PAPER www.rsc.org/softmatter | Soft Matter

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Changes in the internal organization of the cell by microstructured substrates

Maruxa Est�evez,*abd In�es Fern�andez-Ulibarri,c Elena Martı́nez,ab Gustavo Egeac and Josep Samitierabd

Received 4th August 2009, Accepted 5th November 2009

First published as an Advance Article on the web 14th December 2009

DOI: 10.1039/b916038h

Surface features at the micro and nanometre scale have been shown to influence and even determine cell

behaviour and cytoskeleton organization through direct mechanotransductive pathways. Much less is

known about the function and internal distribution of organelles of cells grown on topographically

modified surfaces. In this study, the nanoimprint lithography technique was used to manufacture

poly(methyl methacrylate) (PMMA) sheets with a variety of features in the micrometre size range.

Normal rat kidney (NRK) fibroblasts were cultured on these substrates and immunofluorescence

staining assays were performed to visualize cell adhesion, the organization of the cytoskeleton and the

morphology and subcellular positioning of the Golgi complex. The results show that different

topographic features at the micrometric scale induce different rearrangements of the cell cytoskeleton,

which in turn alter the positioning and morphology of the Golgi complex. Microposts and microholes

alter the mechanical stability of the Golgi complex by modifying the actin cytoskeleton organization

leading to the compaction of the organelle. These findings prove that physically modified surfaces are

a valuable tool with which to study the dynamics of cell cytoskeleton organization and its subsequent

repercussion on internal cell organization and associated function.

Introduction

In vivo, the cellular microenvironment consists of diverse extra-

cellular matrix (ECM) proteins that provide both biochemical

and biophysical cues to cells through three-dimensional surface

topography.1 Cell–substrate mechanical interactions induce

changes in cell function and state, including gene expression,

adhesion, migration, proliferation and differentiation.2,3 In vitro,

cell adhesion to biomaterials is mediated by the chemical and

physical signals that cells receive from neighbouring cells, the

surrounding fluid and extracellular matrices (ECM).4 Both

in vitro and in vivo approaches could benefit from smart

topographical modifications of the substrates, which might

favour tissue integration at the cell–biomaterial interface.5

At the molecular level, cell–material adhesion is mediated by

integrins, which are transmembrane receptors that link the ECM

to the actin cytoskeleton. In the cytoplasmic domain, integrins

associate with a large number of proteins such as a-actinin,

vinculin and paxillin, which are involved in a dynamic associa-

tion with actin filaments.6,7 In the cytoplasm, integrin–actin

interactions are used by the cells as mechanosensors to test the

characteristics of the microenvironment.8,9 When integrins detect

internal or external stresses, intracellular transduction responses

can lead to a different focal adhesion complex configuration and

cytoskeletal organization, which in turn affect the shape of the

aNanobioengineering group, Institute for Bioengineering of Catalonia(IBEC), Baldiri Reixac 10-12, 08028 Barcelona, Spain. E-mail:mestevezf@pcb.ub.es; Fax: +34934037181; Tel: +34934037185bCentro de Investigaci�on Biom�edica en Red en Bioingenierı́a, Biomaterialesy Nanomedicina (CIBER-BBN), SpaincDepartment of Biologia Cel$lular, Immunologia i Neurociencies,IDIBAPS, IN2UB, School of Medicine, University of Barcelona,c/ Casanova 143, 08036 Barcelona, SpaindDepartment of Electronics, University of Barcelona, C/Martı́ i Franqu�es 1,08028 Barcelona, Spain

582 | Soft Matter, 2010, 6, 582–590

cell.10 Changes in cell morphology mediated by mechanical

tension may also cause changes in cytoskeleton organization and

nuclear structure, and thus gene expression and cell cycle

progression are also affected.11

Nanostructures and microstructures are present in the natural

environment of the cells. For example, cell membranes contain

nanosized molecules and the ECM is formed by biomolecules

configured in different arrangements, such as nanopores and

nanofibers. Therefore, it is of particular interest to study the

effect of nano- and microscale topographic structures on cell

behaviour and internal organization of the cell. Indeed, several

techniques derived from the microelectronics industry have been

applied to create topographically modified substrates that are

used in cell culture systems. The aim of these experiments is

a better understanding of the influence of the physical properties

of the substrate on cell morphology, adhesion, alignment,

motility, proliferation and/or differentiation.12 The effects of

micro- and nanostructures on cell orientation and adhesion and

cytoskeleton organization have been widely studied. For this

purpose, a large range of cell types such as fibroblasts,13

keratocytes,14 epithelial cells,4 mesenchymal stem cells3 and

osteoblasts15 have been cultured and studied on a variety of

micro- and nanostructured substrates. Cellular responses to

structured substrates depend on the cell type, shape and size of

the feature. Usually, cells seeded onto artificially-produced

micro- and nanogrooves adapt to them, by adopting an elon-

gated shape in the direction of the groove. This alignment is

accompanied by reorganization of the actin cytoskeleton and

other cytoskeletal elements, which become oriented parallel to

the grooves.16,17 Other topographical features, such as wells or

pits, permit a well-spread morphology and correct development

of cytoskeletal components.5,18

However, very little has been reported about the influence of

different topographies on the localization of subcellular

This journal is ª The Royal Society of Chemistry 2010

Fig. 1 (a) A schematic diagram of the nanoembossing technique. (b) The

oxidized silicon mould layout used for nanoimprinting. The chip size was

60 � 24.5 mm (close to a standard microscope slide) and the different

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membrane organelles. Some studies have examined the effect of

