Acta Biomaterialia 21 (2015) 217–227

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Acta Biomaterialia
journal homepage: www.elsevier.com/locate/actabiomat

Novel tricalcium silicate/magnesium phosphate composite bone cement

having high compressive strength, in vitro bioactivity and
cytocompatibility
Wenjuan Liu a,b, Dong Zhai a, Zhiguang Huan a, Chengtie Wu a, Jiang Chang a,⇑
a
State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai
200050, People’s Republic of China
b
University of Chinese Academy of sciences, 319 Yueyang Road, Shanghai 200050, People’s Republic of China

a r t i c l e i n f o a b s t r a c t

Article history: Although inorganic bone cements such as calcium phosphate cements have been widely applied in
Received 20 December 2014 orthopaedic and dental fields because of their self-setting ability, development of high-strength bone
Received in revised form 23 March 2015 cement with bioactivity and biodegradability remains a major challenge. Therefore, the purpose of this
Accepted 2 April 2015
study is to prepare a tricalcium silicate/magnesium phosphate (C3S/MPC) composite bone cement, which
Available online 15 April 2015
is intended to combine the excellent bioactivity of C3S with remarkable self-setting properties and
mechanical strength of MPC. The self-setting and mechanical properties, in vitro induction of apatite for-
Keywords:
mation and degradation behaviour, and cytocompatibility of the composite cements were investigated.
Magnesium phosphate
Tricalcium silicate
Our results showed that the C3S/MPC composite cement with an optimal composition had compressive
Bone cement strength up to 87 MPa, which was significantly higher than C3S (25 MPa) and MPC (64 MPa). The setting
Orthopaedic time could be adjusted between 3 min and 29 min with the variation of compositions. The hydraulic reac-
Bioactive material tion products of the C3S/MPC composite cement were composed of calcium silicate hydrate (CSH)
derived from the hydration of C3S and gel-like amorphous substance. The C3S/MPC composite cements
could induce apatite mineralization on its surface in SBF solution and degraded gradually in Tris–HCl
solution. Besides, the composite cements showed good cytocompatibility and stimulatory effect on the
proliferation of MC3T3-E1 osteoblast cells. Our results indicated that the C3S/MPC composite bone
cement might be a new promising high-strength inorganic bioactive material which may hold the poten-
tial for bone repair in load-bearing site.
Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

1. Introduction them, silicate-based cements have received much attention for

Bone cements have gained great interest as their self-setting
properties allow for the direct injection and filling to complicated
defect sites [1]. Calcium phosphate cements (CPC) are currently
the most frequently studied inorganic bone cements due to their
good biocompatibility and osteoconductivity [2]. However, CPC
still have some drawbacks which hinder their wider application.
For example, the mechanical strength of CPC is commonly not high
enough for bone repair in load-bearing defect sites [3]. Another
concern over the use of CPC is its relatively low degradation rate
which mismatches with the formation of new bone tissue [1,3,4].
In recent years, inorganic cements other than CPC have been
developed, aiming at avoiding the shortcomings of CPC. Among
⇑ Corresponding author. Tel.: +86 21 52412804; fax: +86 21 52413903.
E-mail address: jchang@mail.sic.ac.cn (J. Chang).
their unique biological properties. As typical silicate bone cement,
tricalcium silicate (C3S) is one of the main active components in
mineral trioxide aggregates (MTA) which has been widely used
as biocompatible and bioactive root-end filling material [5]. Our
previous studies revealed that C3S was bioactive and biodegrad-
able [6–9]. It could induce bone-like apatite mineralization
in vitro [6,7] and its ionic extract solution could stimulate prolifera-
tion and osteogenic differentiation of bone-related cells [6,10], as
well as the odontogenic differentiation of human dental pulp cells
[8,9]. It was proved that Si ion released from silicate-based bio-
materials plays an important role in stimulating proliferation, dif-
ferentiation, osteogenic gene expression of osteo-related cells [11].
In addition, C3S showed moderate in vitro degradation rate [12].
Despite the advantages of bioactivity and degradability, the com-
pressive strength of C3S is relatively low and its setting time is
too long for clinical application [6].

http://dx.doi.org/10.1016/j.actbio.2015.04.012
1742-7061/Ó 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.
218 W. Liu et al. / Acta Biomaterialia 21 (2015) 217–227
Magnesium phosphate cement (MPC) is a kind of inorganic
cement developed in recent years. There were several types of
MPC with different compositions and reaction products reported
in the literature. The first MPC reported in the literature composed
of magnesium oxide (MgO) and phosphate acid [13]. Later devel-
oped compositions of MPC include: (1) MgO and phosphates
((NH4)2HPO4, NH4H2PO4, NaH2PO4, Ca(H2PO4)2  H2O et al.)
