Hydroxyapatite scaffolds hydrothermally grown from aragonitic cuttlefish

bones
J. H. G. Rocha, A. F. Lemos, S. Kannan, S. Agathopoulos and J. M. F. Ferreira*
Received 19th July 2005, Accepted 22nd September 2005
First published as an Advance Article on the web 13th October 2005
DOI: 10.1039/b510122k
Scaffolds of pure AB-type carbonated hydroxyapatite (HA) were successfully produced via
hydrothermal transformation (HT) of aragonitic cuttlefish bones at 200 uC. Beyond low
production cost, worldwide availability and natural biological origin of raw materials, the
produced scaffolds preserved the initial structure of the cuttlefish bone, featuring pore size of
y80 mm in width and y100 mm in height. The transformation was complete after 9 h and no
intermediate products were registered. The kinetics were fast, since, HA was the dominant
crystalline phase after only 1 h of HT. The HA crystallites formed had a size of nanoscale
(y2050 nm) and were randomly oriented.
Introduction
The production of porous scaffolds of hydroxyapatite (HA,
Ca
10
(PO
4
)
6
(OH)
2
) is currently the most demanded challenge
of biomaterials science and technology (e.g., scaffolds for
immediate clinical use or for tissue engineering applica-
tions
13
). Hydrothermal transformation (HT) of aragonitic
(CaCO
3
) corals from the Pacific Ocean to HA, described by
the well known chemical equation
10CaCO
3
+ 6(NH
4
)
2
HPO
4
+ 2H
2
O A
Ca
10
(PO
4
)
6
(OH)
2
+ 6(NH
4
)
2
CO
3
+ 4H
2
CO
3
(1)
has gained interest since the 70s because the resultant porous
scaffolds featured similar composition and microstructure to
the inorganic mineralized structure of natural bones.
46
Several
processing routes, reactors and set-ups have been proposed.
59
Cuttlefish bones (CB) have similar chemistry and crystal-
lography as corals
10
but, compared with corals, they are
available worldwide. Hence, they can be considered as an
inexpensive source of materials of natural biological origin.
Furthermore, the size of their channeling pores (i.e., y80 mm
in width and y100 mm in height, Fig. 1) can host physiological
activities.
11
The formation (i.e., biomineralization) of CBs
(in fact, they are technically not bones but shells) and other
similar sea species, such as nacres etc., is well documented in
the literature.
12
This article reports the evolution of hydrothermal trans-
formation (HT) of aragonite from CB to HA. The suscepti-
bility of nacreous aragonite, whether in powder or in plate
form, to undergo spontaneous transformation to HA, even at
room temperature, has been demonstrated in a recent study.
13
In 1998, Zaremba et al.
14
presented the results of a thorough
investigation on the growth of HA in the stacked aragonite
structure of gastropod (abalone) nacres during HT. That study
presented a novel perspective about HA growth mechanism,
which can hold not via a solid state topotactic ion-exchange
reaction,
15
but via a dissolutionrecrystallization process.
Experimental
Fresh CBs from the species sepia officinalis
16
(fished just a few
hours before the experiments) were gently cut into small pieces
(y1 61 61 cm
3
) using a lancet. To estimate the contents of
water, the organic matter and CaCO
3
, the thermal behavior of
fresh and dried samples of CB were determined by differential
and gravimetric thermal analysis (DTA/TGA, Labsys Setaram
TG-DTA/DSC, France, heating rate 5 K min
21
, in air). The
precise quantitative determination of the weight loss after
drying (110 uC) and after heat treatments at 500 uC and
1000 uC (accounting for the content of free water, organic
matter and CaCO
3
, respectively), was performed by drying and
calcination experiments with larger samples of CB (heating
rate was 5 K min
21
). According to these results, for each batch
of cut pieces of fresh CB, the required volume of an aqueous
solution of (NH
4
)H
2
PO
4
was added to set the molar ratio of
Ca : P at 10 : 6.
The mixtures of cut pieces of fresh (still wet) CB and
(NH
4
)H
2
PO
4
solution were sealed in polytetrafluorethylene
Department of Ceramics and Glass Engineering, University of Aveiro,
CICECO, 3810-193 Aveiro, Portugal. E-mail: jmf@cv.ua.pt;
Fax: +351 234 425300; Tel: +351 234 370242
Fig. 1 Lamellar porous structure of cuttlefish bone (this image
corresponds to a scaffold after 24 h hydrothermal transformation).
