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Effects of alloying elements on the cytotoxic response of titanium alloys
Materials Science and Engineering C 31 (2011) 833–839
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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Effects of alloying elements on the cytotoxic response of titanium alloys Alessandra Cremasco a,⁎, André Dutra Messias a, Andrea Rodrigues Esposito a, Eliana Aparecida de Rezende Duek a,b, Rubens Caram a a b
University of Campinas, School of Mechanical Engineering, C.P. 6122, Campinas, SP, 13083-970, Brazil Pontifical Catholic University of São Paulo, Center of Medical and Biological Sciences, Sorocaba, SP, 18030-230, Brazil
a r t i c l e
i n f o
Article history: Received 3 July 2010 Received in revised form 1 November 2010 Accepted 29 December 2010 Available online 8 January 2011 Keywords: Titanium alloys Biocompatibility In vitro Biomaterials Fibroblast
a b s t r a c t Titanium alloys, especially β-type alloys containing β-stabilizing elements, constitute a highly versatile category of metallic materials that have been under constant development for application in orthopedics and dentistry. This type of alloy generally presents a high mechanical strength-to-weight ratio, excellent corrosion resistance and low elastic modulus. The purpose of this study is to evaluate the cytotoxicity and adhesion of fibroblast cells on titanium alloy substrates containing Nb, Ta, Zr, Cu, Sn and Mo alloying elements. Cells cultured on polystyrene were used as controls. In vitro results with Vero cells demonstrated that the tested materials, except Cu-based alloy, presented high viability in short-term testing. Adhesion of cells cultured on disks showed no differences between the materials and reference except for the Ti–Cu alloy, which showed reduced adhesion attributed to poor metabolic activity. Titanium alloys with the addition of Nb, Ta, Zr, Sn and Mo elements show a promising potential for biomedical applications. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Metallic materials are commonly used in various areas of biomedicine. In orthopedics, they are applied as plates, pins and fixing screws for bone fractures and in some complex devices as parts for total hip prostheses or as femoral and tibial components in total knee arthroplasty [1]. Among the metallic materials used in implants, titanium and its alloys possess properties that render their performance superior to that of Co–Cr alloys and stainless steels. Ti–6Al–4V alloy and CP–Ti (commercially pure) are the most important Ti-based materials used in the orthopedic implant industry. Ti–6Al–4V alloy was initially developed to meet the demands of the aerospace industry, and due to its interesting properties, it has been applied in the biomedical field since the 1960s. The use of metallic devices for the replacement of damaged parts of the human body requires not only mechanical compatibility, which is achieved by a combination of a low elastic modulus, high mechanical strength and fatigue resistance, but also biological compatibility, since the material will be in contact with body fluids and should therefore be atoxic to cells. Williams [2] defines “biocompatibility” as follows: “Biocompatibility is the ability of a material to develop its functions with adequate tissue response in a specific application.” Several studies have highlighted the high biocompatibility of titanium and its alloys [3–6]. However, there are some concerns regarding the biocompatibility of Al and V elements in Ti–6Al–4V alloy. Several studies have shown that such elements are ⁎ Corresponding author. University of Campinas, C.P. 6122, Campinas, SP, 13083-970, Brazil. Tel.: + 55 19 3521 3314; Fax: + 55 19 3289 3722. E-mail address: [email protected] (A. Cremasco). 0928-4931/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.12.013
toxic and can cause neurological disorders and Alzheimer's disease [7,8], as well as accumulation of particulates in adjacent tissues [9]. The abovementioned problems have motivated the constant development of new titanium alloys with nontoxic and nonallergenic elements such as the β-stabilizing elements Nb, Ta, Zr, Mo, Pt, Sn [10–15]. These elements can stabilize the titanium body-centered cubic crystal structure (β phase) at room temperature and the resulting alloys may represent the future for titanium alloys insofar as orthopedic applications are concerned. Beta-phase stabilization through the addition of the aforementioned elements yields titanium alloys with low elastic modulus and high mechanical strength, as well as optimized electrochemical and biological performance. Some metallic materials used in implants may not be toxic, but the presence of dissolved metal ions, corrosion products and wear particles may lead to some level of toxicity when combined with certain types of biomolecules and cells [16]. For instance, wear particles can cause osteolysis due to the natural defense mechanism called fagocytosis [17,18]. The biocompatibility of a material can be evaluated from in vitro and in vivo tests. Because in vivo tests take longer, in vitro tests can be considered preliminary assays to determine certain toxicity-related parameters such as cell death, adhesion on substrate surfaces, changes in cell morphology, cell proliferation and biosynthetic activity. With regard to in vitro tests, the cell culture technique is an important methodology in biomaterials research because it allows for the rapid evaluation of biological performance. According to the protocol of the ISO 10993-5 standard [19] for biological evaluations of medical devices, one of the recommended cytotoxicity tests is the MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium
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bromide]. This procedure allows for a quantitative evaluation by measuring the mitochondrial activity of cells after their exposure to the surrounding toxins, revealing information about the material's cytotoxicity and the functionality of cells on its surface [20]. After eliminating the possibility of cytotoxicity, cell adhesion is another relevant factor that indicates cell/material interaction, mainly in applications that integrate the prosthesis to the bone or articulation. Cell adhesion indicates, in an early stage, the adhesion of cells to substrate, which is essential for cell proliferation and differentiation and the formation of neo-tissue [20]. Moreover, characteristics of the material such as roughness, chemical composition and surface free energy are essential for adhesion and affect cell morphology and function. Evidence has revealed that cell morphology can regulate cell growth, protein secretion, differentiation, proliferation and death. In the case of titanium alloys, polished surfaces (with low roughness) help cell fixation and flattening [1]. Cytocompatibility studies of new titanium alloys develop by biomedical application have been investigated by several researches [21,22]. Koike and co-workers observed slightly elevated mitochondrial activity in Cu–Cr and Cu–Si alloys with highest copper dissolution using Balb/c 3T3 mouse fibroblast. In the same study, the biocompatibility of Ti–6Al–4V, Ti–1Fe, Ti–5Al–11Fe and Ti–16Mo–3.2Nb alloys was evaluated [21]. The results showed similar behavior when compared to pure titanium used as control. According to Watanabe et al., Ti–10Cu alloy showed high level of released copper [22]. Their results also showed slight suppression of mitochondrial activity in the Ti–6Al–7Nb
alloy, which could be associated to the release of Al into the medium [22]. Again, separated studies showed that Ti–Au alloys exhibits 100% cell viability [23]. Similarly, Mn addition to titanium-based alloys in concentration up to 8% (wt) results in acceptable cytocompatibility [24]. The main objective of this work is to evaluate the effects of alloying elements on the cytotoxicity of titanium alloys for biomaterial applications by ascertaining their in vitro cytocompatibility. 2. Materials and methods 2.1. Preparation of titanium alloys Titanium alloys with nominal compositions of Ti–35Nb, Ti–35Nb– 7.5Ta, Ti–35Nb–4Sn, Ti–25Nb–15Zr, Ti–25Nb–8Sn, Ti–6Mo, Ti–7.1Cu, Ti–6Al–4V and Ti–CP (wt.%) were placed in copper crucibles and prepared in an arc furnace under argon atmosphere. The alloys were then homogenized at 1000 °C for 24 h in an inert atmosphere, after which they were subjected to plastic deformation through swaging, solution-treated following water quenched and milled to their final dimension of 5 mm diameter. They were then cut into 5 mm × 200 μm disks using a Buehler cutter (IsoMet 4000). The samples were prepared metallographically by sanding with #800 and #1500 grit sandpaper, followed by polishing through tumbling. This process was carried out in a revolving cylindrical drum at 45 rpm for a period of 12 h, with 8 mm size particles immersed in an aqueous solution of B-5 (Roger Química Ltda) and PL-4 (Roger Química Ltda) tensoactives.
Fig. 1. (a) — Optical micrograph of Ti–35Nb alloy. (b) — Optical micrograph of Ti–35Nb–7.5Ta alloy. (c) — Optical micrograph of Ti–35Nb–4Sn alloy. (d) — Optical micrograph of Ti– 25Nb–8Sn alloy. (e) — Optical micrograph of Ti–25Nb–15Zr alloy. (f) — Optical micrograph of Ti–6Mo alloy. (g) — Optical micrograph of Ti–7.1Cu alloy. (h) — Optical micrograph of Ti–6Al–4V alloy. (i) — Optical micrograph of Ti–CP.
A. Cremasco et al. / Materials Science and Engineering C 31 (2011) 833–839 Table 1 Chemical composition (% mass) and surface roughness of the alloys.
2.2. Materials characterization
2.3.2. Direct cytotoxicity assay Titanium alloy samples were placed on the bottom of 96-well culture plates (TPP — Techno Plastic Products, Switzerland). The polystyrene plate itself was used as a control and its respective results were treated as 100% of cell viability.
20.27 20.74 27.10 21.35 29.09 25.66 20.15 20.33 21.40
The wells were filled with 100 μL of medium 199 (EARLE) without BFS and preincubated in CO2 for 24 h, according to the ISO 10993-5 standard [19]. After preincubation, the medium was removed and 200 μL of 1× 105 cells/mL in medium 199 (EARLE) with 10% of BFS were placed in the wells. The plates were incubated for 24 h in CO2 at 37 °C. After incubation, the medium was removed and the wells were washed immediately with the same incubation medium. After washing, each well was filled with 200 μL of 0.5 mg/mL solution of 3-(4,5-dimethylthiazon2-yl)-2,5 diphenyltetrazolium bromide MTT in medium 199 (EARLE). The wells were dark-incubated for 4 h at 37 °C, after which the MTT was replaced with 200 μL of DMSO solution and 25 μL of Sorensen's Glycine buffer solution. 100 μL of the solutions from the wells were then transferred to a new plate and the absorbance was read in a BioTek microplate reader (ELX 800, USA) at a wavelength of 570 nm. 2.3.3. Cell adhesion test For the cell adhesion tests, the titanium alloy samples were placed on the bottom of 96-well culture plates (TPP — Techno Plastic Products, Switzerland). The polystyrene plate itself was also used as a control and its respective results were treated as 100% of cell adhesion. All the wells were filled with 100 μL of medium 199 without FBS and incubated for 24 h at 37 °C in an atmosphere saturated with water vapor and 5% of CO2. After
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2.3.1. Cell culture Vero cells, a line of African green monkey kidney fibroblast cells (Cercopithecus aethiops) supplied by the Adolfo Lutz Institute in São Paulo, Brazil, were employed in the studies of cytotoxicity and cell– substrate interactions with biomaterials [19,25,26]. The Vero cells were cultured in Medium 199 (EARLE) (Cultilab, Campinas, SP), with 10% of bovine fetal serum (BFS) (Cultilab, Campinas, SP) and incubated in 5% CO2 at 37 °C. The culture medium was changed whenever acidification was detected. To maintain the cell line, cells were detached and dissociated from their adhering substrate by means of an enzymatic treatment using trypsin/EDTA, thus providing more room for these cells to continue proliferating.
Ra (nm)
Ti–34.3Nb Ti–34.5Nb–7.9Ta Ti–33.6Nb–4.3Sn Ti–24.2Nb–8.4Sn Ti–24.1Nb–14.2Zr Ti–5.8Mo Ti–7.4Cu Ti–6.5Al–4.6V Ti–0.16Fe–0.2O
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Chemical composition by XRF
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The morphology of the titanium alloy surfaces was evaluated by atomic force microscopy (AFM) (ThermoMicroscopes AutoProbe) in the tapping mode. The samples' chemical composition was analyzed by X-ray fluorescence (XRF) (Rigaku RIX 3100 spectrometer). Average surface roughness was calculated from an area of 5 × 5 μm2.
