chronic wounds diabetic ulcer

✉e-mail: vfalanga@bu edu

https://doi org/10 1038/ s41572-022-00377-3

Chronic wounds

Vincent Falanga 1,2 ✉, Roslyn Rivkah Isseroff 3,4, Athena M. Soulika 3,4,5 , Marco Romanelli6, David Margolis 7,8, Suzanne Kapp 9, Mark Granick10 and Keith Harding 11,12

Abstract | Chronic wounds are characterized by their inability to heal within an expected time frame and have emerged as an increasingly important clinical problem over the past several decades, owing to their increasing incidence and greater recognition of associated morbidity and socio-economic burden. Even up to a few years ago, the management of chronic wounds relied on standards of care that were outdated. However, the approach to these chronic conditions has improved, with better prevention, diagnosis and treatment. Such improvements are due to major advances in understanding of cellular and molecular aspects of basic science, in innovative and technological breakthroughs in treatment modalities from biomedical engineering, and in our ability to conduct well-controlled and reliable clinical research. The evidence-based approaches resulting from these advances have become the new standard of care. At the same time, these improvements are tempered by the recognition that persistent gaps exist in scientific knowledge of impaired healing and the ability of clinicians to reduce morbidity, loss of limb and mortality. Therefore, taking stock of what is known and what is needed to improve understanding of chronic wounds and their associated failure to heal is crucial to ensuring better treatments and outcomes.

Chronic wounds are characterized by their inability to heal within an expected time frame. However, there are no rigorous prospective studies to address the ‘time frame’ of impaired healing. Therefore, it might be argued that authors may be proposing a definition of chronic wounds that is based on how long it takes for a wound to heal1–3. This approach is artificial but, unfortunately, a better definition that is scientifically acceptable is currently not available. A major reason for the definitional challenge is that chronic wounds are quite heterogeneous in terms of aetiology, pathogenesis, size, body location, morbidity, risk of loss of affected limb, host factors and several other variables (Fig.  1 ) . Chronic wounds generally affect the adult population and are the result of complications from venous insufficiency, diabetes and neuropathies, inability to move and/or spinal cord injury (pressure ulcers (PUs)) and arterial insufficiency. There are several other clinical settings in which the initial injury is the result of genetic factors (for example, the spectrum of epidermolysis bullosa in children) or exposure to radiation (accidental or therapeutic). Also, immune system dysfunction has a greater role in the pathogenesis of some chronic ulcers, such as pyoderma gangrenosum and atypical ulcers (for example, those due to cryofibrinogenaemia or cryoglobulinaemia)4, than in others. It is estimated that 1–2% of the population worldwide will experience a chronic wound in their lifetime3 5 In less developed countries, the aetiologies may also

be quite different from those in developed countries; examples include nutritional deficiencies, parasitic and chronic fungal infestation, and leprosy. However, regardless of the underlying cause, a common denominator of chronic wounds is ‘impaired healing’, which some authors and clinicians refer to as ‘failure to heal’.

In the past few years, there has been renewed interest in the understanding of impaired healing in chronic wounds and their treatment. Progress has been made in applying advances in basic research, mostly cellular and molecular, to the testing of novel treatments for chronic wounds, such as those derived from tissue engineering, stem cells and growth factors. In addition, enhancements in devices (such as better dressings and the use of negative pressure) and surgical approaches (such as greater recognition of the importance of debridement) have contributed to improved management of chronic wounds. Despite this progress, chronic wounds constitute a challenging problem, and gaps remain in knowledge of chronic wounds in general and their characteristic feature of impaired healing. Much of the information about pathogenesis of chronic wounds has been obtained from animal models, predominantly rodents. The mechanisms identified in these studies are not always applicable to human chronic wounds, as the goal of developing valid animal models of chronic wounds has not yet been obtained. Moreover, studies in large animals that could give greater insight into human

Author addresses

1Department of Dermatology, Boston University, Boston, MA, USA.

2Department of Biochemistry, Boston University, Boston, MA, USA.

3Department of Dermatology, University of California at Davis, Davis, CA, USA.

4Dermatology Section, VA Northern California Health Care System, Mather, CA, USA.

5Shriners Hospital for Children, Sacramento, CA, USA.

6Department of Dermatology, University of Pisa, Pisa, Italy.

7Department of Dermatology, University of Pennsylvania, Philadelphia, PA, USA.

8Department of Biostatistics, Epidemiology and Informatics, University of Pennsylvania, Philadelphia, PA, USA.

9Department of Nursing, University of Melbourne, Melbourne, Australia.

10Department of Surgery, Rutgers New Jersey Medical School, Newark, NJ, USA.

11Clinical Innovation Hub Cardiff University, Cardiff, Wales, UK.

12Skin Research Institute of Singapore (SRIS), Singapore, Singapore.

wounds are not always possible and often do not lend themselves to the easier genetic manipulation achievable in rodents. However, while imperfect, animal models still provide opportunities to improve understanding of the pathophysiology of impaired healing and foster the development of better therapeutic approaches1,5

In this Primer, we address the epidemiology of chronic wounds, the pathophysiological mechanisms that underlie their formation and the complexities in treating them. We explore how chronic wounds are best diagnosed and characterized. Furthermore, we focus on targeted treatment modalities in the management of chronic wounds and discuss how these wounds affect patients’ quality of life (QOL). Finally, we discuss the research needs in various areas of mechanistic and clinical approaches.

Epidemiology

Chronic wounds are a worldwide problem3,5–7. Epidemiological discussions of chronic wounds are complicated by variations in terminology, underlying diseases (that is, comorbidities) and geo­demographics, as well as healing propensity or effects of management. First, the term ‘chronic wound’ includes many human ailments for which the aetiologies, outcomes, treatment and prognosis vary widely, hampering a unified definition of which injuries constitute a chronic wound2,8. Chronic wounds are sometimes simplistically described as complex wounds, but this term is misleading because even traumatic and acute wounds are quite complex. However, a chronic wound generally refers to a superficial, partial or full­thickness skin loss wound that heals by secondary intention. Chronic wounds have a point prevalence of 1.47 cases per 1,000 population in the UK9. Second, some chronic wounds are defined as a comorbidity of another illness. For example, for a patient to have a diabetic foot ulcer (DFU), they must have diabetes mellitus. However, individuals with diabetes are also more prone to have other chronic wounds, such as venous leg ulcers (VLUs; also known as stasis ulcers)10. Chronic wound prevalence varies by age, and most are more common in elderly individuals. Third, the incidence and prevalence of chronic wounds can vary by geographical location and community or facility under study11–13. Finally, chronic wounds can heal, be treated with limb­altering interventions and recur, altering estimates of prevalence and incidence.

PUs are a worldwide problem especially among patients who are infirm. Prevalence of PUs is highly dependent on residence (for example, hospital or nursing home), patient characteristics and age14–16. In a worldwide review from 2019, prevalence from cross­sectional studies of PUs ranged from 3% to 31% in long­term care facilities16 and also varied by country; however, many of the studies analysed in this review were more than a decade old16, so new data are needed. A 2019 study in Portugal emphasized the variation in prevalence by residence: 5.8% in hospital, 4.0% in nursing homes and 0.02% in the community17. A similar community prevalence was noted in a study from Spain, and a similar nursing home prevalence was noted in a study from China18,19. An assessment of 51 nursing homes in Switzerland revealed a prevalence range of 0–19%15. Prevalence was highly dependent on patient age and Braden score (a risk score based on six patient factors, including mobility, activity, nutrition and patient’s sensory/perception)15,20,21

VLUs are the most common type of chronic wound after PUs, with an estimated prevalence of 1.5–3 cases per 1,000 persons18,22–24. VLUs are more common in women than in men and prevalence also increases with age22,23. The prevalence in individuals >65 years of age is ~1.5% and the yearly incidence is 1.2%23. The prevalence of VLUs has been somewhat stable over time and by country25 but varies by location (that is, full population or primary care setting)26. However, as for PUs, epidemiological studies for VLUs are generally more than a decade old and new data are needed.

DFUs are probably the best studied type of chronic wound12, likely because of the public health importance of diabetes as well as the association of this wound with an important complication of diabetes, lower extremity amputation (LEA), as well as death27,28. Individuals with diabetes and a DFU are >10­fold more likely to have a LEA than those without a DFU29. Those with a DFU or a LEA have a two­ or threefold increased risk of death, respectively, compared with those with neither condition27,28. The prevalence of DFUs in individuals with diabetes is 1.2–20% in hospital and 0.02–10% in the community 12,26,30. The reported worldwide incidence of DFUs among those with diabetes is 5–41 cases per 100 person­years12,31. However, the prevalence of DFUs also varies by age, other complications of diabetes and region11,12,29,32. For example, in a study of all US Medicare beneficiaries, the yearly prevalence was ~8.0% and incidence was 6 cases per 100 person­years but varied with age, from 6.1% and 4.6 cases per 100 person­years among 65­ to 74­year­old individuals with diabetes to 15.0% and 11.5 cases per 100 person­years among those more than 95 years of age33. Both prevalence and incidence varied three ­ to fivefold by US hospital referral region 7,26,27. Regional variation in prevalence and incidence has also been noted in the UK34

Epidemiological research is expected to continue to have a key role in prevention of chronic wounds and in identifying targeted therapies, both established and novel. For DFUs, the focus may necessarily shift to a concerted effort to prevent leg and foot amputation. The United States Diab etes Sur veillance System

Fig. 1 | Representative clinical photographs of typical chronic wounds. a | Diabetic ulceration due to both neuropathy and arterial insufficiency. The forefoot has been previously amputated. b | Venous ulcer with surrounding lipodermatosclerosis on the medial aspect of the ankles. c | Deep pressure (decubitus) ulcer in the sacral area. d | Neuropathic diabetic ulcer on the sole of a patient with diabetes and Charcot foot. e | Extensive ulceration of the lower leg due to combined venous and lymphatic disease. The deep red granulation tissue is abnormal and may signify bacterial colonization. The edges of the wound (surrounded by indurated and fibrotic skin) and the island of skin (arrow) close to the wound centre are unable to migrate onto the surrounding red granulation tissue of the wound bed.

continues to accumulate epidemiological information on diabetes.

