Surface characteristics and corrosion behaviour of WE43 magnesium alloy coated by SiC film

Applied Surface Science 258 (2012) 3074–3081

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Surface characteristics and corrosion behaviour of WE43 magnesium alloy coated by SiC film M. Li a , Y. Cheng a,∗ , Y.F. Zheng a,b,∗∗ , X. Zhang a , T.F. Xi a , S.C. Wei a,c a b c

Center for Biomedical Materials and Tissue Engineering, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871, China Department of Advanced Materials and Nanotechnology, College of Engineering, Peking University, Beijing 100871, China Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Peking University, Beijing 100871, China

a r t i c l e

i n f o

Article history: Received 6 September 2011 Received in revised form 7 November 2011 Accepted 10 November 2011 Available online 20 November 2011 Keywords: WE43 alloy SiC film PECVD Corrosion Hemocompatibility

a b s t r a c t Amorphous SiC film has been successfully fabricated on the surface of WE43 magnesium alloy by plasma enhanced chemical vapour deposition (PECVD) technique. The microstructure and elemental composition were analyzed by transmission electron microscopy (TEM), glancing angle X-ray diffraction (GAXRD) and X-ray photoelectron spectroscopy (XPS), respectively. The immersion test indicated that SiC film could efficiently slow down the degradation rate of WE43 alloy in simulated body fluid (SBF) at 37 ± 1 ◦ C. The indirect toxicity experiment was conducted using L929 cell line and the results showed that the extraction medium of SiC coated WE43 alloys exhibited no inhibitory effect on L929 cell growth. The in vitro hemocompatibility of the samples was investigated by hemolysis test and blood platelets adhesion test, and it was found that the hemolysis rate of the coated WE43 alloy decreased greatly, and the platelets attached on the SiC film were slightly activated with a round shape. It could be concluded that SiC film prepared by PECVD made WE43 alloy more appropriate to biomedical application. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Magnesium and its alloys may be promising candidates for biodegradable materials in bone tissue engineering and cardiovascular stents due to their suitable mechanical property, biocompatibility and bioabsorbability [1–3]. The Mg–Y–Nd system exhibited good mechanical and corrosion properties, and among them WE43 [4] is the most promising Mg-based alloys for stent strut materials [5]. Basically, a biodegradable cardiovascular stent is expected to provide a temporary mechanical support to prevent the arteries from closing up [6], presenting an effective solution to the in-stent restenosis and thrombosis of the current permanent metallic stents [7]. Encouraging results regarding to the biodegradable Mg alloy stents have been obtained in the animal experiment [8] and clinical human trials [4,9,10]. The results of the clinical trials indicate that no acute stent thrombosis occurred and acceptable local inflammation response was observed, and the stents could be safely degraded after 4 months [10]. However, there lies in a major limitation to the Mg alloys stents clinical practice. The initial degradation rate of Mg alloys in vivo is so rapid and localized, especially in physiological environment with pH value of 7.4–7.6 and biological fluid with chloride ions at levels on the order of

∗ Corresponding author. Tel.: +86 10 62753404; fax: +86 10 62753404. ∗∗ Co-corresponding author. E-mail address: [email protected] (Y. Cheng). 0169-4332/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2011.11.040

150 mmol/L [11]. Consequently, the fast degradation process of Mg alloy stents may result in large amounts of Mg2+ release, hydrogen gas (H2 ) accumulation, local alkalization, and mechanical integrity looseness [1,6]. Therefore, a prolonged degradation period of Mg alloy stents is required, so as to compromise their degradation and mechanical integrity during their service period [6]. A simple and useful way to solve these problems is to coat the Mg alloys with a higher corrosion-resistant and biocompatible material. With this aim, several surface modification methods have been used such as PECVD [12], magnetron sputtering [13], alkaline heat treatment [14], micro-arc oxidation [15], electrodeposition [16], sol–gel method [17], etc. In our study, silicon carbide (SiC) was chosen as the coating material because of its biocompatibility, bioinert to biological tissues and aggressive environment [18,19]. Stuart et al. [20] implanted SiC-coated quartz discs into the subcutaneous space of the New Zealand White rabbit and no chronic inflammatory response was obtained from histological diagnosis. Besides, the amorphous SiC films have fairly well anti-thrombogenic properties due to their semiconducting properties [21–24], and as such it is also a promising coating material for coronary stents [25]. Bickel et al. [26] examined the in vitro thrombogenicity of different coatings used for coronary stents and found that the silicon carbide coated coronary stent led to an improved hemocompatibility compared with the uncoated one. In the present study, amorphous SiC films have been successfully fabricated on WE43 alloys with the purpose to slow down

