Skin Wound Healing

A wound is defined as any interruption in the continuity of the body's tissue [1,2]. After injury, a wound immediately begins a complex process of healing, which involves sophisticated synchronization of molecular and biochemical events at the cellular level, to bring about tissue repair and regeneration. Wound healing is this fine-tuned process that restores anatomic and physiologic integrity to the tissue and culminates in the formation of a scar [3].

Wound healing may be compartmentalized into four stages, i.e., inflammation, debridement, repair/proliferation, and maturation/remodeling [3-6]. These stages exhibit a significant degree of overlap and occur as a continuum from start to finish. Thus, more than one stage of healing can occur at one time and, regardless of wound size, all normally healing wounds undergo all stages of healing.

Etiologic factors for wounds are numerous and varied, ranging from mechanical trauma to physical agents such as extreme temperatures, chemicals, neoplastic processes, microbial infection, and surgery [3].

Inflammation and Debridement Stages

The inflammatory stage of healing is the body's vascular and cellular defensive response [7,8]. It is separated into early and late phases [4,5]. The early phase consists of immediate responses including vascular tone dynamics and hemostasis, whereas the late phase is characterized by vascular responses as well as recruitment and activation of inflammatory cell populations [7,8].

Tissue Reactions

The first response to any injury is hemorrhage, with the magnitude varying in accordance with the insult. Vasoconstriction occurs in an attempt to control hemorrhage. This is accompanied by an immediate enlargement of the defect, owing to inherent cutaneous elasticity and external muscle tension that retracts wound edges, causing wound gaping [4]. As a result, a wound may appear large; however, manipulation of the tissues may reveal that much of the enlargement is a result of tissue retraction.

Hemostasis, Lymphatic Reaction, and Scab

Torn blood vessels have exposed basement membranes containing subendothelial collagen, which when in contact with platelets bring about platelet activation and aggregation and initiate the intrinsic path of the coagulation cascade [3,4,7,9] culminating in thrombus formation and hemorrhage control, i.e., a fibrin clot in the wound. The clot not only attenuates hemorrhage, but also occludes ruptured lymphatics, thereby preventing drainage, promoting edema, and localizing inflammation [4]. Later in the healing process, fibrinolysis dissolves fibrin plugs in the lymphatics, and lymphatic drainage resumes. When exposed to the external environment, the clot desiccates to form a scab [3,4]. A tightly adhered scab acts as a biologic bandage, beneath which wound contraction and epithelialization can progress undisturbed [4,5].

Vascular and Humoral Events and Cellular Movement

The initial vasoconstriction is closely followed by reflex vasodilation and increased vascular permeability, resulting in plasma exudation [4,5] and the escape of inflammatory mediators and cellular components from blood vessels. Initiation of the arachidonic acid cascade results in production of prostaglandins, thromboxanes, and leukotrienes [3,4,7,8]. These humoral factors, along with histamine, serotonin, bradykinin, and complement activation, help perpetuate inflammation [8]. Mast cell-derived serotonin and histamine cause rounding of vascular endothelium, thereby causing loss of tight cellular junctions and promoting extravasation of plasma and other inflammatory mediators into the surrounding tissues [8].

Surface receptors for leukocytes on endothelial cells become activated, with resultant leukocyte margination and eventual migration into interstitial tissues [5,8].

Endothelial cells within blood vessels adjacent to the wounded tissue exhibit cellular adhesion molecules (CAMs), which bind inflammatory cells and assist in their passage between endothelial cells to the wound [7]. Further cellular migration is brought about with the help of proteinases (serine, metallo-, cysteine, and aspartic) which, by catabolizing ground substance and extracellular matrix, create a path for the cells [7]. Fibrin polymers are cross-linked to form a scaffold over which neutrophils and macrophages migrate toward the wound after diapedesis from the blood vessels [3].

Polymorphonuclear leukocytes are the first responders to various chemotactic signals and appear at the site within hours of trauma [4,8,9]. They are followed closely by macrophages, and finally, T-lymphocytes [8]. Neutrophils are generally short-lived whereas macrophages persist for a significantly longer period [5].

Cellular Functions

Besides playing a major role in coagulation, platelets also produce several cytokines and growth factors important to the progression of healing. Platelet activation results in release of platelet-derived growth factor (PDGF), transforming growth factor-α and -β (TGF-α and -β), and tumor necrosis factor-α (TNF-α) [2-4,7] from platelet alpha granules [2,8]. These signaling molecules are chemotactic for neutrophils, macrophages, and T-lymphocytes, which migrate toward the wound [3,7]. Platelet alpha granules contain four adhesive glycoproteins: fibrinogen, fibronectin, von Willebrand factor, and thrombospondin [8]. These glycoproteins participate in coagulation as well as temporary matrix synthesis.

