A variety of insults can result in injury to the skin including mechanical, thermal, chemical, nuclear, and infectious. In all cases, the injury to the skin triggers a response aimed at restoring the integrity and function of the skin. Wound healing is a dynamic process that involves a complex and closely orchestrated interaction between blood cells, cutaneous parenchymal cells, soluble factors, and the extracellular matrix (ECM). Although considerable overlap exists among the various stages of wound heal- ing, it is helpful to divide the healing process into three classical stages: Inflammation, tissue formation, and tissue remodeling. The goals of wound healing include rapid wound closure with restoration of its barrier function, avoiding infection, and achieving an optimal aesthetic and functional result. Increased understanding of the molecular and cellular pathophysiology of wound healing over the last decade has led to the development of new healing devices and dressings. Skin substitutes that contain various combinations of epidermal and/or dermal com- ponents are now commercially available. The current chapter will review the biology of heal- ing and discuss the various skin substitutes available for treating wounds.
The skin is the largest organ in the human body. Although it has multiple functions, its pri- mary role is to serve as a barrier to the external environment. Other functions of the skin include fluid homeostasis, thermoregulation, immune surveillance, and sensory detection. A variety of insults can result in injury to the skin including
mechanical, thermal, chemical, nuclear, and infectious. In all cases, the injury to the skin trig- gers a response aimed at restoring the integrity and function of the skin.
Cutaneous wounds, both acute and chronic, are very common. Each year there are more than 8 million traumatic lacerations1 and 1 million burns treated in the United States alone.2In addi- tion, up to 90 million surgically induced incisions are created each year.3Chronic wounds, such as diabetic ulcers, decubitus ulcers, and venous sta- sis ulcers are also quite common with more than 6 million ulcers reported annually.4 Chronic ulcers are especially problematic because they tend to affect the most vulnerable populations, such as the elderly, the diabetic, and the physi- cally debilitated.
Wound healing is a dynamic process that involves a complex and closely orchestrated interaction between blood cells, cutaneous parenchymal cells, soluble factors, and the ECM.5 Although considerable overlap exists among the various stages of wound healing, it is helpful to divide the healing process into three classical stages: Inflammation, tissue formation, and tissue remodeling. The goals of wound heal- ing include rapid wound closure with restora- tion of its barrier function, avoiding infection, and achieving an optimal aesthetic and func- tional result. Increased understanding of the molecular and cellular pathophysiology of wound healing over the last decade has led to the development of new healing devices and dress- ings. The current chapter will review the biology
27
Wound Healing and Skin Substitutes
Adam J. Singer and Marcia Simon
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of healing and discuss the various skin substi- tutes available for treating wounds.
Growth Factors and Cytokines
Because cytokines and growth factors have such a central role in regulating wound healing, a brief description of the origins and actions of some of the most common ones follows (Table 27.1).
Cytokines and growth factors are members of a large group of regulatory polypeptides that are secreted by many different cell lines, which act through autocrine and paracrine mechanisms to affect other cells.6 In general, growth factors refer to polypeptides that regulate cell matura- tion whereas cytokines are important for host defense. Although essential for wound healing and host defense, in excess, cytokines and growth factors may also be responsible for poor healing and scarring. Most cytokines and growth factors are produced by more than one type of cell and have multiple pleiotropic func- tions. Considerable overlap and redundancy are also common among these substances, ensuring that wound healing proceeds even when any individual cytokine or growth factor is absent.
Individual growth factors do not act alone but in concert, and the overall effect is determined by their relative concentration and timing.
The biological effects of the growth factors and cytokines are mediated by interacting with cell surface receptors. In turn, interaction with these receptors results in the formation of sec- ondary messengers that affect the cell’s struc- ture or function. Although the exact molecular pathways that lead to alterations in function are not completely understood, several have been elucidated. Many of the recognized intracellular signal transduction pathways act through acti- vation of a host of tyrosine kinases.7In contrast, the effects of TGF-β are propagated via a signal transduction network involving receptor ser- ine/threonine kinases at the cell surface and their substrates, the SMAD proteins.8,9
Platelet-derived growth factor (PDGF) is first released from platelet α granules result- ing in recruitment and activation of immune cells and fibroblasts.10Later, PDGF is released by macrophages and stimulates the produc- tion of collagen and proteoglycan. There are three different isoforms of PDGF including AA, AB, and BB that are named after the arrangement of two polypeptide chains, A and B. All three isoforms influence wound healing.
Topical application of recombinant PDGF has resulted in increased wound breaking strength and faster healing in both animals and humans.11–13 To date, PDGF is the only Food and Drug Administration-approved growth factor for clinical use.
