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Hydroxyapatite formation on graphene
oxide modified with amino acids:
arginine versus glutamic acid
M. Tavafoghi, N. Brodusch, R. Gauvin and M. Cerruti
Research
Cite this article: Tavafoghi M, Brodusch N,
Gauvin R, Cerruti M. 2016 Hydroxyapatite
formation on graphene oxide modified with
amino acids: arginine versus glutamic acid.
J. R. Soc. Interface 13: 20150986.
http://dx.doi.org/10.1098/rsif.2015.0986
Received: 12 November 2015
Accepted: 21 December 2015
Subject Areas:
biomaterials, biomimetics,
biomedical engineering
Keywords:
hydroxyapatite mineralization, amino acids,
graphene oxide, surface modification,
arginine, glutamic acid
Author for correspondence:
M. Cerruti
e-mail: [email protected]
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rsif.2015.0986 or
via http://rsif.royalsocietypublishing.org.
Materials Engineering, McGill University, Montreal, Quebec, Canada H3A 0C5
Hydroxyapatite (HA, Ca5(PO4)3OH) is the main inorganic component of hard
tissues, such as bone and dentine. HA nucleation involves a set of negatively
charged phosphorylated proteins known as non-collagenous proteins (NCPs).
These proteins attract Ca2þ and PO432 ions and increase the local supersaturation to a level required for HA precipitation. Polar and charged amino acids
(AAs) are highly expressed in NCPs, and seem to be responsible for the mineralizing effect of NCPs; however, the individual effect of these AAs on HA
mineralization is still unclear. In this work, we investigate the effect of a negatively charged (Glu) and positively charged (Arg) AA bound to carboxylated
graphene oxide (CGO) on HA mineralization in simulated body fluids (SBF).
Our results show that Arg induces HA precipitation faster and in larger
amounts than Glu. We attribute this to the higher stability of the complexes
formed between Arg and Ca2þ and PO432 ions, and also to the fact that
Arg exposes both carboxyl and amino groups on the surface. These can
electrostatically attract both Ca2þ and PO432 ions, thus increasing local
supersaturation more than Glu, which exposes carboxyl groups only.
1. Introduction
The formation of human bone is one of the most well-known examples of biomineralization. Bone is an organic– inorganic hybrid material made of collagen
and carbonated hydroxyapatite (HA, Ca5(PO4)3OH) crystals. Collagen fibres
provide a framework known as extracellular matrix (ECM) for HA nucleation
and growth. HA nucleation and growth is directed by a set of non-collagenous
proteins (NCPs) associated with the ECM [1,2]. To answer fundamental questions about biomineralization, researchers have investigated the effect of
smaller biomolecules, such as amino acids (AAs) and peptides. AAs are the
building blocks of proteins, and negatively charged AAs such as glutamic
acid (Glu) and phosphoserine (PSer) are highly expressed in NCPs. Similar to
NCPs [1,3], charged AAs can either inhibit or induce HA mineralization if
they are dissolved in solution or bound to a surface.
While several researchers have analysed the inhibitory effect of AAs dissolved
in solution on HA mineralization [4–18], the effect of AAs bound to surfaces
has been the subject of just a few studies [19–25]; in most papers, researchers analysed the effect of molecules with functionalities simulating those found in
protein [22–25]. Rautaray et al. investigated HA precipitation in the presence of
aspartic acid (Asp)-capped gold nanoparticles [19] and showed that HA precipitation was promoted in the presence of Asp due to the interaction between the
COOH groups from Asp and the Ca2þ ions. In addition to their mineralization
activity, charged AAs, such as arginine (Arg), glutamic acid (Glu) or Asp
bound to the surface of HA were also shown to promote protein absorption,
osteoblast proliferation and alkaline phosphatase (ALP) activity [26 –28]. Other
researchers have focused on the effect of surface functional groups with different
electrical charges on HA precipitation. Self-assembled monolayers (SAMs) of
silanes on silicon [22], or of alkanethiols on gold [23] were used to investigate
the effect of positively versus negatively charged surfaces on HA precipitation.
Most of the works show that HA precipitation was faster on negatively charged
& 2016 The Author(s) Published by the Royal Society. All rights reserved.
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Details on the materials and purity used in this study can be
found in the electronic supplementary material, SI. 1.
2.1. Surface modification of graphene oxide flakes
The AAs can be grafted onto the GO surface by coupling the
NH2 group from the AAs with the carboxyl groups that are naturally present on the surface of GO flakes. However, to improve
this process, more carboxyl groups were introduced on the GO
surface using a previously described technique [43] with a few
modifications (electronic supplementary material, SI. 2). The
AAs were then grafted onto carboxylated GO (CGO) using the
2.2. Precipitation experiment
Simulated body fluid (SBF) was prepared according to Kokubo
et al. [44]. 1.9 mg of GO, CGO, CGO-Glu or CGO-Arg were dispersed in 40 ml of SBF in centrifugation tubes. The tubes were
placed in an incubator at 378C for 15 days. The samples were
then removed from the incubator, washed three times with DI
water (three cycles of centrifugation at 4000 r.p.m. for 15 min and
re-suspension in DI water) and dried using a VirTis freeze-drier.
