Aligned Electroactive TMV Nanofibers as Enabling Scaffold for Neural

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Aligned Electroactive TMV Nanofibers as Enabling Scaffold for Neural Tissue Engineering Yehong Wu, Sheng Feng, Xingjie Zan, Yuan Lin, and Qian Wang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.5b00884 • Publication Date (Web): 21 Sep 2015 Downloaded from http://pubs.acs.org on September 23, 2015

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Aligned Electroactive TMV Nanofibers as Enabling Scaffold for Neural Tissue Engineering Yehong Wu,a,b Sheng Feng,c Xingjie Zan,c Yuan Lin,a,* and Qian Wang,a,c a

State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry,

Chinese Academy of Sciences, Changchun 130022, P. R. China E-mail: [email protected] b

University of Chinese Academy of Sciences, Beijing, 100049, P. R. China

c

Department of Chemistry and Biochemistry, University of South Carolina, 631 Sumter Street,

Columbia, South Carolina, 29208, United States

Abstract: Electroactive nanofibers were fabricated by in situ polymerization of aniline on the surface of tobacco mosaic virus (TMV) using sodium poly(styrenesulfonate) (PSS) as dopant. These electroactive TMV/PANi/PSS nanofibers were employed to support growth of neuronal cells, resulting in augmentation of the length of neurites. In addition, the percentage of cells with neurites was increased in comparison to cells cultured on TMV-derived non-conductive nanofibers. The TMV-based electroactive nanofibers could be aligned in capillaries that could guide the outgrowth direction of neurites, increase the percentage of cells with neurites, and lead to a bipolar cellular morphology. Our results demonstrate that the electroactivity and topographical cues provided by TMV/PANi/PSS nanofibers can synergistically stimulate neural cells differentiation and neurites outgrowth, which make it a promising scaffolding material for neural tissue engineering.

Keywords: neural tissue engineering, tobacco mosaic virus, conductive nanofibers, polyaniline, cell alignment, contact cue guidance

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Introduction Peripheral nerve injury is a relatively common medical problem caused by trauma, tumor resection or reconstructive surgery. Although the peripheral nerve system has the capacity of regeneration and repair1, 2, Wallerian degeneration often occurs and results in poorly functional recovery3. Recently, the autograft has been considered as a clinical gold standard to solve

peripheral nerve injury4,

however, it is limited by donor site morbidity, need for a second surgery, and size mismatch between the donor nerve and the recipient site5-7. In the process of peripheral nerve regeneration, Schwann cells initially proliferate and align longitudinally following the formation of Büngner bands around fractured axons. The alignment of Schwann cells leads to secretion and deposition of a highly anisotropic extracellular matrix (ECM), which provides indispensable pathways for guiding axonal regeneration and bridging gap of nerve injury8. Fibrous materials fabricated from synthetic9, natural10 and composite materials11 have been extensively studied as promising materials for nerve repair, as their structures are similar to ECM fibers. Various methods have been developed to align fibrous materials for tissue engineering applications12, 13. For example, Jiang et al. reported the alignment of Matrigel to direct and stimulate axonal growth of neural cells via an external shear force14. Rong et al. fabricated thin films with aligned nanogrooves using M13 bacteriophages displaying RGD peptides via a simple shearing method, which can guide cell alignment and orient the cell outgrowth along defined directions15. Li et al. used self-assembly peptide amphiphiles (PA) aligned in the poly(lactic-co-glycolic acid) (PLGA) tube to accelerate the recovery of motor and sensory function in animals16. It is believed that the aligned fibrous materials can mimic the aligned architecture of regenerating native nerve to support and guide axonal extension, promote cell migration, guide neurite extension, and