[3,13–16]; (2) magnesium phosphate (Mg3(PO4)2) and ammonium
phosphates ((NH4)2HPO4 or NH4H2PO4) [17–19]; (3) magnesium
chloride or magnesium hydroxide (MgCl2, Mg(OH)2) and phos-
phate acid [20,21], and so on. The MPCs can form different setting
products with the variation of their compositions. For example,
MgO and NH4H2PO4 form NH4MgPO4  6H2O (struvite) and
Mg(NH4)2H2(PO4)2  4H2O (schertelite) [13], MgO and NaH2PO4
form amorphous phase [13], Mg3(PO4)2 and (NH4)2HPO4 form stru-
vite [17–19], Mg(OH)2 and phosphate acid form Mg3(PO4)2 (far-
ringtonite) [21], et al. Among them, type (1) MPC has been
reported to have the characteristics of fast-setting ability and high
compressive strength which can reach 58 MPa [3,13]. The MPC
which is composed of MgO and NaH2PO4 and formed amorphous
products has a lower temperature rise and avoids harmful ammo-
nia release during setting reaction, compared with the MgO and
ammonium phosphate composition, therefore it is more suitable
to be used as a biomedical material. Both in vitro and in vivo eval-
uations have revealed that MPC had good biocompatibility and
moderate degradation rate [3,22–24]. Although it was reported in
the previous studies that Mg2+ ions could promote proliferation
and differentiation of osteoblast cells [25,26] and MPC composed
of Mg3(PO4)2 with (NH4)2HPO4 and NH4H2PO4 was bioactive in
bone regeneration [19], the bioactivity of Mg-containing bone
cements with different compositions remains to be confirmed
further.
It is therefore clear that neither C3S nor MPC alone is suitable to
be used as a high-strength bioactive bone cement. However, con-
cerning their individual advantages, we assumed that it may be
possible to obtain a bioactive cement if C3S and MPC are reason-
ably combined. In fact, our previous studies confirmed that it
was feasible to combine the advantages of C3S with CPC or calcium
sulphate to obtain improved performance [10]. Therefore, in the
present study, we intend to design a C3S/MPC composite cement
system which combines the bioactive property of C3S and high-
strength and fast-setting property of MPC. It is anticipated that
the composite bone cement may have comparable or higher com-
pressive strength than MPC and shorter setting time than C3S, and
in addition, retain the bioactivity of C3S. The content of this study
includes characterization of the hydration products, mechanical
strength and self-setting properties, and the evaluation of in vitro
bioactivity, degradation rate and cytocompatibility in order to
prove our hypothesis that the combination of C3S and MPC will
result in a fast-setting high strength bioactive bone cements.
2. Materials and methods
2.1. Preparation of C3S and MPC cement powder
C3S powder was synthesised by the sol–gel method according
to previous publication [27]. Briefly, nitric acid (18 mL, 2 mol L1)
and tetraethyl orthosilicate (TEOS, 0.5 mol) were added into
200 mL deionized water and stirred for 0.5 h. Then, Calcium nitrate
tetrahydrate (TEOS, 1.5 mol) was added and stirred for 1 h. The
mixture was transferred to an oven, kept at 60 °C for 24 h, dried
at 120 °C for 48 h and finally sintered at 1450 °C in a high-tempera-
ture furnace (Nabertherm LHT 08/17, Germany). The obtained
powder was ground with a roller type ball mill (170 r min1,
Shanghai Congming Haidao Qinggong Machinery Company) for
24 h. The mass ratio of zirconia balls (served as abrasive) and
C3S powder was 2.5:1. Finally, C3S powder was sieved through a
300-mesh sift. MPC powder was prepared by mixing magnesium
oxide (MgO), sodium dihydrogen phosphate (NaH2PO4), and
sodium borate decahydrate (Na2B4O7  10H2O, borax) as the retar-
dant of the setting reaction of MPC [13]. Less-reactive MgO was
synthesized by sintering the light magnesium oxide at 1500 °C
for 6 h. Commercial NaH2PO4 and borax were directly used. All
source reagents were of analytical grade without further purifica-
tion (Sinopharm chemical reagent Co., Ltd., Shanghai, China). All
the three kinds of powders were ground with the roller type ball
mill for 8 h, respectively, and the mass ratio of zirconia balls
(served as abrasive) and each powder was 2.5:1. After grounding,
they were sieved through a 400-mesh sift and then homoge-
neously mixed (molar ratio of MgO and NaH2PO4:3.8:1, bor-
ax:3 wt.% of MPC) through ball-milling without zirconia balls,
which rendered the MPC powders. Then C3S and MPC powders
were mixed at different mass ratios as follows: 25% C3S and 75%
MPC (C25M75), 50% C3S and 50% MPC (C50M50), 75% C3S and
25% MPC (C75M25). The phase composition of the as-prepared
powders was investigated using the X-ray diffractometer (XRD,
Geigerflex, Rigaku Co., Japan) with Cu (Ka) radiation. The particle
size distribution and specific surface area (SSA) of C3S, MgO,
NaH2PO4 and borax powder (Table 1) were measured by laser
diffraction analysis (Mastersizer 3000, Malvern Instruments Ltd.,
UK).
2.2. Preparation and characterization of the cement paste
The cement pastes were prepared by mixing the powders with
de-ionized water. Based on our preliminary experiments, the liquid
to powder ratios (LPR) were set as 0.5, 0.35, 0.25, 0.18 and
0.13 mL g1 for C3S, C25M75, C50M50, C75M25 and MPC, respec-
tively, in order to obtain optimal cohesion and workability. The
mixtures were stirred for 60 s to obtain homogenous pastes, and
then cast into a cylindrical mould with the size of 6 mm in diame-
ter and 12 mm in height. Cements were de-moulded after reaching
final setting (the state that the heavy needle of the Vicat apparatus
fails to make evident indentations on the surface of the paste, see
Section 2.3. The storing time in the mould was 113, 29, 9, 3 and
10 min for C3S, C75M25, C50M50, C25M75 and MPC, respectively.)
and cured in a shaking water bath with constant temperature of
37 °C, 100% humidity and 120 r min1 for 7 days, and then dehy-
drated with anhydrous ethanol followed by drying in the air. The
phase composition and cross-section of the samples were then
investigated using XRD and scanning electron microscope
(Hitachi S4800, Hitachi Ltd., Japan) equipped with X-ray energy
dispersive spectrum (EDS), respectively.
