**2. Nanofibrous scaffolds applied in CNS regeneration**

#### **2.1. Current fabrication of nanofiber-based bioscaffolds used in CNS regeneration**

The aim of biomedical engineering is the design and development of novel biomaterials that can recapitulate the key characteristics of natural ECM with the associated topographical cues, cellular adhesion sites, biochemical signals and physiological viscoelastic modules. In general, extracellular proteins such as collagen, fibrin and glycosaminoglycans, possess fibrous structures with diameters on the nanometer or sub-micrometer scales. Several bioengineering approaches have been developed for the fabrication of artificial nanofibre constructs with diameters that range from 10 to 100nm.

#### *2.1.1. Self-assembling peptide nanofiber scaffolds (SAPNS)*

Many biological macromolecules such as like phospholipids can readily self-assemble to form highly ordered bio-structures through van der Waals forces, hydrogen bonds, ionic bonds, and hydrophobic interactions. To recapitulate the bioactive motif of laminin, Stupp and coworkers designed a self-assembling peptide amphiphile (PA) that consisted of the laminin-derived peptide IKVAV (Ile-Lys-Val-Ala-Val). The self-assembly of IKVAV could be initiated sponta‐ neously upon the introduction of physiological buffer which leads to the formation of nanofibers with diameter ranging from 6 to 8 nm (Tysseling-Mattiace, 2008).

#### *2.1.2. Eletrospun nanofiber scaffolds*

poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and their copolymers poly(lactic acid-coglycolic acid) (PLGA) have been widely utilized to reconstruct bioscaffold in diverse condi‐ tions of neural repair. To facilitate host-material integration, biomaterial candidates must also possess the appropriate elastic module. For instance, hydrogel made of poly(2-hydroxyethyl methacrylate-co-methyl methacrylate) (p(HEMA-co-MMA)) has similar mechanical proper‐ ties to the mammalian spinal cord (elastic modulus of 200 to 600 kPa), and could be customized by altering the ratio of co-monomers (Dalton et al.,2002). The hydrophobic surface properties in most of the synthetic biodegradable materials such as PLGA, PCL and PHB, may be modified by coating them with ECM proteins components like laminin, fibronectin, collagen. Specific adhesion oligopeptide such as RGD and IKVAV, YIGSR may also be added to improve their adhesion properties for seeded cells. Several studies showed that the cellular adhesion performance of these artificial biomaterials including methyl cellulose, alginate, poly (hy‐ droxyethyl methacrylate) (PHEMA), poly (hydroxybutyrate) (PHB) (Samadikuchaksaraei, 2007), could be significantly improved by surface modifications. Another category of synthetic biomaterial is related to the nanofibre scaffold which will be discussed in later sections.

The use of synthetic nondegradable materials in neural repair is limited by their nondegrada‐ blilty and unbioabsorbility. The majority of reported studies involved PNI. For example, poly (2-hydroxyethyl methacrylate) has been demonstrated to support regeneration of injured axons in a rat SCI model (Tsai et al. 2006). Recently, electroactive polymer like polypyrole or hybrid conduct materials showed neuronal attachment and growth. The major concerns regarding these materials are related to immunorejection, chronic inflammatory responses, fibrous scarring, and the associated problems of neural compression and need for re-operation.

These render nondegradable materials unsuitable for CNS repair (Cullen et al., 2008).

**2.1. Current fabrication of nanofiber-based bioscaffolds used in CNS regeneration**

The aim of biomedical engineering is the design and development of novel biomaterials that can recapitulate the key characteristics of natural ECM with the associated topographical cues, cellular adhesion sites, biochemical signals and physiological viscoelastic modules. In general, extracellular proteins such as collagen, fibrin and glycosaminoglycans, possess fibrous structures with diameters on the nanometer or sub-micrometer scales. Several bioengineering approaches have been developed for the fabrication of artificial nanofibre constructs with

Many biological macromolecules such as like phospholipids can readily self-assemble to form highly ordered bio-structures through van der Waals forces, hydrogen bonds, ionic bonds, and

**2. Nanofibrous scaffolds applied in CNS regeneration**

*1.4.2.2. Nondegradable materials*

188 Advances in Nanofibers

diameters that range from 10 to 100nm.

