https://orcid.org/0000-0002-5995-5747
Figures
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.
Citation: de Almeida JM, Matheus HR, Sendão Alves BE, Rodrigues Gusman DJ, Nagata MJH, de Abreu Furquim EM, et al. (2022) Evaluation of antimicrobial photodynamic therapy with acidic methylene blue for the treatment of experimental periodontitis. PLoS ONE 17(2): e0263103. https://doi.org/10.1371/journal.pone.0263103
Editor: Ewa Tomaszewska, University of Life Sciences in Lublin, POLAND
Received: January 27, 2021; Accepted: January 13, 2022; Published: February 10, 2022
Copyright: © 2022 de Almeida et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the manuscript and its Supporting Information files.
Funding: The author(s) received no specific funding for this work.
Competing interests: 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.
Introduction
Periodontitis is a highly prevalent oral disease in the United States, estimated to affect over 42% of dentate US adults 30 years or older [1], and the primary cause for tooth loss in the world [2]. Periodontal breakdown is the consequence of the combination of complex illnesses, interactive amongst inflammatory and immunologic systems of a host, subgingival microbiota, and environmental factors [3]. Even disorders within this microbiota being capable to result in dysbiosis, a specific microenvironment and susceptibility are essential to trigger the disease [4]. Hence, periodontal therapy will be the most effective when capable to encompass the many variables as possible.
The use of antimicrobial photodynamic therapy (aPDT) has been increasingly investigated as an adjunctive approach to non-surgical scaling and root planing (SRP) [5] for the treatment of periodontitis in sites with impaired access, such as deep periodontal pockets and furcation areas [6, 7]. The impact of photobiomodulation therapy ranges from biomolecules (e.g. nucleic acids, amino acids, carbohydrates) to entire organisms [8]. In aPDT, the reactive oxygen species released from the interaction between light at a specific wavelength and photosensitizer (PS) cause irreversible damage to the cytoplasmic membrane and DNA of microorganisms [9, 10].
Despite the benefits of aPDT [8], the positive effects found in animal experiments do not represent potential clinical relevance in human studies [11]. The complexity of periodontitis, limited reachability to biofilms, and the focus of aPDT in one variable (i.e. microbiota) lead to this scenario, and, hence, the proposition of improvements in aPDT may increase the clinical relevance of the therapy. Therefore, aiming to improve the outcomes obtained with aPDT, the literature reports alterations to its initial protocol, such as multiple aPDT sessions [12], distinct light sources [13, 14] and PS [15], and incorporation of nanoparticles [16].
The rate of growth, cell wall composition, and presence of polysaccharide intercellular adhesin (PIA) differ between cells growing in biofilms and their planktonic forms, which may block the penetration of the photosensitizer and light to the deeper layers and thereby reduce the photosensitizing process [17]. This way, alterations in PS in order to target this topic may enhance its antimicrobial properties. Ruggeri Jr et al. [18] supported the hypothesis that manual or ultrasonic instrumentation alone is not able to expose the sound dentin matrix, whereas a subsequent citric acid conditioning exposes collagen fibrils and associated proteoglycans, indicating deeper penetration into mineralized tissues. Also, in addition to the reduced oral biofilm formation achieved with citric acid etching [19], root conditioning with citric acid promotes the adhesion of the fibrin network onto the dentin surface, which may further enhance connective tissue attachment to the root surface [20].
Animal experimentation is known to be the first step for in vivo validation [21]. In periodontics, rodent models of experimental periodontitis allow not only the understanding of the pathogenesis [22] but also the validation of hypotheses related to therapies for periodontal treatment [7].
Substantiated by the favorable properties of citric acid on mineralized tissues and reduction of oral microorganisms formation (both related with periodontal treatment and repair), and based on the increased biocompatibility of methylene blue (MB) acidulated with citric acid pH 1.0 over conventional MB (pH 7.0) [23], this in vivo experiment aimed to assess the hypothesis that a novel acidic MB could provide additional benefits over conventional MB, used in aPDT as adjunctive therapy to SRP for the treatment of experimental periodontitis (EP).
