Healing Mechanisms in Cutaneous Wounds: Tipping the Balance

Acute and chronic cutaneous wounds pose a significant health and economic burden. Cutaneous wound healing is a complex process that occurs in four distinct, yet overlapping, highly coordinated stages: hemostasis, inflammation, proliferation, and remodeling. Postnatal wound healing is reparative, which can lead to the formation of scar tissue. Regenerative wound healing occurs during fetal development and in restricted postnatal tissues. This process can restore the wound to an uninjured state by producing new skin cells from stem cell reservoirs, resulting in healing with minimal or no scarring. Focusing on the pathophysiology of acute burn wounds, this review highlights reparative and regenerative healing mechanisms (including the role of cells, signaling molecules, and the extracellular matrix) and discusses how components of regenerative healing are being used to drive the development of novel approaches and therapeutics aimed at improving clinical outcomes. Important components of regenerative healing, such as stem cells, growth factors, and decellularized dermal matrices, are all being evaluated to recapitulate more closely the natural regenerative healing process. Impact Statement Acute wounds from thermal injury are common; they exert substantial physical and psychological effects on a patient and result in significant morbidity and mortality. This review provides a detailed overview of the mechanisms of reparative and regenerative wound healing; discusses the key cell types, signaling molecules, and molecular targets that influence these important biological pathways; and highlights current therapeutic approaches aimed at promoting regenerative wound healing. An increased understanding of the underlying mechanisms of reparative and regenerative healing will contribute to the development of innovative strategies for the clinical treatment of patients with severe burns.


Introduction
C utaneous wounds pose a significant health and economic burden. In 2014, 8.2 million Medicare beneficiaries in the United States filed claims for wound care, and total wound costs were estimated at $28.1 billion to $96.8 billion per year. 1 The United Kingdom's National Health Service (NHS) spent an average of £2,151 per patient (catchment population of 250,000 adults) with acute wounds in 2012 and 2013. 2 Although estimates for the global prevalence of chronic wounds vary, studies in Western Europe report that 1% to 2% of the population experience chronic wounds, and the NHS spent £2,870 per patient on chronic wounds in United Kingdom in 2012 and 2013. [2][3][4][5] The primary difference between acute and chronic wounds is the length of time required for wound closure. Acute wounds, including those caused by trauma, iatrogenic wounds (surgical procedures), or accidental wounds (e.g., burns, lacerations, and abrasions), progress in an orderly manner through the healing process. 6,7 Chronic wounds, including skin ulcers of various etiologies (e.g., venous or arterial disease, pressure, and vasculitis or autoimmune disease), can be prolonged at many points of the healing process by underlying factors (e.g., infection, cell senescence, inflammation, and ischemia) that interfere with healing. 6,8,9 Most acute wounds heal with minimal sequelae, but even wounds that completely close can lead to adverse consequences for the patient (e.g., excessive scarring can have long-term negative effects and necessitate additional treatment). 6,10,11 Acute wounds resulting from thermal injury are common and can result in significant morbidity and mortality. [12][13][14] An estimated 9 million injuries and 121,000 deaths from fire, heat, and hot substances were reported in the 2017 Global Burden of Disease study. 13 Morbidity associated with a severe burn injury can persist for 10-20 years after the initial injury. 14,15 In 2019, average hospital charges for a U.S. patient with a burn injury were estimated at $105,000, which increased to $310,000 for nonsurvivors. 12 Scarring from large burn wounds or burn wounds in functionally or cosmetically important areas (e.g., the hands, face, neck, and joints) may impart a significant burden on patients. 16,17 Scars can contribute to contracture in functionally or cosmetically important areas, reduced mobility, pain, itching, disfigurement, reduced quality of life, and psychological consequences (e.g., depression and anxiety). 16,[18][19][20][21] Severe burns can also cause extensive nerve damage and peripheral neuropathy. 