Journal of Biomaterials
Applications
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Mechanical Properties and In Vitro Bioactivity of Ca5(PO4)2SiO4
Bioceramic
Wenhao Lu, Wei Duan, Yaping Guo and Congqin Ning
J Biomater Appl 2012 26: 637 originally published online 28 September 2010
DOI: 10.1177/0885328210383599
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Mechanical Properties and
In Vitro Bioactivity of
Ca5(PO4)2SiO4 Bioceramic
WENHAO LU,1,2 WEI DUAN,1,2 YAPING GUO1
1,
AND CONGQIN NING *
1
State Key Laboratory of High Performance Ceramics and Superfine
Microstructure, Shanghai Institute of Ceramics,
Chinese Academy of Sciences, 1295 Dingxi Road,
Shanghai 200050, China
2
Chinese Academy of Sciences, Graduate School of
Chinese Academy of Sciences, 1295 Dingxi Road,
Shanghai 200050, China
ABSTRACT: Pure Ca5(PO4)2SiO4 bioceramic was first prepared by a sol–gel
method using triethyl phosphate, tetraethoxysilane, and calcium nitrate
tetrahydrate as original materials. Simulated body fluid (SBF) immersion tests
revealed that Ca5(PO4)2SiO4 samples had a greater in vitro apatite-forming
ability than hydroxyapatite (HA). After soaking Ca5(PO4)2SiO4 samples in the
SBF for 1 day, bone-like apatite precipitated on the surfaces and the apatite
layer became thicker with increasing the soaking time. However, few bone-like
apatites precipitated on the HA samples even after soaking in the SBF for
7 days. The good in vitro bioactivity of Ca5(PO4)2SiO4 samples was attributed to
the silanol (Si-OH) groups and greater solubility of Ca5(PO4)2SiO4. In addition,
hot-pressed Ca5(PO4)2SiO4 ceramic exhibited lower bending strength and elastic
modulus than hot-pressed HA, since the former had a lower relative density than
the latter. The results have shown that Ca5(PO4)2SiO4 is a potential candidate
material for bone repair.
*Author to whom correspondence should be addressed. E-mail: cqning@[Link]
JOURNAL OF BIOMATERIALS APPLICATIONS Vol. 26 — February 2012 637
0885-3282/12/06 0637–14 $10.00/0 DOI: 10.1177/0885328210383599
ß The Author(s), 2010. Reprints and permissions:
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638 W. LU ET AL.
KEY WORDS: calcium phosphate silicate, sol–gel synthesis, mechanical
property, apatite formation, bioactivity.
INTRODUCTION
I mplantable materials such as hydroxyapatite (HA, Ca10(PO4)6
(OH)2) and tri-calcium phosphate (TCP, Ca3(PO4)2) have drawn a
great deal of interest recently as potential bone substitute materials due
to their biocompatibility, lack of inflammatory response, and their
similarities to the chemical compositions of hard skeleton tissues [1,2].
TCP are referred to as resorbable materials as they can be gradually
absorbed over a period of time when implanted in bone defects, being
simultaneously replaced by the natural host tissue [3]. However, TCPs
have shown a limited bioactivity in vitro [4] and slow osseous tissue
formation in vivo [5]. It has been proved that a bone-bonding can be
formed between HA and the surrounding bone tissue when implanted,
while the stoichiometric HA can hardly be resorbed by the natural host
tissue [6].
On the other hand, the studies by Carlisle [7] have shown the
importance of silicon for skeletal development in baby chicks and an
increasing concentration of silicon in young, actively growing bones [8],
which implied that silicon is an essential trace element for bone
development. Studies on silicon-based biogalsses and bioceramics have
shown that the high bioactivity of bioactive materials are attributed to
the silicon component, which can form silanol groups (Si-OH) on the
material surface through ion exchanges in physiological solutions [9].
The silanol groups can act as bioactive sites to induce the formation of
bone-like apatite by attracting calcium and phosphorus ions. Inspired by
these studies, many efforts have been devoted to fabricate silicon
substituted hydroxyapatite (Si-HA) and silicon doped tri-calcium
phosphate (Si-TCP). The Si-doped calcium phosphate materials have
shown a great rate of in vivo dissolution [10] as well as an improved
bone formation ability [11–13]. During the sintering process of Si-HA or
Si-TCP, calcium phosphate silicate phase (Ca5(PO4)2SiO4) have been
detected [11–13]. Ca5(PO4)2SiO4 is a compound consisting of calcium,
phosphorus, and silicon which is supposed to have a good bioactivity.
However, no studies have been reported on the pure phase
Ca5(PO4)2SiO4 as material for bone repair. In the present work, pure
phase Ca5(PO4)2SiO4 bioceramic was first prepared by a sol–gel method,
and their mechanical properties and in vitro bioactivity were
investigated.
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Properties and In Vitro Bioactivity of Ca5(PO4)2SiO4 Bioceramic 639
MATERIALS AND METHODS
Sol–Gel Synthesis of Ca5(PO4)2SiO4 Powder
Triethyl phosphate (TEP), tetraethoxysilane (TEOS), and calcium
nitrate tetrahydrate (Ca(NO3)2 4H2O) were used as P, Si, and Ca
sources, respectively. The synthesis procedure is described briefly as
follows: TEOS was added dropwise into a mixture of ethanol and
deionized water (Vwater:VEtOH ¼ 2:1) under constant stirring using 2N
HNO3 as a catalyst. After the complete hydrolysis of TEOS, proper
amount of TEP and calcium nitrate were dissolved gradually into the
above solution under stirring, and then the mixture was aged for 2 h at
ambient temperature (208C for the present experiment) followed by
another aging procedure at 608C for 72 h to form gel. The obtained gel
was dried at 1208C for 48 h and then ground and calcined at 14008C for
6 h at a heating rate of 28C/min.
Preparation of HA Powder
The HA powder was prepared using a precipitation reaction (Equation
(1)) between ammoniated solutions of calcium nitrate and di-ammonium
hydrogen phosphate with pH value greater than 10.8.
10CaðNO3 Þ2 þ 6ðNH4 Þ2 HPO4 þ 8NH4 OH
! Ca10 ðPO4 Þ6 ðOHÞ2 þ 20NH4 NO3 þ 6H2 O ð1Þ
The precipitation was filtered and washed three times with water,
then sintered at 9008C for 2 h with a heating rate of 28C/min.
MECHANICAL PROPERTIES
The synthesized Ca5(PO4)2SiO4 powder was hot-pressed (ZT-60-22Y,
Shanghai Chen Hua Electric Furnace Co. Ltd., China) at 14008C for
0.5 h under a pressure of 20 MPa in an argon atmosphere and the
synthesized HA powder was also hot-pressed at 12008C for 0.5 h under a
pressure of 20 MPa in an argon atmosphere. Three-point bending test
was performed on the universal testing machine (Instron 5566, Instron
Corporation, America) using polished specimens with dimensions of
3 mm in thickness, 4 mm in width, and 36 mm in length. The span was
30 mm and the crosshead speed was 0.5 mm/min. The Young’s modulus
of the specimens was calculated from the slope of the initial linear elastic
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640 W. LU ET AL.
portion of the load–displacement curve. The value was the average of
five measurements.
In vitro Bioactivity
Apatite formation ability in the simulated body fluid (SBF) is always
used to evaluate the in vitro bioactivity of biomaterials for bone repair
[14]. In the present work, the SBF was prepared according to the
procedure described by Kokubo and Takadama [15]. The compositions of
the SBF and human blood plasma are listed in Table 1. The synthesized
powders were uniaxially pressed into disks with a dimension of
10 mm 2 mm. The Ca5(PO4)2SiO4 disks were then sintered at
14008C for 2 h and the HA disks were sintered at 12008C for 2 h. The
as-sintered disks were cleaned ultrasonically in acetone, absolute
alcohol, and deionized water, in turn and then soaked in the SBF for
1 day, 3 days, and 7 days with a geometrical surface area/liquid volume
ratio of 0.1 cm1. The SBF was refreshed every day during soaking. At
each time point, all the specimens were removed from the SBF and
gently rinsed with deionized water and then dried at 608C for 6 h.
