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GUIDED PERIPHERAL NERVE REGENERATION

INTRODUCTION

The nervous system consists of two parts that differ in their physiology and function. Neurons of the peripheral nervous system (PNS) receive information from the external environment and carry signals to and from the brain and spinal cord, which constitute the central nervous system (CNS). Peripheral neurons consist of a cell body and a long process, or axon, which may lead one meter in length. Short segments of the axon are wrapped with an insulating myelin sheath formed by Schwann cells.  Axons are grouped together into fascicles, several of which are enclosed in the epineurium (a sheath of connective tissue) to form a peripheral nerve. The major types of peripheral neurons are sensory or afferent neurons, which are responsible from stimulus reception, and motor or efferent neurons, which control organs and tissues such as glands and muscles (Heath, 1998)

Fig.1: Peripheral motor neuron (a) showing the cell body, axon extention surrounded by myelin sheath and the axon terminals. A cross section of the neuron (b) shows how the myelin sheath, composed Schwann cells, wrap around the axons (Heath, 1998)

More than any other forms of trauma, nerve injuries complicate successful rehabilitation, because mature neurons (many other cells in the body) do not replicate; that is, they do not undergo cell division. Under right conditions, however, axon extension can regenerate over gaps caused by injury, reconnecting with distal stump and eventually reestablishing functional contacts (Heath, 1998).

When a nerve is cut or crushed and nerve function is lost, the portion of the nerve distal to the injury dies and degenerates; the proximal segment may be able to regenerate and reestablish nerve function. A crush often leaves a continuous tubule structure through which the axon can grow, but a cut creates a gap across which the growth cone of regenerating axon must navigate. To improve recovery, severed nerves can be surgically sutured end-to-end over small gaps. However, large gaps must be repaired with a graft inserted between the proximal and distal nerve stumps as a guide for regenerating axons.

A typical graft of choice is the autograft, which is a segment of nerve removed from another part of the body. Disadvantages of the nerve autograft include a second surgical procedure, limited availability, permanent denervation of the donor site and mismatch between nerve and graft dimensions. Allografts have also been used, but these are accompanied by the usual need for immunosuppression and have very poor success rates. Autologous and autogenous blood vessels and muscle fibers have also been used as conduits for nerve regeneration with varying levels of success, but these still suffer from some of the same disadvantages as auto- and allografts. Conduits have also been formed from other biological materials, with collagen showing the greatest potential for success.

Avoiding the problem of availability and immune rejection, a promising alternative for extending the length over which nerves can successfully regenerate is the artificial nerve graft (also known as nerve guidance channel). The artificial graft is a synthetic conduit that bridges the gap between the nerve stumps and direct and supports the nerve regeneration. The conduit may be implanted empty, or it may be filled with growth factors, cells of fibers. Any artificial graft can meet many of the needs of regenerating nerves by concentrating neurotrophic factors, reducing cellular invasion and scarring of the nerve, and providing directional guidance to prevent neuroma formation or excessive branching.

With development of the artificial nerve grafts, it has been possible to study the mechanism of nerve regeneration extensively and much progress has occurred in this area. 

PERIPHERAL NERVE REGENERATION

Peripheral nerve regeneration comprises the formation of axonal sprouts, their outgrowths as regenerating axons and reinnervation of original targets (Ide, 1996). Functional recovery from peripheral nerve injury depends on a multitude of factors both intrinsic and extrinsic to neurons. First, the neuron must survive the injury and mount an effective response to initiate regeneration. Second, the growth environment in the nerve stump distal to the injury site must provide sufficient support for regenerating axons. Third, the successfully regenerated axon must reinnervate the proper target and the target must retain the ability to accept reinnervation and recover from denervation atrophy (Fu, 1997).

The axons of both myelinated and unmyelinated fibers in the PNS are surrounded by Schwann cells, which, in turn, are covered by basal lamina on the outer surface facing the endoneurial connective tissue compartment. In myelinated fibers, each Schwann cell forms its own internodal myelin sheath, which is separated from an adjacent internode by a node of Ranvier. Therefore, Schwann cells are aligned discontinuously along the axon, separated by node of Ranvier at both proximal and distal end of the internode. However, basal lamina continuous between the adjacent Schwann cells even at the node of Ranvier. Unmyelinated fibers have continuous Schwann cell sheaths with no gap at the transition between adjacent Schwann cells.  Therefore each fiber of both myelinated and unmyelinated nerves of PNS is regarded as residing within a continuous basal lamina tube.

When the axon is disconnected from the cell body by injury, its distal segment gradually degenerates and eventually disappears, a feature known as Wallerian degeneration (Waller, 1850).

Fig 2: Wallerian degeneration a) Normal nerve including cell body and peripheral fiber, the axon. Most nerves contain thousands of such fibers. b) Wallerian degeneration following nerve section. c) Regeneration. (Seckel, 1984)

Both Schwann cells and macrophages contribute to phagocytosis. The phagocytic activity of macrophages contributes directly to nerve regeneration by removing inhibitory substances associated with myelin and indirectly by release of plethora of factors that appear to be important for successful cellular repair (Fu, 1997). Factors released by macrophages include mitogens for Schwann cells and fibroblasts, and cytokines that stimulate the synthesis of growth factors and adhesion molecules by nonneuronal cells of the nerve sheath and endothelial cells of the blood vessels.

