nanopatterning of fibronectin and the influence of integrin … · 2017. 12. 23. · adhesion...

Nanopatterning of fibronectin and the influence of integrinclustering on endothelial cell spreading and proliferation

John H. Slater, Wolfgang FreyDepartment of Biomedical Engineering and Center for Nano and Molecular Science and Technology,University of Texas at Austin, 1 University Station C0800, Austin, Texas 78712

Received 11 June 2007; revised 15 August 2007; accepted 27 August 2007Published online 17 December 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.31725

Abstract: Investigating stages of maturation of cellularadhesions to the extracellular matrix from the initial bindingevents to the formation of small focal complexes has beenchallenging because of the difficulty in fabricating thenecessary nanopatterned substrates with controlled bio-chemical functionality. We present the fabrication and char-acterization of surfaces presenting fibronectin nanopatternsof controlled size and pitch that provide well-defined cellu-lar adhesion sites against a nonadhesive polyethylene glycolbackground. The nanopatterned surfaces allow us to controlthe number of fibronectin proteins within each adhesion sitefrom 9 to 250, thereby limiting the number of integrinsinvolved in each cell–substrate adhesion. We demonstratethe presence of fibronectin on the nanoislands, while noprotein was observed on the passivated background. We

show that the cell adheres to the nanopatterns with adhe-sions that are much smaller and more evenly distributedthan on a glass control. The nanopattern influences cellularproliferation only at longer times, but influences spreadingat both early and later times, indicating adhesion size andadhesion density play a role in controlling cell adhesion andsignaling. However, the overall density of fibronectin on allpatterns is far lower than on homogeneously coated controlsurfaces, showing that the local density of adhesion ligands,not the average density, is the important parameter for cellproliferation and spreading. � 2007 Wiley Periodicals, Inc.J Biomed Mater Res 87A: 176–195, 2008

Key words: endothelial cell; nanopattern; fibronectin; focaladhesion; integrin

INTRODUCTION

Integrins mediate cellular interactions with theunderlying extracellular matrix (ECM) and form ad-hesion sites that are key regulators of intracellularsignaling cascades that govern many aspects of cel-lular behavior.1–5 Although individual integrins, acti-vated by ligand binding or conformational changesalone, can form stable adhesions, it is the clusteringof ligated integrins that induces many of the signal-ing events, and which is necessary for strong interac-tions with actin fibers.6–9 By contrast, clustering ofnonligated integrins accumulate a large number ofsignaling molecules, but fail to engage the actin cyto-skeleton.7 Groups of only three integrins, activated

by ligand binding, are needed to form an activatedcluster,8 although these clusters immediately formlarger structures that then link to the cytoskeleton.Activated and clustered integrins therefore act syn-ergistically and recruit a large variety of scaffold andsignaling molecules with spatial and temporal de-pendence, leading to the formation of focal com-plexes that mature into focal adhesions.10–13

Focal complexes and focal adhesions regulate mo-tility,14–17 proliferation,2,18–20 differentiation,18,20 andapoptosis.21,22 While often biochemical in nature,these regulatory processes are also determined bythe endogenous force generated by the cell with itsactomyosin network through the adhesion com-plexes on the substrate.23–25 Adhesions have beenshown to support forces proportional to the adhe-sion size, except for small adhesions mostly at theprotruding edge of motile cells, which show higherforces with little or inverse proportionality to size.26–28

Consequently, the substrate compliance and the den-sity of integrin ligands play important roles in cellmotility, leading to a biphasic dependence of themotility on the ligand density and the elastic modu-lus.29–31 Changes in substrate compliance and liganddensity also lead to an altered distribution of focal

Additional Supporting Information may be found in theonline version of this article.Correspondence to:W. Frey; e-mail: [email protected] grant sponsor: National Institutes of Health

(NIH); contract grant number: R21 EB003038-01Contract grant sponsor: Center for Nano and Molecular

Science and Technology at the University of Texas at Austin

� 2007 Wiley Periodicals, Inc.

adhesions and variations in adhesion size.24,30 Theexact mechanism of the integrin-mediated mechano-transduction process is still unclear, and tools thatallow a systematic variation of the size of individualclusters may lead to a better understanding of theforces exerted in each cluster as well as of the molec-ular processes that drive the maturation process ofcellular adhesions.

ECM protein or ECM fragment-coated micro- andnanobeads have been a very successful technique toexplore the minimum size of a ligand cluster,8 theforce exerted on these small clusters,32 the locality ofthe response to ligand binding,33 and the dynamicsand directionality of integrin movement.34,35 How-ever, only larger surfaces that present ECM proteinsin a controlled density or pattern can systematicallyexplore parameters such as a larger range of forces,the maturation process of adhesions, and the proc-esses associated with cell motility.

Direct control over focal adhesion dimensions canonly be achieved with surfaces that present precisenanometer-sized adhesion areas surrounded by anonadhesive background. Micropatterned functionalsurfaces are easily achieved using microcontact print-ing36,37 or traditional lithography techniques,38–40 andthese have been successfully used to show the influ-ence of spreading area and geometry on cell prolifera-tion, differentiation, and signaling.37,41,42 It is still achallenge, however, to produce nanopatterned dual-functional surfaces that are large enough for statisti-cal cell studies. For controlled chemical functionalityat the nanometer scale, these fabrication processeshave to create surface nanopatterns of ECM compo-nents, either full proteins or peptide sequencesadsorbed or covalently linked to the surface in nano-meter-sized patches surrounded by a passive back-ground that does not support protein adsorption orcellular attachment. Because of these conditions, onlya few nanopatterning strategies exist that can providedefined nanoislands of cell adhesion molecules,43–48

and even fewer have been used for systematic studiesof cellular adhesion and motility.43,46,47,49

Clustering of integrins to within about 60 nm ofeach other is necessary for long-term cell attachmentand focal adhesion formation using micelle nanoli-thography.43 This integrin spacing corresponds tothe typical range of sizes and repeats found in keyadhesion molecules such as fibronectin, collagen,and talin. Also, adhesion strength is increased andcells show higher motility on small clusters of RGDpeptides compared with equivalent densities of ho-mogeneous distribution, although only statisticalaverages of cluster distances could be controlled.46–48

The idea that the local density of ligands is moreimportant than the overall density has been sug-gested by analyzing morphological parameters ofcells seeded on fibronectin (FN) islands down to a

size of 0.1 lm2 with varying island separation cre-ated using microcontact printing.50 However, no ex-perimental investigations exist that analyze the rangeof cluster sizes from 40 to 300 nm, corresponding toabout 9–250 fibronectins per island. This size rangecould be key to understanding the importance of theimmediate aggregation seen with ligand trimers,8

the different levels of integrin activation found atearly adhesions,51 and the early processes in thematuration of focal complexes into adhesions duringmotility52 and spreading.53 One would therefore alsoexpect adhesions restricted to the size range of40–300 nm to allow for an important range of forcesper adhesion cluster and the impact on cell adhe-sion, cytoskeletal development, and cell motility tobe investigated.

Here we introduce a patterning technique basedon nanosphere lithography (NSL),54,55 which hasbeen used as a template for the fabrication of pro-tein56 or chemical arrays, but is here expanded touse orthogonal functionalization to tightly controlthe cells’ ability to build adhesions. Our techniqueproduces large areas (cm2) of nanoislands whosesize and spacing is controlled by the sphere diameterand by monolayer or bilayer mask configura-tions.55,57 We create FN islands surrounded by poly(ethylene glycol) (PEG) passivated areas, which,instead of using RGD-containing peptides, allows usto utilize the stronger binding to the full-length pro-tein that includes, for instance, the synergistic bind-ing site. Because the goal is to present well-definedadhesion sites, we characterized the surfaces usingX-ray photoelectron spectroscopy (XPS), atomic forcemicroscopy (AFM), and cell-seeding studies to showthat FN adsorbs specifically to the nanopatternedislands but not to the PEG-treated background. Wepresent initial characterizations of cell adhesion,spreading, and proliferation to show the influence ofadhesion site size, and to introduce an experimentalplatform that can be used for future cell motility andsignaling studies.

MATERIALS AND METHODS

Nanopattern fabrication

Plain microscope glass slides (Erie Scientific Company,Portsmouth, NH) were cleaned in piranha solution(H2SO4:H2O2 3:1) (Fisher Scientific, Pittsburgh, PA) for 20min at 858C. The slides were thoroughly rinsed with dis-tilled deionized (DI) water (Barnstead International, Dubu-que, IA) to remove residual acid. NSL was used to createthe nanopatterns. Briefly, polystyrene spheres (PS) (DukeScientific, Fremont, CA) of a chosen diameter were dia-lyzed, using a 10,000-MW cut-off membrane, (PierceBiotechnology, Rockford, IL) following the sphere manu-

NANOPATTERNING OF FIBRONECTIN 177

Journal of Biomedical Materials Research Part A

facturer’s instructions to remove unwanted surfactants andthen deposited in either a monolayer or bilayer configura-tion onto a clean glass surface using a custom-built capil-lary deposition machine similar to that in Refs. 58 and 59.Two nanometers of chromium (Cr) (R.D. Mathis, LongBeach, CA) and 8 nm of gold (Au) (Alfa Aesar, Ward Hill,MA) were thermally evaporated onto the surface (DentonVacuum, Moorestown, NJ). The spheres were removed bysonication in methanol and the resulting nanopatternedsurface was immediately functionalized for cell adhesionexperiments.

