Table Of Content

Form Approved REPORT DOCUMENTATION PAGE OMB No. 0704-0188 Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202- 4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE (DD-MM-YYYY) 2. REPORT TYPE 3. DATES COVERED (From - To) 2009 Open Literature 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Medical management of cutaneous sulfur mustard injuries 5b. GRANT NUMBER 5c. PROGRAM ELEMENT NUMBER 6. AUTHOR(S) 5d. PROJECT NUMBER Graham, JS, Stevenson, RS, Mitcheltree, LW, Hamilton, A, Deckert, RR, Lee, RB, FScihliaivpeittaa,k A,M K T, Bloom, JC and 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBER AUNSD A ArDmDyR MESeSd(iEcaSl) Research Institute of Aberdeen Proving Ground, MD Chemical Defense 21010-5400 USAMRICD-P08-005 ATTN: MCMR-CDZ-P 3100 Ricketts Point Road 9. SPONSORING / MONITORING AGENCY NAME(S ) AND ADDRESS(ES) 10. SPONSOR/MONITOR’S ACRONYM(S) US Army Medical Research Aberdeen Proving Ground, MD Institute of Chemical Defense 21010-5400 ATTN: MCMR-CDZ-I 11. SPONSOR/MONITOR’S REPORT 3100 Ricketts Point Road NUMBER(S) 12. DISTRIBUTION / AVAILABILITY STATEMENT Approved for public release; distribution unlimited 13. SUPPLEMENTARY NOTES Published in Toxicology, 263, 47–58, 2009. 14. ABSTRACT See reprint. 15. SUBJECT TERMS Sulfur mustard, Wound healing, Pig, Skin, Treatment, Medical management 16. SECURITY CLASSIFICATION OF: 17. LIMITATION 18. NUMBER 19a. NAME OF RESPONSIBLE PERSON OF ABSTRACT OF PAGES John S. Graham a. REPORT b. ABSTRACT c. THIS PAGE UNLIMITED 12 19b. TELEPHONE NUMBER (include area UNLIMITED UNLIMITED UNLIMITED code) 410-436-1197 Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std. Z39.18 Toxicology263(2009)47–58 ContentslistsavailableatScienceDirect Toxicology journal homepage: www.elsevier.com/locate/toxicol Medical management of cutaneous sulfur mustard injuries JohnS.Grahama,∗,RobertS.Stevensona,LarryW.Mitcheltreeb,TraceyA.Hamiltonb, RobinR.Deckerta,RobynB.Leec,AnnM.Schiavettad aMedicalToxicologyBranchAnalyticalToxicologyDivision,U.S.ArmyMedicalResearchInstituteofChemicalDefense, 3100RickettsPointRoad,AberdeenProvingGround,MD21010,USA bComparativePathologyBranch,ComparativeMedicineDivision,U.S.ArmyMedicalResearchInstituteofChemicalDefense, 3100RickettsPointRoad,AberdeenProvingGround,MD21010,USA cS3,Program,Strategies&OperationsOffice,U.S.ArmyMedicalResearchInstituteofChemicalDefense,3100RickettsPointRoad, 3100RickettsPointRoad,AberdeenProvingGround,MD21010,USA dVeterinaryMedicineandSurgeryBranch,ComparativeMedicineDivision,U.S.ArmyMedicalResearchInstituteofChemicalDefense, 3100RickettsPointRoad,AberdeenProvingGround,MD21010,USA a r t i c l e i n f o a b s t r a c t Articlehistory: Background:Sulfurmustard(2,2(cid:3)-dichlorodiethylsulfide;HD)isapotentvesicatingchemicalwarfare Received18April2008 agentthatposesacontinuingthreattobothmilitaryandcivilianpopulations.SignificantcutaneousHD Receivedinrevisedform25July2008 injuriescantakeseveralmonthstoheal,necessitatelengthyhospitalizations,andresultinlong-term Accepted28July2008 complications.Therearecurrentlynostandardizedoroptimizedmethodsofcasualtymanagement.New Availableonline14August2008 strategiesareneededtoprovideforoptimalandrapidwoundhealing. Objective:Theprimaryaimofthisresearchwastodevelopimprovedclinicalstrategies(treatmentguide- Keywords: lines)foroptimaltreatmentofsuperficialdermal(seconddegree)cutaneousHDinjuries,withthegoalof Sulfurmustard returningdamagedskintooptimalappearanceandnormalfunctionintheshortestperiodoftime. Woundhealing Pig Methods:SuperficialdermalHDinjurieswerecreatedontheventralabdominalsurfaceofweanlingpigs. Skin At48hpost-exposure,lesionswerelaserdebridedandatreatmentadjunctapplied.Culturedepithelial Treatment allograftsand11commercialoff-the-shelf(COTS)productswereexaminedfortheirefficacyinimproving Medicalmanagement woundhealingoftheseinjuries.Clinicalevaluationsandavarietyofnon-invasivebioengineeringmeth- odswereusedat7and14dayspost-surgerytofollowtheprogressofwoundhealingandevaluatevarious cosmeticandfunctionalpropertiesofthewounds.Measurementsincludedreflectancecolorimetryto measureerythema;evaporimetrytoexaminetransepidermalwaterlossasamethodofevaluatingbar- rierfunction;torsionalballistometrytoevaluatethemechanicalpropertiesofskinfirmnessandelasticity; andtwo-dimensionalhighfrequencyultrasonography(HFU)tomonitorskinthickness(e.g.,edema,scar tissue).Histopathologyandimmunohistochemistrywereperformed14daysfollowingsurgerytoexam- inestructuralintegrityandqualityofhealing.LogicalDecisions®forWindowswasusedtorankthe12 treatmentadjunctsthatwerestudied. Results:Themostefficacioustreatmentadjunctsincluded(1)VacuumAssistedClosureTM,V.A.C.®,involv- ingapplicationoftopicalnegativepressure,(2)Amino-Plex®Spray(biO2CosmeceuticalsInternational, Inc.,BeverlyHills,CA),anutritivecosmeceuticalproductthatisdesignedtoincreaseoxygenincells, stimulate ATP synthesis, improve glucose transportation, stimulate collagen formation, and promote angiogenesis,and(3)ReCell®AutologousCellHarvestingDevice(ClinicalCellCultureAmericasLLC,Coral Springs,Florida),aninnovativemedicaldevicethatwasdevelopedtoallowrapidharvestingofautolo- gouscellsfromathinsplit-thicknessbiopsyfollowedbysprayapplicationofapopulationofskincells ontowoundswithin30minofcollectingthebiopsy,withouttheneedofculturingthekeratinocytesina clinicallaboratory. Conclusions:Completere-epithelializationofdebridedHDinjuriesin7daysispossible.Ingeneral,shallow laserdebridementthroughthebasementmembranezone(100(cid:2)m)appearstoprovidebetterresultsthan deeperdebridement(400(cid:2)m)withrespecttoearlyre-epithelialization,cosmeticappearance,functional restoration,andstructuralintegrity.Ofthe12treatmentadjunctsexamined,themostpromisingincluded VacuumAssistedClosureTM,Amino-Plex®Spray,andReCell®AutologousCellHarvestingDevice. PublishedbyElsevierIrelandLtd. ∗ Correspondingauthor.Tel.:+14104361197. E-mailaddresses:[email protected](J.S.Graham),[email protected](R.S.Stevenson),[email protected](L.W.Mitcheltree), [email protected](T.A.Hamilton),[email protected](R.R.Deckert),[email protected](R.B.Lee),[email protected](A.M.Schiavetta). 0300-483X/$–seefrontmatter.PublishedbyElsevierIrelandLtd. doi:10.1016/j.tox.2008.07.067 48 J.S.Grahametal./Toxicology263(2009)47–58 1. Introduction easeControlandPreventionavailableathttp://www.cdc.gov).An in-depth review of the currently recommended treatment reg- Sulfur mustard (2,2(cid:3)-dichlorodiethyl sulfide; HD) is a potent imens as they appear in a variety of military textbooks and vesicatingchemicalwarfareagentthatposesacontinuingthreat handbookshasbeenprovided(Grahametal.,2005). to both military and civilian populations. This agent is inexpen- In spite of the symptomatic management strategies currently sive,easilyobtainableorsynthesized,frequentlystockpiled,easily employed, lengthy healing periods and long-term complications disseminated,cannegativelyimpactcombateffectivenessbyforc- stilloccur.Newstrategiesareneededtoprovideforoptimaland ingmilitaryforcestodonprotectivegear,andhasthepotentialto rapidwoundhealing,andtoamelioratelong-termcomplications. beusedbyterrorists(Khaterietal.,2003;Saladietal.,2006).HD Suchstrategieshaverecentlybeenformulatedbyaninternational primarily affects the lungs, eyes and skin. The chemical proper- working group (Graham et al., 2005). The research described in ties,proposedmechanismsofaction,toxicokinetics,pathogenesis this paper was guided by these strategies. In short, adequate ofinjury,acutetoxiceffects,anddelayedtoxiceffectshavebeen wounddebridementofpartial-thicknessinjuriesisneeded,with extensivelyreviewed(Balali-Moodetal.,2005;Grahametal.,2005; subsequenttreatmentofthelesionsusingcontemporarymedical KeheandSzinicz,2005;McManusandHuebner,2005;Melloretal., approachessimilartothoseappliedforthetreatmentofchronic 1991;Momenietal.,1992;Naraghietal.,2005;SomaniandBabu, cutaneousulcersorpartial-thicknessthermalburns. 