Growth and accelerated differentiation of mesenchymal stem c

1、growth and accelerated differentiation of mesenchymal stem c materialschemistry b paper published on 25 june 2021. downloaded by south china university of technology on 25/05/2021 04:20:30. journalof view article online view journal | view issue citethis:j.mater.chem.b,2021,2,5461 growthandaccelerat

2、eddi erentiationof mesenchymalstemcellsongrapheneoxide/poly-l-lysinecomposite lms weiqi,*wenjingyuan,jingyanandhuawang* anewtypeofcomposite lmsofgrapheneoxide(go)andpoly-l-lysine(pll)havebeenfabricatedforuseasbio-sca oldcoatingswithimprovedmechanicalproperties.themorphology,growthanddi erentiationof

3、mesenchymalstemcells(mscs)onthego/pllcomposite lmshavebeenexamined.theresultsdemonstratedthatthego/pllcomposite lmscouldnotonlysupportthegrowthofmscswithahighproliferationrate,butcouldacceleratetheosteogenicdi erentiationofmscs,showingstrongalkalinephosphatase(alp)andgeneexpression.importantly,thehi

4、ghpre-concentrationcapacityofthego/pll lmsforosteogenicinducerscouldplayakeyroleinacceleratingtheosteogenicdi erentiationofmscsthroughthestrongnon-covalentbindingandelectrostaticinteractionbetweenthem.moreover,themildandinexpensivefabricationstrategycouldbeapplicabletobiomacromoleculesincludingchito

5、san,gelatin,etc.therefore,suchakindofcomposite lmcouldprovideamultifunctionalandhighlybiocompatiblesca oldcoatingtailoredforpotentialapplicationinstemcellresearch. received27thmay2021accepted24thjune2021doi:10.1039/materialsb 1introduction mesenchymalstemcells(mscs)isolatedfrombonemarrowhavebeenshow

6、ntodi erentiateintoavarietyofcelllineagesinvitro,includingadipocytes,chondrocytes,myocytesandosteo-blasts.thecombinationofmscswithsca oldmaterialsprovidesapromisingstrategyforcelltherapyandtissueengi-neering.14somenaturalbiomaterialssuchaspolypeptideshavebeeninvestigatedaspotentialsca oldmaterialsfo

7、rstemcelltransplantation.forexample,poly-l-lysine(pll),withplentifulactiveaminogroupsandpositivesurfacecharges,ismostcommonlyusedinbiomedicaleldssuchasbioactivesurfaces,drugdeliveryandorsoforpromotingcelladhesionandsup-portingcellgrowth.57however,mechanicalpropertiesofpllarenotgoodenoughforsomebiome

8、dicalapplicationssuchasbonetissueengineeringorboneregenerativemedicine.therefore,itisessentialtodevelopnanocompositesbasedonplltoimproveitsmechanicalproperties.grapheneanditsderivative,grapheneoxide(go),isanattractivesubstanceasacompositematerial,especiallyaspolymernanocompositesduetoitsextraordinar

9、ymechanicalpropertiesincludinghighyoung'smodulus,hardnessandexcellentexibility.moreover, asreportedpreviously,theincorporationofgointoapolymermultilayercouldimprovethelmpropertiessignicantly.8ontheotherhand,biointerfacescomposedofgrapheneorgohavebeeninvestigatedforadhesion,proliferationanddi ere

10、ntia-tionofthemscs.915particularly,go,inwhichthebasalplaneandedgespossessepoxide,carboxyl,andhydroxylgroups,enablesgreaterinteractionswithbiomacromoleculesthroughcovalent,electrostaticandhydrogen-bondingthangraphene.herein,anewtypeofgo/pllcompositelmshavebeenbuiltupinthisstudyvialayer-by-layer(lbl)a

11、ssembly,forgrowth,proliferationanddi erentiationofstemcells(fig.1).thelblassemblytechniquehasbeenwellrecognizedas a keylaboratoryoflife-organicanalysis,keylaboratoryofpharmaceuticalintermediatesandanalysisofnaturalmedicine,schoolofchemistryandchemicalengineering,qufunormaluniversity,57jingxuanwestro

12、ad,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. thisjournalistheroyalsocietyofchemistry2021j.mater.chem.b,2021,2,546

