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Wound Healing
Wound healing, or wound repair, is the body's natural process of regenerating dermal
and epidermal tissue. When an individual is wounded, a set of complex biochemical
events takes place in a closely orchestrated cascade to repair the damage. These events
overlap in time and may be artificially categorized into separate steps: the
inflammatory, proliferative, and remodeling phases (Some authors consider healing to
take place in four or more stages, by splitting different parts inflammation or proliferation
into separate steps.). In the inflammatory phase, bacteria and debris are phagocytized
and removed, and factors are released that cause the migration and division of cells
involved in the proliferative phase.
The proliferative phase is characterized by angiogenesis, collagen deposition, granulation
tissue formation, epithelialization, and wound contraction. In angiogenesis, new blood
vessels grow from endothelial cells. In fibroplasia and granulation tissue formation,
fibroblasts grow and form a new, provisional extracellular matrix (ECM) by excreting
collagen and fibronectin.
In epithelialization, epithelial cells crawl across the wound bed to cover it. In
contraction, the wound is made smaller by the action of myofibroblasts, which establish a
grip on the wound edges and contract themselves using a mechanism similar to that in
smooth muscle cells. When the cells' roles are close to complete, unneeded cells undergo
apoptosis.
In the maturation and remodeling phase, collagen is remodeled and realigned along
tension lines and cells that are no longer needed are removed by apoptosis.
However, this process is not only complex but fragile, and susceptible to interruption or
failure leading to the formation of chronic non-healing wounds. Factors which may
contribute to this include diabetes, venous or arterial disease, old age, and infection.
Contents
Inflammatory phase
Clotting cascade
Platelets
Vasoconstriction and vasodilation
Polymorphonuclear neutrophils
Macrophages
Proliferative phase
Angiogenesis
Fibroplasia and granulation tissue formation
Collagen deposition
Epithelialization
Contraction
Maturation and remodeling phase
Estrogen, Testosterone, and DHEA affect wound healing
Inflammatory phase
In the inflammatory phase (lag phase/resting phase), clotting takes place in order to
obtain hemostasis, or stop blood loss, and various factors are released to attract cells that
phagocytise debris, bacteria, and damaged tissue and release factors that initiate the
proliferative phase of wound healing.
Clotting cascade
Coagulation
When tissue is first wounded, blood comes in contact with collagen, triggering blood
platelets to begin secreting inflammatory factors. Platelets also express glycoproteins
on their cell membranes that allow them to stick to one another and to aggregate, forming
a mass.
Fibrin and fibronectin cross-link together and form a plug that traps proteins and particles
and prevents further blood loss. This fibrin-fibronectin plug is also the main structural
support for the wound until collagen is deposited. Migratory cells use this plug as a
matrix to crawl across, and platelets adhere to it and secrete factors. The clot is
eventually lysed and replaced with granulation tissue and then later with collagen.
Platelets
Platelets, the cells present in the highest numbers shortly after a wound occurs, release a
number of things into the blood, including ECM proteins and cytokines, including growth
factors. Growth factors stimulate cells to speed their rate of division. Platelets also
release other proinflammatory factors like serotonin, bradykinin, prostaglandins,
prostacyclins, thromboxane, and histamine, which serve a number of purposes,
including to increase cell proliferation and migration to the area and to cause blood
vessels to become dilated and porous.
Vasoconstriction and vasodilation
Immediately after a blood vessel is breached, ruptured cell membranes release
inflammatory factors like thromboxanes and prostaglandins that cause the vessel to
spasm to prevent blood loss and to collect inflammatory cells and factors in the area.
This vasoconstriction lasts five to ten minutes and is followed by vasodilation, a
widening of blood vessels, which peaks at about 20 minutes post-wounding.
Vasodilation is the result of factors released by platelets and other cells. The main factor
involved in causing vasodilation is histamine. Histamine also causes blood vessels to
become porous, allowing the tissue to become edematous because proteins from the
bloodstream leak into the extravascular space, which increases its osmolar load and
draws water into the area. Increased porousness of blood vessels also facilitates the
entry of inflammatory cells like leukocytes into the wound site from the
bloodstream.
