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Acta Biomaterialia 5 (2009) 3593–3604

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Investigation of surface endothelialization on biomedical nitinol(NiTi) alloy: Effects of surface micropatterning combined

with plasma nanocoatings

Yang Shen a,b, Guixue Wang a,b,*, Liang Chen b, Hao Li b, Ping Yu c, Mengjun Bai c,Qin Zhang a, James Lee d, Qingsong Yu b,*

a Bioengineering College of Chongqing University and ‘‘111 Project” Laboratory of Biomechanics, Tissue Repair of Ministry of Education,

Chongqing 400044, Chinab Center for Surface Science and Plasma Technology, Department of Mechanical and Aerospace Engineering, University of Missouri,

Columbia, MO 65211, USAc Department of Physics and Astronomy, University of Missouri, Columbia, MO 65211, USA

d Department of Bioengineering, University of Missouri, Columbia, MO 65211, USA

Received 31 July 2008; received in revised form 23 April 2009; accepted 14 May 2009Available online 27 May 2009

Abstract

Plasma nanocoated films with trimethylsilane-oxygen monomers showed outstanding biocompatibility in our previous studies. In thisstudy, endothelialization on biomedical nitinol alloy surfaces was systematically investigated. Our study focuses on elucidating the effectsof surface micropatternings with micropores and microgrooves combined with plasma nanocoating. Plasma nanocoatings with con-trolled thickness between 40 and 50 nm were deposited onto micropatterned nitinol surface in a direct current plasma reactor. Bovineaortic endothelial cells were cultured in vitro on these nitinol samples for 1, 3 and 5 days. It was found that rougher surfaces couldenhance cell adhesion compared with the smoother surfaces; the surfaces patterned with micropores showed much more endothelializa-tion than microgrooved surface after a 3 days culture. The cell culture results also showed that plasma nanocoatings significantly furtherincreased cell proliferation and cell adhesion on the micropatterned nitinol surfaces, as compared with non-plasma nanocoated surface ofnitinol samples. The surface micropatternings combined with plasma nanocoatings could improve the cell adhesion and accelerate sur-face endothelialization after implantation of intravascular stents, which is expected to reduce in-stent restenosis.� 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Keywords: Surface micropatterning; Nanoroughness; Intravascular stent; Nitinol alloy; Endothelialization

1. Introduction

In-stent restenosis (ISR) is the major risk followingcoronary stent implantation [1–3]. ISR can be decreasedby surface endothelialization of cardiovascular stent,which is regarded as an important means to prevent

1742-7061/$ - see front matter � 2009 Acta Materialia Inc. Published by Else

doi:10.1016/j.actbio.2009.05.021

* Corresponding authors. Address: Bioengineering College of Chongq-ing University, Chongqing 400044, China (G. Wang).

E-mail addresses: wanggx@cqu.edu.cn (G. Wang), yuq@missouri.edu(Q. Yu).

thrombogenicity, to reduce proliferation and migrationof smooth muscle cells (SMCs). As one kind of vascularstent material, nitinol alloy (NiTi) has been appliedwidely due to its shape-memory property and superelasticcapability [4–7]. However, intravascular stents made ofNiTi alloy have some limitations in vascular stent appli-cations. To name a few, the smooth surface of bare niti-nol stent is difficult for endothelial cells (ECs) to adhereto and form a uniform EC monolayer, the corrosionresistance of NiTi is actively debated, and the toxicand carcinogenic effects of nickel ions have been reported

vier Ltd. All rights reserved.

3594 Y. Shen et al. / Acta Biomaterialia 5 (2009) 3593–3604

a long time ago [8,9]. Obviously, the ideal implantedmaterials should induce rapid, predictable and controlledhealing of host tissues. The surface property of animplant plays a critical role in determining its biocom-patibility and ultimately its integration, because of itsdirect contact with blood and host tissues. Surface mod-ification is an easy and economical way to improve thesurface characteristics and to enhance the biocompatibil-ity of implanted biomedical materials. As for intravascu-lar stents, recent research focused on the followingaspects: (i) changing the surface wettability [10], surfacechemistry [11,12], surface roughness [13–15], and micro-patterns [16–18], coated film structure [19], and chargedistribution [20,21] to improve cell adhesion, spreadingand proliferation, to accelerate the endothelialization rateand to rapidly form a uniform ECs monolayer for pre-venting proliferation of SMCs; (ii) improving the hemo-compatibility of NiTi stent to prevent thrombogenicity;(iii) enhancing corrosion resistance by preparing uniformcoatings to restrain the Ni2+ release into blood and tis-sues [22,23].

