Effects of synthetic micro- and nano-structured surfaces on cell behavior

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*Corresponding author. Tel.: 001 608 265 8171.Biomaterials 20 (1999) 573588Eects of synthetic micro- and nano-structured surfaceson cell behaviorR.G. Flemming!, C.J. Murphy!, G.A. Abrams!, S.L. Goodman", P.F. Nealey#,*!Department of Surgical Sciences, School of Veterinary Medicine, UW-Madison, 2015 Linden Drive West, Madison, WI 53706, USA"Center for Biomaterials MC-1615, University of Conn. Health Center, Farmington, CT 06030, USA#Department of Chemical Engineering, School of Engineering, UW-Madison, 1415 Engineering Drive, Madison, WI, USAReceived 10 June 1998; accepted 30 September 1998AbstractTopographical cues, independent of biochemistry, generated by the extracellular matrix may have signicant eects upon cellularbehavior. Studies have documented that substratum topography has direct eects on the ability of cells to orient themselves, migrate,and produce organized cytoskeletal arrangements. Basement membranes are composed of extracellular matrix proteins and foundthroughout the vertebrate body, serving as substrata for overlying cellular structures. The topography of basement membranes isa complex meshwork of pores, bers, ridges, and other features of nanometer sized dimensions. Synthetic surfaces with topographicalfeatures have been shown to inuence cell behavior. These facts lead to the hypothesis that the topography of the basement membraneplays an important role in regulating cellular behavior in a manner distinct from that of the chemistry of the basement membrane.This paper describes the topography of the basement membrane and reviews the fabrication of synthetic micro- and nano-structuredsurfaces and the eects of such textured surfaces on cell behavior. ( 1999 Elsevier Science Ltd. All rights reservedKeywords: Basement membranes; Cell behavior1. IntroductionFundamental knowledge of cellsubstrate interactionsis important for tissue engineering, in the development ofmedical implants, and the production of pharmaceu-ticals. Cellsubstrate interaction may also explain dier-ences in cell behavior in vivo and in vitro. To gain insightinto these interactions, a logical approach is to investi-gate the substrates on which cells attach and grow inliving systems.Basement membranes are found throughout the verte-brate body and serve as substrata for overlying cellularstructures. Basement membranes consist of extracellularmatrix (ECM) components, including brous collagen,hyaluronic acid, proteoglycans, laminin, and bronectin.The eects of the surface chemistry on cell and tissuefunction has been explored extensively in the past fewdecades. Hyaluronic acid, for example, has been shownto inhibit cellcell adhesion and promote cell migration,and laminin can prevent cell migration [1, 2]. Fibronec-tin has been observed to allow greater translocation ofcells than does laminin [3], and excess ECM has beenshown to inhibit endothelial cell replication by causingincreased cellECM adhesion and cytoskeletal re-arrangements [4]. One well-understood mechanism inwhich components of the basement membrane modulatecell behavior is the activation of plasma membrane integ-rin receptors, such as RGD that bind to ligands on thebasement membrane [57]. In addition to mediating cellattachment, integrin receptors also act as signaling mol-ecules, activating intracellular pathways important in cellgrowth and survival [811]. In the cornea, componentsof the basement membrane have been shown to inuencethe distribution of cytoskeletal elements and of their ownintegrin receptors [12, 13] as well as modulating prolifer-ation [14], migration [15, 16], and dierentiation [17].The mechanical and tensile properties of the basementmembrane also inuence fundamental cell behaviors.Cells can sense restraining forces and respond bystrengthening cytoskeletal linkages [18]. The strength ofthese integrincytoskeleton links depend on both the0142-9612/99/$ see front matter ( 1999 Elsevier Science Ltd. All rights reserved.PII: S 0 1 4 2 - 9 6 1 2 ( 9 8 ) 0 0 2 0 9 - 9Fig. 1. Scanning electron micrograph of a corneal epithelial basementmembrane of Macaque monkey. After the corneal epithelium wasstripped, the cornea was xed in 2% glutaraldehyde, dried by thecritical point method, and imaged on Hitachi S-900 scanning electronmicroscope (Bar"1 lm).matrix rigidity as well as its biochemical composition[18]. When cells bind to ligands on the basement mem-brane, the cell receptors can act as mechanochemicaltransducers, activating signal transduction pathwaysand modulating gene expression [19]. Fibroblasts at-tached to strained collagen matrix produced more tenas-cin and collagen XII than those attached to a morerelaxed matrix [19]. Resilience and deformability alsoinuences in vitro migration and morphology of somecells [3].In addition to biochemical and mechanical properties,basement membranes possess a complex, three-dimen-sional topography consisting of nanometer sized features.Physical topography is known to aect cell behavior. Forreviews on the subject of the inuence of substratumtopography on cells, see Curtis and Clark [20], vonRecum and van Kooten [21], Curtis and Wilkinson [22],Singhvi et al. [23], and Clark [24]. Paul Weiss, amongothers, pioneered the eld of contact guidance duringthe 1930s, 1940s and 1950s [25]. Rosenberg claimed, asearly as 1962 and 1963, that nanometer sized featuresinuenced cells [26, 27]. Despite recognition of the im-portance of substratum topography, relatively little isknown about the eects of topographical features ofnanometer scale on cell behavior. The purpose of thispaper is to: (1) describe the nanometer scale topographyof basement membranes, (2) review the techniques usedto fabricate synthetic micro- and nano-structured surfa-ces, and (3) discuss the eects of micro- and nano-struc-tured synthetic surfaces on cellular behavior.2. Topography of the basement membraneBasement membranes are comprised of a complexmixture of pores, ridges, and bers which have sizes in thenanometer range. A scanning electron micrographof a corneal epithelial basement membrane is shown inFig. 