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Sol–gel deposited TiO2 film on NiTi surgical alloy for biocompatibility improvement
Thin Solid Films 429 (2003) 225–230
Sol–gel deposited TiO2 film on NiTi surgical alloy for biocompatibility improvement Jing-Xiao Liua,b,*, Da-Zhi Yanga, Fei Shia,b, Ying-Ji Caib a
Department of Materials Science and Engineering, The Key National Laboratory of Materials Modification by Three Beams, Dalian University of Technology, Dalian 116024, PR China b Department of Materials Science and Engineering, Dalian Institute of Light Industry, Dalian 116034, PR China Received 7 November 2002; received in revised form 29 January 2003; accepted 4 February 2003
Abstract TiO2 thin films were prepared on NiTi surgical alloy by sol–gel method. The forming process, surface morphology and structure of the films were studied by X-ray diffraction and atomic force microscopy. The results showed that nm-scale TiO2 particles were embedded in the film of 205 nm thickness. The film existed mainly in the form of anatase, and the film was compact and smooth. The electrochemical corrosion measurement indicated that TiO2 thin film, as a protective layer, was effective for improving corrosion resistance of NiTi alloy. Additionally, in vitro blood compatibility of the film and NiTi alloy was evaluated by dynamic clotting time and blood platelets adhesion tests. The results showed that NiTi alloy coated with TiO2 film had improved blood compatibility. 䊚 2003 Elsevier Science B.V. All rights reserved. Keywords: Biomaterials; Sol–gel process; Titanium oxide; Coatings
1. Introduction New biomaterials are being constantly developed to respond to the need for better mechanical properties and biocompatibility. NiTi alloys combine the shape memory effect, superelasticity and other excellent mechanical properties. Thus, NiTi alloys have great potential for biomedical applications. However, metallic biomaterials have the tendency to corrode in physiological environment. Especially, the toxicity and carcinogenesis of Ni ions released from NiTi alloy are a very concerned problem w1x. In vitro studies showed that the cell transformations and chromosome damage depend on dissolved molar Ni2q concentration. Ni2q is capable of substituting itself for divalent metals (Ca2q, Mg2q, Zn2q) in sites in enzymes and proteins and thereby changes the molecular structure w2x. Therefore, it is necessary to modify the surfaces of NiTi alloy implants to improve their corrosion resistance and biocompatibility. *Corresponding author. E-mail address: [email protected] (J.-X. Liu).
In recent years, synthesizing bioceramic film on biomedical metal surface has been attracting considerable attention, since the good mechanical properties of metals and good chemical stability of ceramic films can be combined. Many investigations demonstrated that ceramic films such as TiO2 w3x, Al2O3 w4x and SiC w5x all have good blood compatibility. In fact, the wellknown good biocompatibility of titanium is related to the native TiO2 film on its surface w6x. TiO2 film can be prepared by methods such as ion beam assisted deposition (IBAD) or chemical vapor deposition etc. However, it is difficult and expensive to deposit a uniform layer of TiO2 film on the substrate with complex shapes or geometry by these methods. Sol–gel technology is a low temperature method of preparing film from chemical routes. The advantages of using a sol–gel dipcoating technique are that it is independent of the substrate shape, and can achieve a good control of surface properties such as composition, thickness and topography. The previous work on surface modification of titanium alloy has indicated that sol–gel-derived TiO2 films have good bioactivity w7x. In this paper, the effectiveness of TiO2 film on improving the corrosion
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resistance and blood compatibility of NiTi alloy was investigated. In addition, the relationships between the biocompatibility of TiO2 film and its surface properties were discussed briefly. 2. Experimental details 2.1. Film preparation Tetrabutyl titanate (Ti(C4H9)4, or Ti(OBu)4, from Zhejiang, China) was used as TiO2 precursor. First, 5 ml of Ti(OBu)4 was dissolved in 23 ml of ethylene glycol monomethyl ether (EGME), followed by adding 2 ml of ethyl acetoacetate (EAcAc) to the Ti(OBu)4 solution as a chelating agent. After the solution was stirred for 30 min at room temperature, 2 ml of water solution (pH 1–2) containing ethanol and HNO3 (65 wt.% in water) was added for hydrolysis. The molar ratios of EGME, EAcAc and H2O to Ti(OBu)4 were 20, 1 and 4, respectively. The solution was continually stirred for 2.5 h, and subsequently aged for 18–48 h at room temperature in the air. Then a homogeneous and clear solution was achieved and could be used for coating. In order to investigate phase transformation of TiO2, TiO2 powder used for X-ray diffraction (XRD) analyses was obtained by firing TiO2 dry gel at different temperatures. NiTi alloy (49.3 at.%Ti–50.7 at.%Ni) sheets with 2 mm thickness were used as substrates. Before coating, the samples were ground with successive SiC papers down to grit size 1000 and polished with a 0.2 mm alumina solution w8x, and then ultrasonically cleaned with acetone. The coating was carried out by dipping the NiTi alloy substrate into the sol and then withdrawing it at a speed of 0.3 mmys. The obtained films were annealed in air at 500 8C for 1 h. The temperature was raised slowly, at a rate of approximately 3 8Cymin from room temperature to 500 8C to avoid cracking or flaking off. In order to obtain a compact film, the coating was carried out beside a water bath of 80 8C, so that the enhanced humidity could prevent rapid drying of the coating, which would otherwise crack. 2.2. Surface characterization An elliptical polarization meter (TP-77, Beijing, China) employing He–Ne laser was used to measure the film thickness. Phase composition was determined by XRD measurements using SHIMADZU XRD-6000 diffractometer with Cu Ka. The surface morphology of the film was observed by atomic force microscopy (AFM) operated in contact mode (Digital Instrument, Multimode equipped with a NanoScope IIIa controller). Surface hydrophilicity was evaluated by measuring the contact angle of a sessile drop of deionized water on the samples using a contact angle meter (JJC-1, Beijing,
