13.
Wound Healing
Wound Healing § § §
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Complex and dynamic process of restoring cellular structures and tissue layers. Human adult wound healing process can be divided into 3 distinct phases: inflammatory phase, the proliferative phase, and the remodeling phase. Within these 3 broad phases is a complex and coordinated series of events that includes chemotaxis, phagocytosis, neocollagenesis, collagen degradation, and collagen remodeling. In addition, angiogenesis, epithelization, and the production of new glycosaminoglycans (GAGs) and proteoglycans are vital to the wound healing. The culmination of these biological processes results in the replacement of normal skin structures with fibroblastic mediated scar tissue.
Various categories of wound healing lead to an ultimate repair of a tissue defect. § Different categories exist, and the interactions of cellular and extracellular constituents are similar. Primary healing (first intention): Begins within hours of repairing a full-thickness surgical incision where insult results in the mortality of a minimal number of cellular constituents. Delayed primary healing: If the wound edges are not reapproximated immediately, delayed primary wound healing transpires. This type of healing may be desired in the case of contaminated wounds. By the fourth day, phagocytosis of contaminated tissues is well underway, and the processes of epithelization, collagen deposition, and maturation are occurring. Foreign materials are walled off by macrophages that may metamorphose into epithelioid cells, which are encircled by mononuclear leukocytes, forming granulomas. Usually the wound is closed surgically at this juncture, and if the "cleansing" of the wound is incomplete, chronic inflammation can ensue, resulting in prominent scarring. Secondary healing (secondary intention): Occurs when a full-thickness wound is allowed to close and heal. Secondary healing results in an inflammatory response that is more intense than with primary wound healing. A larger quantity of granulomatous tissue is fabricated because of the need for wound closure. Secondary healing results in pronounced contraction of wounds. Fibroblasts differentiate into myofibroblasts, which resemble contractile smooth muscle, and contribute to wound contraction. Myofibroblasts are maximally present in the wound from the 10th-21st days.
Epithelization: Process by which epithelial cells migrate and replicate via mitosis and traverse the wound. This occurs as part of the phases of wound healing. In wounds that are partial thickness, involving only the epidermis and superficial dermis, epithelization is the predominant method by which healing occurs. Wound Healing Sequence Complex amalgam of coordinated events that include inflammation, the fibroblastic phase, wound contracture, and scar maturation. § The inflammatory phase occurs immediately following the injury and lasts approximately 6 days. § The fibroblastic phase occurs at the termination of the inflammatory phase and can last up to 4 weeks. § Wound contracture is a process that occurs throughout the healing process, commencing in the fibroblastic stage. § Scar maturation (remodeling) begins at the fourth week and can last for 1-2 years. Following tissue injury via an incision, the initial response is usually bleeding. The cascade of vasoconstriction and coagulation commences with clotted blood immediately impregnating the wound, leading to hemostasis, and with dehydration, a scab forms. An influx of inflammatory cells follows, with the release of cellular substances and mediators. Angiogenesis and reepithelization occur and the deposition of new cellular and extracellular
Hemostasis and Inflammation: Immediately through Days 4 to 6 Hemostasis serves as the initiating step and foundation for the healing process. § The first action the body takes immediately after wounding is to control bleeding. The injured blood vessel vasoconstricts, and the endothelium and nearby platelets activate the intrinsic part of the coagulation cascade. The clot that forms is made of collagen, platelets, thrombin, and fibronectin, and these factors release cytokines and growth factors that initiate the inflammatory response. The fibrin clot also serves as a scaffold for invading cells, such as neutrophils, monocytes, fibroblasts, and endothelial cells, to use. The clot also serves to concentrate the elaborated cytokines and growth factors.
Chemotaxis. Activation, and Inflammation §
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Immediately as the clot is formed, cellular signals are generated that result in a neutrophil response. As the inflammatory mediators accumulate, prostaglandins are elaborated and the nearby blood vessels vasodilate to allow for the increased cellular traffic as neutrophils are drawn into the injured area by interleukin (IL)-1, tumor necrosis factor (TNF), transforming growth factor (TGF), PF4. Within the first 6-8 hours, the next phase of the healing process is underway, with polymorphonuclear leukocytes (PMNs) engorging the wound. TGF-b facilitates PMN migration from surrounding blood vessels where they extrude themselves from these vessels. These cells "cleanse" the wound, clearing it of debris. The PMNs attain their maximal numbers in 24-48 hours and commence their departure by hour 72. Other chemotactic agents are released, including fibroblastic growth factor (FGF), transforming growth factors (TGF-b and TGF-a), PDGF, and plasma-activated complements C3a and C5a (anaphylactic toxins). They are sequestered by macrophages or interred within the scab or eschar. Monocytes in the nearby tissue and blood are attracted to the area and transform into macrophages, usually around 48 to 96 hours after injury. Activation of the inflammatory cells is critical, especially for the macrophage. An activated macrophage is important for the transition into the proliferative phase. The macrophages continue the cleansing process and manufacture various growth factors during days 3-4. Macrophages orchestrate the multiplication of endothelial cells with the sprouting of new blood vessels, the duplication of smooth muscle cells, and the creation of the milieu created by the fibroblast. Many factors influencing the wound healing process are secreted by macrophages. These include TGFs, cytokines and interleukin-1 (IL-1), tumor necrosis factor (TNF), and PDGF. Macrophages mediate angiogenesis by producing vascular endothelial growth factor (VEGF), fibroblast growth factor, and TNF Macrophages mediate fibroplasia by synthesizing TGF, epidermal growth factor (EGF), platelet derived growth factor (PDGF), IL-1, TNF, nitric oxide (NO) PDGF is chemotactic for fibroblasts and, along with TGF-b, is a potent modulator of fibroblastic mitosis, leading to prolific collagen fibril construction in later phases. Fibrinogen is cleaved into fibrin and the framework for completion of the coagulation process is formed. Fibrin provides the structural support for cellular constituents of inflammation.
