Materials Science and Engineering C 52 (2015) 121–128

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Biocompatible xanthan/polypyrrole scaffolds for tissue engineering Vania Blasques Bueno, Suelen Harumi Takahashi, Luiz Henrique Catalani, Susana Ines Cordoba de Torresi, Denise Freitas Siqueira Petri ⁎ Instituto de Química, Universidade de São Paulo, P.O. Box 26077, São Paulo, SP 05513-970, Brazil

a r t i c l e

i n f o

Article history: Received 5 November 2014 Received in revised form 16 February 2015 Accepted 20 March 2015 Available online 21 March 2015 Keywords: Xanthan Polypyrrole Scaffolds Fibroblast Magnetic field

a b s t r a c t Polypyrrole (PPy) was electropolymerized in xanthan hydrogels (XCA), resulting in electroactive XCAPPy scaffolds with (15 ± 3) wt.% PPy and (40 ± 10) μm thick. The physicochemical characterization of hybrid XCAPPy scaffolds was performed by means of cyclic voltammetry, swelling tests, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), thermogravimetric analyses (TGA), scanning electron microscopy (SEM), atomic force microscopy (AFM) and tensile tests. XCAPPy swelled ~ 80% less than XCA. FTIR spectra and thermal analyses did not evidence strong interaction between PPy and XCA matrix. XCAPPy presented a porous stratified structure resulting from the arrangement of PPy chains parallel to XCA surface. Under stress XCAPPy presented larger strain than neat XCA probably due to the sliding of planar PPy chains. The adhesion and proliferation of fibroblasts onto XCA and XCAPPy were evaluated in the absence and in the presence of external magnetic field (EMF) of 0.4 T, after one day, 7 days, 14 days and 21 days. Fibroblast proliferation was more pronounced onto XCAPPy than onto XCA, due to its higher hydrophobicity and surface roughness. EMF stimulated cell proliferation onto both scaffolds. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Polysaccharides are important materials to create scaffolds for tissue engineering because they are biocompatible, abundant in nature and they can form hydrogels, which are similar to biological systems [1–4]. Xanthan gum is a high molecular weight polysaccharide with branched chains and acidic characteristics, produced by Xanthomonas campestris and with large industrial applications [5]. It is composed by D-glucosyl, D-mannosyl,

and D-glucuronyl acid residues in a 2:2:1 molar ratio and variable proportions of O-acetyl and pyruvyl residues. Side-chains consist of a trisaccharide composed of mannose (β-1,4) glucuronic acid (β1,2) mannose, attached to alternate glucose residues in the backbone by α-1,3 linkages. The deprotonation of O-acetyl and pyruvyl residues at pH N 4.5 creates negative charges along the xanthan chains, which in the presence of Ca2 + ions build physical networks [6–8]. Xanthan chemical networks easily produced by the reaction with citric acid, an efficient nontoxic crosslinker for polysaccharides [9,10], behave as hydrogels in the pH range between 4 and 10. Such xanthan hydrogels, here coded as XCA, have high negative charge density at pH N 4.5 and are bactericidal, if loaded with lysozymes [11]. In combination with xanthan-nanohydroxyapatite particles or their equivalent strontium substituted, XCA hydrogels were used successfully as scaffolds for osteoblast growth and induced high alkaline phosphatase activity [12]. Fibroblast proliferation onto nanocomposites of XCA hydrogels and ⁎ Corresponding author. E-mail address: [email protected] (D.F.S. Petri).

http://dx.doi.org/10.1016/j.msec.2015.03.023 0928-4931/© 2015 Elsevier B.V. All rights reserved.

magnetite nanoparticles was favored, particularly under magnetic field of 0.4 T [13]. Hydrogels of binary mixtures of xanthan and chitosan (1:1) [14] or quaternary blends of xanthan, konjac gum, iotacarrageenan and kappa-carrageenan [15] have also been successfully used as scaffolds in the treatment of skin lesions. Conductive polymers are also a promising class of materials for biomedical applications, because their electrically modulated properties could be engineered for biomedical devices [16]. Some examples include biosensor technologies [17], drug delivery systems [16,18,19] and substrates for neural implants [20,21]. Polypyrrole (PPy) is an interesting conducting polymer for biological and biomedical use due to its easy production, cytocompatibility, environmental stability and electrical conductivity, which can be controlled by the doping degree [22]. The literature shows reports about successful combinations of PPy with polysaccharides for tissue engineering. For instance, chitosan/PPy– alginate composite scaffolds can serve as substrates for bone tissue regeneration [23], membranes of PPy/heparin/poly(L-lactic acid) under electrical stimulation in the range of 100 mV mm−1 and 300 mV mm−1 favored the proliferation of osteoblasts [24], neuronal cells responded positively to PPy/bacterial cellulose scaffolds [21, 25]. In the view of these reports, in the present work PPy was electropolymerized in xanthan hydrogels (XCA) to produce hybrid functional materials. The resulting XCAPPy hybrid materials were characterized by means of cyclic voltammetry, swelling tests, Fourier transform infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), thermogravimetric analyses (TGA), scanning electron microscopy (SEM), atomic force microscopy (AFM) and tensile tests. The in vitro

