- Email: [email protected]
Surface modification of biomaterials by plasma immersion ion implantation
Surface & Coatings Technology 186 (2004) 218 – 226 www.elsevier.com/locate/surfcoat
Surface modification of biomaterials by plasma immersion ion implantation N. Huang *, P. Yang, Y.X. Leng, J. Wang, H. Sun, J.Y. Chen, G.J. Wan Key Lab. of Biomaterials Surface Modification of Sichuan, Laboratory of Advanced Materials Processing of Chinese Education Ministry, Southwest Jiaotong University, Chengdu 610031, China Available online 28 May 2004
Abstract The present paper presents the results of surface modifications of biomaterials using plasma immersion ion implantation (PIII) in Institute of Surface Modification of Biomaterials in Southwest Jiaotong University. In the research, titanium oxide films and diamond-like carbon (DLC) films doped with different elements, as well as surface modification of polymers, are prepared using PIII. The blood compatibility of the films is investigated. Significant improvement in blood compatibility of the films compared with those untreated substrate materials is confirmed. The mechanisms of the blood compatibility of the films are discussed. The results show that the energy band structure of the material surface may be a controlling factor in the blood compatibility of the material. A film with an n-type semiconductor nature would have a better blood compatibility. Improvement in wear resistance of hip joint and modification of antibacterial property of artificial heart valve suture ring material is also presented in the paper. D 2004 Elsevier B.V. All rights reserved. Keywords: Surface modification; Biomaterials; Biocompatibility; Plasma immersion ion implantation
1. Introduction It has been commonly agreed that the reactions on the interface between a material and biological environment play a key role to the biocompatibility of the material. Surface modification to adjust or control the surface characteristics such as surface structure, composition, functional groups, type of bonding bimolecular or cell, morphological feature of the surface, etc., has been proved to be very effective in improving biocompatibility of the material and therefore becomes one of the most attractive branch in the field of biomaterial researches. Among the techniques of surface modification, the recently developed plasma immersion ion implantation (PIII) has been showing positive potentials for its versatile processes of plasma modification, ion implantation and deposition and non-line-of-light feature. Some research groups have utilized PIII to modify the properties of biomaterials [1 –3]. In the present paper the * Corresponding author. College of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China. Tel./fax: +8628-87600625. E-mail addresses: [email protected], [email protected] (N. Huang). 0257-8972/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2004.04.041
recent researches on application of PIII technique in Key Lab. of Biomaterials Surface Modification in Southwest Jiaotong University to modify biomaterials and cardiovascular devices are presented. Further development in the research such as biomimetic surface preparation by means of PIII is also discussed. 1.1. Plasma immersion ion implantation facilities and processes of material surface treatment Since 2000, a plasma immersion ion implantation and deposition (PIIID) device [4] with a larger vacuum chamber of 1.0 m in diameter and 1.2 m high has been utilized in Institute of Surface Modification of Biomaterials in Southwest Jiaotong University. In the advice, pulsed voltages from 1 to 60 kV can be applied on the work stage. Four filtered pulsed cathodic arc sources for creating metal plasma, a RF device with a power of 4 kW and a set of filaments for generating gas plasma are attached to the device. The density of gas plasma is about 109/cm3, the deposition rate of metal film such as titanium film is about 0.2 nm/s. Different processes such as gas ion implantation, metal ion implantation, synthesis of compound films, plasma sputtering, plasma grafting and plasma enhanced
N. Huang et al. / Surface & Coatings Technology 186 (2004) 218–226
219
and PET for increasing the hemocompatibility and antibacterial property. Heparin, albumin or PEO bindings are carried out by argon plasma treating and then grafting [10]. 1.2. Behavior of blood compatibility of titanium oxide films synthesized by PIII
Fig. 1. The structures of Ti – O films vs. pressure of oxygen during PIIID – Ti – O film process.
chemical vapor deposition, etc., can be carried out using the device. For increasing the efficiency and capability of the system, especially for films synthesis, a new device with a vacuum chamber dimension of 0.6 m in diameter and 0.5 m high has also been installed. The new system has two filtered DC arc sources, an electron cyclotron resonance (ECR) gas plasma generator with the power of 2 kW. A negative pulsed voltage of 0.1 to 5 kV can be applied to the working pieces. The gas plasma density around 1011/cm3 and the deposition rate of 1.5 nm/s can be achieved. Different processes to modify biomaterials are investigated using the above mentioned facilities. For synthesis of titanium based films such as titanium oxide film, metal plasma is introduced into the work chamber from the filtered arc source and oxygen gas plasma is generated by RF or ECR. Oxygen atoms in Ti–O films can be controlled by adjusting the inlet oxygen gas flow or the power of RF or ECR. During the synthesis process, the temperature of the work pieces can be adjusted so that the structure of films can be controlled. Different kinds of diamond-like carbon (DLC) films are obtained using the PIIID facilities mentioned above [5 – 7]. In the processes, a hydrogen free DLC film is first synthesized using filtered vacuum arc plasma deposition. Carbon plasma is produced by igniting graphite cathode and the plasma diffuses into the vacuum chamber via the magnetic duct. The magnetic duct is to eliminate macro-particles. Pulsed bias from several ten voltages to several thousands voltages are applied on the work piece to obtain different bonding characteristics of the carbon films. Furthermore, the doped DLC films are deposited by introducing gas elements such as nitrogen, hydrogen, argon or fluorine into the work chamber. Hydrogenated DLC films are also deposited by acetylene gas plasma deposition. Processes for the surface modification of polymer biomaterials, such as polyurethane (PU) and polyethylene terephthalate (PET) are investigated [8,9]. Acetylene gas plasma immersion ion implantation is used to treat PU
Titanium oxide films with different structures and compositions are synthesized using the PIII facilities. Blood compatibility of the titanium oxide films are investigated in vitro and in vivo. Relationships between material characteristics and the interaction behavior of blood with material are revealed. It is found that the crystalline structure, the oxygen content and the doped elements can affect the interaction between the Ti –O films and blood. Fig. 1 shows the different structures of the Ti– O films obtained by changing the flow rate of oxygen into the vacuum chamber of the PIII device. Increasing oxygen flow, the structure of the films changes from amorphous to mixed crystalline of anatase and rutile, and further becomes Rutile. Composition analysis showed that the atom ratio of oxygen to titanium in the films increases from 1.36:1 to 2:1. As the partial pressure of oxygen reached 10 10 2 Pa, the structure of the film is rutile and its composition becomes stoichiometric TiO2. Table 1 lists the structure characteristics, adhesion behaviors of platelet and the conductive properties of the Ti –O films. As mentioned above, as the pressure of oxygen changes from 4.5 10 3 to 10 10 2 Pa during the deposition, the structure changes. As a result, the electronic properties of the film changes from near conductor to semiconductor and further to near insulator. Correspondingly, the adhesion state of platelet changes significantly. It shows that the adhesion state of platelets improved with increase in oxygen content in the film. However, as the Ti –O film becomes stoichiometric TiO2, the state of platelet adhesion turns bad, as shown in Fig. 2. Comparison of behaviors of platelet adhesion on the surface of Ti– O film of rutile, on amorphous Ti– O film which has about same composition of the rutile one, and on low temperature isotropic pyrolytic carbon (LTIC) (LTIC is considered to
Table 1 PIIID parameters, structure of the films, behavior of platelet adhesion and resistance of the films Samples Pressure Structure of O2 (Pa)
A B
4.5e 2.6e
C D
4.0e 10e
Platelet adhesion Amount/ Deformed 2500 (pseudopodium/ Am2 aggregation)%
3 Amorphous 110 F 22 98/95 2 Amorphous + 44 F 14 68/25 Crystalline 2 Rutile 15 F 11 12/20 2 Rutile 36 F 10 47/21
Resistivity q (V/cm)
2.96 10 9.62 10
4
6.76 10 >104
1
3
220
N. Huang et al. / Surface & Coatings Technology 186 (2004) 218–226
Fig. 2. Morphology of platelets on Ti – O film: (A) amorphous Ti – O film; (B) Amorphous + crystalline Ti – O film; (C) Rutile Ti – O film; (D) Rutile TiO2 film (incubation time 90 min).