2.3. Self-setting properties
C3S, C25M75, C50M50, C75M25 and MPC samples used for the
compressive strength test were with the size of 6 mm in diameter
and 12 mm in height. They were prepared by the same procedures
Table 1
Particle size distribution and specific surface area (SSA) of C3S, MgO, NaH2PO4 and
borax powder obtained by laser diffraction analysis. D10, D50 and D90 mean the
particle size below which the amount of particles takes up 10%, 50% and 90%,
respectively.
SSA (m2 g1) D10 (lm) D50 (lm) D90 (lm)
C3S 0.860 2.80 13.10 190.00
MgO 1.425 1.93 5.97 16.10
NaH2PO4 0.360 10.30 25.90 65.20
Borax 0.485 6.28 17.50 36.60
 W. Liu et al. / Acta Biomaterialia 21 (2015) 217–227
as those in Section 2.2. To investigate the development of compres-
sive strength during the self-setting process, the curing time in
shaking water bath was set as 1 h, 2 h, 1 day, 2 days, 7 days,
14 days, 21 days and 28 days, respectively. To investigate the effect
of LPR on compressive strength, another group of C75M25 samples
were prepared with LPR of 0.30, 0.32, 0.35, 0.38 and 0.40 mL g1,
and cured for 24 h. Different cement samples with a constant LPR
of 0.4 mL g1 and curing time of 24 h was prepared for compressive
strength test. Then the samples were tested with a universal test-
ing machine (AG-1, SHIMADZU Corporation, Japan), at a loading
speed of 0.5 mm min1. Three parallel samples were used for each
test. In addition, the porosity of the as-prepared cement cylinders
was measured by Archimede’s principle.
The setting time of the cements was tested with Vicat apparatus
according to ISO-9597-1989E. The C3S, C25M75, C50M50, C75M25
and MPC cement pastes were prepared by mixing powders with
de-ionized water (LPR = 0.5, 0.35, 0.25, 0.18 and 0.13 mL g1,
respectively). The pastes were filled into a cylindrical container,
and the setting time was defined as the time from de-ionized water
was added into the cement powder to the time when the heavy
needle failed to make evident indentations on the surface of the
paste. Three parallel samples were used for each test. To investi-
gate the effect of LPR on setting time, C75M25 samples were
prepared with LPR of 0.30, 0.32, 0.35, 0.38 and 0.40 mL g1 and
their setting times were further tested. For testing the pH values
of the cement paste, 1 g of cement powder and de-ionized water
(0.5, 0.35, 0.25, 0.18 and 0.13 mL for C3S, C25M75, C50M50,
C75M25, respectively) were mixed homogeneously for 60 s, and
then 5 mL of de-ionized water was added. Finally, the pH value
of the water was measured every 2 min until 30 min later.
2.4. Apatite formation on the surface of cement paste
To evaluate apatite mineralization ability of different cements,
disc samples with the size of 6 mm in diameter and 2 mm in height
were soaked in simulated body fluid (SBF) solution for 7 days in the
37 °C environment. SBF solution was prepared according to the
method reported by Kokubo [28] and its composition is presented
in Table 2. The disc samples were prepared by filling the cement
paste described in Section 2.2 into a cylindrical mould, followed
by de-moulding, curing in the shaking water bath with a constant
temperature of 37 °C, 100% humidity and 120 r min1 for 7 days
and dehydrating. Then each of the discs was soaked in SBF solution
in polyethylene bottle and the ratio of the surface area of the disc
to the volume of the solution was 0.1 cm2 mL1. The bottles with
sample and SBF solution inside were put in the shaking water bath.
SBF solution was refreshed every 2 days. After 7 days’ soaking, the
samples were taken out, gently rinsed with de-ionized water and
dried in the air for 24 h. The composition and morphology of the
as-deposited surfaces layer on the discs were analysed by Fourier
Transform Infrared Spectroscopy (FTIR; Thermo Nicolet nexus-IR
spectrometer; Thermo Fisher Scientific, Waltham, MA, USA), XRD
and SEM. The FTIR test was conducted by making the powder sam-
ple which was shaved from the discs and KBr powder into a tablet
with a tablet pressing machine, and testing the infrared spec-
troscopy of the tablet in the range of 4000–400 cm1, and finally,
Table 2
Composition of SBF (1000 mL) used in the apatite mineralization study.
NaCl NaHCO3 KCl K2HPO4  3H2O MgCl2  6H2O
Amount 8.035 g 0.355 g 0.255g 0.231 g 0.311 g
purity (%) 99.5 99.5 99.5 99.0 98.0
1.0 M–HCl CaCl2 NaSO4 Tris 1.0 M–HCl
Amount 39 mL 0.292 g 0.072 g 6.118 g 0–5 mL
purity (%) – 95.0 99.0 99.0 –
219
the spectroscopy was subtracted by that of the blank KBr tablet.
The cement samples before soaking in SBF solution were also
characterized by FTIR as comparison.
2.5. Weight loss
For the weight loss test, disc samples which were cured in the
shaking water bath with constant temperature of 37 °C, 100%
humidity and 120 r min1 for 7 days and with the size of 6 mm
in diameter and 2 mm in height were soaked in Tris–HCl buffer
solution in a polyethylene bottle and the ratio of the surface area
of the disc to the volume of the solution was 0.1 cm2 mL1 [29].
