Highly aligned bacterial cellulose/PPy gradient conductive membranes for directed cell differentiation under electrical stimulation

Highly aligned bacterial cellulose/PPy gradient conductive membranes for directed cell differentiation under electrical stimulation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Highly aligned bacterial cellulose/PPy gradient conductive membranes for directed cell differentiation under electrical stimulation Li Wang, Fuyu Qi, Hao Wang, Shuangshuang Li, Sanming Hu, Zhijun Shi, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4403218/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Both of the topographical and gradient conductive cues can influence the cellular activity and thereby tissue regeneration. However, they have not been combined simultaneously onto biomaterial with electrical stimulation to demonstrate the synergistic role so far. Herein, we assume that a bacterial cellulose (BC) -based membrane by incorporating aligned nanofibers and a concentration gradient of polypyrrole (PPy) with electrical stimulation treatment will promote cell differentiation in peripheral nerve regeneration. The results showed that PPy were successfully deposited on the aligned BC/PPy with gradient conductive structure, which exhibited good mechanical property, thermal stability, the gradient decrease in surface resistance, gradient increase in surface current from the up to down segments, as well as excellent biocompatibility. Especially, the membranes promoted the gradient proliferation and differentiation of PC12 cells in vitro. Importantly, combined with electric field (EF), the aligned BC/PPy gradient conductive membranes synergistically directed the differentiation of PC12 cells. The overall results suggest the aligned BC/PPy gradient conductive membranes with EF could be a promising therapeutic strategy to guide cellular activities for peripheral nerve regeneration. Gradient conductive Topographical Electrical stimulation Aligned nanofiber Cell differentiation Peripheral nerve regeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Directional cell differentiation plays an important role in peripheral nerve regeneration [ 1 ], especially those long-distance or chronic nerve defects. Loss of cell activity and uncontrolled nerve differentiation can lead to a functional gap between regenerative and normal nerves, potentially hindering successful peripheral nerve regeneration. Currently, although autogenous nerve grafts are the gold standard for repairing peripheral nerve defects, there are several limitations, including the restricted availability of tissue, mismatched nerve diameters, donor site morbidity, and loss of function [ 2 , 3 ]. Artificial substitutes on sale are mainly used to repair short-distance nerve gap or as protectant wrap devices [ 4 ], which may associate with single material, difficulty to imitate the complex anatomical structure, as well as appropriate guided cell differentiation and axonal reinnervation [ 5 , 6 ]. Therefore, it is desirable to develop a novel strategy that can programmatically guide the directional growth, proliferation and differentiation of cells at specific locations for accelerated peripheral nerve regeneration. Biomaterials with aligned nanofibers have been proved to actively guide the orientated growth of neural cells and neurites, which can ensure that neurites across the injured nerve gap to the target tissue, thus promoting nerve regeneration and functional recovery [ 7 ]. Bacterial cellulose (BC), a natural polymer produced by Gluconacetobacter xylinus , has recently received enormous attention because of its high mechanical strength, flexibility, and nonimmunogenicity [ 8 ]. In particular, aligned BC nanofibers could provide contact-guidance for directional neural stem cell elongation, neurite outgrowth [ 1 ], pheochromocytoma (PC12) cell differentiation, and Schwann cell migration along the aligned nanofiber [ 9 ]. Due to other methods of producing aligned cellulose reduced the yield and affected the original structure of BC [ 10 ], our group successfully prepared the highly aligned BC-based membranes through quantitative stretching with excellent mechanical properties, biocompatibility, and guidance for cellular activities [ 11 ]. However, this aligned BC nanofibers did not possess electrical conductivity and cannot support an improvement in biofunction such as delivering electrical and electro-chemical stimulation to nerve cells [ 12 ]. To make the scaffolds conductive, incorporation of polymeric- or metallic-conductive nanoparticles onto the aligned fibers have mainly been applied. Among them, polypyrrole (PPy) have been widely studied for nerve regeneration due to its excellent electrical properties, good biocompatibility, and ease of synthesis [ 13 , 14 ]. For instance, Liu et al. reported that conductive polylactic acid/reduced graphene oxide/PPy composite nanofibers coupled with EF could significantly accelerate PC12 cell proliferation and differentiation [ 15 ]. Sun et al. invented a self-powered electrical stimulation using Pt-BC/PPy-N-CNTs for nerve regeneration [ 16 ]. Whereas, these scaffolds could not provide directional cues for cellular activities and axon growth during the nerve repair. To improve the guidance capability, compositional gradient that enable cells to infer their spatial location and determine their fate accordingly is applied for new conduits [ 17 ]. It is reported that at the tip of the axon, the growth cones can sense gradient guidance cues and modify its shape to guide axon extension responsively [ 18 ]. Previous studies showed that introducing growth factor or peptide gradient onto conduits can promote the neurite extension and accelerate the sciatic nerve regeneration [ 19 , 20 ]. In another study, the middle of the gradient NGF-immobilized film achieves longer neurite length of dorsal root ganglia (DRG) than that on the film without NGF gradient [ 21 ]. Nevertheless, a scaffold having both the aligned nanofibers and PPy gradient conductive have not been tested so far, neither its effects on cellular activities for nerve regeneration. To date, different methods have been developed for preparing gradient material such as electrospinning [ 22 ], 3D printing [ 23 ], and directional diffusion [ 19 ]. However, they need complex design and hard operation. Considering the aligned BC nanofibers allow the in-situ polymerization of PPy onto its matrix, a time-controlled reaction method is easier to operate but loading time is the key to control. Endogenous EF can be detected at the damaged nerve section, which play an important role in directing cellular activities for nerve regeneration [ 24 ]. The long period of nerve regeneration after injury may lead to target organ loss [ 15 , 25 ]. To mimic and prolong the endogenous EF, exogenous EF stimulation could be a unique and promising treatment for a variety of peripheral nerve injury types including long distance injuries [ 6 ]. The EF stimulation could promote the differentiation and proliferation of PC12 cells and axon extension [ 26 ], increase intraneuronal cAMP in DRGs and nerve growth factor (NGF) in Schwann cells [ 6 ]. Besides, the EF could induce microtubule associated proteins in axons, promote blood vessel regeneration, and subsequently facilitate peripheral nerve regeneration [ 6 ]. Therefore, the combination of EF with a scaffold having topographical guidance and gradient conductive signals may be a new option for peripheral nerve regeneration. Herein, we developed a new nerve regeneration system, which combined the electrical cues with topographic and gradient conductive guidance of the BC/PPy gradient conductive membranes for enhanced cell differentiation in peripheral nerve regeneration. The aligned BC/PPy gradient conductive membranes were first prepared by quantitatively stretching technique and hot pressing, followed with introduction of gradient PPy along the nanofibers by a kinetically controlled reaction (Fig. 1 ). The physicochemical properties of the prepared gradient conductive membranes were systematically characterized for their morphology, chemical structures, electrical conductivity, and mechanical strength. In addition, the biocompatibility and morphology of cells on the aligned BC/PPy gradient conductive membranes were investigated. Furthermore, the synergistic effect of coupling EF with the gradient conductive membranes on cell differentiation was examined. 2. Materials and methods 2.1. Materials BC was purchased from Hainan Guangyu Biotechnology Co., Ltd. (Hainan, China). Pyrrole (py) was obtained from Sigma-Aldrich (USA). Ferric chloride hexahydrate (FeCl 3 ·6H 2 O) and para-methylbenzenesulfonic acid (p-TSA) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Dulbecco’s modified eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, fetal bovine serum (FBS), horse serum, bovine serum albumin (BSA), penicillin/streptomycin (P/S), 3 (4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium romide (MTT), and dimethylsul-foxide (DMSO) were acquired from Gibico Life Technologies Co., Ltd (Grand Island, USA). Anti-beta III tubulin monoclonal antibody and rhodamine-conjugated goat anti-mouse IgG (H + L) secondary antibody were obtained from Proteintech Group, Inc. (USA). Dihydrochloride (DAPI), FITC-conjugated phalloidin, and Calcein-AM/PI were purchased from Dojindo Laboratories (Kyushu, Japan). Nerve growth factor (NGF) was acquired from Beijing Yiqiao Shenzhou Technology Co., Ltd. (China). PC12 cells were generously provided by Huazhong University of Science and Technology (Wuhan, China). 2.2. Preparation of BC/PPy gradient conductive membranes BC were purified by boiling in 0.1 M NaOH for 30 min, and washed with deionized water. Then, the obtained BC was cut into 12 cm × 4 cm pieces, and wet-drawn at a speed of 1 mm/min until the stretching strain reached 40%. Thereafter, the BC and stretched BC membranes were hot-pressed at 100°C for 12 hours as our previously described [ 11 ]. The nonstretched and stretched BC/PPy gradient conductive membranes were produced by kinetically controlled reaction using a peristaltic pump. Firstly, the py solution (4.13 g of p-TSA and 1.61 g of py in 300 mL water) was poured slowly into FeCl 3 solution (15.6 g of FeCl 3 ·6H 2 O in 300 mL water) with slow stirring to form a reaction solution. When the solution turned black, the BC membranes were immersed vertically in it until reaching the top segment. Then, the peristaltic pump was activated to extract the reaction solution at flow rates of 10 mL/min, 20 mL/min, and 30 mL/min, respectively. Finally, after the reaction was complete, the membranes were washed three times with deionized water, and dried for 20 minutes to obtain the nonstretched BC/PPy gradient conductive membranes. According to the results of biocompatibility, the flow rates to control the reaction time for PPy gradient deposition on the membranes was fixed at 20 mL/min. Following the same method, the stretched BC/PPy gradient conductive membranes were obtained. 2.3. Characterization of BC/PPy gradient conductive membranes The morphology of BC/PPy gradient conductive membranes was examined using field emission scanning electron microscopy (FESEM, Sirion 200, Holland, 10 kV) after sputtering-coated with gold. The current flowing through the membranes can be measured by conductive atomic force microscopy (C-AFM, -0.1 V) using the current detection mode with the scanning size 5 µm × 5 µm. The chemical composition of the membranes was determined by Fourier-transform infrared spectroscopy (FTIR) spectrometer (Vertex 70, Germany) in the spectral range of 500–4000 cm − 1 . The thermal properties of the membranes were determined by using the thermogravimetric analyzer (TGA, Pyrisl, China) at the rates of 10°C/min from 25°C to 800°C under nitrogen atmosphere. The elemental composition of the membranes was identified by X-ray photoelectron spectroscopy (XPS) spectrum (AXIS-ULTRA DLD-600W, Shimadzu) and elemental analyzer (EA, Vario Micro cube, Elementar). The surface resistivity of the different segments on the membranes was measured using a four-point probe resistivity meter. The mechanical property of membranes was assessed using the Instron Universal Testing Machine (UTM6503, SUNS Industrial Testing System Co., Ltd. China) at a stretching speed of 1 mm/min at 25°C and 50% relative humidity. The Young's modulus was determined from the slope at low strain values, and the maximum tensile strength corresponded to the maximum stress at sample breakage. The contact angle (CA) measurements were performed by a drop-shape analyzer (DSA 100, Krüss). The shape of water drops was captured when 10 µL of deionized water was dropped at different positions of the membranes surface and then the average value of contact angle was reported. 2.4. Biocompatibility evaluation and cell morphology observation 2.4.1. MTT assay and the hemocompatibility The cytotoxicity of BC/PPy gradient conductive membranes was determined through MTT assay. NIH3T3 cells were cultured in DMEM medium containing 1% P/S and 10% FBS at 37°C with 5% CO 2 . PC12 cells, which is widely used in research on nerve regeneration, were cultured in RPMI 1640 medium supplemented with 5% horse serum, 5% FBS, and 1% P/S under the condition of 37°C and 5% CO 2 . Prior to cell seeding, the sterilized samples were pre-incubated in serum-free culture medium for 12 h. Then, cells were seeded onto the membranes at a density of 8 x 10 3 cells/well in 96-well plates. After 1, 3 and 5 days of culture, the culture medium was replaced by the fresh medium containing 0.5 mg/mL MTT, and cells were incubated at 37°C for another 4 hours. Then, 300 µL of DMSO was added and incubated for 10 minutes to dissolve the formazan crystals. After the reaction, the absorbance value was measured at 490 nm using a microplate reader. The hemocompatibility of the membranes was assessed via the hemolytic activity assay as previously reported [ 27 ]. Initially, the materials were cut to fit into a 24-well plate and placed in each well. Subsequently, 1 mL of 0.9% NaCl solution was added to each well, and the samples were pre-treated for 24 hours at 37°C. After pre-treatment, the sample was transferred to test tubes, with 0.9 mL of physiological saline and 0.1 mL of diluted sheep whole blood added to each tube (V_whole blood: V_physiological saline = 1:1.25). After thorough mixing, the tubes were incubated at 37°C for 1 hour, followed by centrifugation at 1000 rpm for 5 minutes. The supernatant was collected, and the absorbance was measured at 545 nm using a multi-functional microplate reader. Physiological saline served as the negative control, and distilled water served as the positive control. Each group included 6 parallel samples. The hemolysis rate of the samples was calculated using the following Equation S1: HR (%) = [(OD S -OD N ) / (OD P -OD N )]×100 (1) where OD S , OD N , and OD P represented the optical density values of the sample, negative control, and positive control, respectively. This test provided insights into the hemocompatibility of the materials by evaluating their impact on red blood cell integrity. 