GENERAL PRINCIPLES OF WOUND HEALING

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WOUND HEALING

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GENERAL PRINCIPLES OF WOUND HEALING Maria B. Witte, MD, and Adrian Barbul, MD, FACS

Injury triggers an organized and complex cascade of cellular and biochemical events that result in a healed wound. For didactic purposes, the wound healing response can be divided into three distinct but overlapping phases: (1)hemostasis and inflammation, (2) proliferation, and (3) maturation or remodeling.l16 Failure or prolongation in one phase may result in delay of healing or nonclosure of the wound. Wound healing failures remain a significant clinical problem with large impact on health care costs. A better grasp of the fundamental physiology of healing results in a clearer understanding of the pathophysiologic processes that impair healing. HEMOSTASIS AND INFLAMMATION

The inflammatory phase is an essential phase of healing, characterized by increased vascular permeability, chemotaxis of cells from the circulation into the wound milieu, local release of cytokines and growth factors, and activation of migrating cells. Hemostasis precedes inflammation. The accompanying obligatory rupture of vessels exposes the subendothelial collagen to platelets and results in aggregation of platelets and activation of the intrinsic part of the coagulation cascade. The contact between collagen and platelets, as This article was, supported 1499/ 1-1).

grant from the Deutsche Forschungsgemeinschaft (Wi

From the Department of Surgery, Sinai Hospital of Baltimore, The Johns Hopkins Medical Institutions, Baltimore, Maryland

SURGICAL CLINICS OF NORTH AMERICA VOLUME 77 * NUMBER 3 * JUNE 1997

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well as the presence of thrombin, fibronectin, and their fragments, results in the release of cytokines and growth factors from platelet a-granules such as platelet-derived growth factor (PDGF), transforming growth factor-@(TGF-P),platelet-activating factor (PAF), fibronectin, and serotonin136 (Table 1).The locally formed fibrin clot serves as a scaffolding for invading cells such as neutrophils, monocytes, fibroblasts, and endothelial cells.83Inadequate clot formation, such as that observed in factor XI11 (the fibrin-stabilizing factor) deficiency, is associated with impaired wound healing,15 secondary to either decreased adhesion of cells into 67 the inflammatory area or decreased chem~taxis.~~, Chemotaxis

Neutrophils are the first wave of migrating cells into the wound. Increased vascular permeability due to inflammation and release of prostaglandins together with a concentration gradient of chemotactic substances such as complement factors, interleukin-1, tumor necrosis factor-a (TNF-a),TGF-P, platelet factor 4, and bacterial products19,60, lo2,129 stimulate neutrophil migration. Selectins,3 receptors on the endothelial cell surface, preferentially help neutrophils to adhere to the endothelium, whereas integrin receptors on neutrophil cell surfaces facilitate binding to the extracellular Table 1. HEMOSTATIC AND PLATELET-DERIVED FACTORS ASSOCIATED WITH WOUND HEALING Function Hemostatic factors Fibrin, plasma fibronectin Factor Xlll (fibrin-stabilizing factor) Circulatory growth factors Complement Platelet-derived factors Cytokines, growth factors Fibronectin Platelet-activating factor (PAF) Thromboxane A, Platelet factor IV Serotonin Adenosine dinucleotide

Coagulation, chemoattraction, adhesion, scaffolding for cell migration Induces chemoattraction and adhesion Regulation of chemoattraction, mitogenesis, fibroplasia Antimicrobial activity, chemoattraction Regulation of chemoattraction, mitogenesis, fibroplasia Early matrix, ligand for platelet aggregation Platelet aggregation Vasoconstriction, platelet aggregation, chemotaxis Chemotactic for fibroblasts and monocytes, neutralizes activity of heparin, inhibits collagenase Induces vascular permeability, chemoattractant for neutrophils Stimulates cell proliferation and migration, induces platelet aggregation

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Table 2. THE EFFECT OF GROWTH FACTORS ON FIBROBLAST PROLIFERATION Factor Platelet-derived growth factor Interferon-? Transforming growth factor-p Fibroblast growth factor Epidermal growth factor Interleukin-1 Tumor necrosis factor-a

PDGF

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Concentration

Effect

ng range max at 5-10 ng/mL 1-100 U/mL 1-1000 U/mL 0.1-1 ng/mL 1-10 ng/rnL 0.1-1 ng/mL

t t 1 t 1 + t t t

max at 20 ng/mL

0-1.2 nM Low High

1 -+ t 1

Mechanism

References

20, 68, 81, 96 29 Lengthening of 104 GdG, and G2 80 Via release of 104 PDGF12’ 104 53, 142 81, 128, 138