Materials and methods
Animals
The study followed a randomized, single-blind, controlled design, and was conducted in accordance with the ARRIVE Guidelines: Animal Research: Reporting In Vivo Experiments [23]. The protocols were approved by the Ethics Committee on Animal Use under protocol the 310–2016 of São Paulo State University, UNESP, School of Dentistry, Araçatuba. Previous experience of our research group [7] determined n = 10 enough to reject the null hypothesis in the histopathological, histometric, and immunohistochemical analyses. One hundred twenty healthy 3‐month‐old male rats (Rattus norvegicus, albinus; Wistar) weighing 250–300 g were kept in plastic boxes with wood shavings, under 12 hr/12 hr light/dark cycles, 22 ± 2°C ambient temperature, 20 air changes per hour, 55 ± 5% humidity, receiving feed and water ad libitum, and monitored daily. A blinded staff external to the study used the Minitab® 17 software (Minitab Inc., State College, PA, USA) to perform simple randomization (1:1 allocation ratio) of the animals to one of the four experimental groups: no treatment (NT), SRP, aPDT-pH7 and aPDT-pH1 (Fig 1). The euthanasia were performed at 14, 22, or 37 days after EP induction in groups SRP, aPDT-pH7, and aPDT-pH1, with an overdose (150 mg/kg) of sodium thiopental (Cristália Ltda., Itapira, SP, Brazil), consisting of ten animals per period.
- Group NT (n = 30): EP induction and no treatment performed (ligatures remained around the mandibular left first molars during the entire experiment);
- Group SRP (n = 30): EP induction and treatment with SRP and subgingival irrigation with physiological saline solution (PSS), 7 days after EP induction;
- Group aPDT-pH7 (n = 30): EP induction and treatment with SRP plus aPDT using conventional MB pH 7.0 (100μg/ml)), 7 days after EP induction;
- Group aPDT-pH1 (n = 30) EP induction and treatment with SRP plus aPDT using a novel citric acid-based MB pH 1.0 (100μg/ml), 7 days after EP induction.
Anaesthesia
For all experimental procedures, the animals were anesthetized via intramuscular injection with xylazine hydrochloride (6 mg/kg of body weight) and ketamine hydrochloride (70 mg/kg of body weight).
Experimental periodontitis induction
At day 0, a #24 cotton thread (Cotton Chain N°. 24, Coats Corrente, São Paulo, SP, Brazil) was placed around the mandibular left first molars to induce EP [7, 24] in groups SRP, aPDT-pH7, and aPDT-pH1. In group NT, EP was induced at day 7 by using the same method.
Preparation of the dyes
The conventional PS was prepared (MB 100 μg/ml) in distilled water at native (not adjusted) pH 7.0. Initially, for the novel PS, was prepared an unsaturated solution of citric acid adjusted to pH 1.0. After preparation, this solution was used as a solvent for the MB in order to achieve a concentration of 100 μg/ml. At the moment of the experiment, the pH of the dye was rechecked and kept stable.
Treatments
Were performed by the same calibrated operator [JMA], masked to the experimental groups, 7 days after EP induction. SRP was conducted in accordance with the protocol described by Almeida et al. (2008) [7]. In groups treated with aPDT, either the MB pH 7.0 or pH 1.0 were carefully inserted and kept within the periodontal pocket for 60 s and then irradiated with a red (AlGaInP) diode laser (TheraLase—DMC, Sao Carlos, SP, Brazil) in one point at the buccal and one point at the lingual surfaces of the mandibular left first molars. Laser parameters for each point were 660 nm, 35mW, 74.2 J/cm2, 2.10 J, spot 0.0283 cm2, contact, continuous mode.
Tissue processing
The left hemimandibles were fixed in buffered 4% formaldehyde for 48 hr and demineralized in 10% ethylenediaminetetraacetic acid (EDTA) solution. Then, were submitted to conventional histological processing and paraffin embedding. Semi-serial 4 μm thick sections were obtained from buccal to lingual progression. Five equidistant sections from each specimen were stained with haematoxylin and eosin (H&E) for histopathological and histometric analyses [7]. Four additional sections from each specimen were subjected to the indirect immunoperoxidase method, using the following primary anti‐bodies: goat anti-osteocalcin (OCN) (Santa Cruz Biotechnology®) and goat anti-tartrate-acid phosphatase (TRAP) (Santa Cruz Biotechnology®). The indirect immunoperoxidase technique was conducted in accordance with Matheus et al. (2018) [25].
Microscopy analysis procedure
Analyses were performed by a calibrated and masked staff using image analysis software (AxioVision 4.8.2; Carl Zeiss MicroImaging GmbH, Jena, TH, Germany). Histopathological analysis was conducted by a certified histologist [EE] based on the criteria described by Gusman et al. (2019) [26]. Each parameter was scored from 0 to 3 and is shown in Table 1. For the histometric analysis, the entire furcation (FA) was delineated and considered 100% of the area to be analyzed. The entire bone area (BA) was delineated within the limits of FA. The ratio of BA to FA was calculated and the results were expressed as the percentage of bone in the furcation (PBF). Remeasurement of the 5 sections stained with H&E was performed 1 week after the first measurement in order to assess the intra-examiner reliability and reproducibility by Kappa statistic.