Because many variables influence interactions betweencells and surface structures, it is difficult to draw conclu-sions and to formulate general principles for nano- andmicrostructured surfaces. To date, most of the researchonly focused on attachment of osteoblast-like cells todesigned titanium surfaces. Zhu et al.’s results [24] indi-cated that porous structures in micron- or submicron-scalecould enhance osteoblast attachment on titanium surface.This phenomenon was attributed to pores acting as positiveattachment sites for the filopodia of osteoblasts. Similarfindings were reported by Zinger [25].

It is known that surface modification of biomaterials is animportant way to tailor the materials’ responses to cellswhilst retaining their bulk properties [26–30]. Plasma depo-sition using low-pressure gas glow discharges has been pro-ven to be a very effective method for materials surfacemodification including surface wettability adjustment, cellsadhesion enhancement, and biocompatibility improvement.In fact, many plasma coatings, including DLC, fluorocar-bon, crystal and amorphous titanium oxide, and plasmapolymer films, have been proved to enhance surface mechan-ical and biological properties of implanted biomaterials[31–35]. SiOx:H films made by radiofrequency magnetronsputtering or plasma enhanced chemical vapor depositiontechniques have been widely studied and applied insemiconductor industries [36–38]. However, there are veryfew studies on the biomedical implanted stents and theirbiocompatibility with SiOx:H plasma coatings. In our veryrecent studies [39,40], low-temperature plasma-coatednitinol alloy with amorphous SiOx:H nanocoatings wasevaluated.

The primary objective of this study was to elucidate theeffects of surface characteristics on endothelialization of bio-medical nitinol alloy, improving the performance of nitinol-based intravascular stents. Surface modifications employedin this study were micropatterning and plasma nanocoa-

tings. Surface chemistry and surface wettability was con-trolled by amorphous SiOx:H gas plasma nanocoatings.

2. Materials and methods

2.1. Materials

Nitinol alloy (CW25-BB-34 � 125) containing 55.83wt.% Ni and balance Ti was kindly provided by Memry Cor-poration (Bethel, CT, USA). Rectangular nitinol coupons(0.3 � 0.3 cm) with thickness of 0.1 cm were prepared andused in this study. Prior to surface treatments, the nitinolcoupons were ultrasonically cleaned in an acetone bathand dried in air at room temperature.

2.2. Surface micropatterning

In this study, mechanical polishing, sandpaper grindingand chemical pickling were used to treat the nitinol cou-pons and to prepare the pre-designed surface topographies.

2.2.1. Mechanical polishing (MP)The nitinol coupons were first mechanically polished to

get a mirror-like surface by using a rotating mechanicalpolisher with 0.3 lm polycrystalline diamond polishingpowders. After mechanical polishing, chemical picklingand sandpaper grinding with #1200 SiC sandpapers wereused to further treat the nitinol coupons.

2.2.2. Chemical pickling (C)Chemical pickling was used to treat the NiTi coupons to

achieve micropores on the surfaces. The coupons weredipped into a beaker with the solution of 30% HNO3 (vol-ume ratio) at 4 �C for 24 h. Following this treatment thecoupons were ultrasonically cleaned in deionized waterand then in acetone for 10 min, respectively. The sampleswere then air dried at room temperature.

2.2.3. SiC sandpaper grinding (S)

One-directional grinding of nitinol coupons using SiCsandpapers (#1200) was used to achieve microgrooves onthe surfaces. Samples were then cleaned in ultrasonic bathsof acetone, followed by rinsing with deionized water andair-drying at room temperature.

2.2.4. SiC sandpaper grinding + chemical pickling (SC)To achieve a surface topography patterned with both

microgrooves and micropores, nitinol coupons were firstground by using SiC sandpaper (#1200) as detailed in Sec-tion 2.2.3 and then chemically treated in 30% HNO3 solu-tion for 24 h as described in Section 2.2.2.

