conducting polymers 
 ( cps ) have gained popularity in recent years 
 as components of complex systems designed to electrically communicate 
 with biological environments .
indeed , 
 cps provide new ways to communicate with cells , tissues , and other 
 multicellular structures as they can avoid the mechanical mismatch 
 of harder inorganic based materials and are also able to exhibit ionic 
 in addition to electronic conductivity .
this is highlighted by the use of cps as active components across 
 a broad range of biotic / abiotic interfaces , with pioneering examples 
 including the electronic plant , organic 
 electrochemical transistor , neural cell 
 membrane mimicking electrode , and multiplex 
 immunoassay biosensor . among the many 
 forms they have been processed into , such as multilayered films , fibers , and sponges ,
cps are being considered as a structural material for the 
 fabrication of electroconductive hydrogels . in tissue engineering ,
mimicking the properties of the native extracellular 
 matrix such as its hydrated nature is essential . in the case of electro - responsive 
 organs such as the heart and nervous system
, the engineered scaffold 
 would also be required to be electrically conductive to reinstate 
 the electronic functionality to the tissue . nevertheless , the fabrication of electroconductive hydrogels based 
 on cps has proven challenging .
cps exhibit ( i ) high stiffness due 
 to their inherently rigid backbone containing conjugated double bonds , ( ii ) a hydrophobic nature due to the aromatic rings in the backbone , 
 and ( iii ) unwanted cross - links induced by  stacking 
 of the chains .
in contrast , a 3d hydrogel 
 network consists of cross - linked hydrophilic polymers with high water 
 content , exhibits elastic behavior , and has porous internal structures . here
, we report a new fabrication approach for a functional poly(ethylene 
 dioxythiophene ) ( pedot ) based hydrogel that overcomes the aforementioned 
 challenges . we chemically polymerized a pedot backbone functionalized 
 with ionic pendant groups to synthesize a water - soluble processable 
 conjugated polymer without compromising its electronic properties . 
 additionally , the side functionalities allowed postfunctionalization 
 of the polymer to be tailored for desired scaffold requirements .
copolymerizing 
 of the functional pedot with acrylic acid led to a 3d hydrogel with 
 high water content , a range of mechanical properties that match that 
 of a number of soft tissues , and electroactivity in physiological 
 conditions .
this is the first report of a conductive network built 
 from covalently linked conjugated and hydrophilic polymers .
initial approaches have shown that hydrogels could be made temporarily 
 conductive either by fabricating hybrid hydrogels in which the cp 
 is grown in a prefabricated network or as single 
 component nonhybrid conductive hydrogels where the cp is the sole polymeric component ; however , challenges 
 remain in their application as components of bioelectronic devices . 

hybrid hydrogels are comprised of cp chains that are physically or 
 ionically entrapped within the conventional hydrogel matrix which 
 limit their functionality in physiological conditions . as the hydrogel 
 swells at physiological ph , physically entrapped cp chains leach out , 
 which triggers toxicity and causes a drop in electroactivity .
ionically bound cps are neutralized at ph = 
 7.4 and eventually leach out of the network , which leads to the same 
 detrimental drop in properties .
nonhybrid 
 conductive hydrogels are developed by self - assembly of the polymeric 
 chains , chemical cross - linking , metal ions chelation , freeze - drying 
 of cp dispersions , or employing a low 
 molecular weight gelator ( lmwg ) . the end product 
 is a hydrogel network with enhanced electron transport between the 
 conjugated chains due to the absence of insulating polymers from the 
 matrix .
however , the chemistry currently employed does not allow for 
 the tuning of properties such as swelling or the mechanical characteristics 
 of these scaffolds and is not easily amenable for tailoring to accommodate 
 specific requirements in tissue engineering .
in particular , the stability 
 of hydrogels fabricated via self - assembly or lmwg in physiological 
 conditions has never been tested .
it could be envisioned that ionic 
 strength will have an effect on the physicochemical properties of 
 a network and its assembly .
a new strategy is presented in this study 
 to avoid the loss of the cp chains followed by a drop in the material s 
 electronic properties .
an ideal precursor for a conductive hydrogel 
 is a cp with the following 
 properties : ( i ) water solubility to enhance swelling capacity , ( ii ) 
 functional groups in the side chains that allow chemical cross - linking 
 to eliminate polymer leaching following swelling in ph 7.4 , and ( iii ) 
 electrical conductivity in physiological buffers . here , we report 
 a functional pedot with pendant carboxylic groups ( pedot - cooh ) that 
 meets all these requirements : water solubility , functionalizable with 
 demonstrated postmodification capabilities , and preserved electronic 
 properties upon gelation .
pedot with pendant carboxylic groups has 
 been previously reported but via electropolymerization ; this limits the scope of application since the 
 polymer can only be used on electrode surfaces .
reports on chemically 
 polymerized pedot have focused on alkylated , alkoxylated , or alkylsulfonate 
 derivatives of edot .
however , these substituents do 
 not offer the potential for the postfunctionalization of the polymer 
 and thus limit their application in fabricating scaffolds .
furthermore , 
 the functional carboxylic group ionizes at physiological ph ( 7.4 ) 
 and will allow enhanced swelling of the developed scaffolds .
we chose 
 pedot due to its high stability in aqueous solutions and its demonstrated biocompatibility . by synthesizing for the first time a functional pedot bearing 
 carboxylic groups via chemical polymerization
, we obtained an electroactive 
 polymer that could be cross - linked with hydrophilic polymers to form 
 3d scaffolds with retained electroactivity , high swelling ratios , 
 and soft mechanical properties .
hydroxymethyl edot ( edot - oh ) was purchased 
 from molekula ltd . , uk .
all other chemicals were purchased from sigma 
 uk and used without any further purification .
edot - oh ( 1.0 g ) was dissolved 
 in anhydrous terahydrofuran ( thf ) ( 25 ml ) under the flow of n2 , and the solution was cooled down by immersing the rb flask 
 in an ice bath .
potassium iodide ( 0.19 g ) and a nah suspension ( 0.278 
 g ) were added , and the solution was left stirring for 30 min .
methyl-3-bromoproprionate 
 ( 0.76 ml ) was then added dropwise , and the reaction was left stirring 
 under n2 for 48 h. deionized water ( di - h2o ) 
 was added to quench the reaction , and thf was removed under reduced 
 pressure .
the aqueous mixture was extracted with ethyl acetate , dried 
 over mgso4 , filtered , and concentrated under reduced pressure . 

