Solid State Ionics 151 (2002) 29 – 34
www.elsevier.com/locate/ssi
Electrovectorial effect of polarized hydroxyapatite on
quasi-epitaxial growth at nano-interfaces
Masato Ueshima, Satoshi Nakamura *, Masataka Ohgaki, Kimihiro Yamashita
Department of Inorganic Materials, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University,
Kanda-Surugadai, Chiyoda, Tokyo, 101-0062, Japan
Received 22 January 2001; accepted 25 June 2001
Abstract
Controllability of crystal growth on polarized hydroxyapatite (HAp) associated with simulated body fluid (SBF) was
studied. Polarizing microscopic observation showed the crystal layer with c-axis vertically oriented to the polarized HAp
surface. Scanning electron microscopic observation (SEM) showed an initial growth of the crystal. Structurally and
micromorphologically oriented particles were precipitated on the surfaces of polarized HAp ceramics. Confocal laser scanning
microscopic observation (CLSM) revealed higher Ca2 + concentration near negatively polarized surfaces. The polarization on
HAp may make the crystal deposition with an orientation, and an electrostatic field to the ambient ions.
D 2002 Elsevier Science B.V. All rights reserved.
PACS: 68.45.D
Keywords: Hydroxyapatite; Electrical polarization; Simulated body fluid; Crystal deposition; Confocal laser scanning microscopy
1. Introduction
It is well known that bioactive materials for
artificial bones can directly bond to living bone
through a layer consisted of apatite (bone-like apatite
[1]) grown on their surfaces when they are implanted
in human body. Calcium phosphate biomaterials have
been noted concerning both the biocompatibility and
ability to simulate tissue formation [2,3]. Regulation
or control of growth of the bone-like apatite layer is
important for implantation to each appropriate part.
* Corresponding author. Tel.: +81-3-5280-8014; fax: +81-35280-8005.
E-mail address: [email protected] (S. Nakamura).
Recently, electrically polarized hydroxyapatite (HAp)
ceramics are noted in terms of electrical controllability. We have illustrated that polarized HAp and outstanding effect on the bone-like apatite layer; acceleration and deceleration of the growth of the
bone-like apatite layer occur on the surface of the
electrically polarized HAp in simulated body fluid
(SBF) and culture medium or implantation [4– 6]. It
has been considered that the poling power can stem
orientation of OH ions in HAp structure [4]. Thus,
polarized material could have some specific characteristics (hereafter termed electrovectorial effect).
However, it is not clear yet what is the conclusive
factor for controllability of the bone-like apatite layer
growth. Therefore, it is important to clarify interaction at the interfaces between polarized HAp ceramics
0167-2738/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 1 6 7 - 2 7 3 8 ( 0 2 ) 0 0 6 0 0 - 8
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M. Ueshima et al. / Solid State Ionics 151 (2002) 29–34
and solutions. In this study, some electrovectorial
effects in an initial stage of HAp soaked in SBF at
nano-levels were illustrated. The electrovectorial effect for crystallization and for ambient ions was investigated.
2. Materials and methods
2.1. Materials
HAp powders were prepared by a precipitation reaction from calcium hydroxide and phosphoric acid.
A suspension of calcium hydroxide was stirred and a
solution of phosphoric acid was dropped to produce a
gelatinous precipitate. The obtained slurry was filtered, dried and calcined at 850 jC for 2 h. The
resulting powders were finely ground to under 200
mesh.
The HAp powders were uniaxially pressed into
pellets, then sintered at 1250 jC for 2 h under a water
vapor stream in order not to dehydrate lattice OH
ions from HAp structure [7,8]. The crushed powders
of the sintered specimens were analyzed by the X-ray
diffractometer (XRD) and Fourier transform infrared
analysis (FT-IR).
2.2. HAp polarization
Polarized HAp specimens were prepared for SBF
immersion experiment. HAp specimens were polarized by the following process [9]. The specimens, had
a disk shape with the size of 10-mm diameter and
0.7-mm thickness, were sandwiched between Pt electrodes. They were heated at a room temperature, 300
and 800 jC in air, then subjected to the electrical
polarization treatment in DC fields of 1 and 10 kV/
cm, for 1 h, and thereafter cooled to room temperature under polarization (Fig. 1). The negatively
polarized surface, contacted with Pt cathode in the
poling, is abbreviated as N-surface; and the positively
polarized surface, contacted with Pt anode in the
poling, is abbreviated as P-surface, respectively
(Fig. 1). The surface of nonpolarized HAp specimens
is abbreviated as 0-surface. The thermal stimulated
depolarization current (TSDC) method [9 –11] was
used as a technique for confirming the polarization
treatment.
