Wound Healing: An Overview of Acute, Fibrotic and Delayed Healing

The following article was written by: Robert F. Diegelmann - Departments of Biochemistry, Anatomy and
Emergency Medicine, Medical Center of Virginia Melissa C. Evans - Pediatric Critical Care, Medical
Center of Virginia
Cytokine
Platelet-derived
growth factor
(including isoforms
AA,AB and BB)
Transforming growth
factor beta (including
B1, B2 and B3)
Epidermal growth
factor
Transforming growth
factor alpha
Fibroblast growth
factor family
Keratinocyte growth
factor (also called
FGF-7)
Insulin-like growth
factor-1
Connective tissue
growth factor
Vascular endothelial
cell growth factor
Tumor necrosis factor
Interleukins
Interferons
Symbol
PDGF
TGFB
EGF
TGFa
FGF
KGF
IGF-1
CTGF
VEGF
TNF
IL-1, etc.
IFN gamma
etc.
Source
Platelets, macrophages,
endothelial cells,
keratinocytes, smooth
muscle cells
Platelets, T-lymphocytes,
macrophages, endothelial
cells,  keratinocytes,  
smooth muscle cells,
fibroblasts
Platelets, macrophages,
saliva, urine, milk, plasma
Macrophages,
T-lymphocytes,
keratinocytes and many
tissues
Macrophages, mast cells,
T-lymphocytes, endothelial
cells, fibroblasts, and many
tissues
Fibroblasts
Liver, macrophages,
fibroblasts and other tissues
Endothelial cells, fibroblasts
Keratinocytes
Macrophages, mast cells,
T-lymphocytes
Macrophages, mast cells,
keratinocytes, lymphocytes
and many tissues
Lymphocytes and fibroblasts
Functions
Chemotactic for PMNs, macrophages,
fibroblasts and smooth muscle cells.
Activates PMNs, macrophages and
fibroblasts. Mitogenic for fibroblasts,
endothelial cells and smooth muscle cells.
Stimulates angiogenesis and wound
contraction. Remodeling. Inhibits platelet
aggregation. Regulates integrin expression.
Chemotactic for PMNs, macrophages,
lymphocytes, fibroblasts and smooth muscle
cells. Stimulates TIMP synthesis,
keratinocyte migration, angiogenesis and
fibroplasia. Inhibits MMPs production and
keratinocyte proliferation. Regulates integrin
expression and other cytokines. Induces
TGFB production.
Mitogenic for keratinocytes and fibroblasts.
Stimulates keratinocyte migration and
granulation tissue formation.
Similar to EGF
Chemotactic for fibroblasts. Mitogenic for
fibroblasts and keratinocytes. Stimulates
keratinocyte migration, angiogenesis, wound
contraction and matrix deposition.
Stimulates keratinocyte migration
proliferation and differentiation.
Stimulates synthesis of sulfated
proteoglycans, collagen, keratinocyte
migration and fibroblast proliferation.
Endocrine effects similar to growth hormone.
Chemotactic and mitogenic for various
connective tissue cells.
Increases vaso-permeability. Mitogenic for
endothelial cells.
Activates macrophages. Mitogenic for
fibroblasts. Stimulates angiogenesis.
Regulates other cytokines.
Chemotactic for PMNs (IL-1,8) and
fibroblasts (IL4). Stimulates MMP-1
synthesis (IL-1), angiogenesis (IL-8), TIMP
synthesis (IL-6). Regulates other cytokines.
Activates macrophages. Inhibits fibroblast
proliferation and MMP synthesis. Regulates
other Cytokines.
Table of Contents:
  1. Abstract
  2. Introduction
  3. Cell Signaling
  4. Normal and Pathological
    Responses to Injury
  5. The Healing Cascade
  6. Fibrosis
  7. Chronic Ulcers
  8. References
1. Abstract
Acute wounds normally heal in a very orderly and efficient manner characterized by four distinct, but
overlapping phases: hemostasis, inflammation, proliferation and remodeling. Specific biological markers
characterize healing of acute wounds. Likewise, unique biologic markers also characterize pathologic
responses resulting in fibrosis and chronic non-healing ulcers. This review describes the major biological
processes associated with both normal and pathologic healing.

