6670 | J. Mater. Chem. B, 2016, 4, 6670--6679 This journal is©The Royal Society of Chemistry 2016

Cite this: J.Mater. Chem. B, 2016,4, 6670

Polypyrrole-coated poly(L-lactic acid-co-e-caprolactone)/silk fibroin nanofibrous membranespromoting neural cell proliferation anddifferentiation with electrical stimulation†

Binbin Sun,‡a Tong Wu,‡a Juan Wang,a Dawei Li,a Jing Wang,a Qiang Gao,a

M. Aqeel Bhutto,a Hany El-Hamshary,bc Salem S. Al-Deyabc and Xiumei Mo*a

Polypyrrole (Ppy), as a conductive polymer, is commonly used for nerve tissue engineering because of

its good conductivity and non-cytotoxicity. To avoid the inconvenience of Ppy processing, it was coated

on electrospun poly(L-lactic acid-co-e-caprolactone)/silk fibroin (PLCL/SF) nanofibers via the in situ

oxidative polymerization of pyrrole monomers in this study. Ppy-coated PLCL/SF membranes were

characterized by scanning electron microscopy (SEM), atomic force microscopy (AFM), Fourier transform

infrared spectroscopy (FTIR), X-ray diffraction (XRD), and thermogravimetric (TG) analysis. The results

confirmed the disposition of Ppy on the PLCL/SF nanofibers, and the nanofibers kept their nanofibrous

morphology and thermal stability, in comparison to the untreated ones. The conductivities and

water contact angles were evaluated as well, and indicated that the conductivity and hydrophilicity of

Ppy-coated nanofibers were increased. Furthermore, this study showed that electrical stimulation (ES)

promoted PC12 cell differentiation and axonal extension on Ppy-coated nanofibers. The MTT assay

suggested that both Ppy and ES could promote Schwann cell (SC) proliferation. Immunofluorescence

staining and real time-qPCR (RT-qPCR) testing demonstrated that ES could induce PC12 cell differentiation

even without nerve growth factor (NGF) treatment, and moreover, Ppy coating increased the inducing

effects on PC12 cell differentiation. The overall results indicated the promising potential of Ppy-coated

PLCL/SF nanofibrous membranes for peripheral nerve repair and regeneration.

1. Introduction

Conductive polymers, as a novel generation of organic materials,were first synthesized in the mid-1970s. In the past two decades,conductive polymers (such as: polypyrrole (Ppy), polyaniline(PANi), polythiophene (PT) and poly(3,4-ethylenedioxythiophene)(PEDOT)) have exhibited excellent electrical and optical proper-ties, which have been explored in a number of applicationsincluding actuators, battery technology, light-emitting diodesand some other microelectronics industries.1,2 Recently, moreconductive polymers have also exhibited significant potential

for tissue engineering applications, because lots of biologicaltissues exhibit electrical activity for modulating cellular fatesand behaviors. As reported, electrical stimulation can promotecell proliferation and differentiation via conductive materials,and then promote tissue regeneration and repair, i.e. of neural,bone, cardiac and muscle tissues,3,4 indicating the importanceof conductive polymers for tissue engineering.

However, these conductive polymers had poor biocompatibility,solubility and processability, as well as high hydrophobicity anduncontrollable mechanical properties,5–7 limiting their wide appli-cation for tissue engineering. Ppy has been extensively studiedfor biological and medical applications, because of its goodbiocompatibility and conductivity, but this conductive polymeris insoluble, infusible and brittle, causing poor processabilityfor tissue engineering scaffolds via traditional technology (e.g.electrospinning, three-dimensional printing, phase separation).8

Electrospinning is a commonly used method to prepare tissueengineering scaffolds for nerves, skin, vascular tissue, bone andso on. The electrospun scaffolds can offer an ECM-mimickingnano structure with a high surface area and porosity, and mostimportantly, various polymers (including natural polymers and

a State Key Lab for Modification of Chemical Fibers and Polymer Materials,

College of Chemistry, Chemical Engineering and Biotechnology,

Donghua University, 2999 North Renmin Road, Shanghai, 201620,

People’s Republic of China. E-mail: xmm@dhu.edu.cnb Department of Chemistry, College of Science, King Saud University, Riyadh 11451,

Kingdom of Saudi Arabiac Departments of Chemistry, Faculty of Science, Tanta University, Tanta 31527,

Egypt

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6tb01710j‡ Binbin Sun and Tong Wu contributed equally to this work.

