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 illustrations in this chapter introduce the interplay of cellular and molecular signaling in conjunction with vascular events that occur during the healing process. The figures demonstrate how cells involved in the repair process are directed, based upon global and local stimuli that 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 hydrophilicity. 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 Phenotypical changes in prominent cells (platelets, macrophages, and fibroblasts) are important in directing healing through the influence and production of many of the chemical messengers. 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 cellular communication and signaling that occur 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 sequence as opposed to an isolated view of cell function. Although exceedingly complex, the elegance of the healing response lies both in the ability of multiple systems to evoke healing and in 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 illustrates four recognized levels of cellular activity and the associated effect on local tissue environment. Cells that exist in a senescent state (defined as resistant 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 upregulated state 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 sequence of cell migration, proliferation, and signaling in the context of cell signaling and vascular events, all working in unison to culminate in tissue 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 subsequent desquamation or 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. Inflammed tissue that fails to progress to healing results in tissue fibrosis. Divisions of wound healing are graphically depicted in FIGURE 2-3.

Categories of Healing Responses

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

Category 1

Category 1 (Primary Intention) healing 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 (FIGURE 2-4).⁵⁵

Category 2

Category 2 (Delayed Primary Intention) healing occurs when wound edges are not approximated because of the concern for the presence of pathogens or debris, an existing abscess, or loss of extensive tissue (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

These wounds frequently undergo delayed surgical closure after surgical removal of the granuloma, abscess, or debris. Once the wound is determined to be ready for closure, surgical intervention (such as suturing, skin graft placement, or flap design) is performed, provided the wound edges can be approximated. If the host-initiated cleansing, termed autolysis, of the wound is incomplete, chronic inflammation can ensue. Without further intervention, the result is likely to be prominent scarring.

Category 3

Category 3 (Secondary Intention) healing is entirely accomplished through an appropriate inflammatory response, granulation tissue formation and re-epithelialization. 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 (FIGURE 2-6).⁵⁵

Category 4

Partial thickness wounding refers to partial loss of the epidermis or loss of the epidermis and superficial dermis (the basement membrane is intact and the hypodermis is not exposed). In this case healing is accomplished by epithelial cell mitosis and migration. Wound contracture is not an expected or common occurrence during the healing of partial thickness 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. (Refer to Chapter 1, Anatomy and Physiology of the Integumentary System.)

Overview of Healing

Acute wound healing is divided into 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 2-14B 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 and 2-9 depict normal intact skin prior to wounding; FIGURE 2-10, hemostasis; FIGURES 2-11A to 2-13B, inflammation; and FIGURES 2-14A to 2-16, proliferation. The interplay between each of the four phases is both complex and transitional such that within any wound, signs of more than one phase may be present. Vascular, cellular, and tissues changes that occur during the four phases include the following:

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 Some cells (platelets, macrophages, and fibroblasts) morph in both 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 the actions of cytokines, chemokines, growth factors, and receptor accessibility on target cells. It is accomplished through the binding of chemical messengers to cell receptors present 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 include changes observed in the local periwound environment (pain, redness, and edema) or systemic symptoms detected in the patient (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 which 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 that are important in wound healing are listed in TABLE 2-4.

Cell-to-wound matrix communication occurs extensively during debridement and angiogenesis. Matricellular proteins (defined as dynamically expressed non-structural proteins in the ECM that are rapidly turned over and have regulatory roles)⁵⁷ 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.

The intricacies of cellular communication involve the components of the extracellular matrix (ECM) including fibrous structural proteins, water-hydrated gels, and adhesive glycoproteins (FIGURE 2-17). 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.

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). 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 and are 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 lysyl oxidase, a process that 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 (present mainly in the skin, large vessels, ligaments, and uterus) 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).

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 also 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 (FIGURE 2-20).

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 also form the critical active junction providing both orientation and a dynamic functioning framework. FIGURE 2-21 depicts the adhesion receptors, which are paramount to ECM function and structure.

Wound healing initially appears so very simple.^58–60 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 elegant, rapid, and efficient. The following four phases of wound healing are delineated by the pertinent vascular, cell-signaling, cellular activity, and clinical response as previously defined (FIGURE 2-10 to 2-14B).

1. 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, composed of various cell types (red blood cells, white blood cells, and platelets), 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 released 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,⁶¹ thereby increasing the number of fibroblasts in close proximity to the wound. Fibrinogen is cleaved into fibrin which 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 infiltrating cells and proteins. (See TABLE 2-5.^3,10,62) Clinically, a full-thickness wound bed with a stable clot functions to mitigate blood loss. Clear proteinaceous exudate may or may not be present.

2. Inflammation

Inflammation presents clinically as rubor (redness), tumor (swelling), calor (heat), and dolor (pain). The orchestrated arrival and departure of important cell mediators are 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.