[PART 1] Introduction Wound healing involves a complex series of events, which encompass chemotaxis, cell division, neovascularization, synthesis of new extracellular matrix (ECM) components, and the formation and remodeling of scar tissue. Soluble mediators, such as growth factors, cytokines, matrix metalloproteinases (MMPs), and their regulators, control many of the processes involved in wound healing via their effects on various cell types and the ECM.[1,2] In acute wounds, these processes, which are triggered by tissue injury, involve the four overlapping but well defined phases of hemostasis, inflammation, proliferation, and remodeling (Figure 1). Physiological events in a nonhealing wound, however, do not follow this traditional model of wound repair. The concept of wound bed preparation addresses this issue and provides a model that is appropriate for understanding and treating chronic wounds. Normal Wound Healing Vasoconstriction in combination with clot formation and platelet aggregation occurs in response to initial injury. Once aggregated, the platelets degranulate and release mediators that help form the fibrin clot together with growth factors and chemo-attractants. During this initial hemostatic phase, platelets release several growth factors, including platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and transforming growth factor-beta (TGF-b) into the wound bed. Neutrophils and macrophages migrate into the wound and secrete additional growth factors, such as transforming growth factor-alpha (TGF-a), heparin-binding epidermal growth factor (HB-EGF), and basic fibroblast growth factor (bFGF), which further stimulate the inflammatory process. These various growth factors are mitogenic and chemotactic for endothelial cells, macrophages, keratinocytes, and fibroblasts, which migrate to the site of the wound to form the “granulation tissue” or provisional matrix.[3] During the earliest stages of repair, neutrophil proteases participate in antimicrobial defense and in the debridement of devitalized tissue at the wound site.[4] Macrophages, activated by CD4+ T helper cells and by the cytokine interferon-g (produced by T-lymphocytes and NK cells), stimulate acute inflammation through the secretion of cytokines, mainly tumor necrosis factor (TNF), IL-1 and chemokines, and short-lived lipid mediators, such as platelet-activating factor, prostaglandins, and leukotrienes. Their collective action is to further enhance local inflammation that is rich in neutrophils, which helps to phagocytose and destroy infectious organisms.[5] The cytokines play a further role in tissue repair: IL-1b stimulates the proliferation of fibroblasts, and TNF-a and IL-1b stimulate fibroblasts to synthesize MMPs. Activated macrophages, along with neutrophils, remove dead tissues to facilitate repair after the infection is controlled. In addition, they induce the formation of repair tissue by secreting growth factors that stimulate fibroblast proliferation (PDGF), collagen synthesis (TGF-b), and new blood vessel formation (FGF). Though many different growth factors contribute in their own way to wound repair, FGF plays a pivotal role in this process, as it is a potent mitogen for endothelial cells, accelerates granulation tissue formation by increasing fibroblast proliferation and collagen accumulation,[6] and provides the initial angiogenic stimulus.[7] Fibroblasts, epithelial cells, and vascular endothelial cells begin to multiply freely as the wound enters the proliferative phase of repair. During the proliferative phase, the number of inflammatory cells in the wound decreases, and fibroblasts, endothelial cells, and keratinocytes secrete further growth factors that are required to mediate the process of wound healing. The development of granulation tissue during wound healing requires the formation of new capillaries. New vessel formation or sprouting of capillaries from pre-existing ones is called angiogenesis, and this plays a pivotal role in the complex process of wound healing. Angiogenesis is tightly regulated by factors that act by either stimulating or inhibiting vessel growth.[8] During wound healing, angiogenic capillary sprouts invade the fibrin/fibronectin-rich wound clot and within a few days organize into a microvascular network throughout the granulation tissue.[9] As collagen accumulates in the granulation tissue to produce scar, the density of blood vessels diminishes. Disturbance of this dynamic process may influence the development of chronic wounds.[10] The rapid onset and predominance of proangiogenic factors optimizes healing in damaged tissues.[11] Among all proangiogenic factors, the one that has been extensively studied in wound vessel angiogenesis is the vascular endothelial growth factor (VEGF). Experimental evidence suggests that a defect in VEGF regulation might be associated with wound healing disorders,[12] and healing-impaired diabetic mice show decreased VEGF expression levels.