Received 9th July 2016,Accepted 22nd September 2016

DOI: 10.1039/c6tb01710j

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synthetic polymers) can be used for electrospinning. Presentstudies have focused on using coating methods via in situpolymerization to solve the non-electrospinning problem ofPpy. Specifically, Ppy coating can be achieved either by in situchemical oxidation or by deposition on the electrospun scaffoldsin the vapor phase followed by oxidant treatment. For example,Lee et al. deposited Ppy on electrospun poly(lactic-co-glycolicacid) (PLGA) nanofibers for in vitro cytocompatibility studies,and it was found that the electrospun PLGA scaffold not onlyshowed good conductivity, but also kept the nanofiber morphologyafter coating with Ppy.9

From previous studies, neurite outgrowth, axon regenera-tion, neural repair and even functional recovery in clinicalsituations are known to benefit from electrical stimulation.10

Electrical stimulation (ES) plays an important role in nerve tissueengineering. Numerous studies have suggested that electricalstimulation can promote Schwann cell (SC) proliferation, ratpheochromocytoma (PC12) cell differentiation, axonal regenerationand nerve tissue repair.11–13 Schmidt and colleagues electricallystimulated PC12 cells on Ppy films and confirmed the promotion ofneurite outgrowth, demonstrating that the conductive polymer canpromote PC12 differentiation with ES.14 However, in that research,the PC12 cells were treated with nerve growth factor (NGF) beforethe ES experiment, which means ES can promote but not inducethe differentiation of PC12 cells. In Zhang’s study, the conductivepolymer PANi was blended with biodegradable materials to prepareelectrospun nanofibrous films. After culturing PC12 cells withES, PANi induced PC12 differentiation with ES without NGFtreatment.15 More importantly, the study of ES for inducingPC12 cell differentiation with a Ppy conductive polymer andwithout NGF treatment has not been explored yet.

Herein, we proceeded to use the promising electrospun compo-site poly(L-lactic acid-co-e-caprolactone) (PLCL)/silk fibroin (SF)(PLCL/SF), which has been proven to have good potential forapplications in nerve tissue engineering.16,17 The Ppy-coated electro-spun PLCL/SF nanofiber membranes were produced by in situpolymerization, and the morphology, conductivity, mechanical,spectroscopic and thermal properties were investigated.Biocompatibility was studied by culturing SCs on the membranes.We also performed the electrical stimulation of PC12 cellswithout NGF treatment on Ppy-coated nanofibers with differentelectrical conductivities to investigate whether conductivitycould induce PC12 cell differentiation behavior.

2. Materials and methods2.1 Materials

The copolymer poly(L-lactic acid-co-e-caprolactone) (PLCL,Mw = 300 kDa, LA : CL = 50 : 50) was purchased from JinanDaigang CO. Ltd (China). Bombyx mori silkworm cocoons weresupplied by Jiaxing Silk Co. Ltd (China) and the regenerated silkfibroin (SF) was prepared as previously reported.18 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was purchased from ShanghaiDarui CO. Ltd (China). Pyrrole, iron(III) chloride (FeCl3) andsodium para-toluene sulfonate (pTS) were purchased from

Sigma (St Louis, MO, USA). All of the cell culture reagents werepurchased from Hyclone (Logan, UT, USA). Rat pheochromo-cytoma cells (PC12) and Schwann cells (SCs) were obtainedfrom the Shanghai Institute of Biochemistry and Cell Biology(SIBCB, CAS, China).

2.2 PLCL/SF nanofiber fabrication

At first, the electrospinning solution (w/v (%) = 8) was preparedby dissolving PLCL/SF (w/w (%) = 75 : 25) in HFIP and stirred atroom temperature for 4 h. Then, the PLCL/SF solution waselectrospun with a syringe equipped with a 21G steel needleusing a voltage of 12 kV (BGG6-358, BMEI Co. Ltd, China). Theelectrospun solution was placed in a plastic syringe and thesyringe was loaded in a syringe pump (789100C, Cole-ParmerInstruments, USA) operating at a rate of 1.5 mL h�1. Aluminumfoil was used as the collector, and the distance betweencollector and steel needle tip was 10 cm. After electrospinning,PLCL/SF nanofiber membranes were dried for 12 h in a vacuumoven. Finally, the prepared PLCL/SF membranes were cut into20 � 20 mm2 pieces.

2.3 Polypyrrole coating via in situ polymerization

The mother solutions of pyrrole, FeCl3 and pTS (the concentra-tions were 2 M) were prepared before the in situ polymerizationreaction. The previously prepared 20 � 20 mm2 membraneswere put into a 10 mL aqueous solution with 25 mL, 37.5 mL and50 mL of pyrrole mother solution. Then the FeCl3 and pTSmother solutions which were used as the oxidizer and dopant,respectively, were added to the reaction system (the volumeratio of pyrrole : FeCl3 : pTS = 1 : 2 : 1). The polymerization anddeposition of polypyrrole (Ppy) on the PLCL/SF nanofibermembranes was achieved under shaking at 4 1C for 12 h.Eventually, the conductive nanofiber membranes were washedwith deionized water and ethanol three times and dried in avacuum oven at room temperature for 12 h. Ppy coated PLCL/SFmembranes with different Ppy amounts were named as PLCL/SF-Ppy-1, PLCL/SF-Ppy-2 and PLCL/SF-Ppy-3.

