Review

Tissue engineering of electrically responsive tissues using polyaniline

based polymers: A review
Taimoor H. Qazi
1
, Ranjana Rai, Aldo R. Boccaccini
*
Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg, Cauerstr. 6, 91058 Erlangen, Germany
a r t i c l e i n f o
Article history:
Received 3 June 2014
Accepted 17 July 2014
Available online 10 August 2014
Keywords:
Polyaniline
Polyaniline oligomers
Conducting polymers
Tissue engineering
Electrical stimulation
a b s t r a c t
Conducting polymers have found numerous applications as biomaterial components serving to effec-
tively deliver electrical signals from an external source to the seeded cells. Several cell types including
cardiomyocytes, neurons, and osteoblasts respond to electrical signals by improving their functional
outcomes. Although a wide variety of conducting polymers are available, polyaniline (PANI) has emerged
as a popular choice due to its attractive properties such as ease of synthesis, tunable conductivity,
environmental stability, and biocompatibility. PANI in its pure form has exhibited biocompatibility both
in vitro and in vivo, and has been combined with a host of biodegradable polymers to form composites
having a range of mechanical, electrical, and surface properties. Moreover, recent studies in literature
report on the functionalization of polyaniline oligomers with end segments that make it biodegradable
and improve its biocompatibility, two properties which make these materials highly desirable for ap-
plications in tissue engineering. This review will discuss the features and properties of PANI based
composites that make them effective biomaterials, and it provides a comprehensive summary of studies
where the use of PANI as a biomaterial component has enhanced cellular function and behavior. We also
discuss recent studies utilizing functionalized PANI oligomers, and conclude that electroactive PANI and
its derivatives show great promise in eliciting favorable responses from various cell lines that respond to
electrical stimuli, and are therefore effective biomaterials for the engineering of electrically responsive
biological tissues and organs.
2014 Elsevier Ltd. All rights reserved.
1. Introduction
The eld of tissue engineering aims to regenerate or repair lost
or damaged tissues with the help of biomaterial scaffolds, cells, and
growth factors [1]. The scaffold should mimic the properties and
structure of the organ it aims to replace and essentially acts as an
articial extracellular matrix to support cell survival and growth.
Over the years, numerous studies have demonstrated that cells
respond to various biomaterial properties. Characteristics such as
wettability, mechanical stiffness [2,3], elasticity [4], and surface
topography [5,6] have been identied as having signicant inu-
ence over the behavior of seeded cells including migration, differ-
entiation, and structural reorganization. In a similar manner,
electrical signals too can inuence cellular behavior and function.
The effects of electrical stimuli on tissues have been known
since the 1960's when Bassett and colleagues showed that low
intensity direct electrical currents inuenced the mechanism of
bone formation in adult dogs [7]. Others have reported that the
application of pulsed electrical eld stimulation on mouse osteo-
blast cells resulted in a signicant increase in DNA synthesis [8],
whereas a uniform electrical eld inuenced the clustering and
distribution of membrane proteins on the cellesubstrate interface,
and was able to control the direction of cell locomotion in two
broblast cell lines [9]. Further studies have also reported on the
effects of applied electric elds on the migratory behavior of ker-
atinocytes, vascular endothelial cells, and corneal epithelial cells
[10e12]. Recently, electrical stimulation of myoblasts seeded on
three-dimensional collagen scaffolds was shown to inuence
myogenic differentiation and deposition of type I collagen in a
skeletal muscle construct [13], whereas electrical stimulation of
cardiomyocytes seeded on collagen/Matrigel scaffolds induced
their alignment and coupling, leading to synchronous contractions
[14]. It is therefore evident that electrical stimuli can evoke desir-
able cellular responses, especially from cells belonging to
* Corresponding author. Tel.: 49 9131 85 28601; fax: 49 9131 85 28602.
E-mail address: aldo.boccaccini@ww.uni-erlangen.de (A.R. Boccaccini).
1
Present address: Julius Wolff Institute, Augustenburger Platz 1, Charit e Uni-
versit atmedizin Berlin, 13353 Berlin, Germany.
Contents lists available at ScienceDirect
Biomaterials
j ournal homepage: www. el sevi er. com/ l ocat e/ bi omat eri al s
http://dx.doi.org/10.1016/j.biomaterials.2014.07.020
0142-9612/ 2014 Elsevier Ltd. All rights reserved.
Biomaterials 35 (2014) 9068e9086
electrically excitable tissues such as skeletal muscle, nerve, cardiac
tissue, and bone. In general, majority of the biomaterial scaffolds
employed in tissue engineering and electrical stimulation studies
are electrically resistant in nature. Indeed literary evidence sug-
gests that utilizing electroactive materials in scaffolds could greatly
improve the functional outcomes of such studies.
Some efforts in this regard have been made through the incor-
poration of conductive particles such as carbon nanobers [15] and
gold nanowires [16] in scaffolds to modulate cellular behavior. The
inclusion of these conductive elements makes it possible for the
transmission of electrical signals (supplied froman external source)
throughout the cell seeded scaffold. The use of gold and carbon
based particles in implantable scaffolds could potentially be prob-
lematic since these materials are non-biodegradable and their
long-term effects in vivo are largely unknown. Owing to the lack of
solubility, a further drawback is the inhomogeneous distribution of
the conducting particles in the two phase composite system. This
issue can be overcome by employing conducting polymers, which
can be dissolved in organic solvents and blended with other poly-
mers before being processed, for example by electrospinning, into
porous scaffolds. Blending of the conducting polymer into another
polymer system ensures homogenous distribution of the con-
ducting polymer molecular chains throughout the composite
blend, which translates into electrical signals being effectively
transmitted throughout the entirety of the composite, more
importantly reaching all seeded cells and consequently modulating
their behavior.
Polyaniline (PANI) offers a viable option to induce electroactivity
in biomaterial scaffolds and substrates, and its popularity for use in
biomedical and tissue engineering applications can be judged from
the increasing number of research publications on the subject in
the past decade. Such signicant interest and available knowledge
in the eld of polyaniline for tissue engineering applications has
motivated the preparation of the present review in which we
discuss different aspects of PANI such as its biocompatibility, con-
ductivity, processability, and antibacterial effect, properties that
make it an attractive biomaterial component. The effect of PANI on
cellular behavior in conjunction with electrical stimulation, and its
application in skeletal, cardiac, and nerve tissue engineering are
also discussed. Additionally, the reviewalso touches upon the novel
area of functionalized aniline copolymers which are simulta-
neously conducting and biodegradable, hence rendering them
desirable for use as biomaterials in the eld of tissue engineering.
2. Polyaniline
An early inherently conducting polymer was reported in 1977
when MacDiarmid, Shirakawa and Heeger recognized an 11 orders
of magnitude increase in the conductivity of polyacetylene upon
doping with iodine [17]. Since then, conducting polymers have
witnessed an immense increase in scientic and technological in-
terest, mainly due to their tunable electrical properties, ease of
synthesis, and environmental stability. Conducting polymers have a
conjugated backbone (alternating single and double bonds), which
gives rise to an extended p network [18]. Movement of electrons
within the p network is what gives the polymer, metal like semi-
conductive properties [19]. Polypyrrole, PANI, polythiophene and
poly (3,4-ethylenedioxythiophene) (PEDOT) are just a few of the
many conductive polymers that are employed in technological
applications today. For instance, PANI nds applications in the
microelectronics industry including photovoltaic cells [20], light
emitting diodes [21], and electrochromic displays [22]. In the bio-
logical eld, conductive polymers were shown to be compatible
with cells and other biological molecules [23], and have thus found
applications as substrates for cellular stimulation, DNA synthesis
and protein secretion, as biosensors and bio-actuators [24], and
recently as tissue engineering scaffolds [25]. Interest in conducting
polymers for application in tissue engineering increased after
Wong et al. used polypyrrole to show that application of an elec-
trical potential can non-invasively control certain aspects of cellular
behavior such as spreading, DNA synthesis, and differentiation [23].
Subsequently, numerous studies have highlighted advantages, and
proposed the use of conducting polymers for nerve [26], bone [27],
and cardiac [28] regeneration, among others [29].
Polyanilines are a class of conducting polymers which can exist
in three different oxidation states, namely the completely reduced
leucoemeraldine base, the completely oxidized pernigraniline base,
and the emeraldine base consisting of alternating oxidized and
reduced repeat units in its structure, as shown in Fig. 1 [30].
PANI is generally synthesized by either chemical or electro-
chemical methods [31]. The advantage of electrochemical methods
is that uniform, high purity lms of PANI can be deposited and
collected on a metal electrode. The techniques used for electro-
chemical synthesis include potentiostatic (constant voltage), and
galvanostatic (constant current) polarization, and cyclic voltam-
metry [32]. However, PANI is most commonly synthesized using
chemical methods by oxidative polymerization of aniline in
aqueous media in the presence of an oxidizing agent. Other
chemical methods for synthesizing PANI include emulsion,
dispersion, solution, interfacial, metathesis, and self-assembling
polymerization [18].
Ever since conducting polymers such as polypyrrole were found
to be compatible with cells and biological tissues, efforts to identify
and establish the feasibility of other conductive polymers such as
PANI for use in biomedical applications have been on the rise. Once
the biocompatibility of PANI was established both in vivo and
in vitro [33], research on PANI focused on designing materials for
applications where it is in direct contact with biological tissues.
Thus, PANI based composites have been employed in various
biomedical applications including scaffolds for tissue engineering
Fig. 1. Oxidation states of polyaniline, (A) the completely reduced leucoemeraldine
base, (B) the completely oxidized pernigraniline base, (C) the half oxidized-half
reduced emeraldine base, and (D) the doped, conductive form of emeraldine base,
emeraldine salt.
T.H. Qazi et al. / Biomaterials 35 (2014) 9068e9086 9069
[29], microspheres for drug delivery (Fig. 2) [34], multifunctional
nanobers with anti-cancer effects [35], anti-bacterial substrates
[36], and nanoparticles for anti-tumor therapy [37].
2.1. Conductivity
The emeraldine base form of polyaniline (PANI-EB) is not
inherently conductive, but can be converted into an electrically
conductive emeraldine salt form (PANI-ES) via doping [38]. Doping
is the process where a neutral polymer is either oxidized (p-
doping) or reduced (n-doping), followed by delivery of a counter
ion (dopant), which introduces charge carriers that can move along
and between the polymer chains producing electricity. Doping
agents are proton donors, and are therefore most usually acids such
as hydrochloric, sulfuric, or sulfonic acids. The electrical conduc-
tivity of the doped PANI can be inuenced by a variety of factors
including the degree of oxidation of the polymer, type of protonic
acid used for doping, degree of protonation, moisture/water con-
tent, morphology (stretching) of the polymer chains, chain length,
and degree of crystallization [39]. For example, a wet polymer
needs a small degree of protonation to register an increase in
conductivity several orders in magnitude. This is because in the
presence of moisture, the charge transport can take place due to
two mechanisms: proton exchange reactions, and intermolecular
electron transport [40]. The inuence of PANI chain alignment
(morphology) and chain length on its conductivity was studied by
Monkman et al. by casting PANI lms and subjecting it to uniaxial
stress at elevated temperatures [41]. Conductivity was found to be
anisotropic because the processes of energy transfer parallel and
perpendicular to chain alignment direction had different energy
barriers. The effect of different dopants on the conductivity of PANI
is caused by the dopant size, since large dopants can increase
interchain separations which hamper the charge carriers' mobility
across different chains hence affecting the conductivity [42].
2.2. Processability
To be useful in technological applications, a polymer should be
processable into different forms such as lms or bers. It was
generally accepted that the doped form of PANI did not dissolve in
common nonpolar or weakly polar organic solvents [43] thereby
hindering its fabrication into various shapes, three dimensional
constructs, or bers. However, Angelopoulos et al. showed that the
emeraldine base form of PANI can be dissolved in N-methyl
pyrrolidinone (NMP), as a result yielding lms which could be cast
from solution and subsequently doped by immersion in hydro-
chloric acid [44]. For a long time, only a few solvents like NMP and
concentrated sulfuric acid were known to dissolve PANI. However
in 1992, Cao et al. discovered that if PANI-EB is doped using a
functionalized protonic acid such as dodecylbenzenesulfonic acid
(DBSA), the resulting PANI-ES is rendered soluble in common
organic solvents such as chloroform [43]. This advancement has
since allowed convenient processing of PANI, and has led to the
development of PANI electrospun bers, nanowires, and nanotubes
[18,45,46], and it has opened new avenues for applications.
2.3. Biocompatibility
Serious interest in using PANI for biomedical and tissue engi-
neering applications soared after Kamalesh et al. successfully
veried the long term in vivo biocompatibility of PANI lms
following subcutaneous implantation in male Sprague Dawley rats
for up to 90 weeks [47]. No signs of toxicity or abnormality in the
surrounding tissues were observed and the presence of PANI did
not cause any undesirable inammatory response. Mattioli-
Belmonte and colleagues also conducted in vivo studies on
several polymers including PANI and did not nd any adverse ef-
fects on tissues surrounding the implant [33]. In all these studies,
the in vivo response to PANI was deemed acceptable as no major
inammatory reaction or tumor formation was observed. As
compared to just a handful of in vivo studies, the in vitro biocom-
patibility of pure PANI or PANI in combinationwith other polymeric
systems has been established with various cell lines. For example,
pure PANI was shown to be cytocompatible with H9c2 cardiac
myoblasts [48], and PC-12 cells [49]. As part of a composite, PANI
has exhibited cytocompatibility with C2C12 myoblasts [50], L929
murine broblasts [51], human mesenchymal stromal cells [52],
and rat nerve stemcells [53], among others. Although these studies
carried out at in vitro and in vivo levels have demonstrated certain
extent of biocompatibility, reservations still exist, especially after
some inammation and brous tissue encapsulation was observed
in an in vivo study by Wang and colleagues [54]. Therefore when
compared to other conductive polymers like polypyrrole, whose
biocompatibility has been well established both in vitro and in vivo
[55e57], biomedical application of PANI is still restricted. This
hindrance stems from the limited number of biological in-
vestigations that have been carried out with PANI, and allegations
that it exhibits selective biocompatibility to few cell lines [48].
