Biocompatibility of corrosion-resistant zeolite coatings for titanium alloy biomedical implants

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Acta Biomaterialia 5 (2009) 3265–3271 www.elsevier.com/locate/actabiomat

Biocompatibility of corrosion-resistant zeolite coatings for titanium alloy biomedical implants Rajwant S. Bedi a, Derek E. Beving a, Laura P. Zanello b, Yushan Yan a,* a

Department of Chemical & Environmental Engineering, University of California, Riverside, CA 92521, USA b Department of Biochemistry, University of California, Riverside, CA 92521, USA Received 23 January 2009; received in revised form 11 March 2009; accepted 17 April 2009 Available online 3 May 2009

Abstract Titanium alloy, Ti6Al4V, is widely used in dental and orthopedic implants. Despite its excellent biocompatibility, Ti6Al4V releases toxic Al and V ions into the surrounding tissue after implantation. In addition, the elastic modulus of Ti6Al4V (110 GPa) is significantly higher than that of bone (10–40 GPa), leading to a modulus mismatch and consequently implant loosening and deosteointegration. Zeolite coatings are proposed to prevent the release of the toxic ions into human tissue and enhance osteointegration by matching the mechanical properties of bone. Zeolite MFI coatings are successfully synthesized on commercially pure titanium and Ti6Al4V for the first time. The coating shows excellent adhesion by incorporating titanium from the substrate within the zeolite framework. Higher corrosion resistance than the bare titanium alloy is observed in 0.856 M NaCl solution at pHs of 7.0 and 1.0. Zeolite coatings eliminate the release of cytotoxic Al and V ions over a 7 day period. Pluripotent mouse embryonic stem cells show higher adhesion and cell proliferation on the three-dimensional zeolite microstructure surface compared with a two-dimensional glass surface, indicating that the zeolite coatings are highly biocompatible. Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Corrosion resistant; Biocompatible; Zeolite; Biomaterial; Stem cells

1. Introduction The most essential properties of a biomedical implant, such as a total joint replacement, include biocompatibility, corrosion resistance and an elastic modulus that closely matches that of the human bone to avoid bone resorption [1–3]. At present, titanium alloy, Ti6Al4V, is the material most widely used for these implant applications because it is readily available and has reasonable biocompatibility and corrosion resistance and a relatively low modulus when compared to other alloys, such as stainless steel and cobalt chromium alloys [4]. However, Ti6Al4V still releases vanadium and aluminum ions [5–7], causing poor * Corresponding author. Address: Department of Chemical & Environmental Engineering, University of California, 900 University Ave., Riverside, CA 92521, USA. Tel.: +1 951 827 2068; fax: +1 951 827 5696. E-mail address: [email protected] (Y. S. Yan).

osteointegration and a limited lifespan of the titanium prosthesis. Vanadium ions are cytotoxic, while aluminum ions can cause neurological disorders [8–10]. To prevent or reduce the release of harmful ions, ceramic and polymer coatings have been applied [5], but below-par material properties and poor adhesion of these biocoatings to the metal substrate have led to their failure. In addition, although the elastic modulus of Ti6Al4V (110 GPa) [11] is among the lowest of metal alloys used for implant applications, it is still much higher than that of the bone (30 GPa) [12], leading to bone resorption. To avoid the release of harmful ions and to reduce the elastic modulus, highly sophisticated titanium alloys (e.g., TiNbZr) have been developed, but they are expensive. Zeolites are aluminosilicates with a uniform microporous structure, and have been exploited commercially for catalysis and separation processes [13]. Researchers have shown zeolites to be non-toxic, viable carriers, controlled

1742-7061/$ - see front matter Ó 2009 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.actbio.2009.04.019