2.4 Characterization of Ppy coated PLCL/SF nanofibermembranes

2.4.1 Morphology. The structures and surface morphologiesof the prepared nanofiber membranes were observed with aCANON-55D digital camera (Japan) and scanning electron micro-scope (SEM, Hitachi TM-100, Japan). Image J software (NationalInstitutes of Health, USA) was used to analyze the average fiberdiameter in the SEM images (100 random measurements perimage). The fiber surface topography was inspected with anatomic force microscope (AFM) (Nanoscope IV, America).

2.4.2 Characterization. The characteristics of the preparednanofiber membranes were evaluated by both Fourier trans-form infrared spectroscopy (FTIR) and powder X-ray diffraction(XRD). Infrared measurements were performed on an Avatar380 FTIR spectrometer, and XRD analysis was completed on aD/Max-2550 PC X-ray diffractometer (Rigaku, Japan) with Cu Karadiation. In addition, the thermogravimetric (TG) propertiesof the prepared samples were characterized using a thermal

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analyzer (TG-209-F1). In the experiment, argon was used asthe carrier gas with a flow rate of 50 mL min�1, and the heatingrate was controlled at 10 1C min�1 from room temperatureto 600 1C.

2.4.3 Conductivity. The conductivities of the Ppy coatedPLCL/SF nanofiber membranes were measured using the cyclicvoltammetry method in a three-electrode system (CHI660D,Chenhua Ltd, China) at room temperature. The constant currentapplied to the surface created a voltage difference and then theconductivity of each membrane was calculated as follows:

s ¼ LRS

where s (S cm�1) is the electrical conductivity, L (cm) is thedistance between the reference electrode and working electrode,R (O) is the ohmic resistance, and S (cm2) is the cross-sectionalarea of the sample.

2.4.4 Contact angle. The surface hydrophilicity of the electro-spun nanofibers was studied by measuring the water contactangle (WCA) using a sessile drop method with distilled water(OCA40, Dataphysics, Germany).

2.5 SC proliferation and morphology

The in vitro cell culture experiment was performed using SCs. SCswere cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)supplemented with 10% fetal bovine serum (FBS) and 1%penicillin–streptomycin formulation at 37 1C and 5% CO2 in ahumidified atmosphere. The samples of the Ppy coated PLCL/SFnanofiber membranes and PLCL/SF membranes (positive control)were sterilized with 75% ethanol and UV irradiation for 2 h,respectively. Then, SCs were detached by 0.25% trypsin–EDTA andseeded with a density of 1 � 104 cells per well on the membranes.After culturing for 1, 3 and 5 days, the viability of the SCs wasdetermined by the MTT assay (Sigma-Aldrich Co., USA) (n = 3)19

with the tissue culture plates (TCP) as the negative control. Duringthe experiments, the medium was changed every two days. ForES treatment evaluation, the cells were treated as previouslyreported15 with a voltage of 100 mV cm�1 and an electricalstimulation time of 1 hour per day (for a total of 5 days).

In order to observe the cell morphology, SCs on the nano-fiber membranes were stained with fluorescent dyes. After5 days of culturing, the samples were washed with PBS toremove the residual medium and unviable cells, and fixed in4% paraformaldehyde for 2 h. After permeabilization in 0.1%Triton X-100 (Sigma, USA) for 5 minutes, blocking in 2% bovineserum albumin (BSA, Sigma, USA) for 30 minutes and rinsing3 times with PBS, the cytoskeletons and nuclei of the SCs onthe membranes were stained with Alexa Fluor 568 phalloidin(Life Technologies, USA) and 40,6-diamidino-2-phenylindole(DAPI, Life Technologies, USA) for 30 min and 5 min, respectively.In the end, the SC morphology was observed with fluorescencemicroscopy (Olympus IX-71, Japan).

The SCs on the nanofiber membranes were dehydrated witha gradient ethanol solution (30%, 50%, 70%, 90%, 95%, and100%) followed by freeze drying for 12 h. The samples were

sputter coated with gold and observed by SEM at an acceleratingvoltage of 15 kV.

