Materials Chemistry and Physics 239 (2020) 122004

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Materials Chemistry and Physics

journal homepage: www.elsevier.com/locate/matchemphys

Calcium phosphates nanoparticles: The effect of freeze-drying on particle

size reduction
Rodolfo Debone Piazza a, *, Thales A.G. Pelizaro a, Jorge E. Rodriguez-Chanfrau b,
Amisel Almirall La Serna c, Yaymarilis Veranes-Pantoja c, Anto ^nio Carlos Guastaldi a
a
Group of Biomaterials, Department of Physical Chemistry, Institute of Chemistry, S~
ao Paulo State University (UNESP), Araraquara, SP, 14800-060, Brazil
b
BioMatTech Inovaç~ ao, Pesquisa e Desenvolvimento LTDA-ME, S~ ao Carlos, SP, Brazil
c
Center of Biomaterials, University of Havana, Havana, CP 10400, Cuba

H I G H L I G H T S G R A P H I C A L A B S T R A C T

� Calcium phosphates (ACP, OCP and HA)

were synthesized by wet chemical
methods.
� Freeze drying is a simple technique to
produce nanocrystalline calcium
phosphates.
� Freeze drying does not alter the
morphological characteristics of the
samples.
� Particles reduction is due to interruption
of the maturation of the crystals.

A R T I C L E I N F O A B S T R A C T

Keywords: Calcium phosphate bioceramics have been widely used in medical applications, such as orthopedics and odon­
Calcium phosphates tology, due to their similarities to the synthesized material with the calcium phosphate present in the mammals.
Nanocrystals The interest on amorphous calcium phosphates (ACP), octacalcium phosphate (OCP) and hydroxyapatite (HA)
Freeze-drying
phases correspond to their ability to transform into the biological phases observed during the formation and
Bone regeneration
remodeling of bone. In the present work, the freeze drying was evaluated as a simple technique to produce
calcium phosphates in a nanoscale. The calcium phosphates phases were synthesized by a wet chemical method
and the results showed that freeze drying do not alter the crystallinity and morphological characteristics of the
samples, however the particles size were drastically reduced when compared with other drying techniques. Thus,
the freeze-dry is shown to be an easy alternative to obtain nanomaterials for tissue engineering and bone
regeneration.

* Corresponding author.
E-mail addresses: rodolfo.piazza@unesp.br, rodolfo.piazza@gmail.com (R.D. Piazza).

