Wound Bed Preparation and the Role of Enzymes: A Case for Multiple Actions of Therapeutic Agents

Introduction

  We are presently in an exciting period of time in the approach to chronic wounds. One can probably identify three distinct phases or revolutions in our therapeutic strategies over the years. The first revolution began almost two decades ago with the realization that moist wound healing principles were applicable to the treatment of chronic wounds. Since then, we have developed a variety of dressings capable of providing optimal coverage for wounds in different situations and actually stimulating wound repair. The second revolution, still ongoing, began about ten years ago with the successful testing of advanced technological products, such as topically applied growth factors and bioengineered skin. Finally, the third revolution began a few years ago with the introduction of the concept of wound bed preparation, which allows us to break down into individual components the critical steps involved in optimizing the clinical aspects and the microenvironment of chronic wounds.¹ This review will discuss wound bed preparation with an emphasis on the principle that certain therapeutic agents, some long established, have more than one critical role in the repair process.   It might be worthwhile to first provide some perspective on the topic of chronic wounds. From an evolutionary standpoint, humans and other organisms were not destined to have chronic wounds. There are many responses to acute injury that are a product of evolutionary forces. These responses range from the relatively simple, i.e., coagulation to limit blood loss, to the very complex, i.e., regeneration of a limb in some animals. However, as far as we can tell, there are no known evolutionary steps that have evolved to handle chronic wounds. Humans were not supposed to live long enough to develop venous ulcers, pressure ulcers, or wounds that become chronic after complex surgical procedures. Simply stated, there has never been a survival advantage for handling chronic wounds. It is partly for these reasons that much of what we have learned about acute injury does not apply to chronic wounds from both a physiologic/pathogenic standpoint as well as from the therapeutic approach.   With increasing realization that the approach to chronic wounds should be more ”tailor made” for the wounds and not rely entirely on what we know about acute wounds, a new frame of reference has emerged in the last two or three years. The term “wound bed preparation” refers to this new frame of reference, and this concept is having a very dramatic impact on how we approach chronic wounds and how we view new and established therapies.¹ Cynically, some would argue that the concept of wound bed preparation is too simple, that there is nothing new about it, or that what we are preparing for is unclear. However, this term is being successful in altering the way we manage chronic wounds and in giving chronic wounds the independence they have long needed from models of acute injury. Wound bed preparation as a strategy is allowing us to break into individual components various aspects of wound care, while at the same time maintaining a global view of what we wish to achieve. In this focused review on certain aspects of wound bed preparation, we will first briefly discuss wound bed preparation in general and its main components. We will then address a class of therapeutic agents, in this case enzymes, as a case study for how this novel approach to chronic wounds introduces new elements in our view of established treatments. We do so because we see a number of opportunities in redefining therapies in the context of wound bed preparation. The emphasis on wound bed preparation allows us to better define the steps involved in the management of chronic wounds and in doing so it sheds additional light on what the clinical problems are and on their basic science underpinning.   Therapeutic agents that were mainly thought to accomplish debridement may now also be viewed as needed in inducing a targeted and beneficial inflammatory response, in facilitating angiogenesis, or in aiding keratinocyte migration.

Overview of Wound Bed Preparation

  Wound bed preparation can be defined as the global management of the wound to accelerate endogenous healing or to facilitate the effectiveness of other therapeutic measures. A critical point is the differentiation of wound bed preparation from wound debridement alone. Indeed, if one starts with the same perspective used for acute wounds, a common error is to view wound bed preparation as the same as wound debridement. In acute wounds, debridement is a good way to remove necrotic tissue and bacteria. After that is done, one should have a clean wound that can heal with relative ease. This is not the case for chronic wounds, where much more than debridement needs to be addressed for optimal results. For one, defining the necrotic material in chronic wounds is not so easy. Chronic wounds have what we have termed “necrotic burden,” consisting of both necrotic tissue and exudate. Exudate from chronic wounds has been shown to inhibit the proliferation and function of key resident cells and to contain proteases that break down extracellular matrix proteins.^2–4 Chronic wounds can be intensely inflammatory, e.g., venous ulcers, and thus produce substantial amounts of exudate that interfere with healing or with the effectiveness of therapeutic products, such as growth factors and bioengineered skin. So, in the context of wound bed preparation, not only do we need to concern ourselves with removal of actual eschars and frankly nonviable tissue, but also with the exudative component.⁵ Moreover, there is increasing realization that the resident cells in chronic wounds, e.g., fibroblasts and keratinocytes, may be phenotypically altered and no longer responsive to certain signals, including growth factors.⁶    Figure 1 shows some of the essential abnormalities that characterize chronic wounds as well as established or potentially corrective treatment modalities or approaches. As Figure 1 indicates, there is a spectrum of pathogenic abnormalities ranging from basic issues to more complex ones. Some abnormalities, like the presence of necrotic tissue and hemodynamic problems, are very basic and have established treatments. Other pathogenic issues, such as the presence of a cellular burden comprised of phenotypically altered cells, are quite complex and still poorly understood. Figure 1 can be viewed as a summary of what is involved in wound bed preparation. We do not have yet a complete understanding of the critical abnormalities and of the most appropriate corrective measures. Also, as shown by the dashed lines between each compartment in Figure 1, there is probably considerable overlap and cross-influences with regard to both the pathogenic abnormalities and treatment strategies. Still, the exciting aspect of Figure 1 is that it is completely different from the type of diagram commonly used for acute wounds, where the process of healing is depicted as proceeding unimpeded through overlapping but well described steps of coagulation, inflammation, proliferation, and remodeling.   In the context of wound bed preparation, some seemingly basic every day procedures acquire new significance, and this same line of thinking will be explored further when we discuss enzymatic treatment later on. Figure 2 shows surgical debridement being carried out in a diabetic neuropathic foot ulcer. The wound is being excised, and in the process, the wound bed is being removed as well as the ulcer’s edge and surrounding callus. Simplistically, in this case debridement is removing the necrotic tissue and decreasing the bacterial burden. However, there is increasing awareness that much more is being accomplished with this type of surgical intervention. Importantly, we may be removing some of the senescent cells that are phenotypically altered—the so-called cellular burden. There is evidence that fibroblasts cultured from chronic wounds are senescent, show decreased proliferative rates, and may be unresponsive to a number of important cytokines, including transforming growth factor-beta1 (TGF-b1) and platelet-derived growth factor (PDGF).^7–10   Also, previous work has shown that the pattern and time frame of certain events in chronic wounds are altered with respect to acute wounds. For example, it has been reported that certain extracellular matrix components, including fibronectin, continue to accumulate within the wound for up to 12 months in the life of a nonhealing diabetic foot ulcer.¹¹ Clearly, this is different from the short proliferative phase we have learned about in acute wounds. Hence, the idea is emerging that some of these chronic wounds are stuck in certain phases of the repair process. Interestingly, it has been stated that the sort of debridement shown in Figure 2 turns a chronic wound in an acute wound. We think this is too simplistic, since debridement does not really remove the underlying problems leading to the ulceration. Rather, we like to think that debridement places an acute wound in a chronic wound. According to this line of thinking, we create an injury response that, at least for a period of time, may recondition and be beneficial to the chronic wound.

Overview of Enzymes

   As stated earlier in the discussion, within the context of wound bed preparation one has the opportunity to reappraise certain therapeutic agents and find new rationale for them as well as new roles. This reappraisal will be an ongoing process for many of the therapies used today. In this review, and as a proof of principle, we have focused on enzymatic agents. It just so happens that enzyme-containing products, previously regarded mainly as debriding agents, have acquired new significance within the context of wound bed preparation. While debridement will certainly remain one of debriding agents’ critical roles, their other properties and our understanding of what is needed for optimizing wound care preparation has the potential to greatly expand their therapeutic benefits. Before we turn our attention to enzymatic agents, it might be worthwhile to first discuss enzymes in general and some of the nomenclature that has evolved.    Table 1 shows an accepted nomenclature for proteases, which are a very extensive family of enzymes.¹² There is potential for several of these agents to be used in the treatment of wounds, but, as we shall see, only two of them, papain and collagenase, have had a substantial and continuous role for this indication. Briefly, a more accepted term for proteases is peptidases, and the whole group of enzymes can be subdivided into exopeptidases, which remove amino acids from the ends (either the N- or C-terminus) of proteins, and endopeptidases (or proteinases), which cleave bonds within protein. We will restrict our discussion to proteinases, which, as indicated in Table 1, comprise four groups. The serine proteinases have very well known members in chymotrypsin, trypsin, plasmin, plasminogen activators, and leukocyte elastases. In general, serine proteinases are potent enzymes with a broad range of catalytic activity and are readily available in tissues when needed. The second group of proteinases is the cysteine proteinases, and certainly papain is the better known member of this group and the best studied. The aspartic proteinases have a number of well-known enzymes, such a renin and pepsin. Finally, the metalloproteinases (MMPs) are well known in the field of wound healing because of their properties of being able to cleave collagens and other extracellular matrix components.¹³ MMPs tend to have a very well-defined range of catalytic activity and are generally produced on demand, e.g., after wounding. These proteinases also participate in normal regulated tissue processes, such as morphogenesis during development. For the purpose of our discussion regarding collagenases, the best known of the MMPs are MMP1 (fibroblast collagenase), MMP2 (gelatinase A), MMP8 (neutrophil collagenase), and MMP9 (gelatinase B). Many of these peptidases require zinc or calcium cations (or both) for their activity. In some cases, cobalt or nickel can be substituted for zinc without loss of activity. There is considerable overlap in substrate specificity among the MMPs, and so Table 1 is an oversimplification. Besides their ability to cleave different collagen types (collagenases) and denatured collagen (gelatinases), some MMPs can cleave other substrates, including elastin (MMP12), fibronectin and proteoglycans (MMP10), and laminin (MMP3).    Proteinases are critical to the repair process. Removal of the necrotic tissue is essential to reduce the bacterial burden, which in turn decreases the amount of exudate produced. However, as we will try to illustrate later by using collagenase as proof of principle, proteinase activity in chronic wounds is not only useful for the purpose of debridement but also for more fundamental aspects of cell migration. In response to wounding, keratinocytes migrate from the edge of the wound and assume a collagenolytic phenotype.^14,15 Indeed, it has been shown that collagenase expression is rapidly induced in wound-edge keratinocytes, persists during healing, and stops at reepithelialization.¹⁶ Moreover, the activity of collagenase is required for keratinocyte migration on a type I collagen matrix.¹⁷ Other work using human burn wounds indicates that both collagenase and its inhibitor, tissue inhibitor of metalloproteinases (TIMP), are expressed during wound repair.¹⁸ Therefore, the situation is quite complex and not fully elucidated. The relationships between proteinase activity and keratinocyte migration appear to be very tightly regulated, and it is clear that the type of proteinase expressed is critical. For example, in experimental studies using injured cornea, it was shown that failure to reepithelialize correlated with the amount of gelatinases, but not with the increase in collagenase and stromelysin.¹⁹ Also, one has to be careful in reviewing data where an entirely artificial situation is created. Thus, transgenic mice overexpressing MMP1 were found to have delayed wound closure.²⁰ This is another instance, however, where most of the information comes from acute wounds. As stated earlier, much more needs to be done to understand the situation in chronic wounds.

Enzymatic Products Used in Wounds

   Having described proteases in general, we will now focus on those enzymes that are available to clinicians for use in chronic wounds.^21–23 Again, the discussion is meant to stimulate our thinking in terms of other biological events that might be targets for enzymatic treatment, rather than simply debriding actions, which are already well established. There may also be room for thinking of debridement from a different perspective, i.e., debridement used in wound healing maintenance.    The concept of using proteolytic enzymes to digest necrotic tissue as an adjunct in the treatment of complex wounds is rather old and probably stems from observing the ageless healing techniques of natives in tropical countries. As an example, for wound debridement, these natives seem to have utilized the papain-rich material obtained by scratching the skin of the green fruit of the papaw tree (Carica papaya). Since then, papain has long been used for the treatment of several skin conditions. As we shall see later, the nonspecific activity of papain offers advantages and disadvantages, and the agent may be less than ideal for what we propose, i.e., a maintenance debridement approach. The search has always been focused on finding more specific enzymatic agents. In time, clinicians and scientists began to recognize that the secretory products of certain strains of hemolytic streptococci could be useful in lysing the major insoluble constituents of wound fluid, fibrin and deoxyribonucleoprotein. The once commercially available preparation of streptokinase and streptodornase was only partially purified and contained a number of other streptococcal enzymes, such as ribonuclease, hyaluronidase, and nucleotidase and nucleosidase. This mixture did not contain proteolytic enzymes in the conventional sense, and much of its activity was probably due to streptodornase, which could rapidly reduce the viscosity of purulent exudate. At one time, preparations containing trypsin or chymotrypsin were also available commercially (Table 1). Trypsin directly hydrolyzes a large number of naturally occurring proteins (nonspecific). Chymotrypsin acts upon different bonds in proteins than does trypsin, but its spectrum of activity is similar.¹² Trypsin and chymotrypsin preparations were prepared from pancreatic sources (usually beef) but are no longer available for the treatment of wounds. Another proteolytic enzyme combination obtained from pancreatic tissue, and comprising fibrinolysin (plasmin) and deoxyribonuclease, was commercially available up to a few years ago, when a controlled, randomized study could not demonstrate its effectiveness.²⁴ This underscores another problem with some of these complex enzyme preparations composed of more than one presumably active or facilitating agent—it is often difficult to know whether the combination is truly effective. One of the difficulties encountered by manufacturers of enzymatic products used as debriding agents is that, in recent years, regulatory agencies have insisted that debridement alone cannot be used as an endpoint but should be tied to wound closure. This regulatory view, of course, renders testing much more difficult and expensive. We are certain that this requirement has prevented the manufacturing and testing of other useful enzymatic products for wounds.    At this point in time two enzymatic preparations are rather prominent in terms of their use in chronic wounds: papain-urea based combinations and collagenase. For practical reasons, we will, therefore, focus on these two types of enzymatic approaches to chronic wounds with the purpose of advancing our view that much more than traditional debridement can be obtained by the use of topically applied proteolytic enzymes. The two preparations are quite different in terms of number of acting agents, specificity, and overall effects.    Papain-urea–based combinations. A well-known and widely used enzymatic system is the papain-urea combination.^21–23 In this system, papain is used to attack and break down any protein containing cysteine residues. This property of papain renders the combination quite nonselective because most proteins, including growth factors, contain cysteine residues. Collagen contains no cysteine residues and is thus unaffected by papain.²⁵ The urea component of the most widely used of these combinations (Accuzyme®, Healthpoint Ltd., Ft. Worth, Texas) will also attack a wide variety of proteins. However, urea’s role in this enzymatic combination is to facilitate the proteolytic action of papain by altering the three-dimensional structure of proteins and disrupting their hydrogen bonds, as well as exposing by solvent action the activators of papain. Urea also plays a role in the reduction of disulfide bridges; as the disulfide bridges are reduced, cysteine residues become exposed and are, therefore, more susceptible to the action of papain.²⁶ It should be noted that the combination of papain and urea is probably twice as effective in protein digestion as papain alone.²⁷ Also, and this may be applicable to other enzyme preparations, hydrogen peroxide can block the effect of papain-urea preparations, as can other commonly used treatments and agents for chronic wounds, such as silver sulfadiazine, gentamicin, and alcohol-based products.    