Accepted Manuscript Title: Reduced platelet adhesion and improved corrosion resistance of superhydrophobic TiO2 -nanotube-coated 316L stainless steel Author: Qiaoling Huang Yun Yang Ronggang Hu Changjian Lin Lan Sun Erwin A. Vogler PII: DOI: Reference:

S0927-7765(14)00651-1 http://dx.doi.org/doi:10.1016/j.colsurfb.2014.11.028 COLSUB 6751

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

8-9-2014 5-11-2014 19-11-2014

Please cite this article as: Q. Huang, Y. Yang, R. Hu, C. Lin, L. Sun, E.A. Vogler, Reduced platelet adhesion and improved corrosion resistance of superhydrophobic TiO2 -nanotube-coated 316L stainless steel, Colloids and Surfaces B: Biointerfaces (2014), http://dx.doi.org/10.1016/j.colsurfb.2014.11.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Reduced platelet adhesion and improved corrosion resistance of

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superhydrophobic TiO2-nanotube-coated 316L stainless steel

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Qiaoling Huanga, Yun Yanga, Ronggang Hub, Changjian Lina, Lan Suna, *, Erwin A. Voglerc a

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State Key Laboratory for Physical Chemistry of Solid Surfaces, and Department of Chemistry, College of Chemistry and

Chemical Engineering, Xiamen University, Xiamen 361005, China b

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College of Chemistry and Material Engineering, Guiyang University, Guiyang 550005, China

c

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Departments of Materials Science and Engineering and Bioengineering, The Pennsylvania State University, University Park, PA

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16802, USA

* Corresponding Author: Lan Sun, Tel: +86 592 2184655, Fax: +86 592 2186657, Email: [email protected] Address: State Key Laboratory for Physical Chemistry of Solid Surfaces, and Department of Chemistry, College of Chemistry and Chemical Engineering, Xiamen University, 422 Siming South Road, Xiamen, Fujian, China. 361005

Highlights  We fabricate TiO2 nanotubes (TNTs) with extreme wettabilities on stainless steel.  Crystal phase and wettability affect corrosion resistance and hemocompatibility.  Superhydrophobic TNTs improve both hemocompatibility and corrosion resistance. Page 1 of 34

 Superhydrophilic amorphous TNTs only improve hemocompatibility.

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 Superhydrophilic anatase TNTs trigger coagulation but enhance corrosion resistance.

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ABSTRACT: Superhydrophilic and superhydrophobic TiO2 nanotube (TNT) arrays were fabricated on 316L stainless steel (SS) to improve corrosion resistance and hemocompatibility of SS. Vertically-aligned superhydrophilic amorphous TNTs were fabricated on SS by

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electrochemical anodization of Ti films deposited on SS. Calcination was carried out to induce

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anatase phase (superhydrophilic), and fluorosilanization was used to convert superhydrophilicity to superhydrophobicity. The morphology, structure and surface wettability of the samples were

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characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), X-ray

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photoelectron spectroscopy (XPS), and contact angle goniometry. The effects of surface wettability on corrosion resistance and platelet adhesion were investigated. The results showed

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that crystalline phase (anatase vs. amorphous) and wettability strongly affected corrosion

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resistance and platelet adhesion. The superhydrophilic amorphous TNTs failed to protect SS

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from corrosion whereas superhydrophobic amorphous TNTs slightly improved corrosion

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resistance of SS. Both superhydrophilic and superhydrophobic anatase TNTs significantly improved corrosion resistance of SS. The superhydrophilic amorphous TNTs minimized platelet adhesion and activation whereas superhydrophilic anatase TNTs activated the formation of fibrin network. On the contrary, both superhydrophobic TNTs (superhydrophobic amorphous TNTs and superhydrophobic anatase TNTs) reduced platelet adhesion significantly and improved corrosion resistance regardless of crystalline phase. Superhydrophobic anatase TNTs coating on SS surface offers the opportunity for the application of SS as a promising permanent biomaterial in blood contacting biomedical devices, where both reducing platelets adhesion/activation and improving corrosion resistance can be effectively combined. Keywords: 316L stainless steel; Superhydrophobic; Superhydrophilic; Platelet adhesion; Page 3 of 34

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Corrosion resistance

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1. Introduction It is well known that 316L stainless steel (SS) is one of the most common biomaterials owing to/by virtue of good hemocompatibility and corrosion resistance. However, when it is used as a

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permanent biomaterial such as vascular stent, released metal (Ni, Fe and Cr) ions can trigger

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coagulation and chronic inflammation leading to restenosis [1-3]. Coating a thin film on SS surface is a simple and effective way to avoid the intimate contact of SS with blood, and further,

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achieve improved corrosion resistance [1, 4-6] and biocompatibility [7, 8] without altering its

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mechanical properties.