Polymorphonuclear neutrophils
Within an hour of wounding, polymorphonuclear neutrophils (PMNs) arrive at the wound
site and become the predominant cells in the wound for the first three days after the
injury occurs, with especially high numbers on the second day. They are attracted to
the site by fibronectin, growth factors, and substances such as neuropeptides and kinins.
Neutrophils phagocytise debris and bacteria and also kill bacteria by releasing free
radicals in what is called a 'respiratory burst'. They also cleanse the wound by
secreting proteases that break down damaged tissue. Neutrophils usually undergo
apoptosis once they have completed their tasks and are engulfed and degraded by
macrophages.
Other leukocytes to enter the area include helper T cells, which secrete cytokines to cause
more T cells to divide and to increase inflammation and enhance vasodilation and vessel
permeability. T cells also increase the activity of macrophages.
Macrophages
Macrophages are essential to wound healing. They replace PMNs as the predominant
cells in the wound by two days after injury. Attracted to the wound site by growth
factors released by platelets and other cells, monocytes from the bloodstream enter the
area through blood vessel walls. Numbers of monocytes in the wound peak one to one
and a half days after the injury occurs. Once they are in the wound site, monocytes
mature into macrophages, the main cell type that clears the wound area of bacteria and
debris.
The macrophage's main role is to phagocytise bacteria and damaged tissue, and it also
debrides damaged tissue by releasing proteases. Macrophages also secrete a number of
factors such as growth factors and other cytokines, especially during the third and fourth
post-wounding days. These factors attract cells involved in the proliferation stage of
healing to the area. Macrophages are stimulated by the low oxygen content of their
surroundings to produce factors that induce and speed angiogenesis. and they also
stimulate cells that reepithelialize the wound, create granulation tissue, and lay down a
new extracellular matrix. Because they secrete these factors, macrophages are vital
for pushing the wound healing process into the next phase.
Because inflammation plays roles in fighting infection and inducing the proliferation
phase, it is a necessary part of healing. However, inflammation can lead to tissue damage
if it lasts too long. Thus the reduction of inflammation is frequently a goal in
therapeutic settings. Inflammation lasts as long as there is debris in the wound. Thus the
presence of dirt or other objects can extend the inflammatory phase for too long, leading
to a chronic wound.
As inflammation dies down, fewer inflammatory factors are secreted, existing ones are
broken down, and numbers of neutrophils and macrophages are reduced at the wound
site. These changes indicate that the inflammatory phase is ending and the proliferative
phase is underway.
Proliferative phase
About two or three days after the wound occurs, fibroblasts begin to enter the wound site,
marking the onset of the proliferative phase even before the inflammatory phase has
ended. As in the other phases of wound healing, steps in the proliferative phase do not
occur in a series but rather partially overlap in time.
Angiogenesis
Also called neovascularization, the process of angiogenesis occurs concurrently with
fibroblast proliferation when endothelial cells migrate to the area of the wound.
Because the activity of fibroblasts and epithelial cells requires oxygen, angiogenesis is
imperative for other stages in wound healing, like epidermal and fibroblast migration.
The tissue in which angiogenesis has occurred typically looks red (is erythematous) due
to the presence of capillaries.
In order to form new blood vessels and provide oxygen and nutrients to the healing
tissue. Stem cells called endothelial cells originating from parts of uninjured blood
vessels develop pseudopodia and push through the ECM into the wound site. Through
this activity, they establish new blood vessels.
To migrate, endothelial cells need collagenases and plasminogen activator to degrade the
clot and part of the ECM. Zinc-dependent metalloproteinases digest basement
membrane and ECM to allow cell proliferation and angiogenesis.