China). Three measurements were made and the average value had a standard deviation of "0.38 in u. 2.3. Electrochemical corrosion test Electrochemical measurements were carried out in order to determine the corrosion resistance of film samples. The working electrodes were NiTi alloy plates either bare or coated with TiO2 film immersed 1 cm into the Tyrode’s solution. The composition of Tyrode’s solution (in aqueous solution) was as follows (gyl) w9x: NaCl 8.00, CaCl2 0.20, KCl 0.20, NaHCO3 1.00, MgCl2 0.10 and NaH2PO4 0.05. An AgyAgCl electrode was used as reference, while a platinum foil served as the auxiliary electrode. A Princeton Model 173 potentiostat linked to a microcomputer for data acquisition was used for the experiments. After immersing the samples for 30 min, the polarization measurement was started from the rest potential at a scan rate of 0.5 mVys. 2.4. In vitro blood compatibility evaluation The in vitro blood compatibility was investigated by dynamic clotting time measurement and blood platelet adhesion tests. For clotting time measurement, a kinetic method similar to the work described by Huang et al. w10x was used. First, 0.1 ml of human blood anticoagulated by acid citrate dextrose was dripped on the sample surface in an open atmosphere at room temperature (25 8C). Clotting was initiated by the addition of 10 ml of 0.2 M CaCl2 solution. After 5, 10, 30, 40, 50 and 60 min, each sample was transferred into a beaker containing 50 ml of distilled water. Then the optical density of the supernatant was measured at 540 nm wavelengths using a UVyVIS spectrometer (JNSCO V-560 UVyVIS, Japan). For each coating, average optical density was obtained for three measurements. The relationship between the optical density and time was plotted as the clotting time curves, which would indicate the relative clotting time for each sample. For platelet adhesion test, platelet-rich plasma (PRP) was obtained from 8 ml of citrated human blood after 10 min centrifugation at 4000 rpm. The test material was incubated in the PRP at 37 8C for 1 h. After incubation, the samples were rinsed with isotonic sodium chloride solution and fixed in 2.5% vyv glutaraldehyde solution. After dehydration by graded alcohol exposure, each sample was prepared for scanning electron microscopy (SEM) (JEOL JSM-5600LV). 3. Results and discussion 3.1. Formation of TiO2 film Table 1 presents the effects of EAcAc and HNO3 on sol and film formation. It was difficult to obtain homogeneous sol without EAcAc, and there were small
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particles in the EAcAc-free sol due to the fast hydrolysis of Ti(OBu)4. When a suitable amount of EAcAc was added, the sol was stable and gelation time was longer. It was also observed that the value of pH played an important role in the stability of sol. In similar conditions, the sol at pH 4 had gelled in 120 h and coating had to be performed when the sol was 12–30 h old. As shown in Table 1, the resultant films were uniform and smooth. By contrast, sol at pH 1–2 did not gel even after 2 months had passed and the films obtained from coating with a 40-day-old were more compact, uniform and smooth. 3.2. Film thickness and phase composition The thickness of the compact film obtained from a sol prepared using EAcAc and pH adjusted to 1–2 after 2 days of aging and subsequent heating at 500 8C in air was measured to be 205 nm by ellipsometry. Studies on the blood compatibility of titanium oxide films prepared by IBAD revealed that blood compatibility became better as the thickness of titanium oxide films increased w10x. However, it was also reported that the uniformity of oxide layer on surface of metallic implants was more important than its thickness for improving the biocompatibility w1x. Fig. 1 shows the XRD pattern of TiO2 powder after being heat-treated at different temperatures for 1 h. It can be seen that the phase composition of TiO2 depends on its heat-treatment temperature. Anatase phase is the main form of TiO2 when heat-treatment temperature is in the region of 400–500 8C. And the content of anatase increases when temperature increases. At temperature of 600–750 8C, anatase and rutile exist simultaneously. After heat-treatment at temperature above 800 8C, rutile is the only form of TiO2. Based on the work of Zhang et al. w11x, TiO2 films with rutile-type structure prepared by IBAD have good blood compatibility. It has been reported that sol–gel-derived titania gel consisting of rutile and anatase phases also has good blood compatibility w12x. The XRD patterns of the powder sample prepared at 400 8C are similar to those prepared at 500 8C. However, in order to obtain the desired compact film morphology and improve NiTi–TiO2 interfacial
Fig. 1. XRD pattern of TiO2 powder obtained by heat-treating sol– gel for 1 h each at different temperatures.
