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Micro-nano-structured Polyaniline Assembled in Cellulose Matrix Via Interfacial Polymerization for Applications in Nerve Regeneration Dingfeng Xu, Lin Fan, Lingfeng Gao, Yan Xiong, Yanfeng Wang, Qifa Ye, Ai-xi Yu, Honglian Dai, Yixia Yin, Jie Cai, and Lina Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03555 • Publication Date (Web): 17 Jun 2016 Downloaded from http://pubs.acs.org on June 18, 2016

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ACS Applied Materials & Interfaces

Micro-nano-structured Polyaniline Assembled in Cellulose Matrix Via Interfacial Polymerization for Applications in Nerve Regeneration Dingfeng Xu,1 Lin Fan,2 Lingfeng Gao,1 Yan Xiong,2 Yanfeng Wang,2 Qifa Ye,2 Aixi Yu,2 Honglian Dai,3 Yixia Yin,3 Jie Cai,1 and Lina Zhang1*

1

College of Chemistry & Molecule Sciences, Wuhan University, Wuhan 430081, P.

R. China 2

Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430081, P. R.

China 3

Department of Pharmaceutical Engineering, Wuhan University of Technology,

Wuhan 430070, P. R. China * Corresponding authore-mail address: [email protected]

ABSTRACT Conducting polymers have emerged as frontrunners to be alternatives for nerve regeneration, showing a possibility of the application of polyaniline (PANI) as the nerve guidance conduit. In the present work, the cellulose hydrogel was used as template to in-situ synthesize PANI via the limited interfacial polymerization method, leading to one conductive side in the polymer. PANI submicrometer dendritic particles with mean diameter of about 300 nm consisted of the PANI nanofibers and nanoparticles were uniformly assembled into the cellulose matrix. The hydrophobic PANI nanoparticles were immobilized in the hydrophilic cellulose via the phytic acid

1

ACS Paragon Plus Environment


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as “bridge” at presence of water through hydrogen bonding interaction. The PANI/cellulose composite hydrogels exhibited good mechanical properties and biocompatibility as well as excellent guiding capacity for the sciatic nerve regeneration of adult Sprague-Dawley rats without any extra treatment. On the basis of fact that the pure cellulose hydrogel was an inert material for the neural repair, PANI played an indispensable role on the peripheral nerve regeneration. The hierarchical micro-nano-structure and electrical conductivity of PANI could remarkably induce the adhesion and guiding extension of neurons, showing its great potentials in biomedical materials.

KEYWORDS: cellulose hydrogel, hierarchical micro-nano-structure, interfacial polymerization, polyaniline, biocompatibility, nerve regeneration.

1. INTRODUCTION Neurological injuries affect up to a billion people worldwide, and this number is estimated to increase considerably as life expectancy continues to rise.1-3 Even though the peripheral nervous system could self-heal to some extent after injury,4,5 it still faces challenges including the obstacle of traversing a large gap between distal and proximal ends of a nerve, and the self-healing is very frequently incomplete with poor functional recovery.6,7 Recently, many approaches including the autologous nerve graft, neurolysis or decompression and nerve transfer are being developed to substitute an inclusion of support cells or growth factors.8-10 A self-healing hydrogel

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(stiffness ≈1.5 kPa) has been developed for healing central nerve system deficits.11 Moreover, much efforts have been made to control cell responses by varying the topography, three-dimensional (3-D) geometry, chemical composition,12 coating with conductive polymers13,14 or carbon materials,15 resulting in substantial increases in the charge transfer area compared to conventional metallic materials.16,17 Recent trends in scaffold design have focused on particular structure materials that can provide appropriate guidance cues,18-20 and the organic conductors provide safe electrical stimulation of tissue. Moreover, the biodegradable materials can obviate the need for secondary