Endothelial cells are also attracted to the wound area by fibronectin found on the fibrin
scab and by growth factors released by other cells. Endothelial growth and
proliferation is also stimulated by hypoxia and presence of lactic acid in the wound. In
a low-oxygen environment, macrophages and platelets produce angiogenic factors which
attract endothelial cells chemotactically. When macrophages and other growth factorproducing cells are no longer in a hypoxic, lactic acid-filled environment, they stop
producing angiogenic factors. Thus, when tissue is adequately perfused, migration and
proliferation of endothelial cells is reduced. Eventually blood vessels that are no longer
needed die by apoptosis.
Fibroplasia and granulation tissue formation
Simultaneously with angiogenesis, fibroblasts begin accumulating in the wound site.
Fibroblasts begin entering the wound site two to five days after wounding as the
inflammatory phase is ending, and their numbers peak at one to two weeks postwounding. By the end of the first week, fibroblasts are the main cells in the wound[1]
Fibroplasia ends two to four weeks after wounding.
In the first two or three days after injury, fibroblasts mainly proliferate and migrate, while
later, they are the main cells that lay down the collagen matrix in the wound site.
Fibroblasts from normal tissue migrate into the wound area from its margins. Initially
fibroblasts use the fibrin scab formed in the inflammatory phase to migrate across,
adhering to fibronectin. Fibroblasts then deposit ground substance into the wound bed,
and later collagen, which they can adhere to for migration.
Granulation tissue is needed to fill the void that has been left by a large, open wound that
crosses the basement membrane. It begins to appear in the wound even during the
inflammatory phase, two to five days post wounding, and continues growing until the
wound bed is covered. Granulation tissue consists of new blood vessels, fibroblasts,
inflammatory cells, endothelial cells, myofibroblasts, and the components of a new,
provisional ECM. The provisional ECM is different in composition from the ECM in
normal tissue and includes fibronectin, collagen, glycosaminoglycans, and
proteoglycans. Its main components are fibronectin and hyaluronan, which create a
very hydrated matrix and facilitate cell migration. Later this provisional matrix is
replaced with an ECM that more closely resembles that found in non-injured tissue.
Fibroblasts deposit ECM molecules like glycoproteins, glycosaminoglycans (GAGs),
proteoglycans, elastin, and fibronectin, which they can then use to migrate across the
wound.
Growth factors (PDGF, TGF-รข) and fibronectin encourage proliferation, migration to the
wound bed, and production of ECM molecules by fibroblasts. Fibroblasts also secrete
growth factors that attract epithelial cells to the wound site. Hypoxia also contributes to
fibroblast proliferation and excretion of growth factors, though too little oxygen will
inhibit their growth and deposition of ECM components, and can lead to excessive,
fibrotic scarring.
Collagen deposition
One of fibroblasts' most important duties is the production of collagen. Fibroblasts
begin secreting appreciable collagen by the second or third post-wounding day, and its
deposition peaks at one to three weeks. Collagen production continues rapidly for two
to four weeks, after which its destruction matches its production and so its growth levels
off.
Collagen deposition is important because it increases the strength of the wound; before it
is laid down, the only thing holding the wound closed is the fibrin-fibronectin clot, which
does not provide much resistance to traumatic injury. Also, cells involved in
inflammation, angiogenesis, and connective tissue construction attach to, grow and
differentiate on the collagen matrix laid down by fibroblasts.
Even as fibroblasts are producing new collagen, collagenases and other factors degrade it.
Shortly after wounding, synthesis exceeds degradation so collagen levels in the wound
rise, but later production and degradation become equal so there is no net collagen gain.
This homeostasis signals the onset of the maturation phase. Granulation gradually ceases
and fibroblasts decrease in number in the wound once their work is done. At the end of
the granulation phase, fibroblasts begin to commit apoptosis, converting granulation
tissue from an environment rich in cells to one that consists mainly of collagen.
