The Mechanism of Wound Healing
The complex process of wound healing normally
proceeds from coagulation and inflammation through fibroplasia, matrix
deposition, angiogenesis, epithelialization, collagen maturation,
and finally wound contraction (Figure 6–1).
Wound healing signals include peptide growth factors, complement,
cytokine inflammatory mediators, and metabolic signals such as hypoxia and accumulated lactate. Many of these cellular signaling pathways
are redundant and pleiotropic.
Acute wound healing normally proceeds from coagulation and inflammation, through angiogenesis, fibroplasia, matrix deposition
(granulation tissue formation), collagen maturation, epithelialization, and finally wound contraction. A chronic wound fails to heal anywhere
along this wound healing pathway.
Hemostasis and Inflammation
Following injury, a wound must stop bleeding in order to heal and for the injured host to survive. It is therefore not surprising
that cellular and molecular elements involved in hemostasis also
signal tissue repair. Immediately after injury, the coagulation products fibrin, fibrinopeptides, thrombin split products, and complement components attract inflammatory cells
into the wound. Platelets activated by thrombin release insulinlike
growth factor 1 (IGF-1), transforming growth factor α (TGF-α), transforming growth factor β (TGF-β),
and platelet-derived growth factor (PDGF), which attract leukocytes, particularly macrophages, and fibroblasts into the wound. Damaged endothelial
cells respond to a signal cascade involving the complement products
C5a, tumor necrosis factor α (TNF-α),
interleukin-1 (IL-1), and interleukin-8 (IL-8) and express receptors
for integrin molecules on the cell membranes of leukocytes. Circulating
leukocytes then adhere to the endothelium and migrate into the wounded
tissue. Interleukins and other inflammatory components, such as histamine,
serotonin, and bradykinin, cause vessels first to constrict for
hemostasis and later to dilate, becoming porous so that blood plasma
and leukocytes can migrate into the injured area.
The very early wound inflammatory cells increase metabolic demand. Since the local microvasculature is damaged, a local energy sink
results, and Pao2 falls while CO2 accumulates.
Lactate in particular plays a critical role, since its source is
mainly aerobic, and its level is tightly regulated by tissue oxygen
levels. Oxidative stress is an important signal for tissue repair. These
conditions trigger reparative processes and stimulate their propagation.
Macrophages assume a dominant role in the synthesis of wound healing molecules as coagulation-mediated tissue repair signals
fall. Importantly, macrophages, stimulated by fibrin, continue to release
large quantities of lactate. This process continues even as oxygen
levels begin to rise, thereby maintaining the “environment
of injury.” Lactate alone stimulates angiogenesis and collagen deposition through the sustained production of growth factors. Unless the wound becomes
infected, the granulocyte population that dominated the first days
diminishes. Macrophages now cover the injured surface. Fibroblasts
begin to organize, mixed with buds of new blood vessels. It has
been shown that circulating stem cells, such as bone marrow–derived
mesenchymal stem cells, contribute fibroblasts to the healing wound, but
the extent of this process is as yet unknown.
Fibroplasia and Matrix Synthesis
Throughout wound healing, fibroplasia (the replication of fibroblasts) is stimulated by multiple mechanisms, starting with PDGF, IGF-1, and TGF-β released by platelets and later by the
continual release of numerous peptide growth factors from macrophages
and even fibroblasts within the wound. Growth factors and cytokines
shown to stimulate fibroplasia and wound healing include fibroblast
growth factor (FGF), IGF-1, vascular endothelial growth factor (VEGF), IL-1, IL-2, IL-8, PDGF, TGF-α, TGF-β,
and TNF-α. Dividing fibroblasts localize near the
wound edge, an active tissue repair environment with tissue oxygen tensions of approximately 40 mm Hg in normally healing wounds. In
cell culture, this Pao2 is optimum for fibroblast
replication. Smooth muscle cells are also likely progenitors because
fibroblasts seem to migrate from the adventitia and media of wound
vessels. Lipocytes, pericytes, and other cell sources may exist
for terminal differentiation into repair fibroblasts.
Fibroblasts secrete the collagen and proteoglycans of the connective
tissue matrix that hold wound edges together and embed cells of
the healing wound matrix. These extracellular molecules assume polymeric
forms and become the physical basis of wound strength (Figure 6–2). Collagen synthesis is not a constitutive property
of fibroblasts but must be signaled. The mechanisms that regulate
the stimulation and synthesis of collagen are multifactorial and
include both growth factors and metabolic inputs such as lactate.