13、15467|5461 journalofmaterialschemistrybpaper simpleandversatilemethodtoimmobilizefunctionalmole-cules.1623herein,theuniquepropertiesofgo,i.e.alargesurfaceareaandexcellentmechanicalproperties,couldbesynergicallycoupledwiththebiocompatibilityofpllresultinginakindofmultifunctionalsca oldcoating,whichca

14、nbeusedintissueengineeringandstemcell-basedtherapy. 25/05/2021 04:20:30. 2experimental 2.1 materials expandablegraphite($180mm)wasobtainedfromqingdaobcsm.co.,ltd.poly-l-lysine(pll)andphosphatebu ersolution(pbs)werepurchasedfromsigma-aldrich(usa).cal-cellswithgreencolor)andethidiumhomodimer(tostainde

15、adcellswithredcolor)wasemployed.forthepurposeofcomparison,cellcountingkit-8(cck-8)assays(dojindo,japan)werecarriedoutforallthestudiedcells.themorphologyofthemscsonallthesurfaceswasobservedbyusingaphasecontrastmicroscope(olympusckx31,japan).thepopulationdoublingtimes(pdt)ofcellsonallthestudiedsurface

16、sweredeterminedwiththegrowthkineticsandthefollowingequation.27 pdtt2t1 lg221 (1) n2:numberofcellsattimet2,andn1:numberofcellsattimet1.cein-amandethidiumhomodimerwereobtainedfromdojindo(japan).antibodiesincludinganti-cd-44andanti-osteocalcin(ocn)werepurchasedfromabcam(hongkong).waterusedinthisworkwas

17、puriedbyamilli-qwatersystem(millipore,usa).2.2 fabricationandcharacterizationofgo graphiteoxidewasobtainedbythehummersmethod.8,24briey,graphiteoxidewasobtainedbyoxidationofgraphitewithh2so4,andkmno4.theformedgraphiteoxidewaswashedthreetimeswith1.0mofaqueoushclsolutionanddeionizedwateruntilaphof4.05.

18、0wasachieved.duringthewashingprocesswithdeionizedwater,agrapheneoxidegelformed.followingthat,afreeze-dryprocedurewasconductedtoobtainthegrapheneoxidesolid.atomicforcemicroscopy(afm,digitalinstruments,usa),uv-visspectroscopy(uv-1601,shimadzu,japan)andirspectroscopy(nexus470,nicolet,usa)wereusedtochar

19、acterizethegonanosheets.2.3 preparationandcharacterizationofthego/plllm glasscoverslips(14mmdiameteror4.5mmdiameter)wereusedasthesubstratesforlblassembly.theywererstcleanedinpiranhasolution(7:3v/vh2so4/h2o2)beforeuse,followedbyrinsingwithwater.pllsolutionswerepreparedataconcentrationof2mgml1inpbs,an

20、dgodispersionwaspreparedataconcentrationof0.1mgml1inwater.then,pllandgowerealternatelyassembledontothecleanedglassslides.therewere1520minutesforeachdepositedlayerandthreerinsesinwateruntil4cycles.theobtained(go/pll)4lmwasalsocharacterizedusingafm,uv-visspectroscopyandirspectroscopy.2.4 cellculture r

21、atbonemarrowderivedmscswereobtainedfromcommercialsources(tianjinweikaibioengltd.,china)andwereculturedinlow-glucosedmemmedium(gibco,usa),supplementedwith10%fetalbovineserum(invitrogen,usa),1%penicillin/streptomycin(invitrogen,usa).herein,themscsatpassage4wereused.25,26themscs(2104cellsperwell,24-wel

22、lplate)wereseededonallthesurfacesseparatelyandculturedunderthesameconditions.allthestudiedsurfacesweresterilizedinadvanceandplacedwithinacellculturepolystyrenewell.forcellviability,live/deadstainingwithcalcein-am(tostainlive 5462|j.mater.chem.b,2021,2,546154672.5 osteogenicdi erentiation forosteogen

23、icdi erentiation,themscsculturedonallthesurfacesfor1dayweretransferredtotheosteogenicmedium(tianjinweikaibioengltd.,china)consistingofdmembasalmediumaddedwithdexamethasone(108m),b-glycerolphosphate(10mm),andascorbicacid(0.2mm).themediumwaschangedevery3daysuntilconuence.25,26alkalinephos-phatase(alp)