1989;Willems,1989). Previous animal studies have shown that surgically aggres- Long-term complications in HD casualties from the Iran–Iraq sive approaches are needed to prevent or minimize significant War (1980–1988) have recently been reported. An extensive cosmeticandfunctionaldeficitsthatresultfromdeepHDinjury review of medical records of Iranian casualties indicated that of (Grahametal.,2002a,b).[Superficial(firstdegree)injuriesinvolve the 100,000 HD casualties one third are suffering effects today only the epidermis, superficial dermal (second degree) injuries (Shohratietal.,2007a).Areviewoflatecomplicationsof34,000 involvetheepidermisandupperthirdofthedermis,deepdermal casualties indicated that the incidences of lung, eye, and skin injuries involve the epidermis and most of the dermis, and full- problems were 42.5%, 39.3%, and 24.5%, respectively (Khateri et thicknessinjuriesinvolvethedestructionofallskinelementsand al., 2003). Common cutaneous problems being reported include sometimesinvolveunderlyingmuscle,tendon,orbone(Arturson, hyperpigmentation, hypopigmentation, atrophy, multiple cherry 1996).]Forthebestoutcome,deepdermal/full-thicknesscutaneous angiomas, sensory loss, burning, pruritis, xerosis, hypohidrosis, HD injuries require full-thickness debridement followed by skin localhairloss,erythematouspapularrash,eczema,scaling,desqua- grafting.WhilepastHDwoundhealingresearchhasconcentrated mation,sensitivitytomechanicalinjurywithrecurrentblistering ondeepdermal/full-thicknessinjuries,superficialandsuperficial and ulceration, and dermal scarring (Balali-Mood et al., 2005; dermalHDinjuriesmayhavegreaterclinicalrelevanceonthebat- Balali-Mood and Hefazi, 2006; Emadi et al., 2008; Hafazi et al., tlefield or in terrorist attacks on civilian populations. Superficial 2006; Khateri et al., 2003; Momeni et al., 1992; Panahi et al., dermalHDinjurieswilllikelynotrequiresuchsurgicallyaggressive 2007;Shohratietal.,2007a,b).Chroniccutaneoussymptomsare approaches(e.g.,serialtangentialexcisionsfollowedbyautologous generallymoresevereduringcolderseasons(Hafazietal.,2006). split-thicknessskingrafting).Theresearchdescribedinthispaper Microscopic examinations have noted mild papillomatosis and focusesonthetreatmentofsuperficialdermalHDinjuries. acanthosisintheepidermiswithpigmentationinthebasallayer, Ingeneral,superficialdermalburnshealwithin21dayswith- atrophy of adnexal structures, non-specific dermal fibrosis and outtreatment.Inweanlingpigs,untreatedsuperficialdermalHD sclerosis,markedepidermalatrophy,orthokeratotichyperkerato- injuriesoftenhaveathindryescharpresent14daysafterexposure sis, perivascular mononuclear infiltrate throughout the papillary thatisjustbeginningtoseparatefromtheunderlying,regenerat- dermis,melanosomeswithintheepidermis,andmelanophagesin ingepithelium.Whilethelesionshaveclinicallyre-epithelialized the upper dermis (Balali-Mood et al., 2005; Emadi et al., 2008; by21days,therearesignificanthistologicalabnormalitiespresent Hafazietal.,2006).Thesepatientshaveahigherincidenceofpso- (Graham et al., 2006). It has not been determined how long it riasisandautoimmunediseasessuchasvitiligoanddiscoidlupus would take tissue architecture to return to normal. The primary erthematosisthanthegeneralpopulation(Balali-MoodandHefazi, aim of this research was to develop improved clinical strategies 2006;Hafazietal.,2006).Lowerincidencesofacnevulgaris,fol- (treatmentguidelines)foroptimaltreatmentofsuperficialdermal liculitis,andtineaversicolorhavebeenreported,probablydueto cutaneous HD injuries, with the goal of returning damaged skin chronicdryskin(Hafazietal.,2006). tooptimalappearanceandnormalfunctionintheshortestperiod Significant cutaneous HD injuries can take several months to oftime.SuperficialdermalHDinjurieswerecreatedontheven- heal (Balali-Mood and Hefazi, 2006; Graham et al., 2005, 2006; tral abdominal surface of weanling pigs. At 48h post-exposure, KeheandSzinicz,2005;Momenietal.,1992;Newmarketal.,2007; selectlesionswerelaserdebridedandatreatmentadjunctapplied. Willems,1989),necessitatelengthyhospitalizations,andresultin Culturedepithelialallograftsandavarietyofcommercialoff-the- considerable cosmetic (e.g., scarring, dyspigmentation, dry skin) shelf(COTS)productswereexaminedfortheirefficacyinimproving and/orfunctional(e.g.,contracturesoverjointsthatlimitmotion, woundhealingoftheseinjuries. fragile skin easily damaged by trauma, hypersensitivity, chronic ulceration) deficits. There are currently no standardized or opti- 2. Materialsandmethods mized methods of casualty management (Graham et al., 2005). Currenttreatmentstrategyconsistsofsymptomaticmanagement 2.1. Animalmodel and is designed to relieve symptoms, prevent infections, and promotehealing.Thecurrentstrategyprimarilyinvolvesderoof- Sixty-sixfemaleYorkshirecrossbredpigs(weanlings),Susscrofa,8–15kg(mean 11.8),wereused(CountryViewFarms,Shanksville,PA).Researchwasconductedin ing large blisters, disinfecting and applying antibiotic creams or compliancewithAnimalWelfareRegulations(7USC,9CFR,Ch1SubchapterAparts ointments, conducting frequent dressing changes, administering 1–4)andotherFederalstatutesandregulationsrelatingtoanimalsandexperiments systemic analgesics and antihistamines, applying topical or sys- involvinganimalsandadheredtoprinciplesstatedintheGuidefortheCareand temicantipruritics,andclosemonitoringoffluidsandelectrolytes UseofLaboratoryAnimals,NationalResearchCouncil,1996.Thefacilitywherethis researchwasconductedisfullyaccreditedbytheAssociationforAssessmentand (Balali-Mood and Hefazi, 2006; McManus and Huebner, 2005; AccreditationofLaboratoryAnimalCareInternational(AAALACInternational). Mellor et al., 1991; Newmark et al., 2007; Saladi et al., 2006; Animalswerequarantinedfor1weekpriortouseonthestudytoscreenforsigns Willems,1989,andrecommendationsbytheU.S.CentersforDis- ofdisease.Pigswerelightlyanesthetizedwithintramuscularinjectionsofxylazine J.S.Grahametal./Toxicology263(2009)47–58 49 HCl(Xyla-Ject®,PhoenixPharmaceutical,Inc.,St.Joseph,MO;2.2mg/kg)andacom- clinicalobservations.Sitesremainedundressedfortheremainderoftheexperi- binationoftiletamineHClandzolazepamHCl(Telazol®,FortDodgeAnimalHealth, ment. FortDodge,IA;6.0mg/kg)forbloodcollection.Blood(8ml)wascollectedfromthe anteriorvenacavaforroutineclinicalpathologybeforeagentexposureandjustprior 2.4. Treatmentadjuncts toeuthanasia. Eachefficacystudyconsistedofsixpigs.Surgicalprocedures(laserdebride- Thefollowingtreatmentadjunctsweretested. mentandapplicationoftreatmentadjunct)wereconductedat48-hpost-exposure. A14-dayhealingperiodfollowedsurgery,duringwhichclinicalobservations,body 1. FlexzanFoamAdhesiveDressing(BertekPharmaceuticalsInc.,Morgantown,WV)is temperaturemeasurements,andnon-invasivebioengineeringmethodswerecon- anultra-thin,semi-occlusivepolyurethanefoamadhesivedressingthatisfre- ductedonaweeklybasis.Attheendofthehealingperiod,animalswerehumanely quentlyusedfollowinglaserfacialresurfacing.Excesswoundfluidisabsorbed euthanizedforhistopathologicalandimmunohistochemicalevaluationofskinsites. intoopencellsofthefoamdressingandevaporatesthroughaclosedcellouter surface.AlightcompressiondressingwasplacedovertheFlexzanusingelastic 2.2. Sulfurmustardexposure tapethatwaswrappedaroundthecircumferenceoftheanimal’storsoandheld inplacebysurgicalstaples.Dressings(includingfreshFlexzan)werechanged Eighteento24hbeforeagentexposure,apre-exposurebodytemperaturewas atfourdayspost-surgery.Thisdressingwastestedinparallelwithfrozencul- measuredandtheventralabdominalskinclippedwithanelectricclipper,andthen turedepithelialporcineallografts(#2below)onthesameanimal,wheretwo