Table 27.1. Cytokines and growth factors that have a role in wound healing
Mediator Origin Major Actions
PDGF Platelets, macrophages, epidermal cells Fibroblast proliferation and chemoattraction, macrophage chemoattraction and activation
FGF-1 and -2 Macrophages, endothelial cells Angiogenesis and fibroblast proliferation
KGF-1 Fibroblasts Keratinocyte proliferation and motility, production of granulation tissue KGF-2 Fibroblasts Fibroblast migration and granulation tissue formation
EGF Platelets, macrophages Reepithelialization, angiogenesis, ECM formation
TGF-β1 and -β2 Platelets, macrophages Epidermal cell motility, chemotaxis of macrophages and fibroblasts, ECM synthesis
TGF-β3 Macrophages Anti-scarring effects
TGF-α Macrophages, keratinocytes Reepithelialization
VEGF Epidermal cells, macrophages Angiogenesis, increased vascular permeability IGF Fibroblasts, epidermal cells Reepithelialization and granulation tissue formation
TNF-α Monocytes Neutrophil chemotaxis and activation, mitogenic activity in fibroblasts, pleiotropic expression of growth factors
IL-1 Neutrophils, lymphocytes, keratinocytes, Neutrophil chemotaxis, fibroblast and ECM proliferation macrophages
IL-6 Keratinocytes, macrophages, fibroblasts Major mediator of acute phase response
IL-8 Keratinocytes, Neutrophil chemoattraction, reepithelialization, angiogenesis
Transforming growth factor (TGF)-β is released by platelets, macrophages, and fibrob- lasts resulting in fibroblast migration, matu- ration, and ECM formation.14 TGF-β exists in three isoforms – TGF-β1, TGF-β2, and TGF-β-3.
Although essential for wound healing, the potent fibrogenic effects of TGF-β have been implicated in several fibroproliferative con- ditions, including scleroderma, keloids, and hypertrophic scarring.15,16In contrast, low levels of TGF-β in the fetus are partially responsible for their ability to heal without scar formation.
Whereas TGF-β1 and TGF-β2 promote ECM formation and scarring, TGF-β3 may prevent scarring. Shah et al.17,18have shown that admin- istration of TGF-β1 and TGF-β2 neutralizing antibodies reduce scarring as does administra- tion of TGF-β3.
The fibroblast growth factor (FGF) family consists of 22 related proteins that regulate cel- lular proliferation, differentiation, migration, and/or survival of a variety of cells, mostly fibroblasts and endothelial cells.19 In contrast, FGF-7 and FGF-10 [also known as keratinocyte growth factors (KGFs) 1 and 2] mostly affect keratinocytes.20The most recent addition to this family is FGF-22, which has also been implicated in reepithelialization.21 Basic FGF and acidic FGF (aFGF or FGF-2) are released by macrophages and endothelial cells and stimulate the proliferation and migration of keratinocytes and fibroblasts. Basic FGF also stimulates the growth and migration of endothelial cells and has an important role in wound contraction and remodeling.22
KGF belongs to the FGF family and contains two isomers that share the same receptor.23 Whereas KGF is secreted by fibroblasts and endothelial cells (of mesenchymal origin), its receptors are only located on epithelial cells of ectodermal origin. Thus, KGF may have a key role in the epidermal–dermal interaction. KGF is an important regulator of keratinocyte pro- liferation and maturation.24KGF may also stimu- late epithelial cells to release other growth factors such as PDGF, TGF, and FGF. Administration of KGF-1 or KGF-2 in animal wound models has resulted in improved reepithelialization, colla- gen content, and wound breaking strength.25
Epidermal growth factor (EGF) is secreted by keratinocytes and acts locally through an autocrine function to direct reepithelialization.26 EGF also stimulates the secretion of fibroblast collagenases that are important in wound
remodeling. Whereas topical application of EGF to human skin donor sites accelerated healing in one study,27another study showed no difference in healing rates between split thickness skin wounds treated with EGF or silver sulfadiazine.28 Vascular endothelial growth factor (VEGF) is primarily released by keratinocytes but also by macrophages and fibroblasts.29 VEGF is one of the most potent stimulators of angiogenesis and is induced by local hypoxia, high lactate levels, and nitric oxide production.30,31Topical admin- istration of VEGF has resulted in increased granulation tissue formation in normal and hypoxic experimental wound models.32VEGF is also responsible for increasing vascular perme- ability at the wound site.
Insulin-like growth factor (IGF) exists as 2 isoforms – IGF-1 and IGF-2 (also known as somatomedins). IGF is primarily produced by hepatocytes and skeletal muscle, but also by wound fibroblasts, neutrophils, and macrophages.33IGF stimulates the proliferation of fibroblast and keratinocytes as well as colla- gen synthesis. IGF is reduced in diabetic and steroid-dependent animals and its administra- tion has improved wound healing in diabetic and steroid-dependent animals.34
Early after injury, interleukin (IL)-1 and tumor necrosis factor (TNF)-α are released and chemoattract multiple inflammatory cells and stimulate them to produce additional growth fac- tors and cytokines.35IL-8, which has been shown to enhance reepithelialization,36is also released secondary to the actions of IL-1.37Fibroblast pro- liferation and angiogenesis are also under the influence of IL-1 and IL-2, respectively.
Hemostasis
Injury to the skin usually results in disruption of the vascular integrity and the extravasa- tion of blood and its products into the site of injury. To stop blood loss, the injured vessels undergo vasospasm and platelets adhere to the injured vessels. After their adherence, platelets are activated and release a host of factors that participate in the wound healing process.
Additionally, the injury results in the release of tissue factor by endothelial cells.38Exposure of subendothelial collagen and the release of extrinsic tissue factor activate the coagulation system through the intrinsic and extrinsic
pathways in an attempt to stop the bleeding by forming a clot.
