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Reactive & Functional Polymers 82 (2014) 8188

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Non-cytotoxic conductive carboxymethyl-chitosan/aniline pentamer

Lin Zhang a, Yan Li a, Longchao Li a, Baolin Guo a,, Peter X. Ma a,b,c,d,e,

Center for Biomedical Engineering and Regenerative Medicine, Frontier Institute of Science and Technology, Xian Jiaotong University, Xian 710049, China
Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109, USA
Department of Biologic and Materials Sciences, University of Michigan, 1011, North University Ave., Room 2209, Ann Arbor, MI 48109, USA
Macromolecular Science and Engineering Center, University of Michigan, Ann Arbor, MI 48109, USA
Department of Materials Science and Engineering, University of Michigan, Ann Arbor, MI 48109, USA

a r t i c l e

i n f o

Article history:
Received 23 April 2014
Received in revised form 2 June 2014
Accepted 12 June 2014
Available online 19 June 2014
Conducting hydrogels
Controlled release
Carboxymethyl chitosan
Aniline oligomers

a b s t r a c t
Cytocompatible electrically conducting hydrogels based on amphoteric carboxymethyl chitosan (CMCS)
and aniline oligomers with bioactive molecule delivery properties were presented. A series of conductive
CMCS hydrogels with different aniline pentamer (AP) content were synthesized by a one-pot reaction
with the combination of grafting and crosslinking reaction via glutaraldehyde. The conductivities of
the swollen hydrogels are between 1.14  10 4 and 4.23  10 4 S/cm by tuning the AP content. Swelling
ratio was controlled by the AP content and crosslinking degree of the hydrogels. The hydrogels showed
absorption capacity of diclofenac sodium (DCS) as a model molecule and released DCS in a controlled
manner. Morphologies and mechanical properties of the hydrogels were characterized by SEM and rheometer, respectively. The biocompatibility of the hydrogels was conrmed by C2C12 myoblast cells using
Live/Dead assay and Alamar blue assay. These conducting hydrogels with controlled release capacity as
bioactive scaffolds have potential application for tissue regeneration.
2014 Elsevier Ltd. All rights reserved.

1. Introduction
Tissue engineering aims to construct live functional substitutes
to repair damaged or dysfunctional tissues by using a combination
of cells and polymer scaffolds. Three dimensional (3D) porous scaffolds provide a temporary support for cell growth and promote
neo-tissue regeneration process [13]. The polymer 3D scaffolds
mimic many roles of extracellular matrices (ECM) which are consisted of various amino acids and sugar-based macromolecules
[4,5]. Various types of scaffolds have been developed for tissue
engineering applications. Hydrogel is one of the materials widely
used in tissue engineering due to their rubbery nature similar to
the soft tissues, their control of diffusion of oxygen, nutrients
and other bioactive molecules and their excellent biocompatibility
[6,7]. Hydrogels from naturally derived polymers have potential
advantages of good biocompatibility, degradability and intrinsic
cellular interaction with tissue [8,9]. As one of the most abundant

Corresponding authors. Address: Center for Biomedical Engineering and

Regenerative Medicine, Frontier Institute of Science and Technology, Xian Jiaotong
University, Xian 710049, China (B. Guo). Tel.: +86 29 83395361; fax: +86 29
E-mail addresses: (B. Guo), (P.X. Ma).
1381-5148/ 2014 Elsevier Ltd. All rights reserved.

biopolymers chitosan has been widely used in biomedical elds

because of its unique polycationic nature [1014]. However, the
poor solubility of chitosan in neutral water limits its application.
Carboxymethyl chitosan (CM-CS), a natural amphoteric polyelectrolyte derived from chitosan, is not only soluble in neutral and
basic water, but also has unique chemical, physical and biological
properties. It has found wide applications in wound healing, tissue
engineering, drug delivery system and gene therapy [1518]. It
contains large amount of ACOOH and ANH2 groups which can be
further functionalized.
The 3D scaffolds should regulate the cell adhesion as well as
migration and differentiation in sophisticated tissue engineering.
Previous studies demonstrated that electrical stimulations can regulate cellular activities, including cell attachment, migration, proliferation and differentiation [19,20]. This indicates that conducting
polymers have great potentials in tissue engineering applications,
since the regulation of cellular activities is critical for the regeneration of damaged tissue. Conducting polymer composites and
hydrogels based on polyaniline [2123], polypyrrole [24,25],
polythiophene and their derivatives [2628] have been developed
[2931]. However, the poor solubility and non-degradability of
conducting polymers greatly restricted their applications in tissue
engineering. In contrast, oligomers of aniline have well-dened