TRAP immunolabeling was evaluated by counting TRAP-positive cells at × 200 magnification within TA. A semiquantitative analysis of the immunolabelling of OCN was performed at × 400 magnification [26]. The immunolabelling was characterized as follows:
- Score 0: no immunolabelling (total absence of immunoreactive [IR] cells);
- Score 1: low immunolabelling (IR in ∼1/4 of cells per area);
- Score 2: moderate immunolabelling (IR in ∼1/2 of cells per area);
- Score 3: high immunolabelling (IR in ∼3/4 of cells per area).
Primary and secondary outcomes
The primary outcome was defined as the bone formation within the furcation. Hence, PBF was considered the primary outcome variable. The secondary outcome was to define the tissues’ response and safety of the novel MB when compared with MB pH 7.0, through histopathological and immunohistochemical analyses.
Statistical analysis
Data were analyzed using BioStat software (BioStat version 5.0, Belém, PA, Brazil). Cohen’s kappa coefficient was used to calculate the agreement between the measurements of PBF. The normality of distribution of the collect data was accessed by Shapiro‒Wilk test. The scores evaluated in the histopathological and immunohistochemical analysis of OCN were submitted to analysis of variance with Kruskal–Wallis test and post-test of multiple comparisons of Dunn (p≤0.05). Parametric data (PBF and TRAP) were analyzed with analysis of variance (One-way ANOVA) and post-test of multiple comparisons of Tukey (p≤0.05).
Results
Histometric analysis of PBF
Cohen’s Kappa coefficient showed 96% agreement between measurements. The results of PBF are shown in Fig 2. In intragroup analysis, aPDT-pH7 showed lower PBF at 14 days (64.97%±5.38) compared with 37 days (72.96%±3.46) (p≤0.05). aPDT-pH1 showed lower PBF at 14 days (66.33%±7.35) compared with 22 (75.05%±2.66) and 37 days (78.4%±3.65) (p≤0.05). In intergroup analysis, group NT showed lower PBF at 14, 22 and 37 days (27.09%±6.85; 31.33%±7.41; 29.67%±5.68) when compared with groups SRP, aPDT-pH7 and aPDT-pH1 at the same time points (p≤0.05). Group SRP showed lower PBF at 22 (57.03%±10.46) and 37 days (63.24%±5.58) when compared with aPDT-pH7 and aPDT-pH1 at the same time points (p≤0.05). aPDT-pH7 showed lower PBF at 22 (68.48%±3.32) and 37 days (72.96%±3.46) when compared with aPDT-pH1 at the same time points (p≤0.05).
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).
Histopathological analysis of periodontal tissues
Experimental periodontitis severe in group NT, as confirmed by the higher extent of the local inflammatory response and progressive damage to periodontium when compared with the other groups. In group SRP was noticed decreased extent of local inflammatory response, as well as more favorable repair process over the experimental periods when compared with group NT. Groups aPDT-pH7 and aPDT-pH1 showed very similar histopathologic features, and when compared with group SRP, both presented reduced extent of local inflammatory response and greater tissue repair process after treatment (Fig 3). The parameters, scores, specimens’ distributions, and statistical analysis of periodontal tissues in NT, SRP, aPDT-pH7 and aPDT-pH1 are shown in Table 1.
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.
Immunohistochemical analysis
The immunoperoxidase technique for detection of TRAP and OCN showed high specificity for these antigens, as confirmed by the absence of immunolabeling in negative control. In intragroup analysis, group NT showed higher number of TRAP-positive cells at 37 (63.5±10.5 cells) days when compared with 14 (41.8±5.80 cells) and 22 days (47.7±5.3 cells) (p≤0.05). In intergroup analysis, group NT (41.8±5.80; 47.7±5.3; 63.5±10.5 cells) showed higher number of TRAP-positive cells at 37 days when compared with group SRP (49.2±7.3 cells), at 22 and 37 days when compared with aPDT-pH7 (30.2±12.6; 26.4±7.9 cells), and in all experimental periods when compared with aPDT-pH1 (24.6±9.2; 22.4±10.6; 34±7.9 cells) (p≤0.05). Group SRP (45.2±4.4; 52.2±9; 49.2±7.3 cells) showed higher number of TRAP-positive cells at 14, 22 and 37 days when compared with aPDT-pH7 and aPDT-pH1 at the same time points (p≤0.05) (Fig 4).
