Bioelectrochemistry 85 (2012) 36–43

Contents lists available at SciVerse ScienceDirect

Bioelectrochemistry

j ourna l homepage: www.e lsev ie r .com/ locate /b ioe lechem

Fabrication of conductive electrospun silk fibroin scaffolds by coating withpolypyrrole for biomedical applications

Salvador Aznar-Cervantes a, Maria I. Roca b, Jose G. Martinez b, Luis Meseguer-Olmo c, Jose L. Cenis a,Jose M. Moraleda c, Toribio F. Otero b,⁎a Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), La Alberca (Murcia), E-30150, Spainb Group for Electrochemistry, Intelligent Materials and Devices (GEMDI), Universidad Politécnica de Cartagena, ETSII, Cartagena (Murcia), E-30203, Spainc Cell Therapy Unit & Orthopedic Surgery Service. University Hospital V. Arrixaca, El Palmar (Murcia), E-30150, Spain

⁎ Corresponding author. Tel.: +34 968325519; fax: +E-mail address: toribio.fotero@upct.es (T.F. Otero).

1567-5394/$ – see front matter © 2011 Elsevier B.V. Alldoi:10.1016/j.bioelechem.2011.11.008

a b s t r a c t

a r t i c l e i n f o

Article history:Received 29 April 2011Received in revised form 22 November 2011Accepted 26 November 2011Available online 8 December 2011

Keywords:Fibroin-polypyrrole meshesElectroactivityAnion storageHuman fibroblastAdult human mesenchymal stem cells

Scaffolds constituted bymicro and nanofibers of silk fibroin were obtained by electrospinning. Fibers of fibroinmeshes were coated with polypyrrole (pPy) by chemical polymerization; chemical linkages betweenpolymers were observed by SEM and IR spectroscopy. Mechanical resistance of the meshes was improvedby polypyrrole coating. Furthermore, coated meshes present a high electroactivity allowing anion storageand delivery during oxidation/reduction reactions in aqueous solutions. Uncoated and pPy coated materialssupport the adherence and proliferation of adult human mesenchymal stem cells (ahMSCs) or humanfibroblasts (hFb). The bioactivity of fibroin mesh overcomes that of the polypyrrole coated meshes.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Scaffolds for cellular growth made of silk fibroin (SF) demonstratediverse applications in the field of tissue engineering [1]. SF is highlybiocompatible and able to support appropriate cellular activitywithout eliciting rejection, inflammation or immune activation inthe host [2–5]. SF structure is porous allowing the growing of cells,the exchange of nutrients and growth factors and the production ofextracellular matrix (ECM) to enable communication between thecells. SF has been studied for tissue engineering in blood vessels[3,6], skin [5,7], bone [8,9] and cartilage [10].

Fibroin is extremely versatile and can be processed in very differentformats, adequate for different tissue engineering needs. Fibroin cellularscaffolds can be constructed predominantly as hydrogels, 3-D spongesand mats of nanofibers obtained by electrospinning [1,11]. Nanofiber-based scaffolds are created through electrospinning with wideapplications in biomedicine. Typical electrospinning setup consistsof three components: a high voltage supplier, a capillary needle, and agrounded collector. During electrospinning, an electric potential isapplied to a jet of a polymer solution, usually delivered with a syringepump [12]. Electrohydrodynamic forces produced by charges in thepolymer jet as well as the attractive forces between the liquid and thecollector work together to exert tensile forces on the solution. Resulting

34 968325915.

rights reserved.

in a thinning of the polymer jet to transverse sizes in the nanometer andmicrometer range, which is collected in a metallic plate as a randomnon woven mat. The physical configuration of this mat mimics theextracellular matrix of animal tissues; and this is the reason of theexcellent performance of these structures as cellular scaffolds. Numerouscomposite nanofibers and functionalized electrospun silk matrices havebeen developed in the last years including: Park et al. successfullyproduced chitin/silk fibroin blend fibers [13], collagen/silk fibroinsolutions in HFIP were electrospun by Yeo et al. [14], Wang et al.encapsulated a silk fibroin core fiber within a poly (ethylene oxide)shell fiber [15] and Li et al. [16] added bone morphogenetic protein-2(BMP-2) into silk fibroin nanofibers mixing it into the spinning solutionand Liu et al. [17] immobilized glucose oxidase in a compositemembrane of regenerated silkfibroin and poly(vinyl alcohol) for sensingapplications.

Electrospun mats of silk fibroin as well as other polymeric biomate-rials are satisfactory for a high number of tissue engineering applications.Thefield of potential applications could behighly expandedby fabricatingelectrospun mats with conductive properties. Scaffolds consisting ofconducting polymers have application in biosensing, controlled drugdelivery and tissue engineering with improved cellular growth [18,19].

