acterizingwww

Non-invasivecharacterizationofstructureandmorphologyofsilkbroinbiomaterialsusingnon-linearmicroscopyWilliamL.
Ricea,ShamarazFirdousb,SharadGuptaa,MartinHuntera,CherylW.
P.
Fooa,YongzhongWanga,HyeonJooKima,DavidL.
Kaplana,IreneGeorgakoudia,*aBiomedicalEngineeringDepartment,TuftsUniversity,4ColbyStreet,Room229,Medford,MA02155,USAbLaserLaboratory,DepartmentofPhysicsandAppliedMathematics(DPAM),PakistanInstituteofEngineeringandAppliedSciences(PIEAS),P.
O.
Nilore,Islamabad,PakistanReceived10October2007;accepted22December2007Availableonline21February2008AbstractDesigningbiomaterialscaffoldsremainsamajorchallengeintissueengineering.
Keytothischallengeisimprovedunderstandingoftherelationshipsbetweenthescaffoldpropertiesanditsdegradationkinetics,aswellasthecellinteractionsandthepromotionofnewmatrixdeposition.
Herewepresenttheuseofnon-linearspectroscopicimagingasanon-invasivemethodtocharacterizenotonlymorphological,butalsostructuralaspectsofsilkwormsilkbroin-basedbiomaterials,relyingentirelyonendogenousopticalcontrast.
Wedemonstratethattwophotonexciteduorescenceandsecondharmonicgenerationaresensitivetothehydration,overallbsheetcontentandmolecularorientationofthesample.
Thus,thefunctionalcontentandhighresolutionaffordedbythesenon-invasiveapproachesofferpromiseforidentifyingimportantconnectionsbetweenbiomaterialdesignandfunctionalengineeredtissuedevelopment.
Thestrategiesdescribedalsohavebroaderimplicationsforunderstandingandtrackingtheremodelingofdegradablebiomaterialsunderdynamicconditionsbothinvitroandinvivo.
2008ElsevierLtd.
Allrightsreserved.
Keywords:Silk;Fibroin;Twophotonexciteduorescence;Secondharmonicgeneration;Spectralanalysis;Non-invasivecharacterization1.
IntroductionDesignoptimizationofthebiomaterialsthatareusedasscaffoldsontowhichneworregeneratedtissuesareexpectedtogrowisoneofthemajorchallengesfacingtissueengineers.
Numerousaspectsofthebiomaterialsdesign,fromthemolec-ularstructureandorganizationtotheoverallmacro-architec-tureofthesesystemsareknowntoimpactsignicantlythedevelopmentofnewtissueinvitroandinvivo[1e4].
Scaf-foldsprovidestructuralsupportandimportantenvironmentalcuestocellsthatpopulatethem,thuscontrollingtoalargeextentcellularproliferation[1,5],differentiation[6e8],depo-sitionofnewstructuralproteins[9,10]andultimatelytheregenerationoffunctionaltissue[4,11,12].
Traditionally,methodssuchasNMR,FTIRandX-rayspectroscopyhavebeenusedtoassessbiomaterialsatthemolecularlevel[13e17],whileSEMandTEMhavebeeninvaluabletoolsforchar-acterizingthethree-dimensionalmorphologyofbiomaterialscaffolds[5,18e23].
Histologyandimmunostainingaswellasassaysfordeterminingtheexpressionlevelsofspecicpro-teinsareoftenusedtoassesshowscaffoldsinteractwithcellsastissuesdevelop[5,10,18,20,22e24].
Whilealloftheseap-proachesprovidesensitiveandspecicdata,theyareinvasive.
Asaresult,theyrevealinformationaboutasingletime-pointalongthedevelopmentofadynamicallychangingspecimen.
Thislimitationhindersfullcharacterizationandunderstandingoftherelationshipsthatexistbetweenthestructural,mechan-ical,architecturalandbiochemicalpropertiesofthescaffoldandthecorrespondingpropertiesofthedevelopingtissue.
