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Blood compatibilities of carbon nitride film deposited on biomedical NiTi alloy
Diamond & Related Materials 18 (2009) 1321–1325
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Diamond & Related Materials j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / d i a m o n d
Blood compatibilities of carbon nitride film deposited on biomedical NiTi alloy Jigang Wang a,⁎, Nan Jiang b a b
Jiangsu Key Laboratory of Advanced Metallic Materials, School of Materials Science and Engineering, Southeast University, Nanjing 211189, PR China Institute of Theoretical and Computational Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, PR China
a r t i c l e
i n f o
Article history: Received 4 January 2009 Received in revised form 20 March 2009 Accepted 8 July 2009 Available online 16 July 2009 Keywords: Carbon nitride Sputtering Biocompatibility Surface characterization
a b s t r a c t Carbon nitride (CNx) film, diamond-like carbon (DLC) film, and titanium nitride (TiN) film were deposited on biomedical NiTi alloy substrates using direct current magnetron sputtering, respectively. In order to improve the adhesive strength between the deposited hard film and the NiTi alloy, a Ti transition layer was pre-deposited firstly. We emphatically evaluated the blood compatibilities of the NiTi alloy substrate and the deposited hard films by haemolysis test and platelet conglutination test. It was shown that the blood compatibilities of NiTi alloy can be improved effectively by the deposition of hard films. In comparison with TiN and DLC film, CNx film had the best surface modification effects covering the minimum haemolysis ratio and the best anticoagulation property. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Biomedical materials are essentially a kind of special functional material that are used and adapted for a medical application. Biomedical materials must be compatible with the body, and there are often issues of biocompatibility which must be resolved before a product can be placed on the market and used in a clinical setting [1]. The metallic biomedical materials including stainless steel, pure titanium (Ti), Ti alloy (Ti6Al4V), and shape memory alloy had already been applied widely. Among the above clinical materials, NiTi alloys have gained much attention in biomedical application such as orthodontics and orthopaedics due to their shape memory effect and super-elasticity. In particular, NiTi alloy is characterized by a specific stress–strain diagram that is different from the deformation behavior of conventional materials but is similar to that of living tissues [2]. But in clinical field, the application of nickel (Ni)-containing materials is quite gingerly. Ni is the necessary nutrient element. But the excess Ni will cause anaphylactic and toxic reactions, even carcinogenesis [3]. Both the in vivo and in vitro test had already demonstrated the toxicity effects of high density Ni, e.g., Ni2+ can replace some divalent metals such as Ca2+, Mg2+, Zn2+ in enzyme and proteins. The in vitro test also demonstrated that there is a close relationship between the deformation of cell, the damage of chromosome, and the molar concentration of liberated Ni2+. In NiTi alloy, the percentage composition of Ni is high up to 50–55 wt.%. Ryhanen et al. [4] found that the liberation amount of Ni of untreated NiTi alloy is far higher than that of stainless steel. So, the liberation of Ni during the application of NiTi alloy is always a noticeable problem, and the surface modification is necessary to improve the ⁎ Corresponding author. Tel.: +86 25 52090628; fax: +86 25 52090669. E-mail address: [email protected] (J. Wang). 0925-9635/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.diamond.2009.07.003
biocompatibility. So far, the surface modifications including surface coating and thin film techniques are widely adopted technologies because they can improve the biological function and compatibility of the embedded materials, and had already been demonstrated to be effective research approaches [5]. For the diamond-like carbon (DLC) film, properties like high hardness, low coefficient of friction and extremely smooth surfaces make them good candidates for wear protection in tribological situations [6–10]. The fact that they are mainly based on carbon also makes them good prospects for biocompatibility. Some reports concerning the modification using titanium nitride (TiN) film was also attempted in the past [11,12]. Similar to DLC film, the carbon nitride (CNx) film also has a potential application in biomedicine due to their excellent hardness, tribological properties, chemical inertness, electrical and optical properties, and biocompatibility. Many studies have been made on the application of DLC film as biocompatible protective coatings [6–10]. However, only very few studies have been achieved on the CNx film [9,10,13]. In this study, CNx film was deposited on the surface of biomedical NiTi alloy using direct current magnetron sputtering. In order to improve the adhesive strength, a Ti transition layer was pre-deposited between the NiTi alloy and the hard film. The blood compatibilities of the NiTi alloy substrate and the deposited hard film were emphatically investigated using haemolysis test and platelet conglutination test, besides the investigation of the mechanical properties including hardness, adhesive strength between the NiTi substrate and deposited film. In order to compare and evaluate the modification effects of CNx film, TiN film and DLC film were also deposited and investigated using the same preparation and test methods. It was shown that the blood compatibility of NiTi alloy can be improved effectively by the deposition of hard films. Among the investigated hard films, CNx film had the best surface modification effects.
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2. Materials and methods
Table 1 Preparation parameters of the gradient films.
2.1. Treatment of NiTi alloy substrate
Parameters
The biomedical NiTi alloy with percentage composition of Ni 50.8 at.% and Ti 49.2 at.% was selected as the substrate. The starting temperature of martensitic phase transformation is −12.8 °C, and the finishing temperature of austenitic phase transformation is 33.4 °C. The NiTi alloy substrate was cut into plates with the size of 10 × 10 × 1 mm using electrospark wire-electrode cutting. The surface defects and contaminants were removed using step sanding by diamond abrasive paper. In order to get mirror surface, the plates were subsequently polished using water milling. In the end, the NiTi plates were successively washed by acetone, alcohol, and de-ionized water using ultrasonic cleaning at a given time of 15 min. Then the plates were dried and stored in dry oven. 2.2. The preparation of specimens The magnetron sputtering system with the type of JGP450A2 (Shenyang Tech. Ltd. Co., Chinese Academy of Sciences) was employed to deposit the gradient films. During the deposition process, circulating water was used to cool the sputtering system. The background degree of vacuum was 1.0 × 10− 3 Pa, and the working pressure was 0.5 Pa. The Ti transition layer was pre-deposited on NiTi alloy substrate for 10 min, and TiN, DLC, and CNx hard films were subsequently deposited for 3 h. The deposition parameters of Ti transition layer and surface hard films were listed in Table 1. All deposition temperatures were room temperature.
Sputtering gas Reaction gas Flow of Ar/N2 Target Substrate Sputtering power Bias voltage Sputtering time
Transition layer and hard films Ti
TiN
DLC
CNx
Ar / 30 sccm/0 Pure Ti NiTi alloy 150 W − 50 V 10 min
Ar N2 30 sccm/2 sccm Pure Ti Ti/NiTi 150 W −50 V 3h
Ar / 30 sccm/0 Pure graphite Ti/NiTi 150 W −50 V 3h
Ar N2 30 sccm/10 sccm Pure graphite Ti/NiTi 150 W −50 V 3h
the contact angles of specimens' surface. The operation procedures were expressed as follows: (i) The specimens were successively supersonically cleaned in acetone and de-ionized water for 10 min, and then dried. (ii) The globules with the volume of 10 µl were dropped on NiTi alloy and hard films' surface using micro-injector attaching to the contact angle measuring apparatus, respectively, and were laid aside for 3 min. (iii) The graphics of globules were then captured by camera and stored. (iv) By means of the software attaching to the contact angle measuring apparatus, the contact angles between globules and specimens' surface were measured, and the data were the average value of four tests under the same condition.
