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CiteULikeDeliciousJreconstrMicrosurg201228(08):501508DOI:10.1055/s00321315768ThiemeMedicalPublishers333SeventhAvenue,NewYork,NY10001,USA.

ANewMicrovascularAnastomosisTechniqueUsingMuscleGraft

HaldunO.Kamburolu1,HakanUzun1,OzanBitik1,ErhanSnmez1,Tunafak1,PerginAtilla2,AyeN.akar2

1DepartmentofPlasticReconstructiveandAestheticSurgery,HacettepeUniversityFacultyofMedicine,Ankara,Turkey2DepartmentofHistologyandEmbryology,HacettepeUniversityFacultyofMedicine,Ankara,Turkey

FurtherInformation

Addressforcorrespondenceandreprintrequests

HaldunO.Kamburolu,M.D.,F.E.B.O.P.R.A.S.DepartmentofPlasticReconstructiveandAestheticSurgeryHacettepeUniversityFacultyofMedicine,AnkaraTurkey,06100Email:halonka@yahoo.com

PublicationHistory

27November2011

21March2012

PublicationDate:18June2012(eFirst)

AbstractFullTextReferencesFigures

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MaterialsandMethodsSurgicalTechniqueLightMicroscopyandImmunohistochemicalAnalysisStatisticalAnalysis

ResultsAnastomosisTimeAmountofSutureUsagePatencyRatesandRateofAneurysmFormationAnastomoticLeakageLightMicroscopicandImmunohistochemicalFindings

DiscussionConclusionReferences

Abstract

Anovelmicrovascularanastomosistechniqueisdescribed.FortyfivemaleSpragueDawleyratsweredividedequallyintothreegroupsbeforeundergoingfemoralarteryanastomosis.Thefirstgroupreceivedstandardeightsutureanastomoticrepair.Group2(musclegroup)receivedthreesuturesplusautogenousmusclegraftwrappedaroundtheanastomosis.Ingroup3(fascialsurfacegroup),amusclegraftwaswrappedaroundtheanastomosiswiththefascialsideofthegraftfacingtheanastomosis.Significantlylesstimeandsutureusagewerenotedusingbothfascialsurfaceandmusclegroupscomparedwithcontrols(p0.05).Additionally,grade2anastomoticleakagewaslessfrequentinthe

studygroupscomparedwiththecontrolgroup(p

Figure1Preparationoftheanastomosis.Threesutureswereplacedaroundthevesselsat0degrees,120degrees,and240degrees.Themusclegraftwasplacedjustbeforewrappingthevessels.

Figure2Viewofthemusclegroupat20minutes.Notethatthereisnoanastomoticleakage.

Figure3Viewofthefascialsurfacegroupat20minutes.Grade1anastomoticleakageisseeninthisanastomosis.

SurgicalTechnique

Therightfemoralarteryofeachratwasexposedthroughaninguinalskinincisionanddisplayedfromtheinguinalligamenttothesuperficialepigastricvesselsfollowingtheremovaloftheoverlyinginguinalfatpadandretractionoftheabdominalwallmuscles,medially.Thedeepbranchesofthefemoralarterywereligatedanddividedundermagnification.Allvesselsweretreatedwith2%lidocaineforreliefofspasm.

Thevesselsweredividedwithscissorsperpendiculartothelongaxisofthevesselbetweendoubleclamps.Thebisectedvesselendswereflushedwithheparinizedsaline(100U/mL)andinspectedundermagnification.Allbloodclotswereremoved.Inallthreegroups,adventitiasofvesselstumpsweretrimmedinstandardfashion.Overlappingadventitiawastrimmed.Vesselendsweredilatedgentlywithjeweler'sforcepsandanastomosiswasperformed.

Forgroup1(thecontrolgroup),anastomoseswereperformedwithinterrupted10/0nonresorbablesuturesinthenormalfashion,startingwithtwosuturesat180degreestoeachother,suturingthefront

wallfollowedbythebackwall.Eightstitcheswereusedfortheanastomosis.

Ingroup2(themusclegroup),thevesselendswerepreparedandthreestitcheswereplacedat120degreestoeachother(0degrees,120degrees,240degrees)with10/0nonabsorbablesutures.A0.51cmmusclegraftwasharvestedtangentiallyfromadductormagnusmuscleoftherightthighoftheratwithascalpel(No:15)andamicroforceps(No:5).Musclegraftthicknesswas1mm.Onesideofthemusclegrafthadfascialsurfaceandtheothersidehadaroughsurface.Themusclegraftwaswrappedaroundtheanastomosiswiththeroughsurfacefacingtheanastomosis([Fig.1]).Tostabilizethemusclegraftaroundtheanastomosis,three6/0nonabsorbablesutureswereplacedatthecenter,proximal,anddistalendsofthemuscle([Fig.2]).