2.6 PC12 cell differentiation with electrical stimulation

PC12 cells were cultured in Roswell Park Memorial Institute(RPMI) medium supplemented with 2.5% fetal bovine serum,15% horse serum and 1% penicillin–streptomycin. When PC12cells were grown to the logarithmic growth phase, they wereseeded on the PLCL/SF-Ppy-1, PLCL/SF-Ppy-2 and PLCL/SF-Ppy-3membranes with a density of 1 � 104 cells per well. Similarly, PC12cells were seeded on PLCL/SF as a control. During 5 days ofculturing, electrical stimulation of the PC12 cells on the membraneswas performed according to the same experimental conditionsas the previous SC culturing (the voltage was 100 mV cm�1, andthe electrical stimulation time was 1 hour per day).

After culturing, the PC12 cells were fixed with 4% para-formaldehyde for 2 h and washed with PBS three times. PC12cells were permeabilized in 0.1% Triton X-100 for 5 minutesand blocked in 2% BSA for 30 minutes, followed by rinsing withPBS three times. Mouse Anti-b-Tubulin III (neuronal) antibody(Sigma, USA) (1 : 200 in 1% BSA solution) was added to thesamples, and the samples were incubated at 4 1C overnight andwashed with PBS three times again. The cells were stained withrhodamine (TRITC)-labeled goat anti-mouse IgG (YuanzhiBiotechnology CO., Ltd, Shanghai, China) (1 : 200 in 1% BSAsolution) and DAPI for 30 minutes and 5 minutes respectively,and then washed with PBS twice. Subsequently, the immuno-fluorescence staining images were gathered using fluorescencemicroscopy.

The immunofluorescence staining images of the PC12 cells wereanalyzed using Image J software. The percentage of differentiatedcells was calculated by counting the number of differentiatedcells (the PC12 cells which have axons were differentiated cells)and total number of cells in a 10 � 10 mm2 area (the area wasselected in the immunofluorescence staining images randomly,n = 3), and it was calculated as follows:

The percentage of differentiated cells

¼ The number of differentiated cellsThe totalnumberof cells

� 100%

Furthermore, 20 differentiated cells were chosen randomlyto calculate the average axon length in each image (n = 20),which was used to characterize the cell differentiation level.

To detect the differentiation of PC12 cells which were culturedon different scaffolds with/without ES, the gene expression levels ofb-Tubulin III, GAP43, and synapsin I were separately examinedusing real time-qPCR (RT-qPCR) after culturing for 5 days. In brief,PC12 cells were fixed with 4% paraformaldehyde for 30 min at 4 1C,and the total RNA was isolated and converted to cDNA usingreverse transcriptase (Applied Systems, Foster City, CA, USA).Real-time polymerase chain reaction (PCR) was performed usingan Applied Biosystems 7300 using Taqman primers and probesspecific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH)and other relative genes. The sequences of the primers used are

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listed in Table S1 (ESI†). The relative gene expression was calcu-lated using the 2�DDCt method with GAPDH as the reference gene,and the PC12 cells cultured on TCP were set as the control.

2.7 Statistical analysis

All the quantitative data were expressed as a mean � standarddeviation (SD). The statistical analysis was carried out usingone-way ANOVA and a value of P o 0.05 was considered to bestatistically significant.

3. Results and discussion3.1 Morphologies of Ppy-coated PLCL/SF membranes

The electrospinning method was chosen to fabricate PLCL/SFnanofiber membranes in this study, because electrospunscaffolds offered nano-sized structures with high surface areaand porosity, which mimicked the size and organization of thenatural extracellular matrix (ECM). The digital photos and SEMimages of the PLCL/SF membranes are shown in Fig. 1A and E,respectively. The diameter of the PLCL/SF nanofiber was975 � 102 nm (Fig. 1E), indicating the nano-sized structure ofthe PLCL/SF membranes.

In order to maintain the aligned nanofiber structure for cellgrowth and solve the problem of Ppy processing, pyrrole has beenpolymerized on the PLCL/SF membranes in situ. With pyrrole beingpolymerized, Ppy aggregated in solution and deposited on thePLCL/SF nanofiber surfaces. During the polymerization process,various polymerization conditions were set to produce Ppy-coatedPLCL/SF nanofiber membranes (Table 1). The PLCL/SF membranewas white (Fig. 1A), while it became obviously black after coatingwith Ppy (Fig. 1B–D), indicating that Ppy was successfullycoated on the PLCL/SF membranes. The PLCL/SF nanofibersurface was smooth as shown in the SEM image (Fig. 1E).However, some Ppy nanoparticles were observed on the fibersurfaces of the Ppy-coated PLCL/SF nanofibers, and the number ofPpy nanoparticles was increased when more pyrrole was added(Fig. 1F–H). In addition, a single nanofiber of PLCL/SF and Ppy-coated PLCL/SF has been characterized by AFM (Fig. 1I–L). Due tothe addition of pyrrole, the roughness of the nanofiber wasincreased, and it indicated that lots of nanoparticles (Ppy polymer)were deposited on the PLCL/SF nanofiber surfaces.

