CHAPTER TWO: Healing Response in Acute and Chronic Wounds

At the end of this chapter, the learner will be able to:

1. Describe the sequence of normal acute wound healing.

2. Identify the cells that direct activity in the healing cascade.

3. Describe the chemical messengers necessary for timely wound healing (including the cells of origin, target cells, and actions).

4. Classify the primary enzymes produced during healing (including the cells of origin and actions).

5. Explain the differences between normal acute and chronic wound healing.

6. Relate the pharmacological impact of common drugs on the healing phases.

Perhaps the hamartia, or the flaw, in the study of wound healing is the tendency to oversimplify the truly elegant system that ensures healing, both anatomically and functionally. The multitude of processes that ensure wound closure and the commensurate return of function are equally marvelous.

The following illustrations introduce the interplay of cellular and molecular signaling in conjunction with vascular events that occur in concert during the healing process. The figures demonstrate how cells involved in the repair process are directed, based upon global and local stimuli. The stimuli may be cytokine, chemokine, pH, or galvanically driven.^1,2 If invaders or pathogens (eg, bacteria, fungi, viruses, or debris) are present, innate immune cells migrate and proliferate to the site of injury.³ These cells include macrophages, neutrophils, natural killer (NK) cells, and gamma delta T cells. If the invader is a repeat offender that the host has successfully fended off previously, adaptive immune responses (B cell clonal expansion) are triggered.^3-5 Simultaneously, debris (necrotic and/or injured cells) is removed and a new wound bed excavated via proteases and extracellular matrix (ECM) degradation. This serves two purposes: (1) the clearance of cell and invader refuse and (2) the provision of pathways for cellular migration and proliferation, which constitute repair.²

The signaling in wound healing, renewal, and regeneration is a product of many factors, including the concentration and timing of chemical signal delivery, target cell receptor availability, active form after cleavage, degradation rate, messenger half-life, pH and presence of enzymes (eg, proteases) in the wound milieu, hydrophobicity, and hydrophyllicity. Scaffold-binding (via heparin activation or other mechanisms), fiber type (whether fibrin or collagen), cell shape, adhesion interfaces (via integrins), and storage of growth factors all contribute to the timing and intensity of cell signaling during wound healing. These factors work together to drive growth factor, cytokine, and chemokine bioavailability, thus resulting in wound healing.

Vascular changes occur as a result of endothelial cell activation, migration, and capillary expansion in response to tissue hypoxia and increased lactic acid concentration.^6-8 These responses, along with the appropriate cells and signaling mechanisms, are depicted in TABLE 2-1.

Phenotypical changes in prominent cells are important in directing healing throughout the influence and production of many of the chemical messengers as illustrated. Highlighted cells presented include the platelet, macrophage, and fibroblast. The macrophages have a pronounced cellular functional metamorphosis and are the central orchestrators of the healing process. Cellular roles pertinent to wound healing are depicted in TABLE 2-1 along with the fluid interconnected communication and cell signaling that occurs to effect progression through the healing process.

The pivotal cells and phases of healing are depicted in TABLE 2-2, which provides a cross-reference of healing phases with key cells and signals, as well as a chronological timeline, thus providing the reader an appreciation of the overlapping and essential functions directed in concert as opposed to an isolated or oversimplified view of cell function. Although exceedingly complex, the elegance lies both in the ability of multiple systems to evoke healing and the use of paracrine, autocrine, and juxtacrine mediators to effect expedient resolution of tissue injury using the resources immediately available.

Cells exhibit various levels of activity in response to many factors.FIGURE 2-1 summarizes four recognized levels of activity and the associated effect on the local environment. Cells that exist in a senescent state (defined as resistance to apoptosis or programmed cell death) disrupt normal tissue differentiation, drain the metabolism, and secrete cell products that negatively impact the wound environment. Cells in a baseline state have normal mitotic and metabolic activity, actively survey and monitor adjacent tissues, and do not impact surrounding tissues negatively. An up-regulated cell has a higher level of metabolic activity and purposefully responds in concert with other cells in reaction to injury, presence of pathogens, or both. A cell that is “out of control” exhibits an overproduction of cellular byproducts, is not coordinated with any other cells, and does not respond to feedback inhibition. The cartoons that represent each level of cell activity are overlaid in important diagrams to help the reader discern the cellular state in normal and disrupted wound healing. Both the correct cells and the appropriate level of cellular activity are required to ensure wound healing.

FIGURE 2-2 provides an illustration of the intricate and exquisite concert of cell migration, proliferation, and signaling in the context of cell signaling and vascular events, all working in unison to culminate in healing.

The healing response occurs by one of the following four mechanisms: (1) continuous cell cycling, (2) cell proliferation, (3) regeneration, or (4) fibroproliferative response. Normal intact skin is representative of continuous cell cycling whereby labile cells are constantly undergoing a balance of proliferation and programmed apoptosis throughout life, thereby resulting in a steady-state. The basal keratinocytes continuously undergo mitosis (cell division), followed by migration to the skin surface, and finally sloughing. Cell proliferation occurs when the damaged or lost tissue is replaced by the expansion of remaining healthy cells that undergo mitosis. The structure is not completely duplicated; however, function is approximated.

Regeneration occurs with the loss of a structure. The acute injury that undergoes regeneration stimulates complete duplication in both structure and function of the lost tissue. The liver, hematopoietic tissue, gastrointestinal tract epithelium, and epidermis are examples of tissue that are capable of regeneration. Fibroproliferative healing typically occurs in dermal wound healing. The lost tissue is not replaced, but rather a “patch” is constructed that restores the skin covering, integrity, and function. Inflammation that fails to progress to healing results in fibrosis. This chapter discusses the phases of wound healing and the involved cells, as well as the mechanisms by which acute and chronic wound healing occur. Divisions of wound healing are graphically depicted in FIGURE 2-3.

