efficacy and safety of human mesenchymal stromal … · 2 summary to evaluate the preclinical...
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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,[email protected]
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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
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highervascularisationintheexperimentalgroupsshowedthatremodellingof
remainingtissueisstillongoing.Tofindoutifsmallerdefectcanhealwiththe
hMSCcontributiononlywhilelargerdefectsrequiresomecaseofscaffold
support,wouldneedtobestudiedinfurtherexperimentsandwithlonger
follow‐up.
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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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Figure1
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Figure6