3.2 Characterizations

In order to characterize whether Ppy was coated on the PLCL/SFnanofiber membranes or not, FTIR and XRD evaluations of rawPpy, PLCL/SF, PLCL/SF-Ppy-1, PLCL/SF-Ppy-2 and PLCL/SF-Ppy-3were performed. FTIR results are shown in Fig. 2A, and absorp-tion peaks at 2942 cm�1 and 1757 cm�1 were observed for PLCL/SF, PLCL/SF-Ppy-1, PLCL/SF-Ppy-2 and PLCL/SF-Ppy-3, whichcorrespond to the –CH2 stretching and ester groups of the PLCLmolecular skeleton. The sharp peaks at 1650–1660 cm�1 (amide I),1535–1545 cm�1 (amide II), and 1235–1240 cm�1 (amide III)confirmed the existence of SF in the Ppy-coated and PLCL/SFmembranes.20,21 In addition, there are three characteristicabsorption peaks for raw Ppy, i.e. 1557, 1025, and 916 cm�1.The strong bands at 1557 cm�1 and 1025 cm�1 were attributedto the existence of a stretching pyrrole ring (C–C band) and thebending deformation of the N–H group, respectively. The band

Fig. 1 (A–D) The digital photos and (E–G) SEM and (I–L) AFM images ofthe different nanofiber membranes.

Table 1 Compositions of the Ppy oxidizing reaction systems

SamplesPLCL/SF electrospun membraneconcentration (w/v)/% Pyrrole volume/mL FeCl3 volume/mL pTS volume/mL

PLCL/SF 10 0 0 0PLCL/SF-Ppy-1 10 25 50 25PLCL/SF-Ppy-2 10 37.5 70 37.5PLCL/SF-Ppy-3 10 50 100 50

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at 916 cm�1 was attributed to the bending deformation of theC–H group belonging to Ppy. The peak at 916 cm�1 appearingfor the Ppy-coated samples confirmed the presence of Ppy onthe PLCL/SF nanofiber membranes. However, after coating withPpy, the bands at 1557 cm�1 and 1025 cm�1 for the Ppy-coatedsamples were not obvious, mainly because of the overlap withthe bands for the amide groups of SF. XRD curves for thespecimens are shown in Fig. 2B. There is no significantcrystallization peak for the Ppy nanoparticles because they wereamorphous. There is a strong peak at 16.71 for the PLCL/SF andPpy-coated membranes, which was attributed to a crystallinediffraction peak for the PCL chain segments.22 This crystallinepeak value (16.71) decreased when more Ppy was deposited,because deposition reduced the crystallinity degree of PLCL/SF.

Thermogravimetric (TG) analysis is a method for determiningpolymer thermal stability. It has a close relationship with thepolymer’s molecular structure, degree of crystallinity, moleculesize, molecule distribution, and so on. The TG and derivativethermogravimetric (DTG) curves of the samples are shown inFig. 2C and D. For raw Ppy, the weight decreased gradually withthe temperature increase, and there was still 40% of the Ppyremaining when the temperature was 900 1C, indicating the goodheat stability of Ppy. In comparison, the main weight loss forPLCL/SF occurred at 300–400 1C (the weight loss ratio of thesamples was 92%), and the temperature of maximum weight losswas 350 1C. The weight loss and derivative of weight loss ofPLCL/SF-Ppy-1, PLCL/SF-Ppy-2 and PLCL/SF-Ppy-3 showed similarresults, indicating that the coating of Ppy will not impact thethermodynamic properties of PLCL/SF nanofiber membranes.

The conductivity of the Ppy-coated PLCL/SF nanofibermembranes was measured by the cyclic voltammetry method,

and the corresponding contact angles were evaluated using asessile drop method with distilled water (Table 2). The PLCL/SFmembrane was non-conductive, but it became conductiveafter Ppy coating. The conductivity of the PLCL/SF-Ppy-3membrane was 1.36 � 10�4 S cm�1, which was higher thanthat of PLCL/SF-Ppy-2 (2.82 � 10�5 S cm�1) and significantlyhigher than that of PLCL/SF-Ppy-1 (8.52 � 10�6 S cm�1)(P o 0.05). It was documented that the Ppy-coated PLCL/SFmembranes exhibited better conductivity when more pyrrolehad reacted in the synthesis.

