1

EFFICACYANDSAFETYOFHUMANMESENCHYMALSTROMALCELLSINHEALINGOFCRITICAL‐SIZEBONEDEFECTSINIMMUNODEFICIENTRATS

Shorttitle:HumanMSCforbonehealinginimmunodeficientrats

RobertPytlík1,ClaudiaRentsch2,TomášSoukup3,LadislavNovotný4,5,BarbaraRentsch2,VeronikaKanderová6,HanaRychtrmocová1,MarkétaKalmárová1,StehlíkDavid7,8,MarekTrněný1andOndřejSlanař9.1FirstDepartmentofMedicine,1stMedicalFaculty,CharlesUniversity,PragueandGeneralUniversityHospital,Prague,Unemocnice2,12808,Praha2,Czechrepublic2TranslationalBone,JointandSoftTissueResearch,MedicalFacultyandUniversityHospitalCarlGustavCarus,TechnischeUniversitätDresden,Fiedlerstrasse42,01307Dresden,3InstituteofHistologyandEmbryology,MedicalFacultyHradecKrálové,CharlesUniversity,Prague,Šimkova870,50038,HradecKrálové,CzechRepublic4RadioisotopeLaboratoryandAnimalHouse,MedicalFacultyHradecKrálové,CharlesUniversity,Prague,Šimkova870,50038,HradecKrálové,CzechRepublic5FinnPathologists,OneEyedLane,Weybread,Norfolk,IP215TT,UnitedKingdom6CLIP‐ChildhoodLeukaemiaInvestigationPrague,DepartmentofPaediatricHaematologyandOncology,2ndMedicalFaculty,CharlesUniversity,PragueandMotolUniversityHospital,Prague,Vúvalu84,15006,Praha5,CzechRepublic7InstituteofAnatomy,1stMedicalFaculty,CharlesUniversity,Prague,Unemocnice3,12800,Praha2,CzechRepublic8DepartmentofOrthopedics,2ndMedicalFaculty,CharlesUniversity,PragueandMotolUniversityHospital,Prague,Vúvalu84,15006,Praha5,CzechRepublic9InstituteofPharmacology,1stMedicalFaculty,CharlesUniversity,PragueandGeneralUniversityHospital,Prague,Albertov4,12800,Praha2,Czechrepublic.Correspondingauthor:RobertPytlík,FirstDepartmentofMedicine,GeneralUniversityHospitalandFirstMedicalFacultyofCharlesUniversity,Unemocnice2,12808Praha2,pytlikr@seznam.cz

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Summary

Toevaluatethepreclinicalefficacyandsafetyofhumanmesenchymalstemcells

(hMSC)rapidlyexpandedingrowthmediumforclinicalusewithhumanserum

andrecombinantgrowthfactors,weconductedacontrolled,randomizedtrialof

plasmaclotswithhMSCvs.plasmaclotsonlyincriticalsegmentalfemoral

defectsinrnu/rnuimmunodeficientrats.X‐ray,microCTand

histomorphometricalevaluationwereperformedat8and16weeks.MSCwere

obtainedfromhealthyvolunteersandpatientswithlymphoidmalignancy.

HumanMSCsurvivedinthedefectfortheentiredurationofthetrial.MSCfrom

healthyvolunteers,incontrasttohMSCfromcancerpatients,significantly

improvedbonehealingat8,butnot16weeks.However,at16weeks,hMSC

significantlyimprovedvasculogenesisinresidualdefect.WeconcludethathMSC

fromhealthydonorssignificantlycontributedtothehealingofbonedefectsat8

weeksandtothevascularisationofresidualconnectivetissueforupto16

weeks.WefoundtheadministrationofhMSCtobesafe,asnoadversereactionto

humancellsatthesiteofimplantationandnoevidenceofmigrationofhMSCto

distantorganswasdetected.

Keywords:Humanmesenchymalstromalcells–boneregeneration–athymic

rats–treatmentefficiacy‐safety

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Introduction

Thehealingoflargebonedefectsremainsoneoftheprinciplechallenges

ofmodernorthopedics.Thebestmaterialforthesurgicaltreatmentoflarge

defectsisautologousbonegraft.However,theamountofmaterialislimitedand

theriskofmorbidityattheharvestsiteisnotnegligible(Banwartetal.1995,

Swanetal.2006).Cadaverousbonegraftsareanotheroption,however,the

potentialfortreatmentfailureishigherthanforautologousgrafts.