https://doi.org/10.1016/j.matchemphys.2019.122004
Received 16 April 2019; Received in revised form 6 August 2019; Accepted 8 August 2019
Available online 12 August 2019
0254-0584/© 2019 Elsevier B.V. All rights reserved.
R.D. Piazza et al.
1. Introduction
The problems associated to diseases and injuries of bones, added to
the increase in life expectancy have increased the efforts to find new
approaches on regenerative medicine and bone tissue engineering [1].
The bone has a complex arrangement of structures that provides me­
chanical, chemical and biological functions and, although it shows a
rigid structure, it is not an inert tissue. Thus, the ideal biomaterial should
mimic the bone structure, allowing its regeneration through the resto­
ration of the biological activity.
Calcium phosphates are the major inorganic materials found in the
hard tissues of mammals, like bones and teeth, and in some pathological
calcification, such as dental calculi, salivary and urinary stones [2]. The
hydroxyapatite (HA) was the main material used in biomedical appli­
cation in bone tissue in the last decades due to its chemical and struc­
tural similarities with biological calcium phosphates, however the
biological material shows calcium and hydroxyl deficiency besides the
substitution by carbonate ions. In this context, the research works were
driven to study other calcium phosphates whose phases are observed
along with HA formation in a biological medium, such as calcium
phosphate (ACP), beta-tricalcium phosphate (β-TCP) and octacalcium
phosphate OCP. The ACP phase is only observed on pathological calci­
fication and shows amorphous structure due to its small crystals,
although it was expected structural similarity between ACP and HA
phases [3]. The β-TCP phase is also observed in pathological calcifica­
tion. The β-TCP as biomaterial has emerged due to its higher solubility
when compared with HA. The control of HA/β-TCP ratio improves the
resorption rate and modulates the biological properties during the bone
regeneration [4]. The OCP phase corresponds to an intermediate phase
of HA, been converted thermodynamically in the bone formation steps.
Although OCP has been widely proposed as metastable phase, its pres­
ence is not always identified during the mineralization process [5,6].
The increase of the area/volume ratio of nanomaterials alters
significantly some of their properties when compared to materials in
their bulk state. Dorozhkin [7] summarized in his review the main ad­
vantages of using nanosized calcium phosphates, such as the improve­
ment of the powders sinterizations, the increase of osteoblast cells
adhesion, proliferation and osteointegration. Calcium phosphate nano­
particles can also be used to improve the biological behavior of different
scaffolds for bone regeneration [8–11]. The physical-chemical features
of nanoapatites synthesized by wet chemical methods lead to an increase
in bioresorption according to in vivo tests.
Several synthetic routes have been described in the literature to
obtain calcium phosphates, such as sol-gel [12], hydrothermal [13] and
wet chemical precipitation [14]. The wet method consists in the use of
calcium precursor and orthophosphoric acid to precipitate the desired
calcium phosphate phase, based on the Ca/P ratio of the reactants. The
morphology and the size of calcium phosphates phases obtained by this
method can be controlled by the speeds of the reactant addition, the
stirring and the drying process. The controls of temperature and the pH
of the medium are also important to obtain the calcium phosphates
phases [15,16].
In this previous work developed by our research group, where the
same synthesis methodology was used to obtain calcium phosphates,
traditional evaporation drying and tray drying techniques were studied,
demonstrating that there was no difference between the particle size
when comparing both drying methods. It was also studied, the influence
that the application of ultrasound had on the size of particles. Two
application variants were studied. The first variant was to apply ultra­
sound at the end of the synthesis process. The second variant was to
apply ultrasound during the synthesis process. The results showed that
the particle size decreased significantly, being more efficient when
applying the second variant [17–21].
In the current work, the influence of freeze-dry on particle size of
calcium phosphate was investigated. Freeze-dry is based on a two steps
procedure. First, the suspension is frozen leading to the inhibition of the
2
Materials Chemistry and Physics 239 (2020) 122004
crystallization. Next, the ice of the frozen product is removed by subli­
mation in a chamber at low temperature and pressure [22]. This simple
technique favors the formation of nanocrystalline calcium phosphates
that show to have a great potential for applications on bone
regeneration.
2. Experimental section
2.1. Materials
All chemicals were used as received from suppliers. Calcium hy­
droxide (Ca(OH)2, P.A. 96.0%), calcium hydrogen phosphate (CaHPO4,
P.A. 98.0%) and phosphate buffer solution (PBS: 0.01 mol L 1 phos­
phate, 0.135 mol L 1 NaCl and 0.002 mol L 1 KCl) were purchased from
Sigma-Aldrich Brazil. Phosphoric acid (H3PO4, P.A. ACS 85.0%) was
purchased from Synth Brazil.
2.2. Synthesis of calcium phosphates
The calcium phosphates were synthesized by a wet chemical method.
To obtain the ACP phase, 200 mL of phosphoric acid solution
(0.6 mol L 1) was dripped (30 drops per minute) over 200 mL of calcium
hydroxide solution (1.0 mol L 1) under mechanical stirring (300 rpm)
during 3 h. Then, the suspension was kept in rest for 2 h, filtrated to
remove the as much of the aqueous medium as possible and freeze-dried.
For the synthesis of HA the same conditions of ACP were used,
however, the HA phase was obtained by the calcination of ACP powder
at 800 � C for 3 h.
The OCP phase was obtained by dripping 30 mL of phosphoric acid
solution (0.6 mol L 1) over 50 mL of calcium hydrogen phosphate so­
