Immune Regulation of Skin Wound Healing: Mechanisms and Novel Therapeutic Targets

Abstract

Significance:

The immune system plays a central role in orchestrating the tissue healing process. Hence, controlling the immune system to promote tissue repair and regeneration is an attractive approach when designing regenerative strategies. This review discusses the pathophysiology of both acute and chronic wounds and possible strategies to control the immune system to accelerate chronic wound closure and promote skin regeneration (scar-less healing) of acute wounds.

Recent Advances:

Recent studies have revealed the key roles of various immune cells and immune mediators in skin repair. Thus, immune components have been targeted to promote chronic wound repair or skin regeneration and several growth factors, cytokines, and biomaterials have shown promising results in animal models. However, these novel strategies are often struggling to meet efficacy standards in clinical trials, partly due to inadequate drug delivery systems and safety concerns.

Critical Issues:

Excess inflammation is a major culprit in the dysregulation of normal wound healing, and further limiting inflammation effectively reduces scarring. However, current knowledge is insufficient to efficiently control inflammation and specific immune cells. This is further complicated by inadequate drug delivery methods.

Future Directions:

Improving our understanding of the molecular pathways through which the immune system controls the wound healing process could facilitate the design of novel regenerative therapies. Additionally, better delivery systems may make current and future therapies more effective. To promote the entry of current regenerative strategies into clinical trials, more evidence on their safety, efficacy, and cost-effectiveness is also needed.

Mikaël M. Martino, PhD

Scope and Significance

Chronic skin wounds and excessive scarring represent large and growing challenges to healthcare systems globally. For example, in 2009, chronic wounds affected 6.5 million people in the United States, leading to US$25 billion in annual treatment costs.¹ With rising rates of obesity and diabetes, and an aging population, these numbers are projected to rise.¹ Ineffectively treated chronic wounds can result in infection, sepsis, amputation, and, in extreme cases, death of the patient. Scarring can also have considerable functional and physiological consequences. In 2004, an estimated 234 million surgeries were performed globally, corresponding to about one operation per 25 people.² These incisions result in a scar, each of which is very difficult to treat and impossible to prevent. Nonetheless, $12 billion is spent annually in the United States to reduce scar size.¹

The immune system is a key player throughout the wound healing process, actively participating to reestablish homeostasis following tissue injury via multiple mechanisms.³ For example, the immune response to tissue injury can mobilize tissue resident stem/progenitor cells, promote cell differentiation, extracellular matrix (ECM) deposition, and stimulate growth factor secretion to promote neoangiogenesis. Since these processes require a coordinated series of immune events, their disruption can lead to a variety of wound pathologies, including chronic wounds and scar formation. Chronic wounds are defined as wounds that fail to resolve after 1 month, often having persistent infections and microbial films. Clinically, they are classified as either venous or arterial ulcers, pressure sores, or diabetic ulcers (Fig. 1).⁴ Alternatively, dermal fibrosis results from increased deposition of ECM and hyper-proliferation of keratinocytes at the wound site. Keloids and hypertrophic (HT) scars both represent clinical pathologies of scarring, though even normal scar formation represents a failure of the dermal layer to regenerate postwounding (Fig. 2).⁴ Not surprisingly, chronic wounds and scarring represent nearly opposite extremes of dysregulated inflammation and immune response to dermal wounding. However, many of the mechanisms through which immune cells act to regulate the wound healing response are still unknown. Elucidating these pathways may aid in the development of novel regenerative strategies for promoting wound resolution and preventing scar formation.

Figure 1.

Clinical presentations of various chronic wound pathologies. Impaired wound healing cascade will lead to the development of nonhealing wound pathologies such as: (A) venous leg ulcer (B) arterial ulcer (C) diabetic foot ulcer (D) pressure sore. Reprinted with adaptation with permission from Ref.⁴ ©AAAS Publishing Group.

Figure 2.

Clinical examples of scarring pathologies. (A) Hypertrophic scar (B) Keloid. Reprinted with adaptation with permission from Ref.⁴ ©AAAS Publishing Group.

Translational Relevance

There has been a recent growing interest in the development of immune-based therapies for both chronic wounds and scar prevention, mostly focusing on the delivery of growth factors, cytokines, and other immunomodulatory factors. However, translating these therapies for chronic wound resolution and scar prevention into the clinic has been particularly difficult, due to challenges related to delivery methods and safety. Most therapeutics currently rely on discrete, bolus doses, given via injections or applied topically to the wound site. Because of natural clearance, doses are given at supra-physiological levels to sustain drug presence throughout the wound healing process. This results in both safety concerns and poor long-term efficacy. Hence, developing new methods to effectively control and sustain drug release over longer periods of time is critical to the success of immune therapies.

Clinical Relevance

Numerous immune-based therapies for both chronic wounds and scar prevention have entered clinical trials in the past decade. For example, macrophage-activating lipopeptide-2 (MALP-2) and platelet-rich plasma (PRP) gels have reached clinical trials to resolve chronic wounds.^5,6 In the case of scar prevention, transforming growth factor (TGF)-β3, interleukin (IL)-10, mannose-6-phosphate (M6P), and nefopam have all reached clinical trials with varying levels of success. TGF-β3 significantly decreased scar size in both phase I and II clinical trials, though complete regeneration was never achieved and it failed to meet efficacy standards in phase III.^7,8 In the case of therapeutics for both chronic wounds and scar prevention, it is believed that improved delivery methods that permit sustained release may facilitate clinical effectiveness.

In this review, we describe the molecular pathophysiology of acute wounds and major etiologies that alter the normal wound healing cascade, leading to development of chronic wounds and scars. Then, we highlight the possible approaches to control the immune system using biomaterial and biologic-based strategies to promote chronic wound resolution and dermal regeneration (scar-less healing).

Background

Role of the immune system in acute skin wound healing

Following injury, an inflammatory response is both normal and critical in restoring tissue homeostasis. Wound healing is typically divided into four phases: hemostasis, inflammation, proliferation, and remodeling. These phases result from a cascade of events, largely mediated by immune cells and signaling molecules (Fig. 3A).⁹ In response to cues from both the damaged ECM and tissue-resident cells, thrombocytes and immune cells accumulate at the wound site in a stepwise manner. Platelets are the first cells to arrive, at which point they help initiate the coagulation cascade to prevent further blood loss and provide a provisional ECM for further cell infiltration. Also, by secreting TGF-β1 and platelet-derived growth factors (PDGFs), they play a critical role in the activation of fibroblasts and mesenchymal cells as well as in the recruitment and activation of neutrophils and macrophages.^10
–12 Unsurprisingly, platelet defects are correlated with impaired wound healing, and delivering autologous PRP improves wound resolution.^13

–16

Figure 3.

Overview of the immune mechanisms in acute and chronic wound healing. (A) Acute wound healing results from a well-coordinated series of events divided into four overlapping phases: hemostasis, inflammation, proliferation/matrix deposition, and tissue remodeling. Neutrophils and macrophages are particularly important in mediating this process, though T cells and platelets also play key roles. (B) High numbers of inflammatory cells and the formation of a biofilm preclude the restoration of tissue homeostasis in chronic wounds. Excess secretion of inflammatory mediators leads to growth factor and ECM degradation and prevents macrophage phenotype conversion, which creates a feed-forward loop preventing resolution. Black arrows indicate differentiation, blue arrows indicate inhibition and red arrows indicate induction. CXCL, C-X-C chemokine ligand; ECM, extracellular matrix; FGF, fibroblast growth factor; IL, interleukin; MMP, matrix metalloproteinase; NET, neutrophil extracellular trap; ROS, reactive oxygen species; TGF, transforming growth factor; TIMP, tissue inhibitor of matrix metalloproteinase; TNF, tumor necrosis factor.