The normal healing response begins the moment the tissue is injured. As the blood components spill into
the site of injury, the platelets come into contact with exposed collagen and other elements of the
extracellular matrix. This contact triggers the platelets to release clotting factors as well as essential
growth factors and cytokines such as platelet-derived growth factor (PDGF) and transforming growth
factor beta (TGF-B). Following hemostasis, the neutrophils then enter the wound site and begin the critical
task of phagocytosis to remove foreign materials, bacteria and damaged tissue. As part of this
inflammatory phase, the macrophages appear and continue the process of phagocytosis as well as
releasing more PDGF and TGF-B. Once the wound site is cleaned out, fibroblasts migrate in to begin the
proliferative phase and deposit new extracellular matrix. The new collagen matrix then becomes
cross-linked and organized during the final remodeling phase. In order for this efficient and highly controlled
repair process to take place, there are numerous cell-signaling events that are required.

In pathologic conditions such as non-healing pressure ulcers, this efficient and orderly process is lost and
the ulcers are locked into a state of chronic inflammation characterized by abundant neutrophil infiltration
with associated reactive oxygen species and destructive enzymes. Healing proceeds only after the
inflammation is controlled. On the opposite end of the spectrum, fibrosis is characterized by excessive
matrix deposition and reduced remodeling. Often fibrotic lesions are associated with increased densities of
mast cells. By understanding the functional relationships of these biological processes of normal compared
to abnormal wound healing, hopefully new strategies can be designed to treat the pathological conditions.

2. Introduction
To understand the underlying mechanisms involved in pathologic conditions such as fibrosis and chronic
non-healing ulcers, it is helpful to first review what is known about normal tissue response to injury. The
human body can sustain a variety of injuries, including penetrating trauma, burn trauma and blunt trauma. All
of these insults set into motion an orderly sequence of events that are involved in the healing response,
characterized by the movement if specialized cells into the wound site. Platelets and inflammatory cells are
the first cells to arrive at the site of injury and they provide key functions and signals needed for the influx of
connective tissue cells and a new blood supply. These chemical signals are known as cytokines or growth
factors. (Fig 1)
Figure 1. Cell signaling by cytokines.

The fibroblast is the connective tissue cell responsible for collagen deposition that is needed to repair the
tissue injury. (Fig 2)
Regeneration
Exact
Replacement
Normal Repair
Reestablished
Equilibrium
Deficient
Healing
Chronic Ulcers
Excessive Healing
Fibrosis and
Contractures
Figure 2. The four possible responses following tissue injury.

Collagen is the most abundant protein in the animal kingdom, accounting for 30% of the total protein in the
human body. (Fig 3)
Figure 3. At the time of injury, the tissue is disrupted and the platelets adhere to the exposed collagen and
to each other. The platelets release clotting factors, PDGF and TGF-8 to initiate the repair process.

In normal tissues collagen provides strength, integrity and structure. When tissues are disrupted following
injury, collagen is needed to repair the defect and restore anatomic structure and function. If too much
collagen is deposited in the wound site, normal anatomical structure is lost, function is compromised and
fibrosis occurs. Conversely, if an insufficient amount of collagen is deposited, the wound is weak and may
dehisce. (Fig 4)
Figure 4. By the first day following injury, neutrophils attach to endothelial cells in the vessel walls
surrounding the wound (margination), then change shape to move through the cell junctions (diapedesis)
and migrate to the wound site (chemotaxis). This is the beginning of the inflammatory phase.

Therefore, to understand fully the process of wound healing, it is essential to understand first the basic
cell biology, immunology and biochemistry involved in the processes of inflammation and collagen
metabolism, and how these pathways are regulated.

