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Materials Chemistry and Physics 78 (2003) 816–824

A novel sol–gel technique for hydroxyapatite preparation


G. Bezzi a , G. Celotti b , E. Landi b,∗ , T.M.G. La Torretta a , I. Sopyan c , A. Tampieri b
a CRNM-ENEA, via Ravegnana 186, Faenza, Italy
b ISTEC-CNR, Institute of Science and Technology for Ceramics, via Granarolo 64, Faenza, Italy
c BPPT-Materials Technology Center, Jl. Mh. Thamrin 8, Jakarta, Indonesia

Received 19 July 2002; received in revised form 11 August 2002; accepted 12 August 2002

Abstract
The concept of biocompatible, osteoconductive material mimicking the structure of natural bone to be used as bone graft has generated
a considerable interest in recent decades. This paper reports on a novel and simple sol–gel technique for fabricating pure hydroxyapatite
powder. Compositional, microstructural, morphological and mechanical characterisations carried out on the powder as well as on dense
and porous sintered bodies, are also reported.
© 2002 Elsevier Science B.V. All rights reserved.
Keywords: Biomaterial; Sol–gel process; Hydroxyapatite

1. Introduction phorus which is capable of improving the chemical homo-


geneity. The sol–gel product is characterised by nano-size
When bone is lost due to injury and/or illness, the defects dimension of the primary particles, and this small domain is
are generally filled with natural bone because artificial bone a very important parameter to improve the contact reaction
materials have the problem of bioaffinity. However, natural and the stability at the artificial/natural bone interface.
bone also has supply and infection problems. Therefore, Moreover, the high reactivity of the sol–gel powder al-
studies are recently dedicated towards an artificial material lows a reduction of the processing temperature and of any
which has the same biological properties as bone and that degradation phenomena occurring during sintering. The ma-
can replace natural bone for grafting. jor limitation to sol–gel technique application is linked to the
In recent years, synthetic hydroxyapatite (HA) has as- possible hydrolysis of phosphates and the high cost of the
sumed substantial interest and importance because of its raw materials [6]. Both problems are solved in the method
chemical similarity to the natural Ca phosphate mineral proposed in the present paper where a new process to pre-
present in a biological hard tissue. Considering the numerous pare a pure HA powder via the sol–gel route is presented.
applications of calcium phosphate compounds in biomedical
fields, numerous HA synthesis techniques have been devel-
oped. The most commonly used technique for the formation 2. Experimental
of HA is the precipitation technique, involving wet chemical
reactions between the calcium and phosphate precursors un- Wet chemical reactions to form calcium phosphate ma-
der controlled temperature and pH conditions [1–5]. How- terials can be generally divided in two kinds of process in
ever, this method has some inherent disadvantages, primarily terms of reagents concentration: non-homogeneous and ho-
the difficult control of the pH value over 9 to avoid the for- mogeneous processes. In the first one, calcium phosphate
mation of a Ca-deficient HA which in sintering undergoes compound synthesis involves a dropwise adding of a donor
to easy decomposition, forming tricalcium phosphates. reagent of phosphorus into a calcium hydroxide or salt slurry.
In this respect, the sol–gel technique is an elective method In the second one, calcium and phosphorus salts (usually ni-
for the preparation of highly pure powder due to the pos- trate for calcium and ammonium phosphate for phosphorus)
sibility of a strict control of the process parameters. This are solubilised in water under a strict pH control. In both
method offers a molecular mixing of the calcium and phos- cases a long ageing time is required to let the precipitation
reactions end, because it is very difficult during the precip-
∗ Corresponding author. Tel.: +39-0546-699-758; fax: +39-0546-46381. itation phase to control that the single ion concentration is
E-mail address: elena@istec.cnr.it (E. Landi). uniform.

0254-0584/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 2 5 4 - 0 5 8 4 ( 0 2 ) 0 0 3 9 2 - 9
G. Bezzi et al. / Materials Chemistry and Physics 78 (2003) 816–824 817

