Freeze-drying synthesis, characterization and in vitro bioactivity of chitosan/graphene oxide/hydroxyapatite nanocomposite

Article abstract of DOI:10.1039/c4ra03534h

Polycrystalline and dispersed hydroxyapatite (HAP) nanoparticles were successfully prepared via a simple precipitation method with the aid of a new capping agent based on Schiff base compounds. After that, a composite of chitosan (CT), graphene oxide (GO)

Full text of DOI:10.1039/c4ra03534h

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Freeze-drying synthesis, characterization and
in vitro bioactivity of chitosan/graphene oxide/
Cite this: RSC Adv., 2014, 4, 25993
hydroxyapatite nanocomposite
a
ab
F. Mohandes and M. Salavati-Niasari*
Polycrystalline and dispersed hydroxyapatite (HAP) nanoparticles were successfully prepared via a simple
precipitation method with the aid of a new capping agent based on Schiff base compounds. After that, a
composite of chitosan (CT), graphene oxide (GO) and HAP nanoparticles was synthesized by a freeze-
drying method. The as-produced Schiff base, HAP nanoparticles, and CT/GO/HAP nanocomposite were
analysed by several techniques including FTIR, SEM, HR-TEM, and XRD. In addition, the in vitro bioactivity
of HAP nanoparticles and the CT/GO/HAP nanocomposite was evaluated by soaking them in simulated
body fluid (SBF). By monitoring the changes of chemical composition of the SBF solutions, it was
concluded that more Ca and P ions were released from the CT/GO/HAP nanocomposite compared to
the pure HAP nanoparticles, indicating a high bioactivity of the nanocomposite. The SEM images showed
that the formation and growth of apatite on the surfaces of the products increased after immersion for
14 days in SBF.
Received 18th April 2014
Accepted 30th May 2014
DOI: 10.1039/c4ra03534h
www.rsc.org/advances
mainly produced by the partially deacetylation of chitin that can
be extracted from crustacea. It is a suitable substrate for bio-
Introduction
Hydroxyapatite (Ca10(PO
4
)
6
(OH)
2
, HAP), which belongs to the ceramics due to its biocompatibility, biodegradability, and
12–14
class of bioceramics, has been widely used in tissue engi- nontoxicity.
In addition, it has a high resistance upon
neering, orthopedics, drug and gene delivery systems, and heating due to the intramolecular hydrogen bonds formed
15
protein separation due to its excellent biocompatibility and between hydroxyl and amino groups. Up to now, some
bioactivity with bone tissues, biodegradation and osteo- improved tricomponent systems for tissue engineering scaf-
1
–3
16
conductivity. Despite these advantages, the application of folds including alginate and collagen,
18
halloysite clay
17
HAP for load bearing implants is restricted because of the (Al
peculiar brittleness and low fracture toughness of HAP.
2
Si
2
O
5
(OH)
4
$nH
2
O; n ¼ 0–2), and gelatin with HAP/chito-
4
san have been reported. As we know an ideal scaffold should not
Generally, there are two strategies to improve the mechanical only have biocompatibility and mechanical properties match-
properties of HAP. In the rst way, HAP powders can be coated able to those of natural bone, but also capabilities for response
5,6
on a metallic implant surface, and in the second way, HAP to specic biological signals expressing and promoting cell
powders can be combined with different polymers. Several attachment, proliferation, differentiation and nally tissue
19
authors have addressed the use of biodegradable polymers regeneration. Due to good electric conductivity, excellent
7
8
including polylactic acid (PLA), polyvinyl alcohol (PVA),
biocompatibility, strong mechanical strength, light density and
poly- high stability, carbon materials in the form of nanotubes
methylmethacrylate (PMMA) was developed to prepare HAP (CNTs), graphite and graphene (GR) sheets are promising
9
10
collagen,
polyethylene
glycol
(PEG)
and
11
nanocomposites.
reinforcing agents for polymeric matrix in bone tissue engi-
20–22
So far, a great attention has focused on the composites of neering.
