Artificial Cells, Nanomedicine, and Biotechnology

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Biocompatible agarose-chitosan coated silver

nanoparticle composite for soft tissue engineering
applications

Nupur Kumar, Dayananda Desagani, Girish Chandran, Narendra Nath

Ghosh, Ganesh Karthikeyan, Sachin Waigaonkar & Anasuya Ganguly

To cite this article: Nupur Kumar, Dayananda Desagani, Girish Chandran, Narendra Nath Ghosh,
Ganesh Karthikeyan, Sachin Waigaonkar & Anasuya Ganguly (2017): Biocompatible agarose-
chitosan coated silver nanoparticle composite for soft tissue engineering applications, Artificial
Cells, Nanomedicine, and Biotechnology, DOI: 10.1080/21691401.2017.1337021

To link to this article: http://dx.doi.org/10.1080/21691401.2017.1337021

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ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY, 2017
https://doi.org/10.1080/21691401.2017.1337021

Biocompatible agarose-chitosan coated silver nanoparticle composite for soft

tissue engineering applications
Nupur Kumara, Dayananda Desaganib, Girish Chandranc, Narendra Nath Ghoshb, Ganesh Karthikeyanc,
Sachin Waigaonkarc and Anasuya Gangulya
a
Department of Biological Science, BITS-Pilani, K.K Birla Goa Campus, Zuarinagar, India; bDepartment of Chemistry, BITS-Pilani, K.K Birla Goa
Campus, Zuarinagar, India; cDepartment of Mechanical Engineering, BITS-Pilani, K.K Birla Goa Campus, Zuarinagar, India

ABSTRACT ARTICLE HISTORY

With increasing gap in the demand and supply of vital organs for transplantation there is a pressing Received 15 December 2016
need to bridge the gap with substitutes. One way to make substitutes is by tissue engineering which Revised 6 May 2017
involves combining several types of synthetic or biomaterials, cells and growth factors cross-linked Accepted 29 May 2017
together to synthesize a functional scaffold for repair or replacement of non-functional organs.
Nanoparticle based composites are gaining importance in tissue engineering due to their ability to KEYWORDS
enhance cell attachment and proliferation. The current study focuses on synthesizing agarose compo- Agarose; chitosan; silver
sites embedded with chitosan-coated silver nanoparticles using glutaraldehyde as the cross-linker. The nanoparticles; tissue
synthesis of chitosan coated silver nanoparticles within the scaffold was confirmed with UV-visible spec- engineering; scaffolds
troscopy. Physical and chemical characterization of the synthesized nanoparticles were done by XRD,
FTIR, TGA and SEM. DMA showed higher mechanical strength of the scaffolds. The scaffolds showed
degradation of
37% within a span of four weeks. The higher physical support provided by the synthe-
sized scaffolds was shown by in-vitro cell viability assay. Broad spectrum anti-bacterial activity and
superior hemocompatibility further showed the advantage it offered for growing cells. Thus a biopoly-
mer based nanocomposite was synthesized, with intended widespread use as scaffold for engineering
of soft tissues due to its enhanced biocompatibility and greater surface area for cell growth.

Abbreviations: XRD: X-ray diffraction; FTIR: Fourier transformation Infrared spectroscopy; TGA:
Thermogravimetric analysis; DMA: Dynamic mechanical analysis; SEM: Scanning electron microscopy;
DAPI: 4',6-diamidino-2-phenylindole; DMSO: Dimethyl sulfoxide; DMEM: Dulbecco's modified eagle
medium; FBS: Fetal bovine serum; MTT: 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide;
NADP: Nicotinamide adenine dinucleotide phosphate; FCC: Face centered cubic; ECM: Extracellular
matrix

Introduction These continuing necessities of organs have made scien-

tists to search for alternatives which have paved way for the
Every single day thousands of people undergo surgery or sur-
field of tissue engineering. Tissue engineering aims to create
gical procedures to replace or repair damaged tissues. Tissue
biological substitutes that can replace defective or damaged
damage due to disease, trauma or injury mostly involves
tissues which will help in restoring or maintaining tissue func-
transplantation of tissue from one site from same person to
another or from another person. While there has been a revo- tion [3]. The developing field aims to regenerate damaged
lution in the field of transplantation, it has its shortfall of tissues by growing cells on the scaffold, which acts as a tem-
being expensive and unavailability of donors. Despite con- plate for tissue regeneration. Tissue engineering is an inter-
stant awareness about organ donation and transplantation, disciplinary field which applies the principles of engineering
the gap between supply and demand is constantly increas- and cell science together for developing substitutes for dam-
ing. As per the united network for organ donation (UNOS) aged organs.
report, till February 2015, there were 123,204 people waiting Scaffolds along with cells and bioactive molecules or
for organ transplant and each day around 22 people die wait- growth factors form the tissue engineering triad [4]. Correct
ing for the transplant [1]. This gap between organ supply and combination of these three components aids in development
demand has raised several ethical, moral and societal issues of a substitute for growing damaged tissues. Tissue engineer-
regarding voluntary donors. Moreover, there are various other ing relies heavily on synthesizing a 3D scaffold which could
problems associated with organ transplantation such as low act as a template for regeneration of tissues. In order for the
availability, high cost, graft rejection and surgical complica- cells to assemble and function properly it is important that
tions because of which live organ transplantation might be the 3D scaffold acts as a natural microenvironment.
unsuccessful [2]. Numerous scaffolds have been synthesized using different