topography on nucleus morphology and centromere positioning

to demonstrate regulation of gene expression.16,19 Given that on

one hand cell adhesion and morphology depend on the topog-

raphy of the substrate, and on the other hand there is a close

relationship between the morphology and function of the Golgi

complex and the cytoskeleton organization and its dynamics,20–22

we postulated that there should be a correlation between

topographically modified substrates and the morphology of this

organelle. The Golgi apparatus in mammalian cells is a ribbon-

like system of stacked cisternae, usually localized in the

juxtanuclear area around the centrosome. It is a key organelle in

post-translational modifications and sorting of lipids and

proteins in the biosynthetic pathway.21

In this study, we used nanoimprint lithography to generate

a variety of physical features on poly(methyl methacrylate)

PMMA in the micrometre size range with geometries of posts

and holes, specifically chosen for their complementary geome-

tries and the same specific area. Normal rat kidney (NRK)

fibroblasts were cultured on the patterned substrates for 24 h

and their morphology was examined by scanning electron

microscopy. Single or double immunostaining assays were per-

formed to analyze focal adhesions, cytoskeleton organization

(microtubules and actin filaments) and the Golgi complex

morphology. The results showed that physical modifications on

polymer surfaces alter the size and spatial distribution of cell

adhesion sites, actin filament organisation and Golgi complex

morphology.

etching regions were 9 � 6.5 mm in size.

Materials and methods

Substrate fabrication

PMMA substrates (125 mm thin sheets from Goodfellow, UK)

were microstructured following a nanoembossing procedure

resulting from the application of a nanoimprinting lithography

technique to free-standing polymer sheets23 (Fig. 1a). For this

procedure, a battery of silicon-based masters, with two different

features (posts and holes of sizes 100, 25 and 4 mm2) were

fabricated by AMO Gesellschaft f€ur Angewandte Micro- und

Optoelektronic GmbH (Fig. 1b). PMMA thin polymer sheets

were used, as supplied, after cutting into pieces the approximate

size of the mould. In order to ensure anti-adhesion of the moulds

to the polymer, they were cleaned in a solution of isopropanol/

absolute ethanol (1 : 1) under sonication for 10 min, then silan-

ized by immersion in 10 mM trichloro(tridecafluoro-octyl)silane

(United Chemical Technologies, USA) in hexane for 1 h, and

baking at 80 �C. Upon removal from the oven, substrates were

briefly sonicated in isopropanol/absolute ethanol (1 : 1) to

remove any excess silane from the monolayer surface. The hard

mould was placed in contact with a thin film of thermoplastic

polymer and both were heated and pressed in a nanoimprint

machine (Obducat AB, Sweden).

The conditions for the nanoembossing of PMMA polymer

sheets were as follows: heating to 130 �C (a temperature higher

than the PMMA Tg) and an imprinting pressure of 3 MPa,

applied for 600 s. The system was then cooled to 80 �C,

below that of the Tg, while preserving the applied force. Upon

reaching this temperature, the pressure was released and the

This journal is ª The Royal Society of Chemistry 2010

polymer/master was allowed to cool down to room temperature

(RT) before the polymer was carefully peeled from the mould.

Topographic characterisation of the moulds and the PMMA

replicas was performed using white light interferometry

(Wyko NT110; Veeco Metrology, USA), optical microscopy

and scanning electron microscopy (Strata 250, FEI CO, The

Netherlands).

Cell culture

NRK cells were thawed and expanded to grow to 90% confluence

in a 75 cm2 flask (approximately 2 days after thawing) in Dul-

becco’s modified Eagle’s medium (DMEM) (Gibco/Brl Life

Technologies, Paisley, UK) supplemented with 10% foetal

bovine serum (FBS) (Gibco, UK), containing 1% penicillin/

streptomycin (Invitrogen, CA, USA), 1% L-glutamine (Invi-

trogen, CA, USA) and 1% sodium pyruvate (Invitrogen, CA,

USA). Cell cultures were maintained at 37 �C in a humidified 5%

CO2 atmosphere.