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release agents and adjuvants for drugs, thus showing their potential for biomedical applications [12–19]. While zeolites are used in composite powder form for catalysis and separation applications, high-silica zeolite coatings have previously been prepared on aluminum alloys and shown to be highly resistant to corrosion [20]. Zeolite coatings act as a barrier between metal and corrosive medium to prevent corrosion. In addition, zeolite coatings have been shown to have an elastic modulus of 30–40 GPa [21], which much more closely matches that of a bone than titanium alloys [22]. In this study we show that a high-silica zeolite MFI1 coating can be deposited on titanium alloys to prevent the dissolution of the underlying metal, thus eliminating the release of harmful ions. More importantly, we demonstrate that zeolite coatings are biocompatible and can better sustain the growth of mouse embryonic stem cells (mESC) on a fibroblast feeder layer than glass. 2. Materials and methods 2.1. Substrate and substrate pretreatment Commercially pure titanium (cpTi, 99.5%, 0.25 mm thick; Alfa Aesar, Ward Hill, MA) and high strength titanium (Ti6Al4V, 6% Al, 4% V, 0.41 mm thick; McMaster Carr, Cleveland, OH) were purchased. The substrates were sized to 15.25  7.62 cm panels and immersed at 70 °C for 1 h in an AlconoxÒ detergent solution prepared with 3.0 g of AlconoxÒ (Sigma– Aldrich, St. Louis, MO) in 400 ml of deionized (DI) H2O. The substrates were then rinsed under DI H2O with mild rubbing. Substrates were dried with compressed air and kept at ambient conditions for less than 1 h before immersion in HSZ-MFI synthesis solution. 2.2. Coating solution formulation High-silica zeolite (HSZ) MFI coatings were prepared by an in situ crystallization method. First, a clear synthesis solution with a molar composition of 0.16TPAOH:0.64NaOH:1TEOS:92H2O:0.0018Al (weight compositions: 17.03 g TPAOH, 5.36 g NaOH, 43.60 g TEOS, 336.00 g H2O, 0.0105 g Al) was prepared by dissolving aluminum powder (200 mesh, 99.95+ wt.%; Aldrich, St. Louis, MO) in sodium hydroxide (99.99 wt.%; Aldrich) and DI H2O followed by the dropwise addition of tetrapropylammonium hydroxide (TPAOH, 40 wt.%, aqueous solution; SACHEM, Austin, TX) and tetraethylorthosilicate (TEOS, 98 wt.%; Sigma–Aldrich, St. Louis, MO) under stirring. The clear solution was aged at room temperature for about 4 h under stirring before use.

1 MFI is a three-letter code assigned to the zeolite used in this study by the International Zeolite Association.

2.3. Coating deposition A 2 l Teflon-lined Parr autoclave (Model # 4622, Parr Instruments, Moline, IL) was used as the synthesis vessel and the substrate was suspended vertically inside the synthesis solution using a TeflonÒ holder and steel wire. Crystallization was carried out in a convection oven at 175 °C for 24 h, after which the autoclave was removed from the oven and quenched with tap water. The coated sample was rinsed with DI H2O and dried in ambient room air before characterization. 2.4. Physico-chemical characterizations Scanning electron microscopy (SEM) micrographs were obtained on a Philips XL30-field emission gun scanning electron microscope operated at between 5 and 20 kV. An Au/Pd coating was applied to MFI-coated cpTi and Ti6Al4V samples by sputtering for 20 s before SEM imaging. Adhesion of the coating to cpTi and Ti6Al4V substrates was determined according to ASTM Test Method D 3359, using PAT-2000 adhesion test kit (Paul N. Gardner Co., Pompano Beach, FL). Following a scratch test, the coating is visually inspected and rated on a scale of 0–5, with 5 being the highest rating. A focused ion beam (FIB; Leo XB1540), equipped with a gallium ion gun, was used to mill a 15 lm deep trench in the zeolite–titanium system. Incorporation of titanium in the zeolite structure was determined by semi-quantitative energy-dispersive X-ray spectroscopy (EDS), using the EDAX (Mahwah, NJ) analytical system attached to the FIB column. X-ray diffraction (XRD) measurements were done on bare and MFI-coated cpTi and Ti6Al4V using a Bruker AXS (Madison, WI) D8 Advance Diffractometer using Cu Ka radiation. 2.5. Corrosion resistance Large coated substrates were cut into 2.5  3.8 cm coupons for direct current (DC) polarization tests. Before immersing the coupons into corrosive medium, the edges of the coated substrate were sealed with five-min epoxy (Grainger, Riverside, CA) while exposing the coating surface on which corrosion-resistance was to be measured. Polarization testing was carried out with a Solartron (Farnborough, Hampshire, UK) potentiostat SI 1287 in a threeelectrode configuration, with the zeolite-coated substrate as the working electrode, a platinum foil as the counter electrode and an Ag/AgCl-saturated KCl electrode as the reference electrode. The corrosive medium was either 0.856 M NaCl (pH 7.0) or 0.856 M NaCl/HCl (pH 1.0) aqueous solution. Zeolite-coated samples were immersed in the corrosive medium for 0–7 days prior to the polarization test (note that immersion for a few minutes up to 30 min is normally employed by others for corrosion testing). Ambient temperature was maintained during all polarization tests. A scan rate of 1 mV s1 was applied

