Colloids and Surfaces B: Biointerfaces 109 (2013) 294–300

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Colloids and Surfaces B: Biointerfaces

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

Biocomposite scaffolds containing chitosan/alginate/nano-silica for

bone tissue engineering
J.A. Sowjanya a , J. Singh a , T. Mohita a , S. Sarvanan a , A. Moorthi a ,
N. Srinivasan b , N. Selvamurugan a,∗
a
Department of Biotechnology, School of Bioengineering, SRM University, Kattankulathur 603 203, Tamil Nadu, India
b
Department of Endocrinology, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras Taramani, Chennai, Tamil Nadu, India

a r t i c l e i n f o a b s t r a c t

Article history: Bone tissue engineering is a promising alternative method for treating bone loss by a combination of bio-
Received 28 November 2012 materials and cells. In this study, we fabricated biocomposite scaffolds by blending chitosan (CS), alginate
Received in revised form 16 March 2013 (Alg) and nano-silica (nSiO2 ), followed by freeze drying. The prepared scaffolds (CS/Alg, CS/Alg/nSiO2 )
Accepted 8 April 2013
were characterized by SEM, FT-IR and XRD analyses. In vitro studies such as swelling, biodegradation,
Available online 19 April 2013
biomineralization, protein adsorption and cytotoxicity were also carried out. The scaffolds possessed a
well-defined porous architecture with pore sizes varying from 20 to 100 ␮m suitable for cell infiltration.
Keywords:
The presence of nSiO2 in the scaffolds facilitated increased protein adsorption and controlled swelling
Chitosan
Alginate
ability. The scaffolds were biodegradable and the addition of nSiO2 improved apatite deposition on these
Nano-silica scaffolds. There was no significant cytotoxicity effect of these CS/Alg/nSiO2 scaffolds towards osteolin-
Biocomposites eage cells. Thus, these results indicate that CS/Alg/nSiO2 scaffolds may have potential applications for
Scaffold bone tissue engineering.
Bone © 2013 Elsevier B.V. All rights reserved.

1. Introduction chief component of shells of crustaceans [4,5]. Its biocompatible

Bone is a complex living connective tissue. It plays a vital role in
providing structural frame work, mechanical support, and flexibil-
ity to the body. Apart from these, bone is also involved in mineral
storage and homeostasis, blood pH regulation and possesses other
secondary functions [1]. Bone defect or loss is often treated with
grafting procedures but these surgical treatments are restricted due
to the limited amount of tissue, lack of biocompatibility, immune
rejection [2] and the risk of transmission of diseases. A biodegrad-
able scaffold serves as a temporary skeletal frame inserted into the
area of bone loss to support and induce bone tissue regeneration
while it gradually degrades and is replaced by new bone tissue.
The scaffolds should not only promote cell adhesion, prolifera-
tion and differentiation, but also be biocompatible, biodegradable,
exhibiting increased surface to volume ratio with certain mechan-
ical strength and casted in to desirable shapes [3].
Chitosan is a natural, semicrystalline, cationic polysaccharide
composed of repeating units of d-glucosamine and N-acetyl-
glucosamine obtained from partial alkaline deacetylation of chitin,
Abbreviations: CS, chitosan; Alg, alginate; nSiO2 , nano-silica; 3D, 3dimensional;
SEM, scanning electron microscope; SBF, simulated body fluid.
∗ Corresponding author. Tel.: +91 9940632335.
E-mail addresses: selvamn2@yahoo.com, Selvamurugan.n@ktr.srmuniv.ac.in
(N. Selvamurugan).
nature is attributed to the structural similarity of glycosaminogly-
cans (GAGs) which is a vital component of extracellular matrix of
cartilage [6,7]. It also possesses antibacterial activity and recruits
neutrophils to the site of bacterial infection [8].
Alginate, an anionic polymer widely used for bone tissue engi-
neering [9], is biocompatible, hydrophilic and biodegradable under
normal physiological conditions [10]. It is a linear copolymer with
homopolymeric block of (1–4)-linked ␤-d-mannurate (M) and its
C-5 primer ␣-l-guluronate (G) residue, respectively by covalent
bond. The monomer can appear in different sequence or blocks
of consecutive G residues (G-block), consecutive M-residues (M-
block), alternating M and G residues (MG-block) or randomly
organized blocks. Sodium alginate increases the structural integrity
or mechanical strength by forming a stable bond with chitosan [11].
Sodium alginate can form scaffold within a relatively short period of
time and can easily be manipulated to regulate the level of porosity.
Silicon dioxide (SiO2 ) or silica exhibits several properties asso-
ciated with an ideal material for grafting and scaffolding [12]. The
nano ranged bioactive silica particles having large surface area can
form a tighter interface with the polymer matrix in composites.
Nano-bioglass ceramic particles possessing silica as its one of the
chief contents not only provide polymer scaffolds with biomin-
eralization capability but also increase the stiffness of polymer
material without greatly decreasing the mechanical strength [13].
Hence, considering the physical, chemical and biological proper-
ties of these biomaterials, in this paper we aimed and reported the

