15.1.1 Anatomy of the nervous system

The nervous system can be divided into the central and peripheral nervous systems (CNS and PNS respectively). The CNS is composed of the brain and the spinal cord which contains the vast majority of neural cell bodies. The PNS consists of all nervous tissue excluding the brain and spinal cord and is divided into four parts:

The PNS is made up of the axons of the afferent sensory and efferent motor neurons that run between the CNS and the rest of the body. Peripheral nerves contain both myelinated and unmyelinated fibres with three functional types. Sensory fibres receive information from the viscera, skin, muscle, tendon and joints. Motor fibres supply the end plates in skeletal muscle and autonomic fibres serve the blood vessels, viscera, sweat glands and arrector pilae muscles (Standring, 2008).

15.1.2 Structural layers

The neuronal tissue is covered by a number of layers, which protect and support the underlying structures. These structures enclose the neuronal and non-neuronal glial cells.

The outermost layer, the epineurium, is condensed loose connective tissue. It contains fibroblasts, collagen, fat, lymphatics and blood vessels – the vasa nervosum – which together provide strength, cushioning and nutrition for the deeper-lying structures. Within the epineurium are multiple fascicles, each surrounded by perineurium and in general, the greater the number of fascicles, the thicker the epineurium.

The perineurium runs from the CNS–PNS junction to the capsules of the muscle spindle and encapsulated sensory endings or opens at unencapsulated endings and neuromuscular junctions. It is composed of flattened perineurial cells alternating with collagen in sheets totalling between 15 and 20 layers. Each fascicle contains bundles of individual nerve fibres each surrounded by endoneurium. The endoneurium is made up of collagen fibres running parallel to the long axis of the axons that condense around individual axon-Schwann cell (SC) units and endoneurial vessels (Standring, 2008). Each peripheral nerve has an extensive blood supply composed of interconnecting epineurial, perineurial and endoneurial plexuses, which are linked with extrinsic regional vessels.

15.1.3 Neurons

The cell bodies of peripheral neurons are located in the ganglion, from where the neural processes, dendrites and axons, originate. The axon is a column of neuronal cytoplasm, or axoplasm, enclosed by a cell membrane (axolemma). Within the axon there is a complex system of axoplasmic reticulum, membranous cisterns, tubes and vesicles, mitochondria, lamellar and multi-vesicular bodies (Birch et al., 1998). Most important is the axonal cytoskeleton consisting of microtubules, neurofilaments and matrix, which provides the apparatus for axoplasmic transport (Hollenbeck, 1989).

The axon carries materials between the cell body and the distal end organs in two forms of transport, fast and slow. Fast axonal transport works in both directions at a rate of 40–200 mm/day. Neurotransmitters synthesised in the cell body are transported anterogradely to the distal end of the axon, while distally uptaken extracellular molecules, such as growth factors, are simultaneously transported retrogradely to the cell body. Slow transport occurs only in an anterograde direction, transporting cytoskeletal components from the cell body to the distal terminus. The rate of slow transport, 0.1–3 mm/day, is believed to correspond to that of peripheral regeneration rates following axotomy (Standring, 2008).

15.1.4 SCs

In the PNS, axons are closely associated with SCs. These wrap along the entire length of the larger axons, juxtaposing one another at the nodes of Ranvier and laying down spiral layers of myelin sheath. Each axon–SCs unit, or nerve fibre, is contained within a basal lamina. The smaller fibres are arranged in bundles surrounded by similar columns of SCs (Birch et al., 1998). Axon fibres greater than 2 μm in diameter are generally myelinated.

SCs develop from the neural crest and appear in mature nerves as two different phenotypes, myelinating and non-myelinating. Both types are derived from the same precursor cell (Jessen and Mirsky, 1999), and together they form a stable non-proliferating cell population. The developmental changes towards a myelinating phenotype are associated with alterations in gene expression and protein synthesis including the up-regulation of myelin proteins P0, myelin basic protein and peripheral myelin protein and down-regulation of neural cell adhesion molecule, p75^NTR and glial fibrillary acidic protein (GFAP). If a mature SC loses contact with axons, it undergoes radical changes in morphology and gene expression leading to developmental regression or de-differentiation of individual SCs and myelin breakdown, followed by proliferation.

