1 Introduction

Our nervous system, consisting of two main components: the central nervous system (CNS) and the peripheral nervous system (PNS), has a vital and complex role in conveying signals for physiological processes in limbs and organs as well as controlling sensory and motor functions. Damage or injury to the nervous system may result in serious dysfunction of limbs or organs causing lifelong disabilities and reduced quality of life accompanied by major economic and social burdens. According to reports, it is expected that people will continue to experience increasing rates of spinal cord injuries, traumatic brain injuries, peripheral nerve injuries, and neurodegenerative disorders along with many other nervous system problems around the globe, causing significant economic burden ranging from $3 to $800 billion in the US alone.1,2,3,4,–5 Among these, peripheral nerve injuries (PNIs) affect ~ 3% of trauma patients (mostly young adults of working age and U.S. veterans) in the U.S alone.6,7,–8 According to estimates, over 200,000 peripheral nerve repair procedures are performed in the U.S,6,7,–8 causing ~ $150 billion in medical expenditures spent annually.9 It is also expected that the U.S. transected peripheral nerve repair market will exceed $1.6 billion by 2022.6,7,–8, 10

The PNS, consisting of neuronal, glial, and stromal cells, has the intrinsic capacity for spontaneous regeneration and regrowth of axons to a certain extent. Besides the neurons, glial and stromal cells play a key role in maintaining the proper function of the peripheral nerves. Injury-induced Schwann cell (SCs) reprogramming, which involves upregulation of trophic factors and cytokines, myelin clearance by activation of myelin autophagy in SCs and macrophage recruitment, and formation of Bungner bands are some of the cellular functions orchestrating spontaneous regeneration and regrowth of axons.11,12,13,14,15,–16 Despite the key responses of SCs to injury, the regenerative capacity of PNS remains insufficient, particularly for large PNIs (> 1 cm), without any additional surgical/therapeutic interference.8 Although it is possible to enable functional recovery using autologous nerve grafts, this strategy has limitations such as multiple surgery requirement, biological complexity, donor site morbidity, and lack of graft tissue.17 Alternatively, cell-based therapies offer promising outcomes for PNIs.18 Implantation of SCs using different platforms (scaffolds, conduits, films, etc.) has shown enhanced axonal regeneration across nerve gaps in many studies;19,20,21,22,–23 however, lack of availability and slow in vitro growth of SCs restrict clinical translation.7, 8, 24 Stem cells, isolated and derived from various connective tissue sources, offer promising translational strategies for PNIs without any ethical concerns due to their accessibility, plasticity, and multipotency.25,26,27,–28 Most studies use autologous transplantation of stem cells for PNIs.18 Although they have reported improved axonal regeneration through synergetic interactions of stem cells with host SCs and enhanced paracrine activity,29,30,31,32,33,–34 the multipotency of stem cells and lack of direct control in complex in vivo environment and the design of a platform mimicking extracellular matrix (ECM) raise concerns regarding the implanted cells’ fate, especially over the duration of the nerve regeneration and limit the clinical use of MSCs.35, 36 Because of these reasons, designing a cell-laden, implantable platform with desired 3D microstructural/mechanical properties that mimics the complex ECM microenvironment is critical in neural tissue regeneration.35, 36 Hence, a multifunctional platform possessing biological, chemical, and physical cues and mimicking cellular microenvironment significantly affects and determines successful control of cellular behaviors such as growth, proliferation, directed migration, differentiation, and in turn, neural tissue regeneration.18, 37,38,39,40,–41

The inherent ability of neuronal cells to send electrical signals along axons42,43,44,–45 and the positive effects of electric fields on paracrine activity,46 cellular alignment and migration47, 48, and recovery from peripheral nerve injuries49,50,–51 are known. It is also previously reported that electrical stimulation is an effective cue in stimulating not only the neuronal cells, but also various other cell types’ behavior such as proliferation or differentiation.52 Therefore, besides the biological, chemical, and physical cues, the designed platforms should also have conductivity to enable electrical stimulation for the enhancement of neural tissue regeneration.

In this review, we first discuss electrically conductive biomaterials-based platforms such as films, hydrogels, fibers, composites, and flexible electronic interfaces. Then, we summarize the application of electrical stimulation through these platforms to control cellular behavior such as neurite extension, directed migration and differentiation, and neural tissue regeneration. Finally, we will conclude this work with future perspectives.

