Wound healing

Editor-in-Chief: Michael D. Caldwell, M.D., Ph.D., FACS, Marshfield Clinic, Department of Surgery [mailto:caldwell.michael@marshfieldclinic.org]

Overview
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 events takes place in a predictable fashion to repair the damage. These events overlap in time and must be artificially categorized into separate steps: the inflammatory, proliferative, and remodeling phases (Some authors consider healing to take place in four 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.

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
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 factor-producing 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 post-wounding. By the end of the first week, fibroblasts are the main cells in the wound 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 (Cohen, 2005).

Growth factors (PDGF, TGF-β) 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. 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.