depilatedaspreviouslydescribed(Grahametal.,2002b).Immediatelyafterhair HD-exposedsiteswerelaserdebridedtoadepthof400(cid:2)mwithonedebrided removal,4exposuresitesweredemarcatedontheventralabdominalsurface,two sitedressedinFlexzanandtheotherinallograft. sitespersideparalleltoandapproximately2.5cmlateraltotheteatlineandlocated 2. Frozenculturedepithelialporcineallograft(LivingSkinBank,StateUniversityof betweentheaxillaryandinguinalareas.Theinguinalfoldandsharplycurvedareas NewYork,StonyBrook,NY)waspreparedonapetrolatumbackingfrompig oftheribcagewereavoided.Aplastictemplatewasusedforevenspacingandcon- keratinocytescollectedfromanaïvesetofanimalsaspreviouslydescribed sistentanatomicalpositioningofthesitesamonganimals.Smalldotswereplaced (Grahametal.,2006;RandolphandSimon,1993;RheinwaldandGreen,1975), ateachcornerusingapermanentmarkertodelineateeachsite.Eachsitewithin andthawedatroomtemperaturepriortouse.Amoderatecompressiondressing thegridmeasuredapproximately5cmby5cm.Fourexposuresitesweredemar- wasplacedovertheallograftbyplacingKerlixTMSuperSponges(TycoHealth- catedontheventralabdominalsurface.Threeofthefoursitesoneachanimalwere care/Kendall,Mansfield,MA)andRestonTMSelf-AdheringFoamPads(3MHealth exposedtoundilutedliquidsulfurmustardfor8mintoproducesuperficialdermal Care,St.Paul,MN)overeachtreatedsite,followedbyelastictapethatwas injuries.Thefourthsitewasshamexposed(noHD).Thelocationoftheshamsite wrappedaroundthecircumferenceoftheanimal’storsoheldinplacewith wasrotatedamonganimalstoprecludesensitivitybiasesbaseduponanatomical surgicalstaples.Dressings(includingfreshallograft)werechangedatfourdays location.Theexposureproceduresusedtogenerate3-cmdiametersuperficialder- post-surgery.ThisgraftmaterialwastestedinparallelwithFlexzanFoamAdhe- malinjuriesinthecenterofeachHD-exposedsitewerethesameasthosepreviously siveDressing(#1above)onthesameanimal,wheretwoHD-exposedsiteswere described(Grahametal.,2002b);however,inplaceofthetilefloatsacalibration laserdebridedtoadepthof400(cid:2)mwithonedebridedsitedressedinFlexzan weight(300g)wasplacedontopoftherubberstopperandmanuallyheldinplace andtheotherinallograft. forthedurationoftheexposuretoensureevendownwardpressureandcomplete 3. DuoDERMSignal(ConvaTec,Princeton,NJ)isahydrocolloiddressingdesigned contactofthewettedfilterpaperwiththeskin.Priortoexposure,animalswere toprovideamoistwoundhealingenvironment,manageexudate,andhavea lightlyanesthetizedwithXyla-Ject®andTelazol®asdescribedaboveandplacedin longerweartimethantypicalhydrocolloiddressings.Thedressingwasapplied theagenthoodindorsalrecumbency,supportedbyastainlesssteelpigsling.Ather- tositeslaserdebridedtoadepthofeither300or400(cid:2)m.Edgesofthedressings apeuticheatingpad(GaymarIndustries,Inc.,OrchardPark,NY),withthecirculating weresecuredwithsurgicaltapethatwasfurthersecuredwithsurgicalstaples. watertemperaturesetat41◦C,wasplacedundertheanimalduringtheexposure For this study, two different debridement depths were chosen (300 and periodtominimizehypothermia. 400(cid:2)m)toascertainifdepthofdebridementaffectedoutcome.Asnodiffer- enceswerenotedinoutcomeusingthesetwodepths,adecisionwasmadeto 2.3. Debridementandtreatmentprocedures widenthedepthrangeto100and400(cid:2)minsubsequentexperimentsusingthe treatmentadjunctsdescribedbelow. At48hpost-exposure,animalswerelightlyanesthetizedasdescribedabove, 4. Amino-Plex®Spray(biO2CosmeceuticalsInternational,Inc.,BeverlyHills,CA)is intubated,andplacedon1.0–2.5%isofluraneinoxygenataflowrateof0.8–1.5L/min anutritivecosmeceuticalproductthatisdesignedtoincreaseoxygenincells, using an Excel 210SE anesthetic machine with an Isotec 5 isoflurane vaporizer stimulateATPsynthesis,improveglucosetransportation,stimulatecollagenfor- (Datex-Ohmeda,Madison,WI).Attheendofthedebridementandtreatmentappli- mation,andpromoteangiogenesis.Itisamixtureofover100low-molecular cations,theconcentrationofisofluranewasreducedgraduallyuntiltheanimalwas weightingredients,includingaminoacids,traceminerals,nucleotides,nucle- on100%oxygenandthenroomair.Rectaltemperature,pulserate,respirations,and osides, oligopeptides, electrolytes, glycosaminoglycans, and glycolipids. The pulseoximetryweremonitoredthroughoutallprocedures. ingredientsaredissolvedinde-ionizedwater,alongwithminutequantitiesof Followinginductionintoadeepplaneofanesthesia,treatedwoundswerefirst preservativesdissolvedinasmallpercentageofpropyleneglycol.Theproductis debridedslightlybeyondthevisiblebordersofthelesionstoadepthof100,300 providedinapumpspraybottlewithoutpropellants.Accordingtothecompany, or400(cid:2)mwithanerbium:yttrium–aluminum–garnet(Er:YAG)laser(ScitonPro- thisproducthasbeenclinicallyshowntoreduceirritationandimproveresultsin fileLaserSurgicalSystemwithScanner,Sciton,Inc.,PaloAlto,CA),thenatreatment laserresurfacing,chemicalpeels,microdermabrasion,hairtransplantation,and adjunctimmediatelyapplied.Thelasersystem,configuredasahigh-powereddual hairremoval.Amino-Plex®waslightlysprayedonthesurfaceofwoundsthat modelongpulseEr:YAGlaser,offersindependentcontrolofbothdepthofcoagula- werelaserdebridedtoadepthofeither100or400(cid:2)m.Treatedsiteswerethen tion(tocontrolbloodloss)anddepthofablation(fortissueremoval)tospecified, coveredwithTegadermTMAgMesh(3MHealthCare,St.Paul,MN).AnArgyle uniformdepthswithminimalresidualthermaldamage.Operatingparametersfor SalemSumpTube(TycoHealthcareGroupLP,Mansfield,MA)wassuturedin thelaserwereasfollows.Percentoverlapofspotswas50%.Scanningpatternwas placeovertheTegadermTMAgMeshtoallowdailyapplicationsofthenutri- settoasquaremeasuring3cm×3cm.Forthosesitesdebridedinmultiplepasses, tivemixturedirectlyontothewoundwithouttheneedtochangedressings. escharwaswipedoffusingdrygauzebetweeneachpass.Forthoselesionsdebrided Thetubingwasroutedtotheareaoverthedorsalspineandheldinplacewith toadepthof100(cid:2)m,onepassofthelaserwasusedwiththefollowingabla- Mefix®self-adhesivefabrictape(MölnlyckeHealthCareInc.,Norcross,GA),fur- tion/coagulationsettings(in(cid:2)m):100/0.Forthosesitesdebridedtoadepthof thersecuredwithsurgicalstaples.Next,afoamdressing(3MHealthCare,St. 300(cid:2)m,fourpassesofthelaserwereusedwiththefollowingsettings(insequen- Paul,MN)wascuttosizeandplacedoverthetubing(whichwassittingonthe tialorder):80/0,80/0,80/50,and60/0(30%overlap,lastpassonly).Forthosesites TegadermAgMesh),andtheentiredressingcoveredbyTegadermTMTranspar- debridedtoadepthof400(cid:2)m,fivepassesofthelaserwereusedwiththefollowing entDressing(3MHealthCare,St.Paul,MN).EdgesoftheTegadermTransparent settings:100/0,80/0,80/0,80/50,and60/50.Ablationsettingsof100,80,and60(cid:2)m Dressingwerefurthersecuredwithsurgicaltapeandstaples.Tenmillilitersof correspondedtofluencesof25,20,and15J/cm2,respectively. Amino-Plex®wereinjectedintoeachtube(5mlonsubsequentdays),followed On each animal, one HD-exposed site was left untreated (positive con- slowlyby5mlairtosaturatethefoamdressingwiththematerial.Fivepiecesof trol), and the other two HD-exposed sites were debrided with the Er:YAG sterile4(cid:3)(cid:3)×4(cid:3)(cid:3)gauzewerethenputovereachtransparentdressing,whichwas laserandatreatmentadjunctapplied.Thefourthexperimental(sham-exposed, helddownwithstripsofsurgicaltape.Lightcompressionwasthenappliedby untreated) site served as a negative control. As the location of the sham site elastictapethatwascircumferentiallywrappedaroundtheanimalandheld was rotated among animals, treatment sites were also rotated; however, the inplacebysurgicalstaples.Every24haftersurgeryfor7days(includingthe two treated sites on any given animal were at the same cranial–caudal level daythatthedressingswereremoved),animalswerelightlysedatedwith0.5ml to facilitate circumferential application of compression bandages. The positive