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Volume: 1 Issue: 4 December 2021

FULL TEXT

ARTICLE
Comparative Evaluation of Polymers Versus Traditional Approaches for Local Treatment of Burn Wounds in an Experimental Rat Model

ABSTRACT

OBJECTIVES: We aimed to morphologically analyze and compare the wound healing process of burns in an animal model using Levomekol ointment or a polymer coating. The polymer coating was made from carboxymethyl cellulose, which contained stabilized silver nanoparticles at various concentrations.
MATERIALS AND METHODS: For our analyses, we used 75 White rats, which were divided into 5 groups, to compare local treatment with Levomekol ointment versus polymer coating containing various concentrations of silver nanoparticles. Biopsy specimens from the central and peripheral sections of treated burn wounds were studied on days 7, 15, and 30 after injury/start of treatment.
RESULTS: In rat groups that had local treatment of burn wounds with Levomekol ointment, the period of wound epithelialization was 37.2 ± 0.7 days. In rat groups that had treatment with polymer coating with silver nanoparticles, the epithelialization period was 30.2 ± 0.6 days. The use of a polymer coating with silver nanoparticles in local treatment stimulated the onset of regenerative processes at an earlier date, which was expressed in the active formation of granulation tissue by day 15.
CONCLUSIONS: The traditional method of burn wound treatment, which in our study was the use of Levomekol ointment, demonstrated a more protracted course of the wound healing process versus treatment with polymer coating with silver nanoparticles in our study animals. As shown morphologically, the use of the polymer coating treatment made it possible to shorten the recovery process.


KEY WORDS: Epithelialization, Experimental study, Morphology, Thermal burn

INTRODUCTION
A burn injury is one of the most common types of peacetime injuries.1,2 Mortality from burns in general ranges from 2.3% to 3.6%. For these types of injuries, 85% to 90% of patients are of working age and children. Of patients who survive, 65% to 78% require long-term medical, social/labor, and psychological rehabilitation.3,4

One of the determining factors in successful treatment is the choice of local treatment for burns. The optimal choice of the method of local treatment and the selection of drugs at the same time are the main components, because this allows complications to be prevented during the wound healing process.5

In this study, we conducted a comparative morphological analysis of the wound healing process of burns in experimental animals treated with Levomekol ointment versus a polymer coating, which was made from carboxymethyl cellulose containing stabilized silver nanoparticles at various concentrations.6

MATERIALS AND METHODS
Our study included 75 male and female White rats (weighing 150-230 g), which were divided into 5 groups. Experimental studies were carried out in accordance with the Declaration of Helsinki of the World Medical Association “ International Recommendations for the Conduct of Biomedical Research Using Animals” (2000). At the time that the burn injury was inflicted, animals were in an immobilized state and under the influence of ether anesthesia. The hair was removed from the back of animals using depilatory cream. After the hairless area was dried, burn injury was inflicted by applying a glass beaker that had been placed in a water bath filled with boiling water. The diameter of the bottom of the beaker was 3.5 cm. Exposure time of the beaker was 11 to 12 seconds. As a result of the inflicted thermal burn, formation of second- and third-degree burn wounds was noted.

Rats were treated either with a polymer film containing silver nanoparticles in various concentrations or with Levomekol ointment, which were applied to the burn area. An aseptic bandage was then applied. On around days 8 to 15, animals underwent necrectomy. Treatment then continued with either Levomekol ointment or a polymer film with silver nanoparticles. Dressings were changed every other day.

For this study, biopsies were taken from the central (deep burn area) and peripheral (superficial burn area) areas of the wound surfaces. To track the development of the dynamics of the ongoing processes with the different treatments, wound samples were analyzed on day 7, day 15, and day 30. After wound areas were sampled, the polymer coating or the Levomekol ointment was reapplied. Animals were again under ether anesthesia during sampling of wound areas for biopsies.

