RECONSTRUCTIVE

Chronic Wound Pathogenesis and CurrentTreatment Strategies: A Unifying Hypothesis

Thomas A. Mustoe, M.D.Kristina O’Shaughnessy,

M.D.Oliver Kloeters, M.D.

Chicago, Ill.

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

Chronic wounds are a health problem ofenormous magnitude affecting many hun-dreds of thousands of patients. In fact, the

health care cost amounts to billions of dollarsannually in the United States alone. Chronicwounds do not occur in animals, and establish-ing a true chronic wound model in animals hasbeen extremely difficult. From a practical stand-point of feasible experimental studies, it hasbeen impossible. Therefore, much of our under-standing of chronic wounds has come from hu-mans and, by necessity, has been limited to ob-servation, biopsy, and analysis of wound fluids.These limitations have resulted in significantgaps in our knowledge of etiology and pathogen-esis. There exists a perception that humanwounds have some unique deficiency resultingin a failure to heal. The difficulty in treatingchronic wounds has been underscored by thelimited success of growth factors in treatment1,2

and the failure of so many clinical trials andbiotechnology companies that pegged their fu-ture on the success of those trials. Finally,chronic wounds have diverse etiologies. Morethan 90 percent of all chronic wounds fall intothree categories: venous ulcers, pressure sores,

and diabetic ulcers. At first consideration, thesethree types of chronic wounds appear to havelittle in common, but examining their common-alities in more detail leads to the hypothesis thathuman chronic wounds do not have a uniquedefect but represent a combination of factors.Each component of pathogenesis is deleterious,but in aggregate they collectively overwhelm thehealing response in many patients.

THE EFFECT OF LOCAL TISSUEHYPOXIA

Tissue ischemia profoundly impairs healing.Clinically, vascular surgeons widely accept theguideline that lower extremities with a partial ox-ygen pressure at the toe of less than 30 mmHg willnot heal. In animal studies of the rabbit ear,3,4

tissue partial oxygen pressure drops from the nor-mal 40 to 45 mmHg in controls to 28 to 30 mmHgin ischemic ears, leading to a wound-healing ratereduction of 80 percent at 7 days. Ischemic woundmodels have been devised in many species, all witha finding of major impairment in wound healing.5However, ischemia and tissue hypoxia are not syn-onymous. Tissue partial oxygen pressure is af-fected by local blood flow, which in turn is affectedby arterial flow, arteriovenous gradients, capillarydensity, and local tissue consumption. Manychronic wounds occur without the presence ofeasily measurable ischemia.

In consideration of the three common types ofchronic wounds, it is widely understood that dia-betic ulcers frequently occur in the setting of un-

From the Division of Plastic and Reconstructive Surgery,Northwestern University, Feinberg School of Medicine.Received for publication December 29, 2005; revised March12, 2006.Copyright ©2006 by the American Society of Plastic Surgeons

DOI: 10.1097/01.prs.0000225431.63010.1b

www.plasreconsurg.org 35S

derlying atherosclerotic large vessel disease withchronic ischemia of varying degrees. Althoughlarge vessel disease is not an issue for venous ulcersand pressure sores, many of the most recalcitrantwounds occur in the setting of a previous historyof ulceration. Subclinical injury is reflected byperiwound fibrosis and local reduction in tissueperfusion. Edema is another extremely importantfactor. By increasing the distance between capil-laries at the local tissue level and therefore thedistance for oxygen diffusion, edema lowers localoxygen levels, although the magnitude of this fac-tor has not been measured. Interestingly, in ve-nous ulcers in which edema plays the largest role,multiple clinical studies using compression ban-dages as a single variable in controlling tissueedema have demonstrated this to have the largesteffect on successful wound closure rates.

Compression bandages or compression gar-ments are now part of every standard care guide-line for venous ulcers. The success of intermittentcompression by a sequential compression deviceto augment compression garments in particularlydifficult cases (personal observations) supportsthe hypothesis that edema by itself results in mean-ingful drops in local tissue perfusion and oxygen-ation. Although the concept that edema plays arole in diabetic foot ulcers and pressure sores isless well accepted, the combination of depen-dency and inflammation results in local peri-wound edema. The therapeutic success of vacu-um-assisted closure, in which pressure is applied toreduce local edema in the treatment of thesewounds, also supports the hypothesis that in allchronic wounds the local environment has re-duced oxygenation.

