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 1. Impairment in wound healing thus often leads to physical disability with potentially severe long-term consequences.
Chronic skin wounds, such as ulcers and non-healing surgical wounds, are defined as persistent tissue injuries that cannot be treated with conventional methods. Its contributors are commonly local (injury, sustained pressure, infection, peripheral vascular disease) and systemic (diabetes, immunodeficiency, nutritional deficiencies) 2, 3. In developed countries, chronic wounds affect up to 3 % of the population and their incidence is expected to increase due to rise in associated chronic conditions.
Common features of chronic wounds include tissue degradation, excessive inflammation, fibrosis, necrosis, and persistent infections 4. Marked by local inflammation, swelling, erythema, or pain, the course of skin infection can range from microbial colonization without affecting healing to sepsis and organ dysfunction 5.
Skin ulcers are lesions involving loss of epidermal, and often dermal tissue, due to pressure, ischemia/venous disease or neuropathy 11. An example of chronic skin ulcer with debilitating consequences is diabetic foot ulcer, that poses an estimated lifetime risk of up to 30% for type 1 and 2 diabetes patients. Frequently recurring after initial healing, diabetic foot ulcers precede the majority of amputations in this patient population 12. Pressure ulcers occur in 3 to 15% of hospitalized patients and is associated with a high mortality rate in the elderly 2.
What do we know about the etiology of chronic skin wounds?
The process of skin wound healing requires a highly coordinated spatiotemporal crosstalk between platelets, fibroblasts, keratinocytes, endothelial and immune cells that engage in overlapping processes of inflammation, angiogenesis, extracellular matrix remodeling and re‑epithelialization 13. The exact molecular and cellular mechanisms that are responsible for failure of these finely tuned processes to produce skin repair and regeneration are currently not fully understood.
Repair and regeneration of the human skin are qualitatively distinct processes. Repair is the rapid response to injury, in which keratinocytes proliferate and migrate to restore the epidermal barrier, while fibroblasts deposit collagen in the dermis, producing fibrotic scar tissue. The repair process ensures rapid closure of the wound, but typically results in altered architecture, loss of appendages, and reduced mechanical and physiological function. By contrast, regeneration entails the restoration of the epidermis together with its appendages and the reconstitution of organized dermal tissue, yielding skin that closely resembles its original structure and function. Of particular relevance for patients with chronic skin wounds, in humans skin regeneration is limited and most injuries heal through repair, producing fragile scarred tissues that are prone to breakdown.
Several biological (genetics, age, comorbidities) and lifestyle (diet, smoking, alcohol) factors contribute to the persistence of skin wounds. For instance, deficiency in macronutrients and micronutrients like amino acids, vitamins, and zinc are believed to play a part in healing impairment through reduced cellular regeneration, dysregulated immune function, and decreased collagen synthesis 14. Similarly, smoking hinders bacterial clearance and limits delivery of oxygen and nutrients necessary for wound healing.
Analysis of differentially expressed genes in patient-derived skin tissues has allowed to identify regulators of gene expression and cellular processes that govern different phases of skin repair. Key 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 15, 16.
Skin ulcers are often distinguished as vascular, diabetic neuropathy, and pressure ulcers. All three are believed to share local tissue hypoxia, bacterial colonization, aging, and repetitive ischemia-reperfusion injury as causative factors.
Chronic vascular insufficiencies, such as lower extremity venous disease (LEVD) and lower extremity arterial disease (LEAD), intervene in skin wounds pathogenesis through distinct mechanisms. In LEVD, chronic slow-healing wounds and often reoccurring venous stasis ulcers appear as result of veinous obstruction or valve failure 17, whereas in LEAD, arterial ulcers are typically related to atherosclerosis that leads to ischemia and tissue necrosis 18.
Impaired wound healing in diabetes is driven by hyperglycemia, chronic inflammation, circulatory dysfunction, hypoxia, autonomic and sensory neuropathy, and impaired neuropeptide signaling 23. Hyperglycemia thickens capillary basement membranes through glycation of structural proteins, excessive ECM deposition, and reduced ECM degradation. This structural change impairs the delivery of oxygen, nutrients, macrophages, and neutrophils to skin repair cells, thereby hindering wound healing 19. Additionally, hyperglycemia increases nerve-damaging sorbitol accumulation and ROS, and drives activation of pro-inflammatory cytokines that enhance fibrosis and vascular remodeling 20. The 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 21.
In chronic non‑healing ulcers, an inappropriately prolonged acute inflammatory response transitions into chronic ineffective inflammation that fails to achieve re‑epithelialization. This persistent inflammatory state is accompanied by sustained tissue remodeling, leading to the development of a fibrotic ulcer bed 16. 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 observed ulcer histology, microarray profiling in venous leg ulcers tissues showed enrichment of fibrosis and inflammatory response pathways.
Microvascular dysfunction (diabetic and vascular ulcers) and tissue compression (pressure ulcers) cause intermittent perfusions, in which vessels constrict (ischemia) and then dilate (partial reperfusion). Repeated cycles of ischemia–reperfusion worsen tissue damage by facilitating ROS production, inflammation, vascular leakage and capillary obstruction 22.
The most frequently found bacterial strains in infected wounds are Staphylococcus aureus and Pseudomonas aeruginosa which, together with other bacterial species, form a structured microbial community embedded in an extracellular polymeric substance, locally forming persistent biofilms. These biofilms enhance bacterial resistance to antimicrobial agents and to immune clearance, leading to chronic inflammation, and impaired wound healing 5.
Patient-specific genetic background, immune status, lifestyle and burden of comorbidities, all play a part in heterogeneity of chronic skin wound phenotype and pathomechanisms 23, underscoring the need for a personalized approach to chronic wound therapy.
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, and predictive 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 24. These human species-features can 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 24. The epidermis of domestic pigs is closer to that of the human skin, as both form ridges at the dermal-epidermal junction and have intersecting lines that form a pattern on their external surface. Contrarily 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 25.
Nonetheless, the pig skin differs from the human skin in several wound healing-relevant aspects. It shows poor vascularization of cutaneous glands, extensive deposition of adipose tissue below the hypodermis, high levels of alkaline phosphatase (ALP) in its supra-basal layers and a much denser stratum corneum compared to humans 24. By regulating phosphate turnover, ALP activity is essential for maintaining skin homeostasis. This regulation directly influences epidermal calcium signaling, and in turn supports keratinocyte maturation, lipid metabolism for stratum corneum formation, and overall skin integrity.