2.4.2. Live/dead staining and cytoskeleton staining The morphology of cells on the membranes were observed by confocal laser scanning microscopy (CLSM, Olympus FV1000, Japan) after incubation for 5 days. For the live/dead staining, NIH3T3 and PC12 cells seeded on the membranes were stained using the Calcein-AM (2 µM) and PI (4 µM) for 15 min at 37°C, and observed and imaged by CLSM. For the cytoskeleton staining, PC12 cells on membranes were first fixed with 4% paraformaldehyde for 30 min and then permeabilized with 0.1% Triton X-100 (v/v) for 5 min. Then, the cells were co-stained with FITC-conjugated phalloidin and DAPI for 30 min prior to CLSM observation. ImageJ software was employed for statistical analysis and calculation of cell orientation angles. A value of 0° indicates a perfect alignment (parallel to the fiber alignment). 2.4.3. β3-tubulin staining β3-Tubulin is considered an early neuronal differentiation marker and used for staining of neurites [ 28 ]. For the β3-tubulin staining, PC12 cells on the membranes were first fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 (v/v) for 5 min and blocked with 2% BSA for 1 hour. The cells were then incubated with β3-tubulin primary antibody at 4°C for 12 hours, followed with co-staining of rhodamine-conjugated goat anti-mouse IgG (H + L) and DAPI for 1 hour at room temperature prior to CLSM observation. 2.5. Electrical stimulation of cells on the m embranes The homemade electrotaxis chamber was built on the culture dish as previously reported [ 11 ]. After one day of PC12 cells cultured on the membranes, the medium was replaced with differentiation medium containing 10% FBS and 50 ng/mL NGF for another 12 h. The samples were then placed in the electrical stimulation chamber. A constant EF magnitude of 50 mV/mm was applied for 1 hour per day over two consecutive days. Finally, the cells were cultured for additional 12 hours before staining. 2.6. Statistical analysis Each experiment was performed in triplicate and all data are presented as mean ± standard error of mean (s. e. m.). A comparison between two groups was carried out by the Student’s t-test, and difference was considered statistically significant at P < 0.05. 3. Result and discussion 3.1 Characterizations of the 10, 20, 30-BC/PPy gradient conductive membranes In order to fabricate and find the most suitable PPy gradient deposition on the membranes, 10, 20, and 30-BC/PPy gradient conductive membranes were obtained with the extraction rates of 10, 20, and 30 mL/min, respectively. PPy gradient were immobilized by differential BC/PPy deposition duration. For PPy gradient characterization, the membranes were divided into three segments (up, middle, and down), where the down segments of the membranes have the longest deposition time. The surface morphology and elemental analysis results confirmed the successful loading of PPy gradient on the 10, 20, and 30-BC/PPy membranes (Fig. S2, Fig. S3). The surface resistance and surface current results (Fig. S4, Fig. S5) indicated that the surface conductivity of BC/PPy gradient conductive membranes increased from the up to down segments. The cell activity of NIH3T3 and PC12 cells on the 20-BC/PPy gradient conductive membranes was higher than that of the other groups (Fig. S4). Therefore, 20-BC/PPy gradient conductive membranes with better biocompatibility and excellent surface resistance gradient characteristics were selected for subsequent experiments if not specified. 3.2. Morphological characterizations of the BC/PPy gradient conductive membranes The surface morphology of up, middle, and down segments of the nonstretched and stretched BC/PPy gradient conductive membranes was shown in Fig. 2 . PPy nanoparticles with a diameter of about 100 nm were successfully deposited onto the surface of BC nanofibers with the increasing concentration gradient distribution from the up to down segments (Fig. 2 a-f). The color of the film became darker as more PPy deposited (Fig. S1 ). Specifically, the fibers on the nonstretched BC/PPy gradient conductive membranes were randomly arranged (Fig. 2 a-c), while the stretched BC/PPy gradient conductive membranes exhibit clear fiber alignment along the stretching direction (Fig. 2 d, f). The cross-sectional morphologies of the stretched BC/PPy gradient conductive membranes also demonstrated its internal oriented structure (Fig. 2 g, h). The similarity in the features of fiber alignment was consistent with our previous study [ 11 ]. Moreover, PPy tends to polymerization along the fiber alignment of the stretched membranes, suggesting the ideal guidance of the aligned BC nanofibers on the PPy deposition. This might be related to the deeper groove of the stretched membranes than the nonstretched membranes (Fig. 3 ), which contributes to the formation of aligned and continuous conductive fiber network paths for the electrons transfer. Overall, the stretched BC/PPy gradient conductive membranes with highly aligned and PPy gradient conductive nanofibers could offer appropriate topographic, and electrical guidance for further exploration in nerve cell behavior studies. 3.3. C-AFM In view of the importance of surface topographies and conductivity variations of biomaterials on the adhesion, proliferation, and differentiation of nerve cells [ 29 ], C-AFM was carried out and the results are shown in Fig. 3 [ 30 , 31 ]. The surface currents flow on all the BC/PPy gradient conductive membranes increased with the increasing concentration gradient distribution of PPy from the up to down segments (Fig. 3 a-c and Fig. 3 g-i), indicating the improved electron transfer efficiency and conductivity after the introduction of PPy [ 32 ]. The surface current density on the up, middle, and down segments of the nonstretched BC/PPy gradient conductive membranes were 0.005, 0.139, and 13.378 nA, respectively (Fig. 3 a-c), while the stretched membranes exhibited higher surface currents on the same segments (0.005, 9.135, and 41.614 nA, respectively, Fig. 3 g-i). Interestingly, the stretched BC/PPy gradient conductive membranes showed aligned surface currents distribution along the fiber alignment, where the down segment exhibited the maximum value of the surface current (Fig. 3 i, 41.614 nA) and the regular nanogrooves depth (Fig. 3 l, 482.09 nm). In contrast, the randomly distributed surface currents with nanogrooves in a small depth were observed for the nonstretched membranes. This could be due to that the stretching and hot-pressing processes decreased the specific volume between the nanofibers, which thereby increased the depth of nanogrooves and aligned PPy deposition for current flow. Therefore, considering the gradient surface current distribution and enhanced current flow along the fiber alignment, the highly aligned stretched BC/PPy gradient conductive membranes could have the potential to transfer the topographic and gradient cues to guide cell behaviors, and thus allowing further applications in peripheral nerve regeneration. 3.4. Fourier transform infrared (FTIR) spectra and Thermogravimetric analysis (TGA) FTIR was conducted on BC and stretched BC/PPy gradient conductive membranes to assess the impact of in-situ PPy polymerization on the chemical structure (Fig. 4 a). The spectrum of BC reveals that bands at 3344, 2896, 1646, and 1051 cm − 1 were attributed to the stretching vibrations of O–H, C–H, and deformation vibrations of O–H, C-O, respectively. And the new characteristic peaks at 1543 and 1453 cm − 1 of BC/PPy gradient conductive membranes were respectively assigned to the stretching vibration peaks of C = C and C = N in py, which confirming successful PPy polymerization on BC membranes [ 32 , 33 ]. In present study, compared to pure BC, the BC/PPy gradient conductive membranes displayed a gradual reduction in the absorption peak at 3344 cm − 1 , accompanied by a slight shift towards 3340 cm − 1 . This change is attributed to hydrogen bonding interactions between BC and PPy, altering the original O-H connections on BC and causing a redshift in the infrared stretching vibration peak. Simultaneously, increased PPy deposition on the gradient conductive membranes fibers led to a decrease in the intensity of the O-H stretching vibration peak. In addition, TGA was conducted to evaluate the thermal stability of nonstretched and stretched BC/PPy gradient conductive membranes at different segments (Fig. S6). Three decomposition stages were observed for all the gradient conductive membranes. Notably, the thermal stability of BC/PPy gradient conductive membranes improved attributed to the increased PPy content. Furthermore, stretched membranes exhibited superior thermal stability compared nonstretched membranes, attributed to the more denser structure of aligned BC/PPy fibers. This aligned structure acted as a barrier for BC molecular chains, which further enhanced the thermal stability for tissue engineering application [ 11 ]. 3.5. X-ray photoelectron spectroscopy (XPS) analysis XPS was used on different segments of nonstretched and stretched BC/PPy gradient conductive membranes. The down segment of aligned BC/PPy gradient conductive membranes showed a new characteristic N peak at 398 eV compared to BC, confirming successful PPy loading (Fig. 4 b). The newly emerged S peak was attributed to the incorporation of p-TSA during the synthesis of PPy. Further quantitative analysis of N content in different membranes was shown in Table S1 . The N content in the up, middle, and down segments of nonstretched BC/PPy gradient conductive membranes increased sequentially, with values of 3.57%, 5.32%, and 9.37%, respectively. The similar results appeared in the stretched BC/PPy gradient conductive membranes, with N content values of 3.9%, 8.3%, and 9.6% in up, middle, and down segments, respectively, indicating effective PPy gradient deposition from the up to down segments. Interestingly, the slightly higher N content in the stretched membrane, even at the same reaction time was higher than nonstretched membranes at the same segments. In present study, this can be attributed to the more aligned and tightly-packed BC nanofibers, which could afford topografic structure for more PPy deposition in-situ . These results align with the FESEM and C-AFM analysis, collectively highlighting the effectiveness of PPy gradient deposition and the influence of aligned nanofiber on the structural and compositional characteristics of BC/PPy gradient conductive membranes. 3.6. Surface resistance analysis and hydrophilicity analysis Surface resistance of nonstretched and stretched BC/PPy gradient conductive membranes were evaluated by surface resistance meter (Fig. 4 c). A gradual increase in surface resistance from the down to the up was observed across all BC/PPy gradient conductive membranes (Fig. S4). And it is worth mentioning that stretched BC/PPy gradient conductive membranes exhibited lower surface resistance compared to the nonstretched BC/PPy gradient conductive membranes counterparts at the same segments. This could be due to that more PPy were deposited on aligned nanofibers because of its larger deposition area, forming a continuous and oriented conductive pathway, which thereby enhanced the electron transport efficiency. Such surface resistance results were consistent with previous results of C-AFM, confirming the successful preparation of BC/PPy gradient conductive membranes for the guidance of cellular activities. Besides, the surface hydrophilicity results show that the BC/PPy gradient conductive membranes exhibited gradient change in surface hydrophilicity (Table S2). This trend can be due to that the gradient increase in PPy deposition on BC nanofibers from the up to down segments lead to the gradient reduction of active hydrophilic groups (-OH) on the nanofibers, suggesting tunable surface properties for cell-material interactions in specific tissue engineering applications. 3.7. Mechanical property analysis Mechanical property is a critical parameter for evaluating the tissue engineering scaffolds made from biomaterials [ 34 ]. The mechanical properties of nonstretched and stretched BC/PPy gradient conductive membranes were shown in Fig. 4 d-f. The tensile strength of pure BC membrane was measured at 5.2 MPa, with a Young's modulus of 238.6 MPa, while the tensile strength of the stretched BC/PPy gradient conductive membranes reached 34.1 MPa, with a Young's modulus of 1849.9 MPa (*** P < 0.005). It has also been reported in previous studies that the tensile strength and Young's modulus increased with the deposition of PPy and the alignment of nanofibers [ 11 , 35 ]. In the present study, the improvement in mechanical properties of stretched BC/PPy gradient conductive membranes could be related to two reasons. Firstly, aligned nanofibers are tightly arranged and have stronger hydrogen bonds. When subjected to tensile forces aligned with its orientation, the inherently stronger fibers in the oriented BC/PPy gradient conductive membranes can bear greater external tensile forces. In contrast, in the nonstretched BC/PPy gradient conductive membranes, only a few fibers aligned with the direction of external force can bear the load, leading to the rupture of most fibers simultaneously, as previously studied [ 11 ]. Secondly, the deposition of PPy on aligned BC was more densely continuous, reinforcing the hydrogen bonding interactions between PPy and BC, as well as between BC fibers. In conclusion, deposited PPy and aligned nanofibers synergistically improved the mechanical properties of the BC/PPy gradient conductive membranes for further nerve regeneration application. 