International Journal of Biological Macromolecules 80 (2015) 445454

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journal homepage: www.elsevier.com/locate/ijbiomac

Bio-mimetic composite scaffold from mussel shells, squid pen

and crab chitosan for bone tissue engineering
Amin Shavandi a, , Alaa El-Din A. Bekhit a , M. Azam Ali b , Zhifa Sun c
a
b
c

Department of Food Sciences, University of Otago, Dunedin, New Zealand

Department of Applied Sciences, University of Otago, Dunedin, New Zealand
Department of Physics, University of Otago, Dunedin, New Zealand

a r t i c l e

i n f o

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.

crustaceans and insect chitin are in alpha () form which has a

two chain antiparallel structure. However, squid pen and some
diatoms have beta () chitin which has one chain parallel structure and in gamma () chitin, the biomolecular chains are arranged
randomly in which two parallel chains and one antiparallel chain
form the polymeric structure [7]. Alpha-chitin and chitosan are
commercially available products and are produced normally from
shrimp or crab shell. Chitin/chitosan from the squid pen has a structure that is low packed and has weak intermolecular hydrogen
bonds. These properties makes it chemically more reactive compared to the heavily packed and strong molecular structure of
-chitin/chitosan from shrimp and crab shells [810]. In addition,
-chitin/chitosan can incorporate water molecules in its structure
and forms crystalline structure leading to higher ability to uptake
and hold water more than the alpha form, which is advantageous in
biomedical applications [11,12]. The source of chitosan can affect
its purity, molecular weight, chain length, degree of deacetylation,
density, viscosity, solubility, water retention capacity and distribution of the amino/acetamide groups. All these characteristics
affect the physicochemical properties of chitosan and therefore, its
application [13]. A proper biocomposite should be prepared strategically in a way to have a suitable geometry and pore size, have a

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required mechanical strength, support cell attachment in vitro, and

have tuneable biodegradable properties [14]. In this work, chitosan
was isolated from endoskeleton of New Zealand arrow squid pen
(Nototodarus sloanii) (CHS). In addition, a commercially available
crab chitosan (CHC) was used for comparison with CHS. The HA
and -TCP were prepared using waste green mussel shells. Then
CHS, CHC and HA, -TCP were processed to produce biocomposites
using freezing at different temperatures (20, 80 and 196 C)
and lyophilization processes. The produced biocomposites were
evaluated to scope their potential for bone tissue engineering applications. To improve the mechanical properties of the composites in
this study, tripolyphosphate (TPP) was used as an ionic cross-linker
and glycerol was used as plasticizing agent.
2. Materials and methods
2.1. Materials
Hydroxyapatite (Ca10 (PO4 )6 (OH)2 ) and -TCP (Ca3 (PO4 )2 ) powders were synthesized from waste mussel shells as previously
reported [15,16]. Acetic acid, ethanol, and NaOH were obtained as
analytical grade from Univar (Ajax Finechem, USA) and Sigma (St.
Louis, USA), respectively. The process of squid pen chitosan (CHS)
preparation and fabrication of the biocomposites are displayed in
Fig. 1. Dried arrow squid pens (Nototodarus sloanii) were used for
the preparation of -chitin (obtained from Independent Fisheries
Co., Christchurch, New Zealand). Commercial crab chitosan (CHC)
purchased from Weseta International (Shanghai, China) was used
as received.
2.2. Preparation of chitosan from squid pen
Isolation of chitosan from the squid pen was performed following a method developed by Chaussard and Domard [17] with minor
modications (Fig. 1). In brief, dried squid pens were grinded to particles 1 mm in diameter using Waring laboratory grinder (Waring
Inc., USA), then deproteinization was carried out using 1 M sodium
hydroxide (15 ml/g) at 60 C and constant stirring using a rotary
shaker for 24 h. Then, the leachate was removed by vacuum ltration and particles were washed extensively until neutral pH was
achieved. The obtained highly moist extracted material was frozen
at 80 C and lyophilized using a freeze drier (Labconco FreeZone
12 Plus). Then the chitin powder was slowly added to a beaker containing 45% NaOH to obtain a solid/solvent ratio of 1:15 (w/v) and
soaked at room temperature for 24 h [18]. The temperature of the
reaction was maintained at 60 C and the mixture was stirred for
10 h.
2.3. Degree of N-deacetylation
The degree of deacetylation (DDA) of chitosan (CHS) was calculated from data of elemental analysis (Carlo Erba Elemental
Analyser EA 1108). DDA was calculated using Eq. (1) proposed by
Xu et al. [19].