3. Cell Signaling
The many diverse activities taking place during wound healing are directed by chemical signals referred to
as growth factors or cytokines (1, 5, 6). Originally these signals were named growth factors because that
was their main observed function. As more information was developed it became apparent that many of
these factored controlled more than just cell growth. Cell migration, matrix production, enzyme expression
and differentiation can also be controlled by these factors. Therefore the term cytokine may be a better
description for these chemical signals. A listing of some of the cytokines important for wound healing is
presented in Table 1.

Table 1 - Cytokines and Chemokines Involved in Wound Healing
These cytokines range in weight from 4 to 60 kD and they direct cellular activity when they are present in
very small quantities. In general these factors are very stable. However in the chronic wound environment
where there are increased numbers of neutrophils releasing proteolytic enzymes, such as a neutrophil
elastase, they can be destroyed. Cytokines can regulate cellular activities and functions via endocrine,
paracrine, autocrine and intracrine mechanisms (Fig 1). In order for a particular cytokine to modulate a
cellular activity the target cell must have a receptor. Once receptor binding takes place then a series of
intracellular signals are activated and eventually result in a specific response. Many of the signal pathways
are mediated via activation of tyrosine kinase (7). The number of receptors expressed on the target cell
can also regulate the cell-signaling cascade to some extent (8)

4. Normal And Pathological Responses to Injury
The term wound has been defined as a disruption of normal anatomical structure and, more importantly,
function. Therefore, healing is the complex and dynamic process that results in the restoration of
anatomical continuality and function (4). There are four basic responses that can occur following an injury
(Fig 2). Normal repair is the response where there is a re-established equilibrium between scar formation
and scar remodeling. This is the typical response that most humans experience following injury. The
pathological responses to tissue injury stand in sharp contrast to the normal repair response. In excessive
healing there is too much deposition of connective tissue that results in altered structure and, thus, loss of
function (9). Fibrosis, strictures, adhesions and contractures are examples of excessive healing. Keloids
and hypertrophic scars in the skin are examples of fibrosis (10, 11). Contraction is part of the normal
process of healing but if excessive, it becomes pathologic and is known as a contracture (12). Deficient
healing is the opposite of fibrosis; it exists when there is insufficient deposition of connective tissue matrix
and the tissue is weakened to the point where it can fall apart. Chronic non-healing ulcers are examples of
deficient healing. Regeneration is the elegant process that occurs when there is loss of structure and
function but the organism has the sophisticated capacity to replace that structure by replacing exactly
what was there before the injury. Lower forms of life, such as the salamander and crab, can regenerate
tissues in this manner. As man has evolved, we have lost this capacity and can only replace a limited
amount of damaged tissues by the process of regeneration. In humans the liver, epidermis and, to some
extent, nerves can be partially regenerated after injury. In addition, our laboratory has examined the
process of fetal tissue repair, and it appears that the fetus has the capacity to repair tissue by a process
that closely resembles true regeneration (13).
Basically, all dermal wounds heal by three basic mechanisms: connective tissue matrix deposition,
contraction and epithelization. Wounds that are simple and can be closed by sutures, tape or staples heal
by Primary Intention (14). The main mechanism of healing during Primary Intention is connective tissue
matrix deposition, where collagen, proteoglycans and attachment proteins are deposited to form a new
extracellular matrix. In contrast, wounds that remain open heal mainly by contraction; the interaction
between cells and matrix results in movement of tissue toward the center of the wound. The underlying
mechanisms responsible for contraction are not fully understood but there appears to be a complex
interaction between contractile fibroblasts sometimes referred to as myofibroblasts and the matrix
components (15). Some work has indicated that nerve growth factor and IL-8 can modulate the
contraction response (16). Epithelization is the process where epithelial cells around the margin of the
wound or in residual skin appendages such as hair follicles and sebaceous glands lose contact inhibition
and begin to migrate into the wound area by the process termed epiboly (17). As migration proceeds,
cells in the basal layers begin to proliferate to provide additional epithelial cells.