In humans, the adipose tissue and sensory innervations are more developed than in other mammals. Adipokines (TNF‑α, IL‑6, leptin, adiponectin, resistin) secreted by human mature adipocytes combined with additional growth factors (VEGF, HGF, FGF, TGF‑β) secreted by adipose-derived stem cells (ADSC) promote angiogenesis, epithelialization, and ECM remodelling. Owing to these properties, ADSC have recently emerged as a promising therapeutic candidate for skin regeneration 26. Yet, inter-species differences in subcutaneous adipose tissue may produce different responses to ADSC therapies.
Dermal hair follicles, sebaceous glands and apocrine glands
In most mammals, sebaceous and apocrine glands are structurally connected and functionally integrated with hair follicles, forming hair follicle complexes or pilosebaceous units 27. However, pilosebaceous units vary across mammalian species in their structure and function 28, which has implications for the skin wound healing dynamics.
Follicle stem cells (FSC), located in the bulge region of the hair follicle, are the key drivers of skin regeneration, and their regenerative potential is often impaired in human chronic disease such as diabetes. Located in the basal layer of interfollicular epidermis, interfollicular epidermal stem cells (IFESC) are primarily geared towards skin repair. In contrast to most mammals, in which FSC drives both skin repair and regeneration, the human skin is mainly reliant on IFESC, resulting in increased scarring and decreased skin restoration compared to other species 29, 30. This human-specific feature of dominant IFESC-driven repair can be explained by inter-species differences in follicle density, follicle-sebaceous gland pairing and synchronization, follicle neogenesis, hair cycle dynamics, sebum composition, apocrine glands distribution, fibrosis-prone dermal response, and stem cell niche signaling differences.
Compared to many other mammals, humans have relatively low follicle density and thus fewer FSC niches available. While rodents can regenerate follicles de novo in large wounds, follicle neogenesis is rarely present in humans. 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, non-human primates (NHP), 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, hair follicles cycle in a synchronized fashion in rodents, producing large-scale shedding and renewal.
Sebum produced by sebaceous glands (SG) 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 lipid-mediated activation of downstream pathways involved in wound healing 31. In humans, the main components of the sebum lipid fraction are triglycerides, diglycerides and free fatty acids, and to a lesser extent wax esters, squalene and cholesterol. However, in other species some of these components, such as squalene, are missing or present in different amounts. Additionally, there are significant inter-species differences in molecular and protein signatures of SG. For instance, keratin 7 and MUC1 are sebaceous markers in human but not in murine SG.
Paracrine growth factors (EGF, FGF, Wnt ligands) and cytokines secreted by apocrine glands (AG) stimulate FSC proliferation and differentiation, supporting wound healing. In contrast to other mammals, such as rats, mice, dogs, and pigs, AG are localized only to a small number of areas in humans 32, which limits their effect on FSC activation and epidermal renewal.
Dermal eccrine sweat glands
Human eccrine sweat glands (EG) house stem cells that migrate into the wound bed where they differentiate into keratinocytes. In addition, EG stem cells secrete paracrine factors (growth factors, cytokines) that act on nearby keratinocytes, fibroblasts, endothelial cells and immune cells, stimulating keratinocyte proliferation, angiogenesis and re‑epithelialization. Furthermore, the secretary coil of EG releases a fluid composed of water, electrolytes, lactate, urea, and antimicrobial peptides which contribute to wound healing by maintaining hydration and providing antimicrobial defense. However, animal species that are commonly employed in skin wound healing studies do not have the same distribution and activity of EG as in humans. 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 footpads 33, 34. 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 secretions are driven mainly by adrenergic stimulation to increase adherence and grip, whereas in humans, EG primarily respond to cholinergic effectors in the context of thermoregulation 30. Therapies that promote the action of neurotransmitter acetylcholine are therefore 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
Compared to many other mammals, human skin is characterized by a dense dermal vascular network. This vascularization supports nutritional capillaries near the epidermis, supplies hair follicles and eccrine sweat glands, and enables rapid immune cell recruitment to wounds 24. At the same time, in cases of persistent inflammation and diabetes-related hyperglycemia, the high density of dermal vascularization in humans may increase vulnerability to chronic wounds, further adding to inter-species differences in skin wound healing.
Pigs are closer to humans than to rodents in terms of dermal vascularization complexity. Nonetheless, porcine skin shows less vasculature in the area surrounding hair follicles and sebaceous glands, which may result in inter-species differences in healing processes, immune cell trafficking, and drug absorption. By contrast, rats and mice have a rudimentary vasculature consisting of two horizontal networks, one located beneath the epidermis and the other near the dermis-hypodermis junction, that produce low basal and stimulated blood flow, limiting their capacity to increase skin blood flow after injury.
Epidermal, dermal and hypodermal extracellular matrix
The ECM microenvironment provides keratinocytes, fibroblasts, and endothelial cells with a resilient support that plays a major role in skin wound repair by serving as a structural substrate for adhesion, proliferation, and migration. In addition, ECM products (collagen fragments, fibronectin peptides) acts as signals for recruitment and activation of immune cells during skin repair. 35, 36.
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.
Comparative analyses of ECM composition and ECM-skin cells interactions in humans, pigs, and rats revealed that the properties of the skin ECM vary by species 37. The human skin ECM appeared to be more similar to pigs than to rats in composition and function. Nonetheless, important inter-species differences between human and pig ECM were noted - the pig skin ECM was significantly deficient in its enzyme systems (matrix metalloproteinases, proteases) and immune regulatory factors (cytokines, chemokines), which alters how porcine skin responds to injury, infections, and therapeutics compared to human skin.
Species-specific differences in skin repair
Wound healing characteristics differ dramatically between humans and model organisms, producing inter-species differences in healing kinetics and functional restoration. Human skin repair relies primarily on re‑epithelialization and granulation. During re‑epithelialization, keratinocytes migrate from the basal epidermis to the wound site, covering the defect and re‑establishing the epidermal barrier. Granulation involves fibroblasts depositing ECM and endothelial cells driving angiogenesis, together forming new connective tissue and vascular networks within the wound bed. By contrast, many other commonly employed model organisms, including rats, mice, rabbits, cats, dogs, macaques, and marmosets, rely on contraction, in which the wound area shrinks as myofibroblasts pull the edges of the wound together. In pigs, partial-thickness wounds (epidermis and part of dermis) heal predominantly through reepithelization and granulation, whereas full-thickness wounds (epidermis and entire dermis) heal with additional involvement of contraction that is less extensive than in rodents but more extensive than in humans 38.
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. The exact role of panniculus carnosus in wound contraction and collagen secretion is, nonetheless, still debated 25. 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 that is characteristic of humans, NHP, and pigs.