CONTACT Anasuya Ganguly [email protected] Department of Biological Science, BITS-Pilani, K.K Birla Goa campus, Goa 403726, India
Supplemental data for this article can be accessed here.
ß 2017 Informa UK Limited, trading as Taylor & Francis Group
2 N. KUMAR ET AL.

biomaterials, but regardless of the tissue to be grown on it In recent years, research has shifted towards understand-
number of key factors determines the suitability of the scaf- ing the biology behind the extracellular matrix that provides
folds for application [3]. The most important of them being the cells with necessary foundation to grow and survive.
biocompatibility of the scaffold, allowing the cells to adhere, Different organs in the body have different compositions of
grow and migrate. Secondly the scaffold should possess time extracellular matrix, but most of the ECM has been shown to
dependent degradation thereby replacing the defective tissue consist of various nanostructures. These nanostructures not
over time [5]. Furthermore, the scaffolds should possess archi- only provide strong frame work for the cells but also help in
tecture with interconnected pores and with high porosity cell attachment and subsequent proliferation due to
allowing diffusion of nutrients and wastes [6]. increased surface area [25,26]. Therefore, including nanopar-
As scaffolds are vital elements of tissue engineering, a var- ticles within the scaffolds not only helps in replicating the
iety of fabrication techniques have been employed for natural ECM, it would also aid in cell attachment and
designing them. These techniques include freeze drying, solv- proliferation.
ent casting, gas foaming, electrospining, micromolding, etc. The current study, taking into consideration the compli-
[7]. Freeze drying or lyophilization is one of the most com- cations in using synthetic polymers has incorporated chito-
monly used fabrication technique. This procedure works on san coated silver nanoparticles into Agarose scaffolds. The
the principle of sublimation where the polymer is mixed with biogenic polymers provide the advantage of reduced tox-
a solvent and frozen. Later this solvent is removed by the icity and increased surface area for cell attachment and
process of lyophilization. Lyophilization reduces the pressure proliferation. Though the usage of nanoparticles is not new
of the system which will allow the water in the frozen poly- to tissue engineering, but the combination of using
mer solution to sublimate directly from solid to gas phase Agarose as the scaffold along with chitosan nano-formula-
thereby giving porous and interconnected structures [8,9]. An tion as the framework for cell attachment with glutaralde-
ideal scaffold should have certain favourable properties such hyde as the crosslinking agent is for the first time. Various
as biocompatibility, biodegradability, mechanical properties cell lines has been shown to attach and grow on the
such that they provide support till the time new ECM is scaffold.
formed, surface that promotes cell growth and attachment,
porosity, etc. [10].
The basis of any scaffold lies in the polymer that is being Materials and methods
used to construct the same. Polymers are of two types: nat- Bacterial strains and reagents
ural polymers and synthetic polymers [11]. Agarose is a natur-
ally occurring polysaccharide isolated from red purple sea Staphylococcus aureus, Bacillus subtilis, Escherichia coli and
weed. It has been extensively used for fabrication of scaffolds Klebsiella pneumonia were grown in Luria-Bertani (LB) broth
because of properties like biodegradability, soft tissue like at 37  C on a shaker at 180 rpm. Chitosan, Silver nitrate and
mechanical property, porosity which helps in cell spreading DAPI mounting media (FluoroshieldTM) were purchased from
and proliferation [11]. Agarose forms strong gels even at low Sigma Aldrich chemical Co. (St. Louis, MO). Agarose
concentrations due to its specific chemical structure. Agarose (UltrapureTM) was purchased from Invitrogen. Glutaraldehyde
has been reported to have application in gene delivery, as solution (25%, for synthesis) was purchased from MERCK.
drug delivery systems, hydrogel for maxillofacial and oral sur- Sodium hydroxide, acetic acid and DMSO were acquired from
geries, etc. [11]. One of the major disadvantages associated Fischer Scientific. Dulbecco’s modified eagle’s media (DMEM),
with agarose is the fact that it lacks the ability of cell attach- foetal bovine serum (FBS), 0.25% trypsin, phosphate buffer
ment, which limits its role in this field [12]. To overcome this, saline, 3-(4,5, di methylthiazol-2-yl)-2,5-diphenyl tetrazolium
various blends of agarose and other polymers including gel- bromide (MTT) were obtained from Hi-media, India. HeLa cell
atin, chitosan, fibrin, etc., have been used for tissue engineer- line (human cervical carcinoma cell line), MiaPaCa2 (human
ing applications [12,13]. Also in combination with other pancreatic epithelial carcinoma cell line) and HEK (human
polysaccharides, agarose gel provides moist environment and embryonic kidney cell line) were procured from National
enhance the stability of the system. Centre for Cell Sciences (Pune, India). All the other chemicals
Chitosan is a positively charged linear polysaccharide used in this study were of analytical grade.
composed of b-(1-4)-linked D-glucosamine (deacetylated
unit) and N-acetyl-D-glucosamine. It is the most commonly
Synthesis of chitosan coated silver nanoparticles
used polymer in the field of tissue engineering because of
its remarkable properties which includes biodegradability, Chitosan-coated silver nanoparticles were synthesized using a
biocompatibility, non-antigenicity, anti-bacterial properties, protocol mentioned by Jena et al. [27]. Chitosan was pre-
etc. [14–17]. Chitosan forms secondary interactions (via pared at a concentration of 1 mg/ml at 45  C with constant
hydrogen bond) with other polymers as it has polar groups stirring. 0.1 N sodium hydroxide followed by 100 mM silver
present in its chemical structures. Therefore, chitosan has nitrate was added to the mixture on a continuous basis.
found application in drug delivery systems, nutrition supple- Immediate colour change to dark yellow suggested the for-
ment and in wound healing. Chitosan blended with other mation of chitosan coated silver nanoparticles. The suspen-
polymers have been used in regeneration of almost all the sion was then allowed to settle and later centrifuged at
organs including liver, kidney, pancreatic islets, heart, bone, 2000 rpm. The pellet was washed with distilled water twice,
etc. [18–24]. dried and dissolved in 0.1% acetic acid.
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 3

