Fabrication of Aligned Conducting PPy-PLLA Fiber Films and Their

College of Materials Science and Engineering, Sichuan University, Chengdu 610065, China. ACS Appl. Mater. Interfaces , 2016, 8 (20), pp 12576–12582...
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Fabrication of Aligned Conducting PPy-PLLA Fiber Films and Their Electrically Controlled Guidance and Orientation for Neurites Yuanwen Zou, Jiabang Qin, Zhongbing Huang,* Guangfu Yin, Ximing Pu, and Da He College of Materials Science and Engineering, Sichuan University, Chengdu 610065, China S Supporting Information *

ABSTRACT: Electrically conductive biomaterial scaffolds have great potential in neural tissue regeneration. In this work, an aligned conductive fibrous scaffold was prepared by electrospinning PLLA on rotating collector and chemical oxidation polymerization of pyrrole (PPy) codoped with poly(glutamic acid)/dodecyl benzenesulfonic acid sodium. The characterization results of composition, structure and mechanics of fiber films show that the existence of weak polar van der Waals’ force between PPy coating and PLLA fibers. The resistivity of aligned rough PPy-PLLA fiber film (about 800 nm of fiber diameter) at the perpendicular and parallel directions is 0.971 and 0.874 Ω m, respectively. Aligned rough PPy-PLLA fiber film could guide the extension of 68% PC12 neurites along the direction of fiber axis. Under electrostimulation (ES) of 100, 200, and 400 mV/cm, median neurite lengths of differentiated PC12 on aligned fiber-films are 128, 149, and 141 μm, respectively. Furthermore, under ES of 100, 200, and 400 mV/cm, the alignment rate of neurite along the electropotential direction (angle between neurite and electropotential direction ≤10°) on random fibers film are 17, 23, and 28%, respectively, and the alignment rate of neurites along the fiber axis (angle between neurite and fiber axis ≤10°) on aligned fibers film reach to 76, 83, and 79%, respectively, indicating that the combination of ES and rough conducting aligned structure could adjust the alignment of cellular neurites along the direction of the fiber axis or electropotential. KEYWORDS: aligned conducting fibers, PPy-PLLA, electrical controlled guidance, neurites orientation, nerve regeneration might cause the failure of axon regeneration.12 Polyglutamic acid (PGlu), as a hydrophilic macromolecule, can improve the ductility and hydrophilicity of the scaffold materials. Doping PGlu into PPy could enhance the cell−scaffold interactions.9 Electrical stimulation (ES) is an effective way to improve neuronal extension and outgrowth,7 because ES could change protein adsorption and nerve cell interactions with conducting biomaterials.13 It was demonstrated that an electrical stimulus to PC12 cells cultured on PPy significantly enhanced neurites extension by 90%, compared to cells without ES.7 In addition, ES could also promote nerve growth factor (NGF)-induced neurite outgrowth and signaling. Y.-J Chang et al. found that 100 mV/mm of ES could promote the neurite outgrowth and increase the percentage of cells with neurites.14 Xie et al. prepared PPy-poly(L-lactic acid) (PLLA) and PPy-PCL (polycaprolactone) nanofibers with core−sheath structures for neural applications, and these aligned nanofibers increased dorsal root ganglia maximum neurite length by 82%, compared to those on random fibers.15 Schmidt et al. prepared the aligned and random PPy-coated polylactic-co-glycolic acid nanoscaffolds for neural tissue applications and examined the combined effect of nanofiber structures and ES, showing that ES on PC12

1. INTRODUCTION Nerve injury is one of the main reasons of human disabilities, which include losting mobility and sensory obstacle.1 The nerve grafts can bridge the transected nerves.2 Autogenous nerve grafts provide ultimate biocompatible and biodegradable nerve scaffolds for nerve tissue grafting; however, this method results in additional scarring and pain, as well as permanent sensory function loss of the donating location.3 Conventional neural repairing materials are commonly fabricated using platinum, gold, or iridium oxide electrode.4−6 However, their mechanical properties are mismatched with ambient tissue, which might lead to inflammation of surrounding tissues. In the conductive polymers for neural regeneration, polypyrrole (PPy) has been investigated because of its good biocompatibility, high electrical conductivity, flexible preparation, easy surface modification, and its ability to support cell adhesion and growth. Schmidt et al. first electrically stimulated PC12 cells with PPy films to promote neurite outgrowth from the cells.7 Subsequent studies in other groups focused on the modification of PPy with various cues (e.g., porous structure,8,9 bioactivity,10 topographical features,11 etc.) to produce multiple signals to cells for the promotion of neural tissue regeneration, demonstrating the potential application of PPys for nerve tissue engineering scaffolds. The conductivity and mechanical property of these polymers could be adjusted by different dopants, and highly stiff graft or too flexible grafts © XXXX American Chemical Society

