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Towards Optimizing Sub-Normothermic Machine Perfusion in Fasciocutaneous Flaps: A Large Animal Study

Authors:
Yanis Berkane at Université de Rennes 1
Yanis Berkane
Alexandre G. Lellouch at Harvard Medical School
Alexandre G. Lellouch
Guillaume Goudot at Université Paris Cité
Guillaume Goudot
Austin Shamlou
Austin Shamlou
Austin Shamlou
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Abstract and Figures

Machine perfusion has developed rapidly since its first use in solid organ transplantation. Likewise, reconstructive surgery has kept pace, and ex vivo perfusion appears as a new trend in vascularized composite allotransplants preservation. In autologous reconstruction, fasciocutaneous flaps are now the gold standard due to their low morbidity (muscle sparing) and favorable functional and cosmetic results. However, failures still occasionally arise due to difficulties encountered with the vessels during free flap transfer. The development of machine perfusion procedures would make it possible to temporarily substitute or even avoid microsurgical anastomoses in certain complex cases. We performed oxygenated acellular sub-normothermic perfusions of fasciocutaneous flaps for 24 and 48 h in a porcine model and compared continuous and intermittent perfusion regimens. The monitored metrics included vascular resistance, edema, arteriovenous oxygen gas differentials, and metabolic parameters. A final histological assessment was performed. Porcine flaps which underwent successful oxygenated perfusion showed minimal or no signs of cell necrosis at the end of the perfusion. Intermittent perfusion allowed overall better results to be obtained at 24 h and extended perfusion duration. This work provides a strong foundation for further research and could lead to new and reliable reconstructive techniques.
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Bioengineering2023,10,1415.https://doi.org/10.3390/bioengineering10121415www.mdpi.com/journal/bioengineering
Article
TowardsOptimizingSub-NormothermicMachinePerfusionin
FasciocutaneousFlaps:ALargeAnimalStudy
Yani s Berkane
1,2,3,4,5,
*,AlexandreG.Lellouch
1,2,4,6
,GuillaumeGoudot
7,8
,Austi nShamlou
1,2,4
,
IrinaFilzvonReiterdank
1,2,4,9,10
,MarionGoutard
1,2,4,5
,PierreTawa
1,2,4
,PaulGirard
3
,NicolasBertheuil
3,5
,
BasakE.Uygun
2,4,9
,MarkA.Randolph
1,2,4
,JérômeDuisit
3,11
,CurtisL.Cetrulo,Jr.
1,2,4
andKorkutUygun
2,4,9,
*
1
DivisionofPlasticandReconstructiveSurgery,Vas cul ar ize dCompositeAllotransplantationLaboratory,
CenterforTransplantationSciences,MassachusettsGeneralHospital,Boston,MA02114,USA;
alellouch@mgb.org(A.G.L.);ifilzvonreiterdank@mhg.harvard.edu(I.F.v.R.);
marion.goutard0@gmail.com(M.G.);
piotawa@gmail.com(P.T.);marandolph@mgh.harvard.edu(M.A.R.);ccetrulo@mgh.harvard.edu(C.L.C.J.)
2
HarvardMedicalSchool,Boston,MA02115,USA;basakuygun@mgh.harvard.edu
3
DepartmentofPlastic,Reconstructive,andAestheticSurgery,CHUdeRennes,UniversitédeRennes,
35000Rennes,France;paul.girard@hotmail.fr(P.G.);nbertheuil@gmail.com(N.B.);
jerome.duisit@gmail.com(J.D.)
4
ShrinersChildren’sBoston,Boston,MA02114,USA
5
SITILaboratory,UMR1236,INSERM,UniversitédeRennes,35000Rennes,France
6
InnovativeTherapiesinHaemostasis,INSERMUMR-S1140,UniversityofParis,F-75006Paris,France
7
CardiologyDivision,MassachusettsGeneralHospital,HarvardMedicalSchool,Boston,MA02115,USA;
ggoudot@mgh.harvard.edu
8
INSERMU970PARCC, UniversitéParisCité,75000Paris,France
9
CenterforEngineeringinMedicineandSurgery,DepartmentofSurgery,MassachusettsGeneralHospital,
Boston,MA02115,USA
10
UniversityMedicalCenterUtrecht,3584Utrecht,TheNetherlands
11
IrisSouthHospitals,1040Brussels,Belgium
*Correspondence:yberkane@mgh.harvard.edu(Y.B.);kuygun@mgh.harvard.edu(K.U.);
Tel .: +1-617-371-4881(K.U.)
Abstract:Machineperfusionhasdevelopedrapidlysinceitsfirstuseinsolidorgantransplantation.
Likewise,reconstructivesurgeryhaskeptpace,andexvivoperfusionappearsasanewtrendin
vascularizedcompositeallotransplantspreservation.Inautologousreconstruction,fasciocutaneous
flapsarenowthegoldstandardduetotheirlowmorbidity(musclesparing)andfavorablefunc-
tionalandcosmeticresults.However,failuresstilloccasionallyariseduetodifficultiesencountered
withthevesselsduringfreeflaptransfer.Thedevelopmentofmachineperfusionprocedureswould
makeitpossibletotemporarilysubstituteorevenavoidmicrosurgicalanastomosesincertaincom-
plexcases.Weperformedoxygenatedacellularsub-normothermicperfusionsoffasciocutaneous
flapsfor24and48hinaporcinemodelandcomparedcontinuousandintermittentperfusionregi-
mens.Themonitoredmetricsincludedvascularresistance,edema,arteriovenousoxygengasdiffer-
entials,andmetabolicparameters.Afinalhistologicalassessmentwasperformed.Porcineflaps
whichunderwentsuccessfuloxygenatedperfusionshowedminimalornosignsofcellnecrosisat
theendoftheperfusion.Intermittentperfusionallowedoverallbetterresultstobeobtainedat24h
andextendedperfusionduration.Thisworkprovidesastrongfoundationforfurtherresearchand
couldleadtonewandreliablereconstructivetechniques.
Keywords:fasciocutaneousflaps;machineperfusion;exvivoperfusion;vascularizedcomposite
allotransplantation;intermittentperfusion;machineperfusion;extracorporealperfusion
Citation:Berkane,Y.;Lellouch,A.G.;
Goudot,G.;Shamlou,A.;
FilzvonReiterdank,I.;Goutard,M.;
Taw a, P.;Girard,P.;Bertheuil,N.;
Uygun,B.E.;etal.Towards
OptimizingSub-Normothermic
MachinePerfusio nin
FasciocutaneousFlaps:ALarge
AnimalStudy.Bioengineering2023,
10,1415.https://doi.org/10.3390/
b
ioengineering10121415
AcademicEditors:RaymundE.
HorchandChiaraGiuliaFontanella
Received:25October2023
Revised:23November2023
Accepted:7December2023
Published:12December2023
Copyright:©2023bytheauthors.
LicenseeMDPI,Basel,Switzerland.
Thisarticleisanopenaccessarticle
distributedunderthetermsand
conditionsoftheCreativeCommons
Attribution(CCBY)license
(https://creativecommons.org/license
s/by/4.0/).