Schwann cells, which are devoid of contact with axons, transiently proliferate, forming a cell strand called Schwann cell column or band of Bungner (Bungner, 1981) within the basal lamina tube. Schwann cell mitosis is temporarily correlated with macrophage invasion into the distal nerve stump. Macrophages that have digested myelin membrane release Schwann cell mitogens including various cytokines, cAMP-dependent mitogens and fibroblast growth factors. Proliferated Schwann cells change their phenotype from myelinated to unmyelinated form by upregulation or downregulation of several proteins, such as transcription factors, neurotrophic factors, cell adhesion molecules and basement membrane components, including laminin, fibronectin, various proteoglycans, and collagen.

Regenerating axons are produced at the node of Ranvier, emerge from the proximal stump of the lesion with growth into the distal nerve segment, i.e. the Schwann cell column, and extend as far as the target organs. The Schwann cell column, which is formed after myelin sheaths are removed by macrophages, is an indispensable pathway by which regenerating axons grow through to the target in the PNS. If regenerating axons somehow evade the Schwann cell column and enter the connective tissue compartment, they cease to grow after elongation of only a few millimeters within the connective tissue. The Schwann cell column thus provides regenerating axons with an environment favorable for growth. It is the source of trophic factors for regenerating axons.  Neurotrophic factors, which include nerve growth factor (NGF), brain-derived nerve growth factor (BDNF), neurotrophin 4/5 (NT 4/5), glial-cell-line-derived neurotrophic factor (GDNF) and insulin-like growth factors (IGFs), are expressed, and released from target tissues and by glial cells, fibroblasts and macrophages in the vicinity of both neuronal cell body and axon. After axotomy, nonneuronal in the distal nerve stump, especially Schwann cells, synthesize many neurotrophic factors.  These neurotrophic factors play an essential role in promoting neuronal survival after injury.

Basal lamina is a specialized extracellular matrix that acts as a scaffold for epithelial and neural cells. It contains a variety of adhesion molecules including laminin, fibronectin, immunoglobulin superfamily, various proteoglycans and collagens. Accordingly, basal lamina can be thought of as a ‘glue’: on one side it is in contact with the cells, and the other side it is in contact with the surrounding connective tissue matrix, regulating the axonal growth in the distal nerve stump.  

There is considerable evidence that extracellular matrix components influence morphogenesis. Longo et al. (1984) have demonstrated that extracellular matrix glycoproteins, fibronectin and laminin, and neurite promoting factors promote neurite elongation in vivo. They observed a fibronectin-containing matrix at seven days of implantation of a silicone chamber. Such a matrix is considered to provide a scaffold, which supports a cell migration into the wound with subsequent formation of granulation tissue. Fibronectin influences the motility of cultured fibroblasts and Schwann cells.

Fig.3: Silicone chamber model showing the progression of events during peripheral nerve regeneration. After bridging the proximal and distal nerve stumps, silicone tube become filled with serum and other extracellular fluids. A fibrin bridge containing a variety of cell types connect the two nerve stumps. Schwann cells and axon processes migrate from the proximal end to the distal stump along the bridge. The axons continue to regenerate through the distal stump to their final contacts (Heath, 1998).

The fibronectin present in the chamber matrix at one week may provide the necessary substratum for cell migration and vascular sprouting occurring during the second week after implantation. This fibronectin may originate from plasma and enter the chamber during the initial bleeding and chamber filling or may be secreted into the chamber by cells such as fibroblasts or endothelial cells emigrating from the nerve stump (Longo, 1984). No laminin was detected in the early matrix. At 14 days, laminin was observed near numerous cell surfaces and budding capillaries. It is indicated that Schwann cells and fibroblasts, as well as vascular endothelial cells produce laminin and it functions in mediating the initial contact between the neurite and the Schwann cell and subsequent elongation along its surface. The fluid bathing the regenerating nerve was found to contain an agent(s) that promote neurite outgrowth. The neurite promoting factor of the fluid increases dramatically after third week, suggesting that the ingrown cells are the source of neurite promoting factors (NPF) (Longo, 1984).

The distal nerve has also been found to be important for the peripheral nerve regeneration as it supplies various neurotrophic factors for axonal regeneration (Seckel, 1984). It is found that regeneration fails when the distal nerve stump was absent from the synthetic biodegradable guide, over a 10 mm or longer gaps. Thus, distal tissues exert an essential trophic effect on axons proximal to the transection site.  

GUIDED PERIPHERAL NERVE REGENERATION

A variety of methods have been proposed for nerve regeneration, depending on how far the stumps are apart from each other. Direct suturing of the damaged nerve is possible, provided that the gap is small.  If the gap is long (longer than 3 cm in human, and 1-2 cm for rat), nerve regeneration is impossible without a guide that would function in bridging the distal and proximal ends, thereby directing the nerve regeneration and concentrating the cells, growth factors, etc. within the regenerating tubule. It also prevents fibrous tissue formation between the distal and proximal ends. Guides for neuronal regeneration also play a role in speeding up the regeneration process because growth potential of regenerating axons has been suggested to be maximal 3 weeks after injury. The sooner the repair, the better the functional recovery (Fu, 1997).

