Experimental models and methods for cutaneous wound healing assessment
Funding information
This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (processes numbers: 2013/20554; 2014/23662-1 and 2016/16437-7), Conselho Nacional de Pesquisa e Desenvolvimento (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Fundação de Apoio ao Ensino, Pesquisa e Assistência do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (FAEPA).
Summary
Wound healing studies are intricate, mainly because of the multifaceted nature of the wound environment and the complexity of the healing process, which integrates a variety of cells and repair phases, including inflammation, proliferation, reepithelialization and remodelling. There are a variety of possible preclinical models, such as in mice, rabbits and pigs, which can be used to mimic acute or impaired for example, diabetic and nutrition-related wounds. These can be induced by many different techniques, with excision or incision being the most common. After determining a suitable model for a study, investigators need to select appropriate and reproducible methods that will allow the monitoring of the wound progression over time. The assessment can be performed by non-invasive protocols such as wound tracing, photographic documentation (including image analysis), biophysical techniques and/or by invasive protocols that will require wound biopsies. In this article, we provide an overview of some of the most often needed and used: (a) preclinical/animal models including incisional, excisional, burn and impaired wounds; (b) methods to evaluate the healing progression such as wound healing rate, wound analysis by image, biophysical assessment, histopathological, immunological and biochemical assays. The aim is to help researchers during the design and execution of their wound healing studies.
1 INTRODUCTION
A wound is defined as a disruption of cellular, anatomical and functional continuity of a living tissue and may be caused by physical, chemical, thermal, microbial or immunological insults. In other words, a wound is a break in the epithelial integrity and may be accompanied by disruption of the structure and function of underlying normal tissue.1-3
To restore the structure of the injured tissue, a complex process involving migration, proliferation, interaction and differentiation of multiple cell types (eg epidermal, dermal, infiltrating inflammatory cells), biomolecular interactions, synthesis of matrix components and a complex signalling network must occur.4-7
Cell interactions with their environment are bidirectional and dynamic, since cells can modulate the structure and composition of the extracellular matrix (ECM) and it, in turn, guides cell morphology and behaviour during wound healing and tissue homeostasis.8 The ECM is a three-dimensional structure comprised of extracellular macromolecules, such as collagen, elastin, fibronectin, vitronectin, integrins and laminins, that provide structural and biochemical support and anchorage to surrounding cells.
Wound ECM molecules and cells interact by the presence of transmembrane cell surface receptors called integrins. They are critical in cell differentiation, migration and proliferation in wound healing and in cell-cell adhesion by their attachment through a bidirectional signal transduction mechanism between cells and the ECM, thus playing an important role in epithelial and connective tissue repair.8-10 The most important integrins in wound healing and cells that express them include β3, β1, α5β1 (fibroblasts, keratinocytes), α9, α11β1 (fibroblasts), α3β1, αvβ5, αvβ6, α2β1, α5β1 (keratinocytes), α3β1 (keratinocytes, endothelial cells), αvβ3 (endothelial cells) and αmβ2 (neutrophils, monocytes.10
Another group of proteins present in the ECM of importance in wound healing are laminins. They are the most abundant glycoproteins of the basement membrane and have essential roles in the establishment of tissue architecture and stability, providing cells with a structural scaffold and contributing to two major aspects of wound repair: angiogenesis and re-epithelialisation.11, 12 Laminin-8 (α4β1γ1), laminin-10 (α5β1γ1) and laminin-5 (α3β3γ2) are the major laminins actively involved in wound repair.11, 13 During angiogenesis, laminin-8 promotes dermal endothelial cell attachment, migration and tubule formation, while laminin-10 is highly expressed in blood vessels around cutaneous wounds.14 During re-epithelialization, laminins provide the substrate for epithelial keratinocytes to migrate and cover the wound, re-establishing the epithelial barrier. Keratinocytes in the migrating front edge deposit laminin-5, the major laminin in epithelial tissues, which functions as a track to allow subsequent keratinocytes to migrate.11, 13
Acute wound healing is a well-regulated process consisting of partially overlapping phases that are determined by interacting events on a molecular, cellular and extracellular matrix level that ends with wound closure within days or weeks.7, 15 For didactic reasons, the course of physiological healing is schematically divided into three phases: inflammation, proliferation and remodelling.15, 16
When the healing process does not occur as expected and wounds get stalled at the inflammatory phase or there is an imbalance between metalloproteinases (MMPs) and the associated tissue inhibitors of metalloproteinases (TIMPs) particularly during the tissue formation phase, they are defined as chronic wounds. The underlying aetiology of chronic wounds is diverse, but more than 80% are associated with vascular insufficiency, high blood pressure or diabetes mellitus. Despite the different aetiology, most chronic wounds show a similar behaviour and progress due to consistent components of the multifactorial pathogenesis: local tissue hypoxia, bacterial colonization/infection, repeated ischaemia-reperfusion injury and cellular and systemic changes.15, 17
The study of wound healing pathophysiology and the development of new tools and protocols to monitor the healing process can certainly contribute to optimize a treatment and obtain better patient outcomes.18 A periodical assessment of wounds will lead to a better chance of providing a more appropriate treatment to the patient. Wound's characteristics, aetiology and other medical history along with regular monitoring could help the healthcare team to learn in which phase of the healing process the wound is and to decide on the best treatment approach.
The investigation of any physiological process is dependent on the use of models, and several in vitro and in vivo wound healing models have been described in the literature.19-28 During wound healing studies, researchers should consider several design-related issues, including the study aims, type of wound, sample characteristics and accessibility, costs, timeframe and facilities available. Moreover, a combination of multiple wound assessment methods would increase the reliability and validity of results and provide a further understanding of mechanisms involved in tissue repair.4 Also, when an appropriate wound healing model is chosen from the beginning of testing of a new product, the timeframe for experimentation could be reduced, fewer animals and supplies needed, thereby reducing costs and aggregating value to the end product.
The topic concerning the use of models and methods to assess wound healing is very complex, and it would be a challenge to describe all options available. Thus, the main goals of this article are to provide, firstly, some frequently used in vivo models (eg incisional, excisional and impaired wound models) and, secondly, a variety of methods such as wound healing rate, image analysis, biophysical assays, histopathological, immunological and biochemical assays. The models and methods outlined in this manuscript are intended to provide a practical approach to researchers in their experimental protocols with the potential to move forward to translational studies.
