Abstract
Peripheral nerve injury (PNI) is a common clinical problem, which due to poor recovery often leads to limb dysfunction and sensory abnormalities in patients. Tissue-engineered nerve guidance conduits (NGCs) that are designed and fabricated from different materials are the potential alternative to nerve autografts. However, translation of these NGCs from lab to commercial scale has not been well achieved. Complete functional recovery with the aid of NGCs in PNI becomes a topic of general interest in tissue engineering and regeneration medicine. Electrical stimulation (ES) has been widely used for many years as an effective physical method to promote nerve repair in both pre-clinical and clinical settings. Similarly, ES of conductive and electroactive materials with a broad range of electrical properties has been shown to facilitate the guidance of axons and enhance the regeneration. Graphene and its derivatives possess unique physicochemical and biological properties, which make them a promising outlook for the development of synthetic scaffolds or NGCs for PNI repair, especially in combination with ES. Considering the discussion regarding ES for the treatment of PNI must continue into further detail, herein, we focus on the role of ES in PNI repair and the molecular mechanism behind the ES therapy for PNI, providing a summary of recent advances in context of graphene-based scaffolds (GBSs) in combination with ES. Future perspectives and some challenges faced in developing GBSs are also highlighted with the aim of promoting their clinical applications.
1 Introduction
Peripheral nerve injury (PNI) is a common and widespread clinical disease. Acute trauma, autoimmune diseases, local lesions, and infections are all triggers for injuries in the peripheral nervous system (). Peripheral nerves form an extensive neural network throughout the entire body, connecting the nerve center with target organs and enabling communication between them (Wieringa et al., 2018). Therefore, when PNI occurs, the lack of information transmission poses a severe threat to the mobility and sensory function in distal target organs. Even though peripheral nerves boast the intrinsic capacity to regenerate over small gaps, the slow growth rate of about 1 mm/day results in a limited regeneration of nerve function for nerve injuries more than 3 mm in length (; ; ). After injury, the recovery of the innervation for the distal target organ takes a significant amount of time, and in the case of severe injuries, the distal end of the injured nerve and target organs will atrophy over time, resulting in long-term sensory and motor dysfunction (; Zhang S. et al., 2022).
The slow regeneration speed of peripheral nerves and the poor recovery of nerve function after regeneration have posed challenges in clinical practice for numerous years. Among various treatment methods, autologous nerve transplantation has been considered the gold standard for treating long-gap injuries, albeit the successful rate of recovery following surgery is only 50%, and its clinical application is limited due to the lack of donors and complications at the donor site (). Electrical stimulation (ES), among the most popular non-surgical treatment methods, has been widely studied in the field of tissue engineering, both in preclinical and clinical settings. Numerous studies indicate that low-frequency ES delivered post-operatively has a certain positive effect on the repair of peripheral nerve damage, including nerve crush (), nerve transection (), and long-gap nerve defects () in various types of rodents. For example, ES was found to have the ability to accelerate nerve regeneration in combination with steroids, such as testosterone propionate (). For another example, combined with an reduced graphene oxide (rGO)-coated poly (l-lactic acid-co-caprolactone) (PLCL) microfiber scaffold, ES enhanced neurite outgrowth and alignment of PC-12 cells and primary mouse hippocampal neurons compared to control without ES stimulation (Wang et al., 2020). However, it is important to note that the directionality of the electric field exhibited little contribution to neurite alignment, especially for the neurites outgrowth on PLCL fibers with higher diameters (Wang et al., 2020), instead, it has been observed to enhance nerve fiber growth in random and lead axonal misdirection to incorrect end organs which consequently impaired functional recovery (). Therefore, other interventions such as tissue-engineered tubular structures, i.e., nerve guidance conduits (NGCs) were employed, which have been widely researched in terms of structural design, materials, and fabrication processes, aiming to provide multi-cuefor neural regeneration (; ). Biocompatible and biodegradable materials with appropriate mechanical properties and desirable conductivity are highly beneficial for the establishment of NGCs in peripheral nerve regeneration. By the application of ES, conductive polymer scaffolds have a good effect on differentiation of nerve stem cells and remyelination of regenerated axons (; ; ). However, the artificial polymers with excellent electrical conductivity are non-biodegradable, non-soluble, or brittleness, which inhibits them from clinical translation.
Graphene (Gr) and its derivatives graphene oxide (GO) and rGO possess numerous extraordinary properties for use in tissue engineering of the nervous system (; ; ; ). As building blocks, they can assemble into various forms of graphene-based scaffolds (GBSs), such as coating, films/membranes, fibers, foams, hydrogen, conduit, 3D printing and bioprinting products. Combined with ES, the GBSs exhibit a particularly brilliant performance for the treatment of PNI (; ; ). However, ES through conductive GBSs remains in its infancy based upon the fact that effective and safety of parameters of ES need to be confirmed; related mechanism by which ES and GBSs enhance nerve regeneration and the limitation in PNI repair need to be clarified and overcame. Therefore, this short review starts with a brief understanding the characteristics of PNI and the role of ES in repairing injured nerves, followed by a discussion on recent research advances in the preclinical phase of combining GBSs with ES in promoting nerve regeneration. The GBSs are categorized in view of the assemble precursors, i.e., Gr [including chemical vapor deposition (CVD)-G], GO, and rGO nanosheets, which are given in Table 1. This review attempts to elucidate the features of ES imposed by GBSs and prospect the application of GBSs combined with ES in the area of PNI.
