Bio-inspired Materials for Biomedical Engineering || Color Plates

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  • FIGURE 1.3 3-DphotopatterningofEGFwithinahyaluronicacidPEGhydro-gel. (A) Creation of a linear immobilized gradient of EGF. From the top of thehydrogel,thenumberofscansbythemultiphotonlaserareincreasedasitpenetratesintothesample,correspondingtoanincreaseinfluorescenceintensity,andhence,anincreaseinproteinimmobilization.(B)Theconcentrationofimmobilizedproteininthegradientwasquantifiedbyfluorescenceintensity,showingachangeincon-centrationfrom25nMatthetopofthehydrogelto250nMatadepthof150mminthe hydrogel. EGF, epidermal growth factor. (Reproduced with permission fromOwen,S.C.,Fisher,S.A.,Tam,R.Y.,Nimmo,C.M.,Shoichet,M.S.Langmuir2013.Copyright2013AmericanChemicalSociety.)

    A BEGF gradient column

    0 100 200Depth (m)

    200 m

    50 m

    250

    200

    150

    100

    50

    0

    EG

    F c

    once

    ntra

    tion

    (nM

    )

    Phase

    -catenin

    20 m

    20 m

    4 integrinDAPI

    PhalloidinBMDAPI

    150 400 675

    Elastic modulus (Pa)

    1050 > 5000

    FIGURE 2.4 MCF10Aacinipolarizationrespondstoextracellularmatrixstiffnessandisdisturbedonbeingculturedonincreasedhydrogelstiffness.(ReproducedwithpermissionfromReference35.)

    Bio-inspired Materials for Biomedical Engineering,FirstEdition.EditedbyAnthonyB.BrennanandChelseaM.Kirschner.2014JohnWiley&Sons,Inc.Published2014byJohnWiley&Sons,Inc.

  • FIGURE 3.2 Effectsofabnormalmuscleforceonskeletogenesisinmousemodels.Redindicateseffectonrudimentorjointduetoabnormalmuscle,greenindicatesnoeffect,stripedredandgreenindicatesfindingsofaffectedandunaffectedaspects,andwhiteindicatesnodataavailable.(ReproducedwithpermissionfromReference69.)

    FIGURE 2.5 Immunofluorescent imagesofembryoniccardiomyocytesculturedondynamicthiolatedHAhydrogelsatdifferentdevelopmentalstages:premyofibrilstage(1),maturingmyofibrils(2),andmaturecardiomyocytes(3).(ReproducedwithpermissionfromReference42.)

    1 2 3

  • FIGURE 4.1 Molecularpathwaysmediatingmechanotransductionsignalinginacell.Inthispathway,mechanicalforcessuchas,stretching,hydrostaticpressure,andshearstressstimulatetheintegrinsonthecellmembraneviaextracellularmatrix.Inturn,thestimuliistransducedintothenucleusbyengagementofanchorageproteinstalin(tal),vinculin(vin),paxillin(pax),anda-actininandsignalingproteinsFAK,Src,andzyxin(zyx).

    Adhered cell

    Fibronectin

    Strain

    Integrin

    Vin

    Tal Zyx

    Pax FAK Src

    Nucleus

    Pressure

    Extracellularmatrix

    Baran E.T.

    -actinin

    FIGURE 5.1 (A)InvivocellsreceivebiochemicalandbiophysicalcuesthroughinteractionswiththeECM,solublefactors,andneighboringcells.(Seetextforfullcaption.)

    A B

  • FIGURE 6.3 Assembly of FN matrix in hMSCs. Confluent layers of hMSCsassembleextensiveFNmatrices.(A)F-actinimmunofluorescence;(B)FNlabeledwithanticellularFNantibodyindicatesthatassembledFNwasexpressedinhMSCs;(C)Compositeimageofcellmatrixinteractions.