In the structure of graphene nanosheets, a single
nano-sized HAP and chitosan (CT). CT is one of the most layer of sp2-hybridized carbon atoms are packed together into a
23
abundant polysaccharides consisting of b-(1-4)-2-deoxy-2- honeycomb two-dimensional (2D) lattice. Compared to pris-
amino-D-glucopyranose units and b-(1-4)-2-deoxy-2-acetamino- tine graphene, graphene oxide (GO) is highly hydrophilic due to
D-glucopyranose units liked through 1-4 glycosidic bonds. CT is the presence of COOH and OH groups on the GO surface,
yielding stable dispersion in water. Li et al. showed that the
surface charges on GO are highly negative when dispersed in
a
Department of Inorganic Chemistry, Faculty of Chemistry, University of Kashan, P. O.
water by measuring the zeta potential due to the ionization of
Box. 87317-51167, Kashan, I. R. Iran. E-mail: salavati@kashanu.ac.ir; Fax: +98 361
24
the carboxylic acid and the phenolic hydroxyl groups. There-
fore, the formation of stable GO colloids in water was attributed
to not only its hydrophilicity but also the electrostatic repulsion.
5
552935; Tel: +98 361 5912383
b
Institute of Nano Science and Nano Technology, University of Kashan, Kashan, P. O.
Box. 87317-51167, I. R. Iran
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2
5
Since graphene induces no obvious toxic effects in vivo,
propylendiamine in 50 mL of methanol. The mixture was
ꢁ
functionalized GO with dopamine has been applied for reuxed and heated at 70 C for 3 h. Aer evaporating the
hydroxyapatite mineralization. On the other hand, the solvent, crystallization was induced by adding petrol ether.
26
biocompatibility and applications of GO/HAP/PLA (polylactic Aerward, the crystals obtained were ltered on Buchner fun-
2
7
28
3 4
acid) nanocomposites, GO–Fe O nanoparticle, and GO– nel. Pore diameter of the Whatman lter papers applied was
29
polyethylenimine hybrid materials as drug and gene carriers 0.45 mm.
were studied.
Freeze-drying has been shown to be a promising technique
Synthesis of HAP nanoparticles
for preparing uniform ne-grained powders which can be sin-
HAP nanoparticles were prepared by reaction between calcium–
Schiff base solution and ammonium hydrogen phosphate
solution. In the experiment, the molar ratios of Ca/Schiff base
and Ca/P were 1.0 and 1.67, respectively. Details of the synthesis
process are described. At rst, 10 mmol of Schiff base was dis-
30,31
tered to very high densities.
This method has been
successful in obtaining homogeneous, ne-grained and highly
31,32
reactive Al O and Fe O powders.
In this work, subsequent
2
3
2 3
freeze-drying was utilized to prepare HAp powders. The
obtained HAp powders were then compacted and sintered to
obtain the Hap ceramics.
By considering the hydrophilicity of GO, hydrophilic
2
biopolymers including NH and OH groups such as CT can be
ꢁ
solved in 40 mL of methanol at 40 C under a constant stirring
of 1000 rpm. When Schiff base was completely dissolved in
methanol, 10 mmol of Ca(NO ) $4H O was added into the
3
2
2
solution. Aer that, additional stirring was performed for 30
min, and nally the yellow clear calcium–Schiff base solution
was obtained. To prepare the ammonium hydrogen phosphate
protonated to polycationic materials in acid media, which is
favor of the interaction between polymer chains and GO sheets.
33
Therefore, a good dispersion of GO in CT solution is expected.
solution, 6 mmol of (NH
distilled water under magnetic stirring. A sodium hydroxide
NaOH) solution (0.1 M) was added dropwise into the ammo-
4 2 4
) HPO was dissolved in 40 mL of
It should be mentioned that the graphene/chitosan composites
show a 200 times increase in the elasticity modulus compared
(
34
to pure chitosan. Keeping the as-mentioned points in view,
present study was aimed to prepare a CT-functionalized GO/
HAP nanocomposite scaffolds by freeze-drying method. A
detailed structural and morphological characterization, as well
as in vitro bioactivity of the nanocomposites were performed.