A plethora of methods have been developed to create different kind of surface coatings. Many

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of these coatings such as diamond-like carbon [9, 10], amorphous hydrogenated carbon [11, 12],

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polyethylene terepthalate (PET), hyaluronic acid (HA) and titanium oxide (TiO2) [7, 13] are

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potentially suitable for biomedical use. Among these, TiO2 ceramic coating has been widely used

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due to its excellent hemocompatibility and low degree of corrosion and toxicity [14]. Nanostructured TiO2 such as TiO2 nanotube (TNT) has been extensively studied because of its high surface-to-volume ratio and the similarity to the physiological nanostructure of native bone [14, 15]. Multiple research studies show enhanced cell adhesion, proliferation and accelerated apatite formation on TNTs [16-20]. However, not much is known about hemocompatibility [21-24] or corrosion resistance [25-28] of TNTs, let alone TNT coatings on SS. Nevertheless, existing studies show that TNTs improve blood compatibility [22, 24] and corrosion resistance [25-28] of Ti. This is indicative of a promising application of TNT coatings on SS for biomedical use. Surface wettability is an integrated factor which affects both hemocompatibility and corrosion Page 5 of 34

resistance. Superhydrophilic (water contact angle below 5°) and superhydrophobic (water contact angle above 150°) surfaces attract extensive interest because of their potential applications [29], such as self-cleaning [30, 31], patterning template [32, 33] and protein/cell

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micropatterning [33-36]. TNTs are usually superhydrophilic [24, 35], and can greatly improve

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hemocompatibility or corrosion resistance as discussed above. On the other hand, superhydrophobic materials have also been reported to improve either hemocompatibility [24,

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37-40] or corrosion resistance [41, 42]. Thus, both superhydrophobic and superhydrophilic TNTs

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could be promising coatings for blood contact materials such as SS.

Our previous research results have shown that both superhydrophilic and superhydrophobic

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TNTs were able to improve blood compatibility of Ti [24]. Herein, we further test whether

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superhydrophobic or superhydrophilic TNT coatings on SS can improve both hemocompatibility

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and corrosion resistance. Superhydrophilic amorphous TNT films were fabricated by

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electrochemical anodization of Ti films which were deposited by DC sputtering on SS. Annealing was carried out to convert amorphous TNTs to anatase TNTs. Further superhydrophobicity was achieved by silanization of superhydrophilic TNTs. Corrosion behavior and platelet adhesion/activation of the samples were evaluated using electrochemical impedance spectroscopy (EIS) and polarization measurements and platelet adhesion test, respectively.

2. Materials and Methods 2.1. Superhydrophilic and superhydrophobic TNTs on 316L SS 316L SS sheets (10 mm × 10 mm) were degreased by sequential washes in acetone, anhydrous ethanol and deionized (DI) water using an ultrasonic bath, and then air-dried. Ti films were Page 6 of 34

deposited on SS substrates (SS-Ti) using DC sputtering technique. Sputtering was conducted from a Ti disk (99.995% purity, Jiangxi Haite Advanced Material Co., Ltd, Jiangxi, China) with chamber pressure lower than 1.8 × 10-3 torr before inflating with high purity argon. 130 ± 10 W

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DC power was applied to internal electronics for 60 min at room temperature.

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TNTs were fabricated by electrochemical anodization using a neutral electrolyte composed of 2.0% (wt) NH4F in glycerol with 2.5–3.5% (vol) of deionized water, at 20 V for 60 min, without

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stirring [43]. The resultant amorphous nanotubes (SS-TNT) were crystallized (SS-TNT-450) by

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annealing at 450 oC in air for 2 h. SS-TNT and SS-TNT-450 samples were optionally treated with a methanol solution of hydrolyzed 1% (wt) 1H,1H,2H,2H-perfluorooctyl-triethoxysilane

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(PTES, Degussa Co. Ltd.) for 1 h and subsequently heated at 140 oC for 1 h [44] to obtain

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superhydrophobic surfaces SS-TNT-PTES and SS-TNT-450-PTES, respectively.