Bioengineering2023,10,14152of16
1.Introduction
Theadventofmicrosurgerywasaturningpointtowardimprovingautologousre-
constructions,allowingvascularizedtissuetransferstooccuratadistancefromthedonor
site.Increasinganatomicalknowledgeandbettermicrosurgerytechniqueshaveledtothe
fasciocutaneousflapsgraduallyreplacingmuscle-basedreconstructions[1,2].Addition-
ally,theadventofperforator-basedflaps[3–6]allowedforthereliabilityoftheserecon-
structionstobeincreasedwhileminimizingmorbidity.Reliableperforator-basedflaps
usedinclinicalpracticeincludethedeepepigastricinferiorperforator(DIEP)flap,which
hassurpassedrectusabdominis-basedflapsforbreastreconstruction[7],andantero-lat-
eraltightorsuperficialcircumflexiliacperforatorflaps,whichhavebecomestandardin
limbreconstruction[8].Nonetheless,reportssuggestthat3to10%offreeflapsstillfail
duetovascularcomplications[9–15].Surgicalrevisionscanbesuccessful,buttheseat-
temptsexposepatientstodelayedcomplications.Machineperfusion(MP)techniques
couldpotentiallyincreasethepossibilityofflapsalvationafteraninitialfailureofconven-
tionalmicrosurgery[16],forexvivothrombolysis[17,18],orevenforfillet-flappreserva-
tionaftermajortraumaleadingtoamputation[19–21].Inaddition,somepatientsawaiting
reconstructionarenoteligibleforfreeflapsurgerybecauseoftheirmedicalhistory.For
instance,patientswithextensivesurgicalandradiationhistoriesofthecervicalregion(i.e.,
frozennecks)ordiabeticpatientswithchronicwoundsoftenpresentwithunsuitablevas-
cularnetworks[22,23].
Onesolutiontocircumventtheseobstacleswouldbetomasterextracorporealperfu-
sionprocesses,therebyprovidinganexogenoussupplyofoxygenandnutrientstothe
flapandbridgingtheperiodnecessarytoreachflapautonomization/neo-vascularization
andavoidingavascularizedtransfer.Masteringamulti-dayperfusionprotocol[24]could
beusedformicrosurgery-freeflapreconstruction,asdescribedbyWolffetal.[16,25,26].
Untilnow,theyhavebeenthefirstandonlyteamtodescribeaclinicalseriesoffree
fasciocutaneousflapreconstructionusingexclusiveextracorporealperfusion.Theywere
abletoperformreconstructionsofcomplexhead,neck,andshoulderdefectsusingfree
flapswithafasciocutaneouscomponentandnovascularanastomoses.Theirinnovative
techniqueusedanterolateralthigh,soleus,medialsural,radialforearm,andfibularvas-
cularizedfasciocutaneouspaddlesplacedontherecipientsitefor4to12daysuntilauton-
omization.However,theirinnovativeapproachwillrequireimportantoptimizationto
overcomethecurrentlimitationsandlowerthecurrentcomplicationrateof67%observed
intheirseriesonpartialflaploss[16,25,26].Ontheotherhand,BrouwersandKruithave
exploredmachineperfusion-basedapproachesinmuscleflapstostudyexvivothrombo-
lysisinflapsalvage[27],aswellasforextendedpreservationasarelevantmodelforvas-
cularizedcompositeallotransplantation(VCA)[18,28].Ourteamlaterexploredsubnor-
mothermicmachineperfusiontechniquesinratlimbs,providingaproofofconceptofthe
useofmachineperfusioninVCA,includingbonecomponents[29–32].Overall,thesetech-
niquesinspiredbysolidorgantransplantationaredevelopingaspromisingapproaches
inplasticandreconstructivesurgery.However,experimentalstudiesfocusingonopti-
mizingmachineperfusioninfasciocutaneousflapsarestillmissing.
Weperformedthisstudyusingaporcinesaphenousflap[33]toassessthepossibility
ofusingfasciocutaneousflapmachineperfusioninaclinicallyrelevantsetting.Wehy-
pothesizedthatacellularsub-normothermicmachineperfusion(SNMP)wouldsuitthe
multidayperfusionoffasciocutaneousflaps.Theobjectivewastooptimizetheexvivo
machineperfusionoffasciocutaneousflaps,describethecriticalmonitoringparameters,
andcomparetheoutcomeswithcontinuousandintermittentperfusion.
2.MaterialsandMethods
Twelvefemale30–35kgYorks h ire pigswereusedfortheseexperiments(12flaps
wereincludedinthedata).TheauthorsfollowedtheARRIVEguidelineschecklist[34].
AnimalswerehousedwithaccesstofoodandwateraccordingtothelocalCenterfor
Bioengineering2023,10,14153of16
ComparativeMedicine(CCM)conditions.Afteranacclimationperiod,theanimalsun-
derwentunilateralprocurementsurgeryundergeneralanesthesia.Thecontralateralside
wasusedbyotherresearchteamsthatwereabletoprocuretissuesandsolidorgansbefore
euthanasia,inordertooptimizethenumberofanimalssacrificedwithintheresearchfa-
cilitywithauthorizationfromtheInstitutionalAnimalCareandUseCommittee(IACUC).
Followingallharvestingprocedures,animalswereeuthanizedaccordingtothelocalvet-
erinarianguidelines.
2.1.FlapProcurementProcedure
Unilateralaxialsaphenousfasciocutaneousflapswereharvestedusingourestab-
lishedmodel[33].Vasculardissectionwasextendedtothefemoralvesselstoallowfor
singlecannulationofthetwosmallveinsintheflap(Figure1A,B).AftersystemicIVad-
ministrationofheparin(singledoseof100UI/kg),thefemoralvesselsweredissectedprox-
imallyanddistallytotheoriginofthesaphenouspedicle,ligated,andthendivided.An
18Gcatheterwasinsertedintothefemoralvesselsandsecuredwith3–0silkligatures.
Theflapwasflushedthroughthearterywith30to50mLofcold(4°C)salineheparin(100
UI/mL)untilclearvenousreturnwasachieved.Finally,theflapwasweighedandtrans-
portedtotheperfusionsystemonice.
Figure1.Surgicalmodelandperfusionsystem.(A)Saphenousfasciocutaneousflapduringtheper-
fusion.(B)Schemaoftheflapvasculature.(C)Intra-operativeultrasoundevaluationoftheflow
(colorDopplerandpulsedDoppler)inthesaphenousartery(whitearrow),withanestimateofthe
Bioengineering2023,10,14154of16
flowrate(redarrow).(D)Perfusionsystem:1:peristalticpump;2:oxygenator;3:oxygenflow;4:
bubbletrap;5:pressuresensorandinflowtap;6:pressuremonitor;7:timer;8:perfusatereservoir;
9:arterialcannula.
2.2.MachinePerfusionSystem
Acustomizedmachineperfusionsystemwasdesignedusingarollerpump(DRIVE
MASTERFLEXL/S,Cole-Parmer,Vernon Hills,IL,USA),ahollow-fiberoxygenator(Af-
finityPixie,Medtronic,Dublin,Ireland),inflowandoutflowsilicontubing(MasterflexL/S
16,Cole-Parmer,VernonHills,IL,USA),abubbletrap(Radnoti130149,RadnotiL.T.D,
Dublin,Ireland),andapressuretransducerlinkedtoaportablepressuremonitor(PM-P-
1,LSI,StAlbansCity,VT,USA).Filtrationofpotentialdebriswasachievedbythehollow-
fiberoxygenator,andnofurtherfiltrationwasperformedduringtheperfusion.Anoxy-
gentank(95%O2,5%CO2)wasconnectedtothededicatedvalveontheoxygenatorby
silicontubing,andtheoxygenflowwassetto0.5L/min.Thepressuretransducerwas
connectedtothesystemupstreamofthearterialcannula.Avalvedownstreamofthepres-
suretransducerwasusedtocollectsamplesforthebiochemicalinflowmeasurements.
Theflapwassuspendedoverastainless-steelbowlfilledwithperfusateandlaidontop
ofaperforatedrack,allowingtheoutflowtocollectinthesolutionreservoirfreely(Figure
1D).Asimilarsetuphasbeendescribedforothermodels[31,32].Samplesoftheoutflow
usedinthebiochemicalanalysiswereprocuredfromthevenouscannula(18G).Theper-
fusionsystem(Figure1D)wascontainedinaClassIIbiosafetycabinet.Thetubingand
thesurgicalbowlwereautoclavedbeforeeachperfusion,andtheflapwasmanipulated
withsterileglovesandinstruments.Perfusatesolutionwasinitiallysterilizedbyfiltration.