Neutrophils and macrophages debride the wound of necrotic, devitalized tissue. Later, macrophages coordinate tissue proliferation.

Neutrophils are not essential to wound healing, despite being the first to arrive in the wound [3,9]. Neutrophils phagocytose bacteria and kill them by generating toxic reactive oxygen species or free radicals [2,7]. Their granules contain various proteinases, among which cathepsin G, urokinase-type plasminogen activator, collagenase, and elastase are predominant [2,7]. These proteinases can digest extracellular matrix components including elastin, fibrin, fibronectin, vitronectin, laminin, collagen, and proteoglycans. Proteinase inhibitors protect normal healthy tissues from the action of proteinases. Once spent, neutrophils are removed from the wound by macrophages or via wound exudate [4].

Monocytes are transformed into macrophages once they are out of circulation and sequestered within tissue. Macrophage numbers reach their peak within 24 hours [8,9]. Macrophages are capable of phagocytosis as well as of discharging proteinases, specifically matrix metalloproteinases (MMPs), both of which are crucial during wound debridement and subsequent healing. Debridement and tissue proliferation are decelerated in wounds devoid of macrophages [7]. Tissue inhibitors of metalloproteinases (TIMPs) protect healthy tissue from the indiscriminate action of MMPs. Macrophages are activated to synthesize signaling molecules, including colony-stimulating factor, TNF-α, PDGF, interleukin-1, TGF-α and -β, fibroblast growth factor (FGF), and insulin-like growth factor-1, which induce cell proliferation, especially of keratinocytes, fibroblasts, and endothelial cells [2,7]. Therefore, macrophages are the bridge between the inflammatory and repair phases of wound healing [7].

T-lymphocytes play a regulatory role in wound healing. They are not vital; however, their absence retards the healing process. Lymphocytes produce growth and regulatory factors that govern functions of other cells. The role of lymphocytes in healing is generally associated with a foreign antigen or secondary infection. They arrive in the wound at the same time as macrophages and interact with them in producing an immune response. Macrophages process foreign antigens, and these modified antigens are presented to the lymphocytes. The lymphocytes produce cytokines such as interferon-gamma (IFN-γ) and interleukins 2 to [2,4] Lymphocytes also produce TGF-β [4].

The role of eosinophils in wound healing has not yet been elucidated. They are present in healing wounds and peak around 1 week post injury [8]. A rabbit cutaneous wound healing model demonstrated that eosinophils appeared to be responsible for secreting TGF-α after day [7,8] however, eosinophils may delay wound re-epithelialization [7].

Repair/Proliferation Stage

The repair/proliferation phase is composed of fibroblast proliferation and migration with production of wound extracellular matrix, neovascularization, and epithelial proliferation and migration [2-6,8]. Fibroblast activity and neovascularization result in granulation tissue formation.

Macrophages play a significant role in this stage of healing. They become activated in response to growth and chemotactic factors released from platelets during the latter part of the inflammatory and debridement phases. Digestion of fibronectin and collagen further potentiates macrophage activation and release of additional mediators of fibroplasia and angiogenesis, such as nitric oxide, which is essential for granulation tissue synthesis [3,4]. Macrophages recruit lymphocytes, which release lymphokines (interleukins and interferons) responsible for further reactivating macrophages, to ensure adequate levels of molecular mediators and vasoactive peptides throughout the proliferative phase.

Granulation Tissue - Production and Functions

Granulation tissue is composed primarily of fibroblasts, capillaries, macrophages, and collagen [3,4,10] and appears in the wound between three and six days after commencement of healing [5]. Granulation tissue provides a mechanical barrier against microbial infection of the wound and a biologic barrier owing to the presence of granulocytes and macrophages on its surface [3]. It is also a source of collagen, which is required for wound repair and remodeling. Granulation tissue brings about wound contraction and serves as a foundation across which epithelial migration takes place [4].

Fibroblasts are a predominant component of this stage of healing and a major constituent of granulation tissue. Sources of wound fibroblasts include pericytes and undifferentiated perivascular mesenchymal cells found in connective tissue [4,6,8,9,11]. Under the influence of factors such as PDGF, epidermal growth factor (EGF), FGF, IGF-1, and TGF-β, this quiescent cell population becomes activated, proliferates, and develops cytoplasmic extensions called ruffled membranes [9]. Fibroblasts secrete IGF-1, β-FGF, TGF-β, PDGF and keratinocyte growth factor (KGF) [4]. Contact guidance, haptotaxis [8,10], chemotaxis, and the use of ruffled membranes assist fibroblast movement along the fibrin meshwork within the wound [4,8]. As fibroblasts repopulate the wound bed, they continue synthesizing intercellular matrix constituents, including fibronectin, proteoglycans, collagen, and elastin [4,8].