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significantly enhance axon regeneration, compared with randomly oriented fibers17, 18. In addition, recent studies indicated that neural tissues exhibit electrical activities for modulating cellular behaviors and electrical stimulation through conductive scaffolds can promote cell adhesion, proliferation and differentiation19, 20. TMV is a kind of anisotropic nanoparticle that consists of 2130 identical protein subunits helically arranged around single strand RNA. An intact TMV is 300 nm in length, 18 nm in diameter and 4 nm in inner diameter. TMV nanoparticles represent monodispersed supramolecular assembles with organized three-dimensional architectures. The surface properties of TMV can be adjusted through chemical and genetic modification and TMV nanoparticle remains stable at temperatures up to 60 °C and at pH values between 2 and 10 21. Due to its uniformly ordered structure, well-defined shape and surface chemistry, and anisotropic shape, TMV becomes an attractive building block for multi-functional nanomaterials development22-24. Our group previously reported that rod-like nanoparticles, including TMV, gold nanorod, and bacteriophage M13, could be aligned inside glass capillaries using shear force generated by high pressure air25. We found that topographic features of aligned TMV can guide the orientation and differentiation of myoblasts. On the other hand, conductive polymers have been used to influence cellular behaviors by electrical stimulation or electroactivity of substances can influence cellular behaviors. For example, Hu and coworkers prepared a new water-soluble electroactive polymer, aniline pentamer cross-linking chitosan. This electroactive polymers could significantly promote differentiation of PC12 cells compared with pure chitosan26. Meanwhile, Sirivisoot and coworkers fabricated 3D conductive hydrogel containing electroactive PANi and collagen, which could promote adhesion and differentiation of PC12 cells without addition of nerve growth factor27. We have previously


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demonstrated that TMV could serve as the template to fabricate water-soluble, monodisperse and electroactive nanofiber with length of micrometres via in situ polymerization of aniline on its surface28. Herein we report the creation of highly aligned electroactive TMV nanofibers in large-scale inside capillary tubes by the air shear force. PC12 cells grown on this aligned nanofibers scaffold exhibited prolonged neurites and the orientation of neurites along the direction of alignment of nanofibers. Our results suggest that the electroactive TMV nanofibers are promising scaffolding materials for neural tissue regeneration.

Experimental Section Materials: The purification of TMV nanoparticles and preparation of non-electroactive or electroactive TMV nanofibers

were

carried

out

according

to

previously

reported

methods28,

29

.

Poly(diallydimethylammonium chloride) (PDDA) aqueous solution, (Mw=200000-350000), sodium poly(styrenesulfonate) (PSS, Mw=70000) and MTT agents were purchased from Sigma-Aldrich Co. Aniline was purchased from Acros Organics Co. Ammonium persulfate (APS) was purchased from Beijing Dingguo Changsheng Biotechnology Co. Ltd. Glass capillary (inner diameter ~0.15 cm, outer diameter ~0.18 cm) was purchased from Kimble Chase Co. Rat pheochromocytoma cells (PC12) was obtained from Shanghai Institute of Biochemistry and Cell Biology (SIBCB, CAS, China). Fetal bovine serum (FBS) was purchased from Biological Industries, Israel. Horse serum (HS), nerve growth factor (NGF 2.5S, murine, natural), and penicillin–streptomycin were purchased from Gibco BRL, Life Technologies. RPMI 1640 medium was purchased from Coring. All reagents were purchased from Beijing Chemical Works without any further purification.


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Substrate preparation Glass coverslips (1×1cm) were cleaned by piranha solution (7 : 3 mixture of 98% H2SO4 and 30% H2O2) at 75°C for 2 h, followed by three times washes with ultrapure water and sonication, and dried with nitrogen gas. The cleaned glass coverslip was immersed alternatively in a PDDA solution (1 mg/mL containing 0.25 M NaCl) and PSS solution (2 mg/mL containing 0.25 M NaCl) for 20 min, until (PDDA/PSS)2.5 was obtained, which the outer layer was coated with PDDA. To fabricate different coating of the surfaces, the coverslips with the PDDA as the outer layer were immersed in 0.1 mg/mL solutions of TMV, TMV/Polyaniline (denoted as TMV/PANi), electroactive TMV nanofibers (denoted as TMV/PANi/PSS) for 20 min, respectively. After that, these substrates were washed thoroughly with ultrapure water, then dried by the nitrogen gas. Mechanical characterization of TMV nanofibers Mechanical characterization of TMV nanofibers were measured according to our previously reported methods30. The solutions containing TMV, TMV/PANi, and TMV/PANi/PSS were first dropped onto a Si wafer and dried under ambient conditions (30% R.H.). For the measurement of elastic modulus, atomic force microscopy (AFM) was used to image/locate individual TMV nanofibers, followed by in situ indentation at a constant sampling rate and velocity to obtain the loading force–displacement curves. The mechanical properties for each of the three samples, 10 well-performed indentation tests were collected for data analysis. Flow assembly of TMV nanofibers in capillaries Flow assembly of TMV nanofibers (TMV, TMV/PANi, TMV/PANi/PSS) in capillaries were performed as previous reported25. Briefly, the PDDA coated capillaries were obtained by the above substrate preparation method. Typically, a 10 mL solution containing TMV nanofibers (0.1 mg/mL)


