Received 00th January 20xx, [306789]

Received 00th January 20xx,

Accepted 00th January 20xx

DOI: 10.1039/x0xx00000x

[anonimizat] (Rusu),a Radu Chifor,a Marioara Moldovan,*b Codruta Sarosi,b Sorina Sava,*c Alexandra Dreanca,d Calin Repciuc,d Andras Nagy,d Ioana Baldea,e Mîndra Eugenia Badeaa and Ariadna G. Paunf

This study aims the characterization of new natural photosensitizers (PS) ([anonimizat]) [anonimizat], [anonimizat], treated with photodynamic therapy (PDT). UV-Vis, EPR (Electron Paramagnetic Resonance) and chromatographic analysis (HPLC, GS-MS) measurements were performed for two different PS formulated with this 3 waters ([anonimizat]-[anonimizat]). The cytotoxicity for new natural PS (oregano, curcumin and arnica oil) on oral keratinocyte (DOK) dysplastic cell cultures with exposures of 1, 2, 6 and 24 hours was also evaluated using the MTT assay. Periodontal disease was induced by ligation of the first mandibular molar of 20 rats, which were divided into 5 groups: [anonimizat], [anonimizat]. The animals were euthanized after 4 weeks of study. [anonimizat], no significant differences were observed between the signals emitted by the 3 types of water used. Hematological and histological values showed a greater magnitude of the inflammatory response and severe destruction of the periodontal ligaments in the untreated group. PDT has been shown to be an effective adjuvant treatment of induced periodontitis in groups treated with new natural photosensitizers.

.

Introduction

There are a number of oral diseases that pose a [anonimizat]. The need to develop an alternative prevention treatment with antibacterial agents is to avoid the side effects of antibiotics and to avoid increasing the bacterial resistance to them. Thus, natural products and photodynamic therapy have become a topical and important research.

Periodontitis, [anonimizat], especially the "red complex", [anonimizat], [anonimizat].1-2. [anonimizat], [anonimizat]. The microbial etiology of periodontitis has been studied extensively and it has been found that it is not associated with a [anonimizat], but rather with a consortium of bacteria involved in the initiation and evolution of the disease. 3-5

Conventional mechanical debridement (scaling and root planing) can achieve a temporary decrease in the subgingival levels of pathogens. However, organisms cannot be removed from the majority of periodontal pockets by mechanical therapy alone. [anonimizat], [anonimizat] improve the results of conventional mechanical therapeutic techniques. Some disadvantages of antimicrobial agents usage (such as antibiotics) include antibiotic resistance, immunosuppression and other unfavorable reactions. Considering the complications above, it is necessary to expand research in an attempt to find alternative antimicrobial techniques, such as natural ingredients for antimicrobial therapy. One of them is the use of lasers and photodynamic therapy, which might be effective in eliminating microbes in local and superficial infections in presence of natural photosensitizers. The literature presents many studies regarding antimicrobial photodynamic therapy (aPDT). Antimicrobial chemotherapy may further suppress the periodontal pathogens and increase the benefits obtained by conventional mechanical treatment.1,6–9

Antimicrobial photodynamic therapy or photodynamic inactivation (PDI) is a new promising strategy to eradicate pathogenic microorganisms such as Gram-positive and Gram-negative bacteria, yeasts and fungi. The search for new approaches that can kill bacteria may have advantages over traditional antibiotic therapy. PDT is a non-thermal photochemical reaction that involves the simultaneous presence of visible light, oxygen and a photosensitizer. Several PS have been studied for their ability to bind to bacteria and efficiently generate reactive oxygen species (ROS) upon photostimulation. In photodynamic therapy, the bactericidal effect is entirely based on the ROS generation. Among different types of ROS generated during PDT, singlet oxygen is considered as the most potential as illustrated in many studies and therefore it is predominantly responsible for photodamage and cytotoxic reactions.6,8,10,11

PDT is a new strategy that involves the combination of a non-toxic PS and a harmless visible light source wich has been demonstrated to have a significant anti-microbial effect and presents as an alternative to treating biofilm-related disease. PDT has been studied as a promising approach to eradicate oral pathogenic bacteria that cause endodontic diseases, periodontitis, peri-implantitis and caries.12–16

The novelty of this study is the formulation and characterization of new photosensitizers used in photodynamic therapy, based on natural extracts and investigated through biological, chemical and cytotoxicity tests. The purpose of these studies was to obtain photosensitizers, using oxygen-enriched waters and essential oils, to enhance the biological properties of the photodynamic antimicrobial therapy used in the treatment of experimental induced periodontitis.

