▶ Previous Artlcle : #8-1. Wound Healing Process
4. Re-epithelization
Epithelialization occurs when epithelial cells proliferate and migrate from wound edge (epithelial tongue). Epithelial cells can migrate above the collagen and fibronectin on wound surface by using contractile protein, until the wound is covered by thickened skin.
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- Cell migration
Cells move by rolling as well as by sliding and crawling, presumably with the help of actomyosin, polymerized actin and actin gel without myosin. The epithelial barrier, which is a group of epithelial cells, inhibit cell migration when it is adjacent to the surrounding cells, and epithelial cells build up when cells are in contact with each other. Cells cannot pass through the fibrin clot without melting the fibrin barrier. In this process, plasminogen activation system, composed of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA), which act as enzymatic proteases, converts plasminogen to plasmin, a substance capable of degrading fibrin. These activators and their receptors are known to be upregulated in keratinocyte.
Plasminogen activator inhibitor (PAI), which inhibits and controls the plasminogen activator system, prevents chronic non-healing wound from excessive plasmin system or hypertrophic scarring from decreased degradation of extracellular matrix due to the inhibition of the system. Recently, a clinical study reported that the zone of hyperemia was converted to the zone of stasis by administering exogenous plasminogen activator to burn wound, which reduced the damage to viable local tissue remaining in the wound (a side effect of prothrombotic state).
- Keratinocyte migration
In normal skin, the basal cell layer of keratinocyte is attached to the lamina basalis by hemidesmosomes (α6β4 Integrin), which anchors the cell to laminin creating a link between cells. Upon sustaining an injury, these hemidesmosomes are removed, and α5β1, αvβ6 fibronectin/tenascin receptor and αvβ5 vitronectin receptor are expressed, replacing α2β1 collagen receptor, in front of moving epithelial cells. This is to propel forward movement by grabbing onto newly-formed matrix and exposed dermis of the wound. It is not yet clear which cells drive the forward movement of keratinocyte, but it is speculated that the cells immediately above the basal cell layer push forward. Recently, it was found that epithelial cells move and proliferate in both hair follicles and basal cell layer. Once the exposed wound is covered by a monolayer of epithelial cells, the cells stop migration and starts to form epithelium, where basal cell layer and cells overlap. At this point, cells immediately above the basal layer stops expressing integrin and basal keratin, and starts the general process of epithelial differentiation, by which epithelial cells gradually grow and are eliminated from the base of the epidermis. At the same time, the synthesis of basal cell layer is started, reproducing hemidesmosomes without expressing MMP.
- Epithelial growth factors
EGFs, which are abundantly present in all typical wounds, act as motogen and mitogen of epithelial cells. Keratinocyte-specific growth factors (KGF or FGF-7) are upregulated in dermal fibroblast by more than 100 times within 24 hours, to take effect as motogen and mitogen of epithelial cells. They also contribute to epithelial migration by stimulating plasminogen activator system and the expression of MMP-10.
Matrix metalloproteinase (MMP) families are upregulated in the keratinocytes in wound edges. MMP-9 (gelatinase), among them, is capable of degrading basement membrane or collagen type IV and VII. MMP-1 (collagenase), which is upregulated only in keratinocytes in basal cell layer, is speculated to regulate MMP expression by cell-extracellular matrix interaction. Most of all, they degrade the original collagen, and helps cell migration by cutting collagen type I and III, attached with keratinocyte, from dermal layer. MMP-10 (stromelysin-2), with broad substrate specificity, is expressed in keratinocytes surrounding the wound, but is increased in chronic wound. Hence, even when exogenous growth factor is administered to a resistant chronic wound, increased proteases may become the major cause of incurable wounds.
5. Angiogenesis
Angiogenesis is an essential process in normal wound healing. Granulation tissue, with its capillary arcade, is the preparation phase for wound closure and epithelization. During angiogenesis, when endothelial cells start migration and proliferation, capillary formation from the existing capillary network can be stimulated by angiogenesis factors, including GF, TGF-α, VEGF and PDGF, which can also stimulate endothelial cell growth. Endothelial cells cannot respond to these mitogenic stimulants without the presence of specific growth factor receptors.
