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Article history:
Received 5 March 2015
Received in revised form 2 July 2015
Accepted 8 July 2015
Available online 15 July 2015
Keywords:
Squid pen
Chitosan
Hydroxyapatite
a b s t r a c t
In the present study, chitosan/hydroxyapatite (HA)/-tircalcium phosphate (-TCP) composites were
produced using squid pen derived chitosan (CHS) and commercial crab derived chitosan (CHC). CHS
was prepared from squid pens by alkaline N-deacetylation. HA and -TCP were extracted from mussel
shells using a microwave irradiation method. Two different composites were prepared by incorporating
50% (w/w) HA/(-TCP) in CHS or CHC followed by lyophilization and cross-linking of composites by
tripolyphosphate (TPP). The effect of different freezing temperatures of 20, 80 and 196 C on the
physicochemical characteristics of composites was investigated. A simulated body uid (SBF) solution
was used for preliminary in vitro study for 1, 7, 14 and 28 days and the composites were characterized
by XRD, FTIR, TGA, SEM, -CT and ICP-MS. Porosity, pore size, water uptake; water retention abilities
and in vitro degradations of the prepared composites were evaluated. The CHS composites were found
to have higher porosity (62%) compared to the CHC composites (porosity 42%) and better mechanical
properties. The results of this study indicated that composites produced at 20 C had higher mechanical
properties and lower degradation rate compared with 80 C. Chitosan from the squid pen is an excellent
biomaterial candidate for bone tissue engineering applications.
2015 Elsevier B.V. All rights reserved.
1. Introduction
In recent years, much attention has been paid to marine byproducts, scoping their cost-effective processing schemes and their
potential for production of high-value products. Natural polymers
like chitin, chitosan and calcium phosphate (CaP) compounds can
be obtained from waste marine products [1]. Because of their
intrinsic properties such as biocompatibility, biodegradation and
antimicrobial properties, these natural materials have important
biomedical applications [2,3]. In the last two decades, many reports
have been published on chitin and chitosan applications in drug
delivery, tissue engineering, skin and bone grafting [2,4,5]. Chitosan
is a biopolymer consisting of (1,4)-2-amino-2-deoxy-d-glucose
units that is obtained by N-deacetylation of chitin under alkaline
condition. Chitin can be sourced and extracted from a diverse range
of natural organisms, including molluscs, fungi, insects, crustaceans
and algae [6]. Chitin exists in three different allomorphic forms
depending on the sources of the compound. Most chitins, including
Corresponding author.
E-mail address: amin.shavandi@postgrad.otago.ac.nz (A. Shavandi).
http://dx.doi.org/10.1016/j.ijbiomac.2015.07.012
0141-8130/ 2015 Elsevier B.V. All rights reserved.
446
DDA (%) = 1
(C/N 5.14)
1.72
100,
(1)
Table 1
Composition of HA/-TCP/CH composites.
Composite
A
B
Compound (%)
HA
-TCP
CHS
CHC
30
30
20
20
50
50
447
interest (ROI). The exhibited regions with white pixels are considered as solid portions and regions with black pixels, which are
surrounded by white pixels, are pores and reported as percentage.
2.8. Water uptake and retention capacities
The water uptake and retention ability of the composites was
measured using the method described by Thein-Han et al. [26].
Sample with a known weight (Wd ) was immersed in distilled water
for 24 h. Then, the sample was gently removed and placed on a
wire rack for 1 min. The sample was weighed (Ww ) to determine
448
(2)
W W
t
0
W0
100
CHC and CHS, i.e. the -chitin and -chitin structure, respectively.
Various DDA values have been reported for CHS in literature, up
to 95%, which was dependent on the extraction conditions and
methods used [11].
3.2. Determination of the components by X-ray crystallography
The phase of the composites was conrmed by X-ray diffraction
(XRD). Samples processed at 196 C were all failed and collapsed
during freeze drying, and so we omit any further characterization of these samples. Despite deacetylation, freezing temperature
does not change crystal structure and chemical bonds of the chitosan; therefore, XRD and FTIR analysis were performed on two
treatments (A280 and B280), instead of all four treatments.