L. Zhang et al. / Reactive & Functional Polymers 82 (2014) 8188

structure, good solubility and electroactivity as well as good biocompatibility [3236]. Moreover, their amine-capped structure
makes them possible to be copolymerized or grafted with other
monomers or polymers to prepare functional materials as shown
in our previous work [3739].
The localized and temporally controlled delivery of bioactive
molecules such as drugs, protein and growth factors is very important to improve the clinical efcacy. In advanced tissue engineering
processes, the biodegradable scaffolds should serve as both a
three-dimensional (3-D) substrate and a bioactive molecules delivery depot to enhance cellular activity [4042]. The development of
temporally and spatially controlled delivery systems that integrates the biodegradable scaffolds is still a clinical challenge. The
goal of this work is to design and synthesize a series of amphoteric
conducting hydrogels with bioactive molecule delivery properties
based on CMCS and aniline pentamer for tissue engineering applications. We used diclofenac sodium (DCS, a nonsteroidal antiinammatory compound with a UVVis absorption at 276 nm) as
a model molecule to study the release property of the conducting
hydrogels. The hydrogel preparation, swelling behavior, conductivity, surface morphology, mechanical properties and release properties of the hydrogels were investigated. C2C12 myoblast cell
adhesion and proliferation for the hydrogel were also evaluated.
2. Materials and methods
2.1. Materials
Chitosan (CS, weight molecular weight 100,000300,000 Da)
was obtained from J&K Scientic Ltd. Glutaraldehyde (GA, Aldrich)
was diluted to 5 wt% aqueous solution before use. Monochloroacetic acid, N-phenyl-1, 4-phenylenediamine, phenylamine, ammonium persulphate ((NH4)2S2O8), ammonium hydroxide (NH4OH),
sodium hydroxide (NaOH), isopropanol, anhydrous ethanol, tetrahydrofuran (THF), diclofenac sodium (DCS) and dimethyl sulfoxide
(DMSO) were all purchased from Aldrich and were used as
received. Carboxymethyl chitosan (CMCS) was prepared as
described previously [43]. The substitution degree of CM-chitosan
was 89% by potentiometric titration [44].
2.2. Synthesis of aniline pentamer
Aniline pentamer was synthesized according to Ref. [45], and
synthesis route is shown in Fig. 1 in Supporting Information (SI).
Briey, 0.2 mol aniline and 0.1 mol p-phenylenediamine was oxidized by 0.2 mol (NH4)2S2O8 in 1 mol/L HCl and acetone mixture
to produce amino-capped aniline trimer (Fig. 1 in SI). Aminocapped aniline trimer: 1H NMR (400 MHz, DMSO-d6) d = 6.96