Fig. 2. PANI containing composites proposed for drug delivery applications. SEM image of hollow PANI and Indomethacin (IND) composite microspheres (A), and switchable drug
release behavior of IND from the composite microspheres at different pH values (B). Release of the drug was triggered at pH 7.4 and ceased at pH 2. Adapted from Ref. [34] with
permission from John Wiley and Sons.
T.H. Qazi et al. / Biomaterials 35 (2014) 9068e9086 9070
The biocompatibility of PANI has also been a subject of debate
due to its non-biodegradability, hence its long termpresence inside
the patient could lead to unwanted side effects such as chronic
inammation, for instance due to wear and debris formation [38].
However, most hesitation to use PANI is caused by the fact that its
monomer, aniline, and other reaction byproducts, especially the
aniline dimer benzidine that is formed during PANI synthesis, are
aromatic amines which have been known to be carcinogenic and
therefore highly dangerous [58e61]. However, judging the poten-
tial application of PANI based on the probable carcinogenicity of
aniline is debatable. This is because aniline and its derivatives have
been successfully used for a wide range of applications. For
example, aniline is used as a precursor in the polymer industry for
the synthesis of polyurethane [62] which is a well-known material
for biomedical applications, and in the pharmaceutical industry to
synthesize drugs like paracetamol. These varied applications of
aniline and its derivatives, therefore, support the potential of using
its polymerized form, PANI, for tissue engineering applications.
However, caution must be exercised and improved biocompati-
bility of PANI warrants further in-depth biological investigations
involving more cell types and in different animal models. One study
that demonstrated measures to improve the biocompatibility of
PANI was carried out by Humpolicek and colleagues [63]. By taking
necessary measures to purify PANI through repeated de-
protonation and re-protonation cycles, the cytotoxicity of PANI
was signicantly reduced [63]. It can therefore be concluded that
the apparent cytotoxicity of PANI is caused by the reaction
byproducts of polymer synthesis rather than PANI itself, and that
the biocompatibility of PANI can be considerably enhanced by
employing additional purication steps during synthesis or by us-
ing commercially available PANI of high purity.
In order to get electrically conducting PANI, the processing re-
quires the addition of a dopant (usually strong acids) to protonate
the PANI backbone [21]. Though not in large quantities, the acid
dopant eventually leaches out fromthe PANI matrix, for example in
an aqueous environment, thereby causing a local acidic environ-
ment surrounding the PANI component [40,48]. Therefore, despite
using highly pure doped PANI as the starting material, the possi-
bility of a localized acidic environment causing some level of
toxicity to its surrounding biological environment will always exist.
This was demonstrated quite elegantly by Cullen et al. when they
assessed neuronal cell viability adjacent to, and some distance
away from polyaniline-polypropylene (PANI-PP) substrates [64].
While complete cell death was observed for neurons present in the
immediate vicinity of the PANI-PP sheets, cells located 3 mm away
had a viability of 60%. PANI-PP sheets were then soaked in media to
allow for leaching before evaluating the viability of cultured neu-
rons. The viability of cells right next to the PANI-PP sheets increased
from <1% to 89% after the soaking step whereas media containing
the leachate reduced the neuronal viability to 5%. Fluorescent im-
ages of cell viability at various distances from the PANI-PP sheets
are shown in Fig. 3. This study conrmed that the cytotoxicity was
caused by the released chemical species (most probably the acid
dopant) which was detrimental for the survival of cells [64].
Fig. 3. Confocal uorescent micrographs depicting the viability and survival of dorsal root ganglia neurons at various distances away from polyaniline/polypropylene composite
sheets at day 7 of culture. Major cell death was observed for neurons cultured adjacent to the PANI-PP sheets (A), whereas the viability improved 1.5 mm away (B), and the highest
number of viable cells was observed 3 mm away (C) from the PANI-PP sheets. These results indicated that a substance which is harmful for cell survival was leaching out from the
PANI-PP sheets, and due to diffusion its effects were not as intensely felt further away from the sheets. Neurons were then cultured adjacent to PANI-PP sheets (D), without any
sheets but in media conditioned by the toxic leachate (E), and adjacent to PANI-PP sheets which had been pre-soaked in medium (F). Pre-soaking before culture was found to
remove the leachate from the PANI-PP sheets and result in high viability of cultured cells.

IOP Publishing. Reproduced from Ref. [64] (http://dx.doi.org/10.1088/1741-2560/5/4/002)
by permission of IOP Publishing. All rights reserved.
T.H. Qazi et al. / Biomaterials 35 (2014) 9068e9086 9071
However, it is unclear what amount of dopant was used as other
studies which involve cell seeding directly on the substrate do not
report such a hostile response. For example, when H9c2 cardiac
myoblasts were seeded on PANI lms, the cells attached readily
onto the surface but displayed a slower proliferation rate between
48 and 100 h most likely due to leaching of residual dopant acid
[48]. Once all the dopant had leached out, the proliferation rate
increased and the nal cell number caught up with that on the TCP
control.
Strategies to make PANI more biocompatible and render it
suitable for tissue engineering applications have involved (1)
combining PANI with biocompatible polymers such as gelatin and
poly--caprolactone (PCL) to form a composite system thereby
mitigating any potential cytotoxic effects of PANI and (2) immobi-
lizing cell adhesive peptide sequences onto the PANI backbone. For
example, in a study by Li and colleagues, the bioactive peptide
sequences Tyr-Ile-Gly-Ser-Arg (YIGSR), and Arg-Tyr-Ser-Gly-Ile
(RYSGI) were grafted onto PANI backbones and the resulting
cellular behavior and biocompatibility was compared with un-
modied PANI [65]. Higher cell adhesion was observed for peptide
modied PANI compared to unmodied ones. The grafting of ad-
hesive peptides also improved proliferation of neuronal PC-12 cells,
and promoted neurite extension and neuronal network formation
without the addition of nerve growth factor (NFG). Similarly, ATQD,
an electroactive oligomer derived from PANI, was modied by
covalently grafting cyclic (Arg-Gly-Asp-D-Phe-Lys) containing the
RGD peptide sequence [66]. The presence of the peptide not only
enhanced PC-12 cell adhesion, but also improved the proliferation
rate and induced neurite outgrowth from the cells. Xu et al.
discovered that PANI and poly(L-lactide-co--caprolactone) (PLCL)
composites exhibited different levels of cytotoxicity to PC-12 cells
when in powder or ber forms [67]. The authors attributed this
nding to the extent of direct contact and exposure of cells to PANI.
In the powder form of the composite, the cells came into direct
contact with PANI particles, but in the ber form, most of the PANI
particles were not at the surface but rather embedded inside the
PLCL matrix on which the cells were attached. Furthermore, the
toxicity was found to be highest at high doses of the composite
(50 mg) and decreased to substantially low levels at doses below
10 mg. In another study, the compatibility of single walled carbon
nanotube-polyaniline (SWCNT-PANI) hybrids with primary im-
mune cells (macrophages and mouse spleen cells) had been
demonstrated to be dose-dependent, with no cytotoxicity being
shown at biologically relevant doses [68].
Biomaterials which are bound to come into direct contact with
blood for an extended period of time in vivo, such as biomaterials
designed for vascular tissue engineering, run the risk of inducing
thrombosis due to their surface properties and hence such bio-
materials need to be hydrophilic so as to avoid protein adsorption
and platelet adhesion [69]. Li et al. grafted poly(ethylene oxide)
(PEO) on the surface of PANI lms via chlorosulfonation to prevent
protein adsorption and platelet adhesion, thereby increasing the
biocompatibility of the PANI lm [70]. The water contact angle
measurement indicated that compared to pristine PANI, the surface
of PEO-PANI lmwas hydrophilic, and allowed 80% less proteins to
adsorb on its surface. Additionally, SEM images (Fig. 4) showed a
lower number of platelets adhering to the PEO-PANI lmrelative to
pristine PANI [70].
In summary, though some initial toxicity to cells which were
exposed to materials containing PANI has been reported, the in-
tensity of a toxic response can vary from slight lowering of the cell
proliferation rate to wide spread cell death. The kind of toxic
response can depend on various factors and hence, there are
numerous ways to make sure the PANI exposed to cells is biocom-
patible and no adverse reaction occurs. As mentioned above, these
include pre-soakinginmediumbeforeexposuretocells, purication
of the PANI after synthesis, immobilization of peptide sequences,
and adjusting surface properties such as wettability.
2.4. Biodegradability
Materials used to develop scaffolds should typically be biode-
gradable in nature in order to avoid the onset of infections associ-
ated with the long term presence of foreign materials in the body,
give control over tissue remodeling, and degrade over time as new
tissue forms in its place. Conducting polymers in general, and those
proposed for tissue engineering applications in particular, are
largely non-biodegradable [29]. Most researchers have tried to
counter this problem by blending conducting polymers like
Fig. 4. SEM images depicting differences in platelet adsorption on pristine PANI lm
surface (A), PEO-grafted PANI lm with a grafting density of 3.3 (B) and 5.1 (C).
Increasing the grafting density of PEO signicantly reduces the number of platelets
adsorbed on the surface. In case of biomaterials that will have long term exposure to
blood, surface treatments such as these make the material more biocompatible in
terms of reducing the chances of thrombus formation. Reproduced from Ref. [70] with
permission from Elsevier.
T.H. Qazi et al. / Biomaterials 35 (2014) 9068e9086 9072
polypyrrole and PANI with other biodegradable and biocompatible
polymers like PCL and poly(lactic acid) [52]. For example, Li et al.
prepared in situ forming gelatin-graft-PANI hydrogels crosslinked
with genipin that showed 80e95% weight loss in vitro, depending
on crosslinker density and PANI content [71]. However, most other
PANI containing composites reported in literature have failed to
show similar degradability.
Zelikin et al. designed erodible polypyrrole that degrades slowly
in physiological conditions by polymerizing b-substituted pyrrole
monomers containing hydrolyzable side segments [72]. The last
few years have seen researchers trying out similar strategies for
aniline based conducting polymers [73].
Even though PANI (in all its oxidation states) is non-
biodegradable, a new variety of electroactive and biodegradable
copolymers containinganiline oligomers coupledwithhydrolyzable
groups has been synthesized to facilitate the application of aniline
containing conductive polymers for in vivo tissue engineering ap-
plications. Guo et al. proposed a universal strategy involving com-
bined ring opening polymerization, and post functionalization via
oxidative coupling reactions to synthesize electroactive and
degradable block copolymers [74]. Zhang et al. synthesized a poly-
phosphazene with aniline pentamer and glycine ethyl ester as side
groups responsible for inducing electroactivity, and biodegrad-
ability, respectively [75]. The copolymer, termed PGAP, recorded a
mass loss of 50% after 70 days of immersion in PBS. SEM images of
samples after various immersion times revealed the formation of
pores and holes at the surface, becoming bigger and deeper with
increasingimmersiontime, indicatingsurface erosion, causedbythe
hydrolysis of the glycine ethyl ester side groups.
Similarly, Huang et al. synthesized an ABA block copolymer, PAP,
consisting of an electroactive aniline pentamer with biodegradable
polylactide segments attached to its two ends [76]. In vitro de-
gradability studies recorded a mass loss of 60% after 40 days, con-
rming the biodegradability of the conductive copolymer.
Relatively quicker degradation was achieved in AB block co-
polymers of analine pentamer and polylactide, PLAAP, where
within 200 h, 60% mass loss was observed [77]. The copolymer was
designed such that the non-toxic degradation products including
low molecular weight block copolymer and lactic acid oligomers
could be released following degradation by hydrolytic cleavage.
Copolymers of polyurethane derived from PANI and PEG were
produced via hydrogen transfer polymerization using, among
others, PANI oligomer as chain extenders [62]. Presence of the PANI
oligomer signicantly enhanced the degradation rate of the
copolymer, resulting in a mass loss of over 60% after incubation for
30 days. The inclusion of PANI oligomers, therefore, had consider-
able inuence on degradation rate, which could be controlled by
changing the length of the oligomer chain and the hydrolyzable
group segments on its ends.
Liu et al. prepared porous copolymer hydrogels of aniline pen-
tamer grafted onto gelatin which showed degradation in PBS,
loosing between 45 and 65% of their mass after 28 days of im-
mersion, depending on the amount of aniline pentamer present
[78]. Copolymers of electroactive tetraaniline grafted onto poly(-
ester amide) (PEA-g-TA), developed by Cui et al. [79], were
immersed in TriseHCl buffer solution containing proteinase K and
allowed to degrade for 6 days. Pure poly(ester amide) lost 45% of its
mass, whereas the mass lost by PEA-g-TA copolymers decreased
from42% to 25% with increasing tetraaniline content. The proposed
reasons for lower degradation rate were the hydrophobic character
of the tetraaniline, and increased steric hindrance.
Though polyaniline itself is not degradable, the use of aniline
based copolymers functionalized with hydrolyzable groups ensures
that the resulting materials have the same electroactive properties
as PANI with the additional benet of being biodegradable.
2.5. Antibacterial efcacy
Bacterial infection and biolm formation on biomaterials are
major issues which affect the performance and lifetime of pros-
thetic implants and scaffolds. Bacterial contamination can be traced
to sources such as the environment in the operating theater, non-
sterile surgical tools, or the resident bacteria inside the patient.
Advanced strategies to inhibit biolm formation involve antibiotic
coatings on implant surfaces [80], anti-microbial wound dressings
[81], and novel drug releasing biomaterials [82,83]. The presence of
PANI in composites has been reported to confer anti-microbial
properties against different bacterial species including Escherichia
coli, Streptococcus sp., Staphylococcus sp., and Klebsiella sp. [84].
The antibacterial effect of conducting PANI under both dark and
visible light conditions was reported by Shi et al. [85]. Cast lms of
PANI-polyvinylalcohol (PVA) were tested against Escherichia coli
(E. coli) and Staphylococcus aureus (S. aureus) by the lmattachment
method. While pure PVA showed no antibacterial behavior, a 100%
reduction in the population of both E. coli and S. aureus was
observed on PANI-PVA lms. The observed antibacterial effects can
be explained by (a) the release of acidic dopant ions from the
conducting PANI which reacts with, and kills the bacteria, or (b) the
electrostatic adherence between the bacteria and PANI, both car-
rying charges of opposite polarity, causing the bacterial cell wall to
break thereby causing its death [86].
