Abstract: Wound healing involves a well-orchestrated, complex process leading to repair of injured tissues. However, chronic wounds do not follow the normal pattern of repair. This is due to underlying physiological problems associated with their development, which unless corrected would continue to cause wound deterioration. The key to effective wound care lies in a combination of three approaches: treatment of underlying medical problems, assessment and treatment of local wound bed, and effective management of any patient-centered concerns. An essential component of this recommended approach is restoration of healthy granulation tissue in the wound bed. Wound bed preparation brings a number of existing procedures, including debridement, treatment of infection, and management of exudate levels, together into a systematic approach to help restore the chronic wound bed environment. The aim of wound bed preparation is to remove the barriers to healing and initiate the repair process. This review explores the scientific rationale behind this concept and examines how wound bed preparation offers healthcare professionals an improved paradigm for the treatment of chronic wounds. By implementing wound bed preparation, the formation of healthy granulation tissue will be optimized and the efficiency of biotechnological therapies improved, which would ultimately reduce the time to wound closure.

[PART 2]

Bacterial Imbalance in Chronic Wounds

All chronic wounds intrinsically contain bacteria, and the process of wound healing can still occur in their presence. Therefore, it is not the presence of bacteria[86] but their interaction with the host that determines the organisms’ influence on chronic wound healing.

The existence of bacteria in the wound bed can be divided into four distinct categories based on the induced host response. These categories are defined as contamination, colonization, local infection, and spreading infection. However, the relative number of microorganisms and their pathogenicity, in combination with host response and factors, such as immunodeficiency, dictate whether a chronic wound becomes infected or shows signs of delayed healing.

Contamination is defined as the presence of nonreplicating microorganisms within a wound. Most organisms entering the wound bed fall into this category and are incapable of replicating in soft human tissue. As a consequence, the host defenses rapidly clear them.

Colonization is categorized as replicating microorganisms that adhere to the wound surface but do not cause cellular damage to the host. Examples of colonizing organisms are those usually found living on the skin, such as Staphylococcus epidermidis and Corynebacterium species. Rather than causing damage to the host, these species of commensal organisms have been shown to enhance wound healing.[87–90]

Recently, the terms local infection and critical colonization have been introduced to describe a situation in which the wound has an increasing bacterial burden, which is intermediate between the category of colonization and infection. Wounds that are locally infected or critically colonized will not heal but may not display classic signs of infection.[32] During this phase, subtle clinical symptoms of local infection may, however, become apparent. The clinical signs and symptoms of critical colonization are delayed healing, pain/tenderness, increased serous exudate, change in color of the wound bed, friable granulation tissue, absent or abnormal granulation tissue, and abnormal odor.[91,92]

Wound infection is defined as the presence of replicating microorganisms within a wound with a subsequent host response that leads to a delay in healing. Because of this it is important that infection is recognized as early as possible. The signs and symptoms of local infection are redness (erythema), warmth, swelling, pain, and loss of function. Foul odor and pus may accompany this. Eventually, the local bacterial burden will increase further and becomes systemically disseminated resulting in sepsis, which if not actively treated could progress to multi-organ failure and death in some instances.

There are several factors known to affect the bacterial burden of chronic wounds and increase the risk of infection. These include the number of microorganisms present in the wound, their virulence, and host factors. For instance, experimental studies have demonstrated that regardless of the type of microorganism, impairment of wound repair may occur when there are more than 1x105 organisms per gram of tissue.[74,93–95] However, other studies have shown that many chronic wounds with a bacterial load greater than 1x105 will heal normally.[96] In view of this, it is clear that the type and pathogenicity of the organisms increase the risk of infection rather than the number of microorganisms alone. For example, the isolation of any highly virulent beta hemolytic streptococci from a chronic wound should be considered significant and the appropriate treatment initiated.[97]

The mix of microorganisms within a wound may also be an issue. Most chronic wounds will usually contain more than three species of microorganism.[98,99] This is significant because some combinations of bacterial species may develop synergy with each other, resulting in previously nonvirulent organisms becoming virulent and causing damage to the host.[74,100–102] Deep in the granulation tissue, chronic wounds may contain resident microflora that may not be reflected by routine culture. However, with the advent of new molecular techniques, like denaturing gradient gel electrophoresis (DGGE), novel organisms like Pseudomonas rhodesiae could be identified and the limitations of conventional techniques for the analysis of complex microbial populations could be overcome in the future.[103]

