Skin wound healing in humans and mice: Challenges in translational research

Journal of Dermatological Science 90 (2018) 3–12

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Review article

Skin wound healing in humans and mice: Challenges in translational research Helena D. Zomera,1, Andrea G. Trentina,b,* a b

Department of Biology, Embryology and Genetics, Federal University of Santa Catarina, Brazil National Institute of Science and Technology for Regenerative Medicine, Rio de Janeiro, Brazil

A R T I C L E I N F O

Article history: Received 1 August 2017 Received in revised form 20 November 2017 Accepted 13 December 2017 Keywords: Comparison Men Mouse Mus musculus Cutaneous repair

A B S T R A C T

Despite the great progress in translational research concerning skin wound healing in the last few decades, no animal model fully predicts all clinical outcomes. The mouse is the most commonly used model, as it is easy to maintain and standardize, and is economically accessible. However, differences between murine and human skin repair, such as the contraction promoted by panniculus carnosus and the role of specific niches of skin stem cells, make it difficult to bridge the gap between preclinical and clinical studies. Therefore, this review highlights the particularities of each species concerning skin morphophysiology, immunology, and genetics, which is essential to properly interpret findings and translate them to medicine. © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2. 3.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mice as model for human skin wound healing Morphofunctional characteristics of human and 3.1. Histopathologic findings . . . . . . . . . . . . . Stem cells niches . . . . . . . . . . . . . . . . . . . 3.2. Immunological specificities . . . . . . . . . . . . . . . . Cellular components . . . . . . . . . . . . . . . . 4.1. 4.2. Molecular components . . . . . . . . . . . . . . Genetic specificities . . . . . . . . . . . . . . . . . . . . . . Strategies to improve translational studies . . . . Strategies to prevent contraction . . . . . . 6.1. Role of specific stem cells populations . 6.2. 6.3. Genetically modified models . . . . . . . . . Final considerations . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author at: Dept. Biologia Celular, Embriologia e Genética, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário, 88040-900, Trindade, Florianópolis, SC, Brazil. E-mail addresses: [email protected] (H.D. Zomer), [email protected] (A.G. Trentin). 1 Dept. Biologia Celular, Embriologia e Genética, Centro de Ciências Biológicas, Universidade Federal de Santa Catarina, Campus Universitário, 88040-900, Trindade, Florianópolis, SC, Brazil. https://doi.org/10.1016/j.jdermsci.2017.12.009 0923-1811/ © 2017 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

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H.D. Zomer, A.G. Trentin / Journal of Dermatological Science 90 (2018) 3–12

1. Introduction Skin, the first protective barrier of all animals, has evolved and specialized differently among fish, reptiles, birds and mammals [1]. Epidermis, the outermost layer, consists of a stratified squamous epithelium of keratinocytes delimited by the basal membrane, and contains melanocytes and Langerhans and Merkel cells. Dermis, the internal layer that provides structural integrity, elasticity, and nutrition, is a connective tissue composed by fibroblasts and extracellular matrix enriched in collagen and elastic fibers [2–4], and also contains blood and lymphatic vessels, sebaceous glands, sweat glands, nerve endings, and hair follicles invaginated from epidermis [5–7]. As skin is constantly challenged by a wide variety of external factors, it is highly susceptible to trauma [8]. Complex intra and intercellular mechanisms are triggered after damage to recover tissue homeostasis [9,10]. In mammals, tissue repair reestablishes skin homeostasis, but not its complete functional activity [11]. The

event cascades triggered after skin lesion and scar formation is very similar to those of myocardial infarction or spinal cord injury [10,12]. In this sense, and due to its accessibility, skin is one of the best models to study tissue repair mechanisms and to develop new strategies in regenerative medicine [9]. Despite the recent progress in stem cell field, no current experimental model fully predicts the outcomes of clinical trials [13,14]. Although in vitro models address repair pathways of specific cell populations, they do not recreate the complexity of the healing process [14,15]. Animal models are, therefore, essential to elucidate the physiological and pathological mechanisms of tissue repair [15,16]. Despite interspecies differences, the murine model has greatly contributed to understanding normal and pathological cutaneous repair. As skin repair in mice does not perfectly mirror that of humans, studies face challenges in bridging the gap between preclinical and clinical studies [9,17]. Knowing the particularities of each species is fundamental to properly interpret the results.