The contact angle of the PLCL/SF nanofiber membrane was82.84 � 2.511 (Table 2), indicating that it was hydrophilic. SFwas chosen to blend with PLCL for fabricating electrospunnanofiber membranes, because it improved the hydrophilicitywhen compared with pure PLCL.23 The Ppy-coated PLCL/SFnanofiber membranes showed much better hydrophilicity (thecontact angles of PLCL/SF-Ppy-1, PLCL/SF-Ppy-2, PLCL/SF-Ppy-3were 32.70 � 2.191, 28.27 � 3.631 and 26.78 � 2.951, respectively).This was mainly attributed to the hydrophilic sulfonic acid groupon the dopant (PT) and the amino group on Ppy. Besides, Ppycoating increased the surface roughness of the PLCL/SF nanofiber

Fig. 2 (A) FTIR, (B) XRD, (C) TG and (D) DTG curves for different samples.

Table 2 Conductivity and contact angles of the PLCL/SF and Ppy-coatednanofiber membranes

Samples Conductivity/S cm�1 Contact angle/1 Image of drops

PLCL/SF — 82.84 � 2.51PLCL/SF-Ppy-1 8.52 � 10�6 32.70 � 2.19PLCL/SF-Ppy-2 2.82 � 10�5 28.27 � 3.63PLCL/SF-Ppy-3 1.36 � 10�4 26.78 � 2.95

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membranes, which increased the hydrophilicity as well. Thehydrophilic surface of the materials is beneficial to cell adhe-sion and proliferation, so the coating of Ppy may improve thebiocompatibility of PLCL/SF nanofiber membranes.

3.3 Mechanical properties

PLCL is the copolymer of L-lactic acid and e-caprolactone,with better biodegradability and mechanical properties.24,25 SFis a natural material, which has good oxygen and water vaporpermeability, biocompatibility, and low inflammatory response.26

Therefore, PLCL and SF were blended to fabricate nanofibermembranes for nerve regeneration. In order to investigate themechanical properties of Ppy-coated PLCL/SF, mechanical testingwas performed and the strain–stress curves are shown in Fig. 3.

The tensile stress, elongation at break and Young’s modulusof the PLCL/SF membrane were 5.9 � 0.9 MPa, 277 � 20% and2.19 � 0.016 MPa, respectively. After coating with Ppy, thetensile stresses of PLCL/SF-Ppy-1 (7.2 � 1.2 MPa), PLCL/SF-Ppy-2(7.2 � 0.8 MPa) and PLCL/SF-Ppy-3 (7.8 � 1.5 MPa) wereincreased, but the elongations of PLCL/SF-Ppy-1 (286 � 15%),PLCL/SF-Ppy-2 (272 � 18%) and PLCL/SF-Ppy-3 (253 � 8%) weredecreased. The Young’s modulus values of the three membraneswere 2.93� 0.06, 2.85� 0.044 and 3.03� 0.11 MPa, respectively.Because Ppy was brittle, the Young’s modulus of the Ppy-coatedPLCL/SF membranes was significantly higher than that ofPLCL/SF (Fig. 3B, P o 0.05).

3.4 Biocompatibility characterization in vitro

When a peripheral nerve is injured, SCs will proliferate andmigrate into the injury site, and form longitudinal Bungnerbands to support axon growth and subsequent myelinization.27

In addition, it has been noted that the cellular responses of SCsare influenced by an external electrical field.28 To study thebiocompatibility of the Ppy-coated PLCL/SF nanofiber mem-branes, SCs were cultured on the membranes, and electricalstimulation treatment was performed with a 100 mV cm�1

voltage for 1 hour per day. The MTT assay was used to studythe toxicity and biocompatibility.

The MTT assay results of the nanofiber membranes areexhibited in Fig. 4. With 5 days of culturing, SCs underwent agood degree of proliferation on the four kinds of sample, whichdocumented their biocompatibility and non-toxicity. Compared

to the PLCL/SF groups, all of the SCs on Ppy-coated PLCL/SFnanofiber membranes showed better proliferation behavior ondays 1, 3 and 5 (P o 0.05), indicating that the Ppy coating layernot only had good biocompatibility but also significantlypromoted SC growth and proliferation. It might be becausethe hydrophilicity of Ppy promoted cell adhesion on the nano-fiber surface, or the conductive effect promoted SC myelin geneexpression, neurotrophin secretion and SC proliferation.29

Electrical stimulation (ES) plays an important role to promoteneural cell growth. The data indicated that SCs had betterproliferation under ES than without ES, especially on day 5.In addition, significantly more SCs proliferated on Ppy-coatedmembranes than the PLCL/SF membrane, and increased Ppycoating contributed to better SC growth. It has been recognizedthat SC proliferation on conductive nanofiber membranes fortissue engineering is strongly attributed to electrical stimula-tion. Leandro et al. investigated the mechanism of interactionbetween SCs and Ppy in the presence of an electric field, and itwas found that SCs’ directional migration was governedby electrically mediated phenomena. Generally, the resultssuggested that both the Ppy coating layer and ES treatmentplay important roles in SC proliferation.