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2.2. Structure and morphology characterization

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A field emission scanning electron microscope (SEM, Hitachi S4800) was used to characterize morphology of SS-Ti and SS-TNT, SS-TNT-450, SS-TNT-PTES and SS-TNT-450-PTES nanotube surfaces. X-ray diffraction (XRD) spectroscopy (Philips, Panalytical X'pert, Cu K radiation) was used to identify crystalline phases of nanostructured surfaces. Chemical composition was analyzed using X-ray photoelectron spectroscopy (XPS, VG, Physical Electrons Quantum 2000 scanning ESCA microprobe, Al Kα radiation). Binding energies were normalized to adventitious carbon at 285.0 eV. Static horizontal water contact angles (CA) were measured with an optical contact angle meter system (JC2000D, Powereach) at ambient temperature using DI water. Experimental data were represented as the average with standard deviations (SD) for N = 4. Page 7 of 34

2.3. Electrochemical measurements The electrochemical measurements were carried out in Tyrode solution (NaCl 8.00 g L-1, KCl 0.20 g L-1, CaCl2 0.20 g L-1, NaHCO3 1.00 g L-1, MgCl2 0.10 g L-1, NaH2PO4 0.05 g L-1, and

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Glucose 1.00 g L-1) using Autolab PGSTAT30 Electrochemical Measurement System in a

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three-electrode cell (saturated calomel reference electrode (SCE), platinum auxiliary electrode, and test sample as working electrode). Working electrodes were enveloped in nail polish with 0.5

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cm×0.5 cm exposed area. The EIS measurements were performed at open circuit potential. The

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applied frequencies were varied between 105 to 10-2 Hz using five points/decade. The impedance data was analyzed by Autolab analysis systems. Tafel polarization curves were measured

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between ±120 mV, at the open circuit potential at the rate of 0.167 mV s-1 and started after 40

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min immersion of samples in Tyrode solution. All experiments were executed at 37 oC. For

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superhydrophobic materials, an ultrasonic method in Tyrode solution was applied to remove

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trapped air from the interstices of nanotube surfaces. Experimental details for de-aeration have been previously detailed in reference [36]. Briefly, superhydrophobic samples were immersed in Tyrode solution and ultrasonication was applied for several seconds. 2.4. Platelet adhesion test

Fresh blood was obtained from adult New-Zealand rabbits, in citrate containing Vacutainers (Becton Dickinson, Franklin Lakes, NJ), in accordance with institutional policies. Platelet-rich plasma (PRP) was prepared by centrifugation at 1500 rpm for 12 min. PRP (100 l ) was placed onto coated (Section 2.1) and uncoated (control) SS samples held in a 24-well plate. PRP was kept in contact with the surface in a CO2 incubator (37 oC, 15% CO2) for selected time periods between one and two hours. At analysis time, samples were rinsed with PBS three times to Page 8 of 34

remove the physically attached platelets. After fixation in glutaraldehyde, and dehydration in a gradient solution of ethanol (0-100%), samples were dried in a critical point drier and sputtered with gold before SEM observation. The number of adherent platelets per unit area was counted

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from SEM images using five randomly chosen locations (N = 5). Number of adherent platelets

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was reported as the average value per unit area ± standard deviations (SD).

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3. Results and discussion 3.1. Surface characterization

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Fig. 1a and b show topographical and cross-sectional SEM images of Ti films deposited on 316L SS, respectively. Ti films are comprised of randomly-placed flakelets with a nominal

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thickness of ~1.6 m. Fig. 1c shows the top view SEM image of electrochemically anodized

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SS-Ti. A dense array of vertically aligned nanotubes with diameter of ~55 nm and wall thickness

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of ~5 nm grows from SS-Ti. The nanotubes are above 400 nm in length leaving approximately

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500 nm unmodified Ti underneath (Fig. 1d). Fig. 1e and 1f show topographical and cross-sectional SEM images of the TiO2 layer after annealing. Apparently, the annealing process thickens the nanotube wall (~10 nm) and decreases the tube diameter (~52 nm) correspondingly. Silanization of TNTs (SS-TNT-PTES or SS-TNT-450-PTES) does not change surface morphology (not shown).

