qazi et al

8112019 qazi et al

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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

copy 2014 Elsevier Ltd All rights reserved

1 Introduction

The 1047297eld 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

arti1047297cial 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 [23] elasticity [4] and surface

topography [56] have been identi1047297ed as having signi1047297cant in1047298u-

ence over the behavior of seeded cells including migration differ-

entiation and structural reorganization In a similar manner

electrical signals too can in1047298uence cellular behavior and function

The effects of electrical stimuli on tissues have been known

since the 1960s when Bassett and colleagues showed that low

intensity direct electrical currents in1047298uenced the mechanism of

bone formation in adult dogs [7] Others have reported that the

application of pulsed electrical 1047297eld stimulation on mouse osteo-

blast cells resulted in a signi1047297cant increase in DNA synthesis [8]

whereas a uniform electrical 1047297eld in1047298uenced the clustering and

distribution of membrane proteins on the cellesubstrate interface

and was able to control the direction of cell locomotion in two

1047297broblast cell lines [9] Further studies have also reported on the

effects of applied electric 1047297elds 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 in1047298uence

myogenic differentiation and deposition of type I collagen in a

skeletal muscle construct [13] whereas electrical stimulation of

cardiomyocytes seeded on collagenMatrigeltrade 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 thorn49 9131 85 28601 fax thorn49 9131 85 28602

E-mail address aldoboccacciniwwuni-erlangende (AR Boccaccini)1 Present address Julius Wolff Institute Augustenburger Platz 1 Charite Uni-

versiteuroatmedizin Berlin 13353 Berlin Germany

Contents lists available at ScienceDirect

Biomaterials

j o u r n a l h o m e p a g e w w w e l s e v i e r c o m l o c a t e b i o m a t er i a l s

httpdxdoiorg101016jbiomaterials201407020

0142-9612copy 2014 Elsevier Ltd All rights reserved

Biomaterials 35 (2014) 9068e9086

8112019 qazi et al


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 studiesSome efforts in this regard have been made through the incor-

poration of conductive particles such as carbon nano1047297bers [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 from an 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 signi1047297cant interest and available knowledge

in the 1047297eld 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 1047297eld 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 upondoping with iodine [17] Since then conducting polymers have

witnessed an immense increase in scienti1047297c 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 (34-ethylenedioxythiophene) (PEDOT) are just a few of the

many conductive polymers that are employed in technological

applications today For instance PANI 1047297nds applications in the

microelectronics industry including photovoltaic cells [20] light

emitting diodes [21] and electrochromic displays [22] In the bio-

logical 1047297eld conductive polymers were shown to be compatiblewith 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-invasivelycontrol 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 1047297lms 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

TH Qazi et al Biomaterials 35 (2014) 9068e9086 9069

8112019 qazi et al


[29] microspheres for drug delivery (Fig 2) [34] multifunctional

nano1047297bers with anti-cancer effects [35] anti-bacterial substrates

[36] and nanoparticles for anti-tumor therapy [37]

21 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 thereforemost usually acids such

as hydrochloric sulfuric or sulfonic acids The electrical conduc-

tivity of the doped PANI can be in1047298uenced by a variety of factors

including the degree of oxidation of the polymer type of protonic

acid used for doping degree of protonation moisturewater con-

tent morphology (stretching) of the polymer chains chain length

and degree of crystallization [39] For example a wet polymerneeds 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 in1047298uence of PANI chain alignment

(morphology) and chain length on its conductivity was studied by

Monkman et al by casting PANI 1047297lms 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]

22 Processability

To be useful in technological applications a polymer should be

processable into different forms such as 1047297lms or 1047297bers 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 1047297bers However Angelopoulos et al showed that the

emeraldine base form of PANI can be dissolved in N-methyl

pyrrolidinone (NMP) as a result yielding 1047297lms 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 1047297bers nanowires and nanotubes

[184546] and it has opened new avenues for applications

23 Biocompatibility

Serious interest in using PANI for biomedical and tissue engi-

neering applications soared after Kamalesh et al successfully

veri1047297ed the long term in vivo biocompatibility of PANI 1047297lms

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 didnot cause any undesirable in1047298ammatory response Mattioli-

Belmonte and colleagues also conducted in vivo studies on

several polymers including PANI and did not 1047297nd 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

in1047298ammatory 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 combination with 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 1047297broblasts [51] human mesenchymal stromal cells [52]

and rat nerve stem cells [53] among others Although these studiescarried out at in vitro and in vivo levels have demonstrated certain

extent of biocompatibility reservations still exist especially after

some in1047298ammation and 1047297brous 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 drugrelease behavior of IND from the composite microspheres at different pH values (B) Release of the drug was triggered at pH 74 and ceased at pH 2 Adapted from Ref [34] with

permission from John Wiley and Sons


8112019 qazi et al


The biocompatibility of PANI has also been a subject of debate

due to its non-biodegradability hence its long term presence inside

the patient could lead to unwanted side effects such as chronic

in1047298ammation 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 theaniline 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 signi1047297cantly 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 puri1047297cation 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 protonatethe PANI backbone [21] Though not in large quantities the acid

dopant eventually leaches out from the PANI matrix for example in

an aqueous environment thereby causing a local acidic environ-

ment surrounding the PANI component [4048] 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-PPsheets increased

from lt1 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 con1047297rmed 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 1047298uorescent micrographs depicting the viability and survival of dorsal root ganglia neurons at various distances away from polyanilinepolypropylene 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 15 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 toremove the leachate from the PANI-PP sheets and result in high viability of cultured cells copyIOP Publishing Reproduced from Ref [64] (httpdxdoiorg1010881741-256054002 )

by permission of IOP Publishing All rights reserved


8112019 qazi et al


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 1047297lms 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 1047297nal 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-ε-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-

modi1047297ed PANI [65] Higher cell adhesion was observed for peptide

modi1047297ed PANI compared to unmodi1047297ed 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 modi1047297ed 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-ε-caprolactone) (PLCL)

composites exhibited different levels of cytotoxicity to PC-12 cells

when in powder or 1047297ber forms [67] The authors attributed this

1047297nding 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 1047297ber form most of the PANIparticles 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 adsorptionand platelet adhesion [69] Li et al grafted poly(ethylene oxide)

