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International Journal of Experimental Pathology logoLink to International Journal of Experimental Pathology
. 2024 Oct 22;105(6):235–245. doi: 10.1111/iep.12520

Histomorphometric analysis of excisional cutaneous wounds with different diameters in an animal model

Janiele Staianov 1, Jeiciele Mayara Rodrigues Struz 1, Rafaela Viana Vieira 1, Rafael Messias Luiz 1, Ana Carla Zarpelon‐Schutz 1,2, Kádima Nayara Teixeira 1,3, Juliana Bernardi‐Wenzel 1,2,✉
PMCID: PMC11576331  PMID: 39439085

Abstract

The skin wound model in rats is a fundamental stage in preclinical trials, but there is a lack of standardization in these trials regarding the initial wound area, making analysis and comparison between studies difficult. Therefore, this study evaluates the healing progression of excisional skin lesions of varying diameters in Wistar rats, aiming to identify the optimal wound size for monitoring treatment effects on wound healing. Excisions of 0.8, 1.5, 2.0 and 3.0 cm in diameter were made on the back of the animals. Thirty animals were used per treatment and evaluated on days 3, 7, 10, 14 and 21 after surgery. The lesions were cleaned daily with saline solution until they were completely closed. The 0.8 cm group showed complete repair on D14, while in the other groups, the wounds persisted until day 21, with a reddened surface and no complete epidermal coverage, but with greater keratinization and presence of appendages in the 1.5 cm lesions. Therefore, as a standardization model for creating skin wounds, we suggest using 1.5 or 2.0 cm excisions, considering that 0.8 cm wounds close very early and 3.0 cm wounds, although behaving similarly to 2.0 cm wounds, are more invasive for the animals. The 1.5 cm model proved to be suitable for closure within 21 days. When evaluating a product intended to accelerate wound healing, 2.0 cm lesions are recommended to assess the effectiveness of the treatment.

Keywords: cutaneous wounds, healing, histological analysis, skin, tissue repair

1. INTRODUCTION

The skin is the largest human organ and acts as an external protective barrier against physical, chemical and biological agents. Therefore, it is susceptible to injuries that impact individuals' quality of life and the economy by burdening the healthcare system. 1 , 2 The loss of skin continuity characterizes a type of injury called a wound, which can be acute or chronic depending on the resolution time. 3 , 4

Wounds can be caused by surgical procedures, physical trauma, burns, chemical agents, venous insufficiency, pressure ulcers and complications of chronic diseases such as diabetes mellitus. 5 , 6 In healthy individuals, cutaneous wounds tend to be self‐limiting; however, certain conditions can induce a failure in the tissue repair process, making it chronic. 7

Cutaneous wounds, especially chronic ones, are accompanied by pain, limited mobility and ability to perform routine activities, social stigma, and influence the individual's self‐perception of body image, predisposing them to the development of depressive and anxious conditions and social isolation. 8 , 9

Complications related to the prolongation of the tissue repair process increase the costs associated with the treatment of cutaneous wounds, as there is a greater demand for supplies and medical care at more complex and high‐cost levels. 10 Additionally, the delay in wound closure tends to worsen the patient's clinical condition, leading to more severe outcomes, such as amputations, which negatively impact quality of life and represent a loss to the economically active population. 11 , 12

Due to the socioeconomic impact, studies are developed to understand the mechanisms of tissue repair and the factors leading to process failure. 13 Thus, studies are conducted using experimental models, such as in vivo preclinical trials using rodents, especially Rattus norvegicus (Wistar strain), since these animals experience less stress from handling and, due to their size, allow for the creation of surgical wounds and the collection of larger biological samples for subsequent analysis and comparison. 14 , 15 Additionally, they are easy to handle and house in animal facilities. In this way, they enable the study of the body's response to a damaging stimulus and the action of new compounds in the closure of cutaneous wounds. 16 , 17