Nanopattern imaging and characterization

AFM images were acquired using an MFP-3D AFM(Asylum Research, Santa Barbara, CA). Standard siliconcantilevers, AC240TS (72 kHz, Olympus Optical, Japan)were used for alternating current mode imaging, and non-conducting silicon nitride sharpened cantilevers (VeecoMetrology, Santa Barbara, CA) were used for contact modeimaging. Image processing, analysis, and 3D enhancementwere performed using MFP-3D software in Igor Pro 5(WaveMetrics, Lake Oswego, OR) and with ImageJ (NIH,Bethesda, MD). The individual island size, island areas,island-to-island spacing, topography, and percent surfacecoverage were measured from the AFM images withoutperforming tip deconvolution.

Dual chemical functionalization

Nanopatterned glass slides were exposed to an airplasma at 300 lTorr at �50 W for 10 min (March Instru-ments, Concord, CA). The nanopatterned slides were thenimmersed in a 26.5 mM hydrochloric acid (Fisher Scien-tific, Pittsburgh, PA), 1 mM hexadecane thiol (Aldrich, St.Louis, MO), and 41 mM 2-methoxy(polyethyleneoxy)–pro-pyltrimethoxysilane (PEG-silane) (Gelest, Morrisville, PA)solution in toluene (Fisher Scientific, Pittsburgh, PA) in aself-standing centrifuge tube (Corning, Corning, NY) for48 h with continuous stirring, adapted and extended fromRef. 60. The samples were vigorously rinsed once in tolu-ene and twice in ethanol, dried with nitrogen, and bakedat 1058C for 1 h. The chemically functionalized patternedsurfaces were exposed to 3 mL of bovine plasma fibronec-tin or human plasma fibronectin (Sigma, Saint Louis, MO)solution at a concentration of 10 lg/mL in 50 mM HEPESfor 20 min, followed by two thorough rinses in HEPES so-lution to remove excess fibronectin. Cells were seeded im-mediately following FN adsorption.

Analysis of surface modification

A PHI 5700 XPS system (Physical Electronics, Chanhas-sen, MN) equipped with a dual Mg and monochromaticAl X-ray source was used at a fixed angle of 458 for ele-mental analysis of the chemically modified surfaces. Thesurfaces were survey scanned to determine the elementalcomposition percentages for Si2p, C1s, N1s, Au4f, and O1son each surface and S2p on some. High-resolution scanswith a dwell time of 500 ms and with a pass energy of

58.7 eV were used to acquire the Au4f, N1s, S2p, and Si2pspectra and a pass energy of 11.75 eV was used for the C1sspectra with charge correction set to 285 eV for noncon-ducting samples. Three distinct areas were measured andaveraged for each sample. PeakFit software (Systat Soft-ware, Point Richmond, CA) and a multipeak fit package inIgor Pro 5 (WaveMetrics, Lake Oswego, OR) were used tofit the high resolution C1s scans with multiple Gaussiancurves and linear baseline correction to analyze the bondtypes present within each peak. Nine different surfacetypes were examined: (1) a dual functionalized gold sur-face, (2) a dual functionalized gold surface with FNadsorbed at concentrations of 2, 5, 10, 25, and 50 lg/mL,(3) a dual functionalized glass surface, (4) a dual function-alized glass surface with adsorbed FN, (5) a plain glasssurface with adsorbed FN at concentrations of 2, 5, 10, 25,and 50 lg/mL, and (6) 0400M, (7) 0400B, (8) 0300B, and(9) 0300M surfaces that were functionalized and exposedto FN at a concentration of 10 lg/mL (see Table I fornanopattern descriptions). It should be noted that evenpure gold or glass surfaces were treated with the fullchemistry, thiol and silane, to insure that no nonspecificinteractions took place on the surfaces.

Cells and reagents

Pooled and nonpooled human umbilical vein endothe-lial cells (HUVECs), passages 2–4, were cultured in endo-thelial growth media (EGM) supplemented with 2 mL ofbovine brain extract, 0.5 mL of human endothelial growthfactor, 0.5 mL of hydrocortisone, 0.5 mL of gentamicin/amphotericin-B, and 10 mL of fetal bovine serum accord-ing to manufacturer’s instructions (all reagents and cells:Cambrex Bio Science Walkersville, Walkersville, MD). Thecells were grown to 90% confluence in T-25 tissue cultureflasks that were coated with 30 lg of bovine or humanplasma fibronectin (Sigma, Saint Louis, MO) at 378C and5% CO2. Before seeding onto sample slides, the cells weretrypsinized with 3.0 mL of 0.25% trypsin and 1 mM ethyl-enediaminotetraacetic acid in PBS at 378C for 5 min. Thecells were collected and centrifuged at 240g for 10 min.The cell pellet was then resuspended in full EGM mediaand seeded onto the desired substrate at a density of �40cells/mm2.

Fibronectin adsorption analysis

Nanopatterned samples functionalized with fibronectinwere analyzed before and after cell seeding. After fibronec-tin adsorption, following a procedure modified from ref.61, the nanopatterned surfaces were incubated in 1 mMbis (sulfosuccinimidyl)suberate (BS3) (Pierce, Rockland, IL),a water-soluble, noncleavable, and membrane-imperme-able crosslinker, at 208C for 10 min. Unreacted crosslinkerwas quenched with 20 mM glycine in PBS. Samples thathad been seeded with cells were treated similarly, with theaddition of a cell removal step using 0.2% SDS (sodiumdo-decylsulfate) in PBS for 10 min after crosslinking. Eachsurface was finally washed three times with PBS andimaged with AFM as described earlier.

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Dual fluorescent-atomic force adhesionsite imaging

After 72 h of incubation, the samples were rinsed twicewith PBS followed by fixation in 5% formaldehyde in PBSfor 20 min at 378C. The samples were rinsed with PBS andblocked with 1.0% BSA solution in PBS for 20 min andrinsed twice with PBS. Samples were then incubated in avinculin stain solution (1% by volume of FITC-conjugatedmouse anti-vinculin in 1.0% BSA and 0.01% Tween 20 inPBS) (all Sigma, Saint Louis, MO) overnight at 48C. Thesamples were rinsed twice with PBS supplemented with0.01% Tween 20 and rocked in PBS with 0.1% Tween 20for 20 min followed by a DI H2O rinse and nitrogen dry-ing. Fluorescent images were acquired using an OlympusIX70 inverted microscope equipped with an X-100 CCDcamera with InstaGater on-chip integration (Dage-MTI,Michigan City, IN) connected to an MFP-3D AFM (AsylumResearch, Santa Barbara, CA). AFM images were collectedover the fluorescently imaged areas as described earlier.The vinculin-containing adhesions in the AFM imageswere identified by matching the location to that observedin the simultaneous fluorescent images and by theirincreased height above the background. Height traceswere drawn and the adhesion heights measured usingMFP-3D software in Igor Pro 5 (WaveMetrics, LakeOswego, OR). For the two cells shown in Figure 5 each ad-hesion site’s length (dash adhesions) or diameter (dotadhesions) and area were determined from the AFM scansusing ImageJ (NIH, Bethesda, MD).

Cell proliferation and cell spreading area studies

HUVECs were sparsely seeded at �40 cells/mm2.Twenty 153 magnification phase contrast images were col-lected 4 h after initial seeding and at 24-h time intervalsfor 3 days for each sample. Additionally, fifty 403 magni-fication phase contrast images were collected every 24 hfor 3 days. Cell densities (number of cells per surface area)were measured over a 21 mm2 area. Cell proliferation rateswere calculated after normalizing the cell densities at eachtime point to its initial average seeding density at 4 h anda heteroscedastic t test assuming unequal variance was

used to determine significant differences in the normalizeddata. Cell proliferation assays, such as BrdU DNA synthe-sis assays or fluorescent-activated cell sorting (FACS), area more specific proliferation test, but could lead to mis-leading results due to incomplete nanopattern coverage insome areas of the sample. The cell spreading areas for aminimum of 100 cells were measured by hand on aMotion Computing LE1600 Tablet PC (Motion Computing,Austin, TX) using the outline feature in ImageJ (NIH, Be-thesda, MD). To separate surface effects from other stim-uli, only cells that had just one nucleus and were not inintimate contact with neighboring cells were analyzed forthe cell spreading area study. Statistical analysis was per-formed using SPSS 12.0 (SPSS, Chicago, IL). ANOVA withpost hoc Tukey and Bonferroni tests and nonparametricKruskal–Wallis with a post hoc Dunn procedure were bothapplied to compare the measured projected cell spreadingareas.

