TiNi shape memory alloy coated with tungsten: a novel ... - LBMD

J Mater Sci: Mater Med (2014) 25:1249–1255 DOI 10.1007/s10856-014-5158-8

TiNi shape memory alloy coated with tungsten: a novel approach for biomedical applications Huafang Li • Yufeng Zheng • Y. T. Pei J. Th. M. De Hosson

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Received: 22 October 2013 / Accepted: 16 January 2014 / Published online: 31 January 2014 Ó Springer Science+Business Media New York 2014

Abstract This study explores the use of DC magnetron sputtering tungsten thin films for surface modification of TiNi shape memory alloy (SMA) targeting for biomedical applications. SEM, AFM and automatic contact angle meter instrument were used to determine the surface characteristics of the tungsten thin films. The hardness of the TiNi SMA with and without tungsten thin films was measured by nanoindentation tests. It is demonstrated that the tungsten thin films deposited at different magnetron sputtering conditions are characterized by a columnar microstructure and exhibit different surface morphology and roughness. The hardness of the TiNi SMA was improved significantly by tungsten thin films. The ion release, hemolysis rate, cell adhesion and cell proliferation have been investigated by inductively coupled plasma atomic emission spectrometry, CCK-8 assay and alkaline phosphatase activity test. The experimental findings indicate that TiNi SMA coated with tungsten thin film shows a substantial reduction in the release of nickel. Therefore, it has a better in vitro biocompatibility, in particular, reduced

H. Li Y. Zheng (&) State Key Laboratory for Turbulence and Complex System and Department of Materials Science and Engineering, College of Engineering, Peking University, Beijing 100871, China e-mail: [email protected] Y. T. Pei J. Th. M. De Hosson Department of Applied Physics, Materials Innovation Institute M2i and Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands Y. T. Pei (&) Department of Advanced Production Engineering, Institute for Technology and Management, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands e-mail: [email protected]

hemolysis rate, enhanced cell adhesion and differentiation due to the hydrophilic properties of the tungsten films.

1 Introduction TiNi shape memory alloys (SMAs) are widely used in biomedical devices and components because of their desirable properties, including good fatigue strength, relatively low elastic modulus, good formability, machinability, corrosion resistance and biocompatibility. Such SMAs have become indispensable for applications ranging from hard tissue replacements and dental materials to cardiac and cardiovascular devices such as stents, and are deployed in ever-increasing quantities in the medical field [1–4]. TiNi has proven being safe and biocompatible in many in vitro and in vivo studies due to the formation of its surface oxide film, which is mainly titanium oxide and prevents the nickel ion from corrosion and leaching. However, the medical profession still has concerns about this alloy because of its high nickel content of 50 % in the bulk [5, 6], which is a potential source of released nickel ion. The latter is known to induce hypersensitive reactions and tissue necrosis [7]. In addition, the poor osteoinductive properties of TiNi SMAs suggest that they are not optimized yet for biomedical applications. The smooth and hydrophobic surface properties of bare TiNi alloys do not afford a cell-friendly scaffold for certain physiological environments. Surface roughness is a key factor that influences the properties of cell adhesion and differentiation, and previous studies have demonstrated that the best design for an implant with desirable osseointegration is a surface with a high roughness [8]. Moreover the ‘‘wettability’’ of the implant surface has a strong influence on cell behavior in the initial

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osseointegration process [9]. In general, a hydrophilic surface displays a better affinity for cells than a hydrophobic surface [10]. For all these reasons, it is desirable to perform surface modifications on TiNi substrate to improve its mechanical, chemical, and osteoinductive properties. In order to further enhance the biocompatibility advanced thin films are usually employed on TiNi SMA [11–14]. On the other hand, surface energy and wettability, and its influence on cell adhesion and proliferation have been evaluated in some previous studies [15–17] and their results indicated that the hydrophilic surfaces with high surface energy are benefit for cell adhesion and proliferation. Tungsten (W) shows significant potential for this application, owing to its excellent biocompatibility, high mechanical strength and hardness as well as its high radiopacity. For this reason tungsten was explored as a versatile embolic material for the occlusion of various aneurysms and tumor-nourishing vessels [18]. In this study therefore we used magnetron sputtering to deposit W films on TiNi alloys intended for biomedical applications, and investigated the morphology, roughness, mechanical property, hydrophilic performance, ion release and biocompatibility of the TiNi SMA with and without tungsten coatings.