(PEO) on the surface of PANI 1047297lms via chlorosulfonation to prevent

protein adsorption and platelet adhesion thereby increasing the

biocompatibility of the PANI 1047297lm [70] The water contact angle

measurement indicated that compared to pristine PANI the surface

of PEO-PANI 1047297lm was 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 1047297lm relative 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 arenumerous ways to make sure the PANI exposed to cells is biocom-

patible and no adverse reaction occurs As mentioned above these

include pre-soaking in medium before exposure to cells puri1047297cation

of the PANI after synthesis immobilization of peptide sequences

and adjusting surface properties such as wettability

24 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 arelargely 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 1047297lm

surface (A) PEO-grafted PANI 1047297lm with a grafting density of 33 (B) and 51 (C)

Increasing the grafting density of PEO signi1047297cantly 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


8112019 qazi et al


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 toshow 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 oligomerscoupledwith hydrolyzable

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

increasingimmersion timeindicatingsurfaceerosion caused by the

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-

1047297rming 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 signi1047297cantly 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 in1047298uence on degradation rate which could be controlled by

changing the length of the oligomer chain and the hydrolyzable

group segments on its endsLiu 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

from 42 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 ensuresthat the resulting materials have the same electroactive properties

as PANI with the additional bene1047297t of being biodegradable

25 Antibacterial ef 1047297cacy

Bacterial infection and bio1047297lm 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 bio1047297lm formation involve antibiotic

coatings on implant surfaces [80] anti-microbial wound dressings

[81] and novel drug releasing biomaterials [8283] 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 1047297lms of

PANI-polyvinylalcohol (PVA) were tested against Escherichia coli

(E coli) and Staphylococcus aureus (S aureus) bythe 1047297lm attachment

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 1047297lms 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-

mersof anilineand aminobenzoic acide ABA-PANI) on Ecoli S aureus

and P aeruginosa [87] It was found that the conductive forms of PANI

and ABA-PANI were more effective in inhibitingbacterial 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 energymetabolism and transport and cell wall and bio1047297lm formation In

another study functional copolymers of PANI and 3-aminobenzoic

acid (3ABAPANI) were shown to be excellent matrices for 1047297bro-

blasts and displayed antibacterial activity against S aureus making

these 1047297ber 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 ef 1047297cacy 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

propose that theantibacterial actionin Gram negative bacteria(E coli)

is caused by the blockage and consequent depletion of nutrients

resulting in cell death while in gram positive bacteria (S aureus) i t i s

caused by disruption of the cell wall due to electrostatic binding andexposure of thecell membraneto osmotic shock leading to lysis [90]

Kucekova et al studied the antibacterial properties of PANI 1047297lms

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 in1047298uence 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

31 Pure polyaniline 1047297lms

While majority of the studies focusing on utilizing PANI for

tissue engineering applications have combined it with other


8112019 qazi et al


biocompatible polymers there have been few studies investigating

the behavior and function of cells on pure PANI 1047297lms Wang et al

produced pure PANI 1047297lms either by casting on a Polytetra1047298uoro-

ethylene (PTFE) substrate or by direct deposition followed by

doping with four different acids [92] All PANI 1047297lms were found to

be biocompatible with PC-12 cells displaying signi1047297

cantly highercell attachment and proliferation on synthesized 1047297lms compared to

the cast 1047297lm The nanostructured surface in synthesized 1047297lms

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 1047297lms and

found both substrates to be biocompatible with cells readily

attaching and proliferating to form con1047298uent monolayers after 6

days [48] Furthermore the conducting PANI 1047297lm which was doped

with 1 M HCl for 15 min was found to maintain suf 1047297cient 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 in1047298uence of acidic leachates on seeded cells

con1047297rming that population doubling time was observed to be lower

for cells grown on conductive PANI 1047297lms for the 1047297rst 100 h but

returned to signi1047297cantly higher values between 100 and 150 h

when the leachates had supposedly diffused away Other studies

have also corroborated this 1047297nding by showing higher cell attach-

ment on non-conducting PANI 1047297lms compared to conducting ones

that release acidic dopants [93]

Using a combined self-assembly and surface polymerization

approach Liu et al produced PANI 1047297lms on silicon substrates and

reported higher long term cell viabilities of PC-12 cells on PANI

1047297lms compared to plain silicon substrates and TCP controls as

shown in Fig 5 [49] Techniques such as this enable surface coatingof materials such as scaffolds with a layer of conducting PANI that

can be used to control cell behavior via electrical signaling

32 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 byvarying 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 00138 S

cm (c) have 1047297bers 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 1047297broblasts NIH-3T3 1047297broblasts and C2C12 myoblasts Not

only were all the cell types viable on the composite 1047297bers but they

exhibited signi1047297cantlyenhanced viability on composites containing

higher concentrations of PANI NIH-3T3 1047297broblasts were then

seeded on PANI-PLCL and pure PLCL 1047297bers 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 andvery low metabolic activity a current of 20 mA signi1047297cantly

increased the mitochondrial metabolic activity of the 1047297broblasts

Fig 5 The culture and survival of PC-12 cells stained with acridine orange on pristine Silicon substrate (Ae

C) and on PANI 1047297lm (De

F) for 1 (AD) and 2 (BE) days C and F showmagni1047297ed regions of B and E respectively Signi1047297cantly higher number of viable PC-12 cells can be clearly seen on PANI 1047297lm compared to silicon substrate Reproduced from Ref

