Nanofibrous electroactive scaffolds from a chitosan-grafted-aniline tetramer by electrospinning for tissue engineering

Article abstract of DOI:10.1039/c4ra00083h

Functional degradable biomimetic scaffolds have great potential applications in tissue regeneration. Nanofibrous electroactive biodegradable scaffolds from chitosan-grafted-aniline tetramer (CS-AT) were fabricated by an electrospinning method. The CS-AT was synthesized by amidation reaction between the carboxyl group of aniline tetramer and the amine group of chitosan. The structure of CS-AT copolymer was characterized by ¹H NMR, FT-IR, TGA and XRD. UV-vis and cyclic voltammetry tests were used to demonstrate the electroactivity of CS-AT. The electrospun nanofibers were created from CS-AT solution. The morphology of the CS-AT nanofibers was observed by employing SEM and the results illustrated that the diameter of the nanofibers of deposited CS and CS-AT samples was controlled by both polymer concentration and the AT content. The biocompatibility of the materials was evaluated by cell adhesion and proliferation of C2C12 myoblasts and dog chondrocyte cells, and the results demonstrated that the CS-AT materials had good biocompatibility and greatly enhanced the cell adhesion and proliferation of C2C12 cells. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ra00083h

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Nanofibrous electroactive scaffolds from a
chitosan-grafted-aniline tetramer by
electrospinning for tissue engineering
Cite this: RSC Adv., 2014, 4, 13652
a
a
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Xiaojie Ma, Juan Ge, Yan Li, Baolin Guo and Peter X. Ma
Functional degradable biomimetic scaffolds have great potential applications in tissue regeneration.
Nanofibrous electroactive biodegradable scaffolds from chitosan-grafted-aniline tetramer (CS–AT) were
fabricated by an electrospinning method. The CS–AT was synthesized by amidation reaction between
the carboxyl group of aniline tetramer and the amine group of chitosan. The structure of CS–AT
1
copolymer was characterized by H NMR, FT-IR, TGA and XRD. UV-vis and cyclic voltammetry tests were
used to demonstrate the electroactivity of CS–AT. The electrospun nanofibers were created from CS–AT
solution. The morphology of the CS–AT nanofibers was observed by employing SEM and the results
illustrated that the diameter of the nanofibers of deposited CS and CS–AT samples was controlled by
both polymer concentration and the AT content. The biocompatibility of the materials was evaluated by
cell adhesion and proliferation of C2C12 myoblasts and dog chondrocyte cells, and the results
demonstrated that the CS–AT materials had good biocompatibility and greatly enhanced the cell
adhesion and proliferation of C2C12 cells.
Received 5th January 2014
Accepted 4th March 2014
DOI: 10.1039/c4ra00083h
www.rsc.org/advances
bers generated from electrospinning exhibit high surface area
and porosity, and their diameters are tunable ranging from
several nanometers to several microns.17,18 Thus, electro-
spinning as a very promising method, plays an important role in
the application of tissue engineering.6,7,14
1. Introduction
The search for biodegradable scaffolds originates from the
growing need to interface living cells, tissues and organs, which
structurally mimic the extracellular matrix (ECM). Collagen as
the major protein of ECM arranges into nanobers,1 which not
only give support to tissue but also help to organize the
communication between cells embedded within the matrix.2,3
To imitate the natural ECM, nanobrous polymer scaffolds have
been fabricated by using several different processing tech-
niques, such as phase separation, molecular assembly and
electrospinning.4,5 Among these methods, the electrospinning
as an elegant and facile way to obtain nanobrous structures is
one of the most common technique.6,7 A variety of natural and
synthetic biodegradable polymers have been prepared as
porous scaffolds with random or aligned morphology, such as
chitosan,8–10 polylactide11–13 and other biomaterials.14–16 The
Recently, an increasing attention has been paid to the design
of specic cellular responsive biomaterials at the molecular
level.19 It has been reported that electrical signals can regulate
cell attachment, proliferation and differentiation.20 Electric
elds and stimulations are very helpful for wound healing,
