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
University of Campinas, School of Mechanical Engineering, C.P. 6122, Campinas, SP, 13083-970, Brazil
b
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 a b s t r a c t

Article history: Titanium alloys, especially β-type alloys containing β-stabilizing elements, constitute a highly versatile
Received 3 July 2010 category of metallic materials that have been under constant development for application in orthopedics and
Received in revised form 1 November 2010 dentistry. This type of alloy generally presents a high mechanical strength-to-weight ratio, excellent corrosion
Accepted 29 December 2010
resistance and low elastic modulus. The purpose of this study is to evaluate the cytotoxicity and adhesion of
Available online 8 January 2011
fibroblast cells on titanium alloy substrates containing Nb, Ta, Zr, Cu, Sn and Mo alloying elements. Cells
Keywords:
cultured on polystyrene were used as controls. In vitro results with Vero cells demonstrated that the tested
Titanium alloys materials, except Cu-based alloy, presented high viability in short-term testing. Adhesion of cells cultured on
Biocompatibility disks showed no differences between the materials and reference except for the Ti–Cu alloy, which showed
In vitro reduced adhesion attributed to poor metabolic activity. Titanium alloys with the addition of Nb, Ta, Zr, Sn and
Biomaterials Mo elements show a promising potential for biomedical applications.
Fibroblast © 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: alessandra@fem.unicamp.br (A. Cremasco).
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

0928-4931/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.msec.2010.12.013
834 A. Cremasco et al. / Materials Science and Engineering C 31 (2011) 833–839
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
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.
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.
 A. Cremasco et al. / Materials Science and Engineering C 31 (2011) 833–839 835

Microstructural characterization was performed by optical microscopy Table 1

(Olympus BX60M) in samples that were etched with Kroll reagent (6 mL Chemical composition (% mass) and surface roughness of the alloys.

HNO3, 3 mL HF and 91 mL of H2O) and X-ray diffraction with the Alloy Chemical composition by XRF Ra (nm)
Panalytical X'Pert Pro instrument operated at 40 kV and 30 mA, using
Ti–35Nb Ti–34.3Nb 20.27
CuKα radiation. Ti–35Nb–7.5Ta Ti–34.5Nb–7.9Ta 20.74
Ti–35Nb–4Sn Ti–33.6Nb–4.3Sn 27.10
Ti–25Nb–8Sn Ti–24.2Nb–8.4Sn 21.35
2.2. Materials characterization Ti–25Nb–15Zr Ti–24.1Nb–14.2Zr 29.09
Ti–6Mo Ti–5.8Mo 25.66
Ti–7.1Cu Ti–7.4Cu 20.15
The morphology of the titanium alloy surfaces was evaluated by
Ti–6Al–4V Ti–6.5Al–4.6V 20.33
atomic force microscopy (AFM) (ThermoMicroscopes AutoProbe) in Ti–CP Ti–0.16Fe–0.2O 21.40
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. 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].
2.3. In vitro tests After preincubation, the medium was removed and 200 μL of
1× 105 cells/mL in medium 199 (EARLE) with 10% of BFS were placed
2.3.1. Cell culture in the wells. The plates were incubated for 24 h in CO2 at 37 °C. After
Vero cells, a line of African green monkey kidney fibroblast cells incubation, the medium was removed and the wells were washed
(Cercopithecus aethiops) supplied by the Adolfo Lutz Institute in São immediately with the same incubation medium. After washing, each well
Paulo, Brazil, were employed in the studies of cytotoxicity and cell– was filled with 200 μL of 0.5 mg/mL solution of 3-(4,5-dimethylthiazon-
substrate interactions with biomaterials [19,25,26]. 2-yl)-2,5 diphenyltetrazolium bromide MTT in medium 199 (EARLE).
The Vero cells were cultured in Medium 199 (EARLE) (Cultilab, The wells were dark-incubated for 4 h at 37 °C, after which the MTT was
Campinas, SP), with 10% of bovine fetal serum (BFS) (Cultilab, replaced with 200 μL of DMSO solution and 25 μL of Sorensen's Glycine
Campinas, SP) and incubated in 5% CO2 at 37 °C. The culture medium buffer solution. 100 μL of the solutions from the wells were then
was changed whenever acidification was detected. To maintain the transferred to a new plate and the absorbance was read in a BioTek
cell line, cells were detached and dissociated from their adhering microplate reader (ELX 800, USA) at a wavelength of 570 nm.
substrate by means of an enzymatic treatment using trypsin/EDTA,
thus providing more room for these cells to continue proliferating.
2.3.3. Cell adhesion test
For the cell adhesion tests, the titanium alloy samples were placed on
2.3.2. Direct cytotoxicity assay the bottom of 96-well culture plates (TPP — Techno Plastic Products,
Titanium alloy samples were placed on the bottom of 96-well Switzerland). The polystyrene plate itself was also used as a control and its
culture plates (TPP — Techno Plastic Products, Switzerland). The respective results were treated as 100% of cell adhesion. All the wells were
polystyrene plate itself was used as a control and its respective results filled with 100 μL of medium 199 without FBS and incubated for 24 h at
were treated as 100% of cell viability. 37 °C in an atmosphere saturated with water vapor and 5% of CO2. After

β (e) α' α' (i)

β β α'
α'
β α' α' α'αα
'
' α ' α'
β (d) (h)
β α

β α
Intensity (a.u.)