Neutrophils are the first immune cells recruited into wounded tissue, and remain for about 24 h before undergoing apoptosis.¹⁷ They play a central role in both killing microbes and promoting wound healing.¹⁸ Neutrophils control invading pathogens by secreting a variety of antimicrobial substances—reactive oxygen species (ROS), antimicrobial peptides, and antimicrobial proteases—and by phagocytosing them with the help of neutrophil extracellular traps (NETs).^19,20 Neutrophils also secrete various cytokines and growth factors, including IL-17 and vascular endothelial growth factor (VEGF). Cytokines and growth factors are both chemotactic for inflammatory cells, and promote the proliferation of fibroblasts, keratinocytes, and endothelial cells.^18,21 Demonstrating the importance of neutrophils during wound healing, perturbation of neutrophil recruitment during the early phase of wound healing by knocking out C-X-C motif chemokine receptor 2 (CXCR2) in mice impairs wound healing.²² CXCR2 is an essential mediator of neutrophil chemotaxis via the release of numerous chemokine ligands, including C-X-C chemokine ligand (CXCL)-1, -5, and -8, by keratinocytes at the wound site.²³ However, CXCR2 knock-out mice also exhibit altered temporal patterns of monocyte infiltration, decreased secretion of IL-1β, and reduced keratinocyte migration and proliferation, so impaired wound healing may be the result of numerous compounding factors.²² Another study found accelerated wound closure and reepithelialization in neutrophil-depleted mice, suggesting a relatively complex role of neutrophils during skin repair.²⁴

Cytokines released by neutrophils during apoptosis are chemotactic for monocytes, which start to arrive 5 to 6 h postinjury. These monocytes differentiate into macrophages, which can remain for several weeks at the wound site.¹⁷ Monocyte-derived macrophages have been widely studied in the context of wound healing, and are often considered to be the most important immune cell type in this process.^25,26 In addition to macrophages deriving from mobilized monocytes, there is a population of tissue-resident macrophages in most tissues that can proliferate upon injury.⁹ While, it has been demonstrated in a murine wound model that tissue-resident macrophages have little impact on the timing of the wound healing process or tissue integrity following resolution,²⁷ their role in skin wound healing is still elusive.

Macrophages undergo phenotypic changes throughout the healing process, which helps transition the wound microenvironment from a proinflammatory into a pro-resolution state.⁹ The most commonly studied phenotypes are the proinflammatory—commonly referred to as “classically activated” or M1—and anti-inflammatory—referred to as “alternatively activated” or M2—macrophages. Indeed, this M1 and M2 classification originated from in vitro characterization, but a modified classification system has been recently proposed to link macrophage populations to activation pathways [e.g., M(IFN-γ), M(IL-4), and so on].²⁸ Nevertheless, it is very difficult to know by which pathways macrophages have been activated in vivo. Hence, in this review, we refer to macrophage phenotypes based on their primary function in vivo (“anti-inflammatory,” “proinflammatory,” “profibrotic,” etc.).

Many studies have confirmed that macrophages are critical for proper wound healing.^29

–32 Upon initial infiltration, proinflammatory macrophages (the so-called M1) remove cellular debris, damaged matrix, microbes, and neutrophils. They also secrete proinflammatory cytokines and growth factors (including IL-1, fibroblast growth factor [FGF]-2, PDGF, and VEGF), which mobilize more immune cells, and promote the proliferation of keratinocytes, fibroblasts, and epithelial cells. During the formation of new tissue, micro environmental cues trigger macrophages to transition into a functionally and phenotypically anti-inflammatory state (the so-called M2). At this point, they begin secreting anti-inflammatory cytokines, such as TGF-β1, which promotes ECM synthesis and wound contraction.³³ In the final stage of wound healing, anti-inflammatory macrophages help reorganize the ECM along tension lines and phagocytose remaining debris.¹⁷ Demonstrating the importance of these immune cells, Lucas et al. depleted macrophages from mice at each phase of tissue repair. Early-stage macrophage depletion significantly reduced granulation tissue formation, impaired epithelialization, and resulted in decreased scar formation. Mid-stage macrophage depletion resulted in severe hemorrhage, while late-stage depletion did not significantly impact the outcome of the healing response.³⁴ Moreover, anti-inflammatory macrophages play an important role in angiogenesis. They indirectly promote angiogenesis by degrading the ECM to create tunnels that guide endothelial proliferation and migration, and they release angiogenic factors, such as FGF and placental growth factor (PlGF).³⁵

T lymphocytes (T cells) are also critical in tissue remodeling and the resolution of inflammation. During the inflammatory phase of wound healing, macrophages secrete chemokines such as interferon gamma (IFN-γ) that attract T cells to the wound site.^36,37 IFN-γ also encourages CD4⁺ type 1 T helper (T_H1) polarization, which contributes to the initial proinflammatory wound microenvironment.³⁸ Tissue resident γδT cells also play a role in regulating skin wound healing. Indeed, γδT cell-deficient mice show defects in skin development due to decreased keratinocyte proliferation.³⁹ These immune cells most likely regulate keratinocyte proliferation and differentiation via their secretion of various growth factors, such as FGF-7, FGF-10, and insulin-like growth factor (IGF)-1.⁴⁰ Importantly, there are significant differences between mouse and human γδT cells, though studies exist that suggest a similar role of human γδT cells in wound healing.^41,42 For example, γδT cells isolated from healthy human skin produce a constituent level of IGF-1, which increases threefold upon stimulation. Additionally, based on an in vitro skin organ culture model, T-cell receptor (TCR)-specific activation of human γδT cells accelerates the rate of early wound closure. This increase in wound closure was dependent on IGF-1 secretion.⁴² Regulatory T cells (Tregs) also play a central role in maintaining skin homeostasis.⁴³ They secrete arginase and anti-inflammatory cytokines (including IL-10 and TGF-β1), which encourages anti-inflammatory macrophage polarization and suppresses the inflammatory response.⁴⁴ A recent study using Foxp3-DTR transgenic mice (where Tregs are depleted following diphtheria toxin injection), showed that wound healing is slower in Treg depleted mice compared with wild-type controls.⁴⁵ This clearly shows the importance of Tregs in cutaneous wound healing. Contrastingly, natural killer T cells have an inhibitory role on wound healing. Indeed, mouse studies have shown that depleting natural killer T cells increases wound closure rate.^46,47

As inflammation is resolved, the wound progresses into the final two stages of the wound healing process: matrix formation with angiogenesis and remodeling. This resolution is largely dependent upon macrophages, Tregs and T_H2 cells.⁹ Anti-inflammatory macrophages are a prominent source of cytokines and growth factors that promote matrix deposition and angiogenesis, including TGF-β, PDGF, FGF-2, IGF1, tumor necrosis factor (TNF)-α, and VEGF.⁹ TGF-β directly promotes ECM synthesis by stimulating mesenchymal cells to differentiate into myofibroblasts. Furthermore, TGF-β and PDGF trigger fibroblasts and myofibroblasts to produce new ECM.⁹ T_H2 cells and Tregs also contribute to matrix formation by secreting TGF-β1, IL-4, -5, -13, and -21, which play a distinct role in fibrinogenesis, encouraging anti-inflammatory macrophage polarization, and suppressing other inflammatory cell types.^3,9,48 For example, studies have suggested that IL-4 is almost twice as potent as TGF-β at stimulating collagen synthesis.⁴⁹

During the final phase of wound healing, antifibrotic macrophages release numerous matrix metalloproteinases (MMPs) (including MMP-2, -12, and -19) and trigger the production of type VIII collagen critical for improving local tissue integrity.⁹ The result is a decrease in cell proliferation and protein synthesis, and an ECM that is remodeled into larger fibrils oriented along tension lines. Eventually, most endothelial cells, macrophages, and myofibroblasts either exit the wound or undergo apoptosis, and recently formed capillaries begin to regress, potentially due to reduced nutrient requirements.⁹ Depending on the size of the initial injury, this entire process usually results in a small scar.⁴

Discussion of Findings and Relevant Literature

Role of the immune system in chronic wounds

While the wound healing cascade is well coordinated in acute wound healing, chronic wounds fail to progress beyond the inflammatory phase (Table 1), which precludes proliferation, matrix deposition, and ultimately, wound resolution.^50,51 Indeed, many studies have shown that there is an imbalance between pro- and anti-inflammatory signals in chronic wounds, which disturbs the microenvironment and hampers the wound healing process.^52,53

Table 1.

Comparison of the immune microenvironment between acute and chronic wounds

Components	Acute Wounds	Chronic Wounds
Inflammation	Controlled	Increased
Neutrophils	Present only during early phase	Prolonged presence
Neutrophils	Controlled NETosis	Increased NETosis
Macrophages	Polarization transition from pro- to anti- inflammatory phenotype	Impaired polarization
Inflammatory cytokines	Low level	Increased level
Anti-inflammatory cytokines	High level	Low level
Granulation tissue	Present	Low amount
MMPs	Low level	High level
TIMPs	Normal level	Low level when compared to increased MMPs
Fibrosis	Low level	Increased
Growth factors/cytokines	Normal degradation rate	Increased degradation rate

MMP, matrix metalloproteinase; NET, neutrophil extracellular trap; TIMP, tissue inhibitor of matrix metalloproteinase.

