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Applied Surface Science 301 (2014) 530–538

Contents lists available at ScienceDirect

Applied Surface Science

journa l h om epa ge: www.elsev ier .com/ locate /apsusc

iocompatibility of Ir/Ti-oxide coatings: Interaction with platelets,ndothelial and smooth muscle cells

ajjad Habibzadeha, Ling Lib, Sasha Omanovica,ominique Shum-Timc, Elaine C. Davisb,∗

Department of Chemical Engineering, McGill University, Montreal, QC, CanadaDepartment of Anatomy and Cell Biology, McGill University, Montreal, QC, CanadaDivisions of Cardiac Surgery and Surgical Research, Department of Surgery, McGill University, Montreal, QC, Canada

r t i c l e i n f o

rticle history:eceived 1 September 2013eceived in revised form 18 February 2014ccepted 20 February 2014vailable online 28 February 2014

eywords:ridium/titanium-oxide coatings

a b s t r a c t

Applying surface coatings on a biomedical implant is a promising modification technique which canenhance the implant’s biocompatibility via controlling blood constituents- or/and cell-surface interac-tion. In this study, the influence of composition of IrxTi1−x-oxide coatings (x = 0, 0.2, 0.4, 0.6, 0.8, 1) formedon a titanium (Ti) substrate on the responses of platelets, endothelial cells (ECs) and smooth muscle cells(SMCs) was investigated. The results showed that a significant decrease in platelet adhesion and acti-vation was obtained on Ir0.2Ti0.8-oxide and Ir0.4Ti0.6-oxide coatings, rendering the surfaces more bloodcompatible, in comparison to the control (316L stainless steel, 316L-SS) and other coating composi-

lateletsndothelial cellsmooth muscle cellsiocompatibility

tions. Further, a substantial increase in the EC/SMC surface count ratio after 4 h of cell attachment to theIr0.2Ti0.8-oxide and Ir0.4Ti0.6-oxide coatings, relative to the 316L-SS control and the other coating com-positions, indicated high potential of these coatings for the enhancement of surface endothelialization.This indicates the capability of the corresponding coating compositions to promote EC proliferation onthe surface, while inhibiting that of SMCs, which is important in cardiovascular stents applications.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Occlusion of coronary arteries is typically attributable to auildup of fatty deposits or calcified plaque, known as atheroscle-osis, under the inner lining of the vessel [1–3]. This phenomenons usually termed as coronary artery disease (CAD), which is the

ajor cause of mortality in the Western world [1,4,5]. Depend-ng upon the severity of the CAD, different treatment strategiesan be chosen. Angioplasty with stenting versus coronary arteryypass surgery is the most frequent treatment method when an

ntervention is deemed necessary [2]. Angioplasty with stentings used to treat approximately one third of CAD patients [6].tents are metallic lattice-like cylindrical ‘tubes’ predominantlyade of 316L stainless steel (316L-SS) [7,8], but titanium nickel

Nitinol®) [9,10] or cobalt–chromium [11] are also used. This med-

cal implant can provide endovascular scaffolding in order to relievehe vascular obstruction and minimize the risk of myocardialnfarction, or otherwise known as a heart attack. Nonetheless, stent

∗ Corresponding author. Tel.: +1 5143985893.E-mail address: elaine.davis@mcgill.ca (E.C. Davis).

ttp://dx.doi.org/10.1016/j.apsusc.2014.02.119169-4332/© 2014 Elsevier B.V. All rights reserved.

implantation is often associated with blood clot formation (throm-bosis) and/or neointima hyperplasia [3,12]. These undesiredresponses are triggered by the vascular injury which subsequentlyleads to thrombosis. This phenomenon is induced by the activa-tion of the intrinsic coagulation system and by the formation ofblood clots when plasma proteins and platelets adhere to the stent’ssurface in the early period after stent deployment [13–15].

In blood vessels, surrounding the inner monolayer of endothelialcells (ECs), a thicker vascular media exists containing concen-tric layers of smooth muscle cells (SMCs) bounded by an outeradventitia containing fibroblasts and collagen bundles [16–19]. ECsregulate vascular tone, permeability, inflammation and thrombo-sis through the expression and secretion of a series of regulatorymolecules [20]. When a stent is deployed inside an artery, disrup-tion (removal) of the EC monolayer occurs, which subsequentlyleaves the underlying SMCs exposed to the surface of the stentand to blood. The SMCs then undergo rapid proliferation which,in turn, results in re-narrowing of the artery or so-called in-stentrestenosis (ISR) [16,18,21–24]. This process can proceed to such an

extent as to block the stent and eventually impede or even stop theblood flow. Prompt re-endothelialization of the arterial wall andendothelialization of the surface of the stent after implantation aretherefore of great importance [17,25–27]. In addition to ISR, the low

urface Science 301 (2014) 530–538 531

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Table 1Molar percentage of Ir in IrxTi1−x-oxide coatings (excluding the contribution of oxy-gen and hydrogen in the oxide film). Nominal values refer to the desired Ir contentin the metal precursor salt. ICP values refer to the actual measured concentration ofIr in the coating precursor solution. XPS values refer to the Ir content on the surfaceof the coating.

Iridium content, mol%

Nominal ICPa XPSb

0 – –20 17 1540 35 2160 54 3780 76 65

100 92 81

S. Habibzadeh et al. / Applied S

adiopacity of currently used stents is a significant problem, whichas addressed in our previous study [28].

In order to minimize ISR, drug-eluting stents (DESs) were devel-ped. The use of these stents, which utilize antiproliferative drugs,as resulted in a decrease in restenosis rate to below 10% as com-ared with a restenosis rate of 20–30% with bare-metal stentsBMS) [29]. However, it has been shown that DESs might cause “latetent thrombosis”, where blood clots can form one or more yearsfter stenting [12,30–34]. Therefore, DESs neither offer long-termenefits in comparison to BMS nor better radiopacity.

Surface properties of metallic stents are key determinants in theormation of acute thrombus and ISR. Therefore, surface modifica-ion techniques have been extensively employed to enhance andegulate biocompatibility for blood and cell interaction with stents.everal studies have revealed that the surface properties of metal-ic stents influence platelet activation and cell behavior, includingell attachment, proliferation and differentiation [19,35–37]. Onef the promising surface modification methods in the developmentf coronary artery stents is the formation of coatings/films on themplant surface to alter the surface properties without interfering

ith the main function of the implant [38–40].The present study aims to address the issue of biocompatibil-

ty of metallic stents. Namely, coatings made of iridium/titaniumIr/Ti)-oxides deposited on titanium (Ti) substrates were used toptimize cell and platelet interaction with the metal surface. In ourrevious study [28], it was shown that these Ir/Ti-oxide coatingsan create a more radiopaque (visible) stent. Thus, using Ir in theoating material may not only enhance the stent radiopacity, butight also make the stent surface to be of equal or better biocom-

atibility than a BMS. Ti-oxide was chosen as a secondary coatingaterial due to its high electrochemical (corrosion) stability and

igh biocompatibility. Cruz et al. also employed mixed Ir/Ti-oxides coatings for electrode applications in neural systems [41].

