Pulsed Laser Deposition method (PLD) was used to grow nitinol (NiTi) thin films with goal of investigating their biocompatibility. High purity Ni and Ti targets ...
Mat. Res. Soc. Symp. Proc. Vol. 780 © 2003 Materials Research Society
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Biocompatibility Studies of the Nitinol Thin Films C.Z. Dinu1, R. Tanasa2, V.C. Dinca1, A. Barbalat1, C. Grigoriu1, E.O. Bucur2, A. Dauscher3, V. Ferrari DeStefano4, M. Dinescu1 1 National Institute for Laser, Plasma and Radiation Physics, PO Box MG–16, RO 76900, Bucharest, Romania, 2 Pasteur Institute S.A, Calea Giulesti 333, Bucharest, 77 826, Romania 3 Laboratories of Materials Physic (LPM), INPL, Ecole des Mines de Nancy, Parc de Saurupt, F-54042 Nancy, France 4 University of Rome “La Sapienza”, Dept. of Electronics, 00186 Rome, Italy ABSTRACT Pulsed Laser Deposition method (PLD) was used to grow nitinol (NiTi) thin films with goal of investigating their biocompatibility. High purity Ni and Ti targets were alternatively ablated in vacuum with a laser beam (λ=355 nm, 10 Hz) and the material was collected on room temperature Ti substrates. X-ray diffraction, scanning electron microscopy, energy dispersive spectroscopy and atomic force microscopy analyses have been performed to investigate the chemical composition, crystalline structure and surface morphology of the NiTi films. The nitinol layers biocompatibility has been tested using as a metric the extent to which the cells adhere during the culture period on the surface of NiTi layers deposited on Ti substrates. Vero and fibroblast cell lines dispersed into MEM (Eagle) solution containing 8% fetal bovine serum, at 370 C, were used for tests. Preliminary studies indicate that the interaction at the interface is specifically controlled by the surface morphology, (especially by surface roughness), and by the chemical state of the surface. Cell behavior after contact with NiTi/Ti structure for different intervals (18, 22 and 25 days for the Vero cells, and after 10 and 25 days for fibroblasts) supports the conclusion that NiTi is a very good candidate as a biocompatible material. 1. INTRODUCTION Nickel-titanium (Ni-Ti, nitinol), a thermo elastic alloy with a composition of approximately 50 atomic % nickel, exhibits special properties like shape memory effect, superelasticity, radiopacity, plateau stresses and transformation temperatures that makes it an attractive candidate for an increasing number of medical applications such as orthopedic implants, needles, guide wires, orthodontic wires, bone substitution material and endoscopic instruments to implant (stents and filters). However, there are still a small amount of biocompatibility data available so far. A number of studies have been performed in vitro in order to prove the nitinol biocompatibility, some of them covering the behavior during and after sterilization [1]. The results of the studies performed in vivo, regarding the cytotoxicity of the Ni-Ti in long term behavior are ambiguous: part of the reports notice good biocompatibility for this alloy [2-4], but there are also reports about the negative potential impact on biological systems [5, 6]. It is well known that the toxic effects of different metals can be well quantified in vitro, although cell culture studies cannot directly mimic the cellular response and environment in vivo. Generally, the cell culture method is considered as a very sensitive method of toxic screening
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[7]. The studies use a method of incubation-cultured cells in metal-immersed media and include test experiments of the alloys discs, wires, particles or thin films. The purpose of this study was to evaluate the use of pulsed laser deposition (PLD) in growing of Ni-Ti alloy thin films with variable compositions and their in vitro biocompatibility, while maintaining the mechanical properties of the material. The films were deposited on Ti substrates using high purity nickel and titanium targets. The biocompatibility studies were focused on the growth of the cells, like Vero and fibroblast animal cell lines dispersed into MEM (Eagle) solution containing 8 % fetal bovine serum, at 370 C, on adhesion and proliferation in vitro of these cells on the NiTi thin films deposited on Ti substrate.
