J O U R N A L O F M AT E R I A L S S C I E N C E : M AT E R I A L S I N M E D I C I N E 1 1 ( 2 0 0 0 ) 4 2 1 4 2 6

Composition and structure of apatite formed on

organic polymer in simulated body uid with a
high content of carbonate ion
H.-M. KIM*, K. KISHIMOTO, F. MIYAJI, T. KOKUBO
Department of Material Chemistry, Faculty of Engineering, Kyoto University, Sakyo-ku,
Kyoto 606-8501, Japan
T. YAO
Department of Fundamental Energy Science, Graduate School of Energy Science Kyoto
University, Sakyo-ku, Kyoto 606-8501, Japan
Y. SUETSUGU, J. TANAKA
National Institute for Research in Inorganic Materials, Tsukuba, Ibaraki 305, Japan
T. NAKAMURA
Department of Orthopaedic Surgery, Faculty of Medicine, Kyoto University, Sakyo-ku,
Kyoto 606-8506, Japan
E-mail: [email protected]

Apatite layer was formed on polyethyleneterephthalate (PET) substrate by the following

biomimetic process. The PET substrate was placed on granular particles of a CaO, SiO2 -
based glass in simulated body uid (SBF) with ion concentrations nearly equal to those of
human blood plasma to form apatite nuclei on their surfaces. The apatite nuclei was then
grown into a continuous layer by subsequently soaking the substrate in SBF under air or CO2
atmosphere in which CO2 partial pressure in the ambient was adjusted to 14.8 kPa to increase
the content of carbonate ion to a level nearly equal to that of blood plasma. The increase in
the content of carbonate ions in SBF changed the Ca/P atomic ratio of the apatite from 1.51 to
1.63, content of CO23 ions from 2.64 to 4.56 wt %, and lattice constants a from 94.32 to
94.23 nm and c from 68.70 to 68.83 nm, respectively. The Ca/P ratio and lattice constants of
the apatite formed in SBF under CO2 atmosphere were approximately identical to those of
bone apatite, i.e. Ca/P atomic ratio 1.65, content of CO2
3 ion 5.80 wt % and lattice constants a
94.20 and c 68.80 nm. This indicates that an apatite with composition and structure nearly
identical to those of bone apatite can be produced in SBF by adjusting its ion concentrations
including the content of carbonate ions to be equal to those of blood plasma.
# 2000 Kluwer Academic Publishers

1. Introduction posites are believed to exhibit mechanical properties

The present authors have previously proposed a analogous to those of natural bone as well as bioactivity,
biomimetic process to form a bonelike apatite layer and thus, be truly useful as bone substitutes.
with a desired thickness on any kind and shape of Bioactivity of the apatite-polymer composite prepared
polymer [16]: a polymer substrate is placed on granular by the above biomimetic process is dominated ultimately
particles of CaO, SiO2 -based glass soaked in simulated by the structure and composition of the formed apatite,
body uid (SBF) with ion concentrations nearly equal to since the whole surface of the polymer should be covered
those of human blood plasma to form a large number of with the apatite. The best choice is assumed to make the
apatite nuclei on its surface, and then the substrate is composition and structure of the apatite more identical to
soaked in another solution highly supersaturated with bone apatite. The present authors recently revealed that
respect to the apatite, to make the apatite nuclei grow on the Ca/P atomic ratio and lattice constants of the apatite
the substrate in situ to form a continuous apatite layer. If vary widely with Ca/P molar ratio, ionic activity products
the above biomimetic apatite formation could be and pH of the solution used for the apatite growth in the
performed on polymer fabrics to build them up into a second soaking [79]. Even the conventional SBF,
three-dimensional structure, such apatite-polymer com- however, could not provide an apatite with Ca/P atomic

* Author to whom all correspondence should be addressed.