Experimental

Materials

Gels based on natural compounds used as photosensitizers in PDT were used in the experimental study. Natural revealers contain nanocapsules, which include an organic phase based on essential oil of oregano (Young Living, 9727 AJ Groningen, Netherlands) and curcumin extract and arnica oil, with the active principle wrapped in a fine film of polycaprolactone to ensure controlled release of the active substance through the diffusion phenomenon. Three different types of water were studied: distilled water, Kaqun water and oxygen-enriched water. Distilled water was obtained by double distillation in a glass apparatus GFL-2102, (Schulze-Delitzsch-Strasse 4, 30938, Burgwedel, Germany); oxygen-enriched water used in the experiments was obtained by electrolysis. Electrolyzed water was obtained with a platinum electrode at 1.25 mA current intensity.

The gels were prepared from a mixture of gelatin: glycerol (Sigma-Aldrich Inc., St. Louis, USA) in a weight ratio of 1:1 and 60 ml Kaqun® water (Harghita, 535600, Romania), distilled water and oxygen-enriched water using the following procedure: gelatine and glycerol with 0.015% salicylic acid solution were added to the water. The gels formed were divided into equal parts, in which Oregano essential oil (Young Living, 9727 AJ Groningen, Netherlands) and curcumin extract were added.

UV-Vis and EPR analysis

UV-Vis analysis of the water was made in order to determine their maximum absorbance on Cary 50 (Varian, Inc., Foster City, CA, USA) and Lambda 25 (PerkinElmer Singapore) spectrophotometers. ESR measurements were scanned using the following parameters: centre field, 3360 G; sweep width, 60 G; power, 2 mW; receiver gain, 1×103; modulation amplitude, 1 G; time of conversion, 15 ms; time constant, 30.72 ms; sweep time 60s. 50 µL of water was transferred in EPR quartz capillary. For samples measurement, were used on a Bruker Elexsys E500 spectrometer operating in X band (~ 9.4 GHz) at room temperature, with 100 kHz modulation frequency.

Chromatographic analysis

The gas chromatography-mass spectrometry method (GC-MS) is specific for the analysis of the composition (volatiles compounds) of the essential oils in the studied gels and the high performance liquid chromatography with the ultraviolet detection method (HPLC-UV) is used to determine curcumin from the studied gels. Two type of gels were used for this study, arnica oil and curcumin gel (Gel-AO-CU) and oregano oil gel (Gel-OR).

GC-MS analysis of gels containing essential oils. By GC-MS analysis we have highlighted the incorporation in gel of the essential oils of arnica, curcumin (GEL-AO-CU) and oregano (GEL-OR) as antibacterial agents with applications in dentistry.

The initial procedure used the GC–MS analysis technique to identify the chemical composition of the essential oils tested.)

GS-MS sample processing: the gel (0.5 g) was dispersed in hexane (10 mL) for 2 hours, then ultrasonicated for 15 minutes and centrifuged at 4400 rpm for 15 minutes. The volatile fraction of hexane was filtered and then dried over sodium sulfate. The essential oil recovered from gel was injected into GC-MS.

GC-MS Method: Agilent GC-MS Gas Chromatograph – 7890A / 5975/2008) (Agilent Technologies, Inc. Europe, Waldbronn, Germany) was used for the analysis; GC-MS analyzes were performed in scan mode on a DB-5MS (30m x 0.25mm x 0.25µm) capillary column (Agilent 19091S-433M), high purity He carrier gas at a flow rate of 1 mL/min. Temperature program: initial temperature 40 °C with a ramp of 8 °C / min up to 220 °C, then with 20 °C up to 280 °C and maintained 5 min, injector temperature 250 °C, injection volume of 1 µL, 100: 1 slides, MS 70eV, mass range u.a.m.30-400. The NIST library was used to identify/confirm the components of the structure. In addition, a standard alkane C8-C20 (Alkane Standard Solution C8-C20, Sigma Aldrich) was used to calculate the linear retention index (RI) and to match the experimental values with those reported in the literature for similar chromatographic columns, under the same conditions.