recovery of the damaged spin cord, nerve regeneration21,22 and
so on. Therefore conductive polymers used in biomedical area
have attracted much attention in the past few decades.23 Mawad
et al. successfully prepared a conducting hydrogel based on
poly(3-thiopheneacetic acid), and broblast and myoblast cells
were found to adhere and proliferate on the hydrogel
substrate.24 Polypyrrole was coated onto random and aligned
electrospun poly(lactic-co-glycolic acid) (PLGA) nanobers and
the polypyrrole-coated PLGA electrospun meshes enhanced the
growth and differentiation of rat pheochromocytoma 12 cells
compared to non-coated PLGA control meshes.25 However,
conducting polymers are not degradable and they are expected
to stay in vivo even small amount was used,26,27 which limits
their applications in tissue engineering. Much more attention
has been therefore given to aniline oligomers (aniline trimer,
tetramer and pentamer) modied polymers due to its good
electroactivity, low toxicity and degradability in body.28–30
Aniline oligomers might be consumed by macrophages and can
undergo renal clearance, avoiding the long term adverse
aCenter for Biomedical Engineering and Regenerative Medicine, Frontier Institute of
Science and Technology, Xi'an Jiaotong University, Xi'an, 710049, China. E-mail:
baoling@mail.xjtu.edu.cn; mapx@umich.edu; Fax: +86-29-83395131; Tel: +86-29-
83395361
bDepartment of Biomedical Engineering, University of Michigan, Ann Arbor, MI 48109,
USA
cDepartment of Biologic and Materials Sciences, University of Michigan, 1011, North
University Ave., Room 2209, Ann Arbor, MI 48109, USA
dMacromolecular Science and Engineering Center, University of Michigan, Ann Arbor,
MI 48109, USA
eDepartment of Materials Science and Engineering, University of Michigan, Ann Arbor,
MI 48109, USA
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response in vivo.29 Aniline pentamer crosslinked chitosan was was obtained upon addition of 0.2 mol ammonium persulfate
prepared and used for PC12 cell culturing.31 Our group as the oxidant at room temperature under stirring for 4 h. The
synthesized a series of degradable conducting copolymer and fully reduced leucoemeraldine (LM) of AT was prepared by
hydrogels based on aniline oligomers and polylactide32,33 and reducing the EM base AT with phenylhydrazine for 2 h at room
polycaprolactone.28,34,35 We also developed a facile way to temperature. The LMAT product was washed thoroughly with
synthesize electroactive degradable chitosan–tetraaniline distilled water, followed by washing in a Soxhlet extractor with
hydrogels by Schiff base reaction.36 However, Schiff base is not 1,2-dichloroethane and THF to remove the excess reducing
stable in acid or alkaline solution. Moreover, the fabrication of agent and byproduct in the reactions. The LMAT powders were
nanobrous scaffolds of aniline oligomers graed biomaterials then dried under reduced pressure. LM AT: 1H NMR (400 MHz,
to mimic the natural ECM has not been reported.
DMSO-d6): d ¼ 12.01 (s, 1H, –COOH), d ¼ 9.73 (s, 1H, –NHCO–),
The object of the present work was to create nanobrous d ¼ 7.78 (s, 1H, –NH–), d ¼ 7.69 (s, 1H, –NH–), d ¼ 7.64 (s, 1H,
electroactive scaffolds by using electrospinning from a chito- –NH–), d ¼ 7.36–7.38 (d, 2H, Ar–H), d ¼ 7.11–7.13 (d, 2H, Ar–H),
san-gra-aniline tetramer (CS–AT) copolymer which was d ¼ 6.99–6.85 (m, 12H, Ar–H), d ¼ 6.69–6.65 (d, 2H, Ar–H), d ¼
synthesized by an amidation reaction between the amine group 2.74–2.72 (t, 4H, –CH2CH2–).
of CS and carboxyl group of AT. We chose chitosan as matrix
because of its unique advantage in biomedical eld, and aniline
tetramer was then introduced to chitosan by simple amidation
reaction. The structure and properties of CS–AT copolymers
were characterized. The nanobrous structures of scaffolds
were generated from CS–AT solution by using electrospinning
technique. We also investigated the feasibility of using CS–AT
for adhesion and proliferation of two cell lines, chondrocyte
and C2C12 myoblast cells.
2.3 Synthesis of aniline tetramer graed chitosan (CS–AT)
Chitosan modied with different amount of AT was prepared as
shown in Scheme 2. The weight percentage of AT in the feed
mixture of CS and AT varied from 5% to 40% (Table 1). The
obtained aniline tetramer graed chitosan was respectively
abbreviated as CS, CS–AT5, CS–AT8, CS–AT10, CS–AT20, CS–
AT30 and CS–AT40, meaning that the feed ratio of AT was 0, 5, 8,
10, 20, 30, and 40 wt%.