Characterization Techniques
The phase composition of the as-synthesized powders and sintered
ceramics was analyzed by X-ray diffraction method (XRD, D/Max
2250 V, Rigaku Co., Tokyo, Japan). The microstructure and the surface
morphology of the ceramics were investigated using scanning electron
microscopy (JSM-6700F, JEOL Ltd., Tokyo, Japan) coupled with field
emission (FE-SEM). The surface elemental analysis was performed by
an energy dispersive spectrometer (EDS, JXA-8100, JEOL Ltd., Tokyo,
Japan). The concentrations of calcium, silicon, and phosphorus in the
collected SBF solution were determined by inductively coupled plasma
atomic emission spectroscopy (ICP-AES, Vista AX, Varian Co.,
America). The density of the sintered samples was measured using
Archimedes’ principle. The elemental composition analysis was
Table 1. Ion concentrations of the SBF and human blood plasma.
Ion concentrations (mmol/L1)
Type Naþ Kþ Mg2þ Ca2þ Cl HCO
3 HPO2
4
SBF 142.0 5.0 1.5 2.5 148.8 4.2 1.0
Blood plasma 142.0 5.0 1.5 2.5 103.0 27.0 1.0
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Properties and In Vitro Bioactivity of Ca5(PO4)2SiO4 Bioceramic 641
evaluated using X-ray Fluorescence Analysis (XRF, PW2404, Philips,
Holland).
RESULTS
Characterization of the Synthesized Ca5(PO4)2SiO4 Powder
and HA Powder
The XRD pattern of the synthesized Ca5(PO4)2SiO4 powder is shown
in Figure 1, which coincides well with the standard Ca5(PO4)2SiO4 data
in the JCPDS database (PDF No. 40-0393). From the sharp and distinct
peaks, it is indicated that pure phase Ca5(PO4)2SiO4 can be successfully
synthesized by the sol–gel method using TEOS, calcium nitrate
tetrahydrate, and TEP as sources of Si, Ca, and P, respectively.
The chemical composition of the synthesized Ca5(PO4)2SiO4 deter-
mined by XRF analysis (as shown in Table 2) showed that the
SiO2:P2O5:CaO ratio of the as-synthesized powder was similar to that
of the stoichiometric Ca5(PO4)2SiO4. The XRF results further confirmed
that pure phase Ca5(PO4)2SiO4 was prepared.
Intensity (a.u.)
Ca5(PO4)2SiO4
PDF No. 40-0393
20 25 30 35 40 45 50 55
2q (deg)
Figure 1. XRD pattern of the synthesized Ca5(PO4)2SiO4 powder.
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642 W. LU ET AL.
Table 2. Chemical composition of the synthesized Ca5(PO4)2SiO4 powder.
Composition Realistic contents (mol%) Theoretical contents (mol%) Realistic/Theoretical
SiO2 14.81 14.29 1.03
P 2O 5 13.43 14.29 0.95
CaO 71.76 71.42 1.02
Intensity (a.u.)
HA
PDF No. 09-0432
20 25 30 35 40 45 50 55 60 65 70
2q (deg)
Figure 2. XRD pattern of the synthesized HA powder.
The XRD pattern of the synthesized HA powder is shown in Figure 2.
Both the 2y position and the intensity of the diffraction peaks are
identical to the standard diffraction pattern of HA (PDF 09-0432). It is
revealed that pure HA powder was prepared via aqueous precipitation
method.
Mechanical Properties
The bending strength and elastic modulus of Ca5(PO4)2SiO4 ceramic
are shown in Figure 3, compared with HA ceramic. The bending
strength and elastic modulus of Ca5(PO4)2SiO4 were 60.4 MPa and
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Properties and In Vitro Bioactivity of Ca5(PO4)2SiO4 Bioceramic 643
140 140
Bending strength
120 Elastic modulus 120
Bending strength (MPa)
100 100
Elastic modulus (GPa)
80 80
60 60
40 40
20 20
0 0
HA Ca5(PO4)2SiO4
Figure 3. Bending strength and elastic modulus of the hot-pressed Ca5(PO4)2SiO4 and
HA ceramics.
Ca5(PO4)2SiO4 HA
Figure 4. SEM images of the polished surface of Ca5(PO4)2SiO4 and HA ceramics
prepared by hot-pressing.