Role of neutrophils in chronic wounds

While neutrophils play a role in reestablishing tissue homeostasis through pathogen phagocytosis and macrophage recruitment, excessive neutrophil activity can contribute to the development of nonhealing wounds.^10,21 For instance, excess neutrophils at the wound site lead to an overproduction of ROS, causing ECM and cell membrane damage, and resulting in premature cell senescence (Fig. 3B).^19,54 Not only do ROS directly damage the ECM, but they also activate proteases (MMPs and serine proteases) and inactivate protease inhibitors.^55
–57 Unsurprisingly, this results in increased proteolysis, further exacerbating ECM degradation.^58

–61 In addition, proteases released by neutrophils are capable of degrading key wound healing growth factors, including PDGF-BB and TGF-β1 (Fig. 3B).^10,62 Finally, although the mechanism is not fully understood, it has been shown that excess NETosis impairs wound resolution in both mice and humans.^63

–66

Because neutrophils are recruited to wound sites in such large numbers and exacerbate the proinflammatory microenvironment through toxic compounds and cytokine secretion, their efferocytosis is critical to inflammation resolution. In fact, dysregulated neutrophil apoptosis is likely a key contributor to the nonhealing state of chronic wounds. Chronic wounds in diabetic mice have increased numbers of apoptotic cells, most of which are neutrophils, resulting in a prolonged inflammatory state.⁶⁷ Interestingly, neutrophils isolated from chronic diabetic mouse wounds undergo less apoptosis in response to S. aureus infection. This also results in sustained secretion of proinflammatory cytokines, including TNF-α.⁶⁸ The discrepancy potentially results from an overall increase in neutrophil recruitment to diabetic wounds or from the lack of bacterial infection in the first study. However, in both cases, dysregulated neutrophil apoptosis contributed to the chronic wound state.

Role of macrophages in chronic wounds

Increased macrophage infiltrate at the wound site can also perturb the normal wound healing process, contributing to the development of nonhealing wounds.⁶⁹ Moreover, macrophage conversion from a proinflammatory to an anti-inflammatory phenotype is critical to wound resolution.⁷⁰ If this phenotypic conversion does not occur or reach completion, it may lead to the development of chronic wounds (Fig. 3B). For example, using iron to induce an incomplete macrophage phenotypic switch impairs healing in both humans and mice.⁷¹ In these studies, 80% of macrophages at the wound margins had a proinflammatory phenotype, contributing to the persistence of the wounds.⁷¹ Other studies in diabetic mouse models have also shown that when macrophages do not undergo the appropriate phenotypic conversion, it leads to a reduction in key growth factors, such as TGF-β1, VEGF, and IGF-1, which are necessary for the progression into the proliferation phase.⁷² Proinflammatory macrophages also secrete inflammatory mediators, such as TNF-α, IL-17 and IL-1β, ROS, and inducible nitric oxide synthase (iNOS), which have negative effects on the wound microenvironment at high concentrations.⁷³ For instance, surplus TNF-α leads to increased secretion of MMP-1 and MMP-3 and decreased tissue inhibitor of matrix metalloproteinase (TIMP)-1 secretion, which directly contributes to wound chronicity through excess ECM proteolysis.⁷⁴ Similarly, excess ROS disturb the oxidant/antioxidant balance. Not only does this enhance the signaling pathways that regulate the secretion of proinflammatory cytokines/chemokines (IL-1, IL-6, and TNF-α) and MMPs, but it is also implicated in premature fibroblast senescence.^75,76 This further contributes to wound chronicity as senescent fibroblasts produce elevated levels of proteases (including MMPs 2, 3, and 9), and fewer protease inhibitors.^4,76 Altogether, changes in macrophage polarization are highly regulated in acute wound healing, and disruptions contribute to the development of wound chronicity.

Beyond secreting cytokines and growth factors to mediate wound healing, macrophages play a critical role in phagocytosing apoptotic cells and other debris in the wound bed. As previously mentioned, efficient efferocytosis is especially critical in the context of chronic wounds due to increased infiltration and apoptosis of neutrophils. However, in chronic wounds, macrophages exhibit a reduced phagocytic capacity, exacerbating the apoptotic cell burden.⁶⁷ In addition, unsuccessful efferocytosis results in a higher ratio of pro:anti-inflammatory cytokines, further compromising resolution of diabetic wounds.⁶⁷

Role of adaptive immune cells in chronic wounds

Compared to our understanding of the role of the innate immune system in exacerbating nonhealing wounds, little is known about the role of the adaptive immune system. In a human ex vivo model, it has been shown that a higher number of Langerhans cells (LCs) (a dendritic cell subtype present in early-phase wound healing) is present in healing diabetic foot ulcers (DFUs) compared with nonhealing DFUs.⁷⁷ One of the main roles of LCs following tissue injury is activating and recruiting T cells.⁷⁷ Interestingly, there are also fewer T lymphocytes in chronic wounds, and those that are present exhibit an unresponsive, functionally impaired state.^39,42 Indeed, γδ and αβ T cells isolated from human chronic wounds fail to secrete IGF-1 and IL-2, even following stimulation with phorbol myristate acetate and ionomycin.⁴² Nevertheless, while chronic wounds have prolonged infiltration and different ratios of CD4 and CD8 T cells compared with acute wounds, depleting mice of CD4 and CD8 T cells does not appear to affect wound closure rates.^69,78

Bacterial colonization in chronic wounds

Bacterial overload is another major factor precluding the resolution of chronic wounds. In certain microenvironments, including those in chronic wounds, bacteria thrive as complex surface-attached communities enclosed in an ECM composed of hydrated polymers and debris. This is known as a biofilm. Interestingly, biofilms are not only immune to destruction by the host's immune system, but they also likely manipulate and depend upon the inflammatory response as a source of sustained nutrients.^79,80 Bacterial pathogens in the biofilm express a range of virulence factors that upregulate the levels of proinflammatory cytokines, ROS and MMPs, while diminishing levels of TIMPs and growth factors. This creates a steady state of hyper-inflammation that is outside of the host's control, contributing to the etiology of chronic wounds and making resolution challenging. Biofilms further impair wound healing by perturbing the tight junctions between epithelial cells. Tight junctions create a fluid-impermeable barrier by joining the cytoskeletons of adjacent epithelial cells. Because of this, they are critical to skin's protective function. Biofilms interfere with this barrier partly by causing the downregulation of proteins responsible for creating the tight junctions, including zona occludens-1 and zona occludens-2.⁸¹ Hence, even though the biofilm-infected wound may eventually appear closed, functionally compromised, leaky skin can lead to future infections and wound healing complications.⁸¹

Regulation of skin scarring by the immune system

Scar formation begins in the third phase of wound healing, when fibroblasts, keratinocytes, and epithelial cells are recruited into the site of injury. At this point, fibroblasts differentiate into myofibroblasts, which secrete and assemble new ECM.⁴⁸ This new ECM is later remodeled to form scar tissue. Not only does the secretion of cytokines by leukocytes play a direct role in the initial recruitment of fibroblasts, but the general inflammatory response also plays a key role in mediating future scar development (Fig. 4). This is most evident in the differences between fetal and adult wound healing. In the first two trimesters of human development, fetuses heal dermal wounds without a scar. This phenomenon is likely dependent upon the absence of immune cells, since the transition into the third trimester is characterized by the development of myeloid lineages and significant scarring following injury in both humans and mice.⁸² Wild-type mice undergo a similar transition, evidenced by scarless wound healing in embryonic day 15 (E15) mice and scar formation in embryonic day 18 (E18) mice.^78,82 Early studies found similar scarless wound healing in neonatal PU.1 null mice, which lack neutrophils, macrophages, and mast cells. They also have reduced mRNA levels of TGF-β1 and PDGF, which act as important profibrotic mediators stimulating myofibroblast differentiation and ECM synthesis. Interestingly, Tcell-deficient athymic nude-nu mice also heal with minimal scarring.^82–83

Figure 4.

Molecular and cellular mechanisms involved in scar formation and dermal regeneration. (A) Immune cells act along numerous, redundant pathways to promote excess ECM deposition and hyperproliferation of keratinocytes, leading to scar formation. Key immune cell types involved in pathologic scar formation include macrophages, mast cells, neutrophils, eosinophils, and T cells. (B) Dermal regeneration is improved by generally decreasing innate and adaptive immune cell recruitment and activation, and encouraging antifibrotic macrophage polarization (e.g. via IL-10). Pathways that induce antifibrotic macrophage polarization in vivo are still very elusive, though research on other tissues suggests that Tregs may help promote antifibrotic macrophage polarization and suppress proinflammatory immune responses. Black arrows indicate differentiation, blue arrows indicate inhibition and red arrows indicate induction. IFN, interferon Treg, regulatory T cells.