A typical example (CS–AT10) was as follows: EMAT of 0.0556
g, NHS of 0.0688 g and EDC$HCl of 0.1145 g were dissolved in 5
mL of DMF (dried by CaH2). The mixture was stirred at room
temperature for 24 h. Aer the reaction, the mixture was added
dropwise into the 1 wt% solution of CS (0.5 g CS was dissolved
in 50 mL of 0.05 M HCl). The stirring was continued for 24 h at
room temperature. Aer nishing the reaction, the pH of the
mixture was adjusted by adding 3 M NaOH until the product
was precipitated. The precipitate was then ltered and re-dis-
solved in 10 mL of 1 wt% acetic acid aqueous solution. Aer
the removal of insoluble things by centrifugation, the pH of the
ltrate was again adjusted by adding 3 M NaOH until the
product was precipitated. The nal product was washed several
times with deionized water and dried in air.
2. Materials and methods
2.1 Materials
Chitosan (CS) with a molecular weight of 100 000–300 000 was
purchased from J&K Scientic Ltd. N-(3-Dimethylaminopropyl)-
N0-ethylcarbodiimide hydrochloride (EDC$HCl), N-hydroxy-
succinimide (NHS), hydrochloric acid (HCl), acetic acid (AcOH),
dimethylformamide (DMF), triuoroacetic acid (TFA),
dichloromethane (DCM) and sodium hydroxide (NaOH) were all
purchased from Aldrich and were used as received.
2.2 Synthesis of aniline tetramer (AT)
0.1 mol N-phenyl-1, 4-phenylenediamine was allowed to react
with 0.105 mol succinic anhydride in DCM to obtain phenyl/
carboxyl-capped p-phenylenediamine (Scheme 1). 0.1 mol p-
2.4 Electrospinning
phenylenediamine and 0.1 mol phenyl/carboxyl-capped Electrospun nanobers of CS and CS–AT were prepared in a
p-phenylenediamine were dissolved in a mixture solution of manner similar to that reported previously.37,38 Briey, CS and
DMF and HCl (Scheme 1). The emeraldine (EM) base form of AT CS–AT solutions with different concentrations ranging from
Scheme 1 Synthesis of carboxyl-capped aniline tetramer.
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Scheme 2 Synthesis of aniline tetramer grafted chitosan (CS–AT).
Table 1 Synthesis of CS–AT and the AT content in the copolymer by
UV-vis test
Electrochemical Workstation (CHI 660D) employing a three-
electrode system with a platinum disk as working electrode, a
platinum wire as auxiliary electrode, and an Ag/AgCl as refer-
ence electrode. The scan rate was 20 mV sꢀ1 for the sample.
Thermogravimetric analysis (TGA) of CS and CS–AT was per-
wt% of AT
in feed solution
wt% of AT
in samples
Sample
CS (g)
AT (g)
ꢀ1
ꢁ
CS
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0
0
5
8
10
20
30
40
0
formed on STARe/TGA/DSC with a heating rate of 10 C min
CS–AT5
CS–AT8
CS–AT10
CS–AT20
CS–AT30
CS–AT40
0.026
0.044
0.056
0.125
0.214
0.334
0.5
2.5
4.4
9.1
25.2
26.3
from 25 to 800 ꢁC under a nitrogen atmosphere. The UV-vis
spectra of EMAT and EMAT graed chitosan were recorded with
a UV-vis spectrophotometer (PerkinElmer Lambda 35) using the
mixture of DMF and 10 vol% aqueous solution of AcOH as
solvent (the volume ratio of DMF to AcOH aqueous solution was
1 : 1). The AT content of the samples in Table 1 was determined
by using UV-vis spectrophotometer according to ref. 31. The
morphology of the as-spun CS and CS–AT nanobrous
membranes were investigated by scanning electron microscope
(FE-SEM, SU-8000, Hitachi, Japan). Each sample was coated
with gold by a sputtering device for 30 s prior to SEM observa-
tion. XRD patterns of CS and CS–AT copolymer were obtained
with a Siemens D5005 diffractometer using CuKa radiation.