In the present investigation, besides the surface characteriza-ion of the Ir/Ti-oxide coatings, the response of ECs and SMCs tohe corresponding coated surfaces was examined. Platelet/surfacenteractions were also investigated in order to assess the bloodompatibility of the coated surfaces.

. Materials and methods

.1. Preparation of samples

IrxTi1−x-oxide coatings (x = 0, 0.2, 0.4, 0.6, 0.8 and 1) were formedn flat titanium substrates employing a thermal method. First, aoating precursor solution was prepared by dissolution of a propermount of IrCl3·3H2O and/or Ti4(OCH3)16 in 37 wt% HCl and iso-ropanol at a total metal-content concentration of 0.5 mol l−1.epending on the desired compositions of Ir and Ti in the coatingaterial, the appropriate amounts of iridium chloride and titanium

sopropoxide were dissolved in 37 wt% HCl to yield half of the finalolume of the coating precursor solution. Deionized water was thendded to restore the final volume. Next, the solution was heated tovaporate one-fourth of its volume, and isopropanol was added toe-establish the final solution volume.

Ti substrates, which were used as a support for oxide films,ere 12.7-mm-diameter discs machined to a thickness of 2 mm.efore the metal oxide film deposition, the Ti substrates were firstolished using 600-grit SiC sandpaper, and then etched in an aque-us HCl solution (1:1, v/v, where a 37 wt% stock HCl was used) atoiling temperature for 30 min. Next, the substrates were rinsed

equentially with acetone, isopropanol and water.

Metal-oxide coatings were formed on such pre-treated Ti sub-trates in the following way. First, a thin layer of the metal precursorolution was brushed on one side of the Ti substrate. The substrate

a SD value: ±0.1.b SD value: ±0.3.

was then heated in an air furnace at atmospheric pressure and500 ◦C for 15 min. This procedure was repeated ten times. Finally,the coated sample was annealed at the same temperature for 1 h toconvert the surface coating into a metal oxide coating.

Energy-dispersive X-ray spectroscopy (EDX) analysis of thecoatings performed at various locations on the surface showed thatthe actual average metal composition of the coatings was in goodagreement with the nominal values. However, variations in thelocal composition of the coatings were noted, as discussed in detailin our previous work [28]. Furthermore, the surface content of irid-ium was calculated from X-ray photoelectron spectroscopy (XPS)measurements and compared to the concentration of iridium inthe coating precursor solution; the latter obtained by inductivelycoupled plasma (ICP) analysis (Table 1). The results demonstratethat there is depletion in iridium at the coating surface. This is inline with the literature [42–44], and can be related to the differentreactivity of two metal precursors toward oxygen [28].

2.2. Surface characterization

Electron micrographs of the sample surfaces were producedusing a Philips XL-30 field emission scanning electron microscope(FE-SEM). The wetting properties of the Ir/Ti-oxide coatings wereanalyzed by measuring the equilibrium contact angle of a dropletof deionized (DI) water on the surface using a Dataphysics ContactAngle System OCA goniometer. The measurement was run in trip-licate per sample and repeated at three different times. An atomicforce microscope (AFM) was used to assess the surface roughnessof the 316L-SS (control) and Ir/Ti-oxide coated surfaces. AFM exam-inations were performed in ambient air with in the semi-contactmode using a SOL VER P47H-PRO scanning probe microscope (SPM,NT-MDT).

2.3. Platelet adhesion

Venous blood (50–100 ml) was obtained from healthy volun-teers (free from medications known to interfere with plateletfunction for at least 10 days before sampling) who gave informedconsent in accordance with the policies of the ethics committee ofthe Montreal Heart Institute. Blood samples were anti-coagulatedwith acid citrate dextrose and processed to yield platelet richplasma (PRP) [45,46]. Then, the PRP was centrifuged, and theresulting platelet pellet was re-suspended in Hanks’ balancedsalt solution (HBSS)–HEPES buffer free from Ca2+ and Mg2+, with0.4 mmol l−1 EDTA and 1 �g ml−1 prostacyclin (PGl2, pH 6.5). Any

remaining red blood cells were removed by centrifugation. Theplatelet pellet was then re-suspended in (HBSS)–HEPES/PGl2 andEDTA-free buffer (pH 7.4), with Ca2+ (1.3 mmol l−1 CaCl2) andMg2+ (0.81 mmol l−1 MgSO4), and adjusted to a physiological

532 S. Habibzadeh et al. / Applied Surface Science 301 (2014) 530–538

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Fig. 1. SEM micrographs of (a) 316L-SS s

oncentration of 2.5 × 1011 l−1 using an automated cell counterMicrodriff 16, Beckman Counter, Inc.).

For adhesion studies, PRP was added on top of samples in 24-ell plates until the entire surface was covered. Samples were

ncubated for 60 min at 37 ◦C in a static system. They were theninsed with phosphate buffer saline (PBS) and fixed with 2.5% glu-araldehyde, followed by dehydration in ethanol–water baths ofradually increasing concentrations to 100% ethanol, and in amylcetate–ethanol baths of gradually increasing concentrations to00% amyl acetate. Samples were dried using critical point dryingCPD, Ladd Research Industries, South Burlington, VT, USA), sputteroated with gold–palladium and imaged using a Philips XL30 FEGeld emission gun scanning electron microscope. The number ofttached platelets was then determined using NIH imageJ version.46r (open source software available at http://rsbweb.nib.gov/ij).

.4. Cell attachment, proliferation and morphology

To determine cell attachment, human umbilical vascularndothelial cells (HUVECs) (Lonza, Walkersville, MD) or humanoronary artery smooth muscle cells (hCASMCs) (Lonza), betweenassage 4 and 6, were plated at a density of 50,000 cells cm−2 andllowed to attach to the control or Ir/Ti-oxide coated surfaces in 24-ell plates, at 37 ◦C for 2 or 4 h. HUVECs were grown in endothelial

ell growth media reconstituted with EGMTM-2 BulletKit (Lonza)nd hCASMCs were grown in EBM-2 (endothelial basal medium)Lonza) reconstituted with SmGM-2 BulletKit (Lonza). The cellsere then washed with PBS and fixed with 2% (v/v) paraformalde-yde in PBS for 15 min. After incubation with PBS containing

wt% BSA and 0.1 wt% saponin for 15 min, the cells were stainedor nuclei with 4,6-diamidino-2-phenylindole (DAPI) (1:5000) for

min. Images were recorded using a Zeiss digital camera mountedn a Zeiss fluorescence microscope and the numbers of nuclei wereounted using NIH imageJ version 1.46r.