3.8. In vitro biocompatibility evaluation and cell morphology Biocompatibility is a fundamental requirement for biomaterials in tissue engineering applications [ 36 ]. The hemolysis ratio (HR) was employed to evaluate the blood compatibility of nonstretched and stretched BC/PPy gradient conductive membranes (Fig. 5 a). The HR of nonstretched gradient conductive membranes at the up, middle, and down segments were 1.38%, 0.28%, and 0.28%, respectively, while the stretched membrane exhibited HR of 0.87%, 0.04%, and 0.40% at the corresponding segments. These results indicated a good hemocompatibility of BC/PPy gradient conductive membranes, as the hemolysis ratio less 5% is permissible for biomaterials [ 37 ]. The cytocompatibility of BC, nonstretched and stretched BC/PPy gradient conductive membranes were assessed through MTT assays using PC12 cells and NIH3T3 cells (Fig. 5 b, Fig. S7). The cell viability on all the BC/PPy gradient conductive membranes was higher than that on pure BC membranes throughout 1, 3, and 5-day culture, suggesting the non-cytotoxic nature of the BC/PPy membranes. Specifically, for the nonstretched gradient conductive membranes, the relative cell viability of middle and down segments (120% and 126%, respectively) exhibited significantly higher than that of the up segment (107%, P < 0.05). This gradient cell viability was also appeared on the stretched BC/PPy gradient conductive membranes, where the relative cell viability of the up, middle and down segments was 98%, 129% ( P < 0.005), and 132% ( P < 0.05). Notably, the down segment of stretched BC/PPy gradient conductive membranes promoted the cell proliferation most, which showed a significant 1.32-fold higher cell viability than that of control (culture dish, P < 0.01). Similar results were shown by the live/dead staining assay (Fig. 5 c). From day 3 to day 5, a noticeable gradient proliferation of live cells (green) was observed on all the gradient conductive membranes from the up to down segments, indicating that PC12 cells could proliferate and grow normally on the BC/PPy gradient conductive membranes, especially on the down segment of the stretched BC/PPy gradient conductive membranes. It is well established that PPy has excellent biocompatibility and could support cell proliferation [ 38 ]. In the present study, the increasing concentration gradient distribution of PPy on the aligned membranes contribute to the enhanced cell viability from the up to down segments. These could be attributed to two aspects. First, the PPy gradient deposited on the stretched BC/PPy gradient conductive membranes was mainly distributed along the fiber alignment (Fig. 2 d), which could provide more effective transmission of endogenous electrical signals between cells and fibers for promoted cell proliferation [ 39 ]. Second, the deeper and oriented nanogrooves on the stretched BC/PPy gradient conductive nanofibers give more space for PPy deposition, which could provide more active functional groups and anchoring points for cell adhesion, growth, and proliferation [ 40 ]. Therefore, these results indicated that stretched BC/PPy gradient conductive membranes can promote gradient proliferation of cells from the up to down segments, providing an effective method to correspond topographic (fiber alignment) and gradient (PPy gradient) guidance with the subcellular responses. 3.9. Evaluation of cell differentiation on BC/PPy gradient conductive membranes 3.9.1. In the absence of EF In the absence of EF, PC12 cells on the culture dish, BC, and up segment of nonstretched BC/PPy gradient conductive membranes exhibited a rounded morphology with no protrusion, whereas cells on the middle and down segments of the nonstretched BC/PPy membranes began to show protrusions (Fig. 6 , 7 a, -EF). Compared with the same segments of the nonstretched BC/PPy gradient conductive membranes, more cell protrusions and longer neurites were observed on the stretched BC/PPy membranes with neurites elongate along the fiber alignment. Significantly higher neurite-bearing cell population ( p < 0.005) and neurites length ( p < 0.005) on the down segment are measured than that on the up segment of BC/PPy gradient conductive membranes (Fig. 7 b-c, -EF). Moreover, the neurite-bearing cell population ( p < 0.05) and the neurites length ( p < 0.005) measured on the down segment of the stretched membranes were significantly higher than that of the nonstretched membranes (Fig. 7 b-c, -EF). These results indicate the effect of BC/PPy gradient conductive membranes from the up to down segments on the gradient cell differentiation, where the down segment of the stretched BC/PPy gradient conductive membranes promoted the most. In addition, as shown in Fig. S8, the stretched BC/PPy gradient conductive membranes could promote neurite elongation along the fiber alignment, especially at the down segment of the stretched BC/PPy gradient conductive membranes. This is consistent with a previous report where PC12 cells and Schwann cells exhibited oriented growth on the patterned materials [ 12 , 41 ]. Another study was found for the aligned conductive scaffold, whereon PC12 cells showed enhanced neurogenesis [ 42 ]. In the present study, the mechanism for the gradient and directed cell differentiation on the stretched BC/PPy gradient conductive membranes may relate to the gradient and aligned PPy deposition, which can transfer the gradient electrical signal in the growth of conducting cells like PC12 influencing the cellular signaling pathways and inducing directional cell differentiation. 3.9.2. In the presence of EF To further study the synergistic effect of BC/PPy gradient conductive membranes and EF on the cell differentiation, the PC12 cells cultured on the membranes were exposed to EF (Fig. 6 , 7 a, +EF). Apparently, “+EF” groups showed typical neuronal differentiation morphology and the neurite was markedly formed (Fig. 6 , 7 a). The neurite-bearing cell population and neurite length measured on all the segments of the BC/PPy gradient conductive membranes with EF were significantly higher than that without EF, except for the up segment of the stretched membrane (Fig. 7 b-c). Moreover, the down segment of the stretched membranes in the presence of EF exhibited the highest neurite-bearing cell population (Fig. 7 b, 63.35%), which is 1.29-fold higher than that measured in the down segment of the nonstretched membranes (49.03%, p > 0.05, +EF) and 2.04-fold higher than that measured in the up segment of the stretched membranes (31.13%, p < 0.05, +EF). This behavior is also consistent with their neurite length where the down segment of the stretched membranes under EF (Fig. 7c, 64.39 µm) exhibited 1.45-fold higher than that measured in the down segment of the nonstretched membranes (44.38 µm, p < 0.005, +EF) and 1.82-fold higher than that measured in the up segment of the stretched membranes (35.45 µm, p < 0.005, +EF). In addition, Fig. S8 shows that there's no obvious influence of EF on the direction of neurite extension. Hence, the combination of stretched BC/PPy membranes and EF could synergistically enhanced neurite-bearing and neurite length in a gradient manner from the up to down segments. Similar enhanced cell differentiation was reported by Tang et al. , where the combination of conductive PPy-coated aligned fibers and EF collectively enhanced the functional expression of PC12 cells, including elongation, gene expression, and protein expression [ 13 ]. Previous studies suggested that the conductive microenvironment and EF promoted the opening of voltage-gated calcium channels and the influx of Ca 2+ , which thereby upregulating expression of neurogenic genes and improving neurogenesis [ 13 , 43 , 44 ]. However, the influence of the gradient conductive and topographic guidance with EF on the cell differentiation was still unknown. The influence of the aligned BC/PPy gradient conductive membranes under electrical stimulation can be attributed to three main factors. First, for the PPy gradient guidance, the conjugated π-electrons clouds on the aligned PPy gradient chain formed a delocalized set of electrons, providing continuous and gradient charge transmission, which thereby effectively promoted intracellular signal transmission between cells and materials, and thus enhanced gradient neuronal differentiation from the up to down segments of the membranes. Secondly, for the topographic guidance, aligned BC nanofibers were used as a core component, which is similar to the nerve bundles consisting of numerous aligned cell-matrix assemblies [ 45 ]. In addition, PPy have acceptable biocompatibility, and could allow the adhesion, growth, and differentiation of neural cells [ 46 ]. Thus, the oriented deposition of PPy along the aligned BC nanofibers can activate relevant cell signaling pathways, converting topographic signals into mechanical signals and thereby inducing cell differentiation. Thirdly, for the application of EF, studies have shown its important role in altering the distribution of extracellular matrix molecules and enhancing protein absorption levels, thus promoting the elongation of neural axons. In the present study, the electrical stimulation can simultaneously induce the transfer of delocalized π electrons along the oriented PPy chains, thereby promoting the transport of bioactive molecules or cell growth factors converting topographic signals into mechanical signals, ultimately synergistically achieving gradient differentiation of PC12 cells on the aligned BC/PPy gradient conductive membranes. Overall, the stretched BC/PPy gradient conductive membranes can effectively enhance the directional neurite-bearing and neurite length in a gradient manner from the up to down segments. Importantly, the cells tend to differentiate most along the fiber alignment on the down segment of the stretched BC/PPy gradient conductive membranes with the application of EF. This study is the first to report the synergistically enhanced gradient cell differentiation by the stretched BC/PPy gradient conductive membranes and EF stimulation, suggesting the potential for combinations of these cues to be exploited in peripheral nerve regeneration. 4. Conclusions In summary, the highly aligned BC/PPy gradient conductive membrane was successfully prepared through quantitatively stretching and hot pressing of BC, followed by a kinetically controlled in-situ polymerization reaction of gradient PPy along the nanofibers. The membranes were composed of highly aligned nanofibers with introduction of gradient PPy along the fiber alignment, which presented good mechanical strength, thermal stability, and surface current gradient. In vitro biological evaluation demonstrated that the aligned BC/PPy gradient conductive membrane possessed excellent cytocompatibility, hemocompatibility, and could efficiently promote the gradient proliferation and differentiation of PC12 cells. Moreover, after the application of EF, the directional gradient cell differentiation on the aligned BC/PPy gradient conductive membrane could be significantly enhanced with the maximum axon length (64.39 μm) and neurite-bearing cell population (63.35%) observed at the down segment of the membrane compared to the nonstretched BC/PPy gradient conductive membrane with EF and the stretched BC/PPy membrane without EF. These results reveal that aligned BC and EF may have synergistic effect for nerve regeneration. Taken together, it is consistent with our hypothesis that the aligned BC/PPy gradient conductive membrane with EF could be a promising candidate in enhancing directional cell differentiation for accelerated peripheral nerve regeneration. Declarations Conflicts of interest The authors declare that they have no conflict of interest. Funding This work was supported by the National Natural Science Foundation of China (Grant No. 52373235), Guangdong Provincial Key Laboratory of Advanced Biomaterials(Grant No. KLAB202404007), the National Natural Science Foundation of Hubei Province of China for Young Scholars (Grant No. 2022CFB749), and the Hubei Provincial Education Department Research Young and Middle-aged Talent Fund (Grant No. Q20222803). Author Contribution Li Wang: Investigation, Methodology, Writing. Fuyu Qi: Writing. Hao Wang: Investigation, Methodology, Data. Shuangshuang Li: Data. Sanming Hu: Visualization, review. Zhijun Shi: Conceptualization, Supervision. Guang Yang: Project administration, Supervision. Hong Chen: Conceptualization, Supervision. All authors reviewed the manuscript. Acknowledgements The authors acknowledge the Research Core Facilities for Life Science and the Analytical and Testing Centre at the HUST for performing characterization of various samples. References Luo Y, Li J, Li B, Xia Y, Wang H, Fu C. Physical Cues of Matrices Reeducate Nerve Cells. Front Cell Dev Biol 2021;9:1–14. https://doi.org/10.3389/fcell.2021.731170. Wang J, Zhu YQ, Wang Y, Xu HG, Xu WJ, Wang YX, et al. A novel tissue engineered nerve graft constructed with autologous vein and nerve microtissue repairs a long-segment sciatic nerve defect. Neural Regen Res 2021;16:143–9. https://doi.org/10.4103/1673-5374.286977. Heinzel JC, Quyen Nguyen M, Kefalianakis L, Prahm C, Daigeler A, Hercher D, et al. A systematic review and meta-analysis of studies comparing muscle-in-vein conduits with autologous nerve grafts for nerve reconstruction. Sci Rep 2021;11:1–12. https://doi.org/10.1038/s41598-021-90956-3. Kehoe S, Zhang XF, Boyd D. FDA approved guidance conduits and wraps for peripheral nerve injury : A review of materials and efficacy. Injury 2012;43:553–72. https://doi.org/10.1016/j.injury.2010.12.030. Grinsell D, Keating CP. Peripheral Nerve Reconstruction after Injury: A Review of Clinical and Experimental Therapies. Biomed Res Int 2014;2014. https://doi.org/10.1155/2014/698256. Javeed S, Faraji AH, Dy C, Ray WZ, MacEwan MR. Application of electrical stimulation for peripheral nerve regeneration: Stimulation parameters and future horizons. Interdiscip Neurosurg Adv Tech Case Manag 2021;24:101117. https://doi.org/10.1016/j.inat.2021.101117. Zhang K, Zheng H, Liang S, Gao C. Aligned PLLA nanofibrous scaffolds coated with graphene oxide for promoting neural cell growth. Acta Biomater 2016;37:131–42. https://doi.org/10.1016/j.actbio.2016.04.008. Manan S, Ullah MW, Ul-Islam M, Shi Z, Gauthier M, Yang G. Bacterial cellulose: Molecular regulation of biosynthesis, supramolecular assembly, and tailored structural and functional properties. Prog Mater Sci 2022;129:100972. https://doi.org/10.1016/j.pmatsci.2022.100972. Ren YJ, Zhang S, Mi R, Liu Q, Zeng X, Rao M, et al. Enhanced differentiation of human neural crest stem cells towards the Schwann cell lineage by aligned electrospun fiber matrix. Acta Biomater 2013;9:7727–36. https://doi.org/10.1016/j.actbio.2013.04.034. Wang L, Adhikari M, Li L, Li S, Mbituyimana B, Li X, et al. Electrically stimulated Acetobacter xylinum for the production of aligned 3D microstructured bacterial cellulose. Cellulose 2023;30:9973–88. https://doi.org/10.1007/s10570-023-05521-0. Wang L, Mao L, Qi F, Li X, Wajid Ullah M, Zhao M, et al. Synergistic effect of highly aligned bacterial cellulose/gelatin membranes and electrical stimulation on directional cell migration for accelerated wound healing. Chem Eng J 2021;424:130563. https://doi.org/10.1016/j.cej.2021.130563. Huang Z, Sun M, Li Y, Guo Z, Li H. Reduced graphene oxide-coated electrospun fibre: effect of orientation, coverage and electrical stimulation on Schwann cells behavior. J Mater Chem B 2021;9:2656–65. https://doi.org/10.1039/d1tb00054c. Tang J, Wu C, Chen S, Qiao Z, Borovskikh P, Shchegolkov A, et al. Combining Electrospinning and Electrospraying to Prepare a Biomimetic Neural Scaffold with Synergistic Cues of Topography and Electrotransduction. ACS Appl Bio Mater 2020;3:5148–59. https://doi.org/10.1021/acsabm.0c00595. Sadeghi A, Moztarzadeh F, Aghazadeh Mohandesi J. Investigating the effect of chitosan on hydrophilicity and bioactivity of conductive electrospun composite scaffold for neural tissue engineering. Int J Biol Macromol 2019;121:625–32. https://doi.org/10.1016/j.ijbiomac.2018.10.022. Liu R, Huang X, Wang X, Peng X, Zhang S, Liu Y, et al. Electrical stimulation mediated the neurite outgrowth of PC-12 cells on the conductive polylactic acid/reduced graphene oxide/polypyrrole composite nanofibers. Appl Surf Sci 2021;560:149965. https://doi.org/10.1016/j.apsusc.2021.149965. Sun Y, Quan Q, Meng H, Zheng Y, Peng J, Hu Y, et al. Enhanced Neurite Outgrowth on a Multiblock Conductive Nerve Scaffold with Self-Powered Electrical Stimulation. Adv Healthc Mater 2019;8:1–11. https://doi.org/10.1002/adhm.201900127. Chen S, McCarthy