2 Conductive Polymers (CPs)

Polymers with loosely held electrons in their backbones are generally classified as conductive polymers (CPs) possessing common polymeric properties along with electrical features similar to metals and semiconductors. The source of electrical conductivity comes from the atoms at the backbone with weak π bond enabling delocalization and free movement of electrons, which in turn results in formation of electrical current through mobile charges.53 Most CPs are synthesized through doping process (oxidation or reduction), in which a charge is transferred from dopant molecules to polymer chains via charge carriers (polarons and bipolarons).52 The efficiency of the doping process and properties of the formed CPs depend on many different factors, including polaron, chain and conjugation lengths, charge transfer efficiency, and type of the dopants and molecular size. Typical CPs are polyacetylene (PA), polythiophene (PT), poly(3,4-ethylenedioxythiophene) (PEDOT), polypyrrole (PPy), poly(p-phenylene) (PPP), and polyaniline (PANI).54

PA, which is also called acetylene black, can be synthesized by Ziegler–Natta catalysis, radiation methods, or by the controlled combustion of acetylene under air.55 This material shows excellent biocompatibility, electrical conductivity, and a large specific surface area, which favor biochemical56 and electrochemical sensor applications.57 PT with high conductivity (103 S cm−1)58 and transparency59 can be synthesized by different electrochemical and chemical methods.60 The most popular derivative of PT, highly conductive and stable, PEDOT, is mostly used in electroanalytical applications.52, 61, 62 PPy, which can easily be synthesized by various chemical methods, has high conductivity, stability, and biocompatibility. The conductivity and biocompatibility of PPy may further be improved through hybridization with other materials, including myocytes, biotin, alginate, and silk fibroin.52, 55, 61,62,63,–64 Because of these features, PPy is eligible for many biomedical applications, including neural tissue engineering.52, 55, 61,62,63,–64 PPP with thermal, optical, electrical, and chemical properties can be synthesized by electrochemical and chemical methods.52, 62, 64 PPP-based thin films can be conventionally used in light-emitting diodes, photodetectors, and other optoelectronic devices as well as dental applications and cellular alignment.64 PANI, which is a nontoxic and stable polymer with high electrical conductivity, has simple and cost-effective chemical and electrochemical synthesis routes. The conductivity of PANI can be controlled through determination of oxidation state,65 increasing crystallinity and conjugation length of the polymer,66 or the polymer chain structure (i.e. 1D: nanofibres, nanorods, and nanotubes, 2D: ribbons, nanobelts, and nanoplates and 3D: microspheres, nanospheres, and granules). The properties of PANI facilitate its application in biosensing, medicine, and tissue engineering.52, 55, 62,63,–64

CPs are attractive biomaterials for tissue engineering applications due to their physical and chemical properties, along with their ability to convey electrical signals to cells and provide favorable platforms controlling and promoting specific cellular responses such as cell adhesion, growth, and proliferation, combination of which enhance tissue regeneration.52, 61,62,–63 Although CPs show good in vitro and in vivo biocompatibility and support the in vitro adhesion, proliferation, and differentiation for different cell types, their non-degradability limits their in vivo application.67 However, these properties of CPs can be controlled based on the dopant type and selected synthesis method and conditions in such a way that CPs can retain their electroactivity, while gaining biodegradability.62, 67 Another limitation of using CPs is their stiff, brittle, and insoluble nature, which makes them mechanically poor and difficult to manipulate and process. However, blending CPs with other degradable synthetic or natural polymers (such as PLA, PLGA, PCL, chitosan, and silk fibroin) is a widely used strategy to fabricate conductive and biodegradable platforms for tissue engineering applications.62, 67

Overall, the chemical, physical, and electrical properties of CPs can be controlled and manipulated by polymer chemistry and synthesis methods. Hence, various CP-based platforms in the form of films, hydrogels, fibers, composites, and flexible electronic interfaces can be obtained by physically compositing or forming co-networks with other polymers for neural tissue engineering applications.