The process described in this paper falls in the homoge- HA powder was investigated through potentiometric titra-
neous category. Good reproducibility, high yield, simplicity tion. An automatic titration software was used to measure
of execution, not use of solvent except water make this syn- the ζ potential and conductivity as a function of pH (HNO3
thesis very competitive related to other wet and sol–gel pro- 1 M and KOH 1 M were used to adjust the pH). The isolec-
cesses and suitable for being scaled up to industrial size. A tric point (IEP) was identified at the pH axis crossing point.
very simple method was set up to obtain pure hydroxyapatite The HA densification behaviour was detected by thermal
[7]. Hydroxyapatite powder was prepared from calcium ni- dilatometry (Netzsch Geratebau E 402) in order to identify
trate tetrahydrate (Aldrich) and ammonium hydrogen phos- the best sintering temperature. Dense samples were prepared
phate (Merck), adding etilendiamminetetraacetic acid (Carlo starting from a spray-dried HA powder. The flexural strength
Erba Reagenti) and urea (Merck) as gelling and ammonium was determined on suitable shaped bars (2 mm × 2.5 mm ×
donor agents. To prevent an immediate precipitate formation 25 mm) by a four-point bending device (Instron mod. 1195,
the calcium ions were chelated by etilendiamminetetraacetic High Wycombe, Bucks, UK) with an outer span of 20 mm, an
acid (EDTA) to permit a very good mixture with the phos- inner span of 10 mm and a crosshead speed of 0.5 mm/min.
phate ions. In this solution, under stirring, urea was solu- Porous samples were prepared by soaking natural cellu-
bilised too. In this way the urea decomposition happens in a losic sponges (Spontex, Verniano, Italy) into 25 vol.% HA
very homogeneous system in terms of ion concentration and suspension, containing Duramax D-3021 (Rohm and Haas,
pH. Nitrate ion removal involves a white gel phase formation USA) as a dispersing agent. The density of the porous bod-
passing through a transparent sol phase in one reactor only, ies was measured by geometrical weight/volume evaluation
to set at zero possible contamination. Pure HA powder was (so-called apparent density).
finally obtained after a proper thermal cycle and a peculiar The compressive strength of the HA porous bodies
chemical correction treatment, involving the hydrothermal was measured on cylindrical specimens 10 mm × 10 mm
conversion of tricalcium phosphate (TCP) into HA. (height × diameter) using an Instron machine mod. 1195
The specific surface area of the powder was evaluated by (High Wycombe, Bucks, UK) with a crosshead speed of
the Brunauer–Emmett–Teller method (Sorpty 1750, Carlo 1.0 mm/min. To avoid a shear stress concentration, a thin
Erba, Milano, Italy). For particle size distribution mea- proper foil was inserted between each end of the cylinder
surement, the powder was analysed by sedimentography and the loading plates of the machine. The compressive
(Sedigraph 5100, Micromeritics, Norcross, GA) after ultra- strength was calculated from the maximum load registered
sonic dispersion for 10 min. In order to evaluate Ca/P ratio, during the test divided by the original area (six specimens
inductively coupled plasma-optical emission spectrometry were tested).
(ICP-OES) analysis was performed (Liberty 200, Varian,
Clayton South, Australia). Fourier transformed infrared
(FTIR) spectroscopy (FT-IR 1750, Perkin-Elmer Instru- 3. Results and discussion
ments, Shelton, CT, USA) was also performed in order to
follow the chemical evolution from the gel to the HA, and 3.1. Sol–gel synthesis and powder characterisation
to determine HA stoichiometry deviations, i.e. the presence
of anions partially substituting PO4 3− and/or OH− groups. 3.1.1. Method A
Simultaneous thermal analysis (Netzsch Geratebau STA The flow sheet of the sol–gel process is shown in Fig. 1. To
409) was used in order to follow the thermal transformation produce about 100 g of pure hydroxyapatite powder (HAsg),
process of the gel into HA and the hydroxyapatite thermal 260 g of Ca(NO3 )2 ·4H2 O (98%), 79.7 g of (NH4 )2 HPO4
stability. (99%), 360 g of EDTA and 90 g of urea are required, which
The morphological evaluation of the powder and mi- were solubilised in 1400 ml of distilled water containing am-
crostructural characterisation of the dense bodies was per- monia (7%), with a molar ratio Ca/P > 1.7. This solution
formed by scanning electron microscopy (SEM; Stereoscan was heated at 90–120 ◦ C under stirring for about 2 h till wa-
360, Leica, Cambridge, UK). The powders as well as the ter evaporation and urea decomposition, passing through an
sintered bodies were also characterised by X-ray diffraction intermediate sol state, to obtain finally a white consistent
(Cu K␣ radiation, Miniflex Rigaku, Tokyo, Japan) in order gel. This gel was slowly dried in air at 340 ◦ C, then fired
to evaluate the crystalline phase compositions. in air at 800 ◦ C for 2 h to obtain a white powder, which
Zeta-potential determination was carried out with an elec- was HA (main phase) together with about 14% ␤-TCP and
troacoustic technique, using an electrokinetic sonic ampli- 5% CaO (Fig. 2). The Ca/P ratio was corrected to 1.667
tude (ESA) measurement apparatus (Acoustosizer, Colloidal by adding a right amount of diammonium hydrogen phos-
Dynamics, Sydney, Australia). The suspension sample was phate to a water suspension of the powder and maintaining
prepared by ball milling the powder for 1 h into a 10−2 M the system under stirring at 90 ◦ C for ≈1 h. After drying at
KCl solution to maintain a constant ionic strength. A fixed 120 ◦ C, the final pure hydroxyapatite powder was obtained,
concentration of 3 vol.% solid was used and the suspension, as shown by X-ray diffractometry (XRD) analysis (Fig. 3).
placed in the measurement cell, was kept under constant The absence of Ca(OH)2 in the as-prepared powder was also
stirring at 25 ◦ C. The aqueous dispersing behaviour of the specifically controlled, analysing the powder after firing at
818 G. Bezzi et al. / Materials Chemistry and Physics 78 (2003) 816–824

