Biomaterials and scaffolds for the treatment of spinal cord injury

Biomaterials and scaffolds for the treatment of spinal cord injury

6

Xiaoxiao Wen, Saijilafu, Zongping Luo, Huilin Yang, Weihua Wang and Lei Yang Orthopaedic Institute, Soochow University, Suzhou, P.R. China

6.1

Introduction

Spinal cord injury (SCI) is a severe traumatic event in the central nervous system, which usually leads to motor and sensory loss, together with the impairments of thermoregulation, urinary bladder, and bowel function, negatively impacting the quality of life and life expectancy of the person involved [1]. Traffic accidents, falls, violence, and sports injuries are major causes of SCI. According to the World Health Organization, each year there are about 250,000 500,000 people who suffer from SCI [2]. In the U.S., the annual incidence of SCI is estimated to be 54 cases per million people [3]. A study showed the mean costs per person in the U.S. were $523,098 in the first year and $79,759 in each subsequent year for the patients suffering from SCI [4]. The pathophysiology of SCI can be divided into primary and secondary injury. The primary injury arises from blunt impact, compression, or penetrating trauma, which leads to contusion of the tissue, inflammatory response, and cellular and axonal damage instantaneously [5]. This damage can induce secondary injuries, which can spread to the surrounding tissues and eventually cause the formation of cavities and glial scars [6]. When cavities are formed, the growth of axons will be hindered by such physical gaps. To reconstruct the damaged tissue, it is essential to use biological entities, biomaterials, or scaffolds to restore the continuity of the spinal cord across the injury region and to stimulate tissue regeneration [7]. The future expectation will be that people with great damage to the spinal cord without residual function would replace a segment of cord with proper scaffolds or artificial tissue to facilitate cord repair or regeneration [8]. In the past few decades, increasing numbers of tissue engineering scaffolds based on advanced biomaterials and new technologies like nanotechnology and stem cell therapy have been developed for the repair of SCI. These novel scaffolds have several advantages for the promotion of neural regeneration and functional recovery compared with conventional strategies. These scaffolds are devised to mimic the natural structures of neural extracellular matrix (ECM) or neural tissue itself and therefore influence the growth, differentiation, and proliferation of neural Biomaterials in Translational Medicine. DOI: https://doi.org/10.1016/B978-0-12-813477-1.00006-2 © 2019 Elsevier Inc. All rights reserved.

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cells [9]. For example, scaffolds composed of biomaterials with unique nano- or microscale structural features can interact with biological systems or with substances at the molecular or cellular level. It is also expected that novel biomaterials have the ability to direct the behavior of neural cells, stimulate axon regeneration, as well as restore synaptic connections [10]. In addition, biomaterials and novel scaffolds can be further combined with drug or growth factors to provide specific advantages for neuroprotection or neuroregeneration. In this chapter, we will discuss these new advances of biomaterial scaffolds for SCI treatment, especially those benefiting from nanotechnology and advanced biomaterials.

6.2

Electrospun scaffolds

Electrospinning is a simple, rapid, and flexible technique capable of fabricating nanofibrous scaffolds. Ultrafine fibers are generated by applying a high-voltage electric field to polymer solution or melt coming out from the tip of a needle, and then deposited on a grounded collector [11]. By adjusting parameters, including polymer concentration, solution viscosity and conductivity, applied voltage, the spinneret-to-collector distance, and humidity, fine fibers with different diameters and architectures can be produced. A variety of synthetic and natural biomaterials can be used to fabricate scaffolds by electrospinning, such as poly(D,L-lactide-coglycolide) (PLGA) [12 14], poly(ε-caprolactone) (PCL) [15 17], poly(propylene carbonate) (PPC) [18], poly(hydroxybutyrate) (PHB) [19], collagen [20 22], chitosan [23,24], and silk fibroin [25 27]. Nano- and microfibers with different strengths, porosities, and surface properties can be obtained by using a variety of materials [28]. To fabricate electrospun scaffolds with aligned orientations, different types of collectors have been used to direct the distribution of the electric field, such as a rotating metal cylinder, frame collector, thin wheel with sharp edge, and auxiliary electrodes separated by insulating gap. In neural tissue engineering applications, electrospun scaffolds have the ability to mimic neural ECM by adjusting their fibrous structure. In addition, neurotrophic factors can be incorporated into the scaffolds during the electrospinning process and then delivered to the site of injury. Recently, a variety of electrospun scaffolds have been used to treat injuries in the peripheral nervous system and have achieved success to different extents [29 32]. Based on such progresses, researchers have developed electrospun scaffolds as an alternative treatment for SCI, as summarized in Table 6.1. Besides, due to the physiological complexity of the spinal cord, several design strategies of biomaterials and scaffolds have also been developed, which are reviewed as follows.