2.3.1. Characterization of film structure and test of mechanical performance The 750 typed Fourier transformer infrared (FT-IR) spectrophotometer (Nicolet Co. U.SA) was used to characterize the chemical bonds. The scanning electron microscopy (SEM) with the type of XL FEGSFEG-SIRION (FEI Ltd., Netherlands) was employed to investigate the structural morphologies of the deposited hard films. The indentation method was usually adopted for the test of hardness due to its unique simplicity and economy [14,15]. In our work, a microhardness tester with the type of HVS-1000 (Laizhou Huayin test instrument Co., China) was used to measure the hardness of the deposited films. The indenter used was a rectangular pyramid diamond with the interfacial angle of 136°. A normal load of 0.245 N (25 gram-force) was applied to produce a tetragonal pyramidal indentation with two clear diagonal indentations on the smooth surfaces of the specimens. The diagonal lengths of the indentations were immediately measured using the microscope attaching to the micorhardness tester. The hardness was derived from the following equation:
2.3.3. Haemolysis test The anticoagulant blood was prepared firstly by the addition of sodium citrate into the fresh human blood. The concentration of sodium citrate was 3.8%, and the volume ratio between fresh blood and sodium citrated was 8:1. The diluted blood was subsequently prepared by mixing the anticoagulant blood and physiological salt water (with concentration of 0.9%) with the volume ratio of 4:5. The NiTi alloy substrate and deposited gradient films were immersed in the physiological salt water and placed in the water-bath at 37 °C for 30 min. The volume of physiological salt water was 10 ml. The diluted blood with the volume of 0.2 ml was successively added into the physiological salt water, and the blended liquid was continuously placed in water-bath for 60 min. Then the blended liquid was centrifugally separated with rotational speed of 1000 r/min for 5 min. The separated supernatant liquor was picked up and was then moved into a clear 96-welled microplate, and was finally measured by enzyme-linked immunosorbent assay (ELISA R&D Systems, Minneapolis, USA). The absorbance optical density of supernatant liquor in each well was read on a microplate reader (CliniBio 128C; Salzburg, Austria). The measuring wavelength was 550 nm. The negative check experiment was carried out using 10 ml physiological salt water and 0.2 ml diluted blood. The positive check experiment was carried out using 10 ml distilled water and 0.2 ml diluted blood. The haemolysis ratio is calculated by Eq. (3)
d = ðd1 − d2 Þ × 0:3
Haemolysis ratio =
2.3. Microstructure characterization and property test
HV =
1845 × Fload d2
ð1Þ ð2Þ
where, d1 and d2 are the lengths of the indentation's diagonals (in unit of µm). HV, vickers hardness, is in the unit of GPa. The adhesive strength between the deposited films and the NiTi alloy substrate was tested by WS-2005 typed automatic scratch tester (Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences). The films' hardness and adhesive strength were determined by averaging four test results. 2.3.2. The measurement of surface contact-angles A Jc 2000 B typed contact angle measuring apparatus (Shanghai Zhongchen Digital Tech. Instrument Co. China) was employed to measure
Dt − Dnc × 100k Dpc − Dnc
ð3Þ
where, Dt, Dnc, and Dpc are the absorbance values of specimens, negative check experiment, and positive check experiment, respectively. 2.3.4. Platelet conglutination test The fresh anticoagulant human blood was centrifugally separated at a speed of 1000 r/min for 10 min. The rich platelet-containing upper plasma was picked up using pipette at room temperature and was then placed in a clean beaker. The pure NiTi alloy substrate and the deposited specimens were respectively immersed in the above rich platelet-containing plasma. After 3 h incubation in a thermostat water bath, the rich platelet-containing plasma was removed by pipette. The NiTi alloy as well as the deposited specimens was subsequently immersed in phosphate buffered saline (PBS) for 10 min,
J. Wang, N. Jiang / Diamond & Related Materials 18 (2009) 1321–1325
and then the PBS was removed. The cleaning operations using PBS were repeated for 2 or 3 times, until all un-conglutinated platelets were removed. Then the surface morphologies were fixed by glutaraldehyde with the concentration of 25% for 12 h at room temperature. Then the specimens was successively dehydrated and dealcoholed by alcohol and acetic isoamyl ester for 10 min, respectively. The concentration gradient of alcohol and acetic isoamyl ester were 50, 75, 95, and 100%. Then the specimens were dried using HCP2 typed critical point drying instrument (Hitachi Co. Japan). After the treatment of gold spraying, the amount and deformation of platelets conglutinated on the dried specimens' surfaces were investigated using SEM apparatus.
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Table 2 Comparison of hardness and adhesive strength of the deposited films. Samples
Ti/TiN film
Ti/DLC film
Ti/CNx film
Hardness (GPa) Adhesion force (N)
24.0 82.3
18.9 37.5
23.1 63.2
3. Results and discussion
737.8 cm−1, corresponding to the in-plane rotation of six-fold rings in graphite layers, is commonly observed in the Raman spectra. In contrast, such band is often observed in N-containing films because N has the enhanced sp2 clustering [21,27]. The bands at 2923.1 and 2851.2 cm−1 can be attributed to the absorption of C–H bond, in which the hydrogen is derived from the absorption of water-vapor of CNx film in the atmosphere [28].