1. Abrams and coworkers used scanning electronmicroscopy, transmission electron microscopy, andatomic force microscopy to measure the sizes of featureson the surface of the corneal epithelial basement mem-brane of the Macaque monkey [28]. The average featureheights were between 147 and 191 nm and the averageber width was 77 nm [28, 29]. They found that poresmade up 15% of the total surface area of the membraneand had an average diameter of 72 nm [28]. Similarfeatures were observed on the human corneal epithelialbasement membrane [30] and on MatrigelTM, a commer-cially available basement membrane matrix [31].Shirato et al. studied the glomerular basement mem-brane of the rat kidney and observed a meshwork ofbrils 59 nm thick and pores 1130 nm wide on thelamina rara interna and 611 nm brils and 1024 nmpores on the lamina rara externa [32]. Yamasaki andcoworkers examined the pores and bers present inbovine glomerular and tubular basement membranes[33]. They found pore diameters near 10 nm in theglomerular membranes and pores near 12 nm in dia-meter in the tubular membranes. Fibrous strands wereseen to have widths of 315 nm. Another study byHironaka et al. found that the glomerular, tubular, andBowmans capsule basement membranes of the rat kid-ney all had mean ber diameters of 67 nm while havingmean pore diameters of 9.7, 14.1, and 13.1 nm, respective-ly [34].3. Fabrication of micro- and nano-structured surfacesA list of the fabrication strategies employed to createsynthetic substrates with topography is given in Table 1.The majority of the studies used photolithography toproduce features with controlled dimensions and specicshapes. Among the rst to employ photolithography tocreate controlled features for the study of cell behaviorwas Brunette et al. [35].A simplied schematic diagram of the photolitho-graphic process is shown in Fig. 2. A substrate is coatedwith a thin polymeric lm called a resist. The resist isexposed to light through a mask such that the lightirradiates only selected regions of the resist. A photo-chemical reaction in the irridiated regions renders thoseregions either more soluble or less soluble in a solventcalled a developer. Hence, immersion in the developeryields either a positive-tone image of the mask, or a574 R.G. Flemming et al. / Biomaterials 20 (1999) 573588Table1TheeectoftexturedsurfacesoncellbehaviorFeatureFabricationMaterialFeatureFeatureCelltypeCellulareectRef.typetechniquedimensionsfrequencystudiedGroovesPhotolithographyandreactiveionetching,UVandglowdischargetreatmentPDMScastofsiliconoriginalSquaregrooves2,5,10lmwidth0.5lmdepthEqualgrooveandridgewidthRatdermalbroblasts2,5lmgroovesinducedstrongerorientationthan10lmgrooves;growthloweronUVtreatedsurfacethanonglowdischargetreatedsurface[62]PhotolithographyandreactiveionetchingQuartz0.5,5,10,25lmwidth0.5,5lmdepthEqualgrooveandridgewidthMurineP388D1macrophageCellsspreadfasteronshallowgrooves,butelongatedfasterondeepergrooves;moreelongationonwidergrooves;orientationdependentondepthduringrst30min;60%moreF-actinincells,ingrooves;LPS-activationenhancedorientation[98]PhotolithographyandreactiveionetchingQuartzSquaregrooves0.984.01lmwidth1.121.17lmdepthGrooveandridgewidthsimilarMesenchymaltissuecellsCellsmigratedalonggrooves;cellsbecamehighlypolarized;highestalignmentonwidestrepeatspacing[97]Photolithographyandanisotropicetching,glowdischargeTitaniumcoatedsiliconV-shaped15lmwidth3lmdepthEqualgrooveandridgewidthPorcineepithelialcellsCellsorientedindirectionofgrooves;actinlamentsandmicrotubulesalignedalongwallsandedges;singlecellsshowedlessvariabilityofalignedcytoskeletalarrangementsthancellclusters;nosignicantellipticalmorphology[100]PhotolithographyandreactiveionetchingEpoxyreplicaofsiliconoriginalSquaregrooves0.5lmwidth1lmdepthEqualgrooveandridgewidthHumangingivalbroblastsCellsshowedstrongalignmenttotopography;cellsbridgedorconformedtofeatures[68]PhotolithographyandreactiveionetchingEpoxyreplicaofsiliconoriginalSquaregrooves0.5lmwidth1lmdepthEqualgrooveandridgewidthHumangingivalbroblastsCellsgrewmostlyinmonolayers;somecellsextendedprocessesintogrooves;innercornersofgroovesnotoccupiedbycellularprocesses;somecellsbridgedgrooves;cytoskeletalelementsorientedparalleltolongaxisofgrooves[69]PhotolithographyandanisotropicetchingTitaniumcoatedepoxyreplicaofsiliconoriginalSquareandV-shapedgrooves,30lmrepeatspacingwith3,10,or22lmdepthor7and39lmrepeatspacingwith3or10lmdepthRegularspacing,butunequalgrooveandridgewidthRatparietalimplantmodelgroovesorientedhorizontallyorverticallytolongaxisofimplantEndothelialcellattachmentobservedonsmoothand3,10lmgrooves;endothelialcellsbridged22lmhorizontalgrooves;broblastsencapsulatedsmoothand3,10lmhorizontalgrooves;broblastsinsertedobliquelyinto22lmhorizontalgrooves;epithelialdowngrowthgreatestonverticalandsmoothsurfacewhileleaston10,22lmhorizontalgrooves[60]PhotolithographyandanisotropicetchingTitaniumcoatedsilicon,epoxyreplicas,photo-resistSquareandV-shapedgrooves0.560lmdepthrepeatspacing30220lmRegularspacing,butgrooveandridgewidthnotlistedPorcineperiodontalligamentepithelialcellsCellsorientedbyallgrooves;highestorientationonsmallestrepeatspacing;somecellscrossedridgesordescendedintogrooves;groovesdirectedmigra-tionofcells;0.5lmdeepgrooveslesseectivethandeepergroovesatdirectingcells;cellobservedtohavelamellaeandliopodiabendingaroundedges[76](continuedonnextpage)R.G. Flemming et al. / Biomaterials 20 (1999) 573588 575Table1(continued)FeatureFabricationMaterialFeatureFeatureCelltypeCellulareectRef.typetechniquedimensionsfrequencystudiedPhotolithographyandreactiveionetchingQuartz5,10,25lmwidth0.5,1,2,5lmdepthSpacingnotlistedBHKcellsF-actincondensationsobservedattopographicdiscontinuities;condensationsoftenatrightanglestogrooveedgewithperiodicityof0.6lm;vinculinorganizationsimilartothatofactin;microtubulesobservedafter30min;colcemidincreasedspreadingandreducedorientationandelongation;cytochalasinDreducedspreading,elongation,andorientation;taxolreducedelongation[99]PhotolithographyandreactiveionetchingPolystyrenecastofsiliconoriginal0.5lmwidth0.5,5.0lmdepthRadialarrayofgrooves5lmlongat1intervalsSprague-DawleyratcalavarialcellsMultiplelayerproteinadsorptionfromserum;cellsgrewtoconuencein4daysandproducedECMafter7days[61]CuttingwithdiamondortungstenPolystyrene,epoxyreplicas2,10lmwidthdepthnotlisted530lmrepeatspacingChickheartbroblasts,murineepithelialcells75%ofcellsalignedon5lmgrooves;60%ofcellsalignedon30lmgrooves;cytoplasmicextensionsnotrelatedtosurfacefeatures;alignmentofcellsnotguidedbylamellaeorlopodia;cellsbridged2and10lmgrooveswithouttouchingsurface[70]PhotolithographyandanisotropicetchingSilicondioxide0.5lmwidth1lmdepthEqualgrooveandridgewidthHumanbroblasts,gingivalkeratinocytes,neutrophils,monocytes,macrophages100%ofbroblastsand20%ofmacrophagesaligned;noorientationoralignmentobservedwithkeratinocytesorneutrophils;somemacrophagesextendedprocessesparalleltolongaxisofgroovesafter2h[101]PhotolithographyandanisotropicetchingTitaniumcoatedsilicon,epoxyreplicas,photoresistSquareandV-shapedmajorgrooves5120lmdeep(widthnotlisted),minorgrooves2lmdeeponoorat54tomajorgrooves580lmrepeatspacingHumangingivalbroblastsAlignmentobservedingroovesandonatridges;cellsorientedpreferentiallytomajorgrooves;minorgroovescausedorientationofcellsinabsenceofmajorgroovesorwhendiscontinuityexistedinmajorgroovepattern[59]PhotolithographyfollowedbyglowdischargePDMScastofsiliconoriginal2.0,5.0,10.0lmwidth0.5lmdepthEqualgrooveandridgewidthRatdermalbroblastsCellson2and5lmgrooveswereelongatedandalignedparalleltogrooves;cellson10lmgroovesweresimilartothoseonsmoothsubstrate[63]Photolithographyandanisotropicetching,glowdischargeTitanium-coatedsiliconV-shaped,3lmdepthwidthnotlisted610lmrepeatspacingHumangingivalbroblastsCellselongatedandorientedalonggrooves;cellheight1.5-foldgreaterongrooves;bronectinmRNAandsecretedbronectinincreasedincellsongrooves;GAPDmRNAnotaected;half-livesofbronectinmRNAaltered;2-foldincreaseinbronectinassembledintoECM[95]PhotolithographyandreactiveionetchingQuartz1.658.96lmwidth0.69lmdepth3.032.0lmrepeatspacingChickheartbroblastsRidgewidthmoreimportantthangroovewidthindeterminingcellalignment;alignmentofcellsinverselyproportionaltoridgewidth[96]576 R.G. Flemming et al. / Biomaterials 20 (1999) 573588Photolithography,anisotropicetchingTi-coatedsiliconV-shapedgrooves70,130,165lmwidth80,140,175lmrepeatspacingHumangingivalcells,porcineepithelialcellsCellsfromsuspensionalignedtolongaxisofgrooves;epithelialcellsdidnotbendaroundridgesbetweencells;groovescausedalignmentofmigrationofexplantedcells;multilayeringofepithelialcellswithinandalonggrooves[35]PhotolithographyandanisotropicetchingEpoxyreplicaofsiliconoriginalV-shapedgrooves17lmwidth10lmdepth22lmridgewidthPorcineperiodontalligamentepithelialcells,ratparietalimplantmodelEpithelialcellsattachedtogroovedsurfacesmorethantosmoothsurfacesandwereorientedbygrooves;shorterlengthepithelialattachmentandlongerconnectivetissueattachmentingroovedpartsofimplantcomparedtosmoothparts;groovesimpededepithelialdowngrowthonimplants[72]PhotolithographyandanisotropicetchingTi-coatedsiliconV-shapedgrooves3lmdepth610lmrepeatspacingHumangingivalbroblastsCellsorientedalonggroovesby16h;cellsongroovesshowedalteredmatrixmetalloproteinase-2mRNAtime-courseexpressionandlevelscomparedtocellsonsmoothTiortissuecultureplastic[102]PhotolithographyandanisotropicetchingTi-coatedsiliconV-shaped15lmwidth3lmdepthGrooveandridgewidthequalHumangingivalbroblastsMicrotubulesweretherstelementtobecomealigned;microtubulesalignedatbottomofgroovesafter20min;actinobservedrstatwall-ridgeedgesafter4060min;after3hamajorityofcellsexhibitedalignedfocalcontacts[103]Photolithographyandreactiveionetching,glowdischargetreatmentPDMScastofsiliconoriginal2,5,10lmwidth0.5lmdepthGrooveandridgewidthequalRatdermalbroblastsMicrolamentsandvinculinaggregatesorientedalong2lmgroovesafter1,3,5,and7days,butwaslessorientedon5and10lmgrooves;vinculinlocatedprimarilyonsurfaceridges;bovineandendo-geneousbronectinandvitronectinwereorientedalonggrooves;groove-spanninglamentsalsoobserved[73]Electron-beamlithographyandwetetching,glowdischargetreatmentPDMScastofsiliconoriginalSquaregrooves1lmwidth1lmdepthGroovesseparatedby4lm-wideridgesHumangingivalbroblastsVinculin-positiveattachmentsitesobserved;cellsalignedtogroovesinPDMS,whichhadbeenmadehydrophilicbyglowdischargetreatment;focaladhesioncontactsalsoalignedtogrooves[56]PhotolithographyandwetetchingPDMScastofsiliconoriginal2,5,10lmwidth0.5lmdepthGrooveandridgewidthequalHumanskinbroblastsCellsonsmoothPDMSenteredSphaseofcellcyclesfasterthancellsontexturedPDMS;cellson10lmtextureproliferatedlessthanthoseon2and5lmtextures[74]CuttingwithdiamondSerum-coatedglass2lmwidth2lmdepthSpacingnotlistedHumanneutrophilleukocytesWhencellsmovingacrossplaneofglassencoun-teredagroove,theywerehighlylikelytomigratealonggrooveratherthancrossit[104]Photolithographyandreactiveionetching,glowdischargetreatmentPDMScastofsiliconoxideoriginalSquaregrooves1.010.0lmwidth0.45,1.00lmdepth1.010.0lmridgewidthRatdermalbroblastsCellsorientedandelongatedalonggrooveswithridgewidths4.0lmorless;protrusionscontactingridgesobservedonorientedcells;cellsrandomlyorientedandweremorecircularongrooveswithridgesmorethan4.0lmwide;groovewidthanddepthdidnotaectcellsize,shape,ororientation[64](continuedonnextpage)R.G. Flemming et al. / Biomaterials 20 (1999) 573588 577Table1(continued)FeatureFabricationMaterialFeatureFeatureCelltypeCellulareectRef.typetechniquedimensionsfrequencystudiedPhotolithographyandreactiveionetchingQuartzandprotein-coatedquartzSquaregrooves2,10lmwidth30282nmdepthEqualgrooveandridgewidthP388D1macrophages,ratperitonealmacrophagesSpreadareaofcellson282nmdeepgrooveswastwicethatofcellsonplainsubstrate;degreeoforientationofcellsincreasedwithincreasingdepthanddecreasingwidthofgrooves;cellsongrooveshadincreasednumberofprotrusionsextendingperpendiculartogrooves;groovescausedincreaseinF-actin;F-actinandvinculinaccumulatedalonggroove/ridgeboundaries;cellsongroovesshowedhigherphagocyticactivity[80]PhotolithographyandreactiveionetchingPMMA2,3,6,12lmwidth0.2,0.56,1.10,1.9lmdepthEqualgrooveandridgewidthBHKcells,MDCKcells,chickembryocerebralneuronAlignmentofBHKcellsincreasedwithdepthbutdecreasedwithincreasingwidth;widthhadnoeectonMDCKcells;alignmentofMDCKcellsincreasedwithdepth;responseofMDCKcellsdependedonwhetherornotcellswereisolated;alignmentofchickembryocerebralneuronsalsoincreasedwithdepth[78]LaserholographictechniqueusedtodenemasksforX-raylithographyandreactiveionetchingQuartzandpoly- L-lysine-coatedquartz130nmwidth100,210,400nmdepthEqualgrooveandridgewidthBHK,MDCK,chickembryocerebralneuronsBHKcellsalignedonallgroovepatterns,butdegreeofalignmentincreasedwithincreasingdepth;MDCKalignedandelongatedtogrooves,butonlyelongationincreasedwithdepth;MDCKcellsingroupsandchickembryocerebralneuronsnotaectedbygrooves[79]LaserholographictechniqueusedtodenemasksforX-raylithographyandreactiveionetchingPoly-D-lysine-coatedchrome-platedquartz0.134.01lmwidth0.11.17lmdepth0.138.0lmspacingRatopticnerveoligodendrocytes,opticnerveastrocytes,hippocampalcerebellarneuronsOligodendrocyteswerehighlyalignedbyfeaturesassmallas100nmdepthand260nmrepeatspacing:astrocyteswerealsoalignedwhilehippocampalandcerebellarneuroncellswerenot;oligodendrocytesshowedlittlehigh-orderF-actinnetworks;alignedastrocytesshowedextensivearrangementofactinstressbers;maximumoligodendrocytealignmentinducedbypatterncorrespondingtodiameterofaxonin7dayopticnerve[81]Electron-beamlithography,wetetching,andreactiveionetchingQuartz,poly-L-lysine-coatedquartzandpolystyrenereplicasSquaregrooves1,2,4lmwidth141100nmdepthSpacingnotlistedEmbryonicXenopusspinalcordneurons,rathippocampalneuronsXenopusneuritesgrewparalleltoallgroovesizes;hippocampalneuritesgrewperpendiculartonarrow,shallowgroovesandparalleltowide,deepgrooves;Xenopusneuritesemergedfromsomaregionsparalleltogrooves;rathippocampalpresumptiveaxonsemergedperpendiculartogrooves,butpresumptivedendritesemergedparalleltogrooves;neuritesturnedtoaligntogrooves[58]Electron-beamlithography,wetetching,andreactiveionetchingQuartz,poly-L-lysine-coatedquartzandpolystyrenereplicasSquaregrooves1,2,4lmwidth141100nmdepthSpacingnotlistedEmbryonicXenopusspinalcordneurons,rathippocampalneuronsOrientationofXenopusandhippocampalneuriteswasunaectedbycytochalasinB,whicheliminatedlopodia;taxolandnocodazoledisruptedhippo-campalmicrotubules,butdidnotaectorientationorturningtowardgrooves;perpendicularalignmentof[57]578 R.G. Flemming et al. / Biomaterials 20 (1999) 573588hippocampalneuriteswasnotinhibitedbyseveralcalciumchannel,Gprotein,proteinkinaseandproteintyrosinekinaseinhibitors;somecalciumchannelandproteinkinaseinhibitorsdidinhibitalignmentGroovesandpitsPhotolithographyandanisotropicetchingTitanium-coatedepoxyreplicasofsiliconoriginalV-shapedgrooves35165lmwidth30,60,and120lmdepthV-shapedpits35270lmwidthand30,60,120lmdepthRepeatspacing40175lmforgroovesand40280lmforpits,unequalgrooveandridgewidthRatparietalboneimplantmodelMineralizationoccurredoftenongroovedorpittedsurfaces,butrarelyonsmoothcontrolsurfaces;frequencyofformationofbonelikefociincreaseddecreasedasgroovedepthincreased;frequencyofmineralizationincreasedasdepthofpitincreased;bonelikefociorientedalonglongaxisofgrooves[66]GroovesandchemicalpatternPhotolithographyandreactiveionetching,silanizationAmino-silaneandmethyl-silane-coatedquartzSquaregrooves2.5,6,12.5,25,50lmwidth0.1,0.5,1.0,3.0,6.0lmdepthGrooveandridgewidthequal,silanetracksequalBHKcellsCellsalignedmostto25lmaminosilanetracksand5lmwide,6lmdeepgrooves;stressbersandvinculinalignedwithadhesivetracksandgroovesandridges;alignmentincreasedwhenadhesivetracksandgroovesparallel;cellsalignedtoadhesivetrackswhichwereperpendiculartogrooves;F-actinorientedtobothadhesivecuesandtopographiccueswithinsamecellonthesubstrateswith3and6lmdepth;adhesivecuesdominant[86]Photolithographyanisotropicetching,polymermicromolding,treatmentwithalkanethiolsTi,Au-coatedpolyurethanetreatedwithbronectin,alkanethiolsV-shapedgrooves25,50lmwidthdepthnotlistedGrooveandridgewidthequalBovinecapillaryendothelialcellsCellsadheredtoregionscoatedwithbronectin,whichadsorbedtoregionssilanizedwithmethylbutnottri(ethyleneglycol)-terminatedsilanes;cellsattachedtoeithergroovesorridges,dependingonwhichpossessedthemethyl-terminatedsilaneandbronectincoatings[77]RidgesPhotolithographyandreactiveionetchingPolystyrenecastofsiliconoriginal0.5100.0lmwidth0.035.0lmheight0.562lmbetweenridgesromycesappendiculatusfungusMaximumcelldierentiationobservedforridgesorplateaus0.5lmhigh;ridgeshigherthan1.0lmorsmallerthan0.25lmwerenoteectivesignals;ridgespacingof0.56.7lmcausedhighdegreeoforientationofthefungus[65]EvaporativecoatingSiliconoxideonpolystyrene4lmwidth50nmheightRadialarrayMurineneuroblastomacellsCellsadheredtolinesandprocessesalignedalongthelines;processesgrewinbipolarmanner[90]StepsPhotolithographyandreactiveionetchingPMMA118lmstepsSpacingnotlistedBHKcells,chickembryonicneural,chickheartbroblast,rabbitneutrophilsCellsexhibiteddecreaseinfrequencyofcrossingstepsandincreasedalignmentatstepswithincreasingstepheightregardlessofdirectionofapproach;rabbitneutrophilsshowedtwicethecrossingfrequencyover5lmstepsasdidtheothercells;presenceofadhesivedierenceresultedindecreaseinfrequencyresultedindecreaseinfrequencyofascentonlyforstepheightsof1and3lm[105]WavesSolutionpolymerizationPDMSgelsofvaryingsoftnessSoftergelshadsmallerwaveswhilehardgelhadlargerwaves3,4,15lmperiodicityHumandermalbroblastsandkeratinocytesFibroblastsproliferatedequallyonallsubstrates;keratinocytesspreadmoreandsecretedmoreECMonsoftgelsthanonhardgel[111](continuedonnextpage)R.G. Flemming et al. / Biomaterials 20 (1999) 573588 579Table1(continued)FeatureFabricationMaterialFeatureFeatureCelltypeCellulareectRef.typetechniquedimensionsfrequencystudiedWellsandnodesPhotolithographyandetchingPDMSreplicasofsiliconoriginal2,5lmdiameterroundnodes,0.38and0.46lmhigh,respectively8lmroundwell,0.57lmdeep4,10,19lmcenter-to-centerspacingforthe2,5,8lmfeatures,respectivelyRabbitimplantmodelmurinemacrophages2and5lmtexturedimplantshadfewermononuclearcellsandthinnerbrouscapsulesthandidsmoothand8lmtexturedimplants;cellsonsmoothPDMSwereroundwithfewpseudopods,butcellson2and5lmtextureswereelongatedwithpseudopods[75]PhotolithographyandetchingPDMScastofsiliconoriginal2,5,8lmdiametervariablespacing2,5,10lmconstantspacingVariablespacingorconstantspacingof20.4lmMurineperitonealmacrophagesCellson5lmtextureshadsmallestdimensionswhilecellsonsmoothsiliconeandglasshadlargestdimensions;mitochondrialactivityhighestoncellson5and8lmvariablepitchsurfacesandonpolysty-rene;PMA-stimulatedcellsonsmallertextureswerelessactivethanunstimulatedcells[71]PhotolithographyandetchingPDMScastofsiliconoriginalSquarenodesorwells2,5,10lmdiameterDepthorheightof0.5lmATCChumanabdomenbroblastsCellson2and5lmnodesshowedincreasedrateofproliferationandincreasedcelldensitycomparedtocellson2and5lmwells;10lmnodesandwellsdidnotdierstatisticallyfromsmoothsurfaces[67]LasermodicationPolycarbonate,polyetherimideSquarenodes7,25,or50lmwidth0.5,1.5,2.5lmheightUniformsquarearrayHumanneutrophils,broblastsNoneofthetexturedsurfacessignicantlystimulatedneutrophilmovementcomparedtochemicalstimul-ators,althoughneutrophilmovementwasgreateronsomeofthetexturedsurfacesthanonanuntexturedsurface;noeectsonbroblastorientation,spreading,orelongation[106]PillarsandporesLaserablationusedinconjunctionwithmasksmadebyelectron-beamlithography,reactiveionetchingPMMA,PET,polystyreneCircularpillarsandpores1,5,10,50lmdiameterUniformarrayHumanosteoblastsandamnioticepithelialcellsCellsengulfedpillarsorstretchedbetweenadjacent1and5lmpillars;cellsattachedtoedgesofpores,especiallyon10lmpores;texturecausedincreaseincelladhesiononallmaterialsbutPMMA;greatestincreaseinadhesionwason50lmPETpillars;10lmporescaused5%increaseinresistancetoshearforce[107]PoresMicroporouslter:Nylondip-coatedwithPVC/PANcopolymerUncoatedandsiliconcoatedlters0.210lmdiameterdepthnotlistedSpacingnotlistedInvivocaninemodelNon-adherent,contractingcapsulesaroundimplantswithporessmallerthan0.5lm;implantswith1.41.9lmporesshowedadherentcapsulesbutnoinammatorycells;poresbiggerthan3.3lmwereinltratedwithinammatorytissue;pores12lmallowedforbroblastattachment[84]SpheresParticlesettlingPoly(NIPAM)particlesonpolystyrenesurface0.860.63lmdiameterwhentemperatureraisedfrom25to37C2Dhexagonallattice,0.96lmavg.distancebetweenspherecentersNeutrophil-likeinducedHL-60cellsCellslooselyadheredbutdidnotspreadonsphere-coatedsurfaceandcouldrolleasily;excessactiveoxygenreleasedwhentemperaturewasincreasedonsphere-coatedsurface,butnotonpoly(NIPAM)graftedsurface[108]CylindersFiber-opticlightconduit-fusedquartzcylindricalbersplacedonagarose-coveredcoverslipsFusedquartz1213or25lmradiiSpacingnotlistedPrimarymouseembryobroblastsandratepithelialcelllinesCellsinthepolarizationstageofspreadingwithstraightactinbundlesbecameelongated,orientedalongcylinder,andresistedbendingaroundcylinders;cellsintheradialstageofspreadingwithcircularactinbundlesorcellswithnoactinbundlestendedtobendaround[109]580 R.G. Flemming et al. / Biomaterials 20 (1999) 573588cylinderandexhibitedlesselongationandorientationtolongaxisofcylinderGeneralroughnessReactiveionetchingfollowedbyphotolithographyandisotropicwetetchingAreasofrougher,reactiveionetchedsiliconandsmoother,wetetchedsiliconReactiveionetchedfeatures:57nmavg.diameter,230nmheightwetetchedfeatures:115nmpeak-to-valleyroughness,depressions100250nminwidthReactiveionetchedfeatures:137/lm2surfacedensitywetetchedfeatures:27/lm2surfacedensityTransformedratastrocytes,primaryratcorticalastrocytesTransformedcellsattachedpreferentiallytowet-etchedregionsratherthanreactiveionetchedcolumnarstructures;transformedcellsonwet-etchedareasspreadinepithelial-likemannerandweresmooth;transformedcellsoncolumnarregionswererounded,looselyattached,andexhibitedcomplexsurfaceprojections;transformedcellspreferredareasexposedtoincreasingamountsofwetetching;primarycellspreferredcolumnarstructuresofreactiveionetchedareasanddidnotspreadonwetetchedareas[87]Acidwashing,electropolishing,sandblasting,plasma-sprayedTiTitanium12lmpits,1lmpits,10lmcraters1020globulesandsharpfeaturesof(0.1lmRandomMG63osteoblastElectropolishedsurfacehadmorecellswhileTI-plasma-sprayedhadlessthanTCPS;sandblastedsurfaceshadthesameasTCPS;thymidineincorporationinverselyrelatedtoroughness;proteoglycansynthesisdecreasedonallsurfaces;alkalinephosphataseproductiondecreasedwithincreasingroughnessexceptoncoarseblastedTi;correlationobservedbetweenroughnessandRNAandCDPproduction[83]Aluminaemulsionpolishing,grindingwithSiCpaperTi,Ti/Al/Valloy,TiTaalloy0.04,0.36,and1.36lmpeak-to-valleyheightsRandomHumangingivalbroblastsCellsalignedtogrindingmarks:10%ofcellsorientedonsurfacewith0.04lmroughness,60%on0.36lmroughness,and72%on1.36lmroughness[89]Electropolishing,sandblasting,acidetchingTitanium0.14,0.41and0.80lmpeak-to-valleyheightsforelectro-polished,etched,andsandblastedTi,respectivelyRandomHumangingivalbroblastsCellsonsmooth,electropolishedsurfacesshowedatmorphologyandgrewinlayers;cellsonetchedTimigratedalongirregulargrooves;cellsonsandblastedTigrewinclusters;roundandatcellsfoundonetchedandsandblastedTi;actinbundlesandvinculin-containingfocaladhesionsobservedinspreadingcellsonelectropolishedandetchedTi,butnotinspreadingcellsonsandblastedTi[88]SandblastingwithdierentgrainsizesandairpressuresPMMASandgrainsizesof50,125,and250lmproducedpeak-to-valleyheightsfrom0.07to3.34lmRandomChickembryovascularandcornealcellsSurfaceroughnesswashighestforsurfacessand-blastedwithlargestsizegrains;migrationareaofcellsincreased2-foldforvascularcellsand3-foldforcornealcellsonroughsurfacescomparedtosmooth;celladhesionincreasedwithsurfaceroughness[91]Industrialpolishing,sandblasting,plasma-sprayingwithTi-6Al-4VTitanium/alumi-nium/vanadiumalloySmooth,rough,porous-coatedsurfaces1001000lmporesonporous-coatedsurfacesRandomChickembryoniccalvarialosteoblastsCellsadheredtosurfaces,usingcellularprocessestobridgeunevenareas;ECMsynthesisandmineralizationwereenhancedonroughandporoustitaniumsurfaces[110]ScratchingwithglassrodPolystyreneandH2SO4-treatedpolystyreneDimensionsnotlistedRandomMurineperitonealmacrophagesbroblastsMacrophagesaccumulatedpreferentiallyonroughenedsurfaceswhilebroblastspreferredsmoothsurfaces[112](continuedonnextpage)R.G. Flemming et al. / Biomaterials 20 (1999) 573588 581Table1(continued)FeatureFabricationMaterialFeatureFeatureCelltypeCellulareectRef.typetechniquedimensionsfrequencystudiedPolymersolutioncastingNitro-cellulose,PVDFSmoothandroughsurfaces,featuresizenotlistedRandomRatsciaticnerveimplantmodelTissuestripsbridgednervestumpsinalloftheroughandinsomeofthesmoothnitrocelluloseandPVDFtubeimplants;bell-shapedtissueadheredtoroughtubeimplants;free-oatingnervecables,containingmyelinatedandunmyelinatedaxonsandSchwanncellsgroupedinmicrofasciclesandsurroundedbyanepineuriallayerobservedinsmoothtubes;macro-phagescomprisedinitialcelllayeronroughpolymers;epineuriallayerthinneronroughPVDFthanonroughnitrocellulose,smoothPVDFshowedmoremyelinatedaxonsthandidsmoothnitrocellulose[113]ProteintracksPhotolithographyfollowedbysilanizationandlaminincoatingQuartz,hydrophobicsilane,laminin2,3,6,12,25lmwidth,thicknessnotlistedFeatureandspacingequal,also2lmtracksseparatedby50lmChickembryoneurons,murinedorsalrootganglianeuronsSmallerspacingcauseddecreasedguidance;isolated2lmtracksstronglyguidedneuriteextensionwhile2lmrepeattracksdidnot;growthconesbridgednarrownon-adhesivetracks;growthconemorphologysimpleronnarrowersingletracks;growthconesspannedmanytracksonnarrowrepeats;neuritebranchingreducedon25lmtracks[94]PhotolithographyfollowedbysilanizationandlaminincoatingQuartz,hydrophobicsilane,laminin25lmwidth,thicknessnotlistedFeatureandspacingequalEmbryonicXenopuslaevisneuritesNeutritogenesisnotaected;65%ofneuritesalign-edtotracksafter5h;afteranorthogonallyopposed100140mV/mmDCeldwasapplied;majorityofcellsremainedaligned;somecellsrespondedtobothcues[93]FibronectincoatingGlasscoatedwithbronectin0.25lmwidthSpacingnotlistedBHKcells,rattendonbroblasts,ratdorsalrootgangliacells,P388D1macrophagesFibersincreasedspreadingandalignmentindirec-tionofber;actinalignedinbroblasts;alignmentoffocalcontactsinbroblastsandmacrophages;increasedpolymerizationofF-actin;bersincreasedspeedandpersistenceofcellmovementandrateofneuriteoutgrowth;macrophageshadactin-richmicrospikesandbecamepolarizedandmigratory[82]CoatingglasswithproteinandwithdrawingliquidtoorientproteinOrientedcollagenorbrinSizeofbersnotlistedSpacingofbersnotlistedHumanneutrophilleukocytesCellstendedtomoveindirectionofberaxisalignment;movementwasbi-directional;nochemotaxisevident[104]Micro-texturedsurfaceECMreplication-PMMApolymerizationcastingfollowedbypolyurethanesolutioncastingPolyurethanepositivecastofPMMAnegativeMicronandnanometerscaletopographySimilartoECMBovineaorticendothelialcellsCellsgrownonreplicasofECMspreadfasterandhadthree-dimensionalappearanceandspreadareasatconuencewhichappearedmorelikecellsintheirnativearteriesthancellsgrownonuntexturedcontrolsurfaces[85]Polymers:PMMA:poly(methylmethacrylate);PVC:poly(vinylchloride);PAN:poly(acrylonitrile);PDMS:poly(dimethylsiloxane);PET:poly(ethyleneterephthalate);NIPAM:N-isopropyl-acrylamide;PVDF:poly(vinylideneuoride);TCPS:tissueculturepolystyrene.Other:PMA:phorbol12-mystrate13-acetate;ECM:extracellularmatrix;BHK:babyhamsterkidney;MDCK:MadinDarbycaninekidney;GAPD:glyceraldehyde-3-dehydrogenase;LPS:lipopolysac-charide;CDP:collagenasedigestableprotein.582 R.G. Flemming et al. / Biomaterials 20 (1999) 573588Fig. 3. Schematic diagram of the pattern transfer process. Regions of the substrate not protected by resist are etched isotropically or anisotropically.Three examples of possible etching results are shown: anisotropic, reactive ion etching of silicon substrate to yield square grooves, anisotropic, wetetching of silicon [1 0 0] in KOH to yield V-shaped grooves, and isotropic, wet etching of glass in HF (that undercuts the resist). After the etching iscomplete, the remaining resist is removed.Fig. 2. Schematic diagram of the photolithographic process. A substra-te coated with a thin polymeric resist in irradiated through a mask.Depending on the type of resist, the irradiated regions become eithermore soluble or less soluble in a developer. Immersion in the developeryields either a positive-tone or negative-tone image of the mask.negative-tone image of the mask. Due to diraction lim-itations, the smallest lateral, or width, dimension that canbe achieved by photolithography without using phase-shift masks are of the same size scale as the wavelength ofthe light source used to make them. Current state-of-the-art photolithography employs UV light of wavelength248 nm and is able to achieve features with lateral dimen-sions of 220250 nm.The regions of the substrate not covered by resist canthen be etched isotropically or anisotropically, as shownin Fig. 3, to transfer the pattern in the resist to theunderlying substrate. A common topographical featureinvestigated by many researchers was grooves, structuresmade by etching the substrate using a photoresist pat-terned in the shape of lines. Depending on the type ofetchant used, grooves can be constructed to have crosssections of square wave, V-shape, or truncated V-shape[36, 37]. A few examples of dierent shapes of etchedstructures are depicted in Fig. 3. Repeat spacing is con-sidered the distance from the beginning of a feature to thebeginning of the next feature. A groove and its associatedridge would thus be considered one repeat unit. The etchdepth and the lateral, or width dimensions are alsoshown in Fig. 3. After the etching is complete, the remain-ing resist is removed. The nal product is a three-dimen-sional positive or negative relief image duplicating theopaque and transparent regions of the light mask.One problem encountered with some of the etchingprocesses is undercutting of the resist, yielding shapeswith dierent dimensions than the patterned resist andwith curved sides. In addition, some etchants produceroughened surfaces. These roughened surfaces sometimessuperimpose random nanometer sized features on top ofthe intended topography [38, 39]. The eects of roughen-ed surfaces on cell behavior have yet to be addressed inthe literature. For a more complete discussion of thelithographic and pattern transfer processes, the reader isdirected elsewhere [36, 37].Other techniques used to produce features with con-trolled dimensions include glancing angle deposition[40, 41], laser ablation [42, 43], laser deposition [44],replica molding of X-ray lithography masters [45, 46],imprint lithography [47, 48], microcontact printing andetching [49, 50], and ink-jet printing [51, 52]. Some ofthese techniques are capable of producing nanometerscale features. Electron-beam lithography, for example, isthe most developed, high-resolution lithographic tech-nique known, and has been used to fabricate featuresas small as 50 nm over large areas [5355]. Thesetechniques, for the most part, are not represented inTable 1 because no data has been reported in the literatureR.G. Flemming et al. / Biomaterials 20 (1999) 573588 583concerning the behavior of cells upon surfaces createdby these techniques. Cell studies have been conductedon surfaces created by electron-beam lithography, but inthese studies the lateral, or width dimension was micronscale [5658].In some of the studies summarized in Table 1, thematerial on which cells were cultured was quartz, silicondioxide, or titanium-coated silicon. In other studies poly-meric replicas of the textured surfaces were created bycasting or embossing techniques [5677]. For example,the fth entry in Table 1 lists the fabrication technique asphotolithography and reactive ion etching, and the mate-rial on which the cells were cultured as an epoxy replicaof the silicon original. This means that the original pat-tern was created in silicon, which was used to makea negative polymeric cast, which, in turn, was used tocreate a nal, positive replica. In some cases, such as inthe rst