6.2.1 Electrospun scaffolds with aligned structures Electrospun scaffolds with aligned structure have been routinely developed to direct the extension of neural processes, using neural cells such as Schwann cells (SCs) and neurons. In pioneering experiments by Corey et al., embryonic rat dorsal root

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Electrospun scaffolds for SCI tested by in vivo spinal injury models

Table 6.1

Materials

Combined molecules

Model

Outcome

References

PCL/ PLGA

Self-assembling peptide /BDNF/CNTF/ VEGF/ChABC

Contusion (chronic)

[33]

PLA

Null

Complete transection

PPC

dbcAMP

Hemitransection

PLGA

Null

Complete transection

PPC

Chitosan microspheres/ ChABC Collagen hydrogel/ NT-3 NGF/ChABC

Hemitransection

Cord reconstruction and new tissue comprising neural and stromal cells formed Infiltration of host tissue into scaffolds, and axonal regeneration Axonal regeneration, motor functional recovery, and glial scar reduction Axonal regeneration and motor and sensory recovery Axon sprouting and functional recovery Extension of axons along with fiber direction Significant functional recovery, robust cellular infiltration, and vascular network formation Extension of astrocyte processes with accompanying descending axon regeneration

[37]

PCLEEP

PDS

PVDFTrFE

SC

Hemitransection

Complete transection

Complete transection

[34]

[18]

[35]

[36]

[38]

[39]

Abbreviations: SCI, spinal cord injury; PCL, poly (ε-caprolactone); PLGA, poly (D,L-lactide-co-glycolide); BDNF, brain derived neurotrophic factor; CNTF, ciliary neurotrophic factor; VEGF, vascular endothelial growth factor; ChABC, chondroitinase ABC; PLA, poly(L-lactic acid); PPC, poly propylene carbonate; dbcAMP, dibutyryl cyclic adenosine monophosphate; NT-3, neurotrophin-3; NGF, nerve growth factor; PVDF-TrFE, polyvinylidene fluoride trifluoroethylene; SC, Schwann cell.

ganglion (DRG) was cultured on aligned PLLA electrospun fibers. Compared with the DRG cultured on randomly organized fibers, the cell body of DRG cultured on aligned fibers were more elongated, with longer neurites extending along the fiber orientation [40]. Many other studies using different types of cells reported similar results. Cells cultured on aligned fibers also displayed a directional growth pattern along the fiber orientation [41 43]. In addition, the cells can extend longer

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processes along the aligned fibers compared with cell processes extension on randomly oriented fibers. One of the potential reasons behind this phenomenon was that there were not many barriers in the aligned fibers compared with randomly oriented fibers [44]. Electrospun scaffolds with aligned structure have also been used in numerous studies to direct the regenerating nerve fibers and to further promote the axonal regeneration and functional recovery. In the study of Hurtado et al., the importance of aligned nanofiber scaffolds for the treatment of acute SCI was investigated in a complete transection rat model [34]. In this study, aligned or randomly-oriented electrospun fibers were placed in a conduit structure to bridge the defect in spinal cord. After 4 weeks, the aligned PLLA fibers were demonstrated to have the greatest extension of pioneering axons, as shown in Fig. 6.1. The result was significantly

Figure 6.1 Electrospun PLA scaffolds with aligned structure promote extensive axonal regeneration. Notes: Scanning electron micrographs of randomly-oriented (A) and aligned (B) PLA fibers. (C) Coronal view of the aligned PLA scaffolds. Neurofilament immunostaining of representative spinal cord sections for random (D) and aligned (E) PLA scaffolds after 4 weeks of implantation. (F) Quantification of the average axonal regeneration at all time points. (G) Percentage of the animals with regenerated axons at 4 weeks. 100% of the animals implanted with aligned PLA scaffolds had long distance axonal regeneration (1.5 mm). Reprinted with permission from [34]. Abbreviations: PLA, poly(L-lactic acid).

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different from the extension of axons in rats implanted with scaffolds fabricated by randomly-oriented scaffolds or PLLA films. In another study, Colello et al. produced cylindrical electrospun scaffolds which matched the shape of the cord [38]. The examination of the scaffolds post-implantation proved that aligned electrospun fibers induced more robust cellular infiltration compared with the randomly-oriented electrospun fibers. These results demonstrated that the electrospun scaffolds with aligned structure have the ability to provide not only directional guidance cues, but also trophic support to promote the regeneration of axons after SCI.