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Microstructural characterization was performed by optical microscopy (Olympus BX60M) in samples that were etched with Kroll reagent (6 mL HNO3, 3 mL HF and 91 mL of H2O) and X-ray diffraction with the Panalytical X'Pert Pro instrument operated at 40 kV and 30 mA, using CuKα radiation.
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Fig. 2. (a) — X-ray diffraction pattern for the Ti–35Nb alloy. (b) — X-ray diffraction pattern for the Ti–35Nb–7.5Ta alloy. (c) — X-ray diffraction pattern for the Ti–35Nb–4Sn alloy. (d) — X-ray diffraction pattern for the Ti–25Nb–8Sn alloy. (e) — X-ray diffraction pattern for the Ti–25Nb–15Zr alloy. (f) — X-ray diffraction pattern for the Ti–6Mo alloy. (g) — X-ray diffraction pattern for the Ti–7.1Cu alloy. (h) — X-ray diffraction pattern for the Ti–6Al–4V alloy. (i) — X-ray diffraction pattern for the Ti–CP.
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2.3.4. Scanning Electron Microscopy (SEM) The cell morphology was analyzed by scanning electron microscopy. Vero cells were cultured under the titanium alloys and observed after 24 h of cultivation. The samples were fixed for 1 h at room temperature
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incubation the medium was removed, inoculated with 200 μL of cell suspension (1×105 cells/mL) in Medium 199 with 10% of FBS per well, and incubated for over 2 h in the same conditions. After cultivation, the medium was removed and the wells washed again with the same medium. 200 μL of a 0.5 mg/mL solution of 3-(4,5-dimethylthiazol-2-yl)2,5 diphenyltetrazolium bromide MTT in Medium 199 (EARLE) was then added and the solution incubated in the same cultivation conditions for 24 h. After this period, the solution in each well was replaced with 200 μL of DMSO and 25 μL of Sorensen's Glycine buffer solution. Samples of 100 μL of the solutions from the wells were then transferred to a new plate. The absorbance of the samples was measured in a BioTek microplate reader, (ELX 800, USA) using a filter with 570 nm wavelength. Through the enzyme succinate dehydrogenase, the mitochondria in living cells are able to reduce the water-soluble yellow tetrazolium salt (MTT), converting it into the water-insoluble compound formazan, which is solubilized by DMSO. The amount of formazan produced, which is measured spectrophotometrically, is directly proportional to the metabolic activity and to the number of living cells. All the experiments involving MTT were analyzed statistically by ANOVA and Tukey's test.
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Fig. 4. Cell cytotoxicity assay of Vero cells after 24 h of cell culture. The data represent the mean±standard deviation. All the results are statistically equal except for the Ti–7.1Cu alloy, which presented the lowest cell viability (pb 0.01).
Fig. 3. (a) — Topographic AFM images (5 μm × 5 μm) of the Ti–25Nb–15Zr alloy. (b) — Topographic AFM images (5 μm × 5 μm) of the Ti–35Nb–7.5Ta alloy.
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0.6
each alloy were confirmed by X-ray diffraction as shown in Fig. 2. The presence of orthorhombic martensite phase in a β matrix is evident for Ti– 35Nb (Fig. 1a) and Ti–6Mo (Fig. 1f) alloys. However, Ti–7Cu alloy (Fig. 1g) when subjected to water quenching produced a mixture of needles of martensitic α′ and α and a fine Ti2Cu precipitates. Again, on one hand, Ti–6Al–4V (Fig. 1h) exhibits fully lamellar microstructure of Widmanstätten of α dispersed within the β matrix, whereas commercially pure (CP) Ti (Fig. 1i) showed martensite hexagonal phase. On the other hand, a complete stabilization of β phase was observed in Ti–35Nb–7.5Ta (Fig. 1b), Ti–35Nb–4Sn (Fig. 1c) and Ti–25Nb–15Zr (Fig. 1e) alloys.
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Fig. 5. Cell adhesion test of Vero cells after 24 h of cell culture. The data represent the mean±standard deviation. All the results are statistically the same, except for the Ti–7.1Cu alloy, which presented very low adhesion, and the Ti–35Nb–4Sn and Ti–6Mo alloys, whose adhesion rates exceeded 100% (pb 0.01).
in a fixing solution of 2.5% paraformaldehyde, 2.5% glutaraldehyde, 0.06%, picric acid and 1% tanic acid dissolved in cacodylate buffer solution 0.1 M, pH 7.4. Later, the samples were post-fixed with 1% osmium tetroxide (OsO4) and dehydrated in an ethanol series. They were then dried using a Critical Point Dryer (Balzers CPD 030), gold sputtered (Balzers SCD 050) and examined in a scanning electron microscope (JEOL JSM-5800 LV). All the experiments were carried out in triplicate. 3. Results and discussion 3.1. Microstructure Characterization Fig. 1 shows optical micrographs of the alloys prepared, illustrating the influence of composition on the phases formed. The phases present in
Cytocompatibility studies of some of the titanium alloys were carried out through the direct cytotoxicity assay (MTT) and cell adhesion assay. Table 1 shows the chemical composition and roughness of the analyzed samples. The complete characterization of a biomaterial should include the investigation of its surface morphology, which is reflected in cell– substrate interactions. Therefore, Fig. 3 shows representative AFM images of the surface of two analyzed alloy compositions, Ti–25Nb–15Zr and Ti–25Nb–8Sn. 3.3. In vitro tests In vitro tests allow for quantitative evaluations of the interaction between cells and materials. Figs. 4 and 5 depict the results obtained in the MTT assays, indicating the viability of fibroblast cells on the alloys in question. The cytotoxicity assays presented similar results for all the materials except for the Ti–7.1Cu alloy, which showed absorbance values 50% lower than that of the control in this test and also weak adhesion. In the Ti–Nb–X system (X = Sn, Ta, Zr), the addition of 4% of Sn (% mass) to Ti–35Nb alloys resulted in a tendency for augmented cell adhesion. The Ti–25Nb–8Sn and Ti–25Nb–15Zr alloys exhibited the opposite behavior in response to a decrease in Nb content and an increase in Sn content or the addition of Zr. No evidence of cytotoxicity was detected in the Ti–Mo system. Despite the controversies concerning Mo cytocompatibility [27], Delvat and co-authors recently performed cell culture tests based on cell adhesion and cell density, and demonstrated the excellent cytocompatibility of Ti–Mo and Ti–Mo–Ta alloy systems [15].