TABLE 1
| GBMs | Biomaterial(s) | Construct | Electrical conductance | Cell(s) | Animal model | ES parameters (frequency/intensity/duration/number) | Effects | Related mechanism | Ref. |
|---|---|---|---|---|---|---|---|---|---|
| Graphene (Gr) | LIG, PPy | Film | ∼0.1–0.5 S cm−1 | PC-12 | NI | 50 Hz/400 mV·cm−1/2 h-4 h-8 h d−1/once | The growth, proliferation and differentiation of PC-12 cells on LIG/PPy electrodes was significantly enhanced by applying ES in view of neurite outgrowth length and neural phenotype | ES induced more protein binding and strengthening cell adhesion and growth | |
| Graphene, AP, PCL, PCLF, ESM | Three-layered conduit with well-defined anisotropy | 37.64 ± 0.4 Ω @ 20 Hz | PC-12 | NI | 0.5 V, 0.03 mA/60 min/once | ES had a positive effect on outgrowth, metabolic activity, the arrangement and morphological changes of PC-12 cells cultured on the tubular scaffolds | NM | ||
| Graphene, SA, PVA | Aligned fibrous scaffold | NI | PC-12 | NI | 1 V/60 min/once | ES facilitated to promote the metabolic activity and proliferation of PC-12 cells | NM | ||
| Graphene, PMMA | Film | NI | PC-12 | NI | Cell viability: 2 V/1 min, 0.1 s (on-time), 0.01–5 s (off-time); Released dopamine: 1 Hz–10 kHz/2 V/10 s (on-time/off-time = 1)/once | Developed a graphene neurointerface device as a new platform for simultaneous neurotransmitter sensing and neurostimulation for therapy; confirmed the feasibility of graphene for electroceutical applications to various central nerve system disorders | ES increased the intracellular calcium level and facilitated the extracellular dopamine release | ||
| Graphene, PANI | Membrane | NI | PC-12 | NI | ±500 mV/3 h d−1/3, 5, and 7 times | PANI-Gr electrode possessed high electro-activity, excellent mechanical and electrical properties, and a high biocompatibility; ES enhanced the axon length of PC-12 and wound regeneration, with no adverse impact on cell density | NM | Zheng et al. (2019) | |
| Graphene, TPU | Membrane | 33.45 ± 0.78 S m−1 | RSC-96 | NI | 10, 50, and 100 mV/1 h d−1/5 times | The conductive composite membrane was favorable for the viability, growth, and proliferation of SCs stimulated under 10 mV DC voltage | NM | ti | |
| Graphene, Collagen, PCL (GCFS) | Conduit | 3.12 ± 0.62 S m−1 | MSCs | Rat sciatic nerve (10 mm) | In vitro: 2 Hz/10, 20, and 50 mV·cm−1/10 min d−1/3, 7 times | Combined with ES, GCFS conduit promoted sciatic nerve regeneration and functional recovery | ES facilitated sciatic nerve regeneration by recruitment of endogenous MSCs and modulation of macrophage phenotypes | ||
| In vivo: 2 Hz/200 mV mm−1/10 min d−1/14 times | |||||||||
| Graphene, PCLF, CNTs, MTAC | Hollow conduit | ∼106–105 Ω @ 103–106 Hz | PC-12 | NI | 20 Hz/100 mV mm−1/2 h d−1/7 times | ES increased number of neurite protrusions in PC-12 cells | NM | ||
| Graphene, PLCL, PDA | Micropatterned film | 0.0035 ± 0.0004 S m−1 | RSC-96 | Rat sciatic nerve | In vitro: 20 Hz/10 mV/1 h d−1/3 times | Conduits with ES supported SCs migration, adhesion, and elongation in vitro; promoted growth of myelin sheath, faster nerve regeneration, and functional recovery in vivo | The groove surface combined with ES enhanced cell adhe-sion and neuronal-specific protein expression | ||
| Hollow conduit | In vivo: 100 Hz (pulse width = 200 μm, on time = 5 s, off time = 10 s)/30 min d−1/14 times | ||||||||
| Graphene, PGSA, PVP, AgNPs | Flat/Microgroove structure film | ∼10–5 S cm−1 (pure PGSA) | PC-12 | NI | 50 mV·mm−1/2 h d−1/7 times | Low cytotoxicity for composites extracts and film with the incorporation of graphene; more and longer neurites outgrowth of PC-12 cells on PGSA-Gr; growth direction of both PC-12 and SW10 cells could be guided by ES; ES enhanced healing rate of cells, the higher electrical conductivity of the PGSA composite films, the higher wound healing rate; faster degradation of the PGSA composite scaffold was observed due to the addition of graphene | NM | ||
| ∼10–4 S cm−1 (PGSA-PVP, PGSA-Gr) | SCs (SW10) | ||||||||
| GO | GO, PPy, DBS, PLLA | Film | 32 S cm−1 | PC-12 | NI | 50 mV cm−1/1 h d−1/2 times | ES significantly promotes axonal elongation and arrangement of PC-12 cells | ES enhanced the activity of filamentous filopodia and provided energy to accelerate the actin assembly of growth cone | |
| Carboxylic-GO (C-GO), PPy, PLLA | Film | 4.6 S cm−1 | PC-12 | Rat sciatic nerve (10 mm) | 20 Hz/1 V/1 h d−1//7 times | The incorporation of C-GO improved hydrophilicity of the PPy/PLLA film, and consequently a higher cytocompatibility; functional recovery of ES and conduit group was closer to the autograft group, superior to that of conduit group without ES | NM | ||
| Conduit | L929 | ||||||||