2.2.5. Surface modification of nitinol alloy by plasma

nanocoatingsLow-temperature gas plasma treatment was performed in

the Center for Surface Science and Plasma Technology, Uni-versity of Missouri–Columbia, USA. A bell jar type plasma

Y. Shen et al. / Acta Biomaterialia 5 (2009) 3593–3604 3595

reactor system was used in this study to modify the surface ofnitinol alloy. Two anodes consisting of stainless steel plates(25.4 � 25.4 � 0.16 cm) with magnetron enhancement wereplaced 15.5 cm apart in parallel. The nitinol samples wereplaced in the middle of the two parallel anodes and used asthe cathode of the plasma system. Plasmas were created andsustained by using a direct current (DC) power supply(MDX-1 K Magnetron Drive, Advanced Energy Industries,Inc.). The nitinol samples were pretreated with oxygen plasmafor 2 min under the condition of 2 sccm oxygen, 25 mtorrpressure, 5 W DC power to clean the surfaces. Subsequently,SiOx:H plasma nanocoatings were deposited from a mixtureof trimethysilane (TMS, (CH3)3SiH) and oxygen under differ-ent conditions. The plasma coating thickness was controlledby adjusting plasma deposition time. Surface patterning ofthe nitinol alloy was detailed and the sample identificationcodes used in this study are summarized in Table 1.

2.3. Surface characterization

Scanning electron microscopy (SEM, Quanta 600F,FEI Company, USA) was used to examine and charac-terize the topography of the prepared nitinol specimens.The plasma coating thickness was detected by a null-seeking type AutoEL-II Automatic Ellipsometer(Rudolph Research Corporation, Flanders, NJ) with a632.8 nm helium–neon laser light source. The surfacewettability of plasma coated film was assessed by usinga contact angle measurement system (VCA 2500XE,USA). The surface topography changes after plasmacoating were studied by SEM (Quanta 600F, FEI Com-pany, USA). Atomic force microscopy (AFM, DI Nano-scope IIIA, Veeco Instruments) was further used to scanthree representative areas of the plasma coated surfacefor each sample with an area of 10 lm, respectively,and then obtain 3-D images by built-in software (Nano-scope IIIA Multimode, V5.30). The surface averageroughness (Ra) was quantified using a surface profilome-ter (Form Talysurf Inductive 120, UK) with 10 mm scan-ning range. The chemical structures of the plasma coatedfilms were characterized using a Nicolet FTIR 460 spec-trometer from Thermo Electron Corporation (Waltham,MA, USA).

Table 1Sample identification codes and preparation conditions.

Identificationcodesa

Preparation conditions

MP Mechanical polishingC 30% HNO3 (24 h, 40 �C) chemical picklingS #1200 SiC sandpaper grindingSC 30% HNO3 (24 h, 40 �C) chemical pickling after 1200 SiC a

P Plasma nanocoating deposited from TMS (1 sccm) + O2 (4of 25 mtorr, 5 W, 4 min

a Mechanical polishing + plasma coating (MP + P); chemical pickling + pl(S + P); chemical pickling and #1200 SiC sandpaper grinding + plasma coatin

2.4. Cell cultures using bovine aortic endothelial cells

(BAECs)

The treated NiTi coupons (5 specimens for each treatmentcondition) were put into a 48-well polystyrene tissue cultureplate. UV light was then used to sterilize the specimens for2 h. BAECs were dissociated mechanically and enzymati-cally in flasks with 0.25% trypsin, and then suspended inserum-free media. Hemocytometry was used to calculateand regulate the accurate cell density at 2.0 � 104 cells ml�1.

One day later, the morphologies of single initial celladhesion on nitinol specimens were examined by SEM(Hitachi S-4700, Japan). 1, 3 and 5 days later, the sampleswere removed from the 48-well polystyrene tissue cultureplate and rinsed by phosphate buffered saline (PBS). Hoe-chst stain (H6024, 1 mg ml�1) was used to dye the nucleusof living cells. After the fluorescent staining, the labeledcells were examined by using a fluorescence microscope(Nikon TE2000, Japan) with 200� magnification. Fivespecimens for each treatment condition, and five represen-tative areas on each specimen, were randomly chosen forpicture-taking. The amounts of the adhered cells werecounted by using built-in Metavue fluorescence analysissoftware. Summing up total cell numbers of five represen-tative areas, the average number of cells on each treatmentcondition was calculated and presented as mean ± SD.