The human skin healing rate diverges significantly from that of other mammals, including NHP 39. Compared to NHP and 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 on type I collagen that forms dense scar tissue 40. 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 skin regeneration.
Species-specific differences in skin regeneration
Due to inter-species differences in cell cycle kinetics, migration and differentiation rates, and regulatory signals (Wnt/β-catenin, growth factors), the epidermal renewal cycle - the process by which keratinocytes in the epidermis divide, migrate upward, differentiate and shed from the skin surface - is about three times faster in rodents than in humans. The density of epidermal stem cells in the basal layer is higher in rodents compared to humans, which can influence niche signaling and thereby the frequency of mitotic divisions 41. These rodent features are likely to contribute to a higher rate and quality of skin regeneration compared to humans.
Unfortunately, much of our understanding of stem cell physiology is derived from lineage tracing studies of mice, posing an obstacle for successful development of innovative stem cell therapies. The patterns of expression of stem cell markers vary across species, as exemplified by 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 mice, rats, pigs, and NHP, but clinical trials have not lead to regulatory approval for wound healing indications 42.
In adult mammals, 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 are directly involved in skin regeneration 43.
Based on insights gleaned from animal experiments, several signaling pathways participate in the regulation of stem cells activity within skin regeneration processes, such as Wnt/β-catenin that promotes keratinocyte proliferation, Notch and BMP that enhance keratinocyte differentiation, TGF-β that regulates fibroblast activation, collagen synthesis and ECM remodeling, and PI3K/Akt that enhances keratinocyte survival 44, 45, 46, 47. Yet, there are major inter-species 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, NHP, 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 compared to humans 42, 48.
Species-specific differences in skin immunity
The human skin possesses elements of both innate and adaptive immunity that participate in protection against infection, skin regeneration and homeostasis. Following injury, resident and circulating immune cells - Langerhans and other dendritic cells, neutrophils, macrophages, mast cells, T cells, and B cells - are mobilized at the wound site to combat infection, clear dead cells, and promote re‑epithelialization and granulation 49, 50, 51.
In addition to release of antimicrobial peptides and phagocytosis of pathogens/debris, neutrophils release cytokines and growth factors (IL‑1β, TNF‑α, CXCL8, VEGF) that activate NF‑κB/MAPK, STAT3, ERK, and AP1 pathways in keratinocytes and endothelial cells, promoting inflammation, re‑epithelialization and angiogenesis. Similarly, in addition to phagocytosis of pathogens/debris by macrophages, by secreting growth factors (TGF-β, VEGF-A, PDGF), the anti-inflammatory M2 macrophages activate the SMAD2/3 pathway in fibroblasts that drive ECM deposition and PI3K/Akt pathway in endothelial cells that promotes angiogenesis. Upon detection of DAMP/PAMP, Langerhans and other dermal dendritic cells migrate to lymph nodes where they present antigens via MHC II to naïve CD4 T cells, prompting differentiation of naïve into Th1/Th2/Th17/Treg cells that orchestrate the adaptive immune response. Activated by this antigen presentation from Langerhans dendritic cells, Th1 secrete IFN‑γ that sustains pro-inflammatory M1 response (STAT1 pathway), Th2 release IL4/IL13 which induce M2 polarisation and support ECM remodeling (JAK/STAT6), while Th17/22 secrete Il22 to enhance keratinocyte proliferation and angiogenesis. The pre-existing skin-resident effector/memory αβ T cells rapidly release IGF-1 upon activation by dendritic cells, to promote keratinocyte proliferation and wound closure. In chronic wounds these skin-resident T cells become unresponsive and fail to produce IGF-1, contributing to poor healing 52.
It follows that inter-species differences in skin immunity can have a major impact on wound healing kinetics and dynamics 53, 54. 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, antigen-presenting function of endothelial cells, and expression of chemokines and chemokine receptors 55. Major inter-species 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 56. For instance, in humans, the vast majority of epidermal CD4 and CD8 T cells are αβ T cells that carry a T cell receptor (TCR) made of α and β chains 57. In contrast, mouse and rat epidermal T cells almost all have γδ chains. As for pigs, the percentage of resident γδ T cells is about five-fold higher than in humans. This inter-species difference has functional consequences, since αβ T cells require classical antigen presentation, whereas γδ T cells have a rapid innate-like activation enabling a faster re-epithelialization. What is more, owing to rodent-specific Skint1 gene duplication, humans do not possess the specialized lineage of γδ T cells, termed dendritic epidermal T cells (DETC), that express invariant Vγ5Vδ1 TCR, and form a permanent dendritic network in the epidermis. In addition to not requiring antigen presentation, rodent DETC detect epithelial stress molecules, release growth factors and enhance re-epithelialization more synchronously and rapidly than human γδ T cells 58.
Beyond its primary role in the innate immune response, neutrophils can also assist in antibody production and tissue reconstruction. Skin neutrophils can be operationally classified as defensive neutrophils, helper neutrophils, and regenerative neutrophils. A higher heterogeneity of neutrophils in humans suggests a diversification in inflammatory, infection control, tissue remodeling, and immune-modulating roles. Understanding the functional heterogeneity of neutrophils in wound healing can thus help stratify patients based on their immune profile, identify biomarkers and develop novel treatments 59, 60. Comparison of human and mouse peripheral blood neutrophils had brought to light distinct transcriptional states, underlining the need for human-based tools to advance personalized wound therapies.
Species-specific differences in skin microbiome
Persistent skin wound infection may underlie up to 90% of amputations in patients with diabetic ulcer 61. Effective treatment of biofilms, that can be formed by commensal and opportunistic bacteria, has proven particularly challenging because of their protective matrix (polysaccharides, proteins, DNA) and immune evasion. Understanding the mechanisms by which microbial species confer pathogenic and protective effects is therefore crucial for effectively treating skin wound infections 62, 63. The skin microbiome also participates in skin barrier homeostasis and wound repair, partly through molecules such as microbial lipoteichoic acids (LTA) that act as signals to keratinocytes 64. The downstream effects are, however, dependant on bacterial species - Staphylococcus epidermidis LTA has been shown to stimulate keratinocytes proliferation and reduce inflammatory cytokine production whereas pathogenic Staphylococcus aureus LTA may inhibit keratinocyte differentiation and drive chronic inflammation 65.