77.5 GPa while HA ceramic were 84.4 MPa and 97.7 GPa. Both the
bending strength and elastic modulus of Ca5(PO4)2SiO4 were lower than
those of HA. The SEM images of the surface of Ca5(PO4)2SiO4 and HA
fabricated by hot-pressing are shown in Figure 4, which shows that
Ca5(PO4)2SiO4 has more pores than HA, indicating that the former has a
lower relative density than the latter. The relative densities measured by
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644 W. LU ET AL.
Archimedes’ principle of Ca5(PO4)2SiO4 and HA plates were 92% and
95%, which coincided with the SEM observations.
In Vitro Bioactivity
SEM images of the surfaces of Ca5(PO4)2SiO4 disks before and after
soaking in the SBF for various periods are shown in Figures 5 and 6,
respectively. Before soaking in the SBF (Figure 5), the surface of
Ca5(PO4)2SiO4 disks were relatively rough and many pores were
observed (as shown in Figure 5(a) and (b)), while the SEM images at
higher magnification (Figure 5(c)) showed that the surface of crystal
grains was smooth and clean. The EDS results showed that the surface
compositions of Ca5(PO4)2SiO4 disks before soaking were coincident
with those of the stoichiometry of Ca5(PO4)2SiO4. The Ca/P/Si atomic
ratio was nearly 5/2/1. After soaking in the SBF for 1 day (Figure 6(a)),
the Ca5(PO4)2SiO4 grains could hardly be seen whereas a layer of worm-
like apatite were observed on the material surface. In addition, the
average Ca/P ratio decreased from 2.50 to 2.28 and the signal of Si
(a) (b)
(c)
Ca/P=2.50
Si
O Ca
Ca
0 1 2 3 4 5
Figure 5. SEM images and EDS spectra of Ca5(PO4)2SiO4 before soaking in the SBF.
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Properties and In Vitro Bioactivity of Ca5(PO4)2SiO4 Bioceramic 645
Ca
(a) SBF 1 day
Ca/P=2.28
Si
Ca
Cl Ca
O Cl
Na
0 1 2 3 4 5
Ca
(b) SBF 3 days Ca/P=1.71
P
O
Ca
Cl Mg Si Ca
Cl
Na
0 1 2 3 4 5
Ca/P=1.65 Ca
(c) SBF 7 days
P
O
Ca
Cl Mg Ca
Cl
Na
0 1 2 3 4 5
Figure 6. SEM images and EDS spectra of Ca5(PO4)2SiO4 after soaking in the SBF for
various periods.
reduced obviously. After immersing in the SBF for 3 days (Figure 6(b)),
a successive and denser apatite layer was formed on the surface of the
material surface and it showed an average Ca/P ratio of 1.71, which was
much closer to that of HA. Interestingly, some channels and pores were
formed on the surface of Ca5(PO4)2SiO4 disks after soaking in the SBF
for 7 days (Figure 6(c)). At this time point, the EDS spectra did not show
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646 W. LU ET AL.
(a) Before SBF (b) SBF 7 days
Figure 7. SEM images of HA before and after soaking in the SBF for 7 days.
350
300
250
Element concentration (ppm)
Ca
200
P
Si
150
100
50
–50
–100
0 1 2 3 4 5 6 7
Time(day)
Figure 8. Element concentration changes in the collected SBF immersed with
Ca5(PO4)2SiO4.
any trace of silicon which implied that the thickness of the formed
apatite layer increased.
In the case of HA disks, the surface of HA are much denser than that
of Ca5(PO4)2SiO4 and grain boundaries are clearly seen before soaking
(as shown in Figure 7(a)). After soaking in the SBF for 7 days, the
surface of HA was almost the same as that before soaking, except for
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Properties and In Vitro Bioactivity of Ca5(PO4)2SiO4 Bioceramic 647
140
Ca
120 P
Element concentration (ppm)
100
80
60
40
20
–20
0 1 2 3 4 5 6 7
Time (day)
Figure 9. Element concentration changes in the collected SBF immersed with HA.
some pits resulted from dissolution (Figure 7(b)). No apatite particles
were observed on its surface.