Humanmesenchymalstromalcells(hMSC)werefirstisolatedfromthe

bonemarrowbyFridensteinroughly50yearsago(Fridensteinetal.1966,

Fridensteinetal.1968),andtheirpotentialfordifferentiationtoseverallineages

wasshownbyPittengeretalin1999.Similarcellscanalsobeharvestedfrom

othertissues(dentalpulp,adiposetissue,Whartonjellyandothers–Jiangetal.

2002,Boquestetal.2006,ShiandGronthos2003,Montanuccietal.2011).

Themaindisadvantagesofbonemarrow‐derivedhMSCaretheirrelative

paucityinthebonemarrowandtheneedforlong‐termcultivationwithserial

passaging.WedevelopedanewmethodforthecultivationofhMSCfrombone

marrowmononuclearcells,whichisfasterandmoreeffectivethantraditional

cultivationinamediumsupplementedbyfetalcalfserum.Inthismethod,

CellGroTMMediumforHematopoieticStemCells(Cellgenix,Germany)

supplementedby10%humanserumandinsulin,dexamethasone,ascorbicacid,

EGF,PDGF‐BB,FGF‐2andM‐CSFisused,aspublishedpreviously(Pytlíketal.

2009).ThismethodresultsinahighnumberofMSCsinjusttwoweeksand

withouttheneedofpassaging.Cellsobtainedbythismethodfulfillconsensus

criteriaformesenchymalstromalcells(Dominicietal.2006)andproducemore

andbettermineralizedtissueafterheterotopicimplantationinimmunodeficient

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mice.Furthermore,hMSCcultivatedbythismethodshowgreaterexpressionof

severalgenesconnectedwithossificationandskeletalandbonedevelopment,

namelyARID5B,CDH11,COL1A1,COL12A1,CTGF,IGFBP3andPRRX2bygene

expressionprofilingandqRT‐PCRanalysis(unpublisheddata).

Duetotheirlowimmunogenicity,mesenchymalstromalcellssurvivefor

sometimeinanxenogenicsetting,however,animmuneresponseeventually

occursandcellsarerejected(Wangetal.2007).Therefore,immunodeficient

animalsprovideabetterenvironmentforpreclinicalexperimentswithhMSC.

Athymicrnu/rnuratsarefrequentlyusedinexperimentalstudiesconcerningthe

roleofhMSCinbonehealing.SeveralstudiesonhMSCincritical‐sizefemoralor

calvarialdefectsinathymicratshavebeenperformed(Petersonetal.2005,Yoon

etal.2007,Cuomoetal.2009,Liuetal.2010,Chenetal.2013,Suenagaetal.

2015),however,inallthesestudies,osteoconductivescaffoldswereusedand

hMSCwerefrequentlypreconditionedinvitroorweregeneticallymanipulated.

Despitethis,completehealingofthedefectoccurredonlyinaminorityof

experiments.

Weconductedarandomized,controlledanimaltrialtoevaluatethesafety

andefficacyofhMSCcultivatedbyourproposedmethodaccordingtothe

requirementsofEuropeanMedicalAgency(EMA).Critical‐sizesegmental

femoralbonedefectsinrnu‐rnuratswerefilledwithhumanplasmaclotseither

withhMSC(experimentalgroups)orwithoutthem(controlgroup).Asmany

candidatesforreconstructiveorthopaedicsurgeryarecancerpatients,

approximatelyonehalfoftheexperimentalcohortwerepatientswithlymphoid

malignancy(lymphomagroup),theotherparthealthyvolunteers(healthy

group).Forbettertemporalevaluationofbonehealing,wefurthersubdivided

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theexperimentalandcontrolgroupsintoshort‐term(8weeks)andlong‐term

(16weeks)cohorts.Safetywasassessedbyevaluatingexplantedfemursand

parenchymatousorgansforpathologiesdirectlyorindirectlyattributableto

hMSC.

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Methods

Experimentalsetting

Atotalof42animalswererandomlyassignedtothree8‐weekcohorts

(healthy,lymphoma,control)andtothreesimilarlynamed16weekcohorts.

