Influence of dietary vitamin C on the wound healing process in rainbow trout (Oncorhynchus mykiss)
This study evaluated the influence of dietary vitamin C as ascorbyl-phosphate on the healing process of experimentally wounded rainbow trout, in order to establish recommendations for salmonid feeding with regard to a reduction of the impact of winter sores. Three duplicate treatment groups of fish (mean initial body weight 56 g) were fed experimental diets containing 20, 150 and 1000 mg ascorbic acid (AA) equivalents/kg feed, respectively, for 4 weeks. Fish were then experimentally wounded, and sampled at 0, 3, 7, 10 and 21 days post-wounding for histological examination (skin and muscle), and AA concentration (skin, muscle, blood plasma, liver and head kidney). Experimental diets were continued during the sampling period. Comparison of the dietary treatments was based on the evaluation of a broad range of 22 histological indices and on the measured AA concentrations. Results showed that skin and muscle AA concentrations analysed at the level of the wound were correlated with dietary intake. Of the 22 histological indices evaluated, 13 exhibited significant differences between the three dietary treatment groups. These indices on which dietary vitamin C intake had the greatest affect were generally those containing fibrous tissue, including the repair of damaged dermal fibres, re-vascularisation and the re-establishment of normal dermal and muscle structure. Of the 13 indices, 7 also showed differences between the 150 and 1000 mg AA/kg feed groups at some point during the healing process. A large majority (11) of the 13 significant indices showed a more rapid healing process in the higher two dietary AA treatments, whereas only three showed a faster initial onset of response. Conclusions: (1) dietary vitamin C intake influences the rate of wound healing in rainbow trout, (2) increasing the dietary level of vitamin C from 150 to 1000 mg AA/kg feed enables the establishment of larger pools of AA in various tissues, and (3) with larger tissue AA pools, the increased AA demand following wounding does not become a rate limiting step, thus healing may proceed more quickly.
Author Keywords: Vitamin C; Rainbow trout; Wound healing
Wound Healing, Skin
Author: Thomas Romo III, MD, FACS, Chief, Clinical Instructor, Department of Otolaryngology, Division of Facial Plastic and Reconstructive Surgery, New York Eye and Ear Infirmary Coauthor(s): James M Pearson, MD, Staff Physician, Department of Otolaryngology – Head and Neck Surgery, New York Eye and Ear Infirmary; Haresh Yalamanchili, MD, Staff Physician, Department of Otolaryngology-Head and Neck Surgery, The New York Eye and Ear Infirmary; Richard A Zoumalan, MD, Staff Physician, Department of Otolaryngology-Head and Neck Surgery, New York University Contributor Information and Disclosures Updated: Feb 18, 2008 * Print ThisPrint This * Email ThisEmail This * References Introduction Healing…is not a science but the intuitive art of wooing nature. W.H. Auden, “The Art of Healing” Wound healing is a natural restorative response to tissue injury. Healing is the interaction of a complex cascade of cellular events that generates resurfacing, reconstitution, and restoration of the tensile strength of injured skin. Healing is a systematic process, traditionally explained in terms of 3 classic phases: inflammation, proliferation, and maturation. A clot forms and inflammatory cells debride injured tissue during the inflammatory phase. Epithelialization, fibroplasia, and angiogenesis occur during the proliferative phase. Meanwhile, granulation tissue forms and the wound begins to contract. Finally, during the maturation phase, collagen forms tight cross-links to other collagen and with protein molecules, increasing the tensile strength of the scar. For the sake of discussion and understanding, the process of wound healing may be considered a series of separate events. In actuality, the entire process is much more complicated, as cellular events that lead to scar formation occur in tandem. Many aspects of wound healing have yet to be elucidated. Surgeons should have an understanding of the process of wound healing to help produce scars that are cosmetically pleasing and do not impair function. For further reading, please see Medscape’s Wound Management Resource Center. Inflammatory Phase The early events of wound healing are characterized by the inflammatory phase, a vascular and cellular response to injury. An incision made through a full thickness of skin causes a disruption of the microvasculature and immediate hemorrhage. Following incision of the skin, a 5- to 10-minute period of vasoconstriction ensues, mediated by epinephrine, norepinephrine, prostaglandins, serotonin, and thromboxane. Vasoconstriction causes temporary blanching of the wound and functions to reduce hemorrhage immediately following tissue injury, aid in platelet aggregation, and keep healing factors within the wound. Endothelial cells retract to expose the subendothelial