Classifications of Healing Response

Wound healing can be classified by category or by depth; both assist clinicians in communicating clearly regarding patient needs. Categorically, four divisions describe wound healing: categories 1 to 3 describe healing of full thickness wounds while category 4 refers to partial skin thickness wounds.^53,54

Category 1

Category 1 (Primary Intention) occurs when a clean surgical incision is created and the resulting wound is free from contamination of bacteria, fungi, or foreign bodies. There is minimal tissue loss and the edges can be safely approximated and secured with sutures, staples, or surgical glue. The clotting cascade at the wound surface is largely not initiated and the resulting fibrous scab is absent because of the minimal mortality of cells central to wound healing. The cell signaling cascades usually launched in an acute penetrating injury are not activated. This incisional wound resolves in an orderly, sequenced manner over the course of approximately two weeks.⁵⁵ (FIGURE 2-4)

Category 2

Category 2 (Delayed Primary Intention) occurs when wound edges are not approximated because of the concern for the presence of pathogens or debris, an abscess, or in the case of extensive tissue loss (FIGURE 2-5). Delayed primary wound healing is set in motion by the release of multiple pro-inflammatory cytokines, chemokines, and growth factors. Foreign debris is walled off by macrophages that may metamorphose into epithelioid cells, which in turn become encircled by layers of mononuclear leukocytes. The layers of mononuclear leukocytes can be compared to the sequential layering of nacre on a pearl—the clam overlays the grain of sand with nacre, smooth and protecting. In a wound, the result is a granuloma, with the foreign body or pathogen at the center, walled off from the host tissue. In these wounds the inflammatory response is more intense and is accompanied by increased granular tissue formation.⁵⁵

Frequently these wounds may undergo a delayed surgical closure after surgical removal of the granuloma, abscess, or debris. Primary closure may also be completed surgically as a follow-up to negative pressure wound therapy. Once the wound is determined to be ready for closure, surgical intervention, such as suturing, skin graft placement, or flap design is performed, provided wound edges can be approximated. If the host-initiated cleansing, termed autolysis, of the wound is incomplete, chronic inflammation can ensue. The result, without further intervention, is likely to be prominent scarring.

Category 3

In Category 3 (Secondary Intention) healing is entirely accomplished through an appropriate inflammatory response, granulation tissue formation and re-epithelization. Left to close without surgical intervention, wound contraction by myofibroblasts plays a significant role. The myofibroblasts have characteristics of smooth muscle cells and, when activated, they contract and thereby assist in consolidating the extracellular matrix and decreasing the distance between the dermal edges. The myofibroblasts are maximally present in the wound from approximately 10 to 21 days post-wounding.¹⁴ Additional time may be required for these wounds to close depending on the surface area and depth (volume) of the wound.⁵⁵ (FIGURE 2-6)

Category 4

Partial thickness wounding refers to the loss or partial loss of the epidermis and or the epidermis and superficial dermis (the basement membrane is intact and the hypodermis is not exposed). In this case healing is accomplished by the process of epithelial cell mitosis and migration. Wound contracture is not an expected or common occurrence during the healing process of these wounds, as the sub-dermal layers are not involved and minimal to no granulation tissue is formed (FIGURE 2-7).⁵⁵

Wounding can also be categorized by the depth and involvement of tissue lost or damaged as a result of the injury. These classifications progress from the most superficial erosion, to partial thickness of the dermis, and full thickness extending into the hypodermis. Chapter 1 explains this in detail with correlation to the appropriate skin structure.

Overview of Healing

Acute wound healing is subdivided to the following phases: hemostasis, inflammation, proliferation, and remodeling. Inflammation is further subdivided into three overlapping phases: kill/contain the invader, inflammation, and neo-angiogenesis. FIGURES 2-8 to 2-14 provide a framework for each of the major healing phases in terms of four important events: (1) vascular, (2) cellular, (3) cell signaling, and (4) clinical signs. FIGURES 2-8, 2-9 depict normal intact skin prior to wounding, FIGURE 2-10 is hemostasis, FIGURES 2-11 to 2-13 depict inflammation, and FIGURES 2-14 to 2-16 represent proliferation. The interplay between each of the four events is both complex and elegant. Each of these four events is explained in detail, along with their contribution to normal healing.

1. Vascular events include hemostasis, transient vasoconstriction, retrograde degradation of damaged vessels, and the transition to endothelial cell differentiation, migration, and proliferation—also termed neo-angiogenesis.

2. Cellular events include the directed migration and accumulation of cells known to be necessary for wound healing (eg, neutrophils and macrophages) to the site of injury.⁵ A fascinating characteristic of some cells includes their ability to morph in phenotype and function, depending on the phase of healing and the surrounding stimuli, whether cytokine, chemokine, or ECM activation.⁵

3. Cell signaling orchestrates healing by these mechanisms: cytokines, chemokines, growth factors, and receptor accessibility on target cells. It is accomplished through the binding of chemical messengers to cell receptors displayed on the target cell surface. The binding of the chemical messenger (eg, cytokine, chemokine, growth factor, or interleukin) activates or depresses target cell DNA transcription and protein translation of important activities that are performed by the target cell. These activities include (a) the production of additional chemical messengers, (b) a change in cell phenotype and function, or (c) binding to adjacent ECM sites. Cell signaling occurs between cells (cell to cell) and between the cell and wound matrix (cell to matrix).⁵⁶

4. Clinical signs and symptoms are those observable changes in the local periwound environment (pain, redness, and edema) or patient systemic symptoms (fever, chills, increased heart rate, or pain).

Cytokines are small proteins or glycoproteins that are secreted by numerous cells and alter the function of the target cell. The target cell for the cytokine/interleukin may be itself (autocrine) or a neighboring cell (juxtacrine). Cytokines can be either pro-inflammatory or anti-inflammatory.

Interleukins are a group of cytokines that were first observed being expressed by white blood cells (leukocytes).¹ The term interleukin derives from inter- as a means of communication, and -leukin, deriving from the fact that leukocytes produce many of these proteins and are the target of their action. The name is something of a relic as it has been determined that interleukins are in fact produced by a wide variety of body cells. The function of the immune system depends in a large part on interleukins. The majority of interleukins are synthesized by helper CD4+ T-lymphocytes, as well as monocytes, macrophages, and endothelial cells.³

TABLE 2-3 lists the cytokines important to wound healing. The pro-inflammatory cytokines necessary for wound healing are: TNF-α, IL-1, IL-2, IL-6, IL-8, and IFN-γ. In general, IL-4 and IL-10 are considered anti-inflammatory. Receptor expression on target cells can be either up- or down-regulated. Each of the signals and the complex interplay serves to enhance, depress, or change entirely the cell function while ensuring that the process culminates in functional wound healing.

Growth factors are soluble polypeptides, produced in both normal and wounded tissues, which stimulate cell migration, proliferation, and alterations in cellular function. They are extremely potent and can exert significant effects in nanomolar concentrations. Growth factors bind to specific cell receptors and have one of two different effects: (1) the stimulation of DNA transcription or (2) the regulation of cell entry in the cell cycle (mitosis). Growth factors important in wound healing are listed in TABLE 2-4.

Cell-to-wound matrix communication occurs extensively during debridement and angiogenesis. Matricellular proteins destabilize the cell–matrix bonds and interactions, in essence creating a more fluid environment for cell migration. Proteinases, both plasminogen activators and matrix metalloproteinases (MMPs), act to dissolve the wound matrix both immediately after injury and during the proliferative and remodeling phases. This is necessary during angiogenesis for the directed migration of endothelial cells.

