Growth and accelerated differentiation of mesenchymal stem cells on graphene oxide

MaterialsChemistry

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Citethis:J.Mater.Chem.B,2014,2,5461

Growthandaccelerateddi erentiationof

mesenchymalstemcellsongrapheneoxide/poly-L-lysinecomposite lms

WeiQi,*WenjingYuan,JingYanandHuaWang*

Anewtypeofcomposite lmsofgrapheneoxide(GO)andpoly-L-lysine(PLL)havebeenfabricatedforuseasbio-sca oldcoatingswithimprovedmechanicalproperties.Themorphology,growthanddi erentiationofmesenchymalstemcells(MSCs)ontheGO/PLLcomposite lmshavebeenexamined.TheresultsdemonstratedthattheGO/PLLcomposite lmscouldnotonlysupportthegrowthofMSCswithahighproliferationrate,butcouldacceleratetheosteogenicdi erentiationofMSCs,showingstrongalkalinephosphatase(ALP)andgeneexpression.Importantly,thehighpre-concentrationcapacityoftheGO/PLL lmsforosteogenicinducerscouldplayakeyroleinacceleratingtheosteogenicdi erentiationofMSCsthroughthestrongnon-covalentbindingandelectrostaticinteractionbetweenthem.Moreover,themildandinexpensivefabricationstrategycouldbeapplicabletobiomacromoleculesincludingchitosan,gelatin,etc.Therefore,suchakindofcomposite lmcouldprovideamultifunctionalandhighlybiocompatiblesca oldcoatingtailoredforpotentialapplicationinstemcellresearch.

Received27thMay2014Accepted24thJune2014DOI:10.1039//MaterialsB

1Introduction

Mesenchymalstemcells(MSCs)isolatedfrombonemarrowhavebeenshowntodi erentiateintoavarietyofcelllineagesinvitro,includingadipocytes,chondrocytes,myocytesandosteo-blasts.ThecombinationofMSCswithsca oldmaterialsprovidesapromisingstrategyforcelltherapyandtissueengi-neering.1–4Somenaturalbiomaterialssuchaspolypeptideshavebeeninvestigatedaspotentialsca oldmaterialsforstemcelltransplantation.Forexample,poly-L-lysine(PLL),withplentifulactiveaminogroupsandpositivesurfacecharges,ismostcommonlyusedinbiomedicaleldssuchasbioactivesurfaces,drugdeliveryandorsoforpromotingcelladhesionandsup-portingcellgrowth.5–7However,mechanicalpropertiesofPLLarenotgoodenoughforsomebiomedicalapplicationssuchasbonetissueengineeringorboneregenerativemedicine.Therefore,itisessentialtodevelopnanocompositesbasedonPLLtoimproveitsmechanicalproperties.Grapheneanditsderivative,grapheneoxide(GO),isanattractivesubstanceasacompositematerial,especiallyaspolymernanocompositesduetoitsextraordinarymechanicalpropertiesincludinghighYoung'smodulus,hardnessandexcellentexibility.Moreover,

asreportedpreviously,theincorporationofGOintoapolymermultilayercouldimprovethelmpropertiessignicantly.8Ontheotherhand,biointerfacescomposedofgrapheneorGOhavebeeninvestigatedforadhesion,proliferationanddi erentia-tionoftheMSCs.9–15Particularly,GO,inwhichthebasalplaneandedgespossessepoxide,carboxyl,andhydroxylgroups,enablesgreaterinteractionswithbiomacromoleculesthroughcovalent,electrostaticandhydrogen-bondingthangraphene.Herein,anewtypeofGO/PLLcompositelmshavebeenbuiltupinthisstudyvialayer-by-layer(LbL)assembly,forgrowth,proliferationanddi erentiationofstemcells(Fig.1).TheLbLassemblytechniquehasbeenwellrecognizedas

a

KeyLaboratoryofLife-OrganicAnalysis,KeyLaboratoryofPharmaceuticalIntermediatesandAnalysisofNaturalMedicine,SchoolofChemistryandChemicalEngineering,QufuNormalUniversity,57JingxuanWestRoad,Qufu,Shandong273165,China.E-mail:qf_qw@;huawangqfnu@;Tel:+865374458208;+865374456306 Electronicsupplementary10.1039/c4tb00856a

information

(ESI)

available.

See

DOI:

Fig.1

SchematicrepresentationofformationoftheGO/PLL

composite lm.