Finally, the likelihood of a wound healing is closely associated with wound­based and patient­based factors or attributes6,12, such as the size, duration and depth of the wound35,36. A published mathematical model that makes use of more detailed attributes (for example, wounds ≤2 cm2, ≤2 months old, ≤2 in grade as a measure of depth) has been used to successfully predict healing and may have an increasing role in determining wound severity and clinical management37.

Mechanisms/pathophysiology

Basic mechanisms of wound repair

The physiological phases of the normal wound healing process, including details of the cellular and molecular processes that are involved in these fundamental events, have been extensively reviewed elsewhere38. The normal wound healing process comprises sequential, overlapping phases of immediate haemostasis, followed by inflammation and proliferative and remodelling phases39. However, the process of tissue repair in chronic wounds is unlikely to easily fit this ‘linear’ paradigm of sequential, overlapping phases38–40 and instead displays a nonlinear organization of the phases (Fig.  2). Another

important consideration is that much of what is known about wound healing in humans comes from studies in animal models, which do not convincingly approximate chronic wounds41 and often suffer from excessive contraction (especially in rodent skin)42. In fact, no recognized valid animal models of chronic wounds exist. Therefore, substantial effort has been expended in developing models that have some features that approximate delayed healing and in minimizing wound contraction41,43–45. Nevertheless, suitable animal models will continue to have a key role in achieving a better understanding of cellular and molecular pathophysiological processes and in the testing of new therapeutic agents46–53. The FDA has published a valuable guide for the use of animal testing in the development of treatments for chronic cutaneous ulcers and burn wounds. This often­cited guide has proved valuable in many aspects of clinical trials and research54. Although porcine models are commonly used, whether larger animal models, such as primates, would be more useful is not clear and involves ethical considerations.

As the overall phases of wound healing have been comprehensively reviewed elsewhere38, we focus on the inflammation phase, as its key role in the repair process during wound healing is being increasingly recognized

Acute wound

Linear sequence of events

Coagulation

Day 1

Time

Days 3–5

Inflammation

Cell proliferation and migration

Weeks

Months

Remodelling

(see below and Fig. 3). In addition, to provide a focused discussion, we concentrate on investigative aspects of impaired healing that do not rely exclusively on animal studies but are derived from evidence obtained from human chronic wounds (Fig. 4).

Innate

immunity in wound healing

Innate immunity is the primary, nonspecific, first­line defence that protects the host against pathogens and tissue damage, unlike adaptive immunity, which is the response to a specific antigen (reviewed elsewhere55–58). Most current knowledge of the effects of innate immunity in wound healing derives from studies in rodents; however, new studies using advanced sequencing techniques are starting to elucidate mechanisms that may be associated with chronic wounds in humans. Here, we review the current literature in both rodent and human wound healing, ensuring to distinguish mechanisms that are clearly applicable to and focused on human chronic wounds (Fig. 4)

Keratinocytes, myofibroblasts and fibroblasts. Injury to the epidermal skin barrier allows invasion by pathogens that activate the innate immune system and pro­reparative functions. Damaged keratinocytes release danger signals, damage­associated molecular patterns (DAMPs)59, which are recognized by cellular pattern recognition receptors (PRRs) such as Toll­like receptors (TLRs). PRRs also respond to pathogen­associated molecular patterns (PAMPs) derived from invading pathogens60. These pathways, including increasing PRR signalling activity, have been investigated in the context of impaired healing 61–63. Responses are also ligand

Chronic wound

Non-linear sequence of events

Coagulation

Remodelling

Wound bed (asynchronous wound healing)

Inflammation

specific: DAMPs, such as host non ­ coding doublestranded RNA (dsRNA), upregulate TLR expression by keratinocytes64, and activation of TLR3 in human keratinocytes then initiates synthesis of components needed for epidermal barrier repair, such as sphingomyelin and transglutaminase 1 (reF 65)

Keratinocytes are also important producers of antimicrobial peptides or proteins (AMPs), including members of the β­defensin (hBDs) and cathelicidin (hCAP18/LL­37) families (Fig. 4). Keratinocytes constitutively express hBD1, and wounding upregulates the expression of LL­37, hBD2 and hBD3 (reF 66–68). LL­37 is upregulated at the edge of acute human surgical wounds but is absent from the wound edge in chronic non­healing wounds, and blocking its function with LL­37 antibodies impairs wound epithelialization 69 . Moreover, treatment with LL­37 accelerates the healing of hard­to­heal venous ulcers68. A study using single­cell RNA sequencing (scRNA­seq) analysis showed that keratinocytes in PUs with poor healing outcomes upregulate major histocompatibility complex class II (MHC­II) in response to increased interferon­γ (IFNγ) levels at the wound site70. Furthermore, upregulation of MHC­II in keratinocytes impairs activation of T cells at the wound edges.

Cell proliferation and migration

Fig. 2 | Wound healing in acute and chronic wounds. Schematic representation comparing the process of healing in acute and chronic wounds. Unlike the linear relationship of the recognized phases of normal healing in acute wounds (left), chronic wounds (right) are characterized by a process in which the different phases occur randomly and without a defined time frame. Parts of a chronic wound can be at different phases of healing, which is challenging because different areas of the chronic wound may require separate or different therapeutic approaches. A common clinical solution to this problem is, where possible, to perform surgical debridement and thus, at least for a few days to a week, synchronize the different areas of the chronic wound’s bed for a more unified therapeutic approach. The surgical debridement may need to be performed frequently, often on a weekly basis.

Other cells may also contribute to innate immunity and tissue repair, for example, a subtype of myofibroblasts that are derived from dermal adipose precursor cells71. Conversely, in mouse wounds, hair follicle cells can signal to myofibroblasts and reprogramme them to differentiate into adipocytes, suggesting new avenues for decreasing scarring72. Fibroblasts play an essential part in wound healing, chronic wounds and regenerative medicine73–75, and fibroblast deregulation has also been associated with impaired wound healing. A scRNA­seq and spatial transcriptomics study using skin from non­diabetic donors and diabetic donors with or without foot ulcers identified a novel fibroblast cell type that expresses IL6, TNFAIP6, MMP1, MMP3, MMP11, HIF1A, CHI3L1 and other genes76. In a mouse model of wound healing, Engrailed 1­positive fibroblasts were associated with increased fibrosis77. In agreement with this finding, a single­cell transcriptomics study of diabetic and normal foot ulcers showed that fibroblast populations were dysregulated in DFUs and had increased expression of fibrotic and inflammatory genes78. These observations suggest that fibroblast diversity is another important avenue to actively pursue in research on the pathophysiology of wound chronicity and impaired healing.

Macrophages. Macrophages have a well ­ established role in wound healing, possibly owing to their plasticity. These cells are particularly important in the healing phenotype and scar formation79,80. Macrophages shift from a pro ­ inflammatory, classically activated phenotype, frequently referred to as M1 and characterized by secretion of IL­1, TNF, IL­6, IL­12, matrix metalloproteinases (MMPs) and other cytokines, to a more anti­inflammatory, pro­reparative, alternatively activated phenotype, often termed M2 and characterized by production of arginase, TGFβ, CCL18,

Keratinocyte

Inflammatory phase

DETC

Langerhans cell

Active proteases and MMPs

Reparative macrophage

• CD206

• CD301b

• IGF1

• VEGF

• YM1

• ARG1

• FIZZ1

• TGFβ

• TIMPa

• IL-10

Activated DETC

• IFNγ

• TNF

• IGF1

• FGF2

• FGF10

DAMP

NET

Dermal

γδ T cell

• FGF2

• IL-17

• IL-22

Chronic wounds do not transition to proliferative phase but instead remain in a deregulated inflammatory phase

Inflammatory mediators

Neutrophil

• Proteases

• MMPs

• NO

• ROS

• LTB4

• IL-1

• TNF

• IL-6

Inflammatory macrophage

• Ly6C

• TNF

• IL-1β

Mast cell

• MC proteases

• Histamines

• Eicosanoids

• iNOS

• ROS

• IL-6

• VEGFA

• FGF2

• PDGF

Normally healing wounds transition to proliferative phase and wound closure

• TGFβ

• IL-4

• IL-8

• IFNγ

• MMP2

• MMP9

Blood vessel

Proangiogenic neutrophil

• VEGFR1

• YM1

• ARG1

• MMP9

• IL-10

Collagen matrix

T reg cell

Fibroblast

Pro-reparative factor

Myofibroblast

Inactive proteases and MMPs

Fig. 3 | The inflammatory component of wound healing in normal healing and chronic wounds. After the inflammatory phase, normal healing wounds can transition to the proliferative phase of wound healing, a hallmark of which is a shift of immune cells to anti-inflammatory and proliferative responses to enable tissue repair (bottom right panel). However, chronic wounds are characterized by a failure to quench local inflammatory responses, resulting in a stagnant and deregulated inflammatory phase, and consequently they do not progress to tissue repair (bottom left panel). DAMP, damage-associated molecular pattern; DETC, dendritic epidermal T cell; MC, mast cell; MMP, matrix metalloproteinase; NET, neutrophil extracellular trap; NO, nitric oxide; ROS, reactive oxygen species; Treg cell, regulatory T cell.

