Background
The general treatment of burns victims is not only delicate, but it leads to long periods of time, which increases the final cost of it1–3.
The local treatment of these patients has not changed since long time ago and it is worldwide standardized, including: serial surgical debridement’s with daily dressing changes which are painful for the patient and the use of topical agents over the wound such as Silver sulphadiazine. Although in the least case, the Eschar must be removed to allow the silver compound to penetrate the tissue. Biological covers are available but are expensive.
Super oxidized water use in wound treatment has been reported in several studies1–4. The electrolysis is a process through which an electrical component is passed through water, causing an electrolyzed solution, separating the positive and negative poles, resulting in free radicals’ production5,6.
The superoxidized water started to be an in vitro antimicrobial agent, without causing irritation, toxicity, or sensitivity in-vivo7.
It is now widely used for sterilization of surgical material, and as a preventive antiseptic agent for wounds in burned patients. Furthermore, some studies have shown that it enhances local tissue vascularization.
But to date, there are no studies that show the effect of epithelialization after a deep second-degree burn.
It would be ideal for a topical agent to have antiseptic properties, epithelialization effect over the wound, and a low cost to obtain an earlier recovery.
General objective: To know and describe the clinical and histological changes on the second-degree burns in rats treated with super oxidized water compared with the application of silver sulphadiazine, as conventional treatment in burnt patients.
Specific goals
- To describe the clinical characteristics, time of evolution and the histological features of partial thickness burns without topical treatment
- To describe the clinical characteristics, time of evolution and the histological features of partial thickness burns treated with silver sulphadiazine, as a conventional topical treatment
- To describe the histological and clinical characteristics and the healing time of partial thickness burns treated with local enforcement of super oxidized water
- To know and compare the clinical and histological differences of partial thickness burns, treated: (a) without topical treatment, (b) with the use of super oxidized water, and (c) with the use of silver sulphadiazine.
Hypotesis
Alternative hypothesis (Ha): The super oxidized water compared to the Silver Sulphadiazine, promotes local neovascularization, avoids overinfection, and allows a faster and better epithelialization of deep second-degree burns.
Null hypothesis (H0): There is no difference between the epithelialization on the deep second-degree burns treated either with silver Sulphadiazine or with super oxidized water.
If the super oxidized water can promote neovascularization and is a good antiseptic agent, then it may be useful in the treatment of the deep second-degree burns, by improving the epithelialization.
Level of significance
For all probability value equal to or < 0.05, ha has been accepted and ho has been rejected. p = 0.05.
Materials and methods
Twenty-seven Wistar male rats of 200 gr weight, nine cages with a 3 compartment division each, two 65 gr y 22 mm diameter bronze cylinders (weights), gas stove, water thermometer (-4ºC to 200ºC), stopwatch, digital camera, special rat shirts, and biopsy Punches (2 mm and 3 mm).
For the histological evaluation: We used glass bottles for the placement of biopsies, slides, microscope, digital photos, hematoxylin and eosin staining, staining fixer, boxes for samples placement, wax, and specific histological sample preparation.
Medications: Silver sulphadiazine, super oxidized water, Pentobarbital, Ketamine, Buprenorphine, Diclofenac, Ranitidine, and xylocaine.
Technique and data processing
Photographic evaluation of the wounds’ evolution will be done on every group, making a numerical assessment of the diameter of the wounds to apply the T Student-Welch test to obtain the level of statistical significance to accept or reject the alternative hypothesis. A qualitative analysis and comparison of histopathological characteristics of each sample of the three different groups of rats will be done.
Schedule of activities
DAY 1
The rats were divided into groups and were kept separate and marked for each group.
Group A, marked in green, were the rats for the control group, Group B marked in pink, were the rats treated with silver sulphadiazine (as conventional treatment of burns), and Group C in blue for rats treated with super oxidized water.
Each of these groups was further subdivided into three groups for study at 7, 14, and 21 days, making a total of nine groups of rats.
