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Nano-Structured Cell-Adhesive andCell-Repulsive Plasma-Deposited Coatings:Chemical and Topographical Effects onKeratinocyte Adhesion

Eloisa Sardella,* Loredana Detomaso, Roberto Gristina, Giorgio S. Senesi,Hossein Agheli, Duncan S. Sutherland, Riccardo d’Agostino, Pietro Favia

Cell-adhesive and cell-repulsive coatings have been plasma-deposited on poly(ethyleneterephthalate) surfaces previously structured with nano-metric conical features by meansof colloidal lithography. Surface analysis revealed that both coatings are conformal on nano-structured substrates, with their wettabilitydepending on the substrate morphology. Theeffect of surface chemistry and surface topogra-phy on cell adhesion has been investigated andclarified. The adhesion of a human keratinocytecell-line was found to be strongly dependent onthe surface topography for plasma-depositedacrylic acid (cell-adhesive), and on the surfacechemistry for poly(ethylene oxide)-like (cell-repul-sive) coatings.

Introduction

Cells respond to micro- and nano-metric surface fea-

tures[1,2] with changes in adhesion, morphology, and gene

E. Sardella, R. Gristina, G. S. SenesiInstitute of Inorganic Methodologies and Plasmas (IMIP) – CNR,70126 Bari, ItalyFax: þ39 80 544 3405; E-mail: [email protected]. Detomaso, R. d’Agostino, P. FaviaDepartment of Chemistry, University of Bari, 70126 Bari, ItalyR. d’Agostino, P. FaviaPlasma Solution Srl, Spin-off of the University of Bari,via Orabona 4, 70126 Bari, ItalyH. Agheli, D. S. SutherlandiNANO Interdisciplinary Research Center, University of Aarhus,Aarhus 8000, Denmark

Plasma Process. Polym. 2008, 5, 540–551

� 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

expression.[3–5] In the last decades such knowledge is steering

a trend in biomedical research, aiming to affordable

techniques for producing micro- and nano-patterned

biomedical surfaces and scaffolds with controlled chemical

composition and morphology.[6–8] Such substrates could

possibly drive cell adhesion, growth and physiology in

many applications, including biomaterials, prostheses,

tissue/cell engineering, regenerative medicine, biosensors,

microfluidics and biochips. So far, the development of

nano-structured biointerfaces was limited by the high

production cost of nano-features with advanced precise

methods (e.g. electron beam lithography[9]), by the

difficulty to pattern large area substrates, and by the long

patterning time. Newer approaches to nano-scale patterns,

such as X-ray lithography[10] and nano-imprint lithogra-

phy,[11,12] are thus being developed for obtaining reliable,

DOI: 10.1002/ppap.200800005

Nano-Structured Cell-Adhesive and Cell-Repulsive . . .

fast and cheap fabrication techniques of nano-structured

large area surfaces, as required in cell culture and tissue

engineering applications. Colloidal lithography (CL),

among other techniques, revealed as inexpensive, well

established nano-fabrication technique able to generate

nano-scale features of different shapes on large areas at

reasonable speed and cost.[13,14] CL-structured interfaces

have been often used to produce substrates featured

ad hoc to investigate cell adhesion and morphology.[14,16]

CL exploits the self-assembly of micro/nano-metric

colloidal particles in hexagonal arrays on properly

prepared surfaces; such arrays act as physical masks,

and can be transferred at the surface of the substrate with

lithographic steps, including sputtering and etching

processes. Plasma-enhanced chemical vapor deposition

(PE-CVD)[15] can also be used to modify the substrate

surface through the openings of the sphere array.

Investigating the effects of nano-topography (rough-

ness, shape/size of features, geometric vs. random

distribution, etc.) on cell behavior requires cell-growth

experiments on flat and nano-structured surfaces char-

acterized by same identical chemical composition and

different topography, or vice versa.[16] Studies performed

on surfaces with different topography and identical

chemical properties show that also topography can control

the organization of adsorbed proteins directly involved in

cell adhesion processes.[17,18]

How to use micro/nano-topography, independently

from surface chemistry, to drive the adhesion and growth

of cells, and to which extent cell functions (e.g., production

of certain proteins) could be stimulated/inhibited by

selected chemical/topographic surface features are still

open issues in this field.

Plasma processes are investigated and utilized in a

growing number of applications in life science, from

biomaterials to tissue engineering, from sterilization to

biosensors.[19] Plasma processes can be coupled with CL

techniques[20–24] to promote the self-assembly of the beads

when the substrate is not able to properly support particle

crystallization, to transfer the pattern of the assembled

layer to the substrate by means of PE-CVD or plasma

etching processes and to modify, in a conformal and

homogeneous way, the chemistry of the patterned surface.