2 Materials and methods

J Mater Sci: Mater Med (2014) 25:1249–1255

received without W coating), Wrk–TiNi (TiNi coated with rough and thick W film as indicated by the subscript letters) and Wsn–TiNi (TiNi coated with smooth and thin W film). 2.2 Surface characterization and hardness test A scanning electron microscope (SEM, Philips XL-30s FEG) operating at 3 kV was employed to reveal the surface morphology and the growth microstructure and thickness of the films on fracture cross sections. Hardness, surface roughness, and atomic force microscopy (AFM) morphology of the films were studied using a TI 900 Triboindenter (Hysitron, USA) equipped with an AFM imaging option. Roughness parameters were calculated from the topographic profiles obtained through scanning probe microscopy conducted with the TI 900. Nanoindentation tests were performed on each sample using a diamond Berkovich indenter probe to determine the hardness. Nine samples were used for the roughness measurements and nanoindentation tests respectively. Contact angles for the TiNi alloy samples before and after W DC-pulsed magnetron sputtering were measured by means of the Automatic contact angle meter instrument (Model SL 200B Series, Kino Industry, USA) using CAST 2.0 software and following the Sessile method for the analysis of water droplets. Ultra-pure water was used as the wetting liquid, with drop sizes of 2 ll. Nine samples were used for the contact angle test.

2.1 Sample preparation 2.3 Ion release and hemolysis test W thin films were deposited on well polished Ti–50.8Ni (at.%) plates (10 9 10 9 1 mm3) with DC magnetron sputtering in a TEER UDP400/4 closed-field unbalanced magnetron sputtering system. The system was configured of four magnetrons; one of them was installed with W target (99.9 %) and the rest three were powered off. The magnetron with W target was powered by a Pinnacle 6 kW DC power supply (Advanced Energy) operating at 1.5 A current control. The substrates were biased at -40 V by a Pinnacle Plus 5 kW pulsed-DC power supply (Advanced Energy) operating at 250 kHz and 50 % duty cycle. The base pressure of the vacuum chamber before deposition was 3.0 9 10-6 mbar and the deposition pressure 3.45 9 10-3 mbar was maintained by a constant flow rate of argon gas. The TiNi substrates were clamped at 80 mm distance to the target. In order to produce W films with different surface morphologies, two deposition conditions were chosen: (1) the substrates were rotated at 3 rpm (revolution per minute) rotational speed of the sample carrousel for a deposition time of 120 min, forming a rougher and thicker W film; and (2) the substrates were kept still facing to the center of the target for a deposition time of 12 min, resulting in a smoother and thinner W film. Henceforth the samples corresponding to different conditions are referred as TiNi (as

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The inductively coupled plasma atomic emission spectrometry (Leeman, Profile ICP-AES) was employed to measure the concentrations of nickel and tungsten ions which had dissolved into Hank’s solution after 60 days immersion of the TiNi alloy samples. The hemolysis evaluation was conducted with healthy human blood from a volunteer containing sodium citrate (3.8 wt%) in the ratio of 9:1; normal saline solution was used as a negative control and deionized water as a positive control. Specific experimental process of hemolysis evaluation is elaborated in [19]. Six samples were used for ion release and hemolysis tests respectively. 2.4 Cell attachment and differentiation assay MG63 human osteoblast-like cells were used to evaluate cell response; the detailed culture process can be found in Ref. [20]. The initial attachment of cells was evaluated by CCK-8 assay. After 4 h of incubation, the cells were rinsed with PBS three times gently to remove unattached cells after which 1 ml of fresh cell culture medium was added. 10 ll CCK-8 was added to each well for a further 4 h incubation. The measurement was carried out spectrophotometrically


at 450 nm by enzyme-linked immunosorbent assay (ELISA) (ELx-800, bio-Tek instruments, America). Six samples were taken for each cell attachment measurement. To obtain results for the alkaline phosphatase activity (ALP) test, the culture medium was removed after 7 days of cultivation after which 900 ll/well 10 mM p-nitrophenylphosphate (Amresco, America) containing 2 mM MgCl2 in a carbonate buffer solution (pH 10.2) was added. After the sample had been incubated at 37 °C for 60 min, 100 ll/well of 1 M NaOH was added to terminate the reaction. The ALP activity was evaluated as the amount of p-nitrophenol (PNP) released through the enzymatic reaction and measured at a 405 nm wavelength using an ELISA (ELx-800) reader. For normalization, the total protein content was measured using a bicinchoninic acid (BCA) assay kit (Sigma). Thus, the ALP activity was expressed in nmol of PNP produced per minute per mg protein. For the ALP activity assay, six samples were taken for each group. 2.5 Statistical analysis Statistical analysis was conducted with SPSS 17.0. Differences between groups were analyzed using one-way

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ANOVA followed by Tukey test. A value of P \ 0.05 was considered to be statistically significant.