HPLC-UV analysis of curcumin from AO-CU gel sample. The analyzes were carried out on a Jasco Chromatograph (Jasco International Co., LTD., Tokyo, Japan) equipped with an HPLC intelligent pump (PU-980, Jasco International Co., LTD., Tokyo, Japan), a ternary gradient unit (LG-980-02, Jasco International Co., LTD., Tokyo, Japan), an intelligent column thermostat (CO-2060 Plus, Jasco International Co., LTD., Tokyo, Japan), an intelligent UV/VIS detector (UV-975, Jasco International Co., LTD., Tokyo, Japan) and an injection valve equipped with a 20 µL sample loop (Rheodyne, Thermo Fischer Scientific, Waltham, MA, USA). The HPLC-UV analysis of curcumin in gel samples was performed on a LiChrosorb RP-C18 column (4.6 x 250 mm, 5 µm) with isocratic mobile phase, methanol: 0.1% formic acid solution (90:10, v/v). Column temperature was 30°C, detection wavelength at 425 nm and flow rate 1.2 ml/min. Methanol was purchased from Merck (Darmstadt, Germany). The water used to prepare the standard solution and gel extracts was Millipore water (18.2 MΩ•cm). Curcumin, (1E, 6E) -1,7-Bis (4-hydroxy-3-methoxyphenyl) -1,6-hepta-diene-3,5-dione, (purity> 98%) was purchased from Abcam ( BioZyme, Romania).

HPLC sample preparation: Curcumin containing gel also contains arnica oil. The extraction of curcumin from studied gel was performed in the mobile phase as an extraction solvent. Weighed about 0.5g of gel and 2 mL of extraction solvent were added. The mixture was ultrasonicated 15 minutes and then centrifuged 20 minutes at 4400 rpm. The supernatant was filtered and injected into HPLC.

Cytotoxiciy test

Cytotoxicity evaluation for experimental photosensitizer was performed on dysplastic oral keratinocyte cell cultures (DOK) with exposures of 1, 2, 6 and 24 hours.

Cell culture. Evaluation was performed on dysplastic oral keratinocytes (ECCAC 94122104, Sigma Aldrich, Heidelberg, Germany) used at their 31st – 32nd passage. DOK were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 5% fetal calf serum, 5μg/ml hydrocortisone, 50 μg/ml gentamicin and 5ng/ml amphotericin (Biochrom Ag, Berlin, Germany) in standard cell culture conditions (37°C, 60% humidity and 5%CO2); medium was changed twice a week.

Extract preparation. The culture medium conditioned with the photosensitizer was obtained according to the method described by Cavalcanti et al, 200517 and complying with the ISO 10993–12:2012 proceedings. Conditioning was performed at room temperature for 30 minutes using 0.2 g of photosensitizer/ml of culture medium and immediately thereafter, the conditioned medium was diluted to 0.001, 0.002, 0.005, 0.01 in medium and applied to cell cultures.

Cytotoxicity assay. The cells were grown at a density of 104/well in flat bottom plates with 96 microtiter wells (TPP, Switzerland) and hosted for 24 hours. Then the cells were exposed to the extract of each developer, prepared as described above, in dilutions of: 0.001, 0.002, 0.005, 0.01 for 24 hours. The cells were then washed and the viability was immediately measured by colorimetric measurement of a dye compound – formazan, generated by metabolically active, viable cells using CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (Promega Corporation, Madison, USA). (Tecan, Männedorf, Switzerland) at 540 nm. All experiments were performed in triplicate. Untreated cell cultures were used as controls. The results are presented as OD540.

Statistical analysis. The statistical analysis was done with two-way ANOVA and Student TTEST using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, California, USA). All data were expressed as the mean of OD540 triplicate measurements ± standard deviation (SD) and a p value less than 0.05 was considered statistically significant.