6.2.2 Establishing 3D fibrous guidance channels To date, most electrospun scaffolds have been fabricated into hollow cylindrical conduits for application in neural tissue engineering. However, inappropriate target reinnervation may occur after the implantation of hollow conduits, due to the regenerating axons being randomly dispersed in the lumen or axons originating from the same neuron connecting to different targets [45]. In order to solve the problem, researchers began to establish three-dimensional (3D) fibrous guidance channels. Liu et al. prepared spiral shaped scaffolds by rolling electrospun collagen mats into tubes of four to five layers [46]. The scaffolds were used to treat acute SCI in a rat hemisection model. 30 days after implantation, the aligned electrospun scaffolds appeared more structurally intact. The result of neurofilament staining demonstrated that there were neural fibers sprouting as early as 10 days after implantation. Astrocytes were only observed at the boundary of the lesion site. Moreover, in the implantation area, no astrocyte aggregations were found at any time points. Zamani et al. fabricated a 3D electrospun nanofiber scaffold using the method combined with a water vortex and a two nozzle system [35]. The core of the scaffolds was composed of microstrands of aligned fibers and the sheath of the scaffolds was composed of dense fibrous mat with aligned structure covering the inner part, as shown in Fig. 6.2. The neural cells can adhere and proliferate on the outer layer of

Figure 6.2 (A) Scanning electron micrographs of 3D PLGA scaffolds. (B) Schematic representation of the PLGA scaffolds with nanorough sheath and aligned core consisting of nanofibers. Reprinted with permission from [35]. Abbreviations: 3D, three-dimensional; PLGA, poly(lactic-co-glycolic acid).

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the scaffolds and then penetrate into the inner layer of the scaffolds, growing along the fiber orientation.

6.2.3 Incorporation of bioactive components Bioactive factors can also be incorporated into fibrous scaffolds as biochemical and topographical cues by the electrospinning method, in order to promote the neurite extension and axon regeneration. During the process of electrospinning, ECM proteins, neuroactive peptides, and other growth factors can be directly added into the spinning solution or incorporated in the scaffolds by coaxial electrospinning, immobilization, and adsorption techniques. In 2005, Chew et al. first incorporated a therapeutic agent into electrospun scaffolds for neural tissue engineering application [47]. The nerve growth factor (NGF) was added in the spinning solution composed of PCL and a copolymer of ε-caprolactone and ethyl ethylene phosphate (PCLEEP). Downing et al. used the PLLA electrospun scaffolds for drug release in a rat hemisection model of SCI [48]. The small organic molecules Rolipram were delivered by the scaffolds. The result of the study revealed that the rats implanted with electrospun scaffolds incorporated with Rolipram had better functional recovery following SCI. In the study of Liu et al., neurotrophin-3 (NT3) and chondroitinase ABC (ChABC) were incorporated into electrospun collagen scaffolds to provide topographical and multiple biochemical cues to overcome the inhibition for axon growth after SCI and to promote the regeneration of axons [49]. Compared with bolus delivery of NT3, NT3 delivered by the electrospun collagen scaffolds were helpful for the neurite outgrowth for a longer time period. Xia et al. prepared PCL electrospun scaffolds as a vehicle for prolonged delivery of dibutyryl cyclic adenosine monophosphate in vivo [18]. The result of the study demonstrated that the scaffold can promote the recovery of motor function after SCI.

6.3

Self-assembling peptide scaffolds

Self-assembly is a process in which components of a disordered state autonomously form an organized structure without external stimulation. This is a very useful strategy to produce scaffolds with nanostructures for tissue regeneration [10]. Due to the unique biological and self-assembly characteristics of peptides, they have been widely studied as self-assembling blocks. Self-assembling peptides (SAPs) are usually composed of hydrophilic heads combined with hydrophobic alkylic tails or alternate repeating hydrophilic and hydrophobic amino acids. Self-assembly of peptides is usually triggered by the changes of pH or temperature or the presence of electrolyte ions. Through a number of noncovalent interactions, such as ionic bonds, van der Waals’ bonds, hydrogen bonds, and hydrophobic interactions, nanofiber scaffolds with well-defined structure and stable organizations were formed by intermolecular self-assembly [50]. The microstructure of the SAP scaffolds looks like