Humanmesenchymalstromalcellcollectionandcultivation

Humanmesenchymalstromalcellswerecultivatedfrombonemarrow

mononuclearcells(BMMC)fromhealthyvolunteerdonorsandpatients

undergoingbonemarrowexaminationforsuspectedorprovenlymphoid

malignancy.Thestudywasapprovedbythelocalethicscommitteeandall

donorsprovidedsignedinformedconsentaccordingtotheHelsinkideclaration.

BMMCwereobtainedafterthecentrifugationof10mlofbonemarrow

bloodonFicoll‐Paquegradientmediaandprocessedasdescribedpreviously

Shortly,2.5x106BMMCwereseededin75cm2plasticflasks,cultivatedin

CellGroTMforHematopoieticCellsmediumwith10%humanserumandinsulin,

ascorbicacidphosphate,dexamehtasone,EGF,PDGF‐BBandFGF‐2(suppliers

werethesameasinPytlíketal.2009).Non‐adherentcellswerenotremoved,

cultivationmediumwasnotchangedandsupplementswereaddedtwiceper

weekforatotaloffourdoses.

Aftertwoweeks,non‐adherentcellswerewashedawayandMSCswere

harvestedbyEDTA‐Trypsin(GibcoLifeTechnologies,GrandIsland,NewYork,

USA)andcountedinBurkerchambers.Viabilitywastestedwithtrypanblue.

Aliquotsofcellswerefrozenforlaterflow‐cytometricanalyses.

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Preparationofplasmaandplasma/hMSCclots.

Asuspensionof360µlhumanplasmaand40µlcalciumchloride(Sigma‐

AldrichCzechRepublic,Prague,CzechRepublic)wasseededinto24wellplates

andallowedtoclot.Forexperimentalgroups,cellswereaddedbefore

recalcification(targetnumber,10x106hMSCforclot).Inpreliminary

experiments,hMSCswerecultivatedinplasmaclotsinvitroforoneweek;after

thisperiod,cellswerealiveandproliferating(Figure1A,B).

Animalprocedures

Athymicrnu/rnurats(CharlesRiverLaboratories,Sulzfeld,Germany)

werekeptinsterilecagesandfedsterilefoodandwater,untiltheyreached

skeletalmaturity(10‐14weeks).Animalswerecaredforaccordingtothe

Europeanguidelinesforthecareanduseoflaboratoryanimals(Directive

24.11.1986,86/609/CEE).TheexperimentwasapprovedbytheDresden

regionalveterinaryboard.

Ratswereanesthetizedwithasolutionofketamine35mg/kg(Kemint,

AlvetraGmbH,Neumunster,Germany)andxylazine5mg/kg(Rompun,Bayer

HealthcarePharmaceuticals,Berlin,Germany).Anincisionwasmadealongthe

leftthighandthediaphysisofthefemurwasdisectedbybluntdissection.A5‐

holeminifragmentplate(246190,Synthes,WestChester,PA,USA)wasfixedto

thefemurusing2screws(210.006,Synthes,Tuttlingen,Germany)aboveand

belowtheproposedosteotomysite.Critical‐sizeddefects(target5mm)were

createdandplasmaclotswithorwithoutcellswereplacedintothedefects.

Muscletissuewasfoldedbackandclosedwitharesorbablesutureandtheskin

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wasclosedwithanon‐resorbablesuture(Mariderm,CatgutGmbH,

Markneukirchen,Germany).

Single‐shotantibioticprophylaxiswithamoxycillin15mg/kg(Duphamox,

PfizerGmbH,Berlin,Germany)wasadministeredandCarprofen1.4mg/kg

(Rimadyl,PfizerGmbH)wasgivenimmediatelyand24hoursaftersurgeryfor

painprevention.After8or16weeks,allanimalswereanesthetized

(ketamine/xylazinemixture)andeuthanizedwithacombinationof

embutramide,mebezoniumandtetracaine(Tanax,InterventGmbH

Unterschleißheim,Germany).

X‐rayandmicroCTmeasurements

X‐rayimagingofthedefectswasperformedonanAMX4machine(GE

Healthcare,Buckinghamshire,UK)postoperatively(Figure2A)and8and16

weeksaftersurgery.DefectsizewasevaluatedusingImageJsoftware

(imagej.nih.gov/ij).Newboneformation(x‐ray)wascalculatedasthe

postoperativedefectminusresidualdefect(Figure2B).Aftersacrifice,microCT

examinationwith3DreconstructionswereperformedonavivaCT75(Scanco,

Brüttisellen,Swizerland;Figure2C).Totalvolumes(TV)oftissuebetweentwo

screwsatbothdefectsitesandnewbonevolumes(BV)werecalculatedin

voxels.Theratioofbonevolumetototalvolume(orbonevolumefractional

value)wascalculatedasBV/TV.