Animals

This study was performed on 25 adult male Wistar rats (14 months of age) that weighed 300–350 g. Animals were housed in the Establishment for breeding and use of laboratory animals of USAMV (Cluj-Napoca, Romania) on standard conditions, temperature 22-23șC, humidity 55% and 12 hours light/dark cycle. The rats were kept in plastic cages with free access to standard rodent granular food (Cantacuzino Institute, Bucharest, Romania) and water ad libitum. The rats were allowed to acclimate to the laboratory environment for a period of 3 weeks. All procedures that involved the use of laboratory animals followed the European guidelines and rules 337 as established by the EU Directive 2010/63/EU, and the Romanian law 43/2014 and have been done by an experienced practitioner. The study protocol was approved by the Research Ethics Committee of the University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Romania and they were authorised by the State Veterinary Authority (aut. No. 52/30.03.2017).

Experimental design

In order to investigate the natural photosensitizers an experimental periodontitis was induced.18,19 For all procedures, the rats were anesthetized with ketamine (60 mg/kg) and xylazine (6 mg/kg), which were administered via intramuscular injection, according to Flecknell et al.20 Access to the oral cavity was achieved with a retractor that provided constant opening of the mouth and that held away the cheeks and the tongue. Left-mandibular first molar from each rat in all surgical groups was selected to receive a cotton 4.0 ligature in a sub marginal position to induce experimental periodontitis. The ligatures were removed after 7 days (Figure 1). Postoperatively, all animals received analgesia with subcutaneous injections of tramadol (10 mg/bw).

Animals were randomly assigned to 5 groups. The groups (n=5) were assigned according to the following treatments applied locally: group 1 was left without surgical intervention representing the control group (M, n=5); group 2 (P, n=5) received surgical intervention and was left untreated, group 3 (GC, n=5) was treated with a curcumin photosensitizer and PDT, group 4 (GO, n=5) was treated with oregano photosensitizer and PDT and group 5 (L, n=5) was treated with PDT without photosensitizer.

Figure 1. The treatment regimen followed in the periodontal disease induced in rats.

Treatment was carried out with the use of low-intensity laser SiroLaser Blue (Sirona, 64625 Bensheim Deutschland) at a wavelength of 445 nm and 200mW, using a periodontal tip attached to the hand piece. In the GC and GO groups, the PS was injected into the pocket with the use of a direct blunt needle in the apical-coronal direction. After 60 seconds, the PS was rinsed with 1 ml of Kaqun® water (Harghita, Romania) using a graded syringe. Then in GC, GO and L groups, the laser beams were directed into the pockets for 40 seconds. Irradiation was maintained for 10 seconds in 4 equidistant sites (2 vestibular and 2 buccal sites). The procedures were repeated one week apart during 4 weeks (Figure 1).

All treatments were applied locally following the procedure derived from the standard human treatment and were carried out by a specialist (Figure 2).

Figure 2. (a,b) Different stages during PDT procedure; (c) Oregano and curcumin based gels used in the study.

The animals were kept under close monitoring on the entire length of the study, focusing on infection prevention and analgesic therapy. At the end of the experiment, blood samples were collected in order to determine hematologic, oxidative stress and biochemical parameters. Also body weight has been closely monitored. The animals were euthanatized after 4 weeks of study by prolonged narcosis followed by cervical dislocation. Then the left mandible and aorta were harvested from each rat for histological analysis.

Complete blood count and Biochemistry

Complete Blood count was determined using the automatic Abacus junior Vet hematology counter. The blood parameters determined were: white blood cell (WBC), lymphocyte (LYM), minimum inhibitory dilution (MID), granulocytes (GRA), red blood cell (RBC), hemoglobin (HGB), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (PLT) .

Histological and hypsometric analysis

For the histological examination, bone samples from the mandibular lesion site were harvested. For decalcification, after fixation, the mandibula of the animals were kept in a mix of 8% formic and 8% clorhidric acid for 24 hours and embedded in paraffin. The samples were fixed in 10% buffered neutral formalin, embedded in paraffin. The sections were made with a high precision microtome Leica RM 2125 RT, at 5μm thick and stained by Haematoxiline–Eosine method (HE). The slides were examined under a BX51 Olympus microscope and images taken with an Olympus UC 30 digital camera and processed using Olympus basic stream softweare. Sections were examined by independent observer blinded to the experimental protocol.