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adhesion and proliferation of fibroblasts onto XCA and XCAPPy were evaluated in the absence and in the presence of external magnetic field (0.4 T).

parameters for electropolymerization of PPy in hydrogels of poly(acrylic acid) [18] or polyacrylamide [19]. 2.4. XCAPPy characterization

2. Experimental 2.1. Materials Commercial xanthan (Kelzan®, CP Kelco, USA, degree of pyruvate = 0.38, degree of acetyl = 0.41, Mv ~ 1. 106 g mol−1, degree of polymerization ~ 1072) was used as received. Citric acid (Synth, Brazil) was recrystallized twice before use. NaNO3 (Synth, Brazil) was used as received. Pyrrole monomer (Sigma-Aldrich) was distilled using fractional distillation method prior to use. Deionized water was used in all experiments. The human fibroblast cells were obtained from the foreskins of University Hospital. The Ethics Committee of HU (HU CEP Case943/09) approved the process. 2.2. Xanthan (XCA) hydrogel preparation Xanthan hydrogels were prepared as follows: xanthan films were produced as described elsewhere [10], by casting a 6 g L−1 xanthan aqueous solution in the presence of citric acid at 0.3 g L−1. The solutions were homogenized with an Ika Turrax® stirrer at 18,000 rpm for 3 min and submitted to centrifugation for 5 min at 3600 rpm in order to remove air bubbles prior to casting. Crosslinking was achieved by heating the films at 165 °C for 7 min. The resulting xanthan hydrogels were swollen in water at 70 °C, for 24 h to remove sol fraction and dried at 45 °C, for 24 h. 2.3. XCAPPy preparation The conducting polymer was electrochemically polymerized into XCA hydrogel matrices, according to scheme in Fig. 1. Swollen XCA samples presented high adhesion on tin-doped indium oxide (ITO) surfaces. Thus XCA swollen samples (5 cm × 2 cm) were deposited on ITO and dried at room temperature during 24 h. After drying the XCA films remained firmly attached to ITO surfaces. XCA covered ITO was immersed into the polymerization medium (pyrrole at 0.4 mol L−1 in sodium nitrate solution 1.0 mol L−1) for 15 min prior to reaction. Then, a constant potential of +0.6 V was applied until the charge achieved a value of 1.54 C cm−2 to form XCAPPy hybrid hydrogel. The reference and counter electrodes used were Ag/AgCl (KCl(sat)) and a platinum sheet, respectively. All the experiments were carried out with a potentiostat/galvanostat Autolab PSTAT 30. After polymerization XCAPPy/ITO system was immersed in deionized water for 24 h to remove unreacted substances. The XCAPPy samples were dried at room temperature and detached from ITO surfaces. The electropolymerization conditions were chosen based on previous reports about the optimization of

The electrical properties of XCAPPy films were analyzed by cyclic voltammetry in NaNO3 1 mol L−1 electrolytic solution, sweeping the potential from −0.75 V to 0.75 V, at different scan rates (5, 25, 50 and 100 mV s− 1). All experiments were carried out with a potentiostat/ galvanostat Autolab PSTAT 30; the working electrodes were the XCAPPy films and the counter and reference electrodes were those used for the polymerization process. Swelling degree at equilibrium (Q) was calculated according to Eq. (1): Q¼