24、stainingwasperformedusingbcip/nbtstocksolution(beyotime,china)andalpquanticationwasdoneusinganalkalinephosphataseassaykit(abcam,hongkong)atday2,7,12,and15.2.6 immunouorescencestaining first,thecellsonallthesurfaceswerexedwith3.7%formal-dehydesolutionover30minseparately.aerwashingwithpbsthreetimes,th

25、ecellswereincubatedin0.2%tritonx-100for10min.then,thecellsweretreatedwith10%fetalbovineseruminpbsfor30min.aerremovingtheblockingagent,theantibodiestocellularmarkers(cd-44formscsandocnforosteoblasts)wereaddedontotheseparatesubstrates.aerbeingincubatedfor1h,thesubstrateswereexclusivelywashedwithpbsand

26、furtherwereincubatedwiththedilutedsecondaryantibody(dyelight488goatanti-mouseantibody)for30mininthedark.subsequently,thecellswerestainedbydapi(sigma-aldrich,usa)for30mintocolorthenuclei.finally,inallthecases,thecellswereanalyzedbyusingaconfocaluorescencemicroscopeusingtheolympussystemanditssowarepac

27、kage(olympusfv1000,japan).2.7 statisticalanalysis fluorescentmicroscopyimageswereevaluatedandanalyzedusingimagejsoware(nih,usa).statisticalanalysiswasper-formedusingspss13.0toevaluateatleast10imagestodeterminestatisticaldi erences(*0.05)amongallsamplesorbetweensamplesandcontrols,respectively.allexpe

28、rimentswererepeatedatleastthreetimesintriplicateandallvalueswerepresentedasmeansd.2.8 adsorptionofthechemicalinducers dexamethasone,b-glycerolphosphate,andascorbicacidwerepreparedseparatelyinpbswiththeconcentrationof1mm,2mm,3mm,6mm,and10mm,respectively.thesolutions thisjournalistheroyalsocietyofchem

29、istry2021 published on 25 june 2021. downloaded by south china university of technology on paperjournalofmaterialschemistryb withtheconcentrationof10mmwereemployedforadsorptionkineticswiththeadditionofthesubstrates.theadsorptionofthechemicalswasevaluatedwithuv-visspectroscopy.theamountoftheadsorbedw

30、asdeterminedfromthechangeinabsorptionbeforeandaertheadsorption. 25/05/2021 04:20:30. manifestedtheabsorptionpeaksat240nm,and310nm.theyarecharacteristicabsorptionofgofromthepp*transitionsofaromaticccbondsandnp*transitionsofcobonds,respectively.theseresultsconrmedthesuccessfulformationofthego/pllcompo

31、sitelm.3.2 mscsviabilityandproliferationonthego/plllm 3resultsanddiscussion 3.1 fabricationandcharacterizationofgoandgo/plllmgocanbeobtainedbychemicaloxidationandexfoliationofgraphite.theproducedgowasconrmedrstbytheatomicforcemicroscopy(afm)technique.itwasfoundthatthegotodeterminewhetherornotthego/p

32、lllmhadasignicante ectonthemscs,uorescencestainingwasperformedusingcalcein-am(tostainlivecellswithgreencolor)andethidiumhomodimer(tostaindeadcellswithredcolor).fluorescencemicroscopyrevealedthatmostofthemscsplatedonthesheetshadlateraldimensionsofonetoseveralhundrednanometerswithathicknessof1.0nmappr

33、oximately(fig.s1 ),whichischaracteristicofthefullyexfoliatedgosheets.28thentheftirspectrumwasusedtocharacterizetheproducedgo.infig.s2, thegospectrumshowedthepresenceofoh(3430cm1),co(1733cm1),cc(1630cm1),andco(1128cm1).29asacontrast,besidesthesesites,theirspectrumofthelbl-assembled(go/pll)4compositel

34、mexhibitedpllabsorptionfeatures,suchasnh(3259cm1),co(1633cm1)andcn(1552cm1).also,thenanotopographyandthicknessofthe(go/pll)4lmweredeterminedbyafm.fig.2ashowsclearlytheexistenceofgosheetsonthelmsurfaceshowingthethicknessofthe(go/pll)4lmof5.51nm(fig.s3 ).thebuildupofthego/plllmswasfurthermoni-toredbyu

35、v-visspectroscopy(fig.2b).comparingwiththeuv-visspectrumofpll,allthespectraofthego/plllms fig.2 (a)afmimageofthe(go/pll)4 lm;(b)1,2,uv-vis3, 4.absorption spectraofgo,pllandthe(go/pll)n lm,nthisjournalistheroyalsocietyofchemistry2021compositelmswerealive,asshowninfig.3a.fromthephasecontrastimage(fig.

