The Basic Science of Wound Healing: Summary

RECONSTRUCTIVE
The Basic Science of Wound Healing

George Broughton II, M.D.,
Summary: Understanding wound healing today involves much more than sim-
Ph.D., COL., M.C., U.S.A. ply stating that there are three phases: “inflammation, proliferation, and mat-
Jeffrey E. Janis, M.D. uration.” Wound healing is a complex series of reactions and interactions among
Christopher E. Attinger, cells and “mediators.” Each year, new mediators are discovered and our un-
M.D. derstanding of inflammatory mediators and cellular interactions grows. This
Dallas, Texas; and Washington, D.C. article will attempt to provide a concise report of the current literature on wound
healing by first reviewing the phases of wound healing followed by “the players”
of wound healing: inflammatory mediators (cytokines, growth factors, proteases,
eicosanoids, kinins, and more), nitric oxide, and the cellular elements. The
discussion will end with a pictorial essay summarizing the wound-healing pro-
cess. (Plast. Reconstr. Surg. 117 (Suppl.): 12S, 2006.)
W
ound healing has traditionally been di- ing. The injured blood vessel vasoconstricts, and
vided into three distinct phases: inflam- the endothelium and nearby platelets activate the
mation, proliferation, and remodeling.1 intrinsic part of the coagulation cascade. The clot
Within each phase, a myriad of orchestrated re- that forms is made of collagen, platelets, throm-
actions and interactions between cells and chem- bin, and fibronectin, and these factors release cy-
icals are put into action. There is considerable tokines and growth factors that initiate the inflam-
overlap for each phase, and lines separating matory response.2 The fibrin clot also serves as a
them are blurred. This article will first provide scaffold for invading cells, such as neutrophils,
the reader with a general overview of the wound- monocytes, fibroblasts, and endothelial cells, to
healing process, followed by a more detailed use.3 The clot also serves to concentrate the elab-
discussion of the cells and inflammatory media- orated cytokines and growth factors.4 The impor-
tors involved in wound healing. tance of hemostasis is illustrated by conditions that
cause inadequate clot formation. Deficiency of fac-
PHASES OF WOUND HEALING tor XIII (the fibrin-stabilizing factor) is associated
Table 1 summarizes the process of wound with impaired wound healing5 secondary to de-
healing. Readers are encouraged to review Table creased chemotaxis or decreased adhesion of cells
1 while reading this article. in the inflammatory area.6,7 Table 2 highlights the
important functions the hemostatic and platelet-
Hemostasis and Inflammation (Immediately upon derived factors have in wound healing.
Injury through Days 4 to 6)
Hemostasis serves as the initiating step and Chemotaxis and Activation
foundation for the healing process. Inflammation Immediately as the clot is formed, cellular sig-
results in vasodilation and increased vascular per- nals are generated that result in a neutrophil re-
meability. However, the first action the body takes sponse. As the inflammatory mediators accumu-
immediately after wounding is to control bleed- late, prostaglandins are elaborated and the nearby
blood vessels vasodilate to allow for the increased
From the Department of Plastic Surgery, Nancy L and Perry cellular traffic as neutrophils are drawn into the
Bass Advanced Wound Healing Laboratory, University of
Texas Southwestern Medical Center, and The Georgetown
injured area by interleukin (IL)-1, tumor necrosis
Limb Center, Georgetown University Medical Center. factor (TNF)-␣, transforming growth factor
Received for publication February 1, 2006; revised March (TGF)-␤, PF4, and bacterial “products.”8,9 Mono-
18, 2006. cytes in the nearby tissue and blood are attracted
The opinions or assertions contained herein are the private to the area and transform into macrophages, usu-
views of the author (G.B.) and are not to be construed as ally around 48 to 96 hours after injury. Activation
official or as reflecting the views of the Department of the Army of the inflammatory cells is critical, especially for
or the Department of Defense. the macrophage. An activated macrophage is im-
Copyright ©2006 by the American Society of Plastic Surgeons portant for the transition into the proliferative
DOI: 10.1097/01.prs.0000225430.42531.c2 phase. An activated macrophage will mediate an-
12S www.plasreconsurg.org
Volume 117, Number 7S • Basic Science of Wound Healing
Table 1. Summary of Wound Healing

Event Description
Hemostasis/Inflammatory Phase
Wounding/hemostasis Injured endothelial cells immediately vasoconstrict. Intrinsic coagulation
pathway is activated and hemostasis is achieved.
Platelets become involved and a clot is formed. Platelets in the immediate
area of the wound aggregate to form a clot. They also release and
synthesize several growth factors, cytokines, and other inflammatory
proteins and activate the intrinsic coagulation pathway. Inflammatory
mediators include thromboxane (to result in further vasoconstriction) and
growth factors and cytokines to recruit more platelets and are
chemoattractant for neutrophils and fibroblasts.
Hemostasis Entrapped platelets within the thrombus release:
Mediator: fibrin, plasma fibronectinResult: coagulation, chemoattraction,

adhesion, scaffolding for cell migration
Mediator: factor XIII (fibrin-stabilizing factor)
Result: induces chemoattraction and adhesion
Mediator: circulatory growth factors
Result: regulation of chemoattraction, mitogenesis, fibroplasia
Mediator: complement
Result: antimicrobial activity, chemoattraction
Mediator: cytokines, growth factorsResult: regulation of chemoattraction,
mitogenesis, fibroplasia
Mediator: fibronectin
Result: early matrix, ligand for platelet aggregation
Mediator: platelet-activating factor
Result: platelet aggregation
Mediator: thromboxane A2 (via platelet COX-1)
Result: vasoconstriction, platelet aggregation, chemotaxis
Mediator: serotonin
Result: induces vascular permeability, chemoattractant for neutrophils
Mediator: platelet factor IV
Result: chemotactic for fibroblasts and monocytes, neutralizes activity of
heparin, inhibits collagenase
Inflammation After a short time (1 hour) the endothelial cell’s COX-2 enzyme is activated
to synthesize prostaglandins, to cause vasodilation and platelet
disaggregation, and leukotrienes, which results in increased vascular
permeability, chemotaxis, and leukocyte adhesion (inflammation).
Role of neutrophil Increased vascular permeability from endothelial prostaglandin and

leukotriene synthesis allows neutrophils to adhere to activated endothelial
cells via binding to the endothelial cell membrane receptor- selectin (which
is expressed from LTB4 influence). The inflammatory effects from
leukotrienes also cause the endothelium to form gaps (causing the capillary
to “leak”), allow the neutrophils to slip through (diapedesis), and allow
proteins to pass through, causing swelling.
Neutrophils trapped in the clot converts CTAP-III to NAP2, a very potent and
early neutrophil chemotactic protein.
Platelets also release complement, IL-1, TNF-␣, TGF-␤, and PF4, all of which
are chemoattractants for neutrophils. IL-1 and TNF stimulate adherence of
neutrophils to endothelial cells by induction of intracellular adhesion
molecules.
Neutrophils attach themselves to the extracellular matrix by binding to the
matrix with their integrin receptors. Other neutrophils are attracted to
other sites and travel through the matrix easily, elaborating proteases and
MMP to form oxygen free-radicals to kill bacteria and clear the
extracellular matrix.
(Table continued)
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Plastic and Reconstructive Surgery • June Supplement 2006
Table 1. Continued
Event Description
Turning inflammation off As the number of neutrophils and platelets increases, neutrophils and
platelets bond. Increasing levels of LTA4 and LTB4 in the neutrophil pass
through gaps between the cells and are acted on by the platelets’ 12-
lipoxygenase enzyme to form lipoxin A4 or lipoxin B5.
The lipoxin is capable of turning off destructive inflammatory acts in a variety