Received: January 24, 2016 Accepted: May 4, 2016

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PLLA fibers film. Then, PPy-PLLA fibers film was washed with sufficient deionized water three times, and placed onto a clean glass slide. Finally, PPy-PLLA fibers film was dried in a vacuum oven at room temperature for 24 h. To characterize composition analysis and electrical property of electrospun PPy-PLLA fiber films, we measured FT-IR and the standard four-point probe method.25 2.3. Cell Culture. Rat PC12 cells were maintained at 37 °C in a humid, 5% CO2 incubator in RPMI1640 culture medium containing 10% heat-inactivated horse serum (Hyclone), 5% fetal bovine serum (Hyclone), and 1% penicillin-streptomycin solution (Sigma). PC12 cells as suspension cells were usually subcultured one time every 3 days. Prior to seeding, the cells were precultured with the differentiation medium including 50 ng/mL NGF for 2 days. 2.4. Design of Cell Culture Device. The assembly of electrical stimulation for PC12 on aligned PPy-PLLA fibers film was similar to the report of Lee et al.16 Each fiber film was stuck on a glass slide with a thin poly(dimethylsiloxane) (PDMS, Dow Corning Corp., Midland, MI, USA) film, then a glass well with 1.5 cm inner diameter was placed on the fiber film. The seam between the glass well and fiber film was tightly sealed with PDMS. Two Au electrodes were placed under two ends of the PPy-PLLA fiber film with the perpendicular direction to the fibers axis,26 and these culture devices of electrical stimulation were sterilized by exposure to UV light overnight. 2.5. PC12 Cell Culture on Aligned PPy-PLLA Fiber Film. Before cells were seeded, the culture device was incubated overnight in a mixed solution of rat tail type I collagen (0.06 mg/mL in 6 mM/L acetic acid) and laminin (10 μg/mL), then washed twice with sterile phosphate buffer solution (PBS). Culture medium and random PPyPLLA fibers film were used for blank control group and negative control group, respectively, and aligned PPy-PLLA fiber film was experimental group. Precultured cells with 1 × 105/well were seeded on tissue culture plate (TCP), random and aligned PPy-PLLA fibers film in a 24-well plate, then PC12 cells were cultured at 37 °C in a humid, 5% CO2 incubator. The experiment of each condition was carried out in triplicate. 2.6. Electrical Stimulation of PC12 Cell on Aligned PPy-PLLA Fiber Film. According to previous research, effect of electrical stimulation highly depends on cell type, substrate condition, and exerted intensity.16,17,27 In our experiment, different voltages (100, 200, 400, and 800 mV/cm, respectively) were exerted between two electrodes placed on aligned fiber film with precultured cells for 4 h through a constant power resource (Qianfeng Electronic Co., Shanghai, China). Then, PC12 cells were further cultured with aligned fibers film for 48 h without ES. PC12 cells on PPy-PLLA fibers film without electrical stimulation were used for control, and cultured directly for 72 h. Likewise, the experimental of each condition was carried out in triplicate. 2.7. Cell Observation. For PC12 cell staining, cells were fixed using 4% paraformaldehyde for 10 min. Cells were permeabilized in 0.1% Triton X-100 for 5 min, followed by incubating with 1% bovine serum albumin in PBS for 30 min to reduce nonspecific background staining. After cells were washed twice with PBS, nuclei were stained with 4′,6-diamidino-2-phenylindole dilactate (DAPI, Invitrogen) for 5 min, then cells were washed twice with PBS. Then PC12 cells were incubated with rhodamine phalloidin (Molecular Probes) for 30 min to stain actin filaments. Finally, cells were washed with enormous PBS and observed directly under fluorescence microscope. Neurite outgrowth was evaluated statistically with median length16 because neurite lengths were not normally distributed. Neurite length was measured as a linear distance between the cell junction and the tip of a neurite. Alignment of PC12 cells on aligned fibers was quantified by neurites major axis angle with respect to fiber axis, and all neurties within less than 10° were considered to be aligned along the fiber axis. Statistical significance between medians were calculated with chisquare distribution (p < 0.05). After 3 days of culturing, aligned PPy-PLLA fibers with cells were observed by SEM. The fibers were rinsed twice with PBS and fixed in 4% glutaraldehyde−water solution at 4 °C for 10 min. Fixed cell samples were rinsed twice with PBS and then dehydrated through a series of graded ethanol solutions (30, 50, 70, 85, 90, and 100%,

