15 Preparation of the Wound Bed of the Diabetic Foot Ulcer

From: The Diabetic Foot, Second Edition

Edited by: A. Veves, J. M. Giurini, and F. W. LoGerfo © Humana Press Inc., Totowa, NJ

299

Preparation of the Wound Bed of the Diabetic Foot Ulcer

Vincent Falanga, MD , FACP

INTRODUCTION

Over the last few years, substantial advances have been made in our understanding of diabetic foot ulcers, the importance of thorough surgical debridement, and how this standard therapeutic modality impacts on wound bed preparation. Importantly, wound bed preparation is revolutionizing the way we approach nonhealing or difficult to heal wounds. Much of what we do clinically, from elimination of bacterial burden, to debridement, and to the use of new technologies to heal diabetic foot ulcers, can now be seen as helping wound bed preparation and facilitating the process of healing. From a therapeutic standpoint, at least in the United States, large multicenter clinical trials have led to the regulatory approval of topically applied platelet-derived growth factor (PDGF)-BB (Regranex, Ortho McNeill, NJ) and bioengineered skin (Apligraf, Organo- genesis, Canton, MA; Dermagraft, Smith & Nephew, Largo, FL), and have dramatically increased the number of available therapeutic options. However, not to be forgotten are advances that these and other clinical trials have brought to the standard of care for treating neuropathic diabetic foot ulcers. Indeed, these improvements in standards of care for diabetic foot ulcers have raised the bar for proving the effectiveness of new treatments. Stated differently, it has become harder to prove the effectiveness of new therapeutic agents. Thus, from now on we may be looking for “quantum” jumps in therapeutic efficacy in the treatment of diabetic foot ulcers.

The aim of this review is to examine certain basic science factors involved in the development of diabetic foot ulcers, critical therapeutic advances, such as topically applied growth factors and dermal (Dermagraft) or bilayered (Apligraf) bioengineered skin, and to discuss these topics in the context of wound bed preparation (discussed later). We will focus on the role of debridement, how it affects wound bed preparation, and how the two are intimately linked.

OVERVIEW OF DEBRIDEMENT AND WOUND BED PREPARATION

It was always assumed that clinicians would uniformly perform surgical debridement

as part of a unified approach to the care of chronic wounds. However, this assumption

may have been wrong, and the meaning of debridement was variable. The extent of

debridement, too, did not reflect the reality of office practice and clinical trials. This is not to say that competent clinicians did not perform debridement of eschars and frankly necrotic tissue in compromised diabetic foot ulcers. However, it has become increasingly clear that regular debridement, to remove the callus surrounding the wound and the fibrinous wound bed (not necessarily frankly necrotic), may not have been widely practiced (1,2). We have called this more thorough and regular approach “maintenance debridement”

for diabetic foot ulcers, in the context of wound bed preparation (3–5). To what extent not following this “standard of care” may have played a role in the failure of showing effectiveness of certain therapeutic products in the past is unclear. The initial pilot study of topically applied PDGF changed the way we conduct clinical trials for diabetic foot ulcers. Indeed, the effectiveness of PDGF in those trials seems to have been highly dependent on concomitant and thorough maintenance debridement, and actually worked synergistically with it. This important relationship between debridement and efficacy of and advanced therapeutic product has been discussed elsewhere, and is part of this review in the way it affects the way we view wound bed preparation. It should be noted here that subsequent trials in diabetic ulcers adopted maintenance debridement of the wound bed and callus, to the point that maintenance debridement is now considered the standard of care. In this review, we will discuss certain abnormalities involved in the pathogenesis of diabetic foot ulcers and their failure to heal and which have a great impact on wound bed preparation. Traditionally, these cellular and molecular abnormalities have been wrongly considered separately from the clinical approach, with debridement being seen mostly as a mundane clinical procedure. However, as we shall see, close interactions are emerging between molecular events and certain critical aspects of wound care. Wound bed preparation is a unifying concept, which embodies many of these interactions. Figure 1 illustrates this important concept.

