influence of systematically varied nano-scale topography on cell morphology and adhesion

Cell Communication and Adhesion, 14: 181–194, 2007Copyright C© Informa Healthcare USA, Inc.ISSN: 1541-9061 print / 1543-5180 onlineDOI: 10.1080/15419060701755594

Influence of Systematically Varied Nano-Scale Topographyon Cell Morphology and Adhesion

SEPIDEH HEYDARKHAN-HAGVALL1, CHANG-HWAN CHOI2, JAMES DUNN3, SANAZ HEYDARKHAN1,KATJA SCHENKE-LAYLAND4, W. ROBB MACLELLAN4, AND RAMIN E. BEYGUI1

1Department of Surgery, Regenerative Bioengineering and Repair Laboratory, University of California Los Angeles, Los Angeles,California USA

2Department of Mechanical & Aerospace Engineering, University of California Los Angeles, Los Angeles, California USA3Department of Bioengineering, University of California Los Angeles, Los Angeles, California USA

4Department of Medicine and Physiology, Cardiovascular Research Laboratory, University of California Los Angeles, Los Angeles,California USA

The types of cell–matrix adhesions and the signals they transduce strongly affect the cell-phenotype. We hypothesized that cells sense and respond to the three-dimensionality of theirenvironment, which could be modulated by nano-structures on silicon surfaces. Human foreskinfibroblasts were cultured on nano-structures with different patterns (nano-post and nano-grate)and heights for 3 days. The presence of integrin α5,β1, β3, paxillin and phosphorylated FAK(pFAK) were detected by western blot and immunofluorescence. Integrin β3 exhibited strongersignals on nano-grates. pFAK and paxillin were observed as small dot-like patterns on the cell-periphery on nano-posts and as elongated and aligned patterns on nano-grates. Collectively,our observations highlighted the presence of focal (integrin β1, β3, pFAK, paxillin), fibrillar(integrin α5, β1) and 3-D matrix (integrin α5, β1, paxillin) adhesions on nano-structures. Thepresented nano-structures offer interesting opportunities to study the interaction of cells withtopographical features comparable to the size of extracellular matrix components.

Keywords cell adhesion, integrins, nano-structure

INTRODUCTION

The interaction of cells with their microenviron-ment plays a central role in many biological phe-nomena. Knowledge of these interactions is crucialfor the understanding of many fundamental biologi-cal questions and for the design of medical devices.Two-dimensional surfaces such as plastic or glass,

Received 27 November 2006; accepted 19 August 2007.The authors thank Dr. Chang-Jin “CJ” Kim (UCLA Mechanical Engineering) for providing the nano-structures, Dr. Benjamin Wu (UCLA

Bioengineering) for technical and scientific advice and Dr. Jens Kreth (UCLA School of Dentistry, Oral Biology and Medicine) for microscopefacility. This work was supported by grants from American Heart Association (grant 0465009Y).

Address correspondence to Ramin E. Beygui, M.D. UCLA School of Medicine, 10833 Le Conte Avenue, Los Angeles, CA 90095-1741.Phone: (310) 267-4385; Fax: (319) 825-7473. E-mail: [email protected]

which are frequently used as conventional cell cul-ture substrates, force the cell to adjust to an arti-ficially flat and rigid environment. These in vitroconditions are useful for understanding the cell-substrate interactions but they do not mimic thephysical three-dimensionality, complexity and pli-ability of extracellular matrix (ECM) that supportcells in vivo. Mechanical interactions with physical

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182 S. HEYDARKHAN-HAGVALL ET AL.

scaffolds, structure and topology of the matrix, sol-uble and diffusible factors drive the different statesand functions of a cell (Chen et al. 2004; Sniadeckiet al. 2006; Alberts et al. 2002; Hay 1991; Burridgeand Tsukita 2001; Lo et al. 2000; Yeung et al. 2005).

It is well known that cells respond in differentways to their surrounding environment as well asto the nature of the environment (3-D versus 2-D),resulting in the formation of a variety of adhesionstructures such as focal, fibrillar and 3-D matrixadhesions (Geiger and Bershadsky 2001; Yamadaet al. 2003; Cukierman et al. 2002; Cukierman et al.2001). Cell–matrix adhesions mediate physiologicalresponses regulating cell growth, migration, differ-entiation, survival, tissue organization, and matrixremodeling (Sniadecki et al. 2006; Schwartz 2001;Geiger and Bershadsky 2001; Hynes 1999; Cukier-man et al. 2001; Humphries et al. 2004; Yamadaet al. 2003). Cells can integrate these interactionsthrough clustering of integrin receptors, recruitingfocal adhesions and/or cytoskeleton proteins to ad-hesion sites, and regulating cytoskeleton tension inorder to appropriately respond to their surroundings(Humphries et al. 2004; Yamada et al. 2003; Mid-wood et al. 2004; Ruoslahti and Obrink 1996; Ru-oslahti and Engvall 1997). The surrounding ECM isorganized and sensed by cells via different types ofintegrins.