Conductive electrospun nanofiber mats are constructed by twoapproaches: the first would be direct electrospinning of the conductingpolymer, aswasmadewith polypyrrole by Chronakis et al. [20]; however,little information exists regarding the reabsorption of this material whenfabricating implantable devices. Secondly, an electrospun mat of a wellknown biocompatible and reabsorbable biomaterial is made and then

37S. Aznar-Cervantes et al. / Bioelectrochemistry 85 (2012) 36–43

the nanofibers are coated with a conducting polymer; the approachfollowed by Lee et al. [21], where a polypyrrole mat was coated withPLGA. The work shows that PC12 cells grown on this hybrid scaffoldand subjected to current produce more and longer neurites than whenthey are not subjected to a stimulated control. Hybrid biocompatiblematerials with new functionalities can be attained by coating polymericnanofibers with conducting polymers. SF-conducting polymer can allowthe application of low electric fields during cell growth. Moreover,biocompatible materials can be oxidized and reduced interchangingions with ambient and generating ionic currents under control [22].These possibilities will open new experimental fields when used asscaffolds for cell proliferation studies.

By flow of low anodic currents the material oxidizes: anions areextracted from the electrolyte and “stored” in the film [23,24]. Byflow of low cathodic currents the material reduces and ions areexpelled towards the solution. Biocompatible conducting polymersinterchanging cations are also available, allowing electron/cationtransductions [25].

Given the well known characteristics of silk fibroin electrospunscaffolds in terms of biocompatibility and reabsortion, it could berelevant to confer them electroconductive functionalities. Here wewill present the generation of a hybrid material made of silk fibroincoated with polypyrrole (pPy-SF), studying its electrochemical andmechanical characterization and the concomitant biocompatibility asscaffold for proliferation of primary culture of adult humanmesenchymalstem cells (ahMSCs) and human fibroblasts (hFb). Comparative studiesbetween uncoated SF and pPy-SFwere performed. In subsequent studieselectric fields and local ionic currents will be applied to the cells duringproliferation.

2. Material and methods

2.1. Preparation of fibroin

Cocoons obtained from silkworms reared in the sericulture facilitiesof the IMIDA, were boiled twice for 45 min in 0.02 M Na2CO3 aqueoussolution and then rinsed thoroughly with water to extract the glue-like sericin proteins. The extracted SF was then dried at 40 °C for 24 hand dissolved in LiBr (Acros Organics, New Jersey, NJ) 9.3 M for 3 h at60 °C to generate a 20% w/v solution. Then it was dialyzed in distilledwater for 3 days and the resultant aqueous solution was freeze driedin order to have the purified silk SF ready to be dissolved in the desiredconcentration. Just before the experiment, a 17% w/v SF solution inHexafluoroisopropanol (HFIP) (Sigma, St. Louis, MO) was generatedby dissolving the lyophilized SF during 24 h at room temperature.

2.2. Electrospinning

The electrospinning setup used in this study (Yflow™ 2.2.S-300Electrospinner, Yflow, Spain) consists of a capillary tube with aninner diameter of 0.45 mm and an outer of 0.80 mm, connected to asyringe pump (charged with 3 mL of SF solution in HFIP), two highvoltage suppliers and a collector composed by a metallic surfacecovered with one piece of aluminum foil (10×10 cm). One of thehigh voltage suppliers is positively polarized and connected to themetallic tube where the polymer solution is projected; the other isnegatively polarized and it should be connected to themetallic collectorin order to improve the efficiency of the process. The setup is groundedto avoid electric discharges to the users. For the electrospinning avoltage of +6 kV was applied to the capillary tube and −5 kV to thecollector. The distance between the tip of the capillary tube and thecollector was 10 cm and the selected injection rate of the polymersolution was 6 mL/h.

After the electrospinning the mats were immersed in methanolduring 10 min as an effective technique to induce an amorphous tobeta-sheet transition of silk fibroin molecular structure. In order to

keep uniform meshes during drying, a low pressure was applied tothe meshes employing filter paper in both sides of the meshes.

Then they were washed in ultrapure water and stored at 4 °C insidea hermetic container until the cell seeding (for less than 2 weeks).

2.3. Polypyrrole coating

Pyrrole (Fluka 97% content) was distilled under vacuum at20 mbar using a vacuum pump MZ 2 C SCHOOT. A SF mesh(16×7×0.085 mm) was put into 30 mL of 0.3 M pyrrole and 0.3 MNaCl (Panreac, >99.00%, used as received) aqueous (ultra pure fromMillipore Milli-Q) solution in a 250 mL beaker. The system wassubmitted to ultrasonication for 30 s to allow the pyrrole meshsaturation. The mesh was incubated at 4 °C in a cryostat Julabo F25(± 0.1 °C) for 1 h. Then, 20 mL of a 0.75 M Ferric Chloride FeCl3(from Aldrich, >97%) aqueous solution was added keeping thesystem at 4 °C. After pPy polymerization for 24 h, the polypyrrolecoated mesh (ahead, pPy-SF) was washed several times usingdistilled water and dried at room temperature.

The pPy contentwas determined bymass difference between coatedand uncoated silk meshes using a Sartorious SC2 balance, with asensibility of 10−7 g. The mesh thickness was measured using anElectronic Digital Micrometer with 1 μm sensibility (Comecta).