cells through the conducting nanofiber scaffolds improved neurites outgrowth and percentage of neurite-bearing cells.16 The exact mechanisms of ES are not fully understood; however, some viewpoints are speculated: redistribution of membrane proteins responding to electrical field/current,17 decrease in membrane potentials more likely to cause membrane depolarization of neurons,18 and preferential adsorption of biomolecules such as laminin and fibronectin onto the conducting nanofiber surface.13 These biocompatible and electroconducting nanofibrous scaffolds are suitable for ES on neurons to potentially promote nerve tissue regeneration. Electrospinning was a cost-effective and versatile technique that essentially employs electrostatic forces to produce polymer fibers, ranging in diameter from a few micrometers down to tens of nanometers,19,20 and different fiber collectors were available to produec the different spatial orientations of fibers (such as aligned and random).21,22 For specific tissue engineering applications, including the guidance and alignment of nerve cell growth, the most common approach has been used in aligned electrospun fibers,23 which improve control over the direction of neurites outgrowth from the cells, such as the elongation and neurites outgrowth of neural stem cells on aligned electrospun PLLA fibers.24 So PLLA could be used as a model material because of its biodegradable and noncytotoxics of the produced nanofiber. However, Effect of ES on neurites outgrowth through aligned fibers is not known. In this work, we developed a novel neural tissue engineering scaffold with electrical conductivity and topographic guidance cues, as shown in Scheme 1: first, aligned PLLA fibers were via Scheme 1. Schematic of Aligned PPy-PLLA Formation, PC12 Cell Adhesion, and Neurite Growth

electrospinning and rotating drum collection; subsequently, pyrrole monomers were polymerized on the surface of PLLA fibers. To obtain good ductility of PPy coating layer, we doped PGLu into PPy, then PC12 cells cultured on the rough aligned PPy-PLLA fibers were eletrically stimulated when exogenous NGF was added to the culture medium, and the alignment and extension of cell neurites under ES condition were explored.

2. EXPERIMENTAL SECTION 2.1. Fabrication of Electrospun Aligned PLLA Fibers. PLLA (inherent viscosity 2.28 dL/g, medical device factory, shandong) was dissolved in a mixed solution of dichloromethane (DCM) and N,Ndimethylformamide (DMF) with a volume ratio of 90/10. The polymer solution (6.8 w/v% PLLA) was electrospun with a syringe equipped with a 23 gauge steel needle using a 10 kV potential, a throw distance of 10 cm, and a syringe flow rate of 1 mL/h. Aligned PLLA fibers were obtained on an Al foil-wrapped rotating drum with 10 cm diameter at a linear velocity of 4.14 m/s, and random PLLA fibers were directly electrospun on an Al foil.16 2.2. Preparation of Conductive PPy-PLLA Fiber Film. DBS/ PGlu-codoped PPy was coated on alighed PLLA fibers by in situ chemical oxidation polymerization, similar to previous literature.25 In brief, a PLLA fibers film (30 × 30 mm2) was immersed in 20 mL of mixed solution containing pyrrole (14 mM), DBS (7 mM), and PGlu (7 mM) by ultrasonication for 30 min, which allowed the fibers film to be filled with pyrrole solution. After 30 min, 20 mL of ferric chloride solution (38 mM) was added into the mixed solution, and incubated with shaking at 4 °C for 4 h in order to deposit PPy on the aligned B