A, John J V., Su Y, Xie J. Converting 2D Nanofiber Membranes to 3D Hierarchical Assemblies with Structural and Compositional Gradients Regulates Cell Behavior. Adv Mater 2020;32:1–10. https://doi.org/10.1002/adma.202003754. Moore K, Sc MA, Macsween M, Sc M, Shoichet M, Ph D, et al. Moore, Macsween, Shoichet - 2006 - Immobilized concentration gradients of neurotrophic factors guide neurite outgrowth of primary neuron 2006;12:19–21. Cai Y, Huang Q, Wang P, Ye K, Zhao Z, Chen H, et al. Conductive Hydrogel Conduits with Growth Factor Gradients for Peripheral Nerve Repair in Diabetics with Non-Suture Tape. Adv Healthc Mater 2022;11:1–12. https://doi.org/10.1002/adhm.202200755. Zhang D, Li Z, Shi H, Yao Y, Du W, Lu P, et al. Micropatterns and peptide gradient on the inner surface of a guidance conduit synergistically promotes nerve regeneration in vivo. Bioact Mater 2022;9:134–46. https://doi.org/10.1016/j.bioactmat.2021.07.010. Tang S, Zhu J, Xu Y, Xiang AP, Jiang MH, Quan D. The effects of gradients of nerve growth factor immobilized PCLA scaffolds on neurite outgrowth invitro and peripheral nerve regeneration in rats. Biomaterials 2013;34:7086–96. https://doi.org/10.1016/j.biomaterials.2013.05.080. Zonderland J, Rezzola S, Wieringa P, Moroni L. Fiber diameter, porosity and functional group gradients in electrospun scaffolds. Biomed Mater 2020;15. https://doi.org/10.1088/1748-605X/ab7b3c. Xue T, Yang Y, Yu D, Wali Q, Wang Z, Cao X, et al. 3D Printed Integrated Gradient-Conductive MXene/CNT/Polyimide Aerogel Frames for Electromagnetic Interference Shielding with Ultra-Low Reflection. Nano-Micro Lett 2023;15:1–14. https://doi.org/10.1007/s40820-023-01017-5. Shan Y, Feng H, Li Z. Electrical stimulation for nervous system injury: Research progress and prospects. Wuli Huaxue Xuebao/ Acta Phys - Chim Sin 2020;36:1–11. https://doi.org/10.3866/PKU.WHXB202005038. Costello MC, Errante EL, Smartz T, Ray WZ, Levi AD, Burks SS. Clinical applications of electrical stimulation for peripheral nerve injury: a systematic review. Front Neurosci 2023;17. https://doi.org/10.3389/fnins.2023.1162851. Zha F, Chen W, Hao L, Wu C, Lu M, Zhang L, et al. Electrospun cellulose-based conductive polymer nanofibrous mats: Composite scaffolds and their influence on cell behavior with electrical stimulation for nerve tissue engineering. Soft Matter 2020;16:6591–8. https://doi.org/10.1039/d0sm00593b. Li X, Ji X, Chen K, Ullah MW, Yuan X, Lei Z, et al. Development of finasteride/PHBV@polyvinyl alcohol/chitosan reservoir-type microspheres as a potential embolic agent: From: In vitro evaluation to animal study. Biomater Sci 2020;8:2797–813. https://doi.org/10.1039/c9bm01775e. Prasannan P, Siney E, Chatterjee S, Johnston D, Shah M, Mudher A, et al. A 3D-induced pluripotent stem cell-derived human neural culture model to study certain molecular and biochemical aspects of Alzheimer’s disease. Vitr Model 2022;1:447–62. https://doi.org/10.1007/s44164-022-00038-5. Liu F, Xu J, Wu L, Zheng T, Han Q, Liang Y, et al. The Influence of the Surface Topographical Cues of Biomaterials on Nerve Cells in Peripheral Nerve Regeneration: A Review. Stem Cells Int 2021;2021. https://doi.org/10.1155/2021/8124444. Mikulik D, Ricci M, Tutuncuoglu G, Matteini F, Vukajlovic J, Vulic N, et al. Conductive-probe atomic force microscopy as a characterization tool for nanowire-based solar cells. Nano Energy 2017;41:566–72. https://doi.org/10.1016/j.nanoen.2017.10.016. Pathak CS, Chang BJ, Song S. Review on scanning probe microscopy analysis for perovskite materials and solar cells. Dye Pigment 2023;218:111469. https://doi.org/10.1016/j.dyepig.2023.111469. Xiong F, Wei S, Wu S, Jiang W, Li B, Xuan H, et al. Aligned Electroactive Electrospun Fibrous Scaffolds for Peripheral Nerve Regeneration. ACS Appl Mater Interfaces 2023;15:41385–402. https://doi.org/10.1021/acsami.3c09237. Thakur A V., Lokhande BJ. Study of RuO2 incorporated PPy hybrid flexible electrodes prepared by soaking and drying technique: a novel approach. Appl Phys A Mater Sci Process 2021;127:1–6. https://doi.org/10.1007/s00339-021-05028-0. Dado D, Levenberg S. Cell-scaffold mechanical interplay within engineered tissue. Semin Cell Dev Biol 2009;20:656–64. https://doi.org/10.1016/j.semcdb.2009.02.001. Guo H, Qiao T, Jiang S, Li T, Song P, Zhang B, et al. Aligned poly (glycolide-lactide) fiber membranes with conducting polypyrrole. Polym Adv Technol 2017;28:484–90. https://doi.org/10.1002/pat.3912. Koons GL, Diba M, Mikos AG. Materials design for bone-tissue engineering. Nat Rev Mater 2020;5:584–603. https://doi.org/10.1038/s41578-020-0204-2. Singhal JP, Ray AR. Synthesis of blood compatible polyamide block copolymers. Biomaterials 2002;23:1139–45. https://doi.org/10.1016/S0142-9612(01)00228-9. Yardimci AI, Aypek H, Ozturk O, Yilmaz S, Ozcivici E, Mese G, et al. CNT Incorporated Polyacrilonitrile/Polypyrrole Nanofibers as Keratinocytes Scaffold. J Biomimetics, Biomater Biomed Eng 2019;41:69–81. https://doi.org/10.4028/www.scientific.net/JBBBE.41.69. Zhao X, Wu H, Guo B, Dong R, Qiu Y, Ma PX. Antibacterial anti-oxidant electroactive injectable hydrogel as self-healing wound dressing with hemostasis and adhesiveness for cutaneous wound healing. Biomaterials 2017;122:34–47. https://doi.org/10.1016/j.biomaterials.2017.01.011. Fahlgren A, Bratengeier C, Gelmi A, Semeins CM, Klein-Nulend J, Jager EWH, et al. Biocompatibility of polypyrrole with human primary osteoblasts and the effect of dopants. PLoS One 2015;10:1–17. https://doi.org/10.1371/journal.pone.0134023. Chen CH, Tsai CC, Wu PT, Wang IK, Yu J, Tsai WB. Modulation of Neural Differentiation through Submicron-Grooved Topography Surface with Modified Polydopamine. ACS Appl Bio Mater 2019;2:205–16. https://doi.org/10.1021/acsabm.8b00556. Wang L, Wu Y, Hu T, Ma PX, Guo B. Aligned conductive core-shell biomimetic scaffolds based on nanofiber yarns/hydrogel for enhanced 3D neurite outgrowth alignment and elongation. Acta Biomater 2019;96:175–87. https://doi.org/10.1016/j.actbio.2019.06.035. Toth AB, Shum AK, Prakriya M. Regulation of neurogenesis by calcium signaling. Cell Calcium 2016;59:124–34. https://doi.org/10.1016/j.ceca.2016.02.011. Hao B, Webb SE, Miller AL, Yue J. The role of Ca2+ signaling on the self-renewal and neural differentiation of embryonic stem cells (ESCs). Cell Calcium 2016;59:67–74. https://doi.org/10.1016/j.ceca.2016.01.004. Yang Y, Sun J, Liu X, Guo Z, He Y, Wei D, et al. Wet-spinning fabrication of shear-patterned alginate hydrogel microfibers and the guidance of cell alignment. Regen Biomater 2017;4:299–307. https://doi.org/10.1093/rb/rbx017. Borges MHR, Nagay BE, Costa RC, Souza JGS, Mathew MT, Barão VAR. Recent advances of polypyrrole conducting polymer film for biomedical application: Toward a viable platform for cell-microbial interactions. vol. 314. 2023. https://doi.org/10.1016/j.cis.2023.102860. Additional Declarations No competing interests reported. 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BC/PPy gradient conductive membranes were obtained using a peristaltic pump by kinetically controlled reaction. Then the PC 12 cells were seeded on membranes and electrically stimulated in an electrostimulated cell culture device.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4403218/v1/f5b9ce82250f561eac280f57.jpg"},{"id":58225072,"identity":"2b168173-cac6-45b2-bc3a-bef82bde61fb","added_by":"auto","created_at":"2024-06-12 17:43:21","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":234722,"visible":true,"origin":"","legend":"\u003cp\u003eFESEM images of the nonstretched-up (a), middle (b), down (c) and stretched -up (d), middle (e), down (f) BC/PPy gradient conductive membranes. The cross-sectional images (perpendicular to the stretching direction) and the side view (parallel to the stretching direction) of the stretched-down BC/PPy gradient conductive membranes (g, h). Scale: 500 nm.\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4403218/v1/4905ad7e91f7191fe0e639de.jpg"},{"id":58225835,"identity":"cf1cb365-a1cb-4eaf-8297-94e247faccc0","added_by":"auto","created_at":"2024-06-12 17:51:21","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":148517,"visible":true,"origin":"","legend":"\u003cp\u003eAFM images of current (a-c, g-i) and height (d-f, j-l) of nonstretched (a-f) and stretched (g-l) BC/PPy conductive gradient membranes.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4403218/v1/f96042accb5587403d6dccee.jpg"},{"id":58225071,"identity":"e6f261c0-b07b-4af8-aa7b-639073bd3dd3","added_by":"auto","created_at":"2024-06-12 17:43:21","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":109272,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterizations of BC/PPy conductive gradient membranes. ATR-FTIR (a) and XPS (b) spectra of BC and stretched BC/PPy conductive gradient membranes. (c) The log (R) values of nonstretched, and stretched BC/PPy conductive gradient membranes. (d-f) Stress–strain, tensile strength, and Young’s modulus curves of BC, nonstretched BC/PPy, stretched BC, and stretched BC/PPy conductive gradient membranes. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.005.\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4403218/v1/2146d2bf39d8b241e6c088cf.jpg"},{"id":58225076,"identity":"dd0b4a6d-3559-4ba2-b551-70db7672fdf7","added_by":"auto","created_at":"2024-06-12 17:43:21","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":164357,"visible":true,"origin":"","legend":"\u003cp\u003eBiocompatibility of BC/PPy conductive gradient membranes and cell morphology observations. (a) Hemolysis test for nonstretched and stretched BC/PPy conductive gradient membranes. PC refers to a positive control (distilled water) and NC refers to negative control (normal saline). (b-c) MTT assay and Live/Dead staining images of PC12 cells cultured on Control (petri dish), BC, nonstretched and stretched BC/PPy conductive gradient membranes. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05,**\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01,***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.005,\u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.005, # when compared with the nonstretched-up group under the same condition. \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u0026amp; when compared with the nonstretched-middle group under the same condition. \u003csup\u003e%\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, % when compared with the nonstretched-down group under the same condition. \u003csup\u003e$$\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, $ when compared with Control group under the same condition. Scale bars are 100 μm.\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4403218/v1/4bffd95994803bb39ecf3e36.jpg"},{"id":58225836,"identity":"18e0279d-88d0-49cd-a783-fac725bf2013","added_by":"auto","created_at":"2024-06-12 17:51:21","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":242543,"visible":true,"origin":"","legend":"\u003cp\u003eThe synergistic effect of BC/PPy gradient conductive membranes and EF on PC12 cells. FITC/DAPI staining images of PC12 cells after culturing on Control (petri dish), BC, nonstretched and stretched BC/PPy gradient conductive membranes in the absence or presence of EF, respectively. Scale bars are 100 μm.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4403218/v1/a3c689998ea20769dae7a267.jpg"},{"id":58225079,"identity":"a6f7ff0d-fe9c-46d7-b41e-b3d8175d059a","added_by":"auto","created_at":"2024-06-12 17:43:22","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":122307,"visible":true,"origin":"","legend":"\u003cp\u003eThe synergistic effect of BC/PPy gradient conductive membranes and EF on differentiation of PC12 cells. (a) Immunofluorescence images of PC12 cells after culturing on Control (petri dish), BC, nonstretched and stretched BC/PPy gradient conductive membranes in the absence or presence of electrical stimulation, respectively. Scale bars are 50 μm. (b-c) Quantitative analysis of the neurite bearing (b), neurite length (c) of PC12 cells after culturing on Control (petri dish), BC, nonstretched-down and stretched-down BC/PPy gradient conductive membranes in the absence or presence of electrical stimulation, respectively. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.005, \u003csup\u003e\u0026amp;\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, \u003csup\u003e\u0026amp;\u0026amp;\u0026amp;\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.005,\u0026amp; when compared with Control group under the same condition. \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05,\u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01,\u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.005,# when compared with “ –EF ” groups under the same condition. \u003csup\u003e$\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, $ when compared with nonstretched groups under the same condition.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4403218/v1/78836104bdb2c285f6ac784c.jpg"},{"id":59454415,"identity":"c19926a1-3a5b-4bc2-9e8c-057ab42455a5","added_by":"auto","created_at":"2024-07-02 03:14:26","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1797764,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4403218/v1/cf53a84b-b21c-4f72-8ae8-3dd20539e49a.pdf"},{"id":58225075,"identity":"1c849355-7871-4bb2-b8f3-258399a73c72","added_by":"auto","created_at":"2024-06-12 17:43:21","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":2104979,"visible":true,"origin":"","legend":"","description":"","filename":"SI20240510.docx","url":"https://assets-eu.researchsquare.com/files/rs-4403218/v1/18a20209bef8d06f10ee4f92.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Highly aligned bacterial cellulose/PPy gradient conductive membranes for directed cell differentiation under electrical stimulation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDirectional cell differentiation plays an important role in peripheral nerve regeneration [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], especially those long-distance or chronic nerve defects. Loss of cell activity and uncontrolled nerve differentiation can lead to a functional gap between regenerative and normal nerves, potentially hindering successful peripheral nerve regeneration. Currently, although autogenous nerve grafts are the gold standard for repairing peripheral nerve defects, there are several limitations, including the restricted availability of tissue, mismatched nerve diameters, donor site morbidity, and loss of function [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Artificial substitutes on sale are mainly used to repair short-distance nerve gap or as protectant wrap devices [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], which may associate with single material, difficulty to imitate the complex anatomical structure, as well as appropriate guided cell differentiation and axonal reinnervation [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, it is desirable to develop a novel strategy that can programmatically guide the directional growth, proliferation and differentiation of cells at specific locations for accelerated peripheral nerve regeneration.