Chronic Skin Wounds
ICD-10 Code E11.621, E11.622, E10.621, E10.622, L89.154, L89.314, L89.622, L89.94, I70.231, I70.261, and L91.0
What is the clinical spectrum of chronic skin wounds?
Skin wound healing is essential for maintaining the skin barrier protection, thermal regulation, sensation, and endocrine functions Grubbs & Mana, StatPearls, May 2023.
Impaired wound healing often leads to chronic pain and physical disability, and its long-term consequences can be severe, ranging from amputation to mortality.
In developed countries, chronic wounds, also referred to as complex wounds, affect up to 3 % of the population and their incidence is expected to increase due to rise in underlying systemic conditions.
Chronic wounds are defined as persistent tissue injuries that cannot be treated with conventional methods, either due to local tissue factors (sustained pressure, infection, peripheral vascular disease) or to systemic factors (diabetes, immunodeficiency, nutritional deficiencies) Labib & Winters, StatPearls, Jul 2023, Han & Ceilley, Adv. in Therap., Jan 2017.
Common features of chronic wounds include tissue degradation, excessive inflammation, fibrosis, necrosis, and persistent infections Tottoli et al., Pharmaceutics, Aug 2020.
A wound infection is characterized by local inflammation, swelling, erythema, or pain, and its course can range from microbial colonization without affecting healing, to a systemic infection with sepsis and organ dysfunction Kaiser et al., Drug Del. and Transl. Res, Feb 2021.
Skin ulcers are open wounds on the skin characterized by the loss of epidermal tissue, caused by injury, poor circulation, pressure, or infection Cavazzana et al., Appl. Sci., Jan 2025.
An example of chronic skin ulcer with debilitating consequences is diabetic foot ulcer, that poses an estimated lifetime risk of up to 30% for T2DM and T1DM patients, that is frequently recurring after initial healing and that precedes the majority of amputations in this patient population Edmonds et al., J. Clin. Orthop. Trauma, Feb 2021.
Pressure ulcers occur in 3 to 15% of hospitalized patients and is associated with a high mortality rate in the elderly Labib & Winters, StatPearls, Jul 2023.
What do we know about the etiology of chronic skin wounds?
Variations in intrinsic (genetics, age, comorbidities) and extrinsic (diet, smoking, alcohol) risk factors that affect inflammation, tissue repair, and regeneration, contribute to inter-individual differences in susceptibility to chronic skin wounds Patel et al., Biomed. & Pharmaco., Apr 2019.
Deficiency in macronutrients and micronutrients, including amino acids, vitamins, zinc, and amino acids, are believed to play a role in impaired wound healing through reduced cellular regeneration, dysregulated immune function, and decreased collagen synthesis Hajj et al., Int. J. Mol. Sci., Sep 2024.
The full progress of temporally overlapping phases of wound-healing, hemostasis, inflammation, proliferation, and tissue remodeling, requires a coordinated spatiotemporal crosstalk between platelets, fibroblasts, keratinocytes, endothelial cells, and immune cells.Chronic wounds can result from dysfunction in any of these healing processes Eming et al., Sci. Transl. Med., Dec 2014.
Analysis of differentially expressed genes in patient-derived skin tissues over different phases of repair, has allowed to identify key regulators in gene expression and cellular processes. These regulators include enzymes involved in melanin synthesis, proteins kinases involved in DNA damage response and mitotic progression, and collagen proteins essential for extracellular matrix (ECM) organization, structural integrity and function Zhu et al., BMC Surg., Jun 2021.
In certain cases, such as in delayed wound healing, repeated skin trauma (deep cuts, burns, surgical incisions), and chronic inflammation (acne, infections), excessive proliferation of fibroblasts may occur, leading to thick raised skin lesions termed hypertrophic scars and keloids.
GWAS, epigenetic and transcriptomics analyses in fibrotic tissue derived from patients with hypertrophic scars and keloids, shows upregulation of genes encoding proinflammatory cytokines (IL-1α, IL-1β, IL-6, TNF-α) that stimulate fibroblasts to increase collagen production, insulin-like growth factor-binding proteins that regulate fibrotic tissue remodeling, hypoxia-Inducible factor 1 α transcription factor that promotes formation of new blood vessels, and FKBP10 chaperone protein that is involved in collagen synthesis and ECM stability Stone et al., Int. J. Mol. Sci., Nov 2020.
Skin ulcers can be classified as vascular ulcers, diabetic neuropathy ulcers, and pressure ulcers.
Chronic vascular insufficiencies, such as lower extremity venous disease (LEVD) and lower extremity arterial disease (LEAD) contribute to skin wounds through different mechanisms.
In LEVD, chronic slow-healing wounds, and often reoccurring venous stasis ulcers, appear as result of veinous obstruction or valve failure Brem et al., Am. J. Surg., Jul 2004.
In LEAD, arterial ulcers are typically related to atherosclerosis, that leads to ischemia and tissue necrosis Norgren et al., J. Vas. Surg., Jan 2007.
Neuropathy, in which nerve damage leads to loss of sensation and unnoticed injuries is a complication of diabetes Forbes & Cooper, Amer. Phys. Sco., Jan 2013. Over half of all limb amputations are due to impaired wound healing, most often triggered by hyperglycemia, chronic inflammation, circulatory dysfunction, hypoxia, autonomic and sensory neuropathy, and impaired neuropeptide signaling.
In chronic non-healing ulcers, an inappropriately prolonged acute inflammatory response transitions to chronic ineffective inflammation that fails to result in re-epithelialization and is associated with a sustained remodeling response and the development of a fibrotic ulcer bed Stone et al., Int. J. Mol. Sci., Nov 2020.
In venous leg ulcers in particular, the wound bed is histologically characterized by disorganized ECM, marked fibrosis, and chronic inflammatory infiltrates, all of which contribute to impaired healing. In confirmation of ulcer histology, microarray profiling in venous leg ulcers tissues showed enrichment of fibrosis and inflammatory response pathways.
Pressure ulcers, venous ulcers, and diabetic ulcers are believed to share causative factors of local tissue hypoxia, bacterial colonization of the wound, aging and repetitive ischemia-reperfusion injury.