[49] with permission from Elsevier (For interpretation of the references to color in this 1047297gure legend the reader is referred to the web version of this article)


8112019 qazi et al


cultured on conductive PANI-PLCL relative to non-conducting pure

PLCL Fig 6 shows the morphology of 1047297broblasts 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 1047297bro-

blast cells were cultured on the PANI-CNT-PNIPAm micro1047297brous

scaffold for up to 7 days and the cell viability and proliferation was

compared to bulk PANI-CNT-PNIPAm and Matrigeltrade The

conductive micro1047297brous scaffolds provided a highly compatible

surface for cell adhesion as the cell viability of seeded 1047297broblasts

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 Matrigeltrade

and bulk composite a signi1047297cantly higher number of live cells were

found on the conductive PANI-CNT-PNIPAm scaffold The compos-

ite scaffold exhibited cell detachment behavior as the temperaturewas 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 cellsheets

By incorporating pH dependent methacrylic acid (MAA) to the

PNIPAm the same group also developed smart nano1047297brous

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 1047297broblasts showed

consistently higher cell growth on nano1047297bers of PANI-CNT-

PNIPAm-co-MAA compared to PNIPAm-co-MAA and control

Furthermore LiveDead 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 nano1047297bers The authors attributed these

observations to the added conductivity and mechanical strength

provided by the PANI and CNTs respectively

Coreshell coaxial 1047297bers comprising a silk 1047297broin inner core andan outer layer of PANI were fabricated via in situ oxidation [51]

L929 murine 1047297broblasts were cultured on the PANI-silk 1047297broin

Table 1

Overview of the different biodegradable polymers combined with polyaniline for tissue engineering applications

Polymer system Max conductivity

recorded [Scm]

Cell line tested Observations and results of cell culture Ref

PANI-PLCL 0296 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 00138 Human dermal 1047297broblasts

NIH-3T3 1047297broblasts

C2C12 myoblasts

-NIH-3T3 1047297broblasts when subjected to low electrical stimulation

(20 mA) responded by increasing metabolic activities

[94]

PANI-PLCL 000641 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 1047297broin 048 L929 1047297broblasts -Cultured 1047297broblasts showed highest survival rate on PANI containing

1047297bers compared to pure silk 1047297broin and TCP controls

[51]

PANI-PNIPAm-CNT e L929 1047297broblasts -Improved viability of cells on microporous

PANI-poly(N-isopropylacrylamide)eCNT composites relative to

Matrigeltrade and bulk composite controls

[96]

PANI-Graphene

PANI-Graphene oxide

e L929 1047297broblasts -Presence of PANI enhanced cell viability and proliferation [97]

PANI-BC 0018 e e [98]

PANI-Gel atin 0 0 21 H9 c2 ca rdiac myob lasts - Pro li ferati on of c ar dia c myob la sts t o n umb er s g reater th an t hose on

TCP controls

[99]

PANI-PLGA 00031 Neonatal cardiomyocytes -Enhanced adsorption of the adhesion proteins 1047297bronectin and laminin

-Cardiomyocytes expressed the gap junction protein Connexin 43

leading to synchronous beating of cell clusters

[100]

PANI-Collagen 027 Porcine skeletal muscle cells -No difference in morphology and cell number was observed between

PANI-collagen and pure collagen groups

[101]

PANI-PGS 0018 C2C12 myoblasts -Statistically signi1047297cant 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 00437 Primary rat muscle cells -Cellular attachment and proliferation showed no signi1047297cant difference

between any of the groups tested

[103]

PANI-PCL-BioSilicontrade e Mouse MSCs

human kidney 1047297broblasts

-Accelerated calci1047297cation of the composites in SBF was observed when

an electrical bias was applied

-Composites showed compatibility to kidney 1047297broblasts

[104]

PANI-PCL 0 0 00 08 h MSCs ca rdiomyoc ytes - Via bil ity of ca rdiomyoc ytes was h igh er o n PANI co nta in in g c omposite

patches relative to pure PCL

[105]

PANI-(PCLGelatin) 002 106 Sa Neural stem cells -Cells cultured on PANI containing composite 1047297bers showed higher

viability and proliferation compared to TCP control-Cells stimulated at 15 V for 60 min showed signi1047297cant

improvement in cell proliferation and neurite length and outgrowth

[106]

Gelatin- graft -PANI 0000454 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


8112019 qazi et al


1047297bers and despite a slow initial rate of proliferation showed the

highest viable cell number compared to pure silk 1047297broin and TCP

controls after 7 days These coreshell 1047297bers also allow for the

possibility of dissolving the inner silk 1047297broin core to obtain hollow

PANI nanotubes which could open up further avenues of research

Yan et al fabricated electroactive and biocompatible hybrid

1047297lms 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 1047297lm of nanorods and

nanoparticles were found on the surface of graphene due to

adsorption of short PANI nano1047297bers Viability of L929 1047297broblasts

was found to be higher on PANI-graphene hybrids compared to

pure graphene and higher on PANI-graphene oxide hybridscompared 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 1047297nd various applications as biosensors bio-

electrodes and forthe 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 nano1047297bers 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 micro1047297brils of bacterial

cellulose coated with PANI with diameters in the range of

80e120 nm (Fig 7) Electrical conductivity of the 1047297nal 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 PHBexhibits excellent biodegradability and biocompatibility and when

combined with PANI to form conductive nano1047297brous scaffolds can

serve as effective scaffolds for tissue engineering applications

Despite using biodegradable and biocompatible polymers both of

Fig 6 Culture of NIH-3T3 1047297broblasts on nano1047297ber scaffolds of polyaniline and poly(L-lactide-co-Ɛ-caprolactone) for 2 days under electrical stimulation of 20 mA (a) and 200 mA

