ARTICLE IN PRESS

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YMVRE-02578; No. of pages: 12; 4C:

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Microvascular Research

Pericyte adhesion and growth onto polyhydroxymethylsiloxane surfaces

nanostructured by plasma treatment and ion irradiation

Giovanna Asseroa, Cristina Satrianob, Gabriella Lupoa, Carmelina Daniela Anfusoa,

Giovanni Marlettab, Mario Alberghinaa,*

aDepartment of Biochemistry, University of Catania, 95125 Catania, ItalybDepartment of Chemical Sciences, University of Catania, 95125 Catania, Italy

Received 25 May 2004

Abstract

The study deals with the adhesion and proliferation of bovine retina pericytes onto surfaces of poly(hydroxymethylsiloxane) (PHMS)

modified either by cold plasma or by low-energy ion beams. The surface treatment was able to convert the original polymer matrix into SiO2-

like phases for O2-plasma or ion-mixed SiCxOy(Hz) phases for ion irradiation, respectively, with different modification levels of the surface

free energy (SFE) and related surface wettability. Pericytes exhibited a negligible adhesion and proliferation onto untreated PHMS, an

enhanced adhesion but not proliferation on plasma-treated PHMS, and great adhesion and proliferation to full confluence on ion-irradiated

PHMS, as measured by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), quartz crystal microbalance, and optical

microscopy. On the other hand, the adhesion and proliferation of GP8.39 endothelial cells (EC), which are strongly associated with pericytes

in microvasculature, were very scarce onto both untreated and surface-modified PHMS. The surface-selective pericytal response was related

to changes of physicochemical properties of PHMS film, from hydrophobic/neutral towards hydrophilic/negatively charged polymer layers,

as well as to short- and long-time events of cell–surface interaction. We propose that surface properties can mediate and modulate cell–

polymer matrix adhesion through the establishment of stereospecific chemical interactions and/or electrostatic repulsion, which can also

explain the different behavior of pericytes compared to EC.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Pericytes; Endothelial cells; Poly(hydroxymethylsiloxane); Ion irradiation; Plasma treatment; Surface free energy; X-ray photoelectron

spectroscopy; Atomic force microscopy

Introduction

The cell adhesion and proliferation on synthetic surfaces

are fundamental processes in view of emerging medical

applications of biomaterials, including scaffolds for tissue

engineering, biosensors, medical devices, and bioelectronics

(Dee and Bizios, 1996; Grayson et al., 2004; Willner and

Willner, 2001). Among the various employed materials, an

increasing interest is addressed to the use of engineered

0026-2862/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.mvr.2004.08.003

* Corresponding author. Department of Biochemistry, Faculty of

Medicine, University of Catania, Viale Andrea Doria 6, 95125 Catania,

Italy. Fax: +39 95 7384220.

E-mail address: malber@unict.it (M. Alberghina).

polymers for controlled cell adhesion due to their inherent

advantages, as for instance the biodegradability, the process-

ability, the low cost, and the versatility in shaping (De Santis

et al., 2003; Hunkeler, 1997). Thus, a very large number of

reports deal with adhesion and proliferation of different cells

such as endothelial cells (EC), aortic smooth muscle cells,

chondrocytes, fibroblasts, astrocytes, glial cells, staminal

cells, osteoblasts, etc., on as different polymers as polylactic

acid (Watanabe et al., 2002), lactide-based polymers (Naj-

man et al., 2004), polyurethanes (Wang et al., 2002),

polyesters (Cenni et al., 1993; McFarland et al., 1999;

Ohsawa et al., 2001; Rizzi et al., 2001; Zreiqat et al., 1999),

polysiloxanes (Satriano et al., 1999, 2002a,b, 2001), and

polystyrene (Teare et al., 2000).

xx (2004) xxx–xxx

ARTICLE IN PRESSG. Assero et al. / Microvascular Research xx (2004) xxx–xxx2

Among the various cell types, pericytes are very interest-

ing in view of their capability to act as progenitor cells

differentiating into a variety of different types including

osteoblasts (Couch, 1990; Diaz-Flores et al., 1992), macro-

phages and fibroblasts (Balabanov and Dore-Duffy, 1998;

Thomas, 1999), and adipocytes (Richardson et al., 1982). In

particular, it has been demonstrated that the process of bone

formation from osteoblasts cells already present in the

periosteum is enhanced by proliferation and differentiation

of pericytes, which contribute a supplementary population of

osteoprogenitor cells (Brighton et al., 1992). Furthermore,

microvascular pericytes exhibit in vitro phenotypic expres-

sions that are similar to that of in vitro bone cells (Reilly et al.,

1998). In this context, numerous studies have been carried out

to optimize culturing of osteoblast-like cells on various

biomaterials, for the development of the tissue engineering

techniques, and (in a larger perspective) also for reconstruc-

tive surgery. The growth of osteoblast-like cells on surface-

modified titanium (Yang et al., 2002), on titanium and titania

or hydroxyapatite surfaces (Ramires et al., 2002), on a

bioactive glass and a glass ceramic (Ohgushi et al., 1996), and

on hydroxyapatite (Cerroni et al., 2002; Dong et al., 2001;

Kilpadi et al., 2001) has been widely investigated, but to the

best of our knowledge, no studies have been reported on the

growth of pericytes on polymeric surfaces.