These receptors are closely regulated by a variety of mechanisms, but they particularly interfere with angiogenesis in the absence of interaction between vitronectin, a type of extracellular matrix, and endothelial cells. This may be due to the fact that integrin, an extracellular binding receptor expressed on cell surface, is connected with cytoskeleton, which gives cells shape and mediates traction during cell migration, and induces intercellular signaling mechanism. Among others, αvβ3 and αvβ5, known vitronectin binding receptors, play an important role in angiogenesis, because they can also bind to other components of extracellular matrix, such as fibronectin, fibrinogen and fibrin.
Endothelial cell connects capillaries and forms capillary network for healing wound. Angiogenesis is very important for wound healing as a mechanism for delivering new healing factors to the wound site. The process of angiogenesis stops when the wound has proper blood supply, which is speculated to be regulated by oxygen tension. In wounds, hypoxia promotes angiogenesis, while normal oxygen tension stops it.
Wound site is replaced by granulation tissues, because of the proliferation of multiple new capillaries. FGF2 and vascular endothelial growth factor (VEGF), released from macrophage or injured endothelial cell, help angiogenesis. αvβ3 integrin, a type of receptor in extra cellular matrix, should be expressed on endothelial cells.
6. Wound Contracture
Contraction is a centrifugal force toward the center of the wound and is important for wound closure . Contracture refers to a hypertrophic scar or scarring developed by wound contraction, causing deformation of the surrounding tissues or joint areas.
The purpose of wound re-epithelization is to reduce wound size by inducing contraction, pulling one side of a wound to another. In 3-4 days after sustaining a wound, fibroblasts start proliferation and a number of various growth factors affect these fibroblasts. Platelet-derived growth factor (PDGF) and TGF-β, in particular, are released from fibroblasts and keratinocytes of surrounding tissues, and are deeply involved in wound contracture.
In order to maneuver out of the fibrin clot, fibroblasts use fibronectin, a component of extracellular matrix, and this is known to be regulated by actinomycin cytoskeleton, which plays a very important role in cell migration. In about 7 days after injury, the wound is replaced by fibroblasts, which releases new collagen, by TGF-β1 and other growth factors, and some fibroblasts, which convert to myofibroblast during this time, express a-smooth muscle actin. These are similar to smooth muscle cell with potent ability of contraction, and TGF-β is involved in this change.
7. Remodelling; Maturation Phase
As mentioned above, plasminogen activator system and MMP system are known as important protease systems that affect the initial process of wound healing of the human body. MMP, among them, is very important because they promote cell migration by dissolving surrounding extracellular matrix in the early phase of wound healing. It is also important for matrix remodeling during the scar maturation phase after wound healing.
Fibronectin, fibrin and vitronectin are replaced by collagen III and, as the wound is matured, are gradually replaced by collagen I. MMP-1, -3 and –8, collagenase groups expressed by keratinocyte, fibroblast, endothelial cell and vascular smooth muscle cell, degrade collagen I and III. MMP-2 and –9, gelatinase groups, degrade collagen IV, denatured collagen, elastin and basement membrane. Stromelysin-1(MMP-3) and Matrilysin(MMP-7) broadly act on proteoglycans, laminin, fibronectin, and collagen IV and collagen IX.
The maturation phase of wound healing entails wound remodeling after wound repair. The scar becomes gradually less hyperemic, by reduction of vascularity, and tissue organization and maturation increase wound strength for up to two years. Despite the increase in wound strength, collagen is not increased in the wound. Hyaluronidase, plasminogen activators, collagenase and elastase participate in wound remodeling. In the matrix, hyaluronate is replaced by dermatan sulfate and chondroitin sulfate, and this process reduces cell migration and differentiation. Plasminogen produces plasmin, which degrades fibrin. Urokinase produced from fibroblast, endothelial cell, keratinocyte and leukocyte activates collagenase and elastase. Collagenase is expressed by macrophage, fibroblast, epithelial cell and leukocyte, and is capable of breaking down the collagen triple helix. Collagenase activity lasts for months after sustaining tissue injury.
Collagen remodeling involving degradation and synthesis lasts for almost 2 years after wound healing. Compared to collagen which is abundant in proline and lysine in orderly strands, elastin, another structural protein, is comprised of a random coil that can contract to a certain degree and is fewer in number compared to collagen.
-To be continued-
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