Porous TiNbZr alloy scaffolds for biomedical applications
Xiaojian Wang, Yuncang Li, Jianyu Xiong, Peter D. Hodgson, Cui’e Wen
*
Institute for Technology Research and Innovation, Deakin University, Geelong, Vic. 3217, Australia
Received 27 January 2009; received in revised form 31 May 2009; accepted 2 June 2009
Available online 6 June 2009
Abstract
In the present study, porous Ti–10Nb–10Zr alloy scaffolds with different porosities were successfully fabricated by a ‘‘space-holder”
sintering method. By the addition of biocompatible alloying elements the porous TiNbZr scaffolds achieved significantly higher strength
than unalloyed Ti scaffolds of the same porosity. In particular, the porous TiNbZr alloy with 59% porosity exhibited an elastic modulus
and plateau stress of 5.6 GPa and 137 MPa, respectively. The porous alloys exhibited excellent ductility during compression tests and the
deformation mechanism is mainly governed by bending and buckling of the struts. Cell cultures revealed that SaOS2 osteoblast-like cells
grew on the surface and inside the pores and showed good spreading. Cell viability for the porous scaffold was three times higher than the
solid counterpart. The present study has demonstrated that the porous TiNbZr alloy scaffolds are promising scaffold biomaterials for
bone tissue engineering by virtue of their appropriate mechanical properties, highly porous structure and excellent biocompatibility.
Crown Copyright
Ó 2009 Published by Elsevier Ltd. on behalf of Acta Materialia Inc. All rights reserved.
Keywords: Titanium alloy; Scaffold; Mechanical properties; Biocompatibility
1. Introduction
Porous materials are of significant importance for bone
tissue engineering applications, as they provide good bio-
logical fixation to the surrounding tissue through bone tis-
sue ingrowth into the porous network
. Although porous
ceramics and polymers have been studied as scaffold mate-
rials, they cannot meet the mechanical requirements for
load bearing conditions
. For this reason, porous Ti
and Ti alloys have been developed. The major benefits of
porous Ti and Ti alloys compared with other bone graft
materials include their good mechanical strength, with an
elastic modulus close to that of bone, porous structure,
providing biological fixation, and good biocompatibility
.
A number of approaches to the fabrication of porous Ti
and Ti alloys have been reported, including sintering loose
Ti powder or fibre
, slurry sintering
and rapid pro-
totyping
. Sintering a mixture of Ti powder and
space-holder particles (e.g. urea or ammonium hydrogen
carbonate) produced porous Ti with a porosity of 70%
which exhibited a plateau stress of 53 MPa and an elastic
modulus of 3.4 GPa
. However, the compressive strength
of porous Ti is still lower than that of cortical bone (180–
200 MPa)
. In order to achieve a porous scaffold com-
bined with high strength and high porosity, some porous
Ti alloy scaffolds were therefore developed
. A porous
Ti–6Al–4V alloy scaffold was reported to achieve higher
strength than unalloyed porous Ti at the same porosity
. However, research on the biological behaviour of
metals has shown that the composition of implant bioma-
terials must be carefully selected to avoid or minimize
adverse reactions
. The release of metal ions from some
metal materials, e.g. aluminium (Al), nickel (Ni), iron (Fe),
vanadium (V) and chromidium (Co), can generate adverse
biological effects
. On the other hand, titanium (Ti), zir-
conium (Zr), niobium (Nb) and tantalum (Ta) are believed
to be non-toxic metals with good biocompatibility
and
they are widely used in low modulus b or a/b Ti implant
materials
. Thus there is an increasing research interest
in developing porous Ti-based alloys using non-toxic
1742-7061/$ - see front matter Crown Copyright
Ó 2009 Published by Elsevier Ltd. on behalf of Acta Materialia Inc. All rights reserved.
doi:10.1016/j.actbio.2009.06.002
*
Corresponding author. Address: Institute for Technology Research
and Innovation, Deakin University, Pigdons Rd., Waurn Ponds, Geelong,
Vic. 3217, Australia. Tel.: +61 3 5227 3354; fax: +61 3 5227 1103.
E-mail address:
(C. Wen).
