Growth and accelerated differentiation of mesenchymal stem cells on graphene oxide(3)

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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