Figure 1. Preparation of Agarose-chitosan-coated silver nanoparticles (AG-CHNp) scaffolds. 3% agarose solution was prepared in deionised water. Different concen-
trations of chitosan (1 mg/ml, 2 mg/ml, 3 mg/ml and 4 mg/ml) were added to it from the stock solution (10 mg/ml) followed by sodium hydroxide and silver nitrate
to a final concentration of 0.1 M and 100 mM, respectively, to give chitosan-coated silver nanoparticles embedded in the agarose solution. The AG-CHNp scaffolds
were prepared by cross linking the gel using glutaraldehyde (final concentration -1%) followed by overnight lyophilization.

Nanoparticle characterization chitosan dissolved in acetic-acid aqueous solution was added

to Agarose and allowed to mix for 15 min under constant stir-
In conjunction with the colour change, various other parame-
ring. The nanoparticles were synthesized as described above,
ters were used to characterize chitosan-coated silver nanopar-
leading to an immediate colour change into brownish yellow.
ticles. Synthesized nanoparticles were characterized by UV
Finally, the scaffold was prepared by using a cross linker, i.e.
spectroscopy using Shimadzu UV-2450 spectrophotometer.
glutaraldehyde (1%) for one hour (Figure 1). The synthesized
For transmission electron microscopy (TEM), a drop of aque-
scaffolds would be denoted as AG-CHNp1, AG-CHNp2, AG-
ous solution of chitosan coated silver nanoparticles was
CHNp3 and AG-CHNp4 according to the increasing concentra-
placed on the carbon-coated copper grids. The samples were
tion of chitosan used. The synthesized scaffolds were frozen
dried and kept overnight under a desiccator before loading
them onto a specimen holder. TEM measurements were per- at 80  C for 24 h, followed by lyophilization for 16–18 h.
formed on JEM-2100, HRTEM, JEOL, JAPAN operating at Freeze-drying will cause sublimation of ice crystals directly
200 kV. The size distribution and zeta potential of nanopar- into vapour phase which will in turn cause formation of por-
ticles were determined by Dynamic Light Scattering (DLS) ous structures.
using Zeta sizer nano-ZS Malvern Instruments, UK at Room
Temperature. 1 ml of sample was mixed well by vortexing, Physical characterization of scaffolds
poured into disposable sizing cuvette for size distribution
analysis. Three different peak readings, size versus percentage The synthesized scaffold were characterized by several
intensity were plotted. Three such readings were taken and physio-chemical methods. UV-visible spectroscopic analysis of
average size distribution was determined. Similarly, zeta the scaffold just before gelation was performed to confirm
potential was analyzed with help of clear disposable zeta cell. the synthesis of embedded nanoparticles. The crystalline
Average of three readings was taken to determine zeta structure of the scaffolds were studied using X-ray diffraction.
potential distribution The XRD patterns were generated using Rigaku MiniFlex II at
room temperature operating at a voltage of 30 kV. The read-
ings were taken at 2h angle range from 5 to 50˚. For analyz-
Synthesis of Agarose scaffolds impregnated with
ing the chemical bonding and functional groups present
chitosan coated silver nanoparticles
within the scaffold, FTIR analysis was performed using
Agarose was dissolved in water to prepare a 3 wt% polymeric Shimadzu IR Affinity-I with the help of an attenuated total
solution. The chitosan coated silver nanoparticles were syn- reflectance (ATR) accessory. The FTIR spectrum was analyzed
thesized with the Agarose solution. Different amounts of from 400-4000 cm1. To examine the thermal stability of the
4 N. KUMAR ET AL.