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was observed by scanning electron microscope (SEM; 1910FE, Amray Co. Ltd.), equipped with energy disperse spectrometer (EDS) attachment; the microstructure of the corrosion products was analyzed and characterized by the X-ray diffraction (XRD; DMAX-2400, Rigaku), respectively. XRD was performed using a Cu K␣ radiation ( = 0.154 nm), and the diffraction patterns were measured between 2 values 10–90◦ with a scanning speed of 4◦ /min. Hydrogen evolution experiment was conducted in accordance to Ref. [29], and the amount of hydrogen reveals the corrosion rate of the samples [30]. 2.4. Cytotoxicity evaluation

Fig. 1. Schematic diagram of PECVD apparatus for depositing SiC film on WE43 alloy.

the degradation rate and improve the hemocompatibility. The surface characteristics, in vitro corrosion behaviour, cytotoxicity and hemocompatibility were investigated. 2. Experimental 2.1. Preparation of the samples The extruded WE43 alloy (91.35 wt.% Mg, 4.16 wt.% Y, 3.80 wt.% RE, 0.36 wt.% Zr, 0.20 wt.% Zn, 0.13 wt.% Mn) was provided by Changchun Zhong-Ke-Xi-Mei Magnesium Alloy Co. Ltd., China, with an extrusion ratio of 10. It was cut into samples of Ø10 mm × 2 mm. Both sides of the surface were mechanically polished by silicon carbon paper up to 2000 grit, ultrasonically cleaned in acetone, alcohol and distilled water, respectively and dried at room temperature. SiC film was deposited on the prepared samples by PECVD technique with a radio frequency of 13.56 MHz and power of 80 W. The schematic diagram of this PECVD apparatus is shown in Fig. 1. Prior to deposition, the samples were etched in situ by argon plasma for 10 min at 250 ◦ C in order to completely remove the surface contaminants. SiH4 (99.999%) and CH4 (99.999%) were directly introduced into the reaction chamber with 1 sccm and 4 sccm gas flow rate, respectively. The deposition time was 30 min, with the substrate temperature maintained at 250 ◦ C and the working pressure at 25 Pa. 2.2. Surface microstructure and characterization The microstructural characteristics of the SiC films were investigated by transmission electron microscopy (TEM; Tecnai F30, FEI Co. Ltd.) operating at 300 kV accelerating voltage and the glancing angle X-ray diffraction (GAXRD; D8-Discover, Bruker Co. Ltd.) with ˚ at an incident angle of 1◦ , using Cu K␣ radiation ( = 1.540598 A) 40 kV, and the scanning rate is 6◦ /min. X-ray photoelectron spectroscopy (XPS; AXIS Ultra, Kratos Analytical Ltd.) was employed to identify the surface composition of the film. 2.3. Immersion test Immersion test was carried out at 37 ± 1 ◦ C in simulated body fluid (SBF) presented by Kokubo without organic species [27] according to ASTM G-31-72 [28]. The composition of the SBF are as follows: Na+ 142.0 mM, K+ 5.0 mM, Mg2+ 1.5 mM, Ca2+ 2.5 mM, HCO3− 4.2 mM, Cl− 147.8 mM, HPO4 2− 1.0 mM and SO4 2− 0.5 mM, which are similar to those of human blood plasma. The surface morphology of the samples before and after the immersion test