Integrins are membrane bounded heterodimericreceptors that mediate communication between theECM and cells. Their extracellular domains bindwith low affinity to the ECM and intracellular do-mains link to the cytoskeleton. These molecules, intheir inactive state, are freely diffusive within the cellmembrane until they recognize an available bind-ing domain in the ECM. Upon ligand binding, in-tegrins undergo a conformational change that leadsto the recruitment of cytoplasmic proteins, i.e. thosethat biomechanically connect the integrins to the cy-toskeleton and those that biochemically initiate orregulate intracellular signaling pathways (Ruoslahtiand Obrink 1996; Ruoslahti and Engvall 1997; Bur-ridge and Chrzanowska-Wodnicka 1996). Throughclustering of multiple integrins, more cytoplasmicproteins are recruited to the adhesion site to in-

crease its size, adhesion strength, and biochemi-cal signaling activity. These larger, clustered struc-tures of integrins and cytoplasmic proteins are focaladhesions which act as sensors of the ECM envi-ronment and play a key role in signal transduction(Cukierman et al. 2001; Yamada et al. 2003; Bur-ridge and Chrzanowska-Wodnicka 1996; Cukiermanet al. 2002).

The types of cell–matrix adhesions organized byintegrins in vitro and the signals they transduce arestrongly affected by the flat, rigid surfaces of tissueculture dishes,;hich suggest that a cell probes thestiffness of the ECM and regulates itself accordingly.The stiffness of ECM dramatically affects many cel-lular processes and cell differentiation (Pelham andWang 1997). It has been reported that whether a cellproliferates or dies is rather determined by the de-gree to which a cell physically extends than by theamount of integrin-ligand interactions (Chen et al.1997). It has also been shown that substrate flexibil-ity affects the size of focal adhesions as well as thestrength of adhesion (Guo et al. 2006).

For the binding interactions between cells andsurfaces, it is obvious that cells are influenced bystructural compositions and mechanical forces atmicroscale and nanoscale. The aim of the presentstudy is to investigate the hypothesis that humanforeskin fibroblasts (HFFs) sense and respond to thethree-dimensionality (patterns and heights) of nano-structures.

MATERIALS AND METHODS

All materials were purchased from Sigma (St.Louis, MO) unless otherwise noted. All tissue cul-ture reagents and fetal bovine serum (FBS) were pur-chased from Invitrogen (Carlsbad, CA).

Fabrication of Nanostructures

Silicon nano-structures with superior control ofpattern regularity were fabricated using interferencelithography and deep reactive ion etching (DRIE) to

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NANO-SCALE TOPOGRAPHY AFFECTS CELLS 183

create nano-structures with low, mid and high as-pect ratios (ARs, ratio of height over width). Sharptips have been obtained by thermal oxidation fol-lowed by buffered oxide etching (BOE) (Choi andKim 2006). The interference lithography system ofLloyds-mirror configuration using the HeCd laser ofthe wavelength of 325 nm (Nanotech, University ofCalifornia, Santa Barbara) was used for the fabrica-tion of nanopatterns in this study. A polished siliconsubstrate (2 × 2 cm2) was cleaned with a Piranhasolution (H2SO4:H2O2, 3:1 in volume) and dehy-drated for 10 minutes at 150 ◦C. SPR3001 photore-sist (Shipley, Co.) was then spin-coated at 5000 rpmfor 1 minute, which gives ∼50 nm film thickness. Af-ter the spin coating, soft-bake was done at 95 ◦C for 1minute on a hot plate. The substrate was then exposedunder the laser interference lithography setup fol-lowed by post exposure bake at 115 ◦C for 1 minute.The exposed substrate was developed by MF701 de-veloper (Shipley, Co). The substrate was rinsed withde-ionized water after the development and blow-dried with N2 gas followed by 1 minute hard-bakeat 110 ◦C on a hot plate. The patterned photoresistwas scanned by atomic force microscopy (AFM)to check the development. The substrate was thenetched by deep reactive ion etching (DRIE) using thepatterned photoresist as an etching mask. After theDRIE, the remaining photoresist was removed andcleaned with the Piranha solution. Nano-structurewith the positively-tapered profile was further sharp-ened by thermal oxidation and buffered oxide etch(BOE). The size of the nano-structures could alsobe controlled by the timed oxidation. The sample of2 × 2 cm2 was then cleaved by using a scriber intofour chips of 1 × 1 cm2, which was used for multipleexperiments guaranteeing the identity of the patterngeometries. Planar silicon samples (1 × 1 cm2) of apolished surface were also prepared as controls. Theroot mean square surface roughness of the smoothcontrol samples was measured to be less than 1 nm byAFM in a 10 × 10 µm2 scan area. Two different pat-terns, i.e. nano-post and nano-grate, were fabricatedwith three different ARs. ARs were systematicallyvaried from low (50–100 nm in height), mid (200–300 nm) to high (500–600 nm), while the pattern