5. The Healing Cascade
The healing cascade begins immediately following an injury when the platelets come into contact with
exposed collagen (Fig 3). As platelet aggregation proceeds, clotting factors are released resulting in the
deposition of a fibrin clot at the site of injury. The fibrin clot serves as a provisional matrix and sets the
stage for the subsequent events of healing (18). Platelets not only release the clotting factors needed to
control the bleeding and loss of fluid and electrolytes but they also provide a cascade of chemical signals,
known as cytokines or growth factors, that initiate the healing response. The two most important signals
are platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-B) (5).The PDGF
initiates the chemotaxis of neutrophils, macrophages, smooth muscle cells and fibroblasts. In addition it
also stimulates the mitogenesis of the fibroblasts and smooth muscle cells.
TGF-B adds another important signal for the initiation of the healing cascade by attracting macrophages
and stimulates them to secrete additional cytokines including FGF (fibroblast growth factor), PDGF, TNFa
(tumor necrosis alpha) and IL-1 (interleukin-1). In addition, TGF-8 further enhances fibroblast and smooth
muscle cell chemotaxis and modulates collagen and collagenase expression. The net result of these
redundant signals is a vigorous response of the matrix producing cells to ensure a rapid deposition of new
connective tissue at the injury site during the Proliferative phase that follows the Inflammatory phase.

Neutrophils are the next predominant cell marker in the wound within 24 hours after injury (Fig 4). The
major function of the neutrophil is to remove foreign material, bacteria and non-functional host cells and
damaged matrix components that may be present in the wound site (19, 20). Bacteria give off chemical
signals, attracting neutrophils, which ingest them by the process of phagocytosis. During bacterial protein
synthesis a waste product represented by a tri-peptide called f-Met-Leu-Phe is released which in turn
attracts inflammatory cells (21). Neutrophils will engorge themselves until they are filled with bacteria and
constitute what is called laudable pus in the wound (22).
The mast cell is another marker cell of interest in wound healing. Mast cells release granules filled with
enzymes, histamine and other active amines and these mediators are responsible for the characteristic
signs of inflammation around the wound site (23). The active amines released from the mast cell causes
surrounding vessels to become leaky and thus allow the speedy passage of the mononuclear cells into the
injury area. In addition fluid accumulates at the wound site and the characteristic signs of inflammation
begin. The signs of inflammation have been well recognized since ancient times: rubor (redness), calor
(heat), tumor (swelling) and dolar (pain).
By 48 hours after the injury, fixed tissue monocytes become activated to become wound macrophages
(Fig 5).
Figure 5. The inflammatory phase continues as fixed tissue macrophages become active and move into the
site of injury and transform into very active wound macrophages. These highly phagocytic cells also release
PDGF and TGF-B to recruit fibroblasts to the site and thus begin the proliferative phase.

These specialized wound macrophages are perhaps the most essential inflammatory cells involved in the
normal healing response (24). Inhibition of macrophage function will delay the healing response (25). Once
activated these wound macrophages also release PDGF and TGF-B that further attracts fibroblasts and
smooth muscle cells to the wound site. These highly phagocytic macrophages are also responsible for
removing nonfunctional host cells, bacteria-filled neutrophils, damaged matrix, foreign debris and any
remaining bacteria from the wound site. The presence of wound macrophages is a marker that the
Inflammatory phase is nearing an end and that the Proliferative phase is beginning. Lymphocytes come into
the wound area at a later stage but are not considered to be major inflammatory cells involved in the
healing response; their precise role in the wound healing process remains unclear.
As the Proliferative phase progresses, the TGF-B released by the platelets, macrophages and T
lymphocytes becomes a critical signal. TGF-B is considered to be a master control signal that regulates a
host of fibroblast functions (26). TGF-B has a three-pronged effect on extracellular matrix deposition (27).
First, it increases transcription of the genes for collagen, proteoglycans and fibronectin thus increasing the
overall production of matrix proteins. At the same time TGF-B decreases the secretion of proteases
responsible for the breakdown of the matrix and it also stimulates the protease inhibitor, tissue inhibitor of
metallo-protease (TIMP) (28). Other cytokines considered to e important are interleukins, fibroblast growth
factors and tumor necrosis factor-alpha (Table 1).
As healing progresses several other important biological responses are activated. The process of
epithelization is stimulated by the presence of EGF (epidermal growth factor) and TGFa (transforming
growth factor alpha) that are produced by activated wound macrophages, platelets and keratinocytes (Fig
6) (29, 30 31).
Figure 6. The remodeling phase is characterized by continued synthesis and degradation of the
extracellular matrix components trying to establish a new equilibrium.