\u003c/p\u003e \u003cp\u003eBiomaterials with aligned nanofibers have been proved to actively guide the orientated growth of neural cells and neurites, which can ensure that neurites across the injured nerve gap to the target tissue, thus promoting nerve regeneration and functional recovery [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Bacterial cellulose (BC), a natural polymer produced by \u003cem\u003eGluconacetobacter xylinus\u003c/em\u003e, has recently received enormous attention because of its high mechanical strength, flexibility, and nonimmunogenicity [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In particular, aligned BC nanofibers could provide contact-guidance for directional neural stem cell elongation, neurite outgrowth [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], pheochromocytoma (PC12) cell differentiation, and Schwann cell migration along the aligned nanofiber [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Due to other methods of producing aligned cellulose reduced the yield and affected the original structure of BC [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], our group successfully prepared the highly aligned BC-based membranes through quantitative stretching with excellent mechanical properties, biocompatibility, and guidance for cellular activities [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, this aligned BC nanofibers did not possess electrical conductivity and cannot support an improvement in biofunction such as delivering electrical and electro-chemical stimulation to nerve cells [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo make the scaffolds conductive, incorporation of polymeric- or metallic-conductive nanoparticles onto the aligned fibers have mainly been applied. Among them, polypyrrole (PPy) have been widely studied for nerve regeneration due to its excellent electrical properties, good biocompatibility, and ease of synthesis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. For instance, Liu \u003cem\u003eet al.\u003c/em\u003e reported that conductive polylactic acid/reduced graphene oxide/PPy composite nanofibers coupled with EF could significantly accelerate PC12 cell proliferation and differentiation [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Sun \u003cem\u003eet al.\u003c/em\u003e invented a self-powered electrical stimulation using Pt-BC/PPy-N-CNTs for nerve regeneration [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Whereas, these scaffolds could not provide directional cues for cellular activities and axon growth during the nerve repair. To improve the guidance capability, compositional gradient that enable cells to infer their spatial location and determine their fate accordingly is applied for new conduits [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It is reported that at the tip of the axon, the growth cones can sense gradient guidance cues and modify its shape to guide axon extension responsively [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Previous studies showed that introducing growth factor or peptide gradient onto conduits can promote the neurite extension and accelerate the sciatic nerve regeneration [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In another study, the middle of the gradient NGF-immobilized film achieves longer neurite length of dorsal root ganglia (DRG) than that on the film without NGF gradient [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Nevertheless, a scaffold having both the aligned nanofibers and PPy gradient conductive have not been tested so far, neither its effects on cellular activities for nerve regeneration. To date, different methods have been developed for preparing gradient material such as electrospinning [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], 3D printing [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], and directional diffusion [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, they need complex design and hard operation. Considering the aligned BC nanofibers allow the \u003cem\u003ein-situ\u003c/em\u003e polymerization of PPy onto its matrix, a time-controlled reaction method is easier to operate but loading time is the key to control.\u003c/p\u003e \u003cp\u003eEndogenous EF can be detected at the damaged nerve section, which play an important role in directing cellular activities for nerve regeneration [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The long period of nerve regeneration after injury may lead to target organ loss [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. To mimic and prolong the endogenous EF, exogenous EF stimulation could be a unique and promising treatment for a variety of peripheral nerve injury types including long distance injuries [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The EF stimulation could promote the differentiation and proliferation of PC12 cells and axon extension [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], increase intraneuronal cAMP in DRGs and nerve growth factor (NGF) in Schwann cells [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Besides, the EF could induce microtubule associated proteins in axons, promote blood vessel regeneration, and subsequently facilitate peripheral nerve regeneration [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Therefore, the combination of EF with a scaffold having topographical guidance and gradient conductive signals may be a new option for peripheral nerve regeneration.\u003c/p\u003e \u003cp\u003eHerein, we developed a new nerve regeneration system, which combined the electrical cues with topographic and gradient conductive guidance of the BC/PPy gradient conductive membranes for enhanced cell differentiation in peripheral nerve regeneration. The aligned BC/PPy gradient conductive membranes were first prepared by quantitatively stretching technique and hot pressing, followed with introduction of gradient PPy along the nanofibers by a kinetically controlled reaction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The physicochemical properties of the prepared gradient conductive membranes were systematically characterized for their morphology, chemical structures, electrical conductivity, and mechanical strength. In addition, the biocompatibility and morphology of cells on the aligned BC/PPy gradient conductive membranes were investigated. Furthermore, the synergistic effect of coupling EF with the gradient conductive membranes on cell differentiation was examined.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eBC was purchased from Hainan Guangyu Biotechnology Co., Ltd. (Hainan, China). Pyrrole (py) was obtained from Sigma-Aldrich (USA). Ferric chloride hexahydrate (FeCl\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO) and para-methylbenzenesulfonic acid (p-TSA) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Dulbecco\u0026rsquo;s modified eagle\u0026rsquo;s medium (DMEM), Roswell Park Memorial Institute (RPMI) 1640, fetal bovine serum (FBS), horse serum, bovine serum albumin (BSA), penicillin/streptomycin (P/S), 3 (4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium romide (MTT), and dimethylsul-foxide (DMSO) were acquired from Gibico Life Technologies Co., Ltd (Grand Island, USA). Anti-beta III tubulin monoclonal antibody and rhodamine-conjugated goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) secondary antibody were obtained from Proteintech Group, Inc. (USA). Dihydrochloride (DAPI), FITC-conjugated phalloidin, and Calcein-AM/PI were purchased from Dojindo Laboratories (Kyushu, Japan). Nerve growth factor (NGF) was acquired from Beijing Yiqiao Shenzhou Technology Co., Ltd. (China). PC12 cells were generously provided by Huazhong University of Science and Technology (Wuhan, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Preparation of BC/PPy gradient conductive membranes\u003c/h2\u003e \u003cp\u003eBC were purified by boiling in 0.1 M NaOH for 30 min, and washed with deionized water. Then, the obtained BC was cut into 12 cm \u0026times; 4 cm pieces, and wet-drawn at a speed of 1 mm/min until the stretching strain reached 40%. Thereafter, the BC and stretched BC membranes were hot-pressed at 100\u0026deg;C for 12 hours as our previously described [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe nonstretched and stretched BC/PPy gradient conductive membranes were produced by kinetically controlled reaction using a peristaltic pump. Firstly, the py solution (4.13 g of p-TSA and 1.61 g of py in 300 mL water) was poured slowly into FeCl\u003csub\u003e3\u003c/sub\u003e solution (15.6 g of FeCl\u003csub\u003e3\u003c/sub\u003e\u0026middot;6H\u003csub\u003e2\u003c/sub\u003eO in 300 mL water) with slow stirring to form a reaction solution. When the solution turned black, the BC membranes were immersed vertically in it until reaching the top segment. Then, the peristaltic pump was activated to extract the reaction solution at flow rates of 10 mL/min, 20 mL/min, and 30 mL/min, respectively. Finally, after the reaction was complete, the membranes were washed three times with deionized water, and dried for 20 minutes to obtain the nonstretched BC/PPy gradient conductive membranes. According to the results of biocompatibility, the flow rates to control the reaction time for PPy gradient deposition on the membranes was fixed at 20 mL/min. Following the same method, the stretched BC/PPy gradient conductive membranes were obtained.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Characterization of BC/PPy gradient conductive membranes\u003c/h2\u003e \u003cp\u003eThe morphology of BC/PPy gradient conductive membranes was examined using field emission scanning electron microscopy (FESEM, Sirion 200, Holland, 10 kV) after sputtering-coated with gold. The current flowing through the membranes can be measured by conductive atomic force microscopy (C-AFM, -0.1 V) using the current detection mode with the scanning size 5 \u0026micro;m \u0026times; 5 \u0026micro;m. The chemical composition of the membranes was determined by Fourier-transform infrared spectroscopy (FTIR) spectrometer (Vertex 70, Germany) in the spectral range of 500\u0026ndash;4000 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The thermal properties of the membranes were determined by using the thermogravimetric analyzer (TGA, Pyrisl, China) at the rates of 10\u0026deg;C/min from 25\u0026deg;C to 800\u0026deg;C under nitrogen atmosphere. The elemental composition of the membranes was identified by X-ray photoelectron spectroscopy (XPS) spectrum (AXIS-ULTRA DLD-600W, Shimadzu) and elemental analyzer (EA, Vario Micro cube, Elementar). The surface resistivity of the different segments on the membranes was measured using a four-point probe resistivity meter. The mechanical property of membranes was assessed using the Instron Universal Testing Machine (UTM6503, SUNS Industrial Testing System Co., Ltd. China) at a stretching speed of 1 mm/min at 25\u0026deg;C and 50% relative humidity. The Young's modulus was determined from the slope at low strain values, and the maximum tensile strength corresponded to the maximum stress at sample breakage. The contact angle (CA) measurements were performed by a drop-shape analyzer (DSA 100, Kr\u0026uuml;ss). The shape of water drops was captured when 10 \u0026micro;L of deionized water was dropped at different positions of the membranes surface and then the average value of contact angle was reported.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Biocompatibility evaluation and cell morphology observation\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. MTT assay and the hemocompatibility\u003c/h2\u003e \u003cp\u003eThe cytotoxicity of BC/PPy gradient conductive membranes was determined through MTT assay. NIH3T3 cells were cultured in DMEM medium containing 1% P/S and 10% FBS at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. PC12 cells, which is widely used in research on nerve regeneration, were cultured in RPMI 1640 medium supplemented with 5% horse serum, 5% FBS, and 1% P/S under the condition of 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. Prior to cell seeding, the sterilized samples were pre-incubated in serum-free culture medium for 12 h. Then, cells were seeded onto the membranes at a density of 8 x 10\u003csup\u003e3\u003c/sup\u003e cells/well in 96-well plates. After 1, 3 and 5 days of culture, the culture medium was replaced by the fresh medium containing 0.5 mg/mL MTT, and cells were incubated at 37\u0026deg;C for another 4 hours. Then, 300 \u0026micro;L of DMSO was added and incubated for 10 minutes to dissolve the formazan crystals. After the reaction, the absorbance value was measured at 490 nm using a microplate reader. The hemocompatibility of the membranes was assessed \u003cem\u003evia\u003c/em\u003e the hemolytic activity assay as previously reported [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Initially, the materials were cut to fit into a 24-well plate and placed in each well. Subsequently, 1 mL of 0.9% NaCl solution was added to each well, and the samples were pre-treated for 24 hours at 37\u0026deg;C. After pre-treatment, the sample was transferred to test tubes, with 0.9 mL of physiological saline and 0.1 mL of diluted sheep whole blood added to each tube (V_whole blood: V_physiological saline\u0026thinsp;=\u0026thinsp;1:1.25). After thorough mixing, the tubes were incubated at 37\u0026deg;C for 1 hour, followed by centrifugation at 1000 rpm for 5 minutes. The supernatant was collected, and the absorbance was measured at 545 nm using a multi-functional microplate reader. Physiological saline served as the negative control, and distilled water served as the positive control. Each group included 6 parallel samples.\u003c/p\u003e \u003cp\u003eThe hemolysis rate of the samples was calculated using the following Equation S1:\u003c/p\u003e \u003cp\u003eHR (%) = [(OD\u003csub\u003eS\u003c/sub\u003e -OD\u003csub\u003eN\u003c/sub\u003e ) / (OD\u003csub\u003eP\u003c/sub\u003e -OD\u003csub\u003eN\u003c/sub\u003e )]\u0026times;100 (1)\u003c/p\u003e \u003cp\u003ewhere OD\u003csub\u003eS\u003c/sub\u003e, OD\u003csub\u003eN\u003c/sub\u003e, and OD\u003csub\u003eP\u003c/sub\u003e represented the optical density values of the sample, negative control, and positive control, respectively. This test provided insights into the hemocompatibility of the materials by evaluating their impact on red blood cell integrity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Live/dead staining and cytoskeleton staining\u003c/h2\u003e \u003cp\u003eThe morphology of cells on the membranes were observed by confocal laser scanning microscopy (CLSM, Olympus FV1000, Japan) after incubation for 5 days. For the live/dead staining, NIH3T3 and PC12 cells seeded on the membranes were stained using the Calcein-AM (2 \u0026micro;M) and PI (4 \u0026micro;M) for 15 min at 37\u0026deg;C, and observed and imaged by CLSM. For the cytoskeleton staining, PC12 cells on membranes were first fixed with 4% paraformaldehyde for 30 min and then permeabilized with 0.1% Triton X-100 (v/v) for 5 min. Then, the cells were co-stained with FITC-conjugated phalloidin and DAPI for 30 min prior to CLSM observation. ImageJ software was employed for statistical analysis and calculation of cell orientation angles. A value of 0\u0026deg; indicates a perfect alignment (parallel to the fiber alignment).