Statistical analysis of the data

For the statistical analyses of the findings obtained from the study. The Student t test, Origine Pro 8 SRO (origine Lab Corporation 2007, Northampton MA 01060, SUA) was performed. Analysis of variance was performed to determine whether there were significant differences between the different test conditions. The significance was evaluated at the level of p≤0.05.

Results and discussion

The UV-Vis analysis and ESR spectra

In Figure 3 is presented the UV-Vis spectra of three water samples and ESR spectra of gels prepared with three types of water (control with distilled water – gel D, oxygen-enriched water trough physical methods – gel F, Kaqun water – gel K). Kaqun water, displays one aditional signal in the UV-Vis spectra, compared to distilled water and molecular oxygen-enriched water. Nevertheless, in the ESR spectra are signals identical to each other, for all three gels tested.

(a)

(b)

Figure 3. UV-Vis and ESR spectra of distilled water, Kaqun water and oxygen-enriched water. Conditions for EPR measurement: center field, sweep width, 60 G; 3360 G; power, 2 mW; time of conversion, 15 ms; time constant, 30.72 ms; sweep time 60s; receiver gain, 1×103; modulation amplitude, 2 G.

Chromatographic results

GC-MS chromatogram (TIC, total ion chromatogram) shows the volatile compounds specific for arnica oil and curcumin contained in GEL-OA-CU (Figure 4) and for oregano oil contained in GEL-OR (Figure 5), while Table 1 presents the volatile compounds recovered from GEL-OA-CU and Table 2 presents the volatile compounds recovered from GEL-OR.

Figure 4. TIC chromatogram of GEL-OA-CU.

Figure 5. TIC chromatogram of GEL-OR.

Table 1. Volatile compounds recovered from GEL-OA-CU.

Table 2. Volatile compounds recovered from GEL-OR.

In Figure 6 is presented the HPLC-UV chromatogram of the gel sample containing curcumin. The amount of curcumin found in the gel sample was 20.19 µg /g of gel.

Figure 6. HPLC chromatogram of Gel-AO-CU sample.

Evaluation of cytotoxicity

The cytotoxicity profiles of the control, the oregano essential oil alone and the curcumin extract are presented at different dilutions. All experiments were performed in triplicate and the results are shown in Figure 7.

Figure 7. Cytotoxicity assay of the tested photosensitizer in different gel extract concentrations – 0.001, 0.002, 0.005, 0.01; M – control photosensitizer; O – photosensitizer with oregano essential oil; CU – curcumin based photosensitizer. Each bar represents mean ± standard deviation (n = 3).

The cytotoxicity test showed no significant alteration of the DOK mitochondrial function in the cells treated with the photosensitizers: control sample (M) and oregano oil (O) at any of the concentrations used, compared with untreated controls. The curcumin based photosensitizer (CU) induced a marginally significant decrease (p=0.0509), compared to untreated control, at the highest concentration used. Moreover, when O gel was compared to the control (M) gel, there was a significant interaction (p=0.0161, alpha=0.05) between the M and O gel, showed by two way ANOVA test, that indicates a significantly better result when the O gel was applied to the cells. The CU gel showed no significant differences when compared to the M gel. These results show that the tested gels did not induce any cytotoxic effect against DOK cells in our experimental environment.

Clinical evaluation

Body weight measurements were assessed in order to correlate the clinical status of the animal with the pathology induced. Statistical differences were interpreted (before and after treatment) by using Student t tests. Even though all groups experienced gain in body weight, groups treated with photosensitizers and PDT showed a more significant degree of weight gain (Table 3).

Table 3. Average and standard deviation (M ± SD) of body weight (g) in each group, before and after treatment.

(Mean ± SD) (t test, n = 5 (* p <0.05, ** p <0.005). In comparison to control group, (^p <0.05, ^^p <0.005) in comparison to P group.