Proliferative Phase: Day 4 through 14 Epithelization, Angiogenesis, and Provisional Matrix Formation This phase consists of different subphases (fibroplasia, matrix deposition, angiogenesis and re-epithelialization) that constitute an overall and ongoing process. §
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Epithelial cells located on the skin edge begin proliferating and sending out projections to reestablish a protective barrier against fluid losses and further bacterial invasion. The stimulus for epithelial proliferation and chemotaxis is EGF and TGF produced by activated platelets and macrophages. Epithelization commences with the spreading of cells within 24 hours. Division of peripheral cells occurs in hours 48-72, resulting in a thin epithelial cell layer, which bridges the wound. This process is first stimulated by inflammatory cytokines. Fibroblasts synthesize and secrete keratinocyte growth factor (KGF)-1, KGF-2, and IL-6, which simulate neighboring keratinocytes to migrate in the wound area, proliferate, and differentiate in the epidermis. Fibroblasts and endothelial cells are the predominant cells proliferating during this phase. In days 5-7, fibroblasts have migrated into the wound, laying down new collagen of the subtypes I and III. Early in normal wound healing, type III collagen predominates but is later replaced by type I collagen. Fibroblasts already located in the wound site (termed “wound fibroblasts”) begin synthesizing collagen and transform into myofibroblasts for wound contraction (induced by macrophage secreted TGF-1). Tropocollagen is the precursor of all collagen types and is transformed within the cell's rough endoplasmic reticulum, where proline and lysine are hydroxylated. Disulfide bonds are established, allowing 3 tropocollagen strands to form a triple left-handed triple helix, termed procollagen. As the procollagen is secreted into the extracellular space, peptidases in the cell wall cleave terminal peptide chains, creating true collagen fibrils. The wound is suffused with GAGs and fibronectin produced by fibroblasts. These GAGs include heparin sulfate, hyaluronic acid, chondroitin sulfate, and keratin sulfate. Proteoglycans are GAGs that are bonded covalently to a protein core and contribute to matrix deposition.
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Angiogenesis is the product of parent vessel offshoots. Endothelial cells located at intact venules under the action of VEGF, secreted by keratinocytes and macrophages, to begin forming new capillary tubes. The formation of new vasculature requires extracellular matrix and basement membrane degradation followed by migration, mitosis, and maturation of endothelial cells. Basic FGF and vascular endothelial growth factor are believed to modulate angiogenesis.
Maturation and Remodeling: Day 8 through 1 Year Clinically, the maturation and remodeling phase is perhaps the most important. The main feature of this phase is the deposition of collagen in an organized and well-mannered network. If patients have matrix deposition problems (from diet or disease), then the woundʼs strength will be greatly compromised. If there is excessive collagen synthesis, then a hypertrophic scar or keloid can result. § §
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Initially, the matrix is composed mainly of fibrin and fibronectin. Glycosaminoglycans, proteoglycans, and other proteins are synthesized next by the fibroblasts with deposition in a disorganized way that provides a preliminary framework for the new matrix. This temporary matrix is replaced by a stronger and organized matrix made of collagen. The collagen in uninjured skin is 8090% type I and 10-20% type III. In granulation tissue, collagen type III comprises 30 %, and in the mature scar, it is back down to 10 %. Early in wound healing, the matrix is thin and compliant and allows fibroblasts, neutrophils, lymphocytes, and macrophages to easily maneuver through it. As the matrix becomes denser with thicker, stronger collagen fibrils, it becomes stiff and less compliant. Net collagen synthesis will continue for at least 4 to 5 weeks after wounding. The increased rate of collagen synthesis during wound healing is from not only an increase in the number of fibroblasts but also a net increase in the collagen production per cell. The collagen that is initially laid down is thinner than collagen in uninjured skin and is orientated parallel to the skin. Over time, the initial collagen threads are reabsorbed and deposited thicker and organized along the stress lines. These changes are also accompanied by a wound with an increased tensile strength, indicating a positive correlation between collagen fiber thickness/orientation and tensile strength. Maximal tensile strength of the wound is achieved by the 12th week, and the ultimate resultant scar has only 80% of the tensile strength of the original skin.