OVERVIEW OF DIABETIC FOOT ULCERS AND PATHOPHYSIOLOGICAL PRINCIPLES

Recent publications provide reviews of diabetic ulcers, their basic pathophysiology, and appropriate treatments (6,7). Besides arterial insufficiency, neuropathy plays a major role in the development of ulceration in patients with diabetes. It is important to realize that our concepts of pathophysiology have important consequences on treat- ment. 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 much needed surgical revascularizing procedures were not performed because of the notion of small vessel disease. Therefore, the realization a few years ago that the concept of a true occlusive microangiopathy in the lower extremities of patients with diabetes is incorrect or not specific for patients with diabetes has brought about a dramatic benefit in how revascularization of the diabetic foot is viewed and practiced (8).

Particularly in the context of diabetic ulcers, in the past clinicians and scientists have talked about “failure to heal” (9). 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 resulting from diabetic compli-

cation, do heal in an appropriate time frame. For example, with uncomplicated diabetic

neuropathic foot ulcers, healing should occur relatively unimpeded once off-loading is appropriately practiced (10). However, truly prospective studies are required to answer the question of whether relatively simple measures, such as contact casting, can greatly improve ulcer healing.

A typical feature of diabetic ulcers is their propensity to become heavily colonized with bacterial and, less commonly, fungal organisms. These factors play a major role in the failure to heal. Bacterial colonization has also been called 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. This has been discussed extensively in other publications (11). However, there is evidence that, regardless of the type of bacteria present, a level greater than or equal to 10

organisms per gram of tissue is associated with serious healing impairment. Federal guidelines developed for the treatment of pressure (decubitus) ulcers (pressure plays a major role diabetic neuropathic foot ulcers) recommend quantitative bacteriology, requiring a wound biopsy, if there is continued healing impairment. In practical terms, it makes theoretic and clinical sense to decrease the bacterial burden of wounds. Nevertheless, we know very little about the mechanisms underlying 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 antimicro- bials (12). At the moment, there is no hard evidence for what role biofilms play in dia- betic foot ulcers. Debridement can remove the bacterial burden and “reset” the diabetic foot ulcer in a mode that is more conducive to healing.

Fig. 1. Clinical photograph showing surgical debridement of the callus and wound bed in a

diabetic neuropathic foot ulcer. The photograph also indicates how the surgical debridement may

remove the necrotic tissue, bacterial burden and, possibly, phenotypically altered cells that may

interfere with healing.

OTHER PATHOPHYSIOLOGICAL FACTORS INTERACTING WITH DEBRIDEMENT AND WOUND BED PREPARATION Growth Factor “Trapping”

Debridement has other important impacts on basic pathophysiological abnormalities.

Thus, removing the callus and the fibrinous wound bed can have consequences on growth factor availability. The concept of growth factor trapping was first developed in the context of venous ulcers (13), but has applicability to a variety of chronic wounds, including diabetic neuropathic foot ulcers. 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 (13,14). Even topically applied growth factors can suffer the same fate of becoming bound to leaked macromolecules and altered matrix components. 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 down- stream. Binding of growth factors by macromolecules that leak into the dermis, such as albumin, fibrinogen, and α-2-macroglobulin may disrupt the healing process. α,-2- macroglobulin is an established scavenger for growth factors. There is also evidence that transforming growth factor- β

, a critical multifunctional polypeptide, is bound within the pericapillary fibrin cuffs in the dermis (13,14).

Wound Fluid and Metalloproteinases

The bacterial burden, the inflammatory process developing in diabetic foot ulcers, alter the balance between appropriate moisture levels in the wound, matrix deposition and remodeling, and the levels of tissue metalloproteinases. A major breakthrough of the last 50 years, in terms of how we manage wounds, was the experimental evidence by George Winter indicating that re-epithelization is accelerated when wounds are kept moist (15). This advance has led to the development of a vast array of moisture-retentive dressings that promote “moist wound healing” (16,17). 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 (18–20).

