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CrystEngComm
COMMUNICATION
Cite this: CrystEngComm,2016,18,
3170
Received 6th March 2016,
Accepted 5th April 2016
DOI: 10.1039/c6ce00521g
www.rsc.org/crystengcomm
Crystallization of citrate-stabilized amorphous
calcium phosphate to nanocrystalline apatite: a
surface-mediated transformation†
Konstantinos Chatzipanagis,‡
a
Michele Iafisco,‡
b
Teresa Roncal-Herrero,
a
Matthew Bilton,
ac
Anna Tampieri,
b
Roland Kröger*
a
and José Manuel Delgado-López*
d
This work explores the mechanisms underlying the crystallization
of citrate-functionalized amorphous calcium phosphate (cit-ACP)
in two relevant media, combining in situ and ex situ characteriza-
tion techniques. Results demonstrate that citrate desorption from
cit-ACP triggers the surface-mediated transformation to nanocrys-
talline apatite (Ap). Our findings shed light on the key role of cit-
rate, an important component of bone organic matrix, and the
medium composition in controlling the rate of transformation and
the morphology of the resulting Ap phase.
Many aspects of the mechanisms underlying the formation of
nanocrystalline apatite (Ap), the main constituent of the
inorganic phase of bone,
1,2
remain under debate. In
particular, the crystallization pathway, starting from an
amorphous precursor and resulting in a well-defined mineral,
is a matter of intensive research.
2–5
It has been previously
demonstrated that in zebra fish bone
6
and in dental enamel,
7
Ap formation does not occur directly by the association of
ions from solution according to the classical nucleation and
growth theory, but follows a “non-classical”crystallization
pathway via an amorphous calcium phosphate (ACP) precur-
sor. On this basis, the formation of ACP as a transient/inter-
mediate phase is currently well accepted in in vivo bone
mineralization.
8–11
ACP crystallization in aqueous solutions has been largely
studied revealing that factors such as pH, temperature and
the presence of foreign ions (e.g. fluoride, magnesium, zinc,
carbonates, and silicates) and additives (e.g. polyelectrolytes,
phospholipids, polyglycols, proteins, etc.) affect the ACP sta-
bility and its transformation rate.
4,5
This process has been
proposed to occur, either directly from ACP to Ap or via the
formation of intermediate CaP phases (mostly octacalcium
phosphate; OCP), through different mechanisms:
4,5
(i) disso-
lution–reprecipitation; (ii) clusters reorganization and, (iii)
solution-mediated solid–solid transformation. Therefore, it
seems reasonable to assume that several processes might oc-
cur even simultaneously.
In this context, the role of citrate in stabilizing the ACP
has received less attention and only few reports can be found
in the literature.
12–14
Citrate is an important component of
mineralized tissues.
15
It accounts for ∼2 wt% in bone,
16
which is a concentration approx. 5–25 times higher than that
occurring in soft tissues. In fact, about 90% of the total cit-
rate found in the body resides in bone.
15
In addition, recent
NMR studies showed that it is strongly bound to the surface
of bone Ap nanocrystals, controlling their shape and mor-
phology.
17
However, its role in bone biomineralization is far
from being clearly understood.
ACP not only plays a pivotal role in bone biomineraliza-
tion but it is also widely used in medicine.
4
Hence, under-
standing its behaviour in aqueous media is of paramount im-
portance for designing advanced biomaterials. However, in
situ characterization of ACP is rarely found in literature likely
due to the fact that it instantaneously transforms into a more
stable crystalline phase and the difficulty of finding a suit-
able technique allowing for its characterization in solution.
This work explores the mechanisms underlying the crystal-
lization of citrate-functionalized ACP (cit-ACP) immersed in
two relevant media; pure water and the physiological
phosphate-buffer saline (PBS) solution. The morphological
evolution of cit-ACP in both aqueous solutions was studied
by ex situ transmission electron microscopy (TEM), whereas
the structural evolution was monitored by in situ time-
resolved Raman spectroscopy.