40, 62, 81, 84, 96 Via release of PDGF Via increase of PDGF

48, 78, 108 80 126 1, 14, 101 101, 128

platelet-derivedgrowth factor.

m a t r i ~The . ~ interplay of these two cell receptors is therefore critical to the margination of cells. The response of cells to a chemotactic signal is mediated also by cell surface receptors. These ensure a certain selectivity between stimulus and response because only cells expressing the specific receptor respond. For instance, PDGF is a very strong chemoattractant for fibroblasts and smooth muscle cells, but the chemotaxis of endothelial cells, epithelial cells, and leukocytes is not affected.68 Cytokines and growth factors often have more than one specific effect on cells. Several factors, for example, stimulate both chemotaxis and proliferation in a concentration-dependent fashion. TGF-P, for instance, is chemoattractive to monocytes in the femtomolar range, whereas the concentration necessary to increase collagen synthesis in 141 The same is true for PDGF, fibroblasts lies in the nanomolar range.136, which is chemotactic to fibroblasts at a 100-fold lower on cent ration'^^ than the gradient necessary to stimulate their proliferation (Table 2). Cell Activation

Chemotaxis of cells into the wound milieu is followed by functional activation. Cellular activation implies the phenotypic altering of cellular, biochemical, and functional properties induced by local mediators. Activation may induce new cell surface antigen expression, increased cytotoxicity, increased production and release of cytokines, and other phenotypic alterations. All cells participating successfully in wound healing must be activated. Neutrophils, macrophages, and lymphocytes predominate during

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inflammation (Fig. l),but the contribution of each cell population to the successful outcome of wound healing is variable. Macrophages and lymphocytes exert critical roles,'2*86 but neutrophils are not essential,'" provided that no bacterial contamination is present, because their role in phagocytosis and antimicrobial defense may be taken over by macrophages. Activation of macrophages has fundamental implications in several aspects of wound healing, such as dkbridement, matrix synthesis, and angiogenesis (Fig. 2). The initial and brief release of factors from platelets is a first and strong stimulus of macrophage activation. The phagocytosis of cellular debris such as fibronectin or collagen also contributes to their activation.16 In fetal healing, for instance, which is characterized by minimal inflammation and scarless healing, otherwise normal fetal platelets have been shown to release fewer cytokines than adult platelets and hence cause less activation of macrophages and less inflammati~n.~~ Activation of macrophages leads to release of cytokines, which mediate angiogenesis and fibr0plasia.6~.Io3 The importance of macrophage regulation of these effects has been shown in studies in which the number of macrophages was either reduced or enhanced, a finding that proves that macrophages are critically needed in wound healing.23,28, 86 Activation of wound macrophages also results in the synthesis of nitric oxide, which has many functions, including antimicrobial properties.24,90 Albina et a16showed that macrophages are activated during the early phase of healing to synthesize nitric oxide and that the hypoxic Maturation Proliferation

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Figure 1. The time course of the different cells appearing in the wound during the healing process. Macrophages and neutrophils are predominant during inflammation, whereas lymphocytes peak somewhat later and fibroblasts are predominant during the proliferative phase.

GENERAL PRINCIPLES OF WOUND HEALING

Phagocytosis, antimicrobial function

Angiogenesis

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Matrix synthesis regulation

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-Growth factors TGF-P, EGF, PDGF

Cytokines TNF-a, IL-1, IFN-y

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1 + -Growth factors b FGF, VEGF Cytokines TNF-a

-Growth factors

PDGF, TGF-P, EGF, IGF

Cytokines TNF-a, IL-I, IL-6

*Fibronectin

collagenase, arginase

*Prostaglandins PGE2

Figure 2. The role of the macrophages in wound healing. The main functions are phagocytosis, cellular recruitment and activation, angiogenesis, regulation of matrix synthesis, and wound debridement. Effector mechanisms with examples are given in the boxes.