operation.21,22

Recently,

conductive

polymers

such

as

poly

(3,4-ethylenedioxythiophene) (PEDOT), polypyrrole (PPy), polyaniline (PANI) have been chosen to prepare scaffold materials of neural repair due to their superior electrical conductivity and biocompatibility.23-25 However, their relatively poor mechanical properties limit their usability and medical translation.26-28 It is noted that during the repair of peripheral nerve injuries, flecked tissue can be formed at the cut ends to prevent the recovery of function of the regenerative nerve fibers.29,30 Thus, the PPy implant with biocompatibility was used to envelop neurons and glial cells under electrical stimulation.31-33 By comparison, PANI also has good environmental stability and biocompatibility, indicating the great applications in biomedical engineering as potentially attractive material.34,35 In our laboratory, polyaniline (PANI) doped with phosphate ester has been successfully dissolved in 4 wt % cellulose-7 wt% NaOH/12 wt% urea aqueous solution at low temperature, and the PANI/cellulose composites, for instance, conductive films, composite hydrogels, microspheres were fabricated

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successfully.36-38 More recently, the PANI/cellulose composite microspheres were prepared via the regenerated cellulose matrix as a template, leading to the formation of the PANI submicroparticles with nanomesh structure, showing good conductivity.39 However, PANI/cellulose hydrogels as a nerve regeneration material has not been previously investigated.

It is worth noting that hierarchical micro-nano-structured can influence cell morphology, adhesion, migration and proliferation,40,41 and Gelain et al42 have used electrospun micro- and nanofiber tubes for functional nervous regeneration in sciatic nerve transections, and it can induce nervous regeneration and functional reconnection of the two severed sciatic nerve tracts. Swaminathan et al43 have also demonstrated that hierarchical patterning of the multifunctional conducting polymer nanoparticles can act as guidance conduits for neurostimulation, whereby the presence of electrical current induces remarkable dendritic axonal sprouting of cells. Particularly, the nanoparticles and nanofibers morphological structure can indeed support cells well, promoting the cells adhesion and growth. 44,45 Furthermore, it has been demonstrated that inorganic and organic nanoparticles embedded in polymer scaffolds can promote the growth of cells.46,47 Thus, the one side conductive micro-nano-structured PANI in cellulose matrix as the scaffold materials of neuron were constructed. In the present work, the cellulose hydrogels were fabricated from cellulose solution in 7 wt% NaOH/12 wt% urea aqueous solvent with cooling, and then which was used as template to in-situ synthesize PANI via the limited interfacial polymerization in the natural phytic acid (PA) solution to obtain one conductive side 4


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in the hydrogel. In this case, PA was used as a “bridge” to firm the connecting between hydrophobic PANI and hydrophilic cellulose at room temperature. The structure and properties of the PANI/cellulose composite hydrogels were characterized, and their biocompatibility and guiding capacity for the sciatic nerve regeneration of adult rats without any extra treatment were evaluated. The hierarchical micro-nano-structured and electrical conductivity of PANI could induce significantly the adhesion and guiding extension of neurons, showing its great potentials in biomedical materials. This work opens up a new avenue for the construction of biomedical materials via embedding the PANI nanofibers and nanoparticles in the natural polymer matrix.

2. EXPERIMENT SECTION 2.1 Materials Cellulose (cotton liner pulp, DP 500) was provided by Hubei Chemical Fiber Co., Ltd. (Xiangfan, China). Its weight-average molecular weight (Mw) was determined to be 9.8×104 according to our previous method.48 Aniline monomer (analytical grade, Tianjin Damao Chemical Reagent Company) was distilled under vacuum and stored in a refrigerator. Ammonium persulfate (APS) was of analytical grade from Tianjin Damao Chemical Reagent Company. Phytic acid and toluene were purchased from Alladin, Shanghai, China. All of the other reagents were of analytical grade from china, and used without further purification. 2.2 Preparation of the regenerate cellulose hydrogels 5



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Cellulose was dissolved in a pre-cooled to -12 0C aqueous 7 wt% NaOH/ 12 wt% urea solution to obtain 4 wt% transparent cellulose solutions within 3 min according to our previous method.48 The solution was then subjected to centrifuge to degas at 7200 rpm for 10 min at 5 oC. The transparent solution was immediately cast on a glass plate to give a solution layer with 0.5 mm thickness, and then immersed into a coagulation bath of ethanol to give regenerate cellulose hydrogels. The cellulose gels were thoroughly rinsed with deionized water to achieve the regenerate cellulose hydrogel. 2.3