Available online at www.sciencedirect.com
Acta Biomaterialia 5 (2009) 3616–3624
www.elsevier.com/locate/actabiomat
alloying elements. New generation porous Ti-based alloy
scaffolds are expected to combine high mechanical strength
and good biocompatibility.
In the present study Ti–10Nb–10Zr was designed with
the addition of a b stabilizer of Nb and a stabilizer of Zr
. The TiNbZr alloy is expected to provide high yield
strength due to the addition of Nb and Zr, which ensure
a duplex microstructure of the a/b phase. The structure
and mechanical properties of the porous TiNbZr were
studied. Cell adhesion and proliferation of human osteo-
blast-like cells cultured on the porous alloy scaffolds were
assessed.
2. Materials and methods
2.1. Specimen preparation and characterization
The fabrication method of the porous TiNbZr alloy was
similar to that used in previous studies
. Elemental
metal powders of Ti (purity >99.7%, particle size <45 lm),
Nb (purity >99.8%, particle size <45 lm) and Zr (purity
>99.9%, particle size <45 lm) were weighed to give a nom-
inal composition of Ti–10Nb–10Zr (hereafter wt.%). The
powders were blended in a planetary ball milling system
with stainless steel containers and balls (PM400, Retsch)
for 4 h with a weight ratio of ball to powder of 1:2 and
rotation rate of 100 rpm. The blended TiNbZr powder
was mixed with ammonium hydrogen carbonate (NH
4
H-
CO
3
), which was used as the space-holder material. The
size of space-holder particles was selected to be 500–
800 lm. The mixture of Ti, Nb, Zr powder and NH
4
HCO
3
was cold pressed into green compacts in a 50 ton hydraulic
press and the green compacts were sintered in a vacuum of
10
4
–10
5
Torr in two steps. The first step was carried out
at 175
°C for 2 h to burn out the space-holder particles. In
the second step the compacts were heated up to 1200
°C
and held for 10 h. Porous TiNbZr alloys with porosities
of 42%, 50%, 59%, 69% and 74% were fabricated by adding
20, 30, 40, 50 and 60 wt.% ammonium hydrogen carbonate
to the powder mixture.
The general porosity of the porous alloys was calculated
by the formula
e
¼
1
q
q
s
100
ð1Þ
where q and q
s
are the density of the porous alloy and its
corresponding theoretical density, respectively. The density
of the porous alloy was determined from its weight and
dimensional measurements. The theoretical density is
5.1 g cm
3
(based on the alloy composition of Ti–10Nb–
10Zr). The general pores inside the porous alloys consist
of two parts, interconnected pores (open pores) and closed
pores. The interconnectivity of the porous TiNbZr was
evaluated by measuring the weight of paraffin penetrating
into the porous structure when they were boiled in paraffin
in a vacuum chamber. The interconnectivity was calculated
by comparing the volume of paraffin penetrating into the
porous structure and overall pore volume. Scanning elec-
tron microscopy (SEM) combined with quantitative image
analyse using Image-Pro Plus (Media Cybernetics, Inc., Sil-
ver Spring, MD) software were used to characterize the
pore structure and pore size distribution. X-ray diffraction
(XRD) was used to characterize the phase constituents of
the porous alloys. Surface roughness was measured using
Surtronic 3+ Roughness checker.
2.2. Mechanical test and simulation
Cylindrical samples with a diameter of 10 mm and
length of 15 mm were used to examine the mechanical
properties. Five samples were tested for each condition.
The samples were cut by electrical discharge machining
(EDM), which ensures plane and parallel end faces of the
samples. Compression tests were carried out in a 30 kN
Instron, equipped with a non-contact extensometer (resolu-
tion 0.5 lm) and the initial strain rate was set at 10
3
s
1
.
Compression tests were also conducted on solid Ti–
10Nb–10Zr
cylinder
samples
prepared
by
powder
metallurgy.
The elastic modulus and yield strength of porous TiN-
bZr from this study were compared with the predictions
of the Gibson–Ashby (G&A) model
and Finite Ele-
ment Method (FEM) model proposed by Roberts and Gar-
boczi
. In the Gibson–Ashby model the structure of
the porous material was simplified and it was assumed that
the open-cell foam consists of periodic arranged beams.
