Rev. bras. ortop. vol.51 número1

w w w . r b o . o r g . b r

Original

article

Mesenchymal

stromal

cells

from

bone

marrow

treated

with

bovine

tendon

extract

acquire

the

phenotype

of

mature

tenocytes

夽

Lívia

Maria

Mendonc¸a

Augusto

_,

_Diego

_Pinheiro

_Aguiar,

_Danielle

_Cabral

_Bonfim,

Amanda

dos

Santos

Cavalcanti,

Priscila

Ladeira

Casado,

Maria

Eugênia

Leite

Duarte

InstitutoNacionaldeTraumatologiaeOrtopedia,RiodeJaneiro,RJ,Brazil

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t

i

c

l

e

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n

f

o

Articlehistory:

Received20October2014 Accepted3February2015 Availableonline6January2016

Keywords:

Tendon

Mesenchymalstromalcellsfrom bonemarrow

Tenocytes

a

b

s

t

r

a

c

t

Objective:Thisstudyevaluatedinvitrodifferentiationofmesenchymalstromalcellsisolated frombonemarrow,intenocytesaftertreatmentwithbovinetendonextract.

Methods:Bovinetendonswereusedforpreparationoftheextractandwerestoredat−80◦_C. Mesenchymalstromalcellsfromthebonemarrowofthreedonorswereusedforcytotoxicity testsbymeansofMTTandcelldifferentiationbymeansofqPCR.

Results:Thedatashowedthatmesenchymalstromalcellsfrombonemarrowtreatedfor upto21daysinthepresenceofbovinetendonextractdilutedatdiminishing concentra-tions(1:10,1:50and1:250)promotedactivationofbiglycan,collagentypeI_{andfibromodulin} expression.

Conclusion:Ourresultsshowthatbovinetendonextractiscapableofpromoting differenti-ationofbonemarrowstromalcellsintenocytes.

©2015SociedadeBrasileiradeOrtopediaeTraumatologia.PublishedbyElsevierEditora Ltda.Allrightsreserved.

Células

mesenquimais

do

estroma

da

medula

óssea

tratadas

com

extrato

de

tendão

bovino

adquirem

o

fenótipo

de

tenócitos

maduros

Palavras-chave:

Tendão

Célulasmesenquimaisdoestroma damedulaóssea

Tenócitos

r

e

s

u

m

o

Objetivo:Oestudo avaliaa diferenciac¸ãoinvitro das célulasmesenquimais isoladasdo estromadamedulaósseaemtenócitosapóstratamentocomextratodetendãobovino.

Métodos:Tendõesbovinosforamusadosparaconfecc¸ãodoextratoeestocadosa−80◦C. Célulasmesenquimaisdoestromadamedulaóssea(BMSCs)detrêsdoadoresforamusadas paraostestesdecitotoxicidadeporMTTediferenciac¸ãocelularporqPCR.

Resultados:Osdadosmostram quecélulasmesenquimais doestroma damedulaóssea tratadasporaté21diasempresenc¸adoextratodetendãobovinodiluídoemconcentrac¸ões

夽

WorkdevelopedattheInstitutoNacionaldeTraumatologiaeOrtopedia(INTO),RiodeJaneiro,RJ,Brazil. ∗ _{Correspondingauthor.}

E-mails:liviiaaugusto@gmail.com,liviaaugusto@hotmail.com(L.M.M.Augusto). http://dx.doi.org/10.1016/j.rboe.2015.12.013

crescentes(1:10,1:50e1:250)promovemaativac¸ãodaexpressãodebiglican,colágenotipo I_{efibromodulina.}

Conclusão:Nossosresultadosmostramqueoextratodetendãobovinoécapazdepromover adiferenciac¸ãodasBMSCsemtenócitos.

©2015SociedadeBrasileiradeOrtopediaeTraumatologia.PublicadoporElsevier EditoraLtda.Todososdireitosreservados.

