Nerve Tissue

Nerve gap, as a common soft tissue defect, affects many people’s normal life and work. Researchers have been working to explore a variety of techniques that can restore neural motion and sensory functions in the spinal cord. However, since the injured axons can’t regenerate themselves, neural remodeling in the central nervous system has always been a serious challenge [93]. Autologous nerve grafts are the standard method for the clinical treatment of nerve injury, when a severely damaged nerve without tension can’t be sutured directly [94]. Although autologous nerve grafts are generally accepted by the medical community as the gold standard, donor tissue availability, surgical trauma, and associated complications can lead to the failure of treatment [95, 96]. Nerve tissue engineering has proposed the use of bioartificial nerve conduits as an alternative of autografts to treat nerve tissue damage, especially for large defects.

SEM of the fiber-reinforced chitosan scaffold for nerve regeneration

Fig. 8.9 SEM of the fiber-reinforced chitosan scaffold for nerve regeneration (a) a well-integrated three-layer structure of the scaffold. The outer surface of the scaffold (b) a well-interconnected porous structure. Scale bars 1 mm (Adapted with permission from Ref. [100]. Copyright 2007 Springer)

As a means of guiding the regeneration of axons to promote nerve reconstruction, nerve guidance conduit (NGC) is one of clinical treatments in nerve tissue engineering. A variety of NGCs have been developed as grafts to repair damaged nerves, such as microchannel and microfilaments, hollow fiber membranes, multilayers tubes and fiber bundles [93]. Compared with autografts and allografts, synthetic NGCs have many advantages, such as improved applicability and effectiveness, complex structures and the excellent ability of regenerating axons [97]. Studies have shown that they have excellent performance in inducing axonal regeneration in vitro, but there are still many limitations to be overcome in vivo. In general, NGCs can’t selectively induce sensory and motor axon growth to the target organs. Therefore, it is necessary to use reasonable materials to synthesize NGC, and the ideal biological material should not only have good histocompatibility, biodegradability and bio-activity but also directional guidance ability. During nerve regeneration, the scaffold wall absorbs body fluids and expands, and the scaffold lumen may be obstructed by such swelling, which is detrimental to axon regeneration. Thus, the ideal biomaterial scaffold should provide sufficient longitudinal mechanical support to avoid significant swelling [98, 99]. Fortunately, the addition of fiber or tube seems to be effective in improving mechanical properties of the nerve guide scaffold.

Wang et al. [100] designed a porous chitosan-based fiber-reinforced guide conduit scaffold for nerve regeneration. The chitosan nerve conduits reinforced by braided chitosan fibers were fabricated by a combination of the mold casting and the lyophilization technique. The reinforced guide conduit scaffold with the porous structure had improved physical properties and biocompatibility. The reinforced guide conduit scaffolds had sufficient permeability that allowed molecules penetration ranging from 180 to 66,200 Da in size (Fig. 8.9). Comparing with the chitosan guide conduit scaffold, the compressive load of the reinforced guide conduit scaffold was obviously increased under the same strain. Likewise, the tensile strength value increased significantly from 0.41 ± 0.17 to 3.69 ± 0.64 MPa. Cytotoxicity assays in vitro demonstrated that Neuro-2a cells in the scaffold were not cytotoxic. Moreover, the scaffold had no effect on the surrounding tissue in vivo in primary implantation testing. The chitosan fibers improve the biocompatibility and mechanical properties of the guide conduit scaffold and make the reinforced scaffold fulfill the requirements of nerve regeneration.

Lu et al. [98] designed a microbraided PLA fiber-reinforced scaffold with a biodegradable multilayer for peripheral nerve regeneration. The scaffold was implanted into the sciatic nerve of rats with 10-nm nerve gap. The PLA fiber-reinforced scaffolds were not significantly swollen or deformed during the 8 week culture period. There was a brown fibrous tissue on the surface of the scaffold, and the new fibrous tissues also could be seen at parts of the nerve gap. After the removal of fibrous tissues and the walls of the conduit, regenerated nerve could be found. As a result, the PLA reinforced scaffold with good biodegradability and effectiveness is successful in repairing a 10 mm nerve gap and provides a promising tool for peripheral nerve regeneration.