Water was heated in the oven gas, maintaining a constant temperature of 90° centigrade, and bronze weights were placed on it for 20 min, before applying them to the rats dorsum.
Each rat was taken and applied a dose of 1 mg/kg xylocaine for anesthetic induction, and 40 mg/kg of Pentobarbital for anesthesia.
Once the rat was sedated, shaving and preparation of the interscapular area on the dorsum was done.
The weight (65 g) was placed in the previous prepared area for a period of 6 s.
After weight removal, immediate silver sulphadiazine was applied to rats on Group B and super oxidized water to Group C, while those on Group A were treated with saline solution. The rats were maintained with IM analgesic every 12 h along with gastric protector.
Digital photographs were taken for assessment of the post-burn wound. Each group of rats received their treatment every 8 h daily.
DAY 7, 14, AND 21
Clinical follow-up and wound measures were made, along with digital photos. With the same protocol for analgesia and sedation, a 3 mm sample was taken from the wound and placed in a bottle with formalin. They were taken immediately for histopathologic study. The animals of the biopsy group were slaughtered.
The histopathological analysis was performed by the same pathologist, who never knew the treatment for each group (Blind study).
Results
Of the total of 27 rats, nine rats died, with digestive tract bleeding secondary to stress ulcers.
18 rats survived, of whom, five rats belonged to Group A or control group; six belonged to Group B, treated with silver sulphadiazine and seven rats in Group C, that were treated with super oxidized water.
Diameter of the wounds was measured in all the groups every 7 days, finding in Group A after 7 days, the following results: A rat with a diameter of 22 mm × 21 mm. At the same control group at day 14, a rat was found with a diameter of 12 mm × 17 MM, and 15 mm × 15 mm in another rat. At 21-day follow-up, the diameter of the wound was 13 mm × 15 mm in one rat and 12 × 13 mm in another one (Fig. 1).
Figure 1. Group A wound and scar after burn. Photograph at day 21 of treatment.
In Group B, the wounds were found as follows: at the 7th day, we assessed a diameter of 20 mm × 21 mm in one rat and 20 mm × 22 mm in a second rat. At 14 days, we measured 16 mm × 14 mm diameter in one rat and 15 mm × 0.9 mm in another one, and 21 days after the lesion, one rat had 14 mm × 0.7 mm and the other one had 10 mm × 0.7 mm (Fig. 2).
Figure 2. Group B wound and scar after burn. Photograph at day 21 of treatment.
Group C diameters were measured and found that: on day 7, a rat had a 19mm x 20mm diameter and the other one had 19 mm × 17 mm, at 14 days we measured the following diameters: 7 mm × 14 mm, 10 mm × 7 mm and 1.0 mm × 1.1 mm, and at 21 days follow up we found: 5 mm × 8 mm, and 0.9 mm × 0.7 mm (Fig. 3).
Figure 3. Group C wound and scar after burn. Photograph at day 21 of treatment.
Clinical assessment of the wounds was made and observed that Group B and C had a better clinical appearance than Group A, where the wounds had a larger Eschar and without hair growth above it. Group C wounds reduced their size in less time and with the earliest hair growth.
All the rats had partial thickness depth burns, not involving deep dermis and some follicles (Fig. 4). There were no rats with full-thickness burns and none with superficial partial thickness wounds. In the control Group A, after 7 days, we did not observe epithelium, with hyalinization of the entire dermis, some fat necrosis areas, but with preservation of the deep hair follicles (Fig. 5). At 21-day follow-up, there was no epithelialization observed, but some immature follicles in the dermis.
Figure 4. Table comparing diameter of the wound after the burn and at 7, 14 and 21 days.
Figure 5. Control group histology after 7, 14, and 21 days.
There were fibroblasts and dense hyaline collagen production, as well as local inflammation. It was until day 21 that we assessed an initial epithelialization from hair follicles, with a single layer of cells in basal epidermis. In the dermis, we found hyaline collagen and local inflammation.
In Group B (treated with silver sulphadiazine), an epithelialization was observed at day 7. It had all dermis layers and partial queratinization in 50% of rats and complete queratinization in the remaining 50%.