2. EXPERIMENTAL DETAILS Thin Films Deposition/ Thin Films Characterization Nitinol thin films were prepared by pulsed laser deposition (PLD) by irradiating two different targets. A frequency tripled Nd:YAG pulsed laser (λ=355nm, τ FWHM =5ns) operating at a 10 Hz repetition rate was used to irradiate in vacuum (10-6 mbar) two high purity (99.99%) Ni and Ti targets mounted on a multi-target system. To achieve a stoichiometric layer, the number of laser pulses irradiating each target was set at 15 pulses for the Ti target and 10 pulses for the Ni target. This process was repeated for 10, 20 and 30 sequences to obtain layers with a thickness in the range of 200-300 nm and a homogeneous distribution of elements inside the layer. During the deposition process the targets were rotated and translated in order to avoid crater formation. Polished Ti plates have been used as substrates and the target-collector distance was set at 5 cm. All depositions have been performed at room temperature. The laser fluence was varied from 3J/cm2 to 10 J/cm2. X-Ray Diffraction (XRD) was used to identify the crystalline structure of the NiTi films. Scanning Electron Microscopy (SEM) and energy dispersive spectroscopy (EDS) analyses were carried out in order to check Ni/Ti ratio in the film and the layers surface topography. Q-ScopeTM 250 (Quesant) atomic force microscope has been used to determine the surface roughness and low scale morphology (in direct physical contact mode using a proportional -integral- derivative feedback control). The film surface was analyzed before and after the sterilization procedure.
Cell Culture Vero (kidney, African green monkey, Pasteur Bucharest Cell Culture Banc) [8] and fibroblast cell line (Pasteur Bucharest Cell Culture Banc), were obtained using the noncontinuous trypsining method at 37 °C [9]. They were cultured and subcultured in 75 cm2 plastic tissue culture flasks (CORNING, Corning Costar Corporation, Cambridge, MA, USA) containing MEM medium (Eagle) (MEME), with L-glutamin and 25 mM HEPES (Gibco BRL, Life Technologies, Paisley, Scotland), suplimentated with 8% bovine fetal serum (SFB) (SIGMA) and antibiotics (penicillinum 200 UI/ml, streptomycin 200 µg/ml, amphotericin B 5 µg/ml), the final solution having a pH in the range of 7,0-7,1. The incubation process was done at 37oC, in dry athmosphere, without CO2 and the subcultures were made after an interval of 2-4 days by splitting the original cultures with a Versen-0,2% / trypsin-0,25% solution [8].
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Cell Culture Experiments/ Cells culture examination Vero and fibroblast cells were extracted from 75 cm2 plastic tissue culture flasks and introduced in 25cm2 plastic tissue culture flasks, at a concentration of 2 x 105 cells/ml medium, following all the previous conditions. The samples of nitinol thin films/Ti substrates were sterilized before immersion in the cells cultures by exposure to a UV sterilizing lamp (model TUNGSRAM 15 w F – Germicid B2Y); the samples were maintained 15 cm from the lamp for 24 hours. After the sterilization procedure, the samples were immersed in the 25 cm2 plastic tissue culture flasks; 72 hours later, the culture medium was replaced with MEME suplemented with 1% SFB and antibiotics (penicillinum 200 UI/ml, streptomycin 200 µg/ml, amphotericin B 5 µg/ml). The flasks with the immersed nitinol/Ti samples and those containing reference cultures were kept under the same experimental cell culture conditions. The cell growth and their behavior at the NiTi film/ cells monolayer interface were examined after 18, 22 and 25 days for the Vero cells, and after 10 and 25 days for fibroblasts, using an inverted microscope (ADMIRAL G.M.C., Japan) equipped with a photo camera (Practika, Jena, Germania) and a microscope (Universal Microscope MU2 Carl Zeiss), equipped with a Epson digital camera. After 18, 25 days of incubation (18 days for Vero and 25 days for fibroblasts), NiTi films were washed three times with phosphate buffer saline – PBS) (137mM NaCl; 2,7mM KCl; 4,3mM Na2HPO4 x 7H2O; 1,4mM KH2PO4; pH 7,2-7,4), fixed with methanol for 20 minutes and stained for 60 minutes with Giemsa 10% solution [10]. After fixation and staining with Giemsa the NiTi samples were examinated using an Q-ScopeTM 250 (Quesant) atomic force microscope. The images were obtained at three different points on the samples in order to evaluate the cell growth.
3. RESULTS AND DISCUSSIONS Biocompatibility of NiTi layers can be improved by a suitable adjustment of characteristics, such as the relative chemical composition (Ni/Ti), crystallinity and roughness, etc.[11-14]. It is well known that by varying PLD deposition parameters such as laser wavelength, fluence, target-substrate distance and deposition pressure, etc. will in turn allow the control of film thickness as well as the film uniformity and roughness [15]. The film composition can be controlled by adjusting the number of laser pulses sent to each target. Ni-Ti ratio in the range of 48/51 and 51/49 have been measured by EDS. X-Ray Diffraction studies indicated a polycrystalline structure for the deposited NiTi layers, with a preferential (111) orientation. Spectra of two samples are presented in Fig.1: the Ti peak coming from substrate is also present.