2.5. Statistical analysis

The data obtained in this study were reported as themeans ± standard deviation. Data obtained under differenttreatment groups were then statistically compared by statis-tical software SPSS 11.5 (SPSS, Inc., Chicago, Illinois). Toreveal differences among the groups, one-way ANOVA fol-lowed by Tukey’s test was used. The differences were consid-ered significant at P < 0.05 and highly significant at P < 0.01.

3. Results

3.1. The surface morphology and roughness of the prepared

nitinol specimens

SEM was employed to examine the surface topographies ofthe prepared nitinol specimens and the images are shown in

Surface morphology

Smooth surfaceSurface patterned with microporesSurface patterned with microgrooves

brasive sandpaper grinding Surface patterned with both microporesand microgrooves

sccm) mixture under conditions Surface being similar to the uncoatedsubstrates

asma coating (C + P); #1200 SiC sandpaper grinding + plasma coatingg (SC + P).

Fig. 1. SEM images along with the surface scanning profiles obtained using a surface profilometer with a scanning range of 10 mm on the prepared nitinolspecimens: (a) mechanically polished (MP specimens); (b) chemically pickled (C specimens); (c) SiC sandpaper grounded (S specimens); (d) SiC sandpapergrounded and then chemically pickled (SC specimens).

3596 Y. Shen et al. / Acta Biomaterialia 5 (2009) 3593–3604

Fig. 2. The average surface roughness, Ra, of the prepared nitinolspecimens measured using a surface profilometer. *Significant differenceP < 0.05; **highly significant difference P < 0.01.

Fig. 3. Oxygen flow rate dependence of the water surface contact angles ofSiOx:H plasma nanocoatings. Other plasma conditions are: TMS flow rateof 1 sccm, system pressure of 25 mtorr, DC plasma power of 5 W, andplasma deposition time of 2 min.

Y. Shen et al. / Acta Biomaterialia 5 (2009) 3593–3604 3597

Fig. 1. The surface profile of each specimen obtained by sur-face profilometer was also depicted in Fig. 1. A mirror-likesurface was observed from mechanically polished (MP)specimens. Uniformly distributed pores were observed withchemically picked specimens (C) in Fig. 1b. As shown in

Fig. 4. The water contact angle changes on the surface of samples treated wTMS:O2 = 1 sscm:3 sccm; (c) TMS: O2 = 1 sscm:6 sccm.

Fig. 1b, uniform micropores were obtained by chemicallypickled nitinol specimens. The diameter of the micropores is�1 lm. It can be seen that the micropores uniformly distrib-uted on the C specimen surface. Comparatively, as shown inFig. 1c, microgrooves were formed on the surface after SiCsandpaper grinding (S). As shown in Fig. 1d, in contrast, sur-faces patterned with both microgrooves and micropores wereobtained on nitinol specimens that were treated with SiCsandpaper grinding and subsequently chemical pickling.

Fig. 2 shows the surface roughness parameter, Ra, of theprepared nitinol specimens, which was measured by a sur-face profilometer. The results showed that Ra of MP, C, Sand SC specimens were 16.7 ± 3.1, 63.8 ± 2.5, 94.4 ± 2.3and 519.2 ± 14.8 nm, respectively. It can be seen that SCspecimens had the roughest surfaces, which had significantdifference from other three specimens (P < 0.01). Similarly,the S and C specimens had similar surface roughness, butshowed significant difference from MP specimens.

3.2. Surface characterization of SiOx:H plasma nanocoatings

The effects of plasma chemistry, i.e. plasma gas composi-tion, on plasma nanocoatings were first studied. Figs. 3 and 4show water surface contact angle changes of the resultantSiOx:H plasma nanocoatings with O2 flow rate with trimeth-ylsilane (TMS) kept constant as 1 sccm.