2.3. Characterization
GO-modified samples were characterized by X-ray photoelectron
spectroscopy (XPS), Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), zeta potential, SEM
and TEM. The concentration of Ca and P during the precipitation
experiments was measured by inductively coupled plasma atomic
emission spectroscopy (ICP-AES). Details of all techniques can be
found in the electronic supplementary material, SI. 4.
3. Results
To study the effect of positively and negatively charged AAs
bound to GO, we first transformed GO into carboxylated
GO (CGO), thus increasing the number of carboxylated
groups on its surface. Then, we bound a positive (Arg) and
a negative (Glu) AA on it via an amidation process achieved
with the aid of EDC and NHS. We then immersed the AAmodified CGO in SBF, to study how AAs influence heterogeneous nucleation of HA on CGO.
3.1. Characterization of graphene oxide surface
modification
3.1.1. X-ray photoelectron spectroscopy analysis
Figure 1a shows O, C, S and N atomic % on GO, CGO and
CGO-AA samples obtained from XPS survey spectra. C and
O mainly originate from GO on all samples; however, all
CGO samples show a significant decrease in O and increase
in C compared with GO. This suggests that the amount of
O in the O-containing groups that are removed from GO
(hydroxyl, epoxy and carbonyl groups) is larger than the
amount of O that is introduced by carboxylation. No significant differences in C and O are observed between CGO
samples before and after AA coupling. However, while
CGO does not show any N and S, as expected, CGO-AA
samples show the presence of some N and S. S can originate
from the physisorbed coupling components, NHS and MES,
and N can originate from both the physisorbed coupling
components, EDC, NHS and MES, or from the AAs. The
unexpected S and N observed on the GO sample may be
attributed to the precursors used in Hummer’s method for
GO production [45]; these are removed after the extensive
washing performed during the carboxylation stage, since no
S and N are observed on the CGO sample. A control
sample prepared by immersion of CGO in EDC/NHS solution without further AA coupling showed a similar
amount of S as the CGO-AA samples, but much less N (see
the electronic supplementary material, table S2), thus confirming that while the presence of S on CGO-AA samples is
2
J. R. Soc. Interface 13: 20150986
2. Methods
well-known EDC coupling technique (electronic supplementary
material, SI. 3).
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than on positively charged SAMs, and that among the negatively charged groups, phosphonate groups are the strongest
HA nucleators [23,24]. In one case, though, it was found that
PO4, COOH and NH2 functional groups promoted the nucleation of calcium phosphate to a very similar extent [25].
However, the Ca/P ratio for the calcium phosphate formed
in the presence of PO4 was similar to that of HA (1.67) while
lower Ca/P ratios were found for COOH (1.49) and NH2
(1.60), indicating the presence of some amorphous calcium
phosphate (ACP) on these samples. Interestingly, recent literature discussing the role of collagen on HA mineralization
showed that the AAs present in the collagen sequence close
to the hole zones (i.e. where HA nucleates intrafibrillarly)
include both positive AAs such as Arg and negative ones
such as Glu [29]. The presence of both negatively and positively charged AAs in these areas seems to be crucial for the
retention of Ca2þ and PO4 3 ions which are necessary for
HA nucleation and growth [29,30].
In this work, we try to shed some light on the effect of positive versus negative charges on HA nucleation using a
positively charged (Arg) and a negatively charged (Glu) AA
bound to a substrate made of graphene oxide (GO). Arg and
Glu are present in NCPs involved in bone mineralization [1]
and have been found to play a key role in collagen mineralization [29 –31]. Several other substrates, such as gold
nanoparticles [19], silicon [22] and titanium foil [24] have
been used before to study HA precipitation. We choose GO
as it contains many different surface functional groups,
making it easy to modify [32]. Its high surface area resulting
from its unique two-dimensional structure is ideal to speed
up HA precipitation [32]. In addition, the graphene families
of nanomaterials (GFNs) are able to promote osteogenic
differentiation in mesenchymal stem cells [33] and are potential candidates for bone tissue engineering applications
[32,34,35]. A few papers have started exploring the formation
of HA on these materials [36 –42]. Also, a few researchers have
explored the modification of GFNs with biomolecules and
investigated the effect of such modifications on in vitro HA
mineralization [20,21,36,38,40,42]. All these studies showed
that HA precipitation was improved in the presence of biomolecules. Our work clearly shows that GO modified with a
positively charged AA, Arg, is the best substrate to nucleate
HA in vitro. As discussed in the paper, this result furthers
our fundamental understanding of the effect of positively
and negatively charged AAs bound to surfaces on HA mineralization, while providing an example of a highly promising
GO-based substrate modified with a simple biomolecule for
bone tissue engineering applications.
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80
(O, C) At%
GO
CGO
CGO-Glu
CGO-Arg
*
60
50
40
30
20
4
+
*
3
2
++
*
1
**
0
(b)
C 1s XPS components
60
*
C
S
N
+
* *
50
+
+ *
**
40
30
20
* *
10
*
** *
0
285
O 1s XPS components
(c)
287
288
289
eV
90
75
*
60
45
30
*
*
*
*
*
* *+
*
15
0
531
533
535
eV
Figure 1. XPS quantitative analysis: (a) O, C, S and N atomic % on GO, CGO,
CGO-Glu and CGO-Arg obtained from survey spectra; (b,c) percentages of the
different components used to fit high-resolution XPS C 1s (b) and O 1s
(c) spectra shown in figure 2. (Online version in colour.)
due to physisorbed components from the coupling solution,
the N is mainly related to the presence of AAs on the surface
of the CGO-AA samples. While Arg has three times more N
atoms than Glu in its molecular structure, CGO-Arg shows
only two times more N than CGO-Glu. This may indicate
that less Arg than Glu was presented on the CGO-AA
samples. However, XPS data are affected by the orientation
of the AAs in the coating and by the coating thickness, and
thus this interpretation needs to be corroborated by results
obtained with other techniques. Results obtained by FT-IR,
which also confirm the presence of AAs on the surface, and
zeta potential, are discussed in the electronic supplementary
material, SI. 6 and SI. 7, respectively.
