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Effectiveness of four topical treatment methods in a rat model of superficial partial-thickness burn injury: the advantages of combining zinc-hyaluronan gel with silver foam dressing

Open AccessPublished:October 02, 2022DOI:https://doi.org/10.1016/j.injury.2022.09.062

      Highlights

      • A new rat model for superficial partial-thickness burns was developed.
      • Comparative testing of four different treatment protocols was performed.
      • Burn wound healing was evaluated at 5, 10, and 22 days post-injury.
      • Combination therapy consisting of zinc-hyaluronan gel with a silver foam dressing was the most advantageous.

      Abstract

      Background

      There are several options available for conservative treatment of partial-thickness burns, however, reliable, affordable, and easily obtainable animal testing models are hard to find for the comparison of the different treatment methods. We aimed at developing a preclinical testing model and at comparing four treatment methods for superficial partial-thickness burns.

      Methods

      Burn injury was induced in 90 adult male Wistar rats by placing the 130°C hot tip of a commercially obtainable soldering device for 30 s on the clipped skin of the interscapular region at a steady pressure. Skin histology was studied on days 5, 10, and 22 after the induction of the burn injury, on which days, respectively, the ratio of the not epithelialized wound (%), the extent of re-epithelialization (score), and the scar thickness (µm) were assessed. We compared 4 groups: silver-sulfadiazine cream, zinc-hyaluronan gel, silver foam dressing, and the combination of zinc-hyaluronan gel with a silver foam dressing.

      Results

      On day 5, the induction of superficial partial-thickness burn injury was confirmed histologically in the rats. The zinc-hyaluronan gel and the combination treatment resulted in a markedly smaller ratio of the non-epithelialized area (29 ± 10% and 28 ± 13%, respectively) than silver-sulfadiazine cream (69 ± 4%; p < 0.01). On day 10, the extent of re-epithelialization was the lowest (∼0.2) in the silver-sulfadiazine cream group, while the other 3 treatments performed significantly better. The combination treatment lead to the maximal score of 2 in all rats, which was higher than in the other 3 treatment groups. On day 22, the scar thickness was the smallest in the combination treatment group (560 ± 42 µm), which was significantly less than in the silver-sulfadiazine cream group (712 ± 38 µm; p < 0.05).

      Conclusions

      We designed and histologically confirmed a reproducible method for induction of superficial partial-thickness burns in rats for preclinical testing. In our model, the combination of zinc-hyaluronan gel with silver foam dressing was more effective than either of its components alone or than silver-sulfadiazine cream.

