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Biomaterials 28 (2007) 3668–3678

www.elsevier.com/locate/biomaterials

Expression of cell adhesion receptors in human osteoblasts cultured onbiofunctionalized poly-(e-caprolactone) surfaces

Ilaria Amatoa, Gabriela Ciapettia, Stefania Pagania, Giovanni Marlettac, Cristina Satrianoc,Nicola Baldinia,b, Donatella Granchia,�

aLaboratory for Pathophysiology, Istituti Ortopedici Rizzoli, via di Barbiano 1/10, 40136 Bologna, ItalybDepartment of Orthopaedic Surgery, University of Bologna, Bologna, Italy

cLaboratory for Molecular Surfaces and Nanotechnology, Department of Chemical Sciences, Universita di Catania and CSGI,

v.le A. Doria 6, 95125 Cata nia, Italy

Received 1 February 2007; accepted 30 April 2007

Available online 3 May 2007

Abstract

This study was aimed to investigate whether the activation of poly-(e-caprolactone) (PCL) surface by low-energy irradiation and/or the

biofunctionalization by absorption of arginine–glycine–aspartic sequences (RGD), can modify the expression of integrins closely related

to the osteoblast activity. For this purpose, we analysed the physicochemical changes induced by irradiation and RGD immobilization,

the consequences on cell adhesion and spreading, and the effects on integrin expression. PCL irradiated with 5� 1015He+/cm2 (10 keV

energy) (irr-PCL) showed an altered surface layer with a partial loss of carboxyl species and the formation of carbonyl groups. Moreover,

irr-PCL showed a small smoothening effect and a less polar character in comparison to the pristine ones. The RGD immobilization was

observed only on irr-PCL (surface coverage: 7.0 pmol/cm2). Human osteoblasts (hOB) were cultured on untreated PCL (ut-PCL), ut-

PCL+RGD, irr-PCL, and irr-PCL+RGD. After 24 h, ut-PCL hindered the cell adhesion, while a discrete layer of hOB with a good

cytoskeleton organization was detected on irr-PCL and irr-PCL+RGD. Before seeding, the single hOB suspension expressed a1, a2, a3,a5, b1, and aVb3; after 24 h, cells cultured on tissue-plastic expressed high levels of b1 and aVb3, while a1 showed a low intensity and a2,a3, and a5 were negative. b1 and aVb3 were selected to evaluate the interaction between cells and PCL samples. The b1 expression was

higher in hOB cultured on irr-PCL than on the other samples. A significant increase in aVb3 expression was observed only in irr-

PCL+RGD, and confirmed by the gene expression analysis. In conclusion, ion irradiation and RGD adsorption on PCL surfaces

modulate the expression of integrin involved in hOB growth and function, indicating the effectiveness of biomimetic surfaces in

promoting cell adhesion. Ultimately, the study of integrin expression may suggest proper changes to the surface structure in order to

improve the osteoconductivity of selected materials.

r 2007 Elsevier Ltd. All rights reserved.

Keywords: Poly-(e-caprolactone); Bioactivity; Osteoblast; Cell adhesion; Integrin

1. Introduction

Biodegradable synthetic polymers offer a number ofadvantages as compared to other materials for developingscaffolds to be used for bone tissue engineering [1,2]including the ability to tailor mechanical properties anddegradation kinetics to suit various applications.

e front matter r 2007 Elsevier Ltd. All rights reserved.

omaterials.2007.04.038

ing author. Tel.: +3951 6366896; fax: +39 51 6366748.

ess: donatella.granchi@ior.it (D. Granchi).

Synthetic polymers are also attractive because they canbe fabricated into various shapes and with the desired porefeatures favouring osteoconduction. Moreover, they can bedesigned with chemical functional groups to induce tissuein-growth [3]. A variety of biodegradable polymers forbone tissue engineering have been investigated. In parti-cular, poly-(e-caprolactone) (PCL) is a promising materialfor bone and cartilage repair [3–6]. It shares goodmechanical properties with a lower degradation rate, whichare excellent requirements for the application in tissueengineering, in particular when the scaffold is thought for

ARTICLE IN PRESSI. Amato et al. / Biomaterials 28 (2007) 3668–3678 3669

considerable bone defects. Previous studies have shownthat osteoblast-like cells grow successfully on PCL matrices[7], although cell adhesion of human primary osteoblasts(hOB) is not so effective [8].

Osteoblasts are attachment-dependent cells and theiradhesion to materials is an essential step that influences allthe events leading to the formation of new bone which willtotally replace the scaffold. These events may be summar-ized in the recruitment of osteoprogenitor cells from thetissue around the implanted material, their proliferationand differentiation into mature osteoblasts, and finally theformation of bone matrix and its mineralization [9]. Celladhesion onto a polymeric material can be closelyexamined through the study of the receptors involved inthis process, i.e., integrins, which play a crucial role inspreading, migration, and signalling by providing trans-membrane links between the intra- and extra-cellularmatrix (ECM) [10]. They are composed of at leasttwenty-four a and nine b different subunits that dimerizein specific combinations. hOB are known to expressvarious integrins, each component having a large extra-cellular domain responsible for ligand binding, a trans-membrane domain, and a short cytoplasmic domainresponsible for interacting with the actin cytoskeleton.Integrin heterodimers, bind to specific amino acid se-quences, such as the arginine–glycine–aspartic acid (Arg–Gly–Asp or RGD) recognition motif that is present inmany ECM proteins, including fibronectin, vitronectin,bone sialoprotein, and osteopontin [9,10]. Small syntheticpeptides (a few hundred Dalton) that contain amino acidsequence RGD can mediate cell attachment as well as thelarge parental molecule (a hundred thousand Dalton).Over the last decade, biomimetic approaches have beendeveloped to immobilize short peptides, such as RGD,onto synthetic or natural surfaces. This opens thepossibility to produce biofunctional materials that promotecell attachment by the binding of adhesion receptors[11,12]. In addition, the characteristics of material surfaces,such as topography, chemistry or surface energy, play anessential role in osteoblast adhesion and growth [13], byinfluencing the integrin localization and structure [14].