of cells.
Macrophages migrate into the Macrophages are attracted to the wound site by chemoattractants released by
wound the platelets and the clot (TGF-␤, PDGF, PF4, LTB4, and IL-1).
Endothelial cells also help to bring macrophages and fibroblasts into the
wound by synthesizing IL-1. IL-1 stimulates macrophages and fibroblasts to
express more of their own chemoattractant cytokines and growth factors.
Macrophages release the chemoattractants PDGF, TNF-␣, IL-6, G-CSF, and
GM-CSF to recruit more macrophages and fibroblasts.
Fibroblasts elaborate IL-6, G-CSF, and GM-CSF to further enhance
macrophage and fibroblast chemoattraction.
Fibroblasts release IFN-␥, which causes monocytes to transform into
macrophages.
Macrophages clear matrix of iNOS is activated by rising concentrations of IL-1 and TNF-␣, resulting in
debris and bacteria nitric oxide being synthesized and released. NO will kill pathogens (especially
Staphylococcus aureus) and combine with free oxygen radicals to form the very
toxic peroxynitrite (ONOO–) and hydroxyl radicals. NO in the wound area
also prevents viral DNA replication and serves as an immune regulator.
Protease transcription is induced by several different inflammatory cytokines.
IL-1 and TNF-␣ induce matrix metalloproteinase transcription in
keratinocytes, fibroblasts, and macrophages (all the way through into the
repair phase of wound healing). Proteases and matrix metalloproteinase clear
the extracellular matrix from inflammatory debris and allow migration of
cells through the matrix.
Macrophages will also phagocytose apoptotic neutrophils, debris, and bacteria.
Proliferative phase
Proliferation/epithelialization Epithelization is initiated by macrophages by stimulating fibroblasts with IL-1
and KGF-2. Fibroblasts, through the expression of KGF-2 and IL-6, cause
keratinocytes to proliferate and migrate. Keratinocytes are then able to self-
express IL-6 and NO to perpetuate the process. collagen replaces
proteoglycan and fibronectin.
Matrix formation Macrophages initiate provisional matrix formation through the expression of
PDGF and TNF-␣.
Fibroblasts perpetuate the process with autocrine and paracrine stimulation
with more PDGF.
Fibroblasts will initially synthesize proteoglycans and fibronectin to create the matrix.
TGF-␤ exists in the matrix as an inactive pro-TGF-␤. Macrophages and
fibroblasts release proteases that activate the TGF-␤, which in turn stimulates
further fibroblast proliferation and collagen synthesis. It also prevents
collagen degradation by causing TIMP secretion. TGF-␤ also causes increased
fibronectin secretion and integrin receptor expression to allow cellular
adhesion to the matrix.
(Table continued)
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Table 1. Continued
Event Description
Angiogenesis Macrophages stimulate keratinocytes to express VEGF with IL-1 and TNF-␣.
Fibroblasts stimulate keratinocytes to express VEGF with KGF-2 and TGF-␤
Keratinocytes direct angiogenesis at the wound edge by expressing VEGF.

VEGF causes the proliferation of endothelial cells and the formation of
capillaries.
VEGF expression is upregulated by the presence of NO
Wound contraction (1) Fibroblasts already located within the injury site are triggered by
macrophages (through TGF-␤1 and PDGF) to transform phenotype into a
myofibroblast (2). The fibroblast must be fully attached to the matrix by
fibroblasts integrin receptors and fibronectin within the matrix. TGF-␤1 and
PDGF (3) both cause myofibroblasts to contract, closing the wound (4).
Maturation and remodeling phase

Remodeling TGF-␤ directs collagen matrix construction. TGF-␤ levels peak in the wound 7
to 14 days in an incisional wound (hence the rationale for the timing of
suture removal). The matrix becomes thicker and stronger as type I
collagen replaces proteoglycan and fibronectin.
TGF-␤ also upregulates expression of TIMP, decreases MMP production, and

increases the expression of tissue adhesion proteins. TIMP production is
also upregulated by IL-6. TNF-␣ stimulates the release of IL-6 by fibroblasts,
further enhancing TIMP production.
giogenesis [by synthesizing vascular endothelial proteins, amino acids, or the metal ion within the
growth factor (VEGF), fibroblast growth factor, enzyme. Serine proteases have broad specificity
and TNF-␣] and fibroplasia [by synthesizing (e.g., elastase), whereas metalloproteinase (which
TGF-␤, epidermal growth factor (EGF), platelet- contains a zinc ion) specifically digests collagen.
derived growth factor (PDGF), IL-1, and TNF-␣] Both types of proteases will destroy the preexisting
and synthesize nitric oxide (NO) (from activation extracellular matrix in the wound area. The ma-
of inducible nitric oxide synthase by IL-1 and trix in unwounded tissue is protected by an “ar-
TNF-␣).10 mor” made of protease inhibitors.11 The “antipro-
Neutrophils enter into the wound site and be- tease armor” can be overwhelmed and penetrated
gin clearing it of invading bacteria and cellular if the inflammatory response is extremely robust
debris. The neutrophil releases caustic proteolytic from a massive release of proteases. Neutrophils
enzymes that will digest bacteria and nonviable can also generate reactive oxygen free radicals
tissue. The neutrophil has several different types (through a myeloperoxidase pathway) that com-
of proteases grouped by their preferred target: bine with chlorine to help sterilize the wound of
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Table 2. Hemostatic and Platelet-Derived Factors Associated with Wound Healing

Factor Function
Hemostatic factors
Fibrin, plasma fibronectin Coagulation, chemoattraction, adhesion, scaffolding for cell migration
Factor XIII (fibrin-stabilizing factor) Induces chemoattraction and adhesion
Circulatory growth factors Regulation of chemoattraction, mitogenesis, fibroplasia
Complement Antimicrobial activity, chemoattraction
Platelet-derived factors
Cytokines, growth factors Regulation of chemoattraction, mitogenesis, fibroplasia
Fibronectin Early matrix, ligand for platelet aggregation
Platelet-activating factor Platelet aggregation
Thromboxane A2 Vasoconstriction, platelet aggregation, chemotaxis
Platelet factor IV Chemotactic for fibroblasts and monocytes, neutralizes activity of heparin,
inhibits collagenase
Serotonin Induces vascular permeability, chemoattractant for neutrophils
Adenosine dinucleotide Stimulates cell proliferation and migration, induces platelet aggregation
Adapted from Witte, M., and Barbul, A. General principles of wound healing. Surg. Clin. North Am. 77: 509, 1997.
bacteria.11 Neutrophils shortly succumb to an un- platelets and leukocytes through a transcellular bio-
known stimulus for apoptosis and are replaced by synthesis. Platelets cannot produce lipoxins on their
macrophages (which phagocytosize the dead neu- own, but when platelets and neutrophils adhere to
trophils). Macrophages do not possess myeloper- one another, the leukocyte produces leukotriene A4
oxidase but do continue in pathogen killing by (via 5-lipoxygenase), which is transferred to the
generating NO. The macrophages’ iNOS is stim- platelet; the platelet’s 12-lipoxygenase converts it to
ulated to synthesize very large quantities of NO by lipoxin A4 and B4.14 Neutrophils alone have also
TNF and IL-1 that react with peroxide ion oxygen been shown to synthesize lipoxins. Clinical and ex-
radicals to yield an even more toxic peroxynitrite perimental wound exudate studies have shown that
and hydroxyl radicals.12 the early appearance of leukotrienes and prostaglan-
The damaged extracellular matrix is also dins coincides with neutrophil infiltration to the site
cleared by matrix metalloproteinase (MMP), of inflammation. This is shortly followed by lipoxin
which is expressed by keratinocytes, fibroblasts, biosynthesis, which is concurrent with spontaneous
monocytes, and macrophages in response to resolution of the inflammation.14 Human neutro-
TNF-␣. MMP clears inflammatory debris and en- phils in peripheral blood were exposed to prosta-
ables migration of individual wound cells through glandin E2 (PGE2), which resulted in a switch in
the extracellular matrix.13 eicosanoid biosynthesis from predominantly LTB4
For many years it was thought that, as with a (a 5-lipoxygenase-initiated pathway) to LXA4, a 15-
fire, the inflammatory phase would just “burn it- lipoxygenase product that “stops” polymorphonu-
self out” when initiating exogenous stimuli or clear neutrophil infiltration. In addition, PGE2 ini-
when signals were depleted.14 There is now evi- tiates 15-lipooxygnease gene expression and RNA
dence that such a well-coordinated, elegant, and processing in vitro in a temporal frame that is con-
destructive process is organized as a series of re- sistent with the “switching on” of lipoxin production
actions to produce “stop signals,” referred to as in vivo.14 As Serhan and Chiang14 point out, “these
“checkpoint controllers of inflammation.”14,15 results indicate that functionally distinct lipid medi-
One may recall the synthesis of the eicosanoid ator profiles switch during acute exudate formation
inflammatory mediators: prostaglandin, prostacy- to ‘reprogram’ the exudate (polymorphonuclear
clin, thromboxane, and leukotrienes. The lipoxy- neutrophil) to promote resolution.” In addition, in-
genase enzyme also synthesizes another lipid me- hibition of prostaglandin products might alter the
diator: lipoxins (LXA4 and LXB4). Lipoxins and duration of resolution.
aspirin-triggered lipoxins are the stop signal for Aspirin-triggered lipoxins are the result of
inflammation. Aspirin acetylates COX-2 enzyme aspirin’s inhibition of the COX-2 enzyme. Aspi-
(the inducible form of COX) and triggers the rin’s ability to regulate neutrophil-mediated in-
formation of 15-epimeric lipoxins.14,16 Serhan’s flammation or cell proliferation continues to
laboratory14 has also identified autacoids synthe- be a topic of interest, with new and alternative
sized by aspirin-acetylated COX-2 from omega-3 therapeutic uses for aspirin (e.g., decreasing
polyunsaturated fatty acids that display potent anti- the incidence of lung, colon, and breast cancer
inflammatory and “proresolving actions,” which and preventing cardiovascular diseases).17 Ser-
they termed “resolvins.” Lipoxins are formed by han’s laboratory14 has uncovered a new action
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Fig. 1. Effect of aspirin of lipoxin synthesis.
of aspirin that involves COX-2– bearing cells, establish a protective barrier against fluid losses
such as vascular endothelial cells or epithelial and further bacterial invasion. The stimulus for
cells, and their co-activation with polymorpho- epithelial proliferation and chemotaxis is EGF
nuclear neutrophils. Inflammatory stimuli and TGF-␣ produced by activated platelets and
(e.g., TNF, lipopolysaccharide) induce COX-2 macrophages (fibroblasts do not appear to syn-
to generate 15R-HETE when aspirin is thesize TGF-␣).18,19 Epithelization begins shortly
administered.16 This intermediate carries a car- after wounding and is first stimulated by inflam-
bon-15 alcohol in the “R” configuration that is matory cytokines (IL-1 and TNF-␣ upregulate KGF
converted rapidly by 5-lipooxygenase in acti- gene expression in fibroblasts). In turn, fibroblasts
vated neutrophils to 15 epimeric-LX or aspirin- synthesize and secrete keratinocyte growth factor
triggered lipoxins that carry their 15 position (KGF)-1, KGF-2, and IL-6, which simulate neigh-
alcohol in the “R” configuration rather than boring keratinocytes to migrate in the wound area,
15S native LX. This lipoxin epimer may provide proliferate, and differentiate in the epidermis.20,21
alternate explanations for aspirin’s new thera-
In humans, it seems that KGF-2 is most important
peutic actions. The cellular effects of lipoxins
for directing this process.22
and aspirin-triggered lipoxins are summarized
in Figure 1. Fibroblasts and endothelial cells are the pre-
dominant cells proliferating during this phase.
Endothelial cells located at intact venules are se-
Proliferative Phase: Epithelization, duced by VEGF (secreted predominantly by ker-
Angiogenesis, and Provisional Matrix Formation atinocytes on the wound edge, but also by mac-
(Day 4 through 14) rophages, fibroblasts, platelets, and other
Epithelial cells located on the skin edge begin endothelial cells) to begin forming new capillary
proliferating and sending out projections to re- tubes. Recall that keratinocytes can be stimulated
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Table 3. Effect of Growth Factors on Fibroblast Proliferation