(b) Fluorescence micrograph on the left shows F-actin 1047297lament formation in the cells which is absent in cells stimulated at a higher current value Adapted from Ref [94] with


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 micro1047297brils 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


8112019 qazi et al


the above studies failed to reportany in vitro cell culture results that

could illustrate the in1047298uence of the conductive scaffolds on the

survival proliferation and behavior of cells

321 Cardiac tissue engineering

The majority of studies employing PANI for tissue engineeringapplications have evaluated the behavior function and compati-

bility of cells derived from naturallyconductivesystems of the body

such as the heart and the nervous system [29] The native

mammalian heart is electrically conductive (in the order of 104 S

cm)and is composedof an underlying assembly of 1047297bers nodes and

cell clusters which make up its electrical system [110111] Electrical

impulses emanating from the sinoatrial node (situated in the right

atrium) are transmitted throughout the rest of the myocardium via

atrioventricular node (bridging the atria and the ventricles) and a

network of Purkinje 1047297bers [112] The propagation of electrical sig-

nals through the cardiac cells in a synchronized fashion results in a

heartbeat via the well-known phenomena 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 methacrylate2-hydroxyethyl methacrylate (thiol-

HEMAHEMA) 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 polymersystems to form composites or blends and evaluated for applica-

tion in cardiac tissue regeneration (Fig 8) H9c2 rat cardiac myo-

blasts cultured on composite nano1047297bers 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 1047297brous scaffold compared to smooth surfaces or

1047297lms 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 577 to 1049 MPa) of the composites

with increase in PANI content which will likely be useful inproviding 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 1047297lms or

electrospun into 1047297bers 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 1047297lms was only 30 whereas at the same stimulation con-

ditions cells grown on the 1047297brous 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 1047297brous samples under similar stimulation conditions un-

derscores the bene1047297ts of using scaffolds with nano1047297brous archi-

tecture for tissue engineering applications

Borriello et al reported the electrospinning of synthesized PANI

(s-PANI) short 1047297bers or lsquonano-needlesrsquo with PCL to form patches for

cardiac muscle regeneration [52] The authors claimed that PANI

short 1047297bers provide a more ef 1047297cient conductive network for charge

transfer within the composite Human mesenchymal stromal cells

(hMSCs) whendifferentiated intoa cardiogenic lineage and cultured

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 signi1047297cantat days1 and 3 [52]

In a recently published report Hsiao et al produced aligned

nano1047297brous 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 Adaptedfrom 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


8112019 qazi et al


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 1047297bronectin and laminin was

observed which promoted subsequent cell adhesion Neonatal rat

cardiomyocytes aligned themselves along the long axis of the1047297

bersand 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 nano1047297brous 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 1047297bers

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 [107117118] However as these studies emphasize

1047297ber orientation alone does not have as signi1047297cant an effect on

cellular morphology and desirable protein expressionupregulation

as that achieved by the synergic in1047298uence of 1047297ber orientation along

with electroactivity that is facilitated by PANI

322 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 occurElectrical stimulation has been shown to affect myoblast prolifer-

ation increase rate of protein synthesis and improve contraction

forces [119120] 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-

1047297bers dispersed in a collagen matrix and validated its biocompat-

ibility by culturing porcine skeletal muscle cells [101] The PANI

nano1047297

bers formed a three-dimensional interconnected network inthe collagen matrix which permitted charge transfer through the

composite resulting in a conductivity value of 001 Scm at the

percolation threshold Interestingly even with use of PANI nano-

1047297bers 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 1047297ber 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 (DL -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 PDLA25 PANI sample) the

conductive scaffolds underwent undesirable levels of shrinkage

which might limit the application of this composite

The in1047298uence 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 togetherand formedinto

nano1047297bers by electrospinning C2C12 myoblasts cultured on the

composite 1047297bers readily attached and proliferated on composites

with different PANI contents Quanti1047297cation of myotube charac-teristics after 8 days of culture revealed a clear trend whereby

increasing the amount of PANI resulted in signi1047297cantly higher

Fig 9 (A) Neonatal rat cardiomyocytes cultured on control tissue culture plates (top) undoped non-conductive (middle) and doped conductive (bottom) 1047297bers of PANI-PLGA Both

1047297ber orientation and electrical signals were vital in providing contact guidance to the cardiomyocytes (B) Immuno1047298uorescence staining of cardiomyocytes on doped conductive

1047297bers stained for cardiac troponin (cTnl) connexin 43 (Cx 43) and nucleus (red) The identi1047297cation of these markers indicates celle

cell coupling which leads to effectivecommunication and subsequent synchronized beating of cardiomyocyte clusters Adapted from Ref [100] with permission from Elsevier (For interpretation of the references to

color in this 1047297gure legend the reader is referred to the web version of this article)


8112019 qazi et al


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 1047297

bers of PANI and PCL were able to differen-tiate into myotubes [121] Though the aligned 1047297ber orientation did

not affect cell attachment and proliferation it did promote

morphological alignment of cultured cells along the major axis of

the 1047297bers Aligned 1047297ber meshes also exhibited signi1047297cantly higher

elastic moduli relative to those composed of randomly aligned 1047297-

bers Cell attachment survival and proliferation were similar on all

samples containing varying amounts of PANI and no signi1047297cant

differences were observed between random and aligned 1047297bers

When stained for MHC a protein essential for formation of myo-

tubes the MHC positive area was found to be signi1047297cantly higheron

aligned 1047297bers relative to random ones Moreover signi1047297cant

enhancement of myotube number length diameter and fusion

index was noticed for aligned 1047297bers 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 alwhere the synergic

effects of topography and electroactivity on the differentiation of

myoblasts was investigated [105] Highly aligned nano1047297brous mats

of PANI and PCL were obtained by using a modi1047297ed electrospinning

setup including a magnetic-1047297eld-assisted collector Low PANI con-

tents (1e3 wt ) resulted in suf 1047297cient levels of conductivity while

increasing the PANI content improved the mechanical properties of

the composite (tensile strength from 7 to 10 MPa Youngs 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

nano1047297brous mats had aligned themselves parallel to the 1047297ber di-

rection and showed higher viability on PANI containing samples

relative to pure PCL Myotube characterization at day 5 revealedsigni1047297cantly 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 1047297bers relative to

samples with randomly oriented 1047297bers Optimal myotube charac-

teristics were achieved for aligned and electrically conductive PANI

containing 1047297ber 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 1047297ber 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 1047297

bers for skeletal muscle regen-eration should assess the functional properties of differentiated

myotubes and report the in1047298uence of conducting polymers on

inducing greater contraction forces

323 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 bene1047297cial 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 ef 1047297cient manner [26126127]