scaffolds, TGA was performed using Shimadzu DTG-60. The Assessment of anti-bacterial activity
samples were heated from 30 to 600 C with a heating rate of
Scaffolds were also analyzed for their anti-bacterial activity
10 C/minute. The storage and loss modulus of the scaffolds
against both Gram positive and Gram negative bacteria by
was examined by dynamic mechanical analyser using
resazurin assay. The scaffolds were casted in a 96 well
Universal VA.5 A TA instruments.
plate (approximately 150 ll) and different concentration of
bacterial cells (104, 105 and 106 cfu of bacteria) were
Determination of degree of swelling of scaffolds added. The plate was incubated at 37 C overnight.
Following day, resazurin dye was added to all the wells
The degree of swelling for a scaffold was studied using phos-
and incubated for 3-4 h. Resazurin levels were quantified
phate buffer saline (pH ¼ 7.4) as described previously by
spectrophotometrically at 570 nm with a reference wave-
Srinivasan et al. [28]. The dry weight of scaffolds was meas-
length of 600 nm [30].
ured before immersing in PBS solution for various time inter-
vals (days 1, 3, and 7). On the respective day, the scaffolds
were removed and excess solution was drained out by blot- MTT assay
ting onto a filter paper. Following which, the wet weight of
To assess the biocompatibility of the scaffolds, MTT assay
the scaffolds were measured and swelling ratio was calcu-
was performed. AG-CHNp scaffolds were analyzed for their
lated as follows
cytotoxicity with the help of HeLa cell line as previously
Wet weight  Dry weight described by Bhat et al. [31]. Scaffolds were placed in an
Swelling Ratio ¼
Dry Weight uncoated 24 well plate and sterilized using increasing con-
centration of ethanol followed by UV radiation for 20 min.
The scaffolds were then equilibrated with complete media
In vitro degradation studies to facilitate gaseous exchange for 2–4 h. Following this,
HeLa cells were seeded at a concentration of 1X105 cells
In vitro degradation of the scaffolds was studied as previously
per well and incubated for a period of 16 days at 37 C
described by Srinivasan et al. [28]. Initial weight of the scaf-
with 5% CO2. For control, same cell density was seeded in
folds was noted down (Wri) and then were incubated in
poly-lysine coated 24 well plates. Media was changed every
phosphate buffer saline containing lysozyme (10,000 units/
alternate day. Media was removed from the well and the
ml) at 37  C for different time periods (7, 14, 21, 28 days).
scaffolds were given a gentle PBS wash before addition of
Scaffolds were respectively removed, washed with deionized
MTT at 0.5 mg/ml for 3–4 h. After incubation, media was
water and lyophilized. The final weight of the scaffolds was
aspirated out and the MTT crystals were dissolved with
noted (Wf).
DMSO and incubated for 15–20 min for colour develop-
Percentage degradation was calculated using the following
ment. Absorbance was measured at 570 nm to calculate
formula:
number of viable cells.
Wi  Wf
Rate of degradation ¼  100
Wi
Microscopic analysis (DAPI staining)

Estimation of hemocompatibility To visualize the attachment and proliferation of cells on the

scaffolds, two cell lines (MiaPaCa2 and HEK) were seeded on
Estimation of hemocompatibility was done using protocol the scaffolds and incubated at 37 C with 5% CO2 for a period
given by Pal et al. [29]. Fresh human blood was collected in of five days. On the day of experiment, the media was aspi-
15 ml centrifuge tube containing sodium citrate (10:1). The rated out and the scaffolds were washed with PBS. Later the
blood was then diluted with normal saline (8 ml blood cells were fixed with 2.5% glutaraldehyde and dehydrated
þ10 ml saline). To study hemolysis, scaffolds were cut into using ethanol gradient. Various sections of the fixed scaffold
5mmX5mm size and placed in a tube containing normal were cut and the best obtained sections were stained with
saline solution and incubated for 30 min at 37 C. Diluted DAPI containing mounting media for 15-20 min. Following
blood was added to the tube and incubated for 60 min at incubation, sections were observed under fluorescence micro-
37 C. The positive control had diluted blood added to scope using excitation (405 nm) and emission wavelength
sodium carbonate solution which caused hemolysis and the (450 nm) of DAPI.
negative solution had blood in normal saline solution.
Following the incubation, all the tubes were centrifuged for
5 min at 3000 rpm. The supernatant was transferred to the
Scanning electron microscopy (SEM) analysis
cuvette and readings were taken at 545 nm.
Percentage hemolysis was calculated with the following The microstructures of the prepared scaffolds were examined
formula: by SEM (FEI Nova Nano FESEM 450; FEI, Hillsboro, OR).
OD ðtestÞ  OD ðNegative ControlÞ Sample preparation is same as above. All the samples were
 100 gold coated and scanning was carried at voltage of 10 kV (for
OD Positive ControlÞ  OD ðNegative controlÞ
ð
visualizing the surface of the scaffolds) and 5 kV (for visualiz-
ing MiaPaCa and HEK cells on the scaffolds).
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 5

