Allograft integration in a rabbit transgenic model for anterior cruciate ligament reconstruction

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Article

Reference

Allograft integration in a rabbit transgenic model for anterior cruciate ligament reconstruction

BACHY, M., et al.

BACHY, M., et al . Allograft integration in a rabbit transgenic model for anterior cruciate ligament reconstruction. Orthopaedics & Traumatology, Surgery & Research , 2016, vol. 102, no. 2, p. 189-195

DOI : 10.1016/j.otsr.2015.12.007

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http://archive-ouverte.unige.ch/unige:80300

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Original article

Allograft integration in a rabbit transgenic model for anterior cruciate ligament reconstruction

M. Bachy

^a,b,c

, I. Sheriﬁ

^a,d

, F. Zadegan

^a,e

, H. Petite

^a

, R. Vialle

^b,∗,c

, D. Hannouche

^a,e

aLaboratoiredebioingénierieetbioimagerieostéo-articulaire(B2OA),CNRSUMR7052,universitéDenis-DiderotParisVII,Paris,France

bUniversitéPierreetMarieCurieParis6,DepartmentofPediatricOrthopaedics,Armand-TrousseauHospital,26,avenueduDr-Arnold-Netter, 75571Pariscedex12,France

cTheMAMUTHHospital-UniversityDepartmentforInnovativeTherapiesinMusculoskeletalDiseases,Armand-TrousseauHospital,26, avenueduDocteur-Arnold-Netter,75571Pariscedex12,France

dTheMountSinaiHospitalOneGustaveL.-LevyPlace,NewYork,USA

eServicedechirurgieorthopédiqueetréparatrice,hôpitalLariboisière,AP–HP,universitéDenis-DiderotParisVII,Paris,France

a r t i c l e i n f o

Articlehistory:

Received25September2015 Accepted15December2015

Keywords:

Anteriorcruciateligament Tendinousallograftboneintegration Ligamentization

Animalstudy

a b s t r a c t

Background:Tissueengineeringstrategiesincludebothcell-basedandcellhomingtherapies.Ligamen- toustissuesarehighlyspecializedandconstitutevitalcomponentsofthemusculoskeletalsystem.Their damagecausessigniﬁcantmorbidityandlossinfunction.

Hypothesis:Theaimofthisstudyistoanalyzetendinousgraftintegration,cellrepopulationandliga- mentizationbyusingGFP+/–allograftsinGFP+/–transgenicNewZealandwhite(NZW)rabbits.

Materialandmethods:Graftimplantationwasdesignedtocloselymimicanteriorcruciateligament(ACL) repairsurgery.Allograftswereimplantedin8NZWrabbitsandassessedat5days,3weeksand6weeks through:(1)arthroCTimaging,(2)morphologicalanalysisofthetransplantedallograft,(3)histologicalanalysis,(4)collagentypeIimmunochemistry,and(5)GFPcelltracking.Collagenremodelingwas appreciatedat3and6weeks.

Results:Graftrepopulationwithhostcells,chondrocyte-likecellsatthetendon-boneinterfaceandgraft corticalizationinthebonetunnelswerenoticedat3weeks.Bycontrastwenoticedacentralnecrosis aspectintheallograftsintra-articularlyat6weekswithacellmigrationtowardsthegraftedgenearthe synovium.

Discussion:Ourstudyhasservedtogainabetterunderstandingoftendinousallograftboneintegration, ligamentizationandallograftrepopulation.Webelievethatbothcell-basedtherapiesandcellhoming therapiesarebeneﬁcialinligamenttissueengineering.Futurestudiesmayelucidatewhethercellrepop- ulationoccurswithpre-differentiatedorprogenitorcells.Webelievethatbothcell-basedtherapiesand cellhomingtherapiesarebeneﬁcialinligamenttissueengineering.

Levelofevidence:LevelV(animalstudy).

1. Introduction

Tissueengineeringsolutionsaimtoregeneratefailedordefec- tiveorgansandtissues.Cell-basedapproachesusedtohelpheal defectsandreplacenativecellshaveconstitutedthemainstayof researchformanyyears[1].Recentadvancesintissueengineering haveevaluatedtheemergingthemeofcellhomingwherebyusing thehost’s endogenous cells, includingstem orprogenitor cells,

∗ Correspondingauthor.UniversitéPierre-et-Marie-CurieParis6,Department ofPediatricOrthopaedics,Armand-TrousseauHospital,26,avenueduDr-Arnold- Netter,75571Pariscedex12,France.Tel.:+33144736125;fax:+33144736324.