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the PLLA fiber with the thicker diameter should result from the relaxation of PLLA chains during PPy coating. The AFM image in Figure S 2d shows that the fibers diameter is 300−500 nm, and the rougher doped PPy coating layer is composed of many small irregular particles (30−100 nm diameter). Figure 1e shows FT-IR spectra of PGlu/DBS-codoped PPy particles (red cure), PPy-PLLA composite fibers (blue curve) and electrospun PLLA fibers (black curve). The strong peaks at 1757 cm−1, 1453 and 1185 cm−1 1088 cm−1 are attributed to CO strength vibration and C−O stretch vibration of PLLA, respectively;28 the strong bands at 1552 and 1033 cm−1 represent the pyrrole ring vibration.29 These characteristic peaks also appear on the FT-IR spectrum of PPy-PLLA composite fibers (shown by red arrows and black arrows, respectively), suggesting that PPy particles were successfully coated the surface of PLLA fibers. Two weak bonds at 1650 and 1710 cm−1 represent stretch vibration of amide I of PGlu on the spectra of PPy particle and PPy-PLLA composite fibers, respectively.25 A much weaker bond at 1610 cm−1 represents stretch vibration of sulfo-acid groups of DBS on the spectra of PPy particle, indicating that DBS molecules were doped into PPy NPs. However, a weaker peak of DBS and a typical peak of pyrrole ring in PPy-PLLA pattern were slightly shifted from 610 to 614 cm−1 and from 1032 to 1037 cm−1, respectively, indicating that there were interadsorption between ester groups of PLLA and sulfo-acid groups near to pyrrole rings. This result indicate that DBS of doping agents played an important role in the adhesion between PPy and PLLA, because these ions pairs of doped PPy NPs might also be adsorbed with ester groups of PLLA, leading to two typical peaks were red-shifted. Figure 1f shows the surface resistivity of random and aligned PPy-PLLA fibers film. The surface resistivity of aligned PPy-PLLA fibers film are 0.971, 0.874 Ω m at the perpendicular and parallel directions of fiber axis, respectively, and there is no significant difference between two resistivity (p > 0.05). However, the surface resistivity of random PPy-PLLA fibers film is 0.427 Ω m, which might result from the lower porosity and greater number of PPy NPs of/onto aligned PLLA fibers. In XRD patterns of three samples in Figure S3a (PLLA fibers, PPy NPs, and PPy-PLLA fibers), a broad peak located in the range of 12−40° of PPy-PLLA pattern was the typical amorphous peak, and three weaker peaks (at 16.8, 19.5, and 22.6°) should arise from the α crystal structure of PLLA, and two very weaker reflections at 21.6 and 25.4° related to fine PPy NPs.30 These results indicate that the immersion during the pyrrole polymerization partly disturbed the PLLA crystal structure through the relaxation of PLLA chains, decreasing of PLLA crystallinity degree during the pyrrole polymerization.31 13 C and 2H NMR spectra of PLLA, PPy-PLLA, and PPy NPs in Figure S3b, c shows that the pyrrole rings of PPy might be bound with PLLA via the weak polar van der Waals’ force to form coating layer. On the basis of these results, a conjugation mechnism between PPy and PLLA is proposed, as shown in Scheme S1: first, longer dodecyl-alcyl chains of DBS, as doping agents, were adsorbed onto PLLA fibers surface via weak dispersion van der Waals’ force, and hydrophilic sulfonate groups were pendant outside PLLA chain; then pyrrole monomers were oxidated by Fe3+ ions and polymerized into PPy; subsequently PPy NPs were conjugated with DBS (or PLLA) via polar van der Waals’ force between oxide of sulfonate groups (or ester groups) and hydrogen proton of amino groups, as shown by red ring in Scheme S1.

respectively, each for 20 min), then these cells were supercritical dried. PC12 cells were sputter-coated with Au prior to observation with SEM. For observation of the cells under Laser Confocal Fluorescence Microscope (LCFM), cellular nuclei were stained with DAPI, and actin filaments were stained by Alexa Fluor 488 phalloidin. After cells were fixed by 4% paraformaldehyde for 10 min, 0.1% Triton X-100 was added for 5 min to extract the cells, then 1% bovine serum albumin (BSA)-PBS solution was added to reduce nonspecific background staining. Alexa Fluor 488 phalloidin was added and kept for 20 min. After cells were washed 3 times with PBS, DAPI was added and stewed for 3−5 min. Finally, cells were washed with enormous PBS, and observed directly under LCFM.