SCs are a major source of neurotrophic factors. Höke et al. (2006) recently showed that mature SCs express either motor or sensory phenotypes. The motor SCs express significantly higher levels of vascular endothelial growth factor-1, insulin-like growth factor-1 (IGF-1) and pleiotrophin whereas sensory SCs express higher levels of brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and hepatocyte growth factor. Other molecules such as neurotrophic growth factor (NGF), IGF-2, fibroblast growth factor-2 and ciliary neurotrophic factor (CTNF) are expressed at similar levels in both cell types.

15.1.5 Extracellular matrix (ECM)

The cellular components of the PNS are supported by an extracellular matrix (ECM) comprising the SCs’ basal lamina and surrounding extracellular space. The ECM contains a diverse set of macromolecules including laminin-2, collagen types IV, VI and P200, tenascin-C, F-spondin, heparan sulphate and chondroitin sulphate proteoglycans, fibronectin and entactin. During peripheral nerve development, SCs synthesise and assemble basal lamina ECM and fibril-forming collagens (Chernousov et al., 1998). This synthesis is dependent upon axonal contact (Bunge et al., 1982). Following injury, many ECM molecules are important in promoting axonal growth and regeneration (Ard et al., 1987; Agius and Cochard, 1998). Thus, peripheral nerve integrity is maintained by the close coordination and complex interactions of both the cellular and extracellular components. It is this complexity that makes the tissue engineering assembly of peripheral nerve components so challenging.

15.3.1 The gold standard: autografting

Repair of a gap injury using an autologous nerve graft was not widely accepted until the 1970s with the advent of microsurgery (Millesi et al., 1976; Matejcik 2002) and to date it remains the gold standard in bridging a nerve gap (Birch et al., 1998; Battiston et al., 2005). A graft is commonly harvested from the sural nerve in the leg or the medial cutaneous nerve in the forearm of the patient, to provide a guidance conduit to the regrowing axons which interact with the basal lamina scaffold and endogenous SCs. As a natural, non-immunogenic, ready-to-use graft, this method has clear advantages, but functional outcome remains poor with only 50% of patients achieving successful outcomes (Pabari et al., 2010, Lee and Wolfe, 2000). The use of a sensory nerve graft can be limiting when used for pure motor or mixed nerve injuries (Nichols et al., 2004; Brenner et al., 2006) due to the morphometric mismatches in environments, axonal alignment, distribution and size (Nichols et al., 2004; Koh et al., 2010); motor axons typically range between 3 and 20 μm and sensory nerves between 0.2 and 15 μm (Kiernan, 1998). More recently, the differences between motor and sensory SC modalities has been noted (Moradzadeh et al., 2008) and if in the wrong environment, the regenerative ability of the graft is impacted. Also, for the patient, there is additional donor site morbidity, scarring, sensory loss and possible neuroma formation (Wu and Chiu, 1999; Evans, 2001). The use of autografts also has a critical distance of 5 cm between the nerve stumps, beyond which they have to be supplemented with allografts (Siemionow and Brzezicki, 2009). Technical modifications such as the vascularised nerve graft have failed to demonstrate significant improvement in outcome (Doi et al., 1992).

15.3.2 Allografting

Nerve allografting circumvents the problems caused by autogenous nerve harvest and has been used clinically in situations where autografting was not possible (Mackinnon et al., 2001). However, the necessity for immune suppression for up to 18 months following this procedure makes it difficult to justify in the majority of cases, where the risk of opportunistic infections and tumour formation is increased (Siemionow and Sonmez, 2007).