3 Carbon-Based Conductive Materials

Carbon-based conductive materials, such as graphene and carbon nanotubes (CNTs), are another group of biomaterials that can be incorporated into non-conducting polymers to provide structural reinforcement and impart novel properties such as electrical conductivity, enhancing cell attachment, directed growth, proliferation, and differentiation.63, 68,69,–70

Graphene-based materials can be obtained through mechanical exfoliation, chemical vapor deposition, and liquid-phase exfoliation. Graphene oxide (GO) is formed through sp2 and sp3 hybridization of carbon atoms and has the ability to be easily dispersed in water and interact with different inorganic and organic materials enabling various conductive ink formulations.71, 72 However, the conductivity of GO-based materials is limited due to the presence of oxides, which can be improved by reducing GO through thermal or laser processing leading to reduced GO (rGO) with enhanced physical and electrical properties.73

These materials offer several advantages, including exceptional electrical and thermal conductivity, mechanical strength, chemical stability, non-toxicity, and biocompatibility, which make them well suited for tissue scaffolds capable of mediating cell growth, proliferation, and differentiation.74,75,76,77,78,79,80,81,82,–83 The intimate cell-graphene-based material interaction is most likely due to the π–π interactions of aromatic amino acids in the cell membrane that orient proteins with the graphene layer.78, 84, 85 The hydrophobic nature of graphene can also potentially facilitate the immobilization of extracellular matrix (ECM) proteins78, 86 and facilitate physicochemical interactions to further enhance cellular attachment and proliferation.78, 79 Therefore, graphene-based materials can be used in the fabrication of conductive and biocompatible platforms; however, non-degradable nature of graphene could be a limitation for in vivo applications.

CNTs, with cylindrical shape, nano-scale dimensions, and high aspect ratio, can be fabricated by laser cutting, arc discharge, or chemical vapor deposition. The high aspect ratio, low density, and electrical and physical properties of CNTs favor their use in biomedical applications.87, 88 The structure of CNTs has the potential to induce oxidative stress in the cells, when applied in the form of suspension causing toxic response.89, 90 However, the toxic effect of CNTs could be eliminated through the surface functionalization or immobilization of CNTs to a platform.52 Nevertheless, graphene and CNTs are promising materials for producing conductive and biocompatible platforms for neural tissue engineering.

4 Conductive Polymer-Based Platforms and Electrical Stimulation

The presence of endogenous electrical fields during the embryonic development91,92,–93 and the inherent ability of neuronal cells to send electrical signals along axons42,43,44,–45 are well known in the literature. Based on this fact, there have been various attempts to control cellular alignment and migration,47, 48 differentiation,79, 94, 95 paracrine activity,46 neurite extension, and recovery from nerve injuries49,50,–51 through the application of electrical stimuli.46, 96,97,98,–99 Electrical stimuli can be applied through different platforms such as films, hydrogels, scaffolds/conduits, fibers, composites, and flexible electronic interfaces (Table 1). In the following sections, we will summarize the use of different platforms to control cellular behavior via applied electrical stimuli with a specific emphasis on neural regeneration (Table 1).

Table 1. Summary of the used platforms to apply electrical stimuli for neural regeneration
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4.1 Films

Recording the electrical and neural signals through implantable electrodes and probes is important to provide direct measurement of cellular function or communication between the brain/nervous system and machines. CPs are considered as potential coating materials, creating thin films on the probe surfaces due to their high surface area and conductivity, which can enable effective ion exchange between recording sites and the surrounding tissue.127, 128 It was reported that platinum electrodes coated with PANI via in situ polymerization enhanced the aggregation of retinal fragments and demonstrated long-term stability along with significant promise for the reduced inflammation and scar formation.129 In another study, microfabricated electrode arrays were coated by PEDOT: PSS providing high charge injection accompanied by safe and efficient stimulation of central auditory system.130

CPs can also be used to fabricate stand-alone films as implantable platforms to electrically stimulate cells. It was reported that PC12 cells seeded on PPy films responded to the applied electrical stimuli (100 mV) by showing significant neurite extension (18.14 μm)100 due to electrical stimuli-induced protein adsorption from serum-containing medium.131 Besides self-standing films, the CPs can also be used as coating materials on biodegradable polymer-based films/membranes. In a study, PC12 cells demonstrated neuronal differentiation and neurite outgrowth as a result of the electrical stimuli applied though PPy-coated poly(D,L-lactide-co-ε-caprolactone) membranes. These platforms also demonstrated significant axonal regeneration on rat sciatic nerve model.101

As an alternative to coatings or composites with other polymers, CPs-based copolymers can also be developed. It was shown that electrically stimulated PC12 cells significantly extended neurites on conductive films made up of PLA and carboxyl-capped aniline pentamer-based copolymer.102 The copolymer-based conductive films can further be modified with biologically active components. N-hydroxyl succinimidyl ester pyrrole; copolymer-based films were further modified with NGF through surface immobilization. The synergistic effect of NGF and electrical stimulation resulted in neurite extension of PC12 cells.103