The composition in bacterial species is uniquely shaped by species-specific factors such as sweat composition, sebaceous glands activity, hair density, immune system, and environmental adaptations. It is therefore not surprising that the human skin microbiome diverges significantly from that of model organisms, including NHP 66, 67. Humans have the particularity of possessing millions of widely distributed eccrine sweat glands that participate in the formation of the acid mantle that protects the epidermis, regulating the growth of skin commensal microorganisms 33. Human eccrine glands continuously secrete lactic acid, urea, and free fatty acids, contributing to a consistently lower skin pH compared to other mammals and supporting microbiome populations that promote skin barrier integrity. Additionally, several factors that can vary across patient populations, such as age, gender, ethnicity, health status, use of antibiotics, and lifestyle are also known to affect the skin microbiome. Unfortunately, our understanding of the role of the microbiome in chronic skin wounds is mainly derived from in vivo experiments that do not capture human-specific features, which may explain why antimicrobials therapies tested in animals often show limited efficacy in humans.
Apart from the above described species-specific characteristics, inter-species differences in nervous system (neuropathy), endocrine system (diabetes) and muscular system (impaired motility) are also likely to affect face, construct, and predictive validity of animal models of chronic skin wounds in ways that are not predictable.
Face validity - How well do animal models replicate the human disease phenotype?
Animal models tend to mimic acute healing wounds but are not representative of human chronic non-healing skin wounds. The most commonly used model organisms in wound healing impairment studies are mice, rats, rabbits, and pigs. Apart from these species, non-human primates, guinea pigs, dogs, sheep, horses, and zebrafish were also subjected to a number of experimental wound inductions.
A wide variety of skin wound induction methods was employed over the last decades, producing animal models that are excisional (full/partial thickness), incisional (surgical wounds), splinted (skin contraction prevention), punch (biopsy-like excisional), magnet-pressure induced (pressure/ischemic ulcers), redox manipulated (oxidative stress induction), ischemic (surgical flap section), diabetic (genetic/chemical induction), aging (aged animals), or infected (biofilm/bacterial) 38, 68, 69, 70. The resulting wounds were assessed using visual (wound size, closure rate, scab formation), histological (angiogenesis, inflammation, re-epithelialization markers), molecular, microbial, and other assays.
Diabetic wound models
In animal models of diabetic wounds, type 1 or type 2 diabetes are induced through chemical, spontaneous/selective breeding, diet, and genetic methods, followed by excisional founds or ischemic flaps 38, 69, 70, 71. Typically, type 1 diabetes is chemically induced in rats, mice or pigs by single or multiple administration of streptozotocin (STZ) that causes pancreatic beta cell death. In certain cases, STZ induction was employed in combination with genetic editing in mice to study the role of key genes in wound healing processes. Developed by selective breeding of mice that present with spontaneous diabetes, the non-obese diabetic (NOD) mouse is a polygenic model susceptible to autoimmune diabetes. The bio-breeding diabetes-prone (BB) rat displays spontaneous autoimmune destruction of beta cells, hyperglycemia and ketoacidosis.
Type 2 diabetes animal models include db/db mice, ob/ob mice, and Zucker diabetic fatty rats. The db/db mouse carries a spontaneous point mutation in the gene encoding the receptor of the fat cell-specific hormone leptin (LEP-R), whereas the ob/ob mouse possesses a spontaneous nonsense mutation in the gene encoding leptin, both mutations causing severe obesity, insulin resistance and hyperglycemia at a young age. The Zucker diabetic fatty (ZDF) rat, selectively bred to develop obesity and diabetes, shows symptoms of insulin resistance, but without inclination to develop hyperglycemia.
However, in contrast to diabetic patients who develop chronic skin ulcers, the experimentally-induced skin wounds healed in mouse models of diabetes. Moreover, none of these models recapitulated the processes of segmental demyelination, axon loss and fiber loss related to diabetic ulcers 72. The Akita mouse, commonly used to study neuronopathy, presents with sensory loss 21. 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 73. Yet, after experimental incisional, splinted excisional and ischemia–reperfusion injury, the Akita strain failed to show significant wound-healing deficits 71. In an attempt to model chronic wounds in aged individuals, antioxidant enzymes catalase and glutathione peroxidase were inhibited in 5-6 months old db/db mice by topical administration of mercaptosuccinic acid immediately after wounding. The resulting mouse model reportedly formed persistent ulcers similar to those in humans, with increased oxidative stress, chronic inflammation, damaged microvasculature, lack of re-epithelialization, and spontaneous development of multi-bacterial biofilm. However, this result was not reproduced in follow-up studies and the current consensus is that inhibitors of antioxidant enzymes do not produce chronic skin wounds in db/db mice 68, 71.
Though diabetic dogs and NHP showed slowing of nerve conduction and corneal hyposensitivity after years of hyperglycemia, degenerative neuropathy was minimal even in these larger animals. In the STZ-induced pig model of diabetic ulcer, a delay in healing was noted, nevertheless, the wounds healed after 18 days, which is not consistent with the persistence of diabetic wounds in humans 71, 74.
Ischemic wound models
Ischemic models were designed to study healing under reduced perfusion and do not model systemic vasculopathy. Though animal models of ischemic wounds produced delayed healing, unlike in human chronic vascular ulcers, the wounds eventually closed. The most frequently used approaches to experimentally induce ischemia in rats, mice, rabbits or pigs include the rabbit ear ulcer model, ischemic flap models, and hindlimb ischemia models.
In the rabbit ear ulcer model, ischemia is created by ear vessel ligation, followed by punch biopsy creating full thickness wounds 38, 69, 70. Because the dermis of the rabbit ear is firmly attached to the cartilage, the avascular wound bed heals by re-epithelization and granulation instead of by contraction. In spite of this procedure, the rabbit ear ischemic ulcer model did not replicate human wounds - the experimentally-induced ischemia was reversible and collateral circulation developed in about 2 weeks.
In rat, mouse and pig ischemic flap models, surgically isolated areas of skin with minimal continued blood supply, termed bipedicle skin flaps, are generated, followed by an excision of full-thickness wounds. In this manner ischemia is created locally in the flap, usually on the back or the abdomen. Wounds made in non-necrotic ischemic zones healed more slowly than those made on normally perfused skin 69, 71. Nevertheless, in these models angiogenesis from the underlying wound bed eventually allowed blood flow to return to the ischemic flap, enabling healing within 10 days 70. In contrast, human chronic ulcers tend to remain ischemic due to persistent vascular disease. Placement of silicon sheets between the flap and subcutaneous tissue prevented reperfusion, delaying about 3-fold the healing in rat 70 and pig 75 models of ischemic wounds.
To model chronic venous insufficiency in rats, skin flap creation was combined with epigastric artery and vein occlusion 70. This model lacked several hallmarks features seen in human chronic venous insufficiency, such as varicose vein formation and chronic ulceration with recurrent breakdown.