0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.colsurfb.2013.04.006
 J.A. Sowjanya et al. / Colloids and Surfaces B: Biointerfaces 109 (2013) 294–300
synthesis and characterization of the biocomposite scaffolds con-
taining chitosan/alginate/nano-silica (CS/Alg/nSiO2 ) for bone tissue
engineering applications.
2. Materials and methods
2.1. Materials
Chitosan (CS) powder (low molecular weight, 75–85% deacety-
lated with a viscosity of 20–300 cPs), sodium alginate (alginic acid,
sodium salt with a viscosity of 15–20 cPs), nano-Silicon dioxide
(nSiO2 ) powder (10–20 nm), Dulbecco’s modified Eagle Medium
(DMEM) and MTT (3-4,5-dimethylthiazol-2yl {-2,5-diphenyl-2H-
tetrazolium bromide) were purchased from Sigma–Aldrich, USA.
Foetal bovine serum (FBS) was purchased from Invitrogen, USA.
NaOH and other reagents used were all of analytical grade.
2.2. Methods
2.2.1. Preparation of chitosan/alginate/nano-silica biocomposite
scaffolds
The biocomposite scaffolds containing chitosan (CS), alginate
(Alg), nano-silica (nSiO2 ) scaffolds were prepared by freeze dry-
ing method. Briefly, chitosan (1%, w/v) was dissolved in 10 ml of 1%
acetic acid solution. Secondly, sodium alginate (1%, w/v) was added
to the above chitosan solution and stirred for 1–2 h at room temper-
ature. After the formation of a homogenous solution, silicon dioxide
nanopowder (1%, w/v) was added to the solution and stirred for an
additional hour. Simultaneously, for fabrication of CS/Alg scaffolds,
the above protocol was followed in where the addition of nSiO2 was
excluded. The resulting mixture was then poured into the culture
plates and frozen overnight at −80 ◦ C. Finally, it was freeze dried in
a lyophilizer to obtain dried scaffolds. The prepared scaffolds were
neutralized by immersing in 0.2 M NaOH solution and washed twice
with deionized water and lyophilized again. The resultant scaffolds
were immersed in 1:5 ratio of ethanol and CaCl2 solution 2% (w/v)
for crosslinking followed by lyophilization. Finally, the obtained
scaffolds were stored in an air tight container for further analysis.
2.2.2. Characterization of the biocomposite scaffolds
2.2.2.1. Scanning electron microscopy (SEM). The surface morphol-
ogy and porosity of CS/Alg and CS/Alg/nSiO2 scaffolds were
examined using scanning electron microscopy. Samples were fixed
on carbon tapes, air dried under vacuum and coated with a thin
layer of gold. The samples were then subjected to the analysis on
HR SEM Quanta 200 FEG instrument, Netherlands.
2.2.2.2. Fourier transform-infrared spectroscopy (FT-IR). The FT-IR
was used to characterize intermolecular interaction between the
components in the prepared scaffolds. The spectra of the individual
components used for scaffold fabrication and the spectra of CS/Alg
and CS/Alg/nSiO2 bio-composite scaffolds were recorded with a FT-
IR spectrophotometer [American Perkin Elmer Co., USA] using KBr
press. The spectra were recorded ranging from 4000 to 400 cm−1 .
2.2.2.3. X-ray diffraction analysis (XRD). The XRD patterns of CS/Alg
and CS/Alg/nSiO2 scaffolds were obtained at room temperature
using an analytical XPERT PRO powder diffractometer (Cu K␣ radia-
tion) operating at a voltage of 40 kV. Initially, scaffolds were placed
in liquid nitrogen at −197 ◦ C for 1 h and then finely powdered using
mortar and pestle. The powdered biocomposite scaffolds were ana-
lysed in the 2 angle of 5–80 at a speed of 2◦ min−1 .
2.2.2.4. Mechanical testing. The tensile properties of the prepared
scaffolds were investigated using a mechanical testing machine
(Instron 3369, USA). The samples were cut into rectangular sized
295
slabs with the dimension of 5 cm × 1 cm × 1 mm. The loading rate
was set at 5 mm/min at room temperature. Five scaffolds of same
geometrical dimensions representing each group were subjected in
this study for calculation of the compressive strength and young’s
modulus.
2.2.2.5. In vitro swelling studies. To determine the amount of fluid
absorbed, swelling studies were performed in 1× PBS (phosphate
buffered saline) at physiological temperature and pH. The scaf-
folds were equally weighed and the dry weight of scaffolds was
recorded as (WD ). Scaffolds were immersed in PBS solution for dif-
ferent time intervals of 24 h, 48 h and 72 h. At the end of incubation
period, the scaffolds were taken out and blotted onto filter paper to
remove surface water, and wet weight was recorded as (Ww ). The
experiment was carried out in triplicates. The swelling ratio was
determined by using the formula.
Ww − WD
swelling ratio =
WD
2.2.2.6. Protein adsorption studies. Equally weighed scaffolds were
subjected to prewetting prior to the study; accordingly the scaf-
folds were immersed in 100% ethanol for 1 h and in 1× PBS for
30 min. Then, the scaffolds were incubated in 500 ␮l DMEM media
(containing 10% FBS) at 37◦ C for 24 h and 48 h. After comple-
tion of the incubation period, the scaffolds were removed from
the solution, blot dried and gently washed with PBS 3× times to
remove the free and loosely bound proteins onto the scaffolds. The
adsorbed proteins were eluted by incubating them in 1% SDS for 1 h
with continuous agitation and this was repeated 2× times for com-
plete elution of proteins adsorbed onto the surface of scaffolds. The
eluted samples were then collected and total amounts of proteins
were measured by Qubit fluorometer (Invitrogen, USA).
2.2.2.7. In vitro biodegradation behaviour. The rate of degradation
of the biocomposite scaffolds was studied by the following pro-
cedure: The scaffolds were equally weighed and initial weight
was recorded as WI , followed by immersion in 1× PBS containing
lysozyme similar to that of circulating level in the body (10,000 U/L).
They were incubated at 37 ◦ C for different time intervals (24 h, 48 h
and 72 h). After completion of each incubation period, the scaffolds
were washed with deionized water to remove ions absorbed on
the surface and then blot dried with filter paper. The dry weights of
the scaffolds were noted as (Wt ). The experiment was performed
in triplicates. The degradation percentage was calculated by using
the following formula:
WI − Wt
degradation % = × 100
WI
2.2.2.8. Exogenous biomineralization studies. The biocomposite
scaffolds were equally weighed and immersed in 1× simulated
body fluid (SBF) and then incubated at 37 ◦ C in closed Falcon tubes
for 7 days and 21 days. The SBF solution that mimics our body fluid
containing all necessary minerals was prepared according to the
previously reported literature [14]. After the defined time interval,
the scaffolds were removed and washed 3× times with deionized
water to remove any excess minerals. The scaffolds were finally
lyophilized and subjected to SEM analysis for examination of min-
eralization. The mineralized CS/Alg/nSiO2 scaffolds were ground in
a mortar with liquid nitrogen and subjected to XRD analysis.
2.2.3. Cytotoxicity studies
The osteoprogenitor cells were obtained from 3 days old neona-
tal Wistar rat’s calvarias by an enzymatic digestive method [15]. The
animal ethical committee from the University of Madras approved
296 J.A. Sowjanya et al. / Colloids and Surfaces B: Biointerfaces 109 (2013) 294–300