Fig. 6. (a) — SEM micrographs of fibroblastic cells on Ti–35Nb after 24 h of cell cultivation. (b) — SEM micrographs of fibroblastic cells on Ti–35Nb–7.5Ta after 24 h of cell cultivation. (c) — SEM micrographs of fibroblastic cells on Ti–35Nb–4Sn after 24 h of cell cultivation.
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Fig. 7. (a) — SEM micrographs of fibroblastic cells on Ti–25Nb–8Sn after 24 h of cell cultivation. (b) — SEM micrographs of fibroblastic cells on Ti–25Nb–15Zr after 24 h of cell cultivation. (c) — SEM micrographs of fibroblastic cells on Ti–6Mo after 24 h of cell cultivation.
In spite of existing doubts about the cytotoxic effects of V and Al ions in Ti–6Al–4V alloy, verified by Woodman in a study using baboons [28] we were unable to detect the toxicity of this alloy in the present study. According to Geurtsen et al., knowing that the biocompatibility of metal alloys can be determined from cations released through corrosion and wear, in solution, the Ti–6Al–4V alloy presents Ti4+, Al3+ and V5+ cations, which are nontoxic in short-term in vitro studies. However, in long-term studies, the same V5+ cation has presented high cytotoxicity. Therefore, short-term in vitro tests do not suffice to determine the long-term behavior of implantable materials. In fact, the Ti–6Al–4V alloy is still used in Europe and US in hip prosthesis as no conclusive toxicity problems have been confirmed. Likewise, albeit controversial, some studies have reported acute cytotoxicity of Cu, Ni and Be elements [29], as was also observed in the present study of the Ti–7.1Cu alloy. Rocher and co-authors checked the biological parameters of cytotoxicity and cell proliferation using three different cell lines on NiTi, Ti–CP and Ti–6Al–4V alloys and obtained results similar to those of this study. The cell proliferation rates of NiTi and Ti–6Al–4V alloys tend to be slightly higher than those of Ti–CP [30]. According to Okazaki and
Gotoh [31], titanium alloys exhibit low ion release rates due to their high corrosion resistance, which is strongly affected by the medium (solution) and by decreasing pH. There is evidence that titanium alloys with β-stabilizing elements such as Zr, Nb and Ta show considerably lower ion release rates than alloys containing Al and V. The presence of Zr, Nb and Ta elements results in oxides that harden the passive TiO2 film and render it suitable for use in long-term implants. 3.4. Cell morphology Figs. 6 (a–c), 7 (a–c) and 8 (a–c) present an analysis of the cell morphology under the substrate after 24 h of cell cultivation. The SEM results prove that titanium alloys and Ti–CP allowed the fibroblasts to settle on the surfaces. In general, the shape of the cells under the substrate was rounded, frequently flattened or elongated, and only a minor amount of particulate material was detected. Cell behavior is influenced by surface properties, including substrate composition, roughness and texture. A number of studies [3,15,20,32] have observed cell adhesion and cell growth in titanium alloys. The results of the cell–substrate interaction and the resulting
Fig. 8. (a) — SEM micrographs of fibroblastic cells on Ti–6Al–4V after 24 h of cell cultivation. (b) — SEM micrographs of fibroblastic cells on Ti–CP after 24 h of cell cultivation. (c) — SEM micrographs of fibroblastic cells on Ti–7.1Cu after 24 h of cell cultivation.
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morphology are consistent with the literature. The cell proliferation observed after 24 h is a sign of the nontoxicity and cytocompatibility of titanium alloys [3]. The results achieved in the MTT assays are therefore consistent with data from the literature, which show the absence of cytotoxic effects of titanium when compared with other metallic elements [1]. Previous studies [12,29,33,34] have reported the high cytocompatibility of Nb, Ta, Sn and Zr elements applied as alloy elements. Because they are materials that present a shape memory effect, the development of alloys containing Nb and Sn, which have proved to be nontoxic and nonallergenic, is an alternative to Ti–Ni alloy, whose use is currently restricted by the allergenic problems its high Ni content causes. Many efforts have focused on the development of Ni-free alloys in response to toxicity and susceptibility caused by Ni [35]. Arciniegas et al. reported high cytocompatibility of low modulus Ti–Nb–Ta and Ti–Nb–Zr alloys that also exhibited shape memory effects [36,37]. 4. Conclusions Based on the results obtained from the cell cultures in titanium alloys containing β-stabilizing elements, it can be concluded that, except for the Ti–7.1Cu composition, the titanium alloys examined here do not cause toxic effects and show good cell adhesion, indicating in vitro cytocompatibility. The analysis of cell morphology indicated good cell–substrate interaction. The presence of Cu in titanium alloys causes cytotoxic effects on Vero cells, possibly due to the release of ions, rendering it unsuitable for biomedical applications. Acknowledgements The authors would like to thank FAPESP, CAPES and CNPq for their financial support, Dental Morelli for polishing the samples, and LME/ LNLS (Brazilian Synchrotron Light Laboratory) for the AFM analyses. References [1] D.M. Brunette, P. Tengvall, M. Textor, P. Thomsen, Titanium in medicine: materials science, surface science, engineering, biological responses and medical applications, Springer, Berlin, 2001. [2] D.F. Williams, Biocompatibility of Clinical Implant Materials. CRC Press, Boca Raton, 1981. apud Brunette, D.M., et al. Titanium in Medicine: materials science, surface science, engineering, biological responses and medical applications, Springer Berlin, 2001.