Fig. 1. Skin wound healing. In the upper panel, the phases of tissue repair (homeostasis, inflammation, proliferation and remodeling) are shown over time. Dotted lines show involvement of platelets, collagen, and cells during the process. The bottom panel highlights cell-secreted proteins involved in healing. Abbreviations: TGF-b: transforming growth factor b; PDGF: platelet-derived growth factor, TNF-a: tumor necrosis factor a, IL-1: interleukin 1, CSF-1: colony stimulating factor 1, TGF-a: transforming growth factor a, VEGF: vascular endothelial growth factor, EGF: Epidermal growth factor, IGFs: insulin-like growth factor, IFNs: interferons, HGFs: hepatocyte growth factor, FGFs: fibroblast growth factor, MMPs: metalloproteinases, TIMPs: metalloproteinase inhibitors.

H.D. Zomer, A.G. Trentin / Journal of Dermatological Science 90 (2018) 3–12

Therefore, this review aims to compare human and murine skin wound healing to achieve adequate translation to medicine. 2. Mice as model for human skin wound healing Mice (Mus musculus) is the most commonly used animal model, especially in studies of physiology and biochemistry, [9,13,15,18] since they are easy to handle and maintain, reproduce rapidly, and are economically accessible [15,18]. They can be standardized by age, sex, history and genetic predisposition, and allows the use of a relatively high number of animals for statistic validation [14]. Furthermore, genetically modified lineages have been developed to investigate the molecular pathways of healing and regeneration [15,16]. However, mice have small bodies, shorter life expectancy, and differences in physiology compared to humans [16,19]. They do not effectively reproduce the whole pathogenesis of certain human diseases, such as diabetes, and can develop obesity and hypertension due to ad libitum feeding [16,17]. In this context, the use of larger mammals that are physiologically closer to humans, such as pigs, have increased in translational studies [13,16]. Porcine skin structure and healing are similar to human. Certain pathological conditions not reproducible in other animals, such as hypertrophic scars, are well described in Red Duroc pigs [13,15]. On the other hand, pigs are expensive, genetically heterogeneous, and require manipulation training, especially concerning anesthetic, surgical, and post-surgical procedures [9,14,15]. Moreover, pigs are not well characterized at cellular and physiological levels when compared to mice, and specific swine reagents, such as antibodies and growth factors, are still not available [16]. Because of these, the vast majority of studies concerning skin wound healing is performed in mice. 3. Morphofunctional characteristics of human and murine skin Skin healing is similar in humans and mice when considering the distinct and overlapping phases of highly complex cellular and molecular events: homeostasis, inflammation, proliferation, and remodeling, [9,10,20–22] summarized in Fig. 1. Much of the molecular processes are poorly understood and are possibly the reason why current therapies do not result in optimal repair [9,15]. Although human and murine skin has the same layers of cells in the dermis and epidermis, they greatly differ in thickness and