PGE 2 and IL ­ 10, and the upregulation of scavenger receptors (such as CD206 and CD163). In vitro, these two polarization states are characterized as being distinct and more easily controlled than in vivo. However, this nomenclature is evolving, and, in the wound microenvironment, macrophage polarization stages may present as a continuum rather than as a dichotomy 81,82. In mouse injury models, generation of the cytokine CCL2 leads to recruitment of CCR2+ macrophages, initially with a more pro­inflammatory Ly6C high phenotype, which subsequently transitions to a pro­angiogenic CCR2lowLy6Clow phenotype that is crucial for revascularization83. Furthermore, CD301b+ macrophages have been associated with myofibroblast proliferation and wound repair, although the continued presence of these macrophages at the wound site might be linked to fibrosis and keloid formation 71 Conditional ablation of macro phages during

various stages of mouse wound healing have revealed stage­specific functions: deletion early in the wound healing trajectory diminishes granulation tissue and myofibroblast formation, whereas depletion during the middle stage of healing destabilizes the existing vasculature and impairs epithelialization84 85. Interestingly, persistence of pro­inflammatory macrophages at the wound site has been associated with impaired wound healing86

The switch from an M1­rich to an M2­rich wound milieu is crucial for the transition from the inflammatory to proliferative phase of wound healing, and this transitional process might be disrupted in chronic non ­ healing wounds or even vary within the same wound. Various molecules have been identified as cues for the phenotypic transition, including cytokines produced by T helper 2 (TH2) cells and efferocytosis by neutrophils87. IFNβ­induced epigenetic changes are

Blocked cathelicidin (LL-37 antibodies)

↓ LCs

• ↓ T reg cells

• ↓ ILCs

Macrophage switch disruption

Biofilm

• Polymicrobial flora

• Quorum sensing

Cathelicidin (hCAP18) Pathogens

Impaired healing and chronic wounds

cells ↓ Local inflammation and IGF1

Persistent inflammation

Antimicrobial peptides

Pathogen-associated molecular patterns

Cellular pattern recognition (PRRs)

Damage-associated molecular patterns

TLR3

Toll-like receptors

Injured keratinocytes

Keratinocytes

β-Defensins

Wound environment

• Molecular leakage

• Ischaemia, poor flow

• Insensate foot

• Pressure injury

• Trauma and comorbidities

• Venous insuffiency

• Arterial insufficiency

• Diabetic neuropathy

• Pressure-induced ulceration

↑ Components of epidermal barrier repair TLR3

Fig. 4 | Key pathway components to impaired healing in chronic wounds. Schematic representation of selected pathophysiological components that underlie impaired healing and chronic wounds. Separating these components (largely studied in chronic wounds) from those that have been studied more extensively or exclusively in acute wounds is difficult. The complexity of the various pathways involved in chronic wounds, such as the general immunological aspects, innate immunity, cellular abnormalities, infection and biofilm formation, and clinical aspects, is very challenging, and further research is required to improve understanding of this complex network of components and their interactions. ILC, innate lymphoid cell; LC, Langerhans cell; PRR, pattern recognition receptor; Treg cell, regulatory T cell.

crucial for the transition of macrophages from the M1 to the M2 phenotype. IFNβ is upregulated in normally healing wounds, which promotes the expression of the histone methyltransferase SETDB2 in macrophages88 SETDB2 trimethylates lysine 9 on histone 3 (H3K9me3) and results in abolition of nuclear factor­κB (NF­κB) binding to DNA and the transition from M1 to M2. Diabetic wounds exhibit reduced IFNβ levels and, therefore, decreased levels of SETDB2, leading to an increased number of M1 macrophages at the wound site. The metabolic status of the wound is also crucial in these events and responses89,90. For example, redox profiling, which affects multiple signalling pathways, leads to differences in molecular patterns between acute and chronic wounds 91. Another example is the role of arginine. Arginase signalling has an important role in the context of the metabolic status of chronic wound92. The nonlinear progression to healing of chronic wounds (Fig. 2) is a further complicating feature; it is quite possible, for example, that different areas of the chronic wound may be in varying metabolic states. These findings and observations present challenges for a full understanding of chronic wounds and also opportunities for much needed research.

Supplementation of diabetic and acute wounds with M2 polarized macrophages not only does not promote but instead delays wound healing93,94. In support of this finding, a scRNA­seq analysis showed that M1 macrophage abundance was higher in healing than in non­healing DFUs76, and other studies found that neutrophil and macrophage abundance was lower in diabetic than in non­diabetic acute wounds95. These seemingly

contradictory results might be due to timing of the investigation relative to the transition from one macrophage subtype to the other and/or a lack of understanding of the specific characteristics of a ‘healing’ macrophage that supports healing and a macrophage that impairs healing96. In fact, a longitudinal study showed that the M1 to M2 ratio was increased in the early phases of healing DFUs compared with non­healing DFUs but, during the later stages, was increased in non­healing DFUs compared with those that healed. This result suggests a relationship between a sustained inflammatory response and healing failure97. The ageing process may indeed lead to further inflammation and impaired healing98

Langerhans cells are tissue­resident macrophages that are the major antigen­presenting cell in the epidermis. These cells express elevated levels of MHC­II and migrate to draining lymph nodes for antigen presentation. In addition, they express C­type lectins, including langerin (also known as CD207), CD205 and CD206, which are PRRs that facilitate Langerhans cell motility99 Increased numbers of Langerhans cells are found at the edges of healing wounds, and reduced numbers are present in diabetic wounds in both mice and humans100,101 In mechanistic support of these findings, depletion of langerin­positive Langerhans cells results in improved healing in a mouse model102.

Neutrophils. Cells of myeloid origin populate the skin after injury and exhibit either pro­inflammatory (often detrimental) or pro ­ reparative functions. Although neutrophils have long been regarded as mainly

fighting microbial infection and removing damaged tissue, they are actually integral to wound repair103–105 However, persistence of neutrophils in a wound is associated with degradation of newly formed collagen and impaired tissue repair; in fact, slow­healing wounds such as diabetic wounds are populated by large numbers of neutrophils 51,106–108. In excisional wounds, pan­neutrophil depletion using a GR1 antibody results in accelerated healing in both diabetic and non ­ diabetic mice 109. Interestingly, healing is impaired in mice deficient for CXCR2 (re F s. 110) , one of the main receptors that mediates neutrophil migration to the wound102,111. Neutrophil extracellular traps (NETs) comprise histone ­ containing nuclear DNA, proteases and other proteins that aid in trapping and killing microorganisms112. Patients with diabetes and nonhealing wounds have increased levels of neutrophils with NET markers, and non­infected diabetic mice in which NET formation is impaired heal more quickly51,108 Neutrophils exhibit phenotypic heterogeneity and functional plasticity: a pro­reparative, pro­angiogenic subpopulation expresses MMP9 for tissue remodelling, which, in turn, liberates VEGFA from the extracellular matrix113. Furthermore, neutrophils also express VEGF themselves114.

Mast cells. Mast cells are another type of granulocyte that has been studied in wound healing. Mast cells have pro­reparative functions, including secretion of growth factors that result in activation of endothelial cell angiogenesis, collagen synthesis by fibroblasts and restoration of the epithelial barrier115. High numbers of mast cells are found in chronic wounds, where they impair tissue repair owing to increased degranulation and protease activity116. In addition, mast cells have been associated with fibrosis in some but not all studies117.

Adaptive immunity in wound healing

Both mouse and human skin contain γδ T cells118 119 , which can promote repair. In the mouse epidermis, resident γδ T cells are also known as dendritic epidermal T cells (DETCs) and are activated upon skin injury120 Following activation, DETCs express the keratinocyte growth factors FGF7 and FGF10, and IGF1, which promotes wound healing118,119. γδ T cells are also found in the dermis and express IL­17 in excisional wounds, which promotes local inflammation and suppresses IGF1, which promotes its effects by binding to specific cell receptors121. In chronic wounds, γδ T cell numbers are reduced and those that remain are dysfunctional122, further supporting a pro­repair role for these cells in normally healing wounds.