collagen surfaces; platelets attach to these surfaces. Adhesion to exposed collagen surfaces and to other platelets occurs through adhesive glycoproteins: fibrinogen, fibronectin, thrombospondin, and von Willebrand factor. The aggregation of platelets results in the formation of the primary platelet plug. Aggregation and attachment to exposed collagen surfaces activates the platelets. Activation enables platelets to degranulate and release chemotactic and growth factors, such as platelet-derived growth factor (PDGF), proteases, and vasoactive agents (eg, serotonin, histamine). The coagulation cascade occurs via 2 different pathways. The intrinsic pathway begins with the activation of factor XII (Hageman factor) when blood is exposed to extravascular surfaces. The extrinsic coagulation pathway occurs through the activation of tissue factor found in extravascular cells in the presence of factors VII and VIIa. Both pathways proceed to the activation of thrombin, which converts fibrinogen to fibrin. The fibrin product is essential to wound healing and is the primary component of the wound matrix into which inflammatory cells, platelets, and plasma proteins migrate. Removal of the fibrin matrix impedes wound healing. In addition to activation of fibrin, thrombin facilitates migration of inflammatory cells to the site of injury by increasing vascular permeability. By this mechanism, factors and cells necessary for healing flow from the intravascular space and into the extravascular space. The result of platelet aggregation and the coagulation cascade is clot formation. Clot formation is limited in duration and to the site of injury. Clot formation dissipates as its stimuli dissipate. Plasminogen is converted to plasmin, a potent enzyme that aids in cell lysis. Clot formation is limited to the site of injury because uninjured nearby endothelial cells produce prostacyclin, an inhibitor of platelet aggregation. In uninjured adjacent areas, antithrombin III binds thrombin, and protein C binds factors of the coagulation cascade, namely, factors V and VII. The vasoconstriction period is followed by a more persistent period of vasodilation mediated by histamine, prostaglandins, kinins, and leukotrienes. Vasodilation is responsible for the erythema, edema, and heat observed after tissue injury. Vasodilation is an important means by which the wound can be exposed to increased blood flow, accompanied by the necessary inflammatory cells and factors that fight infection and debride the wound of devitalized tissue. Alterations in pH (secondary to tissue and bacterial degradation), swelling, and tissue hypoxemia at the injury site contribute to the sensation of wound pain. Following injury, the products of the earliest cellular events activate intricately related inflammatory pathways that modify subsequent events in the wound-healing process. For example, Hageman factor activates the kinin pathway, which produces bradykinin. Bradykinin stimulates vasodilation and increased vascular permeability. Histamine released from platelets and circulating mast cells increases vascular permeability and indirectly stimulates vasodilation through the production of prostaglandins E1 and E2. Prostaglandins cause vasodilation through the activation of the adenylate cyclase pathway via the production of cyclic adenosine monophosphate. Prostaglandins also accumulate at the area of injury through the activation of phospholipases located on injured cell membranes. Phospholipases stimulate the release of arachidonic acid, ultimately leading to the production of prostaglandins, leukotrienes, and other factors. Hageman factor also activates the classic complement pathway during the inflammatory phase. Inactive proteins of the complement system (ie, C1-C9) are activated by means of a cascade of reactions. These proteins stimulate important inflammatory events such as chemotaxis, degranulation of mast cells, and cytolysis. C5a and C567 are chemotactic agents for neutrophil migration. C3a, C4a, and C5a cause degranulation of mast cells, leading to release of histamine and increased vascular permeability. The membrane attack complex, C5b6789, is responsible for cytolysis. The cellular aspect of the inflammatory phase occurs within hours of injury. Neutrophils are the predominant cell type for the first 48 hours after injury but do not appear essential to the wound-healing process. Neutrophils cleanse the wound site of bacteria and necrotic matter and release inflammatory mediators and bactericidal oxygen-free radicals. The absence of neutrophils does not prevent healing. Macrophages are essential to wound healing and perhaps are the most important cells in the early phase of wound healing. Macrophages phagocytose debris and bacteria. Macrophages also secrete collagenases and elastases, which break down injured tissue and release cytokines. In addition, macrophages release PDGF, an important cytokine that stimulates the chemotaxis and proliferation of fibroblasts and smooth muscle cells. Finally, macrophages secrete substances