Levomekol ointment treatment group
Fifteen rats were included in the group that was locally treated with Levomekol ointment. Levomekol ointment (produced by Nizhpharm joint-stock company) consists of the active substances of chloramphenicol (7.5 mg) and methyluracil (40 mg), with auxiliary substances of macrogol (1500-190.5 mg, 400-762 mg). The drug is a combination drug for topical use. It has anti-inflammatory (dehydrating) and antimicrobial effects and is active against gram-positive and gram-negative microorganisms (staphylococci, Pseudomonas aeruginosa, and Escherichia coli). In the presence of pus and necrotic masses, the antibacterial effect persists. Methyluracil, which normalizes nucleic acid metabolism, stimulates regeneration processes in wounds and stimulates growth and maturation of granulations and epithelization.

Polymer coating with silver nanoparticles
The remaining rats (n = 60 rats) were divided into 4 groups treated with a polymer coating with silver nanoparticles at various concentrations (0.00216%, 0.00324%, 0.00432%, and 0.00648%). The polymer coating was prepared at the Institute of Chemistry and Physics of Polymers of the Academy of Sciences of the Republic of Uzbekistan. Industrial samples of sodium carboxymethyl cellulose (Na-CMC) with degree of substitution (DS) of 0.65 to 0.85 and degree of polymerization (DP) of 200 to 600, obtained from cotton cellulose, were used for this agent after purification from accompanying inorganic and organic impurities. For the formation of silver nanoparticles in films based on CMC, aqueous solutions of AgNO3 of various concentrations were used.

For the formation of films, 2% to 4% aqueous solutions of purified samples of Na-CMC of various DS and DP were used, with the gel fraction removed by centrifugation in a laboratory centrifuge at 2500 revolutions/minute for 20 minutes. For this purpose, 0.1 to 0.001 M aqueous solution of AgNO3 salt and 0.1% to 0.5% glycerol as a plasticizer were added by stirring to the Na-CMC solutions freed from the gel fraction; stirring continued until a homogeneous Ag + CMC- hydrogel was obtained.

Photochemical reduction of silver ions in the Ag + CMC- structure to nanoparticles was carried out at 25 °C by irradiation with a DRSh-250 high-pressure mercury lamp. To obtain dispersions of silver nanoparticles, the hydrogel underwent ultrasonic dispersion.

The formation of films was carried out on a degreased glass plate; films were then dried at 35 °C to 400 ºC in air.

Silver nanoparticles, which were stabilized in the NA-CMC solution with DS of 0.85 and DP of 600, were synthesized. The structural, physicochemical, and mechanical properties and bactericidal activity of films obtained from Na-CMC solutions with silver nanoparticles have been studied.

Transmission electron microscopy, atomic force microscopy, and ultraviolet spectroscopy were used to determine the shape, amount, and size of silver nanoparticles present in films based on Na-CMC. We found that, with an increase in the concentration of silver nitrate in Na-CMC solutions, during the photoirradiation process, the size and shape of silver nanoparticles changed, which was also shown in the obtained coating materials (substances that we have obtained).

Histological examination
Biopsy samples were fixed in a 10% solution of neutral formalin (pH 7.2-7.4) and embedded in paraffin according to Loyda and colleagues.7 From the obtained blocks, serial sections were prepared, which were stained with hematoxylin and eosin. The finished histological preparations were placed under a ProgRes CT3 digital video camera mounted on an Axioskop 40 microscope (Zeiss) for serial shooting.

RESULTS
A slow regeneration process in patients with extensive and deep burns can lead to an evident inflammatory process and the subsequent formation of rough scars. To examine ways to avoid the development of severe complications, we analyzed the use of a polymer coating with silver nanoparticles as a local treatment option. Rats treated with a polymer coating with silver nanoparticles were compared with rats that received local treatment of burn wounds with Levomekol ointment. To determine which agent had regenerative capacities of the skin, both clinical and morphological results were compared in the rat groups.