Histologicalprocedures

Femoralexplantswerefixedin4%bufferedformalin(SAVLP,Flintsbach,

Germany)foratleast7days.Thesampleswerewashed,decalcifiedin

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ethylenediaminetetraaceticacid,dehydratedovernightandembeddedinmethyl

methacrylate(Technovit9100New,HeraeusKulzerGmbH,Werheim,Germany).

Threeµmsectionsofeachsectionplanewereprepared,metyl‐methacrylatewas

removedandsampleswererehydratedinagradedseriesofethanol.

Femoralexplantswerestainedwithhematoxylinandeosin(Figure3A),

Goldner(Figure3B,C)andMallorystain.Toprovethepresenceofhumancells,

immunohistochemicaltestsforanti‐humannestin,clone10C2(Chemicon

International,Inc.,Temecula,CA,US).Inseveralcases,immunofluorescent

detectionofhumanmitochondria,clone235‐1(Genetex)wasperformed.

Antigenretrievalwasdoneinacitratebuffersolutionusingamicrowavefor3×5

minutes.ThestandardABC(AvidinBiotinComplex)methodwasusedforthe

detectionwithDAB(3,3'‐Diaminobenzidine),withMason'sgreentrichromeused

asacounterstain(Figure3E).Fibroplasiafoundinoneanimalfromthe

experimentalgroupwasfurtherimmunostainedforvimentin,cloneRV202

(Abcam,Cambridge,UK)andanti‐CD68,cloneED1(Abcam).Immunostainingfor

theevaluationofvesselformationwasperformedusingactindetection,clone

1A4(DAKO),visualizedwithABCandfastred,withMayer'shematoxylinasa

counterstain(Figure3D).

Parenchymatousorgans(liver,spleen,lungs,heartandkidneys)from

eachanimalwerefixedin4%bufferedformalinfor7days.Fixedspecimens

werecutintotissueblocksembeddedinparaffinandsectionedintoslices6–7

μmthick.SectionswerestainedwithhematoxylinandeosinandMasson’sblue

trichrome.

ImageswerecollectedusinganOlympusBX51microscopeequippedwith

aDP25digitalcameraandwereevaluatedusingImageJ(FiJi,NIH).

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Histomorphometricquantificationandvesseldensityinsampleswithresidual

fibroustissueweretestedin10samplesfromdifferentareas(40visualfields

wereobserved).

Flowcytometryanalysis

Afterthawing,cellswerewashedonceinphosphate‐bufferedsaline(PBS)

supplementedwith0.5%(w/v)bovineserumalbumin(BSA,CarlRothGmbH,

Karlsruhe,Germany).Aliquotsof1x105cellswereincubatedwithantibodiesfor

15minutesatroomtemperatureinthedarkandwashedagaininPBS

supplementedwith0.5%(w/v)BSA.ThedatawerecollectedwithanLSRIIor

CantoIIflowcytometer(BDBiosciences,SanJose,CA,USA).Thefollowing

conjugatedmonoclonalantibodieswereused:anti‐CD3APC,cloneUCHT1and

anti‐CD19PE‐Cy7,cloneJ3119,(bothBeckmanCoulter,Miami,FL,USA); anti‐

CD73PE,cloneAD2andanti‐CD90APC,clone5E10(allBiolegend,SanDiego,CA,

USA);anti‐CD16PE,cloneLNK16,anti‐CD45PerCP,cloneMEM‐28,anti‐CD105

FITCcloneMEM‐226,andanti‐CD235aFITC,cloneHIR2(allExbioPraha,a.s.,

Vestec,CzechRepublic).Aminimumof20,000cellswereacquiredfromeach

tube.ThedatawereanalyzedwithFlowJosoftware(Treestar,Ashland,OR,USA).

CellswereclassifiedashMSCaccordingtoCD105,CD90andCD73positivity,and

CD45,CD19,CD235a,CD16andCD3negativity[19].

Statistics

Categoricalvariableswereevaluatedbychi‐squaretests.Continuous

variables,ingeneral,werenormallydistributed,therefore,parametrict‐tests

andparametricANOVAwereusedforcomparisonamonggroupsandcohorts,as

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appropriate.Pairedt‐testswereusedforthecomparisonofpairedsamples.