Fabrication

of

PANI/cellulose

composite

hydrogels

by

interfacial

polymerization The composite hydrogels were prepared through the interfacial polymerization by using APS as the oxidant. Acidic solutions were prepared with 10 wt% phytic acid solution and 0.05 mol L−1 APS, as well as 0.1 mol L−1 aniline of toluene solution. The cellulose hydrogel (7 mm×7 mm×0.5 mm) was sandwiched in the middle of a U tube (Figure 1). The aniline was added into one side of the hydrogels, and then the U tube was placed in an ice bath for 30, 60, 120, 180, and 240 min, respectively. The U tube was removed from the ice bath, subsequently, the PANI/cellulose composite hydrogels were taken out from the U tube and washed thoroughly with ethanol and deionized water to remove the residual chemical reagents and oligomers. 2.4 Characterizations The mechanical properties of the hydrogels were measured on a universal tensile tester (CMT 6503, Shenzhen SANS Test machine Co. Ltd., Shenzhen, China) according to ISO527-3-1995 (E) at a speed of 2 mm/min. Scanning electron 6


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microscopy (SEM) observation was carried out on a Field Emission Scanning Electron Microscopy (FESEM, Zeiss, SIGMA), by using an accelerating voltage of 5 kV. The samples were coated with Au for the SEM observation. Transmission electron microscopy (TEM) images were observed on a JEOL JEM-2010 (HT) electron microscope, using an accelerating voltage of 200 kV. A copper grid coated with a holey carbon support film was used to prepare samples for TEM observation. Solid-state 13C NMR and 13P NMR spectra (BRUKER AVANCE III spectrometer) and ATR-FTIR spectra were taken with a FTIR spectrometer (1600, Perkin–Elmer Co., MA) in the wavelength range from 4000 to 400 cm-1. 2.5 RSC 96 Cell culture The PANI/RC/2 hydrogel was sterilized, and the Schwann cells (RSC96 cell) suspension (100 mL) was added to each triplicate samples. The RSC96 cells, with a cell density of 2 ×105 cells cm-3, were co-cultured with the hydrogels in an incubator for 2 days. The hydrogels with cultured RSC96 cells were then washed with PBS, and then fixed for 2 h in 2.5 wt% glutaraldehyde, and finally post-fixed for 24 h. After being washed again with PBS, the hydrogels with RSC96 cells were treated by dyes, and then were observed and imaged using a fluorescence microscope (Nikon ECLIPSETE 2000-U) at the corresponding excitation wavelength (Ex/Em = 484/501nm). 2.6 Surgical procedure The Sprague-Dawley rats, of 201-230 g, were purchased from Hubei Provincial Center for Disease Control and Prevention (Wuhan, Hubei, China). All animals were

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housed in Specific pathogen free conditions, and the experimental procedures involving animals were in accordance with NIH Guidelines for the Care and Use of Laboratory Animals and under the approval of the Administration Committee of Experimental Animals, Hubei Province, China. The 5 mm defects were made in the right thigh sciatic nerve were removed of the nerve tissue and were wrapped with the composite hydrogels (5 rats). The rats were anesthetized with 3 mg/kg body weight chloralhydrate. The incision was made parallel to the thighbone, 5 mm below. The muscles around the nerve tissues were separated using blunt dissection with Mosquito's pliers. Subsequently, the 5 mm defects were created by surgical resection of the right sciatic nerve, which was severed at its center. One implant received for each rat, and then removed after 3 months. 2.7 Histological assessment The regenerating nerve was harvested after 3 months. The regenerated nerve was taken from the middle regions for two kinds of detections. One part of the nerve sections was fixed in a cold buffered 4 % paraformaldehyde solution. After fixation, the regenerated nerve was embedded in Epon 812 epoxy resin and stained with methylene blue. Then, it was observed under a light microscope (TE2000-U, Nikon, Japan). The others were fixed in a cold buffered 2.5 % glutaraldehyde solution for the TEM observation (JEM-1200 EX, JEOL, Japan). The myelin sheath regeneration was observed by using electron microscopy. 2.8 Statistical analysis SPSS 21.0 statistical software was used for statistical analysis of the relevant data.