Other tissue­resident lymphocytes such as regulatory T (Treg) cells, are involved in acceleration of wound repair123,124. Treg cells are usually identified by their expression of FOXP3, but they also express GATA3 (a transcription factor associated with TH2 cell responses) by which they curb fibrosis123. Non­cytotoxic innate lymphoid cells (ILCs) are also implicated in wound healing in human and experimental animal wounds, and depletion of one ILC subgroup, group 2 ILCs (ILC2s), results in impaired mouse wound re­epithelialization125,126 (Fig. 4)

Immune signalling in wound healing

The ever­expanding network of cytokines and cytokine signalling pathways56,58 presents a major opportunity for further investigation of failure to heal. Some of the most studied cytokines that contribute to healing include CCL2 (which recruits the first wave of macrophages to the wound), IL­8 (which signals through CXCR2 in human epidermis to support re­epithelialization)127 and IL­6 (which is pro­inflammatory in the initial stages of healing and stimulates keratinocyte migration). Interestingly, IL­6­deficient mice have delayed healing, as do mice deficient in IL­17 (a pro­inflammatory cytokine also known as IL­17A) or IL­22 (a cytokine with pro­inflammatory and anti­inflammatory effects)119,128.

Infection and hypoxia in chronic wounds

The pathophysiological mechanisms that result in the transition of an acute wound destined to heal to a chronic wound that does not heal is unclear. Many factors have been associated with wound chronicity, including but not limited to, the above­noted aberrations in innate and adaptive immune cells in the wound as well as sustained inflammation, alterations in angiogenesis, dysregulated matrix deposition, neuropathy and impaired neuropeptide signalling, and cellular senescence; furthermore, bacterial infection and biofilm formation, and wound hypoxia, are also implicated in the pathogenesis of chronic wounds129

Biofilms and their formation are now well documented in chronic wounds, and they likely have a crucial role in impaired healing and the recurrence and, possibly, initial occurrence of chronic wounds (Fig. 4). The biofilm research field is vast, and the role of biofilms in the pathogenesis of chronic wounds has been reviewed elsewhere130–133 Biofilms effectively shield bacteria from systemic antibiotics. Moreover, it is becoming clear that biofilm evolution also includes the formation of polymicrobial communities, making them less susceptible to antibiotics that could eliminate a more uniform bacterial population; examples of biofilm evolution have been documented134

Hypoxia in the wound environment results in the release of mediators that regulate angiogenesis and re­epithelialization, via activation of the transcription factor hypoxia­inducible factor 1 (HIF1)135,136. In vitro and preclinical in vivo studies demonstrate the downstream pro­reparative consequences of HIF1 translocation to the nucleus (extensively reviewed elsewhere137,138), which led to the proposed use of deferoxamine, a HIF1α inducer and stabilizer, as a therapeutic agent for chronic wounds135 136. Indeed, a clinical trial is currently underway to examine whether topically applied deferoxamine can improve healing in DFUs139, and a patch engineered for transdermal delivery of deferoxamine is being trialled for the treatment of ulcers in individuals with sickle cell anaemia140,141. The outcome of these trials might provide novel approaches for both the prevention and treatment of chronic ulcers.

Methodological advances

Non-coding RNA. Since their discovery, the diversity of roles of non­coding RNAs (ncRNAs) has been expanding. Most research has focused on a subset of ncRNAs,

microRNAs (miRNAs), which are ~22–23 nucleotides in length and perform regulatory functions142. Early investigations of the cutaneous role of miRNAs focused on their tissue­ and cell­type­specific expression patterns and roles in maintaining stemness in epithelial and hair stem cell populations143 144. The expression pattern of multiple miRNAs changes during the various phases of wound repair145–148. One of the most studied is miR­21, which is upregulated in both keratinocytes and fibroblasts at the wound margin in mouse wounds, and inhibition of miR­21 impairs healing in mice149. Expression of miR­21 promotes an anti­inflammatory phenotype in human macrophages in vitro and enhances differentiation of rat mesenchymal stem cells to fibroblasts150. However, miR­21 is overexpressed in venous ulcers151, suggesting that it possibly inhibits epithelialization.

Dermal remodelling is modulated by miR­29, which targets several transcripts responsible for generating the extracellular matrix and can decrease wound contraction and collagen deposition152,153. Suppression of the downstream actions of miR­29 by competitive binding to the long non­coding RNA (lncRNA) H19 is associated with increased extracellular matrix synthesis and fibroblast proliferation; however, overexpression of lncRNA H19 may improve healing in diabetic mice154. Another miRNA, miR­127­3p, regulates the transition from the proliferative phase to the remodelling phase of mouse wounds, possibly by inhibiting the proliferation of myofibroblasts155. In human wound healing, miR­127­3p might function as an epigenetic activator that regulates the transition from repair to remodelling during skin wound healing155,156. Finally, miR­210 is induced by tissue hypoxia and targets keratinocyte proliferation157, and inhibition of miR­210 is being investigated as a way to increase keratinocyte proliferation and accelerate healing. Wound inflammation and its resolution might be controlled by specific miRNAs such as miR­132, the expression of which is diminished in diabetic wounds in humans158 159

Members of another class of ncRNAs, lncRNAs (those ≥200 nucleotides in length), target genetic networks and have been found to have epithelial functions160. Notably, two lncRNAs regulate the balance between proliferation and differentiation in human organotypically cultured epidermis: anti­differentiation non­coding RNA (ANCR) maintains the progenitor status of basal keratinocytes in human epidermis; its counterpart, terminal differentiation­induced non­coding RNA (TINCR) promotes differentiation in the upper epidermal compartment161 162. Studies suggest that healing could be accelerated by targeting specific lncRNAs, either by inhibiting the expression of a novel lncRNA, wound and keratinocyte migration­associated lncRNA 2 (WAKMAR2), which is present in non­healing wounds, or by inducing the expression of the pro­proliferative lncRNA Gas5 by the use of statins (HMG­CoA reductase inhibitors), which are orally administered agents that are widely used clinically to decrease high cholesterol levels163 164 Another mechanism by which lncRNAs can regulate cell function is by competitively binding to, or ‘sponging’, multiple other miRNAs to limit their function154,165,166

The microbiota. The microbiota comprises microorganisms either in a free­floating form (the planktonic state) or adherent to the outer mucosa (such as biofilm), and both mostly likely have important roles in chronic wounds167. Understanding of the relationship between microbiota and biofilm has improved in the past decade, largely owing to advances in metatranscriptomics, metabolomics and other technologies168–170. With the advent of new molecular tools, the role of the wound microbiota is being increasingly investigated as a contributor to wound chronicity 171,172. The prolonged inflammatory state of the chronic wound is associated with persistent infection or biofilm formation131. A causal role has been demonstrated in many studies using animal models. For example, application of a bacterial inoculum or transfer of preformed biofilm results in delayed healing in experimental porcine acute wounds173,174. The transfer of the skin microbiota of a mouse with a mutation that impairs the innate immune response and healing to a wild­type mouse results in acquisition of the impaired healing phenotype by the wild type175 Staphylococcus aureus and Pseudomonas aeruginosa are among the most commonly encountered species by either molecular or culture ­ based techniques172,176. However, the classic skin commensal bacterium Staphylococcus epidermidis induces a type 17 immune programme, which is a T H17 cell ­ induced pro­inflammatory immune response generally associated with autoimmune diseases. and accelerated repair through mechanisms linked to antimicrobial peptide production and activation of a subset of skin­resident T cells that recognize specific commensals (‘commensal­specific T cells’)177 S. epidermidis also upregulates the expression of the antimicrobial molecule perforin 2 in keratinocytes172. Thus, the link between specific microorganisms in the wound microbiota and wound chronicity remains unclear.

A better prognosticator of wound chronicity might be the overall bacterial diversity of the wound microbiota178. An inverse linear relationship has been documented between decreasing wound microbiota diversity and wound healing rate in two cohorts (each with ~80 patients) with chronic wounds179. Similar associations have been noted in the wounds of patients with dystrophic epidermolysis bullosa, a genetic disorder that causes the skin and upper gastrointestinal mucosal surfaces to become fragile and blister easily180. Additional mechanistic progress may be achieved in future studies through deeper sequencing afforded by shotgun metagenomics181

In addition, host neuroimmune mechanisms that mediate the inflammatory response to pathogens and affect wound repair have been discovered. Nociceptors in the skin that express transient receptor potential cation channel subfamily V member 1 (TRPV1) are activated by a cutaneous challenge with the yeast Candida albicans and respond by expressing the neurotransmitter calcitonin gene­related peptide (CGRP). The downstream consequences are activation of dermal dendritic cells to generate IL­23, which then induces the production of the protective, antimicrobial and pro­reparative cytokine IL­17A by dermal γδ T cells182. However, skin infection

Table 1 | Characteristics of chronic wounds and common diagnostic features

Pressure ulcers Sacral areas and hips

Arterial ulcers

Venous ulcers

Lower extremities, especially ankle and toes

Medial aspect of lower legs

Inability to move, pressure

Atherosclerosis

Paralysis, aftermath of spinal injury, muscle weakness

Pale skin and poor or absent peripheral arterial pulses

Hair loss on lower extremities

Venous hypertension

Diabetic ulcers

Toes, soles

Atypical chronic wounds Any location, but more common on lower extremities

Neuropathy with or without arterial obstruction

Various: vasculitis, cryofibrinogenaemia, cryoglobulinaemia, immunological (as in pyoderma gangrenosum), dystrophic epidermolysis bullosa

with Streptococcus pyogenes also activates TRPV1+ nociceptors to release CGRP, but the result is inhibition of neutrophil recruitment and function, which can be reversed by administration of a CGRP antagonist183,184 These divergent nociceptor responses may be due to expression of different TRPV splice variants, or may be pathogen specific179. The potential for TRPV1­targeted therapies to improve healing may require a personalized medicine approach that is tailored to the channel splice variant expressed or specific pathogen infecting the wound185