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mats of nanofibers, while the macrostructure usually looks like hydrogel [51]. In biomedical applications, the SAP nanofiber scaffolds reveal great potential since natural ECM components are essentially the self-assembly of cell-derived materials. For the past few years, SAP scaffolds have been widely studied as an alternative treatment for the repair of SCI and have showed much success. The materials made of SAP are usually biocompatible and do not cause significant immunologic reactions as some natural materials. In addition, during the polymerization process of SAP, the chemical cross-linking agents that are often harmful to the cells and tissues are not necessary. The degradation products of the SAP scaffolds can be metabolized by cells as they are natural amino acids. By adjusting the initial concentration of peptides in solution, SAP scaffolds with different fiber density can be achieved [52]. The SAP scaffolds can be readily applied for the repair of SCI due to the good biocompatibility, versatility, and customization. They can be injected to the injured site and fill the cavity in the spinal cord, regardless of the size and shape. In addition, after the gelation of the peptides in solution, the SAP scaffolds can integrate with the host tissues. Several studies have shown that the SAP scaffolds have the ability to promote the differentiation of neural stem cells (NSCs) or neural precursor cells into neurons [53]. For these reasons, many types of SAP scaffolds for SCI repair have been developed and evaluated in vivo, as listed in Table 6.2. Due to the alternating positive and negative charges on the residues, the ionic self-complimentary peptides have also been studied and applied. Different SAP variants, such as KLD12, RADA16-I, LDLK12, have been designed and studied extensively. Similarly to the sequence RGD in laminin, fibronectin, and collagen, RADA16-I has a sequence RAD that can promote cell adhesion [61]. In the work reported by Guo et al., the RADA16-I scaffold was implanted into the spinal cord lesion cavity of rat [54]. Six weeks after treatment, a large number of cells migrated from host tissues to the transplant area. In addition, the RADA16-I scaffolds could also support regeneration of axons and blood vessels, and reduce inflammation of the lesion site and surrounding tissues. SCs and neural progenitor cells (NPCs) were combined with the RADA16-I scaffold to facilitate the repair of spinal cord. The morphological evidence supported that NPCs and SCs could survive in the scaffold, and some of the cells migrated to the host tissues. Some of the SCs were observed to have a matured phenotype similar to the cells in myelin, and expressed characteristic myelin basic protein. Some of the NPCs differentiated into neurons, oligodendrocytes, and astrocytes. However, a drawback of RADA16-I peptides is that the low pH value of RADA16-I must be neutralized before implantation, otherwise the host tissue will be damaged [52]. In order to resolve the problem, researchers have introduced the complementary co-assembly peptides by mixing two self-repulsive complementary charged peptides, so as to eliminate the shifts of pH, ionic strength, or temperature in the gelation process, which is more likely to preserve the native conformation of biomolecules in the hydrogel [62]. Raspa et al. prepared the co-assembly of LDLD12/LKLK12 peptides, which were combined with PCL/PLGA microtubes and showed the ability to guide the development of axon and synapse [56].

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Table 6.2

Biomaterials in Translational Medicine

Self-assembling peptide scaffolds for SCI

Sequence/abbreviation

Additional components

Outcome

References

RAD16-1

SC and NPC

[54]

RADA16-1-BMHP1

Null

RADA16-1-BMHP1

Electropsun PCL/PLGA scaffolds Electropsun PCL/PLGA scaffolds

Axonal regeneration in the scaffold Basement membrane deposition and axon regeneration Cord reconstruction, new tissue comprising neural and stromal cells formed Cyst area reduction, axonal regeneration into the injury site and vascularized scaffold implant Axonal regeneration and reduction of glial scarring and inflammation Increased differentiation of NPCs to neurons and oligodendrocytes, reduced astrogliosis and chondroitin sulfate proteoglycan deposition Axon elongation and glial scar attenuation Axon regeneration and myelination around the cavity

LKLK12/LDLD12

QL6

NPC

QL6

NPC

Peptide amphiphileIKVAV (RADA)4-GG-BMHP1

null hEnSC

[55]

[33]

[56]

[57]

[58]

[59] [60]

Abbreviations: SC, Schwann cell; NPC, neural precursor cell; PCL, poly ε-caprolactone; PLGA, poly D,L-lactide-coglycolide; hEnSC, human endometrial stem cells.

Another type of SAP, multidomain peptide (MDP), composed of A-B-A blocks, has also been studied. In the MDP, the A block consists of charged amino acids and is used to mediate the assembly of the central B block [63], which is composed of alternative hydrophobic and hydrophilic amino acids, forming a hydrophobic A-B-A sandwich in a β-sheet conformation by hiding the hydrophobic residues from water [64]. Dong et al. introduced a typical MDP, K2(QL)6K2 (QL6), which could dissolve in water at the pH of 7.4 [65]. QL6 showed a β-sheet conformation and a nanofibrous structure with a uniform diameter and controlled lengths. There were no amorphous aggregates in the QL6. In the work of Liu et al., QL6 was injected into the injury site in a model of SCI [66]. The results showed that the QL6 helped to reduce the appearance of glial scar, facilitate neurological recovery, and mitigate inflammation (Fig. 6.3). The researchers also demonstrated that QL6 prevented the

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Figure 6.3 Self-assembling peptides QL6 promote the neural repair after clip-compression injury of the rat spinal cord.