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Data were expressed as the mean ± standard deviation. Differences between two groups were compared using the T-test. Differences among several groups were analyzed using one-way analysis of variance. P < 0.05 was considered statistically significant.

3. RESULTS AND DISCUSSION 3.1. Construction of single-sided conductive polyaniline assembled in cellulose matrix by interfacial polymerization As shown in Figure 1, the cellulose hydrogel (7 mm×7 mm×0.5 mm) was sandwiched in the middle of a U tube, 0.1 mol/L aniline solution was added into one side, the phytic acid solution of APS was poured into another side, leading to the formation of the one side conductive PANI/cellulose composite hydrogels. Figure 1 (b and d) shows an intuitive evidence of single-sided conductive, the front surface and back of composite hydrogels were placed, respectively, in a circuit. The light emitting diode (LED) of the conductive side of the composite hydrogels exhibited light emitting performance, whereas the insulation side in the circuit did not emit. This confirmed that PANI was successfully in-situ synthesized on one side of the cellulose hydrogel. According to the reaction time 0.5 h, 1 h, 2 h, 3 h and 4 h, the obtained PANI/cellulose composite hydrogels were coded as PANI/RC/0.5, PANI/RC/1, PANI/RC/2, PANI/RC/3, PANI/RC/4, respectively. Figure 2 shows SEM images of the pure cellulose hydrogel and the PANI/ cellulose composite hydrogels fabricated with different reaction times. The cellulose hydrogel was colorless and transparent

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(Figure S1), and it had three-dimensional network pore structure, the size was about 100 to 400 nm, which supplied full cavities for the polymerization of PANI. As shown in Figure 2 (b-f), with an increase of the reaction time, the mean size of the PANI nanoparticles increased from 25 to 90 nm. Similarly, with an increase of the oxidant concentrations, the amount of the micro-nano-structured PANI increased instantly on the interface between the cellulose hydrogel and the toluene, leading to the aggregation (Figure S2). Clearly, the PANI nanoparticles were firmly embedded into the cellulose matrix of the composite hydrogels. The effects of the PANI polymerization reaction time on the electrical conductivity and physical properties of the composite hydrogels are summarized in Table 1. As the reaction progressed, the electrical conductivity increased and the water content of the composite hydrogels decreased, as a result of the occupied cavities with PANI. The increase of electrical conductivity was owing to the contribution of PANI nanofibers and nanoparticles, which formed a submicrometer architecture. The tensile strength of the PANI/RC/0.5, PANI/RC/1 and PANI/RC/2 composite hydrogels was higher than that of pure cellulose hydrogel as well as PANI/RC/3 and PANI/RC/4. The mechanical properties of the biomaterials are important on the successful surgical suture. Considering with the comprehensive properties, PANI/RC/2 exhibited relatively higher tensile strength (the σb and εb values to be 2.25 MPa and 35.4 %) and good electrical conductivity (0.49 S cm-1). Thus, the PANI/RC/2 hydrogel was chosen to analyze the structure and to evaluate the biocompatibility and bioactivity in the peripheral nerve regeneration.