Further confirmation of the presence of AAs can be
obtained by analysing the high resolution C1s and O1s
spectra (figures 1b,c and 2). The C 1s spectra can be deconvoluted into four components, centred at 284.9, 286.9 + 0.2,
288 + 0.05 and 289 + 0.05 eV (figure 2a). On all samples,
the 284.9 eV component originates from C–C/C¼C bonds
from the benzene rings in GO [46,47]. The components at
287, 288 and 289 eV originate from the oxygenated functional
3.1.2. Thermogravimetric analysis
Two main decomposition temperatures are observed for GO
and CGO, and three for CGO-AA samples (figure 3 and
table 1). The weight loss (WL) observed at T1 (approx.
558C) is attributed to the evaporation of water molecules
[53] trapped inside GO. There are no significant differences
in WL% at this temperature among all samples except for
CGO-Arg, which shows somewhat less water content than
the others. While no significant differences in water content
between CGO and GO are found by TGA, a significant
difference was found by XPS (see figure 1c and its discussion); this discrepancy may relate to the fact that the
differences observed by XPS are related to water adsorption
occurring at the surface of the samples, and this amount of
water is not detectable by TGA. The WL at T2 observed on
all samples can be ascribed to the pyrolysis of oxygencontaining functional groups, such as epoxy, carbonyl,
3
J. R. Soc. Interface 13: 20150986
O
groups, namely epoxy/hydroxyl (C–O), carbonyl (C¼O) and
carboxyl (O –C¼O) groups, respectively [46,47]. Figure 1b
shows a drastic decrease in C –O and C¼O bonds and a significant increase in O –C¼O bonds on CGO compared to GO.
This confirms the replacement of epoxy, hydroxyl and carbonyl groups on GO by carboxyl groups during the
carboxylation stage.
Both CGO-Arg and CGO-Glu show a similarly drastic
decrease in the 287 and 288 eV components, and an increase
in the 285 and 289 eV component compared with GO
(figure 1b). However, on these samples, the 287 eV component is significantly more intense than on CGO; this is
related to the fact that on these samples, this component
can also correspond to C– N [48] and C¼N [49] bonds,
which can originate from the AAs present on their surface.
The higher intensity of the 287 eV component on CGO-Arg
compared to CGO-Glu can be explained by the fact that
Arg has more C– N/C¼N bonds than Glu. The peak at
approximately 289 eV (figure 2a) on these samples can also
be assigned to amide groups (N –C¼O) originating from
the coupling between AAs and carboxyl groups during
EDC coupling [50]. The CGO-AA samples do not show significant changes in this component intensity compared with
CGO (figure 1b); this is to be expected, since the amide
bond formed by EDC coupling substitutes for carboxylate
groups already present on CGO.
The changes in C¼O and C–O bonds are also evident
from the high-resolution O 1s spectra shown in figure 2b
(components centred at 531.4 + 0.2 and 533.1 + 0.1 eV,
respectively) [51]. Consistent with the C 1s results, the C–O
component of O 1s is significantly lower for the CGO
and CGO-AA samples in comparison with the GO
sample, while their C¼O component is significantly higher
(figure 1c). This further confirms the replacement of
oxygen-containing functional groups on GO by carboxyl
groups during the carboxylation stage. The small component
at 535 + 0.1 eV observed on all samples (figure 2b) can be
attributed to water molecules [52]. Its higher intensity
(figure 1c) on CGO and CGO-AA samples compared with
GO confirms the presence of more charged functional
groups on these samples, making them more hydrophilic.
CGO showed somewhat higher water content than
CGO-Arg, but no significant differences between CGO and
CGO-Glu were found.
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**
70
5
+
*
(S, N) At%
(a)
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(a)
(b)
C–O
4
C–O
GO
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GO
intensity
C–C
C=O
C=O
O–H in water
O–C=O
CGO
intensity
J. R. Soc. Interface 13: 20150986
CGO
CGO-Glu
CGO-Glu
intensity
C-N
C=N
N–C=O
CGO-Arg
intensity
CGO-Arg
292
290
288
286
binding energy (eV)
284
282
540
538
538
534
532
530
binding energy (eV)
528
526
weight loss (%)
Figure 2. XPS C 1s (a) and O 1s (b) high-resolution spectra for GO, CGO, CGO-Glu and CGO-Arg samples. (Online version in colour.)