The parallel and perpendicular directions, mentioned in the X-ray
analysis (Figs. 3, 4 and Table 1), are indicated.
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(Teflon) lined stainless steel autoclaves and placed in an
electric furnace. The temperature of the HT was 200 uC
(heating and cooling rates were 5 K min
21
). Extrapolation
of the available literature data of water vapor pressure
17
up to
200 uC estimates a pressure of y15 atm. However, the actual
pressure in the chamber should be lower due to the highly
concentrated solutions used. Different times of HT were tested
between 1 and 72 h in independent experiments. The pH of the
solution was 9.75 (0.2) before sealing the autoclaves and
slightly higher after the HT (9.810.1).
The crystalline phases were identified by X-ray diffraction
analysis (XRD, Rigaku Geigerflex D/Mac, C Series, Cu Ka
radiation, Japan). Copper Ka radiation (l 5 1.5406 nm)
produced at 30 kV and 25 mA scanned the range of diffraction
angles (2h) between 20u and 60u with a 2h-step of 0.02u per
second. Lattice constants were refined by least square
refinements from the well-determined positions of the most
intense reflections. Identification of the phases was realized by
comparing the experimental XRD patterns to standards
complied by the International Centre for Diffraction Data
(ICDD) using the cards 00-009-0432 for HA and 01-071-2396
for aragonite. To register any possible preferential direction of
the crystal phases, the lamellar structures of the scaffolds
shown in Fig. 1 were exposed to the X-ray beam under
different selected directions, i.e., perpendicular and parallel to
the direction of the lamellas (the two directions are defined in
Fig. 1). The results were compared with X-ray patterns of fine
powders produced by first smashing the scaffolds followed by
grinding into a mechanical agate mortar for 10 min.
Elemental analysis for the presence of Ca and P elements
was made using X-ray fluorescence spectroscopy (Philips
PW2400 X-Ray Fluorescence Spectrometer). The vacuum of
the chamber was lower then 2 Pa.
Fourier transform infra-red absorption spectroscopy of
powders obtained after milling of the scaffolds was conducted
using KBr tablets (FT-IR, Mattson Galaxy S-7000, USA,
.32 scans mode, resolution 4 cm
21
). The microstructure of
the produced scaffolds was observed by scanning electron
microscopy (SEM, Hitachi S-4100, Japan, 25 kV acceleration
voltage) under secondary electron mode, equipped with energy
dispersive X-ray analysis (EDS).
Results and discussion
Thermal analysis
Fig. 2a indicates that the fresh (i.e., still wet) CB contains a
great amount of water (y25 wt%), which starts to escape at
about y80 uC, and less amount of organic matter (y5 wt%)
that burns out at y270 uC. The decomposition of CaCO
3
clearly holds at higher temperatures.
Fig. 2b shows the results of thermal analysis of dried (110 uC)
CB. The endothermic peak at y70 uC should result from
residual moisture evaporation. The DTA-plot shows an
exothermic shoulder at around 270 uC followed by a sharper
exothermic peak centered at 305 uC. The thermal analysis of
nacres
14
has indicated two types (1 and 2) of organic matter,
which have different rates of weight loss and separate
exothermic peaks (the weight loss due to the organic matter
type-1 has been registered between y70300 uC while the
type 2 was burnt out between y300400 uC). Accordingly, the
TG-plot of Fig. 2b suggests that the weight ratio of type-1 :
type-2 in CB seems to be closer to corals (1 : 1.3) rather than to
nacres (3.3 : 0.5).
14
The weak exothermic peak at 436 uC (Fig. 2b) should be
assigned to the phase transformation of the metastable
aragonite to the thermodynamically stable calcite (that holds
exothermically at 440 uC
18
). X-Ray analysis of CB samples
calcined at 500 uC (not shown) registered calcite (ICDD card
01-083-0578) as the only calcium carbonate phase. For corals,
our interpretation of earlier published results of DTA/TGA
and XRD analyses
19
suggests that this transition holds with a
small endothermic effect at much lower temperature (310 uC).