| GO, PPy, PDA, PLLA | Film | 17.3 S cm−1 | RSC-96 | NI | 50 mV cm−1/1 h d−1/once | Good adhesion to the neural proteins of RSCs; ES arranges 31% of RSCs on the membrane along the current direction | The movement of the cytomembrane proteins under ES and their linkage with serum proteins immobilized by PDA facilitated the extension of growth cone along the ES direction | ||
| GO, PCL | Fibrous membrane | NI | PC-12 | NI | 3 Hz/0.5 V cm−1/20 min d−1/1, 2, 3, and 6 times | Established triboelectric nanogenerators with excellent output performance based on modification of GO nanosheets; in vitro ES experiments demonstrated considerable proliferation and migration of PC-12 cells from receiving an alternating electrical field | NM | ||
| Annealed GO (a-GO), COL | Coating with crumpled surface morphology | 1 × 106 Ω/sq | PC-12 | NI | 23.6 Hz/30–80 mV·mm−1/1 h d−1/5 times | The coating improved neuronal cell differentiation; facilitated the development of a biohybrid retinal implant that integrated neuronal cells; printed aGO-COL micropatterns supported the creation of neuronal cell microarrays with specific patterns | NM | Yang et al. (2022) | |
| rGO | rGO ink, polyimide | Printed coating | <1 kΩ/sq | MSCs | NI | 50 Hz/100 mV/10 min d−1/15 times | MSCs differentiated into SC like phenotypes by applying ES from rGO-based electrodes | Electrical stimuli provided by the graphene IDE significantly enhanced the paracrine activity of MSCs and the degree of MSCs’ transdifferentiation | |
| rGO, silk fibroin (SF) | Electrospun mat | NI | PC-12 | NI | 100 mV/2 h d−1/2 times | Adhesion and proliferation were improved in PC-12 cells growing on rGO-coated SF mats with cell viability higher than 95%; rGO coating sole without application of ES could induce differentiation of PC-12 cells to neuronal-like phenotypes, while the neurite outgrowth was more pronounced when electric currents were applied | NM | ||
| 100 mV/24 h d−1/once | |||||||||
| rGO, ApF, PLCL | Hollow conduit | 4.05 × 10−2 S m−1 | RSC-96, PC-12 | Rat sciatic nerve (10 mm) | In vitro: 100 mV·cm−1/1 h d−1/5 times | ES promoted the migration, proliferation, and myelin formation of SCs; induced differentiation of PC-12 cells; repair ability of NGC implantation was similar to that of autologous nerve transplantation | The conductive AP/RGO scaffolds under ES were beneficial to SC myelin gene expression and neurotrophin secretion | ||
| rGO, PCL | Fibrous membrane | 0.105 S m−1 | RSC-96 | NI | 10 mV/1 h d−1/5 times | ES combined with orientation topography in rGO-coated scaffolds promoted the expression of local NGF, accelerated the migration of SCs, and improved the proliferation of SCs | NM | ||
| rGO, CS, OHEC, asiaticoside liposome | Hydrogel | 5.27 ± 0.42 ×10−4 S cm−1 | PC-12 | NI | 250 mV·cm−1/8 h d−1/once | The hydrogel was non-toxic and suitable for adhesion and proliferation of PC-12 cells in vitro; ES made nerve cells highly differentiated and accelerated nerve regeneration; significant inhibitory effect on the growth and collagen secretion of fibroblasts | NM | Zheng et al. (2020) | |
| RSC-96 | |||||||||
| rGO, PLA, PPy | Nanofiber membrane | 1.46 × 10−1 S cm−1 | PC-12 | NI | 50 Hz/0, 100, 400, and 700 mV·cm−1/0.5 h d−1/3 times | ES has a significant promoting effect on the proliferation, differentiation, and axonal growth of PC-12 cells under an electric field intensity of 400 mV/cm | When placed in different ES, the protein adsorption is affected by the surface properties and charge of composite nanofibers, which will influence the subsequent adhesion, growth and pro-liferation of nerve cells | ||
| rGO, PDA, PVA | Hydrogel | 4.3 × 10−2 S m−1 | PC-12 | NI | 100 Hz/100 mV·cm−1/4 h d−1/7 times | Successful long-term growth and proliferation of PC-12 cells encapsulated demonstrated the biocompatibility and noncytotoxicity of the hydrogel; highly efficient neuronal differentiation was observed with or without ES | NM | ||
| rGO, PLCL | Microfiber | 0.95 S cm−1 | PC-12, primary mouse hippocampal neurons | NI | 100–150 mV·cm−1/1 h d−1/14 times | ES and rGO-coated microfiber with tailored architecture significantly induced orientated neuronal-like network formation | NM | Wang et al. (2020) | |
| rGO, PCL | Nanofibrils (NF) | 0.0443 ± 0.0004 S m−1 | PC-12 | Rat sciatic nerve (5 mm) | 100 Hz/100 mV·cm−1/1 h d−1/7 times | ES stimulated neurogenic differentiation of PC-12 cells; tailored to repair PNI by NGC filled with rGO-coated NF and ADSC | 30rGO@NF and ES synergistically facilitated the differentiation of the PC-12 cells into the middle and late stages | ||
| Filled conduit |
Graphene-based scaffolds (GBSs) combined with electrical stimulation (ES) for repairing injured peripheral nerve.