have the best blood compatibility among the clinic applied mechanical heart valve materials) is presented in Ref. [11], Fig. 7. The test results show that the platelet adhesion state is significant improved on Ti– O film comparing with that on LTIC, while rutile Ti– O film is the best of them. For quantitative evaluation of the platelet activation on the material surface, approach GMP140 expression on the platelet surfaces is used [12]. GMP 140, also called Pselection (CD62), only exists on activated platelets and thus may be used as a marker of platelet activation on biomaterials [13]. Fig. 3 shows GMP140 expression on platelet surfaces indicated by percentage of positive cells, at the same incubation condition. The GMP140 expression gives a direct indication of the platelets activation as: Ti>LTI-carbon>Ti–O. Investigation on protein adsorption has been carried out by contacting fibrinogen which is radio-labeled with 125I and detected using g-counter. The level of protein adsorption on the Ti–O surface is found to be significantly lower than that on LTIC as shows in Ref. [11], Fig. 10. It is also found that the tendency to activate clotting factors is lower on the Ti– O film than on LTIC [14]. The test results suggest that one of important factors affecting the blood compatibility of the Ti– O films may be its semiconductor nature. The n-type nature and broad band gap of the Ti –O films could prevent the protein
denaturation, which is caused by charges transferring from protein onto the material. The suitable non-stoichiometric state of a titanium oxide film with a more perfect crystalline structure could posses a better semiconducting characteristic which has high electron density in the conduct band and low cavity density in the valence band and forbidden band. So far, all facts about Ti– O films are in good consistent with this consideration. Amorphous Ti–O film which has inevitably a high density of local state in the forbidden band showed bad blood compatibility than rutile TiO2 x [11], even the composition of the amorphous Ti –O film and the rutile TiO2 x is about the same. The same principle may also explain why the stoichimetric rutile TiO2 film has a lower blood compatibility than rutile TiO2 x. The reason is that the TiO2 film is almost insulator with very few electrons in the conduct band. Observation of the state of fibrinogen on TiO2 x films using atom force microscopy also confirms that most of fibrinogen on TiO2 x film surface is in single molecular state (Ref. [11], Fig. 11). Due to the lower level of protein denaturation, the tendency of platelet activating and the clotting factors are lower. Therefore, blood compatibility of the film is improved. The phenomena of the interaction of blood with titanium oxide film doped with 5+ valence element such as tantalum also agrees with this principle [15].
Fig. 3. GMP140 expression of platelets on difference materials.
Fig. 4. The surface state of mechanical heart valve (MHV) cages after in vivo test for 30 days implantation. (A) PIII treated; (B) untreated.
N. Huang et al. / Surface & Coatings Technology 186 (2004) 218–226
221
Fig. 5. Radiographic figures of coronary stents implanted in rabbits abdominal aorta for 20 weeks (no anticoagulant was taken). (A) Modified; (B) untreated.
In vivo test results improve the excellent blood compatibility of n-type Ti –O films further (Ref. [11], Figs. 12 – 14). In vivo tests from 17 to 90 days show that almost no coagulation on Ti samples with Ti– O film surfaces, while thrombus are found on LTIC samples. Fig. 4 shows the in vivo test results of mechanical heart valve (MHV) cages which have been implanted into right ventricle of a dog for 30 days without anticoagulant treatment. It clearly shows that no thrombus on the MHV cage surfaces with Ti –O film coating, but thrombi exist and have encapsulated the stent of the valve cage without Ti–O surface. Twenty weeks in vivo test results on coronary stents is shown in Figs. 5 and 6. With untreated stents, thrombus have formed and caused the constriction in the stent position. On the surface of Ti– O film coated stents, a thin layer of endothelial cells, but no thrombus, is found. These results prove further that Ti –O films have excellent blood compatibility and the films should have potentials for clinic applications.
1.3. Diamond like carbon films Diamond-like carbon (DLC) film has many attractive properties such as extremely high hardness, low friction coefficient and chemical inertness. Investigations on biocompatibility and blood compatibility of DLC film show that DLC film is biocompatible and may have good blood compatibility [16,17]. Recently, DLC film has been suggested for application in blood-contacting devices such as rotary blood pumps, artificial hearts, mechanical heart valves, and coronary stents [18,19]. However, very few investigations have been done on the relationship of the characteristics of DLC film and its biocompatibility or blood compatibility, and to modify the biomedical properties of DLC films. Our work focuses on the effect of characteristics of DLC film on blood compatibility. It is found that the relationship between the DLC film characteristics and blood compatibility is very complicated. Our research shows that it is
Fig. 6. SEM photography of the coagulation state of the coronary stents in Fig. 5.