DDA (%) = 1

(C/N 5.14)
1.72

100,

(1)

where C/N is the ratio (w/w) of carbon to nitrogen in chitosan.

2.4. Preparation of the composite
Squid chitosan production and fabrication of the composites
are shown in Fig. 1. Crab chitosan (CHC) solution (2%) was made
by dissolving 5 g of chitosan in 250 ml of 1% acetic acid solution
[20]. Due to the high hygroscopic nature of squid pen chitosan

Table 1
Composition of HA/-TCP/CH composites.
Composite

A
B

Compound (%)
HA

-TCP

CHS

CHC

30
30

20
20

50

50

(CHS), the solution was very viscous at 1% and so the preparation of

higher concentrations was technically difcult. To prepare 2% CHS
solution, a 1% solution of chitosan dissolved in 1% acetic acid was
subjected to microwave irradiation to remove excess water and
achieving the desired concentration of 2%. The solution was then
mixed by an overhead mixer (IKA T25 Ultra Turrax) for 2 min to
obtain a transparent gel. The HA and -TCP powders were mixed
together based on ratios shown in Table 1. The powders were
weighed and made into a homogeneous paste using ethanol (1:10,
w/v). The paste was added to the chitosan solutions, homogenized
by the overhead mixer. The chitosan solutions were then sonicated
(Elmasonic S40 (H)) for 1 h to remove any air bubbles. The air bubble
free mixtures were transferred to 15 ml Poly-Cons plastic container and frozen at 20 C, 80 C, or 196 C (the latter by direct
immersion of the plastic container into liquid nitrogen for approximately 10 s). Then the samples were freeze-dried for 48 h (Labconco
FreeZone 12 Plus) to form the HA/-TCP/CH (CHC or CHS) composites. The dried HA/-TCP/CH composites were soaked in 2.5%
tripolyphosphate (TPP) aqueous solution at 4 C for 2 h [21]. Then
the composites were rinsed with deionised water for 12 h at 4 C to
remove residual TPP and were freeze dried for 24 h at 40 C. Composites made with crab chitosan and processed at 20 and 80 C
were denoted as A220 and A280 respectively, and those made with
squid pen chitosan were denoted as B220 and B280, respectively.

2.5. Characterization of the composites

The distribution of HA/-TCP in the chitosan matrix was analysed using an X-Ray Diffractometer (XRD; PANnalytical XPert
PRO MPD System) in the range 0 < 2 < 60 with Cu K radiation
(k = 0.15418 nm) with a scan speed of 2.63 s [22]. The functional
groups of the samples were identied using Fourier Transform
Infrared Spectroscopy (FT-IR; Perkin-Elmer #100) in the region
4004000 cm1 with 4 cm1 spectral resolution using the KBr pellet technique [22]. Thermogravimetric analysis of the composites
was carried out using a TGA instrument (TGA; Q 500) up to 1000 C
at a 10 C/min heating rate under a nitrogen ow. Scanning electron microscopy (SEM) (JEOL 6700F FESEM JEOL Ltd, Tokyo, Japan)
was used to examine the microscopic details of the composites.

2.6. Mechanical testing

The mechanical properties of the HA/-TCP/CH composites
were tested according to the guidelines of ASTM D5024-95a (22).
The mechanical properties of composites were determined using
a TA.XTPlus, Texture Analyzer (Texture Technologies Corp., Stable Micro Systems, Godalming, Surrey, UK). The analysis was
carried out on cylindrical samples with dimensions of 25 mm
diameter 12 mm height. A 250 N load cell was operated at a
rate of 0.5 mm min1 until the sample was compressed to 50%
of the original height at room temperature. The compression
stressstrain curves were recorded, and compression modulus,
yield and ultimate strength were calculated using the Exponent
software (version V6.1.5.0) using 5 replicates per each of the composite samples [23].

