Z Kardiol 90: Suppl 3, III/86 – III/91 (2001)

© Steinkopff Verlag 2001

T. Kokubo Process of calcification on artificial materials

H.-M. Kim
M. Kawashita
T. Nakamura

M Summary CaO, SiO2-based
glasses form the bonelike apatite on
their surfaces in an acellular simu-
lated body fluid (SBF) with ion con-
centrations nearly equal to those of
the human blood plasma. The
apatite formation of the former
Prof. Dr. Tadashi Kokubo (Y)
Dr. H.-M. Kim · Dr. M. Kawashita
Department of Material Chemistry
Graduate School of Engineering
Kyoto University
Sakyo-ku, Kyoto 606-8501, Japan
E-mail: kokubo@sung7.kuic.kyoto-u.ac.jp
T. Nakamura, MD
Department of Orthopaedic Surgery
Graduate School of Medicine
Kyoto University
Sakyo-ku, Kyoto 606-8507, Japan
glasses is attributed to the catalytic
effect of the Si-OH groups, which
are formed on their surfaces in SBF,
for the apatite nucleation. The gels
of SiO2, TiO2, ZrO2, Ta2O5, and
Nb2O5 formed the apatite on their
surfaces in SBF, but Al2O3 gel did
not. This indicates that the Ti-OH,
Zr-OH, Ta-OH, and Nb-OH groups
besides the Si-OH groups are also
effective for the apatite nucleation,
but Al-OH groups are not effective.
Apatite formation on self-assem-
bled monolayer terminated with
various functional groups in SBF
showed that COOH and H2PO4
groups are also effective for the
apatite nucleation. All these groups
are negatively charged around pH
7.40. Their apatite nucleating abil-
ity is varied with their arrange-
ments. Among the Ti-OH groups,
those in anatase structure are most
effective for the apatite nucleation.
Transmission electron microscope
attached with energy dispersive X-
ray spectrometer showed that these
functional groups induce the
apatite nucleation not directly, but
through formation of their calcium
compounds and subsequent forma-
tion of amorphous calcium phos-
phate with low Ca/P atomic ratios.
M Key words Apatite nucleation –
functional groups – Si-OH group –
Ti-OH group – amorphous calcium
phosphate
M Abbreviations SBF = Simulated
body fluid; TF-XRD = Thin-film
X-ray diffraction; SEM = Scanning
electron microscope; TEM-EDX =
Transmission electron microscope
attached with energy dispersive
X-ray spectrometer.