Once the epithelial bridge is complete, enzymes are released to dissolve the attachment at the base of
the scab resulting in removal. Due to the high metabolic activity at the wound site, there is an increasing
demand for oxygen and nutrients. Local factors in the wound microenvironment such as low pH, reduced
oxygen tension and increased lacate actually initiate the release of factors needed to bring in a new blood
supply (32, 33). This process is called angiogensis or neovasculatization and is stimulated by vascular
endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF) and TGFB (34, 35).
Epidermal cells, fibroblasts, macrophages and vascular endothelial cells produce these factors. One
interesting signaling pathway involves the role of low oxygen tension that in turn stimulates the expression
of a nuclear transcription factor termed hypoxia-inductible factor (HIF) by vascular endothelial cells (36).
The HIF in turn binds to specific sequences of DNA that regulate the expression of VEGF thus stimulating
angiogenesis. As new blood vessels enter the wound repair area and the oxygen tension returns to normal
level, oxygen binds to HIF and blocks its activity leading to a decreased synthesis of VEGF.
As the Proliferative phase progresses the predominant cell in the wound site is the fibroblast. This cell of
mesenchymal origin is responsible for producing the new matrix needed to restore structure and function
to the injured tissue. Fibroblasts attach to the cables of the provisional fibrin matrix and begin to produce
collagen (18). At least 23 individual types of collagen have been identified to date but type 1 is
predominant in the scar rissue of skin (3). After transcription and processing of the collagen messenger
ribonucleic acid, it is attached to polyribosomes on the endoplasmic reticulum where the new collagen
chains are produced. During this process, there is an important step involving hydroxylation of proline and
lysine residues (37). The collagen molecule begins to form its characteristic triple helical structure and the
nascent chains undergo further modification by the process of glycosylation (38). The procollagen
molecule is then secreted into the extracellular spaces where it is further processed (39). Hydroxyptoline
in collagen is important because it gives the molecule its stable helical conformation (40). Fully
hydroxylated collagen has a higher melting temperature. When hydroxyproline is not present, for example
in collagen produced under anaerobic or Vitamin C-deficient conditions (scurvy), the collagen has an
altered structure and can undergo denatutation much more rapidly and at lower temperature (37, 41).
Finally, the collagen released into the extracellular space undergoes further processing by cleavage of the
procollagen N and C-terminal peptides. In the extra-cellular spaces an important enzyme, lysyl oxidase,
acts on the collagen to form stable cross-links. As the collagen matures and becomes older, more and
more of these intramolecular and intermolecular cross-links are placed in the molecules. This important
cross-linking step gives collagen its strength and stability over time (42).
Dermal collagen on a per weight basis approaches the tensile strength of steel; in normal tissue it is a
strong and highly organized molecule. In contrast, collagen fibers formed in scar tissue are much smaller
and have a random appearance, scar tissue is always weaker and will break apart before the surrounding
normal tissue. The regained tensile strength in a wound will never approach normal. In fact the maximum
tensile strength that a wound can ever achieve is approximately 80% of normal skin.
Finally, in the process of collagen remodeling, collagen degradation also occurs (43, 44). Specific
collagenase enzymes in fibroblasts, neutrophils and macrophages clip the molecule at a specific site
through all three chains, and break it down to characteristic three-quarter and one-quarter pieces. These
collagen fragments undergo further denaturation and digestion by other protcases.
In summary, the normal healing cascade begins with an orderly process of hemostasis and fibrin
deposition, which leads to an inflammatory cell cascade, characterized by neutrophils, macrophages and
lymphocytes within the tissue (45). This is followed by attraction and proliferation remodeling by collagen
cross-linking and scar maturation (Fig 7).
Figure 7. The sequence of events during normal wound healing. Reprinted with permission (45).

