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Structural characterization of Sol-Gel derived
Sr-substituted calcium phosphates with
anti-osteoporotic and anti-inammatory properties.
Guillaume Renaudin, Patrice Laquerriere, Yaroslav Fillinchuk, Edouard
Jallot, Jean-Marie Nedelec
To cite this version:
Guillaume Renaudin, Patrice Laquerriere, Yaroslav Fillinchuk, Edouard Jallot, Jean-Marie Ned-
elec. Structural characterization of Sol-Gel derived Sr-substituted calcium phosphates with anti-
osteoporotic and anti-inammatory properties.. Journal of Materials Chemistry, Royal Society of
Chemistry, 2008, 18, pp.3593-3600. �10.1039/b804140g�. �hal-00360355�
1
Structural characterization of Sol-Gel derived Sr-
substituted calcium phosphates with anti-osteoporotic and
anti-inflammatory properties.
G. Renaudin
1
, P. Laquerrière
2
, Y. Filinchuk
3
,
E. Jallot
4
and J.M. Nedelec
1*
1
Laboratoire des Matériaux Inorganiques CNRS UMR 6002. Université Blaise Pascal &
Ecole Nationale Supérieure de Chimie de Clermont-Ferrand - 24 avenue des Landais, 63177
Aubière Cedex, France.
2
INSERM, U 926, IFR 53, 1 rue du Maréchal Juin, BP 138, 51095 Reims, Cedex, France.
3
Swiss-Norwegian Beam Lines at ESRF, 6 rue Jules Horowitz, BP 220, 38043 Grenoble,
France
4
Laboratoire de Physique Corpusculaire de Clermont-Ferrand CNRS/IN2P3 UMR 6533.
Université Blaise Pascal - 24 avenue des Landais, 63177 Aubière Cedex, France.
*
Corresponding author : J.M. Nedelec
Laboratoire des Matériaux Inorganiques CNRS UMR 6002, Université Blaise Pascal and ENSCCF
j-marie.nedelec@univ-bpclermont.fr
+ 33 4 73 40 71 95
2
Abstract
Sol-Gel chemistry has been successfully used to prepare un-doped and Sr-doped calcium
phosphate ceramics exhibiting a porous structure. The samples composition is very close to
the nominal one. All samples present phase mixtures mainly Hydroxyapatite (HAp) and Tri
Calcium Phosphate (β-TCP). Doping with Sr
2+
ions has a clear effect on the proportions of
the different phases, increasing the amount of β-TCP. An amorphous phase is also observed
incorporating some 40 % of the total amount of strontium. Strontium ions also substitute for
calcium both in HAp and β-TCP in specific sites that have been determined from Rietveld
refinement on synchrotron powder diffraction data. The soluble amorphous and TCP phases
are responsible for a beneficial partial release of strontium ions in solution during interactions
between the material and biological fluids. Preliminary in vitro study demonstrates anti-
inflammatory effect of strontium for human monocytes cultured in contact with calcium
phosphates.
1. Introduction
Due to the global aging of the population, the need for bone substitutes is increasing very
quickly. There is also a strong demand for bioceramics with specific properties such as anti-
inflammatory, bactericidal or anti osteoporotic. Bone mineral mass is dominated by nano-
crystalline non-stoichiometric hydroxyapatite (Hap, Ca
5
(PO
4
)
3
OH) and whitlockite (
β
-TCP,
β
-Ca
3
(PO
4
)
2
) can be found at many different sites in the human body [1-3]. For these reasons,
apatite and whitlockite have been widely used as biocompatible materials for bone
replacement and for bone protheses coating [4]. An interesting alternative is the use of so
called Biphasic Calcium Phosphate (BCP) which is mixture of HAp and β-TCP. Because of
the difference in the solubility of the two phases, the solution behaviour of BCP can be easily
tuned. Another interesting possibility is to perform ionic substitution in calcium phosphates to
improve their properties and to modulate their bioactivity.