Bending of the beams dominated the deformation mecha-
nism. The relationships between plateau stress, elastic mod-
ulus and relative density are given by:
E=E
s
¼ C
1
ðq=q
s
Þ
n
1
ð2Þ
r
pl
=
r
ys
¼ C
2
ðq=q
s
Þ
n
2
ð3Þ
where E is the elastic modulus of the foam, E
s
is the elastic
modulus of the cell surface solid material, r
pl
is the pla-
teau stress of the porous material, r
ys
is the yield strength
of the cell surface solid material and C
1
, C
2
, n
1
and n
2
are
constants, depending on the cell structure. To date, the
complex dependence of C
1
, C
2
, n
1
and n
2
on structure is
not well understood. Experimental evidence suggests that
n
1
= 2, n
2
= 1.5 for open-cell foams, C
1
is a constant of
1 for rigid polymers, elastomers, metals and glasses and
C
2
is a constant of 0.3 for cellular metals and polymers.
Elastic moduli of both open-cell and closed-cell foams
based on the FEM have been proposed by Roberts and
Garboczi
. The ‘‘Gaussian Random Filed” model
was used in the present study, as this model deals with
structures similar to the cellular structures of porous TiN-
bZr scaffolds. The FEM model is assumed to be a highly
irregular structure with curved struts of variable thickness.
Equations to fit the results of their simulations are:
E=E
s
¼ 4:2ðq=q
s
Þ
3:15
for 0:05 <
q
q
s
<
0:20
ð4Þ
X. Wang et al. / Acta Biomaterialia 5 (2009) 3616–3624
3617
Eq.
describes the higher porosity data (relative density
<0.2). For lower porosity, where
q
q
s
> 0.20, the relationship
is:
E=E
s
¼
q=q
s
0:029
0:971
2:15
ð5Þ
2.3. MTT assay
Cell culture was performed on a solid TiNbZr alloy
sample and the porous TiNbZr alloy with a porosity of
69% in 12-well tissue culture plates. SaOS2 osteoblast-like
cells (10,000 per well) were seeded on the top surface of
the samples. The cells were cultured in minimum essential
media (MEM) supplemented with 10% fetal bovine serum,
1% non-essential amino acid, 10,000 U ml
1
penicillin,
10,000 lg ml
1
streptomycin and 0.4% amphostat B at
37
°C in a humidified atmosphere of 5% CO
2
and 95%
air. The media was changed every 3 days.
An MTT assay to measure cell viability and prolifera-
tion was used to quantitatively determine the number of
viable cells that had attached and grown on the samples.
After culture for 14 days the MTT solution was added
and incubated with the cells for 4 h. The cells were then
lysed to release and solubilize purple formazan. Colorimet-
ric analysis and comparison to a standard curve of known
viable cell numbers can be used to calculate viable cell
numbers for each condition. The MTT assay was con-
ducted on a two-dimensional tissue culture-treated surface
(2D Con), cell seeded metal discs (Disc) and the wells that
contained the discs (Well). Disc + Well refers to the sum of
the cell numbers of seeded discs and their respective wells.
n = 3 for all experimental groups. Significant differences in
the cell number were analysed using one-way ANOVA
(P < 0.05). Samples after cell culture for 14 days (n = 2)
were fixed and then rinsed with phosphate-buffered saline,
dehydrated in a graded ethanol series, critical point dried
and examined by SEM.
3. Results and discussion
3.1. Sintering of porous TiNbZr alloy
a shows the morphology of the blended Ti, Nb and
Zr powders. Pre-alloying did not occur during the blending
process, as a low energy ball milling process was adopted in
the present study. The morphology of the blended powder
is similar to that of the raw powders. The size of these pow-
ders is mainly in the range 5–45 lm. XRD showed that the
blended powder is a mixture of Ti, Nb and Zr powders, as
shown in
. After burning off the space-holder parti-
cles the green compacts were then sintered at 1200
°C in
high vacuum.
