Soft Conducting Polymer Hydrogels Cross-Linked and Doped by

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Soft Conducting Polymer Hydrogels Crosslinked and Doped by Tannic Acid for Spinal Cord Injury Repair Lei Zhou, Lei Fan, Xin Yi, Zhengnan Zhou, Can Liu, Ruming Fu, Cong Dai, Zhengao Wang, Xiuxing Chen, Peng Yu, Dafu Chen, Guoxin Tan, Qiyou Wang, and Chengyun Ning ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b04609 • Publication Date (Web): 04 Oct 2018 Downloaded from http://pubs.acs.org on October 4, 2018

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Soft Conducting Polymer Hydrogels Crosslinked and Doped by Tannic Acid for Spinal Cord Injury Repair Lei Zhou,1, 2, # Lei Fan,1, 2, 3, # Xin Yi,1,2 Zhengnan Zhou,4 Can liu,3 Ruming Fu,1, 2 Cong Dai,4 Zhengao Wang,1, 2 Xiuxing Chen,6 Peng Yu,1, 2 Dafu Chen,5 Guoxin Tan,4,* Qiyou Wang,3,* and Chengyun Ning1, 2, * 1

College of Materials Science and Technology, South China University of Technology,

Guangzhou, 510641, China 2

National Engineering Research Center for Tissue Restoration and Reconstruction, South China

University of Technology, Guangzhou, 510641, China 3

Department of Spine Surgery, The Third Affiliated Hospital of Sun Yat-sen University,


School of Chemical Engineering and Light Industry, Guangdong University of Technology,


Laboratory of Bone Tissue Engineering, Beijing Research Institute of Orthopaedics and

Traumatology, Beijing JiShuiTan Hospital, Beijing, 100035, China 6

VIP Inpatient Department, Sun Yat-sen University Cancer Center, Guangzhou, 510060, China

#

These authors contributed equally to this work.

*Correspondence and requests for materials should be addressed to G.T.( [email protected]), Q. W.( [email protected]) or C.N.( [email protected])


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ABSTRACT: Mimicking soft tissue mechanical properties and the high conductivity required for electrical transmission in the native spinal cord is critical in nerve tissue regeneration scaffold designs. However, fabricating scaffolds of high conductivity, tissue-like mechanical properties and excellent biocompatibility simultaneously remains a great challenge. Here, a soft, highly conductive, biocompatible conducting polymer hydrogel (CPH) based on a plant-derived polyphenol, tannic acid (TA), crosslinking and doping conducting polypyrrole (PPy) chains is developed to explore its therapeutic efficacy after a spinal cord injury (SCI). The developed hydrogels exhibit an excellent electronic conductivity (0.05-0.18 S/cm) and appropriate mechanical properties (0.3-2.2 kPa), which can be achieved by controlling TA concentration. In vitro, a CPH with a higher conductivity accelerated the differentiation of neural stem cells (NSCs) into neurons while suppressing the development of astrocytes. In vivo, with relatively high conductivity, the CPH can activate endogenous NSC neurogenesis in the lesion area, resulting in significant recovery of locomotor function. Overall, our findings evidence that the CPHs without being combined with any other therapeutic agents have stimulated tissue repair following an SCI, and thus have important implications for future biomaterial designs for SCI therapy.

KEYWORDS: conducting polymer, hydrogel, electrical cue, mechanical mismatch, spinal cord injury


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The spinal cord transmits information in the form of electrical impulses along highconducting nerve fibers. The endogenous bioelectric signals in the spinal cord are widely accepted to play an indispensable role in maintaining neuronal function, neurite growth and nerve regeneration.1-3 A biomaterial-based treatment has been investigated as a attractive strategy to repair spinal cord injury (SCI) by bridging spinal cord lesions after a SCI.4-6 Mimicking the high electrical transmission properties of the native spinal cord would be highly beneficial for tissue repair.1, 2 This consideration should inspire us to design an innovative implant bridge with a high conductivity that supports the functional restoration of an interrupted conducting neural path, maintaining the endogenous electric microenvironment giving rise to nerve regeneration. Among the existing conducting electroactive materials, conducting polymers (CPs) [e.g., polythiophene, polyaniline (PAni), and polypyrrole (PPy)] have gained popularity as components of complex systems designed to provide a conductive environment for nerve tissues because of their physical characteristics, biocompatibility, and electrical properties similar to those of metals and inorganic semiconductors.7, 8 However, a major limitation of using CPs for treatment of SCI is their poor mechanical matching with the native soft spinal cord tissue, infusibility and difficult processing of complex, three-dimensional (3D) structures.7, 9 Hydrogels consisting of cross-linked, 3D, hydrophilic polymers are typically soft and have moduli similar to those of nervous tissue due to their high hydration level.10-12 These features render them excellent candidates for nerve tissue scaffolds. Therefore, effectively mimicking both the 3D soft mechanical signature and electrical transmission function of the native spinal cord to form nascent circuits by combining the soft mechanical properties of hydrogels with the electrical properties of CPs is of interest. Conducting hydrogels (CHs) can fulfill this role. The CHs used in tissue engineering are typically polymeric blends or co-networks that combine CPs