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was injected into the feeder, whose one end was connected to nitrogen tank and the other end to capillary tube. The solution was driven through the capillary tube and dried by nitrogen gas. The flow rate (5 L/min) was controlled by the gas pressure applied to the flow. Meanwhile, capillaries coated randomly oriented TMV nanofibers were fabricated by immersing in the TMV nanofiber solutions (0.1 mg/mL) for 20 min, and dried by nitrogen gas. The surface morphology was observed under AFM. The orientation of TMV nanofibers were further quantitatively assessed by analyzing angles of more than 200 nanofibers with respect to direction of flow using Image J software and then plotted in angular distribution histograms. Cell culture PC12 cells were cultured in growth medium RPMI1640 supplemented with 10% heat inactivated fetal bovine serum and 2.5% horse serum, 100U/mL of penicillin and 100 µg/mL of streptomycin at 37°C under an atmosphere of 5% CO2 for routine culture. The medium was replaced every two to three days. After reaching about 80-90% confluence, the cells were detached and viable cells were counted by trypan blue assay for subsequent experiments. Cytotoxicity evaluation The MTT assay was used to assess cell cytotoxicity of TMV, TMV/PANi, TMV/PANi/PSS nanofibers. PC12 cells were diluted to 2×104 cells/mL by growth medium, seeding onto glass coverslips coated various fibers in 24 wells plate, and the glass coverslip coated PDDA was chosen as control. The medium was replaced every two to three days. After seeding PC12 cells for 1, 3, and 5 days, MTT solution (0.5mg/mL) was added to each well, incubated for 4 h at 37°C, and then replaced by dimethyl sulfoxide (DMSO, sigma) to dissolve the formazan crystals. The absorbance of the solution was measured by the enzyme-labeled instrument (Infinite M200 PRO, Nanoquant) at


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490 nm. All groups were placed 3 repeat wells. PC12 cells on 2D substrates and within 3D capillaries First, we investigated cell behaviors of neural cells on 2D substrates. PC12 cells were diluted to 5×104 cells/mL by the differentiation medium, which is RPMI1640 supplemented with 2% horse serum +2% FBS and NGF (50 ng/mL). Then, the cells were added to different substrates placed into 24 wells plate, and glass coverslip coated PDDA was chosen as control. Optical microscope was used to observe the morphology and neurite outgrowth of cells at 1 and 3 days, respectively. Furthermore, we investigated cell behaviors of neural cells within capillaries. The PC12 cells were diluted to 1×105 cells /mL by the differentiation medium, and pipetted into the capillary tubes with aligned or randomly oriented TMV nanofibers. After 4 h, the tubes with cells were placed into 24 well plates with differentiation medium. Optical microscope was used to observe the morphology and neurite outgrowth of cells at day 1 and day 3. Three substrates or glass tubes for each condition were employed (n = 3). The neurite length was defined as the straight-line distance from the tip of the neurite to the junction between the soma and neurite base. Data was collected for the neurite lengths greater than 5 µm31. Neurite outgrowth was reported in terms of median length because neurite lengths displayed non-Gaussian distribution19, 32, 33. Also, the percentages of PC12 cells with neurite and the numbers of neurite per cell were calculated. More than 500 PC12 cells were analyzed for each condition. The orientation of neurites was quantitatively assessed by analyzing angles with respect to the direction of the air flow with Image J software and then plotted in angular distribution histograms. In addition, after 3 days culturing, the glass coverslips and capillary tubes carrying PC12 cells were washed one time with PBS, subsequently staining by fluorescein diacetate (FDA, 1 µg/mL) for 5 mins, then the