The PANI/RC/2 displayed a dark green color, and could be curled up into a tube, this 10


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was very important as the nerve conduits (Figure 3a). The TEM images of the ultrathin sections of PANI/RC/2 from the insulative to the conductive side are shown in Figure 3 (d, e and f). Observably, the PANI submicrometer particles displayed dendritic architecture consisted of nanofibers and nanoparticles, and were uniformly assembled into the cellulose matrix. The nanoparticle size significantly reduced from the conductive (the side of aniline solution) to another side. With the reaction process, three-dimensional pores of cellulose hydrogel was filled with polyaniline nanoparticles, leading to the formation single-sided conductive materials. As shown in the ATR-FTIR spectra (Figure 3c), the cellulose absorption bands at 3347 cm−1 and 2899 cm−1 can be assigned to the hydroxyl groups and the C-H stretching of CH2 of the PANI/RC/2 insulative side, respectively. The peak at 3227 cm−1 for the PANI/RC/2 conductive side was assigned to N-H stretching, and that 1558 and 1491 cm−1 can be assigned to the quinone and benzene rings in PANI.49,50 This further confirmed that PANI was successfully in-situ synthesized in the PANI/RC hydrogels. In view of these results, the PANI submicrometer particles with a mean size of about 300 nm exhibited hierarchical micro-nano-structured pattern, and appeared on the surface and in inner of the composite hydrogels. In our findings, PANI was grew in pores of the cellulose hydrogel, which as a microreactor for the polymerization of PANI, but also a backbone to protect and to immobilize their micro- and nano-structure. Therefore, the PANI/RC/2 composite hydrogels had hierarchical micro-nano-structure and single-sided conductive performance.

3.2. The hydrogen bonding between PANI and cellulose Via phytic acid 11



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To confirm the hydrogen bonding between cellulose and PANI via phytic acid, the cellulose hydrogel and PANI/RC/2 were investigated with the and

13

13

C NMR (Figure 3b)

P NMR spectra (Figure S3). The cellulose hydrogel at 106.1, 75.6, and 63.1

ppm displayed characteristic chemical shifts, respectively, corresponding to the cellulose Ⅱof C1, C2 and C6.39,51 However, the chemical shifts of C1, C2 and C6 for cellulose in the PANI/RC/2 hydrogels shifted to upfield by about 1 ppm. These data indicated the hydrogen bonds between the -OH groups of cellulose and the -NH groups of PANI via phytic acid.

13

P NMR spectrum of the pure phytic acid was

compared with that of the PANI/RC/2 to further confirm the hydrogen bonding between phytic acid with cellulose and PANI (Figure S3a). There were five symmetrical signals appeared of

31

P NMR spectrum due to the

31

P-D spin-spin

coupling.52 Nonetheless, the signals of 31P NMR spectrum of the PANI/RC/2 became less, because of the hydrogen bonding of the phytic acid. From the analysis of NMR and

31

13

C

P NMR spectra, it can be concluded that the strong hydrogen bonds

existed between PA, PANI and cellulose of the PANI/RC/2 hydrogels. Therefore, PANI was immobilized in the cellulose backbone through hydrogen bonding. On the basis of the TEM and SEM images in Figures 1c, 2d, 3f and 4a, the approximate mean length and diameter of the PANI nanofibers could be estimated to be ~ 200 nm and ~ 15 nm, and the approximate mean size of nanoparticles to be ~ 25 nm. A schema to describe the polymerization reaction of polyaniline and the hydrogen bonding interactions between PANI, cellulose and phytic acid is shown in Figures 4b. It was not hard to imagine that nucleation followed by PANI growth during

12


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polymerization led to the observed particle morphology. The complex interactions from hydrogen bonding as well as repulsion between the hydrophilic cellulose and the hydrophobic PANI could induce the formation of nanofibers and nanoparticles. Therefore, a scheme of the hierarchical micro-nano-structured PANI embedded in cellulose matrix is proposed in Figure 4c. The PANI submicrometer dendritic particles containing the PANI nanofibers and nanoparticles were assembled homogenously into the cellulose matrix, consistent with the Figures 2 and 3. The PANI nanoparticles were immobilized in cellulose matrix via the hydrogen bonding of PA, which easily formed hydrogen bonds to connect PANI and cellulose, leading to the PAIN growing vertically on the hydrophilic cellulose matrix, resulting in nanoparticles due to the incompatible interactions; or to expand in parallel to form fibrils, in which the PANI could stretch via PA “bridge” to fix on the cellulose backbone. In our findings, the formation of the hierarchical micro-nano-structure of PANI could be relevant to the micro-pores in the cellulose hydrogel, and the hydrophilic and the hydrophobic interactions between PANI, the cellulose matrix and the PA “bridge” in water. Particularly, the presence of abundant water in the hydrogels (Table 1) played a crucial role for the construction of PANI submicrometer dendritic particles with network architecture, as a result of the good disperse action of water. The hierarchical micro-nano-structure is beneficial for the adhesion and proliferation of cells.53-55 3.3. Cell culture evaluation On the basis of the strain-stress curves and the correlative data (Figure S4 and Table 1), the PANI/cellulose composite hydrogels exhibited good tensile strength (σb)

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and elongation at break (εb), which are required for the application as the guidance conduits for peripheral nerve regeneration. The σb and εb values of PANI/RC/2 could meet the requirements of biomedical materials. To evaluate the safety and biocompatibility of the composite hydrogels, the hemolysis compatibilities of the PANI/RC/2 was evaluated with spectrophotometric method. The hemoglobin release from erythrocytes after treatment with the PANI/RC/2 and cellulose hydrogel (RC) was measured (Figure S5). No indication of hemolysis was observed with the hydrogels, and the hemolysis rate of the PANI/RC/2 was only 1.48 %, indicating the integrity of erythrocyte membrane.56 The visual observation of the hemolytic phenomenon (Figure S6) further confirmed the safety and nontoxicity of the PANI/RC/2. We chose the RSC96 cells line as a model system for evaluating the biological performances of PANI/RC/2 as nerve-tissue repair materials, including their in vitro cytotoxicity and neural differentiation abilities. As shown in Figure 5a, the incorporation of PANI into the cellulose hydrogel enhanced RSC96 cells adhesion and proliferation. RSC96 cells exhibited a great proliferation rate on the surface of the conductive side of PANI/RC/2. Therefore, the PANI/RC/2 had good biocompatibility in vitro, and without cytotoxicity (Figure 5b). Moreover, most of the RSC96 cells cultured on PANI/RC/2 displayed shuttle shape (Figure 5c), namely, the normal neuron morphology, and the cell pseudopodiums adhered into the PANI submicrometer dendritic particles of the hydrogel (Figure 5d). One plausible explanation was that the hierarchical micro-nano-structure and conductive properties

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of the composite hydrogels induced the RSC96 cells adhesion and growth. Thus, the PANI/cellulose composite hydrogels were biocompatible, and can be used as nerve guidance conduit for the nerve-tissue repair. 3.4. Neural repairing in vivo The experimental results have demonstrated that the pure cellulose hydrogel was an inert substrate for the neural repair, in which the injured nerve atrophied (Figure 6a, b and c). Moreover, the conductive side was wrapped on the outside of the nerve, leading to grow only along the conductive side, so no nerve regeneration occurred on the non-conductive side (Figure 6d, e and f). This proved strongly the important role of PANI in promoting the neural cell proliferation. To further confirm the clinical applications of the PANI/RC/2 composite hydrogels, a 5 mm defect in the sciatic nerve of adult rats was applied as a model for in vivo nerve regeneration. The defect was wrapped up with PANI/RC/2 conduits, and then the regenerated nerves were harvested after 3 months. Figure 7 shows the reconstruction as well as the corresponding nerve morphological and histological analysis of the sciatic nerve in different periods. Clearly, the PANI/RC/2 accelerated the injured nerve reconstruction at 3 months after surgery (Figure 7c). In particular, the regenerated sciatic nerve became very robust at 6 months after surgery (Figure S7) and the adult SD rats were very agile (see Video S1). The TEM images (Figures 7d) of the repaired sciatic nerve tissues after 3 months and the normal sciatic nerve revealed that the formation of regenerated myelinated fibers occurred at similar levels in both the PANI/RC/2 and normal groups (Figure 7f). The repaired nerve samples stained with methylene blue to