Polypyrrole-silk fibroin meshes having 1.12 cm2 geometric surfacearea (16×7 mm) were used for mechanical tests and as workingelectrodes for electrochemical characterizations.

2.4. Characterization of physical and mechanical properties of electrospunmats

Scanning electron microscopy (SEM) was used to determine themaximum transverse size of the electrospun fibers after and beforethe coating with pPy and to visualize the growing and morphologicalcharacteristics of the cells in the materials. The pictures obtained withSEM were processed with ImageJ software to calculate the fiber size.The samples were fixed with 3% glutaraldehyde in 0.1 M cacodylatebuffer for 1.5 h at 4 °C. Then they were rinsed and post-fixed inosmium tetroxide for 1 h, before being dehydrated through increasingconcentrations of ethanol (30, 50, 70, 90 vol.%), with final dehydrationin absolute alcohol. After this, they were dried by the critical-pointmethod and gold coated. The mats were observed with a scanningelectronic microscope Jeol T-6100 at 15 kV.

Fourier transform infrared spectroscopy (FTIR) was used to analyzethe structural changes of themats after being coated with pPy. Sampleswere compressed into KBr (Sigma, St. Louis, MO) pellets and eachspectrum was acquired in transmittance mode by accumulationof 128 scans with a resolution of 2 cm−1 and spectral range of4000–400 cm−1. The analysis was finally focused in the range of1800–800 cm−1 as the most informative in the IR spectra for theseassays. FTIR data were gathered on a Perkin-Elmer Spectrum 100 IRspectrometer.

Once the pPy-SFmesheswere prepared, theirmechanical propertieswere studied by using a universal test frame machine Qtest from MTSSystems. The Young's modulus of the silk-fibroin and silk-fibroin-polymer meshes was obtained by submitting different samples to asteady stretching force (F) every time.

Young′s modulus ¼F=

SΔL�

L0

ð1Þ

where L0 is the initial length of the mesh film, ΔL is the lengthvariation after application of the force F and S, the transverse averagegeometric surface area of the sample in m2. The mesh thicknesses,76±10 μm for uncoated meshes and 96±7 μm for coated meshes,were measured using an electronic digital micrometer having a

38 S. Aznar-Cervantes et al. / Bioelectrochemistry 85 (2012) 36–43

precision of 1 μm. Sample widths, 5.15±0.93 mm for uncoated meshand 3.97±1.43 mm for coated mesh, and lengths, 12.19±3.21 mmfor uncoated meshes and 10.46±0.58 mm for coated pPy-SF meshes,were measured employing a digital caliper having a precision of0.01 mm.

2.5. Electrochemical characterization of the coated meshes

All the electrochemical experiments were performed in aMetrohm one compartment electrochemical cell connected to a PARM273A potentiostat–galvanostat controlled by computer throughECHEM software. The electrolyte, 0.1 M NaCl aqueous solution,provides electrochemical behaviors similar to that of the biologicalambient. As counter electrode a steel plate having 4 cm2 of surfacearea was used, after polished, cleaned for 15 min in acetone ultrasonicbath and rinsed with Milli-Q water before every characterization. Theworking electrode is a pPy-SF mesh: 15–17 mm length, 6–8 mmwidth and 80–90 μm thickness. The mass of the used meshes rangesfrom 4 to 5 mg. The reference electrode was a Crison Ag/AgCl (3 MKCl) electrode. All the experiments were performed at 22 °C (roomtemperature). Before every experiment the working solutions werede-aerated by bubbling N2 gas for 20 min.

2.6. Sterilization of meshes

Samples (uncoated and polypyrrole coated) were sterilized for60 min employing low temperature hydrogen peroxide gas plasma(LTHPGP) using a STERRAD NX™ (Johnson & Johnson) equipment.The by-products of the plasma sterilization method are primarilywater and oxygen. This method can inactivate a broad spectrum ofmicroorganisms, including resistant bacterial spores. The fibers donot suffer any change in their morphology during sterilization. Theplasma treatment could have changed surface chemistry, e.g. surfaceenergy.

2.7. Cell culture

For adhesion and proliferation studies, undifferentiatedmultipotentahMSCs from bone marrow and hFb from a primary human skinfibroblast culture were used. All the chemicals for cell culture werepurchased from Sigma-Aldrich (St. Louis, MO, USA) and the cultureplates were provided by Nunc (Roskilde, Denmark). The cell extractionand this studywere approved by the ethics committee of our institution(University Hospital V. Arrixaca, Murcia, Spain).

2.7.1. Isolation and culture of ahMSCsThe ahMSCs were isolated from bone marrow obtained by percu-

taneous direct aspiration from the iliac crest of three human malevolunteers, ranging from 27 to 35 years old, in good physical conditions.They were undergoing elective surgery for slipped disc and a vascularnecrosis of the femoral head as result of posterior dislocation. Thevolunteers signed previously an informed consent.

Mononuclear cells were obtained from buffy coat by ficoll gradientthrough a device SEPAX™ System (Biosafe, Eysines, Switzerland).After counting with a Neabauer chamber and estimating the viabilitywith tripan blue, the mononuclear cells were plated out in 75 cm2

culture flasks (Sarsted) with 10 mL of growth culture medium andtheywere incubated at 37 °C, in a 7.5% CO2 and 95% of relative humidityatmosphere to attach undisturbed for 7 days. The culture growthmedium (GM) used was Dulbecco's Modified Eagle's Medium (DMEM)supplemented with 10% fetal bovine serum (FBS) and penicillin andstreptomycin, 100 U mL−1 and 100 μg mL−1, respectively.

After 7 days, the culture medium was renewed removing thus thenon-adherent hematopoietic cells. The confluent ahMSCs cellsattached to the plastic of the culture flasks were subcultured in a 1:3ratio treated with trypsin/EDTA (0.25%/0.25%), in phosphate-buffered

saline (PBS, pH 7.4) solution, for 5 minutes. Only after the second orthe third subculture (P2,P3) process the ahMSCs were employed forthis study.

In order to confirm the identity of those adherent cells they weretested for the expression of cluster differentiation (CD) CD73, CD90and CD105 surface markers and no expression of CD34 and CD45markers (hematopoietic markers), following the criteria of theInternational Society Cell Therapy (ISCT). After the test, the identityof the cultured cells as ahMSCs was corroborated.

2.7.2. Isolation and culture of hFbThe hFb culture was initiated from skin explants obtained from the

same patients. Briefly, the skin biopsy was washed several times inPBS containing 100 U mL−1 penicillin and 100 μg mL−1 streptomycin.Subcutaneous tissue was separated from the skin with sterile scissorsand cut into small pieces (1–2 mm) that were placed in 6-well plateand stuck with a solution of 0.1% w/v collagenase in DMEM for 1 h, at37 °C, 7.5% CO2. The culture medium DMEM was then supplementedwith 10% FBS and routine antibiotics (100 μg/mL−1 streptomycin and100 U/mL−1 penicillin). When the cell confluence reached 80% thehFb were re-plated in 75 cm2

flasks.Viability and cell number was determined by trypan blue staining

in a Neubauer chamber. ahMSCs and hFb were seeded in 75 cm2

flasks at density of 5.0×103 cells/cm2 in expansion medium(DMEM, 10%FBS, 100UmL−1 penicillin and 100 μg mL−1 streptomycin,0.1 mM non essential amino acids) at 37 °C, 7.5% CO2. Non-adherentcells were removed during medium exchange after 4 days. Mediumwas changed twice a week and cells were allowed to grow until theculture reached 80% confluence. Then cells were detached using 0.25%trypsin/1 mM EDTA and replanted at 5.0×103 cells/cm2, under thesame culture conditions. Part of the first passage was trypsinized andfrozen in 10% DMSO/10% FBS/DMEM for its use in the future.Second or third passage (P2, P3) cells were used for seeding on thebiomaterials.

2.8. Plating technique on the mats

For seeding the mats, with hFb and ahMSCs, cultures that hadreached 75–80% confluence were used. Pieces of mats (1 cm×1 cm)were previously placed into 24 wells cell culture plates and 3 dropsof pure FBS were added to each sample to facilitate the initialadhesion and then incubated at 37 °C for one hour before seeding.Cells were detached with 0.25% trypsin/1 mM EDTA, then the cellsolution was concentrated until 1.0×106 cell/ml and, after removingthe excess of FBS in the wells, 4.0×104 cells were seeded in eachpiece of mat, 40 μL of the solution were slowly dropped onto theelectrospun mats in order to prevent solution from draining off thematerial. Following the addition of the droplets, plates with thesamples were placed in an incubator for 1 h to allow cells to settlebefore 1 mL of media was added to each well. Media was carefullyreplaced twice a week during the growing of the cells on the mats.

2.9. Measurement of cell proliferation. MTT assay

Cell proliferation wasmeasured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenil tetrazolium bromide (MTT) staining (Sigma, St. Louis,MO). Proliferation assay was developed at 1, 7, 14 and 21 days afterseeding. Experiments were performed in triplicate. SF and pPy-SFmats were incubated in MTT solution (1 mg/mL, 37 °C/5% CO2) for4 h. MTT is reduced to purple formazan derivatives in living cellsthat were dissolved with dimethyl sulfoxide and the absorbancewas measured with a microplate reader (BMG Fluostar Galaxy) at570 nm and the reference wavelength of 690 nm.

Fig. 1. SEM micrographs showing: (a) Morphology of superficial aspect of non-coatedSF mesh and (b) SF-pPy coated mesh (bar: 30 μm).

Table 1Initial masses of six different uncoated fibroin meshes (mi); final mass of the dry pPycoated meshes (mf) and pPy mass percentage, m (%), in dry samples.

Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6

mi/mg 5.450 5.295 4.880 4.252 4.180 4.028mf/mg 10.104 9.819 9.286 8.553 8.620 8.500m/% 46.06 46.08 47.45 50.29 51.50 52.61

39S. Aznar-Cervantes et al. / Bioelectrochemistry 85 (2012) 36–43

2.10. Statistical analysis

Data are presented as mean±SEM (standard error of the mean).Averages and standard deviations were calculated from 3 samplesper condition (in MTT assay). Fiber transverse size calculations wereperformedmeasuring 100 fibers per treatment. When the assumptionof homocedasticity was satisfied the statistical significance wasdetermined using Student's t-test (pb0.05) or ANOVA (pb0.05) fortwo or more groups comparisons, respectively. If this requirement wasnot satisfied Kruskal–Wallis (pb0.05) test was used for determiningthese differences.

3. Results and discussion

3.1. Electrospinning

The pictures obtained with SEM (Fig. 1) were processed withImageJ software. No significant differences were found betweenuncoated and pPy coated mat fiber transverse sizes (t-test, p>0.05).The average transverse size of the fiber is preserved and the pPy

Fig. 2. Fiber transverse size distribution (n=100) of SF mats (a) and SF-PPY coated mats (b

coating does not diminish the free space between fibers in the meshavailable for cell adherence and proliferation.

The average transverse size of spun SF fibers was 2300 nm, rangingfrom 600 to 6081 nm (Fig. 2), the standard deviation being 1295 nmwhen 100 different fibers were studied. Similar values were obtainedfor SF-pPy coated fibers. In this case, the average transverse size was2630 nm, the standard deviation was 1722 nm and the range was 472to 8670 nm. Fiber sizes (the small ones) could mimic the architecture ofECM [26]. The presence of some thicker fibers may improve mechanicalproperties.

3.2. Polypyrrole coating

Polymerization of pyrrole resulted in black silk fabrics, gettinguniform pPy coated fibers. The pPy mass percentage (W %) in thecoated, rinsed and dried mesh was calculated as:

W %ð Þ ¼ Wf−Wi

Wi⋅100 ð2Þ

whereWi andWf are the mesh film mass before and after polymeriza-tion, obtained following the procedure above described. The attainedpPy mass percentages from 6 different samples are shown in Table 1.

3.3. Voltammetric characterization of the pPy coated mesh

The pPy-SF meshes were studied by three consecutive cyclicvoltammograms between −0.5 V and 0.5 V at different scan rates(different period of the linear potential evolution with time) in0.1 M NaCl aqueous solution. The two initial voltammograms atevery scan rate were performed in order to erase any memory effecton the material and to attain a stationary voltammogram. The thirdof the voltammetric responses are depicted in Fig. 3 for scan ratesranging between 10 and 0.5 mV s−1. The shape of the voltammogramsis similar to those previously reported for free-standing pPy [27] and Ptcoated pPy electrodes [28] in aqueous solutions. Fig. 3 indicates that theobtained pPy-SF mesh can support the flow of a large range of anodicand cathodic currents (b ± 8mA) in the cell proliferation bath, at lowpotentials and before any water dissociation potentials. Thicker coated

).There were no significant differences before and after the coating (ANOVA, p>0.05).

Fig. 3. Voltammetric responses from a pPy-SF mesh (1.12 cm2 geometric surface area),under different scan rates, in 0.1 M NaCl aqueous solution in a potential range between−0.5 and +0.5 V. Positive currents: pPy oxidation storing Cl−. Negative currents: pPyreduction delivering Cl− towards the solution.

Fig. 5. FT-IR spectra of pure SF electrospun fibers (dotted line) and SF-PPY coatedelectrospun fibers (straight line).

40 S. Aznar-Cervantes et al. / Bioelectrochemistry 85 (2012) 36–43

fibers and reproducible voltammograms support uniform pPy coatingwith some pPy dendrites on the top.

Integration of those voltammograms from Fig. 3 leads to theevolution of the charges stored in polypyrrole, or recovered from(Fig. 4). Those charge (Q) evolutions can be transformed, by usingthe Faraday's constant (F=96485 C mol−1) and the mesh surfacegeometric area, to the number of mols (Q/F=n) of counterions (Cl-

ions) stored or delivered per square centimeter at the mesh/solutioninterface (n/S): the ionic flow at the interface [29–31]. Taking intoaccount the pPymass in the coatedmesh, this flow can be transformedinto a specific counter ion concentration mol g−1 in the pPy materialfraction. Figs. 3 and 4 establish that under the studied experimentalconditions (concentration, potential ranges, etc.) no degradation ofthe polypyrrole is present by over oxidation. Ranges indicate thepotential limits for the material polarization during cell proliferation,avoiding side reactions.

3.4. Characterization of physical and mechanical properties of electrospunmats

The molecular structure of SF mats was tested before and afterbeing coated with pPy using FTIR spectroscopy as can be seen inFig. 5. The spun SF mats exhibits the characteristics peaks of amide I

Fig. 4. Chronocoulograms: charge evolution, obtained by integration (Q=∫ I dt) of the vovariation (mol g−1) of Cl- in the pPy of the mesh using the pPy mass. Considering the pPmesh/solution interface is obtained.

(1640 cm−1), amide II (1520 cm−1) and amide III (1228 cm−1).The absence of the peak at 1660 cm−1 corroborates that the chemicalconformation in the SF mats changed from random coil to β-sheet[32].