Introduction

Tendons are a specialized type of tissue composed of tenoblastsandtenocytes,whichareembeddedinan extra-cellularmatrixmostlycomposedoftypeIcollagen.Tenocytes onlyhavelimitedpotentialforproliferationandthusconfer lowregenerativecapacityontendons.1,2

Tendon injuries constitute a serious problem within orthopedicpractice and generate high costs forthe public healthcaresystem,aswellashavinganimpactonthequality oflifeofthepatientsaffected.Althoughregenerationisthe aimofthe clinicaltreatments used,the methodscurrently availablecontinueto beineffective.Thus, tendon dysfunc-tionsleadtodefinitivephysicalincapacity.3–5

Mesenchymal cells isolated from bone marrow stromal cells(BMSCs)areknowntobeapromisingtherapeuticoption withinthefieldofcelltherapyandbioengineeringof muscu-loskeletaltissues.5–8_{Theiruseinassociationwithsynthetic} biomaterials has been proposed as an option for modern treatmentsaimingtotowardtendonreconstruction,usingan allograft,autograftorxenograft.9,10_{UseofautologousBMSCs} biosyntheticgraftshastheaimsofimprovingtheresultsfrom conservativesurgeryandreducingthetimetakenforthe pre-injurybiomechanicalpropertiestoberestored.11_Furthermore, thelowimmunogenicityofBMSCsmakesitpossibletouse themallogeneicallyandminimizestheneedfor immunosup-pressionofthereceptor.12

Despite the significant therapeutic potential of BMSCs, littleisyetknownaboutthemechanismsandsignaling path-waysinvolvedindeterminingthat BMSCswilldifferentiate toward a tenogenic route, or in relation to progression of theirdifferentiation.ConsideringthatBMSCsseemtorespond tostimuli thatarepresentinextractsfromhealthy mature tissuesandhavespecificphenotypiccharacteristics,13,14 _we developedthehypothesisthattendonextractsmightinduce differentiation ofBMSCs into tenocytes. Thus, the present studyhadtheobjectiveofevaluatingtheinfluenceof treat-mentofhumanBMSCswithdifferentconcentrationsofbovine tendonextract,oninvitrodifferentiationtowardatenocytic route.

Material

and

methods

Isolationandexpansionofmesenchymalcellsfromthe stromaofhumanbonemarrow

BMSCs were isolated from surgical waste that originated fromhiparthroplastyproceduresonfivepatients(twomen and three women) aged 45–60years, who did not present

any comorbidities.Informedconsentwasobtainedfrom all of these individuals after approval of the study protocol by the institutional ethics committee. After the samples had been collectedinthe surgicalcenter,theywere stored in sterile flasks containing Iscove’s modified Dulbecco’s medium (IMDM; Sigma–Aldrich, St. Louis, MO, USA), sup-plemented with20% bovinefetal serum(BFS; Gibco,Grand Island, NY, USA),at4◦_{C fornotmorethan 18h. Toisolate}

the total cellular fraction, the bone marrow was resus-pendedinphosphate-bufferedsaline(PBS)solutionandwas mechanicallydissociatedfromanybonefragments.Thecell suspensionsthusobtainedwerecollectedin50mLtubesand werecentrifugedat836×gand4◦Cfor5min.Thecellswere thenresuspendedin50mLofIMDMsupplementedwith20% BFSandwerecountedusingaNeubauerchamber.Following this,6×105mononuclearcellsweredistributedinculturing flasksofvolume75cm2_{,in10mLofIMDMwith20%BFS,and} weremaintainedat37◦_Cunder5%CO

2.Threedayslater,the non-adherentfractionwasremovedbymeansoflavagewith PBSandtheculturingmediumwaschanged.Afterafurther14 days,thecellswereremovedusingasolutionof0.125%trypsin and0.78mMEDTAandwereexpanded.