THE ROLE OF BACTERIAAn open wound becomes contaminated with

bacteria from the surrounding skin within 48hours, and so all chronic wounds have measurablebacteria counts.6 In the absence of cellulitis, it isoften not possible to distinguish normal coloni-zation from excessive bacteria or critical coloni-zation. The host immune response to bacteria isinflammation, chiefly an influx of polymorpho-nuclear leukocytes that release proteases and ox-idants, which not only degrade cytokines and theextracellular matrix but also place stress on localcells.

Classic studies by Robson have demonstratedfailure of skin graft survival when grafts are placedon a wound bed with bacterial counts in excess of100,000/mm3.7 The senior author (T.A.M.) hastreated several chronic pilonidal wounds in young

patients, some with a several-year history of a non-healing wound. When aggressive local measureswere taken to reduce bacterial counts (frequentwashing, water irrigation, hair removal by shavingthe area, and use of topical antibiotics), thesechronic wounds went on to heal rapidly. In otherwords, critical colonization is by itself sufficient toresult in a chronic wound.

Analysis of wound fluid from chronic wounds,as compared with acute surgical wounds, demon-strates increased protease levels, increased proin-flammatory cytokine levels,8–10 and a reduction ingrowth factor levels, supporting the role that bac-teria play in most chronic wounds. The fibronec-tin degradation observed in chronic wounds,11 aswell as other matrix degradation, could explain toa significant degree the delay in epithelializationobserved in chronic wounds in which the woundmargins have heaped up epithelial margins withactively dividing cells but a failure to migrate. Bio-film overlying a chronic wound protects bacteriafrom host defenses.12 The type of bacteria colo-nizing a wound also plays a role. Recently, it hasbeen recognized that anaerobic bacteria arepresent in most wounds when looked for usingcareful culturing techniques.13 Several studies14,15

have reported that healing wounds are character-ized by a reduction in bacteria counts and markersof inflammation.

Although a large body of evidence supportsthe importance of bacteria and the resulting in-flammatory response in the etiology of chronicwounds, there are still substantial gaps in knowl-edge.

THE ROLE OF ISCHEMIA-REPERFUSIONINJURY

Reperfusion, while necessary to limit irrevers-ible cellular injury in the setting of ischemia, canparadoxically result in additional tissue compro-mise. Collectively referred to as ischemia-reperfusion injury, this complex cascade of molec-ular and cellular events has significant clinicalimplications. The adverse effects are evident in or-gan transplantation,16,17 myocardial infarction,18

stroke, 19 free flap transfers,20 aortic aneurysm re-pair,21 and several other pathological entities22,23;however, ischemia-reperfusion injury has not beencommonly discussed in relation to the developmentand potentiation of chronic wounds. In fact, al-though ischemia has been implicated in the patho-genesis of certain chronic wounds, such as pressuresores, 24 only during recent years has reperfusionbeen suggested as an additional etiologic factor.25–27

Despite the expanding depth of knowledge and

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understanding of ischemia and reperfusion, includ-ing signaling transduction pathways28 and molecu-lar events regulating injury,29 the pathophysiologyand its many subtleties have yet to be fully eluci-dated.

The fundamental mechanisms that lead toischemia-reperfusion injury is similar in all organsystems, especially at the molecular level, wherereactive oxygen species and inflammation are keymediators in establishing tissue injury.30 However,there are important distinctions that make isch-emia-reperfusion injury unique in the setting ofchronic wounds. Most importantly, ischemia andreperfusion events occur in a repetitive fashionsuch that the injury is potentiated with each cycle.Clinically overt tissue damage becomes evident inpatients who are unable to avoid this cyclic-de-pendent injury. For example, the patient with ve-nous leg ulcers alternates between recumbent andambulatory leg positioning. In simplistic terms,ischemia occurs during leg dependency andreperfusion occurs during leg elevation. In an am-bulatory patient, this is an unavoidable sequenceof events that can result in severe tissue injury.Similarly, most critically ill patients or persons withparaplegia are often on strict turning regimens.Such a regimen, while well intentioned, results incyclic intervals of variable blood flow that may havedetrimental clinical effects. Cutaneous tissues en-dure a period of pressure-induced ischemia whenthe patient is recumbent followed by hyperemiawhen a change in position provides reestablish-ment of blood flow.

