Introduction

The human brain is analogous to a black box of information and unraveling its mysteries is essential to understand its complex relationship with the various components of the peripheral and central nervous systems. This information is vital to probe the causes for various neural disorders and arrive at a plausible therapy for the treatment of ischemic, metabolic, congenital, or degenerative disorders of the central or peripheral nervous systems. Conventionally, autologous grafts are gold standards and have been used to treat neural defects [1–3]. However, autografts have limitations that include shortage of nerves since it is taken from the patient. Moreover, there is a mismatch of donor-site nerve size with the recipient site, neuroma formation and lack of functional recovery [4, 5]. Allogenic grafts, which are isolated from cadavers, are not limited by supply but suffer from host-graft immune rejection [6]. To overcome immune rejection, several studies have been conducted to examine the potency of acellular nerve grafts [7, 8]. However, as acellular nerve graft lacks viable cells, nerve regeneration and remodeling of extracellular matrix have been delayed [8]. The use of pre-degenerated nerve grafts having high matrix metalloproteinase (MMP) expression shows some potential as it degrades the inhibitory chondroitin sulphate and proteoglycans thereby retaining the ability to promote nerve regeneration even in the absence of cells [8, 9].

Recent advances in nanotechnology [10] and tissue engineering [11, 12] have been found to cover a broad range of applications in regenerative medicine and offer the most effective strategy to repair neural defects. The major determinant in all tissue engineering research is to regulate the cell behavior and tissue progression through the development and design of synthetic extracellular matrix analogues of novel biomaterials to support three-dimensional cell culture and tissue regeneration. Ideal properties of a scaffold for nerve regeneration are biocompatibility, less inflammatory, controlled biodegradability with non-toxic degradative products, porosity for vascularization and cell migration and three-dimensional matrices with appropriate mechanical properties to mimic the extracellular matrix [13–15]. Figure 1 shows the various characteristics desired for an ideal scaffold for neural regeneration.

Figure 1
figure 1

Ideal properties of scaffold.

Full size image

Polymeric biomaterials are widely preferred as scaffolds for peripheral and central nerve regeneration both in vitro and in vivo [16–19]. There is a wide choice of polymers available with programmable biodegradability, non-toxic/non-inflammatory nature, mechanical properties similar to the tissue to be replaced, high porosity that promotes cell attachment and growth, economical and simple manufacturing processes along with a potential for chemical modification leading to increased interaction with normal tissue [20]. Several techniques such as nanofiber self-assembly, solvent casting and particulate leaching, gas foaming, emulsification/freeze-drying, liquid-liquid phase separation, electrospinning and computer aided design and manufacturing techniques have been employed to fabricate tissue engineering scaffolds with varying degrees of success [21–10, 117]. Porous polymeric nanofibrous scaffold using biodegradable poly (L-lactic acid) (PLLA) fabricated by liquid-liquid phase separation method resembles ECM of natural collagen to support neuron differentiation and neurite outgrowth [22]. However, it is very difficult to maintain the fiber diameter and alignment using this technique. Likewise, various techniques have been reported to develop nanofibers namely, template synthesis, phase separation, self-assembly, drawing and electrospinning [115]. Among these techniques, electrospinning offers more advantages due to its ease of fabrication. Nanofibrous conduit comprising poly (D,L-lactide-co-glycolide) and poly(ε-caprolactone) (PCL/PLGA) were found to promote nerve regeneration across 10 mm nerve gap in rat sciatic nerve [118]. PLGA random nano and microfibers, aligned microfibers and films were investigated for C17.2 neural stem cell culture and recognized the differentiation of neurons along the fiber direction [119].

Figure 2
figure 2

Electrospun PLGA-PANi nanofibers.

Full size image

PLLA nanofibrous scaffolds were developed via electrospinning and found to support neural stem cell (NSC) adhesion, outgrowth and differentiation [21]. Suitability of aligned electrospun PLLA nanofibers compared with random nanofibers was evaluated for neural tissue engineering in terms of their fiber alignment and dimension [21]. The aligned nanofibers were found to support the orientation of cells and improve the neurite outgrowth and contact guidance [21, 67]. Based on the experimental results, this study recommended the aligned PLLA nanofibrous scaffold as a potential cell carrier in neural tissue engineering [21, 119]. The parameters such as viscosity, conductivity, surface tension of polymer solution, applied electric potential, flow rate, and distance between the electrodes are to be optimized while carrying on the electrospinning process [120]. It is also observed that the orientation of fiber became disordered at the top layer of the electrospun mesh when the collecting time was longer than thirty minutes due to the residual charges on the collecting fibers [21]. The desirable properties of electrospun nanofiber scaffolds seem to offer a promising alternative towards the treatment of neural defects. Aligned electrospun collagen/PCL fibers supports cell proliferation, glial migration, orientation of neurite outgrowth, suggested its suitability as nerve implants [71]. Aligned electrospun PCL fibers have been found to up regulate specific genes such as P0 and down regulate NCAM - 1 on cultured Schwann cells thereby promoting the Schwann cell maturation [67]. Significantly higher Schwann cell migration and neurite outgrowth was observed on uniaxially aligned fibers of poly (acrylonitrile-co-methylacrylate) (PAN-MA) developed by electrospinning than on random fibers [121].