      Keywords

      Introduction

      A burn wound is an injury to the skin or other organic tissue primarily caused by heat or due to radiation, friction, radioactivity, electricity or contact with chemicals [
      • Jeschke M.G.
      • van Baar M.E.
      • Choudhry M.A.
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      • Logsetty S.
      Burn injury.
      ,]. The severity of the burn is influenced by several factors such as the mechanism of the injury, the contact time, the depth and extent of the burn, the age and general condition of the injured person, as well as, by regional and socioeconomic factors [
      • Jeschke M.G.
      • van Baar M.E.
      • Choudhry M.A.
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      • Gibran N.S.
      • Logsetty S.
      Burn injury.
      , ,
      Burn Incidence and Treatment in the.
      ,
      • Atiyeh B.
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      • Dahai H.
      • Duteille F.
      • Fowler A.
      • Enoch S.
      • et al.
      International Best Practice Guidelines: effective skin and wound management of noncomplex burns.
      ].
      Nowadays, annually 6 million patients seek medical treatment for burns worldwide. In the UK (with a population of more than 60 million), around 250,000 people suffer from burns each year and 300 people die because of the burn injury, which constitutes a serious burden on the healthcare system and economy [
      • Atiyeh B.
      • Barret J.
      • Dahai H.
      • Duteille F.
      • Fowler A.
      • Enoch S.
      • et al.
      International Best Practice Guidelines: effective skin and wound management of noncomplex burns.
      ]. In the United States (with a population of ∼314 million), 1.25 million people suffer from burns every year. Of those, 450,000 receive medical treatment and 5,500 die from burns [
      • Atiyeh B.
      • Barret J.
      • Dahai H.
      • Duteille F.
      • Fowler A.
      • Enoch S.
      • et al.
      International Best Practice Guidelines: effective skin and wound management of noncomplex burns.
      ]. A systematic review found that the mortality rate of burns ranges from 1.4% to 18% across Europe [
      • Brusselaers N.
      • Monstrey S.
      • Vogelaers D.
      • Hoste E.
      • Blot S.
      Severe burn injury in Europe: a systematic review of the incidence, etiology, morbidity, and mortality.
      ].
      Burn injuries are often classified based on the depth of the wound (i.e., superficial, partial-thickness, and full-thickness burns) and the size of the affected skin area, assessed as the percentage of total body surface area (TBSA) [
      • Jozsa G.
      • Vajda P.
      • Garami A.
      • Csenkey A.
      • Juhasz Z.
      Treatment of partial thickness hand burn injuries in children with combination of silver foam dressing and zinc-hyaluronic gel: case reports.
      ,
      • Bittner E.A.
      • Shank E.
      • Woodson L.
      • Martyn JA.
      Acute and perioperative care of the burn-injured patient.
      ,
      • Kessides M.
      • Skelsey M.
      Management of acute partial-thickness bums.
      ]. Superficial burns usually do not require hospital admission and special medical treatment, while surgical intervention is always needed for the most severe, full-thickness burns [
      ISBI Practice Guidelines Committee. ISBI practice guidelines for burn care.
      ]. The therapeutic options and recommendations are more complex in case of partial-thickness (also called as second-degree) burns. Superficial partial-thickness (II/A or II/1) burns affect the epithelium and penetrate the papillary layer of the skin. They are characterized by moist and red surfaces, fluid-filled blisters, and severe pain upon touching. Deep partial-thickness (II/B or II/2) burns affect the deeper, reticular layer of the skin. In such injuries, the skin is usually dry, white or dull red in color, blisters may also be present, and it is relatively less painful [
      • Jozsa G.
      • Vajda P.
      • Garami A.
      • Csenkey A.
      • Juhasz Z.
      Treatment of partial thickness hand burn injuries in children with combination of silver foam dressing and zinc-hyaluronic gel: case reports.
      ,
      • Jozsa G.
      • Toth E.
      • Juhasz Z.
      New dressing combination for the treatment of partial thickness burn injuries in children.
      ]. While deep burns occasionally need surgery for skin grafting, superficial partial-thickness burns generally do not require surgical intervention, however, several different topical treatment options are available, which include silver-sulfadiazine cream, silver foam dressing, and zinc-hyaluronan-containing gel [
      • Markiewicz-Gospodarek A.
      • Koziol M.
      • Tobiasz M.
      • Baj J.
      • Radzikowska-Buchner E.
      • Przekora A.
      Burn wound healing: clinical complications, medical care, treatment, and dressing types: the current state of knowledge for clinical practice.
      ,
      • Rowan M.P.
      • Cancio L.C.
      • Elster E.A.
      • Burmeister D.M.
      • Rose L.F.
      • Natesan S.
      • et al.
      Burn wound healing and treatment: review and advancements.
      ,
      • Wasiak J.
      • Cleland H.
      • Campbell F.
      • Spinks A.
      Dressings for superficial and partial thickness burns.
      ]. Currently, there is no gold standard topical treatment in case of partial-thickness burns. The treatment of choice is mainly based on individual experience and institutional recommendations [
      • Atiyeh B.
      • Barret J.
      • Dahai H.
      • Duteille F.
      • Fowler A.
      • Enoch S.
      • et al.
      International Best Practice Guidelines: effective skin and wound management of noncomplex burns.
      ,
      • Kessides M.
      • Skelsey M.
      Management of acute partial-thickness bums.
      ,
      • Alsbjörn B.
      • Gilbert P.
      • Hartmann B.
      • Kaźmierski M.
      • Monstrey S.
      • Palao R.
      • et al.
      Guidelines for the management of partial-thickness burns in a general hospital or community setting—recommendations of a European working party.
      ]. While the different treatment options were all shown to have certain benefits for the healing of the wound, a tightly-controlled comparison of the effects of several dressings at different time points of the wound healing has not been performed, which may hinder their evidence-based recommendation. Besides the beneficial effects on wound healing, when choosing the topical treatment the need for anesthesia during dressing changes should be also taken into account. Repeated anesthesia (e.g., during regular dressing changes of a burn treatment), especially in childhood, can be associated with the impairment of cognitive functions. Neonates and infants (less than 6 months of age) who were anesthetized multiple times developed impaired cognitive functions compared to their peers who were anesthetized two times or less [
      • Oba S.
      • Işıl C.T.
      • Turk H.
      • Karamursel S.
      • Aksu S.
      • Kaba M.
      • et al.
      Evaluation of neurotoxicity of multiple anesthesia in children using visual evoked potentials.
      ].
      In the present study, our goal was to develop an adequate testing model and to compare the effects of four common topical treatment methods on superficial partial-thickness burns at three different time points (viz., 5, 10, and 22 days post-injury) of the wound healing.

      Materials and methods

      Animals

      The experiments were performed in 90 adult male Wistar rats. The animals were housed in standard plastic cages kept in a room with ambient temperature maintained at 21–23°C and humidity at 30–40%. The room was on a 12 h light-dark cycle (lights on at 5:00 a.m.). Standard rodent chow and tap water were available ad libitum. At the time of the experiments, the rats weighed 298-466 g. All procedures were conducted under protocols approved by the Institutional Animal Use and Care Committee of the University of Pecs (registration no.: BA02/2000–15/2018, approved on 18 April 2018) and were in accordance with the directives of the National Ethical Council for Animal Research and those of the European Communities Council (86/609/EEC).