stability and corrosion resistance of the film, the heat treatment was carried out at 500 8C for 1 h in this study. 3.3. Surface morphology examination Fig. 2 shows the AFM image of the surface of TiO2 film on NiTi substrate. It can be observed that nm-scale TiO2 particles are embedded in the film. The mean surface roughness (Ra) is 3.9 nm. Since the roughness of the polished NiTi substrate was below 5 nm and the TiO2 films were approximately 200 nm thick, the measured roughness should not be affected by the substrate and is mainly related to the TiO2 particles embedded in the film. As is reported, if the biomaterial is used for hard tissue replacement such as bone and tooth, appropriate surface micro-roughness is necessary to improve the bioactivity of implants w13x. An implant with suitable
Table 1 Effects of EAcAc and pH on film formation Results
EAcAc
pH
Adding EAcAc
Without EAcAc
4
1–2
Homogeneous sol
Obtainable
Obtainable
Obtainable
Stability of sol
Stable, gelation time longer Uniform and smooth
Not easy, with small particles in the sol Less stable
Less stable
Usually not smooth
Uniform and smooth
Very stable, gelation time longer More uniform, compact and smooth
Quality of film
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Fig. 2. AFM image of TiO2-coated NiTi plate prepared by sol–gel method. The value of maximum z is 34.5 nm, and the surface roughness is RMSs5.1 nm.
surface roughness will stimulate bone-like apatite formation and achieve strong integration with bone. Usually, surface roughness at the level of cell adhesion (1 mm) is helpful for an implant to have good osseointegration. However, for implants in contact with blood such as artificial heart valve and cardiovascular stents, the surface should be smoother and the roughness at the level of protein adsorption (-50 nm), otherwise blood platelets may adhere and thrombogenesis may occur w14x. In this study, surface roughness of 3–4 nm is enough for blood compatibility. As is well known, if the surface of the implant in a blood flow route is very smooth, it will inhibit thrombogenicity w14x. However, for cardiovascular stents, restenosis after stent placement is a severe problem. In general, restenosis occurs due to abrupt thrombosis and intimal hyperplasia. It was reported that the success of a vascular stent should depend on minimal thrombosis and rapid endothelialization w15x. But whether endothelium can grow on very smooth surfaces needs to be investigated further.
200 mV higher than the value obtained for NiTi alloy substrate specimen. Additionally, the passive current density (ip) of TiO2 film specimen was lower than that of NiTi alloy substrate. These results indicate surface coating enhances the passivity of NiTi alloy. In fact, this enhancement of passivity of the TiO2-coated NiTi alloy benefited from the stability of the protective oxide film. Therefore, it can be concluded that the corrosion resistance of NiTi alloy is improved by synthesizing TiO2 protective film on its surface. On the basis of our previous study w16x, the compactness, the uniformity and the interfacial stability of the film play an important role in their corrosion resistance. If there are pores in the film or the interface adhesion between the substrate and film is poor, then the corrosive ions (Cly) will easily penetrate through the film and react with the metal substrate. As a result, surface pitting will quickly occur. Of course, the breakdown potential also depends on surface roughness and film thickness. A smooth surface will inhibit corrosion due to reduced surface area. However, the compactness, uniformity and strong interface adhesion of the oxide film seem to be the predominant factors to protect the substrate from corrosion. 3.5. Blood compatibility In order to evaluate the effectiveness of TiO2 film on improving the antithrombosis characteristics of NiTi surgical alloy, preliminary in vitro blood compatibility assessment was conducted. Fig. 4 shows the clotting time curves for NiTi alloy and TiO2 film specimens. Clotting time measurement is to test the activated degree of intrinsic coagulation factors. The slower the optical density value decreases with time, the longer the clotting time is. As can be seen from Fig. 4, the optical densities were bigger for TiO2-coated specimen than those for NiTi substrate when the blood had been in contact with the materials for a long time (50 min). This result
3.4. Corrosion resistance When metallic biomaterials are implanted into human body, electrochemical corrosion easily occurs in the blood plasma environment and greatly influences the biocompatibility of the implants. Anodic polarization curves measurement can be used to evaluate the corrosion resistance of metallic implant materials. Fig. 3 shows a comparison of the anodic polarization curves for NiTi alloy coated and non-coated with TiO2 film in Tyrode’s solution at 37 8C. For TiO2 film specimen, the breakdown potential (Eb) was q760 mV, which was
Fig. 3. The anodic polarization curves for the NiTi alloy coated and non-coated with TiO2 film in Tyrode’s solution at 37 8C.