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Materials Science and Engineering C j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / m s e c
Effects of alloying elements on the cytotoxic response of titanium alloys Alessandra Cremasco a,⁎, André Dutra Messias a, Andrea Rodrigues Esposito a, Eliana Aparecida de Rezende Duek a,b, Rubens Caram a a b
University of Campinas, School of Mechanical Engineering, C.P. 6122, Campinas, SP, 13083-970, Brazil Pontifical Catholic University of São Paulo, Center of Medical and Biological Sciences, Sorocaba, SP, 18030-230, Brazil
a r t i c l e
i n f o
Article history: Received 3 July 2010 Received in revised form 1 November 2010 Accepted 29 December 2010 Available online 8 January 2011 Keywords: Titanium alloys Biocompatibility In vitro Biomaterials Fibroblast
a b s t r a c t Titanium alloys, especially β-type alloys containing β-stabilizing elements, constitute a highly versatile category of metallic materials that have been under constant development for application in orthopedics and dentistry. This type of alloy generally presents a high mechanical strength-to-weight ratio, excellent corrosion resistance and low elastic modulus. The purpose of this study is to evaluate the cytotoxicity and adhesion of fibroblast cells on titanium alloy substrates containing Nb, Ta, Zr, Cu, Sn and Mo alloying elements. Cells cultured on polystyrene were used as controls. In vitro results with Vero cells demonstrated that the tested materials, except Cu-based alloy, presented high viability in short-term testing. Adhesion of cells cultured on disks showed no differences between the materials and reference except for the Ti–Cu alloy, which showed reduced adhesion attributed to poor metabolic activity. Titanium alloys with the addition of Nb, Ta, Zr, Sn and Mo elements show a promising potential for biomedical applications. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Metallic materials are commonly used in various areas of biomedicine. In orthopedics, they are applied as plates, pins and fixing screws for bone fractures and in some complex devices as parts for total hip prostheses or as femoral and tibial components in total knee arthroplasty [1]. Among the metallic materials used in implants, titanium and its alloys possess properties that render their performance superior to that of Co–Cr alloys and stainless steels. Ti–6Al–4V alloy and CP–Ti (commercially pure) are the most important Ti-based materials used in the orthopedic implant industry. Ti–6Al–4V alloy was initially developed to meet the demands of the aerospace industry, and due to its interesting properties, it has been applied in the biomedical field since the 1960s. The use of metallic devices for the replacement of damaged parts of the human body requires not only mechanical compatibility, which is achieved by a combination of a low elastic modulus, high mechanical strength and fatigue resistance, but also biological compatibility, since the material will be in contact with body fluids and should therefore be atoxic to cells. Williams [2] defines “biocompatibility” as follows: “Biocompatibility is the ability of a material to develop its functions with adequate tissue response in a specific application.” Several studies have highlighted the high biocompatibility of titanium and its alloys [3–6]. However, there are some concerns regarding the biocompatibility of Al and V elements in Ti–6Al–4V alloy. Several studies have shown that such elements are ⁎ Corresponding author. University of Campinas, C.P. 6122, Campinas, SP, 13083-970, Brazil. Tel.: + 55 19 3521 3314; Fax: + 55 19 3289 3722. E-mail address: [email protected] (A. Cremasco). 0928-4931/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2010.12.013
toxic and can cause neurological disorders and Alzheimer's disease [7,8], as well as accumulation of particulates in adjacent tissues [9]. The abovementioned problems have motivated the constant development of new titanium alloys with nontoxic and nonallergenic elements such as the β-stabilizing elements Nb, Ta, Zr, Mo, Pt, Sn [10–15]. These elements can stabilize the titanium body-centered cubic crystal structure (β phase) at room temperature and the resulting alloys may represent the future for titanium alloys insofar as orthopedic applications are concerned. Beta-phase stabilization through the addition of the aforementioned elements yields titanium alloys with low elastic modulus and high mechanical strength, as well as optimized electrochemical and biological performance. Some metallic materials used in implants may not be toxic, but the presence of dissolved metal ions, corrosion products and wear particles may lead to some level of toxicity when combined with certain types of biomolecules and cells [16]. For instance, wear particles can cause osteolysis due to the natural defense mechanism called fagocytosis [17,18]. The biocompatibility of a material can be evaluated from in vitro and in vivo tests. Because in vivo tests take longer, in vitro tests can be considered preliminary assays to determine certain toxicity-related parameters such as cell death, adhesion on substrate surfaces, changes in cell morphology, cell proliferation and biosynthetic activity. With regard to in vitro tests, the cell culture technique is an important methodology in biomaterials research because it allows for the rapid evaluation of biological performance. According to the protocol of the ISO 10993-5 standard [19] for biological evaluations of medical devices, one of the recommended cytotoxicity tests is the MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium
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bromide]. This procedure allows for a quantitative evaluation by measuring the mitochondrial activity of cells after their exposure to the surrounding toxins, revealing information about the material's cytotoxicity and the functionality of cells on its surface [20]. After eliminating the possibility of cytotoxicity, cell adhesion is another relevant factor that indicates cell/material interaction, mainly in applications that integrate the prosthesis to the bone or articulation. Cell adhesion indicates, in an early stage, the adhesion of cells to substrate, which is essential for cell proliferation and differentiation and the formation of neo-tissue [20]. Moreover, characteristics of the material such as roughness, chemical composition and surface free energy are essential for adhesion and affect cell morphology and function. Evidence has revealed that cell morphology can regulate cell growth, protein secretion, differentiation, proliferation and death. In the case of titanium alloys, polished surfaces (with low roughness) help cell fixation and flattening [1]. Cytocompatibility studies of new titanium alloys develop by biomedical application have been investigated by several researches [21,22]. Koike and co-workers observed slightly elevated mitochondrial activity in Cu–Cr and Cu–Si alloys with highest copper dissolution using Balb/c 3T3 mouse fibroblast. In the same study, the biocompatibility of Ti–6Al–4V, Ti–1Fe, Ti–5Al–11Fe and Ti–16Mo–3.2Nb alloys was evaluated [21]. The results showed similar behavior when compared to pure titanium used as control. According to Watanabe et al., Ti–10Cu alloy showed high level of released copper [22]. Their results also showed slight suppression of mitochondrial activity in the Ti–6Al–7Nb
alloy, which could be associated to the release of Al into the medium [22]. Again, separated studies showed that Ti–Au alloys exhibits 100% cell viability [23]. Similarly, Mn addition to titanium-based alloys in concentration up to 8% (wt) results in acceptable cytocompatibility [24]. The main objective of this work is to evaluate the effects of alloying elements on the cytotoxicity of titanium alloys for biomaterial applications by ascertaining their in vitro cytocompatibility. 2. Materials and methods 2.1. Preparation of titanium alloys Titanium alloys with nominal compositions of Ti–35Nb, Ti–35Nb– 7.5Ta, Ti–35Nb–4Sn, Ti–25Nb–15Zr, Ti–25Nb–8Sn, Ti–6Mo, Ti–7.1Cu, Ti–6Al–4V and Ti–CP (wt.%) were placed in copper crucibles and prepared in an arc furnace under argon atmosphere. The alloys were then homogenized at 1000 °C for 24 h in an inert atmosphere, after which they were subjected to plastic deformation through swaging, solution-treated following water quenched and milled to their final dimension of 5 mm diameter. They were then cut into 5 mm × 200 μm disks using a Buehler cutter (IsoMet 4000). The samples were prepared metallographically by sanding with #800 and #1500 grit sandpaper, followed by polishing through tumbling. This process was carried out in a revolving cylindrical drum at 45 rpm for a period of 12 h, with 8 mm size particles immersed in an aqueous solution of B-5 (Roger Química Ltda) and PL-4 (Roger Química Ltda) tensoactives.