the crude product was purified by flash chromatography ( hexane / ethyl 
 acetate = 5:1 ) to give a clear viscous liquid .
h nmr ( 400 
 mhz , cdcl3 ) :  6.32 ( 2h , s ) , 4.264.33 ( 1h , 
 m ) , 4.22 ( 1h , dd , j = 11.6 , 2.3 hz ) , 4.03 ( 1h , dd , j = 11.6 , 7.5 hz ) , 3.79 ( 2h , t , j = 6.3 
 hz ) , 3.67 ( 3h , s ) , 3.623.76 ( 2h , m ) , 2.62 ( 2h , t , j = 6.3 hz ) ( figure s1 ) .
the functional pedot polymer was synthesized 
 by chemical oxidation using fecl3 as the oxidant . in a 
 2-neck rb flask ,
fecl3 ( 1.45 g , 8.94 mmol ) and anhydrous 
 chloroform ( 32 ml ) were mixed under the flow of n2 .
edot - cooch3 ( 0.64 g , 2.48 mmol ) was dissolved in anhydrous chloroform 
 ( 6 ml ) and added dropwise to the oxidant mixture and left stirring 
 for 24 h at room temperature .
the mixture was poured in methanol ( 500 
 ml ) and centrifuged to collect the solid .
the precipitate was washed 
 thoroughly with methanol and then with water until the solvent was 
 clear . to remove the methyl group on the side chain ,
2 m naoh ( 50 ml ) for 1 day at room temperature and then 
 filtered to remove the insoluble parts .
the filtrate was purple in 
 color and treated with 10% hcl to precipitate the polymer .
the polymer 
 was washed with water and dried under vacuum to obtain a black solid 
 ( 100 mg ) .
the chemical structure of pedot - cooh 
 was confirmed using a bruker avance 111 solid state nmr spectrometer 
 operating at 75 mhz for c and 299.75 for h and fitted with a 4 mm cpmas probe .
sample ( 60 mg ) was 
 packed into a 4 mm zirconia oxide rotor .
spectra were acquired with 
 mas of 12 khz , using a spin echo sequence allowing for direct c detection with high power h decoupling with 
 spinal 64 sequence at 62 khz .
the relaxation delay was set to 20 s , 
 and total instrument time was 13 h. cross - polarization failed 
 to give any signal .
pedot - cooh ( 4.9 
 mg ) was dissolved 
 in dmso - d6 ( 770.93 mg ) , and its nmr spectroscopy 
 was recorded using a bruker avance 111 , hd 600 with 5 mm tci cryoprobe . 