E-mailaddress:raphael.vialle@trs.aphp.fr(R.Vialle).

torepopulatetissueengineeringscaffoldsforuseinregeneration therapies[1].

Injurytotheanteriorcruciateligament(ACL)affectsmorethan 200,000peopleeachyearintheUnitedStates[2].TherepairofACL defectoftenrequirestheharvestofareplacementgraftfromthe patientcausingsigniﬁcantpainandmorbidity[2].Inadditionto autografts,muchresearchhaslookedatallograftreplacementsfor rupturedanddefectivetendons[3,4].Incorporationofthetendon occursthroughtendongraftligamentizationandintegrationinto thebonetunnel[5,6].Duetothisreason,itbecomesincreasingly importanttostudy,understand,andoptimizecellgrowthandheal- inginthebonetunnelafterACLrepair[7].Despitesomeattempts toimprovethebiomechanicalstrengthofthebone-tendoninter- facebyinstillingsubstancessuchasinsulin-growthfactor1and http://dx.doi.org/10.1016/j.otsr.2015.12.007

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2 M.Bachyetal./Orthopaedics&Traumatology:Surgery&Researchxxx(2015)xxx–xxx

Fig.1. A.Photographyofthesurgicalprocedureshowingtendongraftpassageintibialbonetunnel.B.Macroscopicﬁnalaspectofimplantationoftendongraftasnative ACL.C.Sagittaltibialslide.D.AxialtibialslideofarthroCTrevealingcorticalizationsignalallthelengthofbonetunnels.E.Allograftmacroscopicaspect3weeksafterthe surgery.

matrix-metalloproteaseinhibition[8,9],cellgrowthandtendinous graftintegrationinthehost’sbonetissuestillremainsachallenge [10,11].

Considerableresearchhasbeenfocusedongraftosteointegra- tioninbonetunnelsandintra-articulargraftligamentizationafter anteriorcruciateligament(ACL)reconstruction[12–18].Thedevel- opmentofaneasyandreliablemethodthatwouldallowcellfate monitoringintransplantrecipientsisapressingconcernforthe ﬁeldofligamentallografts.Thetransductionofdetectablegenetic markers,suchasthoseencodinggeneﬂuorescentprotein(GFP), allowstableandreliablelong-termlabelingoftransplantedcells andprovideanimportantvehicleforthestudyofcellfateinthe implantedgraft.

We describe here a process used to better understand cell survivalandhealinginabonetunnelfollowingsurgicalgraftrecon- structionofrupturedACLinaGFPtransgenicrabbitmodel[19].The GFPallowsspeciﬁctrackingofcellsderivedfromthehostorganism andthecellspertainingtotheACLallograft.Inaddition,itallows thepossibilityofclearlyfollowingthefateofhostandgraftcells inordertodeterminecellsurvivaland graftincorporationafter allograftimplantation.Itfurtherallowstheopportunitytostudy therelativecontributionsofgraftversushostcellsistendontis- sueallografts.WehypothesizedthatallograftsinGFPtransgenic modelforACLreconstructioncouldgiveabetterunderstandingof graftintegration,cellrepopulationandligamentization.

2. Materialsandmethods 2.1. GFPanimals

Transgenic GFP animals were obtained by the technique of pronuclearmicroinjectionofDNAintonaturallyovulatedandfer- tilizedrabbiteggspreviouslydescribedbyHoudebine[19](INRA, Jouy-en-Josas, France). Eight skeletally immature New-Zealand whiterabbits(averageage=10weeksold,agedistribution=6to14 weeksold,averageweight=2.35±0.6kg)wereused.Ofthese,four wereGFP-expressinganimalmodels(GFP+)and4didnotexpress GFP(GFP–).

2.2. Surgicaltechnique

The technique of ACL reconstruction was performed under general anesthesia. All procedures were approved by the

Animal Experiment Ethics Committee of Lariboisiere-Villemin Number09(CEEA-LV/2010-01-01)andwerepreviouslydescribed [20].Inordertostudygraftintegrationandallowcelltrackingin thefreshallografts,one GFP–(controlspecimen)and oneGFP+

rabbitwereoperatedsimultaneouslybythesamesurgeon.Theallo- grafttakenfromaGFP+rabbitwasimplantedinaGFP–hostand theallografttakenfromaGFP–rabbitwasimplantedinaGFP+

host.