The cell culture wells were delimited on the microstructured

substrates by the FlexiPERM� system (Greiner Bio-One

GmbH, Germany), thus creating multiple growth chambers on

the same polymer sheet. Silicon moulds for nanoembossing were

specifically designed to create culture areas (�1 cm2) on PMMA

that fitted the FlexiPERM� wells. Cells were subsequently

cultured on unstructured and microstructured PMMA

substrates, as well as on 1 cm diameter glass coverslips, which

were used as a control. Immediately prior to culture, all

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substrates were cleaned by sonication in Milli-Q water

(MilliPore, USA) for 10 min. Thereafter, they were rinsed in 70%

ethanol and exposed to UV-light in a cell culture cabin for 20 min

for sterilisation. FlexiPERMs� followed the same sterilisation

procedure. Finally, substrates and FlexiPERMs� were dried

under laminar flow and culture well chambers were mounted.

Cells were trypsinized (Gibco, UK) and then seeded at a density

of 20 000 cells cm�2. Each FlexiPERM� well was filled with

300 ml of culture medium. Cell cultures were maintained for 24 h

at 37 �C in a humidified 5% CO2 atmosphere.

Cell attachment, proliferation and viability

Cell attachment to the different substrates was evaluated 3 h after

cell seeding. Cell culture medium was removed from wells and

cells were detached from the surfaces by adding 100 ml of trypsin

solution for 4 min at 37 �C. Trypsin action was stopped with

200 ml of complete culture medium. Detached cells were then

centrifuged for 5 min at 1000 rpm, resuspended in complete

culture medium and counted using an automatic cell counter

(Innovatis AG Casy� Technology, Germany). This equipment

quantifies the total number of cells and their viability. The

particular response of the cell counter to NRK cells was first

calibrated and cell number and viability were verified by inde-

pendent measurements using the Neubauer chamber and trypan

blue exclusion dye for dead cells examined under the light

microscope.

Cell proliferation and viability on microstructured samples as

well as on unstructured PMMA and glass coverslips were also

determined after 24 h of culture using an analogous procedure to

that described for testing cell adhesion.

Cell morphology: scanning electron microscopy

NRK cells cultured on microstructured PMMA and controls

were prepared for scanning electron microscopy. For this

procedure, cells were fixed with 2.5% glutaraldehyde in

0.1 M phosphate buffer for 2–4 h at 4 �C. Cells were then

rinsed (3 � 10 min) with 0.1 M phosphate buffer before

proceeding to post-fixation with 1% osmium tetroxide

combining potassium ferrocyanide. Thereafter, samples were

freeze-dried overnight, covered with carbon and examined in

a Dual beam FIB/SEM apparatus (DB Strata 235 FIB, FEI

Company, The Netherlands).

Cell adhesion, size and cytoskeleton organization:

immunofluorescence microscopy

Cell adhesion. Immunofluorescence staining of the cell adhe-

sion structures and cell nuclei was performed on cell cultures on

the microstructured, unstructured and control samples. Cells

were fixed in 3% paraformaldehyde in 0.1 M phosphate buffer

containing 60 mM saccharose for 15 min at room temperature

(RT), then cells were rinsed (2 � 5 min) with phosphate buffered

saline-Glycine (PBS-Gly), permeabilized with 0.1% Triton x100

for 15 min and rinsed again with PBS-Gly (2 � 5 min). Blockage

of free aldehyde groups was performed using 1% bovine serum

albumin (BSA) in PBS-Gly for 20 min at RT. Afterwards, mouse

anti-vinculin (Sigma-Aldrich, Germany) (diluted 1 : 400 in 1%

BSA in PBS-Gly) was added and incubated with the cells for 1 h

584 | Soft Matter, 2010, 6, 582–590

at 37 �C, then cells were washed with PBS-Gly (2 � 5 min) and

a final incubation with secondary antibody goat anti-mouse

Alexa A-488 (Sigma-Aldrich, Germany) (diluted 1 : 1000 in 1%

BSA in PBS-Gly) and Hoechst (Invitrogen, USA) for nucleus

staining (1 : 500 also diluted in 1% BSA in PBS-Gly) was carried

out for 1 h at 37 �C. Dried samples were mounted in Mowiol

(Calbiochem, EMD Biosciences CA, USA). Image analysis of

stained cell adhesion sites was performed by ImageJ free soft-

ware. Histograms of stained areas of at least 100 cells per sample

type were performed and analyzed by fitting the envelope curve

as a convolution of three Lorentzian components.

Cytoskeleton elements. For microtubule immunostaining, cells

were fixed in cold methanol (��20 �C) for 5 min, then cells were

rinsed twice in PBS and incubated with primary mouse mono-

clonal antibody against b-tubulin (Sigma-Aldrich, USA) (dilu-

tion 1 : 1000 in 1% BSA in PBS) for 1 h at RT in a humid

chamber. The cells were then rinsed twice with PBS and incu-

bated with secondary antibody goat anti-mouse Alexa A-488

(diluted 1 : 1000 in 1% BSA in PBS) against primary mouse

b-tubulin antibody for 45 min in a humid chamber. PMMA

substrates and glass coverslips were mounted on microscope

slides using Mowiol and imaged with a Nikon Eclipse 1000

(Nikon, Japan). Images were further analyzed by ImageJ soft-

ware to compute cell size and cell circularity (computed as

(4p cell area)/(cell perimeter)2). Histograms of at least 150 cells

per sample were obtained and analyzed by assuming a normal

cell size distribution. Actin microfilaments were labelled with

TRITC-phalloidin (Fluka-Biochemika, Switzerland) (diluted

1 : 500 in 1% BSA in PBS from a stock solution of 1 mg ml�1 in

DMSO) following the same procedure employed for cell adhe-

sion immunostaining. Finally, dried samples were mounted on

microscope slides with Mowiol for further visualization of the

different cytoskeleton elements with fluorescence microscopy.