2.5% to 4% (w/v) were prepared respectively by using the
mixture of TFA and DCM (5 : 1 v/v) as solvent. The as-prepared
CS and CS–AT solution was fed into a 5 mL glass syringe tted
with a gauge 20 stainless steel needle used as the nozzle with an
inner diameter of 0.6 mm. An iron plate was used as collector,
and a positive voltage of 17 kV and negative voltage of 4 kV were
applied. Moreover, the tip-to-collector distance and ow rate of
feed solutions were xed at 15 cm and 0.04 mL hꢀ1, respectively.
2.6 Biocompatibility test of CS–AT polymer
Cell culture. The dog chondrocytes were isolated from a
beagle dog (2 week old) and incubated at 37 ꢁC in an incubator
2.5 Characterization
FT-IR spectra of EMAT, CS and CS–AT20 were recorded on a with 5% CO2. The complete growth medium was Dulbecco's
Nicolet 6700 FT-IR spectrometer (Thermo Scientic Instrument) Modied Eagle Medium (DMEM, GIBCO) supplemented with
1
with a resolution of 4 cmꢀ1. H NMR (400 MHz) spectra of AT 15% fetal bovine serum (FBS, GIBCO), 1.0 ꢂ 105 U per L peni-
and CS–AT were obtained on a Bruker Avance 400 MHz NMR cillin (Hyclone) and 100 mg Lꢀ1 Streptomycin (Hyclone). All
instrument with DMSO-d6 as solvent at room temperature. national and institutional guidelines for the care and use of
Cyclic voltammetry (CV) of AT and CS–AT was conducted on an laboratory animals were followed.
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C2C12 myoblasts were originally obtained from the ATCC C2C12 myoblasts were trypsinized, pelleted, resuspended in
(American Type Culture Collection) and cultured at 37 ꢁC in an complete growth medium and seeded in 96-well plate at a
incubator with 5% CO2. The complete growth medium was density of 1000 cells per well. Aer being cultured for 24 h, the
DMEM supplemented with 10% FBS, 1.0 ꢂ 105 U per L peni- medium was changed into the CS–AT extracts. 10 mL of the
cillin and 100 mg Lꢀ1 streptomycin.
alamaBlue® reagent was then added into each well aer being
Cell adhesion and morphology on CS–AT lm substrate. 80 cultured for another 24 h. Then the plate was incubated for 7 h,
mL of the solution of CS, CS–AT5, CS–AT8, CS–AT10 and CS– and 90 mL of the medium in each well was removed into a 96-
AT20 was coated onto 18 mm ꢂ 18 mm cover slides respectively well black plate. Fluorescence was read by a microplate reader.
and the solvent was evaporated at room temperature for 2 days. The cells were incubated for 1, 4, and 7 days and tested
The cover slides placed into a 6-well plate (Costar) were then respectively. Cells treated with the extract of CS served as the
sterilized with ethylene oxide and washed three times with positive control group. Tests were repeated ve times for each
DPBS and twice with cell culture medium for 30 min each at group. All the experiments were done twice independently.
ꢁ
37 C while rotating at 50 rpm. The chondrocytes and C2C12
Statistical analysis. All the data were expressed as mean ꢃ
myoblasts were respectively seeded on the cover slides at the standard deviation. Statistical comparison of the alamaBlue®
density of 1.0 ꢂ 105 cells per well. The plates were incubated for assay of C2C12 myoblasts proliferation on CS–AT polymer was
48 h at 37 ꢁC in an incubator with 5% CO2. For cell viability test, performed by the variance analysis of repeated measurements
the cells on cover slides were washed with Dulbecco's Phos- between two groups using the SPSS18.0 statistical package. A
phate-Buffered Saline (DPBS) twice and stained with LIVE/ value of p < 0.05 was considered statistically signicant.
DEAD® Viability/Cytotoxicity Kit (Molecular Probes) for 30 min
at room temperature following the protocol of manufacturer.