wound environment further enhances its expression. Many other cells participating in wound healing, including endothelial cells,''* fibrob l a s t ~ , 'monocytes,92 ~~ and 1ymphocytes,la can be activated in vitro to produce nitric oxide. Recent studies demonstrated that nitric oxide synthesis is reduced in impaired wound healing modelsz6;conversely, in vivo inhibition of nitric oxide synthesis in mice impairs wound healing.Il4These data suggest that nitric oxide plays more than an antimicrobial role during healing. Activated macrophages can activate other cells such as lymphocytes via cytokines. The lymphocytes in turn release lymphokines such as interferons (IFN) and interleukins (IL).**,136 Interestingly, released IFN-y acts back on macrophages and monocytes to induce the release of other cytokines such as TNF-a and IL-l.136This is an example of a paracrine mechanism, which ensures a prolonged presence of cytokines in the wound milieu and illustrates the complexity of interactions between cells during healing. As mentioned earlier, activation of cells during wound healing also means profound phenotypic change of certain cell populations. The fibroblast has been the most-investigated cell type in this 74, 122 but epidermal cells also undergo phenotypic changes.31Fibroblasts derived from the wound are characterized by increased collagen synthe-

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sis and contraction but decreased proliferation compared with normal dermal fibroblasts11o;hence, they are referred to as “wound fibroblast.” The trigger for phenotypic alteration of fibroblasts originates mainly lz4 This has been well shown for from macrophage-derived ~ytokines.~~, the myofibroblastic phenotype, which is strongly induced by TGFpl.37,124 The matrix surrounding the cells also influences their phenotype. For example, cell adhesion promoted by synthesis of the extracellular matrix molecule fibronectin can result in phenotypic alteration.=,39 Reduced inflammatory responses profoundly affect subsequent healing, as demonstrated clinically and experimentally in diabetesm, and steroid treatment.86In diabetes, the reduced activation of inflammatory cells coupled with diminished chemotaxis results in less efficient killing of bacteria with more subsequent infections and reduced collagen deposition. The reduced inflammation induced by steroids affects cell migration, proliferation, and angiogenesis. It can be partially reversed by vitamin A administration.” PROLIFERATIVE PHASE

Fibroblasts and endothelial cells are the primary cells proliferating during this phase. Fibroblasts migrate into the wound site from the surrounding tissue. Endothelial cells proliferate from intact venules close to the wound and form new capillaries by the process of angiogenesis. The growth factors and cytokines responsible for the proliferation of these two cell types derive mainly from platelets and activated macrophages. Some of them are stored in the fibrin clot, which is invaded by the cells. Mesenchymal cells themselves can be induced to release growth factors and cytokines in an autocrine manner. Fibroblasts in the surrounding tissue need to become activated from their quiescent state, in which they are nonreplicative. Many of the growth factors mentioned earlier, such as PDGF and EGF, induce chemotaxis and proliferation of fibroblasts and are also strong stimulators of their replication (Table 2). Some of the reported proliferative effects of cytokines are contradictory, indicating that the observed effects depend greatly on assay conditions and on the cell lines used. Direct translation of in vitro results to in vivo conditions is therefore rendered difficult. In excisional wound healing, the role of epithelial cell proliferation in re-establishing a barrier against fluid losses and infections should not be underestimated. Epithelial cells start proliferating a few days after wounding from wound edges or uninjured epithelial islands within the wound. Clinically, mesh-grafting corresponds with implanting ”healthy” epithelial islands on defective epidermal wounds and thus accelerating re-epithelialization and wound closure. The stimuli for epithelial cell proliferation are not yet fully but macrophages as well as the cells themselves8 are a source of cytokines and growth factors acting in an autocrine and paracrine mechanism.

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Although much interest has been focused on signals that turn on the proliferative phase, little is known about the signals that bring this phase to a controlled end. Stimuli for the activation of cells do not have to be long lasting,79but the induced phenotypic alterations are often 135 Negative feedback mechanisms not maintained in vitro over probably play a role in this context as well. The destiny of some cells after they complete their function in the wound-healing cascade remains unclear. Neutrophils undergo apoptosis and are ingested by macrophages. Macrophages themselves have been suspected to share the same destiny while influencing the wound metabRecently, in a model of peritoneal olism through the release of argina~e.~ inflammation, elicited macrophages have been shown to drain via local lymph nodes and not to undergo apoptosis, thus influencing antigen pre~entati0n.l~ MATURATION AND REMODELING PHASE