The coagulation system is composed of a complex enzyme cascade in which the compo- nents are activated by proteolysis. The end result of coagulation is the formation of fibrin, which forms the backbone of the clot. Other components of the clot include ECM proteins such as fibronectin, vitronectin, and throm- bospondin.
In addition to plugging the defect, the aggre- gated platelets serve as a surface for formation and activation of the blood clot.39 This blood clot not only helps stop bleeding and provides a barrier to microbial invasion, but also serves as the provisional matrix for invading cells and as a reservoir of growth factors. Activation of the coagulation system also results in activation of the complement system,40,41 another plasma proteinase cascade system that generates several chemotactic agents, such as C5-derived mono- cyte chemotactic factor C5a.42 The role of the coagulation system in wound healing is further demonstrated by the ability of factor XIII to modulate tissue remodeling and repair by medi- ating interaction between platelets and endothe- lial cells.43Degranulation of platelet α-granules results in the release of multiple cytokines including PDGF, and TGF-β that stimulate chemotaxis of neutrophils, monocytes, and lym- phocytes and enhance their proliferation.39
The Inflammatory Phase
Injury to the skin results in the release of multi- ple inflammatory mediators that initiate the inflammatory response.35 Substances released from the complement system and platelets results in the chemotaxis of inflammatory cells such as the neutrophils and monocytes.23These processes are also enhanced by the release of histamines, bradykinins, and leukotrienes from adjacent mast cells.44 The adherence and pas- sage of neutrophils into the wound site through the injured blood vessels is mediated through adhesion molecules that are expressed on the endothelial cells, partially in response to TGF- α.45 The release of mediators such as PDGF, TGF-β, FGF, and EGF by platelets also helps recruit and activate inflammatory cells.
The main function of the infiltrating neu- trophils is to engulf and destroy any bacteria,
foreign debris, or necrotic tissue in the wound area. The neutrophils also produce angiogenic factors (VEGF, TNF-α, and IL-1) and pro- teinases that are necessary in degrading the matrix of the wound bed. Whereas most of the neutrophils are shed with the wound eschar over the first few days after injury, the entrance of monocytes that are stimulated by monocyte- specific chemoattractants continues for longer periods. Upon entry to the wound, the mono- cytes are transformed into macrophages that release a large number and amount of growth factors and cytokines such as PDGF, TGF-α, TGF-β, IGF-1, VEGF, and TNF-α.46 Thus, macrophages become the predominant leuko- cyte in the wound by day 3 after injury.
Maturation of monocytes into macrophages is regulated by IL-2, TNF-α, and interferon-γ that are secreted by T lymphocytes as well as by PDGF.
The fibrin-rich provisional wound matrix of the resolving clot also has an important role in wound healing and the inflammatory response.47 It serves as a protein reservoir by binding cytokines and growth factors and amplifies their chemotactic properties by increasing local mediator concentration and directing cellular migration through its scaffold by the specific expression of integrins that are recognized by the various cells in the wound.48
Although inflammation is an essential com- ponent of wound healing, excessive inflamma- tion can result in impaired healing, such as in chronic, nonhealing ulcers. Persistent accumu- lation of neutrophils in chronic ulcers results in the release of large amounts of proteolytic enzymes that degrade connective tissue matrix49 and growth factors.50 Furthermore, prolonged inflammation resulting in high prostaglandin levels may result in fibroblast proliferation and excessive collagen production.51,52Indeed, phar- macologic reduction of the early inflammatory phase by topical application of a cyclooxyge- nase-2 inhibitor has been shown to reduce scar formation in full-thickness murine wounds.53
Reepithelialization
Reepithelialization is one of the earliest and most important stages of wound healing aimed at restoring the barrier function of the skin.54 With healing by primary intention, in which
the divided wound edges are approximated, the process of reepithelialization is completed within 48–72 hours of injury. With healing by secondary intention, this process is more pro- longed, requiring dermal reconstitution before being completed. Reepithelialization and epi- dermal maturation is delayed in the presence of infection.55
In order for reepithelialization to proceed, the well-differentiated keratinocytes at the wound edges and within the epidermal appendages of the dermis must detach from their neighboring cells and basement mem- brane, migrate over the wound defect, and pro- liferate. Separation of keratinocytes from neighboring cells occurs after breakdown of the hemidesmosomes that connect neighboring cells.56 Shedding of the keratinocyte cell–cell adhesion molecules is aided by the presence of multiple growth factors such as EGF, TGF-α, TGF-β, granulocyte-macrophage colony-stimu- lating factor, and FGFs. Changes in the ker- atinocyte cytoskeleton occur that result in the exposure of lamellipodia and filopodia, which are structures that adhere to surrounding inte- grins (structures that allow cell–matrix and cell–cell interaction and adhesion), resulting in forward movement of the cells.57 To facilitate keratinocyte migration, the expression of inte- grins on the cell surface is also altered.58,59 Movement of the keratinocytes through the ECM is further aided by the secretion of prote- olytic enzymes, the matrix metalloproteinases (MMPs), which are expressed and secreted from the keratinocytes, helping to pave a path- way for the migrating cells.60–62Collagen types I and IV, fibronectin, and vitronectin, which are components of the ECM, also facilitate ker- atinocyte migration.63–65 Additionally, the MMPs are probably required to detach the ker- atinocytes from the ECM to allow their forward movement. The direction of cellular migration is further dictated by the presence of chemotac- tic factors.57 Cytokines such as TGF-α, TGF-β, IL-1, and IL-8, also have an important role in mediating keratinocyte migration.66–69
Although the exact mechanisms responsible for signal transduction in the keratinocytes are still under investigation, several processes have been elucidated. After injury, rapid activation of various transcription factors [such as activated protein (AP)-1] occurs, probably through increased levels of intracellular Ca2+and mito- gen activated protein kinase.70Increases in the
intracellular Ca2+ levels probably result from direct stimulation of the injured cells as well as from paracrine signals from surrounding unin- jured cells. AP-1 in turn is known to stimulate downstream genes involved in keratinocyte migration and proliferation, such as genes for collagenase, stromelysin, and gelatinase B, that facilitate cell migration through the provisional ECM. AP-1 also regulates genes encoding for cell adhesion molecules such as integrins and laminins that allow migrating cells to attach to and crawl over the provisional matrix. Increased proliferation of basal keratinocytes at the wound edges is also thought to be regulated by AP-1.
