Pelagia Research Library - iMedPub · The interrelationships between diabetes and periodontal disease provide an example of systemic disease predisposing to oral infection, and once

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Der Pharmacia Sinica, 2012, 3 (1):31-40

ISSN: 0976-8688 CODEN (USA): PSHIBD

31 Pelagia Research Library

Evaluation of the Effect of Ozonized Water on Periodontium of Diabetic Rats

B. M. Zaki1, Effat Abbas2, Nagwa Osman3, Atef Elshahat3, Abdelrazek Hussein4

and Shadia El Rafei5*

1Surgery and Oral Medicine Dept., Oral and Dental Medicine Research Division, National Research Center, Cairo 2Oral Pathology at the Basic Sciences Dept., Oral and Dental Medicine Research Division, National Research

Center, Cairo 3Oral Medicine and Periodontology Dept., Faculty of Oral and Dental Medicine, Cairo University

4Pathology Dept, Medical Research Division, NRC, Cairo 5Chemical Engineering Dept, NRC, Cairo

_____________________________________________________________________________________________

ABSTRACT Topical application of ozone has been used in management of gingivitis and periodontitis with no reliable data obtained regarding its direct effect on the periodontal tissues in diabetic rats. Blood glucose level measurements were done before and after ozone application. Cementum thickness measurements showed no significant difference in the diabetic groups before and after ozone application. The number of bone cells was also measured showing a significant increase in number as compared with the controls in some of the groups, followed by decrease in number of bone cells with prolonged ozone application. The same results were obtained after counting the number of fibroblasts. The histopathological examination of the samples revealed presence of some structural alterations with prolonged use of ozone. DNA content was measured using the image analysis system showing different degrees of abnormalities denoting the presence of pathological changes. Key words: Ozonated water, periodontitis tissues, Diabetic rates, histopathology, DNA abnormalities. _____________________________________________________________________________________________

INTRODUCTION

Diabetes mellitus (DM) is a complex metabolic disease characterized by hyperglycemia, diminished insulin production, impaired insulin action, or a combination of both resulting in the inability of glucose to be transported from the blood stream into the tissues. This in in turn results in high blood glucose levels and excretion of glucose in urine [1]. The interrelationships between diabetes and periodontal disease provide an example of systemic disease predisposing to oral infection, and once that infection is established, the oral infection exacerbates the progression of the systemic disease. Both diseases share a common pathogenesis involving the enhancement of the inflammatory process which is mainly caused by the chronic effects of hyperglycemia. It was stated that Diabetes is a risk factor for periodontal disease [2] and [3]. Periodontal disease has been reported as the sixth complication of diabetes, along with neuropathy, nephropathy, retinopathy, and micro and macrovascular diseases [2]. Grossi [4] presented a model in which severe periodontitis increases the severity of diabetes mellitus and complicates its metabolic control.DM is a pathological condition, resulting in severe metabolic imbalances and non-physiological changes in many tissues. Oxidative stress play an important role in the etiology [5]. There is increased production of the reactive oxygen species (ROS) in the pancreas as a result of persistent and chronic hyperglycemia depleting the activity of the antioxidant defense system

Shadia El Rafei et al Der Pharmacia Sinica, 2012, 3(1):31-40 ______________________________________________________________________________


and promoting free radical generation [6] and [7]. Ozone therapy is a modern non-invasive method of treatment; it is a powerful oxidizing agent capable of killing a wide variety of viruses and bacteria. It is also a powerful non-chemical disinfectant [8]. Ozone therapy can activate the antioxidant system, influencing the level of glycemia. It was reported that it improved the glycemic control and decreased the oxidative stress by activation of the antioxidant enzymes such as catalase and superoxide dismutase [9]. It has shown that uncontrolled or poorly controlled diabetic patients have a greater incidence of severe periodontal disease compared with those patients who are well controlled or have no diabetes mellitus. Poorly controlled blood glucose levels will increase the severity and extent of periodontal disease and the progression is more aggressive. The prevalence of diabetes in patients with periodontal diseases is 17% as opposed to 9% in individuals without diabetes (same information) [10]. Studies have provided evidence that control of periodontal infection has an impact on improvement of glycemic control evidenced by a decrease in demand for insulin and decreased glycated hemoglobin levels [2] and [11]. Several researchers investigated the effect of the use of ozonted water or olive oil in the management of diabetic patients reporting the effectiveness, safty and simplicity of using medical ozone treatment is an excellent disinfectant and healing stinulator more than using topical antibiotics [11] and [12]. The topical application of ozone has long been used in management of periodontitis but the literature regarding the direct effect of ozone on the oral tissues is still obscure [13]. This stimulated the idea of the present work in studying the effect of using local ozonized water on the periodontal tissues of diabetic rats in order to detect any pathological changes that may occur.

