Wound Healing

Neil A. Fine
Thomas A. Mustoe
An understanding of wound healing is fundamental to all of surgery and involves a broad
range of cellular actions and interactions. Wound healing is a fundamental homeostatic
process in response to injury. It involves the activation of basic cellular processes of
inflammation, cell proliferation, and growth as well as regulation of these processes once
repair is complete. Increased understanding of the cellular and molecular events involving
growth factors and cytokines, and the realistic prospects for pharmacologic manipulation,
have focused a great deal of research interest from a broad range of disciplines. Although
much new knowledge has been gained, there remain many unanswered questions. This
chapter outlines a basic set of concepts regarding wound healing, with emphasis on the
clinical principles of basic surgical techniques and the care of surgical incisions and open,
acute, and chronic wounds.
NORMAL WOUND HEALING
Wound healing is the body's response to injury. Injury can be acute or chronic and involve
multiple tissues, but wound healing is most clearly illustrated by examining the response to
full-thickness injury (e.g., a cut or an incision) to the epidermis and dermis. This injury sets in
motion a sequence of interrelated reparative forces. Although the events overlap in time, it is
helpful to consider the process as stages or phases of wound healing; these are presented as
separate events. This provides for clear conceptualization of the individual events and
conforms to standard conventions. These events, however, do not occur independently, and
the degree of temporal overlap is significant (Fig. 4.1).1
Every tissue in the body undergoes reparative processes after injury. Bone has the unique
ability to heal without scar. Liver has the potential to regenerate parenchyma and is the only
organ that has maintained that ability in the adult human. Although liver does regenerate, it
often heals with scar (cirrhosis). With these exceptions, all other human tissues heal with
scar. This chapter reviews the healing process of human skin with particular emphasis on
surgical applications. Delineation of the individual mediators is still evolving, so the
emphasis is on the underlying physiologic processes and the patterns of response.
Inflammatory Phase
The inflammatory phase of acute wound healing begins immediately after injury. The initial
response to the disruption of blood vessels is bleeding. The homeostatic response to this is
clot formation to stop hemorrhage. Platelet plug formation initiates the hemostatic process
along with clotting factors activated by collagen and basement membrane proteins exposed
by the injury. Fibrin, converted from fibrinogen by the clotting cascade, binds the platelet
plug and forms the provisional matrix for the cellular responses that follows. After injury,
transient vasoconstriction is mediated by catecholamines, thromboxane, and prostaglandin
F2α (PGF2α). Platelet degranulation, the emptying of the granules into the extracellular space,
provides the contents of α granules and dense granules, most notably platelet-derived growth
factor (PDGF) and transforming growth factor β (TGF-β). These substances initiate
chemotaxis and proliferation of inflammatory cells, beginning
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the inflammatory response that will ultimately heal the wound (Table 4.1). Transient
vasoconstriction is necessary to decrease blood loss at the time of the initial wounding and
also to allow clot formation. Vasoconstriction lasts for 5 to 10 minutes. Once a clot has been
formed and active bleeding has stopped, vasodilation increases local blood flow to the
wounded area, supplying cells and substrate necessary for further wound repair. The vascular
endothelial cells also deform, increasing vascular permeability. The vasodilation and
increased endothelial permeability are mediated by histamine, prostaglandin E2 (PGE2), and
prostaglandin I2 (prostacyclin; PGI2) as well as vascular endothelial cell growth factor
(VEGF). These vasodilatory substances are released by injured endothelial cells and mast
cells and enhance the egress of cells and substrate into the wounded tissue (Fig. 4.2).

FIGURE 4.1 Time line of phases of wound healing with dominant cell types and major
physiologic events.