For cell morphology observation, the C2C12 cells on the cover
slides were washed twice with DPBS and xed with 2.5%
3. Results and discussions
3.1 Synthesis and characterization of CS–AT
glutaraldehyde in DPBS at room temperature for 30 min, then Chitosan (CS) with good biocompatibility, degradability and
washed with DPBS. The C2C12 myoblasts were stained by DPBS mechanical properties has been widely used in biomedical
solution with uorescein isothiocyanate (FITC) labeled phal- elds, such as drug delivery,39–43 gene delivery44 and tissue
loidin (Sigma) at the concentration of 5 mg mLꢀ1 for 90 min at engineering.45,46 In our previous work, we synthesized CS–
room temperature, and then washed with DPBS twice. The cells glutaraldehyde–AT hydrogels by taking glutaraldehyde as
were redyed with DPBS solution of DAPI (Sigma, 0.1 mg mLꢀ1) for crosslinking agent and graing agent.36 However, Schiff base
10 min. Cell adhesion and morphology was observed under the formed in the product is not stable. In this work, we use ami-
inverted uorescence microscope (IX53, Olympus).
dation reaction between the amine group of CS and carboxyl
C2C12 myoblasts adhesion and proliferation on the CS–AT group of AT to gra AT on the main chain of CS. The process of
lms. 100 mL of CS, CS–AT5, CS–AT8, CS–AT10, CS–AT20 and the preparation of aniline tetramer graed chitosan is depicted
CS–AT30 solutions (10 wt%) was added into a 96-well plate schematically in Scheme 2. Briey, aniline tetramer graed
(Costar) respectively and the solvent was removed. The plate chitosan was obtained by reacting the amino groups on chito-
was sterilized with ethylene oxide for 5 h and washed three san with the carboxyl groups of AT which were activated by NHS
times with DPBS and twice with cell culture medium for 30 min before mixing with CS solution. FT-IR and 1H NMR spectra were
1
ꢁ
at 37 C while rotating at 50 rpm.
used to characterize the CS–AT copolymer. Fig. 1 shows the H
100 mL of the rat C2C12 myoblasts suspension containing NMR spectra of AT and CS–AT. In the 1H NMR spectra of CS–AT,
approximately 2000 cells was added in each well. Aer being there were proton signals between 7.39 and 6.68 ppm (multi-
cultured for 24 h, 10 mL of the alamaBlue® reagent was then plet) ascribing to the hydrogen in the benzene ring, and proton
added into each well. The plate was incubated for 7 h at 37 ꢁC in signals at 3.8, 2.7 and 1.8 ppm assigned to the hydroxyl and
an incubator with 5% CO2 protected from direct light. 90 mL of alkyl groups of CS, which conrmed the existence of AT
the medium in each well was carefully removed into a 96-well segment in CS–AT, illustrating that the aniline tetramer was
black plate (Costar). Fluorescence was read using 567 nm as successfully graed onto chitosan. Furthermore, the signals of
excitation wavelength and 594 nm as emission wavelength by –NH– groups between 7.78 and 7.64 ppm from AT segment are
the microplate reader (Molecular Devices). The cells were still present in the CS–AT, indicating that the structural inte-
incubated for 1, 2, 3, and 4 days and tested respectively. Cells grality of AT is remained.
seeded on CS substrate served as the positive control group.
Tests were repeated six times for each group.
The FT-IR spectra of CS, AT and CS–AT were displayed in
Fig. 2. The IR spectrum of non-modied CS in Fig. 2(a) shows
Cytotoxicity of degradation products of CS–AT polymers. To absorption peaks at 1647 and 1590 cmꢀ1 assigned to the C]O
evaluate the cytotoxicity of degradation products of CS–AT5 and stretching (amide) and N–H bending (amine), respectively. In
CS–AT10 for a longer time, C2C12 myoblasts were cultured in Fig. 2(c) of AT, the bands at 1718 cmꢀ1 and 1664 cmꢀ1 are
the CS–AT polymer extracts and the cell viability was deter- assigned to the absorption from the carbonyl groups (–CO–) in
mined by alamaBlue® assay. The sterile CS–AT5 and CS–AT10 –COOH and amide groups –NHCO–, and the peaks at 1570 cmꢀ1
polymers with concentration of 100 mg mLꢀ1 were sucked in and 1490 cmꢀ1 ascribe to the vibration of the quinoid ring and
the complete growth medium at 37 ꢁC in the incubator benzene ring, respectively. By comparing with the spectra of AT
respectively for 14 days to extract the degradation products. and CS, curve b of CS–AT shows both peaks at 1646 cmꢀ1 from
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Fig. 1 1H NMR spectra of (a) AT and (b) CS–AT20.