gpc was performed 
 using phosphate buffered saline ( pbs , 0.1 m ) ( flow rate : 0.5 ml min ) .
a shimadzu modular system was employed comprising 
 a dgu-20a3 solvent degasser , an lc-20at pump , a cto-20a column oven 
 operated at 35 c , and a spd - m20a diode array uv  vis detector 
 ( 200800 nm ) .
the system was equipped with an ultrahydrogel 
 250 column ( 6 m , 7.8  300 mm ) ( waters , wat01152 ) .
calibration 
 was achieved using sodium poly(styrenesulfonate ) standards ( polymer 
 laboratories ltd , uk ) ranging from 1400 to 32  000 g mol .
drop - casted films 
 of pedot - cooh were 
 chemically doped by treating the surface with 0.1 m hclo4 aqueous solution for 24 h. excess dopant was wiped gently off the 
 surface , and the films were rinsed for 30 s with di - h2o , 
 dried with n2 , and stored in the dark until use .
absorbance spectra were 
 recorded using a perkinelmer lambda 25 uv  vis spectrophotometer . 

the spectra of pedot - cooh dissolved in water , 1 m naoh , and dimethyl 
 sulfoxide ( dmso ) as well as that of drop - casted films ( doped and undoped ) 
 were recorded .
the optical band gap , eg , opt , was estimated from the uv  vis spectra of undoped pedot - cooh 
 film . the linear portion of the curve was extrapolated to the wavelength 
 axis , and the intercept was determined to be the absorption edge wavelength , 
 ab .
eg , opt was calculated 
 as follows : where h is the planck constant 
 and c is the speed of light . in situ
spectroelectrochemical 
 measurements were conducted on undoped pedot - cooh films prepared by 
 drop casting on ito slides ( 1  4 cm ) .
the electrolyte solution 
 was 0.1 m tetrabutylammonium tetrafluoroborate ( tbab ) in acetonitrile . 

the applied voltages on the ito modified electrode were rescaled vs 
 ag / agcl by using ferrocene .
the controlled - potential measurements 
 were carried out with an edaq system controlled by echem software .
the counter electrode was 
 a platinum mesh , and the reference electrode was ag / agcl in saturated 
 kcl aqueous solution ( chi instruments ) .
the working electrode was 
 ito coated glass ( delta technologies limited ) on which the pedot - cooh 
 polymer was drop casted .
electrochemical measurements were performed 
 at different scan rates using 0.1 m tbab in acetonitrile as the electrolyte 
 solution .
lumo levels 
 were determined as follows : to introduce 
 a double bond 
 in the side chain of pedot - cooh , the polymer was reacted with n-(3-aminopropyl ) methacrylamide hydrochloride ( apma ) .
pedot - cooh 
 ( 0.355 g ) was dissolved in dmso ( 30 ml ) at 60 c and then cooled 
 to room temperature .
apma ( 0.117 g ) and n-(3-(dimethylamino)propyl)-n-ethylcarbodiimide hydrochloride ( edc ) ( 0.249 g ) 
 were added , and the reaction was left to stir for 24 h. to wash out 
 all the byproducts and unreacted monomer ( apma ) , the solution was 
 poured into excess water , which led to the precipitation of a black 
 solid . the black solid was collected by centrifugation , washed repeatedly 
 with water , and dried under vacuum to give f - pedot in powder form .
f - pedot ( 5.0 mg ) was dissolved in 
 dmso - d6 ( 768.25 mg ) , and its nmr spectroscopy 
 was recorded as described above .
the degree of substitution of apma 
 was quantified using an external reference ( trioxane ( 10.5 mg ) in 
 dmso - d6 ( 771.65 mg ) ) and using topspin 
 eretic software .
elemental analysis 
 was performed 
 at campbell microanalytical laboratory , university of otago , new zealand . 

samples were precisely weighed on a mettler umt2 microbalance into 
 lightweight tin capsules and dropped into a combustion tube of the 
 elemental analyzer ( carlo erba ea 1108 ) through which a constant stream 
 of helium is maintained .
total carbon , hydrogen , nitrogen , and sulfur 
 were determined for both pedot - coona and f - pedot . to fabricate a cross - linked functional 
 pedot hydrogel , f - pedot was copolymerized with acrylic acid ( aa ) in 
 the presence of poly(ethylene glycol ) diacrylate ( peg - da , average 
 molecular number ( mn ) = 700 ) as a cross - linker 
 and 2,2-azobis(2-methyl - proprionitrile ) ( aibn ) as the initiator . 
 in a typical procedure , f - pedot ( 5 mg )
was dissolved in dmso ( 1 ml ) 
 at 60 c to a final concentration of 0.5 wt % polymer . the solution 
 was then cooled to room temperature .
aa ( 95.2 l ) , peg - da ( 10 
 l ) , and aibn ( 2 mg ) were added to the polymer solution , and 
 it was vortexed and left at 60 c for 24 h. gelation of the solutions 
 was confirmed by the test tube inverting method .
the vials containing 
 the mixture were inverted , and gelation was deemed to have occurred 
 when no flow of liquid was observed .
the hydrogels were then removed 
 from the vials , washed with excess 10% ( w / v ) hcl for 1 day , and then 
 soaked in excess di - h2o for 3 days .
poly(acrylic acid ) ( paa ) hydrogels 
 were fabricated according to the same procedure but without adding 
 f - pedot in the solution .
the dry weights of freeze - dried hydrogels 
 were recorded ( md ) before incubating the 
 samples in pbs ( 0.1 m , ph = 7.3 , 37 c ) and then leaving them 
 to swell until equilibrium was reached . at predetermined time points , 