222
N. Huang et al. / Surface & Coatings Technology 186 (2004) 218–226
possible to modify the blood compatibility of DLC film by changing the bonding state, element doping of the film, etc. Hydrogenated DLC films have been synthesized using acetylene gas plasma immersion ion implantation and deposition [20]. In the investigation, a DLC film prepared at a lower bias voltage, such as 75 V, shows a low platelet adhesion and activation, similar as LTIC but better than stainless steel. However, when the bias voltage is high, such as 900 V, the property of platelet adhesion and activation on the DLC film turns bad. This seems that the higher bonding ratio of Sp3/Sp2 may contribute to the blood compatibility of DLC. However, as we dope the DLC film with nitrogen of certain concentration, even if the bonding ratio of Sp3/Sp2 is decreased, the behavior of platelet adhesion is improved significantly and is even superior to that of LTIC, as shown in Fig. 7. Similar phenomenon has been observed in hydrogen free DLC film doped with argon or nitrogen [21]. As argon gas flow into the vacuum chamber changes from 0 to 13 sccm during the deposition of hydrogen free DLC films, the bonding ratio Sp3/Sp2 of the DLC films changes from 78/22 to 56/44, and the platelets adhesion behavior of the films is modified significantly. It is also found that above phenomena may be related to changes in surface tension on the films. The DLC film, which has a more hydrophilic nature, seems to have better blood compatibility than that of the DLC film with more hydrophobic nature [20]. This may be due to the preferential adsorption of albumin which is related to the higher adhesion work Wa of albumin compared that of fibrinogen. If DLC films are annealed at the temperature of 600 jC, the DLC films present a p-type characteristic. The blood compatibility becomes deteriorated [22]. This is another proof that n-type characteristic of the material helps maintaining the configuration of proteins, while carriers in p-type semiconductive films will help charges transferring from proteins to the material and lead to changes of the conformation of protein and activate the coagulation system. Another approach to change the surface properties of DLC films is to synthesize a fluorine contained carbon
Fig. 8. Contact angles water vs. CF4 flow of fluorine contained carbon based films.
based films using CF4 and a filtered vacuum carbon arc source on the PIII facility [23]. Fig. 8 shows the variation of water contact angles as a function of the CF4 flow. As can be seen from the figure, the water contact angles monotonously increase with the increase of CF4 flow. Improvement of the hydrophobicity of the films is obvious. With the highest CF4 flow rate, the contact angle of distilled water on the DLC film increases by one time and is close to that of PTFE. This high hydrophobicity surface possesses good anti-adhesion property. Fig. 9 gives the wear resistance of Ti6Al4V coated and uncoated with DLC. According to the wear trace and the friction coefficient analyses, increase of several orders of magnitude in the wear resistance of the Ti6Al4V can be achieved by DLC coating. This gives a great potential of using DLC film to improve the durability of biomedical device. Fig. 10 shows a DLC coated head of a metal hip joint. A significant improvement of durability of the wear couple can be expected.
Fig. 7. Behavior of platelets adhesion on DLC films with and without nitrogen as well as on LTIC (incubation time: 15 min).
N. Huang et al. / Surface & Coatings Technology 186 (2004) 218–226
223
Fig. 9. (a) The optic photography of (A) DLC film coated TI6Al4V undergo the pin on desk dry wear test of 200 thousands cycles and (B) uncoated Ti6Al4V undergo dry wear test of 10 thousands cycles (SiC pin). (b) The friction coefficient curve of DLC coated and uncoated TI6Al4V.
1.4. Application of PIII surface modification in polymer biomaterials research
Fig. 10. The DLC coated head of a metal hip joint.
Polyurethane (PU) and polyethylene terephthalate (PET) are widely used as biomaterials for cardiovascular implants such as artificial blood vessels, artificial heart valve sewing rings, etc. However, their blood compatibility is insufficient and long-term antithrombotic is required. The incidence of thromboembolic complications or bleeding complications is a serious concern. As an attempt to improve the blood compatibility of the materials, in our lab, acetylene gas plasma immersion ion implantation is used to modify PU and PET. Pulsed voltages from several kV to 10 kV are applied on the work stage to implant carbon and hydrogen ions into polymers samples. Composition and structure analyses show that an amorphous carbon layer is formed
224
N. Huang et al. / Surface & Coatings Technology 186 (2004) 218–226
Fig. 11. Morphology of adherent platelets on the (A) PET, (B) C2H2 PIII treated (incubation time 20 min).
Fig. 12. Statistics of adhesion behavior of human platelets on the untreated PU and surface-modified PUs after 15 min incubation, Ar treated: Argon plasma treated; PU-PEG: Ar treated and PEG greafted; PU-HEP: Ar treated and heparin grafted; PU-PEG-FEP: Ar treated and PEG as well as heparin grafted.
on the polymer samples. The wettability of the material is increased, while platelets adhesion and activation are suppressed, as shown in Fig. 11. By means of plasma grafting we successful bonded bimolecular such heparin, albumin and hydrophilic spacer groups polyethylene glycols (PEG) on the surface of PU and PET. Fig. 12 gives the statistics of platelet adhesion and activation. The result shows that grafting of multiple bimolecular and PEG is significantly beneficial for suppressing platelets adhesion and activation. This result, together with the results of partial thromboplastin time (APTT) testing which shows the bioactivity of intrinsic blood coagulation factors of the surface grafted PU and PET [24], confirms a significant improvement in blood compatibility of PU and PET. The infection from medical devices is a life threatening complication and leads to significant morbidity and mortality [25]. In particular, the mortality of prosthetic valve endocarditis (PVE) could be as high as about 60% in some places in China. The bacterial adhesion to the biomaterial surface is the first event in a series of both host and organismic reactions
that lead to PVE [26]. Therefore, there is an increasing interest in developing new methods to reduce bacteria adhesion onto polymeric materials used in biomedical implants. We investigated the antibacterial behavior of polyethylene terephthalate (PET) treated by acetylene (C2H2) plasma
Fig. 13. The adhered numbers of bacteria SA, SE, EC, PA, CA on the surface of the PET and C2H2 PIII treated PET.
N. Huang et al. / Surface & Coatings Technology 186 (2004) 218–226
Fig. 14. Endothelial cells growth on (A) TiO2
immersion ion implantation –deposition. Fig. 13 shows that the changes of the adhered numbers of Staphylococcus aureus (SA), Staphylococcus epidermidis (SE), Escherichia coli (EC), Pseudomonas aeruginosa (PA), Candida albicans (CA) on the surface of the PET. A significant decrease of bacteria growth on the modified PET surface is verified. The more detail work about the mechanism of suppressing bacteria growth by PIII treatment is under investigation [27]. Construction of Biomimetic surface is an attractive and new field of the biomaterial researches. Many attempts have been made to seed endothelial cells (EC) onto the surface of biomaterials. But few of the work deal with seeding endothelial cells on inorganic surface. Recently, we have developed PIII modified endothelial cell cultured Ti– O surface, and found that EC growth on crystalline Ti– O film is quite well than on amorphous Ti – O film [28], but on LTIC surface it is difficult to seed EC, as shown in Fig. 14. Further work of the long-term activity and the bonding properties are under investigation. 1.5. Conclusions The presented investigation proves that PIII technique is a new and versatile method to modify of biomaterials. Combined with other surface treatments, PIII can be widely used to fabricate films with good biocompatibility and durability. It can also be used to modify a polymer surface to improve the blood compatibility and antibacterial property of the material. PIII can also be used to bind the desired bimolecular and cells to obtain biomimetic surfaces. A great promise of obtaining new biomaterials with good biocompatibility using PIII can be expected.