PE-CVD of thin organic stable films, in particular, can

functionalize surfaces in a specific and controlled way,

with the aim of tuning adhesion, spreading and prolifera-

tion of cells on surfaces from cell-adhesive to cell-

repulsive.[25–27] Surfaces with different oxygen-containing

functionalities affect adhesion and spreading of different

cell lines;[28–31] in particular, plasma-deposited acrylic acid

(pdAA) films with controlled surface density of carboxyl

groups induce attachment and growth of keratinocytes,

osteoblasts and fibroblasts,[32–34] while plasma-deposited

poly(ethylene oxide)-like (PEO-like) coatings with at least



70–80% of ether –CH2CH2O– over all carbon moieties

discourage protein and cell adhesion.[35,36]

For this work pdAA and PEO-like coatings have been

plasma-deposited on flat and conical CL-nano-structured

PET substrates, in order to study simultaneously surface

topographical and chemical effects on adhesion and

morphology of human keratinocytes. This investigation

aims to highlight the role of surface chemistry and of

surface topography in cell adhesion, but also to stress how

surface chemical/morphological properties of materials

can be adjusted to drive the behavior of cells.

Experimental Part

Colloidal Lithography

Poly(ethylene terephthalate), PET, has been utilized. PET-flat and

CL nano-structured (PET-CL) substrates have been plasma coated

with pdAA (PET-flat-pdAA; PET-CL-pdAA) and PEO-like (PET-flat-

PEO; PET-CL-PEO) coatings. The CL technique, described in detail

elsewhere,[10,37,38,39] utilizes electrostatically assembled, dispersed

monolayers of colloidal spherical particles as masks to transfer a

pattern at the surface of a substrate. PET (Goodfellow, UK) substrates

were pretreated with an O2 radio frequency (RF, 13.56 MHz) glow

discharge to improve the electrostatic self-assembly of a multilayer

of polyelectrolytes, i.e., poly(diallyldimethylammonium chloride)

(PDDA, MW 20 000–35 0000 g �mol�1, Aldrich, UK), poly(sodium 4-

styrenesulfonate) (PSS, MW 70 000 g �mol�1, Aldrich, UK), and

aluminum chloride hydroxide (ACH, Reheis). Subsequent assem-

bly of a colloidal mask of sulfate-modified polystyrene (PS) beads

(107�5 nm diameter, IDC, USA) from water solution, followed by

drying, resulted in a dispersed colloidal monolayer with short-

range order. The pattern of the mask was transferred at the PET

surface with a combination of vertical and angled Arþ ion

bombardment. A DC argon ion beam (CAIBE Ion Beam System,

Oxford Ionfab) was used to perform a two-step etching process as

follows: the first step used Arþ ions (250 eV, 0.074 mA � cm�2,

incident angle 158 off normal angle, sample rotation, 560 s) and

3 sccm O2 released at the surface as a chemical etching assistance;

the second step employed Arþ ions (250 V, 0.074 mA � cm�2,

normal incident angle, 240 s) and 3 sccm O2.

The sputter-etching process was performed until the PS beads

were completely removed, resulting in conical pillars and chemical

modifications. More details on the CL step are in ref.[39] PET-CL

samples were blown with N2 to remove any particulate

contamination, and cleaned in 70% ethanol-water. Fabrication

and cleaning were carried out in a class-1000 clean room before

packaging in airtight boxes for transfer.

Plasma Deposition Processes

PdAA coatings were deposited in a stainless steel plasma reactor

with two internal stainless steel ‘‘parallel plate’’ circular (Ø 25 cm)

electrodes; the upper is shielded and connected to a RF (13.56 MHz)

generator through a manual matching network, the lower is

grounded. The reactor was evacuated with a rotary pump

www.plasma-polymers.org 541

E. Sardella et al.

542

equipped with a liquid N2 trap. A mixture of acrylic acid (AA,

Sigma Aldrich, 99%) vapors and argon (Air Liquide) was used as

feed (AA 3 sccm, Ar 5 sccm). The pressure, monitored with a MKS

baratron, was kept constant at 150 mTorr. Discharges were ignited

for 5 min at a power input of 100 W, resulting in pdAA films

30� 5 nm thick on flat silicon samples. PdAA coatings deposited in

such conditions are known to induce good adhesion and growth

of fibroblasts[24] and were used to coat PET-flat and PET-CL

substrates.

