RECONSTRUCTIVE

Chronic Wound Pathogenesis and Current Treatment Strategies: A Unifying Hypothesis

Thomas A. Mustoe, M.D. Kristina OShaughnessy, M.D. Oliver Kloeters, M.D.
Chicago, Ill.

Summary: Most chronic wounds can be classified into three major types: pressure ulcers, venous ulcers, and diabetic ulcers. The authors propose a unifying hypothesis of chronic wound pathogenesis based on four main causative factors: local tissue hypoxia, bacterial colonization of the wound, repetitive ischemiareperfusion injury, and an altered cellular and systemic stress response in the aged patient. Traditional strategies for the treatment of chronic wounds have shown limited success. The authors explore potential treatment regimens specifically aimed at each individual determinant of chronic wound pathogenesis. Furthermore, they explore a combined therapeutic approach that collectively targets all the components of chronic wound pathology. These innovative ideas and therapies could be of substantial interest for clinicians and researchers, while further offering significant benefit to patients with chronic wounds. (Plast. Reconstr. Surg. 117 (Suppl.): 35S, 2006.)

hronic wounds are a health problem of enormous magnitude affecting many hundreds of thousands of patients. In fact, the health care cost amounts to billions of dollars annually in the United States alone. Chronic wounds do not occur in animals, and establishing a true chronic wound model in animals has been extremely difficult. From a practical standpoint of feasible experimental studies, it has been impossible. Therefore, much of our understanding of chronic wounds has come from humans and, by necessity, has been limited to observation, biopsy, and analysis of wound fluids. These limitations have resulted in significant gaps in our knowledge of etiology and pathogenesis. There exists a perception that human wounds have some unique deficiency resulting in a failure to heal. The difficulty in treating chronic wounds has been underscored by the limited success of growth factors in treatment1,2 and the failure of so many clinical trials and biotechnology companies that pegged their future on the success of those trials. Finally, chronic wounds have diverse etiologies. More than 90 percent of all chronic wounds fall into three categories: venous ulcers, pressure sores,
From the Division of Plastic and Reconstructive Surgery, Northwestern University, Feinberg School of Medicine. Received for publication December 29, 2005; revised March 12, 2006. Copyright 2006 by the American Society of Plastic Surgeons DOI: 10.1097/01.prs.0000225431.63010.1b

and diabetic ulcers. At first consideration, these three types of chronic wounds appear to have little in common, but examining their commonalities in more detail leads to the hypothesis that human chronic wounds do not have a unique defect but represent a combination of factors. Each component of pathogenesis is deleterious, but in aggregate they collectively overwhelm the healing response in many patients.

THE EFFECT OF LOCAL TISSUE HYPOXIA

Tissue ischemia profoundly impairs healing. Clinically, vascular surgeons widely accept the guideline that lower extremities with a partial oxygen pressure at the toe of less than 30 mmHg will not heal. In animal studies of the rabbit ear,3,4 tissue partial oxygen pressure drops from the normal 40 to 45 mmHg in controls to 28 to 30 mmHg in ischemic ears, leading to a wound-healing rate reduction of 80 percent at 7 days. Ischemic wound models have been devised in many species, all with a finding of major impairment in wound healing.5 However, ischemia and tissue hypoxia are not synonymous. Tissue partial oxygen pressure is affected by local blood flow, which in turn is affected by arterial flow, arteriovenous gradients, capillary density, and local tissue consumption. Many chronic wounds occur without the presence of easily measurable ischemia. In consideration of the three common types of chronic wounds, it is widely understood that diabetic ulcers frequently occur in the setting of un-