Other transcription factors, such as nuclear fac- tor-κB also have a role in regulating ker- atinocyte migration.71
As the epidermal integrity is restored, the basement membrane proteins reappear in a highly organized, zipper-like manner.72The epi- dermal cells then revert to their original pheno- type and become firmly attached to their underlying dermis.
Angiogenesis
Angiogenesis, or the process of forming new blood vessels, is critical to wound healing, because it allows the delivery of vital cells and nutrients to the wound.73 Angiogenic factors that have an important role in neovasculariza- tion include FGF-1,74 TGF-β,75PDGF-BB,76 and VEGF.77,78 Other angiogenic agents include angiogenin,79 angiopoietin,80 and human mast cell tryptase.81Of the angiogenic factors, VEGF has a central role. Under hypoxic conditions, hypoxia-inducible factor-1 activates the VEGF gene.82 Furthermore, the beneficial effects of hyperbaric oxygen on wound healing83 have been attributed to up-regulation of VEGF.84 Nitric oxide (NO) also has a key role in angio- genesis.85 Indeed, blockage of endothelial NO synthase that produces NO in endothelial cells results in impaired healing.86 Many of the actions of VEGF are dependent on NO, and expression of VEGF by keratinocytes is increased by NO.87
The process of neovascular formation is tightly regulated by a set of antiangiogenic fac- tors such as endostatin88 and the throm- bospondins.89 Disruption of parenchymal cells such as keratinocytes and fibroblast results in
the release of potent angiogenic factors such as VEGF and FGF-1, respectively. Additionally, macrophages are a major source of angiogenic factors. The release of VEGF is also induced by tissue hypoxia and lactic acid.90
One of the critical phases of angiogenesis is the dissolution of the endothelial cell basement membrane. This process is mediated by MMPs (mostly MMP-9 and MMP-2) that normally are not present in the skin.91 Fibroblasts express MMP-9 at wound sites within 1–4 days of injury resulting in secretion of large amounts of MMP- 9 that helps dissolve the basement membrane of vessel walls.92 Fibroblasts also enhance angio- genesis by secreting a host of mediators (such as VEGF, bFGF, PDGF, IL-1β, and granulocyte colony-stimulating factor) that stimulate endothelial cell proliferation.93 Fragmentation of the basement membrane allows the sprouting of capillary buds that migrate into the injured area in response to FGF-1 and VEGF. The endothelial cells at the tips of the capillary buds express specific integrins such as αvβ394 that enable attachment to the fibronectin in the base- ment membrane and the ECM. This integrin also mediates endothelial cell migration.95 The expression of these integrins is also regulated by the fibrin and fibronectin in the provisional ECM96further demonstrating the importance of cell–matrix interactions in the healing process.
Endothelial cell proliferation is stimulated by FGF and VEGF whereas the secretion of fibronectin and proteoglycan by these cells is stimulated by TGF-β. Under the influence of fac- tors such as VEGF, FGF, and mast cell tryptase, the capillary sprouts branch and join to form capillary networks allowing the restoration of blood flow. As the ECM matures and is replaced by scar tissue, the endothelial cells undergo apoptosis and degenerate,97which explains why the color of the scar fades with time.
The Proliferative Phase
During the proliferative phase of wound healing, there is an increase in the number of fibroblasts and blood vessels in the wounded area. The granular appearance of this tissue is the source of its name. The fibroblasts invade the wound in response to a number of chemoattractants and growth factors.92 Their entry into the wound is aided by the presence of extracellular proteins
within the matrix (such as fibrin, fibronectin) that help guide the motion of the fibroblast. The expression of ECM receptors by fibroblasts is further regulated by the ECM proteins them- selves.98This complex interaction between cells and the ECM has been coined “dynamic reci- procity.”99 Once in the wound, the fibroblasts begin to produce new matrix in the form of collagen and glycosaminoglycans. With the arrival of fibroblasts, the wound content of col- lagen, especially collagen type I, increases.