(s, 4H, ArH), d = 6.81 (d, 4H, ArH), d = 6.62 (d, 4H, ArH),
d = 5.43 (s, 4H, NH2). This result agrees well with the Ref. [46].
For the synthesis of aniline pentamer, 2.28 g amino-capped aniline trimer and 3.04 g N, N-diphenylamine were dissolved in 80 mL
DMF. 40 mL water and 20 mL 36% HCl were then added to the above
solution with vigorous stirring. After reaction at room temperature
for 4 h, the HCl-doped aniline pentamer was obtained by ltration,
and then washed by a mixture of DMF/H2O. The product was dedoped in a 100 mL 1 mol/L NH4OH for 30 min to produce aniline
pentamer in emeraldine state. The emeraldine aniline pentamer
was reduced by phenylhydrazine, and was precipitated in a H2O/
ethanol mixture. The leucoemeraldine aniline pentamer was
collected by ltration and washed thoroughly with H2O/ethanol
mixture. The product was nally dried in a vacuum oven. The 1H
NMR spectrum was shown in Fig. 2 in SI. Leucoemeraldine aniline
pentamer: 1H NMR (400 MHz, DMSO-d6) d = 7.73 (s, 1H, ArNH),
d = 7.52(s, 1H, ArNH), d = 7.37 (s, 1H, ArNH), d = 7.14 (s, 1H, Ar
NH), d = 7.137.11 (m, 2H, ArH), d = 6.986.95 (m, 2H, ArH),
d = 6.906.83 (m, 10H, ArH), d = 6.776.75 (m, 4H, ArH),
d = 6.676.65 (m, 1H, ArH), d = 6.516.49 (m, 2H, ArH), d = 4.63
(s, 2H, ArNH). 13C NMR (100 MHz, DMSO-d6), d = 145.61 (ArC),
142.53 (ArC), 139.47 (ArC), 139.39 (ArC), 138.56 (ArC),
135.86 (ArC), 135.71 (ArC), 134.29 (ArC), 133.67
(ArC), 129.04 (ArC), 120.09 (ArC), 120.27 (ArC), 119.16
(ArC), 118.99 (ArC), 117.72 (ArC), 117.04 (ArC), 116.67 (Ar
C), 116.23 (ArC), 114.88 (ArC), 114.38 (ArC). This data agree well
with the previous results [47].
2.3. Synthesis of CMCS-AP hydrogels
The conductive hydrogels were synthesized in a one-pot reaction as shown in Fig. 1. 0.05 g CMCS was dissolved in 2 mL of distilled water. Different amounts of AP (see Table 1) which dissolved
in 1 mL DMSO and appropriate amounts of GA (Table 1) were
added into the above solution and stirred vigorously. The reaction
was then kept for 12 h at room temperature without stirring. The
hydrogel was immersed in H2O/THF mixture (Vol:Vol = 90:10) for
2 days to extract the unreacted AP. The H2O/THF mixture was
changed every 6 h. All the extracted solution was collected and
was used for UVVis test. After that, the hydrogels were soaked
into distilled water to exchange with the THF in the hydrogels with
changing the water for several times. The hydrogels were then
dried in air at room temperature and stored in a desiccator.
Hydrogels with same crosslinking degree but containing different weight ratio of AP were prepared by adding different amounts
of AP into the system, and samples having 0%, 5%, 10%, 15% and 20%
weight ratio of AP in the hydrogel were coded as CMCS hydrogel,
CMCS-AP5 hydrogel, CMCS-AP10 hydrogel, CMCS-AP15 hydrogel
and CMCS-AP20 hydrogel, as listed in Table 1.

Fig. 1. Schematic synthesis of CMCS-AP hydrogels.

L. Zhang et al. / Reactive & Functional Polymers 82 (2014) 8188

Fig. 2. FT-IR spectra of (a) CS, (b) CMCS, (c) AP and (d) CMCS-AP10 hydrogel.

Table 1
Feed composition of the hydrogels with different AP contents.
Sample name





5 wt% GA

CMCS hydrogel
CMCS-AP5 hydrogel
CMCS-AP10 hydrogel
CMCS-AP15 hydrogel
CMCS-AP20 hydrogel
CMCS-AP10G1 hydrogel
CMCS-AP10G2 hydrogel
CMCS-AP10G3 hydrogel






Hydrogels with different crosslinking degree were synthesized

by adding various amounts of crosslinking GA solution and are
respectively called as CMCS-AP10G1 hydrogel, CMCS-AP10G2
hydrogel and CMCS-AP10G3 hydrogel, as shown in Table 1.
2.4. Characterization
FT-IR spectra of CS, CMCS, aniline pentamer (AP) and CMCS-AP
hydrogel were recorded on a Nicolet 6700 FT-IR spectrometer
(Thermo Scientic Instrument) in the range from 4000 cm 1 to
600 cm 1 with average of 16 scans at a resolution of 4 cm 1.
H NMR spectra of aniline pentamer were obtained on a BRUKER Ascend spectrometer (400 MHz) with DMSO-d6 as solvent at
room temperature and internal standard.
UVVis spectra of the solution were carried out on a UVVis
spectrophotometer (PerkinElmer Lambda 35).
Swelling ratio of the hydrogels was determined by swelling
measurements. The dry hydrogels were soaked in 30 mL PBS with
pH value 7.4 in sealed vials. The hydrogels were withdrawn from
the solution after regular periods of time and weighed after the
removal of excess surface water with a lter paper. They were then
returned to the same vial until swelling equilibrium was reached.
Swelling ratio was calculated from the following equation swelling
ratio = (Ws Wd)/Wd  100%, where Ws and Wd represent the
weights of the swollen and dry state samples, respectively.
The dried hydrogels before swelling and the freeze-dried hydrogels after reaching the swelling equilibrium were mounted on
metal stubs and the morphology of them were then observed by
using a eld emission scanning electron microscope (FE-SEM,
SU-8000, Hitachi, Japan).
The hydrogel was immersed in distilled water at room temperature to reach swelling equilibrium, which resulted in the absorption of water inside of the CMCS-AP hydrogels. The unabsorbed
water was removed by lter paper, and the sample was placed in