2 MODELS
Experimental wound healing models have been developed over many decades in attempt to understand the tissue repair process and test new treatment protocols. Such models are usually divided into two groups: in vitro and animal (in vivo or preclinical) models, each offering advantages and shortcomings.6 Figure 1 depicts an overall design to assist researchers on the choice of different wound healing models.
In vitro models such as cell culture, scratch model and skin explant culture, are essential in several of these studies, since important preliminary results will lead to the design of subsequent investigations. However, the authors believe that a discussion on in vitro models is beyond the scope of this present manuscript which is focused on in vivo models.
In vivo models involve wounding a laboratory animal and observing wound closure over time. Physical, chemical or biological modifications of the wound environment can also be incorporated.6, 29 Different species are used for cutaneous wound experiments including rodents (rats, mice), rabbits and pigs. Some small mammals can easily be genetically altered and provide a model capable of approximating defective human conditions such as diabetes, immunological deficiencies and obesity.2, 6, 24
Investigations using animal models should follow the principles of the 3Rs (replacement, reduction and refinement) to ascertain that ethical and humane treatment of the animals will be followed, respecting the animal welfare. The first principle, replacement, means the use of non-sentient animals (eg fish) or materials rather than conscious living animals; the second principle, reduction, means to reduce the number of animals used in an experiment or procedure; and lastly, the third principle, refinement, means to use techniques to decrease the incidence or amount of animal pain and distress.30
In vivo models remain the most predictive models for studying wound healing, allowing a realistic representation of the wound environment including various cell types, environmental cues and paracrine interactions.31 The model chosen should consider characteristics such as the accurate reproducibility of the lesion, the possibility for multiple investigations, the ability to obtain multiple biopsy samples, the compatibility with animal facilities, the ease of handling and the time required to obtain valuable results.32 The ideal model is one that represents certain aspects of human physiology but does not require human volunteers for experiments.33 Small animals usually have accelerated healing time in comparison with humans, and thus experimental duration lasts for days, as opposed to weeks or months in human studies.2
The most widely used species are rats and mice. Although there are documented differences between the structure and physiology of rodent and human skin, wound healing studies designed with these differences in mind can provide valuable translational information.29 Rodents are easy to house, handle and maintain, and a wide variety of specific reagents are available for research purposes.31, 34
Rodents’ skin is unique in having a panniculus carnosus layer (a thin muscle layer that is only found in the platysma of the neck in humans), which produces rapid wound contraction following injury. In contrast, human wounds heal via re-epithelialization and granulation tissue formation, important differences to consider when assessing the translational relevance of rodent studies. Another point to consider when designing the experiments is the gender difference in skin anatomy and physiology. For example, male skin is 40% stronger due to a much thicker dermis, while female skin exhibits a thicker epidermis and subcutaneous layer.35 Despite these differences, these models have contributed significantly to our understanding of skin biology and disease.31 In our experience, when using rodents for wound healing studies, we believe that rats are a better choice than mice. Some of the reasons are the skin difference between them and the small size of mice. Mouse skin is thin and has less layers of keratinocytes when compared to rat skin and wounds will heal in about 7 days, while in rats, it is possible to evaluate healing for about 12-14 days. The wound size should be proportional to the animal size, and thus, wounds in mice are smaller than in rats and will heal fast. However, experiments that require minimal sample sizes for analysis, mice can be more suitable. When a larger sample size is necessary, a larger animal, such as rats or rabbits, will be more suitable since multiple wounds can be inflicted in one animal.
Rabbits have been used for many years as a wound healing model, known as rabbit ear excisional wound model. Wounds are created by a biopsy punch and include damage to the epidermis, dermis and cartilage. The repair occurs from the margin inwards, and there is no contraction as opposed to murine models. Wounds in this model will heal by re-epithelialization, which is one of the advantages of this model.6, 25, 36 Furthermore, this model increases the possibility of using a small number of animals, providing enough data for within-animal replicates: for example, up to 6 wounds per ear can be created.25
Although rat, mouse and rabbit models have been used in experimental dermatological studies, pig skin has been shown to be the most anatomically and physiologically similar to human skin.37
Structurally, epidermal thickness and dermal-epidermal thickness ratios are similar in pigs and humans. Their skin is relatively hairless and has a fixed subcutaneous layer and dermal hair follicles like humans.6, 37 Biochemically, porcine skin contains dermal collagen and elastic content that is more similar to humans than other laboratory animals. Pigs also have similar physical and molecular responses to various growth factors.37 However, they are expensive to house and maintain, and have a greater risk of infection; and molecular reagents are often not validated for swine tissues.31, 32, 34
Overall, in vivo wound models have advantages. They allow the study of multiple cell populations/body system interactions during repair; allow the investigation of multiple elements of the healing process; allow selective depletion of specific genes to determine their effect on wound healing; permit the study of a functional immune system; enable the creation of multiple wounds within one animal; and also can model different wound healing causes (burns, surgery, crushing, etc).6, 32, 38
On the other hand, in vivo models have disadvantages including the complexity of the whole animal that prevents the clear analysis of distinct contribution of tissues/cells during the healing process and could be expensive; animal skin is not an exact replica of human skin; many animals are usually required to reach statistical significance; the immune response might differ from humans; it could be hard to control the size/depth/dimensions of the wound; and no accurate and reproducible chronic wound model representation has been obtained.6, 32, 39
2.1 Incisional wounds
This model can be beneficial for studies investigating surgical incision materials, such as degradation of different suture threads and mechanical properties through the evaluation of tensile strength.40
Incisional wounds can be classified as primary or secondary closure, and sutured immediately after wound infliction or not respectively. Primary closure or first intention is an excellent model for biomechanical analysis of wound strength and less suited for histologic assessment of healing, or evaluation of wound tissue biochemistry or epithelialization, due to the limited volume of wound healing activity. The secondary closure model of incisional wounds, or second intention, can be valuable to investigate scarring at late time points (over 65 days after incision).41
Mice, rats, rabbits and porcine can be used as a model to evaluate healing on incisional wounds.40, 42, 43 Reid et al39 reported several experimental animal models to investigate healing such as incisional injury on mice back allowing tensile strength measurements by tensiometry. Januszyk et al42 used the technique to evaluate stress in surgical incision in pigs. Also, Most et al43 used knockout iNOS mice to explain the cellular mechanism in wound healing in an incisional model.