Transcription of collagen mRNA is followed by hydroxylation of proline and lysine residues within the polyribosomes of the endoplasmic reticulum.100Formation of a triple-helical struc- ture and glycosylation of the collagen is followed by secretion of procollagen into the extracellular space. Further processing of the collagen includes cleavage of the procollagen N and C-terminal peptides and action by the enzyme lysyl oxidase that helps form stable crosslinks.
Wound Remodeling
In response to TGF-β that is secreted during the early and middle phases of wound healing, the fibroblasts differentiate into “myofibroblast-like”
cells that express α-smooth muscle actin.101This enables the cells to exhibit a contractile force in vivo that results in a reduction in the size of the wound that is critical to wound repair.102,103
Wound remodeling is a highly organized process that includes regulation of both the pro- duction of new ECM and its degradation by pro- teases (primarily MMPs). The degradation of the ECM is further controlled by the tissue inhibitors of metalloproteinases that inhibit the action of the proteinases.104 By 3 weeks after injury, the production of collagen is matched by its degradation and restructuring and remodel- ing of the ECM is the primary mode of increased wound strength. As the collagen matures, the number of intramolecular and intermolecular crosslinks increases, giving collagen its strength and stability over time.105In normal tissue, the collagen fibers form a highly organized basket- weave architecture. In contrast, the collagen fibers in scar tissue are smaller, thinner, and randomly arranged, making scar tissue weaker than normal. In fact, even when fully healed, wounds have a tensile strength that is only 70%–80% of the skin’s original strength.106
Apoptosis and Wound Healing
Apoptosis, or programmed cell death, is an active physiologic process that is critical to all stages of wound healing.97,107This process is dis- tinctly different from necrosis, which represents passive cell death. Apoptosis is highly regulated and results in formation of nuclear and plasma membrane blebs, loss of cell volume, detach- ment from cellular matrixes, and structural changes in organelles.108 Resolution of the inflammatory response in the healing wound is critical to wound healing109and is regulated in part by TGF-β that induces apoptosis through mitochondrial signaling.110 Apoptosis is also important in wound maturation and remodeling and is responsible for the removal of the myofi- broblasts.111 Indeed, excessive scarring in keloids may be the result of abnormal control of apoptosis in fibroblasts.112,113 Integrins at the leading edge of the wound may also have an important role in effecting apoptosis.114,115 Enhanced epithelial cell apoptosis may have a role in chronic wounds such as gastric ulcers.116 The ability of growth factors such as PDGF to prevent apoptosis117may help explain its benefi- cial effects in chronic ulcers.11
The Therapeutic Role of Growth Factors
Treatment of wounds with exogenous growth factors or cytokines has been shown to acceler- ate healing, especially in chronic wounds. For example, Steed13 conducted a clinical trial involving patients with diabetic foot ulcers and demonstrated accelerated healing after treat- ment with recombinant PDFG-BB. Similar results were demonstrated by Robson et al.11for pressure ulcers. Other growth factors that have resulted in accelerated healing include bFGF with pressure ulcers, KGF-2 with venous stasis ulcers, and TGF-β1 for diabetic foot ulcers.118 Unfortunately, clinical experience with these growth factors has been very disappointing. One possible explanation for the lack of dramatic clinical benefit using these agents is the presence of bacteria and bacterial proteases that degrade both the growth factors and their receptors.6 Several methods that may overcome the local degradation of growth factors include sustained
release of these agents from a delivery system or introduction of genetic material that results in transient local production of cytokines.
Carboxymethylcellulose suspensions improve the handling and delivery of growth factors to open wounds, but are cumbersome and unreli- able. Fibrin-based carriers can improve growth factor delivery, but act as mechanical barriers to wound healing. Delayed-release polymers aid in the delivery and handling of growth factors.
Priming of wounds with growth factors has been shown to reduce the lag period required prior to increased levels of fibroproliferative growth factors such as TGF-β, thus shifting the wound healing trajectory in a leftward direction.
Recently, Robson et al.119demonstrated reduced rates of incisional hernias in rodents pretreated with TGF-β2, bFGF, and IL-1β. Thus, priming of acute wound sites with growth factors before injury can result in earlier activation of the repair process. This method may prove to be useful in patients undergoing repair of abdomi- nal incisions for which the rates of hernia for- mation are especially high.
The development of gene therapy offers the potential to enhance or inhibit the gene expres- sion of specific cytokines and extracellular mol- ecules.120Because gene therapy generally results in only transient gene expression, it is especially well suited for wound healing that is temporary in itself.
Fetal Wound Healing
During the early phases of gestation, the fetus has the extraordinary capacity to heal without scarring.121Increasing understanding of the dif- ferences between fetal and adult wound healing that are responsible for scarless healing in the fetus is likely to result in knowledge that may ultimately lead to the development of novel therapies aimed at reduced scarring.
In the fetus, reepithelialization occurs by the action of actin fibers within the epidermal cells that draws the wound edges together similar to the function of a purse string.122In contrast, epi- dermal cells in the adult resurface the wound by crawling across it.