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 1047298attened 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 speci1047297c to neurons derived from a single organism or specie or

was triggered by an unfavorable oxidation state of PANI is unclear

Cullen et al used PANI to form composite 1047297bers for a tissue

engineered neural electrical relay [64] PANI was blended with

Fig 10 C2C12 myoblasts seeded on electrospun random polycaprolactone 1047297bers (R-PCL) aligned polycaprolactone 1047297bers (A-PCL) random composite polycaprolactonepolyaniline1047297bers (R-PCLPANi) and aligned composite polycaprolactonepolyaniline 1047297bers (A-PCLPANi) Myogenic differentiation can be observed with stainings for MHC and MHC thorn nuclei

Signi1047297cantly higher numbers of myotubes were detected on 1047297bers containing PANI Adapted from Ref [105] with permission from Elsevier


8112019 qazi et al


polypropylene and either extruded into 1047297bers 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 1047297

bers after just two days of culture The cell viabilityimproved from lt1 adjacent to the PANI-PP sheets to over 60 just

3 mm away from it indicating the leaching of a toxic agent from the

composites This was further substantiated with a detoxifying

treatment by soaking PANI-PP sheets in media for a few days 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

detoxi1047297ed PANI-PP 1047297bers 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 in1047298uence of direct electrical stimulation on nerve stem cells

cultured on electrospun composite 1047297bers of PANI and a PCLgelatin

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-PCLGelatin 1047297bers 8 MPa sciatic nerve 27 MPa) (ii) elec-

trical properties with suf 1047297cient conductivity to perform electrical

stimulation and (iii) physical properties with tunable degradation

rate due to the presence of two biodegradable polymers Nerve

stem cells 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 tostimulate the cellsat 15 V for 15 30 and 60 min whilenon-

stimulated samples served as controls Electrical stimulation for a

duration of 60 min was found to signi1047297cantly improve cell prolif-

eration as well as neurite length and outgrowth relative to non-

stimulated controlsThe same group also carried out a similarstudy investigating the

effects of electrical stimulation on nerve stem cells cultured on

electrospun1047297bers of PANI and poly-L-lactide (PLLA) [53] The nerve

stem cells showed signi1047297cantly 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 1047297eld of 100 mVmm 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 nano1047297bers (Fig 11) The 1047297ndings 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 1047297bers

of PANI and PLCL for use as nerve grafts and demonstrated that the

addition of PANI along with nanoscale topography of the 1047297

bers notonly increased cell viability but also prompted the expression of

neuronal differentiation proteins [128] When cultured on PANI-

PLCL 1047297brous meshes PC-12 cells showed signi1047297cantly 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 1047297bers 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 prostheticrobotic arms [129130]

Highly conductive PANI has found application as nanostructured

1047297lm covering the surface of the electrodes making up the neural

probe [131] Wang et al successfully polymerized nanostructured

PANI 1047297lms 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 09

sodium chloride solution for 1 month after which microscopic

examination showed no cracks or 1047297ssure formation indicating that

the PANI 1047297lm 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 corrosionNo such surface irregularities were found on the PANI-Pt surface

leading to the conclusion that the nanostructured PANI 1047297lm resists

erosion over a long period of time Adsorption of 1047297bronectin 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 1047297lm 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 1047297bers 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


8112019 qazi et al


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

324 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 signi1047297cant enhancement in cell prolif-

eration concentration of extracellular calcium and collagen I

expression [135] Shao et al identi1047297ed 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 (BioSilicontrade) and observed

accelerated calci1047297cation of the composites in simulated body 1047298uid

(SBF) when electrically stimulated [104] Calci1047297cation assays

revealed that the 1047297rst signs of formation of calcium phosphate (Ca

P ~ 11) in SBF without electrical stimulation occurred after 1

month but required only 7 h to achieve improved calci1047297cation (P

Ca ~ 44) when an electrical 1047297eld was applied The scaffolds were

found to be non-cytotoxic to human kidney 1047297broblasts 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 Overallthese results strongly indicate that the rate of calci1047297cation can be

greatly enhanced with the application of electrical bias

In a recent article porous chitosan-gelatinnanohydroxyapatite-

polyaniline (CS-GelnHA-PANI) composite scaffolds fabricated by

Azhar and colleagues showed cytocompatibility with dental pulp

stem cells and exhibited higher mineralization rates in SBF than CS-

Gel and CS-GelnHA 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 proteinaseK the copolymerdegraded andlost 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 signi1047297cantly 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) graftedwith anilinetetramer which waslater dispersed ina poly(lactic acid) (PLA) matrix [139] The surface grafting of aniline

tetramer not only rendered the HA electroactive but also modi1047297ed

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 3days 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