TelazolandadditionalAmino-Plex®treatmentsappliedasdescribedabove.Fol- andnegativecontrolsites(alsoatthesamecranial–caudallevel)weredressed lowinginjections,theportendsofthetubingwerecappedwithParafilmM® with a single layer of sterile gauze secured in place with surgical tape and (AlcanPackaging,Neenah,WI). staples. After all sites were dressed, a protective cotton stockinette was then 5. ReCell®AutologousCellHarvestingDevice(ClinicalCellCultureAmericasLLC,Coral put in place over the animal’s torso, secured with surgical staples and elas- Springs,FL)isaninnovativemedicaldevicethatwasdevelopedtoallowrapid tictape.Alldressingswereremovedonpost-surgicalday7(PS07)justpriorto harvestingofautologouscellsfromathinsplit-thicknessbiopsyfollowedby 50 J.S.Grahametal./Toxicology263(2009)47–58 sprayapplicationofapopulationofskincellsontowoundswithin30minof 100or400(cid:2)m,treatedsiteswerecoveredwithBiobranewithminimalover- collectingthebiopsy,withouttheneedofculturingthekeratinocytesinaclin- lapontoperilesionalskin,andstapledinplace.Fivesingle4(cid:3)(cid:3)×4(cid:3)(cid:3)sterilegauze icallaboratory.Thissingle-usedeviceisdesignedforinjuriesupto2%total padswereplacedovertheBiobrane®,heldinplacewithsurgicaltapeandsta- bodysurfacearea(TBSA).Thisproductisdesignedforuseinsuperficialder- ples.Lightcompressionwasthenappliedbyelastictapethatwaswrapped mal,deepdermal,andfull-thicknessburns,donorsites,scartreatment,chronic circumferentiallyaroundtheanimal’storsoandheldinplacebysurgicalstaples. ulcers,pigmentloss,andcosmeticskinrejuvenationfollowinglaserresurfacing, 8. AQUACEL®Ag(ConvaTec,Princeton,NJ)isasilverimpregnatedhydrofiberdress- dermabrasion,orchemicalpeels.Followingprocessingoftheskinbiopsy,the ingdesignedforuseonpartial-thicknessburns,donorsites,andchronicskin resultingcellsuspensionprovidesautologouskeratinocytes,epidermalstem ulcers.Itmaybeleftinsituforupto14days,hasalargefluidabsorptioncapacity, cells,melanocytes,fibroblasts,andLangerhanscells.Briefly,asplit-thickness andkillsabroadspectrumofwoundpathogens,includingPseudomonasaerugi- biopsy(250(cid:2)mthick)measuringapproximately5cm×5cmwasaseptically nosa,Staphylococcusaureus,methicillin-resistantStaphylococcusaureus(MRSA), removedfromtherightparavertebralareaofeachanimalusinganitrogen- andvancomycin-resistantEnterococcus(VRE).Dressingswereappliedtosites drivendermatome(Zimmer,Inc.,Warsaw,IN)followingsubcutaneousinjection thathadbeenlaserdebridedtoadepthof100or400(cid:2)m.Thedressingswere ofapproximately35mlsterilesalinetostiffenupthearea.Thisdonorsitewas coveredwithsterilegauze.Lightcompressionwasthenappliedbyelastictape dressedinxeroformpetrolatumdressing(SherwoodMedical,St.Louis,MO)and thatwaswrappedcircumferentiallyaroundtheanimal’storsoandheldinplace sterilegauze,securedbysurgicalstaplesandleftinplacefor7days.Thebiopsy bysurgicalstaples. wastrimmedintotwopieceseachmeasuringapproximately1.5cm×1.5cm. 9. ACTICOAT7DayAntimicrobialDressing(Smith&Nephew,Inc.,Largo,FL)consists Lyophilizedtrypsin(0.75%)wasreconstitutedwith10mlofsterilewater,dis- oftwolayersofanabsorbent,rayon/polyesterinnercorebetweenthreelayers pensedintoawarmingchamberintheReCell® kit,andpre-heatedto37◦C. ofsilver-coatedpolyethylenemesh,designedforuseonpartial-thicknessburns, Eachpieceoftrimmedtissuewasplacedintotheheatedtrypsinsolutionfor donorsites,andchronicskinulcers.Theinnercoremaintainsamoistwound 25min(usingseparatekits)toallowtheepidermistobeseparatedfromthe healingenvironment.Thedressingmaybeleftinsituforupto7days,andkills dermispriortofurtherprocessing.After25min,thetissuewasremovedfrom abroadspectrumofwoundpathogens.Followinglaserdebridementtoadepth thetrypsinsolutionandbrieflyrinsedinasterilesodiumlactatesolutionto of100or400(cid:2)m,ACTICOAT7dressingswereplacedoverthepreparedwound stopthetrypsinizationprocess.Usingsterilefinetippedforceps,theepidermis bedsandwettedwithsterilewatertoactivatethedressing.TheACTICOAT7 wasseparatedfromthedermisinthekit’ssterilepetridish.Afewdropsoffresh dressingswerethencoveredwithAllevynAdhesivehydrocellularpolyurethane sodiumlactatesolutionwerethendrippedontothedermal–epidermaljunction dressings(Smith&Nephew,Inc.,Largo,FL).Lightcompressionwasthenapplied ofbothlayers.Thecellsfromthejunctionalsurfaceswerethenscrapedwitha byelastictapethatwaswrappedcircumferentiallyaroundtheanimal’storso sterilescalpeltodevelopaplumeofcells.Thepetridishwasthenrinsedwith andheldinplacebysurgicalstaples. 1.5mloffreshsodiumlactatesolution.Theplumeofcells,suspendedinthe 10. Silon-TSR® TemporarySkinReplacement(BioMedSciences,Inc.,Allentown,PA) sodiumlactatesolution,wasthendrawnupintoasterilesyringefittedwitha isacomplexweaveofbiopolymersthatproduceathinprotectivemembrane, blunt-tipped18-gaugeneedleandaspiratedseveraltimestocreateacellsus- andisdesignedforuseonpartial-thicknessburns,donorsites,andlaserresur- pension.Thefinalcellsuspensionwasthendispensedthroughacellstrainerinto facedskin.Thedressingisperforatedtoallowexudatetoescape.Followinglaser aconicalwellinthekit.Theentirefilteredcellsuspensionfromeachpieceoftis- debridementtoadepthof100or400(cid:2)m,Silon-TSR®dressingswereplacedon suewasdrawnupintoanewsterilesyringefittedwithablunt-tipped18-gauge thepreparedwoundbedsandsecuredwithsurgicaltapeandstaples.Thedress- needleandthendrippedontoanHD-exposedsitethathadbeenlaserdebrided ingswerethencoveredwithKerlixTMsupersponges(TycoHealthcare/Kendall, toadepthofeither100or400(cid:2)m.Immediatelyfollowingapplicationofthe Mansfield,MA).Lightcompressionwasthenappliedbyelastictapethatwas cellsuspension,eachtreatedwoundwascoveredwithSURFASOFTFixativefor wrappedcircumferentiallyaroundtheanimal’storsoandheldinplacebysur- SkinGrafts(MEDIPROF,Holland),securedwithsurgicalstaples,overwhichwas gicalstaples. placedsterilegauzesecuredwithsurgicaltapeandstaples.Lightcompression 11. APLIGRAF®(Organogenesis,Inc.,Canton,MA)isalivingbi-layeredskinsubstitute wasthenappliedbyelastictapethatwascircumferentiallywrappedaroundthe designedforthetreatmentofvenouslegulcersanddiabeticfootulcers.Accord- animalandheldinplacebysurgicalstaples. ingtothemanufacturer,“likehumanskinApligrafconsistsoflivingcellsand 6. Applicationoftopicalnegativepressure(alsoknownasVacuumAssistedClosureTM, structuralproteins.Thelowerdermallayercombinesbovinetype1collagenand V.A.C.®)involvesplacinganopencellfoamintothewoundbed(cuttoconform humanfibroblasts(dermalcells),whichproduceadditionalmatrixproteins.The totheshapeofthewound),sealingitwithanadhesivedrape,andapplying upperepidermallayerisformedbypromotinghumankeratinocytes(epidermal subatmosphericpressure(125mmHgbelowambient)thatistransmittedvia cells)firsttomultiplyandthentodifferentiatetoreplicatethearchitectureofthe anevacuationtubebyacomputerizedvacuumpump.SeveralV.A.C.®therapy humanepidermis.Unlikehumanskin,Apligrafdoesnotcontainmelanocytes, systemsareavailablefromKineticConcepts,Inc.(KCI),SanAntonio,TX.The Langerhans’cells,macrophages,andlymphocytes,orotherstructuressuchas unitusedintheseexperimentswastheV.A.C.ATS® System.Laserdebride- bloodvessels,hairfolliclesorsweatglands.”APLIGRAF®wasappliedtosites mentwasconductedtoadepthof100or400(cid:2)m.Priortoapplicationofthe thatwerelaserdebridedtoadepthof100or400(cid:2)m.Theproductwasstapled V.A.C.®dressingsaccordingtomanufacturer’sinstructions,areassurrounding inplaceusingsurgicalstaplesandthencoveredwithasinglepieceofxero- thetreatedsiteswerecloseshavedwitharazor,washedwith20%soapsolu- formpetrolatumdressing(SherwoodMedical,St.Louis,MO)thatwasfolded tion,andrinsedwithsaline.Mastisol®liquidadhesive(FerndaleLaboratories, intoquarters.Fivesingle4(cid:3)(cid:3)×4(cid:3)(cid:3)sterilegauzepadswereplacedoverthexero- Inc.,Ferndale,MI)wasblottedaroundthetreatedareas(afterdebridement) form,heldinplacewithDuraporetapeandsurgicalstaples.Lightcompression wheretheV.A.C.