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number (Fig. 2). Human skin is relatively thick (over 100 mm), firm, and adhered to the underlying tissues, whereas murine skin is thinner (less than 25 mm) and loose [15,17,18]. Human epidermis is composed of 5 to 10 cell layers, whereas murine skin contains only 2 or 3, [23] which decreases its barrier function and enhances percutaneous absorption [24,25]. Therefore, researchers should be careful when translating results concerning drug delivery and percutaneous absorption efficiency. Skin and subcutaneous tissue thickness also differs according to site, age, sex and nutrition, both in mice and humans. Murine dermis, for instance, is thicker and 40% firmer in males compared to females. Epidermis and subcutaneous tissue, on the other hand, are thicker in females than in males [15]. Because skin thickness influences the biomechanics of healing, it should be considered when analyzing preclinical studies. Furthermore, panniculus carnosus found in murine subcutaneous tissue but virtually absent in humans influences skin biomechanics [18]. Consisting of a thin layer of muscular tissue, it gives great contraction potential to skin, and large wounds heal mainly by contraction and union of edges. Up to 90% of excisional wounds in mice close by contraction. Human dermis, in contrast, is firmly attached to the subcutaneous tissues, and contraction is highly variable and much less pronounced than in mice [14]. Thus, cutaneous wounds in men heal by formation of granulation tissue and reepithelization [13–17]. Although murine skin laxity and contraction make difficult to study these events, [14] they should not be reasons to exclude the murine model in healing studies, but to take care in interpreting results. 3.1. Histopathologic findings Some specific structures of human and mice skin are easily identified by histological routine stains as Hematoxylin and Eosin and Mallory and Fucsin staining (Fig. 3a–d). For instance, the epidermal ridges that intersect dermal papillae are present in human skin and normally absent in mice. Similar ridges, however, were observed in mice with pseudoepitheliomatous hyperplasia, (a condition that follows various stimuli, such as trauma, infection or inflammation) and should not be misunderstood [15,26]. Other stains, such as Orcein and Picrosirius used to visualize elastic fibers and to distinguish collagen types I and III, respectively, are useful tools to assess healing and regeneration in the remodeling phase

Fig. 2. Human versus murine skin morphology. Human skin is thicker and contains more epidermal layers than murine skin and is adherent to the underlying tissues. Rete ridges, ecrine sweat glands and neutrophil defensins are present in human but absent from mice skin. On the other hand, murine skin is richer in hair follicles, presents gd dendritic epidermal T cells (DETCs) and the Panicullus carnosus, a muscle layer with important contraction potential.

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Fig. 3. Human and murine skin histology. Routine stains can distinguish several structural differences between human and mouse skin. a,b) Hematoxylin and Eosin stain highlighting human rete ridges and eccrine glands, mouse panniculus carnosus and other identifiable structures; c,d) Mallory and Fucsin stain showing collagen enriched dermal matrix in blue and epidermis and hair follicles in red; e,f) Orcein stain identifying elastic fibers; g,h) Picrosirius stain under polarized light showing collagen I and III bundles in red and green, respectively. Both Orcein and Picrosirius stain similarly mice and humans skin. Bars: 100 mm.