Tempe raturewasmonitoredusinganexternalthermometer(Cole-Parmer,TraceableIR
Thermometer)andwaskeptinthetargetrange(19–21°C)withoutintervention.Before
theflapperfusion,thesystempressurewasmeasuredatincrementalflowsfrom0.5–4
mL/minusingthesameperfusatetocorrectthemeasurementswiththesystem’spres-
sures.
2.3.PerfusateSolution
Acustom-modifiedacellularSteen+solutionwasused.Ourteampreviouslyopti-
mizedthissolutionforvascularizedcompositeallotransplant(VCA)preservation[28,31].
ThemaindifferencesfromtheoriginalSteensolutionwerethealbuminconcentration(in-
creasedto15%intheSteen+,versus7%)andtheadditionof0.5%of35kDapolyethylene
glycol[28](Sigma-Aldrich,Saint-Louis,MO,USA).ASteensolution(7%bovineserum
albumin),improvedbyaddingbroad-spectrumantibiotics(vancomycin1g/Landpipe-
racillin–tazobactam1g/L),wasusedfortheintermittentperfusionexperiments.Theper-
fusatewasrecirculatedinaclosedloopandexchangedevery24hforthemulti-dayper-
fusion.Sodiumbicarbonate(8.4%)wasaddedtothesolutiontocorrectthepH.Theopti-
malpHlevelsofthesolutionwerebetween7.1–7.4,varyingwiththeCO2levels.
2.4.PerfusionMonitoring
Thefollowingmetricsweremonitoredthroughouttheperfusionperiod:
Wei ghtgainoftheflapevery6h;
Perfusionparameters,includingflow(mL/min)andmeasuredandcorrectedpres-
sures(mmHg);
ResistanceswerecalculatedaccordingtotheformulaR=P/Q(R:resistance
(mmHg.min/mL),P:correctedpressure(mmHg),andQ:flowrate(mL/min));
Biochemicalparameterswererepeatedlymeasuredusingahandheldanalyzer(iStat
1,Abbott,Chicago,IL,USA).Inflowandoutflowsampleswerecollectedforeach
timepointandassessedthefollowingmeasurements:pH,pO2(mmHg),pCO2
(mmHg),lactate(mmol/L),[K+](mmol/L),[Na+](mmol/L),[HCO3−](mmol/L),base
excess(mmol/L),andglucose(mmol/L).Oxygenconsumptionwasmeasuredbased
onthedifferenceinpartialpressurebetweentheinflowandoutflow,theflowrate,
Bioengineering2023,10,14155of16
andtheinitialweightusingamodifiedFickequation[35].Similarly,glucosecon-
sumptionwasestimatedastheinflow–outflowdifference.
2.5.DeterminationofFlowRatesforExperiments
Twopreliminaryflapperfusions(notincludedinthedata)wereconductedfor12h,
allowingforthefinetuningofseveralperfusionparameters.Thebaselineflowrateofthe
saphenousarterywasmeasuredinvivobyultrasound(Figure1C),witharteryidentifica-
tionbycolorDopplerandmeanvelocityquantificationovertimebypulsedDoppler.This
optimizationallowedforsuccessfulsubsequentperfusions.
2.6.ContinuousVersus IntermittentPerfusionProtocols
Twonon-pulsatilesub-normothermicperfusionregimenswerecompared.Thefirst
groupreceivedcontinuousperfusion(CP)withSteen+.Theflowratewasmanually
adaptedthroughouttheperfusiontokeepthemeasuredpressurebetween30and55
mmHgbasedonourpreviousexperienceinothermodels[27,28].Asecondgroupre-
ceivedintermittentperfusion(IP)withSteen.Theperfusionrateschosenwerebasedona
priorworkofWolffetal.[25],assumingatoleranceoftheskintoischemia.Toaddress
theischemiccomplicationsobservedintheirseries,theperfusiontime/ischemictimeratio
wasincreasedto30–45minofperfusionfollowedby75–90minofischemia.Theperfusion
parameterswereassessedevery10minduringtheperfusionphases,andthemeanvalue
percyclewasusedforeachtimepoint.
Forbothgroups,terminationcriteriawereedemagreaterthan50%oftheinitialflap’s
weightorinflowdecreasedto50%oftheinitialvalue[17].
2.7.StatisticalAnalysis
AlldatawererecordedinExcel(Microsoft,Redmond,WA,USA),andallstatistical
analyseswereperformedusingPrism(v.9.5.0,GraphPadSoftware,LaJolla,CA,USA).
Thealphariskwasfixedat5%.Foreachvariablemeasuredduringmonitoring,themean
andstandarderrorofthemeanweredetermined.Linearregressionwasusedtoassessthe
stabilityovertimeineachgroup.Mann–WhitneyUtestswereperformedtocomparecon-
tinuousquantitativevariablesbetweengroups(non-paired,non-Gaussian,non-paramet-
ricrankdistributioncomparison).
3.Results
Sixcontinuousandsixintermittentsub-normothermicporcineflapperfusionswere
performed.Theaveragesurgicaldurationwas2.6±0.5h.Theaverageskinpaddlesurface
beforetheincisionwas55.9cm2intheCPgroupand64.5cm2intheIPgroup.Themean
initialweightwas22.61±3.98gintheCPgroupand30.95±8.28gintheIPgroup.AllCP
werestoppedatt=24hduetoreachingtheterminationcriteria(weightat24h>150%of
theinitialweight).TheIPflapswerekeptintheperfusionsystemfor24to72h.
3.1.PerfusionParameters
TheperfusionparameterresultsarepresentedinFigure2.Themeaninitialflowwas
1.15±0.27mL/minintheCPgroupand1.26±0.48mL/minintheIPgroup.Themean
flowvaluesat24hwere1.60±0.59and1.14±0.40mL/minfortheCPandIPgroups,
respectively.Comparisonbetweengroupsshowednodifference(p-valuesof0.90att=0
and0.33att=24h).Themeaninitialvascularresistancewas45.78±15.30mmHg.min/mL
intheCPgroupand39.72±20.33intheIPgroup.Themeanresistanceat24hwas42.08±
23.17and51.48±33.90fortheCPandIPgroups,respectively.Atrendshowedanincrease
inresistanceafter12h,buttheanalysesat0,6,12,and24hshowednosignificantdiffer-
encesbetweengroups(pvaluesof0.39,0.17,0.07,and0.70,respectively).Inmostflaps,
highresistancewasobservedduringthefirst45minbeforestabilization.Otherperfusion
models,suchastherathindlimbs,showedevidenceofsimilarpatterns[31].Interestingly,
Bioengineering2023,10,14156of16
flapsperfusedwiththeintermittentprotocolshowedlowervascularresistanceduringthe
perfusioncyclesafterrepeatedischemiadurations.Weightgainshowedanonsignificant
increaseafter12hofperfusion(mean:101.5%oftheinitialweight)inbothgroups,fol-
lowedbyadrasticriseat24h(164.1%)intheCPgroup,whereastheIPgroupshowed
verylowedemaat24h(109.92%,p=0.04).At48h,theIPgroupshowedinterestingresults,
withmeanflow,resistance,andweightvaluesof1.14±0.40mL/min,54.72±38.8
mmHg.min/mL,and111.10±80.66%,respectively.
Figure2.Perfusionparameters.Thefirst,second,andthirdrowsdisplaythecontinuousperfusion
group,theintermittentperfusiongroup,andthestatisticalanalysisbetweengroups,respectively.
(a,d,g)Flow(b,e,h),resistance,and(c,f,i)weightvariationareshown.Overallflowandresistances
werecomparablebetweengroups.EdemawasstatisticallylowerintheI.P.groupat24h.Mean
weightgainat36and48hwaslowerintheI.P.groupcomparedtotheC.P.groupat24h.Please
notethatthex-axisissplitin(a,b)(continuous)into0–3hand3–24htoallowforbetterviewingof
theinitialthree-hourresults.FortheI.P.group,theflowandresistancewerethemeanvalueper
perfusioncycle(4valuespercycle).Values fortheI.P.werecollectedfor48hofperfusion.Thethird
rowshowsmeanvalues±S.E.M.*statisticallysignificant.