The XRD patterns of the composites made with CHS and CHC are
displayed in Fig. 2A. The wide peak at 2 = 20 in all patterns was
assigned to chitosan and corresponded to its degree of crystallinity
[33]. The crystallinity was decreased with the increase in DDA [10].
As shown by the broad peaks of XRD graph at about 2 = 10 and
20 , CHC had less ordered structure, and lower crystallinity compared to CHS. The sharp peak at around 2 = 32 represents the
HA/-TCP in the composite, also the peaks at 2 = 33, 34, 40, 47,
48 and 5054 are also assigned to HA/-TCP. These results were
in agreement with previous reports [34]. It has been reported that
HA/-TCP weakens the intramolecular interaction of the chitosan
chain [33]. The crystallinity of the HA/-TCP compound was found
to be lower than either pure HA or -TCP [15,16] due to the presence of the chitosan matrix. However, the XRD patterns of natural
bone also have broad and overlapping peaks [35].
(3)
3.3. FTIR
FTIR is a typical technique to investigate the structure-function
of chitosan biomacromolecule and its interaction with HA/-TCP
[36]. The infrared (IR) spectra of the composites made with CHS
and CHC are shown in Fig. 2B. The peak at 1647 and 1560 cm1 can
be assigned to intracellular hydrogen bond between the carbonyl
groups of amide I and II of chitosan respectively [26,30,37]. The peak
at around 3260 cm1 represents intramolecular hydrogen bonding
between the stretching vibration of the N H bond of chitosan, and
OH group of HA/-TCP. The sharp peak at 1415 cm1 is devoted
to the symmetrical deformation mode of CH3 . The peaks at 1029
and 1100 cm1 are assigned to the C O stretching vibration. The
typical band of PO4 observed at 500700 cm1 . The FTIR spectra of
all composites indicated that the characteristic bands of both CaP
compounds and chitosan were present in the composites. This FTIR
result suggests that the structure of chitosan biomacromolecule
provides a matrix for the HA/-TCP particles and also binds them
together in the composites [26]. However, comparing the spectra
bands of B280 and A280 in Fig. 2B, different intensities are observed
in peaks at 1647 and 1560 cm1 . The C O bands at 1660 cm1 for
A280 is less intense than B280, this is likely due to the deacetylation
process that removed the C C bands.
3.4. Thermal analysis
Thermal degradation behaviour of the composites was studied using TGA. The TGA curves of the four composites are shown
in Fig. 2(C). The weight loss for all composite samples began at
50 C, which could be due to water loss and sharpest decrease was
observed at 50100 C [38]. The second thermogram peak occurs
at 250350 C, which could be due to degradation of deacetylated
molecules and the formation of saccharide molecule structure.
This process includes dehydration of the saccharide ring, polymerization and decomposition of the acetylated units [39]. In
case of composites A and B, which processed at different freezing
449
Fig. 2. X-ray diffraction pattern (A), FTIR spectra (B) and thermograph analysis (C) of composite samples manufactured using two different chitosan (crab (A280) and squid
(B280)) and processed at different freezing temperature (20 and 80 C). A280 = 50% HA/-TCP and 50% crab chitosan (CHC). B280 = 50% HA/-TCP and 50% squid pen
chitosan (CHS).
compare to B280. It is anticipated that, HA/-TCP provides a thermal barrier and hinders the degradation of composites [40]. This
heat-resistant behaviour of chitosan can be assigned to bonds that
formed between hydroxyl (OH) and amino (NH2 ) groups [38]. From
450
Fig. 3. SEM images and photograph of the synthesized biocomposites. (A) Sample processed in liquid nitrogen, (B) Sample processed at 80 C. (C) A280 and B280 samples
that processed at 20 C. A280 = 50% HA/-TCP and 50% crab chitosan (CHC). B280 = 50% HA/-TCP and 50% squid pen chitosan (CHS).
