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Wound Healing
Ziv Peled, M.D.
University of Connecticut School of Medicine, Farmington, Connecticut, U.S.A.

Robert D. Galiano, M.D.

New York University School of Medicine, New York, New York, U.S.A.

Michael T. Longaker, M.D.

Stanford University School of Medicine, Stanford, California, U.S.A.

I. INTRODUCTION A. Wound healing is a complex biologic response that ultimately results in a mature scar. B. Several phases dene healing, namely, inammation, epithelialization, extracellular matrix synthesis and remodeling, and wound contraction. Although in the past these were considered distinct stages, a more accurate conceptualization of tissue repair recognizes that these are overlapping and present in some degree throughout most of the course of a healing wound. C. These processes require the coordinated integration of a variety of cellular activities, including phagocytosis, chemotaxis, mitogenesis, neovascularization, and the synthesis and degradation of collagen and other matrix molecules. D. This complex response to cutaneous injury eventually leads to the formation of a scar. This is in contrast to the regenerative repair seen in early gestation fetal skin repair. II. TYPES OF WOUND HEALING There are four general types of wound healing: primary, delayed primary, secondary, and the healing that occurs in partial-thickness wounds.
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A. Primary Healing Wounds are closed by reapproximation using suture or by some other mechanical means within hours of their creation.

B. Delayed Primary Healing 1. To allow normal host defenses to debride the area, contaminated or poorly delineated wounds are left open and unopposed to prevent infection. 2. By day 3 postinjury, local inammatory cell recruitment into the wound has occurred to destroy contaminating bacteria. Furthermore, granulation tissue composed of inammatory cells, extracellular matrix, and new capillaries begins to form. 3. Following a delay of several days, the wound edges are surgically approximated. Collagen metabolism is undisturbed and tensile strength develops as if closure had been immediate.

C. Secondary Healing 1. An open full-thickness wound is allowed to close by both wound contraction and epithelialization.

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2. The circumference of the wound contracts by yet incompletely understood mechanisms. However, myobroblasts are thought to play a pivotal role. Hypothesized to be derived from broblasts, myobroblasts contain a well-dened actin microlament system. These cells appear in the wound on approximately the third day after wounding and disappear as contraction is completed. 3. Current evidence suggests a direct correlation between the number of myobroblasts and the extent of wound contraction. D. Healing of Partial-Thickness Wounds 1. In this type of wound, only the epithelium and the supercial portion of the dermis is injured. Epithelial cells that remain within the dermal appendages, hair follicles, and sebaceous glands proliferate to close the wound. 2. Thus, epithelialization is the main process by which the exposed dermis is covered in partial thickness wounds, with minimal collagen deposition and wound contraction. III. PHASES OF WOUND HEALING A. Introduction Wound repair involves the regulation of a dened sequential cascade of overlapping processes and the coordinated completion of a variety of cellular activities, including phagocytosis, chemotaxis, mitogenesis, and the synthesis of extracellular matrix components. In order for successful healing to occur, these activities must occur in a carefully regulated and reproducible fashion that correlates with the appearance of dierent cell types present in the wound. Each phase of this complex response to injury is discussed below. B. Tissue Injury 1. Injury initiates a regulated sequence of events, including coagulation, inammation, cell replication, angiogenesis, epithelialization, and matrix synthesis and turnover. 2. Vascular injury leads to rapid constriction of aected vessels and activation of the coagulation cascade in order to limit blood loss. 3. Inammatory cells release vasoactive amines and other mediators, which contribute to vessel