For cell proliferation, 3000 HUVECs cm−2 or000 hCASMCs cm−2 were allowed to attach to the control orr/Ti-oxide coated substrates in 24-well plates at 37 ◦C and incu-ated for 4 or 7 days. Using a Quick Cell Proliferation Assay KitBD Biosciences, K301-2500), cell number was determined using a

icroplate reader set to 450 nm. The assay is based on the cleavagef tetrazolium salt WST-1 to formazan by cellular mitochondrialehydrogenases. To provide adequate nutrition, the culture mediaor both types of cells was changed every 48 h.

To access cell morphology, 50,000 HUVECs cm−2 or0,000 hCASMCs cm−2 were plated in either 4-well culturehamber slides, or in 24-well plates containing the control andr/Ti-oxide coated substrates at 37 ◦C and left for 4 h. The cells were

(control) and (b) Ir0.2Ti0.8-oxide surface.

then washed with PBS and fixed with 2% (v/v) paraformaldehydein PBS for 15 min. After incubation with PBS containing 1 wt% BSAand 0.1 wt% saponin for 15 min, the cells were stained for 3 minwith DAPI for nuclei (1:5000) following by washing with PBS.Then, the cells were stained for F-actin with red-fluorescent phal-loidin (Invitrogen, Burlington, ON) (1:5000) for 15 min and thenwashed thoroughly with PBS. Cell morphology was evaluated andimages recorded using a digital camera mounted on a fluorescencemicroscope (Zeiss) and an interactive microscope software (CarlZeiss, AxioVision 4.8) was used for image processing.

All attachment and proliferation experiments were performedin triplicate. Statistical analyses were carried out using Student’st-test at a significance level of p < 0.05, with the data reported asmean ± standard deviation (SD) values.

3. Results and discussions

3.1. Surface characterization

Fig. 1 shows scanning electron microscopy (SEM) images dis-playing the surface topography of the control 316L-SS surface andIr0.2Ti0.8-oxide coating (images of coatings of other compositionsare presented in our other manuscript [28]). The control surface(Fig. 1a) displays a relatively homogenous morphology, while theimage of the metal-oxide surface (Fig. 1b) shows the presence ofsmall crystallites separated by micro-cracks, rendering the sur-face relatively rough. Results published in [28] reveal that with anincrease in Ir content, the morphology of the surface changes suchthat the size of the cracks diminishes with an increase in Ir contentfrom 0 to 40%, and then slightly increases to yield a “cracked-mud”morphology [43,47] for the pure Ir-oxide coating. The analysis ofcross-section images of the coatings has revealed that all the coat-ings are of a very similar thickness, 1.8 ± 0.4 �m [28].

This visually-observed trend is in disagreement with the mea-sured surface-roughness trend presented in Fig. 2 (symbols).Namely, the graph shows that with an increase in Ir contentin the coating, the surface roughness actually increases andreaches a maximum for the coating containing 40% of Ir, andthen starts decreasing. On the other hand, the correspondingsurface wettability (Fig. 2, bars) continuously decreases with anincrease in Ir content in the coating. Thus, the comparison ofthese three sets of results demonstrates that the observed surfacemorphology/topography-related features do not predominantly

control the surface energy and thus the surface wettability. It mightbe that the latter is rather controlled by the surface chemistry,i.e. coating composition. Nevertheless, both the surface rough-ness and surface wettability are two parameters that considerably

S. Habibzadeh et al. / Applied Surface

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ig. 2. Magnitude of RMS roughness (�) and water contact angle (bar) of 316L-SSurface (control) and IrxTi1−x-oxide coatings (x = 0, 0.2, 0.4, 0.6, 0.8, 1).

nfluence the interaction of biomaterials with the surrounding bio-ogical/biochemical environment [37,48–50].

.2. Surface/platelet interactions

Adhesion and aggregation of platelets are crucial events inhe process of thrombus formation [51,52]. To assess the surfacehrombogenicity of the Ir/Ti-oxide coatings in vitro, platelet adhe-ion experiments were performed. Fig. 3 shows the number oflatelets attached on 316L-SS (control) and Ir/Ti-oxide coatingsfter 60 min of static incubation in PRP. Platelet attachment on allr/Ti-oxide surfaces was significantly lower than that on the 316L-S surface (p < 0.01 for Ir0.2Ti0.8-oxide, Ir0.4Ti0.6-oxide and purer-oxide; p < 0.05 for Ti-oxide, Ir0.6Ti0.4-oxide and Ir0.8Ti0.2-oxide).ewer adherent platelets on the Ir/Ti-oxide surfaces demonstrate

lower degree of activation compared to 316L-SS. A decrease oflatelet attachment by nearly 85% on the Ir0.2Ti0.8-oxide surface,

ith respect to the control, shows a considerable improvement

f blood-compatibility of the former surface. However, there is notatistically significant difference (p > 0.05) between platelets acti-ated on Ir0.2Ti0.8- and Ir0.4Ti0.6-oxide surfaces. Further, the result

ig. 3. Adhesion of platelets on the 316L-SS surface (control) and IrxTi1−x-oxidex = 0, 0.2, 0.4, 0.6, 0.8 and 1) coated surfaces, measured after 60 min of incubation inRP. Results are expressed as mean value ± SD of three samples averaged over thentire sample surface for each bar. *p < 0.05 and **p < 0.01. Note that asterisks aboveach bar represent a significant difference relative to the control.

Science 301 (2014) 530–538 533

in Fig. 3 demonstrates that less thrombogenic surfaces can be pro-duced by applying a coating with 20–40% Ir content instead ofusing 100% precious Ir metal in the coating material, which is alsobeneficial from the financial point of view.

Fig. 4 displays SEM images of platelets attached on 316L-SS andselected Ir/Ti-oxide surfaces (Ir0.2Ti0.8-oxide, Ir0.4Ti0.6-oxide andpure Ir-oxide) after 60 min of incubation in PRP. One can observethat the platelet morphology on 316L-SS and Ir/Ti-oxide surfacesis quite different. Namely, large quantities of platelets were aggre-gated on the surface of 316L-SS, with well-developed pseudopodiaevident (Fig. 4a). This is in agreement with previous observationsof high thrombogenicity of 316L-SS [13,53–55]. However, there areno signs of agglomeration of platelets on the surface of Ir/Ti-oxidecoatings with 20% and 40% Ir content (Fig. 4b and c), indicating awell-preserved, isolated and nearly round platelet morphology.

In comparison, most of the adherent platelets on the surfaceof pure Ir-oxide coating (Fig. 4d) are in a spreading pseudopodiumstate, such that they are interconnected by stretching dendritic pro-cesses or pseudopods. Nevertheless, even this coating clearly showsbetter hemocompatibility than 316L-SS.