RESULTS

Nanopattern fabrication and functionalization

The NSL fabrication process allows us to fabricatepatterns with characteristic lengths ranging from 90to 400 nm with homogenous topography of 10–13 nmover cm2 areas. Figure 1(A) schematically outlinesthe process used to create the surfaces. First, amonolayer or bilayer of densely packed nanospheresis deposited onto the surface with a custom-builtdeposition machine [Fig. 1(B)]. A 2-nm adhesionlayer of chromium and an 8-nm layer of gold arethen thermally evaporated onto the surface, followedby sphere removal using sonication in methanol.This leaves an array of truncated triangular pyra-mids of gold in a hexagonal or orthorhombic latticesurrounded by glass [Fig. 1(C,D)].

The resulting surface is composed of two materialsthat allow for two separate functionalization chemis-tries to be applied, such as thiols for the gold and

TABLE INomenclature, Pattern Size, Pitch, and Pattern Area for the Nanopatterned Surfaces Used

Pattern NameSphere

Diameter (nm)CharacteristicLength (nm)

Center-to-Center Spacing[Rim-to-Rim Spacing] (nm) Island Area (nm2)

1500M 1500 405 6 41 883 6 51 [478] 85,653 6 17,0441000M 1000 305 6 39 589 6 27 [284] 55,923 6 72730820M 820 255 6 24 470 6 22 [215] 37,049 6 49740500M 500 157 6 19 285 6 16 [128] 14,947 6 27780400M/0420M 400/420 97 6 9 253 6 13 [156] 6485 6 6860400B/0420B 400/420 102 6 16 414 6 12 [312] 4675 6 24810300M 300 94 6 11 177 6 13 [83] 5191 6 12980300B 300 92 6 33 308 6 11 [216] 3629 6 1013

M, monolayer mask; B, bilayer mask. The characteristic length refers to the length of the longest bisector of the triangu-lar pattern, the center-to-center spacing (pitch) is measured from the center of one nanoisland to the center of its nearestneighbor, and the rim-to-rim spacing from the rim of one nanoisland to the closest rim of its nearest neighbor. All meas-urements were made from multiple high resolution AFM scans (n � 100, error 5 1 SD).



silanes for the glass areas. After sphere removal thesurface is exposed to the O2 plasma to producehydroxyl groups on the glass surface. The hydroxylsare necessary to create a high density of couplingsites for the silane selfassembled monolayer (SAM).Low-density PEG-SAMs are not effective in prevent-ing protein adsorption and subsequent cellularattachment. For dual SAM functionalization, thefreshly oxidized surface is immersed in a solutioncontaining the hexadecane thiol and the PEG-silanefor 48 h. Since the oxygen plasma treatment woulddestroy the thiol molecules, and with the tendency ofthe silane to nonspecifically adsorb to the gold if nothiol is present, the thiol and silane surface function-

alizations are performed simultaneously. The combi-nation of solvents does not mix completely, and thusmust be stirred vigorously. Shorter incubation timesand different concentrations of PEG-silane in the mix-ture led to increased cell adhesion to the PEG-surfa-ces (see Cell Seeding on Functionalized ½ Au–½ GlassSurfaces). The SAM-functionalized surface is thenexposed to a FN solution, where FN is only able toadsorb to the gold nanopatterns producing FN nano-islands of controlled size and pitch surrounded by anonadhesive background [see the changed texture ofthe pattern in Fig. 1(E) compared with 1(C)]. Finally,cells are seeded onto the surface [Fig. 1(F)].

Table I summarizes the samples used, namedaccording to the size (in nm) of the spheres usedand the type of mask, monolayer (M) and bilayer(B). The table also lists the measured sizes, the bisec-tor of the triangle for monolayer (M) or the diameterof the dots for bilayer (B) samples, the distancesbetween the nanoislands, and the projected areas asa function of the sphere diameter and mask type.The measured dimensions of the nanoislands rangedfrom 12 to 25% larger than the calculated theoreticalsizes, yet the island-to-island spacing was within 2%of the theoretical values.55,57 This could be attributedto inhomogeneities in the sphere size, nonperpendic-ular metal evaporation, or to convolution effectsfrom the AFM tips. The measured percent surfacecoverage of the nanoislands was 7.2% 6 0.4% and2.3% 6 0.4%, for mono- and bilayers, respectively,and closely matched the calculated theoretical valuesof 7.2 and 2.2%.

Surface characterization with XPS

XPS was used to confirm that the surface chemis-try is selective, even on the nanometer scale, andthat FN adsorbs exclusively to the methylated goldareas while leaving the passivated PEG-glass back-ground free of protein, and to determine the FN sur-face concentrations after adsorption. The analysiswas performed before chemical treatment, after si-multaneous functionalization with thiol and silane,and after FN adsorption. Four nanopatterned surfa-ces, 0300B, 0300M, 0400M, and 0400B, were analyzedafter the full modification protocol.

The results from the XPS elemental analysis forthe simultaneous functionalization with thiol and sil-ane SAMs are given in Table II (for the spectral anal-ysis see supplemental data). Several indicators havebeen used to confirm protein adsorption by XPSanalysis62–64: the appearance of a distinct N1s peakindicating the presence of nitrogen in protein, anincrease in the C��N, C��C, C��O, and N��C¼¼Obond contributions to the C1s spectra from FN, and a

Figure 1. (A) Schematic depicting the nanopattern fabri-cation process, chemical functionalization, and directedfibronectin adsorption to the chemically modified nanopat-terns. 3D AFM images with zoomed-in inserts of (B)monolayer mask of 500-nm diameter spheres (scan size5 lm 3 5 lm) (C) 0420M surface (scan size 2 lm 3 2 lm)(D) 1500B surface (scan size 10 lm 3 10 lm) (E) 1500Msurface after dual chemical functionalization and fibronec-tin adsorption (scan size 5 lm 3 5 lm) and (F) HUVECseeded on a 0300M surface after the full surface treatment(scan size 90 lm 3 90 lm). [Color figure can be viewed inthe online issue, which is available at www.interscience.wiley.com.]

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decrease in the background (Au4f, Si2p) signal as theprotein shadows the substrate signal.

We used the most direct and specific indicator forprotein adsorption, the appearance of a nitrogen N1speak, which is absent in all samples before FNabsorption. While a clear N1s peak was observed af-ter protein adsorption on the modified gold [Fig.2(F)] and plain glass surfaces [Fig. 2(E)], no distinctN1s peak was seen on PEGylated glass samples ei-ther before or after FN adsorption [Fig. 2(D)]. Toquantify the adsorption of FN, we established a cali-bration relation of the Gaussian-fit area under theN1s peak at different concentrations of FN in solu-tion. The typical sigmoidal behavior that has beenobserved with 125I-labeled FN61 is reproduced withthe N1s area measurements [Fig. 2(A)]. A quantifica-tion of the amount of FN can be achieved by fittingthe data to a sigmoidal curve and comparing the sat-uration value of the XPS signal of FN on thiolatedgold surfaces to published data of 125I-labeled FNadsorbed to CH3-terminated silane SAMs,

61 which isvery similar to adsorption curves found for tissueculture polystyrene and glass.65 We also examinedthe background signal to quantify the extent of shad-owing as FN is adsorbed to the surfaces. The back-ground signals for both, Au4f for gold and Si2p forglass, exponentially decrease as the FN densityincreases [Fig. 2(B,C)].

A FN saturation value of 190 ng/cm2 (2543 FNmolecules per square lm) occurred on CH3-termi-nated silane SAMs when exposed to a 20 lg/mL so-lution of FN.61 The calibration curves indicate that amonolayer of FN is formed on clean glass at a surfaceconcentration of 2300 FN/lm2. The saturation valuefor a thiolated gold surface is slightly higher at 2605FN/lm2. The higher saturation value is attributed tothe fact that FN has a more compact form whenadsorbed to hydrophobic surfaces thereby inducing ahigher packing density for a monolayer of protein onthiolated gold than would be expected on a glass sur-face.66 Furthermore, we were able to show that themono- and bilayer nanopatterned surfaces, respec-tively, adsorb 8.3% (217 FN/lm2) and 1.6% (41 FN/lm2) of the amount of protein as the nonpatternedthiolated gold surfaces. These values are close to the

measured pattern surface coverage of 7.2 and 2.3%for the mono- and bilayer surfaces, respectively, indi-cating that the FN adsorbs almost exclusively to thethiolated gold nanopatterns and avoids the passivePEG background.