Preparationofbovinetendonextract

Five bovine calcaneal tendons were obtained. They were maceratedmechanicallyandthenweregroundupusingan electricblenderofpower20W,intheproportionsof1gof tis-sueto2mLofIMDM,withoutBFS.15_{Thetissueextractwas}◦_C

centrifugedat836×gand8◦Cfor5minandwasthenstored at−80◦_{Cforamaximumoftwomonths.}

AnalysisofcellviabilityusingtheMTTmethod

BMSCs were cultured on 24-well plates, at a density of 2.5×104cells/well and were treated with bovine tendon extractdilutedintheproportionsof1:10,1:50or1:250(v/v)in IMDMsupplementedwith10%BFS.Cellviabilitywasassessed 24,72,120and168hoursafterthetreatment,inthepresenceof MTT(thiazolylbluetetrazoliumbromide;Sigma–Aldrich)ata concentrationof25mg/mL.Equalconcentrationsofdimethyl sulfoxide(DMSO)wereusedasanegativecontrol. Colorimet-ricevaluationwasperformedatawavelengthof550nm,using theSIRIOSSEACreader(Burladingen,Germany).

AnalysisofgeneexpressionusingqPCR

Table1–Targetgenesanddesignsoftheprimersusedinanalyzinggeneexpression.

Targetgene Accessnumber Sequence(5′_–3′_{) Product(bp)}

COL1A1 NM000088.3 Sense GTGCTCCTGGTATTGCTGGT 150

Antisense GCTCTCCCTTAGCACCAGTGT

BGN NM001711.4 Sense TGAAGTCTGTGCCCAAAGAGA 157

Antisense GCCTTCTCATGGATCTTGGA

FMOD NM002023.4 Sense CAACACCTTCAATTCCAGCA 178

Antisense CTGCAGCTTGGAGAAGTTCA

ACTB NM001101.3 Sense TACAATGAGCTGCGTGTGG 165

Antisense AGAGGCGTACAGGGATAGCA

COL1A1,type1Acollagen;BGN,biglycan;FMOD,fibromodulin;ACTB,betaactin;bp,sizeofamplifiedproductinbasepairs.

was extracted using the Trizol® _{method (Invitrogen Corp.,} Carlsbad,CA,USA)andwastreatedusingDNase(Ambion® DNA-freeTM _{DNase treatment; Life Technologies), in} accor-dancewiththemanufacturer’sinstructions.Theintegrityand quantityoftheRNAwereevaluatedbymeansof electrophore-sison denaturing gel and bymeans ofspectrophotometry (NanodropTM _{1000; Thermo Fisher Scientific, Inc.). Reverse} transcription for synthesis of complementary DNA (cDNA) wasperformed induplicate, from 1.0␮g ofRNA, usingthe ImProm-IITM_{reversetranscriptionsystem(Promega),in} accor-dancewiththemanufacturer’sprotocol.qPCRwasperformed using the Power Sybr Green Master MIX® _detection sys-tem(AppliedBiosystems,MolecularProbes,Inc.)intheStep Oneequipment(AppliedBiosystems,MolecularProbes,Inc.). Primersfromtheconstitutivegenes(rDNA28Sandactin)were usedascontrolsforthe experiment.Theexpressionofthe genesfortypeIcollagen,biglycanandfibromodulinwas nor-malizedinrelationtotheexpressionoftheconstitutivegene for␤-actin(Table1).The2−CT13_{methodwasusedfor} analyz-ingtheexpressionofthetargetgenesofthisstudyinrelation totheconstitutivegene.Thevaluesforrelativeexpressionthat havebeenpresentedtookthefixedcontrol-groupvalueof1.0 asthereference(calibrator).

24h 0

0.1 0.2 0.3 0.4 0.5 0.6 0.7

72h

Absorbance (U.A.)

120h C 1:10 1:50 1:250 B

168h

Fig.1–Evaluationofthecytotoxicityofbovinetendon extractinmesenchymalcellsofthebonemarrowstroma.C representscontrolcondition;1:10,1:50and1:250represent thedilutionsofthebovinetendonextractinIMDM;B representsthecontrolconditionforthetechniqueinwhich therewerenocells.Underallconditions,IMDMwasused withsupplementationwith1%BFS.