The variation in the platelet/surface interactions between theIr/Ti-oxide coatings and the control, observed in Figs. 3 and 4,can partially be related to the surface energy variations, i.e. wet-tability. Namely, Fig. 2 shows that all the Ir/Ti-oxide surfaces aremore hydrophilic than the control 316L-SS surface, and this obser-vation correlates well with previous literature findings of lowerplatelet attachment to hydrophilic surfaces [53,56,57]. On the otherhand, results published in literature demonstrated that there is noinfluence of surface roughness on platelet adhesion, when testedunder static conditions [48,58]. Therefore, the influence of surfaceroughness of samples investigated here (Fig. 2) on the platelet adhe-sion and activation (Figs. 3 and 4) can be excluded. However, amore detailed analysis shows that neither of the two parameterspresented in Fig. 2 (roughness and wettability) can be separatelycorrelated with the trend observed in Fig. 3, and it is thus not pos-sible to conclude which of them actually governs the interaction ofplatelets with the investigated Ir/Ti-oxide surfaces. It is, most likely,that the combined influence of surface wettability, surface rough-ness and surface chemistry control the platelet – Ir/Ti-oxide surfaceinteraction. Unfortunately, due to the complexity of Ir/Ti-oxidecoatings (in terms of their heterogeneous physicochemical proper-ties, surface morphology/topography/structure), it is not possibleto vary one parameter and keep the others constant, and thus sep-arately investigate the influence of each on the platelet/surfaceinteractions.

3.3. Endothelial cell attachment, proliferation and morphology

Cell attachment is the initial step in a cascade of cell–biomaterialinteractions because adhesion precedes other cellular process, suchas spreading, proliferation and often, differentiation. Initial inter-action of the ECs with Ir/Ti-oxide coated surfaces studied here, wasinvestigated by seeding cells on the surfaces. Fig. 5 shows the resultsof EC counts on the surface of Ir/Ti-oxide coatings, together with316L-SS as the control. The experiment was conducted for incu-bation periods of 2 or 4 h. The results demonstrate that there is asignificant increase in EC adhesion to the oxide coatings in caseswhere Ir is added into the coating material, as compared to thecontrol and the Ti-oxide covered surface. Note that no significantdifference in adherent cells was seen for the Ti-oxide coating overthe control, despite the usual belief that Ti-oxide is more biocom-patible than 316L-SS. The number of ECs on the coated surfaces

with an Ir content of 20% and 40% is higher than that on the controlby 31% and 24%, respectively, after 2 h of attachment. In addition,by increasing the incubation time from 2 to 4 h, the correspondingabsolute increase in EC attachment to the Ir0.4Ti0.6-oxide surface is

534 S. Habibzadeh et al. / Applied Surface Science 301 (2014) 530–538

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ig. 4. SEM images of platelets attached to the (a) 316L-SS surface (control), (b) Ir0.2

ncubation in PRP. Inset: zoomed areas of the corresponding main image.

reater than that on the surfaces of the other coating compositionsnd the control. In fact, except for Ir0.2Ti0.8-oxide and Ir0.4Ti0.6-xide coatings and the control surface, no statistically significantncrease in EC attachment was observed on the other coating sur-aces when the culturing time was increased from 2 to 4 h. This

ight indicate that the major attachment of ECs to all the surfaces

ccurs during the first 2 h of incubation.

Results presented so far show that among the investigatedurfaces, the Ir0.4Ti0.6-oxide surface represents the best substrateor desired short-term interactions of ECs and platelets with the

ig. 5. Attachment of ECs on a 316L-SS surface (control) and IrxTi1−x-oxide (x = 0,.2, 0.4, 0.6, 0.8 and 1) coated surfaces. The results are the surface cell count meanalue ± SD of three samples for each bar, *p < 0.05 and **p < 0.01. Note that asterisksbove each bar represent a significant difference relative to the control whereassterisks over brackets indicate a significant difference between 2 and 4 h timeoints.

xide, (c) Ir0.4Ti0.6-oxide, and (d) Ir-oxide coated surfaces, taken after 60 min of static

surface. On the other hand, pure Ir-oxide has been considered asa surface of high biocompatibility and, considering high radiopaci-tiy, has been investigated as a possible stent modification material[39,59]. Therefore, it would be interesting to compare longer-terminteractions of ECs with the Ir0.4Ti0.6-oxide, which has much less

only to the control surface (316L-SS) but also to an Ir-oxide sur-face. Consequently, Fig. 6 shows results on proliferation of ECs on

Fig. 6. Proliferation of ECs on a 316L-SS surface (control) and Ir0.4Ti0.6-oxide andIr-oxide coated surfaces. The results are the surface cell density mean value ± SD ofthree samples for each bar, *p < 0.05 and **p < 0.01. Note that asterisks above eachbar represent a significant difference relative to the control whereas asterisks overbrackets indicate a significant difference between 4 and 7 day time points.

S. Habibzadeh et al. / Applied Surface Science 301 (2014) 530–538 535

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ig. 7. Fluorescence microscopical images of stained ECs cultured on a (a) tissue cur-oxide coated surfaces after 4 h attachment. Actin filaments were stained by FITC-

he three surfaces, after 4 and 7 days (Fig. 6). For this purpose, aower initial cell seeding was selected to give the cells more spaceo grow and proliferate, since high plating density would resultn differentiation rather than proliferation [60]. The graph showshat EC density on the Ir0.4Ti0.6-oxide surface is significantly higherp < 0.05) than on the control surface (316L-SS), after both 4 and

days (by ca. 39% and 35%, respectively). In contrast, a significantecrease (p < 0.05) in EC number on the pure Ir-oxide coating wasbserved at both times point, relative to the control. Contrary to theommon belief, this result indicates that the pure Ir-oxide coatingay not actually be a good substrate for long-term EC growth.To investigate EC morphological differences and cytoskeleton

rrangement on the control and Ir/Ti-oxide coated surfaces, theells were stained for actin and visualized 4 h after attachmentFig. 7). In accordance with the results in Fig. 5, the visual inspec-ion of images in Fig. 7 shows that the Ir0.2Ti0.8- and Ir0.4Ti0.6-oxideurfaces yielded a higher cell density in comparison to the otherurfaces. In addition, on the Ir0.2Ti0.8- and Ir0.4Ti0.6-oxide sur-aces the ECs spread to form a confluent monolayer with a typicalobblestone-like cell morphology (Fig. 7c and d), with similar mor-hology in terms of size, spreading area and cell-cell contact. Theolygonal type ECs attached to these surfaces displayed consider-ble intracellular actin staining, often localized at the cell periphery.n the bare tissue culture plastic surface (Fig. 7a), the control 316L-S surface (Fig. 7b) and the pure Ir-oxide (Fig. 7e), less cells werebserved and reduced actin staining was apparent. The densely-opulated cells on the Ir0.2Ti0.8- and Ir0.4Ti0.6-oxide coated surfaces

ndicate the greater EC attachment to the respective surfaces, whichre in agreement with the results in Fig. 5.