(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.
In intragroup analysis for OCN, aPDT-pH7 and aPDT-pH1 showed lower immunolabeling pattern at 14 days when compared with 22 and 37 days (p≤0.05). In intergroup analysis, group NT showed no statistically significant difference with group SRP and lower immunolabeling pattern when compared with groups aPDT-pH7 and aPDT-pH1 in all experimental periods (p≤0.05). Group SRP showed lower immunolabeling pattern when compared with groups aPDT-pH7 and aPDT-pH1 at 22 and 37 days (p≤0.05) (Fig 5).
(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.
Discussion
To target more than one system and/or tissue enrolled with periodontal treatment and repair (i.e. microbiota, specific microenvironment, host immune and inflammatory systems, bone, root surface, and collagen fibers) may be the key for adjunctive therapies to provide standout additional benefits over traditional forms of treating periodontitis. The present study evaluated the effectiveness and safety of a citric acid-based MB pH 1.0 to be used with aPDT as adjunctive therapy to SRP for the treatment of experimental periodontitis. The results support that the acidic MB is as safe as the conventional MB (pH 7.0) used with aPDT so far (i.e. similar histopathologic features, tissues’ reaction, and expression of bone-related biomarkers). Additionally, our hypothesis that the acidic MB pH 1.0 could provide additional benefits over conventional MB was confirmed by the higher PBF in group aPDT-pH1 at 22 and 37 days, when compared with aPDT-pH7.
The methodology of EP induction by ligature is an experimental model widely used in researches evaluating periodontal diseases [7, 22]. The histopathologic features obtained 7 days after ligature placement are very similar [22] to those observed in humans, and, also, the equivalent complexity of the microbiota is confirmed by the absence of substantial periodontal breakdown in “germ-free” animals [27]. Furthermore, animal studies might mimic the closest situation to the clinical scenario in order to establish valuable cause and effect relationships. On this topic, the reduced dimensions and the impaired access to the furcation of rats’ molars validate the application of adjunctive therapies for the treatment of EP in these areas [6]. Also, neither maintaining (present experiment) nor removing the ligature 7 days after induction would compromise the comparisons in this model, since the removal of the ligature didn’t prevent the occurrence of alveolar bone loss in the furcation [28]. Hence, this experimental model is capable to validate the hypothesis and to confirm the security and effectiveness of novel therapies for the treatment of periodontal disease in humans [22].
The innate host molecules and inflammatory cells are significantly different when healthy and diseased sites are compared. Although bacteria are present in healthy sites, they coexist in a homeostatic relationship with periodontal tissues, in which an orchestrated expression of select innate host-defense mediators are observed [29, 30]. Conversely, periodontitis sites disorderly express more and different mediators compared with healthy sites [3, 31]. aPDT is known to target microorganisms related to the pathogenesis of periodontitis, however, it was hypothesized by Braham et al. [32] that aPDT may promote periodontal healing not only by killing bacteria but also by inhibiting destructive host responses. Hence, the higher biocompatibility of MB pH 1.0 found by Gusman et al. [33] is likely to support some of the better results found in group aPDT-pH1.
Persistence on the expression of prostaglandin-E2 harms bone formation through upregulation of RANKL and inhibition of osteoprotegerin [34]. Osteoblasts and osteoclast are two of the main cells responsible for bone metabolism and maintenance of the hierarchical structure of bone [35]. Osteocalcin regulates bone mineralization and turnover [36], while TRAP is expressed from differentiation until apoptosis of osteoclasts [37], therefore, both proteins are important regulators of the bone tissue. With regard to the expression of both biomarkers, this experiment showed that both aPDT protocols (aPDT-pH7 and aPDT-pH1) reduced the number of TRAP-positive cells and increased the immunolabelling pattern of OCN in the furcation when compared with only SRP.
Noteworthy, there is a strict relationship between alveolar bone loss and the success of periodontal therapies [38]. In spite of a possible positive effect of aPDT with MB acidulated with citric acid at pH 1.0 for the treatment of periodontitis, as evidenced by the increased PBF at 22 and 37 days, when compared with 14 days in group aPDT-pH1, further experimental research pursuing deep knowledge on this topic, as well as researches evaluating other parameters shall be conducted in order to eventually translated their results to the clinical scenario.