We deconvoluted the C1s signal for contributionsfrom chemical bond energies that are expected in thePEG, thiol, and FN chemical structures. The C1sspectra for the functionalized and protein-treated ho-mogenous gold and plain glass samples were fitwith three Gaussian curves corresponding to theexpected bond energies of the C��C (285.0 eV),C��N (286.0 eV), C��O and PEO (286.5 eV), andN��C¼¼O (288.3 eV) bonds.67 Previous XPS studieson proteins, including FN, have shown that it is dif-ficult to differentiate the C��O and C��N peaksfrom one another and that they can be fit as a singlepeak centered in the range of 285.7–286.5 eV.68 Inour data, the peak position occurred in this samerange, usually centered at 286.5 eV. The relative con-tribution of each bond to the total C1s energy for thegold and nanopatterned surfaces is displayed in Ta-ble III; glass data are not shown due to their similar-ity to the gold surfaces.

After protein adsorption, the appearance of a nitro-gen N1s peak on the gold sample is accompanied bythe appearance of two additional peaks in the C1sspectra at 286.5 and 288.3 eV, which indicate the pres-ence of amine, ether, and amide bonds [Fig. 2(F)],similar to previous results.68,69 The characteristic pro-tein peak contributions to the C1s spectra consisted of64.5% C��C bonds from the thiol and the FN back-bone, 23.4% C��N and C��O bonds, and 12.0%N��C¼¼O bonds for the gold surface with a FN den-sity of 1882 FN/lm2. The C��C contribution to theC1s spectra decreases and the C��N, C��O, andN��C¼¼O contributions increase as more protein isadded to the thiolated gold surfaces (Table III). ThePEG-silane functionalized glass samples show theexpected C��C and PEG peaks, but no amide bondpeak [Fig. 2(D)] after FN adsorption. A ratio of C��Cto PEG components of about 1:4 would be expected,but the contribution of the C��C peak is larger [Fig.2(D)]. We attribute this to a small amount of hydro-carbon contamination or possibly to some thiols

TABLE IIMeasured XPS Elemental Composition for Each Test Sample in Percent of the Total Signal from Survey Scans,

Extracted from Gauss-Fits to the Peaks

Surface Au4f Si2p S2p O1s N1s C1s

Glass-PEG-Thiol 0.01 20.84 0.00 60.39 0.14 18.63Glass-PEG-Thiol-10 lg/mL FN 0.01 17.68 0.00 57.97 0.27 24.06Au-Thiol-PEG 15.43 0.00 3.13 46.85 0.51 34.08Au-Thiol-PEG-1,882 FN/lm2 10.93 2.31 1.15 31.33 4.27 50.010400B-Thoil-PEG- 41 FN/lm2 0.20 19.72 ND 49.08 0.75 30.250400M-Thiol-PEG- 217 FN/lm2 0.77 20.28 ND 49.87 0.81 28.26

ND, not determined.



Figure 2.

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entrapped in the PEG layer, although no sulfur wasdetected. Still, the absence of a 288.3 eV amide bondpeak and a nitrogen N1s peak on the PEGylated sur-face indicate a protein-repulsive surface.

On the four nanopatterned surfaces, two small N1speaks appeared after functionalizing with thiol/sil-ane and FN [Fig. 2(G,H)]. Data for 0300M and 0300Bare very similar to the 0400M and 0400B surfaces,respectively (not shown). An evaluation of the C1scurve shows that both nanopatterned surfaces havea noticeable N��C¼¼O signature that is not presenton the PEG-functionalized glass, which, togetherwith the appearance of an N1s peak, indicates FNadsorption as seen on the functionalized nonpat-terned gold surfaces. A comparison of the areaunder the N1s curves of the nanopatterned surfacesto the nonpatterned gold surfaces after FN adsorp-tion shows that the amount of protein surface cover-age (8.3% for M and 1.6% for B surfaces) on thenanopatterns is close to the measured nanopatternsurface coverage (7.2% for the M and 2.3% for the Bsamples). Also, the area under the amide bond peakat 288.3 eV for the monolayer nanopatterned surfa-ces is 7% of the area for the same portion of the C1sspectra of the protein-adsorbed gold sample.

The gold background on the monolayer nanopat-terned surfaces displayed 8.9% the amount of gold

compared with the nonpatterned controls, in goodagreement with the measured 8.3% surface coverage ofFN. As expected, the nanopatterned surfaces had highersignals from the Si2p spectra, indicating that no proteinwas adsorbed to the functionalized glass background.

Taken together, these results indicate selectivebinding of the silane and thiol functionalities to theglass and gold areas, respectively, and selective FNadsorption to the thiolated gold areas. The presenceof signatures for the thiol and PEG and distinct N1speaks on the protein-adsorbed gold, unmodifiedglass surface, and on nanopatterned surfaces that areabsent on the PEGylated glass sample indicate thatthe chemical functionality can be translated to thenanometer scale and FN can be adsorbed to verysmall thiolated gold nanoislands. This precise place-ment of chemical functionality and subsequent pro-tein adsorption is the basis for control of the adhe-sion site dimensions that are designed to influencecell behavior in contact with the biomaterial surface.

Cell seeding on functionalized½ Au–½ glass surfaces

To validate the XPS data, HUVECs were seededon the modified surfaces. Time-dependent functional

TABLE IIIDeconvolution of the XPS C1s Peak into the Chemical Bond Energies for Each Test Sample (in % of total peak area)

Surface C��C 285.0 eV C��N, C��O, PEO 285.7–286.5 eV N��C¼¼O 288.3 eV

Glass-PEG-Thiol 32.93 67.07 –Glass-PEG-Thiol-10 lg/mL FN 25.16 74.84 –Au-Thiol-PEG 92.88 7.12 –Au-Thiol-PEG-537 FN/lm2 81.79 13.53 4.68Au-Thiol-PEG-1,199 FN/lm2 64.35 24.58 11.06Au-Thiol-PEG-1,882 FN/lm2 64.54 23.42 12.04Au-Thiol-PEG-2,474 FN/lm2 56.87 26.29 16.84Au-Thiol-PEG-2,605 FN/lm2 55.55 27.39 17.700400B-Thoil-PEG- 41 FN/lm2 35.88 60.69 3.430400M-Thiol-PEG- 217 FN/lm2 33.35 63.01 3.65

The appearance of C��N and N��C¼¼O bonds was observed after the addition of FN to the modified gold surfaces. Asmall N��C¼¼O peak was also detected on both nanopatterned surfaces and was not observed after FN adsorption to thePEG-functionalized glass surfaces.

Figure 2. (A) Calibration curves depicting the surface density of FN (FN/lm2) on (u-grey) clean glass and (n-black)functionalized gold surfaces as a function of the solution concentration. The surface density of FN is also shown for both(~) M and (l) B nanopatterned surfaces. Calibration curves showing the exponential decay of the background signal(Au4f for gold and Si2p for glass) as protein is adsorbed at higher concentrations for functionalized (B) gold and (C) cleanglass surfaces, (~) M and (l) B show values for the nanopatterned surfaces. (D-H) XPS data comparing C1s and N1s spec-tra for nonpatterned and nanopatterned surfaces after dual functionalization and FN adsorption (10 lg/mL), and phasecontrast images of cells seeded on the respective surfaces. (D) PEGylated glass surface, (E) unmodified glass, (F) thiolatedgold, (G) 0400M, and (H) 0400B surfaces. The FN contribution to the C1s spectra (C��C, C��N, N��C¼¼O) is shown for the(E) glass, (F) gold, (G) 0400M, and (H) 0400B surfaces and the N��C¼¼O is not seen on the (D) PEGylated glass. The char-acteristic N1s peak corresponding to adsorbed FN is detected on the (E) untreated glass sample, (F) gold, (G) 0400M, and(H) 0400B surfaces and not on the (D) PEGylated glass. Cells adhered to, spread, and proliferated on the (E) unmodifiedglass, (F) gold, (G) 0400M, and (H) 0400B surfaces over 3 days whereas almost no cells attached to the (D) PEGylated glasssurface.



controls to show that the surfaces selectively supportcell adhesion and repulsion on the functionalizedgold and glass areas, respectively, were performedon ½Au–½glass slides. Slides underwent full chemi-cal functionalization treatment as described above,but with varying concentrations of PEG-silane (1, 2,3, and 5% by volume) before seeding with HUVECs.Cells adhered to and proliferated on all of the surfa-ces [Fig. 3(B,E)] except for the PEG-silane functional-ized glass surface [Fig. 3(A,C)], again reaffirmingthat the PEG layer prevents protein adsorption tothe glass areas and consequently inhibits cell attach-ment. Figure 3(D) shows a phase-contrast image ofthe intersection of the gold and glass halves after3 days of culture. The HUVECs have grown to nearconfluence on the gold half and align to the bound-ary. A small strip of cells can be seen protrudinginto the lighter glass side, probably caused by imper-fect shadowing of the deposition mask allowing asmall amount of gold to deposit in the transitionzone. The density of adhered cells increased on the

gold-coated side over 3 days for all four silane con-centrations, while very few cells adhered to thePEG-treated glass side. However, the density of cellswas the lowest on the 2% PEG-silane surfaces, whichwe therefore used as our standard concentration forall subsequent experiments.