mswollengel −mdriedgel mwater ¼ mdriedgel mdriedgel

ð1Þ

where mdriedgel is the mass of dried hydrogel, mwater is the amount of water absorbed by the gel and mswollengel is the mass of swollen hydrogel. Fourier transform infrared (FTIR) spectra were obtained in a Bomem MB100 equipment with the resolution of 4 cm−1 and 32 scans per spectrum. Samples were prepared using KBr pellets. Thermal behavior was investigated by differential scanning calorimetry (DSC, TA-DSC Q10V9.0 equipment), according to the procedure described by Guru and coworkers [26] for xanthan. The samples underwent heating/ cooling/heating cycle. In first cycle of measurements, samples were heated up to 90 °C and equilibrated to remove remaining water content in the hybrid material films, then cooled down to −10 °C and reheated up to 250 °C. The heating/cooling/heating rate was set at 10 °C/min. Samples (~3 mg) were placed inside hermetically closed Al pans. Thermogravimetric analyses (TGA) were performed in a TGA-STA i1500 equipment. For TGA analyses the samples were dried in an oven at 50 °C until constant weight and kept in desiccator. They were removed from desiccator just prior to the measurements. The samples (~10 mg) were heated from 25 °C to 900 °C, at heating rate of 10 °C/min, under dynamic N2 atmosphere (50 mL/min), using Pt crucibles. SEM analyses were performed in a Jeol microscope FEG7401F equipped with a FieldEmission Gun. Samples were prepared by cryo-fracturing freeze-dried hydrogels. Resultant surfaces were analyzed after gold coating (sputtering). Atomic force microscopy (AFM) analyses were performed with a PICO SPM-LE (Molecular Imaging) microscope in intermittent contact mode in air at room temperature, using silicon cantilevers with resonance frequency close to 300 kHz. Areas of 1 μm × 1 μm were scanned with a resolution of 512 × 512 pixels. Image processing and the determination of the root mean square (rms) roughness were performed using the Pico Scan software. Mechanical properties were evaluated in a DMA Q800-TA Instruments for dried films 0.010 mm

Fig. 1. Schematic representation of the experimental setup for the production of XCAPPy hybrid materials.

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5 4

j / mA cm-2

Dried hydrogel samples were cut in square scaffold format (36 mm2) and exposed to UV light during 15 min each side for sterilization. Then the samples were placed in 24 well cell culture plates (Costar, Corning, NY, USA) and wet with DMEM supplemented with 10% of fetal bovine serum (FBS) containing penicillin (100 IU mL−1) and streptomycin (100 mg mL−1) for 24 h before cell seeding. The medium was removed and human fibroblasts were seeded at a density of 4.4 × 104 cells cm−2 in 25 μL of supplemented DMEM. After 2 h of incubation, 250 μL of culture medium (supplemented DMEM) was added. The samples were incubated at 37 °C and 5% CO2 atmosphere and the complete media were refreshed every 2 days. For SEM analyses, membranes were rinsed with phosphate buffered saline (PBS), fixed with formalin (10%) for 15 min, dehydrated in aqueous ethanol solution by washing 10 min with concentrations of 25, 50, 70, 90, 95, and 100 vol.% ethanol and dried. For adhesion and proliferation assays [27], scaffolds were rinsed once with PBS solution and placed in a new well plate containing 300 μL of DMEM with MTT (0.5 mg mL−1) in every well. After 3 h, solution was removed and 1 mL of DMSO was added to each well to dissolve MTTformazan crystals. Solutions were diluted to 4 mL to respect Lambert– Beer linearity. Aliquots (500 μL) were taken in order to measure the absorbance at 570 nm (Shimadzu Multispec 1501). MTT assays were done in quadruplicate (n = 4). In order to evaluate the effect of external magnetic field (EMF) on the cellular adhesion and proliferation, the cell culture plates were modified with neodymium magnet (0.4 T) arrays placed under the scaffolds (Supplementary material, Fig. SM1). Experimental data were given as mean values with the corresponding standard deviations for four replicates (n = 4) after subtraction from control experiments (absorbance under the same conditions in the absence of cells). Data significant comparisons were performed using the one-way ANOVA test using Statistics Calculator software. The F-ratio was used to compare the variance between the groups. Significance was defined as a probability of the F-ratio (p-value) b 0.05.

6

3 2 1 0 0

1

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7

time / min

b

12

8

j / mA cm-2

2.5. Cellular adhesion and proliferation assay

a

4

0

-4 XCA XCA/PPy

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-0.6

-0.4

-0.2

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0.2

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E/V

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5 mV s -1 25 mV s -1 50 mV s -1 100 mV s

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j / mA cm-2

thick with rectangular dimensions (2 mm × 5 mm between the grips). The samples were dried in an oven at 50 °C until constant weight and kept in desiccator. They were removed from desiccator just prior to the tensile tests.