Hyaline dense collagen and granulation tissue was also observed (Fig. 6).
Figure 6. Silver sulfadiazine group histology after 7, 14, and 21 days.
On the 14th day, a complete queratinization was observed in the 100% of the samples, with granulation tissue in deep hypodermis, and proliferation of hair follicles in 100%.
But on the 21st day, we observed that in 50% of the samples, an ulceration of the epithelium with acute inflammation was present along with abundant granulation tissue and fibrosis in superficial and deep dermis. In the other 50% of the samples, a band inflammation and abundant collagen hyaline dense was observed.
In Group C treated with super oxidized water, the following results were found: a 7 days, complete epithelialization and queratinization were present in the 100% of the samples. There was hair follicles proliferation in 100% of the rats and dense hyaline collagen formation (Fig. 7).
Figure 7. Superoxidized water group Histology after 7, 14, and 21 days.
At the 14th-day follow-up, we observed a remarkable hyaline collagen production, as well as a large proliferation of hair follicles.
We found focal microcalcifications in the dermis in 33.3% of the samples. On day 21, we assessed abundant fibroblasts proliferation with a very dense hyaline collagen in 50% of the samples.
After doing inferential statistics with the variable quality of the Mann–Whitney “U”, 0 = 0, 1 = +, 2 = ++, 3 = +++,4 = ++++, we found an increased production of collagen fibers, as well as a faster epithelialization on the group that was treated with super oxidized water, compared with the other two groups, with p < 0.05.
Patients with deep second-degree burns, remain in the hospital for long periods of time, with extremely annoying and expensive treatments7. In many cases, they require Eschar resections to place the various topical medicines, as some of them do not penetrate this barrier8. The silver sulphadiazine is expensive and can cause neutropenia, and silver nitrate as well, is an expensive medication, and has a low penetration through the Eschar. Both are standard treatment in many hospitals in Mexico9,10.
Other topical agents, like Mafenide, have a painful application, besides it is expensive too11,12. The super oxidized water in addition to being a good antiseptic agent, has been found to promote wound healing, and its implementation is not painful and has a lower cost13,14.
Discussion
Superoxidized water is a good alternative for disinfection of wounds. Other articles indicate that superoxidized water has been effective and safe for the treatment of diabetic ulcers, chronic wounds, and burns6,15 and is commonly used not only as an antiseptic solution, but for decreasing hospitalization rates, with significant improvement in the appearance of granulation tissue and epithelization16.
We found important changes on hyaline collagen proliferation, after the treatment of second-degree burns with super-oxidized water, so we propose studying now the possibility of resulting in hypertrofic or keloid scars, after the treatment of second-degree burns with superoxidized water.
Conclusions
The super oxidized water is easier to apply and less expensive than other topical agents as well as being an excellent antimicrobial agent.
Histologically, superoxidized water causes an earlier Eschar dropping, and a more complete and faster epithelialization and queratinization, as well as the further proliferation of hair follicles.
According to our findings of a remarkable hyaline collagen proliferation, we propose to conduct a new study to analyze the histological features of the newly produced collagen and also to demonstrate if the application of super oxidized water in burnt patients: (a) increases, (b) decreases, or (c) it is not related to the formation of hypertrophic scars or keloids in this group of patients in a long term period.
Funding
The authors declare that this work was carried out with the authors’ own resources.
Conflicts of interest
The authors declare that they have no conflicts of interest.
Ethical disclosures
Protection of human and animal subjects. The authors declare that the procedures followed were in accordance with the regulations of the relevant clinical research ethics committee and with those of the Code of Ethics of the World Medical Association (Declaration of Helsinki).
Confidentiality of data. The authors declare that no patient data appear in this article.
Right to privacy and informed consent. The authors declare that no patient data appear in this article.
Use of artificial intelligence for generating text. The authors declare that they have not used any type of generative artificial intelligence for the writing of this manuscript nor for the creation of images, graphics, tables, or their corresponding captions.