3. RESULTS AND DISCUSSION 3.1. Surface Morphology of Aligned PPy-PLLA Fiber Film. Figure 1a−d is SEM images of aligned and random PLLA

Figure 1. SEM images of electrospun (a) aligned and (c) random PLLA fibers, and (b, d) PPy-PLLA composite fibers; (e) FT-IR spectra of three samples; (f) surface resistivity of random and aligned PPyPLLA fibers film.

fibers, and PPy-coated PLLA composite fibers. The surface of electrospun PLLA fibers (about 300 nm of fiber diameter) is smooth and uniform (shown in the inset of Figure 1a, c). PPyPLLA fibers (about 800 nm of fiber diameter) have a rough surface, resulting from the deposition of PPy NPs after the polymerization (Figure 1b, d), and good orientation of the aligned fibers is still remained (Figure 1b). Moreover, the obtained fibers are composed of PLLA fiber cores and PPy shells with complete uniformity and some PPy aggregates. Figure S1a shows the composite structure of PLLA fiber cores and PPy shell, and slight stretching leaded to the brittle cracking of PPy shell and the slight ductile extension of PLLA core fibers. The hollow structure in Figure S1b suggests the weaker adhesion force between PLLA core and PPy shell. Some PPy NPs and their aggregations onto/into of PLLA fiber in the TEM image of Figure S 1d indicate the discontinuous shell profile of PPy-PLLA composite fiber, and round pores on/in C

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ACS Applied Materials & Interfaces The results of energy-dispersive X-ray spectroscopy in Figure S4 and Table S1 clearly suggest DBS-doped PPy coating on PLLA fibers. The stress−strain curves and the statistics of yield stress and Young’s modulus in Figure S5a, b indicate that, after PPy coating, yield stress and young’s modulus of aligned PPyPLLA fiber film are about half and one-fourth of aligned PLLA fiber film, respectively. These results suggest that PPy NPs coating could lead to the strength decrease of aligned fiber film, because the disorientation or flabby of PLLA molecule chains might lead to the crystallinity decrease of PLLA fibers during the oxidation polymerization of pyrrole. The load−displacement curves of Figure S5c suggest that PPy-PLLA film was stiffer than PLLA film due to PPy coating on the surface of PLLA fibers. In the inset of Figure S4d, the mean hardness of PPy-PLLA (119 ± 6.3 MPa) is obviously larger that that of PLLA fiber films (99 ± 7.4 MPa), (larger than previous reports on modified fiber films32,33), indicating that PPy coating increased fiber hardness because of the higher strength of PPy. The rheological results in Figure S6 also shows that PPy NPs coating leaded to the increase in both elastic modulus G′ and loss modulus G″ of PLLA film at full frequencies, and the higher viscosity η* and loss modulus G″ of PPy-PLLA sample at the lower frequencies further indicate PPy linking on the PLLA fine fibers via the weaker polar Van der Waal’s force. GPC results in Table S2 show that the 30 days of immersion in PBS with 1.5 μg/mL of lysozyme leads to the decrease of number-molecular weight of PLLA fiber film (from 68 500 into 52 100) because of the PLLA degradation. However, the molecule weights of PPy in the immersion with same condition were not changed (Mn ≈ 2.41), indicating that the degradation of PPy NPs did not occur. The results of immersion test in PBS of Figure S7a further show that, the PBS immersion of 4 weeks led to the degradation of part PLLA chains (the decrease of ∼7%). 3.2. Effect of PPy-PLLA Fiber Film on Cell Neurites. Figure 2a, b shows that the fluorescent images of PC12 cultured on random and aligned PPy-PLLA fibers film, respectively. The neurites differentiated on TCP are short and have many branches, extending toward all directions (Figure S8). The shape of cells cultured on random fibers is similar to that on