Guides for regenerating neurons can be of biological origin such as nerve grafts, muscle grafts, vein grafts, and extracellular matrix components as collagen and fibrin tubes. The artificial nerve grafts can be made up of non-degradable materials such as silicone and polyurethane; or degradable materials such as polygylcolic acids, poly(organo)phosphazine,  poly(L-lactide-co-caprolactone) and Poly-3-hydroxybutyrate (PHB). Unlike the materials with biological origin, the properties of artificial nerve grafts can be modulated. The guides with biological origin have an advantage in that they possess intrinsic growth promoting factors. However, they are mechanically less stable than the artificial growth chambers. Thus, the combinations of autologous and artificial growth chambers have been used with increased success in regeneration of neurons. 

Biological Nerve Grafts

In this technique, a nerve segment is taken out from the patient (autograft) or from a donor (allograft) in order to suture the stumps. Although autologous nerve grafting is a frequent surgical practice, it is detrimental to the donor nerves with possible complications, such as numbness, painful neuroma formation, and unacceptable scarring (Maquet, 2000).

Allografts have been used in reconstruction but require systemic immunosuppression.

Veins, pseudosynovial membranes around a silicone implant, collagen conduits, perineurium tubes, fresh nerve grafts in a vein conduit and acellular muscle grafts are some examples of autologous materials that have been used.

Autologous skeletal muscle grafts have been proved effective for the repair of peripheral nerves. After the sarcoplasm and plasma membrane has been eliminated by macrophages, the basal lamina of the muscle act as a guide for the regenerating axons. Furthermore, the basal lamina of the muscle tissue contains both collagen type IV and laminin (Meek, 1998), which are known to positively influence the outgrowth of damaged nerve fibers, because of their intrinsic neurite promoting effect (Ide, 1996). However, by using a denatured autologous muscle tissue to bridge a nerve gap there is a high risk that nerve fibers can easily grow out of the muscle tissue, thereby forming a neuroma-in-continuity (Meet,1998).  Meet et al. used a biodegradable nerve guide with the insertion of modified denatured muscle tissue for the reconstruction of a 15 mm gap in the sciatic nerve of the rat. They observed fast nerve regeneration; the first myelinated and unmyelinated nerve fibers in the distal nerve stump, as well as the first sign of functional nerve recovery could be observed after three weeks of reconstruction.

 Good revascularization of the graft is essential for a successful regeneration of the nerve. Nerve regeneration seems to be improved by association of the muscle graft with vascular implantation. The blood supply enhances the migration of Schwann cells and the diffusion of neurotrophic factors.

 Vein grafts have been used for many years to repair the peripheral nerves. It has been shown that, when the gaps in the nerve have a similar short length, the results using the vein grafts or nerve grafts are comparable (Chiu, 1982). Although after two or three months the vein graft has a reinnervation percentage lower than the nerve graft, the difference is no longer detectable after six months. The lumen of the vein provides favorable microenvironment for nerve regeneration and coating the luminal epithelium with collagen gel improves regeneration (Wang, 1992).

Some researchers have used the inside-out vein graft for a sensory nerve regeneration with a better success than standard vein grafts (Ferrari, 1999) The vein wall has three layers: the endothelial layer contains a laminin reach basal lamina, the media is a thin muscle layer that is also rich in laminin, and the adventitia is rich in collagen. By pulling the vein graft through itself and then turning it inside-out, these layers become reversed. The resulting conduit exposes regenerating axons directly to the adventitia. This conduit is a nonimmunogenic, permeable to external factors, and lined with abundant trophic and neurite-promoting factors; therefore, it may provide a superior microenvironment for peripheral nerve regeneration (Wang, 1995). Ferrari et al. (1999) concluded that the regeneration of a sensory nerve by the inside-out and standard vein graft techniques consists of three steps: 1) there is an invasion of new capillaries into the vein graft, which come mainly from proximal stump; 2) the neurites follow the vessels and are arranged around them; 3) the connective tissue decreases between the new microfascicles in the different postsurgical times. They observed that the diameters and the myelin sheath thickness of the fibers were not different in two techniques; however, they were smaller in the experimental groups than in the normal group (Fig.4).

The use of combined autologous conduit, consisting of a vein plus acellular muscle grafts have been described by Benedetto and his coworkers (1998) for a 2 cm gap in rat sciatic nerve. The gap was filled with a 2 cm long femoral vein conduit in which two autologous acellular muscle grafts had been previously inserted. Five out of seven rats achieved regeneration and nerve conduction velocity values were very close to one using nerve grafts to bridge gaps of comparable length. Regeneration seemed to progress from the muscle implants to completely fill the vein conduit. Thus, they succeeded in regeneration of a well-formed nerve fascicle and concluded that the effect of acellular muscle graft in promoting regeneration was probably strengthened by the presence of vein conduits acting as a guide.

Fig 4: Electron micrographs of the distal stump: A and C in the inside-out vein graft (IOVG) group; B and D in the SVG group. Panel A shows 12 weeks after surgery (X4,500) and panel C shows 20 weeks after surgery (X4,500). The disposition of the microfascicles is similar to the graft in the IOVG group at same time. Panel B shows 12 weeks after surgery (X4,000) and panel D shows 20 weeks after surgery (X4,000) the regenerated fibers are more rounded in the IOVG group.