Role of neutrophils in skin scarring

Potentially because they only participate in the earliest stages of wound healing, few studies have looked at the role of neutrophils in dermal scarring and they are believed to have a negligible long-term impact. Nonetheless, it is interesting to note that neutrophils are absent from scarless fetal wounds, there is an 80% reduction of neutrophil circulation in Acomys mice (which heal full-thickness excisional skin wounds with complete regeneration of dermal components and without scarring) compared with Mus mice that heal with a scar, and there is a significant reduction in neutrophil recruitment to wounds in the oral mucosa, which also heals rapidly and with minimal scarring.^78,84

Role of macrophages in skin scarring

The role of macrophages in dermal scarring has been extensively studied, and shown to change throughout the stages of wound healing.²⁵ Monocytes are the primary cells that secrete IL-1, which increases collagen synthesis, and promotes fibroblast and keratinocyte proliferation.²³ Ablating or depleting macrophages during the acute and subacute phases of repair results in slower wound closure, epithelialization, and granulation tissue formation, but significantly reduced scarring.^25,34,85 Similarly, reducing macrophage migration, adhesion, and their ability to produce TGF-β1 by diminishing β-catenin levels results in slower wound repair but less scarring.⁸⁶ If macrophage inhibition or ablation is discontinued, wound closure rates are rescued by their infiltration in the later stages of wound repair without affecting scar formation or morphology.⁸⁵ Alternatively, only depleting macrophages in the late stage wound healing has no effect on scar morphology compared to control mice.^25,34 Furthermore, a study by Dardenne et al. showed that wounds treated with high mobility group box-1 (HMGB-1), an alarmin, have increased scarring. These wounds showed a significant increase in macrophages 48 h postinjury and no differences in neutrophil infiltration or mast cell numbers or degranulation.⁸⁷ Contrarily, a study examining HT and normotrophic (NT) scars on human subjects found delayed and prolonged macrophage infiltration in HT scars compared with NT scars, suggesting an important role for initial macrophage infiltration rather than later-stage macrophage presence in reducing fibrosis.⁸⁸

It is possible that changes in macrophage polarization are critical in determining the varying role of macrophages in wound healing. Indeed, in muscle remodeling, increases in the anti-inflammatory population and higher ratios of anti- to proinflammatory macrophages within the remodeling site at 14 days postinjury were associated with reduced scarring.⁸⁹ During early stage wound healing, there is a mixed pro/anti-inflammatory activation phenotype. This transitions to an anti-inflammatory macrophage polarization in late stage wound healing characterized by an upregulation of IL-10.²⁵ Since wound associated macrophages are the main sustained source of TGF-βs (1, 2, and 3) and extensively secrete MMPs to activate TGF-β, differences in TGF-β and MMP secretion between classically and alternatively activated macrophages could account for the time-specific role of macrophages.^85,90 For example, IL-4, a cytokine that promotes profibrotic macrophage polarization, was significantly downregulated in Acomys, which heal without a scar, compared to Mus. Macrophages polarized via IL-4 (M(IL-4)) are known to secrete and activate TGF-β1, so their absence likely reduces collagen deposition.⁹¹ Broek et al. also found increased and prolonged expression of anti-inflammatory macrophages in HT scar compared with NT scar, and decreases in inflammatory gene expression (including IL-10) over a 52-week period.⁸⁸ Hence, it is possible that the macrophages present were remaining in the profibrotic polarization state rather than transitioning to an antifibrotic M(IL-10)-like polarization, leading to the HT scar development.

Role of mast cells in skin scarring

The final immune cell subtype about which there exists substantial literature is the mast cell, though the effect of mast cells in scar formation remains somewhat under debate.⁹² Mast cells are tissue resident and become activated following tissue injury, releasing granules into the extracellular space. They secrete TGF-β1, as well as other proteases that promote fibrotic responses in fibroblasts and collagen fibril formation.⁹² Mast cells also form gap junctions with fibroblasts to stimulate myofibroblast differentiation, proliferation, and ECM contraction.^93

–96 It has been found that mice treated with disodium cromoglycate (DSCG) to inhibit mast cell activation results in a reduced scar width, higher fibrillar density, and collagen fibers that were oriented more similarly to normal tissue compared to controls. DSCG treated mice also showed decreased neutrophil recruitment and fewer proinflammatory cytokines.⁹⁷ There are significantly less dermal mast cells in second trimester fetal wounds compared to third trimester wounds, and almost no degranulation. Interestingly, injecting mast cell lysates into E15 wounds resulted in scarring, and wounds in E18 mast cell-deficient mice healed with significantly less scarring than wild-type littermates.⁹⁸ There is also less mast cell degranulation in oral mucosal wounds compared with cutaneous wounds, and higher mast cell numbers in HT scars compared with normal scars.^82,92 Contrarily, a study by Willenborg et al. showed genetic ablation of mast cells does not alter the amount of scar tissue, or the infiltration of leukocytes into the injury site.⁹⁹ Further research into the role of mast cells in scar formation is needed to resolve this discrepancy.

Role of T cells in skin scarring

Though they are known to play a role in immune cell recruitment and cytokine expression, the role of T cells on scar development is unclear. Recently, Nosbaum et al. found that mice depleted of Tregs have significantly slower wound closure and increased granulation tissue compared to wild-type mice partly due to their suppression of IFN-γ, which reduces the recruitment of proinflammatory macrophages.⁴⁵ An earlier study showed that CD4-deficient mice had reduced neutrophil recruitment to the wound site, whereas CD8-deficient mice had less neutrophils and macrophages. The changes in cytokine expression also differ according to whether mice are CD4 deficient or CD8 deficient.¹⁰⁰ While there are many subsets of CD4 lymphocytes, two main types are T_H1 and T_H2. T_H1 cells primarily secrete IFN-γ, IL-2, and TNF-α, while T_H2 cells primarily secrete IL-4, 5, 6, 9, 10, and 13, many of which are proinflammatory or profibrotic.²³ Additionally, epidermal γδT cells respond within hours to tissue damage, releasing numerous growth factors to promote keratinocyte proliferation, immune cell infiltration, and expedite wound closure in mice.¹⁰¹ The effect of γδT cells on scar formation remains unknown, though data summarized in previous paragraphs correlating early macrophage infiltration with increased scar size suggest that γδT cells may increase scarring.¹⁰¹ Together, these data suggest T cells play an important role in wound repair and scar formation and support the need for controlling the immune system.

Role of other immune cell subtypes in skin scarring

Research into the role of eosinophils, dendritic cells, and B cells on cutaneous scar formation is also lacking. Eosinophils stimulate fibroblasts to upregulate α-smooth muscle actin and collagen secretion in bleomycin-induced models of fibrosis and secrete growth factors that promote keratinocyte migration in vitro, suggesting a possible, but unknown, role in dermal scar formation.⁷⁸ Dendritic cells are known to play a role in the rate of reepithelialization, but they likely do not play an important role in scarring.⁷⁸ Finally, one study showed that mice deficient in CD19, a critical positive regulator of B cells, showed reduced recruitment of neutrophils and macrophages to the wound site, reduced proinflammatory and profibrotic cytokines (including TGF-β), reduced granulation tissue formation, and slower reepithelialization.¹⁰² This suggests that depleting B cells may be an interesting therapeutic avenue for scar prevention, though there is minimal supporting research.

Future Directions

Therapeutic strategies targeting the immune system for chronic wounds

Since chronic wounds are often the result of excessive inflammation, controlling the immune response is an attractive avenue for designing novel regenerative strategies. To this end, numerous material-based and molecular strategies have been explored, including targeting the immune response using cytokines, protease inhibitors, miRNA, small interfering RNA (siRNA) and extracellular vesicles (EVs) (Fig. 5).

Figure 5.

Biomaterial and molecular-based strategies to resolve chronic wounds principally focus on promoting reepithelialization, angiogenesis, progenitor cell recruitment, directing macrophage polarization, and inhibiting the migration of excess inflammatory cells. Current strategies are very diverse, either directly delivering cytokines and growth factors to the wound, or by using siRNAs, miRNAs, stem cells, and EVs to alter cytokine expression and production by cells in the wound bed. Material-based strategies are also being explored, particularly for their potential to direct macrophage polarization. Blue arrows indicate inhibition and red arrows indicate induction. EV, extracellular vesicle; siRNA, small interfering RNA.

