NERVE TISSUE ENGINEERING

Suzan BER

Introduction

The nervous system is a wired communication system of the body. [4] The fundamental component of the human nervous system is the neuron. It consists of a branched cell body, the shorter extensions to be dendrites and the longer one the axon. Signals are conducted from the dendrites to the axon. The various parts of the nervous system are interconnected, but for convenience they can be divided into two parts which differ in their physiology and function:

1) The Central Nervous System (CNS), composed of the brain and the spinal cord;

2) The Peripheral Nervous System (PNS), consisting of the nerves which extend from the brain and the spinal cord out to all points of the body.

Peripheral neurons consist of a cell body and a long process, or axon which may reach one meter in length. Short segments of the axon are wrapped with an insulating myelin sheath formed by Schwann cells, which also serves several important roles in the axon-regeneration process. [1] (Fig.1) In the CNS, however, the supporting cells are astrocytes and olygodendrocytes. (Fig.2) They maintain the extracellular environment to best suit and nourish the neighbouring neurons. [3]

Figure 2: The neuron of CNS

Nerves can be damaged by trauma caused by compression of nerves, or cuts as a result of accident, or by disease like sclerosis, diabetes, polio. [4]

Unlike the robust regenerative response of the peripheral nervous system (PNS) to injury, trauma to the adult mammalian CNS leads to permanent disability with little or no functional regeneration of injured axons.

WHY BODY INHIBITS ITS REGENERATIVE ABILITY?

Classic descriptions of injury to the CNS suggested for many years that regenerative failure was attributable primarily to a structural barrier to axon growth, a glial scar composed of astrocytes and connective tissue. [2]

Glial cells, or astrocytes, are recognized not only for their supportive properties, but also for establishing functional boundaries important for guiding developping neuronal connections. They play an integral role in axon guidence during embryogenesis by providing structural or molecular cues that repel growing axons or redirect them toward other pathways. [2]

Recent studies indicate that astrocytic scar that often forms following injury does not prevent axon growth simply by mechanical mechanism. Both tenascin and certain proteoglycans are upregulated following injury to the CNS. Tenascin levels increase following trauma to the brain and the spinal cord. Similarly, chondroitin sulfate proteoglycans persist in the extracellular matrix of the CNS following injury. Post-injury responses thus include the production of the same type of boundary molecules that have been described as having axon inhibitory functions during the development of NS. The experiments in vivo demonstrate that increases in chondroitin sulfate proteoglycans after injury are associated with breakdown of blood brain barrier and infiltrating macrophages at the lesion site, suggesting that serum factors or inflammatory cytokines play a role in the molecular cascade leading to extracellular matrix production in the immediate vicinity of the developing glial scar. [2]

Proteoglycans may influence the normal environment of the adult CNS to favor the inhibition of axon growth in an attempt to maintain normal synaptic connections. Therefore, the upregulation of inhibitory molecules after injury may be one mechanism that the adult NS uses to prevent aberrant growth of axons and the formation of inappropriate connections They may serve to hinder long distance axon growth by binding and functionally removing important growth signals from injury site. This response of the mature CNS to injury may represent a “walling off” of injured tissue as a protective response for the healthy tissue surrounding the wound epicenter. Since these molecules are known to inhibit the phagocytosis and destruction of b-amyloid protein (which triggers the production of inhibitory ECM molecules) by macrophages, an attractive hypothesis is that proteoglycans have a protective function to prevent secondary damage within the CNS after trauma, thus limitting the devastating process of progressive necrosis. The inhibitory properties of these molecules on axonal growth may simply be an unfortunate side-effect of a normal wound-healing response of CNS tissue. Strategies to modulate the production of these molecules may be one way of approaching the enhancement of the regenerative response by adult neurons in future interdisciplinary approaches to the therapy of CNS injury. [2]

A variety of methods has been proposed for nerve reconstruction, depending on how far the stumps are from each other. Direct suturing of the damaged nerve is possible, provided that the gap is small. If it is large, larger than 3 cm in human, nerve segments can be taken from the patient (autograft) or from a donor (allograft) in order to suture the stumps without tension. [10] Disadvantages of the nerve autograft include a second surgical procedure, limited availability and permanent denervation at the donor site. Allografts, on the other hand, are accompanied by the usual need for immunosuppression and have very poor success rates. [1] Avoiding problems of availability and immune rejection, a promising alternative for extending the length over which nerves can successfully regenerate is the artificial nerve construct. [1]

Two important components in nerve tissue engineering are a scaffold made up of both natural and synthetic materials and provide structural support for axonal growth; and support cells, which offer a highly preferred substrate for axon migration and release bioactive factors that further enhance nerve migration. ( Fig. 3)

Figure 3

SUPPORT CELLS

Although autologous cells are excellent source for use in tissue engineering, they are fully differentiated cells and as such they have a decreased ability to proliferate. A pluripotent, highly proliferative cell source obtained from human fetal tissue from which a variety of cell types could be derived became extremely useful in tissue engineering. Many adult tissues contain stem cell populations with the ability to repair tissue after disease or trauma. Additionally, a cell population isolated from human bone marrow, “the mesenchymal stem cells”, has been induced to differentiate into different kind of cells under different cultural conditions. [5] Using this knowledge of stem cells, scientists are using those cells in nerve degeneration.

Researchers are pursuing two fundamental strategies to exploit this discovery:

1) To grow in a laboratory dish differentiated cells that are suitable for implantation into a patient by starting with undifferentiated neural cells. The idea is either to treat the cells in culture to nudge them toward the desired differentiated neuronal cell type before implantation, or to implant them directly and rely on signals inside the body to direct their maturation into the right kind of brain cell.