As shown in Fig. 3, an increase in O2:TMS ratio inplasma gas mixture gradually reduced the water surfacecontact angle of the resultant plasma coatings. It can beobserved from Fig. 3 that the surface hydrophilicity ofthe SiOx:H plasma nanocoatings can be well controlledthrough adjusting the O2:TMS ratio in plasma gas mixture.

3.3. Water surface contact angle of plasma nanocoated

micropatterned nitinol alloys

Fig. 5 summarizes the change in water contact angles ofthe micropatterned nitinol samples before and after plasmacoating. It can be seen from Fig. 5 that the SiOx:H plasmananocoatings could reduce the water contact angles fromover 80� to nearly 40�. The surface hydrophilicity of micr-opatterned nitinol surfaces was improved.

ith different flow ratio of TMS over O2: (a) 1 sccm TMS without O2; (b)

Fig. 5. Water contact angle of micropatterned nitinol samples with andwithout SiOx:H plasma nanocoatings. Plasma nanocoatings were depos-ited under conditions of TMS flow rate of 1 sccm, O2 flow rate of 4 sccm,system pressure of 25 mtorr, DC plasma power of 5 W.

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3.4. Surface chemistry

Fourier transform infrared spectroscopy with attenu-ated total reflection (FTIR-ATR) was used to study thechemical structure of the SiOx:H plasma nanocoatingsand the spectra were displayed in Fig. 6. There were somesimilarities in all the coatings prepared with differentTMS:O2 ratios. The peaks at about 3500 cm�1 were due

Fig. 6. FTIR spectra of SiOx:H plasma nanocoatings deposited from gas mixtpressure of 25 mtorr, DC plasma power of 5 W.

Fig. 7. The typical (a) digital picture and (b) SEM image (1000�) of

to the –OH vibrations of intermolecularly bonded OHgroup. The adsorption bands at 1050 cm�1 were due toSi–O–Si bonds absorbance similar to what was observedby other researchers [41]. The peaks at about 600 cm�1

were the Si–H [42]. The absorption bands at 1500 cm�1

stretch were due to C–C stretching.

3.5. The surface morphology of the micropatterned nitinol

surfaces after plasma nanocoating

For observing the difference between groups with orwithout plasma nanocoating, a strip of adhesive tape wasused to cover half the nitinol samples during plasma depo-sition process. One typical digital picture and one SEMimage obtained from such sample surfaces were respec-tively shown in Fig. 7. In Fig. 7b, there are no obviouschanges in surface morphology of the nitinol sample beforeand after plasma nanocoatings.

AFM was used to further examine the plasma nanocoat-ed nitinol surfaces and the AFM images are shown inFig. 8. As seen from Fig. 8, AFM examination presentedan agreement with the SEM results. The MP + P sampleshowed a smoother surface than that of MP (Fig. 8a).

ures of TMS + O2 with different ratios under plasma conditions of system

nitinol samples with and without SiOx:H plasma nanocoatings.

Fig. 8. AFM images of the micropatterned nitinol surfaces before and after plasma nanocoatings: (a) MP and MP + P samples; (b) C and C + P samples;(c) S and S + P samples; (d) SC and SC + P samples.

Y. Shen et al. / Acta Biomaterialia 5 (2009) 3593–3604 3599

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According to AFM analysis, as compared with theuncoated samples showed in Fig. 8, however, the plasmananocoating showed almost no influence on the surfacetopographies of the C, S, SC samples.

3.6. Cell adhesion of BAECs

Cell attachment was assessed by seeding BAECs on thespecimens with a density of 2.0 � 104 cells ml�1 and thenculturing the cells for 1, 3 and 5 days. After Hoechst stain-ing, five areas of each sample, and five samples of eachtreatment, were examined under the fluorescence micros-copy with 200� magnification. To elucidate the effects ofsurface roughness and micropatterns, the MP specimensthat had very smooth surface were used as controls. Thefluorescence images of BAECs adhered to the preparednitinol specimens were shown in Fig. 9. It can be seen thatmore BAECs grew and adhered to the C, S and SC speci-mens than that on the smooth MP controls. Based on thefluorescence images which were taken by fluorescencemicroscopy, the numbers of cells attached to each specimenwere counted and the data is shown in Fig. 10.