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Fig. 4. A comparison of the optical densityytime curves for uncoated and TiO2-coated NiTi.
indicates that clotting time is longer for TiO2 film specimen. Fig. 5 shows platelet adhesion SEM micrographs obtained after 1 h of incubation in PRP. Fig. 5a shows several platelet aggregations and obvious pseudopod formations of some platelets on NiTi alloy substrate. While on the sol–gel TiO2 film (Fig. 5b), the number of adhered platelets decreases and pseudopod formation is not evident. This also indicates that TiO2-coated samples have a better blood compatibility than the NiTi substrate. Numerous studies have shown that surface structure and properties such as surface roughness, surface hydrophilicity and surface charges have a decisive role in increasing the blood compatibility of the implants. Surface roughness and surface hydrophilicity are beneficial to the blood compatibility. In this study, the contact angle of water on NiTi alloy was measured to be 69.58, while the contact angle of water on TiO2 film specimen was approximately 568. Obviously, TiO2 film is more hydrophilic than NiTi alloy substrate. As is well known, protein will adsorb to the surface as soon as the
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biomaterial are implanted in living system. And the formation of thrombus on the artificial biomaterial is correlated with electron transfer from the inactive state of fibrinogen to the surface of the biomaterial. When fibrinogen adsorbed on the surface is oxidized and transforms to fibrin monomer, it will quickly cross-link to irreversible thrombus w10x. TiO2 film has higher dielectric constant and is semiconductive, so it can inhibit the electron transfer, preventing fibrinogen from denaturing and decreasing thrombus formation w3x. In addition, the isoelectric point of titania is pH 6.2 w17x. When placed in the blood (pH 7.4), titania will be negatively charged, thus the blood elements with negative charges such as blood platelets will not adhere to the negatively charged surface and blood clotting on the surface will not easily occur. 4. Conclusions Preliminary tests on the clotting time, platelet adhesion, corrosion resistance and contact angle for blood compatibility were performed on a (49.3 at.% Ti–50.7 at.%Ni) NiTi surgical alloy plate coated with a 200 nm anatase phase TiO2 film with embedded nm-scale TiO2 particles using a sol–gel process. It was found that the coated sample had better biocompatibility than the bare NiTi plate. Further work on statistical assaying of the observed biocompatibility parameters will be conducted to establish the validity of these preliminary findings. Acknowledgments The authors wish to acknowledge the financial support from the National Natural Science Foundation of China (No. 50081001) and the State Science and Technology Department of China (No. 96-907-04-05). References
Fig. 5. SEM micrographs of platelets adhered on (a) NiTi alloy substrate and (b) TiO2 film after 1 h of incubation in PRP.
w1x C. Trepanier, M. Tabrizian, L’H. Yahia, J. Biomed. Mater. Res. 43 (1998) 433. w2x S.A. Shabalovskaya, Supplement au J. de Physique III 5 (1995) 1199. w3x F. Zhang, Z.H. Zheng, Y. Chen, J. Biomed. Mater. Res. 42 (1998) 128. w4x T. Yuhta, Y. Kikuta, Y. Mitamura, J. Biomed. Mater. Res. 28 (1994) 217. w5x M. Amon, A. Bolz, M. Schaldach, J. Mater. Sci. Mater. Med. 7 (1996) 273. w6x M. Shirkahanzaeh, J. Mater. Sci. Mater. Med. 6 (1995) 206. w7x T. Peltola, M. Patsi, H. Rahiala, I. Kangasniemi, A. Yli-Urpo, J. Biomed. Mater. Res. 41 (1998) 504. w8x S. Trigwell, R.D. Hayden, K.F. Nelson, G. Selvaduray, Surf. Interface Anal. 26 (1998) 483. w9x S.Y. Pu, Y.S. Wu, Chinese J. Biomed. Eng. 7 (1988) 125, in Chinese.