ISCHEMIA AND REPERFUSION:MOLECULAR BIOLOGY

Ischemia-reperfusion injury is characterizedby a sequence of biochemical and cellularevents that causes extensive cellular damagethrough pathways that lead to leukocyte andcomplement activation, oxidative stress, andmicrovasculature dysfunction.23 The process be-gins with an inciting ischemic event that, on acellular level, reduces ATP production throughimpairment of mitochondrial oxidative phos-phorylation. A reduction in ATP sources resultsin loss of the transmembrane potential and aninflux of various ionic species, leading to cellswelling. Increased levels of calcium ion in thecytosol lead to activation of signal transductionpathways that activate enzymes responsible forcell membrane disruption.30 In addition, isch-emia induces the expression of endothelial ad-hesion molecules and cytokines that prime thetissue for further insult by creating a proinflam-

matory state. Upon reperfusion, leukocytes be-come activated and interact with the endothe-lium through rolling, adherence, andsubsequent transmigration into the interstitialcompartment. Regulated by preformed media-tors, the inflammatory cascade ensues, causingdeleterious cellular reactions and tissuedamage.29 Reoxygenation further impairs bothvascular and cellular processes through theoverproduction of reactive oxygen species, aprocess termed oxidative stress, which over-whelms the endogenous defense mechanisms,thereby causing cytotoxic injury to surroundingtissues.28 Impaired vasorelaxation due to a de-crease in nitrous oxide expression duringreperfusion contributes significantly to micro-vascular dysfunction.31 This process, along withleukocyte trapping, leads to the no-reflow phe-nomenon, described as failure to restore flowto an ischemic organ despite reperfusion.23 Inthe setting of chronic wounds, these events re-cur with each cycle of ischemia and reperfu-sion. The additive effects of leukocyte and com-plement activation, oxidative damage, andmicrovascular dysfunction cause repetitive tis-sue injury and eventual tissue necrosis.

ISCHEMIA AND REPERFUSIONPATHOGENESIS IN CHRONIC WOUNDS

The sequence of events leading to venous skinulcerations and the mechanisms that maintaintheir chronic nature continue to cause contro-versy. Historically, physicians and scientists havefocused on incompetent valves, venous stasis, andultimately ischemia as the etiology of ulcerformation.32 Although the development of isch-emia through venous hypertension, edema, fibrindeposition, and microvascular changes is well doc-umented, these factors do not fully explain theetiology of chronic venous stasis.33–35 Only in re-cent years have repeated ischemia/reperfusioncycles34,36 and inflammatory mechanisms37,38 beenhypothesized to occur in the development andpersistence of chronic venous wounds. A humanstudy using laser Doppler scanning techniquesshowed postural vasoregulation to cause relativeischemia and reperfusion in leg ulcers.39 Venoushypertension leads to leukocyte sequestration,and upon reperfusion, as seen with leg elevation,the leukocytes are activated and reactive oxygenspecies cause further injury to the already isch-emic tissues.37 Further support is reflected by theoverabundance of neutrophils and vulnerability toinfection seen with venous stasis ulcers37 and bythe clinical efficacy of anti-inflammatory drugs re-

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ported in both humans with venous disease40 andin animal models of ischemia-reperfusion injury.41

Furthermore, the leukocyte trapping seen duringvenous hypertension42 and subsequent release ofinflammatory mediators invoke not only a localinflammatory response but also a systemic inflam-matory response, which has been documented byblood sampling in humans38 and is described inother ischemia-reperfusion injury models.23

It is agreed that the etiology of pressure soresis multifactorial and dependent on a variety offactors, including the overall health of the pa-tient, pressure-induced ischemia, and shearinjury.43– 45 Some authors have recently sug-gested that reperfusion injury is a major con-tributing determinant.26,27,46 Again, it is impor-tant to emphasize the repetitive nature ofischemia and reperfusion in relation to pres-sure sore development. In both rat and mousemodels, reproducing ischemia- and reperfu-sion-mediated pressure sores through con-trolled periodic magnetic force compressionand subsequent reperfusion indicate that theextent of tissue damage is attributable to thetotal number of ischemia-reperfusion cycles.27,47

This has been demonstrated in our laboratorywith the mouse ischemia-reperfusion–mediatedpressure sore model. More importantly, whenischemia is held constant, a greater number ofreperfusion events result in increased tissuedamage, and authors have concluded that re-peated ischemia-reperfusion injuries are moredamaging to tissue than prolonged ischemiaalone.27,47 These animal studies support the hy-pothesis that reperfusion is an important com-ponent of pressure sore etiology.