Bioengineering2023,10,14157of16
3.2.BiochemicalParameters
Lactatewasmeasuredinthevenousoutflow,andtherecirculatinglactatemeasured
intheinflowwassubtracted.IntheCPgroup,flaps#5and#6showedhigherlactatevalues
(upto1.9mmol/L),andtheseflapswereconsideredischemic(Figure3a).Thedropin
thesetwocurveswasduetothepartialperfusateexchange.Themeaninitiallactatevalues
were0.60±0.49mmol/LintheCPand0.18±0.22mmol/LintheIPgroup(Figure3e).The
meanvaluesat24hwere0.55±0.48and2.47±3.93mmol/Lforthegroups,respectively.
Acomparisonbetweengroupsshowedstatisticallysignificantlyhigherlactatevaluesin
theIPgroup,relatedtointermittentoxygenation(Figure3i).TheinitialpHvaluesvaried
between7.1and7.4dependingonthepCO
2
ofthesolution.Interestingly,thepHtended
tostabilizeovertimetoaround7.2inbothgroups,exceptfortheischemicflaps(Figure
3b),duetometabolicacidosis.Thepotassiumconcentration(Figure3c,g)measuredinthe
outflowslightlyincreasedduringthefirst24hofperfusion,butnotoutsideofaverage
physiologicalvalues(3.5–5mmol/L),apartfromoneflap(#1)intheCPgroup,whichwas
determinedtobecontaminatedwithcleaningsolutionresidues.Themeaninitialpotas-
siumconcentrationwas4.75±0.75mmol/LintheCPgroupand4.56±0.24mmol/Linthe
IPgroup.Themeanpotassiumconcentrationsat24hwere5.23±1.07mmol/Land5.61±
0.50mmol/LintheCPandIPgroups,respectively.Nostatisticaldifferencewasfound
betweengroupsduringthe24hperiod(Figure3k).Figure3dshowsanincreasedO
2
con-
sumptionbeyond12hofperfusionintheCPgroup,butnostatisticaldifferencewasfound
betweenthegroups(Figure3l).Oxygenconsumptiondroppedafter8hinflap#4(CP
group)duetobacterialgrowthinthebubbletrap.Glucoseconsumptiontypicallyde-
creasedduringthefirsthourofperfusionbeforestabilizingatlowvalues.Theglucose
consumptioninflap#4(CPgroup)reachedhighvaluesafter7h,andthiswasassociated
withbacterialinfection.
Figure3.Biochemicalanalyses.Thefirst,second,andthirdrowsrepresentthecontinuousperfusion
group,theintermittentperfusiongroup,andthestatisticalanalysisbetweengroups,respectively.
(a,e,i)LactatereleaseshowedhighervaluesintheI.P.group,whichwasexpectedbecauseofthe
ischemicperiods.(b,f,j)pHlevelswerecomparablebetweengroupsuntilt=18h,wherethepH
washigherintheI.P.group,whichwaslinkedtobicarbonateadjunctioninoneischemicreplicate.
Bioengineering2023,10,14158of16
(c,g,k)Potassiumlevelswerecomparablebetweengroups.(d,h,l)Oxygenconsumptionwasmeas-
uredwiththefollowingformula:O
2
cons=100×(InflowO
2
-OutflowO
2
)×Flowrate×0.0314/[initial
weightoftheflap]withO
2
consinmlO
2
/min/g,InflowO
2
andOutflowO
2
inmmHg,flowratein
mL/min,andinitialweightingrams.Pleasenotethatthex-axisissplitin(a–d)(continuous)(0–3h
and3–24h),toallowforbetterviewingoftheinitialthree-hourresults.Val ues fortheI.P.were
collectedfor48hofperfusion.Thethirdrowshowsmeanvalues±SEM.
4.Discussion
Inthiswork,wepresentedthesetupandthemainparametersfortheexvivoperfu-
sionoffasciocutaneousflaps.Continuousperfusionwasperformedfor24hinthefirst
group(CP).Intermittentperfusion(IP)wasstudiedinthesecondgroupuntilperfusion
failurewasreached.Thisstudydesignallowedforacomparisonofthetwodifferent
groupsinthefirst24h(Figure4).Akeygoalwastoshowthatacellularperfusioncanbe
usedforperfusingthistypeofflap.Preservingfasciocutaneousflapsforshortdurations
(12–24h)couldbeusedforimmediateclinicalapplications,suchascomplexfreeflapsur-
geriesorrevisionsurgeries,todecreasetheischemictimeduringpreparationoftherecip-
ientsite[36].Exvivoperfusioncouldalsobeusedtopreservefilletflapsprocuredon
amputatedlimbsfollowingmajortraumasasanexampleofatissue-sparingprocedure
[19–21].Anotherapplicationisexvivothrombolysisincompromisedflaps,aspreviously
describedinaswinemusculocutaneousflapmodel[17].Ourworkcanalsoinformfuture
exvivoperfusionstudies,whichisacurrenttrendinthefieldofreconstructivesurgery
[37].
Figure4.Macroscopicaspectofthecannulatedfemoralvesselsfollowing24hofintermittentper-
fusion.Theveinwascannulatedtofacilitatetheprocurementoftheoutflowsamplefrombothveina
comitans.Notethatthepositioningofthefemoralvesselswasadjustedwhilemonitoringthesys-
tem’spressuretoallowforperfusionwithminimalmechanicalresistance.
Toassessthemicro-vascularizationoftheskinpaddle,fluoresceinangiographywas
performed(FigureA1,AppendixA) .Itisinterestingtonotethattheinitialfluorescent
surfacewasnot100%.Themodelitselfcouldexplainthis:themicrovesselsdedicatedto
theflapareknowntobehighlydynamic,asdescribedbySaint-CyrandRohrichwiththe
perforasometheory[38].Machineperfusionleadstoincreasedresistanceinothermodels
[31,39],andthiscouldexplainthelimitedareareachedbythefluorescence.Itisalsolikely
Bioengineering2023,10,14159of16
thatthefluoresceinangiographyitselfmaycausethisresult,asithasbeenreportedthat
fluoresceinonlyassessesthedeepdermalplexus[40].Itwouldbeinterestingtoimprove
thismicrovasculatureassessmentbyperformingindocyaninegreenangiography(ICG)
[40].Histologyshowednodifferencebetweenanyoftheflapsineitherperfusedgroup
(FigureA2,AppendixA) ,revealingedema,butnosignsofapoptosis.
Thesepreliminaryexperimentsshowedustheimportanceoftheinitialflowvalueon
resistanceintheflaps,reflectiveofthemicrovasculature.Sinceeachflaphasitsownspe-
cificvascularcomplianceandanatomy,werecommendanintra-operativeultrasoundex-
aminationforeachnewflapmodeltoconfirmitsqualityandestimatetheinitialarterial
rate[41].Biochemicalmeasurementsindicatedpotentialischemia,eveninflapswithno
muscle.Elevatedlactatemayalsosuggestabacterialinfectionconsumingoxygen,asseen
intwopreliminaryflapperfusionswhereantibioticscorrectedadropininflowoxygen
(datanotincluded).Untreated,prolongedlowoxygenationoftheinflowcouldresultin
lacticacidosisduetoanaerobicmetabolism[42].Thislastpointshowsthatcriticalcare
mustbetakentopreventtheperfusatefrombecomingcontaminated.Therefore,forclin-
icalapplications,theperfusateshouldbemicro-filtratedanddiscarded,andshouldnot
berecirculated.Weusedbiochemicalmetricssuchaspotassium,lactate,pH,andoxygen
consumptionbytranslationfromotherVCAmodels[31,32,43].Thesemetabolicoutcomes
havebeenshowntoberelevantinsolidorganpreservation[35,44],butotherparameters
maybesuitableforfasciocutaneousflaps.ChangesinATPlevelshavebeendescribedin
severalmodels[45,46]andcouldbeofinteresttoimproveperfusedflapmonitoring,but
thisseemstobedifficulttoimplementinclinicalsettings.Allparametersdescribedinour
methodscanbemonitoredusinghandheldandlightdevices,makingthemrelevantfor
bedsideapplications.