Kim et al. [101] prepared an aligned polymer fiber-based NGC for repair the long peripheral nerve gaps. Aligned fibers differing from randomly oriented ones can establish topographical cues. Even if there was no exogenous growth to promote protein, the endogenous neural repair mechanisms could be triggered. The reinforced conduit guided and enhanced neurite outgrowth and axonal regeneration to repair neuromuscular junctions with a 17 mm nerve gap. Significantly, enhanced electrophysiological and behavioral results demonstrated that aligned fibrous structures had a role in promoting sensory and motor nerve regeneration. Additionally, sub-micron scale topographical cues of the aligned oriented fiber reinforced NGC were more conducive to encourage endogenous nerve repair mechanisms, according to the quantitative results in vivo of DRG outgrowth and nerve regeneration between randomly oriented and aligned fiber films. Thus, the aligned polymer nanofiber-based NGCs as a new synthetic scaffold have great potential for facilitating the regeneration of peripheral nerves.

Bini et al. [102] initially studied random poly PLGA nanofiber-reinforced scaffolds and formed 11 NGCSs though the electrospinning for peripheral nerve regeneration. Five of these scaffolds had the ability to support nerve regeneration of nerve defects in 10 mm based on chemical and histomorphometric results, but there was no obvious characterization function providing for recovery. Five PLA-fiber- reinforced scaffolds with single, double and triple layer structures all had the ability to induce nerve tissue growth to complete nerve repair and regeneration. In addition, the regenerative nerve had a structure similar to that of thick fibrous tissue, including cells and vascularized endomembranes. The clusters schwann cells also could be observed under the condition of nerve cuff bridging the bridging, which is a unit of regeneration around the unmyelinated axons, a common tissue structure [103]. As a result, the PLGA nanofiber-reinforced NGCSs can be used as nerve substitutes to promote the nerve regeneration.

Gao et al. [104] deisigned a 3D new-typed fiber-reinforced scaffold encapsulated Schwann cells and vascular endothelial cells in vivo to support the vascularization and induce the nerve regeneration for the repair of long-distance sciatic nerve defects in rabbit. The fiber-reinforced scaffolds were composed of a composite of nano-hydroxyapatite, collagen and poly(L-lactic acid) (PLLA) (nHACP) reinforced by chitin fibers, and prepared by using a previously method published by Li et al. [105]. In order to explore the role of vascularized grafts in the early recovery of neurological function, two groups of the scaffolds, nonvascularized and vascularized nerve scaffolds, were implanted into the rabbit sciatic nerve. The experiment group was implanted the prevascularized nerve scaffold, while the control group only cultured with Schwann cells. According to the results of neurological functional recovery at 4, 8, and 16 weeks after surgery, the experimental group had more significant growth in the recovery of ulcer healing, nerve conduction velocity, nerve fiber number, and ultrastructure of myelin sheath and regenerating nerve, as compared to the control group. Thus, the fiber-reinforced nHACP scaffolds cultured with Schwann cells and vascular endothelial cells can induce vascular sciatic nerve formation. The prevascularization of the scaffold can effectively stimulate the recovery of nerve function in sciatic nerve regeneration. Therefore, the study demonstrates that the fiber-reinforced nHACP scaffolds cultured with Schwann cells and vascular endothelial cells can induce vascularized sciatic nerve formation. Prevascularized scaffolds can effectively promote the restoration of neurological function in sciatic nerve defects regeneration. This study provides not only a new idea for finding a method to promote vascularization of tissue engineered nerve but also an ideal nerve substitutes for the regeneration of vessel and nerve.

In the other work, carbon nanotubes (CNTs) as a new kind of materials have attracted enormous attention as the most commonly used tubes reinforced materials for unique applications in the nerve tissue or other soft tissues repair [106]. CNTs have excellent structures and properties that are unmatched by other materials, such as high aspect ratio [107, 108], high conductivity [109, 110], strength [111], and chemical stability. It is interesting to note that aligned CNTs within the scaffold provide a key advantage of directional electrical conductivity, which helps to promote effective and rapid healing of damage nerve tissue [112-114]. Therefore, the researchers should spare no efforts to develop more extensive research and study on CNTs in the future.