entry, the negative cast was simply used in thecell assays. If the entry lists a polymer cast in theMaterial column, then the cells were cultured on a nega-tive replica of the original surface. If the entry lists a poly-mer replica, then cells were cultured on a positive rep-lica. Polymers used for making such casts and replicasinclude PDMS, epoxy, and polystyrene. Other polymersused in studies include PMMA, PET, polycarbonate,polyetherimide, poly (NIPAM), nitrocellulose, and PVDF.Most of the studies to date address the eects ofsurface features with dimensions of 0.5 lm and greater.The relevance of these studies to topography found onbasement membranes is unclear since membrane topo-graphy is comprised of much smaller nanometer scalefeatures, as discussed in the previous section. A few inves-tigators have fabricated structures with control over atleast one dimensional less than 500 nm [57, 58, 64, 65, 75,7889]. These studies are highlighted in bold type inTable 1. In most of those studies, the lateral dimension ofthe features was greater than 0.5 lm, and the depthdimension was less than 0.5 lm. The depth dimension iseasily controlled in the etching process. Using reactiveion etching, Webb et al. [81] and Clark et al. [79]achieved grooves with depths of 100 nm. In anotherstudy, Clark et al. fabricated grooves with depths of200 nm, but with lateral dimensions of 212 lm on a sur-face of PMMA using photolithography (for patterningthe lateral dimensions) and reactive ion etching (to con-trol the etch depth) [78]. Wojciak-Stothard and cowork-ers used photolithography and reactive ion etching toproduce silicon grooves 210 lm in width and withdepths of 30282 nm [80]. Several series of polystyreneridges with heights of 30 nm and widths of 0.5100 lmwere made by Hoch et al. via electron-beam lithographyand reactive ion etching [65]. Grooves as shallows as14 nm and 1, 2, or 4 lm wide were achieved by Rajniceket al. [57, 58]. Britland and coworkers also used photoli-thography followed by reactive ion etching to producegrooves of 100 nm in depth, but 2.550 lm in width [86].Cooper et al. were able to create radial arrays of groovesonly 50 nm in height [90]. Schmidt and von Recumreport round nodes only 380460 nm in height [75].Only two studies were found in which cell behaviorwas investigated on surfaces with features of controlledshape and size, and lateral dimensions less than 0.5 lm.Webb et al. [81] and Clark et al. [79] both used a laserholographic method developed for fabrication of masksfor X-ray lithography to create patterns of grooves inquartz with widths of 130 nm and depths of 100400 and1001170 nm, respectively.Other methods have been used to fabricate surfaceswith nanoscale features, but with no direct control overthe topographical shapes and dimensions. Turner et al.used reactive ion etching to produce columnar structureswith average diameters of 57 nm [87]. Martin et al.sandblasted then acid-etched titanium surfaces to createne and coarse structures with sharp features (100 nmas well as larger pits and craters [83]. Kononen et al.,Lampin et al., and Eisenbarth et al. have used sandblast-ing [88, 91] and sanding [89], respectively, to producefeatures less than 0.5 lm in size. Polymer-coated micro-porous lters [84] and bronectin-coated glass [82] havealso been used as textured surfaces and have been foundto have features as small as 200 nm. Goodman et al.replicated the surface topography of extracellular matrixvia poly(methylmethacrylate) polymerization casting,which produced an inverse replica, followed by poly-urethane solution casting, which yielded the nal positivereplica [85]. The microtextured surface produced by thismethod contained micron and nanometer scale topo-graphical features. Sims and Albrecht have also achievedsome success in replicating the surface structure of bril-lar collagen, but interactions of cells with such surfaceshave not been reported [92].Protein tracks have been used as guidance cues forcells. Laminin tracks have been patterned by adsorptionto hydrophobic silane tracks, which had been patternedusing photolithography [93, 94]. In another study, bersof bronectin as small as 200 nm in diameter were ad-hered to glass [82].4. Eects of micro- and nano-structured surfaceson cell behaviorStudies of the interactions between substrate topogra-phy and cells have encompassed a wide variety of celltypes and substratum features including grooves, ridges,steps, pores, wells, nodes, and adsorbed protein bers.Table 1 summarizes the literature, providing a list offeature type, fabrication technique employed, substratummaterial, feature size and spacing, cell type studied, andthe cellular eect generated by the surface features.Grooves are the most common feature type employedin the study of the eects of surface structure on cells584 R.G. Flemming et al. / Biomaterials 20 (1999) 573588[35, 5664, 6870, 7274, 76, 7881, 90, 95104]. Typi-cally, the grooves are arrayed in regular, repeating pat-terns, often with equal groove and ridge width. The crosssections of the groove are often of the square wave,V-shape, or truncated V-shape [59, 80, 95].In general, investigations of grooved surfaces revealedthat cells aligned to the long axis of the grooves [35,5664, 6870, 7274, 76, 7881, 95104], often with or-ganization of actin and other cytoskeletal elements in anorientation parallel to the grooves [56, 69, 73, 80, 81, 99,100, 103]. The organization of cytoskeletal elements wasobserved to occur in some cases with actin and micro-tubules aligned along walls and edges [69, 98, 100]. Woj-ciak-Stothard et al. noted that F-actin condensationsappeared at topographic discontinuities, often at rightangles to groove edges [98]. They also observed thatmicrotubules formed after 30 min and that vinculin or-ganization was similar to that of actin. In addition, somecells were observed to have lamellae and lipodia be-nding around edges. Similar results were obtained byOakley and Brunette, who found that microtubules werethe rst element to align to grooves, followed by actin[103]. Many studies found that the depth of grooves wasmore important than their width in determining cellorientation [7880, 96]. Orientation often increased withincreasing depth, but decreased with increasing groovewidth. Repeat spacing also played a role, with orientationdecreasing at