The collagen gene promoter has regulatory binding sites to stress
corticoids, the TGF-β signaling pathway, and retinoids,
which control collagen gene expression. Other growth factors regulate
glycosaminoglycans, tissue inhibitors of metalloproteinase (TIMP),
and fibronectin synthesis. The accumulation of lactate in the extracellular
environment is shown to directly stimulate transcription of collagen
genes as well as posttranslational processing of collagen peptides.
It is clear that the redox state and energy stores of repair cells
occupying the wound regulate collagen synthesis.
The fundamental cellular and molecular elements activated
during normal wound healing.
The increase in collagen messenger RNA (mRNA) leads to an increased
procollagen peptide. This, however, is not sufficient to increase
collagen deposition because procollagen peptide cannot be transported
from the cell to the extracellular space until, in a posttranslational step,
a proportion of its proline amino acids are hydroxylated. In this
reaction, catalyzed by prolyl hydroxylase, an oxygen atom derived
from dissolved O2 is inserted (as a hydroxyl group) into selected
collagen prolines in the presence of the cofactors ascorbic acid,
iron, and α-ketoglutarate. Thus, accumulation of lactate, or any
other process that decreases the nicotinamide adenine dinucleotide (NAD+) pool, leads to production of collagen mRNAs,
increased collagen peptide synthesis, and (provided enough ascorbate and
oxygen is present) increased posttranslational modification and
secretion of collagen monomers into the extracellular space.
Another enzyme, lysyl hydroxylase, hydroxylates many of the procollagen lysines.
A lysyl-to-lysyl covalent link then occurs between collagen molecules, maximizing mature collagen fiber strength. This process, too, requires adequate
amounts of ascorbate and oxygen. These oxygenase reactions (and therefore collagen deposition) are rate limited by tissue oxygen
level, Pao2. The rates are half-maximal
at about 20 mm Hg and maximal at about 200 mm Hg. Hydroxylation
can be “forced” to supernormal rates by tissue hyperoxia. Collagen deposition, wound strength, and angiogenesis rates may be increased and accelerated as tissue Pao2 is elevated.
Angiogenesis is required for wound healing. It is clinically evident
about 4 days following injury but begins earlier when new capillaries
sprout from preexisting venules and grow toward the injury in response
to chemoattractants released by platelets and macrophages. In primarily
closed wounds, budding vessels soon meet and fuse with counterparts
migrating from the other side of the wound, establishing blood flow across
the wound. In wounds left open, newly forming capillaries connect
with adjacent capillaries migrating in the same direction, and granulation
tissue forms. Numerous growth factors and cytokines are observed
to stimulate angiogenesis, but animal experiments indicate that
the dominant angiogenic stimulants in wounds are derived first from platelets
in response to coagulation and then from macrophages in response
to hypoxia or high lactate, fibrin and its products.
Epithelial cells respond to several of the same stimuli as fibroblasts
and endothelial cells within the mesenchymal area of a wound. A
variety of growth factors also regulate epithelial cell replication.
TGF-α and keratinocyte growth factor (KGF), for
instance, are potent epithelial cell mitogens. TGF-β tends
to inhibit epithelial cells from differentiating and thus may potentiate
and perpetuate mitogenesis, though it is itself not a mitogen for
these cells. During wound healing, mitoses appear in the epithelium
a few cells away from the wound edge. The new cells migrate over
the cells at the edge and into the unhealed area and anchor to the
first unepithelialized matrix position encountered. The Pao2 on the underside of the cell at the anchor point is usually low. Low
Pao2 stimulates squamous epithelial cells
to produce TGF-β, likely suppressing terminal differentiation
and again supporting further mitosis. This process of epidermal-mesenchymal communication
repeats itself until the wound is closed.
Squamous epithelialization and differentiation proceed maximally
when surface wounds are kept moist. It is clear that even short
periods of drying impairs the process, and therefore wounds should not
be allowed to desiccate. The exudates from acute, uninfected superficial wounds
also contain growth factors and lactate and therefore recapitulate
the growth environment found at the base of the wound.
