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This journal is ©The Royal Society of Chemistry 2015 J. Mater. Chem. B, 2015, 3, 9157--9167 | 9157
Cite this: J. Mater. Chem. B, 2015,
3, 9157
A potential mechanism for amino acid-controlled
crystal growth of hydroxyapatite
Ziqiu Wang,
a
Zhijun Xu,*
a
Weilong Zhao
a
and Nita Sahai*
abc
The mineral component of bone, dentin and calcified parts of avian tendon, hydroxyapatite (HAP), has
non-stoichiometric composition (idealized as Ca
10
(PO
4
)
6
(OH)
2
), plate-like morphology and nanometer size.
This unique crystal morphology contributes to the physico-chemical and biochemical properties of bone.
Thus, understanding the mechanism for the controlled growth of plate-like HAP nanocrystals is significant
in the study of bone biomineralization. Previous studies have shown that acidic non-collagenous proteins
(ANCPs), which are enriched in the residues of acidic amino acids, may play an important role in HAP
crystal growth modulation. In this study, glutamic acid (Glu) and phosphoserine (Ser-OPO
3
) were used as
model compounds to modify the synthesis of HAP nanocrystals. To identify the mechanisms of amino
acids as regulators, X-ray diffraction (XRD), transmission electron microscopy (TEM) and solid state nuclear
magnetic resonance (ssNMR) were used. The crystals obtained in the inorganic controls were needle-like,
while crystals synthesized in the presence of the amino acids presented a plate-like morphology. The
plate-like crystals had a preferred crystal orientation on (300) face, which was lacking in the inorganically
grown crystals, indicating preferential adsorption and suppression of growth in specific crystal directions.
Ser-OPO
3
was more efficient than Glu in modulating HAP nucleation and crystal growth. Furthermore,
NMR revealed interactions between the charged side chain groups in amino acids and the crystal surfaces.
These results were successfully explained through our MD simulations for the free energy calculation of
amino acid binding on HAP crystal faces. The present study revealed that amino acids may act as effective
regulators of HAP morphology without the need to invoke large NCPs in bone biomineralization and in
designing bioinspired materials for orthopaedic and dental applications.
1. Introduction
Bone is a hierarchical, compositematerialcomposedprimarilyof
an insoluble collagen matrix, non-stoichiometric hydroxyapatite
(HAP) mineral phase idealized as Ca
10
(PO
4
)
6
(OH)
2
,solubleacidic
non-collagenous proteins (ANCPs) as well as small molecules
such as citrate.
1,2
HAP has been considered as one of the most
biocompatible materials for orthopedic and dental applica-
tions.
3,4
In bone, HAP is Ca
2+
- and PO
43!
-deficient (with a Ca : P
ratio less than 1.67, which is the theoretical value for pure HAP),
and Na
+
-, Mg
2+
-andCO
32!
-enriched. Because of these substitutions,
the HAP crystals in bone are often poorly crystalline. The crystals are
plate-shaped, with average lengths and widths of B30–200 nm and
thickness of only B1.5 nm for mineralized avian tendon and up to
about 4.0 nm for mature bone.
5,6
These HAP crystals are aligned
with [001] orientation along the long axis of the collagen fibrils.
7
The uniaxially oriented, plate-like HAP nanocrystals contribute,
in part, to the remarkable biomechanical properties of bone.
6,8,9
Therefore, it is important to understand the fundamental
mechanisms for crystal nucleation and growth at the atomic
level. The knowledge of normal mineralization pathways could
benefit efforts in developing treatment for the bone diseases, in
which mineralization is defective such as osteomalacia, osteo-
genesis imperfecta, osteopetrosis, etc. Also, the improved design
of scaffolds for bone tissue engineering desperately requires
more details about how uniaxially aligned plate-shaped crystals
grow within the organic matrix.