permeability and the leak of plasma and proteins into the wound and permit eector cells to enter the environment. C. Coagulation 1. Platelets. Hemostasis, achieved by trapping platelets in the clot, eventually leads to coagulation. These trapped platelets are also essential for the normal inammatory response by releasing vasoactive amines. These include serotonin, from dense bodies, and cytokines, including platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-h), epidermal growth factor (EGF), and platelet factor IV, from their a granules. The released serotonin increases microvascular permeability, while the growth factors initiate the wound-healing cascade by attracting and activating macrophages, broblasts, endothelial cells, and by stimulating granulation tissue formation. 2. Fibrin is the end product of both the intrinsic and extrinsic coagulation pathways. Derived from factor I, also known as brinogen, brin is essential to early wound healing because it provides the matrix foundation into which cells can migrate. In addition, brin can also serve as a reservoir for peptide growth factors. This infrastructure consists of bronectin and traps platelets, bloodborne cells, and plasma proteins. Removal of the brin provisional matrix is known to impede wound repair. D. Early Inflammation 1. Complement. The inammatory phase of wound healing is characterized by the activation of complement and the initiation of the classical molecular cascade, which leads to inltration of polymorphonuclear cells (PMNs) into the wound milieu within 2448 hours. 2. PMNs. A number of chemical messengers released from the damaged tissue, platelets, and bacteria attract PMNs into the wound site. These biochemical agents include complement components such as C5a, formylmethionyl peptide products from bacteria, and TGF-h. Upon entering the wound environment, PMNs begin to adhere to the endothelial cells in the adjacent blood vessels by a process called margination. Thereafter, they begin to actively move through

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the vessel wall, a process known as diapedesis. The major function of PMNs is to remove bacteria and foreign debris from the wound. However, depletion of these cells is not thought to signicantly alter healing in normal wounds. 3. Epithelialization results in the formation of a barrier between the internal and external environments. Within hours of the injury, migration of epithelial cells across the wound (i.e., reepithelialization) begins. The cells at the leading edge begin to lose their basement membrane adhesion, atten, send out cytoplasmic projections, secrete proteases, and phagocytize a path for the impending keratinocyte migration. One to 2 days after injury, epithelial cells at the wound edges begin to proliferate behind the migrating epithelium.


poor brosis. Upon entering a wound through a blood vessel wall, monocytes dierentiate into wound macrophages that act as phagocytic cells. By 4872 hours postwounding, macrophages are the predominant cell type within the wound. In addition, macrophages are the primary producers of growth factors responsible for both the production and accumulation of the extracellular matrix (ECM) by broblasts and proliferation of smooth muscle and endothelial cells resulting in angiogenesis. Chemoattractants for macrophages include complement, clotting components, immunoglobulin G (IgG) fragments, collagen and elastin breakdown products, and cytokines, such as leukotriene B4, platelet factor IV, PDGF, and TGF-h. 2. Lymphocytes. Attracted by interleukin-1 (IL-1), IgG, and complement products, lymphocytes are the last cells to enter the wound during the inammatory phase (>72 h after wounding). Although the role of lymphocytes in wound healing has not been clearly dened, IL-1 is believed to have a role in the regulation of collagenase, implicating lymphocyte involvement in collagen and ECM remodeling. F. Proliferation 1. Fibroblasts. By day 7, broblasts are the predominant cell type in the wound. Stimulated by cytokines, they migrate through the ECM into the wound. Between 5 and 7 days postwounding, broblasts begin to synthesize collagen, which increases in a linear fashion for 23 weeks. 2. Collagens are the most abundant ECM in the human body. To date, at least 19 dierent types of collagen have been discovered.


Full-thickness woundsthe edge of the wound is the sole source of the migrating epithelium. As a result, these types of wounds heal more slowly and with more inammation; these delays are thought to result in a greater propensity for hypertrophic scar formation. Partial-thickness woundsthese include skin graft donor sites, which epithelialize more rapidly in a moist environment. The rate of epithelialization is slowed by dry eschars (scabs). In addition, as in all open wounds, as long as the dermis is not completely destroyed, epithelialization can occur through the migration of epithelial cells from the remaining dermal appendages, sweat glands, and hair follicles. An example of this type of healing is a seconddegree burn or partial-thickness skin graft donor site. Growth factorsthese are potent stimulators of epithelial mitogenesis and chemotaxis. Key players include EGF, basic broblast growth factor (bFGF), and keratinocyte growth factor (KGF).