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with nonconducting hydrogels.13-15 These hybrid CHs have been promoted for application to soft tissue engineering, but still have a series of limitations. Due to the presence of a nonconducting hydrogel matrix, these gels generally exhibit poor conducting electroactivity, which does not meet the requirements for a highly conductive nerve bridge.8 Because hydrogels swell under physiological conditions, physically or ionically entrapped CP components can leach out, leading to toxicity and a decline in conductivity.16 Thus, strategies to develop suitable CHs for nerve tissue engineering are needed. Herein we report a facile strategy to prepare biocompatible conducting polymer hydrogels (CPHs) with both appropriate mechanical properties and relatively high conductivities by using tannic acid (TA), a polyphenol widely found in many plants, as the dopant and crosslinker. TA has been used in many biomedical products due to its anti-oxidant, antibacterial, antiinflammatory, and anti-carcinogenic properties.17 Moreover, TA is capable of supramolecular interactions with neutral polymers and proteins via multiple reaction pathways, including electrostatic, hydrogen bonding and hydrophobic interactions.18-20 However, until now, few studies have focused on the interaction between TA and PPy. Here, we show that TA is a natural crosslinking agent for PPy hydrogel formation because there are significant intermolecular interactions between the molecules. Compared with traditional CHs, the TA cross-linked CPHs are free of insulating polymer and the hydrogel backbone is conducting PPy, thus exhibit relatively high conductivities. In addition to its excellent electroactivity, the resultant CPHs has other potential functions that render it an ideal biomimetic electroactive bridge for SCI repair, including good tissue adhesion, high swelling ratio and appropriate mechanical properties. Furthermore, we demonstrated that CPHs can direct NSC differentiation in vitro by promoting neuronal differentiation while suppressing astrocyte differentiation. Finally, and most


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importantly, the highly conductive hydrogel bridges implanted in a severe mouse SCI model promoted endogenous neurogenesis and functional recovery by restoring the interrupted spinal circuit.

RESULTS AND DISCUSSION Formulation for CPHs. Figure 1a presents a schematic of the material preparation and the chemical structure of TA. The simple addition of an oxidative initiator (FeCl3) to a solution containing the pyrrole (Py) monomer and TA leads to rapid gelation (within 2 s). All three reagents (FeCl3, TA, and Py) were required for gelation to occur. As shown in Figure 1b, without all three reagents, no gelation occurs. TA crosslinks with PPy chains by intermolecular electrostatic interactions between the protonated nitrogen groups on PPy and the phenol hydroxy groups on TA. Protonation of the nitrogen groups makes the PPy conductive and thus TA is also considered as a dopant.7 An excess amount of Fe3+ plays a key role in the gelation process because the Fe3+ ions promote the polymerization of Py via oxidation and act as ionic crosslinkers to further coordinate interactions with TA to form ionically cross-linked networks.18 To confirm the importance of Fe3+, ammonium peroxydisulfate (APS) was used as an oxidant instead of FeCl3, and only homogeneous solutions were obtained. Notably, the PPy hydrogel can form at very low molar ratios of the TA monomer to Py (≈1:425) with fast gelation speeds (within 2 s), which are significantly less than the phytic acid:AN molar ratio (1:7) and gelation time (3 min) required to create the phytic acid-crosslinked Pani hydrogel.21 These results indicate that TA has a stronger supramolecular interaction with PPy. Physicochemical and Electrical Characterization of CPHs. The FT-IR spectrum of the CPHs was in good agreement with that of the pristine PPy, which indicated that the main


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component of the hydrogel is PPy (Figure S1). A number of characteristic broad bands were observed, particularly the peaks at 1174 and 903 cm−1, which can be attributed to the stretching vibrations of the C−N+ bonds and C=N+−C bonds, respectively,22 confirming the high dopant concentration in this material. Based on the reversibility of the dopant and coordination bonds, the developed CPHs demonstrate electrical self-recovery properties based on the observation of cut hydrogel pieces quickly reintegrating into a monolithic hydrogel and its conductivity recovery (Figure S2). After lyophilizing CPHs, the cross-sectional morphology of the CPHs was obtained using a scanning electron microscopy (SEM) analysis. The CPHs exhibited a 3D, microporous foam network similar to that of typical hydrogels (Figure 1c and d). The microporous structures consisted of interconnected globular nanoparticles (Figure 1d and Figure S3). These micro/nanoporous structures offer large effective surface areas for substance exchange and cell adhesion and facilitate cellular penetration and tissue formation within the 3D hydrogel structures.23 Moreover, a free-standing CPHs film can be obtained by peeling from superhydrophobic glass substrates, and therefore easily plastered onto irregular objects, such as fingers (Figure 1f). Because of the abundant presence of catechol in TA, the CPHs also can adhere to mouse spinal cord tissue (Figure 1f). Both free-standing nature and tissue adhesiveness of the CPHs are important properties for the envisioned in vivo applications. A series of CPHs with three different TA compositions were prepared by varying the TA concentration as follows: TA-PPy-1, 0.15 wt%; TA-PPy-2, 0.6 wt%; and TA-PPy-3, 2.5 wt%. Despite the hydrophobic characteristic of the PPy backbone because of the existence of pyrrole ring, our PPy hydrogel displayed a relatively high swelling ratio, which facilitates highly efficient mass transfer and is beneficial for applications in soft tissue engineering. At a fixed Py concentration, the TA concentration influenced the swelling behavior of the fabricated CPHs.


