PROLIFERATIVE PHASE

The proliferative phase (Figure 2.25) of wound healing over- laps with and succeeds the infl ammatory phase, beginning 3 to 5 days postinjury and continuing for 3 weeks in acute wounds that heal by primary intention⁵⁰ (see Figure 2.12). The goals of this healing phase are to fi ll in the wound defect with new tissue and restore the integrity of the skin. New tissue formation is the benchmark for the start of this phase. The processes involved in the proliferative phase are angiogenesis (growth of new blood vessels), collagen synthesis (ECM formation), and wound con- traction (the drawing together of the wound edges as shown in Figures 2.17 to 2.19.²

Oxygen and nutrition demands remain very high to support the cells of repair (i.e., fi broblasts, myofi broblasts, endothelial cells, and epidermal cells), which reproduce at a rapid rate to create the collagen matrix. Nutrients, including zinc, iron, cop- per, vitamin C, and oxygen, are essential for fi broblast synthesis of the collagen matrix. The macrophages and neutrophils work to control infection as long as the wound remains open. The combination of these activities raises tissue temperatures. The respond to signals from the macrophages, neutrophils, and the

current of injury by entering an activation cycle.⁸⁸ Keratinocyte activation is transmitted to other neighboring cells (primarily fi broblasts), which in turn release multiple growth factors and initiate the wound healing cascade.⁸⁸

Keratinocytes respond to signals from released growth factors by advancing in a sheet to resurface the open space. The leading edges of the advancing keratinocytes become phagocytic; they clean the debris, including clotted material, from their path.

Cell sheets continue to migrate until the wound is covered and a new basement membrane is generated. Multilayered epithelial cells appear to migrate either as a moving sheet or in a complex

“leapfrog” manner (epiboly). The wound environment speeds the migration of keratinocytes toward one another from the edges of the wound and the dermal appendages.

Full-thickness wounds involve loss of the dermal append- ages, which are an important source of new keratinocytes. As a consequence, epithelial cells can migrate only from the wound edges. The advancing front of epidermal cells cannot cover a cavity, so they dive down and curl under at the edges. For example, full-thickness pressure wounds develop a buildup of epithelial cells at the wound edges, forming an epidermal ridge that curls under the edges and slows closure. The situation is as though the epithelial cells get tired of waiting for granulation tissue to fi ll in the wound defect, so they prematurely prolif- erate and migrate over the edge, as shown in Figure 2.17 and form and epidermal ridge. The migration of epithelial cells is also oxygen-dependent; when there are low levels of oxygen, epithelial migration cannot debride the wound.

In surgical wounds that are sutured, epidermal migration begins within the fi rst 24 hours. In healthy adults, it is usu- ally complete within 48 to 72 hours postoperatively. In other wounds, skin trauma results in tissue degeneration, with broad, indistinct areas and edges that are diffi cult to see. This forms a shallow lesion, with more distinct, thin, separate edges. As tissue trauma progresses, the reaction intensifi es, with a thick- ening and rolling inward of the epidermis. The edge is well- defi ned, sharply outlining the ulcer, with little or no evidence of new tissue growth. Repeated trauma and attempts to repair the wound edges result in fi brosis and scarring. The edges of the wound become indurated and fi rm,⁸⁹ a condition that can impair the migratory ability of the keratinocytes.⁹¹

Once the wound has been resurfaced by epithelial cells, the cells begin the process of differentiating and maturing forming scar tissue. Tissue properties of elasticity and tensile strength of the replaced epidermal layers affect the function of the skin as it overrides bony prominences and moving muscles or tendons because it is less elastic and more friable, easily torn, than the original. The tensile strength, breaking strength of the fi bers, of the remodeled skin will not exceed 70% to 80% of the original.

The quality of the scar tissue is an indication of the fi nal outcome.

The fact that closure has been achieved by reepithelialization does not mean that the wound is fully healed. At this time, the new skin has a tensile strength of approximately 15% of normal compared to the 70% to 80% of the original that it will possess when it is remodeled and mature. It must be treated carefully to avoid trauma, which can cause edema and infection, and can lead to reinfl ammation. Chronic infl ammation compounds the problems of impaired scar quality since it causes a thickening of the skin and less elastic remodeled tissue.⁹²

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FIGURE 2.25 A. Acute proliferative phase. Note the attached wound edges from the 12:00 to 6:00 positions and how well vascularized granulation tissue fi lls up one side of the ulcer. A new pink border of epithelium surrounds the granulation tissue. B: Same wound as in (A). All of the wound edges are now attached to the wound base. Note the presence of fi brin (yellow) within the granulation tissue. Ready for epithelialization phase. (Copyright © B.M. Bates-Jensen.)

wound needs warmth at this time to promote cellular division and manage infection.