22 In a systematic review, patients with burns reported the lowest scores in domains of work and heat sensitivity, bodily pain, physical role limitations, and pain/discomfort over the short term. 23 Burn injuries are also associated with a high prevalence of posttraumatic stress disorder, ranging from 11% to 50% across studies. 24 Burn wounds heal through the reparative wound healing process and result in the formation of scar tissue, which is mainly composed of dense collagen, and lacks sweat glands, hair follicles, and other appendages. 10,25 In contrast, regenerative healing is a process that can restore the wounded skin to an uninjured state, with minimal or no scarring. 26 This type of healing occurs in early-development fetal wounds (up to 24 weeks of gestation in humans) and results in skin that has a structure and function similar to the surrounding uninjured skin, including regenerated epidermal appendages. 26,27 While the goal of true regenerative healing has not yet been realized with current therapies, it is the aspirational objective of burn wound treatment. 17,28,29 This review provides an overview of the mechanisms of reparative and regenerative wound healing; discusses the key cell types, signaling molecules, and molecular targets that influence these pathways; and highlights current and future therapeutic approaches aimed at promoting regenerative healing in acute burn wounds. Recent reviews by Zhao et al. and Han and Ceilley provide a robust overview of the current landscape for chronic wounds. 9,11 The Wound Healing Process Most postnatal wounds heal through reparative healing, which is a complex biological process involving cells, signaling molecules, and the extracellular matrix (ECM) that occurs in four overlapping, highly coordinated stages: hemostasis, inflammatory, proliferation, and remodeling. 25,29 Fetal wounds heal in utero through regenerative healing; postnatal microenvironments with an attenuated inflammatory response (e.g., the oral mucosa) also show healing with regenerative characteristics, including a reduced immune response and scarring. 30 Regenerative healing occurs in the same four stages as reparative healing, with some key differences. The following sections summarize the wound healing process and the important biological components that differ between the two wound healing mechanisms; Table 1 provides more detail on the specific cells and signaling molecules involved in each type of wound repair.
It is important to note that many studies on cutaneous wound healing, including those on burns, use animal models. 31,32 Although animal models may be preferred for reproducibility, control of factors that affect wound healing, costs, and ethical considerations, such models are not completely representative of human wound healing. 31,32 An additional factor making regenerative healing difficult to study and characterize in humans is that it happens primarily in utero. 33 While this review focuses on human cutaneous wound healing, much of the primary literature cited uses animal models in an attempt to better characterize the process.

Hemostasis phase
Burn wounds result in significant damage to the surrounding vasculature, which extends out from the initial injury zone and into the zone of stasis, leading to low oxygenation and vessel leakage. 34 Reparative healing begins with the hemostasis phase. Immediately following a cutaneous injury, a blood clot comprising platelets, cross-linked fibrin, and fibronectin starts to form. 25,35 Initial clotting prevents excessive blood loss and helps protect the wound from infection. 36,37 The clot also serves as a temporary ECM that stores growth factors and facilitates the movement of vascular cells, leukocytes, and fibroblasts during the inflammatory stage. 35,38 In early-stage embryos, hemostasis begins with the formation of a fibronectin clot. 39 Fibrin is not present in the clot, and platelets have not yet differentiated. 40,41 Reepithelialization also starts immediately and is completed rapidly. 33,39,40 Inflammatory phase In postnatal healing, the inflammatory phase is initiated by the innate immune response; this phase can last several days and, in cases of severe burns, lead to a hypermetabolic state. 28,42,43 The response of toll-like receptors to damage-associated molecular patterns released by injured cells triggers the innate immune response and leads to the production of signaling molecules, including tumor necrosis factor alpha (TNF-a), and interleukin (IL)-1, IL-6, and IL-8, which promote the migration of immune cells to the wound. [43][44][45] Leukocytes infiltrate the injured tissue by extravasation and help protect the wound from infection. 