Collagen is also biological molecule used as nerve guidance channel. It is an extracellular matrix component, longitudinally oriented after the initial fibrin matrix is lysed by endogenous mechanisms. Together with laminin and fibronectin (extracellular matrix components), it acts as a tropic factor that guides the growth of cones. Prefilling an artificial tube with dialyzed plasma, which forms a fibrin gel, or with collagen gel or a laminin containing gel supports axonal growth across longer gaps than when chambers are filled with saline solution. Recently, prefilling a tube with magnetically aligned type I collagen gels has been introduced in which the desired local direction would be provided by fibrils aligned along the tube axis. Therefore, using magnetically aligned collagen gel, achieved by exposing the forming collagen gel to a high-strength magnetic field, has been found as an attractive approach to improve the regeneration that has been obtained using collagen gel thus far (Ceballos, 1999). However, collagen is proteolytically degraded and the local direction conferred by the aligned fibrils is lost prematurely during nerve regeneration. In order to decrease the rate of degradation, they used glycation method. The nonenzymatic cross-linking of proteins with free amine groups, such as lysine and hydroxylysine residues of type I collagen, by reducing sugars such as glucose and ribose, which normally occur during aging, particularly in diabetics. Their results demonstrated that the collagen tube with magnetically aligned type I collagen gel significantly improved the nerve regeneration over 6 mm gap, over tubes filled with control collagen gel. This result was presumably due to contact guidance of regenerating axons and nonneuronal cells, with Schwann cells being considered key in this respect. By cross-linking the collagen gel, they attempted to increase its structural stability by reducing its rate of resorption. However, the ribosylation conditions used for cross-linking in this study decreased the likelihood of nerve regeneration and number of regenerated fibers. It is known that the intratubular exogenous gels, even if containing neurotrophic factors, may impair the regeneration process by physically impeding the diffusion of soluble factors, the migration of nonneuronal cells, or the growth of axons. The nerve regeneration has been shown to be dependent on the viscosity, density and the concentration of the gels.

Longitudinally aligned fibrin gel has also been shown to increase the nerve regeneration because of the same story. With the tubes filled with fibrin clot, or dialyzed plasma, where longitudinal alignment of fibrin fibrils occur in an uncontrolled manner, the degree of regeneration improve due to contact guidance, with maximal structural arrangement of regenerated axons and minimal neuroma formation. In fact, if the fibrin fibrils are not longitudinally aligned, regeneration can be impaired (Williams, 1987).

Artificial Nerve Guides

An attractive alternative to autografts consists in bridging the two nerve stumps by a tube, which have an advantage of directing the axonal growth properly and which can be loaded with biologically active compounds such as growth factors. Using a conduit, the need to remove a nerve segment is avoided and loss of function in the donor site is prevented.

However, it is necessary that the artificial guide can ensure a satisfactory nerve repair, at least as good as the autologous grafts. To be successful, a nerve guide must be integrated into the surrounding tissues and must guide the nerve fibers toward the distal nerve stump. It is also expected to prevent fibrous tissue from invading the nerve gap, to be stable as long as the regeneration nerve fibers are not mature enough, and finally to disappear (bioabsorbable) rather than being removed, to avoid the risk of injuring the repaired nerve. Tube dimension, biocompatibility, permeability, flexibility, and degradation rate, as well as surface texture and porosity of walls are other important parameters that should be controlled. The orientation of matrix filling the tube is also known to have beneficial effect on the nerve regeneration. It is thought that, increasing the conduit diameter increases the concentration of NGF in the proximity of regenerating axons, which could increase their rate of extention.   

Many synthetic polymer conduits made of silicon, acrylic copolymers, polylactic acid, polyglycolic acid, poly(lactide-co-ε-caprolactone) and polyorganophosphazenes are used for nerve regeneration with a various degrees of success. Unlike materials of biological origin, synthetic materials can be fabricated and modified to exhibit a wide array of well-controlled properties including permeability, biodegradability, flexibility, growth factor release and surface microgeometry.  

Permeability of Conduits

Regenerating axons span a much longer gap if the tube that encases the nerve stump is made permeable (Jeng, 1987). The mechanism that leads to this enhancement of regeneration is that macroscopic holes in the wall of the tube result in mingling of the contents of the tube with the contents of the extracellular space in general and that this mingling presumably facilitates the migration of non-neural connective tissue cells and the formation of extracellular matrices that promote or support the axon growth across relatively long gaps (Jeng, 1987).  When bridging short or medium length gaps, such as 4 mm in the mouse and 4-8 mm in the rat, tubulization with impermeable materials is usually successful, provided that the physical parameters of the nerve guide are adequate and the lumen is not occluded. However, when the gap is longer, semipermeable tubes improve the regeneration success.

When using porous materials, the nominal pore size will determine which molecules will pass through the graft between the surrounding tissue and the regenerating nerve. A molecular weight cut-off of approximately 50,000 has been found suitable to allow diffusional transport of nutrients and other molecules while preventing cells from entering the conduit (Heath, 1998).