E. Late Inflammation 1. Macrophages are the key regulatory cells present in the healing wound. When depleted of circulating monocytes and tissue macrophages, wounds in experimental animal models have been shown to heal with poor debridement, delayed broblast proliferation, inadequate angiogenesis, and

Collagen types include (a) type I collagen, the major structural component of bones, skin, and tendons; (b) type II collagen, found predominantly in cartilage; (c) type III collagen, found in association with type I collagen in varying ratios, depending on the type of tissue; (d) type IV collagen, found in the basement membrane; and (e) type V collagen, found in the cornea.  Upon injury, the exposed collagen elicits the chemotactic response of the woundhealing cells and promotes platelet aggregation. Eventually, the migrating broblasts synthesize and secrete types I and

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III collagen to form the foundation of the wound extracellular matrix.



Collagen metabolism begins with the synthesis of the procollagen a chains on membrane-bound ribosomes. Chromosomes 17 and 7 encode the a1 and a2 chains of type I collagen, respectively. Type I collagen consists of two a1 and one a2 chains. Through hydroxylation of proline and lysine amino acids, the a chains then interact to form a triple helical molecule, which is important for thermal stability. In addition, without the triple helix the collagen cannot be exported from the cell. Within the cell, cross-linking between the chains by means of disulde bonds also occurs. Procollagen is then packaged into secretory vesicles that move to the cell surface. At the cell membrane, procollagen is cleaved into collagen by procollagen peptidase, and subsequently the collagen is released extracellularly into the wound. The importance of collagen in wound healing is dramatically exemplied by scurvitic wounds, which heal poorly.

of their inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs), are increased. Thus, TGF-h promotes matrix accumulation. 2. Fibronectin is a matrix molecule found in the tissue stroma and the basal lamina that acts as a scaold for collagen deposition. Produced by broblasts, epithelial cells, and macrophages, bronectin is involved in wound contraction, cell-cell and cell-matrix interaction, cell migration, collagen matrix deposition, and epithelialization. Fibronectin has the ability to bind a wide variety of molecules involved in wound healing, including collagen types I and IV, actin, brin, hyaluronic acid, dermatan and heparan sulfates, bronectin itself, and broblast surface receptors. Among the rst proteins laid down in a fresh wound, the main function of bronectin is to promote cell-cell and cell-matrix interactions, cross-linking to brin clot and facilitating broblast attachment. As the wound matures, bronectin decreases and type I collagen replaces type III collagen. 3. Stroma (ground substance) is another vital component of the wound architecture, consisting of proteoglycans and glycosaminoglycans (GAGs). Although proteoglycans are known to create a charged, hydrated environment that facilitates cell mobility and provides viscoelastic properties of normal connective tissues, their role in wound healing remains unclear. However, proteoglycans are covalently linked to a special type of polysaccharide that may be important to wound matrix, namely, GAGs. There are four known types of GAGs involved in wound healing: chondroitin sulfate, heparan sulfate, keratan sulfate, and hyaluronic acid (HA). Unlike other GAGs, HA is a repeating sequence of nonsulfated disaccharides without a protein core and appears earlier in the wound than the other GAGs, all of which are sulfated and have protein cores. These proteoglycans have been shown to bind and in some cases present extracellular growth factors to their cell surface receptors. IV. GROWTH FACTORS IN WOUND HEALING Growth factors (GFs) are proteins that aect a myriad of cellular processes, including migration, proliferation, dierentiation, and ECM production. In addition, they are responsible for modulating the inammatory re-

3. Angiogenesis. Ongoing throughout the previously mentioned phases, angiogenesis describes the process of forming new blood vessels.