MATERIALS AND METHODS

One Hundred and twenty male adult albino rats weighing 100-150 gwere used inthis study. The animal wre kept in plastic cages with access to food and water and libitium in temperature-controlled room with a standard 12/12h light-dark illumination cycle. The cages were kept under hygienic conditions and away from any source of chemical contamination according to the animal house protocol approved by the Ethical ommittee of the National Research Center, Cairo, Egypt. Before the surgical procedures, all the animals were allowed to acclimatize to the laboratory environment for a period of 5 days. Experimental Design The rates were divided into subgroups: Subgroup I: (consisted of 30 rats): This group represented the control group for the streptozocin induced diabetic models. The rats of this group did not receive ozone and each ten rats were sacrificed after 1, 3, and 6 months to obtain the diabetic control groups at different periods. Subgroup II: (consisted of 30 rats): The rats of this group received local subgingival irrigation of the periodontal tissues once weekly for a period of either 1, 3 or 6 months after induction of diabetes. Each ten rats were sacrificed after 1, 3 and 6 months intervals after ozone application. Subgroup III: (consisted of 30 rats): The rats of this group received local subgingival irrigation of the periodontal tissues twice weekly for a period of either 1, 3 or 6 months after induction of diabetes. Each ten rats were sacrificed after 1, 3 and 6 months intervals after ozone application. Subgroup IV: (consisted of 30 rats): The rats of this group received local subgingival irrigation of the periodontal tissues three times weekly for a period of either 1, 3 or 6 months after induction of diabetes. Each ten rats were sacrified after 1, 3 and 6 months intervals after ozone application. Diabetes was induced by intraperitoneal injection of streptozocin to overnight fasted rats, streptozocin was used in a multiple low doses (40 mg/kg for 3 days) which resulted in slow immune-mediated form of type 1 diabetes. Streptozocin was freshly prepared immediately before injection [6] and [14]. Blood samples were collected from the retro-orbital plexus of veins under ether anesthiesia once after 72 hours to determine the blood glucose levels. Glucose levels measurement was done with a calibrated blood glucose meter to confirm the establishment of diabetes. The normal blood glucose level in rate is between 50-135 mg/dl. Blood glucose levels above 250 mg/dl or higher were considered hyperglycemic [15]. Seventy two hours after streptozotocin injection, glucose solution was administered to the diabetic aimal to prevent secondary hypoglycemia whichis fstsl. Experimental diabetes was induced before induction of periodontal disease because diabetes takes about 48-72 hours to occur [14]. Blood samples were collected from all subgroups before starting the experiment and recollected again before animal



scarification after 1, 3 and 6 months interval to estimate any changes in the blood sugar level. Periodontal pockets were irrigated with 2.7 ml of ozonized water for 11 seconds according to the equivalent calculated dose and time period in rats [16]. This amount of ozone (2.7 ml) was applied 1, 2 or 3 times per week for one, three or six month period, for 11 seconds per application with ozone concentration (36 µg /L). The desired ozone concentration in water was attained by adjusting the flow rate of gaseous ozone in the double distilled water for a specific time period using the semi-patch method to obtain the desired concentration [17]. Ten rats from each subgroup were sacrified using cervical dislocation method after using chloroform following the first, the third and the sixth month’s intervals after ozone application was completed. Formalin fixed followed by 10% EDTA (Ethylene Diamine Tetra Acetic acid) fixation for 6-8 weeks for decalcification and paraffin embedded tissue specimen were prepared. Paraffin sections of 4µm thickness were obtained by using a rotary microtome and mounted on clean slides in order to be stained with Feulgen stain for DNA analysis [18]. Other paraffin sections of 5µm thickness were obtained to be stained with routine hematoxyine and eosin stain for histopathological studies [19]. Histopathological evaluation was done 1, 3, 6 months intervals for the different subgroups in order to detect any pathological changes. The morphometeric measurements and the DNA analysis were performed at the Pathology Department, National Research Center using the Leica Qwin 500 Image Analyzer (LEICA Imaging Systems Ltd, Cambridge, England).DNA content analysis was performed on real- time image from the microscope visualized on the video monitor as a black-and-white image with optical magnification of 400x on the Feulgen stained slides. For DNA content analysis optical density differences were used for calculation of DNA content of the different samples. The morphometeric measurements were carried out with optical magnification of 200x on the routine hematoxyine and eosin slides. The thickness of cementum was measured for the different samples in different positions namely cervical, middle and apical third portions of the whole length of the root cementum and given in micrometers as a unit. The number of fibroblasts and bone cells was counted from a real-time image displayed on the monitor at three different positions namely cervical, middle and apical portion with optical magnification of 200x on the routine hematoxyine and eosin slides to detect any possible changes that might occur.