air for 30 min. The electrical conductivity of CMCS-AP hydrogel

was determined by a Pocket Conductivity Meter (HANNA8733).
80 mg of the model molecule diclofenac sodium was dissolved
in 10 mL water. 10 mg of dry hydrogel of CMCS and CMCS-AP20
was then submerged into the above solution for 60 h until the
swelling equilibrium was reached. The hydrogel was taken out
and dried at 37 C in an oven until reaching a constant weight.
For the release experiment, the dried hydrogel with loaded-drug
was placed into conical ask containing 100 mL buffer solution
(pH 7.4). The ask was then place into a shaking incubator at
37 C with a rotation speed of 100 rpm. At predetermined time
intervals, 1 mL of solution was taken out and replaced with equal
volume of the same release media. The amount of drug loaded
and released from the hydrogel was spectrophotometrically determined at 276 nm using a UVVis spectrophotometer (PerkinElmer
Lambda 35) with a standard linear curve of diclofenac sodium.
The rheological properties of the CMCS-AP hydrogels were evaluated with a TA rheometer (DHR-2) at 25 C with the parallel-plate
geometry (plate diameter of 20 mm, gap of 1000 lm). The measurement was performed using a dynamic frequency sweep test,
which covered a range of frequencies from 1 to 100 rad/s at a constant shear amplitude of 0.1%.
2.5. Cell culture
2.5.1. In vitro cytotoxicity and cell proliferation assay
Cytotoxicity and cell proliferation of CMCS, CMCS-AP5, CMCSAP10, CMCS-AP15 and CMCS-AP20 hydrogels were assessed by
the alamarBlue (Invitrogen) assay. The dry hydrogels were rstly
sterilized by ethylene oxide, and they were then respectively incubated into the culture medium (Dulbeccos Modied Eagle Medium
(DMEM), Gibco)) supplemented with 10% fetal bovine serum (Gibco), 100 U mL 1 penicillin and 100 lg mL 1 streptomycin
(Hyclone) for 24 h at 37 C to obtain their extract liquid with the
concentration of 10, 5, 2.5, and 1.25 mg mL 1, respectively.
100 lL of C2C12 myoblast cells were seeded in 96-well plates
(Costar) at a density of 1  104 cells per cm2 and were incubated
in the culture medium under standard conditions (a humidied
incubator, 37 C, 5% CO2) for 18 h. Then various concentrations of
extract liquid were added to the corresponding wells. The wells
lled with the culture medium instead of the extract liquid were
considered as the control group (TCPS). After incubation for 24 h,
10 lL of alamarBlue reagent was respectively added into each
well. The system was incubated at 37 C under 5% CO2 for another
6 h. 90 lL of the medium in each well was transferred into 96-well
black plates (Costar). The uorescence of the medium was read by
a microplate reader (Molecular Devices) with excitation and emission wavelengths of 565 nm and 590 nm.
For the control group and CMCS-AP10 hydrogel sample, equivalent volume of fresh culture medium or extract liquid was added
into the wells and the plate was returned to the incubator for further incubation. The cell proliferation data were collected after
incubating for 24 h, 48 h and 72 h, respectively.
The living and the dead cells in CMCS-AP20 hydrogel extraction
after incubation for 72 h were stained with LIVE/DEAD Viability/
Cytotoxicity Kit (Molecular Probes). The nal concentration of
the calcein AM and the EthD-1 reagents was 1 lM. The stained
cells were observed under the inverted uorescence microscope
(IX53, Olympus).
2.5.2. In vitro cell morphology observation
The C2C12 cells previously incubated for 72 h in 96-well plates
under standard conditions (a humidied incubator, 37 C, 5% CO2)
in the various concentrations of extracts of CMCS-AP10 were
stained with uorescein isothiocyanate (FITC) labeled phalloidin
(Sigma) and DAPI (Sigma). After removing the extract liquid from


L. Zhang et al. / Reactive & Functional Polymers 82 (2014) 8188

the wells, the wells were rapidly washed twice with Dulbeccos
Phosphate Buffered Saline (DPBS) (Gibco). The cells were xed
with 2.5% glutaraldehyde for 20 min, and then rapidly washed
twice with DPBS. After soaked in 0.1% Triton X-100, FITC labeled
phalloidin solution was respectively added into the wells and incubated for 90 min at room temperature. After removing the stain,
the wells were then washed twice with DPBS. 100 lL of DAPI
(Sigma) was respectively added into the wells and incubated for
10 s. The stained cells were observed under the inverted uorescence microscope (IX53, Olympus).