A typical powder sintering process comprises three
stages: stage 1 – where the growth of the sintering bond
from initial loose powder occurs and the sintering neck
Fig. 1. (a) Powder morphology of the blended TiNbZr powders. (b)
Backscattering image of porous TiNbZr alloy sintered at 1200
°C for 10 h.
Fig. 2. XRD pattern of the blended powder mixture (a) and the sintered
porous TiNbZr (b).
3618
X. Wang et al. / Acta Biomaterialia 5 (2009) 3616–3624
forms; stage 2 – where pore rounding, densification and
grain growth occur simultaneously (densification in this
stage is accomplished by diffusion of the alloying elements,
grain boundary diffusion and growth); stage 3 – where den-
sification is slower than in stages 1 and 2. There are simul-
taneous grain coarsening events that impede densification
. In general, the densification rate increases with
decreasing particle size and increasing sintering tempera-
ture and time, where the particle size and sintering temper-
ature are much more efficient than the sintering time in
influencing the sintering rate
. It is shown that Nb
and Zr diffused into the Ti matrix leading to the formation
of a + b phases after sintering for 10 h. This was confirmed
by XRD, as shown in
. As shown in
b, there
were no obvious elemental Nb- and Zr-rich areas remain-
ing after sintering and the sintered porous TiNbZr alloy
consists of lamellar a and b phases.
3.2. Structure and pore distribution in porous TiNbZr alloys
shows the morphologies of the porous TiNbZr
alloys with porosities ranging from 42% to 74%. The por-
ous alloys were prepared using the same space-holder par-
ticles. Statistical analysis of pore size reveals that the
porous TiNbZr alloy sintered using space-holder particles
of 500–800 lm gave pore sizes ranging from 300 to
800 lm, accounting for 52.4% of the total pores. Beside
macropores, there were also some micropores present on
the cell wall of the porous alloys (
e). The size of the
micropores was in the range 5–20 lm. It is believed that
the presence of micropores is essential for porous scaffolds
to achieve osteoinduction
By adjusting the amount of space-holder particles the
porosity of the resulting porous TiNbZr alloy can be
altered. The interconnectivity of the porous alloy also
Fig. 3. Morphology of porous TiNbZr alloys with different porosities: (a) 42%; (b) 50%; (c) 59%; (d) 69%; (e) 74% and (f) Statistical analysis of pore size
distribution in (e).
X. Wang et al. / Acta Biomaterialia 5 (2009) 3616–3624
3619
increases with porosity. As shown in
, the intercon-
nectivity was 40% for the sample with a total porosity of
42%, and the porous sample with a porosity >69% achieved
nearly full interconnectivity. The interconnectivity is very
important for porous biomaterials, as the connected pores
will allow cells to grow inside the material and body fluid to
circulate
. Thus, high porosity is preferable for porous
scaffold biomaterials. However, high porosity causes a
decrease in the mechanical properties of porous materials
, which will be discussed in the next section.
3.3. Mechanical properties of porous TiNbZr alloys
The mechanical properties of porous TiNbZr alloys
were investigated by compression tests on cylindrical sam-
ples. The compressive stress–strain curves of the porous
TiNbZr alloys with porosities of 42–74% are shown in
. The stress–strain curves are smooth, indicating
excellent ductility of the porous alloys during the compres-
sion test. The calculated elastic moduli and plateau stresses
(yield stresses) are shown in
. The elastic modulus
and yield strength of dense TiNbZr (porosity 4%) were
68 GPa and 1438 MPa, respectively. The porous TiNbZr
alloy scaffold with a porosity of 59% exhibited an elastic
modulus and plateau stress of 5.6 GPa and 137 MPa,
respectively, which is close to the mechanical properties
of natural bone
. The plateau stress of the porous TiN-
bZr alloy was significantly higher than that of porous pure
Ti. The compressive strengths of porous TiNbZr scaffolds
with porosities of 59% and 69% were 137 and 67 MPa,
while at the same porosity the compressive strengths for
porous pure Ti were 122 and 53 MPa, respectively
shows the predicted elastic modulus (E) of the
porous TiNbZr alloy normalized to the elastic modulus
of the solid alloy (E
s
) as a function of the relative density
(q/q
s
). For comparison, the Gibson–Ashby and FEM mod-
els are also included. It can be seen that the experimental
data are better predicted by the FEM model than the Gib-
son–Ashby model in the whole range of relative densities.
