Journal of Asian Ceramic Societies 4 (2016) 85–90

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Journal of Asian Ceramic Societies

journal homepage: www.elsevier.com/locate/jascer

Synthesis of hydroxyapatite from eggshell powders through ball

milling and heat treatment
Shih-Ching Wu a , Hsueh-Chuan Hsu a , Shih-Kuang Hsu a , Ya-Chu Chang b , Wen-Fu Ho c,∗
a
Department of Dental Technology and Materials Science, Central Taiwan University of Science and Technology, Taichung, Taiwan, ROC
b
Department of Materials Science and Engineering, Da-Yeh University, Changhua, Taiwan, ROC
c
Department of Chemical and Materials Engineering, National University of Kaohsiung, Kaohsiung, Taiwan, ROC

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

Article history: Every day, several million tons of eggshells are being generated as bio-waste across the world. This study
Received 26 September 2015 demonstrates the synthesis of HA powder using dicalcium phosphate dehydrate (CaHPO4 ·2H2 O, DCPD)
Received in revised form and eggshell powders via ball milling and subsequent heat treatment. The formation of HA phase can be
28 November 2015
initiated by sintering the 1 h milled sample at 1000 ◦ C for 1 h, while pure HA phase can be obtained upon
Accepted 3 December 2015
Available online 30 December 2015
sintering the 10 h milled sample. Additionally, the final products composed of biphasic calcium phosphate
(HA + ␤-TCP crystals) can easily be prepared by ball milling for 5 h followed by heat treatment at 1000 ◦ C
for 1 h. The carbonate peaks observed in the FTIR analysis of the as-prepared HA closely matched those
Keywords:
Eggshell of A- and B-type carbonates, which is typical of the biological apatite. The elemental composition of the
Hydroxyapatite as-synthesized HA showed the presence of Ca, P, Mg, and Sr.
␤-Tricalcium phosphate © 2015 The Ceramic Society of Japan and the Korean Ceramic Society. Production and hosting by
Ball milling Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
Heat treatment licenses/by-nc-nd/4.0/).

1. Introduction turn depend on the synthesis precursors and processing conditions

Hydroxyapatite (Ca10 (PO4 )6 (OH)2 , HA) is being actively inves-
tigated as a potential catalyst or a catalyst support for a variety of
chemical reactions [1,2]. Besides, HA is also being analyzed as an
adsorbent for environmental protection, owing to its strong affin-
ity toward heavy metal ions [3,4]. Furthermore, synthetic HA finds
application in various medical and dental applications as a promis-
ing material for healing damaged bones and teeth, for implant and
scaffold, and as a drug delivery agent, due to its biocompatibility
and bioactivity as well as its similarity to the inorganic component
of the hard tissues in natural bones [5,6]. However, the properties of
natural apatite crystals and conventional synthetic stoichiometric
HA are significantly different [7]. For example, the natural apatite
in the human body contains significant amounts of carbonate and
trace elements [8,9]. Natural bone is clearly a non-stoichiometric
HA [10]. The properties of synthetic HA are largely determined by its
particle size, morphology, crystallinity, and composition, which in
∗ Corresponding author at: Department of Chemical and Materials Engineering,
National University of Kaohsiung, 700 Kaohsiung University Rd., Nanzih District,
Kaohsiung 81148, Taiwan, ROC. Tel.: +886 7 591 7276; fax: +886 7 591 9277.
E-mail addresses: fujii@nuk.edu.tw, titi0918@yahoo.com.tw (W.-F. Ho).
Peer review under responsibility of The Ceramic Society of Japan and the Korean
Ceramic Society.
[11].
To this end, a variety of synthesis techniques, including sol–gel
[12], aqueous precipitation [13], hydrothermal technique [14–16],
and solid-state reactions [17–19] have been reported for the syn-
thesis of HA. Over the past years, biologically derived natural
materials, such as fish bone, bovine bone, corals, oyster shell, and
eggshells, have been converted into useful biomaterials like HA.
For instance, the study reported by Lemos et al. [20] demonstrates
the transformation of natural aragonite from cuttlefish bone into
HA via hydrothermal treatment at 200 ◦ C. Ooi et al. [21] reported
the synthesis of porous HA from bovine bone via heat treatment
in the temperature range of 400–1200 ◦ C. Sivakumar et al. [22]
have demonstrated the conversion of coral into monophasic HA
via a low temperature hydrothermal process. A recent study has
demonstrated the synthesis of HA from oyster shell powders mixed
with calcium pyrophosphate (Ca2 P2 O7 ) or dicalcium phosphate
dehydrate (CaHPO4 ·2H2 O, DCPD) by ball milling and subsequently
heat treatment [18]. More recently, Ho et al. [16] synthesized
nanosized HA via hydrothermal treatment of eggshell and several
biomolecules obtained from waste materials (pomelo, grape, and
sweet potato peel extracts). These materials offer promising oppor-
tunities, given the fact that the raw materials are wastes. Besides,
the use of ‘biological apatite’ containing some trace elements as
bone substitutes, instead of ‘chemical apatite’, would be much ben-
eficial for bone defect healing [23].

http://dx.doi.org/10.1016/j.jascer.2015.12.002
2187-0764 © 2015 The Ceramic Society of Japan and the Korean Ceramic Society. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
86 S.-C. Wu et al. / Journal of Asian Ceramic Societies 4 (2016) 85–90

Eggshells generated after breaking eggs represent a significant

waste because they become typically useless after the use of egg
contents and its derivatives. Such eggshell wastes are commonly
disposed of in landfills without any pre-treatment. Such activi-
ties are highly undesirable to the environment, especially from
the viewpoint of the odor generated during the biodegradation.
Eggshells constitute 11% of the total weight of the egg and are
mainly composed of calcium carbonate (CaCO3 ) [24]. In the present
investigation, an attempt has been made to synthesize pure and
bone-like HA powder from eggshells. The eggshell powder, which
was used as the Ca source, was combined with DCPD powder. In the
typical process, the powder mixture was ball-milled for 1, 5, and
10 h, followed by heat treatment at 1000 ◦ C for 1 h. Although there
are several reports on the synthesis of HA from eggshells, most pro-
cesses are done by wet chemical methods. Clearly, this technique
is lengthy, complicated and require pH adjustment and control. To
the best of our knowledge, no study has reported the synthesis of
HA powder using DCPD and eggshell powders via ball milling and Fig. 1. XRD patterns of the powders produced by milling DCPD and eggshell powders
subsequent heat treatment. This study shows a great potential for for various durations (a) 1, (b) 5, and (c) 10 h.
the conversion of eggshell waste into highly valuable compounds
using simple yet effective processes.

The average crystallite size of the synthesized HA particles was

2. Materials and methods calculated by using the Scherrer’s formula as follows [26]:

In this study, DCPD (Yakuri Chemicals Co., Ltd., Japan) and
eggshell powders were used as starting materials for the syn-
thesis of HA. Raw membrane-bound hen eggshell was collected
from a breakfast shop in the university campus and immediately
stored in a refrigerator. The eggshells were pre-treated by strip-
ping the membrane off the eggshell, followed by rinsing with
water, drying, and then crushing and powdering using an agate
mortar. The eggshell powders thus obtained were sieved using a
325-mesh sieve. Subsequently, the hand-ground eggshell powder
and DCPD were homogeneously mixed with deionized water in
a zirconia container. The ratio of eggshell powders to DCPD was
4:3 (mole ratio). The resulting mixture was wet-milled in a plan-
etary ball-milling machine (QM-3SP4J, Nangjing, China) for 1, 5,
or 10 h at a speed of 170 rpm in a zirconia bottle. After milling,
the slurry was dried completely in a convection oven at 150 ◦ C
for 24 h. Following that, the dried powder was heated to 1000 ◦ C
at a rate of 10 ◦ C/min, and subsequently held at that temperature
for 1 h.
The crystalline phases of the ball-milled powders before and
after heat treatment were analyzed by using powder X-ray diffrac-
tion with Cu K␣ radiation (XRD; XRD-6000, Shimadzu, Japan).
The phases were identified by comparing the experimental X-ray
diffractograms with the standards compiled by the Joint Committee
on Powder Diffraction Standards (JCPDS). The microstructure of the
powders was observed using scanning electron microscope (SEM;
JSM-6700F, JEOL, Japan) under secondary electron mode. Further-
more, the Fourier transform infrared (FTIR; Bio-Rad, FTS-40, USA)
spectra of the powdered samples were obtained in the wavenum-
ber range of 600–4000 cm−1 . The composition of the synthesized
powders was analyzed by using inductively coupled plasma-atomic
emission spectroscopy (ICP-AES; ICAP 9000, Jarrell-Ash Co., USA).
From the XRD data, the crystallinity (Xc ) of the HA particles was
calculated according to the following equation [25]:
V112/300
Xc = 1 −
I300
where I300 is the intensity of (300) diffraction peak and V112/300
is the intensity of the hollow between (112) and (300) diffraction
peaks of HA.
0.9
Xs =
FWHM cos 
where Xs is the average crystallite size (nm);  is the wavelength of
the X-ray used for the analysis (1.5406 Å); FWHM is the full width
at half maximum for the diffraction peak under consideration (rad)
and  (◦ ) is the Bragg’s angle. In this study, the (002) diffraction
peak was chosen for calculation of the crystallite size since it was
isolated and sharper than the other peaks.
3. Results and discussion
3.1. Characterization of ball-milled powders
Fig. 1 shows the XRD patterns of the DCPD and eggshell powder
mixtures milled for 1, 5, and 10 h. As can be seen from the diffrac-
tion pattern of the 1 h milled powder, the peaks mainly correspond
to dicalcium phosphate anhydrous (DCPA) and CaCO3 with trace
amounts of ␤-TCP and HA phase. The DCPD precursor completely
decomposed to DCPA because of the increased temperature and
vigorous stirring during ball milling [27]. Upon milling for 5 h, the
intensities of DCPA and CaCO3 decreased with the obvious appear-
ance of diffraction peaks corresponding to ␤-TCP phase, together
with trace amounts of HA phase. With further increase in milling
time to 10 h, the diffraction peaks indicated the formation of HA
phase with small amounts of DCPA and CaCO3 from precursors.
When compared to 5 h milled sample, the XRD pattern of the 10 h
milled sample shows a decrease in ␤-TCP content, which indicates
the gradual replacement of ␤-TCP by HA.
The microstructure of the DCPD and eggshell powders milled
for 1, 5, and 10 h showed remarkable differences in size and shape
(Fig. 2). The reduction in the particle size was greater with increase
in milling time. The microstructure of the powders milled for 1 and
5 h showed larger particles of irregular shape and non-uniform size
distribution, which could be related to the unreacted precursors.
The submicron-sized particles observed in the 5 h milled sample
could possibly be related to the ␤-TCP and HA phase. However,
with increase in milling time to 10 h, the microstructure revealed
smaller particles with relatively uniform size distribution.
 S.-C. Wu et al. / Journal of Asian Ceramic Societies 4 (2016) 85–90
Fig. 2. SEM images of the powders produced by milling DCPD mixed with eggshell powders for various durations (a) 1, (b) 5, and (c) 10 h.
Fig. 3. XRD patterns of the products synthesized by milling DCPD and eggshell pow-
ders for various durations (a) 1, (b) 5, and (c) 10 h, followed by heat treatment at
1000 ◦ C for 1 h.
3.2. Characterization of the powders after heat treatment
Fig. 3 shows the X-ray diffraction patterns of the samples syn-
thesized by milling DCPD and eggshell powders for 1, 5, and 10 h
followed by subsequent heat-treated at 1000 ◦ C for 1 h. The XRD
pattern of the 1 h milled and sintered sample shown in Fig. 3a indi-
cates the presence of unreacted Ca2 P2 O7 (pyrophosphate derived
from DCPD after heating) and CaO (obtained from eggshell pow-
ders after heating). Besides, this stage had large amounts of ␤-TCP
phase. However, with further increase in milling time to 5 h and
subsequent heat-treatment, there were no peaks corresponding
to CaO or Ca2 P2 O7 . This indicates that the unreacted precursor
has been incorporated into the HA and ␤-TCP lattice through
high temperature diffusion. Furthermore, the intensity ratio, ␤-
TCP(0210)/HA(211), also decreased with increase in ball-milling
time, indicating that the HA phase is preferred over the ␤-TCP
phase. With further increase in milling time to 10 h and subsequent
87
heat treatment, the XRD peak corresponding to ␤-TCP phase dis-
appeared completely and replaced by HA. The final products (10 h
milled and heat-treated sample) were composed entirely of HA
crystals. An earlier study [28] has indicated the formation of HA
powder via a one-step ball milling process without any heat treat-
ment. However, in the current study, it seems that the milling time
of 10 h is not sufficient to obtain a pure HA sample without sinter-
ing the powder at elevated temperatures. It is evident from Fig. 3
that the formation of HA phase has been initiated through sinter-
ing the 1 h milled sample at 1000 ◦ C for 1 h, while pure HA phase is
formed only upon sintering the 10 h milled sample. The 1 and 5 h
milled samples had additional XRD peaks corresponding to ␤-TCP
phase.
The final products composed entirely of HA crystals or biphasic
calcium phosphate (HA + ␤-TCP crystals) can easily be prepared
by varying the ball milling time, as shown in Fig. 3b and c. The
formation of ␤-TCP phase under certain experimental conditions
can be considered beneficial, although preparation of phase-pure
HA from eggshell is the original intention of the present work.
Recently, biphasic calcium phosphate (HA/TCP) composite has been
studied to overcome the low biodegradability of HA. Tetsuya et al.
[29] demonstrated that HA ceramics containing TCP induced better
osseointegration than pure HA ceramics when implanted in sheep
femora. In addition, biphasic calcium phosphate ceramics have
been evaluated in both osseous and non-osseous tissues [30,31].
Results have shown that the biphasic ceramics are biologically
more active than pure HA ceramics, and that the biological behavior
of the biphasic ceramics is superior in the formation of new bone.
Fig. 4 shows the SEM images of the products synthesized from
DCPD and eggshell powders milled for 1, 5, and 10 h and then
heat-treated at 1000 ◦ C for 1 h. As is seen, the powders have spher-
ical shaped particles of nano to submicron size range. The average
particle size decreased from 2.21 to 1.25 ␮m with increase in ball-
milling time. Upon subsequent heat treatment, the agglomeration
of the particles became more prominent due to high temperature
sintering and high surface areas.
Table 1 summarizes the crystallinity and crystallite size of the
as-prepared HA powders milled for 1, 5, and 10 h, followed by heat-
treatment at 1000 ◦ C for 1 h. The crystallinity of the synthesized HA
88 S.-C. Wu et al. / Journal of Asian Ceramic Societies 4 (2016) 85–90

Fig. 4. SEM images of the products synthesized by milling DCPD and eggshell powders for various durations (a) 1, (b) 5, and (c) 10 h, followed by heat treatment at 1000 ◦ C
for 1 h.

particles, as calculated from the XRD data, was found to increase

with ball-milling time, although it was slightly lower than that
of commercial HA. According to the XRD results (Fig. 3c), the as-
synthesized HA powder exhibited good crystallinity (96.4%) and
high phase-purity. The crystallite sizes of the HA particles, which
were obtained by milling DCPD and eggshell powders for 1, 5, and
10 h followed by heat-treatment at 1000 ◦ C for 1 h, were 37.1, 40.8,
and 36.0 nm, respectively. According to previous reports, bone crys-
tals can be 30–50 nm in length, 15–30 nm in width, and 2–10 nm in
thickness [32]. The nanostructure of bone-substituting materials
is closely related to their good bioactivity and osteoconductivity.
That said, synthetic HA composed of nanosized crystals could offer
increased osteoblast functions [33].
Furthermore, FTIR analysis was performed to identify the func-
tional groups present in the HA particles. This in turn provided
information about the constitution and phase composition of the
products synthesized by milling DCPD and eggshell powders for Fig. 5. FTIR spectrum of the HA prepared by milling DCPD and eggshell powders for
10 h followed by heat treatment at 1000 ◦ C for 1 h. The FTIR spec- 10 h, followed by heat treatment at 1000 ◦ C for 1 h.
tra of the samples shown in Fig. 5 had bands corresponding to
OH− , PO4 3− and H2 O. Further analysis of the IR spectrum revealed
CO3 2− substitution, as identified by characteristic peaks of CO3 2− (B-type) in the crystal lattice [36]. Accordingly, it can be concluded
at around 888 and 1521 cm−1 , which are attributed to the vibra- that the prepared HA is chemically and structurally analogous to
tional modes of the carbonate ions substituted at the hydroxide ion apatite of natural bone. The apatite in natural bone contains sig-
(A-type) [34]. Moreover, the carbonate ion band at 1465 cm−1 can nificant amounts of carbonate ions, ranging from about 4 to 6 wt%
be ascribed to B-type carbonate substitution on phosphate ion sites [32]. In recent years, several in vitro studies [37,38] have demon-
[35]. In this experiment, the carbonate peaks observed for the spec- strated the outstanding properties of carbonated HA for use as a
imens closely matched those of A- and B-type carbonates. Indeed, bioresorbable bone substitute, as compared to pure stoichiomet-
anionic sites within the HA crystals are very susceptible to carbon ric HA. Also, in vivo study indicated that the dissolution rate of
dioxide originated from the atmosphere, and hence the CO3 2− gen- subcutaneously implanted sintered carbonated HA ceramics was
erated from CO2 can substitute hydroxyls (A-type) or phosphates intermediate between ␤-TCP and pure HA [39]. The carbonated HA

Table 1
Effect of ball milling on the crystallinity, crystallite size, and particle size of as-prepared HA powders obtained by milling for 1, 5, or 10 h, followed by heat treatment at
1000 ◦ C for 1 h.

Ball milling time (h) Crystallinity, Xc (%) Crystallite size, Xs (nm) Particle size (␮m)

1 85.0 37.1 2.21

5 94.1 40.8 1.98
10 96.4 36.0 1.25
 S.-C. Wu et al. / Journal of Asian Ceramic Societies 4 (2016) 85–90
has been found to be more effective with a shorter post-operative
rehabilitation program [40]. Thus, among the variety of HA-based
bioceramics, carbonated HA has attracted significant attention.
Given this fact, the awareness of the contribution of carbonated
HA to bone and dental enamel health has been greatly increasing
[41].
In the current study, pure HA is the main constituent of the prod-
uct obtained from DCPD and eggshell powders ball-milled for 10 h
followed by heat treatment at 1000 ◦ C for 1 h (Fig. 3c). Elemental
composition of the synthesized HA, as analyzed by using ICP-AES,
showed the presence of Ca (39.2 wt%), P (16.8 wt%), Mg (0.648 wt%),
and Sr (0.031 wt%), with an average Ca/P molar ratio of around 1.81.
The higher Ca/P molar ratio observed in the present study, as com-
pared to that of stoichiometric HA, can be attributed to the presence
of carbonate ions substituting the phosphate. This type of HA is typ-
ical of the mineral phase of apatite in natural bone [42], where the
carbonates strongly contribute to the variation of Ca/P ratio. Other
studies have observed similar high value of Ca/P ratio in HA from
bovine bones [43].
Eggshells are essentially composed of calcium carbonate (94%),
calcium phosphate (1%), organic matter (4%), and magnesium
carbonate (1%) [24]. The natural biological origin of eggshells,
containing several trace elements that remain in the crystalline
structure of as-prepared HA making its composition alike human
bone, will benefit the overall physiological functioning after
implantation [23]. Mg is known to be an important trace element
in bone and teeth. Moreover, Mg plays an important role in bone
metabolism and hence its depletion causes bone fragility and bone
loss [44]. Consequently, the incorporation of Mg ions into the HA
structure is of great interest for the development of artificial bones.
Besides, in conventional biomimetic coatings on Ti metals, the
presence of Mg greatly influences both the physical and biologi-
cal properties of the deposited coatings. Barrere et al. studied the
role of Mg2+ ions in the deposition of apatite coating on Ti using
concentrated SBF solution [45]. It appeared that the relatively high
interfacial concentration in Mg2+ favors heterogeneous nucleation
of tiny Ca–P globules onto the Ti-6Al-4V substrate, thereby enhanc-
ing physical adhesion at the early stages of the coating formation.
In addition, in vitro results showed higher cell adhesion of Mg2+
apatite coating than apatite coating, promoting proliferation and
expression of collagen type I with respect to bare Ti [46]. Similarly,
Sr is reported to improve bone strength and provide benefits in the
treatment of osteoporosis [47]. Sr-containing HA has also exhib-
ited better mechanical properties than those of stoichiometric HA,
and has shown to enhance the proliferation and differentiation of
osteoblast cells in vitro [48]. Moreover, Xue et al. [49] demonstrated
that plasma-sprayed Sr-HA coatings on Ti-6Al-4V substrate exhib-
ited good biocompatibility with human osteoblasts, favoring the
survival and proliferation of cells on the surface of the coatings.
4. Conclusions
This work demonstrates the synthesis of HA from eggshell
powders mixed with DCPD via ball milling and subsequent heat
treatment. The following key results can be summarized based on
the results obtained in this study:
(1) The XRD pattern of the 1 h milled sample indicated a small
amount of ␤-TCP and HA phases. With increase in milling time
up to 10 h, the ␤-TCP phase decreased, resulting in predomi-
nantly HA phase with small amounts of DCPA and CaCO3 from
precursors.
(2) The formation of HA phase can be initiated by sintering the
1 h milled sample at 1000 ◦ C for 1 h, while pure HA phase can
be obtained upon sintering the 10 h milled sample. Even after
89
sintering, the presence of ␤-TCP phase was observed in the XRD
patterns of the 1 and 5 h milled samples. No peaks correspond-
ing to the starting materials were found in the powders milled
for 5 and 10 h followed by heat treatment at 1000 ◦ C for 1 h.
(3) The final products composed entirely of pure HA crystals or
biphasic calcium phosphate (HA + ␤-TCP crystals) can easily
be prepared by using DCPD and eggshell powders after 5 and
10 h of ball-milling, respectively, and subsequent sintering at
1000 ◦ C for 1 h.
(4) The FTIR analysis of 10 h milled and sintered sample showed
bands corresponding to OH− , PO4 3− and H2 O. The carbonate
peaks of the specimen closely matched those of A- and B-type
carbonates.
(5) Based on the XRD results, it can be concluded that the as-
synthesized HA powder obtained by milling DCPD and eggshell
powders for 10 h followed by heat treatment at 1000 ◦ C for 1 h
exhibited good crystallinity (96.4%) and high phase-purity. The
elemental composition of the synthesized HA, as evaluated by
the ICP-AES method, showed the presence of Ca (39.2 wt%), P
(16.8 wt%), Mg (0.648 wt%), and Sr (0.031 wt%).
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