Acknowledgements The authors sincerely thank the help and cooperation from Prof. P.K. Chu of City University of Hong Kong. Ian G. Brown of Lawrence Berkeley National Laboratory, California, USA. Prof. Guangjun Cai of Tongji University,
x
225
film and (B) on LTIC incubation time 85 h.
Shanghai, China. Prof. W. Moeller and Dr. M. Maitz of Research Center of Rossendorf, Germany, Prof. Xi Wu of Medical College, Southeast University of China and Prof. Tingfei Xi of National Institute for Control of Pharmaceutical and Biological Products of China. This work is jointly financially supported by Key Basic Research Program of China (G1999064705) and Natural Science Foundation of China (30270392, 50203011). References [1] A. Chen, J.T. Scheuer, C. Ritter, R.B. Alexander, J.R. Conrad, Appl. Phys. 70 (1991) 6757. [2] M. Ababneh, J.K. Gregory, U. Holzwarth, Proceedings of the 1st Tagung des DVM-Arbeitskeises ‘Biowerkstoffe, Deu tscher Verband fur Materialforschung und -prufung, Berlin’, 1998, p. 120. [3] S. Mandl, B. Rauschenbach, Surf. Coat. Technol. 156 (2000) 276. [4] P.K. Chu, B.Y. Tang, L.P. Wang, X.F. Wang, S.Y. Wang, N. Huang, Rev. Sci. Instrum. 72 (3) (2001) 1660. [5] Y.X. Leng, J.Y. Chen, P. Yang, H. Sun, G.J. Wan, H. Huang, Surf. Sci. 531 (2) (2003) 177. [6] P. Yang, N. Huang, Y.X. Leng, J.Y. Chen, R.K.Y. Fu, S.C.H. Kwok, Y. Leng, P.K. Chu, Biomaterials 24 (17) (2003) 2821. [7] Y.X. Leng, J.Y. Chen, P. Yang, H. Sun, G.J. Wan, N. Huang, Surf. Coat. Technol. 173 (1) (2003) 67. [8] J. Wang, N. Huang, P. Yang, J.Y. Chen, Z.B. Yang, P.K. Chu, R. Guenzel, The 6th International Workshop on Plasma Based Ion Implantation, Gernoble, France, 2001 June 28 – 30, p. 22. [9] J. Wang, S.C.H. Kwok, N. Huang, P. Yang, P.K. Chu, 7th World Biomaterials Congress, May, 2004, Sydney, Australia, 2004. [10] J. Wang, Q.L. Liu, H. Sun, P. Yang, Z.B. Yang, Y. Leng, N. Huang, The 5th Asian Symposium on Biomedical Materials, Hongkong University of Science and Technology, 2001 9 – 12 December, p. 18. [11] N. Huang, P. Yang, Y.X. Leng, J.Y. Chen, H. Sun, J. Wang, G.J. Wan, P.D. Ding, T.F. Xi, Y. Leng, Biomaterials 24 (13) (2003) 2177. [12] P. Yang, N. Huang, Y.X. Leng, J.Y. Chen, H. Sun, J. Wang, G.J. Wan, 7th International Workshop on Plasma Based ion Implantation, Sept. 16 – 20, San Antonio, USA, 2003. [13] T.E. Mollnes, V. Videm, D. Christiansen, J.R. Bergseth, T. Hovig, Thromb. Haemost. 82 (1999) 1132. [14] P. Yang, N. Huang, Y.X. Leng, J.Y. Chen, H. Sun, J. Wang, F. Chen, P.K. Chu, Surf. Coat. Technol. 56 (1 – 3) (2002) 84. [15] J.Y. Chen, Y.X. Leng, X.B. Tian, L.P. Wang, N. Huang, P.K. Chu, P. Yang, Biomaterials 23 (2) (2002) 2545. [16] R. Hauert, Diam. Relat. Mater. 12 (3 – 7) (2003) 583.
226
N. Huang et al. / Surface & Coatings Technology 186 (2004) 218–226
[17] M.I. Jones, I.R. McColl, D.M. Grant, K.G. Parker, T.L. Parker, Diam. Relat. Mater. 8 (2 – 5) (1999) 457. [18] K. Gutensohn, C. Beythien, J. Bau, T. Fenner, P. Grewe, R. Koester, K. Padmanaban, P. Kuehnl, Thromb. Res. 99 (6) (2000) 577. [19] L.K. Krishnan, N. Varghese, C.V. Muraleedharan, G.S. Bhuvaneshwar, F. Derange`re, Biomol. Eng. 19 (2 – 6) (2002) 251. [20] P. Yang, S.C.H. Kwok, P.K. Chu, Y.X. Leng, J.Y. Chen, J. Wang, N. Huang, Nuclear Instruments and Methods in Physics Research. Section B, Beam Interact. Mater. Atoms 206 (2003) 721. [21] Y.X. Leng, J.Y. Chen, P. Yang, H. Sun, G.J. Wan, N. Huang, Surf. Sci. 531 (2) (2003) 177. [22] P. Yang, J.Y. Chen, Y.X. Leng, H. Sun, N. Huang, P.K. Chu, 7th International Workshop on Plsama Based Ion Implantation, Sept. 16 – 20, 2003, San Antonio, USA, 2003. [23] Z.Q. Yao, P. Yang, H. Sun, J. Wang, Ch.J. Pan, F. Wen, Y.X.
[24]
[25] [26] [27]
[28]
Leng, G.J. Wan, J.Y. Chen, N. Huang, Appl. Surf. Sci. 230 (1 – 4) 171 – 177. N. Huang, P. Yang, Y.X. Leng, J.Y. Chen, J. Wang, H. Sun, G.J. Wan, P.K. Chu, Y. Leng, 11th International Congress on Plasma Physics, July 14 – 18, 2002, Sydney, Australia, 2002. R.A. Weinstein, Clin. Infect. Dis. 33 (8) (2001) 1386. S.B. Calderwood, L.A. Swinski, C.M. Waternaux, A.W. Karchmer, M.J. Buckley, Circulation 72 (1) (1985) 31. J. Wang, N. Huang, P. Yang, Y.X. Leng, H. Sun, Z.Y. Liu, P.K. Chu, 7th International Workshop on Plasma Based Ion Implantation, Sept. 16 – 20, 2003, San Antonio, USA, 2003. J.Y. Chen, G.J. Wan, Y.X. Leng, P. Yang, H. Sun, J. Wang, N. Huang, 7th International Workshop on Plasma Based Ion Implantation, Sept. 16 – 20, 2003, San Antonio, USA, 2003.
