Depth (PPD) in mm, were recorded. Clinical Attachment Level (in mm) were recorded.

Surgical Procedure: In the Control Group, adequate anaesthesia was achieved by administering 2 % Xylocaine HCl with adrenaline 1:80,000. After giving crevicular incisions with a Bard Parker knife (blade no. 12), full-thickness mucoperiosteal flaps were reflected using the periosteal elevator. Complete debridement of the defect was done, and a thorough root planing was carried out using the universal curettes (2-R and 2-L, 4-R, and 4-L). The surgical area was thoroughly irrigated with saline and 1 % povidone iodine. Finally, sutures were given.

For the test group, after completing the debridement, the exposed root and bone surface were treated with photodynamic therapy using a diode laser (Sunny Gold®) at a wavelength of 680 nm, a power of 150 mW, and methylene blue solution. The solution at a concentration of 100 μg/mL was applied using a 1 mm syringe. After 5 min, irradiation was done by laser with a photodynamic tip for 60 s, moving anterior to posterior on both the buccal and lingual part.⁸ The site was then washed with physiological saline after the laser irradiation, and the flaps were approximated with the sutures. (Fig. 1).

Fig. 1.

Photodynamic Therapy done with Diode laser.

After surgery, postoperative instructions were given, and prophylactic antibiotics for 7 days were prescribed. After 10 days, the patient was recalled for suture removal and reinforcement of oral hygiene instructions. Patients were also recalled after 3 months post-operatively for statistical analysis (Fig. 2).

Fig. 2.

3 months Post-operative Pocket Probing depth.

Statistical analysis: All data were transferred and stored in Microsoft Excel 2009. Data analysis was performed using statistical test files. The calculations that were performed using the statistical program SPSS version 23, IBM Corporation, NY, USA, were.

• 1.Descriptive analysis: For comparison between the baseline reading and the subsequent readings, the level of significance was kept at 5 %.
• 2.Unpaired t-test for analysis within the same group. For comparison between the two groups, we used a paired t-test. P < 0.05 was considered significant and <0.01 as highly significant. Graphs were drawn using the Microsoft Excel 2009 program.
• 3.Plaque index, CAL, PPD, and Gingival Index scores of two groups were presented using mean and SD and compared between two groups using an independent t-test.

3. Results

Comparison of plaque index between the control and test groups showed that there was no difference in plaque index between the two groups at baseline, after 1 month, and after 3 months. Also, there was no significant difference in change in plaque index from baseline to 1 month, from 1 month to 3 months, and from baseline to 3 months between the two groups (Table 1).

Table 1.

Comparison of plaque index between two groups.

Interval	Control Group		Test Group		Difference	p value
	Mean	SD	Mean	SD
Baseline	1.25	0.20	1.18	0.14	0.07	0.414
1 month	1.23	0.21	1.12	0.08	0.09	0.137
3 months	1.17	0.12	1.17	0.13	0.00	1.000
0–1 month	−0.02	0.24	−0.06	0.11	0.04	0.569
1–3 months	−0.02	0.24	−0.06	0.11	0.04	0.569
0–3 months	−0.08	0.19	−0.01	0.22	−0.07	0.488

Independent t-test.

Comparison of probing depth between the control and test groups showed that there was no significant difference in probing depth between the two groups at baseline and after 1 month; however, the test group showed a significantly lesser probing depth than the control group after 3 months. Also, there was no significant difference in change in probing depth from baseline to 1 month, from 1 month to 3 months, and from baseline to 3 months between the two groups (Table 2). Also, there was no significant difference in change in clinical attachment level from baseline to 1 month, from 1 month to 3 months, or from baseline to 3 months between the two groups (Table 3).

Table 2.

Comparison of pocket probing depth between two groups.

Interval	Control Group		Test Group		Difference	p value
	Mean	SD	Mean	SD
Baseline	5.60	0.55	5.41	0.44	0.19	0.410
1 month	3.65	0.37	3.65	0.29	0.00	0.995
3 months	3.08	0.11	2.95	0.16	0.13	0.050∗
0–1 month	1.95	0.64	1.76	0.48	0.19	0.463
1–3 months	0.57	0.31	0.70	0.27	−0.13	0.347
0–3 months	2.52	0.60	2.46	0.53	0.06	0.804

Independent t-test; ∗ indicates significant difference at p ≤ 0.05.