Histological analysis

Following the histological analysis, the tooth and the periodontal ligament, respectively the dental alveolar bone (the dental support device) showed a normal appearance in M group (Figure 8).

Figure 8. Control group: (A) histopathological images from the dental crown and partially from the dental root area (hematoxylin-eosin); (B,C) a higher magnification of the area demarcated by the rectangle.

In the cervical and interdental space, moderate gingival retractions associated with chronic and superficial focal gingivitis were observed (group P). The superficial area of ​​the inflammatory outbreak is covered by an abundant serum-leukocyte crust mixed with tissue and fodder debris. Also, a moderate-segmental osteoclastic resorption of the alveolar bone was observed combined with suppurative (moderate) periodontitis extending from the previously described gingival defect. At the level of the sub gingival area, abundant granulation tissue that partially delimits the septic focal point and replaces the dental ligament focal point was observed (Figure 9).

Figure 9. Periodontisis group : (A, B) the superficial area of ​​the inflammatory outbreak covered by tissue and fodder debris; (C) detail of the dental support device, with moderate-segmental osteoclastic resorption of the alveolar bone and suppurated periodontitis; (hematoxylin-eosin).

In the L group (Figure 10), an important hyperplasia and hyperkeratosis (orthokeratotic) of the gingival epithelium with the formation of irregular, anatomic epithelial papillae, separated by a fibro-vascular inflammatory stroma (primarily neutrophils), moderate-segmental osteoclastic resorption of the alveolar bone and suppurated periodontitis was observed. The inflammatory process was represented by bands and degenerated neutrophils in mixture with rarely mononuclear cells and reactive fibroblasts.

Figure 10. Laser group: (A) hyperplasia and marked hyperkeratosis of the gingival epithelium (hematoxylin-eosin); (B) a higher magnification of the area demarcated by the rectangle; (C) numerous neutrophils with rare mononuclear and reactive fibroblasts.

In the GC group (Figure 11), an important hyperplasia and hyperchoratosis (orthokeratosis) in the gingival epithelium was noticed. Therefore, the formation of irregular, anastomising epithelial papillae, separated by an abundant (inflammatory granulation tissue) fibro-vascular inflammatory stroma (primarily neutrophils and macrophages) was observed. Furthermore, the superficial gingival area presented a focal ulcer (minimal) covered by a serum cellular crust mixed with cellular debris and fodder. In Figure 11C, a detailed aspect of inflammatory granulation tissue with abundant neutrophils (viable and degenerate) mixed with rarely mononuclear cells and reactive fibroblasts was highlighted.

Figure 11. GC group: (A) hyperplasia and marked hyperkeratosis of the gingival epithelium. The superficial gingival area presents a focal ulcer (minimal), covered by a serocellular crust mixed with cellular debris and forage (the area demarcated by the rectangle); (B) a higher magnification of the area demarcated by the rectangle; (C) detail of inflammatory granulation tissue, with the abundance of neutrophils (viable and degenerate) in combination with rare mononuclear and reactive fibroblasts.

In the GO group, marked gingival epithelial hyperplasia and hyperkeratosis (orthokeratosis) and partial replacement of the dental ligament with partially oriented fibro-vascular connective tissue was observed (Figure 12).

Figure 12. GO group: (A, B) hyperplasia and marked hyperkeratosis of the gingival epithelium, and partial replacement of the dental ligament with partially oriented fibrosvascularized connective tissue; (C) a higher magnification of the area demarcated by the rectangle.

Complete blood count

A significant increase in the total WBC count in the P group (11.16 ± 2.06) was observed compared to the M group (8.46 ± 1.13) (Fgure 13) (p <0.05), however, both recorded values ​​were within the physiological limits of the species (4 -12×109 / l). At the same time, a statistically significant decrease (p <0.05) in the number of average cells (monocytes) in both the P group (0.13 ± 0.11) and in the L group (0.12 ± 0.10) compared to the M group (0.50 ± 0.28) was observed (Figure 13). Nevertheless, the values ​​evidenced by monocytes are within the species physical limit (0-0.98×109 / l).