Factor Effect Comment
PDGF Increases
IFN-␥ Increases (low concentration)/decreases (high concentration)
TGF-␤ Increases (low concentration )/decreases (high concentration) Via release of PDGF
FGF Increases
EGF Increases
IL-1 Increases Via release of PDGF
TNF-␣ Increases (low concentration)/decreases (high concentration) Via increase of PDGF
to express VEGF by IL-1, TNF-␣, TGF-␤1, and KGF. fibroblasts for wound contraction (induced by mac-
NO is made by endothelial cells (from endothelial rophage-secreted TGF-␤1). They have less
nitric oxide synthase eNOS) in response to hyp- proliferation compared with the fibroblasts coming
oxia, and this in turn stimulates more VEGF pro- in from the wound periphery.23–25 In response to
duction. The increased concentrations of NO also PDGF, fibroblasts begin synthesizing a provisional
protect the new tissue from the toxic effects of matrix composed of collagen type III, glycosamino-
ischemia and reperfusion injury12 and cause en- glycans, and fibronectin.26 Integrins are a matrix
dothelium to vasodilate.10 component that serves to anchor cells to the provi-
Fibroblasts migrate into the wound site from the sional matrix and is upregulated by TNF-␣.27 In a
surrounding tissue, become activated and begin syn- normal incisional wound, TGF-␤ peaks around day
thesizing collagen, and proliferate. PDGF and EGF 7 to 14 and directs extracellular matrix production
are the main signals to fibroblasts and are derived and a decrease in its degradation. TGF-␤ causes fi-
from platelets and macrophages (Table 3). PDGF broblasts to synthesize type I collagen, decrease pro-
expression by fibroblasts is amplified by autocrine duction of MMP, enhance production of tissue in-
and paracrine signaling. Fibroblasts already located hibitors of metalloproteinase, and increase
in the wound site (termed “wound fibroblasts”) be- production of cell adhesion proteins.12 The signal to
gin synthesizing collagen and transform into myo- turn off activity seems to come from interferon-in-
Fig. 2. The deposition of wound matrix components over time. Although fibronectin and
collagen type III constitute the early matrix, collagen type I accumulates later, corresponding
to the increase in wound-breaking strength. Adapted from Witte, M., and Barbul, A. General
principles of wound healing. Surg. Clin. North Am. 77: 509, 1997.
18S
ducible protein (IP-10), which inhibits EGF-induced Grinnell7 wrote an elegant discussion on the
fibroblast motility and thereby limits fibroblast re- relationship of fibroblasts and other dermal cells
cruitment, interferons themselves, and PF4, which to the matrix as a three-dimensional structure.
has a negative mitogenic effect on fibroblasts.28 Early in wound healing, the matrix is thin and
Larger wounds healing by secondary intention compliant and allows fibroblasts, neutrophils, lym-
are still directed, in part, by TGF-␤, which causes phocytes, and macrophages to easily maneuver
wound contracture (transforming “wound fibro- through it. As the matrix becomes denser with
blasts” into myofibroblasts) and epithelization.29 thicker, stronger collagen fibrils, it becomes stiff
Components of the wound matrix at different and less compliant. The fibroblasts are capable of
points are summarized in Figure 2. “adaptive response” to the changing mechanical
loading on the matrix as it matures. Before iso-
metric tension develops, remodeling of the com-
Maturation and Remodeling (Day 8 through 1 pliant matrix depends on the cell migration
Year) throughout the matrix and proteolysis of the ma-
Clinically, the maturation and remodeling trix proteins. Isometric tension is defined as a
phase is perhaps the most important. The main situation in which internal and external mechan-
feature of this phase is the deposition of collagen ical forces are balanced such that cell contraction
in an organized and well-mannered network. If occurs without cell shortening or lengthening. At
patients have matrix deposition problems (from an early point, cellular adhesion to the matrix is
diet or disease), then the wound’s strength will be not possible. As the matrix stiffness increases and
greatly compromised. If there is excessive collagen isometric tension develops, lysophosphatidic ac-
synthesis, then a hypertrophic scar or keloid can id–stimulated remodeling switches to a Rho-ki-
result. nase-dependent myosin-light-chain phosphoryla-
The building of the wound matrix follows a tion mechanism of contraction.
pattern. Initially, the matrix is composed mainly of PDGF stimulates the fibroblast dendritic net-
fibrin and fibronectin (arising from the efforts for work to swell and reach out, which it can do when
hemostasis and by macrophages).3 Glycosamino- the matrix is compliant. Lysophosphatidic acid, the
glycans, proteoglycans, and other proteins (such simplest of all glycerol phospholipids, causes the
as secreted protein acidic rich in cysteine, or dendritic branches of the fibroblasts to retract.
SPARC) are synthesized next by the fibroblasts.2 Lysophosphatidic acid is widely distributed in
This haphazard and disorganized collection of gly- mammalian tissues and serum and is generated
cans provides a preliminary framework for the new by cleavage from membranes of stimulated cells
matrix. This temporary matrix is replaced by a (most likely from platelets for wound healing).31
stronger and organized matrix made of collagen. Cellular effects of lysophosphatidic acid can be
The collagen in uninjured skin is 80 to 90 percent categorized as “growth-related” or “cytoskele-
type I and 10 to 20 percent type III. In granulation ton-dependent,” resulting in the modulation of
tissue, collagen type III comprises 30 percent, and adhesion, chemotaxis, contraction, or aggrega-
in the mature scar, it is back down to 10 percent.24 tion. As a mitogen for fibroblasts and with ad-
The appearance of collagen type III also coincides ditional effects on endothelial cells, macro-
with the presence of fibronectin. It has been pro- phages, and vascular smooth muscle cells,
posed that the coating of denatured collagen with lysophosphatidic acid has been implicated in
fibronectin facilitates its phagocytosis.30 The role wound healing,32 because it activates its associ-
for the early and increased deposition of type III ated G-protein– coupled receptors, three of
collagen (which does not significantly contribute which have been identified. Lysophosphatidic
to the strength of the wound) is unclear. The acid receptors couple to at least three distinct
matrix remodeling proteinases, MMPs (there are G-proteins and thereby activate multiple signal
several different ones, each specific for a type(s) transduction pathways, particularly those initi-
of collagen and under the influence and control ated by the small GTPase Ras, Rho, and Rac.
of different cytokines), are influenced by chang- To increase contractility more, fibroblasts dif-
ing concentrations of TGF-␤, PDGF, IL-1, and ferentiate into myofibroblasts under the influence
EGF. MMP activity is further suppressed by tissue of TGF-␤. Differentiation is signaled by cell inter-
inhibitors of metalloproteinase, whose produc- action with an alternatively spliced form of fi-
tion by fibroblasts is upregulated by TGF-␤ and bronectin that causes the fibroblast to increase its
IL-6; TNF-␣ stimulates the release of IL-6 by expression of ␣-smooth muscle actin isotype,
fibroblasts.28 which has been shown to be linked to cell
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contractility.7 Because the fibroblasts are anchored soluble factors released by damaged and nearby
and not free floating, they can organize to form focal cells, platelets, and leukocytes in an attempt to
adhesions that give the myofibroblasts the mechan- control the damage and begin healing. Inflam-
ical leverage to contract. TGF-␤ only stimulates the matory mediators include, but are not limited to,
differentiation of fibroblasts in a restrained matrix; cytokines, growth factors, proteases, eicosanoids,
therefore, switching between mechanically loaded kinins, and cellular metabolites.
and unloaded conditions regulates the differentia- Eicosanoids
tion and regression of myofibroblasts. Unloading of Eicosanoids comprise a family of biologically
mechanical contraction results in apoptosis and de- active, oxygenated arachidonic acid metabolites,
creased collagen synthesis, with the net effect of an including prostaglandins, prostacyclin, thrombox-
improved scar. Persistent mechanical loading cre- anes, leukotrienes, and lipoxins. A phospholipase
ates the pathological condition of contracture and (usually phospholipase A2, but others exist) in
results in hypertrophic or widened scars caused by lysosomes or bound to cell membranes is released
the persistence of fibroblasts and collagen synthesis. in response to specific and nonspecific stimuli
Interestingly, fibroblast-collagen matrix remodeling (e.g., cellular trauma, including ischemia and
results in plasma membrane tears from mechanical hypoxia,37,38 oxygen free radicals,39 or osmotic
changes that accompany rapid remodeling upon stress).40,41 Phospholipase A2 (PLA2) moves to the
release of restrained collagen. These membrane cell membrane and hydrolyzes arachidonic acid
tears result in activation of phospholipid and mito- off of a cellular membrane lysophospholipid (usu-
gen-activated protein-kinase signaling pathways. The ally phosphatidylcholine). This cleavage step is
significance of this is not known.7 rate-limiting in the production of biologically rel-
Net collagen synthesis will continue for at least evant arachidonate metabolites. Other hormones
4 to 5 weeks after wounding. The increased rate of and growth factors, including EGF and PDGF, ac-
collagen synthesis during wound healing is from tivate PLA2 directly through tyrosine residue ki-
not only an increase in the number of fibroblasts nase activity. After deesterification, arachidonic
but also a net increase in the collagen production acid is rapidly reesterified into membrane lipids or
per cell.33,34 The collagen that is initially laid down avidly bound by intracellular proteins,42 in which
is thinner than collagen in uninjured skin and is case it becomes unavailable for further metabo-
orientated parallel to the skin (instead of the bas- lism. Should it escape reesterification and protein
ket weave pattern seen in uninjured skin). Over binding, free arachidonic acid becomes available
time, the initial collagen threads are reabsorbed as a substrate for one of three major enzymatic
and deposited thicker and organized along the transformations, the common result of which is
stress lines. These changes are also accompanied the incorporation of oxygen atoms at various sites
by a wound with an increased tensile strength, of the fatty acid backbone and ring formation42– 44
indicating a positive correlation between collagen to result in the formation of biologically active
fiber thickness/orientation and tensile strength.2 molecules, or “eicosanoids.” Arachidonic acid can
The collagen found in granulation tissue is bio- be converted into biologically active compounds
chemically different from collagen from unin- by cyclooxygenase-, lipoxygenase-, or cytochrome
jured skin. Granulation tissue collagen has a P-450 –mediated metabolism. The cytochrome
greater hydroxylation and glycosylation of lysine P-450 – dependent oxygenation of arachidonic
residues, and this increase of glycosylation corre- acid mediates the formation of epoxyeicosatrie-
lates with the thinner fiber size.35 The collagen in noic acids, their corresponding diols, mono-, di-,
the scar (even after a year of maturing) will never and tri-hydroxyeicosatetraenoic acids, and mono-
become as organized the collagen found in unin- oxygenated arachidonic acid derivatives.45 No fur-
jured skin. Wound strength also never returns to ther discussion will follow on the cytochrome
100 percent. At 1 week, the wound only has 3 P-450 – dependent oxygenation of arachidonic
percent of its final strength; at 3 weeks, it is 30 acid. The cyclooxygenase and lipoxygenase path-
percent; and at 3 months (and beyond), it is ap- ways are discussed in more detail below.
proximately 80 percent.36 Cyclooxygenase pathway. The generation of pros-
taglandins is mediated by two different enzymes,
THE ELEMENTS OF WOUND HEALING COX-1 and COX-2. Prostaglandins are divided
into series based on structural features, as coded
Inflammatory Mediators by a letter (PGD, PGE, PGF, PGG, and PGH) and
“Inflammatory mediator” is an all-encompass- a subscript numeral (e.g., 1, 2) that indicate the
ing and confusing label given to a collection of number of double bonds in the compound.46 The
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most important ones in inflammation are PGE2, response to generate prostaglandins if the con-
PGD2, PGF2-␣, PGI2 (prostacyclin), and TxA2 centration of arachidonic acid is high.50 COX-2,
(thromboxane), each of which is derived by the on the other hand, can generate prostaglandin
action of a specific enzyme. Some of these enzymes when arachidonic acid concentrations are low. In
have restricted tissue distribution. For example, response to injury, COX-2 is induced in keratin-
platelets contain the enzyme thromboxane syn- ocytes, macrophages, and endothelium in the
thetase, and hence TxA2 is the major product in granulation tissue50 (Fig. 3).
these cells. TxA2, a potent platelet-aggregating The prostaglandins are also involved in the
agent and vasoconstrictor, is itself unstable and pathogenesis of pain and fever in inflammation.
rapidly converted to its inactive form, TxB2. Vas- PGE2 is hyperalgesic, causing the skin to become
cular endothelium lacks thromboxane synthetase hypersensitive to painful stimuli.51 It causes a
but possesses prostacyclin synthetase, which leads marked increase in pain from suboptimal concen-
to the formation of prostacyclin (PGI2) and its trations of intradermal histamine and bradykinin
stable endproduct, PGF1␣. Prostacyclin is a vaso- and interacts with cytokines to cause fever during
dilator and a potent inhibitor of platelet aggrega- infection. PGD2 is the major metabolite of the
tion. It also markedly potentiates the permeability- cyclooxygenase pathway in mast cells; along with
increasing and chemotactic effects of other PGE2 and PGF2␣ (which are more widely distrib-
mediators.47 uted), it causes vasodilation and potentiates
Cyclooxygenase has received a lot of attention edema formation.
recently because of select COX-2 inhibitors. Lipoxygenase pathway. The initial products are
COX-1 is a constitutive form and is considered a generated by three different lipoxygenases, which
“housekeeping enzyme” involved in physiological are present in only a few types of cells. 5-Lipoxy-
reactions such as regulating renal and vascular genase (5-LO) is the predominant enzyme in
homeostasis and gastric mucosa protection.29 The neutrophils.52 On cell activation, it translocates to
COX-2 enzyme is considered an “immediate early” the nuclear membrane and interacts with a mem-
gene that can be synthesized rapidly in response brane-associated regulatory protein (5-lipoxygen-
to a wide variety of growth factors, cytokines, and ase–activating protein) to form the active enzyme
hormones, particularly in the course of the in- complex. The main product, 5-HETE, which is
flammatory process.48,49 COX-1 is thought to be chemotactic for neutrophils, is converted into a
involved in normal skin homeostasis and does not family of compounds collectively called leukotri-
respond to inflammatory mediators.48 However, enes. LTB4 is a potent chemotactic agent and ac-
COX-1 can respond and trigger an inflammatory tivator of neutrophil functional responses, such as
Fig. 3. COX-1 and COX-2 enyzmes. The COX-1 enzyme is stimulated by physiologic stimuli
and is a housekeeping enzyme involved in maintaining hemostasis and gastric mucosa pro-
duction. COX-2 enzyme is found in keratinocytes, macrophages, and endothelium and is
induced by growth factors and cytokines released in response to injury. It also initiates in-
flammation.
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aggregation and adhesion of leukocytes to venular contact enhances transcellular metabolism, and
endothelium, generation of oxygen free radicals, blocking adhesion inhibits lipoxin production. Li-
and release of lysosomal enzymes. The cysteinyl- poxins have a number of proinflammatory and
containing leukotrienes C4, D4, and E4 (LTC4, anti-inflammatory actions. They inhibit neutro-
LTD4, and LTE4) cause intense vasoconstriction, phil chemotaxis and adhesion but stimulate
bronchospasm, and increased vascular permeabil- monocyte adhesion.56 LXA4 stimulates vasodila-
ity. The vascular leakage, as with histamine, is re- tion and attenuates the actions of LTC4-stimulated
stricted to venules. Cell-cell interactions are im- vasoconstriction. There is an inverse relationship
portant in the biosynthesis of leukotrienes.53 between the amount of lipoxin and the amount of
Arachidonic acid products can pass from one cell leukotriene formed, suggesting that the lipoxins
type to another, and different cell types can co- may be endogenous negative regulators of leuko-
operate with each other to generate eicosanoids triene action. The inflammatory synthesis and ac-
(termed transcellular biosynthesis).54 In this way, tions of eicosanoids are shown Figure 4.
cells that are not capable of generating a partic- Cytokines
ular class of eicosanoid can produce these medi- Cytokines are extremely potent small regula-
ators from intermediates generated in other cells, tory peptides or glycoproteins with a molecular
thus expanding the array and quantities of eico- weight of 5 to 30 kDa that are released by nucle-
sanoids produced at sites of inflammation. One ated cells. They act to modulate immune or repair
example of transcellular biosynthesis is the gen- processes by controlling cellular growth, differen-
eration of lipoxins. tiation, metabolism, and protein synthesis. Cyto-
Lipoxins are the most recent addition to the kines are related more directly to the control of
family of bioactive products generated from ara- cell immune responses and have hematopoietic
chidonic acid, and transcellular biosynthesis is key cells for targets (growth factors, which are often
to their production. Platelets alone cannot form confused with cytokines, have nonhematopoietic
lipoxins, but when they interact with leukocytes cells as targets). They can be subcategorized as
they can form the metabolites from neutrophil- chemokines, lymphokines, monokines, interleu-
derived intermediates. Lipoxins A4 and B4 (LXA4 kins, colony-stimulating factors, and interferons.
and LXB4) are generated by the action of platelet Chemokines. Chemokines are a subset of cyto-
12-lipoxygenase on neutrophil LTA4.55 Cell-cell kines that are soluble proinflammatory factors
Fig. 4. Inflammatory actions of eicosanoids and their synthesis.
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that attract and activate leukocytes. Chemokines The Cxxxc chemokines are associated with
are further subdivided into four families charac- natural killer cell activation.
terized by a conserved amino acid pattern at the Lymphokines, Monokines, and Interleukins.
first two cysteine residues near the N terminus.57 Lymphokines are a subset of cytokines that
This pattern is important in that different cys- are produced by activated T lymphocytes.
teine patterns are chemoattractive to different Monokines are a subset of cytokines that are
types of leukocytes. The nomenclature for these produced by mononuclear phagocytes.
families is descriptive: C represents cysteine and Interleukins are a subset of cytokines origi-
X represents a nonconserved residue. The four nally thought to be secreted by one type of leu-
families and their significance are as follows: CXC kocyte that acts on another type of leukocyte.
chemokines, C-C chemokines, C chemokines, Now it is known that these mediators are also
and Cxxxc chemokines. released from nonhematopoietic cells and have a
The CXC family of chemokines is further di- myriad of effects. The progressive numbering sys-
vided into two subtypes separated by the presence tem for interleukins was started in 1978 and
or absence of a glutamic acid-leucine-arginine ranges from IL-1 through IL-23.58 Many of the
sequence near the N terminus (this is referred to interleukins belong to other cytokine families.
as the ELR motif).57 In the first subtype, CXC Interleukins with numbers higher than 10 seem
chemokines with the ELR motif attract neutro- to have no recognizable role in wound healing
phils only. This subtype includes the chemokines but are involved in immunity. A summary of im-
important for wound healing [IL-8, (GRO)-␣, portant interleukins and their affects on wound
(GRO)-␤, (GRO)-␥, NAP-2, and ENA-78). In the healing is given in Table 4.
second subtype, CKC chemokines without the Colony-Stimulating Factors. Colony-stimulating
ELR motif attract activated lymphocytes. The im- factors are a subset of cytokines that have a stim-
portant chemokines in this group include IP-10 ulatory wound-healing effect. CSF-1 is secreted by
and MIG. macrophages, is an autocrine mediator, and “aids
C-C chemokines are chemoattractant for lym- in self-preservation.”28 Once activated, the macro-
phocytes, monocytes, eosinophils, and basophils, phage releases granulocyte-macrophage CSF
but not neutrophils. The important chemokines which has generalized chemotactic, cellular pro-
belonging to this family include MCP-1 through liferation, and activation properties.
-5, RANTES, MIP, and MDC. Interferons. There are three members of the
The C chemokines are known to stimulate interferon (IFN) family (␣, ␤, and ␥), and their
neutrophils. nomenclature is based on their ability to “inter-
Table 4. The Role of Interleukins during Wound Healing