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3. β3-tubulin staining\u003c/h2\u003e \u003cp\u003eβ3-Tubulin is considered an early neuronal differentiation marker and used for staining of neurites [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. For the β3-tubulin staining, PC12 cells on the membranes were first fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.1% Triton X-100 (v/v) for 5 min and blocked with 2% BSA for 1 hour. The cells were then incubated with β3-tubulin primary antibody at 4\u0026deg;C for 12 hours, followed with co-staining of rhodamine-conjugated goat anti-mouse IgG (H\u0026thinsp;+\u0026thinsp;L) and DAPI for 1 hour at room temperature prior to CLSM observation.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Electrical stimulation of cells on the \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003em\u003c/span\u003eembranes\u003c/h2\u003e \u003cp\u003eThe homemade electrotaxis chamber was built on the culture dish as previously reported [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. After one day of PC12 cells cultured on the membranes, the medium was replaced with differentiation medium containing 10% FBS and 50 ng/mL NGF for another 12 h. The samples were then placed in the electrical stimulation chamber. A constant EF magnitude of 50 mV/mm was applied for 1 hour per day over two consecutive days. Finally, the cells were cultured for additional 12 hours before staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical analysis\u003c/h2\u003e \u003cp\u003eEach experiment was performed in triplicate and all data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of mean (s. e. m.). A comparison between two groups was carried out by the Student\u0026rsquo;s t-test, and difference was considered statistically significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Result and discussion","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Characterizations of the 10, 20, 30-BC/PPy gradient conductive membranes\u003c/h2\u003e \u003cp\u003eIn order to fabricate and find the most suitable PPy gradient deposition on the membranes, 10, 20, and 30-BC/PPy gradient conductive membranes were obtained with the extraction rates of 10, 20, and 30 mL/min, respectively. PPy gradient were immobilized by differential BC/PPy deposition duration. For PPy gradient characterization, the membranes were divided into three segments (up, middle, and down), where the down segments of the membranes have the longest deposition time. The surface morphology and elemental analysis results confirmed the successful loading of PPy gradient on the 10, 20, and 30-BC/PPy membranes (Fig. S2, Fig. S3). The surface resistance and surface current results (Fig. S4, Fig. S5) indicated that the surface conductivity of BC/PPy gradient conductive membranes increased from the up to down segments. The cell activity of NIH3T3 and PC12 cells on the 20-BC/PPy gradient conductive membranes was higher than that of the other groups (Fig. S4). Therefore, 20-BC/PPy gradient conductive membranes with better biocompatibility and excellent surface resistance gradient characteristics were selected for subsequent experiments if not specified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Morphological characterizations of the BC/PPy gradient conductive membranes\u003c/h2\u003e \u003cp\u003eThe surface morphology of up, middle, and down segments of the nonstretched and stretched BC/PPy gradient conductive membranes was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. PPy nanoparticles with a diameter of about 100 nm were successfully deposited onto the surface of BC nanofibers with the increasing concentration gradient distribution from the up to down segments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-f). The color of the film became darker as more PPy deposited (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Specifically, the fibers on the nonstretched BC/PPy gradient conductive membranes were randomly arranged (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea-c), while the stretched BC/PPy gradient conductive membranes exhibit clear fiber alignment along the stretching direction (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed, f). The cross-sectional morphologies of the stretched BC/PPy gradient conductive membranes also demonstrated its internal oriented structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eg, h). The similarity in the features of fiber alignment was consistent with our previous study [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moreover, PPy tends to polymerization along the fiber alignment of the stretched membranes, suggesting the ideal guidance of the aligned BC nanofibers on the PPy deposition. This might be related to the deeper groove of the stretched membranes than the nonstretched membranes (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), which contributes to the formation of aligned and continuous conductive fiber network paths for the electrons transfer. Overall, the stretched BC/PPy gradient conductive membranes with highly aligned and PPy gradient conductive nanofibers could offer appropriate topographic, and electrical guidance for further exploration in nerve cell behavior studies.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.3. C-AFM\u003c/h2\u003e \u003cp\u003eIn view of the importance of surface topographies and conductivity variations of biomaterials on the adhesion, proliferation, and differentiation of nerve cells [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], C-AFM was carried out and the results are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The surface currents flow on all the BC/PPy gradient conductive membranes increased with the increasing concentration gradient distribution of PPy from the up to down segments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c and Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-i), indicating the improved electron transfer efficiency and conductivity after the introduction of PPy [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The surface current density on the up, middle, and down segments of the nonstretched BC/PPy gradient conductive membranes were 0.005, 0.139, and 13.378 nA, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea-c), while the stretched membranes exhibited higher surface currents on the same segments (0.005, 9.135, and 41.614 nA, respectively, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eg-i). Interestingly, the stretched BC/PPy gradient conductive membranes showed aligned surface currents distribution along the fiber alignment, where the down segment exhibited the maximum value of the surface current (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ei, 41.614 nA) and the regular nanogrooves depth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003el, 482.09 nm). In contrast, the randomly distributed surface currents with nanogrooves in a small depth were observed for the nonstretched membranes. This could be due to that the stretching and hot-pressing processes decreased the specific volume between the nanofibers, which thereby increased the depth of nanogrooves and aligned PPy deposition for current flow. Therefore, considering the gradient surface current distribution and enhanced current flow along the fiber alignment, the highly aligned stretched BC/PPy gradient conductive membranes could have the potential to transfer the topographic and gradient cues to guide cell behaviors, and thus allowing further applications in peripheral nerve regeneration.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.4. Fourier transform infrared (FTIR) spectra and Thermogravimetric analysis (TGA)\u003c/h2\u003e \u003cp\u003eFTIR was conducted on BC and stretched BC/PPy gradient conductive membranes to assess the impact of \u003cem\u003ein-situ\u003c/em\u003e PPy polymerization on the chemical structure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). The spectrum of BC reveals that bands at 3344, 2896, 1646, and 1051 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e were attributed to the stretching vibrations of O\u0026ndash;H, C\u0026ndash;H, and deformation vibrations of O\u0026ndash;H, C-O, respectively. And the new characteristic peaks at 1543 and 1453 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of BC/PPy gradient conductive membranes were respectively assigned to the stretching vibration peaks of C\u0026thinsp;=\u0026thinsp;C and C\u0026thinsp;=\u0026thinsp;N in py, which confirming successful PPy polymerization on BC membranes [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In present study, compared to pure BC, the BC/PPy gradient conductive membranes displayed a gradual reduction in the absorption peak at 3344 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, accompanied by a slight shift towards 3340 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. This change is attributed to hydrogen bonding interactions between BC and PPy, altering the original O-H connections on BC and causing a redshift in the infrared stretching vibration peak. Simultaneously, increased PPy deposition on the gradient conductive membranes fibers led to a decrease in the intensity of the O-H stretching vibration peak. In addition, TGA was conducted to evaluate the thermal stability of nonstretched and stretched BC/PPy gradient conductive membranes at different segments (Fig. S6). Three decomposition stages were observed for all the gradient conductive membranes. Notably, the thermal stability of BC/PPy gradient conductive membranes improved attributed to the increased PPy content. Furthermore, stretched membranes exhibited superior thermal stability compared nonstretched membranes, attributed to the more denser structure of aligned BC/PPy fibers. This aligned structure acted as a barrier for BC molecular chains, which further enhanced the thermal stability for tissue engineering application [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e\u003cem\u003e3.5.\u003c/em\u003e X-ray photoelectron spectroscopy (XPS) analysis\u003c/h2\u003e \u003cp\u003eXPS was used on different segments of nonstretched and stretched BC/PPy gradient conductive membranes. The down segment of aligned BC/PPy gradient conductive membranes showed a new characteristic N peak at 398 eV compared to BC, confirming successful PPy loading (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The newly emerged S peak was attributed to the incorporation of p-TSA during the synthesis of PPy. Further quantitative analysis of N content in different membranes was shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e. The N content in the up, middle, and down segments of nonstretched BC/PPy gradient conductive membranes increased sequentially, with values of 3.57%, 5.32%, and 9.37%, respectively. The similar results appeared in the stretched BC/PPy gradient conductive membranes, with N content values of 3.9%, 8.3%, and 9.6% in up, middle, and down segments, respectively, indicating effective PPy gradient deposition from the up to down segments. Interestingly, the slightly higher N content in the stretched membrane, even at the same reaction time was higher than nonstretched membranes at the same segments. In present study, this can be attributed to the more aligned and tightly-packed BC nanofibers, which could afford topografic structure for more PPy deposition \u003cem\u003ein-situ\u003c/em\u003e. These results align with the FESEM and C-AFM analysis, collectively highlighting the effectiveness of PPy gradient deposition and the influence of aligned nanofiber on the structural and compositional characteristics of BC/PPy gradient conductive membranes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.6. Surface resistance analysis and hydrophilicity analysis\u003c/h2\u003e \u003cp\u003eSurface resistance of nonstretched and stretched BC/PPy gradient conductive membranes were evaluated by surface resistance meter (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). A gradual increase in surface resistance from the down to the up was observed across all BC/PPy gradient conductive membranes (Fig. S4). And it is worth mentioning that stretched BC/PPy gradient conductive membranes exhibited lower surface resistance compared to the nonstretched BC/PPy gradient conductive membranes counterparts at the same segments. This could be due to that more PPy were deposited on aligned nanofibers because of its larger deposition area, forming a continuous and oriented conductive pathway, which thereby enhanced the electron transport efficiency. Such surface resistance results were consistent with previous results of C-AFM, confirming the successful preparation of BC/PPy gradient conductive membranes for the guidance of cellular activities. Besides, the surface hydrophilicity results show that the BC/PPy gradient conductive membranes exhibited gradient change in surface hydrophilicity (Table S2). This trend can be due to that the gradient increase in PPy deposition on BC nanofibers from the up to down segments lead to the gradient reduction of active hydrophilic groups (-OH) on the nanofibers, suggesting tunable surface properties for cell-material interactions in specific tissue engineering applications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.7. Mechanical property analysis\u003c/h2\u003e \u003cp\u003eMechanical property is a critical parameter for evaluating the tissue engineering scaffolds made from biomaterials [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The mechanical properties of nonstretched and stretched BC/PPy gradient conductive membranes were shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed-f. The tensile strength of pure BC membrane was measured at 5.2 MPa, with a Young's modulus of 238.6 MPa, while the tensile strength of the stretched BC/PPy gradient conductive membranes reached 34.1 MPa, with a Young's modulus of 1849.9 MPa (***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.005). It has also been reported in previous studies that the tensile strength and Young's modulus increased with the deposition of PPy and the alignment of nanofibers [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In the present study, the improvement in mechanical properties of stretched BC/PPy gradient conductive membranes could be related to two reasons. Firstly, aligned nanofibers are tightly arranged and have stronger hydrogen bonds. When subjected to tensile forces aligned with its orientation, the inherently stronger fibers in the oriented BC/PPy gradient conductive membranes can bear greater external tensile forces. In contrast, in the nonstretched BC/PPy gradient conductive membranes, only a few fibers aligned with the direction of external force can bear the load, leading to the rupture of most fibers simultaneously, as previously studied [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Secondly, the deposition of PPy on aligned BC was more densely continuous, reinforcing the hydrogen bonding interactions between PPy and BC, as well as between BC fibers. In conclusion, deposited PPy and aligned nanofibers synergistically improved the mechanical properties of the BC/PPy gradient conductive membranes for further nerve regeneration application.