Fig. 1. HA sol–gel synthesis flow sheet.

Fig. 2. XRD analysis of the powder obtained after the thermal treatment at 800 ◦ C.
G. Bezzi et al. / Materials Chemistry and Physics 78 (2003) 816–824 819

Fig. 3. XRD analysis of the HAsg powder.

600 ◦ C: X-ray diffractometry did not evidence CaO forma- size about ten times lower than 0.8 ␮m, as well as shown in
tion; such a finding was also confirmed by the high precision Fig. 4.
phenolphtalein test. To study the thermal transformation process of the gel, a
Fig. 4 shows the morphology of the synthesised powder: thermogravimetric analysis was performed on the gel using
even after the treatment at 800 ◦ C, the primary particles a heating rate of 10 K/min. A total multistep weight loss of
resulted submicrometric in size, with a tendency to ag- ≈80% was recorded (Fig. 5): the first drop at temperature
glomerate. Granulometric evaluation has been performed T ≈ 100 ◦ C is due to absorbed water, the second one starting
on the as-prepared powder by X-ray sedimentography and at T ≈ 200 ◦ C (≈50% of the total weight loss) can be
the mean particle size turned out to be ≈0.8 ␮m. Such a attributed to nitrates and urea decomposition, then at higher
value refers to the agglomerate dimension, surviving the temperatures the decomposition of acetates (from EDTA)
ultrasound treatment performed before the analysis, in or- and hydroxides occurs (weight loss ≈ 25%). The DTA curve
der to disaggregate the powder. The specific surface area, is characterised by a sequence of peaks at T ≈ 300, 330
evaluated by the Brunauer–Emmett–Teller method, was and 480 ◦ C, corresponding to the decomposition processes
7–11 m2 /g, which value is consistent with an average particle of the compounds described above.
Such a behaviour is in accordance with the FTIR analy-
sis which was used to follow the evolution of the chemical
compounds contained in the initial gel up to the final HA
powder. In particular, the FTIR spectrum of the dried gel

Fig. 5. Thermogravimetric and differential thermal analysis (TG–DTA) of


Fig. 4. SEM image of the HAsg powder. the gel.
820 G. Bezzi et al. / Materials Chemistry and Physics 78 (2003) 816–824

Fig. 6. FTIR analysis of the gel after thermal treatment at 340 ◦ C.

(Fig. 6) yet shows broad peaks, typical of amorphous prod- The FTIR spectrum of the final powder shows the charac-
ucts, but anyway the characteristic bands of carboxylic and teristic peaks corresponding to OH− (630 and 3560 cm−1 )
carbonate (1400–1600 cm−1 ), phosphate (900–1100 cm−1 ), and PO4 3− (960, 1050, 1090 cm−1 ) vibrations, together with
amino (≈1400, 1600 and 3200 cm−1 ) and acetate (≈2800, the weak bands of the CO3 2− group (870, 1415, 1450 and
2300 cm−1 ) groups are still distinguishable. very weak 1540 cm−1 ), which indicates that it is slightly
The stoichiometric purity of the final powder in term of B-carbonated hydroxyapatite, as expected since the sol–gel
chemical substitutions (CO3 2− and HPO4 2− can be present process involves organic reagents (Fig. 7). The ICP anal-
as partially substituting groups of PO4 3− and/or OH− in the ysis performed on the final powder gave a Ca/P molar ra-
HA structure) have been also controlled by means of ICP tio of 1.66, in accordance with the XRD evaluation (1.67),
and FTIR analysis. considering the experimental errors. This value is also in

Fig. 7. FTIR analysis of the HAsg powder.