Among the various possible surface treatments, ionbeam irradiation of polymer surfaces is known to inducethe formation of a drastically modified altered layer,strongly affecting the cell-surface interaction [15]. Inparticular, low-energy ion irradiation in itself has beenshown to markedly enhance the adhesion of hOB [8], andto improve the performance of PCL through the differ-entiation of osteoprogenitors into mature osteoblasts [16].Because the high hydrophobicity has a detrimental effecton cell adhesion, PCL represents a proper material fortesting the changes in cell reactivity due to surfacetreatment. In the present study, 10 keV He+ has beenemployed to investigate in vitro whether such enhanced celladhesion and differentiation onto irradiated surfaces canbe related to the modification of the integrin expressioninvolved in the hOB function. The comparison has

been extended to the same surfaces treated with RGDsolutions.In fact, as integrin expression and osteoblast biology are

closely related, the behaviour of these molecules in theearly phases of the adhesion could represent an indicator ofthe effectiveness of biomimetic surfaces in promotingadhesion, proliferation and function of osteoblast.For this purpose, hOB were cultured for 24 h on He-

irradiated PCL, with or without RGD adsorption. At thatendpoint, cell adhesion, morphology of cytoskeleton, andintegrin expression were analysed by confocal microscopyand quantified by image analysis.

2. Materials and methods

2.1. PCL film preparation and modification

Thin films of PCL (about 100 nm) were prepared as previously

described [8]. The ion irradiation of samples was performed at room

temperature by using 10 keV He+ ions from a differentially pumped AG61

ion gun (VG Ionex). The ion dose, controlled by means of a Faraday cup,

was set at a fluence of 5� 1015 ions/cm2, with a current density of 1.5 mA/

cm2, and the chamber pressure during irradiation was at maximum

10�5 Pa.

RGD sequence was purchased as a powder (Sigma) and solubilized in

deionized water at a final concentration of 2.9mM at a pH values of 5.35,

which is consistent with the estimated value for the isoelectric point. The

RGD adsorption was performed by soaking the PCL samples in the RGD

solution at room temperature for 1 h, then rinsing with Millipore water,

and drying in the laboratory atmosphere.

Four types of PCL samples were prepared and coded as follows:

untreated PCL film: ut-PCL; untreated PCL film with RGD moieties:

ut+RGD-PCL; irradiated PCL film: irr-PCL; irradiated PCL film with

RGD moieties: irr+RGD-PCL.

X-ray photoelectron spectroscopy (XPS) and Atomic Force Micro-

scopy (AFM) were employed to characterize chemical structure and

surface morphology, while the Surface Free Energy (SFE) detection was

measured by the sessile drop method of contact-angle technique.

XPS analysis were performed by means of a small spot AXIS Kratos

apparatus, equipped with hemispherical analyser and dual Al/Mg anode.

Spectra were acquired at photoelectron take-off angles (y) with respect to

the sample surface of 901, with an estimated sampling depth of 7.5 nm.

The X-ray source (MgKa1/2) was run at a reduced power of 150W

(15 kV, 10mA) which did not caused any appreciable damage to the

sample during the time required to acquire the full set of XPS data, as

core-level line shapes of the sample did not change significantly in a

repeated scan. The pressure in the analysis chamber was maintained at

10�9mbar or lower during each measurement. All binding energies were

referenced to C 1 s neutral carbon peak at 285.0 eV. The peak fitting

analysis was performed by maintaining constant the full-width at half-

maximum (FWMH) for all the components in the spectra. Surface

elemental stoichiometry was determined from peak area ratios, after

correcting with sensitivity factors, and were reliable to 72%.

Contact-angle measurements were performed by using a half automatic

video-based contact-angle meter (OCA30, Dataphysics): liquid drops of

2ml of volume were applied with a microsyringe on the sample surface and

measurements of contact angle were made on both sides of the two-

dimensional projection of the droplet. At least five measurements were

made on different areas of each sample and then averaged. In order to

evaluate the SFE parameters the three liquid technique was employed,

according to the Good-van Oss approach [17].

The total SFE (gTOT) and its components were evaluated, in terms of

apolar Lifshitz-van der Waals (gLW), electron-donor Lewis base (g�) andelectron-acceptor Lewis acid (g+). The probe liquids were: Milli-Q water,

trycresylphosphate and glycerol (Sigma).