Several notable differences between fetal and adult wound healing may be responsible for the reduced tendency to form scars. First, lack of proinflammatory signals may limit the amount
of inflammation during the early phases of heal- ing.123 The reduction in inflammation in turn reduces the stimulus for fibroblast proliferation and collagen production. Second, fetal fibrob- lasts have the capacity to produce collagen in a more rapid and orderly manner than in adults.
Also, more collagen type III than type I is pro- duced in the fetus.124Because type III collagen fibers are smaller and finer than type I fibers, the higher proportion of type III fibers in the fetus may result in a more reticular pattern of fiber distribution. Furthermore, the highly hydro- scopic, hyaluronic acid-rich environment char- acteristic of the fetus may help proliferating cells avoid inhibitory signals.125 Third, reduced expression of TGF-β1 and increased expression of TGF-β3 reduce the amount of scar tissue for- mation.126Other growth factors, such as PDGF and FGF-2 also differ between the fetus and the adult.127Fourth, in the fetus, there is an altered balance between the MMPs and their inhibitors that favors rapid turnover of the ECM that is required for scarless healing.128Finally, there is now evidence that differences in gene expres- sion and cell signaling in the fetus may also be responsible for the reduced tendency for scarring.129
Tissue Engineering and Skin Biology
The genesis of tissue engineered products as skin substitutes began more than 20 years ago with improved culture methods for the growth keratinocytes, and the production of natural and synthetic matrices used either as delivery sys- tems for cells or cell products. The goal is the recapitulation of normal skin and normal skin function. Any epidermal replacement must restrict transepidermal water loss, limit bodily damage induced by environmental chemical and physical insults, and minimize microbial load. Any dermal replacement must provide epi- dermal support, neovascularization, and func- tional pliability.
Normal epidermis turns over once a month during which time cells from the outermost layer are sloughed. This necessitates a lifelong process of self-renewal fueled presumably by keratinocyte stem cells, which by definition are capable of both self-maintenance and of produc- tion of differentiated cells.130 We might there- fore view our normal physiologic state as one of
subclinical wound healing in which stem cells are accessed to support tissue maintenance. To obtain the requisite cell numbers while limiting the accumulation of replicative errors in the stem cell population, stem cells give rise to prog- eny, transit amplifying cells, that undergo rapid cell division and then terminally differenti- ate.131–137
Access to populations of keratinocytes that can be grown and expanded in vitro, and still serve as stem cells subsequent to transplant is a requirement for producing epidermal skin sub- stitutes. This is independent of whether the cul- tures are to be used as epidermal replacements, biologic dressings, or genetically modified epithelia that deliver therapeutically required factors. It has therefore been of interest to iso- late and identify stem cell populations. To date, the identity and genetic signature of epidermal stem cells have remained elusive. It is generally agreed that they have a high nucleus/cytoplas- mic ratio and can be enriched by density gradi- ent centrifugation or by their rapid adherence to a variety of ECMs.138–140 They have been reported to either express high levels of integrin β1140 or alternatively to express high levels of integrin α6 coupled with low levels of transfer- rin receptors.141 Among other markers, stem cells have been associated with localized keratin 19 expression,142,143reduced connexin 43 expres- sion with decreased gap junction function144 (see, however, Bjerknes et al.145), and, under cer- tain circumstances, associated with keratin 15 expression.146 The actual complexity of the genetic signature is undoubtedly greater, as has been indicated by the detection of clusters of genes correlating stem cell functions and their modulation by environmental cues.147The loca- tion of the stem cell population has also been a matter of contention. Putative stem cell popula- tions have been identified in the bulge of the pilosebaceous unit,148 in the region below the bulge,149both in the follicular and interfollicular epidermis,150 and in the eccrine duct.151,152 Whereas these differences may simply distin- guish differences in the endpoint assays or model systems used, they may reflect the inher- ent plasticity of the keratinocyte. In the context of wound healing, it is fortunate that follicular keratinocytes can be accessed for reepithelial- ization,153that interfollicular keratinocytes can give rise to follicular and sebaceous gland ele- ments,154–157and that transit amplifying cells can exhibit stem cell functions.137,156,157
Epidermal Substitutes: Delivering Autologous Epithelial Tissue
The extensive capacity for keratinocyte in vivo proliferation predicts that, with proper signals, the proliferative potential of keratinocytes might be accessed in vitro. The first report validating this prediction appeared in the mid-1970s. In this early work, Rheinwald and Green158 described a technique using serum-containing medium together with lethally irradiated 3T3 cells to serially cultivate human keratinocytes.
With continued growth, individual keratinocytes produce colonies that fuse (confluence) and form a multilayered epithelium, containing dif- ferentiated cell layers suprabasal to a prolifera- tive cell layer adherent to the surface of the culture dish; the irradiated 3T3 cells are dis- placed and removed during successive feedings.
The discovery that incubation of confluent ker- atinocyte cultures with Dispase II results in detachment of an intact epithelial tissue from the surface of the culture vessel159promoted the use of epithelial sheets for treatment of burn injury.160,161 By 1988, cultured epithelial auto- grafts (CEA) were being commercialized with indications for deep partial-thickness injury, full-thickness burn injury, and congenital nevi.