Although bacterial quantity and virulence are important factors in wound infection, host resistance is also of equal significance. Systemic factors also increase the risk of infection in chronic wounds (Table 3). Factors, such as immunosuppression, concomitant disease, medication, and age, can all influence whether bacteria present in a wound impair healing.[74,104]



More recently, the importance of biofilms has become apparent in the context of wound infection, and these may contribute to delayed healing in chronic wounds. When bacteria proliferate they form microcolonies that become attached to the wound bed and secrete a glycocalyx or biofilm that helps to protect the microorganisms from antimicrobial agents.[105] Organisms may exist as clusters of individual bacterial types or as mixed bacterial colonies. These bacterial colonies undergo several changes, expressing different genes, which can then alter the organisms’ antimicrobial sensitivity. The periodic release of motile bacteria from these colonies may result in infection. Thus, biofilms are bacterial colonies that may be resistant to the effects of antimicrobial agents, such as antibiotics and antiseptics, and could contribute to delayed healing.[105]

Infection and Wound Bed Preparation

Effective wound bed preparation depends on the management of microbial balance as well as the treatment of infection. The concept of wound bed preparation recognizes that a patient with a chronic wound may display all the classic signs of infection but that infection could equally be clinically less obvious. The reasons for this lie in the immunological response to chronic wound infection.

The continual presence of a bacterial infection stimulates the host immune defenses leading to the chronic production of inflammatory mediators, such as prostaglandin E2 and thromboxane. Neutrophils continue to migrate into the wound, releasing cytotoxic enzymes and free oxygen radicals.[106] Thrombosis and vasoconstrictive metabolites cause wound hypoxia, leading to enhanced bacterial proliferation and continued tissue damage. With prolonged bacterial presence in chronic wounds, the bacteria change their pattern of behavior and alter their phenotype and their immune expression. All these factors help them to evade detection by the body’s immune system, thus making it difficult to be negated by the host defenses. This development of “immune tolerance” can create the spurious impression of no infection[74] and may prevent the eradication of microorganisms from the wound.

Wound bed preparation transforms the way infection is viewed and treated because it recognizes that wound healing may be delayed by bacteria, even in the absence of classic signs of infection. Instead, subtle secondary signs of infection, such as lack of healthy granulation tissue, change in color of the wound bed, and friable granulation tissue, could be present. A common feature of locally infected wounds will be failure to heal and progressive deterioration. Wound bed preparation expands our existing view and recognizes that critical colonization as well as infection may need to be addressed so that healing can be optimized.

Treating Infected Wounds

It is important that treatment is initiated as soon as bacteria have been identified as a reason for the impaired healing of a chronic wound. Treating infected wounds will help to reduce the bacterial burden and hence remove one of the barriers to healing. There are a number of means by which bacterial burden can be reduced, which include the use of antibiotics and antiseptics. Though antibiotic therapy is useful to treat infected wounds and helps to prevent infection spreading in the soft tissues beyond the wound (e.g., cellulitis and ascending lymphangitis), repeated use in patients with chronic wounds could lead to the development of bacterial resistance. Therefore, great caution needs to be exerted in the use of antibiotics, and they should be avoided as a first-line management.[107]

The resolution of microbial imbalance with slow-release antiseptics is regaining recognition as an important adjunct to antibiotic treatment.[38] The use of antiseptic agents may be essential for effective wound bed preparation, since inadequate removal of bacteria delays wound healing.[108] In contrast to antibiotics that have a specific mode of action, antiseptics target bacteria at the cell membrane, cytoplasmic organelle, and nucleic acid level. These multitarget antibacterial effects mean that bacterial resistance is unlikely. Commonly applied antiseptics include slow-release antimicrobials, such as cadexomer iodine.[109] Cadexomer iodine is a slow-release antimicrobial capable of absorbing excess wound exudate while providing a sustained level of iodine in the wound bed.[110,111] Evaluation of its benefits have indicated that it is well tolerated and accelerates the healing of chronic leg ulcers.[112–118] Cadexomer iodine has also demonstrated efficacy in vivo against Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA).[119,120]

