The chronic wound: impaired healing and solutions in the context of wound bed preparation

Blood Cells, Molecules, and Diseases 32 (2004) 88 – 94 www.elsevier.com/locate/ybcmd

The chronic wound: impaired healing and solutions in the context of wound bed preparation Vincent Falanga * Department of Dermatology and Department of Biochemistry, Boston University School of Medicine, Boston, MA 02118, USA Department of Dermatology, Roger Williams Medical Center, Providence, RI 02908, USA Submitted 22 September 2003 (Communicated by M. Lichtman, M.D., 15 October 2003)

Abstract In the past few years, a different paradigm for the understanding and treatment of chronic wounds has emerged. The term used to describe this new context in which failure to heal is viewed is ‘‘wound bed preparation’’. This term is revolutionizing the way we approach chronic wounds, and has allowed chronic wounds to gain independence from established models of acute injury. Within the context of wound bed preparation, impaired healing and solutions to it are being addressed in novel ways. In this report, we make use of the diabetic ulcer as an example of a chronic wound, and emphasize the pathophysiological principles, the cellular and molecular abnormalities, and the solutions offered by the new approaches of gene therapy and stem cells. The emerging view is that chronic wounds are characterized by resident cells that have undergone phenotypic changes that need to be corrected for optimal healing to occur. We have established in animal models and in humans that stem cells have the potential to bring about fundamental changes in the repair process and, ultimately, a ‘‘quantum’’ jump in our therapeutic success. D 2003 Elsevier Inc. All rights reserved. Keywords: Chronic wound; Healing; Diabetic ulcer

Introduction In the field of wound healing, most of the emphasis has been on the mechanisms underlying the normal repair process. Much has been learned about wound healing even in the last few years, given the technical opportunities brought about by molecular science. For example, numerous growth factors, thought to play a role in wound healing, have been isolated, cloned, produced as recombinant molecules, and tested for their ability to accelerate wound closure [1,2]. Another dramatic example is our ability to grow cells in vitro, including what were previously rather fastidious cells such as keratinocytes and microvascular endothelial cells [3,4]. These tissue culture techniques, together with increased understanding and more effective manipulation of extracellular matrix components, have * Department of Dermatology and Skin Surgery, Roger Williams Medical Center, Elmhurst Building, 50 Maude Street, Providence, RI 02908. Fax: +1-401-456-6449. E-mail address: [email protected]. 1079-9796/$ - see front matter D 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bcmd.2003.09.020

helped spawn the field of tissue engineering in wound repair [5,6]. We have also learned some important lessons from fetal wound healing, where scarring is noticeably downregulated or absent [7 –10]. In addition, our understanding of the mechanisms underlying tissue repair is benefiting from transgenic and knockout animal models, which are beginning to point to particular proteins that are critical to healing [11]. The above advances have also brought to the field a rather sophisticated array of new therapeutic products. However, in chronic wounds, the efficacy of many advanced therapeutic agents has been less than what had been predicted from in vitro studies or from animal models and human acute wounds. Several reasons for this discrepancy become apparent when one examines the underlying pathophysiology and complicating features of chronic wounds. In this discussion, we will discuss the phenomenon of impaired healing, its scientific and clinical underpinnings, and how the field of wound healing can benefit from greater recognition of these issues. The clinical emphasis will be on the diabetic ulcer and how these advances in our under-

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standing of barriers to healing can benefit this type of chronic wound. The cellular and molecular abnormalities of chronic wounds will be discussed in the context of wound bed preparation, a new paradigm for our understanding of impaired healing.

Diabetic foot ulcers and pathophysiological principles The cost of diabetic ulcers to the Medicare system in the United States was $1.5 billion in 1995. Amputation is the major driver for costly care in patients with diabetic ulcers [12]. In other countries, like Sweden, where financial data are more easily retrievable, the cost of a single episode of diabetic ulceration has been reported to be $7850, rising to $52,920 if an amputation is required. Clearly, therefore, diabetic ulcers are a serious economic problem, even when one does not consider other parameters related to loss of productivity and quality of life. Additionally, the incidence and prevalence of chronic wounds, including diabetic ulcers, are expected to rise due to the increasing number of elderly individuals. Several publications provide in-depth reviews of diabetic ulcers, their basic pathophysiology, and appropriate treatments [1,13] (Phillips, 2001 #78; Vowden P, 2001 #79). Besides arterial insufficiency, the neuropathy plays a major role in the development of ulceration in diabetic patients. It is extremely important to note that our notions of pathophysiology have important consequences on treatment and the adoption of surgical and other therapeutic modalities. For example, for decades, it was thought that the main problem in diabetes was ‘‘small vessel disease’’, a rather poorly defined term that was offered as an explanation for many of the complications of this disease, including foot ulcers. It is likely that this terminology and pathophysiological point of view may have adversely affected the care of diabetic ulcers for decades, as it introduced a therapeutic nihilism in the approach to these chronic wounds. Therefore, the realization a few years ago that the concept of a true occlusive microangiopathy in the lower extremities of diabetic patients is incorrect or not specific for diabetic patients has brought about a dramatic benefit in how revascularization of the diabetic foot is viewed and practiced [14,15].

Impaired healing In the past, clinicians and scientists have talked about ‘‘failure to heal’’ when referring to chronic wounds [16]. However, this term does not accurately describe what is observed clinically, and it may be preferable to use the term ‘‘impaired healing’’. The fact is that many chronic wounds, including those due to diabetic complications, do heal in an appropriate time frame. For example, with uncomplicated diabetic neuropathic foot ulcers, healing should occur

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relatively unimpeded once off-loading is appropriately practiced [17]. Bacterial burden and biofilms A typical feature of chronic wounds, including diabetic ulcers, is their propensity to become heavily colonized with bacterial, and sometimes fungal, organisms. The fact that these wounds remain open for a prolonged period of time, in addition to other factors such as poor blood flow and hypoxia, plays an important role here [18,19]. A variety of names have been given to this process of colonization, including bioburden or bacterial burden. There remain questions regarding what constitutes an unacceptable level of organisms in the tissues and one that would disrupt the healing process. However, there is evidence that, regardless of the type of bacteria present, a level greater than or equal to 106 organisms per gram of tissue is associated with serious healing impairment [20 – 24]. Federal guidelines developed for the treatment of pressure ulcers indicate the need for quantitative bacteriology, requiring a wound biopsy, if there is continued healing impairment. In practical terms, it makes theoretical and clinical sense to decrease the bacterial burden of wounds. Nevertheless, we know very little about the mechanistic underpinning of impaired healing in the presence of large number of organisms. More recently, there has been increasing interest in the possible presence of biofilms in chronic wounds and their role in impaired healing or even ulcer recurrence. Biofilms represent bacterial colonies surrounded by a protective coat of polysaccharides; such colonies become more easily resistant to the action of antimicrobials [25 – 27]. At the moment, there is no hard evidence for what role biofilms play in chronic wounds. Growth factor ‘‘trapping’’ This concept was first developed in the context of venous ulcers, but has applicability to a variety of chronic wounds. The hypothesis is that certain macromolecules and even growth factors are bound or ‘‘trapped’’ in the tissues, which could result in unavailability or maldistribution of critical mediators, including cytokines [28]. Trapping of growth factors and cytokines, as well as matrix material, however limited, has the potential to cause a cascade of pathogenic abnormalities. For example, in the well-coordinated process of wound healing, disruption of some key mediators could have adverse consequences well downstream. Binding of growth factors by macromolecules that leak into the dermis, such as albumin, fibrinogen, and h-2macroglobulin, may disrupt the healing process [28,29]. Alpha-2-macroglobulin is an established scavenger for growth factors. There is also evidence that transforming growth factor-1 (TGF-h1), a critical multifunctional polypeptide, is bound within the pericapillary fibrin cuffs in the dermis [30].

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Wound fluid and metalloproteinases A major breakthrough of the last 50 years, in terms of how we manage wounds, was the experimental evidence indicating that reepithelization is accelerated when wounds are kept moist [31,32]. This advance has led to the development of a vast array of moisture-retentive dressings that promote ‘‘moist wound healing’’ [33,34]. The experimental evidence for moist wound healing was mainly developed in acute wounds, and its lessons were quickly extrapolated to chronic wounds. Contrary to what had always been conventional wisdom, keeping the wound moist has not resulted in increased infection rates [35 – 37]. However, that remains a fear of many clinicians. It is not entirely clear whether moisture-retentive dressings work mainly by keeping the wound fluid in contact with the wound. One of the reasons for this uncertainty is that wound fluid appears to have distinctly different properties in acute and chronic wounds. For example, it has been shown that fluid collected from acute wounds will stimulate the in vitro proliferation of fibroblasts, keratinocytes, and endothelial cells [38 – 40]. Conversely, fluid obtained from chronic wounds will block cellular proliferation and angiogenesis [39,41] and contains excessive amounts of matrix metalloproteinases (MMPs) [42,43] capable of breaking down critical extracellular matrix proteins, including fibronectin and vitronectin [44]. Undoubtedly, MMPs play a key role in the wound healing [45]. For example, interstitial collagenase (MMP-1) is important for keratinocyte migration [46]. In contrast, it has been suggested that excessive activity (or maldistribution) of other enzymes (MMP-2, MMP-9) contribute to healing impairment [47,48]. Taken together, however, the emerging evidence is that control of MMPs and their localization could have important therapeutic implications, including the enhanced survival of topically applied growth factors. Impaired blood flow and hypoxia Ischemia will lead to tissue necrosis, and that will often result in wounds that are more easily compromised by infection. There is a substantial body of data indicating that low levels of oxygen tension as measured at the skin surface (transcutaneous oxygen measurements or TcPO2) correlate with inability to heal [49 – 51]. These data are most relevant in the treatment of diabetic ulcers, and can often guide therapy and even the level of amputation. However, it should be noted that ischemia is not the same as hypoxia. Interestingly, from a biological standpoint, low levels of oxygen tension can stimulate fibroblast proliferation and clonal growth and can actually enhance the transcription and synthesis of several growth factors [52 –55]. However, there is also evidence that exposure to hyperbaric oxygen can increase the production of VEGF [56]. It is possible that low oxygen tension can serve as a potent initial stimulus after injury but that prolonged hypoxia, as seen in chronic

wounds, can lead to several abnormalities including scarring and fibrosis [57]. The pathophysiological status of the wound and phenotypic alteration of wound cells The normal process of wound repair goes through welldefined stages that are well studied. However, as stated, chronic wounds do not seem to have defined time frames for healing. This is apparent from both clinical and the pathophysiological standpoint. It has been stated that the diabetic ulcer is ‘‘stuck’’ in the proliferative phase of wound repair. Indeed, there is evidence that the accumulation and remodeling of diabetic foot ulcers are impaired with respect to certain matrix proteins, including fibronectin [58]. There is also increasing evidence that the resident cells of chronic wounds have undergone phenotypic changes that impair their capacity for proliferation and movement. To what extent this is due to cellular senescence is unknown, but the response of diabetic ulcer fibroblasts to growth factors seems to be either impaired or requiring a sequence of growth factors [59,60]. Similar observations have been made in other types of chronic wounds. For example, it has been reported that fibroblasts from venous and pressure ulcers are senescent, show diminished ability to proliferate [61 – 63], and that their decreased proliferative capacity correlates with failure to heal [61 – 64] and respond to platelet-derived growth factor (PDGF) [65]. At the moment, it is not known whether this phenotypic abnormality of wound cells is only observed in vitro and whether it plays a role in impaired healing.

Barriers to healing: solutions An advantage of thinking more comprehensively about the pathogenic abnormalities that impair healing is that one can view existing therapies and procedures in a different light, and with different justifications. For example, the process of surgical debridement of diabetic foot ulcers becomes more than simply removing necrotic tissue; at the same time, one is also removing the excessive bacterial burden and, possibly, the phenotypically abnormal cells that may be present in and around the wound. Another example is the removal edema, which is also critical in the management of diabetic ulcers. Edema removal decreases the chronic wound fluid that has been shown to be deleterious to resident cells and that may enhance bacterial colonization. Therefore, existing therapies can be integrated better with pathophysiological principles. A different paradigm for chronic wounds: wound bed preparation Advances in the treatment of chronic wounds have relied heavily on concepts developed for acute wounds. In the last

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few years, however, a new paradigm for addressing chronic wounds is emerging. The concept is much more important than the terminology in this case. For example, the term ‘‘wound bed preparation’’ has been used, trying to emphasize the importance of optimizing the appearance and readiness of the wound bed [66]. The main point, however, is that this approach represents a way for chronic wounds to gain independence from models of acute injury. Once appropriate steps have been taken to optimize the wound bed, then the normal endogenous process of wound healing is going to be facilitated. It is important to note that wound bed preparation is more than debridement alone, but rather a very comprehensive approach aimed at reducing edema and exudate, eliminating or reducing the bacterial burden and, importantly, correcting the abnormalities discussed earlier as contributing to impaired healing. There are both basic and more advanced approaches to wound bed preparation and optimization. Basic aspects, as with compromised acute wounds, include debridement, infection control, edema removal, and surgical correction of underlying defect [67,68]. More advanced aspects, for which we may not have all the answers yet, include attempts at dermal reconstitution, as with the use of biological agents. Recently, an international advisory panel has recommended the introduction of the TIME concept to crystallize our thinking about wound bed preparation. TIME is an acronym for the following: T = tissue buildup; I = infection and inflammation; M = matrix material; E = epithelialization. We will use these concepts throughout as we discuss impaired healing and therapeutic solutions for diabetic ulcers. Growth factors Over the last two decades, several recombinant growth factors have been tested for their ability to accelerate the healing of chronic wounds. Among others, some promising results have been obtained with the use of epidermal growth factor (EGF) [69] and keratinocyte growth factor-2 [70] for venous ulcers, fibroblast growth factor (FGF) [71], and platelet-derived growth factor (PDGF) [72,73] for pressure ulcers. However, for the most part, the only topically applied growth factor that is commercially approved for use is PDGF. In randomized controlled clinical studies, PDGF has been shown to accelerate the healing of neuropathic diabetic foot ulcers by approximately 15% [74 – 76]. One may wonder why we do not have a greater number of growth factors approved for clinical use, and why the results of clinical trials have not been of greater magnitude, as we might have predicted from preclinical data. A number of explanations can be given for this, and perhaps all of them apply. It has been hypothesized that the dosage and mode of delivery of topically applied growth factors may have been wrong, or that actually combinations of growth factors are required to bring about a better response [77 – 79]. It is also possible, however, that closer attention should

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have been paid to appropriately preparing the chronic wound before treatment with the growth factor being tested in clinical trials [66]. Notably, there is evidence that the aggressive approach to surgical debridement in the initial PDGF trial for diabetic neuropathic ulcers seems to have worked synergistically with the application of the growth factor [75]. It is important to note that the treatment of chronic wounds has been evolving in the last few years, and it might be argued that the increased number of randomized clinical trials for chronic wounds has improved standard wound care. If this is so, it is expected that in the future new products will need to perform much better than control to show efficacy. Bioengineered skin A number of bioengineered skin products or skin equivalents have become available for the treatment of acute and chronic wounds, as well as for burns. Since the initial use of keratinocyte sheets [4,80,81], several more complex constructs have been developed and tested in human wounds. Skin equivalents may contain living cells, such as fibroblasts or keratinocytes or both [6,82 – 84], while others are made of acellular materials or extracts of living cells [85,86]. Some allogeneic constructs consisting of living cells derived from neonatal foreskin have been shown to accelerate the healing of neuropathic diabetic foot ulcers in randomized controlled trials, and are available for clinical use [87,88]. The clinical effect of these constructs is between 15% and 20% over conventional ‘‘control’’ therapy. One of the important arguments relates to what constitutes an appropriate control. In US trials, saline-soaked gauze and off-loading have been accepted by the Food and Drug Administration as the control. However, the methods for off-loading differ in many countries, and the wound dressings to be used are also subject to controversy. As a result, in spite of notable successes with the use of bioengineered skin in the treatment of diabetic neuropathic foot ulcers, acceptance of this type of therapy by clinicians will likely be less than desirable. Bioengineered skin may work by delivering living cells, which are said to be ‘‘smart’’ in engineering terms, and thus capable of adapting to their environment. There is evidence that some of the living constructs are able to release growth factors and cytokines [89,90], but this cannot yet be interpreted as being their mechanism of action. It should be noted that some of these allogeneic constructs do not survive for more than a few weeks when placed in a chronic wound [91]. Gene therapy For some time now, there has been ample technology for introducing certain genes into wounds by a variety of physical means or biological vectors, including viruses. There are ex vivo approaches, where cells may be manip-

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ulated before reintroduction into the wound, to more direct in vivo techniques, which may rely on simple injection or the use of the gene gun [92 –94]. Gene therapy as a whole is a very active area of research. As of 1999, 320 clinical protocols for gene therapy had been submitted to regulatory bodies around the world [95]. Inability to achieve stable and prolonged expression of a gene product, which has been a problem in the gene therapy treatment of systemic conditions, can actually be an advantage in the context of nonhealing wounds, where only transient expression may be required [93]. Most of the work with gene therapy of wounds has been done in experimental animal models [96]. However, there are promising indications that certain approaches may work in human wounds. For example, the introduction of naked plasmid DNA encoding the gene for vascular endothelial growth factor (VEGF) has been reported to enhance healing and angiogenesis in selected patients with ulcers from arterial insufficiency [97]. Undoubtedly, the area of gene therapy for chronic human wounds will become very active in the next few years. The introduction of the gene, rather than its product, that is, a growth factor, is seen as a less expensive and potentially more efficient delivery method.

sion criteria in those trials. Such purely neuropathic ulcers are relatively straightforward, and many clinicians believe that they can be effectively treated with sound surgical debridement and off-loading. While it may be argued that accelerating the healing of even these relatively simple ulcers may prevent complications from infection, more needs to be done to show cost-effectiveness to our society as a whole. Still, considerable progress has been made, and many therapeutic approaches, including improved standard care, are now available. It is hoped that continued advances will come about, which, when combined with basic medical and surgical approaches, will accelerate healing of chronic wounds to an extent that is still not possible with present therapeutic agents.

Acknowledgments This paper is based on a presentation at a Focused Workshop on ‘‘Stem Cell Plasticity’’ held in Providence, RI, April 8 – 11, 2003, sponsored by The Leukemia and Lymphoma Society, Roger Williams Medical Center, and the University of Nevada, Reno. This work was supported by NIH grants AR42936 and AR46557.

Stem cell therapy An extension of the hypothesis that cell therapy may be required to recondition chronic wounds and to accelerate their healing is the notion that stem cells might perhaps offer greater advantages. Pluripotential stem cells are capable of differentiating into a variety of cell types, including fibroblasts, endothelial cells, and keratinocytes, which are critical cellular components required for healing. Although the subject of some controversy, pluripotential mesenchymal stem cells may be present in the bone marrow [98]. A recent uncontrolled report suggests that direct application of autologous bone marrow and its cultured cells may accelerate the healing of nonhealing chronic wounds [99]. This type of report needs to be confirmed in a larger controlled trial. Nevertheless, when considering the pathophysiological abnormalities present in chronic wounds, there is the potential that stem cells may reconstitute dermal, vascular, and other components required for optimal healing. Summary A rational strategy for addressing diabetic foot ulcers will likely require greater understanding of the clinical factors involved as well as the pathophysiological components that underlie their impaired healing. Greater therapeutic boldness is required as well. For example, existing advanced therapeutic products tested in diabetic foot ulcers, such as growth factors and skin equivalents, have focused entirely on neuropathic ulcers of the metatarsal heads; arterial insufficiency and the more complex heel ulcers have been exclu-

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