Fig. 1. SEM images of CS/Alg scaffolds (A) and (B), CS/Alg/nSiO2 scaffolds (C) and (D). CS/Alg/nSiO2 exhibited a well organized porous network, while CS/Alg with less defined
pore architecture.

this protocol. To determine the cytotoxicity of prepared scaffolds,
we used MTT assay. Rat osteoprogenitor cells with the concentra-
tion of 3 × 104 cm−2 were seeded into the wells. Scaffolds weighing
2.5 mg were soaked in 500 ␮l of the DMEM for 24 h. The conditioned
medium was taken at different volume ranges (10, 50 and 100 ␮l)
and made up to 1 ml with medium and added to the wells contain-
ing cells. 0.1% of Triton-X-100 was included as positive control and
DMEM alone used as negative control. They were also incubated for
24 h. After the treatment period the media were drained from the
cells loaded wells, and 500 ␮l of MTT (3-(4, 5-dimethylthiazole-2-
yl)-2,5-diphenyl tetrazolium) solution (0.05%) was added to each
well. Following 4 h incubation at 37 ◦ C, DMSO was used to dissolve
the formazan crystals and optical densities (OD) were determined
at 570 nm using the spectrophotometer (ULTRA SPEC 2100 pro,
Amersham Life Sciences, USA). The positive control was also treated
in the same manner and the results were compared with control
and were finally statistically analysed.
scaled pores. The surface was rough and no well defined pores
were observed. In contrast, the addition of nano-silica to chitosan
and alginate matrix yielded heterogeneous and well defined pores
(Fig. 1C and D) ranging from 20 to 100 ␮m. A minimal decrease in
the porosity of CS/Alg/nSiO2 scaffold was observed when compared
with the CS/Alg scaffolds. Simultaneously a highly interconnected
porosity was also produced by the addition of nano-silica. The
interconnected feature of porosity suits the scaffolds for cell attach-
ment, migration in to the deeper struts and neo-vascularization
[16,17].
Chitosan is a cationic polymer which possesses strong electro-
static interaction with its counterpart anionic polymer alginate.
The interaction is further strengthened by the Ca2+ ions through
calcium chloride crosslinking. These molecular interactions were
studied by FTIR analysis. Fig. 2 indicates the FTIR spectra of CS/Alg

2.2.4. Statistical analysis

In all the experiments performed, a minimum of five samples
were utilized (n = 5). Results are expressed as means of five repli-
cates in each experimental group ± SD. The statistical analysis was
carried out using one-way analysis of variance (ANOVA). The P
value less than 0.05 was considered as significant.