PEO-like coatings were deposited in a stainless steel plasma

reactor equipped with two vertical ‘‘parallel plate’’ asymmetric

stainless steel electrodes The small (Ø 8 cm) one is connected to a

RF (1356 MHz) generator trough a matching network, the large (Ø

18 cm) electrode is grounded. The reactor was evacuated with a

rotary/root pump system. A mixture of diethylene glycol dimethyl

ether (DEGDME, Sigma Aldrich) vapors and Ar was used as feed

(0.4 sccm DEGDME, 5 sccm Ar). The pressure, monitored with a

MKS baratron, was kept constant at 400 mTorr. Discharges were

ignited for 30 min at a power input of 5 W, resulting in PEO-like

films 30�5 nm thick on flat silicon samples. Only this set of

experimental conditions was used, in this work, to coat PET-flat

and PET-CL substrates; PEO-like coatings deposited in such

conditions show net protein and cell repulsive ‘‘non fouling’’

properties[25,33,34] on flat surfaces.

AA and DEGDME were degassed with freeze/thaw cycles and

used without further purification; vapors of both liquids were fed

from glass reservoirs with a needle valve, Ar was fed through MKS

mass flow controllers. For both PE-CVD processes samples were

positioned on the ground electrode of the reactors.

Chemical and Morphological Analyses

Processed surfaces were characterized with X-ray photoelectron

spectroscopy (XPS) and water contact angle (WCA) measurements.

XPS analyses were performed with a PHI 5300 ESCA instrument

with non-monochromatized Mg Ka X-rays. Wide scan [0–1000 eV

binding energy (BE)] and high-resolution (C1s, O1s) spectra were

acquired at 458 electron take-off angle within 1 h after the

deposition. Error bars in the graphs are the standard deviations on

3 replicated samples. C1s spectra of pdAA and PEO-like coatings

were best fitted with four peak components corresponding to C-

atoms with zero, one, two and three carbon-oxygen bonds,

namely: C0 (C–H, C–C; 285.0�0.2 eV, BE reference); C1 (C–OH,

C–O–C; 286.6� 0.2 eV); C2 (O–C–O, C––O; 288.1�0.2 eV), C3

(COOH, COOR; 289.1�0.2 eV). For PET-flat and PET-CL surfaces the

following C1s peak components were also used: Ca (aromatic C–H,

C–C, 284.7�0.2eV); C0a (C–H in the terephthalate moiety,

286.2�0.2 eV); C3a (COOR, 288.6� 0.2 eV).[40] The best fitting

procedure was performed with a fixed full width at half maximum

(FWHM) of 2.00 eV and a 80–100% Gaussian for all peak

components. Sample charging was corrected by positioning the

hydrocarbon C1s peak component at 284.7 eV (Ca) for PET-flat and

at 285.0 eV (C0) for the other substrates.

Static and dynamic WCA measurements were performed soon

after each deposition at room temperature, with double distilled

water, using a manual optical goniometer (Rame-Hart mod100-

00). Advancing WCA (ua) measurement were performed by

progressively increasing the volume of the water drop by stepwise



addition of �2mL per step until a constant angle, ua, was observed;

the volume was progressively reduced till the last WCA value, the

receding angle ur, could be read before the substrate area wetted

by the drop was reduced. To avoid vibrations and/or deformations,

the needle of the micro-syringe was kept immersed in the drop

during the measurements. WCA values reported are the mean of at

least 5 individual measurements on 3 replicated samples; the

corresponding standard deviations are reported in the graphs as

error bars.

The thickness of the coatings was measured with an Alpha-

Step1 500 (KLA TENCOR) surface profiler with a vertical resolution

of 25 A. The morphological characterization of the nano-structured

surfaces was performed with an Autoprobe CP-Research (CP-R)

scanning probe microscope. Images were acquired in the non-

contact mode of an atomic force microscope (nc-AFM) with

commercial, unmodified silicon nitride tips. Topographical para-

meters such as root mean square roughness (RRMS) and average

height (AH) of the nano-features were calculated with a

ThermoMicroscopes SPMLab NT Ver. 5.01 software on three

different points (3 replicated samples). The frequency of each

measured height of the investigated surface area was represented

with a normalized histogram.

To evaluate the stability in water of the plasma deposited films,

duplicate samples were XPS, WCA and AFM analyzed before and

after 50 h of soaking in 7 mL of doubly distilled, non stirred water,

followed by overnight drying at room temperature.