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.8. In vitro biocompatibility evaluation and cell morphology\u003c/h2\u003e \u003cp\u003eBiocompatibility is a fundamental requirement for biomaterials in tissue engineering applications [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. The hemolysis ratio (HR) was employed to evaluate the blood compatibility of nonstretched and stretched BC/PPy gradient conductive membranes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The HR of nonstretched gradient conductive membranes at the up, middle, and down segments were 1.38%, 0.28%, and 0.28%, respectively, while the stretched membrane exhibited HR of 0.87%, 0.04%, and 0.40% at the corresponding segments. These results indicated a good hemocompatibility of BC/PPy gradient conductive membranes, as the hemolysis ratio less 5% is permissible for biomaterials [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. The cytocompatibility of BC, nonstretched and stretched BC/PPy gradient conductive membranes were assessed through MTT assays using PC12 cells and NIH3T3 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, Fig. S7). The cell viability on all the BC/PPy gradient conductive membranes was higher than that on pure BC membranes throughout 1, 3, and 5-day culture, suggesting the non-cytotoxic nature of the BC/PPy membranes. Specifically, for the nonstretched gradient conductive membranes, the relative cell viability of middle and down segments (120% and 126%, respectively) exhibited significantly higher than that of the up segment (107%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This gradient cell viability was also appeared on the stretched BC/PPy gradient conductive membranes, where the relative cell viability of the up, middle and down segments was 98%, 129% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.005), and 132% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Notably, the down segment of stretched BC/PPy gradient conductive membranes promoted the cell proliferation most, which showed a significant 1.32-fold higher cell viability than that of control (culture dish, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Similar results were shown by the live/dead staining assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). From day 3 to day 5, a noticeable gradient proliferation of live cells (green) was observed on all the gradient conductive membranes from the up to down segments, indicating that PC12 cells could proliferate and grow normally on the BC/PPy gradient conductive membranes, especially on the down segment of the stretched BC/PPy gradient conductive membranes.\u003c/p\u003e \u003cp\u003eIt is well established that PPy has excellent biocompatibility and could support cell proliferation [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In the present study, the increasing concentration gradient distribution of PPy on the aligned membranes contribute to the enhanced cell viability from the up to down segments. These could be attributed to two aspects. First, the PPy gradient deposited on the stretched BC/PPy gradient conductive membranes was mainly distributed along the fiber alignment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), which could provide more effective transmission of endogenous electrical signals between cells and fibers for promoted cell proliferation [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Second, the deeper and oriented nanogrooves on the stretched BC/PPy gradient conductive nanofibers give more space for PPy deposition, which could provide more active functional groups and anchoring points for cell adhesion, growth, and proliferation [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Therefore, these results indicated that stretched BC/PPy gradient conductive membranes can promote gradient proliferation of cells from the up to down segments, providing an effective method to correspond topographic (fiber alignment) and gradient (PPy gradient) guidance with the subcellular responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.9. Evaluation of cell differentiation on BC/PPy gradient conductive membranes\u003c/h2\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.9.1. In the absence of EF\u003c/h2\u003e \u003cp\u003eIn the absence of EF, PC12 cells on the culture dish, BC, and up segment of nonstretched BC/PPy gradient conductive membranes exhibited a rounded morphology with no protrusion, whereas cells on the middle and down segments of the nonstretched BC/PPy membranes began to show protrusions (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, -EF). Compared with the same segments of the nonstretched BC/PPy gradient conductive membranes, more cell protrusions and longer neurites were observed on the stretched BC/PPy membranes with neurites elongate along the fiber alignment. Significantly higher neurite-bearing cell population (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.005) and neurites length (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.005) on the down segment are measured than that on the up segment of BC/PPy gradient conductive membranes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb-c, -EF). Moreover, the neurite-bearing cell population (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and the neurites length (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.005) measured on the down segment of the stretched membranes were significantly higher than that of the nonstretched membranes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb-c, -EF). These results indicate the effect of BC/PPy gradient conductive membranes from the up to down segments on the gradient cell differentiation, where the down segment of the stretched BC/PPy gradient conductive membranes promoted the most. In addition, as shown in Fig. S8, the stretched BC/PPy gradient conductive membranes could promote neurite elongation along the fiber alignment, especially at the down segment of the stretched BC/PPy gradient conductive membranes. This is consistent with a previous report where PC12 cells and Schwann cells exhibited oriented growth on the patterned materials [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Another study was found for the aligned conductive scaffold, whereon PC12 cells showed enhanced neurogenesis [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. In the present study, the mechanism for the gradient and directed cell differentiation on the stretched BC/PPy gradient conductive membranes may relate to the gradient and aligned PPy deposition, which can transfer the gradient electrical signal in the growth of conducting cells like PC12 influencing the cellular signaling pathways and inducing directional cell differentiation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.9.2. In the presence of EF\u003c/h2\u003e \u003cp\u003eTo further study the synergistic effect of BC/PPy gradient conductive membranes and EF on the cell differentiation, the PC12 cells cultured on the membranes were exposed to EF (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, +EF). Apparently, \u0026ldquo;+EF\u0026rdquo; groups showed typical neuronal differentiation morphology and the neurite was markedly formed (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). The neurite-bearing cell population and neurite length measured on all the segments of the BC/PPy gradient conductive membranes with EF were significantly higher than that without EF, except for the up segment of the stretched membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb-c). Moreover, the down segment of the stretched membranes in the presence of EF exhibited the highest neurite-bearing cell population (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb, 63.35%), which is 1.29-fold higher than that measured in the down segment of the nonstretched membranes (49.03%, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, +EF) and 2.04-fold higher than that measured in the up segment of the stretched membranes (31.13%, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05, +EF). This behavior is also consistent with their neurite length where the down segment of the stretched membranes under EF (Fig.\u0026nbsp;7c, 64.39 \u0026micro;m) exhibited 1.45-fold higher than that measured in the down segment of the nonstretched membranes (44.38 \u0026micro;m, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.005, +EF) and 1.82-fold higher than that measured in the up segment of the stretched membranes (35.45 \u0026micro;m, \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.005, +EF). In addition, Fig. S8 shows that there's no obvious influence of EF on the direction of neurite extension. Hence, the combination of stretched BC/PPy membranes and EF could synergistically enhanced neurite-bearing and neurite length in a gradient manner from the up to down segments. Similar enhanced cell differentiation was reported by Tang \u003cem\u003eet al.\u003c/em\u003e, where the combination of conductive PPy-coated aligned fibers and EF collectively enhanced the functional expression of PC12 cells, including elongation, gene expression, and protein expression [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Previous studies suggested that the conductive microenvironment and EF promoted the opening of voltage-gated calcium channels and the influx of Ca\u003csup\u003e2+\u003c/sup\u003e, which thereby upregulating expression of neurogenic genes and improving neurogenesis [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. However, the influence of the gradient conductive and topographic guidance with EF on the cell differentiation was still unknown.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe influence of the aligned BC/PPy gradient conductive membranes under electrical stimulation can be attributed to three main factors. First, for the PPy gradient guidance, the conjugated π-electrons clouds on the aligned PPy gradient chain formed a delocalized set of electrons, providing continuous and gradient charge transmission, which thereby effectively promoted intracellular signal transmission between cells and materials, and thus enhanced gradient neuronal differentiation from the up to down segments of the membranes. Secondly, for the topographic guidance, aligned BC nanofibers were used as a core component, which is similar to the nerve bundles consisting of numerous aligned cell-matrix assemblies [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In addition, PPy have acceptable biocompatibility, and could allow the adhesion, growth, and differentiation of neural cells [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Thus, the oriented deposition of PPy along the aligned BC nanofibers can activate relevant cell signaling pathways, converting topographic signals into mechanical signals and thereby inducing cell differentiation. Thirdly, for the application of EF, studies have shown its important role in altering the distribution of extracellular matrix molecules and enhancing protein absorption levels, thus promoting the elongation of neural axons. In the present study, the electrical stimulation can simultaneously induce the transfer of delocalized π electrons along the oriented PPy chains, thereby promoting the transport of bioactive molecules or cell growth factors converting topographic signals into mechanical signals, ultimately synergistically achieving gradient differentiation of PC12 cells on the aligned BC/PPy gradient conductive membranes.\u003c/p\u003e \u003cp\u003eOverall, the stretched BC/PPy gradient conductive membranes can effectively enhance the directional neurite-bearing and neurite length in a gradient manner from the up to down segments. Importantly, the cells tend to differentiate most along the fiber alignment on the down segment of the stretched BC/PPy gradient conductive membranes with the application of EF. This study is the first to report the synergistically enhanced gradient cell differentiation by the stretched BC/PPy gradient conductive membranes and EF stimulation, suggesting the potential for combinations of these cues to be exploited in peripheral nerve regeneration.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn summary, the highly aligned BC/PPy gradient conductive membrane was successfully prepared through\u0026nbsp;quantitatively stretching and hot pressing of BC, followed by a kinetically controlled \u003cem\u003ein-situ\u003c/em\u003e polymerization reaction of gradient PPy along the nanofibers. The membranes were composed of highly aligned nanofibers with introduction of gradient PPy along the fiber alignment, which presented good mechanical strength, thermal stability, and surface current gradient.\u003cem\u003e\u0026nbsp;In vitro\u003c/em\u003e biological evaluation demonstrated that the aligned BC/PPy gradient conductive membrane possessed excellent cytocompatibility, hemocompatibility, and could efficiently promote the gradient proliferation and differentiation of PC12 cells. Moreover, after the application of EF, the directional gradient cell differentiation on the aligned BC/PPy gradient conductive membrane could be significantly enhanced with the maximum axon length (64.39 \u0026mu;m) and neurite-bearing cell population (63.35%) observed at the down segment of the membrane compared to the nonstretched BC/PPy gradient conductive membrane with EF and the stretched BC/PPy membrane without EF. These results reveal that aligned BC and EF may have synergistic effect for nerve regeneration. Taken together, it is consistent with our hypothesis that the aligned BC/PPy gradient conductive membrane with EF could be a promising candidate in enhancing directional cell differentiation for accelerated peripheral nerve regeneration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eConflicts of interest\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no conflict of interest.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grant No. 52373235), Guangdong Provincial Key Laboratory of Advanced Biomaterials(Grant No. KLAB202404007), the National Natural Science Foundation of Hubei Province of China for Young Scholars (Grant No. 2022CFB749), and the Hubei Provincial Education Department Research Young and Middle-aged Talent Fund (Grant No. Q20222803).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLi Wang: Investigation, Methodology, Writing. Fuyu Qi: Writing. Hao Wang: Investigation, Methodology, Data. Shuangshuang Li: Data. Sanming Hu: Visualization, review. Zhijun Shi: Conceptualization, Supervision. Guang Yang: Project administration, Supervision. Hong Chen: Conceptualization, Supervision. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors acknowledge the Research Core Facilities for Life Science and the Analytical and Testing Centre at the HUST for performing characterization of various samples.