Statistical analysis measured by zeta potential was found be positive

(Supplementary Figure S(d)).
Statistical analysis was performed with GraphPad Prism v 5.0
(GraphPad Software, La Jolla, CA, (http://www.graphpad.com).
Significance was referred as  for p < .0001,  for p < .001, Synthesis and characterization of AG-CHNp scaffolds
 for p < .05, ns for non-significant.
Using a 3% polymeric solution of Agarose and varying con-
centration of chitosan coated silver nanoparticles as the base
four scaffolds were synthesized. The chitosan nanoparticles
Results
were synthesized inside the scaffolds to obtain a uniform and
Synthesis and characterization of chitosan coated silver porous nanoparticle base. The synthesis was confirmed by UV
nanoparticles spectroscopy and visible colour change (Figure 2(a)).
Glutaraldehyde, shown to be an effective crosslinking agent
Ultraviolet visible spectroscopy showed a prominent and
for stabilization of several kinds of biomaterials was used to
characteristic peak at 420 nm, suggesting the formation of obtain a cross-linked Agarose scaffold.
chitosan-coated silver nanoparticles (Supplementary Figure To identify the components of the synthesized scaffold
S(a)). Shape, size and crystalline nature of the synthesized and its crystallinity, powder XRD was performed. Obtained
nanoparticles were determined by TEM studies. TEM micro- peaks were analyzed to identify individual components of the
graphs showed monodispersed and spherical nanoparticles synthesized scaffold. A peak at 2h ¼ 20 was identified to be
[32] (Supplementary Figure S(b)). Size distribution profile of of chitosan, which corresponds to previous literature and the
the synthesized nanoparticle were determined by dynamic single peak indicates its semi-crystalline nature (Figure 2(b))
light scattering. Three peaks corresponding to 23.12, 353.1 [33]. Pure agarose also exhibits a single peak at 2h ¼ 20 . This
and 4841 nm were obtained suggesting that most of the par- can be due to the amorphous nature of agarose. XRD pattern
ticles synthesized were below 500 nm (Supplementary Figure for silver nitrate shows distinct diffraction peaks at around
S(c)). But a fine peak at 4800 nm suggested agglomeration of 25 , 37 , 45 , 70 and 76 which are characteristics of (110),
a small proportion of nanoparticles. The degree of stability of (111), (200), (220) and (311) planes of Face centred cubic
the colloidal dispersion and degree of surface potential as (FCC) silver, respectively [18,34]. Presence of peak at 2h ¼ 20

Figure 2. (a) UV-visible absorption spectrum of AG-CHNp scaffolds. AG-CHNp scaffolds showed a prominent peak at 420 nm (characteristic of silver nanoparticles) as
compared to agarose control (AG control) and agarose-chitosan control (AG-CH control). (b) X-ray diffraction analysis of the AG-CHNp scaffolds and controls. The dif-
fraction pattern of all the four scaffold (AG-CHNp1 (iv), AG-CHNp2 (v), AG-CHNp3 (vi), AG-CHNp1 (vii)) shows characteristic peaks of the polymers, i.e. agarose and
chitosan at 2h ¼ 20˚. It also showed peaks for silver nitrate suggesting that all the polymers and silver nitrate were present in the scaffolds. (i), (ii), (iii) shows diffrac-
tion profile for the silver nitrate, chitosan and agarose respectively.
6 N. KUMAR ET AL.

Figure 3. (a) FTIR spectrum of all the four AG-CHNp scaffolds and controls (agarose, chitosan and silver nanoparticles). Typical peaks for –OH stretching, –CH axial
deformation, –C–O axial deformation and 3,6-anhydrogalactose were visible in the agarose spectrum. Chitosan spectrum showed specific peaks for –OH group and
–NH2 group and O–C–O group. All the AG-CHNp scaffolds showed peaks from both the polymers and presence of peak at 1645 cm1 attributed to presence of imine
group (C¼N) confirms bond formation between aldehyde group of glutaraldehyde and amino group of chitosan. (b) TGA profile of AG-CHNp scaffolds and agarose
control. Thermal profile for scaffolds showed two transition temperatures First at 75–100  C due to moisture vaporization and second 200–250  C due to polymer
degradation. As compared to control, AG-CHNp scaffolds showed improved thermal stability.