The nucleation and growth of HAP is closely associated to
the extracellular matrix environment. Blood plasma is super-
saturated with respect to HAP, but mineralization only occurs
at specific sites in the body under normal physiological condi-
tions. Furthermore, in bone and dentin, the earliest calcium
phosphate nucleation and plate-shaped crystal growth occurs
within a matrix of collagen fibrils, which are themselves
composed of collagen molecules self-assembled in a pseudo-
hexagonal array. It is believed, therefore, that some proteins
existing in extracellular fluids such as fetuin and osteopontin,
and small molecules such as citrate, may act as inhibitors of
a
Department of Polymer Science, University of Akron, 170 University Ave, Akron,
OH 44325-3909, USA. E-mail: sahai@uakron.edu, zxu@uakron.edu;
Tel: +1 330-972-5795
b
Department of Geology, University of Akron, OH 44325, USA
c
Integrated Bioscience Program, University of Akron, OH 44325, USA
Received 28th May 2015,
Accepted 30th October 2015
DOI: 10.1039/c5tb01036e
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HAP nucleation.
10–12
A possible mechanism for the formation
of plate-like crystals is that the growth modulating molecules
may adsorb preferentially on specific faces of HAP crystals. This
process would limit growth rates in specific directions, ultimately,
controlling the crystal shape as well as size. The predominant
crystal face of HAP crystals from mineralized turkey tendon and
bone is the (100) face, and this face is oriented along the length
of the collagen fibril. The (100) face also has the largest surface
area, followed by the (010), and the (001) face, which has the
smallest surface area.
5
Because the interactions occur at the organic-
mineral interface, it is implicit that understanding the atomic-level
details of biomolecule adsorption onto mineral surfaces is of vital
significance. It has been proposed in the literature that acidic NCPs,
which are enriched in negatively charged acidic amino acid
residues of Glu, Aspartate (Asp) and Ser-OPO
3
residues, are active
in mediating biologically directed mineral formation.
13–20
For
example, osteopontin and bone sialoprotein can affect HAP
crystal formation and growth in vitro.
11,21
However, it is not
known whether these acidic NCPs can be accommodated inside
the small space between collagen molecules within a fibril, so
their role in intra-fibrillar mineralization is unclear.
10,22,23
Furthermore, the elucidation of the adsorption mechanisms of
those macromolecules on HAP is hindered by the complexity
of the molecular structures and diversity of functional groups
usually found in NCPs.
Taking into account the above considerations of NCPs, it has
been proposed in some studies that some small molecules such
as amino acids and citrate may be biologically relevant to HAP
crystal growth.
24–30
Early studies focused on the amount of
amino acids adsorbed on HAP surfaces. Kresak et al. measured
the adsorption isotherms of different amino acids on HAP
surfaces and found that both Asp and Glu had a higher affinity
for HAP compared to that of Arg, which has a basic negatively
amine group side chain.
27
Koutsopoulos and Dalas
31–34
used
constant composition technique to synthesize HAP crystals in the
presence of amino acids under conditions of low supersaturation.
Their results suggested that amino acids with different side chain
groups may have various abilities to inhibit crystal growth rate.
The crystal growth rates of HAP were found to decrease markedly
compared with the inorganic control.
35
This effect was attributed
to adsorption and subsequent blocking of the growth sites on the
surface of the HAP crystals. Some active sites such as charged
carboxyl terminus as well as hydrogen bonds between hydroxyl
group and phosphate ion were successfully found by using
ssNMR at interface and benefit the binding of biomolecules on
crystal surface.
36,37
Furthermore, some studies suggested that
small acidic amino acids are capable of affecting HAP crystal
morphology.
35,38–41
Most of the previous attempts to synthesize
HAP crystals have failed to present an plate-like morphology that
resembles bone apatite. Also, a big remaining challenge is to
elucidate the detailed inhibitory effects of amino acids on HAP
growth molecular level.
Molecular dynamic simulations have been employed in
recent studies as another approach to identify the binding sites
between amino acids or small peptides and HAP faces in recent
studies.