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The maximum swelling ratios of 2472 ± 32%, 2875 ± 10%, and 1960± 19% for TA-PPy-1, TAPPy-2 and TA-PPy-3, respectively, were achieved after 24 h (Figure 2 a). These ratios are even higher than the swelling ratio of some reported conducting hybrid hydrogels.13, 24 When the TA concentration increased, the water uptake amount increased in TA-PPy-2 but decreased in TAPPy-1. This may be attributed to the fact that the TA-PPy-2 hydrogels contain more hydrophilic polyphenol groups than TA-PPy-1. Although TA-PPy-3 has a more hydrophilic polyphenol moiety, higher crosslinking density likely results in the lower hydration capacity of TA-PPy-3 compared to that of TA-PPy-2. The mechanical properties of a matrix environment are known to be key to affect cell function and differentiation.25 As shown in Figure 2b, the storage modulus (elastic modulus, G’) value was higher than loss modulus (viscous modulus, G”) for all three hydrogel samples over the entire frequency range (0.1 to 10 Hz), which indicated that these hydrogels were stable and behaved as viscoelastic solids (Figure 2b). As expected, the average storage moduli measured at 1 Hz increased from 394±14 Pa for TA-PPy-1 to 2260±20 Pa for TA-PPy-3 as the TA concentration increased (Figure 2c). The hydrogel modulus value is in the range reported for the modulus of spinal cord tissues (100–3000 Pa).25 This change in the mechanical behavior could be attributed to the different crosslinking densities in these gels. The electronic properties of CPHs anchored on ITO were investigated using cyclic voltammetry (CV) in phosphate-buffered saline (PBS; 0.1 M, pH 7.4) versus Ag/AgCl (3 M KCl). Figure 2d the ITO bare electrode exhibited a negligible current response, whereas the anodic and cathodic currents both significantly improved for the pristine PPy and hydrogel coating. All three hydrogels have a higher current response than that of pristine PPy, which can be attributed to their porous structures facilitating fast electron and ion transport. The TA-PPy-2


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hydrogel had the highest current values for both oxidation and reduction. This indicated that the TA-PPy-2 hydrogel networks are more conductive in PBS than the other two hydrogels, which is likely due to their high doping level and moderate crosslinking density. The drop in electrical properties of TA-PPy-3 could be explained by the higher cross linking density, which in turn causes a decrease in the swelling thus hinder ionic transport. The conductivity of the three water swollen CPHs is from 0.05 to 0.18 S·cm−1 (Table 1), which was determined using the I-V curves at room temperature, and these values exceed the reported value for some typical conductive hybrid hydrogels (10-6–10-5 S·cm−1).8,

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Importantly, the values fall in the range of

conductivities reported for spinal cord tissue (10 –2 to 10 −1 S/cm),27 which indicated that this gel is suitable for use as a conducting scaffold for nerve cell cultures. The conductivity and electroactivity of the CPHs was further assessed by electrochemical impedance spectroscopy (EIS) in PBS. The Nyquist plots of both the pristine PPy and PPy hydrogel samples showed a quasi semi-circular arc in the high frequency region, which implies good redox activity (Figure 2e). The diameter of the semicircles corresponds to the charge transfer resistance (RCT). In agreement with the CV results, the RCT of TA-PPy-2 was relatively low, which indicated the ease of charge transfer. The CPH exhibited lower impedance at medium-to-high frequencies compared to that of the pristine PPy, which suggested its fast electron transfer kinetics. Neural cell usually communicate with each other at frequencies between 300 Hz and 1 kHz (Figure 2f).28 The TA-PPy-2 hydrogel showed excellent transportation behavior, as evidenced by a low impedance of 10 Ω at 300 Hz, which is lower than that of pristine PPy (19 Ω) (Table 1). The low resistance is beneficial for in vivo, rapid cellto-cell communication by endogenous bioelectrical signals produced by the activity of ion channels and pumps between neighboring cells.29 In addition, our CPH also exhibited good


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electronic stability under physiological conditions (Figure S4), which is another key requirement for the long-term use of CPs in biomedical applications.30 Cellular Behaviour on CPHs. The cytotoxicity of the developed CPHs was first evaluated using neural stem cells (NSCs). The cell viability was measured using CCK-8 assay and live/dead staining (Figure S5). The cell viability was close to or exceeded 80% for all three hydrogels 1, 3, and 7 days after seeding (Figure S5a), which suggested that the engineered CPHs are not cytotoxic against NSCs. In agreement with the CCK-8 assay, the live/dead staining of the NSCs showed excellent viability and firm attachment to the hydrogel surface 24 h after seeding (Figure S5b-e). These results demonstrated that this conductive hydrogel provides a biocompatible substrate for NSCs. We next investigated the capacity of these hydrogels to enhance the differentiation of NSCs into neurons while inhibiting astrocyte differentiation in vitro. Immunostaining was used to determine the differentiation of stem cells after 1 and 7 days in vitro (DIV) (Figure 3a). The acquisition of neuronal and astrocyte type cells was evaluated by the expression of the neuronspecific class III β-tubulin (Tuj1) and astrocyte-specific glial fibrillary acidic protein (GFAP), respectively. Confocal microscopy images revealed clear differences in the percentages and growths of the neurons and glial cells differentiated from NSCs on four different conductive surfaces (Figure 3a). The neuron-specific Tuj1 expression was significantly higher in cells cultured on the hydrogels relative to that of the cells cultured on the pristine PPy substrates. Notably, the primary neurons showed poor neurite outgrowth on pristine PPy, which was in contrast to the results observed for the hydrogels. These result can be explained by the fact that the NSCs optimally differentiated into neurons on the soft, hydrogel surfaces, whereas the stiff, pristine PPy surfaces promote astrocytic differentiation.31 Among the hydrogels, TA-PPy-2,