Cytotoxicity of SiC coated and uncoated samples to L-929 fibroblast line was evaluated by methyl thiazolyl tetrazolium (MTT) assay. The test was conducted by using an indirect method according to ISO 10993-5:2009 [31]. All the samples were sterilized by ultraviolet-radiation for at least 2 h, and then they were incubated in the DMEM serum free medium for 72 h in a humidified atmosphere with 5% CO2 at 37 ◦ C. After that, the supernatant culture solution was withdrawn and centrifuged to prepare the extraction medium, then refrigerated at 4 ◦ C before the cytotoxicity test. Cells were seeded at 1 × 104 cells/100 ␮L per well (96-well plates) and incubated in the Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U mL−1 penicillin and 100 ␮g mL−1 streptomycin in a humidified atmosphere with 5% CO2 at 37 ◦ C. Then the culture medium in each well was replaced with the prepared extracts supplemented with 10% fetal bovine serum after 24 h. In the control groups, DMEM medium was used as negative control and DMEM with 10% dimethylsulfoxide (DMSO) as positive control. After 24 h, 72 h and 120 h incubation, 10 ␮L MTT was added to each well for 4 h incubation, and then 100 ␮L of formazan solubilization solution was added to each well overnight. The measurement of the solution optical density (OD) was performed by a spectrophotometer (Elx-800, bio-Tek instruments) at 570 nm wavelength, with a reference wavelength of 670 nm. 2.5. In vitro hemocompatibility evaluation The in vitro hemocompatibility evaluation was investigated by hemolysis test and blood platelets adhesion test, using a method as previously described [32,33]. For hemolysis test, health human blood from a volunteer was mixed with sodium citrate (3.8 wt.%) in the ratio of 9:1 for resisting coagulation and the mixture was diluted with physiological saline at a volume ratio of 4:5. The SiC coated and uncoated WE43 alloys in triplicate were dipped in centrifuge tubes containing 10 mL of physiological saline, and they were incubated at 37 ◦ C for 30 min. Then 0.2 mL of saline diluted blood was added to these tubes respectively and the mixtures were incubated at 37 ◦ C for 60 min. As a positive control for hemolysis, 0.2 mL of blood was diluted in distilled water; whereas saline diluted blood was added to an empty tube served as a negative control. After this period, the samples were removed and all the tubes were centrifuged at 800 × g for 5 min. The supernatant from each tube was transferred to a cuvette where the absorbance was measured by ultraviolet spectrophotometer (UNIC-7200, China) at 545 nm. The hemolysis was calculated as follows: hemolysis=

OD(test) − OD(negative control) × 100% OD(positive control) − OD(negative control)

where OD = optical density at 545 nm. For blood platelets adhesion test, platelet-rich plasma (PRP) was prepared from citrated human blood by centrifuging at 200 × g for 15 min. The yellow supernatant was transferred to a tube, kept at

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Fig. 2. The TEM images of the SiC film with low (a) and high (b) magnification, the electron diffraction patterns (c) of the SiC film shown in (b) and the GAXRD patterns of the uncoated and the SiC coated WE43 alloys (d).

room temperature, and used within 4 h of preparation [34]. The experimental samples were covered by PRP and incubated at 37 ◦ C for 60 min. After that the samples were gently rinsed with phosphate buffer saline (PBS), and then fixed in 2.5% glutaraldehyde solutions for 60 min at room temperature followed by dehydration in graded ethanol/distilled water mixture from 50% to 100% with increasing by 10% for 10 min each and finally dried at room temperature for 2 days. The surfaces of samples attached by platelets were observed by SEM. 3. Results and discussion

4:1 during the deposition. Therefore, the probability of the carbon atoms bonded to carbon atoms was expected greater than that to silicon atoms in the carbon rich region [38]. The oxygen atoms were detected at the surface due to the presumably reaction of dangling bond with the atmospheric oxygen [37,38] when exposed the film to the ambient. The high-resolution XPS spectra of C1s and Si2p core levels are shown in the inset figures in Fig. 3. Both of the C1s (283.5 eV) [39] and Si2p (100.3 eV) [40] peaks are assigned to SiC. As a result, the XPS combined with the GAXRD analysis indicate that amorphous SiC film was successfully prepared on the WE43 alloy by PECVD.