In order to understand the intricacies of the communication, the components of the extracellular matrix (ECM) in their entirety must be understood. Components of the ECM include fibrous structural proteins, water-hydrated gels, and adhesive glycoproteins. The fibrous structural proteins include the collagens and elastins, which confer tensile strength and recoil to the tissue. Water-hydrated gels that permit resilience and lubrication are categorized as proteoglycans and hyaluronans. Adhesive glycoproteins connect the matrix elements to one another and to cells. The common components of the ECM are illustrated in FIGURE 2-17.

Collagen, one of the two fibrous structural proteins, is composed of three separate polypeptide chains braided into a rope like a triple helix. FIGURE 2-18 depicts the triple helix structure of collagen. There are approximately 50 types of identified collagen. Some collagen types (eg, I, II, III, V) form fibrils by virtue of lateral cross-linking of the triple helix. These collagens form a major portion of connective tissue in healing wounds and particularly in scars. The cross-linking is a result of a covalent bond catalyzed by the enzyme lysly-oxidase. This process is dependent on vitamin C. Types of collagen important to wound healing include: Type I—skin and bone, Type IV—basement membrane, and Type VIII—dermal–-epidermal junction.

Elastin is present mainly in the skin, large vessels, ligaments, and uterus. Morphologically, elastin consists of a central core of elastin surrounded by a mesh-like network of fibrillin glycoprotein. Fibroblasts secrete fibrillin into the ECM where it becomes assimilated into insoluble microfibrils and provides a platform for elastin deposition. FIGURE 2-19 shows the structure and ability of elastin to stretch and recoil.

Proteoglycans form extremely hydrated compressible gels that provide both resilience and lubrication (eg, in the skin, cartilage, and joints). Proteoglycans consist of glycosaminoglycans (GAGs) and hyaluronan. GAGs are long polysaccharide chains like heparin sulfate and dermatan sulfate. Hyaluronan binds water and forms a very viscous, gelatin-like matrix. Proteoglycans function to provide compressibility and serve as a reservoir for growth factors that are secreted into the ECM. Proteoglycans are also an important component of cell membranes and as such have roles in proliferation, migration, and adhesion. See FIGURE 2-20 for examples and for the schematic structure of proteoglycans.

The adherent components of the ECM are the adhesive glycoproteins and adhesion receptors. The adhesive glycoproteins include fibronectin and laminin. The adhesion receptors include immunoglobulin, selectin, cadherins, and integrins. Together laminin and fibronectin, by adjoining to collagen and connecting to the cellular plasma membrane of cells primary to tissue healing, reestablish both strength and function to the new replacement tissue. Laminin and fibronectin form the critical active junction providing both orientation and a dynamic functioning framework. See FIGURE 2-21 depicting the adhesion receptors, which are paramount to ECM function and structure.

Wound healing initially appears so very simple.^57-59 The human body is designed to heal, repair, and in some cases regenerate lost tissue through a well-orchestrated sequence of events. When it proceeds as planned—though infinitely complex—healing is at once elegant, rapid, and efficient. The following phases of wound healing are delineated by the pertinent vascular, cell-signaling, cellular activity, and clinical response as previously defined.

Hemostasis—Clot Formation

With an acute injury, the small blood vessels respond initially with vasoconstriction to stem further blood loss and tissue injury. (FIGURE 2-22) Activated platelets adhere to the endothelium and eject adenosine diphosphate (ADP), which promotes the clumping of thrombocytes and further ensures clot formation. The clot is composed of various cell types, including red blood cells, white blood cells, and platelets. The clot is stabilized by fibers of fibrin.¹⁷ (See FIGURES 2-23 and 2-24).

Alpha granules containing platelet-derived growth factor (PDGF), platelet factor IV, and transforming growth factor beta (TGF-β) are liberated from the platelets. Dense bodies contained within the thrombocytes release vasoactive amines, including histamine and serotonin. PDGF is chemotactic for fibroblasts, and in coordination with TGF-β modulates mitosis of fibroblasts,⁶⁰ thereby increasing the number of fibroblasts in close proximity to the wound. Fibrinogen is cleaved into fibrin. Fibrin undergirds the structural support for the completion of the coagulation process and provides an active lattice for the important cellular components during the inflammatory phase. The fibrin will further serve as a scaffold for other infiltration cells and proteins. (See TABLE 2-5.^3,10,61) Clinically, a full thickness wound bed with a stable clot functions to mitigate blood loss. Clear proteinaceous exudate may or may not be present.

Inflammation

Inflammation presents clinically as rubor (redness), tumor (swelling), calor (heat), and dolor (pain). These signs along with the cellular mechanisms responsible are highlighted in the tables, figures, and text that follow. At the immune cellular level several cells arrive, depart, up-regulate, or down-regulate during the phases of healing. The orchestrated arrival and departure of important cell mediators is depicted in FIGURE 2-25. Important changes that permit the initiation of inflammation and result in the clinical findings associated with inflammation are summarized in TABLE 2-6.

Kill and Contain Invader

Within the first six to eight hours after injury, polymorphonuclear leukocytes (PMNs) or neutrophils flood the wound. TGF-β (released from platelets) facilitates PMN migration and extrusion from surrounding intact blood vessels to the interstitial wound space. PMNs are phagocytic cells, functioning to cleanse the wound of debris, including both necrotic cells and pathogens. The highest number of PMNs within the wound is observed between 24 and 48 hours post injury. By 72 hours, PMN numbers are significantly reduced, just as macrophages are infiltrating.^56,62,63 FIGURE 2-26 summarizes factors that promote neutrophil adherence and migration. These factors are a combination of cellular activity, chemokines, cytokines, and proteases. TABLE 2-7 summarizes PMN function and cellular events in healing.

Mast cells are activated by antibodies and move rapidly from vascular circulation to the site of injury. Upon activation, mast cells become “sticky” and adhere to the endothelial surface. (FIGURE 2-27) Mast cells can influence the local environment by degranulation or through the products that they synthesize. Histamine, a product of degranulation, increases the permeability of endothelial cells, thus facilitating extravasation of PMNs and macrophages. The three major product categories synthesized by mast cells are prostaglandins, thromboxanes, and leukotrienes.³

Neutrophils play a noteworthy role in the early phagocytosis of pathogens and in the recruitment of macrophages by programmed apoptosis. Neutrophils are also capable of expelling a neutrophil extracellular trap (NET) that is composed of DNA and loosely aggregated chromatin. The NET is somewhat like flypaper, trapping would-be invaders and allowing increased clearance by recruited macrophages or neighboring neutrophils.⁴⁵

Wound Debridement

Autolytic wound debridement begins immediately post-injury and includes the use of cells and enzymatic proteins to break down necrotic tissue. Cells typically involved in debridement include neutrophils, macrophages, and mast cells.^36,64 Enzymatically active proteins, including proteinases and collagenases, degrade the damaged tissue and ECM, thus providing a migratory path to the site of injury for the key cells needed to complete the healing process.^17,19