The main feature of the maturation phase is the deposition of collagen in the wound. From a clinical viewpoint this is the most important phase of healing because the rate, quality, and total amount of matrix deposition determine the strength of the scar. Many healing deficiencies become clinically manifest secondary to poor collagen deposition, although the underlying cause may vary. The poor matrix deposition in diabetes, for instance, is in part due to reduced inflammation. On the other hand, excessive collagen synthesis such as in hypertrophic scar or keloid remains a clinical problem with few therapeutic choices.77.85.120 The Deposition of Matrix in the Wound

Changes in wound matrix composition follow a certain pattern over time: Initially it is composed mainly of fibrin and fibronectin originating from hemostasis and macrophagess3;another early expressed protein is thrombospondin 1, which also supports cellular recruitment in the wound milieu.lwGlycosaminoglycans,proteoglycans, and other proteins like SPARC (secreted protein acidic rich in cysteine) are synthesized next, and they support future matrix deposition and remodeling.'**l8, lo9 Subsequently, collagens become the predominant scar protein (Fig. 3). The intact dermis is composed predominantly of collagen I (80% to goo/,) and I11 (loo/, to 20%). In granulation tissue type I11 collagen is increased (30%), whereas in the mature scar type I11 collagen is rather low (lo%).'", 47, 95 The temporary presence of collagen type VI has also been dem~nstrated.~ The early appearance of collagen type I11 coincides with the appearance of fibronectin,@j,83 and it has been proposed that the coating of denatured collagen with fibronectin facilitates its phagocytosis.66However, the role of the early deposition of collagen type 111,32,61

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Maturation

Proliferation

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Collagen I

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Days Postwounding Figure 3. The deposition of wound matrix components over time. Although fibronectin and collagen type Ill constitute the early matrix, collagen type I accumulates later, corresponding to the increase in wound-breaking strength. Data compiled from a review of the literat~re.30, 32.43.61.87

which does not significantly contribute to the strength of the wound, remains unclear. How long does the wound display an increased collagen synthetic rate after wounding? Net collagen synthesis is increased for at least 4 to 5 weeks after ~ounding.'~, 42, 89, *I5 Because fibroblasts are the main collagen-synthesizing cells, it is noteworthy that the increased rate of collagen synthesis during healing is due not only to an increased number of cells but also to a net increase of collagen production per cell. The structure of the matrix changes with time as well. Normal dermis shows a basket weave-like pattern, whereas in the scar the thinner collagen fibers are arranged parallel to the skin. These thinner collagen fibers gradually thicken after wounding and organize along the stress line of the wound. This change is accompanied by increased scar tensile strength, indicating a positive correlation between fiber thickness and orientation with tensile strength.43Biochemically, the collagen derived from granulation tissue is different from collagen derived from nonwounded skin, with greater hydroxylation and glycosylation of lysine residues. Greater glycosylation correlates with thinner fiber diameter.55But its role remains unclear. Despite a long, ongoing remodeling phase (up to 1 year), the collagen fibers in the healed scar tissue never become as organized as in the intact dermis. As a corollary, scar breaking strength never equals the strength of the skin. A time line of breaking strength shows that after 1 week the wound has only 3%47and after 3 weeks 20% of its final strength.'* After 3 months it shows approximately

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80% of the strength of unwounded skin but no further increase thereafter.87 Collagen Metabolism

The synthesis of the 19 known collagens occurs as for any other protein within the cell. The collagen molecule is characterized by the repeating sequence Gly-X-Y, with X often being proline and Y often being hydroxyproline. The molecule undergoes eight post-translational steps until its secretion as procollagen. Those steps are (1) cleavage of the signal peptides, (2) hydroxylation of the proline or lysine amino acids in the X-position to 4-hydroxyproline or 4-hydroxylysine, (3) hydroxylation of some proline residues to 3-hydroxyproline, (4) glycosylation of some hydroxylysine molecules with galactose or glucose, (5) addition of oligosaccharides to the propeptides, (6) association of the Cterminal propeptides, (7) formation of interchain and intrachain disulfide bonds, and (8) formation of the triple helix, which starts at the Cterminal end and goes to the N-terminal end.lo6 After post-translational modifications are complete, the triple helix is secreted as procollagen into the extracellular environment, where the propeptide ends are specifically cleaved by procollagen-C-proteinases and procollagen-N-proteinases. This cleaving process is directly responsible for the decrease in the solubility of the molecule. Then the process of fibril formation begins. The cross-linking of fibrils occurs after several lysine and hydroxylysine residues have their free amino acid group transformed to aldehyde residues by the enzyme lysyl oxidase. Crosslinking occurs between these aldehyde groups and amino acid groups of the nontransformed lysine or hydroxylysine residues.55 The group of Ehlers-Danlos syndromes is a clinical example of impaired wound healing due to defects in these post-translational 132 On the other hand, inhibition of these specific postmodifications.100, translational enzymes or of lysyl oxidase could be one future therapeutic option for fibrotic diseases such as hypertrophic scar and k e l o i d ~ . ~ ~ Collagen breakdown during healing begins early and is very active during inflammation. Sources of collagenase in the wound are the inflammatory cells137and endothelial cells7Ias well as the fibroblastsz5and keratinocyte~.~~ Collagens are almost exclusively digested extracellularly by the specific collagenases. These specific enzymes are able to degrade the normally very stable triple helical structure of the collagen at specific sites, rendering the molecule more susceptible to degradation by other pro tease^.^^, 97 The activity of collagenases is tightly controlled by cytokines. Many cytokines actually exert their effect on matrix metabolism in the wound not only by inducing new gene transcription but also by decreasing collagenase activity (for example TGF-P,). Based on in vitro work, it has been suggested that collagenase is also regulated by the organization of the cytoskeleton of the cell134and by the extracellular matrix.130,139