*2.1.1. Self-assembling peptide nanofiber scaffolds (SAPNS)*

Electrospinning was a traditional industrial fabrication technique used widely in 1930s. Due to its effectiveness of producing microfibers with diameters of sub-micron or nanometer scale, it has been utilized to process a number of natural and synthetic polymers such as collagen, fibroin, PLLA, PLGA, and PCL. The electrospun nanofiber matrices resemble the structural morphology of ECM with a high surface area–to-volume ratio, which has been shown to greatly facilitate cellular attachment, proliferation and differentiation. Recent studies continue to report the development of novel electrospun nanofibers, and the introduction of bioactive molecules such as growth factors during the fabrication process of nanofibers for peripheral nerve regeneration (Prabhakaran et al., 2008). The intrinsic limitations of this technique include the degradation of bioactive factors during procedure, the inability to fabricate complex 3D structures or specific microstructure with designed internal pore size.

#### *2.1.3. Phase separation*

Phase separation, or thermally induced liquid–liquid phase separation, was developed by Ma and Zhang to produce a nanofibrous foam materials. Polymer scaffolds generated by phase separation normally have a sponge-like porous morphology with spherical pores 50–500 nm in diameter. Phase separation system consists of a polymer-rich component and a polymerlean/solvent-rich component, by which the polymer morphology can solidify by quenching under low temperature. A few nanofibre scaffold from artificial biodegradable polymers have been produced with phase separation including PLLA, which was studied as a suitable matrices in which NSCs can grow and differentiate (Yang et al., 2004). Compared with other techniques previously discussed phase separation has simpler and the need for specialized equipment is minimal. However, due to the small number of candidate polymers suitable for *in vivo* study, reports on nanofiber scaffold generated by phase separation in neural tissue engineering are limited.

#### **2.2. Current application of SAPNS for the repair of injured CNS**

#### *2.2.1. Traumatic brain injury*

The applications of RADA16-I, a representative SAPNS, in 3D cell culturing, wound hemo‐ stasis and healing has been well described in a series of studies. The initial research of RADA16- I in experimental TBI was conducted by Ellis-Behnke et al. (Ellis-Behnke et al. 2006). Using an acute TBI model in which the midbrain of P2 hamsters was injured surgically with a knife wound (1.5 mm deep and 2.0 mm wide), 10µl of 1% SAPNS was applied to bridge the injury gap. The central traumatic lesion showed restoration in all SAPNS-treated animal subjects within the first 24-hour and at all other timepoints up to 30 days post-injury. Compared with saline-treatment, SAPNS created a seamless connection across lesion site and appropriate hostscaffold interfaces which led to significantly improved repair. Further studies were conducted by Guo et al. using a rodent TBI model (Guo et al., 2009). Immediately after the infliction of severe mechanical injury to the sensory-motor cortex, 20 µL of 1% RADA16-I SAPNS was implanted at the lesion sites to bridge the injury gap. Histological, immunohistochemical and apoptosis studies were performed at 2 days, 2 weeks, and 6 weeks after injury. The SAPNStreated lesion sites had no cyst formation after injury and showed integrated host-scaffold interfaces; saline-treatment resulted in significant cyst formation. Moreover, SAPNS signifi‐ cantly reduced apoptosis in the perilesional area and effectively mitigated reactive gliosis and inflammation. Currently, a few integrative strategies of SAPNS incorporated with bioactive factors have been conducted with an aim to improve functional recovery after severe TBI.

progenitor cells *in vitro,* and enhance neural regeneration *in vivo*. With the aid of phage display technology, a functionalized SAPNS was found to demonstrate high *in vitro* stem cell viability and neural differentiation, as well as significantly promoted axonal regrowth and locomotor

Use of Self-Assembly Nanofibre Biomaterials for Neural Repair After Injury

http://dx.doi.org/10.5772/57098

191

Collectively, these in vivo studies with various SCI models evidenced the significant potential of SAPNS in the repair of SCI from different aspects that SAPNS provide. Based on the current advances of fabrication and biochemistry techniques, future directions would consider introduction of updated topographical cues and more bioactive motifs or growth factors into the scaffold design to induce more robust and organized regeneration for injured central neural