TABLE 4.1 PLATELET GRANULES AND MEDIATORS OF PLATELET
AGGREGATION PLATELET GRANULES
PLATELET GRANULES
α Granules: Contain Platelet-specific Proteins
Platelet factor 4
β-Thromboglobulin
Platelet-derived growth factor
Transforming growth factor-β

Dense Granules
Adenosine diphosphate
Serotonin
Calcium

MEDIATORS OF PLATELET AGGREGATION

Thomboxane A2
Thrombin
Platelet factor 4
At this stage, the wound is full of debris from the initial injury. This material consists of a
mixture of injured, devitalized tissue (fat, muscle, epithelium), clot (platelets, erythrocytes,
fibrin), bacteria (from the skin surface and external environment), extravasated serum
proteins (glycoproteins and mucopolysaccharides), and foreign material introduced at the
time of injury (suture, dirt). During the next several days, the wound is cleared of bacteria,
devitalized tissue, and foreign material by recruited and activated phagocytic cells.
Polymorphonuclear leukocytes (PMNs) begin to arrive immediately, attaining large numbers
within 24 hours. The process of clearing the wound of debris usually takes several days, but
the time varies depending on the amount of material to be cleared. The PMNs are followed
temporarily by macrophages, which appear in wounds in significant numbers within 2 or 3
days. The macrophages are mononuclear phagocytic cells derived from circulating
monocytes or resident tissue macrophages. They complete the process of removing all
material not necessary for the ensuing steps of wound healing.
In the absence of significant bacterial contamination, macrophages promptly replace PMNs
as the dominant cell type during the inflammatory phase. The role of the macrophages is not
limited to phagocytosis.2 In addition, macrophages are the sources of more than 30 different
growth factors and cytokines. These growth factors induce fibroblast proliferation,
endothelial cell proliferation (angiogenesis), and extracellular matrix production, and they
recruit and activate additional macrophages. The result is the induction of a wound healing
amplification cycle as growth factors recruit macrophages and elicit additional growth factor
release. Specific antibodies that destroy PMNs or block certain aspects of amplification
function (such as adhesion) have shown that wounds heal normally in the absence of bacteria,
but that healing is significantly impaired without functional macrophages (even in the
absence of bacteria). These studies confirm the dominant role of the macrophage in the
inflammatory phase of wound healing. The amplification process is so powerful that only
scattered fibroblasts and endothelial cells are seen in a surgical incision or at the edge of an
open wound at day 3, but by day 5 the wound is very cellular with active proliferation. By
day 7, the incisional wound is strong enough to allow suture removal, and proliferating
fibroblasts and angiogenesis give the wound an appearance similar in many respects to the
stroma surrounding tumors.
Lymphocytes also appear in wounds in small numbers during the inflammatory phase. The
role of lymphocytes in the wound healing process remains to be clarified, but they are
thought to be related more to chronic inflammatory processes than to the initial response to
wounding. Because recombinant growth factors are available in sufficient quantities for
clinical use, the prospect of using growth factors as pharmacologic agents to stimulate wound
healing (and potentially modulate abnormal scarring) has been the focus of much research.
Dozens of growth factor studies have been performed in humans, based on animal studies
that have demonstrated wound healing activity by about a dozen different growth factors
[PDGF, TGF-β1, TGF-β2, TGF-β3, fibroblast growth factor (FGF)2, FGF7, keratinocyte
growth factor (KGF), GMCSF, insulin-like growth factor (IGF1) plus IGFBP1, and
epidermal growth factor (EGF) represent a partial list), and to date only PDGF has been
approved.3 After a 7-year process, involving 1,000 patients, PDGF (Regranex) was approved
in 1997 by the Food and Drug Administration (FDA) for use in diabetic foot ulcers. The
failure of so many clinical trials is a reflection of both the complexity and variability of
human wounds, which make it difficult to demonstrate efficacy in all but the largest of
studies, and also the multiple factors that result in chronic wounds, which are discussed later.
Another product, an artificial skin equivalent made of a dermis-like matrix covered with
cultured epithelium from heterologous human foreskins (Apligraf) was FDA approved in
1998 for use in venous ulcers of more than 1-year duration that had failed other therapies.