Description Source
IL-1 Induces fever, adrenocorticotrophic hormone release, Macrophages, mast cells, keratinocytes, lymphocytes
enhances TNF-␣ and IFN-␥, activates granulocytes
and endothelial cells, and stimulates hematopoiesis
IL-2 Activates macrophages, T cells, natural killer cells, Macrophages, mast cells, keratinocytes, lymphocytes
and lymphokine-activated killer cells; stimulates
differentiation of activated B cells; stimulates
proliferation of activated B and T cells; and
induces fever
IL-6 Is released in response to IL-1; induces fever; Macrophages, mast cells, keratinocytes, lymphocytes
enhances release of acute-phase reactants by the
liver; and is important in inhibiting extracellular
matrix breakdown during proliferation
IL-8 Enhances neutrophil adherence, chemotaxis, and Macrophages, mast cells, keratinocytes, lymphocytes
granule release; and enhances epithelization
IL-4 Early: stimulates fibroblast proliferation; later (72 Mast cells
hours): downregulates cytokine expression
IL-10 Early: unknown; later (72 hours): downregulates Unknown
cytokine expression
Sources: Henry, G., and Garner, W. Inflammatory mediators in wound healing. Surg. Clin. North Am. 83: 483, 2003; Lawrence, W., and
Diegelmann, R. Growth factors in wound healing. Clin. Dermatol. 12: 157, 1994; Cross, K., and Mustoe, T. Growth factors in wound healing.
Surg. Clin. North Am. 83: 531, 2003; and Bennet, N., and Schultz, G. Growth factors and wound healing: Biochemical properties of growth factors
and their receptors. Am. J. Surg. 165: 728, 1993.
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fere” with viral growth. IFN-␣ is primarily derived Table 5 lists the five growth factor superfami-
from monocytes, macrophages, B lymphocytes, lies and includes examples pertinent to this dis-
and natural killer cells. It has significant antiviral cussion on wound healing.18,19,64 – 82 A summary of
activity mediated through its ability to inhibit viral the growth factors and cytokines is shown in Ta-
replication within virus-infected cells, protect un- bles 5 and 6.
infected cells from infection, and stimulate anti- Nitric Oxide
viral immunity by cytotoxic lymphocytes and nat- NO is a small radical formed from the amino
ural killer cells. The most important interferon acid L-arginine by three distinct isoforms of nitric
for wound healing is IFN-␥. It is primarily pro- oxide synthase (NOS). Two of the isoforms are
duced by T-helper lymphocytes but is also derived called cNOS because they are constitutively
from cytotoxic T cells and natural killer cells. expressed.83 Neuronal NOS (nNOS, ncNOS,
IFN-␥ stimulates antigen presentation and cyto- NOS1), the first to be discovered, is found in neu-
kine production by monocytes and also stimulates rons, skeletal muscle, the pancreas, and the
monocyte effector functions, including adher- kidneys.84,85 The other constitutive enzyme, endo-
ence, phagocytosis, secretion, the respiratory thelial NOS (eNOS, ecNOS, NOS3), is predomi-
burst, and nitric oxide production. The net result nantly membrane-bound in endothelial cells, but
is the accumulation of macrophages and destruc- it can also be found in other cell types (e.g., neu-
tion of intracellular pathogens. In addition to its rons and cardiac myocytes).86 Intracellular cal-
effects on mononuclear phagocytes, IFN-␥ stimu- cium concentrations are the dominant mecha-
lates killing by natural killer cells and neutro- nisms for activation, leading to low-level NO
phils. It stimulates adherence of granulocytes to production in the span of just a few minutes.87,88
endothelial cells through the induction of intra- The third isoform, inducible NOS (iNOS, NOS2),
cellular adhesion molecules, an activity shared is not typically expressed in cells in the basal
with IL-1 and TNF.59 state.89 First isolated from activated macrophages,
Growth Factors this enzyme can be expressed in virtually all tissues
Growth factors are constitutively present me- under the appropriate conditions.15,90 iNOS is syn-
diators that act on nonhematopoietic cells to mod- thesized in the early phase of wound healing de
ulate wound healing by stimulating protein pro- novo in response to cytokines, microbes, microbial
duction, extracellular matrix synthesis, matrix products, and hypoxia, resulting in the sustained
turnover, and cellular death. Growth factors are production of NO.89,90 Once formed, iNOS is
proteins weighing between 4000 and 60,000 kDa. maintained in an active state by calmodulin bound
They can affect cellular function through endo- to the enzyme, allowing it to operate independent
crine (released from a distant organ or cluster of of calcium concentrations.91 This leads to a much
cells through the blood stream), paracrine (af- larger release of NO, limited only by substrate and
fecting a neighboring cell), autocrine (affecting cofactor availability and enzyme concentration.
itself by stimulating a membrane receptor), or Although the in vitro signals of iNOS induc-
intracrine (affecting itself intracellularly) mecha- tion are well described, little is known of the in vivo
nisms. Their actions are enhanced by their ability signals during wound healing. Among the numer-
to act on target cells in an autocrine fashion.60 ous cytokines and growth factors secreted and re-
Growth factors typically have the words “growth leased into the wound environment, IL-1, TNF-␣,
factor” in their name; the only notable exception and IFN-␥ are the most likely inducers of iNOS.
is TGF-␤, which is more like a cytokine owing to its Wound fluid, as a biological reflection of the
smaller molecular weight and its selective effect on wound environment, induces NO synthesis in a
multiple inflammatory processes.28 It is the only variety of cells.92 Although iNOS expression is high
known growth factor to use a serine/threonine during the early phases of wound healing, little is
kinase instead of a tyrosine kinase transduction known about the downregulation of iNOS activity
system.61 at the wound site during the later phases of heal-
There are five known superfamilies of growth ing. Presumably, iNOS activity can be downregu-
factors.62 Most growth factors originate from large lated by resolution of the inflammatory response
proteins that have undergone posttranslational or by cytokine signaling. It is likely that colonized
modification before being released in an active or infected wounds with continued inflammatory
state. Growth factor receptors are transmembrane responses would continue to generate large
glycoproteins that exert their effect through a ty- amounts of NO, although this has not been stud-
rosine kinase enzyme and phosphorylation ied directly. TGF-␤1 is one of the strongest iNOS
reactions.63 inhibitors during wound healing.93 However, even
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Table 5. Growth Factor Superfamilies and Their Role in Wound Healing