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derlying atherosclerotic large vessel disease with chronic ischemia of varying degrees. Although large vessel disease is not an issue for venous ulcers and pressure sores, many of the most recalcitrant wounds occur in the setting of a previous history of ulceration. Subclinical injury is reflected by periwound fibrosis and local reduction in tissue perfusion. Edema is another extremely important factor. By increasing the distance between capillaries at the local tissue level and therefore the distance for oxygen diffusion, edema lowers local oxygen levels, although the magnitude of this factor has not been measured. Interestingly, in venous ulcers in which edema plays the largest role, multiple clinical studies using compression bandages as a single variable in controlling tissue edema have demonstrated this to have the largest effect on successful wound closure rates. Compression bandages or compression garments are now part of every standard care guideline for venous ulcers. The success of intermittent compression by a sequential compression device to augment compression garments in particularly difficult cases (personal observations) supports the hypothesis that edema by itself results in meaningful drops in local tissue perfusion and oxygenation. Although the concept that edema plays a role in diabetic foot ulcers and pressure sores is less well accepted, the combination of dependency and inflammation results in local periwound edema. The therapeutic success of vacuum-assisted closure, in which pressure is applied to reduce local edema in the treatment of these wounds, also supports the hypothesis that in all chronic wounds the local environment has reduced oxygenation. patients, some with a several-year history of a nonhealing wound. When aggressive local measures were taken to reduce bacterial counts (frequent washing, water irrigation, hair removal by shaving the area, and use of topical antibiotics), these chronic wounds went on to heal rapidly. In other words, critical colonization is by itself sufficient to result in a chronic wound. Analysis of wound fluid from chronic wounds, as compared with acute surgical wounds, demonstrates increased protease levels, increased proinflammatory cytokine levels,8 10 and a reduction in growth factor levels, supporting the role that bacteria play in most chronic wounds. The fibronectin degradation observed in chronic wounds,11 as well as other matrix degradation, could explain to a significant degree the delay in epithelialization observed in chronic wounds in which the wound margins have heaped up epithelial margins with actively dividing cells but a failure to migrate. Biofilm overlying a chronic wound protects bacteria from host defenses.12 The type of bacteria colonizing a wound also plays a role. Recently, it has been recognized that anaerobic bacteria are present in most wounds when looked for using careful culturing techniques.13 Several studies14,15 have reported that healing wounds are characterized by a reduction in bacteria counts and markers of inflammation. Although a large body of evidence supports the importance of bacteria and the resulting inflammatory response in the etiology of chronic wounds, there are still substantial gaps in knowledge.

THE ROLE OF ISCHEMIA-REPERFUSION INJURY

Reperfusion, while necessary to limit irreversible cellular injury in the setting of ischemia, can paradoxically result in additional tissue compromise. Collectively referred to as ischemiareperfusion injury, this complex cascade of molecular and cellular events has significant clinical implications. The adverse effects are evident in organ transplantation,16,17 myocardial infarction,18 stroke, 19 free flap transfers,20 aortic aneurysm repair,21 and several other pathological entities22,23; however, ischemia-reperfusion injury has not been commonly discussed in relation to the development and potentiation of chronic wounds. In fact, although ischemia has been implicated in the pathogenesis of certain chronic wounds, such as pressure sores, 24 only during recent years has reperfusion been suggested as an additional etiologic factor.2527 Despite the expanding depth of knowledge and

THE ROLE OF BACTERIA

An open wound becomes contaminated with bacteria from the surrounding skin within 48 hours, and so all chronic wounds have measurable bacteria counts.6 In the absence of cellulitis, it is often not possible to distinguish normal colonization from excessive bacteria or critical colonization. The host immune response to bacteria is inflammation, chiefly an influx of polymorphonuclear leukocytes that release proteases and oxidants, which not only degrade cytokines and the extracellular matrix but also place stress on local cells. Classic studies by Robson have demonstrated failure of skin graft survival when grafts are placed on a wound bed with bacterial counts in excess of 100,000/mm3.7 The senior author (T.A.M.) has treated several chronic pilonidal wounds in young