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w10x N. Huang, Y.R. Chen, J.M. Luo, J. Yin, R. Lu, J. Xiao, Zh.N. Xue, X.H. Liu, J. Biomater. Appl. 8 (1994) 404. w11x F. Zhang, Zh.H. Zheng, Y. Chen, X.H. Liu, A.Q. Chen, Zh.B. Jiang, J. Biomed. Mater. Res. 42 (1998) 128. w12x Sh. Takemoto, K. Tsuru, S. Hayakawa, A. Osaka, S. Takashima, J. Sol–Gel Sci. Tech. 21 (2001) 97. w13x M. Jokinen, M. Patsi, H. Rahiala, J. Biomed. Mater. Res. 42 (1998) 295.
w14x D.R. Buddy, S.H. Allan, J.S. Frederick, E.L. Jack, Biomaterials Science: An Introduction to Materials in Medicine, Academic Press, Inc, 1996. w15x A.S. Richard, Circulation 79 (1989) 445. w16x J.X. Liu, J.H. Chen, D.Z. Yang, Y.J. Cai, J. Mater. Sci. Tech. 11 (Suppl. 1) (2001) s35. w17x M. Shirkhanzadeh, J. Mater. Sci. Mater. Med. 9 (1998) 335.
Sol–gel deposited TiO2 film on NiTi surgical alloy for biocompatibility improvement Jing-Xiao Liua,b,*, Da-Zhi Yanga, Fei Shia,b, Ying-Ji Caib a
Department of Materials Science and Engineering, The Key National Laboratory of Materials Modification by Three Beams, Dalian University of Technology, Dalian 116024, PR China b Department of Materials Science and Engineering, Dalian Institute of Light Industry, Dalian 116034, PR China Received 7 November 2002; received in revised form 29 January 2003; accepted 4 February 2003
Abstract TiO2 thin films were prepared on NiTi surgical alloy by sol–gel method. The forming process, surface morphology and structure of the films were studied by X-ray diffraction and atomic force microscopy. The results showed that nm-scale TiO2 particles were embedded in the film of 205 nm thickness. The film existed mainly in the form of anatase, and the film was compact and smooth. The electrochemical corrosion measurement indicated that TiO2 thin film, as a protective layer, was effective for improving corrosion resistance of NiTi alloy. Additionally, in vitro blood compatibility of the film and NiTi alloy was evaluated by dynamic clotting time and blood platelets adhesion tests. The results showed that NiTi alloy coated with TiO2 film had improved blood compatibility. 䊚 2003 Elsevier Science B.V. All rights reserved. Keywords: Biomaterials; Sol–gel process; Titanium oxide; Coatings
1. Introduction New biomaterials are being constantly developed to respond to the need for better mechanical properties and biocompatibility. NiTi alloys combine the shape memory effect, superelasticity and other excellent mechanical properties. Thus, NiTi alloys have great potential for biomedical applications. However, metallic biomaterials have the tendency to corrode in physiological environment. Especially, the toxicity and carcinogenesis of Ni ions released from NiTi alloy are a very concerned problem w1x. In vitro studies showed that the cell transformations and chromosome damage depend on dissolved molar Ni2q concentration. Ni2q is capable of substituting itself for divalent metals (Ca2q, Mg2q, Zn2q) in sites in enzymes and proteins and thereby changes the molecular structure w2x. Therefore, it is necessary to modify the surfaces of NiTi alloy implants to improve their corrosion resistance and biocompatibility. *Corresponding author. E-mail address: [email protected] (J.-X. Liu).