Platelets. Upon entering the wound, platelets release numerous cytokines, including TGF-h and PDGF, that promote angiogenesis by attracting macrophages and PMNs. Macrophages, in turn, release a number of angiogenic substances including vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha (TNF-a), and bFGF.

G. Remodeling By approximately 21 days, equilibration of collagen synthesis and breakdown is reached. More specically, a steady state of collagen synthesis and breakdown is achieved as the ECM is continually remodeled. The increase in tensile strength of a skin wound plateaus at around 67 weeks. 1. Matrix Metalloproteinases (MMPs) are produced by broblasts, granulocytes, and macrophages. TGF-h is thought to decrease the activity of matrix metalloproteinases, while the activity

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sponse. Weighing between 4,000 and 60,000 daltons, GFs exert their eects via membrane receptors, which in turn initiate a cascade of intracellular activity. Growth factors exert their eects through endocrine, paracrine, autocrine, and intracrine mechanisms. Endocrine GFs are produced by a specic cell type and, thereafter, transported via the bloodstream to act on a cell at a distant site. Paracrine GFs are produced by a specic cell type and act on an adjacent cell. Autocrine GFs exert their eects on the same cell that produced them. Finally, intracrine GFs act within the cell that produced them. The majority of the GFs involved in the woundhealing process act via the paracrine or autocrine mechanisms. Growth factors were initially named for their rst known actions or the rst cells known to produce them. With further study, scientists have discovered that many dierent cell types may produce any one GF, and virtually all growth factors are known to have more than one function. Thus, the names of various growth factors can often be misleading. The following is a brief discussion of some of the more common growth factors known to be involved in the adult wound-healing process. To date, although many growth factors have begun clinical trials, only one, PDGF, has received FDA approval for the treatment of chronic wounds. A. Epidermal Growth Factor EGF is a 53-amino-acid polypeptide that is released from platelets during platelet degranulation. EGF is mitogenic and chemotactic for several cell types, including epithelial cells, endothelial cells, and broblasts, all of which have receptors for this molecule. In addition, EGF stimulates angiogenesis and collagenase activity at the wound site. B. Fibroblast Growth Factor FGFs are a large family of GFs. FGF-2, in particular, is a well-established angiogenic factor and mitogen for broblasts. FGF-2 is produced primarily by macrophages and endothelial cells. FGF-2 also stimulates collagen and ECM synthesis and promotes wound contraction and epithelialization. C. Keratinocyte Growth Factor KGF, also known as FGF-7, is a protein produced by broblasts early in the course of tissue repair. Epithelial cells, such as keratinocytes, are the only known cells with receptors for KGF. As such, this molecule is believed to play an important role in mesenchymal-

epithelial signaling and reepithelialization during wound healing. D. Platelet-Derived Growth Factor PDGF is a 3032 kDA glycoprotein made up of two subunits called A and B. These two subunits form a heterodimer that is produced primarily by platelets and stored in the a granules. Other cells such as macrophages and epidermal cells release similar PDGF-like substances. PDGF is known to modulate numerous functions such as broblast and smooth muscle cell proliferation/chemoattraction, activation/chemoattraction of macrophages, and stimulation of neutrophils. It is also thought that PDGF participates in the process of neovascularization, but this function may be secondary to its eects on the other cells involved in the wound-healing process. E. Transforming Growth Factor-B TGF-B exists as a homodimer with a molecular weight of 25,000 kDA and has been isolated from nearly all types of tissues. TGF-B is one of the most well-studied growth factors. Consisting of three dierent isoforms (B1, B2, B3) in humans, TGF-B is chemotactic for macrophages and broblasts and is involved in extracellular matrix synthesis and remodeling. Produced initially by platelets (and stored within the A granules) and macrophages, TGF-B acts via the autocrine mechanism on monocytes to stimulate the secretion of other growth factors such as FGF, PDGF, TNF-A, and IL-1. Furthermore, TGF-B is perhaps the most potent stimulator of collagen and extracellular matrix synthesis and has, therefore, been implicated in scar formation. Several experimental studies have successfully employed strategies to attenuate the eects of TGF-B (i.e., antibodies to TGF-B1 and TGFB2) and have demonstrated reduced scar formation as a result. Not all members of the TGF-B family have detrimental eects. Actually, TGF-B1 has been shown, like vitamin A, to reverse the inhibition of wound healing caused by glucocorticoids in an animal incisional model. Also, recent studies suggest that TGF-B3 may have antiscarring activity, in contrast to TGF-B1 and TGFB2. This is, however, controversial. F. Transforming Growth Factor-A A TGF-A is a molecule that is produced by macrophages, platelets, keratinocytes, and other epidermal cells. Similar to EGF, it binds the EGF receptor and acts in an autocrine manner to exert its eects. It is chemotactic and mitogenic for epithelial, endothelial, and mesen-