The elastic modulus predicted by the FEM model is lower
than that predicted by the Gibson–Ashby model. It should
be noted that the FEM model deals with a highly irregular
structure with curved struts of variable thickness. Since the
elastic modulus of a porous structure is determined by the
thinnest struts, the mass in the thickest regions of the struts
contributes little to the overall elastic modulus. This has
the effect of reducing the elastic modulus of a porous struc-
ture for a given density, compared with models having
struts with a uniform cross-sectional area
. It is clear
that the experimental data in the present study are slightly
lower than the value predicted by the FEM model in the
higher porosity range (
). It can deduced from this
Table 1
Summary of the mechanical properties of porous TiNbZr alloys as a function of porosity.
TiNbZr
Elastic modulus (GPa)
Plateau stress (MPa)
Interconnectivity (%)
Ultimate stress (MPa)
Elongation (%)
Solid TNZ
68 (3.5)
1438 (39)
N/A
1830 (30)
15.3 (0.5)
42% porosity
21 (0.3)
368 (5.5)
40
50% porosity
7.9 (0.7)
235 (4.6)
82
59% porosity
5.6 (0.3)
137 (2.3)
91
69% porosity
3.9 (0.3)
67 (2.3)
98
74% porosity
1.6 (0.2)
27 (1.5)
99
a
Yield stress; standard deviations are shown in parentheses.
Fig. 4. Compressive stress–strain curves for the porous TiNbZr alloy
scaffolds with various porosities from 42% to 74%.
Fig. 5. Elastic modulus (E) normalized to the solid metal elastic modulus
(E
s
) as a function of relative density for the porous TiNbZr alloys.
Predictions from the Gibson–Ashby model and Finite Element Method
(FEM) model of Roberts and Garboczi.
3620
X. Wang et al. / Acta Biomaterialia 5 (2009) 3616–3624
that there are highly non-uniform struts in the porous TiN-
bZr alloy with high porosity and this leads to a decrease in
the elastic modulus of the porous alloy. The elastic modu-
lus of the porous alloys with higher porosities is very sensi-
tive to the homogeneity of strut thickness of the porous
structure.
shows the plateau strength of the porous TiNbZr
(r) normalized to the yield strength of the solid metal (r
s
)
as a function of relative density (q/q
s
) and that predicted by
the Gibson–Ashby model. It can be seen that the plateau
strength of a porous material depends on the porosity (rel-
ative density). According to Eq.
, C is a constant for the
geometric effect and n is the density exponent. The varia-
tion in the value of C is probably due to heterogeneity in
pore shape and size. The value of n in the literature varies
over a wide range from 1.0 to 6.3
. It is reported that the
value of n in Eq.
is governed by the deformation mode
of the individual cell struts, i.e. yielding, bending and buck-
ling
. Yamada et al.
suggested the value of n for cell
wall or strut bending is 2 and that for struts buckling is 3,
while the value of n is 1 for cell strut yielding. In the present
study the value of n is 3 for the porous TiNbZr alloy.
Therefore, this suggests that the deformation mode of the
porous TiNbZr alloy with a porosity ranging from 42%
to 74% mainly comprises bending and buckling of the cell
struts.
3.4. In vitro biocompatibility of porous TiNbZr alloys
The MTT assay was used to determine cell viability and
the proliferation of human osteoblast-like cells on the solid
and porous TiNbZr alloys. For comparison, solid pure
titanium was also assessed.
shows the cell numbers
on the solid Ti and solid TiNbZr alloy and porous TiNbZr
scaffold after cell culture for 14 days. It can be seen that the
solid TiNbZr alloy shows slightly higher biocompatibility
than pure Ti. However, the total cell number (Well + Disc)
of the porous TiNbZr scaffold is significantly higher than
the solid TiNbZr alloy and solid pure Ti plates. In partic-
ular, the cell number on the samples (Disc) of the porous
TiNbZr scaffold is three times higher than that on the solid
Fig. 6. Plateau stress (r) normalized to the solid metal yield stress (r
s
) as a
function of relative density and the prediction of Gibson–Ashby model for
the porous TiNbZr alloy.