Jenq et al. studied the nerve regeneration through holey silicone tubes to see the effect of permeability on nerve regeneration. Initially they thought that impermeable tubes are advantageous because they will keep the fluid and cells that come from the nerve stump around the regenerating axon. Then, making the tube permeable by putting holes in it might change the patterns of regeneration. They opened two large rectangular holes on the 11 mm long silicone tube, and implanted it into one sciatic nerve of the rat. As a control, they implanted one normal silicone tube of same length and diameter into the other sciatic nerve. Rendering the silicone tube permeable by making large holes in its walls markedly changes both the cytologic nature of the regenerate and the number of axons that regenerate through the tube compared to a similar surgical paradigm where the tube is impermeable. This what they expect, however, what is surprising is that the regeneration in the holey tube is superior. They observed more individual fascicles, more cells line each fascicle and lining cells are coated with more prominent external lamina in holey tube. For axonal numbers, there are more myelinated and unmyelinated axons in the gap and more unmyelinated axons in the distal stump than after regeneration in a regular silicone tube.

It is not known that whether the extended regeneration with the use of permeable chambers are due to 1) metabolic exchange across the tube wall (diffusion of nutrients such as glucose, oxygen, etc. and elimination of waste products), 2) diffusion into the guide lumen of growth or trophic factors generated in the external environment (wound healing factors), 3) retention of growth or trophic factors secreted by the nerve stumps, 4) a combination of the above. Aebischer et al. (1988) compared perm-selective acrylic copolymer (AC) channels and impermeable silicone elastomer (SE) channels in terms of regeneration in the absence of a distal nerve stump, in the mouse sciatic nerve of 6 mm length. The distal end of the polymer tube is capped in half of the animals and it is left open in the other half. The perm-selective acrylic polymer channels without distal nerve stump contained regenerated nerve cables, which extended fully to the distal end of the channel. Non-permeable silicone silicone elastomer channels with distal nerve stump present showed regenerated nerve cables bridging the proximal and distal nerve stumps, however, capped SE channels contained only fine threads of connective tissue which extended for no more than 1 mm from the proximal nerve stump. It is known that growth and trophic factors secreted from distal nerve stump is essential for nerve regeneration. However, the result of this study showed that a perm-selective guidance channel may support regeneration in the absence of distal nerve stump by allowing the inward passage of nutrients and growth or trophic factors from the external wound environment while preventing the inward migration of scar-forming cells.  Growth or trophic factors, secreted in the wound-healing process external to the guidance channel, diffuse into the regenerating environment and are capable of supporting nerve regeneration. Controlled exchange between the intra- and extra-channel environment is important for the regeneration of severed peripheral nerves through synthetic guidance channels (Aebischer, 1988).

Bioabsorbability of Conduits

Non-absorbable conduits exhibit unfavorable behavior when used in nerve repair, because they should be removed after regeneration is complete (Aldini, 1996). Among the nonabsorbable conduits, silicone has been the most widely used for experimental tubulization and has also been applied clinically. However, as remains in situ after nerve has regenerated, it may cause a chronic foreign body reaction and late nerve compression with secondary complaints and impaired nerve function.

Because of inert and elastic properties, silicone tubing is one of the first and most frequently used synthetic material for nerve grafts. Clinical intubulation of regenerating nerves, however, often leads to long-term complications including fibrosis and chronic nerve compression, requiring surgical removal of the conduit. Despite diminishing clinical use, the silicone chamber, and other nonabsorbable materials as such as polyethylene, has been a tremendously useful model for studying nerve regeneration in vivo, and has allowed spatial and temporal examination of the regeneration process (Heath, 1998). The chronological sequence of nerve regeneration was also elucidated by silicone chamber experiments.

 Nerve guides made of biodegradable materials might overcome the problems non-absorbable conduits if, after allowing the outgrowth and maturation of the nerve, they are gradually degraded without significant deformation. The most important problem related to the achieving of a synthetic resorbable nerve prosthesis, besides the biocompatibility of the material, is to obtain a conduit with a rate of resorption that is not too fast, and a stability of the wall for a time adequate to warrant a space in which the regeneration and reorganization of the nerve will be effective (Aldini, 1996). If the nerve guide breaks down at an early stage, fibrous tissue can be formed inside the tube and impair further maturation of the regenerated nerve (Rodriguez, 1999). Moreover, if degradation occurs too fast, the regenerated axons may suffer from mechanical or biochemical damage. The time required for regeneration will be a function of the nerve location, the species and age of the patient, and the gap length (heath, 1998). For example, based on information gained from silicone-chamber model, a biodegradable graft for 10 mm gap in the rat sciatic nerve should maintain its strength for eight weeks or longer in order to ensure that axons have entered the distal stump and been myelinated (Heath, 1998). In addition the graft should be flexible and its wall should have thickness sufficient to hold a suture connecting the nerve epineurium and the graft (Heath,1998). To avoid nerve compression, the inner diameter of the graft should be large enough to accommodate polymer swelling during degradation.    

Another important problem with resorbable nerve guides is the inflammatory response of body against the degradation products. The products of degradation should not cause foreign body reaction or inflammation in the host. Cellular activity during the process of degradation may cause deleterious effects on the regenerated nerve.