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L

breakdown of tissues around the injured site and relieved the pathophysiology of secondary injury. It was observed that the QL6 reduced the inflammation and astrogliosis 8 weeks after transplantion. In addition, QL6 improved axonal conduction, showing functional recovery of locomotion. In a recent work of Zweckberger et al., QL6 and NPCs were injected into the spinal cord of rat at 1 day and 14 days after SCI, respectively [58]. The number of surviving cells and differentiated cells were significantly increased, while scar tissue and astrogliosis were reduced. Meanwhile, there was more tissue preserved around the injured site in the rat treated with QL6. The results also demonstrated that QL6 could mitigate the pathophysiology of secondary injury and improve neuroregeneration. Although many SAP nanofiber scaffolds can promote cell adhesion and some minor functions, other desirable functions have to be induced by bioactive motifs, which are usually designed to be as small as possible so that they do not adversely affect the assembly process. A number of bioactive motifs have been derived from functional proteins, which are beneficial for synaptogenesis, reduction of neuroinflammation, regulating cell viability, and regeneration of spinal cord [61]. IKVAV is one of the most studied motifs derived from laminin, which is capable of promoting and directing the growth of neurite [67]. Zhang et al. designed RADA16-IKVAV scaffolds by inducing the functional motif IKVAV at the C-termini of RADA16 [68]. Compared with RADA16 scaffolds, the RADA16IKVAV scaffolds can significantly improve cell proliferation, facilitate the migration of cells to scaffolds, and promote the differentiation of NSCs into neurons. IKVAV has also been combined with peptide amphiphiles (PAs) in several in vivo applications for neural tissue regeneration. The PAs sequences have an alkyl tail and a peptide head, and the head group is increasingly hydrophilic away from the tail. The self-assembly of nanofibers is the process of separating the hydrophobic alkyl chains from the aqueous solution [62]. This allows any bioactive motifs connected to the alkyl chains outwards, so as to promote the hydrophilicity and potential cell interaction of the scaffolds. Tysseling et al. showed that the injection of IKVAV-PA could encourage functional recovery and the elongation of axon in compression and contusion models of SCI, respectively [59,69]. IKVAV-PA could inhibit astrogliosis while promoting remyelination of axons, and does not affect the hypertrophic changes of astrocytes, which are necessary to repair the blood brain barrier and restore homeostasis. In addition, the injectable IKVAV-PA can increase the serotonergic fibers caudal to the lesion significantly, and improve the cell viability. Another type of peptide motif derived Notes: (A1) Transmission electron micrographs of QL6 peptide in water. (A2) Scanning electron micrographs of QL6 scaffolds (1% w/v) in PBS at 2 h. (B) Schematic showing the procedure of the intraspinal microinjection. A total of 10 μL QL6 were injected bilaterally rostral and caudal to the injury site. (C) LFB-HE staining of representative spinal cord sections from animals treated with QL6 and saline control. (D) Percent cross-sectional area of residual tissue at the injury site. Animals treated with QL6 exhibited significantly increased residual tissues. Reprinted with permission from [66]. Abbreviations: PBS, phosphate buffer saline; LFB, luxol fast blue; HE, hematoxylin-eosin.

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from bone marrow homing peptides, PFSSTKT (BMHP1), has been used to promote the viability and differentiation of NSCs [70]. Tavakol et al. encapsulated human endometrial-derived stromal cells (hEnSCs) by a sandwich method into the nanofiber scaffold (RADA)4-GG-BMHP1 [60]. The results showed that the scaffolds could induce the differentiation of hEnSCs, regeneration of axons, and myelination. Furthermore, there was improved motor neuron function with less inflammatory response in the rat SCI model after the treatment using the scaffolds.

6.4

Scaffolds based on carbon nanomaterials

With the advances of nanotechnology, a large number of nanomaterials have been employed for tissue repair. Conductive carbon nanomaterials were proposed as promising candidates for nerve stimulation and regeneration, due to their unique mechanical, electrical, and physicochemical properties. This section discusses the application of carbon nanomaterials-based scaffolds for the treatment of SCI. Currently, the most commonly used carbon nanomaterials are carbon nanotubes (CNTs) and graphene.

6.4.1 Carbon nanotubes CNT is the allotrope of carbon with a tubular structure, which is formed by chiral rolling of a sheet of carbon atoms arranged in a hexagonal array. The radius of the tube influences the properties of the CNTs. CNTs can be classified as single-walled nanotubes (SWNTs) and multiwalled nanotubes (MWNTs). SWNTs have a tubular structure rolled up by a single sheet of carbon, and MWNTs consist of multiple walls of carbon sheets [71]. CNTs can be prepared by many different methods, such as arc discharge, chemical vapor deposition, laser ablation, and catalysis cracking [72 74]. CNTs are reported to serve as scaffold material for neural tissue engineering due to their unique mechanical properties and high electrical conductivity. The affinity of neurons linked to the surface containing CNTs can be altered by adjusting the properties of CNT scaffolds including charge, chemistry, roughness, and polarity [75]. For example, Hu et al. proved that there were more growth cones and neurite branches of neurons on the surface of MWCNTs with positive charge compared to the MWCNTs that were negatively charged [76]. Several studies based on CNTs have been performed to restore the connections between neurons. In CNT scaffolds, the formation of synaptic contacts can be promoted with increasing efficacy of neural signal transmission. The study of Lovat et al. showed that CNTs increased the network activity of neuronal circuits and improve the cell adhesion [77]. In addition, the neurons on the surface of CNTs were associated with enhanced excitability, due to the increase in the discharge frequency of action potential. The unique electrical conductivity of the CNT scaffolds may be the potential cause of the