36、3b),thecellsdisplayedthepolygonalandattenedcellmorphologyonthego/plllmandpresentedtheelongatedstructurewiththeirspindleshape.themorphologyofcellsontheotherstudiedsurfacesisshowninfig.s4. additionally,acellcountkit-8(cck-8)assaywascarriedouttoconrmthecellviabilitydataaer1dayand7dayofcellcultureonthec

37、ompositelmrespectively.glasscoverslips(treatedwithpiranhasolution),go-coatedcoverslips(go-coverslips)fabricatedwiththespincoatingtechnique,pllcoatedglasscoverslips(pll-coverslips)andtissueculturepolystyrene(tcps)wereemployedforcomparison.asshowninfig. 3c, fig.3 (a)fluorescenceimagesofmscswereobtaine

38、donthe(go/ pll)4 lmbylive/deadstainingofcellsafterincubationfor3days.livecellswerestained uorescencegreen,anddeadcellsappearedred.(b)phasecontrastimagesofmscsculturedfor3daysonthe(go/pll)4 lminnormalcellculturemedia.(c)cellviabilitiesofmscsculturedonallthestudiedsurfacesincellculturemediabyday1andda

39、y7.(d)cellproliferationonallthestudiedsurfacesasestimatedbytheratio(d7/d1).(e)growthkineticsofcellsonthetcpsandgo/pllcomposite lms.(f)thepopulationdoublingtimesofcellsonallthestudiedsurfaces.thetotalnumberofcellswasdeterminedatdi erenttimepointstoobtainthedoublingtime.j.mater.chem.b,2021,2,54615467|

40、5463 published on 25 june 2021. downloaded by south china university of technology on journalofmaterialschemistrybpaper 25/05/2021 04:20:30. almostnodi erencewasnotedinthecellviabilityonallthestudiedsurfacesaerbeingculturedfor1day(d1).whileonthe7thdayofculture,thecellviabilityofmscsonthecompositelmw

41、as186.0%,142.7%,105.8%and120.7%greaterthanthoseontheglasscoverslips,go-coverslips,pll-coverslips,andtcps(d7),respectively.cellproliferationwasestimatedbytheratioofabsorbancevalued7/d1andthevalueswereshowninfig.3d.itcouldbeseenthattheproliferationofcellsonthepll-coverslipswasnearlyequaltothoseontcps,

42、andthatofthego-coverslipswasabout20%lower.whilesignicantproliferationwasobservedforthe(go/pll)4lm.thehighproliferationmaybeattributedtothesynergisticcouplinge ectsofgoandpll,whichwillbeinvestigatedpositivelyandthecytoplasmwasstainedblue-black.alpstainingresultsofmscsonthe7thdayofdi erentiationontheo

43、therstudiedsurfacesareshowninfig.s5. also,quantitativealpstainingwasusedtostudythedi erentiationofmscsonallthestudiedsubstrates(fig.4b).thecellsonthego-cover-slipsandthepll-coverslipsexpressedalpobviouslyfromday7,andthealpactivityincreasegraduallytillday15.thealpactivityofcellsonthepll-coverslipswas

44、alittlehigherthanthatofgo-coverslipsandtcps,respectively.again,signicantalpproductionbycellswereseenonthe(go/pll)4lmbyday7.importantly,fromday7onward,thecellsgrownonthelmshowedhigheralpactivitythanthoseonthesurfacesascontrolsobviously.and,theactivityofcellsonthego/plllmfurtherinthiswork.infig.3e,gro

45、wthkineticsofcellsonthetcpsandthego/plllmwasrecorded.accordingly,thepopulationdoublingtimewasanalyzedatmultipletimepointsoverthecultureperiod.incomparison,thedoublingtimeofcellsonthecompositelmwas28.07h,anddecreasedfromthoseontcps(32.82h)(fig.3f).theseresultssuggestedthatthego/plllmdidnothampertheno

46、rmalgrowthofstemcellsbutratherprovidedasuitableenvironmentfortheprolif-erationofmscs. 3.3osteogenicdi erentiationofmscsonthego/plllm then,whenculturedinosteogenicmedia,themscsweredeterminedandanalyzedosteogenesisusingalkalinephos-phatase(alp)stainingasamarker.30onthe2nddayofdi er-entiation,cellscult