    A B C

    FIGURE6.4 AssemblyofFNmatrixonamicropillarscaffold.Immunofluores-cenceimagesofa layerofhumanmesenchymalstemcellsgrownonasurfaceofmicropillars for10days. (A)Fluorescently labeledpillars; (B)Actincytoskeleton(red)(highermagnificationshowninC);(D)Assembledfibronectinfibrils(highermagnificationshowninE);(F)Compositeimage.Notethatwhiletherearevisiblespacesbetweencellsintheactinimage,theyhaveformedacompletelayerofECMacrossthetopsurfaceofthepillars.Scalebaris50mm.

    A

    D E F

    B C

  • FIGURE9.3 A 65-year-oldwoman treatedwith resection and application of adermalregenerationtemplate.(Seetextforfullcaption.)

    FIGURE9.1 Applicationofallogenicproducttolegwound.(PhotoscourtesyofLaurenR.Bayer,PA-C.)

  • FIGURE10.3 Engineeringapproachesthatmimicepithelialmorphogenesis.(A)Micropatternedadhesivesubstratumforinvestigatingepithelialsheetmigration.(B)MimickingwoundedepitheliumusingPDMSpillars.(C)Switchablesubstratumforexpansionofepithelialsheets.(D)3-Dprintingforconstructingabiologicaltube.(E,F)3-Dmicropatternedtubesforinvestigatingbranchingmorphogenesis.(AdaptedfromReferences55,57,59,71,and72.)

    A B

    C D

    E

    a

    a

    a

    Stamp

    0.5 0 1

    2 3 4 h

    Liquidcollagen

    Gelledcollagen

    Gelledcollagen

    Tubule0

    100%

    Cells

    Cell aggregates

    Migrating sheet

    PDMS pillarPDMS stamp

    UV flood exposure

    Remove Wound closure

    37C

    50

    b c d e

    b c

    b

    F

    A B

    E

    E

    DD

    50m50m

    FIGURE 11.4 H&E-stained cross-sections of bilayer skin tissues composed ofepidermal(E)anddermal(D)layersandformedbyculturingL-b-Lassembledcell/fiberconstructsfor3days(A)and7days(B).GreenbrokenlineoutlinestheborderbetweenEandD.(ReprintedfromReference98,withpermissionfromMaryAnnLiebert,Inc.Publishers.)

  • 400

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    B

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    CIRCLONG

    2.0

    0 0.05 0.10

    Str

    ess

    (kP

    a)S

    tres

    s (k

    Pa)

    Strain

    0.15 0.20

    LONG

    FIGURE12.3 Mechanicalandstructuralcharacteristicsofthemyocardium.(Seetextforfullcaption.)

  • FIGURE12.4 Thecardiacconductionsystem.(Seetextforfullcaption.)

    FIGURE12.5 Thewholevasculatureofanadultratheartwasreconstructed(topleft) frommicro-CT data (top right).Transverse sections obtained in four planes(below) show the penetrating network of capillaries. The color of the renderedvesselscorrespondstotheintensityofthevoxelsintheoriginaldataset.(ReprintedwithpermissionfromReference142.)

    1

    2

    3

    4

    4321

  • AB D

    9090

    90

    T

    10 m T90

    C

    FIGURE12.9 Alternativestoelectrospinningtocreatemicro-andnanofiberscaf-folds.Rotary jet spinning uses a high-speed rotating spindle to drawfibers fromsyntheticandnaturalmaterials.(A)SEMimagesofgelatinfibersshowahighdegreeofalignment.Surface-initiatedassembly isa technique thatmimicscell-mediatedassembly and provides control of the scaffold nano- tomacroscale structure andcomposition.(B)Schematic(left)andopticalphaseimage(right)oftwopatternsof20-mmwidthby20-mmspacingfibronectinlinesmicrocontact-printedorthogonallyontoPIPAAm.Aftersometime(T)incoolingwater,themeshtermednanofabricisreleasedandmaintainsitsshape.(C)Thesamepatternwascreatedwithfibronectin(green) and laminin (red) and was released as a bicomponent nanofabric (right)showingthatSIAcanbeusedtocontrolthearchitectureandcompositionofbiomi-meticECMnanofabrics.(D)Three-dimensional,false-coloredrenderingofafibro-nectin mesh with 20-mm wide elliptical holes observed by scanning electronmicroscopy.Thenanofabric showsfishnet-like ripples.Scalebars are40mm inBandCand100mmforthereleasedbicomponentnanofabrics.X,Yaxesare360mminD.(AdaptedwithpermissionfromReferences77and78.)