Collagen Fiber Remodeling and Wound Contraction
Remodeling of the wound extracellular matrix is also a well-regulated
process. First, fibroblasts replace the provisional fibrin matrix
with collagen monomers. Extracellular enzymes, some of which are
Pao2-dependent, quickly polymerize these monomers,
initially in a pattern that is more random than in uninjured tissue, predisposing
early wound to mechanical failure. Progressively, the very early
provisional matrix is replaced with a more mature one by forming
larger, better organized, stronger, and more durable collagen fibers.
The very early wound provisional matrix usually mechanically fails
within the matrix itself (days 0–5). Next, mechanical failure
occurs at the matrix-tissue interface or fusion point (Figure
6–3). The mechanism for connecting the wound matrix
to the uninjured tissue border is poorly understood.
The very early wound matrix is weak and susceptible to
mechanical failure, especially in load-bearing tissues like the abdominal
wall. After 5 days, mechanical failure occurs at the interface of
the wound matrix and the uninjured surrounding tissue.
Reorganization of the new matrix is an important feature of healing,
and fibroblasts and leukocytes secrete collagenases that ensure
the lytic component. Turnover occurs rapidly at first and then more
slowly. Even in simple wounds, wound matrix turnover can be detected
chemically for as long as 18 months. Healing is successful when
a net excess of matrix is deposited despite concomitant lysis. Lysis,
in contrast to anabolic synthesis, is less dependent upon energy
and nutrition. If synthesis is impaired, however, lysis weakens wounds.
During rapid turnover, wounds normally gain strength and durability
but are vulnerable to contraction or stretching. Fibroblasts exert
the force for contraction. Fibroblasts attach to collagen and each
other and pull the collagen network together when the cell membranes shorten
as the fibroblasts migrate. The wound myofibroblast, a specialized
phenotype, expresses intracellular actin filaments that also contribute
force to fibroblast-mediated wound contraction. The collagen fibers
are then fixed in the packed positions by a variety of cross-linking
mechanisms. Both open and closed wounds tend to contract if not subjected
to a superior counterforce. The phenomenon is best seen in surface wounds,
which may close 90% or more by contraction alone in loose
skin. For example, the residual of a large open wound on the back
of the neck may be only a small area of epithelialization. On the
back, the buttock, or the neck, this is often a beneficial process,
whereas in the face and around joints, the results may be disabling
or disfiguring. Pathological wound contraction is usually termed
a contracture or a stricture. Skin grafts, especially thick ones,
may minimize or prevent disabling wound contractures. Dynamic splints,
passive or active stretching, or insertion of flaps containing dermis
and subdermis also counteract contraction. Prevention of a stricture often depends on ensuring
that opposing tissue edges are well perfused so that healing can
proceed quickly to completion and contraction stops. Healing wounds
may also stretch during active turnover when tension overcomes contraction.
This may account for the laxity of scars in ligaments of injured
but unsplinted joints and the tendency for incisional hernia formation
in abdominal wounds of obese patients.
Healing of Specialized Tissues
Tissue other than skin heals generally by the same fundamental
pathways. Although tissue structure may be specialized, the initial
repair processes are shared. It does appear that the rate and efficiency
of wound healing in different tissue types depends in large part
on total collagen content, collagen organization, and blood supply.
The rate of repair varies from one part of the intestine to the
other in proportion to blood supply. Anastomoses of the colon and
esophagus heal least reliably and are most likely to leak, whereas
failure of stomach or small intestine anastomoses is rare. Intestinal
anastomoses regain strength rapidly when compared to skin wounds.
After 1 week, bursting strength may exceed the uninjured surrounding intestine.
However, the surrounding intestine also participates in the reaction to
injury, initially losing collagen by lysis, and as a result may
lose strength. For this reason, leakage can occur a few millimeters
from the anastomosis. A tight suture line causing ischemia will
exacerbate this surgical problem.
The mesothelial cell lining of the peritoneum also is important
for healing in the abdomen and GI tract. The esophagus and retroperitoneal
colon lack a serosal mesothelial lining, which may contribute to
failed wound healing. There is evidence that mesothelial cells signal
the repair of peritoneal linings and are a source of repair cells.
Comorbidities that delay collagen synthesis or stimulate collagen
lysis are likely to increase the risk of perforation and leakage.