In recent years, synthesizing bioceramic film on biomedical metal surface has been attracting considerable attention, since the good mechanical properties of metals and good chemical stability of ceramic films can be combined. Many investigations demonstrated that ceramic films such as TiO2 w3x, Al2O3 w4x and SiC w5x all have good blood compatibility. In fact, the wellknown good biocompatibility of titanium is related to the native TiO2 film on its surface w6x. TiO2 film can be prepared by methods such as ion beam assisted deposition (IBAD) or chemical vapor deposition etc. However, it is difficult and expensive to deposit a uniform layer of TiO2 film on the substrate with complex shapes or geometry by these methods. Sol–gel technology is a low temperature method of preparing film from chemical routes. The advantages of using a sol–gel dipcoating technique are that it is independent of the substrate shape, and can achieve a good control of surface properties such as composition, thickness and topography. The previous work on surface modification of titanium alloy has indicated that sol–gel-derived TiO2 films have good bioactivity w7x. In this paper, the effectiveness of TiO2 film on improving the corrosion
0040-6090/03/$ - see front matter 䊚 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0040-6090(03)00146-9
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resistance and blood compatibility of NiTi alloy was investigated. In addition, the relationships between the biocompatibility of TiO2 film and its surface properties were discussed briefly. 2. Experimental details 2.1. Film preparation Tetrabutyl titanate (Ti(C4H9)4, or Ti(OBu)4, from Zhejiang, China) was used as TiO2 precursor. First, 5 ml of Ti(OBu)4 was dissolved in 23 ml of ethylene glycol monomethyl ether (EGME), followed by adding 2 ml of ethyl acetoacetate (EAcAc) to the Ti(OBu)4 solution as a chelating agent. After the solution was stirred for 30 min at room temperature, 2 ml of water solution (pH 1–2) containing ethanol and HNO3 (65 wt.% in water) was added for hydrolysis. The molar ratios of EGME, EAcAc and H2O to Ti(OBu)4 were 20, 1 and 4, respectively. The solution was continually stirred for 2.5 h, and subsequently aged for 18–48 h at room temperature in the air. Then a homogeneous and clear solution was achieved and could be used for coating. In order to investigate phase transformation of TiO2, TiO2 powder used for X-ray diffraction (XRD) analyses was obtained by firing TiO2 dry gel at different temperatures. NiTi alloy (49.3 at.%Ti–50.7 at.%Ni) sheets with 2 mm thickness were used as substrates. Before coating, the samples were ground with successive SiC papers down to grit size 1000 and polished with a 0.2 mm alumina solution w8x, and then ultrasonically cleaned with acetone. The coating was carried out by dipping the NiTi alloy substrate into the sol and then withdrawing it at a speed of 0.3 mmys. The obtained films were annealed in air at 500 8C for 1 h. The temperature was raised slowly, at a rate of approximately 3 8Cymin from room temperature to 500 8C to avoid cracking or flaking off. In order to obtain a compact film, the coating was carried out beside a water bath of 80 8C, so that the enhanced humidity could prevent rapid drying of the coating, which would otherwise crack. 2.2. Surface characterization An elliptical polarization meter (TP-77, Beijing, China) employing He–Ne laser was used to measure the film thickness. Phase composition was determined by XRD measurements using SHIMADZU XRD-6000 diffractometer with Cu Ka. The surface morphology of the film was observed by atomic force microscopy (AFM) operated in contact mode (Digital Instrument, Multimode equipped with a NanoScope IIIa controller). Surface hydrophilicity was evaluated by measuring the contact angle of a sessile drop of deionized water on the samples using a contact angle meter (JJC-1, Beijing,
China). Three measurements were made and the average value had a standard deviation of "0.38 in u. 2.3. Electrochemical corrosion test Electrochemical measurements were carried out in order to determine the corrosion resistance of film samples. The working electrodes were NiTi alloy plates either bare or coated with TiO2 film immersed 1 cm into the Tyrode’s solution. The composition of Tyrode’s solution (in aqueous solution) was as follows (gyl) w9x: NaCl 8.00, CaCl2 0.20, KCl 0.20, NaHCO3 1.00, MgCl2 0.10 and NaH2PO4 0.05. An AgyAgCl electrode was used as reference, while a platinum foil served as the auxiliary electrode. A Princeton Model 173 potentiostat linked to a microcomputer for data acquisition was used for the experiments. After immersing the samples for 30 min, the polarization measurement was started from the rest potential at a scan rate of 0.5 mVys. 2.4. In vitro blood compatibility evaluation The in vitro blood compatibility was investigated by dynamic clotting time measurement and blood platelet adhesion tests. For clotting time measurement, a kinetic method similar to the work described by Huang et al. w10x was used. First, 0.1 ml of human blood anticoagulated by acid citrate dextrose was dripped on the sample surface in an open atmosphere at room temperature (25 8C). Clotting was initiated by the addition of 10 ml of 0.2 M CaCl2 solution. After 5, 10, 30, 40, 50 and 60 min, each sample was transferred into a beaker containing 50 ml of distilled water. Then the optical density of the supernatant was measured at 540 nm wavelengths using a UVyVIS spectrometer (JNSCO V-560 UVyVIS, Japan). For each coating, average optical density was obtained for three measurements. The relationship between the optical density and time was plotted as the clotting time curves, which would indicate the relative clotting time for each sample. For platelet adhesion test, platelet-rich plasma (PRP) was obtained from 8 ml of citrated human blood after 10 min centrifugation at 4000 rpm. The test material was incubated in the PRP at 37 8C for 1 h. After incubation, the samples were rinsed with isotonic sodium chloride solution and fixed in 2.5% vyv glutaraldehyde solution. After dehydration by graded alcohol exposure, each sample was prepared for scanning electron microscopy (SEM) (JEOL JSM-5600LV). 3. Results and discussion 3.1. Formation of TiO2 film Table 1 presents the effects of EAcAc and HNO3 on sol and film formation. It was difficult to obtain homogeneous sol without EAcAc, and there were small