There is some concern and controversy with antiseptic use on wounds because of its cytotoxic effect on cells involved in wound healing (fibroblasts, keratinocytes, and leukocytes) in vitro.[121,122] This effect appears to be concentration dependent, as several antiseptics in low concentrations are not cytotoxic, besides retaining their antibacterial activity.[123] In addition, there is insufficient evidence at present to show that antiseptics have a deleterious effect on healing. On the contrary, nine clinical trials (in humans) comparing the effects of cadexomer iodine with other forms of treatments on chronic ulcers have shown enhanced healing along with decreased pus, debris, pain, and erythema with the use of cadexomer iodine. No negative effect on healing with cadexomer iodine was observed in these trials.[124]

Nanocrystalline silver dressings are a new variety of antimicrobial barrier dressings that may help reduce infection in partial- and full-thickness wounds. These are comprised of a silver-coated, high-density. polyethylene mesh with an absorptive gauze core, which slowly releases silver into the dressing and maintains an effective antimicrobial barrier for up to seven days. Nanocrystalline silver has been shown to be effective against a broad spectrum of bacterial strains in vitro, including MRSA and vancomycin-resistant Enterococcus.[125]

Noble metals like silver have been in use since the middle of the last century in the treatment of both acute and chronic wounds. They have been shown to be effective in combating antibiotic-resistant strains in colonized wounds.[126] Bishop, et al.,[127] investigated the effect of silver sulfadiazine (AgSD) cream in a prospective, randomized, controlled trial of 86 patients with chronic venous ulcers. They found a statistically significant reduction in their size using one-percent AgSD compared to tripeptide copper complex 0.4-percent cream or the placebo. Similarly, Kucan, et al.,[128] observed rapid healing and significant reduction in the bacterial counts of chronic pressure ulcers (reduced to 105 or less per gram of tissue within the three-week test period) using one-percent AgSD compared to povidone-iodine and physiological saline. These studies would probably not meet today’s standards required to convincingly demonstrate efficacy. However, more recently, Mi, et al.,[129] demonstrated long-term inhibition of the growth of Pseudomonas aeruginosa and Staphylococcus aureus at an infected wound site using AgSD incorporated in a bilayer chitosan wound dressing.

Debridement of necrotic tissue is another means by which bacterial burden can be reduced to optimize wound healing and enhance wound closure. Research has demonstrated that the presence of necrotic tissue in the wound bed is associated with wound infection,[92,130,131] and its removal works on several levels to reduce bacterial burden. Firstly, debridement enhances local host defense mechanisms and prevents active infection by reducing the amount of devascularized tissue and removing foreign bodies.[45,46] Secondly, by activating the release of growth factors and tissue cytokines, debridement helps to promote the formation of well vascularized granulation tissue.

The diagnosis of infection in a chronic wound is a complex clinical skill, but the most important indicators of infection are both local and systemic host characteristics. Wound bed preparation as a clinical strategy helps clinicians address the issues of increased bacterial burden and remove this barrier to healing using advanced antibacterial agents and debridement.

Exudate Levels in Chronic Wounds

Several studies indicate that chronic wounds become “stuck” in the inflammatory phase of the wound healing process.[85,132,133] Due to their inflammatory state, chronic wounds often produce copious amounts of exudate, and this may be increased further in wounds that are heavily colonized or infected by bacteria. The amount of fluid produced by a chronic wound can be a barrier to healing.[18,33,36,134] Furthermore, it is not just the amount of exudate produced by a chronic wound that is important but also an understanding of the altered molecular and biochemical changes in the fluid. Hence, studies comparing acute and chronic wound fluid have focused on analyzing the molecular and biochemical environment in order to understand more about chronic ulcers.