G. Bezzi et al. / Materials Chemistry and Physics 78 (2003) 816–824 821

Fig. 8. XRD analysis of the HAsg powder after thermal treatment at 1250 ◦ C.

agreement with the FTIR analysis, keeping also into account 3.2. Characterisation of water suspensions of
the opposite influence on the Ca/P value of the hydrogen HAsg powder
phosphate and carbonate ions as partially substituting groups
of PO4 3− in the HA structure. The isoelectric point of the HAsg powder dispersed in
To evaluate the thermal stability of the powder, XRD H2 O was evaluated, by measuring the ζ potential versus
analysis was performed after thermal treatment at 1250 ◦ C, pH, in comparison with that of a powder (coded as pow-
showing the absence of decomposition phases (Fig. 8). This der N) prepared by a classical precipitation technique via
was in agreement with the result of a simultaneous thermal the neutralisation route (powder N: specific surface area
analysis which detected no degradation phenomena of the 35 m2 /g, 80% of crystallinity degree, molar Ca/P = 1.65
powder up to such a temperature. [8]). Being H+ , OH− and lattice ions the potential deter-
mining ions for apatite solids [9], the surface charge is the
3.1.2. Method B result of pH and preferential dissolution or adsorption of
The same procedure described in method A was applied calcium and phosphate ions. The formation of surface com-
using a molar ratio of calcium nitrate and ammonium hy- plexes between the potential determining ions further com-
drogen phosphate to respect the HA theoretical Ca/P ratio plicates the estimation of the ion activity product and surface
of 1.667. All attempts did not give good results, because potential.
the final HA powder contained about 15 vol.% of TCP. The The natural pH of the sol–gel and N powder suspension,
process was then modified, starting with a higher value of 6.7 and 6.8, respectively, are higher than pH = 5.9 of the
Ca/P ratio and correcting the excess by adding the phosphate surrounding medium solution (KCl 10−2 M, such a dispers-
reagent during the gel formation. XRD showed that at the ing medium is necessary to ensure a constant ionic strength
end of the whole process, inclusive of the thermal treatment during the acid or base addition), as a consequence of the
of the gel, the main product was TCP (62 vol.%) together HA basic hydrolysis in aqueous solutions, which is also in-
with HA (38 vol.%). fluenced by the powder surface area. The ζ potential and
In order to produce HA powder via a sol–gel route avoid- conductivity measurements as a function of pH are shown
ing the calcination at 800 ◦ C, the Ca/P ratio correction was in Fig. 9. The slightly negative ζ potential exhibited by the
finally attempted on the product after the thermal treatment sol–gel powder, at natural pH, is probably due to a prevailing
at 340 ◦ C. Anyway, the XRD analysis on the calcined pow- adsorption of HPO4 2− ions as regards to Ca2+ . The anionic
der pointed out the presence of 8–16% ␤-TCP as a secondary specific adsorption determines a shift of pHiep towards acid
phase. Also calcination pre-treatments and variations of the pH. This shift is not evident for the powder prepared via
cooling rate of the thermal cycle did not allow to obtain the neutralisation route, which shows a pHiep ≈ 7, close
pure HA powder as final product. On the basis of these re- to values reported in literature [10,11]. The suspension con-
sults, the postponed Ca/P ratio correction was definitively ductivity showed a strong increase, which is attributed to
adopted, and the 100% pure HA powder produced and fully HA dissolution phenomena, at pH ≈ 4.5 and 6 for the HA
characterised, as previously reported. powder prepared via the sol–gel and neutralisation route,
822 G. Bezzi et al. / Materials Chemistry and Physics 78 (2003) 816–824

Fig. 9. Electroacoustic analysis of HAsg powder compared to HA powder prepared via classical neutralisation method.

respectively, indicating a lower solubility at pH close to neu- 3.4. Preparation and characterisation of dense
trality for the sol–gel HA. HAsg bodies