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 (21–23) Conversely, fluid obtained from chronic

wounds will block cellular proliferation and angiogenesis (24) and contains excessive

amounts of matrix metalloproteinases (MMPs) capable of breaking down critical extra-

cellular matrix proteins, including fibronectin and vitronectin (25–27). Undoubtedly,

MMPs play a key role in the wound healing (28). For example, interstitial collagenase

(MMP-1) is important for keratinocyte migration (29). In contrast, it has been suggested

that excessive activity (or maldistribution) of other enzymes (MMP-2, MMP-8, MMP-9)

contribute to healing impairment (30,31). Taken together, however, the emerging

evidence is that control of MMPs and their localization could have important therapeu- tic implications, including the enhanced survival of topically applied growth factors.

Impaired Blood Flow and Hypoxia

Of particular importance in diabetic ulcers is the level of oxygen tension at the wound site. In the case of ulcers because of poor tissue perfusion and consequent hypoxia, revascularization is required. However, in ulcers whose pathogenesis is mainly pressure in the presence of adequate blood flow, hypoxia may be playing some interesting roles.

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 meas- urements or TcPO

) correlate with inability to heal (32). 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 (33) and can actually enhance the transcription and synthesis of a number of growth factors (34–36). However, there is also evidence that exposure to hyperbaric oxygen can increase the production of vascular endothelial growth factor (37). 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 a number of abnormalities including scarring and fibrosis.

The Pathophysiological Status of the Wound and Phenotypic Alteration of Wound Cells

Another critical relationship can be established between the value of maintenance debridement and the removal of resident wound cells that have become phenotypically abnormal. The normal process of wound repair goes through well-defined stages that are well studied. However, as stated, chronic wounds do not seem to have defined time frames for healing. This is apparent both clinical and from the pathophysiological standpoint. It has been stated that the diabetic ulcers 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 protein, including fibronectin (38–40).

There is also increasing evidence that the resident cells of chronic wounds have undergone phenotypic changes that impair their capacity for proliferation and movement (38–40). To what extent this is because of 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. 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 (41–44), and that their decreased prolif-

erative capacity correlates with failure to heal and lack of response to PDGF (45,46). There

is also substantial evidence that ulcer fibroblasts are insensitive to the action of trans-

forming growth factor- β

, and that this is the result of decreased receptor expression (47)

and phosphorylation of key signaling proteins, including Smads and MAPK (48). 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.

WOUND BED PREPARATION AS A NEW PARADIGM FOR DIABETIC FOOT ULCERS

Advances in the treatment of diabetic foot ulcers have relied heavily on the concepts developed for acute wounds. In the last few years, however, a new paradigm for address- ing chronic wounds, including diabetic foot ulcers, is emerging. We have applied the term “wound bed preparation” to this paradigm, trying to emphasize the importance of optimizing the appearance and readiness of the wound bed (3). The main point, however, is that this approach represents a way for us to emphasize that and their way to achieve complete closure is different than from acute wounds (4,5). As new insights and thera- peutic modalities become available, for example, stem cells, we may be talking more about “wound bed reconstitution.” The critical concept is that, once appropriate steps have been taken to optimize the wound bed, then the normal endogenous process of wound healing is going to be facilitated (5). It is important to note that wound bed preparation is more than surgical 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/optimization. Basic aspects, as with compromised acute wounds, include debridement, infection control, edema removal, and surgical correction of underlying defect. More advanced aspects, for which we may not have all the answers yet, include attempts at wound bed reconstitution, as with the use of biological agents.

THERAPEUTIC ADVANCES INTERACTING WITH WOUND BED PREPARATION

As stated throughout this review, important and intimate links exist between debride- ment, wound bed preparation and reconstitution, and therapeutic advances. In the last few years, we have seen the development and marketing of very sophisticated products for impaired healing. These products, whether they are growth factors or tissue engi- neering constructs, represent the culmination of decades of basic research. As we have already mentioned, the efficacy of many advanced therapeutic agents has not matched the expectations, partly because the standards of care for some chronic wounds, including diabetic foot ulcers, were not adequate. In a way, wound bed preparation was not born yet. 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 (Fig. 1). Another example is the removal of 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 which may enhance bacterial colonization. Therefore, existing therapies can be integrated better with pathophysiological principles (3–5).