CS and at 1580 cmꢀ1 from AT. While the peak at 1718 cmꢀ1 was very close. That might be because with the addition of
corresponding to –COOH group was absent in curve c, illus- increased amount of AT, the concentration of carboxyl groups
trating that the AT was chemically connected to CS main chains was no more a key factor effecting on the AT content in CS–AT
by the coupling reaction between the carboxyl group in AT and samples. However, the viscosity of CS–AT solution increased
amine group in CS. Moreover, the characteristic peaks of along with the increment of AT content, due to the strong
residual amino groups and hydroxyl group on chitosan in CS– attraction among AT segments in CS–AT. Especially for CS–AT30
AT emerged at 3354 cmꢀ1 which shied to lower wavenumber and CS–AT40, they were really not appropriate for fabrication of
compared to pristine CS at 3362 cmꢀ1, indicating that the CS–AT nanobers using electrospinning method in the
hydrogen bonds were formed between the CS and AT.
following work.
The compositions of the CS–AT copolymers, prepared under
different feed weight percentages of AT, are summarized in
Table 1. It is obvious that the content of AT in the CS–AT
samples increased with the increment of feed weight fraction of
AT. It is reasonable that the active sites increased with the
addition of AT in feed solution from 0% to 40% and the amount
of AT graed on CS increased accordingly. In addition, we can
see that in Table 1 the AT content in CS–AT30 and in CS–AT40
3.2 XRD of CS–AT copolymers
Fig. 3 shows XRD pattern of CS and CS–AT copolymers. We can
see that the XRD of CS showed two strong peaks in the dif-
fractogram at 2q at 10.2ꢁ and 19.8ꢁ which are characteristics of
the crystalline structure of chitosan, indicating the high degree
of crystallinity of chitosan. The rst peak at 2q ¼ 10.2ꢁ ascribes
to the crystal forms I and stronger reection appeared at 2q ¼
19.8ꢁ assigns to crystal forms II according to ref. 47 and 48. The
introduction of AT in the CS–AT was conrmed by the decrease
Fig. 2 FT-IR spectra of (a) CS, (b) CS–AT20 and (c) EMAT.
13656 | RSC Adv., 2014, 4, 13652–13661
Fig. 3 XRD pattern of CS–AT copolymer.
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of peak intensity at 2q of 10.2ꢁ because the AT segment might
interrupt the formation of inter- and intra-molecular hydrogen
bonds between chitosan and decrease the structure order of
chitosan aer modication.
3.3 Thermal stability of CS–AT copolymers
The thermal properties of CS and CS–AT were studied by TGA,
and the representative curves are shown in Fig. 4. For CS, there
is an obvious weight loss between 50 and 150 ꢁC assigned to the
weight loss of water moisture in the CS. The weight loss of CS–
AT copolymer was much less between 50 and 150 ꢁC compared
to CS, because the hydrophobic AT segment was introduced to
CS and the amine groups on CS main chain was consumed by
the amidation reaction with AT segment. When the temperature
ꢁ
increased to 800 C, the weight loss of CS was 80%, indicating
Fig. 5 UV-vis spectra of AT and CS–AT20 in the mixture of DMF and
10% aqueous solution of AcOH (v/v ¼ 1 : 1).
the degradation of the CS main chain. In case of the CS–AT
copolymer, the weight loss decreased with increasing the AT
content in the copolymer due to the better thermal stability of
AT segment, illustrating that the introduction of AT into CS
strengthened the heat resistance of CS.
3.4 Electroactivity of CS–AT copolymer
The electrochemistry of the samples was investigated by UV-vis
and CV measurements. Noticeably, there were three absorption
peaks in the UV spectrum for samples in Fig. 5. The two peaks
around 308 and 590 nm was attributed to the p–p* transition
of the aromatic benzene ring and the benzenoid to quiniod
(pB–pQ) excitonic transition, respectively. Doping with acid is a
key factor for electroactive polymers, which was veried by the
peak appearing at 430 nm due to the formation of polarons.