lution (2.0 mol L 1). The experimental procedure was the same as for
the ACP phase.
2.3. Characterization
Crystalline phases were evaluated by X-ray diffraction (XRD) tech­
nique. XRD powder diffractions were acquired using a Simiens D5005
diffractometer with Cu Kα radiation source. The samples were recorded
in 5–80� of 2θ, with a step of 0.02� . Crystallographica Search-Match
software was used to index the samples. Infrared spectroscopy was
carried out to evaluate the stretching modes on calcium phosphate
samples. FTIR measurements were performed in a PerkinElmer, Dual
frontier, equipped with a diffuse reflectance infrared Furrier trans­
formed (DRIFT) collector accessory, using the resolution set at 4 cm 1
and 128 scans. The morphology and size of synthesized samples were
investigated by Scanning Electron Microscopy (SEM) in a JEOL micro­
scopy, model JSM-7500F. An electron beam with an acceleration
voltage of 2 kV was used. The software ImageJ (version 1.45s) was used
for image treatment. The samples were dispersed in isopropanol and
deposited in a silicon grid before being coated by C or Au. The
morphology was also analyzed by Transmission Electron Microscopy
(TEM), whose images were obtained in a Phillips microscopy, model CM
200, operating at 200 kV. Zeta potential and hydrodynamic diameter
were measured in a Zetasizer ZSNano, from Malvern Instruments. The
samples were dispersed in PBS (pH 7.4) before the measurements. All
characterizations were carried out using the equipment from the Insti­
tute of Chemistry, on Sa
~o Paulo State University.
3. Results and discussion
3.1. XRD analysis
The diffractograms of synthesized samples are shown in Fig. 1. The
ACP sample corresponds to the mixture of different calcium phosphate
phases, whose peaks were indexed as amorphous calcium phosphate
(PDF:18-303), dicalcium phosphate dihydrate (DCPD) (PDF:11-293),
R.D. Piazza et al.
Fig. 1. XDR patterns of synthesized calcium phosphates samples. A: DCPD; B:
OCP, C: Calcium phosphate carbonate, D: HA, E: ACP and F: β-TCP.
HA (PDF: 86-740) and calcium phosphate carbonate (PDF: 35-180). The
presence of carbonate ions occurs due to the substitution of hydroxyl
groups by air carbon dioxide during the synthesis [25]. The calcination
of ACP sample resulted in a crystalline sample based on a mixture of HA
(PDF: 86-740) and β-TCP (PDF: 9-169) phases, which was denominated
as HA sample. The absence of β-TCP signals on ACP sample is due to its
low instability in water, where this phase does not easily precipitate.
However, the calcination treatment of ACP sample leads to the forma­
tion of a β-TCP and, consequently, a mixture of both phases [26]. The
crystallinity index of HA sample was calculated according to the Person
et al. [27] method, whose calculated value was 1.09. The OCP sample
also resulted in a mixture of phases, whose patterns were assigned to the
DCPD and octacalcium phosphate (PDF:74-1301). The formation of
these phases is expected due to the change on the pH of the solution
during the pH of the reaction, which reached, at the end, the value of 6.7
[28]. At lower pH, DCPD phase precipitates before OCP phase, however,
its conversion to OCP occurs gradually.
3.2. FTIR analysis
The FTIR spectra of calcium phosphates are shown in Fig. 2. The
signals assigned to 571, 600 and 970 cm 1 correspond to the PO34 vi­
bration modes, while the bands at 1032 and 1095 cm 1 correspond to
the P – OH stretching. The difference between ACP and HA phases
corresponds to the presence of peaks at 1417 and 1475 cm 1 which are
assigned to residual carbonate, whose origin occurs during the synthesis
process, as expected [25]. These peaks are suppressed on HA phase due
to the decomposition of the carbonate during the calcination process.
3
Materials Chemistry and Physics 239 (2020) 122004
Fig. 2. DRIFT spectra of ACP, HA and OCP samples.
The presence of water and/or free O-H can be noticed by the signals at
3450 cm 1 and 1641 cm 1, which are assigned to the stretching and
banding vibrations, respectively. The shoulder at 642 cm 1 on HA
spectrum is also attributed to O-H vibration. Analyzing the phosphate
region on OCP sample, the bands at 877 and 1217 cm 1 were not
observed on ACP or HA phases. These bands were assigned to HPO24
ions on OCP lattice [29].
3.3. Zeta potential measurements
The synthesized samples were evaluated through Zeta potential
measurements. At PBS solution (pH 7.4), the values of 9.22 � 0.37 mV,
12.3 � 0.61 mV and 14.8 � 0.7 mV were determined for the ACP, HA
and OCP samples, respectively. The negative charge observed for the
samples corresponds to the external position of phosphates and hydroxyl
groups in the crystal structure. The increase of proteins absorption on
implanted materials that have a negative surface has proved to increase
bone regeneration [30–32].
3.4. The morphological and size analysis
The morphology of synthesized samples are shown in Fig. 3. The low
magnification micrographs resulted in flocculate morphology for ACP
and HA samples and a plate-like to the OCP sample. The high magnifi­
cation micrographs revealed that the flocculate is composed of aggre­
gates of needles and practically spherical particles, whose average size
were of 36.2 nm and 70.7 nm for ACP and HA samples, respectively,
according to the histogram fitting. the presence of larger spheres over
the plates of OCP sample was observed, with an average diameter of
205.4 nm. This spherical morphology is characteristic of DCPD phase,
that was formerly identified by XRD analysis [5].
In a previous report, Pelizaro et al. [20] evaluated the evaporation
and the tray dry as drying techniques using the same methodology of the
calcium phosphates phase synthesis. The tray dry resulted in smaller
particles than conventional evaporation, whose observed size values for
the ACP, HA and OCP were 5.96 μm, 7.38 μm and 1.97 μm, respectively.
R.D. Piazza et al. Materials Chemistry and Physics 239 (2020) 122004