Superfamily Members Discussion
Platelet-derived PDGF PDGF affects cells of mesodermal origin and VEGF has its primary effect on
growth factor endothelial cells.64 PDGF was originally isolated from platelets, but it is now known
that it is secreted by many different cells types, including macrophages, monocytes,
fibroblasts, smooth muscle cells, and endothelial cells. PDGF secretion results in
chemotaxis, proliferation, and new gene expression in these cells.26
VEGF VEGF receptors are found almost exclusively on endothelial cells and act as an
effective mitogen; once stimulated, they result in angiogenesis. VEGF does not
act on macrophages, fibroblasts, or smooth muscle cells.65 Although VEGF has
little direct effect on most cells of the skin, many cells either produce it or
release factors that regulate its expression. FGF-4, PDGF, TNF-␣, IGF, and
some interleukins stimulate VEGF production and others inhibit it.66
Epidermal EGF EGF is found in saliva, urine, milk, plasma, and platelets. Platelets release it
growth factor when they degranulate. Most cells have receptors for it, but epithelial cells
have the largest number of receptors. Significant numbers of receptors are
found on endothelial cells, fibroblasts, and smooth muscle cells. EGF is
chemotactic and a potent mitogenic stimulant for epithelial cells, endothelial
cells, and fibroblasts. It also stimulates angiogenesis and collagenase activity.18,19
TGF-␣ TGF-␣ has 30% structural homology with EGF and may represent a variant of EGF
that functions more in an autocrine fashion.67 It is produced by activated
macrophages, platelets, keratinocytes, and other tissues. It stimulates mesenchymal,
epithelial, and endothelial cell growth and endothelial cell chemotaxis. 18,19
Fibroblast aFGF, bFGF FGF has two different forms: an acidic FGF (aFGF) and a basic FGF (bFGF);
growth factor both have 50% homology, although bFGF is 10 times more potent as an
angiogenic stimulant. Both aFGF and bFGF stimulate endothelial cell
proliferation and motility and contribute to wound angiogenesis. bFGF
stimulates collagen synthesis, wound contraction, epithelialization, and
fibronectin and proteoglycan synthesis.18,68
KGF-1, KGF-2 KGF is found at very low levels in normal (undamaged) skin; however, after
tissue damage, fibroblasts produce high quantities. KGF-1 is the most potent
mediator of keratinocyte proliferation and motility. It also results in
production of glutathione peroxidase, a DNA repair enzyme that helps to
protect the keratinocyte from damaging reactive oxygen species released into
the wound by neutrophils to sterilize the wound.69 KGF-2 shares 57% homology
with KGF-1 and has been shown to increase granulation tissue formation by
directly stimulating the migration of fibroblasts into wounds.60
Transforming TGF-␤1, ␤2, ␤3 TGF-␤ got its name because of the initial and now erroneous belief that it was
growth factor capable of transforming normal cells into malignant ones. Several subtypes
have been identified, but there are no known major differences in terms of
function.70 TGF-␤ has been isolated from platelets, macrophages, lymphocytes,
bone, and kidneys.18 Like PDGF, it is released by platelets during
degranulation71 (in case you were wondering, they are found in the alpha
granules). It stimulates monocytes to secrete other growth factors (FGF, PDGF,
TNF-␣, and IL-1)72 and is chemotactic for macrophages and regulates its own
production within macrophages in an autocrine fashion.18 TGF-␤ stimulates
fibroblast chemotaxis and proliferation. At different concentrations, it can
either stimulate or inhibit cellular proliferation, and this effect may be
regulated or driven by what other growth factors are present.18,73 It may be the
most potent stimulant for collagen synthesis,73 but it also decreases the
stimulatory effect of other factors on collagenase activity.74 TGF-␤ also
stimulates fibronectin and proteoglycan synthesis by fibroblasts75,76 and
fibronectin synthesis by keratinocytes.77 It also has the ability to organize the
extracellular matrix and may be involved in scar remodeling and wound
contracture.78 It stimulates epithelial cell proliferation and inhibits endothelial
cell proliferation, but with a cofactor it will stimulate angiogenesis.18
Insulin growth IGF-I and IGF-II IGF-II is most prominent during fetal development, whereas IGF-I persists
factor throughout life and is synthesized in the liver, heart, lung, kidney, pancreas,
cartilage, brain, and muscle. IGF-I (also known as somatomedin C) is stimulated
by human growth hormone (especially in the liver) and the two together stimulate
skeletal cartilage and bone growth.79 Platelets will release IGF-I during degranulation
and fibroblasts will also produce it when stimulated. IGF-I is a potent chemotactic
agent for endothelial cells, resulting in neovascularization.80 It also stimulates
mitosis of fibroblasts, osteocytes, and chondrocytes and may act with PDGF to
enhance epidermal and dermal growth.81 It reversibly binds to an IGF-binding
protein in the plasma.82 When IGF is bound it is inactive; therefore, the affect IGF-
I has on wound healing depends on the amount of available free IGF-I.
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Table 6. Cytokine and Growth Factor Summary by Source and Function
Source Function
Leukocyte
Mast chemo- Leukocyte Wound
Class Platelet Neutrophil Macrophage Lymphocyte Fibroblast Endothelium Keratinocyte cell attractant activator Epithelialization Angiogenesis fibroplasia
IL-1 CY/IL X X X X ⫹⫹⫹ ⫹ ⫹⫹