and silver nitrate peak in the AG-CHNp scaffolds confirms the characteristic peaks at around 3300 cm1 attributed to –OH
presence of chitosan coated silver nanoparticles. To study the stretching, 2913 cm1 attributed to –CH axial deformation,
conformational changes in the functional groups of the poly- 1083 cm1 for –C-O axial deformation and 946 cm1 for 3,6
mers present in the scaffolds, FTIR analysis was performed. anhydrogalactose [12,35]. Chitosan showed its characteristic
The FTIR spectrum for all the scaffolds obtained from ATR- peaks at around 3400 cm1 (–OH and –NH2 groups),
FTIR spectrometry is depicted in Figure 3(a). Agarose showed 1553 cm1 and 1370 cm1 (amino groups) and 1080 cm1
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 7

Figure 4. (a) Dynamic mechanical analysis of the AG-CHNp scaffolds. The mechanical strength of the scaffolds was found to be in the range of 5–8 MPa which shows
its application in soft tissue engineering. (b) Swelling profile of Agarose control and AG-CHNp scaffolds. All the AG-CHNp scaffolds showed increase in swelling till
the final time point. As compared to the control, scaffolds showed lower swelling ratio possibly because of additional cross-linking resulting in strong interaction
between the polymer and nanoparticle thereby restricting entry of water.

(O–C–O group) [36]. The presence of combination of func- and chitosan. The profile also suggests improved thermal sta-
tional groups from both the polymers is clearly visible in bility of the scaffolds as compared to the controls as there is
spectrum of all the four AG-CHNp scaffolds. The presence of a significant difference in the complete degradation rates
peak at around 1645 cm1 (C¼N) can be attributed to gluta- which can be attributed to the presence of chitosan-coated
raldehyde crosslinking within the composite [18,35]. silver nanoparticles.
Thermal stability of the compounds was assessed by
Thermogravimetric analysis, with the profile showing two
prominent stages (Figure 3(b)). First transition occurs Mechanical strength and elasticity of the AG-CHNp
between 75 and 100 C, which can be attributed to moisture scaffolds
vaporization causing the weight loss. The second transition
occurs between 200 and 250 C. This can be due to decom- When a specific amount of force is applied to a scaffold, it
position of both the polysaccharide polymers, i.e. agarose tends to deform which can cause changes in its storage
8 N. KUMAR ET AL.

modulus and loss modulus. A scaffold should be able to pro-

vide adequate mechanical strength so that it can sustain the
wear and tear in the body. For soft tissues, mechanical
strength in the range of 0.4–350 MPa has been found to be
suitable [37]. The results were shown in the form of graph
plotted between storage modulus, loss modulus and percent-
age strain against applied force. The value of all the four
AG-CHNp scaffolds was found be in the range of 5–8 MPa
(Figure 4(a)). A decrease in elastic deformation (indicated by
storage modulus) and viscous response of the scaffold (as
indicated by loss modulus) is evident. The strength of the
scaffold was found to increase with strain, thereby showing
the strain hardening effect. With the excellent mechanical Figure 5. In-vitro degradation profile of all AG-CHNp scaffolds. The degradation
strength and spongy-like property, AG-CHNp scaffolds show profile for all four scaffolds showed optimum rate of 35–39%. This profile shows
additional advantage in tissue engineering of soft tissues sustained and continuous degradation of the scaffolds which is also important
for the cells to secrete their own ECM.
such as pancreas, liver, heart, etc.

Table 1. Hemocompatibility assay of AG-CHNp scaffolds.