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Figure 1: X-Ray Diffraction Spectra of the nitinol thin film deposited on a Ti substrate
SEM images (not presented here) indicated a uniform and dense surface structure without any cracks or pores. We have tested the assumption that the cells adhere during the culture period on the surface of NiTi layers deposited on Ti substrate, the adhesion being an important factor for the future nitinol biomedical applications such as spreading, proliferation, migration and biosynthetic activity [16]. It is known that cell adhesion to the nitinol is mediated through preadsorbed proteins, which forms local contacts with the cell membrane [17, 18]. A certain amino acid sequence of these proteins binds to integrins that are connected with the cytoskeleton [19, 20]; in this way, several other proteins inside the cell respond to cell signals [16]. The cell growth on the nitinol thin films in culture flasks was observed (and compared) with respect to those from control cultures (Fig. 2). Ater 18 days immersion in the medium, the nitinol samples have shown no significant differences between the number of cells from the test flask with respect to those from the reference flask, for both types of cells. The fibroblast and Vero cell monolayers thrive as naturally as the reference ones and a tendency of the interface cells to adhere to the samples edges has been observed. The same characteristics were maintained and visualized by direct microscopic examination after 25 days in the case of the fibroblast cells stained with Giemsa. No macroscopic changes of cells cultured on NiTi samples deposited on the Ti have been noticed.
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Figure 2: a. b. c.
Vero and fibroblast cell cultures fixed stained with Giemsa on the nitinol thin films Vero reference culture after10 days of incubation Fibroblast references cell culture after 10 days of incubation Vero cell cultures on NiTi films after 25 days incubation
The cells adhered to the nitinol thin film; their cytoplasmic prolongations are well developed and they did not show any tendency to change their (round) shape. The junctions are also preeminent, this being a good prove of the fact that the nitinol thin film form a good support for the growing processes. In addition to this, the results indicate the in vitro noncytotoxicity of NiTi deposited films. We have applied AFM to study the dynamic behavior of the fixed cells and their adhesion to the nitinol deposited samples. The cells morphological features and the adhesion areas on material surface have been analyzed. The nitinol/Ti structures have been first investigated as deposited and then after the sterilization procedure since it is known that this process could induce changes [1]. Subsequently, cellular behavior at the interface after NiTi/Ti immersion have been compared with the reference cultures.
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Figure 3: 3-D images of fibroblast cells on nitinol thin films by using AFM direct contact mode. The maximum height is 3 µm. a) Ti substrate, b) NiTi film, c) NiTi film sterilized, d) NiTi with cells, e) Reference culture onto the flask It can be shown (Fig. 3) that the cell adhesion followed the profile of the nitinol thin films (and its roughness). In previous studies it was observed that the surface roughness has an important effect on the cell orientation [10, 11]. This effect was not noticed in our case: cells are chaotic oriented, without a preferential orientation along the axes or grooves. This could be a consequence of the homogenous roughness of surface structure of NiTi thin films obtained by PLD. The sterilization procedure and its influence on the alloy properties is another important aspect, less studied [1, 21]. No considerable difference between the morphology of the nitinol thin films after the sterilization procedure, performed with the protocol described in the Cell examination section, and the unsterilized films has been noticed. Another way to test the biocompatibility of a material is the detection of the apoptosis of the living cells that adhere to the samples. It was evidenced that the number of the cells on the nitinol thin films is not changed in comparison with the number of the cells from the control culture flasks. The apoptosis can be also checked by measuring the Ni ions concentration in solution, since it is known that the released nickel ions could have a toxic effect on the cell culture [22]. They could act like an inhibitor of the enzymatic processes involved in cell development and