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6 0 -6 -12

3. Results 3.1. Production and characterization of XCAPPy XCAPPy hybrid materials were produced by electrochemical polymerization of pyrrole inside the XCA membranes. Initially, colorless membranes (Fig. 1) acquired a grayish color, which became black (Fig. 1) as the formation of polypyrrole took place. The mean thickness of dried XCAPPy films amounted to 40 ± 10 μm and the content of PPy amounted to (15 ± 3) wt.%, as evidenced by gravimetric analyses. Samples with lower PPy contents were not homogeneous (visually) and those with larger PPy contents presented very low swelling degree. Therefore, all results refer to XCAPPy samples with (15 ± 3) wt.% PPy. Fig. 2a shows a typical chronoamperogram obtained for electropolymerization of pyrrole inside the XCA hydrogel. Initially the current density increased with time, indicating the formation of an electroactive material, until reached jmax (5.55 mA cm−2) at tmax (1.8 min). After this, current density decreased due to the decrease of the electroactive area caused by the coalescence of polypyrrole nucleation sites. Then, current density stabilized and from this point on, the reaction was predominantly controlled by mass transport [28]. Fig. 2b shows the j/E potentiodynamic profiles of XCA and XCAPPy hydrogels. Whereas no electroactivity could be observed for XCA hydrogels, the profile obtained for XCAPPy was similar to the voltammetric profiles of pure polypyrrole, already reported in the literature [29]. The oxidation and reduction

-18 -0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

E/V Fig. 2. (a) Chronoamperogram obtained during polypyrrol deposition onto XCA hydrogel in NaNO3 1.0 mol L−1 and pyrrole 0.4 mol L−1 solution. (b) Potentiodynamic profiles of XCA hydrogel and XCAPPy in NaNO3 1.0 mol L−1 solution. Scan rate: 0.05 V s−1. (c) Potentiodynamic profiles of XCAPPy in NaNO3 1.0 mol L−1 solution at various scan rates.

peaks of PPy, observed at 0.2 and −0.4 V, respectively, were observed at about 0.2 and − 0.3 V, respectively, in the XCAPPy hybrid material. These peaks are associated with the oxidation/reduction processes that are accompanied with the expulsion/injection of anions, cations and water from the hybrid material [18]. The modification in the reduction peak suggests that xanthan chains affect the electrochemical behavior of polypyrrole, as observed for nanocomposite particles made Table 1 Swelling degree Q, Young's modulus (E), stress at break (σ) and elongation at break (ε) measured for XCA and XCAPPy films. Sample

Q

E (GPa)

σ (MPa)

ε (%)

XCA XCAPPy

31 ± 3 4.7 ± 0.9

1.7 ± 0.3 0.35 ± 0.08

14 ± 4 16 ± 4

0.5 ± 0.1 3±1

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a

b

100

96

transmittance (%)

transmittance (%)

PPy

XCAPPy

80

60 XCA 4000

PPy XCAPPy

88 80 XCA

72 64

3000

2000

1750

1000

Wavenumber (cm-1)

1500

1250

Wavenumber (cm-1)

Fig. 3. FTIR spectra obtained for PPy (black line), XCA (red line) and XCAPPy (blue line). Spectral ranges (a) from 500 cm−1 to 4000 cm−1 and (b) from 1250 cm−1 to 1875 cm−1, the dash lines indicate the characteristic bands. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

molecules, which were not removed by drying in the oven; (ii) a second stage with small weight loss at ~230 °C, attributed to the removal of small volatile molecules; and (iii) a major weight loss at ~ 310 °C, due to chain degradation, which continued up to 800 °C. The amounts of ashes resulting from XCA and XCAPPy degradation were similar, namely, 35% and 29%, respectively. A similar thermal behavior was observed for composites of bacterial cellulose [21] or xanthan [30] and chemically polymerized PPy. The thermal degradation of pure PPy presented three significant weight losses, namely, at

a

100

PPy XCA XCAPPy

weight loss (%)