The effect of surface roughness on cell behavior remainsuite controversial. However, a considerable amount of litera-ure claim that cell attachment and proliferation are improved

s the surface roughness increases [13,37,61,62]. Furthermore,t has been observed that cells more favorably attach to sur-aces with moderate hydrophilicity, than to more hydrophilic or

ore hydrophobic surfaces [49,50,63,64]. This feature might be

plastic, (b) 316L-SS surface (control), (c) Ir0.2Ti0.8-oxide, (d) Ir0.4Ti0.6-oxide and (e)odin and cell nuclei were stained by DAPI. Bars indicate 20 �m.

attributed to the preferential adsorption of cell adhesive proteins,such as fibronectin and vitronectin, from the culture medium ontothe moderately hydrophilic surfaces [65]. Therefore, the positiveinteraction observed for ECs with Ir0.2Ti0.8- and Ir0.4Ti0.6-oxidesurfaces might be ascribed to the surfaces’ high roughness andmoderate hydophilicity (see Fig. 2).

3.4. Smooth muscle cell attachment, proliferation andmorphology

Following EC injury after stent implantation, SMCs in the vascu-lar media proliferate and migrate to the intimal layer of the bloodvessel [66]. Normally, SMCs show a contractile phenotype, with alow rate of proliferation. However, after angioplasty, due to the lossof contact with the EC monolayer and due to the exposure to themechanical stress and mitogen release, the SMC phenotype changesfrom a low to high proliferative state, often referred as syntheticphenotype [67]. The result of this SMC proliferation leads to intimalhyperplasia which compromises vascular function. Therefore, theinteraction of SMCs with the stent surface is a key considerationin biocompatibility evaluation. Fig. 8 shows the number of SMCson Ir/Ti-oxide coating and control surfaces after 2 and 4 h incuba-tion periods. The graph shows that after 2 h of attachment, the Ircontent of 20% and 40% in the oxide coating yields respectively ca.33% and 32% fewer SMCs attached to the surface in comparison tothe control. No significant difference with respect to the number ofcells was observed between the Ir0.2Ti0.8- and Ir0.4Ti0.6-oxide sur-faces, when culturing time increased from 2 to 4 h. In contrast, asthe incubation time increased from 2 to 4 h, a significantly highernumber of cells attached to coatings with only Ti (Ti-oxide coating)and with an Ir content of more than 40%.

Further investigations showed that the Ir0.4Ti0.6-oxide surface

minimizes long-term interactions with SMCs so that the num-ber of adherent cells decreased by nearly 62% and 65% after 4and 7 days of incubation, respectively (Fig. 9), relative to the con-trol surface. Thus, SMCs appear to show lower affinity toward the

536 S. Habibzadeh et al. / Applied Surface Science 301 (2014) 530–538

Fig. 8. Attachment of SMCs on the 316L-SS (control) and IrxTi1−x-oxide (x = 0, 0.2, 0.4,0.6, 0.8 and 1) coated surfaces. The results are the surface cell count mean value ± SDof three samples for each bar, *p < 0.05 and **p < 0.01. Note that except for Ti-oxide, allother coatings are significant (p < 0.05) over control after 2 h attachment. However,at 4 h, only Ir0.2Ti0.8- and Ir0.4Ti0.6-oxides are significant (p < 0.01) as compared to thecontrol and no significant difference was observed for the other coatings (p > 0.05)oa

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Fig. 9. Proliferation of SMCs on the 316L-SS (control) and Ir0.4Ti0.6-oxide and Ir-oxide coated surfaces. The results are the surface cell density mean value ± SD ofthree samples for each bar, *p < 0.05 and **p < 0.01. Note that asterisks above each

FI1

ver the control. Asterisks over brackets indicate a significant difference between 2nd 4 h time points.

r0.4Ti0.6-oxide coating surface during both short- and long-termnteractions.

Fig. 10 depicts the morphology of SMCs on control and Ir/Ti-xide coated surfaces after 4 h of attachment. SMCs attached to theontrol surface (Fig. 10b) had a multilayered spindle-shaped mor-hology, similar to SMCs on a bare tissue cultured plastic (Fig. 10a).

n comparison, the SMCs on Ir0.2Ti0.8- and Ir0.4Ti0.6-oxide surfacesFig. 10c and d) showed a reduced density and a migratory phe-otype due to the presence of numerous lamellipodia. The pure

r-oxide coated surface (Fig. 10e) showed fewer SMCs than the

ig. 10. Fluorescence microscopical images of stained SMCs cultured on a (a) tissue culturr-oxide coated surfaces after 4 h attachment. Actin filaments were stained by FITC-phalli0 �m.

bar represent a significant difference relative to the control whereas asterisks overbrackets indicate a significant difference between 4 and 7 day time points.

control, although, more than Ir0.2Ti0.8- and Ir0.4Ti0.6-oxide coatingsurfaces. Hence, as it was previously shown statistically in Fig. 8,fewer SMCs attached to the surfaces of Ir0.2Ti0.8- and Ir0.4Ti0.6-oxide coatings. This, thus, indicates the lower affinity of SMCs tothe corresponding surfaces (see Section 3.5)

3.5. Ability of endothelialization

During post-stent deployment, the density of ECs on theluminal surface of the stent gradually increases until complete re-endothelialization is achieved. Rapid endothelialization of stentsprevents platelet activation, thrombus formation and excessive

e plastic, (b) 316L-SS surface (control), (c) Ir0.2Ti0.8-oxide, (d) Ir0.4Ti0.6-oxide and (e)odin and cell nuclei were stained by DAPI. Arrows point lamellipodia. Bars indicate

S. Habibzadeh et al. / Applied Surface

Fig. 11. The ratio of the attached endothelial to smooth muscle cells (EC/SMC) onthe 316L-SS (control) and IrxTi1−x-oxide (x = 0, 0.2, 0.4, 0.6, 0.8 and 1) coated sur-faces during attachment experiments, *p < 0.05 and **p < 0.01. Note that except forTi-oxide, all other coatings yielded a significant difference (p < 0.05) over controlafter 2 h attachment. At 4 h, Ir0.2Ti0.8- and Ir0.4Ti0.6-oxides yielded a significant dif-ffs

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erence (p < 0.01) compared to the control and no significant difference was observedor the other coatings (p > 0.05) over the control. Asterisks over brackets indicate aignificant difference between 2 and 4 h time points.

issue growth. Thus, an effective stent would involve a coatinghat accelerates EC regeneration, while inhibiting SMC proliferationnd platelet aggregation, thereby resulting in a non-thrombogenictent with early re-endothelialization. The potential of stent sur-ace endothelialization can be estimated by the ratio of ECs to SMCshat attach on the stent surface, under the same experimental con-itions [66,68,69].