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understanding of ischemia and reperfusion, including signaling transduction pathways28 and molecular events regulating injury,29 the pathophysiology and its many subtleties have yet to be fully elucidated. The fundamental mechanisms that lead to ischemia-reperfusion injury is similar in all organ systems, especially at the molecular level, where reactive oxygen species and inflammation are key mediators in establishing tissue injury.30 However, there are important distinctions that make ischemia-reperfusion injury unique in the setting of chronic wounds. Most importantly, ischemia and reperfusion events occur in a repetitive fashion such that the injury is potentiated with each cycle. Clinically overt tissue damage becomes evident in patients who are unable to avoid this cyclic-dependent injury. For example, the patient with venous leg ulcers alternates between recumbent and ambulatory leg positioning. In simplistic terms, ischemia occurs during leg dependency and reperfusion occurs during leg elevation. In an ambulatory patient, this is an unavoidable sequence of events that can result in severe tissue injury. Similarly, most critically ill patients or persons with paraplegia are often on strict turning regimens. Such a regimen, while well intentioned, results in cyclic intervals of variable blood flow that may have detrimental clinical effects. Cutaneous tissues endure a period of pressure-induced ischemia when the patient is recumbent followed by hyperemia when a change in position provides reestablishment of blood flow. matory state. Upon reperfusion, leukocytes become activated and interact with the endothelium through rolling, adherence, and subsequent transmigration into the interstitial compartment. Regulated by preformed mediators, the inflammatory cascade ensues, causing deleterious cellular reactions and tissue damage.29 Reoxygenation further impairs both vascular and cellular processes through the overproduction of reactive oxygen species, a process termed oxidative stress, which overwhelms the endogenous defense mechanisms, thereby causing cytotoxic injury to surrounding tissues.28 Impaired vasorelaxation due to a decrease in nitrous oxide expression during reperfusion contributes significantly to microvascular dysfunction.31 This process, along with leukocyte trapping, leads to the no-reflow phenomenon, described as failure to restore flow to an ischemic organ despite reperfusion.23 In the setting of chronic wounds, these events recur with each cycle of ischemia and reperfusion. The additive effects of leukocyte and complement activation, oxidative damage, and microvascular dysfunction cause repetitive tissue injury and eventual tissue necrosis.

ISCHEMIA AND REPERFUSION PATHOGENESIS IN CHRONIC WOUNDS

The sequence of events leading to venous skin ulcerations and the mechanisms that maintain their chronic nature continue to cause controversy. Historically, physicians and scientists have focused on incompetent valves, venous stasis, and ultimately ischemia as the etiology of ulcer formation.32 Although the development of ischemia through venous hypertension, edema, fibrin deposition, and microvascular changes is well documented, these factors do not fully explain the etiology of chronic venous stasis.3335 Only in recent years have repeated ischemia/reperfusion cycles34,36 and inflammatory mechanisms37,38 been hypothesized to occur in the development and persistence of chronic venous wounds. A human study using laser Doppler scanning techniques showed postural vasoregulation to cause relative ischemia and reperfusion in leg ulcers.39 Venous hypertension leads to leukocyte sequestration, and upon reperfusion, as seen with leg elevation, the leukocytes are activated and reactive oxygen species cause further injury to the already ischemic tissues.37 Further support is reflected by the overabundance of neutrophils and vulnerability to infection seen with venous stasis ulcers37 and by the clinical efficacy of anti-inflammatory drugs re-

ISCHEMIA AND REPERFUSION: MOLECULAR BIOLOGY

Ischemia-reperfusion injury is characterized by a sequence of biochemical and cellular events that causes extensive cellular damage through pathways that lead to leukocyte and complement activation, oxidative stress, and microvasculature dysfunction.23 The process begins with an inciting ischemic event that, on a cellular level, reduces ATP production through impairment of mitochondrial oxidative phosphorylation. A reduction in ATP sources results in loss of the transmembrane potential and an influx of various ionic species, leading to cell swelling. Increased levels of calcium ion in the cytosol lead to activation of signal transduction pathways that activate enzymes responsible for cell membrane disruption.30 In addition, ischemia induces the expression of endothelial adhesion molecules and cytokines that prime the tissue for further insult by creating a proinflam-

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ported in both humans with venous disease40 and in animal models of ischemia-reperfusion injury.41 Furthermore, the leukocyte trapping seen during venous hypertension42 and subsequent release of inflammatory mediators invoke not only a local inflammatory response but also a systemic inflammatory response, which has been documented by blood sampling in humans38 and is described in other ischemia-reperfusion injury models.23 It is agreed that the etiology of pressure sores is multifactorial and dependent on a variety of factors, including the overall health of the patient, pressure-induced ischemia, and shear injury.43 45 Some authors have recently suggested that reperfusion injury is a major contributing determinant.26,27,46 Again, it is important to emphasize the repetitive nature of ischemia and reperfusion in relation to pressure sore development. In both rat and mouse models, reproducing ischemia- and reperfusion-mediated pressure sores through controlled periodic magnetic force compression and subsequent reperfusion indicate that the extent of tissue damage is attributable to the total number of ischemia-reperfusion cycles.27,47 This has been demonstrated in our laboratory with the mouse ischemia-reperfusionmediated pressure sore model. More importantly, when ischemia is held constant, a greater number of reperfusion events result in increased tissue damage, and authors have concluded that repeated ischemia-reperfusion injuries are more damaging to tissue than prolonged ischemia alone.27,47 These animal studies support the hypothesis that reperfusion is an important component of pressure sore etiology. The diabetic population with neuropathy is at risk for ulcerations on weightbearing portions of the foot, secondary to a lack of ability to perceive excessive pressure or pain; the altered foot mechanics secondary to loss of intrinsic musculature; and a shortened Achilles tendon.48 After even a seemingly minor initial injury, these wounds can quickly progress because of an impaired leukocyte response to bacteria49 and repeated trauma and ischemic injury.50 As with the other types of chronic wounds, we hypothesize that both reperfusion, upon release of pressure, and the additive effect of repeated ischemiareperfusion cycles would further escalate the amount of tissue injury. Many of the main mediators of ischemia-reperfusion injury are already altered in these patients, which likely delays wound healing even further.