46 Neutrophils are involved in phagocytosis and protect the wound from infection by secreting proteases. [46][47][48] Neutrophils also secrete cytokines with immunomodulatory functions and chemokines, which signal for additional inflammatory cells to clear debris from the wound. 47,[49][50][51] The infiltration of immune cells is helped by mast cells, which release histamine and heparin, reducing blood coagulation and increasing fluid accumulation. 45,52,53 Later in the inflammatory phase, angiogenic growth factors secreted by neutrophils help promote the formation of blood vessels. 49 27,200 Does not occur in regenerative healing; reepithelialization starts immediately after wounding (murine and rat models) 33,40 Cells Platelets are activated and drive clot formation, which prevents excessive blood loss and protects the wound from infection 25,[35][36][37] Not well characterized in the published literature Signaling molecules VEGF is released by platelets 201 Not well characterized in the published literature ECM Cross-linked fibrin and fibronectin contribute to clot formation and provide an initial structure for cell movement 25,35,38 A fibronectin clot forms 39 Tenascin is present in the tissue surrounding the wound and helps with rapid reepithelialization 33,62 There are high levels of hyaluronic acid 30 Inflammatory phase

Time point
Starts on day 1 during hemostasis and can last up to day 8 27,200 Although it is known that this phase is attenuated in fetal wounds, the timing of the appearance of cells and cytokines associated with inflammation has not been characterized in human or other large mammalian fetuses 61 Signaling molecules Inflammatory cytokines and chemokines (e.g., TNF-a, TGF-b1, IL-1, IL-6, and IL-8) promote the migration of immune cells to the site of inflammation 49,51,133,203,206,207 Proteases debride the wound and eliminate toxins from damaged tissue 46,47 Growth factors (e.g., HGF, VEGF, and FGF) promote angiogenesis 49,54,55 Histamine and heparin reduce blood coagulation and increase fluid accumulation 52,53 Type I interferons contribute to wound healing and antimicrobial activity 56,57 Expression of inflammatory cytokines and chemokines, including IL-6 and IL-8, is reduced or absent 65-67 Anti-inflammatory IL-10 expression is increased 68,69 ECM New blood vessels start to form 49,54,55 Angiogenesis does not increase, and does not contribute to, inflammation 40,62 Proliferation phase Time point Starts 3-10 days after wounding and can last until day 25 27,200 Reepithelialization starts immediately and wound closure is achieved 2-3 days after wounding (murine, rat, and lamb models) 33 Endothelial progenitor cells originate from the bone marrow and contribute to angiogenesis and increased blood circulation 99 (continued) Natural killer cells and plasmacytoid dendritic cells are involved in antimicrobial activity during the innate response, and contribute to angiogenesis and tissue repair during the adaptive response. [56][57][58][59] For more on the role of the immune response in wound healing, see the recent reviews by Cañedo-Dorantes and Cañedo-Ayala and Ellis et al. 45,60 Compared with reparative healing, the inflammatory response in regenerative healing is attenuated. 61,62 Many of the cells involved in both innate and acquired immunity (e.g., mast cells, macrophages, and neutrophils) are not yet differentiated or are not responsive to the wound. 40,41,[62][63][64] Therefore, levels of inflammatory cytokines and chemokines MMPs break down the granulation tissue and remodel the ECM into a more permanent structure [111][112][113] Higher levels of antifibrotic TGF-b3 than profibrotic TGF-b1 and TGF-b2 30,103,105,106 IL-10 downregulates the expression of collagen type I 105,106,116,117 ECM Collagen is laid down in parallel bundles to form the more permanent ECM and scar tissue 30,114,115 The ratio of fibrillar collagen types I to III increases and shifts to that of normal skin; fibril size increases to that of a healthy dermis over time 83 The number of blood vessels in the granulation tissue regresses to the density of unwounded skin 86,110 Collagen is laid down in a basket-weave pattern similar to that of uninjured skin 114,118,119 Lower ratio of collagen type I to III 105,106,116,117 Blood vessel density is also reduced to levels similar to the surrounding tissue 101 a Most of the research into regenerative healing has been done in nonhuman fetuses, which have different gestational lengths in comparison to humans; thus, the chronology of fetal wound healing in humans is not well established. The animal models used for the time points given are provided.