Results

With the aim of evaluating the toxicity of bovine tendon extract toward BMSCs, the MTT test was performed. The resultsshowedthatthebovinetendonextractdidnotalter theviabilityoftheBMSCsatanyoftheconcentrationstested (Fig.1).Thus,wecaninferthatthebovinetendonextractdid nothaveanycytotoxiceffectonthemesenchymalstemcells. Giventhattherewasnocytotoxiceffect,thepotentialofthe bovinetendonextractfordifferentiationwastestedwiththe aimofevaluatingwhetherthegrowthfactorspresentinthe extractwouldstimulatedifferentiationofthemesenchymal progenitorcells.WeobservedthatintheBMSCs,theextract was capableof activatingexpressionofthe genes fortype I collagen(Fig. 2), biglycan(Fig. 3)and fibromodulin (Fig. 4) overperiodsof7,14and21days.Theseresultsshowedthat thebovinetendonextractwasnotcytotoxicandthatitwas capable of inducingexpression ofthe genes implicatedin tenocyticdifferentiationofmesenchymalstemcells. Further-more, the tendon protein extractpromoted inductionin a

1:10 0

10 20 30 40

1:50

Relative levels of mRNA expression (type I collagen)

1:250

7 days

14 days

21 days

1:10 0

10 20 30 40

1:50

Relative levels of mRNA expression (biglycan)

1:250 7 days

14 days

21 days

Fig.3–EvaluationofbiglycanexpressionthroughqPCRon mesenchymalstromalcellstreatedwithincreasing concentrationsofbovinetendonextract(1:10,1:50and 1:250).

1:10 0

10 20 30 40 50

1:50

Relative levels of mRNA expression (fibromodulin)

1:250 7 days

14 days

21 days

Fig.4–Evaluationoffibromodulinexpressionthrough qPCRonmesenchymalstromalcellstreatedwith increasingconcentrationsofbovinetendonextract(1:10, 1:50and1:250).

dose-dependentmannerandasafunctionofthedurationof exposuretotheextract.

Discussion

Ourstudy showed that the bovine tendonextract had the potentialtoinducetenocyticdifferentiationofBMSCsandthat thisextractdidnothavecytotoxicityatany ofthe concen-trationsused.Inouranalyses,theresultsshowedthatthere was a spatial and temporal window for success regarding differentiationofBMSCsintotenocytes.Weemphasizethat fortheproceduretobeefficient,theBMSCs(at70% conflu-ence)shouldbetreatedwithtendonextractat1:50forseven days.Weobservedthatthepeakexpressionofbiglycanand

fibromodulin(whicharemarkersfortenocytes)16–21_wasatthis time.ThisindicatedthattheBMSCsbecamecommittedtoa tenocyticlineage. Thetreatmentprotocolneedstoproceed withincreasedconcentrationoftheextract,whichshouldbe 1:10foranother10days.ThisallowstheBMSCstomaintain typeIcollagenexpressionandprovideanefficient extracellu-larmatrix,soasalsotomaintaincellviability.

Ourresultssuggestthatintendons,therearegrowth fac-tors that stimulate differentiation ofpluripotent cells into tendoncells.Thisopensupapossibilityforthefieldofcell therapy,fortreatingtendinopathy.Finally,thepotentialwas evaluatedinaninvitromodelandthereforethereisaneedfor validationinaninvivomodel,inordertoconfirmtheresults. Moreover,weraisethepossibilitythat,inthefuture,the poten-tial oftendonextracts originating from humantendonsin cadaverdonorsmightbeevaluated.

Conclusions

Theset ofresultsshowed thattreatment ofBMSCswitha proteinextractfrombovinetendontissuepromoted differen-tiationintotenocytes.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

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