XRD patterns of TNTs before and after annealing are presented in Fig. 2. The as-prepared TNT on SS is purely amorphous, and only peaks from Ti and SS can be observed. In contrast, strong diffraction peaks (101), (004), (200) and (220) corresponding to anatase crystalline phase (JCPDS No. 21-1272) are identified after calcination, indicating that amorphous TiO2 has been converted to anatase by annealing. Page 9 of 34

Fig. 3a and b compare XPS survey spectra and the high-resolution spectra of C 1s and F 1s of different samples, respectively. It is noteworthy that the intensities of the F1s and the FKLL auger signal increase greatly after PTES modification. The peak at 684.5 eV corresponds to F-

(294.1

eV)

appeared

in

the

high-resolution

spectra

of

SS-TNT-PTES

and

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-CF3

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residue from electrolyte. After calcination, this peak weakens greatly. Peaks -CF2 (291.8 eV) and

SS-TNT-450-PTES derive from PTES silanization. It is well known that the outermost of oxide

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film is covered by hydroxyl group and water vapor adsorbed on the hydroxylated layer by

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forming hydrogen-bonded network on the surface [45]. In order to investigate the effect of calcination on hydroxylated layer, the high-resolution spectra of O 1s for SS-TNT and

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SS-TNT-450 are curve-fitted by non-linear least squares fittings with a Lorentz–Gauss ratio

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using the XPSpeak fit software and shown in Fig. 3c and 3d, respectively. The O1s region could

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be decomposed into three peaks spaced ~1.2 eV apart. The primary peak at ~530.0 eV is

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attributed to Ti-O in TiO2. The peaks at ~531.2 and ~532.4 eV are assigned to chemical hydroxyl and physically adsorbed water (or other O-containing species), respectively. The atomic concentration of the above three components are shown in Fig. 3e. The results show that the amount of chemical hydroxyl group and physically adsorbed water decreases after calcination. It could be attributed to the consumption of hydroxyl group during the transformation of amorphous TiO2 to anatase [46, 47].

Wettability is a key factor that influences hemocompatibility [7, 48]. It is important to define the boundary between hydrophilic and hydrophobic materials. In this study, we follow the conventional definition that surfaces with CA smaller than 90° are considered as hydrophilic, and surfaces with CA larger than 90° are defined as hydrophobic. In particular, CA above 150° is Page 10 of 34

defined as superhydrophobic, and CA below 5° as superhydrophilic [49]. TiO2 is well known as a hydrophilic material due to hydroxyl group on oxide films [7, 45]. On the contrary, functional groups such as methyl and fluorocarbon are known to exhibit a hydrophobic character (low

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wettability) [50]. Static horizontal water contact angles of samples are collected in Fig. 4. After

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Ti thin film deposition on SS surface, the CA remains unchanged. However, after SS-Ti is anodized, the CA decreases from 77.4° ± 8.4° (Ti-coated SS, SS-Ti) to 3.8° ± 2.3°. It indicates

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that SS-TNT exhibits superhydrophilicity, which results from the combination of hydrophilicity

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of hydroxyl group and wicking property of the high rugosity of nanotubular structure [51]. Further annealing does not affect the superhydrophilicity of SS-TNT and maintains CA of 4.6° ±

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1.1° for SS-TNT-450. After silanization, CA increases dramatically to 152.2° ± 0.8° and 151.8°

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± 2.8° respectively for SS-TNT-PTES and SS-TNT-450-PTES, implying that both of them are

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superhydrophobic. The hydrophilicity of SS-TNT and SS-TNT-450 decreases over time by the

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deposition of alkane/organic contaminants [52], however, the superhydrophilicity could be retrieved by UV irradiation. SS-TNT-PTES and SS-TNT-450-PTES could be maintained in desiccator without changing superhydrophobicity for up to a year. 3.2. Corrosion resistance of superhydrophilic and superhydrophobic coatings It is widely accepted that trapped air is an important property of superhydrophobic materials [36, 42]. In this study, it was unable to collect stable EIS data due to air trapped within interstices of superhydrophobic nanotubes (Fig. 5a). Therefore, electrochemical tests of superhydrophobic materials were carried out after removal of trapped air by ultrasonic vibration. Fig. 5b shows Tafel polarization curves for different samples in Tyrode solution. By means of the analysis program of GPES software, the electrochemical parameters obtained from Tafel Page 11 of 34

curves are given in Table 1. The corrosion potential (Ecorr) of SS negatively shifts from -0.086 to -0.279 V for the superhydrophilic amorphous TNTs (SS-TNT). The corrosion density, Icorr, increases by more than 5 times (Table 1, compare row 2 to 1) and the corrosion resistance