Fig (1) Experimental Set-up of the ozone System

Ozone preparation The desired ozone concentration in water was attained by adjusting the flow rate of gaseous ozone in the double distilled water for a specific time period using the semi-patch method to obtain the desired concentration [17]. Ozone is produced by Fisher ozonator by passing 20 L/h of oxygen at 5 Atm., through the generator Fig (1). Oxygen is supplied by compressed bottled gas. Electrical input is changed from 120 – 240 MA in order to vary ozone concentration output. Leakage of ozone from reactor is detected by potassium iodide trap (1). Ozone concentration in the gas stream is measured by iodine / thiosulphate, using Standard iodometric method.



Ten rats from each subgroup were sacrificed using cervical dislocation method after using chloroform following the first, the third and the sixth months intervals after ozone application was completed.

RESULTS

Group No. of applications Duration

Non-diabetic One per week Two per week Three per week

1 month 3 months 6 months

Diabetic One per week Two per week Three per week

1 month 3 months 6 months

Table (1) The key for this study

Table (2) Blood glucose measurements in the diabetic group

Before After Application Time Mean SD Mean SD

One 1 month 3 months 6 months

268.5 23.6 270.4 22.0 281.9 29.6 286.6 29.7 289.8 19.8 293.9 19.2

Two 1 month 3 months 6 months

285.6 23.5 290.6 24.5 290.8 25.2 295.4 24.2 297.4 20.5 298.5 21.6

Three 1 month 3 months 6 months

294.1 14.4 294.9 14.3 302.4 33.0 307.0 32.3 306.2 12.7 310.9 12.6

Analysis of Variance results: Effect on the blood glucose level measurements P-value Significance Time (before and after ozone application) 0.001 Yes Number of applications 0.156 No Period (1, 3 and 6 months intervals) 0.012 Yes Time*number of applications interaction 0.315 No Time*period interaction 0.027 Yes Number of applications*period interaction 0.995 No Time*number of applications*period interaction 0.212 No

� It should be noted that P-values ≤ 0.05 are considered significant. � SD= Standard deviation.

Table (3) The cementum thickness measurements in the diabetic group:

� It should be noted that P-values ≤ 0.05 are considered significant. � SD= Standard deviation.

Applications Time Cervical Middle Apical Mean SD Mean SD Mean SD

No O3 application 1 month 8.31 0.63 15.59 0.73 26.45 1.65 3 months 8.53 0.61 15.76 0.79 26.95 1.45 6 months 8.89 0.70 15.96 0.81 27.45 1.55

1 O3 application 1 month 8.37 0.86 15.63 0.77 26.50 1.43 3 months 8.66 0.65 15.81 0.72 26.99 1.17 6 months 8.97 0.65 16.05 0.72 27.55 1.23

2 O3 applications 1 month 8.44 0.78 15.70 0.80 26.65 1.27 3 months 8.73 0.76 15.86 0.65 27.08 1.24 6 months 9.03 0.77 16.20 0.71 27.62 1.54




The significant change detected was between the blood glucose values measured before and after ozone application for the different groups (P = 0.001). Also, there was a significant difference in 1, 3, and 6 months intervals of the experiment (P = 0.012). On looking to the interaction between the different time intervals and the measurements of blood glucose level taken before and after ozone application a significant difference was found with P = 0.027. Analysis of variance showed no statistical significant difference in the cementum thickness measurements in the different positions (cervical, middle &apical) with the different number of applications and along the different time periods.

Table (4)The number of bone cells in the diabetic group

� It should be noted that P-values ≤ 0.05 are considered significant. � SD= Standard deviation. Statistical analysis of variance showed a statistical significant difference in the number of bone cells in the three measured positions (cervical, middle and apical) with the number of applications for the different time periods (1, 3 & 6 months) P value 0.016. Also the interaction between the number of applications and the time period showed significant change in the number of bone cells as compared with the different control groups (P=0.005). When ozone was used three times per week for three or six months there is a decrease in the number of bone cells starts to occur.

Table (5)The number of fibroblast cells in the diabetic group: � P value is considered significant ≤ 0.05. � SD= Standard deviation. Statistical analysis of variance showed a statistical significant difference in the number of fibroblast cells in the three measured positions (cervical, middle and apical) with the number of applications for the different time periods (1, 3 & 6 months). Also the interaction between the number of applications and the time periods showed significant change in the number of fibroblast cells as compared with the different control groups. P values obtained <0.05.


