Introduction

Low and non-uniform cell density, disorganization in nerve cell arrangements like nerve coils, and instability of seeded cells can result in sensory-motor problems in healing peripheral nerve tissues [1]. Therefore, rapid repair of neural tissue with grafts or conduits is not the only treatment option. Regenerative methods should prioritize the seeding site, proliferation, differentiation, and cell shape for cell orientation and alignment to ensure that develo** neurons resemble natural structures [2]. Alignment and orientation of neurons during the repair process have been demonstrated to have a positive impact on the function of neural tissue, including increased NCV, reduced neuroma, and improved CMAP [3,4,5].

Nanomedicine-based methods allow tissue engineering to regulate the topography [6], biological or chemical gradients of the microenvironment [35]. This triggers PI3-kinase, phospholipase C, and MAPK signaling pathways in Schwann cells, resulting in neuronal cell growth, migration, and myelination.

Cell-ECM interaction

The ECM is essential as the main structural support in the body, performing specialized functions tailored to the needs of each organ. Despite its diverse composition, collagen, elastin, fibronectin, and laminin are the main components of the ECM [36]. Collagen is plentiful in peripheral nerve tissue, which is associated with the layer formed by Schwann cells [36]. The ECM plays a crucial role in regulating neuronal functions, including differentiation, migration, alignment, adhesion, cell–cell communication, and the dispersion of mature neurons. This is achieved through interactions between cell integrins and laminin, fibronectin, collagen, and ECM peptides [37]. Integrins are transmembrane receptors composed of α and β subunits that form noncovalent interactions [38]. These interactions are observed in four ways in neuron-extracellular matrix interactions: RGD (Arg-Gly-Asp) ligands bind to αV, α5β1, α8β1, and αIIbβ3 integrins, LDV (Leu-Asp-Val) ligand binds to integrins α4β1, α4β7, and α9β1, Laminin/collagen binds integrins α1, α2, α10, α11, and laminin binds integrins β1 (α3, α6, and α7) and α6β4 [38, 39]. Integrin-mediated neuron-ECM interactions facilitate two-way signaling (outside-in and inside-out) between cells and ECM (Fig. 1C). These interactions involve talin, paxillin, zyxin, and vinculin, along with actin filaments, and contribute to the formation of different adhesive structures like nascent adhesions, focal complexes, focal adhesions, and fibrillar adhesions [40]. Paxillin-rich, short-lived nascent point-like adhesions are found beneath the lamellipodia at the front of the migrating cell. Focal adhesions containing low levels of paxillin and high levels of vinculin form at the ends of stress fibers containing actin and myosin [41]. This cytoskeleton-derived adhesion is effective in force transmission. The most stable adhesions, fibrillar adhesions containing large amounts of tensin and β1-integrin, form along matrix fibrils beneath the cell center [39]. During cell migration, the cytoplasm expands by filopodia and lamellipodia, adhesions form, creating focal complexes rich in FAK and paxillin, and actin polymerization also takes place. Actomyosin contractile forces release mature focal adhesions on the opposite side, separating the cell from the environment [38, 40, 41].

Supporting structures

Efforts have been made to develop support structures containing neural cells to alleviate the limitations of autograft and allograft in peripheral nerve regeneration, with the most important being biological or polymeric conduits. Despite the significant impact of construction methods and materials on the healing process through biocompatibility, biodegradability, permeability, and mechanical capabilities (tensile strength, pressure resistance, flexibility, etc.) (Table 1), this study specifically examines the role of neuron function (adhesion, proliferation, differentiation, growth) in the physical structure of conduits, including orientation and topology patterns. While it may be challenging to establish criteria based on the physical properties of conduits, a review of existing literature may offer valuable insights to enhance and expedite the repair process of neural tissue.