Even though the mechanisms by which the acidic MB increased the amount of alveolar bone when compared to neutral MB are not completely elucidated, it is known that the interaction of citric acid with mineralized surfaces leads to ionic-exchange processes, increasing the release of Ca++ [39] and precipitation of Ca3(citrate)2.4H20 onto apatite surface [40], plausible triggers for bone formation. Furthermore, this beneficial property over molecules that positively affect bone might not be understood as limited to bone tissue, since calcium and calcium citrate regulate fundamental cellular events, such as mitochondrial activity [41]. Also, despite the distinct purpose and methods from this experiment, de Rezende et al. [42] observed that demineralization of bone surfaces with citric acid pH 1.0 provided an appropriate substrate for pre-osteoblast proliferation and bone mineralization.
Innumerous PS have been used with aPDT for the treatment of periodontitis [43]. Curcumin [44], toluidine blue [45], chlorin [46], and MB [47] are able to promote a significant antifungal and antibacterial effect against microorganisms. However, the impaired penetration of PS deep in the biofilm matrix decreases the effectiveness of the few PS molecules that are able to penetrate the biofilm matrix [48]. Such drawbacks can be overcome by the development of modified PS [49]. Most experiments confirmed the efficacy of the optimization of MB derivatives to kill bacteria [50, 51] but usually fail to assess their effects on periodontal tissues.
Negatively charged citric acid is believed to inactivate bacteria by either destabilizing the outer membrane or by sequestering essential metals from the growth environment [52, 53]. Recently, Burel et al. [54] evaluated the impact of pH on citric acid antimicrobial activity against Gram-negative bacteria. Their [54] results indicate that tribasic citric acid was the most effective in damaging the bacterial membrane. However, a reduction in the absolute number of bacteria and damages to cell walls were found regardless of the tested pH. No similar experiment has been carried in order to evaluate the effectiveness of citric acid on targeting the membrane or killing periodontal pathogens. Hence, the promising findings of Burel et al. [54] encourage further research on the plausible efficacy of using citric acid-based therapies for reducing bacterial colonization related to the pathogenesis of periodontitis.
The harm to tissues is reasoned by the term “acid strength”, described as the potential of acid to lose protons H+. For instance, this property can be adjusted by topology structure and morphology, and crystallinity in addition to the chemical composition [55]. Although in a contaminated environment no statistical significance in inflammatory parameters has been observed between acidic and neutral MB, a subcutaneous test showed the best biocompatibility for MB pH 1.0 [22] as compared with MB pH 7.0.
The use of citric acid for decontamination and repair has being the focus of in vitro and in vivo experiments in implantology. Souza et al. [19] demonstrated that citric acid significantly reduced the biofilm related with peri-implantitis, as well as enhanced the electrochemical stability of titanium. Htet et al. [56] observed increased bone formation following the combination of mechanical and chemical treatment with citric acid for disinfection of anodized implant surface, which may encourage further research using the acidic MB for the treatment of peri-implantitis as well.
To the best of our knowledge, this is the first experiment to investigate the influence of an acidic dye used in aPDT for the treatment of EP in an in vivo model. As with any animal model, the data presented by this experiment shall be interpreted with caution, since the contact of acidic substances with denuded root surface is frequently related with dentin hypersensitivity in humans.
Collectively, our data indicate that aPDT using the citric acid-based MB pH 1.0 is as secure for periodontal tissues as the conventional MB (pH 7.0) used in aPDT so far. Our findings suggest an additional benefit of stimulating bone formation in the furcation following aPDT with the acidic MB.
Acknowledgments
It was conducted at the Department of Diagnosis and Surgery—Division of Periodontics, São Paulo State University (UNESP), School of Dentistry, Araçatuba, São Paulo, Brazil. The authors thank the laboratory of Osteobiology Applied to Dentistry for assistance in the process of histological and immuhistochemical samples.