39,42–45
However, the reliability of results obtained by
MD depends on accurate benchmarking of the applied force fields
to ab initio quantum chemical results and to experimental
results,
46
which has been largely neglected in most MD studies
of biomineralization. In our previous state-of-the-art MD simula-
tions, we developed properly benchmarked force-fields and
examined interactions between Glu or Ser-OPO
3
and HAP crystal
surfaces.
47
Here we used Glu and Ser-OPO
3
as model of NCPs to
examine the role of small molecules in HAP crystal nucleation
and growth. Results of the present study showed that, in the
presence of amino acids, a morphology change occurred from
needle-like to plate-like crystals with preferential directions of
crystal growth. Both Glu and Ser-OPO
3
showed inhibitory effect
on HAP crystal growth and nucleation, and the interactions
between charged side chains in amino acids and HAP surface
ions were observed. All these results were consistent with our
MD simulations
47,48
and can be combined to elucidate a novel
mechanisms of face-specific organic-mediated HAP crystal
nucleation and growth at the atomic level, which could rationalize
the role of small molecules as effective regulators in HAP
morphology regulation.
2. Materials and methods
2.1 Materials
All solutions used in the experiments were prepared with ultra-pure
water (18.2 MOcm, Nanopore UV, Barnstead). Calcium chloride
(CaCl
2
,anhydrous,96.0%purity,molecularweight110.98gmol
!1
),
ammonium phosphate dibasic ((NH
4
)
2
HPO
4
, ACS reagent grade,
98% purity, molecular weight 132.06 g mol
!1
), and sodium
hydroxide solution (50% in H
2
O) were used to synthesize HAP
crystals. O-Phospho-L-serine (molecular weight 185.07 g mol
!1
)
and L-glutamic acid (molecular weight 147.13 g mol
!1
) were used
as additives. All chemicals were purchased from Sigma-Aldrich.
2.2 Synthesis of HAP crystals
2.2.1 Preparation of control solution. 55.5 mg calcium chloride
and 39.6 mg ammonium phosphate dibasic were dissolved sepa-
rately in 10 mL water in 50 mL beakers. The ammonium phosphate
dibasic solution (30 mM) was pipetted into the calcium chloride
solution (50 mM) at a rate of 3 mL min
!1
in a 125 mL Erlenmeyer
flask. The suspension was kept stirred using a magnetic stir bar on a
heating plate at 37 "0.5 1C. The pH was kept constant at B9.5 "0.1
by drop-wise addition of 1 M NaOH using a pipette. The HAP crystals
formed in the solution as follows:
10Ca
2+
+ 6HPO
42!
+ 8OH
!
-Ca
10
(PO
4
)
6
OH + 6H
2
O
The suspension was subsequently placed and aged for three
days in an incubator-shaker at 37 "0.2 1Cwithashakingspeedof
130 rpm. Then, a small aliquot was used for TEM analysis, and the
remaining solution was centrifuged and dried to obtain powders.
2.2 Preparation of solution with small organic molecules
Various concentrations Glu or Ser-OPO
3
were used to examine their
effects on HAP crystal morphology (Table 1). 111 mg calcium
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chloride was dissolved in 20 mL water in 50 mL beakers. Then,
Glu or Ser-OPO
3
was dissolved in calcium chloride solution to
yield concentrations of 20, 50, 80 and 200 mM respectively, and
the solution was stirred using a magnetic stir bar on a heating
plate at 37 "0.5 1C. 79.2 mg Ammonium phosphate dibasic
was dissolved in 20 mL water and added to the mixed solution
by pipette at a rate of 3 mL min
!1
. The pH was kept constant at
B9.5 using 1 M NaOH solution added by pipette. The suspension
was subsequently placed and aged in an incubator-shaker at
37 "0.2 1C with a shaking speed of 130 rpm for two weeks.
Then, few aliquot was used for TEM, and the remaining
solution was centrifuged and dried to obtain powders.