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simpleandversatilemethodtoimmobilizefunctionalmole-cules.16–23Herein,theuniquepropertiesofGO,i.e.alargesurfaceareaandexcellentmechanicalproperties,couldbesynergicallycoupledwiththebiocompatibilityofPLLresultinginakindofmultifunctionalsca oldcoating,whichcanbeusedintissueengineeringandstemcell-basedtherapy.

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2Experimental

2.1

Materials

Expandablegraphite($180mm)wasobtainedfromQingdaoBCSM.CO.,LTD.Poly-L-lysine(PLL)andphosphatebu ersolution(PBS)werepurchasedfromSigma-Aldrich(USA).Cal-cellswithgreencolor)andethidiumhomodimer(tostaindeadcellswithredcolor)wasemployed.Forthepurposeofcomparison,cellcountingkit-8(CCK-8)assays(Dojindo,Japan)werecarriedoutforallthestudiedcells.ThemorphologyoftheMSCsonallthesurfaceswasobservedbyusingaphasecontrastmicroscope(OlympusCKX31,Japan).Thepopulationdoublingtimes(PDT)ofcellsonallthestudiedsurfacesweredeterminedwiththegrowthkineticsandthefollowingequation.27

PDT¼ðt2Àt1Þ

lg221

(1)

N2:numberofcellsattimet2,andN1:numberofcellsattimet1.cein-AMandethidiumhomodimerwereobtainedfromDojindo(Japan).Antibodiesincludinganti-CD-44andanti-osteocalcin(OCN)werepurchasedfromAbcam(HongKong).WaterusedinthisworkwaspuriedbyaMilli-Qwatersystem(Millipore,USA).2.2

FabricationandcharacterizationofGO

GraphiteoxidewasobtainedbytheHummersmethod.8,24Briey,graphiteoxidewasobtainedbyoxidationofgraphitewithH2SO4,andKMnO4.Theformedgraphiteoxidewaswashedthreetimeswith1.0MofaqueousHClsolutionanddeionizedwateruntilapHof4.0–5.0wasachieved.Duringthewashingprocesswithdeionizedwater,agrapheneoxidegelformed.Followingthat,afreeze-dryprocedurewasconductedtoobtainthegrapheneoxidesolid.Atomicforcemicroscopy(AFM,DigitalInstruments,USA),UV-Visspectroscopy(UV-1601,Shimadzu,Japan)andIRspectroscopy(NEXUS470,Nicolet,USA)wereusedtocharacterizetheGOnanosheets.2.3

PreparationandcharacterizationoftheGO/PLLlm

Glasscoverslips(14mmdiameteror4.5mmdiameter)wereusedasthesubstratesforLbLassembly.Theywererstcleanedinpiranhasolution(7:3v/vH2SO4/H2O2)beforeuse,followedbyrinsingwithwater.PLLsolutionswerepreparedataconcentrationof2mgmLÀ1inPBS,andGOdispersionwaspreparedataconcentrationof0.1mgmLÀ1inwater.Then,PLLandGOwerealternatelyassembledontothecleanedglassslides.Therewere15–20minutesforeachdepositedlayerandthreerinsesinwateruntil4cycles.Theobtained(GO/PLL)4lmwasalsocharacterizedusingAFM,UV-VisspectroscopyandIRspectroscopy.2.4

Cellculture

RatbonemarrowderivedMSCswereobtainedfromcommercialsources(TianjinWeikaiBioengLtd.,China)andwereculturedinlow-glucoseDMEMmedium(Gibco,USA),supplementedwith10%fetalbovineserum(Invitrogen,USA),1%penicillin/streptomycin(Invitrogen,USA).Herein,theMSCsatpassage4wereused.25,26TheMSCs(2Â104cellsperwell,24-wellplate)wereseededonallthesurfacesseparatelyandculturedunderthesameconditions.Allthestudiedsurfacesweresterilizedinadvanceandplacedwithinacellculturepolystyrenewell.Forcellviability,live/deadstainingwithcalcein-AM(tostainlive

5462|J.Mater.Chem.B,2014,2,5461–54672.5

Osteogenicdi erentiation

Forosteogenicdi erentiation,theMSCsculturedonallthesurfacesfor1dayweretransferredtotheosteogenicmedium(TianjinWeikaiBioengLtd.,China)consistingofDMEMbasalmediumaddedwithdexamethasone(10À8M),b-glycerolphosphate(10mM),andascorbicacid(0.2mM).Themediumwaschangedevery3daysuntilconuence.25,26Alkalinephos-phatase(ALP)stainingwasperformedusingBCIP/NBTstocksolution(Beyotime,China)andALPquanticationwasdoneusinganalkalinephosphataseassaykit(Abcam,HongKong)atday2,7,12,and15.2.6