Fluorescence images of SCs stained for F-actin and nucleiafter being cultured for 5 days are shown in Fig. 5A–H. TheSCs exhibited an elongated and spreading morphology on the

Fig. 3 (A) Stress–strain curves and (B) Young’s moduli of different nanofiber membranes.

Fig. 4 The MTT results for SCs cultured on different nanofiber membranes.

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nanofiber membranes. The SEM images also showed that SCson the nanofiber membranes stretched along the nanofibers(Fig. 5I–P). More SCs proliferated on Ppy-coated PLCL/SF nanofibermembranes than PLCL/SF, and SC growth with ES performedmuch better than that without ES. This finding was consistentwith the previous MTT results, that the conductive polymer Ppyenhanced SC proliferation with electrical stimulation treatment.

3.5 PC12 cell differentiation with electrical stimulation

Rat pheochromocytoma 12 (PC12) cells, which exhibit a numberof neuronal properties including morphological differentiation,electrophysiological responsiveness, and neurotransmittersynthesis, are widely used as a model for primary neuronalcells. Nerve growth factor (NGF) is a kind of nerve growthregulator, which promotes both neuron nutrition and neuriteoutgrowth, and even induces PC12 cell differentiation andaxonal outgrowth.30–32 Conductive polymers were verified topromote PC12 cell axonal outgrowth with ES treatment. It wasconcluded that the conductive polymer enhanced neurons’myelin gene expression and sustained neurotrophin secretionunder ES which was beneficial to promoting PC12 celldifferentiation.29 However, in the studies, the function of theconductive polymer for PC12 differentiation was performedwith NGF; this means that ES could promote PC12 cell differ-entiation, but we cannot draw the conclusion that ES alonecould induce the differentiation. In our previous work, Zhanget al. blended the conductive polymer PANi into nanofibers,and the results indicated that even without the treatment withNGF, the conductive nanofibrous membranes could inducePC12 cell axonal outgrowth by ES.15 Therefore, in order todetermine whether Ppy-coated PLCL/SF nanofibrous membranes

can induce PC12 cells to differentiate or not, we cultured PC12cells on four kinds of membrane with ES but without NGFtreatment.

PC12 cells were cultured on the nanofiber membranes for5 days. Then the cytoplasm and nucleus of the PC12 cellswere stained with rhodamine-labeled b-Tubulin III and DAPIfor analysis. The fluorescence staining results for PC12 cellscultured on the four kinds of membrane without ES are shownin Fig. S1 (ESI†). It was observed that PC12 cells showed betterproliferation in the Ppy-coated PLCL/SF groups, comparedto the PLCL/SF group. However, no differentiated PC12 cellswere found, and the cells were relatively small and rounded. Itindicated that the conductive polymer cannot induce PC12 celldifferentiation in the absence of ES. However, in Fig. 6B–D,PC12 cells showed great differentiation (the PC12 cell morphologywas irregular, long neurites and many branches were observed,and the extended axons are marked with white arrows) in thePLCL/SF-Ppy-1, PLCL/SF-Ppy-2 and PLCL/SF-Ppy-3 groups with ES,in comparison to the control group (PLCL/SF, Fig. 6A). To quantifythe neurite differentiation, the proportion of differentiatedPC12 cells and axon length was calculated for several cases(Fig. 6E and F). 7.67 � 1.01% PC12 cells were differentiated onthe PLCL/SF nanofiber membrane, and the average axon lengthwas 5.16 � 1.76 mm. It indicated that even without NGF or theconductive polymer (Ppy), PC12 cells still differentiated withES, because the medium was conductive and the electric fieldinduced PC12 differentiation. The proportion of differentiatedcells for the conductive (Ppy-coated) membranes was muchhigher than for the nonconductive membranes, and similarresults were obtained from the axon length results (P o 0.05). Itis concluded that the Ppy coating layer can significantly induce

Fig. 5 Fluorescence images of SCs cultured on different nanofiber membranes without ES (A–D) and with ES (E–H); SEM images of SCs cultured ondifferent nanofiber membranes without ES (I–L) and with ES (M–P). (Bar = 100 mm.)

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PC12 differentiation under an electric field. The proportion ofdifferentiated cells on PLCL/SF-Ppy-3 (99.67 � 0.47%) washigher than on PLCL/SF-Ppy-2 (91.37 � 0.59%), and significantlyhigher than on PLCL/SF-Ppy-1 (22.19 � 4.69%) (P o 0.05). Theaxon length on PLCL/SF-Ppy-3 (53.28 � 19.48 mm) was signifi-cantly longer than on PLCL/SF-Ppy-2 (30.93 � 10.58 mm) and onPLCL/SF-Ppy-1 (22.37 � 7.49 mm) as well.