Dry powder cit-ACP was synthesized by the batch precipi-
tation method described elsewhere
12,18,19
(see ESI,†for fur-
ther details). A negative zeta potential of −10.5 ± 3.9 mV was
3170 |CrystEngComm,2016,18, 3170–3173 This journal is © The Royal Society of Chemistry 2016
a
Department of Physics, University of York, York, UK
b
Institute of Science and Technology for Ceramics (ISTEC), National Research
Council (CNR), Faenza, Italy
c
Department of Chemistry, Simon Fraser University, Burnaby, Canada.
E-mail: roland.kroger@york.ac.uk
d
Laboratorio de Estudios Cristalográficos, Instituto Andaluz de Ciencias de la
Tierra (IACT, CSIC-UGR), Armilla, Granada, Spain. E-mail: jmdl@iact.ugr.csic.es
†Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ce00521g
‡Both authors equally contributed to this work.
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obtained for cit-ACP. This value is in excellent agreement
with that obtained for citrate-stabilized ACP by Chen et al.
14
These authors related such a negative value to the adsorption
of negatively charged citrate on ACP surface. However, fur-
ther studies are necessary to confirm or exclude the possible
presence of citrate also inside the ACP particles. Fig. 1A
shows the Raman spectrum of cit-ACP particles (red line). It
exhibits bands related to the OCO bending (845–847 cm
−1
),
OCO stretching (1400–1600 cm
−1
) and CH
2
stretching (2928–
2933 cm
−1
) modes of citrate.
18
Fig. 1A also shows the Raman
spectrum of the particles obtained after immersing cit-ACP in
water for a total of 5 days (blue line). The same citrate vibra-
tions are still noticeable. In addition, phosphate vibrational
modes are clearly visible in both spectra. The assignments of
the corresponding vibrational modes are summarized in Ta-
ble S1 (ESI†). cit-ACP evolution was studied in situ by moni-
toring the symmetric vibration of phosphate groups (v
1
PO
4
).
In water (Fig. 1B), a single symmetric band centred at 952
cm
−1
, assignable to ACP,
4,20
is observed at the early stages.
Upon maturation, this band progressively becomes asymmet-
ric due to the formation of the crystalline Ap. Indeed, after
49 hours (2950 min), a distinct Raman peak at 959 cm
−1
was
observed. This value is in accordance with the Raman shift
reported for Ap.
18,21
In situ time-dependent Raman spectra of
cit-ACP immersed in PBS (Fig. 1C) indicate that the cit-ACP-
to-Ap conversion is greatly accelerated in this ionic medium.
The normalized ratio of the corresponding v
1
PO
4
Raman
band of Ap and ACP (A
959
/A
952
,i.e. fraction of the transformed
phase, Φ) (Fig. S1, ESI†) was used to study the extent of the
conversion in both media. The time dependence of Φis repre-
sented in Fig. 2A. From these curves, we estimated an induc-
tion time in PBS of 10 minutes whereas the tapering off period
was reached after 110 minutes. Conversely, the induction time
in water was 830 minutes (ca. 14 hours), and the conversion
gradually developed up to 3260 minutes (ca. 54 hours), when
the steady state was achieved. Therefore, the appearance of Ap
was significantly accelerated (by a factor of 75) in PBS.
The kinetics of the crystallization were studied in more de-
tail using the Avrami model, which describes phase transfor-
mation in terms of volume fraction changes. This model is
characterized by the generalized expression for the time de-
pendence of the fraction of the transformed phase (Φ)as
22,23
Φ=1−exp(−kt
n
)
where the parameter kentails information on nucleation
density and growth rates whereas nrepresents the dimen-
sionality of the growth and the possible impact of diffusion.
We assume here that A
959IJAp)
/A
952IJcit-ACP)
∝Φand upon com-
plete transformation Φ= 1, which means that the data were
normalized. However, the data reveal that signal of v
1IJcit-ACP)
never completely vanishes indicating that an amorphous/dis-
ordered layer remains at the crystallite surface even after full
crystallization, as previously reported.