the samples were removed and gently blotted , and their swollen weight 
 ( ms ) was recorded .
the percentage - swelling 
 ratio was calculated according to the following equation : a total of 9 specimens were measured 
 at each 
 time point .
the compression 
 modulus of cylindrical 
 hydrogels was measured at room temperature using a bose instrument 
 equipped with a 3 n load cell .
samples were removed from the swelling 
 media and blotted dry to get rid of excess water . the thickness and 
 radius ( r ) of each sample
the samples 
 were placed between parallel plates and compressed at 0.1%/s strain 
 rate .
the strain under compression was defined as the change in 
 the thickness relative to the thickness of the freestanding specimen . 

the compression modulus was calculated from the slope of the linear 
 region of the stress 
results were reported for hydrogels in two different states : swollen 
 in pbs ( n = 5 ) and hclo4-doped hydrogels 
 ( n = 4 ) .
hydrogels were 
 soaked in 0.1 m hclo4 aqueous solution for 24 h. this was 
 followed by rinsing with water and freeze - drying the samples .
freeze - dried hydrogels ( undoped and 
 doped ) were anchored on gold mylar surfaces and soaked in pbs for 
 1015 min to allow for water uptake .
cvs were then recorded 
 as described above but using pbs ( ph = 7.4 ) as the electrolyte .
the electrochemical impedance ( eis ) spectra 
 of paa ( control ) and f - pedot hydrogels were measured using solartron 
 si 1287 electrochemical interface coupled with an si 1260 frequency 
 response analyzer ( solectron analytical , hampshire , england ) from 
 10 khz to 1 hz with an applied ac potential of 10 mv versus the reference 
 electrode potential . before recording the measurements , hydrogels 
 were anchored on gold mylar and incubated in pbs for 24 h to ensure 
 swelling to equilibrium .
ag / agcl and pt electrodes were used as the 
 reference and counter electrodes , respectively .
the applied dc voltage 
 was 290 mv , and the electrolyte solution was pbs ( 0.1 m ) .
cells were maintained in dulbecco s modified eagle s 
 medium ( dmem ) supplemented with 20% ( v / v ) fetal bovine serum ( fbs ) 
 and antibiotics ( 100 units / ml penicillin and 100 g / ml streptomycin ) , 
 under standard mammalian cell culture conditions .
the high concentration 
 of fbs provides a rich source of mitogenic growth factors required 
 for optimal cell growth and proliferation while the addition of antibiotics 
 prevents the growth of potential bacterial contaminants .
hydrogels were transferred 
 into 24 well 
 plates and sterilized by cell culture - grade uv light irradiation for 
 1 h. the samples were then soaked in di - h2o overnight prior 
 to cell seeding in order to wash excess acid from the doped samples . 
 the next day , to meet the required environment for cell culture , water 
 was removed from the wells and replaced with media for 1 h which was 
 then removed before seeding the cells .
c2c12 cells were detached from 
 the culture plates using trypsin , followed by centrifugation ( 6000 g , 5 min ) .
cells ( 1.5  10 cells in 30 l ) were dispensed 
 on the surface of the hydrogels and left for 1 h to allow for adhesion . 

adherence of c2c12s to the surface is necessary for the cells to remain 
 viable and for all cell function ( cell division , migration , and maturation ) . 