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which has the best electroactivity, showed the best performance in terms of neuronal differentiation and growth ability. Most of the TA-PPy-2-cultured neurons at 7 DIV remained healthy with high Tuj1 expression levels and maintained their neuronlike morphologies; dendrites of single neurons spread in all directions and formed an extensive network with other neurons. In contrast, very little GFAP-positive astrocyte differentiation was observed after 7 DIV; it is believed that inhibition of astrocyte differentiation is important in preventing glial scar formation, which is a known barrier to axonal growth after SCI.32 A gene expression analysis was also performed to quantitatively evaluate the NSCs differentiation behavior. For comparison, a standard poly(D-lysine) (PDL)-coated glass was used as a positive control in the test. The TA-PPy-2 hydrogel promoted faster and more differentiation of the stem cells into neurons than previously studied PDL-coated substrates. The Tuj1 expression of the hydrogel was four times greater than that on the PDL substrate at 3 DIV (Figure 3b). In accordance with the immunocytochemistry, the gene expression data also confirmed that NSCs seeded on TA-PPy-2 had significantly higher percentages of neurons and dramatically lower percentages of glial cells than those on the other hydrogels after 3 and 7 DIV without any induction (Figure 3b and c). Because the TA-PPy-2 gel has a medium elastic modulus and the highest electroactivity, it is concluded that the high electroactive properties enhanced neuronal activities and neural differentiation, even though previous work has demonstrated that the hydrogel stiffness can also mediate NSCs differentiation.31 The high electrical properties of the gels can enhance neuronal activities and neural differentiation by promoting cell-cell communication using bioelectric signals.29 In addition, the porous 3D structures of the gels may be beneficial for the accelerated neuronal differentiation.


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CPHs for SCI Repair. To examine the therapeutic efficacy of the CPHs in vivo, a ‘C’-shape, semi-tubular, free-standing CPHs patch was implanted as a highly conductive bridge across a spinal cord hemisection gap (Figure 5a). The CPHs patch tightly bound to the spinal cord tissue due to its good tissue adhesion and remained attached for 6 weeks after transplantation (Figure S6). As shown in Figure 4a and c, severe fibrosis (fibroblasts and collagen, blue) and few neuronal cells were observed around the pristine PPy, which was randomly scattered in the fibrous tissue due to the lack of crosslinking 2 weeks post-implantation. In contrast, the CPH did not cause obvious fibrous encapsulation after 2 weeks of implantation (Figure 4b). Moreover, nerve tissue adhered to and grew longitudinally along the hydrogel (Figure 4b and d). Six weeks after the SCI, the hydrogel implantation lowered CD68-positive microglia/macrophage infiltration in lesion regions compared to that of the SCI group (Figure S7). The reduced inflammatory reaction may be attributed to the mechanical compatibility between the the soft nervous tissue and the hydrogel implants,9, 33 as well as the anti-inflammatory effect of TA in the hydrogel.17 Furthermore, we also observed that a large amount of the Tuj1-positive neurons were gathered around the bulk hydrogel (Figure S8). Six weeks after implantation, a small part of hydrogel became granular, and nerve cells, including neurons and astrocytes, infiltrated the hydrogel cavities, implying that the hydrogels are sufficiently porous to allow cells around the gels grow into the interior of the hydrogel (Figure S8 and S6). The cavitations of the hydrogels were likely a result of both crosslinker TA degrading (TA contains many hydrolyzable ester bonds) and break down caused by host cells (e.g., inflammatory cells). The ultimate breaks down products of the hydrogels are likely PPy nanoparticles because the hydrogel is constructed with interconnected nanoparticles (observed in the SEM images). PPy shell nanoparticles have been reported to be a relatively safe conducting polymer material with low toxicity.34 Future work will


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include long-term in vivo studies to evaluate the complete degradation of CPHs, and definitely rule out potential toxicity of break down products. To confirm that electroactivity is required for in vivo SCI repair, we examined cavity formation via Tuj1 immunohistochemical staining of SCI animals untreated or treated by two CPHs with significantly different electrical activities. As expected, large cystic cavities filled with fibrotic scarring were observed in the lesion area in the SCI group animals (Figure 4b and S9). After the hydrogel treatment, the lesion region was mostly filled with Tuj1-positive nerve tissue instead of cystic cavities. Moreover, the higher-electroactivity gel (TA-PPy-2) group promoted more nerve tissue preservation (Figure 5b) and exhibited a significantly reduced cavity size than the control group, which was supported by the quantification data (Figure 5c). This trend is consistent with the in vitro results. Taken together, these data suggest that the conducting electroactive properties of these hydrogels can promote tissue repair in vivo. To further investigate the therapeutic effect of the CPHs following SCI, we assessed the neuron growth and astrocytic response at the injury site by immunofluorescence staining. The GFAP immunostaining levels significantly increased around the injury site in the SCI group in comparison with the sham group, which indicated that a compact glial scar formed around the cavity (Figure 5d and e). In contrast, the GFAP immunostaining around the lesion site in the animals treated with the CPH was less intense (Figure 5f1-4). Thus, animals treated with the hydrogel had significantly inhibited astrocytic responses and glial scar formation after an SCI. In addition, in animals treated with the hydrogel, numerous neurons (positively stained Tuj1) were observed by Hoechst nuclear staining to invade the lesion site. Interestingly, nestin-specific positive NSCs were synchronously observed in the corresponding regeneration sites, which suggested that endogenous NSCs migrated into the lesion area and differentiated into new