This product's main effect is most likely due to the delivery of growth factors produced by
the neonatal cells. Increased knowledge of the role of growth factors in wound healing has
also provoked the evaluation of strategies using other pharmacologic agents to indirectly
modulate growth factor levels in wounds
Proliferative Phase
The proliferative phase begins with the formation of a provisional matrix of fibrin and
fibronectin as part of initial clot formation. Initially, the provisional matrix is populated by
macrophages; however, by day 3, fibroblasts appear in the fibronectin–fibrin framework
and initiate collagen synthesis. Fibroblasts proliferate in response to growth factors to
become the dominant cell type during this phase. Growth factors produced by macrophages
simultaneously induce angiogenesis, which induces the ingrowth and proliferation of
endothelial cells, forming new capillaries. This neovascularity is visible through the
epithelium and gives the wound a pink or purple-red appearance. Capillary ingrowth provides
the fibroblasts with oxygen and nutrients to sustain cell proliferation and support the
production of the permanent wound matrix. This matrix is composed of collagen and
proteoglycans or ground substance and replaces the provisional fibronectin–fibrin matrix.
FIGURE 4.2 Schematic representation of wound healing processes. ADP, adenosine
diphosphate; TXA2, thromboxane A2; TGF, transforming growth factor; PDGF, platelet-
derived growth factor; PF4, platelet factor 4; TNF-α, tumor necrosis factor α; FGF,
fibroblast growth factor; PAF, platelet-activating factor; KGF, keratinocyte growth factor.
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Collagen is the dominant structural molecule in the wound matrix and in the final scar. This
is not surprising because collagen is the principal structural protein in skin, bone, and indeed
all human tissue. Collagen is synthesized into an organized cable-like network in a multistep
process with both intracellular and extracellular components (Fig. 4.3). The collagen
molecule has abundant quantities of two unique amino acids, hydroxyproline and
hydroxylysine. The hydroxylation process that forms these amino acids requires ascorbic acid
(vitamin C) and is necessary for the subsequent stabilization and cross-linkage of collagen.
Procollagen is formed within the fibroblasts as a long linear amino acid segment with regular
repeats of hydroxyproline every third amino acid and with terminal extension peptides.
Procollagen molecules aggregate in the case of type 1 collagen (the most common) as three
α chains to form a triple-helical complex (see Fig. 4.3A). The triple helix is maintained by
intramolecular disulfide bonds between specific cystine residues. Procollagen is secreted in
its triple-helical form; extracellular peptidases cleave the extension peptides at the amino and
carboxy termini, leaving the central collagen molecule. Collagen cross-linking (see Fig. 4.3B)
then occurs in the extracellular space as the collagen molecules aggregate into larger
structures. Lysyl oxidase catalyzes the conversion of lysine and hydroxylysine into aldehyde
forms. These aldehydes form intermolecular bonds by undergoing spontaneous
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condensation. This produces stable, cross-linked collagen fibrils. These intramolecular and
intermolecular bonds provide strength and stability. As the wound matures, fibrils cross-link
to form large cables of collagen, providing increased tensile strength (see Fig. 4.3C). The
wound is now more appropriately considered a scar.
Although there are many types of collagen, type I predominates throughout the body. The
principal collagen in scar is type I, with lesser amounts of type III collagen present. Other
collagen types make important contributions to the basement membrane, cartilage, and other
structures (Table 4.2).
Remodeling Phase: The Formation of Scars
The transition from the proliferative phase to the remodeling phase is defined by reaching
collagen equilibrium. Collagen accumulation within the wound reaches a maximum within 2
to 3 weeks after wounding. Although supranormal rates of synthesis and degradation
continue throughout remodeling, there is no further change in total collagen content.4 Tensile
strength gradually increases as random collagen fibrils are replaced by organized fibrils with
more intermolecular bonds. During the initial phase of wound healing, there is a relative
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abundance of type III collagen in the wound. With remodeling, the normal adult ratio of 4:1
(type I to type III) collagen is restored. Under normal wound healing conditions, the capillary
density gradually diminishes, and the number of fibroblasts is reduced. The wound loses its
pink or purple vascular color and becomes progressively pale. The collagen undergoes
constant remodeling. New collagen is formed, and collagen degradation by specific
collagenases is ongoing. Collagenase activity is balanced against new production of collagen
to produce a steady state. As equilibrium is achieved, the collagen fibrils align themselves in
a longitudinal arrangement as dictated by stress placed on the wound. Scars never achieve the
degree of order achieved by collagen in normal skin or tendons, but they do increase in
strength for 6 months or longer, eventually reaching 70% of the strength of unwounded skin
(Fig. 4.4).