®drapewastobeadheredtothesurroundingskin.Debrided wasthenappliedbyelastictapethatwaswrappedcircumferentiallyaroundthe siteswerecoveredfirstwithTELFATMclearnon-adhesivewounddressing(Tyco animalandheldinplacebysurgicalstaples. Healthcare/Kendall,Mansfield,MA)thenaGranuFoamsilverdressing(KCI,San 12. Promogran*MatrixWoundDressing(Johnson&JohnsonWoundManagement, Antonio,TX)thathadbeenbisectedwidth-wise.ThepurposeoftheTELFATM Somerville,NJ)isafreezedriedcompositeof45%oxidizedregeneratedcellu- dressingwastopreventingrowthofcellsintotheV.A.C.’sGranuFoamdressing. lose(ORC)and55%collagenforuseonpartial-thicknessburns,donorsites,and Afterbothtreatedlesionshadbeendressed,abridgewasconstructedofV.A.C.® chronicskinulcers.Itisdesignedtobindmatrixmetalloproteasesandprotect drapematerialandGranuFoamsilverdressingtoconnectthetwosites.Both growthfactors.Treatedsiteswerelaserdebridedtoadepthof100or400(cid:2)m. treatmentareasandthebridgewerecoveredwithasingleV.A.C.®drape,which Promogran*dressingswerethencuttoshape,wettedwithsterilewater,placed hadbeentrimmedtoproperwidthbeforeapplication.Theedgesofthedrape onthepreparedwoundbeds,andcoveredwithAllevynAdhesivehydrocellu- werefurthersecuredwithMefix®self-adhesivefabrictape(MölnlyckeHealth larpolyurethanedressings(Smith&Nephew,Inc.,Largo,FL).Lightcompression CareInc.,Norcross,GA).Asmallholewascutinthedrapeinthecenterofthe wasthenappliedbyelastictapethatwaswrappedcircumferentiallyaroundthe bridge,andaV.A.C.®tracpadputinplaceovertheholewithtubingroutedto animal’storsoandheldinplacebysurgicalstaples. thebackinamannersimilartothatdescribedfortheAmino-Plex®experiment. 3MTMVetrapTMbandagingtape(3MHealthCare,St.Paul,MN)wasthencoiled overthedrapedareausinglightcompression,furthersecuredwithstripsofelas- 2.5. Pharmacologictreatment tictape.Followingdressingofthepositiveandnegativecontrolsiteswithsterile gauzeandplacementofaprotectivecottonstockinetteovertheanimal’storso, Thirty minutes prior to surgery, animals were administered a prophylac- eachanimalwasthenplacedinaspeciallydesignedV.A.C.® harnesstopre- tic antibiotic (cefazolin sodium, Ranbaxy Pharmaceuticals Inc., Jacksonville, FL; ventdamagetoorentanglementintheevacuationtubes.Thetubeswerethen 20mg/kgi.m.).Foranalgesia,buprenorphineHCl(Buprenex® Injectable,Reckitt connected,andtheindividualpumpsstartedat125mmHg(pumpandwound BenckiserPharmaceuticalsInc.,Richmond,VA;0.01mg/kgi.m.)wasadministered pressure).Thepumpswererun24hperday.Thedressingswerechangedat48 immediately after HD exposure, the morning following agent exposure, and at and96hpost-surgerypermanufacturerrecommendation,andwereremoved theconclusionofthesurgicalprocedures.Beginningonthemorningfollowing onPS07justpriortoclinicalobservations. surgery,animalswereprovided160mgacetaminophenoncedailyfor7daysper 7. Biobrane®(BertekPharmaceuticalsInc.,Morgantown,WV)isabi-layeredcompos- os. iteconsistingofasyntheticepidermalanalogandabiologic(collagen-based) Foranyrequireddressingchangesorjustpriortoconductingclinicalobser- dermalanalogdesignedforpartial-thicknesswounds.Itiscommonlyusedin vationsonPS07andPS14,animalsweresedatedwithXyla-Ject®(2.2mg/kg)and treatingpartial-thicknessthermalburns.Followinglaserdebridementateither Telazol®(6.0mg/kg).Repeatedinjectionswereprovidedifneeded.Forthoseani- J.S.Grahametal./Toxicology263(2009)47–58 51 malsintheAmino-Plex®study,animalswerelightlysedatedwith0.5mlTelazol® Table1 i.m.fordailyadministrationoftheAmino-Plex®. Histomorphologicscale EuthanasiaonPS14(followingclinicalobservations)wasaccomplishedunder Parameter Score Xyla-Ject®/Telazol®sedation(asdescribedabove)byaninjectionofanoverdose ofsodiumpentobarbital-basedeuthanasiasolution(Fatal-Plus®Solution,Vortech Re-epithelialization 1=complete Pharmaceuticals,Ltd.,Dearborn,MI;78mg/kgi.v.)administeredintotheanterior 0=notcomplete venacava. Epidermal 1=absent hyperplasia 0=present 2.6. Post-surgicalprocedures Epidermal/dermal 1=absent 2.6.1. Clinicalevaluations separation 0=present Lesionsweregentlycleansedwithsterilesalineandgauzetoremovedried andlooselyadheredexudatepriortoclinicalevaluationofeachtreatedsitefor Inflammatorycells 1=absent re-epithelialization.Re-epithelializationofdebridedHDwoundswassubjectively 0=present scoredonPS07andPS14usingthefollowingscale:0=none,1=lessthan25%ofthe Hairfollicles 1=present originalHD-exposedareahadre-epithelialized,2=atleast25%butlessthan50% 0=absent oftheoriginalareahadre-epithelialized,3=atleast50%butlessthan75%ofthe originalareahadre-epithelialized,4=atleast75%butlessthan100%oftheoriginal Glands 1=present areahadre-epithelialized,and5=100%oftheoriginalareahadre-epithelialized.An 0=absent areawasconsideredtobere-epithelializedonlyifitwasvisible(i.e.,notcoveredby Elasticfibers 1=normal adherentescharorscab)andpetechialhemorrhagingwasabsent. 0=reducedsizeandnumber 2.6.2. Non-invasivebioengineeringmethods Smoothmuscles 1=present A variety of non-invasive bioengineering methods were used to follow the 0=absent progressofwoundhealingandevaluatevariouscosmeticandfunctionalproper- tiesofthewounds.Measurementsincludedreflectancecolorimetry(RC)tomeasure Collagenorientation 3=normaldermis 2=abnormalpapillarydermis erythema;evaporimetrytoexaminetransepidermalwaterloss(TEWL)asamethod 1=abnormalupperreticulardermis ofevaluatingbarrierfunction;torsionalballistometry(TB)toevaluatethemechan- 0=abnormallowerreticulardermis icalpropertiesofskinfirmnessandelasticity;andtwo-dimensionalhighfrequency (20MHz)ultrasonography(HFU)tomonitorskinthickness(e.g.,edema,scartis- Fibroplasia 1=absent sue).MeasurementsweremadebeforeagentexposureandonPS07andPS14for 0=present allmethodsexceptHFUandTB,whichwereonlyconductedbeforeexposureand onthelastdayofeachstudy(PS14).HFUandTBwerenotconductedonPS07to Vascular 1=absent avoiddisruptionofthefragileneoepidermis.HFUwasnotconductedintheDuo- proliferation 0=present DERMstudyduetoequipmentfailure.Bioengineeringmethodswereperformedas Hemorrhage 1=absent previouslydescribed(Grahametal.,2002b). 0=present 2.6.3. Histopathology Tissuesectionsweretrimmed,paraffinembedded,cutonamicrotomeinto5- Onthelastdayofeachstudy,animalswereeuthanizedwithanoverdoseof um thick sections, and stained with hematoxylin and eosin (H&E) for routine Fatal-PlusSolution®.Full-thicknessexcisions(includingpanniculuscarnosa)ofeach histopathology.SerialsectionswerealsostainedwithMasson’strichrometohigh- entirelesionplussurroundingskinwereremoved,stapledontolabeled,laminated light dermal collagen, and Movat’s pentachrome to highlight elastic fibers. A indexcards,andplacedinto10%neutralbufferedformalin.Sectionswerelater veterinary pathologist scored multiple parameters in each section in a blinded trimmed,paraffinembedded,cutonamicrotomeinto5-(cid:2)mthicksections,and fashion.Foreachtissuesection,scoresforeachindividualparameterwereadded stainedwithhematoxylinandeosin(H&E)forroutinehistopathology.Serialsec- together.Maximumtotalscoreforbestpossibleoutcome(e.g.,normalcy)was14. tionswerealsostainedwithMasson’strichrometohighlightdermalcollagen,and Movat’spentachrometohighlightelasticfibers.Aveterinarypathologistscoredthe sectionsinablindedfashionbasedonapublishedhistomorphologicscaleforrat- otherstains,thepercentareaofnormallocalizationfoundacrosstheentiresec- ingburnscars(Singeretal.,2000)modifiedforevaluationofthetissues(Table1). tionwassubjectivelygradedusingthefollowingscale:0=none,1=5%,2=10–40%, Maximumtotalscoreforbestpossibleoutcome(e.g.,normalcy)was14. 3=50–80%,4=90–95%,and5=100%. 