To study the cell adhesion on polymeric surfaces two

main strategies are currently pursued: the first one involves

the chemical modification of the surfaces (Craighead et al.,

2001; Zhang et al., 1999), while the second one points to

topographical structuring of the surfaces (Curtis and

Wilkinson, 1997). A relatively unconventional way to

induce controlled chemical modifications on the polymer

surfaces involves the use of high-energy density deposition

methods, like keV-MeV ion beams (Curtis and Wilkinson,

1997; Kusakabe et al., 1995; Marletta and Satriano, 2004;

Satriano et al., 2003a,b,c) and RF or microwave cold

plasmas (Dewez et al., 1999; Pu et al., 2002).

Previous studies have shown that cell adhesion enhance-

ment may be related to as different surface properties as the

increase of polar basic character of the irradiated surfaces

(Satriano et al., 2003b), the formation of very complex

conductive hydrogenated amorphous carbon layers for

carbon-based polymers (Marletta and Satriano, 2004), or

the formation of an insulating silica-like phase for silicon-

based polymers (Satriano et al., 2002a,b). This large spread

of physical properties and chemical surface structures has

not yet been unified in a single exhaustive model, but it

appears that the response of the various cell lines is

substantially affected by the nature and structure of the

adsorbed protein layer, acting as the mediating agent

between surface and cell membrane (Satriano et al.,

2003a). Thus, different cell types could exhibit different

adhesion and spreading behavior on a surface of given

composition and properties.

Accordingly, the present study reports the comparison

between the cell response of pericytes from bovine retina

microcapillaries and immortalized endothelial cells

(GP8.39) from rat brain microcapillaries onto surfaces of a

poly(hydroxymethylsiloxane) (PHMS) modified by O2-

plasma treatments and 50 keV Ar+ ion beams. It appeared

very promising to study the different sensitivity of these two

types of cells with respect to a model activated polymeric

surface due to the fact that microcapillary pericytes are

strongly associated with EC and share a common basement

membrane with them in microvasculature where muscular

cells are absent, playing the key role of maintaining the

vasal tone.

The polysiloxane has been chosen as model polymer to

test the basic process of cell–surface interaction for these

specific cells because previous works in our laboratory

demonstrated that irradiated PHMS exhibit a dramatic

enhancement of adhesion and proliferation of fibroblasts.

Furthermore, the properties of surface free energy and the

related wettability of PHMS may be easily modified in a

controlled and graded way by means of surface irradiation

techniques (Satriano et al., 1999, 2002a,b, 2003a, 2001), at

variance of what it has been found for other polymer surfaces

such as poly(ethyleneterephtalate) and poly(caprolactone),

which do not exhibit such a behavior (Satriano et al., 2003d).

Materials and methods

Polymer film preparation and modification

Poly(hydroxymethylsiloxane) (PHMS, HoneyWell) thin

films were deposited by spin coating (3000 rpm, 60 s, room

temperature) from solutions on either p-doped silicon (100)

wafers, glass or gold-covered quartz crystals. The structure

formula of the polymer is reported below.

The thickness of the deposited films was 500 F 50 nm,

as measured from an alpha-step profilometer. The surfaces

modification was performed with low-energy ion irradiation

or cold plasma treatment. The ion irradiation was done with

a Danfysik ion implanter, with Ar+ ions at an energy of 50

keV. The beam was rastered over the samples to keep the

thermal load as low as possible and the ion dose was

controlled at 1015 ions/cm2. Plasma treatments were carried

out in a March Instrument solid-state PlasmodR unit

(Concord, CA, USA) supplied with an RF generator with

an excitation frequency of 13.56 MHz. The treatment

conditions were as follows: 99.95% minimum purity oxy-

gen; power, 100 W; pressure, 66.6 Pa; treatment time, 1

min. After the plasma exposure the samples were aged in

ARTICLE IN PRESSG. Assero et al. / Microvascular Research xx (2004) xxx–xxx 3

laboratory atmosphere for periods ranging from 24 h up to 1

week, that is, the periods observed for the surfaces to exhibit

a steady behavior. The effective thickness of the modified

layer can be estimated to be higher than roughly 9 nm

because the XPS analysis of the fresh plasma-modified

samples does not show any significant traces of the

characteristic carbon signal coming from the original methyl

groups of the polymer.

Surface characterization

X-ray photoelectron spectroscopy (XPS)

XPS analysis was carried out with a Kratos HX AXIS

spectrometer equipped with a dual Al/Mg anode and a

hemispherical analyzer. The spectra were obtained in fixed

analyzer transmission mode (pass energy 40 eV) by using

the Mg Ka1,2 radiation. The estimated sampling depth is

about 9 nm, according to an attenuation length of 3.0 nm for

Si 2p peak in organic materials (Suzuki et al., 1997). Such

value is actually comparable to the estimated thickness of

the ion- and plasma-modified layers (see above). XPS

spectra were analyzed by using an iterative least squares

fitting routine based on Gaussian peaks and the Shirley

background subtraction (Seah and Brown, 1999). Binding

energies (BEs) of all the spectra were referenced to the

intrinsic (before irradiation treatment) hydrocarbon-like C

1s peak set at 284.6 eV or to the adventitious one set at

285.0 eV (after the plasma treatments) (Hongbing and

Hamers, 1998).