Table 3.

Comparison of clinical attachment level between two groups.

Interval	Control Group		Test Group		Difference	p value
	Mean	SD	Mean	SD
Baseline	4.16	0.38	4.23	0.42	−0.07	0.698
1 month	3.10	0.18	3.14	0.21	−0.04	0.650
3 months	2.55	0.41	2.68	0.38	−0.13	0.495
0–1 month	1.05	0.44	1.09	0.51	−0.04	0.886
1–3 months	0.55	0.39	0.47	0.35	0.08	0.618
0–3 months	1.60	0.54	1.55	0.60	0.05	0.837

Independent t-test indicates non-significant difference at p ≥ 0.05.

4. Discussion

The results of this study showed that diode laser irradiation (680 nm) combined with Methylene Blue dye reduced pocket probing depth and gingival bleeding by up to 95.85 % on the group treated with PDT with 5 min of irradiation (Table 1, Table 2, Table 3).

PDT requires two components: the laser or light and a photosensitiser. The photosensitiser is activated by light of a given wavelength with a high absorbance for dye, thereby producing singlet oxygen or other reactive toxic agents to certain bacteria. Lethal photosensitization of the periodontopathogen bacteria must involve changes in membranes and/or plasma membrane proteins and DNA damage mediated by singlet oxygen.⁹

Clinical studies have shown that the use of PDT alone is as effective as scaling and root planing. Successful PDT depends on several parameters, such as the photosensitiser, its concentration, and the energy of the light source. The photosensitiser must not be toxic to tissues, must absorb a laser beam at the compatible wavelength, and must produce a high-excitation efficiency (a high probability of triplet state formation per photon absorbed) and a long-lived triplet state.¹⁰

A split-mouth double-blind randomized controlled clinical trial by Bundidpun et al¹¹ evaluate the clinical effects of photodynamic therapy as an adjunct to full-mouth ultrasonic scaling and root planing in treatment of chronic periodontitis. Twenty patients with moderate to severe chronic periodontitis were treated with subgingival piezoelectric ultrasonic device alone in control group and adjunct treated with PDT in the test group. Probing pocket depth (PD), clinical attachment level (CAL), plaque index (PI), gingival bleeding index (GBI) and gingival inflammation index (GI) were evaluated at baseline, 1 month, 3 and 6 months after treatment. All parameters in test group were better than that control group, with statistically significant differences of GBI and GI (P < 0.05) at 3 and 6 months after treatment but no statistically significant differences of PD, CAL and PI.

Lipopolysaccharide is the principal constituent of the outer membrane of Gram-negative bacteria. The low permeability of the membrane acts as a barrier against antibiotics. The advantage of using cationic dyes (like Methylene Blue) is the interaction with the anionic lipopolysaccharide macromolecule of Gram-negative bacteria, resulting in the generation of Methylene Blue dimers. Penetration of photosensitisers through the epithelium and connective tissues is also important because periodontopathogens may infiltrate through the epithelial barrier into the periodontal tissue. The photodynamic activity of the photosensitiser is based on photo-oxidative reactions, which induce multiple consecutive biochemical and morphologic reactions.¹²

When a photosensitiser molecule absorbs light from a resonant energy, it may undergo an electronic transition to the singlet excited state. Following light absorption, the photosensitiser, initially at the ground state, is activated to a short-lived excited state that may convert into a long-lived triplet state. This triplet state is the photoactive state, which may generate cytotoxic species, such as free radicals and singlet oxygen. These reactive species produced during PDT act on various constituents of bacterial cells, resulting in cell death.¹³

PDT is also beneficial during the maintenance of periodontal therapy because it may act on the biofilm and eliminate the need for the removal of additional root substance by mechanical retreatment.¹⁴ Thus, the patient may experience less dentin hypersensitivity. This therapy also serves as an adjunct to mechanical therapy in sites with difficult access. Additional advantages of PDT include the reduced need for flap procedures and shorter treatment time, as local therapy, with a lack of microflora disturbance in other sites of the oral cavity.

A randomised controlled clinical study by De Oliveira et al¹⁵ compared the effects of PDT alone without subgingival SRP to subgingival SRP in subjects with aggressive periodontitis. At three months following the therapy, both treatments yielded comparable outcomes in terms of reduction of bleeding on probing and probing depth (PD), gains in clinical attachment level (CAL), thus suggesting potential clinical benefits of PDT.