Cutaneous wounds can be made using different techniques, depending on the study's objective. In total excision wounds, all layers of the skin are removed, allowing the evaluation of the tissue repair process as a whole, differing from partial excisions that aim to assess only the repair of the epidermis. Other methodologies include creating wounds by puncture, suction, burn or induction of chronic disease. 18 Although the rat skin wound model is widely explored in preclinical trials, there is a lack of standardization in these trials in terms of the initial wound area, making it difficult to carry out a reliable and thorough analysis of the stages of the tissue repair process. Studies have reported diameters ranging from 0.6 cm 19 to 3.0 cm 20 for the excision of lesions, with many variations between these two values, 21 , 22 , 23 which prevents comparison between the available studies. Therefore, there is a need to standardize this experimental model to reduce bias in research into the acceleration of tissue repair, which can be misinterpreted due to the incorrect choice of wound area, preventing a complete assessment of the process.

This study assessed the progression of tissue repair in excisional cutaneous wounds with different diameters in Wistar rats to determine the most suitable initial area for this type of study. It is expected to provide relevant information so that future preclinical research can more accurately assess the mechanisms involved in the pathophysiology of wound closure.

2. MATERIALS AND METHODS

2.1. Animals and ethical procedures

The study was approved by the Animal Use Ethics Committee of the Universidade Federal do Paraná‐Setor de Ciências Biológicas (Approval 1498; 13‐12‐2022 ‐ Appendix A). The experimentation was conducted on healthy male and female Wistar rats (Rattus norvegicus), 90 to 120 days old, weighing from 250 to 350 g, randomly distributed into four experimental groups (n = 20 animals/group), according to the diameter (area) of the cutaneous wound‐0.8 cm; 1.5 cm; 2.0 cm and 3.0 cm. The animals were kept in the animal facility of the Federal University of Paraná‐Toledo Campus, received pelleted feed specific for rodents and chlorinated water ad libitum, and were maintained at a temperature of 22°C, on a 12 h light/dark cycle.

2.2. Surgical procedure and experimental design

The animals received diazepam (2 mg/kg, i.p.), and after 10 min, anaesthetic induction and maintenance were performed with isoflurane at 3%–4% and 1.5%–2% in 100% oxygen respectively. A trichotomy was performed on the dorsal region of the animals, 1.5 cm below the base of the neck; local antisepsis was done with 10% topical iodopovidone, and local anaesthesia was administered with lidocaine hydrochloride (5 mg/kg, s.c.). A 0.8 cm diameter wound was created using a circular metal punch. The other circular wounds of 1.5, 2.0 and 3.0 cm were demarcated with a marker, with a paper template in each measurement and the excisions made using a scalpel; the skin was removed, and the wound was cleaned with 0.9% (w/v) sodium chloride (NaCl). After the surgery, each animal received a single administration of cefalotin (60 mg/kg, s.c.) and meloxicam (2 mg/kg, s.c.). No treatment was administered to the animals; only daily wound cleaning with 0.9% (w/v) NaCl was performed once a day and the lesions kept open during the healing process until the 21st day (D21). The animals were observed daily for complete wound closure.

2.3. Measurement of cutaneous wound areas

On D3, D7, D10, D14 and D21 after the surgical excision, six animals from each group were randomly subjected to euthanasia by isoflurane overdose, followed by visual and morphometric evaluation of the wounds. Photographs of the wounds were taken using a camera Canon EOS 80D fixed at distance of 20 cm from the lesions, without adding a zoom. A 30 cm ruler was inserted next to the animals showing the lesions to determine the scale for calculating the lesion areas using ImageJ® software (National Institutes of Health‐NIH). The degree of wound contraction was calculated using the equation: % contraction = 100 (Wo‐Wi)/Wo, where Wo = initial wound area and Wi = wound area on the observed experimental day, according to Oliveira et al. 24

2.4. Macroscopic parameters of cutaneous wounds

After euthanasia, the wounds were analysed for the presence of oedema, necrosis (scab) and exudate, with a score assigned according to the absence (0), slight presence (1 = 1%–25%), moderate presence (2 = 26%–50%) and significant presence (3 = above 50%) of the finding, according to Oliveira et al., 25 with modifications.