Swelling test
Percentage
Sample O.D at 545nm hemolysis Remarks
The ability of the scaffolds to retain liquid or water is an
Positive control 0.785
important property for tissue engineering applications. The Negative control 0.0125
swelling studies conducted on the scaffold are presented in AG-CHNp1 0.0255 1.68 Highly hemocompatible
Figure 4(b). Dry weight of the scaffolds was noted and then AG-CHNp2 0.031 2.39 Highly hemocompatible
AG-CHNp3 0.020 0.97 Highly hemocompatible
soaked in PBS for a period of 7 days. Readings were taken at AG-CHNp4 0.020 0.97 Highly hemocompatible
three different time intervals and swelling ratio was calcu- Hemocompatibility for all the four scaffolds was tested using fresh human
lated. All the samples showed increase in swelling till the blood. Positive control used was blood diluted with sodium carbonate caus-
final time point. The scaffolds showed lesser swelling ratio as ing haemolysis and negative control had blood diluted with normal saline.
Samples after equilibration were incubated with blood and then absorbance
compared to the control. This can be due to strong inter- was taken. Percentage haemolysis was calculated using absorbance of the
action between agarose and chitosan-coated silver nanopar- controls and samples (AG-CHNp scaffolds). For all the four AG-CHNp scaf-
ticles. This strong interaction can result in the formation of folds, the percentage haemolysis was found to be less than 5%. Therefore,
the scaffolds are highly hemocompatible.
additional cross links which can restrict the entry of water.
Our results were in accordance with what has been previ-
ously observed [18,38]. The strong water retaining capacity Therefore, it is important that the scaffolds degrade with
of the synthesized scaffolds could be attributed to their time and degradation should be optimum. The above result
hydrophilicity and a 3-D architecture. Understanding the suggests that these formulated scaffolds are biodegradable
degree of swelling is very critical for tissue engineering as and have an optimum rate of degradation which can allow
swelling will cause increase in pore size thereby helping in cells to secrete their own extracellular matrix.
movement of oxygen and nutrient in and out of the
tissue [34].
Acceptable hemolysis of the scaffolds
The degree of hemocompatibility of the biomaterials refers to
In vitro biodegradability
the degree of mutual interaction between the components of
The in vitro degradation profile of all the AG-CHNp scaffolds the scaffolds and blood. Though the scaffolds were found to
is shown in Figure 5. Rate of degradation was measured be porous, it is important that the scaffolds should not alter
using phosphate buffer saline (pH 7.4) containing lysozyme the integrity of blood when they come in contact with it. To
for a period of 28 days. The degradation rate was measured check the degree of haemolysis of the synthesized scaffolds,
in terms of change in dry weight of the scaffolds at a given the scaffolds along with appropriate controls were incubated
time interval. All the scaffolds showed a gradual rate of deg- with equal amount of diluted blood. Hence, hemocompatibl-
radation with time and were about 35–39% after 4 weeks of ity of AG-CHNp scaffolds was analyzed and is shown in
incubation. This result is in accordance with the previously Table 1. Hemolysis is calculated in terms of percentage and
published reports by Rad et al. and Frydrch et al. where the can be placed in one of the following categories [29]:
scaffolds showed a degradation rate of 36.85% (7 days) and
1. Highly hemocompatible (<5% haemolysis)
55–56% (31 days) respectively [39,40]. The degradation can be
2. Hemocompatible (5–10% haemolysis)
primarily because of lysozyme action on the macromolecules
3. Non-hemocompatible (>20%)
and degrading it to smaller chains. When the scaffold is
implanted in the body, cells grow on its surface and simul- The percentage hemocompatiblity of the synthesized
taneously secretes its own extra cellular matrix, i.e. rate of scaffold was found to be less than 5% suggesting that it lies
degradation should match the rate of tissue formation. within the acceptable range and is highly compatible.
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 9

Figure 6. Estimation of anti-bacterial activity of AG-CHNp scaffolds using resazurin assay. (A) Scaffolds were tested for their anti-bacterial activity using resazurin
dye. All the four scaffolds showed no change in resazurin as compared to the control. (a) Escherichia coli2345, (b) Bacillus subtilis, (c) Klebsiella pneumoniae and (d)
Staphylococcus aureus737. (B) Graph showing the resazurin reduction for all the bacterial cultures; (a) Escherichia coli2345, (b) Bacillus subtilis, (c) Klebsiella pneumo-
niae and (d) Staphylococcus aureus737.

This indicates that the scaffold is safe for tissue engineering was determined using resazurin assay. Different concentra-
applications. tions of bacteria were treated with scaffolds along with
necessary controls and resazurin was added to observe colour
change (Figure 6(A)). Live bacterial cells convert resazurin dye
Effect of scaffolds on bacterial growth
into resorufin, which is pink in colour. If bacterial growth is
Certain biological scaffolds have been surprisingly resistant to not there, resazurin does not get converted and hence will
bacterial infections [41]. Moreover, scaffolds made of natural remain blue in colour. Evident colour change in case of con-
polymers showed resistance to even deliberate infections in trols as compared to the scaffolds indicated at significant
clinical set ups. Thus, the antibacterial activity of the scaffolds antibacterial activity. The absorbance values were compared
10 N. KUMAR ET AL.

Figure 7. Cell viability and proliferation of HeLa cells in AG-CHNp scaffolds evaluated by MTT assay. All the four scaffolds showed pronounced cell growth with time
as compared to 2D control where cell death was seen after day 6. Out of the four scaffolds AG-CHNp4 showed best cell proliferation capability, possibly due to
higher chitosan content.