Gizdavic-Nikolaidis et al. investigated the mode of antibacterial
action of PANI and functionalized aniline based polymers (co-poly-
mers of anilineandaminobenzoicacideABA-PANI) onE. coli, S. aureus,
and P. aeruginosa [87]. It was found that the conductive forms of PANI
and ABA-PANI were more effective in inhibiting bacterial growth than
the non-conductive forms, and between the two, ABA-PANI induced
bacterial inhibition at a much lower concentration than PANI. Quan-
titative RT-PCR analysis revealed that exposure of E. coli to function-
alized PANI down-regulates the expression of genes whose products
are involved in processes vital to bacterial survival such as energy
metabolism and transport, and cell wall and biolm formation. In
another study, functional copolymers of PANI and 3-aminobenzoic
acid, (3ABAPANI), were shown to be excellent matrices for bro-
blasts, and displayed antibacterial activity against S. aureus, making
these ber mats an attractive option for use as wound dressings [88].
Riaz et al. developed nanostructured copolymers of poly(-
naphthylamine) (PNA) and aniline (PNA-co-PANI) in a colloid form,
and evaluated their antibacterial efcacy against S. aureus and E. coli
[89]. Results revealed that PNA-co-PANI caused greater bacterial in-
hibition than pure PNA and the control drug ampicillin. The authors
proposethat theantibacterial actioninGramnegativebacteria(E. coli)
is caused by the blockage and consequent depletion of nutrients
resulting in cell death, while in gram positive bacteria (S. aureus) it is
caused by disruption of the cell wall due to electrostatic binding, and
exposure of the cell membrane to osmotic shock leading to lysis [90].
Kucekova et al. studied the antibacterial properties of PANI lms
containing silver nanoparticles [91]. Conducting PANI and its
composites with silver were found to have a greater antibacterial
effect on both S. aureus and E. coli, whereas non-conducting PANI
and its composites with silver did not inuence E. coli and had only
minimal effect on S. aureus. Based on these results, the authors
propose that the antibacterial activity of PANI is most likely a result
of the presence of acidic dopants on the PANI backbone.
3. Polyaniline for tissue engineering applications
3.1. Pure polyaniline lms
While majority of the studies focusing on utilizing PANI for
tissue engineering applications have combined it with other
T.H. Qazi et al. / Biomaterials 35 (2014) 9068e9086 9073
biocompatible polymers, there have been few studies investigating
the behavior and function of cells on pure PANI lms. Wang et al.
produced pure PANI lms either by casting on a Polytetrauoro-
ethylene (PTFE) substrate, or by direct deposition followed by
doping with four different acids [92]. All PANI lms were found to
be biocompatible, with PC-12 cells displaying signicantly higher
cell attachment and proliferation on synthesized lms compared to
the cast lm. The nanostructured surface in synthesized lms
caused a change in surface hydrophobicity which resulted in an
enhancement of cell attachment and proliferation [86].
Bidez et al. studied the adhesion and proliferation of H9c2 car-
diac myoblasts on non-conductive and conductive PANI lms and
found both substrates to be biocompatible, with cells readily
attaching and proliferating to form conuent monolayers after 6
days [48]. Furthermore, the conducting PANI lm, which was doped
with 1 M HCl for 15 min, was found to maintain sufcient levels of
electrical conductivity for up to 100 h in an aqueous physiologic
environment. Based on surface resistivity measurements, the au-
thors propose that the dopant acid leaches out completely by the
100 h time point, which not only results in de-doping of the PANI
and hence loss in conductivity, but also causes an appreciable in-
crease in the rate of cellular proliferation. This study therefore also
sheds light on the inuence of acidic leachates on seeded cells,
conrming that population doubling time was observed to be lower
for cells grown on conductive PANI lms for the rst 100 h, but
returned to signicantly higher values between 100 and 150 h
when the leachates had supposedly diffused away. Other studies
have also corroborated this nding by showing higher cell attach-
ment on non-conducting PANI lms compared to conducting ones
that release acidic dopants [93].
Using a combined self-assembly and surface polymerization
approach, Liu et al. produced PANI lms on silicon substrates and
reported higher long term cell viabilities of PC-12 cells on PANI
lms compared to plain silicon substrates and TCP controls, as
shown in Fig. 5 [49]. Techniques such as this enable surface coating
of materials such as scaffolds with a layer of conducting PANI that
can be used to control cell behavior via electrical signaling.
3.2. Polyaniline composites and blends
PANI has been combined with other biocompatible and biode-
gradable polymers to form conductive composites or blends with
tunable mechanical and physicochemical properties. The ability to
alter mechanical properties such as elongation and strength by
varying polyaniline content would allow the composite to mimic as
closely as possible the properties of the native tissue that it is
designed to replace or regenerate. Because PANI is a rather brittle
material, combining it with elastic polymers should result in ma-
terials which are more mechanically compatible with native tis-
sues. Table 1 gives an overview of the numerous polyaniline
containing polymer composite systems developed for tissue engi-
neering applications, their electrical conductivities, and the cell
lines tested.
Jeong et al. developed three-dimensional electrospun compos-
ites of PANI and PLCL which were found to be (a) surface-active e
which could enhance initial protein adsorption and subsequent cell
adhesion, (b) conductive e with a highest conductivity of 0.0138 S/
cm, (c) have bers in the range 300e400 nm e allowing cells to
sense and respond to the nanoscale topography, and (d) possess
mechanical properties which could be tuned by changing PANI
content [94]. For instance, an increase in PANI content reduced the
tensile strain of the composite from 390 to 200% and, upon further
addition of PANI, the tensile strain was further decreased to a range
which matches the strain typically exhibited by native soft tissues
such as skin and blood vessels (35e115%) [107]. For cytotoxicity
assessment, three different cell lines were tested including human
dermal broblasts, NIH-3T3 broblasts, and C2C12 myoblasts. Not
only were all the cell types viable on the composite bers, but they
exhibited signicantly enhanced viability on composites containing
higher concentrations of PANI. NIH-3T3 broblasts were then
seeded on PANI-PLCL and pure PLCL bers and subjected to elec-
trical stimulation for two days. Evaluation of the results showed
that while an electrical stimulus of 200 mA caused cell death and
very low metabolic activity, a current of 20 mA signicantly
increased the mitochondrial metabolic activity of the broblasts
Fig. 5. The culture and survival of PC-12 cells stained with acridine orange on pristine Silicon substrate (AeC), and on PANI lm (DeF), for 1 (A,D) and 2 (B,E) days. C and F show
magnied regions of B and E, respectively. Signicantly higher number of viable PC-12 cells can be clearly seen on PANI lm compared to silicon substrate. Reproduced from Ref.
[49] with permission from Elsevier. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of this article.)
T.H. Qazi et al. / Biomaterials 35 (2014) 9068e9086 9074
cultured on conductive PANI-PLCL relative to non-conducting pure
PLCL. Fig. 6 shows the morphology of broblasts after being stim-
ulated with different electrical currents for two days.
By combining PANI, carbon nanotubes (CNTs) and poly(N-
isopropylacrylamide) (PNIPAm) using coupling chemistry, Tiwari
et al. developed smart three-dimensional electrospun matrices that
allowed temperature dependent cell detachment [96]. L929 bro-
blast cells were cultured on the PANI-CNT-PNIPAm microbrous
scaffold for up to 7 days, and the cell viability and proliferation was
compared to bulk PANI-CNT-PNIPAm, and Matrigel. The
conductive microbrous scaffolds provided a highly compatible
surface for cell adhesion, as the cell viability of seeded broblasts
was found to be more than two folds higher when compared to the
bulk and control samples at each time point tested. Moreover, while
the percentage of live cells was more than 90% on the Matrigel
and bulk composite, a signicantly higher number of live cells were
found on the conductive PANI-CNT-PNIPAm scaffold. The compos-
ite scaffold exhibited cell detachment behavior as the temperature
was lowered from 37

C to 20

C (lower critical solution tempera-
ture of PNIPAm) caused by the hydration of the PNIPAm chains.
Such smart composites have great potential in tissue engineering
applications because they are biocompatible, electrically conduc-
tive, have the possibility of carrying bioactive factors (inside the
carbon nanotubes), and could be used to culture detachable cell
sheets.
By incorporating pH dependent methacrylic acid (MAA) to the
PNIPAm, the same group also developed smart nanobrous
matrices of PANI-CNT-PNIPam-co-MAA which exhibited dual
functionalities, responding to both temperature and pH changes
[108]. Cell culture on the samples using L929 broblasts showed
consistently higher cell growth on nanobers of PANI-CNT-
PNIPAm-co-MAA compared to PNIPAm-co-MAA, and control.
Furthermore, Live/Dead staining of the cells on day 7 of culture
revealed the highest percentage of live cells to be present on PANI-
CNT-PNIPAm-co-MAA nanobers. The authors attributed these
observations to the added conductivity and mechanical strength
provided by the PANI and CNTs, respectively.
Core/shell coaxial bers comprising a silk broin inner core and
an outer layer of PANI were fabricated via in situ oxidation [51].
L929 murine broblasts were cultured on the PANI-silk broin
Table 1
Overview of the different biodegradable polymers combined with polyaniline for tissue engineering applications.
Polymer system Max. conductivity
recorded [S/cm]
Cell line tested Observations and results of cell culture Ref.
PANI-PLCL 0.296 C2C12 myoblasts -Increase in myotube number, length, and area were observed with
increasing PANI content.
-Overexpression of myogenin, troponin T, and myosin heavy chain
(MHC) on PANI containing composites relative to pure PLCL.
[50]
PANI-PLCL 0.0138 Human dermal broblasts,
NIH-3T3 broblasts,
C2C12 myoblasts
-NIH-3T3 broblasts, when subjected to low electrical stimulation
(20 mA), responded by increasing metabolic activities.
[94]
PANI-PLCL 0.00641 PC-12 cells -Compared to pure PLCL, cells cultured on PANI-PLCL meshes showed
higher viability, lower apoptotic activity, and triggered the expression
of neuronal differentiation markers GAP-43 and b-tubulin.
[95]
PANI-Silk broin 0.48 L929 broblasts -Cultured broblasts showed highest survival rate on PANI containing
bers compared to pure silk broin and TCP controls.
[51]
PANI-PNIPAm-CNT e L929 broblasts -Improved viability of cells on microporous
PANI-poly(N-isopropylacrylamide)eCNT composites, relative to
Matrigel and bulk composite controls.
[96]
PANI-Graphene
PANI-Graphene oxide
e L929 broblasts -Presence of PANI enhanced cell viability and proliferation. [97]
PANI-BC 0.018 e e [98]
PANI-Gelatin 0.021 H9c2 cardiac myoblasts -Proliferation of cardiac myoblasts to numbers greater than those on
TCP controls.
[99]
PANI-PLGA 0.0031 Neonatal cardiomyocytes -Enhanced adsorption of the adhesion proteins bronectin and laminin.
-Cardiomyocytes expressed the gap junction protein Connexin 43
leading to synchronous beating of cell clusters.
[100]
PANI-Collagen 0.27 Porcine skeletal muscle cells -No difference in morphology and cell number was observed between
PANI-collagen and pure collagen groups.
[101]
PANI-PGS 0.018 C2C12 myoblasts -Statistically signicant increase in cell numbers (proliferation) on
PANI-PGS composites containing 20 and 30 vol. % PANI, relative to pure
PGS after 3 days in culture.
[102]
PANI-PDLA 0.0437 Primary rat muscle cells -Cellular attachment and proliferation showed no signicant difference
between any of the groups tested.
[103]
PANI-PCL-BioSilicon e Mouse MSCs,
human kidney broblasts
-Accelerated calcication of the composites in SBF was observed when
an electrical bias was applied.
-Composites showed compatibility to kidney broblasts.
[104]
PANI-PCL 0.00008 hMSCs, cardiomyocytes -Viability of cardiomyocytes was higher on PANI containing composite
patches relative to pure PCL.
[105]
PANI-(PCL/Gelatin) 0.02 10
6
S
a
Neural stem cells -Cells cultured on PANI containing composite bers showed higher
viability and proliferation compared to TCP control.
-Cells stimulated at 1.5 V for 60 min showed signicant
improvement in cell proliferation and neurite length and outgrowth.
[106]
Gelatin-graft-PANI 0.000454 Bone marrow stromal cells,
C2C12 myoblasts
-Presence of PANI allowed for increased intercellular communication
leading to higher cell viabilities and proliferation rates.
-Cell proliferation on PANI containing hydrogels was even better
than gelatin.
[71]
PANI, polyaniline; PLCL, poly(l-lactide-co- 3 -caprolactone); BC, bacterial cellulose; PLGA, poly(lactic-co-glycolic) acid; PGS, poly(glycerol-sebacate); PDLA, poly(D-lactic acid);
PCL, polycaprolactone; PNIPAm, poly(N-isopropylacrylamide).
a
Conductance reported in Siemens.
T.H. Qazi et al. / Biomaterials 35 (2014) 9068e9086 9075
bers, and despite a slow initial rate of proliferation, showed the
highest viable cell number compared to pure silk broin and TCP
controls after 7 days. These core/shell bers also allow for the
possibility of dissolving the inner silk broin core to obtain hollow
PANI nanotubes which could open up further avenues of research.
Yan et al. fabricated electroactive and biocompatible hybrid
lms of PANI and graphene, and PANI and graphene oxide, for
potential biomedical applications [97]. Through rapid mixture
polymerization, PANI was deposited on the surfaces of graphene
and graphene oxide papers. SEM observation revealed that
compact nanoparticle clusters of PANI were formed on the surface
of graphene oxide, whereas a continuous lm of nanorods and
nanoparticles were found on the surface of graphene due to
adsorption of short PANI nanobers. Viability of L929 broblasts
was found to be higher on PANI-graphene hybrids compared to
pure graphene, and higher on PANI-graphene oxide hybrids
compared to graphene oxide, indicating that the presence of PANI
can enhance cell survival and proliferation of both graphene, and
graphene oxide substrates. Though not ideally suited for tissue
regeneration due to their non-biodegradable characteristic, these
hybrid materials can nd various applications as biosensors, bio-
electrodes, and for the in vitro analysis of the behavior of electrically
excitable cells.