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evaluate the numbers and diameters of regenerated axons for PANI/RC/2 conduits groups. The numerous bundles of regenerated nerve fibers were identified in the sections of the regenerated tissues after 3 months. (Figure 7e), demonstrating that the inner layer of the PANI/RC/2 hydrogels was sufficiently compact to basically avoid connective tissue ingrowth after 3 months, suggesting that the injured nerve has basically recovered. It was confirmed that the integrity of the severed nerves was well recovered by the PANI/RC/2 conduits without electrical stimulation and growth factor. On the basis of the above results, the hierarchical micro-nano-structured PANI played a decisive role for nerve repair. In our findings, the cell pseudopodiums easily adhered to the PANI submicrometer dendritic particles of the composite hydrogel, inducing the proliferation of the nerve cells, as shown in Figure 4c (right). Thus, in the future, if we use a biodegradable natural polymer as a basement, which can be biodegraded completely within 6-12 months after the nerve repair, the PANI nanoparticles will remove out from the body at last. Thus, the secondary operation for the nerve repair would be avoided. Therefore, the hierarchical micro-nano-structured PANI materials would open new pathway in the nerve regeneration applications.

4. CONCLUSIONS PANI/cellulose composite hydrogels with hierarchical micro-nano-structure were constructed in cellulose matrix via the limited interfacial polymerization method to obtain one conductive side in the polymer. The three-dimensional network structure of the cellulose hydrogel provided not only full cavities for the polymerization of PANI,

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but also as a backbone to protect and to immobilize their micro-nano-structure. The interactions between the hydrophilic cellulose, the phytic acid “bridge” and the hydrophobic PANI at presence of water induced the formation of hierarchical micro-nano-structure. The PANI submicrometer dendritic particles were tightly assembled on the cellulose matrix. The PANI/cellulose composite hydrogels exhibited good electrical conductivity, soft physical characteristic and excellent biocompatibility. The results of the sciatic nerve regeneration of adult SD rats indicated that the pure cellulose hydrogel was an inert material for the neural repair, whereas PANI in the PANI/cellulose composite hydrogels played an indispensable role on the peripheral nerve repair. The hierarchical micro-nano-structure and conductivity of the nerve guidance conduit were beneficial to the adhesion and guiding extension of neurons. This work provided important information for the nerve repair and the design of bionic devices capable of in vivo applications.

ACKNOWLEDGMENT This work was supported by the Major Program of National Natural Science Foundation of China (21334005) and the National Natural Science Foundation of China (20874079).

ASSOCIATED CONTENT Supporting information

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The SEM image of the hydrogel from the freezing-dried process. This information is available free of charge via the Internet at http://pubs.acs.org/. AUTHOR INFORMATION Corresponding Author * Fax: +86-27-68754067. E-mail: [email protected] Notes The authors declare no competing financial interest. REFERENCES (1) Theodore, L. R.; Debasis, N.; Tatjana, A.; Alan, P. K.; Lawrence, L. L.; Dorian, B. M. Transcranial Amelioration of Inflammation and Cell Death After Brain Injury. Nature 2014, 505, 223-228. (2) Georgiou, M.; Golding, J. P.; Loughlin, A. J.; Kingham, P. J.; Phillips, J. B. Engineered Neural Tissue with Aligned, Differentiated Adipose-d Erived Stem Cells Promotes Peripheral Nerve Regeneration Across a Critical Sized Defect in Rat Sciatic Nerve. Biomaterials 2015, 37, 242-251. (3) Cattin, A. L.; Burden, J. J.; Emmenis, L. V.; Mackenzie, F. E.; Hoving, J. A. N.; Calavia, G.; Guo, Y.; McLaughlin, M.; Rosenberg, L. H.; Quereda, V.; Jamecna, D.; Napoli, I.; Parrinello, S.; Enver, T.; Ruhrberg, C.; Lloyd, A. C. Macrophage-Induced Blood Vessels Guide Schwann Cell-Mediated Regeneration of Peripheral Nerves. Cell 2015, 162, 1127-1139. (4) Wang, Y.; Zhao, Z.; Ren, Z.; Zhao, B.; Zhang, L.; Chen, J.; Xu, W.; Lu, S.; Zhao, Q.; Peng, J. Recellularized Nerve Allografts with Differentiated Mesenchymal Stem Cells Promote Peripheral Nerve Regeneration. Neurosci. Lett. 2012, 514, 96-101. (5) Das, S.; Sharma, M.; Saharia, D.; Sarma, K. K.; Sarma, M. G.; Borthakur, B. B.; Bora, U. In Vivo Studies of Silk Based Gold Nano-composite Conduits for Functional Peripheral Nerve Regeneration. Biomaterials 2015, 62, 66-75. (6) Xue, C.; Hu, N.; Gu, Y.; Yang, Y.; Liu, Y.; Liu, J.; Ding, F.; Gu, X. S. Joint Use of 18