Biomaterial-based strategies to resolve chronic wounds

Various biomaterials have shown interesting results for promoting wound resolution by modulating the immune system. Current biomaterial-based strategies focus either on macrophage adhesion and recruitment, or on directing their polarization to encourage inflammation resolution. One method of controlling macrophage polarization is to modify the surface chemistry of the biomaterial. Indeed, macrophage adhesion is enhanced on hydrophobic surfaces. However, even though fewer macrophages and foreign body giant cells (FBGCs) adhere to hydrophillic/neutral surfaces, those that do adhere secrete more cytokines than macrophages/FBGCs adhered to hydrophobic and ionic surfaces.¹⁰³ This may result from the unique activation of biomaterial-adherent macrophages and a phenotypic switch that occurs over time.⁸² Furthermore, it has been demonstrated that monocyte-derived macrophages change their surface protein expression depending upon the chemical composition of the biomaterial surface they are in contact with.¹⁰⁴ These findings highlight the role of biomaterial surface chemistry in altering macrophage response and activation.

Another important factor to biomaterial design is the surface topography. Indeed, various studies have shown the importance of surface topography in altering macrophage response.^{105

–110} For example, surface topography has been altered to mimic the natural ECM structure by imprinting small patterns on the surface, which improves cell adhesion, proliferation, and migration.¹¹¹ These patterns not only affect the function of epithelial cells, endothelial cells, and fibroblasts, but they also promote anti-inflammatory macrophage polarization by encouraging a specific cell shape.¹¹¹ While the effect of incorporating complex matrix topography onto micro-patterned gels has shown enhanced skin regeneration and graft performance in animal models of chronic wounds, its effect on immune cells in this context remains elusive.¹¹²

The biomaterial origin also affects its immunomodulatory properties. Synthetic biomaterials can be advantageous because they usually avoid an undesirable host immune response driven by the antigens in naturally derived materials.¹⁷ For example, treatment of wounds in diabetic mice with poly(methacrylic acid-co-methyl methacrylate) (PMMA) beads accelerated chronic wound healing, presumably by increasing sonic hedgehog signaling (Shh).¹¹³ Shh is implicated in CD4 T cell activation and increased proliferation of hematopoietic stem cells (HSCs) and keratinocytes.^114,115 On the other hand, naturally derived biomaterials can more effectively replace the lost components of the original ECM. Naturally derived biomaterials that are commonly used for chronic wound resolution include hyaluronic acid (HA), chitosan, fibrin, and collagen.¹¹³ For example, using chitosan-based biomaterial N-carboxymethyl chitosan, 5-methyl pyrrolidinone chitosan (MPC) to deliver neurotensin to diabetic wounds in mice induced rapid healing (50% wound area reduction) by reducing levels of TNF-α, inflammatory cells, and MMP-9 at the site of injury.¹¹⁶ Several other studies have shown similar results using chitosan-based biomaterials for chronic wound repair.^117
–119 Additionally, a clinical trial delivering PRP in a HA scaffold accelerated wound closure by 48% in 3 days, and lead to complete closure after two doses.⁵ PRP is a known source of numerous proresolution growth factors and cytokines, including PGDF, TGF-β1, TGF-α, granulocyte macrophage colony-stimulating factor (GM-CSF, also known as colony-stimulating factor 2), and others.¹²⁰

Among naturally derived biomaterials, decellularized ECM possesses immunomodulatory properties. Decellularized ECM is isolated from donor tissues by removing the cellular components using mechanical, chemical, or enzymatic methods.¹²¹ A common origin for ECM-based biomaterials is porcine small intestinal submucosa, which has been shown to modulate the wound healing microenvironment in rats via macrophage polarization.¹²² Interestingly, one mechanism by which decellularized ECM modulates macrophage polarization is through matrix-bound nanovesicles (MBVs), which survive the chemical and enzymatic processes of decellularization. These MBVs contain miRNA that polarizes macrophages to an anti-inflammatory phenotype.¹²³ Decellularized and dehydrated human amniotic membrane (DDHAM) products have also demonstrated potential for resolving chronic wounds and are clinically available. Indeed, numerous clinical trials have shown that DDHAM products successfully promote the resolution of chronic wounds that are unresponsive to traditional therapies.^124

–127 Moreover, in a clinical trial evaluating DDHAM allograft safety, patients with various types of chronic wounds did not experience any product-related adverse effects.¹²⁴ DDHAM promotes wound resolution by stimulating the secretion of important growth factors, cytokines and proteases, including PDGFs, TGF-α, TGF-β1, FGF-2, epidermal growth factor, placental growth factor, granulocyte colony-stimulating factor (also known as colony-stimulating factor 3), IL-4, IL-10, and various TIMPs.¹²⁸ DDHAM also increases the release of stromal cell-derived factor (SDF)-1, which is reduced in diabetic wounds and aids in the recruitment of progenitor cells to the site of injury.^129,130 Therefore, DDHAM appears to be an effective option for designing regenerative strategies for chronic wounds.

Cytokine-based strategies to resolve chronic wounds

Cytokines are obviously critical during the time course of wound healing, making them an attractive target for therapeutic development. For example, IL-1β is part of a proinflammatory positive feedback loop that sustains a persistent proinflammatory wound macrophage phenotype, contributing to impaired healing of diabetic wounds.^62,131 A study by Mirza et al. showed a significant increase in wound closure in diabetic mice using an antibody to inhibit IL-1β. This downregulated proinflammatory macrophages and upregulated prohealing factors in the wound.¹³¹ Using IL-1 receptor antagonist (IL-1Ra) is another means of blocking IL-1 signaling pathways. Indeed, interaction between IL-1β and IL-1Ra with IL-1R is critical for epithelial wound healing, and the IL-1Ra:IL-1 ratio is known to be higher in fluids from healing wounds than those from chronic wounds (480:1 vs. 7:1).^62,132 Not surprisingly, blocking IL-1 using IL-1Ra has been shown to accelerate wound healing in diabetic mouse corneas. TNF-α is a second proinflammatory cytokine that contributes to a chronic wound state. Neutralizing TNF-α at the incision site significantly accelerated chronic wound healing in mice by decreasing inflammatory cell infiltrates and suppressing proinflammatory macrophage activation.¹³³

SDF-1 is another promising chemokine to promote wound healing. For example, delivering nanosized SDF-1 liposomes to diabetic mouse wounds promotes dermal cell proliferation, increases granulation tissue formation, and accelerates wound closure.¹³⁴ Moreover, the liposomes protect SDF-1 from degradation by proteases and serine exopeptidase. However, heparin sulfate present in the wound bed causes dimerization of the SDF-1 liposomes, thereby reducing their bioavailability. A more effective delivery method could probably increase the effectiveness of SDF-1 treatments.

GM-CSF, which facilitates differentiation of HSCs to granulocytes and macrophages, also plays a key role in wound healing. GM-CSF has been shown to accelerate wound closure in diabetic mice by increasing reepithelialization, recruiting leukocytes, increasing angiogenesis, and upregulating proinflammatory mediators, such as IL-6 and macrophage chemoattractant protein (MCP)-1.¹³⁵ Considering proinflammatory mediators are known to exacerbate chronic wounds when present for long periods of time, this study is somewhat paradoxical. However, numerous other studies support GM-CSF effectiveness in resolving chronic wounds.^136–137 Clinical translation has still not been achieved, though, because most of these studies used intradermal or subcutaneous injections to deliver GM-CSF to the wound site, which raised concerns over injection pain, toxicity, side effects, and uneven distribution.¹⁴⁰ As an alternative, GM-CSF has been delivered using alginate. In clinical trials, this delivery method accelerated wound healing and relieved pain.¹⁴⁰ Altogether GM-CSF is a potent cytokine for chronic wounds, but more efficient delivery methods are still required for clinical translation.

Delivery of other cytokines has also shown promise in resolving chronic wounds. Surprisingly, IL-22, a proinflammatory cytokine, helps resolve chronic diabetic wounds in mice by inducing keratinocyte proliferation and signal transducer and activator of transcription 3 (STAT3) activation.¹⁴¹ Additionally, anti-inflammatory cytokines have been widely used for macrophage polarization. For example, recombinant IL-10 has been incorporated into dextrin nanogel matrix to resolve chronic wounds, though clinical trial results were not promising.^142,144 Treatment of skin wounds in diabetic mice using TGF-β1 also rescued wound healing and normalized macrophage polarization.⁷² Though not itself a cytokine, using MALP-2 in diabetic mice promoted wound healing by increasing macrophage infiltration and directing anti-inflammatory polarization.¹⁴⁴ MALP-2 stimulates in vivo synthesis of macrophage inflammatory protein (MIP)-1α, MIP-2, and MCP-1.¹⁴⁵ Phase I clinical trial results using MALP-2 to treat diabetic wounds in 12 patients were also promising.⁶

Since prolonged presence of proinflammatory cytokines may prevent resolution, and both pro- and anti-inflammatory cytokines are necessary for acute wound healing, sequential delivery of pro- and anti-inflammatory cytokines could be an interesting strategy for improving chronic wound healing. This concept has been explored in other tissues with some success. For example, to improve bone repair, decellularized bone was engineered to sequentially release IFN-γ and IL-4 and implanted in mice at the site of injury. The sequential release promoted macrophage polarization to switch from a pro- to an anti-inflammatory phenotype, resulting in improved wound healing.¹⁴⁶ Similar strategies for releasing cytokines in a biphasic manner have been reviewed by Alvarez et al. ¹⁴⁷ Considering crosstalk between IFN-γ and TGF-β is essential for resolving inflammation during skin wound healing, a similar strategy may be useful for chronic wounds.¹⁴⁸ However, no cytokine-based, chronic wound therapies have reached clinical trials; we need a more effective delivery method to facilitate clinical translation.