[13] Initial angiogenic stimulus in wound healing might be provided by FGF-2, followed by a subsequent and more prolonged angiogenic stimulus mediated by VEGF.[7] During the phases of inflammation and proliferation, proteolytic enzymes, such as MMPs and serine proteases, play an essential role in tissue repair and remodeling. Once in the tissue, the inflammatory cells and fibroblasts stimulate the production of MMP-1, -2, -3, and -9 so that they can degrade the damaged ECM in preparation for macrophage phagocytosis of the ECM debris. Cytokines, growth factors, and tissue inhibitors of metalloproteinases (TIMPs) tightly regulate the production and activity of MMPs. The final phase of wound healing involves a balanced process that degrades old ECM and synthesizes new ECM in order to remodel the scar that was formed during proliferation and repair. Among the most important cells during this phase are the fibroblasts, as they synthesize matrix molecules, such as collagen, elastin, and proteoglycans. They also produce MMPs that degrade the matrix and TIMPs, which regulate the activity of MMPs. This phase can take several months and results in a mature scar. In summary, normal wound healing is a complex and finely tuned process that is mediated by growth factors, cytokines including angiogenic mediators, and MMPs.[14] Although the phases involved in normal wound healing overlap, they occur in a timely manner and are tightly regulated. The Chronic Wound A chronic wound can be defined as a wound in which the normal process of healing has been disrupted at one or more points in the phases of hemostasis, inflammation, proliferation, and remodeling.[15] Chronic wounds have several distinctive features (Table 1) and in contrast to acute wounds, they fail to heal in a timely and orderly manner. Chronic wounds are often regarded as being “stuck” in the inflammatory or proliferative phases of wound healing. Since growth factors, cytokines, and proteases all play important roles in each phase of the wound healing process, alterations in one or more components of these factors may account for the impaired healing observed in chronic wounds. Analyses of the molecular and cellular environments of acute and chronic wounds have revealed several important differences. The cytokine environment of chronic wounds is substantially altered, and the levels of IL-1a, a pro-inflammatory cytokine, have been shown to be elevated in chronic wounds.[16] Trengove, et al.,[17] found that the levels of other pro-inflammatory cytokines (IL-1b and TNF-a), along with IL-1a, were significantly elevated (p = 0.01 for IL-1a, p = 0.005 for IL-1b, and p = 0.013 for TNF-a) in nonhealing wound fluids from leg ulcers compared to those from healing wounds. Moreover, the levels of these cytokines decreased substantially as the chronic wound healed, indicating a significant correlation between nonhealing wounds and increased levels of pro-inflammatory cytokines. They also found that there was a statistically significant (p = 0.002) decrease in the mitogenic activity in nonhealing wounds compared to healing wounds. Similarly, when chronic wound fluid is added to cultures of fibroblasts, keratinocytes, or vascular endothelial cells, it fails to stimulate DNA synthesis in these cells, which is in direct contrast to the DNA-synthesizing ability of acute wound fluid.[18,19] Oxygen-derived free radicals have been implicated in the causation of venous ulceration and their persistence. Scavenging such radicals using antioxidants expedites healing of venous ulcers.[20] Nitric oxide (NO) is known to combine with hydroxyl free radicals forming peroxynitrate, a potent free radical, which causes tissue destruction. NO overexpression in chronic venous ulcers may be involved directly or indirectly (through production of peroxynitrate) in the pathogenesis and delayed healing of chronic venous ulcers through its effects on vasculature, inflammation, and collagen deposition.[21] In a study of 44 patients with chronic venous disease, Howlander and Coleridge Smith[22] observed that the total plasma NO levels were elevated in patients with severe skin damage. Similarly, Jude, et al.,[23] found that diabetic patients with recurrent neuropathic and neuroischemic foot ulcers had significantly higher plasma NO levels compared to patients with nonrecurrent foot ulcers (46.9 +/- 6.3 microm/L versus 30.2 +/-2.[4] microm/L respectively, p Surgical debridement. Surgical or sharp debridement is not a new technique, and historical texts show that ancient civilizations often made surgical changes to the wound bed.