In addition to the initial ability of the PEG surfa-ces to reject cell adhesion, we tested whether ECMadsorption from the serum-containing media couldsupport cell attachment. After 3 days of culture inserum-containing media the ½Au–½glass surfaces(3% PEG) were reseeded with four times the numberof cells used for the original seeding. On the fourthday, 24 h after the samples had been seeded again, alarge spike in cell density was observed on the goldhalf, while almost no cells were seen on the PEG-functionalized glass half [Fig. 3(A,B)]. This indicatesthat the PEG-functionalized glass retained its passi-vating properties against protein adsorption andsubsequent cellular attachment for at least 4 days inserum-containing media.

Figure 3. Number of cells per surface area for HUVECs seeded on ½ Au–½ glass slides after chemical functionalizationand FN adsorption. (A) Density of adherent cells on the PEG-treated glass half, and (B) on the functionalized gold halfmeasured at 24-h intervals. The fourth bar on the 3% PEG sample shows the cell density 24 h after reseeding cells at Day3 to test the stability of the chemical pattern contrast. (C–E) Phase contrast images of HUVECs on the ½Au–½glass slidesafter 3 days in culture. (D) HUVECs grow on and align with the gold-glass intersection. Representative images of cell den-sity on Au half (E) and glass half (C) after 3 days (n 5 20, error bars 5 1 SD).

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FN adsorption analysis with AFM

To control the clustering of integrins it is importantto know the number of adhesions per nanoisland.Nanopatterned surfaces were functionalized and theFN crosslinked with cell-impermeable amine-reactiveBS3 before and after cell seeding. AFM images of thesurfaces before cell seeding show distinct orderedarrays of protein on the gold nanopatterns [Fig.4(A,C)]. The protein clusters have an approximatesize and height of 36 and 7 nm, respectively, which isin agreement with lateral dimensions of globular FNusing electron microscopy.70,71 The protein clustersalso have a rounded or compact shape typical of FNadsorbed to hydrophobic surfaces,66 and practicallyno protein was observed on the PEG-modified glasssurface between the nanoislands, which is in agree-ment with our XPS and cell-seeding experiments. Tak-ing each spherical entity as a single FN, there are�60–70 FN proteins on each nanopattern for the1500M surface [Fig. 4(A)] and 2 to 5 on the 0420M pat-tern [Fig. 4(C)], resulting in an approximate density of714 FN/lm2 on the pattern for each sample or an av-erage of 55 FN/lm2 for the composite surface, whichincludes the surrounding glass-PEG background.However, the XPS data indicate that a composite aver-age of 217 FN/lm2 should be measured for the mono-layer surfaces, which is in agreement with the esti-

mated total volume of 0.72 mL/g of FN.72 This dis-crepancy may be induced by the BS3 crosslinker usedto secure the protein before AFM analysis by artifi-cially enlarging the imaged proteins during the cross-linking process, or by making it difficult to distinguishbetween two separate proteins and thereby inducing alower-than-actual protein count. Even though ourmeasured FN dimensions appear in agreement withAFM measurements by others,66 we have used thehigher surface concentration of FN derived from theXPS-radiolabeling comparison for the following analy-sis. Still, the density of FN, averaged over the pat-terned and nonpatterned areas of a nanopatternedsurface is very low. In comparison, endothelial cell ad-hesion has been shown on homogenous surfaces dis-playing as low as 250 FN/lm2, but these surfaces ledto reduced proliferation or cell death over time.73

We compared the FN morphology before cellseeding to its morphology after HUVECs adheredto and spread on the nanopatterned surfaces. After1 day, all surface proteins were crosslinked and rinsedwith SDS to remove the majority of each cell, leavingbehind only cell sections that are crosslinked to thesurface, or bare ECM in areas where no cells wereattached at the time of crosslinking. In some areas,instead of displaying ordered arrays of clustered FN,the FN was rearranged or removed (Figures 4B and4D). A reorganization of FN could be expected as

Figure 4. The 3D images from AFM scans (inserts) displaying crosslinked FN on a nanoisland. FN on a 1500M surfacebefore (A) and after (B) cell attachment, and FN on a 0420M surface before (C) and after (D) cell attachment. In both casesthe FN is clearly seen in a compact shape before cell seeding and is rearranged into a more elongated shape after cellseeding and attachment. Cells have been removed in (B) and (D) (see text). (Scale bars: (A) 143 nm, (B) 158 nm, (C) 47 nm,(D) 57 nm).



the cells remodel and stretch FN as stress is appliedduring focal adhesion maturation and cell move-ment.74

Adhesion analysis

For proof of principle and to validate our claimthat surfaces with nanoislands of FN surrounded bya passive background limit adhesion site size, weperformed dual fluorescence and AFM imaging on

vinculin-containing adhesion sites. Figure 5 showsan example of cells fixed and immunostained forvinculin after 72 h on a nanopatterned (average den-sity of 217 FN/lm2) and a nonpatterned glass sur-face (2092 FN/lm2). While the vinculin-containingadhesions on the glass control surface could be eas-ily correlated between fluorescence and AFM images[Fig. 5(A-C)], on the nanopattern this matching wasmore difficult due to the small size of the adhesions.To positively confirm the location of vinculin-

Figure 5. (A–E) Fixed HUVECs at 72 h on glass control. Corresponding images of (A) vinculin fluorescence of adhesionsand (B) AFM topographic image of the same area, showing large adhesions located mainly at the cell periphery; (C) shows(B) in a 3D view. A dot and dash adhesion are indicated by a small and large arrow, respectively. (D) High-resolutionAFM image of the two adhesions marked in (B). (E) Height trace corresponding to the line across the dash adhesion in(D) from bottom to top shown left to right. (F–K) Fixed HUVECs at 72 h on the 0400M pattern. (F) Vinculin fluorescenceof adhesions and (G) AFM topographic image of the same area with the fluorescent adhesions from (F) overlaid in red(dark dots). The adhesions are much smaller and more evenly distributed than on the control surface. (H) and (I) show azoomed-in view of the box area of (F) and (G), respectively, with the fluorescent areas in (H) overlaid in red and outlinedin (I). (J) High-resolution AFM image of the same area as in (I) with the arrow marking an adhesion terminating on ananopattern. (K) Height trace corresponding to the dotted line across the raised adhesion in (J) from bottom to top shownleft to right. The adhesion structure is about 1.7-lm long, stretching from 0.5 to 2.2 lm along the trace. Adhesions in con-trol and patterned samples are about 13–17 nm high after background correction (see text for details). Scale bar for H–J is1 lm. [Color figure can be viewed in the online issue, which is available at www. interscience.wiley.com.]

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containing adhesions in the AFM images, the fluo-rescence image was slightly scaled and overlaid onthe AFM image using multiple points on the cell rimas alignment markers [Fig. 5(F)]. Fluorescently la-beled adhesions closely matched increased heightfeatures in the AFM image across the entire cell [Fig.5(G)]. The matched features were terminations of upto 20 lm long, thin nonfluorescent raised lines thatcould be traced across the cell, presumably actinmicrofilaments. High-resolution AFM scans [Fig. 5(J),white box in Fig. 5(F)] were then correlated usingzoomed portions of Figure 5(F,G) as reference [Fig.5(H,I)]. Although the adhesions incorporate a defectline in some instances, they most often do not alignwith the direction of defect lines and the over-whelming majority of the vinculin-stained areas arenot on defects. The large, optically resolution-limitedfluorescence areas decompose into single or doublethin elongated features in the AFM images, as seento the left and center in Figure 5(I,J), respectively.The optical resolution limitation does not allow us todetermine whether the vinculin is localized exclu-sively over the nanopatterns, or whether it bridgesbetween patterns.

Detailed AFM height measurements can give fur-ther insight into the structuring of the adhesions. Thedash adhesion on the control glass surface [long arrowin Fig. 5(A–D)] had a measured height of �25 nmabove the glass background, including an 8–10 nmcontribution from the remainder of the cell mem-brane [Fig. 5(E)]. The adhesion on the 0400M [arrowin Fig. 5(H–J)] had a measured height of about 32nm [Fig. 5(K)], including about 19 nm due to theheight of the nanopattern, FN, and the cell mem-brane (measured independently). The adhesion siteis therefore about 13–17 nm higher than the back-ground in both the glass and the 0400M cases. Inde-pendent measurements on fiber structures on thesesamples give 7–9 nm height above background,which is close to the thickness of an actin microfila-ment of 7 nm in diameter.