that attract endothelial cells to the wound and stimulate their proliferation to promote angiogenesis. Macrophage-derived growth factors play a pivotal role in new tissue formation, as evidenced by the fact that new tissue formation in macrophage-depleted animal wounds demonstrates defective repair. In studies in which experimental wounds are rendered monocytopenic, subsequent stages of fibroplasia and granulation tissue formation are impaired and the overall rate of wound healing is delayed. T lymphocytes migrate into the wound during the inflammatory phase, approximately 72 hours following injury. T lymphocytes are attracted to the wound by the cellular release of interleukin 1, which also contributes to the regulation of collagenase. Lymphocytes secrete lymphokines such as heparin-binding epidermal growth factor and basic fibroblast growth factor. Lymphocytes also play a role in cellular immunity and antibody production. Proliferative Phase Formation of granulation tissue is a central event during the proliferative phase. Inflammatory cells, fibroblasts, and neovasculature in a matrix of fibronectin, collagen, glycosaminoglycans, and proteoglycans comprise the granulation tissue. Granulation tissue formation occurs 3-5 days following injury and overlaps with the preceding inflammatory phase. Epithelialization Epithelialization is the formation of epithelium over a denuded surface. Epithelialization of an incisional wound involves the migration of cells at the wound edges over a distance of less than 1 mm, from one side of the incision to the other. Incisional wounds are epithelialized within 24-48 hours after injury. This epithelial layer provides a seal between the underlying wound and the environment. The process begins within hours of tissue injury. Epidermal cells at the wound edges undergo structural changes, allowing them to detach from their connections to other epidermal cells and to their basement membrane. Intracellular actin microfilaments are formed, allowing the epidermal cells to creep across the wound surface. As the cells migrate, they dissect the wound and separate the overlying eschar from the underlying viable tissue. In superficial wounds (eg, wounds due to laser resurfacing, dermabrasion, chemical peel treatments) adnexal structures (eg, sebaceous glands, hair follicles)contribute to reepithelialization. Epidermal cells secrete collagenases that break down collagen and plasminogen activator, which stimulates the production of plasmin. Plasmin promotes clot dissolution along the path of epithelial cell migration. The extracellular wound matrix over which epithelial cells migrate has received increased emphasis in wound-healing research. Migrating epithelial cells interact with a provisional matrix of fibrin cross-linked to fibronectin and collagen. The matrix components may be a source of cell signals to facilitate epithelial cell proliferation and migration. In particular, fibronectin seems to promote keratinocyte adhesion to guide these cells across the wound base. Wounds in a moist environment demonstrate a faster and more direct course of epithelialization. Occlusive and semiocclusive dressings applied in the first 48 hours after injury may maintain tissue humidity and optimize epithelialization. When epithelialization is complete, the epidermal cell assumes its original form, and new desmosomal linkages to other epidermal cells and hemidesmosomal linkages to the basement membrane are restored. Fibroplasia The fibroblast is a critical component of granulation tissue. Fibroblasts are responsible for the production of collagen, elastin, fibronectin, glycosaminoglycans, and proteases Fibroblasts grow in the wound as the number of inflammation cells decrease. The demand for inflammation disappears as the chemotactic factors that call inflammatory cells to the wound are no longer produced and as those already present in the wound are inactivated. Fibroplasia begins 3-5 days after injury and may last as long as 14 days. Skin fibroblasts and mesenchymal cells differentiate to perform migratory and contractile capabilities. Fibroblasts migrate and proliferate in response to fibronectin, platelet-derived growth factor (PDGF), fibroblast growth factor, transforming growth factor, and C5a. Fibronectin serves as an anchor for the myofibroblast as it migrates within the wound. The synthesis and deposition of collagen is a critical event in the proliferative phase and to wound healing in general. Collagen consists of 3 polypeptide chains, each twisted into a left-handed helix. Three chains of collagen aggregate by covalent bonds and twist into a right-handed superhelix, forming the basic collagen unit. A striking structural feature of collagen is that every third amino acid is glycine. This repeating structural feature is an absolute requirement for triple-helix formation. Collagen is rich in hydroxylysine and hydroxyproline moieties, which enable it to form strong cross-links. The hydroxylation of proline and lysine residues depends on the presence of