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447

Fig. 1. Diagram for the fabrication of the HA/-TCP/CH composites.

2.7. Micro architectural analysis of composite

X-ray microtomographic (-CT) systems have been widely used
as a non-destructive technique to study or characterize microstructural morphology of various biomaterials [24,25]. -CT images
of the HA/-TCP/CH composites were obtained using a Skyscan
1172 system (Bruker-Micro CT, Kontuch, Belgium) to quantify the
3-D microstructure of the composite samples. The CT-analyzer software v.1.14.4 (Bruker-Micro CT, Kontuch, Belgium) was used to
calculate morphometric parameters and the calculations were performed using a segmented image from a rectangular region of

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

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the water uptake. To measure the retention ability of water, the

wet composite sample was transferred to a falcon tube with a lter
paper at the bottom of the tube was centrifuged using a Beckman
GPR centrifuge at 500 RPM for 3 min, and measure its ultimate
 ). The weight of the sample was averaged from tripweighed (Ww
licate measurements (n = 3) for each group of samples. The water
uptake and retention percentage of the composites were calculated
using the following Eq. (2) [23,26]:
EA = [(Ww Wd )/Wd ] 100
 W )/W ] 100
ER = [([(Ww
d
d

(2)

2.9. In vitro degradation of composites

A simulated body uid that considered a physiological buffer
solution consisting of -lactate and lactic acid (Lac-SBF) with an
ionic concentration similar to human blood plasma was prepared
as described by Cuneyt Tas [27]. The degradation ability of the composites was studied by incubating dry and weighed (W0 ) samples
in Lac-SBF (pH 7.4 at 37 C) for up to 28 days. Approximately 25 mg
samples (n = 3 for each group) were placed in glass liquid scintillation vials (volume 20 ml). A 10 ml of Lac-SBF solution were added
to each of the vials and were then incubated at 37 C. The samples
were removed at time intervals (1, 7, 14, and 28 days), washed ve
times with distilled water, and then dried at 60 C for overnight.
The degradation was monitored in terms of changes in the weight
of the samples over the time period. The degradation ratio (D) was
calculated using the following Eq. (3):
D = 100

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)

where W0 represent the original weight and Wt is the weight of the

sample at time t. Changes in the pH of Lac-SBF solution was also
measured over the testing time using a pH meter (HI 2211, HANNA
instruments, Rhode Island, USA) [28].
2.10. Pore size determination
The pore structure of composites was examined using scanning
electron micrograph (SEM) and the average pore size was calculated using ImageJ [29]. The mean pore diameter was estimated by
measuring about 100 different pores for each composite and three
images were tested for each composite.
2.11. Statistical analysis
Triplicate experiments were performed for each sample, and
results were expressed as the mean of at least three replicates SEM. General linear Model (GLM) was performed between
different composites, incubation times and/or temperatures using
Minitab 16.2.4 Statistical Software and the differences were considered statistically signicant at P < 0.05. The graphs were generated
using the GraphPad Prism software (version 6.00 for Windows,
GraphPad Software, San Diego, CA, USA, www.graphpad.com).