CorrelationswerecalculatedbythePearsontest.Flowcytometrydatawerenot

normallydistributed,therefore,theMann‐WhitneyUtestwasusedfor

comparisonbetweengroups.Resultsarereportedasmean±standarddeviation,

exceptforflowcytometrydatawheremediansandrangesarereported.Pvalues

≤0.05wereconsideredstatisticallysignificant.Allcalculationswereperformed

withSTATISTICAsoftwarev.12(StatSoft,Tulsa,Minnesota,USA).

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Results

MSCdonorsandMSCcultivation

DonorcharacteristicsareshowninTable1.Intotal,13donorswereused

(5malesand8females),withameanageof47±14years(range27–65years).

Sixdonorswerepatientswithlymphoidmalignancy,however,onlyintwocases

bonemarrowwasinfiltratedbylymphoma(1.4%and3.4%byFACS,

respectively).Sevendonorswerehealthyvolunteers,significantlyyoungerthan

lymphomadonors(38±11yearsv.55±10years,p=0.008).Fromallbutone

donor,therewereenoughcellsfortwoclotsandin19of25clots(76%)the

targetnumberof10x106cellswasimplanted.Theviabilityofimplantedcellswas

consistentlyabove90%.ThemediannumberofcellsCD105+CD90+CD73+was

82.9%(range57.8–94.9%),whilethemedianCD45,CD235a,CD16,CD19and

CD3positivitieswere1.7%,1.2%,1.4%,3.1%and0.5%.

Surgicaloutcomes

SurgicaloutcomesareshowninTable2.Fourtyoffourty‐fouranimals

couldbeevaluated(91%).Twoanimalsdiedduringthesurgicalprocedure(both

ininthe16‐weekcontrolgroup)andosteosynthesisfailedintwoanimals(one

8‐weeklymphomagroup,onein16weekhealthygroup).Inexperimental

groups,9pairedsamples(8+16weekfromthesamedonor)wereavailablefor

statisticalanalyses.

Onpostoperativex‐rayimages,themeansizeofthedefectwas4.2±0.5mm,

withnodifferencesbetweenthesixcohorts(p=0.59;Figure2D).

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X‐rayandmicroCTmeasurements

8and16weeksx‐rays

Inallsixcohorts,someanimalshealedwellandotherslesswell(Figure

4A‐D).After8weeks,differencesbetweenthreegroupswerenotsignificantby

ANOVA(p=0.21),however,healthygrouphadsignificantlybetterhealingthan

controlgroup(newboneformation2.7±1vs.1.6±1mminthecontrolgroup,p

=0.05,Figure4E).Therewassomenegativecorrelationbetweenageandnew

boneformation,whichwasnotstatisticallysignificant(r=‐0.48,p=0.19).After

16weeks,therewasnodifferencebetweenhealthy,lymphomaorcontrol

cohorts(p=0.33)andneitherhealthynorexperimentalgroupwerebetterthan

controls(Figure4F).

8‐and16‐weekmicroCTmeasurements.

MicroCTmeasurementscorrelatedwellwithx‐raymeasurements(r=‐

0.73,p=0.03),however,differencesat8or16weekswerenotstatistically

significant(p=0.29and0.60byANOVA,respectively,Figure5A,B).Onlya

trendforhigherBV/TVratiointhehealthyversuscontrolgroupwasobserved

at8weeks(22±5%vs.18±3%,p=0.09).

Histopathology

Femoralexplants,generalhistology

Goodandpoordefectbridgingswereseenbothintheexperimentaland

controlgroups,andinboththe8and16weekcohorts(Figure3A‐C).Incasesof

goodbridging,afibrouscalluswithfociofchondroiddifferentiationandwoven

boneformationwasfound(Figure3A,B).Incasesofpoorbridging,residual

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spacewasgenerallyfilledwithadiposetissuewithnumerousbloodvessels

(Figure3C,D).Scatteredhumancells,detectedbyanti‐humannestin,anti‐

humannucleiandanti‐humanmitochondrialantibodieswerefoundinfibrous

andadiposetissueinallexaminedcases(Figure3E),unfortunately,theycould

notbequantifiedduetotechnicalreasons.Nohumancellswerefoundinnewly

formedboneorcartilaginoustissue.Nosignsofinflammatoryreactionortumor

formationwerefound.