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samples were rinsed three times with PBS. After that, the samples were observed by confocal laser scanning microscopy (LSM700, Zeiss, Germany). Characterization The morphology of TMV, TMV/PANi and TMV/PANi/PSS nanofibers were observed by transmission electron microscopy (TEM) performed on a JEOL JEM-1011 electron microscope operating at an acceleration voltage of 100 kV. The samples for TEM observation were prepared by dropping 7 µL solutions of fibers (0.2 mg/mL) on carbon-coated Cu grids. After drying, the sample was negatively stained with 2% uranyl acetate. UV-Vis-spectrum (TU-1901 spectrometer, Beijing Purkinje General Instrument Co., Ltd.) was analyzed to study conductivity of TMV/PANi/PSS fibers. The coverage of nanofibers on the glass coverslips were characterized using AFM with tapping-mode by a NanoScopeIIIA MultiMode (Veeco). The water contact angle was measured by using a contact-angle meter (Krüss DSA 10-MK2). Statistical analysis Averages and standard deviations were calculated and reported from three samples per each condition. Statistical analyses (SPSS software package (version 13.0)) between two groups or multi-groups were performed using one-way ANOVA or Student’s t-test, respectively, and a value of p< 0.05 was considered to be statistically significant.

Results and Discussion Synthesis and characterization of TMV based nanofibers As shown in Figure 1b-e, conductive TMV nanofibers with several micrometers length can be readily fabricated via two steps as reported28. Here we assess conductivity of TMV nanofibers in the


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solution of ultrapure water by using UV-vis spectrum. As shown in Figure 1f, the absorption peak of TMV/PANi at 427 nm was attributed to oligoaniline in its branched form, which indicated its nonconductive nature34. For the TMV/PANi/PSS, two absorption peaks at about 410 and 800 nm were attributed to polaron band transitions, and another peak at about 335 nm was due to ð-ð* transition of the benzenoid rings28, 35. These peaks were consistent with the emeraldine salt form of PANi when using PSS as dopant, indicating that the TMV/PANi/PSS nanofiber was in its conductive nature. Moreover, the electronic property could be directly measured using scanning spreading resistance microscopy (SSRM) indicated a conductivity of 10-5 S/cm for the TMV/PANi/PSS nanofibers, which has been reported in our previous publication28.

Figure 1. (a) Structural illustration of TMV. (b) Scheme of the preparation of electroactive TMV nanofibers. TEM images of (c) wild type TMV, (d) TMV/PANi, and (e) TMV/PANi/PSS. (f) UV-vis spectra of TMV, TMV/PANi and TMV/PANi/PSS in the solution at pH 5.5. The scale bars: (c) 200 nm; (d, e) 500 nm.

PC12 cells on 2D substrates coated random TMV fibers


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TMV has been shown to be an effective building block for scaffolds that accelerates differentiation of bone marrow mesenchymal stem cells (BMSCs) when coated on 2D substrate36. Because electroactive substrates can promote the neurite outgrowth of neuron26, we hypothesized our electroactive TMV/PANi/PSS nanofibers should enhance the neurite outgrowth of neural cells. To test our hypothesis, PC12 cell was chosen to be cultured on our designed substrates, as PC12 cell is one of the most widely used cells type for neurogenesis studies37. To obtain a uniform thin film of TMV, TMV/PANi and TMV/PANi/PSS coating, the layer-by-layer (LbL) assembly technique was employed38. AFM images showed that, the wild type TMV, TMV/PANi and TMV/PANi/PSS nanofibers were homogeneously and fully covered on glass substrates. The water contact angles for substrates coated with wild type TMV, TMV/PANi and TMV/PANi/PSS nanofibers were 52.7±0.6o, 55.1±0.6o and 50.1±0.7o, respectively (Figure 2). Prior to cell culture experiments, the conductivity of TMV/PANi/PSS nanofibers at pH 7.4 was studied. As shown in Figure S1, two absorption peaks at about 410 and 800 nm were observed, which indicated that TMV/PANi/PSS nanofiber kept its conductive nature. Furthermore, the stability of the TMV nanofibers on the 2D substrates was investigated, showing that TMV nanofibers remained on all surfaces after incubation in PBS for 3 days (Figure S2). PC 12 cells were seeded on TMV nanofibers coated surfaces at a concentration of 2×104 cell/mL. As shown in Figure 3, although cells on nanofibers coated surfaces all displayed lower viability than cells on glass, these substrates showed significant proliferation over the tested period. In addition, there was no substantial difference regarding initial cell proliferation among three kinds of nanofibers on day 1. However, on day 3, the numbers of viable cells cultured on TMV and TMV/PANi/PSS coated scaffolds were higher than that on TMV/PANi scaffolds (p＜0.05). After 5