Following the profile along the line in Figure 5(J)from top to bottom [right to left in Fig. 5(K)], startingwith an adhesion-free area (�7- to 8-nm thick) with ahole in the membrane and two nanopatterns, then ris-ing to the adhesion on a nanopattern at 2.2 lm, con-tinuing at about 20–22 nm height towards the nextpattern at 1.5 lm, crossing a defect line at 1.0 lm, andone more nanopattern at 0.6 lm, before continuing ata reduced height of about 15 nm as actin fibers. Theseheights agree with the 13–17 nm for adhesions andthe actin height of 7 nm above background. Theheight between each of the nanopatterns is close tothe thickness of adhesions, which probably resultsfrom firmly crosslinked bridges between the raisedadhesions on the patterns built from cytoplasmiccomponents of adhesion complexes. The length of the

adhesion structure is about 1.7 lm, but it is onlyabout 120-nm wide, which leads to a total area of0.2 lm2. The two adhesions in Figure 5(J) that to-gether compose the vinculin fluorescence area arecompletely separated, as the height between themreaches background levels (not shown). The two linesjoin at around the defect line.

We also find a striking difference in the size anddistribution of adhesions between control and nano-patterned surfaces. HUVECs on nonpatterned glasssurfaces have large adhesions mostly at the cell pe-riphery [Fig. 5(A,B)] ranging in size, for the cell ana-lyzed here, from about 1–6 lm and in area from 6.8 to7.3 lm2 for a dot adhesion [short arrow Fig. 5(A,D)]and a dash adhesion [long arrow Fig. 5(A,D)], respec-tively, as determined from the AFM images. Theseadhesions act as terminating sites for multiple actinfibers as seen in the AFM images [Fig. 5(B,C)]. In con-trast, HUVECs on the 0400M surface displayed amore homogenous distribution of small adhesions[Fig. 5(F)]. The adhesion site area for the adhesionindicated in Figure 5(H) is 0.036 lm2 or 0.2 lm2,depending on whether only the pattern or the fullarea indicated by vinculin staining is counted. In anycase, this is far smaller than adhesions formed on thecontrol surface. The smaller adhesions on the nano-patterned surfaces were coupled to fewer actin fibers[Fig. 5(I,J)], as opposed to multiple fibers seen in cellsseeded on the glass surfaces. Although we providehere just a snapshot, more detailed investigations ofcombined AFM and fluorescence imaging are in pro-gress, and we can conclude that HUVECs react to thenanopatterned surfaces by forming smaller and moredistributed adhesions on the supporting patternareas. Most importantly, the adhesions terminate inthe pattern and can limit adhesion site growth. How-ever, it appears likely that cytoplasmic components ofadhesions can bridge between individual patterns, atleast if the distance is not too great.

Influence of nanopatterned surfaceson cell proliferation

HUVECs were seeded on nanopatterned and con-trol surfaces and their cell densities measured (Fig.6). Proliferation rates were calculated by normalizingthe measured density at each time point to the aver-age cell seeding density at 4 h; these rates were usedfor statistical analysis. Figure 6(A) compares celldensities and proliferation rates for different patternsizes. HUVECs were seeded on several nanopat-terned surfaces with varying adhesion site sizes butwith a constant average FN surface coverage of7.2%. Initial attachment densities were similar for allpatterns, as were the initial proliferation rates, whichwere in good agreement with literature values for



pooled HUVECs.75 However, beginning after 48 h,but clearly after 72 h, HUVECs on the 0820M and1500M surfaces displayed statistically significantincreased proliferation compared with the 0300M

(p < 0.003) and 0420M (p < 0.04) surfaces, indicatinga trend of increased proliferation with increasingadhesion site size.

Figure 6(B) compares cell density and proliferationof HUVECs on the two smallest pattern sizes with 2.2and 7.2% average FN surface coverage to cells seededon nonpatterned FN-coated gold slides with FN den-sities ranging from 537 to 2605 FN/lm2 at 4, 24, 48,and 72 h in culture. HUVECs were also seeded on aclean glass surface at a FN concentration of 10 lg/mL(data not shown), and the cells responded similarlyto cells on the gold surface at the same FN concentra-tion. Initial cell-seeding densities after 4 h were sig-nificantly higher (p � 0.001) on both nanopatternedsurfaces compared with the homogenously coatedgold surface with the lowest FN surface density (537FN/lm2), even though the homogenously coated sur-face presents an order of magnitude higher averageFN density compared with the 0300B nanopatternedsurface. There was no significant difference in initial cellseeding densities between the nanopatterns and thenonpatterned higher ligand densities (>1199 FN/lm2),implying that the ability to quickly cluster integrinsto form well-defined adhesion sites is more influen-tial in promoting initial cell attachment than theoverall ligand surface density.

After 24 h, cells on 0300B proliferated at a statisti-cally significant lower rate than on homogenouslycoated surfaces displaying at least 1199 FN/lm2,and these differences were maintained for 3 days inculture (p � 0.03). The 0300M surface induced prolif-eration that was not statistically different from thehomogenously coated surfaces of at least 1199 FN/lm2

within a p-value of 0.3, but the proliferation differ-ence compared with the highest nonpatterned den-sity reached a p-value of 0.06 at 48 and 72 h. The1199 FN/lm2 sample is somewhat inconsistent, as ithas higher proliferation than both the 0300M sampleat 72 h and the highest density nonpatterned surfaceat 24 h [Fig. 6(B)]. This behavior could be caused bya partial breakdown of the BSA block and serumproteins adsorbing at later time points. The fact thatthe 0300M and 0300B samples are not significantlydifferent, even though the 0300B sample is differentfrom the higher density nonpatterned samples andthe 0300M is not, suggests that there may be a grad-ual difference that may be resolved with a largernumber of samples. We conclude that the adhesionsite size and to some degree the pattern pitch areimportant regulators of proliferation, and this de-pendence is much more pronounced at later timepoints. However, the local ligand density is the mostimportant determining parameter, as adhesion andproliferation are supported at average FN densitieson the nanopatterns that are much lower than thelowest nonpatterned surface density that supportsproliferation with 537 FN/lm2.

Figure 6. (A) Influence of the pattern size: PooledHUVEC densities as a function of increasing nanopatternsize for M-type samples, each at 24, 48, and 72 h. Statisticaldifferences are given for proliferation, that is the cell den-sity relative to the cell density at 4 h. Initial seeding den-sities are very similar for all patterns. A significantincrease in cell proliferation (p < 0.05) was observedbetween the smaller and larger nanopatterned surfaces (n5 20, error bars 5 1 SD). *Statistically higher proliferationrate compared with 0300M. **Statistically higher prolifera-tion rate compared with 0420M. (B) Influence of the aver-age FN density: Nonpooled HUVEC densities as a functionof FN surface concentration (FN/lm2) at 4, 24, 48, and72 h of incubation. Surfaces displaying nanoclusters of FNcan support adhesion and proliferation at orders of magni-tude lower FN surface concentrations than homogenouslycoated surfaces. All surfaces induce significantly higher(p < 0.002) proliferation than the nonpatterned 537 FN/lm2

surface at all times, including the initial seeding. While the0300M surface shows statistically similar proliferation tothe higher-density nonpatterned surfaces, the 0300B sur-face induces statistically significantly less proliferation atlater time points than the higher-density nonpatterned sur-faces (n 5 20, error bars 5 1 SD). *Statistically higher pro-liferation rate compared with 0300B (p < 0.03). **Statisti-cally higher proliferation rate compared with 0300M (p <0.03). #Statistically higher proliferation rate than the 2474and 2605 FN/lm2 surfaces (p < 0.03).

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Cell spreading area

The projected cell spreading area was measured at24-h intervals for 3 days for glass control (2092 FN/lm2) and four nanopatterned surfaces. A lognormaldistribution was used to cope with the high levels ofskewness towards higher spreading areas.76 ANOVAand a nonparametric statistical analysis were per-formed to take the skewed distribution into account.Results from both ANOVA (ln) and nonparametric(np) analysis techniques indicate that after 24 h theHUVECs on the nanopatterned surfaces spread sig-nificantly less (pln � 0.07, pnp � 0.003) than cells onnonpatterned control surfaces (Fig. 7). After 3 days inculture, the cell spreading area on the nanopatternedsurfaces was no longer as significantly different (pln �0.45, pnp � 0.05) from the glass control, yet theHUVEC spreading area on the largest nanopattern,1500M, was significantly higher than cells on any ofthe other surfaces (pln � 0.006, pnp < 0.0001) (Fig. 7).The higher spreading on the 1500M nanopattern withan adhesion site area close to 0.1 lm2 and a pitch ofnearly 1 lm compared with the nonpatterned glasscontrol could indicate the influence of the relativelylarge distance between individual adhesions or thebeginning of a qualitatively different behavior thatstarts at around 0.1 lm2 per adhesion, as suggestedby the proliferation data (see: Influence of Nanopat-terned Surfaces on Cell Proliferation).