3. Results and discussion

The surface morphology of CS/Alg and CS/Alg/nSiO2 scaffolds

were studied to evaluate pore distribution and their architecture
by SEM analysis. Inspection of the SEM image of CS/Alg scaf-
folds (Fig. 1A and B) showed the porous nature of scaffold with Fig. 2. FT-IR spectra of chitosan, alginate, nano-silica, CS/Alg scaffolds and
pore size ranging from 20 to 150 ␮m with an average of 60 ␮m CS/Alg/nSiO2 scaffolds.
 J.A. Sowjanya et al. / Colloids and Surfaces B: Biointerfaces 109 (2013) 294–300
Fig. 3. XRD patterns of CS/Alg and CS/Alg/nSiO2 scaffolds.
and CS/Alg/nSiO2 scaffolds and represents the spectrum of individ-
ual components used in scaffold fabrication. The alginate spectrum
showed its characteristic peak at 1632 cm−1 corresponding to the
carboxyl group (C O); while chitosan spectrum showed charac-
teristic peaks corresponding to amide I at 1651 cm−1 and amide
II at 1324 cm−1 respectively. The peak at 1080 cm−1 is indicative
of the amine group vibration. Since the chitosan used is 75–85%
deacetylated, thus double amide peaks occurred at 1651 cm−1 and
1324 cm−1 may be due to the partial deacetylation of chitin. The
COOH group of alginate interacts with the NH2 group of chi-
tosan hence there should be a shift in the amino groups and amide
groups of chitosan. But the bands at 1651 cm−1 and 1324 cm−1 were
replaced by a new band at 1313 cm−1 due to interaction of two
COOH groups linking the adjacent chains due to CaCl2 crosslink-
ing. Nano-silica exhibited its characteristic peak at 1088 cm−1
corresponding to symmetric Si-O Si bond vibration. The peak at
3448 cm−1 proved the presence of OH functional group on the
nSiO2 surface. The band shift from 1651 cm−1 to 1631 cm−1 in the
CS/Alg/nSiO2 scaffolds indicates a possible interaction between the
Si OH group of nano-silica and carbonyl group of chitosan.
The XRD analysis of CS/Alg and CS/Alg/SiO2 scaffolds are shown
in Fig. 3. Crystalline nature of the scaffolds determines its mechan-
ical and tensile strength. The XRD of CS/Alg and CS/Alg/nSiO2
scaffolds showed peaks at 22◦ , 24◦ corresponding to 2 values
which attribute to the semi crystalline nature of chitosan ranging
from 20◦ to 25◦ . Both alginate and nano-silica are amorphous in
nature. The coacervation of these polymers may lead to the change
in the phase of the system. From the results it is clear that the semi
crystalline nature of chitosan is unaltered even after addition of
amorphous alginate and nano-silica.
Swelling refers the ability of fluid uptake by the scaffold mate-
rials under physiological conditions. The swelling behaviour of
CS/Alg and CS/Alg/nSiO2 scaffolds was studied by immersing these
scaffolds in 1× PBS solution for 24, 48 and 72 h of incubation
periods (Fig. 4A). The result showed that there was no signifi-
cant change in the swelling ratio between these scaffolds at 24 h
incubation; whereas the inclusion of nSiO2 in CS/Alg scaffolds
decreased the swelling ratios during 48 and 72 h (Fig. 4A). It was
reported that alginate absorbs water very quickly and is capable
of absorbing 200–300 times of its own weight [18]. Due to elec-
trostatic interaction of alginate with chitosan, and crosslinking of
chitosan and alginate with calcium chloride could have resulted
the availability of only few free functional groups to form hydro-
gen bonding with water in CS/Alg scaffolds. Furthermore, addition
of nano silica results in the unavailability of free functional groups
in the CS/Alg/nSiO2 scaffolds, thus the decreased swelling ability
was more prominently observed. The swelling results in the loos-
ening of coiled chitosan chains and leads to an increase in the
pore size of the scaffolds as this enhances better cell infiltration,
more nutrients availing and vascularization. Swelling generally
297
Fig. 4. (A) Swelling behaviour of CS/Alg and CS/Alg/nSiO2 scaffolds in PBS at 37 ◦ C
for 24 h, 48 h and 72 h. Swelling ratio was found to be decreased by addition of
nano-silica particles. *Significant difference compared to CS/Alg scaffolds (P < 0.05)
(n = 5). (B) Mechanical testing of the CS/Alg and CS/Alg/nSiO2 scaffolds indicating
compressive stress and Young’s modulus. *Significant difference compared to CS/Alg
scaffolds (P < 0.05) (n = 5).