It should be taken into account that short-term interactionsf ECs and SMCs predominantly determine their competitivettachment and proliferation on an implant (e.g. stent) surface13,70,71]. Consequently, the initial attachment behavior ratherhan a proliferation response of both cells was considered to esti-

ate endothelialization potential. The data in Figs. 5 and 8 wereombined in Fig. 11 to show the EC-to-SMC attachment ratio onhe control and Ir/Ti-oxide coated surfaces after incubation peri-ds of 2 and 4 h. The EC/SMC ratio on the control surface is around.2, indicating that ECs have a slightly higher affinity toward thisurface. However, on the Ir/Ti-oxide coatings with 20% and 40%f Ir, the EC/SMC ratio increases to ca. 2.5, which implies that thenvestigated surfaces are much better substrates for the attachmentf ECs than for the attachment of SMCs. Further, because of theigher initial attachment of ECs on Ir0.2Ti0.8- and Ir0.4Ti0.6-oxideoatings, it is possible that SMC attachment and then prolifera-ion could be inhibited in a competitive environment, thus enablingaster endothelialization of these two surfaces, relative to the con-rol. This would not be the case for Ir/Ti oxide coatings with an Irontent of zero (Ti-oxide coating) and those greater than 40%, since

significant decrease in the EC/SMC ratio can be observed whenrolonging the attachment time from 2 h to 4 h (Fig. 11), indicatinghe potential of the SMCs to overrun the ECs.

Interestingly, preliminary in vivo experiments with animalodels (unpublished data) have also demonstrated that the

r0.4Ti0.6-oxide oxide coating formed on a commercial 316L-SS stents the most biocompatible coating in terms of restenosis rates and,hus, the best candidate, among the investigated coatings, for the

odification of commercial coronary stent surfaces.

. Conclusions

The outcome of platelet, EC and SMC interactions with Ir/Ti-xide coating surfaces of various compositions was presented in

[

Science 301 (2014) 530–538 537

this paper. It was found that all the metal-oxide surfaces are lessthrombogenic than the 316L-SS control surface; the metal-oxidecoatings containing 20% and 40% of Ir yielded a minimum inter-action with platelets. The metal-oxide coated surfaces were notonly found to be more blood-compatible, but also showed a sig-nificantly higher EC attachment combined with lower affinity ofSMCs toward proliferation. Furthermore, in our previous work [28].Ir0.4Ti0.6-oxide coating was found to be the most uniform, corro-sion resistant and radiopaque (visible) coating. Thus, based on allthe results, it could be concluded that the Ir0.4Ti0.6-oxide coating isthe coating of choice for the modification of coronary stents since itoffers a significantly higher bio/hemocompatibility and radiopacitythan currently used 316L-SS stent surfaces. The application of thiscoating could, thus, potentially reduce in-stent restenosis.

Acknowledgments

The authors gratefully acknowledge the financial support fromthe Natural Science and Engineering Research Council of Canada(NSERC), the Canadian Institutes of Health Research (CIHR), and theFonds de recherche du Québec – Nature et technologies (FRQ NT).

References

[1] E. Alt, D.O. Reip, Vascular and Endoluminal Stents, Inflow Dynamics Inc.,Alexandria, VA, United States, 2002.

[2] H.S. Lett, J.A. Blumenthal, M.A. Babyak, A. Sherwood, T. Strauman, C. Robins,et al., Depression as a risk factor for coronary artery disease: evidence, mech-anisms, and treatment, Psychosom. Med. 66 (2004) 305–315.

[3] A. Andukuri, M. Kushwaha, A. Tambralli, J.M. Anderson, D.R. Dean, J.L. Berry,et al., A hybrid biomimetic nanomatrix composed of electrospun polycapro-lactone and bioactive peptide amphiphiles for cardiovascular implants, ActaBiomater. 7 (2011) 225–233.

[4] H.M. Krumholz, E.D. Peterson, J.Z. Ayanian, M.H. Chin, R.F. DeBusk, L. Gold-man, et al., Report of the National Heart, Lung, and Blood Institute WorkingGroup on Outcomes Research in Cardiovascular Disease, Circulation 111 (2005)3158–3166.

[5] J. Bogaert, S. Dymarkowski, A. Taylor, Coronary artery disease, in: J.Bogaert, S. Dymarkowski, A. Taylor (Eds.), Clinical Cardiac MRI, Springer,Berlin/Heidelberg, 2005, pp. 381–437.

[6] A.D. Michaels, K. Chatterjee, Angioplasty versus bypass surgery for coronaryartery disease, Circulation 106 (2002) e187–e190.

[7] S. Windecker, I. Mayer, G. De Pasquale, W. Maier, O. Dirsch, P. De Groot, et al.,Stent coating with titanium-nitride-oxide for reduction of neointimal hyper-plasia, Circulation 104 (2001) 928–933.

[8] S. Windecker, R. Simon, M. Lins, V. Klauss, F.R. Eberli, M. Roffi, et al., Random-ized comparison of a titanium-nitride-oxide-coated stent with a stainless steelstent for coronary revascularization: the TiNOX Trial, Circulation 111 (2005)2617–2622.

[9] S.W. Robertson, V. Imbeni, H.R. Wenk, R.O. Ritchie, Crystallographic texturefor tube and plate of the superelastic/shape-memory alloy Nitinol used forendovascular stents, J. Biomed. Mater. Res. Part A 72A (2005) 190–199.

10] S.A.V. Holmes, P.D. Miller, P.R. Crocker, R.S. Kirby, Encrustation of intraprostaticstents – a comparative study, Br. J. Urol. 69 (1992) 383–387.

11] D.J. Kereiakes, D.A. Cox, J.B. Hermiller, M.G. Midei, W.B. Bachinsky, E.D. Nukta,et al., Usefulness of a cobalt chromium coronary stent alloy, Am. J. Cardiol. 92(2003) 463–466.

12] U. Baber, R. Mehran, S.K. Sharma, S. Brar, J. Yu, J.-W. Suh, et al., Impact of theeverolimus-eluting stent on stent thrombosis: a meta-analysis of 13 random-ized trials, J. Am. Coll. Cardiol. 58 (2011) 1569–1577.

13] A. Shahryari, F. Azari, H. Vali, S. Omanovic, The response of fibrinogen, platelets,endothelial and smooth muscle cells to an electrochemically modified SS316LSsurface: towards the enhanced biocompatibility of coronary stents, Acta Bio-mater. 6 (2010) 695–701.

14] C.-C. Shih, C.-M. Shih, Y.-Y. Su, L.H.J. Su, M.-S. Chang, S.-J. Lin, Quantitative evalu-ation of thrombosis by electrochemical methodology, Thromb. Res. 111 (2003)103–109.

15] K.C. Dee, D.A. Puleo, R. Bizios, An Introduction to Tissue–Biomaterial Interac-tions, John Wiley & Sons, Inc., New York, 2002.

16] X. Wang, X. Zhang, J. Castellot, I. Herman, M. Iafrati, D.L. Kaplan, Controlledrelease from multilayer silk biomaterial coatings to modulate vascular cellresponses, Biomaterials 29 (2008) 894–903.

17] J. Li, G. Li, K. Zhang, Y. Liao, P. Yang, M.F. Maitz, et al., Co-culture of vascularen-

dothelial cells and smooth muscle cells by hyaluronic acid micro-pattern ontitanium surface, Appl. Surf. Sci. 273 (2013) 24–31.

18] Q. Lin, X. Ding, F. Qiu, X. Song, G. Fu, J. Ji, In situ endothelialization of intravascu-lar stents coated with an anti-CD34 antibody functionalized heparin–collagenmultilayer, Biomaterials 31 (2010) 4017–4025.