Cell Culture

Cell culture experiments were performed with NCTC 2544 human

keratinocyte cell lines from stocks routinely grown in Dulbecco’s

modified eagle medium (DMEM) supplemented with 10% fetal

bovine serum, 50 IU �mL�1 penicillin, 50 IU �mL�1 streptomycin

and 200� 10�3M glutamine, under 5% CO2/95% air atmosphere at

37 8C. All samples were placed, modified side up, in 24 well culture

plates (Iwaki 24 wells). Cells were obtained after trypsinization of

confluent or near-confluent culture, seeded (1�104 cells per well)

in suspension on all test materials and incubated at 37 8C under

5%CO2/95% air atmosphere. After different periods of incubation

(30 min, 180 min and 24 h) cells were fixed with formaldehyde

(4 wt.-% in PBS for 15 min, then stained with Comassie blue. Cell

attachment, distribution and morphology on flat and nano-

structured surfaces were measured as a function of culture time

using digital images acquired with a phase contrast light micro-

scope (Leica DM IL). The number of adhered cells was determined

in at least 10 areas of 0.8 mm2 per sample. At least three repeated

samples per experiment were analyzed. The two-way ANOVA and

the Bonferroni post test were used to evaluate statistical

significant differences among samples. Variations were consid-

ered significant when p<0.05 was obtained.

Results and Discussion

PET-Flat and Nano-Structured PET-CL Substrates

Conical nano-features were formed on PET with the CL

technique, as described in ‘Colloidal Lithography’. Figure 1

DOI: 10.1002/ppap.200800005


Figure 1. 3D AFM micrographs and height distribution of PET-flat (a) and of PET-CL (b) surfaces.

Figure 2. Best fitted C1s XPS signal of PET-flat (a) and PET-CL(b) surfaces. Refer to Table 4 for XPS C1s components.

shows the nc-mode AFM 3D topography images of PET-flat

(a) and PET-CL (b) substrates with their normalized height

distribution. Only one height distribution was recorded on

PET-flat surfaces, with AH value of 4� 2 nm. The conical

shape of PET-CL structures is well evident; two height

distributions were observed on PET-CL, the first peak

(lower value) refers to the AH of the background, the

second (higher) value refers to the AH of the conical nano-

features. The distance between the two peaks is the AH

value of the nano-cones, 117� 5 nm in this work. C1s XPS

signals shown in Figure 2 evidence the different surface

composition of PET-flat and PET-CL surfaces, due to the

chemical changes (broken/re-arranged bonds) induced by

the Ar/O2 etching process in the CL procedure. The typical

shake-up feature (292 eV) due to the aromatic rings in PET

disappears from the outer layer of PET-CL surfaces, the

COOR/H (C3) component becomes less intense, and new

components C1 and C2 (ether/alcohol and carbonyl

groups) appear. A slight decrease of the O/C XPS ratio is

also observed, from 0.40� 0.01 (PET-flat) to 0.37� 0.2 (PET-

CL). These results attest for a high crosslinking of the

outermost layers (up to 10 nm) of PET due to the Ar/O2

sputter/etching process.[37,41]

Data in Table 1 show no significant difference of static

WCA values between PET-flat and PET-CL surfaces; a

marked difference is found, instead, for their WCA

hysteresis, i.e. the difference between ua and ur values.

The WCA hysteresis is typical for non-ideal surfaces, due to



E. Sardella et al.

Table 1. Static and dynamic WCA values of PET-flat and PET-CLsurfaces.

Type WCA

-

PET-flat PET-CL

Static 76W 2 78W 3

ua 87W 2 96W 2

ur 54W 2 7W 2

544

non-homogeneous distribution of chemical groups, rota-

tion of chemical groups above/below the liquid/material

interface, and surface roughness.[42,43] The hysteresis was

found to be much higher on PET-CL (where, in particular, ur

is extremely low) than on PET-flat surfaces, likely due to

the presence of the conical nano-structures rather than to

the chemical compositional change induced with CL.[44]

Due to their high WCA hysteresis, PET-CL can be considered

to be a Wenzel-type surface.[45]

In order to differentiate the chemical and topographic

effect of nano-structures on cell behavior in cell growth

experiments, pdAA and PEO-like 30� 5 nm thick coatings

were conformally deposited on flat and nano-patterned

substrates, as described in ‘Plasma Deposition Processes’,

Figure 3. 3D AFM micrographs and height distribution diagrams of P



to obtain surfaces with different topography and identical

chemical composition.

Cell-Adhesive pdAA Surfaces

The stability in water of plasma deposited coatings on flat

and nano-structured surfaces was tested; Figure 3 shows

nc-AFM 3D topography images and normalized height

distribution of a PET-CL-pdAA surface before (a) and after

(b) 50 h of immersion in water. The data show clearly that

no relevant shape/dimension changes occurred to the CL

conical structures after the pdAA deposition, beside a very

slight AH increase, from 117� 5 nm (PET-CL, see Figure 1)

to 124� 5 nm (PET-CL-pdAA), thus attesting for a

conformal coating. This situation is practically not altered

by 50 h immersion in water. RRMS values reported in

Table 2 show that both the deposition of the pdAA coating

and the immersion in water do not alter the roughness of

PET-CL surfaces, thus attesting the stability of pdAA

coating in water when deposited either on flat or on nano-

structured PET.