Repetitive ischemia-reperfusion injury occurs when tissues experience cycles of restricted blood flow (ischemia) followed by restoration (reperfusion), leading to progressive damage instead of recovery Przykaza, Front. Immunol., Nov 2021.
As a result of chronic cycles of ischemia-reperfusion, oxidative stress, inflammatory responses, vascular leakage and capillary obstruction, are repeatedly exacerbated, impairing healing.
Comorbidities commonly associated with ischemia-reperfusion injury include coronary artery disease, hypertension, hyperlipidemia, ischemic stroke, neurodegenerative diseases with vascular dysfunction, and diabetes.
In patients with diabetes, causes of impaired wound healing are multifactorial, and include microvascular dysfunction, neuropathy, metabolic dysregulation and/or chronic inflammation.
The precise mechanisms that lead to nerve cell damage in diabetic neuropathy in T1DM and T2DM are not fully understood.
Based on currently available evidence, chronic hyperglycemia damages small blood vessel that supply nerves, through formation of advanced glycation end products which stiffen walls of blood vessels and trigger inflammation, through increase in ROS that leads to endothelial cell damage and impaired vasodilatation, through disruption of production of vasoprotective nitric oxide, and through repeated activation of pro-inflammatory cytokines that enhances fibrosis and vascular remodeling Strand et al., Curr. Pain Head. Rep., Jun 2024.
In addition, reduced blood flow impairs skin wound healing by restricting the delivery of oxygen and nutrients necessary for wound closure Xue et al., Cell Comm. & Sign., Oct 2023.
Resulting nerve damage produces loss of pain sensation and pressure detection in diabetic neuropathy patients, which subsequently tend to become more vulnerable to skin injuries and more unaware of its degradations, which, if left untreated, progress to diabetic ulcers.
The most frequently found bacterial strains in infected wounds are Staphylococcus aureus and Pseudomonas aeruginosa, while the most common fungi genus is Candida spp Kaiser et al., Drug Del. and Transl. Res, Feb 2021. Together with other bacterial species, Staphylococcus aureus and Pseudomonas aeruginosa form a structured microbial community which, embedded in an extracellular polymeric substance, locally forms persistent biofilms that enhance bacterial resistance to antimicrobial agents and to immune clearance, leading to chronic inflammation, and impaired wound healing.
Patient-specific genetic background, immune status, lifestyle and burden of comorbidities, all contribute to inter-individual differences in phenotype and pathomechanisms of impaired skin wound healing. A personalized approach to wound healing therapy would allow to take account of these inter-individual variations by administering therapies that are most adapted to the wound type, patient profile and underlying healing impairment mechanisms.
How similar are human and animal skins?
This is not an exhaustive list of species-specific differences, nor can one be made given their unknown full extent, but rather an example of how these differences impact the face, construct, predictive, and intrinsic validity of animal models.
Not all species-specific differences can be accounted for in animal models, as there are hundreds of them, their relevance to chronic disease wounds in humans is unclear, and their interaction with other organ systems in the animal model makes it difficult to predict how they would have behaved within the human system.
Species-specific differences in skin anatomy and physiology
The human skin differs anatomically, physiologically, and biochemically from skin in other mammals and possesses several aspects that are unique to humans Meyer et al., Curr. Problem. Dermatol., 1978.
These species-specific differences are likely to create major divergencies between humans and animal models in skin wound repair and regeneration, hindering our understanding of mechanisms that underpin impaired wound healing in humans.
Epidermis, Dermis and Hypodermis
The human epidermis is five-fold thicker than that of mice, rats, and rabbits Kim et al., Lab. Anim. Res, Oct 2022.
The epidermis of domestic pigs is closer to that of human skin, as both form ridges at the dermal-epidermal junction and have intersecting lines that form a pattern on their external surface. However, the pig skin presents with several divergencies from the human skin that are likely to affect wound healing, such as poor vascularization of cutaneous glands, extensive deposition of fat below the subcutis, high levels of alkaline phosphatase (ALP) in its supra-basal layers and a much denser stratum corneum compared to humans Meyer et al., Curr. Problem. Dermatol., 1978.
The ALP activity is relevant for skin homeostasis since it is linked to keratinocyte maturation, regulation of lipid metabolism influencing the formation of the stratum corneum and skin permeability, and modulation of phosphate turnover, affecting epidermal calcium signaling and skin integrity.
In humans, the adipose tissue and sensory innervations are more developed than in other mammals. The human adipose tissue can have a positive impact on wound healing as it contains adipose-derived stem cells (ADSC), which promote angiogenesis, epithelialization, and extracellular matrix (ECM) formation. However, in individuals with obesity, excess adipose cells may be a contributing factor to prolonged inflammation, leading to wound healing impairments.
Lately, ADSC have emerged as a promising therapeutic candidate for skin regeneration Suchanecka et al., Biomed. Pharmaco., May 2025.
In contrast to most mammals, including mice, rats and rabbits, humans and pigs are tight-skinned. Loose-skinned animals possess a layer of striated muscle lying beneath the adipose layer, termed panniculus carnosus, that is believed to participate in the contraction mechanism of skin wound healing, which is fundamentally different from skin wound healing mechanisms in humans Naldaiz-Gastesi et al., J. Anat., Jun 2018.
Dermal Hair Follicles, Sebaceous Glands and Apocrine Glands
In most mammals, hair follicles associated with sebaceous and apocrine glands form the hair follicle complex or the pilosebaceous unit Martel et al., StatPearls, Jun 2024. However, the structure and the function of the pilosebaceous unit vary across mammalian species Philpott, Exp. Dermat., Apr 2018.
Inter-species difference in function of the pilosebaceous unit have implications for skin wound healing since, in contrast to humans, follicle stem cells (FSC) play a major role in skin regeneration in most mammalian species.
In rodents, each hair follicle is paired with two sebaceous glands, whereas, in pigs and humans, there is one sebaceous gland per hair follicle.
In humans, pigs, and guinea pigs, each hair follicle operates independently, meaning that follicles enter growth (anagen), regression (catagen), and rest (telogen) phases at different times, ensuring continuous hair renewal. In contrast, rodent follicles are synchronized, producing large-scale shedding.
Sebaceous glands (SG) produce sebum, which contains bioactive lipids, such as linoleic acid, that influence FSC proliferation and differentiation by interacting with peroxisome proliferator-activated receptors (PPAR) that function as transcription factors.