Integrins

In unwounded tissues, cells that are involved in repair are sta- tionary; however, after wounding, they change into migrating cells. Migrating cells express specialized receptors called inte- grins.⁵² Integrins serve a critical function in cell adhesion and signaling during wound healing. They are critical for a duality of events: reepithelialization and formation of granulation tis- sue because of their ability to recognize and respond to various components during formation of the ECM.⁹⁴

Growth Factors—Proliferative Phase

As wound healing progresses from the infl ammatory to pro- liferative phase, the keratinocytes, fi broblasts, and endothelial cells synthesize growth factors. As we will see, GFs function to promote cell migration, proliferation, angiogenesis, and syn- thesis of the ECM components.⁵³ AGFs, TGF-β, TNF-a, IL-1, and bFGF are the GFs involved.³⁹

Angiogenesis

Restoration of vascular integrity begins in the infl ammatory phase but becomes a major activity during the proliferative phase. During this phase, angiogenesis, growth of new blood vessels by the endothelial cells (also known as neovasculariza- tion), takes place. Angiogenesis occurs as new capillary buds, arising from intact vessels adjacent to the wound, extend into the wound bed. As endothelial cells proliferate, grow into the wound space, and create capillaries, they connect to form a new network of vessels to fi ll the tissue defect. In the early stages of vessel growth, the vessels have loose junctions and gaps in the endothelial lining.⁵¹ As a result, the initial capillaries are fragile

and permeable, allowing passage of fl uids from the intravascular to the extravascular space; thus, new tissue is often edematous in appearance.⁵¹ The thick capillary bed, which fi lls the matrix, supplies the nutrients and oxygen necessary for the wound to heal. To the naked eye, the capillary loops look like small gran- ules, explaining the name granulation tissue.²⁷ Granulation tis- sue fi rst appears as pale pink buds; as it fi lls with new blood vessels, it becomes bright, “beefy,” and red, as shown in Figure 2.25. In Figure 2.25A,B, the granulation begins at one side of the wound and “marches” across the wound bed.

At this time, the granulation tissue is very fragile and unable to withstand any trauma. Trauma to the new tissue will cause bleeding that can reinitiate the infl ammatory process and cause the laying down of excessive collagen; this results in poor elas- ticity and a less desirable scar. Protection of the new granula- tion tissue is very important.

Even when mature, granulation tissue will remain structur- ally and functionally different from the tissues it replaces. It will not differentiate into nerves, muscles, tendons, or other tissue.²⁴

Fibroblasts

Fibroblasts mainly participate in the biosynthesis of collagen to form the ECM, which acts as a mold, precursor, plastic mate- rial, and cementing substance in the wound healing process.

Fibroblasts secrete collagen until the wound is fi lled; then, col- lagen production ceases as the fi broblast is downregulated. This process of fi brous tissue formation is called fi broplasia. Wound healing by fi broplasia requires that the wound be shaped like a boat or bowl to ensure that granulation tissue fi lls the base before the epithelial edges of the wound meet. Wounds that are not of this shape are at risk for premature surface healing, leaving a cavity under the skin that will subsequently break

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down.^2,95 The optimal wound conditions for supporting fi bro- blast production of collagen and ground substances includes an acidic, low-oxygen wound bed.

Fibroblasts synthesize three polypeptide chains that coil to form a right-handed triple helix.⁹² Now called procollagen, these spiraled chains are extruded from the fi broblast into the extracellular space. The next step is cleavage of the triple-helical molecule at specifi c terminal sites. Now the helix is referred to as a tropocollagen molecule. Tropocollagen, thus transformed, amassed and convolving with other tropcollagen molecules, form a collagen fi bril, then combine with ground substance to form the ECM or scaffolding of repair that will support blood vessel growth by the endothelial cells.^24,96 The endothelial cells migrate and proliferate along the scaffolding, building new cap- illaries that are capable of providing oxygen and nourishment to the new collagen.^51,66

Tropocollagen, a precursor to collagen, is a soluble substance that is transformed into hydroxyproline (HoPro), insoluble collagen, by a process called hydroxylation of praline. Ferrous iron, a reducing agent (e.g., ascorbic acid), alpha ketoglutarate (a collagen boosting agent), and oxygen are required to achieve this transformation. In research, the measurement of HoPro is used to assess collagen deposition, with collagen estimated as seven times the value of HoPro.⁹⁷ In vitro and in vivo studies show that collagen hydroxylation, cross-linking, and deposi- tion are proportional to the arterial partial pressure of oxygen.