[47] Surgical debridement is the fastest way to remove dead tissue. It causes considerable pain, and hence, it was earlier restricted to the treatment of neuropathic diabetic ulcers where the use of anesthesia and pain management was not necessary. However, this problem could be overcome with the use of topically applied local anesthetics (e.g., combination of lidocaine [lignocaine] and prilocaine), applied 30 to 45 minutes prior to debridement.[48] Although surgical debridement is thought to be selective, there may be some damage to viable tissue, and bleeding is likely. Nevertheless, this may help to revitalize the wound and encourage healing by inundating the wound bed with growth factors and cytokines. Mild to moderate bleeding could be controlled by the application of pressure and a hemostatic calcium alginate dressing. Autolytic debridement. All wounds experience some level of autolytic debridement, which is the natural and highly selective process by which endogenous proteolytic enzymes break down necrotic tissue. These endogenous enzymes are mainly produced by neutrophils and include elastase, collagenase, myeloperoxidase, acid hydrolase, and lysosomal enzymes.[49] Autolytic debridement may not take place fast enough to encourage rapid wound healing and closure, but the use of occlusive dressings can enhance this natural process, while maintaining a moist wound bed and managing excess exudate.[50] This allows painless, selective debridement and promotes the formation of healthy granulation tissue.[51] Autolytic debridement can result in the production of significant quantities of exudate. Typical practice for autolytic debridement involves the use of a hydrogel to soften and break down necrotic tissue covered with an absorptive, occlusive dressing to absorb the excess exudate. With an increase in antibiotic-resistant pathogens, there has been a renaissance in recent times in the use of honey for the treatment of wounds and ulcers.[52] As well as having an antibacterial action, honey provides rapid autolytic debridement and deodorizes wounds, in addition to having anti-inflammatory properties[53] and stimulating immune responses.[54] Though the exact mode of action remains unclear, Tonks, et al.,[55] observed that reactive oxygen intermediate production was significantly decreased (p Enzymatic debridement. Enzymatic debridement is a highly selective method of wound debridement that uses naturally occurring proteolytic enzymes that are manufactured by the pharmaceutical and healthcare industry specifically for wound debridement. These exogenously applied enzymes work alongside the endogenous enzymes in the wound. Several enzyme debriding agents have been developed including bacterial collagenase, papain/urea, fibrinolysin/DNAse, trypsin, streptokinase-streptodornase combination, and subtilisin. Only the first three products are widely available commercially in those markets where they are registered, although availability varies geographically. Collagenase-based debridement. Collagenase, derived from Clostridium histolyticum, is the best characterized of all of the enzyme debriding agents. It specifically digests all triple helical collagen and will not degrade any other proteins lacking the triple helix. This is a unique feature of bacterial collagenase, since none of the other available proteases can digest collagen.[56] It has been used for over 25 years and has a number of clinical advantages, including selectively removing dead tissue, being painless, and causing the least amount of blood loss.[57] This type of debridement can be appropriate to use in long-term care facilities and in the home care setting.[58] Clinical research has shown that bacterial collagenase is an effective and selective enzyme debriding agent in a range of wound types.[59–65] Papain-based debridement. Papain is a nonspecific proteolytic enzyme derived from the fruit of the papaw tree (Carica papaya). Papain breaks down fibrinous material in necrotic tissue and requires the presence of sulfhydryl groups, such as cysteine, for its activity. It does not digest collagen,[66] and it requires specific activators that are present in necrotic tissue in order to be stimulated. Urea is combined with papain because urea is able to expose the activators of papain in necrotic tissue. Urea also denatures proteins, making them more susceptible to proteolysis by papain. The combination of papain and urea is approximately twice as effective at digesting protein compared with papain alone.[67] Papain use is known to produce an inflammatory response and possibly as a result of this, considerable pain is often experienced with the use of this method.[68,69] Therefore, chlorophyllin, an anti-agglutinin, has been added to preparations of papain/urea in an attempt to reduce the pain.