Christodoulides et al¹⁶ evaluated the clinical and microbiologic effects of the adjunctive use of PDT to non-surgical periodontal treatment. 24 subjects with chronic periodontitis were randomly treated with scaling and root planing followed by a single episode of PDT. The additional application of a single episode of PDT to scaling and root planing failed to result in an additional improvement in terms of pocket depth reduction and clinical attachment level gain, but it resulted in a significant reduction in bleeding scores compared to scaling and root planing alone.¹⁵

A split-mouth double-masked randomized controlled clinical study by Bassir et al¹⁷ evaluated the effectiveness of photoactivated disinfection (PAD) using light-emitting diode (LED) as an adjunct in the management of patients affected by moderate to severe chronic periodontitis and concluded that the application of PAD using LED with the current setting did not have additional effects on clinical parameters.

A double-blind, randomized, controlled clinical trial by Cadore et al¹⁸ assessed the efficacy of multiple sessions of antimicrobial photodynamic therapy (aPDT) as an adjunct to surgical periodontal treatment (ST) in patients with severe chronic periodontitis (SCP) and found that there was a significant reduction in PD and CAL gain after 150 days. Changes in the subgingival microbiota were similar between the groups (P > 0.05), but the TG revealed a larger number of bacteria associated with periodontal disease at the end of the experiment compared with the CG (P < 0.05).

Several studies have demonstrated bactericidal and detoxification effects of high-level lasers on contaminated dental implant surfaces. High-level lasers have been used successfully in the surgical management of peri-implantitis. Hass et al¹⁹ examined the efficacy of PDT in killing bacteria associated with peri-implantitis that adhered to titanium plates with different surface characteristics. Scanning electron microscopic analysis showed that antimicrobial photodynamic therapy led to bacterial cell destruction without damaging the titanium surface.

4.1. Strength of the study

A key strength of PDT studies is their demonstration of a substantial reduction in microbial load, particularly targeting pathogenic bacteria such as Porphyromonas gingivalis and other anaerobes commonly associated with chronic periodontitis. This bactericidal effect contributes to the rationale behind PDT's use in managing periodontal infections. PDT is a relatively non-invasive approach. This is particularly beneficial for patients who may have contraindications for surgery or who prefer less invasive treatment options.

4.2. Limitation of study

The limitations of this studies include 1) smaller sample Size, 2) PDT protocols, such as the type of photosensitiser, light source, and dosage which vary significantly between studies. The lack of standardization makes it difficult to compare results and assess the consistency of PDT's effectiveness across different settings and 3) relatively short follow-up periods, ranging from a few weeks to a few months may not provide sufficient evidence on the long-term efficacy of PDT in preventing disease recurrence or maintaining improved clinical outcomes.

5. Conclusion

With the results drawn from the present study, it can be concluded that adjunctive use of photodynamic therapy with Diode 680 nm Laser and Methylene Blue Photosensitiser can be beneficial in reducing pocket probing depth, plaque index, and gingival bleeding index. Antimicrobial photodynamic therapy may hold promise as a substitute for currently available chemotherapy in the treatment of periodontal and peri-implant diseases and in endodontic therapy.²⁰ The available knowledge should enable and encourage steps forward into more clinically orientated research and development.

Ethical

The Institutional Ethical Committee of the Subharti Dental College and Hospital approved this study. All participants signed an informed consent form.

Summary

It can be concluded that adjunctive use of photodynamic therapy with Diode 680 nm Laser and Methylene Blue Photosensitiser can be beneficial in improving periodontal health status of the patient.

Author contribution

• •Manisha Rout, Shilpa Sharma, Aprajita Srivastava: Data Curation, Writing – Review & Editing
• •Soundarya Singh, Manisha Rout:Conceptualization, Funding Acquisition, Methodology
• •Mayur Kaushik, Shilpa Sharma:Methodology, Investigation
• •Mayur Kaushik, Soundarya Singh:Supervision, Project Administration, Visualisation

Funding

This research was financially supported by author's institution. The funding bodies had no role in the design of the study, data collection, analysis, interpretation of data, writing of the manuscript, or the decision to publish.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.