Hydroxyproline deposition is proportional to wound tensile strength in rats.⁶¹ Another reason is that the oxygen gradient is critical to the wound healing process.

Collagen Types

Tropocollagen polymerizes (joins with many small particles to create a large molecule) to form various types of colla- gen.²³ Types I and III collagen are important to our discussion, because they are found in the dermis and are involved with wound healing. New tissue growth in the wound follows a char- acteristic pattern:^51,66

• Production of type III collagen. Type III collagen is the pre- dominant type of collagen synthesized in the wound.⁶⁶

• Type III collagen is poorly cross-linked and not aligned in the same manner as type I collagen; as such, it provides minimal tensile strength to the new tissue.⁵¹ Type III collagen fi brils are small (40–60 nm), but elastic. They help the tissue withstand a load over time, a quality referred to as creep resistance. The proportion of small to large fi brils changes over the life span, with type III gradually replaced by type I.

• Production of type I collagen. Early immature type III collagen is gradually replaced with the normal adult type I collagen throughout the proliferation phase of healing and continu- ing through the remodeling phase of healing. Type I collagen fi brils have a larger diameter (100–500 mm) than type III, and are less elastic. These fi bers are organized and arranged in parallel lines along mechanical stress points, giving tissue strength.⁵¹ Approximately 80% of dermal collagen is type I, and these fi bers provide the tensile strength to the tissue. The proportion of large-diameter to small-diameter fi brils deter- mines the overall dermal tensile strength.⁹⁶

At this point, about 3 weeks after wounding, the greatest mass of collagen assembled by the tensile strength is roughly only 15% of normal. This new scar will not tolerate mobiliza- tion or rough handling, both of which will ultimately lead to creation of a new wound and further scarring.⁹² Wound dehis- cence (separation of all the layers of an incision or wound) occur most frequently during this phase.²³

Matrix Formation

Fibrous connective tissue elements that give strength to the ECM include collagen, elastin, fi bronectin (an adhesive gly- coprotein secreted during the infl ammatory phase as part of the process of ECM formation) and reticulin (young collagen fi bers).⁹⁸ Elastin derives its name from its elastic properties.

It is found in skin, lungs, blood vessels, and the bladder, and functions to maintain tissue shape.²³ Matrix proteins, including collagen, form the basal lamina over which the keratinocytes migrate. Laminin is a component of the basement membrane that functions to inhibit keratinocyte migration.⁵³

Cross-Linking of Collagen

Once the collagen fi brils are formed, they are very disorganized.

This disorganization is why early scar tissue is weak with poor tensile strength. Cross-linkage is necessary for wound tensile strength. As the proliferative phase gives way to the remodeling phase of wound healing, the collagen is remodeled into ordered, structured formations, which increase the tensile strength of the scar tissue. The number of collagen fi laments does not give the collagen matrix durability or tensile strength; rather, tensile strength depends on the microscopic welding, or bonding, of one fi lament to another. The sites at which these bonds occur are called cross-links. Intermolecular bonds are the major force holding the collagen molecules together. The greater the num- ber of these bonds, the greater the strength of the collagen fi la- ment. In an anoxic wound, cross-linking is inhibited.

Ground Substance

Ground substance is primarily water, salts, and GAGs. GAGs are polysaccharides that contain amino acids, sugars, and gly- coprotein (called proteoglycans).⁹⁶ GAGs are hydrophilic sub- stances, so they attract large amounts of water and sodium. The turgor (feeling of fullness) normally associated with connective tissue is a manifestation of the accumulation of fl uid by the GAGs. Ground substance has semiliquid gel properties²⁴ Turgor will be discussed as part of wound assessment in Chapter 3.