09574530 # 2000 Kluwer Academic Publishers 421

ratio and lattice constants equal to those of bone apatite. The solution for apatite nucleation and growth on the
These results were attributed to the much smaller content substrate was an acellular SBF with pH (7.25) and ion
of carbonate ions in the uid than in blood plasma. In the concentrations nearly equal to those of human blood
present study, an apatite was formed on an organic plasma (Table I) [11]. The SBF was prepared by
polymer in SBF with a high content of carbonate ions. dissolving reagent grade chemicals of NaCl, NaHCO3 ,
The composition and structure of the apatite were KCl, K2 HPO4 ? 3H2 O, MgCl2 ? 6H2 O, CaCl2 and
discussed in comparison with those of the apatite Na2 SO4 into distilled water, and buffered at pH 7.25
formed in SBF under normal conditions and the bone with tris-hydroxymethylaminomethane NH2 C(CH2 -
apatite. OH and hydrochloric acid at 36.5  C.
The PET substrate was placed on 1.5 g particles of
glass G in 30 mL of SBF at 36.5  C for two days to form
apatite nuclei on its surface. The substrate was
2. Materials and methods subsequently soaked to make the apatite nuclei grow in
2.1. Apatite formation on organic polymer 30 mL of SBF at 36.5  C for six days merely under air
substrate atmosphere or under CO2 atmosphere in which the CO2
Organic polymer substrate was polyethyleneterephtha- partial pressure was adjusted to 14.8 kPa (Fig. 1). The
late (PET) 1561061 mm3 in size whose surface was SBF was renewed every two days during the second
abraded with no. 400 diamond paste, washed with soaking period. As shown in Table I, the SBF contains
ethanol and dried in air. The PET substrate was subjected much smaller amounts of HCO23 ions (4.2 M M ) under
to O2 glow-discharge (GD) treatment in a plasma normal conditions than blood plasma (27.0 M M ).
polymerization system (Model BP-2, SAMCO Ltd, Assuming that CO2 dissolves in water with CO2 =H2 O
Japan). This GD treatment was previously shown to volume ratio of 0.592 [12], it is estimated that adjusting
accelerate apatite nucleation on the substrate [4]. the CO2 partial pressure in the ambient to 14.8 kPa could
The agent for apatite nucleation was the mother glass increase the content of HCO23 ion in SBF to 27.0 M M by
of glass-ceramic A-W (glass G), whose nominal the following equilibria [13]:
composition is MgO 4.6, CaO 44.7, SiO2 34.0, P2 O5
16.2, CaF2 0.5 wt % [10]. Glass G was prepared by
conventional melting-quenching technique, and pulver- H2 CO3 ?
/ HCO3 H

K1 4:46107 1
ized to be sieved into granular particles 150-300 mmf in
size. HCO3 ? 2
/ CO3 H

K2 5:661011 2

Figure 1 The biomimetic method used in the present study to form an apatite layer on PET substrate in SBF with a high carbonate ion concentration.

T A B L E I Ion concentrations of human blood plasma and SBF

Concentration / M M

Na K Mg2 Ca2 Cl HCO3 HPO42 SO42

Blood plasma SBF (in air) 142.0 5.0 1.5 2.5 103.0 27.0 1.0 0.5
142.0 5.0 1.5 2.5 148.8 4.2 1.0 0.5

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2.2. Characterization of apatite formed on
PET substrates
Apatite formation on the PET substrate was examined
by scanning electron microscopy (SEM; S-2500CX,
Hitachi Co. Ltd, Japan) and thin-lm X-ray diffraction
(Tf-xRD; RINt-1400, Rigaku Co., Japan).
Lattice constants a and c of the apatite were
determined by the Rietveld method [14]. XRD data
was collected by step-scanning measurement with 0.01
stepping and 10 sec of count time in the 2y range of 20-
60 . The individual hkl diffraction peak was expressed by
the product of calculated integrated intensity Icalc hkl
and prole function Xhkl 2y.
Figure 2 SEM photographs of the surfaces of PET substrates after
Icalc hkl ? Xhkl 2y 3 soaking in SBF under air and CO2 atmospheres.