33 Polyaniline copolymers

Because PANI is non-biodegradable even transplanting small

amounts can cause potential in1047298ammation 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

nano1047297ber 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 1047297bers

makes functionalized PANI copolymers an attractive option as a

substitute material for PANI The suitability of these functional

electrospun 1047297bers as scaffolds for the culture of human adipose

derived stem cells (hASCs) was evaluated in another study by thesame group [141] On seeding hASCs attached readily onto the

electrospun 1047297ber 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 1047297bers Microscopic analysis

revealed that the cells adopted a 1047297broblastic morphology and

formed abundant focal adhesion points on all samples tested This

was attributed to the high substrate stiffness of the nano1047297bers

measured by nanoindentation to be in the GPa range which has

been reported to have a major in1047298uence on development and

maturation of focal adhesion points

Gizdavic-Nikolaidis et al investigated conductive electrospun

nano1047297bers 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] Thebiocompatibility was established by seeding COS-1 1047297broblasts on

1047297ber mats containing varying amounts of 3ABAPANI and moni-

toring the proliferation over a period of four days The 1047297broblasts

proliferated with a similar rate on all samples and when compared

to cells cultured on TCP and glass substrates a signi1047297cantly higher

number of cells was found to be viable on the 4555 3ABAPANIPLA

1047297ber 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

1047297bers

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 ethylester as side groups which rendered the copolymer both electro-

active (a conductivity of 2 105 Scm was achieved) and


8112019 qazi et al


biodegradable (50 mass loss after 70 days in PBS in vitro) RSC96

Schwann cells were cultured on thin 1047297lms 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 49 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 1047297lm when higher

contents of AP are incorporated The copolymer containing 49 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

5 106 Scm which is suf 1047297cient 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 1047297lms The area fraction covered by C6 cells on

PAP1047297lms 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 survivalSimilarly 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

105e106 Scm 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

in1047298

uence 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 in1047298ammation

Blends of poly (aniline-co-ethyl-3-aminobenzoate) and PLA

(3EABPANI-PLA) were electrospun to form electrically conductive

(7 103 Scm) nano1047297brous meshes [144] The cytocompatibility

was assessed by using COS-1 1047297broblast cells on the nano1047297bers The

number of viable cells surviving on the (3EABPANI-PLA) 1047297bers after

4 days of culture was higher compared to TCP and glass controls

Moreover the viability of the 1047297broblasts increased with increasing

3EABPANI content

Apart from thin 1047297lms 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 thesehydrogels 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

Fig12 S chematic of the synthesis route adopted for the development of PLAAP copolymers consisting of poly(L-lactide) and aniline pentamer Reprinted with permission from Ref

[77] Copyright (2008) American Chemical Society


8112019 qazi et al


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 toattach 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 castingsalt leaching method for nerve

tissue regeneration [146] The electrical conductivity was measured

to be in the range 105e106 Scm whereas the mechanical

properties could be varied over a wide range (eg 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 PANInanotubes [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 1047297lm 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 1047297bers compared to cast 1047297lms 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-GAwith 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) P CLhyperbranched conducting polymer consisting of 9 aniline pentamer Adapted from Ref [146]

with permission from Elsevier


8112019 qazi et al


4 Concluding remarks and outlook

This review highlighted the bene1047297ts 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 conductivityprocessability and antibacterial ef 1047297cacy have resulted in an ever

increasing scienti1047297c 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 1047297elds 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 1047298exible 1047297lms and multi-scale

1047297bers but has also resulted in materials possessing a range of

physical chemical and mechanical properties which can be

highly relevant for satisfying speci1047297c biomaterial needs Recent

studies have spearheaded efforts to enhance the biocompatibility

of polyaniline by improving on the polymerization and puri1047297ca-

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 con1047297dence in the biological and

clinical communities Limited biodegradability has been identi1047297ed

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 modi1047297ed to become biodegradable Polyaniline and its

copolymers have already opened up exciting new possibilities inthe 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 1047297eld

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[11] Li X Kolega J Effects of direct current electric 1047297elds on cell migration andactin 1047297lament distribution in bovine vascular endothelial cells J Vasc Res200239391e404

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[39] Boara G Sparpaglione M Synthesis of polyanilines with high electricalconductivity Synth Met 199572135e40

[40] Focke WW Wnek GE Wei Y In1047298uence of oxidation state pH and counterionon the conductivity of polyaniline J Phys Chem 1987915813e8

[41] Monkman AP Adams P Optical and electronic properties of stretch-orientedsolution-cast polyaniline 1047297lms Synth Met 19914087e96

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[45] Chiou NR Epstein AJ Polyaniline nano1047297bers prepared by dilute polymeri-zation Adv Mater 2005171679e83

[46] Shadi L Karimi M Entezami A Safa K A facile synthesis of polyanilinepolyethylene glycolpolyaniline terpolymers preparation of electrospunconducting nano1047297bers by blending of the terpolymers with poly-caprolactone Polym Bull 2013703529e45

[47] Kamalesh S Tan P Wang J Lee T Kang ET Wang CH Biocompatibility of

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electroactive polymer supports adhesion and proliferation of cardiac myo-blasts J Biomater Sci Polym Ed 200617199e212

[49] Liu S Wang J Zhang D Zhang P Ou J Liu B et al Investigation on cellbiocompatible behaviors of polyaniline 1047297lm fabricated via electroless surfacepolymerization Appl Surf Sci 20102563427e31

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8112019 qazi et al


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[134] McLeod KJ Rubin CT The effect of low-frequency electrical 1047297elds on osteo-genesis J Bone Joint Surg e Ser A 199274920e9

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8112019 qazi et al


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 studiesSome efforts in this regard have been made through the incor-

poration of conductive particles such as carbon nano1047297bers [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 from an 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 signi1047297cant interest and available knowledge

in the 1047297eld 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 1047297eld 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 upondoping with iodine [17] Since then conducting polymers have

witnessed an immense increase in scienti1047297c 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 (34-ethylenedioxythiophene) (PEDOT) are just a few of the

many conductive polymers that are employed in technological

applications today For instance PANI 1047297nds applications in the

microelectronics industry including photovoltaic cells [20] light

emitting diodes [21] and electrochromic displays [22] In the bio-

logical 1047297eld conductive polymers were shown to be compatiblewith 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-invasivelycontrol 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 1047297lms 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