the wells were then filled with growth media and allowed to proliferate 
 for 3 days .
although the greatest care was taken , some cells could have 
 been dislodged during this process .
the cells attached to the hydrogels 
 were then placed in a differentiating media ( dmem with 2% ( v / v ) horse 
 serum , 100 units / ml penicillin , and 100 g / ml streptomycin antibiotics ) 
 and incubated for 5 days .
horse serum was included in the differentiation 
 medium to provide a minimal level of nutrients which are needed to 
 keep the cells healthy and viable . at various time points ,
differentiation 
 media was removed and the cells / hydrogels were washed in pbs before 
 being submerged in freshly prepared fixation buffer ( 4% ( v / v ) paraformaldehyde 
 in pbs ) for 10 min at room temperature .
fixed hydrogels were 
 cut into 250 m 
 cross - sectional and longitudinal slices using a high precision vibrating 
 microtome ( 7000 smz , campden instruments ltd . , uk ) .
the slices were 
 blocked in 5% ( v / v ) fbs in pbs for 2 h and then incubated overnight 
 at 4 c with primary antibodies that specifically bind to the 
 proteins ki67 and desmin ( abcam ; ab8470 and ab15580 ) or myosin heavy 
 chain iib ( mhc iib , clone mf20 ; rnd systems ) .
unbound primary antibodies 
 were removed by 3  20 min washes in pbs . to visualize the bound 
 antibodies ,
the cells were incubated in secondary antibodies labeled 
 with fluorophores ( goat antimouse alexa 488 and goat antirabbit alexa 
 488 ; invitrogen ) .
for some experiments , cells / hydrogels were also 
 incubated in alexa fluor 594-phalloidin ( invitrogen ) .
phalloidin is 
 a peptide that binds to actin in cells and allows visualization of 
 cell shape .
we achieved the synthesis of pedot - cooh ( scheme 1a ) by oxidation polymerization 
 of edot - cooch3 ( figure s1 ) followed 
 by deprotection 
 in naoh ; this yielded a polymer with carboxylic groups in the backbone 
 ( figure s2a ) .
the chemical structure of 
 the polymer was also confirmed by solution nmr ( broadened peaks , figure s2b ) and elemental analysis ( table s2 ) .
the resultant polymer had a weight - average 
 molecular weight ( mw ) of 10  102 
 gmole   and a polydispersity of 2.49 as determined 
 by gpc .
the polymer was soluble in dmso ( 1 wt % ) , water ( 0.6 
 wt % ) , and basic aqueous ph solutions ( 10 wt % at ph 12 ) .
this conferred 
 solubility provides an advantage over the more commonly used form 
 of pedot , which is a dispersion in water stabilized with high concentrations 
 of poly(styrenesulfonate ) ( pss ) .
figures 1a and s3 show 
 the absorption spectra of pedot - cooh film and solutions , respectively . 

a broad absorption band , max  519 nm for 
 the films and 480 nm for solutions , was detected corresponding to 
 the  * transition in the conjugated backbone .
the optical band gap ( eg , opt ) determined from the film spectra was 1.70  0.09 
 ev .
this value is in the range ( 1.52.0 ev ) of other chemically 
 synthesized derivatives of pedot , which indicated that edot - cooch3 polymerization 
 proceeded efficiently to produce a functional pedot polymer with sufficient 
 conjugation length .
( a ) spectroelectrochemistry of a pedot - cooh film drop - casted on ito - coated 
 glass as a function of applied potential between 0.54 and 
 0.86 v. ( b ) uv  vis absorption spectra of reduced (  ) 
 and hclo4 doped ( - - - ) pedot - cooh films .
( c ) cyclic voltammogram 
 ( 25 mvs ) of pedot - cooh film drop - casted 
 on ito - coated glass in tbab acetonitrile solution ( 0.1 m ) .
we conducted in situ spectroelectrochemistry to 
 elucidate the electronic 
 properties of the modified pedot ( figure 1a ) . in the absence of a bias ,
the film exhibited 
 a broad peak at 513 nm , which suggested that the film was 
 in the anticipated reduced state following treatment in naoh .
as the 
 potential exceeded 0 v , the intensity of the peak decreased along 
 with the appearance of a new absorption feature in the near - infrared 
 region ( > 800 nm ) , which is representative of polaronic states . 

this 
 suggested a substantial change in the oxidation state of the pedot - cooh 
 film from reduced to oxidized as the positive bias was applied .
chemical 
 treatment of the films with hclo4 caused a similar switch 
 between the reduced ( undoped ) and oxidized form ( doped ) ( figure 1b ) .
hence , it could 
 be inferred that the pedot - cooh is a conjugated polymer with intrinsic 
 electroactivity . to further confirm the conjugated nature of 
 the polymer , we examined 
 the electrochemical behavior of the modified pedot in tetrabutylammonium 
 tetrafluoroborate ( tbab ) electrolyte solution ( figure 1c ) .
the film exhibited good redox activity 
 with a broad anodic ( 0.3140.735 v ) and a broad cathodic peak 
 ( 0.320.19 v ) .
this indicated that the electron transfer ability 
 of the chemically polymerized pedot was not hindered by the carboxylic 
 group in the side chain .
a linear relation was also established between 
 the peak current and the potential scan rate ( figure s4 ) , which denoted that the electron transfer process 
 was not diffusion limited and that the pedot 
 film was in intimate contact with the ito electrode . from the experimental 
 results of cyclic voltammetry ( cv ) and uv spectrophotometry , the calculated 
 values of the homo and lumo of pedot - cooh were 5.28 and 3.51 
 ev , respectively , which was comparable to other reported values for 
 pedot polymers . introducing a functional 
 group in the side chain of pedot enabled 
 postfunctionalization of the polymer .
we reacted pedot - cooh with n-(3-aminopropyl ) methacrylamide hydrochloride ( apma ) to 
 introduce a double bond in the chain ( f - pedot ) ( figures s5 and s6a ) . using an external reference ( figure s6b ) ,
this was confirmed by elemental analysis that 
 showed the presence of nitrogen in f - pedot as opposed to pedot - cooh 
 ( table s2 ) and a degree of substitution 
 approximated at 16 wt % .
f - pedot was then polymerized for 
 24 h with acrylic acid ( aa ) and poly(ethylene 
 glycol ) diacrylate ( peg - da ) to fabricate a 3d cross - linked gel comprised 
 of poly(acrylic acid ) ( paa ) chains covalently linked to f - pedot via 
 the peg - da ( scheme 1b ) .
each of the three components works synergistically to produce 
 a conductive 3d hydrogel , namely : paa is a polyelectrolyte that ionizes 
 in physiological ph and causes water uptake ; peg - da is a cross - linker 
 that covalently binds the polymeric chains to produce the 3d structure ; 
 pedot is a conjugated polymer that introduces redox and electronic 
 activity into the scaffold .
the gel was removed from the glass 
 vial and sectioned into specimens 
 5 mm in thickness .
the gel is dark in color ( scheme 1b ) . while this could be a limiting factor 
 for external bioapplications , many of the electroresponsive tissues 
 that could benefit from the conductive gel are internal organs such 
 as the heart , spinal cord , or the brain .
the specimens were washed 
 repeatedly with 10% hcl , which ensured the exchange of dmso with acidic 
 water without causing uncontrolled swelling of the gels if soaked 
 immediately in water .
the specimens were then transferred into excess 
 di - h2o and left to soak for 48 h. of significance here 
 is that the chemically produced pedot can be functionalized with a 
 plethora of compounds other than apma or copolymerized with other 
 monomers or polymers bearing double bonds .
considering the hydrophobic 
 nature of the f - pedot backbone due 
 to the presence of aromatic rings , it was important to investigate 
 whether the cp in the network hindered the swelling behavior of the 
 hydrogel .
freeze - dried specimens were incubated in pbs ( ph = 7.4 at 
 37 c ) and weighed at different times .
hydrogels were found to 
 exhibit a remarkable water uptake after 48 h ( 5000% ) , which 
 was comparable to the swelling ratio of other paa copolymer hydrogels 
 ( figure 2a ) .
the swelling of the hydrogels was due to the 
 ionization at ph = 7.4 of the pendant carboxylic groups present on 
 both polymers ( paa and f - pedot ) .
as the proton of the carboxylic groups 
 dissociates in ph = 7.4 , electrostatic repulsion between the polymer 
 chains caused the network to uptake water .
compared to previously 
 reported conductive hydrogels , our f - pedot hydrogel displayed significantly higher ratio of swelling , 
 which is an attractive property for applications in tissue engineering 
 and drug delivery . with their high water content ,
strong 
 water uptake promises for efficient mass transfer , which is advantageous 
 for applications of the hydrogel in cell proliferation and tissue 
 formation .
( b ) young s 
 modulus of hydrogel networks swollen in different media ( p < 0.005 ) .
it is well established 
 that the stiffness of any scaffold dictates 
 the cell  biomaterial interaction .
since the conductive hydrogel is designed to function as a scaffold 
 for cell seeding and attachment , we characterized its mechanical properties . 

additionally , the redox state of the polymer ( doped versus undoped ) 
 might alter the mechanical properties of the network ; hence , we measured 
 the compressive moduli of both doped and undoped scaffolds ( figure 2b ) .
the young modulus 
 of pbs treated samples was 8.2  3.1 kpa , respectively .
in contrast , 
 doped hydrogels displayed an increased stiffness ( 45.14  18.78 
 kpa , p < 0.005 , tukey s multiple comparison 
 test , one - way anova ) . monitoring the swelling of the samples in the 
 dopant solution ( 0.1 m hclo4 ) revealed minimal water uptake 
 compared to those in pbs ( figure s7 ) . in 
 a low ph media ,
the carboxylic groups are protonated and the polymeric 
 chains come into closer vicinity , which could lead to secondary bonds 
 between the aromatic rings , and in turn increases the mechanical strength 
 of the network .
having established that the f - pedot hydrogel 
 displayed desirable 
 physical characteristics of a network applicable to tissue engineering 
 ( high swelling ratios and mechanical integrity ) , we characterized 
 its electronic properties to examine whether f - pedot retained its 
 electroactivity in the presence of the insulators paa and peg - da . 

redox activity of the scaffold is important to show that the material 
 being designed is capable of providing an electrical stimulus , which 
 will ultimately allow for direct stimulation of cells and the transduction 
 of biological signals to be promising .
figure 3a shows the electrochemistry of the f - pedot 
 hydrogel in pbs as an electrolyte .
the cvs exhibited characteristic 
 features comparable to those of the pedot - cooh polymer ( figure 1c ) ; broad anodic and reduction 
 peaks at 0.48 and 0.35 v , respectively , with a half - wave 
 potential , e1/2 , of 0.415 v , which indicated 
 that a clear effective charge transport is occurring in the f - pedot 
 hydrogel despite the swollen state and the presence of insulating 
 polymers ( paa and peg ) in the network .
furthermore , hydrogels subject 
 to repeated cycles exhibited a stable electrochemical response ( figure s8 ) .
( b ) doped hydrogels in 
 0.1 m pbs conducted at different potential scan rates .
after doping the hydrogels with 0.1 m hclo4 , we observed 
 a shift in the e1/2 to lower potentials 
 ( e1/2= 0.29 v ; anodic peak at 0.31 v and 
 cathodic peak at 0.27 v ) ( figure 3b ) .
this indicated that the oxidation of the doped 
 network required a lower potential compared to the undoped network . 

the cvs of the undoped system also showed a quasi - reversible process 
 with oxidation and reduction peak separation increasing with potential 
 scan rate .
following doping , the cvs became more reversible ( figure 3b ) , which indicated 
 a faster charge transport kinetics due to the higher electrical conductivity 
 in the doped system .
we could also infer from the linear relation 
 between the potential 
 scan rate and anodic peak current that both the doped and undoped 
 hydrogels were in good contact with the electrode surface ( figure s9 ) .
in contrast , paa hydrogels displayed 
 no redox activity as shown by the absence of oxidation / reduction waves 
 in their cyclic voltammogram ( figure s10 ) .
the redox activity of the doped hydrogels was further confirmed 
 by electrochemical impedance spectroscopy ( eis ) conducted at 290 mv 
 ( figure s11 ) . unlike the nyquist plot of 
 the paa system ,
the nyquist plot of the doped hydrogel showed a quasi - semicircular 
 arc in the high frequency regime ( inset figure s11 ) .
this implies a redox activity as opposed to a capacitive 
 behavior of the nonredox active paa system .
this was further evidence 
 that the modified pedot scaffold retained its electroactive properties 
 in aqueous electrolytes and despite the attachment of functional groups 
 to its side chains .
envisioned applications of the pedot - cooh 
 as a component of bioelectronic 
 devices will require the polymer to be in contact with electroresponsive 
 tissue . additionally , the conductive and electroactive nature of our 
 developed f - pedot hydrogel in the swollen state , along with its favorable 
 swelling ratio and mechanical integrity , make it suitable as a 3d - scaffold 
 for the proliferation and differentiation of electro - responsive cells . 