2.5. Histological examination of cutaneous wounds

A fragment of the wound, with a 0.5 cm margin and depth down to the muscular fascia, was removed and fixed in 3.7% (v/v) buffered formalin for histopathological tissue analysis. Histological slides were prepared with 5 mm sections and stained with haematoxylin–eosin (HE) to analyse granulation tissue parameters, according to Oliveira and colleagues, 25 with modifications—collagenization, inflammatory infiltrate, vascular proliferation, fibroblastic proliferation and re‐epithelialization. Masson's trichrome (MT) staining was used to evaluate the organization and maturation of collagen fibres. 26

2.6. Statistical analysis

The areas of the lesions obtained by the ImageJ software were analysed using GraphPad Prism 9.5.0 software by means of analysis of variance (ANOVA) followed by the Tukey test, with significance considered at p < .05.

3. RESULTS

3.1. Cutaneous wound area

Considering that the wound area increases after excision, the initial areas measured were 0.692 ± 0,075 cm2 (0.8 cm); 3.371 ± 0,395 cm2 (1.5 cm); 6.709 ± 0,654 cm2 (2.0 cm); and 11.039 ± 1227 cm2 (3.0 cm), in D0. Then the areas were measured on D3, D7, D10, D14 and D21 of the experiment. On D3, the wound areas for each group were 0.430 ± 0.06 cm2 (0.8 cm); 2.190 ± 0.15 cm2 (1.5 cm); 4.582 ± 0.38 cm2 (2.0 cm); and 8.452 ± 1.04 cm2 (3.0 cm); and these gradually reduced over the observation days. The wounds with an initial diameter of 0.8 cm closed completely by day 14; wounds with a diameter of 1.5 cm closed completely by day 21, while in animals with wounds of 2.0 and 3.0 cm in diameter, complete wound closure was not observed (Figure 1).

FIGURE 1.

FIGURE 1

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Cutaneous wound area of the experimental groups on D3, D7, D10, D14 and D21. *Different from 0.8 cm; #Different from 0.8 and 1.5 cm; **Different from 0.8, 1.5 and 2.0 cm.

3.2. Macroscopic analysis of the cutaneous wounds

Macroscopic analysis of the cutaneous wounds allowed for the evaluation of the scarring process, as well as the presence of oedema, exudate and necrosis, assessed by the presence of a scab (Figure 2). Oedema was observed exclusively in animals with 3.0 cm wounds, predominantly on D3. Necrosis was observed only in the groups with 2.0 and 3.0 cm wounds, identified by the presence of a blackened scab, with some localized areas.

FIGURE 2.

FIGURE 2

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Macroscopic appearance of the cutaneous wounds in the experimental groups on D0, D3, D7, D10, D14 and D21. Measurements in centimetres. Bar size: 1 cm.

During the experiment, all groups showed exudate, primarily from D7 onwards. Animals with 0.8 cm wounds exhibited less exudate, with incidence detected only on D7. In the other groups, exudate was observed on D3, D7 and D10, with higher amounts in the 3.0 cm wound group on all days except D3, where the 2.0 cm wound group showed more pronounced exudate.

On D0, all groups exhibited red colouration and similar appearance in the wounds. From D3 to D7, the beginning of the tissue repair process was observed with the presence of reddish‐brown scabs covering the wounds, seen in all groups, except in some animals from the 3.0 cm group. Throughout the experiment, the 0.8 cm and 1.5 cm wounds did not show adherence of cage debris, while the 2.0 cm and 3.0 cm wound groups had bedding material adhered to the wound bed. No wounds exhibited apparent inflammatory response reactions such as oedema and secretion.

On D7, compared to D3, there was a reduction in the wound area. The 0.8 cm and 1.5 cm groups showed scab detachment, and the wounds became homogeneous and pink in colour. On this day, five animals in the 3.0 cm group exhibited an accumulation of purulent secretion in the wound, suggesting an infectious process. The main macroscopic variations between the groups were observed from D10 onwards, especially in the animals with 0.8 cm wounds, which showed a significant reduction in wound area and the presence of epithelial restructuring compared to the other groups.