3. Results and discussion

3.1. Synthesis of the conducting hydrogels
We developed a facile synthesis approach to degradable conducting hydrogels based on chitosan and aniline tetramer in the
previous work [48]. However, chitosan is not soluble in neutral
water, and the hydrogels showed low swelling ratio (about 200%)
in physiological conditions. Furthermore, the hydrogels showed a
low conductivity (10 6 S/cm) when the aniline tetramer is less
than 20 wt%. In this work, we prepared CMCS-AP hydrogels to
enhance the swelling ratio and conductivity of the hydrogels, and
the release properties and cytotoxicity of the CMCS-AP hydrogels
were also evaluated. Carboxymethyl chitosan (CMCS) is a water
soluble amphoteric polyelectrolyte with both amine and carboxyl
groups on its main chains, and carboxylate group on the main
chain of CMCS would greatly increase its hydrophilicity at pH 7.4
solution. Aniline pentamer having a higher conductivity than aniline tetramer contains amine groups as shown in Fig. 1. In this
work, glutaraldehyde was used as crosslinking agent to crosslink
the amine groups from CMCS to form a degradable network. At
the same time, glutaraldehyde was employed to graft AP to the
CMCS main chain (Fig. 1). In this way, we obtained a series of conductive hydrogels with different AP contents from a one-pot reaction. After drying at 37 C for 48 h, a representative black hydrogel
was obtained as shown in Fig. 3(a) in SI. We can see that the hydrogel after swelling as shown in Fig. 3(b) in SI has a much bigger volume than the dried hydrogel, and the integrity of the hydrogel was
The FT-IR spectra of chitosan, carboxymethyl chitosan, aniline
pentamer and CMCS-AP10 hydrogel were shown as curves ad in
Fig. 2, respectively. FT-IR spectrum of CS exhibited typical adsorption peaks at 32703370 cm1 owning to the partially overlapped
characteristic peaks of amine (ANH2) and hydroxyl group (AOH)
stretching vibrations. The absorption peaks at 1648 cm1 and at
1590 cm1 correspond respectively to the amide (ACONHA) and
amine (ANH2) stretching and bending vibrations. Band at
1027 cm1 was assigned to ACAOA stretching vibrations from
CS. Compared to the IR spectrum of CS, a new peak appeared at
1595 cm 1 corresponding to the carboxyl sodium group (ACOONa)
[49], indicating that the CMCS was successfully obtained. The
absorption of aniline pentamer (curve c) showed peaks at
1597 cm 1 and 1495 cm 1 assigned to the absorption of benzene
ring and the quinoid ring. The 1305 cm 1 band was attributed to
CAN stretching in the proximity of quinoid rings [50]. Compared
to curves b and c, curve d of CMCS-AP10 hydrogel has all the characteristic peaks of curve b, and also an absorption peak at
1499 cm 1 corresponding to the quinoid ring, demonstrating that
the CMCS-AP hydrogels were obtained.
The crosslinking and grafting reaction took place between the
amine groups of CMCS, amine groups of AP and aldehyde groups
of GA. We used UVVis spectrum to determine the amount of AP
which was connected to the hydrogel network. The UVVis of
the extract solution was tested and shown in Fig. 4 in SI. Two

Fig. 3. Swelling kinetic of hydrogels with different AP content (a) and hydrogels
with different crosslinking degree (b) at pH 7.4 solution.

Fig. 4. Diclofenac sodium release curves from CMCS-AP hydrogels.

absorbance peaks at about 308 nm and 618 nm appeared, which

were attributed to the pp transition of the benzene ring and
the benzenoid to quinoid excitonic transition. The amount of AP
in the extraction solution was calculated according to a linear standard curve of AP. The reacted percentage of AP was determined by
the following equation: Reacted AP% = (total amount of AP
amount of AP in the extraction solution)/total amount of
AP  100% and the results were listed in Table 2. It was found that
the reacted AP percentage was between 60.2% and 66.4% for all the
systems. The actual AP contents in the hydrogels were also listed in
Table 2.
3.2. Swelling of the hydrogels
The swelling properties of the hydrogels are important for their
applications, and swelling ratio is the criterion of describing water