Epithelialization
The formation of granulation tissue in an open wound allows the reepithelialization phase
to take place, as epithelial cells migrate across the new tissue to form a barrier between
the wound and the environment. Basal keratinocytes from the wound edges and dermal
appendages such as hair follicles, sweat glands and sebacious (oil) glands are the main
cells responsible for the epithelialization phase of wound healing. They advance in a
sheet across the wound site and proliferate at its edges, ceasing movement when they
meet in the middle.
Keratinocytes migrate without first proliferating. Migration can begin as early as a
few hours after wounding. However, epithelial cells require viable tissue to migrate
across, so if the wound is deep it must first be filled with granulation tissue. Thus the
time of onset of migration is variable and may occur about one day after wounding.
Cells on the wound margins proliferate on the second and third day post-wounding in
order to provide more cells for migration.
If the basement membrane is not breached, epithelial cells are replaced within three days
by division and upward migration of cells in the stratum basale in the same fashion that
occurs in uninjured skin. However, if the basement membrane is ruined at the wound
site, reepithelization must occur from the wound margins and from skin appendages such
as hair follicles and sweat and oil glands that enter the dermis that are lined with viable
keratinocytes. If the wound is very deep, skin appendages may also be ruined and
migration can only occur from wound edges.
Migration of keratinocytes over the wound site is stimulated by lack of contact inhibition
and by chemicals such as nitric oxide.[31] Before they begin to migrate, cells must
dissolve their desmosomes and hemidesmosomes, which normally anchor the cells by
intermediate filaments in their cytoskeleton to other cells and to the ECM.
Transmembrane receptor proteins called integrins, which are made of glycoproteins and
normally anchor the cell to the basement membrane by its cytoskeleton, are released from
the cell's intermediate filaments and relocate to actin filaments to serve as attachments to
the ECM for pseudopodia during migration. Thus keratinocytes detach from the
basement membrane and are able to enter the wound bed.
Before they begin migrating, keratinocytes change shape, becoming longer and flatter
and extending cellular processes like lamellipodia and wide processes that look like
ruffles.Actin filaments and pseudopodia form. During migration, integrins on the
pseudopod attach to the ECM, and the actin filaments in the projection pull the cell
along. The interaction with molecules in the ECM through integrins further promotes
the formation of actin filaments, lamellipodia, and filopodia.
Epithelial cells climb over one another in order to migrate. This growing sheet of
epithelial cells is often called the epithelial tongue. The first cells to attach to the
basement membrane form the stratum basale. These basal cells continue to migrate across
the wound bed, and epithelial cells above them slide along as well. The more quickly
this migration occurs, the less of a scar there will be.
Fibrin, collagen, and fibronectin in the ECM may further signal cells to divide and
migrate Like fibroblasts, migrating keratinocytes use the fibronectin cross-linked with
fibrin that was deposited in inflammation as an attachment site to crawl across.
As keratinocytes migrate, they move over granulation tissue but underneath the scab (if
one was formed), separating it from the underlying tissue. Epithelial cells have the
ability to phagocytize debris such as dead tissue and bacterial matter that would
otherwise obstruct their path. Because they must dissolve any scab that forms,
keratinocyte migration is best enhanced by a moist environment, since a dry one leads to
formation of a bigger, tougher scab. To make their way along the tissue,
keratinocytes must dissolve the clot, debris, and parts of the ECM in order to get
through. They secrete plasminogen activator, which activates plasmin to dissolve
the scab. Cells can only migrate over living tissue, so they must excrete collagenases
and proteases like matrix metalloproteinases (MMPs) to dissolve damaged parts of the
ECM in their way, particularly at the front of the migrating sheet. Keratinocytes also
dissolve the basement membrane, using instead the new ECM laid down by fibroblasts to
crawl across.
As keratinocytes continue migrating, new epithelial cells must be formed at the wound
edges to replace them and to provide more cells for the advancing sheet. Proliferation
behind migrating keratinocytes normally begins a few days after wounding and occurs
at a rate that is 17 times higher in this stage of epithelialization than in normal tissues.
Until the entire wound area is resurfaced, the only epithelial cells to proliferate are at the
wound edges.