Immunouorescencestaining

First,thecellsonallthesurfaceswerexedwith3.7%formal-dehydesolutionover30minseparately.AerwashingwithPBSthreetimes,thecellswereincubatedin0.2%TritonX-100for10min.Then,thecellsweretreatedwith10%fetalbovineseruminPBSfor30min.Aerremovingtheblockingagent,theantibodiestocellularmarkers(CD-44forMSCsandOCNforosteoblasts)wereaddedontotheseparatesubstrates.Aerbeingincubatedfor1h,thesubstrateswereexclusivelywashedwithPBSandfurtherwereincubatedwiththedilutedsecondaryantibody(Dyelight488goatanti-mouseantibody)for30mininthedark.Subsequently,thecellswerestainedbyDAPI(Sigma-Aldrich,USA)for30mintocolorthenuclei.Finally,inallthecases,thecellswereanalyzedbyusingaconfocaluorescencemicroscopeusingtheOlympussystemanditssowarepackage(OlympusFV1000,Japan).2.7

Statisticalanalysis

FluorescentmicroscopyimageswereevaluatedandanalyzedusingImageJsoware(NIH,USA).Statisticalanalysiswasper-formedusingSPSS13.0toevaluateatleast10imagestodeterminestatisticaldi erences(*<0.05)amongallsamplesorbetweensamplesandcontrols,respectively.AllexperimentswererepeatedatleastthreetimesintriplicateandallvalueswerepresentedasmeanÆSD.2.8

Adsorptionofthechemicalinducers

Dexamethasone,b-glycerolphosphate,andascorbicacidwerepreparedseparatelyinPBSwiththeconcentrationof1mM,2mM,3mM,6mM,and10mM,respectively.Thesolutions

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withtheconcentrationof10mMwereemployedforadsorptionkineticswiththeadditionofthesubstrates.TheadsorptionofthechemicalswasevaluatedwithUV-Visspectroscopy.Theamountoftheadsorbedwasdeterminedfromthechangeinabsorptionbeforeandaertheadsorption.

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manifestedtheabsorptionpeaksat240nm,and310nm.TheyarecharacteristicabsorptionofGOfromthep–p*transitionsofaromaticC–Cbondsandn–p*transitionsofC]Obonds,respectively.TheseresultsconrmedthesuccessfulformationoftheGO/PLLcompositelm.3.2

MSCsviabilityandproliferationontheGO/PLLlm

3Resultsanddiscussion

3.1

FabricationandcharacterizationofGOandGO/PLLlmGOcanbeobtainedbychemicaloxidationandexfoliationofgraphite.TheproducedGOwasconrmedrstbytheatomicforcemicroscopy(AFM)technique.ItwasfoundthattheGOTodeterminewhetherornottheGO/PLLlmhadasignicante ectontheMSCs,uorescencestainingwasperformedusingcalcein-AM(tostainlivecellswithgreencolor)andethidiumhomodimer(tostaindeadcellswithredcolor).FluorescencemicroscopyrevealedthatmostoftheMSCsplatedonthesheetshadlateraldimensionsofonetoseveralhundrednanometerswithathicknessof1.0nmapproximately(Fig.S1 ),whichischaracteristicofthefullyexfoliatedGOsheets.28ThentheFTIRspectrumwasusedtocharacterizetheproducedGO.InFig.S2, theGOspectrumshowedthepresenceofO–H(3430cmÀ1),C]O(1733cmÀ1),C]C(1630cmÀ1),andC–O(1128cmÀ1).29Asacontrast,besidesthesesites,theIRspectrumoftheLbL-assembled(GO/PLL)4compositelmexhibitedPLLabsorptionfeatures,suchasN–H(3259cmÀ1),C]O(1633cmÀ1)andC–N(1552cmÀ1).Also,thenanotopographyandthicknessofthe(GO/PLL)4lmweredeterminedbyAFM.Fig.2AshowsclearlytheexistenceofGOsheetsonthelmsurfaceshowingthethicknessofthe(GO/PLL)4lmof5.51nm(Fig.S3 ).ThebuildupoftheGO/PLLlmswasfurthermoni-toredbyUV-Visspectroscopy(Fig.2B).ComparingwiththeUV-VisspectrumofPLL,allthespectraoftheGO/PLLlms