2.3 HAP characterization
2.3.1 Transmission electron microscopy. Aliquots of about
40 mL was extracted from the suspension and dropped onto a
formvar/carbon film-supported copper grids (300 mesh, Ted
Pella supplies) and dried in air. The sample was then examined
by TEM (JEOL JSM-1230, 120 kV; FEI/Philips CM-200T, 200 kV) to
obtain bright-field images and selected area electron diffraction
(SAED) patterns. An energy-dispersive X-ray spectrometer (EDX)
connected to the TEM was used to get calcium to phosphorus ratio.
In order to ensure that artifacts were not introduced during
the drying process, cryo-TEM (Tecnai G2 F20) images were
obtained on selected samples for comparison. The solution
was dropped on the TEM grid by using FEI vitrobot. A thin layer
(B100 nm) remained on the grid after the excess liquid was
removed by blotting with filter paper. The grid was then transferred
into liquid ethane and carried by a cryo TEM holder. The cryo
holder worked at B!170 1C. Images were taken by TEM equipped
with a 4 k #4kUltraScanCCDcamera.
2.3.2 X-Ray diffraction. After aging for 3 days (inorganic
control) or 2 weeks (amino acid-containing solutions), the
precipitates in solution were collected by centrifugation and
rinsed three times with ultra-pure water. Sample powder was
collected and scanned by XRD (Bruker SMART Apex) over a 2y
range from 2–701. Results were analyzed by MDI Jade 5.0 and
compared with standard HAP card (JCPDS 09-0432) to find
corresponding peaks.
2.3.3 Solid-state nuclear magnetic resonance. About 30 mg
sample powder was used for NMR. The experiments were
performed on a Bruker Avance 300 equipped with a 4 mm
double-resonance probe. Temperature was controlled at 298 K. The
carrier frequencies of
1
Hand
13
Cwere300MHzand75.5MHz,
respectively. The frequency for magic-angle sample spinning was
12 kHz. In
13
C{
1
H} cross polarization (CP), the radio-frequency
field (rf) strength was set to 62.5 kHz for
13
C and a ramped field
was used for
1
H. During
13
C signal acquisition,
1
H two-phase
pulse modulation (TPPM) was applied with a rf strength of
62.5 kHz. The contact time and the recycle delay were 2 ms
and 2 s, respectively. The 2D
13
C{
1
H}hetero-nuclear correlation
(HETCOR) NMR spectra with cross polarization were then recorded.
3. Results and discussion
3.1 Inorganic control
The crystal structure of calcium phosphate solid synthesized
inorganically in solution was analyzed by XRD, and the pattern
showed diffraction peaks corresponding to the characteristic
peaks of synthetic standard HAP (Fig. 1a). The peaks occurred
at 261, 321, 401, 471, 501, 531and 641referring respectively to
(002), (211), (310), (222), (321), (004) and (304) faces of HAP
crystals.
Crystal morphology and structure were analyzed using TEM.
The morphology of the HAP crystals was needle-like, with
dimensions of B100–150 nm length and B20–30 nm width.
The crystals were observed to form large aggregates (Fig. 1c).
Several diffraction rings were obtained in the SAED pattern
(Fig. 1d), indicating random orientation of the nanocrystals in
the aggregates. The diffraction rings can be referred to HAP
(310), (004), (211), (210) faces. The EDX element analysis
(Fig. 1b) showed calcium peaks at B3.75 keV and B4.0 keV
and phosphorus peak at B2.0 keV. By integrating the area
Table 1 Conditions for HAP crystal synthesis
Solution Time Calcium
concentration (mM) Phosphate
concentration (mM) Amino acid
concentration (mM) Charge
Control 37 1C, pH 9.5 3 days 50 30 0
Glu 37 1C, pH 9.5 2 weeks 50 30 20, 50, 80, 200 !1
Ser-OPO
3
37 1C, pH 9.5 2 weeks 50 30 20, 50, 80, 200 !2
Fig. 1 Characterization of HAP crystals synthesized inorganically. (a) XRD
spectrum, (b) EDX spectrum, (c) TEM bright field image, and (d) SAED
pattern.
