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Study contribution
Due to industrial development associated changes in environmental conditions, contact risk to chemical agents increases in animals. Therefore, chemical contact burn is high and common in animals. Ozone is a medical gas and used for clinical treatment in recent years, proper different concentration to improve oxygen supply in local tissue, inhibit inflammatory and stress factors. This study aimed to investigate the efficacy of ozone therapy in experimentally induced hydrofluoric skin burns. The results demonstrated that ozone therapy could be a viable option for treating acidic skin burn. Ozone repairs degenerated tissues by increasing fibroblast activation and promoting re-epithelization.
Introduction
Burns are still a frequent trauma worldwide. With the change in the environmental conditions due to industrial development, the risk of contact with chemical agents and, therefore, the risk of chemical burns increases both in humans and animals. Numerous experimental studies are still being conducted on burn treatments.1) Hydrofluoric acid (HF) is a chemical compound widely used in the industry as a metal and glass cleaner. HF skin burns are physiopathologically different from other acid burns. The hydrogen ions of HF have a corrosive effect on the skin, allowing the fluoride ions to enter the circulatory system through the skin, where they cause poisoning by binding the calcium and magnesium present in the serum. Also, the release of potassium from red blood cells is increased, producing liquefaction necrosis and bone decalcification in deep tissues. Due to the mentioned effects, the skin burn induced by HF causes severe pain and a systemic toxic effect.2
Ozone (O3) is widely used for medical purposes as antibacterial, antiviral, antifungal and for healing wounds.3 The systemic effects of O3 are the increase of blood flow and oxygen transfer in ischemic tissues, the release of growth factors by the activation of the immune system, the disinfection when topically applied and the stimulation of the neuroendocrine system. It is especially the positive effect on the transportation and release of oxygen to the tissues, the preferred feature for the treatment of different wound types.4,5
Despite the fact that HF is extensively used in the industry and that related skin burns are frequently encountered, in the literature reviews, no study has been found on the investigation of the therapeutic effects of O3 in HF skin burns. In this study, it was aimed to investigate the efficacy of O3 therapy in experimentally induced HF skin burns in rats.
Materials and methods
Ethical statement
The experimental protocol was approved by the Animal Ethical Committee of the University of Erciyes (approval number: 21/28).
Animal grouping
The study was conducted on a total of 20 healthy male Wistar Albino rats (weighing 250-300 g, aged 16 weeks), which were divided into two groups (experimental and control) of 10 rats housed individually and fed ad libitum.
Anaesthesia and experimental HF burn
After routine examination, for general anaesthesia, xylazine [5 mg(kg)-1] and ketamine [60 mg(kg)-1] were applied intraperitoneally. All animals were positioned dorso-ventrally, and a thoracolumbar skin region of 3 × 2 cm2 was prepared for HF burn. The modified experimental procedure of Cavallini and Casati was used to induce skin burn. 0.1 mL HF (38 % Merck, USA) was dropped on the skin and left for 2 min to produce the burn.6 After that time, the area was washed with saline solution for 3 min, after which Doxycycline [5 mg(kg)-1] (Peradoks 100 mg/mL, Alke, Tokat, Turkey) for 5 days and Calpol [2 mg (kg)-1] (Paracetamol, 24 mg/mL, GlaxoSmithKline, Turkey) for 2 days were administered orally as an analgesic and to avoid the risk of secondary infection, respectively.
Ozone administration
The ozone generator used was (Evozone Basic Plus GMBH Germany) adjusted to 80 µg/mL for O3 concentration and fed with pure oxygen at a flow rate of 500 mL/min. The liquid vaseline (20 mL) was ozonized for 10 min at 20 °C, and the maximum O3 concentration was obtained. The ozonized (20 µg O3/mL) for liquid vaseline was applied topically for seven days to the experiment group, while, parallelly, a saline solution was applied to the control group. The ozonization procedure for liquid vaseline was repeated before each application in order to apply the same amount of O3. All animals were sacrificed under general anaesthesia on the 8th day, the last day of application.
Histopathological evaluations
The skin samples obtained from the experimental and control groups were fixed in 10 % formaldehyde solution for 48 h. After the fixation process, the tissues were washed in tap water and passed through graded alcohol series. Then the tissues were cleared with xylol and embedded in paraffin, and the blocking process was performed. Five micrometres thickness sections were taken from paraffin blocks, then cleaned with xylene and dehydrated by passing through graded alcohol series. The sections were stained with Hematoxylin&Eosin (H&E) and Masson’s trichrome (MT) and evaluated according to the histopathological scoring parameters under the Olympus BX51 (Japan) light microscope. The criteria set for evaluation are as follows: epithelial proliferation, collagenization and infiltration (0: no detection, 1: poor detection, 2: intermediate detection, 3: prominent detection).