increases the surface area of the scaffolds resulting in more cellular
attachment and infiltration. Though the CS/Alg/nSiO2 scaffolds
exhibited decreased swelling, the presence of nano-silica in
the scaffolds could have exhibited more surface area. Increased
swelling also leads to implant loosening, decrease in mechanical
properties and cause stress to the surrounding tissue.
The prepared scaffolds should exhibit certain mechanical
strength for their in vivo applications at load bearing sites. Thus,
the scaffolds were subjected to determine their compressive
stress and young’s modulus. The CS/Alg scaffolds exhibited a
compressive stress and young’s modulus of 0.66 ± 0.02 MPa and
8.99 ± 0.016 MPa, respectively, while the CS/Alg/nSiO2 scaffolds
exhibited in the order of 0.59 ± 0.405 Mpa and 8.16 ± 0.567 MPa,
respectively (Fig. 4B). The addition of nanosilica did not alter the
young’s modulus whereas there was a significant decrease in the
compressive stress.
Implanted scaffolds adsorb various key proteins including
fibronectin, vitronectin and other signalling molecules on their
surface from the circulating body fluids which possess a key role
in regulating the cell attachment and differentiation [19,20]. The
cell binding to the adsorbed proteins triggers various intracellu-
lar events leading to cell attachment, spreading and proliferation.
The amounts of proteins adsorbed by CS/Alg and CS/Alg/nSiO2 scaf-
folds with respect to different incubation periods were determined.
The scaffolds were incubated in DMEM supplemented with FBS
which serves as the source of different serum proteins. As shown
in Fig. 5, CS/Alg/nSiO2 scaffolds showed a marked increase in the
proteins adsorption at the initial period of 1 h incubation. The
initial adsorption of proteins is necessary for attachment of the
cells on to the scaffolds, thereby triggers various cell signalling
pathways and promotes cell spreading and proliferation. On
increasing the incubation period to 4 h and 8 h, the similar trend of
298 J.A. Sowjanya et al. / Colloids and Surfaces B: Biointerfaces 109 (2013) 294–300
Fig. 5. Protein adsorption studies of CS/Alg and CS/Alg/nSiO2 scaffolds in FBS con-
taining DMEM at 37 ◦ C for 1 h, 2 h, 4 h and 8 h. The CS/Alg/nSiO2 scaffolds exhibited
an increased protein adsorption due to the presence of nano-silica. *Significant
difference compared to CS/Alg scaffolds (P < 0.05) (n = 5).
increased protein adsorption was found on CS/Alg/nSiO2 scaffolds.
The significant increase in the proteins adsorbed is due to the
increased surface area achieved by the presence of nano silica par-
ticles. The addition of silica tailored at nano range seems to be
essential for enhancement of the attachment of osteoblasts on the
scaffolds and improve bone formation as reported earlier [21]. The
results are in concordance with our previous report that the addi-
tion nano scaled particles in the scaffolds could increase surface
area, thus increasing the number of sites of protein interaction on
to the scaffolds [22].
The crucial parameter to be considered in bone tissue engi-
neering is controlling the degradation rate of scaffolds as it
provides space for tissue ingrowth and matrix deposition, which
is essential for quality and quantity of bone regeneration [23].
Lysozyme is the primary enzyme responsible for in vivo degra-
dation and it acts on the N-acetyl glucosamine (NAG) group in
the chitosan chain [24]. Scaffolds were incubated in PBS solu-
tion containing lysozyme for 24 h, 48 h and 72 h. A significant
increase in degradation of CS/Alg/nSiO2 scaffolds was observed
compared to CS/Alg scaffolds at all incubation time periods (Fig. 6).
The increased degradation of CS/Alg/nSiO2 than CS/Alg scaffolds
may be due to crosslinking of alginate with calcium ions and
chitosan, and that might have occurred in two different planes
bringing the alginate chains to a much fine compaction. Multiple
crosslinking of alginate with Ca2+ ions and chitosan may lead to
Fig. 6. In vitro biodegradation of CS/Alg and CS/Alg/nSiO2 scaffolds in PBS contain-
ing lysozyme at 37 ◦ C for 24 h, 48 h and 72 h. Addition of nano-silica to CS/Alg matrix
increased susceptibility to lysozyme scission and hydrolytic action. *Significant dif-
ference compared to CS/Alg scaffolds (P < 0.05) (n = 5).