periodicity (230 nm) and the tip sharpness (needle-or blade-like sharp tip) were fixed in our samples.

Cell Culture on Nano-Structures

Human foreskin fibroblasts (HFFs, AmericanType Culture Collection [ATCC, Manassas, VA])were cultured in Dulbecco’s Modified Eagle Medium(DMEM) supplemented with 10% FBS and peni-cillin/ streptomycin (100 U/ml, Invitrogen). The cellswere kept at 37◦C and 5% CO2. At confluence, HFFswere detached from the culture dishes using trypsin/EDTA followed by centrifugation (1000 rpm, 5 min).The pellet was re-suspended in the culture medium.HFFs were seeded at the density of 1 × 104/cm2 onthree nano-turf samples and two smooth control sur-faces (silicon and gelatin-coated silicon). Cells werekept in culture for 3 days.

Immunocytochemistry

The cell-seeded samples were rinsed with PBSand fixed in fresh 4% paraformaldehyde for 20 min,followed by 3 washes with PBS 5 min. each. Thecells were then permeabilized with 0.5% Triton X-100 for 10 min, followed by 3 washes with washbuffer (WB, PBS/0.1% Tween-20), for 5 min each.Non-specific antibody binding sites were blocked byincubating the samples for 30 min in blocking buffer(1% BSA, 2% goat serum and 0.5% Triton X-100in PBS). Primary antibodies to integrin subunits α5(1:50, BD Pharmingen, San Jose, CA),β1, (1:50, BDPharmingen), β3 (1:50, Abcam, Cambridge, MA),paxillin (1:50, BD Pharmingen) and phosphorylatedFAK (1:70, Abcam) were diluted in antibody buffer(PBS 1% BSA, 0.5% Triton X-100). The sampleswere then incubated for 60 min at room temperature,followed by several washes. Labeled secondary an-tibodies (Alexa Fluor 488, Alexa Fluor 594, Invitro-gen) were diluted in PBS, applied to the samples andincubated for 45 min at room temperature. After sev-eral washes, the nuclei were stained with DAPI. TheF-actin was stained with Alexa fluor 488 phalloidin

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(Molecular Probes, CA) diluted in PBS. Digital im-ages were captured of the stained cultures with adigital camera (Optronics, CA) from a Leica DMIRB inverted microscope equipped with 20x (0.40numerical aperture (NA)), 40x (0.75 NA) and 100x(1.25 NA) objectives.

Western Blot

Western blot was performed on HFFs cultured onthe nano-structures for 3 days. In order to obtain asufficient amount of protein, cells from three paral-lel samples were pooled together. Cell lysates wereobtained by adding lysis buffer containing 20 mMTris, 1% Triton X-100, 0.1% SDS, 1 mM NaF,1 mM Na3VO4, 150 mM NaCl directly to each sam-ple, followed by centrifugation for 10 min at 4◦C.The protein concentrations were determined usinga standard curve of BSA (Bradford dye reagent,Bio-Rad Laboratories, Inc, Richmond, CA). Pro-tein molecular weight markers and equal amountsof protein (40 µg) from each sample were loaded tosodium dodecyl sulphate polyacrylamide gel elec-trophoresis (SDS–PAGE, 4–20%, Bio-Rad). Afterelectrophoresis, the separated proteins were trans-ferred from the gel onto a PVDF membrane (Bio-Rad). The membrane was treated with 5% non-fatmilk in TBST to block nonspecific binding and thenprobed with primary antibodies against integrin sub-units α5 (1:1000, BD Pharmingen), β1, (1:2500, BDPharmingen), β3 (1:2500, Abcam), paxillin (1:1000,BD Pharmingen), phosphorylated FAK (1:1000, Ab-cam) and the internal standard, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:1000, SantaCruz Biotechnology Inc, CA), respectively. Afterseveral washes, a diluted horseradish peroxidase(HRP) conjugated secondary antibody (anti rabbitHRP-conjugated IgG or anti mouse HRP-conjugatedIgG, 1:2000, Cell Signaling, Danvers, MA) wasadded to the membranes and incubated for 60 minat room temperature. The positive signals weredetected by Enhanced ChemiLuminescence (ECL,Bio-Rad). The membranes were exposed to a light-image film (Kodak, Sigma) to visualize the bands.