Ingroup3(thefascialsurfacegroup),thesameprocedureasingroup2wasperformed,exceptthatthemusclegraftwaswrappedaroundtheanastomosiswiththefascialsurfacefacingtowardtheanastomosis([Fig.3]).

Followingcompletionofeachprocedure,theinguinalincisionswereclosedinasinglelayerwithcontinuous3/0nonresorbablesuture(silk).Theanimalswereallowedtorecoverfromsedationandthenwerereturnedtotheircages.

Timeperiodfromthebeginningofanastomosis(clampplacement)totheendoftheanastomosis(clamprelease)wasrecorded.Ingroup2andgroup3,muscleharvestingwasperformedduringtheanastomosisperiod.Inaddition,thelengthsoftheremainingsutures(10/0polyamidesutures)wererecordedaftercompletionofeachanastomosis.Aftertheclampswerereleased,theleakagewasassessedusingthefollowinggradingsystem:GradeI:immediatebleedingthatstoppedwithoutanyinterventionGradeII:somebleedingthatwasinhibitedbylightpressureGradeIII:heavybleedingthatrequiredreclampingandadditionalsutures.[2]

Patencywasevaluatedonallanastomosesat20minutes,1day,15days,and60daysaftersurgeryusingthemilkingtest.Anythrombus(vascularocclusion)oraneurysmformationwasalsorecorded.

#

LightMicroscopyandImmunohistochemicalAnalysis

Onday60,afterthepatencycheck,allratsweresacrificedandspecimensmeasuring1cminlengthwereharvestedfromthefemoralartery.Specimenswerefixedin10%formalinsolutionbeforetissueprocessingandthenembeddedinparaffinwax.Horizontalsectionsof~5mwereobtainedfromeachspecimenandstainedwithhematoxylinandeosin(H&E).Tenspecimensfromeachgroupwererandomizedandsubsequentlyexaminedbytwoexperiencedpathologistsunderlightmicroscopyusingthefollowingparameters:Eachspecimenwasgradedforendothelization,subintimalhyperplasia,andinflammatoryresponseattheadventitia.Thegradesgivenwereabsent,mild,moderate,orintense.Inaddition,thepresenceofanythrombus,occlusivephenomena,oraneurysmaldilatationwasrecorded.

Inaddition,fivefreshfrozenspecimensfromgroup2andfivefreshfrozenspecimensfromgroup3wereexaminedusingRatAntiMouseCD31(PECAM1)Antibody(BDPharmingen,SanDiego,California,USA)at1:50dilutionforendothelialstaining.

#

StatisticalAnalysis

OnewayANOVAtestwasusedtocomparealldata.Inallcases,a5%levelofsignificance(p=0.05)wasadopted.

##

Results

AnastomosisTime

Group1(controlgroup)anastomosestookthelongesttimetoperform(p0.05[Tables1]and[2]).

Table1

MeanAnastomosisTime,MeanSutureUsage,PercentageofAnastomoticLeakage,PercentageofAneurysmFormationandPatencyRatesAmongAllGroups

Group1 Group2 Group3

MeanAnastomosisTime(Seconds) 91052 78350 78668

MeanAmountofSutureUsage(millimeter) 193 72 72

Patency(onday60) 93.3% 86.6% 93.3%

Aneurism 6.6% 6.6% 6.6%

AnastomoticLeakage GradeI 33.3% 93.3% 86.6%

GradeII 66.6% 6.6% 13.3%

GradeIII

Table2PValuesforAnastomosisTime,SutureUsage,AnastomoticLeakage,AneurysmFormationandPatencyRatesAmongAllGroups

Table2

Table2

PValuesforAnastomosisTime,SutureUsage,AnastomoticLeakage,AneurysmFormationandPatencyRatesAmongAllGroups

Group1Versus2pValue

Group1Versus3pValue

Group2Versus3pValue

AnastomosisTime 0.001 0.001 0.960

AmountofSutureUsage

0.001 0.001 0.874

Patency(onday60) 0.808 1 0.808

Aneurism 1 1 1

AnastomoticLeakage 0.001 0.006 0.914

#

AmountofSutureUsage

Similartoanastomosistime,thenumberofsuturesusedingroups2and3waslessthanthenumberusedingroup1(p0.05[Tables1]and[2]).