3 Results and discussion The SEM micrographs of plan view and cross sectional view in Fig. 1 show the surface morphology and growing microstructure of the W films coated on TiNi SMA substrates. As seen from Fig. 1a, b, the Wrk–TiNi film exhibited a sharp columnar microstructure with the growing surface of faceted and pyramid-like morphology. Due to the shadowing effects that are associated with the angular dependence of the atomic flux to the substrate in the closedfield configuration of magnetrons and rotational movement of sample carrousel [21], the groove network that surrounds the pyramids and intersects with the columnar boundary is rather deep and wide, providing a potential channel for outward diffusion of nickel ions. In contrast, by keeping the substrates still and facing to the center of the target where the flux density of the impinging Ar ions is much higher and the sputtered W flux reaches the growing film in its normal direction, this restrains shadowing and makes the growing

Fig. 1 SEM micrographs of top view and cross sectional view showing the surface morphology and growth microstructure of a, b Wrk film and c, d Wsn film coated on TiNi substrates

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surface of the Wsn–TiNi film rather smooth as seen in Fig. 1c, d. The columnar boundaries become diffused, though the Wsn–TiNi film is characterized by columnar microstructure. In addition, the Wrk–TiNi film is 1.85 lm in thickness and the Wsn–TiNi film is 1.20 lm thick although

Fig. 2 XRD spectra of Wrk film and Wsn film coated on TiNi substrates Fig. 3 2D and 3D AFM micrographs of Wrk film (a, b) and Wsn film (c, d) coated on TiNi substrates

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the deposition time was ten times shorter. X-ray diffraction spectra shown in Fig. 2 reveal a h110i growth texture of Im3m bcc phase in the Wrk–TiNi film but no texture is observed in the Wsn–TiNi film. Figure 3 shows the AFM micrographs of top view of Wsn film (a) and Wrk film (b) coated on TiNi substrates. From the AFM observation, it is clear that the Wsn film (a) is consisted of small spheres and the Wrk film (b) is consisted of pyramid-shaped grain. Similar to the SEM micrographs, it can be seen from Fig. 2 that the surface of the Wsn film (a) is much smoother than the Wrk film (b) and the diameter of the grain increased with the increasing deposition time. Figure 4a shows the roughness change of TiNi alloy before and after W coatings by magnetron sputtering. The average roughness (Ra) of TiNi substrate was 0.030 ± 0.005 lm, while the average roughness of the Wsn film and the Wrk film was considerably increased to 0.130 ± 0.010 lm and 0.403 ± 0.052 lm, respectively. There was a significant difference in roughness among the tungsten film groups and TiNi substrate (P \ 0.05). Surface roughness is a key factor that influences the properties of cell adhesion and differentiation [22, 23], and the appropriate design for an


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Fig. 4 a Roughness and b hardness of TiNi substrate, Wsn–TiNi and Wrk–TiNi samples, *P \ 0.05 compared with bare TiNi substrate group

implant with desirable osseointegration is a surface of rough microtopography. Figure 4b displays the hardness of the TiNi alloy and W film coated TiNi alloy. Unlike the uncoated TiNi alloy which showed a hardness of 5.2 ± 0.4 GPa, the hardness of the W coated specimens was 15.3 ± 0.3 GPa for the smooth-thin Wsn–film coated TiNi sample and 15.5 ± 0.4 GPa for the rough-thick Wrk–film coated TiNi sample, almost triple the hardness of the uncoated sample. It is anticipated that the wear resistance of W-coated TiNi SMAs will be effectively improved with enhancing the surface hardness of TiNi implants [24]. Water contact angle measurements (Fig. 5) indicated that the bare TiNi alloy exhibited a contact angle of 90.7 ± 1.0° and thus hydrophobic surfaces. After being coated with W films, the surfaces showed significantly enhanced hydrophilic behavior (P \ 0.05), with the contact angles being 35.2 ± 0.9° for the smooth Wsn–film coated sample and 23.9 ± 1.3° for the rough Wrk–film coated sample, respectively. Figure 6a shows the amount of Ni2? ion release into the Hank’s solution after the specimens were immersed for 60 days. The results showed that, with 1–2 lm thick W