Fig. 3. Low magnification (a), high magnification (b) SEM images of ACP sample and particle size distribution (c). Low magnification (d), high magnification (e)
SEM images of HA sample and particle size distribution (f). Low magnification (g), high magnification (h) SEM images of OCP sample and particle size distribution
(i). The histograms were fitted by a lognormal distribution.

However, the freeze-drying showed to be a powerful technique to reduce

the particle size of calcium phosphate to the nanoscale. In conventional
evaporation and tray dry, the calcium phosphates are kept in contact
with the solvent throughout the drying process leading to the growth of
their crystals, while in the freeze-dry the maturation is interrupted
during the freezing of the sample. Itatani et al. [33] obtained various
calcium phosphates phases by ultrasonic spray freeze drying technique
with particle size varying from 300 nm to 400 nm. Despite the reduced
size of particles, the complexity of the apparatus for dispersing and
freezing the drops of calcium phosphate makes this technique less viable
than just the freeze-dry. Brangule et al. [34] evaluated the influence of
dry techniques only for amorphous calcium phosphates and concluded
that freeze-dry gives better results, although it consumes more time to
carry out the drying. Ramachandran et al. [35], obtained calcium
phosphate nanoparticles in the order of 50–100 nm by ultrasound before
lyophilizing the samples. These works support the use of freeze-drying to
reduce the size of calcium phosphates.
The size distribution was also evaluated by dynamic light scattering
measurements and the hydrodynamic diameter distributions are shown Fig. 4. Hydrodynamic diameter distribution of ACP, HA and OCP samples.
in Fig. 4. The value of the average distribution was 342.0 nm, 396.1 and
1106.0 nm for the ACP, HA and OCP samples, respectively. These values flocculate morphology of ACP sample was also observed on bright field
are higher than the ones observed in SEM histograms since the calcium images. The high-resolution image does not reveal crystalline domains
phosphate nanoparticles tend to form aggregates, which were the ones and spots were neither observed on fast Fourier transformed (FFT),
measured by this technique instead of the nanocrystals, in addition, the corroborating the low crystallinity index [6]. For the HA sample, a
electric double layer should also be considered in the hydrodynamic spherical morphology was observed on bright field images and the
diameter. high-resolution images show a crystalline sample, with lattice fringes
Fig. 5 shows TEM micrographs of synthesized samples. The

4
R.D. Piazza et al.
Fig. 5. TEM images of synthesized samples. Bright field (a) and high resolution (b) micrograph of ACP sample. Bright field (c) and high resolution (d) micrograph of
HA sample. Bright field (e) and high resolution (f) micrograph of OCP sample. The insert of figure (b) corresponds to the FFT of micrograph and the insert of (d) and
(f) correspond to the electron diffraction images.
corresponding to the planes (0 0 2) and (2 1 1) are 2.67 Å and 2.72 Å,
respectively [36,37]. The planes (2 1 1), (3 0 0) and (1 1 2) are over­
lapping on electron diffraction images due to the similarity among
interplanar distances [6]. The plate-like morphology was observed on
bright field images on OCP sample. Although the XRD analysis resulted
in a mixture of OCP and DCPD phases, it was only possible to identify the
lattice fringes of (0 2 0) and (0 4 0) planes, which correspond to the
interplanar distances of 3.89 Å and 7.46 Å, respectively. This behavior is
characteristics of the DCPD phase on electron diffraction.
4. Conclusions
The calcium phosphates phases of ACP, HA and OCP were success­
fully synthesized by wet chemical method. Different from other drying
techniques, where the material remains in contact with the mother li­
quor until complete removal of the solvent, freeze drying results in
smaller particles due to interruption of the maturation of the crystals by
the freezing step, while the solid solvent is then removed by sublimation.
The freeze drying does not modify the crystalline phase, vibrations mode
and morphological aspects of samples, acting only on reducing the
particle size to the nanoscale. Thus, freeze dry showed to be an adequate
and simple technique to obtain nanocrystalline calcium phosphates for
bone regeneration applications.
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Materials Chemistry and Physics 239 (2020) 122004
Acknowledgments
The authors thank the CAPES-MES Project (project 186/13), the S~ao
Paulo Research Foundation - FAPESP (project 2017/15487-3), Labora­
tory of Magnetic Materials and Colloids (Zeta and DLS measurements) X-
ray laboratory and the Scanning Electron Microscopy Laboratory of the
Institute of Chemistry, UNESP. Araraquara, Brazil, for the measurements
made.
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