Levomekol ointment treatment group
In the Levomekol ointment treatment group, in the early period after thermal burn injury, rats were assessed as having moderate conditions; that is, animals were lethargic and drowsy. We viewed a clear limitation of the zone of necrosis with moderate inflammation in some of the animals.

On day 7 after burn injury and start of treatment, animals remained in discomfort and were adynamic and showed a lack of interest in food. The wound displayed a dirty brown necrosis zone with indistinct borders. Microscopic examination in the affected area of the stratified squamous epithelium (SSE) was not detected due to necrosis. Under the epithelium, in the dermis, both in the papillary and in the reticular layer, coagulation necrosis, fiber homogenization, and extensive hemorrhage fields were noted. In the vessels at the border of the papillary and reticular layers, as well as in the hypodermis, plethora, erythrocyte stasis with sludge formation, and erythrocyte hemolysis were observed.

The appendages of the skin in some areas of the study samples were preserved; other areas were in a state of necrosis. In the subcutaneous adipose tissue, there was edema, dilatation, and plethora of blood vessels, focal hemorrhages, and accumulations of leukocyte-infiltrating foci. At the same time, myocytes in the muscle tissue were necrotic, and a large number of leukocytes had accumulated in the intermuscular space (Figure 1).

In the zone bordering with necrosis, the following changes were noted: epidermal exfoliation with the formation of “bubbles” and pronounced dystrophy of SSE cells. In addition, in some areas of the transition from necrosis to the border zone, only the basal and thorny layers remained intact. In the dermis, there was a pronounced edema, with small focal hemorrhages. In the vessels of the dermis, plethora was observed. A zone of edema was formed around the skin appendages that had preserved the structure. Body fat was shown to be preserved. In the adjacent muscle tissue, intercellular edema was determined, but the structure of myocytes remained stable.

On day 15 after burn injury and start of treatment, the animals behaved actively, showing interest in the surrounding relatives, and maintained a good appetite. Examination of the wound showed sluggish necrolysis and sluggish marginal epithelialization with small areas of granulation tissue formation. Microscopic examination for the epidermis over a larger area of the investigated tissue was not detected, and multiple areas of epidermal necrosis with leukocyte infiltration were noted. Edema persisted in the dermis, with necrosis of the papillary layer, homogenization of the fibers of the reticular layer, and foci of hemorrhage, which were located mainly under the epithelium. Vessels of the dermis and hypodermis were deserted; along the periphery, in some areas of tissue samples, an accumulation of lymph and plasma cells was observed. Proliferative activity was observed in the skin appendages located in the immediate vicinity of the SSE. The phenomena of edema in the subcutaneous fat and muscle tissue continued to persist, myocytes were in a state of dystrophy, and necrosis was observed in certain groups of muscle cells (Figure 2a).

In the zone bordering on necrosis, edema persisted, and only the thorny and basal layers were preserved in the SSE structure, with focal proliferation of cells observed in the basal epidermis (Figure 2b). The connective tissue fibers of the dermis were edematous, with signs of moderate swelling of the fibers. Expansion of the lumen of the vessels with their plethora was noted in the vessels of the dermis and hypodermis; skin appendages showed signs of proliferative activity. Edema and degenerative changes in adipose and muscle tissue were less pronounced than in the central sections.

On day 30 after burn injury and start of treatment, the animals remained active without showing any signs of discomfort. Wounds in most of the animals displayed clear boundaries; flaccid granulations were observed on the wound surface. It was impossible to determine the structure of the SSE by microscopy; necrotic detritus with massive leukocyte infiltration was detected on the surface of the tissue fragments under study in the central zone. In the subepithelial layer, in the area of the papillary layer of the dermis, a sluggish formation of granulation tissue took place. In these areas, there were large numbers of newly formed vessels and inflammatory infiltration, consisting of lymphocytes, plasma cells, and a small number of leukocytes (Figure 3a). The edema of the connective tissue fibers still persisted in dynamics in the reticular layer. We could not determine the skin appendages in the studied preparations as well as in adipose and muscle tissue.