12,13,18,24–26
The solid
lines in Fig. 2A represent Avrami fits to the data. A plot of
ln[−ln(1 −Φ)] vs. ln(t) as shown in Fig. 2B reveals how closely
the transformation follows Avrami kinetics (in comparison to
the straight line fits shown in the figure). Overall we observed
an approximate –albeit not perfect –fit of the data to an
Avrami type kinetics with a goodness of fit varying between
97% and 99%. Most notably, the transformation in PBS is in-
dicative for an Avrami transformation. Whereas in water, the
initial part of the curve shows a changing slope during the
transformation. This indicates an enhanced role of a change
of nucleation rates during the process. This difference can be
Fig. 1 (A) Raman spectra of dry cit-ACP (red line) and dry Ap (blue line, obtained after 5 days of cit-ACP immersion in water). The phosphate vibra-
tional modes are denoted in blue whereas those involving bonds of citrate are marked in red. In situ time-dependent Raman spectra (v
1
PO
4
vibra-
tions) collected during the transformation of cit-ACP in (B) water and in (C) PBS.
Fig. 2 (A) Avrami fits to the data obtained for PBS and water. (B) Plots
of the linearized Avrami equation.
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explained by the approximations made in the Avrami equa-
tion, namely assuming spherical crystal growth and ignoring
diffusion and a time dependence of the nucleation. The slope
of the lines provides information on the parameter n, which
differs significantly between the two cases. We find values of
n= 2.1 and 3.8 for PBS and pure water, respectively. Assum-
ing interface-controlled phase transformation Wong and
Czernuszka
27
relate values of nabove 3 to either zero nucle-
ation (n= 3), decreasing nucleation rate (n=3–4), or constant
nucleation rate (n= 4) for solvent mediated re-dissolution
and re-crystallization processes. Values below 3 indicate
diffusion-controlled growth. Therefore, in PBS our observa-
tions suggest a significant role of species diffusion in solu-
tion leading to a rapid crystal growth. To evaluate the corre-
sponding transformation rates the time derivative of the
Avrami-fit, namely dΦ(t)/dt, was determined for cit-ACP trans-
formation in the two media (Fig. S2C, ESI†). This plot reveals
a rapid transformation for pure PBS with a maximum trans-
formation rate of 2.0 ×10
−2
min
−1
, which reduces to 1.2 ×
10
−3
min
−1
for water. The significant difference in transfor-
mation rates is also reflected by the values for the parameter
k, which are 9.4 ×10
−14
min
−n
for water and 2.9 ×10
−4
min
−n
for PBS. In our analysis we have assumed a single nucleation
event but in particular for water it is highly likely that further
nucleation events occur during the transformation affecting
the value for k. As a control experiment, we also studied the
impact of a PBS/water mixture (1 : 1 volume) on the transfor-
mation kinetics (Fig. S2, ESI†). This revealed an intermediate
timescale for full transformation between that of pure water
and PBS.
The morphological amorphous-to-crystalline evolution was
studied ex situ by TEM. Fig. 3A shows TEM micrograph of
the as-prepared cit-ACP nanoparticles and the corresponding
selected area electron diffraction (SAED) pattern, which con-
firms their amorphous nature. The particles are aggregated
exhibiting round shaped morphology with average diameters
of ca. 50 nm, as previously reported.
12,13
In PBS, after 10
min, which corresponds to the estimated induction time by
Raman, formation of crystalline domains of about 3 nm in
diameter within the aggregated cit-ACP particles were ob-
served (Fig. 3B). The SAED pattern (insert in Fig. 3B) from
these particles indicates that they are Ap (STM card file no.
09-432). Similar domains have been previously observed by
high-resolution TEM during the ACP-to-Ap transforma-
tion,
28,29
and has been proposed that Ap crystallization oc-
curs from multiple nuclei within the ACP nano-
particles.