Histomorphometry–newboneformation

After8weeks,theamountofthenewbonewassignificantlydifferent

betweenthreegroups(p=0.03byANOVA).Betterboneformationwasnoted

againforhealthyv.controlgroup(1.61±0.25vs.0.88±0.33mm2,p=0.015;

Figure3F).After16weeks,nodifferencesinboneformationwereseenbetween

thethreegroups(p=0.64),Figure3G).Again,correlationsbetween

histomorphometryandnewboneformationmeasuredbyx‐rayandmicroCT

werestrongandstatisticallysignificant(histomorphometryvs.x‐ray,r=0.68,p

=0.0009,histomorphometryvs.BV/TV,r=0.64,p=0.002).

Histomorphometry‐newvesselformation

Therewasatrendforbettervesselformationat8weeksinthe

experimentalgroups(121±46vs.71±26vessels,p=0.07),whichreached

statisticalsignificanceat16weeks(177±56vs.92±13vessels,p=0.01;Figure

3H).Inthesemeasurements,nodifferencesbetweenhealthyandlymphoma

sampleswerefound.

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Histologyofotherorgans

InthemyocardiumoftheleftventricleofanimalNo.44(DonorNo.13in

the8weekcohort),weobserveddensecellularproliferationofspindle‐shaped

topleomorphicmesenchymalcellsreplacingcardiomyocytes.Thecellswere

arrangedindisorganizedstreamsandbundlesseparatedbyasmalltomoderate

amountofcollagenousmatrix,andhadalowtomoderateamountofpale

eosinophiliccytoplasmandlargeovalvesicularnucleiwithprominentmedium‐

sizednucleoli.Anisocytosisandanisokaryosisweremoderate.

ImmunohistochemicalevaluationrevealedvimentinpositivityandCD68

negativity.Themorphology,arrangementandimmunohistochemistryofcells

suggestedreactiveorreparativefibroplasia(Figure6).Cellsofhumanorigin

werenotdetectedhere.Inotheranimals,nomicroscopicchangesandno

evidenceofhumancellswerefoundinparenchymatousorgans.

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Discussion

Cellulartherapyandtissueengineeringholdgreatpromisefor

regenerativemedicineandmaychangethefutureofclinicalpractice.Various

materialswithosteoconductivepropertiescanbecombinedwithosteoinductive

factorsandhMSC.Theseadvancedtherapymedicinalproducts(ATMP)must

complywithregulatoryrequirementsincludinggoodmanufacturingpractice

(GMP)andproofofproductsafetyandefficacy.Themorecomplextheproduct,

themorecomplextheregulatoryissues(Salmikangasetal.2015).

HumanMSCcontributetothehealingofvariousorgansbothbydirectand

indirectmechanismsinvolvingtheproductionofcytokinesandgrowthfactors

(HorwitzandPrater,2009).Furthermore,theimmunomodulatorypropertiesof

hMSCinhibitinflammatoryreactionsindamagedtissues(Urdzíkováetal,2014).

Twobasicmodelsareusedinboneregenerationexperimentsinrats:

calvarialdefectsandsegmentalfemoraldefects.Incalvarialdefects,apunch‐hole

of5–8mmindiameterisconsideredtobeofcriticalsize.Yoonetal.(2007)used

undifferentiatedorinvitroosteogenicallypre‐differentiatedadiposetissue‐

derivedstemcells(ATSC)onpoly‐lactic‐co‐glycolic‐acid(PLGA)scaffolds.

Fourteendaypre‐differentiatedATSCproducedthebestresults,however,only

35–72%ofthedefectwashealed.Chenetal.(2013)usedbonemarrowhMSC

(BMMSC)andumbilicalcordbloodstemcells(UCBSC)withcalciumphosphate

andbothimprovedhealingcomparedtocontrols,however,evenafter24weeks

thenewlyformedbonecoverednomorethan35%ofthedefectinbothhMSC

groups.AlsointheSuenagaetal.(2015)study,3D‐assembledhMSC(hMSC

spheroids)combinedwithTCPsignificantly,butonlypartially,contributedto

theregenerativeprocess.