Meyersetal.haverecentlyshownthatweightgainisanearlymarkerofperfusion
failure[47].Theoverallanalysisofourpresenteddatasuggeststhatbothcontinuousand
intermittentoxygenatedacellularperfusioncanbesuccessfulforshortdurationsofless
than12h,andthatintermittentperfusionseemsbetterforlongerdurations,potentially
becauseitpreservesthevasculartree,allowingforlowervascularresistanceandedema.
AcorrelationbetweenthesetwoparametershasbeendescribedpreviouslybyDr.Poma-
hac’steaminapighindlimbmodel[48].Ourfindingsconfirmedtheirresultsbyshowing
agradualparallelincreaseinweightgainandvascularresistance.Inordertoreachseveral
daysofoptimizedperfusion,furtherstudiesshouldthereforefocusonbetterprotecting
themicrovasculaturetopreventinterstitialedemaandincreasedresistance.
Wechosetocomparecontinuousandintermittentperfusionregimensforseveralrea-
sons:Currentmachineperfusiontechniquesinsolidorgans,butalsoinvascularizedcom-
positeallografts,allusecontinuousperfusiontoconstantlyprovideoxygenandnutrients
whileconstantlyclearingtoxicmetabolites.Ontheotherhand,intermittentperfusionin
thespecificcaseoffasciocutaneousflapsisinterestingtoexplore:(i)theabsenceofmuscle
makestheischemicphasesacceptable;(ii)theintermittentperfusionallowsforischemic
preconditioningontheflap,whichcanexpeditetheneo-vascularizationprocessanden-
sureautonomizationattheendofthemachineperfusionperiod;and(iii)thelogisticsat
thepatient’sbedsidewouldgainconvenience,sinceintermittentperfusionwouldallow
formobilizationandwalkingduringtheOFFphases,helpingtodecreasedecubituscom-
plications.Therefore,itseemedcriticaltocomparebothperfusionsettings.
Toourknowledge,thisworkisthefirstdescriptionofexvivoperfusionoffasciocu-
taneousflapsinalargeanimalmodel.Muscle-sparingflapsseemtobethemostclinically
relevanttomodernreconstructivetechniquesinplasticsurgery[1,2,49].Kruitetal.[28,50]
firstdemonstratedperfusionsuccessinporcinemusculocutaneousflaps,allowingfor18
hofpreservationbeforereplantation.Theycomparedtwodifferentcommercializedper-
fusates,buttheirworkdidnotfocusontheperfusionparameters.Moreover,musculocu-
taneousflapsdifferfrompurefasciocutaneousflapsduetothepresenceofmultipleper-
foratorvesselsthatprovideadequatevascularizationtotheskinpaddle,butwithalower
toleranceofthemuscletoischemia.Performingmachineperfusionoffasciocutaneous
Bioengineering2023,10,141510of16
flapsappearstobesafeforreconstructivesurgeryapplications,andthiswasthefocusof
ourstudybecauseofthepotentialforimmediateimplementationinplasticsurgery
[2,51,52].Ozturketal.describedtheperfusionoffivefreshlyharvestedDIEPflapsonpa-
tientsundergoingabdominoplasty[53].Theyusedfreshwholebloodandwereableto
keeptheflapsperfusedfor4to5days.However,theydidnotaddresstheidealflowrate
orpressureparameters,whicharecriticalforreproducibility.Additionally,theuseof
wholebloodcouldbelimiting,bothintermsofsafetyandlogisticsforclinicaluse.We
expectthatacellularperfusionwouldbepreferableforfasciocutaneousflapperfusion,
limitingthecostandriskofinfectiousdiseasetransmission,asshownbyWolffetal.in
vivo[25].Toaddressthislastpoint,itseemsnecessarytocomparedifferentperfusate
solutions,includingtestingofpotentialartificialoxygencarriers.
Thispreliminarystudyhasseverallimitationsthatshouldbeaddressedinthefuture.
Firstly,alargercohortwouldhaveprovidedbetterpowerforstatisticalanalysis.Addi-
tionally,thecontributionsofangiography(FigureA2,AppendixA)wereminorandlim-
itedtoconfirmingarterialflow.UsingICGforsuchexvivoflapperfusionscouldpermit
bettermonitoringofskinperfusion.Anotherpointisthetemperature,whichwassetas
sub-normothermic(19–21°C)andcouldhaveinfluencedtheflap’smicro-vascularization
[54].Normothermicperfusioncouldimprovetheskinpaddle’svascularization.However,
choosingasub-normothermicperfusionpermitsusinganacellularperfusatesolutionbe-
causeofthelowermetabolism[55,56],avoidingsafety-relatedconcernsregardingblood
products.Moreover,thebacterialhazardneedstobeaddressedcarefully.We modified
ourprotocolbyusingpiperacillin–tazobactamandvancomycininourperfusatebasedon
preliminarycasesinwhichlikelybacterialinfectionswereobserved.Anotherlimitationis
theabsenceofmicroscopicassessmentofendothelialinjuriesfollowingperfusion,which
couldpotentiallyexplaintheedemaandtheperfusiondurationlimitation.Addingase-
quenceofnormothermicbloodreperfusionattheendofthepreservationperiodcould
unveilischemia–reperfusioninjuriesandincreasethesignificanceofthiswork,and
shouldbeexploredinsubsequentstudies.Todate,onlyafewpublicationshavefocused
onendothelialcellsduringMP[57,58].Finally,comparingextracorporealperfusionpro-
tocolswithconventionalmicrosurgerycouldbeinteresting(outcomes,safety,cost-effec-
tiveness…),butitseemsthatthisinnovationshouldbe,atleastinitially,exclusivelyre-
strictedtopatientsdisqualifiedformicrosurgicalfreeflapsorforfreeflapsalvageat-
tempts(thrombolysis).Therefore,nocomparisonshouldbeperformedyetbetweenthese
approachesduringtheoptimizationprocess.
ThisstudywasinspiredbypioneeringworksbyWolffetal.,whodescribedthecases
ofsixpatientswhobenefitedfromextracorporealperfusiontechniquesforreconstruction
oftheneckwithfasciocutaneousflaps[16,25,26].Thepatientsintheirserieseventually
healed,butfourofsixexperiencedpartialorsubtotalflaploss.Thisstudydidnotevaluate
certainparameters,suchasperfusionrhythm,frequency,solutetype,andtotalperfusion
duration,whichcouldhaveaddedbenefitstoavoidpartialischemiccomplications.Our
objectivewastooptimizeperfusioninaclinicallyrelevantmodel.Wefoundthatintermit-
tentperfusionseemedmoresuitablethancontinuousperfusionformulti-dayperfusion
basedonvascularresistanceandedemamonitoring.
Tofurtheroptimizethepromisingapproachofintermittentflapperfusion,itiscru-
cialtoinvestigatetheimpactofperfusion/ischemiaratesonflapperfusionquality.Several
preclinicalmodelsalreadyexist[59–61],andthenewperspectivesonreconstruction
shouldpushresearcherstodelveintothismatter.Furtherresearchshouldalsoexplorethe
healingcapacityofflapsfollowingextendedperfusionpreservation,aswellastheendo-
thelialinjuriesprovokedbymachineperfusionshearstress,whichcouldexplainthecur-
rentlimitationinperfusiondurationduetoweightgainbyextravascularperfusateleak-
age.Thisstudyactsasastrongfoundationformorestudies,whichwillbeneededinorder
toprovideareliableprotocolallowingfasciocutaneousflapperfusionsforextendeddu-
rations,thereforeenablingmicrosurgery-freereconstructionwithoutischemiccomplica-
tions.