100% 49
100% 50
164
100% 56
169
100% 56
161
303
CGO-Arg
CGO-Glu
CGO
GO
304
51%
49%
213
44%
42%
0
150
300
450
600
temperature (°C)
750
900
Figure 3. TGA curves collected under N2 atmosphere on GO, CGO, CGO-Glu
and CGO-Arg samples. The empty circles correspond to the temperatures at
which peaks were observed on the dmass/dT derivative curves. (Online version in colour.)
hydroxyl and carboxyl groups, present on the GO surface or in
the AA structure [53,54]. T2 is lower for CGO (163 + 38C),
CGO-Glu (167 + 28C) and CGO-Arg samples (165 + 18C)
than for GO (206 + 98C). This can be attributed to the higher
concentration of carboxyl groups on CGO and CGO-derived
samples, because carboxyls have lower decomposition temperatures than carbonyl, epoxy or hydroxyl groups [55,56].
The WL at this temperature is significantly higher for GO
(38 + 1%) than on CGO (20 + 1), CGO-Glu (19 + 1) and
CGO-Arg (21 + 0). This once again confirms that there are
more hydroxyls/epoxy/carbonyl groups on GO than carboxylic groups on CGO and CGO-derived samples. The WL
observed at T3 only on CGO-AA samples can be attributed
to the decomposition of CN bonds, either formed during
EDC coupling [57], or present in the AAs, as shown by the presence of similar temperatures on the TGA graphs of Arg and
Glu powders (electronic supplementary material, figure S4).
As the WL observed at T3 is about the same on both
samples (approx. 11%), we can conclude that there are probably fewer Arg molecules on CGO-Arg than Glu molecules
on CGO-Glu because Arg (i) has more CN than Glu; (ii) can
form more amide bonds than Glu and (iii) has a higher
MW than Glu (174.2 and 147.1 g mol21, respectively) [57].
This is in agreement with XPS survey data discussed before
(figure 1a).
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(a)
(b)
** *
SBF
+
*
2.1
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2.4
+
*
0.8
GO
1.8
1.5
1.2
+
*
CGO
+
*
+
*
CGO-Glu
+
*
CGO-Arg
[P] mM
[Ca] (mM)
5
1.0
0.6
+
*
0.4
+
*
+
*
+
*
0.2
HA
0.9
3
8
incubation time (d)
15
0
3
8
incubation time (d)
15
Figure 4. [Ca] (a) and [P] (b) measured by ICP on negative control sample (SBF, grey bars) and in SBF containing GO (solid black), CGO (striped black), CGO-Glu (solid
red), CGO-Arg (striped red) and positive control (HA powder, blue bars) after 0, 3, 8 and 15 days of immersion in SBF. Asterisk and plus signs indicate values that are
statically significantly different from the correspondent values measured on GO and CGO samples, respectively, with p , 0.05. (Online version in colour.)
Table 1. WL temperature (8C) and corresponding weight loss WL% observed on the TGA curves (figure 3) for GO, CGO, CGO-Glu and CGO-Arg samples. Asterisk
and plus signs indicate values that are statically significantly different from the correspondent values measured on GO and CGO samples, respectively, with p ,
0.05.
samples
GO
CGO
CGO-Glu
CGO-Arg
WL (T1)
WL (T2)
5 + 1 (57 + 1)
38 + 1 (206 + 9)
6 + 1 (57 + 2)
20 + 1* (163 + 3)
4 + 1 (53 + 3)
19 + 1* (167 + 2)
3 + 0*,þ (53 + 6)
21 + 0* (165 + 1)
11 + 1*,þ (340 + 15)
11 + 1*,þ (302 + 1)
WL (T3)
0 + 0 (n.a.)
0 + 0 (n.a.)
3.2. Mineralization assay
We immersed all samples in SBF to evaluate the mineralization potential of the modifications introduced on GO. The
initial degree of supersaturation in SBF solution has been previously calculated by several authors [58,59]; for an initial
total [Ca] of 2.5 mM, total [P] of 1 mM and pH of 7.4, at
378C, the initial supersaturation with respect to HA is 1.33
E12, corresponding to a thermodynamic driving force for
HA precipitation (DG) of approximately 28 kJ mol21 [59].
During our experiment, the pH is maintained constant due
to the presence of tris buffer (this was confirmed by pH
measures performed throughout the experiment). Changes
in total [Ca] and [P] measured by ICP are shown in
figure 4. The initial negative DG value implies that HA precipitation is thermodynamically favoured in the starting
SBF. However, the sample labelled as ‘SBF’ in figure 4 (grey
bars), shows that no significant changes in total [Ca] and
[P] were measured in the absence of GO substrates. This
implies that the initial supersaturation degree was low
enough to prevent homogeneous nucleation over the period
of time considered in our experiment. The sample labelled
as ‘HA’ (blue bars) is, instead, a positive control sample, i.e.
HA particles that act as nucleation seeds to promote HA precipitation. The rapid decrease in both [Ca] and [P]
concentrations measured after 3 days of immersion on this
sample clearly shows that the presence of HA seeds strongly
favours heterogeneous nucleation of HA. On the GO and
CGO samples, a decrease in total [Ca] was observed after 3
days of immersion in SBF (although lower than on the positive control, HA sample); and a decrease in total [P] was
detected only after 15 days on the CGO-Arg sample. These
results show that heterogeneous nucleation on GO and
CGO samples happens starting from day 3. Significant
differences related to the presence of AAs are visible after
15 days of incubation, when all CGO-AA samples show significantly lower [Ca] and [P] than both GO and CGO. CGOArg shows the strongest decrease.