Atomic Force Microscopy (AFM)

The surface microtopography and the morphology of the

surfaces were measured with a Multimode/Nanoscope IIIA

Atomic Force Microscope (Veeco) in tapping mode in air

with a standard silicon tip. The relative room humidity was

30% and the room temperature was 238C. Data were

acquired on square frames having edges of 10 Am, 1 Am,

and 350 nm. Images were recorded using height, phase-

shift, and amplitude channels with 512 � 512 measurement

points (pixels). Measurements were made twice or three

times on different zones of each sample.

Surface free energy (SFE) measurements

Measurements of surface free energy were performed by

evaluating both static and dynamic contact angles of three

different liquids onto the untreated and treated surfaces. Half

automatic video-based measurements of contact angle were

performed at 258C and 65% relative humidity by using an

OCA30 instrument (Dataphysics). By using the sessile drop

method, liquid drops of 2 Al of volume were applied on

different zones of each sample surface; and by digital image

analysis, the static contact angles (hs) were measured on

both sides of the two-dimensional projection of the droplet.

The advancing (hadv) and the receding (hrec) contact angleswere measured by the needle-syringe method (Erbil et al.,

1999). At least five measurements were made for each

sample and then averaged. The surface free energies, in

terms of apolar Lifshitz–van der Waals (cLW) and polar

Lewis acid (c+) and basic (c�) components, were evaluated

by using the Good-van Oss model (van Oss, 2002), with the

three following liquids: ultrapure Millipore water, glycerol,

and tricresyl phosphate (Aldrich).

Pericytes isolation and in vitro culture

Microvessel pericytes were extracted from bovine retinas

as previously described (Lupo et al., 2001). Cells were

characterized by negative staining for factor VIII-related

antigen, positive staining for smooth muscle a-actin

monoclonal antigen, morphological features including

absence of contact inhibition and g-glutamyltranspeptidase

activity (Lupo et al., 2001). The isolated cells were then

cultured in DMEM supplemented with 10% Fetal Calf

Serum (FCS), 100 U/ml penicillin, and 100 Ag/ml strepto-

mycin. Cells were incubated at 378C in a 5% CO2 incubator

and the medium was changed every 2 days. When the cells

reached the plateau phase of growth, they were harvested by

trypsinization, followed by addition of fresh culture medium

to create a new single cell suspension with desired seeding

cell number per cm2. Pericytes were identified by their

characteristic polygonal shape and through smooth muscle

a-actin staining.

QCM-D experiments and early events of cell–surface

interaction studies

In situ and real time studies of the early events, that is, till

2 h of incubation, of cell–surface interaction in PBS solution

were undertaken by using a Quartz Crystal Microbalance

with Dissipation Monitoring (QCM-D) instrument (Q-Sense

AB, Gothenburg, Sweden), which allowed the simultaneous

measurements of both frequency ( f) and energy dissipation

(D) of the sensor consisting of 5 MHz-crystals (Q-Sense),

spin-coated with PHMS thin films. Baseline curves were

measured with sensors oscillating in phosphate buffer saline

(PBS) solution; then the changes in D and f due to the

addition of a PBS solution containing 104 cells/ml were

monitored for both the fundamental frequency (n = 1, i.e., f

approximately 5 MHz), and the first three overtones (n = 3,

5, and 7, corresponding to f approximately 15, 25, and 35

MHz, respectively). The experiment was performed at 378C.

Pericytes growth studies on various PHMS substrates

Third passage pericytes were used to seed various PHMS

substrates at an initially cell density of 2 � 104 cells/cm2.

Incubation was performed in polystyrene culture dishes. The

PHMS samples were placed in the center of the dishes and

added with 1 ml of cell suspension to allow full attachment

of cells to polymers. Cultivation was conducted for 2, 24, 48

h up to 6 days. Culture media were changed every 2 days.

The cultures were carried out up to 5 days. All experiments

ARTICLE IN PRESSG. Assero et al. / Microvascular Research xx (2004) xxx–xxx4

were performed in triplicate. After each incubation, cells

were washed with Dulbecco Phosphate Buffer Saline

(DPBS) to remove nonadherent cells, and samples were

observed using an inverse phase light microscope (Zeiss

Axiovert 100) for analysis of cell morphology, spreading

and distribution on the investigated substrates, and collec-

tion of photomicrographs. In order to determine the number

of cells grown on various substrates, at least five random

visual fields per sample were photographed, and cells were

counted on five areas of each sample at 100� magnification.

Quantification of cells was obtained using an image analysis

software (Scion Image) after taking pictures of spreading

cells, evaluating the cell coverage in terms of integrated

density (ID). Cell viability was assessed using a trypan blue

assay. In another set of experiments, various PHMS

substrates were seeded with pericytes and cells were at the

end of the same incubation period washed three times with

DPBS and then treated with trypan blue for 15 min at 378C.Trypan blue assay was employed in this study to quantita-

tively assess the number of viable pericytes attached and

grown on tested polymers.