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days, the viable number of cells cultured on the surface of TMV/PANi/PSS coated substrate was significantly higher than that on TMV and TMV/PANi (p ＜ 0.05). Considering similar hydrophobicity and morphology of the surfaces of TMV/PANi and TMV/PANi/PSS coated substrates, these results indicated that incorporation of electroactive form of PANi through doping with PSS onto TMV may enhance cell proliferation compared with non-conductive TMV nanofibers. These results were consisted with literature reports that electroactive PANi could promote adhesion and proliferation of cells, such as PC12 cells39, H9c2 cardiacmyoblasts40 and C2C12 myoblasts41.

Figure 2. AFM images of TMV (a), TMV/PANi (b), and TMV/PANi/PSS (c) adsorbed on the glass coverslip coated PSS/PDDA2.5. Insert figures showed the water contact angle of substrates. All scale bars indicate 1 µm.


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Figure 3. MTT assay of PC12 cells cultured on various fibers of TMV, TMV/PANi/, and TMV/PANi/PSS coated substrates. Values expressed are mean (n = 3)±S.D.,* indicate p ＜0.05.

We next examined the effect of electroactivity of TMV/PANi/PSS on cell behaviors of PC12 cells. As shown in Figure S3c and e, after 1 day, there were no neurite outgrowth in PC12 cells cultured on both TMV and TMV/PANi coated surfaces. However, on the surface of TMV/PANi/PSS, PC12 cells extended neurites from the cell bodies (Figure S3g). After 3 days, the fluorescence and optical images showed that cells cultured on all of surfaces extended neurites with a branching phenotype from the cell bodies (Figure 4a-d, Figure S3b, d, f, h). The median neurite lengths are 32.7, 34.1, and 41.2 µm on the surfaces of TMV, TMV/PANi and TMV/PANi/PSS, respectively (Figure 4e, Figure S4). The percentage of cells with neurites 15.1 ±0.9% on the TMV/PANi/PSS (Figure 4f), significantly higher than that of TMV 6.9±1.0%, TMV/PANi 7.5±1.5% or glass 11.0±1.7% (p＜ 0.05). As shown in Figure S5, the mechanical properties of all samples are very similar, and the


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elastic modulus was determined to be 1.08±0.20, 1.20±0.15, and 1.27±12 GPa for TMV, TMV/PANi, TMV/PANi/PSS fiber, respectively. The TEM results (Figure 1c-e) showed the thicknesses of PANi and PANi/PSS coated TMV are 0.5 nm and 1.5 nm. Therefore, we speculated that the eletroactive layer plays an important role in promoting neurite outgrowth of neural cells.

Figure 4. Representative fluorescence (FDA staining) images of PC12 cells cultured for 3 days on (a) glass, (b) TMV randomly oriented, (c) TMV/PANi randomly oriented, and (d) TMV/PANi/PSS randomly oriented. (e) The median neurite length and (f) the percentage of cells with neurites cultured on different substrates. All scale bars indicate 50 µm. At least 200 neurites were analyzed from three substrates for each condition. *p ＜0.05.

PC12 cells in the capillaries with aligned electroactive TMV nanofibers In our previous studies, we have developed a robust and facile method for large scale alignment of one dimensional (1D) nanoparticles (NPs) in capillaries, in which 1D NPs were aligned using an


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high pressure air shear force25. Here, we applied this method to align TMV-based electroactive nanofibers inside glass capillary tubes. As shown in the Figure 5a, over 85% TMV/PANi/PSS nanofibers were aligned within ±20o along the direction of the air flow. Without air flow, TMV/PANi/PSS nanofibers were randomly distributed (Figure 5b).

Figure 5. AFM images of aligned (a) or randomly oriented (b) TMV/PANi/PSS nanofibers in the capillaries coated PDDA/PSS2.5. The white arrow represented the direction of the flow. The insets are the histograms of TMV/PANi/PSS angular distribution with respect to the direction of air flow. Scale bars: 1 µm.