Intensity (a.u.)

β (c) α α α α β α
α'/α

β
β β (g)
α'/α Ti2 Cu

α'/α

β β β (b)
Ti2 Cu
α'/α

α'/α
Ti2 Cu

α'/α

α'/α

α'/α

α" (f)
β
β
(a) α" β

α" α" α" α"α"

α" β α" β

30 40 50 60 70 80 90 30 40 50 60 70 80 90
2θ (Degrees) 2θ (Degrees)

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.
836 A. Cremasco et al. / Materials Science and Engineering C 31 (2011) 833–839

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, 1.0
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)- 0.8

Absorbance (570 nm)

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 0.6
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. 0.4
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 0.2
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 0.0

PS - control

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
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.

Fig. 4. Cell cytotoxicity assay of Vero cells after 24 h of cell culture. The data represent the
2.3.4. Scanning Electron Microscopy (SEM)
mean±standard deviation. All the results are statistically equal except for the Ti–7.1Cu
The cell morphology was analyzed by scanning electron microscopy. alloy, which presented the lowest cell viability (pb 0.01).
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

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) 833–839 837

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)
0.5
when subjected to water quenching produced a mixture of needles of
martensitic α′ and α and a fine Ti2Cu precipitates. Again, on one hand,
Absorbance (570 nm)

0.4 Ti–6Al–4V (Fig. 1h) exhibits fully lamellar microstructure of Widman-

stätten of α dispersed within the β matrix, whereas commercially pure
(CP) Ti (Fig. 1i) showed martensite hexagonal phase. On the other hand, a
0.3 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.

0.2
3.2. Surface morphology

0.1 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
0.0 samples.
PS - control

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
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.
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
3.3. In vitro tests
alloy, which presented very low adhesion, and the Ti–35Nb–4Sn and Ti–6Mo alloys, whose
adhesion rates exceeded 100% (pb 0.01).
In vitro tests allow for quantitative evaluations of the interaction
between cells and materials. Figs. 4 and 5 depict the results obtained
in a fixing solution of 2.5% paraformaldehyde, 2.5% glutaraldehyde, in the MTT assays, indicating the viability of fibroblast cells on the
0.06%, picric acid and 1% tanic acid dissolved in cacodylate buffer alloys in question.
solution 0.1 M, pH 7.4. Later, the samples were post-fixed with 1% The cytotoxicity assays presented similar results for all the
osmium tetroxide (OsO4) and dehydrated in an ethanol series. They materials except for the Ti–7.1Cu alloy, which showed absorbance
were then dried using a Critical Point Dryer (Balzers CPD 030), gold values 50% lower than that of the control in this test and also weak
sputtered (Balzers SCD 050) and examined in a scanning electron adhesion. In the Ti–Nb–X system (X = Sn, Ta, Zr), the addition of 4% of
microscope (JEOL JSM-5800 LV). All the experiments were carried out in Sn (% mass) to Ti–35Nb alloys resulted in a tendency for augmented
triplicate. 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
3. Results and discussion increase in Sn content or the addition of Zr. No evidence of
cytotoxicity was detected in the Ti–Mo system. Despite the contro-
3.1. Microstructure Characterization versies concerning Mo cytocompatibility [27], Delvat and co-authors
recently performed cell culture tests based on cell adhesion and cell
Fig. 1 shows optical micrographs of the alloys prepared, illustrating the density, and demonstrated the excellent cytocompatibility of Ti–Mo
influence of composition on the phases formed. The phases present in 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.
838 A. Cremasco et al. / Materials Science and Engineering C 31 (2011) 833–839