3.1. The microstructure analysis of deposited CNx film
3.2. The comparison of mechanical properties of the deposited films
Fig. 1 shows the IR spectrum of the CNx film. The absorption band at 2200 cm− 1, corresponding to the triple C≡N stretching mode, has been reported until recently [16–20]. In those previous reports a broad band having a single absorption peak has been observed for almost all of the CNx films. Usually, the absorption peaks of C–N single-bond and CfN double-bond are overlapping with that of CfC double-bond, whereas the peak of C≡N triple-bond is independent. So, the appearance of C≡N triple-bond is the characteristic identification of carbon–nitrogen structure existing in the CNx film. On the other hand, the bands in the zone of 1100–1700 cm− 1, especially around 1300 and 1530 cm− 1, are associated with the stretching modes of sp2 and sp3 hybrid C in graphitic and disordered structures, as well as with the formation of sp3 C–N and sp2 CfN bonds [21]. These bonds are normally IR-invisible. However, their contributions to the spectra are the result of the N incorporation in the graphitic rings, which distorts its planar geometry [21]. It can be seen from Fig. 1 that the peak groups at 1110.9–1297.7 cm− 1, which represent the characteristic sp3 C–N bond's absorption, are clearly shown in the IR spectra. According to the theoretical calculation, a characteristic peak will be appeared at approximate 1250 cm− 1 if the C and N atoms are bonded in the mode of β-C3N4 crystal [22]. But this peak usually cannot be observed due to the influence of strong broad band of sp2 hybrid structure [23]. Among these peaks shown in Fig. 1, the peak at 1251.5 cm−1, the characteristic absorption of sp3 C–N bond in β-C3N4 crystal, is clearly shown. So, one can presume that some β-C3N4 microcrystals exist in the deposited CNx film. The bands at 1379.1 and 1530–1570 cm−1 are the sensitized sp2 hybrid structure in disordered and ordered states, respectively. A main absorption band centered at 1616 cm−1 is observed, which is attributed to the CfN vibrations [24,25] exiting in aromatic system or conjugated structure [26]. And the band at 1463.8 cm−1 is characteristic absorption of sp3 C–N bond in non-crystal. A band at
As expressed in Section 2.2, the substrate is biomedical NiTi alloy, on which the Ti transition layer is previously deposited. The hard films including CNx, DLC, and TiN were subsequently deposited, respectively. The thickness of Ti transition layer is around 486 nm after being deposited for 10 min. And the thickness of DLC, CNx, TiN are 1.64, 1.71, and 1.77 µm, respectively, after beding deposited for 3 h. Table 2 exhibits the vickers hardness and adhesive strength of the deposited films. Generally, the hardness of commercial TiN products is approximately 16–20 GPa. The hardness of TiN film obtained by Park J H et al. [12] were approximately 18 and 20 GPa using low and high radio frequency plasma-assisted metal-organic chemical vapor deposition system, respectively. In our work, the hardness of Ti/TiN and Ti/DLC gradient films are 24 and 18.9 GPa, respectively. As to the CNx films, the experimentally achieved CNx films are amorphous, which are embedded with small amount of micro-crystals. So, the hardness of the synthesized CNx films is obviously lower than the theoretically obtained value. The data reported in references are usually around 20–30 GPa [29–31]. In our work, the hardness of Ti/ CNx gradient film is 23.1 GPa, which is close to that of TiN film, and is somewhat higher than that of DLC film. The hardness and elastic modulus of NiTi alloy substrate are about 3.5 and 63.4 GPa [7]. Therefore, the deposition of gradient films can effectively improve the surface hardness of NiTi alloy. It also can be seen from Table 2 that the adhesive strength between Ti/CNx gradient film and NiTi alloy is only 37.5 N, while the adhesive strength between Ti/TiN gradient film and NiTi alloy is high up to 82.3 N. The purpose of the pre-deposition of Ti transition layer is to reduce the interface stress that is derived from the mismatch of crystal lattices, and the difference of elastic modulus between the NiTi alloy substrate and the deposited films. In the case of the deposition of Ti/ TiN gradient film, the elastic modulus, crystal lattice parameters are more matchable with those of NiTi alloy substrate. So, the adhesive strength between Ti/TiN gradient films and NiTi alloy substrate is the strongest among the studied three specimens. As far as Ti/CNx film is
Fig. 1. IR spectrum of CNx film.
Fig. 2. Measurement results of surface contact angles.
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Table 3 Haemolysis ratios of specimens. Samples
Optical density (550 nm)
Haemolysis ratios (%)
The NiTi alloy The Ti/TiN film The Ti/DLC film The Ti/CNx film Positive check Negative check
0.234 ± 0.008 0.217 ± 0.009 0.226 ± 0.010 0.208 ± 0.005 2.240 ± 0.050 0.185 ± 0.010
2.39 1.58 1.99 1.12 – –
concerned, the residual stress at the interior of CNx film is decreased, owing to the transformation of carbon hybrid from sp3 to sp2 with the introduction of nitrogen element [32]. So, in comparison with Ti/DLC gradient film, the adhesive strength between Ti/CNx gradient films and NiTi alloy substrate is stronger (with value of 63.6 N). 3.3. The comparison of the blood compatibilities
of surface contact angle, the surface energy of materials can be deduced. As a result, the platelet conglutination is closely related to the surface contact angle. Tamada and Ikada [33] have already studied the platelet conglutination on polythene surface, and found that the amount of the conglutinated platelets was increased with the elevation of surface contact angles. As shown in Fig. 2, the surface contact angle of untreated NiTi alloy is 50.77°, which is lower than those of Ti/TiN and Ti/DLC (68.24 and 72.32°, respectively). In Refs. [34] and [8], the contact angles of TiN and DLC films are 68.78 and 80–100°, respectively, are close to our results. Since the contact angles of Ti/TiN and Ti/DLC gradient films are larger than 65°, which is the crossover between hydrophilic materials and hydrophobic materials, these films belong to hydrophobic materials. It also can be seen from Fig. 2 that the contact angle of Ti/CNx gradient films is the minimum with value of 32.68°. The achievement of satisfactory hydrophilicity of CNx film can be attributed to the formation of C–N, CfN, especially C≡N bond [35]. The improvement of hydrophilicity will result in the decrease of free energy of deposited film, leading to satisfactory anticoagulation property. So, it is predictable that the CNx film possesses the best anti-thrombus property which will be demonstrated in the following discussion.
The biomedical materials with satisfactory blood compatibility should have excellent anticoagulation property besides conventional compatibility requirements. The embedded materials should neither cause the platelets to be deformed and collapsed, and nor have toxicity on the blood. In general, the blood compatibilities will involve many biological reactions when the biomedical materials contact directly or indirectly with the blood. The detailed biological reactions include the conglutination, aggregation, deformation of platelets, as well as the adsorption of plasma protein. The materials with unsatisfactory blood compatibilities will cause coagulation, activation of fibrinolytic system, and eventually resulting in thrombus. In the following subsection, the blood compatibilities of the NiTi alloy and the hard films were investigated and evaluated from the aspects of the conglutination, aggregation, and deformation of platelets.
3.3.2. The comparison and evaluation of films' haemolysis ratios As shown in Table 3 that the haemolysis ratios of all specimens are smaller that 5%, indicating that all these materials meet the criterion of biomedical materials (the haemolysis ratio is lower than 5%). Among these materials, the NiTi alloy has the maximum haemolysis ratio of 2.39%, and will damage the erythrocyte to some extent. From Table 3 one can also find that the haemolysis ratios of Ti/TiN, Ti/DLC, and Ti/ CNx films are 1.58, 1.99, and 1.12%, respectively, which are obviously lower than that of the NiTi alloy (2.39%). These results indicate that the surface modification can effectively restrain the release of Ni element, and consequently reducing the haemolysis ratio.
3.3.1. The measurement of surface hydrophilicity of the deposited hard films The surface energy of biomedical materials has some effects on the platelet conglutination level. By means of the measurement and analysis
3.3.3. The comparison and evaluation of platelet conglutination The conglutination and activation of platelets on the materials surface will lead to the coagulation reaction, and subsequently result in the release of procoagulant substance [36]. By means of SEM, the
Fig. 3. SEM morphologies of platelet conglutination on the samples' surface (5000×). (a) NiTi alloy (b) Ti/TiN (c) Ti/DLC (d) Ti/CNx.