Moreover, we have now found that even advanced therapies may be thought of as methods for improving wound bed preparation, because ultimately we are accelerat- ing the endogenous process of wound healing. There are many examples of this.

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 for venous ulcers (49), fibroblast growth factor (50), and PDGF for pressure ulcers (51). However, at least in the United States, the only topically applied growth factor that is commer- cially approved for use is PDGF. In randomized controlled clinical studies, PDGF has been shown to accelerate the healing of neuropathic diabetic foot ulcers by approx 15% (1,52).

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. Inadequate dosage, mode of delivery of the peptide, and possibly the need for combinations of growth factors are some of the problems hypothesized to explain the lack of more dramatic clinical effects. It is also possible however, that closer attention should have been paid to appropriately preparing the chronic wound before treatment with the growth factor being tested in clinical trials.

Notably, there is evidence that the aggressive approach to surgical debridement in the ini- tial PDGF trial for diabetic neuropathic ulcers seems to have worked synergistically with the application of the growth factor (2). 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

Another major advanced treatment that can improve wound bed preparation is cell therapy, such as with the use of 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 (53,54), several more complex constructs have been developed and tested in human wounds. Skin equiva- lents may contain living cells, such as fibroblasts or keratinocytes or both (55–59), whereas others are made of acellular materials or extracts of living cells. 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 (60,61). 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 the 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 (10). Truly prospective trials need to be done to determine the contribution of off-loading done with contact casting and how this approach can improve the outcome in patients treated with advanced biological products.

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, but this

cannot yet be interpreted as being their mechanism of action (62). 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.

Gene Therapy and Stem Cells

At the forefront of advanced treatments, stand gene therapy and the use of stem cells.

As we have discussed, a spectrum is likely to evolve, going from wound bed preparation to wound bed reconstitution. Gene therapy and stem cells have the potential to drastically alter the wound bed and may thus be at the right end of the spectrum, toward wound bed reconstitution. For some time now, there has been ample technology for introducing cer- tain genes into wounds by a variety of physical means or biological vectors, including viruses. There are ex vivo approaches, in which cells may be manipulated before rein- troduction into the wound, to more direct in vivo techniques, which may rely on simple injection or the use of the gene gun (63). 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, in which only transient expression may be required. Most of the work with gene therapy of wounds has been done in experimental animal models. 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 has been reported to enhance healing and angiogenesis in selected patients with ulcers from arterial insufficiency (64). 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, i.e., a growth factor, is seen as a less expensive and potentially more efficient delivery method.

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 (65–67).

A recent uncontrolled report suggests that direct application of autologous bone marrow and its cultured cells may accelerate the healing of nonhealing chronic wounds (68).

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 aggressiveness 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 exclusion 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. Although 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 a number of 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. Wound bed preparation is changing the approach to diabetic foot ulcers and other types of chronic wounds. Debridement is seen as more than removal of unwanted necrotic tissue and callus. As illustrated in Fig. 1, surgical debridement may be a way to also decrease the bacterial burden and to remove phenotypically altered cells that may be interfering with the healing process. As stated earlier, debridement in diabetic foot ulcers is preferably done by surgical means, although there is a need for better topical debriding agents. Presently, there is also reliance on slow release antiseptics (silver or iodine based) and, to a lesser extent, on autolytic debridement with occlusive dressings.

The emphasis by regulatory agency to determine the efficacy of debriding agents in terms of healing endpoints has not helped; industry has been reluctant to embark on such expensive studies, and patients may be suffering from this regulatory link between debridement and healing outcome. Hopefully, this regulatory policy will change in the future. What has also become clear is that therapeutic agents may actually work by improving wound bed preparation, thus allowing the endogenous process of wound healing to move forward. As a result of many advances, including the concepts of main- tenance debridement and wound bed preparation, the standards of care for diabetic foot ulcers have improved. As stated earlier, because the control group will receive better care in therapeutic trials, it will become more difficult to prove the effectiveness of novel therapeutic agents. However, we should strive for a “quantum” jump in the way we deal with diabetic foot ulcers and other chronic wounds. Perhaps, the use of gene therapy, stem cells, may provide a quantum advance in accelerating diabetic foot ulcer healing.