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specific electrophysiological effect, since no studies have proven there are chemical interaction between the CNTs and neurons [78]. However, it is still not so clear how the high conductivity of CNTs can affect the growth of neuron. There was a hypothesis that a direct electrical coupling exists between CNTs and neurons [79]. Besides, CNT scaffolds have been studied in more complex tissue models. In the study of Fabbro et al., organotypic spinal slices representing a biological model of segmental microcircuits were interfaced with CNT scaffolds for weeks [80]. The results showed that there were more neuronal fibers expanding from the spinal cord explants interfaced to CNT scaffolds. Furthermore, the CNT scaffolds can mediate neuronal signaling sensed in synaptic networks spatially far from the scaffolds’ interface. Recently, Usmani et al. cultured organotypic spinal explants on 3D CNT scaffolds, which were composed of 3D meshes of self-assembled, interconnected large MWCNTs [81]. The scaffolds guided the formation of neural webs, as the regrown neurite bundles formed a dense random net. More importantly, the 3D CNT scaffolds possessed an ideal morphology for the successful reconnection of segregated spinal explants (Fig. 6.4). Although considerable progress has been made in the biomedical research of CNTs, the CNT scaffolds are still limited in their clinical application due to unknown toxicity. One of the possible solutions is the incorporation of appropriate

Figure 6.4 3D CNT scaffolds direct the reconnection of segregated organotypic spinal explants. Notes: (A) Scanning electron micrographs of the CNT scaffolds (left) and 3D reconstruction of the scaffolds (right) from confocal image stacks. (B) β-tubulin III (green), neurofilament H (red) and DAPI (blue) staining of the spinal slices cultured in plasma gel control and 3D CNT scaffolds for 14 days. Reprinted with permission from [81]. Abbreviations: 3D, three-dimensional; CNT, carbon nanotube.

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chemical or biological functionalities onto the CNT scaffolds to modify the properties of CNTs. As an example of functionalization, covalently decorating the surface of CNTs by hydrophilic polymers, such as poly(ethylene glycol) (PEG), can increase the solubility and biocompatibility of CNT scaffolds. In addition to simple surface modifications using polymer additives, CNTs can also be biofunctionalized with biological and bioactive molecules. During the processes of biofunctionalization, bioactive molecules such as peptides, proteins, targeting ligands, and growth factors, can be conjugated to CNTs to improve the biocompatibility of CNTs scaffolds and the functionality of the neural cells. In the study of Gaillard et al., the surfaces of MWCNTs were coated with two types of cell-adhesion-promoting peptides, respectively [82]. Results demonstrated that MWNTs functionalized with GRGDSPC or IKVAVC peptide sequences did not affect neuronal morphology, viability, and basic function. Since the application of CNT scaffolds for SCI is still at a nascent stage, most of the in vivo studies have used functionalized CNTs-based scaffolds. Roman et al. tested SWNTs functionalized with PEG (SWNT-PEG) in a transection model of SCI [83]. The SWNT-PEG was injected into the injury site 1 week after the spinal cord transection. The lesion volume was decreased while the neurofilament-positive fibers and corticospinal tract fibers were increased in the SWNT-PEG treatment group. Additionally, the improvement in hindlimb locomotor recovery was observed. Another study using CNTs functionalized with the Nafion nanocomposite at 1 week post-SCI revealed similar results, in which increased neurofilamentpositive fibers and corticospinal tract fibers and decreased lesion volume without increase in reactive gliosis were observed after the administration of the CNTs/ Nafion [84]. Recently, Sang et al. found that the hydrogel using copolymerization of n-isopropylacrylamide and SWNTs could promote the regeneration of injured spinal nerve endings and reduce the formation of scar tissue in a 1 mm3 cavity of SCI model [85].

6.4.2 Graphene Graphene is a basic building block for graphitic materials and consists of sp2bonded carbon atoms arranged in a two-dimensional hexagonal lattice. Because of its unique structure and geometry, graphene has extraordinary physical and chemical properties, such as high Young’s modulus, high intrinsic mobility, large theoretical specific surface area, and ultra-high thermal and electrical conductivity [86]. For the past few years, graphene has been widely studied in the field of electronics and chemistry, and has also attracted much attention for potential biomedical applications [87]. Recently, the derivatives of graphene such as graphene oxide (GO) and partially reduced graphene (rGO) have also been considered promising materials for potential applications in regenerative medicine. These derivatives not only retain some of the properties of graphene, such as mechanical ductility and stiffness, but also have other interesting features including higher hydrophilicity and versatility for surface modification with biological molecules [88]. However, there are biocompatibility concerns about the application of graphene, especially the use