2.2 Excisional wounds
Excisional wounds are one of the most commonly used wound healing models29 and are considered to resemble acute clinical wounds, which require healing by second intention, that is, the skin edges are not sutured together. These wounds are generated by the surgical removal of all skin layers (epidermis, dermis and subcutaneous fat) from the animal. This model allows the investigation of haemorrhage, inflammation, granulation tissue formation, reepithelialization, angiogenesis and remodelling. Wound area can be recorded (photographed regularly) over time, and wound closure is calculated based on wound size relative to the original dimensions (wound healing rate). For histological analysis, animals can either be euthanized (eg mice, rats) or locally anesthetized (eg rabbit ear model) and biopsies collected, processed and examined for both the epithelial gap (the quantifiable distance between the epithelial wound margins), granulation bed characteristics (recruited cell populations, vascularity and matrix alterations) and collagen organization.4, 6, 31, 32, 41
The material and techniques necessary to use this model are relatively simple and practical. The wound bed can be easily accessed to apply topical agents (eg pharmaceuticals, cells, biomaterials, wound dressings) and investigate their effect on the repair process.31, 41
Different animals can be used, such as mice, rats, rabbits and pig, and a different number of wounds may be created on each animal. It is possible to inflict for instance: up to two excisional wounds in mice,22, 44-48 four wounds in rats,23, 26, 27 four or more in pig34, 49 and six wounds in rabbits25, 36, 50 (Figure 2). The number of wounds per animal will depend on the type of excision performed and on the biopsy punch diameter.
It is noteworthy that wounds in some of the classic excisional models, especially mice, will heal by contraction, which accounts for a large part of wound closure. Wound contraction is one of the most significant limitations of using loose-skinned animals to model human wounds. In attempt to overcome this situation, approaches include choosing anatomical sites with firmly attached dermis and subcutaneous tissue (eg rabbit ear) or performing mechanical fixation of the skin by using certain devices or splints.51
The use of splinted wound model has been proposed and discussed to inhibit dermal contraction and increase the relevancy of the murine model to wound healing in humans.22, 51, 52 This technique involves the creation of two full-thickness wounds extending through the panniculus carnosus on the mouse dorsum, followed by the placement of silicone splints centred and fixed to the skin with immediate-bonding adhesive and nylon sutures.22 In the mouse excisional splinted wound model, the splinting ring tightly adheres to the skin around the wound, preventing contraction. Therefore, the wound will heal through granulation tissue formation and reepithelialization, similar to the process in humans.48
2.3 Burn wounds
Burn wound healing models can be generated by either water scalding the skin or by thermal damage. In the first model, blisters are created by exposing a fixed area of skin to hot water, and the second consists of direct application of heat to the skin by a hot metal plate. In both models, a blister can be de-roofed to expose the dermis and leave an open wound.6
Burn wounds by thermal injury can be used for measuring re-epithelialization, granulation tissue formation, angiogenesis, contraction, scarring and wound tissue biochemistry, depending on the depth of burn. The extension of the burned area will depend on the study goals as well as on the required time to follow-up on the effects of an experimental treatment. A small area certainly will heal fast and may not be appropriate to acquire enough data.
Caliari-Oliveira et al53 described the use of an extensive and severe burn model in rats, comparable to human third-degree burns, to evaluate the potential of xenogeneic mesenchymal stromal cells as a potential treatment of severe burns and to accelerate the healing process. The extensive burn wounds (2cm x 3cm) affecting the three skin layers were created with light pressure of a metal plate heated to 200°C against the animal's dorsal area for 25 seconds. Wound healing was assessed by digital photographs of the wounds over time, histopathological analyses (vascularization, granulation tissue, total polymorphonuclear inflammatory cells and collagen fibres) and by myeloperoxidase assay (MPO).
2.4 Impaired wound models: diabetes, marasmus and obesity
In order to study chronic wounds and develop potential treatments, models that can replicate the chronicity associated with a non-healing condition are often used. The goal of using such models is to mimic the pathology associated with chronic wounds as seen in patients. There are different approaches to obtain impaired wound models. For instance, vascular to metabolic conditions can be inflicted as well as genetic modifications. It is noteworthy that most models of impaired acute wound healing can provide useful information on delayed wound healing; however, they are not considered models of true chronic wounds.54, 55 Some of the most frequently used models to mimic an impaired healing process are diabetes and nutritional-related conditions frequently observed in the wound care clinic.
2.4.1 Diabetes
The impairment in diabetic wound healing is a multifactorial process affecting numerous mechanisms necessary for healing. Diabetic wounds in humans and laboratory animals have been shown to display an irregular inflammatory response and decreased neovascularization, as compared to non-diabetic wounds.56
Diabetic animal models can be obtained by chemical, dietary induction, surgical manipulations or through systemic mutations such as diabetes/diabetes (db/db) and obese/obese (ob/ob) mice.