Although most studies of the wound microbiota have focused on bacteria, emerging work implicates fungi (the ‘mycobiome’) and viruses (the ‘virome’) in the wound as potential contributors to wound chronicity. An increased abundance of fungal species in the wound microbiota is associated with longer healing times186. Fungal activation of a fungus­specific TH17 cell response can exacerbate local inflammation and impair healing187. Although viruses are recognized as members of the wound microbiota, and viral modulation of inflammatory responses in wounds is documented, at present there are few studies of the effects of viruses in the microbiota on wound outcomes or chronicity62

Diagnosis, screening and prevention

Chronic wounds are heterogeneous in their aetiology, pathogenesis, appropriate management approach and prevention. Thus, clinical evaluation is an essential step to reach a final diagnosis and to establish a specific therapeutic strategy. The most common types of chronic wound (arterial, venous, diabetic and pressure ulcers), as well as atypical wounds, which are less common but have

Lower leg varicosities, hard indurated and hyperpigmented skin (lipodermatosclerosis), hyperpigmentation, oedema

Insensate foot, lower extremity and foot arterial pulses

Livedo net-like skin pattern, purpura, signs of previous radiation

Undermined wound edges

Rule out osteomyelitis

Doppler ultrasonography of ankle and/or brachial index measurements

Vascular studies, angiography

Duplex (Doppler and B-mode) ultrasonography

If suspected, biopsy to rule out malignancy

Doppler ultrasonography of ankle and/or brachial index measurement

Neuropathy testing

Vascular studies if pulses are not adequate

Serum cryoglobulin and plasma cryofibrinogen

Biopsy to rule out skin cancer, occluded dermal blood vessels or vasculitis

proved difficult to heal, differ in their typical body site, cause, features observed on examination and the appropriate diagnostic tests to undertake (Table  1). Atypical wounds and their diagnosis and management have been reviewed elsewhere4. The term ‘atypical’ is used because these wounds do not present with typical clinical findings. These wounds are often the result of immunological factors, micro­occlusion of blood vessels of the lower extremities (as with cryofibrinogenaemia or cryoglobulinaemia), vasculitis from various aetiological causes (such as collagen vascular diseases, calcium deposition in and around blood vessels (for example, calciphylaxis in the setting of renal disease)), breakdown of metastases to the skin and primary skin cancers. Regardless of the type of wound and underlying cause or aetiology, a comprehensive assessment of the patient’s condition, including the medical and pharmacological history, is essential. Various diagnostic features must be analysed from the medical history, physical examination and imaging or laboratory testing (Table  1). In addition to immediate surgical considerations that arise during the diagnostic phase, such as surgical debridement and planning for grafting or vascular and orthopaedic procedures, the fundamental clinical criteria involve three major components of the wound: the wound bed, wound edges and the surrounding skin. Figure  5 outlines the major diagnostic and interventional approaches. Of note, consortia are increasingly being established to develop a better understanding of biomarkers in chronic wounds188. Given the medical and surgical complications of DFUs, government agencies are establishing consortia, such as the Diab etic Fo ot C ons or tium, a US NIH­funded six­institution network tasked with finding ways to treat

Chronic wound evaluation

• Identify the need for immediate surgical intervention

• Assess social and systemic medical factors

Diagnostic considerations and re-evaluation of diagnosis

• Analyse medical, vascular and neuropathic factors and pressure-induced ischaemia

Anatomical and vascular ulcers

• Poor arterial pulses, cold skin, ischaemia, necrosis

• Varicosities, venous disease, hyperpigmentation

• Swelling and/or lymphoedema

Standard treatment and management

Pressure-induced ulcers

• Peripheral neuropathy

• Immobility, decubitus ulcers

• Combined pressure and vascular disease

Atypical and unusual ulcers

• Purpura, livedo

• Vasculitis, necrosis, pathergy

• Pyoderma gangrenosum

• Cryoproteinaemia

• Ulcerated skin cancer

Final diagnosis, classification and ulcer staging

• Vascular laboratory evaluation

• Imaging studies

• Tissue biopsy

• Debridement: enzymatic, dressings, scalpel, hydrosurgery

• Wound bed preparation

• Infection control: antiseptics (silver, iodine) and/or antibiotics (topical or systemic)

• Pressure relief

• Dressings: hydrocolloids, foams or gels

• Compression: wraps (self-adherent) or pumps

• Vascular surgery: stenting, bypass, venous surgery or grafing

• Systemic agents: for atypical wounds; steroids, other immunomodulators

Maintenance debridement and periodic assessment of improvement

Advanced treatments (individualized, multiple-modality therapies)

• Skin substitutes: bioengineered skin, placental tissue, living and non-living skin constructs

• Growth factors (i.e. topical PDGF)

• Stem cells: multipotent, from bone marrow or adipose tissue

• Biologic or other immunosuppressants

• Hyperbaric oxygen

Fig. 5 | General algorithm of evaluation, diagnosis and treatment of chronic wounds. Proposed algorithm that outlines the available therapeutic options for the management of chronic wounds. Initial consideration is essential to identify wounds that require immediate treatment, such as diabetic foot ulcers. The progression of therapy leads from basic to more complex treatment modalities. Debridement is important, and some aspects of therapy are more applicable to certain types of chronic wound than others. PDGF, platelet-derived growth factor.

and reduce complications of DFUs. The Diabetic Foot Consortium aims to identify biomarkers, biological and molecular mechanisms related to DFUs, and prevention of severe complications of DFUs, such as amputation.

Clinical features of chronic wounds

The most common chronic wounds are vascular ulcers (including those due to venous and/or arterial insufficiency), DFUs (of neuropathic, vascular or mixed aetiology from neuropathy and vascular obstruction), arterial insufficiency and PUs (also known as decubitus ulcers). The aetiology of these chronic wounds is easily established by clinical inspection owing to such characteristics as body location, detection and measurement of arterial pulses, neuropathic findings, the state of the wound bed, unusual features in the surrounding skin and other features (Table 1)

The features of VLUs reflect the main pathophysiology, which involves incompetent perforator (or communicating) veins between the deep and venous system and failure of the venous ambulatory pressure to decrease when walking or exercising (venous hypertension)189,190. The clinical–aetiology–anatomy–pathophysiology (CEAP) classification for VLUs has been found to be useful in various guidelines 191 192. In this classification system, each parameter is divided into several

subclasses, which enables the clinician to score the complexity of the patient’s condition. VLUs are typically located above the medial malleolus, more commonly on the medial side of the leg, have irregular edges and tend to be superficial (that is, not involving muscle or bone). Surrounding varicosities (visible varicose veins upon clinical examination) and oedema are common. The wound bed is typically yellow or pale red owing to fibrinous material deposition, and there can be considerable exudate even in the absence of infection. These ulcers are typically not very painful, notably except when the ulceration is close to or over the malleoli and, therefore, near the nerve­rich periosteum. The surrounding skin of the lower leg is often characterized by indurated (hardened) and hyperpigmented skin, which is termed lipodermatosclerosis and represents a deeply fibrotic process50,190,193–195. Arterial leg ulcers tend to occur more commonly on the lateral side of the lower leg, are often distally located (such as on the dorsum of the foot or toes and on the heel), and display poor proximal (femoral or popliteal) or distal (dorsalis pedis or posterior tibialis) pulses by palpation or Doppler ultrasonography examination. Arterial ulcers are smaller in size than VLUs, are deeper (often down to muscle and even tendons or bone), more painful and more regular in configuration (punched­out appearance). The wound bed often

displays black necrotic tissue. The surrounding skin may show atrophy, with pale skin colour and loss of hair. DFUs are categorized as being neuropathic, ischaemic or neuro­ischaemic, according to their preponderant aetiological and pathogenetic mechanisms196 197 . Neuropathic diabetic ulcers usually occur in areas of high pressure on the plantar surface of the foot and, typically, over the metatarsal heads. They may be preceded by an inflammatory process that leads to distortion of the underlying bones (referred to as Charcot foot)198,199 Callus formation is common and contributes to the pressure on the insensate foot. Diabetic ulcers in which ischaemia has a substantial role show an absence of or poor pedal pulses, typical ischaemic atrophic changes in the skin and a necrotic wound bed at an early stage. Importantly, small diabetic ulcers may involve a much larger portion of the foot and may become easily infected, develop tunnelling (the formation of subdermal sinus tracts) and lead to the need for amputation198. Thus, diabetic ulcers are always in danger of causing devastating clinical effects. Crucial classification and treatment approaches for DFUs have been reviewed elsewhere200 PUs are located over bony prominences, such as the heel, hip and sacral areas. These ulcerations may exhibit skin redness (stage I) that becomes non­blanchable during digital palpation. PUs may rapidly advance to deep ulcers involving the dermis (stage II) and subcutaneous tissues (stage III), and even tendons and bone (stage IV) (extensively reviewed elsewhere)196. The National Pressure Ulcer Advisory Panel (NPUAP) served as an independent nonprofit organization for the many aspects of PUs that would foster improvement in the care, prevention and research efforts dedicated to these chronic wounds. In 2019, realizing that PUs constitute chronic wounds that actually begin with normal and intact skin that succumbs to injury from external pressure forces and immobility of patients, the organization changed its name to National Pressure Injury Advisory Panel (NPIAP). Thus, the organization emphasizes the role of pressure injury in the pathogenesis of PUs, pointing out the fact that, ideally and with proper care, these ulcers are technically completely preventable. In Europe, the European Pressure Ulcer Advisory Panel (EPUAP) has chosen to acknowledge that, regardless of the initial cause, the presence of pressure­induced ulcers remains the central component requiring staging and attention, even though avoidance of pressure is paramount.