Bioengineering2023,10,141511of16
5.Conclusions
Fasciocutaneousflapscanbepreservedusingcontinuousacellularsubnormothermic
machineperfusionfor12h.Intermittentperfusionpermittedupto48hofflappreserva-
tion.Thisstrategycanallowforflapsalvageusingexvivothrombolysis,orevenflap
preservationbeforereplantationincomplexcases.Furtherresearchshouldaimforlonger
perfusiondurations,eventuallyleadingtooptimizinganastomoses-freeflaptransferre-
constructions.
6.Patents
TheauthorsdeclareU.S.PatentApplicationNo.63/377,519,filed28September2023,
asrelevanttotheworkincludedinthismanuscript.
Aut ho rContributions:Design:Y.B.,A.G.L.,P.G.,B.E.U.,J.D.,C.L.C.J.andK.U.;performedresearch:
Y.B.,G.G.,A.S.,I.F.v.R.andM.A.R.;collecteddata:Y.B .,M.G.andP.T.;analysis:Y.B.,B.E.U.and
N.B.;writingandproofreading:Y.B.,A.G.L.,P.G.,J.D.,K.U.,B.E.U.,C.L.C.J.,N.B.,A.S.,I.F.v.R.,
M.A.R.,M.G.,P.T.andG.G.;supervision:K.U.,C.L.C.J.,J.D.,N.B.andA.G.L.Allauthorshaveread
andagreedtothepublishedversionofthemanuscript.
Funding:Y.B. receivedfundingfromCHUdeRennes(France,CORECTUF8946-07andPrixmobil-
ité2021),FondationdesGueulesCassées(France,Grants06-21,07-21and09-22),andShrinersChil-
drenBoston(#84308-BOS-22).PrizebytheFrenchSocietyofPlasticSurgery(SOFCPRE,Prix
Zagamé2022)toY.B .isgreatlyacknowledged.ThisworkwaspartiallyfundedbyShrinersHospi-
talsforChildrengrants#85127and#84702(B.E.U.,C.L.C.J.,A.G.L.)#85105-BOS-23(K.U.)andby
FondationdesGueulesCassées(France,Grants06-21,07-21and09-22).G.G.wasfundedbythe
FrenchFederationofCardiologyandtheServierInstitute.I.F.v.RwasfundedbyShrinersChildren
Boston(#84302-BOS-21).TheU.S.ArmyMedicalResearchAcquisitionActivity,820ChandlerStreet,
FortDetrick,MD21702-5014istheawardingandadministeringacquisitionoffice.Thisworkwas
supportedbytheOfficeofAssistantSecretaryofDefenseforHealthAffairsthroughtheReconstruc-
tiveTransplantResearchProgram,Techno logyDevelopmentAwardunderAwardsNo.W81XWH-
17-1-0437andW81XWH-17-1-0440(C.L.C.J.,A.G.L.,K.U.).Opinions,interpretations,conclusions,
andrecommendationsarethoseoftheauthorandarenotnecessarilyendorsedbytheDepartment
ofDefense.ThismaterialispartiallybaseduponworksupportedbytheNationalScienceFounda-
tionunderGrantNo.EEC1941543.PartialsupportfromtheUSNationalInstitutesofHealth
(R01EB028782andR56AI171958)isgratefullyacknowledged.
InstitutionalReviewBoardStatement:Allexperimentswereperformedwithintheauthor’slabor-
atoryandtheresearchhospital’sfacilities.Allanimalcareandprocedureswereapprovedbythe
IACUC(Protocol2022N000046“2FEP”)oftheauthor’sinstitutionandwerecompliantwiththe
GuidefortheCareandUseofLaboratoryAnimals,editedbytheInstituteofLaboratoryAnimal
Resources,NationalResearchCouncil,andpublishedbytheNationalAcademyPress.
InformedConsentStatement:Notapplicable.
DataAvailabilityStatement:Datacanbeprovidedbythecorrespondingauthorsondemand.
Acknowledgments:WethankMichaelDuggan,JessicaBurke-Pallotta,NickDeLuca,AnetCalisir,
NelsonMarquezCarvajal,ElijahSmith,andErinMarxfortheirtechnicalhelpintheanesthesiaof
theanimals,andEloideClermont-Tonnerre,andClaireGuinierfortheirparticipation.Theauthors
wouldliketothanktheFrenchSocietyofPlas ticSurgery(SOFCPRE)foritssupport,whichwill
allowforfurtherresearchtobeconductedinthisfield.
ConflictsofInterest:Someauthorsdeclarecompetinginterests:K.U.,C.L.C.J.,Y.B.andA.G.L.have
patentapplicationsrelevanttothisfield.K.U.hasafinancialinterestinandservesontheScientific
AdvisoryBoardforSylvaticaBiotechInc.(Charleston,SouthCarolina,USA),acompanyfocusedon
developinghighsubzeroorganpreservationtechnology.Nootherauthorhasanyfurthercompet-
inginteresttodeclare.CompetinginterestsforMGHinvestigatorsaremanagedbytheMGHand
MGBinaccordancewiththeirconflict-of-interestpolicies.
AppendixA
Perfusionqualityassessmentthroughvascularangiography.
Bioengineering2023,10,141512of16
Methods:Tomonitortheflapmicrovasculature,angiographieswereperformedby
manuallyinjecting0.3mLof5%fluoresceinthroughthearterialcannulaandexposingthe
flaptoaWoodUVlight(365nmpeakwavelength).High-resolutionphotographsofthe
skinpaddlefluorescencewerethentakenatt=0,t=12h,andt=24h.Pixel-analyzing
software(v.2.10.,GNUImageManipulationProgram,Berkeley,CA,USA)wasusedto
measurethepercentageoffluorescentpixelsinsidetheflapateachtimepoint.Apre-
injectionphotographwastakenbeforeeachangiographinordertoexcluderesidualfluo-
rescencefromtheprevioustimepoint.Theoutflowwasdiscardedfor30saftertheangi-
ographytominimizefluoresceinrecirculation.Flapangiographywasperformedinthe
continuousperfusiongroupandwasnotrepeatedintheintermittentgroup.
FigureA1.Flapfluoresceinangiography.Aspectsoftheflapangiographiesat(A)t=0;(B)t=12h
and(C)t=24h.(D)Anon-pairedt-testshowednostatisticallysignificantdifferencebetweenthe3
timepoints.MeaninitialangiographiesintheC.P.group(t=0)showed55.80±14.39%greenfluo-
rescentpixelsonthewholeflapskinpaddle,confirmingthatthearterialflowreachedtheskinpad-
dle.Meanvaluesat12hand24hwere48.30±16.90%and32.20±18.10%,respectively.
TissuesamplesandHistology:
Methods:Full-thicknesspunchbiopsieswerecollectedatt=0(initialskincontrols)
andt=endofperfusion(24,48,or72h)ontheperfusedflaps.Biopsieswerefixedin10%
formalin,embeddedinparaffin,andstainedwithhematoxylinandeosin(H&E)forbasic
pathologyassessment.Furthercontrolflapsconsistedofnativeskin(t=0),staticcold-
storedflaps(4°C,inCustodiol,EssentialPharmaceuticalsLLC,Durham,NC,USA),and
non-perfusedflapskeptatroomtemperature(19–21°C)for24h.Theslideswereanalyzed
byanexperimentedpathologist.Apathologiccomponentscoringsystemwasusedto
comparethesamples[62].Apoptoticcellsperfield(C.P.F)werecountedandshowedneg-
ativeresultsinallflapsofbothexperimentalgroupsattheendoftheperfusion(24h
Bioengineering2023,10,141513of16
(FigureA2(1)),48or72h),instaticcold-storedflapsat4°C(n=3),andinnativecontrol
flaps(n=3).Onlythenon-perfusedcontrolflaps(n=3)thatwerestaticandstoredatroom
temperature(21°C)showedminorapoptosis(FigureA2(2)).Thecomponentpathology
scoreshowednodifferencebetweenanyofthegroups.Thislimitedcontributionofhis-
tologymaybeduetotheabsenceofnormothermicbloodreperfusion,whichwillbein-
corporatedintoourfutureexperiments.