Chemically induced type 1 diabetes is usually obtained by the administration of drugs such as alloxan or streptozotocin. Alloxan-induced β-cell necrosis has been used for inducing experimental diabetes since the initial findings in 1943. This drug destroys the pancreatic β islets leading to insulin deficiency, hyperglycaemia and ketosis.57 Like alloxan, streptozotocin causes hyperglycaemia mainly by its direct cytotoxic action on the pancreatic β-cells. In susceptible rodents, it induces an insulinopenic diabetes in which immune destruction plays a role, as in human type 1 diabetes.58-60
Although chemically induced diabetes provides a simple and relatively cheap diabetic model, there is some evidence that it is not completely representative of diabetes in humans. On the other hand, genetic models allow the investigation of the natural mechanisms of diabetes without the potential side effects associated with chemical treatment. Models such as db/db, ob/ob of type 2 diabetes are close to the human disease, as these animals naturally develop hyperglycaemia as a consequence of obesity and exhibit many of the symptoms observed in type 2 diabetic patients. In particular, the db/db mice become hyperinsulinaemic at around the age of 2 weeks and develop resistance to insulin, eventually exhibiting advanced hyperglycaemia due to beta-cell failure.61
Diabetes and insulin resistance can greatly increase the risk of cardiovascular disease and cutaneous ulcerations in patients presenting with these disorders. Oxidative stress has also been postulated to cause microvascular complications including retinopathy, nephropathy and neuropathy as a result of hyperglycaemia and diabetes.62
2.4.2 Malnutrition
During wound healing, an increase in metabolic demand for nutrients occurs as restoration of the structural and functional integrity of damaged tissues progresses.63, 64 Protein-energy malnutrition (PEM) affects the skin, causing significant morphological and functional changes predisposing it to damage (ulceration) with a consequent impairment in healing. Malnutrition results in changes in the inflammatory reaction, immune function and tissue repair, leading to an increase in pro-inflammatory cytokines, a delay in the healing process and a greater risk of infection.65, 66
Leite et al67 investigated the changes that occur in the trophic skin of rats in an experimental model of marasmus (type of PEM). The animals were randomly assigned to receive rat chow ad libitum (well-nourished group) or half of the daily diet (malnourished group) and were followed up for two months. Histological results showed that the dermis of the malnourished animals was significantly thinner and the percentage collagen was lower compared to that of the well-nourished animals. In another study, Leite et al23 used the same experimental model to analyse the effects of malnutrition on healing of cutaneous wounds, by the ulcers healing rates (UHRs) and histological analysis of collagen. Undernourishment significantly impaired healing, and a low percentage of collagen was observed histologically. Their findings corroborate previous studies, showing that malnutrition impairs wound healing and delays collagen synthesis.63, 65, 66
2.4.3 Metabolic syndrome/obesity
Other systemic factors such as metabolic diseases (metabolic syndrome, obesity) increase the concentration of reactive oxygen species (ROS) and alter the wound healing process.67-69 In these conditions, the inflammatory phase suffers prolonged disturbances in the process of angiogenesis and reduction of granulation tissue, and there is a delay in epithelialization. In addition, the cellular redox environment is altered leading to oxidative stress, which can cause important cellular changes. The development of metabolic diseases may be triggered by a high-calorie diet,70 and many studies have used a high-fat diet in animals to mimic these metabolic changes in humans.
Leite et al24 used an experimental model of high-fat diet to investigate the wound healing process in rats with the purpose of mimicking chronic wounds in patients with metabolic disorder/obesity. After receiving normal and high lipidic diet for 45 days, the nutritional status was measured and excisional wounds created on the back of the rats. Animals that received the high-fat diet showed increased blood glucose, triglycerides and total cholesterol when compared with animals receiving a control diet. Besides, the high-fat diet group demonstrated lower hydroxyproline content, elevated levels of malondialdehyde (MDA) and lower levels of reduced glutathione (GSH), which are reflections of the oxidative stress related to the dysmetabolic condition (condition of altered metabolism by hyperlipidaemia).71
Along with the selection of an appropriate model, which should aim for reproducibility, clinical relevance, humane treatment and quantitative interpretation, it is important to determine which methods will be suitable for a particular model and the purpose of the study. In the next section, we will present some of the common methods used in wound healing studies.
3 METHODS
Progressive changes in wounds during the healing process can be assessed by several techniques, each analysing specific parameters. Appropriate wound assessment is dependent on the understanding of the healing pathophysiology, factors that delay the process and optimal conditions required at the wound bed to maximize healing and therapeutic effectiveness. It is important that a comprehensive analysis of the healing progression of the wound itself and surrounding tissue is carefully performed and documented during follow-up.72, 73
Measurements in wound healing generally refer to wound size, characteristics of the wound bed and whether they correlate with tissue growth, extent of scarring, and many of the underlying vascular and pathophysiological disorders that may be causing impaired healing.74 Methods to evaluate the wound progression include qualitative and quantitative protocols, which should be performed until the tissue is restored.
In the next sections, we will present some methodologies that range from clinical standard visual inspection, evaluation of re-epithelialization by wound healing rate, to histopathological analyses, immunological (cytokines, growth factors) and biochemical (collagen metabolism, myeloperoxidase, N-acetylglucosaminidase and oxidative stress) assays to physical wound assessments, as outlined in Figure 3.
3.1 Wound healing rate
The endpoint of a successful treatment is the complete and permanent wound closure. In clinical practice, the rate of change in wound surface area, also known as wound healing rate, is the best way to quantify the healing progress, since the most established clinical marker during a wound follow-up is its size.75, 76 Several ways of measuring the surface area are available, ranging from measuring the length and width by a ruler to image analysis algorithms.
Another approach is to understand how cellular repair behaviours are dynamically coordinated at the single-cell level and how a tissue orchestrates the cellular mechanisms necessary to re-epithelialize a wound, while maintaining homeostasis in a live mammal. To assess these behaviours that result in tissue-scale changes underlying wound repair, Park et al77 have proposed an intravital imaging (live cell imaging) method to study wound repair in real time using live mice genetically labelled with fusion protein of histone H2B with green fluorescent protein (GFP) driven by the keratin 14 promoter (K14-H2BGFP mice). A circular full-thickness wound on the dorsal side of mouse ear, stated as a convenient region for imaging and for its similarity with respect to epithelial functions to other regions of mouse skin, was created and imaged in live mice under anaesthesia. Image stacks and serial optical sections were acquired with a laser scanning microscope, and migration and proliferation (re-epithelialization) rates were determined by imaging software.