8112019 qazi et al





21 Conductivity




























22 Processability


















23 Biocompatibility
































8112019 qazi et al





























































8112019 qazi et al









control















































pristine PANI [70]










24 Biodegradability
















8112019 qazi et al


























































































































8112019 qazi et al


















































lines tested



























8112019 qazi et al






































Table 1



recorded [Scm]






[50]



C2C12 myoblasts



[94]




[95]



[51]




[96]

PANI-Graphene

PANI-Graphene oxide


PANI-BC 0018 e e [98]


TCP controls

[99]




[100]



[101]




[102]



[103]






[104]



[105]




[106]


C2C12 myoblasts




than gelatin

[71]




8112019 qazi et al























excitable cells























8112019 qazi et al

































[115]













































8112019 qazi et al


















the native heart























nano1047297





































8112019 qazi et al






















T and MHC


































































8112019 qazi et al







sheets and 1047297











[64]































growth factors














































8112019 qazi et al























and repair





























































































1047297bers







8112019 qazi et al













































in1047298






















3EABPANI content















8112019 qazi et al


























applications













on both substrates






materials






8112019 qazi et al







































this 1047297eld

References


















































8112019 qazi et al















































Aspects 201342951e


























8112019 qazi et al





































2011322821e














8112019 qazi et al





21 Conductivity




























22 Processability


















23 Biocompatibility
































8112019 qazi et al





























































8112019 qazi et al









control















































pristine PANI [70]










24 Biodegradability
















8112019 qazi et al


























































































































8112019 qazi et al


















































lines tested



























8112019 qazi et al






































Table 1



recorded [Scm]






[50]



C2C12 myoblasts



[94]




[95]



[51]




[96]

PANI-Graphene

PANI-Graphene oxide


PANI-BC 0018 e e [98]


TCP controls

[99]




[100]



[101]




[102]



[103]






[104]



[105]




[106]


C2C12 myoblasts




than gelatin

[71]




8112019 qazi et al























excitable cells























8112019 qazi et al

































[115]













































8112019 qazi et al


















the native heart























nano1047297





































8112019 qazi et al






















T and MHC


































































8112019 qazi et al







sheets and 1047297











[64]































growth factors














































8112019 qazi et al























and repair





























































































1047297bers







8112019 qazi et al













































in1047298






















3EABPANI content















8112019 qazi et al


























applications













on both substrates






materials






8112019 qazi et al







































this 1047297eld

References


















































8112019 qazi et al















































Aspects 201342951e


























8112019 qazi et al





































2011322821e














8112019 qazi et al





























































8112019 qazi et al









control















































pristine PANI [70]










24 Biodegradability
















8112019 qazi et al


























































































































8112019 qazi et al


















































lines tested



























8112019 qazi et al






































Table 1



recorded [Scm]






[50]



C2C12 myoblasts



[94]




[95]



[51]




[96]

PANI-Graphene

PANI-Graphene oxide


PANI-BC 0018 e e [98]


TCP controls

[99]




[100]



[101]




[102]



[103]






[104]



[105]




[106]


C2C12 myoblasts




than gelatin

[71]




8112019 qazi et al























excitable cells























8112019 qazi et al

































[115]













































8112019 qazi et al


















the native heart























nano1047297





































8112019 qazi et al






















T and MHC


































































8112019 qazi et al







sheets and 1047297











[64]































growth factors














































8112019 qazi et al























and repair





























































































1047297bers







8112019 qazi et al













































in1047298






















3EABPANI content















8112019 qazi et al


























applications













on both substrates






materials






8112019 qazi et al







































this 1047297eld

References


















































8112019 qazi et al















































Aspects 201342951e


























8112019 qazi et al





































2011322821e














8112019 qazi et al









control















































pristine PANI [70]










24 Biodegradability
















8112019 qazi et al


























































































































8112019 qazi et al


















































lines tested



























8112019 qazi et al






































Table 1



recorded [Scm]






[50]



C2C12 myoblasts



[94]




[95]



[51]




[96]

PANI-Graphene

PANI-Graphene oxide


PANI-BC 0018 e e [98]


TCP controls

[99]




[100]



[101]




[102]



[103]






[104]



[105]




[106]


C2C12 myoblasts




than gelatin

[71]




8112019 qazi et al























excitable cells























8112019 qazi et al

































[115]













































8112019 qazi et al


















the native heart























nano1047297





































8112019 qazi et al






















T and MHC


































































8112019 qazi et al







sheets and 1047297











[64]































growth factors














































8112019 qazi et al























and repair





























































































1047297bers







8112019 qazi et al













































in1047298






















3EABPANI content















8112019 qazi et al


























applications













on both substrates






materials






8112019 qazi et al







































this 1047297eld

References


















































8112019 qazi et al















































Aspects 201342951e


























8112019 qazi et al





































2011322821e














8112019 qazi et al


























































































































8112019 qazi et al


















































lines tested



























8112019 qazi et al






































Table 1



recorded [Scm]






[50]



C2C12 myoblasts



[94]




[95]



[51]




[96]

PANI-Graphene

PANI-Graphene oxide


PANI-BC 0018 e e [98]


TCP controls

[99]




[100]



[101]




[102]



[103]






[104]



[105]




[106]


C2C12 myoblasts




than gelatin

[71]




8112019 qazi et al























excitable cells























8112019 qazi et al

































[115]













































8112019 qazi et al


















the native heart























nano1047297





































8112019 qazi et al






















T and MHC


































































8112019 qazi et al







sheets and 1047297











[64]































growth factors














































8112019 qazi et al























and repair





























































































1047297bers







8112019 qazi et al













































in1047298






















3EABPANI content















8112019 qazi et al


























applications













on both substrates






materials






8112019 qazi et al







































this 1047297eld

References


















































8112019 qazi et al















































Aspects 201342951e


























8112019 qazi et al





































2011322821e














8112019 qazi et al


















































lines tested



























8112019 qazi et al






































Table 1



recorded [Scm]