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and all potentials were measured with respect to the reference electrode. After substrates were removed from the solution, the solution was tested for the presence of Al, Ti and V ions using a Perkin Elmer (Waltham, MA) 3000DV inductively coupled plasma optical emission spectrometer. 2.6. Biocompatibility Large coated substrates were then cut into 1.5  1.5 cm for cell culture. All zeolite surfaces were ultraviolet irradiated overnight before cell culture. A fibroblast cell (MEF cell line STO from ATCC, Manassas, VA) culture was prepared in Dulbecco’s modified Eagle’s medium (DMEM) with a 10% fetal bovine serum concentration. MFI-coated Ti6Al4V substrates as well as glass coverslips were placed in Petri dishes and immersed in DMEM medium, then aliquots of fibroblast cells were added to the medium and incubated at 10 °C for 24 h. After a 100% confluent fibroblast layer was obtained on the substrates, mouse embryonic stem cells (mESC line D3 from ATCC, Manassa, VA) were cultured on the substrates with a fibroblast layer in a medium containing 122 ml of DMEM, 22 ml of fetal bovine serum (inactive, 15%), 1.5 ml of L-glutamine (4 mM), 1.5 ml of sodium pyruvate (1%), 1.5 ml of nonessential amino acids (1%), 750 ll of penicillin/streptomycin (0.5%), 150 ll of leukemia inhibitory factor (LIF, 1000 units ml1) to maintain pluripotency and 15 ll of 2mercaptoethanol (0.1 mM). After 1 day of culture, cells were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate for 1 h, incubated in 1% osmium tetraoxide for

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another hour and dehydrated in an ethanol series. Samples were dried using Balzar’s critical point dryer, and subsequently sputter coated with Au/Pd layer for SEM imaging. 3. Results and discussion Continuous and polycrystalline coatings on Ti6Al4V (Fig. 1A and C) were successfully synthesized. The crosssectional SEM images revealed that the coating thickness is uniform and about 8 lm (data not shown). EDS analyses confirmed the composition of the bare metal substrates specified by the manufacturer (Fig. 1B), and showed that the MFI coating was primarily composed of Si and O (Fig. 1D), which is consistent with the previously published literature [20,23]. XRD results confirmed that the coating had a pure MFI structure (data not shown). Adhesion of the MFI coating to the Ti6Al4V substrates was tested according to American Society of Testing and Materials (ASTM) test protocol D 3359 and the highest possible rating of 5B was obtained. No delamination or flaking of the coating was observed after scratches were made on the coating. To understand the excellent adhesion observed, FIB milling was carried out to visualize the coating–substrate interface and study the possible transition of elemental composition across the interface. There is no observable gap between the metal surface and the MFI coating (Fig. 2A). Some cracks are visible in the coating, but their penetration into the substrate at the same location indicates that the cracks are not innate to the coating but, rather, are a result of the FIB milling process. The crack observed in the zeolite coating (Fig. 2) starts at the top sur-

Fig. 1. (A) SEM micrograph of the Ti6Al4V surface; (B) EDS analysis of the Ti6Al4V surface; (C) SEM micrograph of the MFI coating on Ti6Al4V; (D) EDS analysis of the MFI coating on Ti6Al4V.

