Evaluation of the effect of topical and systemic ozone application in periodontitis: an experimental study in rats*

Abstract Objective: The goal of the present study was to determine the effect of systemic and topical ozone application on alveolar bone loss (ABL) by evaluating the effect of Hypoxia-inducible factor −1 alpha (HIF-1-α) and receptor activator of NF-kB ligand (RANKL)-positive cells on histopathological and immunohistochemical changes in a rat periodontitis model. Methodology: Thirty male Wistar rats were divided into three groups: 1) Group C (control group); 2) Group SO (systemic ozone group) and 3) Group TO (topical ozone group). Experimental periodontitis was induced with a 3/0 silk suture placed at the mandibular left first molars of rats, and the suture was removed 14 days later. Ozone gas was injected intraperitoneally (0.7 mg/kg) in SO group. Topical ozone application protocol was performed using an ozone generator at 80% concentration (4th grade) 90- degree probe for the duration of 30 s. Both ozone applications were carried out for two weeks at intervals of two days. Histomorphometric and immunohistochemical analysis were performed. Results: ABL was significantly lower in Group SO compared to Group C (p: 0.0052). HIF-1α- positive cells were significantly lower in Group TO than in Group C (p: 0.0043). RANKL-positive cells were significantly lower in Group SO and in Group TO compared to the control group (p: 0.0033, p: 0.0075, respectively). Conclusion: Both ozone applications decreased RANKL-positive cell counts, TO application decreased HIF-1-α positive cells counts, and SO application was found to be more effective in reducing ABL compared to control group.


Introduction
Chronic periodontitis is an infectious disease of the periodontium and is characterized by the complex destruction between pathogenic microorganisms and the host response. 1  Hypoxia, a sign of chronic inflammation, is the insufficient oxygen supply to cells and tissues. 3 The tissue oxygen requirement is increased due to metabolic activation of inflamed tissue and infiltration of the inflammatory cells in chronic inflammation.
Furthermore, vasoconstriction and microthrombosis cause tissue perfusion to weaken it and a decrease in oxygen supplementation. This results in low-oxygen environment and the accumulation of hypoxiainducible factor alpha in the inflamed area. 4 Hypoxiainducible factor -1 alpha (HIF-1-α) is one of the HIFalfa subunits that are the main sensors of hypoxia. 5 HIF-1-α is the main regulatory protein that provides adaptation to hypoxic conditions.6 HIF-1-α has been shown to be activated by proinflammatory signals in periodontal cells. 7,8 It has been reported that hypoxia may play an important role in the progression of periodontal disease and the destruction of periodontal tissue. 9 Yu, et al. 10 (2012) stated that the lack of oxygen in periodontal tissue may accelerate the development of periodontitis. It is also known that hypoxia increases the release of receptor activator of NF-kB ligand (RANKL). 10 RANKL is a tumor necrosis factor ligand superfamily member. It is responsible for bone resorption by stimulating osteoclastic differentiation. 11 The binding of RANKL to the RANK receptor on the pre-osteoclast surface induces bone resorption by stimulating mature osteoclastic differentiation. 11 Ozone is a naturally occurring compound containing three oxygen atoms. Ozone has various effects, such as antimicrobial, anti-hypoxic, immune-modulator, biosynthetic and analgesic. It has been medically used in both gaseous and aqueous forms and can be dissolved in either water or oil. 12 Ozone allows oxygen to move inside the tissues more easily by increasing the amount of 2,3-diphosphoglycerate in erythrocytes. Ozone increases the release of nitric oxide, leading to vasodilatation and increase blood flow in tissue. An increase in free oxygen radicals leads to a change in antioxidant enzyme levels and immune system activity. It induces the production of interferon, interleukin, tumor necrotising factor and growth factors in leukocyte and endothelial cells. As a result, ozone therapy can be used for treatment in physio-pathological conditions in which the inflammatory process is intense and the immune system is triggered. 13 There are several studies evaluating the application of local ozone in chronic periodontitis and aggressive periodontitis. 14 The sample size was calculated to provide 80% power (1-β) with a 95% confidence interval (α = 0.05); ten animals per group were required. 18 Thirty male Wistar rats (Rattus norvegicus albinus; initial body weights ranging between 320-350 g) were dived into three groups as follows; 1) Group C (control group, n:10 rats); 2) Group SO (systemic ozone group, n:10 rats) and 3) Group TO (topical ozone group, n:10 rats). The rats were housed under standard laboratory conditions in accordance with the National Institute of Health throughout the study. 19 The rats were housed in a room with a 12-hour light/dark cycle, temperature of 22±2° C and humidity of 45±15 %. The animals were fed with selected solid diet and water ad libitum.

Examiner Calibration
All analyses were performed by the same examiner (UPH) who was blind to the treatments. The second measurement was performed seven days after the first measurement to estimate the intraexaminer error. 23 The student t-test was used to determine the examiner calibration in the analyses of the immunolabeling.
Calibration was accepted if the difference between two measurements were not statistically significant (p>0.05).

Statistical Analyses
Statistical analysis was performed using a software program (SPSS, version 20.0; IBM, Chicago, IL, USA). The Kolmogorov-Smirnov test was used for distributional adequacy. The significance of differences between groups was determined by Kruskal-Wallis tests, followed by Dunn multiple comparison post hoc test. The significance level was set at 5%.

Results
A total of 30 Winstar rats (ten rats in each group) were evaluated. ABL, HIF-1-α and RANKL-positive    Figure 4.
The highest ABL was in the control group. ABL was statistically and significantly lower in the SO group than in the control group (p: 0.0052). In the TO group ABL was lower than in the control group but this difference was not statistically significant. Ozone therapy improves inflamed tissue oxygenation and reduces total inflammatory process.
In that way, it positively affects some infectious disease outcomes. Ozone therapy can be applied via mixing a range of gases and liquids, and injecting these solutions into the body, including the vagina, rectum, muscle, beneath the skin or by autohemotherapy. 13,28 There are some studies evaluating the effect of ozone therapys, anti-hypoxic and anti-inflammatory feature, in periodontal tissues. 14,17,29 Although there are several studies indicating that topical ozone application with nonsurgical periodontal treatment has no additional benefit in clinical periodontal parameters, 14,[30][31][32] few studies have reported significant improvement in periodontal parameters healing compared to the control group. 38 In another study investigating the effect of SO therapy in diabetic nephropathy rats, HIF-1-α gene expression levels was reported to be decreased in the ozone therapy group. 39 In the present study, HIF-1-α values were lower in both ozone application groups than in the control group. There was no statistically significant difference between SO and TO groups. However, a statistically significant decrease was observed in the TO group compared to the control group. The reason HIF-1-α was lower in the TO group may be because local application reached a more effective concentration than systemic application in the inflammation area.
RANKL leads to osteoclastic differentiation by binding to RANK. 40 RANKL-mediated osteoclastogenesis plays a crucial role in inflammatory bone resorption. It has been reported that RANKL levels increase in the case of hypoxia in human periodontal ligament cells. 10 In this study, RANKL-positive cells were significantly lower in both ozone application groups compared to control group. There was no statistically significant