15.4.1 Regeneration in a hollow tube

Essentially the basic concept of a NGC is to use a hollow tube to connect the proximal and distal stumps. The process of regeneration through a hollow tube can be divided into five phases (Daly et al., 2012; Fig. 15.1):

In the fluid phase, there is influx of exudates from the proximal and distal stumps into the conduit. This is rich in neurotrophic factors and ECM precursor molecules which reach peak concentrations between 3 and 6 h after injury and repair. Following this over the next week acellular bands of fibrin, formed from the converted ECM precursor molecules, connect the proximal and distal stumps. In the second week, the ECM framework provides a pathway for the SCs, fibroblasts and endothelial cells to migrate along. SCs start to proliferate and align to form the bands of Büngner. This provides the guidance for the axonal sprouting to the distal stump, a process that occurs over next 2–4 weeks. The fibrin cable then degrades once cellular migration is complete. After axonal regeneration to the distal stump, the SCs switch from a proliferative to myelinating phenotype, leading to some degree of functional recovery at 6–16 weeks post repair. Using simple hollow tubes this process is limited to gap injuries of 4 cm in humans and 1.5 cm in rats. Beyond this distance of injury, there is limited to no regeneration (Daly et al., 2012).

15.4.2 Development of NGCs for repair

A major development in the construction of bioengineered NGCs has been the progression from the simple concept of tubulisation to the creation of a conduit that more closely mimics the nerve environment. The NGC has a number of criteria to fulfil, namely to:

Therefore, the ideal NGC should be biocompatible, biodegradable, permeable and exhibit certain biomechanical and surface properties such as flexibility, limited swelling, a predictable degradation rate and if possible be transparent (de Ruiter et al., 2009; Gu et al., 2011). This has meant a threefold approach to construction of a NGC with developments in (1) the NGC body; (2) the intraluminal structure and (3) the intraluminal contents (Fig. 15.2).

The construction and use of biological NGC bodies based on harvesting and manipulation of other areas of the body has been investigated since the 1880s when hollow bone was used for a 30 mm gap injury in a dog (reviewed in Ijpma et al., 2008). The formation of conduits based on naturally occurring materials, such as collagen, was then explored in the 1990s (Archibald et al., 1991). A number of Food and Drug Administration (FDA)-approved, commercially available NGCs based on type I bovine collagen including Neuromatrix®, Neuroflex® and Neurogen® were subsequently developed (Meek and Coert, 2008). More recently, an EU-approved porcine collagen NGC named Revolnerv® has been successfully used in bridging a 10 mm gap in a rat peroneal nerve injury (Alluin et al., 2009). However, due to relative limitations in the availability and unpredictable reactions of the body to biological options, there has been a drive towards the research and use of synthetic NGCs (de Ruiter et al., 2009). NGC body construction has focused previously on the use of hollow bodies but with increasing knowledge of the complexity of nerve regeneration, the considerations related to body construction can be broken down into multiple parts. These include natural or synthetic materials with biodegradable or non-biodegradable properties. They may also exhibit differential permeability, electrical conductivity and ability to integrate growth factors or regenerative cells.

15.4.3 NGCs made of natural materials

NGCs have been developed from non-nerve tissue and include muscle, vein, muscle-in-vein, small intestinal submucosa, tendons and epineural grafts (Gu et al., 2011). Acellular grafts of allogenic or xenogenic materials can be created by various physical, chemical or enzymatic decellularisation methods (Gilbert et al., 2006).

Muscle grafts have been shown to support regeneration comparable to nerve grafts over a 2 cm rat sciatic nerve gap (Bryan et al., 1993). In vivo, the graft was penetrated by SCs, fibroblasts, perineural and endothelial cells and axon regeneration was apparent within 3 weeks, while the graft itself ultimately disappears (Hall, 1997). Reasonable clinical outcomes have been reported with the use of vein grafts to bridge sensory nerve lesions in the hand (Risitano et al., 2002). Modifications such as turning the vein inside-out (Wang et al., 1993a) and coating the vein with collagen gel (Wang et al., 1993b) have also had moderate success. However, this technique appears inferior in comparison to muscle grafts (Fansa et al., 2001). Small intestinal submucosa has been proposed as a potential biological nerve conduit (Smith et al., 2004) and, stripped of its mucosal and serosal layers to leave an acellular collagen matrix, it can be fashioned into a roll to bridge a nerve gap (Hadlock et al., 2001).