Abbreviations: AO/EB, acridine orange/ethidium bromide; ADSC, adipose-derived stem cell; AP, alginate-polyvinyl alcohol; ApF, antheraea pernyi silk fibroin; AP/RGO, scaffold, coated the rGO, onto an ApF/PLCL, nanofiber; CCFs, conductive composite film; CFGO, carboxyl functionalized graphene oxide; CGO, carboxylic graphene oxide; CNTs, carbon nanotubes; COL, collagen; CS, chitosan; CV, cyclic voltammograms; DBS, sodium dodecyl benzenesulfonate; DC, direct current; ES, electrical stimulation; ESM, eggshell membrane; GBMs, graphene-based materials; GCFS, graphene-based conductive fiber scaffold; GO, graphene oxide; IDE, interdigitated electrode; LIG, laser-induced graphene; LSCM, laser scanning confocal microscope; MSCs, mesenchymal stem cells; MTAC, [2-(methacryloyloxy)ethyl]trimethylammonium chloride; NGC, nerve guidance conduit; NGF, nerve growth factor; NI, not investigated; NM, not mentioned; OHEC, oxidized hydroxyethyl cellulose; PANI, polyaniline; PCL, poly (ε-caprolactone); PCLF, polycaprolactone fumarate; PC-12, rat pheochromocytoma cell line; PDA, polydopamine; PLA, polylactic acid; PLCL, Poly (L-lactic acid-co-caprolactone); PLLA, poly-L-lactic acid; PMMA, Poly (methyl methacrylate); PPy, polypyrrole; PVA, polyvinyl alcohol; rGO, reduced graphene oxide; RSCs, rat Schwann cells; SA, sodium alginate; SCs, Schwann cells; TPU, thermoplastic polyurethane; 3D, three-dimensional.
2 Characteristics of peripheral nerve injury
In the peripheral nervous system, a peripheral nerve is wrapped by three layers with different constituents and functions, namely, the endoneurium, perineurium, and epineurium (). According to the Sunderland grading system (), PNI is categorized into five types which sorts the nerve injury into five different degrees, and provides a reference for whether surgical intervention is needed: (I) temporary or reversible block (no surgical intervention required/-); (II) axons are damaged, but the endoneurium, perineurium and epineurium entire (−); (III) axons and the endoneurium are damaged, but the perineurium and epineurium are complete (−); (IV) axons, endoneurium, and the perineurium are damaged, but the epineurium is intact (surgical intervention is necessary); (V) severe nerve injury, with nerves divided into two parts (surgical intervention and nerve transplantation are necessary). The selection of repair methods after injury is usually based on the type of nerve injury, the type of target organ, the selectivity of transplanted nerve donors, the location of nerve injury, and the time interval of nerve injury ().
After PNI, various metabolic, genomic, and biological mechanisms involved in the regeneration of damaged nerve’s structure and function take place (). The destruction of the integrity of the axonal plasma membrane causes a large influx of extracellular calcium and sodium ions into the cytoplasm, leading to the generation of high-frequency action potentials in the proximal axonal region of the cell body, which can retrograde to the cell body (; ). Under the mediation of calcium ions (Ca2+), upregulation of regeneration-associated genes (RAGs) occurs through the cyclic adenosine monophosphate (cAMP) signaling pathway, which is crucial for the formation of growth cones (; ; ). Simultaneously, Wallerian degeneration, a series of molecular and cellular changes, providing a microenvironment conducive to axonal regeneration and reinnervation is crucial for nerve repair after PNI (; ) (Figure 1). The cell fragments generated by Wallerian degeneration are cleared by Schwann cells (SCs) and macrophages. Besides, activated SCs form Bands of Büngner through the injury gap, guiding the proximal growth cone to reach the neural tube, thereby achieving regenerative innervation of the target organ (; ). The inflammatory response is likewise an important aspect of peripheral nerve regeneration (). In the early stage of injury, M1 macrophages (pro-inflammatory) are mainly recruited, which can enhance the inflammatory response and promote tissue necrosis; in the later stage, M2 macrophages (anti-inflammatory) play a crucial role in effectively responding to hypoxia, increasing the expression of vascular endothelial growth factor A (VEGF-A), and leading to the proliferation and migration of endothelial cells to the injured site (; ).