The sebum composition is remarkably species-specific, affecting downstream pathways Schneider & Zouboulis, Exp. Dermat., Feb 2018.
In humans, the main components of the sebum lipid fraction are triglycerides, diglycerides and free fatty acids, with lower levels of wax esters, squalene and cholesterol. In other species, however, some of these components, like squalene, are missing or are present in different amounts.
In addition, there are significant species-specific differences in SG markers. For instance, keratin 7 and MUC1 are sebaceous markers in human but not in murine SG. These dissimilarities could explain why SG-associated diseases, such as acne vulgaris, are not faithfully recapitulated in animal models.
Due to species-specific differences in apocrine glands (AG) distribution, the role of AG in FSC activations differs between humans and other mammals.
Since in humans AG are localized only to a small number of areas, they do not play a major role in epidermal regeneration. In contrast, AG in other mammals, such as rats, mice, dogs, and pigs, participate in widespread FSC activation and epidermal renewal.
Dermal Eccrine sweat Glands
In humans, eccrine sweat glands (EG) play an important role in skin wound healing since they contain adult stem cells that can be recruited for skin regeneration and release molecules that promote keratinocyte migration and tissue remodeling.
However, animal species that are commonly used in skin wound healing studies lack EG and, as a result, the human-specific role of EG in wound healing was long underestimated and understudied.
In humans and Catarrhini primates, EG are distributed all over the body only, whereas in rodents, they are found on foot pads Rittie et al., Am. J. Pathol., Jan 2013, Quick et al., Anatom. Rec., Apr 1984.
Upon wounding, the turnover of human EG dramatically increases. In contrast, mouse paw EG remained quiescent during repair after epidermal injury, suggesting that stem cell characteristics of mouse EG are not translatable to humans.
In rodents, EG primarily respond to adrenergic stimulation to increase adherence and grip, whereas in humans, EG regulate body temperature and primarily respond to cholinergic effectors Rittie, J. Cell Comm. Sign., May 2016.
As a result, therapies that promote the action of neurotransmitter acetylcholine are likely to be inconclusive in rodent models of skin wounds.
In pigs, EG are histologically distinct, and are found only on the snout, lips and carpal organ. On the rest of the body surface, pigs have apocrine sweat glands, which in humans are restricted to only a few areas such as armpits and genital areas.
Dermal and hypodermal Vascularization
The human skin is highly vascularized compared to other mammals. Human skin vascularization supplies a superficial plexus that allows increased skin blood flow, nutritional capillaries that feed the epidermis, deeper vascularization connected with hair follicles and eccrine sweat glands, and rapid immune cell recruitment to wound site Meyer et al., Curr. Problem. Dermatol., 1978.
Such organization of vascularization is likely to accelerate skin wound repair. At the same time, it may represent a vulnerability in case of chronic wound-related persistent inflammation and diabetes-related hyperglycemia, contributing to inter-species differences in skin wound healing.
The vasculature of porcine skin resembles that of humans to a lesser degree. For instance, porcine skin shows less vasculature in the area surrounding hair follicles and sebaceous glands, which may produce inter-species differences in skin healing, immunological responses, and drug absorption studies.
In comparison to humans and pigs, rats and mice have a rudimentary vasculature consisting of two horizontal networks that produce low basal and stimulated blood flow.
Epidermal, dermal and hypodermal Extracellular Matrix
The ECM microenvironment provides skin cells with a resilient support that plays a major role in skin wound repair by serving as a structural substrate for cell adhesion, proliferation, and migration Rousselle et al., Matr. Biol., Jan 2019, Pfisterer et al., Front. Cell Dev. Biol., Jul 2021.
The dermal ECM scaffold is rich in type I and III collagens, hyaluronic acid, fibronectin, and elastin, while the basement membrane ECM is rich in collagen type IV and VII, laminin, and perlecan. The heterogeneity in ECM composition both within and between skin layers is believed to support distinct processes of wound healing, including stimulation of activity of keratinocytes and fibroblasts, cell adhesion, ECM remodeling, and immune cell recruitment.
Proteomic and bioinformatic analyses of ECM composition, as well as of interactions between ECM components and skin cell types, in humans, pigs, and rats, showed that the properties of the skin ECM were species-specific Liu et al., ACS Biomat. Sci. Eng., Sep 2020.
For example, the composition and function of the ECM of the human skin were more similar to those of pigs than rats. However, the skin ECM of the pig was significantly deficient in its enzyme systems and immune regulatory factors compared with that of humans, likely contributing to inter-species differences in wound healing dynamics.
Species-specific differences in skin wound healing and tissue repair
Wound healing characteristics differ dramatically between humans and model organisms, producing inter-species differences in speed and quality of wound healing.
Humans rely primarily on reepithelization and granulation healing processes. In reepithelization, keratinocytes migrate from the epidermal basal layer to the site of injury, covering the wound and restoring the epidermal barrier. In granulation, fibroblasts participate in ECM deposition and endothelial cells in angiogenesis, forming new connective tissue and vascularisations in the wound bed.
In contrast, rats, mice, rabbits, cats, dogs, certain non-human primates (NHP) like macaques and marmosets, and several other mammals, rely on contraction, in which the wound area shrinks as myofibroblasts pull the edges of the wound together. In pigs, partial-thickness wounds were found to heal with reepithelization and granulation, while full-thickness wound healed with contraction Grada et al., J. Inv. Dermat., Oct 2018.
This difference is believed to be related to an extensive presence in rodents of a subcutaneous striated muscle layer called the panniculus carnosus, that is mostly absent in humans, although the exact role of panniculus carnosus in wound contraction and collagen secretion is still debated Naldaiz-Gastesi et al., J. Anat., Jun 2018.
In rodents, panniculus carnosus allows the skin to move independently of the deeper tissues, hence the rodent skin is called loose skin, as opposed to tight skin in humans, NHP, and pigs.
The human skin healing rate diverges significantly from that of other mammals, including NHP Matsumoto-Oda et al., Proc. Biol. Sci., Apr 2025. Compared to NHP and to rodents, the skin wound healing is approximately three times slower in humans.
Of note, the human epidermis is three to four times thicker than that in NHP, implying that a thicker epidermis is associated with a slower re-epithelialization-based wound-healing rate.