3.1. Microstructure and composition of the SiC film It has been reported that SiC film can be deposited on different substrates at lower temperatures (usually 200–400 ◦ C) by PECVD and the microstructure of the prepared SiC film is usually amorphous [35,36]. The TEM images of the SiC film are presented in Fig. 2(a and b) and the electron diffraction patterns (Fig. 2(c)) of the SiC film (Fig. 2(b)) indicate that the film is in an amorphous state. It can be seen from Fig. 2(d) that the diffraction peaks for SiC are not observed in the coated samples. All the peaks of the two curves coincide with each other well, and correspond with the diffraction peaks for magnesium (PDF # 65-3365), so it can be deduced that an amorphous SiC film was obtained. Fig. 3 displays survey XPS spectra of SiC film, indicating that only Si, C, and O existing on the surface (at.% Si:C:O = 32.2:57.1:10.7). Moreover, the ratio of Si to C (Si:C = 0.56) is smaller than the stoichiometric value. Similar results were obtained in Ref. [37]. One possible explanation to the high content of C is that C–C bond is easier to form than Si–C [35] and the methane to silane ratio was

Fig. 3. Survey XPS spectra of SiC film. The C1s and Si2p core level spectra of SiC film are shown in the insert figures.

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Fig. 4. The pH value (left panel) of SBF incubating the uncoated and SiC coated WE43 alloys as a function of immersion time and the hydrogen evolution volume (right panel) of the uncoated and SiC coated WE43 alloys as a function of immersion time in SBF.

3.2. Corrosion behaviour of the SiC film coated samples The degradation rate of Mg alloy in aqueous solution at pH value below 11.5 can be predicted and monitored by hydrogen evolution reaction as below [41]: Mg + 2H2 O → Mg(OH)2 + H2

(1)

According to Eq. (1), the corrosion of one gram of metallic magnesium results in the production of 1 L of hydrogen gas. The hydrogen evolution experiment was conducted in triplicate with moderate reproducibility at a certain immersion time. The hydrogen evolution volume and pH value of SBF as a function of time are depicted in Fig. 4. It can be seen that hydrogen evolution of WE43 displayed approximately linear relationship with the immersion time, quite higher than that of the SiC coated one (p < 0.05). Contrary to the smooth and compact surface before the immersion test (Fig. 5(a, (1))), many deep and large pits can be observed on the corroded surface (Fig. 5(c)), which is quite undesirable for loadbearing implants, since it may cause local stress concentration and rupture. For the SiC coated WE43 alloy sample, it should be noted that the hydrogen evolution increases slowly at initial immersion time and keeps almost constant at low value after a certain period of time as illustrated in Fig. 4. Such slow degradation rate is just crucial for degradable implants to match the mechanical integrity, especially for stent which is made of very thin strut (about 70–150 ␮m). The protective efficiency of SiC film can also be verified by the morphology observed after the coated samples immersed in SBF at 37 ◦ C for 240 h. The degradation surface was relatively flat with small black pits (Fig. 5(4)) without detached corrosion products. Mildly localized corrosion product can be seen from SEM image (Fig. 5(d)). From the EDS analysis, the deposited corrosion product was composed of high concentration of O, Ca, P, Mg and Si elements, and this kind of degradation products can further slow down corrosion attack. From the results of the immersion test, it can be deduced that the mechanism of the corrosion for the SiC coated sample is as follows: due to the strong covalent bonding between silicon and carbon and its tetrahedral coordination, SiC film has been used as a protective coating to improve the lifetime or the performance of metallic substrates when exposed to aggressive environments. In the case of SiC coated magnesium, at the beginning of immersion, the bioinert SiC films can properly protect magnesium substrate