Along the periphery of the wound, the structure of the epidermis was preserved, with pronounced irritation of the growth layer. In some sample areas, acanthotic strands were noted. Under the SSE, in the papillary layer, the formation of granulation tissue was noted, in which mixed inflammatory infiltration was present (Figure 3b). The structure of the connective tissue fibers of the mesh layer was preserved, and the tissue fibers were edematous. Skin appendages were not detected, and signs of edema were observed in adipose and muscle tissue.

Polymer coating with silver nanoparticle treatment groups
In groups of rats treated with a polymer coating with silver nanoparticles, in the early period after receiving thermal burns, the animals were mostly active, with good appetites. The wound surface showed necrotic tissue, without signs of a pronounced inflammatory process.

On day 7 after burn injury and start of treatment, the animals were active and showed interest in food and in relatives. Wounds were covered with dry coagulation scab with the beginning of its rejection at the periphery. Microscopic examination in the affected area showed edema of all layers of the examined tissues: dermis, adipose tissue, and muscle tissue. From the side of the epidermis, in different areas, either focal or total coagulation necrosis with extensive hemorrhage fields and massive leukocyte infiltration in the necrosis zone itself was noted. In the papillary dermis, changes such as edema, lymph-leukocyte infiltration, fiber homogenization, focal hemorrhages, and areas of coagulation necrosis were observed. Identical processes were present in the reticular layer. Vessels of the dermis and hypodermis were paretic dilated, with stasis of erythrocytes in their lumen. In the areas where the skin appendages were preserved, edema formed around them; at the same time, necrotic skin derivatives were also found. In the subcutaneous adipose tissue, edema was observed with a visible expansion of the boundaries between the dermis and the muscle layer. In the muscle tissue, edema was expanding, including expansion in the intermuscular space; in the zone of myocyte necrosis, neutrophilic leukocytes contracted and accumulated (Figure 4).

Along the periphery of the burn zone, the SSE structure was preserved; dystrophic changes and edema were noted in the epidermis. The dermis showed moderate edema of the fibers, with an expansion of the space between the fibers and homogenization of individual fibers. Microcirculation vessels with expansion of the lumen and stasis of erythrocytes in their lumen were shown. Edema zones had formed around the skin appendages and in the subcutaneous fat. Myocytes showed signs of dystrophic changes; in the muscle layer, due to edema, wide light areas were formed between the groups of fibers.

On day 15 after burn injury and start of treatment, animals remained active and showed no discomfort. Examination of the wound showed active epithelialization with the formation of granulation tissue like large islands. Microscopy in 80% of cases did not detect the epidermis due to necrosis and massive leukocyte infiltration; in the other 20% of cases, the epidermis was determined partially in the form of a narrow band, with only cells of the basal layer being visible. From the side of the dermis, the phenomena of edema were noted, and a large number of leukocytes had accumulated in small areas with coagulation necrosis. Most of the study area was occupied by granulation tissue with a large number of newly formed capillaries and an accumulation of lymph and plasma cells and macrophages (Figure 5a). Hair follicles and sweat glands were not detected due to necrosis. Edema was observed in the subcutaneous fat. In this case, the granulation tissue grew not only in the dermis but also in the form of small foci in the subcutaneous fat layer. A polymorphic pattern was observed in muscle tissue, from zones with myocyte necrosis, with presence of leukocytes and edema, to zones with dystrophic changes and focal growths of granulation tissue.

At the periphery of the affected areas in the epidermis, necrosis of the stratum corneum, cell dystrophy, and proliferation of the basal sections of the epidermis were observed. The fibers of the dermis retained edema, proliferation of cells of the basal layer of the SSE was noted, and the epithelium had an “irritated” appearance with the formation of acanthotic strands. The formation of islets of granulation tissue accompanied the formation of a large number of vessels of the capillary type, with an accumulation of lymph and plasma cells proceeded in its turn. In most samples, the appendages were not detected; however, when preserved, their structures corresponded to normal structures (Figure 5b). Changes in muscle tissue were mainly reduced to edema and dystrophy of myocytes.