The amount of precipitates formed on the samples after
15 days of immersion in SBF was measured by TGA
(figure 5a and table 2). These TGAs are performed in air to
fully burn the organic components and leave only the inorganic component as residues at the end of experiment. The
WL at T1, T2 and T3 correspond to the removal of water molecules, C/O bond destruction and carbon combustion to CO
and CO2, respectively [54]. The amounts of residues in table 2
indicate that CGO-Arg (44 + 4%) contained the highest
amount of precipitate, followed by CGO-Glu (21 + 2%). The
GO (16 + 2%) and CGO (14 + 1%) samples showed comparable amounts of inorganic component as residual mass. The
residual mass (9 + 1%) found on GO not soaked in SBF
(GO-0d) is attributed to contamination.
We analysed the nature of the precipitates by FT-IR (electronic supplementary material, figure S5 and table S4) and
XPS (figure 5b and electronic supplementary material,
table S5) spectroscopy, and X-ray diffraction (XRD; electronic
supplementary material, figure S6). Both FT-IR and XRD can
detect the formation of HA only on CGO-Arg (electronic supplementary material, figures S5 and S6). A more detailed
discussion of these data is found in the electronic supplementary material, SI. Using XPS, a small amount of Ca can be
detected on all samples, but CGO-Arg is the only sample
where both Ca and P are found (figure 5b); the Ca/P ratio is
1.9 + 0.2, i.e. not significantly different from what is expected
for HA (1.67). The higher value may be due to carbonate-tophosphate substitutions in HA [60,61]. No P is found on
samples other than CGO-Arg; this is due to the fact that since
J. R. Soc. Interface 13: 20150986
0
0
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samples
GO 0d
GO 15d
CGO 15d
CGO-Glu 15d
CGO-Arg 15d
WL% (T1)
3.0 + 0.3 (45)
2.0 + 0.2 (53)
2.0 + 0.2 (61)
2.0 + 0.2 (60)
2.0 + 0.2 (60)
WL% (T2)
WL% (T3)
40 + 4 (202)
48 + 5 (516)
27 + 3 (190)
55.0 + 5.5 (455)
18 + 2* (184)
66 + 7 (491)
19 + 2* (182)
57 + 6 (525)
13 + 1*,þ (170)
41 + 4*,þ (482)
residue
9+1
16 + 2
14 + 1
21 + 2*,þ
44 + 4*,þ
weight loss (%)
CGO-Arg 15d
CGO-Glu 15d
CGO 15d
GO 15d
GO 0d
100%
100%
100%
100%
44%
100%
21%
14%
16%
9%
0
150
300
450
600
temperature (°C)
750
900
(b)
5
+
*
At %
4
3
+
*
2
GO
CGO
CGO-Glu
CGO-Arg
* *
1
0
P
Ca
Mg
Figure 5. Analysis of precipitates formed on GO, CGO, CGO-Glu and CGO-Arg
after 15 day immersion in SBF: (a) TGA curves collected in air. GO 0d is a GO
sample not immersed in SBF. The empty circles correspond to the temperatures at which peaks were observed on the dmass/dT derivative curves. (b) P,
Ca and Mg atomic % measured from XPS survey spectra. Complete survey
results are shown in the electronic supplementary material, table S5. Asterisk
and plus signs indicate values that are statically significantly different from
the correspondent values measured on GO and CGO samples, respectively,
with p , 0.05. (Online version in colour.)
the Ca levels are very low on these samples, the P level,
expected to have a similar ratio to Ca than what is found on
CGO-Arg, goes below the XPS detection limit. This confirms
what was previously observed at ICP (figure 4), i.e. a larger
amount of calcium phosphate precipitates on CGO-Arg.
The amount of Ca detected on CGO and CGO-Glu is comparable and significantly higher than on GO, which again is in
line with the observed depletion of Ca in solution measured by
ICP. Some Mg is observed on GO and CGO. Other researchers
noticed the presence of Mg in HA precipitating from SBF
on titanium [62]. Some N is found on GO and CGO after
immersion in SBF (between approx. 1.4 and 1.7%; electronic
supplementary material, table S5). Since almost no N was present on these samples before SBF immersion (figure 1a), this
may indicate the incorporation of tris, used as a buffer in
SBF, in the precipitates formed on these samples.
Back-scattered electron (BSE) images obtained by SEM are
shown in figure 6. Before immersion in SBF, randomly
oriented GO flakes are observed (figure 6a), with no impurities or particles on their surface (figure 6b,c). Similar images
were collected on functionalized GO samples before immersion in SBF (electronic supplementary material, figure S7).
After immersion in SBF for 15 days, some particles are
observed on all samples (figure 6d –q). The largest amount
of precipitation on CGO-Arg is clearly confirmed by the
low-magnification SEM images shown in figure 6a,d,g,j,m.
On GO, CGO and CGO-Glu irregularly shaped aggregates
are found (average size of 1.5 + 1, 1.0 + 0.5 and 2.0 + 1 mm,
respectively). The aggregates consist of spherical particles
with an average diameter of approximately 100 + 20 nm on
GO and CGO (figure 6f,i), and of a more wide variety of
particles on CGO-Glu (figure 6l). On CGO-Arg, instead,
two different sets of particles are observed (figure 6n–q):
many micrometre-sized spherulites of 3.5 + 0.5 mm diameter
made of nano-sized platelets (20 + 3 nm) (figure 6n,o), and a
few spherical particles of 60 + 5 nm diameter (figure 6p,q).