75

50

25

0 0

200

400

600

800

o

T ( C)

b

0.8

XCA PPy XCAPPy

0.6

dTG

for PPy and xanthan [30]. This experiment is a clear indication of the electroactivity of the XCAPPy material and that the conducting polymer chains formed an interpenetrating network with the XCA matrix. Fig. 2c shows the j/E potentiodynamic profiles at different sweep rates. The difference between the anodic and cathodic current peak potentials (ΔEp) increased with the sweep rate, which is characteristic of a quasireversible reaction and its dependence on the diffusion of ions inside the polymeric matrix to compensate the electrical charge injected. Hydrogel ability to absorb water and biological fluids depends on the chain flexibility, hydrophilic nature of the polymeric network and the crosslinking density [31]. The diffusion mechanism of water into XCA was determined by means of tensiometry; initially the diffusion was stereoselective, controlled by wicking properties, and then it changed to anomalous (XCA) behavior [10]. The swelling degree values (Q) determined for XCA and XCAPPy hydrogels amounted to 31 ± 3 and 4.7 ± 0.9, respectively, as presented in Table 1. The presence of PPy, a more hydrophobic polymer [32] decreased ~ 80% the swelling ability of XCA. The FTIR spectra of PPy, XCA and XCAPPy are presented in Fig. 3a and b. The PPy spectra presented the characteristic absorption bands in the region 3600–3000 cm− 1 (NH vibrational stretching), at 1632 cm− 1 and 1385 cm− 1, which were assigned to the C_C stretching and C\N bending [33], respectively. XCA spectra presented the typical bands in the region 3600–3000 cm−1 (OH vibrational stretching), at 2896 cm−1 (asymmetrical CH stretching), 1726 cm−1 and 1647 cm−1 (C_O stretching acidic and ester groups present in xanthan network), and 1427 cm−1 (CH bending), respectively. The spectra obtained for the XCAPPy presented the characteristic bands of XCA and PPy. The broadening of the band at ~ 1640 cm− 1 (Fig. 3b) is probably due to the overlapping of XCA carbonyl absorption band and PPy C_C stretching vibrations. The spectra obtained for XCAPPy films presented no shift of the characteristic band positions in comparison to the spectra of pure XCA or PPy, indicating (i) the absence of H bonding between both polymers and (ii) only physical interaction between both polymers. In a similar way FTIR spectra obtained for nanoparticles of xanthan and PPy, which were chemically synthesized, presented typical absorption bands of each polymer, but there was no evidence of strong interaction between PPy and xanthan [30]. The melting (peak) temperature (Tmelting) of XCAPPy was observed at 104 °C, which lies between the Tmelting of PPy (97 °C) and XCA hydrogel (123 °C), as determined by DSC analyses (Supplementary material, Fig. SM2). Thermogravimetric (TGA) and the corresponding dTG curves determined for neat XCA, neat PPy and XCAPPy hybrid material are presented in Fig. 4a and b, respectively. XCA and XCAPPy underwent similar thermal decompositions, which can be described as follows: (i) an initial stage (up to 100 °C), where the weight loss corresponded to the release of residual adsorbed water

0.4

0.2

0.0 0

200

400

600

800

T (oC) Fig. 4. (a) TG and (b) dTG curves of XCA (red), PPy (black) and XCAPPy (blue). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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a

b

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c 15

z (nm)

10

5

0

10 µm

0

500

1000

cross line (nm) Fig. 5. (a) Typical SEM image of cryofracture surface of XCA, (b) topographic AFM image (1.1 μm × 1.1 μm) of XCA with (c) the corresponding cross section (white line in panel b).

b

a

c 150

z (nm)

100

50

10 µm 0 0

500

1000

cross line (nm)

10 µm

Fig. 6. (a) SEM images of cryofracture surface of XCAPPy at two different magnifications, (b) topographic AFM image (1.0 μm × 1.0 μm) of XCAPPy (c) the corresponding cross section (white line in panel b).