Diagnostic work-up

The clinical guidelines for vascular wound assessment are well established201. In VLUs, the initial evaluation is clinical and is based on clinical findings and concomitant exclusion of arterial insufficiency. However, detection of incompetent perforators on Doppler duplex scanning ultrasonography is helpful in the diagnosis. The vascular diagnostic work­up starts with pedal pulse palpation, which might be difficult in patients with obesity, those with leg oedema and those with concomitant diabetes and extensive vascular calcification196 202. Diagnostic tests for ulcers due to arterial insufficiency include continuous wave Doppler (CWD) ultrasonography, ankle brachial pressure index (ABI) and transcutaneous oximetry

(TcPO2). In patients with diabetes, the Doppler probe must be used according to systolic toe pressure (TP) and toe brachial index (TBI)203. The results and, particularly, test interpretation guide the medical and surgical management and possible complications. The ABI involves the measurement of the Doppler signal, by dividing the ankle systolic blood pressure over the brachial systolic blood pressure; a value of 1.0 or 1.1 is considered normal, whereas a ratio of ≤0.8 is abnormal and raises increasing suspicion about the level of poor perfusion with a decreasing ratio198. However, because of calcification in blood vessels, the ABI is unreliable in the assessment of diabetic ulcers. Palpation of the pedal pulses is a subjective test for the detection and intensity of the pulse of the dorsalis pedis and posterior tibialis arteries; the detection depends on the clinician’s skills and experience. The use of a Doppler probe for the CWD test helps by recognizing the arterial signal with either triphasic or biphasic sounds in normal patients, and monophasic or absent sounds in those with peripheral arterial disease1. Measurements of TcPO2 are time­consuming, require specialized equipment and are most valid for diabetic foot assessment, where a value of <40 mmHg indicates poor oxygen perfusion and may even be used by surgeons to determine the leg level at which amputation may be required204. PU assessment is used mainly to understand the true wound size and depth, and has evolved from pure clinical evaluation to imaging techniques in more difficult cases205. The main problem with assessment of PUs is to completely assess the depth and size, as these wounds typically have undermined edges; several 2D and 3D assessment options are available, ranging from high­frequency ultrasonography to laser scanner imaging or optical coherence tomography206

The role of the bacterial swab in chronic wound management is now clear: it should be considered only if clinical signs of infection are present207. The simple swab results may not reflect the organisms causing the infection, but there has been controversy as to whether the quantitative number of organisms, not necessarily the presence of specific species, constitutes infection and the need for clinical intervention196,197. Nevertheless, there is some evidence that, especially in DFUs, a swab culture is not totally irrelevant for determining infection208 More research needs to be done with the diagnosis of infection, also in view of the importance of biofilms. Symptoms and signs of wound infection are more crucial in DFUs and PUs than in VLUs. These signs and symptoms include increased exudate and foul odour from the wound, increased wound pain (often not helpful because of the neuropathy and thus diminished pain sensation), localized swelling, increased heat of the surrounding skin, friable and hypergranulating wound bed, and increased erythema and cellulitis. Increasing evidence suggests that biofilm formation and persistence have an important role in chronic wound formation, chronicity and possibly recurrence131,132,207.

Wound biopsy

When wounds fail to heal despite appropriate management, it is important to perform a wound biopsy, which is essential to confirm the clinical wound diagnosis, to

understand the failure to heal and respond to treatment and to rule out neoplasia. The biopsy should be performed at the wound edge and should include a portion of the surrounding skin209. Peculiarly, the biopsy site usually heals more quickly than the actual wound bed tissue210. Wound biopsy is also helpful in the diagnostic work­up in atypical wounds, which include inflammatory wounds, such as vasculitis and those wounds due to occlusion of microvascular channels from cryoglobulins and cryofibrinogens211. Atypical ulcers due to cryoproteins are surrounded by small blood vessels that give the appearance of linear hyperpigmentation often termed ‘micro livedo’211. In pyoderma gangrenosum, a diagnosis of exclusion, the biopsy can exclude other vasculopathies, including calciphylaxis212.

Biomarkers

Levels of TNF in pyoderma gangrenosum and in non­healing VLUs show a good correlation with prognosis in specific groups of patients213,214. TNF is elevated in non­healing VLUs, and, when elevated, the therapeutic use of blocking TNF may prove helpful in decreasing inflammation and accelerating healing of chronic wounds213. Another biomarker is osteopontin, which is used to detect the presence of calciphylaxis215, as vascular smooth muscle cell expression of osteopontin is upregulated in calciphylaxis. Furthermore, the levels of specific metalloproteinases have shown promise as biomarkers in non­healing chronic wounds216. Elevated level of MMPs in wound fluid from non­healing wounds has been correlated with reduced healing rates217. The field of biomarkers for chronic wounds is likely to see advances. For example, GM­CSF and MMP13 as biomarkers of venous ulcers may predict healing status with >80% accuracy218. Genetic markers have been found that are linked to chronic wound microbiota composition and as risk factors for impaired healing179.

Prevention

Various guidelines and recommendations have been proposed for the primary and secondary prevention of chronic wounds219. An increasingly recognized, important component of prevention and ulcer recurrence is that patients with chronic wounds are generally not able to adhere to effective self­management strategies, owing to the absence of proper and continued guidance and teaching220. Prevention of VLUs is achieved mainly by using compression leg bandages and stockings. Continued use of compression stockings helps to reduce the rate of recurrence. Modern textile materials have helped in increasing compliance (adherence) from patients and practitioners. There is evidence that enhanced comfort level and improved compression are achieved with textile material that relies less on nylon and more on cotton and collagen peptide bonds in the structure of stockings221. Control of leg oedema is best achieved using stockings and leg elevation196,197. A UK initiative called the Lindsay Leg Club Foundation has helped in reinforcing compliance and/or adherence222. Prevention of DFUs is very complex and demanding and is best achieved using a multidisciplinary foot care unit. The risk of developing these foot ulcers has been

stratified into low, moderate and high according to the extent of neuropathy and limb ischaemia223. Assessment of foot biomechanics and sequential use of orthoses, together with constant follow­up, reduces the risk of foot ulceration in patients with diabetes224. Prevention of PUs is challenging but essential, and is initially achieved by using a risk assessment scale, such as the widely used Braden scale196,225, although several scales exist to assess risk in different categories of patient. The approach in patients with PUs is to minimize pressure, shear and moisture by repositioning the patient according to scheduled protocols, and by avoiding contact of the chronic wound with urine, faeces and other fluids196,197. In patients who are not able to receive proper repositioning, the use of multi­layered silicone dressings in areas at risk decreases the incidence of PUs226. Furthermore, a more proactive approach is needed in identifying populations that are at risk of different types of ulceration, which would help in determining how to best prevent occurrence of ulcerations in vulnerable groups.

Management

The scale of the clinical problem

The extent and cost of treating wound problems in modern­day practice are not completely known, but research suggests that chronic wounds may affect up to 5% of the adult population and consume up to 10% of health­care expenditure. For example, the number of patients with wounds in the UK National Health Service increased by 71% between 2012/2013 and 2017/2018, which was associated with an increase in cost of care of 48%3,227,228. In the past, clinicians may have been slow to recognize the nature and extent of the challenge of this problem, and there was a general lack of appreciation of the need to develop a system of optimal clinical delivery and care that takes advantage of emerging treatments and technologies. This situation is compounded by the fact that ‘precision medicine’ for chronic wounds is still in its infancy; improved molecular diagnostic and therapeutic tools are needed to focus on individual patients. Despite these considerations, the management of chronic wounds has improved substantially in the past two or three decades. Wound dressings are a very pertinent example of basic wound care that can clearly improve the management of chronic wounds. Several types of wound dressing exist, and the choice of dressing depends on several clinical parameters, including wound exudate, skin fragility and the development of favourable granulation tissue after debridement229 . The multiple definitions of chronic wounds in use can make it difficult to compare results from different studies2,230,231. Therapeutic options vary according to the type of wound and include dressings, devices, systemic drugs, and surgical and biological approaches131,232–235 (Table 2 and Fig. 5). A systemic drug (pentoxifylline) has been shown to accelerate VLU healing236,237. Importantly, basic clinical approaches also rely on appropriate surgical management. In addition to vascular reconstruction, grafting and limb salvage, debridement is crucial for removing necrotic tissue. Indeed, ‘maintenance debridement’ might be required to keep the process of healing going in the right direction238. Another valuable

Type of chronic wound Clinically well-established basic care

Venous leg ulcers

Compression bandagesa

Layered compression bandagesb

Diabetic foot ulcers

Pressure ulcers

Off-loading for the insensate foot and for Charcot deformity

Padding and protection

Regular callus removal and surgical debridement

Reducing walking activity

Avoidance of pressure

Turning patient frequently (repositioning)