FIGURE 1
The rate of axonal regeneration is quite slow. Furthermore, because of Wallerian degeneration, the nerve fiber tube lacks an internal structure for a long time, leading to collapse of the nerve tube and an increase in collagen fibers inside it, which results in a smaller diameter and increased difficulty of nerve regeneration (). The staggered growth pattern of nerve regeneration, where the regenerated axons germinate from the proximal stump at different times rather than growing all at once (), further delays the regeneration of neurons post-PNI. Prolonged denervation impairs target organ function, leading to target muscle atrophy and persistent sensory disturbance (neuralgia or neurosensitivity), which causes long-term distress for patients (). Therefore, accelerating axonal regeneration speed and reducing the mismatch of regenerated nerve fibers represent the focal points of current research.
3 Application of ES for treatment of PNI
As soon as the PNI occurs, endogenous electric fields are generated correspondingly, which may take part in regulating the rate of nerve sprouting, growth, and regeneration. Applied electric fields have likewise been found to influence the regeneration of nerves after injury, i.e., promote survival, migration, and axonal elongation of neurons, either applied immediately following nerve repair or a perioperative ES (Zarrintaj et al., 2020; ; ). In 1952, Hoffman was the first to apply ES to injured nerve. In his study, a 50–100 Hz sine-wave ES was utilized to the injured sciatic nerve of rats for 10–60 min. Results showed that germination was accelerated in the nerves of partially denervated gastrocnemius and soleus muscles (). Subsequently, Pocket and Gavin subjected the sciatic nerve of rats to compression injury and applied ES with a frequency of 1 Hz for 15 min to 1 h, resulting in faster recovery of the toe extension reflex in the ES group (). To date, there have been few reports of translation of intraoperative ES therapy to the clinic (). In post-operative intervention, for instance, neuromuscular ES, transcutaneous nerve ES), and functional ES have demonstrated potential to alter neuromuscular activity through an electric field ().
The mechanism by which ES promotes nerve regeneration is not completely understood. Nevertheless, it is widely believed that it may be related to ES promoting intracellular Ca2+ waves, cell membrane potential, membrane receptors, and gap junctions, etc. At axonal injury sites (; Zuo et al., 2020). The possible pathways related to biological responses to ES are given in Figure 2. For example, In vivo studies show that upregulated brain-derived neurotrophic factor (BDNF) due to the increase in Ca2+ concentration caused by ES and their high affinity receptor tropomyosin receptor kinase B (TrkB) receptor interactions increase the expression of RAGs, such as T-α-1 tubulin and GAP-43, through the cAMP pathway (; Wang et al., 2011; ). Subsequently, ES activates cAMP response element binding protein (CREB) through phosphokinase A (PKA), inhibits Rho protein expression in the p75-Nogo receptor (p75-NgR) pathway, and upregulates T-α-1 tubulin, hence enhancing cytoskeleton assembly (; Yan et al., 2016). Meanwhile, ES can activate CREB to promote axonal extension through another pathway, namely, the p38 mitogen-activated protein kinase (MAPK) pathway. One study applied ES to PC-12 cells with nerve growth factor (NGF) induced axon growth impairment, and found that the CREB activation pathway could be induced by p38 MAPK to promote axon growth (). Another study has shown that ES also promoted the induction of pluripotent stem cells into neurons, which may be related to the production of novo ciliary neurotrophic factor (CNTF) (). In addition, in vitro experiments indicated that the application of ES (1 Hz, 5 V cm−1) promoted the secretion of neurotrophins by SCs, including NGF and NT-3, via the Ca2+ influx. Moreover, it has been observed that ES supported the transition of macrophages from M1 to M2, effectively clearing myelin debris, alleviating local inflammatory reactions, and providing a favorable microenvironment for axonal regeneration ().
FIGURE 2
In recent years, the application of electroactive materials in the field of peripheral nerve repair has received increasing attention. These materials not only are capable of connecting damaged nerves in the form of scaffolds, providing mechanical support and physical cues, but also simulate the electrophysiological microenvironment of damaged peripheral nerves and transmit biochemical signals through their own electroactive properties (Wang et al., 2022). However, there still is a long way ahead to repair long-gap PNI under ES conditions. Some possible reasons include 1) The stimulation mode (direct current, alternating current, or capacitive coupling), appropriate timing (pre-, peri-, or post-operative), and parameters of ES protocols including frequency, intensity, time, and number of ES have not been standardized (; ), 2) The exact mechanism by which ES and electroactive materials enhance nerve regeneration is relatively unknown, 3) The misdirection of regenerated axons by ES demands of other interventions such as conduits (), and 4) the complex circumstances with spatial-temporal evolution of nerve injury in vivo become an evident challenge for nervous tissue engineering and regenerative medicine.