Between D14 and D21, the groups with 2.0 cm and 3.0 cm wounds exhibited significant tissue repair, with greater asymmetry in edge approximation observed in the 3.0 cm group, which showed regular contraction edges.

Based on the wound contraction rate and restoration of epithelial coverage, the 0.8 cm group showed complete repair on D14. In the other groups, the wounds persisted until day 21, with a reddened surface and no complete epidermal coverage, but with greater keratinization and presence of appendages in the 1.5 cm lesions.

3.3. Wound contraction rate

The progression of wound area in each group was evaluated by monitoring the contraction rate over the 21 experimental days. On D3, all groups showed a significant contraction rate compared to the initial wound area (D0). The 0.8 cm group had a contraction rate of 37.1 ± 11.9%, followed by 36.3 ± 8.7%, 31.5 ± 12.2% and 25.6 ± 14.2% for the other groups respectively.

Over the 21 experimental days, the 0.8 cm group maintained the highest contraction rate compared to the other groups. The 2.0 cm and 3.0 cm groups exhibited similar contraction rates in D3. In D7, the 2.0 lesions showed a contraction rate of less than 3.0, but from D10 onwards, the 3.0 cm group showed a higher contraction rate than the 2.0 cm group, and this difference persisted until the end of the experiment. On D21, the 0.8 cm and 1.5 cm groups exhibited a contraction rate of 100%, while the 2.0 cm and 3.0 cm groups showed contraction rates of 97.8% and 98.5% respectively (Figure 3).

FIGURE 3.

FIGURE 3

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Wound contraction rate of the experimental groups on D3, D7, D10, D14 and D21. *different from 0.8; #different from 0.8 and 1.5; **different from 0.8; 1.5 and 2.0.

3.4. Microscopic analysis of the cutaneous wounds

The images obtained from HE‐stained histological sections of the wounds on D3, D7, D10, D14 and D21 are shown in Figure 4. On D3, it was observed that the 0.8 cm wounds had a scab and an inflammatory infiltrate with non‐homogeneous distribution, mainly concentrated in the more superficial regions of the wound bed (Figure 4). Neovascularization was slight, and collagen was present but disorganized, making it difficult to clearly distinguish its fibres from other elements of the extracellular matrix.

FIGURE 4.

FIGURE 4

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Microscopic appearance of the cutaneous wounds in the experimental groups on D3, D7, D10, D14 and D21. HE staining, magnification of 100X. Bar size: 0.25 mm.

Still on D3 in HE, the 1.5 cm wounds exhibited a scab, moderate inflammatory infiltrate and discrete angiogenesis, with dispersed red blood cells in the tissue, indicating recent bleeding. Regarding collagenization, irregular and disorganized collagen fibres were observed, with dispersed distribution of fibroblasts throughout the tissue. The 2.0 cm and 3.0 cm wounds showed microscopic characteristics similar to the 1.5 cm group. Vascular proliferation was slight in all groups, with dispersed presence of fibroblasts and collagen fibres, which were disorganized. No group showed re‐epithelialization.

From D7 onwards, using TM staining, histological evaluation indicated the presence of structured collagen. The images of the histological sections on D7, D14 and D21 show differences in collagen deposition (Figure 5 and Appendix A).

FIGURE 5.

FIGURE 5

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Microscopic appearance of the cutaneous wounds in the experimental groups on D7, D14 and D21. TM staining, magnification of 100X. Bar size: 0.25 mm.

On D7, the 0.8 cm group did not present a scab, and collagenization was moderate, with collagen arranged in a disorganized manner and a large amount of inflammatory infiltrate and fibroblasts present (TM). There was an increase in vascular proliferation, with frequent visualization of dispersed vessels throughout the tissue. No epithelialization was observed (HE).

The other groups shared similar characteristics to those of the 0.8 cm group. Differences were found in the following aspects in HE: the 1.5 cm wounds showed a discrete onset of epithelialization and had a thicker scab compared to the other groups; the 2.0 cm wounds exhibited a scab and pronounced vascular proliferation; and the 3.0 cm wounds had a scab, pronounced vascularization, red blood cells distributed throughout the tissue, moderate fibroblast disposition and a layer of forming epithelium. Thus, on D7, it was possible to observe in all groups a higher quantity of fibroblasts, neovessels and inflammatory infiltrate compared to D3.

On D10, the 0.8 cm group exhibited complete re‐epithelialization: organized dermis with thick and well‐defined collagen fibres and all layers of the epidermis (TM). There was a decrease in inflammatory infiltrate and fibroblast proliferation compared to the previous analysis, a characteristic that was also observed in the other experimental groups (HE).

The 1.5 cm group in HE showed progression of re‐epithelialization, with formation of skin appendices and a greater quantity of epithelial cells. It was noted that epithelial layer development was more advanced in the 0.8 cm group, but the 1.5 cm group showed a higher level of organization of the epithelial layer. Additionally, a pronounced increase in vascularization compared to D7 was identified.

On D10, the 2.0 cm group did not show the onset of reepithelialization and had moderate collagenization with initial organization in fibres (HE). Tissue repair of the 3.0 cm wounds occurred similarly to the 2.0 cm group, but differed by having the onset of epithelial layer development. It is worth noting that all groups had scabs over the wound bed or the area of re‐epithelialization on D10.

On D14, histological analysis showed that the 0.8 cm group exhibited clear differentiation of the dermis into layers of loose and dense unmodeled connective tissue, as well as absence of scab. Vascular proliferation, as well as the distribution of fibroblasts and collagenization, was pronounced (TM), and the amount of inflammatory infiltrate decreased to isolated mononuclear cells visualized throughout the tissue (HE).

Unlike the 0.8 cm group, the 1.5 cm group remained with incomplete re‐epithelialization and presence of scab on D14, with moderate fibroblast proliferation, neovascularization with smaller calibre vessels compared to D10, little extravasation of red blood cells and collagenization (TM). Additionally, there was a reduction in inflammatory infiltrate to mononuclear cells (HE).

On D14, in the 2.0 cm group, the beginning of reduction in the quantity and calibre of blood vessels was observed, along with the formation of new epithelium and organization of collagen fibres, while maintaining moderate fibroblast proliferation and collagenization. In contrast to the 0.8 cm and 1.5 cm groups, there was no reduction in inflammatory infiltrate to mononuclear cells.

The progression of the 3.0 cm group regarding neovessels and inflammatory infiltrate was similar to the 2.0 cm group, except that in the 3.0 cm group there was pronounced distribution of fibroblasts and collagen, as well as greater epithelial layer development. Both groups showed scab in microscopic evaluation.

On D21, all experimental groups achieved complete re‐epithelialization, except for the 2.0 cm group, which still had partial epithelium. No group had scab, and all had reduced vascular proliferation to blood vessels sparsely distributed throughout the tissue. The quantity of fibroblasts in the tissue and collagenization, similarly, was pronounced for all groups (TM). The inflammatory infiltrate of the 0.8 cm group remained discrete, differing only from the 3.0 cm group, which did not present a significant amount of these cells (HE).

4. DISCUSSION

Experimental models of cutaneous wound tissue repair have been developed over the years in an attempt to understand the process and test new treatments. 27 In vivo models are recognized as the most predictive for studying wound repair, allowing a realistic representation of its microenvironment 28 including various cell types and paracrine interactions. 29

The phases of the cutaneous wound healing process are completed in about 21 days. 30 , 31 In this study, animals with wounds of 0.8 and 1.5 cm showed complete tissue repair in less than 21 days, indicating limitations of wounds of these dimensions in faithfully mimicking the stages of the healing process.

Similar results were observed by Guirro et al., 32 who evaluated cutaneous healing in animal models with 0.8 cm diameter wounds treated only with saline solution, and complete repair occurred on the 14th day of the experiment. The authors described that for 0.8 cm wounds, the use of 0.9% (w/v) NaCl twice a day was sufficient to keep the wound clean and infection‐free; in this study, treatment with 0.9% (w/v) NaCl occurred once a day, but it was still sufficient to keep the wounds clean and infection‐free.