    FIGURE12.10 Examplesofhydrogelsforcardiactissueengineering.MyocardialECMgelscanbeobtainedbydecellularization,lyophilization,andenzymaticdiges-tion.(A)ThesolubilizedECMcomponentsgelunderphysiologicalconditionsintofibrousmulticomponenthydrogelswithECM-likestructurerevealedbySEM.Scalebaris1mm.(B)Fibrinmatrigelhydrogelscastaroundanarrayofmicropillarsareremodeledbymyocytestoformacontractilecardiacconstructwithlocalanisotropy.Scale bars are 500mm(top) and200mm(bottom). (C)Synthetic polypeptides aredesignedtoself-assembleintonanofibroushydrogelsandtomimicVEGFtoinduceangiogenesis.(AdaptedwithpermissionfromReferences89,94,and100.)

  • Cell sheets stacked orthogonallyUpper layer

    Lower layer

    Y (m

    )

    Y (m

    )

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    m)X (m)

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    X (m)

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    FIGURE12.12 Cellsheetengineeringisascaffold-freeapproachtocardiactissueengineering.HumanfibroblastswereseededonananisotropicPIPAAmlayer.Afterrelease,thecellsheetsproducedtheirownanisotropicECM,revealedbyobservationof highly aligned collagen type I fibers (green, left).Aligned cell sheets can bestackedatdifferentanglestocreatemultilayerconstructs(right)withF-actin(red)andnuclei (blue).Scalebar is100mm.(Adaptedwithpermission fromReference110.)

    PDMS stamp

    PDMS stamp

    A B C

    Inkingwith

    adsorbedprotein

    Substrate

    pattern cells

    100 m

    50 m

    Capture molecules

    Target molecules Inked stamp

    A C

    E

    B inking of the stamp

    D Microcontact printing

    Microcurray of printed proteins

    Flow cell or biomoleculesuspension

    Dispense cell orbiomolecule suspension

    -Stamp

    FIGURE13.4 Schematicofsoftlithographytechniquesusedtocreatemicropat-terning on substrates using (A) blocking methods and solution dispensing withmicrochannels or stencils, (B) microcontact printing of adsorbed proteins usingconformalcontact,and(C)affinitycontactprintingusingimmobilizedligandsforconformalcontactprintingoftargetbiomolecules.(PanelAadaptedfromParkandShuler[136];panelBadaptedfromWilliamset al.[143,258];panelCfromRenaultet al.[141].)

  • FIGURE14.2 Underultravioletlight,thebonesofthesetransgenicmousepupsfluoresce green, indicating the successful incorporation of both theGFP reportergeneandthelinkedfunctionalgeneofinterest.(CourtesyofProfessorDavidRoweoftheUniversityofConnecticutHealthCenter.)

    FIGURE14.4 Fluorescencemicroscopy imagesof culturesexpressing3.6Col/GFPassociatedwithpreosteoblastsjustpriortomineralization,andnontoxicxylenolorange(XO)stainingofmineraltakenfromthesameareainthecellcultureplateatmultipletimepoints.Thecellreportertechnologyallowscontinuousmonitoringof cell differentiationwithout requiring the use of dyes or antibody staining thatrequirecellculturetermination.(CourtesyofYu-HsiungWangoftheUniversityofConnecticutHealthCenter.)