a-SMA, alpha-smooth muscle actin; ECM, extracellular matrix; FGF, fibroblast growth factor; HGF, hepatocyte growth factor; IL, interleukin; MMP, matrix metalloproteinase; PlGF, placental growth factor; TGF, transforming growth factor; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor. are reduced or absent in regenerative healing. [65][66][67] In addition, increased expression of anti-inflammatory cytokine IL-10 in postnatal regenerative healing helps decrease the inflammatory response. 68,69 Although the role of inflammation in regenerative wound healing is still not well understood, inflammation is associated with a fibrotic response, and reduced inflammation is thought to be more conducive to reduced fibrosis and less scarring. 64,69,70 Proliferative phase During the proliferative phase in reparative healing, resident cells in the tissue migrate and proliferate to replace damaged tissue and close the wound.
Macrophages secrete cytokines, chemokines, and growth factors (including vascular endothelial growth factors [VEGFs] and transforming growth factor [TGF]-b) to attract fibroblasts and keratinocytes to the wound. 49,71 Dermal fibroblasts proliferate and produce an ECM of collagen types I and III, fibronectin, hyaluronic acid, and proteoglycans. [72][73][74][75][76] Signals from macrophages and other immune cells cause the migration of endothelial cells and endothelial progenitor cells to the wound, where they form new blood vessels. 77,78 These vessels vascularize the ECM formed by fibroblasts, and together form a highly vascularized stroma of granulation tissue. 74 Stem cells or mesenchymal progenitor cells from hair follicles, injured nerves, and the bone marrow, and dedifferentiated cells from underlying fat, also contribute to the generation of new tissue. [79][80][81][82] Induced by TGF-b1, fibroblasts in the granulation tissue differentiate into myofibroblasts. 83,84 The myofibroblasts are rich in alpha-smooth muscle actin (a-SMA) stress fibers, and contract to decrease the wound area. 85 The contraction of the wound contributes to the vascularization of the granulation tissue by pulling in pre-existing vascular tissue, which increases the size and length of vessels already present. 74,86 As the wound contracts, keratinocytes from the surrounding tissue and stem cells from the interfollicular epidermis and hair follicles migrate across the wound bed between the granulation tissue and the fibrin clot, epithelializing the wound. 81,[87][88][89][90][91] In fetal regenerative healing, the proliferative phase is initiated quickly after wounding, potentially due, in part, to the early appearance of tenascin, which initiates cell migration and reepithelialization. 33,39,62,92 Granulation tissue does not form. 93,94 Instead, fetal wounds show higher levels of proliferating fibroblasts and keratinocytes than postnatal wounds, which produce an organized ECM of fibronectin, tenascin, chondroitin sulfate, and hyaluronic acid, which is similar to the surrounding tissue. 33,39,62,95 High levels of IL-10 upregulate hyaluronic acid by increasing protein synthesis and decreasing degradation, while also increasing invasion of fibroblasts, which raise the hyaluronic acid content of the ECM. 39,[95][96][97][98] Endothelial progenitor cells originate from the bone marrow and contribute to angiogenesis and increased blood circulation. 99 Both fetal and oral wounds have lower levels of VEGF and associated angiogenesis than in reparative healing; this is possibly because the vessels are more organized and efficient, so fewer are required. 62,[100][101][102] In fetal wound healing, the levels of antifibrotic TGF-b3 are higher relative to those of profibrotic TGF-b1 and TGF-b2, and fibroblasts do not differentiate into myofibroblasts. 30,41,[103][104][105][106] Instead, the fetal fibroblasts contract the ECM to decrease the wound area, but with lower contractile force than postnatal myofibroblasts; this is thought to contribute to the scarless phenotype. 104,107,108 Reepithelialization happens simultaneously through an actin cable running through the basal cells around the wound edge. 107,109 This cable contracts and brings the edges of the wound closer together until they seamlessly close the wound. 107,109 Unlike in postnatal healing, the epidermis moves over the damaged tissue in fetal healing. 33

Remodeling phase
In the remodeling phase of reparative wound healing, granulation tissue is replaced with a more organized ECM, resulting in scar tissue that has mechanical properties similar (although not identical) to tissue in the preinjury state. 60 The density of blood vessels in the granulation tissue regresses to that of unwounded skin. 86,110 Matrix metalloproteinases secreted by fibroblasts, keratinocytes, and inflammatory cells break down the collagen in the granulation tissue and fibroblasts remodel it into a more permanent structure. [111][112][113] The types and arrangement of collagens expressed impact the reparative wound healing process.