L. Zhang et al. / Reactive & Functional Polymers 82 (2014) 8188

Table 2
Conductivity of the hydrogels with different AP content.
Sample code
CMCS hydrogel
CMCS-AP5 hydrogel
CMCS-AP10 hydrogel
CMCS-AP15 hydrogel
CMCS-AP20 hydrogel

Conductivity (S/cm)
1.14  10
2.52  10
3.04  10
3.55  10
4.23  10


Theoretical AP content in hydrogel (%)

Reacted percentage of AP (%)

Actual AP content in hydrogel (%)




absorption capacity of the hydrogels. The swelling kinetic of the

CMCS-AP hydrogels was shown in Fig. 3a. The swelling ratio of
CMCS-hydrogel increased dramatically to 808% in the rst 24 h.
It then increased slowly to 1062% in the next 24 h due to the repulsion between the ACOOA groups from CMCS. The CMCS-AP hydrogel showed a similar swelling kinetic compared to CMCS hydrogel.
However, the swelling ratio of CMCS-AP hydrogel decreased
accordingly with increasing the amount of AP in the hydrogels.
This might be because the increasing AP contents in the hydrogels
consumed more amine group of CMCS which decreased the hydrophilicity of the network. On the other hand, the AP itself is a hydrophobic segment, and hydrogen bonds would form between the
AOH, ANH2 groups from CMCS and ANHA groups from AP. This
further decreased the hydrophilicity of the hydrogels. The chitosan/aniline tetramer hydrogels in our previous work showed a
swelling ratio between 70% and 195% in pH 7.4 solution which
may restrict their application in tissue engineering since the swelling ratio is very low [48]. The swelling ratio of the hydrogel in this
work in the range of 5521062% is much higher that of chitosan
aniline tetramer hydrogel [48], and this means these hydrogels
may have greater opportunities in tissue engineering applications,
since the large pores of the hydrogels are benecial for cell, nutrition, oxygen, and waste transportation in scaffolds during tissue
The crosslinking density is another important factor to determine the swelling ratios of hydrogel. We prepared the conducting
hydrogels with different crosslinking density by adding different
amounts of crosslinking agent, as listed in Table 1. The swelling
kinetic of these hydrogels was shown in Fig. 3b. The sample of
CMCS-AP-G1 showed the fastest swelling speed and its swelling
ratio reached 908% after 48 h. The swelling ratio of CMCS-AP10
hydrogel, CMCS-AP-G2 hydrogel and CMCS-AP-G3 hydrogel
decreased accordingly as the crosslinking density increasing. This
is because the GA consumed more amine groups and formed more
condensed network which hindered the swelling of hydrogels.
Therefore, we could tune the swelling ratio of these hydrogels to
meet a specic tissue engineering application by changing the AP
content and crosslinking density of the hydrogels.
3.3. Morphology of the hydrogels
The morphology of the hydrogels before and after swelling was
observed by SEM and the results are shown in Fig. 5 in SI. The
hydrogels generally showed a smooth surface before swelling,
although the roughness increased slightly with increasing AP content in the hydrogels. However, a large amount of pores and cracks
appeared on the surface after swelling and the size of the pores
from Fig. 5(f) to (j) in SI decreased with increasing AP content in
the hydrogels. This agrees well with the swelling ratio results as
shown in Fig. 3a. The pore size was between 30 and 100 lm for
most of the hydrogels, so the cells could migrate into the pores
and to the inner part of the hydrogels, probably leading to a more
uniform tissue when these hydrogels were used for tissue engineering [51]. The morphology of the hydrogels with different
crosslinking density was shown in Fig. 6 in SI. The pore size
decreased with the crosslinking agent increasing in the hydrogels,

Fig. 5. Mechanical properties of the hydrogels.