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under the peaks, the atomic percentages of calcium and
phosphorus were calculated to be 12.28% and 7.25%. Thus,
the Ca/P atom ratio was 1.69, which is close to the ideal Ca/P
ratio of 1.67 in stoichiometric HAP.
3.2 Hydroxyapatite synthesized in the presence of amino acids
3.2.1 Transmission electron microscopy. The morphology
of HAP nanocrystals in the presence of different Glu (Fig. 2) or
Ser-OPO
3
(Fig. 3) concentrations was observed using TEM.
When synthesized in the presence of 20 mM Glu, the crystals
had a needle-like morphology (Fig. 2a and b) and the crystal
size was similar to the inorganic control (Fig. 1c). However, the
aggregates obtained in the presence of Glu were generally
smaller in size. At Glu 50 mM concentration (Fig. 2d and e),
there was no obvious morphology change compared to 20 mM.
The crystal morphology changed when concentration was
increased to 80 mM, with reduced width and greater length
(Fig. 2g and h). Most crystals were less than 100 nm in length
and more than 20 nm in width. There were also some large
crystals of length greater than 100 nm. At the 200 mM Glu
concentration, fewer crystals were observed when examined
at different areas on the TEM grid. Also, theses crystals had
plate-like morphology, with dimensions of B300–500 nm
(Fig. 2j and k).
Fig. 2 TEM bright field images and SAED patterns of HAP crystals synthesized in the presence of Glu at different concentrations. (a)–(c) 20 mM; (d)–(f)
50 mM; (g)–(i) 80 mM and (j)–(l) 200 mM.
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At low concentration (20 mM), the SAED pattern showed
diffraction rings (Fig. 4c), indicating that the nanocrystals were
randomly orientated. The rings could be referred to the same
HAP faces as in the inorganic sample. Arcs corresponding to
(006) and (200) faces were obtained at 50 mM (Fig. 2f). The
appearance of arcs suggested that the crystals began to have
some preferential alignment directions on these faces. As the
crystals changed to plate-like morphology, the diffraction pattern
presented spots instead of arcs (Fig. 2i). The spots corresponded
to (004) and (400) faces, which are in the same crystal zone as
the (004), (006), and (600) faces that were observed at lower
concentration. However, at high concentration, some high lattice
index faces such as (502) and (602) appeared.
The crystals synthesized in the presence of 20 mM Ser-OPO
3
also presented as needle-shaped and in large aggregates (Fig. 3a
and b). The ability of Ser-OPO
3
to regulate HAP crystal morphology
was evident at 50 mM (Fig. 3d and e), which was at a lower
concentration compared to the 80 mM offered for Glu. Also, large
crystal aggregates were not seen at this condition. Thus, Ser-OPO
3
was more effective at suppressing nucleation as well as modifying
crystal growth. At the concentration of 80 mM, crystal morphology
became closer to plate-like (Fig. 3gandh),andthewidthincreased
Fig. 3 TEM bright field images and SAED patterns of HAP crystals synthesized in the presence of Ser-OPO
3
at different concentrations. (a)–(c) 20 mM;
(d)–(f) 50 mM; (g)–(i) 80 mM and (j)–(l) 200 mM.
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from B30 nm to B50–100 nm. Similar to 200 mM Glu concen-
tration, large plate-like crystals appeared with B300–500 nm in
dimensions in 200 mM Ser-OPO
3
system (Fig. 3j and k).
From the diffraction pattern, it was evident that with 20 mM
Ser-OPO
3
, HAP nanocrystals were randomly oriented (Fig. 3c).
At 50 mM concentration, diffraction spots could already be
observed, indicating that the nanocrystals were more oriented
compared to those at the same Glu concentration which still
showed arcs (Fig. 3f). The brighter spots corresponded to (002)
and (004) faces of HAP. When Ser-OPO
3
was increased up to
200 mM, the (300), (102) and (512) faces were also seen in
Fig. 3l. This difference in diffraction patterns from samples
with various concentrations suggested the change in preferential
crystal growth orientations among different HAP crystals.