Eachrightextensordigitorumlongus(EDL) tendonwashar- vestedandpreparedforimplantation.AfterexcisionoftheACL, tibialandfemoralbonetunnelswerecreatedtoimplanttheallo- graftintheACLisometricnativeposition(Fig.1A).Thegraftwas ﬁxedwithnon-absorbable3.0polypropylenesutures(Fig.1B).All operatedanimalswereallowedtomovefreelyafterthesurgical technique.

2.3. Specimencollection

OneGFP+andoneGFP–rabbitweresacriﬁced5daysafterACL reconstruction.TwoGFP+andtwo GFP-rabbitweresacriﬁced 3 weeksafterACLreconstructionprocedure.OneGFP+andoneGFP–

rabbitweresacriﬁced6weeksafterACLreconstructionprocedure.

2.4. Imagingtechnique

ArthroCT(GE;lightspeed64slice)wasusedtoimagetheposi- tionoftheallograftinthekneejointandtheintegrationofthegraft inthebonetunnel.

2.5. Histologyandimmunochemistry

Attheendofeachpredeterminedtime,therabbitsweresac- riﬁced and the femur-graft-tibiacomplexes were removed and processed for immunohistochemistry. Frozen sections (14␮m thick)werecollectedonslidesandprocessedforGFPandtypeI collagenimmunohistochemistryusingstandardprocedureswith mousemonoclonalantibodies(anti-GFPClontechref:632381and anti-collagen-I Sigma ref: C2456). In order tocheck the speci- ﬁcity of the immunostaining,alternative sections were treated similarly but incubated without the primary antibody. More- over,noGFP-immunostainingwasobservedinnon-GFPrabbits.

Morphologic analysis was performed on serial sections with hematoxylin/safranin-O/fastgreenstaining.

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Fig.2. Temporalmorphologicalchangesintendongraftafter5days(A,B),3weeks(C,D)and6weeks(E,F,H)inthebonetunnel(A,C,E)andclosetothesuturepoints(B, D,F)comparedtonormaltendon(G).D,F,H.Suturelevels.Inﬂammatoryresponse(M:macrophagesactivés,E:erythrocytes);∅:acellularregions;IF:interfacemembrane;

Ca:cartilaginoustissu;Ch:chondrocytes;B:endochondralboneformation.(hematoxylin/safranine-O/fastgreenstaining);scalebar100␮m.

3. Results 3.1. ArthroCT

Acorticalizationsignalwasseenalongthewholelengthofthe bonetunnelsstartingat3weeksaftersurgery(Fig.1C,D).Thiswas observedinboththesagittalandaxialcross-sectionsimaged.

3.2. Grossmorphology

TheallograftsappearedmorphologicallysimilartonativeACLat 3(Fig.1E)and6weeks:glisteningwhitishcolorationandﬁrmness topalpation.

3.3. Histologicalanalysis

Atday5(Fig.2A,B),nointerfacewasdiscernedbetweenthe graftand the bonetissue in thetunnel. We didnot appreciate tendontoboneadherenceatthispoint.However,ahemorrhagic phenomenonwasnoticedinthebonetunnelatthistimepossibly relatedtothebeginningofthehealingprocess.

At3weeks(Fig.2C,D),cartilaginouscellswerediscernedatthe interfacewithafibroustissuecomponentsurroundingthegraftin thebonetunnel.Inaddition,macrophageswereseenattheattach- mentpointssuggestingthepossibilityofaninflammatoryprocess, howevernoossificationwaspresent.

At6weeks(Fig.2E,F,H),chondrocyte-likecellswerenoticed intheintra-articularportionoftheallograft.Thesecellswerenot present at5 daysor 3weeks. In addition,theﬁbrous interface wasdenserinthebonetunnelcomparedto3weeks.Hypertrophic chondrocytesattheattachmentpointsandmedullarycavitywithin thegraft were noticedat 6 weeks suggesting thebeginning of

endochondralossiﬁcation.Thisprocesswasnewatthistimepoint andwasnotpreviouslyseenattheearliertimepoints.

3.4. Immunochemistry

Animmunohistochemistryanalysiswasundertakeninorderto betterstudygrafthealingandintegration.Asimilaranalysiswas doneat5 days,3 weeksand 6 weeks.Aninitialimmunohisto- chemistry analysisofa controltendonwasdone(Fig.3A,4).A multitudeofcollagenType1fiberswerediscernedwiththeirtypi- calcrimppattern.At5days,nomicroscopicchangeswerenoticed betweenthegraftandcontrol(Fig.3B).At3weeks,thecollagen fibersappearedalteredandexhibitedalossofthecrimppattern (Fig.3C).Attheendpointattachmentareas,bloodvessels were noticedin thecollagenmatrixof theallograft.Themicroscopic analysisat6weeksshowedafurtheralterationofthecollagencom- positioninthebonetunnel(Fig.3D),howevertheintra-articular portionremained unchanged.Most interestingly,Sharpey’s like fibres(Fig.3E)anchoringthegrafttothebonetunnelandacar- tilaginoustendon-boneinterface(Fig.3F)werenoticedatthis6 weekstimeperiod.