Fig. 5 Water contact angle of bare TiNi substrate (a), Wsn–TiNi (b) and Wrk–TiNi (c) samples

film, the release of Ni2? ions can be greatly reduced (P \ 0.05), from 1.03 lg/ml (TiNi alloy)–0.02 lg/ml (Wsn–TiNi) and 0.01 lg/ml (Wrk–TiNi), respectively. After 60 days immersion, the dissolution quantity of Ni2? ion of the tungsten-coated samples was found to be fifty to one hundred times lower than that of the as-received TiNi alloy. It is considered that the columnar boundaries are the primary channel of Ni2? ions outwards diffusion [25]. Although the column boundaries in the smooth Wsn film are narrower and denser than those in the rough Wrk film (see Fig. 1c vs a), the diffusion of Ni2? ions is dominated by the length of the diffusion path (i.e. the thickness of the W films). The hemolysis rate of all the specimens are smaller than 5 % as shown in Fig. 6b, indicating that all these materials meet the criterion of biomedical materials (the hemolysis

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Fig. 6 a Nickel ion release into Hank’s solution after 60 days immersion, b hemolysis rate of TiNi alloys before and after W coating, *P \ 0.05 compared with bare TiNi substrate group

rate \5 % according to ISO 10993-4 [26] ). Among these materials, the TiNi alloy has the maximum hemolysis rate of 0.33 %. After coating with W thin films, the hemolysis rate was significantly reduced to 0.15 % and 0.13 % respectively (P \ 0.05). These results prove that the surface modification with W thin films can effectively restrain the release of Ni element, and thus reduce the hemolysis rate. The MG63 cell attachment and the ALP results are shown in Fig. 7a, b. From Fig. 7a, it can be seen that after 4 h incubation, the number of MG63 cells attached to W-coated surfaces was greater than that of cells on uncoated TiNi alloy surface (P \ 0.05). Moreover, the rough W-film sample had more MG63 cells attached than appeared on the smooth W-film sample. These cell adhesion results confirm that a rougher W film coated on the TiNi alloy results in a favorable surface for cell adhesion. In addition, it can be seen from Fig. 6b that the ALP activity of MG63 cells on W-film coated TiNi was significantly higher than that on uncoated TiNi alloy (P \ 0.05). The rough W-film coated TiNi sample showed the best ability to enhance ALP activity, in agreement with the cell attachment tests.

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Fig. 7 a Spectrophotometry of MG63 cell attachment and b ALP activity cultured on TiNi alloy surfaces before and after W coating, *P \ 0.05 compared with bare TiNi substrate group

It is well known that the surface roughness can significantly alter cell-material interactions [27]. Cell attachment and differentiation are primarily associated with the material surface characteristics, such as chemistry, topography, wettability, and surface energy [28]. Previous studies have shown that the highest level of cell attachment is on moderately hydrophilic surfaces [29]. TiNi surfaces with a high contact angle and a hydrophobic nature tend to show poor cell attachment compared with those hydrophilic surfaces. In contrast, the low contact angles measured on W film coated surfaces suggests higher surface tensions, which can contribute to improving cell attachment and proliferation on TiNi alloy surfaces.

4 Conclusions In summary, two tungsten thin films of different roughness were deposited on TiNi alloy with magnetron sputtering. The experimental results demonstrated that W films transformed the substrate from a hydrophobic surface to an increasingly hydrophilic surface with enhancing the roughness of the tungsten films. The hardness of the TiNi alloy


was significantly improved by tungsten coating. The amount of nickel ion release was substantially reduced by the tungsten films, and the hemolysis rate of W films coated TiNi alloy was lower than that of TiNi alloy substrate owing to the reducing nickel ion release. The differences in surface morphology and surface energy between uncoated TiNi and tungsten coated surfaces were found to have a direct influence on osteoblast cell adhesion, growth and differentiation. Tungsten thin films significantly improved cell-material interactions on the TiNi surfaces. From this study, we can draw the conclusion that surface modification of TiNi alloy with magnetron sputtered tungsten film can be employed to improve the hardness, surface roughness, wettability and biocompatibility performance of TiNi alloys for biomedical applications. Acknowledgments This work was supported by the National Basic Research Program of China (973 Program) (Grant Nos. 2012CB619102 and 2012CB619100), National Science Fund for Distinguished Young Scholars (Grant No. 51225101) and the China Exchange Programme of the Royal Netherlands Academy of Arts and Sciences (KNAW) (project ‘‘Surface modification of novel metallic biomaterials’’).

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