Fig. 1. Schematic representations of electrode configurations to Nand P-surfaces for electrical polarization.
2.3. Thermal stimulated depolarization current
(TSDC) measurement
The TSDC measurement may be a good index to
know their electrostatical power [9 – 11]. To estimate
stored electrical charge during the polarization treatment, the TSDC was measured for both the polarized
specimens and blank test (i.e. without specimen).
The polarized specimens were sandwiched between
Pt electrodes. The polarized specimens with Pt electrodes were heated at the heating rate of 5 jC/min up
to 850 jC. TSDC of the blank test was also measured in order to know specific background of the
instrument. The stored electrical charge ( Q ) was
estimated by integration of the current on the measurement.
2.4. Simulated body fluid (SBF) immersion
The bioactivity on the surfaces of polarized HAp
was evaluated by soaking in simulated body fluid
(SBF). The SBF with ion concentrations nearly equal
to those of human blood plasma [12] was prepared
using the technique described by Kokubo [13] for
M. Ueshima et al. / Solid State Ionics 151 (2002) 29–34
31
precipitation of bone-like apatite. Specimens were
immersed in 10 cm3 of SBF in glass bottles at 37
jC up to 9 days. These were then removed from the
SBF solutions and washed with distilled –deionized
water. The specimens were dried at a room temperature for microstructural observations.
2.5. Microscopic observations
To know an electrovectorial effect for precipitates
on HAp surfaces, morphological and crystallographic
orientation of precipitates on polarized and nonpolarized HAp surfaces in SBF was noted by microscopic
observations. Microstructural development in an initial stage after immersion in SBF was observed by
scanning electron microscope (SEM). Crystallographic orientation of the precipitated layer was evaluated
by observation of thin-sectioned specimen (0.03 mm
in thickness) using polarizing microscopy. To know
an electrovectorial effect for ambient phase of polarized surfaces, Ca2 + ion distribution near polarized
HAp surfaces was observed by confocal laser scanning microscopy (CLSM). For the CLSM preparation,
HAp ceramics were soaked in 4.8 cc of SBF added
with 49.5 mg of agarose and 8 mg of calcein for 3
days.
3. Results and discussion
3.1. Electrical characteristics of polarized HAp
ceramics
The HAp ceramics used in this study were confirmed as the pure HAp phase by XRD and FT-IR.
The relative density of the HAp ceramics was approximately 96%. The grain size was identified as 1– 3 Am
based on SEM observation.
Fig. 2. Polarizing micrographs of cross-sectioned HAp and the
precipitation layer of bone-like apatite. In the precipitation layer,
oriented microcrystals are ubiquitously polarizing (arrows).
3.2. Structural characteristics of precipitate layer on
polarized HAp surface
Fig. 2 shows polarizing micrographs of a crosssectioned precipitate layer on N-surface of polarized
HAp soaked in SBF for 7 days. The precipitate layer
partially showed interference colors (orange and
blue, the colors not shown in Fig. 2) every 90j
rotation which indicate that the precipitate layer
partially has crystallographic orientation. Apatite
layer usually formed on bioactive ceramics by SBF
soaking [14,15]. Although the precipitate has not
identified mineralogically, the precipitates suggest
that c-axis of HAp particles was oriented vertical
to HAp surface (i.e. parallel to electrically polarized
vector).
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M. Ueshima et al. / Solid State Ionics 151 (2002) 29–34
3.3. Micromorphological characteristics of precipitate layer on polarized HAp surface
Fig. 3 shows an initial stage of precipitation on Nsurface of polarized HAp ceramics soaked in SBF for
2 days. Particles with the size of ca. 50 nm were
precipitated on some grains. Each precipitation can be
seen on each grain. Some grains were covered with
particles with the size of ca. 50 nm. On the other hand,
other grains were not covered with the particles at all
(Fig. 3A). Morphology of the particles on each grain
was uniform (Fig. 3B). Morphology of the particles
on the major grains was spheroidal, whereas that of
the aggregates on the minor grains were apparently
Fig. 4. SEM images of the precipitation on P-surface of polarized
HAp ceramics soaked in SBF for 8 days. Individual precipitation
was apparently observed on each grain, suggesting that the grain
boundaries prevent the next grain from conducting the poling power
for the precipitation.
oriented, suggesting that most of the particles were
affected by electrovectorial effect of polarized HAp
and that the growth was oriented parallel to the poling
vector in their precipitation. Moreover, it seems provable that the difference is due to the presence of
polarization in each grain and that the grain boundaries prevent the next grain from conducting the
electrostatical power for the precipitation.
Fig. 3. SEM images of an initial stage of the precipitation on Nsurface of polarized HAp ceramics soaked in SBF for 2 days. (A)
Particles with the size of ca. 50 nm were precipitated on some
grains. (B) Morphology of the particles on the major grains was
spheroidal, whereas that of the aggregates on the minor grains were
apparently oriented.
Fig. 5. SEM images of the precipitation on 0-surface of nonpolarized
HAp ceramics soaked in SBF for 7 days. Spheroidal particles with
the sizes of various diameters were randomly aggregated on the
surface.
M. Ueshima et al. / Solid State Ionics 151 (2002) 29–34
Similar tendency of the precipitation was recognized on P-surface of polarized HAp ceramics soaked
in SBF for 8 days (Fig. 4). Individual precipitation
apparently occurred on each grain (Fig. 4). On the
contrary, neither individual precipitation nor orientation on each grain was observed on 0-surface (Fig. 5).
Further investigation of the particle sizes and morphology of the precipitates on each surface will be
needed in order to know the role of electrovectorial
effect on nucleation.
3.4. Confocal laser scanning microscopy (CLSM)
A CLSM image of Ca2 + ion distribution between
N- and P-surfaces of polarized HAp immersed in SBF
for 3 days was shown in Fig. 6. This figure indicates
N-surface attracted Ca2 + ions, whereas P-surface and
Ca2 + ions repelled each other. Because N-surface has
33
more precipitation rate than P- and 0-surfaces, Ca2 +
distribution may have a key to the precipitation of
bone-like apatite.
3.5. Relation between stored electrical charge and
thickness of the precipitation layer
In the present study, the polarized HAp specimen
had more stored electrical charge ( Q ), resulted in
precipitating thicker layer of bone-like apatite on the
N-surface. Moreover, the difference between N- and
P- surfaces of polarized specimen with high Q were
more drastic than that with low Q. It will be needed to
evaluate a quantitative relation between poling power
and electrovectorial effects for crystal growth and
ambient solutions.
4. Conclusions
Controllability of the crystal growth of bone-like
apatite on polarized HAp associated with SBF was
studied. In the present study, the phenomena due to
the electrovectorial effect at nano-interfaces of polarized HAp were observed.
The polarizing microscopic observation showed
the crystal layer with c-axis vertically oriented to the
polarized HAp surface. The SEM showed an initial
growth of bone-like apatite. Structurally and micromorphologically oriented particles were precipitated
on the surfaces of polarized HAp ceramics.
The CSLM revealed higher Ca2 + concentration near
negatively polarized surfaces. The polarization on HAp
may make the crystal deposition with an orientation,
and an electrostatic field to the ambient ions.
Acknowledgements
Fig. 6. CLSM image of Ca2 + ion distribution between N- and Psurface of polarized HAp immersed in SBF for 3 days, showing
high intensity of fluorescence from Ca2 + ions near N-surface,
whereas low intensity of fluorescence from Ca2 + ions near Nsurface. The intensity indicates the Ca2 + ion distribution.
I gratefully thank Olympus for lending polarization
microscope and confocal scanning laser microscope,
and Dr. S. Ichinose of Tokyo Medical and Dental
University for analyzing SEM, respectively. This
work was supported by Grants-in-Aid for Scientific
Research from the Ministry of Education, Science,
Sports, and Culture of Japan (Nos. 10305047 and
11650855), the Inamori Foundation, and the Murata
Science Foundation.
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M. Ueshima et al. / Solid State Ionics 151 (2002) 29–34
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