We discuss this in more detail below when we analyse the
TEM results for this sample.
EDS analysis showed the presence of C, N, O, P, Ca and
Mg in all samples. This discrepancy with XPS, which showed
P only on CGO-Arg, is likely related to the lower amount of
sample analysed by XPS, and thus its overall lower sensitivity. A small amount of S was also present on the smaller
particles observed on CGO-AA samples, possibly due to
the S originally present on these samples (figure 1a). The
Ca and Mg content in each sample is normalized to P and
shown in table 3. The Ca/P ratio measured on the micrometre-sized particles on CGO-Arg (figure 6o) is 1.8 + 0.3,
which is very close to that of HA (1.67). This confirms that
these particles are HA, as hypothesized before based on
both XPS and IR (figure 5b and electronic supplementary
material, figure S5). Both we and other researchers previously
reported the formation of micrometre-sized HA spherulites in
the presence of AAs with morphology very similar to that
shown in figure 6 [7,44,63]. The Ca/P ratios measured on
the small spherical particles observed on CGO-Arg
(figure 6q) have large variability (1.7 + 1.2), probably due
to the fact that it was difficult to isolate them during the
analysis. More definite results on these particles are provided
by TEM, discussed below. Much higher Ca/P ratios are
found on GO and CGO (10.1 + 3.3 and 9.8 + 1.2, respectively). These values indicate that on these samples there
is no significant amount of HA, and the high amount of
Ca may be related to physisorbed Ca ions or Ca(OH)2
J. R. Soc. Interface 13: 20150986
(a)
6
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Table 2. WL percentages and temperatures (8C) for GO not immersed in SBF (GO 0d), and for GO, CGO, CGO-Glu and CGO-Arg samples immersed in SBF for 15
days. Asterisk and plus signs indicate values that are statically significantly different from the correspondent values found on GO 15d and CGO 15d, respectively,
with p , 0.05.
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(b)
7
(c)
20 µm
(d )
3 µm
(e)
1 µm
3 µm
20 µm
(g)
(h)
0.5 µm
CGO, 15d
(i)
10 µm
( j)
(l)
CGO-Arg, 15d
(k)
20 µm
(m)
1 µm
3 µm
5 µm
1 µm
(o)
CGO-Arg, 15d
(n)
100 µm
10 µm
(p)
1 µm
CGO-Arg, 15d
(q)
2 µm
0.5 µm
Figure 6. BSE-SEM images of GO sample not immersed in SBF (a – c) and GO (d – f ), CGO (g – i), CGO-Glu ( j – l) and CGO-Arg (m – q) sample immersed in SBF for
15 days. (Online version in colour.)
derivatives [64]. A Ca/P ratio closer to HA is found on CGOGlu (4.0 + 1.8). Similarly to what found by XPS, EDS shows
the presence of significant amounts of Mg only on GO and
CGO (error bars on the Mg/P found on the other samples
make them not statistically significant). Finding Mg only on
the samples that show the highest Ca/P ratios and lowest
amounts of HA may be related to the role of Mg as an
inhibitor of HA crystallization [65].
CGO-Arg immersed in SBF for 15 days was further
analysed by TEM (figure 7). Again, we observed both spherulitic particles with diameters of approximately 3 + 1 mm
(figure 7a) and much smaller nanoparticles with diameters
of approximately 50 + 5 nm (figure 7d). Both types of particles contained mainly Ca and P (see EDS spectra shown
in the insets). Traces of Mg are detected on the larger particles
only, although its absence on the nanoparticle spectrum may
J. R. Soc. Interface 13: 20150986
GO, 15d
(f)
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GO, 0d
(a)
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8
(b)
(a)
(002)
(211)
(112)
(300)
Ca
2 µm
Ca
Cu Mg
0.8 1.4 2.0 2.6 3.2 3.8 4.4 keV
(c)
20 µm
(d )
(002)
(211)
C
O
Cu
200 nm
P
Ca
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
keV
20 µm
Figure 7. TEM images (a,c), and corresponding SAED patterns (b,d), and EDS spectra (insets) for CGO-Arg sample immersed in SBF for 15 days. (Online version
in colour.)
Table 3. Mg/P and Ca/P ratio calculated from the SEM-EDS spectra on GO,
CGO, CGO-Glu and CGO-Arg after 15 day immersion in SBF.
samples
Ca/P
Mg/P
GO
CGO
10.1 + 3.1
9.8 + 1.2
0.64 + 0.1
9.13 + 3.18
CGO-Glu
CGO-Arg (spherulite)
4.0 + 1.8
1.8 + 0.3
0.64 + 0.45
0.04 + 0.02
CGO-Arg (sphere)
1.7 + 1.2
0.14 + 0.17
be due its overall lower intensity. The selected area electron
diffraction (SAED) pattern shown in figure 7b shows that
the spherulitic particles are poorly crystalline and mostly
composed of HA, consistent with IR and XRD results and
previous SEM discussion based on Ca/P ratios. The main diffraction rings observed on the TEM SAED (figure 7b) are in
agreement with the most intense peaks observed by
XRD on this sample (electronic supplementary material,
figure S6d), overall similarly to what was observed by
Zhou et al. [66]. These SAED on the nanoparticles, instead,
show mostly amorphous material; only weak diffraction
rings corresponding to the (004) and (211) reflections are visible. Most probably, then, these smaller particles are mostly
ACP precursors not yet transformed into HA. The presence
of these particles on CGO-Arg may be interpreted in two
different ways. Nucleation may be continuously occurring
on this sample. The ACP nanoparticles are formed first,
and with time they conglomerate and reorganize into the
micrometre-size spherulitic crystalline HA particles observed
in figures 6o and 7a. Throughout SBF immersion, ACP nanoparticles keep nucleating, and thus after 15 days some of
them are still visible. Alternatively, it is possible that some
precipitates remain in the form of ACP nanoparticles and
never transform into HA. A study performed at different
time points will be the subject of a forthcoming publication,
to help elucidate which of these mechanisms is correct, and
to attempt to explain the similar shape but different composition observed for the nanoparticles found on CGO-Arg
and all other samples.