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neurons (Figure S10). NF200 expression is associated with neurite outgrowth. Some NF200positive nerve fiber infiltration was observed around the edge of the regeneration site (Figure 5g and h). These new fibers may have originated from newly regenerated neuronal cells, which suggested the formation of neural networks that might connect severed ascending and descending axons in the injured spinal cord. Moreover, the diminished astrocytic reaction and enhanced endogenous neurogenesis after the hydrogel implantation were also observed by the Western blot analysis of the GFAP, Tuj1 and NF levels (Figure 5i). The possible mechanism of promoting new endogenous neurogenesis for CPH is illustrated in Figure S11. The electrical transmission property of the CPH may contribute to the beneficial effects of the CPH by restoring the interrupted spinal circuit in the lesion area; thus, the spinal cord can deliver endogenous electrical signals to the interrupted cells.35, 36 Furthermore, the infiltration of cellular elements and axon regeneration in the lesion area are substantially promoted due to the presence of endogenous bioelectric cues. To evaluate whether endogenous neurogenesis after the CPH treatment could improve functional recovery, Basso, Beattie and Bresnahan (BBB) rating scale scores were obtained for 6 weeks after the SCI (Figure 6a). For the sham group, the hindlimb locomotion was approximately 21, which indicated the spinal cord was not harmed and was in accordance with the above histological examinations. As expected, all animals in each groups showed complete right-hindlimb paralysis (BBB score = 0) at day 0 after undergoing a right-sided hemisection injury. Although animals in all groups exhibited a certain degree of functional recovery over 6 weeks, the animals treated with the hydrogel showed significant improvements in functional motor skills relative to those of the SCI control group (beginning in week 1 (p < 0.01), and the improvements further expanded at 2 weeks and thereafter (p < 0.0001, Figure 6a, Video S1,


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Supporting Information). A footprint analysis was also used to test the locomotor recovery (Figure 6b). The footprints of the hindpaws almost overlap with those of the forepaws during walking for the sham group, which showed good coordination. The SCI resulted in nonoverlapping of fore- and hindpaw footprints, and the right hindlimbs did not have the ability to support weight. Both the rotation angle of the hindpaw and the relative position significantly increased in comparison with those of the sham group (Figure 6 c and d). The hydrogel treatment significantly reduced the relative position and rotation angle, which indicated that the hydrogel treatment resulted in enhanced coordination between the forepaw and hindpaw. These results suggested that implanting CPHs into injured spinal cords can form functional neural networks, resulting in motor behavioral recovery of the hindlimbs.

CONCLUSIONS In summary, we have developed a supramolecular strategy to prepare a porous, highly conductive, soft, and biocompatible CPH suitable for SCI repair by simply mixing TA, Py, and Fe3+. This hydrogel possesses an appropriate mechanical properties and excellent electronic conductivity, which are similar to the soft mechanical properties and high electrical conductivity of the native spinal cord, respectively. These outstanding properties of the CPH accelerated the differentiation of NSCs into neurons while suppressing the development of astrocytes in vitro. Furthermore, our in vivo data showed that the hydrogel implantation as a highly conductive bridge can stimulate new neurogenesis and subsequent functional neural network formation. These results suggest that the potential of the CPH bridges with relatively high conductivity to repair the injuried spinal cord, hence our works are of great importance for the future development of high-performance SCI repair biomaterials.


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MATERIALS AND METHODS CPH Fabrication. In a typical synthesis procedure, a 4 mg of tannic acid (TA, Aladdin, Shanghai, China) was first dissolved in 0.6 ml of deionized (DI) water, and then a 35 uL (0.5 mmol) pyrrole (py, 99%, Aldrich, St. Louis, USA) was added into the TA solution under stirring to form solution A. Solution B was prepared by 0.316mg of ferric chloride hexahydrate (FeCl3·6H2O, 98%, Aladdin, Shanghai, China) was dissolved in 0.6 ml water. The solution A and solution B were cooled to 4 °C and then mixed quickly for gelation. The resulting CPH were incubated in a large amount of DI water for 3 days to remove excess ions and by-products. As comparison, pristine PPy films were prepared as follows: The solution A (No TA added) and solution B were mixed and immediately dropped evenly onto the glass substrates (1cm×1cm), let it sit and react for 12 h at 4 °C and were immersed into DI water for 3 days for purification. Characterization. Physicochemical Properties: The morphology of lyophilized CPHs was analyzed by scanning electron microscopy (Quanta 200, FEI, USA). Fourier-transform infrared (FT-IR) spectra were obtained over 32 scans at a resolution of 4 cm−1 using a Nicolet IS10 spectrometer (Thermo Scientific, USA). Rheological experiments were performed on a Physica MCR-301 rheometer (Anton Paar, Austria) with parallel plate (PP-10 probe, 10 mm diameter, flat). Dynamic oscillatory frequency sweep measurements were conducted at a constant strain (1 %) and frequency ranging from 10 to 0.1 Hz. The electrical conductivity of water swollen PPy hydrogel was tested by 2-probe current (I)–voltage (V) measurements using a Keithley 2400 sourcemeter (USA). To assess hydrogels swelling, the CPHs were first lyophilized and weighed to obtain the dry weight of sample (md), then were soaked in PBS (pH=7.4) at 37 ℃ for 24 h to reach equilibrium. The swollen weight (ms) of the gels was measured. The mass swelling ratio was then calculated according to the following equation:


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Swelling ratio % =

− × 100

Electrical Properties: Cyclic voltammetry (CV) and electrochemical impedance (EIS) measurements were performed with an electrochemical workstation (Zennium Zahner, Germany). A platinum counter electrode (CHI Instruments) and an Ag/AgCl reference electrode (CHI Instruments) were used in the measurements. The electrolyte solution was PBS (pH=7.4, 0.1 M). The working electrode was prepared by the hydrogel or pristine PPy anchored on indium tin oxide (ITO) coated glass substrates. CV measurements were performed in the potential range of 0.8-1.0 V with a scan rate of 10 mV/sec. EIS spectra were measured over the frequency range of 100 kHz to 0.01 Hz at open-circuit potential. Cell Culture and Viability. NSCs were isolated from the the hippocampi of E14.5 mouse embryos as previous described.37 Briefly, the dissociated NSCs were seeded on low-attachment dishes and expanded in maintenance medium containing DMEM/F12 medium (Gibco), B27 neural supplement (2%, Gbico), 2 mM GlutaMAX (Sigma–Aldrich), 20 ng/mL epidermal growth factor (EGF, PeproTech), and 20 ng/mL basic fibroblastic growth actor (bFGF, PeproTech). After cultured for 7 days, the obtained neurospheres were passaged three or four times and were used for further experiment. The dissociated NSCs and undissociated neurospheres were plated onto appropriate substrates (e.g. CPHs, pristine PPy, or poly-D-lysine coated cover slips) in growth media. After 12 hours, the maintenance medium was replaced with mixed differentiation medium consisting of 1:1 mix of neurobasal medium (Gibco) and DMEM/F12 (Gibco), B27 (1%, Gibco), N2 (1%, Gibco), 2 mM GlutaMAX (Gibco), and 100 mg/mL penicillin-streptomycin (Gibco). Half of the medium was replaced every other day. The viability/cytotoxicity of CPHs was assessed using a Cell Count Kit-8 (CCK-8) cell viability assay (Dojindo Laboratories, Kumamoto, Japan). The absorbance at 450 nm was measured using


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a microplate reader (Multiskan FC, Thermo) with pure medium as a blank. The cell viability (mean ± SD, n = 6) was expressed as a percentage of the absorbance of the cells cultured on hydrogel to that of the control group. Additionally, cell viability/cytotoxicity was also assessed by live/dead staining in which live cells were stained with Calcein-AM (Sigma-Aldrich, USA) and dead cells were stained with propidium iodide (PI, Sigma-Aldrich, USA). Images were obtained using an Olympus IX73 inverted microscope (Shinjuku, Tokyo, Japan). Immunostaining and Imaging. To investigate the extent of neuronal differentiation, at 3 and 7 d, the NSCs cultured on PDL-coated glass, pristine PPy or CPH were fixed with 4% formaldehyde for 30 min at room temperature. Cells were incubated in 0.1% TritonX-100 in PBS for 15min at room temperature and blocked with 1% BSA in PBS for 2 h at room temperature. The fixed samples were incubated overnight at 4°C in solutions of primary antibodies (anti-Tuj1 (ab7751, 1:1000; Abcam) and anti-GFAP (ab7260, 1:1000; Abcam) in PBS containing 5% BSA. After washing three times with PBS, the samples were exposed to secondary goat anti-mouse IgG conjugated with Alexa-Fluor 488 (1:500; Abcam) and donkey anti-rabbit IgG conjugated with Alexa Fluor 594(1:800: Sigma) in PBS containing 5% BSA for 2 h in the dark at room temperature, respectively. Finally, cell nuclei were stained by Hoechst33342. The images were captured using a Leica TCS SP8 confocal microscope. Gene Expression Analysis. For comparison, a standard poly(D-lysine) (PDL)-coated glass was used as a positive control in the test. To quantitatively determine the differentiation status of NSCs in cultured on PDL-coated glass, pristine PPy or CPH, the expression level of neuronal and astrocytic marker genes was analyzed using the quantitative reverse transcription polymerase chain reaction (RT-qPCR). Total RNA was extracted from cells by the Trizol™ reagent (Invitrogen) at 3 days and 7 days. RNA was reverse transcribed into cDNA using a Reverse