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.
 A. Cremasco et al. / Materials Science and Engineering C 31 (2011) 833–839
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.
839
[3] C. Wirth, V. Comte, C. Lagneau, P. Exbrayat, M. Lissac, N. Jaffrezic-Renault, L.
Ponsonnet, Mater. Sci. Eng. C. 25 (2005) 51.
[4] T. Jinno, V.M. Goldberg, D. Davy, S. Stevenson, J. Biomed. Mater. Res. 42 (1998) 20.
[5] J. Ryhänen, M. Kallioinen, J. Tuukkanen, J. Junila, E. Niemela, P. Sandvik, W. Serlo, J.
Biomed. Mater. Res. 41 (1998) 481.
[6] Y.M. Lee, E.J. Lee, S.T. Yee, B.I. Kim, E.S. Choe, H.W. Cho, J. Mater. Sci. Mater. Med. 19
(2008) 1851.
[7] C.R.M. Afonso, G.T. Aleixo, A.J. Ramirez, R. Caram, Mater. Sci. Eng. C. 27 (2007) 908.
[8] C.M. Lee, C.P. Ju, J.H. Chern-Lin, J. Oral Rehabil. 29 (2002) 314.
[9] M. Karthega, V. Raman, N. Rajendran, Acta Biomater. 3 (2007) 1019.
[10] Y.F. Zheng, B.L. Wang, J.G. Wang, C. Li, L.C. Zhao, Mater. Sci. Eng. A. 438–440 (2006) 891.
[11] A. Choubey, R. Balasubramaniam, B. Basu, J Alloys Compd. 381 (2004) 288.
[12] Y. Okazaki, Y. Ito, K. Kyo, T. Tateishi, Mater. Sci. Eng. A. 213 (1996) 138.
[13] B.L. Wang, Y.F. Zheng, L.C. Zhao, Mater. Sci. Eng. A. 486 (2008) 146.
[14] M. Niinomi, Sci. Technol. Adv. Mater. 4 (2003) 445.
[15] E. Delvat, D.M. Gordin, T. Gloriant, J.L. Duval, M.D. Nagel, J. Mech. Behav. Biomed.
Mater. 1 (2008) 345.
[16] T. Hanawa, Sci. Technol. Adv. Mater. 3 (2002) 289.
[17] V. Borsari, G. Giavaresi, M. Fini, P. Toricelli, M. Tschon, R. Chiesa, L. Chiusoli, A.
Salito, A. Volpert, R. Giardino, Biomaterials 26 (2005) 4948.
[18] C.N. Kraft, O. Diedrich, B. Burian, O. Schmitt, M.A. Wimmer, J. Bone Joint Surg. Br.
85 (2003) 133.
[19] ISO 10993–5, Biological evaluation of medical devices — part 5: Tests for in vitro
cytotoxicity (1992).
[20] E.T. Uzumaki, C.S. Lambert, A.R. Santos Jr., C.A.C. Zavaglia, Thin Solid Films 515
(2006) 293.
[21] M. Koike, P.E. Lockwood, J.C. Wataha, T. Okabe, J. Biomed. Mater. Res. B Appl.
Biomater. 83B (2007) 327.
[22] I. Watanabe, J.C. Wataha, P.E. Lockwood, H. Shimizu, Z. Cai, J. Oral Rehabil. 31
(2004) 185.
[23] Oh. KT, D.K. Kang, G.S. Choi, K.N. Kim, J. Biomed. Mater. Res. B Appl. Biomater. 83B
(2007) 320.
[24] F. Zhang, A. Weidmann, J. Barbara Nebe, U. Beck, E. Burkel, J. Biomed. Mater. Res. B
Appl. Biomater. 94B (2010) 406.
[25] C.J. Kirkpatrick, Regul. Aff. 4 (1992) 13.
[26] A.R. Santos Jr., S.H. Barbanti, E.A.R. Duek, H. Dolder, R.S. Wada, M.L.F. Wada, Artif.
Organs 25 (2001) 7.
[27] E. Eisenbarth, D. Velten, M. Müller, R. Thull, J. Breme, Biomaterials 25 (2004) 5705.
[28] J.L. Woodman, J.J. Jacobs, J.O. Galante, R.M. Urban, J. Orthop. Res. 1 (1983) 421.
[29] W. Geurtsen, Crit. Rev. Oral Biol. Med. 13 (2002) 71.
[30] P. Rocher, L. El Medawar, J.C. Hornez, M. Trainsnel, J. Breme, H.F. Hildebrand, Scr.
Mater. 50 (2004) 255.
[31] Y. Okazaki, E. Gotoh, Biomaterials 26 (2005) 11.
[32] D.J. Wever, A.G. Veldhuizen, M.M. Sanders, J.M. Schakenraad, J.R. Van Horn,
Biomaterials 18 (1997) 1115.
[33] M. Niinomi, Metall. Mater. Trans. A 33 (2002) 477.
[34] T. Ozaki, H. Matsumoto, S. Watanabe, S. Hanada, Mater. Trans. 45 (2004) 2776.
[35] M. Fini, N. Nicoli Aldini, P. Torricelli, G. Giavaresi, V. Borsari, H. Lenger, J. Bernauer,
R. Giardino, R. Chiesa, A. Cigada, Biomaterials 24 (2003) 4929.
[36] M. Arciniegas, J. Peña, J.M. Manero, J.C. Paniagua, F.J. Gil, Philos. Mag. 17 (2008)
2529.
[37] M. Arciniegas, J.M. Manero, J. Peña, F.J. Gil, J.A. Planell, Metall. Mater. Trans. A 39
(2008) 742.