95 °C, 350 °C and 707 °C, which were assigned to moisture and volatile molecules, and chain degradation processes, respectively, remaining at 800 °C 25% of ashes. SEM images of cryofracture surface of freeze-dried XCA and XCAPPy hybrid materials are shown in Figs. 5a and 6a, respectively. XCA presented micrometric pores, whereas XCAPPy revealed a porous stratified structure. The latter might be resulting from the combination of two features, namely, the XCA pristine porous structure and the arrangement of PPy chains parallel to XCA surface, which is favored by the planar nature of PPy chains [34]. The structures observed for XCAPPy are more compact than those observed for neat XCA, corroborating with the low Q value (Table 1). AFM images taken from the top side of XCA and XCAPPy, Figs. 5b and 6b, respectively, revealed that after electropolymerization of PPy the surfaces became rougher, since the rms values increased from (6 ± 2) nm to (40 ± 2) nm and the peak to valley distances increased one order of magnitude, as shown in Figs. 5c and 6c, respectively. Fig. 7 shows typical stress–strain curves determined for XCA and XCAPPy films. The Young's modulus (E) values determined as the slopes of linear fits (dash lines) are presented in Table 1. The mean E value of pure XCA was determined as (1.7 ± 0.3) GPa, in agreement with previous report [10]. It is fivefold higher the mean E value found for XCAPPy hybrid materials, namely, (0.35 ± 0.08) GPa, as shown in Table 1. The stress–strain curves in Fig. 7 shows that under the same stress XCAPPy films elongated much more than XCA films; at break they presented mean elongation (ε) values of (3 ± 1) % and (0.5 ± 0.1) %, respectively. The increase in ε values might be attributed to the sliding of planar PPy chains in the physical network, evidenced in the stratified structure (Fig. 6a). The mean values of stress at break (σ) measured for XCA hydrogels and XCAPPy hydrogel were similar, (14 ± 4) MPa and (16 ± 4) MPa, respectively, revealing that the crosslinked xanthan chains preserve their structure after PPy polymerization. The tensile

properties of neat PPy electropolymerized in the absence of XCA were not evaluated because PPy chains did not form continuous films. 3.2. Cell adhesion and proliferation The adhesion and proliferation of fibroblasts onto XCA and XCAPPy were tested in the absence and in the presence of external magnetic field (EMF) of 0.4 T after one day, after 7 days, 14 days and 21 days, as shown in Fig. 8. Cell adhesion was evaluated after one day. In the absence of EMF the cell adhesion on XCA was very low. XCA has high

Fig. 7. Stress–strain curves determined for XCA and XCAPPy films. The error bars correspond to the standard deviation of mean stress values (n = 4). The dash lines correspond to the linear fits.

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Absorbance / 570 nm

1.5

d1 d7 d14 d21

With EMF

Without EMF

1.0

0.5

0.0

After 21 days, the general tendency for fibroblast proliferation onto investigated scaffolds was XCAPPy + EMF N XCA + EMF N XCAPPy N XCA. SEM images obtained for cells grown onto XCA and XCAPPy after 7 days are presented in Fig. 9. In the absence of EMF the cells were scarcely distributed on XCA; the few observed cells had spherical format (Fig. 9a). On the contrary, many cells with a more elongated format could be observed on XCA scaffolds, which were under EMF (Fig. 9b). Fig. 9c and d shows cells grown onto XCAPPy in the absence and under EMF, respectively. Analyses of cell morphology and sizes were avoided because cell agglomeration might take place upon drying. The protuberant regions observed in Fig. 9c and d stem from the interpenetrating network formation (Supplementary material, SM4). 4. Discussion

XCA

XCA/PPy

XCA

XCA/PPy

Fig. 8. Mean values with corresponding standard deviations determined for fibroblast proliferation (MTT test) onto XCA and XCAPPy in the absence and in the presence of EMF. All values were subtracted from control values.

negative charge density [11], which might cause electrostatic repulsion to proteins responsible for cell attachment. Cells adhered better onto XCAPPy scaffolds than on XCA because they are rougher and more hydrophobic than XCA. On the other hand, the cell adhesion onto XCA under EMF was similar to that onto XCAPPy scaffolds. The EMF might interact with the XCA electric field, stimulating cell attachments. In the absence of EMF, fibroblast proliferation onto neat XCA or XCAPPy hydrogels in the absence of EMF increased considerably after 7 days. Nevertheless, on XCA or XCAPPy it did not increase significantly any more after 14 days or longer, probably due to confluency. The differences between fibroblast proliferation onto neat XCA or XCAPPy hydrogels were statistically analyzed by ANOVA (Supplementary material, SM3). The fibroblast proliferation was always significantly larger (p b 0.05) on XCAPPy than on XCA hydrogels because the former are rougher and more hydrophobic than the latter. Under EMF, after 7 days there was no significant difference (p N 0.05) between cell proliferation onto XCAPPy and neat XCA. The differences became significant after 14 or 21 days of proliferation.