References
- 1. Eke PI, Thornton-Evans GO, Wei L, Borgnakke WS, Dye BA, Genco RJ (2018) Periodontitis in US Adults: National Health and Nutrition Examination Survey 2009–2014. J Am Dent Assoc 149:576–588. pmid:29957185
- 2. Kassebaum NJ, Bernabé E, Dahiya M, Bhandari B, Murray CJ, Marcenes W (2014) Global burden of severe periodontitis in 1990–2010: a systematic review and meta-regression. J Dent Res 93:1045–1053. pmid:25261053
- 3. Bartold PM, Van Dyke TE (2013) Periodontitis: a host-mediated disruption of microbial homeostasis. Unlearning learned concepts. Periodontol 2000 62:203–217. pmid:23574467
- 4. Darveau RP, Hajishengallis G, Curtis MA (2012) Porphyromonas gingivalis as a potential community activist for disease. J Dent Res 91:816–820. pmid:22772362
- 5. Mizutani K, Aoki A, Coluzzi D, Yukna R, Wang CY, Pavlic V, et al (2016) Lasers in minimally invasive periodontal and peri-implant therapy. Periodontol 2000 71:185–212. pmid:27045437
- 6. Renvert S, Persson GR (2002) A systematic review on the use of residual probing depth, bleeding on probing and furcation status following initial periodontal therapy to predict further attachment and tooth loss. J Clin Periodontol 29:82–89. pmid:12787209
- 7. de Almeida JM, Theodoro LH, Bosco AF, Nagata MJ, Bonfante S, Garcia VG (2008) Treatment of experimental periodontal disease by photodynamic therapy in rats with diabetes. J Periodontol 79:2156–65. pmid:18980525
- 8. Arany PR (2016) Craniofacial Wound Healing with Photobiomodulation Therapy: New Insights and Current Challenges. J Dent Res 95:977–984. pmid:27161014
- 9. Romanova NA, Brovko LY, Moore L, Pometun E, Savitsky AP, Ugarova NN, et al (2003) Assessment of photodynamic destruction of Escherichia coli O157:H7 and Listeria monocytogenes by using ATP bioluminescence. Appl Environ Microbiol 69:6393–6398. pmid:14602591
- 10. Jori G, Fabris C, Soncin M, Ferro S, Coppellotti O, Dei D, et al (2006) Photodynamic therapy in the treatment of microbial infections: basic principles and perspective applications. Lasers Surg Med 38:468–81. pmid:16788934
- 11. Chambrone L, Wang HL, Romanos GE (2018) Antimicrobial photodynamic therapy for the treatment of periodontitis and peri-implantitis: An American Academy of Periodontology best evidence review. J Periodontol 89:783–803. pmid:30133749
- 12. Cadore UB, Reis MBL, Martins SHL, Invernici MM, Novaes AB Jr, Taba M Jr, et al (2019) Multiple sessions of antimicrobial photodynamic therapy associated with surgical periodontal treatment in patients with chronic periodontitis. J Periodontol 90:339–349. pmid:30383298
- 13. Nikinmaa S, Alapulli H, Auvinen P, Vaara M, Rantala J, Kankuri E, et al (2020) Dual-light photodynamic therapy administered daily provides a sustained antibacterial effect on biofilm and prevents Streptococcus mutans adaptation. PLoS One 15:e0232775. pmid:32374766
- 14. Allaker RP, Douglas CW (2009) Novel anti-microbial therapies for dental plaque-related diseases. Int J Antimicrob Agents 33:8–13. pmid:18804350
- 15. Ghorbani J, Rahban D, Aghamiri S, Teymouri A, Bahador A (2018) Photosensitizers in antibacterial photodynamic therapy: an overview. Laser Ther 27:293–302. pmid:31182904
- 16. Qi M, Chi M, Sun X, Xie X, Weir MD, Oates TW, et al (2019) Novel nanomaterial-based antibacterial photodynamic therapies to combat oral bacterial biofilms and infectious diseases. Int J Nanomedicine 14:6937–6956. pmid:31695368
- 17. Sharma M, Visai L, Bragheri F, Cristiani I, Gupta PK, Speziale P (2008) Toluidine blue-mediated photodynamic effects on staphylococcal biofilms. Antimicrob Agents Chemother 52:299–305. pmid:17967908