Bioabsorbable grafts also have significant advantage in that, as they degrade, they can they can be made to release growth and trophic factors trapped in adsorbed to the polymer.  

Ideally, a nerve guide should be composed of a biocompatible, bioresorbable material that degrades at a controlled rate in accordance with the rate of axonal growth and maturation, maintaining mechanical continuity and lumen stability for longer time than required for axons to cross the gap (Rodriguez, 1999).

There are varieties of resorbable synthetic polymeric materials used in nerve regeneration as guidance channel. Among them are polygylcolic acids, poly(organo)phosphazine and poly(L-lactide-co-caprolactone) have yielded good results.

 Poly-3-hydroxybutyrate (PHB) is one of the resorbable materials used as nerve conduit as an alternative to autograft in nerve gap repair (Hazari, 1999). PHB occurs within bacterial cytoplasm as granules. It is available as bioabsorbable sheets, which are non-antigenic, easy to handle have good tensile strength. PHB undergoes hydrolytic degradation and is completely absorbed in 24-30 months. It elicits a low macrophage reaction comparable to that of a nerve graft. They used the material to repair a 10 mm gap in the rat sciatic nerve by comparison with an autologous nerve graft of the same length and position. They observed slower rate regeneration (measured by regeneration distance) in the first 1-2 weeks after implantation, however, after 30 days, all regenerating fibers reached the distal end. Also, there was a linear increase in the amount of axonal regeneration through time, and level of regeneration was slower in PHB, the difference being significant by 30 days. The inflammatory reaction to PHB was measured by macrophage counts and no significant difference was observed at all stages of regeneration process. The rate and amount of regeneration in PHB conduits does not fully compare with that observed in nerve graft, but this difference is due to the cellular elements inherently present in nerve grafts which aid regeneration. It is stated that level of regeneration in a PHB conduit may be further improved by the addition of growth factors.  

Polylactides are a class of polymers with a broad spectrum of properties, as biocompatibility and biodegradability, which make them suitable for many different applications. It has been used extensively as a nerve conduit in peripheral nerve regeneration. It offers several advantages including 1) a fully degradable conduit that will be replaced by myelinated axons at variable times, 2) a porous scaffold to allow vascularization, 3) a conduit that can be varied in length and luminal diameter while maintaining structural integrity and flexibility, 4) a unique fabrication process that reproduces consistent geometric conformity and added stability, and 5) a functional model to test the conduit in an attempt to restore normalcy (Evans, 1999). Evans et al. (1999) used poly(L-lactic acid) PLLA conduits to repair a 10 mm sciatic nerve defect in rats. They have seen an increasing regeneration over the 16 weeks of placement in both the conduit and isograft control groups, with control values significantly greater. The nerve fiber density in the distal sciatic nerve for PLLA conduits was similar to that for control isografts at 16 weeks. The number of axons/mm² for the PLLA conduits was lower than that of isografts at 16 weeks (Fig.5).

However the results for PLLA were significantly improved over those for 75:25 poly(DL-lactic-co-glycolic acid) (PLGA) of a previous study that they carried out. The results for PLGA demonstrated adequate distal nerve innervation and axonal growth was not inhibited by polymer degradation, however, elongation and partial collapse have led them to search for a different polymer as an alternative conduit for guided nerve regeneration. They have found PLLA tissue guidance conduits 10 times stronger and stiffer.    

Fig.5: Light micrographs demonstrating (a) the native sciatic nerve in the Sprague Dawley rats, (b) distal sciatic nerve at 16 weeks in control isografts, and (c) distal sciatic nerve at 16 weeks in the PLLA group.

In order to modulate the mechanical properties of the lactic acid polymers, a suitable comonomer appears to be 6-caprolactone (ε-caprolactone), by which it is possible to prepare a diversified family of biodegradable materials, comprising soft and gummy compositions (Perego, 1994). They have produced a biocompatible, biodegradable nerve guide form the copolymer of poly(L-lactide-co-6-caprolactone) (PLC). The guide has good mechanical properties, easy application, high biocompatibility and complete and benign bioabsorbability. Gardino et al. (1999) used a PLC conduit in peripheral nerve regeneration and they obtained a good axonal regeneration and the restoration of the nerve trunk continuity, similar to that observed with autologous grafts. The conduit slowly degrades in about 6 months, a time sufficient for complete regeneration of the nerve trunk.

Rodriguez and his coworkers (1999) compared regeneration and functional reinnervetion after sciatic nerve resection and tubulization repair with bioresorbable guides poly(L-lactide-co-ε-caprolactone) (PLC) and permanent guides of polysulfone (POS) with different degrees of permeability leaving a 6 mm gap in different groups of mice. They used five groups of animals with: a) bioresorbable non-permeable tubes of PLC, b) bioresorbable tubes of PLC of high permeability, c) bioresorbable tubes of PLC of low permeability, d)durable tubes of POS with MW cut-off of 30 kDa, and e) durable tubes of polysulfone with MW cut-off of 100 kDa. They have observed that bioresorbable PLC guides provide better conditions for nerve regeneration than durable POS guides. Moreover, by using PLC tubes of high permeability regeneration and reinnervation was considerably improved with respect to impermeable or low-permeable tubes. The reason for this improvement are still unknown, although it may be hypothesized that resorbable tubes allow for better nutrient supply to the regenerated nerve, enhance the constitution of the initial matrix and the subsequent nerve cable, and increase their flexibility as they degrade, thus avoiding secondary damage to the maturing regenerating nerve. The PLC guides that they implanted are well tolerated by the host tissue, elicit only a mild foreign body reaction, and have a low resorption rate for over 6 months, a period long enough for regenerated nerve to be well formed over a long gap. The surface of the PLC tubes, although not as smooth as POS or silicone did not exert a negative influence. They observed that regenerated cable was centered in the guide lumen and not adhered to the wall, and number of regenerated axons was considerably higher than in POS and silicone guides. PLC tubes of high permeability also maintained good mechanical stability. In fact, the results obtained by highly permeable PLC tubes are similar to those found with a sciatic autograft of the same gap length.

Fig. 6: Micrographs of transverse sections of successfully regenerated nerves through nerve guides. (a) impermeable PLC, (b) highly permeable PLC, (c) low permeable PLC, (d) 30 kDa semipermeable POS guides. Note the fiber grouping in small fascicles and the changes in fiber density and myelin thickness.

Grafts may also be constructed of two layers of the same or different material, to achieve the desired support and retention properties (Heath, 1998). Hoppen et al. constructed a synthetic biodegradable nerve guide of two polymeric layers: an inner microporous layer prepared from a copolymer of L-lactide and ε-caprolactone (pore size rage 0,5-1μm) and an outer macroporous layer prepared from a polyurethane / poly(L-lactide) mixture (pore size range 30-70 μm). Microporous inner layer was used to allow the transport of tissue fluids and to prevent ingrowth of perigraft scar tissue. The outer layer was made up of a macroporous polymeric material to facilitate the biodegradation of the nerve guide. The material possess good elastic properties and biocompatible. This nerve guide was used to bridge a 7 mm gap in the right sciatic nerve of the rats. The regenerated nerve cable was observed eight weeks after implantation and it contained myelinated axons as well as unmyelinated axons interspersed by Schwann cells, endothelial collagen and capillaries. The nerve guide was also found to prevent neuroma formation. The guide was effective in re-establishing the contact between the proximal and distal nerve ends and motor functions are recovered after 8 weeks. It functioned as effective as an autograft.

Incorporation of Neurite-Promoting Factors into the Guides

Nerve conduits can be divided into ‘inert’ and ‘active’ on the basis of their behavior. Inert conduits solely assure a structure in which a process of nerve regeneration can develop, while active conduits release various factors by diffusion that may enhance regeneration.

Polyphosphazenes are also used as nerve conduit with their degradability and good biocompatibility (Aldini, 2000). Poly-(organo)-phosphazenes are prepared from aminoacid substituents slowly degrade in water with the release of non-toxic products that are excreted or metabolized. It is also possible to incorporate drugs into the polymer and thus obtain their controlled release during polymer degradation. The experimental use of promoting factors in association with the nerve conduit has been widely studied. Among these substances there are nerve growth factors, insulin-like growth factors, laminin and neurotrophic factors. The entrapment of a promoting factor in the polymer mesh and release during degradation allows a prolonged bioavailability of the factor during the regeneration process. Poly-[organo]-phosphazene conduits therefore give new possibilities when compared with the commonly used nerve guides. However the physico-chemical features of phosphazenes do not permit a reduction in the thickness of wall below 500 μm (compared with that of 175 Um in PLC guides). This made it harder to insert the suture threads under the operating microscope.

Poly-[bis-(ethylalanate)-phosphazene], a poly-[organo]-phosphazene, is used as a nerve guide to repair 10 mm sciatic nerve defect in rats. The conduit was found to be as effective as polylactide-caprolactone copolymer for guided nerve regeneration, while polyphosphazenes allowed the use of neurite-promoting factors (Aldini, 2000). The conduit has demonstrated an initial stability followed by progressive and slow degradation, which is enough to allow reorganization of the nerve trunk in its lumen. No sign of local or general toxicity are observed and it allows regeneration of nerve fibers in its lumen to occur.

Many of the neurotrophic factors that have been considered for controlled release are made by Schwann cells, which serve several important roles in nerve regenration, as discussed earlier. Thus, a logical alternative to controlled release of growth factors is to add Schwann cells directly into the lumen of a biodurable or bioabsorbable artificial nerve graft. The first experiments to explore artificial nerve grafts seeded with Schwann cells were carried out in the rat sciatic nerve. Although the heterelogous cells elicited a strong immune response that hindered regeneration, the syngeneic Schwann cells increased the number of myelinated axons in a densitydependent fashion (Heath, 1998). Bioartificial nerve grafts containing Schwann cells are being used to overcome the 10 mm gap barrier beyond which conduits have not been successful in regenerating axons. Schwann cells forming a monolayer on the inner wall of the polyethylene conduit permitted the regeneration of the axon over a 20 mm gap in the rat sciatic nerve. Schwann cells suspended in gelatin within the lumen of polyglycolic acid conduits have supported nerve regenration over a 30 mm gap in the peroneal nerve of the rabbit (Heath, 1998).    