A variety of biodegradable materials have been processed into nanofibrous scaffold using eletrospining technique for PNI repair (Xie et al., 2010). In an early study, a bilayer chitosan conduit with inner layer of nano/microfibrous structure modified with oligopeptide was generated to repair a 15mm sciatic nerve gap in rats (Wang et al., 2008). This novel integrative chitosan conduit effectively promoted the axonal regeneration that was comparable to that of autologous nerve grafting? on histological assessment. Recently, a blend of biodegradable polymers PLGA/PCL was used to produce electrospun tubes to bridge a 10mm long sciatic nerve lesion gap in rat. Four months after surgery, most of the electrospin conduit-treated animals showed neural regeneration and functional restora‐ tion on immunohistochemial studies and electrophysiological assessment (Panseri et al., 2008). More interestingly, a novel bi-layer nanofibrous nerve conduit made of poly (Llactide-co-caprolactone) and poly(propylene glycol) has been fabricated with electrospin‐ ning technique for PNI repair. The electrospin nerve conduit was designed as the luminal layer composed of longitudinally aligned nanofibers to promote axon regeneration, while the outer layer was equipped with random- organized nanofibers for mechanical support. After being implanted to bridge a 10mm gap of sciatic nerve, the nanofibrous nerve conduit significantly improved the regeneration of injured peripheral axons and motor functional recovery at 2 and 12 month post-surgery (Zhu et al., 2011). More recently, the effects of fibre diameter of electrospun conduits on peripheral nerve regeneration was analyzed with a 15mm sciatic nerve injury model. These fibrous conduits consisted of aligned electro‐ spun poly (ε-caprolactone) (PCL) nanofibers (251±32 nm) and microfibers (981±83 nm). The nanofiber-treated group showed significantly greater total number of myelinated fibers and thicker myelin sheaths when compared with groups that received Microfiber and Film conduits at 3 month post-treatment. The number of regenerated dorsal root ganglion neurons in animals that received nanofiber conduits was increased significantly by retrograde labeling with fluorogold. On electrophysiological testing including compound

functional recovery in acute spinal cord injury (Gelain et al., 2006; 2012).

**3.1. Development of nanofiber biomaterials used for PNS regeneration**

system, achieving more significant functional recovery.

**3. Nanofibrous scaffolds applied in PNS repair**

#### *2.2.2. Spinal cord injury*

A variety of biodegradable hydrogel have been extensively studied in the treatment of experimental SCI. IKVAV peptide amphiphile, which consists of neuroactive pentapeptide epitope from laminin, has been applied in a moderate spinal cord contusion model in which 1% aqueous solution of IKVAV-SAPNS was injected into the lesion sites 24h after injury. IKVAV-SAPNS significantly reduced the degree of oligodendroglial apoptosis perilesion and enhanced their survival rate with cleaved caspase-3 immunohistochemistry at 10 d after SCI. Additionally, astrogliosis was reduced significantly and the regeneration of motor-sensory axons were improved remarkably on BDA-labeling 11 week after treatment. Moreover, at 9wks after treatment, the mean locomotor score of IKVAV-SAPNS group was significantly better than that of the control group on BBB score measurement, and dorsal stepping was observed with IKVAV-treatment, indicating functional return in hindlimb movement (Tysseling-Mattiace, 2008).

One of the most important advantages of SAPNS is its ability to provide a 3-D matrice in which neural cells can survive and differentiate. Guo and coworkers incorporated neural stem cells (NSCs) and Schwann cells in SAPNS, and transplanted them into dorsal column lesion of the cervical spinal cord. At 6 wks after implantation, there was excellent integration between the implant and the host tissue. Moreover, extensive axonal regrowth was observed with immu‐ nohistochemistry staining with NF, 5-HT, and CGRP (Guo et al., 2007). Recent reports highlighted the further applications of controlled release of bioactive factors incorporated into the SAPNS *in vitro* and *in vivo*. For instance, CT04, a cell permeable RhoA inhibitor, was incorporated into RADA16-I-SAPNS and implanted in a complete transection lesion at T9 level of the spinal cord. This novel integrative SAPNS not only reconstructed the injured nerve gap, but also elicited significant axonal regeneration and motor functional recovery. Additionally it also effectively reduced the infiltration and apoptosis of activated macrophages within the injured spinal cord. The SAPNS-based delivery of RhoA inhibitor is a potentially effective therapeutic strategy by reknitting lesion gap, attenuating secondary injury and improving axonal regeneration (Fig.1). Moreover, Gelain and Zhang's group has developed functional‐ ized SAPNS that can improve the engraftment and neural differentiation of seeding neural progenitor cells *in vitro,* and enhance neural regeneration *in vivo*. With the aid of phage display technology, a functionalized SAPNS was found to demonstrate high *in vitro* stem cell viability and neural differentiation, as well as significantly promoted axonal regrowth and locomotor functional recovery in acute spinal cord injury (Gelain et al., 2006; 2012).

Collectively, these in vivo studies with various SCI models evidenced the significant potential of SAPNS in the repair of SCI from different aspects that SAPNS provide. Based on the current advances of fabrication and biochemistry techniques, future directions would consider introduction of updated topographical cues and more bioactive motifs or growth factors into the scaffold design to induce more robust and organized regeneration for injured central neural system, achieving more significant functional recovery.