Ulagesan, Sankaranarayanan and Kuppusamy 33 analysed wound healing in Wistar rats aiming to evaluate the influence of natural treatments compared to a 5% iodopovidone control on the tissue repair process. The authors used Wistar rats with circular wounds of 0.8 and 1.5 cm in diameter. Their results demonstrate that within 21 days, the wounds were completely healed, regardless of the initial size. Additionally, it was found that in 0.8 cm wounds, re‐epithelialization was complete by the 14th day, and for 1.5 cm wounds, by the 21st day, corroborating the perspective that the initial wound size can affect tissue repair in the evaluation of interventions that accelerate the process. In our study, the 0.8 cm wounds showed a similar time for complete tissue repair; however, it required more time for repair, which was completed on the 21st day.

Dwivedi et al. 34 and Selvam et al. 35 present similar results for 1.5 cm wounds; their research demonstrated complete wound closure by the 21st and 20th days, respectively, for the control groups. Although the total time for complete repair was similar, Selvam et al. 35 found a reduction in wound diameter to 0.7 cm on the 10th experimental day, while in our study, a reduction to 0.47 cm was observed during the same period.

In contrast, Ebbo et al. 36 found that 1.5 cm wounds treated with carboxymethylcellulose, which stimulates autolytic debridement and favours tissue repair, showed complete closure by the 15th day. These results indicate that for evaluating if a treatment is effective in accelerating the tissue repair process, the repair time will be less than 21 days, suggesting that this diameter can be used for treatment evaluation.

In this study, the 2.0 cm and 3.0 cm groups were the only ones with remaining wound area on the 21st day. Goorani et al. 37 and Dilmann et al. 38 evaluated 2.0 cm diameter wounds in Wistar rats and found similar results to each other.

Goorani et al. 37 evaluated 2.0 cm wounds and, for groups treated with saline solution, they identified a small lesion extension on the 21st day and a contraction rate of 97.12%, corroborating the findings of this study. Dilmann et al. 38 investigated 2.0 cm diameter wounds, observing that, after 15 days, untreated wounds remained open with a contraction rate of 93.25%. In a similar study, Vyas et al. 39 found that wound contraction in the control group was 92.21% after 15 days and 96.28% after 21 days, results that closely matched those identified in this study, where the retraction rates were 91.2% and 97.8% on days 14 and 21 respectively.

In this study, the 3.0 cm wounds exhibited contraction rates similar to those of the 2.0 cm wounds during the 21 days experiment. However, the macroscopic appearance of the wounds was different, particularly regarding the symmetry in the contraction of the edges. Davidson et al. 40 highlight that wounds in some classic excisional models, especially in murines, heal primarily by contraction, which accounts for a significant part of wound closure. This occurs because the skin of rodents has a layer of panniculus carnosus, a thin muscular layer similar to the platysma in the human neck, which produces rapid wound contraction after injury. Therefore, the regularity of wound edges during the tissue repair process represents an important aspect of analysis in animal models.

Regarding the edges, the 3.0 cm wounds exhibited greater asymmetry in edge approximation compared to the other experimental groups, corroborating the results of Claro et al. 20 In their study, Claro et al. evaluated wounds of the same diameter treated with a commercial dressing and described residual lesions larger than 1 cm after 14 days, with irregular and asymmetrical contraction of the edges. Additionally, they reported increased inflammation and necrosis in these wounds.

Yassine et al. 41 evaluated the healing properties of a topical ointment containing Lawsonia inermis leaf, a shrub naturally cultivated in Northeast Africa and India, on excisional wounds in Wistar rats. They used a rectangular excisional wound model of 700 mm2, created by removing the skin from the dorsal neck area of the animal with a scalpel. The progressive change in wound area was monitored on days 0, 3, 6, 9, 12, 15, 18, 21 and 24. On the 3rd day post‐wounding, the percentage of wound closure (contraction rate) was 39.81% in the group treated with Lawsonia inermis, compared to 13.99% in the control group. This trend continued over the following days, with the Lawsonia inermis treated group showing 78.28% closure on the 9th day, compared to 60.17% in the control group at the same time. By day 24, the wounds in the animals treated with Lawsonia inermis were completely healed.