At first, the numbers of cells attached on four kind ofmicropatterned surface were compared. Statistic analysisshowed that, with 1 day (24 h) incubation, there is no sig-

Fig. 9. The fluorescence images (scale bar = 200 lm) of BAECs that were cucontrols; (b) C specimens; (c) S specimens; and (d) SC specimens. White arrow

nificant difference in the cell adhesion among the four typesof nitinol specimens. After a 3 day cell culture, the numbersof cells adhering to C, S and SC specimens was significantlymore than that on the smooth MP control (P < 0.01). Inaddition, the number of cells adhering to the surface of Cand SC specimens was also significantly more than thaton S specimens. The results mean that the surfaces pat-terned with micropores showed much more cell adhesionthan the surfaces patterned with microgrooves. After5 day cell culture, BAECs formed a confluent layer on allthe nitinol specimens including the smooth MP controls.However, statistical analysis still showed that there was adifference between the SC specimens and the MP controls(P < 0.05). There was no significant difference either amongthe C, S samples and MP controls or among the C, S andSC specimens.

The numbers of cells attached on micropatterned sur-face with and without plasma nanocoatings after 3 day cellculture were then compared. It can be concluded fromFig. 11 that the SiOx:H plasma nanocoatings significantlyenhanced cell proliferation and adhesion as compared withthe uncoated samples.

After 1 day cell culture, the single cell initial adhesion oneach of the nitinol specimens was explored by SEM. Asshown in Fig. 12, the SEM images clearly showed the mor-

ltured for 1, 3, and 5 days on the prepared nitinol specimens of: (a) MPs indicate the alignment direction of the microgrooves on the surfaces.

Fig. 10. The cell numbers of BAECs adhered on the prepared nitinol specimens. *Significant difference P < 0.05; **highly significant difference P < 0.01.

Fig. 11. The cell numbers of BAECs adhered to micropatterned nitinolsurfaces with and without plasma nanocoatings obtained after 3 day cellculture. *Significant difference P < 0.05; **highly significant differenceP < 0.01.

Y. Shen et al. / Acta Biomaterialia 5 (2009) 3593–3604 3601

phologies of the cell adhering to the nitinol surfaces with dif-ferent surface topographies. As compared with the cell shapefound on the smooth MP control shown in Fig. 12a, it canbeen seen that the cells adhering to C and SC specimen sur-

Fig. 12. SEM images (scale bar = 20 lm) of single BAEC after 1 day of cnanocoating: (a) MP; (b) C; (c) S; (d) SC; (e) MP + P; (f) C + P; (g) S + Pmicrogrooves on the surfaces.

faces, which had micropores, showed many microvillus/infoldings and extended many thin and long filopodia asshown in Fig. 12b and d. The microvillus and infoldingscould increase the surface areas of the cells, and enhance sub-stance exchanging between the cells and environment. Thisphenomenon was observed from the cells adhering to bothC and SC specimens, but not from those adhering to the Sspecimens and the smooth MP controls. The SEM resultsindicated that of C and SC specimens with a microporousstructure provided more contact sites and were favorablefor BAECs adhesion with healthy growth. In addition,according to the SEM and fluorescence images, the surfaceof S and SC specimens had obvious grooves and ridges,where the contact guidance was most likely to occur. Theamounts of the attached BAECs on S and SC specimensrevealed contact guidance morphologies because these cellsspread along the grooves over the specimen surfaces (as indi-cate by arrows in Fig. 12c). This phenomenon of contactguidance was also evidently identified from fluorescencemicroscopic images shown in Fig. 9c and d, in which the

ell culture on the prepared nitinol specimens with and without plasmaand (h) SC + P White arrows indicate the alignment direction of the

3602 Y. Shen et al. / Acta Biomaterialia 5 (2009) 3593–3604

BAECs adhered to and grew on the surfaces in the samedirection as the microgrooves.

Similar results could be found in Fig. 12e–h; moremicrovillus/infoldings could be found on the surface ofC + P and SC + P specimen surfaces compared withMP + P and S + P samples. Besides, more filopodia werefound in C + P and SC + P samples, meaning cells coulddistinguish surfaces patterned with micropores which pro-vided more contact sites for cell adhesion. In addition,according to the SEM images, the surface of S + P andSC + P specimens had obvious grooves and ridges, wherethe contact guidance for cell spreading was most likely tooccur (as indicate by arrows in Fig. 12g and h). In addition,cells spread more easily on the plasma-coated surfaces thanthe uncoated nitinol surfaces. These phenomena could beattributed to the changes in surface hydrophilicity afterplasma coating.