PDGF, released by platelets, is chemotactic for monocytes, causing the monocytes to exude from adjacent vessels and transform into wound-activated macrophages (WAMs). WAMs continue the process of debris removal, and more importantly, begin the signaling that will orchestrate the remaining transitions and events central to healing. (FIGURE 2-28)^27,60 Macrophage numbers will remain high for approximately three to four days, releasing various tissue growth factors, cytokines, interleukin-1 (IL-1), tumor necrosis factor (TNF), and PDGF.⁶³ In addition to the phagocytic function, the macrophages provide a unifying script for the multiplication of endothelial cells and the sprouting of new blood vessels, both of paramount importance for continued cell migration and proliferation.^3,5,65-67

The controlled liquefaction of the ECM continues during the debridement process until all of the damaged cells are removed and cells central to repair are in place. The rate of autolytic debridement will decline as debris is cleared, new cells proliferate, and healing ensues. The pro-inflammatory cytokines and their effects are found in TABLE 2-3 by cell and in TABLE 2-2 by phase of wound healing.

Angiogenesis (Early)

Just as the re-epithelialization process begins a few hours after wounding, so, too, does neo-angiogenesis.⁶⁸ Keratinocytes from the wound edges migrate beneath the fibrin clot and over the wound. Activated fibroblasts migrate to the site of injury in response to TGF-β1,^11,60,69 and, in combination with macrophages, form granulation tissue.^16,68 Both macrophages and fibroblasts produce vascular endothelial growth factor (VEGF). In addition, fibroblasts produce connective tissue growth factor (CTGF),⁷⁰ which results in their proliferation via an autocrine loop.¹² The formation of new vasculature requires both extracellular matrix and basement membrane degradation followed by migration, mitosis, and maturation of endothelial cells. Basic FGF and VEGF are believed to be central in modulating angiogenesis.^10,28,71-74 As a critical component of healing, new vessel formation will both (1) supply the oxygen and nutrients required and (2) remove the waste products of autolysis. FIGURES 2-29 and 2-30 provides a broad overview of the key events involved in angiogenesis while clinically a well-vascularized, early granulating wound bed.

Proliferation

The proliferation phase of healing consists of four subphases: angiogenesis, fibroplasia, matrix deposition, and re-epithelialization.^59,68 FIGURE 2-16 illustrates a broad overview of the events occurring in the proliferation phase, which is characterized by the formation of granulation tissue. This tissue consists of a rich capillary bed, fibroblasts, macrophages, and a loose arrangement of collagen, fibronectin, and hyaluronic acid.

Angiogenesis is mandatory to supply necessary nutrients and to remove waste products. Angiogenesis, although discussed as part of the inflammatory and proliferative phases, begins immediately after injury and is paramount to ensuring endothelial cell migration, which results in capillary sprouting. Injured endothelial cells, adhering blood cells, and numerous soluble factors trigger a cascade of events resulting in matrix remodeling and concurrent endothelial cell growth and differentiation. Capillary tube formation is a complex process involving cell-to-cell and cell-to-matrix interactions, many of which are modulated by progenitor endothelial cell adhesion molecule (PECAM-1) and stimulated by VEGF. ?1 Integrin acts to stabilize the cell-to-cell and cell-to-matrix contacts. As new capillaries sprout, they are differentiated into arterioles and venules, while others undergo involution and apoptosis with subsequent ingestion by macrophages.

Fibroplasia is characterized by the presence of fibroblasts, specialized cells that differentiate from resting mesenchymal cells located in connective tissue. FIGURE 2-31 provides a broad overview of fibroplasia. By approximately the fifth day, fibroblasts have migrated into the wound and are laying down new collagen, either sub-type I or III. Early in normal wound healing, type III collagen predominates and is later replaced by type I collagen. The process of collagen formation begins within the fibroblast cell's rough endoplasmic reticulum, where tropocollagen (the precursor to all collagen types) has its proline and lysine hydroxylated. Establishing disulfide bonds allows three tropocollagen strands to form a triple left-handed helix, termed procollagen. The procollagen is secreted into the extracellular space, passing through the cell wall where peptidases cleave the terminal peptide chains, creating true collagen fibrils.^{16,15,65,75,76} (FIGURE 2-32)

The wound is permeated with glycosaminoglycans (GAGs) and fibronectin produced by the fibroblasts. The GAGs include heparin sulfate, hyaluronic acid, chondroitin sulfate, and keratin sulfate. Proteoglycans are GAGs covalently bonded to a protein center. All of these constitute and contribute to matrix deposition. The ECM provides both the lattice infrastructure and key binding sites for cells and chemical messengers alike.^{14,16,19,77-79} (FIGURE 2-33) Although labile and stable cells are capable of regeneration, injury to these tissues results in restitution of the normal structure only if the ECM is not damaged. Disruption of the ECM leads to collagen deposition and scar formation.

Re-epithelialization of the wound begins within hours of injury. The wound is rapidly sealed by clot formation and subsequently by the migration of epithelial (epidermal) cells across the defect. Keratinocytes located within the basal layer of the remaining and undamaged epidermis migrate to resurface the wound. Keratinocytes undergo a sequence of changes to c-omplete re-epithelialization, which is depicted in FIGURES 2-34 and 2-35 and summarized in TABLE 2-8.

Epidermal cells express integrin receptors, thus allowing them to interact with ECM proteins. The migrating epidermal cells dissect the wound, separating desiccated eschar from living or viable tissue. The migratory path is determined by both the degradation of injured tissue and the integrins expressed on the epidermal cell surfaces. Epidermal cells also secrete collagenases and metalloprotease-1 (MMP-1) to degrade the ECM ahead of their migratory path. Studies have also demonstrated that the edge-leading epidermal cells can phagocytize debris. Cells behind the leading cells are stimulated to proliferate, thus resulting in epithelial cells moving in a tumbling motion across the wound surface until contact is established with the opposite edge. If the basement membrane is not intact, it is repaired prior to migration. Local release of epidermal growth factor (EGF), tissue growth factor alpha (TGF-α), and keratinocyte growth factor (KGF), along with the increased expression of their receptors, may also stimulate this process. TABLE 2-9 summarizes in detail the growth factors secreted and the cells responsible for their production, as well as the cells influenced by those growth factors.