Using an in situ nano-assembly approach, PANI was synthesized
on the surface of bacterial cellulose nanobers to form electrically
conductive hydrogels [98]. The excellent biocompatibility and
biodegradability of bacterial cellulose, combined with the electro-
activity of PANI can result in hydrogels with desirable properties for
biomedical applications. The hydrogel fabricated by Shi et al. con-
sisted of a three-dimensional network of microbrils of bacterial
cellulose coated with PANI, with diameters in the range of
80e120 nm (Fig. 7). Electrical conductivity of the nal composite
hydrogel was found to depend on reaction time, and the type and
concentration of the dopant used. In a separate study, three-
dimensional scaffolds of PANI and poly(3-hydroxybutyric acid)
(PHB) were obtained by electrospinning [109]. The polyester PHB
exhibits excellent biodegradability and biocompatibility, and when
combined with PANI to form conductive nanobrous scaffolds, can
serve as effective scaffolds for tissue engineering applications.
Despite using biodegradable and biocompatible polymers, both of
Fig. 6. Culture of NIH-3T3 broblasts on nanober scaffolds of polyaniline and poly(L-lactide-co--caprolactone) for 2 days under electrical stimulation of 20 mA (a), and 200 mA
(b). Fluorescence micrograph on the left shows F-actin lament formation in the cells, which is absent in cells stimulated at a higher current value. Adapted from Ref. [94] with
permission from John Wiley and Sons.
Fig. 7. Schematic diagram illustrating the process of aniline polymerization on the bacterial cellulose (BC) hydrogel. (a) SEM image of a BC membrane, and (b) SEM image of a BC-
PANI membrane. The microbrils in (b) consist of an inner BC core, and an outer coating of PANI. Adapted from Ref. [98] with permission from the Royal Society of Chemistry.
T.H. Qazi et al. / Biomaterials 35 (2014) 9068e9086 9076
the above studies failed to report any in vitro cell culture results that
could illustrate the inuence of the conductive scaffolds on the
survival, proliferation, and behavior of cells.
3.2.1. Cardiac tissue engineering
The majority of studies employing PANI for tissue engineering
applications have evaluated the behavior, function, and compati-
bility of cells derived fromnaturally conductive systems of the body
such as the heart and the nervous system [29]. The native
mammalian heart is electrically conductive (in the order of 10
4
S/
cm) and is composed of an underlying assembly of bers, nodes and
cell clusters which make up its electrical system[110,111]. Electrical
impulses emanating from the sinoatrial node (situated in the right
atrium) are transmitted throughout the rest of the myocardiumvia
atrioventricular node (bridging the atria and the ventricles) and a
network of Purkinje bers [112]. The propagation of electrical sig-
nals through the cardiac cells in a synchronized fashion results in a
heartbeat via the well-knownphenomena of excitation-contraction
coupling [113]. The porous scaffolds currently employed for cardiac
tissue engineering applications are electrically resistant and
thereby hinder communication between cells on the scaffold, as
well as prevent effective electrophysiological coupling between
seeded cells and native tissue. To overcome this issue, many recent
studies have resorted to the addition of conductive elements in
their scaffolds. For example, You et al. impregnated thiol-2-
hydroxyethyl methacrylate/2-hydroxyethyl methacrylate (thiol-
HEMA/HEMA) scaffolds with gold nanoparticles to render it
conductive and detected the upregulation of the gap junction
protein Connexin 43 which is vital for cellecell communication and
contractile behavior [114]. More recently, carbon nanotubes were
incorporated into Gelatin methacrylate hydrogels which not only
resulted in improved cardiomyocyte attachment, organization, and
coupling, but also exhibited spontaneous synchronous contractions
[115].
PANI has been combined with other biocompatible polymer
systems to form composites or blends, and evaluated for applica-
tion in cardiac tissue regeneration (Fig. 8). H9c2 rat cardiac myo-
blasts cultured on composite nanobers of PANI and gelatin
attached readily, and proliferated to numbers greater than those on
TCP controls by day 6 [99]. The high surface area for cell attachment
offered by the brous scaffold compared to smooth surfaces or
lms allowed proliferation to high cell numbers. The authors re-
ported an increase in the elastic modulus (from 499 to 1384 MPa)
and tensile strength (from 5.77 to 10.49 MPa) of the composites
with increase in PANI content, which will likely be useful in
providing robust structural support to the infarcted heart during
left ventricular remodeling.
In another study, hyperbranched Poly-L-Lysine dendrimers
were combined with PANI nanotubes and either cast into lms, or
electrospun into bers with diameters less than 100 nm [116]. The
cytotoxicity of the composite was evaluated by exposure to Chinese
hamster ovary cells which maintained almost perfect cell viability
even at high PANI concentrations. Next, neonatal rat car-
diomyocytes were cultured on the cast and electrospun samples
and subjected to electrical stimulation (Voltage: 10e40 V, Fre-
quency 5 Hz, Pulse duration: 5 ms). 72 h after electrical stimulation
at 40 V, the highest observed viability of cardiomyocytes cultured
on cast lms was only 30%, whereas at the same stimulation con-
ditions cells grown on the brous scaffolds were 75% viable [116].
Although this study lacked controls which could help draw com-
parisons between stimulated and non-stimulated cells, the un-
usually high difference between the viabilities of cells cultured on
cast and brous samples under similar stimulation conditions un-
derscores the benets of using scaffolds with nanobrous archi-
tecture for tissue engineering applications.
Borriello et al. reported the electrospinning of synthesized PANI
(s-PANI) short bers or nano-needles with PCL to form patches for
cardiac muscle regeneration [52]. The authors claimed that PANI
short bers provide a more efcient conductive network for charge
transfer within the composite. Human mesenchymal stromal cells
(hMSCs), whendifferentiatedinto a cardiogenic lineage andcultured
on the sPANI-PCL composite patch, initially showed a relatively low
survival rate of ~40% which increased to almost 100% at day 5.
Moreover, at all the time points evaluated, the survival of car-
diomyocytes was greater on sPANI-PCL composite patches than on
PCL, the difference being statistically signicant at days 1 and 3 [52].
In a recently published report, Hsiao et al. produced aligned
nanobrous meshes of PANI and PLGA for improved coupling and
Fig. 8. Macroscopic images of polyaniline containing composites for tissue engineering applications. Patches of PGS (A), and PANI-PGS (D) obtained via solvent casting. Adapted
from Ref. [102] with permission from Elsevier. Fibrous patches of PCL (B), and PANI-PCL (E) obtained via electrospinning. Adapted from Ref. [52] with permission from Springer.
Hydrogels of BC (C), and PANI-BC (F) obtained via in-situ nanoassembly. Adapted from Ref. [98] with permission from the Royal Society of Chemistry.
T.H. Qazi et al. / Biomaterials 35 (2014) 9068e9086 9077
synchronization between seeded cardiomyocytes during culture
[100]. Due to electrical attraction between the negatively charged
proteins and positively charged conductive mesh, enhanced
adsorption of the adhesion proteins bronectin and laminin was
observed which promoted subsequent cell adhesion. Neonatal rat
cardiomyocytes aligned themselves along the long axis of the bers
and expressed the gap junction protein Connexin 43, as a result of
which separate clusters of coupled cardiomyocytes were observed
beating synchronously (Fig. 9). Furthermore, the group applied
external electrical stimulation, designed to mimic native heart
rates, to control the rate of synchronous contractions of car-
diomyocyte clusters on PANI-PLGA meshes. As an implication of
this study, conductive nanobrous scaffolds could be used to
stimulate seeded cardiomyocytes into beating synchronously
before being applied as a cardiac patch in vivo, which would in-
crease the chances of achieving electrophysiological coupling with
the native heart.
All of the above studies demonstrate the effectiveness of uti-
lizing composite meshes of randomly oriented or aligned bers,
which are able to mimic certain aspects of the native extracellular
matrix such as topography and architecture, and results in an open
pore scaffold allowing for cell attachment and migration while also
retaining the potential to accommodate blood vessels during
angiogenesis [107,117,118]. However, as these studies emphasize,
ber orientation alone does not have as signicant an effect on
cellular morphology and desirable protein expression/upregulation
as that achieved by the synergic inuence of ber orientation along
with electroactivity that is facilitated by PANI.
3.2.2. Skeletal muscle tissue engineering
Native skeletal muscle responds to electrical stimuli received via
neuromuscular junctions (NMJs) by contracting and generating
forces [113], but because skeletal muscle, unlike the heart, is
voluntarily controlled, spontaneous contractions do not occur.
Electrical stimulation has been shown to affect myoblast prolifer-
ation, increase rate of protein synthesis, and improve contraction
forces [119,120], suggesting that electrical signals can be a potent
trigger to enhance skeletal muscle regeneration.
Kim et al. produced a novel conductive hybrid of PANI nano-
bers dispersed in a collagen matrix and validated its biocompat-
ibility by culturing porcine skeletal muscle cells [101]. The PANI
nanobers formed a three-dimensional interconnected network in
the collagen matrix which permitted charge transfer through the
composite, resulting in a conductivity value of 0.01 S/cm at the
percolation threshold. Interestingly, even with use of PANI nano-
bers with high aspect ratios, the percolation threshold was
reached at a rather high PANI concentration of 50 wt. % when
compared to relatively lower percolation thresholds of 1 wt. % [105]
and 15 wt. % [50] reported in other studies. The authors cite the
curing of collagen, which can limit ber connectivity in the matrix,
as the reason for this observation. Porcine skeletal muscle cells
were grown on PANI-collagen composites for two days, and
showed morphology and cell number similar to cells grown on pure
collagen samples [101].
McKeon and colleagues produced electrospun composites of
PANI and poly (D,L-lactide) for use as tissue engineering constructs
for the revival of muscle contractility following trauma to NMJs
[103]. Primary rat muscle cells harvested from the soleus muscle
were cultured on the electrospun composites and were found to
attach and proliferate on all scaffolds containing varying amounts
of PANI for up to 14 days. However, due to degradation (up to 19%
weight loss by day 14 for 75% PDLA/25% PANI sample) the
conductive scaffolds underwent undesirable levels of shrinkage
which might limit the application of this composite.
The inuence of electrically conducting PANI-PLCL substrate on
the myogenic differentiation of myoblasts without any supple-
mentary electrical stimulation was investigated by Jun and col-
leagues [50]. PANI and PLCL were blended together and formed into
nanobers by electrospinning. C2C12 myoblasts cultured on the
composite bers readily attached and proliferated on composites
with different PANI contents. Quantication of myotube charac-
teristics after 8 days of culture revealed a clear trend whereby
increasing the amount of PANI resulted in signicantly higher
Fig. 9. (A) Neonatal rat cardiomyocytes cultured on control tissue culture plates (top), undoped non-conductive (middle) and doped conductive (bottom) bers of PANI-PLGA. Both
ber orientation and electrical signals were vital in providing contact guidance to the cardiomyocytes. (B) Immunouorescence staining of cardiomyocytes on doped conductive
bers, stained for cardiac troponin (cTnl), connexin 43 (Cx 43), and nucleus (red). The identication of these markers indicates cellecell coupling which leads to effective
communication and subsequent synchronized beating of cardiomyocyte clusters. Adapted from Ref. [100] with permission from Elsevier. (For interpretation of the references to
color in this gure legend, the reader is referred to the web version of this article.)
T.H. Qazi et al. / Biomaterials 35 (2014) 9068e9086 9078
myotube number, length, and area. Moreover, real time polymerase
chain reaction (RT-PCR) revealed that the presence of PANI stimu-
lated the upregulation of pro-myogenic genes including myogenin,
troponin-T, and myosin heavy chain. Ku et al. came to a similar
conclusion when they discovered that myoblasts cultured on
aligned electrospun bers of PANI and PCL were able to differen-
tiate into myotubes [121]. Though the aligned ber orientation did
not affect cell attachment and proliferation, it did promote
morphological alignment of cultured cells along the major axis of
the bers. Aligned ber meshes also exhibited signicantly higher
elastic moduli relative to those composed of randomly aligned -
bers. Cell attachment, survival, and proliferation were similar on all
samples containing varying amounts of PANI and no signicant
differences were observed between random and aligned bers.
When stained for MHC, a protein essential for formation of myo-
tubes, the MHC positive area was found to be signicantly higher on
aligned bers relative to random ones. Moreover, signicant
enhancement of myotube number, length, diameter, and fusion
index was noticed for aligned bers. Increase in the PANI content
also stimulated an increase in the expression of myogenin, troponin
T, and MHC.
A similar study was carried out by Chen et al. where the synergic
effects of topography and electroactivity on the differentiation of
myoblasts was investigated [105]. Highly aligned nanobrous mats
of PANI and PCL were obtained by using a modied electrospinning
setup including a magnetic-eld-assisted collector. Low PANI con-
tents (1e3 wt. %) resulted in sufcient levels of conductivity, while
increasing the PANI content improved the mechanical properties of
the composite (tensile strength from 7 to 10 MPa, Young's modulus
from 8 to 55 MPa) and resulted in high strength scaffolds with
appropriate elasticity suitable for use in tissue engineering appli-
cations. At day 3 of culture, C2C12 myoblasts cultured on the
nanobrous mats had aligned themselves parallel to the ber di-
rection, and showed higher viability on PANI containing samples
relative to pure PCL. Myotube characterization at day 5 revealed
signicantly greater myotube number, length, fusion index, and an
overall higher maturation index for (a) samples containing PANI
relative to pure PCL, and (b) samples with aligned bers relative to
samples with randomly oriented bers. Optimal myotube charac-
teristics were achieved for aligned and electrically conductive PANI
containing ber mats (Fig. 10).