DISCUSSION

Nanopattern fabrication and functionalization

Integrin binding to its ECM ligand and integrinclustering are synergistic events needed to induce sig-naling and the full engagement with the cytoskeleton.Cluster size is an indicator of the maturation ofadhesions, which is driven by the applied force of theactomyosin machinery of the cell. The size of eachcluster can range from about 30 nm for a trimer, tosubmicron-sized for motile cells, to many microns forfully matured adhesions. Because of this wide rangeof adhesion sizes, restrictions in the growth processinduced by chemical patterns or nanotopographicalsurface features are expected to influence both the ad-hesion of adhesion-dependent cells and the eventsinitiated and controlled by adhesion.

Nanotopographic surfaces created by a variety oftechniques have been shown to influence cell adhe-sion in several ways, including promoting alignmentto grooves,77 decreasing seeding efficiency, adhesionstrength, and spreading on nanopits,78–80 as well asincreasing cell adhesion, spreading area, and cytoske-letal formation on raised nanopatterns.81,82 The mech-

anism of nanotopography action is not clear, butincreased filopodia formation,83,84 Rac localization tothe cell periphery,81 and an upregulation of genesassociated with cell signaling, proliferation, cytoskele-tal components, and ECM production82 have all beendocumented. While ECs and fibroblasts showincreased cellular responses with decreasing nanoto-pography,81 osteoblasts behave oppositely, displayingincreased adhesion, proliferation, and deposition ofextracellular calcium with increasing topography.85–89

Nanotopographic surfaces have been suggested toinduce selective adsorption of ECM proteins,89 andwhile they demonstrate the importance of nanome-ter-scale features on cell behavior, they have so farnot provided systematic insight into the connectionsbetween adhesion site properties and downstreamcell behavior.

Chemically defined patterns of cell adhesionligands, on the other hand, provide direct molecularcontrol over the processes of cell adhesion as well asover the mechanics of force transduction. This con-trol has been used very successfully to investigatecell adhesion, spreading, and differentiation on themicron scale with such tools as microcontact printingof thiols on gold. Cell adhesion size control down to0.1 lm2 using thiol stamping has shown that cellscan adhere and spread well using such areas forintegrin binding, unless the distance between the ad-hesion sites exceeds 2 lm.50 The extension into nano-meter-scale adhesion size control has been muchmore difficult, as serial lithography techniques, such

Figure 7. Measured average cell spreading areas on fournanopatterned surfaces and a glass control with 2092 FN/lm2. HUVECs on nanopatterned surfaces are significantlyless spread than those on control surfaces after 1 day, butthe significance decreases after 3 days. Cells on the 1500Msurface spread much more than cells on nanopatternedand control surfaces after 3 days (n 5 107, error bars 5 1SD (based on lognormal)). *Statistically significant (pln �0.45, pnp � 0.05) less spreading than cells on control; **Stat-istically significant (pln � 0.07, pnp � 0.003) less spreadingthan cells on control; **Statistically significant (pln � 0.006,pnp � 0.0001) less spreading than cells on 1500M.



as dip-pen nanolithography,44,90–93 other scanningprobe lithography techniques,94–97 and electron beamlithography,98–100 are too time consuming in theirfabrication to allow for systematic cell studies. Paral-lel patterning techniques such as imprint lithogra-phy,101,102 micelle nanolithography,103,104 particlelithography,56 and capillary lithography45 are bettersuited for quickly fabricating nanostructured surfa-ces over large areas.

Several nanopatterning techniques have been usedto demonstrate cell adhesion,105–107 but have so farnot been used for systematic adhesion studies. Theseinclude capillary or imprint lithography,45 molecularassembly patterning by lift-off (MAPL),108,109 andvariations of NSL.110,111 Protein and cell adhesionhave been observed on PEG pillars, but due to therepulsive properties of PEG, the level of cell adhe-sion was significantly lower than adhesion to simi-larly treated protein-coated glass surfaces.45 Othertechniques such as micelle lithography or starpolymers have provided significant insights into theimportance of integrin proximity in the clusteringprocess, but they do not allow for the formation oflarger adhesion sites except when used in conjunc-tion with a serial technique such as e-beam lithogra-phy. Additionally, most techniques rely on RGDsequences for cell adhesion, and investigations ofcell interactions with the full FN protein have notpreviously been possible on nanopatterns.

Experimental tools for systematic investigations tocontrol the adhesion size from about 40 nm to about300 nm, a size range in which a number of criticalmolecular and mechanical processes in cell adhesionmay occur, have been missing so far. We have shownthat NSL, a highly parallel self-assembly process, canbe used to create large, cm2-sized areas of nanopat-terns of exactly this pattern size range, with definedsize, spacing, and topography. These nanopatternedsurfaces are a composite of two materials and cantherefore be chemically modified with two distinctchemical functionalities. Our results with XPS, AFM,and cell-seeding experiments consistently show thatmodification of the glass background with PEG andthiolization of the gold nanostructures directs FNadsorption exclusively to the gold nanostructures. Byperforming the functionalization for both materialssimultaneously, we minimized the PEG adsorption tothe gold areas, and the lack of sulfur on the glassareas indicates no or little thiol on the PEG surface.The ability to immobilize the full FN on the goldareas enables the use of the synergistic sites in FN.The terminal functionality used here presents FN in acompact form similar to that found on nonpatternedhydrophobic surfaces,66 but changing the thiol func-tionality to a hydrophilic end group would induceFN to appear in an extended form.66 This flexibilitywould allow us to test the preferential engagement of

different integrins based on the presentation of FN ondifferent thiol terminal functionalities.112,113 Althoughwe have used noncovalent immobilization of FN onthe nanopattern, the use of other functional thiolswould allow specific and covalent anchoring by bio-conjugation procedures. Of course, the presentationof small peptides tethered to thiols could also be eas-ily achieved.

NSL has clear limitations: only two pattern size tospacing ratios can be achieved, resulting in a constantaverage FN surface density of 7.2% or 2.2% for M andB samples, respectively. Pattern size and spacing arecoupled, but pattern pairs from M and B samples canbe created that display similar adhesion site areaswith varying interadhesion site spacing and viceversa. Since spheres are used as a mask, the choice ofpattern shapes is strongly limited and mostly consistsof triangles and disks, a limitation that, however, isunlikely to be important for the goal of adhesion sizelimitation. Also, as a self-assembly method, NSL hasdefects that possibly could present additional adhe-sion sites. However, we have not found an effect oncell behavior attributed to the inherent defects in thepatterns fabricated by NSL, and Figure 5(I,J) showthat the extensions terminate on small patterns, eventhough the possibility for attachment to defects ispresent. Although we have not found the limitationsof NSL to have a significant influence on cell behaviorin our experiments, a second generation of large-scalenanopatterned surfaces using nanoscale orthogonalbiofunctionalization imprint lithography to createdefect-free surfaces with a wide range of patternproperties is being developed.102

Nanopattern influence on endothelial cell behavior

Studies using homogenous surfaces have shown adirect correlation between FN surface density andendothelial and epithelial cell proliferation andspreading.73,114 The selective adhesion of FN tonanopatterned surfaces changes the overall averagedensity of FN, and allows comparative studies ofcell attachment, spreading, and proliferation betweenoverall and local densities of FN. As confirmed byXPS, M-type and B-type nanopatterned surfaces con-tain 7–8% and 2%, respectively, of the saturationamount of FN on a homogenous gold surface, inagreement with the average surface coverage of thepattern. We could show that seeding density as wellas proliferation are mostly not determined by the av-erage FN density, as cells on both the 7% and the2% coverage surfaces seeded and proliferated farbetter than on the �20% (537 FN/lm2) coveragenonpatterned surface, and at average FN concentra-tions more than 10 times lower than would support

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cell proliferation on homogeneously coated FN sur-faces found in earlier studies.73

At later time points, pattern size and possibly pitchbecome important, as indicated by the significantlylower proliferation of cells on 0300M and 0420Msamples relative to cells on the larger pattern sizes,and by the lower proliferation of cells on the 0300Bsample compared with cells on the high-density non-patterned samples. The time dependence of our pro-liferation results suggests that initially the local FNdensity is important to support fast cluster formationof integrins regardless of adhesion site size. Since ho-mogenous substrates with low levels of FN are ran-domly covered and the nanopatterns induce the for-mation of discrete adhesion sites of densely packedFN, nonpatterned surfaces would need a far densercoating to support the same local density of adhe-sions, as has been suggested recently with experi-ments on larger cluster sizes.43,46,50 At later timepoints proliferation is influenced not only by the localFN density, but also by adhesion size and possiblyby the distance between adhesions. On the high-den-sity nonpatterned surfaces adhesions are not limitedin size, while on the small nanopatterns the restric-tion of an adhesion to below a threshold pattern sizebetween 250 nm (0800M) and 100 nm (0420M) leadsto slower proliferation. This corresponds to some-where between 20 and 90 densely packed FNs pernanoisland. Also, it is interesting to note that the low-est nonpatterned FN surface concentration, whichhas an average density of 537 FN/lm2 and very lowcell proliferation rates, has an average distancebetween FN molecules of 43 nm. This value is belowthe 58–70 nm separation between cyclic RGDs foundto be the threshold to support fibroblast adhesionusing nanopatterned cyclic RGD.43 This discrepancycould be explained either by the fact that in thatstudy adhesion and spreading but not proliferationwere tested, or otherwise by an inhomogeneous dis-tribution of FN in our nonpatterned experiments orby a significant portion of the adsorbed FN havinginaccessible binding sites.