Basement membrane proteins, like laminin, appear in a highly ordered sequence proliferating from the wound margin inward. Laminins are a family of glycoproteins that provide an integral part of the structural scaffolding of the basement membrane in almost every animal tissue. Each laminin is a heterotrimer assembled from alpha, beta, and gamma chain subunits, secreted and incorporated into cell-associated extracellular matrices. The laminins are unique in that they can self-assemble, bind to other matrix macromolecules, and have shared cell interactions mediated by integrins and other receptors. Through the interactions with other cells, laminins critically contribute to cell differentiation, cell shape and movement, and maintenance of tissue phenotypes, as well as promote tissue survival. After the wound is completely re-epithelialized, the cells again become columnar, stratified, and firmly attached to the newly constructed basement membrane and underlying dermis.^17,80 Although the wound may be fully re-epithelialized and closed, it is not referred to as healed until the next, final stage of wound healing is completed.

Maturation and Remodeling

Restored Function

The final stage of the wound healing process consists of dermal regeneration, wound contraction, and programmed involution of the granulation tissue. Wound contraction occurs in a centripetal fashion, involving the full thickness of the wound and periwound tissue (FIGURE 2-36). The primary goal of wound contraction is to aid in the reduction of wound size and reduce the amount of disorganized scar tissue. Wound contraction occurs through complex interactions between the ECM and fibroblasts; however, the interactions have not been completely elucidated. It is understood that aborted cell locomotion, phenotypic change from fibroblast to myofibroblast, and the expression of TGFβ-1 and MMP3 are all important. Aborted cell locomotion results in the bunching and contraction of collagen fibers. Phenotypically fibroblasts undergo cellular changes and become myofibroblasts, cells that express α-smooth muscle actin (αSMA). TGFβ-1 promotes wound contraction by increasing the expression of β1 integrin. Stromyelysin, or MMP3, through the utilization of integrin β1, allows for the modification of attachment sites between fibroblasts and collagen fibers.

During remodeling, the number of fibroblasts and myofibroblasts decrease, the dense capillary network regresses, and wound strength gradually increases. The early epidermal–-dermal interface is fragile because it lacks the interlocking rete pegs. Without rete pegs, the newly healed wound is at risk for avulsion with minor trauma. Apoptosis of myofibroblasts, endothelial cells, and macrophages results in tissue composed primarily of ECM proteins, particularly collagen type III. Metalloproteinases produced by the epidermal cells, endothelial cells, fibroblasts, and macrophages that remain in the scar will continue the process of remodeling, replacing collagen type III with collagen type I.^58,59,61,81 The resulting tissue “patch” will have approximately 80% of the tensile strength of the original tissue but will have completely reestablished function.^82-85

Clearing and Containing the Invader

Most pathogenic microorganisms have evolved mechanisms to overcome innate immune responses and thus continue to grow. An adaptive immune response is required to eliminate the pathogenic invader and prevent subsequent re-infection. Both the innate and adaptive immune responses are discussed as they pertain to each phase of wound healing.

Immediately During Clot Formation

The formation of a clot is critical for two reasons. The first obvious reason is to stem the loss of blood as discussed previously. The second more obtuse but equally important reason is to recruit the innate immune system to clear debris and pathogens. Platelets are central in the following three areas: (1) initiation of the clotting cascade, (2) formation of a platelet plug (the outer surface of activated platelets becomes sticky with a mucopolysaccharide coating), and (3) release of cytokines and alpha granules. The fibrin provides an infrastructure for the clot and mechanical entrapment of debris and invaders. TGF-β1 and TGF-β2, released by the platelets, attract macrophages to the site of injury. Macrophages in turn express IL-1, which stimulates antigen-presenting cells (APCs) to produce IL-8. IL-8 both signals and directs neutrophil migration and margination while simultaneously up-regulating “sticky factors” and vascular permeability to expedite the process. The recruitment of both macrophage and neutrophils (PMNs) immediately to the site of injury begins the process of invader identification and clearing.³

Innate Immunity

The innate immune response occurs with the migration and extravasation of PMNs from the neighboring vasculature. The extravasation, migration, and proliferation are facilitated by TGF-β1, a growth factor released by the platelets and macrophages that have migrated to the wounded tissue. Neutrophils are the first inflammatory, innate immune system cells to infiltrate a wound; they do so in large numbers in response to the numerous inflammatory cytokines produced by endothelial cells, activated platelets, and degradation products from invading pathogens.⁶⁴ Neutrophils undergo programmed cell death (apoptosis) shortly after infiltration, thus releasing additional cytokines that recruit macrophages to the wound site.⁶³

Neutrophils, or PMNs, exhibit several important defense mechanisms. Three of the many defense mechanisms are highlighted here: (1) neutrophil extracellular traps (NETs), (2) the release of azurophilic pore-forming granules, and (3) phagocytosis and programmed apoptosis.⁴ NETs are the result of the neutrophils expelling chromatin, a very sticky substance, and thus serve to mechanically entrap the pathogen. (FIGURE 2-37) Once entrapped, proteases, elastases, pore-forming granules, and other cytotoxic molecules lyse the invader. Both the entrapment and the lysis of the invader serve to increase the visibility of the intruder to the host immune system.⁸⁶ Neutrophils are also capable of direct phagocytosis of an invader. The programmed apoptosis of neutrophils releases pro-inflammatory cytokines, attracts additional macrophages, and up-regulates the key activities of macrophages. Th1 (innate system) neutrophils produce interferon gamma (IFN-γ), which also up-regulates macrophage activity, and alters the macrophage phenotype to a WAM, which characteristically produces increased nitric oxide and H₂O_2.Both nitric oxide and H₂O₂acidify the environment and the lower pH serves to increase phagocytosis. In addition, the WAMs display increased MHCII for the presentation of antigens specific to the invader to the adaptive immune system.^87,88 Both neutrophils and macrophages are important to the immediate containment of the invader and the activation of the adaptive (memory/B-cell) immune system of the host.

T-lymphocytes are another population of inflammatory immune cells that routinely invade the wound. Although less numerous than macrophages, they bridge the transition from the inflammatory to proliferative phase. Depletion of most wound T-lymphocytes decreases wound strength and collagen content. T-lymphocytes affect fibroblast function by producing stimulatory cytokines like IL-2, fibroblast activating factor, and inhibitory cytokines (including TGF-β, TNF-α, and IFN-γ). The role of IFN-γ secreted by T-lymphocytes is illustrated in FIGURE 2-38. The effects of IFN-γ are far-reaching and may be an important mediator of chronic non-healing wounds.

Adaptive Immunity

Adaptive immunity, or learned immunity, is triggered by the first responders, namely macrophages. Macrophages enhance APC's activity, thereby aiding in the presentation of antigens (antibody generator) to the host immune system T cells. Bacterial proteins degraded by host proteases are ingested by macrophages and become antigens, which are then presented to T cells via Class II MHC receptors on the macrophage surface. Once a T cell binds to the loaded Class II MHC receptor, the T cell becomes activated and begins secreting IL-2. IL-2 simulates T cells to divide and activates the T cell phenotypic expression of IL-2 receptors, thus resulting in autocrine lymphocyte proliferation and activation. This is illustrated in FIGURE 2-39.