Local Cytokines Â
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Growth factors represent the intercellular signaling that orchestrates the complex sequence of cell migration, division, differentiation, and protein expression during wound healing. The major families of growth factors are expressed in varying levels by cells involved with Growth Factor
Production
Epidermal Growth Platelets, macrophages Factor (EGF)
Known Effects Stimulates fibroblasts to secrete collagenase to degrade the matrix during the remodeling phase. Stimulates keratinocyte and fibroblast proliferation. May reduce healing time when applied topically.
Transforming Growth Platelets, macrophages, TGF-a: Mitogenic and chemotactic for keratinocytes Factor (TGF) lymphocytes, hepatocytes and fibroblasts TGF-b1 and TGF-b2: Promotes angiogenesis, upregulates collagen production and inhibits degradation, promotes chemoattraction of inflammatory cells. TGF-b3 (antagonist to TGF-b1 and b2): Has been found in high levels in fetal scarless wound healing and has promoted scarless healing in adults experimentally when TGF-b1 and TGF-b2 are suppressed. Vascular Growth (VEGF)
Endothelial Endothelial cells Factor
Fibroblast Factor (FGF)
Promotes angiogenesis during tissue hypoxia.
Growth Macrophages, mast cells, Promotes angiogenesis, granulation, and T-lymphocytes epithelialization via endothelial cell, fibroblast, and keratinocyte migration, respectively.
Platelet-Derived Platelets, macrophages, Attracts macrophages and fibroblasts to zone of Growth Factor and endothelial cells injury. Promotes collagen and proteoglycan (PDGF) synthesis. Interleukins
Macrophages, keratinocytes, endothelial cells, lymphocytes, fibroblasts, osteoblasts, basophils, mast cells
Colony-Stimulating Factors
Stromal cells, fibroblasts, Granulocyte colony stimulating factor (G-CSF): endothelial cells, Stimulates granulocyte proliferation. lymphocytes Granulocyte Macrophage Colony Stimulating Factor (GM-CSF): Stimulates granulocyte and macrophage proliferation.
Keratinocyte factor
growth Fibroblasts
IL-1: Proinflammatory, chemotactic for neutrophils, fibroblasts, and keratinocytes. Activates neutrophils IL-4: Activates fibroblast differentiation. Induces collagen and proteoglycan synthesis. IL-8: Chemotactic for neutrophils and fibroblasts.
Stimulates keratinocyte migration, differentiation, and proliferation.
Wound Healing Optimization
Creating conditions that allow for proper wound healing can make all the difference in various wounds. When approaching an injured patient, the following list can guide the thought process of the physician or caretaker in optimizing healing conditions. §
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Perfusion: Tissues cannot heal without the cells, oxygen, and nutrients that the cardiovascular system delivers. Patients with severe vascular disease may experience enhanced wound healing via increased perfusion after a vascular bypass or related procedure. Patients who smoke should cease smoking immediately in the event of major surgery or injury. Nicotine causes severe vasoconstriction, and the toxins in cigarette smoke can greatly decrease the ability of tissues to heal. Paraplegics and diabetics with neuropathy must cease all substance abuse and be continually educated and reinforced on the need for pressure relief to avoid pressure ulcers. In the event of pressure sore discovery, absolute pressure relief to increase perfusion is paramount. Infection: Infection is defined as having quantitative bacterial counts of 105 colony forming units per gram of tissue. Infected wounds do not heal because of decreased epithelialization and increased collagen breakdown. These wounds should be appropriately cleared of infection by drainage, debridement, and the administration of appropriate antibiotics. Nutrition: When assessing nutritional status, certain serum nutritional markers can be helpful. Albumin is a good marker of overall long-term nutritional status over the last month; ideally, it should be at least 3.5 g/dL to optimize wound healing. Prealbumin can offer a more recent nutritional status picture and should be maintained above 17 g/dL. Caloric needs of the severely injured patient can exceed 35 kcal/kg/d and 0.8-2 g/kg/d of protein and should be continually assessed and adjusted according to the stage of healing and injury. Steroids: Corticosteroids can blunt the response of macrophages, the most essential cell in wound healing. Vitamin A, insulinlike growth factor (IGF), and oxandrolone (anabolic steroid) can be given to reverse the effects of corticosteroids. Dressing: Numerous dressings are available on the market. The wound should be kept moist (but not wet) at all times. Desiccated tissue is dead tissue and must be sharply debrided. With the advent of negative pressure wound dressing, wound healing for even chronic wounds can be greatly increased.
Suggested Reading: Gurtner G. (2007) Wound Healing: Normal and Abnormal. In: Thorne C, et al: Grabb and Smithʼs Plastic Surgery, 6th ed (pp. 15-22). Philadelphia: Lippincott Williams & Wilkins. Galiano R, Mustoe T. (2007) Wound Care. In: Thorne C, et al: Grabb and Smithʼs Plastic Surgery, 6th ed (pp. 23-32). Philadelphia: Lippincott Williams & Wilkins.