Common and preferred wound dressings

Foamsb

Hydrocolloidsc

Alginatesd

Saline dressings

Foamsb

Foamsb Alginatesd

Other local dressings or devices

Slow-release iodine or silver dressingse

Negative pressure

Saline dressings

Contact casting (especially for Charcot foot deformity): series of leg/foot casts, a removable cast or a Charcot restraint orthotic walker

Crutches, knee walker and wheelchair

Hyperbaric oxygen

Slow-release iodine or silver dressingse

Support surfaces (mattresses, cushioning)

Negative pressure

Pharmacological agents and nutritional approaches

Oral pentoxifylline

Weight control

Diabetes control with diet, oral agents or insulin

Weight control

Common surgical management Biological approaches

Debridement

Autologous grafting

Subfascial endoscopic perforator surgery

Debridement

Removal of callus around the wound

Achilles tendon lengthening

Stenting, revascularization when required

Weight control

Nutritional support to normalize blood albumin

Pain control, especially during repositioning (ibruprofen, paracetamol (acetomenophen))

Debridement

Autologous grafting

Surgical flap in the absence of healing

Tissue engineering treatments: acellular or cellular constructs

Platelet-derived growth factor

Bioengineered skin and tissue engineering

Platelet-rich plasma

Bioengineered skin and tissue engineering

aLayered bandaging may be more effective for healing. bFor fluid absorption. cFor antibacterial protection and painless debridement. dAntibacterial and absorption activity. eSlow-release antiseptics of iodine or silver.

therapy consists of the use of negative pressure. Also called vacuum­assistive wound closure, the principle of negative pressure for accelerating wound healing relies on an airtight dressing placed on a draining wound and connected to tubing and an electrical device that generates negative pressure. Chronic wound fluid has been shown to inhibit cellular proliferation in vitro239,240, and its removal has been shown to enhance wound closure in vivo241,242

Antimicrobial approaches remain important in management of chronic wounds. The common occurrence of antibiotic­resistant bacteria in wounds171 has prompted the search for non­antibiotic antimicrobial treatment approaches. Indeed, a study demonstrated that application of topical antibiotics to a wound markedly impairs healing in mice and, possibly, in humans243. Multiple antimicrobial modalities are being investigated, including therapeutic bacteriophages, antimicrobial peptides, repurposed drugs, cold plasma treatment, photodynamic therapy, probiotics and bioelectric dressings244,245. Trials of both topically administered probiotics in preclinical models of wounds and orally administered probiotics in patients have been reported246. In a randomized controlled study, oral probiotic supplementation improved healing in the treated cohort of patients with a DFU247. The gut–skin axis and gut dysbiosis are implicated in the pathogenesis of skin diseases such as atopic dermatitis and psoriasis, and ongoing investigation might reveal that this signalling axis also affects wound healing248

Other basic approaches to the most common types of chronic wound are addressed in Table  2 ; some of the approaches are specific for the chronic wound type.

Evidence-based treatment selection

The C o chrane Wounds systemat ic re v ie ws d at ab as e details many useful reviews of studies focused on dressings, devices, drugs, surgical and biological approaches and provides valuable insights into the evidence base for these treatments but also highlights the paucity of data from which to draw conclusions. The inability to draw definite conclusions is multifactorial and may have to do with poor statistical power analysis when designing clinical research trials, among other clinical and treatment factors. At present, several treatment recommendations are still based on expert opinion and clinical guidelines.

The stimulus for establishing chronic wounds management guidelines came largely from an emphasis on wound bed preparation131,232–235. There are basic approaches to the treatment of chronic wounds that are rooted in both experience and evidence: compression for venous ulcers, off­loading (keeping pressure off the wound site) for diabetic neuropathic ulcers and PUs, debridement of necrotic tissue, stenting and revascularization to restore blood flow and oxygen, and hyperbaric oxygen for chronic wounds that are unresponsive to treatment. Wound dressings currently available are better than those of even 10–20 years ago. Occlusive dressings

or moisture­retentive dressings, as they are often called, are based on the recognition that moist wound healing can also aid healing of chronic wounds or at least the formation of granulation tissue189 229. Film wound dressings are preferred for less exudative wounds, foams to absorb excessive exudate, hydrocolloids to initiate debridement and alginates to provide moisture to very dry wound beds229,249. Composites of these dressings and variations have been developed, such as the delivery of antiseptics and antimicrobials from the dressings250. Specific considerations for standard and basic therapies in the different types of chronic wound are discussed below.

Biologically based treatments

Despite the difficulties in identifying and studying specific treatments, enormous progress has been made over the past two or three decades in developing advanced therapeutic approaches that might benefit healing of chronic wounds. One of the first approaches was the use of topical growth factors that are important in the healing cascade39,251. Platelet­derived growth factor (PDGF) was the first topically applied recombinant growth factor approved by a regulatory agency (the FDA) for the treatment of diabetic neuropathic foot ulcers39,252–256 . This approval also helped to highlight the importance of wound debridement252 257. However, at present, there are limitations to topical growth factors, including PDGF. The potential reasons for the general lack of effectiveness of topically applied growth factors have been reviewed elsewhere39,258. Among the reasons for the unexpected ineffectiveness or failure of growth factors to stimulate healing in chronic wounds are inadequate topical formulation, lack of penetration into the wound bed, inability to use a growth factor in concert with others that might be synergistic because of regulatory obstacles and breakdown of the protein in the hostile and bacterially colonized microenvironment of the wound bed. Other publications suggest that growth factors may be effective259

At the same time, research activity into tissue engineering to treat chronic wounds has been growing. In this approach, epidermal and dermal cells cultured in the laboratory were considered to be another way to enhance healing260–263. The use of this bioengineered or ‘artificial’ skin has led to improved understanding of the mechanism of action of cell therapy. For example, in the case of constructs consisting of living cells, it has become clear that when applied to chronic wounds, even if autologous, the cells are no longer detectable a few weeks after application. Therefore, bioengineered skin has been considered a stimulus for wound healing and not a true cell replacement, like grafted skin264,265. Some of the bioengineered skin constructs have been shown to accelerate healing and have received regulatory approval for the treatment of VLUs and DFUs 202,260,261,266–269 However, the percentage of improved healing over control treatment is less than ~20%. Many of these bioengineered constructs have been approved and marketed with the suffix ‘graft’, which differs from the understanding by clinicians, especially surgeons, of grafts as a more permanent solution to wound resurfacing. Moreover, repeated treatments may be required, diminishing their

cost­effectiveness. Another important consideration is that these advanced therapies are mostly not available to lower­income patients and those in lower­income countries.

Despite these setbacks, progress in elucidating the more mechanistic aspects of wound healing is likely to lead to improved and more cost­effective treatments. It could still be stated that expenses are focused on technologies that might have theoretical benefits in assisting healing rather than identifying the aberrations that are present in an individual patient and that are preventing healing270. The bacterial burden and the formation of biofilms are important considerations in wound chronicity, albeit their contribution might often be subtle. Surface organisms are generally misleading and non­specific, thus making a simple swab often not relevant and causing the unnecessary use of systemic antibiotics. Instead, the number of organisms in the wound and the formation of biofilms play a greater part in impaired healing271

In addition to biological factors that influence healing, social and psychological factors can influence both choice of treatment and disease outcome272 Another challenge in researching chronic wounds is the assessment of effective wound healing; for example, in patients with terminal disease from other comorbidities, it is clearly inappropriate to expect full wound closure. However, it is both appropriate and desirable to evaluate interventions that influence pain, smell, discharge and patients’ health­related QOL (HRQOL) by measures other than complete wound closure or speed of healing273

Quality of life

Impact of chronic wounds

Chronic wounds have a negative effect on HRQOL, owing to the physical injury, the treatment required, the chronicity of the condition and the likelihood of recurrence. More than two decades ago, research indicated that patients with leg ulcers experienced considerably poorer QOL than matched individuals in the areas of emotional, social and physical health274. Today, it is well established that chronic wounds are painful and affect physical role and function7, can cause mental health concerns275 and may limit social and workforce participation276. Chronic wounds can present a financial burden to those who must self­fund their treatment277. Wound treatment and prevention have improved in recent decades; however, mitigation of the negative effect of chronic wounds on QOL remains an unresolved challenge. Chronic wounds affect many facets of QOL in three main domains (physical, emotional and social), often in a fundamental way ( b ox  1 ) . Of note, these domains overlap, so that changes in one domain almost invariably affect the others. These considerations and the multiple needs of patients highlight the need for a multidisciplinary approach to patients with chronic wounds. Some advances are also being made in the interactions of social genomics and wound healing. Although a clear picture has not yet emerged, it is likely that this field will become increasingly important and provide opportunities for exciting research278,279

Box 1 | Quality of life domains affected in patients with chronic wounds

Physical domain

• Physical deformity

• Wound pain, odour and exudate

• Reduced mobility

• Suboptimal sleep

• Impaired activities of daily living

• Feeling unwell from adverse effects of treatment modalities

• Worsening of comorbidities

Emotional domain

• Stress, anxiety and depression

• Frustration, worrying and helplessness

• Dissatisfaction with health-care givers

• Fear of trauma to the wound or of recurrence

• Stigma and worsening self-esteem

• Lack of life enjoyment

Social domain

• Withdrawal from family and friends

• Loss of independence

• Decreased levels of sexual activity

• Difficulty with domestic duties, hobbies and recreation

• Lack of acceptable clothing and footwear

• Difficulty with work, studying and shopping

• Time required for wound treatment and medical appointments

Measuring quality of life

The introduction of patient­reported outcomes and patient­centred care has required that health­care providers identify and respond to the patient’s most important concerns. Measuring QOL in research can be achieved with generic instruments, such as the 36­item short form (SF­36)280 and the EuroQol five­dimensional (EQ­5D) questionnaires281. Disease­specific measures include the Cardiff Wound Impact Schedule282 and the Diabetic Foot Scale283, which are clinically useful and responsive, as they include items that are specific to the condition of interest. QOL is often a secondary outcome measure, and research that investigates QOL as the primary purpose of the study should be conducted if QOL improvement is to be realized by people with chronic wounds.