Fluorescence microscopy was performed with a Nikon Eclipse

E1000 and E800 (Japan) and analyzed with image processing

software (Metamorph, Imaging and ImageJ).

Golgi complex morphology evaluation: immunostaining

An indirect immunofluorescence assay for observation of the

Golgi complex under fluorescence microscopy was carried out as

previously described.21 Briefly, NRK cells cultured on the studied

substrates were fixed in paraformaldehyde (4% in PBS) at RT for

15 min. Thereafter, cells were rinsed twice in PBS and free

aldehyde groups were blocked with 50 mM ammonium chloride

during 15 min. The cells were then washed in PBS and per-

meabilized with PBS containing 0.1% saponin and 1% BSA in

PBS for 10 min in a humid chamber. The cells were further

processed for a double-label immunofluorescesce assay by using

TRITC-phalloidin (Fluka-Biochemika, Switzerland) (diluted

1 : 500 in 1% BSA in PBS from a stock solution of 1 mg ml�1 in

methanol) and mouse monoclonal anti-giantin (G1/133) (Alexis,

Switzerland) (diluted 1 : 750 in 1% BSA in PBS from a stock

solution of 1 mg ml�1). The morphology of the Golgi complex in

NRK cells was defined as normal or compact and was quanti-

tatively evaluated according to Valderrama et al.24 Briefly,

normal Golgi morphology was defined as the subcellular

structure immunolabelled with the Golgi marker giantin,

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showing a characteristic perinuclear reticular morphology and

a minimum extension around half of the nucleus profile. A

compact Golgi was thus defined either as a juxtanuclear compact

structure or a relatively reticular structure in which the Golgi

extended around less than half of the nucleus profile. Golgi

complexes were imaged with fluorescence microscopy Nikon

E1000. The quantification of both defined Golgi morphologies

was carried out using six independent sample replicates of

unsynchronized cells from the same passage and same flask. Two

hundred cells per sample in randomly chosen microscopic fields

were analyzed by using Metamorph software.

Statistical analysis

All measurements were obtained from datasets of six

independent experiments. Parametric one-way ANOVA or

t-tests were performed on the statistical analysis of variables

plotted. All graphical data are reported as the mean �standard deviation (SD). Significance levels were established

at p < 0.05.

Results

Polymer microstructure characterization

All the PMMA microstructured replicas were checked to deter-

mine the reproducibility of the nanoembossing method used.

Measurements performed by white light interferometry micros-

copy showed 3D reconstructed images and X and Y sectional

profiles of each sample that faithfully replicated the mould

features, posts and holes sized 100 and 25 mm2. However, square

posts and holes of 4 mm2 in size seemed to have a rounded shape

rather than a square configuration after the embossing process.

Accurate measurements on the moulds proved that the problem

was not in the replication process but in the lithographic and

etching processes used for the master fabrication. Therefore,

samples were used while taking into account the rounded-shape

of the smaller structures.

Cell attachment, viability and proliferation

The number of cells that adhered to control surfaces (glass and

unstructured PMMA) and microstructured substrates is shown

in Table 1. 3 h after cell plating, between �30 to 50% of NRK

cells were attached to all the substrates, including both controls.

Cell attachment was not significantly affected either by the

polymer material (when compared to glass coverslips) or by the

post microstructures tested. Hole-shaped microstructures of

Table 1 Cell adhesion at 3 h, percentage of proliferation with respect to initiaexpressed as mean (SD) for n ¼ 6 independent experiments

Substrate Cell adhesion 3 h % Viability

Holes 4 mm2 8802 (1448) 83.6 (2.5)Holes 25 mm2 6798 (2270) 80.4 (3)Holes 100 mm2 6662 (1182) 79.8 (1)Posts 4 mm2 8537 (1917) 76.5 (1.3)Posts 25 mm2 9639 (1905) 80.9 (3.6)Posts 100 mm2 8555 (1410) 80.5 (2.1)Unstructured PMMA 7686 (2962) 79.7 (2.1)Glass 9416 (740) 83.9 (1.6)

This journal is ª The Royal Society of Chemistry 2010

100 and 25 mm2, however, produced a slight but still significant

decrease in cell attachment efficiency.

The viability of NRK cells after 3 h of seeding was around 80%

for all substrates, with viability percentages being slightly

decreased by the microstructures with respect to glass control

surfaces. However, when comparing microstructured PMMA

substrates with the unstructured polymer substrates, there was

only a significant decrease in the viability percentage for 4 mm2

posts (76.5%).