FigureA2.Tissuesamplesandhistology.Legend:H&Estainingofthefasciocutaneousflapskin
after24hofcontinuousperfusion(1)andafter24hofstaticstorageat21°C(2).Thisphotographof
H&E-stainedtissuerevealstheepidermisandpartialdermis.Thedifferentcellularcomponentscan
bevisualized:epidermalstratumcorneum(a),epidermalstratumspinosum(b),epidermalstratum
basale(c),anddermiscontainingfibroblasts(d).Thestratumspinosumandstratumbasaleinthe
controlgroup(2)showminorapoptosis(blackarrow).Nonecrosisisshowninanyofthegroups.
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... The variety of tissues composing VCAs brings a substantial challenge when translating solid organ preservation protocols. Kruit et al. and later our team described the first machine perfusion protocols applied to large animal VCA 27,28,36,39,[44][45][46] . Nonetheless, current dynamic hypothermic and SNMP techniques are limited to preservation periods of 24 to 36 h due to factors such as edema and vascular resistance 34,[47][48][49] . ...
VCA supercooling in a swine partial hindlimb model
Article
Full-text available
  • Jun 2024
Vascularized composite allotransplantations are complex procedures with substantial functional impact on patients. Extended preservation of VCAs is of major importance in advancing this field. It would result in improved donor-recipient matching as well as the potential for ex vivo manipulation with gene and cell therapies. Moreover, it would make logistically feasible immune tolerance induction protocols through mixed chimerism. Supercooling techniques have shown promising results in multi-day liver preservation. It consists of reaching sub-zero temperatures while preventing ice formation within the graft by using various cryoprotective agents. By drastically decreasing the cell metabolism and need for oxygen and nutrients, supercooling allows extended preservation and recovery with lower ischemia–reperfusion injuries. This study is the first to demonstrate the supercooling of a large animal model of VCA. Porcine hindlimbs underwent 48 h of preservation at − 5 °C followed by recovery and normothermic machine perfusion assessment, with no issues in ice formation and favorable levels of injury markers. Our findings provide valuable preliminary results, suggesting a promising future for extended VCA preservation.
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... In contrast, the use of subnormothermic "room" temperatures (20°C-25°C) in machine perfusion offers several advantages. First, it simplifies the perfusion device setup, eliminating the need for a heating or cooling system [29,38]. Second, it allows clinicians to reach a balance between oxygen needs and subdued tissue metabolism resulting in perfusate simplifications [28]. ...
Machine Perfusion Enables 24-h Preservation of Vascularized Composite Allografts in a Swine Model of Allotransplantation
Article
Full-text available
  • May 2024
  • TRANSPL INT
The current gold standard for preserving vascularized composite allografts (VCA) is 4°C static cold storage (SCS), albeit muscle vulnerability to ischemia can be described as early as after 2 h of SCS. Alternatively, machine perfusion (MP) is growing in the world of organ preservation. Herein, we investigated the outcomes of oxygenated acellular subnormothermic machine perfusion (SNMP) for 24-h VCA preservation before allotransplantation in a swine model. Six partial hindlimbs were procured on adult pigs and preserved ex vivo for 24 h with either SNMP (n = 3) or SCS (n = 3) before heterotopic allotransplantation. Recipient animals received immunosuppression and were followed up for 14 days. Clinical monitoring was carried out twice daily, and graft biopsies and blood samples were regularly collected. Two blinded pathologists assessed skin and muscle samples. Overall survival was higher in the SNMP group. Early euthanasia of 2 animals in the SCS group was linked to significant graft degeneration. Analyses of the grafts showed massive muscle degeneration in the SCS group and a normal aspect in the SNMP group 2 weeks after allotransplantation. Therefore, this 24-h SNMP protocol using a modified Steen solution generated better clinical and histological outcomes in allotransplantation when compared to time-matched SCS.
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... These multi-step procedures could benefit from simplification resulting from a reduction in the necessary delay. Another benefit that may result from these data is the improvement in the reliability of extracorporeal machine-perfused flap reconstruction techniques [117,118], as initially described by Wolff [102]. Their pioneering work requires optimization to decrease the observed complication rate, yet the total perfusion time (4 to 6 days) deserves attention. ...
The Autonomization Principle in Vascularized Flaps: An Alternative Strategy for Composite Tissue Scaffold In Vivo Revascularization
Article
Full-text available
  • Dec 2023
Autonomization is a physiological process allowing a flap to develop neo-vascularization from the reconstructed wound bed. This phenomenon has been used since the early application of flap surgeries but still remains poorly understood. Reconstructive strategies have greatly evolved since, and fasciocutaneous flaps have progressively replaced muscle-based reconstructions, ensuring better functional outcomes with great reliability. However, plastic surgeons still encounter challenges in complex cases where conventional flap reconstruction reaches its limitations. Furthermore, emerging bioengineering applications, such as decellularized scaffolds allowing a complex extracellular matrix to be repopulated with autologous cells, also face the complexity of revascularization. The objective of this article is to gather evidence of autonomization phenomena. A systematic review of flap autonomization is then performed to document the minimum delay allowing this process. Finally, past and potential applications in bio- and tissue-engineering approaches are discussed, highlighting the potential for in vivo revascularization of acellular scaffolds.
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A rat model of dual-flow liver machine perfusion system
Article
Full-text available
  • Oct 2023
  • ACTA CIR BRAS
Purpose As clinical liver perfusion systems use portal vein and artery flow, dual perfusion techniques are required even in small animal models in order to reproduce clinical setting. The aim of this study was to construct a new dual-flow perfusion system in rat model and optimized the oxygen supply to ensure the aerobic metabolization. Methods The dual-flow circuit was fabricated using rat liver and whole blood samples as perfusates. The oxygen supply was controlled according to the amount of dissolved oxygen in the perfusate. Perfusate parameters and adenosine triphosphate (ATP) levels were analyzed to evaluate organ function and metabolic energy state. Stored whole blood also tested the suitability as perfusate. Results Stored blood showed decrease oxygen delivery and liver function compared to fresh blood. Using fresh blood as perfusate with air only, the dissolved oxygen levels remained low and anaerobic metabolism increased. In contrast, with oxygen control at living body level, anaerobic metabolism was well suppressed, and tissue ATP content was increased. Conclusions We developed a new dual-flow system that enable to reproduce the clinical settings. The perfusion system showed the possibility to improve the energy metabolic state of the perfused organ under appropriate partial pressure of oxygen. Key words Oxygenation; Perfusion; Rats; Ischemia; Liver
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Axillary Hidradenitis Suppurativa: A Comparison between Two Perforator Flap Reconstructive Approaches after Radical Surgical Management
Article
Full-text available
  • Oct 2023
Background Axillary hidradenitis suppurativa (HS) can result in significant functional impairment in both personal and professional lives. Stage 3 HS requires radical surgical treatment. Flap reconstruction allows for faster healing and better functional and aesthetic outcomes. We compared the results of thoracodorsal artery perforator (TDAP) and propeller inner arm artery perforator (IAAP) flap reconstructions after radical surgical treatment of axillary HS. Methods We conducted a retrospective study that included 13 consecutive patients who underwent stage 3 axillary HS treatment between August 2015 and January 2023. Seven patients underwent reconstruction by islanded TDAP flaps, whereas six patients underwent reconstruction by propeller IAAP flaps, with one patient undergoing bilateral reconstruction. The data collected from the patient records included age, gender, smoking status, body mass index, comorbidities, operative time, defect size, flap size, hospital stay, and complications. Results Although not statistically significant ( P = 0.1923), a higher rate of flap complications is reported here with propeller IAAP flaps (42.86 %), whereas islanded TDAP flaps had no flap complications (0%). We found a statistically significant difference in operative time ( P = 0.0006), defect size ( P = 0.0064), and flap size ( P = 0.0012) between the two groups. All patients exhibited satisfactory functional and aesthetic outcomes. Fourteen flaps were performed in total; only one case exhibited recurrence (7.14%). Conclusion After radical surgical management, both islanded TDAP and propeller IAAP flap reconstructions offer excellent outcomes for stage 3 axillary HS. We strongly encourage our peers to consider performing perforator flaps over secondary healing for these patients with a major functional impairment.