STRATEGIES TO OVERCOME ISCHEMIA-REPERFUSION INJURY

Some of the current treatment strategies utilized in chronic wounds include offloading, compression, warming, vacuum-assisted closure devices, irrigation, de bridement, and topical antibiotics or growth factors. Although in combination these treatments may demonstrate success, persistence or recurrence of chronic wounds remains a significant problem. The recent advances in understanding the pathophysiology of ischemia-reperfusion injury in other organ systems have led to innovative strategies for treatment, such as ischemic preconditioning, anti-inflammatory agents, antioxidants, and complement therapy.23 Although many of these therapies to overcome general ischemia-reperfusion injury have been described in the literature and are used to some degree, human clinical trials for efficacy are lacking. Furthermore, cyclic ischemia-reperfusion injury as it relates to venous ulcers, pressure sores, and diabetic wounds remains a novel concept that has not been widely discussed or emphasized. Therefore, continued efforts utilizing in vivo models and human studies are necessary. Currently, the potential for development of effective, fast-acting, and long-term therapeutics is dependent on a better understanding of the pathophysiological processes underlying chronic wound etiology.

THE ROLE OF AGING AND THE IMPAIRED STRESS RESPONSE

Most chronic wounds occur in the elderly. In reviewing clinical studies on venous ulcers, diabetic ulcers, and pressure sores, the average age of patients with these types of chronic wounds is over 60 years.25 Cells in the local environment of a chronic wound are subject to significant stresses, including chronic inflammation, the harmful presence of reactive oxygen species,51 and a proteolytic environment secondary to bacteria, underlying ischemia, and recurrent ischemia-reperfusion cycles. Clinical observations have long supported the ability to safely operate on the elderly, but when the stress is severe, such as with a major burn, the elderly are unable to respond. For example, in patients with a 50 percent total body surface area burn, the survival rate in major burn centers is high and relatively constant from the second through the fifth decade, when survival falls off. Over the age of 80, the survival rate from a 50 percent burn is unprecedented. Even in young patients stress results in impaired healing.

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In a group of students undergoing the psychological stress of examinations, experimental wounds in the oral cavity had a significant delay in healing compared with control wounds.52 There has been increasing experimental evidence detailing the impaired response of aging cells to stress that involves broadly altered gene expression. Microarray analysis of human fibroblasts from aged patients compared with young adults reveals a baseline increase in expression of stress response genes, as well as a broad range of genes in other signal transduction pathways. However, the aged human fibroblasts fail to upregulate under hypoxic conditions.53 A more detailed analysis of the human aged fibroblast response to oxidant stress plus ischemia, which replicates conditions found in chronic wounds, reveals increased cell death when compared with young adult cells. These findings are coupled with a failure to upregulate heat shock protein 70; however, transfection with heat shock protein 70 partially reversed the impaired response of aged fibroblasts, suggesting a causative effect.54 One theory for chronic wound etiology concentrates on cells in the local wound environment. Subjected to chronic stress and prolonged cytokine exposure, cells constantly replicate and turnover, ultimately entering into a replicative senescence where they become unable to divide. This is in part a potential rationale for the benefits of aggressive de bridement in diabetic ulcers, which converts a chronic wound to an acute wound by removing the wound margins in which many cells may have lost their proliferative potential. An in vitro study in our group simulated the effect of stress-induced premature senescence by the exposure of young versus aged human dermal fibroblasts to hydrogen peroxide. We found an increased entry rate into senescence, along with a reduction in cell viability and phosphorylation of prosurvival kinases.55 Other in vitro studies have confirmed the theory of an altered stress response in aged cells, as they found decreased proliferative capacity and prolonged doubling times subsequently ending in replicative senescence.56 These in vitro data add support to the theory that the prevalent stress in chronic wounds is a deleterious factor, especially in the elderly. In addition, transfecting cell lines that approach replicative senescence with the telomerase gene indefinitely prolonged senescence in vitro, with persistence of the phenotype of nontransformed cell lines.57 Employing an adenoviral construct overexpressing the telomerase gene in aged rabbits improved wound healing significantly (J. E. Mogford and T. A. Mustoe, unpublished observations), suggesting a new, promising therapeutic strategy. In summary, both clinical and laboratory observations support the hypothesis that the altered response in the aged to ischemic stress,58 or the combined ischemic and oxidant stress of ischemiareperfusion injury,59 contributes a great deal to the impaired healing response.