4. Discussion

The effects of surface roughness and topography on celladhesion to implantable materials have been unclearbecause the change in surface roughness was often accom-panied with surface chemistry change. For example, Yaoet al. [43] formulated nanometer features on titanium sur-faces through anodization, in which both chemistry andsurface roughness are altered due to increases in the per-centage of oxygen on titanium surfaces. Chung et al. [44]studied Gly-Arg-Gly-Asp (GRGD)-grafted PU-PEG sur-faces and demonstrated that the increased surface rough-ness of biomaterials at 101–102 nm scale could enhancethe surface adhesion and growth of HUVECs. However,it has been difficult to clarify the independent contributionsof surface roughness versus chemistry changes to promot-ing cell adhesion and growth.

With the development of photolithography and dryetching techniques, introducing extra coating films couldeasily produce different surface topographies as expected,but at the same time would also change the surface chem-istry. Surface topography of materials is also recognized asa major factor to affect cell adhesion. Micropatterned sur-faces with parallel grooves based on plasma modificationof PEO-like coating, micron- and submicron-scale porousstructures of titanium surface for osteoblastic cells adhe-sion, have been studied [24,25]. The problem is the diffi-culty in preparing patterned surface structures insubmicron and nanometer scale on biometallic implantedmaterials without extra coating films. In this study, sur-faces patterned with microgrooves and micropores wereobtained on biomedical nitinol alloy by using mechanicaland chemical methods. The surface roughness of the nitinolspecimens prepared by SiC sandpaper grinding (to formmicrogrooves) and chemical treatment (to form microp-ores) was in nanoscale with Ra as 63.8 ± 2.5 and94.4 ± 2.3 nm, respectively. However, the Ra of the nitinolspecimens subsequently prepared by these two techniquesincreased significantly to a submicron scale of

519.2 ± 14.8 nm, which is very likely due to the combiningeffects of microgrooves and micropores on the same surfaceas shown in the images in Figs. 1 and 8. Using 30% HNO3

chemical pickling, the diameter of the micropores was inthe range of �1 lm, which, however, can be changed andcontrolled by changing the concentration of HNO3, treat-ment duration and temperature. Of course, the size of themicrogrooves can be easily controlled by using differentsizes of SiC sandpapers and polishing powders.

In this study, it was found that nitinol surfaces patternedwith both microgrooves and micropores in nanometer levelcould significantly enhance cell adhesion of BACEs ascompared with the controls with a very smooth surface.The microporous surfaces showed even better cell adhesionresults than the surfaces patterned with microgrooves. It isimportant to understand the mechanism by which BAECs’responses were improved on these nitinol surfaces withmicroporous structure as compared with that on the sur-faces with microgrooves. It is understandable that arougher surface could provide more contact sites for cellinitial attachment when compared with a smoother surface.Extracellular matrices (ECMs), i.e. some proteins includingfibronectin, laminin and vitronectin, could also be moreeasily absorbed and fixed on a rougher surface. Cell–sub-strate interactions depend on cytoskeletal organization,transmembrane integrin receptor expression and ECMs.Focal contacts are the main adhesion structures in thecell–substrate interaction. One side of cell filopodia linkstightly to actin of cytoskeleton, another side combines withabsorbed protein on the surface of samples via fibronectin.Microstructured pores, acting as a favorable environment,supply positive guidance cues for anchorage-dependentcells to attach, leading to enhanced cell attachment. Then,filopodia adapts to the surface structures to attach, and toenable optimum anchorage by using specific points alongthe filopodia as well as their tips. The contact guidancephenomenon, which was provided by material surface withmicrogrooves for osteoblast-like cell adhesion, has beendiscussed in some previous studies [13,45–47]. Cell orienta-tion reflects the influence of the microgrooves in producing‘‘contact guidance”, which can control the direction andspeed of cell migration.