higher repeat spacing [76, 96]. In otherwords, as ridge width or groove width increased, theorientation phenomena of cells on grooves diminished.Dunn and Brown reported that ridge width is a moreimportant factor than groove width [96].Certain hierarchical eects have been encountered aswell. Brunnette exposed cells to a pattern of major andminor grooves, in which the minor grooves were placedon the oor of the major grooves at an angle of 54 to themajor grooves [59]. Brunnette found that the cellsoriented preferentially to the major grooves, althoughcells did align to the minor grooves when major grooveswere not present or when a discontinuity existed in thepattern of major grooves. Britland et al. investigated thebehavior of cells upon grooves that were overlaid withadhesive tracks of silane and observed that the cellsresponded to both the topographical and adhesive cues,but that the adhesive cues had the dominant eect[86].Although most cell types studied exhibited alignmentto grooves, some did not align. One study conducted byMeyle et al. found that, while 100% of broblasts alignedto grooves, alignment was observed in only 20% ofmacrophages and not at all in keratinocytes or neuro-phils [101]. Webb et al. noticed that oligodendrocytesand astrocytes were aligned by grooves, but that hippo-campal and cerebellar neuron cells were not aligned [81].In addition, although the oligodendrocytes were aligned,they exhibited little high-order F-actin organization. Onthe other hand, the aligned astrocytes showed extensiveorganization of actin stress bers. Rajnicek and McCraigobserved that Xenopus neurites grew parallel to grooves,but that rat hippocampal neurites grew perpendicular togrooves [57].When ridges are placed far apart, relative to cellulardimensions, they may be considered individually as stepsor clis which the cell must encounter and traverse. Onlya few studies are available in the literature that deal withthis type of substratum topography: the study by Hochet al., which investigated the eect of ridges on funguscells [65], and the study by Clark et al., in which severaldierent cell types were exposed to steps of varyingheights [105]. As might be expected, cell alignment in-creased with increasing step height.There are some studies in the literature investiga-ting the behavior of cells on other synthetic features,including wells and nodes [67, 71, 75, 106, 107], pores[84, 107], spheres [108], and cylinders [109]. Green andcoworkers found that nodes of 2 and 5 lm resulted inincreased cell proliferation compared to 10 lm nodes andsmooth surfaces [67]. Cambell and von Recum examinedthe eects of pore size and hydrophobicity in their studyinvolving a canine in vivo implant model [84]. They usednylon mesh coated with polyvinylchloride/polyacrylonit-rile with or without an additional silicone coating andfound that pore size played a larger role than materialhydrophobicity in determining tissue response, withpores of 12 lm allowing for direct broblast attachment[84]. Fujimoto et al. investigated the behavior of cells onspheres and observed that cells responded to a change insphere size produced by an increase in temperature[108]. The cells released excess active oxygen whensphere diameters shrunk as a result of the temperaturebeing increased from 25 to 37C.There were several studies in the literature in whichtextured surfaces were created by techniques which yieldtopographical features that were less denable than thosepreviously discussed, but nevertheless equally interesting.The behavior of cells on sandblasted surfaces has beenstudied, although the observed trends seem less clearthan those on controlled morphologies, such as grooves.In general, adhesion, migration areas, and ECM produc-tion were greater on rougher surfaces, or those surfacessandblasted with larger grain sizes [83, 91, 110]. Turneret al. compared cell behavior on reactive ion and wetetched surfaces and found that primary cells behaved ina manner opposite that of a transformed cell line [87].The primary cells preferred the narrow, columnar struc-tures created by the reactive ion etching, but the trans-formed cells preferred the smoother, wet etched surfaces.A few studies were found in which protein tracks wereemployed as guidance cues for several cell types, includ-ing neural cells [82, 93, 94]. Isolated tracks were found toprovide stronger guidance than repeated tracks [94].One interesting result from these studies was that cellsR.G. Flemming et al. / Biomaterials 20 (1999) 573588 585remained aligned to laminin tracks even when an ortho-gonal DC eld was applied [93].Lastly, Goodman et al. used polymer casting to repli-cate the topographical features of the extracellular matrix[85]. Goodman and coworkers observed that endothelialcells cultured on the ECM textured replicas spread fasterand had appearance more like cells in their native arteriesthan did cells grown on untextured surfaces [85].5. ConclusionsTopographical cues, independent of biochemistry, gen-erated by the ECM may have signicant eects uponcellular behavior. Clearly, substratum topography hasdirect eects on the abilities of cells to orient themselves,migrate, and produce organized cytoskeletal arrange-ments, as documented by the studies summarized here.However, the relevance of such studies to the behavior ofcells adhering to basement membranes is unclear sincethe topographical features of basement membranes havebeen shown to possess much smaller features in thenanometer size range [2834]. The fact that basementmembranes are composed of unique and intricate topo-graphies into which cells adhere and extend processes,coupled with the fact that topographical features havebeen shown to inuence cell behavior, leads to the hy-pothesis that the topography of the basement membraneis important in regulating cellular behavior in a mannerdistinct from that of the chemistry of the basement mem-brane.It is therefore concluded, that, in order to fully under-stand the role that substrate topography plays in theregulation of cell behavior, smaller, denser, nanometerscale features must be fabricated. Techniques such aslaser holography and X-ray lithography oer the clearestway to synthesize topographical features with controlledlateral dimensions in the nanometer size range. 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