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transcription Kit (Takara, Japan). The RT-PCR was performed using the MaximaTM SYBR Green/ROX qPCR Master Mix (Thermo). Each individual PCR experiment was repeated at least four times, and all expression data were normalized to GAPDH expression. The relative gene expression was quantified using the 2-∆∆Ct method. The primers used in this study are shown in Table S1. Surgical Procedures and CPHs Implantation. All animal procedures approved by and carried out in accordance with the standards of the Experimental Animal Ethics Committee of Sun Yat-sen University. Adult male C57BL/6N mice (6-8 weeks old, supplied by Experimental Animals Center of Sun Yat-Sen University) were used in this study. Animals were randomly allocated to the following there groups: Sham, SCI, and CPH (n = 15). Before surgery, animals were anesthetized by injection of a mixture of 70 mg/kg ketamine and 5 mg/kg xylazine via intraperitonea. To prepare the hemisection model, we performed a laminectomy under a stereo microscope, followed by hemisection at the T9-10 level, with removal of a 2-mm right side segment of the spina cord tissue. After hemostasis, the semi-tubular CPH was transplanted (2.5 mm length and 1.2 mm semi-diameter) to attach to the lesion sites in the hydrogel groups. Then, the paravertebral muscles and skin were closed in layers by using sutures. Control animals underwent sham surgery with exposing spinal cord then closed (Sham group), and spinal cord hemisection surgery without materials implantation (SCI group). After surgery, the animals were kept warm and free access to food and water. Histological staining. At 2 weeks after the operation, animals were anesthetized deeply with sodium pentobarbital. After transcardial perfusion with 0.9% NaCl plus 4% paraformaldehyde, and the implants as well as the surrounding tissue were explanted and further processed for histological analysis. The samples were fixed with 4% paraformaldehyde (PFA) at 4 °C


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overnight, followed by rinsing with PBS for 3 times, and then dehydrated with gradient alcohol and acetone and embedded in epoxy resin. The histological sections were stained by using a standard protocol for Mason’s trichrome stain, and imaged with a Nikon E800 upright microscope. The degree of inflammation was analyzed by a blinded histopathologist. Fibrosis was determined by collagen deposition. Immunohistochemistry (IHC) Staining. IHC staining was used to evaluate cavitations of spinal cord in different groups. Sections were prepared as described above. The paraffinembedded spinal cord tissues were dewaxed and dehydrated. The samples were incubated with 3% hydrogen peroxide in methanol solution for 30 min to inhibit endogenous peroxidase activity. Samples were then thoroughly washed with PBS and blocked with 5% goat serum in PBS for 30 min at room temperature. Subsequently, the samples were incubated with anti-Tuj1 (ab18207, 1:2000; Abcam) at 4 °C overnight. After washing with PBS, the samples were incubated with the goat anti-rabbit IgG second antibodies (PV-6001, ZSGB-Bio, China) for 20 min at 37 °C. The peroxidase activity was visualized by reaction with 3, 3′-diaminobenzidine (DAB) for 5 min. The samples were finally counterstained with hematoxylin. The cavity areas were quantified using ImageJ software (NIH). Immunohistofluorescence (IHFC) staining. At 2 and 6 weeks after the operation, the spinal cord tissue were excised and fixed in 4% paraformaldehyde at 4 °C for 6-8 h and then incubated in sucrose solutions for cryoprotection. Transverse sectioning of frozen spinal cord (15 µm thick) was obtained using a cryostat microtome (CM 1900, Leica) and placed onto Superfrost® Plus Microscope Slides (Thermo). After blocking with 10% goat serum, the samples were incubated with the respective primary antibodies: NF (ab8135, 1:1000, abcam), Nestin (ab11306, 1:200, abcam), CD68 (ab31630, 1:200, abcam), and doubly labeled for Tuj1 (ab7751, 1:200, abcam)


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and GFAP (ab7260, 1:500, abcam). Alexa 488 or Alexa 594 was used as the secondary antibody. Cell nuclei were stained with Hoechst33342. Finally, the slides were observed under a Leica TCS SP8 confocal microscope. Western blot. At 6 weeks after the operation, the spinal cord lesion site was dissolved into protein. The proteins were separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes. 5% nonfat milk powder was added on the PVDF membranes to block for one hour. After that, the membranes were then incubated with primary antibodies: rabbit anti-NF200 (NF) antibody (ab8135, 1:10000, abcam), rabbit anti-GFAP antibody (ab7260, 1:10000, abcam), and rabbit anti-Tuj1 (ab18207, 1:1000, abcam) at 4 °C overnight. After washing with PBS, membranes were incubation with anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibody (ZSGB Bio, China), and using an ECL kit to enhance chemiluminescence assay. The optical density of each band was calculated by using ImageJ software. Functional Analysis. Hindlimb locomotor function recovery was evaluated weekly using the BBB rating scale scores and footprint analysis. The BBB open-field locomotion score was performed by two examiners blinded to group identity. Animals were placed in an open field and the locomotion score was evaluated after a 5 min of observation. For footprint analysis, the foreand hindlimbs of the animals were dipped in blue and red ink, respectively. Subsequently, the animals were allowed to walk on a straight strip of white paper. The rotation angle of hindpaw measured as the angle formed between the line connecting the third toe and the central axis of the runway. The relative position of the forelimb and hindpaw was defined by measuring the distance between center pads of the ipsilateral forelimb and hindpaw.


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Statistical Analysis. Statistical analysis was performed using one-way ANOVA with a Bonferroni’s post-hoc test using software SPSS v19.0.0 (IBM, USA). All data were expressed as mean ± standard deviation. Tests were conducted with a 95% confidence interval (α = 0.05).

ASSOCIATED CONTENT The authors declare no competing financial interest Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: FT-IR

spectra;

self-recovery

properties;

electronic

stability

in

PBS;

cytotoxicity;

immunofluorescence staining; evaluation of CPHs hydrogel decomposition in vivo; Mason’s trichrome staining; schematic repair mechanism (PDF) Video of restoration of locomotor function for mouse (AVI)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (G.T), [email protected] (Q. W), or [email protected] (C.N.) Author Contributions #

L. Zhou and L. Fan contributed equally to this work.

ACKNOWLEDGMENT The authors acknowledge financial support from the National Natural Science Foundation of China (Grant Nos.51772106, 31771080, 51702104) and the Natural Science Foundation of Guangdong Province (Grant No. 2016A030308014, 2015A030313493).