c2c12 cells are a mouse myoblast cell line commonly used as an in vitro model to study muscle differentiation .
we seeded 
 c2c12 cells on both undoped and doped scaffolds and assessed qualitatively 
 for adhesion and viability at day 3 .
as shown in figure s12 , the f - pedot hydrogels promoted cell adhesion without 
 the need to precoat the scaffold with any cell - adhesion protein ( such 
 as fibronectin or laminin ) .
significantly , other conjugated based 
 systems normally require modification of their surfaces with adhesive 
 molecules to enhance their cytocompatibility and cell attachment . 
 of particular interest
was the migration of the cells from the surface 
 into the 3d highly swollen and porous structure of the hydrogels ( figure s13 ) .
we found that cells penetrated to 
 a depth of 2 mm , which confirmed the cytocompatibility of 
 the conductive hydrogel ( figure s13e 
the structural composition of the c2c12 cells was confirmed by 
 phalloidin stain for actin ( figure 4a , b ) .
actin is a critical component in many structural 
 and functional processes in the cell .
the phalloidin staining confirmed 
 that the structural network remained undamaged on both doped and undoped 
 hydrogels .
proliferation of c2c12 cells was confirmed by ki67 staining 
 on both undoped and doped hydrogels ( figure 4a , b ) .
the ki67 protein is present in the 
 cell nucleus during all active phases of the cell cycle ( g1 , s , g2 , 
 and mitosis ) but is absent from resting cells ( g0 ) .
detection of the 
 ki67 protein by immunostaining indicates that the cells are proliferating ; 
 thus , the hydrogels support muscle cell expansion . when c2c12 myoblasts , 
 immature muscle cells , are grown to a high density
, the cells are placed 
 in low serum conditions as the growth factor withdrawal initiates 
 a differentiation program . in this multistep process ,
the myoblasts 
 stop proliferating , elongate , and align next to each other before 
 they are then in a position to fuse together to form multinucleated 
 tubes capable of contraction .
cells grown in the hydrogel were observed 
 to align at day 3 ( figures 4c and s12 and s14 ) , and multinucleated 
 tubes were present by day 7 ( figure 4c ) indicating that cells were able to differentiate 
 in the hydrogels .
in addition to morphological changes , maturing muscle 
 myoblasts such as c2c12s begin to produce proteins required for their 
 contractile function , such as mhc iib ( figure 4c ) .
mhc iib is a motor protein that generates 
 force to drive muscle contraction and is an essential component of 
 functional muscle .
the presence of mhc iib in cells grown 
 in both doped and undoped hydrogels shows that both forms of the hydrogel 
 are conducive to the maturation of muscle cells .
c2c12 cultured in f - pedot 
 hydrogel ; proliferation of cells in undoped 
 ( a ) and doped hydrogels ( b ) at day 3 .
proliferation is indicated by 
 positive nuclear ki67 staining ( green ) , while phalloidin staining 
 for actin ( red ) allows visualization of the cell shape .
c ) staining for the mature muscle 
 marker myosin heavy chain iib ( mhc iib ) ( green ) and dapi ( blue ) in 
 undoped and doped hydrogel at day 3 and day 7 .
the presence of mhciib 
 shows that the c2c12s , which are immature muscle cells and normally 
 have low expression of mhciib , are becoming mature muscle cells when 
 seeded on the hydrogel .
while the c2c12s exist as single cells at 
 day 3 , multicellular tubes are observed by day 7 showing increased 
 maturation with continued culture time . in summary , we found that c2c12 cells grown in both the doped 
 and 
 undoped samples formed myotube structures and expressed mhc iib ( figure 4c ) indicating that 
 they were differentiating . coupled with their expression of ki67 ( figure 4a ,
b ) , this showed 
 that our hydrogels were capable of sustaining the differentiation 
 of c2c12 cells in addition to their proliferating precursors .
this in vitro study was conducted as evidence of biocompatibility 
 of the newly fabricated pedot based hydrogels .
future studies will 
 investigate the mechanistic insight into the role of the hydrogel 
 on cellular differentiation .
in this study , we successfully 
 produced an electroactive hydrogel 
 network composed of pedot covalently bound to a hydrophilic polymer . 

this led to the formation of a scaffold combining both the appropriate 
 physical properties of a hydrogel and the electronic characteristics 
 of a conjugated polymer .
we achieved this by synthesizing a processable 
 pedot with functional groups tethered on the side chains that could 
 be further modified to fabricate a covalently linked polymeric network . 

the pedot - cooh was shown to be electroactive as both a pristine polymer 
 and a component of the hydrogel .
finally , we demonstrated the suitability 
 of these scaffolds as hydrated 3d structures for cell adhesion , proliferation , 
 and differentiation . the next step will be to employ these electroactive 
 hydrogels in bioelectronics devices and test their capacity to either 
 electrically stimulate electroresponsive cells or probe the bioelectric 
 signals .
covalently linking pedot with hydrophilic polymers to produce 
 scaffolds of tailored properties represents a major advance in tissue 
 engineering that paves the way for the smart design of conductive 
 functional scaffolds .
 poly(ethylene 
 dioxythiophene ) with functional pendant groups bearing 
 double bonds is synthesized and employed for the fabrication of electroactive 
 hydrogels with advantageous characteristics : covalently cross - linked 
 porous 3d scaffolds with notable swelling ratio , appropriate mechanical 
 properties , electroactivity in physiological conditions , and suitability 
 for proliferation and differentiation of c2c12 cells . 
 this is a new 
 approach for the fabrication of conductive engineered constructs . 
Introduction
Experimental
Section
Results
and Discussion
Conclusion