The IR spectra of the pPy coated mats are similar to those of spunSF (Fig. 5). The coated mesh presents a peak characteristic of pyrrolering fundamental vibration at 1544 cm−1, and a second peak at1038 cm−1, corresponding to=C–H in-plane vibration. Similarresults were obtained by Xia and Lu [33] after coating SF native fiberswith pPy. The peak for amide II seems to disappear after pPy-coating.This fact doesn't mean that the structure of the SF has changed: it isoverlapped by pPy bands [34]. The peak for amide I shifts to a lowerwavenumber (1631 cm−1), attributed to an increase of the crystallinityin the mats (understood as a higher β-sheet content). As reported byXia and Lu, these spectral changes indicate that the conjugated polymerhave some interactions with the peptide linkage affecting to SF macro-molecular chains. The interactions are mainly covalent bonding,hydrogen-bond interactions and electrostatic attraction.

For rising external preloads (forces) the extension of different SFuncoated or pPy coated meshes samples strips was followed asdepicted in Fig. 6. The uncoated fibroin mesh brakes for externalforces comprised between 2 and 4 N and extensions from 0.12 and0.25 mm. The mechanical resistance was increased after pPy coating,

ltammograms from Fig. 4. Charge evolution is transformed into specific concentrationy mesh geometric surface area the density of Cl− ions flow (mol cm−2) through the

Fig. 6. Mechanical test. Length variation (extension) of: (a) a SF mesh, (b) a polypyrrole-SF mesh, as a function of the applied external loads (forces).

41S. Aznar-Cervantes et al. / Bioelectrochemistry 85 (2012) 36–43

supporting breaking forces higher than 5 N and extensions over0.3 mm; and the corresponding average Young's modulus rangebetween 266.7±17.3 MPa for the uncoated meshes and 310.5±37.6 MPa for the polypyrrole coated meshes.

Fig. 7. SEMmicrographs of ahMSCs (2a, 2b, 2c and 2d) and hFb (2e, 2f, 2g and 2h) growing oInitial cell response showing: some ahMSCs (2a,2c) and hFB (2e,2g) with flattened-polygomats surface at 3 days in culture. Finally, at 21 days, ahMSCs (2b, 2d) and hFb (2f, 2h) prol

3.5. Cell culture experiments. Adhesion and proliferation assay

In an effort to study the potential use of pPy-SF coated electrospunmats in tissue engineering we have cultured hFb and ahMSCs on these

n pure SF mats surface and pPy-coated mats at 3 and 21 days respectively after seeding.nal shape and steady adhesion by broad cytoplasmic extensions (philopodia) on bothiferated forming a layer with cytoplasmic connections. (bar 100 μm).

Fig. 8. (a) MTT results for fibroblasts growing on SF and SF-PPY coated mats at different times (seeding density 2×105 cells cm−2). Data are expressed as mean±SEM (n=3)Fig. Z2. (b) MTT results for hMSCs growing on SF and SF-PPY coated mats at different times (seeding density 2×105 cells cm−2). Data are expressed as mean±SEM (n=3).

42 S. Aznar-Cervantes et al. / Bioelectrochemistry 85 (2012) 36–43

materials. This constitutes a first step, previous to the application ofelectrical stimulation during seeding and proliferation.

Proliferation was followed by cell's visualization of images obtainedby SEM. Cell growth was followed by absorbance measurements usingMTT assays. Adhesions on the tested materials (SF and pPy-SF mats)were observed for both, hFb and ahMSCs cells, 3 days after the seedingas can be seen in Fig. 7. Significant absorbance differences (t-test,pb0.05) were found (Fig. 8) from hFb between coated and un-coatedSF mats after 1 day and at the end of the cell culture period (21 days).After 7 and 14 days no differences were detected between bothmaterials (t-test, p>0.05), which can be due to the high standarddeviation for these samples. Related with the growing of hFb significantdifferences (Kruskal–Wallis, pb0.05)were detectedwith theMTT assayat different times for both kind of mats.

No significant differences (ANOVA, p>0.05) were found forahMSCs growing on pPy coated or un-coatedmats (Fig. 8) for differentproliferation times; however they always grew better on uncoated SFmats. When compared period by period significant differences weredetected after 7, 14 and 21 days (t-test, pb0.05), indicating that pPycoating reduces the bioactivity of the silk fibroin meshes, as expectedfrom an artificial polymer related to a natural material, reducing alsoadhesion and proliferation. Alternatively the polypyrrole may havehad contaminants from the synthesis causing depressed growth.

The bioactivity of the pPy coated fibroin and its electrochemicalactivity are still high enough to go on with the next step of adhesion,proliferation and differentiation studies under applied local electricfields, or under local ionic currents at low potentials.

Our results agree with those proposed by other authors [8,35]confirming the ability of electrospun silk matrices to support ahMSCsattachment, spreading and growth in vitro.

4. Conclusions

We have succeeded coating silk fibroin nano and microfibers withpolypyrrole uniform films by chemical polymerization.