Cell Viability

Viability in HFF cultures was assessed by doublestaining with calcein-AM and propidium iodide (PI,both dyes from Molecular Probes, OR). Calcein-AMis a non-fluorescent, cell-permeable molecule that iscleaved into fluorescent products by nonspecific es-terases. These products are retained in cells with in-tact plasma membranes. In practice, calcein-AM in-dicates both cell vitality (via enzymatic activity) andmembrane integrity (via localization inside the cell).PI is a cell-impermeable, DNA-binding fluorescentdye that stains only the DNA of cells with compro-mised membranes. For HFF cultures, the sampleswere washed once in PBS, then stained for 15 minin pre-warm DMEM containing 2 µM calcein-AMand 5 µM PI. Digital images were captured of thestained cultures with a digital camera from a LeicaDM IRB inverted microscope through green and redfluorescent filters at 20× (0.40 NA) magnification.

Cell Proliferation and Alamar Blue

HFFs were seeded at the density of 1 × 104/cm2 onthe nano-structure samples as well as on polystyreneculture plates. After 24 hours, a mixture of alamarblue (Serotec, Raleigh, NC; in an amount equal to10% of the total culture volume) and culture mediumwas aseptically added to the nano-structure samples.Samples were incubated with alamar blue for 2 hoursup to 3 days. The metabolism levels were evalu-ated on a spectrofluorometer at wavelengths of 570and 600nm (the amount of reduced alamar blue isAbsorbance570–Absorbance600) for each time point.

Scanning Electron Microscopy (SEM)

Cell-seeded samples were rinsed with SEM buffer(0.1M sodium cacodylate buffer, pH 7.2, supple-mented with 5% sucrose) for 10 min. The sampleswere then fixed for 30 min in 2% paraformalde-hyde/2% glutaraldehyde in SEM buffer, followed bydehydration through grades of ethanol, 30, 50, 70,

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80 and 95% for 10 min each, followed by 3 incu-bations in 100% ethanol for 10 min and a final in-cubation in 100% ethanol for 40 min. The sampleswere dried by incubating in one-half volume 100%ethanol and one-half volume hexamethyldisilazanefor 20 min followed by 100% hexamethyldisilazanefor 20 min. Finally, the 100% hexamethyldisilazanesolution was evaporated during 20 min air-drying.Once dry, the samples were mounted onto stubs andsputter coated by gold/palladium (Au/Pd, thicknessof ∼10nm) using Denton Desk II sputtering beforescanning (Hitachi S-4700).

RESULTS

Fabricated Silicon Surfaces with SystematicallyVaried Nano-Scale Topography

To determine if three-dimensionality of the envi-ronment affects cellular adhesion and phenotype, wefabricated solid surfaces with sharp posts (needle-like) or sharp grates (blade-like) with differentheights in a regular pattern. Scanning electron mi-crographs of the engineered nano-surfaces demon-strated the presence of uniform sharp-tip nano-patterns, i.e. nano-posts and nano-grates, on thesamples (Fig. 1). The height was systematically var-

Figure 1. Scanning electron micrographs of silicon-based nano-structures with two different patterns, nano-post (a–c; needle-like) andnano-grate (d–f; blade-like), and with three heights, Low (a, d; 50–100 nm), Mid (b, e; 200–300 nm) and High (c, f; 500–600 nm). The patternperiodicity (230 nm) and the tip sharpness were maintained with no variations.

ied on the samples from Low (50–100 nm), Mid(200–300 nm) to High (500–600 nm). The rangegiven for each height reflects sample-to-sample vari-ation in fabrication. For a given sample, however, theheight deviates less than 10% over the sample area(1 cm × 1 cm). On the other hand, the pattern peri-odicity (230 nm) and the tip sharpness (less than 10nm in tip apex radius of curvature) deviate negligiblyand are also repeatable sample-to-sample.

Silicon-Based Nano-Structures ReduceProliferation Rates of Human Fibroblasts

Calcein and propidium iodide (PI) staining wasused to compare the viability of HFFs cultured onsilicon surfaces with nano-pattern versus on flatsurfaces. The efficiency of attachment of HFFs topolystyrene (PS, Fig. 2a) in a tissue culture dish, non-coated smooth silicon (Fig. 2b) and gelatin-coatedsmooth silicon (Fig. 2c) was indistinguishable. How-ever, HFFs exhibited distinct morphological featureson the surfaces with nano-post structures (Fig. 2d–f), especially on nano-post Mid and nano-post High.Cells on these surfaces were smaller than on nano-post Low. On the surfaces with nano-grate structures(Fig. 2g–i), the cells were longer and they alignedwith the gratings. Positive PI staining of HFFs (dead

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Figure 2. Calcein and propidium iodide staining of cultured HFFs on different surfaces for 3 days. Green color in all micrographs (20×)shows viable HFFs cultured on polystyren culture dish (a), smooth silicon (b), gelatin coated smooth silicon (c), nano-post Low (d), nano-postMid (e), nano-post High (f), nano-grate Low (g), nano-grate Mid (h) and nano-grate High (i). HFFs exhibited a smaller morphology on thesurfaces with nano-post structures (d–f), especially on nano-post Mid and nano-post High. Elongation and alignment of HFFs on nano-gratestructures were clearly observed (g–i). Positive PI-staining of HFFs (dead cells, arrows (→) in a-i) was relatively Low on all surfaces after 3days in culture. The arrows (↔) indicate the direction of the gratings on the nano-structures (g–i). The metabolic activity of HFFs on nano-post(j) and nano-grate (k) was investigated by alamar blue assay. The measured absorbance values follow an exponential trend. Significantly lowerproliferation rate was observed with higher nano-topographical structures.

cells) was relatively low on all surfaces after 3 daysin culture (Fig. 2a–i).

To determine if metabolic activity of the cells wasalso affected by three-dimensionality of the envi-ronment, we measured metabolic activity of HFFsplated on different nano-patterned silicon surfacesusing the alamar blue assay. A significantly lowerproliferation rate was observed with higher nano-topographies, more specifically nano-post-High andnano-grate-High. A significant difference was ob-served on the surfaces with High nano-post patternscompared to nano-grate. No significant differenceswere observed between proliferation rates of HFFscultured on surfaces with lower heights (Fig. 2j–k).

HFFs Attach to Silicon Nano-Structure andOrient Along the Axis of the Nano-Pattern

To investigate the effects of 3-D nano-environment on cell morphology, we cultured HFFs

on surfaces with two different nano-patterns, i.e.nano-post and nano-grate, and three height ranges.SEM revealed that HFFs cultured on smooth control-surfaces with no nano-patterns (gelatin-coated andnon-coated) were spread with a flattened morphol-ogy (Fig. 3Aa–b). HFFs were elongated on nano-post samples with Low and Mid. The cells on nano-post structures with High, were sparse with a roundand small morphology and less cell-surface adhe-sions (Fig. 3Ba–c). HFFs cultured on the surfacewith nano-gratings adapted with an elongated mor-phology and were mostly parallel to one anotherwith increasing structure height (Fig. 3Bd–f). Nano-patterns initiated elongation of HFFs, compared tosmooth control surfaces with no nano-structures;however, this morphological alteration was clearlyobserved on samples with nano-grate pattern. Theorientation of the cells along the axis of the gratingswas observed by SEM (Fig. 3Bd–f) and F-actin stain-ing (Fig. 4). The F-actin fibers were mostly stretchedalong the long axis of the cells. The orientation of

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Figure 4. Immunofluorescence micrographs (40×) of HFFs cultured on silicon surfaces: A) smooth (a), gelatin coated smooth silicon (b),B) nano-post Low (a), Mid (b), High (c), nano-grate Low (d), Mid (e) and High (f) stained for F-actin (green). Nuclei were stained with DAPI(blue).

the cells along the axis of the gratings could be seenmore clearly in HFF cultured on nano-structureswith Mid and High grating patterns (Fig. 4e–f). Be-sides elongation of cell bodies, alignment and elon-gation of nuclei along the long axis of the cell werealso observed on structures with nano-gratings inmost cases (Fig. 4d–f). In addition, the alignmentand elongation could be easily observed when vi-able HFFs, after 3 days in culture, were stained withCalcein and PI (live/dead staining, Fig. 2g–i).

HFFs Adhere to the Nano-Patterned SurfacesThrough Integrin Complexes

In order to determine the effects of a 3-D nano-environment on cell adhesions, the composition ofadhesions between HFFs and surrounding nano-structures was investigated by immunofluorescencestaining for a panel of adhesion molecules. Inte-grin α5 was detected in HFFs cultured on all nano-structures as well as on controls, i.e. smooth sili-con with and without coating, (Fig. 5). Integrin β3

exhibited stronger signals in HFFs cultured on sur-faces with nano-grate pattern compared to nano-postand controls (Fig. 6). pFAK (Fig. 7Aa-b, 7Ba–c) and

paxillin (Fig. 8a–e) were mostly observed as smalldot-liked pattern at the periphery of attached cells onthe surfaces with nano-post structures as well as oncontrols (smooth silicon with and without coating).pFAK (Fig. 7Bd–f) and paxillin (Fig. 8f–g) were ob-served as elongated patterns on surfaces containingnano-grate patterns. These attachment points werealigned with the direction of nano-gratings. Integrinα5, β3, and paxillin could not be detected on Highnano-grate structures due to light interference by thetall structures. Integrin β1was detected in HFFs cul-tured on all nano-structures as well as on smoothsilicon with and without coating (Fig. 9). HFFs cul-tured on the surface with nano-gratings adapted withan elongated morphology and were mostly parallelto each other and to the nano-structures (Fig. 4Bd–f).

To more thoroughly characterize the adhesion ofHFFs to the surrounding nano-structures, the pro-tein expressions of the adhesion molecules were an-alyzed by western blot (Fig. 10). Consistent with theimmunofluorescence data, the presence of differentadhesion molecules such as integrin subunits α5, β1,β3, paxillin and pFAK were detected by western blotin HFFs cultured on the nano-structures with post orgrating pattern with different heights as well as oncontrols.

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Figure 5. Immunofluorescence micrographs (100×) of HFFs cultured on silicon surfaces: smooth (a), gelatin coated smooth silicon (b),nano-post Low (c), Mid (d), High (e), nano-grate Low (f) and Mid (g) stained for integrin α5 (green). Nuclei were stained with DAPI (blue).Integrin α5 could not be detected on nano-grate structures with High due to light interferences.

DISCUSSION

When cells form interfaces with biomaterials,such as scaffolds used in the field of tissue engi-neering or implants, biomaterial surfaces provideextracellular signals. Cells sense, communicate, andrespond to these signals from biomaterial surfacechemistry, topography, charge, surface energy, andwettability (Cukierman et al. 2002; Cukierman et al.2001; Lim and Donahue 2004; Grayson et al. 2004;Boyan et al. 1996). Understanding the mechanismsby which cells sense and respond to chemical andphysical signals from biomaterials will facilitateidentification of novel biomaterial properties thatcontrol cell behavior. Exploiting biomimetic prop-erties of biomaterials is an attractive strategy in de-veloping novel cell-stimulating cues. One importantconsideration is the interaction of cells with nano-scale topographic interfaces in vivo.

In the current study, we fabricated silicon nano-structures of varying height with controlled patternregularity in order to investigate adhesions and re-sponses of HFFs to these topographical nano-scalefeatures. It has been reported that micro- and nano-patterning alter cell shape and influence cellular be-havior. Changes in cell shape can alter the cell cy-toskeleton and intracellular signaling pathways anddiverse cellular processes such as apoptosis, pro-liferation and differentiation (McBeath et al. 2004;Dalby et al. 2003; Itano et al. 2003; Thomas et al.2002). The incorporation of such patterning ap-proaches to biomedical devices could be used to di-rect cell differentiation or behavior to induce, forinstance, stem cell differentiation, and generate de-sired cell types or regulate cell function within 3-Dscaffolds.

Our results demonstrate that HFFs were elongatedon nano-post samples with Low and Mid structures.

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Figure 6. Immunofluorescence micrographs (100×) of HFFs cultured on silicon surfaces: smooth (a), gelatin coated smooth silicon (b),nano-post Low (c), Mid (d), High (e), nano-grate Low (f) and Mid (g) stained for integrin β3 (green). Nuclei were stained with DAPI (blue).Integrin β3 could not be detected on nano-grate structures with High due to light interferences.

Figure 7. Immunofluorescence micrographs (100×) of HFFs cultured on silicon surfaces: A) smooth (a), gelatin coated smooth silicon (b),B) nano-post Low (a), Mid (b), High (c), nano-grate Low (d), Mid (e) and High (f) stained for pFAK (red). Nuclei were stained with DAPI(blue). pFAK was observed as elongated patterns on surfaces containing nano-grate patterns (Bd–f).

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Figure 8. Immunofluorescence micrographs (100×) of HFFs cultured on silicon surfaces: smooth (a), gelatin coated smooth silicon (b),nano-post Low (c), Mid (d), High (e), nano-grate Low (f) and Mid (g) stained for paxillin (green). Nuclei were stained with DAPI (blue).Paxillin was observed as elongated patterns on nano-grate (f, g). Paxillin could not be detected on High nano-grate structures due to lightinterferences.

Figure 9. Immunofluorescence micrographs (100×) of HFFs cultured on silicon surfaces: A) smooth (a), gelatin coated smooth silicon (b),B) nano-post Low (a), Mid (b), High (c), nano-grate Low (d), Mid (e) and High (f) stained for integrin β1 (green). Nuclei were stained withDAPI (blue).

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Figure 10. Western blot of adhesion molecules in HFFs culturedon silicon surfaces: smooth (S); nano-post Low (NPL), Mid (NPM),High (NPH); nano-grate Low (NGL), Mid (NGM) and High (NGH).

The cells showed a rounder and smaller morphologywith less cell-surface adhesions on High nano-poststructures. The observed decreases in cell spread-ing in these structures can be consistent with de-creased available area for adhesion. HFFs culturedon the surface with nano-gratings adapted with anelongated morphology and they were mostly par-allel to the gratings with increasing height. Theelongated morphology and alignment, which resem-ble the natural state of fibroblasts in vivo, suggestthat the nano-patterned surface would be a morefavorable substrate for the culture of these cellsand it might facilitate native self-assembly of ECMmolecules to mediate cell attachment and orientationprocesses. This topography-related alignment andguidance might be used for further mimicking of invivo conditions. Attachment of cells to a surface re-lies on orientational control of ECM protein surfaceadsorption and molecule conformation. Interactionbetween substrate and cells directly influences cellbehavior, communicated from membrane (integrin)receptors via cytoskeletal and biochemical networksto influence cell growth, ECM formation and ori-entation (Mrksich and Whitesides 1996; Burmeisteret al. 1996; Wilson et al. 2001). It has been shownthat aggregation of ECM molecules such as collagenis important in formation of a fibrillar network in vivo(Bozec and Horton 2005), though the mechanism isnot well understood. Surface patterning might bean important issue in the formation and regulationof matrices for tissue engineering and implantablemedical devices.

Nuclei elongation and alignment was observed inthis study on the samples with nano-grate patterns.

The elongation of cells and nucleus has been corre-lated with changes in gene and protein expressionprofile (Maniotis et al. 1997). The nucleus is me-chanically integrated with the physical entity of thecell. Forces are transferred to the nucleus throughactin-intermediate filament system during changesin cell shape (Dalby et al. 2003). The mechanicaltension causing alignment of cells can rearrange thecentromere through deformation of the nucleus. Mi-crotubule mass in the cell can also be affected viaintegrin–cytoskeleton coupling, which in turn in-fluences the cellular structure and phenotypes ofthe cells (Cukierman et al. 2001; Cukierman et al.2002). A significantly lower proliferation rate wasobserved on the surfaces with High nano-post pat-terns, which could be due to the decrease in cellspreading and change in mechanical force exertedon the cells. We observed the molecular composi-tion and function of the in vitro cell-matrix adhesionsformed by fibroblasts on the nano-structures. It hasbeen reported that in 2-D versus 3-D cell culture, fo-cal, fibrillar and 3-D matrix adhesions have distinctmolecular compositions. Focal adhesions character-istically contain integrin αvβ3 as well as plaque pro-teins such as paxillin, vinculin, and FAK, whereasfibrillar adhesions are composed prominently of in-tegrin α5β1 and tensin. In 3-D, it has been shown thatpaxillin and integrin α5 colocalized rather than local-izing separately (Cukierman et al. 2002, Cukiermanet al. 2001, Yamada et al. 2003). The expression ofintegrins is related to expression of different ECMproteins. Grayson et al. have reported that integrinα2β1 was expressed to a greater extent by humanmesenchymal stem cells in 3-D polymer constructsthan on 2-D surfaces (Grayson et al. 2004). Theyhave also shown that the expression of integrin αvβ3

and integrin α5β1 was lower in their reported 3-Dconstructs. Also, paxillin expression was localizedand concentrated to fewer attachment points. Lim etal. have shown that in human fetal osteoblasts, inte-grin subunits α5, β1 and β3 did not exhibit significantvariation with nono-topographies (Lim et al. 2007).They have also shown that the expression of paxillin,FAK and pFAK were decreased with higher nano-topographies. We detected integrin subunits α5, β1,

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β3, paxillin and pFAK in all cultures in different in-tensities by immunocytochemistry and by westernblot. Fibroblasts initially require culture for longerthan three days to generate 3-D matrices and evolve3-D-matrix adhesions, yet when cultured on the pre-sented nano-structures, they began regenerating 3-Dmatrix adhesions molecules such as integrin α5, β1

and paxillin. Integrin α5 and β1 are components ofthe fibronectin receptors and they are strongly ex-pressed in both 2-D and 3-D cultures. Furthermore,integrin α5and β1 bind to collagen type I and it isreported that they play an important role in orga-nizing its structure (Klein et al. 1991). The expres-sion of integrin subunits α5and β1 in HFFs culturedon the structures may be a result of the presence offibronectin, which is produced by these cells. Theexpression of integrin αvβ3 in endothelial cells is re-lated to cell proliferation (Meerovitch et al. 2003).Integrin β3 exhibited stronger signals in HFFs cul-tured on surfaces with nano-grate pattern. This maybe linked to HFF survival on these structures. pFAKand paxillin were mostly observed as a small dot-like pattern at the periphery of attached cells onthe surfaces with nano-post structures and on con-trol surfaces. The cell attachments were elongatedon surfaces containing nano-grate patterns, and alsoaligned with the direction of nano-gratings. Paxillinis an intracellular protein confined to adhesion pointson the inner cell surface and it is one of severaladaptor proteins that connect integrins to the cy-toskeleton (Turner et al. 1998). The large availablesurface area on smooth surfaces without any nano-topography facilitates many points of attachmentsas shown in Fig 8 compared to nano-structures. Thechanges in the paxillin expression pattern suggest avariation in the manner HFFs influence and interactwith their environment when cultured on the pre-sented nano-structures. Consequently, the types ofadhesion molecules formed under these conditionsvary and cellular responses differ. It has been re-ported that the size of focal adhesions increases withstronger connections between the substrate and thecytoskeleton (Tsuruta and Jones, 2003). In the pre-sented study detached HFFs, mostly in the cell bodyarea, were detected on high nano-structures (data not

shown), which indicates a weak adherence of HFFsto the structures. However, after one week in cul-ture, HFFs formed a “sheet” of elongated and alignedcells, which could easily be peeled off from the grat-ing substrates with high nano-structures (data notshown). High nano-grate structures may offer in-put for design of tissue-engineered scaffolds. For in-stance, orientation of smooth muscle cells (SMC) isessential in providing a strong mechanical propertyfor tissue engineered vascular constructs. The nano-structures reported in this study can direct SMC ori-entation.

Collectively, our observations highlighted thepresence of focal, fibrillar and 3-D adhesions in thepresent nano-structures. Local fibrillar adhesions areimportant for maintaining the dynamic and regener-ation of in vivo matrices. Moreover, although focaladhesions appear to be relatively rare in vivo, smalldot-like structures containing integrin β3 and pFAKmay fulfill anchorage functions in 3-D systems invivo (Wang et al. 2000; Engler et al. 2004). Substratestiffness can also regulate cell migration, adhesion,as well as the strength of adhesion, cell growth andapoptosis which are closely related to cell shape andadhesion and are equally important in tissue forma-tion (Yeung et al. 2005). Furthermore, a report in-dicated that integrin α5 was expressed at a higherlevel on stiff substrates, which may contribute to theenhancement of adhesion (Yeung et al. 2005). In thecurrent study, HFFs may sense the nano-structurestiff since the expression of integrin α5 has been de-tected. HFFs grown on the presented silicon basednano-structures may develop a distinct cell adhesionapparatus and mechanotransduction mechanism thataffects the subsequent tissue development, basedon the hypothesis that surface nano-topography al-ters nuclear morphology in adherent cells leadingto changes in gene transcription. Cells sense forcesduring adhesion to a substrate, which in turn aredirectly transmitted to the nucleus via the cytoskele-tons and altered cytoskeletal tension then feeds backto induce local changes in focal adhesion assembly.Cell-substrate interactions are central to many bio-logical phenomena. Knowledge of these interactionsis important for understanding many fundamental

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biological questions and for design of medical de-vices. By understanding the response of different celltypes to well defined topographies, it may be possi-ble to tailor scaffolds for use with different cell typesto elicit specific responses which control the forma-tion of tissue. Additionally, the application of nano-structures to biomedical devices could be used formodification and improvement of the texture of im-plantable devices such as pacemakers, defibrillators,ventricular assist devices, and vascular grafts. Withthe knowledge that much of the cellular environ-ment in vivo involves nano-scale features, our nano-structures offer interesting opportunities to study theinteraction of cells with topographical features com-parable to the size of ECM components.

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influence of systematically varied nano-scale topography on cell morphology and adhesion

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