5 urface

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

38 S. Habibzadeh et al. / Applied S

19] M. Wang, Y. Wang, Y. Chen, H. Gu, Improving endothelialization on 316L stain-less steel through wettability controllable coating by sol–gel technology, Appl.Surf. Sci. 268 (2013) 73–78.

20] R.V. Stan, 6.06 – in vitro vascular cell culture systems–endothelial cell culturesystems, in: Editor-in-Chief: A.M. Charlene (Ed.), Comprehensive Toxicology,second ed., Elsevier, Oxford, 2010, pp. 97–111.

21] G. Dangas, F. Kuepper, Restenosis: repeat narrowing of a coronary artery: pre-vention and treatment, Circulation 105 (2002) 2586–2587.

22] R. Hoffmann, G.S. Mintz, Coronary in-stent restenosis – predictors, treatmentand prevention, Eur. Heart J. 21 (2000) 1739–1749.

23] R.E. Kuntz, C.M. Gibson, M. Nobuyoshi, D.S. Baim, Generalized model ofrestenosis after conventional balloon angioplasty, stenting and directionalatherectomy, J. Am. Coll. Cardiol. 21 (1993) 15–25.

24] G.S. Mintz, J.J. Popma, A.D. Pichard, K.M. Kent, L.F. Satler, S. Chiu Wong, et al.,Arterial remodeling after coronary angioplasty: a serial intravascular ultra-sound study, Circulation 94 (1996) 35–43.

25] J.J. Castellot, M.L. Addonizio, R. Rosenberg, M.J. Karnovsky, Cultured endothelialcells produce heparinlike inhibitor of smooth muscle cell growth, J. Cell Biol.90 (1981) 372–379.

26] S.A. Biela, Y. Su, J.P. Spatz, R. Kemkemer, Different sensitivity of human endothe-lial cells, smooth muscle cells and fibroblasts to topography in the nano–microrange, Acta Biomater. 5 (2009) 2460–2466.

27] Y.X. Leng, J.Y. Chen, P. Yang, H. Sun, J. Wang, N. Huang, The biocompatibility ofthe tantalum and tantalum oxide films synthesized by pulse metal vacuum arcsource deposition, Nucl. Instrum. Methods Phys. Res. Sect. B 242 (2006) 30–32.

28] S. Habibzadeh, D. Shum-Tim, S. Omanovic, Surface and electrochemical charac-terization of IrTi-oxide coatings: towards the improvement of radiopacity forcoronary stent applications, Int. J. Electrochem. Sci. 8 (2013) 6291–6310.

29] G.W. Stone, J.W. Moses, S.G. Ellis, J. Schofer, K.D. Dawkins, M.-C. Morice, et al.,Safety and efficacy of sirolimus- and paclitaxel-eluting coronary stents, N. Engl.J. Med. 356 (2007) 998–1008.

30] A.T.L. Ong, E.P. McFadden, E. Regar, P.P.T. de Jaegere, R.T. van Domburg, P.W.Serruys, Late angiographic stent thrombosis (LAST) events with drug-elutingstents, J. Am. Coll. Cardiol. 45 (2005) 2088–2092.

31] E.P. McFadden, E. Stabile, E. Regar, E. Cheneau, A.T.L. Ong, T. Kinnaird, et al., Latethrombosis in drug-eluting coronary stents after discontinuation of antiplatelettherapy, Lancet 364 (2004) 1519–1521.

32] R. Virmani, A. Farb, G. Guagliumi, F.D. Kolodgie, Drug-eluting stents: cau-tion and concerns for long-term outcome, Coron. Artery Dis. 15 (2004)313–318.

33] A.A. Bavry, D.J. Kumbhani, T.J. Helton, P.P. Borek, G.R. Mood, D.L. Bhatt, Latethrombosis of drug-eluting stents: a meta-analysis of randomized clinical trials,Am. J. Med. 119 (2006) 1056–1061.

34] R. Virmani, G. Guagliumi, A. Farb, G. Musumeci, N. Grieco, T. Motta, et al.,Localized hypersensitivity and late coronary thrombosis secondary to asirolimus-eluting stent: should we be cautious? Circulation 109 (2004)701–705.

35] P.B. Wachem van, A.H. Hogt, T. Beugeling, J. Feijen, A. Bantjes, J.P. Detmers, et al.,Adhesion of cultured human endothelial cells onto methacrylate polymers withvarying surface wettability and charge, Biomaterials 8 (1987) 323–328.

36] F.J. Jing, N. Huang, L. Wang, R.K.Y. Fu, Y.F. Mei, Y.X. Leng, et al., Behavior of humanumbilical vein endothelial cells on micro-patterned amorphous hydrogenatedcarbon films produced by plasma immersion ion implantation & depositionand plasma etching, Diamond Relat. Mater. 16 (2007) 550–557.

37] T.-W. Chung, D.-Z. Liu, S.-Y. Wang, S.-S. Wang, Enhancement of the growth ofhuman endothelial cells by surface roughness at nanometer scale, Biomaterials24 (2003) 4655–4661.

38] M.N. Babapulle, M.J. Eisenberg, Coated stents for the prevention of restenosis:Part I, Circulation 106 (2002) 2734–2740.

39] C. Di Mario, E. Grube, Y. Nisanci, N. Reifart, A. Colombo, J. Rodermann, et al.,MOONLIGHT: a controlled registry of an iridium oxide-coated stent with angio-graphic follow-up, Int. J. Cardiol. 95 (2004) 329–331.

40] J. Al Suwaidi, P.B. Berger, D.R. Holmes Jr., Coronary artery stents, J. Am. Med.Assoc. 284 (2000) 1828–1836.

41] A.M. Cruz, N. Casan-Pastor, Graded conducting titanium–iridium oxide coat-ings for bioelectrodes in neural systems, Thin Solid Films 534 (2013)316–324.

42] Y.E. Roginskaya, O.V. Morozova, E.N. Loubnin, A.V. Popov, Y.I. Ulitina, V.V.Zhurov, et al., X-ray diffraction, transmission electron microscopy and X-rayphotoelectron spectroscopic characterization of IrO2+ Ta2O5 films, J. Chem. Soc.Faraday Trans. 89 (1993) 1707–1715.

43] Silva LAd, V.A. Alves, Silva MAPd, S. Trasatti, J.F.C. Boodts, Morphological, chem-ical, and electrochemical properties of Ti/(TiO2 + IrO2) electrodes, Can. J. Chem.75 (1997) 1483–1493.

44] L.A. da Silva, V.A. Alves, S.C. de Castro, J.F.C. Boodts, XPS study of the state of

iridium, platinum, titanium and oxygen in thermally formed IrO2 + TiO2 + PtOx

films, Colloids Surf. A: Physicochem. Eng. Aspects 170 (2000) 119–126.45] A. Caron, J.F. Théorêt, S.A. Mousa, Y. Merhi, Anti-platelet effects of GPIIb/IIIa

and P-selectin antagonism, platelet activation, and binding to neutrophils, J.Cardiovasc. Pharmacol. 40 (2002) 296–306.