Fig.2

(A)AFMimageofthe(GO/PLL)4 lm;(B)1,2,UV-Vis3,

4.absorption

spectraofGO,PLLandthe(GO/PLL)n lm,n¼Thisjournalis©TheRoyalSocietyofChemistry2014compositelmswerealive,asshowninFig.3A.Fromthephasecontrastimage(Fig.3B),thecellsdisplayedthepolygonalandattenedcellmorphologyontheGO/PLLlmandpresentedtheelongatedstructurewiththeirspindleshape.ThemorphologyofcellsontheotherstudiedsurfacesisshowninFig.S4. Additionally,acellcountkit-8(CCK-8)assaywascarriedouttoconrmthecellviabilitydataaer1dayand7dayofcellcultureonthecompositelmrespectively.Glasscoverslips(treatedwithpiranhasolution),GO-coatedcoverslips(GO-coverslips)fabricatedwiththespincoatingtechnique,PLLcoatedglasscoverslips(PLL-coverslips)andtissueculturepolystyrene(TCPS)wereemployedforcomparison.AsshowninFig.

3C,

Fig.3

(A)FluorescenceimagesofMSCswereobtainedonthe(GO/

PLL)4 lmbylive/deadstainingofcellsafterincubationfor3days.Livecellswerestained uorescencegreen,anddeadcellsappearedred.(B)PhasecontrastimagesofMSCsculturedfor3daysonthe(GO/PLL)4 lminnormalcellculturemedia.(C)CellviabilitiesofMSCsculturedonallthestudiedsurfacesincellculturemediabyday1andday7.(D)Cellproliferationonallthestudiedsurfacesasestimatedbytheratio(D7/D1).(E)GrowthkineticsofcellsontheTCPSandGO/PLLcomposite lms.(F)Thepopulationdoublingtimesofcellsonallthestudiedsurfaces.Thetotalnumberofcellswasdeterminedatdi erenttimepointstoobtainthedoublingtime.J.Mater.Chem.B,2014,2,5461–5467|5463

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almostnodi erencewasnotedinthecellviabilityonallthestudiedsurfacesaerbeingculturedfor1day(D1).Whileonthe7thdayofculture,thecellviabilityofMSCsonthecompositelmwas186.0%,142.7%,105.8%and120.7%greaterthanthoseontheglasscoverslips,GO-coverslips,PLL-coverslips,andTCPS(D7),respectively.CellproliferationwasestimatedbytheratioofabsorbancevalueD7/D1andthevalueswereshowninFig.3D.ItcouldbeseenthattheproliferationofcellsonthePLL-coverslipswasnearlyequaltothoseonTCPS,andthatoftheGO-coverslipswasabout20%lower.Whilesignicantproliferationwasobservedforthe(GO/PLL)4lm.Thehighproliferationmaybeattributedtothesynergisticcouplinge ectsofGOandPLL,whichwillbeinvestigatedpositivelyandthecytoplasmwasstainedblue-black.ALPstainingresultsofMSCsonthe7thdayofdi erentiationontheotherstudiedsurfacesareshowninFig.S5. Also,quantitativeALPstainingwasusedtostudythedi erentiationofMSCsonallthestudiedsubstrates(Fig.4B).ThecellsontheGO-cover-slipsandthePLL-coverslipsexpressedALPobviouslyfromday7,andtheALPactivityincreasegraduallytillday15.TheALPactivityofcellsonthePLL-coverslipswasalittlehigherthanthatofGO-coverslipsandTCPS,respectively.Again,signicantALPproductionbycellswereseenonthe(GO/PLL)4lmbyday7.Importantly,fromday7onward,thecellsgrownonthelmshowedhigherALPactivitythanthoseonthesurfacesascontrolsobviously.And,theactivityofcellsontheGO/PLLlmfurtherinthiswork.InFig.3E,growthkineticsofcellsontheTCPSandtheGO/PLLlmwasrecorded.Accordingly,thepopulationdoublingtimewasanalyzedatmultipletimepointsoverthecultureperiod.Incomparison,thedoublingtimeofcellsonthecompositelmwas28.07h,anddecreasedfromthoseonTCPS(32.82h)(Fig.3F).TheseresultssuggestedthattheGO/PLLlmdidnothamperthenormalgrowthofstemcellsbutratherprovidedasuitableenvironmentfortheprolif-erationofMSCs.