In wounds that heal without incident, the ratio of fibrillar collagen type I to collagen type III increases and shifts to that of normal skin, and fibril size increases to that of a healthy dermis over time. 83,114 Scar tissue is made up primarily of collagen type I arranged in parallel bundles, which is weaker and less pliable than tissue in healthy skin. 30,114,115 In regenerative healing, IL-10 downregulates the expression of collagen type I through the TGF-b signaling pathway, leading to a lower ratio of collagen type I to collagen type III deposition than in reparative healing. 105,106,116,117 Blood vessel density is reduced to levels similar to the surrounding tissue. 101 Furthermore, collagen is deposited in a basket-weave pattern similar to that of uninjured skin, which reduces or eliminates scarring. 114,118,119 Understanding the key molecular differences between reparative and regenerative wound healing contributes to the identification of factors and therapeutic approaches that may tip the balance toward regenerative wound healing.

Contraction and Scarring
All reparative healing results in scarring, and large or severe burn wounds can lead to scar contracture and pathological scarring. 120 The contraction of granulation tissue by myofibroblasts during the proliferative phase is a natural part of the reparative wound healing process. 85 However, persistence of the myofibroblasts can lead to scar contracture, resulting in pain, physical limitations, and adverse cosmetic results. [120][121][122][123] Pathological scars (i.e., hypertrophic scars [HTSs] and keloids) are also a result of dysregulated healing. HTSs result from an accumulation of fibroblasts and increased collagen production due to reduced apoptosis and collagenase activity and have increased angiogenesis. 49,[124][125][126][127] The resulting scar is a mass of cross-linked collagen aligned with the epidermal surface. 49,126,127 HTSs have a higher ratio of collagen type I to collagen type III than in normal skin, but have more collagen type III than non-HTSs. 49,114,128,129 There is a high chance of HTS formation if wound healing is delayed by more than 3 weeks. 28 Unlike HTSs, which typically stay within the wound edges, keloids exhibit uncontrolled growth beyond the borders of the initial wound. 130 They have multiple layers with varying ratios and levels of organization of collagen types I and III and are difficult to treat. 130,131 Burn progression, extensive inflammation, and increased mechanical forces are all characteristics of severe burn wounds that contribute to scarring and contracture.