To explore the possibility of any artifacts introduced by sample
dehydration during TEM sample preparation, cryo-TEM was also
used to observe the crystal morphology of selected samples (Fig. 4).
Bright field images of both Glu and Ser-OPO
3
samples presented
similar morphology as well as similar dimensions compared to
those taken by traditional TEM at the corresponding concentrations.
The results proved that detectable artifacts were not produced during
dehydration of the sample preparation on the grid.
To further characterize the crystal structure in detail, HRTEM
was employed to obtain the lattice fringes of the crystals (Fig. 5). For
HAP synthesized with 80 mM Glu (Fig. 5a) or 50 mM Ser-OPO
3
(Fig. 5c), interplanar distances (d) of B0.34 nm were seen,
which corresponded to the HAP (002) face. The d spacing
showed that [100] is the preferred crystal growth direction. In
samples synthesized at 200 mM Glu (Fig. 5b) or 80 mM Ser-OPO
3
(Fig. 5d), an additional dof B0.269 nm was found, indicating a
second crystal growth orientation on the (300) face. Thus, TEM
analysis showed that, at different amino acids concentrations,
the preferential crystal growth directions changed, leading to a
change in overall morphology of the crystal.
3.2.2 Solid-state nuclearmagneticresonance.The interactions
between the amino acids and HAP crystal surfaces were examined
by applying solid-state NMR. Note that NMR collected and averaged
signals from atoms on all HAP faces, thus no face-specific informa-
tion could be provided. The cross polarization magic angle spin-
ning (CP/MAS) spectra of HAP synthesized in the presence of Glu
and Ser-OPO
3
are shown in Fig. 6a and b. The chemical shift for
carbon at B180 ppm assigned to carboxyl groups in Glu appeared
(peaks 1 and 2, Fig. 6a), while the peak assigned to the carboxyl
group in Ser-OPO
3
sample showed at B170 ppm in (peak 1,
Fig. 6b). The
13
C{
1
H} HETCOR spectra for Glu and Ser-OPO
3
(Fig. 6c and d) provided information about interactions between
C atoms in amino acid molecules with H atoms on HAP surfaces
or with H atoms in amino acids. For Glu (Fig. 6c), the major
correlation peaks appeared at
1
H peaks of 0 ppm, which corre-
spond to hydroxyl group in HAP, suggested interactions between
Glu and HAP. For example, the correlation peak at
13
C chemical
shift of B180 ppm indicated that the carboxyl groups in Glu
have interactions with HAP surfaces. For Ser-OPO
3
(Fig. 6d),
broad correlation peaks observed at
1
HB0 ppm was due to the
contribution of protons both in amino acids and HAP.
1
H peaks
appearing near 0 ppm and
13
C at 170 ppm revealed possible
interactions between carbon atoms in Ser-OPO
3
and protons on
crystal surfaces (Fig. 6d). In addition, correlation peaks at
1
H peaks of 15 ppm were observed for both amino acids, which
was the chemical shift in pure amino acids (no more than
10 ppm, not shown in this result), implicating possible hydrogen
bond formation between amino acids and phosphate groups on
HAP surface. These results suggest that Glu and Ser-OPO
3
may
Fig. 4 Cryo-TEM images of HAP crystals synthesized in the presence of
(a) 20 mM Glu; (b) 80 mM Glu; (c) 20 mM Ser-OPO
3
and (d) 80 mM Ser-OPO
3
.
Fig. 5 Lattice fringes observed in HRTEM images of HAP crystals synthesized
in the presence of (a) 80 mM Glu; (b) 200 mM Glu; (c) 50 mM Ser-OPO
3
and
(d) 80 mM Ser-OPO
3
.
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adsorb on HAP and interact with the outermost surface atoms
via electrostatic force.
3.2.3 Bulk precipitation. The effect of amino acids on HAP
precipitation was also examined at the bulk level, by determining
the mass of precipitates formed in each experiment relative to
the inorganic control (Fig. 7). The amount of HAP synthesized in
the presence of amino acids was less compared to the control for
both Glu and Ser-OPO
3
sample, and the weight of precipitates
further decreased with increasing concentration of amino acids.