The dendritic cells, another important APC, traffic to local lymph nodes and present the antigens to resident cells. If the antigen presented is recognized by existing memory B cells, those B cells are activated and clonal expansion is triggered. In addition, macrophages produce IL-2, a powerful cytokine that drives T cells to become Th2 helper cells, and thereby increases the effectiveness of B cells.³

The B cells produce and release antibodies resulting in an increase in the number of circulating antibodies specific to the pathogen (eg, MRSA, MSSA). As a result, either neutralization or opsonization can more effectively occur. Neutralization occurs with the binding of an antibody to the pathogen, thus blocking access to host cells. Opsonization refers to the coating of the pathogen with antibody, thereby increasing the ease of identification and subsequent clearance and phagocytosis.³

Introduction

Typically a cell is thought of as having a unique phenotype and set of correlating functions; however, cells necessary for wound healing undergo phenotypic changes as wound healing progresses to completion. A well-known example of cellular phenotypic change is found in the red blood cell. A red blood cell begins as a myeloid cell, and undergoes phenotypic differentiation to become and remain a red blood cell (RBC) whose function primarily is to transport and facilitate the exchange of dissolved gases (eg, CO₂ and O₂). In the elegance of wound healing, several cells demonstrate the ability to morph in function. Three prevalent cell chameleons are reviewed: platelets, macrophages, and fibroblasts.

Platelets, although anucleate, provide chemical messengers at the point of initial injury to achieve hemostasis. Alpha granules contained within the platelets release PDGF, platelet factor IV, and TGF-β1, which function during the proliferative phase of healing to accelerate granulation and connective tissue proliferation.⁴ Macrophages, usually thought of as phagocytes, transition from APCs to phagocytes, to chief-air-traffic-controllers, adapting their cytokine phenotype expression profile to move the healing cascade forward and thus accelerate closure. Fibroblasts are vital early during clot construction, granulation tissue formation, and neo-angiogenesis. Uniquely, during the later stages of proliferation and early remodeling, under the influence of TGF-β,⁷⁰ fibroblasts truly change phenotype, becoming myofibroblasts. The myofibroblasts facilitate centripetal, full-thickness, wound edge approximation, thus decreasing the overall size of the wound. Each of these unique cell metamorphoses is reviewed in the following section. Their specific characteristics and their effects are in TABLES 2-1 and 2-9, which list the growth factors and signaling that occurs throughout wound healing.

Platelets

Platelets at the site of an acute injury are activated, become “sticky,” and adhere to the adjacent endothelium while simultaneously ejecting adenosine diphosphate (ADP). Together these two actions promote the initiation of the clotting cascade and the clumping of thrombocytes. The initial goal is clot formation. The further release of alpha granules from the platelets transforms the clot to an “active” state. Alpha granules release PDGF in two forms (small- and large-molecule PDGF) and platelet factor IV, as well as transforming growth factor –β (TGF-β), into the surrounding tissues. PDGF, a chemotactic substance for fibroblasts, draws increased numbers of fibroblasts to the wounded tissue. In coordination with TGF-β, PDGF modulates mitosis of fibroblasts. Both the attraction and increased mitotic activity elevate the number of fibroblasts in direct proximity to the wound.⁴ PDGR and TGF-β simultaneously facilitate the migration and extrusion of phagocytic cells from nearby capillaries. PDGF is specifically chemotactic for monocytes (which transform into macrophages) and TGF-β for PMNs. This initiates the cleaning of the wound.

During the proliferative phase when new tissue formation begins, large-molecule PDGF is again key by promoting neo-angiogenesis. Hence the platelets perform the following functions: (1) serve as a component of the physical clotting structure, (2) release chemical messengers important for the initiation of autolytic debridement by macrophage and PMNs, and (3) continue to function during the proliferative phase by stimulating angiogenesis via PDGF release.

Macrophages

The morphing of macrophages begins when monocytes leave the vessels adjacent to injured tissue and transition into macrophages when they enter the interstitial tissue. Macrophages then demonstrate myriad phenotypes, levels of activation, and differentiation based on the phase of wound healing. Their ability to differentiate into either M1, WAMs, or M2 repair macrophages highlights their complex plasticity and responsiveness to the wound. (TABLE 2-10 and FIGURE 2-40) The phenotypic changes are not merely a passive result of the wound milieu or environment, but macrophages are actively directed and either delay or advance the healing process. Macrophages transition from an innate immune cell (phagocyte and APC) and from there differentiate into a pro-inflammatory cell, or an immunoregulatory cell (during proliferation and re-epithelization), or a phagocytic cell aiding in tissue remodel. See TABLE 2-11 for a summary of the important pro-inflammatory and anti-inflammatory cytokines produced by macrophages to direct wound healing in both the inflammatory and resolution phases.

Only a few macrophages are typically located in the dermis and function as surveillance macrophage. Neutrophil apoptosis during the first 24 hours after injury signals macrophages to massively infiltrate the wound. The numbers of macrophages are the greatest and most elevated beginning approximately two days after injury. During this phase, macrophages demonstrate increased antigen presentation and phagocytic activity.⁶³

The WAMs produce free radicals (NO, H₂O₂, O₂^-) and pro-inflammatory cytokines, including TNF-α, IL1β, IL-6, or IL-12. These M1 macrophages also overexpress MHC class II molecules. MHC class II molecules present antigens to T cells and B cells to activate both innate and adaptive immune system responses.⁸⁸ The antigen presentation is largely via major histocompatibility complex class II (MHC class II) complex. Antigens presented by MHC class II molecules are derived from extracellular proteins of the pathogen, for example, a bacterium that may be infecting the wound. The loading of the MHC class II complex occurs after phagocytosis by the macrophages. The extracellular proteins are endocytosed and ingested by lysosomes, and signature peptides unique to the specific invader are loaded onto the MHC class II complex and then presented on the macrophage cell surface. Thus, in the early phase of healing the macrophages are both phagocytic and important in stimulating the immune system. (FIGURE 2-41)

During the inflammatory and proliferative phase when new tissue formation and angiogenesis are paramount, macrophages produce VEGF, thus stimulating the recruitment of endothelial progenitor cells and the proliferation of existing endothelium.⁸⁹ In addition, macrophages are also vital to lymphangiogenesis, as VEGF_c and VEGF_D modulate this process. It has been demonstrated that decreased macrophage numbers are associated with decreased lymphangiogenesis. Remodeling begins with the gradual involution of granulation tissue and simultaneous dermal renewal. During this phase, apoptosis of a significant percentage of macrophages occurs. The remaining macrophages will facilitate the remodeling process by cleaning up the cellular and matrix debris. This ECM debris is largely a result of the cellular apoptosis and the degradation of collagen type III by metalloproteinases produced by epidermal and endothelial cells.⁶⁸ (FIGURE 2-40)

Although the phenotype of undifferentiated macrophages that infiltrate dermal wounds is not fully characterized, the evidence supports the changing role of the macrophage during the phases of healing.^5,90 This suggests that the “same cell,” the macrophage, has different and diverse roles throughout the phases of skin or wound regeneration.^27,89 FIGURE 2-40 and TABLE 2-12 summarize macrophage functions.