An advantage of the papain-urea combination may be nonspecific bulk debridement within a broad pH range (3.0–12.0). However, perhaps because of the nonselective feature of this enzymatic preparation, a prominent inflammatory response is associated with its use in chronic wounds. This inflammatory response, together with breakdown of still viable components of the wound bed, is perhaps the reason for the considerable pain often associated with the use of these agents. To remedy this situation, another approach first used in the 1950s has been the modification of the papain-urea combination by the addition of chlorophyllin.^26,28 This extra ingredient is an antiagglutinin, and its mechanism of action is thought to be prevention of agglutinated erythrocytes, which may increase thrombus formation and fibrin deposition and plugging of capillaries and lymphatic vessels.^28,29 The effect of chlorophyllin on viable tissue is not known, but it is felt that this additional ingredient in the final combination of papain-urea (Panafil®, Healthpoint Ltd., Ft. Worth, Texas) has no detrimental effect and does not increase pain. The papain-urea preparations have been used clinically for decades, especially in pressure ulcers. The available literature indicates that these debriding systems are effective when properly used,^26,28–31 especially if one keeps in mind that they cannot substitute for surgical debridement when that is required.    The question at this point is: What role do these papain-urea–based combinations play in the new world of wound bed preparation? In summary, the nonselective features of these combinations offer advantages and disadvantages. Bulk and quick debridement, without having to worry about affecting viable tissue, might be an advantage in certain situations, particularly when the affected area is insensate and thus not able to experience the pain associated with this preparation. The addition of the chlorophyllin may have improved the product by reducing pain. There have been concerns that these papain-based enzyme preparations can destroy locally active growth factors, such as PDGF.³² In experimental wounds in animals, the papain-urea combination has been shown to be quite effective for debridement.³³ However, in both experimental and human burns, these preparations may behave too aggressively, both in terms of affecting viable tissue and in causing pain.^34,35 Later, we will discuss whether there are opportunities for these preparations other than debridement of frank necrotic tissue.    Collagenase preparations. Collagenase is another well known and established enzyme preparation used for debridement. Its development as a debriding agent as well as for other applications came to a peak in the early 1970s.³⁶ The commercially available preparation of collagenase (Collagenase Santyl®, Smith and Nephew Inc., Largo, Florida) is derived from bacteria (Clostridium histolyticum). Collagenase is a water-soluble proteinase that specifically attacks and breaks down collagen.^37,38 Collagenase is reported to be most effective in a pH range of 6 to 8. It has been shown^40,43 that collagenase can hydrolyze native collagen and thereby facilitate rapid debridement and healing of chronic wounds. The mechanism of action of collagenase is to degrade collagen and convert it to gelatin, upon which less specific enzymes can then act. However, until collagenase cleaves collagen, no other enzyme is capable of breaking it down. An interesting observation is that the collagenase preparation may be selective for nonviable collagen. This effect needs to be studied further, but it is thought that viable collagen is surrounded and protected by mucopolysaccharide sheaths.³⁹ How does collagenase enter the necrotic tissue and thus aid in debridement? One hypothesis is that collagenase may cleave the collagen molecules at the boundary of the necrotic tissue, thus freeing up the necrotic tissue from the wound. Thus, it has been shown that necrotic tissue is anchored to the wound by strands of undenatured collagen.⁴⁰ Until these fibers are severed, debridement cannot take place. This attractive explanation may be applicable to other debriding agents, for it is difficult to understand how these topically applied agents can effectively penetrate thick eschars and other necrotic areas of the wound.    Collagenase has been found to be remarkably gentle on viable cells. For example, cell suspensions prepared with collagenase (this enzyme is widely used in tissue culture for this purpose), then stored at low temperature, were found equal to trypsinized cells in their viability and growth. Similarly, collagenase can be used as a permanent ingredient of culture media without loss of cell viability.⁴¹ In more recent work, the addition of collagenase derived from Clostridium histolyticum to keratinocyte cultures enhanced their proliferation and migration up to 10-fold.³⁷ Figure 3 is a diagrammatic representation of how collagenase might work, both in terms of debridement and in terms of stimulating other aspects of wound bed preparation. Figure 3 suggests that some potentially underestimated effects of collagenase, such as angiogenesis and epithelialization, occur at the same time wound debridement is being accomplished by this enzyme. As is the case with the papain-based debriding systems, there is considerable published information detailing the effectiveness of collagenase for wound debridement for all types of wounds.^{36,38,42–45}