#

PatencyRatesandRateofAneurysmFormation

Ineachgroup,oneof15anastomoseswasthrombosedat20minutes.Onedayaftersurgery,anotherthrombosedanastomosiswasfoundingrouptwo([Table3]).Thecalibersoftheproximalanddistalvesselsandanastomoticregionswereallwellmatchedatday60([Figs.47]).Oneaneurysmwasobservedineachgroup.Asaresult,therewasnosignificantdifferenceregardingpatencyratesoraneurysmformationamongthesethreegroups(p>0.05[Tables1]and[2]).

Figure4Viewofthemusclegrouponthe60thday.Notethatmuscletissueistotallyabsorbed.

Figure5Viewofthefascialsurfacegrouponthe60thday.

Figure6Viewofthemusclegrouponthe60thdayaftertheloosetissuedissection.Itisclearthattheadherenceofthemusclegraftaroundtheanastomosisisfirmandtheanastomosisishealthywithoutocclusion.

Figure7Viewofthefascialsurfacegrouponthe60thdayaftertheloosetissuedissection.Allofthemusclegrafthasresorbed,leavingastablestructurearoundtheanastomosis.

Table3

PatencyRatesofallGroupsat20Minutes,1Day,15Days,and60

Days

Group1 Group2 Group3

20thminute 93.3% 93.3% 93.3%

1stday 93.3% 86.6% 93.3%

15thday 93.3% 86.6% 93.3%

60thday 93.3% 86.6% 93.3%

#

AnastomoticLeakage

Bleedingwasevaluatedateachanastomoticsite.AlthoughGradeIanastomoticleakagewashigheringroups2and3comparedwithgroup1,grade2anastomoticleakagewassignificantlyloweringroups2and3comparedwithcontrols(p0.05[Tables1]and[2]).

#

LightMicroscopicandImmunohistochemicalFindings

Smoothendothelizationcoveredthemusclefibersofgroups2and3,similartothecontrolgroup([Figs.8]and[9]).Inaddition,theendotheliallayerwascontinuousandlaiddowninamonolayerpattern([Figs.10]and[11]).However,mildintimalhyperplasiawasnotedinthreeofthecontrolgroup'sspecimensaswellasinthreespecimensofgroup2andfourspecimensofgroup3.Tunicaintimaandtunicamedialayerswereobservedwithnormalhistologicpatternsintherestofthesegroups.Internalandexternalelasticmembraneswereofnormalthicknessinallgroups.Moderateinflammatoryreaction,withgiantcellsnotedagainstthesuturematerial,wasfoundinallgroups,butthisreactionwasmildingroups2and3becausethenumberofsutureswerereducedinthosetwogroupscomparedwithcontrols.Stenosisduetothrombusformationwasobservedinfivespecimensincludingoneingroup1,twoingroup2,andoneingroup3.Oneaneurysmformedineachgroup.

Figure8Hematoxylinandeosinstainoftheanastomosiswithmusclegraft,60daysaftertheprocedure.Muscleremnantandforeignbodyactivityaroundsuturematerialcanbeseen(x100).

Figure9Hematoxylinandeosinstainoftheanastomosiswithfascialsurfacefacingtheanastomosis,60daysaftertheprocedure.Theremnantcanbeseenwithendothelizationcoveringit(x100).

Figure10Hematoxylinandeosinstainoftheanastomosiswithroughsurfaceofmusclegraftfacingtheanastomosis,60daysaftertheprocedure(x400).

Figure11Hematoxylinandeosinstainoftheanastomosiswithfascialsurfacefacingtheanastomosis,60daysaftertheprocedure(x400).

CD31immunoreactivitywasseeninendothelialcellsofallgroupsinacontinuousmonolayerpattern([Figs.12]and[13]).

Figure12CD31immunohistochemicalstainoftheendotheliuminthemusclegroupatday60(x630).Immunohistochemicalactivityoftheendotheliumcaneasilybeseen.

Figure13CD31immunohistochemicalstainoftheendotheliuminfascialsurfacegroupatday60(x630).Immunohistochemicalactivityoftheendotheliumcanbeseen.