TCP, and the neurites extend randomly (Figure 2a). Cells cultured on aligned fibers almost differentiate into the spindle shape with polarization and their neurites stretch along the fiber axis direction, indicating their good parallel distribution due to the inductivity of aligned fibers (Figure 2b). In Figure 2c, the median lengths of neurites from PC12 cultured on random and aligned fibers film are 65.44 and 114.73 μm, respectively. Compared with those on TCP, the shorter neurites on random fiber film should result from their weak hydrophilicity and the more porosity. The neurites lengths of aligned fibers film are significantly larger than those of culture medium and random fibers film (p < 0.05), suggesting aligned fibers film could guide cells outgrowth and their neurites extension along the direction of fiber axis. There was 68% of neurites extension along the direction of the fiber axis (angle between neurite and fiber axis direction ≤10°) on aligned fibers film, compared with 3.6% of neurites within less than 10° on random fiber film. 3.3. Effect of Electrostimulation on Cell Neurites. 3.3.1. Growth of PC12 Neurites. Figure S9b−d shows the fluorescent images of PC12 cultured on random PPy-PLLA fiber films under electrostimulation of 100−400 mV/cm. Although the neurite lengths under electrostimulation groups (67.54, 75.42, and 65.44 μm, respectively, in Figure S9e) have no significant enhancement compared with the nonstimulated group (61.41 μm), the extended rate of neurites along the direction of electropotential under these electrostimulation (angle between neurite and electropotential direction ≤10°) are 17, 23, and 28%, respectively (Figure S9f), suggesting that ES could adjust the alignment of cell neurites along the direction of electropotential. Figure 3a−d shows that the fluorescent images of PC12 cells cultured on aligned fibers film

Figure 2. Fluorescence images of PC12 cultured on (a) random and (b) aligned PPy-PLLA fibers, and median length (c) and extended distribution (d) of neurites from PC12 cultured on random and aligned PPy-PLLA fibers. Scale bars are 100 μm.

Figure 3. Fluorescence images of PC12 with electrostimulation of (a− d) 100, 200, 400, and 800 mV/cm, respectively. (e) Median length and (f) extended distribution of neurites from PC12 with under different electrostimulations. Scale bars are 100 μm. D

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ACS Applied Materials & Interfaces under different electrostimulation. There exist more spindle cells under the electrical stimulation (marked by arrows), and PC12 cells differentiated much more neurites extending along the cell polarization, compared with nonstimulated group. The median lengths of PC12 neurites under 100, 200, 400, and 800 mV/cm electrostimulation are 128.45, 149.39, 141.48, and 119.8 μm, respectively, indicating that electrical stimulation through alighed fiber film is favor of the growth of PC12 neurites. The neurites lengths of 200 and 400 mV/cm electrostimulation groups have significant enhancement, compared with nonstimulated group. The alignment rate of neurites along the fiber axis (angle between neurite and fiber axis ≤10°) on aligned fibers film reached to 76, 83, 79, and 78%, respectively, suggesting that ES enhance the rate of neurites extending along the fiber axis. 3.3.2. Effect of ES on the Neurites through Aligned Fiber Film. To investigate the synergic effect of guidance cues and electrical stimulation on neurites, we observed cells by SEM and LCFM. PC12 cultured on aligned fibers film under nonstimulation differentiates into the spindle shape with polarization and their neurites stretch along the fiber axis direction of aligned fiber films (Figure 4a, e), whereas spindle PC12 cells grow and extend along the perpendicular direction of major fibers (Figure 4b), and the filopodia adhere to the surface of parallel fiber, indicating this fiber could guide the extension of neurites. The spindle PC12 applied with ES of 200

mV/cm still grows and there are two neurites which differentiated from the polarization stretching along the direction of fiber axis (Figure 4c, f). As a leading edge detector, the filopodia (marked by white arrows in Figure 4c, f) adhere to the surface of fiber closely and guide the axon extend forward, and black arrows in Figure 4d show that some microtubule in the neurite are closely adhered on the fiber surface. In Figure 4e, only one neurite with two branches is observed, suggesting that the neurite slowly protruded without electrostimulation. However, after electrostimulation, longer axons are observed (Figure 4f). There are some slender filopodia (marked by white arrows) at the leading edge of P domain in growth cones (Figure 4f), and the red arrows in Figure 4f point out the position of the swelling central domain in the growth cone, suggesting the engorgement of organelles, which is the energy resource of complex chemical and physical reactions during axon elongation because of the electrostimulation.34 3.3.3. Mechanism Analyses of Neurites Extension under Electrical Stimulation. On the basis of previous literature16,25 and these results, elongation mechanism of differentiated neurites on rough aligned PPy-PLLA fiber film under electrical stimulation is proposed. The guidance cue of NGF induces PC12 cultured on aligned fiber film to differentiate two neurites with the polarization, which mainly stretch along the direction of fiber-axis (Figure 5a). Figures 5b−d show the cytoskeletal