IL-2 CY/IL X X X X ⫹
IL-4 CY/IL X ⫺ ⫺ ⫹⫹
IL-6 CY/IL X X X X ⫹⫹
IL-8 CY/CK X X X X ⫹⫹ ⫹⫹
IL-10 CY/IL ? ⫺ ⫺
TNF-␣ CY/GF X X X X ⫹⫹⫹ ⫺
TGF-␣ GF X X X ⫹ ⫹
TGF-␤ CY/GF X X X ⫹⫹ ⫹
(GM)-CSF CY/CSF X ⫹⫹⫹
(G)-CSF CY/CSF X ⫹⫹⫹
(M)-CSF CY/CSF X ⫹⫹⫹
MDC CY/CK ⫹⫹
MIP CY/CK X ⫹⫹⫹
MCP 1-5 CY/CK X ⫹⫹⫹
IFN-␥ CY/IFN X X ⫹ ⫹ —
PDGF GF X X X X ⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹
VEGF GF X X X X ⫹⫹⫹
EGF GF X X ⫹ ⫹⫹⫹ ⫹ ⫹
FGF GF X X X ⫹ ⫹⫹⫹ ⫹
KGF GF X ⫹⫹⫹ ⫹
IGF-I GF X X ⫹⫹
PF4 X ⫺
⫹, positive effect; ⫺, negative effect; GF, growth factor; CY, cytokine; CY/CK, chemokine cytokine; CY/CSF, colony-stimulating factor cytokine; CY/IFN, interferon cytokine; CY/IL, interleukin
cytokine.
Sources: Henry, G., and Garner, W. Inflammatory mediators in wound healing. Surg. Clin. North Am. 83: 483, 2003; Lawrence, W., and Diegelmann, R. Growth factors in wound healing. Clin.
Dermatol. 12: 157, 1994; Cross, K., and Mustoe, T. Growth factors in wound healing. Surg. Clin. North Am. 83: 531, 2003; and Bennet, N., and Schultz, G. Growth factors and wound healing:
Biochemical properties of growth factors and their receptors. Am. J. Surg. 165: 728, 1993.
during the inflammatory phase of wound healing, smooth muscle cells is inhibited by low does of
there is counterregulation of NO synthesis, pos- NO95; low doses of NO stimulate endothelial cells
sibly by the presence of an unknown factor that and keratinocytes to proliferate, but higher levels
reduces iNOS activity but not by substrate (in vitro) are inhibitory.98 NO has also been shown
depletion.94 to protect endothelials cells from apoptosis99 and
to arbitrate VEGF-induced endothelial cell
Role of NO in Wound Healing proliferation.100
NO exerts itself in a variety of mechanisms. Angiogenesis
Some of its effects are due to its chemical reaction Neovascularization is critical for successful
with oxygen, leading to formation of reactive rad- wound healing, and NO plays a pivotal role. VEGF
ical species, or its interaction with heme- or metal- is the most potent angiogenic factor and it appears
containing enzymes. A complete review of NO to be dependent on upon NO. VEGF helps itself
chemistry is beyond the scope of this discussion, by increasing NO production by upregulating
but highlights of its role in wound healing are eNOS.101 VEGF’s other effects–-endothelial cell
reviewed in Figure 5. migration, decreased adhesion, and organiza-
The First 72 Hours tion–-are also dependent on NO. These effects
Expression of iNOS may peak as early 48 hours may also rely on NO produced from iNOS in ad-
(Fig. 17). During this time, many of the primary dition to eNOS.95 NO also increases VEGF expres-
effects of NO are especially relevant to wound sion in stimulated keratinocytes,102 resulting in a
healing: vasodilation, antimicrobial activity, anti- rapid accumulation of VEGF and NO.
platelet aggregation activity, and induction of vas- Matrix Deposition and Remodeling
cular permeability.95 As NO concentrations rise, In animal studies and in vitro, the link between
they cause the downregulation of RANTES (a NO and collagen deposition has been well de-
monocyte-attracting chemotactic cytokine)96 and scribed. In most studies, treatment with NO do-
MCP-1 (a macrophage chemoattractant expressed nors, dietary arginine, or iNOS overexpression via
by hyperproliferating keratinocytes located on the gene therapy increases the collagen content of
wound edge).97 The net effect of this is to move the experimental wounds.95,103 Likewise, NOS inhibi-
wound from an inflammatory state toward one of tion has been found to decrease collagen and
regeneration and repair.95 granulation tissue formation in experimental
Cell Proliferation burn wounds.104 One study had the opposite re-
The capacity of NO to regulate proliferation is sult, that is, decreased wound collagen content
dependent on the level of NO and the sensitivity following a topical NO donor treatment or argi-
of the cell to NO. Proliferation of fibroblasts and nine and improved wound collagen with NOS
Fig. 5. Phases of wound healing and the generation of wound nitric oxide. Adapted from
Witte, M., and Barbul, A. Role of nitric oxide in wound repair. Am. J. Surg. 183: 406, 2002.
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inhibition.105 It is likely that the timing and level help neutrophils to adhere to the endothelium) in
of NO production in the healing wound must be a process called margination. Neutrophils next
carefully balanced to ensure a beneficial effect.95 move through the vessel wall (diapedesis) to re-
In vitro, both wound-derived and normal skin- side at the wound site. The neutrophil attaches
derived fibroblasts produce increased collagen af- itself to the extracellular matrix with integrin re-
ter NO donor treatment and decreased collagen ceptors found on the neutrophil cell surface.2 The
after NOS inhibition.95,106 This appears to be pri- neutrophil’s role is phagocytosis and wound dé-
marily due to posttranslational enhancement of bridement. During the inflammatory stage, neu-
collagen synthesis and not to increased transcrip- trophils phagocytize invading organisms and de-
tion of relevant collagen genes.95 bris and release proteolytic enzymes to destroy the
invading organisms and digest nonviable tissue.
Cells There are several protease classes, depending on
preferred target protein. All the protease types
Platelets destroy preexisting extracellular matrix. Matrix in
Platelets play a pivotal role in wound healing. unwounded tissue is protected by protease
To achieve wound hemostasis, the coagulation inhibitors.11 This protection can be overwhelmed
cascade is initiated and platelet ␣-granules are by the massive release of proteases by neutrophils
opened, releasing large quantities of TGF-␤ sometimes seen in the inflammatory phase of
and more (see below). This early concentration wound healing. Neutrophils also generate (via the
of TGF-␤ stimulates the chemotaxis of macro- myeloperoxidase pathway) reactive oxygen free
phages and lymphocytes and enhances their radicals that combine with chloride and assist in
proliferation.107 Lymphocytes and monocytes bacterial killing within acute wounds.11 As time
are also attracted to the wound by other plate- passes, neutrophils are replaced by macrophages.
let-derived inflammatory products (i.e., PDGF, Neutrophils are not required for wound healing or
TGF-␤, PF4, C5a complement, PAF, and leuko- collagen synthesis.110 Through a mechanism that
triene B4).18,107,108 The thrombus itself also has a is not yet fully understood, neutrophils receive a
role to play, as the fibrin provisional matrix of signal to end their destructive débridement of the
the resolving thrombus serves as a protein res- wound, undergo apoptosis, and are ingested by
ervoir by binding cytokines and growth factors, macrophages.2
locally concentrating and magnifying their Macrophages
affects.4 MCP-1 is closely associated with newly Macrophages migrate into the wound 48 to 96
formed thrombus and levels increase even hours after injury and are the predominant cell
more with thrombus resolution.109 type before the fibroblasts migrate and begin rep-
The following factors are found in the plate- licating. Macrophages are important and neces-
let’s ␣-granules: PDGF, TGF-␤, FGF, EGF, sary for wound healing. Macrophages are the “or-
␤-thromboglobulin, PF4, platelet-derived angio- chestra leader”18 of wound healing because of
genesis factor, histamine, serotonin, bradykinin, their important role directing the wound-healing
prostaglandins, prostacyclin, and thromboxane. process (Fig. 6). Macrophages complete the neu-
Neutrophils trophil’s job of débridement and conclude the
Neutrophils are the first immune cells to arrive inflammatory response with the release of cyto-
at the wound site, peaking at 24 hours. Increased kines and growth factors. Macrophages use phago-
vascular permeability due to inflammation and cytosis and reactive radicals (nitric oxide, oxygen,
release of prostaglandins, together with a concen- and peroxide) to sterilize the wound and enzymes
tration gradient of chemotactic substances re- (collagenase and elastase) to débride the wound.2
leased by platelets such as complement factors, Macrophages, unlike neutrophils, lack myelo-
IL-1, TNF-␣, TGF-␤, PF4, and bacterial products,2,8 peroxidase but do assist in proteolysis and patho-
stimulate neutrophil migration into the injured gen killing. Their major contribution to wound
area. Neutrophils attached or caught up in the healing is the secretion of cytokines and growth
thrombus at the wound site transform chemokine factors. These cytokines act in a paracrine manner
connective tissue–activating peptide III into neu- to activate and recruit other cells involved in
trophil-activating peptide-2; this is one of the first wound healing, such as other macrophages or lym-
potent signals for neutrophil chemotaxis.28 Neu- phocytes. Macrophages secrete many different
trophils will adhere to the endothelium at the types of metalloproteinases that degrade the col-
wounded site by binding to selectins (receptors on lagen matrix.28 The cytokines TNF-␣ and IL-1 may
the endothelial cell surface that preferentially activate iNOS in macrophages to synthesize large
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Fig. 6. The main functions of macrophages in wound healing are phagocytosis, cellular
recruitment and activation, angiogenesis, regulation of matrix synthesis, and wound dé-
bridement. Effector mechanisms with examples are shown in the boxes. Reprinted with per-
mission from Witte, M., and Barbul, A. General principles of wound healing. Surg. Clin. North
Am. 77: 509, 1997.
amounts of NO.10 Macrophage-synthesized NO re- they are referred to as “wound fibroblasts.”23 Mac-
acts with peroxide ion-oxygen radicals to yield the rophage-derived cytokines trigger the phenotypic
more toxic peroxy nitrite and hydroxyl radicals for transformation of fibroblasts.24 This has been well
pathogen killing. NO helps kill Staphylococcus au- documented for the myofibroblastic phenotype,
reus, prevents the replication of DNA viruses which is strongly induced by TGF-␤1.25 The sur-
within cells, and serves as an immune regulator.12 rounding matrix also influences the fibroblast’s
Cytokines and growth factors also regulate fibro- phenotype. Cell adhesion promoted by synthesis
blast chemotaxis, proliferation, and collagen syn- of the extracellular matrix molecule, fibronectin,
thesis, as well as other cells involved in the repair can also result in phenotypic alteration.114,115 Fi-
process, such as endothelial cells.111,112 Through broblasts and endothelial cells are the primary
these various functions, macrophages influence cells in the proliferative phase. Fibroblasts migrate
angiogenesis, fibroplasia, and extracellular matrix
into the wound site from the surrounding tissue.
synthesis.
Fibroblasts in the surrounding tissue need to be-
Monocytes come activated from their quiescent state. The
Upon arrival to the wound site, blood and growth factors and cytokines responsible for their
tissue monocytes are stimulated to transform into activation and proliferation are mainly from plate-
macrophages by IL-2, TNF-␣, PDGF, and IFN-␥ lets and activated macrophages. Some of them are
(released by T lymphocytes).28 stored in the fibrin clot and the fibroblasts them-
Fibroblasts selves can be induced to release growth factors and
The fibroblast undergoes phenotypic changes cytokines in an autocrine manner. The most im-
during wound healing.113 Fibroblasts derived from portant growth factor for fibroblast proliferation
the wound are characterized by increased collagen is PDGF. Table 3 summarizes the effect different
synthesis and contraction but decreased prolifer- growth factors and cytokines have on fibroblast
ation compared with normal dermal fibroblasts; proliferation.
29S
Table 7. Various Collagen Types Grouped by Major Collagen Families