3.3Osteogenicdi erentiationofMSCsontheGO/PLLlm

Then,whenculturedinosteogenicmedia,theMSCsweredeterminedandanalyzedosteogenesisusingalkalinephos-phatase(ALP)stainingasamarker.30Onthe2nddayofdi er-entiation,cellsculturedonallthesubstratesshowednodi erenceinALPproduction.TherewasnegativestainingandabsolutelynoALPproductionwasobservedfromtheimagesshowninFig.4A.Byday7,cellsonthelmwerestained

Fig.4

(A)ALPstainingresultsofMSCsonthe(GO/PLL)4 lmfor2,7,

and12days,respectively.Thescalebaris100mm.(B)ActivityofALPproducedfromcellsculturedfor2,7,12,and15daysonallthestudied

surfaces.5464|J.Mater.Chem.B,2014,2,5461–5467onthe12thdayofdi erentiationwasnearlyequaltothatof15thday.Therefore,theGO/PLLlmsupportedandacceleratedtheosteogenicdi erentiationofMSCsshowingtheapparentosteogenesisduringtheosteogenicdi erentiationprocess.Furthermore,osteocalcin(OCN)asoneoftheosteogenicgeneswasexaminedtoconrmtheosteogenicdi erentiationofMSCsonthe(GO/PLL)4lm.SinceCD-44isoneofthecharac-teristicgenesforstemcells,itsexpressionwasalsocheckedfortheMSCsculturedonthesamples.15Herein,theproteinexpressionwascomparedbytheintensityofuorescenceviaimmunouorescencestaining,wherethecellswerestainedwithDAPI(blue),CD-44andOCN(green).AsveriedinFig.5A,theCD-44couldbevisibleclearlystillbyday2,however,theintensityofuorescencedecreasedsignicantlybyday7andcompletelydisappearedbyday12.Ontheotherhand,aprogressiveenhancementofuorescencewasobservedbytheOCNrecognitionfromday7today12(Fig.5B).TheproteinexpressionofcellsontheTCPSandblankglasscoverslipswereshownascomparisoninFig.S6andS7, respectively.

Also,thedi erentiationofMSCsonallthestudiedsurfaceswascomparedbytheratioofCD-44(Fig.6A),OCN(Fig.6B)positivecellsinallthecellsbyday2,7,12,respectively.AsdemonstratedinFig.6B,celldi erentiationwasaccelerated

on

Fig.5

Immunostainingofcellsgrowingonthe(GO/PLL)4 lmwas

performedfrom2daysto12days.CellsarestainedwithDAPI(blue)andCD-44orosteocalcin(OCN)asindicated(green).(A,A0andA00)CD-44,markerforstemcells,decreasedovertimeandcompletelydisappearedbyday12.(B,B0andB00)OCN,markerforosteoblasts,becamevisibleatday7andveryintensebyday12.Thisjournalis©TheRoyalSocietyofChemistry2014

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Fig.6Immuno uorescent(A)CD-44and(B)OCNstainingofMSCsgrownonthe(GO/PLL)4 lm(left)andontheTCPS(right).Thehistogramistheratioofpositivelystainedcellsandthesumofposi-tivelyandnegativelystainedcellsonallthestudiedsubstratesbyday2,7,and12,

respectively.

theGO/PLLlmbyday7,ascomparedtothoseonTCPS,showingnearly55%positiveOCNwhilethelattershowed35%positive.Takentogether,theseresultsindicatedthattheGO/PLLlmcouldpromotetheosteogenesisofMSCssignicantly.Herein,thepredominanceoftheGO/PLLcompositelmsovertheGOsurfaceandthePLLsurfacemightbetheresultsofcoupledcombinationoftwocomponents,whichcouldbeevi-dencedbythefollowingstudy.

Sincetheconventionalosteogenicmediacandirectlymediatethedi erentiation,adirectcorrelationwiththeadsorptionofthesubstratefortheosteogenicchemicalinducerswasthoughttobeinvolved.15,31Andwhatismore,previousstudieshaverecognizedthatGOhastheremarkableloadingcapacitiesforproteins,DNAandorso,viaitslarge

Thisjournalis©TheRoyalSocietyofChemistry2014Fig.7Loadingcapacityof(A)dexamethasone,(B)b-glycerolphos-phate,(C)ascorbicacidindi erentconcentrationonallthestudiedsurfaces.Theinsetsarethechemicalstructures,respectively.

