Growth and accelerated differentiation of mesenchymal stem c(4)

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

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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