J. Wang, N. Jiang / Diamond & Related Materials 18 (2009) 1321–1325
conglutination phenomena of platelets, such as the conglutination density and deformation level, on the NiTi alloy and the deposited films can be investigated, and the blood compatibilities can consequently be compared and evaluated. The smaller the conglutination density and deformation level are, the better the blood compatibility will be. Fig. 3 shows the platelet conglutination morphologies of the NiTi alloy and Ti/TiN, Ti/DLC, and Ti/CNx gradient films with magnification ratio of 5000 times (5000×). Many platelets are conglutinated on the surface of NiTi alloy, and obvious aggregation phenomenon also can be easily found (Fig. 3a). In such field of vision, no separate platelets can be found due to the serious aggregation. Nearly all platelets are linked with each other by lots of stretching dendritic pseudopods, and the platelets are deformed in a large degree. While in the case of deposited films, surface modification effects including the decreased amount and homogeneous distribution of conglutinated platelets are achieved, especially for Ti/TiN and Ti/CNx films. As far as the Ti/TiN film is concerned, although the conglutinated platelets are very few, they are aggregated to some extent (Fig. 3b). As to the Ti/ DLC film (Fig. 3c), there is no obvious aggregation. However, the conglutinated platelets on the Ti/DLC film are obviously more than those on the Ti/TiN film. In addition, the platelets on Ti/DLC film are deformed in a larger degree than those on Ti/TiN film. As shown in Fig. 3c, most of platelets stretched out pseudopods, even some cellular stroma can be found between the pseudopods. It is worth noting that, among the deposited films, the amount of the conglutinated platelets on Ti/CNx film is the minimum (Fig. 3d), with no aggregation being found. The original shape of the platelet is well maintained, without the stretched pseudopods. It is demonstrated that the Ti/CNx film has the best blood compatibility due to its best effects on the inhibition of conglutination and activation of platelets. In a word, the conglutination and deformation of NiTi alloy is quite serious, while the surface modification by deposited gradient films can improve the blood compatibility. Among the above tested gradient films, the Ti/CNx film has the best anticoagulation property, as indicated by both the minimum amount of conglutinated platelets and the least deformation. 4. Conclusions Ti/TiN, Ti/DLC, and Ti/CNx gradient films were deposited on the NiTi alloy substrate using magnetron sputtering. The mechanical properties and blood compatibilities of the biomedical NiTi alloy substrate and the above gradient films were investigated and compared. It was concluded that the Ti/CNx gradient film had the best surface modification effect. It is conceivable that CNx has a great potential as a very attractive coating material for biomedical applications in the future. (i) The deposition of Ti interlayer realized the satisfactory combination between NiTi alloy and hard films. In the case of Ti/CNx film, satisfactory mechanic properties were achieved with the surface hardness of 23.1 GPa and the adhesive strength of 63.6 kN.
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(ii) The surface contact angle of CNx film was only 32.68°, and the deposition of CNx film can improve the surface hydrophilicity of NiTi alloy. In comparison with CNx film, TiN and DLC films belong to the hydrophobic materials with the contact angles of 68.24 and 72.32°, respectively. (iii) CNx film had the best blood compatibilities. Compared with the TiNi alloy and other deposited films, CNx films had the minimum haemolysis ratio of 1.12%. In addition, the amount and the deformation level of conglutinated platelets were the least. References [1] D.F. Williams, J. Black, P.J. Doherty, in: P.J. Doherty, R.L. Williams, D.F. Williams, A.J.C. Lee (Eds.), Biomaterial-tissue interfaces, vol. 10, Elsevier, Amsterdam, 1992, p. 525. [2] T. Duerig, A. Pelton, D. Stockel, Mater. Sci. Eng. A 273–275 (1999) 149. [3] F. Villermaux, M. Tabrizian, L. Yahia, G. Czeremuszkin, D.L. Piron, Biomed. Mater. Eng. 6 (1996) 241. [4] J. Ryhanen, E. Niemi, W. Serlo, E. Niemela, P. Sandvik, H. Pernu, et al., J. Biomed. Mater. Res. 35 (1997) 451. [5] X.Y. Liu, P.K. Chu, C.X. Ding, Mater. Sci. Eng. R 47 (2004) 49. [6] J.H. Sui, W. Cai, Diamond Relat. Mater. 15 (2006) 1720. [7] J.H. Sui, W. Cai, L.C. Zhao, Nucl. Instrum. Methods B 248 (2006) 67. [8] Y. Yin, L. Hang, J. Xu, D.R. McKenzie, M.M.M. Bilek, Thin Solid Films 516 (2008) 5157. [9] F.Z. Cui, D.J. Li, Surf. Coat. Technol. 131 (2000) 481. [10] S.E. Rodil, R. Olivares, H. Arzate, S. Muhl, Diamond Relat. Mater. 12 (2003) 931. [11] Y. Cheng, Y.F. Zheng, Surf. Coat. Technol. 201 (2007) 6869. [12] J.H. Park, C.K. Jung, D.C. Lima, J.H. Boo, Tribol. Int. 40 (2007) 345. [13] H. Sjöström, L. Hultman, J.E. Sundgen, S.V. Hainsworth, T.F. Page, G.S.A.M. Theunissen, J. Vac. Sci. Technol. A 14 (1996) 56. [14] J. Musil, H. Zeman, F. Kunc, J. Vlček, Mater. Sci. Eng. A 340 (2003) 281. [15] S. Chowdhury, M.T. Laugier, I.Z. Rahman, Diamond Relat. Mater. 13 (2004) 1543. [16] S.L. Sung, T.G. Tsai, K.P. Huang, J.H. Huang, H.C. Shih, Jpn. J. Appl. Phys. 37 (1998) L148. [17] W.T. Zheng, H. Sjostrom, I. Ivanov, et al., J. Vac. Sci. Technol. A 14 (1996) 2696. [18] M. Therasse, M. Benlahsen, Solid State Commun. 129 (2004) 139. [19] S. Kennou, S. Logothetidis, L. Sygellou, A. Laskarakis, D. Sotiropoulou, Y. Panayiotatos, S. Kennou, et al., Diamond Relat. Mater. 11 (2002) 1183. [20] L.Y. Chen, C.Y. Cheng, F.C.N. Hong, Diamond Relat. Mater. 11 (2002) 117. [21] J.H. Kaufman, Phys. Rev. B. 39 (1989) 13053. [22] M.R. Wixom, Ceram. Soc. 73 (1990) 1973. [23] D. Li, S. Lopey, Y.W. Chung, M.S. Wong, J. Vac. Sci. Technol. A13 (1995) 1063. [24] J.W. Robinson, Practical Handbook of Spectroscopy, CRC. Press, Boston, MA,1991, p. 551. [25] P. González, R. Soto, E.G. Parada, X. Redondas, S. Chiussi, J. Serra, J. Pou, B. León, M. Pérez-Amor, Appl. Surf. Sci. 109–110 (1997) 380. [26] C.B. Cao, Q. Lv, H.S. Zhu, Diamond Relat. Mater. 12 (2003) 1070. [27] N. Nakayama, Y. Tsuchiya, S. Tamada, et al., Appl. Phys. 32 (1993) 1465. [28] S.R.P. Silva, J.G.A. Robertson, et al., Appl. Phys. 81 (1997) 2626. [29] P.Y. Tessier, L. Pichon, P. Villechaise, P. Linez, B. Angleraud, N. Mubumbila, et al., Diamond Relat. Mater. 12 (2003) 1066. [30] D. Li, Y.W. Chung, S.T. Yang, M.S. Wong, F. Abidi, W.D. Sproul, J. Vac. Sci. Technol. A 12 (1994) 1470. [31] H.S. Myung, Y.S. Park, B. Hong, J.G. Han, L.R. Shaginyan, Thin Solid Films 506–507 (2006) 87. [32] C.B. Wang, S.R. Yang, J. Zhang, J. Non-Cryst. Solids 354 (2008) 1608. [33] Y. Tamada, Y. Ikada, Polymer 34 (1993) 2208. [34] L. Yin, T.W. Guo, W. Zhao, Y. Yue, Y.Y. Wang, Chin. J. Prosthodont. 8 (2007) 287. [35] H. Riascos, J. Neidhardt, G.Z. Radnóczi, J. Emmerlich, G. Zambrano, L. Hultman, et al., Thin Solid Films 497 (2006) 1. [36] Y.J. Weng, R.X. Hou, G.C. Li, J. Wang, N. Huang, H.Q. Liu, Appl. Surf. Sci. 254 (2008) 2712.