No O3 application 1 month 45.6 6.1 47.4 3.6 48.6 3.2 3 months 46.2 5.2 49.4 4.3 49.6 4.2 6 months 48.6 3.5 48.1 6.0 50.6 4.6





No O3 application 1 month 95.9 5.1 95.8 7.6 96.2 4.4 3months 95.1 4.3 94.2 5.4 95.2 6.1 6 months 91.4 5.7 91.6 6.3 92.2 3.5






When ozone was used three times per week for three or six months there is a decrease in the number of bone cells starts to occur.

Table (6) DNA content analysis in the diabetic group

Sample % 2c cells % 4c cells % >4c cells Diabetic control 77.95 0.537 ------ 1 O3 application for 1 month 75.325 6.494 1.948 2 O3 applications for 1 month 71.154 5.769 1.923 3 O3 applications for 1 month 40.385 3.205 0.641 1 O3 application for 3 months 56.051 5.732 3.185 2 O3 applications for 3 months 40.127 12.102 3.185 3 O3 applications for 3 months 29.375 8.75 4.375 1 O3 application for 6 months 31.25 7.954 9.091 2 O3 applications for 6 months 25.333 20 35.333 3 O3 applications for 6 months 0.658 19.079 73.684

The DNA content measurements showed that the normal diploid value of the control diabetic group was 77.95 and that the nearest normal value was obtained in the group which received ozone one time per week for one month. The rest of the groups showed different degrees of abnormalities with the worst results obtained when ozone was used three times per week for six months. Histopathological examination of the tissue samples revealed different pathological changes including presence of irregular epithelial ridges with increased hyperkeratosis and areas of degeneration in the connective tissues when ozone was applied once per week for three months. When ozone was received once per week for one month a slight increase in the number of fibroblasts & osteoblasts occurred. The group of diabetic rats that received two ozone applications per week for six months showed depressed epithelial ridge with areas of degeneration in the connective tissues. There is also degeneration in the periodontal ligament in the diabetic group that received one ozone application for six months. Concerning the histopathological examination of the gingiva of the diabetic group that received three ozone applications for six months incomplete epithelial ridges and areas of degeneration of lamina propria was detected. The only normal histopathological samples obtained were when ozone was applied once per week for one month and twice per week for one month. histopathological samples obtained were when ozone was applied once per week for one month and twice per week for one month.

Figure (1a): A photomicrograph of the gingiva of diabetic rat which received one ozone application for one month showing normal histological features of surface epithelium and numerous chronic inflammatory cells (H&E x200).



Figure (1b): A photomicrograph showing alveolar bone (B), periodontal ligament (P) and cementum (C) of diabetic rat which received one ozone application for one month showing mild increase in number of fibroblasts (H&E x200).

Figure (2a): A photomicrograph of the gingiva of diabetic rat which received one ozone application for three months showing numerous irregular epithelial ridges and areas of degeneration in the lamina propria (H&E x100).

Figure (2b): A photomicrograph showing areas of degeneration in periodontal ligament (P) of diabetic rat which received one ozone application for three months (H&E x200).

Figure (3a): A photomicrograph of the gingiva of diabetic rat which received two ozone applications for six months

showing hyperkeratosis and depressed epithelial ridges with areas of degeneration in the lamina propria (H&E x200).



Figure (3b): A photomicrograph showing degeneration and decreased number of fibroblasts in periodontal ligament (P) of diabetic rat which received two ozone applications for six months (H&E x200).

Figure (4a): A photomicrograph of the gingiva of diabetic rat which received three ozone applications for six months showing incomplete epithelial ridges and areas of degeneration of lamina propria (H&E x200).

Figure (4b): A photomicrograph showing degeneration of collagen fibers in the periodontal ligament (P) of diabetic rat which received three ozone applications for six months (H&E x200).

