Growth and accelerated differentiation of mesenchymal stem c(2)

JournalofMaterialsChemistryBPaper

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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