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The Influence of the Matrix

Matrix accumulation in wound healing is the balance between new deposition and degradation. The cells themselves mainly regulate that balance. But more and more data demonstrate that an interaction also occurs between the matrix and the cells and thus point toward a role for the matrix in tissue repair59,75, Ifl7 (Fig. 4). For example, the clot initially contains large amounts of plasma fibronectin, which serves as a scaffolding for migrating cells. With the onset of cellular invasion, the clot is lysed and cells start synthesizing cellular fibronectin. Whereas fibronectin and its breakdown products are chemotactic and adhesive and induce proliferation, matrix consisting of incomplete fibronectin particles can induce collagenase activity in fibroblasts. Thus, the extracellular matrix can regulate its own turnover by influencing in situ cellular activity. These cell-matrix interactions take place partially through integrins, which are classes of transmembrane cell surface receptors composed of two subunits (01 and P).If17 Although little is known about the signal transduction pathway triggered by integrins, it is noteworthy that fibroblasts change their integrin receptor pattern during healing.3"During the initial phase, a pattern promoting cell migration dominates, and the later pattern favors cell attachment and matrix synthesis. alpl Integrins

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I/

Migration synthesis proliferation I \

\ \ \ \

--

'

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Matrix synthesis remodeling

Figure 4. The interaction between the fibroblasts as the main matrix-synthesizing cells and the matrix itself. Whereas the fibroblasts are activated through soluble products from platelets and macrophages, which leads to increased matrix synthesis, the matrix is regulating fibroblast parameters such as migration or proliferation and therefore influencing the outcome of healing.

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are expressed mainly by mesenchymal cells and mediate collagen-depen65, 117 all main features of dent adhesion, migration, and gel ~ontraction,3~, wound healing. alplIntegrin expression is therefore critical for matrix remodeling after injury. The expression of different integrin patterns is possibly regulated by cytokines present at the wound site. Candidates are TGF-P, PDGF, and TNF-a.30,49 The expression of a certain integrin receptor can code for very different function, depending on the cell type. The collagenase-secreting phenotype of keratinocytes, for example, is characterized by the a5P1 integrin expression, whereas in fibroblasts, blocking that integrin receptor induces collagenase expression during wound healing.39,113 This variability demonstrates once more the complexity of wound healing. WOUND CONTRACTION

Wound contraction is the approximation of the wound edges, and wound contracture is the shortening of the scar itself. Healing by primary or secondary intention determines the role of wound contraction in the healing process. Several theories have been proposed for the mechanisms of wound contraction.112One proposes that a special cell-the myofibroblast-is responsible for contraction, whereas another theory suggests that the locomotion of all fibroblasts leads to a reorganization of the matrix and therefore to contraction. The myofibroblast is different from the normal fibroblast in regard to its cytoskeletal structure. Typically this cell expresses a-smooth muscle actin in thick bundles called stress fibers.119This a-smooth muscle actin is nondetectable until day 6 and then is expressed progressively for the next 15 days of wound healing.36After 4 weeks this expression fades and the cell is believed to undergo a p o p t ~ s i s This . ~ ~ process transforms the cell-rich granulation tissue into a cell-poor scar tissue. a-Smooth muscle actin renders the myofibroblast competent to contract, in contrast to the normal dermal fibroblast. The summation of all contracting cells may therefore induce the wound edges to approach. Interestingly, the appearance of the myofibroblast does not correspond perfectly to the time course of wound contraction, which starts almost immediately after wounding and continues for the next 2 to 3 weeks.36 On the other hand, the facts that fibroblasts placed in a collagen lattice can contract it without expressing stress fibers and that these fibroblasts are actively moving in the lattice led Ehrlichg6to hypothesize that the movement of the cells with a concomitant reorganization of the cytoskeleton is responsible for contraction. In summary, little is known about the exact mechanisms of wound contraction, and further investigation is needed. ANALYSIS OF THE WOUND FLUID