The ability to produce mature aligned myotubes is highly
desirable for the development of in vitro tissue engineered muscle
constructs. The studies discussed in this section identify two major
stimuli, namely ber orientation and electroactive PANI content,
which could enable the creation of reproducible functional muscle
substitutes. However, an essential property that all muscle con-
structs should exhibit is contractility. Therefore future studies
employing electroactive aligned bers for skeletal muscle regen-
eration should assess the functional properties of differentiated
myotubes and report the inuence of conducting polymers on
inducing greater contraction forces.
3.2.3. Nerve tissue engineering and neural prosthetics
The nervous system relies on neurons, which are electrically
excitable cells, to transmit signals at a rapid pace. Numerous stra-
tegies for the repair and regeneration of defects to the brain, spinal
cord, and the peripheral nervous system have been proposed,
which make use of several non-conductive scaffolds [122]. Elec-
trical stimulation has long been known to be an effective cue for
neuronal function and several theories have been put forward
explaining the benecial effects of electrical stimulation on neurite
growth and nerve regeneration [123e125]. With the emergence of
conducting polymers in biomedical applications, researchers have
started using conducting polymers such as polypyrrole and PANI as
a scaffold component to accommodate and promote the growth
and regeneration of nerve tissue without the need for nerve growth
factor, while concurrently being able to deliver electrical signals to
the cells in an efcient manner [26,126,127].
One of the earlier studies performed by Oren et al. involved
culturing Aplysia neurons on a two-dimensional PANI layer syn-
thesized on sulfonated polystyrene templates [106]. The neurons
cultured on 2D PANI showed an unusual morphology which con-
sisted of large, attened lamellipodia with a tendency to collapse
spontaneously leading to neuronal disintegration. For the purpose
of comparison, neurons were also cultured on cover slides coated
with poly(L-lysine) and were found to maintain typical neuronal
morphology and exhibited good viability. Micro contact printing
(mCP) was used to form alternate strips of 2D PANI and poly(L-
lysine) on the same glass slide. Cultured neurons formed neurite
outgrowths which preferred to grow along the poly(L-lysine) strips
while mostly avoiding PANI. Whether this unusual cellular behavior
was specic to neurons derived froma single organismor specie, or
was triggered by an unfavorable oxidation state of PANI, is unclear.
Cullen et al. used PANI to form composite bers for a tissue
engineered neural electrical relay [64]. PANI was blended with
Fig. 10. C2C12 myoblasts seeded on electrospun random polycaprolactone bers (R-PCL), aligned polycaprolactone bers (A-PCL), random composite polycaprolactone/polyaniline
bers (R-PCL/PANi), and aligned composite polycaprolactone/polyaniline bers (A-PCL/PANi). Myogenic differentiation can be observed with stainings for MHC, and MHC nuclei.
Signicantly higher numbers of myotubes were detected on bers containing PANI. Adapted from Ref. [105] with permission from Elsevier.
T.H. Qazi et al. / Biomaterials 35 (2014) 9068e9086 9079
polypropylene and either extruded into bers or formed into
sheets. The biocompatibility of the PANI-PP composites, when
evaluated with neurons derived from dorsal root ganglia (DRG) of
rats, showed a distance-dependent relationship. Complete cell
death was observed for neurons cultured adjacent to the PANI-PP
sheets and bers after just two days of culture. The cell viability
improved from<1% adjacent to the PANI-PP sheets to over 60% just
3 mmaway fromit, indicating the leaching of a toxic agent fromthe
composites. This was further substantiated with a detoxifying
treatment by soaking PANI-PP sheets in media for a fewdays before
cell culture. Interestingly, the viability of the neurons adjacent to
the pre-soaked composite sheets improved to over 89%. The group
was able to optimize neural adhesion and network distribution on
detoxied PANI-PP bers and provide a protective environment by
low concentration agarose hydrogel encapsulation to render this
conductive composite suitable for use as neural electrical relays
[64].
The inuence of direct electrical stimulation on nerve stem cells
cultured on electrospun composite bers of PANI and a PCL/gelatin
blend was investigated by Ghasemi-Mobarakeh et al. [95]. The
combination of these materials resulted in favorable (i) mechanical
properties, with tensile strength similar to that of a rat sciatic nerve
(PANI-PCL/Gelatin bers: 8 MPa, sciatic nerve: 2.7 MPa), (ii) elec-
trical properties, with sufcient conductivity to perform electrical
stimulation, and (iii) physical properties, with tunable degradation
rate due to the presence of two biodegradable polymers. Nerve
stemcells cultured on the electrospun composite scaffolds attached
and survived for up to 7 days and showed higher viability and
proliferation than cells cultured on TCP controls, indicating the
absence of any cytotoxic effects. A direct current (DC) source was
used to stimulate the cells at 1.5 V for 15, 30, and 60 min, while non-
stimulated samples served as controls. Electrical stimulation for a
duration of 60 min was found to signicantly improve cell prolif-
eration as well as neurite length and outgrowth relative to non-
stimulated controls.
The same group also carried out a similar study investigating the
effects of electrical stimulation on nerve stem cells cultured on
electrospun bers of PANI and poly-L-lactide (PLLA) [53]. The nerve
stemcells showed signicantly enhanced viability and proliferation
when cultured on PANI-PLLA scaffolds relative to pure PLLA scaf-
folds as well as TCP controls. Furthermore, electrical stimulation
carried out at an electric eld of 100 mV/mm for 60 min resulted in
considerable neurite outgrowth compared to non-stimulated con-
trols with neurite lengths greater than the length of cells cultured
on pure PLLA nanobers (Fig. 11). The ndings of these studies
reveal that essential indicators for nerve regeneration such as
neurite outgrowth and length can be substantially enhanced by the
use of electrical cues even in the absence of additional bioactive
growth factors.
Bhang et al. produced conductive electrospun composite bers
of PANI and PLCL for use as nerve grafts, and demonstrated that the
addition of PANI along with nanoscale topography of the bers not
only increased cell viability but also prompted the expression of
neuronal differentiation proteins [128]. When cultured on PANI-
PLCL brous meshes, PC-12 cells showed signicantly enhanced
cell adhesion and viability on days 1 and 3, besides having the
lowest apoptotic activity among all the groups tested. PANI con-
taining samples were also found to promote enhanced neurite
outgrowth, and to modulate neuronal differentiation as a higher
expression of the neuronal differentiation markers GAP-43 and b-
tubulin were observed on PANI-PLCL bers relative to pure PLCL.
Neural prosthetics such as neural probes and implantable
electrodes require an interface which promotes intimate contact
between the tissue and the electrode to effectively transmit signals,
and is an area of research where conducting polymers are
increasingly being used [25]. Neural probes generally consist of an
array of electrodes which are implanted into the brain in order to
stimulate and record signals of the surrounding neurons, thereby
allowing information contained in neuronal signals to eventually
control external devices such as prosthetic/robotic arms [129,130].
Highly conductive PANI has found application as nanostructured
lm covering the surface of the electrodes making up the neural
probe [131]. Wang et al. successfully polymerized nanostructured
PANI lms on the surface of platinum electrodes, and characterized
it in terms of protein adsorption and long term stability [132]. The
Pt-PANI electrode was subjected to electrical stimulation in 0.9%
sodium chloride solution for 1 month, after which microscopic
examination showed no cracks or ssure formation, indicating that
the PANI lm acts as a protective layer for the underlying Pt elec-
trode. Evaluation of naked Pt electrode surface following electrical
stimulation for 1 month revealed black holes caused by corrosion.
No such surface irregularities were found on the PANI-Pt surface
leading to the conclusion that the nanostructured PANI lm resists
erosion over a long period of time. Adsorption of bronectin and
bovine serum albumin was higher on the PANI-Pt electrode surface
relative to naked Pt electrode, whereas electrical stimulation was
found to boost protein adsorption by nearly 2 times. The higher
protein adsorption on PANi-Pt surface was most likely a result of
the higher surface area and roughness caused by the nano-
structures in the PANI lm, creating a more favorable environment
for protein adsorption. Furthermore, the PANI nanostructures also
inhibited aggregation of the adsorbed proteins whereas
Fig. 11. Nerve stem cells cultured on composite electrospun bers of polyaniline and poly(L-lactide) without (A) and with (B) electrical stimulation. Electrically stimulated cells were
able to extend neurites of longer lengths. Reproduced from Ref. [53] with permission from Elsevier.
T.H. Qazi et al. / Biomaterials 35 (2014) 9068e9086 9080
aggregation was observed on naked Pt electrode surface. Di and
colleagues carried out a similar study on PANI coated platinum
electrodes, and reported long term stability of the PANI coating
after being subjected to electrical stimulation for 6 months [133].
The PANI coating exhibited excellent anti-corrosive properties, and
showed inactivity towards lipid peroxidation.
3.2.4. Bone tissue engineering
Bone has been known to respond favorably to electrical signals
[134], and previous studies carried out on electrical stimulation of
osteoblasts cultured on conductive substrates have revealed
excellent outcomes such as signicant enhancement in cell prolif-
eration, concentration of extracellular calcium, and collagen I
expression [135]. Shao et al. identied a range of electrical currents
which produce optimum results in terms of osteoblast attachment,
alignment, and proliferation [136]. However, these studies utilized
carbon nanotube (CNTs) based composites as electrically conduc-
tive substrates, which might raise concerns due to potential
nanocytotoxicity [137]. Despite only a handful of studies employing
PANI based composites as substrates for bone tissue engineering,
there is great potential for developing biocompatible, biodegrad-
able, and electroactive scaffolds for improved bone regeneration
and repair.
Whitehead et al. fabricated conductive composites of PANI, PCL,
and bioactive mesoporous silicon (BioSilicon) and observed
accelerated calcication of the composites in simulated body uid
(SBF) when electrically stimulated [104]. Calcication assays
revealed that the rst signs of formation of calcium phosphate (Ca/
P ~ 1.1) in SBF without electrical stimulation occurred after 1
month, but required only 7 h to achieve improved calcication (P/
Ca ~ 4.4) when an electrical eld was applied. The scaffolds were
found to be non-cytotoxic to human kidney broblasts after being
sterilized for 72 h. Mouse stromal cells seeded onto electrospun
scaffolds of the same composite proliferated to form a thick
network of cells resembling the bone extracellular matrix. Overall,
these results strongly indicate that the rate of calcication can be
greatly enhanced with the application of electrical bias.
In a recent article, porous chitosan-gelatin/nanohydroxyapatite-
polyaniline (CS-Gel/nHA-PANI) composite scaffolds fabricated by
Azhar and colleagues showed cytocompatibility with dental pulp
stemcells and exhibited higher mineralization rates in SBF than CS-
Gel and CS-Gel/nHA samples due to lowering of the surface energy
threshold required for nucleation of minerals [138].
A polyaniline oligomer, tetraaniline (TA) was grafted onto
poly(ester amide) to form electroactive and biodegradable co-
polymers (PEA-g-TA) [79]. Upon immersion in TriseHCl buffer
containing proteinase K, the copolymer degraded and lost up to 43%
of its mass after 144 h. The cytotoxicity was assessed by seeding
osteoblastic MC3T3-E1 cells on substrates with different TA con-
centrations. The viability of cells decreased slightly with increase in
concentration for all samples. At the highest concentration, the
viability of cells seeded on pure TA was 80%, which was the lowest
observed among all substrates including PEA-g-TA copolymers,
showing that the grafting to PEA signicantly improved biocom-
patibility. Moreover, the cells readily adhered to the surface of the
substrates and adopted an elongated, spindle-like morphology
which was not observed in pure PEA and TCP controls. On being
electrically stimulated using a pulsed signal, an increase in the
intercellular free calcium concentration and ALP enzyme activity
was observed, suggesting that the electroactive PEA-g-TA co-
polymers promoted osteogenic differentiation.
Liu et al. developed electroactive nanoparticles of hydroxyapa-
tite (HA) grafted with aniline tetramer, which was later dispersed in
a poly(lactic acid) (PLA) matrix [139]. The surface grafting of aniline
tetramer not only rendered the HA electroactive, but also modied
the surface properties such that the nanoparticles dispersed
homogenously throughout the PLA substrate, whereas the HA
nanoparticles without surface grafting aggregated to form large
clusters on the PLA substrate. The biocompatibility of the hybrid
composites was evaluated by seeding bone marrow cells, which
adhered and adopted the desirable spindle-like phenotype. After 3
days of culture, the marrow cells had proliferated to viable cell
numbers higher than those on TCP control, making these hybrid
composites suitable for application in bone tissue engineering.
3.3. Polyaniline copolymers
Because PANI is non-biodegradable, even transplanting small
amounts can cause potential inammation in the long term. Aniline
oligomers, on the other hand, possess similar electrical conduc-
tivities with the added advantage of being biodegradable, made
possible by functionalizing the aniline backbone with hydrolyzable
end groups. The degradation byproducts of oligomers can be taken
up by macrophages and can subsequently undergo renal clearance
to exit the body and avoid any adverse long term effects.
Abdul Rahman and colleagues prepared functional electrospun
nanober mats from a solution of biocompatible PLA combined
with either PANI or poly(aniline-co-m-aminobenzoic acid) (P(ANI-
co-m-ABA)) [140]. Owing to the presence of COOH groups and the
short polymer chain lengths, the solubility of (P(ANI-co-m-ABA)) in
common solvents like DMF is enhanced, which results in conve-
nient processing of the composite. An additional advantage of acid
functionalized PANI is that these copolymers are self-doping,
where the acid group acts as the dopant. The fact that enhanced
solubility and self-doping ability can be achieved without
compromising on electrical conductivity of the composite bers
makes functionalized PANI copolymers an attractive option as a
substitute material for PANI. The suitability of these functional
electrospun bers as scaffolds for the culture of human adipose
derived stem cells (hASCs) was evaluated in another study by the
same group [141]. On seeding, hASCs attached readily onto the
electrospun ber surfaces, and proliferated for 7 days, with prolif-
eration rates on PANI and (P(ANI-co-m-ABA)) containing compos-
ites similar to that on pure PLLA bers. Microscopic analysis
revealed that the cells adopted a broblastic morphology and
formed abundant focal adhesion points on all samples tested. This
was attributed to the high substrate stiffness of the nanobers,
measured by nanoindentation to be in the GPa range, which has
been reported to have a major inuence on development and
maturation of focal adhesion points.