In contrast to rodents, human wound healing relies heavily on fibroblast-driven collagen synthesis, particularly type I collagen, which forms dense scar tissue Haukipuro, Br. J. Surg., Jun 1991. Unlike humans, mice and rats do not naturally present with hypertrophic scars and keloids. This suggests an evolutionary adaptation in humans that may prioritize slow fibrotic repair over rapid regeneration.
Inter-species differences in mechanisms of fibrous healing are numerous and include increased mitosis in animal wounds, subcutaneous fat as the main location of fibrous healing in humans, larger vessels in animal wounds compares to humans, divergent morphology of macrophages, and gradual contraction of granulation tissue as the wound bed fills.
Species-specific differences in skin regeneration
Due to species-specific differences in stem cell activity and Wnt/β-catenin signaling pathways, epidermal renewal cycle in rodents is three times faster than in humans. In comparison to humans, rodents have a higher density of epidermal stem cells in the basal layer, leading to more frequent mitotic divisions Liu et al., Int. J. Mol. Sci., May 2013.
These rodent-characteristic features are likely to contribute to inter-species differences in speed and efficiency of skin tissue regeneration.
Unfortunately, much of our understanding of stem cell physiology is derived from lineage tracing studies of mice, which is likely to hinder successful development of innovative stem cell therapies. For instance, there are inter-species differences in pattern of expression of stem cell markers, such as mouse CD34 that in humans is also expressed by non-stem cells, such as fibroblasts and dendritic cells. Various stem cell therapies had shown remarkable wound healing effects in mouse models, only to disappoint in clinical trials Garg et al., Curr. Pharmac. Rep., Feb 2024.
In adult mammals, stem cells directly involved in skin regeneration include epidermal stem cells located in basal layer of the epidermis, hair follicle stem cells located in the bulge region of hair follicles, dermal stem cells, and adipose-derived stem cells present in subcutaneous fat Strong et al., Clin. Plast. Surg., Apr 2017.
According to animal experiments, key pathways involved in regulation of stem cells activity in the context of skin wound repair and regeneration include Wnt/β-catenin that promotes keratinocyte proliferation, Notch and BMP signaling that enhance keratinocyte differentiation, TGF-β signaling that regulates fibroblast activation, collagen synthesis and ECM remodeling, and PI3K/Akt signaling that enhances keratinocyte survival Gumede et al., Cell Comm. Sign., Apr 2024, Rangarajan et al., EMBO J., Jul 2001, Calautti et al., JBC, Sep 2005, Ren et al., Chem. Bio. Inter., Jan 2023.
However, there are major species-specific differences in expression and role of these stem cells systems. For instance, while rodents rely heavily on hair follicle stem cells for epidermal regeneration, humans depend more on basal epidermal stem cells.
In rats, mice, rabbits, primates, cats, and dogs, the hair follicle-derived mesenchymal stem cells exhibit higher proliferative capacity than in humans, leading to faster tissue remodeling, accelerated epidermal regeneration and reduced fibrotic scars in these animal species compared to humans Plotczyk & Jimenez, Hair Foll. Reg., Jun 2022, Garg et al., Curr. Pharm. Rep., Feb 2024.
Species-specific differences in skin immune system
Inter-species differences in composition of epidermal immune cells contribute to inter-species differences in speed of wound healing, in scarring, and in epithelial regeneration Pasparakis et al., Nature Rev. Imm., Apr 2014, Smithey et al., Bioger., Jul 2014.
In human epiderma, immune cells types, such as Langerhans cells (LC) and αβ T cells, that initiate adaptive immune responses are particularly represented, whereas in mice it is the rapid γδ dendritic epidermal T cells (DETC)-mediated innate immunity response that predominates.
For instance, in humans, specialized dendritic LC detect skin injury-generated damage-associated molecular patterns (DAMP) and pathogen-associated molecular patterns (PAMP), that activate NF-κB pathways, MAPK signaling, and lymph node migration, leading to release of release of proinflammatory cytokines, and keratinocyte proliferation. Activated by antigen-presenting cells, including LC, αβ T cells induce JAK-STAT pathways, that drive differentiation into effector T cells, and TGF-β signaling pathways, that regulate fibroblast activity and ECM deposition.
In mice, γδ DETC induce PI3K-Akt signaling that favors cells proliferation, as well as IGF-1 and KGF secretion, that directly stimulates keratinocyte migration and re-epithelialization. In addition, murine immune cells appear to shift faster from inflammatory (M1) to pro-repair (M2) macrophages, enabling quicker tissue remodeling.
Importantly, the human skin possesses elements of both innate and adaptive immunity that participate in protection against infection, skin regeneration and homeostasis. The human immune system possesses species-specific features that cannot be faithfully recapitulated in animal models. Discrepancies in both innate and adaptive immunity include the balance of leukocyte subsets, defensins, Toll receptors, inducible NO synthase, cytokines and cytokine receptors, Th1/Th2 differentiation, Ag-presenting function of endothelial cells, and chemokine and chemokine receptor expression Mestas & Hughes, J. of Imm., Mar 2004.
Major species-specific differences were also found in transcriptional regulation, chromatin state and higher order chromatin organization. Notably, cis-regulatory sequences next to immune-system-related genes have shown the most divergence, as they have apparently undergone more rapid evolution Yue et al., Nature, Nov 2014.
Beyond its primary role in the innate immune response, neutrophils can also assist in antibody production and in tissue reconstruction. Skin neutrophils can be therefore operationally classified as defensive neutrophils, helper neutrophils, and regenerative neutrophils.
A higher heterogeneity of neutrophils in humans suggest a diversification in their inflammatory, infection control, tissue remodeling, and immune-modulating roles in healing of human chronic wounds.
Understanding the functional heterogeneity of neutrophils in wound healing can help in stratifying patient based on their immune profiles, in identifying biomarkers of disease status and in developing treatments to manage excessive inflammation and enhance wound healing Reno et al., Biomed., Mar 2025.
However, comparison of human and mouse peripheral blood neutrophils has revealed distinct transcriptional states, meaning that insights on neutrophil activity obtained from animal models are unlikely to be relevant for humans.