- 18. Ruggeri A Jr, Prati C, Mazzoni A, Nucci C, Di Lenarda R, Mazzotti G, et al (2007) Effects of citric acid and EDTA conditioning on exposed root dentin: An immunohistochemical analysis of collagen and proteoglycans. Arch Oral Biol 52:1–8. pmid:17098210
- 19. Souza JGS, Cordeiro JM, Lima CV, Barão VAR (2019) Citric acid reduces oral biofilm and influences the electrochemical behavior of titanium: An in situ and in vitro study. J Periodontol 90:149–158. pmid:30088827
- 20. Subramanian S, Appukuttan D, Tadepalli A, Gnana PPS, Athmarao RT (2017) Root conditioning with citric acid and ethylenediaminetetraacetic acid and their effect on fibrin clot adhesion to dentin-a scanning electron microscopic study. J Clin Diagn Res 11:ZC82–ZC85. pmid:28969280
- 21. Hollinger JO, Kleinschmidt JC (1990) The critical size defect as an experimental model to test bone repair materials. J Craniofac Surg 1:60–68. pmid:1965154
- 22. de Molon RS, de Avila ED, Boas Nogueira AV, Chaves de Souza JA, Avila-Campos MJ, de Andrade CR, et al (2014) Evaluation of the host response in various models of induced periodontal disease in mice. J Periodontol 85:465–477. pmid:23805811
- 23. Percie du Sert N, Hurst V, Ahluwalia A, Alam S, Avey MT, Baker M, et al (2020) The ARRIVE guidelines 2.0: Updated guidelines for reporting animal research. PLoS Biol 18:e3000410. pmid:32663219
- 24. Johnson IH (1975) Effects of local irritation and dextran sulphate administration on the periodontium of the rat. J Periodontal Res 10:332–345. pmid:129558
- 25. Matheus HR, Ervolino E, Faleiros PL, Novaes VCN, Theodoro LH, Garcia VG, et al (2018) Cisplatin chemotherapy impairs the peri-implant bone repair around titanium implants: An in vivo study in rats. J Clin Periodontol 45:241–252. pmid:28965362
- 26. Gusman DJR, Ervolino E, Theodoro LH, Garcia VG, Nagata MJH, Alves BES, et al (2019) Antineoplastic agents exacerbate periodontal inflammation and aggravate experimental periodontitis. J Clin Periodontol 46:457–469. pmid:30854670
- 27. Rovin S, Costich ER, Gordon HA (1966) The influence of bacteria and irritation in the initiation of periodontal disease in germfree and conventional rats. J Periodontal Res 1:193–204. pmid:4225530
- 28. De Almeida J, Ervolino E, Bonfietti LH, et al (2015) Adjuvant Therapy With Sodium Alendronate for the Treatment of Experimental Periodontitis in Rats. J Periodontol 86:1166–1175. pmid:26062841
- 29. Moughal NA, Adonogianaki E, Thornhill MH, Kinane DF (1992) Endothelial cell leukocyte adhesion molecule-1 (ELAM-1) and intercellular adhesion molecule-1 (ICAM-1) expression in gingival tissue during health and experimentally-induced gingivitis. J Periodontal Res 27:623–630. pmid:1281230
- 30. Tonetti MS (1997) Molecular factors associated with compartmentalization of gingival immune responses and transepithelial neutrophil migration. J Periodontal Res 32:104–109. pmid:9085218
- 31. Ren L, Leung WK, Darveau RP, Jin L (2005) The expression profile of lipopolysaccharide-binding protein, membrane-bound CD14, and toll-like receptors 2 and 4 in chronic periodontitis. J Periodontol 76:1950–1959. pmid:16274315
- 32. Braham P, Herron C, Street C, Darveau R (2009) Antimicrobial photodynamic therapy may promote periodontal healing through multiple mechanisms. J Periodontol 80:1790–1798. pmid:19905948
- 33. Gusman DJR, Cintra LTA, Novaes VCN, Matheus HR, de Araujo NJ, de Almeida JM (2018) pH influences the biocompatibility of methylene blue solutions. Clin Oral Investig 22:361–367. pmid:28536783
- 34.
Horowitz M, Kacena M, Lorenzo J. Genetics and mutations affecting osteoclast development and function. In Bronner F., Farach-Carson M. & Rubin J. (Eds.), Bone resorption. London: Springer-Verlag; 2006:91–107.
- 35. Adamopoulos IE (2018) Inflammation in bone physiology and pathology. Curr Opin Rheumatol 30:59–64. pmid:29016371
- 36. Ivaska KK, Hentunen TA, Vääräniemi J, Ylipahkala H, Pettersson K, Väänänen HK (2004) Release of intact and fragmented osteocalcin molecules from bone matrix during bone resorption in vitro. J Biol Chem 279:18361–18369. pmid:14970229
- 37. Minkin C (1982) Bone acid phosphatase: tartrate-resistant acid phosphatase as a marker of osteoclast function. Calcif Tissue Int 34:285–290. pmid:6809291
- 38. Polimeni G, Xiropaidis AV, Wikesjö UM (2006) Biology and principles of periodontal wound healing/regeneration. Periodontol 2000 41:30–47. pmid:16686925
- 39. Sterrett JD, Bankey T, Murphy HJ (1993) Dentin demineralization. The effects of citric acid concentration and application time. J Clin Periodontol 20:366–370. pmid:8501277
- 40. Misra DN (1996) Interaction of citric acid with hydroxyapatite: surface exchange of ions and precipitation of calcium citrate. J Dent Res 75:1418–1425. pmid:8831638
- 41. Bertero E, Maack C (2018) Calcium Signaling and Reactive Oxygen Species in Mitochondria. Circ Res 122:1460–1478. pmid:29748369
- 42. de Rezende ML, Coesta PT, de Oliveira RC, Salmeron S, Sant’Ana AC, Damante CA, et al (2015) Bone demineralization with citric acid enhances adhesion and spreading of preosteoblasts. J Periodontol 86:146–154. pmid:25272980
- 43. Carrera ET, Dias HB, Corbi SCT, Marcantonio RAC, Bernardi ACA, Bagnato VS, et al (2016) The application of antimicrobial photodynamic therapy (aPDT) in dentistry: a critical review. Laser Phys 26:123001. pmid:29151775
- 44. Araújo NC, Fontana CR, Bagnato VS, Gerbi ME (2014) Photodynamic antimicrobial therapy of curcumin in biofilms and carious dentine. Lasers Med Sci 29:629–635. pmid:23793414
- 45. Qin YL, Luan XL, Bi LJ, Sheng YQ, Zhou CN, Zhang ZG (2008) Comparison of toluidine blue-mediated photodynamic therapy and conventional scaling treatment for periodontitis in rats. J Periodontal Res 43:162–167. pmid:18302617
- 46. Pfitzner A, Sigusch BW, Albrecht V, Glockmann E (2004) Killing of periodontopathogenic bacteria by photodynamic therapy. J Periodontol 75:1343–1349. pmid:15562911
- 47. de Oliveira RR, Schwartz-Filho HO, Novaes AB Jr, Taba M Jr (2007) Antimicrobial photodynamic therapy in the non-surgical treatment of aggressive periodontitis: a preliminary randomized controlled clinical study. J Periodontol 78:965–973. pmid:17539707
- 48. Shih MH, Huang FC (2013) Repetitive methylene blue-mediated photoantimicrobial chemotherapy changes the susceptibility and expression of the outer membrane proteins of Pseudomonas aeruginosa. Photodiagnosis Photodyn Ther 10:664–671. pmid:24284125
- 49. Dascalu Rusu LM, Moldovan M, Prodan D, Ciotlaus I, Popescu V, Baldea I, et al (2020) Assessment and Characterization of Some New Photosensitizers for Antimicrobial Photodynamic Therapy (aPDT). Materials (Basel) 13:3012. pmid:32640635
- 50. Klepac-Ceraj V, Patel N, Song X, Holewa C, Patel C, Kent R, et al (2011) Photodynamic effects of methylene blue-loaded polymeric nanoparticles on dental plaque bacteria. Lasers Surg Med 43:600–606. pmid:22057487
- 51. de Freitas LM, Calixto GM, Chorilli M, Giusti JS, Bagnato VS, Soukos NS, et al (2016) Polymeric Nanoparticle-Based Photodynamic Therapy for Chronic Periodontitis in Vivo. Int J Mol Sci 17:769. pmid:27213356
- 52. Beuchat LR, Golden DA (1989) Antimicrobials occurring naturally in foods. Food Technol 43, 134–142.
- 53. Buchanan RL, Golden MH (1994) Interaction of Citric Acid Concentration and pH on the Kinetics of Listeria monocytogenes Inactivation. J Food Prot 57:567–570. pmid:31121707
- 54. Burel C, Kala A, Purevdorj-Gage L (2021) Impact of pH on citric acid antimicrobial activity against Gram-negative bacteria. Lett Appl Microbiol 72:332–340. pmid:33099798
- 55. Zhang X, Zhao Y, Xu S, Yang Y, Liu J, Wei Y, et al (2014) Polystyrene sulphonic acid resins with enhanced acid strength via macromolecular self-assembly within confined nanospace. Nat Commun 5:3170. pmid:24463793
- 56. Htet M, Madi M, Zakaria O, Miyahara T, Xin W, Lin Z, et al (2016) Decontamination of anodized implant surface with different modalities for peri-implantitis treatment: lasers and mechanical debridement with citric acid. J Periodontol 87:953–961. pmid:26966876