4 GBSs combined with ES for PNI repair
Since Andre Geim and Konstantin Novoselov first isolated single-layer graphene in 2004, graphene has been a popular material for modern chemistry and physics applications (). In recent years, graphene and its derivatives have received extensive attention as a biomaterial for use in the field of tissue engineering and regenerative medicine owing to their variety of extraordinary properties: 1) Controllable mechanical and electrical properties, either as an enhanced coating/blending in a composite or alone, providing mechanical support and physical guidance of extracellular matrix (ECM); 2) Oxygen-containing functional groups or chemical functionalization, endowing them with excellent chemical properties and hydrophilicity, which provide more chemical cues to interacting with cells of PNS; 3) High specific surface area and unique surface features, providing favorable topography and more bioactive sites for cell anchorage and cytoskeletal remodeling; 4) Antibacterial activity, preventing bacterial growth and formation of biofilm on the surface of an implant; 5) Defects and oxidation-dependence of biodegradation; 6) Facilitated fabrication of two-dimensional coatings or three/four-dimensional architectures.
The biocompatibility of graphene and its derivatives has been widely studied, and there are several related reviews on the topic (; ; ; ; ). The toxicity of suspended graphene-based materials was found to be highly dependent on their concentration, size of nanosheets, time of exposure, and surface chemistry. Meanwhile, either as a supporting substrate or an implantable medical device, the parameters of the material surface that influence the cellular response are significantly different from their counterparts in suspension (). Reports on the application of GBSs for tissue regeneration as a promising approach found negligible toxicity on cells in vitro and in rats for periods as long as 18 months (; ). Besides the biocompatibility of graphene and its derivatives for biomedical applications, their biodegradability has also been investigated. In recent in vitro and in vivo experiments, the biodegradation of GO and rGO was demonstrated to be defect- and oxidation-dependent, which may pave the way for their applications in nanomedicine and biomedical fields (). However, GO film is unstable in biological solutions and may lead to uncontrollable biosafety issues. Therefore, the incorporation of GO into a matrix-forming composite would be one of necessary choices. Compared with pristine graphene and GO, rGO-based scaffolds are more favorable for electrical active tissue regeneration owing to their high stability in aqueous solutions and remarkable electrical conductivity ().
The conductive nature of graphene and its derivatives has generated significant interest in neural tissue engineering in recent years (). They were found to improve proliferation rate of neural stem cells and induce neuronal differentiation, even without the addition of growth factors. In particular, besides electrical conductivity, GO and rGO have the ECM characteristics of PNS, which enables maintenance of high cellular viability, simulates the neurite outgrowth, regulates the degree of neurite extension and number of neuronal branches, and induces axonal alignment with or without adsorbed proteins such as poly (D-lysine) or laminin. Even though there is a great enthusiasm in exploring GBSs in this field and several review papers have been published on the study of GBSs for PNI repair, (; ; ; ; ; ), the discussion of ES and graphene-based materials remains only a footnote in the mentioned reviews above. Therefore, considering the significance of ES for neural scaffolds in tissue engineering and regenerative medicine, our review mainly focuses on the current exploration of GBSs combined with ES in PNI, summarizing their respective characteristics and impact on peripheral nerve repair, aiming to give guidance to current clinical potential.
4.1 Graphene
Graphene is usually prepared by a mechanical and chemical exfoliation technique, and graphene films/foams with a single, few-, or multi-layer structure are prepared by CVD method (). Besides, a 3D printing technique has also been studied to fabricate 3D graphene for neuronal networks or conduit (). Because of its two-dimensional atomic structure (Figure 3A) and unique electron distribution character, graphene has excellent physical and chemical properties, including a large specific surface area (∼2,630 m2 g−1), higher-than-diamond hardness and elastic modulus almost reaching 1 TPa, good optical behaviors (∼97.4% transmittance), high thermal conductivity (∼5,000 W m−1 K−1, higher than copper), and high electron mobility (∼2 × 105 cm2 V−1 s−1), even higher than that of carbon nanotubes and monocrystalline silicon (Yin et al., 2015; Zhang F. et al., 2022). The unique electrical properties and excellent electrochemical stability make graphene a good candidate for neuronal applications, including neural regeneration (; ). In particular, graphene-based composite materials combined with ES accelerate the growth rate of neurons via induction of Ca2+ influx, exhibiting high levels of Tuj1 and MAP2 expressions, which led to the investigation of their potential for neural tissue engineering applications ().
FIGURE 3
The combination of graphene and ES has shown significant advantages in the proliferation and differentiation of PC-12 cells, where the graphene was either used as a coating (; ) or mixture (; ; Zheng et al., 2019; ; ) in the polymer matrix to improve mechanical, electrical or biological properties. Along with an oriented topography, the graphene-based scaffold could further improve cell proliferation and growth direction in vitro (; ; ; ). The in vivo implantation of PLCL/PDA/GN conduit into rat sciatic nerve defects, which exhibits both electrical conduction and an axon-guiding surface structure, promoted neural regeneration, myelination, and recovery of motor and sensory functions under the synergistic stimulation of ES (Figure 3B). Besides neurons, applying ES (10 mV, 1 h d−1, 3 times) through graphene-based conductive polymers yields a positive influence on morphologies and proliferation of SCs, which plays an important role in the process of peripheral nerve repair. Li et al. prepared a conductive composite membrane composed of graphene and TPU (). The presence of graphene significantly improved the mechanical properties and conductivity of the membrane. By applying various voltages (10, 50, and 100 mV) of direct current (DC) ES (1 h d−1, 5 times) to the conductive composite containing SCs, a DC voltage of 10 mV was found to be most suitable for survival, synaptic stretching, and the proliferation of SCs (Figure 3C).