Surface modification of biomaterials by plasma immersion ion implantation N. Huang *, P. Yang, Y.X. Leng, J. Wang, H. Sun, J.Y. Chen, G.J. Wan Key Lab. of Biomaterials Surface Modification of Sichuan, Laboratory of Advanced Materials Processing of Chinese Education Ministry, Southwest Jiaotong University, Chengdu 610031, China Available online 28 May 2004
Abstract The present paper presents the results of surface modifications of biomaterials using plasma immersion ion implantation (PIII) in Institute of Surface Modification of Biomaterials in Southwest Jiaotong University. In the research, titanium oxide films and diamond-like carbon (DLC) films doped with different elements, as well as surface modification of polymers, are prepared using PIII. The blood compatibility of the films is investigated. Significant improvement in blood compatibility of the films compared with those untreated substrate materials is confirmed. The mechanisms of the blood compatibility of the films are discussed. The results show that the energy band structure of the material surface may be a controlling factor in the blood compatibility of the material. A film with an n-type semiconductor nature would have a better blood compatibility. Improvement in wear resistance of hip joint and modification of antibacterial property of artificial heart valve suture ring material is also presented in the paper. D 2004 Elsevier B.V. All rights reserved. Keywords: Surface modification; Biomaterials; Biocompatibility; Plasma immersion ion implantation
1. Introduction It has been commonly agreed that the reactions on the interface between a material and biological environment play a key role to the biocompatibility of the material. Surface modification to adjust or control the surface characteristics such as surface structure, composition, functional groups, type of bonding bimolecular or cell, morphological feature of the surface, etc., has been proved to be very effective in improving biocompatibility of the material and therefore becomes one of the most attractive branch in the field of biomaterial researches. Among the techniques of surface modification, the recently developed plasma immersion ion implantation (PIII) has been showing positive potentials for its versatile processes of plasma modification, ion implantation and deposition and non-line-of-light feature. Some research groups have utilized PIII to modify the properties of biomaterials [1 –3]. In the present paper the * Corresponding author. College of Materials Science and Engineering, Southwest Jiaotong University, Chengdu 610031, PR China. Tel./fax: +8628-87600625. E-mail addresses: [email protected], [email protected] (N. Huang). 0257-8972/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.surfcoat.2004.04.041
recent researches on application of PIII technique in Key Lab. of Biomaterials Surface Modification in Southwest Jiaotong University to modify biomaterials and cardiovascular devices are presented. Further development in the research such as biomimetic surface preparation by means of PIII is also discussed. 1.1. Plasma immersion ion implantation facilities and processes of material surface treatment Since 2000, a plasma immersion ion implantation and deposition (PIIID) device [4] with a larger vacuum chamber of 1.0 m in diameter and 1.2 m high has been utilized in Institute of Surface Modification of Biomaterials in Southwest Jiaotong University. In the advice, pulsed voltages from 1 to 60 kV can be applied on the work stage. Four filtered pulsed cathodic arc sources for creating metal plasma, a RF device with a power of 4 kW and a set of filaments for generating gas plasma are attached to the device. The density of gas plasma is about 109/cm3, the deposition rate of metal film such as titanium film is about 0.2 nm/s. Different processes such as gas ion implantation, metal ion implantation, synthesis of compound films, plasma sputtering, plasma grafting and plasma enhanced
N. Huang et al. / Surface & Coatings Technology 186 (2004) 218–226
219
and PET for increasing the hemocompatibility and antibacterial property. Heparin, albumin or PEO bindings are carried out by argon plasma treating and then grafting [10]. 1.2. Behavior of blood compatibility of titanium oxide films synthesized by PIII
Fig. 1. The structures of Ti – O films vs. pressure of oxygen during PIIID – Ti – O film process.
chemical vapor deposition, etc., can be carried out using the device. For increasing the efficiency and capability of the system, especially for films synthesis, a new device with a vacuum chamber dimension of 0.6 m in diameter and 0.5 m high has also been installed. The new system has two filtered DC arc sources, an electron cyclotron resonance (ECR) gas plasma generator with the power of 2 kW. A negative pulsed voltage of 0.1 to 5 kV can be applied to the working pieces. The gas plasma density around 1011/cm3 and the deposition rate of 1.5 nm/s can be achieved. Different processes to modify biomaterials are investigated using the above mentioned facilities. For synthesis of titanium based films such as titanium oxide film, metal plasma is introduced into the work chamber from the filtered arc source and oxygen gas plasma is generated by RF or ECR. Oxygen atoms in Ti–O films can be controlled by adjusting the inlet oxygen gas flow or the power of RF or ECR. During the synthesis process, the temperature of the work pieces can be adjusted so that the structure of films can be controlled. Different kinds of diamond-like carbon (DLC) films are obtained using the PIIID facilities mentioned above [5 – 7]. In the processes, a hydrogen free DLC film is first synthesized using filtered vacuum arc plasma deposition. Carbon plasma is produced by igniting graphite cathode and the plasma diffuses into the vacuum chamber via the magnetic duct. The magnetic duct is to eliminate macro-particles. Pulsed bias from several ten voltages to several thousands voltages are applied on the work piece to obtain different bonding characteristics of the carbon films. Furthermore, the doped DLC films are deposited by introducing gas elements such as nitrogen, hydrogen, argon or fluorine into the work chamber. Hydrogenated DLC films are also deposited by acetylene gas plasma deposition. Processes for the surface modification of polymer biomaterials, such as polyurethane (PU) and polyethylene terephthalate (PET) are investigated [8,9]. Acetylene gas plasma immersion ion implantation is used to treat PU
Titanium oxide films with different structures and compositions are synthesized using the PIII facilities. Blood compatibility of the titanium oxide films are investigated in vitro and in vivo. Relationships between material characteristics and the interaction behavior of blood with material are revealed. It is found that the crystalline structure, the oxygen content and the doped elements can affect the interaction between the Ti –O films and blood. Fig. 1 shows the different structures of the Ti– O films obtained by changing the flow rate of oxygen into the vacuum chamber of the PIII device. Increasing oxygen flow, the structure of the films changes from amorphous to mixed crystalline of anatase and rutile, and further becomes Rutile. Composition analysis showed that the atom ratio of oxygen to titanium in the films increases from 1.36:1 to 2:1. As the partial pressure of oxygen reached 10 10 2 Pa, the structure of the film is rutile and its composition becomes stoichiometric TiO2. Table 1 lists the structure characteristics, adhesion behaviors of platelet and the conductive properties of the Ti –O films. As mentioned above, as the pressure of oxygen changes from 4.5 10 3 to 10 10 2 Pa during the deposition, the structure changes. As a result, the electronic properties of the film changes from near conductor to semiconductor and further to near insulator. Correspondingly, the adhesion state of platelet changes significantly. It shows that the adhesion state of platelets improved with increase in oxygen content in the film. However, as the Ti –O film becomes stoichiometric TiO2, the state of platelet adhesion turns bad, as shown in Fig. 2. Comparison of behaviors of platelet adhesion on the surface of Ti– O film of rutile, on amorphous Ti– O film which has about same composition of the rutile one, and on low temperature isotropic pyrolytic carbon (LTIC) (LTIC is considered to
Table 1 PIIID parameters, structure of the films, behavior of platelet adhesion and resistance of the films Samples Pressure Structure of O2 (Pa)
A B
4.5e 2.6e
C D
4.0e 10e
Platelet adhesion Amount/ Deformed 2500 (pseudopodium/ Am2 aggregation)%
3 Amorphous 110 F 22 98/95 2 Amorphous + 44 F 14 68/25 Crystalline 2 Rutile 15 F 11 12/20 2 Rutile 36 F 10 47/21
Resistivity q (V/cm)
2.96 10 9.62 10
4
6.76 10 >104
1
3
220
N. Huang et al. / Surface & Coatings Technology 186 (2004) 218–226
Fig. 2. Morphology of platelets on Ti – O film: (A) amorphous Ti – O film; (B) Amorphous + crystalline Ti – O film; (C) Rutile Ti – O film; (D) Rutile TiO2 film (incubation time 90 min).