Burn wound progression and inflammation
Although the inflammatory phase is an essential part of the wound healing process, excessive inflammation during the early stages of wound healing and prolonged inflammation can lead to scarring, fibrosis, and delayed healing. 25,49 Second-degree (partial thickness) burn wounds can progress to third-degree (full thickness) burn wounds in a matter of days, and this progression is associated with increased inflammation and cell death. [132][133][134][135] In addition, burn wounds with greater total body surface area (TBSA) have greater and more prolonged inflammation than those with smaller TBSA. 42 The inflammation associated with severe burn wounds leads to a hypermetabolic response. 42,136 Increased inflammation and the hypermetabolic response delay wound healing, including reepithelialization; delayed reepithelialization can increase scarring and lead to HTSs. [137][138][139][140][141] There is evidence that preventing burn wound progression and reducing inflammation and the hypermetabolic response can lead to accelerated reepithelialization and wound closure in burn wounds, reducing scarring. 132,138,142,143 Studies using fetal lamb and human fetal ex vivo models have shown that fetal thermal wounds are able to heal without scars or an inflammatory response, although this may be dependent on the size and extent of the burn. 33,63,144 Mechanical forces Although the focus of this review is on the biological mechanisms of wound healing, mechanical forces also play a role and need to be briefly discussed. While the role of mechanical load in wound repair is still not well characterized, it has been associated with an increase in the inflammatory response, the conversion of fibroblasts to myofibroblasts, the specific orientation of myofibroblasts and associated collagen bundles, and fibrosis. 120,125,[145][146][147][148] Greater mechanical force is associated with the formation of HTSs and keloids, and repeated mechanical tension can lead to scar contracture. 124,125,[149][150][151] In addition, the tension caused by contracture may lead to pathological scarring. 146 Pathologic scarring and contracture are common in burn wounds. 120,152 Treatment for large burn wounds may involve physical therapy to prevent muscle contraction and stretching or splinting of scars to prevent contracture; this increases mechanical forces and may facilitate the conversion of fibroblasts into myofibroblasts, which can lead to further scarring. 120,153 Although it is difficult to study the role of mechanical forces in fetal skin, fetal mouse skin has a much lower resting tension than adult skin. 124 Adult mouse skin has lower resting tension than human skin, and when the tension of human skin is applied to mouse skin, pathologic scarring occurs. 124 Because fetal skin has a low resting tension, a severely reduced inflammatory response, and no myofibroblasts, it is thought that reduced mechanical tension contributes to scarless healing. 150,153 Wound healing studies have shown that a reduction of mechanical forces reduces scarring, although therapies that can reduce mechanical forces during burn wound healing are still in development. 145,154 For an in-depth discussion of mechanical forces and their potential applications in the goal of wound healing, see the recent reviews by Yannas and Tzeranis and Barnes et al. 148,153 Therapeutics Aimed at Promoting Regenerative Healing in Burn Wounds The aspirational objective of burn wound treatment is regenerative healing. 17,28,29 One of the key differences between regenerative and reparative healing is the reduced inflammatory stage in fetal wound healing, secondary to decreased angiogenesis and expression of proinflammatory cytokines and the increased expression of the anti-inflammatory cytokine IL-10. 30,40,62,68,105,155 Optimal therapeutics for cutaneous wound healing aim to combine cells and cell signaling molecules that modulate inflammation with matrices that allow these cells to respond to endogenous signals in a spatiotemporal manner. 29,156 Although regenerative healing may never be achieved for burn wounds, basic research continues to provide novel approaches and products aimed at restoring normal skin architecture and reducing adverse outcomes, including infection, delayed reepithelialization, and scarring. 29,157 The following section will discuss skin substitutes for burn wound healing, give examples of products currently available in the United States, and describe the components being researched for new therapeutic development.

Skin substitutes for burns
Although autografts are preferred for the treatment of acute burn wounds, they create another wound that can lead to pain, scarring, and reduced quality of life. 158,159 Additionally, in the case of extensive burn wounds, an individual may not have enough uninjured skin to provide sufficient coverage, creating a need for skin substitutes. 159 There are many different ways to classify skin substitutes. The categories in Table 2 depend on the origin of the components (human, xenogeneic, and synthetic), the types of components (cells and ECM/scaffold), whether the product contains human cells or tissue, and whether the product is autologous or allogeneic. Depending on the product composition, some products are intended to provide temporary coverage, while others can be used in place of autografts or allografts.