Reis et al. 42 conducted an experimental study on the effects of amniotic membrane or topical propolis on cutaneous wound healing in Wistar rats. Male Wistar rats were used in the experiment, divided randomly into three groups, and a rectangular surgical wound measuring 1.5 × 1.5 cm and 0.5 cm deep was made on the dorsal region of the animals. Seven days post‐surgery, the wounds exhibited similar morphology across all experimental groups, with an average total area of 0.756 cm2. Fourteen days post‐procedure, the wounds showed differing morphologies among the groups. By 21 days post‐surgery, the wounds of the animals treated with propolis were fully repaired. However, those treated with the amniotic membrane still had around 25% (0.123 cm2) of the wound area remaining. The group treated with propolis was the only one to achieve complete tissue repair, on average, by the 19th day of the study, while the other comparative groups did not achieve complete repair by the 21st post‐operative day.

It is important to note that Yassine et al. 41 and Reis et al. 42 used a rectangular wound model, which limits direct comparison with the circular model used in this study. The primary mechanism of skin repair is contraction, in which the wound area reduces centripetally from the edges 43 due to the action of actin filaments in myofibroblasts 44 and collagen rearrangement. 45

Wound contraction depends on skin mobility, which is determined by the direction of Langer's lines (tension lines), primarily dictated by the arrangement of skin elastic fibres. Contraction occurs transversely to Langer's lines. 46 Therefore, the orientation of the wound in relation to the skin's tension lines is crucial for tissue repair, as wounds that cross Langer's lines have more difficulty healing. 47 Consequently, rectangular or square wounds tend to have a delayed repair process and exhibit hypertrophy.

Indeed, data on the wound healing process from preclinical assays can vary significantly depending on the chosen animal model, age, gender, wound location 48 and wound shape. Therefore, standardization and validation of the model are crucial. Critical processes underlying wound repair were initially described based on animal models, 49 and although the process in animals does not fully resemble that in humans, animal models have so far provided important insights into the principles of tissue repair. 50

5. CONCLUSION

As a standardization model for creating skin lesions, the use of 1.5 or 2.0 cm excisions is suggested, taking into account that 0.8 cm lesions closed very early and 3.0 cm lesions, despite behaving similarly to 2.0 cm lesions in D21, are more invasive for the animals. The 1.5 cm model proved to be suitable for closure within 21 days. In the case of evaluating a product that accelerates healing, the 2.0 cm lesions can be used to check whether healing occurs by the 21st day, differentiating them from untreated lesions that have not yet finished healing.

AUTHOR CONTRIBUTIONS

Janiele Staianov, Jeiciele Mayara Rodrigues Struz and Rafaela Viana Vieira performed all the experimental steps and the writing of the manuscript. Rafael Messias Luiz collaborated with conducting animal experiments. Ana Carla Zarpelon‐Schutz collaborated with performing histological analysis and contributed to the design of the manuscript. Kádima Nayara Teixeira and Juliana Bernardi‐Wenzel wrote the project, supervised all stages of the study and contributed to the design and writing of the manuscript.

FUNDING INFORMATION

FM and this study were supported by Universidade Federal do Paraná‐Campus Toledo, coming from the national treasury.

CONFLICT OF INTEREST STATEMENT

All the authors have no conflicts with the manuscript to disclose.

APPENDIX A.

A.1.

A.1.

Staianov J, Struz JMR, Vieira RV, et al. Histomorphometric analysis of excisional cutaneous wounds with different diameters in an animal model. Int J Exp Path. 2024;105:235‐245. doi: 10.1111/iep.12520

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are openly available in Doi at https://doi.org/10.1111/iep.12520.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are openly available in Doi at https://doi.org/10.1111/iep.12520.

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