have the best blood compatibility among the clinic applied mechanical heart valve materials) is presented in Ref. [11], Fig. 7. The test results show that the platelet adhesion state is significant improved on Ti– O film comparing with that on LTIC, while rutile Ti– O film is the best of them. For quantitative evaluation of the platelet activation on the material surface, approach GMP140 expression on the platelet surfaces is used [12]. GMP 140, also called Pselection (CD62), only exists on activated platelets and thus may be used as a marker of platelet activation on biomaterials [13]. Fig. 3 shows GMP140 expression on platelet surfaces indicated by percentage of positive cells, at the same incubation condition. The GMP140 expression gives a direct indication of the platelets activation as: Ti>LTI-carbon>Ti–O. Investigation on protein adsorption has been carried out by contacting fibrinogen which is radio-labeled with 125I and detected using g-counter. The level of protein adsorption on the Ti–O surface is found to be significantly lower than that on LTIC as shows in Ref. [11], Fig. 10. It is also found that the tendency to activate clotting factors is lower on the Ti– O film than on LTIC [14]. The test results suggest that one of important factors affecting the blood compatibility of the Ti– O films may be its semiconductor nature. The n-type nature and broad band gap of the Ti –O films could prevent the protein
denaturation, which is caused by charges transferring from protein onto the material. The suitable non-stoichiometric state of a titanium oxide film with a more perfect crystalline structure could posses a better semiconducting characteristic which has high electron density in the conduct band and low cavity density in the valence band and forbidden band. So far, all facts about Ti– O films are in good consistent with this consideration. Amorphous Ti–O film which has inevitably a high density of local state in the forbidden band showed bad blood compatibility than rutile TiO2 x [11], even the composition of the amorphous Ti –O film and the rutile TiO2 x is about the same. The same principle may also explain why the stoichimetric rutile TiO2 film has a lower blood compatibility than rutile TiO2 x. The reason is that the TiO2 film is almost insulator with very few electrons in the conduct band. Observation of the state of fibrinogen on TiO2 x films using atom force microscopy also confirms that most of fibrinogen on TiO2 x film surface is in single molecular state (Ref. [11], Fig. 11). Due to the lower level of protein denaturation, the tendency of platelet activating and the clotting factors are lower. Therefore, blood compatibility of the film is improved. The phenomena of the interaction of blood with titanium oxide film doped with 5+ valence element such as tantalum also agrees with this principle [15].
Fig. 3. GMP140 expression of platelets on difference materials.
Fig. 4. The surface state of mechanical heart valve (MHV) cages after in vivo test for 30 days implantation. (A) PIII treated; (B) untreated.
N. Huang et al. / Surface & Coatings Technology 186 (2004) 218–226
221
Fig. 5. Radiographic figures of coronary stents implanted in rabbits abdominal aorta for 20 weeks (no anticoagulant was taken). (A) Modified; (B) untreated.
In vivo test results improve the excellent blood compatibility of n-type Ti –O films further (Ref. [11], Figs. 12 – 14). In vivo tests from 17 to 90 days show that almost no coagulation on Ti samples with Ti– O film surfaces, while thrombus are found on LTIC samples. Fig. 4 shows the in vivo test results of mechanical heart valve (MHV) cages which have been implanted into right ventricle of a dog for 30 days without anticoagulant treatment. It clearly shows that no thrombus on the MHV cage surfaces with Ti –O film coating, but thrombi exist and have encapsulated the stent of the valve cage without Ti–O surface. Twenty weeks in vivo test results on coronary stents is shown in Figs. 5 and 6. With untreated stents, thrombus have formed and caused the constriction in the stent position. On the surface of Ti– O film coated stents, a thin layer of endothelial cells, but no thrombus, is found. These results prove further that Ti –O films have excellent blood compatibility and the films should have potentials for clinic applications.
1.3. Diamond like carbon films Diamond-like carbon (DLC) film has many attractive properties such as extremely high hardness, low friction coefficient and chemical inertness. Investigations on biocompatibility and blood compatibility of DLC film show that DLC film is biocompatible and may have good blood compatibility [16,17]. Recently, DLC film has been suggested for application in blood-contacting devices such as rotary blood pumps, artificial hearts, mechanical heart valves, and coronary stents [18,19]. However, very few investigations have been done on the relationship of the characteristics of DLC film and its biocompatibility or blood compatibility, and to modify the biomedical properties of DLC films. Our work focuses on the effect of characteristics of DLC film on blood compatibility. It is found that the relationship between the DLC film characteristics and blood compatibility is very complicated. Our research shows that it is
Fig. 6. SEM photography of the coagulation state of the coronary stents in Fig. 5.