ARTICLE IN PRESSI. Amato et al. / Biomaterials 28 (2007) 3668–36783670

2.2. Cell cultures

Primary cultures of hOB were obtained from normal bone removed

during surgery for primary hip replacement, as previously described [8]. A

signed informed consent was obtained from patients, all of whom agreed

to the use of biological materials for research studies. Cells were cultured

in alpha modification of Eagle’s Medium (a-MEM) supplemented with

10% FBS, L-glutamine, penicillin and streptomycin (Gibco BRL, Life

Technologies, Scotland), and maintained at 37 1C in a 5% CO2 humidified

atmosphere. At confluence (mean 3–4 weeks), hOB were washed with

phosphate buffered saline (PBS), detached from the flask by trypsin-

EDTA digestion (0.05%, Sigma Chemicals Poole, UK) and splitted 1:2.

Two- or three-passage hOB were detached and seeded at 15,000 cells onto

30–50mm2 PCL samples, which were previously put in borosilicate glass

Lab-Tek Chamber SlideTM System (area: 1 cm2/well, VWR International,

Milan, Italy). Before cell seeding, the PCL samples were treated for 2 h

with a solution containing 10,000U/mL penicillin, 10mg/mL streptomy-

cin, and 25mg/mL amphotericin, then washed in PBS, and finally pre-

wetted for 2 h in a-MEM+10% FBS. Cells were maintained at 37 1C in a

5% CO2 humidified atmosphere for 24 h and then used for integrin

detection. Integrin expression of hOB seeded directly onto tissue-culture

plastic surface and on the borosilicate glass of empty wells was used as a

control.

2.3. Integrin detection

2.3.1. Flow cytometric analysis

Before seeding onto PCL samples, hOB were fixed in 3.7%

paraformaldehyde in 2% sucrose for 20min, and then washed in PBS.

100,000 cells were incubated with 100ml of anti-a1, -a2, -a3, -a5, -avb3, and-b1 monoclonal antibody (0.5–1mg/mL diluted in PBS 2% BSA) (BD

Bioscience Pharmigen, San Diego, CA) for 45min at room temperature;

IgG1k, monoclonal antibody was used (BD Bioscience) as negative

control. After washing, cells were incubated for 30min at room

temperature with a fluorescein isothiocyanate (FITC)-conjugated goat

anti-mouse antibody (BD Bioscience Pharmigen), which was diluted 1:100

(volume/volume) in PBS 2% BSA. Finally, samples were resuspended in

300ml of PBS, and analysed with a flow cytometer equipped with an argon

laser (EPICS XL-MCL; Beckman Coulter, Hialeah, FL). The percentage

of positive cells was calculated on 10,000 events.

2.3.2. Confocal microscopy

The confocal microscopy was performed in hOB cultured in wells

containing the PCL samples as well as in empty wells of the chamber

slides.

Cells were fixed with PBS containing 3.7% paraformaldehyde and 2%

sucrose for 20min at 4 1C. The plasma membrane was permeabilized with

Hepes 0.5% Triton X-100 for 5min. Cells were washed in PBS and

incubated in 100ml (1:100) of anti-a1, -a2, -a3, -a5, -aVb3, and b1antibodies for 45min followed by immunostaining with FITC-goat anti-

mouse antibody (1:10) (BD Bioscience Pharmigen) and rhodamine-

conjugated phalloidin (10mg/ml) (Sigma-Aldrich, Saint Louis, MO) in

PBS. The specimens were kept in the dark for 30min, washed twice with

PBS, mounted in glycerol/PBS, and protected by a glass coverslip. Cells

were viewed with a confocal microscope (Nikon, Tokio, Japan), and laser

scanning was performed in order to investigate the co-localization of

integrins and actin filaments; the optical sections were collected with a step

size of 1.2 mm from the top to the bottom of the cell (depth of 13.8mm).

Image analysis was employed to quantify both integrin expression and

cell adhesion on PCL samples. Samples were observed with a Nikon

Eclipse E800M microscope, equipped with a Nikon Plan Fluor 20x/0.50

objective and the appropriate excitation/emission filters (494 nm/518nm).

Three non-overlapping fields were chosen within the inner sample area

in order to avoid edge effects. Images were analysed by using a dedicated

software (LUCIA Measurement, version 4.60, Nikon Instruments,

Florence, Italia) which detects changes in image colour intensity based

upon the fluorescence signal per pixel, and calculates the area occupied by

fluorescence on the total area (170,600mm2 with 20� magnification). The

quantification of osteoblast spreading was obtained by the ratio between

the area occupied by red fluorescence (rhodamine fluorescent emission)

and the total area analysed. The quantification of integrin expression was

represented by the ratio between green-fluorescent area (FITC emission)

and area of spreading.

2.3.3. Gene expression analysis

RNA isolation and reverse transcription were performed as previously

described [18]. Five microlitres of cDNA (1.5mg) were amplified to detect

the expression of aVb3. The b-actin (house-keeping gene) was used as

control for the reverse transcription effectiveness, and as a reference for

the semi-quantitative analysis of the gene of interest. Each sample was

added to 45ml of a master mix solution containing 1.25 units of TAQ

polymerase (Invitrogen, San Giuliano Milanese, MI, Italy) and 0.1 mM of

each primer (b actin: sequence number NM_001101, sense: 50-ATC TGG

CAC CAC ACC TTC TAC AAT GAG CTG CG-30, antisense: 50-CGT

CAT ACT CCT GCT TGC TGA TCC ACA TCT GC-30; aV: sequence

number NM_002210, sense 50-GGC TTC GCC GTG GAT TTC TT-30,

antisense 50-TGC TGT AAA CAT TGG GGT CGT-30). The amplifica-

tion was performed in a thermal cycler (iCycler, Bio-Rad Laboratories,

Hercules, CA, USA) under the following conditions: denaturation at 94 1C

for 10min; 30 cycles including denaturation at 94 1C for 30 s, annealing at

65 1C for 45 s for b actin and 59.4 1C for 30 s for aV, extension at 72 1C for

10min, and final extension at 72 1C for 10min.