Type Tissue Distribution
Fibril-forming collagens
I Bone, dermis, tendon, ligaments, cornea
II Cartilage, vitreous body, nucleus pulposus
III Skin, vessel wall, reticular fibers of most tissues (lungs, liver, spleen, and so on)
V Lung, cornea, bone, fetal membranes; together with type I collagen
XI Cartilage, vitreous body
Basement membrane collagen
IV Basement membranes
Microfibrillar collagen
VI Widespread: dermis, cartilage, placenta, lungs, vessel wall, intervertebral disc
Anchoring fibril
VII Skin, dermal-epidermal junctions; oral mucosa, cervix
Hexagonal network-forming collagens
VIII Endothelial cells, Descemet’s membrane
X Hypertrophic cartilage
Fibril-associated collagens with interrupted
triplet helices (FACIT)
IX Cartilage, vitreous humor, cornea
XII Perichondrium, ligaments, tendon
XIV Dermis, tendon, vessel wall, placenta, lungs, liver
XIX Human rhabdomyosarcoma
XX Corneal epithelium, embryonic skin, sternal cartilage, tendon
XXI Blood vessel wall
Transmembrane collagens
XIII Epidermis, hair follicle, endomysium, intestine, chondrocytes, lungs, liver
XVII Dermal-epidermal junctions
Multiplexins
XV Fibroblasts, smooth muscle cells, kidney, pancreas,
XVI Fibroblasts, amnion, keratinocytes
XVIII Lungs, liver
Adapted from Gelse, K., Pöschl, E., and Aigner, T. Collagens: Structure, function, and biosynthesis. Adv. Drug Deliv. Rev.55: 1531, 2003.
Keratinocytes production. As the capillaries form, endothelial