Collagen is a biological material that can be shaped into a NGC and collagen tubes obtained from rat tail tendons have been shown to support moderate nerve regeneration across a 1 cm nerve gap (Brandt et al., 1999). Nerve regeneration was also demonstrated in collagen tubes stabilised by microwave crosslinking, whilst the absence of cross-linkage resulted in swelling of the tube, obstruction of the lumen and lack of regeneration (Ahmed et al., 2004). Similarly, type I bovine collagen tubes, strengthened by UV irradiation, showed regeneration comparable to that of autografts over 1.5 cm nerve gaps whereas untreated tubes obstructed axonal growth (Itoh et al., 2002). Over longer (2 cm) gaps, purified bovine collagen tubes failed to bridge a nerve defect (Yoshii and Oka, 2001).

Another constituent of the ECM is hyaluronic acid, a linear polysaccharide. NGCs composed of this molecule have been shown to be non toxic, biocompatible and biodegradable (Avitabile et al., 2001). Also we have used fibrin in the construction of NGCs (Kalbermatten et al., 2009; di Summa et al., 2011; Pettersson et al., 2011).

Chitin and its derivative, chitosan, are polysaccharides derived from cellulose and the outer shells of crustaceans, insect exoskeleton and fungal cell walls. Structurally similar to glycosaminoglycans, they interact with ECM molecules and demonstrate biological compatibility (Y. M. Yang et al., 2009). Since chitin and chitosan are fragile when dry, they must be chemically cross-linked with other materials to be used to create a NGC, a process that has been used successfully (Kim et al., 2008). Gelatin was used to create one of the first biodegradable NGC (Ijkema-Paassen et al., 2004). Both chemical and mechanical properties of this molecule can be controlled by cross-linking.

15.4.4 NGCs made of synthetic materials

The choice of materials is an important step in the fabrication of synthetic NGC. Ideally, a biodegradable material would be used so that once regeneration is complete, the NGC is reabsorbed, negating the risk of compression of the treated nerve or need for removal of the NGC.

Silicone NGCs have been one of the most extensively used materials, both in experimental models of nerve regeneration and clinically (Fields et al., 1989; Dahlin et al., 2001b; Li et al., 2004). The non-absorbable, inert silicone acts as a biological chamber, allowing the accumulation of growth factors, ECM molecules and SCs which promote nerve regeneration across short nerve gaps (Lundborg et al., 1997). However, a clinical trial using silicone NGCs has shown that the conduit can cause symptoms of mild irritation and nerve compression, occasionally necessitating removal (Lundborg et al., 2004). Other non-resorbable materials used for NGCs include polysulphone (Aebischer et al., 1989), polyvinyl chloride (Scaravilli, 1984) and polytetrafluoroethylene (Gore-tex) tubing (Pitta et al., 2001) but the disadvantage of this group in general is the foreign body reaction and scar formation that can negatively impact the regenerated nerve (Braga-Silva, 1999).

More recently, attention has focused on the increasing availability of biodegradable synthetic materials which have the distinct advantage of unlimited supply and reproducible properties (Wan et al., 2001). Polyesters and their copolymers have been the main materials studied in this area (Bell and Haycock, 2012). Examples of these are polyglycolic acid (PGA), poly(lactic acid) (PLA), polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA). An advantage of these materials is their degradation through hydrolysis of the ester bond and subsequent resorption through metabolic pathways, resulting in minimal toxicity to the host (Cao et al., 2009; Jiang et al., 2010).