Anumberofopticalmethodshavebeendevelopedtomon-itornon-invasivelydifferenttissuecomponentsinthecontextofdiseasediagnosisandmonitoring[25e29].
Suchmethods*Correspondingauthor.
Fax:16176273231.
E-mailaddress:irene.
georgakoudi@tufts.
edu(I.
Georgakoudi).
0142-9612/$-seefrontmatter2008ElsevierLtd.
Allrightsreserved.
doi:10.
1016/j.
biomaterials.
2007.
12.
049Availableonlineatwww.
sciencedirect.
comBiomaterials29(2008)2015e2024www.
elsevier.
com/locate/biomaterialshaveonlyrecentlystartedtobeexploitedastoolsforassessingdifferentpropertiesofthecellandmatrixcomponentsofengineeredtissues[30e33].
Thegoalofthisstudywastode-terminethetypeofmorphologicalandstructuralinformationthatcouldbeacquiredaboutsilk-basedbiomaterialscaffoldsusingspectraltwophotonexciteduorescence(TPEF)andsecondharmonicgeneration(SHG)imaging.
Thisinformationwillbeessentialindevelopinganopticalbiomarkertoolkitthatwillallowustomonitordynamicallyhowsuchscaffoldsinteractwithandaremodiedbycellsasengineeredtissuesgroweitherinvitroorinvivo.
Furthermore,theseopticaltoolkitscanalsobeextendedtootherbiomaterialmatricesasthesystemsaredevelopedandoptimized.
Silkisanaturalproteinpolymervaluedforitsbiocompat-ibility,lightweight,andstrength[34].
Processingmethodsforthispolymerarewellestablishedforcontrolofmorphology,mechanicalpropertiesandenvironmentalstability[35e38].
Duetotheseproperties,silkisanexcellentcandidateforgen-eratingbiomaterialscaffoldsforengineeredtissues.
TheBombyxmorisilkwormsilkprotein,broin,canbedescribedbytwostructuralmodels:SilkI,consistingoftypeIIbturn,randomcoildomains,andmixedstructuresincludingalphahelices,andSilkII,consistingmostlyofantiparallelbpleatedsheets[14,15].
Thebsheetcontentandthealignmentofthesebsheetcrystals,alongwiththenon-crystallinedomainsoftheprotein,areimportantdeterminantsofthebulkmechanicalpropertiesanddegradationkineticsofbiomaterialsgeneratedfromsilk[14,36,37,39e41].
Mostofthebsheetcontentandorientationofthesecrystallinedomainsislostduringthepro-cessingofsilkbroinintoaqueoussolutions,asteprequiredfortheregenerationofnewbiomaterialscaffoldsfortissueculture[42].
Thebsheetcontentandorientationcanberecon-stitutedtodifferentextentsdependingonthemodeofmaterialpreparation[14,38,42].
Thus,thenon-invasive,opticalassess-mentofbsheetcontentandorientationofsilkbroinduringbiomaterialscaffoldformationwasoneofthespecicgoalsofthisstudy.
Linearopticalapproaches,suchasuorescenceandRamanspectroscopyhavebeenusedpreviouslytocharacterizemate-rialsmadefromsilkbroin[39,43,44].
However,therearenostudiestoourknowledgeonthenon-linearopticalpropertiesofsilk.
Non-linearopticalmethodssuchasTPEFandSHGofferadditionaladvantagesfornon-invasiveimaging,includingexcitationinthenearinfraredregionofthespectrum,wherescatteringistypicallylowerthaninthevisibleregion,andreducedphotobleaching[45].
InTPEFandSHG,twophotons,typicallyofthesameenergy,interactsimultaneouslywithamoleculeandyieldeitheruorescenceemission(TPEF)orscattering(SHG)ofasinglephoton.
InthecaseofSHG,thescatteredphotonhasthesameenergyasthecollectiveenergyofthetwoincidentphotons(i.
e.
thereisnonetenergylossandthewavelengthofthescatteredphotonisatexactlyhalfthewavelengthofeachoneoftheincidentphotons).