As reported in Table 3, the 30� 5 nm pdAA deposited

layer confers a similar O/C ratio (0.29) to both PET-flat and

PET-CL surfaces, after 50 h in water. The C1s XPS signal of

PET-flat (a) and PET-CL (b) surfaces before (black) and after

(dotted line) pdAA deposition are shown in Figure 4, and

ET-CL-pdAA surfaces before (a) and after (b) 50 h soaking in H2O.

DOI: 10.1002/ppap.200800005


Table 2. RRMS value of PET-flat and PET-CL surfaces uncoated,pdAA-coated, and after 50 h soaking in H2O.

Surface RRMS

nm

PET-flat PET-CL

Uncoated 1.0W 0.5 42.6W 1.9

pdAA 1.4W 0.5 46.5W 1.9

pdAA/H2O 1.8W 0.5 47.9W 1.9

Table 3. XPS O/C ratio of PET-flat and PET-CL substrates uncoated,pdAA-coated before and after 50 h in H2O.

Surface O/C ratio

PET-flat PET-CL

Uncoated 0.47W 0.03 0.37W 0.03

pdAA 0.29W 0.03 0.29W 0.03

pdAA/H2O 0.30W 0.03 0.29W 0.03

Figure 4. C1s XPS signal of PET-flat (a) and PET-CL (b) surfaces; as it(black), pdAA coated (dotted line), pdAA coated after 50 hin H2O (light grey).

evidences how the same surface composition is imparted

by pdAA and kept after 50 h in water on PET-flat and PET-

CL samples. The same information is read from the C1s XPS

components in Table 4. All data confirm that the pdAA

deposition was homogeneous on the two different

surfaces; since no compositional changes were detected

after water soaking (Figure 4 and Table 4), the high

stability of the pdAA coating is attested for both flat and

structured surfaces.

It is worth to stress here that coatings intended for

biomedical use, thus engineered to perform in water-based

fluids/tissues, should be always tested for their stability

(change of composition, re-arrangement, and leach of

compound) in water media, to optimize their synthesis

Table 4. Percentages (�1) of C1s XPS components observed on uncoatein H2O.

Surface PET-flat

Ca C0 C0a C1 C2

% % % % %

Uncoated 65 – 21 – –

pdAA – 72 – 17 6

pdAA/H2O – 71 – 18 6



and prevent failure and cytotoxicity. As a general rule of

thumb for PE-CVD processes with organic monomers, an

extended fragmentation of the monomer in the plasma

(high RF power density, low pressure, low flow rate of

the monomer) and an intense ion-bombardment of the

growing film (asymmetric configuration, high RF power

density, low pressure, substrate bias applied to the

substrate) increases both density and cross-linking in

d and pdAA-coated PET-flat and PET-CL before and after 50 h soaking

PET-CL

C3a C3 C0 C1 C2 C3

% % % % % %

14 – 70 19 5 6

– 5 73 17 5 5

– 5 75 15 6 4


E. Sardella et al.

Table 5. Static and dynamic WCA values of PET-flat-pdAA andPET-CL-pdAA substrates.

WCA

-

Static ua ur

PET-flat-PdAA 59W 2 74W 2 38W 2

PET-CL-pdAA 78W 3 96W 2 10W 4

PET-flat-pdAA/H2O 59W 2 74W 2 38W 2

PET-CL-pdAA/H2O 77W 2 95W 2 9W 4

546

the coatings, and lead to solid, reliable coating-substrate

interfaces highly stable in water. As a drawback, a reduced

density of the chemical functionalities of the monomer is

found in the corresponding coating. This is exactly what

happens to the pdAA coating used in this research,[32] with

reduced O/C ratio (Table 3) and density of COOH groups

(%C3, Table 4), but high stability in water.

Static and dynamic WCA values for pdAA-coated

surfaces are reported in Table 5. The static WCA value of

PET-CL-pdAA remained unchanged with respect to

uncoated PET-CL surfaces (see Table 1). On the contrary,

the WCA value of PET-flat-pdAA is lower than PET-flat

surfaces (Table 1), as it occurs also for pdAA coatings

deposited in the same conditions on glass, silicon, PS,[24]

and other substrates. According to the Wenzel equation, in

effect, a flat surface characterized by a WCA value lower

than 908, like our PET-flat-pdAA samples, should become

even more hydrophilic when its roughness increases.[46–48]

The static WCA values show, instead, that PET-CL-pdAA

surfaces become slightly less hydrophilic than correspond-

ing flat surface. Very likely, as also reported in the

literature,[49] this deviation is due to the fact that, besides

chemical composition and roughness, other factors

influence the wettability of a surface, such as shape,

density and distribution of the micro/nano-structures

themselves. In our case, although slight, the change of

wettability of PET-CL-pdAA with respect to PET-flat-pdAA

surfaces goes in the opposite direction with respect to

what is predicted by the Wenzel equation.