The combination of graphene and ES is likewise of great significance for stem cells orienting into specific cell linages. reported that graphene-based conductive fiber scaffolds (GCFS) prepared by combining different concentrations of graphene with COL and PCL exhibited concentration-dependent conductivity. After studying the effect of GCFS with different graphene contents on neural differentiation of MSCs, they found that a concentration of 1.0 wt% was more conducive to MSCs differentiation into mature neurons; however, higher graphene contents exhibited potential toxicity to the cells. ES promoted the secretion of MSCs and neurotrophic factors. With external stimulation (2 Hz, 20 mV cm−1) of MSCs cultured on the 1.0 wt% GCFS surfaces, a significant promotion of the migration and differentiation into neurons was achieved, even though the promoting effect could not be enhanced with increasing ES intensity. For the in vivo study, they applied a nerve guidance conduit (NGC) made of 1.0 wt% GCFS, combined with ES (2 Hz, 200 mV mm−1, 10 min d−1, 14 times), as a bridging material to the sciatic nerve defect site in rats, achieving satisfactory recovery results. ES was found to promote the regeneration and functional recovery of the sciatic nerve after nerve-guided tube implantation. Current in vitro and in vivo research on ES combined with GBSs with different electrical conductivity provides a reference for their neural tissue engineering applications (Figure 3D) ().
4.2 GO
GO is an oxidized graphene derivative produced by oxidizing graphite with sulfuric acid and potassium permanganate under acidic conditions (). The surface of GO sheets contains oxygen-containing functional groups such as epoxy, carboxyl, and hydroxyl groups (Figure 4A), which give it good hydrophilicity and colloidal stability, making them more suitable for adhesion, proliferation, and differentiation of cells. Simultaneously, oxygen-containing groups enable it to interact with biological molecules such as peptides, DNA, or proteins through physical adsorption or chemical binding that can be modified or functionalized (; ). As one of the derivatives of graphene, GO also has unique physicochemical properties for biomedical applications, including as scaffolds in regenerative medicine (). Even though GO is considered to be an electrically insulating material, its electrical conductivity and piezoelectric property can be regulated by its density and types of oxygen-containing groups, and combining with ES, GO has also been explored for use in peripheral nerve repair ().
FIGURE 4
Huang et al. prepared polypyrrole (PPy) conductive composite films (CCFs) doped with GO nanosheets on aligned poly-l-lactic acid (PLLA) fibers using the electrochemical deposition method (; ). PC-12 cells cultured on the surface of CCFs with administered ES at an intensity of 50 mV cm−1 showed significantly higher neurite length and percentage of alignment than those without ES (Figures 4B, C). The enhanced promotion of neurite elongation and orientation was ascribed to GO sheets that coated the surface of the film, providing electrical and topographical cues for regulating PC-12 cells behavior. In another study, in vitro experiments were carried out on RSC-96 cells (). The results indicated that the film had a promoting effect on the expression of neural proteins, and the application of 1 h ES (50 mV cm−1) arranged SCs along the current direction, which was of great significance for peripheral nerve repair (Figure 4D). Furthermore, the study confirmed the synergistic stimulation of ES and conductive conduit with high tensile strength and aligned surface morphology on nerve regeneration and functional recovery ().
4.3 rGO
rGO can be produced by reducing GO via chemical or thermal treatment. Therefore, rGO exhibits a similar structure to GO, a two-dimensional nanomaterial comprising single-layer sheets of sp2 and sp3 hybridized carbons, with the exception of the decreased amount of oxygen-containing functional groups (Figure 5A) (). Due to the removal of oxygen-containing groups, rGO exhibits higher thermal stability, higher electrical conductivity, and lower cytotoxicity compared to GO, which are important properties for neural tissue engineering and regenerative medicine (). In recent years, the application of rGO combined with ES in PNI repair has gained increasing attention.