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decreases by nearly 10 times. It indicates that the coated superhydrophilic amorphous TNT film

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is more susceptible to corrosion than SS in Tyrode solution. It could be ascribed to the high surface-to-volume ratio of nanotubular structure in which the practical surface area is much

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higher. However, Icorr reduces remarkably (10 times lower) along with enhanced corrosion

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resistance Rp (10 times higher) and positively shifted Ecorr after calcination, meaning that SS-TNT-450 enhances corrosion resistance of SS-TNT. The enhancement of corrosion resistance

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of SS-TNT-450 can be attributed to the conversion of the amorphous phase to a more stable

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anatase phase with larger crystalline size [26, 53]. Interestingly, after PTES silanization, Ecorr of

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the SS-TNT-PTES surface significantly shifts to the positive direction (-0.009 V) compared to

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that of SS-TNT, whereas Icorr decreases remarkably. Accordingly, superhydrophobic conversion highly improves corrosion resistance of superhydrophilic SS-TNT. Likewise, silanization increases Ecorr for SS-TNT-450-PTES while Icorr and Rp remain effectively unchanged. It has been reported that trapped air within the interstices of superhydrophobic nanostructure is an effective corrosion protector [42, 54]. In this study, trapped air have been removed by ultrasonication due to the low conductivity of superhydrophobic surfaces. Therefore, the improved

corrosion

resistance

of

superhydrophobic

surfaces

(SS-TNT-PTES

and

SS-TNT-450-PTES) might be ascribed to water repellency and physical diffusion barriers introduced by C-F bonds in the silanized layer that any hydrophilic molecules/ions, such as corrosive ions Cl-, are inhibited for entering into and contact with the surface [41, 55]. On the Page 12 of 34

other

hand,

superhydrophilic

anatase

SS-TNT-450

and

superhydrophobic

surfaces

(SS-TNT-PTES and SS-TNT-450-PTES) show reduced corrosion density and enhanced corrosion resistance compared to SS, indicating that superhydrophilic anatase TNTs and

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superhydrophobic TNTs provide good corrosion resistance. Among the modified samples,

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superhydrophobic amorphous TNTs improve the anticorrosion property of SS slightly while both superhydrophobic and superhydrophilic anatase TNTs enhance corrosion resistance more notably.

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It is worth pointing out that the corrosion resistance of superhydrophobic SS-TNT-PTES is lower

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than superhydrophilic SS-TNT-450, and consequently there is no direct correlation between corrosion resistance and surface wettability in this study.

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Corrosion resistance could be further confirmed by Nyquist plot as shown in Fig. 5c. The

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Nyquist diagrams are interpreted on the basis of two circuits as presented in Fig. 5d and e. Fig.

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5d shows the equivalent circuit model of unmodified SS surface which has one time constant,

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and Fig. 5e represents equivalent circuit model of modified SS surfaces. Rs is the resistance of solution and Rct value is an indicator of corrosion reaction occurring at the electrode/solution interface. Qct in parallel represents the constant phase element CPE. In the case of coated SS surface, a pair of Rf and Qf in parallel denotes the resistance and CEP of the coating. Fig. 5f compares Rct values of different samples. For superhydrophilic amorphous TNT, Rct reduces significantly compared to SS. However, this value increases dramatically after PTES modification (SS-TNT-PTES) or calcination (SS-TNT-450) and reaches the highest by combination of calcination and PTES modification (SS-TNT-450-PTES). The Rct value indicates total corrosion resistance performance and it is consistent with the Tafel polarization results. Hence, both superhydrophilic and superhydrophobic anatase TNT coatings could enhance Page 13 of 34

anticorrosion property of SS.