There has been some interest in investigating the effects of chronic wound fluid on the proliferation of the various cell types involved in wound healing. Bucalo and colleagues[18] collected wound exudate from the venous ulcers of six patients in order to investigate the effects of chronic wound fluid on the proliferation of human dermal fibroblasts, microvascular endothelial cells, and keratinocytes in culture. The results of this study showed chronic wound fluid inhibited or failed to stimulate the proliferation of dermal fibroblasts, endothelial cells, and keratinocytes. The study also found that the chronic wound fluid was cytotoxic and that this may have contributed to the effect on cell proliferation. In contrast, fluid from acute wounds has been found to stimulate fibroblast proliferation, and this is likely to be mediated in part by platelet-derived growth factor-like peptides.[135]

Other studies have focused on analyzing how pro-inflammatory cytokines and growth factors can contribute to delayed wound healing. Clinical research comparing exudate from acute mastectomy wounds and exudate from chronic wounds demonstrated that the chronic wound fluid had an imbalance of the pro-inflammatory cytokines IL-1b, TNF-a, and IL-6.[133] The high levels of pro-inflammatory cytokines found in this exudate are responsible for promoting a chronic inflammatory response. Growth factors from acute and chronic wound fluid also show considerable variability. For example, chronic wound fluid was shown to contain lower levels of EGF and higher levels of TGF-a, TGF-b, and IGF-1 compared with fluids from acute wounds.133

Similar studies have confirmed that other growth factors, such as TGF-b1 and PDGF, are degraded by proteases present in chronic wound exudate.[24,28] Moreover, the degradation of these growth factors was shown to occur when serine proteases were not inhibited, suggesting that growth factor degradation as a result of protease action is more likely to be due to serine proteases, such as neutrophil elastase, rather than MMPs. Significantly, the reduced activity of proteases present in chronic wound ulcers was directly correlated with improved healing. In contrast to the above, another inhibitor study examining protease activity in chronic wounds indicated that MMPs rather than serine proteases are crucial in delaying wound repair.[136] Likewise, Weckroth, et al.,[137] found MMPs (gelatinase and collagenase) in chronic leg ulcer exudates and not serine proteases (elastase and cathepsin G). The reported differences observed in the above studies could be due to the heterogeneity of the patients involved in the studies or variations in the components of the chronic wound fluid from wounds at different stages of the healing process.

Chronic wounds of different etiologies may display varied levels of protease activity depending on the underlying pathophysiology. Disordered proteolytic activity is almost certainly associated with the failure of chronic wounds to heal.[137–139] Whatever the precise mechanisms, collectively published data demonstrate that exudate from chronic wounds contains an imbalance of growth factors and pro-inflammatory cytokines, excessively high levels of proteases, and a decreased level of TIMPs. All these factors will contribute to a delay in wound healing.

Likewise, degradation of angiogenic mediators (cytokines) might be an underlying cause in chronic wounds. Drinkwater, et al.,[140] compared wound fluids collected from 16 venous ulcers and acute wound fluids collected from subcutaneous drains in seven patients using VEGF as control. They found that venous ulcer exudates significantly inhibited angiogenesis compared with acute wound (p < 0.002) and VEGF (p = 0.01). Furthermore, among the 16 venous ulcers, five ulcers were slow to heal (> 1 year) and they inhibited angiogenesis more significantly (p = 0.03) than the five rapidly healing (< 3 months) ulcers from the same group.

Analysis of wound exudate presents many technical difficulties, and different studies are unlikely to follow standardized methodologies. This makes it difficult to interpret the data and draw definitive conclusions at this stage.[141] Despite this reservation, it is clear that exudate from chronic wounds is biochemically different to that of acute wounds. However it seems likely that disruption of the subtle balance between the cellular and biochemical components of the microenvironment within a chronic wound may contribute to delayed wound healing.

Wound Bed Preparation and Management of Exudate

Controlling exudate levels is an important aspect of the management of chronic wounds. The concept of wound bed preparation recognizes this but also highlights the fact that chronic wound exudate is interlinked with other barriers to healing, such as necrotic burden and microbial imbalance. Debriding and cleaning the wound is the first step in managing excessive exudate, but long-term management requires the use of modern dressing materials. These are available in a range of absorptive capacities to suit the volume of exudate produced by the wound. If excess exudate results from local edema, compression therapy may be the treatment of choice. Effective management of chronic wound fluid is an essential element of wound bed preparation and will also help to address the issues of cellular dysfunction and biochemical imbalance.[18,33,34,36]

Cellular Dysfunction and Biochemical Imbalance

The process of normal wound healing involves the complex interaction between several cell types, ECM molecules, and soluble mediators, such as growth factors, cytokines, and MMPs. However, in chronic wounds, the ordered cellular and molecular processes that are present in acute wounds are disrupted and incorrectly regulated. These differences, termed cellular dysfunction and biochemical imbalance, respectively, are likely to be of great importance in explaining why the healing process in chronic wounds is impaired.