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particles in the EAcAc-free sol due to the fast hydrolysis of Ti(OBu)4. When a suitable amount of EAcAc was added, the sol was stable and gelation time was longer. It was also observed that the value of pH played an important role in the stability of sol. In similar conditions, the sol at pH 4 had gelled in 120 h and coating had to be performed when the sol was 12–30 h old. As shown in Table 1, the resultant films were uniform and smooth. By contrast, sol at pH 1–2 did not gel even after 2 months had passed and the films obtained from coating with a 40-day-old were more compact, uniform and smooth. 3.2. Film thickness and phase composition The thickness of the compact film obtained from a sol prepared using EAcAc and pH adjusted to 1–2 after 2 days of aging and subsequent heating at 500 8C in air was measured to be 205 nm by ellipsometry. Studies on the blood compatibility of titanium oxide films prepared by IBAD revealed that blood compatibility became better as the thickness of titanium oxide films increased w10x. However, it was also reported that the uniformity of oxide layer on surface of metallic implants was more important than its thickness for improving the biocompatibility w1x. Fig. 1 shows the XRD pattern of TiO2 powder after being heat-treated at different temperatures for 1 h. It can be seen that the phase composition of TiO2 depends on its heat-treatment temperature. Anatase phase is the main form of TiO2 when heat-treatment temperature is in the region of 400–500 8C. And the content of anatase increases when temperature increases. At temperature of 600–750 8C, anatase and rutile exist simultaneously. After heat-treatment at temperature above 800 8C, rutile is the only form of TiO2. Based on the work of Zhang et al. w11x, TiO2 films with rutile-type structure prepared by IBAD have good blood compatibility. It has been reported that sol–gel-derived titania gel consisting of rutile and anatase phases also has good blood compatibility w12x. The XRD patterns of the powder sample prepared at 400 8C are similar to those prepared at 500 8C. However, in order to obtain the desired compact film morphology and improve NiTi–TiO2 interfacial
Fig. 1. XRD pattern of TiO2 powder obtained by heat-treating sol– gel for 1 h each at different temperatures.
stability and corrosion resistance of the film, the heat treatment was carried out at 500 8C for 1 h in this study. 3.3. Surface morphology examination Fig. 2 shows the AFM image of the surface of TiO2 film on NiTi substrate. It can be observed that nm-scale TiO2 particles are embedded in the film. The mean surface roughness (Ra) is 3.9 nm. Since the roughness of the polished NiTi substrate was below 5 nm and the TiO2 films were approximately 200 nm thick, the measured roughness should not be affected by the substrate and is mainly related to the TiO2 particles embedded in the film. As is reported, if the biomaterial is used for hard tissue replacement such as bone and tooth, appropriate surface micro-roughness is necessary to improve the bioactivity of implants w13x. An implant with suitable
Table 1 Effects of EAcAc and pH on film formation Results
EAcAc
pH
Adding EAcAc
Without EAcAc
4
1–2
Homogeneous sol
Obtainable
Obtainable
Obtainable
Stability of sol
Stable, gelation time longer Uniform and smooth
Not easy, with small particles in the sol Less stable
Less stable
Usually not smooth
Uniform and smooth
Very stable, gelation time longer More uniform, compact and smooth
Quality of film
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Fig. 2. AFM image of TiO2-coated NiTi plate prepared by sol–gel method. The value of maximum z is 34.5 nm, and the surface roughness is RMSs5.1 nm.
surface roughness will stimulate bone-like apatite formation and achieve strong integration with bone. Usually, surface roughness at the level of cell adhesion (1 mm) is helpful for an implant to have good osseointegration. However, for implants in contact with blood such as artificial heart valve and cardiovascular stents, the surface should be smoother and the roughness at the level of protein adsorption (-50 nm), otherwise blood platelets may adhere and thrombogenesis may occur w14x. In this study, surface roughness of 3–4 nm is enough for blood compatibility. As is well known, if the surface of the implant in a blood flow route is very smooth, it will inhibit thrombogenicity w14x. However, for cardiovascular stents, restenosis after stent placement is a severe problem. In general, restenosis occurs due to abrupt thrombosis and intimal hyperplasia. It was reported that the success of a vascular stent should depend on minimal thrombosis and rapid endothelialization w15x. But whether endothelium can grow on very smooth surfaces needs to be investigated further.