osteoblast is activated (5) to preferentially proliferate and

Introduction
Calcification on the vascular wall is considered to be sim-
ilar to deposition of the apatite in the bone. In the field of
bone-repairing materials, it has been revealed that some
ceramics, such as Bioglass® (1), sintered hydroxyapatite
(2) and glass-ceramic A-W (3), spontaneously bond to
the bone in the living body. The key issue in this bonding
of the ceramics to the bone is the formation of bonelike
apatite layer on their surfaces in the living body (4). Once
the bonelike apatite layer is formed on their surfaces,
differentiate on this apatite layer (6). Consequently, the
neighboring bone can come into direct contact with the
apatite layer on the surface of the ceramics. When this
occurs, a tight chemical bond is formed between the bone
apatite and the surface apatite in order to reduce the
interface energy between them (7).
In order to develop new kinds of bone-bonding mate-
rials, it is important to reveal the kind of materials and
how they form the bonelike apatite layer on their surfaces
in the living body. Fortunately, the formation of the
bonelike apatite layer on the bone-bonding ceramics in
 T. Kokubo et al.
Calcification on material
the living body can be reproduced in an acellular SBF
with ion concentrations (Na+ 142.0, K+ 5.0, Mg2+ 1.5, Ca2+
2.5, Mg2+ 1.5, Cl– 148.8, HCO3– 4.2, HPO42– 1.0, SO42–
0.5 mM, and pH 7.40) nearly equal to those (Na+ 142.0, K+
5.0, Mg2+ 1.5, Ca2+ 2.5, Mg2+ 1.5, Cl– 103.0, HCO3– 27.0,
HPO42– 1.0, SO42– 0.5 mM, and pH 7.20 – 7.40) of the
human blood plasma (8). The problems described above
can be investigated in SBF. In the following, the results of
the studies in SBF on the problems described above will
be reviewed. These fundamental findings might provide
some valuable information on the calcification of the
vascular wall.
What kind of materials form the apatite
In order to investigate what kind of materials form the
bonelike apatite layer on their surfaces in the living body,
various kinds of glasses with different compositions in
the simple ternary system CaO-SiO2-P2O5 were prepared
by a conventional melt-quenching technique. They were
soaked in SBF at 36.5 °C for various periods. Apatite for-
mation on their surfaces was examined by TF-XRD and
SEM (9).
The bonelike apatite layer was formed on the surfaces
of CaO, SiO2-based glasses within 28 days in SBF, but not
on those of CaO, P2O5-based glasses, contrary to the con-
ventional expectation, as shown in Fig. 1. The former
glasses released mainly the calcium ion into SBF, whereas
the latter glasses released mainly the phosphate ion. Both
the released ions increased almost equally the ionic activ-
ity product of the apatite in SBF. Despite this, only the
CaO, SiO2-based glasses formed the apatite on their sur-
faces. This indicates that their surfaces provided sites
effective for the apatite nucleation. When the CaO, SiO2-
based glasses are soaked in SBF, they release the calcium
ions from their surfaces via exchange with H3O+ ion in
SBF, and the silica networks of the glasses are hydrated.
As a result, a lot of Si-OH groups are formed on their sur-
faces. These Si-OH groups might be responsible for the
apatite nucleation.
Functional groups effective for apatite nucleation
In order to confirm the catalytic effect of the Si-OH
groups for the apatite nucleation, a pure silica gel was
prepared by hydrolysis and polycondensation of
tetraethoxysilane, and soaked in SBF at 36.5 °C. Apatite
formation on its surface was examined by TF-XRD and
SEM. The silica gel formed the apatite on its surface in
SBF within 14 days, as shown in Fig. 2 (10). This indicates
that Si-OH groups abundant on the surface of the gel are
effective for the apatite nucleation.
III/87
Fig. 1 Apatite formation on the surfaces of CaO-SiO2-P2O5 glasses in SBF within 28
days.
In order to investigate other kinds of functional
groups effective for the apatite nucleation, various kinds
of metal oxide gels were prepared by hydrolysis and poly-
condensation of metal alkoxides, and soaked in SBF.
Gels of TiO2 (11), ZrO2 (12), Ta2O5 (13), and Nb2O5
(14) besides the SiO2 gel also formed the apatite on their
surfaces in SBF within 14 days, as shown in Fig. 2, but
Al2O3 gel did not (11). This indicates that Ti-OH, Zr-OH,
Ta-OH, and Nb-OH groups abundant on their surfaces of
the gels are also effective for the apatite nucleation, but
Al-OH group is not effective. All the functional groups
effective for the apatite nucleation described above are
negatively charged around pH 7.40, but the Al-OH group
is positively charged at the same pH.
It should be noted here, however, that all the Si-OH
groups are not equally effective for the apatite nucle-
ation. A silica gel prepared by hydrolysis and polycon-
densation of tetraethoxysilane in an aqueous solution
containing polyethylene glycol formed the apatite on its
surface in SBF within 14 days, but those prepared in pure
water and in aqueous solution containing polyacrylic
acid did not (15). This indicates that the apatite nucleat-
ing ability of the Si-OH group is varied with the arrange-
ment of the Si-OH groups. Even the latter silica gels
formed the apatite on their surfaces in SBF added with
the excessive Ca2+ ion (16).
Titania gels prepared by hydrolysis and polyconden-
sation of tetraisopropyl titanate in an aqueous solution
containing diethanolamine take an amorphous, anatase,
and rutile structure at 500, 600, and 800 °C, respectively.
Among them, the gel in amorphous structure did not
form the apatite on its surface within 14 days, whereas
that in anatase structure formed a lot of the apatite and
that in rutile structure formed only a small number of the
apatite (17), as shown in Fig. 3. This indicates that Ti-OH
III/88 Zeitschrift für Kardiologie, Band 90, Supplement 3 (2001)
© Steinkopff Verlag 2001

Fig. 2 SEM photographs of apatites

formed on gels of SiO2, TiO2, ZrO2, Ta2O5,
and Nb2O5 in SBF within 14 days.

Fig. 3 SEM photographs of apatites for-

med on titania gels in amorphous, ana-
tase, and rutile structures in SBF within
14 days.