The electrically conductive platforms can also be used in combination with Schwann cells, which are the key players of the peripheral nervous system contributing to myelination and axonal regeneration. Schwann cells, seeded on polypyrrole–chitosan based composite films, demonstrated enhanced cell viability and paracrine activity (secretion of NGF and brain-derived neurotrophic factor (BDNF) (Fig. 1a), along with upregulated gene expression (Fig. 1b) upon applied constant potential gradient (100 mV/mm).104

Figure 1
figure 1

Reproduced with permission from Huang et al.104 Copyright 2009 Wiley Periodicals, Inc.

a Representative images of DAPI staining of Schwann cells. Magnification: × 200. The cell number count and MTT values in each group (*p < 0.05, one‐way ANOVA). b mRNA levels of NGF and BDNF with or without electrical stimuli (ES) 12 and 24 h after ES. (*p < 0.05 and **p < 0.01, one‐way ANOVA)

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Overall, conductive polymer-based films demonstrated potential to control cell behavior; however, lack of a natural 3D microstructure is limiting complete mimicry of the ECM. For this reason, 3D hydrogels or scaffolds could be a better alternative.

4.2 Hydrogels and Scaffolds/Conduits

Besides electrical conductivity, the platforms should also have certain properties such as 3D porous structure, optimal mechanical properties, and topographical/physical cues to ensure complete mimicry of naturel ECM of the cells.132 CPs-based hydrogels, scaffolds, or conduits with proper 3D microstructural and mechanical properties are considered as promising platforms enabling electrical stimuli-based cell manipulation within a 3D microstructure to promote neuronal proliferation and differentiation.

Hydrogels have a hydrated, flexible, and soft nature that makes them ideal for soft tissue applications. The added property of electrical conductivity through polymerization or cross-linking with CPs, further facilitates their use in neural tissue engineering.61, 132 For example, Guarino et al. (2013) developed macroporous hydrogel platforms with a conductivity of 1.1 × 10−3 mS cm−1 through in situ precipitation of PANI in polyethyleneglycol diacrylate (PEGDA) matrix, which was then followed by UV cross-linking and sodium chloride particle leaching, leading to formation of microporous structure (136–158 μm pore size). These platforms provided improved biological response of PC12 and human mesenchymal stem cells (hMSCs) due to their favorable 3D microstructure and electrical conductivity.105 Similarly, Shi et al. (2014) developed conductive PPy and cellulose-based nanoporous hydrogels with a conductivity of 80 mS cm−1, through in situ polymerization of PPy monomers within nanoporous cellulose gel matrix. Their results indicated that PC12 cells showed enhanced viability, adhesion, proliferation, and induced neurite extension on the developed conductive hydrogel platforms.106 In another study, Yang et al. (2016) developed PPy/alginate hydrogels with enhanced mechanical and electrical properties via chemical polymerization of PPy within ionically cross-linked alginate hydrogel network as multifunctional neural tissue engineering platforms. Their in vitro studies showed that the developed PPy/alginate hydrogel platforms improved the adhesion and growth of hMSCs as well as inducing the expression of neural differentiation markers (i.e., Tuj1 and MAP2) (Fig. 2a). In addition, the in vivo studies demonstrated that the platforms are sufficiently biocompatible, suggesting the promise of further examining the influence of electrical and mechanical signals on stem cells and/or neural cells using these platforms (Fig. 2b).107

Figure 2
figure 2

Reproduced with permission from Yang et al.107 Copyright 2016 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim.

a In vitro hMSC culture onto PPy/Alg hydrogels for 4, 24, and 72 h. Scale bars: 200 μm. Cell numbers on the samples. Relative Tuj1 and MAP2 gene expression levels of the cells cultured on the PPy[10]/Alg and TCP for 14 days. Individual bars indicate the average ± the standard error of the mean. An asterisk (*) denotes a statistical significance between two groups (p < 0.05). b HE staining of histological sections subcutaneously implanted with various PPy/Alg gels for 1, 4, and 8 weeks. Scale bars represent 100 μm

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Using a different approach, Bu et al. (2018) synthesized sodium alginate, PPy, and carboxymethyl chitosan-based hydrogel with a conductivity of 2.41 mS cm−1 as a supporting platform to be used in the conduit systems for peripheral nerve regeneration. They used a different synthesis approach, where calcium cross-linked alginate/carboxymethyl chitosan hydrogels with controlled release properties were further coated with conductive PPy particles, to easily manipulate the hydrogel properties, such as swelling, gelation, elasticity, porosity, and electrical conductivity. These platforms provided improved cell adhesion and proliferation properties for PC12 and bone marrow-derived MSCs as well as high in vivo biocompatibility.108