Fig. 7. MTT assay of cells on solid Ti, TiNbZr plates and porous TiNbZr alloy.
*
Significant difference from TiNbZr plates (P < 0.05).
X. Wang et al. / Acta Biomaterialia 5 (2009) 3616–3624
3621
samples. This mainly results from the larger surface area of
the porous material over the solid material. The results
indicate that the porous TiNbZr scaffolds are more favour-
able for cell adhesion and proliferation than their solid
counterparts.
After cell culture for 14 days, cells on the porous TiNbZr
were observed to be spreading well, both on the surface of
the porous scaffold and inside the pores. Cells were
observed growing across the scaffold edge into the pores
(
a). However, different cell growth behaviours were
observed on the surface of the porous scaffold and inside
the pores. As shown in
e, the surface of the porous
scaffold had been ground with 600 grit sand paper, resulting
in a rather smooth surface (Ra = 0.21 ± 0.01 lm), while the
surface inside the pores was rougher (Ra = 2.4 ± 0.37 lm).
It is now clear that cell attachment and proliferation are
sensitive to the surface topography and roughness
After culture for 14 days the surface of the porous scaffold
was covered by a monolayer of flat, spread cells (
a and
b). The cells were observed to grow along the grinding
grooves. Cell growth inside the pores was randomly orien-
tated. Cells started to cover the space between two particles.
It can be seen that fibrous extracellular matrix (ECM) for-
mation was found on the surface of the porous scaffold,
which appears to be collagen (
d), while on the surface
of the solid samples less ECM could be found. It can be con-
cluded that a rough surface promotes osteoblast-like cell
differentiation towards an osteoblastic phenotype. Previous
studies also found a positive correlation between cell differ-
entiation and roughness
. An increase in alkaline
phosphatase activity and increased ECM formation were
found on rough surfaces compared with smooth surfaces.
Confocal micrographs confirmed that cells grew through-
out the entire porous structure, both on the surface and
inside the pores (
). It can be seen that the surface of
the porous scaffold was covered by a monolayer of flat
and spread cells, as shown in
a. Cell growth inside
the pores was randomly orientated and the density of cell
inside the pores was lower than that on the surface of the
porous scaffold (
b).
4. Conclusions
Porous Ti–10Nb–10Zr alloy scaffolds were successfully
fabricated in the present study. The porous structure,
mechanical properties and in vitro biocompatibility of the
porous alloy scaffolds were investigated. The conclusions
reached are as follows.
The porous TiNbZr scaffolds had a bimodal porous
architecture, with macropores of 300–800 lm and microp-
ores of several micrometres. By adjusting the amount of
space-holder particles, the porosity of the resulting porous
Fig. 8. Morphology of osteoblast cells after 14 days culture. (a) Cell growth across the pore and the surface; (b) a confluent cell layer covering the surface
of porous scaffolds; (c) cell growth inside the pores of the porous TiNbZr alloy scaffold and (d) inside the pores, where cells cover the space between
particles. ECM formation was also found.
3622
X. Wang et al. / Acta Biomaterialia 5 (2009) 3616–3624
TiNbZr alloy could be changed over the range 42–74%.
The porous scaffolds with porosity over 69% achieved
nearly fully interconnectivity.
The porous TiNbZr scaffolds presented higher strength,
compared with porous unalloyed Ti scaffolds, for the same
porosity. The porous alloys exhibited excellent ductility
during compression tests and the compressive deformation
was mainly governed by bending and buckling of the struts
of the porous structure.
The SaOS2 osteoblast-like cells grew on the surface and
inside the pores and showed good spreading. The cell via-
bility for the porous TiNbZr scaffold was three times
higher than the solid counterpart.
Acknowledgements
The authors acknowledge financial support for this re-
search through the ARC Discovery Project DP0770021
(Australian Research Council). P. Hodgson was also sup-
ported by the ARC through a Federation Fellowship.
Appendix
Figures with essential color discrimination. Certain
figures in this article, particularly Figure 9, are difficult to
interpret in black and white. The full color images can be
found
in
the
on-line
version,
at
.
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