The lack of surface homogeneity introduced by conical

nano-structures introduces also WCA hysteresis; a higher

hysteresis value was found (see Table 5) on PET-CL-pdAA

with respect to the corresponding flat surfaces, as observed

(see Table 1) for PET-flat and PET-CL surfaces. These results

suggest a predominant effect of topography on the

wettability of the surfaces investigated, that could likely

influence also the behaviour of cells seeded and grown

there. Static and dynamic WCA values remained

unchanged after water soaking (see Table 5), thus



confirming once more the stability of all pdAA-coated

surfaces.

Cell-Repulsive PEO-Like Surfaces

Also the stability in water of flat and nano-structured

surfaces coated with the PEO-like film (PET-flat-PEO and

PET-CL-PEO, respectively) was tested; Figure 5 shows

nc-AFM 3D surface topography images and the related

normalized height distribution of 30� 5 nm thin non-

fouling PEO-like coatings deposited on conical PET-CL

surfaces before (a) and after (b) 50 h of soaking in distilled

water. After a comparison with Figure 1, it can be stated

that, also in this case, no shape change occurred on the

conical nano-structures after the deposition of the PEO-like

coating. The average height of PET-CL (117� 5 nm) and of

PET-CL-PEO (125� 5 nm) surfaces practically remained

unchanged after the PEO-like deposition, as it occurs for

the pdAA layer. The RRMS values of PEO-like coated

substrates, shown in Table 6, were found to be very

similar to those measured on uncoated PET-CL. Further, as

the conformal pdAA layers described previously, also the

PEO-like coating resisted unaltered in shape and roughness

after 50 h of immersion in water, both on flat and on nano-

structured surfaces.

After the PEO-like deposition, the XPS O/C ratio of PET-

flat and PET-CL surfaces was found increased to the same

value (0.43� 0.03), as shown in Table 7, with respect to the

uncoated substrates. Figure 6 shows the C1s XPS signals of

(a) PET-flat and of (b) conical PET-CL surfaces before (black)

and after (dark grey) the deposition of 30� 5 nm PEO-like

coating; from these spectra and from the best-fitting C1s

data in Table 8 we can state that flat and structured PET

surfaces exhibit the same PEO-like composition, very

different from the uncoated substrates. Eighty percent of

PEO character (i.e., the relative importance of the C1 ether

component at 286.5 eV in the C1s spectrum) was measured

on both coated surfaces, that is a marker of non-fouling

character for protein and cells for plasma-deposited PEO-

like coatings.[50–52]

No alteration in the shape of the C1s signal (see Figure 6,

and Table 8) was detected after water soaking, attesting for

the resistance of also PEO-like coatings to water.

Static and dynamic WCA data of PEO-like films on flat

and conical structured surfaces before and after immersion

in water are shown in Table 9; PET-CL-PEO exhibited a

higher static WCA value than that found on PET-flat-PEO

surfaces. A higher WCA hysteresis value was measured on

PET-CL-PEO substrates with respect to PET-flat-PEO ones,

due to the very low value of the receding ur angle, as found

also for PET-CL-pdAA surfaces (see previous section), which

can be ascribed also in this case to the nano-topography.

Also for PEO-like surfaces static and dynamic WCA values

remained unchanged after water soaking, attesting for

DOI: 10.1002/ppap.200800005


Figure 5. 3D AFM micrographs and height distribution diagrams of PET-CL-PEO surfaces before (a) and after 50 h soaking in H2O (b).

their stability in water media. No difference in contact

angle hysteresis was observed between coated and

uncoated substrates, which confirms the prevalent effect

of surface topography on wettability properties.

Keratinocytes Culture

NCTC-2544 human keratinocyte cells were seeded on PET-

flat, PET-CL, PET-flat-pdAA, PET-CL-pdAA, PET-flat-PEO and

PET-CL-PEO surfaces, according to the procedure described

in ‘Cell Culture’ paragraph, in order to evidence any

possible difference in cell growth to be ascribed to

chemical composition or morphology of surfaces. It is

Table 6. RRMS values of PET-flat and PET-CL surfaces in uncoatedform, PEO-like coated and after 50 h soaking in H2O.