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with similar results in mice and men (Fig. 3e–h) [27,28]. However, it should be noticed that the presence of elastic fibers was previously reported in mice scars but absent in humans [29]. 3.2. Stem cells niches Murine skin is covered by hair follicles, a complex structure composed by columns of keratinized and pigmented epithelial cells associated with sebaceous glands, erector muscles, nervous endings and follicular papillae (Fig. 3b,d). Human skin, in contrast, displays sparse and uneven hair follicle distribution, and most of human epidermis is classified as interfollicular, [18,23] a significant difference, since areas with high hair density heal faster than those with few follicles [30]. Consistent with this observation, hair follicle stem cells were shown to play important role in cutaneous repair [31–33] and up to 25% of newly formed epidermis is derived from hair follicle stem or progenitor cells [31]. Furthermore, since mice hair follicles are regenerated after skin healing while in humans this process is limited to the fetal stage (up to 23 weeks) [9,10,34], it would be critical to determine if differences in human and mouse hair follicle stem cell populations influence cutaneous repair. The basal layer of the epidermis is also a stem cell niche responsible for skin renewal and healing [35]. In mice, two types of epidermal stem cells were described: (1) progenitor cells heterogeneously arranged in the basal layer and able to generate epidermal keratinocytes during skin renewal and (2) quiescent and undifferentiated stem cells located near hair follicles and recruited after wound [36]. In humans, however, the locations of epidermal stem cell populations and their role in skin wound healing are still under debate [3]. Another stem cells niche in skin is the sweat glands (Fig. 3a) [37]. In men, eccrine sweat glands are responsible for body temperature control and play a role in innate immune response [18]. They also harbor stem cell populations that participate in cutaneous repair [37]. Mice, however, lack eccrine glands and contain only apocrine sweat glands in breasts [3,15]. Skin stem cell populations and stem cells from other body locations, as bone-morrow-derived mesenchymal stem cells, are recruited during wound repair [9]. While epidermal stem cells from hair follicle, epidermis and eccrine gland generate only epithelial phenotypes, mesenchymal stem cells differentiate into a wide variety of mesodermal and non-mesodermal cell types [8,32,35]. However, the differentiation of mesenchymal stem cells are highly regulated by their niche and particularities between humans and mice are still unknown [12]. Better understandings of the biology of specific stem cell populations in human and murine skin would contribute to proper translate studies. 4. Immunological specificities Tissue repair is initiated by platelet secretion of inflammatory cytokines which attract inflammatory cells to the wound site and trigger the cascade events that lead to healing [9]. Immune differences between mice and men concerning the innate and acquired immune systems were described at both tissue and systemic levels [38]. Here, we will focus on those that interfere with cutaneous wound healing. 4.1. Cellular components Although the percentages of peripheral blood leukocytes differ between humans and mice (50–70% neutrophils and 30–50% lymphocytes in humans, and 10–25% and 75–90%, respectively, in mice), [39] it is not clear if they affect skin healing.

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Macrophages, mast cells, and innate lymphoid cells are the major immune cells found in mammalian skin [23]. Specialized lymphoid and myeloid cells have been described in cutaneous repair [23]. In this context, macrophages are highly plastic, and might inhibit or stimulate cell proliferation and tissue repair corresponding to their polarization to either M1, pro-inflammatory (classical pathway), or M2 pro-repair (alternative pathway) phenotypes [38–40]. The M1/M2 phenotype tracking is a useful way to understand mechanisms of cutaneous repair. The phenotypic markers of macrophages, however, differ between species and must be selected accordingly. F4/80 adhesive glycoprotein, for instance, identifies both murine macrophages and human eosinophils [41]. By contrast, the macrophage mannose receptor found in murine M2 macrophages is also present in human dermal fibroblasts and keratinocytes [42–44]. Langerhans and CD8-positive T cells populate human epidermis. Murine epidermis, in addition to these cell types, contains a specific population of gd dendritic epidermal T cells (DETCs) fundamental for skin homeostasis and tissue repair [23,38,45]. As DETCs secrete FGF-9 in injured microenvironment promoting WNT activation and additional secretion of FGF-9 by dermal fibroblasts leading to hair follicle regeneration [46], their presence could explain hair follicles in mice scars but not in humans. 4.2. Molecular components Skin contains defensins, a family of antimicrobial peptides (AMPs) responsible for preventing pathogen entry in skin wound and for stimulating keratinocyte migration, proliferation, and production of proinflammatory cytokines and chemokines [47– 49]. Despite defensins, such as dermocidin, are greatly expressed in neutrophils of human skin and sweat glands, but absent from mouse skin [17,18,50] murine model has been suitably used to test AMP application in infected wounds [48,49,51]. Inflammatory cytokines and cytokine receptors play a role in wound healing by stimulating inflammation and tissue repair. Interleucin-1 (IL-1), for instance, regulates human and murine inflammatory cytokines important in pain and hyperalgesia following injuries. In response to IL-1, keratinocytes and fibroblasts produce keratinocyte-derived-chemokine in mice and IL-8 in humans in addition to other inflammatory and nociceptive mediators [38,52]. Moreover, IL-8, CXCL-7, CXCL-11, and monocyte chemoattractant were identified in humans but not in mice, and regulate re-epithelialization, tissue remodeling and angiogenesis [17,38,53]. Human skin also specifically expresses CCL-13, CCL-14, CCL-15 and CCL-18, whereas CCL-6, CCL-9, CXCL-15, CXCL-14 and CCL-12 are only expressed in mice. The role of these ligands is not well elucidated in cutaneous repair [38,53,54] and differences between mice and humans regarding the immune system are evident at the gene transcription level, as described below. 5. Genetic specificities Molecular differences between humans and mice are not surprising since they evolutionarily diverged over 90 million years ago [55,56]. Although genetic identity between humans and mice is over 95%, murine genome is 14% smaller than human, with 40% alignment between the two species [57]. A recent comparative gene array study revealed 30.2% identity between murine and human skin [18], suggesting strong evolutionary modulation [55]. Most conserved genes are related to barrier structure or function, such as keratin, cell-to-cell junctions, structural proteins, and cell proliferation-related molecules [18]. In addition, 15 conserved genes display unknown function [18]. Among the differentially expressed genes with key roles in skin wound healing are WNT-4 and WNT-16, [18] both