FIGURE 5
The combination of rGO and ES promotes the differentiation of MSCs into SCs. electrically stimulated (50 Hz, 100 mV, 10 min d−1, 15 times) MSCs using inkjet-printed rGO-based electrodes. The results showed that ES enhances cellular differentiation more than conventional chemical strategies (). The circuit made of rGO ink after pulsed-laser processing displayed high conductivity, with a sheet resistance lower than 1 k Ω/sq (Figure 5B). Research has shown that combining rGO-based biomaterials with ES effectively promotes the maturation and differentiation of neurons and regulates the repair function of SCs, which plays an important role in the formation of a peripheral nerve regeneration microenvironment. coated the rGO onto the surface of an ApF/PLCL nanofiber scaffold through an in-situ redox reaction of GO. In vitro, the scaffold significantly promoted SC migration, proliferation, and myelin formation, including myelin specific gene expression and secretion of neurotrophic factors. PC-12 cells cultured on the conductive scaffolds also exhibited high differentiation ability with the aid of ES. The in vivo performance of implanting AP/rGO NGC into the sciatic nerve defect of rats was similar to that of autologous nerve transplantation (Figure 5C). Liu et al. prepared conductive PLA/rGO/PPy composite nanofibers by the incorporation of rGO into PLA (). Owing to the presence of conductive PPy and rGO, the conductivity of the composite achieved 1.46 × 10−1 S cm−1, which is higher than some of the graphene-based composites, as shown in Table 1. To study the effect of ES on the proliferation and differentiation of nerve cells seeded on scaffolds with high electrical conductivity, the authors applied electric field strength of 0, 100, 400, and 700 mV cm−1 and 50 Hz for 0.5 h d−1 for 3 days. The electric intensity of 400 mV cm−1 was found to be most favorable for cell proliferation, differentiation, and neurite growth. Most recently, developed a conductive NGC for better nerve regeneration compared to their previous study through filling with PCL NF that was coated by rGO layers. In vitro study demonstrated the excellent cytocompatibility of rGO@NF with 30 layers of rGO, which exhibited the highest electrical conductivity and promoted PC-12 cells extension and neurite outgrowth in the presence of ES. Further, transplantation of the NGC in vivo to bridge the nerve defect in a Sprague Dawley rat model accelerated nerve regeneration to a greater extent compared to bridging the fractured nerve by a hollow NGC (Figure 5D). All the results above indicate that ES combined with rGO is an efficient strategy to develop an artificial implant for long-gap PNI repair.
5 Summary and perspectives
As a common clinical disease, the incidence rate of PNI has experienced an upward trend in recent years. ES is considered an effective treatment for PNI, and has been extensively studied in the preclinical stage. GBSs represent promising media and carriers for ES, owing to their excellent electrical conductivity and mechanical properties. The combination of GBSs and ES has shown encouraging effects in promoting stem cell differentiation, inducing neuronal repair, and promoting the proliferation, migration, and maturation of SCs. However, their practical applications have certain limitations that must be overcome. First, research to date is still limited to the preclinical stage, and most reports concern in vitro studies. Meanwhile, a large amount of in vivo and clinical translation data is needed to support a next-generation scaffold. Furthermore, there are still challenges in the biocompatibility of GBSs, and long-term safety in vivo is of particular significance for GBSs in future clinical applications. In addition, low-frequency or direct current electric fields are currently chosen to administer post-operational ES through GBSs. However, given the importance of balance between conductivity and ES for a proper neural regeneration, stimulation settings of the intensity of the applied ES through GBSs—in particular 3D-graphene or 3D-reduced graphene oxide with superior electrical conductivity must be carefully taken into account. In addition, the potential for GBSs piezoelectricity is still to be extensively explored to provide an effective platform for a wireless or non-invasive repair of PNI.
GBSs have been reported to assist in regulating neuronal excitability, which has a significant impact on neuronal repair and axonal regeneration. Simultaneously, studies indicate that GBSs can serve as a bridge to connect nerve defect sites and help transmit chemical signals between cells, promoting nerve regeneration. Further, reports showed their ability to improve the microenvironment of nerve repair, promote angiogenesis, and regulate immune responses. The GBSs with their unique topography and surface structure exhibit strong effect on the morphology and differentiation of stem cells into neurons. Even though the biodegradation of graphene and its derivatives is still a challenging issue for applications in tissue engineered grafts, the development of neural prosthesis or non-degradable flexible electronics for long-term applications for next stage of nerve guidance conduit or neural electrodes may have opened up numerous opportunities to GBSs for an optimal recovery in patients with PNI.
Statements
Author contributions
YZ: Formal Analysis, Resources, Writing–original draft, Writing–review and editing. YL: Data curation, Funding acquisition, Project administration, Writing–original draft. SK: Methodology, Software, Writing–original draft. DS: Data curation, Writing–review and editing. YL: Data curation, Writing–original draft. XW: Supervision, Writing–review and editing. LL: Supervision, Writing–review and editing.
Funding
The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was financially supported by the Project of the Natural Science Foundation of Jilin Province (20210101381JC), the Department of Science and Technology of Jilin Province (YDZJ202201ZYTS673), and the National Natural Science Foundation of China (No. 81901245).
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Publisher’s note
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Summary
Keywords
peripheral nerve injury, electrical stimulation, graphene-based scaffolds, neural tissue engineering, nerve regeneration
Citation
Zhao Y, Liu Y, Kang S, Sun D, Liu Y, Wang X and Lu L (2024) Peripheral nerve injury repair by electrical stimulation combined with graphene-based scaffolds. Front. Bioeng. Biotechnol. 12:1345163. doi: 10.3389/fbioe.2024.1345163
Received
27 November 2023
Accepted
14 February 2024
Published
28 February 2024
Volume
12 - 2024
Edited by
Víctor Carriel, University of Granada, Spain
Reviewed by
Óscar Darío García García, University of Granada, Spain
Claudio Cordova, Universidad de Valparaiso, Chile
Updates
Copyright
© 2024 Zhao, Liu, Kang, Sun, Liu, Wang and Lu.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Xin Wang, wang_xin@jlu.edu.cn; Laijin Lu, ljlu@jlu.edu.cn
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