The whole diffraction pattern Ycalc 2y was then given 3.2. Tf-XRD analysis
by summing all the peaks and adding background Fig. 3 shows the TF-XRD patterns of the surfaces of PET
(Q(2y)). substrates before and after soaking in SBF under air and
Ycalc 2y SIcalc hkl
Xhkl 2y Q2y 4 CO2 atmospheres. XRD peaks appeared after the
soaking, and were all ascribed to a crystalline apatite,
Pseudo-voigt function [15] was used to describe the indicating that the deposits on the substrates in Fig. 2 are
prole shape. The background was obtained from the apatite.
diffraction data for the original substrate. The lattice Fig. 4 shows the Rietveld renement plot of the TF-
constants included in the prole function as variable XRD pattern of the apatite formed on PET substrate in
parameters and other prole/structure parameters were SBF under CO2 atmosphere. The reliability factor
then rened to minimize the reliability factor Rwp calculated by the method described above was 0.0626,
dened as which was small enough to provide a good tting for
( )1=2
SYobs 2yi Yobs 2yi 2
Rwp 5
SYobs 2yi 2
where Yobs 2yi is the observed intensity at 2yi . The
Rietveld calculation was performed by using a computer
program named RIEVEC [16].
The apatite was also analyzed by Fourier transform
infrared (FTIR) absorption spectroscopy (System 2000,
Perkin-Elmer Ltd, UK). The apatite was scratched from
the substrates, heated at 150  C for 3 h to remove
adsorbed water, mixed with KBr powder at 1 : 100 in
weight ratio, and pressed into pellet form to be subjected
to FTIR measurement.
In order to analyze the composition of the apatite, the
apatite was dissolved in 6M -HCl solution and subjected
to inductively coupled plasma (ICP) atomic emission
spectroscopy (SPS-1500 VR, Seiko Instruments Inc.,
Japan).
The content of water and carbonate in the apatite were
analyzed by a carbon and water determinator (RC-412,
LECO Co., USA). About 50 mg of the apatite was
gathered from the substrate, heated at a rate of 3  C/min.
The amounts of CO2 and H2 O gases released at given
temperatures were measured from IR absorption peaks at
670 and 3010-3020 cm 1, respectively.

3. Results
3.1. SEM observation
Fig. 2 shows the SEM photographs of the surfaces of PET
substrates after soaking in SBF under air and CO2
atmospheres. It can be seen from Fig. 2 that dense and
uniform layers of deposits are formed on the surface of Figure 3 TF-XRD patterns of the surfaces of PET substrates before and
the substrate in SBF under both conditions. after soaking in SBF under air and CO2 atmospheres.

423
Figure 4 Rietveld renement plot of the TF-XRD pattern of the apatite
formed on PET substrate in SBF under CO2 atmosphere. The calculated
and observed patterns are shown on the top; the positions of calculated
Bragg reections with vertical marks in the middle; a plot of the
difference between the observed and the calculated at the bottom.

assuring precision of lattice parameters. The lattice

constants a and c calculated from this result were 94.23
and 68.83 nm, respectively. Those of the apatite formed
on the PET substrate under air atmosphere were obtained
as a 94.32 and c 68.70 nm by the same method.