Unlike autograft transplantation, skin substitutes can elicit both innate and adaptive immune responses that can lead to rejection of the substitute. 159 Skin substitutes with bioactive keratinocytes and fibroblasts generate growth factors and cytokines that can elicit a host response that aids in the wound healing process. [159][160][161][162] These substitutes may avoid rejection because they do not contain antigen-presenting immune cells. 160,162 Substitutes that are acellular, or that have synthetic components, are also less likely to be rejected. 159 Although many of these products can reduce the 1156 SINGER   (1) and Section 361 of the Public Health Service Act. 164 Previously, the FDA exercised enforcement discretion for certain regenerative medicine products so that they did not require premarket review and approval. 166 As of May 31, 2021, all HCT/P manufacturers were required to file an Investigational New Drug application or a BLA to legally market their products. 165 e Apligraf is used off-label to treat burns. BLA, biologics license application; DRM, dermal regeneration matrix; DRT, dermal regeneration template; FDA, U.S. Food and Drug Administration; FRO/KGN, product code for medical devices; HCT/P, human cells, tissues, and cellular and tissue-based product; HDE, Humanitarian Device Exemption; PMA, premarket approval.
need for autografting and improve burn wound repair, consideration of the product composition and its potential immunogenicity are important for clinical practice.

U.S. Food and Drug Administration regulation of skin substitutes
The products in this section and in Table 2 were chosen because they are U.S. Food and Drug Administration (FDA) cleared and marketed in the United States. While the products may all be considered skin substitutes in clinical practice and for insurance billing purposes, the specific indications for use (and subsequent cost) of these products are determined by the FDA. The FDA regulatory category of a product is determined by the product's components and its level of risk to the patient. 163 The categories, from lowest to highest level of risk, are human cells, tissues, and cellular and tissue-based products (HCT/Ps), humanitarian use device (HUD), 510(k), premarket approval (PMA), and biologics license application (BLA). 163 The least-rigorous regulatory category is HCT/Ps. 163,164 This categorization applies to products that are minimally manipulated and intended for homologous use. 163 Until recently, the FDA exercised enforcement discretion for some HCT/Ps to give manufacturers time to determine if an application for more rigorous regulation was needed. 165 As of May 31, 2021, products that do not meet all the requirements for HCT/Ps are required to have a BLA or an investigational new drug application to be marketed. 165,166 Products that are derived from human and/or animal tissue are regulated under an HUD or PMA, and xenogeneic and synthetic products are regulated under the 510(k) pathway. 163 A BLA is used for products using human cells and tissues that make a specific action claim. 163 Tissue-engineered products without live cells may be considered medical devices, which are classified from Class I (lowest risk) to Class III (highest risk), with the level of regulation increasing with the class. 163 Acellular products are then regulated through two pathways: Class I devices are generally exempt from the 510(k) pathway, Class II devices are usually regulated using the 510(k) pathway, and Class III devices usually require a PMA. 163 For a more in-depth discussion of FDA regulation of skin substitute products for burns, see Belsky and Smiell. 163 These products may also be regulated differently outside the United States, and there are additional products that are not indicated for use in U.S. settings. Oberweis et al. discussed various regulatory frameworks worldwide for tissue-based products, and the alliance for regenerative medicine lists skin substitutes and where they are approved on their website (https://alliancerm.org/ available-products). 167,168 Components of regenerative healing used for burn wound research One of the components of regenerative healing that is being explored for wound healing is stem cells. In regenerative healing, stem cells help mediate wound repair through a variety of molecular signals that promote angiogenesis and ECM formation, recruit endogenous progenitor cells, induce cell differentiation, and reduce inflammation and scarring. [169][170][171][172][173][174][175][176][177] Preclinical burn studies in murine models have found that treatment with bone marrowderived mesenchymal stem cells accelerated wound closure, improved mobility, and reduced fibrosis, and may mediate inflammation, myofibroblast differentiation, and collagen deposition. [178][179][180] There are few skin substitute products for burns in clinical trials or on the market that are made with stem cells; the use of autologous or allogeneic cells is more common ( Table 2 and Supplementary Table S1). In a realworld study, cultured human keratinocyte autografts, used alone or adjunctively with a wide-meshed autograft, improved survival in patients with large (mean TBSA, 67.5%) burn wounds. 