Conductive carbon-based materials, such as GO or CNTs, can also be used in hydrogel formulations as an alternative to CPs. Considering this, Jafarkhani et al. (2018) mixed conductive GO with chitosan powder, and further reacted with lactic acid to synthesize conductive hydrogels possessing different mechanical and structural properties. Their results suggested that the optimum mechanical and structural properties enhanced the nerve cell growth by 20%, demonstrating the potential benefit of these platforms.109 Similarly, Zhao et al. (2018) developed polyacrylamide, GO, gelatin, and sodium alginate-based composite hydrogel for peripheral nerve regeneration applications. They reported that the microstructural and mechanical properties of hydrogels can be controlled by the amount of GO, which in turn determines the adhesion and proliferation of Schwann cells along with the expression of myelination markers (Sox10, GAP43, and myelin basic protein).110

As an alternative to GO, CNTs can also be used as potential conductive carbon-based material. In a study by Koppes et al. (2016), the researchers used single-walled carbon nanotubes (swCNTs) to optimize the electrical properties of collagen-based hydrogels. They reported that the electrical conductivity of hydrogels was increased without any negative effect on hydrogel matrix properties (such as microstructure or elasticity) as the swCNTs among increased, which in turn improved neurite outgrowth by sevenfold through the applied electrical stimuli compared to all other control groups.111 In a different study, Shin et al. compared the influence of swCNTs and/or PPy-incorporated hyaluronic acid (HA) hydrogels on human neural stem/progenitor cells (hNSPCs) differentiation.

The hydrogels (with swCNTs and/or PPy) provided dynamic and electrically conductive 3D microenvironments that are supportive for neuronal differentiation of hNSPCs. The platforms improved electrophysiological cellular function through the upregulation of calcium channel expression, activation of depolarization, and increase in calcium influx, hence demonstrating the future promise of these platforms to improve neuronal regeneration.112 As an alternative to swCNTs, Imaninezhad et al. (2018) developed multi-walled CNTs (mwCNTs) incorporating polyacrylamide/polyethylene glycol hydrogels, which provided significant alignment and directional extension in neurite outgrowth of PC12 cells in response to electrical stimulation.113 In another study Liu et al. (2017) demonstrated the potential of combined use of GO and CNTs through cross-linkable bonds. They covalently embedded the cross-linked GO/CNTs into oligo(poly(ethylene glycol) fumarate) hydrogel to provide surface charge and electrical conductivity of ~ 5.75 × 10−2 mS cm−1. The composite hydrogel platforms showed good biocompatibility accompanied by enhanced proliferation and spreading of PC12 cells. The electrical stimuli applied through the hydrogel platforms in the form of conduits promoted the neurite extension of PC12 cells depicting potential for neural tissue engineering.114

Scaffolds can be considered in the form of a template with defined 3D geometrical shapes and internal configuration, filled with conductive material-based hydrogels, and further freeze-dried to obtain 3D porous microstructure.132 For example, Wang et al. (2017) developed PEDOT/chitosan/gelatin-based scaffolds through in situ interfacial polymerization of PEDOT nanoparticles on porous chitosan/gelatin scaffolds. The electrical conductivity, hydrophilicity, mechanical properties, and thermal stability of the scaffolds were improved through the incorporation of PEDOT, which at on the other hand, reduces the water absorption and biodegradability of scaffolds. Nevertheless, their results indicated that these platforms significantly promoted PC12 cell adhesion, proliferation, and neurite growth by upregulating protein and gene expression levels through their conductive nature.115 In another study, Gupta et al. (2019) investigated the influence of morphological differences between graphene nanoplatelets (GNP) and multi-walled carbon nanotubes (MWCNT) on neural cell regeneration. They developed GNP- and MWCNT-incorporated chitosan scaffolds with different electrical conductivity and mechanical properties. They reported that the cellular responses, such as protein adsorption, cell adhesion, cytotoxicity, and alignment, change with respect to the material properties.133 The same group developed MWCNT-aligned chitosan scaffolds to control the directional neuronal growth and cellular alignment via electrical cues. Their results indicated that this platform can successfully control the alignment in 50–60% of neurons.134