The combination of xanthan, a non-toxic, biocompatible and biodegradable polysaccharide [35], with PPy, a cytocompatible conducting polymer, gave rise to more hydrophobic hydrogels with smaller swelling degree, but with larger elasticity, in comparison to neat XCA hydrogels. Circular dichroism measurements revealed that in swollen XCA hydrogels xanthan chains assume disordered conformation (coils), exposing a large number of negative charges stemming from carboxylate groups [11]. The hydrophilicity of XCA favors its adhesion to ITO surface. The pyrrole positively charged repeating units tend to polymerize close to ITO surface and to the XCA negatively charges, which act as dopants for PPy. The planar nature of PPy favors the formation of porous stratified structures, as observed in Fig. 6a. In a similar way PPy electropolymerized in hydrogels of poly(acrylic acid) [18] or polyacrylamide [19] formed interpenetrating networks. In such networks the concentration of PPy tends to be closer to the electrode (ITO) surface, then it decreases as the distance from the electrode surface increases [18,19], as depicted in Fig. 10. Studies about the effects of electrical stimulation on cell proliferation using scaffolds made of conductive PPy in combination with poly(Llactide) and heparin showed that the intensity and duration of electrical stimulation modulated cell proliferation [23,36,37]; for instance, significant increase in osteoblast proliferation could be observed after 6 h of electric stimulation at 200 mV/mm, but higher electrical stimulation

a

b

c

d

Fig. 9. SEM images obtained for fibroblasts grown after seven days onto (a) XCA without EMF, (b) XCA under EMF, (c) XCAPPy in the absence of EMF and (d) XCAPPy under EMF. The scale bar corresponds to 100 μm.

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Fig. 10. Schematic representation of xanthan chains (blue) crosslinked with citric acid (red) and electropolymerized PPy chains (black). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

suppressed cell viability [24]. Chondroitin sulfate (CS)-doped PPy was used to coat poly-D-lactide (PLA) scaffolds; under electrical stimulation the proliferation and osteogenic differentiation of human adipose stem cells were superior to that observed onto neat PLA scaffolds [38]. The results presented in Fig. 8 evidenced that the proliferation of fibroblasts onto XCA or XCAPPy was stimulated by EMF of 0.4 T. After 21 days the fibroblast proliferation onto XCAPPy stimulated by EMF was the largest, followed by cell proliferation on pure XCA hydrogels stimulated by EMF, which achieved the same level as XCAPPy without EMF. The less efficient proliferation was on neat XCA without EMF. One relevant issue is how to correlate the type of scaffold, EMF and cell proliferation. The negative charge density in the XCA hydrogels is larger than in XCAPPy because part of the negative charges acts as dopants for PPy. One important observation is that in comparison to the situation without EMF, the fibroblast viability onto neat XCA doubled under the EMF stimulation, whereas onto XCAPPy the EMF caused an increase of 42%. These findings indicate that the magnetic stimulus for cell proliferation was observed for both scaffolds, but it was more efficient in scaffolds with higher charge density. The Lorenz force, which results from the sum of the electric and the magnetic forces, might act on the polarizability of ions, enhancing their hydration [39,40]. Thus, one hypothesis is that Lorenz force enhances the hydration of crucial ions (Ca2+, K+) present in the medium, altering their diffusion through the cell membrane, resulting in an increased cell proliferation. Experiments are under progress to test this hypothesis.

5. Conclusions The electropolymerization of polypyrrole (PPy) in xanthan (XCA) hydrogels yielded electroactive XCAPPy scaffolds with (15 ± 3) wt.% PPy. The stratified structure of XCAPPy films revealed the arrangement of PPy chains parallel to the surface, which caused strain enhancement in comparison to neat XCA hydrogels. XCAPPy presented larger hydrophobicity and larger surface roughness than XCA, making them better scaffolds for fibroblasts than neat XCA. The proliferation of fibroblasts onto XCA or XCAPPy was clearly stimulated by EMF of 0.4 T. These results demonstrated that scaffolds made of natural and conducting polymers in combination with EMF offer new strategies for cell proliferation.

Acknowledgments We thank Brazilian Funding Agency FAPESP (grants # 2010/13034-2 and # 2010/51219-5), CNPq (grant # 305178/2013-0), Rede Nanobiotec CAPES and Provost's Office for Research of the University of São Paulo, NAPMI-USP, for the financial support. The authors thank Dr. Ricardo Bentini for assistance during the DMA measurements and Prof. Silvya Stuchi Maria-Engler (Universidade de São Paulo, Faculdade de Ciências Farmacêuticas) for supplying the human fibroblasts.

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