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is shared between two adjacent tetrahedra. A significant portion of the Si atoms can be replaced by Al and Si/Al ratio from 1 to infinity has been observed. Therefore the significant incorporation of Al into the zeolite framework previously observed is understandable. SiO4 tetrahedra can also be replaced by Ti tetrahedra, although usually to a much lesser degree than AlO4. Specific to the MFI framework, when Ti is incorporated the resultant material is called TS-1. TS-1 has been extensively studied for catalysis and other applications [24–27]. Corrosion resistance of bare and MFI-coated Ti6Al4V was determined using the DC polarization method (Fig. 3). A much lower corrosion current density was observed for the MFI-coated samples (8.6E-8 A cm2 in pH 7.0 and 1.7E-7 A cm2 in pH 1.0) than the bare Ti6Al4V (8.0E-6 A cm2 in pH 7.0 and 9.3E-5 A cm2 in pH 1.0) samples after 7 days of immersion in neutral and acidified salt solutions (Fig. 3). MFI-coated samples showed no change in current density over 7 days of immersion, while bare Ti6Al4V samples showed an increase in current density over time of several orders of magnitude. For the bare Ti6Al4V samples, the increase in current density over time is more significant in the acidified medium Fig. 2. (A) SEM micrograph of the MFI–Ti6Al4V interface; (B) SEM micrograph overlaid with an EDS linescan indicating th eincorporation of Ti (green) into the MFI framework (Si, yellow). Below the interface is metal substrate, above it is MFI. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

face of the coating and does not end at the coating–substrate interface, but penetrates further into the substrate until the bottom of the trench. We did not observe cracks in the coating when trenches were milled using very small amounts of current. This procedure deposited debris of gallium (the source of the ion beam) in the trenched area, which confounded the EDS results. Therefore, a high current milling approach was used, by which the gallium ions bombarded the coating/ substrate with higher force, causing a few cracks. These cracks were therefore not inherently present in the coating but were a result of the FIB milling process, which affected the zeolite coating and the titanium alloy substrate equally. An EDS linescan of the FIB-milled cross-section of the metal–zeolite interface revealed that Ti was incorporated into the zeolite coating up to 4 lm deep (Fig. 2B). Incorporation of element from the substrate has been previously observed with a high-silica MFI coating on Al substrates [23] and contributed to the excellent coating adhesion. Specifically, in the previous study, EDS measurements showed a low Si/Al ratio (1) at the interface, which continuously increased away from the interface to the external surface of the coating (Si/Al  40). X-ray photoelectron spectroscopy showed the external surface to have a pure silica composition. At the atomic level, zeolites can be considered to be constructed from SiO4 tetrahedra (Si is called a tetrahedral atom); each apical oxygen atom

Fig. 3. Direct current polarization curves of MFI-coated (solid lines) and bare Ti6Al4V (dashed lines) in (A) 0.856 M NaCl solution at pH 7.0 and (B) 0.856 M NaCl solution at pH 1.0. The legend shows the immersion time from 5 min (5 m) to 7 days (7d); the asterisk indicates MFI-coated Ti6Al4V.

