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. 2022 Feb 10;17(2):e0263103.
doi: 10.1371/journal.pone.0263103. eCollection 2022.

Evaluation of antimicrobial photodynamic therapy with acidic methylene blue for the treatment of experimental periodontitis

Juliano Milanezi de Almeida  1   2 , Henrique Rinaldi Matheus  1   2 , Breno Edson Sendão Alves  1   2 , David Jonathan Rodrigues Gusman  1   2 , Maria José Hitomi Nagata  1   2 , Elisa Mara de Abreu Furquim  1   2 , Edilson Ervolino  2   3
Affiliations

Evaluation of antimicrobial photodynamic therapy with acidic methylene blue for the treatment of experimental periodontitis

Juliano Milanezi de Almeida et al. PLoS One. .

Abstract

Objective: To investigate the security and effectiveness of antimicrobial photodynamic therapy (aPDT) with a citric acid-based methylene blue (MB) on the periodontal repair following the treatment of ligature-induced experimental periodontitis (EP) in rats.

Material and methods: Were used 120 male rats, randomly divided into 4 experimental groups (n = 30): no treatment (NT), SRP alone (SRP), SRP plus aPDT using conventional MB pH 7.0 (aPDT-pH7), SRP plus aPDT using acidic MB pH 1.0 (aPDT-pH1). EP was induced at day 0 by the placement of a ligature around the mandibular left first molars. Ten animals per group/period were euthanized at 14, 22 and 37 days. Histopathological, histometric (percentage of bone in the furcation [PBF]) and immunohistochemical (for tartrate-resistant acid phosphatase [TRAP] and osteocalcin [OCN]) analyses were performed. Data were statistically analyzed.

Results: aPDT-pH1 showed the highest PBF as compared with the other treatments. Collectively, tissues' reaction to both dyes were controlled and healthy for the periodontium. Both aPDT protocols reduced the extent and intensity of the local inflammatory response, reduced the alveolar bone resorption, and promoted a better structural arrangement of the connective tissue as compared with SRP. TRAP expression was downregulated while OCN expression was upregulated by aPDT as compared with SRP alone.

Conclusion: Our data implicate that the novel MB pH 1.0 is as safe as the conventional MB for use in aPDT and raises its additional benefit of increasing the amount of alveolar bone in the furcation.

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Conflict of interest statement

The author Juliano Milanezi de Almeida declares that has no conflict of interests related to this study. The author Henrique Rinaldi Matheus declares that has no conflict of interests related to this study. The author Breno Edson Sendão Alves declares that has no conflict of interests related to this study. The author David Jonathan Rodrigues Gusman declares that has no conflict of interests related to this study. The author Elisa Mara de Abreu Furquim declares that has no conflict of interests related to this study. The author Maria José Hitomi Nagata declares that has no conflict of interests related to this study. The author Edilson Ervolino declares that has no conflict of interests related to this study. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. Scheme illustrating the experimental procedures performed during the study for each group.
Fig 2
Fig 2. Graph showing the quantification (M±SD) of the percentage of bone in the furcation (PBF) of the mandibular left first molars for each group and period.
Statistical tests: ANOVA and Tukey. Symbols: *, statistically significant difference with 14 days at the same group (p≤0.05); †, statistically significant difference with NT at the same time points (p≤0.05); ‡, statistically significant difference with SRP at the same time points (p≤0.05); §, statistically significant difference with aPDT-pH7 at the same time points (p≤0.05).
Fig 3
Fig 3. Histopathologic features of the periodontal tissues in the furcation region of the mandibular left first molars.
Abbreviations and symbols: ab, alveolar bone; *, inflammatory infiltrate. a- h: photomicrographs showing the features of the periodontal tissues at 14 in NT (A, B), SRP (C, D), aPDT-pH7 (E, F) and aPDT-pH1 (G, H). I—P: photomicrographs showing the features of the periodontal tissues at 37 in NT (I, J), SRP (K, L), aPDT-pH7 (M, N) and aPDT-pH1 (O, P). A, C, E, G, I, K, M, and O allow an overview of the furcation, while B, D, F, H, J, L, N, and P provide a closer view of the inflammatory infiltrate, fibroblasts, arrangement of collagens fibers and alveolar bone in the furcation. Staining: Haematoxylin & Eosin. Scale Bars: A, C, E, G, I, K, M, and O: 500 μm; B, D, F, H, J, L, N, and P: 75 μm.
Fig 4
Fig 4. TRAP immunobelling in the furcation region of the mandibular left first molars at 22 days.
(A) Means and standard deviations (M±SD) of the number of TRAP-positive cells in the furcation for each group and period. Statistical tests: ANOVA and Tukey. Symbols: *, statistically significant difference with 37 days at the same group (p≤0.05); †, statistically significant difference with NT at the same time points (p≤0.05); ‡, statistically significant difference with SRP at the same time points (p≤0.05). (B—I) Photomicrographs showing the TRAP-positive cells (black arrowheads) at 22 days in groups NT (B, C), SRP (D, E), aPDT-pH7 (F, G) and aPDT-pH1 (H, I). Counter-staining: Harris’ haematoxylin. Scale bars: B, D, F, and H: 75μm; C, E, G, and I: 25 μm.
Fig 5
Fig 5. OCN immunobelling in the furcation region of the mandibular left first molars at 22 days.
(A) Graph showing the median and interquartile range of the scores for OCN in the furcation for each group and period. Statistical tests: Kuskal-Wallis and Dunn. Symbols: ‡, statistically significant difference with 14 days at the same group (p≤0.05); †, statistically significant difference with NT at the same time points (p≤0.05); §, statistically significant difference with SRP at the same time points (p≤0.05). B–E: photomicrographs showing the immunolabelling pattern for OCN and OCN-positive cells (black arrowheads) at 22 days in groups NT (B), SRP (C), aPDT-pH7 (D) and aPDT-pH1 (E). Counter-staining: Harris’ haematoxylin. Scale bars: 75 μm.
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