The obtained results showed that the CT/GO/HAP nano-
composites have a much higher bioactivity rather than that of
the pure HAP nanoparticles.
nium hydrogen phosphate solution to adjust the pH of the
solution to 12. At this time, the ammonium hydrogen phos-
ꢀ
1
phate solution was added slowly at a rate of 5 mL min into the
calcium–Schiff base solution. During the addition of the
ammonium hydrogen phosphate solution into the calcium–
Schiff base solution, a milky suspension was obtained. A round-
bottom ask loaded with the resulting mixture was kept in an
ꢁ
oil bath. The reaction mixture was heated at 120 C for 5 h. Aer
cooling the reaction mixture to room temperature, the nal
precipitates were collected by centrifugation at 4500 rpm for 5
min, washed three times with distilled water, methanol, and
chloroform, sequentially. The white precipitates obtained were
dried at 50 C for 5 h. For comparison, the synthesis was carried
out under the same preparation conditions without using any
Experimental
Materials
All the chemicals with analytical grade were used as received.
Salicylaldehyde, 2,2-dimethyl-propylendiamine, and methanol
with analytical grade were used for the synthesis of Schiff base.
ꢁ
Schiff base. This experiment is named as blank test.
Calcium nitrate tetrahydrate (Ca(NO
3 2 2
) $4H O) and diammonium
hydrogen phosphate ((NH HPO ) as calcium and phosphate
4
)
2
4
Synthesis of GO nanosheets
sources were applied. In the synthesis method of HAP nano-
particles, NaOH was used for adjusting pH. Chitosan powder was
purchased from Acros Organics (CT, degree of deacetylation ¼
According to the previous work, GO was prepared by using
35
graphite powder. In this method, the improved Hummer
method was applied to oxidize graphite for the synthesis of
ꢀ
1
8
2%, M
w
¼ 100 000–300 000 g mol ). To prepare CT solution,
36
GO. At rst, 3 g of graphite powder, 18 mL of HNO
of H SO
3
, and 46 mL
acetic acid (100%, glacial) from Merck Company was used. To
synthesize graphene oxide (GO), graphite powder, sulfuric acid
ꢁ
2
4
were mixed and strongly stirred in the range of 0–5 C
for 15 min in a 500 mL reaction ask immersed in an ice-water
bath. Then 6 g of KMnO4 was added slowly into the above
solution within 15 min. Aer this, the suspended solution was
stirred continuously for 2 h in an ice-water bath and maintained
(
98%), nitric acid (67–70%), potassium permanganate (KMnO
4
)
and H (30%) were commercially available and employed
without further purication. To prepare the SBF solution, NaCl,
NaHCO , KCl, K HPO $3H O, MgCl $6H O, CaCl , Na SO ,
tris(hydroxymethyl)aminomethane ((CH OH) CNH ) and HCl
with analytical or reagent grades were purchased from Merck
Company.
2 2
O
3
2
4
2
2
2
2
2
4
ꢁ
the temperature in the range of 10–15 C, and then the sus-
2
3
2
ꢁ
pended solution was stirred continuously at 35 C for half an
hour. Subsequently, 138 mL of distilled water was a slowly
added into the suspension within 10 min, and then the
ꢁ
temperature was kept in the range of 95–98 C for 30 min.
Synthesis of Schiff base
Immediately, the suspension was diluted by 200 mL of warm
ꢁ
In this method, Schiff base was prepared by reaction between 2 distilled water (40 C) and treated with 18 mL of H
2
O
2
(30%) to
mol of salicylaldehyde and
1
mol of 2,2-dimethyl- reduce residual permanganate to soluble manganese ions.
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ꢁ
ꢀ1
Finally, the resulting suspension was ltered, washed with under air atmosphere at a heating rate of 10 C min from
ꢁ
ꢁ
distilled water and dried in a vacuum oven at 60 C for 24 h to room temperature to 600 C. Chemical analysis of the SBF
obtain GO.
solutions were determined by inductively coupled plasma-
optical emission spectrometry (ICP-OES; Liberty 200, Varian,
Clayton South, Australia).