oxygen, vitamin C, ferrous iron, and a -ketoglutarate. Deficiencies of oxygen and vitamin C, in particular, result in underhydroxylated collagen that is less capable of forming strong cross-links and, therefore, is more vulnerable to breakdown. Collagen is secreted to the extracellular space in the form of procollagen. This form is then cleaved of its terminal segments and called tropocollagen. Tropocollagen can aggregate with other tropocollagen molecules to form collagen filaments. Filaments consist of tropocollagen molecules arrayed in a staggered fashion, joined by intermolecular cross-links. Filaments aggregate to form fibrils. Collagen fibrils, in turn, aggregate to form collagen fibers. Filament, fibril, and fiber formation occur within a matrix gel of glycosaminoglycans, hyaluronic acid, chondroitin sulfate, dermatan sulfate, and heparin sulfate produced by fibroblasts. Intermolecular cross-links within the collagen fiber stabilize it, making it resistant to destruction. Age, tension, pressure, and stress affect the rate of collagen synthesis. Collagen synthesis begins approximately 3 days after injury and may continue at a rapid rate for approximately 2-4 weeks. Collagen synthesis is controlled by the presence of collagenases and other factors that destroy collagen as new collagen is made. Approximately 80% of the collagen in normal skin is type I collagen; the remaining is mostly type III. In contrast, type III collagen is the primary component of early granulation tissue and is abundant in embryonic tissue. Collagen fibers are deposited in a framework of fibronectin. An essential interaction seems to exist between fibronectin and collagen; experimental wounds depleted of fibronectin demonstrate decreased collagen accumulation. Elastin is also present in the wound in smaller amounts. Elastin is a structural protein with random coils that allow for stretch and recoil properties of the skin. Angiogenesis A rich blood supply is vital to sustain newly formed tissue and is appreciated in the erythema of a newly formed scar. These blood vessels disappear as they become unnecessary, as does the erythema of the scar. The macrophage is essential to the stimulation of angiogenesis and produces macrophage-derived angiogenic factor in response to low tissue oxygenation. This factor functions as a chemoattractant for endothelial cells. Basic fibroblast growth factor secreted by the macrophage and vascular endothelial growth factor secreted by the epidermal cell are also important to angiogenesis. Fibronectin is chemotactic for endothelial cells. Capillaries bud from existing capillaries in response to these growth factors. Endothelial cells coalesce and bind fibrin, which adds support to the vessel wall. Angiogenesis results in greater blood flow to the wound and, consequently, increased perfusion of healing factors. Angiogenesis ceases as the demand for new blood vessels ceases. New blood vessels that become unnecessary disappear by apoptosis. New blood vessel formation is a complex process that relies on several angiogenic factors such as vascular endothelial growth factor, angiogenin, and angiotropin. Contraction Wound contraction begins almost concurrently with collagen synthesis. Contraction, defined as the centripetal movement of wound edges that facilitates closure of a wound defect, is maximal 5-15 days after injury. Contraction results in a decrease in wound size, appreciated from end to end along an incision; a 2-cm incision may measure 1.8 cm after contraction. The maximal rate of contraction is 0.75 mm/d and depends on the degree of tissue laxity and shape of the wound. Loose tissues contract more than tissues with poor laxity, and square wounds tend to contract more than circular wounds. Wound contraction depends on the myofibroblast located at the periphery of the wound, its connection to components of the extracellular matrix, and myofibroblast proliferation. Radiation and drugs, which inhibit cell division, have been noted to delay wound contraction. Contraction does not seem to depend on collagen synthesis. Although the role of the peripheral nervous system in wound healing is not well delineated, recent studies have suggested that sympathetic innervation may affect wound contraction and epithelialization through unknown mechanisms. Contraction must be distinguished from contracture, a pathologic process of excessive contraction that limits motion of the underlying tissues and is typically caused by the application of excessive stress to the wound. Maturation Phase Collagen Collagen remodeling during the maturation phase depends on continued collagen synthesis in the presence of collagen destruction. Collagenases and matrix metalloproteinases in the wound assist removal of excess collagen while synthesis of new collagen persists. Tissue inhibitors of metalloproteinases limit these collagenolytic enzymes, so that a balance exists between formation of new collagen and removal of old collagen. During remodeling, collagen becomes increasingly organized. Fibronectin