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Figure 1. Formation of the supramolecular CPHs. a) An illustration of the fabrication of a crosslinked CPH following the addition of TA, Py, and Fe3+. TA acts as a crosslinker and dopant, and Fe3+ acts as an oxidant and ionic crosslinker. b) Photographs showing the occurrence of gelation with different reagent combinations. Vial 1 has no TA; vial 2 has no FeCl3; vial 3 has no Py; vial 4 has APS but no FeCl3. Vial 5 has all three reagents, and gelation occurs. c) SEM image and d) a higher magnification SEM image of the CPH. e) A photograph of free-standing CPH films plastered on finger. f) Demonstration of the adhesion of the CPH to spinal cord tissue in vitro. The tissue is hanging from the hydrogel, which is pasted on the glass on top.


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Figure 2. The physical and electrical properties of CPHs with different TA concentrations. a) Swelling ratio percentage for different hydrogels. b) Frequency-dependent oscillatory shear rheology of different hydrogels. c) Elastic modulus (G’) at 1 Hz of different hydrogel networks. Insert: a photograph of the CPHs after the template removal. d) Cyclic voltammograms of the ITO bare electrode, pristine PPy, and different hydrogels in 0.1 M PBS at a scan rate of 10 mV s−1. e) Nyquist curves and f) Bode plots of pristine PPy and different hydrogels in 0.1 M PBS.


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Figure 3. In vitro NSC differentiation on the control samples and different CPHs. a) Confocal microscopy images of NSC differentiation on pristine PPy and different hydrogel samples into Tuj1-positive cells (for neurons, green) and GFAP-positive cells (for astrocytes, red) for 3 and 7 DIV. All cell nuclei were Hoechst stained (in blue). Scale bars = 50 µm. Normalized gene expression levels of Tuj1 b) and GFAP c) of the NSCs cultured on PDL, pristine PPy, and different hydrogel samples. Error bars indicate the SD from five different experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.


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Figure 4. Biocompatibility of the CPH in vivo. Histology images of (a) pristine PPy and (b) the CPH with the surrounding tissue stained with Mason’s trichrome (MT) 2 weeks after implantation. Severe fibrosis (fibroblasts and collagen, blue) and few neuronal cells were observed around the pristine PPy (red arrows). In contrast, the conducting hydrogel caused little fibrosis relative to the pristine PPy, which was randomly scattered in the fibrous tissue due to a lack of crosslinking. Representative immunohistochemical (IHC) staining images for Tuj1 at the interface between the tissue and (c) pristine PPy and (d) the CPH 2 weeks after implantation, confirming the CPH (black arrows) was tightly surrounded by nerve tissue, while no obvious nerve tissue was observed around the pristine PPy (scale bars, 100 µm).


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Figure 5. CPH implantation promoted new endogenous neurogenesis in a hemisection model of SCI. a) Graphical representation of a ‘C’-shape, semi-tubular CPH that was implanted as a bridge to cover the spinal cord hemisection gap. b) Representative images of transverse spinal cord sections stained with Tuj1. Scale bars = 150 µm. c) Quantification graphs showing the average cystic cavity area of animals with SCI and different hydrogel treatments. * p < 0.05 and ** p < 0.01, *** p < 0.001. Immunohistofluorescence images of transverse spinal cord sections obtained from animals in the sham (d1-d4), SCI (e1-e4), and hydrogel groups (f1-f4) at 6 weeks. Boxed regions in d4, e4, and f4 are magnified in d4’, d4”, e4’, e4”, f4’, and f4”. Scale bars indicate 200 µm (d1-4–f1-4) and 100 µm (d4'–f4”). g and h) NF staining of the regenerated nerve fibers in the hydrogel group. g) Note that the NF-positive regenerating fiber growth was observed around the lesion area; h) beginning to infiltrate the lesion site. Scale bars = 200 µm. i) Western blot analysis of the spinal cord protein extracts showing the Tuj1, GFAP and NF protein bands in the sham, SCI and hydrogel groups.


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Figure 6. Implantation of the CPH promotes locomotor function recovery. a) The locomotor recovery of the animals was measured using the standard BBB scale in an open field. ** p < 0.01, *** p < 0.001, **** p < 0.0001. n = 5 animals in each group. Error bars represent the SD. d) Representative footprints of the animals in the sham, SCI, and hydrogel groups. The fore- and hindlimbs of the animals were inked in blue and red, respectively. c) The rotation angle and d) relative position were used to quantify the locomotion quality at 6 weeks. * p < 0.05, ** p < 0.01, *** p < 0.001.

Table 1. Conductivity and impedance at frequencies f = 300 Hz of pristine PPy and three CPHs Samples

Conductivity (S/cm)a)

Impedance (Ω)b)

pristine PPy

0.02

19

TA-PPy-1

0.05

17

TA-PPy-2

0.18

10

TA-PPy-3

0.12

11

a) Conductivity was determined using the I-V measurements. b) Impedance was obtained from EIS data


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Table of Contents


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Soft Conducting Polymer Hydrogels Cross-Linked and Doped by

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