Fig. 1. (a) — Optical micrograph of Ti–35Nb alloy. (b) — Optical micrograph of Ti–35Nb–7.5Ta alloy. (c) — Optical micrograph of Ti–35Nb–4Sn alloy. (d) — Optical micrograph of Ti– 25Nb–8Sn alloy. (e) — Optical micrograph of Ti–25Nb–15Zr alloy. (f) — Optical micrograph of Ti–6Mo alloy. (g) — Optical micrograph of Ti–7.1Cu alloy. (h) — Optical micrograph of Ti–6Al–4V alloy. (i) — Optical micrograph of Ti–CP.
A. Cremasco et al. / Materials Science and Engineering C 31 (2011) 833–839 Table 1 Chemical composition (% mass) and surface roughness of the alloys.
2.2. Materials characterization
2.3.2. Direct cytotoxicity assay Titanium alloy samples were placed on the bottom of 96-well culture plates (TPP — Techno Plastic Products, Switzerland). The polystyrene plate itself was used as a control and its respective results were treated as 100% of cell viability.
20.27 20.74 27.10 21.35 29.09 25.66 20.15 20.33 21.40
The wells were filled with 100 μL of medium 199 (EARLE) without BFS and preincubated in CO2 for 24 h, according to the ISO 10993-5 standard [19]. After preincubation, the medium was removed and 200 μL of 1× 105 cells/mL in medium 199 (EARLE) with 10% of BFS were placed in the wells. The plates were incubated for 24 h in CO2 at 37 °C. After incubation, the medium was removed and the wells were washed immediately with the same incubation medium. After washing, each well was filled with 200 μL of 0.5 mg/mL solution of 3-(4,5-dimethylthiazon2-yl)-2,5 diphenyltetrazolium bromide MTT in medium 199 (EARLE). The wells were dark-incubated for 4 h at 37 °C, after which the MTT was replaced with 200 μL of DMSO solution and 25 μL of Sorensen's Glycine buffer solution. 100 μL of the solutions from the wells were then transferred to a new plate and the absorbance was read in a BioTek microplate reader (ELX 800, USA) at a wavelength of 570 nm. 2.3.3. Cell adhesion test For the cell adhesion tests, the titanium alloy samples were placed on the bottom of 96-well culture plates (TPP — Techno Plastic Products, Switzerland). The polystyrene plate itself was also used as a control and its respective results were treated as 100% of cell adhesion. All the wells were filled with 100 μL of medium 199 without FBS and incubated for 24 h at 37 °C in an atmosphere saturated with water vapor and 5% of CO2. After
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2.3.1. Cell culture Vero cells, a line of African green monkey kidney fibroblast cells (Cercopithecus aethiops) supplied by the Adolfo Lutz Institute in São Paulo, Brazil, were employed in the studies of cytotoxicity and cell– substrate interactions with biomaterials [19,25,26]. The Vero cells were cultured in Medium 199 (EARLE) (Cultilab, Campinas, SP), with 10% of bovine fetal serum (BFS) (Cultilab, Campinas, SP) and incubated in 5% CO2 at 37 °C. The culture medium was changed whenever acidification was detected. To maintain the cell line, cells were detached and dissociated from their adhering substrate by means of an enzymatic treatment using trypsin/EDTA, thus providing more room for these cells to continue proliferating.
Ra (nm)
Ti–34.3Nb Ti–34.5Nb–7.9Ta Ti–33.6Nb–4.3Sn Ti–24.2Nb–8.4Sn Ti–24.1Nb–14.2Zr Ti–5.8Mo Ti–7.4Cu Ti–6.5Al–4.6V Ti–0.16Fe–0.2O
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Chemical composition by XRF
Ti–35Nb Ti–35Nb–7.5Ta Ti–35Nb–4Sn Ti–25Nb–8Sn Ti–25Nb–15Zr Ti–6Mo Ti–7.1Cu Ti–6Al–4V Ti–CP
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The morphology of the titanium alloy surfaces was evaluated by atomic force microscopy (AFM) (ThermoMicroscopes AutoProbe) in the tapping mode. The samples' chemical composition was analyzed by X-ray fluorescence (XRF) (Rigaku RIX 3100 spectrometer). Average surface roughness was calculated from an area of 5 × 5 μm2.