Protease inhibitor-based strategies to resolve chronic wounds

The microenvironment of chronic wounds not only has reduced growth factor levels, but also has an imbalance of MMPs and TIMPs. This can lead to the degradation of exogenously delivered growth factors, exacerbating the problem.¹⁴⁹ Thus, one way to overcome this issue is to deliver protease inhibitors. For example, delivering ND-336, which selectively inhibits MMP-2, MMP-9, and MMP-14, accelerates wound closure in diabetic mice by decreasing inflammation, enhancing reepithelialization, and increasing angiogenesis (Table 2).¹⁵⁰ Incorporating protease inhibitors into wound dressings has also been explored. For example, combining collagen and oxidized regenerated cellulose matrix into wound dressings improves wound repair by binding to and inactivating MMPs.¹⁵¹ Silver dressings and hydrogels, which are antimicrobial have also shown promising results in porcine models and clinical trials.^152,153 In addition, silver inactivates proteases by displacing zinc from MMPs, which promotes faster granulation tissue formation.¹⁵² Finally, incorporating oleic acid/albumin formulations into topically applied cotton fiber dressings targets neutrophil elastase in vitro, suggesting that it may help resolve chronic wounds.¹⁵⁴

Table 2.

List of potential immunomodulatory therapeutics for chronic wounds

Pharmaceutical	Effects	Outcome	Species	References
PMMA	Activates T cells	Increases wound closure	Diabetic mouse	¹¹³
	Increases HSC and keratinocyte proliferation
IL-1Ra	Downregulates inflammation triggered by IL-1R	Increases wound closure	Diabetic mouse	^62,131,132
SDF-1	Increases dermal cell proliferation promotes granulation tissue formation	Increases wound closure	Diabetic mouse	¹³⁴
GM-CSF	Increases re-epithelialization and angiogenesis	Increases wound closure	Diabetic mouse	^{135 –140}
	Promotes leukocyte recruitment
	Increases collagen deposition
	Increases proinflammatory cytokine production
IL-22	Increases keratinocyte proliferation	Accelerates wound healing	Diabetic mouse	^141 –143
TGF-β1	Promotes ECM deposition	Improves diabetic wound healing	Diabetic mouse	⁷²
	Promotes granulation tissue formation directs macrophage polarization
MALP-2	Macrophage infiltration and activation	Promotes early wound closure	Diabetic mouse	^6,144,145
			Phase I
ND-336	Inhibits MMPs	Accelerates wound closure	Diabetic mouse	¹⁵⁰
	Increases reepithelialization and angiogenesis Reduces inflammation
miR-21	Modulates TGF-β pathway	Accelerates wound closure	Diabetic mouse	¹⁵⁶
miR-99	Regulates PI3K/AKT pathway	Accelerates wound closure	Diabetic mouse	¹⁵⁷
miR-132	Decreases chemokine secretion reduces leukocyte recruitment	Promotes transition into proliferative phase	Diabetic mouse	¹⁵⁸
miR-126-3p	Promotes reepithelialization and angiogenesis	Accelerates wound closure	Diabetic mouse	¹⁶²
siRNA	Regulates immune cell infiltration decreases protease activity	Accelerates wound closure	Diabetic mouse	^163 –165
Fibrocyte-derived exosomes	Increases keratinocyte proliferation activates fibroblasts	Accelerates wound closure	Diabetic mouse	¹⁶⁷
PRP gels	Source of cytokines and growth factors	Improves healing in diabetic patients	Phase I	^5,120
DDHAM	Increase cytokine/growth factor production Increases progenitor cell recruitment	Improves healing in diabetic patients	Phase I	^{124 –130}
Silver dressings	Antimicrobial	Accelerates wound closure	Clinically available	^152,153
	Inhibits MMPs
	Promotes granulation tissue formation

DDHAM, dehydrated human amniotic membrane; ECM, extracellular matrix; GM-CSF, granulocyte macrophage colony-stimulating factor; HSC, hematopoietic stem cell; IL, interleukin; IL-1Ra, IL-1 receptor antagonist; MALP, macrophage-activating lipopeptide; PMMA, poly(methacrylic acid-co-methyl methacrylate); PRP, platelet-rich plasma; SDF, stromal cell-derived factor; siRNA, small interfering RNA; TGF, transforming growth factor.

miRNA- and siRNA-based strategies to resolve chronic wounds

miRNAs are short, noncoding RNA molecules that repress gene expression by binding to mRNA targets, either causing degradation or inhibiting translation. Interestingly, miRNAs can modulate multiple genes, and studies in diabetic mouse models have shown that they play a key role in orchestrating the inflammatory response during wound healing. An imbalance of these signals may result in an improper inflammatory cascade, leading to the development of chronic wounds.¹⁵⁵ Indeed, many have been implicated in the regulation of chronic wounds, including miR-21, -99, and -132.^156
–158 For example, miR-21 promotes fibroblast recruitment by modulating the TGF-β pathway, which is significantly reduced in diabetic wounds.¹⁵⁶ miR-99 overexpression in diabetic wounds increases the migration and proliferation of keratinocytes by regulating the PI3K/Akt pathway, which is implicated in proinflammatory cytokine secretion and the inhibition of Tregs.^157,159,160 Finally, miR-132 promotes the transition from the inflammatory phase to the proliferation phase of wound healing by decreasing chemokine secretion and suppressing the NF-κB pathway, thereby reducing leukocytes recruitment.¹⁵⁸ Altering miRNA levels at the site of injury is therefore a promising strategy for designing chronic wound therapies (Table 2).

Unfortunately, despite the pivotal role of miRNA in regulating chronic wounds, this path has not been extensively explored and miRNA-based therapeutic strategies are scarce due to the lack of optimal delivery systems. Current delivery systems are based on soluble injections and oligonucleotide conjugation, both of which are inefficient due to bio-fluid degradation. To overcome this issue, Li et al. developed a peptide-based, self-assembled, three-dimensional (3D), supramolecular hydrogel that facilitated miRNA delivery to encapsulated cells, even in the presence of fetal bovine serum.¹⁶¹ In another study, miR-126-3p was overexpressed in synovium mesenchymal stem cells (MSCs), which promoted angiogenesis and reepithelization of full-thickness skin defects in a diabetic rat model.¹⁶² However, these miRNA delivery methods have yet to be translated into the clinic and more efficient delivery strategies are still needed.

siRNA has been widely used for gene silencing purposes to improve wound healing (Table 2). For instance, siRNA against the kelch-like ECH associated protein 1 improved wound closure in a diabetic mouse model by repressing NF-E2-related factor 2. This siRNA was delivered in a lipoproteoplex nanoparticle based system and helped maintain normal levels of inflammatory cells and ROS.¹⁶³ MMP-9 siRNA delivered by β-CD-(D3)7, a cationic, star-shaped polymer, to diabetic rat wounds also accelerated wound closure rate by decreasing MMP-9 expression.¹⁶⁴ Though not yet tested in the context of chronic wounds, delivering MMP-9 siRNA using liposomes may be more effective as they have higher rates of interference.¹⁶⁵ Overall, miRNAs and siRNAs have shown promising potential to modulate the immune system and accelerate wound healing, though none have reached clinical trials. Improvements in delivery methods are still required for their use as therapeutics in the clinic.