from corrosion. But with the increase of immersion time, SBF, especially the chloride ions, can diffuse into WE43 alloy substrate through the small defects, such as voids or micro cracks in the films formed during the deposition (as illustrated in Fig. 6(a-1)), then initial degradation corrosion happened. Fig. 6(b) shows the metallographic structure of the extruded WE43 alloy and as we known, the microstructure of the WE43 alloy consists of Mg-rich grains (␣ phase) and eutectic RE-rich Mg compounds (the second phase) [42]. Usually the second phases Mg24 Y5 and Mg12 Nd in WE43 alloy [43] have higher corrosion potentials than the Mg-rich ␣ phases, and the potential differences would lead to microgalvanic corrosion [33,44]. When the substrate Mg alloy was exposed to the aqueous solution, corrosion initiated in the ␣ phase with the production of Mg2+ ions, and spread over the grain boundaries with a thick white corrosion product precipitated on the surface, which was assumed to be Mg(OH)2 (as it is shown in Fig. 5(c(2)) and illustrated in Fig. 6(a-2)) [45,46]. The corrosion products Mg(OH)2 could be transformed partly into the easily soluble MgCl2 by Cl− ions in SBF, and therefore it is proposed that the high Cl− concentration in SBF accelerates the corrosion of magnesium alloys [47]. As the immersion experiment proceeded in SBF, biomimetic process was induced due to the highly supersaturated of the solution with respect to apatite [27] and the hydroxyapatite (HA) was precipitated on the undissolved Mg(OH)2 [44] and the SiC film. The hydroxyapatite formation would be accelerated with the increase of the solution alkalinity by absorbing the calcium and phosphate ions from SBF. The X-ray diffraction (XRD) analysis of the corrosion products are shown in Fig. 6(c), and both of the Bragg diffraction peaks for Mg(OH)2 (PDF # 44-1482) and HA (PDF # 09-0432) are observed. The resulting precipitated hydroxyapatite on the SiC film could retard the penetration rate of SBF through the SiC film defects and further slow down the corrosion rate of the substrate, due to its high thermodynamic structural stability [48] and its barrier action between the film defects and the outside aqueous environment (as it is illustrated in Fig. 6(a-3)). Moreover, the pH value exhibited a similar variation in comparison with the change of the hydrogen evolution volume. During the immersion process, the pH value of the SBF for the SiC coated sample firstly increased slowly and reached 8.5 after 100 h. While for the bare WE43 alloy, the pH value initially increased rapidly and kept unchanged about 10.5 h after 216 h, quite higher than the coated sample. This result indicates that SiC film can efficiently alleviate local alkalization after implantation.

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Fig. 5. The SEM images (from a to d) and the photos (from 1 to 4) of the uncoated WE43 alloys before (a, (1)) and after 240 h (c, (2)) immersion in SBF, and SiC coated one before (b, (3)) and after 240 h (d, (4)) immersion in SBF, and the EDS analysis (e) of the corrosion products on the surface of the SiC coated WE43 alloy (d).

Fig. 6. . Schematic diagram of the corrosion process of the SiC coated WE43 alloy in SBF (a), (a-1) the microstructure of the SiC film and WE43 substrate; (a-2) the initial corrosion process of the substrate; (a-3) the deposition of hydroxyapatite), the optical micrograph of the extruded WE43 alloy (b) and the X-ray diffraction patterns of the corrosion products on the SiC film (c).

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Fig. 7. Cytotoxicity of L929 cells cultured in the uncoated and SiC coated WE43 alloys extraction medium.

Fig. 9. Hemolysis percentage of the uncoated and SiC coated WE43 alloys. The insert SEM images show the morphology of blood platelets attached to the uncoated (a and b) and SiC coated (c and d) WE43 alloys with different magnification.