Other Approaches

Fabrication of a multi-channel scaffold using injection molding with solvent evaporation technique has been demonstrated to promote spinal cord axon regeneration [122]. A new facile method named 'fiber stimulating technique' used to fabricate oriented PHEMA scaffolds successfully for neural tissue engineering, promises to be more effective and reproducible [77]. Melt compression and melt extrusion are also considered to be viable techniques to prepare nerve guides [14]. Innovative fabrication techniques such as wire mesh method and mandrel adhesion method are used to prepare multi-channel biodegradable nerve guides without the requirement of complex instrumentation, acidic conditions or exposure to extreme temperatures [123]. Designing of biodegradable PLGA hollow fiber by wet phase inversion technique has been attempted for the development of nerve tract guidance conduit [13]. Micropatterning is a novel patterning technique for biodegradable polymers and is reported to enhance peripheral nerve regeneration by controlling the alignment of Schwann cells [124–126]. Fabrication techniques continue to evolve novel routes to provide the most suitable nanostructure topography for adequate neural growth.

Electrical Cues

Human body responds to electrical fields and the key component of neural communication in the body is the action potential generated at the synapse. This implies that an ideal neural scaffold should also possess electrical conductivity to promote neurite outgrowth and thereby enhance nerve regeneration in culture. The use of electrically conducting polymers in biomedical applications has become more attractive due to its tailor-made specificities [127]. Polypyrrole (Ppy), a well-known conducting polymer used in biomedical applications has been found to enhance the nerve regeneration by electrical stimulation [128, 129]. Moreover, the antioxidant property of polypyrrole and polyaniline makes them more attractive substrates for tissue engineering applications [130, 131] as they could scavenge any free radicals at the site of injury minimizing scar formation which is a bane of neural regeneration. The structures of polythiophene, polyaniline and polypyrrole are shown in figure 3.

Figure 3
figure 3

Chemical structure of conducting polymers.

Full size image

A comparative study was made on the efficacy of two different laminin fragments p20 and p31 as dopants in conducting polypyrrole surfaces for in vitro growth of neurons. The results indicated that p20 as dopant supported the highest neuronal density than p31 dopant [132]. Conducting Ppy/PDLLA/PCL composites have been implanted to bridge the gap of 8 mm in rat sciatic nerves and shown to promote the nerve cell proliferation and axon regeneration using electrical cues [69]. The rats were gradually recovered the mobility in operated limb over the period of 2 months [69]. Moreover, the immunohistological analysis and transmission electron microscopy of harvested implants demonstrated the presence of newly formed myelinated axons and Schwann cells similar to that of native nerve [69]. Recently the cell adhesion property of polypyrrole has been improved by chemical conjugation of a functionalized carboxylic acid group with RGD peptide [133]. The suitability of poly ethylene dioxythiophene (PEDOT) as a biomaterial was evaluated by studying the adhesion and proliferation of epithelial cells and was demonstrated that the electroactive substrate favors cell adhesion [134]. Though the mechanism in which electrical stimulation promotes nerve regeneration is not clearly understood, several possible hypothesis have been postulated to elucidate their role in nerve regeneration. Some of the plausible reasons include electrophoretic redistribution of cell surface receptors, activate growth controlling transport process and altered adsorption of adhesive proteins [135]. The later hypothesis has been proved by stimulating the adsorption of fibronectin (ECM adhesive glycoprotein) from serum to polypyrrole surface via electrical stimulation, which subsequently facilitate the neuronal attachment and neurite outgrowth [128].

However, these conducting polymers are non-biodegradable and questions on their safety in biological systems have delayed their wide-spread use in neural conduits.

Future Prospective

Many Challenges still remain unwrapped. Though the researchers have found different strategies to achieve the functional recovery to some extent, regaining the maximal or full function remains unexplored. There are some issues listed below have to be addressed in future; (1) the first inescapable conclusion arising over various reports on nerve tissue engineering by super positioning of all these approaches is crucial for promoting the neural regeneration on multiple levels (2) the probable hazards of long term usage of such novel biomaterials on human health yet to be revealed (3) the need for novel Biomaterials and approaches has to be established in order to treat the delayed nerve injuries in patients who have neurological disorders.

Conclusion

An ideal nerve conduit requires a suitable porous, biocompatible, biodegradable, neuroconductive, neuroinductive, infection resistant, compliant three-dimensional biomaterial scaffolds. The engineered construct should also mimic the ECM architecture and porosity, desirable for cell attachment and other vital functions. Biomedical nanotechnology, electrospinning techniques and tissue engineering methods give us exciting insights to the design of a scaffold with good electrical, mechanical, biological properties and compliance match closely resembling the native ECM. Such scaffolds can also avoid infections, multiple surgeries and additional cost to the patient. An array of methods has been used for polymer scaffold preparation but electrospinning scores high due to its ease of operation, better control of fiber properties and desirable results. Lot of synthetic biodegradable polymers has been used till date but at the same time suffer from the demerits of release of acidic degradation products, hydrophobicity, poor processability and loss of mechanical properties. Also they support elongation and partial collapse of nerves. Hydrogels mimic soft tissue properties but are very difficult to sterilize and handle due to their fragile nature. A new strategy using polymers like polypyrrole, polyaniline etc., having conducting properties are being investigated for neural tissue engineering to stimulate neurite extension. However, the biocompatibility of these polymers has not been conclusively proved till now. It is seen that though different classes of biomaterials are available, no single material is enough to improve the scaffold properties for nerve regeneration. Hence, the major challenge in develo** a scaffold lies primarily in the choice of a blend of biomaterials with the correct combination of properties. The field is still wide open to design the most appropriate polymer scaffold with all the vital conditions and properties for effective neural applications in vivo.