surfaceareaandintermolecularinteraction.32–34Therefore,theadsorptionofthe(GO/PLL)4lmforthechemicalinducerswasinvestigatedusingUVspectrophotometry.First,theloadingcapacitiesofthelmfortheosteogenicchemicals,dexameth-asone,b-glycerolphosphate,ascorbicacid,weredeterminedwiththeadsorptionkineticsrecordedinFig.S8. Furthermore,theadsorptionofthechemicalsonthestudiedsubstrateswasalsodeterminedwithincreasingconcentrationof

the

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adsorbents(Fig.7).TheadsorbedamountwasexpressedinmgcmÀ2ofsubstrates.Itwasfoundthatthe(GO/PLL)4lmcouldlargelyadsorbdexamethasone(10mM),glycerolphos-phate(10mM),ascorbicacid(10mM),withtheloadingamountof629,384and194mgcmÀ2ofsubstratebyday3,respectively.ThehighadsorptioncapacityoftheGO/PLLlmandtheGO-coverslipfordexamethasonemaybeattributedtothep–pstackingbetweenthearomaticringsintheorganicmoleculesandthegraphenebasalplane.Asfortheadsorptionofb-glyc-erolphosphate,theelectrostaticattractionbetweentheanionsandthepositivelychargedPLLmoleculesfromtheGO/PLLlmaswellasthePLL-coverslipcouldplayamajorroleduringtheadsorptionprocess.Whileinthecaseofascorbicacid,the2H.Lu,N.Kawazoe,T.Kitajima,Y.Myoken,M.Tomita,A.Umezawa,G.ChenandY.Ito,Biomaterials,2012,33,6140.3M.F.Pittenger,A.M.Mackay,S.C.Beck,R.K.Jaiswal,R.Douglas,J.D.Mosca,M.A.MoormanandD.W.Simonetti,Science,1999,284,143.

4X.Wang,W.Song,N.KawazoeandG.Chen,J.Biomed.Mater.Res.,PartA,2013,101,3388.

5J.Zhu,Y.Zhang,N.Xu,L.Wang,X.XiangandX.Zhu,J.Biomater.Sci.,Polym.Ed.,2013,24,1721.

6C.Zheng,M.Zheng,P.Gong,J.Deng,H.Yi,P.Zhang,Y.Zhang,P.Liu,Y.MaandL.Cai,Biomaterials,2013,34,3431.

7Y.LiuandL.Yobas,Biosens.Bioelectron.,2013,50,78.

hydrogen-bondbetweenascorbicacidandGOshouldbeconsideredtopushtheadsorptionofascorbicacidintotheGO/PLLlmandtheGO-coverslips.Therefore,itmaybededucedthatthehighadsorptioncapacityoftheGO/PLLlmforthesechemicalinducerscouldcausetheinducerstobelocallyconcentratedonthelmtofacilitatecell–substrateinteractionsresultingingreatenhancementofosteogenicdi erentiationofMSCs.

4Conclusions

Insummary,anewtypeofGO/PLLcompositelmshasbeenfabricatedviaLbLassemblyinamildandinexpensiveway,whichcouldbeapplicabletootherkindsofbiomacromoleculessuchaschitosan,gelatin,etc.IthasbeendemonstratedthattheGO/PLLcompositelmcouldenhancethedi erentiationofMSCstotheosteoblastlineagewithoutinuencingtheshapeandthegrowthofMSCs.Moreover,comparedtothecommer-cialmaterialssuchasTCPSorglasscoverslips,theGO/PLLlmcouldobviouslypreconcentratetheosteogenicinducers,i.e.dexamethasone,b-glycerolphosphateandascorbicacid.Itmightplayakeyroleinthesignicantenhancementofosteo-genicdi erentiationofMSCsonthecompositelm.Overall,theGO/PLLlmaswellasthelmfabricationstrategycouldbepromisingforclinicalapplicationsintissueengineeringorregenerativemedicinewitheasyfabrication,biocompatibility,andversatility.

Acknowledgements

W.Q.thanksDrQingminJiandDrKatsuhikoAriga,Interna-tionalCenterforMaterialsNanoarchitectonics(MANA),NationalInstituteforMaterialsScience(NIMS),fortheirtech-nicalsupportsandvaluablediscussions.Thisworkwasnan-ciallysupportedbytheNationalNatureScienceFoundationofChina(no.21003084,21375075),theStudentResearchTrainingProgramofQufuNormalUniversity(no.2013A046),andtheTaishanScholarFoundationofShandongProvince,China.

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