In clinical studies it was shown that NGCs made from PGA were superior to autograft (Weber et al., 2000). However, since it was reported that the NGCs collapsed at 12 weeks, use of these conduits was recommended for low load areas only (Shin et al., 2009).

PCL, predominantly manufactured as a co-polymer with either natural or synthetic blends, has been used with success in both experimental and clinical studies (Bini et al., 2004; Chan-Park et al., 2004; Bertleff et al., 2005; Schnell et al., 2007; Gupta et al., 2009; Ribeiro-Resende et al., 2009; Sun et al., 2010; Cooper et al., 2011).

Poly(L-lactide) (PLLA), when used alone, degrades over a 3 year period, but the crystalline nature of the material makes stiffness of this NGC is an issue (Bergsma et al., 1995). When lactic acid and glycolic acid are copolymerised to form PLGA in various proportions, the material becomes more usable with faster degradation times and enhanced flexibility (Engelberg and Kohn, 1991) but when comparing functional outcomes, PLLA rather than PLGA shows results closer to autografts (Evans et al., 2000).

Poly-3-hydroxybutyrate (PHB) is a thermoplastic polyhydroxyalkanoate which has been shown to have good biocompatibility and has been used for a number of years in the manufacture of sutures and dressings (Chen and Wu, 2005). Clinical studies have shown that PHB has a degradation time of 18–24 months and gives better results than epineural suturing (Young et al., 2002; Åberg et al., 2009). PHB may also have anti-apoptotic properties (Xiao et al., 2007).

Poly(2-hydroxyethyl methacrylate) (PHEMA) is a hydrogel NGC (Jiang et al., 2010). Results at 4 and 8 weeks showed promising regeneration comparable with autografts (Belkas et al., 2005a), but at later time points there was evidence of tube collapse (Belkas et al., 2005b). Subsequently, these NGCs have been redesigned with reinforcement, and this has given similar good performance and patency 16 weeks after surgery (Katayama et al., 2006).

The construction of a synthetic NGC body has moved away from a solid, inert construct to one with a greater number of properties. One method is to introduce pores into the walls to allow for diffusion of waste products out, and nutrients into, the NGC to support the indwelling cells at the early stages of regeneration, prior to the establishment of active blood supply (Zhao et al., 1993). It is important to note that the ability to modify the NGC wall is influenced by the hydrophilic nature of the material used (Busscher et al., 1983). It has also been shown both in vitro and in vivo that the use of weak electrical stimulation can aid nerve recovery by enhancing axonal regrowth and increasing the blood supply (Schmidt, 1997; Mendonça et al., 2003). Examples of materials from which NGCs can be used to produce electrically conductive walls are polyaniline, polypyrrole, polythiophene and polyacetylene (Rajaram et al., 2012).

Regenerating axons can also be guided using topographical cues and gradients within the inner wall of the conduit (J. Yang et al., 2009). For instance, NGF and laminin gradients stimulate axon growth to similar levels of autografts (Dodla and Bellamkonda, 2008). Laminin can both direct axonal growth and promote proliferation of SCs on biomaterials (Wang et al., 1992; Armstrong et al., 2007). Synthetic polymer NGCs coated by plasma polymerisation have demonstrated increased biocompatibility (Murray-Dunning et al., 2011). Topographic signals can be divided into anisotrophic and isotrophic cues such as grooved surfaces or nanorough surfaces respectively (Hoffman-Kim et al., 2010). It is also possible to use ECM proteins to coat the surface of the NGC. When topographical cues and chemical gradients have been used together, they have been found to have positive effects on regeneration although their mechanisms of action have yet to be elucidated (Hoffman-Kim et al., 2010).

Given the importance of neurotrophic factors during the regeneration process, a number of studies have explored the use of these molecules integrated into the NGC wall. The method of integrating growth factors, rather than through adsorption, has given enhanced biological effects (Cho et al., 2010). Glial derived neurotrophic factor (GDNF), BDNF, NGF and NT-3 have all been found to increase myelination, particularly when used in combination (Sterne et al., 1997; Fine et al., 2002; Boyd and Gordon, 2003). However, the limiting factor for their inclusion in clinical treatment is deemed to be cost (Bell and Haycock, 2012).