Conduits are considered a special form of hydrogels and scaffolds with cylindrical tube geometry, and particularly used for peripheral nerve regeneration applications. For example, electrical stimulation via PANI-based conduits, providing electrical field of 10 mV/cm to 2 V/cm, demonstrated enhanced hMSCs growth and proliferation through the alteration of cytoskeletal arrangement producing long filopodial extensions and leading to formation of neural-like cells.116 Similarly, electrical stimuli applied through a blend of PANI/Poly-l-Lactic acid (PLLA) conduit resulted in neurite extension of 24 ± 4 μm in rat nerve cells.117 In another work, Xu et al. (2014) developed PPy/poly(dl-lactic acid) (PDLLA)-based conduits with the conductivity range of 5.65 to 15.56 mS cm−1 via oxidative polymerization. The PC12 cells seeded on PPy/PDLLA conduits showed increased neurite extension upon the applied 100 mV electrical stimuli for 2 h (Fig. 3a). The same conduits were used for the in vivo treatment of 1 cm long rat sciatic nerve transection model and the results indicated that the used conduits provided functional recovery (Fig. 3b) similar to that of autologous nerve grafts, a gold standard for the peripheral nerve injury treatment.98

Figure 3
figure 3

Reproduced with permission from Xu et al.98 Copyright 2013 Elsevier Ltd.

a Percentage of neurite-bearing PC12 cells and median neurite length on PPY/PDLLA composite films with varying PPY composition (n = 4, *p < 0.05). b Intraoperative photographs of the PPY/PDLLA nerve conduits. “P” signifies the proximal end and “D” signifies the distal end. Histology images stained with methylene blue and transmission electron microscopy (TEM) micrographs. Recovery of sciatic nerve function. Sciatic function index (SFI) as a function of implantation time (top). Footprint stamps in walking track analysis after 6 months of implantation (bottom). (A) PPY/PDLLA. (B) PDLLA. (C) Autograft. (D) Normal left leg

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As a brief summary, the 3D microstructured platforms with multiple functionalities in the form of hydrogels, scaffolds, and conduits hold significant promise to pave the way for the development of new strategies to enhance neural tissue regeneration.

4.3 Electrospun Fibers

Fibers can be considered as another type of 3D scaffolds possessing inherent characteristics to mimic the ECM microenvironment for tissue engineering applications. Although most of the fibers used are non-conductive, electrical conductivity can be provided through CPs coating or direct fabrication of fibers using CPs. Electrospinning is the most common process enabling fiber fabrication using a wide range of polymers, including CPs, at nano- and micrometer-scale, supporting adhesion, and guiding extension of neurons for nerve regeneration.132, 135 For instance, Lee et al. (2009) developed electrically conductive random or aligned fiber structures through the coating of PPy on electrospun poly(lactic-co-glycolic acid) (PLGA) nanofibers. Their random nanofibers provided a favorable microenvironment for the growth and differentiation of PC12 cells and hippocampal neurons, while application of electrical stimuli (10 mV/cm) through the nanofibers resulted in formation of high number of neurites with longer neurite size. They further reported that the same electrical stimuli applied via the aligned nanofibers led to even longer neurites and more neurite-bearing cells than that of the random ones.118 In a similar study, Xie et al. (2009) developed electrospun poly(ε-caprolactone) (PCL) and poly(l-lactide) (PLA)-based nanofibers, further coated with PPy through in situ polymerization. They observed that dorsal root ganglia (DRG) cells attached to grown and extended neurites in the direction of nanofiber alignment in the presence of electrical stimuli revealing the potential of these platforms for neural tissue engineering applications.119 In the work of Shafei et al. (2017), a novel approach based on electrospinning and vapor-phase polymerization, was used to fabricate highly conductive (1.9 S cm−1) PCL/PPy nanofibers supporting PC12 cell viability and neurite extension.120 In similar work, Ghasemi-Mobarakeh et al. (2009) designed conductive nanofiber scaffolds made of PANI–PCL–gelatin, which enhanced PC12 cell proliferation and neurite outgrowth (~ 30 μm) upon electrical stimulation (DC voltage of 100 mV/mm for 1 h).121 In a different study, Wu et al. (2015) used a different material to fabricate electroactive nanofibers. They applied in situ polymerization of conductive PANI on the surface of non-conductive scaffold material, tobacco mosaic virus (TMV), in the presence of dopant, poly(styrenesulfonate) (PSS). They concluded that the synergetic effect of electrical and topographical cues promoted the directed neurite outgrowth of PC12 cells.122 In another study, Wang et al. (2018) developed surface modified, electrically conductive, aligned nanofibrous scaffolds composed of poly(lactic-co-glycolic acid) (PLGA) and multi-walled carbon nanotubes (MWCNTs) in the form of conduits for neural regeneration. They observed that the topographically aligned nanofibers provided directed cellular alignment and neurite outgrowth for PC12 (Fig. 4a) and DRG (Fig. 4b) cells in the presence of electrical stimulation (voltage of 40 mV). In addition, the synergetic effect of nanofiber alignment and electrical stimuli also enhanced the cell attachment, proliferation, and myelination of Schwann cells.123