Endothelial cell culture

The immortalized rat brain endothelial cells (GP8.39)

used were generously provided by Dr. J. Greenwood

(Department of Clinical Ophthalmology, University Col-

lege, London). The cell line was already characterized and

our cell cultures were prepared following the procedures

previously described (Anfuso et al., 1999; Greenwood et al.,

1996). EC were grown at confluency in F-10 Ham medium

Fig. 1. X-ray photoelectron spectroscopy (XPS) photoelectron peaks of C 1s (le

(PHMS) thin film: (a) untreated, (b) plasma treated, and (c) 50 keV Ar+ irradiate

containing 10% plasma-derived serum (PDS), 2 mM

glutamine, 80 Ag/ml heparin, 100 unit/ml penicillin, and

100 Ag/ml streptomycin. EC were seeded on different

PHMS substrates (untreated, plasma-air-treated, and ion-

beam-irradiated PHMS) at a density of 2 � 104 cells/cm2

and cultured for 2, 24, and 48 h. At the end of these

incubation times, cells were washed twice with DPBS to

remove nonadherent cells and observed using an inverse

phase light microscope (Zeiss Axiovert 100) to evaluate

cells adhesion and morphology. Photomicrographs were

taken to quantify the cell number.

Results

Chemical modification of surfaces and related properties

Surface chemical structure and composition

The XPS analysis of surface chemical composition of

PHMS samples modified, respectively, by O2-plasma and

1 � 1015 ions/cm2 50 keV Ar+ beams indicates that the

original stoichiometry [Si1C1.2O3.2] of the untreated PHMS

changes, respectively, to [Si1C0.3O3.4] for plasma-treated and

to [Si1C0.8O3.0] for ion-irradiated PHMS. In particular, both

treatments induce the loss of the methyl groups, indicated by

decreases in the carbon content at surfaces, from an initial

value of approximately 22% to about 7% and 16%,

respectively, for plasma and beam-irradiated PHMS. The

detailed analysis of the photoelectron peak shape and

binding energy (BE) showed differences among the various

samples. Fig. 1 displays both C 1s and Si 2p peaks for

ft hand side) and Si 2p (right hand side) for poly(hydroxymethylsiloxane)

d.

ARTICLE IN PRESS

Table 1

Root mean square (RMS) and average roughness (Ra) for the various

PHMS surfaces measured in air by AFM in tapping mode on (1 � 1) Am2

scan regions

PHMS RMS (nm) Ra (nm)

Untreated 0.546 F 0.007 0.440 F 0.009

Plasma treated 0.520 F 0.06 0.428 F 0.04

Ion irradiated 0.544 F 0.06 0.434 F 0.04

G. Assero et al. / Microvascular Research xx (2004) xxx–xxx 5

untreated, plasma-treated, and ion-irradiated PHMS. For the

untreated PHMS surfaces (Fig. 1a), the C 1s peak can be

fitted by using only two basic components, both having full

width at half maximum (FWHM) of approximately 1.5 eV.

The main one (C1) is centered at 284.6 eV of BE and

assigned to NC–Si bonds, in agreement with literature data

(Satriano et al., 2001); the second peak component (C2) is

found at approximately 286.6 eV of BE, being assigned to

NC–OH and NC–O–C groups belonging either to the

terminal polymer groups or to the solvent residues (Satriano

et al., 2001). The Si 2p peak analysis evidences a symmetric

band, which is well fitted by using a single gaussian

component (Si1) of FWHM approximately 1.7 eV, centered

at about 102.2 eV of BE, assigned to SiO3C clusters, in

agreement with literature (Satriano et al., 2001).

For the plasma-treated and aged PHMS surfaces (Fig.

1b), the C1 component is dramatically reduced with respect

the C2 one; these components basically remain at the same

BE as in unirradiated PHMS, whereas in the freshly treated

samples (not shown) the carbon peak is completely

eliminated. This fact suggests that a partial recovery process

occurs with exposure to atmosphere, basically involving a

small but significant surface segregation of bulk chains

(Satriano et al., 2002a,b). As to Si 2p peak, a symmetric and

narrow band (FWHM approximately 1.5 eV) has been

found at approximately 103.6 eV (Si2 component), charac-

teristic of the formation of an amorphous SiO2-like phase,

predominantly formed by randomly interlinked [SiO4]

clusters.

At variance of the plasma case, for the 50-keV Ar+-

irradiated PHMS (Fig. 1c), two new components, centered at

286.1 eV (C3) and 288.0 eV (C4), were found for the C 1s

peak and respectively assigned to the formation of C–O–Si

moieties (C4 component) and NC = O groups (C5 compo-

nent) (Satriano et al., 2001). Furthermore, traces of newly

formed NCOO� groups could be identified in the high BE

tail of the peak, confirmed by TOF-SIMS measurements

(data not shown). These new components replaced the

pristine C2 component. It is to stress that in this case, the

freshly irradiated samples have similar composition of the

aged ones, showing no evidence of any carbon recovery at

surfaces. Furthermore, Si 2p peak fitting evidences the

coexistence of two components, Si1 and Si2, with a relative

ratio of 4:1 between [SiO3C] and [SiO4] clusters.