Gizdavic-Nikolaidis et al. investigated conductive electrospun
nanobers of PLA blended with poly(aniline-co-3-aminobenzoic
acid), (3ABAPANI), as wound dressings, allowing for cell growth
and proliferation and also exhibiting antibacterial activity [88]. The
biocompatibility was established by seeding COS-1 broblasts on
ber mats containing varying amounts of 3ABAPANI and moni-
toring the proliferation over a period of four days. The broblasts
proliferated with a similar rate on all samples, and when compared
to cells cultured on TCP and glass substrates, a signicantly higher
number of cells was found to be viable on the 45:55 3ABAPANI:PLA
ber mat owing to its high surface area and rough three-
dimensional morphology. Moreover, the cell viability was found
to increase with increasing 3ABAPANI content in the composite
bers.
Zhang and colleagues successfully synthesized the copolymer
poly[(glycine ethyl ester) (aniline pentamer) phosphazene] (PGAP)
as an electroactive biomaterial for nerve regeneration [75]. The
novel polyphosphazene had aniline pentamer and glycine ethyl
ester as side groups, which rendered the copolymer both electro-
active (a conductivity of 2 10
5
S/cm was achieved) and
T.H. Qazi et al. / Biomaterials 35 (2014) 9068e9086 9081
biodegradable (50% mass loss after 70 days in PBS in vitro). RSC96
Schwann cells were cultured on thin lms of the cast PGAP
copolymer to determine cytocompatibility. Cells on the PGAP had
an elongated and spread out morphology, showing good adhesion
compared to cells cultured on pure PDLLA after 3 days of culture.
In another study, the frequently used biomaterial chitosan (CS)
was cross-linked with conductive aniline pentamer (AP) to produce
a water-soluble electroactive polymer which was found to induce
differentiation of PC-12 cells [142]. The biocompatibility was
assessed using C6 glioma cells, and the CS-AP copolymer was found
to be highly cytocompatible, supporting the survival of more viable
cells compared to chitosan and TCP controls. Accelerated differen-
tiation of nerve cells was achieved on all conductive polymers
without any electrical stimulation compared to pure CS, and the
length of neurite extensions was found to increase with higher
concentrations of AP up to 4.9%, after which the length of neurite
extensions was found to be shorter. This effect was observed due to
possible changes in the surface properties of the lm when higher
contents of AP are incorporated. The copolymer containing 4.9% AP
was found to have the optimum biocompatibility, and induced the
formation of an intricate neurite network. Due to its amphiphilic
nature (hydrophobic AP and hydrophilic CS), the copolymer was
found to spontaneously self-assemble into spherical micelles,
opening new possibilities for the application of this copolymer as a
carrier for drug delivery. The same group also synthesized (PLA-b-
AP-b-PLA) (PAP), an ABA block copolymer of polylactide and ana-
line pentamer exhibiting conductivity, biocompatibility, and
biodegradability [76]. The conductivity was measured to be
5 10
6
S/cm which is sufcient for the conduction of micro-
currents to stimulate nerve cell proliferation and differentiation.
The biocompatibility was assessed by seeding C6 glioma cells on
the copolymer thin lms. The area fraction covered by C6 cells on
PAP lms at 4 and 48 h was almost equal to that on TCPS, indicating
that the PAP copolymer is non-toxic and supported cell adhesion
and survival.
Similarly, Huang et al. synthesized an AB block copolymer of
polylactide and analine pentamer, PLAAP, which acted as a favor-
able substrate for adhesion and proliferation of cells and aided in
the differentiation of neuronal cells [77]. The synthesis route
employed for the development of PLAAP copolymer is illustrated in
Fig. 12. Electrical conductivity was reported to be in the range of
10
5
e10
6
S/cm, which is higher than that achieved in the PAP
block copolymer. The copolymer was found to be biocompatible
when C6 cells adhered and proliferated, ending with a higher
percentage area fraction compared to pure PLA. To investigate the
inuence of a conductive substrate on neuronal differentiation, PC-
12 cells were cultured on the PLAAP copolymer for 5 days. The cells
adopted a neuronal phenotype, but only very few neurite exten-
sions were observed. However, upon electrical stimulation, almost
all cells exhibited neurite extensions, the lengths of which were
higher on the PLAAP copolymer compared to TCP control.
Liu et al. prepared a diblock copolymer consisting of poly(-
ethylene glycol) methyl ether (mPEG), and tetraaniline (TEA), with
improved solubility in water and organic solvents [143]. The
copolymer was electrically conductive, supported the adhesion and
survival of seeded cells, and improved the neuronal differentiation
of C6 glioma cells. The authors also claim that the degradation
products of the copolymer could be consumed by macrophages
during normal wound healing response, reducing the occurrence of
unwanted inammation.
Blends of poly (aniline-co-ethyl-3-aminobenzoate) and PLA,
(3EABPANI-PLA), were electrospun to form electrically conductive
(7 10
3
S/cm) nanobrous meshes [144]. The cytocompatibility
was assessed by using COS-1 broblast cells on the nanobers. The
number of viable cells surviving on the (3EABPANI-PLA) bers after
4 days of culture was higher compared to TCP and glass controls.
Moreover, the viability of the broblasts increased with increasing
3EABPANI content.
Apart from thin lms, these conductive and degradable co-
polymers can also be produced in the form of hydrogels, as
demonstrated by Guo et al. [145]. Hydrogels based on analine
pentamer (AP), chitosan (CS), and glutaraldehyde, were obtained
by a one-pot synthesis reaction. The resulting hydrogels were
electrically conductive and degraded slowly in physiological
conditions (up to 13% mass loss was observed). Even though these
hydrogels possess properties that are desirable for tissue engi-
neering applications, their biocompatibility towards cells remains
to be established. Liu et al. also fabricated biodegradable and
electroactive hydrogels consisting of aniline pentamer grafted
onto gelatin (Fig. 13) [78]. The cytotoxicity was assessed by
Fig. 12. Schematic of the synthesis route adopted for the development of PLAAP copolymers, consisting of poly(L-lactide) and aniline pentamer. Reprinted with permission fromRef.
[77]. Copyright (2008) American Chemical Society.
T.H. Qazi et al. / Biomaterials 35 (2014) 9068e9086 9082
exposing RSC96 cells to samples of pure gelatin, aniline pentamer,
and their copolymers and its degradation products at different
concentrations. Increase in the AP concentration by up to two
orders of magnitude only slightly decreased the viability of the
cells. Osteoblasts seeded onto the copolymers were found to
attach and proliferate, and after 7 days a higher number of viable
cells survived on the composites compared to pure gelatin and
TCP controls. The synergic contribution of biocompatible gelatin
and electrical signals from the aniline pentamer resulted in an
improvement of cell behavior.
A hyperbranched copolymer, based on three-armed PCL and
aniline pentamer was blended with linear PCL and processed into
tubular scaffolds via solvent casting/salt leaching method for nerve
tissue regeneration [146]. The electrical conductivity was measured
to be in the range 10
5
e10
6
S/cm, whereas the mechanical
properties could be varied over a wide range (e.g. tensile strength:
2e18 MPa, strain at break: 6e900%, and elastic modulus:
77e324 MPa) based on the oxidation state and weight content of
aniline pentamer in the blend. The surface of the scaffold was hy-
drophilic in nature, with a water contact angle of 30

, which is
expected to facilitate cell attachment. The cytocompatibility was
evaluated using the extracts from the soaked scaffolds, and results
indicated that all samples tested were non-cytotoxic. The tubular
morphology (Fig. 14), together with degradability and conductivity
make this scaffold suitable for nerve tissue engineering
applications.
Moura and De Queiroz combined PANI and polyglycerol den-
drimers (PGLDs) to develop electrospun functionalized PANI
nanotubes [147]. PGLDs are hydrophilic and biocompatible, two
properties which are vital for the attachment and survival of cells
on a scaffold. The cytotoxicity was determined by exposing Chinese
hamster ovary cells to serially diluted extracts of PGLD-PANI
nanotubes, and the viability of the cells remained over 90% over
the entire range of extract concentrations tested (0e100%). More-
over, cardiomyocytes seeded on cast lm and electrospun nano-
tubes of PGLD-PANI were electrically stimulated (0e12 mV), which
resulted in the survival of a higher number of viable car-
diomyocytes on the PGLD-PANI bers compared to cast lms. In-
crease in the applied electrical potential promoted cellular survival
on both substrates.
To summarize, PANI oligomers have shown great promise for
use in biomedical applications. Despite their slight cytotoxicity in
the pure state, the ability to functionalize the oligomer chain with
biocompatible and hydrolyzable side segments has proven to be
an excellent way to develop electroactive yet biodegradable
materials.
Fig. 13. SEM images of aniline pentamer grafted gelatin (AP-g-GA) hydrogels in the porous state after lyophilization, pure gelatin (a), AP-g-GA with 10 (b), 20 (c), and 30 (d) wt. % of
aniline pentamer in reaction. Reproduced from Ref. [78] with permission from John Wiley and Sons.
Fig. 14. Macroscopic images of porous tubular scaffolds of (a) linear PCL, and (b) PCL/hyperbranched conducting polymer consisting of 9% aniline pentamer. Adapted from Ref. [146]
with permission from Elsevier.
T.H. Qazi et al. / Biomaterials 35 (2014) 9068e9086 9083
4. Concluding remarks and outlook
This review highlighted the benets of employing polyaniline,
a conducting polymer, as a biomaterial component exclusively for
tissue engineering applications. The many positive attributes of
polyaniline such as its biocompatibility, tunable conductivity,
processability, and antibacterial efcacy have resulted in an ever
increasing scientic interest in this material. The use of such
conducting polymers as polyaniline, in combination with external
electrical stimulation can result in control over cellular response
and improvement in cellular function. The elds of tissue engi-
neering and regenerative medicine, especially those of electrically
excitable tissues and organs, stand to gain immensely from
conductive scaffolds comprising materials such as polyaniline and
aniline oligomers. The combination of electrically conducting
polyaniline with a host of biodegradable polymers to form com-
posites or blends has not only paved the way for shaping these
composites into application relevant exible lms and multi-scale
bers, but has also resulted in materials possessing a range of
physical, chemical, and mechanical properties which can be
highly relevant for satisfying specic biomaterial needs. Recent
studies have spearheaded efforts to enhance the biocompatibility
of polyaniline by improving on the polymerization and purica-
tion steps. Even though polyaniline has been shown to be cyto-
compatible with numerous cell lines, more studies involving
in vivo transplantation of these conducting composites need to be
carried out to instill further condence in the biological and
clinical communities. Limited biodegradability has been identied
as one of the key issues hindering the application of polyaniline in
a biological environment such as the human body, but in recent
years the development of novel aniline oligomers and copolymers
has generated much hope and excitement. These materials are not
only electrically conductive and biocompatible, but can also be
chemically modied to become biodegradable. Polyaniline and its
copolymers have already opened up exciting new possibilities in
the areas of nerve, skeletal muscle, and cardiac tissue engineering,
and promise to become key biomaterial components in the repair
and regeneration of lost or damaged tissues in the future. The
authors hope that the present review article will generate further
interest and open new avenues for research and development in
this eld.
References
[1] Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920e6.
[2] Discher DE, Janmey P, Wang Y-l. Tissue cells feel and respond to the stiffness
of their substrate. Science 2005;310:1139e43.
[3] Lo CM, Wang HB, Dembo M, Wang YL. Cell movement is guided by the ri-
gidity of the substrate. Biophys J 2000;79:144e52.
[4] Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell
lineage specication. Cell 2006;126:677e89.
[5] Stevens MM, George JH. Exploring and engineering the cell surface interface.
Science 2005;310:1135e8.
[6] Chou L, Firth JD, Uitto VJ, Brunette DM. Substratum surface topography alters
cell shape and regulates bronectin mRNA level, mRNA stability, secretion
and assembly in human broblasts. J Cell Sci 1995;108:1563e73.
[7] Bassett CA, Pawluk RJ, Becker RO. Effects of electric currents on bone in vivo.
Nature 1964;204:652e4.
[8] Ozawa H, Abe E, Shibasaki Y, Fukuhara T, Suda T. Electric elds stimulate
DNA synthesis of mouse osteoblast-like cells (MC3T3-E1) by a mechanism
involving calcium ions. J Cell Physiol 1989;138:477e83.
[9] Brown MJ, Loew LM. Electric eld-directed broblast locomotion involves
cell surface molecular reorganization and is calcium independent. J Cell Biol
1994;127:117e28.
[10] Pullar CE, Isseroff RR, Nuccitelli R. Cyclic AMP-dependent protein kinase A
plays a role in the directed migration of human keratinocytes in a DC electric
eld. Cell Motil Cytoskeleton 2001;50:207e17.
[11] Li X, Kolega J. Effects of direct current electric elds on cell migration and
actin lament distribution in bovine vascular endothelial cells. J Vasc Res
2002;39:391e404.
[12] McBain VA, Forrester JV, McCaig CD. HGF, MAPK, and a small physiological
electric eld interact during corneal epithelial cell migration. Invest Oph-
thalmol Vis Sci 2003;44:540e7.
[13] Park H, Bhalla R, Saigal R, Radisic M, Watson N, Langer R, et al. Effects of
electrical stimulation in C2C12 muscle constructs. J Tissue Eng Regen Med
2008;2:279e87.
[14] Radisic M, Park H, Shing H, Consi T, Schoen FJ, Langer R, et al. Functional as-
sembly of engineered myocardium by electrical stimulation of cardiac myo-
cytes cultured on scaffolds. Proc Natl Acad Sci U S A 2004;101:18129e34.
[15] Stout DA, Yoo J, Santiago-Miranda AN, Webster TJ. Mechanisms of greater
cardiomyocyte functions on conductive nanoengineered composites for
cardiovascular application. Int J Nanomedicine 2012;7:5653e69.
[16] Dvir T, Timko BP, Brigham MD, Naik SR, Karajanagi SS, Levy O, et al. Nano-
wired three-dimensional cardiac patches. Nat Nanotechnol 2011;6:720e5.
[17] Chiang CK, Fincher Jr CR, Park YW, Heeger AJ, Shirakawa H, Louis EJ, et al.