222
N. Huang et al. / Surface & Coatings Technology 186 (2004) 218–226
possible to modify the blood compatibility of DLC film by changing the bonding state, element doping of the film, etc. Hydrogenated DLC films have been synthesized using acetylene gas plasma immersion ion implantation and deposition [20]. In the investigation, a DLC film prepared at a lower bias voltage, such as 75 V, shows a low platelet adhesion and activation, similar as LTIC but better than stainless steel. However, when the bias voltage is high, such as 900 V, the property of platelet adhesion and activation on the DLC film turns bad. This seems that the higher bonding ratio of Sp3/Sp2 may contribute to the blood compatibility of DLC. However, as we dope the DLC film with nitrogen of certain concentration, even if the bonding ratio of Sp3/Sp2 is decreased, the behavior of platelet adhesion is improved significantly and is even superior to that of LTIC, as shown in Fig. 7. Similar phenomenon has been observed in hydrogen free DLC film doped with argon or nitrogen [21]. As argon gas flow into the vacuum chamber changes from 0 to 13 sccm during the deposition of hydrogen free DLC films, the bonding ratio Sp3/Sp2 of the DLC films changes from 78/22 to 56/44, and the platelets adhesion behavior of the films is modified significantly. It is also found that above phenomena may be related to changes in surface tension on the films. The DLC film, which has a more hydrophilic nature, seems to have better blood compatibility than that of the DLC film with more hydrophobic nature [20]. This may be due to the preferential adsorption of albumin which is related to the higher adhesion work Wa of albumin compared that of fibrinogen. If DLC films are annealed at the temperature of 600 jC, the DLC films present a p-type characteristic. The blood compatibility becomes deteriorated [22]. This is another proof that n-type characteristic of the material helps maintaining the configuration of proteins, while carriers in p-type semiconductive films will help charges transferring from proteins to the material and lead to changes of the conformation of protein and activate the coagulation system. Another approach to change the surface properties of DLC films is to synthesize a fluorine contained carbon
Fig. 8. Contact angles water vs. CF4 flow of fluorine contained carbon based films.
based films using CF4 and a filtered vacuum carbon arc source on the PIII facility [23]. Fig. 8 shows the variation of water contact angles as a function of the CF4 flow. As can be seen from the figure, the water contact angles monotonously increase with the increase of CF4 flow. Improvement of the hydrophobicity of the films is obvious. With the highest CF4 flow rate, the contact angle of distilled water on the DLC film increases by one time and is close to that of PTFE. This high hydrophobicity surface possesses good anti-adhesion property. Fig. 9 gives the wear resistance of Ti6Al4V coated and uncoated with DLC. According to the wear trace and the friction coefficient analyses, increase of several orders of magnitude in the wear resistance of the Ti6Al4V can be achieved by DLC coating. This gives a great potential of using DLC film to improve the durability of biomedical device. Fig. 10 shows a DLC coated head of a metal hip joint. A significant improvement of durability of the wear couple can be expected.
Fig. 7. Behavior of platelets adhesion on DLC films with and without nitrogen as well as on LTIC (incubation time: 15 min).
N. Huang et al. / Surface & Coatings Technology 186 (2004) 218–226
223
Fig. 9. (a) The optic photography of (A) DLC film coated TI6Al4V undergo the pin on desk dry wear test of 200 thousands cycles and (B) uncoated Ti6Al4V undergo dry wear test of 10 thousands cycles (SiC pin). (b) The friction coefficient curve of DLC coated and uncoated TI6Al4V.
1.4. Application of PIII surface modification in polymer biomaterials research
Fig. 10. The DLC coated head of a metal hip joint.
Polyurethane (PU) and polyethylene terephthalate (PET) are widely used as biomaterials for cardiovascular implants such as artificial blood vessels, artificial heart valve sewing rings, etc. However, their blood compatibility is insufficient and long-term antithrombotic is required. The incidence of thromboembolic complications or bleeding complications is a serious concern. As an attempt to improve the blood compatibility of the materials, in our lab, acetylene gas plasma immersion ion implantation is used to modify PU and PET. Pulsed voltages from several kV to 10 kV are applied on the work stage to implant carbon and hydrogen ions into polymers samples. Composition and structure analyses show that an amorphous carbon layer is formed
224
N. Huang et al. / Surface & Coatings Technology 186 (2004) 218–226
Fig. 11. Morphology of adherent platelets on the (A) PET, (B) C2H2 PIII treated (incubation time 20 min).
Fig. 12. Statistics of adhesion behavior of human platelets on the untreated PU and surface-modified PUs after 15 min incubation, Ar treated: Argon plasma treated; PU-PEG: Ar treated and PEG greafted; PU-HEP: Ar treated and heparin grafted; PU-PEG-FEP: Ar treated and PEG as well as heparin grafted.
on the polymer samples. The wettability of the material is increased, while platelets adhesion and activation are suppressed, as shown in Fig. 11. By means of plasma grafting we successful bonded bimolecular such heparin, albumin and hydrophilic spacer groups polyethylene glycols (PEG) on the surface of PU and PET. Fig. 12 gives the statistics of platelet adhesion and activation. The result shows that grafting of multiple bimolecular and PEG is significantly beneficial for suppressing platelets adhesion and activation. This result, together with the results of partial thromboplastin time (APTT) testing which shows the bioactivity of intrinsic blood coagulation factors of the surface grafted PU and PET [24], confirms a significant improvement in blood compatibility of PU and PET. The infection from medical devices is a life threatening complication and leads to significant morbidity and mortality [25]. In particular, the mortality of prosthetic valve endocarditis (PVE) could be as high as about 60% in some places in China. The bacterial adhesion to the biomaterial surface is the first event in a series of both host and organismic reactions
that lead to PVE [26]. Therefore, there is an increasing interest in developing new methods to reduce bacteria adhesion onto polymeric materials used in biomedical implants. We investigated the antibacterial behavior of polyethylene terephthalate (PET) treated by acetylene (C2H2) plasma
Fig. 13. The adhered numbers of bacteria SA, SE, EC, PA, CA on the surface of the PET and C2H2 PIII treated PET.
N. Huang et al. / Surface & Coatings Technology 186 (2004) 218–226
Fig. 14. Endothelial cells growth on (A) TiO2
immersion ion implantation –deposition. Fig. 13 shows that the changes of the adhered numbers of Staphylococcus aureus (SA), Staphylococcus epidermidis (SE), Escherichia coli (EC), Pseudomonas aeruginosa (PA), Candida albicans (CA) on the surface of the PET. A significant decrease of bacteria growth on the modified PET surface is verified. The more detail work about the mechanism of suppressing bacteria growth by PIII treatment is under investigation [27]. Construction of Biomimetic surface is an attractive and new field of the biomaterial researches. Many attempts have been made to seed endothelial cells (EC) onto the surface of biomaterials. But few of the work deal with seeding endothelial cells on inorganic surface. Recently, we have developed PIII modified endothelial cell cultured Ti– O surface, and found that EC growth on crystalline Ti– O film is quite well than on amorphous Ti – O film [28], but on LTIC surface it is difficult to seed EC, as shown in Fig. 14. Further work of the long-term activity and the bonding properties are under investigation. 1.5. Conclusions The presented investigation proves that PIII technique is a new and versatile method to modify of biomaterials. Combined with other surface treatments, PIII can be widely used to fabricate films with good biocompatibility and durability. It can also be used to modify a polymer surface to improve the blood compatibility and antibacterial property of the material. PIII can also be used to bind the desired bimolecular and cells to obtain biomimetic surfaces. A great promise of obtaining new biomaterials with good biocompatibility using PIII can be expected.