Table 1 A summary of the features and construction methods of conduits used in peripheral nerve repair
Full size table

Aligned fiber structures

Repairing injured peripheral nerves is often challenging, and sometimes even impossible, due to the misdirection of nerve cells and or improper suturing of the two nerve endings. Leveraging the inherent properties of neural tissue, such as its striped structure and directional cell distribution, conduits with aligned fibers have been found to expedite the healing process by facilitating the directional growth of neurons. Numerous studies in this area have indicated that the use of aligned fibers promotes peripheral nerve regeneration and enhances performance (Table 2). Nevertheless, achieving the precise extent and shape of cell alignment remains problematic due to the adverse effects of excessively aligned fibers on cell junctions and the unbalanced growth of axons.

Table 2 The performance of aligned or random fibers in the peripheral nerve regeneration
Full size table

Kim JI et al. [58] demonstrated that altering the conduit fibers from random to aligned within PLGA and PU (from electrospinning: voltage: 16 kV, flow rate: 1 mL/h) conduits containing NGF enhances the orientation of PC12 cells, aiming to improve nerve cell function. Aligned fibers were found to significantly improve the directional growth of PC12 cells by tripling the length and volume of intracellular actins parallel to the fibers. This enhancement was attributed to the reinforcement of cell adhesion, resulting from increased hydrophilicity that altered the cell contact angle from 128.3 to 120.2°. The alteration of PC12 cell morphology from flat or spherical in random fibers to spindle-shaped and oriented with aligned fibers validates this [58]. In next study, Chen S et al. [59] demonstrated that applying decellularized peripheral nerve matrix gel (pDNM gel) on PLLA fibers (diameter: 650 ± 90 nm) not only enhances axon growth by 30% through increased adhesion and interaction between DRG cells and fibers, but also leads to a 3–sixfold rise in Schwann cells migration in aligned fibers, significantly impacting the orientation of DRG cells. Schwann cell migration on aligned fibers and secretion of biological signals like NGF or neurotrophin-3 on them, have influenced the directional growth of neurons and axons extension. Moreover, it was demonstrated that the growth orientation of DRG cells significantly depends on the substrate pattern. It was observed that augmenting the thickness of the pDNM gel coating (from 0.25 to 1%) and decreasing the resolution of the substrate pattern lead to a more random growth of DRG cells. Surprisingly, the utilization of pDNM gel led to the emergence of fascicle-like axonal bundling in neural tissue, likely due to the increased presence of neuronal ECM molecules [59]. To study the impact of fiber alignment on nerve tissue function, Du J et al. [6C). Despite the potential toxicity of glutaraldehyde, no toxicity was detected in this system. Additionally, the peptide gradient was found to enhance synergistic activity, leading to improved polarization of M1 to M2 macrophages and angiogenesis (Fig. 6C). Furthermore, the synergism of grooves with the peptide gradient was observed to boost the regeneration rate of the transected sciatic nerve (Fig. 6C), resulting in larger fiber diameter, thicker myelin membrane, longer neurites, and increased SFI, NCV, and CMAP [122].