3.3. Platelet adhesion

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Platelet adhesion/activation is one of the primary indicators of hemocompatibility of

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biomaterials [7, 56, 57]. The platelet adhesion experiment on different samples was performed to preliminarily evaluate the blood compatibility. Adherent platelet shapes are classified into five

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categories according to activation [58, 59]: round, dendritic, spread dendritic, spreading and fully

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spreading. Fig. 6a-e shows the SEM images of adherent platelets on different surfaces after 1 h incubation. Abundant platelets adhere on SS surface (Fig. 6a-1), and the adherent platelets are

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mainly dendritic or spread dendritic (Fig. 6a-2). In contrast, the adherent platelet number on

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SS-TNT surface is significantly reduced (Fig. 6b-1) and the platelet shape is primarily dendritic

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(Fig. 6b-2), which is consistent with previous work [24]. It is remarkable that adherent platelets

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are barely found on PTES modified superhydrophobic surfaces (Fig. 6f, SS-TNT-PTES and SS-TNT-450-PTES) with highly suppressed round or dendritic shape (Fig. 6c-1, c-2 e-1, e-2). Interestingly, superhydrophilic SS-TNT-450 surface activates the formation of fibrin network into which platelets become entrapped (Fig. 6d). Two discernable fiber types, i. e., major and minor fiber type [60, 61], are distinguished. The major fiber type (Fig. 6d-2, A) is thicker and dominates the network. The minor type (Fig. 6d-2, B) cross-links the network into a threadlike thin net with the minor fraction distributed between the major fractions. Platelets become entrapped within this network, as shown in Fig. 6d-1. After 2 h incubation, adherent platelets display a dendritic morphology on SS and SS-TNT surfaces, whereas, adherent platelets remain a mainly round/dendritic morphology on superhydrophobic surfaces (not shown). Page 14 of 34

Fig. 6f compares the number of adherent platelets on different surfaces incubated for 1 and 2 h. Adherent platelet numbers on SS and SS-TNT increase with incubation time. On the contrary, there is no discernable change on SS-TNT-PTES or SS-TNT-450-PTES.

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A comparison of platelet adhesion and fibrin formation of different samples is presented in

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Table 2. SS-TNT and SS-TNT-450 exhibit similar superhydrophilicity (Fig. 4) but completely different platelet adhesion behavior (Table 2; compare column 2 to 3). We ascribe it to the

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annealing process which transforms amorphous (SS-TNT) into anatase crystalline (SS-TNT-450).

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Anatase crystalline phase is reported to be a stronger activator of the blood plasma coagulation cascade [23, 62]. Moreover, hydroxyl groups and associated water are reduced on anatase

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SS-TNT-450 in comparison to amorphous SS-TNT. It is speculated that even though lower

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density of hydroxyl group does not apparently change the superhydrophilicity of SS-TNT-450,

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hydrated outer layer with higher water content on SS-TNT is more difficult for proteins such as

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fibrinogen to adsorb to [63, 64]. Fibrinogen (Factor I) is one of the key factors that induces platelet aggregation and activation. Furthermore, the absorbed fibrinogen can be converted into fibrin fibers comprising the fibrin network through oligomerization [65]. Therefore, it is not surprising that fibrin network forms on SS-TNT-450 surface due to the reduced hydroxyl group and water content.

Reduced platelet adhesion and activation on superhydrophobic surfaces can be imputed to hydrophobic PTES coating (Fig. 4). Hydrophobicity of PTES molecular combined with vertically oriented nanotubular structure traps air within interstices of nanostructure and reduces the interaction of fibrinogen/platelet with surface [24, 63, 66]. The precise mechanism of hemocompatibility of superhydrophilic and superhydrophobic TNTs needs further investigation Page 15 of 34

to substantiate all of this speculation. 4. Conclusions Amorphous superhydrophilic TiO2 nanotube (TNT) arrays on 316L stainless steel (SS) were

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successfully fabricated through electrochemical anodization of Ti films deposited on SS. Anatase

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superhydrophilic TNT arrays were obtained by annealing amorphous superhydrophilic TNTs. Fluorosilanization was further used to convert superhydrophilicity to superhydrophobicity. The

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crystalline phase of TNTs on SS (anatase vs. amorphous) strongly affects platelet adhesion and

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fibrin formation in contact with platelet-rich-rabbit-plasma. Amorphous superhydrophilic TNT layers reduce platelet adhesion, but fail to protect SS from corrosion. An annealing process

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dramatically increases the anatase crystalline phase in surface layers which simultaneously

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increase corrosion resistance and potentiates the formation of fibrin. The superhydrophilic

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anatase TNT on SS implants may be a useful material in dental or orthopedic prosthetics.