Figure 4
figure 4

Reproduced with permission from Wang et al.123 Copyright 2018 Elsevier B.V.

a The expression of NF200 on poly-l-lysine-coated random and aligned nanofibers. b The neurite extension of DRG neurons on poly-l-lysine-coated random and aligned nanofibers

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Besides the in vitro work on PC12 or DRG cells, the studies on the stem cells also show great potential for neural tissue repair. In that sense, the use of fibers providing topographical, biochemical, and electrical cues to control the stem cell behaviors such as differentiation into desired cell lineages, including neuronal or glial cells, has also been investigated. In the work of Yow et al. (2011), PPy-incorporated collagen fibers with significant mechanical and electrical properties were prepared using interfacial polyelectrolyte complexation. After applying 10 days of electrical stimuli to the hMSCs seeded on the developed fibers in proliferating medium, they observed that hMSCs upregulated the expression of neural markers, such as noggin, MAP2, neurofilament, β tubulin III, and nestin, indicating their differentiation into neuronal-like phenotypes, accompanied by slight reduction in hMSCs viability.124 In addition to the CPs, carbon-based conductive materials can also be used in combination with biodegradable and biocompatible non-conductive polymers in the form of nanofibers for the differentiation of stem cells. For instance, Zhu et al. (2018) developed polyacrylonitrile and conductive carbon-based nanofibrous scaffolds with high flexibility and conductivity using high temperature annealing method. They reported that the neural stem cells seeded on the scaffolds demonstrated neuronal differentiation and maturation through the upregulation of neuronal gene expression levels and MAP2 protein upon the application of biphasic electrical stimulation (AC voltage of 5 V for 4 h during 7 days).125 In a different study, Guo et al. (2016) developed graphene–PEDOT hybrid microfibers to differentiate MSCs into neural-like phenotypes through a self-powered electrical stimulation without bio/chemical cues.76

Overall, these studies demonstrated the potential use of conductive CPs-based fibers as promising platforms for neural tissue engineering applications.

4.4 Flexible Electronic Interfaces

The graphene is a well-known material due to its excellent mechanical and electric properties, allowing its use in combination with other biomaterials as mechanical support or as conductive fillers to design functional and biocompatible hydrogel or scaffold platforms for tissue engineering applications.64 Alternatively, conductive graphene has also been widely used in the development of implantable and flexible bioelectronic interfaces to be used in neural tissue engineering.69 These flexible electronic interfaces with high resolution and low feature size conductive graphene circuits are mostly fabricated via inkjet printing, 3D printing/bioprinting, chemical vapor deposition (CVD), or various other graphene pattern transfer methods and used to control cellular function. For instance, Lee et al. (2015) used CVD-grown graphene substrate to enhance the neuronal differentiation of hMSCs under low-frequency electromagnetic fields (ELF-EMF; 50 Hz, 1 mT). They demonstrated that successful differentiation was obtained as a result of ELF–EMF induced cell adhesion, upregulation of intracellular calcium influx, and activated focal adhesion kinase signaling pathway, producing extracellular matrix molecules. However, the success of this approach was relying on not only the electrical stimuli, but also the use of many different chemicals and growth factors triggering neuronal differentiation in their cell culture media. These chemicals and growth factors are expensive and not suitable for use in in vivo applications, limiting the clinical translation of this approach.79 To address this issue, for the first time, we used inkjet printer and laser-annealed graphene-based interdigitated circuits on polyimide substrates as flexible bioelectronic interface to control the stem cell differentiation via sole electrical stimuli, free from any additional chemical induction. Our results indicated that the MSCs were successfully differentiated into Schwann cells by applying voltage of 100 mV at 50 Hz for 10 min per day for 15 days. We reported high immunolabeling of Schwann cell markers along with enhanced paracrine activity.95 Considering the fact that the used polyimide substrate was not porous and biodegradable, we developed a novel method, enabling effective fabrication of biodegradable and flexible electronic interfaces via simple polymer casting method.126 In this method, first conductive graphene-based micropatterns (or circuits) were developed on molds via microfluidic filling, inkjet printing or CVD. Then, by applying a polymer casting method, we were able to transfer the graphene patterns (or circuits) to the target substrate, by which we can control the microstructure, mechanical properties and also biodegradability. This is a versatile method that can be used for various synthetic or natural biodegradable polymers. The obtained biodegradable and implantable and flexible bioelectronic interfaces that demonstrated successful differentiation of MSCs into Schwann cells under same electrical stimuli conditions.126 Although these flexible electronic platforms are porous and biodegradable, they still do not possess actual 3D microstructure creating a favorable microenvironment for the formation of 3D cellular network. Considering this, we created a 3D printed graphene-based circuit and embedded it into a gelatin matrix possessing actual 3D microstructure and desired mechanical properties (Fig. 5a, b). Following the application of electrical stimuli, we observed MSCs to Schwann cell differentiation accompanied by significantly enhanced paracrine activity (Fig. 5c), which was due to the synergistic effort of 3D microstructural/mechanical properties and electrical stimuli.94