To asses wound re-epithelialization clinically and experimentally (in vivo and in vitro, eg scratch assay), wound healing rate (WHR) or ulcer healing rate (UHR) index is used and can be calculated following the equation: [(Ai − Af)/Ai], where Ai represents the initial wound area and Af represents the final area/measurement. The results from WHR can be expressed in arbitrary units, usually ranging from −1.0 to 1.0 or in percentage closure. In this case, the result from the equation is multiplied by 100. WHR equal to 1 or 100% means complete reepithelialization, while WHR equal to 0 or 0% means no signs of reepithelialization; WHR >0 or 0% means a decrease in area and WHR <0 or 0% an increase.20, 23, 25-27, 45, 46, 78-80
Wound tracing is an inexpensive, accessible and conventional method utilized both in the clinical and research settings for measuring wound size and sequential comparisons of tracings permit monitoring of healing progression. A transparent film is placed over the wound surface and the wound perimeter traced with a permanent marker. From the tracing, the wound surface area measurement can be performed. Though wound tracing is relatively non-invasive, there is a risk of patient discomfort, wound contamination and wound bed damage from taking the measurements.4 As an alternative to wound tracing, digital photographs of the wound surface can be taken and wound parameters calculated using different image software.76, 81, 82
3.2 Wound analysis by image
Photography is a valuable tool in medicine; especially in dermatology, since an image can provide information that is often synonymous with diagnosis, it is non-invasive and can help with the case documentation and follow-up.83 Images can also provide important information on morphological changes, colour variations and so forth, during wound healing progression in both clinical and experimental settings.83, 84 Furthermore, digital photographs can be electronically transferred, ideal for remote wound management and interdisciplinary analyses.4
When taking digital photographs is important to use a camera with high resolution,4 the number of pixels must be high enough to produce an original digital representation; the more pixels per image, the more detail the camera has captured per image.83 Moreover, high-resolution images can clearly identify epithelial growth at the wound margins improving outcomes reliability.4
Special care must be taken during the capture of images such as the standardization of the distance where the camera is positioned, preventing inaccurate visual perception; photographs should be captured perpendicularly to the wound, as non-perpendicular images may significantly underestimate surface area; lighting should not focus directly on the wound bed avoiding reflection; the use of camera flash is not recommended, as it could cause glare or shadows; as a standard of measurement, a calliper or ruler should be placed next to the wound. A standardized photographic protocol will enable the subsequent computer analysis and quantification of the wound area—after the image is acquired (that is photographed), the image will be opened with software that will convert the number of pixels to cm2, allowing the wound area to be quantified.4
The use of digital photography and the subsequent assessment of healing by image analysis software has been widely used and provides accuracy in monitoring patients’ response to treatment,78-80, 83, 85 as well as in research settings using animal models.23-27, 44, 45
3.2.1 Image analysis by software
The integration of commonly used digital cameras, personal computers and computerized planimetry software makes wound image analysis affordable, user-friendly and practical in clinical and research settings.4, 19, 20, 67, 78 Once the wound image is in the computer, the wound perimeter is manually traced using a mouse. A variety of software is available to compute and analyse wound dimensions, surface area and volume (if wound depth is provided), and also provide data about clinical aspects of wound healing by the colorimetric analyses for monitoring changes in surface area by tissue type and its respective colour such as granulation tissue (red), fibrin/sphacelus (yellow), necrosis (black), among others.4, 19, 20, 67, 80, 86 Also, there are many software designed to assist image analysis from histological, immunohistochemical, enzymatic and fluorescent methods, from slides with tissue biopsies, cell counting (eg fibroblasts, blood vessels) and collagen analysis can be performed. This technology is non-invasive; there is no discomfort, risk of contamination or chance of damaging the wound bed during measurements.4, 19, 20, 67, 78
An example of an image analysis software package is ImageJ, freely available from the National Institutes of Health (NIH) website. It can quantify perimeter and surface area from digital images and perform colour analyses. ImageJ is widely utilized in research and clinical practice and has been used to evaluate changes in diabetic foot ulcers, chronic diabetic leg ulcers,85 chronic venous ulcers4, 78 and in many other applications such as those shown by our group.19, 20, 24-27, 45, 67, 79, 80
Several reports have shown the application of image analysis methods to assess wound healing. Geer et al33 used ImageJ to examine wound reepithelialization and angiogenesis. Chiquetti-Jr et al87 used Image-Pro® plus 4.5 (Media Cybernetics, Inc) for histological analysis (cell count and quantification of collagen fibres) to evaluate the effect of immunosuppressive drugs in wound healing in rats. Mendonça et al5 described the application of two programmes: Scion Image (Scion corp.) for histomorphometrical photomicrographs of sections stained with Masson's trichrome for the inflammatory cells’ quantification and measurement of epidermis and dermis area, and Image-Pro® plus 4.5 for collagen quantification (total collagen and type I and III collagen) in Picrosirius stained slides. Gonçalves et al88 used Sigma-pro® image (St. Louis, MO, USA) for inflammatory cells counting, collagen formation and reepithelialization.
Other software described in the literature for wound area/re-epithelialization measurements, and changes in wound dimensions and colour include the following: AutoCAD® 14 software (Autodesk®, Inc),89 Image House Program 5.11,90 DigiSkinTM,91 Verge Videometer—VeV (Verg, Inc),76 Image-Pro Plus,92 Visiopharm93 and LEICA QWin Image.94
3.3 Biophysical assessment of wound healing
Another non-invasive approach to assess wound healing consists of the use of biophysical techniques such as optical coherence tomography (OCT), confocal laser scanning microscopy (CLSM) and diffuse near-infrared spectroscopy (DNIRS) among others.
Optical coherence tomography is an emerging technology in diagnosis and monitoring of inflammatory dermatological conditions that generates high-resolution real-time images of cutaneous architecture. Greaves et al95 compared OCT with histological assessment of in vivo acute wound healing to determine the level of agreement in terms of inflammation, proliferation and remodelling. The results were comparable, and the authors suggested that OCT might represent a diagnostic alternative to punch biopsies.
Tsai et al96 used OCT for in vivo wound healing studies after non-ablative fractional lasers or ablative fractional laser treatments. To monitor the wound healing process, the treated areas were scanned at different time points and an algorithm developed to quantitatively evaluate the morphological changes at different tissue depths during healing. Sattler et al97 performed a study to determine whether OCT could be used to quantify the kinetics of the dynamic wound healing process. In this study, they also evaluated the use of confocal laser scanning microscopy (CLSM) and concluded that OCT made it possible to visualize skin changes and the depth of visualization using OCT was superior to CLSM.
Another biophysical method is the diffuse near-infrared spectroscopy (DNIRS), a technology that measures the level of oxygen in the blood at the wound site. DNIRS uses 70-MHz modulated light in the diagnostic window (650-900 nm) to quantify levels of oxy- and deoxy-haemoglobin in the wound bed, which when measured over time, can show a trend towards or away from healing.98
In many clinical and experimental wounds, other methodologies might be necessary to evaluate the healing process. These methods are invasive and would require biopsies. However, they will provide more thorough information on the healing progression at the histological, immunological, molecular and biochemical levels.