Similar to proliferation, cell spreading area is com-monly used to determine cellular reaction to a sub-strate. Endothelial cell spreading and proliferationhave been linked: as the FN concentration on a sur-face increases, so do both the extent of cell spreadingand the proliferation rates.73 Nanotopographic surfa-ces have shown the ability to increase cell spreadingin both endothelial cells86 and fibroblasts,82 whilevery high nanocolumns (160 nm) can reverse thiseffect in fibroblasts.84 We show reduced spreadingwith HUVECs seeded onto FN nanopatterns withminimal topography of 10–13 nm compared with thenonpatterned controls after 24 h. Our HUVEC resultsshow the opposite tendency to endothelial cells iso-lated from human tendon granuloma on 13-nm high

random topography.86 This discrepancy could be aresult of different cell types or due to differences inthe surface properties and ECM adsorption. The factthat HUVECs on the 1500M surfaces with an individ-ual adhesion site area of �0.1 lm2 and an interadhe-sion site spacing of 0.9 lm spread more after 72 hthan on a glass control could be attributed to the largespacing between the adhesion site areas. However,the cells on 1500M surfaces also spread more than onsurfaces with smaller nanopatterns, which correlateswith the increase in proliferation starting around0.04 lm2, suggesting that there is a different qualityto the adhesion for adhesion sizes above �0.04 lm2.Experiments to systematically probe the cell adhesivebehavior for adhesion site areas of 0.01–0.05 lm2 arein progress.

A threshold of 0.01–0.05 lm2 for a single adhesionarea is interesting, as this area is less than half thesmallest pattern of 0.1 lm2 investigated by Lehnertet al.50 Their results showed that such a patternleads to cell behavior indistinguishable from homo-geneously coated surfaces as long as the distancebetween adhesions (pitch) was below 2 lm. Such athreshold is far smaller than the 1 lm2 thresholdfound in force studies. Cellular traction and forcestudies have shown a linear relationship between ad-hesion site size and the force applied to the surfacefor adhesion sites larger than 1 lm2, with largeradhesions applying higher forces per adhesionarea.27,115,116 Similar studies have also shown aninverse trend, with adhesions smaller than 1 lm2 atthe leading edge of cell movement applying higherforces per attachment area than larger adhe-sions.27,117 Additionally, it has been shown thatlarger well-spread cells exert more force on the sur-face than smaller rounded cells.118

Vinculin and AFM imaging suggest a differencebetween the size and distribution of adhesions, withlarge adhesions mainly along the cell perimeter on aglass control, and small adhesions distributed moreevenly across the cell for the nanopatterned surfaces.These data taken from a few cells need to be studiedsystematically and a study of the adhesion size anddistribution induced by the nanopattern is in prepa-ration. Our analysis of individual adhesions, as char-acterized by vinculin stains and AFM height scans,has shown that cellular adhesions are located at nano-patterns, although the adhesion complex may bridgebetween patterns. The complexes can therefore reachlengths in the micron range, but their width is onlyaround 120 nm, leading to adhesion areas well below1 lm2. This corresponds to the nanoisland size usedhere, and preliminary data suggest that this correla-tion between width of the adhesion and nanoislandsize holds for other patterns; further studies into thisrelationship are under way. The adhesion sites arelinked to long elongated structures which are most



likely actin microfilaments. Although we have beenunable to perform dual-label experiments for adhe-sions and actin with the current fluorescence micro-scope AFM combination, we have consistently seenwith double-label experiments that adhesions on allnanopatterns are endpoints of actin fibers. Addition-ally, the height measured with AFM matches thethickness of microfilaments of 7 nm. The 13–17 nmheight of the adhesions is independent of their lateraldimensions on nanopattern and control surfacesalike. Our AFM results of the HUVEC adhesions onglass agree qualitatively with earlier studies, exceptthat our adhesion height is lower than found for largeadhesions of rat embryo fibroblasts after 72 h on glasssurfaces that had been de-roofed to allow for AFManalysis of the adhesions in cytosolic buffer.119 We at-tribute this to our much more rigid crosslinking andimaging in air that could lead to a more collapsedstructure.

While the nanopattern clearly influences the sizeand distribution of adhesions, it appears that theadhesions can sometimes bridge several patterns.The question therefore arises whether integrins arelimited to the nanopatterns and the cytoplasmiccomponents, such as vinculin, are able to bridge, orwhether the integrins are also anchoring to the surfa-ces between the patterns. Although we do not havea positive proof at this time, XPS analysis has shownalmost no FN between the nanoislands, cell reseed-ing after 3 days in medium containing culture hasshown little increase in the cells’ ability to adhere toPEGylated surfaces, and AFM scans have shown lit-tle evidence for FN outside the pattern before andafter cell seeding. However, further experiments onthe distribution of integrins and other cytoplasmicadhesion proteins are ongoing to determine whichadhesion components can bridge the gap betweentwo individual nanoislands, and what maximum dis-tance can be bridged. Furthermore, the coupling offewer actin fibers to the adhesions on nanopatternedsurfaces than on nonpatterned surfaces supports ourview that mechanotransduction and the applicationof force to the surface is key to the modulation ofcell behavior by adhesion size restriction.

It is interesting to further compare cellular mecha-notransduction on homogeneous but elastic sub-strates with our rigid but nanopatterned substrates.ECs on elastic substrates show a strong correlationbetween elastic modulus and spreading, while pro-liferation is unaffected.24 Our findings of a relativelysmall proliferation decrease and decreased cellspreading of ECs on smaller nanopatterns suggeststhat restricting integrin clustering with nanopatternsand reducing force generation at adhesion sites withelastic surfaces may induce changes in cell behaviorthrough a similar pathway. Considering the impor-tance of cellular mechanotransduction for cell differ-

entiation and phenotype,120 the nanopatterned surfa-ces presented here to control integrin clusteringcould provide a complementary tool to elastic sub-strates for understanding the molecular mechanismsof integrin-mediated mechanotransduction. Furtherstudies relating focal adhesion properties to cytoske-letal formation and cell motility will elucidate thesequestions.

CONCLUSIONS

We have demonstrated a fabrication technique tocreate nanopatterned surfaces with dual chemicalfunctionality that allow for directed protein adsorp-tion on nanoislands from 90 to 400 nm in size. Cellsadhere to the fibronectin residing on these nanois-lands and fibronectin is rearranged after cell adhe-sion. The cells build anchor sites that are directed tothe nanoislands, but the cytoplasmic complex of theadhesions may be able to bridge between close adhe-sions. Adhesions are much smaller and more homo-genously distributed on the nanopattern than oncontrol surfaces. Proliferation at all times and on allnanopattern sizes tested is far higher than on non-patterned surfaces of equivalent or up to 10 timeshigher average FN surface concentration. The nano-patterned surfaces induce changes in proliferationthat depend on time. Early proliferation is independ-ent of pattern size and equivalent to that seen onhigh surface density nonpatterned surfaces. Later,the proliferation is influenced by pattern pitch, oraverage FN surface density, and pattern size below�0.05 lm2. The FN nanoclustering also induceschanges in the HUVEC spreading area, with initiallyless spreading on all patterned surfaces than on con-trol. After 72 h the difference becomes less signifi-cant, but again the �0.1 lm2-sized pattern induces ahigher spreading then all other samples. Theobserved changes in cell behavior on the nanopat-terned surfaces are not merely a result of the averagedensity of ECM proteins, but indicate a balance oflocal FN density, adhesion size, and distancebetween adhesions. The results for cells seeded onchemically nanopatterned surfaces suggest an impor-tant role of the forces the cell can exert onto the sur-face via its adhesions, and paint a complementarypicture on rigid nanopatterned surfaces to cellsseeded on elastic substrates. Because of the differen-ces seen for cells adhering to nanoislands below asize of 0.1 lm2, the results highlight the importanceof this size range of very small adhesions for cellsignaling. Our approach may therefore provide anew view on the molecular mechanisms of cellularmechanotransduction and the development of tissueengineering constructs.

192 SLATER AND FREY


The authors thank Dr. Martin Poenie (UT Austin) andDr. David Boettiger (U Penn) for insightful discussions.JHS is a Graduate Research Fellow of the National ScienceFoundation and a Thrust Fellow of the College of Engi-neering of UT Austin.

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