Furthermore, we can see from Fig. 5 that the absorption peak of
p–p* band has a slight blue shi from 308 nm to 299 nm by
comparing with the spectra of AT and CS–AT samples. This
phenomenon may be caused by the large steric hindrance in
CS–AT copolymer which restricts the non-planar conformation
of the AT segments, leading to a decrease in the effective
conjugation length of AT segment in the copolymer.49 The
Fig. 6 Cyclic voltammograms of AT (a) and CS–AT30 (b) in the
mixture of DMSO and HCl.
electroactivity of AT and CS–AT30 were also observed by CV test,
as shown in Fig. 6. AT shows two pairs of reversible redox peaks
at 0.38 and 0.58 V, respectively. The rst pair of redox peaks
around 0.38 V was due to the transition from the leucoemer-
aldine state to the emeraldine state. The second pair of redox
peaks with the redox potential at around 0.58 V was attributed
to the transition from the emeraldine state to the pernigraniline
state50 (as shown in Scheme 3). While the CV curve of CS–AT30
only shows one pair of reversible redox peaks around 0.67 V
which may ascribe to the transition of AT segment in CS–AT
from the leucoemeraldine state to the emeraldine state. Aer AT
was graed on CS, the large steric hindrance in CS–AT polymers
hampered the conjugation of the system and increased the
Fig. 4 Representative TGA curves of CS–AT copolymers.
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Scheme 3 Molecular structure of aniline tetramer at various oxidation states.
Fig. 7 SEM images of nanofibers from (a) CS, (b) CS–AT5, (c) CS–AT8 and (d) CS–AT10 with polymer concentration of 3 wt%.
oxidation energy barrier, which was in concordant with the concentration of 3 wt%, by varying samples from CS to CS–
result of UV-vis results. These data conrmed the good elec- AT10 (as seen in Fig. 7a–d). It was evident that the nanobers
troactivity of CS–AT copolymer and provided more evidence that were with few defects and the diameter of them ranged from
the AT segment was successfully graed onto CS main chains. around 100 nm to 600 nm. However, no bers but bead-like
structures for sample CS–AT20 were exhibited (data not
shown). That was caused by the increased affinity among the
chains of CS–AT with the increase of AT content as shown in
3.5 Nanobrous scaffolds from CS–AT by electrospinning
Table 1. The solubility of the CS–AT in the solvent decreased
compared to that of chitosan because of the hydrophobicity of
the AT segments. The viscosity of the CS–AT copolymer solu-
tion is higher than that of chitosan at the same concentration.
The introduction of AT into CS resulted in a tight entangle-
ment among polymer chains, which requires for a high voltage
to overcome the resistance caused by entanglement. In addi-
tion, dramatic morphology changes of samples varying from
CS to CS–AT10 were observed in Fig. 8a–d with a higher
concentration of 4 wt%. By increasing the CS–AT polymer
concentration, the entanglement among polymer chains
become deeper and the viscosity of the polymer solution
increased. Hence, the morphology of deposited CS and CS–AT
nanobers depended both on polymer concentration and the
AT content. A higher or lower concentration was not benecial
for the fabrication of nanober structures.
We employed the solution of CS and CS–AT using AcOH as
solvent for electrospinning in our initial work. A series of
concentrations from 1 wt% to 4 wt% of CS and CS–AT
were tested for electrospinning, but none of them produced
bers (data not shown). However, nanobers were success-
fully fabricated by taking TFA instead of AcOH as solvent
(Fig. 7). According to Ohkawa et al.,37 the triumphant elec-
trospinning of CS in TFA resulted from the formation of salts
between TFA and the amino groups on CS chain. Moreover,
another advantage of TFA is its property of volatility and
the volatility of solvent system was further ensured by
adding DCM.
The SEM pictures of the nanobers from CS and CS–AT
solution in the mixture of TFA and DCM, with different
concentration of AT content were represented in Fig. 7. Obvi-
ously, nanobers were deposited onto the collector at a xed
Fig. 8 SEM images of nanofibers from (a) CS, (b) CS–AT5, (c) CS–AT8 and (d) CS–AT10 with polymer concentration of 4 wt%.
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Fig. 9 Fluorescent images of chondrocytes (top) and C2C12 myoblasts (bottom) on CS–AT polymer films stained by live/dead assay after
culturing for 2 days. Most cells observed on CS, CS–AT5, CS–AT8 and CS–AT10 substrates were live (green) cells while some dead (red) cells
appeared on CS–AT20 substrate. Scale bars represent 50 mm.
Fig. 10 Fluorescent images of C2C12 myoblasts on CS–AT films stained by FITC labeled phalloidin and DAPI after being cultured for 48 h. The
C2C12 myoblasts exhibited a normal spindle-like morphology. Scale bars represent 50 mm.