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significantly expressed in humans and associated with epithelialmesenchymal interactions and keratinocyte differentiation, respectively, but absent from mice [58]. Moreover, the epidermal growth factor receptor (EGFR), strongly expressed in human skin but not in mice, [18] has a key role in homeostasis, inflammatory control, microbial response, and barrier function [59,60]. For this reason, no side effects were observed in skin when anti-cancer drugs targeting EGFR where first tested in rodents. Therefore, considering the several described skin human alterations [61,62], genetic specificities between mice and men should not be disregarded.

Most human skin-specific genes including dermocidin and IL37 are related to pathogen defense [18]. IL-37 was associated with human inflammatory diseases such as psoriasis, systemic lupus erythematosus, and rheumatoid arthritis [63,64] and considered as a possible therapeutic target. In contrast, the majority of micespecific genes are related to the immune system, such as the Skint genes, which reflects the predominance of gd DECTs in mouse skin [18]. These observations highlight the strong immunological differences between these species in skin. Despite this, biotechnological strategies have developed a large variety of transgenic

Table 1 Differences between human and mouse skin structure and wound healing. Morphofunctional, immunological and genetic specificities between humans and mice skin and wound healing are summarized. Feature

Humans

Morphofunctional differences Skin thickness <100 mm Epidermis layers 5–10 Yes Adherence to underlying tissues Panniculus Virtually absent carnosus Granulation tissue Wound closure formation Present Epidermal ridges Elastic fibers Absent in scars Hair follicles Location-dependent; mainly sparse Unclear Epidermal stem cells Present Eccrine sweat glands Immunological differences Peripheral blood 50–70% neutrophils and leukocytes 30–50% lymphocytes Eosinophil marker F4/80 adhesive glycoprotein Dermal fibroblasts and Macrophage keratinocytes mannose receptor gd DETCs Absent Neutrophil Present defensins Response to IL-1 IL-8 production IL-8 Present

Mice

Consequence

Ref.

>25 mm 2–3 No

Influence in biomechanics. Influence in percutaneous absorption. Influence in biomechanics.

[15,17,18] [23] [14]

Present

Enhanced contraction.

[18]

Mainly by contraction

Careful to analyze results.

[13–17]

Absent Present in scars High density

Unknown. Tissue elasticity Role of hair follicle stem cells in wound repair. Role of epidermal stem cells in wound repair.

[15,28] [29] [30–33]

Role of eccrine sweat gland stem cells in wound repair.

[18,37]

10–25% neutrophils and 75–90% lymphocytes

Unclear.

[39]

Macrophage marker

Careful in inflammation tracing.

[42]

M2 macrophages

Careful in inflammation tracing.

[42–44]

Present Absent

Hair follicle neogenesis. Influence in pathogen entry and keratinocyte stimulation. Careful to analyze results. Influence in re-epithelialization, tissue remodeling and angiogenesis. Influence in re-epithelialization, tissue remodeling and angiogenesis. Influence in re-epithelialization, tissue remodeling and angiogenesis. Influence in re-epithelialization, tissue remodeling and angiogenesis. Unknown. Unknown. Unknown. Unknown. Unknown. Unknown. Unknown. Unknown. Unknown.