Growth factors, stimulated by integrins and MMPs, cause cells to proliferate at the
wound edges. Keratinocytes themselves also produce and secrete factors, including
growth factors and basement membrane proteins, which aid both in epithelialization and
in other phases of healing.
Keratinocytes continue migrating across the wound bed until cells from either side meet
in the middle, at which point contact inhibition causes them to stop migrating. When
they have finished migrating, the keratinocytes secrete the proteins that form the new
basement membrane. Cells reverse the morphological changes they underwent in order
to begin migrating; they reestablish desmosomes and hemidesmosomes and become
anchored once again to the basement membrane. Basal cells begin to divide and
differentiate in the same manner as they do in normal skin to reestablish the strata found
in reepithelialized skin.
Contraction
Around a week after the wounding takes place, fibroblasts have differentiated into
myofibroblasts and the wound begins to contract In full thickness wounds, contraction
peaks at 5 to 15 days post wounding. Contraction can last for several weeks and
continues even after the wound is completely reepithelialized. If contraction continues
for too long, it can lead to disfigurement and loss of function.
Contraction occurs in order to reduce the size of the wound. A large wound can become
40 to 80% smaller after contraction. Wounds can contract at a speed of up to 0.75
mm per day, depending on how loose the tissue in the wounded area is. Contraction
usually does not occur symmetrically; rather most wounds have an 'axis of contraction'
which allows for greater organization and alignment of cells with collagen.
At first, contraction occurs without myofibroblast involvement. Later, fibroblasts,
stimulated by growth factors, differentiate into myofibroblasts. Myofibroblasts, which are
similar to smooth muscle cells, are responsible for contraction. Myofibroblasts contain
the same kind of actin as that found in smooth muscle cells.
Myofibroblasts are attracted by fibronectin and growth factors and they move along
fibronectin linked to fibrin in the provisional ECM in order to reach the wound edges.
They form connections to the ECM at the wound edges, and they attach to each other and
to the wound edges by desmosomes. Also, at an adhesion called the fibronexus, actin in
the myofibroblast is linked across the cell membrane to molecules in the extracellular
matrix like fibronectin and collagen. Myofibroblasts have many such adhesions, which
allow them to pull the ECM when they contract, reducing the wound size. In this part
of contraction, closure occurs more quickly than in the first, myofibroblast-independent
part.
As the actin in myofibroblasts contracts, the wound edges are pulled together. Fibroblasts
lay down collagen to reinforce the wound as myofibroblasts contract. The contraction
stage in proliferation ends as myofibroblasts stop contracting and commit apoptosis.
The breakdown of the provisional matrix leads to a decrease in hyaluronic acid and an
increase in chondroitin sulfate, which gradually triggers fibroblasts to stop migrating and
proliferating. These events signal the onset of the maturation stage of wound healing.
Maturation and remodeling phase
When the levels of collagen production and degradation equalize, the maturation phase of
tissue repair is said to have begun. The maturation phase can last for a year or longer,
depending on the size of the wound and whether it was initially closed or left open.
During Maturation, type III collagen, which is prevalent during proliferation, is gradually
degraded and the stronger type I collagen is laid down in its place. Originally
disorganized collagen fibers are rearranged, cross-linked, and aligned along tension
lines. As the phase progresses, the tensile strength of the wound increases, with the
strength approaching 50% that of normal tissue by three months after injury and
ultimately becoming as much as 80% as strong as normal tissue. Since activity at the
wound site is reduced, the scar loses its erythematous appearance as blood vessels that
are no longer needed are removed by apoptosis.
The phases of wound healing normally progress in a predictable, timely manner; if they
do not, healing may progress inappropriately to either a chronic wound such as a
venous ulcer or pathological scarring such as a keloid scar.
Estrogen, Testosterone, and DHEA affect wound
healing
In humans and mice, estrogen and DHEA promote wound healing and testosterone
inhibits healing except as to severe burns.
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