greater alginate stability. But, this interaction could have been
affected by the addition of nano-silica thus weaking the interaction
in the system leading to increased degradation of CS/Alg/nSiO2 scaf-
folds. In this study, the degradation behaviour of the scaffolds was
performed in PBS containing lysosyme. The scaffolds would interact
with many other factors under in vivo environment which would
influence the degradation in vivo. Thus, there is possibility that the
degradation as seen in in vitro would not be the same under in
vivo.
Exogenous biomineralization of CS/Alg and CS/Alg/nSiO2 scaf-
folds was studied by immersing the scaffolds in 1X SBF solution
(Fig. 7). The scaffolds were incubated for 7 days, and 21 days and
mineral deposition was analysed by SEM. CS/Alg scaffolds showed
fewer deposition of apatite layer after completion of 7 days incu-
bation period (Fig. 7A), whereas CS/Alg/nSiO2 scaffolds deposited
comparatively more apatite crystals (Fig. 7C). After 21 days of incu-
bation period, CS/Alg/nSiO2 scaffolds showed more prominent and
increased deposition of apatite crystals (Fig. 7D) than CS/Alg scaf-
folds (Fig. 7B). The small round inset in the figures indicates the
magnification of a small portion of regions containing apatite crys-
tals. The presence of nano-silica in CS/Alg/nSiO2 scaffolds may
initiate formation of a calcium phosphate layer due to electro-
static interactions of negatively charged Si O units (formed by
dissociation of the Si OH groups) with the positively charged cal-
cium ions in the SBF solution. The formed calcium silicate layer
exhibits a positive charge which may interact with the negatively
charged phosphate ions in the SBF leading to the formation of
amorphous calcium phosphate (ACP). Then, it stabilizes to form
a nano-crystalline apatite layer which may possess significant role
in recruiting cells to the site of implantation. The rate of apatite
formation depends on scaffolds’ morphology, and pores may act as
nucleation sites where the degree of supersaturation for precipita-
tion of apatite layer is attained more rapidly [25]. The exogenous
mineralized CS/Alg/nSiO2 scaffolds (Fig. 7) were also subjected to
XRD analysis for determining the phase of crystals deposited on
to the scaffolds. The XRD patterns of CS/Alg/nSiO2 scaffolds col-
lected at the end of 7 days (Fig. 7C, inset) and 21 days (Fig. 7D,
inset) of biomineralization exhibited the presence of hydroxyap-
atite deposition. The crystalline peaks at 26.7◦ , 28.8◦ , 31.9◦ , 32.7◦ ,
45.7◦ indicated the presence of nanohydroxyapatite growth. These
peaks corresponded to the (2 1 1), (1 1 2) and (3 0 0) lattice planes of
HAp which is in strong agreement with the JCPDS card # 09-0432.
Thus, this result indicated the deposition of hydroxyapatite growth
on to the scaffolds.
Biomaterials intended for in vivo applications should not exhibit
toxicity to mammalian cells. Thus, the prepared CS/Alg/nSiO2
scaffolds were subjected to cytotoxicity using MTT assay on rat
primary calvarial osteoprogenitor cells and human osteosarcoma
cell lines (HOS). This is a colorimetric study based on ability of
cellular mitochondrial dehydrogenase in the reduction of yel-
low coloured tetrazolium salt to blue coloured formazan crystals.
The scaffolds were incubated for overnight in DMEM and the
conditioned media were collected. 10, 50 and 100 ␮l of con-
ditioned media were incubated with either rat osteoprogenitor
(Fig. 8A) or human osteoblastic cells (Fig. 8B) for a period of
24 h and at the end of incubation period, MTT assay was per-
formed as mentioned in methods section. No significant toxicity
of these scaffolds towards these two types of cells was observed.
A long term MTT assay was also performed for an incubation
period of 72 h with human osteoblastic cells (MG63) (Fig. 8C).
The results indicated that there was no significant loss of cell
viability; instead there was a significant increase in cell via-
bility observed at three different concentrations of conditioned
media. A significant loss of cell viability was observed when cells
were incubated with 0.1% Triton X-100 which served as positive
control.
 J.A. Sowjanya et al. / Colloids and Surfaces B: Biointerfaces 109 (2013) 294–300 299