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than in the neutral medium. Bending of the anodic branch of the polarization curve of MFI-coated samples towards lower current density in acidified medium indicates the formation of a passivation layer, thus conferring a higher corrosion resistance. MFI coating is a barrier coating which prevents the electrolyte from penetrating to the metal surface. This causes a decrease in the oxidation reaction and enhances cathodic protection of the metal, thus lowering the corrosion potential (Ecorr). Ti6Al4V has a highly passive TiO2 surface layer, but this layer is not as protective as a zeolite MFI coating. Synthesis and characterization of MFI coatings on commercially pure titanium (cpTi) was also examined to show technological applicability on pure titanium implants. Changing the substrate did not change either the morphology or the physico-chemical properties of the MFI coating. Uniform polycrystalline coatings with high corrosion resistance and excellent adhesion were formed on cpTi substrates (data not shown). This indicates the robustness of the coating deposition procedure, and shows the adaptability of the zeolite coating. The higher corrosion rate of bare Ti6Al4V suggests that there is a possibility of toxic ions being released into the solution. Thus the neutral and acidified salt solutions in which the bare and MFI-coated Ti6Al4V samples were immersed were tested for the presence of Al, V and Ti ions (Table 1). No release of Ti, Al or V ions was detected when bare Ti6Al4V was immersed in 0.856 M NaCl solution at pH 7. However, initial tests of the MFI-coated Ti6Al4V

Table 1 Release of metal ions from bare and MFI-coated Ti6Al4V surfaces after immersion in neutral and acidified salt solution for 1–7 days. Immersion time (days)

Neutral/0.856 M NaCl

pH 1.0/0.856 M NaCl/HCl

Bare

MFIcoated

Bare

MFIcoated

Mean Al Conc.a (lg l1)

1 1# 2 4 7

– N/A – – –

10 – – – –

251 N/A 552 529 570

13 – – – –

Mean Ti Conc.b (lg l1)

1 1# 2 4 7

– N/A – – –

– – – – –

203 N/A 266 681 809

– – – – –

Mean V Conc.c (lg l1)

1 1# 2 4 7

– N/A – – –

– – – – –

13 N/A 21 37 39

– – – – –

N/A, bare metal alloy was not flushed with fresh solution after day 1. # MFI-coated coupons flushed with fresh solution after day 1 immersion. a Detection limit = 6 lg l1. b Detection limit = 2 lg l1. c Detection limit = 5 lg l1.

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showed a low level of release of Al ions (barely above the detection limit of 6 lg l1). The concentration of Al was approximately constant from 1 to 7 days of immersion, and no release of Ti and V ions was observed. These observations suggested that the release of Al ions from MFIcoated substrates was due to the presence of loose Al ions trapped on the coating’s surface after the coating synthesis process was stopped. To confirm this, we soaked the MFIcoated substrates in the salt solution for 1 day to remove the loose Al, then flushed the solution (day 1  indicates 1 day of immersion after flushing). No release of Al ions was detected after flushing the substrates with fresh solution. A significant release of Al, Ti and V ions was detected from bare Ti6Al4V when immersed in 0.856 M NaCl at pH 1.0, and the concentrations increased with immersion time. By contrast, MFI coatings successfully prevented the release of cytotoxic V ions as well as Ti ions for up to 7 days of immersion. Similar to the results in the neutral salt medium, initial tests of MFI-coated Ti6Al4V showed that the release of Al ions was at the same low level, but this release was not seen after the solution was flushed after 1 day of immersion with fresh medium. The chemical dissolution data suggest that MFI coatings may be able to protect Ti6Al4V implants from releasing of toxic Al and V ions into the surrounding tissue in the harsh acidic environment of the oral cavity (created by the consumption of soft drinks, and bacterial adhesion to teeth). The increase in the release of ion concentrations from bare Ti6Al4V in the acidified medium parallels the increase in its corrosion current density with increasing immersion time. Hence, the determination of chemical stability of bare and MFI-coated Ti6Al4V can potentially be achieved quickly by measuring the corrosion current density instead of performing the long immersion studies and tedious analysis of metal ions. An increase in current density of the alloy after immersion in corrosive medium indicates decreased corrosion resistance and chemical stability, and thus greater release of metallic ions from the substrate can be expected over time. While chemical inertness, a useful indicator for biocompatibility, can be determined by DC polarization or chemical analysis, direct determination of biocompatibility requires cell culture studies. Our preliminary cell culture data indicate that MFI coatings are biocompatible. To test the hypothesis that MFI coatings can be used in hard tissue regeneration, we cultured pluripotent mouse embryonic stem cells on MFI-coated Ti6Al4V and glass coverslips, and studied the effect of the topography of the material surface on cell growth. A monolayer of mitotically inactivated fibroblasts was first seeded on the substrates to investigate cell adhesion, followed by culture of pluripotent mouse embryonic stem cells. We found that zeolites not only sustained, but also favored the growth and attachment of both fibroblasts and embryonic stem cells compared to glass (Fig. 4). Even though both surfaces—zeolites and the control surface, glass—were composed mainly of silica, cells attached sig-