Hindlimb ischemia models reduce or eliminate blood flow to the distal limb, typically by ligating or excising femoral, iliac and saphenous arteries, producing ischemia of the leg and paw. In rodent models of hindlimb ischemia, the healing of ischemic wounds was reported to be delayed compared to non-ischemic wounds 70. To study vascular occlusion and diabetic angiopathy, an ischemic foot rat model was created by hyperglycemia induction through STZ, followed by ischemia in the limb induction through resection of the external iliac, femoral, and saphenous arteries. This approach produced acute ischemic necrosis but without open wounds 70. In contrast, humans with diabetic foot ulcers present with chronic non-healing ulcers, often due to a combination of diabetes, chronic peripheral arterial disease, neuropathy, and repeated trauma. Alternatively, hindlimb ischemia was also generated by chemical endothelial injury. In an arterial insufficiency peripheral artery disease model, sodium laurate solution was injected into the right femoral arteries of rats to trigger ischemia, producing necrosis but still without skin ulcers 70. A systematic review of animal studies in which ischemia was induced through ligation of the femoral/iliac/saphenous arteries suggested that the majority of hindlimb ischemia animal studies was not clinically relevant 76. Specifically, the existence of wounds in the paw - which would have aligned better with the anatomical location in diabetic ulcer patients - was reported in only a minority of rodent studies.
Pressure wound models
Pressure ulcers in elderly patients were modelled in loose-skinned animals such as rats and mice by applying weight-induced, piston-induced, and magnet-induced pressure 69, 70. In the weight-induced model, the skin tissue is compressed between an implanted metal plate and an external mechanical indenter. The indenter is attached to a loading device to apply controlled mechanical pressure, causing ischemia, reperfusion injury and pressure ulcer formation.
To simulate repeated ischemia–reperfusion cycles using magnets, Stadler and team placed folded mouse back skin between two magnetic plates that were repeatedly removed and reapplied 77. After three 12-hour removal-reapplication cycles, the tissue breakdown had progressed into pressure‑ulcer–like open wounds, however, it is unclear whether the ulcer ultimately resolved. In another magnet-induced rodent model of pressure ulcer, a metal plate was surgically implanted under the skin, and periodic compressions of the skin was applied using an external magnet. The most severe, stage IV pressure ulcer, that involves full-thickness tissue loss and exposes bone, tendon or muscle, was obtained after prolonged magnet compression for up to 6 days 70.
In a pig model of pressure ulcers, a cast was placed over a bony prominence (trochanter, shoulder, sacrum) to cause pressure, ischemia, reperfusion injury and friction on the skin surface 38. Yet, in contrast to human chronic pressure ulcers, the wounds created in the porcine model eventually healed.
Biofilm-infected wound models
To study the effects of bacterial infection and biofilm formation on wound healing dynamics, typically a monomicrobial suspension of Pseudomonas aeruginosa or Streptococcus aureus is applied after creating a full-thickness biopsy punch/incision wound in mice, rats, rabbits or pigs 38. To create a combined infection-pressure ulcer rat model, several strains of Pseudomonas aeruginosa were inoculated in weight-induced model of ischemic wounds 70. In a combined infected-diabetic wound rat model, bacterial suspensions of either Staphylococcus aureus or Pseudomonas aeruginosa were inoculated in full-thickness wounds of a STZ-induced diabetic rat 70.
A similar experiment was conducted in pigs, where STZ injection was followed by full-thickness excisional wounding and wound inoculation with Staphylococcus aureus 78. In this pig model, the degree of epithelialization was significantly delayed in inoculated diabetic wounds compared to non-inoculated diabetic wounds. Nonetheless, in contrast to human non-healing diabetic ulcers, inoculated wounds showed substantial re-epithelialization.
While biofilm-infected animal models capture acute infections, they are not representative of the chronic, multi-bacterial infections found in human ulcers. Human ulcer polymicrobial biofilms are thick, stratified and highly structured, whereas in animals biofilms tend to be simplified, immature and more easily cleared. Unlike in patients with chronic skin wound infections, infections in animal models of skin wounds did not show persistent inflammation and resolved spontaneously.
Construct validity - How well do the mechanisms of disease induction in animals reflect the currently understood etiology of the human disease?
Animal models of chronic skin wounds do not recapitulate the complex mechanisms of chronic skin wound pathogenesis in humans, further diminishing their translational value. To better approximate the multifactorial nature of comorbidity-driven human chronic wounds, such as diabetic foot ulcers, experimental induction of diabetes, ischemia, and infection were sometimes combined within the same model 38, 68, 69, 70, 71. As to prevent wound healing by contraction in rodent models, a rigid ring or frame was placed around the wound to mechanically restrict skin movement (splinting). In certain cases, wounding was applied during telogen or early exogen phases of the hair cycle, or alternatively in hairless mice, to minimize the impact of rodent hair follicles on healing. Despite these efforts to minimize, where possible, the effects of inter-species differences, the human relevance of animal models was not significantly improved.
Diabetic wound models
Owing in great part to inter-species differences in physiology and inherent limitations of experimental induction methods, animal models are not representative of the processes that give rise to human type 1 and type 2 diabetes. The existence of significant inter-species differences in genetics, pancreatic islets structure and function, glucose regulation, metabolic rates, feeding patterns, diet, immunity, and microbiome makes it very difficult to recapitulate in animal models of diabetes the complex processes responsible for human diabetic ulcer chronicity 79, 80, 81, 82, 83, 84.
Chemical induction of diabetes through direct cytotoxic action of glucose analogue STZ does not replicate the underlying pathophysiology of either type 1 (autoimmunity) or type 2 (insulin resistance) diabetes 85, 86. Another major limitation of the use of chemicals to model human diabetes is the difficulty in producing irreversible symptoms in model organisms. Spontaneous recovery from chemically-induced hyperglycemia was reported in several species and strains, including in pigs who showed partial correction of hyperglycemia several weeks after STZ injection, despite the use of high STZ dose 87. Employing gene editing in mice to determine the role of individual genes in human diabetic ulcer pathogenesis 71 is likely to produce misleading hypothesis. In contrast to human who have one insulin gene, rodents have two non-allelic insulin genes that can potentially amplify its effects through transcriptional regulation of over 150 genes 82, 89. Furthermore, the overall homology of the insulin promoter between humans and rodents is only around 45 to 48% 88. Trans‑acting factors (activators and repressors) that bind to cis‑regulatory elements in the insulin promoter, are regulated differentially in human and mouse islets. Consequently, the regulatory mechanisms by which genes involved in glucose metabolism regulate their own expression and expression of other related genes through cis and trans inputs, diverge between humans and other species.