Measuring of surface proteins and intracellular transcripts at the single-cell level, indicates that transcriptional subsets are independent of the canonical surface proteins that are commonly used to characterize human neutrophils Wigerblad et al., J. Immunol., Aug 2022, which has implications for identification of reliable biomarkers of neutrophil activities and potentially for identification of therapeutic targets.
Species-specific differences in skin microbiome
Persistent skin wound infection is believed to underlie up to 90% of amputations in patients with diabetic ulcer Boulton et al., Lancet, Nov 2005. It is therefore important to understand pathogenic and protective mechanisms of action of microbial species in skin wound infection Glatthardt et al., Antibiotics, Jan 2024, Chen et al., Front. Immunol., Apr 2023.
Beyond its role in skin infection, the skin microbiome also appears to participate in skin barrier homeostasis and wound repair, by stimulating keratinocytes proliferation through microbial lipoteichoic acids.
Crucially, the microbiome composition, its immune interactions, and environmental adaptations vary across animal species.
In addition, inter-individual differences in age, gender, ethnicity, use of antibiotics, and lifestyle factors are also know to affect the skin microbiota composition.
Unfortunately, experiments that study the role of microbiome in skin ulcers rely heavily on mouse models that do not recapitulate human-specific comorbidities and human-specific skin microbiota, impeding our understanding of the human-specific role of skin microbiome.
Metagenomics comparison of mouse and human gastrointestinal microbiota showed an overlap of 62% at the genus but only 10% at the species level, demonstrating that human and mouse gut microbiota are largely distinct Kieser et al., Plos, Mar 2022.
It was also found that mouse and human strains of the same species can have divergent gene content and function, as is exemplified by the species Limosilactobacillus reuteri, which has mouse-adapted and human-adapted strains, however, with very different functions.
These results suggest that skin microbiota composition and function are also likely to be human-specific, explaining the limited efficacy of systemic and topical antimicrobials that are commonly used to treat skin wound infections.
Environmental factors, like the skin pH, moisture, and lipid composition, shape microbial communities uniquely in each animal species.
Humans have the particularity of possessing millions of widely distributed eccrine sweat glands that also participate in the formation of the acid mantle that protects the epidermis, regulating the growth of skin commensal microorganisms Rittie et al., Am. J. Pathol., Jan 2013.
In humans, eccrine glands continuously secrete lactic acid, urea, and free fatty acids, contributing to a consistently lower skin pH compared to other mammals. The acidic pH of human skin supports specific microbiome populations, such as Staphylococcus epidermidis, which promotes skin barrier integrity and facilitates infection resistance via the release of antibacterial molecules.
The resulting species-specific skin microbiome interacts differentially with the host immune system, shaping immune-mediated response and susceptibility to chronic local inflammations that are not recapitulated in animal models of skin ulcers.
Apart from the above species-specific differences in skin, species-specific difference in nervous system (neuropathy), endocrine system (diabetes) and muscular system (impaired motility) are also likely to contribute to poor face, construct and predictive validity of animal models of chronic skin wounds.
Face validity - How well do animal models replicate the human disease phenotype?
Animal models do not recapitulate the phenotype, the heterogeneity and the complexity of chronic skin wounds.
The most commonly used animal species to model skin wound healing impairment are mice, rats, rabbits, and pigs.
It was suggested that different animal models of skin wounds could be used to answer specific questions, alone or in combination with other animal models. However, since animal models were never validated and standardized based on their similarity to humans, comparative analysis of animal studies is unlikely to yield reliable and human-relevant results.
Mouse models may be excisional, incisional, splinted, punch, polyvinyl alcohol sponge inserted, magnet-pressure induced, redox manipulated, bleomycin induced, ischemic, diabetic, aging, infected, humanized, or xenografted and may involve dorsal, ear, or tail skin. Resulting wounds are assessed based on gross wound size, planimetry, presence of the scab, re-epithelialization by histology, tensile/breaking strength, tensile stiffness, fibrosis, cellular infiltrate, microbial composition, inflammation, granulation tissue formation, and transcutaneous water loss Elliot et al., J. Inv. Dermat., Apr 2018.
In order to prevent wound healing by contraction in rodent models, a more human-like healing process by reepithelization is experimentally induced by placing a rigid ring or frame around the wound to mechanically restrict skin movement (splinting).
Also, as to minimize the impact of hair follicles on healing in rodent skin wound models, wounding is created during telogen or early exogen phases of the hair cycle, or alternatively in hairless mice.
However, it is unclear how effective these experimental procedures are in minimizing inter-species difference in wound healing, tissue repair, and tissue regeneration.
Diabetic wound models
No animal model of diabetes-related microvascular complications, metabolic dysfunction, neuropathy, and chronic inflammation faithfully recapitulates symptoms of human
and Type 2 diabetes mellitus (T1DM and T2DM).
Examples of inadequate diabetic wound healing models includes chemically-induced STZ mouse model of T1DM, associated with microvascular dysfunction, and spontaneous ob/ob and db/db mouse models of T2DM, associated with chronic inflammation.
Depending on the dose of STZ employed, hyperglycemia may develop in STZ through direct cytotoxic action on beta cells in STZ rather than through autoimmunity mechanisms.
The ob/ob mouse model possesses a spontaneous nonsense mutation in the obese gene coding for leptin, and displays severe obesity and insulin resistance. However, leptin deficiency is rare in humans with T2DM and the ob/ob mouse does not recapitulate the much more prevalent non-leptin obesity phenotype. Despite its severe obesity and hyperlipidemia, the db/db mouse does not develop atherosclerotic lesions, unless they are given a highly atherogenic diet or crossed into a vulnerable genetic background Pandey et al., Biomed., Oct 2023, Chandrasekera & Pippin, Altex, May 2014.
Numerous other species-specific differences, such as less prolonged inflammatory response and more efficient tissue regeneration in rodents contribute to poor face and construct validity of animal models of diabetic wounds.
As a result, in contrast to diabetic patients who go on to develop chronic skin ulcer, the wounds heal in mouse models of diabetes.
In order to artificially delay wound healing, additional inducing factors, such as inhibitors of catalase and glutathione peroxidase, were used in db/db mice. Catalase and glutathione peroxidase are antioxidant enzymes that neutralize ROS and their inhibition leads to chronic inflammation and impaired skin repair. However, there is no evidence that catalase and glutathione peroxidase inhibitors play a role in skin wound healing in diabetic patients Elliot et al., J. Inv. Dermat., Apr 2018.