Cell viability of C2C12 myoblasts on the CS–AT substrates
was further quantied by alamarBlue assay, and the results are
shown in Fig. 11. A continuous increase of cell number was
found from day 1 to day 4 for all groups (p < 0.01). The cell
viability of C2C12 myoblasts of samples CS–AT5, CS–AT8 and
CS–AT10 showed much higher values than that of the control
group CS which has good biocompatibility. The cell numbers
3.6 Cell adhesion, proliferation and morphology on CS–AT
polymers
The dog chondrocytes and C2C12 myoblasts were used for the
cytotoxicity test of the CS–AT polymers. Chondrocytes and
C2C12 myoblasts were cultured on the CS–AT substrates for
48 h and LIVE/DEAD® Viability/Cytotoxicity Kit was used to
qualitatively evaluate the biocompatibility of the materials. Live
and dead cells were respectively stained green and red in the
test, and the representative images were shown in Fig. 9. The
chondrocytes and C2C12 cells adhered well on all the substrates
and a majority of the live cells (green) were found for CS–AT5,
CS–AT8 and CS–AT10 except the TCP and CS, illustrating that
CS–AT5, CS–AT8 and CS–AT10 are generally non-toxic. More-
over, the CS–AT5, CS–AT8 and CS–AT10 showed improvement
in cell adhesion and proliferation compared to CS, due to the
good biocompatibility of CS and the electroactivity of AT
segment. However, some dead cells (red color) appeared on the
CS–AT20 substrate indicating that the CS–AT20 is weakly toxic.
Both the chondrocytes and C2C12 cells showed a similar result
on the CS–AT substrates.
The toxicity of the materials could affect the morphology of the
cells. Cell adhesion and morphology of C2C12 myoblasts on CS–
AT polymers was evaluated by cell staining. Aer being incubated
for 48 h and then stained by FITC labeled phalloidin and DAPI,
C2C12 myoblasts spreaded well and showed a normal, healthy
spindle-like shape on all the CS–AT polymer substrates (Fig. 10).
Fig. 11 Cell viability results of C2C12 myoblasts on the CS–AT
films. These cell culturing results showed that the CS–AT5, CS–AT8
and CS–AT10 enhanced the cell proliferation of C2C12 myoblasts
compared to CS.
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4. Conclusions
Nanobrous electroactive scaffolds were created by electro-
spinning technique from chitosan-gra-aniline tetramer (CS–
AT) copolymer. CS–AT copolymers with different AT content was
synthesized by the amidation reaction between the carboxyl
group and amine group of CS, and their structure was
conrmed by NMR, XRD and FT-IR. The thermal stability of the
CS–AT copolymer compared to CS was strengthened by the
introduction of AT segment. The electroactivity of the CS–AT
copolymer was veried by UV and CV measurements. The
nanobers were fabricated by electrospinning from the CS–AT
copolymer, and the diameter of the nanobers depended on the
polymer concentration and AT content. The CS–AT substrates
and their degradation products are not cytotoxic and could
improve the cell adhesion and proliferation of C2C12 myoblasts
compared to chitosan. All these results indicated that these
biocompatible electroactive materials could be potentially used
as tissue scaffolds that require electroactivity.
Fig. 12 Cell viability of C2C12 myoblasts in CS–AT extracts.
were increased in order for the CS–AT5, CS–AT8 and CS–AT10
groups probably owing to enhancement of chemical and signal
exchanges between the cells as a result of incorporating AT
segments in the copolymers.49 These results indicated that the
CS–AT copolymers enhanced the cell adhesion and prolifera-
tion of C2C12 myoblasts compared to CS. But the cell viability of
CS–AT20 and CS–AT30 group (data not shown) were obviously
lower than that of CS group (p < 0.05). This is because the
electroactivity that could improve the cell proliferation and
toxicity that hinders the cell proliferation were both introduced
into the materials along with AT segment.49,51 The AT segment
should be in a favorable content to maximize the effect of
electroactivity on promoting the cell proliferation and offset its
toxicity. Therefore, a high concentration of AT segment in the
copolymers may not be good for the materials for tissue engi-
neering application.
Acknowledgements
The authors gratefully acknowledge the National Natural
Science Foundation of China (grant number 21304073) for
nancial support of this work.
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