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Gluteal Propeller Perforator Flaps: A Paradigm Shift in Abdominoperineal Amputation Reconstruction
Article
Full-text available
  • Jun 2023
Abdominoperineal amputation (AAP) is a gold standard procedure treating advanced abdominal and pelvic cancers. The defect resulting from this extensive surgery must be reconstructed to avoid complications, such as infection, dehiscence, delayed healing, or even death. Several approaches can be chosen depending on the patient. Muscle-based reconstructions are a reliable solution but are responsible for additional morbidity for these fragile patients. We present and discuss our experience in AAP reconstruction using gluteal-artery-based propeller perforator flaps (G-PPF) in a case series. Between January 2017 and March 2021, 20 patients received G-PPF reconstruction in two centers. Either superior gluteal artery (SGAP)- or inferior artery (IGAP)-based perforator flaps were performed depending on the best configuration. Preoperative, intraoperative, and postoperative data were collected. A total of 23 G-PPF were performed—12 SGAP and 11 IGAP flaps. Final defect coverage was achieved in 100% of cases. Eleven patients experienced at least one complication (55%), amongst whom six patients (30%) had delayed healing, and three patients (15%) had at least one flap complication. One patient underwent a new surgery at 4 months for a perineal abscess under the flap, and three patients died from disease recurrence. Gluteal-artery-based propeller perforator flaps are an effective and modern surgical procedure for AAP reconstruction. Their mechanic properties, in addition to their low morbidity, make them an optimal technique for this purpose; however, technical skills are needed, and closer surveillance with patient compliance is critical to ensure success. G-PPF should be widely used in specialized centers and considered a modern alternative to muscle-based reconstructions.
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Development of a rat forelimb vascularized composite allograft (VCA) perfusion protocol
Article
Full-text available
Vascularized composite allografts (VCAs) refer to en bloc heterogenous tissue that is transplanted to restore form and function after amputation or tissue loss. Rat limb VCA has emerged as a robust translational model to study the pathophysiology of these transplants. However, these models have predominately focused on hindlimb VCAs which does not translate anatomically to upper extremity transplantation, whereas the majority of clinical VCAs are upper extremity and hand transplants. This work details our optimization of rat forelimb VCA procurement and sub-normothermic machine perfusion (SNMP) protocols, with results in comparison to hindlimb perfusion with the same perfusion modality. Results indicate that compared to hindlimbs, rat forelimbs on machine perfusion mandate lower flow rates and higher acceptable maximum pressures. Additionally, low-flow forelimbs have less cellular damage than high-flow forelimbs based on oxygen uptake, edema, potassium levels, and histology through 2 hours of machine perfusion. These results are expected to inform future upper extremity VCA preservation studies.
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Microvascular assessment of fascio-cutaneous flaps by ultrasound: A large animal study
Article
Full-text available
  • Dec 2022
Objectives: Blood perfusion quality of a flap is the main prognostic factor for success. Microvascular evaluation remains mostly inaccessible. We aimed to evaluate the microflow imaging mode, MV-Flow, in assessing flap microvascularization in a pig model of the fascio-cutaneous flap. Methods: On five pigs, bilateral saphenous fascio-cutaneous flaps were procured on the superficial femoral vessels. A conventional ultrasound evaluation in pulsed Doppler and color Doppler was conducted on the ten flaps allowing for the calculation of the saphenous artery flow rate. The MV-Flow mode was then applied: for qualitative analysis, with identification of saphenous artery collaterals; then quantitative, with repeated measurements of the Vascularity Index (VI), percentage of pixels where flow is detected relative to the total ultrasound view area. The measurements were then repeated after increasing arterial flow by clamping the distal femoral artery. Results: The MV-Flow mode allowed a better follow-up of the saphenous artery’s collaterals and detected microflows not seen with the color Doppler. The VI was correlated to the saphenous artery flow rate (Spearman rho of 0.64; p = 0.002) and allowed to monitor the flap perfusion variations. Conclusion: Ultrasound imaging of microvascularization by MV-Flow mode and its quantification by VI provides valuable information in evaluating the microvascularization of flaps.
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Abstract #436 - 24h Acellular Subnormothermic Machine Perfusion for VCA Preservation
Poster
  • Jun 2023
  • AM J TRANSPLANT
Purpose: The gold standard for preserving vascularized composite allotransplantation (VCA) is 4°C Static Cold Storage (SCS). Muscles are being susceptible to ischemia, and ischemia-reperfusion injuries can be described as early as after 2hrs of SCS. Machine perfusion is growing in the world of organ preservation. We describe here the use of acellular subnormothermic machine perfusion (SNMP) for 24-hr VCA preservation before transplantation. Methods: Six partial hindlimbs were procured on 35kgs Yorkshire pigs under general anesthesia. 3 limbs were preserved with SNMP, and 3 control limbs were cold stored for 24hrs before heterotopic allotransplantation. The follow-up period lasted 14 days, during which immunosuppression was provided by IV administration of tacrolimus and steroids. Animals were clinically monitored twice daily, and transplant biopsies and blood samples were regularly collected (K+, Lactate, CBC, WBC, and Proinflammatory cytokines). At the end of the study, pigs were humanely euthanized. Two blinded pathologists assessed skin and muscle samples. Results: Overall survival was higher in the SNMP group. Early euthanasia of 2 pigs in the SCS group was linked to significant graft degeneration. Overall, CBC and WBC were comparable between groups. Potassium and lactate concentrations were higher in the SCS group without reaching statistical significance. Macroscopic analysis of the grafts after necropsy showed massive muscle degeneration in the SCS group and a normal aspect in the SNMP group. Conclusions: SNMP with Steen+ demonstrated better results for the preservation of VCA when compared with 4°C SCS. We showed that this technique allows transplantation after 24h preservation. This data following in-vivo application in a large animal is essential for clinical application.
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Breast Reconstruction Free Flap Failure: Does Platelet Count Matter?
Article
  • Nov 2022
  • ANN PLAS SURG
Background: Microvascular free tissue transfer is an increasingly popular modality for autologous tissue breast reconstruction. However, flap failure remains an ominous risk that continues to plague surgeons and patients even in the setting of meticulous surgical technique and monitoring. Venous and arterial thromboses are the leading causes of free flap failure. The purpose of this study was to determine whether thrombocytosis is associated with breast free flap failure. Methods: A retrospective study was conducted of breast reconstruction with free flaps in North America between 2015 and 2020 using the National Surgical Quality Improvement Program database. Patient comorbidities and preoperative laboratory tests were used to determine risk factors for free flap failure. Results: During the study interval, 7522 female patients underwent breast reconstruction with free flaps, and flap failure occurred in 2.7% patients (n = 203). In multivariate regression analysis, breast free flap failure was significantly higher in patients smoking cigarettes within the past year (P = 0.030; AOR, 1.7) and dyspnea on moderate exertion or at rest (P = 0.025; AOR, 2.6). Furthermore, each 50 K/mcL elevation in platelet count was independently associated with an increased odds of flap failure (P < 0.001; AOR, 1.2). Patients experienced significantly higher rates of flap failure with platelet counts greater than 250 K/mcL (P = 0.004), which remained significant through progressively increasing thresholds up to 450 K/mcL. Conclusions: Platelet count greater than 250 K/mcL is associated with progressively increasing risk of free flap failure in breast reconstruction. Future studies of personalized patient anticoagulation protocols based on hemostatic metrics may improve free flap survival after autologous tissue breast reconstruction.
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