Figure 5
figure 5

Reproduced with permission from Uz et al.94 Copyright 2019 American Chemical Society.

a 3D printed circuit embedded in gelatin matrix. b MSCs attachment and formation of 3D cellular network. c Immunolabeling of Schwann cell marker after electrical stimuli

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The application of biodegradable and implantable bioelectronic interfaces for the control of stem cell differentiation and fate commitment is a promising approach for future neural regeneration strategies. This is a new hot topic in the field and more research is needed in this aspect.

5 Potential Cellular Mechanisms

The influence of electrical fields during the embryonic development along with the existing electrical signals throughout our body, particularly in our nervous system, has been known for centuries. However, the exact role of electrical fields and signaling such as time-dependent voltage gradients, ion fluxes or regulation of synapses in developmental processes have only recently been investigated.46, 47, 50 In the literature, there have been some efforts to connect cellular behaviors, such as proliferation,136 migration,137 protein/growth factor secretion,138 adhesion, and differentiation,139 with the applied electrical stimuli through the regulation of various signaling pathways such as FAK and p3875, 76, 79, 140,141,–142 ion channels and ERK pathway,143,144,–145 MAPK, PI3K, and ROCK,97, 146 and ROS.97, 147 Some studies have also suggested that the electrically induced intracellular Ca2+ signaling along with the signaling pathways related to ferritin serve as a novel regulatory mechanism controlling the neural differentiation of MSCs.148 For the CPs, it is claimed that the expulsion of negative ions through the neutralization of CPs or uptake of positive ions such as Na+ from the medium has several effects on cellular processes, including protein adsorption and the cell cycle.64 Despite the promising attempts to elucidate the effect of electrical stimuli on cellular processes, further investigation needs to be performed to understand the physicochemical mechanisms of this phenomenon.

6 Conclusions and Future Perspectives

The conductive polymer or carbon-based platforms hold significant potential to manipulate cellular behavior and promote neural regeneration. However, most of the ongoing research is at pre-clinical level as opposed to clinical trials for the application of conductive platforms for neural tissue engineering. Thus, the properties of these platforms need further improvements to provide precise control of cellular mechanisms to enable clinical translation. The improvements in microstructural and mechanical properties of these platforms along with the electrical conductivity and biodegradability can enable better mimicry of the ECM, which in turn allows enhanced control of implanted cells and facilitate clinical translation. There are still number of unknown questions regarding the relationship between the electrical properties and cellular functions, particularly from a mechanistic perspective. Therefore, elucidating the regulated genes, proteins, and cellular pathways upon electrical stimuli with a desirable mimicry of ECM environment will pave the way for the development of novel and functional platforms. In addition, most of the conductive materials are not biodegradable. Therefore, the use of conductive platforms that are also biodegradable, in tissue engineering applications, can potentially open the way for minimally invasive implantation, which can prevent additional surgical interventions improving the comfort of patients. Furthermore, development of conductive and biodegradable polymeric platforms that are capable of manipulating cells through remotely applied electrical stimuli will be the next generation approach to provide a minimally invasive strategy for patients, not only for neural tissue engineering, but also for other tissue engineering areas, including skin, muscle, and cardiac and even brain–computer interfaces. In conclusion, the conductive polymer and hydrogel-based platforms are promising and have a lot of potential to address neural tissue engineering problems, but there are still certain gaps in this field that need immediate attention to accelerate clinical translation.