##

Discussion

Theconventionaltechniqueofendtoendmicrovascularanastomosisusinginterruptedsutureshasbeenwelldescribed.[3][4]Althoughitiscurrentlyconsideredasthegoldstandard,[5]thetechniquehascertainlimitations.Themajorproblemoftheconventionaltechniqueisthetimeconsumedtoperformanastomosis.[6][7][8]Thisbecomesacriticalfactorwhenperformingmultipleanastomoses,suchasinmultipledigitreimplantations.[6][7][8][9]

Also,increasednumberofsuturesisanotherdisadvantagefortheconventionaltechnique.Ifthenumberofsuturesisincreased,thiscouldinducevascularwalldamage,whichaffectsthehealingresponseandincreasestheriskofthrombosis.[7][10][11][12][13]Inaddition,nonresorbablesuturematerialmayinduceaninflammatoryreactionifleftasanintraluminalforeignbody.Itmayalsoprecipitatethrombocyticaggregation,impairedendothelialfunction,intimalhyperplasia,orstenosis.[7][10][11][12][13]Thereisalsoanincreasedbackwallbitingriskwithanincreasednumberofsutures,eveninexperiencedhands.Ontheotherhand,ifthesuturenumberdecreases,anastomosisleakagecouldbeaproblem.

Toovercomethesedisadvantages,variousmethodssuchascontinuoussuture,mattress[14]suturing,

sleeve[15]suturingandfishmouthanastomosistechnique,[7]autogenouscuffs(arterial,[16]venous,[17]fatpad[18]),vesselclips,[19][20][21]vesselcouplingdevices,[22][23][24][25][26][27]stents[28][29][30],biological[8][31][32][33]andnonbiological[6][34][35]adhesives,andlaserwelding(NeodymiumYAG,[36]carbondioxide,[37]argon,[38]excimer,[39]1.9mdiode[40])havebeenperformedwithvaryingsuccess.Nonehavegainedworldwideacceptancebecauseofvariousassociatedproblemsthatincludereducedrateofpatency,increasedcost,shortenedvessels,ornarrowedanastomosis.

Themattresssuturetechnique(asdefinedbySasaki[14])andthefishmouthtechnique(introducedbyTuran[7])havetheadvantageofreducinganastomosistime,butvesselshorteningisrequiredtoperformtheanastomosis.Anothertechniqueusedforanastomosisisthesleevetechnique,whichcanresultinanastomoticnarrowing.[15]Also,thistechniquerequiresthatthedistalpediclebelargerthantheproximalone.

Anattempttosolvetheseproblemsresultedinautogenouscuffs,whichrequirelesssutureusageand,subsequently,shorteranastomosistimes,butrequirescarificationoftherecipientvessel.Also,thereishighriskofaneurysmformationwithveincuffs.[17]Investigatorshaveusedfatpadandfourstitchestoperformthistypeofanastomosiswithvaryingsuccessduetothehighriskofadhesionsafteranastomosis.[18]

Vascularclipshavealsobeenusedtoperformanastomosis.ThefirstvascularclipsweredescribedbyBikfalviandDubeczin1953.[41]Kirschintroducedmoremodernclipsandtheirclinicalusagein1992,[19]butthesedeviceshadtheproblemsofhighcost,reducedavailability,andlimitedapplication,especiallywhenusedinatheroscleroticvessels.[20][21][41]

Anotheranastomoticoption,couplingdevicesandcouplingcircles,werefirstdescribedHoltandLewisin1960.[22]Theuseofamicrovascularcouplingdeviceforperformanceofvenousanastomosishasrecentlyregainedpopularity.Severalpublishedserieshavedemonstratedtheeaseandreliabilitywithwhichthesedevicescanbeusedwithanastomoticpatencyratesequaltoorsurpassingthoseforsuturedanastomoses.[42][43]Thesedevicesmaybeusedwithequalfacilityforbothendtoendandendtosideanastomosesandhavebeenusedtoanastomoseveinswithsignificantsizediscrepancies.[44][45]Also,ithasbeenshownthatthistechniqueisusefulandsafeforarterialanastomosis.[46]Topreventforeignbodyreaction,absorbabledeviceshavebeenusedforvesselcoupling.[26]Buttheirlimitedringdiametersizes,diametermismatch,highcost,limitedavailability,[41]andsizemismatchesinarterialanastomosesarestillproblems.[46]

Soluble[29]andinsoluble[30]tubesorstentswereinvestigatedsinceDr.Abbefirstusedglasstubestoperformvesselanastomosis.[28]Theirresultingpatencyrateswereverylow,however.[41]