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Fig. 4. SEM micrographs of mouse embryonic stem cells on a fibroblast monolayer on the MFI coating on Ti6Al4V (A and B) and on glass coverslips (C and D).

nificantly better to the zeolite surface, indicating that the microtopography of zeolites offers higher proliferative properties. Round stem cell colonies were found on zeolite coatings, while a smaller number of flat cell colonies were observed on the glass surface. Retaining their round shape could allow cells to function as if they were in their natural environment. Stem cell colonies on the zeolite surface were larger in size (100 lm long) than those on the glass surface (50 lm long), indicating that the zeolite surface was better for cell proliferation. A greater number of elongations protruding out of the stem cell colony and attaching to the fibroblasts were found on the zeolite compared with the glass. In addition, a higher density of fibroblasts was seen on the zeolite surface. This indicates that zeolite coatings support proliferation of both fully differentiated (fibroblasts) and undifferentiated mitotically active mESCs, with the potential for use in tissue regeneration. Higher cell densities, and smaller gaps between cells, were observed on zeolite than on glass, indicating that the three-dimensional microstructure of zeolite crystals is favored for cell growth over the two-dimensional flat glassy surface. No cytotoxic effects on the stem cells or fibroblasts were observed on either the zeolite or the glassy surfaces. 4. Conclusions A novel application of zeolite coatings as biocompatible coatings for biomedical implant use has been described. A uniform and strongly adhered coating with high corrosion resistance is obtained on Ti6Al4V. The high corrosion resistance of MFI coatings makes them ideal for preventing

toxic Al and V ions from leaching from Ti6Al4V prosthetic implants into the surrounding tissue. The incorporation of titanium within the zeolite framework provides excellent adhesion to the substrates and may prevent implant loosening, which are highly desirable traits for bioimplants. Adding zeolite coatings to titanium implants reduces the modulus mismatch with bone tissue, and can potentially enhance osteointegration of implants. Cell culture studies showed that mouse embryonic stem cells favored growth on the three-dimensional microstructure of zeolite coatings over a two-dimensional glassy surface, indicating the biocompatible nature of zeolite coatings. The good fibroblast adhesion to zeolite surfaces indicates that zeolite coatings could be seen as biocompatible, and could be applied to dental and orthopedic implants to increase their lifespan as well as enhance patient recovery after surgery. Zeolite coatings have the potential in the future to become a suitable scaffold for hard tissue regeneration. References [1] Piveteau LD, Girona MI, Schlapbach L, Barboux P, Boilot JP, Gasser B, et al. Thin films of calcium phosphate and titanium dioxide by a sol–gel route: a new method for coating medical implants. J Mater Sci Mater Med 1999;10:161. [2] Long M, Rack HJ. Titanium alloys in total joint replacement – a materials science perspective. Biomaterials 1998;19:1621. [3] Williams DF. Biomaterials and tissue engineering in reconstructive surgery. Sadhana 2003;28:563. [4] Haynes DR, Crotti TN, Haywood MR. Corrosion of and changes in biological effects of cobalt chrome alloy and 316L stainless steel prosthetic particles with age. J Biomed Mater Res 2000; 49:167.