200 mV higher than the value obtained for NiTi alloy substrate specimen. Additionally, the passive current density (ip) of TiO2 film specimen was lower than that of NiTi alloy substrate. These results indicate surface coating enhances the passivity of NiTi alloy. In fact, this enhancement of passivity of the TiO2-coated NiTi alloy benefited from the stability of the protective oxide film. Therefore, it can be concluded that the corrosion resistance of NiTi alloy is improved by synthesizing TiO2 protective film on its surface. On the basis of our previous study w16x, the compactness, the uniformity and the interfacial stability of the film play an important role in their corrosion resistance. If there are pores in the film or the interface adhesion between the substrate and film is poor, then the corrosive ions (Cly) will easily penetrate through the film and react with the metal substrate. As a result, surface pitting will quickly occur. Of course, the breakdown potential also depends on surface roughness and film thickness. A smooth surface will inhibit corrosion due to reduced surface area. However, the compactness, uniformity and strong interface adhesion of the oxide film seem to be the predominant factors to protect the substrate from corrosion. 3.5. Blood compatibility In order to evaluate the effectiveness of TiO2 film on improving the antithrombosis characteristics of NiTi surgical alloy, preliminary in vitro blood compatibility assessment was conducted. Fig. 4 shows the clotting time curves for NiTi alloy and TiO2 film specimens. Clotting time measurement is to test the activated degree of intrinsic coagulation factors. The slower the optical density value decreases with time, the longer the clotting time is. As can be seen from Fig. 4, the optical densities were bigger for TiO2-coated specimen than those for NiTi substrate when the blood had been in contact with the materials for a long time (50 min). This result
3.4. Corrosion resistance When metallic biomaterials are implanted into human body, electrochemical corrosion easily occurs in the blood plasma environment and greatly influences the biocompatibility of the implants. Anodic polarization curves measurement can be used to evaluate the corrosion resistance of metallic implant materials. Fig. 3 shows a comparison of the anodic polarization curves for NiTi alloy coated and non-coated with TiO2 film in Tyrode’s solution at 37 8C. For TiO2 film specimen, the breakdown potential (Eb) was q760 mV, which was
Fig. 3. The anodic polarization curves for the NiTi alloy coated and non-coated with TiO2 film in Tyrode’s solution at 37 8C.
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Fig. 4. A comparison of the optical densityytime curves for uncoated and TiO2-coated NiTi.
indicates that clotting time is longer for TiO2 film specimen. Fig. 5 shows platelet adhesion SEM micrographs obtained after 1 h of incubation in PRP. Fig. 5a shows several platelet aggregations and obvious pseudopod formations of some platelets on NiTi alloy substrate. While on the sol–gel TiO2 film (Fig. 5b), the number of adhered platelets decreases and pseudopod formation is not evident. This also indicates that TiO2-coated samples have a better blood compatibility than the NiTi substrate. Numerous studies have shown that surface structure and properties such as surface roughness, surface hydrophilicity and surface charges have a decisive role in increasing the blood compatibility of the implants. Surface roughness and surface hydrophilicity are beneficial to the blood compatibility. In this study, the contact angle of water on NiTi alloy was measured to be 69.58, while the contact angle of water on TiO2 film specimen was approximately 568. Obviously, TiO2 film is more hydrophilic than NiTi alloy substrate. As is well known, protein will adsorb to the surface as soon as the
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biomaterial are implanted in living system. And the formation of thrombus on the artificial biomaterial is correlated with electron transfer from the inactive state of fibrinogen to the surface of the biomaterial. When fibrinogen adsorbed on the surface is oxidized and transforms to fibrin monomer, it will quickly cross-link to irreversible thrombus w10x. TiO2 film has higher dielectric constant and is semiconductive, so it can inhibit the electron transfer, preventing fibrinogen from denaturing and decreasing thrombus formation w3x. In addition, the isoelectric point of titania is pH 6.2 w17x. When placed in the blood (pH 7.4), titania will be negatively charged, thus the blood elements with negative charges such as blood platelets will not adhere to the negatively charged surface and blood clotting on the surface will not easily occur. 4. Conclusions Preliminary tests on the clotting time, platelet adhesion, corrosion resistance and contact angle for blood compatibility were performed on a (49.3 at.% Ti–50.7 at.%Ni) NiTi surgical alloy plate coated with a 200 nm anatase phase TiO2 film with embedded nm-scale TiO2 particles using a sol–gel process. It was found that the coated sample had better biocompatibility than the bare NiTi plate. Further work on statistical assaying of the observed biocompatibility parameters will be conducted to establish the validity of these preliminary findings. Acknowledgments The authors wish to acknowledge the financial support from the National Natural Science Foundation of China (No. 50081001) and the State Science and Technology Department of China (No. 96-907-04-05). References
Fig. 5. SEM micrographs of platelets adhered on (a) NiTi alloy substrate and (b) TiO2 film after 1 h of incubation in PRP.
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