4. Discussion
Overall the results presented before show that both a positive
(Arg) and a negative (Glu) AA increase HA precipitation rate
compared to CGO and GO; however, despite the fact that we
probably had fewer Arg molecules bound on CGO-Arg than
Glu on CGO-Glu, Arg increased the HA precipitation rate
much more than Glu, and much more HA was found on
CGO-Arg than CGO-Glu after 15 days of immersion in SBF.
This result contradicts many previous studies, which
showed that the interaction between Ca2þ and negatively
charged residues of biomolecules, such as carboxylate and
J. R. Soc. Interface 13: 20150986
P
CO
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(004)
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(b)
9
HO
Ca
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O
O
C
HN
O
Ca
O
HO
(a)
HO
Ca
OH
O
HO Ca
NH2
O
O
C
HO
O
Ca
C OH
J. R. Soc. Interface 13: 20150986
HN
Glu
O Ca
HO Ca
O
O
C
HN
O
O Ca
HO
Ca
C OH
(c)
Ca
O
HO
O
O
N
H
C
C OH
P
NH
HN
carboxylated GO
H2N
NH2
H
N
P
NH2
OH
NH
O
(d)
O
Ca
C
Arg
HN
H
N
OH
Ca
O
NH
(e)
P
NH2
O
C
O
NH
NH
HN
HN
C
OH
O
Ca
Figure 8. Schematic showing possible interactions of Glu and Arg with CGO surface and Ca2þ (circle) and PO4 3 (triangle) ions. The ions are shown in red or blue if
they are interacting with the AAs by forming complexes or electrostatically, respectively. (Online version in colour.)
phosphorylated groups play a key role in HA precipitation
[20,22 –24,38,40]. In this section, we first attempt to provide
an explanation for this result, and then we compare the effectiveness of CGO-Arg on HA precipitation with previously
reported GFN substrates.
4.1. Effect of Arg versus Glu bound to carboxylated
graphene oxide on hydroxyapatite precipitation
Precipitation happens when the concentration of precursor
ions in solution increases above a critical level. This can be
achieved locally on a surface, if there are some functionalities
that can strongly interact with the ions. Our results show that
both Arg and Glu are able to locally increase Ca2þ and PO4 3
surface concentration, and induce early nucleation of HA
compared to what is observed on both GO and CGO. As
we had somewhat fewer Arg molecules on CGO-Arg than
Glu on CGO-Glu, Arg must be able to interact with the
ions more strongly than Glu. Figure 8 shows a few possible
ways in which Arg and Glu may bind to CGO, and the subsequent interactions between the remaining functional
groups exposed by the AAs and Ca2þ and PO4 3 ions in solution. With the help of this schematic, we discuss here two
reasons why Arg is a more effective HA nucleator than Glu.
As shown in figure 8b, after binding to CGO, Glu exposes
two carboxylate groups, one alpha and one gamma, which
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In this section, we compare our precipitation results with the
few previous studies on in vitro precipitation of HA on GFNs
[20,38,40]. In general, GFNs are good HA nucleators due to
their high surface area, and the presence of biomolecules
always increase the amount and rate of HA precipitation
[20,21,38,40,42].
Liu et al. [20,38] showed the formation of HA on reduced
GO (rGO) modified with a layer of in situ polymerized dopamine ( polydopamine, PDA) after one week and gelatinmodified GO after two weeks of immersion in 1.5 SBF.
On rGO-PDA, a layer of HA nanoparticles was formed, in
an amount corresponding to approximately 50% in weight
of the composite, as evaluated by TGA [20]. This result is
comparable with what we observed after two weeks of
immersion in SBF (figure 5a); however, we used 1.5 less
5. Conclusion
We studied the effect of a positively (Arg) and a negatively
(Glu) charged AA bound to GO flakes on the precipitation
of HA at physiological conditions. Both AAs were bound to
GO after transforming oxygenated GO functionalities in carboxyl groups (CGO). We showed that while both AAs
increased the HA precipitation rate on CGO compared with
GO and CGO, Arg increased the GO mineralization rate
much more than Glu, giving rise to larger amounts of HA
precipitates, including both micrometre-sized spherulitic
aggregates and smaller nanoparticles. We explained these
results based on two factors: Arg can form more stable complexes with Ca2þ and PO4 3 ions than Glu; and, the presence
of both carboxyl and amino groups exposed to the solution
on CGO-Arg may favour local supersaturation with respect
to HA by electrostatically attracting both Ca2þ and PO4 3
ions. This result may not be generalized to all positive and
negative functionalities; for example, groups able to form
even stronger complexes with either ion, such as phosphonate groups, which can attract Ca2þ ions very strongly, may
change this balance. Indeed, when comparing our results
with previous work studying the mineralization of surfacemodified rGO, we showed that the amount of HA deposited
on CGO-Arg was much lower than that observed in the
presence of heavily phosphonated peptides like CPP.