R.S. Bedi et al. / Acta Biomaterialia 5 (2009) 3265–3271 [5] Okazaki Y, Gotoh E. Comparison of metal release from various metallic biomaterials in vitro. Biomaterials 2005;26:11. [6] Lacefield WR. Materials characteristics of uncoated/ceramic-coated implant materials. Adv Dent Res 1999;13:21. [7] Yoruc ABH, Gulay O, Sener BC. Examination of the properties of Ti–Al–4V based plates after oral and maxillofacial application. J Optoelectron Adv Mater 2007;9:2627. [8] Silva HM, Schneider SG, Neto CM. Study of nontoxic aluminum and vanadium-free titanium alloys for biomedical applications. Mater Sci Eng C 2004;24:679. [9] Cortizo AM, Salice VC, Vescina CM, Etcheverry SB. Proliferative and morphological changes induced by vanadium compounds on Swiss 3T3 fibroblasts. BioMetals 1997;10:127. [10] Shi X, Hengguang J, Yan M, Jianping Y, Umberto S. Vanadium(IV)mediated free radical generation and related 20 -deoxyguanosine hydroxylation and DNA damage. Toxicology 1996;106:27. [11] Gunawarman B, Niinomi M, Akahori T, Souma T, Ikeda M, Toda H. Mechanical properties and microstructures of low cost [beta] titanium alloys for healthcare applications. Mater Sci Eng C 2005;25:304. [12] Rho J-Y, Tsui TY, Pharr GM. Elastic properties of human cortical and trabecular lamellar bone measured by nanoindentation. Biomaterials 1997;18:1325. [13] Szostak R. Handbooks of molecular sieves. New York: Van Nostrand Reinhold; 1992. [14] Balkus Jr KJ, Shi J. Synthesis of hexagonal Y type zeolites in the presence of Gd(III) complexes of 18-crown-6. Microporous Mater 1997;11:325. [15] Balkus Jr KJ, Gabrielov AG. Zeolite encapsulated metal complexes. J Incl Phenom Macrocyclic Chem 1995;21:159.

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[16] Bresinska I, Balkus Jr KJ. Studies of Gd(III)-exchanged Y-type zeolites relevant to magnetic resonance imaging. J Phys Chem 1994;98:12989. [17] Domingo C, Garcia-Carmona J, Fanovich MA, Saurina J. Study of adsorption processes of model drugs at supercritical conditions using partial least squares regression. Anal Chim Acta 2002;452:311. [18] Dyer A, Morgan S, Wells P, Williams C. The use of zeolites as slow release anthelmintic carriers. J Helminthol 2000;74:137. [19] Pavelic´ K et al. Natural zeolite clinoptilolite: new adjuvant in anticancer therapy. J Mol Med 2001;78:708. [20] Cheng X, Wang Z, Yan Y. Corrosion-resistant zeolite coatings by in situ crystallization. Electrochem Solid State Lett 2001;4:B23. [21] Li Z et al. Mechanical, dielectric properties of pure-silica-zeolite lowk materials. Angew Chem Int Ed 2006;45:6329. [22] Cachinho S, Correia R. Titanium scaffolds for osteointegration: mechanical, in vitro and corrosion behaviour. J Mater Sci Mater Med 2008;19:451. [23] Beving DE, McDonnell AMP, Yang W, Yan Y. Corrosion-resistant high-silica-zeolite MFI coating. J Electrochem Soc 2006;153:B325. [24] Mihailova B, Valtchev V, Mintova S, Konstantinov L. Characterization of water in microporous titanium silicates. J Mater Sci Lett 1997;16:1303. [25] Saxton RJ. Crystalline microporous titanium silicates. Top Catal 1999;9:43. [26] Zhang S, Kobayashi T, Nosaka Y, Fujii N. Photocatalytic property of titanium silicate zeolite. J Mol Catal A Chem 1996;106:119. [27] Corma A, Navarro MT, Perez Pariente J. Synthesis of an ultralarge pore titanium silicate isomorphous to MCM-41 and its applications as a catalyst for selective oxidation of hydrocarbons. J Chem Soc Chem Commun 1994:147.