The long-term persistence of grafted CEA indicative of functional stem cell transfer has been demonstrated in two types of studies. The first followed patients who had received CEA comprising plantar-derived keratinocytes grafted onto nonpalmoplantar surfaces. The grafted areas were found to contain cells with plantar keratinocyte specific markers162,163; similar graft maintenance was later demonstrated using human xenografts on athymic mice.164 In the second type of study, telomere length was com- pared in keratinocytes isolated from engrafted CEA with those isolated from engrafted split- thickness skin grafts (STSG). It was found that the CEA contained cells with shortened telom- eres.165Although these observations are consis- tent with CEA persistence, some concern is raised as to the remaining lifespan of the engrafted CEA. However, whereas the telomere length in the CEA was typical of aged epidermal keratinocytes, this cell type has an extraordinary proliferative potential that extends beyond the requirements for lifelong tissue maintenance.
The clinical significance of these findings is not yet known.
Despite the demonstrated lifesaving potential of CEA and the relatively high percentage of engraftment (60%–65%) reported in a large multicenter and a large single center study,166,167 and although CEA may be the preferred thera- peutic option in certain clinical settings, three factors place this product as an adjunct therapy rather than as a replacement for split-thickness grafting. First, reports from the late 1980s and through the late 1990s document large varia- tions in the percentage engraftment (“take”).168 The variations were observed even in studies in which wound beds were prepared with de-epi- dermized dermis to promote engraftment and that excluded patients whose wounds contained excessive microbial loads.169–172Second, during the first 2 weeks posttransplant, the grafts are delicate and particularly sensitive to loss because of infection. Lastly, using the suggested 2-cm2 skin biopsies, the time between biopsy and graft production is 14–21 days, dependent on donor and surface area to be covered.
Although larger biopsies can be used to decrease the production time, the first cell expansion requires 7–10 days. Obviously, with early exci- sion, more rapid coverage is preferable. In an effort to speed CEA production, consideration has been given to the use of allogeneic-syn- geneic cultures.173However, there have not been multicenter trials to demonstrate safety and effi- cacy. CEA are currently marketed as Epicel™
(Genzyme Biosurgery) and can be used in con- junction with de-epidermized cadaveric skin, with Alloderm® and with Integra® (see below).
Epidermal Substitutes: Delivery of Autologous Epithelia Before Tissue Formation
To shorten the time between biopsy and trans- plantation of autologous epithelia, two addi- tional approaches have been evaluated. One utilizes keratinocytes grown on matrices that do not require enzymatic release from the culture surface to allow culture transplant before the keratinocyte confluence. Autologous ker- atinocytes that have been grown on perforated matrices of benzyl esterified hyaluronic acid, currently marketed as Laserskin™ (Fidia Advanced Biopolymers), have been used to heal diabetic foot ulcers,174and those grown on fibrin
glue have been used to successfully treat deep partial and full-thickness burn injuries.175–177 Histologically normal epidermis has been shown to develop in situ.
A second approach utilizes keratinocyte cell suspensions delivered directly to the wound bed. Keratinocytes delivered within a fibrin spray have been shown to promote wound clo- sure of deep partial and full-thickness burns, and of chronic wounds; the fibrin helps secure even placement of the keratinocytes.178–182 The requirement for fibrin is therefore dependent on the architecture of the wound.183,184 The preclinical investigations of these alternative approaches to delivering autologous ker- atinocytes have been reviewed elsewhere.185The keratinocyte spray technology is being commer- cialized for human use pending clinical trials and regulatory approvals (ReCell®, CellSpray®, CellSpray®XP; see also Grant et al.186
Epidermal Dressings: Use of Cultured Epithelial Allografts
Because keratinocytes can also serve as a source of growth factors and specific matrix molecules, cultured epithelial allografts (CEAllo) have been evaluated for their ability to promote the healing of donor sites, leg ulcers, and partial-thickness burn wounds. The preparation of grafts with allogeneic material avoids the delays inherent in autologous graft production, whereas serial cul- tivation attenuates the expression of the MHC class I and II antigens in keratinocytes.187,188 Typically, allogeneic keratinocytes are replaced rather than rejected.189–196 Using CEAllo, the time required for reepithelialization of donor sites was reduced 18%–52% in studies described for both pediatric and geriatric burns. Healing times for the control groups ranged from 9.2 to 15.3 days, whereas healing times in the treatment groups ranged from 6.2 to 8.4 days.197–201 Comparative evaluations between Op-site and CEAllo have alternatively demonstrated significant improvement with CEAllo or no difference.202,203
Similar to the early reports using autologous keratinocytes,204 results on the treatment of venous ulcers and diabetic leg ulcers with allo- geneic keratinocytes have shown promise.205 Complete reepithelialization has been noted in some patients,206,207 and most patients have
experienced reductions in wound size because of healing from the indolent edges.208–215Efficacy has been reported to be independent of whether CEAllo have been prepared immedi- ately before use or have been cryopre- served,210–212 frozen,213,214 or lyophilized.215 Freshly prepared and banked CEAllo have also shown promise for treatment of partial-thick- ness burn injury presumably by promoting reepithelialization from the wound edges and remaining epidermal appendages. Time to wound closure was reduced by 18%–60%.216–220 Despite these successes, CEAllo are not yet com- mercially available.