181 Treatment with autologous epidermal cells on a fibrin matrix reduced contraction and helped maintain skin pliability in patients with burn wounds. 182 In addition, the incorporation of allogeneic progenitor cells with an acellular matrix may, under appropriate conditions, form an epidermal structure that can respond to local signaling. 161,183,184 Dermal matrices are another approach to harnessing properties of regenerative healing. 185,186 Made of synthetic materials or decellularized tissue (human or xenogeneic) ( Table 2), these products aim to provide a structured scaffold to guide the development of nonfibrotic tissue. 187 A study of patients with severe burns found that a decellularized dermal scaffold used over joints helped prevent scarring, and resulted in better conservation of joint function. 188 In addition, a case study of a patient with extensive HTSs from a burn wound demonstrated that scar excision followed by treatment with a decellularized dermal scaffold and a split-thickness skin graft resulted in limited scar formation, supple skin, and increased range of motion. 189 Signaling molecules are difficult to incorporate into skin substitutes. 156,190 When applied without a scaffold, exosomes from mesenchymal stem cells have been shown to reduce inflammation in burn wounds in rats, mirroring the low inflammation seen in regenerative healing. 191 Plateletrich plasma, which has high levels of growth factors, is also being explored as a burn treatment. [192][193][194] Rats with burn wounds treated with platelet-derived biomaterials showed accelerated healing and fewer inflammatory cells than controls. 195 While some skin substitute products contain bioactive cells that secrete signaling molecules (Table 2), there are no acellular products for burns with published data showing that they provide signaling molecules at optimal dosages in a relevant spatiotemporal manner. 156,162,190,196 This may be one aspect for future research to address.
Shaping the future of regenerative medicine for burn wound healing requires further understanding of the processes that have led to the currently available products. The principles and components of regenerative wound healing have not changed over time, but how researchers understand and apply them to the development of new therapeutics has continued to shift. For example, inflammation and a strong immune response are linked to reparative healing, but recent research has linked both processes as also being important to regeneration in animal models. 25,43,49,197,198 Continued improvement in the understanding of pathways, cells, and signals involved in regenerative healing will allow for the identification of new targets that can be used to drive the development of new therapeutics. 199

Summary
Scarring is a natural consequence of reparative healing, including for wounds that heal quickly with minimal interference. All scarring-even in the absence of pathological scarring-results in tissue that does not have the same appearance, strength, or function as the surrounding skin and contributes to physical and psychological burdens for patients with severe burn wounds. 29,42,111 To ensure long-term patient well-being and quality of life, burn care approaches necessitate advancements that move toward regenerative healing, reduced scarring, and restored strength and function. 29,42 An increased understanding of the underlying mechanisms of regenerative and reparative healing will contribute to the development of innovative strategies that better incorporate aspects of regenerative healing and improve outcomes for patients with severe burns. 17,29 Acknowledgments Medical writing and editorial support, conducted in accordance with Good Publication Practice 3 (GPP3) and the International Committee of Medical Journal Editors (ICMJE) guidelines, were provided by Caroline Leitschuh, PhD, of Oxford PharmaGenesis Inc., Newtown, PA.

Author's Contributions
A.J.S. conceived the original ideas of this article, reviewed all drafts, and provided critical feedback throughout the development of this article, and has read and approved the final article. The sponsor, Mallinckrodt Pharmaceuticals, plc., Hampton, NJ, did not contribute to the content development, but did participate in the review of the final article and the decision to submit.

Disclosure Statement
No competing financial interests exist.

Funding Information
Medical writing and editorial support were provided by Oxford PharmaGenesis Inc., and funded by Mallinckrodt Pharmaceuticals, plc.

Supplementary Material
Supplementary Table S1