EV-based strategies to resolve chronic wounds

EVs are spherical particles enclosed by a phospholipid bilayer that range from 30 to 2,000 nm in diameter. They are very important in cell–cell communication, facilitating the exchange of biomolecules such as cytokines, DNA, non-coding RNAs, MMPs, miRNAs, and mRNAs. These biomolecules modify recipient cell protein production, gene expression, and behavior in response to local environmental factors.¹⁶⁶ EVs are released by every cell type in the ECM and are rapidly taken up by the targeted cells. Thus, because of these properties, EVs can potentially be used as immunomodulatory molecules to promote wound healing. For example, delivering human fibrocyte-derived exosomes has been shown to accelerate wound closure in diabetic mice by promoting angiogenesis, increasing keratinocyte migration and proliferation, and activating fibroblasts. This was a result of increased expression of heat shock protein-90α, STAT3, and certain miRNAs, including miR-124 (anti-inflammatory), miR-126 (proangiogenic), and miR-21 (regulates collagen deposition).¹⁶⁷ EVs can also promote wound healing by directing macrophage polarization. For example, exosomes from supernatant of lipopolysaccharide-preconditioned mesenchymal stromal cells promote wound closure by regulating the TLR4/NF-κB/STAT3/AKT signaling pathway in diabetic rats.¹⁶⁸ These studies demonstrate the therapeutic potential of EVs for promoting wound resolution by modulating the immune system. However, like other therapeutic strategies, EV-based therapies have yet to enter clinical trials and more research is required to improve delivery methods.

Therapeutic strategies targeting the immune system for skin scar prevention

Since the immune system has been shown to play such a critical role in modulating scar formation, recent research has explored both pharmaceuticals and the development of biomaterials that target immune cells to promote scarless wound healing. Leukocytes, namely neutrophils, macrophages, mast cells, and Tregs, are the most common targets for many of these therapeutics (Fig. 6).

Figure 6.

Therapeutic strategies for cutaneous wound regeneration that are targeting the immune system. Therapeutics for scar prevention should, in general, promote antifibrotic macrophage polarization, and prevent mast cell degranulation and inflammatory macrophage polarization/recruitment. Pending future studies confirming the role of Tregs in skin regeneration, therapeutics should also promote their recruitment. This is currently being done via delivery of cytokines, chemokines, other antifibrotic mediators, and stem cells, though delivering siRNA, miRNA, and EVs may also be promising. Biomaterials present a promising means of delivering antifibrotic modulators, and their properties can also encourage macrophage polarization. Black arrows indicate differentiation, blue arrows indicate inhibition, and red arrows indicate induction. M6P, mannose-6-phosphate.

Material-based strategies to reduce scarring

Clinically, even when administering a pharmaceutical to promote wound healing, large dermal wounds require bandaging to protect exposed tissue and prevent excessive fluid loss. Hence, immunomodulatory biomaterials that facilitate scarless healing are very desirable. Many recent studies have focused specifically on immunomodulatory biomaterials that reduce scar formation by delivering MSCs or adipose-derived stem cells (ASCs) to the wound area (Table 3). MSCs have been successfully encapsulated in and delivered to wounds in animal models by gelatin microspheres and microcryogels, or loaded into a 3D graphene foam.¹⁶⁹ It has been shown that they release prostaglandin E2 (PGE2), which suppresses the release of proinflammatory cytokines (TNF-α, IFN-γ, IL-6, IL-8, and IL-12p70) and increases the release of anti-inflammatory cytokines (IL-10 and IL-12p40) and TGF-β1 by macrophages.^170,171 Additionally, PGE2 attenuates the proliferation of T cells in the wound and is a cofactor in the transition from T_H1 to T_H2 cells, which favor inflammation resolution and, most likely, tissue regeneration.¹⁷⁰ The altered cytokine release profile inhibits further neutrophil invasion and respiratory burst, which causes local tissue damage and likely increases scar size.¹⁷¹ Delivering ASCs in polyhydroxybutyrate-co-hydroxyvalerate constructs achieves a similar outcome in rats and may be more clinically appropriate since the yield following isolation is much higher than MSCs.^169,172

Table 3.

Biomaterial-based immunomodulatory strategies to reduce scarring

Material Property	Function	References
Deliver stem cells	Reduces neutrophil recruitment, prevents respiratory burst	^169 –172
	Encourages antifibrotic macrophage polarization
	Attenuates T cell proliferation, encourages T_H2 transition
Deliver neonatal cells	Attenuates inflammatory response	^169,173
HA-based	Reduces TGF-β1 secretion	^113,169
Stiffness	Softer gels encourage anti-inflammatory polarization and reduced inflammation	¹⁵⁵
Surface topography	1–5 μm gratings encourage anti-inflammatory macrophage polarization and reduce inflammation	¹⁷⁴
Chemical cross-linking	Triggers chronic foreign body response (increases scarring)	⁸⁹

HA, hyaluronic acid.

Aside from the delivery of stem cells, a few other immunomodulatory biomaterial strategies have been explored, some of which are also being explored to promote chronic wound resolution (Table 3). Treatment of dermal wounds in children with neonatal foreskin tissue cultures leads to wound closure and tissue regeneration with minimal scarring.¹⁷³ Since scarless fetal wound healing is dependent upon the reduced inflammatory response, it is likely that treatment of skin wounds with fetal tissue cultures also reduces scarring in adult wounds by attenuating the inflammatory and immune response.¹⁶⁹ Alternatively, delivering ECM proteins that are more abundant in fetal wounds has shown potential for decreasing scar formation. For example, HA hydrogels minimize scarring by reducing TGF-β1 secretion in rabbit and rat models.¹⁶⁹ Furthermore, incorporating hyaluronan and type III collagen into collagen-heparin scaffolds in an attempt to recapitulate the fetal ECM may also aid in scar prevention.¹¹³

The chemo-physical properties are also an important parameter. Though not studied in relation to dermal scar formation, softer gels and materials with 1–5 μm wide gratings reduce the inflammatory response and encourage anti-inflammatory macrophage polarization.¹⁷⁴ In addition to increasing stiffness, cross-linking changes the ultrastructure, composition and surface topology, and prevents material degradation and release of important bioactive matricryptic peptides from naturally derived materials.¹⁵⁵ Indeed, when using naturally derived materials, chemical cross-linking triggers a chronic foreign body response. Finally, large amounts of cellular material resulting from incomplete decellularization of naturally derived materials elicit detrimental immune responses to promote scar development.¹⁵⁵ Further elucidating the mechanisms by which these biomaterials reduce the inflammatory response, and developing novel materials that exploit numerous of these properties could lead to considerable improvements in wound care for scar-free dermal regeneration.

Molecular-based strategies to reduce scarring

Recently, many different molecular-based immunomodulatory strategies for preventing scar formation have been explored with some success (Table 4). For example, Chemerin15 (C15), a resolution mediator, has been shown to reduce both neutrophil and macrophage infiltration in mice by around 70% and 40%, respectively, and restricted their area of infiltration nearly 10-fold, reducing scar size.¹⁷⁵ Mechanistically, C15 competitively inhibits its proinflammatory precursor, full-length chemerin. Both proteins interact with the ChemR23 receptor upregulated on neutrophils, macrophages, and keratinocytes following wounding, which plays a role in cell recruitment and adhesion to activated platelets.¹⁷⁵ Similarly, injecting alpha-melanocyte-stimulating hormone (αMSH), a neuropeptide with strong anti-inflammatory and immunomodulatory activity, into mice 30 min before wounding resulted in significantly smaller scars and a reduction of leukocytes and mast cells. Though the mechanism by which αMSH reduced scarring was not specifically studied, previous research has shown αMSH suppresses IL-1, TNF-α, IFN-γ, and IL-6 mRNA expression, and induces Tregs via IL-10.^176,177 Mast cells can be specifically targeted to reduce scar formation by administering stabilizers to prevent degranulation, such as cromoglicic acid and ketotifen, using tyrosine kinase inhibitors to reduce mast cell numbers following injury, or by neutralizing chymase activity.⁹² Another potential therapeutic target for reducing leukocyte recruitment is Phospholipase Cɛ (PLCɛ). PLCɛ knockout mice have significantly reduced levels of proinflammatory cytokines and chemokines, including IL-6, CXCL-1, CXCL-2, and C-C chemokine ligand (CCL)-20, and heal dermal wounds with less scarring.¹⁷⁸ SDF-1, which is potently chemotactic for lymphocytes and monocytes, binds to the CXCR4 and CXCR7 receptors. Targeting that pathway using CXCR4 agonist CTCE-9908 decreased cell recruitment and reduced scarring in mice.¹⁷⁹ Similarly, administration of polydeoxyribonucleotide (PDRN) to excisional skin wounds in rats significantly reduced scarring. PDRN inhibits mast cell degranulation, decreases inflammatory cell recruitment, and reduces the amount of proinflammatory mediators (including TNF-α, IL-6, and HMGB-1) at the wound site.¹⁸⁰ Interestingly, exploiting oral tolerance to common dietary proteins, specifically zein, which is found in corn, can also result in less prominent scarring in mice. Since reexposure to orally tolerated proteins blocks nonspecific inflammation, mice that received a parenteral injection of zein immediately before wounding showed decreased amounts of proinflammatory cytokines, increased amounts of anti-inflammatory cytokines, and had smaller scars 40 days postwounding.¹⁸¹ Curcumin is a natural material that also reduces proinflammatory cytokines, including IL-1β, IL-6, and IL-8 to reduce scarring in rabbits.¹⁶⁹ Finally, though its mechanism of action is still somewhat unclear, fibromodulin, a proteoglycan that participates in the assembly of collagen fibers in the ECM, was recently found to restore scar-free wound healing in late-stage gestational murine wounds by decreasing TGF-β1 expression.¹⁸²

Table 4.