The gene expression levels of b-Tubulin III, GAP43, andsynapsin I by the RT-qPCR technique are shown in Fig. 7 andFig. S2 (ESI†). GAP43 is a kind of growth associated proteinof PC12 cells, and synapsin I is a presynaptic membrane-associated protein, both of which will be increased in levelwith the formation of mature synapses. b-Tubulin III is themain expression protein of neurites, so these three kinds ofgene could be used as markers for PC12 cell differentiationin vitro. In Fig. S2 (ESI†), there was no significant difference inthe three kinds of gene expression between the nanofibermembranes and the TCP group. It was documented that in theabsence of ES, PC12 cells were not differentiated on Ppy-coatednanofiber membranes, the same as the TCP and PLCL/SF groups.

However, the level of gene expression of the PC12 cells on thePpy-coated nanofiber membranes was significantly higher thanthat of the cells in the TCP and PLCL/SF groups with ES, andthe differentiation of the PC12 cells increased the expression ofb-Tubulin III, GAP43, and synapsin I with increasing conduc-tivity (Fig. 7). The relative gene expression values for b-TubulinIII, GAP43, and synapsin I in cells on the PLCL/SF group withES were 1.39 � 0.10, 1.34 � 0.05 and 1.44 � 0.09 respectively(Fig. 7), which were higher than the PLCL/SF group without ES(1.08 � 0.06, 1.01 � 0.01 and 1.03 � 1.02 respectively, Fig. S2,ESI†) (P o 0.05). This indicated that ES could induce PC12 celldifferentiation when the cells were cultured on non-conductivenanofiber membranes, even on TCP.33

As a result, the Ppy-coated PLCL/SF nanofiber membranessupported the attachment and differentiation of PC12 cells incomparison to the nonconductive PLCL/SF nanofiber membrane.It may be attributed to the electric stimulation, and the effect wasincreased with the enhancement of conductivity. The axon lengthsof PC12 cells under electrical stimulation on the conductingnanofiber membranes were similar to the results of previous

Fig. 6 Immunofluorescence images of PC12 cells cultured on (A) PLCL/SF, (B) PLCL/SF-Ppy-1, (C) PLCL/SF-Ppy-2 and (D) PLCL/SF-Ppy-3 with ES;(E) proportion of differentiated cells and (F) axon length statistical results which were calculated from the immunofluorescence images.

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studies. In the study of Schmidt et al., it was observed that themedian axon length of electrically stimulated PC12 cells was18.14 mm on Ppy membranes after 2 days of culturing, whereasthe median length for an unstimulated control was 9.5 mm.14

However, in the previous study, the PC12 cells were induced todifferentiation with NGF before ES, which just indicated that ESon conductive membranes stimulated PC12 cell differentiation.Herein, NGF was not used to induce PC12 cell differentiationbefore culturing. It was found that ES induced PC12 celldifferentiation even on nonconductive membranes (PLCL/SF),because the electrical field in the medium (the medium wasalso conductive) can induce PC12 cells to differentiate, and thePpy coating enhanced this function. These non-cytotoxic andelectro-conducting nanofiber membranes were suitable forelectrically stimulating neuron outgrowth and potentially enhan-cing nerve tissue regeneration.

4. Conclusion

In the present study, conductive nanofiber membranes weredeveloped by coating Ppy on PLCL/SF nanofibers via in situoxidative polymerization. SEM and AFM results indicated thatPpy nanoparticles were deposited on the PLCL/SF nanofibermembranes. FTIR results confirmed the existence of Ppy as well.The crystallinity of the Ppy-coated nanofiber membranes decreasedwith more Ppy deposition. However, the thermal properties ofthe conductive nanofiber membranes were similar in stabilitywhen compared with untreated nanofibers. The conductivityand hydrophilicity of the Ppy-coated samples were improvedwith increasing pyrrole monomer amounts. In addition, after

culturing SCs and PC12 cells on the scaffolds with ES, it wasfound that ES could enhance SC proliferation and induce PC12cell differentiation without NGF treatment, and the effectwas enhanced with increasing conductivity. Thus, Ppy-coatedPLCL/SF electrospun nanofibers have a promising potential topromote peripheral nerve tissue regeneration.

Acknowledgements

This research was supported by the National Natural ScienceFoundation of China (31470941, 31271035), Science and Tech-nology Commission of Shanghai Municipality (15JC1490100,15441905100), National Major Research Program of China(2016YFC1100200), PhD Programs Foundation of Ministry ofEducation of China (20130075110005) and Light of TextileProject (J201404). The authors extend their appreciation tothe International Scientific Partnership Program ISPP at KingSaud University for its funding research through (ISPP-0049).This work was financially supported by the Opening Project ofState Key Laboratory of Polymer Materials Engineering (SichuanUniversity) (sklpme2016-4-29).

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