Due to the direct contact of intravascular stents withblood after its implantation, hemocompatibility is a keyfactor that should be taken into account. Micro-rough sur-face could enhance the cells’ initial adhesion, but it mayalso accelerate some components in blood such as fibrino-gen and blood platelets to deposit on the stent surface andinduce thrombogenicity eventually. A rougher surface thatcould activate thrombus formation had been reported insome researches [48–50]. In contrast, the submicron and/or nanoroughness obtained in this study through micropat-terning could potentially eliminate the aforementionedproblems with microlevel roughness. The adhesion kineticsof albumin and fibrinogen on nitinol surface with differentroughness and micropatterns will be further explored inour future studies.

Y. Shen et al. / Acta Biomaterialia 5 (2009) 3593–3604 3603

Plasma nanocoating technology showed its capability insurface modification and offering a wide range of wettabil-ities simply through adjusting plasma chemistry or plasmagas compositions. In this study, SiOx:H plasma nanocoa-tings on nitinol alloy with controlled surface chemistryand properties were investigated with regard to surfaceendothelialization. The changes in surface morphology,thickness of SiOx:H coating, surface chemistry, and hydro-philicity were characterized and assessed. SiOx:H plasmananocoatings deposited from TMS + O2 mixture couldeffectively reduce the water surface contact angle of themicropatterned nitinol surface from 80–100� to about40�. Cellular behaviors, e.g. adhesion, spreading and prolif-eration, are greatly affected by surface wettability of bio-materials [10]. Surface wettability depends on surfacechemistry, i.e. hydrophilic and hydrophobic chemicalgroups, of materials. Various functional groups such as –CH3, –OH, –NH2 and –COOH groups have been investi-gated by some other researchers [51,52]. A balance betweenhydrophilic and hydrophobic surfaces was regarded asbeing suitable for cell proliferation and adhesion [53].

Due to the nanoscale thickness of SiOx:H plasma nano-coatings, there is no obvious changes in surface topogra-phies and roughness of the designed micropatternednitinol surfaces. The plasma nanocoating technology pro-vides an effective method to modify micropatterned nitinolsurfaces without affecting the well-designed surface pat-terns. Integration of plasma nanocoating with the well-designed micropatterned nitinol surface enables us to fur-ther enhance the cell proliferation and adhesion of BAECsand consequently improve surface endothelialization,which is known to be useful in preventing and reduceISR in intravascular stents. This method is also expectedto be applicable to other implanted biomaterials.

5. Conclusions

Surfaces patterned with uniformly distributed microg-rooves and micropores were designed and obtained in acontrollable manner on biomedical nitinol alloys throughmechanical and chemical treatments. Our experimentalresults showed that the patterned nitinol surface gave riseto controllable submicron and/or nanoscale roughness,which could enhance cells adhesion and growth of BAECs,and as a result the surface endothelialization. The surfaceof nitinol patterned with micropores showed better celladhesion densities than that on the surface patterned withmicrogrooves. The surface of nitinol specimens patternedwith both microgrooves and micropores gave rise to themost cell attachment. The surface hydrophilicity of theplasma nanocoated nitinol can be controlled with watersurface contact angle in the range from 100� to 40�. Withwell-controlled surface chemistry and surface hydrophilic-ity, SiOx:H plasma nanocoated nitinol surfaces showed sig-nificantly enhanced cell proliferation and adhesionperformance. Due to the nanoscale thickness of SiOx:H

plasma nanocoatings, there is no obvious changes in sur-face topographies and roughness of the well-designed micr-opatterned nitinol surfaces. It is expected that, whencombined with a well-controlled surface morphology, sub-micron/nanoscale roughness and biocompatible plasmananocoatings, biomedical nitinol alloys will be much moreeffective in surface endothelialization, in eliminatingthrombosis and consequently in reducing the ISR thatoccurs in existing nitinol intravascular stents.

Acknowledgments

This study was supported by grants from China Scholar-ship Council, the Chinese Ministry of Science and Technol-ogy (2004DFA06400) and the Chongqing Municipality,China (CSTC2006AA5014-3), and the Bioprocessing andBiosensing Center at University of Missouri, Columbia,Missouri, USA. The authors are grateful to Mr YoungJo Kim and Mr Andrew Ritts for their helpful discussionand assistance in this study.

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