Figure 5. Schematic of axon elongation from PC12 cells on aligned fibers (a) after differen- tiation, (b−d) the change of growth cone, and (e−g) their inner change of filopodia during the elongation.

strucrue schematic of neural growth cone, and C, T, and P represent central domain, transition zone, and peripheral domain, respectively. The filopodia at the leading edge of P domain are very dynamic, and could continuously extend and retract, thus they promote the growth cone to perceive extracellular guidance cues.35,36 ES in the surrounding environment (such as conducting fiber film) likely brings about the depolarization of membrane, leading action potential.7,13 The exerted ES could result in the enrichment of electric charge on the tops of the protruding PPy particles of the rough fibers surface, further enhancing the extension of filopodia actin in the P domain of neural growth cone. Moreover, the filopodia still detect the cues of adhesion molecules and growth factor, which combine with adhesion molecules (Col and LN) and NGF

Figure 4. SEM images of PC12 cells cultured on aligned PPy-PLLA fibers (a, b) without and (c, d) with electrostimulation of 200 mV/cm, and inset images show the magnified parts of corres- ponding square frames, respectively. Fluorescent images of PC12 cells cultured on aligned PPy- PLLA fibers (e) without and (f) with electrostimulation of 200 mV/cm, insets are images of two growth cones, in which white arrows show filopodia in the P zone, and red arrows show the consolidated C zone of growth cones. E

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receptors. Since filopodia are immobilized on aligned fibers by binding adhesion molecules, continuous self-assembly and disassembly of microfilament produce a force to guide the neurites stretch along the fiber-axis.35,37 The leading-edge filopodia are supplied with fresh membrane and adhesion molecules by exocytosis of cell membrane (marked by arrows). When the electrical stimulation is exerted on aligned rough fibers, electrical charge would enrich at the tops of the aligned NPs of the rough PPy shell, and further improve the aggregation of adhesion receptors and the interaction of adhesion receptors at the leading edge with adhesion molecules on the fibers.16 In addition, a great deal of chondriosome aggregated in microtubule growth channel provides enough energy and nutriment for further extension of axon (Figure 5c, d). Assembled actins result in microtubules pulled by actinmicrotubule linker, accompanied by continuously self-assemble, and microtubules elongation and filopodia extension appeared.38 Finally, the microtubules were bundled, and growth cone collapsed at the neck to form new cytoplasmic domain of the axon.35 Thus, the multiple stimuli cues, including aligned PPy particles arrangement, the rough fiber surface, and electrical stimulation, are combined to improve neurite elongation.

ACKNOWLEDGMENTS This work has been supported by the National Natural Science Foundation of China (Projects 51273122, 51173120, and 51202151).

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CONCLUSION Aligned conductive PPy-PLLA fiber film is fabricated using a simple method, involving deposition of DBS/PGlu-codoped PPy particles on electrospun aligned PLLA fibers. The results of XRD, NMR, rheological behavior, and mechanics on fiber film show PPy linking on the PLLA fibers via the weak polar van der Waals’ force. The surface resistivity of PPy-PLLA fibers film are 0.971 and 0.874 Ω m at the perpendicular and parallel directions, respectively. In vitro cells culture of PC12 cells demonstrated that the fabricated PPy-PLLA fibers film was appropriate for neuronal applications and present topographies for modulating celluar interactions. Finally, electrical stimulation of PC12 cells on aligned fibers film improved the outgrowth and extended distribution of neurites, and extended distribution of neurites could be adjusted through aligned conducting fibers film under electrical stimulation of 0−400 mV/cm, especially under the electrical stimulation of 200 mV/ cm, longer neurite outgrowth and guided the differentiated neurites stretching along the direction of fiber-axis were obtained. In addition, a preliminary mechanism of two guidance cues inducing neurites elongation and arrangement is proposed. Thus, this aligned PPy-PLLA fiber film would be a promising scaffold material for neural tissue engineering. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b00957. Methods, discussion of results, Figures S1−S9, and Table S1 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: 86-28-85413003. Fax: 8628-85413003. Notes

The authors declare no competing financial interest. F

DOI: 10.1021/acsami.6b00957 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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DOI: 10.1021/acsami.6b00957 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX