Evaluation of the effects of photodynamic therapy with hypericin-glucamine in the treatment of periodontal disease induced in rats
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The purpose of this study was to evaluate the effectiveness of a photosensitizer hypericin-glucamine, activated by LED amber (34.10 J/cm2) as an adjuvant to scaling and root planing (SRP), on the treatment of experimental periodontal disease (PD) in rats.
In a 15-day period, PD was induced in the mandibular molars through ligature placement. The animals (n = 60) were randomly divided into four groups: control (with induction of periodontal disease), scaling and root planing (SRP), antimicrobial photodynamic therapy (aPDT), and SRP + aPDT. At 7, 15, and 30 days after treatments, animals were euthanized. Digital microtomography, histometric, and stereometric analyses were performed to calculate the bone loss of mandibular second molars, and the tissue repair was analyzed histologically. The data were subjected to statistical analysis (α = 5%).
Histologically, the control group periodontium showed several morphological changes, but an evident gradual reduction in the inflammatory process was observed in the subsequent periods. The SRP, aPDT, and SRP + aPDT groups presented the same, but in less intensity. The stereometric analysis showed a significantly higher proportion of fibroblasts in SRP group (p < 0,001) and SRP + aPDT group (p < 0.0001) compared to that in the control group at 7 days post treatment.
We conclude that scaling and root planing with or without photodynamic therapy with hypericin-glucamine increased density of the fibroblast and cell density. However, there is no statistically significant difference between SRP and SRP + aPDT.
KeywordsHypericin-glucamine Scaling and root planing Periodontal disease
The most appropriate treatment for periodontal disease is scaling and root planing (SRP) . However, in clinical evaluation exams, deep pocket or those involving furcation areas, invaginations, and root curvatures do not seem to be able to recover and maintain their periodontal health [12, 26, 31, 35]. For this reason, there is a constant commitment to developing treatments auxiliary to periodontal therapy.
Antimicrobial photodynamic therapy (aPDT) has shown a bactericidal effect against periodontopathogens such as Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Capnocytophaga gingivalis, Fusobacterium nucleatum, Prevotella intermedia, and Streptococcus sanguis [7, 15, 16, 30]. In this light-based treatment, a photosensitizer is applied to the diseased site, left for a period of incubation in the dark, and then a source of light with a specific wavelength activates it. The photosensitizer used in aPDT should be selected carefully since it is influenced by the site in which it will be deposited. The type, concentration, and mode of action of the photosensitizer are essential factors for the effectiveness of the therapy [10, 19].
Hypericin is a natural photosensitizer, present in herbaceous plants known as St. John’s wort (genus Hypericum perforatum) . It is used currently as an herbal medicine in psycho-vegetative disorders and depressive states and for fear and/or anxiety . It has anti-inflammatory, antiseptic, anti-infective, and antiviral properties and stimulates blood circulation and eliminates bruising [30, 38].
Studies have shown the great phototoxic power of hypericin in the treatment of psoriasis and other skin diseases  as well as in anticancer activity by inducing both apoptosis and necrosis of tumor cells . This photosensitizer has also the ability to inactivate microorganisms , making it one of the most powerful photo-agents found in nature [28, 33]. Nonetheless, the hypericin loses its photosensitizing action when submitted to a biological medium due to its hydrophobic characteristic. Its ability to generate singlet oxygen decreases as consequence to the auto-aggregation in an aqueous medium, thus affecting the effectiveness of the aPDT [22, 32].
A strategy proposed by chemical scholars is inducing the formation of supramolecular species, which are composed of two or more molecules joined by secondary molecular bonds to obtain a certain property or functionality. In the case of hypericin, a reaction is induced from the phenolic group of the molecule which yields the proton H+. The result is the formation of hypericin-glucamine. The presence of several hydroxyl groups in the hypericin-glucamine supramolecule provides sites where hydrogen bonds will be established, thus increasing its hydrophilicity .
The aPDT benefits mechanical treatment in residual pockets and reduces the indications of surgical debridement and the risk of bacteremia during the treatment . Given these benefits summed with the hypericin-glucamine properties [2, 5, 9, 10, 23], we aim at evaluating the influence of aPDT using the photosensitizer hypericin-glucamine activated by LED (34.10 J/cm2) in the treatment of periodontal disease induced in rats.
This study was performed using 60 male adult Wistar rats (Rattus norvegicus albinus, Holtzman). All experimental procedures involving the rats were performed under anesthesia, with a combination of ketamine chlorhydrate (0.08 mL/100 g; Syntec, Sao Paulo, Brazil) and xylazine chlorhydrate (0.04 mL/100 g; Syntec, Sao Paulo, Brazil). The experimental periodontal disease model was randomly implemented by ligature insertion (Corrente Algodão n” 24; Coats Corrente, São Paulo, SP, Brazil) around one of the upper second molars (randomly chosen) in the subgingival sulcus for a 15-day period. The study was approved by the Institutional Animal Care and Use Committee (07/2012).
Hypericin-glucamine was obtained from a stock solution (200 μmol L−1 in polar aprotic solvent, dimethyl sulfoxide (DMSO), sterilized by filtration using a 0.22-μm membrane and kept out of the light). The working solution was prepared by diluting the stock solution in sodium phosphate buffer (pH 7.2) to a final concentration of 10 μg/mL, where the final percentage of DMSO was less than 1%.
An amber-colored LED (MM Opto, São Carlos/SP, Brazil) with 700 mA, the wavelength of 590 nm—coincident with the maximum absorption band of hypericin-glucamine and converted the power of 90 mW—was used in this study. The LED was applied for 6 min at a dosage of 34.10 J/cm.
Induction of periodontal disease (control): No treatment was performed.
Scaling and root planing (SRP): Supra- and subgingival scaling and root planning (SRP) using manual curettes (n. 7–8/ mine five, 11–12/mine and 13–14/mine; Hu-Friedy, Co.) was performed by a single operator. Mechanical removal of mature plaque present on the free faces of the tooth was performed by repeated movements in the apical-coronal direction.
Antimicrobial photodynamic therapy (aPDT): Ten microliters of the photosensitizer, which was stored in light-protected falcons, transferred to a blunt tip syringe and coated cylinder. The tip of the needle was inserted between the tooth and gingival tissue, and photosensitizer (PH) was deposited in the gingival sulcus. After incubating for 10 min, the LED was applied for 6 min on the occlusal surface (34.10 J/cm2); consequently, the transmucosal irradiation of all faces of the second molar was given in only one application.
Scaling and root planing + antimicrobial photodynamic therapy (SRP + aPDT): both protocols were carried out, respectively. After the corresponding experimental period (7, 15, and 30 days after treatments), the animals were euthanized with an anesthetic overdose, and the maxillas were removed for the proposed analyses.
Microtomography of X-rays (μ-CT)
The hemimaxillas were analyzed by X-ray beam scanning in a 3D radiography system (computerized digital microtomography - microCT) . The samples were scanned by the SkyScan microtome (SkyScan 1176 Bruker MicroCT, Aartselaar, Belgium, 2003) using cuts of 18 μm (50 Kv and 500 μm), and the area of interest was delimited by standardized dimensions (1.26 mm high × 0.56 mm wide) in the proximal (mesial and distal) areas of the second molar . Next, the volume percentage of the bone tissue present in these areas was obtained with a threshold radiodensity of 55–250 Hounsfield units in the grayscale scale . For the analysis, the mean values obtained for the mesial and distal areas were used. Measurements were performed by a blinded and calibrated examiner (SC).
After decalcification in 7% EDTA buffered, the samples were dehydrated embedded in paraffin and histological serial sagittal sections (thickness of 4 μm) were stained with hematoxylin-eosin (HE) technique. The images were captured using an optical microscope (DIASTAR Microscope, Leica Reichert & Jung products, Wetzlar, Hessen, Germany). Evaluations were performed by a trained, calibrated, and blinded examiner (SC). The following parameters were evaluated: the inflammatory reactions of the connective tissue, bone resorption processes, and tissue neoformation.
To perform the histometry, a blinded and calibrated examiner selected two slides of each animal considering the presence of the central portion of the pulp (SC). The furca region was delimited according to the methodology of Cesar Neto et al. 2006 using a software (Image J Launcher 1.42q, Bethesda, MD, USA) . The measurement values of bone loss in the proximal area were obtained from images with a tenfold increase, in which lines were drawn between the height part of the cementum-enamel junction and the height of the bone crest.
The microscopic stereologic point-counting technique was employed on sections stained with HE to the experimental periods of 7 and 15 days. The images were captured in an optical microscope (DIASTAR Microscope, Leica Reichert & Jung products, Wetzlar, Hessen, Germany) at × 10 magnification. A square-lattice grid micrometer was positioned over two regions of interest: (a) interproximal and (b) furca. For each grid, 50 coincident points were counted on the histological structures, which indicated the relative volume density of the following tissue components: bone, fibroblasts, inflammatory cells, and blood vessels. A percentage analysis of each tissue component was carried out considering the total number of points counted per experimental period . Evaluations were performed by a trained, calibrated, and blinded examiner (FK).
A software (GraphPad Prism version 5.00 for Windows, GraphPad Software, La Jolla, CA, USA) was used for statistical analysis. The Shapiro-Wilk normality test was applied and ANOVA test was used, followed by Tukey’s post-test for the intra- and inter-group analysis. A significance level of 95% was applied to all analysis.
Microtomography of X-rays (μ-CT)
Descriptive histological analysis
Analysis of the SRP, aPDT, and SRP + aPDT groups (Fig. 2) presented the same, but in less intensity, histological characteristics were found for the control group. Thereby, the histological descriptions were similar for all experimental groups. The images illustrate the periodontal tissues after each treatment in different periods.
This study aims at evaluating the efficacy of photodynamic therapy using the photosensitizer hypericin-glucamine as an adjuvant to scaling and root planing (SRP) in the treatment of periodontal disease. The mechanical treatment of scaling and root planing with the application of photodynamic therapy showed promising results in the treatment of periodontal disease [2, 5, 9, 10, 14, 34].
The scaling and root planning (SRP) model, although frequently used, has some limitations to fully represent the clinical reality. There is a lack of clinical instruments suitable for reduced size of the animal tooth, and consequently, the treatment is restricted to removing the mature biofilm in the accessible regions. Nonetheless, the mechanical treatment in clinical practice cannot always reach hard-accessible regions of patient’s teeth, which compensate in part those limitations mentioned before.
As already described previously , the rats show a rapid tissue repair; therefore, we evaluated its inflammatory profile by stereometry analysis only for the periods of 7 and 15 days after removing the ligatures and treating the animals. Seven days after the treatment, the SRP and aPDT + SRP groups had a significantly higher proportion of fibroblasts than the control group. Fibroblasts are able to modulate connective tissue response since they synthesize proteins such as collagen and elastin [20, 27], and their increased density indicates the connective tissue repair in the post-treatment period.
The histological, histometric, and X-ray microtomography findings did not show a statistically significant difference between the experimental groups. The groups in which treatment with photodynamic therapy (aPDT and SRP + aPDT) was applied had similar results to those obtained in the group where only scaling and root planing (SRP) was performed. These results demonstrate that the hypericin-glucamine photosensitizer did not cause any damage to the periodontal tissues. On the other hand, Melo et al.  evaluated hypericin in relation to hypericin-glucamine in cultures of epithelial cells, and the results demonstrated that hydrophilicity increases the efficiency of the photosensitizer, preserving its selectivity. Another justification for these results is the photosensitizer time of action where the singlet oxygen formed in the reaction during the activation of hypericin with light has a short action time and therefore a limited range of action (20 nm), which means that it will only act where hypericin-glucamine is exposed to light. This is due to the high selectivity of photodynamic therapy with hypericin-glucamine that avoids interactions with adjacent tissues.
The principle of photodynamic therapy action is the formation of reactive oxygen species. Hypericin is a polycyclic quinone that reacts with neighboring molecules when activated by transferring energy to oxygen, inducing the production of singlet oxygen (1O2) even at low concentrations (< 0.1 μg/mL); thus, it is considered more powerful  and photostable than most photosensitizers [4, 24, 36]. The hypericin-glucamine photosensitizer used in our study shows that hypericin has the advantage of being effective at lower concentrations due to its hydrophilic characteristics, allowing the photosensitizer to penetrate the target cell easily and with less aggregation [17, 25]. Since hypericin-glucamine is a modification of the hypericin photosensitizer, we maintained the 10 μg/mL consensus for hypericin-glucamine used in this study.
In 2014, Melo et al.  assessed the action of hypericin in comparison to hypericin-glucamine accounting its most water-soluble derivative in the selective inactivation of Staphylococcus aureus and Escherichia coli bacteria. The study concluded that hydrophilicity increases the photodynamic efficiency of the photosensitizers, suggesting that this inactivation is related to the greater intracellular accumulation of the hydrophilic photosensitizer by the bacteria. This fact could be observed in the photoinactivation assays of E. coli and S. aureus since hypericin-glucamine caused greater inhibition of the bacteria than hypericin without prejudice to the selectivity of the photosensitizer.
Although the results from the digital microtomography and histometry for the groups treated with photodynamic therapy were similar to the SRP group, some factors should be discussed. The methodology used in this study consisted of only one section of aPDT. Although other studies claim positive results with one application [9, 14], most aPDT reports in dentistry and in cancer treatments presented a gradual improvement only after consecutive sections were performed . Other factors include the concentration of the photosensitizer and the expected incubation time for light application. The periodontal disease occurs locally as a chronic inflammatory process. Thereby, the photosensitizer may have interacted with the hemoglobin from the gingival sulcus before it could react with the microorganisms in the site. In other words, the timespan of 1 day between removing the ligature and treating the rats with the LED lights may have affected the effectiveness of the aPDT .
No significant differences were found in the analysis by computerized microtomography; however, we observed that the control group had a tendency toward lower bone volume. The SRP and aPDT groups showed similar results for the three experimental periods. A trend toward a higher percentage of bone volume in the proximal groups of the SRP treated after 7 and 15 days was observed. This same trend occurred in the aPDT group at 30 days.
The differences in the results between the histometric analysis and microCT may be related to the characteristics of the evaluations. Microtomography promotes a 3D scan of the entire sample, obtaining a total bone volume around the tooth while, in the histometric evaluation, two slides were chosen to represent the entire tooth, which may not represent what was occurring throughout the furca .
Finally, the results corroborate the literature and show the importance of the mechanical treatment for periodontal disease. The aPDT with hypericin-glucamine could accentuate the repair response of the connective tissue. The method is promising, and indeed, there is a plenty of space for more studies, such as changing the concentration and the incubation time of the photosensitizer as well as the dose of light employed.
We conclude that scaling and root planing with or without photodynamic therapy with hypericin-glucamine increased density of the fibroblast and cell density. However, there is no statistically significant difference between SRP and SRP + aPDT. Additional studies are needed to better understand the mechanisms involved in the process of recovering and improve the therapy routine.
This work was supported in part by a grant from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (BR) (AUXPG 655/2014) and by a grant from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (BR) (133436/2012-8).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical board approval was obtained for this study by the Ethics in Animal Research Committee of the School of Dentistry of Araraquara (UNESP, Brazil CEEA/FOAr 07/2012).
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