The danger of leakage is greatest from the fourth to seventh days,
when tensile strength normally would rise rapidly but may be impeded
by impaired collagen deposition or increased lysis. Local infection,
which most often occurs near esophageal and colonic anastomoses,
promotes lysis and delays synthesis, thus increasing the likelihood
Bone healing is controlled by many of the same mechanisms that
control soft tissue healing. It too occurs in predictable, morphologic
stages: inflammation, fibroplasia, and remodeling. The duration
of each stage varies depending on the location and extent of the
Injury (fracture) causes hematoma formation from the damaged
blood vessels of the periosteum, endosteum, and surrounding tissues.
Within hours, an inflammatory infiltrate of neutrophils and macrophages
is recruited into the hematoma as in soft tissue injuries. Monocytes
and granulocytes debride and digest necrotic tissue and debris, including
bone, on the fracture surface. This process continues for days to
weeks depending on the amount of necrotic tissue. As inflammation
progresses to fibroplasia, the hematoma is progressively replaced
by granulation tissue that can form bone. This bone wound tissue, known
as callus, develops from both sides of the fracture and is composed
of fibroblasts, endothelial cells, and bone-forming cells (chondroblasts,
osteoblasts). As macrophages (osteoclasts) phagocytose the hematoma
and injured tissue, fibroblasts (osteocytes) deposit a collagenous
matrix, and chondroblasts deposit proteoglycans in a process called
enchondral bone formation. This step, prominent in some bones, is
then converted to bone as osteoblasts condense hydroxyapatite crystals
at specific points on the collagen fibers. Endothelial cells form
a vasculature structure characteristic of uninjured bone. Eventually the
fibrovascular callus is completely replaced by new bone. Unlike
healing of soft tissue, bone healing has features of regeneration,
and bone often heals without leaving a scar.
Bone healing also depends on blood supply. Following injury,
the ends of fractured bone are avascular. Osteocyte and blood vessel
lacunae become vacant for several millimeters from the fracture.
New blood vessels must sprout from preexisting ones and migrate
into the area of injury. As new blood vessels cross the bone ends,
they are preceded by osteoclasts just as macrophages precede them
in soft tissue repair. In bone, this unit is called the cutting
cone because it bores its way through bone in the process of connecting
with other vessels. Excessive movement of the bone ends during this
revascularization stage will break the delicate new vessels and
delay healing. Osteomyelitis originates most often in ischemic bone
fragments. Hyperoxygenation optimizes fracture healing and aids
in the cure (and potentially the prevention) of osteomyelitis. Acute
or chronic hypoxia slows bone repair.
Bone repair may occur through primary or secondary intention.
Primary repair can occur only when the fracture is stable and aligned
and its surfaces closely apposed. This is the goal of rigid plate fixation
or rod fixation of fractures. When these conditions are met, capillaries
can grow across the fracture and rapidly reestablish a vascular
supply. Little or no callus forms. Secondary repair with callus
formation is more common. Once the fracture has been bridged, the
new bone remodels in response to the mechanical stresses upon it,
with restoration to normal or near-normal strength. During this process,
as in soft tissue, preexisting bone and its vascular network are
simultaneously removed and replaced. Increased bone turnover may
be detected as long as 10 years after injury. Although remodeling
is efficient, it cannot correct deformities of angulation or rotation
in misaligned fractures. Careful fracture reduction is still important.
Bone repair can be manipulated. Electrical stimulation, growth
factors, and distraction osteogenesis are three tools for this purpose.
Electrical currents applied directly (through implanted electrodes)
or induced by external alternating electromagnetic fields accelerate
repair by inducing new bone formation in much the same way as small
piezoelectric currents produced by mechanical deformation of intact
bone controls remodeling along lines of stress. Electrical stimulation
has been used successfully to treat nonunion of bone (where new
bone formation between bone ends fails, often requiring long periods
of bed rest). Bone morphogenetic protein (BMP)-impregnated implants
have accelerated bone healing in animals and have been used with encouraging
results to treat large bony defects and nonunions, including during spinal
The Ilizarov technique, linear distraction osteogenesis,
can lengthen bones, stimulate bone growth across a defect, or correct
defects of angulation. The Ilizarov device is an external fixator
attached to the bones through metal pins or wires. A surgical break
is created and then slowly pulled apart (1 mm/d) or slowly
reangulated. The vascular supply and subsequent new bone formation
migrate along with the moving segment of bone.
Franz MG et al: The use of the wound healing trajectory
as an outcome determinant for acute wound healing. Wound Repair Regen
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