Effect of topical ozonetherapy on gingival wound healing in pigs: histological and immuno-histochemical analysis

Abstract In this study, the effects of ozonetherapy on secondary wound healing were evaluated histologically and immuno-histochemically. Material and Methods: 8 healthy pigs were used in this study. Six wounds with 10 mm in diameter were created through the punch technique on the palatinal gingiva of each pig. Ozone gas was applied on only 3 wounds (test group) and the remaining 3 were left to natural healing (control group). Biopsy samples were taken from one of the wounds in each group on the third day, from another wound of each group on the seventh day, and from another one on the tenth day. Routine histological analysis and immuno-histochemical staining were performed to investigate transforming growth factor-beta (TGF-β) and (VEGF) expressions. Results: No statistical difference was found between the test and control groups in terms of collagen fibers, epithelial formation and inflammation scores. A VEGF expression found in the test group was statistically higher than control group samples taken on the 3rd and 7th day. There was no statistical difference between the test and control groups in terms of TGF-β expression on any of the sampling days. Conclusion: The topical application of ozone gas could be effective in the early stages of wound healing by increasing the amount of VEGF expression. Clinical Relevance: Topical application of ozone gas may be effective in the early stages of oral wound healing.


Introduction
Oral wound healing is a dynamic and complex process of restoring cellular structures and tissue layers 1 . During this process, coordination occurs among epithelial cells, platelets, endothelial cells, fibroblasts and macrophages 2 .
Wound healing occurs in overlapping phases, which are inflammation, re-epithelialization, granulation tissue formation, matrix formation, and tissue remodeling 2 .
Re-epithelialization or epithelial healing is the most important and most complex of these processes.
It includes cellular movement, proliferation and differentiation that provide functional and structural tissue repair 3 .
Secondary healing following surgical procedures, such as free gingival autograft, laterally sliding flap, gingivectomy and gingivoplasty, is always slower than primary healing.
The oral cavity provides a unique environmental challenge for healing wounds produced during various periodontal surgical procedures. Trauma from mastication, relatively large commensal oral flora and elevated levels of dental plaque can impair the normal sequence of the healing process 4,5 . Therefore, there is concern regarding the delayed healing of oral cavity wounds 2 .
In previous studies, it was observed that antimicrobials such as hydrogen peroxide, chlorhexidine, sodium hypochlorite, and povidone iodine cause cytotoxic effects on epithelial cells and gingival fibroblast cells 6,7 .
Another alternative agent that has recently been used in dentistry because of its effect on wound healing is ozone.
Ozone (also known as triatomic oxygen and trioxygen) is an allotropic form of oxygen occurring naturally in the Earth's atmosphere. It is created in nature when ultraviolet rays cause oxygen atoms to temporarily recombine in groups of three 8 .
It is an unstable gas and it quickly gives up nascent oxygen molecules to form oxygen gas. Due to its property of releasing nascent oxygen, it has long been used in human medicine to kill bacteria and fungi, to inactivate viruses and to control hemorrhages 8 .
Moreover, under the influence of ozone, improved rheological properties, activated cellular metabolism, raised intracellular ATP concentrations and expressions of cytokines relevant to wound healing, especially transforming growth factor-β1 (TGF-β1), have been observed 1,9 . Medical grade ozone is produced commercially in ozone generators and these machines provide the topical administration of the ozone gas 8
Impressions were taken from the palatal region of each pig with silicone impression material (Zeta Plus ® , Zhermack, Italy) before creating the wound Three of the six wounds created on each pig were selected randomly and topical ozonetherapy was applied onto them through the ozone generator for ten days straight (test group). 60 μg/μl for 120-s ozone gas plasma (gingival healing stimulator mode of applicator) was applied, similar to the study of Akdeniz, et al. 11 (2018). No ozonetherapy was applied on to the other three wounds (control group).
During the ozone application, the glass probe (10 mm in diameter) of the ozone generator was inserted through the perforations on the impression and the ozone was applied only to the selected zone.
Enough space was created between perforation on the impression and probe for air circulation ( Figure 2).
The animals were given free access to water and food (ad libitum) but they were fed with soft food.  control groups were not included in the normal distribution, the Mann Whitney U test was used.
The Wilcoxon Sign Test was used for normal, nonscattering variables for testing the differences between TGF-β and VEGF values on the third, seventh, and tenth days in the test and control groups. The Spearman Correlation Analysis was used to investigate the relations among inflammation, TGF-β and VEGF values on days 3, 7 and 10 in the test and control groups.
Differences were considered significant as p<0.05.