Animal models of diabetic microvascular complications do not recapitulate features of segmental demyelination, axon loss, and fiber loss Sharma & Thomas, J. Neuro. Sci., Sep 1974. They are therefore inadequate for studying mechanisms that underly impaired wound healing in diabetic neuropathy.
The Akita mouse, commonly used to study neuronopathy, presents with sensory loss Forbes & Cooper, Amer. Phys. Sco., Jan 2013. It is a spontaneous model that carries a heterozygous missense mutation in the insulin 2 gene that ultimately results in endoplasmic reticulum stress, pancreatic beta cell destruction, insulin deficiency, and hyperglycemia.
However, unlike in human T1DM, the Akita mouse pathophysiology is not related to an autoimmune process and does not show insulitis. This means that the effect of dysregulated immune response on tissue modelling and angiogenesis in diabetic ulcers cannot be studied in Akita mice.
Although diabetic dogs and NHP develop slowing of nerve conduction and corneal hyposensitivity after years of hyperglycemia, degenerative neuropathy is minimal even in these larger animals.
In the pig model of diabetic ulcer, the skin wounds healed after 18 days, which is inconsistent with the chronic nature of diabetic wounds in humans Grada et al., J. Inv. Dermat., Oct 2018.
Pressure wound models
Pressure ulcers in elderly patients are modelled in loose-skinned animals such as rats and mice by surgically implanting a metal plate under the skin, followed by intermittent and periodic compressions of the skin using an external magnet. Owing to their tight skin, pigs are used to model pressure ulcers of young humans by reperfusion injury and friction on skin surface Grada et al., J. Inv. Dermat., Oct 2018. However, because of inter-species differences in skin anatomy and skin regeneration capacity, wounds heal much faster is these animal models than in humans.
Ischemic wound models
In the commonly used rabbit ear ulcer model, ischemia is created by ear vessel ligation Grada et al., J. Inv. Dermat., Oct 2018. Because the dermis of the rabbit ear is firmly attached to the cartilage, the avascular wound bed heals via epithelization and granulation tissue formation instead of by contraction.
However, in spite of this procedure, the rabbit ear ischemic ulcer model does not replicate human hypoxic wounds since the experimentally-induced ischemia is reversible and collateral circulation develops in about 14 days.
Hypertrophic scars and keloids models
Since mice and rats do not spontaneously develop hypertrophic scars or keloids, implanting human keloid tissue into immunodeficient mice is a widely used approach to study fibroblast behavior and test therapeutic candidates. However, the experimentally-induced phenotype does not fully replicate human hypertrophic scars and keloids Seo et al., Biomed. Res. Int., Sep 2013, Ramos et al., J. Burn Care Res., Mar 2008.
The cross talk between immune cells and fibroblast are believed to play a key role in formation of hypertrophic scars and keloids Zhang et al., Int. J. Mol. Sci., Oct 2023. Yet, chronic inflammation and the crosstalk between immune and non-immune cells were not recapitulated in immunodeficient mice, and even if they were, they would not match the human-specific immune system composition and function.
Construct validity - How well do the mechanisms of disease induction in animals reflect the currently understood etiology of the human disease?
Due to numerous species-specific differences between humans and model organisms, the mechanisms that underpin impaired wound healing in humans is poorly understood, limiting therapeutic options.
The impaired wound healing modeled in animals also fails to capture inter-individual differences in genetic background, age, immunity, lifestyle, and comorbidities, representing a barrier for working towards a personalized approach to treatment.
The use of splinting to avoid wound healing through contraction introduces foreign material to the wound site and it is unclear how this procedure affects underlying healing mechanisms and to which extent mechanisms triggered in this manner are similar to wound healing mechanisms in humans.
The fact that human cardiovascular, renal and endocrine diseases cannot be faithfully recapitulated in animals represents an important limitation for identifying mechanisms by which hyperglycemia, inflammation, dysregulation of immune response, metabolic dysfunction, and vascular complications, contribute to impaired wound healing.
For instance, chemically-induced and spontaneous mouse models of diabetes are not representative of disease causes in real world patient populations.
The inter-individual variability in comorbidities type, age, disease duration, immune status, and genetic predisposition, likely to produce unique combinations of healing impairment mechanisms, is not reflected in animal models of diabetic and ischemic skin ulcers either Przykaza, Front. Immunol., Nov 2021.
Experiments that study the role of microbiome in skin ulcers rely heavily on mouse models that do not recapitulate human-specific skin microbiota, hindering our understanding of human-specific composition and function of pathogenic and protective bacterial species. In addition, animal models of microbial biofilm formation that employ bacterial isolates from patients’ wounds do not capture complex interactions between the human-specific immune system and the environment (microbes, pollutants, oxygen, nutrients). As a result, the role of microbial species in skin ulcer outcomes is poorly understood.
Transgenic and knockout mouse models have been used to study the impact of a single gene in wound healing. However, because of mouse-specific compensatory changes in gene expression, genetically engineered mouse models cannot be relied upon to draw conclusions on the role of genes in wound healing Grada et al., J. Inv. Dermat., Oct 2018.
The above listed fundamental species-specific differences, also pose important limitations for understanding human-relevant mechanisms of formation of hypertrophic scars and keloids Limandjaja et al., Front. Cell Dev. Biol., May 2020. In addition, animal models do not reflect patient-specific characteristics that influence susceptibility to this condition, including in ethnicity and in genetic background.
Predictive validity - How well do animal models predict safety and efficacy of therapies in patients?
Animal studies of skin wound healing consistently result in poor efficacy of wound healing therapies in humans.
As a result, to date there is no specific pharmacological treatment with demonstrated efficacy in enhancing wound healing.
Depending on the severity of wound infection, antibiotics may be used Han & Ceilley, Adv. in Therap., Jan 2017.
The use of antibiotics may have adverse consequences such as bacterial resistance that may lead to superinfections. One such example is methicillin-resistant Staphylococcus aureus that has acquired resistance to beta-lactam antibiotics, including methicillin, oxacillin, and many cephalosporins, making it difficult to treat Choo & Chambers, Infect. Chemother., Dec 2016. Another example of adverse effect of antibiotics in treatment of skin wounds is delayed hypersensitivity reaction, that can lead to contact dermatitis.