Surface RRMS

nm

PET-flat PET-CL

Uncoated 1.0W 0.5 42.6W 1.9

PEO 1.3W 0.5 46.0W 1.9

PEO/H2O 1.6W 0.5 47.5W 1.9



worth to stress again that all nano-structured surfaces

used in this research for cell-growth experiments exhibit

almost the same roughness and same average height, but

different pdAA and PEO-like surface chemical composition,

characterized by cell-adhesive[34] and cell-repulsive[27,36]

properties, respectively. This evidence comes useful when

cell growth results are compared. Since it is well known

that completely different cell-materials interactions can

arise from different chemical functionalities distributed on

the surface of a material probed by cells, a different cell

behavior on pdAA and PEO-like coated surfaces is expected.

Cell-materials interactions are strongly mediated by

proteins adhering at the surface of the material from

the culture media, and/or produced by the cells in contact

Table 7. XPS O/C ratio of PET-flat and PET-CL substrates inuncoated form and PEO-like coated before and after 50 h in H2O.

Surface XPS O/C ratio

PET-flat PET-CL

Uncoated 0.47W 0.03 0.37W 0.03

PEO 0.42W 0.03 0.45W 0.03

PEO/H2O 0.43W 0.03 0.44W 0.03


E. Sardella et al.

Figure 6. C1s XPS signal of PET-flat (a) and conical PET-CL(b) surfaces; uncoated (black), PEO-like coated (dotted), PEO-likecoated after 50 h in distilled water (light grey).

Table 9. Static and dynamic WCA values of PET-flat-PEO-like andPET-CL-PEO-like substrates.

Surface WCA

-

Static ua ur

PET-flat-PEO 47W 2 58W 2 41W 2

PET-CL-PEO 60W 2 85W 2 5W 5

PET-flat-PEO/H2O 50W 2 64W 1 46W 1

PET-CL-PEO/H2O 65W 2 90W 2 10W 5

548

with the same surface and this event is strongly influenced

by chemical composition and properties (e.g., polar/non

polar; acid/base; hydrophilic/hydrophobic, etc) of the

surface of the material.[36] Generally speaking, it can be

stated that moderately hydrophilic surfaces characterized

by polar groups and affinity with water allow cell adhesion

and spreading.

Quantitative data shown in Figure 7 were obtained by

counting the number of keratinocytes adhering to the

Table 8. Relative content of C1s XPS components observed on uncoated and PEO-like coated PET-flat and PET-CL before and after 50 hsoaking in H2O.

Surface PET-flat PET-CL

Ca C0 C0a C1 C2 C3a C3 C0 C1 C2 C3

% % % % % % % % % % %

Uncoated 65 – 21 – – 14 – 70 19 5 6

PEO – 16 80 4 – 17 80 3 –

PEO/H2O – 16 80 4 – 16 80 4 –



different surfaces during the first 24 h of culture at

different time intervals (30 min, 180 min and 24 h). Within

the first 24 h most cells remained isolated and in direct

contact only with the substrate, with no cell/cell interac-

tion. This condition allowed us to study the direct effect of

the substrate on cell adhesion and spreading.

At a very first glance, it is evident that no growth is

present on both flat and structured samples plasma-coated

with the PEO-like layer (PET-flat-PEO and PET-CL-PEO in

Figure 7); less than 3 cells per area were found at all the

considered time span for these surfaces. In fact, the PEO-

like surface prevents any adhesion of keratinocytes, due to

its high affinity to water molecules, that is bound at its

surface in a sort of hydrogel layer.[58] With such a surface

proteins first, then cells, do not find any thermodynamic

advantage to adhere at the surface from the medium, and

the non-fouling effect results, as Figure 8 shows. Here

clusters of round and dead cells float in the cell-culture

medium both on PET-flat-PEO and on PET-CL-PEO surfaces,

and very few round cells adhere. These results suggest that

the cell unfouling characteristics of the plasma-deposited

PEO-like coating were not altered by the presence of the

nano-structures underneath. Moreover, the presence of

spread cells adhering at the surface of the tissue-culture

polystyrene (TCPS) well, were the sample is located,

indicates that nontoxic chemicals are leached in the cell

culture medium from PEO-like coatings. A marked effect of

DOI: 10.1002/ppap.200800005


Figure 7. Number of NCTC 2544 human keratinocytes (mean� SD)adhered on conical and flat samples without and with pdAA andPEO coatings at different incubation time. Statistical significancehas been assessed by means of a two-way Anova and a Bonfer-roni post-test (*:p<0.05 vs. PET-flat-PEO, &: p<0.05 vs. PET-flat, ~: p<0.05 vs. PET-flat-pdAA, &: p<0.05 vs. PET-CL, �:p<0.05 vs. PET-CL-PEO).