Ten microlitres of each product were mixed with 2ml of 6� loading

buffer (Sigma). The mixture was applied to a 2% agarose gel containing

0.5% ethidium bromide, then it was run at 90V per 30min and analysed

by using a UV transilluminator. The molecular weights of the products

were 838 bp for b-actin, and 547 bp for aV. The gel image was captured by

a digital photocamera and the semi-quantitative analysis was performed

by using dedicated software for the densitometric evaluation of the bands

(Gel-Doc, Biorad). For each experiment, the ratio between aV and b-actinwas calculated.

2.3.4. Statistical analysis

Statistical analyses were performed using StatView 5.01 for Windows

(SAS Institute Inc., Cary, NC). Data were expressed as arithmetic mean

plus or minus the standard error of the mean (SEM) of three separate

experiments. A paired analysis of the data (Wilcoxon signed rank test) was

applied to detect the effects of the treatments. The p-values equal or below

0.05 were considered significant.

3. Results

3.1. Surface characterization

Both the He+-beam-induced modification as well as theefficiency of RGD immobilization has been controlled byusing XPS technique. Fig. 1 shows that He+ irradiation ofPCL induced a drastic change of the chemical structure ofthe polymer surface, with the significant decrease of estergroups (C3 component), appearance of newly formedcarbonyl bonds (C2

0 component), and evidence of–OHgroups due to water adsorbed onto the irradiated surfaces(O3 component).Quantitative information about surface atomic composi-

tions for the various components of oxygen is shown inFig. 2, where the decrease of total oxygen content onto thesurfaces of irradiated PCL is evident. In parallel the totalcarbon content increased, and the surface stoichiometrychanged from C3.0O1 for unirradiated PCL to C4.1O1 forHe+-irradiated PCL (data not shown).

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292 288 284

C1(285.0±0.2eV): C-C, C-H

C2 (286.4±0.2 eV): C-O-C

C2' (288.0±0.2 eV):C=O

C3 (289.0±0.2 eV): C(=O)O-

O1 (532.2±0.2 eV): C=O

O2 (533.5±0.2eV): C-O-C

O3 (534.2±0.2 eV): O-H

N1 (400.7±0.2 eV): N-CO

C1

C3 C2

C1a

536 532 528

O2 O1

dc

b

292 288 284

C3

C2' C2

C3C2

'C2

C1e

binding energy (eV)

inte

nsi

ty (a.u

.)

536 532 528

O3

O2

O1

f

404 400 396

N1g

536 532 528

O3

O2

O1

292 288 284

Fig. 1. XPS peaks for surfaces of: ut-PCL ((a) C 1 s and (b) O 1 s); irr-PCL ((c) C 1 s and (d) O 1 s); irr+RGD-PCL ((e) C 1 s, (f) O 1 s and (g) N 1 s). N 1 s

regions for ut-PCL and irr-PCL are not reported as no detectable N signal has been found.

30

25

20

15

10

5

0

ut-PCL irr-PCL irr+RGD-PCLut+RGD-PCL

O3 O2 O1

O c

om

p. (a

t%)

Fig. 2. Surface content in atomic percentage of oxygen with the related components for the different PCL surfaces.

I. Amato et al. / Biomaterials 28 (2007) 3668–3678 3671

Fig. 3 shows the relative ratio between the carbonoxidized-related components (i.e., C2+C2

0+C3) and theapolar carbon component (C1). The ratio significantlydecreased for irradiated PCL surfaces with respect to theunirradiated ones, suggesting the ion-induced formation ofa surface layer less polar than the pristine polymer. Inparticular, the ratio C2

0/C1, diagnostic of carbonyl presenceand absent from unirradiated PCL, was found as anirradiation effect on the irr-PCL surfaces and it signifi-cantly increased onto the irr+RGD-PCL ones, indicatingthat RGD-carbonyl immobilization occurred to someextent on this surface.

Indeed, it is to note that only for irradiated PCL surfacesa noticeable peak of nitrogen, corresponding to a surfaceatomic percentage of about 1.0%, was found after theadsorption step (see Fig. 1(g)), the estimated RGD contentwithin the XPS sampling depth being about 4%, which,according to the procedure described in ref. [8], corre-sponds to about 7.0� 10�12mol/cm2 adsorbed onto thesurface.The analysis of SFE components (Fig. 4) clearly

indicated that 10 keV He+ irradiation of PCL induced areduction of the total SFE, in corresponding to the smallincrease (�7%) of the hydrophobic character of the

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0.8

0.6

0.4

0.2

0

Inte

nsity r

atios

ut-PCL irr-PCLut+RGD-PCL irr+RGD-PCL

C2/C1 C3/C1 Cox/C1C2′/C1

Fig. 3. Intensity ratios of carbon components for the different PCL surfaces. Cox corresponds to the sum of C2, C20, and C3 components, i.e., the oxidated

carbon species.