Overall, this report provides new insights into the effect of
single AAs bound to surfaces on HA mineralization in physiological conditions, and some guidelines on how to improve
the mineralization of GO for bone regeneration applications.
If our insight on the importance of both complex formation
and the presence of positive and negative groups to increase
local supersaturation is correct, future attempts to modify
10
J. R. Soc. Interface 13: 20150986
4.2. Arg-carboxylated graphene oxide as a strong
nucleator of hydroxyapatite
concentrated SBF than Liu et al. Thus, we conclude that Arg
and PDA are similarly effective in promoting HA nucleation,
despite their large differences in size. On rGO-gelatin, Liu
et al. show spherulitic particles similar to what we found on
CGO-Arg [38]. SEM images show a more extensive coverage
on this sample after two weeks of immersion in 1.5 SBF
than what we observed, but no quantitative TGA results
are reported.
Fan et al. showed that the surface of casein phosphopeptide (CPP)-modified CGO was partially covered with
HA nanoparticles after only 1 day incubation in 1.5 SBF
and was completely covered with HA after 3 days, corresponding to an HA content of approximately 80 wt% by
TGA [40]. This amount of precipitation achieved in such a
short time is significantly larger than what we observed
after 15 days (figure 5a), and thus cannot be solely related
to the higher concentration of SBF used in [40]. Evidently,
the high concentration of phosphonate groups on CPP
makes these peptides much stronger HA nucleators than Arg.
Overall, this comparison shows that although a small,
positively charged molecule like Arg cannot compete with
highly phosphonated biomolecules, its effectiveness at promoting HA nucleation once bound to GO or CGO is not
drastically different from that of large, negatively charged
biomolecules such as PDA or gelatin. This confirms the
importance of the large local supersaturation achieved in
the presence of Arg, due to both complex formation and
electrostatic reasons, as discussed in the previous section.
rsif.royalsocietypublishing.org
can interact with the ions present in solution. While Ca2þ
ions can interact with these groups (stability constants of
log K ¼ 1.4 [67] and 1.7 [67] are reported for Glu/Ca2þ complexes at pH 7.4), PO4 3 ions do not have strong interactions
with carboxylate groups [63]. Therefore, Glu/PO4 3 complexes are not shown in figure 8. Arg can bind to CGO
through both its a-amino (figure 8c) and guanidyl group
(figure 8d), which implies that it exposes its a-carboxyl
group and either its guanidyl group (figure 8c) or a-amino
group to the solution (figure 8d). Arg can also form more
than one amide bond to CGO (figure 8e shows an example
of this). At pH 7.4, Arg can interact with Ca2þ ions through
its a-amino group, forming complexes whose stability constant (log K ) is 2.21 [68]. Arg can interact with PO4 3 ions
through its guanidyl side chain, forming a complex with a
reported stability constant of log K ¼ 1.9 [69]. The stability constants of complexes formed between Arg and both Ca2þ and
PO4 3 are higher than between Glu and Ca2þ. Thus, the
faster precipitation of HA on CGO-Arg than on CGO-Glu
may be partially explained by the stronger Arg/Ca2þ and
Arg/PO4 3 interactions leading to more stable complexes
formed on CGO-Arg, which result in a higher concentration
of Ca2þ and PO4 3 ions on the CGO-Arg surface.
In addition to complex formation, electrostatic interactions between surface functional groups and Ca2þ and
PO4 3 ions could play a role in HA precipitation. As shown
by zeta potential measures (electronic supplementary material,
figure S3), all surfaces are overall negatively charged. However, locally, CGO-Arg exposes both positive and negative
charges (figure 8c,d), whereas CGO-Glu exposes only negative charges (figure 8b). This implies that locally, CGO-Arg
is more likely to attract both Ca2þ and PO4 3 ions than
CGO-Glu, which is likely to attract only Ca2þ ions. This
factor may contribute to a higher increase in local supersaturation with respect to HA for CGO-Arg than the
CGO-Glu sample, and thus a faster precipitation of HA on
CGO-Arg. This hypothesis is confirmed by the EDS
(table 3) and XPS (figure 5b) results, which showed higher
Ca/P ratios on CGO-Glu than on CGO-Arg, thus confirming
the greater tendency for CGO-Glu to attract positively
charged ions than CGO-Arg. Thus, the presence of both positively and negatively charged groups close to each other on
CGO-Arg may be another justification for the stronger HAnucleating effect of this sample compared with CGO-Glu.
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Authors’ contributions. As the first author of the paper, M.T. designed and
Competing interests. We declare we have no competing interests.
Funding. This research project is supported by the McGill Engineering
Doctoral Award, the Canada Research Chair foundation, the Natural
Science and Engineering Research Council of Canada, the Canada
Foundation for Innovation, the Center for Self-Assembled Chemical
Structures and the Fonds Quebecois de la Recherche sur la Nature et les
Technologies.
Acknowledgement. We thank Mr Gul Zeb for his help with drawing the
schematic in figure 8.
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