Despite this orderly sequence of events responsible for normal wound healing, pathologic responses
leading to fibrosis or chronic ulcers may occur if any part of the healing sequence is altered.

6. Fibrosis
Fibrosis can be defined as the replacement of the normal structural elements of the tissue by distorted,
non-functional and excessive accumulation of scar tissue. This is perhaps the most significant biological
marker for fibrosis. Many clinical problems are associated with excessive scar formation (46). For
example, keloids and hypertrophic scars in the skin, tendon adhesions, transmission blockage following
nerve injury, scleroderma, Chrohn's disease, esophageal strictures, urethral strictures, capsules around
breast implants, liver cirrhosis, atherosclerosis and fibrotic non-union in bone.
Keloids can be used as a clinical example of fibrosis to define some of the biochemical and cellular
markers characteristic of fibrosis (11, 47). Fibroblasts isolated from keloids produce about 2 to 3 times
more collagen compared to fibroblasts isolated from normal skin in the same patients (48). It appears that
keloids have increased expression of TGFB and also an up-regulation of receptors for TGFB (49, 50).
Hypertrophic scars are also characterized by excessive accumulation of scar collagen and are frequently
misdiagnosed as keloids. There is one very significant biological marker that distinguishes keloids from
hypertrophic scars and that is the absence of myofibroblasts in keloids and an abundance of these
contractile cells in hypertrophic scars (1). It is also interesting to note that most conditions of fibrosis are
characterized by an increased density of mast cells (52, 53). Mast cells contain specialized enzymes
capable of processing procollagen and it has been suggested that abnormal peptides are produced that
can actually stimulate collagen synthesis thus producing fibrosis (54).
7. Chronic Ulcers

Chronic non-healing dermal ulcers such as pressure ulcers contribute significantly to the morbidity and
even mortality of many patients (55). Pressure ulcers are a serious and frequent occurrence among the
immobile and debilitated patients. Spinal cord injury patients are particularly vulnerable to pressure ulcer
formation. There are approximately 225,000 spinal cord injury patients in the United States, with
approximately 9,000 new patients each year. Approximately 60% of these patients develop pressure
ulcers, and the annual cost estimate ranges from $14,000 to $25,000 per patient for medical, surgical and
nursing care. If the elderly nursing home population with pressure ulcers is added to the spinal cord injury
population, then the figure for the care of all pressure ulcers is enormous. The national expenditure for
costs related to the care of patients with pressure ulcers is over $1.3 billion per year (56)! In addition, is it
estimated that in the next 15 yeas the population over age 85 will increase from 4 million to over 17 million
individuals! Therefore, this health care problem is increasing at a dramatic rate.
Excessive infiltration of these ulcers by neutrophils appears to be a significant biological marker. The
over-abundant neutrophil infiltration is responsible for the chronic inflammation characteristic of non-healing
pressure ulcers. The neutrophils release significant amounts of enzymes such as collagenase (matrix
metalloproteinase-8) that is responsible for destruction of the connective tissue matrix (57, 58). In
addition, the neutrophils release an enzyme called elastase that is capable of destroying important healing
factors such as PDGF and TGF-B (59). Another marker of these chronic ulcers is an environment
containing excessive reactive oxygen species that further damage the cells and healing tissues (60).
These chronic ulcers will not heal until the chronic inflammation is reduced. These wounds will not respond
to the current high tech materials such as skin substitutes and topical cytokines such as PDGF until the
wound be is properly prepared by the skills of the wound care specialist (61, 62).

8. Conclusion
As we continue to develop new information about the unique biological markers associated with normal
and pathologic wound healing responses, the better prepared we will be to develop new strategies to
treat these costly clinical problems. In addition, understanding this basic biological information will allow
wound care specialists greater insight into the importance of how their skills can impact the healing
response.

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