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of granular graphene nanoparticles, in biomedical applications. Existing investigations indicate that graphene and its derivatives exhibit a dose-dependent toxicity. Nevertheless, the published results also demonstrate that the physicochemical properties of graphene and its derivatives such as the size, surface charge, and structural defect influence their toxicity in biological systems [89]. In fact, graphene with low concentrations, ultrasmall sizes, and proper surface modification usually displays favorable biocompatibility [90]. In the study reported by Tu et al., primary rat hippocampal neurons were cultured on chemically functionalized GO with different surface charges, and results showed that positively charged GO was more favorable for the extension of neurites and branching of neurons compared with negatively, neutral, or zwitterionic charged GO [91]. Since graphene has unique electroconductive properties, 2D structure, and chemical stability, there is great interest in the use of graphene and its derivatives as electroactive scaffolds to enhance neuroregeneration. In numerous studies, graphene-based scaffolds have showed the ability to promote the differentiation of stem cells specifically into the neuronal lineage, which is crucial for neural regeneration. Parker et al. reported that there was a more rapid human NSCs attachment onto graphene scaffolds compared with glass [92]. The neural differentiation of NSCs in a long-term ( . 2 weeks) culture was also enhanced on graphene scaffolds. A recent study compared the NSCs cultured on GO, ginseng-rGO and hydrazinerGO scaffolds, respectively, and it found that more NSCs differentiated into neurons rather than glial cells on the hydrazine-rGO and especially the ginseng-rGO scaffolds than those on the GO scaffolds after 3 weeks of culture [93]. Graphenederived scaffolds can also serve as a platform to support and direct neural growth. Bendali et al. cultured adult retinal ganglion cells directly on graphene substrates and found that the cells survived and grew neurites on graphene without any protein coating on the scaffold [94]. In a mouse hippocampal culture model, the number of neurites and the average neurite length of neurons were significantly increased after 2 7 days on graphene scaffolds compared with polystyrene substrates [95]. Based on the findings above, recently researchers have begun to investigate graphene and its derivatives as potential scaffolds for repairing SCI in vivo. In an early study, Lo´pez-Dolado et al. implanted 3D porous rGO scaffolds into hemisected rat spinal cord and then thoroughly investigated the early tissue responses in the spinal cord and other major organs at 10 days post-injury [88]. The results revealed the filler effect of the rGO scaffolds on facilitating the regaining of tissue integrity. The capacity of the scaffolds to prevent additional scars due to the establishment of a tissue interface and to prevent further damage was also demonstrated. In addition, the implantation of the rGO scaffolds did not induce local and systemic toxic responses. In a follow-up study, they investigated the chronic tissue responses to the rGO scaffolds at 30 days post-injury [96]. Injured animals without the rGO scaffolds showed more cavities and badly structured lesion zones than those implanted with rGO scaffolds. The results suggest the important role of the rGO scaffolds in injury sealing and stabilization. In addition, the rGO scaffolds promote axon growth (Fig. 6.5) and angiogenesis both around and inside the material. There were abundant new blood vessels inside the scaffolds where several regenerated axons were

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Figure 6.5 Regenerating axons grown inside the rGO scaffolds after 30 days of implantation. Notes: (A) Immunostaining of representative spinal cord section. Axon were stained positively with β-tubulin III (TUB, green) and neurofilaments (NF, white). Laminin (LAM) from basal membranes was stained in red. MERGE 1, TUB and NF; MERGE 2, TUB and LAM. (B) Bright field image of the spinal cord sections. The rGO scaffolds are visualized in the image. Reprinted with permission from [96]. Abbreviations: rGO, reduced graphene oxide.

found. In contrast, there were a decreased quantity of blood vessels at the lesion site where no regenerated axons were found in the experimental group without using scaffolds. In another study, graphene scaffolds combined with a hydrogel were implanted to bridge the lesion of Wistar rats after hemispinal cord transection [97]. Results showed that the graphene scaffolds adhered well to the spinal cord tissue. No area of pseudocyst was found around the scaffolds, suggesting that the scaffolds did not cause any toxic responses. The histological evaluation showed an ingrowth of SCs, neurofilaments, blood vessels, and connective tissue elements around the graphene scaffolds. The results summarized above give clear evidence of the usefulness of graphene scaffolds for spinal cord repair.

6.5

Scaffolds combined with nanoparticles

In spite of a large number of experimental studies on scaffolds, there are some limitations in the studies using a single material. In order to overcome the multiple hurdles of SCI, combined strategies which take advantage of different therapies can be employed to promote the regeneration and functional recovery of the injured spinal cord. The candidate strategies include controlled and sustained delivery of both biomolecular therapeutics and cells to regenerate damaged tissues and are promising for the regeneration of SCI [98]. One of the common and