[70,71] Current preparations containing the above combination tend to cause less pain. Papain/urea preparations, however, may be particularly useful in patients with pressure ulcers combined with a loss of sensation (e.g., spinal injuries), as pain may not be a limiting factor for its use in such instances.[33] Mechanical debridement. Mechanical debridement is a nonselective, physical method of removing necrotic tissue and debris from a wound using mechanical force. This debridement method is generally easy to perform and is more rapid than autolytic and enzymatic debridement. However, this nonselective method can damage healthy granulation tissue both in the wound bed and at the margins of the wound thus causing significant discomfort to the patient. Despite these disadvantages, there are a number of mechanical debridement methods that are in use. Wet-to-dry dressings are the simplest method of mechanical debridement, but due to the frequent dressing changes, it can require considerable nursing time and hence is costly.[72] Wet gauze dressings are placed onto the wound bed and allowed to dry, trapping the necrotic debris within the gauze. Upon removal of the dressing, embedded necrotic tissue and debris are mechanically separated from the wound bed.[73] Pressurized irrigation involves applying streams of water, delivered at either high or low pressure, to wash away bacteria, foreign matter, and necrotic tissue from the wound. However, if the pressure is too great, there may be a risk of forcing bacteria and debris deeper into the wound or damaging viable tissue.[74] Whirlpool therapy uses powered irrigation and can be very effective at loosening and removing surface wound debris, bacteria, necrotic tissue, and exudate from the wound. Ultrasound treatment has been used to remove necrotic tissue and has been shown to effectively debride wounds and reduce infection caused by bacteria.[75] Vacuum-assisted closure is a noninvasive form of mechanical or physical debridement that exposes the wound bed to negative pressure (approximately 125mmHg below ambient pressure) by way of a closed system. It helps healing of chronic wounds by minimizing exudate and slough in the wound bed, reducing tissue edema,[76,77] increasing peripheral blood flow, improving local oxygenation, and promoting angiogenesis and good quality granulation tissue.[78] Biosurgery (myiasis). For a decade since its introduction in 1931, fly maggots have been known to help debride and heal wounds. This technique uses sterile maggots, which digest sloughy and necrotic material from the wound without damaging the surrounding healthy tissue.[37] In the study by Mumcuoglu, et al.,[79] complete debridement was achieved using maggots in 38 of the 43 patients (88%) with chronic leg ulcers and pressure ulcers. Among them, five patients had their limbs salvaged after being referred for amputation of the leg. Likewise, Sherman[80] in a cohort of 103 patients with pressure ulcers observed that 80 percent of maggot-treated wounds were completely debrided compared to only 48 percent of wounds that were treated by conventional therapy alone (p = 0.021). The precise mechanism by which maggots debride the wound and promote wound healing remains unclear. However, there is speculation that they probably act by ingesting and killing bacteria, exerting a bacteriostatic effect by increasing wound pH,[81] secreting proteolytic enzymes that are important in eschar degradation,[82] and increasing tissue oxygenation.[83] Nevertheless, despite recent encouraging reports,[80] some patients complain of increased pain with maggot therapy.[84] Likewise, the potential psychological and aesthetic considerations cannot be ignored. Maintenance Debridement and Wound Bed Preparation Traditionally, debridement has been undertaken as a single therapeutic step within defined time frames. This procedure is usually only repeated if necrotic tissue reappears, since it is assumed that healthy granulation tissue will form after complete debridement. Although this may be applicable for acute wounds, it is unlikely to remove the necrotic burden that continually accumulates in a chronic wound. In the case of nonhealing chronic wounds, it may be more appropriate to perform regular or even continuous debridement. An important aspect of wound bed preparation is the recognition that chronic wounds have underlying pathogenic abnormalities that cause necrotic tissue to accumulate. Therefore, in order to facilitate wound progression, repeated removal of necrotic tissue will be necessary throughout the lifespan of the chronic wound. In this way an extended “maintenance” phase of debridement has been proposed.[33] This is more likely to be effective than a single intervention. Wound bed preparation advocates that clinicians should consider a steady state removal of the necrotic burden and that regular and efficient debridement is necessary to reduce necrotic burden and obtain healthy granulation tissue.[85]