The Akita mouse is a widely used model of type 1 diabetes. However, unlike in human type 1 diabetes, the Akita mouse pathophysiology is not related to an autoimmune process 73, hindering investigation of the effect of dysregulated immune response on tissue modelling and angiogenesis in diabetic ulcers.
While the BB rat displays spontaneous autoimmune destruction of beta cells, it is also associated with T-lymphopenia 90, which is not a hallmark of type 1 diabetes nor of diabetic skin ulcers in humans. Though NOD mice recapitulate the autoimmune components of human type 1 diabetes, they are poorly predictive of responses to immunotherapies in humans 91, 92. NOD mice exhibit a stronger Th1 response, leading to direct cytotoxic CD8+ T-cell-mediated beta-cell destruction, whereas in humans, Th17 plays a more significant role, contributing to chronic islet inflammation and beta cell dysfunction, rather than immediate cytotoxicity. The mutations that cause diabetes-like symptoms in ob/ob and db/db mice are not representative of the genetic causes of human diabetes. Human type 2 diabetes is polygenic and lifestyle-driven. In humans, leptin or LEP-R mutations are extremely rare and their occurrence does not lead to type 2 diabetes 93. Additionally, the fact that animal models of diabetic microvascular complications do not recapitulate features of segmental demyelination, axon loss, and fiber loss 72 makes them inadequate for studying the mechanisms that drive impaired wound healing in diabetic neuropathy.
Ischemic wound models
In humans, chronicity of wounds arises from a complex interplay of chronic low-grade ischemia, peripheral arterial disease, microvascular dysfunction, neuropathy, repeated trauma, biofilm-dominated infection, and systemic diseases. These factors cannot be simultaneously reproduced in model organisms and not in a manner that faithfully recapitulates human-relevant mechanisms of each systemic condition. It is therefore not surprising that in rabbit ear ulcer model, ischemic flap models, and hindlimb ischemia models, wounds do not become chronic 38, 69, 70, 71, 76. Despite etiological similarity to human ischemic foot ulcers in terms of compromised distal perfusion, paw ischemia tends to cause acute ischemia with rapid necrosis rather than prolonged, partial ischemia. While hyperglycemia was reported in many combined diabetes-ischemia animal models, diabetic neuropathy was systematically absent.
Inter-species differences in skin physiology and regeneration capacity further prevent the development of persistent, open, non‑healing ulcers. As a result, animal models cannot be meaningfully used to establish the mechanisms underpinning ischemic wound healing in humans.
Pressure wound models
In humans, pressure ulcers develop when deep tissues are repeatedly compressed between the bone and an external object. In the long-term, repeated cycles of ischemia–reperfusion causes deep tissue ischemia, muscle and fat necrosis, microvasculature collapse and eventual breakdown of the skin. However, experimental models of pressure ulcers, such as magnet-induced pressure models, often compress only the skin layer, giving rise to minimal muscle ischemia and superficial skin necrosis 69, 70, 77. While the pig model in which a cast was placed over a bony prominence 38 showed endothelial damage and microvascular collapse, it failed to capture other chronic conditions in the context of which human pressure ulcers develop - prolonged immobility, diabetes, vascular disease, malnutrition, and biofilm-driven chronic inflammation.
Biofilm-infected wound models
In human skin wounds, the polymicrobial community, composed of commensal and opportunistic bacterial species, form structured biofilms that are particularly challenging to treat 62, 63. These communities are difficult to reproduce in animal experiments given that the human skin microbiome is influenced by a multitude of human-specific features (sweat, sebaceous glands, immune system) 66, 67. Animal models of bacterial infection often rely on monomicrobial inoculations that do not mimic human-relevant polymicrobial synergy and pathogen-host interactions. The mechanisms of human chronic conditions that can influence biofilm formation, whether it is through poor oxygenation, impaired immune cell function, or chronic inflammation, are not faithfully recapitulated in animal models. In addition, inter-species differences in healing dynamics and kinetics can weigh heavily on the ability of microbial communities to cause deep tissue infections and mature over extended periods of time into highly perseverant biofilms. Consequently, the molecular and cellular pathways responsible for complex, persistent biofilms in humans may differ from those responsible for simplified, immature biofilms in animals, highlighting the need to study the dynamics of biofilm formation in a human-based in vitro system.
Predictive validity - How well do animal models predict safety and efficacy of therapies in patients?
Because animal models do not reproduce the complex pathophysiology of human chronic skin wounds, drugs that perform well preclinically have rarely translated into clinical efficacy. In the past three decades of research, only one drug for this indication, Regranex, was clinically approved by the FDA. Novel, effective medication remains badly needed to improve the quality of life of patients with ulcers, prevent amputations and reduce mortality.
To date, the standard of care for chronic wounds typically comprises debridement, wound dressings, pressure relief and managing underlying comorbidities. Based on meta-analyses and systemic reviews of randomized control trials, supplementary treatment options, such as advanced impregnated dressings, drugs, negative pressure wound therapy, and skin substitutes, enable in average 10-30% absolute increase in complete healing rates compared to standard of care alone.
Despite initial healing, recurrences remain high in a significant portion of patients with diabetic foot, venous and pressure ulcers. For instance, 40% of patients diabetic ulcers have a recurrence within 1 year, almost 60% within 3 years, and 65% within 5 years 94.
Wound debridement (surgical, mechanical, biological, enzymatic) removes necrotic tissue, slough, and biofilms to prepare the wound bed. Topical or systemic antibiotic therapy may be applied in case of infection 95, 96. The use of antibiotics is, nonetheless, not routine due to risk of 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 97. Research to develop agents that would be able to eradicate or disrupt established biofilms in chronic skin wounds is still ongoing 98.
Wound dressings help maintain optimal moisture, manage exudate, protect against infection, and promote healing. Dressings like DuoDerm (hydrocolloid), Aquacel (hydrofiber), and Purilon (hydrogel) provide moisture balance, while impregnated antimicrobial dressings such as Mepilex Ag, Allevyn Ag (silver) and Contreet (iodine) are commonly applied over infected wounds 95, 96, 100.
Negative pressure wound therapy (NPWT), which applies sub-atmospheric pressure to remove exudate, promote granulation, and reduce bacterial load, has shown increase in granulation tissue in both animal models and patients 95, 99. Nonetheless, NPWT is not a stand alone solution, has limited impact on ulcer recurrence, can pose therapy adherence challenges, and is contraindicated in cases of necrosis, bleeding or malignancy.
