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Chronic Wound Healing: Molecular and Biochemical Basis

Sophia Tate1, and Keith Harding2

1University Hospital of Wales, Cardiff and Vale University Health Board, Cardiff, UK

2Division of Population Medicine, Cardiff University School of Medicine, Cardiff, UK

1.1 Introduction

A wound can be defined as a break in the epithelial integrity of the tissue, or a disruption of normal anatomical structure and function [1]. Usually a wound progresses through several sequential, though overlapping, stages of cellular and biochemical activity to achieve healing. A chronic wound may be defined as one that is failing to progress through the wound healing process in an anticipated time frame [2]. A wound that does not show significant improvement within 4 weeks, or heal completely in 8 weeks, may be considered a chronic wound [3]. There are four stages described in normal wound healing: haemostasis, inflammation, proliferation, and remodelling. The healing of a chronic wound may be arrested in any of these stages, but most commonly during inflammation or proliferation [4]. This chapter will briefly describe normal wound healing, consider some subtypes of chronic wound, and then examine the different molecular and biochemical processes that occur.

1.2 Acute Wound Healing

The process of acute wound healing is well described and widely reported in the literature, and is summarised in Figure 1.1.

Figure 1.1 A summary of acute wound healing. EGF, epidermal growth factor; FGF, fibroblast growth factor; IGF, insulin-like growth factor; IL-1, interleukin 1; MMP, matrix metalloproteinase; PDGF, platelet-derived growth factor; TGF, transforming growth factor; TNF-a, tumour necrosis factor a; VEGF, vascular endothelial growth factor.

The first step, haemostasis, is characterised by vasoconstriction and coagulation; it takes place soon after injury and is complete within hours. The tissue in the wound is exposed to blood because of disruption of the blood vessels and lymphatics during injury. Platelets are activated when they come into contact with collagen and initiate the coagulation cascade, resulting in the deposition of a haemostatic 'plug' [5]. A number of cytokines are released by the degranulation of activated platelets. Of particular importance are platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-ß). PDGF is a chemoattractant of neutrophils, macrophages, smooth muscle cells, and fibroblasts [1]. TGF-ß is also involved in the chemotaxis of macrophages, fibroblasts, and smooth muscle cells, and has a role in activating these cells to express other cytokines and enzymes which are crucial to enable the wound healing to progress [1].

After the initial vasoconstriction during haemostasis, there is vasodilation and increased vascular permeability as the stage of inflammation begins. This is regulated by mast cell degranulation, which releases histamine and other vasoactive mediators [1]. Debris, dead cells, and bacteria are cleared from the tissue by neutrophils, and later by macrophages. Inflammation is usually complete after 48-72?h, but may last as long as 5-7 days [6].

The next stage is proliferation, which continues for weeks. The hallmark of the proliferative phase is the migration of fibroblasts into the wound, where they are activated to produce collagen III, fibrin, fibronectin, and hyaluronic acid in the new extracellular matrix [7]. Granulation tissue is deposited to fill the defect. Keratinocytes, stimulated by epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-a), migrate to the wound edges, and eventually close the defect [1]. Angiogenesis is important to support the increased metabolic activity in the wound. A number of growth factors stimulate the neovascularisation, including vascular endothelial growth factor (VEGF). Epidermal cells, fibroblasts, macrophages, and vascular endothelial cells produce these factors in response to conditions in the wound environment, such as low pH and reduced oxygen tension [1].

The final stage, remodelling, begins after about a week and may last for years. This phase is characterised by the removal of type III collagen from the extracellular matrix and the deposition of mature type I collagen [8]. Collagenase enzymes from fibroblasts, neutrophils, and macrophages are important in this stage [1]. Wound contraction is mediated by differentiated fibroblasts (myofibroblasts) in response to TGF-a, and the presence of matrix proteins such as extra-domain-A fibronectin and tenascin C [9]. Once remodelling has occurred, there is apoptosis of fibroblasts, leaving relatively acellular scar tissue [9].