Similarly to the case of Ca, an estimated 99% of the total amount of Sr in the body is confined
in bone [1]. The total amount of Sr in human skeleton is small but not insignificant; the Sr/Ca
ratio is in the range 0.1-0.3 weight ‰ [5]. Sr is readily taken up in bone [6]. Various studies
investigating the therapeutical and detrimental effects of Sr have been carried out on animals
and humans [1, 7-12]. In vitro and in vivo studies have indicated that oral strontium intake not
only increases bone formation, the number of bone-forming sites and the bone mineral
3
density, but also reduces bone resorption [1,10-12]. Sr is used for treatment of osteoporosis
[13,14], and was found to induce osteoblast activity when introduced in biocompatible bone
cements [15-19]. A better osteointegration has been observed when Sr is present at low
concentration level in biomaterials. The role of strontium in human pathology has attracted
less attention than the other two important alkaline earths calcium and magnesium. However
there has been an increasing awareness of the potential biological role of strontium and its
incorporation in calcium phosphate cements has been largely studied in the last decade
[15,16,20-30]. The purpose of this study was to examine the incorporation of Sr
2+
ions in
hydroxyapatite sample at the atomic level: identification and quantification of the crystallised
phases, identification of the Sr-occupied crystallographic sites, identification of an amorphous
phase and determination of the chemical composition of the different phases.
2. Experimental
2.1 Sol-Gel elaboration of Sr-substituted hydroxyapatite
The sol-gel route previously proposed by the authors [31, 32] has been used. Briefly, to
produce 2 g of pure HAp powder, 4.7 g of Ca(NO
3
)
2
·4H
2
O (Aldrich), 0.84 g of P
2
O
5
(Avocado Research chemicals) were dissolved in ethanol under stirring and refluxed at 85°C
during 24 h. Then, this solution was kept at 55°C during 24 h, to obtain a white consistent gel
and further heated at 80°C during 10 h to obtain a white powder. Finally, the powder was
heated at 1100°C during 15 h. To prepare Sr-substituted hydroxyapatite, the required amount
of Sr(NO
3
)
2
(Aldrich) was added to the solution.
Two samples have been studied: pure hydroxyapatite sample (named ‘HAp’ in this paper) of
nominal composition Ca
5
(PO
4
)
3
·OH and 5% Sr-doped hydroxyapatite sample: named
‘Sr:HAp’ in the following. The doped sample corresponds to an hydroxyapatite where 5
atomic % of calcium have been replaced by strontium atoms thus achieving the nominal
composition Ca
4.75
Sr
0.25
(PO
4
)
3
·OH.
The chemical composition of the hydroxyapatite powders were determined by ICP-AES
(Inductively Coupled Plasma-Atomic Emission Spectrometry). The nominal and experimental
compositions of the two HAp samples are listed in Table 1. As usually observed for sol-gel
derived ceramics [33], the nominal compositions have been readily achieved.
2.2 Characterization of HAp samples
4
Scanning Electron Microscopy Analysis
The hydroxyapatite powders were deposited on carbon disks and sputter-coated with
20 nm of gold. Then, the samples were examined using a JEOL JSM-5910 LV scanning
electron microscope at an accelerating voltage of 15 kV. All the images were recorded using
the secondary electrons detector. A representative SEM image is shown in Figure 1
demonstrating the porosity of sol-gel derived HAp samples.
[Figure 1 around here]
Specific Surface Area(SSA) measurements
SSAs were calculated with the BET model from the adsorption isotherm measured by
nitrogen sorption analyses at 77 K using an Autosorb MP1 Quantachrome equipment. SSAs
are found to be equivalent for both samples around 0.5 m
2
.g
-1
. These values were considered
for in vitro release studies keeping a constant surface/volume ratio.
In vitro Sr
2+
release studies
The interactions between the ceramics and a biological medium have been carried out at 37°C
for 1 to 6 days by immersing about 10 mg of powder in a standard Dulbecco’s Modified
Eagle Medium (DMEM), which composition is almost identical to the one of human plasma.
The specific surface area to DMEM volume ratio was fixed at 500 cm
-1
. These in vitro assays
were conducted under static conditions: biological fluids were not renewed and therefore
contained only limited amount of P and Ca. Namely, this is a good approach for further
development and to compare different doping elements and compositions. ICP-AES was used
to analyze the biological fluid before and after bioceramics interactions.
2.3 X-rays powder diffraction
Synchrotron measurement
Synchrotron powder diffraction data of both samples were obtained at ESRF (Grenoble,
France) on the SNBL. Finelly grinded powders were introduced into a 0.3 mm diameter glass
capillary. Data collections were performed at 295 K with a MAR345 Image Plate detector by
using a monochromatic wavelength of λ = 0.710130 Å. The calculated absorption coefficient
was m
µ
R = 0.68 (m = powder packing factor and
µ
= linear absorption coefficient, R = radius
of the capillary, 0.15 mm). Two sample-to-detector distances were used (150 and 350 mm) in