and it was in accordance with the swelling ratio results of the

hydrogels as shown in Fig. 3b.
3.4. Conductivity of the hydrogels
The conductivity of the hydrogels plays an important role in tissue engineering application. It can enhance the signal transfer and
chemical exchange between the cells to benet the cell growth
[52]. The conductivity of the hydrogels was usually determined
in a dried state by most researchers. However, the conductivity
values would change when these hydrogels were used in a swollen
state. We tested the conductivity of the hydrogels in a swollen
state with a conductivity meter in this work. This would reect
the actual conductivity of the hydrogels when they are used as
scaffold for tissue engineering. The conductivity of CMCS hydrogels
as shown in Table 2 is about 1.14  10 4 S/cm due to ionic conductivity of carboxyl groups and amine groups on the main chain of
CMCS. By connecting 3.3 wt% AP segment to the CMCS main chain,
the conductivity of CMCS-AP5 increased to 2.52  10 4 S/cm. The
conductivity of the CMCS-AP10, CMCS-AP15 and CMCS-AP20
hydrogels ranged from 3.04  10 4 to 4.23  10 4 S/cm with
increasing AP content in the hydrogel, because the high AP content
would form a conductive network which benets the electron
transfer. The conductivity value was lower than those of the polyaniline hydrogels with acrylamide which was between 1.5 and
2.4  10 3 S/cm [53]. However, they are 10 times higher than that
of chitosananiline tetramer hydrogels which is in the order of
10 510 6 S/cm [48].
3.5. Bioactive molecules absorption and release from the hydrogels
Hydrogels are widely used as scaffolds for tissue engineering. It
is desirable for hydrogels to have the controlled release properties
of bioactive molecules, such as drugs, proteins and growth factors
[54,55]. We took diclofenac sodium (DCS) as a model molecule and
incorporated it into the conductive hydrogels and studied its
release behavior from the hydrogels to demonstrate the release
properties of the materials. The amount of loaded DCS in CMCS,


L. Zhang et al. / Reactive & Functional Polymers 82 (2014) 8188

Fig. 6. The Live/Dead staining results of CMCS-20AP group at day 3. A large amount of live (green) cells were observed in different concentration of CMCS-20AP hydrogel
extract solutions. Concentration: (a) 1.25 mg/mL; (b) 2.5 mg/mL; (c) 5 mg/mL and (d) 10 mg/mL. Scale bar means 50 lm. (For interpretation of the references to color in this
gure legend, the reader is referred to the web version of this article.)

CMCS-AP5 hydrogel, CMCS-AP10 hydrogel, CMCS-AP15 hydrogel

and CMCS-AP20 hydrogel was 0.085 mg/mg, 0.064 mg/mg,
0.054 mg/mg, 0.044 mg/mg and 0.035 mg/mg, respectively. The
incorporation process was controlled by the swelling and interaction between DCS and hydrogel network. The hydrogels would
have a higher porosity and bigger pore size with a higher swelling
ratio as indicated by SEM in Fig. 5 in SI. The DCS molecules would
thus more easily penetrate into the CMCS hydrogel and absorbed
by the network with hydrogen bonds between ANHA from DCS
and AOH, ANH2 groups from CMCS and ANHA from AP segment.
Moreover, the extraction between ACOOA from DCS and ANH2
from CMCS increased the interaction of DCS and the hydrogel network. Therefore, the absorbed amount of DCS decreased with
decreasing the swelling ratio of the hydrogels.
The hydrogels loaded with DCS were then dried in a vacuum
oven and then placed in a 100 mL PBS to determine the release
behavior of DCS. It was found that the CMCS hydrogel showed a
burst release of 81% of DCS in 20 min (Fig. 4), which is attributed
to the release of drug loaded on the surface of the hydrogel. However, the DCS release amount is 3272% for the CMCS-AP hydrogels
in 20 min, indicating that the addition of AP to the network would
greatly reduce the burst release of the molecules. The higher concentration of aniline pentamer in the hydrogels showed more
effective reduction of the burst release of the bioactive molecules.
The DCS then released from CMCS-AP hydrogel matrix gradually
with the hydrogel swelling. About 88% of DCS was released from
the hydrogels at 8 h, and it reached over 93% at 11 h. Generally,
the incorporation of aniline pentamer into the carboxymethyl
chitosan hydrogels greatly reduced the burst release and prolonged the release time of diclofenac sodium in these hydrogels
compared to CMCS hydrogel. Therefore, the system is useful for
an anti-inammatory drug release because there might be infection after the hydrogels as scaffolds were implanted into the
human body.