Alloy
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Microstructural characterization was performed by optical microscopy (Olympus BX60M) in samples that were etched with Kroll reagent (6 mL HNO3, 3 mL HF and 91 mL of H2O) and X-ray diffraction with the Panalytical X'Pert Pro instrument operated at 40 kV and 30 mA, using CuKα radiation.
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Fig. 2. (a) — X-ray diffraction pattern for the Ti–35Nb alloy. (b) — X-ray diffraction pattern for the Ti–35Nb–7.5Ta alloy. (c) — X-ray diffraction pattern for the Ti–35Nb–4Sn alloy. (d) — X-ray diffraction pattern for the Ti–25Nb–8Sn alloy. (e) — X-ray diffraction pattern for the Ti–25Nb–15Zr alloy. (f) — X-ray diffraction pattern for the Ti–6Mo alloy. (g) — X-ray diffraction pattern for the Ti–7.1Cu alloy. (h) — X-ray diffraction pattern for the Ti–6Al–4V alloy. (i) — X-ray diffraction pattern for the Ti–CP.
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2.3.4. Scanning Electron Microscopy (SEM) The cell morphology was analyzed by scanning electron microscopy. Vero cells were cultured under the titanium alloys and observed after 24 h of cultivation. The samples were fixed for 1 h at room temperature
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incubation the medium was removed, inoculated with 200 μL of cell suspension (1×105 cells/mL) in Medium 199 with 10% of FBS per well, and incubated for over 2 h in the same conditions. After cultivation, the medium was removed and the wells washed again with the same medium. 200 μL of a 0.5 mg/mL solution of 3-(4,5-dimethylthiazol-2-yl)2,5 diphenyltetrazolium bromide MTT in Medium 199 (EARLE) was then added and the solution incubated in the same cultivation conditions for 24 h. After this period, the solution in each well was replaced with 200 μL of DMSO and 25 μL of Sorensen's Glycine buffer solution. Samples of 100 μL of the solutions from the wells were then transferred to a new plate. The absorbance of the samples was measured in a BioTek microplate reader, (ELX 800, USA) using a filter with 570 nm wavelength. Through the enzyme succinate dehydrogenase, the mitochondria in living cells are able to reduce the water-soluble yellow tetrazolium salt (MTT), converting it into the water-insoluble compound formazan, which is solubilized by DMSO. The amount of formazan produced, which is measured spectrophotometrically, is directly proportional to the metabolic activity and to the number of living cells. All the experiments involving MTT were analyzed statistically by ANOVA and Tukey's test.
Absorbance (570 nm)
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Fig. 4. Cell cytotoxicity assay of Vero cells after 24 h of cell culture. The data represent the mean±standard deviation. All the results are statistically equal except for the Ti–7.1Cu alloy, which presented the lowest cell viability (pb 0.01).
Fig. 3. (a) — Topographic AFM images (5 μm × 5 μm) of the Ti–25Nb–15Zr alloy. (b) — Topographic AFM images (5 μm × 5 μm) of the Ti–35Nb–7.5Ta alloy.
A. Cremasco et al. / Materials Science and Engineering C 31 (2011) 833–839
0.6
each alloy were confirmed by X-ray diffraction as shown in Fig. 2. The presence of orthorhombic martensite phase in a β matrix is evident for Ti– 35Nb (Fig. 1a) and Ti–6Mo (Fig. 1f) alloys. However, Ti–7Cu alloy (Fig. 1g) when subjected to water quenching produced a mixture of needles of martensitic α′ and α and a fine Ti2Cu precipitates. Again, on one hand, Ti–6Al–4V (Fig. 1h) exhibits fully lamellar microstructure of Widmanstätten of α dispersed within the β matrix, whereas commercially pure (CP) Ti (Fig. 1i) showed martensite hexagonal phase. On the other hand, a complete stabilization of β phase was observed in Ti–35Nb–7.5Ta (Fig. 1b), Ti–35Nb–4Sn (Fig. 1c) and Ti–25Nb–15Zr (Fig. 1e) alloys.
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Fig. 5. Cell adhesion test of Vero cells after 24 h of cell culture. The data represent the mean±standard deviation. All the results are statistically the same, except for the Ti–7.1Cu alloy, which presented very low adhesion, and the Ti–35Nb–4Sn and Ti–6Mo alloys, whose adhesion rates exceeded 100% (pb 0.01).
in a fixing solution of 2.5% paraformaldehyde, 2.5% glutaraldehyde, 0.06%, picric acid and 1% tanic acid dissolved in cacodylate buffer solution 0.1 M, pH 7.4. Later, the samples were post-fixed with 1% osmium tetroxide (OsO4) and dehydrated in an ethanol series. They were then dried using a Critical Point Dryer (Balzers CPD 030), gold sputtered (Balzers SCD 050) and examined in a scanning electron microscope (JEOL JSM-5800 LV). All the experiments were carried out in triplicate. 3. Results and discussion 3.1. Microstructure Characterization Fig. 1 shows optical micrographs of the alloys prepared, illustrating the influence of composition on the phases formed. The phases present in
Cytocompatibility studies of some of the titanium alloys were carried out through the direct cytotoxicity assay (MTT) and cell adhesion assay. Table 1 shows the chemical composition and roughness of the analyzed samples. The complete characterization of a biomaterial should include the investigation of its surface morphology, which is reflected in cell– substrate interactions. Therefore, Fig. 3 shows representative AFM images of the surface of two analyzed alloy compositions, Ti–25Nb–15Zr and Ti–25Nb–8Sn. 3.3. In vitro tests In vitro tests allow for quantitative evaluations of the interaction between cells and materials. Figs. 4 and 5 depict the results obtained in the MTT assays, indicating the viability of fibroblast cells on the alloys in question. The cytotoxicity assays presented similar results for all the materials except for the Ti–7.1Cu alloy, which showed absorbance values 50% lower than that of the control in this test and also weak adhesion. In the Ti–Nb–X system (X = Sn, Ta, Zr), the addition of 4% of Sn (% mass) to Ti–35Nb alloys resulted in a tendency for augmented cell adhesion. The Ti–25Nb–8Sn and Ti–25Nb–15Zr alloys exhibited the opposite behavior in response to a decrease in Nb content and an increase in Sn content or the addition of Zr. No evidence of cytotoxicity was detected in the Ti–Mo system. Despite the controversies concerning Mo cytocompatibility [27], Delvat and co-authors recently performed cell culture tests based on cell adhesion and cell density, and demonstrated the excellent cytocompatibility of Ti–Mo and Ti–Mo–Ta alloy systems [15].