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also can disrupt the intracellular organelles, change cell morphology and decrease the cells number. One of the main questions was whether the amount of nickel dissolved from the NiTi thin films is high enough to affect the cell proliferation in a cell culture and what is the dissolution rate for nickel. We have checked the amount of nickel dissolved in solution by using a (Solar 92 AA) spectrometer. The control sample concentration was 0.0294 mg/l; after three days the concentration was 0.0524 mg/l and no a further increase was noticed after 25 days; the limit in the literature is reported to be less then 0.1 mg/l [14]. In our experiments, the volume of media and cells was quite small compared to the volume and surface area of the tested metal thin films. Thus, the corrosion effect on cells is expected to be much more prominent with respect to the in vivo case. The release of Ni ions in solution is also related to the corrosion; the key to this alloy biocompatibility resides in improvement of the corrosion behavior and also improvement of the surface properties. The surface homogeneity of NiTi films prepared by PLD and the deposition finishing with laser pulses on Ti target is thus an advantage for the adhesion of the cells and also for the low concentration of the Ni ions in solution. 4. CONCLUSIONS We have demonstrated that the nitinol thin films obtained using PLD are suitable materials for biological applications. In vitro proliferation experiments showed good biocompatibility of the nitinol/Ti structures to the Vero and fibroblast cell culture. 5. ACKNOWLEDGMENTS The authors gratefully acknowledge help from D. Matei, I. Vrejoiu, and C. Ghica for experimental assistance and D. Dumitras for the scientific advices. Research was partially supported by EC IST 2001-33326- PISSARO Project. 6. REFERENCES 1. Thierry B., Tabrizian M., Savadago O., Yahia L., Effects of sterlization processes on NiTi alloy: surface characterization, J. Biomed Mater Res 2000, 49:88-98. 2. Wever D.J., Velduizen A.G., Sanders M.M., Schakenraad J.M., van Horn J.R., Cytotoxic, allergic and genotoxic activity of a nickel- titanium alloy, Biomaterials 1997; 18: 11151120. 3. Trepanier C., Tabrizian M., Yahia L., Bilodeau L., Piron D.L., Effect of modification of oxide layer on NiTi stent corrosion resistance,J. Biomed. Mater. Res. (Appl. Biomater.) 1998; 43:433-440. 4. Trepanier C., Leung T. K., Tabrizian M., Yahia L., Bienvenu J.G., Tanguay J.F., Piron D.L.,Bilodeau L., Preliminary investigation of the effects of surface treatments on biological response to shape memory NiTi stents,J. Biomed. Mater. Res. (Appl. Biomater.) 1999; 48:165-171 5. Wataha J.C., Lockwood P.E., Marek M., Ghazi M., Ability of Ni-containing biomedical alloys to active monocytes and endothelial cells in vitro, Biomed. Mater. Res. 1999; 45: 251-257.
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6. Assad M., Lemieux N., Rivard C.H., Yahia L., Comparative in vitro biocompatibility of nickel- titanium, pure nickel, pure titanium and stainless steel: genotoxicity and atomic absorption evaluation, Biomed. Mater Eng. 1999; 9 :1- 12. 7. Rae, T. (1986) In: Techniques of Biocompatibility Testing, Volume II, Williams, D.F., ed., CRC Press, Boca Raton, FL, 81. 8. *** Flow Manual. Flow Laboratories Ltd, Victoria Park, Irvine, 1974, 108 9. Freshney RI. Culture of animal cells. A manual of basic technique. Third edition, WileyLiss, Inc., New York, 1994, p. 127 – 147. 10. Tanasa R. Contributions to the study of some animal lentiviroses. DSc (PhD) Thesis. Bucharest University of Agronomic Sciences and Veterinary Medicine, 2001, p. 124 – 138 (in Romanian). 11. Brailovski, V., Trochu, F., 1996. Review of shape memory alloys medical applications in Russia. Bio-Medical of Materials & Engineering 6 (4), 291–298. 12. Rutner B., Surface properties of biomaterials, Biomaterial Science, Academic Press, 1996, 21-34. 13. Wen X., Wang X., Zhang N., Micro-rough surface of metallic biomaterials, Review, BioMed. Mater. Eng., 1996; 6: 173-189. 14. Shabalovskaya A. S., Surface, corrosion and biocompatibility aspects of Nitinol as an implant material, Bio- Medical Materials and Eng. 12, 2002: 69-109. 15. Chrisey D.B., Hubler G.H., Pulsed Laser Deposition of Thin Films, Eds. John Wiley, INC. 229 1994. 16. Gumbiner B.M., Proteins associated with the cytoplasmic surface of adhesion molecules, Neuron 11, 1993, 551-564. 17. Anderson JM, Bonfield TL & Ziats NP (1990) Protein adsorption and cellular adhesion and activation on biomedical polymers. Int.J.Artif.Organs 13: 375-382. 18. Groth T, Altankov G & Klosz K (1994) Adhesion of human peripheral blood lymphocytes is dependent on surface wettability and protein preadsorption. Biomaterials 15: 423-428. 19. Pytela R, Pierschbacher MD & Ruoslahti E (1985) Identification and isolation of a 140 kd cell surface glycoprotein with properties expected of a fibronectin receptor. Cell 40: 191-198. 20. Ruoslahti E & Pierschbacher MD (1987) New perspectives in cell adhesion: RGD and integrins. Science 238: 491-497. 21. Shabalovskaya S., Anderagg J., Sachdeva R., Harmon B., Preliminary XPS spectroscopic characterization of autoclaved NiTi shape memory alloys for implants, Biomaterials for Drug and Cell Delivery, MRS, Vol.331, 1994:239-244. 22. Gerber H & Perren SM (1980) Evaluation of tissue compatibility of in vitro cultures of embryonic bone. In: Winter GD, Leray JL & de Groot K (Eds) Evaluation of Biomaterials, Wiley, New York, p 307-314.