The wound fluid is believed to reflect the wound environment at any time during the healing process. Therefore, wound fluid has been

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subjected to many investigations for analysis of growth substances, amino acid composition, and functional effects. It is noteworthy that wound fluid reflects the sum of all specific activities at the time of harvest. Nevertheless, very few comprehensive data are available about the composition of the wound fluid, partially because of different approaches to harvesting and analysis. Wound fluid harvested from polyvinyl alcohol sponges as well as Gore-Tex implants or Schilling chambers is well established as a model. Attempts to explain by means of wound fluid why some wounds heal and others do not are not yet 127, 131 conclu~ive.~~, The Presence of Cytokines in Wound Fluid

Many cytokines have been shown to be present in wound fluid (Table 3). However, detection of a cytokine does not always correlate with biologic activity. The detected cytokines may be inactive because they are degraded or because they are secreted in an inactive form (e.g., TGF-P). Similaritiesexist in the appearance after wounding of IL-1 and TNFa [also called cachectin], both proinflammatory cytokines. IL-lP has been detected in wound fluid early after wounding, with a gradual decrease thereafter; this corresponds to the presence of mRNA as well as protein in cells isolated from the wound.51IL-1 exists in two forms, a and P, which bind to the same receptor and provoke the same cellular response. IL-1 stimulates the proliferation of fibroblasts by increasing the production of PDGF in these cells (see Table 2). IL-1 also stimulates collagen as well as collagenase synthesis in fibroblasts, the net effect being an increase in matrix turnover. IL-1 acts also on endothelial cells to promote the synthesis of vasodilating factors such as prostaglandin E2 (PGE,), which may influence hemostasis and angiogenesis during repair. Macrophages and monocytes are the main source of IL-1, but endothelial cells and fibroblasts also contribute to its elaboration in the wound. TNF-a activity appears early during inflammation, with a peak at day 3 after wounding.54Protein levels of TNF-a decline quickly after wounding despite prolonged mRNA presence, suggesting a post-translational regulatory m e ~ h a n i s mMain . ~ ~ sources of TNF-a are macrophages Table 3. GROWTH FACTORS AND WOUND HEALING

Growth Factor

Biologic Effect

Platelet-derived growth factor Epidermal growth factor Transforming growth factor-a Fibroblast growth factor Transforming growth factor-p

Induces proliferation, chemotaxis, matrix synthesis Stimulates epithelialization, proliferation Induces angiogenesis, epithelialization Stimulates proliferation, angiogenesis Increases matrix synthesis, proliferation

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and monocytes. The effects of TNF-a on fibroblast collagen production are not uniform. Depending on the culture conditions, TNF-a has been shown to increase or decrease collagen synthesis in fibroblasts.@* 77 Collagenase activity is downregulated by TNF-a, whereas proliferation of fibroblasts and angiogenesis are both increased. It is noteworthy that TNF-a is responsible for the release of other cellular mediators that might exert antagonistic effects. For example, TNF-a increases the production of PGE, in many cell types; PGE, in turn modifies collagen, 52 collagenase, and proliferative activity.41, Highest levels of PDGF are found in wound fluid directly after wounding, with a rapid decline within days.45,91 The mitogenic and chemotactic activity of purified PDGF from wound fluid also decreases over time, indicating a decrease in potency of the growth factor.45In an excisional porcine wound model, the expression of PDGF protein and PDGF receptor mRNA in fibroblasts and epithelial cells corresponded to the stage of tissue repair: indicating that PDGF acts in an autocrine or paracrine manner on cellular functions such as proliferation, chemotaxis, and matrix synthesis. FGF is known to be a chemoattractant, a mitogen, an angiogen, and a stimulus for matrix synthesis for several cell types, including fibroblasts, endothelial cells, smooth muscle cells, and keratinocytes.21Studies showed beneficial effects of FGF application in normal and impaired wound models.5,22, 93, 94, 133 Platelets and mesenchymal cells are potential sources of FGF, which is not only induced after injury but also expressed constitutively in dermal tissue.63FGF has great affinity to extracellular matrix. Bound to it, FGF loses its ability to stimulate proliferation."' Nevertheless, the expression of FGF in granulation tissue is not fully elucidated.82 Epidermal growth factor (EGF), the first-discovered growth factor, is known to stimulate fibroblast replication by facilitating the cells going through the G, phase of the cell cycle. EGF also stimulates collagen formation and re-epithelialization. EGF shares several features with TGF-P, such as certain sequence homology and binding to the same receptor.70EGF has been demonstrated to be beneficial in a steroidimpaired wound