[50]



C2C12 myoblasts



[94]




[95]



[51]




[96]

PANI-Graphene

PANI-Graphene oxide


PANI-BC 0018 e e [98]


TCP controls

[99]




[100]



[101]




[102]



[103]






[104]



[105]




[106]


C2C12 myoblasts




than gelatin

[71]




8112019 qazi et al























excitable cells























8112019 qazi et al

































[115]













































8112019 qazi et al


















the native heart























nano1047297





































8112019 qazi et al






















T and MHC


































































8112019 qazi et al







sheets and 1047297











[64]































growth factors














































8112019 qazi et al























and repair





























































































1047297bers







8112019 qazi et al













































in1047298






















3EABPANI content















8112019 qazi et al


























applications













on both substrates






materials






8112019 qazi et al







































this 1047297eld

References


















































8112019 qazi et al















































Aspects 201342951e


























8112019 qazi et al





































2011322821e














8112019 qazi et al






































Table 1



recorded [Scm]






[50]



C2C12 myoblasts



[94]




[95]



[51]




[96]

PANI-Graphene

PANI-Graphene oxide


PANI-BC 0018 e e [98]


TCP controls

[99]




[100]



[101]




[102]



[103]






[104]



[105]




[106]


C2C12 myoblasts




than gelatin

[71]




8112019 qazi et al























excitable cells























8112019 qazi et al

































[115]













































8112019 qazi et al


















the native heart























nano1047297





































8112019 qazi et al






















T and MHC


































































8112019 qazi et al







sheets and 1047297











[64]































growth factors














































8112019 qazi et al























and repair





























































































1047297bers







8112019 qazi et al













































in1047298






















3EABPANI content















8112019 qazi et al


























applications













on both substrates






materials






8112019 qazi et al







































this 1047297eld

References


















































8112019 qazi et al















































Aspects 201342951e


























8112019 qazi et al





































2011322821e














8112019 qazi et al























excitable cells























8112019 qazi et al

































[115]













































8112019 qazi et al


















the native heart























nano1047297





































8112019 qazi et al






















T and MHC


































































8112019 qazi et al







sheets and 1047297











[64]































growth factors














































8112019 qazi et al























and repair





























































































1047297bers







8112019 qazi et al













































in1047298






















3EABPANI content















8112019 qazi et al


























applications













on both substrates






materials






8112019 qazi et al







































this 1047297eld

References


















































8112019 qazi et al















































Aspects 201342951e


























8112019 qazi et al





































2011322821e














8112019 qazi et al

































[115]













































8112019 qazi et al


















the native heart























nano1047297





































8112019 qazi et al






















T and MHC


































































8112019 qazi et al







sheets and 1047297











[64]































growth factors














































8112019 qazi et al























and repair





























































































1047297bers







8112019 qazi et al













































in1047298






















3EABPANI content















8112019 qazi et al


























applications













on both substrates






materials






8112019 qazi et al







































this 1047297eld

References


















































8112019 qazi et al















































Aspects 201342951e


























8112019 qazi et al





































2011322821e














8112019 qazi et al


















the native heart























nano1047297





































8112019 qazi et al






















T and MHC


































































8112019 qazi et al







sheets and 1047297











[64]































growth factors














































8112019 qazi et al























and repair





























































































1047297bers







8112019 qazi et al













































in1047298






















3EABPANI content















8112019 qazi et al


























applications













on both substrates






materials






8112019 qazi et al







































this 1047297eld

References


















































8112019 qazi et al















































Aspects 201342951e


























8112019 qazi et al





































2011322821e














8112019 qazi et al






















T and MHC


































































8112019 qazi et al







sheets and 1047297











[64]































growth factors














































8112019 qazi et al























and repair





























































































1047297bers







8112019 qazi et al













































in1047298






















3EABPANI content















8112019 qazi et al


























applications













on both substrates






materials






8112019 qazi et al







































this 1047297eld

References


















































8112019 qazi et al















































Aspects 201342951e


























8112019 qazi et al





































2011322821e














8112019 qazi et al







sheets and 1047297











[64]































growth factors














































8112019 qazi et al























and repair





























































































1047297bers







8112019 qazi et al













































in1047298






















3EABPANI content















8112019 qazi et al


























applications













on both substrates






materials






8112019 qazi et al







































this 1047297eld

References


















































8112019 qazi et al















































Aspects 201342951e


























8112019 qazi et al





































2011322821e














8112019 qazi et al























and repair





























































































1047297bers







8112019 qazi et al













































in1047298






















3EABPANI content















8112019 qazi et al


























applications













on both substrates






materials






8112019 qazi et al







































this 1047297eld

References


















































8112019 qazi et al















































Aspects 201342951e


























8112019 qazi et al





































2011322821e














8112019 qazi et al













































in1047298






















3EABPANI content















8112019 qazi et al


























applications













on both substrates






materials






8112019 qazi et al







































this 1047297eld

References


















































8112019 qazi et al















































Aspects 201342951e


























8112019 qazi et al





































2011322821e














8112019 qazi et al


























applications













on both substrates






materials






8112019 qazi et al







































this 1047297eld

References


















































8112019 qazi et al















































Aspects 201342951e


























8112019 qazi et al





































2011322821e














8112019 qazi et al







































this 1047297eld

References


















































8112019 qazi et al















































Aspects 201342951e


























8112019 qazi et al





































2011322821e














8112019 qazi et al















































Aspects 201342951e


























8112019 qazi et al





































2011322821e














8112019 qazi et al





































2011322821e














Documents

qazi et al