The coated material presents a high electroactivity supportingpolypyrrole oxidation and reduction in chloride aqueous solutionsat lower potentials than those required for Cl2 evolution or waterdissociation.

The oxidized material stores Cl− ions that can be liberated duringculture of cells generating local ionic flow on cells during prolifera-tion, if the material is bioactive enough. The ionic flow will be undercontrol of the cathodic current flowing through the oxidized material.

Our results confirm the ability of electrospun silk matrices tosupport ahMSCs attachment, spreading and growth in vitro.

Silk fibroin and polypyrrole coated silk fibroin fibers show quitesimilar bioactivity for hFb attachment and growth, in average thebioactivity of the polypyrrole coated material being a few lower.Similar results were obtained by seeding ahMSCs on both materials.

Moreover the open possibility of subsequent “in vivo” studies, theproved bioactivity of pPy-SF meshes open two ways for new “in vitro”proliferation studies by applying: local electric fields, without currentflow; or local ionic current flow without water discharge.

This will be the subject of our subsequent work.

Acknowledgements

Authors acknowledge financial support from Spanish Government(MCI) Projects MAT2008-06702, Seneca Foundation Project 08684/PI/08, FEDER Operative Program of the Region of Murcia 2007–2013from the UE. The work of S. Aznar-Cervantes was financed by apredoctoral research fellowship of the INIA (Instituto Nacional deInvestigación y Tecnología Agraria y Alimentaria), Government ofSpain. The authors are also grateful to Jonah Riddell for the criticalreading of the manuscript.

References

[1] Y.Z. Wang, H.J. Kim, G. Vunjak-Novakovic, D.L. Kaplan, Stem cell-based tissue en-gineering with silk biomaterials, Biomaterials 27 (2006) 6064–6082.

[2] Y. Yang, X. Chen, F. Ding, P. Zhang, J. Liu, X. Gu, Biocompatibility evaluation of silkfibroin with peripheral nerve tissues and cells in vitro, Biomaterials 28 (2007)1643–1652.

[3] L. Soffer, X.Y. Wang, X.H. Mang, J. Kluge, L. Dorfmann, D.L. Kaplan, G. Leisk, Silk-based electrospun tubular scaffolds for tissue-engineered vascular grafts, J. Bio-mater. Sci. Polym. Ed. 19 (2008) 653–664.

[4] M. Santin, A. Motta, G. Freddi, M. Cannas, In vitro evaluation of the inflammatorypotential of the silk fibroin, J. Biomed. Mater. Res. 46 (1999) 382–389.

[5] A. Schneider, X.Y. Wang, D.L. Kaplan, J.A. Garlick, C. Egles, Biofunctionalized elec-trospun silk mats as a topical bioactive dressing for accelerated wound heating,Acta Biomater. 5 (2009) 2570–2578.

43S. Aznar-Cervantes et al. / Bioelectrochemistry 85 (2012) 36–43

[6] X.H. Zhang, C.B. Baughman, D.L. Kaplan, In vitro evaluation of electrospun silk fi-broin scaffolds for vascular cell growth, Biomaterials 29 (2008) 2217–2227.

[7] B.M. Min, G. Lee, S.H. Kim, Y.S. Nam, T.S. Lee, W.H. Park, Electrospinning of silk fi-broin nanofibers and its effect on the adhesion and spreading of normal humankeratinocytes and fibroblasts in vitro, Biomaterials 25 (2004) 1289–1297.

[8] H.J. Jin, J.S. Chen, V. Karageorgiou, G.H. Altman, D.L. Kaplan, Human bone marrowstromal cell responses on electrospun silk fibroin mats, Biomaterials 25 (2004)1039–1047.

[9] K.J. Mcleod, H.J. Donahue, P.E. Levin, M.A. Fontaine, C.T. Rubin, Electric-fieldsmodulate bone cell-function in a density-dependent manner, J. Bone Miner.Res. 8 (1993) 977–984.

[10] H.S. Baek, Y.H. Park, C.S. Ki, J.C. Park, D.K. Rah, Enhanced chondrogenic responsesof articular chondrocytes onto porous silk fibroin scaffolds treated withmicrowave-induced argon plasma, Surf. Coat. Technol. 202 (2008) 5794–5797.

[11] X. Zhang, M.R. Reagan, D.L. Kaplan, Electrospun silk biomaterial scaffolds for re-generative medicine, Adv. Drug Deliv. Rev. 61 (2009) 988–1006.

[12] M.M. Hohman, M. Shin, G. Rutledge, M.P. Brenner, Electrospinning and electrical-ly forced jets. I. Stability theory, Phys. Fluids 13 (2001) 2201–2220.

[13] K.E. Park, S.Y. Jung, S.J. Lee, B.M. Min, W.H. Park, Biomimetic nanofibrous scaf-folds: preparation and characterization of chitin/silk fibroin blend nanofibers,Int. J. Biol. Macromol. 38 (2006) 165–173.