Neovascularization

The second component of the repair stage is neovascularization. Endothelial cell proliferation and subsequent migration occur in the presence of interstitial matrix or the absence of an intact basement membrane, resulting in capillary buds sprouting from pre-existing venules [3,4,6,10] and capillary loop formation [8,10]. Once canalized, blood flow begins and circulation is established. New blood vessels grow at an approximate rate of 0.4 to 1.0 mm per day. In vitro studies have demonstrated that endothelial cells cultured on collagen types IV and V, the components of basement membranes [8,12] form tubular structures instead of confluent monolayers, which are observed when cells are cultured on collagen types I and II [8]. Capillary buds that do not form lumens regress [3,4,6]. Lymphatics, although much slower to re-establish, follow a pattern similar to that of the capillaries and, therefore, lymphatic drainage during the early phases of wound healing is inadequate, contributing to edema formation [3,4,8].

Wound pH and oxygen are important factors for fibroblast function and neovascularization. Optimal collagen synthesis is achieved when the microenvironment of the wound maintains a mildly acidic pH [8]. Oxygen tension at the wound periphery is almost 90 mm Hg, whereas in the center of the wound bed it falls to almost zero [2,11]. Under these hypoxic conditions, glycolysis-derived energy is sufficient to initiate fibroplasia. However, to sustain collagen production, molecular oxygen is vital for post-translational hydroxylation of proline and lysine moieties and cross-linkage of collagen fibrils [11]. Twenty mm Hg appears to be the critical oxygen tension for fibroplasia and collagen synthesis [8,9]; however, another report suggests that oxygen tension below 40 mm Hg impairs fibroplasia [11].

Despite its deleterious effects on wound healing, a hypoxic environment and the oxygen tension gradient act as a stimulus for fibroblast mitosis, angiogenesis, and sustained secretion of growth factors from macrophages [3,4,11]. An oxygen tension gradient is a prerequisite for neovascularization [8].

Angiogenic growth factors include PDGF, vascular endothelial growth factor (VEGF), tumor necrosis factor (TNF), and endothelin-1 [2,11]. Upregulation of these angiogenic factors in endothelial cells increases cellular mitotic activity and thereby promotes neovascularization. Endothelial cells demonstrate a phenomenon called dynamic reciprocity, whereby they respond to environmental influences, such as cellular and chemical signals, and are capable of transforming their environment to induce changes beneficial to wound healing [11].

As healing progresses, early components of the process regress as repair components increase. Fibrinolysis by plasmin disrupts the clot in the wound bed [4]. Gradually, neutrophils and lymphocytes are cleared from the wound by apoptosis or phagocytosis by macrophages. Fibrin, proteoglycans, tropocollagen, and protocollagen are substituted by collagen fibrils synthesized by the wound fibroblasts.

Collagen is a major component of granulation tissue [10]. Fibroblasts, under the influence of TGF-α and -β, EGF, PDGF, β-FGF, IL-1, and TNF, synthesize collagen fibrils and the viscoelastic extracellular matrix [9,12]. Collagen biosynthesis is a complicated process that involves gene transcription and translation, intracellular, intercellular, and extracellular transformation, and fibril assembly and cross-linkage [12]. Collagen includes two specific amino acids, hydroxylysine and 4-hydroxyproline [12,13]. These amino acids, along with glycine, form alpha peptide chains. These alpha peptide chains are intertwined to form a triple helix [2,6,12,13] which is further convoluted to form a supercoiled helix [13]. Galactose is added to the superhelix before being extruded from the fibroblast as procollagen, which is further cleaved to produce tropocollagen.

Increase in wound strength is due to collagen deposition and collagen maturation. Tropocollagen is aggregated and cross-linked to produce collagen filaments. Collagen filaments are cross-linked to form collagen fibrils, which, when cross-linked together, form collagen fibers. On maturation, collagen type III becomes collagen type I [3,4,6,10] which as it matures and reorients within the wound increases the tensile strength of the wound [4]. In the first 2 weeks of wound healing, fibroblasts, fibrin, and collagen are vertically arranged between the wound edges, and the increase in tensile strength of the wound is due to an increase in collagen deposition [9]. In the late repair stage, the fibers are aligned horizontally [3,4,8]. Early strength of the wound is attributed to its collagen content, whereas late wound strength is associated with collagen type, maturation, and three-dimensional remodeling. As the collagen content of a wound increases, collagen synthesis balances collagen lysis. Extracellular proteoglycan matrix decreases proportionately as collagen increasingly occupies space [4]. This signals the beginning of the maturation phase.

Wound Contraction

Granulation tissue also contributes to wound contraction. Wound contraction is the centripetal force by which a wound achieves a reduction in size [3,4,6,10], which may or may not culminate in complete wound closure. This phenomenon is especially important in open wound healing. Wound contraction does not involve synthesis of new skin, but rather centripetal movement of skin around the wound [4,10].