Figure 8. NCTC 2544 human keratinocytes floating on PEO-likecoatings (a) and adhered and spread on TCPS surface around thePEO-like sample (b).

surface chemistry was, thus, detected on PEO-like coated

surfaces and the absence of adhered cells on PEO-like films

can be exclusively ascribed to the presence of protein

repellent ether groups on the coating.[53]

Concerning the cell adhesion results of Figure 7 on all

other samples, very interesting observation can be done on

the behavior of cells on flat and on nano-structured

surfaces.

After 30 min of incubations, the only statistically

significative difference in cell adhesion was observed

between cells grown on PET-flat and those grown on all the

other surfaces. This first piece of data clearly shows that

both topographical and chemical modification added to

the substrate confer an higher cell adhesive character to

PET. The pdAA coating support cell adhesion and spreading

due to O-containing polar functionalities (hydroxyl,

carbonyl, carboxyl etc.) on its surface,[21] as it occurs also

on commercial TCPS plates, where the same chemical

groups are grafted on PS by means of corona discharges in

air.[54]

From a comparison of growth data on PET-flat and PET-

CL, whose surface composition is very similar while the

roughness is different on a nano-metric scale, it can be

observed that the higher roughness of PET-CL induces more

cells to adhere with respect to the corresponding PET-flat

surfaces, at all culture times. The deposition of pdAA

coating on PET-flat surfaces increases cell growth at all

times, and this effect is clearly due to the new pdAA

composition of the surface. A further increase of cell

growth, respect to PET-flat-pdAA, is found for PET-CL-pdAA



surfaces, after 180 min of culture, and this effect is clearly

due to nano-topography. Another detail that can be

noticed is that, after 24 h, cell growth on PET-CL and

PET-CL-pdAA nano-structured surfaces reaches almost the

same value, although the growth rate on PET-CL-pdAA

surfaces is faster (compare data after 180 min).

These results allow us to attest that the presence of the

pdAA coating does not increase cell adhesion on nano-

structured samples.

From 180 min on, that is after the initial cell attachment

event, when cells start to proliferate on the samples, a

positive effect of the nano-features on cell adhesion is

observed with respect to the corresponding flat substrates.

This is well evident in comparison with PET-flat and PET-

flat-pdAA surfaces. This phenomenon may be due to the

fact that conical structures enhance the surface area of the

sample, and provide a higher number of sites able to

stimulate cell adhesion, much more than on the corre-

sponding flat surfaces.


E. Sardella et al.

550

Conclusion

When a material is in contact with a physiological cell

culture medium, surface chemistry and topography play

an important synergistic role in controlling all events

occurring at the interface between the material and the

biological milieu. For this reason, the fabrication of

differently shaped surfaces having the same chemistry,

or vice versa, is of great importance to understand the

fundamentals of cell-material interactions. Many nano-

structuring procedures can produce chemically and

topographically different substrates where it is almost

impossible, though, to discriminate the effect of chemical

composition from that of topography on cell behavior. In

this paper, a simple and cost effective method is described,

which allows to tune the chemical composition of

topographically different surfaces. Plasma deposition

processes are used to deposit thin conformal layers

without alteration of the substrates nano-topography; in

this way chemical and topographical effects on cell

behavior can be discriminated.

It is worth to state here that the approach followed in

this work with one topography and two different plasma

deposited coatings, pdAA and PEO-like, can be easily

extended to other topographies and other coatings of

many different properties, that plasma chemistry can

easily produce, provided that a conformal deposition is

achieved.[55–57]

For pdAA coated surfaces an improvement of cell-

adhesion was observed on nano-patterned substrates with

respect to the flat ones. Further, the inertness of PEO-like

coatings to protein adsorption and cell adhesion[28]

was confirmed, on flat and nano-patterned substrates.

We can conclude that surface conical nano-topography

plays an important role only when the surface chemistry is

suitable for supporting protein adhesion, cell adhesion and

spreading.

Acknowledgements: The EC project Nanobiotechnology andMedicine ‘‘NANOMED’’ EC 5thFP Quality of Life, and the MIUR-FIRB RBNE012B2K and RBNE01458S projects are acknowledged forfinancially supporting this research. A special acknowledgementis dedicated to S. Cosmai for his technical support. Prof. A. Curtisand Dr. M. J. Dalby (University of Glasgow, UK) are gratefullyacknowledged for a fruitful collaboration and for stimulatingdiscussions.

Received: January 15, 2008; Revised: May 2, 2008; Accepted: May16, 2008; DOI: 10.1002/ppap.200800005

Keywords: acrylic acid; cell adhesion; nanostructures; poly(ethyl-ene oxide); plasma deposition; soft lithography

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