groups in anatase structure are most effective for the

apatite nucleation, but those in amorphous structure are
not effective.
Tanahashi et al. prepared self-assembled monolayers
terminated with various functional groups on a gold film
deposited on a slide glass and soaked in SBF (18). Those
terminated with COOH and PO4H2 formed the apatite on
their surfaces, but those terminated with CONH2, OH,
NH2, and CH3 groups did not. The former functional
groups form negatively charged COO– and PO42– groups
in an aqueous solution.
Not only functional groups combined with a solid
substrate but also those combined with ions dissolved in Fig. 4 Atomic force microscopic picture of apatite nuclei formed on a poly(ethy-
an aqueous solution can induce the apatite nucleation lene terephthalate) faced to a CaO, SiO2-based glass in SBF.
when they sit on a solid substrate. For example, when a
solid substrate was faced to a CaO, SiO2-based glass
(4.6MgO, 44.7CaO, 34.0SiO2, 16.2P2O5, 0.5CaF2 mass%) in
SBF, a lot of apatite nuclei were formed on the solid sub- Mechanism of apatite nucleation
strate, as shown in Fig. 4, since silicate ions terminated
with Si-OH groups were dissolved from the glass and In order to investigate mechanism of apatite nucleation
adsorbed on the surface of the solid to induce the apatite induced by Si-OH groups, collodion films supported by
nucleation in situ (19). titanium grids were faced to the CaO, SiO2-based glass
 T. Kokubo et al.
Calcification on material
Fig. 5 TEM-EDX pictures of a collodion
film supported by a Ti grid faced to a
CaO, SiO2-based glass in SBF for 12 h (a)
and 2 days (b).
taking a distance of 0.5 mm in SBF at 36.5 °C. They were
observed under TEM-EDX after soaking in SBF for vari-
ous periods (20). Silicate ions dissolved from the glass
were attached on the surface of the collodion film within
6 h, and combined with calcium ions in SBF to form an
amorphous calcium silicate within 12 h, as shown in Fig.
5 (a). These calcium ions combined with the phosphate
ions in SBF to form an amorphous calcium phosphate
with Ca/P atomic ratio of 1.53 within 2 days, as shown in
Fig. 5 (b). This amorphous calcium phosphate trans-
formed into a crystalline apatite with Ca/P ratio of 1.64,
which is nearly equal to that of the bone apatite, within
4 days. This apatite contained small amounts of Na+,
Mg2+, and Cl– ions similar to the bone apatite.
III/89
In order to investigate the mechanism of apatite
nucleation induced by Ti-OH groups, titanium grids
were previously subjected to a 5.0M-NaOH treatment at
60 °C for 24 h and, subsequently, to a heat treatment at
600 °C for 1 h to form an amorphous sodium titanate on
their surfaces (21). They were soaked in SBF at 36.5 °C
and observed under TEM-EDX after soaking for various
periods. The titanium grids released the Na+ ions from
the surface sodium titanate layer via exchange with H3O+
ions in SBF to form Ti-OH groups on their surfaces
within 30 min. The Ti-OH groups combined with the cal-
cium ions in SBF to form an amorphous calcium titanate
immediately after they were formed, as shown in Fig. 6
(a) (22). These calcium ions combined with the phos-
III/90 Zeitschrift für Kardiologie, Band 90, Supplement 3 (2001)
© Steinkopff Verlag 2001

Fig. 6 TEM-EDX pictures of a Ti grid

which was subjected to NaOH and heat
treatments, and then soaked in SBF for
30 min (a) and 3 days (b).

phate ions in SBF to form an amorphous calcium phos-
phate with Ca/P ratio of 1.40 within 1.5 days. This amor-
phous calcium phosphate transformed into a crystalline
apatite with Ca/P ratio of 1.62 within 3 days, as shown in
Fig. 6 (b). Its Ca/P ratio increased up to 1.65, which is
nearly equal to that of the bone mineral, within 5 days.
The resultant apatite contained small amounts of Mg2+
ion, similar to the bone mineral.
These results indicate that both the Si-OH and Ti-OH
groups induce the apatite nucleation not directly, but
through formation of their calcium compounds and an
amorphous calcium phosphate with low Ca/P ratio. Sim-
ilar process of apatite nucleation was observed also for
Ta-OH groups (23).
 T. Kokubo et al. III/91
Calcification on material

Conclusion nucleating ability is, however, varied with their arrange-

CaO, SiO2-based glasses formed the apatite on their sur-
faces in SBF, but CaO, P2O5-based glasses did not. The
apatite formation on the former glasses is attributed to
the catalytic effect of the Si-OH groups, which were
formed on their surfaces in SBF, for the apatite nucle-
ation. Gels of SiO2, TiO2, ZrO2, Ta2O5, and Nb2O5 formed
the apatite on their surfaces in SBF. This indicates that
Ti-OH, Zr-OH, Ta-OH, and Nb-OH groups besides the
Si-OH groups are also effective for the apatite nucleation.
They are all negatively charged at pH 7.40. Their apatite
ments. Among the Ti-OH groups, those in anatase struc-
ture is most effective for the apatite nucleation. These
functional groups induce the apatite nucleation not
directly, but through formation of their calcium com-
pounds and subsequent formation of an amorphous
calcium phosphate with low Ca/P atomic ratio.
Acknowledgments This study was supported by Grant-in-Aid 11490020
for Scientific Research, the Ministry of Education, Science, Sports, and
Culture, Japan.

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