Wound management and patient care

QOL must be assessed early in the care episode so that needs are identified and interventions can be targeted. Interdisciplinary teamwork is necessary284 and a genuine partnership with the patient is essential. Traditional models of treatment and care (provided in the doctor’s clinic and in the home by nurses) have evolved to better meet the holistic need of patients. Advances include specialist wound clinics that bring together multidisciplinary expertise284, community­based interventions such as Lindsay Leg Clubs that offer opportunity for social support222 and self­management approaches to optimize the individual’s involvement in their care285. Engagement with informal care ­ givers who support individuals affected by chronic wounds and their participation in wound care may improve QOL286

Comorbidities and chronic wounds

As chronic wounds are a symptom, manifestation or consequence of the health conditions that cause the greatest disease burden in society (for example, vascular disease and diabetes), they should be considered a health policy priority287. People who experience chronic wounds often present with multiple comorbidities98 and, therefore, many interrelated factors might affect their QOL. Understanding of QOL is predominantly informed by

research conducted with patients who are the easiest to access but who are not necessarily the most vulnerable. Future research should target people who experience less prevalent types of wounds, live in developing countries, are from diverse backgrounds, experience cognitive impairment and/or are at the end of life.

Outlook

Considerable progress has been made in our understanding of the healing process of the skin and the impaired healing that characterizes chronic wounds. However, many opportunities exist for improving the outlook in this challenging field, especially with regard to meaningful treatment and diminished recurrence rates. Understanding of the pathophysiological mechanisms that lead to impaired healing is still inadequate and much work remains, despite the clear progress discussed earlier. The potential avenues to better understand this challenging and vast subject are many, but an example of an important area of research that should be addressed is the role of lipid mediators in diabetes and possible cellular senescence288,289. Below, we discuss particularly promising areas.

Stem cells

Although all tissues, including skin, have resident stem cells, bone marrow­derived mesenchymal stem cells (BM­MSCs) have been widely investigated for their potential to accelerate and/or improve aspects of wound healing. In vitro and in vivo studies demonstrate that BM­MSCs exhibit multiple pro­reparative functions, including migrating to sites of injury, stimulating the proliferation of wound­resident cells and promoting wound angiogenesis; these effects are mediated by secretion of growth factors and cytokines, suppression of inflammation and generation of antimicrobial peptides290–292. In addition, BM­MSCs are easily harvested and cultured, and autologous or allogenic BM­MSCs can be administered without much risk of immune rejection. Adipose tissue is another easily accessible source of MSCs with some multipotent properties like those of BM­MSCs293,294. Tissue­resident stem cells, such as dermal or epidermal stem cells, have also been proposed as therapeutic mediators of repair295,296 but require autologous sources to prevent immune rejection, limiting their availability. Other sources of stem cells that have been investigated less fully include the umbilical cord and Wharton’s jelly (a gelatinous substance within the umbilical cord)297.

An emerging concept termed priming of advanced therapeutic agents298 is supported by several lines of evidence. For example, the pro­reparative efficacy of stem cells might be amplified by their conditioning in hypoxia, TGFβ1 or other drugs, or by lentiviral transduction of MSCs for VEGF expression298–301. This priming approach resembles that proposed for bioengineered skin before its clinical use in chronic wounds298. Additional strategies to improve therapeutic efficacy of MSCs include co­administration with other cell types, such as fibroblasts, or modulation of the co­administered extracellular matrix ‘niche’300 302 303. For example, a promising approach is the delivery of well­characterized cultured autologous

Black eschar (remove for evaluation)

Eczema and/or dermatitis

Depth

Scarring (fibrosis and/or callus) Colour of wound bed (may require removal of dark eschar for evaluation)

edema and/or swelling

Fig. 6 | Wound bed scoring system. Representation of crucial clinical components of wound bed preparation with a scoring system, the wound bed score (WBS), which may be useful for bedside assessment without complex devices. The scoring system requires evaluation to determine its validity in terms of weekly or biweekly follow-up clinical monitoring and decision-making regarding therapeutic agents and alteration in clinical management. At this time, in its current form, the WBS may communicate the state of the wound. The best possible score with this system is 16. It is predicted that as the validation process takes place, some components of the score may be more important and informative for outcome than others. For example, black eschar, wound depth and resurfacing epithelium are likely to have a greater weight in predicting healing or lack thereof than other components of the WBS. Adapted with permission from reF 317, Elsevier.

BM­MSCs in a fibrin spray to hard­to­heal wounds265. In subsequent trials, autologous BM­MSCs improved healing of chronic wounds304,305. Although promising, MSC therapy has limitations, including the limited number of clinical trials from which to draw conclusive evidence and the lack of uniform protocols for clinical administration. Ongoing work will address these issues, as in a 2022 prospective randomized controlled trial306

Exosomes

One approach to standardization of cellular therapy may be through the use of MSC­generated exosomes, which have similar pro­reparative functions to the cells from which they originate, primarily in animal models of wound repair and in vitro. Their cargo of miRNAs, WNT ligands, growth factors, cytokines and signalling lipids provides the mechanism for paracrine stimulation of wound­resident cells307–310. Exosomes also orchestrate cell migration, including that of mouse dermal fibroblasts311

Exosomes from many variations of cellular sources improved healing in numerous preclinical studies312 The lack of standardization currently precludes identification of the optimal exosome approach. In addition, the large numbers of cells required to generate sufficient exosomes to obtain pro­reparative effects and the variable cargo depending on parental cell strain might limit therapeutic use of these extracellular vesicles308

The concept of wound bed preparation

The concept of wound bed preparation was first proposed to provide initial diagnostic steps and/or therapeutic management, a framework for unifying mechanisms of tissue repair, and for maximizing available and future treatments. The concept of wound bed preparation also recognized the need to address biochemical and molecular factors232. This all­encompassing concept was later refined to better address biochemical and therapeutic considerations233–235

system for wound bed preparation, focusing on certain parameters to be addressed and improved.

Perspectives

Despite established progress and emerging technologies, challenging obstacles remain but might also be fertile ground for research. The finite speed at which keratinocytes can migrate from the edge and resurface the wound is a perplexing phenomenon313,314, suggesting the existence of a so­called keratinocyte speed limit39,315. Indeed, a rate of healing (advancement of keratinocytes from the edge towards the centre of the wound) of <0.75 mm per week is associated with a poor prognosis and failure to heal315. Conversely, a rate of >1.4–1.5 mm per week has not been demonstrated, even in healing wounds315. Another major challenge is that chronic wounds have anatomical and physiological abnormalities that cannot be adequately corrected with present knowledge and approaches. The use of surgical approaches is still very much needed, including attention to debridement, revascularization and the use of negative pressure.

Regrettably, at least until a breakthrough in our capabilities is achieved, we might need to attenuate our expectations of ideal tissue regeneration. Evolution into complex

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organisms resulted in loss of the ability to heal by fully regenerative processes. Instead, healing mechanisms rely on stopping bleeding after injury and achieving rapid closure of acute wounds, even if that entails scarring. This approach is beneficial as long as lifespans are limited; however, with increased ageing, diseases such as diabetes, circulatory problems and pressure injury became common. These conditions associated with ageing lead to decreased blood flow and the development of heart failure, immobility from strokes, neuropathy and poor healing from diabetes, and immunosuppression. Furthermore, early experimental evidence indicates that chronic wounds may not be as dormant as previously thought and the undesirable additional tissue stimulation may actually increase their energy requirements316. Finally, approaches to render cellular components and extracellular matrix more resistant to the effects of pressure and ischaemia might also deserve exploration. Thus, although much focus has been given to finding new ways to stimulate and accelerate the healing process, opposite and unusual laboratory and clinical strategies may be required to improve outcomes of patients with chronic wounds.

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Author contributions

Introduction (V.F.); Epidemiology (D.M.); Mechanisms/pathophysiology (R.R.I. and A.M.S.); Diagnosis, screening and prevention (M.R. and M.G.); Management (K.H.); Quality of life (S.K.); Outlook (V.F.).

Competing interests

The authors declare no competing interests.

Peer review information

Nature Reviews Disease Primers thanks R. D. Galiano, L. Téot and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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RElatED links

Cochrane Wounds systematic reviews database: https:// wounds cochrane org/news/reviews

Diabetic Foot Consortium: http://diabeticfootconsortium org/ Lindsay Leg Club Foundation: https://www legclub org/ United States Diabetes Surveillance System: https://gis cdc gov/grasp/diabetes/DiabetesAtlas html

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