0.5

-0.5

-1

-2

-3

-2.5

-1.5

0

TOTLW

ABAcid

BaseΔγ (m

J/m

2)

Fig. 4. SFE values, taken as difference between irr-PCL and ut-PCL values. LW and AB respectively indicate the dispersive (Lifshitz-van der Waals) and

the acid–base component of the total SFE.

I. Amato et al. / Biomaterials 28 (2007) 3668–36783672

unirradiated PCL surfaces, which exhibited a static watercontact angle of about 701. Furthermore, the figureindicates that, notwithstanding the evidence of the increaseof acid character of the irradiated PCL, the total SFEdecreases mainly due to the decrease of both dispersive LWand Lewis electron-donor (base) contributions.

Finally, the morphology at the nanometer scale wasinvestigated by means of AFM and showed a reduction ofabout 30% of the roughness of the unmodified PCL and novisible changes after RGD immobilization (data notshownn). Details on the AFM analysis for the He+-irradiated PCL surfaces has been reported elsewhere [16].

3.2. Integrin expression on HOB

Flow cytometric analysis was used to measure theexpression of integrin-specific subunits on a single cellsuspension of hOB before the seeding onto a substrate:high levels of b1 (90%), a5 (87%), a1 (80%), avb3 (79%),

and a3 (72%) were detected, while a2 (21%) was expressedwith a low intensity.After 24 h of culture on tissue-plastic surface, the same

integrins were analysed by confocal microscopy: HOB werepositive for b1, a1, and aVb3, integrin subunits, while a2,a3, and a5 were not expressed (Fig. 5). Confocal analysis ofthe immunostained cells were used to study the spatialrelationship between integrins and actin filaments.A diffuse expression of the b1 chain was found on theapical surface, while a co-localization between b1 andF-actin was found at levels of the focal contacts (Fig. 6).

3.3. HOB adhesion on PCL and integrin expression

The cell adhesion of hOB seeded onto PCL samples wasvisualized after 24 h of culture. The number of adherentcells on ut-PCL (Fig. 7a and i) was very low and thecytoskeleton architecture was completely disrupted. Theaddition of RGD (Fig. 7c and k) partially improved

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Fig. 5. Integrin expression in hOB for b1 (a), avb3 (b), a1 (c), a3 (d), a5 (e), and negative control (f) after 24 h on tissue-culture plastic surface. b1 and avb3were strongly expressed, a1 showed a low intensity, while a2, a3, and a5 were not found. Arrows indicate the co-localization (yellow) between integrin

(green-fluorescence) and F-actin (red-fluorescence) (original magnification, 60� ).

Fig. 6. Laser confocal microscopy of hOB stained for F-actin and b1integrin. The optical sections were collected with a step size of 1.2mm and

are directed from the top to the bottom of the cell (depth of 13.8mm).

Optical sections reveal that the b1 subunit is diffusely localized on the cell

apex. The arrows show the presence of focal adhesion contacts which are

evident on the bottom of the cell (original magnification, 60� ).

I. Amato et al. / Biomaterials 28 (2007) 3668–3678 3673

the hOB adhesion, although the cytoskeleton organizationwas affected, and the actin filaments were assembled as athick layer at the cell borders. In contrast, the irr-PCL,with (Fig. 7g and o) or without RGD (Fig. 7e and m),favoured the cell adhesion and the actin filaments werewell-defined, with marked stress fibers oriented with thelong axis of the cell. Moreover, actin concentration infocal adhesion contacts was seen in both irr-PCL andirr+RGD-PCL.

The quantitative analysis of the spreading showed thatthe area occupied by hOB was significantly lower in ut-PCL in comparison to irr-PCL, ut+RGD-PCL, irr-PCLand irr+RGD-PCL (po0.01) (Fig. 8a), as well as inut+RGD-PCL in comparison to irr-PCL, irr+RGD-PCL(po0.05).The expression of the b1 chain was higher in hOB grown

on irr-PCL, with (Fig. 7h) or without RGD (Fig. 7f), thanin cells seeded onto others samples (Fig. 7b and d). The b1expression on PCL samples correlated with the osteoblastadhesion. The staining pattern was thoroughly diffused oncell surface, while the co-localization of the b1 subunit withthe F-actin was observed only on irradiated PCL. Thequantitative analysis of the b1 expression confirmed thequalitative data, even though no significant differenceswere observed (Fig. 8b).The highest expression of aVb3 was found in hOB plated

on irr+RGD-PCL samples (Fig. 7p), while in othersamples the vitronectin receptor was absent (Fig. 7j andl) or poorly expressed (Fig. 7n). Moreover, cells seededonto irr+RGD-PCL showed a diffuse co-localization ofavb3 with cytoskeletal actin, compatible with the initialformation of focal contacts. The quantitative imaging ofthe aVb3 expression supported the morphological data,and the statistical analysis highlighted significant differ-ences between irr+RGD-PCL and ut-PCL, RGD-PCLand irr-PCL (po0.01).As a further confirmation of the significant results

obtained with the immunofluorescence image analysis,the expression of the transcripts related to the vitronectinreceptor was analysed. Fig. 8d shows a representativeexperiment where the expression of b actin house-keepinggene was similar in all samples, while the lack of aV

transcripts was evident in hOB seeded onto ut-PCL. Thevitronectin-receptor expression showed a slight increase inRGD-PCL and irr-PCL, but in irr+RGD PCL becamesimilar to that observed in cells seeded onto tissue-culture