List of potential immunomodulatory therapeutics to reduce skin scarring

Pharmaceutical	Target	Function	Species	Reference
C15	Neutrophils	Reduces recruitment, restricts area of infiltration	Mouse	¹⁷⁵
C15	Macrophages	Reduces recruitment, restricts area of infiltration	Mouse	¹⁷⁵
αMSH	Leukocytes	Suppresses TNF-α and IL-6 mRNA expression	Mouse	^176,177
	Lymphocytes	Suppresses TNF-α and IL-6 mRNA expression
	Mast cells	Induces IL-10 generation of Tregs
PLCɛ	Leukocytes	Diminishes IL-6, CXCL-1, CXCL-2, and CCL-20	Mouse	¹⁷⁸
CXCR4 agonist	Lymphocytes	Reduces recruitment	Mouse	¹⁷⁹
CXCR4 agonist	Monocytes	Reduces recruitment	Mouse	¹⁷⁹
PDRN	Leukocytes	Reduces recruitment	Rat	¹⁸⁰
PDRN	Mast cells	Inhibits degranulation	Rat	¹⁸⁰
Curcumin	Leukocytes	Reduces IL-1β, IL-6, and IL-8	Rabbit	¹⁶⁹
FM	Cytokine profile	Reduces TGF-β1 expression	Mouse	¹⁸²
TGF-β3	Lymphocytes	Induces Tregs	Phase III clinical trial	^7,8
TGF-β3	Macrophages	Affects polarization	Phase III clinical trial	^7,8
M6P	Cytokine profile	Inhibits TGF-β1 and TGF-β2	Phase II clinical trial	¹⁸⁵
IL-10	Lymphocytes	Induces Tregs	Phase II clinical trial	^3,185
IL-10	Macrophages	Encourages antifibrotic macrophage polarization	Phase II clinical trial	^3,185
Nefopam	Macrophages	Reduces migration and adhesion	Phase I clinical trial	¹⁸⁶

αMSH, alpha-melanocyte-stimulating hormone; C15, Chemerin 15; CCL, C-C chemokine ligand; CXCL, C-X-C chemokine ligand; CXCR, C-X-C motif chemokine receptor; FM, fibromodulin; M6P, mannose-6-phosphate; PDRN, polydeoxyribonucleotide; PLCɛ, phospholipase Cɛ; TNF, tumor necrosis factor; Tregs, regulatory T cells.

While the strategies above are still in the phases of initial exploration and preclinical development, four molecular-based strategies for reducing scar formation have reached clinical trials: TGF-β3 (planned trade name Avotermin), M6P, IL-10 (planned trade name Prevascar), and nefopam (planned trade mane ScarX). TGF-β3 is a cytokine whose presence is associated with scar-free wound healing. While its role on the immune response has yet to be carefully evaluated, there is evidence that it induces Tregs and affects macrophage polarization.^90,183,184 Renovo Ltd. translated numerous preclinical studies showing a decrease in scarring following local administration of TGF-β3 before and after operation into phase I and II clinical trials.⁸ Further evaluation was halted after Avotermin failed to meet its efficacy endpoints during phase III, potentially because the dose was halved. However, since temporal variation critically affects healing, continuing studies are exploring the efficacy of continuously delivering TGF-β3 in decreasing scarring.⁷ M6P is a potent inhibitor of TGF-β1 and TGF-β2. While it was successful in phase I clinical trials, in the phase II exploratory trial neither topical nor intradermal application showed statistical significance.¹⁸⁵ A phase II trial injecting human recombinant IL-10 into surgical wound margins showed a statistically significant decrease in scar development. IL-10 not only induces Tregs, but also leads to an anti-inflammatory, alternatively activated macrophage polarization.³ In a second phase II trial that extended for longer time points following treatment, there was actually more scarring following IL-10 treatment compared with placebo controls.¹⁸⁵ Finally, topical nefopam, was first developed as a non-narcotic analgesic drug and attenuates β-catenin levels, thereby affecting the migration and adhesion of macrophages. Phase I clinical trials for its effectiveness in reducing dermal scarring have just begun, though around 30 years of data exist to demonstrate its systemic safety.¹⁸⁶

Finally, interesting directions for future development of molecular-based scar prevention strategies may focus on chemokine receptor pathways. There exist considerable data supporting both positive and negative roles of different chemokines and cytokines in wound healing and scar formation,¹⁷⁹ yet few of these pathways have been explored as therapeutics. Some chemokine receptor pathways that show promise for successful therapeutic developments for skin regeneration include C-X3-C chemokine motif receptor (CX3CR)1, C-C chemokine receptor (CCR)2 and CXCR3.^179,187,188

As previously emphasized, a major letdown of many current strategies is their reliance on very high concentration (supra-physiological) doses, which do not remain at the wound site for long enough without performing multiple deliveries. Hence, a critical aspect for these strategies is to develop appropriate delivery systems. For example, various protein engineering strategies have been developed to confer a high affinity of therapeutics to the endogenous ECM.¹⁸⁹ These approaches allow the retention of the therapeutic at the delivery site without using biomaterials. Perhaps, using a similar delivery strategy would improve the clinical efficacy of protein-based therapeutics for scar prevention. Extensive research has also been done developing controlled release polymers, though many remain untested in the context of wound healing and scar formation.¹⁹⁰ Ongoing research in this area will likely potentiate therapeutic effectiveness of the molecular-based targets discussed herein.

Summary

Recent findings have greatly improved our understanding of the roles of the immune system in acute wound healing. The immune response is either dysregulated in chronic wounds, or leads to undesirable scar development. Macrophages clearly play an important role, especially through their phenotypic changes. Further clarifying activation pathways could yield therapies that directly impact their polarization to improve wound outcomes. Additionally, Tregs have been shown to have regenerative properties in other tissues, yet there is a lack of information explaining their role in wound resolution and scar formation.³

Ultimately, translating current and future immune-modulatory therapeutics hinges on the development of improved drug delivery methods. Many therapies that have reached clinical trials failed because current delivery methods rely on supra-physiological bolus doses rather than controlled, sustained release. Because of natural drug clearance, meeting both safety and efficacy benchmarks in this way is incredibly challenging. This is especially evident in chronic wound therapies, which have fallen short largely because of inefficient delivery methods. Hence, novel delivery systems are critical for successful translation of therapeutics currently being explored and those to be explored in the future. Perhaps certain strategies that failed clinical trials in the past may become successful using novel delivery strategies.

Footnotes

Acknowledgments and Funding Sources

This work was supported in part by the research grant of Astellas Foundation for Research on Metabolic Disorders and by the Australian Research Council (DE170100398) to M.M.M. and a Whitaker International Fellowship awarded to J.L.

Author Disclosure and Ghostwriting

No competing financial interests exist. The authors listed expressly wrote the content of this article. No ghostwriters were used to write this article.

About the Authors

Jacqueline Larouche, BS, received her degree in Biomedical Engineering from the Georgia Institute of Technology. She is currently a Whitaker Fellow at the Australian Regenerative Medicine Institute (ARMI) in Monash University, where she focuses on growth factor engineering to enhance regeneration. Sumit Sheoran, MSc, received his degree from the Manipal University School of Regenerative Medicine and is currently a research fellow at ARMI. Kenta Maruyama, MD, PhD, obtained his degrees from Keio University and Osaka University School of Medicine. After clinical residency at the National Hospital Organization Tokyo Medical Center, he joined the Immunology Frontier Research Center at Osaka University as a research fellow and became Assistant Professor in 2014. His research focuses on bone-biology, pain-biology, molecular-immuno-physiology, and geriatric medicine. Mikaël M. Martino, PhD, obtained his degree in Biotechnology and Bioengineering at the Ecole Polytechnique Fédérale de Lausanne. Then, he became a postdoctoral fellow and an Assistant Professor in the Immunology Frontier Research Centre at Osaka University, focusing on how the immune system influences tissue regeneration. In 2016, he became a European Molecular Biology Laboratory (EMBL) Australia group leader at ARMI. His long-term goal is to work at the interface between bioengineering and immunology for designing efficient regenerative medicine strategies.

Abbreviations and Acronyms

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