This result provided evidence that both Glu and Ser-OPO
3
had an
Fig. 6
13
C CP/MAS ssNMR spectra of HAP crystals synthesized in the presence of (a) 200 mM Glu and (b) 200 mM Ser-OPO
3
and corresponding
13
C{
1
H}
HETCOR 2D spectrum of (c) 200 mM Glu and (d) 200 mM Ser-OPO
3
.
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overall inhibitory effect on the formation of HAP even at the bulk
level. At 20 mM, the mass of HAP showed only a small decrease
for both cases, thus the crystal morphology under TEM was
similar to the inorganic control (Fig. 1c). When amino acid
concentration was increased to 50 mM, a distinct difference
between Glu and Ser-OPO
3
was observed. A stronger inhibition
effect of Ser-OPO
3
compared to Glu was reflected on the basis of
the remarkable decrease in HAP amount, which was consistent with
the TEM result that the plate-like morphology began to occur with
50 mM Ser-OPO
3
(Fig. 3d and e). The sharp decrease of HAP amount
in the presence of Glu appeared when concentration was increased
to 80 mM, and that was the concentration at which plate-like HAP
was observed in TEM data (Fig. 2g and h). Based on the above
results, it can be concluded that Ser-OPO
3
was a stronger inhibitor
compared to Glu. However, when the concentration of amino
acids was high enough (e.g. 200 mM), the inhibition effect
between Glu and Ser-OPO
3
was at the same level.
3.3 Potential growth mechanism
In general, the final morphology of a crystal depends on the
relative growth rate of various faces. When crystal growth rate is
inhibited in a particular direction, growth can still occur in the
other two directions. The mechanism for the transformation
from needle-like to plate-like morphology in the presence of
amino acids may involve the preferential adsorption of amino
acids on specific crystallographic faces of a growing crystal.
Growth could be inhibited in the perpendicular direction, while
continuing parallel to the face, ultimately, resulting in the
largest surface area of that face (Fig. 8a). It has been found
here that needle-like HAP crystals formed in the absence of Glu
and Ser-OPO
3
, and the morphology changed to plate-like in the
presence of the amino acids. Also, preferred orientation on
(300) face was found in the plate-like HAP crystals compared to
that of need-like crystals. Based on the NMR results, we infer
that the electrostatic interactions between carboxylic acid side
chain of amino acids and HAP crystal surface contributed to the
regulation of crystal growth. The orientation on (300) face was
found to be preferred, indicating that crystal growth in the [100]
direction was inhibited the most compared to other directions
such as [001]. Previously, our group used MD simulations to
calculate the binding free energies of Glu and Ser-OPO
3
to
different faces of HAP.
47
The binding free energy profile can be
calculated as a function of the distance between the amino acid
and HAP face chosen as a reaction coordinate. This profile is
defined as the potential of mean force (PMF), which includes
both enthalpic and entropic contributions. We used umbrella
sampling method to calculate the PMF for adsorption of simple
organic molecules on HAP (001) and (100) face, together with
the weighted histogram analysis method. The separation dis-
tance along the z-direction (which is perpendicular to the (100)
face) between the center of mass of the amino acid and the
outermost Ca
2+
ion layer at each HAP surface was defined as the
reaction coordinate. The simulation results showed that the
adsorption of Glu and Ser-OPO
3
on the growing crystal nuclei
was driven by electrostatic interactions between the charged
functional groups of the amino acid and the ions on the crystal
faces. Thus, the –OPO
32!
and –COO
!
functional groups inter-
acted with Ca
2+
and the –NH
3+
groups formed hydrogen bonds
with –PO
43!
on the HAP faces. The distance between amino
acids bound on the surface and the adjacent hydroxyl group
(binding distance) was 7.6 Å, indicating no hydrogen bond
formation between them. Considering the fact that in the MD
simulation, a flat-surface crystal model (the most widely used
model in MD studies) was applied while the crystals synthesized
Fig. 7 Mass of HAP precipitates synthesized in the presence of Glu and
Ser-OPO
3
compared to the inorganic control. *Po0.05.