Several dozen advanced dressings/biomaterials with integrated active compounds - iron(II) scavenger deferoxamine, stromal cell derived factor-1, heparin-binding domains of laminin, tethered laminin-derived peptide A5G81, peptidic derivative of heat-shock protein 90α, and others - had improved healing in various mouse, rat and pig models of chronic wounds 100. However, this preclinical pipeline is mostly at proof-of-concept stage and has not so far translated to clinically-approved, marketed products for chronic wounds 101.
Over seventy skin substitutes, grafts, and tissue products that provide scaffolds, cells, or matrices to stimulate healing in deep or non-responsive wounds were so far approved by the FDA, including AmnioExcel (dehydrated human amniotic membrane), Grafix (cryopreserved placental membrane), EPIFIX (dehydrated human amnion/chorion), Dermagraft (cryopreserved fibroblast-derived), OASIS (porcine small intestine submucosa), MatriStem MicroMatrix (porcine urinary bladder matrix), DermACELL/Graftjacket/AlloPatch (human acellular dermal matrices), Integra (dermal regeneration template for deep wounds), and Theraskin (cryopreserved human skin allograft) 96, 102. Several barriers to a more widespread application of these devices need to be balanced against potential therapeutic benefits, such as lack of comparative analysis of efficacy, availability, high cost, fragility, disease transmission or potential of rejection 102.
Since 1997, Regranex (becaplermin) remains the only FDA‑approved medication for chronic skin wounds. Indicated for lower‑extremity diabetic neuropathic ulcers, it is a topical gel containing recombinant human platelet‑derived growth factor‑BB (rhPDGF‑BB). By promoting the chemotactic recruitment and proliferation of cells involved in wound repair, rhPDGF‑BB enhanced the formation of granulation tissue in several animal wound‑healing models, including in db/db mouse, pig, and rabbit ear skin excision models 103, 104. When combined with good standard care (debridement, infection control), in about 50% of cases versus 35% with placebo, Regranex increased the speed of healing and the percentage of diabetic foot ulcers that fully close. Of particular relevance for patients with cancer, this treatment is associated with an increase in death rate among patients who had pre-existing malignancy 105, 106.
Despite encouraging results in rodent and pig models of chronic skin wounds, the evidence of therapeutic benefit for epidermal growth factor (EGF), keratinocyte growth factor 2 (KGF-2), vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) was inconclusive 107. For instance, in model organisms epidermal growth factor (EGF) stimulated keratinocyte migration and proliferation, leading to accelerated wound closure in various chronic wound simulations. However, clinical trials did not demonstrate a major clinical benefit with exogenous EGF and it was discovered much later that human cell membranes have a much lower expression of EGF receptors 108. In the same manner, while human KGF-2 repifermin therapy accelerated wound healing in preclinical animal models of venous leg ulcers, it did not meet the endpoint of complete healing in human phase 2/3 trials, leading to discontinuation for chronic wound indications. Delivery of cathelicidin antimicrobial peptide LL‑37 increased conversion of HaCaT immortalized human keratinocytes to a migratory phenotype and accelerated re‑epithelialization and granulation tissue formation in ob/ob diabetic mouse excisional wounds 109. However, phase 2 trials in patients with hard-to-heal venous leg ulcers did not demonstrate any significant differences in healing of venous lower leg ulcers with LL-37 compared to placebo 110. The angiotensin peptide analogue DSC127 (aclerastide, NorLeu3-A(1-7) accelerated re-epithelialization and improved healing in diabetic db/db mouse and rat full-thickness excisional wound models 111. Yet, it was terminated in phase 3 clinical trials for diabetic foot ulcer after failing to meet its primary efficacy endpoint of complete wound closure within 12 weeks 112.
Since there are no adequate animal models for chronic wounds, the FDA guidance suggested that wound healing products should be assessed using multiple models (chicken chorioallantoic membrane for angiogenesis, rabbit ear model for re-epithelialization, etc.) 100. However, this approach has not resulted in an improved predictivity of animal models. Skin wound healing is not merely the sum of individual processes, but the outcome of highly complex spatiotemporal interactions. Because of inter-species physiological differences, the mechanisms underlying these interactions cannot be meaningfully deduced from comparative analyses of individual animal models. Moreover, chronic wound induction methods were not standardized, and existing animal models were not validated for the human relevance of their wound healing biology, further limiting their predictive value.
Advanced human-based systems, that recapitulate more closely human-specific features of skin physiology and chronic wound pathophysiology, are needed to develop new therapeutic solutions that translate more reliably to complete wound healing in patients. These human-relevant systems would also enable to identify the molecular and cellular drivers of human systemic diseases responsible for high recurrence rates of ulcers. Integrating patient-specific tissue would pave the way for personalized wound healing solutions 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. Humans assume the right to conduct scientific experiments on animals, despite the fact that animals clearly express non-consent through their behaviour (fleeing, vocalizing, defecating, defense).
Animal research 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, experiments inflict severe clinical harm in animals 113:
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.
The 3Rs principle rests on the unfounded assumption that animal models possess robust face, construct and predictive validity. It also implies that animal cruelty is acceptable unless alternative methods are applicable. In practice in vitro methods are used alongside, rather than in place of, animal models. Switching to 100% human-based research enables biomedical science without animal suffering, and invites a shift to a conceptually separate framework that excludes animal use entirely.
Intrinsic validity - How well do animal models capture the clinical heterogeneity of the human disease?
Animal models do not capture the inter‑individual variability in comorbidities, age, disease duration, immune status and do not recapitulate the heterogeneity of human chronic skin wounds phenotype, pathophysiology and responses to therapies 23.
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 114. 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 115. Nonetheless, and in spite of significant investment in dissemination, various incentives and training of animal researchers, the Arrive guidelines remain poorly implemented 116, 117. 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 118.
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 119.
While in vitro methods are not immune to issues of reproducibility, the moral weight of irreproducible animal studies is not the same. Animals are not inanimate, disposable objects - they are sentient beings who suffer. Their value lies not in how they might serve human interests, but in their intrinsic worth as living, feeling individuals.
In Summary
Chronic skin wound is 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.
Modelling of persistent wounds in vivo produces particularly severe symptoms in model organisms. Yet, animal models of chronic skin wounds are widely recognized as inadequate and have rarely translated to effective therapies for humans. This result can be explained by numerous human-specific features of skin anatomy, physiology, repair, regeneration, microbiome and immune system, as well as human-specific susceptibility of systemic diseases that underly wound healing impairment.