2.6.4. Immunohistochemistry 2.7. Dataanalysis Eight-mmbiopsieswerecollectedfromeachexcisedsiteandprocessedfor immunohistochemicalstaining.Theyweretrimmedofexcesssubcutaneousfat, Initial comparisons of treatment groups were made on the two principal placedincottongauze,flashfrozeninchlorodifluoromethanerefrigerant(Freon22) histopathological(re-epithelializationandcollagenorientation)andimmunohis- forapproximately30s,placedinfoilpackages,andstoredat−70◦C.Immunohisto- tochemical(alpha6andcollagenIV)variablesmeasured.Thiswasperformedusing chemistrywasconductedonfrozentissuesectionstocharacterizetheprogression ananalysisofvarianceandaKruskal–Wallistestonthescores.Ifsignificanttreat- of cutaneous wound healing by localizing proteins associated with granulation menteffectswereobserved,thenaTukey’sorMann–Whitney’stestwasusedto tissueformation,neovascularization,basementmembranezoneremodeling,and comparepairsoftreatmentgroups.Forbinomialresponsedata,aChi-squareanaly- re-epithelialization.Sectionsoffrozentissueswerecutat10(cid:2)minacryostatand siswasusedtocomparethetreatmentgroupsandifsignificant,furtherChi-square stainedusingtheavidin–biotin–peroxidasecomplex(ABC)methodforlabelingof orFisher’sexacttestswereusedforpairsoftreatmentgroupcomparisons.Statistical primaryantibodies(VECTASTAIN®EliteABCkits,VectorLaboratories,Burlingame, significancewasdefinedasp<0.05foralltests.Theseinitialanalysesprovidedlit- CA).Thechromagenusedwas3,3-diaminobenzidinetetrahydrochloride(DAB).The tleinformationregardingconsistenttreatmentgroupdifferences(datanotshown). followingcross-reactivehumanantibodieswereused(allwitha30-minincuba- Sincethereweremanymorehistopathological,immunohistochemical,bioengineer- tionperiod):CD49f(forstainingalpha6integrininthebasementmembranezone, ing,andclinicalvariablesmeasured,adecisionanalysiswasconsideredthebest 1:50dilution,ChemiconInternational,Inc.,Temecula,CA),collagenIV(forstaining methodtoincorporatealltheinformationcollectedtodeterminethebestover- anchoringplaquesandthelaminadensainthebasementmembranezone,1:50dilu- alltreatmentgroups.Therefore,furtherstatisticalanalysescomparingtreatment tion,ICNBiomedicals,Inc.,Irvine,CA),collagenVII(forstaininganchoringfibrilsin groupsforindividualvariableswerenotperformed. thebasementmembranezone,1:100dilution,SigmaChemicalCompany,St.Louis, LogicalDecisions®forWindowsV6.0(Fairfax,VA)wasusedtorankthe12treat- MO),laminin5(forstaininganchoringfilamentswithinthebasementmembrane mentadjunctsthatwerestudied.Fourmainareasofexaminationandscoringwere zone,1:40dilution,ChemiconInternational,Inc.,Temecula,CA),filaggrin(forstain- defined,routinehistopathology,bioengineering,clinicaljudgment,andimmuno- ingepitheliuminthestratumgranulosum,1:50dilution,LabVisionCorp.,Fremont, histochemistry.Ineachoftheseareas,therewereatleasttwoandupto12distinct CA),laminin(forstainingthelaminalucidaofthebasementmembranezoneand parametersrecorded.ParameterswererecordedatPS07and/orPS14.Themean basallaminaofendothelium,1:100dilution,SigmaChemicalCompany,St.Louis, ormedianscoreforeachparameterwasusedforeachtreatment.Whenscores MO),vimentin(tostainintermediatefilamentproteinsoffibroblasts,endothelial weremissing,eitherthepositivecontrolvalueorthemeanofallscoreswasused. cells,andmacrophages,1:50dilution,BioGenexLaboratories,Inc.,SanRamon,CA), VimentinandvonWillebrandfactormeasurementswerenormalizedbasedonthe andvonWillebrandfactor(tostainendothelialcells,1:100dilution,ChemiconInter- totalscoreacrossalltreatmentstobringthelargecontinuousmeasurementsmore national,Inc.,Temecula,CA).ForthevonWillebrandfactorandvimentinstains, inlinewiththecategoricalscoresgiventotheotherparametersunderimmuno- theareaofstainingwasquantifiedbyimageanalysisusingImage-Pro® Plus5.0 hisotchemistry.Tomaintainconsistencyacrossparameters,allparameterswere forWindows2000/XPProfessional(MediaCybernetics,SilverSpring,MD).Forall scoredsimilarly;ahighscorewasconsideredthebestresponse.Ifaparameter’s 52 J.S.Grahametal./Toxicology263(2009)47–58 originalscoringdidnotfollowthis,theparameter’sscorewastransformedusingan judgment,datacollectedonPS14wereconsideredofhighervaluethanthosecol- inverseofthescore(e.g.originalscore0.5,inverseis1dividedby0.5whichequals lectedonPS07immediatelyafterbandageremoval. 2).Thiswasusedforbioengineeringcategoriesalpha(TB),deltaa*anddeltaE∗ ab (RC),TEWLandskinthickness(HFU).Itwasalsousedforthenormalizedvimentin 3. Results andvonWillebrandfactormeasurements. EachparameterwasassignedaweightbytheLogicalDecisions®softwarebased onthenumberofparametersintheirrespectiveareaandtheparameter’srel- All 66 animals remained healthy in appearance and behav- ativeimportance.Thesumoftheparameterweightsequaledoneforeacharea. iorthroughoutthecourseofthestudy.Meanpre-exposurebody Thefourmainareaswerealsoweightedbasedontheirorderofimportanceusing temperaturewas101.2◦F.Post-exposure,temperaturesremained thesoftware.Table2displaystherelativeimportanceofeachareaandeachdis- within two standard deviations of the mean with the exception tinctparameterwithineachareaalongwiththeirrespectiveweights.Structural integritywasconsideredthemostimportantindicatorofwoundhealing;thus, ofslight,transientelevationsnotedontwoseparateoccasionsin routinehistopathologyandimmunohistochemistrywereassignedarelativeimpor- different animals. No signs of infection within the experimental tanceofone.Functionaldata(bioengineeringresults)wereconsideredsecondin sites were seen. Occasional loose stools or diarrhea, likely stress importance,andsubjectiveclinicalassessmentstheleastimportant. related,werenoted.Skinscratchesandmildinflammationaround Regardingroutinehistopathology,parametersrelatedtothemainstructural componentsoftheskin(epidermis,elasticfibers,collagenfibers,andbloodvessels) somesurgicalstapleswereoccasionallynoted.Thesesignsdidnot wereconsideredofhighestimportance.Ofleastimportancewereparametersrelated correlatetoelevatedtemperatures. toadnexalstructures(hairfollicles,glands,smoothmuscles)thatcouldpotentially Clinical pathology examinations indicated that several clini- beabsentinanygivensectionduetotheexactlocationofthehistologicalsection cal chemistry parameters were elevated or depressed (data not thatwascutfromtheparaffinblock.Ofintermediateimportancewasthepresence shown).Depressionswerenotclinicallysignificant.Clinicallysig- ofinflammatoryorredbloodcellsoutsideofthevasculature. Regardingimmunohistochemistry,antigensrelatedtothebasementmembrane nificantincreasesweredefinedasathree-to-five-foldincreaseover zone(BMZ)wereconsideredofhighestimportance;withoutanintactBMZ,recur- publishedreferenceranges.Alkalinephosphatase(ALP),ananalyte rentblisteringoftheskinismorelikely.Regardingbioengineeringandclinical whoseincreasemaybeassociatedwithhepatobiliarydisease,was elevatedin39pigs.Anincreasedlevelofthisanalyteiscommonin weanlingpigs,andincreasedlevelswerenotconsistentlyrelatedto Table2 treatment.Alaninetransaminase(ALT),oftenusedasanindicator Areas/parametersmeasured,relativeimportance,andweights ofliverfunction,wasslightlyincreasedin60animalsandtended Mainareas/parameters Relativeimportance Weights tobehigherafterexposure.However,noneofthemeasuredlev- Routinehistopathology(PS14) 1 0.361 els were clinically significant. Aspartate aminotransferase (AST), Re-epithelialization 1 0.107 another indicator of liver function, was elevated in only six ani- Epidermalhyperplasia 1 0.107 mals,andinallcasestherewasalsoanincreasebetweenpre-and Epidermal/dermalseparation 1 0.107 post-exposurelevels,butnochangewasclinicallysignificant.Lac- Inflammatorycells 2 0.071 Hairfollicles 3 0.036 tatedehydrogenase,anotherimportantindicatorofliverfunction Glands 3 0.036 inthisspecies,waselevatedin56animals,butlevelswerenever Elasticfibers 1 0.107 clinicallysignificant,andchangesthatoccurredbetweenpre-and Smoothmuscles 3 0.036 post-exposurevaluesdidnottrendinonedirection.Whentaken Collagenorientation 1 0.107 together, liver enzyme values (ALT, AST, LDH) reflect no notable Fibroplasia 1 0.107 Vascularproliferation 1 0.107 effects on liver function during the study. Amylase, a pancreatic Hemorrhage 2 0.071 