Owing to the metastable features of aragonite, kinetics
factors evidently affect the temperature at which this
transformation occurs.
The precise weight loss measurements, assessed with big
dried samples (not powders), showed that after heat treatment
at 110 uC the weight loss due to free water was 30%. After
heating at 500 uC, the weight loss due to the organic matter
was 5%. The weight loss between 500 uC and 1000 uC was
exactly 44%, as expected for pure calcium carbonate.
Microstructure
The scaffolds obtained after HT were strong enough to be
safely handled. Fig. 1 shows that the channelled microstructure
of the initial CB was preserved after HT. This is not, however,
Fig. 2 Thermal analysis of cuttlefish bone performed under two
conditions: (a) wet, (b) dry. (DTA: thin line; TG: thick line).
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an obvious result. For instance, aragonite structures from
bivalve and cephalopod nacres usually collapse to HA powder,
even when HT occurs at lower temperatures.
14
The preserva-
tion of the scaffold configuration of the CB (Fig. 1) might be
attributed to the quantity of the organic matter, its composi-
tion and physical properties and its structural arrangement
throughout the composite material. The reader is addressed to
ref. 14, where a thorough explanation on this issue is provided
for the case of gastropod (abalone) nacre.
The importance of the organic matter on scaffold structural
preservation has been evidenced in an earlier study,
20
where
Indian corals were first heat treated to burn off the organic
phase and then were subjected to HT. The resultant product
was HA but the coral structure collapsed to powder.
Phase composition
The fresh CB exclusively comprises pure aragonite (Fig. 3a, b),
in close agreement with the results of the thermal analysis
(Fig. 2). Nevertheless, the relative intensity of the peaks
significantly differs from the standard aragonite, plotted at the
bottom side of these diagrams (ICDD card 01-071-2396).
There is also a great difference between the intensities of the
aragonite peaks when the X-ray incidence beam was parallel
(Fig. 3a) or perpendicular (Fig. 3b) to the plane of the lamellas
direction of the CB (see Fig. 1). The observed differences are
plausibly due to the layered configuration (Fig. 1) that
obviously results from the bio-mineralization process of the
CB in the sea.
One hour of HT was enough to obtain predominantly HA,
suggesting fast reaction kinetics. Only weak evidences of
aragonite were registered (Figs. 3a, b). Table 1 presents the
lattice parameters calculated from the relevant X-ray spectra.
Although in the present study, there is incorporated carbonate
in the structure (as will be discussed later), the XRD results
show only a single phase HA. The Ca/P molar ratio of 1.69
0.02 was confirmed by XRF analysis. Similar results have been
reported by Gibson and Bonfield
21
for synthetic AB type
carbonated HA even for Ca/P ratios in the range of 1.681.76.
These deviations from stoichiometric HA resulted in signifi-
cant variations in the lattice parameters. The relative intensity
of HA peaks, whether the spectra were taken under parallel or
perpendicular scans, perfectly match the X-ray diffraction
patterns of standard HA, also plotted in these diagrams
(ICDD card 00-009-0432). This suggests that the orientation of
aragonite does not affect the orientation of the HA crystallites
developed after 1 h HT, which seem to be randomly orientated
in the hydrothermally transformed scaffold. 100% randomly
oriented HA crystallites were observed in the case of HT of
corals.
14
The HA crystallites developed in nacres were some
50% randomly oriented while the rest 50% was preferentially
oriented perpendicularly to the original aragonite lattice,
especially after prolonged HT.
14
The monitoring of the crystallographic development of HA
during HT between 1 and 72 h (Fig. 3a, b) indicated that
the transformation is complete after 9 h because there is
no evidence of aragonite. Moreover, all the X-ray spectra
obtained for HT longer than 9 h were similar (in terms of the
phases identified). According to Table 1, there is a negligible
deviation (,1%) of the values of the lattice parameters,
calculated from the spectra for each HT time, from standard
HA (00-009-0432).
With regard to the intensity of the HA-peaks, the spectra
obtained under parallel scan (Fig. 3a) feature higher intensities
than those obtained along the perpendicular direction (Fig. 3b).