600 C to 1000 C the curve was stable and about 50% of the samples
weight remained for all composites, which reected the HA/-TCP
%.
3.5. Microstructure morphology
Scanning electron microscopy (SEM) was used to examine the
microstructure of the composites (Fig. 3). The composites processed
in liquid nitrogen (196 C) illustrated radial and aligned channels. This microstructural formation is likely occurred due to water
crystallization in chitosan droplets and subsequent ice sublimation
(Fig. 3A). These needle-like channels clearly show the ice crystal
shapes [41] since the composites had very compact and almost non
porous structure (Fig. 3A), which could be due to its very fast freezing and solidication. The composites prepared at 196 C were
incoherent and collapsed over freeze-drying processes, which possibly as a result of evaporation of water from the compact region
and from channelled cavities. In a recent study, Ouyang et al. [41]
reported that freezing chitosan beads in liquid nitrogen resulted in
width channel size of 1020 m. However, in that study chitosan
drops were immersed in liquid nitrogen and therefore, no structural
collapse was observed. Due to disjoint structure and low porosity of 196 C samples, we were unable to examine the structural
morphology of the composites any further. The composite samples
451
Fig. 4. (A) Pore size of composites fabricated using CHC (A) and CHS (B) that are processed at the freezing temperature 20 and 80 C, respectively. (B) Two dimensional
(2D) cross-section image of composites processed at 20 C, where composite B220 (left) and composite A220 (right).
452
Table 2
Micro-architectural parameters for the composites.
Type of composite
Degree of anisotropy
A220
A280
B220
B280
1.34
1.31
1.36
1.09
0.25
0.23
0.26
0.08
Porosity (%)
45.43
79.10
62.79
66.40
2.54
1.62
4.36
5.70
Fig. 5. Porosity of composites fabricated using different chitosan (A) CHC and (B)
CHS and freezing temperature (20 and 80 C).
composites is critical for the use in bone-tissue engineering applications. Porosity facilitates cell migration, blood circulation and
vascularization during tissue repairing or regeneration. It has been
reported that composite construction via lyophilization technique
has demonstrated better pore size compared to solgel or precipitation method [50]. As shown in Table 2, composites frozen at 20 C
had lower porosities compared to composites frozen at 80 C. For
example, composite (A220), which was frozen and processed at
20 C, exhibited lower porosity compared to its counterpart composite (B220). In general, the crab chitosan composites processed
at 20 C exhibited more shrinkage compared to squid chitosan
composites frozen at the same temperature. This behaviour can be
due to structure loss of composites frozen at 20 C compared to
compact structure of composites at 80 C. The higher the freezing
temperature is the slower the freezing rate of the chitosan solution is, therefore at a slower freezing rate, chitosan phase has more
time to grow its grains size and ice crystals were bigger [42]. Therefore, the nal dried structure was looser compared to 80 C. On
the other hand, the lower porosity of crab chitosan composites A
can be due to its lower initial viscosity compared to squid chitosan
composites. Accordingly, TPP appears to be working better in composites processed at 20 C. At 80 C, composites A280 showed
higher porosity compared to B280; this can be due to unstable and
non-uniform structure of A280, which cannot hold all HA/-TCP in
its structure and so due to loss of material it has higher porosity.
3.8. Mechanical properties of composites
The mechanical properties of composites are shown in Fig. 6.
The mechanical behaviour of the composites is a critical factor
in their biomedical application and bone healing properties. It is
documented that incorporation of CaP into the biomacromolecule
matrix enhanced the mechanical properties of the composite [3].
Furthermore, cross-linking of composites with 2.5% TPP caused
microstructure changes, which had signicant effects on the
mechanical properties of the composites. As shown in Fig. 6,
composites frozen at 20 C showed better mechanical properties. In particular, composite B220, which was frozen at 20 C,
recorded highest compression modulus, ultimate strength and
yield strength. This can be due to formation of bigger ice crystals
during freezing at low freezing temperature (i.e. 20 C compared
to 80 C); so enabled better TPP cross-linking. Enhanced tensile properties were also reported by Hsieh et al., for composites
frozen and processed at 20 C [42]. In case of the composites that
were frozen and processed at 80 C, there were no signicant
differences between the composites (A280 and B280) and both
showed lower mechanical properties compared to the composites frozen/processed at 20 C. These ndings were in accordance
Fig. 6. Mechanical properties of the composites. (A) Compression modulus and (B) ultimate strength and yield strength of the composites.