      Induction of superficial partial-thickness burn injury

      Rats were anesthetized with the intraperitoneal administration of a ketamine-xylazine cocktail [78 mg/kg (Calypsol; Gedeon Richter Plc., Budapest, Hungary) and 13 mg/kg (Sedaxylan; Eurovet Animal Health B.V., Bladel, The Netherlands), respectively]. The fur on the nape was clipped in a 3 × 3 cm area, and then the rat was placed in a ventral position on a surgery board.
      The burn injury was induced with a soldering device (model Industa HF-5100; Stannol Inc., Velbert, Germany) in the center of the clipped skin area. The handpiece of the device weighed 46 g and it had a wedge-shaped iron tip with a 4 × 4 mm flat surface on each side (model M-4,2-HF; Stannol Inc., Velbert, Germany). Based on our literature search, contact burn injuries were induced in experimental models by heating the tip of the device to 60-200°C and keeping it in direct contact with the skin for 2-60 seconds [
      • Guo H.F.
      • Ali R.M.
      • Hamid R.A.
      • Zaini A.A.
      • Khaza'ai H.
      A new model for studying deep partial-thickness burns in rats.
      ,
      • Gurfinkel R.
      • Singer A.J.
      • Cagnano E.
      • Rosenberg L.
      Development of a novel animal burn model using radiant heat in rats and swine.
      ,
      • Kaufman T.
      • Lusthaus S.N.
      • Sagher U.
      • Wexler M.R.
      Deep partial skin thickness burns: a reproducible animal model to study burn wound healing.
      ,
      • Tavares Pereira Ddos S.
      • Lima-Ribeiro M.H.
      • de Pontes-Filho N.T.
      • Carneiro-Leão A.M.
      • Correia M.T.
      Development of animal model for studying deep second-degree thermal burns.
      ,
      • Priya K.S.
      • Gnanamani A.
      • Radhakrishnan N.
      • Babu M.
      Healing potential of Datura alba on burn wounds in albino rats.
      ,
      • Sakamoto M.
      • Morimoto N.
      • Ogino S.
      • Jinno C.
      • Kawaguchi A.
      • Kawai K.
      • et al.
      Preparation of partial-thickness burn wounds in rodents using a new experimental burning device.
      ,
      • Singer A.
      • Berruti L.
      • Thode H.
      • McClain S.
      Standardized burn model using a multiparametric histologic analysis of burn depth.
      ,
      • Venter N.G.
      • Monte-Alto-Costa A.
      • Marques R.G.
      A new model for the standardization of experimental burn wounds.
      ,
      • Branski L.K.
      • Mittermayr R.
      • Herndon D.N.
      • Norbury W.B.
      • Masters O.E.
      • Hofmann M.
      • et al.
      A porcine model of full-thickness burn, excision and skin autografting.
      ,
      • Eldad A.
      • Simon G.A.
      • Kadar T.
      • Kushnir M.
      Immediate dressing of the burn wound–will it change its natural history?.
      ,
      • Kim J.Y.
      • Dunham D.M.
      • Supp D.M.
      • Sen C.K.
      • Powell H.M.
      Novel burn device for rapid, reproducible burn wound generation.
      ,
      • Lotter O.
      • Held M.
      • Schiefer J.
      • Werner O.
      • Medved F.
      • Schaller H.E.
      • et al.
      Utilization of laser Doppler flowmetry and tissue spectrophotometry for burn depth assessment using a miniature swine model.
      ]. In our experiments, we heated the device to 130°C and touched it to the skin of the rats for 30 seconds. To minimize the variability of the impact, the full (4 × 4 mm) flat side of the tip of the handpiece was steadily held by hand in direct contact with the skin without applying extra pushing or pulling force.

      Treatment groups

      The rats were assigned to one of four treatment groups: silver-sulfadiazine cream (Dermazin; LEK Pharmaceuticals, Ljubljana, Slovenia); silver foam dressing (Aquacel Ag; ConvaTec Ltd., Deeside, UK); zinc-hyaluronan gel (Curiosa; Gedeon Richter Plc., Budapest, Hungary); and the combination of zinc-hyaluronan gel with silver foam dressing. In all groups, the wound was covered with a cohesive conforming bandage (Peha-haft; Paul Hartmann AG, Heidenheim, Germany) and a perforated plastic sheet. The latter was needed to prevent the animal from scratching the wound and removing the bandage, while also maintaining the ventilation of the wound.