Myofi broblasts and Contraction

Wound contraction pulls the wound edges together for the purpose of closing the wound. In effect, this reduces the open CLINICAL WISDOM

Granulation Tissue Complications

A change from healthy red granulation tissue to dark red or a dusky pink color is a sign that warrants further investigation and possible intervention (Figs. 2.15 and 2.16). Wound fl uid can also change in color and/or quantity. Pain may increase;

this is often a sign of infection.

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5. Myofi broblasts appear to attempt wound contraction, but their efforts may be compromised by cellular senescence and/or necrosis.

The literature review associated with this study pointed out that diabetic patients show premature fi broblast senescence and decreased fi broblast division; moreover, senescent fi broblasts lose their ability to bind to collagen.⁹⁹

Fibronectin Composition

Fibronectin is a matrix protein critical in the laying down of collagen. Wysocki demonstrated differences in the composi- tion of fi bronectin in chronic wounds, as compared with acute wounds.¹⁰⁰ The fi bronectin in chronic wounds was partially degraded, whereas it remained intact in acute wounds. The small fi bronectin fragments found in chronic wounds may per- petuate the activity of matrix proteases and inhibit healing.¹⁰¹ Indeed, the excess activity of proteases, which breaks down connective tissue faster than it is formed and destroys impor- tant polypeptide-signaling molecules that coordinate healing, may play a role in persistent nonhealing wounds.¹⁰²

Parks¹⁰³ has shown that chronic wounds exhibit produc- tion of stromelysins (metalloproteinases that do not lyse col- lagen), which may represent the unregulated production of proteinase that contributes to the inability of some chronic wounds to heal.

Chronic Wound Fluid

The desire to learn more about the wound microenvironment has led researchers to look at wound fl uid as a refl ection of the microenvironment from which it was collected. Human stud- ies of wound fl uids are complicated by the inability to carefully control the variables related to the wound, the patient, and the way wound fl uids are collected, leading to varied results.

However, considerable efforts have been made by investigators to study fl uid from both acute and chronic wounds.

It is fairly well-accepted that fl uid from acute wounds is mitogenic for wound-associated cells, and fl uid collected from chronic wounds is inhibitory to these cells of regenera- tion.^104,105 Analysis and study of wound fl uid is providing an important insight into the healing of chronic wounds. It has been suggested that chronic wound fl uid analysis could be used to identify potential biomarkers of wound chronicity, leading to new treatment strategies and possibly customized treatment based on the identifi ed biomarkers. This concept has already been applied to secreted biofl uids of other chronic infl amma- tory conditions such as osteoarthritis and periodontal disease to determine disease and metabolic activity.⁵⁴

area and, if successful, results in a smaller wound, with less need for repair by scar formation.

Myofi broblasts are important in wound contraction. They contain an actin and myosin contractile system similar to that found in smooth muscle cells,⁶¹ which allows them to contract and extend. The myofi broblast connects itself to the wound skin margins and pulls the epidermal layer inward. The myofi bro- blast ring forms what has been described as a “picture frame”

beneath the skin of the contracting wound. The contracting forces start out equal in all wounds, but the shape of the “pic- ture frame” predicts the resultant speed of contraction. Linear wounds contract rapidly, square and rectangular wounds con- tract at a moderate pace, and circular wounds contract slowly.

One characteristic of pressure ulcers is that they take on a circu- lar shape, which is an indicator that they will contract slowly (see Figure 2.17).⁹² Wound contraction is manifested by a change in wound shape and reduction in the open area of the wound. This occurs at the fi nal stage of wound repair (see Figure 2.19).

Partial-thickness wounds heal with very little wound con- traction. However, in full-thickness wounds, contraction can account for up to a 40% decrease in wound size.^51,93 For suc- cessful healing, contraction needs to be balanced. A diminished level of contraction leads to delayed healing, with possible excess bleeding and infection. Conversely, excess contraction can lead to loss of function from tissue contractures.⁵¹

Wound contraction can be extremely benefi cial in the clo- sure of wounds in areas such as the buttocks and trochanter, but it can be harmful in areas such as the hand and around the neck and face, where it can cause disfi gurement and excessive scarring.

Rapid, uncontrolled wound contraction in these areas must be avoided. Tissue that draws together too tightly can cause defor- mity of the repaired scar and impairment of tissue function.

Skin grafting is used to reduce contraction in undesirable loca- tions. The thickness of the skin graft infl uences the degree of contraction suppression. Pressure garments are another method of controlling wound contraction (see Chapter 16).