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effective strategies is combining scaffolds with neuroprotective and neuroregenerative factors, which can reduce the loss of sensorimotor neurons and improve the neurite outgrowth via direct local influence or by overpowering the neuroinhibitory microenvironment. Nanoparticle-enabled drug delivery strategies for sustained and local delivery of bioactive molecules from scaffolds have shown great potential. Nanoparticles can serve as vehicles due to their capacity to pass biological barriers, to enter and diffuse within cells. Numerous nanoformulations have been developed for the sustained release of different types of neuroprotective or neuroregenerative factors, combined with scaffolds for SCI repair. For instance, biodegradable PLGA nanoparticles are commonly used to encapsulate a variety of biomolecular therapeutics and the drug-loaded PLGA nanoparticles are further dispersed in hydrogels to form drug delivery systems. One early study showed that the implantation of methylprednisolone-loaded PLGA nanoparticles embedded in agarose hydrogel to the site of contusion injury significantly reduced the early inflammation and lesion volume at 7 days after injury [99]. Donaghue et al. developed an injectable hyaluronan/methyl cellulose hydrogel combined with PLGA nanoparticles for the delivery of NT3 [100]. The study showed that the in vitro release of NT3 sustained for up to 50 days. The nanoparticle/hydrogel composite was injected into the intrathecal space in a clip-compression model of SCI to achieve acute local delivery of NT3 to the spinal cord. In comparison with the vehicle control of bull serum albumin (BSA) encapsulated in PLGA nanoparticles in hydrogel and the injury-only control group, the injection of the nanoparticle/ hydrogel for delivery of NT3 resulted in significant axon regeneration and promoted functional recovery without increased lesion size at 21 days post-injury. Moreover, vascular endothelial growth factor (VEGF) has also been encapsulated in PLGA nanoparticles to stimulate spinal cord regeneration. VEGF-loaded PLGA nanoparticles were mixed with alginate hydrogels supplemented with fibrinogen and injected into hemisected rat spinal cord [101]. Increased angiogenesis and neurite growth were observed after 4 weeks. The local delivery of VEGF improved the fiber growth into the lesion site. Although functional improvement was limited, proregenerative effects supporting spinal cord plasticity such as fiber growth into the lesion site were confirmed. In another study, PLGA nanoparticles were embedded in Pluronic F-127 hydrogels for sustained release of hirudin, a thrombin inhibitor [102]. The oligodendrocyte formation was promoted and the recovery in coordinated stepping was accelerated after direct injection of hirudin-loaded PLGA/ F-127 at the injury site in a model of spinal cord contusion-injury, demonstrating that the functional recovery from a demyelination lesion was achieved. In addition, biomolecule-loaded nanoparticles can also be incorporated to scaffolds with complex architectures to promote neural regeneration after SCI. In the study reported by Tuinstra, hydroxylapatite nanoparticles were complexed with lentivirus encoding for neurotrophic factors NT3 and brain-derived neurotrophic factor (BDNF) and incorporated into multiple-channel poly(lactide-co-glycolide) scaffolds before implantation into a hemisected rat spinal cord [103]. The scaffolds could provide physical support to stabilize the injury and more importantly, axonal

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growth was observed along the channels of the scaffolds, whereas the number of regenerating axons and the myelination as a function of time and location were clearly enhanced by the delivery of lentivirus encoding for NT3 and BDNF.

6.6

Summary

Treatment of SCI remains a significant challenge for both clinicians and scientists. Although cell therapy has been applied for the repair of SCI, this treatment using cells alone has achieved limited effect to date. Strategies of combining biomaterial scaffolds with bioactive molecules and/or cells appears to be more effective, especially for the injured spinal cord with large-sized physical gaps. Driven by the continued advances of tissue engineering, new strategies based on the design of biomaterial scaffolds have been developed for the repair of SCI. In this chapter we summarize the recent progress in biomaterial scaffolds for SCI treatment, especially those benefiting from nanotechnology and advanced biomaterials. Electrospun scaffolds and SAP scaffolds have revealed promising results both in vitro and in vivo. These scaffolds can mimic the natural structure of ECM of neural cells and influence the growth, differentiation, and proliferation of neural cells, and thus are able to promote functional recovery and nerve fibers extension through the injured site. Also, the scaffolds composed of nanomaterials which have the unique size features can interact with neural tissue at the molecular level. For example, CNTs and graphene are actively studied as structural and functional materials for SCI scaffolds due to their unique mechanical, electrical, and physicochemical properties. It is also worth mentioning that, although these scaffolds containing carbon nanomaterials demonstrate a promising ability to restore interconnections between neurons and to promote (NSCs) differentiation, the toxicity concerns currently hinder their applications in human. Moreover, scaffolds combined with nanocarriers to deliver drugs/growth factors have also demonstrated possibly better therapeutic effect for neuroprotection or neuroregeneration, as the sustained release of cargoes from the carriers in the scaffolds can serve as complex temporal biochemical cues to promote the axon regeneration. For further study in the near future, combined strategy is a possible direction. For example, SAP could be incorporated into the electrospun scaffolds, together with neural cells and/or neuroprotective and neurogenerative agents to better mimic the native microenvironment of neural ECM. Another example is that electroactive graphene could be blended with other natural components and fabricated into bioactive scaffolds to provide axonal guidance and attract migrating neuroblasts. However, as the combined strategy increases the complexity of the material system, proper or precise control of the variables are also highly desired for the construction of the scaffolds, ensuring they play appropriate roles in every step of the SCI repair process. In summary, the design and development of the scaffolds with optimal integration of directional, chemical, and structural cues are expected to pave a new road for the regeneration and repair of injured spinal cord.

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