      Tissue sample collection and histology

      The sample collection was conducted 5, 10, and 22 days after the induction of the burn injury. On the day of the sample collection, the rat was anesthetized with intraperitoneal administration of ketamine and xylazine (for doses, see section 2.2). The bandage was removed from the nape, and then the wound was excised in the center of a 2 cm × 2 cm area (in toto). The excised tissue samples contained all layers of the skin and part of the underlying muscular layer. After the removal of the tissue sample, the rat was euthanized with an intraperitoneal injection of sodium thiopental [400 mg/kg (Tiobarbital; B. Braun Medical S.A., Barcelona, Spain)].
      The removed tissue samples were immediately placed in 10% buffered formalin. After 48 hours of fixation, the biopsy has been serially sectioned and submitted entirely for histopathological examination (5-8 slices). The tissue samples were dehydrated in a graded series of ethanol solutions, embedded in paraffin, and cut into approximately 3-µm sections. The hematoxylin and eosin staining was performed according to routine procedure by a Leica ST 4040 linear automatic stainer (Leica Microsystems GmbH, Wetzlar, Germany). Histological changes were evaluated under a light microscope (DM500; Leica Microsystems GmbH, Wetzlar, Germany). The sections were evaluated by an expert pathologist blinded to the treatment of the rat.
      Histological evaluations included the assessment of the degree of re-epithelialization and final wound contraction. On day 5, the ratio of the unhealed burned surface was calculated as the percentage of the not epithelialized distance compared to the total length of the wound (Fig. 1). On day 10, the re-epithelialization of the wound was evaluated by a 3-score system (0: no re-epithelialization; 1: partial re-epithelialization; and 2: complete re-epithelialization that is multiple epithelial layers over the entire length of the wound) (Fig. 2). On day 22, the scar thickness was measured as the distance between the basal cell layer of the epidermis and the lowest cell layer of the dermis (Fig. 3).
      Fig 1
      Fig. 1Representative photomicrograph of the excised skin tissue on day 5 post-burn injury. The extent of the re-epithelialization was assessed by measuring the distance from the edge of the wound to the furthest newly formed keratinocyte (arrow). For orientation purposes, the insert on top right shows the whole tissue section with a red box indicating the magnified area.
      Fig 2
      Fig. 2Representative photomicrograph of the partially re-epithelialized skin tissue on day 10 post-burn injury. Loose granulation tissue (indicated by the red bracket) is present beneath the re-epithelialized surface, which contains newly-forming capillaries and plump fibroblasts. Scab is attached to the re-epithelialized surface from the top (arrow). For orientation purposes, the insert on top right shows the whole tissue section with a red box indicating the magnified area.
      Fig 3
      Fig. 3Representative photomicrograph of the completely healed skin tissue on day 22 post-burn injury. A slightly more cellular dermis and the lack of adnexal structures are present in the center of the skin section (indicated by the red bracket) as remnants of the prevoius burn. The scar thickness of the burn wound is shown by the arrow.

      Statistical analysis

      Data on unhealed wound percentage, scores of re-epithelialization, and scar thickness were compared by one-way ANOVA. As in previous studies [
      • Banki E.
      • Pakai E.
      • Gaszner B.
      • Zsiboras C.
      • Czett A.
      • Bhuddi P.R.
      • et al.
      Characterization of the thermoregulatory response to pituitary adenylate cyclase-activating polypeptide in rodents.
      ,
      • Olah E.
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      • Kormos V.
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      • Pakai E.
      • Wilson H.V.
      • et al.
      The hypothermic effect of hydrogen sulfide is mediated by the transient receptor potential ankyrin-1 channel in mice.
      ], ANOVA was followed by the Fisher's LSD post hoc test. Sigmaplot 11.0 (Systat Software, San Jose, CA, USA) software was used for statistical analyses. Differences were considered significant when p < 0.05. All data are reported as mean ± standrard error (SE).

      Results

      Percentage of the non-epithelialized wound area on day 5 post-burn injury

      Five days after the induction of the burn injury, the depth of the burn and the ratio of the not epithelialized wound surface to the whole wound diameter was analyzed histologically. In all of the studied rats, it was confirmed that the applied method for burn induction resulted in a superficial partial-thickness burn injury (also see Fig. 4, for a representative photo).
      Fig 4
      Fig. 4The ratio of the not epithelialized surface and the burn wound diameter was calculatued to assess the healing of the burn on day 5 post-injury. The diameter of the entire burn wound, as well as, the epithelial invasion from both sides, and the not epithelialized surface are marked with black, blue, and red arrows, respectively. For orientation purposes, the insert on top right shows the whole tissue section with a red box indicating the magnified area.
      When we compared the four used treatment options (viz., silver-sulfadiazine cream, silver foam dressing, zinc-hyaluronan gel, and the combination of zinc-hyaluronan gel with silver foam dressing), we found that the treatment had a significant effect on the wound healing [ANOVA, F(3,26) = 4.837, p = 0.08], as assessed on day 5 by the percentage of the not epithelialized surface to the whole diameter of the burn (for an example, see Fig. 4). With post hoc analysis, we found that the zinc-hyaluronan gel and the combination treatment resulted in significantly smaller ratio of the not epithelialized area (29 ± 10% and 28 ± 13%, respectively) than the silver-sulfadiazine cream (69 ± 4%; p < 0.01 compared to both) (Fig. 5). The not epithelialized area tended to be decreased (47 ± 8%) also with silver foam treatment as compared to silver-sulfadiazine, but the difference did not reach the level of significance (p = 0.080).
      Fig 5
      Fig. 5The percentage of the open (not epithelialized) surface compared to the whole diameter of the burn wound 5 days after the induction of the burn injury in rats (treatment groups and number of animals are indicated). **p < 0.01 vs. silver-sulfadiazine cream as determined by one-way ANOVA followed with Fisher LSD test. Data are presented as mean ± SE.

