Functionalization strategies of electrospun nanofibrous scaffolds for nerve tissue engineering

1. Introduction

The nervous system regulates many physiological functions, including breathing, muscle contraction, and sensory. However, severe nerve injury is usually difficult to recover from. Nerve injury includes central nerve injury (CNS) and peripheral nerve injury (PNI). The CNS mainly refers to the nerve injury of the brain and spinal cord, whereas the nerve injury outside the central nervous system is called PNI. Degenerative nerve diseases and injuries of the CNS may result in the breakdown of neural circuits and a loss of neuronal function, even leading to death [1]. Besides, 3% of injuries are accompanied by PNI, and there are more than five million new cases of PNI each year [2]. PNI causes sensory or motor deficit, innervation area dysfunction, and neuropathic pain [3]. In the repair of PNI, several strategies have been investigated to guide nerve fiber regeneration to the distal nerve, thereby improving axonal regeneration. Conventionally, autogenous nerve transplantation is most commonly used for nerve repair. However, it suffers from secondary injury and donor nerve deficiency [[4], [5], [6], [7], [8]]. Therefore, tissue engineering scaffolds have been studied as alternative methods for guiding nerve regeneration.

Various methods for preparing nanofibers have been reported, such as template synthesis, self-assembly, and phase separation. As one of the most widely-used techniques to fabricate tissue engineering scaffolds, electrospinning has attracted much attention because of its advantages compared with other methods. Specifically, template synthesis technology can well control the fiber diameter, while it is a time-consuming process and can not produce continuous nanofibers [9]. Self-assembly technology makes it possible to make successive and uniformly-shaped nanofibers, but its main drawback lies in the uncontrolled nanofiber size and the narrow selection of materials [10]. Phase separation technology widely used in the preparation of nanofibers is simple and inexpensive, and it enables successive nanofibers one after another and mass production. However, it has some major limitations, such as time-consuming, lack of structural stability, difficulty in maintaining porosity, and therefore is not suitable for all polymers [11]. In clear contrast, electrospinning possesses many virtues including easy operation, governable scaffold structure and fiber diameter, and is suitable for all sorts of materials [12]. Micro/nanofibers within electrospun scaffolds mimic the ultrastructure of natural extracellular matrix (ECM) [8,13]. Moreover, it owns a high surface area to volume ratio for cell adhesion, porous structure for cell infiltration, and adjustable mechanical properties to guide optimal mechanoresponses of residing cells [14].

The detailed electrospinning process mainly includes the following stages. First, polymer solution is entered through a thin needle and then a suspended conical droplet is formed. The surface tension of the droplet is balanced with the applied electric field between the spinneret and collector. Subsequently, a tiny jet is ejected from surface of the droplet when the electrostatic field is strong enough to overcome surface tension of the liquid. As the solvent evaporates and the nanofibers move towards the collecting plate, a thin film eventually forms on the collecting plate. Thus, the polymer solution in the syringe is converted into fibers. However, the conventional electrospun scaffolds still cannot optimize the efficient healing of large-scale nerve defects, mainly due to the following challenges: disordered cell migration, absence of electrical signal stimulation, lack of growth factors, and unfavorable immune-inflammatory response [15].

As a result, a series of approaches have been used to optimize electrospinning scaffolds for nerve regeneration. Electrospinning fibers with controlled alignment were especially fit for neural tissue engineering owing to their spatial guidance of neurite growth and axon elongation [16]. Nerve cell migration, adhesion, and polarization could be regulated by the topological cues of residing ECM [17]. In the previous studies, aligned electrospun nanofibers were found to promote the migration of Schwann cells (SCs) for the regeneration of axon growth cones [18]. Moreover, conducive materials were often used to transmit electrical signals and stimulate neuron growth. In the nervous system, electrical signals were transmitted through axons to relay nerve impulses. In turn, the nerve ​impulse builds up as the nerve fibers are electrically stimulated. Therefore, electrical stimulations were often applied to promote the growth and proliferation of nerve processes and accelerate nerve regeneration [19]. Furthermore, surface modifications of the electrospun scaffolds were often performed to promote cell proliferation and reduce the unfavorable immune-inflammatory response. For example, the addition of growth factors could promote the infiltration and survival of SCs, and enhanced the differentiation of proximal neurons of damaged nerves, thus effectively promoted nerve regeneration [20,21]. The interaction between SCs and stem cells indirectly promoted nerve regeneration through paracrine growth factors [22,23]. Finally, the nerve injury attracted macrophages. Nogo-66 receptor expressed in the infiltrated macrophages increased phagocytosis of the macrophages, to promote CNS regeneration in vitro [[24], [25], [26]]. However, as macrophages differentiated into the M1 phenotype, the elevated concentration of reactive oxygen species (ROS) in the local micro-environment as well as released pro-inflammatory signals exerted negative impacts on the repair of nerve tissue. Therefore, some scaffolds loaded with antioxidants are designed to neutralize ROS, reducing nerve scarring and promoting nerve regeneration [27].

In this review, we have introduced electrospinning as a promising method for the fabrication of aligned fibrous scaffolds for neural tissue engineering, as shown in Scheme 1. The natural, synthetic, and composite materials used in electrospinning have been reviewed. Moreover, we have also summarized key modification strategies, including the induction of aligned topologies, bioelectricity, surface functionalization with bioactive molecules, and the addition of anti-inflammatory drugs to optimize nerve regeneration. Lastly, we have discussed the current challenges and future directions of designing more functionalized electrospun nanofibrous scaffolds for nerve repair. The primary goals of the present review are to evaluate and consolidate the findings of the latest studies that used electrospun neural tissue-engineering scaffolds, which provide effective strategies and contribute to the development of functionalized scaffolds in the field of nerve regeneration.

Scheme 1. Schematic representation of functionalized electrospun nanofibrous scaffolds [[28], [29], [30], [31], [32]].

2. Materials for electrospun nanofibrous scaffolds

The characteristic of electrospinning nanofiber scaffolds is that they can regulate biological behaviors such as cell adhesion, migration, and proliferation by controlling the changes of biochemical signals [33]. The high specific surface area enables the scaffold to carry and release a variety of biochemical substances such as drugs, proteins, and nucleic acids, and at the same time increases the contact area between cells and fibers so that cells fully absorb these substances, thereby regulating cell behavior [34,35]. In addition, nanofibrous scaffolds are porous, which is permeable to nutrient and O2 penetration, cell uptake, and metabolite excretion [36]. A wide variety of materials are used for electrospinning, which can be generally split into three categories: natural materials, synthetic materials, and composite materials [37]. Natural materials that simulate ECM composition generally have good biocompatibility and biodegradability [38]. However, they can suffer from batch-to-batch variances and relatively weak mechanical strength. Besides, some natural materials have immunogenicity causing the unfavorable immune response of the recipient, which limits its application in nerve tissue regeneration. Compared with natural materials, synthetic materials are engineered for better mechanical strength and key material properties can be fine-tuned to deliver reproducible results [39]. Synthetic materials are often used together with naturally derived materials to create composites for better natural cell affinity and optimized guidance for nerve regeneration