On day 30 after burn injury and start of treatment, rats displayed adequate behavior, showing interest in food, contact with each other, and no expression of any aggression. On visual examination, burn wounds on the trunk of most rats had completely epithelialized. In some rats, a small residual granulation zone was preserved. In rats with complete wound healing, the structure of the epidermis was restored, as shown by microscopic examination. In animals with residual granulation, SSE was not detected; small foci of necrosis with leukocyte infiltration were noted. Under the epithelium, the overgrown connective tissue was fully formed, appearing like a wide layer. In some cases, when the granulation had not yet been completed, samples showed areas of granulation tissue together with large areas of growth of connective tissue. In the granulation tissue, a large number of vessels were determined and their lumen was neglected. In other cases, erythrocyte stasis was noted. Skin appendages were not detected in the preparations. In the subcutaneous fat, the proliferation of connective tissue was noted, which penetrated from the overlying layers. In the intermuscular space of the muscle layer, the proliferation of connective tissue, moderate edema, focal lymph, and plasmacytic infiltration were also noted. The structure of muscle fibers in most preparations was preserved, while slight edema and degenerative changes in the fibers were noted (Figure 6a).

Along the wound periphery, the SSE structure was preserved, and irritation of the basal layer and the formation of acanthotic outgrowths were noted (Figure 6b). Connective tissue with a large number of vessels grew under the SSE. In the dermis and subcutaneous fat, the proliferation of connective tissue occurred in the form of foci surrounding the normal dermis. No muscle tissue was detected.

Comparison of treatment groups
Table 1 shows the timing of complete epithelialization of burn wounds with different approaches to the local treatment of burn wounds.

In the Levomekol ointment treatment group, epithelialization occurred in 37.2 ± 0.7 days. In the polymer coating with silver nanoparticle group, epithelialization occurred in 30.2 ± 0.6 days. At the same time, relatively short periods of epithelialization were noted in groups with the lowest and highest content of silver nanoparticles; this observation requires further study.

On day 7 of treatment after burn injury, the morphological picture did not differ much in both groups, but significant differences in the wound-healing process were noted on days 15 and 30. The use of a polymer coating with silver nanoparticles as local treatment stimulated the onset of regenerative processes at an earlier date, which was expressed in the active formation of granulation tissue by day 15 and with its transition to connective tissue by day 30. At the same time, in the Levomekol ointment treatment group, the regeneration processes were significantly delayed due to sluggish inflammatory processes.

DISCUSSION AND CONCLUSIONS

Our study compared the results of local treatment of burn wounds with different approaches. The traditional method of treating burn wounds, which in our work was represented by the use of Levomekol ointment with a water-soluble base, demonstrated a more protracted course of wound healing versus the use of polymer coating with silver nanoparticles. Our morphological studies showed that the use of a polymer coating resulted in a shortened recovery process; this treatment allowed a more physiologically acceptable method for closing the wound surface, resulting in achievement of its main goal, which is early formation of granulation tissue and reduction of secondary inflammatory processes.

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Volume : 1
Issue : 4
Pages : 179 - 185


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From the 1Republican Research Center of Emergency Medicine and the 2Institute of Chemistry and Physics of Polymers of the Academy of Sciences of the Republic of Uzbekistan, Tashkent, Republic of Uzbekistan
Acknowledgements: The authors express their sincere gratitude for all personnel of the Republican Research Center of Emergency Medicine and Institute of Chemistry and Physics of Polymers of the Academy of Sciences of the Republic of Uzbekistan for their daily efforts and assistance. This work would not be possible without you. The authors have not received any funding or grants in support of the presented research or for the preparation of this work and have no declarations of potential conflicts of interest.
Corresponding author: Utkur R. Kamilov, Republican Research Center of Emergency Medicine, 2, Kichik Halqa Yoli Street, 100115, Tashkent, Republic of Uzbekistan
E-mail: vladimir-1986@internet.ru