Growth and accelerated differentiation of mesenchymal stem cells on graphene oxide(6)

JournalofMaterialsChemistryBPaper

25/05/2015 04:20:30.

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

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