3.4 Histopathological analysis
Histopathology of wounds is a very helpful tool to exclude a malignancy cause, monitor healing progress in the course of treatment, better understand the pathophysiology of non-healing wounds, assess morphological changes and help with diagnosis.18, 99 Clinically, the best site for biopsy is the edge of the wound because it enables the comparison between the ulcerated area and the surrounding skin.18 In the laboratory setting, a biopsy for histopathological analysis will encompass the whole wound, including the edges.44-46
Immediately after harvesting, tissue samples should be placed into specific solutions such as 10% buffered formaldehyde, one of the most common fixatives, to maintain their integrity without cellular structure changes. Biopsies can also be frozen, by placing the specimens in Tissue Tek® Optimal Cutting Temperature (OCT), and stored at −80ºC until processing. These embedding and storage conditions are advised if molecular analyses may be required. The tissue will then be subjected to the several steps of histological processing, including embedding, sectioning and staining.100 The most widely used stain in wound pathology as in general dermatopathology is haematoxylin and eosin (H&E). Special stains (non-H&E), trichromes and immunohistochemical markers are used to highlight tissue components or foreign materials non-visible or less visible on the H&E sections.18
Histopathological analysis of wounds usually includes quantification of white blood cells (macrophages, mast cells, lymphocytes and neutrophils) to evaluate the inflammatory phase; blood vessels to evaluate angiogenesis; fibroblasts and collagen. The assessment of collagen fibres is also important, since the arrangement and orientation of collagen play a vital role during the remodelling phase and consequently on the final scar appearance after wound closure. Picrosirius red is a special stain that demonstrates both thin and thick collagen fibres.45, 101
Trichrome stains consist of three different colours (red, green and blue) and normally highlight collagen fibres and muscle. Some of the most common trichromes are as follows: (a) Gomori's trichrome, which stains muscle fibres in red, collagen in green and nuclei in blue to black; collagen may also be stained in blue when fast green or light green from the trichrome are replaced by Aniline Blue24, 27, 44, 67 and (b) Masson's trichrome, which stains muscle and intercellular fibres, cytoplasm and keratin in red, collagen in blue and nuclei in black.102
3.5 Immunological methods
Within the complex cascade of biological events in wound healing and other processes, a vitally important concept is how cells communicate with each other and with the extracellular matrix. One mechanism of cell-to-cell communication in vivo is via soluble or membrane-bound factors (cytokines and growth factors), which stimulate endogenous repair mechanisms by providing the signals to cells and thereby leading to a functional restoration of damaged tissues. These signalling molecules can be identified and quantified by different techniques such as immunohistochemistry and enzyme-linked immunosorbent assay (ELISA).103-107
3.5.1 Immunohistochemistry
Immunostaining of wound tissue is helpful to identify molecular surface markers, cytokines and growth factors of value to predict the wound progression. The staining may be undertaken using either cryopreserved or paraffin-embedded tissue sections. Immunohistochemistry (IHC) involves the binding of a primary antibody to the antigen of interest and the detection of the bound antibody by a detection/visualization system. A variety of enzymatic [eg avidin-biotin peroxidase plus 3,3′-diaminobenzidine tetrahydrochloride (DAB)] or fluorescent systems are available, and the selection by the researcher will depend upon affordability, reliability, sensitivity, the signal-to-noise ratio and convenience.19, 79, 108, 109
Paraffin-embedded tissue sections will require processing prior to immunostaining procedure to unmask the antigenic sites of the tissue proteins. The antigen retrieval may be performed enzymatically by using pepsin, trypsin and K-proteinase, or by using heat treatment with citrate buffer. Antigen retrieval pretreatment is not necessary for cryopreserved tissue sections.110
The use of a polymeric labelling (eg NovoLinkTM Polymer Detection System, Novocastra Laboratories Ltd) that uses a controlled polymerization technology to prepare polymeric horseradish peroxidase (HRP) antibody conjugates can avoid non-specific staining that could occur with streptavidin/biotin detection systems due to endogenous biotin. This system allows identification of antigens in sections of formalin-fixed and paraffin-embedded tissue. After immunological reactions are completed, sections are counterstained with haematoxylin, coverslipped and analysed.111
Immunohistochemical monitoring of wound healing involves a variety of markers that can be determined based on the phase of healing that is being assessed. Some of these markers can be investigated by IHC and by other protocols, as described in other sections of this review. Suitable markers to monitor healing using IHC include collagen I, III and IV, IL-1β, IL-10, IL-17, TGF-β, VEGF, cytokeratins 10, 14, vimentin, fibronectin, laminin, α-SMA, CD31, CD34, among others.19, 112-116
Investigation of angiogenesis by IHC using endothelial markers such as CD31 and CD34 is a common approach described in wound healing studies. Tissue sections are incubated with primary (eg anti-CD31, anti-CD34) and secondary antibodies (biotinylated or HRP-coupled, or fluorescence antibodies) and stained. After immunostaining, the quantification of blood vessel density can determined by counting the stained endothelial cells and/or calculate the percentage of total vessel stained area.112 The average number of blood vessels counted in all fields provides a microvascular density.117 Tissue sections are usually imaged using an optical or fluorescence microscope (when the secondary antibody has a fluorescent conjugate), and an average of 10 images is captured in determined regions of interest (ROIs) with the highest density of blood vessels. The counting of blood vessels can be performed on the acquired images by computerized image analysis systems, such as ImageJ.19, 114, 117 With this software, blood vessels can be counted using the plug-in ‘cell counter’ and the percentage stained area obtained using the plug-in ‘colour deconvolution’.118 This last plug-in unmixes an RGB image produced by subtractive mixing (eg histological dyes) into separate channels corresponding to up to three determined colours. In IHC images, when tissue is stained with DAB the plug-in will separate brown/orange colour.
3.5.2 Enzyme-Linked Immunosorbent Assay
ELISA is a common laboratory technique used to measure the concentration of an analyte (usually antibodies or antigens) in solution and can provide important information on wound healing progress by quantification of important components of repair such as cytokines and growth factors from tissues samples (homogenates) or cell culture supernatants.
After collecting a tissue biopsy, it is transferred to a tube with protease inhibitor buffer for ELISA, homogenized and centrifuged and the tissue homogenates or cell culture supernatants are used for the assays.119 Among the most investigated cytokines and growth factors in wound healing studies are as follows: pro-inflammatory cytokines such as tumour necrosis factor (TNF-α), interleukin-6 (IL-6), IL-1β, interferon-gamma (IFN-γ), anti-inflammatory cytokines such as IL-10; epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β1) vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF).28, 45
3.6 Biochemical methods
Several biochemical assays such as hydroxyproline, myeloperoxidase assay (MPO), N-acetylglucosaminidase (NAG) and oxidative stress profile can be performed to monitor the progression of the wound healing process.