One theory on wound contraction states that a sub-population of fibroblasts from the granulation tissue develop a higher percentage of actin-rich fibers, thereby transforming them into myofibroblasts [2]. These fibers are called stress fibers and contain myosin and tropomyosin [8]. TGF-β is thought to play a role in differentiation [3,4,10]. This response is an attempt to counteract by the inherent elastic nature of skin the retractive forces exerted by the wound edges [4,10]. Fibronexus is the interconnection between myofibroblasts and extracellular matrix. It plays a role in mediating wound contraction by reorienting the ground substance [8]. Additionally, fibronexus links myofibroblast stress fibers with collagen via fibronectin [8].

A second theory on wound contraction suggests that regular fibroblasts use cell membrane tractional forces to reorganize and rearrange collagen fibrils [4,6,10]. As the fibroblast traverses the collagen, the cell membrane pulls collagen fibrils toward the center of the wound. This is best explained by the "toy tank moving over loose carpet" comparison [4,10]. This process is enhanced by PDGF, which stimulates extracellular matrix contraction, whereas FGF and IFN-γ inhibit the process.

Wound contraction is terminated by contact inhibition, that is, when one edge of the wound meets the other or when tensions exerted by skin elasticity at the wound edges and fibroblasts and myofibroblasts within the wound are equal [4,10]. The number of myofibroblasts present in any wound is proportional to the size of the wound and the tension on the wound edges; large, open wounds demonstrate higher numbers, whereas wounds that have undergone primary closure have significantly fewer myofibroblasts [9].

Epithelialization

Epithelialization protects against external infection and fluid loss [3]. For epithelialization to occur, the wound must be free of infection and must maintain a moist, oxygen-rich microenvironment [8]. Epithelialization occurs independently of wound contraction and, in open wounds, occurs after granulation tissue has been laid down, i.e., after a lag phase of four to five days [4]; whereas in closed-wound healing, it begins almost immediately and is complete within two days.

Epithelial cells are influenced by β-FGF, EGF, IL-1, PDGF, and TGF-α and β. When up-regulated, these factors induce accelerated mitotic activity of epithelial cells. Mitotic activity and migration are not interdependent factors [8]. Epithelial cells develop microvilli within cell membranes. They then extend pseudopodia that facilitate migration [3]. Two theories elucidate epithelial migration. One theory proposes that desmosomal and hemidesmosomal attachments of basal epithelium to the basement membrane and adjacent epithelial cells at the wound edge are lost, thereby promoting migration of cells across the wound bed in monolayers [2,3]. This is known as the sliding theory [4,8]. The second hypothesis is called the leapfrog theory. This states that basal epithelial cells at the wound edge migrate on to the granulation tissue and implant. The epidermal cells just behind them migrate over the newly implanted epithelial cells and attach once they contact granulation tissue [4,8]. Epithelial migration is terminated by contact inhibition and desmosomal attachments are re-formed [3,8]. Epithelial cells also possess phagocytic activity, which is potentiated by fibronectin. This facilitates epithelialization over the wound bed under the scab or through wound exudate [8].

Maturation/Remodeling Stage

Early wound strength increases as quickly as within the first 24 hours of wounding. The fibrin clot is the first contributor toward wound strength; however, strength does not reach a significant degree until 6 days [4]. Between days 5 and 15, continued deposition of collagen fibrils is reflected in a gain in wound tensile strength [9]. As healing progresses, extracellular matrix, collagen fibrils, capillary growth, and adhesive forces of epithelial cells add to building early wound strength.

Late wound strength starts only after wound healing has continued undisturbed for at least 21 days [4]. Once the rate of collagen synthesis is on par with that of collagen lysis, collagenases and matrix metalloproteinases digest extraneous material and preserve appropriately oriented collagen fibers. Collagen type III is replaced by collagen type I, which increases the tensile strength of the wound. As maturation progresses, the newly formed collagen bundles and the preexisting dermal collagen coalesce such that differentiation between the two is extremely difficult. No matter how complete the wound healing or how minimal the scar formation, it is always about 20% weaker than the adjacent uninjured tissue [3,4,11].

As practitioners and their clients observe wound healing progress, certain visible processes will appear that have significance as to what is occurring. These can be helpful to the practitioner in explaining wound healing to the client and in management of the wound (Table 61-1).

Conclusion

The physiology of wound healing is an intricately woven, delicately balanced process involving fine-tuned communication between cellular players and various cytokines, vasoactive peptides, and chemical signals. The process of wound healing can be compared to the performance of a symphony orchestra. When each instrument plays its part in conjunction with other instruments at the appropriate time, beautiful music is the result. The same is true of wound healing. When each component performs its function in conjunction with other components at the proper time, uncomplicated wound healing takes place. The slightest upset of this fine-tuned equilibrium is capable of impairing it. Although we have come to learn so much about wound healing, more discoveries await around the corner.