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Thesurgicalcreationofcriticalsizefemoraldefects(5–6mm)andtheir

stabilizationbyinternalfixationislikelyamorephysiologicallyrelevant

experimentalmodelforbonehealing.Cuomoetal.(2009)foundbonemarrow

bloodenrichedwithhMSCsseededondemineralizedbonetissuenobetterthan

demineralizedbonetissuealone(1/10defecthealedat12weeks),while

demineralizedbonetissueoracollagenspongewithrhBMP‐2healed6mm

defectseffectively(9/10defectshealed).Liuetal.(2010)showedthathuman

UCBSCincombinationwithpartiallydemineralizedbonematrixmaycompletely

healcriticalfemoraldefects.Incontrast,Petersonetal.(2005)showedthatat8

weeks,nohealingoccurredwithadiposetissue‐derivedstemcellsloadedona

collagen‐ceramiccarrier.ThesecellshadtobetransfectedtoexpressBMP‐2to

fullyhealcritical‐sizedefects.Altogether,publisheddataareheterogenous,as

variousstudiesdifferwithregardtothesourceofhMSC,theirpre‐differentiation

orgeneticmodification,thetypeofscaffold,andthelengthoftheexperiment.

Ourstudydiffersfrompreviousstudieswithregardtothemethodof

hMSCpreparationandlackofanosteoconductivescaffold.Rather,hMSCswere

deliveredinplasmaclots,whileplasmaclotswithouthMSCformedthecontrol

group.Aswehaveshownpreviously,hMSCgrowninCellGromediumwith

humanserumandsupplementsproducedmoremineralizedtissuethanhMSC

grownwithfetalcalfserum,therefore,wedidnotconsidernecessarytousethe

latterasfurthercontrol.Ourexperimentaimedatevaluationofperformanceof

hMSCalone,withoutothersupportivemeasures.

WeobservedsignificantlybetterbonehealingwithhMSCateightweeks,

butonlyindonorswithoutmalignantdisease.Bonemarrow‐derivedhMSCfrom

patientswithhematologicalmalignancyhavedifferentfunctionalproperties

18

thanhMSCfromhealthydonors(Feietal.2014),andevenmayharborkaryotype

abnormalities(Blauetal.2011).Alsolymphomapatientswereolderthan

healthydonorand,bonehealingpropertiesofhMSCmaydecreasewithage

(Stenderupetal.2003).However,ourcohortwastoosmallforstatistically

meaningfulmeasurementofinteractionbetweenageandhealthstatusofdonors.

Inanycase,hMSCwereabsentinnewlyformedboneandcartilaginoustissues.

ThisisinagreementwiththehypothesisthattheroleofhMSCinthe

regenerativeprocessesisrathertodeliver"software"(i.e.,cytokinesandgrowth

factors)ratherthan"hardware"(directcontributiontohealing).

Aftereightweeks,bonehealingseemedtoceaseinboththecontroland

experimentalgroups,buthMSCexertedfurtheractivityinthesecondeight

weeksoftheexperiment,assignificantlymorevesselswerefoundinthe

remainingconnectivetissueintheexperimentalgroupscomparedtothecontrol

group.Therefore,weconcludethatremodellingofthenonunionsiteisstillgoing

onandthatwithlongerfollow‐up,thehealinginexperimentalgroupswill

continue.

TheimplantationofhMSCtothedefectwassafe.Humancellswerenot

foundinanyoftheexaminedparenchymatousorgans.Theonlypathologyfound

insacrificedanimalswasonecaseofproliferationofratmesenchymalcellsin

themyocardium,whichissuggestiveofreactiveorreparativefibroplasia,which

isaphenomenonwelldescribedinlaboratoryrats(Jokinenetal.2011).Noother

pathologies,werefoundmacroscopicallyormicroscopically.

Inconclusion,wehavefoundthathMSCevenwithoutosteoconductive

scaffoldcancontributetobonehealingincritical‐sizefemoraldefectin

immunodeficientrats.Thiscontributionseemedtobelimitedtoeightweekbut

19

highervascularisationintheexperimentalgroupsshowedthatremodellingof

remainingtissueisstillongoing.Tofindoutifsmallerdefectcanhealwiththe

hMSCcontributiononlywhilelargerdefectsrequiresomecaseofscaffold

support,wouldneedtobestudiedinfurtherexperimentsandwithlonger

follow‐up.

20

Acknowledgements

ThisworkwassupportedexclusivelybyagrantfromMinistryofPublicHealth,

Czechrepublic,IGANT13531/4,2011.

Theauthorsdeclarethatthereisnoconflictofinterestregardingthepublication

ofthispaper.