3.6.1 Hydroxyproline assay
Collagen is the major protein component of connective tissue and is composed primarily of glycine, proline and hydroxyproline. Collagen synthesis requires hydroxylation of lysine and proline, and co-factors such as ferrous iron and vitamin C.69 Breakdown of collagen liberates free hydroxyproline and its peptides. Hence, measurement of hydroxyproline can be used as a biochemical marker for tissue collagen and an index for collagen turnover.2, 120 Increase in the hydroxyproline content indicates increased collagen synthesis, which corresponds to an enhanced wound healing.121
Hydroxyproline can be analysed in tissue (biopsies) and in cell culture supernatants.20, 24, 28, 45, 122-124 It is an important quantitative biochemical marker for collagen in wound healing and has been widely used as an indicator of both the presence and metabolism of collagen.4, 120, 125 Measurement of hydroxyproline can be carried out by colorimetric methods,45, 126 high-performance liquid chromatography (HPLC), gas chromatography/mass spectrometry and enzymatic methods.126, 127 All methods have two common steps: (a) hydrolysis of sample with either a strong acid or alkali to release hydroxyproline and (b) detection of free imino acid by either colorimetric, fluorimetric or HPLC techniques.
Stegemann and Stadler128 originally introduced a method using acetate-citrate buffer, chloramine-T and Ehrlich's reagent, with assessment at 550 nm using a spectrophotometer, that was later modified by Huszar et al.129 Modifications include a change on hydrolysis procedure, reduction of the sample volume and omission of the neutralization step, other minor modifications have been made to optimize the conditions for the measurement of hydroxyproline in biological samples or cell culture supernatants. The method is essentially based on the acidic or alkaline hydrolysis of samples and subsequent quantification of free hydroxyproline in the hydrolysate. The entire procedure comprises three steps: (a) hydrolysis, (b) oxidation and (c) development of chromophores.24, 26, 44, 45, 122-124
A study has shown that a simple, convenient and reliable computer-aided histomorphometrical method for determining total collagen in cutaneous wound specimens was comparable to the gold standard hydroxyproline assay. The methods proved to be equivalent, and therefore, the methodology of choice will depend on the laboratory resources. Noteworthy, both methods are considered useful tools in clinical and biomedical research.122
3.6.2 Myeloperoxidase assay
Myeloperoxidase (MPO) assay can estimate the recruitment and accumulation of neutrophils in the tissue and evaluate the inflammatory phase of wound healing. MPO is a member of the peroxidase-cyclooxygenase superfamily that includes lysozymes, elastases, cathepsins, acid hydrolases and lactoferrins. It is a proteolytic enzyme present in cytoplasmatic granules of the polymorphonuclear neutrophils and participates in innate immune defence mechanisms through the formation of ROS.19, 20, 45, 130, 131
After a tissue is biopsied, the fragment is immediately frozen in phosphate buffer and stored at −80°C until the assay is performed. The fragment is weighed and homogenized, and the pellet goes through hypotonic lysis. After centrifugation, the pellet is re-suspended in buffer containing hexadecyltrimethylammonium bromide and re-homogenized. Aliquots undergo two cycles of freezing-thawing using liquid nitrogen and are centrifuged, and the supernatant diluted in sodium phosphate to be used for the assay. The myeloperoxidase activity is measured using a chromogenic substrate (3,3′,5,5′-Tetramethylbenzidine) at 450 nm. Results are expressed as total number of neutrophils × 103 per milligram of tissue comparing to controls in a standard curve19 or also as optical density per milligram of tissue.132, 133
3.6.3 N-acetylglucosaminidase assay
Neutrophils also have a role of recruiting macrophages to the wound site, which in turn are present in all stages of wound healing, phagocytizing pathogens and dead cells at the wound bed and producing important cytokines and chemokines.131 N-acetylglucosaminidase (NAG) is a lysosomal enzyme highly expressed in activated macrophages, being an important inflammatory marker. Indirect measurements can estimate the levels of NAG present in activated macrophages as follows: the supernatant (part of the supernatant used to measure MPO activity can be used for this assay) is incubated with a solution of p-nitrophenyl-2-acetamide-β-d-glucopyranoside and citrate buffer, and after incubation, the reaction is terminated by the addition of glycine buffer. Hydrolysis of the substrate is determined by measuring the colour absorption at 405 nm, and NAG activity is expressed as the change in optical density per milligram of tissue.134
3.6.4 Oxidative stress
Reactive oxygen and nitrogen species (ROS, RNS) are fundamental in many pathophysiological and biochemical processes, maintaining the cell homeostasis, as long as a fine balance between their formation and removal is present. However, when there are significant changes in this balance, that is tissue injury, a pro-oxidant state is generated leading to an oxidative stress.135
Among the ROS formed in the skin are the radicals hydroxyl (HO•), superoxide (O2•−), alkoxyl (RO2• and RO•), the singlet oxygen (1O2),136, 137 hydrogen peroxide (H2O2) and the organics (ROOH). Besides ROS, other intermediate species, such as •NO (nitric oxide), are involved in redox processes with significant biological importance.