Fibroblasts

Fibroblasts begin to infiltrate the wound during the first steps of granulation tissue formation. Fibroblasts are involved in granular tissue formation, cytokine production to increase keratinocyte proliferation/migration, and differentiation of fibroblasts to myofibroblasts. The primary function of fibroblasts is synthesis of collagen, a process that begins in the inflammation phase and continues throughout the healing process. After the fourth week, the rate of collagen synthesis declines, approaching equilibrium with the demolished collagen as a result of the presence of MMP-1. At this point the wound enters a phase of collagen maturation.

During the maturation phase, fibroblasts undergo a phenotypical change and become activated myofibroblasts, expressing α smooth muscle actin (αSMA). During wound contraction, myofibroblasts uniquely generate centripetal forces across the wound and throughout the entire depth by contracting the αSMA fibers. The contractile force generated by the myofibroblasts serves to approximate wound edges and continually decrease the wound area during healing.⁹¹

Proposed mechanisms that may be responsible for causing the phenotypic change from fibroblast to myofibroblast include interstitial flow and TGFβ₁. Interstitial flow through shear stress on the cells directly or via strain on fibroblast-to-ECM attachment may further trigger TGFβ1 expression.⁹² TGFβ1 expression drives αSMA expression as the fibroblasts differentiate into myofibroblasts and align the matrix fibers.^14–16,93 See FIGURE 2-42for various mechanisms that trigger the phenotypic change from fibroblast to myofibroblast.

Cutaneous wounds result from a breech in the skin or epithelium and are generally classified as either acute or chronic. Acute wounds are those that resolve without incident in an anticipated time frame, typically 21 days or less, and restore the skin structural and functional integrity.⁵⁹ Chronic wounds, by contrast, result from an inadequate or disrupted healing process and are defined as those wounds that neither follow a normal trajectory nor restore the skin functional and structural integrity.⁹⁴ Chronic wounds or ulcers may take months or years to heal,⁹⁵ disrupting patients' lives, interfering with their ability to work, contributing to disfigurement or amputation, and costing the healthcare system billions of dollars each year.^96,97

If the skin fails to progress through an orderly reparative process to intact epithelium, the wound becomes chronic.⁹⁶ Chronic wounds are often cyclical with periods of healing followed by reoccurrence.⁹⁸ The reoccurring presentation of chronic ulcers, confirmed in large population studies,⁹⁹ contributes to the high cost of care, decreases individual's quality of life,⁹⁸ and underscores the lack of understanding regarding the exact pathology of chronic wounds.⁵⁹ Chronic wounds are known to have multifactorial etiology with risk factors, including but not limited to, prolonged standing, advancing age, previous lower extremity injury or surgery, peripheral arterial disease, peripheral vascular disease, diabetes, phlebitis, and a history of varicose veins or deep vein thrombosis.¹⁰⁰

Although the body is designed to heal with directional focus and decisiveness, employing cells that offer flexibility in function and redundancy in signaling, chronic or non-healing wounds are occurring with increased prevalence and incidence. The chronic wounding environment pervades and is sustained despite the body's best efforts. (TABLE 2-13) Factors that impede wound healing are discussed in depth in Chapter 11.

Chronic wounds usually arrest in a pro-inflammatory, proteolytic-hostile milieu that does not favor cell proliferation, migration, or differentiation. The three primary categories that provide the backdrop for the development of chronic wounds are the presence of foreign bodies, pathogenic invaders (eg, bacteria, virus, fungi, mycobacterium), or underlying disease processes (eg, diabetes, circulatory disorders).¹⁰¹ Removal of foreign bodies and sharp debridement can convert the stalled wound to an acute wound and innate healing proceeds.¹⁰² The presence of an invader or an underlying disease process complicates healing advancement and is discussed in more detail in Chapter 11. Chronic wounds associated with disease processes may or may not be associated with an acute trauma. The lack of an acute trauma and the resulting cascade of promoted and invited cells to the site of injury that should occur during acute injury may explain the senescence of important cells. This cell senescence would include macrophages and the associated chemical signaling cascades that normally occur in response to tissue damage. A chronic wound may be likened to a “sneak attack” wherein the normal signaling mechanisms are not fired in response to injury. Senescent cells are particularly susceptible and resulting cell-to-cell and cell-to-matrix signaling does not occur, and the normal progression of wound healing fails to ensue.

Bacteria infect their host using two seemingly opposite strategies—either the immediate direct kill of the host cell to secure a nutrition source^103-105 or construction of biofilm^106-110 to establish a semi-permanent microbial community. Direct kill requires the up-regulation of planktonic bacterial genes, which supports “free floating,”¹¹¹ while biofilm construction requires expression of genes used to produce or manufacture attachments (integrins, adhesion molecules) to host or ECM structures.^107,112 In the biofilm community, the bacteria secrete a polysaccharide matrix construct to cover themselves, neighbors, and the progeny of both. This community evades host detection, antibiotic delivery, and innate and adaptive immune responses, thus providing fertile ground for the sharing and swapping of gene cassettes encoded with antibiotic resistance strategies (eg, efflux pump, deactivation).^113-115 The biofilm community dines on the increased plasma exudate that is a result of the localized hyper-inflammatory state produced by cellular responses largely initiated and sustained by macrophages and neutrophils.^112,113 The very cells designed to identify and eliminate the invaders¹¹⁶ (macrophage and neutrophils) have become the pathogenic invaders' “short-order-cook,” supplying an ongoing stream of nutrition via inappropriate and indiscriminate cell, protein, and plasma destruction.¹¹⁷

The climate of the chronic wound is known to be highly proteolytic, having elevated levels of MMP2, MMP8, MMP9, and elastase.^59,118,119 In this proteolytic environment, protein degradation proceeds in random chaos, failing to set a course of the orderly cell migration and proliferation that is a necessary component of healing. Furthermore, the presence of pro-inflammatory cytokines (eg, TNFα, If-γ, IL-1, IL-6, IL-8)¹¹⁸ serves to continue indiscriminant recruitment of neutrophils and macrophages.^5,23,49 The increased concentration of neutrophils^47,120 and macrophages in the context of pro-inflammatory cytokines also prevents the necessary cellular morphologic changes in key cells required to move the wound environment from the inflammatory phase to the proliferation phase.^1,2,121

As if the polysaccharide biofilm cloak and the up-regulation of proteases, pro-inflammatory cytokines, and chemokines along with the excessive accumulation of neutrophils and macrophages were not sufficient, biofilm also induces host cell senescence, thereby protecting the biofilm's attachment anchor to the host. Host cell senescence renders the cells unable to undergo cell division, migration, or complete apoptosis. The lack of apoptosis may be the most harmful effect as the controlled, programmed host cell apoptosis initiates anti-inflammatory pathways and cellular cascades that move the healing process from one of inflammation to proliferation.^{21,48-51,81,141,142} The resulting host inflammation is nonfunctional, repetitive, and detrimental to host tissues; however, it serves to nourish the biofilm and the resident inhabitants.