15.5.1 Intraluminal structure

As described earlier, fibrin cables that bridge the proximal and distal stumps are only able to do so over relatively short distances. Therefore, modern NGC design has attempted to provide a preliminary structure that the regenerating nerve can use to aid growth and supplement this process.

One method is the use of a multi-channel NGC, which aims to mimic the architecture of nerve fascicles (de Ruiter et al., 2008). The presence of multichannels increases the surface area within the lumen to allow for greater likelihood of cell adhesion as well as extra surface area from which growth factors can be released. However, the extra layers or thickness of the NGC may interfere with permeability to nutrients, essential at the early stages. In addition, moulding of the multi-channels does not exactly mimic the in vivo environment and therefore, may force mismatching of motor and sensory axons. Although multichannel NGCs reduce dispersion of regenerating axons within the lumen of the conduit, they have been found to have no overall functional advantages (de Ruiter et al., 2008). However, in future it might be possible to improve these NGCs by constructing channels small enough to replicate the bands of Büngner.

The lumen of NGCs can also be filled with filaments, fibres, gels or sponges (Gu et al., 2011). Again these materials are used with the aim to mimic the fibrin cables formed during the matrix phase of regeneration as closely as possible. When placed longitudinally, addition of fibres has been shown to improve regeneration when compared with a hollow conduit (Cai et al., 2005; Newman et al., 2006). Application of ECM protein fibres has also demonstrated the possibility to enhance regeneration beyond the normal critical distance (Matsumoto et al., 2000). The benefits of fibre addition are affected by their ease of alignment and density of the surrounding scaffolding. Alignment can be achieved by using magnetic fields and has shown to be effective in enhancing axonal regeneration both in vitro and in vivo (Chamberlain et al., 1998; Ceballos et al., 1999; Dubey et al., 1999). Increased fibre density beyond a certain point occludes regrowth of the nerve, but if there are too few fibres, inadequate support is provided and regrowth does not occur (Labrador et al., 1998). In order to overcome the issue of density, nanofibres have been used as they mimic natural ECM proteins more closely (Cao et al., 2009). Manufacturing methods include electrospinning, phase separation and self-assembly (Gu et al., 2011). Although in theory addition of fibres is a good principle, insufficient research has been carried out to make solid conclusions regarding their efficacy.

15.5.2 Matrix materials

With advances in the understanding of the mechanisms of regeneration, the importance of the intraluminal environment has been shown. As described previously, the ECM plays an important role in the early phases of regeneration through a hollow tube. The provision of the insoluble contents of ECM such as collagen, laminin and fibronectin, and other proteoglycans and glycosaminoglycans play a significant role in the stimulation of nerve regeneration by encouraging the body to switch from the fluid to matrix phase sooner (Rustihauser, 1993; Bovolenta and Fernaud-Espinosa, 2000; Asher et al., 2001; Grimpe and Silver, 2002; Xu et al., 2011). Materials commonly used to make matrices include hydrogel-forming collagen, fibrin, laminin, alginate, heparin and heparin sulphate (Pabari et al., 2011).

When using matrices it is important to use low density materials to minimise obstruction to regenerating axons (Labrador et al., 1998). It is also necessary to understand the interactions between growth factors and the ECM, since various elements of the matrix can work together in many different ways through ligand–receptor binding, ionic, electrostatic, hydrophobic and covalent interactions (Pabari et al., 2011). Understanding these principles can be used to manipulate the nerve microenvironment by delaying degradation and enzymatic breakdown of growth factors thereby promoting regeneration to levels similar to autografting (Han et al., 2010). A suitable matrix also provides the option of incorporating regenerative cells into the NGC (Keilhoff et al., 2005).