3.3. Preparation and characterisation of porous HAsg Considering that the optimal sintering temperature for HA
bodies powders depends on the starting powder features, and that
the lower temperature is defined by the excess in residual
A water-based slurry was prepared with 25 vol.% HA porosity at the grain boundaries and the higher one by the
powder and using Duramax D-3021 as dispersing agent at advancement of grain growth and of the degradation process,
the fixed concentration of 0.34 mg/m2 . Duramax D-3021 is sintering tests were performed.
a 40 wt.% solution of an ammonium salt of a polycarboxy- To prepare dense samples, HA powder was added with
late polyelectrolyte with a low molecular weight of approx- 5% polyvinylalcohol and spray-dried at 190 ◦ C. Fig. 11a
imately 2500 Da, pH = 7–8. shows the morphology of the spray-dried product formed
To prepare porous bodies, to be used as bone substitute, by spheric micrometric granules, which are constituted of
following the procedure elsewhere reported [12], cellulosic submicronic primary particles (50–200 nm, Fig. 11b).
sponges were soaked in the HA suspension and then dried at The shrinkage of cold isostatically pressed bars (100 MPa)
room temperature for 72 h. Finally, the porous samples were was recorded as a function of temperature by a Netzsch
thermally treated at 600 ◦ C for 1 h, using an heating rate of
50 K/h in flowing air to eliminate the organic matrix. The
sintering was performed at 1250 ◦ C for 1 h. The apparent
density of the porous bodies after sintering is 1.29 g/cm3 ,
with 59% of porosity. The linear sintering shrinkage was
about 20%.
The compressive strength was measured on cylindrical
specimens about 10 mm height × 10 mm diameter, using an
Instron machine 1195 with a crosshead speed of 1.0 mm/min.
The compressive strength value was 1.3 ± 0.3 MPa, which
is low in relation with the apparent density found, and in
comparison with previously reported data [12–14]. Even if
the slurry seemed to have a good fluidity, and it was kept in
an ultrasound bath during the soaking process, sedimentation
occurred giving rise to a disomogeneous impregnation of
the cellulosic matrix. Some big pores of the sponge were
completely filled with HA while the struts between them Fig. 10. SEM image of a portion of the porous sample: the original
appeared as scarcely loaded with HA, showing debris aspect porous structure of the sponge is lost; some macropores are completely
(Fig. 10). filled with HA and the struts between them show a debris aspect.
G. Bezzi et al. / Materials Chemistry and Physics 78 (2003) 816–824 823

E402 dilatometer, using a heating rate of 5 K/min (Fig. 12).


The densification process starts at ≈1000 ◦ C and shows its
highest rate at about 1220–1250 ◦ C.
The flexural strength was determined on suitable shaped
bars (2 mm × 2.5 mm × 25 mm) by a four-point bending
device (Instron) with an outer span of 20 mm, an inner span
of 10 mm and a crosshead speed of 0.5 mm/min. The bars
were cut from a pellet, which was produced by cold press-
ing at 100 MPa the spray-dried powder, and then calcining
at 800 ◦ C (heating rate of 50 K/h) and finally sintering at
1250 ◦ C for 1 h (heating rate of 250 K/h). The sintering
temperature was chosen on the basis of the HA thermal
stability (beyond 1250 ◦ C the amount of ␣- and ␤-TCP
increases greatly over 2–3%). Two series of samples were
tested: (a) bars arising from uniaxially (100 Ma) pressed
powder and (b) bars arising from uniaxially (80 MPa)
and isostatically (100 MPa) pressed powder. The flexu-
ral strength resulted in 58 ± 7 MPa and 80 ± 10 MPa, in
agreement with the sintering density values 90 and 95%,
for samples (a) and (b), respectively. The SEM analysis
of the fracture surfaces sometimes evidenced macropores
(20 ␮m in size) or low-sintered areas together with micro-
porosity (1–2 ␮m in size), in the samples only uniaxially
pressed. The sintered surface of the isostatically pressed
samples (Fig. 13) showed limited intergranular porosity
together with a slight grain growth (grain size ≈ 2–5 ␮m).
X-ray diffractometric analysis pointed out that a nearly
undetectable amount (less than 1 vol.%) of decomposition
products formed in the sintered samples. The fracture anal-
ysis showed that the rupture was caused by the presence of
localised inhomogeneities in the microstructure (Fig. 14),
probably arising from the forming process, not yet well
Fig. 11. SEM image of the HAsg powder after spray drying treatment:
spheric micrometric granules (a) are constituted of submicronic primary
optimised.
particles (b).

Fig. 12. Thermal dilatometry of HAsg cold isostatically pressed sample, showing that the densification starts at about 1000 ◦ C, reaching the its highest
rate at 1220–1250 ◦ C.
824 G. Bezzi et al. / Materials Chemistry and Physics 78 (2003) 816–824

ment at 800 ◦ C, which reduces the potentiality arising from


nanometric powder, i.e. the reactivity in sintering, some pos-
itive results remain.
The uselessness of the control of temperature and pH
value during the synthesis and the absence of repeated fil-
tration and washing steps, which can easily cause loss of
material, are both important goals.
From the first tests the performance of the powder is
promising. The sintering temperature can be probably low-
ered also in function of an optimised pressing process, which
seems the most critical step.
Regarding the porous bodies production, a powder
pre-treatment and secondarily the determination of the sta-
bility conditions of the powder suspension seem inevitable
in order to prepare suitable slurries for the sponge impreg-
nation method.

Fig. 13. SEM image of the fracture surface of a dense sample. References

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