[45,46] [17,47– 50] [38,52] [38,53]

Influence in epithelial-mesenchymal interactions. Influence in keratinocyte differentiation. Influence in homeostasis, inflammatory control, microbial response, and barrier function. Influence in pathogen defense. Influence in pathogen defense. Presence of gd DECTs in mouse skin.

[18]

Epidermal progenitors in the basal layer and quiescent undifferentiated stem cells near hair follicles. Absent

keratinocyte-derived-chemokine production Absent

CXCL-7

Present

Absent

CXCL-11

Present

Absent

Monocyte chemoattractant CCL-13 CCL-14 CCL-15 CCL-18 CCL-6 CCL-9 CXCL-15 CXCL-14 CCL-12

Present

Absent

Present Present Present Present Absent Absent Absent Absent Absent

Absent Absent Absent Absent Present Present Present Present Present

Genetic differences WNT-4 Expressed

Not expressed

WNT-16 EGFR

Expressed Expressed

Not expressed Not expressed

Dermocidin IL-37 Skint genes

Expressed Expressed Not expressed

Not expressed Not expressed Expressed

[3,36]

[17,38,53] [17,38,53] [17,38,53] [53,54] [53,54] [53,54] [53,54] [53,54] [53,54] [53,54] [53,54] [53,54]

[18] [18,60,61]

[18] [18,63,64] [18]

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murine lineages useful in studying certain physiologic and pathologic conditions. 6. Strategies to improve translational studies Despite the differences between murine and human skin (Table 1), mice are still very useful in skin wound healing research. Different methods of wound induction and a wide variety of genetically modified models have been developed to improve the translation of results to medicine. As any model, each strategy displays advantages and disadvantages, as discussed below. The combined use of different tools might increase result accuracy and reduce their limitations (Fig. 4). 6.1. Strategies to prevent contraction One main problem in reproducing human wound healing is the contraction of mouse skin caused by the panniculus carnosus. The widely used full-thickness excisional wound model keeps the skin loose, and the wound closes quickly by the union of edges. In mouse, little granulation tissue is formed, which is substantially different from the human skin healing [13,65]. Despite this, Chen

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and collaborators (2015) recently demonstrated the gradually increase in contraction rate over time after wound closure using the murine excisional model. Granulation tissue and re-epithelization were observed in the first days after wound suggesting that this model could be useful for the analysis of early healing process [22]. However, if a longer investigation is desired, adapted models can be used. Some strategies, such as the splinted model that uses a silicon ring attached around the wound, can prevent contraction [13,15,65]. This model are convenient to analyze all phases of wound healing since it effectively avoids the panniculus carnosus contraction, and therefore wounds takes longer time to close in comparison to not-splinted models [22]. On the other hand, splinting is suggested to cause stress shielding, interfering in healing process [22]. Alternatives include creating a wound in scalp, ear or tail, body locations where the underneath bone or cartilage prevent contraction (Fig. 5) [14]. 6.2. Role of specific stem cells populations Specific endogenous stem cell niches, including human eccrine sweat glands and murine hair follicles, play a key role in skin wound healing, [32,37,66] and its potential therapeutic use are widely reported in literature [67–70]. However, translational studies demonstrating the activity of specific stem cell populations are scarce. Mouse tail presents sparse hair follicles and could be advantageous as well as the use of hairless mouse strains [32,71]. Lineage tracing and live imaging technologies are also helpful [72– 74]. Using lineage tracing, Aragona et al. (2017) recently clarified the clonal dynamics and individual contribution of epidermal stem cell populations during mice wound healing [75]. In this study, the self-renew potential of mice epidermal progenitors was not enhanced after wound healing as previously reported in human keratinocytes in vitro [75]. In the same way, live imaging studies using two-photon laser scanning fluorescent microscopy have been used in interfolicular and hair follicles stem cell tracing with minimal and non-invasive approaches [76]. More studies to further understand differences between mice and men stem cell populations during skin wound healing are encouraged. 6.3. Genetically modified models

Fig. 4. Strategies to improve translational studies. The diagram summarizes the murine models adapted wound, genetic modified and humanized models. Limitations of individual methods can be overcome by using a combination of different models.