The size of the HA crystallites, calculated from the analysis of
these spectra (Fig. 4), are quite small and close to the size of
bone-like apatite (i.e., ,50 nm).
Fig. 3 XRD spectra acquired from cuttlefish bone in two orienta-
tions, (a) parallel and (b) perpendicular to the plane of the lamellas
(see Fig. 1), and from (c) milled scaffold powder as a function of
hydrothermal transformation time. The plots of the pure phases
correspond to the ICDD cards of aragonite (01-071-2396) and
hydroxyapatite (HA, 00-009-0432). The intensities of the original
spectra have not been normalized. The full scales of the intensity axis
are 6500 cps for (a) and (b), and 24 000 cps for (c).
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The crystallinity improves with time, as indicated by the
intensity of the X-ray spectra of powders obtained from the
scaffolds, (Fig. 3c). For the scaffolds, however, the intensity of
the X-ray spectra reached a maximum after 24 h and then
decayed over prolonging the HT (Fig. 3a, b). This observation
may suggest that some re-orientation of HA crystallites
intrinsically occur inside the lamellar structure of the scaffolds
during maturation time (i.e., .24 h) under the hydrothermal
conditions. In the case of nacres, the parallel oriented HA
crystallites dissolve and re-crystallize with perpendicular
orientation.
14
Fig. 5 shows bi-layered features observed in
fracture surfaces of CB scaffold walls after HT, which strongly
resemble configurations of bi-layered nanocrystallites of HA
observed in nacres after HT.
14
This structural coincidence
might indicate that the developed HA crystallites are not
absolutely independent on the initial structure of aragonite
in the CB.
Infrared spectroscopy
Fig. 6 presents the evolution of the FT-IR spectra (of powders
obtained after milling of the scaffolds) over increasing HT
time. It is clearly seen that the characteristic bands of the
functional groups of HA phase are absent. After 1 h of HT,
evidence of formation of a mostly amorphous apatite phase
can be detected from the corresponding weak shoulders of the
phosphate group (1042 cm
21
: n
3
). Prolonged HT (more than
9 h) caused an increase of the resolution of the characteristic
peaks of HA assigned to the PO
4
tetrahedra (1042, 1088 cm
21
:
n
3
; 601, 566 cm
21
: n
4
; 960 cm
21
: n
1
; and 470 cm
21
: n
2
) and
the OH group (3570, 631 cm
21
; the peak at 3570 cm
21
is not
shown but it was immediately apparent in the spectra). The
Fig. 4 Size of crystallites calculated from the XRD spectra of Fig. 3a
and 3b obtained for the X-ray beam incidence under (a) parallel and
(b) perpendicular to the plane of the lamellas of the cuttlefish bone as
defined in Fig. 1.
Fig. 5 Typical bi-layers of HA nanocrystallites as observed at
fracture surfaces of scaffolds walls after 72 h HT and sintering
at 1250 uC.
Fig. 6 FT-IR spectra of milled powders obtained from fresh
cuttlefish bone scaffolds and after hydrothermal treatment for
different times.
Table 1 Lattice parameters (,0.0005 A

) and unit cell volumes (,0.05 A

3
) of hydroxyapaptite (HA, hexagonal-space group, i.e., a 5 b)
formed after hydrothermal transformation (HT) for different times, calculated from the X-ray spectra obtained after parallel (// ) and perpendicular
(H) scanning to the direction of the lamellas (Figs. 1, 3a, 3b). The lattice parameters of standard HA (00-009-0432) and aragonite (01-071-2396) as
well as those calculated from the spectra of the fresh cuttlefish bone (CB) (Figs. 3a, b) are also shown
HT time/h
a-Axis/A

b-Axis/A

c-Axis/A

Volume/A

3
// H // H // H // H
1 9.4268 9.4249 6.8744 6.8484 1581.59 1574.97
3 9.4102 9.4344 6.8756 6.8712 1576.30 1583.13
9 9.4101 9.4291 6.8948 6.8802 1580.67 1583.70
12 9.4087 9.3676 6.8640 6.9009 1573.14 1567.81
24 9.4179 9.4126 6.8539 6.8796 1573.90 1578.02
36 9.4299 9.4239 6.8744 6.8588 1582.63 1577.03
48 9.3988 9.4480 6.8766 6.8438 1572.71 1581.64
72 9.4485 9.4124 6.8487 6.8788 1582.94 1577.77
HA 9.418 6.884 1586.3
Aragonite 4.961 7.967 5.741 226.90
CB 4.9381 4.9387 7.9705 7.9962 5.741 5.7372 225.960 226.57
5010 | J. Mater. Chem., 2005, 15, 50075011 This journal is The Royal Society of Chemistry 2005
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increasing peak resolution, in conjunction with the increase of
peak intensity in the XRD spectra (Fig. 3c), indicate increasing
crystallinity of HA-phase under prolonged HT.