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Superhydrophobic TNTs on SS improves both corrosion resistance and blood compatibility, and further extends the application of SS as a promising candidate in blood contacting biomedical devices.

Acknowledgements

Authors gratefully acknowledge financial supports from the National Natural Science Foundation of China (21321062) and the National Scientific Support Program of China (2012BAI07B09). Authors thanks Dr. Avantika Golas for a careful check of English language style and accuracy of the manuscript.

References Page 16 of 34

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Page 24 of 34

Table 1 Electrochemical parameters from Tafel curves for different electrodes Ecorr

Rp

nA∙cm-2

V (SCE)

MΩ∙cm2

SS

13.9

-0.086

2.91

SS-TNT

81.8

-0.279

0.38

SS-TNT-PTES

8.67

0.06

3.95

SS-TNT-450

5.65

0.09

7.26

SS-TNT-450-PTES

4.66

0.126

cr

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Icorr

10.2

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te

d

M

an

us

Samples

Page 25 of 34

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te

d

M

an

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cr

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Table 2 Summary of platelet adhesion and fibrin formation comparing to titanium surface Superhydrophilic Superhydrophobic Amorphous Anatase Amorphous Anatase Platelet adhesion ? ~2×lower ~10×lower ~10×lower Fibrin formation + Note: “?” denotes number of adherent platelet is unknown due to fibrin formation. “+” denotes significant fibrin whereas “-“ denotes firbin not detectable.

Page 26 of 34

Figure Captions Figure 1. Surface and cross-sectional SEM images of titanium coated 316L stainless steel (SS-Ti) (a, b), TiO2 nanotube (TNT) coated stainless steel (SS-TNT) (c, d) and anatase nanotube coated stainless steel (SS-TNT-450) (e, f).

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Figure 2. XRD spectra of SS-TNT (a) and SS-TNT-450 (b).

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Figure 3. (a) XPS survey spectra of samples. (b) The high-resolution spectra of C 1s and F 1s regions. (1) SS-TNT, (2) SS-TNT-PTES (SS-TNT silanized by PTES), (3) SS-TNT-450, (4) SS-TNT-450-PTES (SS-TNT-450 silanized by PTES). (c, d) The high-resolution spectra of O 1s for SS-TNT (c) and SS-TNT-450 (d), showing the O2-, OH and H2O components. (e) Relative contents of the O species in sample SS-TNT and SS-TNT-450 (atomic percentage according to XPS analysis).

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Figure 4. Water contact angle of stainless steel (SS), SS-Ti, SS-TNT, SS-TNT-PTES, SS-TNT-450 and SS-TNT-450-PTES.

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Figure 5. (a) Nyquist diagram of the SS-TNT-PTES with air trapped within interstices of nanotubes in Tyrode solution, at the open circuit potential, the fequency range is 105~10-2 Hz. (b) Polarization curves

d

for electrodes in Tyrode solution: (1) SS, (2) SS-TNT, (3) SS-TNT-PTES, (4) SS-TNT-450, and (5)

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SS-TNT-450-PTES. (c) Nyquist diagrams of the electrodes in Tyrode solution, at the open circuit

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potential, the frequency range is 105~10-2 Hz. (d, e) Equivalent circuit for the bare SS (d) and modified SS (e). (f) Charge transfer resistance Rct of the electrodes in Tyrode solution. Superhydrophobic SS-TNT-PTES and SS-TNT-450-PTES were treated with ultrasonication to get rid of air trapped within interstices before test.

Figure 6. SEM images of adherent platelets on the surface of SS (a-1, a-2), SS-TNT (b-1, b-2), SS-TNT-PTES (c-1, c-2), SS-TNT-450 (d-1, d-2) and SS-TNT-450-PTES (e-1, e-2). (a-2), (b-2), (c-2). (d-2) and (e-2) are magnified images of (a-1), (b-1), (c-1), (d-1) and (e-1) respectively. (f) Number of adherent platelets on different samples. **p