Surface free energy

After irradiation, the water contact angle changes from

the original value of 93.08 F 1.78 of the hydrophobic

untreated PHMS to 12.88 F 0.58 (i.e., very hydrophilic) for

plasma-treated and 51.78 F 0.98 (i.e., mildly hydrophilic)

ion-irradiated PHMS surfaces, respectively. It is worthy to

stress that the contact angle modification for the various

irradiated surfaces could in principle be due to radiation-

induced morphology changes. Accordingly, the average

roughness of all the investigated surfaces has been measured

by AFM. Table 1 shows the results in terms of root mean

square (Rrms) and mean (Ra) roughness. It appears that the

various treatments do not induce any significant change in

roughness for both O2-plasma-treated and 50 keV Ar+-

irradiated samples, respectively.

The surface free energy of each sample was evaluated in

terms of the apolar or Lifshitz–van der Waals component,

including the dispersive, inductive, and orientational con-

tributions to the van der Waals interactions and acid and

basic Lewis polar components. Fig. 2 shows the total SFE,

as well as the abovementioned dispersive and polar

components for untreated, plasma-, and ion-irradiated

PHMS surfaces. Both irradiation treatments generally

increase the polar components much more than the

corresponding apolar cLW term.

Short-term events in cell–surface interaction

Fig. 3 shows the frequency and dissipation plots for cell

attachment events onto untreated (a), O2-plasma-treated (b),

and 50 keV Ar+ ion-irradiated PHMS (c), respectively. The

experiments have been ran for 2 h in PBS solution, allowing

just an early interaction of the cells on the various polymer

surfaces. The frequency ( f) and dissipation (D) plots show

that PHMS surfaces trigger different pericyte responses

according to various treatments. In fact, both untreated and

ion-irradiated PHMS surfaces exhibited a huge frequency

shift of approximately �70 H �80 Hz due to a quite high

beffectiveQ cellular mass attachment on the electrode surface

and a corresponding dissipation shift of approximately 6 �10�6, that is, a large dissipative response of the attached

cellular matter, corresponding to a very pronounced cell

viscoelastic character. A different cell behavior was found in

the plasma-treated PHMS surfaces where the observed shift

in frequency was about�25 Hz, and the dissipation of about

approximately 1 � 10�6, that is, a lower effective cell mass

on the surface and a lower viscoelasticity of the adhered

matter. It is to point out that the frequency shift observed in

the case of cell–substrate interaction in the QCM-D experi-

ments is not directly proportional to real mass uptake

because the shear stress wave measuring the adsorbed mass

penetrates no more than 0.25 Am in the overlaying matter.

Therefore, the technique does not bsenseQ the whole cellularbody, but only the interaction of cellular membrane region

with the substrate (Fredriksson et al., 1998), and provides

just an estimation of beffectiveQ mass, which is not directly

proportional to the number of adhered cells, but to the

relative substrate coverage.

ARTICLE IN PRESS

Fig. 3. QCM-D shift normalized curves of frequency (upper panel) and

dissipation (lower panel) for the third overtone after addition of 104 pericyte

cells/ml in PBS, up to 2 h at room temperature, to untreated (a), plasma-

treated (b), and 50 keV Ar+ ion-irradiated PHMS (c).

Fig. 2. Total surface free energy (SE) and corresponding Lifshitz–van der Waals (LW), Lewis acid, and basic components for the various PHMS surfaces (*P b

0.05; **P b 0.01 compared to untreated surface by Student’s t test).

G. Assero et al. / Microvascular Research xx (2004) xxx–xxx6

Fig. 4 shows the dissipation versus frequency shifts in

characteristic D/f plots. This graph allows to drop the time as

explicit variable; in turn, it allows to interpret the temporal

homogeneity of the investigated attachment process as due to

fast kinetics for sparse data points and slow attachment

kinetics, reaching the steady state, for overlapping dense

points (Fredriksson et al., 1998). Fig. 4 clearly shows that a

very different cell–surface interaction occurred for the three

investigated surfaces. Thus, the attachment process of

pericytes on the unirradiated PHMS is characterized by a

relative high apparent mass uptake and a relatively soft

adsorbed layer. In this case, the adsorption kinetics consists

in a peculiar curve shape exhibiting an inversion of slope

around the half of the adsorption process and the saturation

of both frequency and dissipation with the time. At variance

of this, the ion-irradiated surfaces exhibit a faster adsorption

kinetics with a large apparent mass uptake and a relatively

stiff adsorbed layer, without slope inversion and slowly

saturating behavior. Finally, for the plasma-treated surfaces

the apparent mass uptake is very low, with a very fast and

linear adsorption kinetics, reaching the saturation in a few

minutes, the adsorbed layer being very rigid.

Long-term events in cell–surface interaction

Fig. 5 reports the optical micrographs for the pericyte

growth on the PHMS substrates treated in the above

discussed way. Only sporadic cells were found on untreated

PHMS surfaces at all investigated incubation times (panel

A); these cells have a characteristic spherical or polygonal

shape. On plasma-treated samples (panel B) a significantly

ARTICLE IN PRESS

Fig. 4. Dissipation (D) versus frequency ( f) plots (D/f plots) measuring cell attachment. The graph allows to drop the time as explicit variable. Pericytes

suspended in PBS (104 cells/ml) were allowed to attach on the various PHMS surfaces up to 2 h at room temperature.