The diabetic population with neuropathy isat risk for ulcerations on weightbearing portionsof the foot, secondary to a lack of ability toperceive excessive pressure or pain; the alteredfoot mechanics secondary to loss of intrinsicmusculature; and a shortened Achilles tendon.48

After even a seemingly minor initial injury, thesewounds can quickly progress because of an im-paired leukocyte response to bacteria49 and re-peated trauma and ischemic injury.50 As with theother types of chronic wounds, we hypothesizethat both reperfusion, upon release of pressure,and the additive effect of repeated ischemia-reperfusion cycles would further escalate theamount of tissue injury. Many of the main me-diators of ischemia-reperfusion injury are al-ready altered in these patients, which likely de-lays wound healing even further.

STRATEGIES TO OVERCOMEISCHEMIA-REPERFUSION INJURYSome of the current treatment strategies uti-

lized in chronic wounds include offloading, com-pression, warming, vacuum-assisted closure de-vices, irrigation, debridement, and topicalantibiotics or growth factors. Although in combi-nation these treatments may demonstrate success,persistence or recurrence of chronic wounds re-mains a significant problem. The recent advancesin understanding the pathophysiology of isch-emia-reperfusion injury in other organ systemshave led to innovative strategies for treatment,such as ischemic preconditioning, anti-inflamma-tory agents, antioxidants, and complementtherapy.23 Although many of these therapies toovercome general ischemia-reperfusion injuryhave been described in the literature and are usedto some degree, human clinical trials for efficacyare lacking. Furthermore, cyclic ischemia-reper-fusion injury as it relates to venous ulcers, pressuresores, and diabetic wounds remains a novel con-cept that has not been widely discussed or em-phasized. Therefore, continued efforts utilizing invivo models and human studies are necessary. Cur-rently, the potential for development of effective,fast-acting, and long-term therapeutics is depen-dent on a better understanding of the pathophys-iological processes underlying chronic wound eti-ology.

THE ROLE OF AGING AND THEIMPAIRED STRESS RESPONSE

Most chronic wounds occur in the elderly. Inreviewing clinical studies on venous ulcers, dia-betic ulcers, and pressure sores, the average age ofpatients with these types of chronic wounds is over60 years.25 Cells in the local environment of achronic wound are subject to significant stresses,including chronic inflammation, the harmfulpresence of reactive oxygen species,51 and a pro-teolytic environment secondary to bacteria, un-derlying ischemia, and recurrent ischemia-reper-fusion cycles. Clinical observations have longsupported the ability to safely operate on the el-derly, but when the stress is severe, such as with amajor burn, the elderly are unable to respond. Forexample, in patients with a 50 percent total bodysurface area burn, the survival rate in major burncenters is high and relatively constant from thesecond through the fifth decade, when survivalfalls off. Over the age of 80, the survival rate froma 50 percent burn is unprecedented. Even inyoung patients stress results in impaired healing.

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In a group of students undergoing the psycholog-ical stress of examinations, experimental woundsin the oral cavity had a significant delay in healingcompared with control wounds.52

There has been increasing experimental evi-dence detailing the impaired response of agingcells to stress that involves broadly altered geneexpression. Microarray analysis of human fibro-blasts from aged patients compared with youngadults reveals a baseline increase in expression ofstress response genes, as well as a broad range ofgenes in other signal transduction pathways. How-ever, the aged human fibroblasts fail to upregulateunder hypoxic conditions.53 A more detailed anal-ysis of the human aged fibroblast response to ox-idant stress plus ischemia, which replicates con-ditions found in chronic wounds, revealsincreased cell death when compared with youngadult cells. These findings are coupled with a fail-ure to upregulate heat shock protein 70; however,transfection with heat shock protein 70 partiallyreversed the impaired response of aged fibro-blasts, suggesting a causative effect.54