15.6.1 SCs

Since SCs are essential for nerve regeneration they are an obvious choice. The regenerative effects of SCs are twofold; namely physical by forming paths for the axon to grow into via the bands of Büngner, and chemically by the synthesis and secretion of increased amounts of neurotrophic factors (Li et al., 2006). They have been shown to enhance both axon elongation and sprouting (Armstrong et al., 2007) and these mechanisms may contribute to increased probability for successful nerve repair. However, peripheral nerve biopsy is needed to obtain autologous SCs for culture, with an additional surgical procedure that the concept of a bioengineered graft aspires to avoid. The generation of sufficient quantities of SCs for transplantation from the patient’s peripheral nerve biopsy requires at least 3–6 weeks according to established protocols (Vroemen and Weidner, 2003). This represents a significant delay in managing an acute injury – a factor widely recognised to impair overall outcome in peripheral nerve repair (Sulaiman and Gordon, 2000; McKay Hart et al., 2003). However, it was shown that a new method to rapidly isolate SCs could give a sufficient number of cells to be seeded in a nerve conduit and provided good regeneration (Brandt et al., 2005).

When SCs were transplanted into a PLGA conduit, the percentage of neural tissue per cross sectional area was statistically similar to autografts (Hadlock et al., 2000). This result has been replicated in many other conduit materials with regeneration shown to approach the levels of an autograft (Mosahebi et al., 2001; Cheng and Chen, 2002; Sinis et al., 2005; di Summa et al., 2011). The addition of SCs to acellular vein and muscle grafts also improved regeneration in comparison to the same conduits left empty, although control autografts performed superiorly to all experimental alternatives (Fansa and Keilhoff, 2004).

In SC transplantation studies, autologous or syngeneic cells were used to avoid immune reactions. Conceptually, allogenic SCs are an alternative and their use would allow some of the problems with autologus cell use to be overcome. However, there is conflict regarding their effectiveness. Allogenic SCs have been shown to be effective in improving axonal regeneration when transplanted in PHB conduits with alginate matrix (Mosahebi et al., 2002). Although there was an increased immune reaction in terms of lymphocyte and macrophage count in transplants containing allogenic SCs, axonal ingrowth into the conduits was comparable to that observed with syngeneic SCs, suggesting that the immune response was not deleterious to regeneration. This suggested that despite rejection of the allogenic cells, the neurotrophic factors they produce may be long lasting and remain functional within the matrix after the SCs’ demise. While there is progressive death of transplanted cells without immunosuppression, simultaneous infiltration of host SCs occurs in greater numbers than after injury alone. Previous studies have suggested that heterologous transplanted SCs within a nerve conduit provoke an intense immune reaction (Guenard et al., 1992) and that only autologous SCs promote regeneration rates approaching that of standard nerve grafts (Rodriguez et al., 2000). Evans et al. (2002) found no positive effect when comparing allogenic SCs with an empty PLLA conduit.

15.6.2 Stem cells

Recently, the possibility of using stem cells as an alternative source of intraluminal support has been proposed. These are progenitor cells with the capacity both to self-renew and to generate differentiated progeny (Morrison et al., 1997). Three classes of stem cells can be used in nerve regeneration therapy. These are totipotent cells – those that can differentiate into all cell types of a particular organism and into a complete organism; pluripotent cells – those that are able to form cells from different lineages but not a complete organism; and multi-potent cells – those that can differentiate into many kinds of cells but with restricted potential (Temple, 2001). Stem cells isolated from a number of different sources including the embryo, neural tissue, bone marrow, adipose tissue, amniotic fluid, hair follicles and skin have demonstrated the ability to aid neural regeneration. A number of mechanisms broadly divisible into two parts; replacement and supportive roles have been postulated for the stem cell presumed mode of action (Dadon-Nachum et al., 2011). These include direct cell replacement, trophic factor production, ECM molecule synthesis, axonal guidance, remyelination, microenvironmental stabilisation and modulation of the immune environment.