21

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Table1.MSCdonorcharacteristicsDonornumber Donortype Sex Age Experimental

groupDiagnosis BMinvolvement Cellsimplanted

1 Patient Male 66 8weeks DLBCL No oneclot10x1062 Patient Male 62 8+16weeks FL No twoclotsa10x1063 Volunteer Male 47 8+16weeks Healthy Notapplicable twoclotsa10x1064 Patient Female 55 8+16weeks FL No twoclotsa8.75x1065 Volunteer Female 31 8+16weeks Healthy Notapplicable twoclotsa10x1066 Volunteer Female 27 8+16weeks Healthy Notapplicable twoclotsa10x1067 Volunteer Female 52 8+16weeks Healthy Notapplicable twoclotsa8x1068 Patient Female 59 8+16weeks FL Yes twoclotsa10x1069 Patient Male 39 8+16weeks FL No twoclotsa10x10610 Patient Male 62 8+16weeks FL Yes twoclotsa10x10611 Volunteer Female 51 8+16weeks Healthy No twoclotsa10x10612 Volunteer Female 28 8+16weeks Healthy No twoclotsa10x10613 Volunteer Female 32 8+16weeks Healthy No twoclotsa7.5x106Abbreviations:FL=follicularlymphoma,DLBCL=diffuselargeB‐celllymphoma

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Table2‐surgicaloutcomes Groups 8‐week 16week healthy lymphoma control healthy lymphoma controlAnimalsoperated 7 6 9 7 5 10Perioperativemortality 0 0 0 0 0 2Osteosynthesisfailure 0 1 0 1 0 0Animalsevaluated 7 5 9 6 5 8

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FigurecaptionsFigure1:HumanMSCinplasmaclot.A‐hematoxyllin‐eosin,B‐anti‐Ki67immunostainingFigure2:A,B‐evaluationofdefectsizebyx‐ray,C‐evaluationofdefectsizebymicroCT,D‐defectsizeaftersurgeryinexperimentalandcontrolgroups.Figure3:microscopyofbonehealingandhistomorphometricanalyses.A‐ControlNo.3,8weekcohort:exampleofgoodhealingwithfibrouscallus(asterisk)andresidualadiposetissue(middleright).B‐DonorNo.12,8weekcohort:exampleofgoodhealingwithfibrouscallus,fociofchondroiddifferentiation(leftarrow)andwovenboneformation(rightarrow).Linesforhistomorphometricalevaluationareshowninred.C‐ControlNo.4,16weekcohort:exampleofpoorhealingwithvascularizedadiposetissuefillingmostofthedefect.D‐DonorNo.3,16weekcohort,stainingfornewvesselsinfibrouscallus(mostofthedefect)andadiposetissue(middleleft).E‐Stainingforhumanmitochondria.F,G‐bonetissueformationbyhistomorphometry.H‐newvesselformationbyhistomorphometry.Experimentalgroupmeansthathealthyandlymphomapatientsarecountedtogether.Stainings:A‐hematoxylin‐eosin,B,C‐Goldner'strichrome,D‐anti‐actin,fastred,Mayer'shematoxylincounterstain,E‐anti‐humannestin,DAB,Mallorytrichromecounterstain.Figure4:A‐D‐examplesofgoodandpoorhealinginexperimentalandcontrolgroupsasevaluatedbyx‐ray:A‐8weekexperimentalcohort,B‐8weekcontrolcohort,C‐16weekexperimentalcohort,D‐16weekcontrolcohort;E,F‐comparisonofnewboneformationbyx‐raymeasurementsafter8and16weeksinexperimentalandcontrolgroupsandinhealthydonorsubgroupsandcontrolgroups:E‐8weekcohorts,F‐16weekcohorts.Figure5:comparisonofnewboneformationexpressedasbonevolumefractionalvalue(BV/TV)inmicroCTmeasurementsinexperimentalandcontrolgroupsandhealthydonorsubgroupsandcontrolgroups:A‐8weekcohorts,B‐16weekcohorts.Figure6:Fibroplasiainleftventricularmyocardiumofanimalno.44,healthydonorno.13,8‐weekexperimentalgroup.A‐hematoxylin‐eosin,lowmagnification,B‐hematoxylin‐eosin,highmagnification,C‐anti‐vimentinimmunostaining,lowmagnification.

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Figure6