Fig. 8 Mechanism for amino acid mediated HAP crystal growth. (a) Schematic
showing preferential adsorption of amino acids on specific HAP faces, which
leads to different crystal growth rates, thus, modulating the final morphology.
The direction of slowest growth results inthatfacehavingthelargestsurface
area. Black arrows indicate passage oftime.Aminoacids=purple;crystal=
yellow and blue. Yellow color of crystal indicates original crystal form and blue
color indicates intermediate and finalforms;crystalgrowth=redarrow.The
length of arrows represents growth rate. (b) Adsorption free energy profiles of
Glu and Ser-OPO
3
on HAP (100) face with corresponding snapshots of
molecular structures. H = white; C = grey; N = dark blue; O = red; P = tan;
Ca = cyan spheres.
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in the experiment were non-ideal, this simulation HAP model
cannot precisely reflect the properties of the synthesized HAP in
real system. The presence of defects or steps on the crystal
surface would decrease the binding distance between amino
acids and hydroxyl groups, thus the interactions between them
became easier to be detected by NMR. Besides, the adsorption
free energy profiles showed that both Ser-OPO
3
and Glu adsorb
more strongly on the (100) face compared to (001).
47
Notably,
multiple free energy minima of !8.1, !9.5, and !9.8 kcal mol
!1
at around 2.0 Å to 4.0 Å were observed for Ser-OPO
3
binding on
the (100) face. The PMF curve of Glu on (100) face showed only
two free energy minima of !8.2 and !6.5 kcal mol
!1
are
obtained at 3.2 and 4.7 Å, respectively. The observation that
compared to Glu, Ser-OPO
3
has much stronger binding inter-
action with (100) face is reasonable, considering the fact that the
electrostatic interaction between the Ser-OPO
3
with two negative
charges and HAP surface is stronger with respect to Glu with one
negative charge. Here the PMF results show that the amino acids
may specifically interact with the HAP (100) face and further
provide thermodynamics information to understand the mecha-
nism of HAP regulation morphology, for example, why Ser-OPO
3
was more efficient than Glu in modulating HAP nucleation and
crystal growth. The results of the present study with the MD
simulation provide complementary and consistent information
for understanding the mechanism of amino-acid mediated
crystal growth.
For bone HAP crystals, the (100) face is the dominant face,
indicating that the growth rate perpendicular to this face is the
slowest, and the (001) face is the smallest, indicating that
growth along the [001] direction is the fastest. Thus, a similar
mechanism as proposed in the present study may be involved
in ANCP- or small molecule-mediated crystal growth in vivo.
4. Conclusions
HAP crystals synthesized without additives were needle-like and
formed large aggregates. In the presence of Glu and Ser-OPO
3
,
the nucleation and growth of crystals was inhibited compared
to the inorganic control. The amino acids adsorbed to the
growing crystals by electrostatic interactions, and preferential
adsorption of each amino acid on (300) faces lead to a significant
change in crystal morphology from needle-like to plate-like, which is
similar to bone and dentin mineralcrystals.Amongthetwoamino
acids, Ser-OPO
3
was found to be a more effective morphology
regulator compared to Glu. These results are consistent with our
MD simulation results for free energies of adsorption of these
amino acids on HAP surfaces.
Acknowledgements
We would like to thank Prof. William Landis for productive
discussion in this study. Also, we acknowledge Wei Chen and
Prof. Toshikazu Miyoshi for help and use of ssNMR and Mr Thomas
P. Quick for training on the XRD. Thanks to Mr Hendrik Colijn,
Ohio State University, and Dr Min Gao, Kent State University, for
their assistance on TEM. Financial support was provided by
start-up funds from University of Akron and NSF DMR Biomat
grant 0906817 to Nita Sahai. Computational resources were
provided by the Ohio Supercomputer Center grant PBS0286 to
Z. X. and N. S.
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