In recent years, many studies have been conducted on the resulting properties of carbon nanotubes as scaffold reinforcement materials in never tissue engineering. Based on previous studies on the use of CNTs for nerve repair, Hu et al. [115] used carbon nanotubes as substrates for successful cultured hippocampal neuronal. Mattson et al. [116] successfully cultured embryonic rat brain neurons on the nanotubes, and the neurons survived and continued to grow for 8 days. Shin et al. [114] designed a CNT-reinforced gelatin methacrylate (GelMA) hybrid to create a 3D constructs with biocompatibility and cellular responses. This study also presented a simple and direct method to fabricate a porous CNT-reinforced scaffold. Through the use of GelMA hydrophobic interaction between the polypeptide chain and the sidewall of carbon nanotubes, a thin layer of GelMA were coated to CNTs, and their complete physical properties were still maintained. The addition of CNTs successfully enhanced the performance of GelMA hydrogel without the reduction of the

Digital images of the fabricated scaffolds (A colour version of this figure can be viewed online) (Adapted with permission from Ref. [107]. Copyright 2015 Elsevier Ltd)

Fig. 8.10 Digital images of the fabricated scaffolds (A colour version of this figure can be viewed online) (Adapted with permission from Ref. [107]. Copyright 2015 Elsevier Ltd)

porosity and inhibition of cell growth. NIH-3T3 cells encapsulated in the CNT/ GelMA scaffold kept their viability in 48 h of the culture, and readily spread and proliferated in the cytotoxicity test. As for the results of the mesenchymal stem cells (hMSCs) embedded into the CNT/GelMA scaffold, they appeared good biocompatibility. The nanofiber CNTs with a web-like structure enhanced mechanical properties of the GelMA hydrogel without interfering with their beneficial bioactive properties. Interestingly, the mechanical properties of the CNT/GelMA composite can be adjusted via controlling the content ratio of CNTs. CNT-reinforced GelMA composites provide the hope to generate 3D scaffolds to guide cell deposition into nervous tissue in a manner.

In the other study, Gupta et al. [107] fabricated macroscale aligned carbon nanotube (MWCNT) reinforced chitosan scaffolds by using electric field alignment technique for neural tissue regeneration (Fig. 8.10). This study provides an efficient platform for adjusting the mechanical and electrical properties of CNT-reinforced scaffolds by changing the alignment of CNTs. Compared with the random CNT- chitosan and pure chitosan scaffolds, the yield strength, elastic modulus and ultimate tensile strength of MWCNT-chitosan scaffolds were increased by 21.9%, 12.7% and 11.2%, respectively. This was because carbon nanotubes in the latter had a uniform distribution, good interface and aligned network. The MWCNT-chitosan scaffolds exhibited an anisotropic conductivity of 100,000 times higher in the alignment direction than that of the transverse direction, and had the ability to guide the direction of neurites growth and cell migration. The fabricated scaffolds were evaluated by incubating HT-22 mouse hippocampal neurons in vitro (Fig. 8.11). HT-22 hippocampal neurons on the MWCNT-chitosan had a significantly increased viability, which proved the scaffolds with a good biocompatiblity. Moreover, 50-60% of neurons were arranged in the alignment direction of the scaffold, which was also evidenced that the addition of aligned carbon CNTs supplied mechanical and electrical clues in favor of regular regeneration of the nerves.

Acridine orange-stained fluorescent images of HT-22 rat hippocampal neuron on chito- san

Fig. 8.11 Acridine orange-stained fluorescent images of HT-22 rat hippocampal neuron on chito- san (a, d, g), hybrid chitosan-random MWCNT film (b, e, h), and hybrid chitosan-aligned MWCNT film (c, f, i), respectively, after cultured for 1, 3 and 5 days (A colour version of this figure can be viewed online) (Adapted with permission from Ref. [107]. Copyright 2015 Elsevier Ltd)

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