Figure 13. Means and standard deviations for all groups and variables without outliers: (a)WBC; (b) LYM; (c) MID; (d) GRA; (e) RBC; (f) HGB; (g) MVC; (h) MCH; (i) MCHC; (j) PLT; (Physiological values: WBC: 4-12×109/l, LYM: 2-14.1×109/l, MID: 0-0.98×109/l, GRA: 0.1-5.4×109/l, RBC: 9-15×1012/l, HGB 90-150 mg/dl, HCT: 24-45%, PLT: 250-750×109/l; * p<0,05; ** p<0,01; *** p<0,001).

Photodynamic therapy has demonstrated promising results in the treatment of several clinical pathologies through the photochemical reaction caused by the combination of a photosensitizer and a light source.21,22

The antimicrobial effect of the photodynamic therapy is based on an oxidative explosion due to the light therapy and is based on the deterioration of the cellular structures and of the biomolecules, thus making a nonspecific mechanism. aPDT requires the presence of three components: (I) a non-toxic dye, the so-called photosensitizer, (II) visible light of a corresponding wavelength and (III) molecular oxygen. The absorption of light by the PS leads to a transition to its triple state, through which there are two reaction mechanisms to allow the PS to regain its baseline state. In the type I mechanism, the charge is transferred to a substrate or to molecular oxygen generating reactive oxygen species like hydrogen peroxide and oxygen radicals such as superoxide ions or free hydroxyl radicals. In the type II mechanism, only energy – not charged – is transferred directly to molecular oxygen, from which simple reactive oxygen (O2) comes.23-25

Therefore, combining photosensitizers with light and molecular oxygen species derived from enhanced water could generate a mechanism of cellular death by provoking cytotoxity to bacterial etiological factors, which are implicated in periodontitis.

Using chromatographic analysis, our study confirmed the existence of compounds with biological, antioxidant and antimicrobial properties that can have synergy with small oxygenated components. These compounds have been identified, especially for oregano oil, and have been isolated: thymol, caryophyllene, caryophyllene oxide, α-Pinene, Limonen, γ-Terpinen, Linalool, Terpinen-4-olL, α-Terpineol (Table 2).

Turmeric constituents include the three curcuminoids: curcumin (diferuloylmethane, the primary constituent and the one responsible for its vibrant yellow color), demethoxycurcumin and bisdemethoxycurcumin, as well as volatile oils (tumerone, atlantone and zingiberone), sugars, proteins and resins.26

Curcumin has been shown to possess significant antimicrobial, antiinflammatory, antioxidant, anticarcinogenic, antimutagenic, anticoagulant and anti-infective effects and has also been shown to have significant wound healing properties. The recent literature on the wound healing properties of curcumin also provides evidence for its ability to enhance granulation tissue formation, collagen deposition, tissue remodeling and wound contraction favoring this way the healing process.27-31

Two of the major bioactive constituents (tumerone and atlantone) were identified in our study as well, possibly exhibiting the above mentioned properties. Combined with the natural compounds isolated from Arnica Montana (table 1), a medicinal plant widely used as an herbal remedy, containing terpenoids, sesquiterpene lactones, flavonoids and tannins with anti-inflamatory, antifungal, antimicrobial and antibiotic properties32 could provide a potential mechanism of action highlighting the benefits of our implemented therapy.

Essential oils of oregano (Origanum vulgare) are widely recognized for their antimicrobial activity, as well as their antiviral and antifungal properties. It is one of the most used aromatic plant, whose essential oils are particularly rich in mono- and sesquiterpenes.33

Nevertheless, recent ivestigations have demonstrated that these compounds are also potent antioxidant and antiinflammatory agents. Carvacrol (CV), the main compound found in essential oils of oregano, is a phenolic monoterpenoid, which possesses a wide range of bioactive properties34 and has been isolated within our experimental compound. These properties of oregano essential oils are of potential interest to the food, cosmetic and pharmaceutical industries.35

The procedures described in this article are consistent and efficient in determining and quantifying induced cytotoxicity in vitro for two experimental photosensitizers based on oregano essential oil and curcumin extract. To achieve optimal cytotoxicity in response to PDT, it is crucial to choose the optimal parameters of laser light and incubation times.