453
can take up and hold water more than its own weight (values are
higher than 100%). However, maximum water uptake recorded in
this study is almost 1200%, which is lower than the Chitosan/CaP
composites (1600% and 2500%) reported in literature [26,52]. This
lower water uptake recorded in this study might be due to the cross
linking and shrinkage of the composites. As shown in Fig. 7A, composite A280 recorded highest water uptake, which could be due to
its loose structural morphology that allows more water uptake and
retention. On the other hand, lower water uptake and retention
of composite B280 might be due to its degradation from 14 days
onward. It is also noteworthy to mention that, the stiffer and rigid
structure of composites processed at 20 C hindered their water
uptake and retention ability.
3.10. In vitro degradation of composites
Biodegradation properties of composites are an important factor
on the long term functionality of the bone regeneration, including,
grafting or repairing. The degradation prole of composite samples
as a function of weight loss vs soaking time in SBF is presented in
Fig. 7C. As shown the weight loss gradually increased by increasing
time, which indicates composites degraded with soaking time. It is
envisaged that the backbone of the chitosan bio-macromolecule is
hydrolytically unstable [53], which enhance its degradation. After
28 days of in vitro degradation, the structural stability of all composite composites seems to be stable except for B280 samples, which
degraded by about 40%. This high degradation might be due to two
reasons: rst the composite processed at 80 C (B280) did not
cross-linked adequately compared to its counterpart B220. This
could be due to the loss and weaker structure morphologies of
B220 composite. Second, CHS chitosan that used for B composites
had smaller DDA which promotes its degradation, as the structural
stability of chitosan is inversely related to the DDA [54]. Considering these factors, composites processed at 20 C and cross-linked
afterwards are durable and these composites maintained their
structural stability over the tested period.
3.11. Change in pH of physiological solution
Chemical stability of the composite is importance. Solubility of
composite compartments can lead to change in pH and may affect
the cell response [55]. The pH variation of SBF solution of composites monitored over a four-week period (Fig. 8). During the rst two
week of incubation in SBF, the pH value of all composites increased
slightly and then decreased slightly for the following week (Fig. 8).
This increase might be due to alkalescent nature of chitosan, which
has alkaline groups, and also alkaline ions from degradation of CaP
compounds [56,57]. The decrease of pH can be due to consumption
of calcium and phosphate ions and their deposition on the composite surface [58]. The pH change pattern of the SBF solution might be
Fig. 8. The pH values of the SBF solution in which samples were incubated.
454
related to the dissolution characteristic of HA/-TCP and degradation prole of chitosan. Overall, the composites processed at 80 C
showed higher chemical stability than 20 C composites and their
pH of SBF solution only changed slightly and decreased to 7.20 from
its original value of 7.45, and remained close to physiological value.
4. Conclusions
The squid derived chitosan composites have shown to be more
hydrophilic compared to commercial crab chitosan. Additionally,
the SEM images and -CT results revealed that squid composites
have homogeneous and uniform lamellar structure. The results of
this study indicated that composites produced at 20 C had higher
mechanical properties and lower degradation rate compared with
80 C and in overall, squid chitosan has better mechanical properties. These results indicated that chitosan isolated from the squid
pen could be an alternative for existing commercial chitosan to be
considered for biomedical applications.
Acknowledgements
The authors acknowledge the facilities as well as scientic
and technical assistance from staff at Otago Centre for Electron
Microscopy (OCEM) at the University of Otago. We would also like
to thank Mr Damian Wallas for his help and technical support for
XRD. The rst author acknowledges the PhD scholarship by University of Otago, New Zealand.
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