Embryonic stem (ES) cells are totipotent cells that can be differentiated into any of the neuronal and non-neuronal cells. They have been shown to differentiate into motor neurons in a spinal cord injury and extend axons into the peripheral nerve that contribute to formation of new neuromuscular junctions (Deshpande et al., 2006). The use of ES cells has been shown to stop muscular degeneration in a denervated muscle through the formation of axons and their cholinergic terminals in the muscle (Erb et al., 1993; Thomas et al., 2003). However, use of ES cells is limited for a few significant reasons. Firstly, obtaining these cells means sacrificing an embryo, which causes various legal and ethical issues. Secondly, ES cells may be tumourigenic. And thirdly, since the cells are not autologous, treatment would have to be supplemented with immunosuppressive treatment (Dadon-Nachum et al., 2011).

Neural stem cells (NSCs) are multi-potent stem cells found in the hippocampal and subventricular zone in foetal and adult CNS. When used in a collagen conduit, they have been shown to differentiate into SC-like cells and survive transplantation for up to 2 months, so aiding nerve regeneration (Murakami et al., 2003). NSCs have also been reported to differentiate into functional motor neurons after transplantation into a transected peripheral nerve (MacDonald et al., 2003), and synthesise and secrete synaptophysin suggesting high neuronal activity (Gu et al., 2010). However, being sourced from another individual, treatment with NSCs would again have to be supplemented with immunosuppression.

Bone marrow mesenchymal stem cells (BMSCs) are multi-potent stem cells found in the bone marrow where they provide support for the haematopoietic system (Coronel et al., 2009). They have demonstrated hypo-immunogenicity and are thought to home to an injury site (di Nicola et al., 2002; Rombouts and Ploemacher, 2003; Aggarwal and Pittenger, 2005). The criterion for identification of BMSCs was set out by the International Society for Cellular Therapy using three sets of characteristics:

BMSCs have been shown to differentiate into glial-like cells (Dezawa et al., 2001; Caddick et al., 2006; Brohlin et al., 2009) and have extensively been shown to enhance nerve regeneration when transplanted in various types of NGCs (Tohill et al., 2004; Pereira Lopes et al., 2006; di Summa et al., 2011; Yang et al., 2011; McGrath et al., 2012; Rodrigues et al., 2012) and allografts (Wang et al., 2008).

Adipose derived stem cells (ADSCs) are multi-potent stem cells, which as the name suggests, are derived from adipose tissue (Zuk, 2010). As a source of stem cells, adipose tissue has a number of distinct advantages over bone marrow in that it contains a higher density of stem cells (Strem et al., 2005), the isolated ADSCs grow faster in culture and they are more easily harvested from a patient (Kingham et al., 2007; Choi et al., 2010; Witkowska-Zimny and Walenko, 2011).

ADSCs are likely to be of benefit for neural regeneration thanks both to their ability to differentiate into neural cells as well as through the secretion of a variety of growth factors and cytokines (Erba et al., 2010). We have shown that ADSCs can differentiate into glial-like cells (Kingham et al., 2007) and when transplanted in NGCs made from fibrin they enhance sciatic nerve regeneration (di Summa et al., 2011). We also showed that ADSCs transplanted in PCL NGCs could enhance early nerve regeneration (Fig. 15.3) and promote the survival of neurons in the dorsal root ganglion (Reid et al., 2011). Other research groups have also shown encouraging results using ADSCs in various types of NGCs (Santiago et al., 2009; Scholz et al., 2011; Mohammadi et al., 2013; Wang et al., 2012). Research into the use of these cells is rapidly expanding due to their distinct advantages for translation into clinical practice. Current topics being addressed are studies to correlate tissue donor demographics (age, sex, depot site) with cell function, methods to produce these cells under Good Manufacturing Practice conditions including identification of animal serum alternatives and improved methods for cryopreservation and more extensive animal testing to monitor risks of tumorgenesis.