THERAPEUTIC IMPLICATIONS FOR THE TREATMENT OF CHRONIC WOUNDS

From the above discussion, we can conclude that chronic wounds are multifactorial in etiology. The lack of success of single-agent growth factor therapy is, therefore, not surprising. One therapy that has achieved broad acceptance, despite a lack of prospective randomized studies, is vacuum-assisted closure (negative pressure wound therapy). In fact, this single-modality therapy addresses simultaneously many of the issues discussed above. (1) Bacterial counts are substantially reduced by continuous removal of the exudates that serve as a culture medium, and there is a documented reduction in inflammation. (2) The negative pressure reduces edema and therefore increases oxygen and nutrient delivery to the local area of therapy. In the best-studied chronic wound type (venous ulcers), in terms of edema reduction, the single most important therapy for improving healing rates is appropriate use of compression with edema control. In addition, although not well documented for negative pressure wound therapy, other studies demonstrate a beneficial effect of local elevation of temperature on healing in chronic wounds, and it is likely that negative pressure wound therapy has this effect secondary to the closed system. (3) As long as the vacuumassisted closure device is in place, its bulk almost certainly prevents the patient from putting pressure on the area, and thus cycles of ischemia and reperfusion are prevented. (4) Theoretically, it is possible that the beneficial effects of negative pressure on blood flow also increase the likelihood that circulating mesenchymal progenitor cells will repopulate a wound in which some of the cells have entered into replicative senescence. In the future, major advances in therapy are likely to come from addressing multiple factors at one time. The recent advances in understanding the pathophysiology of ischemia-reperfusion injury in other organ systems have led to innovative strategies for treatment, such as ischemic preconditioning, anti-inflammatory agents, antioxidants, and complement therapy.23 Although many of