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Blood compatibilities of carbon nitride film deposited on biomedical NiTi alloy Jigang Wang a,⁎, Nan Jiang b a b
Jiangsu Key Laboratory of Advanced Metallic Materials, School of Materials Science and Engineering, Southeast University, Nanjing 211189, PR China Institute of Theoretical and Computational Chemistry, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing, 210093, PR China
a r t i c l e
i n f o
Article history: Received 4 January 2009 Received in revised form 20 March 2009 Accepted 8 July 2009 Available online 16 July 2009 Keywords: Carbon nitride Sputtering Biocompatibility Surface characterization
a b s t r a c t Carbon nitride (CNx) film, diamond-like carbon (DLC) film, and titanium nitride (TiN) film were deposited on biomedical NiTi alloy substrates using direct current magnetron sputtering, respectively. In order to improve the adhesive strength between the deposited hard film and the NiTi alloy, a Ti transition layer was pre-deposited firstly. We emphatically evaluated the blood compatibilities of the NiTi alloy substrate and the deposited hard films by haemolysis test and platelet conglutination test. It was shown that the blood compatibilities of NiTi alloy can be improved effectively by the deposition of hard films. In comparison with TiN and DLC film, CNx film had the best surface modification effects covering the minimum haemolysis ratio and the best anticoagulation property. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Biomedical materials are essentially a kind of special functional material that are used and adapted for a medical application. Biomedical materials must be compatible with the body, and there are often issues of biocompatibility which must be resolved before a product can be placed on the market and used in a clinical setting [1]. The metallic biomedical materials including stainless steel, pure titanium (Ti), Ti alloy (Ti6Al4V), and shape memory alloy had already been applied widely. Among the above clinical materials, NiTi alloys have gained much attention in biomedical application such as orthodontics and orthopaedics due to their shape memory effect and super-elasticity. In particular, NiTi alloy is characterized by a specific stress–strain diagram that is different from the deformation behavior of conventional materials but is similar to that of living tissues [2]. But in clinical field, the application of nickel (Ni)-containing materials is quite gingerly. Ni is the necessary nutrient element. But the excess Ni will cause anaphylactic and toxic reactions, even carcinogenesis [3]. Both the in vivo and in vitro test had already demonstrated the toxicity effects of high density Ni, e.g., Ni2+ can replace some divalent metals such as Ca2+, Mg2+, Zn2+ in enzyme and proteins. The in vitro test also demonstrated that there is a close relationship between the deformation of cell, the damage of chromosome, and the molar concentration of liberated Ni2+. In NiTi alloy, the percentage composition of Ni is high up to 50–55 wt.%. Ryhanen et al. [4] found that the liberation amount of Ni of untreated NiTi alloy is far higher than that of stainless steel. So, the liberation of Ni during the application of NiTi alloy is always a noticeable problem, and the surface modification is necessary to improve the ⁎ Corresponding author. Tel.: +86 25 52090628; fax: +86 25 52090669. E-mail address: [email protected] (J. Wang). 0925-9635/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.diamond.2009.07.003
biocompatibility. So far, the surface modifications including surface coating and thin film techniques are widely adopted technologies because they can improve the biological function and compatibility of the embedded materials, and had already been demonstrated to be effective research approaches [5]. For the diamond-like carbon (DLC) film, properties like high hardness, low coefficient of friction and extremely smooth surfaces make them good candidates for wear protection in tribological situations [6–10]. The fact that they are mainly based on carbon also makes them good prospects for biocompatibility. Some reports concerning the modification using titanium nitride (TiN) film was also attempted in the past [11,12]. Similar to DLC film, the carbon nitride (CNx) film also has a potential application in biomedicine due to their excellent hardness, tribological properties, chemical inertness, electrical and optical properties, and biocompatibility. Many studies have been made on the application of DLC film as biocompatible protective coatings [6–10]. However, only very few studies have been achieved on the CNx film [9,10,13]. In this study, CNx film was deposited on the surface of biomedical NiTi alloy using direct current magnetron sputtering. In order to improve the adhesive strength, a Ti transition layer was pre-deposited between the NiTi alloy and the hard film. The blood compatibilities of the NiTi alloy substrate and the deposited hard film were emphatically investigated using haemolysis test and platelet conglutination test, besides the investigation of the mechanical properties including hardness, adhesive strength between the NiTi substrate and deposited film. In order to compare and evaluate the modification effects of CNx film, TiN film and DLC film were also deposited and investigated using the same preparation and test methods. It was shown that the blood compatibility of NiTi alloy can be improved effectively by the deposition of hard films. Among the investigated hard films, CNx film had the best surface modification effects.
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2. Materials and methods
Table 1 Preparation parameters of the gradient films.
2.1. Treatment of NiTi alloy substrate
Parameters
The biomedical NiTi alloy with percentage composition of Ni 50.8 at.% and Ti 49.2 at.% was selected as the substrate. The starting temperature of martensitic phase transformation is −12.8 °C, and the finishing temperature of austenitic phase transformation is 33.4 °C. The NiTi alloy substrate was cut into plates with the size of 10 × 10 × 1 mm using electrospark wire-electrode cutting. The surface defects and contaminants were removed using step sanding by diamond abrasive paper. In order to get mirror surface, the plates were subsequently polished using water milling. In the end, the NiTi plates were successively washed by acetone, alcohol, and de-ionized water using ultrasonic cleaning at a given time of 15 min. Then the plates were dried and stored in dry oven. 2.2. The preparation of specimens The magnetron sputtering system with the type of JGP450A2 (Shenyang Tech. Ltd. Co., Chinese Academy of Sciences) was employed to deposit the gradient films. During the deposition process, circulating water was used to cool the sputtering system. The background degree of vacuum was 1.0 × 10− 3 Pa, and the working pressure was 0.5 Pa. The Ti transition layer was pre-deposited on NiTi alloy substrate for 10 min, and TiN, DLC, and CNx hard films were subsequently deposited for 3 h. The deposition parameters of Ti transition layer and surface hard films were listed in Table 1. All deposition temperatures were room temperature.
Sputtering gas Reaction gas Flow of Ar/N2 Target Substrate Sputtering power Bias voltage Sputtering time
Transition layer and hard films Ti
TiN
DLC
CNx
Ar / 30 sccm/0 Pure Ti NiTi alloy 150 W − 50 V 10 min
Ar N2 30 sccm/2 sccm Pure Ti Ti/NiTi 150 W −50 V 3h
Ar / 30 sccm/0 Pure graphite Ti/NiTi 150 W −50 V 3h
Ar N2 30 sccm/10 sccm Pure graphite Ti/NiTi 150 W −50 V 3h
the contact angles of specimens' surface. The operation procedures were expressed as follows: (i) The specimens were successively supersonically cleaned in acetone and de-ionized water for 10 min, and then dried. (ii) The globules with the volume of 10 µl were dropped on NiTi alloy and hard films' surface using micro-injector attaching to the contact angle measuring apparatus, respectively, and were laid aside for 3 min. (iii) The graphics of globules were then captured by camera and stored. (iv) By means of the software attaching to the contact angle measuring apparatus, the contact angles between globules and specimens' surface were measured, and the data were the average value of four tests under the same condition.