InthecaseofTPEF,thewavelengthoftheincidentphotonsisapproxi-matelytwiceaslongasthewavelengthofaphotonrequiredforlinearexcitation,whiletheuorescentlyemittedphotonshavenearlyidenticalspectralfeaturesasthoseresultingfromsinglephotonexcitation.
Becausetheprobabilityofsi-multaneousinteractionwithtwophotonsisordersofmagni-tudelowerthansinglephotoninteractions,TPEFandSHGprocessesrequirethepresenceofhighphotondensities.
Asaresult,theseeventsareconnedwithinasmallvolumeintheapexofafocusedconeoflightandautomaticallyyieldopticallysectioned,depth-resolvedimages.
Theconnementoftheopticaleffect,andtheuseoflowenergy(longerwave-length)photonsresultsinreducedthermalandphotodamagewithinandoutsidetheplaneoffocus[45].
Exploitingsuchprocessesinmicroscopicimagingplatformsallowsfrequentsampleassessmentoverlongperiodsoftime,withoutdamageorcontaminationfromelementsoutsidethetissuecultureenvironment.
2.
MaterialsandmethodsAllchemicalswereobtainedfromSigmaAldrich(St.
Louis,MO)unlessotherwisenoted.
2.
1.
PreparationofsilkbroinCocoonsofB.
moriwerekindlyprovidedbyM.
Tsukada(InstituteofSericulture,Tsukuba,Japan).
Thecocoonswereinitiallyprocessedtoremovetheglue-likesericinproteinsandtoextractthepuresilkbroincomponentaspreviouslydescribed[39].
Theresultingaqueoussilkbroinsolutionhadanapproximateconcentrationof8%(wt)silkbroin.
Forhexauoro-2-propanol(HFIP)silkbroinsolutions,aqueoussilkwaslyophilizedandredissolvedinHFIPresultingina6%(w/v)broinsolution.
2.
2.
PreparationoflmsandgelsSilkbroinlmsweremadebypipettingvolumesofaqueousorHFIPsilksolutionsonto35mmglassbottomdishes(MatTek,ActonMA)andallowingthesolventtoevaporate.
Gelsweremadefromaqueoussolutionsin35mmglassbottomdishesatsilkconcentrationsof8and4%with6%(vol)0.
1MHCl.
Thesilksolutionswereplacedonthedishesrstandtheacidwastitratedintothesolutionforanalvolumeof2.
5mL.
Disheswerethensealedwithparafnlmandplacedat37Cfor72h[42].
bsheetcontentwasinducedinsilkbroinlmsbyimmersioninmethanolfor30min.
Filmswerecom-pressedandstretchedbyplacinglmsinopposingclamps,applyingpressuretosecurethelmandthendrawingtheclampsapart.
2.
3.
PreparationofscaffoldsThree-dimensionalsilkscaffoldswerepreparedaspreviouslydescribed[39].
Thescaffoldswerecutintodiscs(5mmindiameterand3mminthick-ness)driedat60C,andautoclavedforfurtherexperiments.
Theporesizeofthescaffoldswas55030mm.
Humanmesenchymalstemcells(Cambrex,EastRutherfordNJ)wereseededontoanaqueoussilkscaffoldasdescribedbyKimetal.
[18].
Theseededscaffoldswereplacedonglassbottomdishesandimagedonday21.
2.
4.
TwophotonexciteduorescenceandsecondharmonicgenerationmicroscopyTPEFandSHGmicrographswereacquiredonaLeicaDMIRE2micro-scopewithaTCSSP2scanner(Wetzlar,Germany).
Thesystemwasequippedwitha10(NA0.
3)dry,20(NA0.
7)dryanda63(NA1.
2)waterimmer-sionobjective.
TheexcitationlightsourcewasaMaiTaitunable(710e920nm)titaniumsapphirelaseremitting100fspulsesat80MHz(SpectraPhysics,MountainViewCA).
Sampleswereplacedonculturedisheswithnumber1.
5coverglassbottoms(MATTEK,AshlandMA)andexcitedat2016W.
L.
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