Fig. 7. Exogenous biomineralization of CS/Alg scaffolds (A), (B) and CS/Alg/nSiO2 scaffolds (C), (D). Apatite deposition was more prominently seen on to the CS/Alg/nSiO2
scaffolds after 7 days (C) and 21 days (D) of incubation in the simulated body fluid solution. Inset shows apatite crystal formation and XRD patterns.

Fig. 8. Cytotoxicity assay of CS/Alg/nSiO2 scaffolds using (A) rat primary calvarial osteoblasts (osteoprogenitor cells), (B) human osteoblastic cells (HOS) and (C) human
osteoblastic cells (MG63). *Significantly decreased compared to control. **Significantly increased compared to control (P < 0.05) (n = 5).

4. Conclusions Furthermore, nano-scaled silica was added to improve the

bioactivity of scaffolds. The well-defined 3D-porous architec-
In this study, we prepared the biocomposite scaffolds contain- ture and increased protein adsorption proved the advantages of
ing chitosan and alginate by utilizing strong ionic interaction of CS/Alg/nSiO2 over CS/Alg scaffolds. The controlled biodegrada-
the amine groups of chitosan and carboxyl groups of alginate. tion and enhanced apatite deposition of CS/Alg/nSiO2 scaffolds by
300 J.A. Sowjanya et al. / Colloids and Surfaces B: Biointerfaces 109 (2013) 294–300
nano-silica would definitely promote bone formation that is essen-
tial in bone tissue engineering.
Acknowledgements
We thank S. Balaji and S. Vimalraj for their technical help during
preparation of the manuscript. We also thank the Nanotechnology
Research Centre and School of Pharmacy, SRM University for pro-
viding instrumental facilities. This work was, in part, supported by
the Indian Council of Medical Research, India (grant nos. 5/13/2009-
NCD-III; 80/10/2010-BMS to N.S.) and the Council of Scientific and
Industrial Research, India (grant no. 37(1574)/12/EMR-II to N.S.).
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