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

3. Results and discussion

3.1. Degree of deacetylation
The degree of deacetylation (DDA) represents the number of
glucosamine units in a chitosan biopolymer chain [30]. DDA determines some of the properties of the polymer such as solubility,
water uptake, biodegradation behaviour and its crystallinity [31].
The DDA of CHS and CHC was 72.8 2.9 and 86.9 1.7, respectively
[32]. These different DDA values appear to be due to different efciencies of deacetylation resulted from intramolecular structure of

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

A. Shavandi et al. / International Journal of Biological Macromolecules 80 (2015) 445454

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).

temperatures, composites of A (A220 and A280) showed a similar

trend, while in case of B composites, B220 was slightly more thermally stable than B280. This higher stability might be attributed to
its denser and robust structure as a result of its better cross linking

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

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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

processed at 80 C exhibited elongated pore structure, which is

also showed extended irregular shape (Fig. 3B). However, the composites processed at 20 C appeared more irregular in shape, the
pore structures were layered, and morphological geometry were
more collapsed compared to samples processed at 80 C (Fig. 3C).
This phenomenon can be attributed to a weaker structure of composites processed at 20 C compared to 80 C, which can be due
to its slower freezing rate, bigger water crystals and consequently
bigger pores and loser structure of 20 C samples compared to
80 C processed samples. Therefore, more intense cross-linking
with TPP occurred that caused the breakdown of the internal structure. The presence of HA/-TCP in the composite was also observed
on the surface of pore walls.
3.6. Pore size
The average diameter of the pores was 122.2 m for the
B220 (squid chitosan composite containing 50% HA/-TCP and
frozen at 20 C), 456.2 m for B280 (squid chitosan composite containing 50% HA/-TCP and frozen at 80 C) composite,
93.1 m for A220 (crab chitosan composite containing 50% HA/TCP and frozen at 20 C) and 322.13 m for A280 (crab chitosan
composite containing 50% HA/TCP and frozen at 80 C) composites. It was observed that the pore size and pore distribution of

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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).

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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 cross-linked with TPP and processed at 20 C

appeared less homogeneous compared to composites processed at
80 C (Fig. 4A). It has been reported by Hsieh et al., that temperature of 20 C generated porous chitosan composites with larger
pore-sized [42]. The two dimensional (2D) cross-section images of
the A220 and B220 (Fig. 4B) revealed that B220 had a more uniform and regular internal structure compared to A220, suggesting
CHS is a good candidate for fabrication of composites with homogeneous microstructure. The composite morphology is an important
feature of the composite for considering it for bone-tissue engineering. The structure has to have enough porosity and proper pore size
that allow blood vascularization and cell proliferation. Wide range
of pore sizes, from 30 to 1000 m, has been reported in literature
for adequate bone tissue engineering [43,44]. It has been reported
that pore diameter greater than 300 m is essential for vascularization of composites and bone ingrowth [4548]. The results from
our study are in accordance with those previously reported pore
sizes for chitosan/hydroxyapatite composites [20,49].
3.7. Porosity
In this study, the fabricated composites showed porosity values from 45 to 79% (Table 2 and Fig. 5). The porosity of the

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.

A. Shavandi et al. / International Journal of Biological Macromolecules 80 (2015) 445454

with previously literature by Reys et al., who reported CHS to have

better mechanical properties than CHC composites [32]. In addition, when comparing different composites at same temperature of
20 C, composite A had collapsed internally and was not as strong
as composite B (Fig. 4B).
3.9. Water uptake and water retention test
The water retention ability of the composite is a critical factor for
its efcacy for bone-tissue engineering; in particular, bone grafting.
It has been reported that water uptake ability of the composite can
signicantly effect on cell proliferation and differentiation [51]. In
this study, the results of the water uptake and retentions are shown
in Fig. 7A and B. All the composites started swelling rapidly in the
rst day, indicating good water uptake characteristic [26]. From
this water uptake data, it can be conferred that 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. 7. Water uptake, water retention and degradation properties of HA/-TCP/CH

composites. (A) Water uptake and (B) retention ability of the composites. Water
uptake for composite B220 was signicantly higher than A220 for all the tested
times (P < 0.05) and Water uptake of composite A280 was signicantly higher than
B280 from day 14 onward. There was not signicant differences for water retention
between the composites. (C) In vitro degradation prole of the composites with
various time period (1, 7, 14 and 28 days). Degradation of composite B280 was
signicantly higher than other composites.

Fig. 8. The pH values of the SBF solution in which samples were incubated.

454

A. Shavandi et al. / International Journal of Biological Macromolecules 80 (2015) 445454

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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