Effect of Surface Microgeometry

The luminal surface microgeometry of tubes used to repair transected nerves also exert strong influences on the patterns of regeneration across the nerve gaps. Differences in surface microgeometry are likely to produce different nerve cable reactions as regards the contacting biomaterial surface and, therefore, a different nerve regeneration outcome can be expected. Rough inner surface tubes elicit an intense inflammatory response in comparison with tubes featuring a smooth inner surface. Macrophages, fibroblasts, red blood cells and degenerating white blood cells are found in the rough tube. A fibrin matrix arranged with a longitudinal orientation, which favors regeneration of the nerve cable, is found in the smooth tube. In this case, the nerve cable is centered, free from attachment to the tube wall and contains blood vessels; also the axons are grouped in microfascicles and surrounded by an epineurium (Aebischer, 1990). It may be hypothesized that the microgeometry of the inner surface of the nerve guidance channel modulate the nerve regeneration process by altering the protein and cellular components of the regenerating tissue bridge. Channels with rough inner surface may alter the mechanism of fibrin gel formation. The fibrin, instead of forming a longitudinally oriented loose matrix connecting the nerve ends; draw together the particles of the dispersed phase of the gel. As a result, cells, migrating in the fibrin gel from the nerve stump disperse in the entire lumen rather than a discrete central structure (Soldani, 1998).

 In a study made by Aebischer and his coworkers (1990), synthetic guidance channels (acrylonitril vinylchloride copolymer) with identical polymeric composition and molecular weight cut-offs but with smooth, rough or alternating smooth-rough or rough-smooth inner surfaces were used to bridge a 4-mm sciatic gap in adult mice. The results show that, smooth-walled tubes support the regeneration of a nerve cable containing numerous myelinated axons  grouped into microfascicles. The cable never contacts the tube’s smooth inner wall and the neoepineurium is composed of a continuous ring of interdigitating fibroblast-like cells and collagen fibrils. In contrast, rough-walled tubes contain only a loose connective tissue stroma and few or no myelinated axons. The regenerated tissue completely fills the tube lumen and no cellular sheath delineates its outer perimeter. A critical event in regeneration across a gap is the formation of a fibrin cable bridge, which serves as a scaffold for migrating cells and elongating axons. Fibrin strands orient according to the luminal structure of the tubes. A longitudinal stump-to-stump matrix coalesces in the smooth tubes and an unorganized matrix intermingled with the rough trabecular network forms in the rough tubes. Thus, cells migrating into rough-walled tubes encounter a random substrate and never organize into a nerve-like cable. Other important factors regulating cable formation include cytokines, which are synthesized and released by host cells, especially macrophages in response to nerve injury, the surgical wound and implanted biomaterial. In the rough-walled tubes used in this study, greater numbers of macrophages and reactive cells are seen within the trabecular structure as well as within the regenerated tissue. This may be result of host tissue reaction to the complex physical structure and/or larger surface area of the rough tubes as compared to smooth tubes. Activated macrophages have been shown to release various factors including NGF, FGF and interleukine 1, which is a primary mediator of inflammatory response, in particular the formation of granulation tissue. In rough tubes excessive release of interleukin 1 may favor the loose granulation tissue, which impedes the inward migration neuronal elements. According to these results, they suggest that surface microgeometry of guidance channels influence the outcome of peripheral nerve regeneration, potentially by effecting the early arrangement of fibrin matrix and/or inducing different cellular responses.

Soldani et al. (1998) have manufactured a polyurethane nerve guidance channels featuring a highly smooth internal surface by the method of sequential ‘dipping’ and ‘spray thermal-heating’ (STH). The surface of the material they obtained was much more smooth than the silastic (silicone elastomer) samples.

            CONCLUSION

            By the development of artificial nerve guides, the recovery from the nerve injuries, especially if the gap is long, are made possible. Various polymeric materials have been tried for the improvement of nerve regeneration. The success of the regeneration varies according to the properties of the material used. Biodegradable materials have been proved to improve nerve regeneration better than the non-degradable polymers, as there is no need for the second surgical step to remove a biodegradable material. However, the material should degrade after the nerve is completely regenerated. Moreover, durable nerve guides may cause chronic inflammatory response over long term presence in situ. Another important property shown to be effective in facilitating nerve regeneration is permeability. It is proved that permeable tubes improve nerve regeneration by permitting the exchange of material between the inner and outer compartment of the nerve guide chamber. These materials include nerve growth factors, fibroblast growth factors, Schwann cells etc. that are shown to promote axonal regeneration. Recently, artificial nerve guides are started to be used in conjunction with the biological nerve guides, which possess intrinsic nerve promoting agents. Artificial nerve guides seeded with Schwann cells have also given good results.

            With the addition of neuritis promoting factors into a biodegradable nerve guide, the nerve regeneration has been facilitated. As the material degrades, it releases its content into the guide lumen, promoting the neuritis elongation.

            It seems that a best choice for a nerve guide would be a composite of biological and artificial materials, having good mechanical properties, biocompatible, biodegradable (with an optimum rate) and supplying neuritis-promoting factors to the outgrowing cells.             

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