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Table 1. Mechanical properties, electrical conductivity and water content of the cellulose and PANI/cellulose composite hydrogels Sample number

Tensile strength Elongation at break (MPa) (%)

Conductivity (S﹒ ﹒cm-1)

Moisture content (wt%)

Cellulose

1.94±0.04

45.7±0.1

92.0±0.1

PANI/RC/0.5

2.71±0.06

48.8±0.5

91.4±0.1

PANI/RC/1

2.27±0.03

38.1±0.3

2.5×10-2

91.1±0.1

PANI/RC/2

2.25±0.05

35.4±0.2

4.9×10-1

88.7±0.2

PANI/RC/3

1.69±0.02

26.4±0.3

6.4×10-1

87.5±0.2

PANI/RC/4

1.08±0.05

14.1±0.4

6.8×10-1

86.5±0.1

24


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Figure 1. Apparatus of the preparation of the PANI/cellulose composite hydrogels, the SEM images (a, c) and conductive performances of the two sides (b, d).

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Figure 2. SEM images of pure cellulose hydrogels (a) and the conductive side PANI/cellulose composite hydrogels formed with the reaction time of 0.5 h (b), 1 h (c), 2 h (d), 3 h (e) and 4 h (f), respectively.

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Figure 3. Photograph of the PANI/RC/2 (a), solid-state

13

C NMR and FTIR-ATR

spectra for the pure RC hydrogel and the PANI/RC/2 (b, c); and TEM images of the cross-section of the PANI/RC/2 composite hydrogels from the insulative to the conductive side (d, e, f).

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Figure 4. TEM image of the cross-section of the PANI/RC/2 of the conductive side (a); descriptions of the polymerization reaction of polyaniline and hydrogen bonding between PANI, cellulose and phytic acid (b); the schema of the hierarchical micro-nano-structured PANI embedded in cellulose matrix as well as the adhesion for RSC96 cell (c).

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Figure 5. Fluorescence micrographs and SEM images of the RSC 96 cultured on PANI/RC/2 (a, c, d), and MTT assay showing differences in cell viability of RSC 96 on RC and PANI/RC/2 (b).

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Figure 6. Intraoperative photographs of the cellulose hydrogel; the sutured cellulose hydrogel has a transparent blank (defected nerve) (a, b), 3 months postoperatively of cellulose hydrogel with no nerve regeneration (c); and intraoperative photographs of the PANI/RC/2 of conductive side wrap on the outside of the nerve, 3 months postoperatively of the nerve growth along the conductive (d, e), and non-conductive side with no nerve regeneration (f).

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Figure 7. Intraoperative photographs of the PANI/RC/2 composite hydrogels, immediately after snipping (a) and suturing (b) by using PANI/RC/2 composite hydrogels, after 3 months (c) in PANI/RC/2, the sheath and histology images stained with methylene blue; TEM images of PANI/RC/2 ultrathin cross sections charged with regenerated sciatic nerves in rats at 3 months and the average thickness of regenerated myelinated (d, f); optical images represent cross sections of regenerated nerves taken from types of nerve conduits implanted in rats after 3 (e).

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Table of Contents

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Micro-Nanostructured Polyaniline Assembled in ... - ACS Publications

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