ARTICLE IN PRESS

Fig. 7. HOB adhesion on PCL, and integrin expression. The rhodamine–phalloidin staining (red fluorescence) shows the number of adherent cells on ut-

PCL (a, i) is very low and the cytoskeleton architecture appears completely disrupted. The addition of RGD alone partially improves the hOB adhesion (c, k),

even if the cell morphology is poor with the actin filaments assembled as a thick layer at the cell borders. The cell adhesion increases on irradiated PCL,

without (e, m) or with RGD (g, o), and the actin filaments are well-defined, with marked stress fibers oriented with the long axis of the cell. The actin

concentration in focal adhesion contacts (red spots) is observed in both irr-PCL and irr+RGD-PCL. The b1 expression (green fluorescence, ut-PCL (b),

ut+RGD-PCL (d), irr-PCL (f), and irr+RGD-PCL (h)) correlates with the number of adherent cells. In irr-PCL, the arrows indicate the yellow spots

representing the co-localization of the b1 subunit with F-actin, which is consistent with the presence of focal contact adhesion. The aVb3 expression

(green-fluorescence, ut-PCL (j), ut+RGD-PCL (l), irr-PCL (n), and irr+RGD-PCL (p)) was found only in hOB plated on irr-RGD-PCL (p). The

presence of yellow ‘dots’ (arrows) in irr+RGD-PCL indicates the diffuse co-localization of avb3 with cytoskeletal actin (original magnification, 60� ).

I. Amato et al. / Biomaterials 28 (2007) 3668–36783674

polystyrene plate which was used as control. Twoexperiments were performed and the semi-quantitativeanalysis with the aV-to-b actin ratio confirmed the aboveresults (ut-PCl 0.0370.03; RGD-PCL 0.1170.09; irr-PCL0.1570.04, irr+RGD PCL 0.2970.03; control0.2070.12).

4. Discussion

The interaction between bone cells and ECM is anessential step for the development and remodelling of bonetissue. Often synthetic materials do not support osteoblastadhesion and this may result in poor cell differentiationand limited bone formation [9,10]. Generally, the effect ofsurface properties on cellular response depends on differ-ences in species, concentration, and biological activity ofadsorbed proteins, which may be obtained from differentsources, i.e., biological fluids and cell-mediated synthesisand deposition [19,20]. Recently, bioadhesive motifsengineered on biomaterial surfaces have been used tofavour cell adhesion. RGD are the most employedsequences for stimulating the cell adhesion on syntheticsurfaces, even if their attachment strictly depends on thetopography, chemistry, or surface energy [13,14,21,22].

Because the adhesion process is closely related to theadhesion receptors [19], we aimed to investigate whether alow-energy irradiation (He+10keV) and/or RGD absorp-tion on PCL surfaces could modify the expression ofintegrins remarkable for the hOB function. For thispurpose following ion and/or RGD treatment of the PCLfilms, we analysed (i) the physicochemical changes of thesurfaces, (ii) the consequences on cell adhesion andspreading, and (iii) the effects on the integrin expression.He irradiation may be defined as a mild modification of

surface which leads to a slight smoothening as well as to adifferent nano-structured surface. In fact, total SFE andthe related components of irradiated samples were found tobe almost unchanged in comparison to untreated PCL,while the contact-angle hysteresis measurements and AFMphase contrast imaging revealed the formation of chemi-cally heterogeneous nanometric domains. Moreover, RGDpeptides seemed to be strongly attached only to theirradiated PCL films, resisting to the adopted severewashing procedure involving two rinsing steps and airdrying. In spite of the low proportion (about 10%) ofRGD coverage onto ion-irradiated surface, we couldcalculate a relevant absolute amount of adsorbed peptideranging between 1.7� 10�11 and 5.0� 10�11mol/cm2,

ARTICLE IN PRESS

1 2 3 4 5

β-actin 838 bp

αV 547 bp

0

20

40

60

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are

a c

ove

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by c

ells

(%

)

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ubunit (

%)

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ut-PCL ut+RGD-PCL irr-PCL irr+RGD-PCL Control

are

a a

lfa

(v)

be

ta 3

(%

)

*

**

*

a

b

c

Fig. 8. Quantitative analysis of cell adhesion and integrin expression on PCL samples. In panel (a), bars represent the mean7SEM of the area occupied by

cells (ratio ‘red fluorescence area/total area’� 100). **po0.01 ut-PCL vs. ut+RGD-PCL, irr-PCL, and irr+RGD-PCL; *po0.05 ut+RGD-PCL vs. irr-

PCL, irr+RGD-PCL. In panel (b), the quantification of b1 expression is represented by the mean7SEM of the ratios between area covered by b1staining’ and ‘area covered by cells’. In panel (c), bars represent the mean7SEM of the ratios between ‘area covered by avb3 staining’ and ‘area covered by

cells’. *p ¼ 0.01 vs. ut+RGD-PCL and irr-PCL. Panel (d) shows a representative experiment confirming the significant differences observed in panel (c).

The expression of b-actin house-keeping gene is similar in all samples; no aV transcripts are observed in hOB seeded onto ut-PCL; a poor expression is

found in RGD-PCL and irr-PCL, while in irr+RGD PCL the aV expression becomes similar to that observed in cells seeded onto tissue-culture

polystyrene (control).