Electrical conductivity in doped polyacetylene. Phys Rev Lett 1977;39:
1098e101.
[18] Bhadra S, Khastgir D, Singha NK, Lee JH. Progress in preparation, processing
and applications of polyaniline. Prog Polym Sci (Oxford) 2009;34:783e810.
[19] MacDiarmid AG. Synthetic metals: a novel role for organic polymers (Nobel
Lecture). Angew Chem Int Ed 2001;40:2581e90.
[20] Liu Z, Zhou J, Xue H, Shen L, Zang H, Chen W. Polyaniline/TiO2 solar cells.
Synth Met 2006;156:721e3.
[21] Gaponik NP, Talapin DV, Rogach AL. A light-emitting device based on a CdTe
nanocrystal/polyaniline composite. Phys Chem Chem Phys 1999;1:1787e9.
[22] Rodrigues MA, De Paoli M-A, Mastragostino M. Electrochromic properties of
chemically prepared polyaniline. Electrochim Acta 1991;36:2143e6.
[23] Wong JY, Langer R, Ingber DE. Electrically conducting polymers can non-
invasively control the shape and growth of mammalian cells. Proc Natl Acad
Sci U S A 1994;91:3201e4.
[24] Peng H, Zhang L, Soeller C, Travas-Sejdic J. Conducting polymers for elec-
trochemical DNA sensing. Biomaterials 2009;30:2132e48.
[25] Guimard NK, Gomez N, Schmidt CE. Conducting polymers in biomedical
engineering. Prog Polym Sci 2007;32:876e921.
[26] Schmidt CE, Shastri VR, Vacanti JP, Langer R. Stimulation of neurite
outgrowth using an electrically conducting polymer. Proc Natl Acad Sci U S A
1997;94:8948e53.
[27] Meng S, Zhang Z, Rouabhia M. Accelerated osteoblast mineralization on a
conductive substrate by multiple electrical stimulation. J Bone Miner Metab
2011;29:535e44.
[28] Ravichandran R, Sundarrajan S, Venugopal JR, Mukherjee S, Ramakrishna S.
Applications of conducting polymers and their issues in biomedical engi-
neering. J R Soc Interface 2010;6:7.
[29] Bendrea AD, Cianga L, Cianga I. Review paper: progress in the eld of con-
ducting polymers for tissue engineering applications. J Biomater Appl
2011;26:3e84.
[30] MacDiarmid AG, Epstein AJ. Polyanilines: a novel class of conducting poly-
mers. Faraday Discuss Chem Soc 1989;88:317e32.
[31] Huang W-S, Humphrey BD, MacDiarmid AG. Polyaniline, a novel conducting
polymer. Morphology and chemistry of its oxidation and reduction in
aqueous electrolytes. J Chem Soc Faraday Trans 1: Phys Chem Condens
Phases 1986;82:2385e400.
[32] Anand J, Palaniappan S, Sathyanarayana DN. Conducting polyaniline blends
and composites. Prog Polym Sci (Oxford) 1998;23:993e1018.
[33] Mattioli-Belmonte M, Giavaresi G, Biagini G, Virgili L, Giacomini M, Fini M,
et al. Tailoring biomaterial compatibility: in vivo tissue response versus
in vitro cell behavior. Int J Artif Organs 2003;26:1077e85.
[34] Zhang L, Zhang Z, Kilmartin PA, Travas-Sejdic J. Hollow polyaniline and
indomethacin composite microspheres for controlled indomethacin release.
Macromol Chem Phys 2011;212:2674e84.
[35] Konwarh R, Pramanik S, Devi KSP, Saikia N, Boruah R, Maiti TK, et al. Lyco-
pene coupled 'trifoliate' polyaniline nanobers as multi-functional bioma-
terial. J Mater Chem 2012;22:15062e70.
[36] Nand AV, Swift S, Uy B, Kilmartin PA. Evaluation of antioxidant and anti-
microbial properties of biocompatible low density polyethylene/polyaniline
blends. J Food Eng 2013;116:422e9.
[37] Ibarra LE, Yslas EI, Molina MA, Rivarola CR, Romanini S, Barbero CA, et al.
Near-infrared mediated tumor destruction by photothermal effect of PANI-
Np in vivo. Laser Phys 2013:23.
[38] Chiang J-C, MacDiarmid AG. Polyaniline: protonic acid doping of the
emeraldine form to the metallic regime. Synth Met 1986;13:193e205.
[39] Boara G, Sparpaglione M. Synthesis of polyanilines with high electrical
conductivity. Synth Met 1995;72:135e40.
[40] Focke WW, Wnek GE, Wei Y. Inuence of oxidation state, pH, and counterion
on the conductivity of polyaniline. J Phys Chem 1987;91:5813e8.
[41] Monkman AP, Adams P. Optical and electronic properties of stretch-oriented
solution-cast polyaniline lms. Synth Met 1991;40:87e96.
[42] Sinha S, Bhadra S, Khastgir D. Effect of dopant type on the properties of
polyaniline. J Appl Polym Sci 2009;112:3135e40.
[43] Cao Y, Smith P, Heeger AJ. Counter-ion induced processibility of conducting
polyaniline and of conducting polyblends of polyaniline in bulk polymers.
Synth Met 1992;48:91e7.
[44] Angelopoulos M, Asturias GE, Ermer SP, Ray A, Scherr EM, Macdiarmid AG,
et al. Polyaniline: solutions, lms and oxidation state. Mol Cryst Liq Cryst
Incorporating Nonlinear Opt 1988;160:151e63.
T.H. Qazi et al. / Biomaterials 35 (2014) 9068e9086 9084
[45] Chiou NR, Epstein AJ. Polyaniline nanobers prepared by dilute polymeri-
zation. Adv Mater 2005;17:1679e83.
[46] Shadi L, Karimi M, Entezami A, Safa K. A facile synthesis of polyaniline/
polyethylene glycol/polyaniline terpolymers: preparation of electrospun
conducting nanobers by blending of the terpolymers with poly-
caprolactone. Polym Bull 2013;70:3529e45.
[47] Kamalesh S, Tan P, Wang J, Lee T, Kang ET, Wang CH. Biocompatibility of
electroactive polymers in tissues. J Biomed Mater Res 2000;52:467e78.
[48] Bidez PR, Li S, Macdiarmid AG, Venancio EC, Wei Y, Lelkes PI. Polyaniline, an
electroactive polymer, supports adhesion and proliferation of cardiac myo-
blasts. J Biomater Sci Polym Ed 2006;17:199e212.
[49] Liu S, Wang J, Zhang D, Zhang P, Ou J, Liu B, et al. Investigation on cell
biocompatible behaviors of polyaniline lm fabricated via electroless surface
polymerization. Appl Surf Sci 2010;256:3427e31.
[50] Jun I, Jeong S, Shin H. The stimulation of myoblast differentiation by elec-
trically conductive sub-micron bers. Biomaterials 2009;30:2038e47.
[51] Xia Y, Lu X, Zhu H. Natural silk broin/polyaniline (core/shell) coaxial ber:
fabrication and application for cell proliferation. Compos Sci Technol
2013;77:37e41.
[52] Borriello A, Guarino V, Schiavo L, Alvarez-Perez MA, Ambrosio L. Optimizing
PANi doped electroactive substrates as patches for the regeneration of car-
diac muscle. J Mater Sci Mater Med 2011;22:1053e62.
[53] Prabhakaran MP, Ghasemi-Mobarakeh L, Jin G, Ramakrishna S. Electrospun
conducting polymer nanobers and electrical stimulation of nerve stem cells.
J Biosci Bioeng 2011;112:501e7.
[54] Wang CH, Dong YQ, Sengothi K, Tan KL, Kang ET. In-vivo tissue response to
polyaniline. Synth Met 1999;102:1313e4.
[55] Ramanaviciene A, Kausaite A, Tautkus S, Ramanavicius A. Biocompatibility of
polypyrrole particles: an in-vivo study in mice. J Pharm Pharmacol 2007;59:
311e5.
[56] Williams RL, Doherty PJ. A preliminary assessment of poly(pyrrole) in nerve
guide studies. J Mater Sci Mater Med 1994;5:429e33.
[57] Wang X, Gu X, Yuan C, Chen S, Zhang P, Zhang T, et al. Evaluation of
biocompatibility of polypyrrole in vitro and in vivo. J Biomed Mater Res A
2004;68:411e22.
[58] Vineis P, Pirastu R. Aromatic amines and cancer. Cancer Causes Control
1997;8:346e55.
[59] Harrison Jr JH, Jollow DJ. Role of aniline metabolites in aniline-induced he-
molytic anemia. J Pharmacol Exp Ther 1986;238:1045e54.
[60] Bus JS, Popp JA. Perspectives on the mechanism of action of the splenic
toxicity of aniline and structurally-related compounds. Food Chem Toxicol
1987;25:619e26.
[61] Hand RL, Nelson RF. Anodic oxidation pathways of N-alkylanilines. J Am
Chem Soc 1974;96:850e60.
[62] Luo YL, Nan YF, Xu F, Chen YS, Zhao P. Degradation behavior and biocom-
patibility of PEG/PANI-derived polyurethane co-polymers. J Biomater Sci
Polym Ed 2010;21:1143e72.
[63] Humpolicek P, Kasparkova V, Saha P, Stejskal J. Biocompatibility of poly-
aniline. Synth Met 2012;162:722e7.
[64] Cullen DK, Patel AR, Doorish JF, Smith DH, Pster BJ. Developing a tissue-
engineered neural-electrical relay using encapsulated neuronal constructs
on conducting polymer bers. J Neural Eng 2008;5:374e84.
[65] M-y Li, Bidez P, Guterman-Tretter E, Guo Y, MacDiarmid AG, Lelkes PI, et al.
Electroactive and nanostructured polymers as scaffold materials for neuronal
and cardiac tissue engineering. Chin J Polym Sci 2007;25:331e9.
[66] Guo Y, Li M, Mylonakis A, Han J, MacDiarmid AG, Chen X, et al. Electroactive
oligoaniline-containing self-assembled monolayers for tissue engineering
applications. Biomacromolecules 2007;8:3025e34.
[67] Preparation and cytocompatibility of polyaniline/PLCL conductive nano-
bers. In: Xu P, Hussain AM, Xu X, Cui J, Li W, Wang G, editors. 3rd Inter-
national Conference on Biomedical Engineering and Informatics (BMEI), vol.
4; 2010. p. 1719e22.
[68] Ben-Valid S, Dumortier H, Decossas M, Sfez R, Meneghetti M, Bianco A, et al.
Polyaniline-coated single-walled carbon nanotubes: synthesis, character-
ization and impact on primary immune cells. J Mater Chem 2010;20:
2408e17.
[69] Ruckenstein E, Gourisankar SV. A surface energetic criterion of blood
compatibility of foreign surfaces. J Colloid Interface Sci 1984;101:436e51.
[70] Li ZF, Ruckenstein E. Grafting of poly(ethylene oxide) to the surface of pol-
yaniline lms through a chlorosulfonation method and the biocompatibility
of the modied lms. J Colloid Interface Sci 2004;269:62e71.
[71] Li L, Ge J, Guo B, Ma PX. In situ forming biodegradable electroactive hydro-
gels. Polym Chem 2014;5:2880e90.
[72] Zelikin AN, Lynn DM, Farhadi J, Martin I, Shastri V, Langer R. Erodible con-
ducting polymers for potential biomedical applications. Angew Chem Int Ed
2002;41:141e4.
[73] Guo B, Glavas L, Albertsson A-C. Biodegradable and electrically conducting
polymers for biomedical applications. Prog Polym Sci 2013;38:1263e86.
[74] Guo B, Finne-Wistrand A, Albertsson A-C. Universal two-step approach to
degradable and electroactive block copolymers and networks from com-
bined ring-opening polymerization and post-functionalization via oxidative
coupling reactions. Macromolecules 2011;44:5227e36.
[75] Zhang Q, Yan Y, Li S, Feng T. The synthesis and characterization of a novel
biodegradable and electroactive polyphosphazene for nerve regeneration.
Mater Sci Eng C 2010;30:160e6.
[76] Huang L, Hu J, Lang L, Wang X, Zhang P, Jing X, et al. Synthesis and char-
acterization of electroactive and biodegradable ABA block copolymer of
polylactide and aniline pentamer. Biomaterials 2007;28:1741e51.
[77] Huang L, Zhuang X, Hu J, Lang L, Zhang P, Wang Y, et al. Synthesis of
biodegradable and electroactive multiblock polylactide and aniline pentamer
copolymer for tissue engineering applications. Biomacromolecules 2008;9:
850e8.
[78] Liu Y, Hu J, Zhuang X, Zhang P, Wei Y, Wang X, et al. Synthesis and char-
acterization of novel biodegradable and electroactive hydrogel based on
aniline oligomer and gelatin. Macromol Biosci 2012;12:241e50.
[79] Cui H, Liu Y, Deng M, Pang X, Zhang P, Wang X, et al. Synthesis of biode-
gradable and electroactive tetraaniline grafted poly(ester amide) copolymers
for bone tissue engineering. Biomacromolecules 2012;13:2881e9.
[80] Zilberman M, Elsner JJ. Antibiotic-eluting medical devices for various appli-
cations. J Control Release 2008;130:202e15.
[81] Leaper DJ. Silver dressings: their role in wound management. Int Wound J
2006;3:282e94.
[82] Kim K, Luu YK, Chang C, Fang D, Hsiao BS, Chu B, et al. Incorporation and
controlled release of a hydrophilic antibiotic using poly(lactide-co-
glycolide)-based electrospun nanobrous scaffolds. J Control Release
2004;98:47e56.
[83] Mourino V, Boccaccini AR. Bone tissue engineering therapeutics: controlled
drug delivery in three-dimensional scaffolds. J R Soc Interface 2010;7:
209e27.
[84] Boomi P, Prabu HG. Synthesis, characterization and antibacterial analysis of
polyaniline/AuePd nanocomposite. Colloids Surfaces A Physicochem Eng
Aspects 2013;429:51e9.