3.7. In vitro cytocompatibility assay

The biomaterials should be not cytotoxic for the biomedical
applications. The cytotoxicity assay was conducted using the
extracts from soaked hydrogel samples. The cytotoxicity of the
CMCS-AP20 hydrogel extract solutions was tested by a Live/Dead
assay, and the results are shown in Fig. 6. After cultured for 72 h,
as expected, dominant live (green color) cells were observed for
all the groups with different concentrations of extract solution,
and few dead cells (red color) were found, indicating that the
CMCS-AP20 hydrogel is not cytotoxic. We also employed Alamar
blue test to quantify the cell viability of the CMCS-AP hydrogels
as shown in Fig. 7. It is well-known that CMCS is non-toxic and
is widely used in biomedical elds. It was found that all the
CMCS-AP hydrogel exhibited higher cell viability than CMCS, and
the ratios of the cell viability between the hydrogel group and control group (TCPS) are higher than 80%, indicating that all the hydrogels are non-cytotoxic. CMCS-AP5 and CMCS-AP15 groups even
showed much higher cell viability than control group (TCPS).
C2C12 cell proliferation in different concentrations of CMCSAP10 hydrogel extract solution was determined by Alamar blue
assay, and the results are displayed in Fig. 8. A continuous increase
of uorescence intensity was observed for all the groups, indicating
that cells proliferated well in the hydrogel extraction medium. At
the rst and second day of culturing, the C2C12 cells in the hydrogel extract solution showed higher uorescence intensity than the
control group (TCPS). On the third day, the uorescence intensity
of groups of 5 mg/mL and 10 mg/mL of hydrogel extract solution
was somewhat lower than control group (TCPS). However, the ratio
between them was higher than 85%. Furthermore, the uorescence
intensity of groups of 2.5 mg/mL and 1.25 mg/mL of hydrogel

3.6. Mechanical properties of the hydrogels

The storage modulus of the hydrogels was evaluated by a rheometer and the results are shown in Fig. 5. It was found that the
storage modulus of the CMCS hydrogel is 1511 Pa, and the storage
modulus of the hydrogels decreased from 1511 Pa to 770 Pa with
increasing the AP content in the materials. This could be due to
more aniline pentamer in the reaction system would consume
more amine groups on the CMCS main chain, which reduced the
electrostatic attraction between the amine group and carboxylate
groups. The mechanical properties of the hydrogels with different
crosslinking densities are also depicted in Fig. 5. The storage modulus of hydrogels increases from 929 Pa to 1545 Pa with increasing
the amount of crosslinkers in the reaction as shown in Table 1,
because the higher crosslinker concentration would result in more
condensed network which would increase the strength of the

Fig. 7. The cell viability at 24 h in different concentrations of CMCS-AP hydrogel

extract solutions, expressed as percentage of viability of control group (TCPS). Mean
for n = 4 SD.

L. Zhang et al. / Reactive & Functional Polymers 82 (2014) 8188


Appendix A. Supplementary material

Supplementary data associated with this article can be found, in
the online version, at

Fig. 8. The C2C12 cells proliferation in different concentrations of CMCS-AP10
hydrogel extract solutions (mg/mL) during 72 h culturing, expressed as percentage
of uorescence intensity of control group (TCPS). Mean for n = 4 SD.


extract solution was still much higher than that of the control
group, indicating that they could improve the proliferation of
C2C12 cells, probably because the charges associated with aniline
pentamer introduced into the materials.
The cell morphology of C2C12 cells was stained to evaluate the
effect of CMCS-AP10 hydrogel on the cell morphology. The C2C12
cells exhibited spindle-like cell morphology and spread well in
the medium for all the hydrogel extraction groups (Fig. 7 in SI),
which is quite similar to the control group (TCPS).


4. Conclusions
A series of amphoteric conducting hydrogels with bioactive
molecules delivery capacity for tissue engineering applications
were designed and synthesized. These hydrogels were prepared
in a one-pot reaction with the combination of crosslinking and
graft reaction based on carboxymethyl chitosan (CMCS) and aniline
pentamer. The swelling ratio of the hydrogels was between 552%
and 1062% tuned by the AP content and crosslinking degree of
the hydrogels. The hydrogel formation was conrmed by FT-IR
and the AP content in the hydrogels was determined by UVVis
measurement. The conductivity of the hydrogels in a swollen state
is in the range of 1.14  10 4 S/cm and 4.23  10 4 S/cm tuned by
AP content. The hydrogels absorbed diclofenac sodium and
released it in a controlled manner. The CMCS-AP hydrogels greatly
reduced the burst release of the bioactive molecules. The porous
structure of the hydrogels after freeze-drying was conrmed by
SEM observation. The hydrogels were non-cytotoxic and supported
the adhesion and proliferation of rat C2C12 myoblast cells. All
these results indicated that these conductive bioactive hydrogels
with release properties are promising scaffolds for tissue


The authors gratefully acknowledge the National Natural Science Foundation of China (Grant Number: 21304073) and the
Fundamental Research Funds for the Central Universities (Grant
No. xjj2013029), and the Scientic Research Foundation for the
Returned Overseas Chinese Scholars for nancial support of this


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