      Extent of wound re-epithelializatzion on day 10 post-burn injury

      By day 10 after the induction of the burn injury, the whole wound diameter was to some extent epithelialized in the majority of the rats (in 20 of the 28 animals), therefore we used a simple scoring system to assess the healing of the wound. If at least some part of the wound was not re-epithelialized the score was 0; if the entire wound was re-epithelialized but only partially (i.e., in a single layer) the score was 1; if the wound was closed completely in multiple layers then the score was 2.
      The effect of the treatment was significant on the re-epithelialization score [ANOVA, F(3,24) = 13.868, p < 0.001]. We found that the extent of re-epithelialization was the lowest (0.2 ± 0.2) in the silver-sulfadiazine cream group, while the other 3 treatments performed significantly better than that with scores of 1.0 ± 0.2 for silver foam (p = 0.008), 1.0 ± 0.4 for zinc-hyaluronan (0.012), and 2.0 ± 0.0 for the combination treatment (p < 0.001) (Fig. 6). It should be noted that the combination treatment lead to the maximal score of 2 in all rats, which was higher than the scores in the other treatment groups (p < 0.001 vs. silver foam and p = 0.002 vs. zinc-hyaluronan).
      Fig 6
      Fig. 6The score for the re-epithelialization of the burn wound 10 days after the induction of the burn injury in rats (treatment groups and number of animals are indicated). 0: no re-epithelialization; 1: partial re-epithelialization; and 2: complete re-epithelialization in multiple layers over the entire length of the wound. **p < 0.01 and ***p < 0.001 vs. silver-sulfadiazine cream; ###p < 0.001 vs. silver foam dressing; §§p < 0.01 vs. zinc-hyaluronan gel as determined by one-way ANOVA followed with Fisher LSD test. Data are presented as mean ± SE.

      Scar thickness of the wound on day 22 post-burn injury

      On day 22, the burn wounds were already completely re-epithelialized in all of the studied rats. Therefore, to further evaluate the healing process, we analyzed if there is a difference in the scar thickness among the treatment groups, because an increased scar thickness can be an indicator of hyperthrophic scarring, which remains a major challenge following burn injury [
      • Finnerty C.C.
      • Jeschke M.G.
      • Branski L.K.
      • Barret J.P.
      • Dziewulski P.
      • Herndon D.N.
      Hypertrophic scarring: the greatest unmet challenge after burn injury.
      ].
      We found that the scar thickness was the smallest in the combination treatment group (560 ± 42 µm), which was significantly less than in the silver-sulfadiazine cream group (712 ± 38 µm; p = 0.024) (Fig. 7).
      Fig 7
      Fig. 7The scar thickness of the burn wound 22 days after the induction of the burn injury in rats (treatment groups and number of animals are indicated). *p < 0.05 vs. silver-sulfadiazine cream as determined by one-way ANOVA followed with Fisher LSD test. Data are presented as mean ± SE.
      For macroscopic visualization of the burned skin, digital photographs of the burn wound in randomly selected rats at the beginning and at the end of the experiment are shown in Fig. 8.
      Fig 8
      Fig. 8Digital photographs of four randomly selected rats on day 0 briefly after the induction of the burn wound before any of the treatments were applied (top row) and a picture of one randomly selected rat from each treatment group at the end of the experiment on day 22 post-burn injury (bottom row).