With the acceptance of wound bed preparation of which debridement is an integral part, we will move toward wound bed reconstitution, which will require more active and innovative therapeutic approaches.

ACKNOWLEDGMENT

This work was supported by NIH grants AR42936, AR46557, and DK067836.

REFERENCES

1. Steed DL. Clinical evaluation of recombinant human platelet-derived growth factor for the treatment of lower extremity diabetic ulcers. Diabetic Ulcer Study Group. J Vasc Surg 1995;21(1):71–78 (Discussion 79–81).

2. Steed DL, et al. Effect of extensive debridement and treatment on the healing of diabetic foot ulcers. Diabetic Ulcer Study Group. J Am Coll Surg 1996;183(1):61–64.

3. Falanga V. Classifications for wound bed preparation and stimulation of chronic wounds.

Wound Repair Regen 2000;8(5):347–352.

4. Falanga V. Wound bed preparation: future approaches. Ostomy Wound Manage 2003;49(5A Suppl):30–33.

5. Saap LJ, Falanga V. Debridement performance index and its correlation with complete closure of diabetic foot ulcers. Wound Repair Regen 2002;10(6):354–359.

6. Jeffcoate WJ, Price P, Harding KG. Wound healing and treatments for people with diabetic

foot ulcers. Diabetes Metab Res Rev 2004;20(Suppl 1):S78–S89.

7. Boulton AJ, Kirsner RS, Vileikyte L. Clinical practice. Neuropathic diabetic foot ulcers.

N Engl J Med 2004;351(1):48–55.

8. LoGerfo FW, Coffman JD. Current concepts. Vascular and microvascular disease of the foot in diabetes. Implications for foot care. N Engl J Med 1984;311(25):1615–1619.

9. Pecoraro RE, et al. Chronology and determinants of tissue repair in diabetic lower-extremity ulcers. Diabetes 1991;40(10):1305–1313.

10. Armstrong DG, et al. Off-loading the diabetic foot wound: a randomized clinical trial.

Diabetes Care 2001;24(6):1019–1022.

11. Robson MC, et al. Maintenance of wound bacterial balance. Am J Surg 1999;178(5):399–402.

12. Edwards R, Harding KG. Bacteria and wound healing. Curr Opin Infect Dis 2004;17(2):91–96.

13. Falanga V, Eaglstein WH. The “trap” hypothesis of venous ulceration. Lancet 1993;

341(8851):1006–1008.

14. Higley HR, et al. Extravasation of macromolecules and possible trapping of transforming growth factor-beta in venous ulceration. Br J Dermatol 1995;132(1):79–85.

15. Falanga V. Occlusive wound dressings. Why, when, which?. Arch Dermatol 1988; 124(6):

872–877.

16. Helfman T, Ovington L, Falanga, V. Occlusive dressings and wound healing. Clin Dermatol 1994;12(1):121–127.

17. Ovington LG. The evolution of wound management: ancient origins and advances of the past 20 years. Home Healthc Nurse 2002;20(10):652–656.

18. Hutchinson JJ. Infection under occlusion. Ostomy Wound Manage 1994;40(3):28–30, 32, 33.

19. Hutchinson JJ, Lawrence JC. Wound infection under occlusive dressings. J Hosp Infect 1991;17(2):83–94.

20. Smith DJ, Jr, et al. Microbiology and healing of the occluded skin-graft donor site. Plast Reconstr Surg 1993;91(6):1094–1097.

21. Katz MH, et al. Human wound fluid from acute wounds stimulates fibroblast and endothe- lial cell growth. J Am Acad Dermatol 1991;25(6 Pt 1):1054–1058.

22. Drinkwater SL, et al. Effect of venous ulcer exudates on angiogenesis in vitro. Br J Surg 2002;89(6):709–713.

23. Schaffer MR, et al. Stimulation of fibroblast proliferation and matrix contraction by wound fluid. Int J Biochem Cell Biol 1997;29(1):231–239.