Synergism between support structures and the electrical stimulation

Several studies confirm that electrical stimulation enhances nerve cell proliferation, differentiation, migration, and integration by activating ion channels and influencing growth patterns [123]. The electric field influences the directional growth of neurites/axons, while the electromagnetic field promotes neurogenesis [124]. Hence, the synergism of electrical stimulation with supporting structures is expected to enhance the directional growth of nerve cells and their incorporation into healthy tissue. For instance, it was found that aligned fibers (diameter: 800 nm) composed of PPy-coated PLLA, when subjected to electrical stimulation of 100, 200, and 400 mV/cm, increased neurite extension from 68% along the fiber axis to 76%, 83%, and 71%, respectively [24]. The highest directional growth rate of neurites/axons and cell orientation was observed with electrical stimulation of 200 mV/cm (Fig. 6D). Moreover, the results show increased directional growth of neurites/axons in random fibers with electrical stimulation, indicating a positive response of nerve cells along the electropotential direction (Fig. 6D). Furthermore, the rise in longitudinal fiber growth from 65.44 and 114.73 µm in random and aligned fiber groups to 128.45 µm (100 mV/cm), 149.39 µm (200 mV/cm), and 141.48 µm (400 mV/cm) in aligned fiber groups with electrical stimulation demonstrates the beneficial impact of electrical stimulation on nerve development (Fig. 6D). Electrical stimulation induces cell membrane depolarization on aligned fibers, enhancing the electrical charge in PPy nanoparticles. This leads to the accumulation of adhesive receptors, actin expansion in filopodia, and an increase in growth cones [24]. While actin concentration and self-assembly on one side of the cell dictate neurites/axons directional growth and cell orientation, the main impact of electrical stimulation is the promotion of cell adhesion, which negatively affects their migration. However, Zhang J et al. [53] showed that combining PCL/CNT aligned fibers with electrical stimulation (20 Hz and 100 mV) in the nerve conduit improves the directional growth of neurites/axons and cell orientation more effectively. This also enhances parameters like SFI, amplitude, latency, the number of axons, and myelin sheet thickness in sciatic nerve regeneration, similar to the gold standard autograft. Recently, instead of using electrical stimulation, which faces the challenge of reducing migration due to higher induction of adhesion foci, the use of piezoelectric polymers such as aligned PVDF fibers [125] is of interest. In this regard, after loading 400 nm PLLA nanofibers obtained by electrospinning (Flow rate: 0.5 mL/h, voltage: 12 kV) on PLLA films, Jiang F et al. [126] demonstrated that piezoelectric stimulation (~ 260 mV), not only enhanced cell differentiation (11% more than the non-stimulated group), but also led to a twofold increase in cell length compared to the non-stimulated group (142.7 µm vs. 70.2 µm). It was revealed that piezoelectric stimulation through double activation of Ca2+ channels initiates downstream signaling by increasing intracellular calcium. However, piezoelectric stimulation did not significantly affect cell orientation; instead, cell orientation was primarily determined by aligned fibers.

Challenges and future perspective

Management of PNI treatment remains inadequate despite advances in drugs and surgical techniques. Reports indicate that fewer than 25% of patients undergoing nerve repair achieve optimal sensory and motor function recovery after 5 years. The scientific community is focused on improving nerve cell growth and guidance through new technologies to create conduits to overcome treatment obstacles. Utilizing conduits significantly enhances repair processes for damaged peripheral nerves by promoting nerve cell growth, neurite and axon directional growth, and nerve cell orientation. However, challenges exist in using conduits in medical settings, including:

  • One of the main challenges in using conduits is ensuring biological safety in humans. A thorough comprehension of cell behaviors within conduits, as well as the behavior of nanoparticles loaded and released from conduits, can only be assessed through cell viability, migration, growth, proliferation, adhesion, and differentiation in short-term laboratory settings. The fluctuating nature of conduit degradation in the body, the range of inflammatory responses, the variety of cells affected, the extended human treatment process, the diverse immune system components, patient lifestyle, and medical history all contribute to the challenge of ensuring optimal conduit function. Thus, considering the constraints of the human model, focusing on models such as human organoids that closely resemble original tissue may aid in predicting potential immunogenic strategies.

  • Conduit studies typically aim to promote the directional growth of neurons and align them during development or differentiation. However, the challenge of linearly distributing conductive materials and biological/chemical gradients hinders the targeted transmission of electrical-neural signals and the induction of directional growth of nerve cells, posing a significant issue for peripheral nerve repair. Focusing on accurately regulating the reception of electric-nerve pulses from the proximal end to the distal stump in conduits, based on determining the precise path of conductive material loading, can offer a clearer perspective on repairing damaged peripheral nerves. Additionally, relying solely on the detection of gradients in conduits is concerning. Thus, focusing on the extent of arrangmenet and diffusion of gradients, evaluating the specific quantity and quality of gradients, and considering their potential side effects on other parts or cells can offer valuable insights for their application.

  • Despite the wide range of conduits available (biological, natural, synthetic), they are not yet a dependable choice for axon growth and nerve tissue regeneration over long distances, unlike autologous nerve grafts. To achieve this goal, a thorough comprehension of the physicochemical characteristics of conduits larger than 2.5 cm is essential, particularly in motor-sensory models resembling humans.