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chymal cells. TGF-A may also be mitogenic for broblasts in vivo. G. Interleukin-1 Initially described as a mitogen for lymphocytes, IL-1 is produced by macrophages and neutrophils and is now known to be chemotactic for epithelial cells, monocytes, neutrophils, as well as lymphocytes. IL-1 is thought to play an important role in the processes of wound repair and remodeling, based on its mitogenic eects on broblasts and its ability to synthesize collagen, to promote collagenase and to promote hyaluronidase activity. H. Tumor Necrosis Factor-A A TNF-A is a 157-amino-acid molecule produced by macrophages and neutrophils. Its primary functions are to stimulate broblast proliferation and to promote collagen and collagenase synthesis. I. Vascular Endothelial Growth Factor A family of at least three dierent isomers (VEGF-A, -B, and -C), the VEGFs act on dened tyrosine kinase receptors on endothelial cells and are extremely important in angiogenesis. They have been used in ischemic ap models to stimulate neovascularization and in tissue engineering research to improve tissue vascularity. J. Insulin-like Growth Factors IGF-I is the main member of this family involved in wound healing. It is released by many cells involved in wound healing, including broblasts and macrophages. Its actions are modulated by so-called IGF-binding proteins found in the extracellular matrix. They are potent anabolic agents that have also been demonstrated to enhance cell migration and proliferation. V. CHRONIC WOUNDS Chronic wounds are one of the most costly medical problems encountered in the United States. Among the most common types of chronic wounds are pressure ulcers, venous stasis ulcers, and diabetic foot ulcers. In 1997, 6.5 million people in the United States suered from one of these chronic conditions. The estimated annual cost of treating these wounds was in excess of $3.3 billion dollars and continues to rise. The key to wound healing is the balance between tissue formation and tissue degradation. In successful wound healing, the timing and character of ECM deposition and turnover as well as protein synthesis and proteolysis is well

synchronized and specic. In chronic wounds, the normal sequence of repair or the imbalance between tissue production and turnover is disrupted, resulting in either delayed or absent repair. The microenvironment created by chronic wounds is often hostile to the repair process, being decient in some growth factors. In support of this, wound uid derived from acute wounds has been shown to stimulate proliferation of broblasts and keratinocytes, whereas wound uid derived from chronic wounds decreases proliferation of these same cell types. In addition, most chronic wounds also are characterized by varying degrees of infection, host malnutrition or a catabolic state, and local tissue hypoxia. A. Matrix Metalloproteinases MMPs are a family of zinc-dependent endopeptidases that include gelatinases, collagenases, and stromelysins. During wound healing, MMPs modulate much of the ECM turnover. Thus, an imbalance between matrix deposition and degradation in favor of tissue turnover has been postulated as one possible mechanism for the delayed healing seen in chronic wounds. MMP-2 and MMP-9 have been described in chronic wound uid but are noticeably absent from acute wound uid. In addition, tissue inhibitor of MMP (TIMP-1) has been found to be decreased in chronic wounds, while active gelatinases have been found in increased amounts when compared with acute wounds. B. Cytokine Function Even if certain growth factors are present in chronic wound uid, they may not function normally. Recent data about chronic wound uid suggest that peptide growth factors may not function optimally due to the presence of excess proteolysis; this may be due to bacterial enzymes in contaminated wounds that degrade peptides, including growth factors. C. Hypoxia While tissue hypoxia is present in all wounds to some degree or another, the persistent lack of oxygen and nutrients to a wound will serve as a major impediment to successful healing. For example, it has been shown experimentally that cells exposed to a hypoxic environment respond by proliferating, by migrating more rapidly, and by synthesizing growth factors such as VEGF. In this way, the cells are directing their own repair and recruiting endothelial cells to reverse their hypoxic environment. However, cells exposed to chronic, longstanding hypoxia (as seen in most chronic wounds) stop