2.3.1. Characterization of film structure and test of mechanical performance The 750 typed Fourier transformer infrared (FT-IR) spectrophotometer (Nicolet Co. U.SA) was used to characterize the chemical bonds. The scanning electron microscopy (SEM) with the type of XL FEGSFEG-SIRION (FEI Ltd., Netherlands) was employed to investigate the structural morphologies of the deposited hard films. The indentation method was usually adopted for the test of hardness due to its unique simplicity and economy [14,15]. In our work, a microhardness tester with the type of HVS-1000 (Laizhou Huayin test instrument Co., China) was used to measure the hardness of the deposited films. The indenter used was a rectangular pyramid diamond with the interfacial angle of 136°. A normal load of 0.245 N (25 gram-force) was applied to produce a tetragonal pyramidal indentation with two clear diagonal indentations on the smooth surfaces of the specimens. The diagonal lengths of the indentations were immediately measured using the microscope attaching to the micorhardness tester. The hardness was derived from the following equation:
2.3.3. Haemolysis test The anticoagulant blood was prepared firstly by the addition of sodium citrate into the fresh human blood. The concentration of sodium citrate was 3.8%, and the volume ratio between fresh blood and sodium citrated was 8:1. The diluted blood was subsequently prepared by mixing the anticoagulant blood and physiological salt water (with concentration of 0.9%) with the volume ratio of 4:5. The NiTi alloy substrate and deposited gradient films were immersed in the physiological salt water and placed in the water-bath at 37 °C for 30 min. The volume of physiological salt water was 10 ml. The diluted blood with the volume of 0.2 ml was successively added into the physiological salt water, and the blended liquid was continuously placed in water-bath for 60 min. Then the blended liquid was centrifugally separated with rotational speed of 1000 r/min for 5 min. The separated supernatant liquor was picked up and was then moved into a clear 96-welled microplate, and was finally measured by enzyme-linked immunosorbent assay (ELISA R&D Systems, Minneapolis, USA). The absorbance optical density of supernatant liquor in each well was read on a microplate reader (CliniBio 128C; Salzburg, Austria). The measuring wavelength was 550 nm. The negative check experiment was carried out using 10 ml physiological salt water and 0.2 ml diluted blood. The positive check experiment was carried out using 10 ml distilled water and 0.2 ml diluted blood. The haemolysis ratio is calculated by Eq. (3)
d = ðd1 − d2 Þ × 0:3
Haemolysis ratio =
2.3. Microstructure characterization and property test
HV =
1845 × Fload d2
ð1Þ ð2Þ
where, d1 and d2 are the lengths of the indentation's diagonals (in unit of µm). HV, vickers hardness, is in the unit of GPa. The adhesive strength between the deposited films and the NiTi alloy substrate was tested by WS-2005 typed automatic scratch tester (Lanzhou Institute of Chemical Physics, Chinese Academy of Sciences). The films' hardness and adhesive strength were determined by averaging four test results. 2.3.2. The measurement of surface contact-angles A Jc 2000 B typed contact angle measuring apparatus (Shanghai Zhongchen Digital Tech. Instrument Co. China) was employed to measure
Dt − Dnc × 100k Dpc − Dnc
ð3Þ
where, Dt, Dnc, and Dpc are the absorbance values of specimens, negative check experiment, and positive check experiment, respectively. 2.3.4. Platelet conglutination test The fresh anticoagulant human blood was centrifugally separated at a speed of 1000 r/min for 10 min. The rich platelet-containing upper plasma was picked up using pipette at room temperature and was then placed in a clean beaker. The pure NiTi alloy substrate and the deposited specimens were respectively immersed in the above rich platelet-containing plasma. After 3 h incubation in a thermostat water bath, the rich platelet-containing plasma was removed by pipette. The NiTi alloy as well as the deposited specimens was subsequently immersed in phosphate buffered saline (PBS) for 10 min,
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and then the PBS was removed. The cleaning operations using PBS were repeated for 2 or 3 times, until all un-conglutinated platelets were removed. Then the surface morphologies were fixed by glutaraldehyde with the concentration of 25% for 12 h at room temperature. Then the specimens was successively dehydrated and dealcoholed by alcohol and acetic isoamyl ester for 10 min, respectively. The concentration gradient of alcohol and acetic isoamyl ester were 50, 75, 95, and 100%. Then the specimens were dried using HCP2 typed critical point drying instrument (Hitachi Co. Japan). After the treatment of gold spraying, the amount and deformation of platelets conglutinated on the dried specimens' surfaces were investigated using SEM apparatus.
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Table 2 Comparison of hardness and adhesive strength of the deposited films. Samples
Ti/TiN film
Ti/DLC film
Ti/CNx film
Hardness (GPa) Adhesion force (N)
24.0 82.3
18.9 37.5
23.1 63.2
3. Results and discussion
737.8 cm−1, corresponding to the in-plane rotation of six-fold rings in graphite layers, is commonly observed in the Raman spectra. In contrast, such band is often observed in N-containing films because N has the enhanced sp2 clustering [21,27]. The bands at 2923.1 and 2851.2 cm−1 can be attributed to the absorption of C–H bond, in which the hydrogen is derived from the absorption of water-vapor of CNx film in the atmosphere [28].
3.1. The microstructure analysis of deposited CNx film
3.2. The comparison of mechanical properties of the deposited films
Fig. 1 shows the IR spectrum of the CNx film. The absorption band at 2200 cm− 1, corresponding to the triple C≡N stretching mode, has been reported until recently [16–20]. In those previous reports a broad band having a single absorption peak has been observed for almost all of the CNx films. Usually, the absorption peaks of C–N single-bond and CfN double-bond are overlapping with that of CfC double-bond, whereas the peak of C≡N triple-bond is independent. So, the appearance of C≡N triple-bond is the characteristic identification of carbon–nitrogen structure existing in the CNx film. On the other hand, the bands in the zone of 1100–1700 cm− 1, especially around 1300 and 1530 cm− 1, are associated with the stretching modes of sp2 and sp3 hybrid C in graphitic and disordered structures, as well as with the formation of sp3 C–N and sp2 CfN bonds [21]. These bonds are normally IR-invisible. However, their contributions to the spectra are the result of the N incorporation in the graphitic rings, which distorts its planar geometry [21]. It can be seen from Fig. 1 that the peak groups at 1110.9–1297.7 cm− 1, which represent the characteristic sp3 C–N bond's absorption, are clearly shown in the IR spectra. According to the theoretical calculation, a characteristic peak will be appeared at approximate 1250 cm− 1 if the C and N atoms are bonded in the mode of β-C3N4 crystal [22]. But this peak usually cannot be observed due to the influence of strong broad band of sp2 hybrid structure [23]. Among these peaks shown in Fig. 1, the peak at 1251.5 cm−1, the characteristic absorption of sp3 C–N bond in β-C3N4 crystal, is clearly shown. So, one can presume that some β-C3N4 microcrystals exist in the deposited CNx film. The bands at 1379.1 and 1530–1570 cm−1 are the sensitized sp2 hybrid structure in disordered and ordered states, respectively. A main absorption band centered at 1616 cm−1 is observed, which is attributed to the CfN vibrations [24,25] exiting in aromatic system or conjugated structure [26]. And the band at 1463.8 cm−1 is characteristic absorption of sp3 C–N bond in non-crystal. A band at
As expressed in Section 2.2, the substrate is biomedical NiTi alloy, on which the Ti transition layer is previously deposited. The hard films including CNx, DLC, and TiN were subsequently deposited, respectively. The thickness of Ti transition layer is around 486 nm after being deposited for 10 min. And the thickness of DLC, CNx, TiN are 1.64, 1.71, and 1.77 µm, respectively, after beding deposited for 3 h. Table 2 exhibits the vickers hardness and adhesive strength of the deposited films. Generally, the hardness of commercial TiN products is approximately 16–20 GPa. The hardness of TiN film obtained by Park J H et al. [12] were approximately 18 and 20 GPa using low and high radio frequency plasma-assisted metal-organic chemical vapor deposition system, respectively. In our work, the hardness of Ti/TiN and Ti/DLC gradient films are 24 and 18.9 GPa, respectively. As to the CNx films, the experimentally achieved CNx films are amorphous, which are embedded with small amount of micro-crystals. So, the hardness of the synthesized CNx films is obviously lower than the theoretically obtained value. The data reported in references are usually around 20–30 GPa [29–31]. In our work, the hardness of Ti/ CNx gradient film is 23.1 GPa, which is close to that of TiN film, and is somewhat higher than that of DLC film. The hardness and elastic modulus of NiTi alloy substrate are about 3.5 and 63.4 GPa [7]. Therefore, the deposition of gradient films can effectively improve the surface hardness of NiTi alloy. It also can be seen from Table 2 that the adhesive strength between Ti/CNx gradient film and NiTi alloy is only 37.5 N, while the adhesive strength between Ti/TiN gradient film and NiTi alloy is high up to 82.3 N. The purpose of the pre-deposition of Ti transition layer is to reduce the interface stress that is derived from the mismatch of crystal lattices, and the difference of elastic modulus between the NiTi alloy substrate and the deposited films. In the case of the deposition of Ti/ TiN gradient film, the elastic modulus, crystal lattice parameters are more matchable with those of NiTi alloy substrate. So, the adhesive strength between Ti/TiN gradient films and NiTi alloy substrate is the strongest among the studied three specimens. As far as Ti/CNx film is
Fig. 1. IR spectrum of CNx film.