Novel pharmaceutical agents are needed to enhance wound healing and prevent relapse in patients with diabetic, pressure and vascular ulcers. Research using advanced human-based in vitro systems will allow to more faithfully recapitulate the molecular and cellular drivers of chronic wounds, better understand its heterogenous pathophysiology, identify new therapeutic targets and develop personalized therapies.
How is Human-Based In Vitro the Answer to Advance Biomedical Research into Chronic Skin Wounds
*To study human-specific healthy skin physiology, by gene expression mapping and in situ protein spatial profiling in donor-derived human skin biopsies 120. To generate a proteomic atlas of the human healthy skin, including functional proteins that are specific of cellular subsets involved in wound healing 121. To identify the molecular markers of skin ulcers by comparing multi-omics data across healthy and healing-impaired skin samples from individuals with endocrine/vascular comorbidities, nutrient deficiencies, and other lifestyle and biological risk factors.
*To investigate the mechanisms of chronic skin wound healing dynamics and kinetics by leveraging human ex vivo skin models that replicate the human-specific skin architecture (epidermis, dermis, immune cells, vasculature, appendages).
*To dissect the contribution of specific human skin components to wound healing by engineering human 3D skin constructs that incorporate select components (keratinocytes, fibroblasts, ECM, adipocytes, immune cells, endothelial cell, pericytes, appendages) 122, 123.
*To study the dynamics of tissues repair in 3D organotypic full-thickness human skin equivalents/ex-vivo human skin and other 3D human wound-healing models, by employing computational simulation 124.
*To study the effects of chronic wound-specific milieu (growth factors, proteases, cytokines) on tissue repair processes, by measuring proliferation of fibroblasts and keratinocytes in an engineered 3D human skin wound model challenged with fluid derived from patients with skin ulcer 125.
*To investigate the role of human hair‑follicle stem cells and interfollicular epidermal stem cells in skin repair and regeneration, by employing hiPSC-derived hair-bearing skin organoids 126.
*To identify the cellular and molecular mechanisms responsible for high skin regeneration capacity in human foetuses and develop therapies that promote tissue regeneration in skin ulcers, by comparing transcriptional profiles of prenatal human skin fibroblasts to those of healthy adult human skin fibroblasts 127.
*To determine which fibroblast subtypes are most likely to enhance wound healing, by mapping transcriptional signatures of human skin fibroblast populations (scRNA seq, GO-term analysis, pseudotime trajectory analysis, immunostaining) in donor-derived skin biopsies 128, 129. To develop anti-fibrotic and pro-regenerative therapies that focus on specific human fibroblast subpopulations.
*To study the mechanisms of diabetic peripheral neuropathy (DPN) by simulating hyperglycemic conditions in a human immunocompetent microvascularized peripheral nervous system microfluidic device (PNS-on-chip). To test neuroprotective effects of therapeutic candidates by measuring reversibility in vitro of axonal degeneration, demyelination, neuronal excitability, metabolic dysfunction, inflammatory signatures.
*To uncover the mechanisms of DPN-induced wound healing impairment, by engineering a dual immunocompetent microvascularized human PNS-wound-on-chip in which the skin compartment and the PNS compartment communicate through axons, neuropeptides and immune cells. Both compartments are exposed to the same hyperglycemic milieu via the shared microvasculature. To distinguish the effects of systemic metabolic changes (glucose, lipids, vitamins) on wound healing by integrating a liver compartment to form a liver-DPN-wound-on-chip.
*To model type1/type 2 diabetes-induced skin ulcer, study its mechanisms and test candidate treatments, by engineering immunocompetent diabetic foot ulcer in vitro platforms containing diabetes patient-derived ex-vivo skin tissues or fibroblasts that carry intrinsic diabetic phenotypes (altered metabolism, abnormal ECM deposition, increased MMP) and blood-derived monocytes 130.
*To model type 2 diabetes-induced non-healing diabetic ulcers by leveraging 3D cell printing to engineer vascularized full-thickness skin constructs containing type 2 diabetic patient-derived dermal fibroblasts and preadipocytes. To study the mechanisms by which insulin resistance, vascular dysfunction, adipose hypertrophy and inflammation impair wound healing. To test the efficacy of drugs for diabetic skin ulcer by applying compounds into the vascular channel 131.
*To study the effects of repetitive ischemia-reperfusion injury on wound healing in a microfluidic device, by simulating stop/restart flow in endothelialized channels and/or microvascular networks connected to a skin compartment through programming of syringe pumps, peristaltic pumps, or valves.
*To model human-relevant drivers that produce and sustain pressure ulcers, by engineering a microfluidic device in which the skin tissue, nerve fibers and vasculature are positioned between a rigid channel and a flexible membrane that is pressured by a pneumatic control/indentor in a manner that simulates several hours long compression cycles. To investigate the pathophysiology of pressure ulcers by measuring ROS generation, vessel density and morphology, permeability of basement membrane, necrosis, ECM deposition, inflammation and other readouts.
*To investigate the mechanisms by which lifestyle and environmental factors – tobacco smoke, cosmetics, solvents, steroids, antiseptics, gels, dressings, nanoparticles, micronutrients etc. – contribute to wound healing, by engineering an immunocompetent skin wound-on-chip in which the topical route is mimicked via air-liquid interface and the systemic route is mimicked either via an endothelial channel 132 or via bioprinted perfusable microchannels 133. The same model can be used to assess cumulative effects of combined exposure to several external factors on wound closure, and to test toxicity and bioavailability of skin wounds treatments.
*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 or skin ulcer-on-chip.
*To study wound infection, biofilm development, and host-pathogen crosstalk by inoculating human ex vivo skin wound models with polymicrobial/chronic wound-associated bacterial species 134. To assess responses to anti-biofilm treatments by measuring biofilm architecture, metabolic activity, biomass, cytotoxicity, and skin inflammatory response. A biofilm–wound‑on‑chip can be engineered to include polymicrobial communities and immune cells under vascular‑like perfusion.
*To test the ability of novel dressings, medications, devices and products to disrupt biofilms, manage infections, and enhance wound healing in immunocompetent ex vivo human skin biopsies 135, 136.
*To analyse inter-patient heterogeneity, to assess the effects of patient-specific features (age, ethnicity, lifestyle, comorbidity), and to segment patient populations according to their profile, wound features and responses to treatments, through large-scale morphological, immunohistochemical and multi-omics comparison of patient-derived ex vivo skin models.
*To test safety and efficacy of individual and combined treatments in patient-derived skin tissue/wound-on-chip, in a personalized medicine approach.
Last Updated: December 2025
Even though 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. As the above examples show, the full extent of inter-species differences is unknown and new species-specific features are discovered every day, often only once the drug has failed to show safety and efficacy in patients. 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!
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