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proliferating and may even enter apoptosis. It is axiomatic in surgery that a wound needs a reliable blood supply to heal; as clinicians, this is one of the variables that plastic surgeons can aect. By decreasing local tissue edema (which increases the diusion distance of oxygen and nutrients from capillaries to wound cells) and enhancing local blood ow (which may mean something as simple as warming the patient or as drastic as a free ap or bypass to restore vascular ow), the delivery of oxygen to a healing wound should be a goal to be reached when dealing with any recalcitrant wound. VI. FETAL WOUND HEALING Over the past decade, our experience with fetal surgery has led to some remarkable advances in the treatment of previously fatal conditions. Observations from these procedures indicate that the human fetus is capable of healing without a scar. This discovery has led to intense research, with the ultimate goal of repairing adult wounds without scars. The fundamental question in elucidating this phenomenon is: Are the dierences between scarless fetal wound repair and scarring adult-type wound repair a function of the fetal environment (extrinsic dierences) or are they a unique function of the fetal cells themselves (intrinsic dierences)? A. Extrinsic Differences 1. Amniotic uidThe fetus is continuously bathed in warm amniotic uid that is rich in growth factors and ECM components such as hyaluronic acid (HA) and bronectin. 2. SterilityThe amniotic uid and entire fetal environment are sterile. B. Intrinsic Differences 1. HypoxemiaThe fetus is profoundly hypoxemic compared with the adult. Although high tissue oxygenation is necessary for wound healing in the adult, the relative hypoxemia in fetal tissues may promote greater production of certain growth factors and for matrix molecules, resulting in a reduction or elimination of scar formation.

2. Immune responseMajor dierences between fetal and adult acute inammatory responses include the degree of the response, the character of the response, and the function of inammatory cells, once recruited into the wound. Histologically, there are few granulocytes in fetal wounds. For example, it has been noted that fetal lamb wounds showed less acute inammatory response with a large number of macrophages, while having relatively few neutrophils. This is contrast to a large number of neutrophils acutely seen in adult wounds. Furthermore, the amounts and types of cytokines and GFs produced by the various inammatory cells during fetal wound healing may dier markedly from those seen during adult wound repair. These dierences would certainly inuence the phenotypic outcome of the wound repair mechanism. 3. ECMThe ECM of fetal wounds is rich in HA, an important structural and functional component of wound matrix whenever rapid tissue proliferation occurs. Although present early in both fetal and adult wound healing, HA deposition is sustained in fetal wound matrix, which may provide the local environment that promotes regeneration rather than scar formation. In the adult, subsequent to the initial deposition of HA, hyaluronidase is produced to degrade the HA, and the wound matrix is replaced by collagen. As alluded to, this process of early HA degradation may not occur in the fetus. In addition, there is an enhanced production of bronectin and deposition of highly organized collagen architecture in the dermis of fetal cutaneous wounds.

C. Growth Factors The exact role of growth factors in scarless fetal wound healing remains unknown. Nevertheless, given the prominent role that growth factors play in overall fetal development, it is highly suggestive that these cytokines may also play an important role in scarless fetal wound healing.

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