The bottles with sample and Tris–HCl buffer inside were put in
the shaking water bath. After soaking for different periods (7, 14,
21, 28 days), and the discs were taken out, dried in the 60 °C oven
for 24 h, and weighed on an analytical balance with an accuracy of
0.0001 g. The Tris–HCl solutions were daily refreshed. The weight
loss ratio was calculated as follows:
Weight loss ð%Þ ¼ ðm0  mt Þ=m0  100%
where m0 denotes the original mass of the discs, and mt the mass
after soaking in Tris–HCl solution for different periods. The ion
release and pH of each sample in Tris–HCl solution was tested by
Inductively Coupled Plasma Optical Emission Spectrometer (ICP-
OES) and pH meter (PHSJ-4A, INESA Instrument, China), respec-
tively. Three parallel samples were used for each test.
2.6. In vitro cytocompatibility
In vitro cytocompatibility of the materials was evaluated by the
MTT method according to the international standard (ISO/
EN10993-5) [30], with the MC3T3-E1 osteoblast cell as the experi-
mental cell. The extract solutions were prepared as follows. Firstly,
the cement paste was cured in the shaking water bath for 7 days,
ground and sieved through a 300-mesh sift. Secondly, 1 g of the
as-prepared powder was mixed with 5 mL cell culture medium
(a-MEM) and kept in a CO2 incubator at 37 °C for 24 h. Thirdly,
the mixture was centrifuged at 4000 rpm for 10 min and the super-
natant was collected and filtered through a bacterial filter to obtain
the bacteria-free cement extract. Finally, the extract was diluted to
different concentrations (0.78, 1.56, 3.125, 6.25, 12.5, 25, 50, 100
and 200 mg mL1). 100 ll MC3T3-E1 cells with the density of
4  104 cells/mL were seeded in each hole of 96-hole plate and
cured in the CO2 incubator for 1 day. Then the culture medium
was replaced with the cement extracts of different concentrations.
After culturing for 1, 3, and 7 days, the cement extract was
removed and 100 ll MTT solution (0.5 mg mL1) was added into
each hole. After 4 h incubation in the CO2 incubator, the MTT solu-
tion was replaced by 100 ll dimethyl sulphoxide (DMSO). The
plate was shaken for 5 min and tested for optical density (OD) on
plate reader (ELX800; Bio-Tek, Winooski, VT, USA).
2.7. Statistical analysis
The data were denoted as mean ± standard deviation (SD). The
significant differences between the two groups of data were tested
by student’s t-test and if the obtained p < 0.05, the two groups of
data were considered significantly different.
3. Results
3.1. Characterization of the bone cement powder and paste
The XRD patterns of the C3S, C25M75, C50M50, C75M25 and
MPC powders and paste are shown in Fig. 1. In Fig. 1A, the C3S
220 W. Liu et al. / Acta Biomaterialia 21 (2015) 217–227
powder was pure crystalline phase (ICDD PDF No. 49-0442) (a). In
MPC powders, there were mainly MgO (ICDD PDF No. 45-0946)
and NaH2PO4 (ICDD PDF No. 11-0659) phases. For the composite
groups of C75M25 (b), C50M50 (c) and C25M75 (d), the crystalline
phases included C3S, MgO and NaH2PO4. The XRD patterns for
cement paste changed compared with the powders (Fig. 1B). For
C3S (a), the characteristic peaks in its XRD pattern were attributed
to calcium silicate hydrate (CSH, ICDD PDF No. 33-0306) which can
be denoted as xCaO  SiO2  yH2O in which x ranges from 1.2 to 2.3
according to literature report [31–33], calcium carbonate (CaCO3,
ICDD PDF No. 05-0586) and unhydrated C3S. In the XRD pattern
for MPC, there was only unreacted MgO identified (e) and its
hydration product was not discernible as it was an amorphous
phase according to previous studies [13]. The hydration products
of the composite cements (C25M75, C50M50, C75M25) were com-
posed of CSH and CaCO3 from C3S and the amorphous phases, and
no newly formed phase was identified (Fig. 1B, b–d). Moreover, for
all the cements, the intensity of diffraction peaks increased with
the amount of its corresponding component.
The cross-sectional surface morphology of cements with vari-
ous weight ratios of C3S after hydration for 7 days is shown in
Fig. 2. It can be seen that C3S presented a microporous structure
(Fig. 2a), and SEM image with higher magnification showed that
the hydrated C3S was composed of aggregates of needle-like parti-
cles (Fig. 2b). In comparison, MPC presented a smoother and more
compact structure which was composed of gel-like bulks (Fig. 2i)
Fig. 1. XRD patterns of (a) C3S, (b) C75M25, (c) C50M50, (d) C25M75 and (e) MPC
powders (A), and bone cements after curing under the environment of 37 °C and
100% humidity for 7 days (B). The phases were identified according to the following
PDF number: MgO (ICDD PDF No. 45-0946), NaH2PO4 (ICDD PDF No. 11-0659), C3S
(ICDD PDF No. 49-0442), CSH (ICDD PDF No. 33-0306), CaCO3 (ICDD PDF No. 05-
0586).
with notable cracks (Fig. 2j). In the composite samples (Fig. 2c–
h), both the particles from C3S and the gel-like bulks from MPC
coexisted, and the surface morphologies changed as the weight
ratio of C3S varied. It can be seen from the lower magnification
images that the structure became smoother and more compact
with the increase in MPC content (Fig. 2c, e and g), and C25M75
possessed the most compacted structure with least defects such
as pores and cracks (Fig. 2g). From the higher magnification
images, it can be seen that the particles were wrapped by the
gel-like substance gradually with the increase in MPC content,
and in C25M75 it was obvious that the particles was almost com-
pletely wrapped in the gel-like matrix.
To investigate the distribution of components and possible
interaction between C3S and MPC within the composite cements
during hydration, EDS-element mapping was conducted on all
the cement samples and the results are presented in Fig. 3. The
analysed area is shown by the back-scattered electron image
(Fig. 3a) which showed the composition contrast of the composite
cements. For C25 M75, it can be seen that there are mainly two
kinds of substances in this image: one is the isolated particle (num-
bered 1), and the other is the continuous matrix (numbered 2)
Fig. 2. SEM images of (a, b) C3S, (c, d) C75M25, (e, f) C50M50, (g, h) C25M75 and (i,
j) MPC bone cements after curing under the environment of 37 °C and 100%
humidity for 7 days.
 W. Liu et al. / Acta Biomaterialia 21 (2015) 217–227
Fig. 3. (a) Atomic number contrast image of the C25M75 bone cement using the back-scattered electron mode of FESEM; (b) elemental mapping images of the C25M75 bone
cement after curing under the environment of 37 °C and 100% humidity for 7 days. The numbers 1 and 2 represent the distribution area of Ca–Si elements and Na–P–Mg
elements, respectively.
which wrapped the isolated particles. From the individual element
mapping images (Fig. 3b), it can be judged that the isolated particle
corresponded to Ca and Si elements, and the continuous matrix
corresponded to Na, P and Mg elements. The boundaries between
the Ca–Si area and the Na–P–Mg area were evident. However, for
C75M25 and C50M50, the distribution of Ca, Si elements and Na,
P and Mg elements overlapped and the phenomenon of element
enrichment was not obvious as compared with C25M75
(Figs. S1–S4).
3.2. Mechanical and self-setting properties
The change of compressive strength of different cements with a
curing time from 1 h to 7 days is presented in Fig. 4A. The testing
point for C3S started from 1 day after hydration because it was
too weak to obtain a measurable value before that time point.
Among all specimens, C25M75 showed the highest compressive
strength among all the cements and increased with prolonged cur-
ing time. The compressive strength of C25M75 reached 41 MPa
and 87 MPa after curing for 1 h and 7 days, respectively, which
was significantly higher than C3S (beyond measurement and
25 MPa after 1 h and 7 days, respectively) and MPC (9 MPa and
64 MPa after 1 h and 7 days, respectively). For C50M50 and
C75M25, the compressive strength was much lower as compared
with C25M75.
The effect of LPR on the compressive strength of C75M25 sam-
ples is presented in Fig. 4B. The compressive strength of C75M25
with LPR of 0.35 mL g1 was significantly higher than with LPR of
0.30, 0.32, 0.38 and 0.40 mL g1.
The compressive strength of different cements with a constant
LPR of 0.4 mL g1 and a curing time of 24 h is presented in
Fig. 4C. It can be seen that the compressive strength of MPC and
composite cements was significantly lower than C3S. Compared
with the compressive strength for 1 day with the LPR ratio of
0.5 mL g1 (Fig. 4A), the compressive strength of C3S with LPR ratio
of 0.4 mL g1 (Fig. 4C) increased, while other formulations
decreased. Especially for MPC, the compressive strength with LPR
221
of 0.4 mL g1 decreased to 0.7 MPa, as compared with 53 MPa with
LPR of 0.13 mL g1.
The porosity of C3S, C75M25, C50M50, C25M75 and MPC
cement samples decreased gradually as shown in Fig. 4D.
The setting time of C3S, C75M25, C50M50, C25M75 and MPC
with different LPR is shown in Fig. 5A. The setting time of C3S
and MPC was 113.3 and 9.7 min, respectively. For the composite
bone cements, C75M25 had a medium setting time of 29.3 min
as compared with C3S and MPC. However, it was noted that the
C50M50 and C25M75 components had setting times lower than
those of MPC, which were 9 and 2.9 min, respectively. The
C75M25 composite cement was further studied to investigate the
influence of LPR on setting time. The results showed that setting
time increased with the increase in LPR (Fig. 5B).
The pH values in cement pastes during setting reaction were
measured to investigate possible influences of pH on the setting
reaction. The results (Fig. 6) showed that the pH of C3S paste
was around 12. The pH of MPC increased from 7.1 to 8.3 after
30 min of setting reaction. The pH of C75M25, C50M50, C25M75
was between that of C3S and MPC and increased gradually with
the proceeding of setting reactions.
3.3. Apatite mineralization
Fig. 7 shows the results of FTIR analysis on C3S, C25M75,
C50M50, C75M25 and MPC cements before (A) and after (B) soak-
ing in SBF solution for 7 days. The FTIR results showed the forma-
tion of crystalline hydroxyapatite (HA) on C3S and all the C3S/MPC
samples, which was identified by the characteristic double peaks
(563 and 603 cm1) of PO3 4 in the crystalline apatite [34]. In the
FTIR spectra of MPC, the absorption peak with the wave number
of 603 cm1 was not found in MPC.
The cements after soaking in SBF solution for 7 days were fur-
ther characterized by XRD (Fig. 8). It can be seen that the diffrac-
tion peaks of HA (ICDD PDF No. 09-0432) appeared in the XRD
pattern of C3S after soaking in SBF. Although a wide dispersing
peak in the similar position appeared in the spectra of MPC, it
222 W. Liu et al. / Acta Biomaterialia 21 (2015) 217–227

Fig. 4. Compressive strength of the C3S, C75M25, C50M50, C25M75 and MPC bone cements with the LPR of 0.5, 0.35, 0.25, 0.18 and 0.13 mL g1, respectively, after curing for
different periods (A); compressive strength of C75M25 with the variation of LPR, after curing for 24 h (B); compressive strength of C3S, C75M25, C50M50, C25M75 and MPC
with a constant of LPR (0.4 mL g1), after curing for 24 h (C), and porosity of C3S, C75M25, C50M50, C25M75 and MPC bone cements, after curing for 7 days. The curve for C3S
in (A) starts from 1 day because its compressive strength was too low to be measured before 1 day. The symbol ‘‘⁄’’ in (A) denotes existence of a significant difference between
MPC and other groups with the same LPR, and (B) it denotes existence of a significant difference between the C75M25 with LPR of 0.35 mL g1 and with other LPRs; The
symbol ‘‘’’ in (C) and (D) denotes non-existence of a significant difference between two groups.

Fig. 5. Setting time of the C3S, C75M25, C50M50, C25M75 and MPC bone cements with the LPR of 0.5, 0.35, 0.25, 0.18 and 0.13 mL g1, respectively (A) and the setting time of
C75M25 with the variation of LPR (B). The symbol ‘‘’’ in (A) and (B) denotes non-existence of significant difference between two groups.

cannot be assigned to apatite as the typical HA-related PO3 4 was
not presented in FTIR measurement of MPC. For C75M25,
C50M50 and C25M75, the diffraction peaks of HA were obvious,
which further confirmed their apatite formation ability as has been
indicated by the results of FTIR. It was found that the diffraction
peaks of MgO became stronger as the content of MPC increased.
The SEM images of the cements after soaking in SBF solution for
7 days are shown in Fig. 9. It can be seen that for all the cements a
layer of minerals formed on the surface. The microstructure of the
mineral layer for C3S was flake-like (Fig. 9b), which, according to
the IR and XRD results, should be crystalline HA. For MPC, it was
small particles (Fig. 9j). The surfaces of the composite cements
were completely covered with flake like minerals, which were
similar to those on C3S.
3.4. Weight loss
The in vitro weight loss behaviour of the samples during soaking
in Tris–HCl solution is presented in Fig. 10. When soaked in Tris–
HCl solution, all the cements dissolved gradually with prolonged
soaking time. However, they showed different degradation pro-
files. The degradation rate of C3S remained nearly constant within
28 days, with the weight loss of 4.42% and 16.16% for 7 days and
28 days, respectively. In comparison, MPC degraded faster within
the first 7 days (11.74%), and slowed down afterward with the
weight loss of 15.9% after 28 days of soaking. The degradation pro-
files of C25M75, C50M50 and C75M25 were similar to those of
MPC. It can be seen that, the weight loss of the composite cements
were between that of C3S and MPC within the first 21 days during
 W. Liu et al. / Acta Biomaterialia 21 (2015) 217–227
Fig. 6. pH values of the C3S, C75M25, C50M50, C25M75 and MPC bone cement
pastes with prolonged hydration time.
soaking. However, after 21 days, they were exceeded by C3S
because of the higher degradation rate of the latter. Comparison
between the composite cements showed that their weight losses
were close to each other, and no significant differences were
identified.
The concentrations of ions released in Tris–HCl solution during
different soaking times are presented in Fig. 11. It can be seen that
Ca2+, SiO4
4 and Mg
2+
ions showed sustained release and the release
of Mg , Na and PO3
2+ + 2
4 and B4O7 was much more rapid in the ini-
tial 7 days than that in the following 21 days. The pH values of
Tris–HCl solution mainly varied from 7.2 to 8.2 during the first
7 days of soaking time (Fig. 12).
3.5. In vitro cytocompatibility
The C25M75 composite cement was selected for the evaluation
of in vitro cytocompatibility because it showed the highest com-
pressive strength and maintained good apatite mineralization abil-
ity. C3S and MPC were selected as control groups. From the cell
culture results (Fig. 13), it can be seen that when the concentration
of ionic extracts was below 12.5 mg mL1, MC3T3-E1 cells showed
good viability for all the cements, as compared with the blank con-
trol group. However, when the ionic extract concentration was
above 12.5 mg mL1, cell proliferation decreased for all the
cements, as compared with the blank control group. In particular,
at some concentrations, C25M75 showed a significantly higher
Fig. 7. FTIR spectra of the C3S, C75M25, C50M50, C25M75 and MPC bone cements before (A) and after (B) soaking in SBF solution for 7 days.
223
level of cell proliferation as compared with other groups, suggest-
ing the stimulatory effect of C25M75 on the cell proliferation.
4. Discussion
Mechanical property is one important consideration in the clini-
cal application of bone cements. As the hydration of C3S is a grad-
ual process, the short-term compressive strength of C3S cements is
quite low. In contrast, MPC possesses a high initial compressive
strength owing to its fast-setting and relatively compact structure
after hydration. Our results confirmed our assumption that the
combination of C3S and MPC resulted in cements with high
mechanical strength. The most interesting finding is that the
C3S/MPC composite cements revealed an even higher compressive
strength than MPC cements if the ratio of the C3S and MPC compo-
nents was optimally selected. In the present study, the compres-
sive strength of C25M75 after curing for 7 days reaches 86 MPa,
which is much higher than both components [35–39] and also
comparable to the widely used PMMA [40]. An explanation for this
enhancement phenomenon is that, in C25M75 the main morphol-
ogy observed in SEM images is the gel-like bulks generated and
CSH particles are wrapped in it, functioning as reinforcement fil-
lers, and the microstructure is compact with less defects. In con-
trast, with a higher content of C3S, the original integrated bulk
matrix of MPC becomes discontinuous and disintegrated and thus,
the whole structure becomes porous and the porosity increases as
shown in Fig. 4D, which leads to a decrease in compressive
strength. Therefore, it is assumed that the improved mechanical
strength is mainly attributed to the compact structure and the
enhancement effect of CSH fillers, and the lower compressive
strength for C50M50 and C75M25 can be explained by the loose
microstructure due to the poor match of the two components. In
addition, the amorphous phases as the setting products of
C75M25, C50M50, C25M75 and MPC may be different, which is
another possible explanation for the improved compressive
strength of C25M75. Our results indicate that the microstructure
of the composite cements is critical for the mechanical strength
of the composite cements, and it can be controlled by relative
weight ratios of C3S and MPC, and the optimal weight ratio of
C3S should be around 25%. Thus, the content of C3S must be con-
trolled within a certain range to obtain high compressive strength.
The LPR ratio influences the mechanical strength of bone
cements by directly influencing porosity. With the composition
constant, compressive strength of C75M25 increased first and then
decreased with the increase in LPR, which suggests that an optimal
LPR exists for composite cements cement to reach high
224 W. Liu et al. / Acta Biomaterialia 21 (2015) 217–227

crystals [42]. Previous studies on the calcium silicate-containing

composite bone cements also revealed similar results, in which
C3S contributed to the apatite formation in composite cements
[43]. Through this apatite layer, bone and material can form a
chemical bond which may lead to good bone-material integration
[44,45].
For successful bone regeneration, the implant material should
be biodegradable so that they can be replaced by regenerated
new bone tissue gradually [46]. In the present study, all of the five
cements degraded gradually in Tris–HCl buffer solution with a pro-
longed soaking time, and their degradation rates varied with differ-
ent chemical compositions of the cements. In the composite
cements, degradation behaviour is dominated by MPC as their
degradation curves are similar to those of MPC cements.
However, the degradation rates of the composites are lower than
those of MPC because of the relatively slow degradation of C3S.
Consequently, it is possible to acquire composite cements with dif-
ferent degradation rates by adjusting the composition.
Fig. 8. XRD patterns of the C3S, C75M25, C50M50, C25M75 and MPC bone cements
after soaking in SBF solution for 7 days. The phases were identified according to the
following PDF number: HA (ICDD PDF No. 09-0432), CSH (ICDD PDF No. 33-0306),
CaCO3 (ICDD PDF No. 05-0586), MgO (ICDD PDF No. 45-0946).

compressive strength. When the LPR was kept as 0.4 mL g1, the
compressive strength of MPC and the composite cements
decreased significantly. Therefore, both composition and LPR are
important factors to consider in optimizing the mechanical
property of C3S/MPC composite bone cement.
Besides compressive strength, setting time is another important
factor that influences the clinical applications of bone cement. The
setting process for C3S mainly involves a hydraulic reaction
(3CaO  SiO2 + (3-x + y)H2O ? xCaO  SiO2  yH2O + (3-x)Ca(OH)2, in
which x ranges from 1.2 to 2.3 according to literature report)
[31–33,41]. The hydration product CSH is in the form of a solid net-
work which is the main reason for cement setting and strength
development. As the hydration of C3S is a gradual process, it takes
relatively long time for the cement to set. While for MPC, the set-
ting process is a rapid acid-base reaction between the acid reactant
of NaH2PO4 and the basic reactant of MgO [13], which is responsi-
ble for the fast-setting behaviour of MPC. In the C3S/MPC compos-
ite cements, the hydration products were the combination of the
corresponding ingredients, and the amorphous products of the set-
ting reaction may be different, leading to difference in setting time.
Besides, there may be inter-reactions between the components in
the C3S/MPC composite cements. The hydration of C3S induces for-
mation of Ca(OH)2 which may further react with NaH2PO4 to form
amorphous calcium phosphates. The pH values of C3S and MPC
cement pastes differ significantly. Thus, the setting reaction of each
component in the composite cement may be further affected by
the distinct discrepancy of pH between them. The setting time
for the composite cements can be modified by adjusting their
compositions in the present study. For example, with high
amounts of MPC (above 50%), the setting time of the composite
cements is close to that of MPC, which may meet the clinical
requirement regarding the setting time of a bone cement.
The formation of a layer of apatite is important for the binding
of orthopaedic implant materials to living bone tissue, and this
bone binding ability of materials can be evaluated in vitro in SBF
solution [28]. The present study shows that C3S and C3S/MPC com-
posite cements induced the formation of apatite layer on their sur-
face in SBF solution. As there was no apatite formed on the surface
of MPC after soaking, the apatite layers formed on the surface of
composite cements can be attributed to the presence of C3S within
the materials. The mechanism of the apatite mineralization is that
the Ca2+ ions release firstly from C3S to form a Si-rich layer, which Fig. 9. SEM images of (a, b) C3S, (c, d) C75M25, (e, f) C50M50, (g, h) C25M75 and (i,
then induces the formation of Ca–P nucleation and further apatite j) MPC bone cements after soaking in SBF solution for 7 days.