\u003c/p\u003e"},{"header":"References ","content":"\u003col\u003e\n\u003cli\u003eLuo Y, Li J, Li B, Xia Y, Wang H, Fu C. Physical Cues of Matrices Reeducate Nerve Cells. Front Cell Dev Biol 2021;9:1\u0026ndash;14. https://doi.org/10.3389/fcell.2021.731170.\u003c/li\u003e\n\u003cli\u003eWang J, Zhu YQ, Wang Y, Xu HG, Xu WJ, Wang YX, et al. A novel tissue engineered nerve graft constructed with autologous vein and nerve microtissue repairs a long-segment sciatic nerve defect. Neural Regen Res 2021;16:143\u0026ndash;9. https://doi.org/10.4103/1673-5374.286977.\u003c/li\u003e\n\u003cli\u003eHeinzel JC, Quyen Nguyen M, Kefalianakis L, Prahm C, Daigeler A, Hercher D, et al. A systematic review and meta-analysis of studies comparing muscle-in-vein conduits with autologous nerve grafts for nerve reconstruction. Sci Rep 2021;11:1\u0026ndash;12. https://doi.org/10.1038/s41598-021-90956-3.\u003c/li\u003e\n\u003cli\u003eKehoe S, Zhang XF, Boyd D. FDA approved guidance conduits and wraps for peripheral nerve injury : A review of materials and efficacy. Injury 2012;43:553\u0026ndash;72. https://doi.org/10.1016/j.injury.2010.12.030.\u003c/li\u003e\n\u003cli\u003eGrinsell D, Keating CP. Peripheral Nerve Reconstruction after Injury: A Review of Clinical and Experimental Therapies. Biomed Res Int 2014;2014. https://doi.org/10.1155/2014/698256.\u003c/li\u003e\n\u003cli\u003eJaveed S, Faraji AH, Dy C, Ray WZ, MacEwan MR. Application of electrical stimulation for peripheral nerve regeneration: Stimulation parameters and future horizons. Interdiscip Neurosurg Adv Tech Case Manag 2021;24:101117. https://doi.org/10.1016/j.inat.2021.101117.\u003c/li\u003e\n\u003cli\u003eZhang K, Zheng H, Liang S, Gao C. Aligned PLLA nanofibrous scaffolds coated with graphene oxide for promoting neural cell growth. Acta Biomater 2016;37:131\u0026ndash;42. https://doi.org/10.1016/j.actbio.2016.04.008.\u003c/li\u003e\n\u003cli\u003eManan S, Ullah MW, Ul-Islam M, Shi Z, Gauthier M, Yang G. Bacterial cellulose: Molecular regulation of biosynthesis, supramolecular assembly, and tailored structural and functional properties. Prog Mater Sci 2022;129:100972. https://doi.org/10.1016/j.pmatsci.2022.100972.\u003c/li\u003e\n\u003cli\u003eRen YJ, Zhang S, Mi R, Liu Q, Zeng X, Rao M, et al. Enhanced differentiation of human neural crest stem cells towards the Schwann cell lineage by aligned electrospun fiber matrix. Acta Biomater 2013;9:7727\u0026ndash;36. https://doi.org/10.1016/j.actbio.2013.04.034.\u003c/li\u003e\n\u003cli\u003eWang L, Adhikari M, Li L, Li S, Mbituyimana B, Li X, et al. Electrically stimulated Acetobacter xylinum for the production of aligned 3D microstructured bacterial cellulose. Cellulose 2023;30:9973\u0026ndash;88. https://doi.org/10.1007/s10570-023-05521-0.\u003c/li\u003e\n\u003cli\u003eWang L, Mao L, Qi F, Li X, Wajid Ullah M, Zhao M, et al. Synergistic effect of highly aligned bacterial cellulose/gelatin membranes and electrical stimulation on directional cell migration for accelerated wound healing. Chem Eng J 2021;424:130563. https://doi.org/10.1016/j.cej.2021.130563.\u003c/li\u003e\n\u003cli\u003eHuang Z, Sun M, Li Y, Guo Z, Li H. Reduced graphene oxide-coated electrospun fibre: effect of orientation, coverage and electrical stimulation on Schwann cells behavior. J Mater Chem B 2021;9:2656\u0026ndash;65. https://doi.org/10.1039/d1tb00054c.\u003c/li\u003e\n\u003cli\u003eTang J, Wu C, Chen S, Qiao Z, Borovskikh P, Shchegolkov A, et al. Combining Electrospinning and Electrospraying to Prepare a Biomimetic Neural Scaffold with Synergistic Cues of Topography and Electrotransduction. ACS Appl Bio Mater 2020;3:5148\u0026ndash;59. https://doi.org/10.1021/acsabm.0c00595.\u003c/li\u003e\n\u003cli\u003eSadeghi A, Moztarzadeh F, Aghazadeh Mohandesi J. Investigating the effect of chitosan on hydrophilicity and bioactivity of conductive electrospun composite scaffold for neural tissue engineering. Int J Biol Macromol 2019;121:625\u0026ndash;32. https://doi.org/10.1016/j.ijbiomac.2018.10.022.\u003c/li\u003e\n\u003cli\u003eLiu R, Huang X, Wang X, Peng X, Zhang S, Liu Y, et al. Electrical stimulation mediated the neurite outgrowth of PC-12 cells on the conductive polylactic acid/reduced graphene oxide/polypyrrole composite nanofibers. Appl Surf Sci 2021;560:149965. https://doi.org/10.1016/j.apsusc.2021.149965.\u003c/li\u003e\n\u003cli\u003eSun Y, Quan Q, Meng H, Zheng Y, Peng J, Hu Y, et al. Enhanced Neurite Outgrowth on a Multiblock Conductive Nerve Scaffold with Self-Powered Electrical Stimulation. Adv Healthc Mater 2019;8:1\u0026ndash;11. https://doi.org/10.1002/adhm.201900127.\u003c/li\u003e\n\u003cli\u003eChen S, McCarthy A, John J V., Su Y, Xie J. Converting 2D Nanofiber Membranes to 3D Hierarchical Assemblies with Structural and Compositional Gradients Regulates Cell Behavior. Adv Mater 2020;32:1\u0026ndash;10. https://doi.org/10.1002/adma.202003754.\u003c/li\u003e\n\u003cli\u003eMoore K, Sc MA, Macsween M, Sc M, Shoichet M, Ph D, et al. Moore, Macsween, Shoichet - 2006 - Immobilized concentration gradients of neurotrophic factors guide neurite outgrowth of primary neuron 2006;12:19\u0026ndash;21.\u003c/li\u003e\n\u003cli\u003eCai Y, Huang Q, Wang P, Ye K, Zhao Z, Chen H, et al. Conductive Hydrogel Conduits with Growth Factor Gradients for Peripheral Nerve Repair in Diabetics with Non-Suture Tape. Adv Healthc Mater 2022;11:1\u0026ndash;12. https://doi.org/10.1002/adhm.202200755.\u003c/li\u003e\n\u003cli\u003eZhang D, Li Z, Shi H, Yao Y, Du W, Lu P, et al. Micropatterns and peptide gradient on the inner surface of a guidance conduit synergistically promotes nerve regeneration in vivo. Bioact Mater 2022;9:134\u0026ndash;46. https://doi.org/10.1016/j.bioactmat.2021.07.010.\u003c/li\u003e\n\u003cli\u003eTang S, Zhu J, Xu Y, Xiang AP, Jiang MH, Quan D. The effects of gradients of nerve growth factor immobilized PCLA scaffolds on neurite outgrowth invitro and peripheral nerve regeneration in rats. Biomaterials 2013;34:7086\u0026ndash;96. https://doi.org/10.1016/j.biomaterials.2013.05.080.\u003c/li\u003e\n\u003cli\u003eZonderland J, Rezzola S, Wieringa P, Moroni L. Fiber diameter, porosity and functional group gradients in electrospun scaffolds. Biomed Mater 2020;15. https://doi.org/10.1088/1748-605X/ab7b3c.\u003c/li\u003e\n\u003cli\u003eXue T, Yang Y, Yu D, Wali Q, Wang Z, Cao X, et al. 3D Printed Integrated Gradient-Conductive MXene/CNT/Polyimide Aerogel Frames for Electromagnetic Interference Shielding with Ultra-Low Reflection. Nano-Micro Lett 2023;15:1\u0026ndash;14. https://doi.org/10.1007/s40820-023-01017-5.\u003c/li\u003e\n\u003cli\u003eShan Y, Feng H, Li Z. Electrical stimulation for nervous system injury: Research progress and prospects. Wuli Huaxue Xuebao/ Acta Phys - Chim Sin 2020;36:1\u0026ndash;11. https://doi.org/10.3866/PKU.WHXB202005038.\u003c/li\u003e\n\u003cli\u003eCostello MC, Errante EL, Smartz T, Ray WZ, Levi AD, Burks SS. Clinical applications of electrical stimulation for peripheral nerve injury: a systematic review. Front Neurosci 2023;17. https://doi.org/10.3389/fnins.2023.1162851.\u003c/li\u003e\n\u003cli\u003eZha F, Chen W, Hao L, Wu C, Lu M, Zhang L, et al. Electrospun cellulose-based conductive polymer nanofibrous mats: Composite scaffolds and their influence on cell behavior with electrical stimulation for nerve tissue engineering. Soft Matter 2020;16:6591\u0026ndash;8. https://doi.org/10.1039/d0sm00593b.\u003c/li\u003e\n\u003cli\u003eLi X, Ji X, Chen K, Ullah MW, Yuan X, Lei Z, et al. Development of finasteride/PHBV@polyvinyl alcohol/chitosan reservoir-type microspheres as a potential embolic agent: From: In vitro evaluation to animal study. Biomater Sci 2020;8:2797\u0026ndash;813. https://doi.org/10.1039/c9bm01775e.\u003c/li\u003e\n\u003cli\u003ePrasannan P, Siney E, Chatterjee S, Johnston D, Shah M, Mudher A, et al. A 3D-induced pluripotent stem cell-derived human neural culture model to study certain molecular and biochemical aspects of Alzheimer\u0026rsquo;s disease. Vitr Model 2022;1:447\u0026ndash;62. https://doi.org/10.1007/s44164-022-00038-5.\u003c/li\u003e\n\u003cli\u003eLiu F, Xu J, Wu L, Zheng T, Han Q, Liang Y, et al. The Influence of the Surface Topographical Cues of Biomaterials on Nerve Cells in Peripheral Nerve Regeneration: A Review. Stem Cells Int 2021;2021. https://doi.org/10.1155/2021/8124444.\u003c/li\u003e\n\u003cli\u003eMikulik D, Ricci M, Tutuncuoglu G, Matteini F, Vukajlovic J, Vulic N, et al. Conductive-probe atomic force microscopy as a characterization tool for nanowire-based solar cells. Nano Energy 2017;41:566\u0026ndash;72. https://doi.org/10.1016/j.nanoen.2017.10.016.\u003c/li\u003e\n\u003cli\u003ePathak CS, Chang BJ, Song S. Review on scanning probe microscopy analysis for perovskite materials and solar cells. Dye Pigment 2023;218:111469. https://doi.org/10.1016/j.dyepig.2023.111469.\u003c/li\u003e\n\u003cli\u003eXiong F, Wei S, Wu S, Jiang W, Li B, Xuan H, et al. Aligned Electroactive Electrospun Fibrous Scaffolds for Peripheral Nerve Regeneration. ACS Appl Mater Interfaces 2023;15:41385\u0026ndash;402. https://doi.org/10.1021/acsami.3c09237.\u003c/li\u003e\n\u003cli\u003eThakur A V., Lokhande BJ. Study of RuO2 incorporated PPy hybrid flexible electrodes prepared by soaking and drying technique: a novel approach. Appl Phys A Mater Sci Process 2021;127:1\u0026ndash;6. https://doi.org/10.1007/s00339-021-05028-0.\u003c/li\u003e\n\u003cli\u003eDado D, Levenberg S. Cell-scaffold mechanical interplay within engineered tissue. Semin Cell Dev Biol 2009;20:656\u0026ndash;64. https://doi.org/10.1016/j.semcdb.2009.02.001.\u003c/li\u003e\n\u003cli\u003eGuo H, Qiao T, Jiang S, Li T, Song P, Zhang B, et al. Aligned poly (glycolide-lactide) fiber membranes with conducting polypyrrole. Polym Adv Technol 2017;28:484\u0026ndash;90. https://doi.org/10.1002/pat.3912.\u003c/li\u003e\n\u003cli\u003eKoons GL, Diba M, Mikos AG. Materials design for bone-tissue engineering. Nat Rev Mater 2020;5:584\u0026ndash;603. https://doi.org/10.1038/s41578-020-0204-2.\u003c/li\u003e\n\u003cli\u003eSinghal JP, Ray AR. Synthesis of blood compatible polyamide block copolymers. Biomaterials 2002;23:1139\u0026ndash;45. https://doi.org/10.1016/S0142-9612(01)00228-9.\u003c/li\u003e\n\u003cli\u003eYardimci AI, Aypek H, Ozturk O, Yilmaz S, Ozcivici E, Mese G, et al. CNT Incorporated Polyacrilonitrile/Polypyrrole Nanofibers as Keratinocytes Scaffold. J Biomimetics, Biomater Biomed Eng 2019;41:69\u0026ndash;81. https://doi.org/10.4028/www.scientific.net/JBBBE.41.69.\u003c/li\u003e\n\u003cli\u003eZhao X, Wu H, Guo B, Dong R, Qiu Y, Ma PX. Antibacterial anti-oxidant electroactive injectable hydrogel as self-healing wound dressing with hemostasis and adhesiveness for cutaneous wound healing. Biomaterials 2017;122:34\u0026ndash;47. https://doi.org/10.1016/j.biomaterials.2017.01.011.\u003c/li\u003e\n\u003cli\u003eFahlgren A, Bratengeier C, Gelmi A, Semeins CM, Klein-Nulend J, Jager EWH, et al. Biocompatibility of polypyrrole with human primary osteoblasts and the effect of dopants. PLoS One 2015;10:1\u0026ndash;17. https://doi.org/10.1371/journal.pone.0134023.\u003c/li\u003e\n\u003cli\u003eChen CH, Tsai CC, Wu PT, Wang IK, Yu J, Tsai WB. Modulation of Neural Differentiation through Submicron-Grooved Topography Surface with Modified Polydopamine. ACS Appl Bio Mater 2019;2:205\u0026ndash;16. https://doi.org/10.1021/acsabm.8b00556.\u003c/li\u003e\n\u003cli\u003eWang L, Wu Y, Hu T, Ma PX, Guo B. Aligned conductive core-shell biomimetic scaffolds based on nanofiber yarns/hydrogel for enhanced 3D neurite outgrowth alignment and elongation. Acta Biomater 2019;96:175\u0026ndash;87. https://doi.org/10.1016/j.actbio.2019.06.035.\u003c/li\u003e\n\u003cli\u003eToth AB, Shum AK, Prakriya M. Regulation of neurogenesis by calcium signaling. Cell Calcium 2016;59:124\u0026ndash;34. https://doi.org/10.1016/j.ceca.2016.02.011.\u003c/li\u003e\n\u003cli\u003eHao B, Webb SE, Miller AL, Yue J. The role of Ca2+ signaling on the self-renewal and neural differentiation of embryonic stem cells (ESCs). Cell Calcium 2016;59:67\u0026ndash;74. https://doi.org/10.1016/j.ceca.2016.01.004.\u003c/li\u003e\n\u003cli\u003eYang Y, Sun J, Liu X, Guo Z, He Y, Wei D, et al. Wet-spinning fabrication of shear-patterned alginate hydrogel microfibers and the guidance of cell alignment. Regen Biomater 2017;4:299\u0026ndash;307. https://doi.org/10.1093/rb/rbx017.\u003c/li\u003e\n\u003cli\u003eBorges MHR, Nagay BE, Costa RC, Souza JGS, Mathew MT, Bar\u0026atilde;o VAR. Recent advances of polypyrrole conducting polymer film for biomedical application: Toward a viable platform for cell-microbial interactions. vol. 314. 2023. https://doi.org/10.1016/j.cis.2023.102860.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Gradient conductive, Topographical, Electrical stimulation, Aligned nanofiber, Cell differentiation, Peripheral nerve regeneration","lastPublishedDoi":"10.21203/rs.3.rs-4403218/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4403218/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBoth of the topographical and gradient conductive cues can influence the cellular activity and thereby tissue regeneration. However, they have not been combined simultaneously onto biomaterial with electrical stimulation to demonstrate the synergistic role so far. Herein, we assume that a bacterial cellulose (BC) -based membrane by incorporating aligned nanofibers and a concentration gradient of polypyrrole (PPy) with electrical stimulation treatment will promote cell differentiation in peripheral nerve regeneration. The results showed that PPy were successfully deposited on the aligned BC/PPy with gradient conductive structure, which exhibited good mechanical property, thermal stability, the gradient decrease in surface resistance, gradient increase in surface current from the up to down segments, as well as excellent biocompatibility. Especially, the membranes promoted the gradient proliferation and differentiation of PC12 cells \u003cem\u003ein vitro. \u003c/em\u003eImportantly, combined with electric field (EF), the aligned BC/PPy gradient conductive membranes synergistically directed the differentiation of PC12 cells. The overall results suggest the aligned BC/PPy gradient conductive membranes with EF could be a promising therapeutic strategy to guide cellular activities for peripheral nerve regeneration.\u003c/p\u003e","manuscriptTitle":"Highly aligned bacterial cellulose/PPy gradient conductive membranes for directed cell differentiation under electrical stimulation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-12 17:43:17","doi":"10.21203/rs.3.rs-4403218/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8cd7c6f6-f55c-4f07-bea4-73a86240114c","owner":[],"postedDate":"June 12th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-02T03:06:18+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-12 17:43:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4403218","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4403218","identity":"rs-4403218","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}