Statistical analysis
Statistical Package for Social Sciences (SPSS) 22.0 for Windows was used for statistical analysis. The results of the study were shown as mean ± standard deviation (X ± SD), and P values below 0.05 were considered statistically significant. Shapiro-Wilk test was used for the normality of values. T-test was used for comparisons between groups.
Results
After the application of HF, redness, erythema and white vesicles were seen macroscopically in all animals. The macroscopic findings showed 2nd degree burns in the skin. The clinical picture started to turn normal on the 3th day of applying O3 to the experimental group. In contrast, the clinical findings started to turn into local necrosis and crusted wounds in the same period in the control group. On the 8th day, seven animals in the experimental group and three animals in the control group were seen macroscopically completely recovered. In the histopathologic evaluation, inflammation, vascularization, epithelial regeneration and fibrosis were evaluated at 4× and 10× in sections stained with H&E and MT in the control group, irregular dermal wound areas, cell loss of the stratified squamous epithelium and low re-epithelialization were detected.
In addition, some keratinocyte migration on the stratified epithelial tissue in the burned area and scar tissue were distinguished. The presence of scabs in this area was a sign of serious damage and delayed healing in the control group. In some areas, a thin layer formed by mitotic cells was seen in the stratum basale, and an irregular dermis layer was also observed. The dense infiltration and enlarged blood vessels were seen beneath the connective tissue of the burned areas. In addition, foci of haemorrhage with collagen loss and angiogenesis formation were also detected in the general structure. In the necrotic area of the dermis under the burned region, no hair follicle or sebaceous gland was found (Figure 1).
Figure 1 General view of the wound area of the control group. 
: Stratified squamous epithelium, C; Collagen,→; Enlarged blood vessels, HR; Hair root, ^; lymphovascular structure, ⊳; Scab, *; Infiltration, F; Fibroblast proliferation, SG; Sebaceous gland, 
;Bleeding.
In the experimental group, an increased cell loss on stratified epithelial tissue and a significant increase in re-epithelialization were detected. Contrary to the control group, keratinocyte migration and scab were seldom seen in the experimental group. The shrinkage of the scab on the ozonized area showed that the healing in the burn area was faster than in the control group. It was determined that the cells in the stratum basale layer formed by mitosis stratum spinosum, stratum granulosum, stratum lucidum, and even stratum corneum layers. A decrease was observed in the areas of irregularity and ulceration and neutrophil infiltration in the dermis layer.
An increase was observed in the amount of collagen compared to the control group. There were also reduced foci of haemorrhage, fibrosis, and necrotic areas with fewer dilated blood vessels (Figure 2). The hair follicles and sebaceous glands were prominent under the burn area. The statistical comparison of epithelial proliferation, collagenization and infiltration between groups is shown in Table 1. The epithelial proliferation and collagenization were higher and statistically significant in the experimental group, while the infiltration was higher and statistically significant in the control group.
Figure 2 General view of the wound area of the experimental group. 
: Stratified squamous epithelium, C; Collagen, →; Enlarged blood vessels, HR; Hair root, ^; lymphovascular structure, ⊳; Scab, *; Infiltration, F; Fibroblast proliferation, SG; Sebaceous gland, 
; Bleeding, 
; Reepithelialization.
Table 1 Epithelial proliferation, collagenization and infiltration scores of the control and experimental groups
| Control | Experimental | P | |
|---|---|---|---|
| Epithelial proliferation | 0.30 ±0.48a | 1.80 ±1.13b | 0.0010 |
| Collagenization | 0.70 ±0.67a | 1.90 ±0.87b | 0.0030 |
| Infiltration | 2.20 ±0.78a | 0.50 ±0.52b | 0.0140 |
The same letters on the same line indicate the similarity between the groups, and different letters indicate the difference.
Discussion
It has been reported that HF burns are physiopathologically different from other acidic burns due to the rapid access of highly permeable fluoride ions to deep tissues. Different treatment methods have been suggested for HF burns, and the primary goals of these methods are to prevent damage to deep tissue by disrupting the effects of fluoride ions.2,7 Ozone is used medically for the treatment of many diseases such as anaerobic infections, abscesses, decubitus wounds, fistulas, skin wounds and gingivitis.8,9 It stimulates the release of Hydrogen Peroxide from the immune system cells and activates the immune system.