[

Science 301 (2014) 530–538

46] J.F. Théorêt, J.G. Bienvenu, A. Kumar, Y. Merhi, P-selectin antagonism withrecombinant P-selectin glycoprotein ligand-1 (rPSGL-Ig) inhibits circulatingactivated platelet binding to neutrophils induced by damaged arterial surfaces,J. Pharmacol. Exp. Ther. 298 (2001) 658–664.

47] A. de Oliveira-Sousa, M.A.S. da Silva, S.A.S. Machado, L.A. Avaca, P. de Lima-Neto,Influence of the preparation method on the morphological and electro-chemical properties of Ti/IrO2-coated electrodes, Electrochim. Acta 45 (2000)4467–4473.

48] T. Hasebe, T. Ishimaru, A. Kamijo, Y. Yoshimoto, T. Yoshimura, S. Yohena, et al.,Effects of surface roughness on anti-thrombogenicity of diamond-like carbonfilms, Diamond Relat. Mater. 16 (2007) 1343–1348.

49] W. Zhou, X. Zhong, X. Wu, L. Yuan, Z. Zhao, H. Wang, et al., The effect of surfaceroughness and wettability of nanostructured TiO2 film on TCA-8113 epithelial-like cells, Surf. Coat. Technol. 200 (2006) 6155–6160.

50] Y. Arima, H. Iwata, Effect of wettability and surface functional groups on pro-tein adsorption and cell adhesion using well-defined mixed self-assembledmonolayers, Biomaterials 28 (2007) 3074–3082.

51] Z. Shi, Y. Wang, C. Du, N. Huang, L. Wang, C. Ning, Silicon nitride films for theprotective functional coating: blood compatibility and biomechanical propertystudy, J. Mech. Behav. Biomed. Mater. 16 (2012) 9–20.

52] W.M. Yang, Y.W. Liu, Q. Zhang, Y.X. Leng, H.F. Zhou, P. Yang, et al., Biomed-ical response of tantalum oxide films deposited by DC reactive unbalancedmagnetron sputtering, Surf. Coat. Technol. 201 (2007) 8062–8065.

53] Y. Yuan, C. Liu, M. Yin, Plasma polymerized n-butyl methacrylate coating withpotential for re-endothelialization of intravascular stent devices, J. Mater. Sci.:Mater. Med. 19 (2008) 2187–2196.

54] P. Yang, N. Huang, Y.X. Leng, J.Y. Chen, R.K.Y. Fu, S.C.H. Kwok, et al., Activationof platelets adhered on amorphous hydrogenated carbon (a-C:H) films synthe-sized by plasma immersion ion implantation–deposition (PIII-D), Biomaterials24 (2003) 2821–2829.

55] L. Chenglong, Y. Dazhi, L. Guoqiang, Q. Min, Corrosion resistance and hemo-compatibility of multilayered Ti/TiN-coated surgical AISI 316L stainless steel,Mater. Lett. 59 (2005) 3813–3819.

56] L.-C. Xu, J. Runt, C.A. Siedlecki, Dynamics of hydrated polyurethane biomate-rials: surface microphase restructuring, protein activity and platelet adhesion,Acta Biomater. 6 (2010) 1938–1947.

57] J. Wang, N. Jiang, Blood compatibilities of carbon nitride film deposited onbiomedical NiTi alloy, Diamond Relat. Mater. 18 (2009) 1321–1325.

58] W. Zingg, A.W. Neumann, A.B. Strong, O.S. Hum, D.R. Absolom, Platelet adhesionto smooth and rough hydrophobia and hydrophilic surfaces under conditionsof static exposure and laminar flow, Biomaterials 2 (1981) 156–158.

59] E. Alt, Stotts, J. Lawrence, Vascular and Endoluminal Stents with Iridium OxideCoating, 1999, United States, US patent.

60] D. Yaffe, O. Saxel, A myogenic cell line with altered serum requirements fordifferentiation, Differentiation 7 (1977) 159–166.

61] J.C. Palmaz, A. Benson, E.A. Sprague, Influence of surface topography onendothelialization of intravascular metallic material, J. Vasc. Intervent. Radiol.10 (1999) 439–444.

62] A. Shahryari, S. Omanovic, J.A. Szpunar, Enhancement of biocompatibility of316LVM stainless steel by cyclic potentiodynamic passivation, J. Biomed. Mater.Res. Part A 89A (2009) 1049–1062.

63] P.B. van Wachem, T. Beugeling, J. Feijen, A. Bantjes, J.P. Detmers, W.G. vanAken, Interaction of cultured human endothelial cells with polymeric surfacesof different wettabilities, Biomaterials 6 (1985) 403–408.

64] T. Tanaka, Y. Suzuki, K. Tsuchiya, H. Yajima, Improvement of cell attachmentcapabilities of poly-l-lactic acid films by modification of surface properties withion-beam irradiation, Surf. Coat. Technol. 218 (2013) 162–166.

65] J.H. Lee, G. Khang, J.W. Lee, H.B. Lee, Interaction of different types of cells onpolymer surfaces with wettability gradient, J. Colloid Interface Sci. 205 (1998)323–330.

66] M.R. Williamson, A. Shuttleworth, A.E. Canfield, R.A. Black, C.M. Kielty, The roleof endothelial cell attachment to elastic fibre molecules in the enhancement ofmonolayer formation and retention, and the inhibition of smooth muscle cellrecruitment, Biomaterials 28 (2007) 5307–5318.

67] K.A. Martin, E.M. Rzucidlo, M. Ding, B.L. Merenick, Z. Kasza, R.J. Wagner, et al.,6.05 – in vitro vascular cell culture systems–vascular smooth muscle, in: Editor-in-Chief: A.M. Charlene (Ed.), Comprehensive Toxicology, second ed., Elsevier,Oxford, 2010, pp. 69–96.

68] S. Choudhary, M. Berhe, K.M. Haberstroh, T.J. Webster, Increased endothelialand vascular smooth muscle cell adhesion on nanostructured titanium andCoCrMo, Int. J. Nanomedicine 1 (2006) 41–49.

69] S. Choudhary, K.M. Haberstroh, T.J. Webster, Enhanced functions of vascu-lar cells on nanostructured Ti for improved stent applications, Tissue Eng. 13(2007) 1421–1430.

70] M.F. Fillinger, S.E. O’Connor, R.J. Wagner, J.L. Cronenwett, The effect of endothe-

lial cell coculture on smooth muscle cell proliferation, J. Vasc. Surg. 17 (1993)1058–1068.

71] H.-I. Yeh, S.-K. Lu, T.-Y. Tian, R.-C. Hong, W.-H. Lee, C.-H. Tsai, Comparison ofendothelial cells grown on different stent materials, J. Biomed. Mater. Res. PartA 76A (2006) 835–841.