Adhesivesbecamepopularoverthelast10years,thoughtheywerefirstintroducedin1977byMatrasetal.[31]Sincethattime,severalstudieshavebeenperformedwithfibrin[32][33]andcyanoacrylateglues.[6][34][35]Allergicandtoxicreactions,leakage,aneurysmformation,andpossiblethrombusformationwerethemaindrawbacksoftheseproducts,althoughtheycanprovideshorteranastomosistimes.[41][47][48]

Anothermethodtoshortenanastomosistimeistheuseoflasers.Laserswerefirstintroducedin1979byJainandGorish.[36]NeodymiumYAG,[36]carbondioxide,[37]argon,[38]excimer[39]and1.9mdiode[40]lasershaveallbeenusedwithvaryingsuccess.Themainissuesafterlaserassistedanastomosisappeartoberelatedtothereducedstrengthoftheanastomosisandincreasedriskofaneurysmformation.[37][38]Also,thecostoftheequipmentandriskofthermalinjuryareimportantadditionaldrawbacks.[41]Recentresultswithlowpower1.9mdiodelasershavebeenmore

satisfactory.[40]Ontheotherhand,theneedforexpensiveequipmentstillexists.

Inthisstudy,wepresentanewanastomosistechniqueusingautogenousmusclegrafts.Shortenedsurgicaltimesandreducednumberofanastomoticsuturesareimprovementsoverconventionaltechniques.Therewouldbeaquestionaboutthecosteffectivenessofanextrasutureusageof6/0prolene.Intermsofsavingsuturecost,one100sutureof10cmlengthcouldbeusedforfourvesselrepairs,usingthestandardmethodofanastomosisthisisgenerallyenoughsutureformostmicrovascularcases.Ontheotherhand,themusclegrafttechniquewouldrequiretwosutures,notone:one10/0forthevesselsandone6/0sutureforthemusclegraftwrapping.Wethinkthatitiscosteffectiveformultipleanastomosisbecausethe10/0suturesarethreetimesmoreexpensivethan6/0sutures.But,insingleordoubleanastomosis(uptofour),likeflapsurgery,itisnot.

Usingthisnewmethod,lessgradeIIanastomoticleakagewasnotedascomparedwithmoreconventionaltechniques,thoughriskofaneurysmformationandpatencyratesweresimilar.Histologically,asmoothmonolayerofendothelizationwasnotedtooccuroverthemusclegrafts,asconfirmedbyimmunohistochemicalanalysis.

Anotheradvantageofthisnewtechniqueisthereductionintheamountofintraluminalforeignbodyandthereducedintimaldamage.Inaddition,itisalmostimpossibletocauseabackwallbiteduringanastomosiswiththisnewmethod.

Sincethematerialusedisautogenous,itisalwaysavailableandabsorbable,ascomparedwithcouplingdevices,lasers,andadhesives.Allergicreactionsandtoxicity,asseenwithadhesives,isavoided.Thereisnoneedtoshortenthevesselslikethemattressandfishmouthtechniques.Also,thereisnoriskofvesselnarrowingasseenusingthesleevetechnique.Novesselscarificationisneededforthisanastomosis,likeotherautogenousmethods.

Thedisadvantagesofthismethodareitssteeplearningcurveandthelackofavailabilityofdonorsitesforthemusclegraft.Butasinmosthandsurgerycases,itisrelativelyeasytofindamuscletouseforasuperficialmusclegraft.Ontheotherhand,whileperformingthistechnique,therearetwoimportantstepsthatthesurgeonshouldlearn.Thefirstistheplacementofthe6/0nonresorbablesuturesincorrectposition.Theyshouldbeplacedproperlytopreventtheslidingofthemuscleoverthevessel.Theotherimportantstepismanagingthetightnessofthesesutures.Iftheyaretootight,theymaysqueezethevesseliftheyaretooloose,leakagemayoccur.

Routineuseofthistechniqueinclinicalanastomosismaynotbepreferredbecauseofthedisadvantagesmentioned.Butinselectcases,likemultipleanastomosisinthesettingofhandtraumaorespeciallyinanastomoticleakage,thismethodshouldbeconsidered.

#

Conclusion

Wedescribedanewmicrovascularanastomotictechniqueusingthreesuturesandanautogenousmusclegraft.Usingthismethod,thenumberofsutures,totalcost,andoperatingtimewerereducedwiththesimilarpatencyratesastheconventionaltechnique.Wealsodemonstratedthatthismethodissafefrombothanastomoticleakageandbackwallbite.

#

#Acknowledgment

ThisstudywassupportedbyHacettepeUniversityScientificResearchCenter.

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