FIGURE 4.3 (A) Type I collagen showing triple helix and intramolecular cross-links. (B)
Intermolecular cross-links provide tensile strength. (C) Assembly of collagen fibrils, fibers,
and fiber bundles.
The other important component of the extracellular matrix is the ground substance or
proteoglycans. These substances are composed of a protein backbone with long hydrophilic
carbohydrate side chains. The hydrophilic nature of these molecules accounts for much of the
water content of scars. In the early immature wound, there is a disproportionately large
amount of proteoglycans (particularly hyaluronic acid). During the maturation phase, the
proteoglycan content returns to a level that closely approximates that of normal skin.
Until recently, the extracellular matrix (predominantly collagen and proteoglycans) was
thought to be inert. It is becoming
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increasingly clear, however, that extracellular matrix signals certain cells through cell
attachment receptors (integrins) and serves as a reservoir for growth factors. Via integrin
receptors, the extracellular matrix influences intracellular signal transduction with an impact
on cell motility and migration, cellular proliferation, and growth factor production. The role
of proteoglycans as signaling molecules is just beginning to be understood. In addition,
mechanical forces on tissue are transmitted from the matrix to the cells that are attached to
the matrix, and increasing evidence indicates that mechanical forces result in signal
transduction, which can result in cellular proliferation, apoptosis, and other signal
transduction processes.5
TABLE 4.2 COLLAGEN TYPES
TypeProperty of aggregate unitTissue distribution
I Rigid fibrils All connective tissue except cartilage
II Rigid fibrils Cartilage and vitreous
III Elastic fibrils Elastic tissue (e.g., fetal skin, blood vessels, uterus)
IV Sheet Basement membrane
V Fibril Widespread
VI Beaded filaments Widespread
VII Anchoring fibrils Interface of basement membrane and underlying stroma
VIII Sheet Descemet's membrane
IX Fibril Hyaline cartilage
X Sheet Hypertrophic cartilage
XI Fibril Hyaline cartilage
XII Fibril Similar to type I
Epithelialization
The skin is composed of two layers, the epidermis and the dermis. The outermost layer, the
epidermis, is the protective barrier that forms the external interface between the body and the
environment. The epidermis protects against water loss, allowing the other cells of the body
to live in a liquid environment, as well forming a barrier to bacteria and other environmental
factors. Reconstruction of the epithelial barrier (epithelialization) begins within hours of the
initial injury. As an initial step, epithelial cells from the basal layer at the wound edge flatten
and migrate across the wound, completing wound coverage within 18 to 24 hours in a
coapted surgical wound. The cells along the margin are also dividing to re-form the
characteristic basilar to apical differentiation of multilayered mature epithelium (see later).
Epithelial cells exhibit contact inhibition; that is, they continue to migrate across an
appropriate bed until a single continuous layer is formed. Epithelial cell migration occurs by
a process in which the epithelial cells send out pseudopods, attaching to the underlying
extracellular matrix by integrin receptors. Bacteria, large amounts of protein exudate from
leaky capillaries, and necrotic tissue all compromise this process, delaying epithelialization.
Delayed epithelialization results in a more profound and prolonged inflammatory process,
thereby contributing to unsatisfactory or hypertrophic scarring. There is increasing evidence
that reestablishment of a mature epithelium with a multilayered keratin layer (stratum
corneum), which completely restores the normal water barrier, is an important component of
the scar resolution process. Cell signals from the epithelium have been implicated in the
pathogenesis of keloids, and reestablishment of an epithelial barrier