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these therapies to overcome general ischemiareperfusion injury have been described in the literature, human clinical trials are lacking. Novel strategies for reversal of some of the deleterious effects of aging, such as gene delivery of telomerase or anti-apoptotic genes, are potential directions. The role of growth factors may well be enhanced by multimodality therapy. In animal studies in ischemic wounds, growth factors by themselves modestly enhance healing, and hyperbaric oxygen by itself modestly enhances healing, but in combination, healing rates are dramatically increased, suggesting at least an additive-if not a synergistic-effect.60 Currently, the potential for development of effective, fast-acting, and longterm therapeutics is dependent on a better understanding of the pathophysiological processes underlying the etiology of chronic wounds.
Thomas A. Mustoe, M.D. Division of Plastic and Reconstructive Surgery Northwestern University 675 North St. Clair, Suite 19-250 Chicago, Ill. 60611 tmustoe@nmh.org
13. Wall, I. B., Davies, C. E., Hill, K. E., et al. Potential role of anaerobic cocci in impaired human wound healing. Wound Repair Regen. 10: 346, 2002. 14. Trengove, N. J., Stacey, M. C., Macauley, S., et al. Analysis of the acute and chronic wound environments: The role of proteases and their inhibitors. Wound Repair Regen. 7: 442, 1999. 15. Trengove, N. J., Bielefeldt-Ohmann, H., and Stacey, M. C. Mitogenic activity and cytokine levels in non-healing and healing chronic leg ulcers. Wound Repair Regen. 8: 13, 2000. 16. Ng, C. S., Wan, S., and Yim, A. P. Pulmonary ischaemiareperfusion injury: Role of apoptosis. Eur. Respir. J. 25: 356, 2005. 17. Kupiec-Weglinski, J. W., and Busuttil, R. W. Ischemia and reperfusion injury in liver transplantation. Transplant. Proc. 37: 1653, 2005. 18. Moens, A. L., Claeys, M. J., Timmermans, J. P., et al. Myocardial ischemia/reperfusion-injury, a clinical view on a complex pathophysiological process. Int. J. Cardiol. 100: 179, 2005. 19. Schaller, B., and Graf, R. Cerebral ischemia and reperfusion: The pathophysiologic concept as a basis for clinical therapy. J. Cereb. Blood Flow Metab. 24: 351, 2004. 20. Siemionow, M., and Arslan, E. Ischemia/reperfusion injury: A review in relation to free tissue transfers. Microsurgery 24: 468, 2004. 21. Norwood, M. G., Brown, M. J., and Sayers, R. D. Ischaemiareperfusion injury and regional inflammatory responses in abdominal aortic aneurysm repair. Eur. J. Vasc. Endovasc. Surg. 28: 234, 2004. 22. Kong, S. E., Blennerhassett, L. R., Heel, K. A., et al. Ischaemia-reperfusion injury to the intestine. Aust. N. Z. J. Surg. 68: 554, 1998. 23. Eltzschig, H. K., and Collard, C. D. Vascular ischaemia and reperfusion injury. Br. Med. Bull. 70: 71, 2004. 24. Daniel, R. K., Priest, D. L., and Wheatley, D. C. Etiologic factors in pressure sores: An experimental model. Arch. Phys. Med. Rehabil. 62: 492, 1981. 25. Mustoe, T. A. Understanding chronic wounds: A unifying hypothesis on their pathogenesis and implications for therapy. Am. J. Surg. 187: 65, 2004. 26. Thompson, D. A critical review of the literature on pressure ulcer aetiology. J. Wound Care. 14: 87, 2005. 27. Peirce, S. M., Skalak, T. C., and Rodeheaver, G. T. Ischemiareperfusion injury in chronic pressure ulcer formation: A skin model in the rat. Wound Repair Regen. 8: 68, 2000. 28. Toledo-Pereyra, L. H., Toledo, A. H., Walsh, J., et al. Molecular signaling pathways in ischemia/reperfusion. Exp. Clin. Transplant. 2: 174, 2004. 29. Anaya-Prado, R., and Toledo-Pereyra, L. H. The molecular events underlying ischemia/reperfusion injury. Transplant. Proc. 34: 2518, 2002. 30. Toledo-Pereyra, L. H., Lopez-Neblina, F., and Toledo, A. H. Reactive oxygen species and molecular biology of ischemia/ reperfusion. Ann. Transplant. 9: 81, 2004. 31. Seal, J. B., and Gewertz, B. L. Vascular dysfunction in ischemia-reperfusion injury. Ann. Vasc. Surg. 19: 572, 2005. 32. Mannarino, E., Pasqualini, L., Maragoni, G., et al. Chronic venous incompetence and transcutaneous oxygen pressure: A controlled study. Vasa 17: 159, 1988. 33. Browse, N. L., and Burnand, K. G. The cause of venous ulceration. Lancet 2: 243, 1982. 34. Chant, A. The biomechanics of leg ulceration. Ann. R. Coll. Surg. Engl. 81: 80, 1999. 35. Fagrell, B. Microcirculatory disturbances: The final cause for venous leg ulcers? Vasa 11: 101, 1982.