Wound Bed Preparation and Enzymes

   As we examine the established properties of enzymatic agents, their potential capabilities for other aspects of wound bed preparation begin to emerge. Table 2 outlines the areas of opportunity for papain-based and collagenase enzymatic systems within the new scenario of wound bed preparation. As indicated earlier, both enzymatic systems have a long history of effectiveness in clinical practice. There are some indications that papain-based preparations may work faster but with much more pain, although comparative studies are few and consisting of small numbers of patients.³¹ In some clinical situations, the pain has been proven to be unacceptable, such as in shallow wounds of split-thickness burns.³⁵ Also, the non-selective feature of papain-based preparation may exaggerate the debridement effects.³⁴ We believe that clinicians need to use these agents with proper knowledge of their use and side effects. There are situations where papain-urea preparations may be quite useful, particularly in patients with deep pressure ulcers and loss of sensation. On the other hand, collagenase may work a little slower but is a more selective enzyme system. As Table 2 suggests, collagenase is more likely to be useful for a very prolonged period of time in the wound in what we call maintenance debridement.    The concept of maintenance debridement is an interesting one and is the result of the emphasis on wound bed preparation. We have generally thought of debridement sessions as single events in specified timeframes. However, as we approach wound bed preparation in a more global sense, cognizant of the plethora of events that are occurring at all times, we become aware of new opportunities for enzymes. For example, we think of necrotic tissue as a clinically visible component of the wound, which in turn leads us to think about debridement as a single event or single phase of the healing process. There is an initial debridement phase to be sure, but it may need to be followed by a maintenance phase to keep the ongoing necrotic tissue from accumulating. Programmed cell death (apoptosis) is a constantly occurring process in wounds, and periods of adequate blood flow or decreased edema cycle with periods of borderline ischemia (i.e., from pressure) and increasing edema. What we are proposing is that necrotic material continues to accumulate and that it needs to be removed. Having agents that can be used for extended periods of time, within the context of maintenance debridement, could be very beneficial.    As indicated by other features of the enzymes outlined in this review, the opportunity is real for these preparations to directly help other components of tissue repair in the context of wound bed preparation. In some cases, there may need to be modifications of existing enzymatic agents if they prove to be too harsh for continuous use. For example, Table 2 suggests that both papain-urea–based and collagenase preparations can stimulate granulation tissue and possibly angiogenesis. Moreover, as we have previously discussed, there is considerable rationale for wanting to use collagenase in wounds for the purpose of enhancing or accelerating reepithelialization.^4,37 Figure 3 shows an ideal scenario in which collagenase is used as a possible “pluripotential” wound bed preparation agent. In Figure 3, we see collagenase cutting collagen at the border of viable and necrotic tissue, thus freeing up the necrotic plug for easy removal. At the same time, the presence of exogenously applied collagenase may directly stimulate the process of reepithelialization of the wound. The bottom panel shows that the process of reepithelialization, sustained by angiogenesis, is well advanced after debridement has been accomplished.

Conclusions

   Sometimes, new terms and new concepts can lead us to think in different ways about what we have long regarded as familiar and established notions. In this review, we suggest that wound bed preparation, as a new concept, will likely lead us to reevaluate emerging agents as well as established treatments for chronic wounds. We will ask whether the assigned role of our therapies and their effectiveness are justified and supported by evidence. Armed with greater understanding of what chronic wounds need, we will also start asking whether the properties of certain therapeutic agents support additional and innovative uses. Here we have used enzymatic preparations as a case in point or proof of principle that this type of approach could work. We could have chosen other existing therapies to ask similar questions. Several therapies come to mind in this context. For example, compression therapy of venous ulcers is likely to do a lot more for wound bed preparation than just compressing the leg and may help in edema removal. It might be that compression of tissues leads to different effects on wound bed preparations, such as in stimulating angiogenesis and epithelialization. If that is the case, we would then start thinking of different ways of applying compression and different cycles of compression. Another possibility would be that wound dressings, with their semiocclusive nature, might help wound bed preparation when applied intermittently. It was perhaps logical to use them from the beginning to the end in acute wounds. But is it equally logical to use them that way in chronic wounds? These are just examples, of course. The main purpose of our review is to suggest that we must remain alert to the concept of wound bed preparation and start reevaluating even our commonly used therapeutic agents for opportunities to explore their other properties.

References

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