Fig. 2. Measurement results of surface contact angles.
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Table 3 Haemolysis ratios of specimens. Samples
Optical density (550 nm)
Haemolysis ratios (%)
The NiTi alloy The Ti/TiN film The Ti/DLC film The Ti/CNx film Positive check Negative check
0.234 ± 0.008 0.217 ± 0.009 0.226 ± 0.010 0.208 ± 0.005 2.240 ± 0.050 0.185 ± 0.010
2.39 1.58 1.99 1.12 – –
concerned, the residual stress at the interior of CNx film is decreased, owing to the transformation of carbon hybrid from sp3 to sp2 with the introduction of nitrogen element [32]. So, in comparison with Ti/DLC gradient film, the adhesive strength between Ti/CNx gradient films and NiTi alloy substrate is stronger (with value of 63.6 N). 3.3. The comparison of the blood compatibilities
of surface contact angle, the surface energy of materials can be deduced. As a result, the platelet conglutination is closely related to the surface contact angle. Tamada and Ikada [33] have already studied the platelet conglutination on polythene surface, and found that the amount of the conglutinated platelets was increased with the elevation of surface contact angles. As shown in Fig. 2, the surface contact angle of untreated NiTi alloy is 50.77°, which is lower than those of Ti/TiN and Ti/DLC (68.24 and 72.32°, respectively). In Refs. [34] and [8], the contact angles of TiN and DLC films are 68.78 and 80–100°, respectively, are close to our results. Since the contact angles of Ti/TiN and Ti/DLC gradient films are larger than 65°, which is the crossover between hydrophilic materials and hydrophobic materials, these films belong to hydrophobic materials. It also can be seen from Fig. 2 that the contact angle of Ti/CNx gradient films is the minimum with value of 32.68°. The achievement of satisfactory hydrophilicity of CNx film can be attributed to the formation of C–N, CfN, especially C≡N bond [35]. The improvement of hydrophilicity will result in the decrease of free energy of deposited film, leading to satisfactory anticoagulation property. So, it is predictable that the CNx film possesses the best anti-thrombus property which will be demonstrated in the following discussion.
The biomedical materials with satisfactory blood compatibility should have excellent anticoagulation property besides conventional compatibility requirements. The embedded materials should neither cause the platelets to be deformed and collapsed, and nor have toxicity on the blood. In general, the blood compatibilities will involve many biological reactions when the biomedical materials contact directly or indirectly with the blood. The detailed biological reactions include the conglutination, aggregation, deformation of platelets, as well as the adsorption of plasma protein. The materials with unsatisfactory blood compatibilities will cause coagulation, activation of fibrinolytic system, and eventually resulting in thrombus. In the following subsection, the blood compatibilities of the NiTi alloy and the hard films were investigated and evaluated from the aspects of the conglutination, aggregation, and deformation of platelets.
3.3.2. The comparison and evaluation of films' haemolysis ratios As shown in Table 3 that the haemolysis ratios of all specimens are smaller that 5%, indicating that all these materials meet the criterion of biomedical materials (the haemolysis ratio is lower than 5%). Among these materials, the NiTi alloy has the maximum haemolysis ratio of 2.39%, and will damage the erythrocyte to some extent. From Table 3 one can also find that the haemolysis ratios of Ti/TiN, Ti/DLC, and Ti/ CNx films are 1.58, 1.99, and 1.12%, respectively, which are obviously lower than that of the NiTi alloy (2.39%). These results indicate that the surface modification can effectively restrain the release of Ni element, and consequently reducing the haemolysis ratio.
3.3.1. The measurement of surface hydrophilicity of the deposited hard films The surface energy of biomedical materials has some effects on the platelet conglutination level. By means of the measurement and analysis
3.3.3. The comparison and evaluation of platelet conglutination The conglutination and activation of platelets on the materials surface will lead to the coagulation reaction, and subsequently result in the release of procoagulant substance [36]. By means of SEM, the
Fig. 3. SEM morphologies of platelet conglutination on the samples' surface (5000×). (a) NiTi alloy (b) Ti/TiN (c) Ti/DLC (d) Ti/CNx.