One theory for chronic wound etiology con-centrates on cells in the local wound environment.Subjected to chronic stress and prolonged cyto-kine exposure, cells constantly replicate and turn-over, ultimately entering into a replicative senes-cence where they become unable to divide. This isin part a potential rationale for the benefits ofaggressive debridement in diabetic ulcers, whichconverts a chronic wound to an acute wound byremoving the wound margins in which many cellsmay have lost their proliferative potential. An invitro study in our group simulated the effect ofstress-induced premature senescence by the ex-posure of young versus aged human dermal fibro-blasts to hydrogen peroxide. We found an in-creased entry rate into senescence, along with areduction in cell viability and phosphorylation ofprosurvival kinases.55 Other in vitro studies haveconfirmed the theory of an altered stress responsein aged cells, as they found decreased proliferativecapacity and prolonged doubling times subse-quently ending in replicative senescence.56 Thesein vitro data add support to the theory that theprevalent stress in chronic wounds is a deleteriousfactor, especially in the elderly. In addition, trans-fecting cell lines that approach replicative senes-cence with the telomerase gene indefinitely pro-longed senescence in vitro, with persistence of thephenotype of nontransformed cell lines.57 Em-ploying an adenoviral construct overexpressingthe telomerase gene in aged rabbits improvedwound healing significantly (J. E. Mogford and

T. A. Mustoe, unpublished observations), suggest-ing a new, promising therapeutic strategy.

In summary, both clinical and laboratory ob-servations support the hypothesis that the alteredresponse in the aged to ischemic stress,58 or thecombined ischemic and oxidant stress of ischemia-reperfusion injury,59 contributes a great deal tothe impaired healing response.

THERAPEUTIC IMPLICATIONS FORTHE TREATMENT OF CHRONIC

WOUNDSFrom the above discussion, we can conclude

that chronic wounds are multifactorial in etiology.The lack of success of single-agent growth factortherapy is, therefore, not surprising. One therapythat has achieved broad acceptance, despite a lackof prospective randomized studies, is vacuum-as-sisted closure (negative pressure wound therapy).In fact, this single-modality therapy addresses si-multaneously many of the issues discussed above.(1) Bacterial counts are substantially reduced bycontinuous removal of the exudates that serve asa culture medium, and there is a documentedreduction in inflammation. (2) The negative pres-sure reduces edema and therefore increases oxy-gen and nutrient delivery to the local area of ther-apy. In the best-studied chronic wound type(venous ulcers), in terms of edema reduction, thesingle most important therapy for improving heal-ing rates is appropriate use of compression withedema control. In addition, although not well doc-umented for negative pressure wound therapy,other studies demonstrate a beneficial effect oflocal elevation of temperature on healing inchronic wounds, and it is likely that negative pres-sure wound therapy has this effect secondary tothe closed system. (3) As long as the vacuum-assisted closure device is in place, its bulk almostcertainly prevents the patient from putting pres-sure on the area, and thus cycles of ischemia andreperfusion are prevented. (4) Theoretically, it ispossible that the beneficial effects of negative pres-sure on blood flow also increase the likelihoodthat circulating mesenchymal progenitor cells willrepopulate a wound in which some of the cellshave entered into replicative senescence.

In the future, major advances in therapy arelikely to come from addressing multiple factors atone time. The recent advances in understandingthe pathophysiology of ischemia-reperfusion in-jury in other organ systems have led to innovativestrategies for treatment, such as ischemic precon-ditioning, anti-inflammatory agents, antioxidants,and complement therapy.23 Although many of

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these therapies to overcome general ischemia-reperfusion injury have been described in the lit-erature, human clinical trials are lacking. Novelstrategies for reversal of some of the deleteriouseffects of aging, such as gene delivery of telomer-ase or anti-apoptotic genes, are potential direc-tions. The role of growth factors may well be en-hanced by multimodality therapy. In animalstudies in ischemic wounds, growth factors bythemselves modestly enhance healing, and hyper-baric oxygen by itself modestly enhances healing,but in combination, healing rates are dramaticallyincreased, suggesting at least an additive–-if not asynergistic–-effect.60 Currently, the potential fordevelopment of effective, fast-acting, and long-term therapeutics is dependent on a better un-derstanding of the pathophysiological processesunderlying the etiology of chronic wounds.

Thomas A. Mustoe, M.D.Division of Plastic and Reconstructive Surgery

Northwestern University675 North St. Clair, Suite 19-250

Chicago, Ill. 60611tmustoe@nmh.org

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