3.5. GFPanalysis

GFPwasusedasacellularmarker.Cellsurvivalintheallograft wasdeterminedbyanalyzingGFPexpressionofanallografttaken fromaGFP+rabbit(Fig.4A,B,C,D)implantedinaGFP–host(Fig.4 E,F).Repopulationoftheallograftwasdeterminedbyconversely studyingGFPexpressionofanallografttakenfromaGFP–animal andimplantedinaGFP+host.

At 5 days, diffusion of the GFP stain was discerned extra- cellularly in the bone tunnel portion of the graft raising the

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Fig.3.TemporalalterationoftypeIcollagenfibresinthetendongraftedintothebonetunnelafter5days(B),3weeks(C)and6weeks(D)comparedtonormaltendon(A).Sharpey-likefibres(E)andcartilaginoustendon-boneinterfaceafter6weeks(F).Ca:cartilaginoustissu;G:graftedtendon;B:bone;S:Sharpey’slikefibres.

(Immunohistochemistryforcollagen-I).

possibilityofcelldamageatthisportioncomparedtocontrol.In addition,lesscellswereseencentrallyintheportionofthegraft locatedinthebonetunnelascomparedtocontrol.

At3weeks(Fig.5A,C),GFP+cellswerenoticedatthebone- tendoninterfaceas well asa number ofcells withinthe GFP–

allograft.Thissupportedthehypothesisthatcellrepopulationof thegraftoccurredwithcellsderivedfromthehost.Moreover,new host-derivedGFP+bloodvesselswerenoticedattheattachment pointsoftheGFP–graft.

At6weeks(Fig.5B,D),GFPstainingwasstillnoticedintheintra- articularportionoftheGFP+graftsimilartocontrolreinforcing theideaofimplantedgraftsurvivalinthehostchondrocyte-like featureswereobservedinanumberofthesecells(Fig.6).

4. Discussion

Ourstudyhasservedtogainabetterunderstandingoftendinous allograftboneintegration,ligamentizationandallograftrepopulation.Inordertostudyallograftrepopulationandintegrationwe haveusedaGFPanimalmodelpreviouslydescribedbyHoudebine [19].Aspreviouslymentioned,thismethodofproducingatrans- genicanimalbyDNAmicroinjectionsometimesresultsinavariable yieldoftransgenicanimalsandvariableexpressionoftransgenes [21].Howeverthisphenomenondoesnotaffecttheresultsofour studybecausewewereabletoseeGFP+cellsderivedfromthehost inallGFP–allografts.Thefractionofcellexpressionwouldbecome criticalinstudiesthatareassessingtheabsenceofGFP+cellsinneg- ativeallografts.Asourstudydoesnotuseameasureofquantitative GFPexpression,thislimitationdoesnotapplytoourresults.

Aswithotherallograft transplantsusedfor tissue engineer- ingapplications,matrixandcellbehaviourinthissystemmaybe affectedbytheimmuneresponseofthehostandsurgicalfailure [22].Forinstance,wenoticedanearlyalterationintheGFPexpres- sionofthegraftinthebonetunnelassociatedwithcelllossinthis

area.Thismaybearesultofanearlyacuterejectionsyndromein theallograftoranischemiccompressionofthegraftduringintro- ductioninthebonetunnel.

Ourresultsalsoshowacentralareaofnecrosiswithintheintra- articularportionofthegraft.Oneexplanationforthis maybea preferentialmigrationofthecellsfromacentrallocusintheallo- grafttowardstheperipherywherecontactwiththesynovialﬂuid allowsnutritionalenrichment.Thiscentralnecrosisintheintra- articularportionhas beenpreviously described in an autograft modelbyPannietal.[23].Thisalsoraisesthepossibilitythatthere isdecreasedsurvivalofthecellsinthecentralportion,whereas cellsattheedgeofthegraftstillsurviveintra-articularlybasedon nutrientssuppliedbythesynovialﬂuid.