I. Amato et al. / Biomaterials 28 (2007) 3668–3678 3675

depending on the RGD configuration, i.e., ‘stretched’RGD (average area�1 nm2) or ‘loop-structure’ RGD(average area�0.35 nm2), respectively. According to

literature data, the amount of adsorbed RGD ismuch higher than the threshold quantity sufficient topromote cell spreading of fibroblasts (1.0� 10�15mol/cm2)

ARTICLE IN PRESSI. Amato et al. / Biomaterials 28 (2007) 3668–36783676

or the formation of focal contacts (1.0� 10�14mol/cm2)[22].

These data suggest that ion beam treatment of thepolymer surfaces allows to obtain physicochemical mod-ifications increasing protein adsorption, and from atheoretical point of view the osteoblast adhesion shouldbe improved. After 24 h of culture, the adhesion rate ofhOB on untreated PCL, with or without RGD, was verypoor, but irradiated surfaces attracted a high number ofosteoblasts which showed a well-organized cytoskeletonwith marked stress fibers oriented with the long axis of thecell and focal adhesion contacts. The addition of RGD onirradiated surface did not enhance cell adhesion andspreading, either qualitatively or quantitatively.

These results agree with previous findings that havedemonstrated how the presence of RGD on high-energyirradiated PCL do not significantly influence the hOBadhesion, even if a slight increase in cell viability wasobserved [8].

A possible explanation is that the adsorbed RGDundergoes conformational changes which alter the recep-tor–ligand interaction. More simply, it is reasonable toassume that whether 10% of PCL surface is covered byRGD, the remaining area may be occupied by the proteinsof the medium which was used for pre-wetting the samples.In this condition, the effect of the adsorbed RGD could bemasked by the other proteins; nevertheless, if the peptidesare functionally active a modulation of the integrinexpression is expected, and the analysis of these moleculesmay be useful to solve the doubt.

Before the seeding onto PCL sample, we defined theexpression of different integrin subunits, i.e., a1, a2, a3, a5,and b1 and aVb3 heterodimer on a single cell suspension ofconfluent hOB by means of flow cytometry. In agreementwith literature data [23], we found that all the testedmolecules were positive. The integrin expression was foundto be uniform in the whole cell population except for the a2chain which was detectable in only 30–40% of the cells.After the seeding on tissue-culture plastic, hOB werepositive for the b1, a1, and aVb3, integrin subunits, whilea5, a2, and a3 were not expressed. These data suggest thathOB are equipped with a wide pattern of adhesionreceptors, but their expression is modulated according tothe type of substrate and it is probably related to theculture time and cell function.

For studying the molecular mechanisms of osteoblastadhesion on biofunctionalized PCL surfaces, two mole-cules were selected: b1 and aVb3. b1 is associated to themost part of the integrins expressed by osteoblastsrepresenting the dominant adhesion mechanism to ECMligands, such as collagen, fibronectin, and laminin [24]. Inaddition to support adhesion, spreading, and migration, b1activates various intracellular signalling pathways, geneexpression, and protein activity that regulate cellularfunctions, including osteoclastogenesis [25,26]. The aVb3integrin, also known as vitronectin receptor, expressesRGD-dependent binding and mediates the adhesion of

hOB to the major non-collagenous proteins, i.e., osteo-pontin, bone sialoprotein, vitronectin, and fibronectin [23].aVb3 overexpression appears to promote hOB prolifera-tion over differentiation by influencing integrin–matrixinteractions, signal transduction and ECM protein expres-sion [27]. Recent data show that the initial attachment ofhOB to RGD surfaces is mediated by the b1 chainassociated with the a2 subunit (collagen receptor), whileaVb3 integrin governs longer-term adhesion [28]. Further-more, in our experimental condition, a noticeable reasonfor selecting b1 and aVb3 was the formation of focaladhesion contacts mediated by these molecules in thecontrol cultures. The focal adhesion complex is composedof a cluster of integrins which connects the ECM to theactin filaments through the binding of b chain withcytoskeletal proteins, i.e., talin–paxillin–vinculin subunits[29]. The adhesion plaque represents an essential structuralunit which directs the flow of information from outside thecell to the inside, promoting specific signalling downstreaminvolved in the control of cell motility, shape, growth, andsurvival [30,31].Our results showed a high expression of b1 on irradiated

PCL surfaces, which was not increased by the addition ofRGD peptides. On the opposite, the aVb3 expression wassignificantly higher on irr+RGD-PCL than on the othersamples, even if the number of attached osteoblasts did notchange.These findings confirm that ion irradiation produced

changes in surface properties and favoured the adsorptionof RGD peptides and proteins released from osteoblasts orderived from the sample ‘pre-wetting’ with culture medium.The pre-wetting has been performed in order to mimic thein vivo condition of an implanted material, which iscovered before by blood and biological fluids and later bycells. It is widely accepted that differences in proteinslayered onto PCL may be responsible for different cellularresponses, such as the modulation of integrin expression[8–17,20,32]. Generally, the cell spreading seems to beclosely related to the b1 modulation. This subunit is sharedby a number of adhesion molecules which can bind theirligands in a RGD-dependent or -independent manner[10,28]. For instance, the recognition of type-1 collagenby a2b1 and a1b1 largely depends on GFOGER sequence,which is found in triple-helical collagens [33]. Although theamount of RGD peptides on irradiated PCL surfaces wascalculated as sufficient to promote cell spreading and theformation of focal contacts [22], we have previouslyhypothesized some conformational changes of adsorbedRGD which could alter the receptor–ligand interaction andaffect cell adhesion. The behaviour of vitronectin receptorcancelled these doubt and confirmed that the RGD peptidewas functionally active. The aVb3 integrin was stronglyexpressed by hOB cultured on irr+RGD-PCL, which wasthe only one sample where the RGD adsorption wasdetectable. Like us, other Authors demonstrated the pivotalrole of aVb3 in mediating the cell adhesion on substratespresenting RGD or RGD-containing sequences [34].