24. Falanga V, et al. Workshop on the pathogenesis of chronic wounds. J Invest Dermatol 1994;102(1):125–127.

25. Wysocki AB, Staiano-Coico L, Grinnell, F. Wound fluid from chronic leg ulcers contains elevated levels of metalloproteinases MMP-2 and MMP-9. J Invest Dermatol 1993;101(1):64–68.

26. Trengove NJ, Bielefeldt-Ohmann H, Stacey MC. Mitogenic activity and cytokine levels in non-healing and healing chronic leg ulcers. Wound Repair Regen 2000;8(1):13–25.

27. Trengove NJ, et al. Analysis of the acute and chronic wound environments: the role of proteases and their inhibitors. Wound Repair Regen 1999;7(6):442–452.

28. Madlener M, Parks WC, Werner S. Matrix metalloproteinases (MMPs) and their physiolog- ical inhibitors (TIMPs) are differentially expressed during excisional skin wound repair. Exp Cell Res 1998;242(1):201–210.

29. Pilcher BK, et al. The activity of collagenase-1 is required for keratinocyte migration on a type I collagen matrix. J Cell Biol 1997;137(6):1445–1457.

30. Weckroth M, et al. Matrix metalloproteinases, gelatinase and collagenase, in chronic leg ulcers. J Invest Dermatol 1996;106(5):1119–1124.

31. Nwomeh BC, et al. MMP-8 is the predominant collagenase in healing wounds and non- healing ulcers. J Surg Res 1999;81(2):189–195.

32. Fife CE, et al. The predictive value of transcutaneous oxygen tension measurement in diabetic

lower extremity ulcers treated with hyperbaric oxygen therapy: a retrospective analysis of

1,144 patients. Wound Repair Regen 2002;10(4):198–207.

33. Falanga V, Kirsner RS. Low oxygen stimulates proliferation of fibroblasts seeded as single cells. J Cell Physiol 1993;154(3):506–510.

34. Kourembanas S, Hannan RL, Faller DV. Oxygen tension regulates the expression of the platelet-derived growth factor-B chain gene in human endothelial cells. J Clin Invest 1990;86(2):670–674.

35. Kourembanas S, et al. Hypoxia induces endothelin gene expression and secretion in cultured human endothelium. J Clin Invest 1991;88(3):1054–1057.

36. Helfman T, Falanga V. Gene expression in low oxygen tension. Am J Med Sci 1993;306(1):37–41.

37. Sheikh AY, et al. Effect of hyperoxia on vascular endothelial growth factor levels in a wound model. Arch Surg 2000;135(11):1293–1297.

38. Loot MA, et al. Fibroblasts derived from chronic diabetic ulcers differ in their response to stimulation with EGF, IGF-I, bFGF and PDGF-AB compared to controls. Eur J Cell Biol 2002;81(3):153–160.

39. Loots MA, et al. Cultured fibroblasts from chronic diabetic wounds on the lower extremity (non-insulin-dependent diabetes mellitus) show disturbed proliferation. Arch Dermatol Res 1999;291(2–3):93–99.

40. Loots MA, et al. Differences in cellular infiltrate and extracellular matrix of chronic diabetic and venous ulcers versus acute wounds. J Invest Dermatol 1998;111(5):850–857.

41. Bruce SA, Deamond SF. Longitudinal study of in vivo wound repair and in vitro cellular senescence of dermal fibroblasts. Exp Gerontol 1991;26(1):17–27.

42. Hehenberger K, et al. Inhibited proliferation of fibroblasts derived from chronic diabetic wounds and normal dermal fibroblasts treated with high glucose is associated with increased formation of l-lactate. Wound Repair Regen 1998;6(2):135–141.

43. Hehenberger K, et al. Fibroblasts derived from human chronic diabetic wounds have a decreased proliferation rate, which is recovered by the addition of heparin. J Dermatol Sci 1998;16(2):144–151.

44. Mendez MV, et al. Fibroblasts cultured from distal lower extremities in patients with venous reflux display cellular characteristics of senescence. J Vasc Surg 1998;28(6):1040–1050.