Synthesis of CT/GO/HAP nanocomposite
Since natural bone contains 70% HAP (inorganic phase) and
ꢂ30% collagen as connective protein, 70% of HAP, 29.9–30% of
CT and 0.1% of GO was used as content of nanocomposite
scaffold. CT/GO/HAP nanocomposites was synthesized by mix-
ing three aqueous solutions. The preparation stages of the
solutions are as follows: CT powder (1.50 g) was dissolved in 2%
In vitro bioactivity tests
Bioactivity of the as-produced HAP nanoparticles and CT/GO/
HAP nanocomposites were evaluated in vitro by soaking in the
SBF solution reported by Kokubo and Takadama.
solution was buffered at pH 7.40 by 50 mM tris(hydroxymethyl)
3
7–39
The SBF
aminomethane ((CH
2
OH)
3
CNH
2
) and 45 mM HCl solution at
(v/v) aqueous acetic acid solution, and then the solution was
ꢁ
37 C. In the bioactivity tests, 0.3 g of the powders were pressed
stirred for 24 h to achieve a homogeneous suspension (solution
I). This viscous solution was sonicated for 15 min to remove the
air bubbles trapped in it. The as-synthesized GO powder
into a pellet with thickness about 3 mm and diameter about
5 mm. The pellets washed, and dried were studied aer
1
soaking for 3, 7, 14, and 28 days. Besides, the concentrations of
Ca, and P of the SBF solutions were analyzed by ICP
spectroscopy.
(
0.025 g) was dispersed in 50 mL DI water by sonication for 30
min (solution II). The as-synthesized HAP nanoparticles (3.0 g)
was dispersed in 50 mL DI water by sonication for 30 min
(solution III). By mixing the solutions I, II and III, a gray
suspension was obtained, which was stirred for 24 h at room Results and discussion
ꢁ
temperature (25 C). The gray suspension was separated by
In this work, a new capping agent based on Schiff base
compounds was synthesized and characterized by FTIR and H-
NMR spectroscopy. Fig. 1a shows chemical structure and
centrifuging the suspension at 4500 rpm for 45 min, and dried
1
ꢁ
in a freeze-dryer at ꢀ45 C for 12 h.
starting reagents of the Schiff base used as capping agent. The
FTIR and H-NMR spectra of the Schiff base are seen in Fig. 1b
Characterization
1
Fourier transform infrared (FTIR) spectra of the as-synthesized
Schiff base, HAP nanoparticles and nanocomposites before
soaking were obtained on a Magna-IR, spectrometer 550 Nicolet
and c, respectively. The FTIR spectrum exhibits a strong peak
ꢀ1
corresponding to the v(C$N) at 1630 cm , which proves the
40
1
formation of Schiff base. Details of H-NMR spectrum of the
Schiff base are as follows: (CDCl as solvent): 1.084 ppm (s, 6H, 2
), 6.895 ppm (t, 2H aromatic),
ꢀ
1
in KBr pellets in the range of 400–4000 cm . In addition, the
pellet forms soaked in the SBF solution were characterized by
diffuse reectance FTIR spectrometer (ATR-FTIR, Tensor 27,
BRUKER) equipped with ATR system (reection mode) in the
3
ꢃ CH
3
), 3.498 ppm (s, 4H, 2 ꢃ CH
2
6
.979 ppm (d, 2H aromatic), 7.271 ppm (d, 2H aromatic),
ꢀ
1
range of 600–4000 cm . Proton nuclear magnetic resonance
1
(
H-NMR) spectrum was obtained on a BRUKER (400 MHz)
spectrometer. Proton chemical shis are reported in ppm
relative to an internal standard of Me Si. Powder X-ray diffrac-
4
tion (XRD) patterns were collected from a diffractometer of
Philips Company with X'PertPro monochromatized Cu Ka
˚
radiation (l ¼ 1.54 A, operated on 35 mA and 40 kV current).
ꢁ
ꢁ
The diffraction angles (2q) were scanned from 10 to 80 . SEM
micrographs were taken by using a eld-emission scanning
electron microscope (HITACHI S4160, Japan). Energy dispersive
spectrometry (EDS) analyses were studied by XL30, Philips
microscope. Transmission electron images (TEM and HR-TEM)
of HAP nanoparticles were recorded on a JEM-2100 with an
accelerating voltage of 200 kV equipped with a high resolution
CCD Camera. TEM images of CT/GO/HAP nanocomposite were
obtained on a Philips EM208S transmission electron micro-
scope with an accelerating voltage of 100 kV. In order to
disperse GO powders in water, ultrasonic device was used. The
dispersion was carried out at atmospheric pressure and
ambient temperature with a multiwave ultrasonic generator
(Sonicator 3000; Bandeline, MS 72, Germany), equipped with a
converter/transducer and titanium oscillator (horn), 12.5 mm in
diameter, operating at 20 kHz. Thermogravimetric analyses
1
Fig. 1 (a) Chemical structure, (b) FTIR and (c) H-NMR spectra of Schiff
(
TGA/DTG) were carried out with Pyris Diamond Perkin Elmer base.
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1
.328 ppm (t, 2H aromatic), 8.346 ppm (s, 2H, 2 ꢃ HC$N),
3.602 ppm (s, 2H, 2 ꢃ OH). No characteristic peaks of impu-
rities like salicylaldehyde and 2,2-dimethyl-propylendiamine
used as starting reagents are observed in the FTIR and H-NMR
1
spectra, indicating high purity of the as-synthesized Schiff base.
SEM image and EDS spectrum of HAP synthesized by Schiff
base are seen in Fig. 2a and b, respectively. Morphology of HAP
is composed of homogeneous nanoparticles. The EDS analysis
clearly shows the presence of Ca, P and O elements in the
product. The carbon signal comes from the EDS grid. Fig. 3a
and b depict TEM and HR-TEM images of HAP nanoparticles.
TEM observations show that the morphology of HAP crystals is
nearly ellipsoid-like with particle sizes between 15-25 nm. The
diffraction rings in the SAED pattern inserted in Fig. 3a indi-
cates the polycrystalline nature of HAP nanoparticles. Moreover,
the HR-TEM images show the clear (222) lattice fringes with the
interplanar spacing of 0.194 nm. The typical TEM image of the
product obtained from the blank test is seen in Fig. 3c. As
shown in this gure, the poor crystalline particles are highly
agglomerated, and it is difficult to measure the individual
particle sizes. Therefore, it is found that the use of capping
agent is a key factor to form dispersed and crystalline HAP
nanoparticles.
Fig. 4 shows SEM images of GO nanosheets. The fully exfoli-
ated GO sheets are different from graphitic platelets or pristine
graphene sheets because of their low electrical conductivity due
to presence of several oxygen containing groups on the surface of
GO. Unlike graphene, GO sheets are expected to be “thicker” due
to the presence of carbonyl, carboxyle, hydroxyle and epoxy
35
groups above and below the original graphene planes.
Fig. 5a–c display low and high magnication SEM images of
CT/GO/HAP nanocomposite. The nanocomposite is highly
porous with good interconnectivity, which is a favorable
Fig. 3 (a) TEM and (b) HR-TEM images of HAP nanoparticles synthe-
sized by Schiff base (SAED pattern is in the inset), (c) TEM image of HAP
synthesized without using any Schiff base.
40
scaffold for cell attachment and new-bone formation. The
pore sizes of nanocomposite measured by SEM are between 20–
50 mm. At higher magnication (Fig. 5c), the effective deposition
and distribution of HAP nanoparticles onto CT-functionalized
GO nanosheets can be clearly observed. The EDS spectrum of
CT/GO/HAP nanocomposite in Fig. 5d shows the presence of Ca,
P, O and C elements in the chemical composition of nano-
composite. To obtain further insight into the details of nano-
composite, TEM micrographs were taken and presented in
Fig. 6. These images revealed appreciable aggregation of HAP
nanoparticles on the surface of CT-functionalized GO nano-
sheets. The aggregation of HAP nanoparticles may be due to the
electrically charged nature of chitosan network.
Thermal properties of GO nanosheets and CT/GO/HAP
nanocomposite were investigated by TGA, and the TGA/DTG
proles are shown in Fig. 7. The weight loss of GO starts upon
ꢁ
heating below 100 C, which is attributed to the removal of
water molecules adsorbed on the surface of GO (Fig. 7a).
Moreover, there are two drops in weight around 150 and 250 C.
Fig. 2 (a) SEM image and (b) EDS pattern of HAP nanoparticles
synthesized by Schiff base.
ꢁ
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Fig. 4 (a and b) SEM images of GO nanosheets.
2
The former can be related to the evolution of CO and CO from
GO caused by the destruction of functional groups including
oxygen atoms, and the latter is assigned to the combustion of
41
the carbon skeleton of GO. The TGA/DTG curves of CT/GO/
HAP nanocomposite are seen in Fig. 7b. The initial drop in
ꢁ
weight below 100 C is attributed to the loss of adsorbed water.
ꢁ
The signicant weight loss around 310 C can be due to the
41,42
thermal decomposition of chitosan and combustion of GO.
It is noted that no notable occurrence of weight loss has been
found for HAP nanoparticles, and they are very stable even at
high temperature.
Fig.
5
(a–c) SEM images and (d) EDS pattern of CT/GO/HAP
12
nanocomposite.
The phase structure of the product was investigated by XRD.
Fig. 8 displays the XRD patterns of GO nanosheets, pure CT,
HAP nanoparticles and CT/GO/HAP nanocomposite before and appeared in the XRD pattern of nanocomposite, which may be
aer soaking in SBF. The reection peaks in the XRD pattern of attributed to the GO-graed-CT network. When the –COOH
GO can be attributed to hexagonal phase carbon with space groups of GO react with the –NH groups of CT to form amide
2
˚
group of P6
˚
3
/mmc and cell constants of a ¼ 2.4640 A, b ¼ 2.4640 groups (–CONH–), the gra of GO and CT occurs. The XRD
˚
A, and c ¼ 6.7360 A (JCPDS no. 08-0415). This pattern includes patterns of pure HAP and CT/GO/HAP nanocomposites aer
ꢁ
the characteristic reection close to 12.5 and those observed at soaking for 14 days indicate the presence of hydroxyapatite. The
ꢁ
ꢁ
ꢁ
43
approximately 16.6 , 26.5 and 42.9 , the strong and sharp low crystallinity of the new-formed hydroxyapatite crystals are
ꢁ
˚
peak at 2q ¼ 11.7 corresponds to an interlayer distance of 7.6 A responsible for the broadness of their diffraction peaks in the
(
d001). The XRD pattern of pure CT shows the characteristic XRD pattern of HAP nanoparticles and CT/GO/HAP nano-
ꢁ
44
peak of chitosan with maximum intensity at 2q ¼ 20.4 . All the composite aer soaking in SBF.
diffraction peaks in the XRD pattern of HAP nanoparticles are
FTIR spectroscopy was employed to study the surface of GO,
well-matched to hexagonal phase hydroxyapatite with space CT, HAP nanoparticles and CT/GO/HAP nanocomposite before
˚
group of P63/m and cell constants a ¼ b ¼ 9.4320 A, and c ¼ and aer soaking in SBF. The spectrum of GO (Fig. 9a) shows an
ꢀ
1
˚
6
.8810 A, the apatite peaks were seen to occur at 2q of 24.38, intense peak at 3437 cm due to the OH stretching vibration of
3
1.64, 33.52, 36.12, 41.40, 54.81, 65.04 and 72.73, which are absorbed water molecules and structural OH groups. Two
ꢀ
1
consistent with the standard XRD peaks for apatite (JCPDS-24- adsorption peaks at 2922 and 2855 cm are related to the
033). By comparing the XRD patterns of CT/GO/HAP nano- asymmetric and symmetric stretching of C–H aliphatic. Also,
0
ꢀ
1
composite, pure CT, GO, and HAP, it can be seen that the this spectrum shows the presence of v(C–O) at 1060 cm , v(C–
characteristic peaks of HAP at 25.9 and 32.0 are available in O–C) at 1120 cm , v(C–OH) at 1380 cm , v(C$O) of carboxylic
the XRD pattern of nanocomposite. In addition, the character- acid and carbonyl moieties at 1625 cm . The adsorption peak
istic peaks of CT and GO disappeared and a new peak at 29.6
ꢁ
ꢁ
ꢀ1
ꢀ1
ꢀ1
ꢁ
ꢀ1
at 1581 cm may be attributed to the skeletal vibrations of
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Fig. 6 (a and b) TEM images of CT/GO/HAP nanocomposite.
Fig. 8 XRD patterns of the products before and after soaking in SBF.
Fig. 7 TGA/DTG curves of (a) GO nanosheets and (b) CT/GO/HAP
nanocomposite.
ꢀ
1
1
030 cm are visible. The characteristic adsorption peaks of
ꢀ1
chitosan observed at 1637, 1593 and 1322 cm are assigned to
the amide I (C$O), amide II (–NH), and amino (NH ). Fig. 9c
2
35
unoxidized graphitic domains. In the FTIR spectrum of CT
Fig. 9b), the stretching vibrations of v(NH ) at 3427 and 1423
cm , v(C–H) at 2878 and 1380 cm , v(O–C–O) at 1083 and
40
(
2
shows the FTIR of HAP nanoparticles. In this spectrum, the
ꢀ
1
ꢀ1
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during the dissolving and stirring processes. This evidence may
improve the biological activity of the carbonated HAP, which is
46
essential for bonding with natural bone. FTIR spectrum of CT/
GO/HAP before soaking in SBF includes the characteristic peaks
of all the raw materials like GO, chitosan and HAP (Fig. 9d). By
considering the FTIR of GO, CT and CT/GO/HAP nano-
composite, it can be seen that the adsorption peak attributed to
the carboxylic acid (–COOH) in GO disappeared in the spectrum
ꢀ
1
of CT/GO/HAP nanocomposite, and a new peak at 1655 cm
corresponding to amide group was detected, indicating the
reaction of the –COOH groups of GO with the groups NH of CT
2
to form amide groups (–CONH–). On the other hand, the
frequency of NH band shied from 1593 (in pure CT) to
ꢀ
1
1
566 cm (in CT/GO/HAP nanocomposite) due to the forma-
tion of the amide groups. These phenomena prove the func-
tionalization of GO by CT (CT-functionalized GO). On the basis
of our experimental results, a scheme has been presented to
describe the functionalization of GO by CT and formation of 3-D
nanocomposite scaffold, as illustrated in Scheme 1. The
formation of new HAP on the surface of the as-produced nano-
sized HAP and composite pellets aer soaking in SBF for 14
days was studied by ATR-IR. In the spectrum of the HAP
nanoparticles immersed in SBF (Fig. 9e), the strong peak of the
3
ꢀ
ꢀ1
2ꢀ
ꢀ1
v
3
(PO
4
) at 1026 cm and the v(CO
3
) at 1454 cm are
observed. The presence of the phosphate and carbonate vibra-
tions indicates the formation of carbonated hydroxyapatite on
the surface of the immersed pellets. The ATR-IR spectrum of
CT/GO/HAP nanocomposite (Fig. 9f) the vibrations of OH,
2
ꢀ
3ꢀ
–
CONH–, CO
3
, C–OH, C–O–C and PO
4
groups at 3724, 1637,
ꢀ
1
1
544, 1375, 1307, and 1022 cm , respectively. These results
show the presence of carbonated HAP and CT-graed-GO
nanocomposite. By comparing the IR frequency of the amide
Fig. 9 FTIR spectra of (a) GO, (b) CT, (c) HAP nanoparticles, (d) CT/GO/
ꢀ1
HAP nanocomposite in the range of 4000–400 cm . ATR-IR spectra
of (e) HAP nanoparticles and (f) CT/GO/HAP nanocomposite after
ꢀ1
soaking in SBF in the range of 4000–600 cm
.
ꢀ
1
weak peak at 3570 cm is due to the stretching vibration of OH
groups in the structure of HAP. The adsorption bands attributed
to the symmetric stretching (v
bending vibrations (v ) of the PO
and around 566–603 cm , respectively. The broad band at 3432
2
), asymmetric stretching (v
3
) and
3
ꢀ
4
4
ions are clear at 472, 1029,
ꢀ
1
ꢀ
1
ꢀ1
cm (stretching vibration) and the weak band at 1635 cm
bending vibration) are attributed to the crystal water and
(
2
ꢀ
surface adsorbed water. The presence of the v(CO ) vibration
at 1454 and 1415 cm are observed in these spectrum, indi-
cating the replacement of some PO
ions. The carbonate ions might come from the atmosphere
3
ꢀ
1
3
ꢀ
2ꢀ
ions in HAP by CO3
4
4
5
Scheme 1 Schematic representation of CT-functionalized GO/HAP
carbon dioxide which combined into the crystal structure scaffold as a 3-D nanocomposite.
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precipitation of apatite on the surface of the powders. Aer 14
days of immersion, the ion concentrations show a negligible
changes. By considering the concentration changes of Ca and P,
it can be seen that more Ca and P ions are releasing from the
CT/GO/HAP nanocomposite compared to the pure HAP nano-
particles. Because thermodynamical stability of HAP nano-
particles is more than that of HAP nanocomposites, the
dissolution rate of pure HAP nanoparticles is lower than that of
12
HAP nanocomposites. Besides, based on the XRD results,
lower crystallinity of the CT/GO/HAP nanocomposite due to the
presence of CT polymer leads to the increase of dissolution rate
in composite.
New-formed apatite on the surface of the powder pressed
into pellets was studied by visualizing change in morphologic
features aer immersing in SBF for 14 days using SEM. The SEM
images of CT/GO/HAP nanocomposite (Fig. 11a and b) and HAP
nanoparticles (Fig. 11c and d) show an increased growth of
apatite aer immersion for 14 days. This was due to the disso-
lution of HAP, and simultaneous precipitation of apatite. The
Fig. 10 (a) Ca and (b) P concentrations of SBF solutions versus in vitro grain sizes of the new-formed nanoparticles on the surface of
aging time.
the products are about 20–25 nm (Fig. 11b) and 10–15 nm
(Fig. 11d).
ꢀ1
ꢀ1
group before (1655 cm ) and aer soaking in SBF (1637 cm ),
it is concluded that this frequency decreased aer soaking due
Conclusions
to the interaction of the amide groups in CT/GO/HAP nano- Because of the hydrophilicity of GO, hydrophilic biopolymers
composite with the new-formed HAP nuclei.
including NH2 and OH groups such as CT, the interaction
Fig. 10a and b correlate the Ca and P concentrations of SBF between polymer chains and GO sheets is desirable and a good
solutions at different aging times, respectively. When the pellets dispersion of GO in CT solution is expected, so, the graphene/
of pure HAP nanoparticles and CT/GO/HAP nanocomposite are chitosan composites show a 200 times increase in the elasticity
immersed in SBF, two main phenomena occur at the same time. modulus compared to pure chitosan.
They are the dissolution of the powders and precipitation of
In current work, at the rst, Schiff base derived from sali-
47
new apatite. In case of CT/GO/HAP nanocomposite, from the cylaldehyde and 2,2-dimethyl-propylendiamine was synthe-
beginning of immersion to 7 days, the dissolution process sized, and then applied as surfactant to fabricate HAP
overcomes to the precipitation process due to the increase of nanoparticles. Aer that, composite of chitosan, graphene
the Ca and P concentrations in the SBF solutions. Aer 7 days of oxide and hydroxyapatite nanoparticles were prepared by
immersion, the Ca and P concentrations decrease due to the simple freeze-drying method and obtained results showed that
the CT/GO/HAP nanocomposites have a much higher bioactivity
rather than that of the pure HAP nanoparticles. The in vitro
bioactivity of HAP nanoparticles and CT/GO/HAP nano-
composite was evaluated by soaking them in simulated body

uid (SBF). By monitoring the changes of chemical composition
of the SBF solutions, it was concluded that more Ca and P ions
released from the CT/GO/HAP nanocomposite compared to the
pure HAP nanoparticles, indicating high bioactivity of the
nanocomposite. The SEM images showed that the formation
and growth of apatite on the surfaces of the products increased
aer immersion for 14 days in SBF.
Acknowledgements
Authors are grateful to council of University of Kashan for
providing nancial support to undertake this work.
Notes and references
Fig. 11 Surface micrographs of (a and b) CT/GO/HAP nanocomposite
and (c and d) HAP nanoparticles after immersing in SBF for 14 days.
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26000 | RSC Adv., 2014, 4, 25993–26001
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