Acknowledgements The authors sincerely thank the help and cooperation from Prof. P.K. Chu of City University of Hong Kong. Ian G. Brown of Lawrence Berkeley National Laboratory, California, USA. Prof. Guangjun Cai of Tongji University,
x
225
film and (B) on LTIC incubation time 85 h.
Shanghai, China. Prof. W. Moeller and Dr. M. Maitz of Research Center of Rossendorf, Germany, Prof. Xi Wu of Medical College, Southeast University of China and Prof. Tingfei Xi of National Institute for Control of Pharmaceutical and Biological Products of China. This work is jointly financially supported by Key Basic Research Program of China (G1999064705) and Natural Science Foundation of China (30270392, 50203011). References [1] A. Chen, J.T. Scheuer, C. Ritter, R.B. Alexander, J.R. Conrad, Appl. Phys. 70 (1991) 6757. [2] M. Ababneh, J.K. Gregory, U. Holzwarth, Proceedings of the 1st Tagung des DVM-Arbeitskeises ‘Biowerkstoffe, Deu tscher Verband fur Materialforschung und -prufung, Berlin’, 1998, p. 120. [3] S. Mandl, B. Rauschenbach, Surf. Coat. Technol. 156 (2000) 276. [4] P.K. Chu, B.Y. Tang, L.P. Wang, X.F. Wang, S.Y. Wang, N. Huang, Rev. Sci. Instrum. 72 (3) (2001) 1660. [5] Y.X. Leng, J.Y. Chen, P. Yang, H. Sun, G.J. Wan, H. Huang, Surf. Sci. 531 (2) (2003) 177. [6] P. Yang, N. Huang, Y.X. Leng, J.Y. Chen, R.K.Y. Fu, S.C.H. Kwok, Y. Leng, P.K. Chu, Biomaterials 24 (17) (2003) 2821. [7] Y.X. Leng, J.Y. Chen, P. Yang, H. Sun, G.J. Wan, N. Huang, Surf. Coat. Technol. 173 (1) (2003) 67. [8] J. Wang, N. Huang, P. Yang, J.Y. Chen, Z.B. Yang, P.K. Chu, R. Guenzel, The 6th International Workshop on Plasma Based Ion Implantation, Gernoble, France, 2001 June 28 – 30, p. 22. [9] J. Wang, S.C.H. Kwok, N. Huang, P. Yang, P.K. Chu, 7th World Biomaterials Congress, May, 2004, Sydney, Australia, 2004. [10] J. Wang, Q.L. Liu, H. Sun, P. Yang, Z.B. Yang, Y. Leng, N. Huang, The 5th Asian Symposium on Biomedical Materials, Hongkong University of Science and Technology, 2001 9 – 12 December, p. 18. [11] N. Huang, P. Yang, Y.X. Leng, J.Y. Chen, H. Sun, J. Wang, G.J. Wan, P.D. Ding, T.F. Xi, Y. Leng, Biomaterials 24 (13) (2003) 2177. [12] P. Yang, N. Huang, Y.X. Leng, J.Y. Chen, H. Sun, J. Wang, G.J. Wan, 7th International Workshop on Plasma Based ion Implantation, Sept. 16 – 20, San Antonio, USA, 2003. [13] T.E. Mollnes, V. Videm, D. Christiansen, J.R. Bergseth, T. Hovig, Thromb. Haemost. 82 (1999) 1132. [14] P. Yang, N. Huang, Y.X. Leng, J.Y. Chen, H. Sun, J. Wang, F. Chen, P.K. Chu, Surf. Coat. Technol. 56 (1 – 3) (2002) 84. [15] J.Y. Chen, Y.X. Leng, X.B. Tian, L.P. Wang, N. Huang, P.K. Chu, P. Yang, Biomaterials 23 (2) (2002) 2545. [16] R. Hauert, Diam. Relat. Mater. 12 (3 – 7) (2003) 583.
226
N. Huang et al. / Surface & Coatings Technology 186 (2004) 218–226
[17] M.I. Jones, I.R. McColl, D.M. Grant, K.G. Parker, T.L. Parker, Diam. Relat. Mater. 8 (2 – 5) (1999) 457. [18] K. Gutensohn, C. Beythien, J. Bau, T. Fenner, P. Grewe, R. Koester, K. Padmanaban, P. Kuehnl, Thromb. Res. 99 (6) (2000) 577. [19] L.K. Krishnan, N. Varghese, C.V. Muraleedharan, G.S. Bhuvaneshwar, F. Derange`re, Biomol. Eng. 19 (2 – 6) (2002) 251. [20] P. Yang, S.C.H. Kwok, P.K. Chu, Y.X. Leng, J.Y. Chen, J. Wang, N. Huang, Nuclear Instruments and Methods in Physics Research. Section B, Beam Interact. Mater. Atoms 206 (2003) 721. [21] Y.X. Leng, J.Y. Chen, P. Yang, H. Sun, G.J. Wan, N. Huang, Surf. Sci. 531 (2) (2003) 177. [22] P. Yang, J.Y. Chen, Y.X. Leng, H. Sun, N. Huang, P.K. Chu, 7th International Workshop on Plsama Based Ion Implantation, Sept. 16 – 20, 2003, San Antonio, USA, 2003. [23] Z.Q. Yao, P. Yang, H. Sun, J. Wang, Ch.J. Pan, F. Wen, Y.X.
[24]
[25] [26] [27]
[28]
Leng, G.J. Wan, J.Y. Chen, N. Huang, Appl. Surf. Sci. 230 (1 – 4) 171 – 177. N. Huang, P. Yang, Y.X. Leng, J.Y. Chen, J. Wang, H. Sun, G.J. Wan, P.K. Chu, Y. Leng, 11th International Congress on Plasma Physics, July 14 – 18, 2002, Sydney, Australia, 2002. R.A. Weinstein, Clin. Infect. Dis. 33 (8) (2001) 1386. S.B. Calderwood, L.A. Swinski, C.M. Waternaux, A.W. Karchmer, M.J. Buckley, Circulation 72 (1) (1985) 31. J. Wang, N. Huang, P. Yang, Y.X. Leng, H. Sun, Z.Y. Liu, P.K. Chu, 7th International Workshop on Plasma Based Ion Implantation, Sept. 16 – 20, 2003, San Antonio, USA, 2003. J.Y. Chen, G.J. Wan, Y.X. Leng, P. Yang, H. Sun, J. Wang, N. Huang, 7th International Workshop on Plasma Based Ion Implantation, Sept. 16 – 20, 2003, San Antonio, USA, 2003.