Figure 8. Scanning electron micrographs of AG-CHNp scaffolds. SEM micrographs of all the four scaffolds showed interconnected pores and an average pore size of
175–300 lM (a) AG-CHNp1, (b) AG-CHNp2, (c) AG-CHNp3, (d) AG-CHNp4, (e) SEM image of AG-CHNp scaffold showing the pore, (f),(g),(h) Image showing HEK cells
growing the on the scaffold, (i),(j),(k) Image showing MiaPaCa2 cells growing on the scaffold.

with blank wells containing resazurin reagent without cells. dehydrogenase enzymes which reduces yellow coloured MTT
Six hundred nanometres absorbance readings were sub- salt (3-(4, 5-dimethylthiazolyl-2)–2, 5-diphenyltetrazolium
tracted from average absorbance at 570 nm of experiment bromide) to NADP and NADPH. The resulting purple forma-
wells. Finally, 570–600 nm or resazurin reduction readings zan crystals are solubilized in DMSO and quantified spectro-
were plotted against bacterial number (Figure 6(B)). As shown photometrically at 570 nm. Thus, an increase in optical
in the graphs, all the scaffolds showed significant reduction density helps to assess cell’s proliferative capacity within the
in the bacterial number as compared to the controls. This scaffold. The viability of HeLa cells was studied for a period
suggests strong anti-bacterial activity of all the four scaffolds of 16 days. As compared to the 2D control, all the four scaf-
against both Gram positive and Gram negative bacteria. folds showed increased and sustained growth of cells for a
longer period. In the 2D controls, HeLa cells grew and
reached confluence by day 6. With no more surface area
Biocompatibility of AG-CHNp scaffolds
available, there is immediate reduction in the cell number
The viability of cells was assessed by MTT assay using HeLa owing to cell death. On the other hand, as the scaffolds have
cell line shown in Figure 7. Metabolically active cells contain 3D morphology and provide better surface area for growth,
 ARTIFICIAL CELLS, NANOMEDICINE, AND BIOTECHNOLOGY 11

Figure 9. Fluorescent images of DAPI staining for AG-CHNp scaffolds showing MiaPaCa2 and HEK cells. Sections of scaffolds with MiaPaCa2 (a) and HEK (b) cells
were stained with DAPI and visualized under fluorescence microscope. The images showed proper cell attachment and rounded morphology of the cells on the
scaffolds.

Figure 10. Schematic representation of fabrication and characterization of AG-CHNp scaffolds. AG-CHNp scaffolds were synthesized using the polymers agarose and
chitosan by freeze drying technique. Chitosan-coated silver nanoparticles were synthesized within the scaffolds. All the four scaffolds were then characterized on
various parameters (physical, chemical, mechanical, biological and morphological).

there is a visible sustained growth during all the time points. important for cell attachment, nutrient and oxygen diffusion
Furthermore, AG-CHNp4 showed better growth of cells as [42]. Scaffolds with similar pore size have been used before for
compared to all the other scaffolds. This could be attributed tissue engineering of pancreas, liver, etc. [43,44] (Figure 8). The
to higher concentration of chitosan in AG-CHNp4. The above- cell adhesion and attachment property was analyzed using
mentioned results show excellent biocompatibility of all the DAPI staining of MiaPaCa2 and HEK cell lines. As DAPI is a
four scaffolds. nuclear stain, rounded morphology of cell’s nucleus was vis-
ible. Furthermore, post-attachment cells were found to grow
in an optimal and sustained manner (Figure 9).
Microstructure analysis and cell attachment
The scaffolds were designed in such a manner to accommo-
Discussion
date cell attachment, growth and migration. The porous struc-
ture allows for diffusion of nutrients and removal of toxic Most of the tissue engineering applications demand the need
compounds. To ascertain the gross morphology of the 3D of biodegradable materials with potential to serve multiple
structure and to determine the pore size SEM analysis was per- purposes. The most common approach for tissue engineering
formed. The scaffolds were found to have an average pore size is seeding cells onto a biomaterial matrix. The design of the
of 175–300 lM. Pore size between 100 and 500 lM is scaffold prior to cell application is of prime importance. The
12 N. KUMAR ET AL.

scaffold used must provide a surface for the attachment and Monsoor sheikh (Department of Biological Science) for FTIR and XRD ana-
growth of cells, also providing tissue growth. Thus the choice lysis. NK would also like to acknowledge Dr. Mainak Banerjee for help
with microscopy.
of material is of great importance. The material should be
biocompatible and biodegradable, and should also have the
required mechanical strength to offer a structural support. Disclosure statement
Moreover it must be able to allow smooth diffusion of
No potential conflict of interest was reported by the authors.
nutrients and also discharge of toxic compounds released. To
achieve all of this, most designed scaffold deliberately mimic
the structure of the naturally occurring extracellular matrix. Funding
Chitosan, derivative of chitin is one such biomaterial and
AG would like to thank The Department of Science and Technology (SR/
has been extensively used to prepare porous scaffolds allow-
FT/LS-137/2011) for financial support, NK thanks Indian Council of
ing the growth of several tissues. Agarose a saccharide poly- Medical Research (ICMR) for Senior Research Fellowship (SRF), (BMS/FW/
mer originating from sea weed possesses significant SCR/2015–20430/MAR-2015/06/GA/PVT).
mechanical strength in aqueous solution. It forms a transpar-
ent colourless gel, forming a porous three-dimensional struc-
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