J. Wang, N. Jiang / Diamond & Related Materials 18 (2009) 1321–1325
conglutination phenomena of platelets, such as the conglutination density and deformation level, on the NiTi alloy and the deposited films can be investigated, and the blood compatibilities can consequently be compared and evaluated. The smaller the conglutination density and deformation level are, the better the blood compatibility will be. Fig. 3 shows the platelet conglutination morphologies of the NiTi alloy and Ti/TiN, Ti/DLC, and Ti/CNx gradient films with magnification ratio of 5000 times (5000×). Many platelets are conglutinated on the surface of NiTi alloy, and obvious aggregation phenomenon also can be easily found (Fig. 3a). In such field of vision, no separate platelets can be found due to the serious aggregation. Nearly all platelets are linked with each other by lots of stretching dendritic pseudopods, and the platelets are deformed in a large degree. While in the case of deposited films, surface modification effects including the decreased amount and homogeneous distribution of conglutinated platelets are achieved, especially for Ti/TiN and Ti/CNx films. As far as the Ti/TiN film is concerned, although the conglutinated platelets are very few, they are aggregated to some extent (Fig. 3b). As to the Ti/ DLC film (Fig. 3c), there is no obvious aggregation. However, the conglutinated platelets on the Ti/DLC film are obviously more than those on the Ti/TiN film. In addition, the platelets on Ti/DLC film are deformed in a larger degree than those on Ti/TiN film. As shown in Fig. 3c, most of platelets stretched out pseudopods, even some cellular stroma can be found between the pseudopods. It is worth noting that, among the deposited films, the amount of the conglutinated platelets on Ti/CNx film is the minimum (Fig. 3d), with no aggregation being found. The original shape of the platelet is well maintained, without the stretched pseudopods. It is demonstrated that the Ti/CNx film has the best blood compatibility due to its best effects on the inhibition of conglutination and activation of platelets. In a word, the conglutination and deformation of NiTi alloy is quite serious, while the surface modification by deposited gradient films can improve the blood compatibility. Among the above tested gradient films, the Ti/CNx film has the best anticoagulation property, as indicated by both the minimum amount of conglutinated platelets and the least deformation. 4. Conclusions Ti/TiN, Ti/DLC, and Ti/CNx gradient films were deposited on the NiTi alloy substrate using magnetron sputtering. The mechanical properties and blood compatibilities of the biomedical NiTi alloy substrate and the above gradient films were investigated and compared. It was concluded that the Ti/CNx gradient film had the best surface modification effect. It is conceivable that CNx has a great potential as a very attractive coating material for biomedical applications in the future. (i) The deposition of Ti interlayer realized the satisfactory combination between NiTi alloy and hard films. In the case of Ti/CNx film, satisfactory mechanic properties were achieved with the surface hardness of 23.1 GPa and the adhesive strength of 63.6 kN.
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(ii) The surface contact angle of CNx film was only 32.68°, and the deposition of CNx film can improve the surface hydrophilicity of NiTi alloy. In comparison with CNx film, TiN and DLC films belong to the hydrophobic materials with the contact angles of 68.24 and 72.32°, respectively. (iii) CNx film had the best blood compatibilities. Compared with the TiNi alloy and other deposited films, CNx films had the minimum haemolysis ratio of 1.12%. In addition, the amount and the deformation level of conglutinated platelets were the least. References [1] D.F. Williams, J. Black, P.J. Doherty, in: P.J. Doherty, R.L. Williams, D.F. Williams, A.J.C. Lee (Eds.), Biomaterial-tissue interfaces, vol. 10, Elsevier, Amsterdam, 1992, p. 525. [2] T. Duerig, A. Pelton, D. Stockel, Mater. Sci. Eng. A 273–275 (1999) 149. [3] F. Villermaux, M. Tabrizian, L. Yahia, G. Czeremuszkin, D.L. Piron, Biomed. Mater. Eng. 6 (1996) 241. [4] J. Ryhanen, E. Niemi, W. Serlo, E. Niemela, P. Sandvik, H. Pernu, et al., J. Biomed. Mater. Res. 35 (1997) 451. [5] X.Y. Liu, P.K. Chu, C.X. Ding, Mater. Sci. Eng. R 47 (2004) 49. [6] J.H. Sui, W. Cai, Diamond Relat. Mater. 15 (2006) 1720. [7] J.H. Sui, W. Cai, L.C. Zhao, Nucl. Instrum. Methods B 248 (2006) 67. [8] Y. Yin, L. Hang, J. Xu, D.R. McKenzie, M.M.M. Bilek, Thin Solid Films 516 (2008) 5157. [9] F.Z. Cui, D.J. Li, Surf. Coat. Technol. 131 (2000) 481. [10] S.E. Rodil, R. Olivares, H. Arzate, S. Muhl, Diamond Relat. Mater. 12 (2003) 931. [11] Y. Cheng, Y.F. Zheng, Surf. Coat. Technol. 201 (2007) 6869. [12] J.H. Park, C.K. Jung, D.C. Lima, J.H. Boo, Tribol. Int. 40 (2007) 345. [13] H. Sjöström, L. Hultman, J.E. Sundgen, S.V. Hainsworth, T.F. Page, G.S.A.M. Theunissen, J. Vac. Sci. Technol. A 14 (1996) 56. [14] J. Musil, H. Zeman, F. Kunc, J. Vlček, Mater. Sci. Eng. A 340 (2003) 281. [15] S. Chowdhury, M.T. Laugier, I.Z. Rahman, Diamond Relat. Mater. 13 (2004) 1543. [16] S.L. Sung, T.G. Tsai, K.P. Huang, J.H. Huang, H.C. Shih, Jpn. J. Appl. Phys. 37 (1998) L148. [17] W.T. Zheng, H. Sjostrom, I. Ivanov, et al., J. Vac. Sci. Technol. A 14 (1996) 2696. [18] M. Therasse, M. Benlahsen, Solid State Commun. 129 (2004) 139. [19] S. Kennou, S. Logothetidis, L. Sygellou, A. Laskarakis, D. Sotiropoulou, Y. Panayiotatos, S. Kennou, et al., Diamond Relat. Mater. 11 (2002) 1183. [20] L.Y. Chen, C.Y. Cheng, F.C.N. Hong, Diamond Relat. Mater. 11 (2002) 117. [21] J.H. Kaufman, Phys. Rev. B. 39 (1989) 13053. [22] M.R. Wixom, Ceram. Soc. 73 (1990) 1973. [23] D. Li, S. Lopey, Y.W. Chung, M.S. Wong, J. Vac. Sci. Technol. A13 (1995) 1063. [24] J.W. Robinson, Practical Handbook of Spectroscopy, CRC. Press, Boston, MA,1991, p. 551. [25] P. González, R. Soto, E.G. Parada, X. Redondas, S. Chiussi, J. Serra, J. Pou, B. León, M. Pérez-Amor, Appl. Surf. Sci. 109–110 (1997) 380. [26] C.B. Cao, Q. Lv, H.S. Zhu, Diamond Relat. Mater. 12 (2003) 1070. [27] N. Nakayama, Y. Tsuchiya, S. Tamada, et al., Appl. Phys. 32 (1993) 1465. [28] S.R.P. Silva, J.G.A. Robertson, et al., Appl. Phys. 81 (1997) 2626. [29] P.Y. Tessier, L. Pichon, P. Villechaise, P. Linez, B. Angleraud, N. Mubumbila, et al., Diamond Relat. Mater. 12 (2003) 1066. [30] D. Li, Y.W. Chung, S.T. Yang, M.S. Wong, F. Abidi, W.D. Sproul, J. Vac. Sci. Technol. A 12 (1994) 1470. [31] H.S. Myung, Y.S. Park, B. Hong, J.G. Han, L.R. Shaginyan, Thin Solid Films 506–507 (2006) 87. [32] C.B. Wang, S.R. Yang, J. Zhang, J. Non-Cryst. Solids 354 (2008) 1608. [33] Y. Tamada, Y. Ikada, Polymer 34 (1993) 2208. [34] L. Yin, T.W. Guo, W. Zhao, Y. Yue, Y.Y. Wang, Chin. J. Prosthodont. 8 (2007) 287. [35] H. Riascos, J. Neidhardt, G.Z. Radnóczi, J. Emmerlich, G. Zambrano, L. Hultman, et al., Thin Solid Films 497 (2006) 1. [36] Y.J. Weng, R.X. Hou, G.C. Li, J. Wang, N. Huang, H.Q. Liu, Appl. Surf. Sci. 254 (2008) 2712.