47、uredonallthesubstratesshowednodi erenceinalpproduction.therewasnegativestainingandabsolutelynoalpproductionwasobservedfromtheimagesshowninfig.4a.byday7,cellsonthelmwerestained fig.4 (a)alpstainingresultsofmscsonthe(go/pll)4 lmfor2,7, and12days,respectively.thescalebaris100mm.(b)activityofalpproduced

48、fromcellsculturedfor2,7,12,and15daysonallthestudied surfaces.5464|j.mater.chem.b,2021,2,54615467onthe12thdayofdi erentiationwasnearlyequaltothatof15thday.therefore,thego/plllmsupportedandacceleratedtheosteogenicdi erentiationofmscsshowingtheapparentosteogenesisduringtheosteogenicdi erentiationproces

49、s.furthermore,osteocalcin(ocn)asoneoftheosteogenicgeneswasexaminedtoconrmtheosteogenicdi erentiationofmscsonthe(go/pll)4lm.sincecd-44isoneofthecharac-teristicgenesforstemcells,itsexpressionwasalsocheckedforthemscsculturedonthesamples.15herein,theproteinexpressionwascomparedbytheintensityofuorescence

50、viaimmunouorescencestaining,wherethecellswerestainedwithdapi(blue),cd-44andocn(green).asveriedinfig.5a,thecd-44couldbevisibleclearlystillbyday2,however,theintensityofuorescencedecreasedsignicantlybyday7andcompletelydisappearedbyday12.ontheotherhand,aprogressiveenhancementofuorescencewasobservedbythe

51、ocnrecognitionfromday7today12(fig.5b).theproteinexpressionofcellsonthetcpsandblankglasscoverslipswereshownascomparisoninfig.s6ands7, respectively. also,thedi erentiationofmscsonallthestudiedsurfaceswascomparedbytheratioofcd-44(fig.6a),ocn(fig.6b)positivecellsinallthecellsbyday2,7,12,respectively.asd

52、emonstratedinfig.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

53、,b0andb00)ocn,markerforosteoblasts,becamevisibleatday7andveryintensebyday12.thisjournalistheroyalsocietyofchemistry2021 published on 25 june 2021. downloaded by south china university of technology on paperjournalofmaterialschemistryb 25/05/2021 04:20:30. fig.6immuno uorescent(a)cd-44and(b)ocnstaini

54、ngofmscsgrownonthe(go/pll)4 lm(left)andonthetcps(right).thehistogramistheratioofpositivelystainedcellsandthesumofposi-tivelyandnegativelystainedcellsonallthestudiedsubstratesbyday2,7,and12, respectively. thego/plllmbyday7,ascomparedtothoseontcps,showingnearly55%positiveocnwhilethelattershowed35%posi

55、tive.takentogether,theseresultsindicatedthatthego/plllmcouldpromotetheosteogenesisofmscssignicantly.herein,thepredominanceofthego/pllcompositelmsoverthegosurfaceandthepllsurfacemightbetheresultsofcoupledcombinationoftwocomponents,whichcouldbeevi-dencedbythefollowingstudy. sincetheconventionalosteoge

56、nicmediacandirectlymediatethedi erentiation,adirectcorrelationwiththeadsorptionofthesubstratefortheosteogenicchemicalinducerswasthoughttobeinvolved.15,31andwhatismore,previousstudieshaverecognizedthatgohastheremarkableloadingcapacitiesforproteins,dnaandorso,viaitslarge thisjournalistheroyalsocietyof

57、chemistry2021fig.7loadingcapacityof(a)dexamethasone,(b)b-glycerolphos-phate,(c)ascorbicacidindi erentconcentrationonallthestudiedsurfaces.theinsetsarethechemicalstructures,respectively. surfaceareaandintermolecularinteraction.3234therefore,theadsorptionofthe(go/pll)4lmforthechemicalinducerswasinvest

58、igatedusinguvspectrophotometry.first,theloadingcapacitiesofthelmfortheosteogenicchemicals,dexameth-asone,b-glycerolphosphate,ascorbicacid,weredeterminedwiththeadsorptionkineticsrecordedinfig.s8. furthermore,theadsorptionofthechemicalsonthestudiedsubstrateswasalsodeterminedwithincreasingconcentrationof the j.mater.chem.b,2021,2,54615467|5465 published on 25 june 2021. downloaded by south china universi