45. Stanley A, Osler, T, Senescence and the healing rates of venous ulcers. J Vasc Surg 2001;33(6):1206–1211.

46. Agren MS, et al. Proliferation and mitogenic response to PDGF-BB of fibroblasts isolated from chronic venous leg ulcers is ulcer-age dependent. J Invest Dermatol 1999;112(4):463–469.

47. Hasan A, et al. Dermal fibroblasts from venous ulcers are unresponsive to the action of transforming growth factor-beta 1. J Dermatol Sci 1997;16(1):59–66.

48. Kim BC, et al. Fibroblasts from chronic wounds show altered TGF-beta-signaling and decreased TGF-beta type II receptor expression. J Cell Physiol 2003;195(3):331–336.

49. Falanga V, et al. Topical use of human recombinant epidermal growth factor (h-EGF) in venous ulcers. J Dermatol Surg Oncol 1992;18(7):604–606.

50. Robson MC, et al. The safety and effect of topically applied recombinant basic fibroblast growth factor on the healing of chronic pressure sores. Ann Surg 1992;216(4):401–406 (Discussion 406–408).

51. Robson MC, et al. Platelet-derived growth factor BB for the treatment of chronic pressure ulcers. Lancet 1992;339(8784):23–25.

52. Smiell JM, et al. Efficacy and safety of becaplermin (recombinant human platelet-derived growth factor-BB) in patients with nonhealing, lower extremity diabetic ulcers: a combined analysis of four randomized studies. Wound Repair Regen 1999;7(5):335–346.

53. Gallico GG, 3rd. Biologic skin substitutes. Clin Plast Surg 1990;17(3):519–526.

54. Phillips TJ, Gilchrest BA. Clinical applications of cultured epithelium. Epithelial Cell Biol 1992;1(1):39–46.

55. Bell E, et al. Living tissue formed in vitro and accepted as skin-equivalent tissue of full

thickness. Science 1981;211(4486):1052–1054.

56. Sabolinski ML, et al. Cultured skin as a ‘smart material’ for healing wounds: experience in venous ulcers. Biomaterials 1996;17(3):311–320.

57. Boyce ST. Design principles for composition and performance of cultured skin substitutes.

Burns 2001;27(5):523–533.

58. Falanga V, et al. Rapid healing of venous ulcers and lack of clinical rejection with an allo- geneic cultured human skin equivalent. Human Skin Equivalent Investigators Group. Arch Dermatol 1998;134(3):293–300.

59. Hansbrough JF, et al. Clinical trials of a biosynthetic temporary skin replacement, Dermagraft-transitional covering, compared with cryopreserved human cadaver skin for temporary coverage of excised burn wounds. J Burn Care Rehabil 1997;18(1 Pt 1):43–51.

60. Marston WA, et al. The efficacy and safety of Dermagraft in improving the healing of chronic diabetic foot ulcers: results of a prospective randomized trial. Diabetes Care 2003;26(6):1701–1705.

61. Veves A, et al. Graftskin, a human skin equivalent, is effective in the management of non- infected neuropathic diabetic foot ulcers: a prospective randomized multicenter clinical trial.

Diabetes Care 2001;24(2):290–295.

62. Falanga V, et al. Wounding of bioengineered skin: cellular and molecular aspects after injury. J Invest Dermatol 2002;119(3):653–660.

63. Badiavas EV, Falanga V. Gene therapy. J Dermatol 2001;28(4):175–192.

64. Comerota AJ, et al. Naked plasmid DNA encoding fibroblast growth factor type 1 for the treatment of end-stage unreconstructible lower extremity ischemia: preliminary results of a phase I trial. J Vasc Surg 2002;35(5):930–936.

65. Badiavas EV, et al. Participation of bone marrow derived cells in cutaneous wound healing.

J Cell Physiol 2003;196(2):245–250.

66. Quesenberry, PJ, et al. Stem cell plasticity: an overview. Blood Cells Mol Dis 2004;32(1):1–4.

67. Quesenberry, PJ, et al. Developmental biology: Ignoratio elenchi: red herrings in stem cell research. Science 2005;308(5725):1121, 1122.

68. Badiavas EV, Falanga V. Treatment of chronic wounds with bone marrow-derived cells.

Arch Dermatol 2003;139(4):510–516.