We noted a neovascularisation occuring in thebone tunnel portionof theallograft ﬁrstat3weeks.Aroundthesametime, host-derivedcellsappearattheinterfacewithcartilaginousmor- phologyraisingtheideathatgraftrepopulationissourcedfromthe host.Eitherthesecellshavemigratedtotheinterfacealreadydif- ferentiatedgiventheformationofnewbloodvesselsorprogenitor cellswerebroughttotheinterfacebytheneovascularisationand thendifferentiatedgiventhegrowthfactorsintheallograft.Further studiesareneededtoanswerthisquestionforexamplebystudying cellproliferationmarkerssuchasKi67orPCNA[24].

WhethertouseanautograftoranallograftinACLreconstruction isstillcontroversialinclinicalpractice.Althoughpatellatendon autograftandthenhamstringtendonshaslongbeenconsideredthe goldstandard,allograftsafetyhasimprovedsigniﬁcantlyinthelast 15years,makingitaviableoptioninsomepatientsasforrevision ACL surgeries. The use of cadaveric allograft has been recom- mendedforrevisionsurgeryandinmulti-ligamentreconstructions [25].Itminimizesdonorsitemorbidityandavoidsfurtherweaken- ingoftheknee.Howeveranallograftdoeshavepotentialproblems ofdiseasetransmission[26].Toavoidtheriskofinfectiousdisease transmissionallograftsshouldberadiation-sterilised.Asradiation- sterilisation supposedly decreases the mechanical strength of

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Fig.4. A.GFPexpressioninnormaltendonofGFPrabbits(A,B,C,D).B.Regionofboneinsertion.C.Centralregion.D.Regionofmuscleinsertion.Normaltendonofcontrol rabbit(Non-GFP)E,F.NoGFPimmunoreactivecells(immunohistochemistryforGFP).

Fig.5. Comparisonofthebone-tendoninterface(IF)at3weeks(A,C)and6weeks(B,D)inthebonetunnel.Non-GFPgraftintoGFPrabbit:athickﬁbrousmembraneat3 weeksandcartilaginoustissueat6weeks.AfewGFPpositivecellsseemtoinﬁltratethetendongraft(C).GFPtendongraftinnon-GFPrabbit:DnoGFPpositivecellswere detectable.IF:interfacemembrane;B:bone;G:tendon-graft(immunohistochemistryforGFP).

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Fig.6.Tendongraftatthetunnelexit(tibialtunnel)inthearticularspace.A,B.GFPtendoninnon-GFPrabbitafter6weeks,proliferationofGFP+andGFP–chondrocytes-like cellsinperiphericareas.C.Non-GFPtendoninGFPrabbitafter3weeks,GFP+vascularrecrutementinthehosttissueadjacenttothetendongraft.G:tendon-graft,∅:acellular regions,AC:articularcartilage,V:vessels(immunohistochemistryforGFP).

tendonitisimportanttoestablishmethodsofallograftpreserva- tionandsterilisationassuringthebestqualityofgraftsandtheir safetyatthesametime.

Therearesomelimitationstothisthestudy.First,theextensor tendonwasusedasgraftinsteadofthehamstringtendoncom- monlyusedinhuman.Biomechanicalligamentstrengthovertime wasnotinvestigated,ascollagentypeIIIcontent.Thisstudyalso hadotherweaknessescommontoanimalstudies,andsomeﬁnd- ingsmaynotbedirectlytransferrabletoclinicalpractice[27,28].

5. Conclusion

Thisstudyprovidesimportantconclusionwithregardstocell fateintissue-engineeredscaffoldsforligamentreplacements.Our resultshaveshowndecreasedcellsurvivalcentrallyintheintra- articularportionofthescaffoldandrepopulationofthescaffold withhostcellsdistallyinthebonetunnels.Theseﬁndingssupport thefactthatbothcellbasedandcellhomingtherapiesareimpor- tant.Thetype ofcells repopulatingthematrixis unknownand furtherstudiesmaybedesignedtoidentifytheprogenitorpotential ofthesecells.Furthermore,giventhisknowledge,therepopulation ofthescaffoldcanbeoptimizedwiththeultimategoalofincreasing graftintegration.

Disclosureofinterest

Theauthorsdeclarethattheyhavenocompetinginterest.

Funding

NofundingfromNationalInstitutesofHealth(NIH),Wellcome Trust,HowardHughesMedicalInstitute(HHMI).

Acknowledgment

The authors would like to thank Prof. Bertrand Mace, Mrs Yolande Calando,Mrs GenevièveJolivet and theJouy-and-Josas AnimalLaboratoryteamfortheirvaluablehelpcommentsandsug- gestionstoimprovethequalityofthiswork.

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