Fibroblasts play a pivotal and multifaceted role in wound healing ranging from the synthesis of ECM to mediating its remodeling by cytokine and metalloproteinase activity.[142] Fibronectin, synthesized by fibroblasts, promotes keratinocyte migration, and this glycoprotein was shown to be completely degraded in chronic wounds.[27,138] Grinnell and Zhu[27] also demonstrated a correlation between the degradation of fibronectin and the increased levels of the serine protease, neutrophil elastase. Moreover, naturally occurring protease inhibitors, such as a2-macroglobulin and a1-proteinase inhibitor, were shown to be degraded and nonfunctional in chronic wound fluid.[27,64] In summary, degradation of proteins involved in ECM synthesis and increased protease levels, due in part to degradation of their inhibitors, also contribute to delayed wound healing.

Degradation of adhesion proteins in the wound bed may also be an important factor in the inability of chronic wounds to close. When cell adhesion assays were performed on varying chronic venous ulcers, the cell adhesion-promoting activity was significantly reduced, and this correlated with degraded vitronectin and fibronectin.[143] These data may explain why chronic wounds become “stuck” in the inflammatory phase and why impaired keratinocyte migration is seen in the proliferative phase of repair.

Cells within the chronic wound bed may exhibit altered phenotypes, which could contribute to the failure of a wound to heal. Levels of MMPs and their endogenous inhibitors TIMPs are expressed differently in chronic and acute wounds. In stromal cells, MMP-13 is abundantly expressed in chronic wounds but to a much lesser extent in acute wounds. MMP-13 is involved in the degradation of type I and type III collagen in the wound bed, and deregulated expression of this MMP may play a role in the pathogenesis of chronic ulcers.[144] Furthermore, TIMP-3 expression levels were absent from the epidermis of chronic venous ulcers, even though it was abundantly expressed in acute wounds.[145]

Another element pertinent to chronic wounds is the proliferation rate of cells together with their altered morphology.[26,146] In an actively healing wound, fibroblasts are highly proliferative, and if they lose this proliferative capacity, for example by becoming senescent, this would have major implications for wound repair. Fibroblasts taken from the edges of chronic venous leg ulcers are less proliferative than fibroblasts taken from normal healthy skin.146 In addition, fibroblasts from the wound edges were larger and responded poorly to growth factors.[30,146] This suggests that cellular senescence is related to a delay in healing.[147] Since fibroblasts from chronic wounds display signs of senescence,[30,148] this may explain why these cells show a reduced response to PDGF-b and TGF-b in vitro.[29,149] If fibroblasts within chronic wounds have been altered and become senescent, the addition of exogenously applied growth factors may not be sufficient to stimulate proliferation and wound repair.

Conclusion

Wound bed preparation is already changing the way clinicians view the management of chronic wounds. If healthcare professionals and patients are to benefit from optimal use of wound treatments, a greater understanding of the basis of good wound bed preparation is essential.

The therapeutic rationale for the treatment of chronic wounds has for many years been based on the acute wound model. However, the acute model of wound healing, which proceeds through the well-defined steps of inflammation, proliferation, and remodeling, is not completely representative of the chronic nonhealing wound. Wound bed preparation is a practical and evidence-based approach to the management of chronic wounds, since it reassesses the science behind the barriers to healing. By defining what it is that prevents chronic wounds from progressing to wound closure, wound bed preparation provides a clinical strategy that will ultimately lead to the removal of all local barriers to the healing process, so that wound repair can progress normally. Furthermore, since wound bed preparation addresses issues, such as cellular dysfunction and biochemical imbalance, a more integrated approach to wound care can be achieved (Figure 2).

Figure 2

This is a representation of the differences in the microenvironment of the wound bed in healing and nonhealing wounds and the concept of wound bed preparation.

The concept of wound bed preparation provides the means by which chronic wounds can be considered in a new and appropriate manner and allows the clinician to identify and address the barriers to wound healing. Furthermore, wound bed preparation is the basis upon which more effective strategies can be developed to address the reasons why chronic wounds do not heal.