The presence of biofilm microenvironment layers that adhere to the wound, protecting bacteria and enhancing their proliferation, poses a particular challenge in treatment of infected wounds. Currently, effective anti-biofilm treatments are still lacking Patra et al., Microb. Pathog., Jan 2025.
Extensive injuries that involve the dermis require much more time to heal and leave a noticeable scar. Treatments of chronic wounds are mainly related to management of processes that limit the wound repair, rather than those that restore tissue integrity. Accordingly, therapeutic avenues for regenerative medicine, such as with autologous cells and stem cells to re-establish the skin tissue without scarring, are also actively explored Tottoli et al., Pharmaceutics, Aug 2020.
Since diabetic peripheral neuropathy is associated with multiple factors, such as metabolic and microvascular diseases, it was suggested that its treatment needs to be based on a combination of drugs that address multiple pathogenic mechanisms. Currently, the evidence on effectiveness of combination therapy remains incomplete, and feedback on these drugs is inconsistent among patients Zhu et al., Front. Endocrinol., Jan 2024. Cardiovascular, metabolic and neurological diseases also lack effective treatments, making it difficult to reverse the underlying causes of chronic skin wounds.
Over the last decades multiple therapeutic approaches for hypertrophic scar and keloids had shown promise in animal studies, only to disappoint in clinical trials. Improved treatments are needed as current medical options for hypertrophic scar and keloids have a low efficacy and a high recurrence rate Zhang et al., Int. J. Mol. Sci., Oct 2023.
Patient-specific genetic background, immune status, lifestyle and burden of comorbidities, all contribute to inter-individual differences in phenotype and pathomechanisms of impaired skin wound healing. A personalized approach to wound healing therapy would allow to take account of these inter-individual variations by administering therapies that are most adapted to the wound type, patient profile, and underlying healing impairment mechanisms.
Ethical validity - How well do animal experiments align with human ethical principles?
Preclinical
Ethics is a human-specific philosophical concept. Animal experimentation is unethical in essence by human standards, since it involves physical constraint, psychological suffering and deprivation of freedom, social interactions, natural environment and life purpose.
In addition to this baseline, animal experiments inflict severe clinical harm in animals De Vleeschauwer et al., Animals, Aug 2023:
Table S5: Severity classification of chemical disease models
Diabetes: causing up to severe clinical signs
Table S13: Severity classification of genetically altered (GA) lines
GA lines with diabetes like NOD mice, BB rats: Severe
GA lines resulting in long-term moderate pain or short-term severe pain: Severe
GA lines resulting in long-term moderate anxiety or short-term severe anxiety: Severe
Table S4: Severity classification of clinical signs
Skin wounds - Causing up to severe clinical signs: Mice: >10 mm body, >3 mm face - Rats: >30 mm body, >10 mm face; Skin open with signs of infection (wet discharge of blood or pus) or open to muscle or bone.
Scratching - Causing up to severe clinical signs: Constant scratching (even when disturbed)
Table S3: Severity classification of surgery and surgical induction of disease
Surgery (intentionally) causing severe infection or sepsis: Severe
Surgical complications resulting in lethality
Table S6: Severity classification of infectious diseases
Bacterial or fungal infections causing severe clinical signs, long-lasting moderate clinical signs or infections causing lethality: Severe
Clinical
While there is no consensus on whether an unethical act can be justified by a pursuit of a hypothetically ethical outcome, it was suggested that animal research was necessary to advance treatments for human diseases.
However, statistics consistently show that clinical success rates of skin wound healing drugs developed and tested in animals is very low, raising the question of whether it is ethical to put the health of patients at risk.
Intrinsic validity - How well do animal models capture the clinical heterogeneity of the human disease?
Animal models do not recapitulate the complexity, the pathophysiology, and the heterogeneity of human chronic skin wounds.
Extrinsic validity - How well does animal experimentation generate reliable and reproducible outcomes?
It is often argued that although animal models have severe limitations, animal research enables to gather insights that may be valuable. However, the basic precondition for a hypothetical benefit is not met since the majority of animal experiments is irreproducible Freedman et al., PLOS Biol., Jun 2015.
Contributing factors include flawed experimental design, variation in animal strains and experimental conditions, and lack of transparency on methodology and results of animal studies.
In a bid to improve the quality of reporting of animal experiments, the ARRIVE - Animal Research: Reporting of In Vivo Experiments - guidelines, were published in 2010 and updated to ARRIVE 2.0 in 2020 Arrive guidelines website.
Nonetheless, and in spite of significant investment in dissemination, various incentives and training of animal researchers, the Arrive guidelines remain poorly implemented Percie du Sert et al., BMC Vet. Res., Jul 2020, Bazoit, BioRxiv, Feb 2025.
As a result of weak relevance, rigor and reliability of animal studies, erroneous and misleading hypothesis are generated, animal and human lives are needlessly sacrificed and dozens of billions of dollars are annually wasted Yarborough et al., PLOS Biol., Jun 2018.
Beyond the problem of experimental design and reporting of animal studies, there are deep-seeded cultural reasons that are not likely to be addressed any time soon, such as the "publish or perish" culture that discourages from unbiased analysis and reporting of negative results and rewards exaggerated and overhyped claims Smaldino & McElreath, Roy. Soc. Op. Sci., Sep 2016.
In Summary
Chronic skin wounds are a debilitating condition with potentially severe long-term consequences, ranging from amputation to mortality. Its heterogeneity in etiology and pathophysiology is linked to a multitude of intrinsic and extrinsic contributing factors that play a part in impairment of normal wound healing processes.
Extensive studies in animal models of severe chronic skin wounds have not translated to successful therapies for humans.
This poor result can be explained by numerous species-specific differences, including in skin anatomy and physiology, in skin repair and regeneration, and in immune system. In addition, animal models do not recapitulate inter-individual variations in patients’ genetic background, immune status, lifestyle, and burden of comorbidities, that lead to heterogeneous presentations of human chronic skin wounds.
A human-based and personalized approach to wound healing is necessary to better understand the human-specific and patient-specific mechanisms that underly skin wound healing impairments and to develop innovative combination therapies for skin repair and skin regeneration.