[14] I.S. Yeo, J.E. Oh, L. Jeong, T.S. Lee, S.J. Lee,W.H. Park, B.M. Min, Collagen-based biomi-metic nanofibrous scaffolds: preparation and characterization of collagen/silk fibro-in bicomponent nanofibrous structures, Biomacromolecules 9 (2008) 1106–1116.

[15] M. Wang, J.H. Yu, D.L. Kaplan, G.C. Rutledge, Production of submicron diametersilk fibers under benign processing conditions by two-fluid electrospinning, Mac-romolecules 39 (2006) 1102–1107.

[16] C.M. Li, C. Vepari, H.J. Jin, H.J. Kim, D.L. Kaplan, Electrospun silk-BMP-2 scaffoldsfor bone tissue engineering, Biomaterials 27 (2006) 3115–3124.

[17] H. Liu, J. Qian, Y. Liu, T. Yu, J. Deng, Immobilization of glucose oxidase in the compos-ite membrane of regenerated silk fibroin and poly(vinyl alcohol): application to anamperometric glucose sensor, Bioelectrochem. Bioenerg. 39 (1996) 303–308.

[18] N.K. Guimard, N. Gomez, C.E. Schmidt, Conducting polymers in biomedical engi-neering, Prog. Polym. Sci. 32 (2007) 876–921.

[19] R. Ravichandran, S. Sundarrajan, J.R. Venugopal, S. Mukherjee, S. Ramakrishna,Applications of conducting polymers and their issues in biomedical engineering,J. R. Soc. Interface 7 (2010) S559–S579.

[20] I.S. Chronakis, S. Grapenson, A. Jakob, Conductive polypyrrole nanofibers via electro-spinning: electrical and morphological properties, Polymer 47 (2006) 1597–1603.

[21] J.Y. Lee, C.A. Bashur, A.S. Goldstein, C.E. Schmidt, Polypyrrole-coated electrospunPLGA nanofibers for neural tissue applications, Biomaterials 30 (2009)4325–4335.

[22] J.E. Collazos-Castro, J.L. Polo, G.R. Hernandez-Labrado, V. Padial-Canete, C. Garcia-Rama, Bioelectrochemical control of neural cell development on conducting poly-mers, Biomaterials 31 (2010) 9244–9255.

[23] M.N. Akieh, W.E. Price, J. Bobacka, A. Ivaska, S.F. Ralph, Ion exchange behaviourand charge compensation mechanism of polypyrrole in electrolytes containingmono-, di- and trivalent metal ions, Synth. Met. 159 (2009) 2590–2598.

[24] T.F. Otero, Soft, wet, and reactive polymers. Sensing artificial muscles and confor-mational energy, J. Mater. Chem. 19 (2009) 681–689.

[25] L.X. Wang, X.G. Li, Y.L. Yang, Preparation, properties and applications of polypyr-roles, React. Funct. Polym. 47 (2001) 125–139.

[26] K.S. Rho, L. Jeong, G. Lee, B.M. Seo, Y.J. Park, S.D. Hong, S. Roh, et al., Electrospin-ning of collagen nanofibers: effects on the behavior of normal human keratino-cytes and early-stage wound healing, Biomaterials 27 (2006) 1452–1461.

[27] T.F. Otero, M.J. Ariza, Revisiting the electrochemical and polymeric behavior of apolypyrrole free-standing electrode in aqueous solution, J. Phys. Chem. B 107(2003) 13954–13961.

[28] X.M. Ren, P.G. Pickup, Ion-transport in polypyrrole and a polypyrrole polyanioncomposite, J. Phys. Chem. 97 (1993) 5356–5362.

[29] T.F. Otero, R. Abadias, Poly(3-methylthiophene) oxidation under chemical con-trol. Rate coefficients change with prepolarization potentials of reduction, J. Elec-troanal. Chem. 610 (2007) 96–101.

[30] T.F. Otero, F. Santos, Polythiophene oxidation: rate coefficients, activation energyand conformational energies, Electrochim. Acta 53 (2008) 3166–3174.

[31] T.F. Otero, J.M.G. de Otazo, Polypyrrole oxidation: kinetic coefficients, activationenergy and conformational energy, Synth. Met. 159 (2009) 681–688.

[32] H. Wang, H.L. Shao, X.C. Hu, Structure of silk fibroin fibers made by an electro-spinning process from a silk fibroin aqueous solution, J. Appl. Polym. Sci. 101(2006) 961–968.

[33] Y.Y. Xia, Y. Lu, Fabrication and properties of conductive conjugated polymers/silkfibroin composite fibers, Compos. Sci. Technol. 68 (2008) 1471–1479.

[34] I. Cucchi, A. Boschi, C. Arosio, F. Bertini, G. Freddi, M. Catellani, Bio-based conduc-tive composites: preparation and properties of polypyrrole (PPy)-coated silk fab-rics, Synth. Met. 159 (2009) 246–253.

[35] A.J. Meinel, K.E. Kubow, E. Klotzsch, M. Garcia-Fuentes, M.L. Smith, V. Vogel, H.P.Merkle, et al., Optimization strategies for electrospun silk fibroin tissue engineer-ing scaffolds, Biomaterials 30 (2009) 3058–3067.