Histological results
In the test and control groups, it was observed that the surface epithelium and the lamina propria were completely separated from the wound area on day 3.
The wound area had reached the submucosal adipose tissue ( Figure 3A-F).
The wound area was filled with extensive fibrin In the test and control groups, it was observed that the wound surface was covered with exudates on day 7. The wound area was filled with fibro vascular granulation tissue, and the inflammation had declined,  When the changes in the VEGF-staining intensity of the test group were evaluated on different days, there was no statistically significant difference between VEGF values taken on days 3, 7, and 10 (p>0.05).

Immuno-histochemical results
When VEGF staining intensity of the test and control groups was compared with days 3, 7, and 10, the ones from the samples of the 3 rd and 7 th day in the test group were significantly higher than the control samples (p<0.05). No statistically significant differences were found between the test and control groups for VEGF values on day 10 (p>0.05) ( Figure 6).

TGF-β
On day 3, TGF-β staining was observed in both groups in the wound area, which was filled with exudates. Positive TGF-β was not detected in the vascular structures surrounding the submucosal connective tissue, or in the surrounding inflammatory cells.
TGF-β staining was observed in the endothelial and inflammatory cells at the base of the exudates on days 7 and 10 ( Figure 7A-F). No statistically significant differences were observed (p>0.05) when TGF-β staining intensity within the test and control groups were compared on days 3, 7, and 10. Although TGF-β staining intensity was higher in the test group than in the control group on days 3, 7, and 10, this was not statistically significant ( Figure 8).

Discussion
Because of its antimicrobial properties and positive effects on cutaneous injuries, one of the chemotherapeutic agents thought to be effective on surgical wounds in the oral cavity is ozone. However, there were no histological or immuno-histochemical studies evaluating the topical effect of ozonetherapy on wound healing around the gingival region. Accordingly, our study evaluated the healing effect of ozonetherapy produced by an ozone generator when applied to a created wound in the palatinal region.
The study was carried out using pigs because they have anatomical and physiological skin characteristics that are similar to humans 9,14 .
Prestudy power analysis determined that 7 animals should be randomized in order to attain an 80% statistical power, and we used 8 adult, healthy pigs in this experiment. Ozone increases endogenous growth factors, and it was thought that these could be used to evaluate the efficiency of ozone on early wound healing 9,15 . Because of these reasons, this study examines VEGF and TGF-β.
Yildirim, et al. 16   and no treatment (control). As a result of the study, it was seen that the VEGF and TGF-β values increased in all groups on days 7 and 11. Similarly, in our study, VEGF values of day 10 were significantly higher than the values of day 3 and 7 (p<0.05), however, there was no statistically significant difference between VEGF values on days 3 and 7 (p>0.05) in the control group. In the test group, when the changes of the VEGF staining intensity of the different days were evaluated, it was seen that VEGF values increased within days, but the increase was not statistically significant (p>0.05). No statistically significant difference was shown between TGF-β levels on days 3 and 7 (p>0.05), and the 3 rd and 7 th day values were significantly higher than the ones from the 10 th day (p<0.05) in the control group. In the test group, the 3 rd day values were significantly higher than the 10 th day values (p<0.05). There was no statistically significant difference between TGF-β levels on days 7 and 10 (p> as compared with the oil group, but no statistical information was given. In our study, when VEGF staining intensities of the test and control groups were compared on days 3, 7 and 10, the VEGF staining densities of the test group samples of the 3 rd and 7 th day were significantly higher than the control ones (p<0.05). There was no statistically significant difference between VEGF values of the test and control groups on day 10 (p>0.05). When the TGF-β staining intensity of the test and control groups were compared on days 3, 7 and 10, it was observed that the staining intensity of the test group was higher, but this difference was not statistically significant (p>0.05).

Conclusion
Our study is the first research in literature that histologically and immuno-histochemically examines Funding Information