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36. Cheatle, T. R., Sarin, S., Coleridge-Smith, P. D., et al. The pathogenesis of skin damage in venous disease: A review. Eur. J. Vasc. Surg. 5: 115, 1991. 37. Angel, M. F., Ramasastry, S. S., Swartz, W. M., et al. The causes of skin ulcerations associated with venous insufficiency: A unifying hypothesis. Plast. Reconstr. Surg. 79: 289, 1987. 38. Coleridge-Smith, P. D. Deleterious effects of white cells in the course of skin damage in CVI. Int. Angiol. 21: 26, 2002. 39. He, C. F., Cherry, G. W., and Arnold F. Postural vasoregulation and mediators of reperfusion injury in venous ulceration. J. Vasc. Surg. 25: 647, 1997. 40. Coleridge-Smith, P. D. From skin disorders to venous leg ulcers: Pathophysiology and efficacy of Daflon 500 mg in ulcer healing. Angiology 54: 45, 2003. 41. Bouskela, E., Cyrino, F. Z., and Lerond, L. Microvascular reactivity after ischemia/reperfusion in the hamster cheek pouch: Beneficial effects of different oral doses of S-5682 (Daflon 500 mg). Angiology 48: 33, 1997. 42. Thomas, P. R., Nash, G. B., and Dormandy, J. A. White cell accumulation in dependent legs of patients with venous hypertension: A possible mechanism for trophic changes in the skin. Br. Med. J. 296: 1693, 1988. 43. Lowthian, P. Notes on the pathogenesis of serious pressure sores. Br. J. Nurs. 6: 907, 1997. 44. Schubert, V., and Heraud, J. The effects of pressure and shear on skin microcirculation in elderly stroke patients lying in supine or semi-recumbent positions. Age Ageing 23: 405, 1994. 45. Woolsey, R. M., and McGarry, J. D. The cause, prevention, and treatment of pressure sores. Neurol. Clin. 9: 797, 1991. 46. Salcido, R., Donofrio, J. C., Fisher, S. B., et al. Histopathology of pressure ulcers as a result of sequential computer-controlled pressure sessions in a fuzzy rat model. Adv. Wound Care. 7: 23, 1994. 47. Reid, R. R., Sull, A. C., Mogford, J. E., et al. A novel murine model of cyclical cutaneous ischemia-reperfusion injury. J. Surg. Res. 116: 172, 2004. 48. Jeffcoate, W. J., and Harding, K. G. Diabetic foot ulcers. Lancet 361: 1545, 2003. 49. Delamaire, M., Maugendre, D., Moreno, M., et al. Impaired leucocyte functions in diabetic patients. Diabet. Med. 14: 29, 1997. 50. Brem, H., Sheehan, P., and Boulton, A. J. Protocol for treatment of diabetic foot ulcers. Am. J. Surg. 187: 1, 2004. 51. Wlaschek, M., and Scharffetter-Kochanek, K. Oxidative stress in chronic venous leg ulcers. Wound Repair Regen. 13: 452, 2005. 52. Marucha, P. T., Kiecolt-Glaser, J. K., and Favagehi, M. Mucosal wound healing is impaired by examination stress. Psychosom. Med. 60: 362, 1998. 53. Mogford, J. E., Sisco, M., Bonomo, S. R., et al. Impact of aging on gene expression in a rat model of ischemic cutaneous wound healing. J. Surg. Res. 118: 190, 2004. 54. Tandara, A. A., Kloeters, O., Kim, I., Mogford, J. E., and Mustoe, T. A. Age effect on HSP70: Decreased resistance to ischemic and oxidative stress. J. Surg. Res. 132: 32, 2006 (Epub Nov. 16, 2005). 55. Gurjala, A., Liu, W. R., Mogford, J. E., Procaccini, P. S., and Mustoe, T. A. Age-dependent response of primary human dermal fibroblasts to oxidative stress: Cell survival, pro-survival kinases, and entrance into cellular senescence. Wound Repair Regen. 13: 565, 2005. 56. Wright, W. E., and Shay, J. W. The two-stage mechanism controlling cellular senescence and immortalization. Exp. Gerontol. 27: 383, 1992. 57. Bodnar, A. G., Ouellette, M., Frolkis, M., et al. Extension of life-span by introduction of telomerase into normal human cells. Science 279: 349, 1998. 58. Wu, L., Xia, Y., Siddiqui, A., et al. TGFB1 fails to stimulate wound healing and impairs its signal transduction in an aged ischemic ulcer model: The importance of oxygen and age. Am. J. Pathol. 154: 301, 1999. 59. Rosenberg, D. S., Lu, L., Gurjala, A., et al. Aged rats display increased ischemia perfusion skin injury in the in vivo magnet model. Wound Repair Regen. 12: A12, 2004. 60. Zhao, L., Davidson, J. D., Wees, S. C., et al. Total reversal of ischemic healing deficit by hyperbaric oxygen plus growth factors in a rabbit ear model. Arch. Surg. 129: 1045, 1994.

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