Functional Analysis of Wound Fluid Experiments looking at the function of wound fluid found that the addition of 10% wound fluid from early stages of healing (until day 10) increases the proliferation of fibroblasts and endothelial cells, whereas fluid from later stages (day 15) decreases the proliferation of cells.76,lo5,110 Fractional analysis showed that a molecular weight fraction larger than 300 kD is responsible for the stimulatory effect, whereas the inhibitory effect is found in a fraction smaller than 10 kD. Caldwell et alZ7analyzed extensively the amino acid composition of the wound fluid. They found

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that most of the amino acids from the early healing period are present in optimal concentrations for cell proliferation. Wound fluid stimulates collagen synthesis,'05, enhances wound contraction in an in vitro and induces angiogenesis independently of the endothelial cell proliferative stimulus.11 In human burn injuries, wound fluid contains metalloproteinase activity with a maximum at day 4, decreasing gradually thereafter.lM This corresponds to the activity of gelatinases extracted from fresh granulation tissue of excisional wounds.2On the other hand, the expression of collagenase in human keratinocytes in vivo is maximal at day 1, with a gradual decrease thereafter.73The metalloproteinase activity in the compartment of keratinocytes might be regulated differentially from the activity in the fibroblastic compartment, the former being responsible for the migration of keratinocytes and the latter for the degradation of injured matrix. SUMMARY

Wound healing is a complex process involving different biologic and immunologic systems. Despite improvements in diagnostics and therapy, wound failures remain a clinical problem. The approach to a nonhealed wound is an interdisciplinary challenge that should not be underestimated. Better understanding of the complex wound-healing cascade helps our approach to wound healing and its possible failure. Manipulations of the involved immunologic features offer future therapeutic strategies. References 1. Agganval BB, Totpal K, LaPushin R, et al: Diminished responsiveness of senescent normal human fibroblasts to TNF-dependent proliferation and interleukin production is not due to its effect on the receptors or on the activation of a nuclear factor NFkappa B. Exp Cell Res 218:381,1995 2. Agren M S Gelatinase activity during wound healing. Br J Dermatol 131:634, 1994 3. Alberts B, Bray D, Lewis J, et al: Cell in their social context: Cell junctions, cell adhesion and the extracellular matrix. In Molecular Biology of the Cell, ed 3. New York, Garland Publishing, 1994, p 950 4. Alberts B, Bray D, Lewis J, et al: Internal organization of the cell: Membrane structure. In Molecular Biology of the Cell, ed 3. New York, Garland Publishing, 1994, p 478 5. Albertson S, Hummel RP, Breeden M, et a1 PDGF and FGF reverse the healing impairment in protein-malnourished diabetic mice. Surgery 114:368, 1993 6 . Albina JE, Henry WL Jr, Mastrofrancesco 8, et a1 Macrophage activation by culture in an anoxic environment. J Immunol 155:4391,1995 7. Albina JE, Mills CD, Henry WL Jr, et al: Temporal expression of different pathways of L-arginine metabolism in healing wounds. J Immunol 144:3877, 1990 8. Ansel JC, Tiesman JP, Olerud JE, et al: Human keratinocytes are a major source of cutaneous platelet-derived growth factor. J Clin Invest 92:671, 1993 9. Antoniades HN, Galanopoulos T, Neville-Golden J, et al: Injury induces in vivo expression of platelet-derived growth factor (PDGF) and PDGF receptor mRNAs in

GENERAL PRINCIPLES OF WOUND HEALING

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