gradually disappears, and hyaluronic acid and glycosaminoglycans are replaced by proteoglycans. Type III collagen is replaced by type I collagen. Water is resorbed from the scar. These events allow collagen fibers to lie closer together, facilitating collagen cross-linking and ultimately decreasing scar thickness. Intramolecular and intermolecular collagen cross-links result in increased wound bursting strength. Remodeling begins approximately 21 days after injury, when the net collagen content of the wound is stable. Remodeling may continue indefinitely. The tensile strength of a wound is a measurement of its load capacity per unit area. The bursting strength of a wound is the force required to break a wound regardless of its dimension. Bursting strength varies with skin thickness. Peak tensile strength of a wound occurs approximately 60 days after injury. A healed wound only reaches approximately 80% of the tensile strength of unwounded skin. Cytokines Cytokines have emerged as important mediators of wound healing events. By definition, a cytokine is a protein mediator, released from various cell sources, which binds to cell surface receptors to stimulate a cell response. Cytokines can reach their target cell by endocrine, paracrine, autocrine, or intracrine routes. Some important cytokines are described as follows: * Epidermal growth factor was the first cytokine described and is a potent mitogen for epithelial cells, endothelial cells, and fibroblasts. Epidermal growth factor stimulates fibronectin synthesis, angiogenesis, fibroplasia, and collagenase activity. * Fibroblast growth factor is a mitogen for mesenchymal cells and an important stimulus for angiogenesis. Fibroblast growth factor is a mitogen for endothelial cells, fibroblasts, keratinocytes, and myoblasts. This factor also stimulates wound contraction and epithelialization and production of collagen, fibronectin, and proteoglycans. * PDGF is released from the alpha granules of platelets and is responsible for the stimulation of neutrophils and macrophages and for the production of transforming growth factor-b. PDGF is a mitogen and chemotactic agent for fibroblasts and smooth muscle cells and stimulates angiogenesis, collagen synthesis, and collagenase. Vascular endothelial growth factor is similar to PDGF but does not bind the same receptors. Vascular endothelial growth factor is mitogenic for endothelial cells and plays an important role in angiogenesis. * Transforming growth factor-b is released from the alpha granules of platelets and has been shown to regulate its own production in an autocrine manner. This factor is an important stimulant for fibroblast proliferation and the production of proteoglycans, collagen, and fibrin. The factor also promotes accumulation of the extracellular matrix and fibrosis. Transforming growth factor-b has been demonstrated to reduce scarring and to reverse the inhibition of wound healing by glucocorticoids. * Tumor necrosis factor-a is produced by macrophages and stimulates angiogenesis and the synthesis of collagen and collagenase. Tumor necrosis factor-a is a mitogen for fibroblasts. Abnormal Wound Healing: Keloids and Hypertrophic Scars Keloids and hypertrophic scars are characterized by an accumulation of excess collagen and are distinguished from each other by their appearance. Keloids grow beyond the borders of the original wound and do not tend to resolve spontaneously. Hypertrophic scars stay within the limit of the original wound and do tend to regress spontaneously. Hypertrophic scars are generally seen soon after tissue injury, whereas keloids can form as late as a year after injury. Hypertrophic scars tend to be associated with a contracture across a joint surface. Upon histological examination, keloids and hypertrophic scars differ from normal skin by a rich blood supply, high mesenchymal density, and a thick epidermal layer. The collagen fibers in hypertrophic scars are loosely arrayed in a wavy pattern. Keloids demonstrate a disorganized pattern of large, irregularly shaped collagen fibers with a lower content of collagen cross-links compared to normal skin. Keloids also contain a greater amount of type III collagen than a mature scar, which suggests a failure in scar maturation. Little is known about the etiology of keloid and hypertrophic scar formation. Abnormalities in cell migration, proliferation, inflammation, and the synthesis and secretion of extracellular matrix proteins, cytokines, and remodeling have all been associated with keloid and hypertrophic scar formation. Increased activity of fibrogenic cytokines and exaggerated responses to cytokines have been described. Transforming growth factor-b (TGF-b) appears to play an integral role in aberrant healing. More recently, abnormal interactions between epidermal-mesenchymal cells and regulatory genes, such as TP53, have been proposed. That certain patients and conditions may predispose to hypertrophic scar formation is well known. Both hypertrophic scars and keloids occur more commonly in dark-skinned