Dermal Substitutes: Acellular Dermal Matrices
Acellular dermal substitutes have been evalu- ated for their ability to jump start neodermis formation, limit wound contracture associated with deep dermal injuries, and to provide a matrix compatible with reepithelialization and neovascularization.221 Currently, there are two types of acellular materials available that fulfill at least some of these requirements. The first material, de-epidermized dermis, is structurally identical to dermis but requires cellular repopu- lation. Although a variety of methods are avail- able to produce the material, an “off-the shelf”
form, sold as Alloderm®, is available. The prod- uct is decellularized to limit antigenicity and subsequently preserved by freeze-drying. It can be used in conjunction with either STSG or CEA.
It is often meshed to allow release of exudates and development of granulation tissue within the interstices. De-epidermized dermis offers the advantage of being able to support engraft- ment of thin rather than thick STSG. This is particularly useful for repair of extensive full- thickness burn injuries that may require repeated harvesting of donor sites. In addition, the presence of an intact, organized basement membrane predicts better adhesion of CEA with rapid deposition of laminin 5 and hemidesmo- some formation.222,223Successful use of the com- mercial product has been reported for patients with burn injuries and with cutis aplasia.224–227 The second type of dermal substitute comprises dermal matrix molecules, but extensive remodel- ing is required to achieve a normal dermal struc- ture. The material, sold as Integra®, is composed
of collagen-chondroitin-6-sulfate covered by a nonporous protective silastic layer.228–231 The pore size of the matrix is designed to be suffi- cient to allow fibroblast and endothelial cell migration to occur, and for the product to be remodeled by invading host cells.232Neodermis is formed in about 3 weeks, at which time the silastic layer readily detaches. As with de-epi- dermized dermis, to achieve permanent wound closure, the treated area must be grafted with either a thin STSG or with CEA. In multicenter clinical trials with more than 200 burn patients, it was found that Integra® was both safe and effective, and that the neodermis formed within 2–3 weeks of application was capable of sup- porting the “take” of STSG.233,234
Dermal Substitutes: Fibroblast- Containing Matrices
Dermal matrices have also been generated that incorporate dermal fibroblasts as a source of cytokines, growth factors, and matrix molecules all of which can reciprocally modulate other skin cells and which can potentially augment tis- sue repair.235The results of clinical evaluations of products containing metabolically viable and nonviable fibroblasts are consistent with critical differences between the needs of acute wounds and chronic ulcers. Thus, distinct fibroblast- containing products are indicated for different wound types. For example, Transcyte® is indi- cated for the treatment of deep partial-thickness burns and excisional wounds, but not for venous ulcers. This product is a temporary dressing that contains human neonatal fibrob- lasts cultured on a bovine collagen-coated nylon mesh bonded to silicone. During production, the fibroblasts secrete new matrix molecules and cytokines; cell viability is lost during a subse- quent freezing process. It is assumed that fibroblast-derived factors promote the observed healing of partial-thickness burn injuries and excisional wounds.236–240As a temporary dress- ing, it differs from the acellular dermal products that become engrafted and are modified by host cells. A second product produced by the same manufacturers, Dermagraft®, contains viable fibroblasts on a bioabsorbable fiber. It is indi- cated for use on chronic ulcers, and has been found effective when used in combination with standard of care for diabetic wounds.241,242
Because chronic wounds may have aberrations in both the resident matrix and cellularity, fibroblast viability and continued cytokine pro- duction may be significant factors in clinical outcome.243,244
Composite Skin Substitutes and Skin Replacements
In 1983, Eugene Bell and colleagues245described construction of a human “living skin equiva- lent” (LSE) comprising a fibroblast-contracted collagen gel overlaid with keratinocytes.
Cultures require a number of different media formulations and placement of the construct at the air–liquid interface for final epithelial matu- ration. The system has been optimized for neonatal keratinocytes and fibroblasts and has been subject to multiple improvements.246–248 Although it resembles skin having dermal and epidermal components, it is allogeneic and is not used as a permanent wound covering but as a biologic dressing that promotes wound heal- ing.249To date, “LSE” has been used effectively as treatment for chronic ulcers250–253and is mar- keted under the name of Apligraf® (formerly called Grafskin®) for that purpose. A second allogeneic product, OrCel®, uses a collagen sponge with keratinocytes and fibroblasts seeded on opposite faces. Although initially used for the treatment of mitten formation in patients with epidermolysis bullosa, it is also used on donor sites.254
Use of autologous fibroblasts and ker- atinocytes together with capillary-forming endothelial cells has been proposed.255 Such changes could potentially transform the LSE from a biologic dressing into a skin substitute.
Other promising modifications include a new skin equivalent combining de-epidermized der- mis with a fibroblast-populated collagen matrix as a support for epidermal production.256
An autologous composite skin substitute, composed of a stratified epithelium produced on a fibroblast-containing matrix of collagen and glycosaminoglycan (GAG), was first described in the late 1980s. It has demonstrated sufficient “take” to help reduce requirements for harvesting of donor skin.257,258 Although it appears to provide an epidermal replacement, de novo dermal formation is required because the construct’s matrix is lost within 2 weeks of