      Discussion

      In the present study, we introduce a novel, accessible rat model of superficial partial-thickness burn injury and evaluation of the wound healing, which can be used for the preclinical testing of different treatment options. In this model, we compared the effects of four treatments on different indicators of the wound healing and showed that the combination of zinc-hyaluronan gel with silver foam dressing was the most advantageous compared to the other treatments, while silver foam or zinc-hyaluronan alone was superior to silver-sulfadiazine cream.
      Different experimental designs were used to study the pathomechanism and therapeutic options in burns [
      • Abdullahi A.
      • Amini-Nik S.
      • Jeschke M.G.
      Animal models in burn research.
      ,
      • Schmauss D.
      • Rezaeian F.
      • Finck T.
      • Machens H.G.
      • Wettstein R.
      • Harder Y.
      Treatment of secondary burn wound progression in contact burns-a systematic review of experimental approaches.
      ], however an easily accessible and reproducible, cost-effective, in vivo animal model for preclinical studies remains to be established. In the current study, we developed a rat model of superficial partial-thickness burns, which fulfills the listed criteria. By applying standardized preparations (adult male Wistar rats, nape skin, anesthesia, shaving, disinfection), burning methods (commercially available soldering device with 4 × 4 mm flat surface on the iron tip, 130°C heating, 30 s contact time, and steady pressure), as well as post-intervention procedures (covering the wound with a cohesive conforming bandage and a perforated plastic sheet), we were able to reliably reproduce histologically confirmed superficial partial-thickness burn wounds that penetrated into the dermo-epidermal papillary region of the skin but did not extend to deeper layers.
      The rat – as a widely available, affordable experimental model – was already used previously for the study of burns [
      • Guo H.F.
      • Ali R.M.
      • Hamid R.A.
      • Zaini A.A.
      • Khaza'ai H.
      A new model for studying deep partial-thickness burns in rats.
      ,
      • Gurfinkel R.
      • Singer A.J.
      • Cagnano E.
      • Rosenberg L.
      Development of a novel animal burn model using radiant heat in rats and swine.
      ,
      • Tavares Pereira Ddos S.
      • Lima-Ribeiro M.H.
      • de Pontes-Filho N.T.
      • Carneiro-Leão A.M.
      • Correia M.T.
      Development of animal model for studying deep second-degree thermal burns.
      • Sakamoto M.
      • Morimoto N.
      • Ogino S.
      • Jinno C.
      • Kawaguchi A.
      • Kawai K.
      • et al.
      Preparation of partial-thickness burn wounds in rodents using a new experimental burning device.
      ,
      • Venter N.G.
      • Monte-Alto-Costa A.
      • Marques R.G.
      A new model for the standardization of experimental burn wounds.
      ]. Among those studies, only two reported the successful induction of superficial partial-thickness burns [
      • Sakamoto M.
      • Morimoto N.
      • Ogino S.
      • Jinno C.
      • Kawaguchi A.
      • Kawai K.
      • et al.
      Preparation of partial-thickness burn wounds in rodents using a new experimental burning device.
      ,
      • Venter N.G.
      • Monte-Alto-Costa A.
      • Marques R.G.
      A new model for the standardization of experimental burn wounds.
      ], while in the others the depth of the burn was deep partial-thickness [
      • Guo H.F.
      • Ali R.M.
      • Hamid R.A.
      • Zaini A.A.
      • Khaza'ai H.
      A new model for studying deep partial-thickness burns in rats.
      ,
      • Tavares Pereira Ddos S.
      • Lima-Ribeiro M.H.
      • de Pontes-Filho N.T.
      • Carneiro-Leão A.M.
      • Correia M.T.
      Development of animal model for studying deep second-degree thermal burns.
      ], full-thickness [
      • Gurfinkel R.
      • Singer A.J.
      • Cagnano E.
      • Rosenberg L.
      Development of a novel animal burn model using radiant heat in rats and swine.
      ], or unknown [
      • Priya K.S.
      • Gnanamani A.
      • Radhakrishnan N.
      • Babu M.
      Healing potential of Datura alba on burn wounds in albino rats.
      ]. However, the device used for the induction of superficial partial-thickness burns in both earlier studies was manufactured or modified by the authors [
      • Sakamoto M.
      • Morimoto N.
      • Ogino S.
      • Jinno C.
      • Kawaguchi A.
      • Kawai K.
      • et al.
      Preparation of partial-thickness burn wounds in rodents using a new experimental burning device.
      ,
      • Venter N.G.
      • Monte-Alto-Costa A.
      • Marques R.G.
      A new model for the standardization of experimental burn wounds.
      ], which limits their widespread accessibility. In our current study, for the first time to our knowledge, we used a commercially available soldering device without any modifications, and described how it was used for the induction of burns, which enables its application for scientific research worldwide. It should be noted, however, that although the rat skin is also composed of the major layers (epidermis, dermis) as the human skin, it does not perfectly mimic the human skin architecture because of its unique skin morphology [
      • Abdullahi A.
      • Amini-Nik S.
      • Jeschke M.G.
      Animal models in burn research.
      ]. Therefore, despite the use of rats for burn research in the present and in previous studies [
      • Guo H.F.
      • Ali R.M.
      • Hamid R.A.
      • Zaini A.A.
      • Khaza'ai H.
      A new model for studying deep partial-thickness burns in rats.
      ,
      • Gurfinkel R.
      • Singer A.J.
      • Cagnano E.
      • Rosenberg L.
      Development of a novel animal burn model using radiant heat in rats and swine.
      ,
      • Tavares Pereira Ddos S.
      • Lima-Ribeiro M.H.
      • de Pontes-Filho N.T.
      • Carneiro-Leão A.M.
      • Correia M.T.
      Development of animal model for studying deep second-degree thermal burns.
      • Sakamoto M.
      • Morimoto N.
      • Ogino S.
      • Jinno C.