Belinello-Souza et al. found that the animals treated by aPDT showed bone gain of approximately 30% compared to the scaling and root planing (SRP) group following 7 days after periodontal intervention.36

Animal models provide valuable information in investigating the pathogenesis of periodontal disease as well as treatment methods. In addition to the immunological and microbiological characteristics of rats, the histological features of parodontal collagen fibrils, alveolar bone, cellular cement, connective tissue, junctional epithelium, oral gingival epithelium and sulcular epithelium are similar to human periodontal tissues37,38 which is why this experimental protocol was chosen.

According to IACUC Standards, body weight is considered an indicator of health and well-being of laboratory animals. This demonstrates that dental therapy with our materials tested in combination with photodynamic therapy does not negatively affect food intake, prehension and mastication. The animals in the treated groups (GO, GC, L) have gained approximately 80 g of weight during the 4 week period, whereas those untreated gained only 40 g. Thus, indicating a variable period of caloric restriction most likely induced by the pain caused by the paradontotic disease, as well as dental and gingival mobility and bleeding.39 According to Toth et al. weight gain is a positive indicator for animal welfare.40

Complete blood count may reveal general pathological conditions of the body as evidenced by anemia, systemic infections or blood neoplasms. This is also done for monitoring a disease or a medical treatment.41

At the end of the experiment, all values recorded in haematological analysis are within the physiological range of species.42 This indicates the lack of an inflammatory or infectious chronic systemic process. After 4 weeks, an inflammatory reaction due to periodontisis or local treatment cannot be observed. The lack of adverse systemic haematological reactions is correlated with the clinical symptoms of the rats and they have a good maintenance status. Different studies have investigated inflammatory conditions after application of PDT in the gingival tissues of Wistar rats with ligature-induced periodontal disease. Carvalho et al. concluded that PDT may be useful in the treatment of periodontal disease, because of immunomodulatory effects that decrease the inflammatory response and consequently, bone resorption.43

Histopathological analysis in this study demonstrated a greater magnitude of inflammatory response and severe destruction of periodontal ligaments in rats who did not receive treatment after ligation removal. It became obvious that ligation was effective in developing experimental periodontal disease. Our experimental protocol is reminicent with Graves et al., which observed that the ligation favors bacterial plaque accumulation, epithelial ulceration and periodontal tissue invasion by bacteria.19,44

In the periodontitis group was observed bone loss due to the osteoclastic activity conferred by the local infections and inflammatory processes. The same bone resorption mechanism but on a smaller scale was observed in the laser treated group.

Remarkably, no bone resorption and minimal inflammatory local reactions were observed in the two experimental photosensitizers groups. However, it is worth mentioning that oregano based treatment (protocol) has the most beneficial impact based on its capacity to provoke regeneration and total absence of inflammation.

Conclusions

This study demonstrates that PDT was an effective adjuvant treatment of induced periodontitis in groups treated with natural photosensitizers based on oregano essential oil and curcumin extract. Histopathological analysis suggested the anti-inflammatory effect of PDT in the presence of natural PS tested in this study.

Following the UV-Vis analysis, no significant differences were observed between the signals emitted by the 3 types of water used.

Following the GS-MS analysis on the investigated gels, the specific compounds of the anti-microbial and anti-oxidant effect were identified.

The encouraging results of this preliminary study suggest that further investigations of this approach to antimicrobial therapy are worth undertaking.

Conflicts of interest

The authors declare that they have no conflict of interest.

Acknowledgements

This work was supported by Romanian Project PNIII no 142-PED/2017.

Author Contributions: Conceptualization, M.M. and M.E.B; Investigation, C.S, A.B., C.R., I.B. and A.P.; Methodology, R.C. and A.N.; Writing – original draft, M.L.D.(R.); Writing – review & editing, S.S.

Ethical approval: All procedures that involved the use of laboratory animals followed the European guidelines and rules 337 as established by the EU Directive 2010/63/EU, and the Romanian law 43/2014 and have been done by an experienced practitioner. The study protocol was approved by the Research Ethics Committee of the University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, Romania and they were authorised by the State Veterinary Authority (aut. No. 52/30.03.2017). This article does not contain any studies with human participants performed by any of the authors.

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