enzymeandpotentialindicatorofpancreaticorrenalcompromise, wasclinicallyelevatedbutwithintherangetypicallyreportedat Bioengineering(PS07andPS14) 2 0.194 TEWL–PS07 2 0.067 thisinstituteforthisspeciesandageanimal,andmaybeareflec- TEWL–PS14 1 0.133 tionofthefeedingregimen.In32cases,levelsincreased5–20%over RRCC––ddeellttaaEEa∗∗bPPSS0147((sskkiinnccoolloorr)) 21 00..013637 baselineaftertreatment.However,becausesamplesweretaken2 ab weeksapart,theincreasesseencouldalsohavebeenage-related, RC–deltaa*PS07(erythema) 2 0.067 RC–deltaa*PS14(erythema) 1 0.133 sincethisenzymeincreaseswithageinthisspecies.Creatinekinase TB–indentationPS14(hardness) 1 0.133 (CK)isanindicatorofmuscledamage.Whilelevelswereincreased TB–alphavaluePS14(elasticity) 1 0.133 in51animals,inallexcept7caseslevelswerenotclinicallysig- HFU–skinthicknessPS14 1 0.133 nificant;increasesin5ofthose7caseswereseenafterexposure. Clinicaljudgment(PS07andPS14) 3 0.083 Allofthesewerewithintherangeofincreasepotentiallycaused HDareare-epithelializationPS07 2 0.33 byintramuscularinjection-associatedtrauma,sincemultiplenee- HDareare-epithelializationPS14 1 0.67 dlesticksoccurredtomaintainlightanesthesiabetweendepilation Immunohistochemistry(PS14) 1 0.361 andeuthanasia.Allotherclinicalchemistryvaluesoutofreference Filaggrin 8 0.028 rangewerefewandnotclinicallysignificant. Laminin 5 0.111 Eight pigs displayed a mild anemia (hematocrit, HCT, of less Alpha6 1 0.222 CollagenVII 4 0.139 than30%)thatdidnotchangewhenmeasuredafterexposure.One CollagenIV 2 0.194 pig’shematocritdecreasedafterexposuretoamildlyanemiclevel. Laminin5 3 0.167 Fourteen pigs had a mild-moderate anemia (HCT less than 25%) vonWillebrandfactor 6 0.083 thatinallcaseseitherremainedthesameorimprovedwhenmea- Vimentin 7 0.056 suredafterexposure.Fouranimalshadamoderateanemia(HCT Logical Decisions® for Windows V6.0 (Fairfax, VA) was used to assign relative less than 20%), which in all cases had increased to above 22% in weightstovariousmeasuredparameters.Fourmainareasofexaminationandscor- post-exposuresamples.Allanemiasnotedweremicrocyticandnor- ingweredefined,routinehistopathology,bioengineering,clinicaljudgment,and immunohistochemistry.Ineachoftheseareas,therewereatleasttwoandupto12 mochromicinnatureandnotconsideredclinicallysignificantsince distinctparametersrecorded.Parameterswererecordedat7daysand/or14days noanimalsappearedpale,weakorillinanywayatanytime.Total post-surgery(PS).Eachparameterwasassignedaweightbasedonthenumberof protein was slightly low for all animals. These combined results parametersintheirrespectiveareaandtheparameter’srelativeimportance.The mayreflectaresidualmildirondeficiency,commoninweanling sumoftheparameterweightsequaledoneforeacharea.RC,reflectancecolorimetry; pigs,whichcausednoclinicaleffects.Theleukogramrevealedthat TEWL,transepidermalwaterloss;TB,torsionalballistometry;HFU,highfrequency ultrasound. 61 animals had a mild monocytosis, and in half of these cases a J.S.Grahametal./Toxicology263(2009)47–58 53 Fig.1. Treatmentrankings.LogicalDecisions®forWindowsV6.0(Fairfax,VA)wasusedtorankthetreatmentsappliedtosulfurmustardinjuriesat48hpost-exposure, utilizingtherelativeimportanceandranksofeachmeasuredparameterlistedinTable1.Themeanormedianscoreforeachparameterwasusedforeachtreatment. lymphopeniaalsooccurred;thesechangesprobablyreflectastress mal by 14 days post-surgery. Lesions treated with ReCell® were responsetohandlingand/orexposures,sincenotrendassociated similarly erythematous but had fully re-epithelialized by 7 days with exposure was seen. No neutrophilia was seen, correspond- post-surgery,andappearancewasalsonearnormalby14dayspost- ingtothelackofclinicalsignsofsignificantinflammationinthese surgery.Ingeneral,forHD-exposedsitestreatedwithanyadjunct, animals. thosethatweredeeplydebrided(300–400(cid:2)m)werenotasnear Thereisthepossibilitythattheacutenatureofthisstudypre- normalincosmeticappearanceaswerethoseshallowlydebrided ventedfullmanifestationofanyclinicalchemistryorhematological (100(cid:2)m,notshown);skintonetendedtobedarkerovertheentire changesdirectlyrelatedtothetreatments. areatreatedwiththelaser. NoTBorHFUdatawerecollectedfrom15ofthe264siteseval- Routine H&E stains of HD-exposed tissues collected on PS14 uatedonPS14duetothepresenceofhardened,adherentescharor fromthelesionsshowninthegrossphotographsareseeninFig.3a scab.Thesesiteswereprimarilyuntreatedpositivecontrols. (untreatedpositivecontrol),b(treatedwithAmino-Plex®),andc Results of the decision analysis comparing all treatments are (treatedwithReCell®).Epidermalhyperplasiaandabnormalcol- displayed in Fig. 1, listed from the most efficacious to least effi- lagen orientation limited to the papillary dermis were noted in cacious method. The top six treatments (in decreasing order allthreesections.Re-epithelializationwascompleteinthetreated of efficacy) were V.A.C.® (400(cid:2)m debridement), Amino-Plex® sites.Incompletere-epithelialization,abnormalcollagenorienta- (400(cid:2)m debridement), Amino-Plex® (100(cid:2)m debridement), tionextendingintothelowerreticulardermis,fibroplasia,vascular V.A.C.® (100(cid:2)m debridement), Aquacel® AG (100(cid:2)m debride- congestion,andhemorrhagecanbeseenintheuntreatedpositive ment,andReCell®(100(cid:2)mdebridement).AsuseofV.A.C.®devices control. would not be practical in mass casualty scenarios and a silver Immunohistochemical localization of collagen type VII and impregnateddressingwasincludedintheAmino-Plex®treatment, CD49f (alpha 6) is shown in Figs. 4 and 5. Untreated sites the remaining figures and subsequent discussion are focused on showed an overall decrease in immunospecificity for collagen Amino-Plex®andReCell®. typeVIIwithinthesublaminadensaofthebasementmembrane Sites treated with many of the adjuncts had not fully re- zone (Fig. 4a). In addition, portions of the basement membrane epithelialized by PS07. Most sites treated with Amino-Plex® and zone were also found to be diffusely localized or absent of col- ReCell® had re-epithelialized by that time point. Some pinpoint lagen VII staining altogether. Fig. 4b (Amino-Plex® treatment) petechialhemorrhagingwasoccasionallynotedwiththesetreat- shows robust immunolocalization of collagen type VII (arrows) ment adjuncts where the primary dressings in contact with the toanchoringfibrilswithinthesublaminadensaofthebasement woundbedremainedadherenttotheneoepidermisandhairthat membranezone,whichischaracteristicofnormalcytoarchitecture. wasregrowing;removalofthedressingsdenudedminutepatches Untreated sites also showed diffuse or interrupted immunolo- ofskin.Grossphotographsofsitesthatwereshallowlydebrided calization of alpha 6 within the basement membrane zone (to 100(cid:2)m) and treated with Amino-Plex® or ReCell® vs. posi- (Fig. 5a). Localization of alpha 6 to cell membranes of hyper- tive controls can be seen in Fig. 2a and b, respectively. Lesions trophic basal and supra basal epithelial cells was also observed. treatedwithAmino-Plex® weretypicallyerythematousbutfully Fig. 5b (ReCell® treatment) shows normal immunolocalization re-epithelializedby7dayspost-surgery.Appearancewasnearnor- patterns of alpha 6 to the cell membranes of basal and supra 54 J.S.Grahametal./Toxicology263(2009)47–58 Fig.2. Clinicalphotographs.(a)HDexposed,100(cid:2)mdebridement,treatedwithAmino-Plex®Sprayvs.nottreated.Treatedlesionwaserythematousandhadanindentation fromthetubingusedtodelivertheAmino-Plex®,butwasfullyre-epithelializedby7dayspost-surgery(leftpanel).Appearancewasnearnormalby14dayspost-surgery (rightpanel).NT:nottreated,100:debridedto100(cid:2)mandtreated.