Changes in element concentrations of calcium, silicon, and phos-
phorus in the collected SBF immersed with Ca5(PO4)2SiO4 ceramics are
shown in Figure 8. There is a rapid increase in calcium and silicon
element concentrations at the early stage of immersion, while the
phosphorus concentration showed a monotonic decrease with the
increase in immersion time. In the case of HA (as shown in Figure 9),
both the calcium and phosphorus concentrations increased during the
first day of immersion, but the calcium concentration was much lower
than that for Ca5(PO4)2SiO4 case. During the following six days, no big
changes were detected in the calcium and phosphorus concentrations.
DISCUSSION
Previous studies have reported the formation of Ca5(PO4)2SiO4 phase
in crystallized Si-containing HA [12]. However, it existed only as a by-
product. Both XRD and XRF results indicated that pure Ca5(PO4)2SiO4
could be successfully synthesized by the sol–gel method using TEOS,
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648 W. LU ET AL.
calcium nitrate tetrahydrate, and TEP as sources of Si, Ca, and P
respectively.
Bioactive ceramics, including bioglass, and A-W glass-ceramic, can
spontaneously form a bone-like apatite layer on their surface in the
living body, and can bond to bone through the apatite layer [16]. Based on
a number of studies, Kokubo et al. [17] pointed out that the pre-requisite
for apatite formation on an implantable material in a living body is the
presence of a type of functional group that could act as effective sites for
apatite nucleation on its surface. It has been proved that the silicon
component in the material can form silanol (Si-OH) groups on its surface
in the physiological solution through ion exchanges between the material
and surrounding solution. The silanol (Si-OH) groups on the material
surface are responsible for its bioactivity. In the present work,
Ca5(PO4)2SiO4 exhibited a greater in vitro apatite-forming ability than
HA, which might be attributed to the silanol (Si-OH) groups and great
solubility of Ca5(PO4)2SiO4 as shown in Figure 8.
After soaking Ca5(PO4)2SiO4 in the SBF, Ca2þ, PO3 4
4 , and SiO4 ions
2þ
were released into the surrounding solution. The released Ca and
PO34 ions could improve the supersaturation of the SBF with respect to
apatite. As stated above, the released SiO4 4 might form silanol groups
on the surface of Ca5(PO4)2SiO4, which could provide low-energy sites
for apatite nucleating. Once the apatite nucleated, it could grow
spontaneously by consuming the calcium and phosphate ions from
the surrounding SBF. The decrease in phosphorus concentration in
the SBF implied that the apatite growth rate was higher than the
Ca5(PO4)2SiO4 dissolution rate, since the Ca/P in Ca5(PO4)2SiO4 is
higher than that in HA.
Channels and pores in the apatite layer were due to the dissolution of
apatite crystals. These channels could provide pathways for the ion
exchange and keep a sustained Ca2þ and SiO4 4 release, which could be
confirmed by the increase of Si in the collected SBF and no trace of Si on
the surface of the Ca5(PO4)2SiO4 ceramic. Although the SBF used in the
present work was highly supersaturated with respect to apatite, few
bone-like apatite precipitated on the HA samples, which indicated that
Ca5(PO4)2SiO4 had a superior bioactivity than HA.
Many pores in the hot-pressed Ca5(PO4)2SiO4 ceramic observed by
SEM analyses indicated that Ca5(PO4)2SiO4 powder synthesized by the
sol–gel method was difficult to densify, which might be due to the
organic residuals in the calcined powder. The lower bending strength
and elastic modulus of the hot-pressed Ca5(PO4)2SiO4 ceramic were
mainly attributed to the lower density.
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Properties and In Vitro Bioactivity of Ca5(PO4)2SiO4 Bioceramic 649
CONCLUSIONS
Ca5(PO4)2SiO4 powders were successfully synthesized by the sol–gel
method using TEOS, calcium nitrate tetrahydrate, and TEP as sources
of Si, Ca, and P, respectively. Ca5(PO4)2SiO4 ceramic showed a superior
in vitro bioactivity by inducing bone-like apatite in the SBF. The hot-
pressed Ca5(PO4)2SiO4 ceramic exhibited lower bending strength and
elastic modulus than hot-pressed HA, since the former had a lower
relative density than the latter.
ACKNOWLEDGMENTS
This work was financially supported by the Shanghai Pujiang
Program (Grant No. 07pj14092) and the National Natural Science
Foundation of China (Grant No. 50732002).
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