  • In vitro studies and clinical trials have been widely conducted on rats, rabbits, dogs, monkeys, pigs, etc. While the generalization of their results to humans based on the complete non-compliance of the aspects of the biology of the repairing nerve, the path of research has become a concern. Focusing on human trials or human organoids that closely resemble original tissue can reduce some of the existing challenges.

  • Conduits are typically designed and created by considering biocompatibility, biodegradability, enhancing cell adhesion, biomarker loading, creating topographical features, and adjusting mechanical properties for nerve tissue. However, the complexity and high cost of production methods pose challenges for commercialization. Focusing on cost-effective designs and simpler production techniques can facilitate their use in both general and personalized clinical applications.

  • Conduit studies mainly rely on autologous cells to prevent transplant rejection, complicating the commercialization and treatment process. The pursuit of a universal neuron or neuron-like cell discovery will enhance the potential for clinical treatments.

  • Conduits are commonly employed to direct axonal growth, inhibit fibrous tissue infiltration and scarring, and discourage axon reinnervation or nerve bundling. Prioritizing the development of the vascular network, promoting the movement of neural stem cells from neighboring areas, and averting uncontrolled swelling of hydrogels within the cavities can enhance treatment outcomes.

Conclusion

Despite the inherent regeneration of peripheral nerves, many nerve injuries encounter challenges like prolonged recovery, inflammation, neuroma, and decreased tissue function. Tissue engineering aims to address these obstacles by incorporating nanotechnologies and cell science to mimic the supportive structures of the ECM. This enhances cell proliferation, migration, and orientation, facilitating the directional growth of neurites/axons. This review suggests that promoting cone growth in a specific direction, achieving even cell distribution through targeted migration, enhancing the longitudinal growth of neurites/axons within conduits, and controlling cell organization to mimic natural tissue structure could be promising strategies. Studies have shown that incorporating micro- or nano-patterns in conduits (such as aligned fibers, grooves, channels, pillars, pits, etc.) along with therapeutic approaches like electrical stimulation and nano-carriers can have a substantial impact on nerve cell behavior, ultimately aiding in the restoration of damaged peripheral nerve functions. Conduits with surface topographies play a significant role in guiding axonal growth and cell orientation, inhibiting fibrous tissue penetration, and preventing axon re-innervation during peripheral nerve regeneration. The utilization of conduits with aligned fibers > multi-channels > grooves has garnered considerable interest due to reduced manufacturing complexity, enhanced ability to direct growth and cell migration, improved accessibility, decreased nerve compression, and control over fiber diameter and myelin thickness. However, the mechanism of action of topography on the development and expansion of peripheral nerves is currently being elucidated. Therefore, thorough research is necessary to understand the mechanism of micro- or nano-patterns and cellular responses based on their physicochemical characteristics, enabling the development of clinical applications with greater confidence. In the following, the focus should be on understanding the mechanism of micro- or nano-patterns in nerve cells to predict cell orientation, neurite/axon growth, and tissue function restoration efficiently. Also, despite various conduit manufacturing techniques, establishing a specific standard or tactic for creating micro or nano patterns is essential to enhance their operational capability in nerve tissue reliably, while addressing issues such as construction complexity, high cost, low reproducibility, and solvent presence. Therefore, besides evaluating conduit surface size and resolution, it is vital to investigate the commercialization process of conduits through biomedical models. This implies that the manufacturing technique should account for the mechanical and chemical changes on conduit surfaces that affect cell migration and adhesion strength, mimicking cell behaviors in the ECM. Recent research indicates that cells develop more focal adhesions on patterned surfaces, which could potentially hinder cell migration and morphological expansion processes, going against their natural tendencies. Finally, this review demonstrates that creating micro- or nano-patterns in the nerve conduit can enhance the peripheral nerve regeneration process by improving motor performance in model mice and tissue structures. Surprisingly, this approach also matches the efficiency of nerve tissue regeneration seen with the gold standard of autograft.