(b)HDexposed,100(cid:2)mdebridement,treatedwithReCell®vs.nottreated.Thetreatedlesionwas erythematousbuthadfullyre-epithelializedby7dayspost-surgery(leftpanel),andappearancewasnearnormalby14dayspost-surgery(rightpanel).NT:nottreated,100: debridedto100(cid:2)mandtreated. basalcellswithintheepitheliumandtothebasementmembrane of topical nutrients supports keratinocyte viability during graft zone. vascularization of cultured skin substitutes and inhibits wound contraction.Inthisstudy,Amino-Plex® showedgreatpromisein 4. Discussion improvingwoundhealingofsuperficialdermalHDinjuriesatboth depths of laser debridement studies (100 and 400(cid:2)m). While Theimportanceofwounddebridementinthehealingprocess TegadermTMAgMeshwasutilizedinconjunctionwiththisnutri- iswellestablished.Thisprincipalshouldapplytocutaneoussulfur tiveproduct,thesilverionsreleasedintothewoundbedbythis mustardinjuriesasitdoestothermalburns,chroniclegulcers,dia- dressingwerelikelynottheprimaryinducerofimprovedwound beticfootulcers,anddecubitisulcers.Powereddermabrasion(Rice, healing,asothersilver-iondeliveringdressingstested(AQUACEL® 1995;Riceetal.,2000)andlaserdebridement(Evisonetal.,2006; Ag and ACTICOAT 7) did not perform as well. Additional testing Grahametal.,1997,2002a,b)havebeenparticularlysuccessfulin without the incorporation of the TegadermTM would be needed improvingtherateofhealingofcutaneousHDinjuriesinpigs.A todeterminewhetherAmino-Plex®wassolelyresponsibleforthe reviewofpreviousresearchinvolvingvariousmethodsofdebride- improvedwoundhealingnoted. mentofvesicantinjuriesisavailable(Grahametal.,2005).Thetype Inthisstudy,debridementtoadepthof100(cid:2)mfollowedimme- oflaser(Er:YAG)usedinthisstudyfordebridementisparticularly diatelybyapplicationofasuspensionofskincellsprocessedbythe suitedforcutaneousresurfacing(Grahametal.,2005). ReCell®kitshowedgreatpromiseinimprovingwoundhealingof VacuumAssistedClosureTMisbecomingwidelyusedfortheclo- superficial dermal HD injuries. Cells applied to lesions debrided sure of chronic wounds such as stage III and IV pressure ulcers; toadepthof400(cid:2)mdidnotproduceresultsbyPS14asgoodas venous,arterial,andneuropathiculcers;andsubacuteandacute thoseproducedusingtheshallowdebridement,butperformedbet- wounds such as dehisced incisions, split-thickness meshed skin terthanmanyoftheothertreatmentadjunctstested.Application grafts,andmuscleflaps(BanwellandTeot,2003;Josephetal.,2000; of keratinocytes in suspension has been shown to improve epi- Mendez-Eastman,1998).V.A.C.® isalsogainingpopularityinthe dermal wound healing in pig (Currie et al., 2003; Navarro et al., managementofcomplexorthopedicwounds.Whileresultswere 2000; Svensjo et al., 2001) and mouse (Horch et al., 1998; Voigt promising, use on the battlefield or in a mass casualty scenario et al., 1999) models. Keratinocyte suspension technology shows is not practical; application and maintenance of the dressings is promise in that it does not require the length of time necessary verylaborintensive.WhileGranuFoamsilverdressingswereused toproduceculturedepidermalsheets.Useofthistechnologyhas aspartoftheV.A.C.® dressingprocedure,thesilverionsreleased proven efficacious in the treatment of thermal burns in humans intothewoundbedbytheGranuFoamwerelikelynottheprimary (Gravanteetal.,2007;Wood,2003;Woodetal.,2006)andappears inducers of improved wound healing, as the V.A.C.® procedure tobeasefficaciousasconventionalmelanocyte–keratinocytetrans- performedbetterthanAQUACEL® AgandACTICOAT7.Additional plantation for the treatment of vitiligo (Mulekar et al., 2008). testing with a non-silver ion delivering foam dressing would be CollectionofautologousbiopsymaterialforuseinReCell® kitsis neededtodeterminewhetherthesubatmosphericpressureapplied lessinvasivethanharvestingofclassicskingraftsyetprovidessim- bytheV.A.C.®procedurewassolelyresponsiblefortheimproved ilaraestheticandfunctionaloutcomes(Gravanteetal.,2007).Use healingnoted. of spray keratinocyte technology has also been shown to reduce Amino-Plex® is a nutritive cosmeceutical product that is totallength-of-stayper%TBSAoverthatseenforpatientstreated designed to increase oxygen in cells, stimulate ATP synthesis, with confluent sheets of cultured epithelial autografts (Wood et improveglucosetransportation,stimulatecollagenformation,and al., 2006). More recently, its concurrent use in conjunction with promote angiogenesis. Boyce et al. (1995) noted that application Integra®DermalRegenerationTemplate(IntegraLifesciencesCorp., J.S.Grahametal./Toxicology263(2009)47–58 55 Fig.4. ImmunohistochemistryofcollagentypeVIIforAmino-Plex®Spray,14days post-surgery.(a)LightmicrographofpigskinexposedtoHDandleftuntreated showinganoveralldecreaseinimmunospecificityforcollagentypeVIIwithinthe sublaminadensaofthebasementmembranezone.Inaddition,portionsthebase- mentmembranezonewerealsofoundtobediffuselylocalizedorabsentofcollagen VIIstainingaltogether(arrows).Epidermis(E).Dermis(D).(b)Lightmicrographof pigskinexposedtoHD,laserdebridedtoadepthof100(cid:2)m,andtreatedwithAmino- Plex®SprayshowingrobustimmunolocalizationofcollagentypeVII(arrows)to anchoringfibrilswithinthesublaminadensaofthebasementmembranezone, whichischaracteristicofnormalcytoarchitecture.Epidermis(E).Dermis(D). notedinareaswheresevereHDdamageinducedlocaldestruction of melanocytes. Otherwise, post-inflammatory hyperpigmenta- tionpredominates(KeheandSzinicz,2005;Khaterietal.,2003). Hyperpigmentation can be treated with laser resurfacing or pharmacologicallytreatedunderUVA/UVBprotectionwithhydro- quinone,kojicacid,azelaicacid,ascorbicacid,tretinoin,ortopical glucocorticoids.Treatmentforhypopigmentationisamuchmore challenging task. Utilization of ReCell®, which provides living Fig.3. (a)Histopathology,14dayspost-surgery,HD-exposed,untreatedpositive control(H&Estain).Epidermalhyperplasia(EH),vascularcongestion(VC),hem- and functional melanocytes in addition to other skin cells, may orrhage(H),andfibroplasiaareevident.Abnormalcollagenorientationfromthe restorepigmenttohypopigmentedordepigmentedskinpreviously papillary dermis to the lower reticular dermis was noted. Re-epithelialization exposed to HD. If the primary aim of using this technology on a wasnotcomplete(arrow).(b)Histopathology,14dayspost-surgery,HD-exposed, patient is to address hyperpigmentation by applying autologous 100(cid:2)m debridement, treated with Amino-Plex® Spray (H&E stain). Epidermal melanocytes (along with other skin cells), as in treating vitiligo hyperplasia was noted in portions of this tissue section, along with abnormal collagenorientationlimitedtothepapillarydermis.Re-epithelializationwascom- (Mulekaretal.,2008),thebiopsymaterialshouldbetakenfrom plete(arrow).(c)Histopathology,14dayspost-surgery,HD-exposed,HD-exposed, an area of the patient’s body with similar pigmentary qualities 100(cid:2)mdebridement,treatedwithReCell®(H&Estain).Epidermalhyperplasiawas asisfoundinunaffectedskinimmediatelysurroundingthetreat- notedinportionsofthistissuesection,alongwithabnormalcollagenorientation mentsite.PatientsshouldbeprotectedfromUVA/UVBandundergo limitedtothepapillarydermis.Re-epithelializationwascomplete(arrow). periodicexaminationbyadermatologist. In this study we noted that complete re-epithelialization of Plainsboro, NJ) for the treatment of experimental full-thickness debrided superficial dermal HD injuries in 7 days is possible. excisionalwoundsinpigsdemonstratedenhancedepithelialization DebridedHDwoundsweremoderatelyexudative,moresointhe atearlytimepoints(1–2weeks)comparedtocontrols,facilitating lesions debrided to 300 or 400(cid:2)m than the lesions shallowly one-stepprocessskinreconstruction(Woodetal.,2007). debrided(100(cid:2)m).Thedressingsappliedinthisstudywereade- Long-term complications of cutaneous HD injury include quatelyabletomanagetheexudate.Ingeneral,shallowEr:YAGlaser hypopigmentation and hyperpigmentation. Hypopigmentation is debridement through the basement membrane zone (100(cid:2)m)