 W. Liu et al. / Acta Biomaterialia 21 (2015) 217–227
Fig. 10. Weight loss of the C3S, C75M25, C50M50, C25M75 and MPC bone cements
after soaking in Tris–HCl solution for different periods. Significant differences exist
between the following groups: (7 days) C3S and all the other four cements, and MPC
and all the other four groups; (14 days) C3S and C75M25, C3S and MPC, C75M25
and MPC, C25M75 and MPC; (14 days) MPC and C3S, MPC and C75M25, MPC and
C25M75; (28 days) C50M50 and MPC;
Previous studies have shown that C3S and MPC had good
cytocompatibility [3,6,12]. Our in vitro results showed that the
extracts of the composite cements have good cytocompatibility
at the concentration lower than 12.5 mg L1. Moreover, the
extracts of C25M75 composite cement showed stimulatory effects
on the proliferation of MC3T3-E1 cells as compared with all other
groups. The higher level of cell proliferation of C25M75 may be
attributed to synergistic stimulatory effects of Si and Mg ions
released from the C25M75 cement on the proliferation of
osteoblast-related cells, as the case in akermanite, a Mg and
Si-containing bioceramic with osteostimulation property [47].
However, since C25M75 is a multicomponent system, other ions,
such as Ca and B may have synergistic effects on cell proliferation.
Our results also showed that higher concentrations resulted in a
certain degree of cytotoxicity in all three groups such as pure
MPC, C3S and the composite cements. Considering that in the
physiological conditions, the actual concentration of the dissolved
Fig. 11. Ions concentrations in Tris–HCl buffer solutions with different soaking time and cement samples.
225
Fig. 12. pH values in Tris–HCl buffer solutions with different soaking time and
cement samples.
ions from the implanted cement will not be the same as the ion
release obtained by the extraction protocol in the current study,
this result does not mean that the cements are not biocompatible
when they are implanted in vivo. Some previous studies with
bioactive glasses, calcium silicate bioceramics and MPC cements
have demonstrated good in vivo biocompatibility, although cell
culture studies also revealed a certain degree of cytotoxicity
[19,24,48–50]. An explanation of the different cytocompatibility
between our results and those from the literature for MPC could
be the difference in cement composition and cell types, which
we know they react differently to different compositions of mate-
rials. Nevertheless, our results indicate that we do need to consider
the cytotoxicity effect of the cements if we apply the materials for
some applications such as tissue engineering approach, in which a
direct contact of cells with the material scaffolds is required. The
MTT assay result suggests that the C25M75 composite cement
may have the activity to stimulate bone regeneration as compared
with C3S and MPC. However, further investigations are needed to
evaluate its bioactivity and biocompatibility both in vitro and
in vivo.
226 W. Liu et al. / Acta Biomaterialia 21 (2015) 217–227
Fig. 13. MTT assay of osteoblast-like (MC3T3-E1) cells cultured in a-MEM medium conditioned with different concentrations of C3S, C25M75 and MPC powder extracts. The
symbols ⁄, § and h represent significant difference as compared with blank, C3S and MPC, respectively.
Although previous studies reported that Mg ions could stimu-
late proliferation and differentiation of osteoblast cells [25,26,51–
53], the MPC cements in the present study did not show stim-
ulatory effects on cell proliferation. Therefore, the function of Mg
ions may be different in different Mg-containing materials. In addi-
tion, it remains to be confirmed whether the bioactive mechanism
of the MPC/C3S composite cements is different from that of the
pure MPC or C3S.
5. Conclusion
In the present study, novel C3S/MPC composite cements with
high compressive strength and short setting time were developed.
The compressive strength and setting time of the composite
cements could be modulated by the weight ratio of C3S and MPC.
Among the composite cements, C25M75 showed the highest com-
pressive strength of 86 MPa, which is close to the lower limit of
human cortical bone (90–209 MPa) and much higher than C3S
and MPC. The composite bone cement has good apatite mineraliza-
tion ability in SBF solution and degraded gradually with a moderate
degradation rate in vitro. Moreover, the composite cement showed
good in vitro cytobiocompatibility and stimulated the proliferation
of MC3T3-E1 osteoblast cells. These results suggest that, through
the combination of C3S and MPC, it is possible to obtain composite
cements which keep or even surpass the advantages of high com-
pressive strength and bioactivity of its individual components.
The bioactive high-strength C3S/MPC composite cement in the pre-
sent study might have potential for orthopaedic applications.
6. Disclosures
There is no potential conflict of interest for this study.
Acknowledgements
We acknowledge the financial support from the National
Natural Science Foundation of China (Grant No. 81190132), the
External Cooperation Program of the Chinese Academy of
Sciences (Grant No.GJHZ1211), One-hundred Talent Program of
SIC-CAS (Y36ZB1110G), and the Funds of the Clinical Reseach
Center for Biomaterials, SIC-CAS (Grant No. BMCRC2010002).
Appendix A. Figures with essential colour discrimination
Certain figures in this article, particularly Figs. 1, 3, 4, 6–8, 10–
13, are difficult to interpret in black and white. The full colour
images can be found in the on-line version, at doi: http://dx.doi.
org/10.1016/j.actbio.2015.04.012.
Appendix B. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.actbio.2015.04.
012.
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