3.3. FTIR analysis

Fig. 5 shows the FTIR transmission spectra of the apatite
formed on PET substrates in SBF under air and CO2 Figure 5 FTIR transmission spectra of the apatite formed on PET
substrates in SBF under air and CO2 atmospheres.
atmospheres. Peak assignments were based on the
previous reports by Hench [17] and Gee and Deitz
[18]. Peaks ascribed to CO2
3 were observed at about 871
and 1450 cm 1 besides the main peaks ascribed to the
apatite [19, 20]. Peaks ascribed to P2 O47 were also content of carbonate ions of the apatite formed on PET
observed at about 730 and 1200 cm 1. The P2 O4 substrate in SBF under air atmosphere was estimated to
7 ions
are speculated to be formed by heat treatment of HPO2 be 2.64 wt % by the same method.
4
ions. This indicates that the apatite formed on the
substrates contains both the CO2 2
3 and HPO4 ions.
4. Discussion
Table II summarizes the lattice constants and composi-
tions of the apatite formed on the substrate in SBF under
3.4. ICP atomic emission spectroscopy air and CO2 atmospheres, in comparison with those of
analysis stoichiometric hydroxyapatite and bone apatite. It is
The Ca/P atomic ratios of the apatite formed on PET apparent from the above results that the composition and
substrate in SBF under air and CO2 atmospheres were structure of the apatite produced in SBF widely differ due
measured to be 1.51 and 1.63, respectively. The Mg/Ca to the content of carbonate ions in the SBF.
and Na/Ca ratios were the same under both conditions, It is well known that composition of an apatite
0.040 and 0.030, respectively. deposited in an aqueous solution is largely dependent
upon the content of its constituent components in the
solution. Although all the other compositions, e.g. Ca/P,
3.5. Thermal analyzes Mg/Ca and Na/Ca molar ratio, excepting the content of
Fig. 6 shows the release of CO2 and H2 O gases from the carbonate ions in SBF, were unchanged under both the
apatite formed on PET substrate in SBF under CO2 conditions, compositional differences were shown
atmosphere on heating at a rate of 3  C/min. The total clearly for Ca/P atomic ratios as well as the content of
amounts of CO2 and H2 O gases released from the apatite CO23 ions of the apatite formed in SBF under air and
were calculated to be 3.86 and 15.40 wt %, respectively. CO2 atmospheres. Stoichiometric apatite is given by a
The amount of H2 O gas due to absorbed water released at chemical formula Ca10 PO4 6 OH2 with Ca/P atomic
92  C was 15.35 wt %. The net content of carbonate ions ratio of 1.67. When an apatite is deposited from an
of the apatite was thus estimated to be 4.56 wt %. The aqueous solution, some sites for PO3 4 ions are often
424
 responsible for the larger Ca/P atomic ratio of the apatite
formed in the former condition.
Stoichiometric apatite takes a hexagonal structure and
its lattice constants a and c are 94.20 and 68.80 nm,
respectively [21]. Substitution of PO3 2
4 ions with CO3 is
known to expand the hexagonal lattice along the c axis,
since the O-C-O angle is larger than the O-P-O angle
[22]. The differences in the lattice constants between the
apatite formed in SBF under air and CO2 atmospheres
correspond well to this account. The larger CO2 3 ion
concentration in the latter apatite is attributed to the
smaller lattice constant a and larger c than those of the
former apatite.
It should be noted in Table II that the composition and
structure of the apatite formed in SBF under CO2
atmosphere is fairly similar to those of bone apatite.
These results correspond well to our previous prediction
that an SBF with ion concentrations, including its
carbonate ion content, more identical to the blood
plasma will produce the more bone-like apatite.

5. Conclusions
An apatite layer with composition and structure
approximately the same as those of bone apatite could
be formed on an organic polymer in SBF by increasing
its carbonate ion content to a level equal to that of blood
plasma. This indicates that an apatite with composition
Figure 6 Release of CO2 and H2 O gases from the apatite formed on
PET substrate in SBF under CO2 atmosphere on heating at a rate of and structure nearly identical to those of bone apatite can
3  C/min. be produced in SBF by adjusting its ion concentrations
including the carbonate ion content to be equal to those
of blood plasma.
substituted by HPO42 and CO2 3 ions. Vacancies are thus
produced in the sites for Ca2 for maintaining electrical
neutrality. It is clear from Fig. 5 that the apatite formed in
References
SBF also susbtitutes HPO2 2
4 and CO3 ions for PO4
3
1. M . TA N A H A S H I , T. KO K U B O , M . M I N O D A , T. M I Y A M OT O ,
1
sites; IR absorption peaks at 871 and 1450 cm and T. N A K A M U R A and T. Y A M A M U R O , J. Am. Ceram. Soc. 77
those at 730 and 1200 cm 1 are respectively due to the (1994) 2805.
CO2 2 3
3 and HPO4 ions occupying the PO4 sites [19].
2. Idem., J. Appl. Biomater. 5 (1994) 339.
2 3
One substitution of CO3 for PO4 results in one P and 3. Idem., J. Mater. Sci.: Mater. Med. 6 (1995) 319.
4. M . TA N A H A S H I , T. Y AO , T. KO K U B O , M . M I N O D A , T.
half Ca deciencies to give a Ca/P ratio larger than 1.67, M I Y A M O T O , T. N A K A M U R A and T. Y A M A M U R O , J.
while one HPO2 4 for PO3
4 results in only half Ca Biomed. Mater. Res. 29 (1995) 349.
deciency to give a Ca/P ratio lower than 1.67. This 5. K . H ATA and T. KO K U B O , J. Am. Ceram. Soc. 78 (1995) 1049.
means that the Ca/P ratio of the apatite could be the 6. G . J . L I U , F. M I Y A J I , T. KO K U B O , H . TA K A D A M A , T.
N A K A M U R A and A . M U R A K A M I , J. Mater. Sci.: Mater. Med.
larger when more CO2 3
3 ions occupy PO4 sites instead
2 9 (1998) 285.
of the HPO4 . It is therefore speculated that much larger 7. F. M I Y A J I , K . K I S H I M O T O , T. KO K U B O , T. Y AO , T.
CO3 2 ion concentration in the apatite formed in the SBF N A K A M U R A , Y. S U E T S U G U and J . TA N A K A , Biomaterials
under CO2 atmosphere than under air atmosphere is (submitted).