Furthermore, it causes serious damage to the cell wall of viral and bacterial pathogens by oxidizing the phospholipids of the pathogens along with the stimulation of growth factors.4,5,10 In the literature review, no study was found on the effectiveness of O3 in treating HF burn. For this reason, it was aimed to investigate the effectiveness of O3 in experimental HF skin burn treatment and therefore contribute to clinical practice and literature. During the healing period of a skin wound, hemopoietic, endothelial, neural cells and growth factors play important roles; nevertheless, it has been stated that keratinocytes and fibroblasts have the most important role in the healing process by migrating to the wound area. Additionality, fibroblasts have been reported to modulate immune cell functions by mediating fibrosis.11
Roblin et al. induced HF skin burns in rats and applied 2.5 % calcium gluconate gel topically for 17 days. At the end of that study, it was suggested that 2.5 % calcium gluconate should be used as first aid in HF burn wounds.12 In another study, Kodik et al. induced HF skin burns in rats and applied epithelial growth factor subcutaneously. They emphasized that epithelial growth factor could be used in cases of HF skin burn for first aid, but its obtention is difficult and expensive. Therefore, they suggested using magnesium sulfate and calcium gluconate instead of epithelial growth factor.13 For the treatment of HF skin burn, the researchers focused on the binding of magnesium and calcium by HF.12,13
Researchers agree that after correcting hypomagnesemia and hypocalcemia, the cardiovascular and circulatory systems return to normality, and then recovery occurs. In the present study, the medical effects and therapeutic properties of O3 were investigated in HF skin burns.12,13 Data show that O3 has potentially positive effects on the healing of HF skin burn. The epithelial proliferation and collagenization in the experimental group supported this condition. Pchepiorka et al. created surgical wounds on the buccal mucosa of rats and used O3 for the treatment. It was found that the collagen fibres accumulated widely in the ozone group, whereas infiltration was more intense in the control group.
This result was attributed to O3 effects such as angiogenesis, increased tissue oxygen, and increased expression of cytokines and growth factors.14 Guven et al. created caustic oesophagal burn and used O3 therapy in rats. They found that O3 therapy has a beneficial effect on the healing process in caustic esophagitis by improving antioxidant defence mechanisms and reducing tissue damage, and ameliorating histopathological injuries.8 Similar results were observed in the present study. Especially the decrease in neutrophil infiltration, the increase of collagen fibres and epithelial proliferation in the experimental group, along with the intensive infiltration in the control group, supported the O3 effects on healing.
Ozone therapy has been used to accelerate the healing process, reducing inflammatory response and oedema. There are different O3 therapy routes such as gas, ozonized oil and ozonized water that could be used for treatment.15 Kim et al. reported that the topical use of ozonized oil was effective in increasing wound healing in the epithelium of rats, promoting the proliferation and synthesis of collagen on injury sites, and increasing growth factors expression such as PDGF, TGF-β and VEGF.16 In this study, ozonized liquid vaseline was used topically for HF skin burn in the experimental group, and the recovery was found to be better and faster than in the control group. Therefore, this study showed that ozonized liquid vaseline could be used topically in HF skin burn and obtained similar results to related studies.15,16
The concentration, dose and application frequency of O3 are essential for the treatment. The standardization of the O3 dose is not clearly revealed in animals. In humans, a dose of 27-50 µg O3/mL O3 was reported for satisfactory results. It is reported that 60 µg O3/mL improves the effects without producing toxicity and accelerates the healing process.14 Concurrently, some researchers emphasized that the size and width of the lesion affect the dose of O3 needed for the treatment. Some studies have developed methodologies with greater lesions and more sessions of O3 application.16,17 In the present study, 20 µg O3/mL was used daily in the experimental group, and it was observed that 20 µg O3/mL was adequate for the treatment. However, even though the use of 20 µg O3 in the study was sufficient for the treatment, the size of the lesion created, the width, and the blood supply of the tissue could affect the treatment.
This condition may cause changes in the dose of O3. During the period of healing of a chemical skin burn, the degree of burn, the timing, and the management of the treatment affect the chances of success of the treatment. The risks of infection and systemic complications could be seen in this period. Especially, inflammatory processes in the epidermis, dermis and subcutaneous tissue can lead to more serious problems.18 In the present study, no infection or complication was detected. Although it is stated that O3 has an antimicrobial effect, it should not be ignored that no infection has occurred due to the use of antibiotic in the study. On the other hand, compared with previous studies, it was observed that O3 accelerated and shortened the healing process. This statement was supported by the detection of healing activity on a cellular basis.12-14
Conclusion
As a result of this study, it was observed that O3 plays an important role in the tissue repair process in chemical burns. The increase of fibroblast activation and re-epithelization in the experimental group demonstrated that O3 therapy could be suggested as first aid in HF skin burn. It was concluded that more ozone-related studies should be done to enlighten the subject.