  • FIGURE 14.7 Fluorescencemicroscopy images of the calvarial cells from thetransgenicmiceonTCPSandcHAat21dayswithDAPIstainingtoshowallcells(A,D),osteoblastsrevealedbyGFPexpression(B,E),andXOstainingfordepositedmineral(C,F).Scalebar=100mm.(ReprintedwithpermissionfromReference10.)

    14 days

    GFP XO

    FC

    TC

    PS

    NF

    C

    FIGURE 14.10 Fluorescence microscopy images of GFP positive (green) andxylenolorangestaining(red)ofmineralizedmatrixafter14and21daysinculture.Scalebars=2mm.

  • FIGURE 14.12 Merged fluorescence images showing colocalization of GFPexpressionandXOstaininginculturesgrownonnonfibrillarcollagen(NFC).Theinset imageofXOstaining shows isolated islandsofmineralnot associatedwithGFPpositivedifferentiatedcells.

    FIGURE15.3 Picrosirius redstained free-floatingfibroblastpopulatedcollagenlatticeundercircularlypolarizedlighttoshowbirefringentcollagen.Centralregion(left) showsdense, randomlyoriented collagenfiberswhile theouter edgeof thelattice (right) shows alignedfibers for a lattice that has reached steady state (seeFigure15.2).Scalebar=50mm.

  • FIGURE16.1 ConceptsoftheHSCniche.(A)SchemeofHSCregulationinsidethenichemicroenvironmentdepicting thedifferentHSC fatedecisionswhichareorchestratedby thenichecomponents. (B)Schemeof thedifferentmicroenviron-mentalcuescontrollingHSCfateincludingbiochemical,biophysical,andmetabolicsignals.

  • FIGURE 16.3 Protein immobilization on a microstructured surface for HSCculture.(A)Poly(dimethylsiloxane)(PDMS)microstructuredwithoxygenplasmaactivationarecoatedbyaminosilanefunctionalizationandmaleicanhydridecopo-lymercoating to immobilizecomponentsof theECM. (B)Fluorescent imagesofECM-modifiedPDMSmicrostructures.

    FIGURE17.1 Summaryofpathwaysforrecognitionofallograftbytheadaptiveimmunesystem.(Seetextforfullcaption.)

    T Cell receptor MHCI

    MHCIIAlloantigens

    T cell activationto effector cells

    Allograft cell surface

    Antigen-presenting

    cell

    CD4+ T cell

    CD4+ T cell CD8+ T cell

  • T cell a

    ctivation

    to effe

    ctor cells

    T cell a

    ctivation

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    ctor cells

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    ctivation

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    ctor cells

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    Toleroge

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    leroge

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    ulatory ag

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    Eluted im

    mun

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    ulatory ag

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    Antigen

    -pres

    entin

    gce

    llAntigen

    -pres

    entin

    gce

    ll

    Antigen

    -pres

    entin

    gce

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    Antigen

    -pres

    entin

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    ll

    CD4+ T cell

    CD4+ T cell

    CD4+ T cell

    Enc

    apsu

    latin

    g Polym

    er

    Enc

    apsu

    latin

    g Polym

    er

    Allograft c

    ell surface

    Allograft c

    ell surface

    Allograft c

    ell s

    urface

    Allograft c

    ell s

    urface

    Immun

    omod

    ulatory

    bioa

    ctive motifs

    CD4+ T cell

    CD8+ T cell

    CD8+ T cell

    Helpe

    rce

    ll

    Helpe

    rce

    ll

    Helpe

    rce

    ll

    Drug eluting

    biom

    aterial

    FIG

    UR

    E1

    7.2

    Engineeringofim

    muneresponsetoallogeneictransplantscanbeconductedvia(A

    )polymericencapsulatio

    n,

    (B)cotransplantationwith

    protectivecells,(C)surfacefunctio

    nalizationoftransplantedbiom

    aterials,and(D

    )codeliv

    eryof

    solublefactors.(Seetextforfullcaption.)

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