ARTICLE IN PRESSI. Amato et al. / Biomaterials 28 (2007) 3668–3678 3677

On irradiated PCL, a high number of b1-mediated focalcontacts were detectable. The presence of the adhesionplaques supports the effectiveness of the biofunctionaliza-tion strategy, because the structural links between thecytoskeleton and ECM mediate stable adhesion and cellmigration [10]. Furthermore, focal adhesions activate thesignalling pathways, such as MAPK and JNK, thatregulate transcription factor activity and direct cell growthand differentiation [30,31]. The b1-mediated focal contactswere not found on irradiated/RGD surfaces, while adiffuse co-localization of aVb3 with cytoskeletal actinwas observed. This morphological variant of the focaladhesion, so-called ‘dot-like’, represents the initial step ofthe contact which is constituted of both transmembraneand some linker proteins, but is not associated with actinbundles [29]. Initial formation of ‘dot’ evolves by matura-tion into ‘dash’ contact, that is the morphological variantfound in b1-mediated focal adhesion.

Our results showed that surfaces presenting RGDpeptides do not enhance the early steps of osteoblastadhesion. Garcia and Reyes [10] describe the critical factorslimiting the potential of this biomolecular strategy for bonerepair purposes: (a) the biological activity of short adhesivepeptides is significantly lower than that of the completeprotein, (b) RGD peptides exhibit limited specificity amongintegrins, and although required, in some cases may be notsufficient for an efficient binding, and (c) osteogenic cellsrequire signals from non-RGD-binding integrins for robustdifferentiation.

We demonstrated that structural changes due to surfaceirradiation help the adsorption of peptides, and they aresufficient and functionally active to modulate the expres-sion of RGD-dependent integrins, i.e., aVb3. Nevertheless,the aVb3 binding competes with the formation ofb1-mediated focal contacts, which in the early steps ofthe adhesion seems to be induced in an RGD-independentmanner [10,28,33]. Both aVb3 and b1 subunit are essentialin regulating the osteoblast activity, as their neutralizationretards matrix mineralization by osteoblasts [35]. On onehand, the b1 integrins appear to be the predominantadhesion receptor subfamily utilized by hOB to adhere tocollagen, fibronectin, laminin [24], and on the other handavb3 seems to be mainly involved in the binding of non-collagenous proteins [23], as well as in increasing the cellproliferation rate [27].

In summary, it is desirable that bioactive materialsdesigned for bone tissue repair do not hamper the early cellattachment because that could influence long-term events,i.e., matrix deposition and mineralization. In this study, wehave further pointed out the importance of integrinanalysis to elucidate the molecular mechanisms of cellularadhesion on biofunctionalized surfaces.

The ability to induce integrin expression throughbiomimetic strategies may be a key step in determiningwhich modifications are suitable for application in bonetissue engineering and other orthopaedically relatedcircumstances.

5. Conclusion

The biofunctionalization of biodegradable syntheticpolymers represents one of the most promising strategiesfor the development of biologically active implants andgrafting substrates for bone tissue repair. As integrinexpression and osteoblast biology are closely related, thestudy of these molecules in the early phases of the adhesioncould represent an indicator that proves the effectiveness ofbiomimetic surfaces in promoting adhesion, proliferation,and function of osteoblasts. In this study, we investigatedthe effect of two well-defined strategies based on theirradiation-induced surface engineering, triggering the‘spontaneous’ reorganization of the ECM matrix, and themimicking of ECM by the engrafting/adsorption ofsuitable cell-adhesive sequences onto the PCL surface. Celladhesion and spreading were dramatically enhanced onirradiated PCL surfaces, and the addition of RGD did notchange the result. The expression of adhesion receptorsvaried according to the type of substrate: a high expressionof b1 on irradiated PCL surfaces was not increased by theaddition of RGD peptides, while a high aVb3 expressionwas found only on irradiated and RGD-treated surfaces,even if the number of attached osteoblasts did not change.We conclude that the low-energy He irradiation (10 keV)produces changes in surface properties which favour thepeptides/proteins adsorption; in turn, differences in thestructure of the adsorbed protein film can significantlymodify the expression of integrins relevant to the hOBfunction. Our findings add new knowledge on themolecular mechanisms of the cell/material interactionsand could be exploited to engineer surfaces provided withbioactive sites, which could elicit specific cell responsesenhancing the performance of implantable devices.

Acknowledgements

This study was funded by grants from the ItalianMinistry of University and Research (MIUR) throughthe projects FIRB 2001 RBAU01N79B and FIRB 2001RBNE01458S_003.

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