1.3 Categories of Chronic Wound

Although chronic wounds may seem varied in their presentation and characteristics, often the underlying aetiological processes are similar. Some common chronic wound categories are considered here. Ultimately, the final common pathway is an open wound that has been colonised with bacteria, initiating a damaging inflammatory response that impedes healing [10].

1.3.1 Pressure Ulcers

Pressure ulcers are an example of chronic ischaemia-reperfusion injury. Repeated tissue trauma occurs in insensate areas when the pressure in the tissue exceeds capillary perfusion pressure [10]. This results in skin breakdown, which is followed by bacterial colonisation, often compounded by the location of such ulcers near to the perineum. There is failure of the processes of angiogenesis, extracellular matrix deposition, and wound contraction, resulting in the development and persistence of a chronic ulcer [11]. These steps in wound healing are usually driven by growth factors, and the destruction or reduced synthesis of these proteins in pressure ulcers has been investigated. In a study using an enzyme-linked immunosorbent assay technique to quantify the levels of growth factors in wound fluid from pressure ulcers, Cooper et al. [12] found that PDGF, fibroblast growth factor (FGF), EGF, and TGF-ß levels were variable, and decreased compared with the levels of growth factors in acute wounds.

1.3.2 Venous Stasis Ulcers

Venous stasis ulcers occur when damaged or defective leg vein valves result in venous hypertension and oedema. Eventually the venous pressure exceeds the capillary perfusion pressure of the skin, and the tissue becomes ischaemic. The increase in intraluminal pressure affects the permeability of the vessel walls, and the veins leak fibrin and other plasma components into the perivascular space [9]. Accumulation of fibrin impairs healing by impairing collagen synthesis, and by forming peri-capillary fibrin cuffs that impede normal vessel function [9]. Often a venous ulcer is precipitated by minor trauma, for example a scratch or insect bite. The skin breakdown is accelerated by the hypoxic conditions, and secondary bacterial colonisation. This increases the tissue injury and inflammation at the wound site, and impairs epithelialisation [11].

1.3.3 Ischaemic Ulcers

Atherosclerosis and/or embolism in leg arteries leads to narrowing of the lumens of the vessels and ischaemia of distal tissue. Minor trauma may then result in an ulcer. Healing is slow because of the low oxygen concentration in the tissue, and the resultant open wound is colonised by bacteria. This increases inflammation in the wound, and the tissue defect persists. The effects of hypoxia are described in more detail in Section 1.5.4.

1.3.4 Diabetic Foot Ulcers

Diabetic foot ulcers are another category of wounds which are commonly chronic in their course. The diabetic foot may be subject to repeated trauma as a result of sensory loss. There may also be a degree of ischaemia because of microvascular arteriopathy. Once the skin barrier is breached, low-grade bacterial colonisation is common. Tissue fragments and bacterial products perpetuate the inflammatory response. The effects of hyperglycaemia are described in more detail in Section 1.5.2.

1.4 How a Chronic Wound Develops: Intrinsic Components

There are several hallmarks of chronic wounds when compared with normal acute wounds [9]. In a normal wound bed, there will be a high concentration of growth factors, with healthy cell populations in an organised extracellular matrix. By comparison, chronic wound beds tend to have low concentrations of growth factors and a disorganised extracellular matrix. This is because of excessive proteolysis driven by a persistent inflammatory state, often a response to a bacterial biofilm or low-grade infection. Impaired angiogenesis and neovascularisation mean that cells in the wound environment are starved of oxygen and nutrients. The result is impaired fibroblast and epithelial cell proliferation and migration, and delayed healing. Figure 1.2 summarises these components.

Figure 1.2 The local environment in the chronic wound. MMP, matrix metalloproteinase; ROS, reactive oxygen species.

1.4.1 Cell Phenotype

The cells in chronic wounds have an altered phenotype, with fewer growth factor receptors and less mitogenic potential [9]. They do not therefore...