Genetically modified mice expand the experimental possibilities. A vast catalog of mice strains is available at http://www. findmice.org/and https://www.mmrrc.org/. Transgenic and knockout lineages are powerful tools to identify the molecular pathways

Fig. 5. Wound models to prevent contraction. a) Regular full-thickness skin wound model: the loose mouse skin is free to contract by the panniculus carnosus; b) Ear punch model: the cartilage avoid skin contraction, as the skull does in the scalp model (c) and the tail bones in the tail wound model (d); e) Splinted wound model: a silicon ring is sutured around the wound to prevent contraction.

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and signal transduction cascades involved in wound healing. Diabetic transgenic mice, such as db/db,akita, and NONcNZO10, have been effectively used in studies of impaired skin wound healing [77–80] and for testing drugs and targets [77,80–82] despite differences in the pathogenesis of types I and II diabetes between humans and mice [14,83]. For example, the peptide thymosin b4, naturally secreted by platelets in the early stage of wound healing, was effective in treating skin ulcers in db/db diabetic mice and also in human clinical trials [84,85]. Furthermore, Nakagami and colleagues (2012) identified and tested the efficacy of AG30 peptide to treat diabetic mice tail wounds. They observed antibacterial and angiogenic effects in mice and later in humans [86–88]. Together, these results demonstrate the importance of mice to discover new drugs and therapies, even when the animal model does not perfect mirror the human disease. Nevertheless, personalized development of new mutant mice could reproduce the human disease in a molecular basis rather than simply phenotype. In future approaches, the patient genomic description should be assessed to define the human disease, and disease pathway would guide the development of the model [89]. The knockout technology has been used to identify the role of specific genes in a great variety of diseases and conditions. In this sense, the improved quality of skin wound healing observed in IL-1 receptor (IL-1R) knockout mice indicates that it is an eligible target for future therapeutic approaches [90]. Many target genes with key-roles during embryonic development, however, are lethal if knocked out. Strategies to make conditioning knockouts are possible, but very time and money consuming. Furthermore, signal transduction cascades can be redundant, and a specific knocked out factor might be compensated by others [14,91]. Thus, despite the wide potential, the knockout technology still carries some setbacks. Future strategies to improve translational studies include the development of humanized mice carrying functional human genes, cells, or organs. These chimeras have been successfully used for several clinical disorders such as infectious diseases, cancer, graft versus host disease and allergies [92]. Combining human keratinocytes with retroviral vector encoding green fluorescent protein to generate humanized mice models allows tracing a wide variety of parameters involved in wound healing, as tissue architecture, cell proliferation, epidermal differentiation, dermal remodeling, and basement membrane regeneration [93]. However, as the resulting healing is a combination of human and murine factors, it is still difficult to reproduce standardized chimeras due to the limitation of human cell types suitable to xenotransplantation, host residual immunoreactivity and the translation barrier [16,94]. Thus, advances in humanized mice development are required to overcome these obstacles and promote the popularization of such useful technology. 7. Final considerations The significant differences in skin wound healing between humans and mice should not prevent the use of the murine model. Despite numerous morphofunctional, immune, and genetic differences, mice have greatly contributed to the knowledge of human wound healing. Combinations of different approaches and technologies can properly surpass the limitations of each model. Nevertheless, knowing the disparities is critical to correctly analyze and translate results to human applications. Acknowledgments We are grateful to Dr. Alex Chen for revising the language of this manuscript and Marina Rosa da Fonseca e Sousa for artwork support. This work was supported by the Ministério da Saúde (MS-

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