      • Kawaguchi A.
      • Kawai K.
      • et al.
      Preparation of partial-thickness burn wounds in rodents using a new experimental burning device.
      ,
      • Venter N.G.
      • Monte-Alto-Costa A.
      • Marques R.G.
      A new model for the standardization of experimental burn wounds.
      ], care should be taken when translating the results obtained in rats for human applications. Nevertheless, the developed model can be very well applied to study burn treatment options that are already available for human patients, but, to our knowledge, their parallel comparison under standardized circumstances (i.e., in a unified model) has not been reported.
      In superficial partial-thickness burns, conservative therapy is the primary choice, while surgical interventions are usually not required [
      • Wasiak J.
      • Cleland H.
      • Campbell F.
      • Spinks A.
      Dressings for superficial and partial thickness burns.
      ]. In the case of conservative treatment, it is crucial to rinse the wound with a disinfecting agent prior to removing the dead tissue. This process, called as debridement, is considerably painful, hence analgesic and anxiolytic drugs or general anesthesia are often administered. During the healing of the wound, epithelial cells originating in the remaining epithelial appendages (e.g., the lining of sebaceous and sweat gland ducts) travel from the uninjured to the damaged areas to begin the healing process [
      • Pastar I.
      • Stojadinovic O.
      • Yin N.C.
      • Ramirez H.
      • Nusbaum A.G.
      • Sawaya A.
      • et al.
      Epithelialization in wound healing: a comprehensive review.
      ]. One of the major aims of conservative treatments is to facilitate the epithelialization process and thereby promote the healing of the wound [
      • Wasiak J.
      • Cleland H.
      • Campbell F.
      • Spinks A.
      Dressings for superficial and partial thickness burns.
      ]. Conservative treatments can be used effectively in superficial partial-thickness burns, as they involve the covering of the affected areas in order to maintain a moist environment, as well as, the delivery of antimicrobial compounds, which prevent the infection and progression (i.e., deeper penetration) of the burn wounds. Several conservative treatment methods can be used [
      • Markiewicz-Gospodarek A.
      • Koziol M.
      • Tobiasz M.
      • Baj J.
      • Radzikowska-Buchner E.
      • Przekora A.
      Burn wound healing: clinical complications, medical care, treatment, and dressing types: the current state of knowledge for clinical practice.
      ,
      • Rowan M.P.
      • Cancio L.C.
      • Elster E.A.
      • Burmeister D.M.
      • Rose L.F.
      • Natesan S.
      • et al.
      Burn wound healing and treatment: review and advancements.
      ], but direct, parallel comparisons of the treatments under standardized conditions are scarce, hence the results of different trials can be compared indirectly by meta-analyses, which are, however, hindered by the methodological quality and heterogeneity of the analyzed studies [
      • Nherera L.M.
      • Trueman P.
      • Roberts C.D.
      • Berg L.
      A systematic review and meta-analysis of clinical outcomes associated with nanocrystalline silver use compared to alternative silver delivery systems in the management of superficial and deep partial thickness burns.
      ,
      • Nimia H.H.
      • Carvalho V.F.
      • Isaac C.
      • Souza F.A.
      • Gemperli R.
      • Paggiaro A.O.
      Comparative study of silver sulfadiazine with other materials for healing and infection prevention in burns: a systematic review and meta-analysis.
      ].
      In the present study we selected four treatment options (see below) and compared their effects on different parameters of wound healing in our newly developed rat model of superficial partial-thickness burns.
      • I) Silver-sulfadiazine (e.g., Dermazin) evokes antibacterial effects and promotes re-epithelialization; its low cost and easy application contributes to its widespread use in clinical practice, which also explains why it could be used as a comparator treatment in previous studies [
        • Nimia H.H.
        • Carvalho V.F.
        • Isaac C.
        • Souza F.A.
        • Gemperli R.
        • Paggiaro A.O.
        Comparative study of silver sulfadiazine with other materials for healing and infection prevention in burns: a systematic review and meta-analysis.
        • Yarboro D.D.
        A comparative study of the dressings silver sulfadiazine and Aquacel Ag in the management of superficial partial-thickness burns.
        ]. However, its use requires daily dressing changes and creates a yellowish plaque on the burn, which makes the assessement of burn depth difficult [
        • Mehta M.A.
        • Shah S.
        • Ranjan V.
        • Sarwade P.
        • Philipose A.
        Comparative study of silver-sulfadiazine-impregnated collagen dressing versus conventional burn dressings in second-degree burns.
        ,
        • Hernández R.
        • G M.
        Silver sulfadiazine creame versus topical application in second degree burns.
        ,
        • Chung J.Y.
        • Herbert M.E.
        Myth: silver sulfadiazine is the best treatment for minor burns.
        ].
      • II) Hydrofiber (e.g., Aquacel Ag foam) is a newer dressing type, which contains an external polyurethane waterproof film layer that surrounds a multilayer absorbent surface with a silver ion content of 1.2 %. The multilayer cushion contains a foam sheet and a plate with hydrofiber technology. Absorption of the wound discharge leads to the gelification of the hydrofiber layer, which helps to keep the wound moist, and promotes wound healing while also preventing infections. The bandage is comfortable and its removal painless, without requiring anesthesia [
        • Yarboro D.D.
        A comparative study of the dressings silver sulfadiazine and Aquacel Ag in the management of superficial partial-thickness burns.
        ,
        • Verbelen J.
        • Hoeksema H.
        • Heyneman A.
        • Pirayesh A.
        • Monstrey S.