Several biomarkers of cellular stress and antioxidant defence mechanisms can be of a great value during wound healing investigations. Among these markers are malondialdehyde (MDA) and hydrogen peroxide.19, 24
MDA is a secondary product of lipid peroxidation, a potential biomarker of oxidative damage. It is the main indicator of lipid peroxidation determined by titration against thiobarbituric acid (TBA), which is a cell damage indicator.138 Hydrogen peroxide is another marker of cellular stress and when overproduced would impair healing. Its presence in biological samples can be determined by the ferrous oxidation xylenol orange assay (FOX).139
Quantification of oxidative stress in the skin can also be assessed by chemiluminescence, a technique that allows the detection of radical levels (when they are increased) by the emission of produced photons. However, this technique can only be used during oxidative stress, since under normal circumstances, the amount of ROS generated by physiological processes is too low to be detected.140
3.7 Flow cytometry
Flow cytometry is another technique that can provide information during investigations involving cellular recruitment in response to a treatment in wound healing. This technology simultaneously measures and analyses multiple physical characteristics of single particles, usually cells, as they flow in a fluid stream through a beam of light. Particle's relative size, relative granularity or internal complexity, and relative fluorescence intensity are determined by an optical-to-electronic coupling system that records how the cell or particle scatters incident laser light and emits fluorescence.141
Investigation of wound healing by flow cytometry has been focused on endothelial and immune cells that are particularly relevant to re-establishing capillary networks and wound bacterial defence. The most used human markers are for endothelial progenitor cells (CD133), endothelial cells (CD31, CD34 and VEGFR-2), macrophages (CD68), T cells (CD3, CD4, CD8) and B cells (CD20).141 The same cells can be studied in an animal wound healing model, but other antibodies must be used depending on the animal species (mouse, rat, rabbit).44
3.8 Macrophage polarization
Macrophages are one of the key regulators of the wound healing process and as it progresses, their phenotype changes, reflecting a differentiation process that shifts their functions from inflammation to proliferation and it is called polarization.142
During acute wound healing, inflammatory macrophages, traditionally referred to as M1, infiltrate after injury to clean the wound of microorganisms, foreign debris and dead cells. As the tissue begins to repair, the overall macrophage population transitions to reparative macrophages (M2) along with migration and proliferation of fibroblasts, keratinocytes and endothelial cells to restore the dermis, epidermis and vasculature.143
Classically activated or inflammatory macrophages (M1) are recognized as CCR2high Ly6C+ in mice and are typically induced by Th-1 cytokines, such as IFN-γ and TNF-α, or by bacterial lipopolysaccharide (LPS) recognition. These macrophages produce and secrete high levels of pro-inflammatory cytokines TNF-α, IL-1α, IL-1β, IL-6, IL-12, IL-23 and cyclooxygenase-2 (COX-2), and low levels of IL-10. M1 macrophages have robust antimicrobial and anti-tumoral activity and mediate ROS-induced tissue damage, and impair tissue repair. The inflammatory response is inhibited by regulatory mechanisms driven by anti-inflammatory function of M2 macrophages in order to protect against tissue damage.144, 145
Alternatively activated, anti-inflammatory or reparative macrophages (M2) are recognized as CCR2low Ly6C−.145 M2 macrophages polarize by Th-2 cytokines, IL-10, TGF-β, IL-4, IL-13, IL-33 and IL-21 via activating STAT6 through the IL-4 receptor alpha (IL-4Rα). M2 cells orchestrate the promotion of tissue remodelling,146 angiogenesis, immunoregulation, tumour formation and progression.147
Depending on the activating stimulus received, M2 macrophages can be further divided into four different subsets: M2a, M2b, M2c and M2d. M2a subset can be induced, for example, by IL-4 and IL-13, stimulate fibroblasts and participate in the formation of ECM, collagenesis and angiogenesis. M2b subset can be induced by stimulation with immune complexes (ICs) and Toll-like receptor (TLR) agonists or IL-1 receptor ligands.148, 149 They produce both anti- and pro-inflammatory cytokines IL-10, IL-1β, IL-6 and TNF-α, acting on immunoregulation. M2c subset is induced by glucocorticoids and IL-10 and strongly exhibits anti-inflammatory activities against apoptotic cells by releasing high amounts of IL-10 and TGF-β.149 M2d is induced by TLR agonists through activation of adenosine receptors, followed by suppression of pro-inflammatory cytokines and induction of anti-inflammatory cytokines (IL-10high IL-12low) and vascular endothelial growth factor (VEGF).148
The change in phenotype—polarization—is an important step in wound healing and both M1 and M2 macrophages are critical for a proper repair process.142 To investigate macrophages phenotypes and subclasses, different methods can be used to detect specific cell surface markers, secretion of cytokines, chemokines and other secreted factors, such as immunohistochemistry, Western Blotting or real-time PCR.
A summary of biological and physiological features of M1 and M2 macrophages phenotypes, including stimuli, cell expression markers, cytokines and chemokines, are presented in Table 1, published by Shapouri-Moghaddam et al.150
| Phenotype | Stimuli | Cell expression markers | Cytokines, chemokines and other secreted mediators | Functions |
|---|---|---|---|---|
| M1 | IFN-γ, TNF-α, LPS | CD80, CD86, CD68, MHC-II, IL-1R, TLR-2, TLR-4, iNOS, IL- 10 low, IL-12 high | TNF-α, IL-1β, IL-6, IL-12, IL- 23, IL-27, CXCL9, CXCL10, CXCL11, CXCL16, CCL5, Arg- 2 (mouse), iNOS (mouse), ROS | Pro-inflammatory Th1 response, tumour resistance |
| M2a | IL-4, IL-13 | Human: MMR/CD206, IL1Ra; IL-1R II Mouse: Arg-1, FIZZ1, Ym1/2 |
IL-10, TGF-β, CCL17, CCL18, CCL22, CCL24 | Anti-inflammatory, Tissue remodelling |
| M2b | Immune complexes, TLR ligands, IL-1β | IL-10 high, IL-12 low, CD86 | TNF-α, IL-1β, IL-6, IL-10, CCL1 | Th2 activation, Immunoregulation |
| M2c | IL-10, TGF-β, glucocorticoids | Human: MMR/CD206, TLR-1, TLR-8 Mouse: Arg-1 |
IL-10, TGF-β, CCL16, CCL18, CXCL13 | Phagocytosis of apoptotic cells |
| M2d | TLR ligands, adenosine receptor ligands | VEGF, IL-12 low, TNF-α low, IL-10 high | IL-10, VEGF | Angiogenesis, tumour progression |
- Abbreviations: Arg-1, arginase-1; FIZZ1, found in inflammatory zone 1; IL1-ra, IL-1 receptor antagonist; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MMR (CD206) , macrophage mannose receptor; ROS, reactive oxygen spices; TGF-beta, transforming growth factor-beta; TLR, Toll-like receptor; TNF-alpha, tumour necrosis factor-alpha; VEGF, vascular endothelial growth factor; Ym1 or chitinase-3-like protein-3 (Chi3l3150).
4 CONCLUSION
Several models and methods to assess wound healing have greatly improved our understanding of tissue repair over the years. Each model has its specific advantages and drawbacks, and a familiarity with common models/methods along with an awareness of their limitations will enable scientists to develop research strategies with a greater translational potential. The use of reliable wound assessment methods will contribute to obtain knowledge of the mechanisms of wound healing, and maximize efforts into potential effective interventions to improve healing.
In this study, we reviewed some of the common experimental animal models and methods in wound healing and believe the information can guide researchers in tissue repair, serving as a starting point in the search for models and methods to evaluate new therapeutic approaches, before they can be translated from bench to bedside and contribute to help millions of wounded patients, especially those with hard-to-heal, chronic wounds.
ACKNOWLEDGMENTS
The authors are thankful to their colleagues from the Wound Healing Group at the Dermatology Division of the Department of Internal Medicine, Ribeirao Preto Medical School—University of Sao Paulo, Brazil.
CONFLICT OF INTEREST
The authors have no conflict of interest.