individuals. Wounds that cross skin tension lines or wounds that are located on the ear lobes or presternal and deltoid areas are common sites for these forms of abnormal healing. The role of wound tension has been examined and found to be related to the exaggerated response to tension of keloid fibroblasts relative to normal fibroblasts with regard to production of pro-fibrotic growth factors. A single, optimal treatment technique for hypertrophic scars and keloids has not been developed, and the recurrence rate of these abnormal scars is high. Surgical management is reserved for cases that are unresponsive to conservative management. Conservative management includes pharmacologic therapy, pressure, laser, and radiotherapy. Each method has varying degrees of reported success. Recent work has demonstrated that treatment of fibroblasts with the combined CO2 and Er:YAG lasers alters the differential expression of mediators responsible for fibrosis and collagen formation and organization. Laser therapy may play a role in the prevention of keloid and hypertrophic scar formation. Fetal Wound Healing Studies and the Future The study of fetal wound healing is intriguing and may result in the discovery of an optimal method that would allow wounds to heal without scar formation. Wounds occurring in fetuses of early gestational age can heal without any scar formation. The difference in the wound environment in fetal wounds occurring in early gestation has been speculated to account for the absence of scar formation. Proposed contributing factors to scarless healing in fetal wounds are the presence of fewer neutrophils and more monocytes during the inflammatory period, different concentrations of cytokines, and a greater proportion of type III collagen in contrast to adult wounds. Transforming growth factor-b (TGF-b—specifically, low levels of TGF-b1 and TGF-b2 and high levels of TGF-b3—probably has a central role in scar formation, and studies of its role are ongoing. Low levels of platelet-derived growth factor (PDGF), a greater amount of epidermal growth factor (a mitogen for epithelialization), a faster rate of wound healing, and a greater amount of hyaluronic acid in the extracellular matrix has been documented and suggests a more efficient process of wound healing in fetal models. Notably, collagen deposition in fetal wounds displays a fine reticular pattern that is indistinguishable from uninjured skin. In addition, fibronectin is more abundant in fetal wounds and has been noted to accelerate wound healing in fetal rat models. In neonates, fibroblasts have a potent collagen-producing response to TGF-b. In fetal skin, this response is blunted and short lived. There is less differentiation of fibroblasts into myofibroblasts, and this differentiation is briefer. Downstream mediators of the TGF-b pathway such as Smad2/3 and c-Jun N-terminal kinase were decreased compared with neonate skin.1 Although evidence indicates that early fetal wound models heal more efficiently than adult wounds, the explanation of this more-efficient process is under investigation. The explanation may also lie in alterations of the wound environment to mimic fetal wound models. Evidence shows that the healing environment may be manipulated in other ways to enhance or to accelerate the healing process. Recent animal studies demonstrate beneficial effects of both laser and electrical stimulation of tissues during the inflammatory, proliferative, and maturation phases of wound healing. Based on the assumption that proteins found in the blood can promote healing, the application of platelet-rich plasma has been investigated and found to foster increased levels of inflammatory cells during the healing process without affecting final cell counts in healed tissue. In healthy individuals, platelet-rich plasma may accelerate wound closure in full-thickness dermal wounds. However, it does not alter the long-term architecture of a healed wound.2 Platelet-derived growth factor D alone may increase the rate of macrophage recruitment and angiogenesis, but its overall long-term effect is not established.3 Pulsed magnetic field therapy is also being investigated as a means to help wound healing. In rats, it has been shown to accelerate cutaneous wound healing.4 The used of these novel modalities and techniques in the clinical setting remains to be determined. Keywords wound healing, skin wounds, healing, scars, scarring, tissue healing, tissue injury, skin injury, skin healing, keloids, hypertrophic scars, hypertrophic scarring, abnormal healing, scars, abnormal scars, healing process, wound healing process, fetal wound healing, inflammatory phase, proliferative phase, maturation phase, epithelialization, fibroplasia, angiogenesis, vasoconstriction, fibrin matrix, fibrin activation, platelet aggregation, coagulation cascade, clot formation, cell lysis, vasodilation, Hageman factor, chemotaxis, degranulation of mast cells, cytolysis, macrophages, fibroblasts, collagen, cytokines More on Wound Healing, Skin References