G. Assero et al. / Microvascular Research xx (2004) xxx–xxx 7

higher number of adhered cells were already found at 2 h of

incubation time, while for longer incubation time, up to 5

days, the number of cells showed only a weakly increase in

adhesion. In this case, only part of the adhered cells looked

healthy and well shaped, indicating a partial cytocompati-

bility of these surfaces. Finally, pericytes adhered massively

on 50 keVAr+-irradiated surfaces (panel C), exhibiting since

the beginning a large stellate shape, diagnostic of an optimal

Fig. 5. Photomicrographs of pericytes on various PHMS substrates. Adherence and

100) on PHMS surfaces. Pericytes were plated at a density of 2 � 104 cells/cm2

PHMS (B), and on ion-beam-modified PHMS (C) for 2, 24, 48 h, and 5 days in

adhesion, producing the full confluence of the cell layer at 5

days of incubation. At this stage, the cell morphology was

consistent with fully healthy cells (Balabanov and Dore-

Duffy, 1998).

Fig. 6 shows the quantitative evaluation of cell coverage.

For short incubation time (2 h), the number of adhered cells

on both plasma- and ion-irradiated PHMS is higher than the

one observed onto the untreated polymer. For longer

spreading of pericytes were observed by optical microscopy (Zeiss Axiovert

and cultured on control unmodified PHMS surface (A), on plasma-treated

cubation times in DMEM containing 10% fetal bovine serum.

ARTICLE IN PRESS

Fig. 6. Number of adhered pericytes on various PHMS substrates. Cells viability was determined by trypan blue vital staining. Pericytes were plated on various

PHMS-modified surfaces at a density of 2 � 104 cells/cm2. After 2, 24, and 48 h, the number of cells on control untreated PHMS surface, on plasma-treated

PHMS and on ion-beam-modified PHMS was determined. Data shown are representative of three separate experiments (*P b 0.05; **P b 0.01 compared to

untreated control by Student’s t test).

G. Assero et al. / Microvascular Research xx (2004) xxx–xxx8

incubation times (24 and 48 h), a significant increase in the

number of adhered cells occurred on the Ar+-irradiated, but

not on plasma-treated, samples, indicating that a real

proliferation process was arising only on the ion-modified

surfaces.

The results of adhesion of rat brain endothelial cells, used

for comparison sake, on the same PHMS surfaces are

reported in Fig. 7. No significant cell adhesion was found

for any of the PHMS substrates, and no differences were

observed neither in proliferation nor in typical morpholog-

ical differentiation of EC. In preliminary experiments, we

used endothelial cells isolated from bovine brain micro-

capillaries, but they did not attach as did immortalized cells

from rat.

Fig. 7. Rat brain GP8.39 endothelial cells (EC) growth and adhesion after 2, 24, a

PHMS surfaces (B). No differences were detected over controls as function of time

substrates.

Discussion

The results basically indicate that pericytes do not

significantly adhere onto untreated PHMS surfaces, while

they adhere, but do not proliferate, on plasma-treated

surfaces and adhere and proliferate massively onto ion-

irradiated surfaces. Since all the incubation experiments in

the present work were performed on the bare substrate

surfaces, that is, without the routinely used precoatings of

gelatin, the observed different cell behavior on the various

surfaces must be related to the specific surface properties on

one side, and the response of the complex environment

formed by culture medium and cell membranes on the other

side.

nd 48 h incubation times onto control untreated (A) and ion-beam-modified

. No cell proliferation and phenotypical differentiation were observed in both

ARTICLE IN PRESSG. Assero et al. / Microvascular Research xx (2004) xxx–xxx 9

In particular, the results of the physicochemical charac-

terization indicate that the untreated PHMS surfaces consist

of a hydrophobic layer, formed by methyl groups oriented

towards the solid-water interface, not charged in solution at

pH 7.4. At variance of this, both the plasma treatment and

the Ar+ irradiation induced the formation of an altered

surface layer basically formed of Si-enriched phases. The

XPS results show clearly that two different nanostructures

of the altered layers were produced depending on the type of

treatment. Indeed, the Ar+-irradiation produced a specific

amorphous SiCxOy(Hz) phase, with a high intrinsic content

of carbon-containing domain and Si–C linkages, while the

plasma treatment essentially produced an SiO2-like phase,

that is, without Si–C linkages (Fig. 1). Both surfaces show a

negative charge in solution at pH 7.4 (Satriano et al.,

2003d).

The related surface free energy measurements are in

agreement with the XPS data, as irradiated surfaces

exhibited a strong modification of water contact angles h,from the hydrophobic character of the unirradiated, methyl-

terminated PHMS surface (h approximately 938) to a mildly

hydrophilic character (h approximately 528) for Ar+-

irradiated surfaces and a strongly hydrophilic one (happroximately 138) for the plasma-treated surfaces. Related

components were also dramatically changed, as the dis-

persive Lifshitz–van der Waals parameter increased from

about 24 mJ/m2 for the hydrophobic untreated PHMS

surface to approximately 36 mJ/m2 for Ar+-irradiated

surfaces and to approximately 40 mJ/m2 for the plasma-

treated ones, respectively, the magnitude being roughly the

same for the two modified samples. In addition, the polar

Lewis base component underwent a dramatic increase due

to the irradiation treatments. In fact, the initial value of

about 5 mJ/m2 becomes about 21 mJ/m2 for Ar+-irradiated

surfaces and about 45 for the plasma-treated one, that is, the

difference in the nanostructure of irradiated surfaces marked

a factor two in the polar Lewis base terms.

In view of the synergic action of culture medium and cell

membranes related to the various surfaces, we discuss the

pericyte behavior discriminating between short-term events,

essentially involving the mere adhesion process (physical

contact) for incubation time up to 2 h, and long-term events

consisting in cell spreading and proliferation phenomena,

for incubation times from 24 h up to 6 days. In short-term

events, we have performed both serum-free and serum-

containing experiments to discriminate between cell mem-

brane–surface interactions and serum-mediated cell adhe-

sion. As shown in the previous section, the short-terms

adhesion events in serum-free experiments could be

analyzed in terms of the QCM-D and optical microscopy

results, whereas the cell response in the serum-containing

experiments has been basically investigated by optical

microscopy only.

In general, QCM-D measurements allow a simultaneous

estimate of the effective mass uptake, given by the measured

frequency shift, and of viscoelastic properties of the sensed

adsorbed layer, that is, the cell–substrate interface reflected

by the energy dissipation factor (Nimeri et al., 1998). In the

case of cell–polymer interactions, the frequency shift is no

longer related in a straightforward way to the mass uptake,

as far as the Sauerbrey equation, stating the proportionality

between quartz frequency shift and the adsorbed mass, is no

longer valid (Fredriksson et al., 1998). However, the

measured frequency shift still contains an indication of the

strength and dynamic character of the interaction between

adhered cells and polymer surfaces, discriminating succes-

sive steps of cell–surface interaction. In fact, the measured

shift is mostly depending on the cell–surface contact area,

that is, both on the rate of cell attachment on the surface and

the spreading process with respect to the initial physical cell

contact. To this purpose, it is to point out that the evanescent

wave produced by the oscillating sensor has a typical

penetration length of approximately 0.25 Am in the over-

laying matter, that is, a sampled thickness much lower than

the typical average vertical size of a single cell.

On the other hand, the energy dissipation measurement

provides valuable information about the viscoelastic proper-

ties, which are originated within the thin cellular region and

the related cell–substrate interface. In particular, the rigidity

of the sampled layer can relate the strength of adhesion to

factors like the number of binding sites, types of expressed

adhesion proteins, changes in the cytoskeleton in proximity

of the cellular wall, etc. (Marx et al., 2003). The data

reported in Fig. 3 have to be interpreted in terms of the

above explanation as due to the characteristics of the cell–

substrate interaction and not to the mere number of cells.

The analysis of D/f plots (see Fig. 4), together with the

optical microscopy (OM) pictures, allows to spot the strong

differences among the features of pericyte adhesion onto

untreated, plasma-treated, and ion-irradiated PHMS. Due to

the fact that OM pictures showed (data not reported) that in

serum-free conditions a comparable number of few cells

was attached on the different surfaces—even if most of them

still did not exhibit the typical pericyte phenotype—the

observed differences in D/f plot must be mostly related to

the differences in the cell–surface interaction features.

In turn, the D/f plot for the plasma-treated PHMS, an

indication of a linear slow kinetics of a small mass uptake

providing a rigid layer, can be interpreted as an evidence of

a very strong interaction between the cell membrane and the

very hydrophilic surfaces, blocking the cell and preventing

the subsequent spreading. On the contrary, the initial fast

adsorption processes followed by a second slower step,

involving a large apparent mass uptake and the formation of

viscoelastic layer for the very hydrophobic untreated PHMS

surfaces, can be interpreted as diagnostic of the sticking of

loosely bound cells, ready to detach from the surface and

therefore not suitable for the spreading and proliferation

steps. Finally, the intermediate behavior of the D/f plot for

the ion-irradiated surfaces, characterized also by an inter-

mediate degree of hydrophilicity, is in agreement with the

occurrence of efficient attachment process of cells relatively

ARTICLE IN PRESSG. Assero et al. / Microvascular Research xx (2004) xxx–xxx10

well spread on the surfaces, as it is suggested by dissipative

behavior characteristic of a more rigid adsorbed layer with

respect to the unirradiated surfaces. In this case, the cells

still would have sufficient degrees of freedom to prompt the

subsequent spreading and proliferation processes.

Thus, the whole results for serum-free experiments could

be understood in terms of the different strength of cell

attachment as a function of the surface free energy, which is

in turn related to the strong increase of Lewis base groups.

Accordingly, the very polar nature of the plasma-treated

surfaces prompts pericyte attachment. However, the cell

viability was hindered by the strong sticking, whereas the

almost exclusive dispersive character of the unirradiated

PHMS surfaces prevents any effective cell binding. In

conclusion, only ion-irradiated PHMS surfaces have the

right density of polar groups prompting a suitable attach-

ment of the cells, without preventing the subsequent

proliferation process.