12,28,29
After 100 min (corresponding to the post-
crystallization stage) platelet-like nanoparticles of Ap were ob-
served which confirms that the transformation to Ap oc-
curred (Fig. 3C). However, a different evolution was found in
water. After one day (early stage of crystallization), the partial
dissolution of ACP was observed (Fig. 3D) since amorphous
particles appeared smaller than those shown in Fig. 3A. Sub-
sequently, during the second day (intermediate stage of crys-
tallization), we observed indistinctly either poorly crystalline
aggregates providing very tenuous 002 reflections in the SAED
pattern (Fig. 3E) or amorphous particles with diffuse borders
(Fig. S3, ESI†). Finally, after 3 days (corresponding to the
post-crystallization stage) smaller Ap nanoparticles than
those grown in PBS were observed (Fig. 3F).
The large differences in the conversion kinetics of both
media cannot be attributed to pH variations (Fig. S4, ESI†).
Actually, Boskey et al.
30
reported much lower differences be-
tween the conversion kinetic parameters at pH values similar
to those measured in this work. The hydrolysis of ACP has
been proposed to be the responsible for triggering its instan-
taneous crystallization to Ap in aqueous media.
31
However,
in the case of cit-ACP, adsorbed citrate, blocking active sites,
delays this transformation. In PBS, the citrate is rapidly re-
leased from the surface (Fig. 4A), most probably by ionic ex-
change with the phosphate of the medium,
32
which in turn
reduces the ACP stability and increases the phosphate con-
centration within ACP. This leads to the rapid transformation
to Ap, starting from small nuclei (as those appearing close to
the surface in Fig. 3B). Conversely, a gradual release of citrate
was found in water (Fig. 4B). After one day, when most of the
citrate has been released, the partial dissolution of the ACP
and the further re-crystallization of Ap occurred. However,
the complete dissolution of cit-ACP can be excluded as
Fig. 3 (A) TEM micrograph and the corresponding SAED pattern of the
as-prepared dry cit-ACP. (B–C, red square) morphological evolution of
the particles immersed in PBS (time: B, 10 min; C, 100 min) and (D–F,
green square) water (time: D, 1 day: E, 2 days; F, 3 days). SAED pattern
in B corresponds to the crystalline domains (dark spots, as the marked
with the white circle). The scale bars correspond to 50 nm except for
B, where is 5 nm.
Fig. 4 Time-dependent evolution of the normalized peak of citrate
(A(δ
OCO
)/A(v
1
PO
4
)) in (A) PBS and (B) water.
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suggested by the time-lapse video recorded during the trans-
formation (Movie S1, ESI†).
Conclusions
The combination of ex situ (TEM and SAED) and in situ
(time-dependent Raman spectroscopy) experiments allowed
for monitoring the crystallization of cit-ACP in two relevant
media; i.e. ultrapure water and PBS. Results demonstrate that
cit-ACP directly transforms to Ap without involving the for-
mation of any other intermediate calcium phosphate phase.
Citrate desorption from ACP triggers the Ap crystallization,
which occurs through a surface-mediated process. This pro-
cess involves the ionic-exchange between labile ions from the
surface of cit-ACP nanoparticles and ionic species in solution.
The exchange between adsorbed citrate and phosphate pro-
motes the rapid Ap crystallization in PBS. Indeed, the pres-
ence of phosphates in the media greatly accelerates such con-
version (by a factor of 75) as determined by in situ Raman.
Overall, our results highlight two important aspects of cit-
ACP to Ap transformation: the role of citrate (or analogous
organic additives) in stabilizing ACP and preventing the in-
stantaneous transformation, and the impact of ionic species
concentration (e.g., phosphate) in controlling crystallization
rates and mechanisms. These aspects are highly relevant for
gaining a better understanding in bone biomineralization
process and for designing advanced biomaterials.
Acknowledgements
This work has been carried out in the framework of the pro-
jects SMILEY (FP7-NMP-2012-SMALL-6-310637), BioBone
(Andalucía Talent Hub, co-funded by Junta de Andalucía and
EU-FP7 within the Marie-Curie Actions), the UK Engineering
and Physical Sciences Research Council (EPSRC) (Grant No.
EP/I001514/1), funding the Material Interface with Biology
(MIB) consortium, and the short-term mobility program (STM
2015) from the National Research Council of Italy (CNR) for
J. M. D.-L. We would like also to thank York JEOL Nanocentre
for the use of their facilities.
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