3.3. In vitro cell toxicity of SiC film coated samples

coated WE43 alloy showed no inhibitory effect to the growth of L929 cells.

Fig. 7 shows the L929 cell viability cultured in the uncoated and SiC coated WE43 alloys extraction medium for 1 day, 3 day and 5 day. The cells cultured in the SiC coated samples extraction medium showed higher absorbance than that of the negative control. Fig. 8 shows the optical micrographs of L929 cells that were cultured in negative control, uncoated and SiC coated WE43 alloy extracts, and positive control after 3 days incubation. The cells cultured in the negative control and SiC coated sample extracts exhibited healthy morphology of cells with a flattened spindle shape. Both the MTT test and microscopic observation indicated that the SiC

3.4. Hemocompatibility of SiC film coated samples Fig. 9 shows the hemolysis percentage of the uncoated (4.9 ± 2.0%) and SiC coated (0.6 ± 0.4%) WE43 alloys. Obviously, the SiC coated samples show a significant low hemolysis percentage compared to that of the uncoated one, which is quite lower than 5% proposed by ISO 10993-4 standard. This result indicates that amorphous SiC film can greatly improve the hemolysis property of WE43.

Fig. 8. Optical micrographs of L929 cells that were cultured in (a) negative control, (b) uncoated and (c) SiC coated WE43 alloys extracts, and (d) positive control for 3 days.

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Since in vitro morphology and attachment of the platelets have a close relationship with the in vivo thrombogenicity of materials, the platelet shape change can be used to indicate the thrombogenicity of materials [49]. According to the criteria proposed by Grunkemeie, the platelet morphology can be categorized into five types: round (R), dendritic (D), spread dendritic (SD), spreading (S) and fully spread (FS) [50]; and the morphologies of these five different stages of platelet activation are illustrated in Ref. [51]. Typical SEM images of the SiC coated and uncoated WE43 alloys with the adhesion of platelets are shown in the insert image in Fig. 9. The platelets on the uncoated WE43 alloy were at the inactivated stage with a round shape (R type) [52], and no spread dendritic platelets were observed, implying that WE43 has good antithrombotic properties. However few thin pseudopods were observed around the platelets on the SiC coated WE43 alloys indicating that they were slightly activated. Christopher [53] investigated the platelet adhesion on amorphous SiC, and non activated resting platelets were observed on the SiC film. The disparity of the platelets activating stage may be due to the different microstructure of the SiC film prepared by different preparation technology. The exact reason for the disparity is unclear and further experiments should be taken to clarify this problem. Although the platelets on the SiC film were slightly activated, they still exhibited a comparable round shape to those on the uncoated alloys and cannot be classified them into the D (dendritic) type platelets compared with the D type ones being illustrated in Ref. [51], which demonstrated that the amorphous SiC, prepared by PECVD method in our study, still exhibited well antithrombogenicity within the acceptable level. The results of the hemolysis test and blood platelets adhesion test suggest that amorphous SiC film can improve the blood compatibility, especially the hemolysis property, of the WE43 alloy.

4. Conclusions Amorphous SiC film was successfully deposited on the surface of WE43 magnesium alloys by PECVD method using CH4 and SiH4 . The immersion test result indicated that the SiC coated samples had much lower degradation rate than the uncoated ones. The indirect toxicity experiment results showed that the extraction medium of SiC coated WE43 alloys exhibited higher cell viabilities than the uncoated ones. The hemolysis rate of the coated WE43 alloy decreased greatly, and the platelets attached on the SiC film were slightly activated with a round shape within an acceptable level. Based on our preliminary study, it could be concluded that the amorphous SiC film prepared by PECVD may be a potential candidate for the application of magnesium stent.

Acknowledgements This work is supported by National Natural Science Foundation of China (no. 30870623, no. 31070846), National Basic Research Program (973) of China (no. 2009CB930004 and 2012CB619102) and National High Technology Research and Development Program of China (2011AA030103).

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