Chronic Wound Prevalence—a Growing Concern

Chronic wounds are typically classified by etiology both as a means to guide plan of care using disease-specific algorithms, and also to gather and report wound-specific data, including healthcare costs. Wound-specific data are tabulated and categorized broadly as acute, cancer-related, or chronic. Acute wound prevalence and cost serve as a point of reference for chronic wounds. Acute wounds include surgeries, trauma, burns, and amputations; cancer-related wounds include basal cell carcinoma, skin cell carcinoma, melanoma, and radiation-associated wounds.

The prevalence of chronic wounds is increasing at a compound annual growth rate that is two to three times that of any of the other wound categories, including surgical, burns, and acute.¹²¹ The estimated worldwide prevalence of venous and diabetic ulcerations is 11 and 11.3 million, respectively. As of 2008, the estimated annual increase in prevalence is 8 to 9% in the developed world, with the increase attributed to advancing population age and increased awareness and incidence of both Type 1 and Type 2 diabetes.¹²¹ Both globally and in the United States, the prevalence of the four major categories of chronic wounds (arterial, venous, pressure, and diabetic) is estimated to affect 2% of the population. The expense of treating those wounds approaches 3% of the healthcare budget,^97,143 representing a significant burden. (FIGURE 2-43)

Venous ulcers, the most common of the chronic wounds, affect 1% of the world's population.¹⁴⁴ Freiburg et al. in 2002 estimated the annual cost incurred for 192 patients with venous leg ulcers to be $1.26 million.¹⁴⁵ In a study of patients with pressure ulcers in the United Kingdom, Bennett found that their management accounted for greater than 4% of the total national health service expenditure and estimated cost at $3 to $4 billion annually.¹⁴⁶ Heyneman tabulated the cost and occurrence for diabetic foot ulcers. He found diabetes was responsible for over 100,000 major limb amputations; the total cost for each with associated charges was $100,000 in 2005, and approximating $10 billion in direct costs.¹⁴⁷

Five Major Chronic Wound Types

Chronic wounds are often categorized by etiology as arterial, diabetic/neuropathic, venous insufficiency, pressure ulcers, or non-healing surgical wounds. However, the variability in the local chronic wound environment is largely due to one of five common factors: diminished perfusion, decreased oxygenation, increased mechanical forces, malnutrition, or systemic disease.^58,148 It is the presence of these factors and not the age or etiology of the wound that determines its chronicity. Nonetheless, the division of chronic wounds by etiologic category allows capture of the primary pathophysiologic factors and statistics by disease process common to each wound type. For example, diabetic foot ulcers have complications related to neuropathy, poor perfusion, white cell dysfunction, poor nutritional status, and abnormal peak pressures or mechanical forces (a complication of neuropathy and the insensate foot). Venous insufficiency ulcers are complicated by perivascular cuffing, edema, and venous hypertension. It is interesting to note that regardless of the etiologic category assigned to a non-healing wound, all chronic wounds possess similar cellular and biochemical impairments.^58,149,150 Data are segregated according to four major chronic wound categories, which account for approximately 90% of all chronic wounds, and summarize the most common etiologies for each of the chronic wounds. Each of these wound types is discussed in more detail in subsequent chapters.

Normal wound healing, although intricate, proceeds in an organized fashion progressing from injury through hemostasis to inflammation, proliferation, and remodeling. During the hemostasis phase, coagulation of platelets and the formation of a clot prevent excessive bleeding and form a mechanical trap for pathogens, thus initiating the transition to a pro-inflammatory environment. A hallmark indicating transition to the inflammatory phase is the recruitment of neutrophils, leukocytes, and macrophages and the production of growth factors and pro-inflammatory cytokines. Completion of the proliferative phase leads to the formation of well-vascularized granulation tissue and extracellular matrix. Remodeling continues the process of scar contraction by myofibroblasts, tissue replacement, and controlled collagen replacement. The resulting tissue has physical properties approximating unwounded skin.

Wound healing is not an isolated event of merely reconstructing the physical skin barrier; rather it is integrated and orchestrated with both the innate and adaptive immune system. Both the innate immune system (eg, neutrophils, macrophages, and leukocytes) and the adaptive immune system (eg, B cells and antibodies) are triggered by early responder cells to protect the host from potentially harmful pathogens. Working together, these cells produce and regulate proteases, growth factors, chemokines, and cytokines to accomplish the regeneration of the skin barrier and avert infection.

In each phase of healing important events transpire, which can be described as vascular, cellular, cell signaling, or cell-to-matrix interactions. Initially, during hemostasis, vasoconstriction occurs. However, during the inflammatory and proliferative phases, angiogenesis and vasodilation predominate, to both supply the nutrients for the repair process and remove the waste and debris associated with autolytic debridement. Although the same cast of characters are largely consistent during the phases of healing, their phenotypes, cellular activities, and numbers vary greatly. Platelets are of primary importance for hemostasis and angiogenesis, macrophages for the inflammatory process, the protein laminin for proper ECM construction and storage of growth factors via ECM attachment, and fibroblasts/myofibroblasts for proliferation and remodeling. The macrophage takes on the most varied phenotypic changes during the healing process, moving from a pro-inflammatory, wound-activated macrophage (WAM) in the inflammatory phase to a repair, M2 macrophage, during proliferation and remodeling. Finally, remaining macrophages function as a component of immune surveillance after healing is complete.

Chronic wounds occur as a result of either a pathophysiological progression (eg, arterial, venous, or diabetic disease) or an acute injury that fails to heal due to infection, intrinsic processes, or extrinsic inhibiting factors. One or more of the primary pathways traversing the healing process are disrupted. The majority of chronic wounds are characterized by excessive or persistent inflammation, infections, presence of biofilm, and the inability of dermal or epidermal cells to respond to reparative stimuli. Clinical observations include chronic inflammation, edema, increased levels of necrotic tissue, bioburden, a poorly developed ECM, and epithelial overgrowth.