How is Human-Based In Vitro the Answer to Advance Biomedical Research into Chronic Skin Wounds
*To model human skin wounds by using full-thickness human skin equivalents in an automated wounding device Safferling et al., JCB, Nov 2013, Rossi et al., J. Vis. Exp., Feb 2015.
*To investigate the role of individual skin components (fibroblasts, ECM, adipocytes) in skin repair and regeneration in human full-thickness skin tissues/organoids/organ-on-chip Workman et al., In Vitro Mod., Sep 2023, Chaudhuri et al., Nature, Aug 2020.
*To study the human-specific skin physiology, by mapping the gene expression of human skin cells and the quantitative distribution of functional and structural proteins, using transcriptomics and proteomics in donor-derived human skin biopsies Edqvist et al., J. Histochem. & Cytochem., Nov 2014, Dyring-Andersen et al., Nature Comm., Nov 2020.
*To study the difference between acute and chronic skin wounds, by assessing the impact of growth factors, proteases, and cytokines-containing acute/chronic wound fluids, on proliferation of fibroblasts and keratinocytes, using a 3D human skin wound tissue/organoid/skin wound-on-chip Manuela et al., J. Tiss. Rep. Reg., Dec 2017.
*To investigate human-specific contribution of eccrine sweat glands to skin wound healing in 3D human skin wound tissue/organoid/skin wound-on-chip.
*To determine distinct roles of human skin fibroblasts in tissue repair and regeneration, by identifying human fibroblast populations, functions, expressions, and markers, using single cell RNA sequencing, gene ontology-term analysis, pseudotime trajectory analysis, and immunostaining, in donor-derived skin biopsies. To develop anti-fibrotic and pro-regenerative therapies that target specific human fibroblast populations Vorstandlechner et al., Faseb J., Jan 2020, Nauroy et al., J. Invest. Dermat., Aug 2017.
*To study the role of human-specific sebum composition in fibroblast proliferation and differentiation, in a 3D seboskin model, using patient-derived sebocytes Zouboulis et al., Pharmac., Feb 2023.
*To uncover molecular and cellular mechanisms of diabetic peripheral neuropathy in healthy/patient-derived peripheral nervous system organoids/(multi) organs-on-chip, using CRISPR editing, eQTL, epigenomics, transcriptomics, proteomics. To identify new therapeutic targets by combining information from multi-omics datasets. To test efficacy of drug candidates by in vitro measurement of biomarkers of neuropathy progression.
*To model diabetes-induced impaired wound healing, by bioprinting diabetes donor-derived fibroblasts, normal keratinocytes, and perfusable vascular channels, in a fill thickness skin construct. To study the effect of insulin resistance, vascular dysfunction, adipose hypertrophy, and pro-inflammatory responses on skin wound healing. To test the efficacy of drugs for diabetic skin ulcer by measuring inflammatory responses and epidermal repair Kim et al., Biomat., May 2021.
*To investigate differences between T1DM and T2DM in pathophysiology of diabetic ulcer, and identify new therapeutic targets, using immunocompetent T1DM and T2DM patient-derived 3D skin tissues/organoids/diabetic ulcer-on-chip Smith et al., Tiss. Eng., Feb 2021, Ejiugwo et al., Tiss. Eng., Feb 2021.
*To study the impact of human-specific intrinsic risk factors (cardiovascular, endocrine, genetic) on severity of chronic wounds by large scale comparative analysis of multi-omics data in tissues derived from patients with skin ulcers, and integration with clinical data.
*To study human-specific long-term cumulative effects of extrinsic factors (skincare, chemicals, smoke) on pathogenesis of chronic wounds, by air-liquid interface exposure of human skin cells to toxicants, using healthy human/chronic wound patient-derived skin tissues/skin wound-on-chip.
*To study the effect of human skin microbiota on skin ulcer pathogenesis by controlled in vitro microbial colonization and real-time measurement of immune activation and skin barrier disruption in healthy human/skin ulcer patient-derived immunocompetent 3D skin tissues/organoids/ulcer-on-chip.
*To model growth of biofilm in wound-like conditions, by combining 3D collagen gel matrix with a drip flow reactor system and continuous perfusion of simulated wound fluid Slade et al., BMC Microb., Dec 2019. To be coupled with metabolomics for real-time detection of bacterial metabolite production.
*To study the crosstalk between microbial biofilms and host’s skin cells, and to investigate the effects of antibiofilm agents, using human epidermis organoid/skin-on-chip model with a biofilm Wu et al., Npj Biof. Micr., Jan 2021. To be coupled with other in vitro skin models as to diversify skin cell types, to include immune cells and to simulate the blood flow.
*To investigate molecular mechanisms and signaling pathways of biofilm formation in a biofilm-on-chip system, by employing CRISPR, transposon mutagenesis, and multi-omics, to determine the role of microbial genes in virulence and antibiotics resistance.
*To identify early biomarkers of biofilm formation, fibroblast senescence, persistent oxidative stress and other endpoints, for rapid diagnosis, patient stratification, and measuring responses to treatments, using chronic skin wound-on-chip.
*To study mechanisms that underly formation of hypertrophic scars and keloids, and to test efficacy of drug candidates, using healthy and hypertrophic scar/keloid patient-derived organoids/keloid-on-chip Lee et al., Wound Rep. & Regen., Dec 2012, Limandjaja et al., Arch. Dermat. Res., Oct 2018, Chawla & Ghosh, Acta Biomat. Mar 2018.
*To test safety and efficacy of individual and combined treatments in patient-specific skin organoids/skin wound-on-chip, in a precision medicine approach.
Although in vitro methods have inherent limitations, their relevance to human biology far exceeds that of animal research.
Animal model organisms were never comprehensively compared to humans and scientifically validated. Complementing in vitro methods with animal experiments is not effective for human patients, because species-specific differences prevent reliable integration and translation of results to humans.
While animal research benefits from experimenting on a complete organism, model organisms fail to replicate the interplay of thousands of human-specific features, from molecular level to organism level, and are therefore not representative of the complete human organism.
To address the challenges of individual human-based in vitro models, they can be integrated with other human-based in vitro methods, AI-driven analysis, clinical data, and real-world patient data.
Have you leveraged in vitro methods in unique ways? We would love to hear how! Join the conversation to exchange ideas, collaborate and inspire new directions in human-based science!