T A B L E I I Lattice constants, Ca/P, Mg/Ca and Na/Ca atomic ratios, and content of CO3 2 of the apatite formed on PET substrates in SBF under
air and CO2 atmospheres, in comparison with those of stoichiometric hydroxyapatite and bone apatite

Lattice constant/nm Ca/P Mg/Ca Na/Ca CO3 2 / wt%

a c

Apatite formed in SBF under air 94.32 68.70 1.51 0.040 0.030 2.64
Apatite formed in SBF under CO32 94.23 68.83 1.63 0.040 0.030 4.56
Stoichiometric hydroxyapatite* 94.20 68.80 1.67
Bone apatite+ 94.19 68.80 1.65 0.016 0.022 5.80
*
[21].
+
[22].

425
 8. H . - M . K I M , K . K I S H I M O T O , F. M I Y A J I , T. KO K U B O , T. Y AO , 16. T. Y AO , ``Report of Grant-in-aid for Scientic Research (No.
Y. S U E T S U G U , J . TA N A K A and T. N A K A M U R A , J. Biomed. 02650560) from the Ministry of Education, Science, Sports and
Mater. Res. 46 (1999) 228. Culture'' (Japan, 1992).
9. H . - M . K I M , K . K I S H I M O TO , F. M I Y A J I , T. KO K U B O
and T. 17. L . L . H E N C H , J. Am. Ceram. Soc. 74 (1991) 1487.
NAKAMURA, J. Ceram. Soc. Japan. (submitted). 18. A . G E E and V. D E I T Z , ibid. 77 (1955) 2961.
10. T. KO K U B O , M . S H I G E M AT S U and T. Y A M A M U RO , J. Mater. 19. C . R E Y, B . C O L L I N S , T. G O E H L , I . R . D I C K S O N and M . J .
Sci. 21 (1986) 536. G L I M C H E R , Calcif. Tissue Int. 45 (1989) 157.
11. T. KO K U B O , H . K U S H I TA N I and S . S A K K A , J. Biomed. Mater. 20. C . Y. K I M , A . E . C L A R K and L . L . H E N C H , J. Non-cryst. Solids
Res. 24 (1990) 721. 113 (1989) 195.
12. S . N A G A K U R A , H . I G U C H I , H . E A Z AWA , S . I WA M U R A , F. 21. R . A . Y O U N G and J . C . E L L I O T, Archs. Oral. Biol. 11 (1966)
S AT O U and R . K U B O ``Iwanami-rikagaku-ziten,'' 5th Edn 699.
(Iwanami-syouten, Tokyo, 1987) p. 975. 22. R . Z . L E G E R O S and J . P. L E G E R O S , in ``An Introduction to
13. R . C . W E A S T, D . R . L I D E , M . J . A S T E L and W. H . B E Y E R , Bioceramics'', edited by L.L. Hench and J. Wilson (World
``CRC Handbook of Chemistry and Physics,'' 70th Edn (CRC Scientic, Singapore, 1993) p. 139.
Press, Boca Raton, FL, 1989) p. d-165.
14. H . M . R I E T V E L D , J. Appl. Cryst. 2 (1969) 65.
15. R . A . Y O U N G , ``The Rietveld Method'' (Oxford University Received 11 December 1998
Press, London, 1993). and accepted 14 January 1999

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