After that, we cultured PC12 cells in capillariescoated with aligned or random TMV/PANi/PSS nanofibers. The fluorescence and optical images showed that PC12 cells cultured on both scaffolds extended neurites from the cell body at day 1 and day 3 (Figure 6a-d, Figure S6). When cells were cultured on aligned TMV/PANi/PSS nanofibers, the orientation of neurites aligned along flow direction with 93% at day 1 and 88% at day 3 as shown in Figure 6e, f (defined as the neurites with an orientation angle less than ±20o). After 3 days, the median neurite length increased from 26.4 µm to 51.9 µm on aligned scaffold (Figure 6g, Figure S7a, c), which represented an almost two-fold


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increment in the median neurite length for cells on the random 3D scaffold 25.8 µm at day 3 (Figure 6g, Figure S7d). In addition, the percentages of cells with neurite were calculated. As shown in Figure 6h, the percentage of cells with neurite cultured on aligned TMV/PANi/PSS group was 31± 2% and 35±3% at day 1 and day 3, respectively, which is significantly higher than the cells cultured on randomly distributed fibers 17±2% and 20±2% at day 1 and day 3. Moreover, the number of neurites per cell was chosen to further compare cellular behaviors of cells on both groups. An average of 1.8 neurites per cell was observed on the aligned nanofibers, in comparison to an average of 2.9 neurites per cell on the random nanofibers after 3 days of incubation (Figure 6i). This data suggest that the high degree of aligned fibers makes the cell grown with the bipolar morphology, which is consistent with literature report42. Similar tendency was also observed on the wild-type TMV and TMV-PANi nanofibers. After three days, the orientation of neurite along flow direction with 86% and 87% of neurites have an orientation angle less than ±20o. However, the median neurites length of cells cultured on aligned TMV and TMV/PANi nanofibers were 35.7 and 37.5 µm, which were higher than the unaligned nanofibers 22.1 and 24.2 µm (Figure S8), but much lower than the aligned TMV/PANi/PSS nanofibers. Taken together, TMV/PANi/PSS nanofibers synergistically stimulate neurites outgrowth induced by electroactivity and topography.


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Figure 6. Representative fluorescence (FDA staining) images of PC12 cells cultured on aligned (a, c) or randomly oriented (b, d) TMV/PANi/PSS for 1 (a, b) and 3 (c, d) days. The angular distribution of neurites with respect to the long axis of glass capillary on the randomly oriented or aligned TMV/PANi/PSS nanofibers at day 1 (e) and day 3 (f).The median neurite length (g), the percentage of cells with neurites (h) and the number of neurites per cell (i) for PC12 cells cultured on the aligned or randomly oriented TMV/PANi/PSS nanofibers. All scale bars indicate 50 µm. At least 200 neurites were analyzed from three substrates for each condition. *p ＜ 0.05.

Conclusions In summary, we successfully synthesized the plant virus template electroactive nanofibers, which


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could be aligned within capillary by air flow. We found that the electroactive TMV/PANi/PSS nanofibers could enhance cell proliferation and neurite outgrowth in comparison to non-conductive TMV nanofibers. The orientation and length of neurites outgrowth of cells were significantly affected by the orientation and electroactivity of nanofibers. We envision that this scaffold will offer a facile way to fabricate functional nerve conduit and the electroactive TMV/PANi/PSS nanofibers may be used as a potential scaffolding material for neural tissue engineering. Supporting Information Including UV-vis spectra of TMV/PANi/PSS; stability of various substrates; optical images of PC12 cells cultured on 2D substrates, aligned or randomly oriented of TMV/PANi/PSS, the neurite length distributions of PC12 cells cultured, plots of loading force versus vertical displacement of Z-piezo on nanofibers and Si wafer. This material is available free of charge via the Internet at http://pubs.acs.org.

Notes The authors declare no competing financial interest.

Acknowledgement The authors would like to thank the financial support from the State Key Laboratory of Polymer Physics and Chemistry of Changchun Institute of Applied Chemistry, Chinese Academy of Sciences and the National Natural Science Foundation of China (Programs 21429401, 21374119).

References


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