After 24 h, cells had proliferated on all substrates except on

samples with 4 mm2 holes, as shown in Table 1 (percentages are

relative to the amount of seeded cells). The percentage of cell

viability after 24 h increased up to 90% which is more than

a 10% increase with respect to the values obtained after 3 h of

culture. The use of polymer PMMA material for the cell culture

did not produce significant differences in cell viability with

respect to the control (glass). In contrast, the presence of

microstructures significantly decreased viability, although the

values obtained (between 87–93%) were good enough to make

comparisons.

Cell size and morphology

Under SEM, cells cultured on flat substrates (either PMMA or

glass) showed a spread out (Fig. 2d), flattened and quite circular

shape (Fig. 2a and 2e). However, cell morphology differed when

cells were grown on microstructured substrates (Fig. 2b and 2c).

In particular, when growing on post-shaped surfaces (Fig. 2c),

cells presented polarized, elongated and spindle-shaped

morphologies (Fig. 2e), with a rounded three-dimensional

morphology because they were much less spread out (Fig. 2d).

The SEM images also revealed the sensitivity of the NRK cells

to the assayed topographies, as their shape was adaptive to the

relief as well as to the outlines of the posts and holes. Cells

cultured on surfaces with holes had a tendency to use the ‘‘flat’’

areas between the features for spreading, sorting or bridging the

hole topography depending on their size. In contrast, cells

cultured on post-shaped substrates tended to grow on the top

surface of the post features, in this case the distance between

one post and its neighbours being an important parameter for

cell spreading.

Cell size was evaluated in terms of the projected surface area

occupied by the cells (Fig. 2d). Cells cultured on all PMMA

surfaces were much smaller than those on the control glass (by

about 60%) and cells cultured on microstructured substrates

showed a significant decrease in size when compared to

unstructured PMMA. Significant differences in the projected

l seeded cells at 24 h and percentage of viability at 3 and 24 h. Results are

3 h % Proliferation 24 h (%dD) % Viability 24 h

�0.8 (25.7) 91.3 (2.2)36.4 (19.4) 89.7 (1.6)25.9 (34.6) 90.1 (2.6)16.5 (43.3) 89.2 (3.1)27.9 (21.5) 87.9 (4)48.4 (18.7) 93.4 (1.6)55.9 (17.1) 94.0 (1.4)36.9 (29.6) 95.1 (0.7)

Soft Matter, 2010, 6, 582–590 | 585

Fig. 2 Scanning electron microscopy images of NRK cells cultured on (a) flat PMMA, (b) holes of 4 mm2*, (c) posts of 4 mm2*. (d) A graph showing

whole cell area on unstructured and microstructured PMMA surfaces. Glass data as number on the top of the graph. (e) The circularity of the cells

compared to control samples. All graph data are expressed as mean� SD. *SEM images of microstructured surfaces with holes and posts 4 mm2 present

round-shaped features rather than square-shaped due to the lithographic and etching processing during master fabrication.

Fig. 3 Fluorescence microscopy images of NRK cells cultured on: (a) glass, (b) flat PMMA, (c) holes of 100 mm2, (d) posts of 4 mm2, (e) holes of 100 mm2,

(f) posts of 4 mm2 stained for vinculin. (g) Plot of the number of areas stained by vinculin per cell onto the different microstructured, unstructured and

glass samples. Significant differences (noted by *) were found between the number of stained areas in microstructured samples with 100 mm2 and 25 mm2

post features and the unstructured PMMA samples, this number being lower for these post-structured samples. (h) Cell adhesion structures are classified

in function of the size: FC (focal complexes) < 1 mm2, FA (focal adhesions) > 2 mm2 and forming FA 1 < x < 2 mm2 and they were quantified. A graph

showing the distribution in size of the vinculin stained areas per cell onto the different substrates. The increase of FC for the samples with the smallest

post features and the decrease of large FA in these samples can be seen.

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cell area were also found between hole and post structures of

25 and 4 mm2 in size, with the posts inducing the smallest cell

sizes.

586 | Soft Matter, 2010, 6, 582–590

Cell morphology was quantified in terms of cell circularity

from ‘‘1’’ for perfectly round cells to close to ‘‘0’’ for elongated

and spindle-like shapes). Results obtained are plotted in

This journal is ª The Royal Society of Chemistry 2010

Fig. 4 Fluorescence microscopy images of cells stained against b-tubulin

onto (a) glass, (b) unstructured PMMA, (c) holes of 100 mm2, (d) posts of

100 mm2 to visualize the microtubular network. The microtubules were

observed to adapt to the surface topography: panels (c) and (d) show

microtubule signals (see the arrows) that are out of the microscope focal

plane, either inside the holes or in the upper surfaces of the posts of the

microstructures.

Fig. 5 Fluorescence microscopy images of cells stained with TRITC-phalloid

holes of 100 mm2, (d) posts of 25 mm2, (e) holes of 4 mm2, (f) posts of 100 mm2, (g

on the glass and the unstructured PMMA surfaces, while some fiber formation

25 mm2), especially for hole structures. Post features of 4 mm2 (panel h) show

around the post feature edges.

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Fig. 2e. Cells on substrates with holes did not show significant

differences in cell morphology with respect to controls (glass or

unstructured PMMA surfaces), in agreement with the SEM

observations. However, cells cultured on post-shaped

substrates showed decreasing circularity as a function of the

post size. This is also in accordance with SEM pictures that

showed that cells on post features were more spindle-like (so

with larger perimeters and, therefore, less circularity).

Cell adhesion

Cells cultured both on glass and unstructured PMMA surfaces

showed vinculin-stained areas mainly in the cell periphery

(Fig. 3a and 3b). On microstructured substrates (post and holes),

the adhesion sites were particularly arranged along the ridges of

the features, and were not randomly distributed (Fig. 3c to 3f).

Quantification of vinculin-stained areas per cell (Fig. 3g) showed

no significant differences between the unstructured substrates

(both PMMA and glass) and the cells cultured on substrates with

holes. However, measurements demonstrated that post features

decreased the number of vinculin-stained areas per cell compared

to holes (statistically significant for all post sizes) or unstructured

surfaces (statistically significant for the 25 and 100 mm2 post

sizes).

in to visualize actin cytoskeleton on (a) glass, (b) unstructured PMMA, (c)

) holes of 25 mm2, and (h) posts of 4 mm2. Actin stress fibers are clearly seen

can still be seen on the surfaces with the largest microstructures (100 and

an actin disrupted network with round small aggregates, in particular

Soft Matter, 2010, 6, 582–590 | 587

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Quantitative analysis of the distribution of the vinculin-stained

areas according to their size was performed by following the

criteria described by Nobes and Hall25 and Geiger et al.8

Accordingly, the areas of vinculin-stained sites were classified as:

small dot-like contacts (<1 mm2), also known as focal complexes

(FC) and elongated structures (2–10 mm in length, corresponding

to 2–10 mm2 in area) known as focal contacts or focal adhesions

(FA). Stained areas with sizes between 1–2 mm2 were also

computed and could correspond to forming FA or growing FC.

The results obtained are depicted in Fig. 3h, which plots the

percentages corresponding to each type of adhesion structure per

cell for all samples tested. Most fibroblast adhesions on glass

were FA (z70%) whilst on PMMA (either unstructured or

microstructured), the percentage of adhesions with sizes smaller

than 2 mm2 was higher, and FC smaller than 1 mm2 were present.

Surfaces with holes of all sizes tested had the same vinculin-

stained area pattern size distribution, which was also similar to

that on the unstructured PMMA. However, surfaces with post

features had dramatically different patterns that showed an

evolution in function of the post size. Thus, posts of 25 and 4 mm2

produced an increase in the number of FC and a decrease in FA,

while the percentages for structures of intermediate sizes

(between 1 and 2 mm2) remained constant.

Fig. 6 The Golgi complex was labelled with anti-giantin antibody. A

schematic representation of an immunolabelled Golgi complex when (a)

it has a common perinuclear shape and (b) it is compacted and presents

a restricted location. (c) Quantitative analysis of the percentage of Golgi

complex with collapsed morphology. The results are the mean � SD of n

¼ 6 independent experiments (p # 0.05); between 100 and 300 cells,

randomly chosen, were counted per microstructured substrate. Signifi-

cant differences were found for all the microstructured surfaces (except

for holes of 25 mm2). In particular, surfaces with post features showed up

to 65% of cells with Golgi compacted morphologies, compared to the

values of 30% found in glass samples.

Cell cytoskeleton organization

Microtubule organization. Immunofluorescence images of

microtubules on control glass slides (Fig. 4a) showed the

cylinder-shaped polymer network that grows from the centro-

some towards the periphery in a characteristic radiating pattern.

This characteristic microtubule organisation was not apparently

changed by culturing the cells on unstructured polymer

substrates (Fig. 4b). However, this component of the cytoskel-

eton showed some rearrangements in cells cultured on the

microstructured samples that can be attributed to the topo-

graphical modification of the surface. In fact, microtubules

accommodated to the shape of the surface features relatively

well, this being more obvious for the samples with the features

with the largest areas (100 mm2) (Fig. 4c and 4d). These images

evidence that the microtubule network was able to bend into the

holes or rise onto the posts, adapting to the physical features,

mainly for those of larger sizes. Microtubules were not able to

totally enter into the 4 mm2 holes, sending out cytoskeleton

elements.

Actin cytoskeleton. Immunofluorescence images of NRK cells

grown on both the glass control and the unstructured polymer

surfaces showed a network of well-formed stress fibres of normal

filamentous morphology (Fig. 5a and 5b). From this image,

which is representative of the general behaviour observed on all

samples, it can be inferred that actin stress fibres are not signif-

icantly altered by cell culture on the unstructured PMMA

material. On the microstructured samples, however, the actin

cytoskeleton was partially depolymerised, showing very weakly

stained and incomplete stress fibres that appeared to be affected

by the microstructured features, either posts or holes (Fig. 5c to

5h). Although staining was weak, sometimes actin stress fibres

were barely visible (particularly in the samples with the smaller

holes and posts i.e. 25 and 4 mm2), and when identified, they were

588 | Soft Matter, 2010, 6, 582–590

thinner and shorter than those found on the unstructured

polymer.

Overall, the staining of the different components of the cell

cytoskeleton showed no significant changes in cytoskeleton

organization due to culturing the cells on unstructured PMMA.

In contrast, the microstructures of the assayed areas and patterns

induced changes in the location and distribution of microtubules

within the cells, while the actin cytoskeleton was strongly

affected, with poorly-formed actin stress fibres, particularly on

the substrates with the smallest features.

3.6. The Golgi complex morphology

The morphology and positioning of the Golgi with respect to cell

nuclei were also studied, revealing that in glass control samples

around 70% of the NRK cells had a Golgi complex with the

typical extended ribbon-like shape and perinuclear localization

(Fig. 6a), while around 30% of the cells showed a Golgi complex

with a compacted shape and a more confined location in refer-

ence to the cell nucleus (Fig. 6b). Quantitative analysis showed

that the percentage of compacted Golgi complexes in NRK cell

cultures was higher (up to 35%) for unstructured PMMA poly-

mer surfaces, although significant differences with the glass

control could not be established with respect to the effect of the

polymer material on the Golgi complex morphology. However,

when cells were cultured on micropatterned surfaces, either with

posts or holes, significant differences were observed in the

percentage of cells with a compacted Golgi (Fig. 6c).

Cells cultured on surfaces with hole-shaped features showed up

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to 45–50% of compacted Golgi complexes which was signifi-

cantly higher than the values obtained in cells grown on both

unstructured PMMA and glass surfaces. The most relevant

differences, however, were found in the cells cultured onto the

post-shaped surfaces, where the percentage of cells with a com-

pacted Golgi complex increased up to 60%. The smallest sizes

(4 and 25 mm2 posts) induced the most significant loss of the

characteristic perinuclear extension of the Golgi complex of

mammal cells.

Discussion

From the results obtained, it can be concluded that cell attach-

ment, viability and proliferation for the first 24 h in culture were

not significantly altered by either the PMMA polymer or the

microstructures, so its influence was discarded in this discussion.

However, cell size and morphology showed changes in these two

characteristics. Cells were almost 50% smaller on PMMA than

on the glass controls, they were also smaller when cultured on

microstructured materials and they were much smaller still when

cultured on surfaces with post-shaped features. Moreover, cells

were more rounded when cultured on glass, unstructured

PMMA and microstructured PMMA with hole-shaped features,

while post-shaped microfeatures, and particularly those of

smaller sizes (25 and 4 mm2), induced spindle-like cell shapes

(computed as a loss in circularity). This could be explained by the

tendency of NRK cells to spread on the flat areas of the hole-

shaped microstructures, whereas they only used the top part of

the post-shaped features to grow, therefore having less effective

area to spread and being confined by the edges of the posts. The

same tendency has also been reported for MG63 cells on PMMA

microstructured substrates.26

The results from the study of cell adhesion sites on the different

substrates support the changes observed in cell morphology.27

The results presented here showed that vinculin-stained areas

were closely associated with the edges of the features on the

microstructured PMMA surfaces, which is consistent with the

literature.2,27,28 Thus, surfaces with hole-shaped features, on

which cells were bigger than on post-shaped microstructures,

have a larger amount of ‘‘edges’’ (number of features per cell

area) on which to attach, and therefore have more adhesive areas

when staining. More interestingly, the size distribution of the

vinculin-stained areas was also affected by the presence of the

microstructures on the surface. Post-shaped features induced

a remarkable increase in the percentage of focal complexes

(<1 mm2 in area) compared to focal contacts (>2 mm2 in area).

This phenomenon is dependent on the post size, such that posts

of 4 mm2 in size had the smallest cell attachment areas. It is

known that the maturation of FCs by the activation of the Rho

GTPase Rac29,30 induces the formation of FAs, and the forma-

tion of stress fibres.31 The higher percentage of FCs with respect

to FAs in cells cultured on the post-shaped features suggests that

these substrates inhibit the complete maturation of FAs and the

correct development of the actin cytoskeleton. It has been

reported that the size of the corresponding contact depends on

the tension exerted by the substrate, in which physical properties,

such as elasticity and rigidity, play a key role.8,32–34 Actin staining

showed clearly visible and well-formed stress fibres on the

control glass and unstructured PMMA surfaces. For the

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microfeatures with the largest areas (both for holes and posts), it

was still possible to distinguish some tiny stress fibres that could

also be visualized for the other samples with hole-shaped

features. However, cells cultured on post-shaped features of

25 and 4 mm2 in area did not show continuous stress fibres, but

more of a bundled structure, with small, stained, round areas

spread around the cytoplasm.35,36 The loss of actin stress fibers

can be directly linked to the observed lack of cell ability to form

mature FAs. The other element of cell cytoskeleton studied,

microtubules, has shown the ability to bend through the gaps of

the hole-shaped microstructured substrates or to sit on top of the

posts on the substrates with post-shaped features, a phenomenon

that has also been reported previously37 and described by Karuri

et al.18 with corneal epithelial cells cultured on nano and

microscale holes on silicon.

The mechanical-stress induced alterations in cell shape and

internal cytoskeleton can be explained by the so-called tensegrity

model,38 which links cellular response to mechanical stress to the

existence of discrete networks of interconnected actin microfila-

ments, microtubules and intermediate filaments. These extend

through the cytoplasm and link to adhesion receptors (integrins).

In living cells, the microtubules bear compressive forces, which

are balanced by tensile forces generated within the contractile

actin cytoskeleton.39 The cells used in this work, when exposed to

an external mechanical stress produced by the substrate micro-

structures, exhibited poor actin organisation but a well-formed

microtubular network, a behaviour which suggests a shift in the

tensegrity model. One possible explanation for this shifted

behaviour could rely on the fact that punctuate actin in response

to topography can be caused by alterations in the Rho-GTPases

signalling pathway,40 which is needed in the formation of stress

fibres and focal contacts.31 Thus, the ability of a cell to form

mature focal adhesions will affect the ability of the cell to form

cytoskeleton tensegrity structures.41 The microtubules, instead,

remain well organised, maybe due to their other multiple func-

tions within the cell, for instance being involved in exocytosis and

endocytosis, and the formation of the spindle for chromosome

separation during mitosis.42 Indeed, the fact that microtubules do

not suffer dramatic modifications due to topography may

explain that even if the cells present modified internal organisa-

tion, they are metabolically active.40

Little is known about the distribution and morphology of cell

organelles when cells are cultured on topographically structured

substrates. Some studies report an alteration in the nuclear

morphology,16,43 but no studies concerning other organelles, such

as the Golgi complex, were found in the literature. The results

obtained here show that the percentage of cells with a compacted

Golgi complex is altered by the microstructures, achieving

compaction ratios that are double (60%) than those found on

glass control surfaces (30%). The studied microstructures and, in

particular, the smaller post features alter cell adhesions and actin

microfilaments. As some authors relate the mechanical stability

of the Golgi complex to actin microfilaments,24 the poor actin

network development found on the post-shaped microstructured

samples could be the reason for the high rate of Golgi complex

collapse. The mechanistics of this Golgi compactation are

currently little understood, but it is reasonable to hypothesize

that an actomyosin system could be acting as a force that helps

the Golgi complex acquire the typical ribbon-like morphology.

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The decrease in such a force as a consequence of an actin cyto-

skeleton could facilitate the compactation of the Golgi, as

observed in cells cultured on microstructured surfaces.

Naturally derived toxins that either depolymerise (latruncu-

lins, cytochalasins) or stabilize (jasplaknolide) actin have been

used as a valid experimental tool with which to study the

dynamics of the actin cytoskeleton and their effects in the Golgi

complex morphology.44 Concomitantly, the use of drugs as

stimulatory agents of the Rho family of small GTPases’

signalling pathway that specifically induce actin rearrangements

in several cell types25,45,46 have been applied to prove that the

cytoplasmic 3D arrangement of the microfilaments is directly

involved in the shape of the GC and that actin cytoskeleton,

microtubules, and the morphology of the Golgi complex are

interdependent phenomena.46 In order to study these depen-

dences, the use of a non-chemical-based system for actin

disruption, such as the one proposed here (microstructured

substrates) could give a better insight into the Rho-GTPases

signalling pathway.

Conclusion

Our work shows that the nanoembossing technique, which has

been adapted from nanoimprint lithography, is a suitable tool for

creating structured surfaces at the microscale for use in cell-

surface interaction studies. In this paper, topographically struc-

tured surfaces with microsized features induced changes in NRK

cell adhesion sites, cell morphology, cytoskeleton network and

internal organelles. The results evidence that cells react to their

physical microenvironment through cytoskeletal elements and

hence these elements may play a significant role in mechano-

sensing. Due to the direct involvement of actin filaments in Golgi

complex morphology, the studied microstructured substrates

alter the mechanical stability of the actin cytoskeleton network

and lead to its collapse.

Acknowledgements

Maruxa Est�evez and Elena Martinez acknowledge the financial

support from the Spanish Ministry of Education for the provision

of grants through the FPU and Ramon y Cajal grant systems,

respectively. The technical support of Miriam Funes and the

scientific advice of Francisco L�azaro-Di�eguez are also recognized.

This work has received the financial support of the ISCIII through

the FIS project PI071162 (to J. Samitier) and CONSOLIDER

IMAGENIO 2010 and BFU 2006-00897 grants (to G. Egea).

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