DISSCUSION

Several researchers have investigated the use of ozone in management of inflammatory periodontal diseases [20]. Diabetes mellitus is an extremely important disease from the periodontal standpoint provides an example of systemic disease predisposing to oral infection caused by accumulation of dental plaque, modifying the tissue response to the presence of this infection and upon establishment of this infection it exacerbates the progression of the systemic condition and complicates its metabolic control as reported by [2] and [21]. Hyperglycemia was reported to increase the production of oxides with decrease in the antioxidant defense system involving reduction of superoxide dismutase and catalase, resulting in decrease in the scavenger capacity of radicals in plasma. It was also proved that there is a close association between oxidative stress and insulin sensitivity; thus improvement of the antioxidant capacity in turn improved insulin resistance [9]. Several studies were conducted investigating the effect of ozone therapy on the hyperglycemic state in diabetic patients. Rectal insufflation or autohemotherapy using ozone



were done. The groups treated with ozone showed a significant increase in the enzyme activity participating in the antioxidant defense mechanism compared to the initial activity recorded in the control groups. The increase in the enzymes activity obtained with ozone treatment could be the result of stimulation of the genes expression encoding these enzymes. Meanwhile in the same study in another group of diabetic patients only topical ozonized olive oil was applied on leg ulcers with no reported effect on the glycemic state. The significant change which was found in the present work is related to the time period showing significant increase in blood glucose level in the diabetic groups with increased time period indicating the ongoing process of pancreas destruction as a result of streptozocin injection. Turk [22] examined the effect of local ozone therapy in treatment of stomatitis and gingivitis. The results showed considerable reduction in the number of bacteria, with positive influences on the disease, acceptance by the patients with no recorded side effects after performing a single application per week for one month using ozonized water. The same results were also proved by [23] which is in agreement with the present study. Stubinger [24] explained the therapeutic effect of ozone in management of periodontitis on the bases of its strong oxidation properties and its capability in directly destructing almost all pathogenic microorganisms. Some structural alterations of the surface epithelium were recorded in the present study such as hyperplasia and hyperkeratosis observed in diabetic groups which received two or three ozone applications for three months. These alterations can result from disturbance in the process of physiological turnover of the epithelium. These alterations are associated with change in the normal pattern of epithelial ridges showing loss of the slender pattern and sometimes incomplete epithelial ridges with increased inflammatory cells which can be explained on the basis of a study conducted by[25]. in which the authors documented that ozone can activate monocytes and lymphocytes together with increased production of various cytokines such as interleukin 1, 2, 6, 7, 8, interferon-γ, tumor necrosis factor -α , granulocyte and macrophage colony-stimulating factors. This was confirmed by a study performed by [26] showing the association between the increased vascularity and ozone application as a result of increased expression of the vascular endothelial growth factor (VEGF). This is also in accordance with [27] which reported that ozone application increased the growth factors which are important in the inflammatory process. The present work showed no statistical significant difference in cementum thickness measurements in the diabetic groups with the different numbers of ozone applications for the different periods in the three different positions namely cervical, middle and apical parts of the root which is coinciding with the results obtained by[28]. In addition the present study showed the periodontal ligament with increased collagen fibers condensation and increased fibroblastic activity in the diabetic groups which received one, two or three ozone applications for one and three months which is coinciding with the results of [29] which reported that ozone is capable of stimulating the fibroblastic activity initiating the repair process by stimulating deposition of collagen. Furthermore the decreased condensation of collagen fibers and decreased number of fibroblasts found in the diabetic groups which received two or three ozone applications for six months period can be explained on two different bases. The first one is related to the dose dependency and cumulative effect of ozone after a prolonged exposure that can kill the fibroblastic cells as reported by [13] and [30]. The second one is related to the persistent chronic hyperglycemia which might adversely affect the synthesis, maturation, and maintenance of collagen [31]. In the present study the number of bone cells was significantly increased in the groups which received 3 ozone applications for three and six months as compared to the control groups. This can be explained on the basis that ozone could increase the osteoblastic activity after its application.Any reported decrease of the number of bone cells can be related to dose dependency [30] and long period of exposure that is capable of killing the cells [13]. Increasing values of abnormal DNA was found related to the severity of dysplasia [32]. In the present work the results showed the normal DNA content (2c) measurements in the diabetic control group (77.95%). After one application of subgingival irrigation with ozonized water for one month the DNA content measurements changed to be 78.57 75.33% for the diabetic group proving that ozone therapy did not produce a significant harmful effect on the DNA content of the cells of theses groups. These results are in agreement with a study conducted by [33] and confirmed by [34] concluding that using ozone is effective and well endured by the patients and does not cause side effects. Abnormalities of the DNA content starts to appear with increased time period whereas the worst results obtained when ozone was applied three times per week for a period of six months. These results are in agreement with [35] in a study conducted on experimental rats reporting that ozone in high doses is capable of inducing cleavage of deoxyreibose of double stranded DNA [36].

CONCLUSION

In the present study the unusual complications found requires further investigations of the benefits and adverse side effects of the topical application of medical ozone on the oral tissues for prolonged time periods.



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Pelagia Research Library - iMedPub · The interrelationships between diabetes and periodontal disease provide an example of systemic disease predisposing to oral infection, and once