        Aquacel(®) Ag dressing versus Acticoat™ dressing in partial thickness burns: a prospective, randomized, controlled study in 100 patients. Part 1: burn wound healing.
        ].
      • III) Zinc-hyaluronan gel (e.g., Curiosa) helps to maintain a moist environment due to the large molecular weight and negative charge of the hyaluronan content, which also facilitates the healing process and reduces pain in second-degree burn injury [
        • Fotso Kamdem A.
        • Parmentier A.-L.
        • Mauny F.
        • Soriano E.
        Assessment of care protocol using hyaluronic acid dressing in second-degree skin burns in children.
        ]. The addition of zinc contributes with an anti-inflammatory and antimicrobial effect, which makes it a suitable alternative for topical wound care therapy [
        • Juhasz I.
        • Zoltán P.
        • Erdei I.
        Treatment of partial thickness burns with Zn-hyaluronan: lessons of a clinical pilot study.
        ].
      • IV) The combination of of zinc-hyaluronan gel with silver foam dressing, which was found to perform better than other conservative methods in previous clinical trials [
        • Jozsa G.
        • Vajda P.
        • Garami A.
        • Csenkey A.
        • Juhasz Z.
        Treatment of partial thickness hand burn injuries in children with combination of silver foam dressing and zinc-hyaluronic gel: case reports.
        ,
        • Jozsa G.
        • Toth E.
        • Juhasz Z.
        New dressing combination for the treatment of partial thickness burn injuries in children.
        ].
      In our experiments, we found that silver-sulfadiazine was less beneficial than the combination treatment at all 3 evaluation points, than zinc-hyaluronan on days 5 and 10, and than silver foam on day 10. The combination treatment performed better than the other 3 interventions on day 10 and it was the only method that caused a significant decrease in scar formation on day 22 compared to silver-sulfadiazine. These results are in accordance with previous studies that question the routine use of silver-sulfadiazine in the modern treatment of burn injuries [
      • Nherera L.M.
      • Trueman P.
      • Roberts C.D.
      • Berg L.
      A systematic review and meta-analysis of clinical outcomes associated with nanocrystalline silver use compared to alternative silver delivery systems in the management of superficial and deep partial thickness burns.
      ,
      • Nimia H.H.
      • Carvalho V.F.
      • Isaac C.
      • Souza F.A.
      • Gemperli R.
      • Paggiaro A.O.
      Comparative study of silver sulfadiazine with other materials for healing and infection prevention in burns: a systematic review and meta-analysis.
      ]. Moreover, our findings highlight that newer treatment options such as silver foam dressing and zinc-hyaluronan or the combination of them can results in improved burn wound healing compared to silver-sulfadiazine. The mechanism by which the combination treatment was superior compared to silver foam dressing or zinc-hyaluronan alone remains subject to future studies, but it can be suggested that the simultaneous presence of silver and zinc ions in the dressing exerts additional advantageous effects on wound healing as compared to the two components alone. Indeed, the combination of silver and zinc resulted in enhanced antibacterial effect [
      • Kyomuhimbo H.D.
      • Michira I.N.
      • Mwaura F.B.
      • Derese S.
      • Feleni U.
      • Iwuoha E.I.
      Silver–zinc oxide nanocomposite antiseptic from the extract of Bidens pilosa.
      • Blanchard C.
      • Brooks L.
      • Ebsworth-Mojica K.
      • Didione L.
      • Wucher B.
      • Dewhurst S.
      • et al.
      Zinc pyrithione improves the antibacterial activity of silver sulfadiazine ointment.
      ], anti-inflammatory and antioxidant responses [
      • Borges Rosa de Moura F.
      • Antonio Ferreira B.
      • Helena Muniz E.
      • Benatti Justino A.
      • Gabriela Silva A.
      • de Azambuja Ribeiro R.I.M.
      • et al.
      Antioxidant, anti-inflammatory, and wound healing effects of topical silver-doped zinc oxide and silver oxide nanocomposites.
      ], as well as improved wound healing, re-epithelialization, and collagen deposition when used in vivo as a dressing for mechanical (not burn) wounds [
      • Lu Z.
      • Gao J.
      • He Q.
      • Wu J.
      • Liang D.
      • Yang H.
      • et al.
      Enhanced antibacterial and wound healing activities of microporous chitosan-Ag/ZnO composite dressing.
      ,
      • Borges Rosa de Moura F.
      • Antonio Ferreira B.
      • Helena Muniz E.
      • Benatti Justino A.
      • Gabriela Silva A.
      • de Azambuja Ribeiro R.I.M.
      • et al.
      Antioxidant, anti-inflammatory, and wound healing effects of topical silver-doped zinc oxide and silver oxide nanocomposites.
      ].
      In conclusion, we developed an easily accessible rat model for the study of superficial partial-thickness burn injuries. We showed that this model is feasible for the preclinical testing of different treatment options by comparing four treatment methods. Among the studied treatments, the combination of silver foam dressing and zinc-hyaluronan was superior compared to the other methods as assessed by different parameters of wound healing.

      Funding

      This work was supported by the National Research, Development and Innovation Office (grant FK 138722 to AG).

      Conflict of interest

      The authors do not have any conflict of interest to declare.

      Acknowledgements

      The authors thank Aniko Varnagyne Rozsafi and Kata Fekete for the excellent technical assistance.

      Declaration of Competing Interests

      The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
      The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

      Acknowledgements

      This work was supported by the National Research, Development and Innovation Office (grant FK 138722 to AG ). The funders had no role in the preparation of the manuscript. The authors thank Aniko Varnagyne Rozsafi and Kata Fekete for their excellent technical assistance.

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