[85] Shi NL, Guo XM, Jing HM, Gong J, Sun C, Yang K. Antibacterial effect of the
conducting polyaniline. J Mater Sci Technol 2006;22:289e90.
[86] Kohanski MA, Dwyer DJ, Collins JJ. How antibiotics kill bacteria: from targets
to networks. Nat Rev Microbiol 2010;8:423e35.
[87] Gizdavic-Nikolaidis MR, Bennett JR, Swift S, Easteal AJ, Ambrose M. Broad
spectrum antimicrobial activity of functionalized polyanilines. Acta Biomater
2011;7:4204e9.
[88] Gizdavic-Nikolaidis M, Ray S, Bennett JR, Easteal AJ, Cooney RP. Electrospun
functionalized polyaniline copolymer-based nanobers with potential
application in tissue engineering. Macromol Biosci 2010;10:1424e31.
[89] Riaz U, Ashraf SM. Evaluation of antibacterial activity of nanostructured
copolymers of poly (Naphthylamine). Int J Polym Mater Polym Biomater
2012;62:406e10.
[90] Chiu H-T, Chen R-L, Wu P-Y, Chiang T-Y, Chen S-C. A study on the effects of
the degree of deacetylation of chitosan lms on physical and antibacterial
properties. Polymer-Plastics Technol Eng 2007;46:1121e7.
[91] Kucekova Z, Kasparkova V, Humpolicek P, Sevcikova P, Stejskal J. Antibac-
terial properties of polyaniline-silver lms. Chem Pap 2013;67:1103e8.
[92] Wang HJ, Ji LW, Li DF, Wang JY. Characterization of nanostructure and cell
compatibility of polyaniline lms with different dopant acids. J Phys Chem B
2008;112:2671e7.
[93] Humpolicek P, Kasparkova V, Stejskal J, Kucekova Z, Sevcikova P. Cell pro-
liferation on a conductive polymer, polyaniline. Chem Listy 2012:380e3.
[94] Jeong SI, Jun ID, Choi MJ, Nho YC, Lee YM, Shin H. Development of electro-
active and elastic nanobers that contain polyaniline and poly(L-lactide-co-
epsilon-caprolactone) for the control of cell adhesion. Macromol Biosci
2008;8:627e37.
[95] Ghasemi-Mobarakeh L, Prabhakaran MP, Morshed M, Nasr-Esfahani MH,
Ramakrishna S. Electrical stimulation of nerve cells using conductive nano-
brous scaffolds for nerve tissue engineering. Tissue Eng Part A 2009;15:
3605e19.
[96] Tiwari A, Sharma Y, Hattori S, Terada D, Sharma AK, Turner AP, et al. Inu-
ence of poly(n-isopropylacrylamide)-CNT-polyaniline three-dimensional
electrospun microfabric scaffolds on cell growth and viability. Biopolymers
2013;99:334e41.
[97] Yan X, Chen J, Yang J, Xue Q, Miele P. Fabrication of free-standing, electro-
chemically active, and biocompatible graphene oxide-polyaniline and
graphene-polyaniline hybrid papers. ACS Appl Mater Interfaces 2010;2:
2521e9.
[98] Shi Z, Zang S, Jiang F, Huang L, Lu D, Ma Y, et al. In situ nano-assembly of
bacterial cellulose-polyaniline composites. RSC Adv 2012;2:1040e6.
[99] Li M, Guo Y, Wei Y, MacDiarmid AG, Lelkes PI. Electrospinning polyaniline-
contained gelatin nanobers for tissue engineering applications. Bio-
materials 2006;27:2705e15.
[100] Hsiao CW, Bai MY, Chang Y, Chung MF, Lee TY, Wu CT, et al. Electrical
coupling of isolated cardiomyocyte clusters grown on aligned conductive
nanobrous meshes for their synchronized beating. Biomaterials 2013;34:
1063e72.
[101] Kim H-S, Hobbs HL, Wang L, Rutten MJ, Wamser CC. Biocompatible com-
posites of polyaniline nanobers and collagen. Synth Met 2009;159:1313e8.
[102] Qazi TH, Rai R, Dippold D, Roether JE, Schubert DW, Rosellini E, et al.
Development and characterization of novel electrically conductive PAN-
IePGS composites for cardiac tissue engineering applications. Acta Biomater
2014;10:2434e45.
[103] McKeon KD, Lewis A, Freeman JW. Electrospun poly(D,L-lactide) and poly-
aniline scaffold characterization. J Appl Polym Sci 2010;115:1566e72.
[104] Whitehead MA, Fan D, Akkaraju GR, Canham LT, Coffer JL. Accelerated
calcication in electrically conductive polymer composites comprised of
T.H. Qazi et al. / Biomaterials 35 (2014) 9068e9086 9085
poly(-caprolactone), polyaniline, and bioactive mesoporous silicon.
J Biomed Mater Res Part A 2007;83A:225e34.
[105] Chen MC, Sun YC, Chen YH. Electrically conductive nanobers with highly
oriented structures and their potential application in skeletal muscle tissue
engineering. Acta Biomater 2013;9:5562e72.
[106] Oren R, Sfez R, Korbakov N, Shabtai K, Cohen A, Erez H, et al. Electrically
conductive 2D-PAN-containing surfaces as a culturing substrate for neurons.
J Biomater Sci Polym Ed 2004;15:1355e74.
[107] Li WJ, Laurencin CT, Caterson EJ, Tuan RS, Ko FK. Electrospun nanobrous
structure: a novel scaffold for tissue engineering. J Biomed Mater Res
2002;60:613e21.
[108] Sharma Y, Tiwari A, Hattori S, Terada D, Sharma AK, Ramalingam M, et al.
Fabrication of conducting electrospun nanobers scaffold for three-
dimensional cells culture. Int J Biol Macromol 2012;51:627e31.
[109] Fryczkowski R, Kowalczyk T. Nanobres from polyaniline/poly-
hydroxybutyrate blends. Synth Met 2009;159:2266e8.
[110] Potse M, Dube B, Vinet A. Cardiac anisotropy in boundary-element models
for the electrocardiogram. Med Biol Eng Comput 2009;47:719e29.
[111] Roth BJ. Electrical conductivity values used with the bidomain model of
cardiac tissue. IEEE Trans Biomed Eng 1997;44:326e8.
[112] Keith A, Flack M. The form and nature of the muscular connections between
the primary divisions of the vertebrate heart. J Anat Physiol 1907;41:172e89.
[113] Sandow A. Excitation-contraction coupling in muscular response. Yale J Biol
Med 1952;25:176e201.
[114] You J-O, Rafat M, Ye GJC, Auguste DT. Nanoengineering the heart: conductive
scaffolds enhance connexin 43 expression. Nano Lett 2011;11:3643e8.
[115] Shin SR, Jung SM, Zalabany M, Kim K, Zorlutuna P, Sb Kim, et al. Carbon-
nanotube-embedded hydrogel sheets for engineering cardiac constructs and
bioactuators. ACS Nano 2013;7:2369e80.
[116] Fernandes EGR, Zucolotto V, De Queiroz AAA. Electrospinning of hyper-
branched poly-l-lysine/polyaniline nanobers for application in cardiac tis-
sue engineering. J Macromol Sci Part A 2010;47:1203e7.
[117] Huang ZM, Zhang YZ, Kotaki M, Ramakrishna S. A review on polymer
nanobers by electrospinning and their applications in nanocomposites.
Compos Sci Technol 2003;63:2223e53.
[118] Zhang Y, Chwee TL, Ramakrishna S, Huang ZM. Recent development of
polymer nanobers for biomedical and biotechnological applications. J Mater
Sci Mater Med 2005;16:933e46.
[119] Donnelly K, Khodabukus A, Philp A, Deldicque L, Dennis RG, Baar K. A novel
bioreactor for stimulating skeletal muscle in vitro. Tissue Eng Part C Methods
2010;16:711e8.
[120] Pedrotty DM, Koh J, Davis BH, Taylor DA, Wolf P, Niklason LE. Engineering
skeletal myoblasts: roles of three-dimensional culture and electrical stimu-
lation. Am J Physiol Heart Circ Physiol 2005;288:18.
[121] Ku SH, Lee SH, Park CB. Synergic effects of nanober alignment and elec-
troactivity on myoblast differentiation. Biomaterials 2012;33:6098e104.
[122] Schmidt CE, Leach JB. Neural tissue engineering: strategies for repair and
regeneration. Annu Rev Biomed Eng 2003;5:293e347.
[123] Patel N, Poo MM. Orientation of neurite growth by extracellular electric
elds. J Neurosci 1982;2:483e96.
[124] Sisken BF, Kanje M, Lundborg G, Herbst E, Kurtz W. Stimulation of rat sciatic
nerve regeneration with pulsed electromagnetic elds. Brain Res 1989;485:
309e16.
[125] Freeman JA, Manis PB, Snipes GJ, Mayes BN, Samson PC, Wikswo Jr JP, et al.
Steady growth cone currents revealed by a novel circularly vibrating probe:
a possible mechanism underlying neurite growth. J Neurosci Res 1985;13:
257e83.
[126] Ghasemi-Mobarakeh L, Prabhakaran MP, Morshed M, Nasr-Esfahani MH,
Baharvand H, Kiani S, et al. Application of conductive polymers, scaffolds and
electrical stimulation for nerve tissue engineering. J Tissue Eng Regen Med
2011;10:10.
[127] Li GN, Hoffman-Kim D. Tissue-engineered platforms of axon guidance. Tissue
Eng Part B Rev 2008;14:33e51.
[128] Bhang SH, Jeong SI, Lee TJ, Jun I, Lee YB, Kim BS, et al. Electroactive elec-
trospun polyaniline/poly[(L-lactide)-co-(epsilon-caprolactone)] bers for
control of neural cell function. Macromol Biosci 2012;12:402e11.
[129] Polikov VS, Tresco PA, Reichert WM. Response of brain tissue to chronically
implanted neural electrodes. J Neurosci Methods 2005;148:1e18.
[130] HajjHassan M, Chodavarapu V, Musallam S. NeuroMEMS: neural probe
microtechnologies. Sensors 2008;8:6704e26.
[131] Li D-F, Wang W, Wang H-J, Jia X-S, Wang J-Y. Polyaniline lms with nano-
structure used as neural probe coating surfaces. Appl Surf Sci 2008;255:
581e4.
[132] Wang LP, Wang W, Di L, Lu YN, Wang JY. Protein adsorption under electrical
stimulation of neural probe coated with polyaniline. Colloids Surf B Bio-
interfaces 2010;80:72e8.
[133] Di L, Wang LP, Lu YN, He L, Lin ZX, Wu KJ, et al. Protein adsorption and
peroxidation of rat retinas under stimulation of a neural probe coated with
polyaniline. Acta Biomater 2011;7:3738e45.
[134] McLeod KJ, Rubin CT. The effect of low-frequency electrical elds on osteo-
genesis. J Bone Joint Surg e Ser A 1992;74:920e9.
[135] Supronowicz PR, Ajayan PM, Ullmann KR, Arulanandam BP, Metzger DW,
Bizios R. Novel current-conducting composite substrates for exposing oste-
oblasts to alternating current stimulation. J Biomed Mater Res 2002;59:
499e506.
[136] Shao S, Zhou S, Li L, Li J, Luo C, Wang J, et al. Osteoblast function on elec-
trically conductive electrospun PLA/MWCNTs nanobers. Biomaterials
2011;32:2821e33.
[137] Lam CW, James JT, McCluskey R, Arepalli S, Hunter RL. A review of carbon
nanotube toxicity and assessment of potential occupational and environ-
mental health risks. Critical Rev Toxicol 2006;36:189e217.
[138] Farshi Azhar F, Olad A, Salehi R. Fabrication and characterization of chito-
sanegelatin/nanohydroxyapatiteepolyaniline composite with potential
application in tissue engineering scaffolds. Des Monomers Polym 2014;17:
654e67.
[139] Liu Y, Cui H, Zhuang X, Zhang P, Cui Y, Wang X, et al. Nano-hydroxyapatite
surfaces grafted with electroactive aniline tetramers for bone-tissue engi-
neering. Macromol Biosci 2013;13:356e65.
[140] Abdul Rahman N, Gizdavic-Nikolaidis M, Ray S, Easteal AJ, Travas-Sejdic J.
Functional electrospun nanobres of poly(lactic acid) blends with polyani-
line or poly(aniline-co-benzoic acid). Synth Met 2010;160:2015e22.
[141] Abdul Rahman N, Feisst V, Dickinson ME, Malmstr om J, Dunbar PR, Travas-
Sejdic J. Functional polyaniline nanobre mats for human adipose-derived
stem cell proliferation and adhesion. Mater Chem Phys 2013;138:333e41.
[142] Hu J, Huang L, Zhuang X, Zhang P, Lang L, Chen X, et al. Electroactive aniline
pentamer cross-linking chitosan for stimulation growth of electrically sen-
sitive cells. Biomacromolecules 2008;9:2637e44.
[143] Liu Y, Hu J, Zhuang X, Zhang P, Chen X, Wei Y, et al. Preparation and char-
acterization of biodegradable and electroactive polymer blend materials
based on mPEG/tetraaniline and PLLA. Macromol Biosci 2011;11:806e13.
[144] Gizdavic-Nikolaidis M, Ray S, Bennett J, Swift S, Bowmaker G, Easteal A.
Electrospun poly(aniline-co-ethyl 3-aminobenzoate)/poly(lactic acid) nano-
bers and their potential in biomedical applications. J Polym Sci Part A Polym
Chem 2011;49:4902e10.
[145] Guo B, Finne-Wistrand A, Albertsson AC. Facile synthesis of degradable and
electrically conductive polysaccharide hydrogels. Biomacromolecules
2011;12:2601e9.
[146] Guo B, Sun Y, Finne-Wistrand A, Mustafa K, Albertsson AC. Electroactive
porous tubular scaffolds with degradability and non-cytotoxicity for neural
tissue regeneration. Acta Biomater 2012;8:144e53.
[147] Moura RM, de Queiroz AA. Dendronized polyaniline nanotubes for cardiac
tissue engineering. Artif Organs 2011;35:471e7.
T.H. Qazi et al. / Biomaterials 35 (2014) 9068e9086 9086