Keratinocytes located next to the wound re- cells express endothelial nitric oxide synthase,
ceive their movement orders from the fibroblasts. which generates even more NO that protects the
Cytokines IL-1 and TNF-␣ upregulate KGF gene tissue from hypoxia and ischemia by inducing va-
expression in fibroblasts.99 In response, fibroblasts sodilation and protecting against reperfusion
secrete KGF-1, KGF-2, and IL-6; this causes kera- injury.10
tinocytes to proliferate, migrate into the wound,
and then differentiate into the epidermis.20 Ker- Collagen
atinocyte migration is sensitive to the extracellular There are 21 known collagens. Their synthesis
matrix environment. Collagen types I and IV, fi- occurs as it does for any other protein within the
bronectin, and vitronectin all seem to facilitate cell. The collagen molecule is characterized by the
keratinocyte migration. Collagen, in the absence repeating sequence Gly-X-Y, with X often being
of cytokines, can still drive keratinocyte proline and Y often being hydroxyproline. The
migration.28 The stimulated keratinocytes also se- molecule undergoes the following eight posttrans-
crete IL-6 and NO, which provides additional pos- lational steps until it is secreted as procollagen116 :
itive stimulation for other keratinocytes to migrate (1) cleavage of the signal peptides; (2) hydroxy-
and proliferate, thereby perpetuating the process. lation of the proline or lysine amino acids in the
As the keratinocytes proliferate to “fill in the hole,” x-position to 4-hydroxyproline or 4-hydroxylysine;
they will need a new capillary network. Keratino- (3) hydroxylation of some proline residues to 3-hy-
cytes initiate neovascularization by secreting droxyproline; (4) glycosylation of some hydroxy-
VEGF, which is synthesized by keratinocytes at the lysine molecules with galactose or glucose; (5)
wound edge.12 Recall that VEGF expression is stim- addition of oligosaccharides to the propeptides;
ulated by IL-1, TNF-␣, KGF, and TGF-␤. (6) association of the c-terminal propeptides; (7)
Endothelial Cells formation of interchain and intrachain disulfide
Dermal endothelial cells respond to VEGF by bonds; and (8) formation of the triple helix, which
proliferating and forming capillary tubes. Endo- starts at the c-terminal end and goes to the n-
thelial cells synthesize NO, which increases VEGF terminal end.
30S
Fig. 7. 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. From
Witte, M., and Barbul, A. General principles of wound healing. Surg. Clin. North Am. 77: 509, 1997.
After posttranslational modifications are com- intraperitoneal thymic grafts at the time of
plete, the triple helix is secreted as procollagen thymectomy. Interestingly, neonatal or intrauter-
into the extracellular environment, where the ine thymectomies, which prevent T-cell matura-
propeptide ends are specifically cleaved by pro- tion, have no effect on wound-healing parameters.
collagen C-proteinases and procollagen N-pro- Postnatal or adult thymectomies have a more se-
teinases. This cleaving process is directly respon- lective effect by preventing induction of suppres-
sible for the decrease in the solubility of the sor T cells. The administration of thymic hor-
molecule. Then the process of fibril formation mones (thymopentin and thymulin) to nude mice
begins. The cross-linking of fibrils occurs after decreases wound-breaking strength and collagen
several lysine and hydroxylysine residues have levels.120 This suggests that the thymus has an in-
their free amino acid group transformed to alde- hibitory effect on wound healing and that this
hyde residues by the enzyme lysyl oxidase. Cross- effect may be mediated by T-suppressor lympho-
linking occurs between these aldehyde groups and cytes.
amino acid groups of the nontransformed lysine Studies of CD4⫹ immature effector T cells
or hydroxylysine residues.35 The collagen types have the potential to differentiate into an inflam-
and tissue distribution are shown in Table 7. matory T cell or a helper T cell; each has distinct
T Lymphocytes cytokine profiles. Both T cell types express IL-3
T lymphocytes migrate into the wound after and granulocyte-macrophage CSF.121,122 Inflam-
inflammatory cells and macrophages on the fifth matory T cells also express IL-2, IFN-␥, and TNF-␤,
day after injury, during the proliferative phase, whereas helper T cells express IL-4, IL-5, IL-6,
and peak at day 7.117 It was long thought that IL-10, and IL-13.123 Histological studies of healing
lymphocytes, although predictably present in the wounds comparing CD4⫹ and CD8⫹ cells have
wound, made no significant contribution to convincingly demonstrated that T lymphocytes do
wound healing. However, a series of experimental regulate wound healing. The inflammatory T cells
studies indicated a significant role for T lympho- are proinflammatory, helper T cells are suppres-
cytes in this process.118 Adult thymectomy in rats sive, and there is a relationship of CD4⫹ to CD8⫹
increases wound maturation and cross-linking of ratios that shows increased CD4⫹ is upregulatory
collagen.119 This effect is reversed by placement of and CD8⫹ is downregulatory.124,125 Therefore, it
31S
appears that T lymphocytes may control the pro- 15. Nathan, C. Points of control in inflammation. Nature 420:
liferation phase of wound healing. 846, 2002.
16. Clària, J., and Serhan, C. Aspirin triggers previously undescribed
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17. Levy, G. Prostaglandin H synthases, nonsteroidal anti-in-
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important pathways have been abbreviated and sim-
20. Smola, H., Thiekotter, G., and Fusenig, N. Mutual induc-
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sion grows, our current perceptions and hierarchies dermal cell interaction. J. Cell Biol. 122: 417, 1993.
of importance will undoubtedly need to change. 21. Xia, Y., Zhao, Y., Marcus, J., et al. Effects of keratinocyte
growth factor-2 (KGF-2) on wound healing in an ischemia-
George Broughton II, M.D., Ph.D., COL., M.C., U.S.A. impaired rabbit ear model and on scar formation. J. Pathol.
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RECONSTRUCTIVE

The Basic Science of Wound Healing

Table 1. Summary of Wound Healing

Hemostasis Entrapped platelets within the thrombus release:

Mediator: fibrin, plasma fibronectinResult: coagulation, chemoattraction,

Role of neutrophil Increased vascular permeability from endothelial prostaglandin and

The lipoxin is capable of turning off destructive inflammatory acts in a variety

Fibroblasts stimulate keratinocytes to express VEGF with KGF-2 and TGF-␤

Keratinocytes direct angiogenesis at the wound edge by expressing VEGF.

VEGF expression is upregulated by the presence of NO

Maturation and remodeling phase

TGF-␤ also upregulates expression of TIMP, decreases MMP production, and

Table 2. Hemostatic and Platelet-Derived Factors Associated with Wound Healing

Fig. 1. Effect of aspirin of lipoxin synthesis.

Table 3. Effect of Growth Factors on Fibroblast Proliferation

Fig. 4. Inflammatory actions of eicosanoids and their synthesis.

Table 4. The Role of Interleukins during Wound Healing

Table 5. Growth Factor Superfamilies and Their Role in Wound Healing

IL-1 CY/IL X X X X ⫹⫹⫹ ⫹ ⫹⫹

Table 7. Various Collagen Types Grouped by Major Collagen Families

Keratinocytes production. As the capillaries form, endothelial

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