Another interesting feature of the FT-IR spectra of Fig. 6 is
the evolution of CO
3
bands over HT time. In the spectrum
of the dried matter of CB (fresh), the presence of CO
3
resulted in the broad band in the region 14001600 cm
21
. The
hydrothermal conditions should favor the orientation of
carbonates to the HA lattice, as the sharpening of the CO
3
bands centered at 872 cm
21
for n
2
and at 1632, 1547, 1458 and
1412 cm
21
for n
3
suggests.
There are two types of carbonate substitutions on apatites
at the OH site (A-type) and at the PO
4
site (B-type).
22
The
FT-IR spectra of Fig. 6 show evidences of bone-like AB
mixed type carbonated apatite, Ca
10
[(PO
4
)
62x
(CO
3
)
x
)]-
[(OH)
22y
(CO
3
)
y
].
21,23
Earlier studies have assigned the bands
peaked at 870, 1430, 1450 cm
21
to B-type and those centred
at 880 and 1540 cm
21
to A-type.
24,25
Other studies have
pointed the bands centred at 1450 cm
21
,
24
1515 cm
21
,
26
and
1550 cm
21 27
as evidence of AB type carbonated apatite.
Therefore, CO
3
has been seemingly incorporated into the
apatite lattice during the HT process partially replacing OH
and PO
4
sites forming AB type apatite. The occupancy of n
2
sites occurs competitively between OH
2
and CO
3
22
groups,
whereas in the n
3
sites it depends on the competition between
PO
4
32
and CO
3
22
.
28
Accordingly, the present study proposes
that the peak at 1547 cm
21
witnesses the formation of A-type
and that at 872 cm
21
the formation of B-type carbonated
substituted HA. It is also reasonable to explain the incorpora-
tion of carbonates in the apatite lattice by the observed
discrepancies in the lattice parameter values in all the HT
conditions with respect to that of HA (ICDD 00-009-0432),
shown in Table 1. Consequently, the produced scaffolds
should perfectly match the implantation aims, since AB-type
carbonated apatite spontaneously forms in situ during the HT.
Conclusions
Natural aragonitic cuttlefish bones (CBs) were hydrothermally
and completely transformed into AB-type carbonated hydro-
xyapatite at 200 uC. The highly channelled structure and the
use of fresh CBs both favor the diffusion of the reacting
solution towards the aragonite structural units, resulting in
fast kinetics. The crystallites of hydroxyapatite formed after
1 h of hydrothermal transformation were predominantly
randomly oriented.
The most important features of the hydrothermally grown
HA scaffolds, which confer to the as produced scaffolds a high
potential for biomedical applications, are:
1. Preservation of the initial structure of the CB, which
exhibits pore size of y80 mm in width and y100 mm in height,
seemingly suitable for supporting biological activities.
2. Similar composition to human bones, i.e., AB-type
carbonated hydroxyapatite.
3. The size of the HA crystallites are in the range of 20
50 nm, similar to the size of bone-like apatite.
4. Low production cost and worldwide availability of a
natural resource.
Acknowledgements
The financial supports of the Portuguese Foundation for
Science and Technology in the framework of the project
POCTI/CTM/60207/2004 and the fellowships of A. F. Lemos
(BD/8755/2002), S. Agathopoulos (BDP/1619/2000) and
S. Kannan (BPD/18737/2004) are gratefully acknowledged.
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This journal is The Royal Society of Chemistry 2005 J. Mater. Chem., 2005, 15, 50075011 | 5011
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