The results obtained in the serum-containing experiments

add more insight to the picture of pericyte behavior on the

various polymer surfaces. In fact, the OM pictures 2 h after

incubation onto plasma- and ion-irradiated surfaces showed

that pericytes were able to reconstruct their phenotype ex-

pression already at this short incubation time, whereas such

an effect was not seen for the unirradiated PHMS. The

average number of adhered cells for different surfaces in this

case was higher than in the corresponding serum-free

experiments, suggesting that the effect of proteins adsorp-

tion on the surfaces from the serum might play a role in

prompting the cell attachment. However, the number of

adhered cells was significantly different among surfaces, the

higher number of adhered cells being observed on the ion-

irradiated surfaces, followed by the plasma-treated and by

the unirradiated ones (see Fig. 6). It is noteworthy that this

quantitative trend corresponded to the above-discussed

findings from D/f plots in terms of relative interaction

strength.

Taking into account long-term events, which surely

involve cell spreading and proliferation processes in

serum-containing culture medium, our results deserve a

special comment. Cell proliferation essentially reflects the

trend already observed for cell attachment. In fact, for

untreated surfaces, there was no proliferation occurring; for

plasma-treated surfaces, the number of attached cells was

almost unchanged, while proliferation took place only for

ion-irradiated surfaces.

Several hypotheses on the role of peculiar cell processes

occurring on a long scale of time contact, that is, protein

adsorption from the serum and cell expression of adhesion

proteins, may be made. A first hypothesis concerns the

differential interaction of adhesion molecules with surfaces.

Integrins, cadherins, intercellular adhesion molecule-1

(ICAM), and vascular cell adhesion molecule-1 (VCAM-

1), some of which are present constitutively and others that

can be up-regulated in response to chemotactic stimuli, play

a key role in cell–cell and cell–extracellular matrix

interactions. Different expression of adhesion molecules

may reflect different culture and experimental conditions

(Wong and Dorovini-Zis, 1995), and it could be influenced

either by wettability or electric charge of solid substrate

available to cells in culture, such as the polymer we used.

However, adhesion molecules are expressed and modulated

in both cultured EC and pericytes (Balabanov et al., 1996;

Ivanov et al., 2001; Daxecker et al., 2002), so this

hypothesis cannot explain the differential behavior of these

two cell types against PHMS.

A second hypothesis concerns the expression of a variety

of extracellular matrix (ECM) components like fibronectin

and laminin (Mandarino et al., 1993; Tilling et al., 2002),

collagen (Cohen et al., 1980), and glycosaminoglycans

(Stramm et al., 1987) by pericytes and EC. These

components enhance, for instance, pericyte adhesion and

growth and play an important role in regulating cell growth,

as demonstrated by coculture (pericytes and EC) experi-

ments (Antonelli-Orlidge et al., 1989) and in the main-

tenance of vessel integrity (Hirschi and D’Amore, 1996).

Expression of ECM components, however, is not able to

explain temporally the early mechanism of cell–polymer

interaction because it occurs within 2 h, while ECM

molecules expression requires longer times.

A further and more consistent hypothesis is that the

different response of pericytes and EC to the interaction

with PHMS could be probably due to their cellular

properties of in vitro growth. EC need an extracellular

matrix to proliferate, usually collagen, and a medium with

high serum percentage to grow and to spread out. Collagen

is designed as a glycoprotein since it contains significant,

but highly variable, amounts of covalently linked carbohy-

drates. All carbohydrate units are linked O-glycosidically to

hydroxylysine residues in a unique way. The absence of this

right substrate in culture dish determines the loss of the

phenotype and makes EC very susceptible of detachment

from polymeric surfaces. Pericytes are instead able to grow

even in absence of matrix support, usually gelatin, which is

an heterogeneous mixture of water-soluble proteins present

in collagen, maintaining cellular characteristics and pheno-

type. The presence of carbohydrate groups in collagen could

play an essential role in EC adhesion to culture dishes in in

vitro conditions.

Based on this premises, we believe that adhesion

molecules or secretion of ECM components appear not to

contribute to the control of cell–substrate interaction on

PHMS slides. We thus propose that surface properties (for

instance, the presence of specific glycoconjugates) can

mediate and modulate cell–polymer matrix adhesion

through the establishment of stereospecific chemical inter-

actions and/or electrostatic repulsion.

In conclusion, our findings highlight the ability of

untreated and irradiated PHMS surface to act as a selective

modulator of cell adhesion, inducting or inhibiting

adhesive interactions depending on the specific properties

of microvascular cells and the matrix around them. In

ARTICLE IN PRESSG. Assero et al. / Microvascular Research xx (2004) xxx–xxx 11

addition, the present study establishes an experimental

framework to analyze adhesive mechanism controlling

cell–surface interactions and provides a general strategy

of surface-directed control to manipulate cellular spreading

in biomaterial and biotechnological applications.

Acknowledgments

The authors wish to acknowledge the financial support of

COFIN 2002 (University of Catania) and CIB (University of

Catania). The authors are also grateful to Dr. F. Rossi (IHCP,

Joint Research Centre, European Commission, Ispra) for the

ion-beam irradiation facilities.

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