Fig. 6. (a) — SEM micrographs of fibroblastic cells on Ti–35Nb after 24 h of cell cultivation. (b) — SEM micrographs of fibroblastic cells on Ti–35Nb–7.5Ta after 24 h of cell cultivation. (c) — SEM micrographs of fibroblastic cells on Ti–35Nb–4Sn after 24 h of cell cultivation.
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Fig. 7. (a) — SEM micrographs of fibroblastic cells on Ti–25Nb–8Sn after 24 h of cell cultivation. (b) — SEM micrographs of fibroblastic cells on Ti–25Nb–15Zr after 24 h of cell cultivation. (c) — SEM micrographs of fibroblastic cells on Ti–6Mo after 24 h of cell cultivation.
In spite of existing doubts about the cytotoxic effects of V and Al ions in Ti–6Al–4V alloy, verified by Woodman in a study using baboons [28] we were unable to detect the toxicity of this alloy in the present study. According to Geurtsen et al., knowing that the biocompatibility of metal alloys can be determined from cations released through corrosion and wear, in solution, the Ti–6Al–4V alloy presents Ti4+, Al3+ and V5+ cations, which are nontoxic in short-term in vitro studies. However, in long-term studies, the same V5+ cation has presented high cytotoxicity. Therefore, short-term in vitro tests do not suffice to determine the long-term behavior of implantable materials. In fact, the Ti–6Al–4V alloy is still used in Europe and US in hip prosthesis as no conclusive toxicity problems have been confirmed. Likewise, albeit controversial, some studies have reported acute cytotoxicity of Cu, Ni and Be elements [29], as was also observed in the present study of the Ti–7.1Cu alloy. Rocher and co-authors checked the biological parameters of cytotoxicity and cell proliferation using three different cell lines on NiTi, Ti–CP and Ti–6Al–4V alloys and obtained results similar to those of this study. The cell proliferation rates of NiTi and Ti–6Al–4V alloys tend to be slightly higher than those of Ti–CP [30]. According to Okazaki and
Gotoh [31], titanium alloys exhibit low ion release rates due to their high corrosion resistance, which is strongly affected by the medium (solution) and by decreasing pH. There is evidence that titanium alloys with β-stabilizing elements such as Zr, Nb and Ta show considerably lower ion release rates than alloys containing Al and V. The presence of Zr, Nb and Ta elements results in oxides that harden the passive TiO2 film and render it suitable for use in long-term implants. 3.4. Cell morphology Figs. 6 (a–c), 7 (a–c) and 8 (a–c) present an analysis of the cell morphology under the substrate after 24 h of cell cultivation. The SEM results prove that titanium alloys and Ti–CP allowed the fibroblasts to settle on the surfaces. In general, the shape of the cells under the substrate was rounded, frequently flattened or elongated, and only a minor amount of particulate material was detected. Cell behavior is influenced by surface properties, including substrate composition, roughness and texture. A number of studies [3,15,20,32] have observed cell adhesion and cell growth in titanium alloys. The results of the cell–substrate interaction and the resulting
Fig. 8. (a) — SEM micrographs of fibroblastic cells on Ti–6Al–4V after 24 h of cell cultivation. (b) — SEM micrographs of fibroblastic cells on Ti–CP after 24 h of cell cultivation. (c) — SEM micrographs of fibroblastic cells on Ti–7.1Cu after 24 h of cell cultivation.
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morphology are consistent with the literature. The cell proliferation observed after 24 h is a sign of the nontoxicity and cytocompatibility of titanium alloys [3]. The results achieved in the MTT assays are therefore consistent with data from the literature, which show the absence of cytotoxic effects of titanium when compared with other metallic elements [1]. Previous studies [12,29,33,34] have reported the high cytocompatibility of Nb, Ta, Sn and Zr elements applied as alloy elements. Because they are materials that present a shape memory effect, the development of alloys containing Nb and Sn, which have proved to be nontoxic and nonallergenic, is an alternative to Ti–Ni alloy, whose use is currently restricted by the allergenic problems its high Ni content causes. Many efforts have focused on the development of Ni-free alloys in response to toxicity and susceptibility caused by Ni [35]. Arciniegas et al. reported high cytocompatibility of low modulus Ti–Nb–Ta and Ti–Nb–Zr alloys that also exhibited shape memory effects [36,37]. 4. Conclusions Based on the results obtained from the cell cultures in titanium alloys containing β-stabilizing elements, it can be concluded that, except for the Ti–7.1Cu composition, the titanium alloys examined here do not cause toxic effects and show good cell adhesion, indicating in vitro cytocompatibility. The analysis of cell morphology indicated good cell–substrate interaction. The presence of Cu in titanium alloys causes cytotoxic effects on Vero cells, possibly due to the release of ions, rendering it unsuitable for biomedical applications. Acknowledgements The authors would like to thank FAPESP, CAPES and CNPq for their financial support, Dental Morelli for polishing the samples, and LME/ LNLS (Brazilian Synchrotron Light Laboratory) for the AFM analyses. References [1] D.M. Brunette, P. Tengvall, M. Textor, P. Thomsen, Titanium in medicine: materials science, surface science, engineering, biological responses and medical applications, Springer, Berlin, 2001. [2] D.F. Williams, Biocompatibility of Clinical Implant Materials. CRC Press, Boca Raton, 1981. apud Brunette, D.M., et al. Titanium in Medicine: materials science, surface science, engineering, biological responses and medical applications, Springer Berlin, 2001.
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