2) The other repair strategy relies on finding growth hormones and other “trophic factors” -growth factors, hormones and other signalling molecules that help cells survive and grow- that can fire up a patient’s own stem cells and endogenous repair mechanisms, to allow the body to cope with damage from disease or injury. [3]

Efforts to develop stem cell therapies for Parkinson’s Disease provide a good example of research aimed at rebuilding the central nervous system. Parkinson’s is a progressive movement disorder that usually strikes after age 50. Symptoms begin with uncontrollable hand tremor, followed by increasing rigidity, difficulty in walking. The symptoms result from the death of a particular set of neurons connecting a structure in the brain called the substantia nigra to another structure called the striatum. These “nigro-striatal” neurons release the chemical transmitter dopamine onto their target neurons in the striatum. Dopamine’s major role is to regulate the nerves that control body movement. As a result of the death of those neurons less dopamine decreased leading to movement difficulties. (Figure 4) [3]

Figure 4: Neuronal Pathways that Degenerate in Parkinson’s Disease

By the early 1980s Anders Bjorkland and others had shown that transplantation of fetal tissue into the damaged areas of the brains of rats and monkeys used as models of the disease could reverse their Parkinson’s-like symptoms. The promising animal results led to human trials in several centers worldwide, starting in the mid-1980s. Although not all patients improved, in the best cases patients receiving fetal tissue transplants showed a clear reduction in the severity of their symptoms. Also, researchers could measure an increase in dopamine neuron function in the striatum of these patients by using a brain-imaging method called position emission tomography (PET). (Fig. 5) [3] Also, autopsies done on the few patients who died from causes unrelated to either Parkinson’s or the surgery revealed a robust survival of the grafted neurons, the grafted neurons sent outgrowths from the cell body that integrated well into the normal target areas in the substriatu

Figure 5: PET images from a Parkinson’s patient. Twelve months after surgery an image from the same patient( right) reveals increased dopamine function

Brain is one of the organs that have stem cells located within it. Stem cells in adult brain occur in two locations. One, the subventricular zone, is an area under fluid-filled spaces called ventricles. The other is the dentate gyrus of the hyppocampus. Researchers showed that (the mid-1990s) when the brain is injured, stem cells in these two areas proliferate and migrate toward site of the damage. James Fallon and his colleagues studied the effects on rat brain of a protein called transforming growth factor alpha (TGF a) – a natural peptide found in the body from the very earliest stages of embryonic development onward that is important in activating normal repair processes in several organs. Fallon’s studies suggest that the brain’s normal repair processes may never be adequately triggered in a slowly developping degenerative disease like Parkinson’s and that providing more TGF a can turn it on. They make injections of TGF a into rats in which they first damage the nigro-striatal neurons and two important things take place. After several days they observed stem cells migration to the damaged areas, where these differentiate into dopamine neurons. Second and most importantly, the treated rats do not show the behavioral abnormalities associated with the loss of neurons. [3]

SCAFFOLDS

To be successful, the nerve graft must be integrated into the surrounding tissues and must guide the nerve fibers between two ends of damaged nerve. It’s also expected to prevent fibrous tissue from invading the nerve gap, to be stable as long as the regenerating nerve fibers are not mature enough, and finally to disappear rather than being removed, to avoid the risk of injuring the repaired nerve. [10]

To constitute an artificial nerve grafts both biodegradable and non-biodegradable materials have been used:

Non-biodegradable Artificial Nerve Grafts: Because of its inert and elastic properties, silicon tubing was one of the first and most frequently used synthetic materials for nerve grafts. Early experiments using silicon chambers indicated that regeneration could occur. During this process, initially the fluid from the nerve stumps, containing neurotrophic compounds fills the chamber, within a week, a fibrin bridge forms between stumps, and fibroblasts begin to infiltrate the bridge from both ends. By the second week, Schwann cells and axons begin migrating along the bridge. Finally, over a period of two to eight weeks, Schwann cells myelinate the axons. In order to enhance regeneration further, the artificial nerve graft may be filled with neurotrophic substances, also with phosphate-buffered saline (PBS) or dialysed plasma. Supplementing the fluid with growth factors and exogenous matrix precursors, many of which are produced by Schwann cells, has also been known to promote regeneration across larger gaps. Clinical intubulation of regenerating nerves, however, often leads to long-term complications including fibrosis and chronic nerve compression, requiring surgical removal of conduit. [1]

Biodegradable Artificial Nerve Grafts: A graft made of biodegradable materials is a promising alternative for promoting successful long-term recovery, because, after serving as an appropriate scaffold for regeneration, the conduit eventually degrades.

Polyglactin grafts resulted in an inflammatory response and poor nerve regeneration. Better results have been obtained with poly (L-lactide-co-caprolactone), polyglycolic acid, poly (organo) phosphazine conduits. A microporous polyglycolic acid mesh coated with cross-linked collagen to promote tissue proliferation, also successfully supported nerve regeneration.

The most important concern in designing a resorbable graft, apart from biocompatibility, is choosing a material and processing conditions that will result in a graft that degrades slowly enough to maintain a stable support structure for the entire regeneration process but will not remain in the body much longer than needed. The graft should be flexible and its wall should have a thickness sufficient to hold a suture connecting the nerve epineurium and the graft.To avoid nerve compression, the inner diameter of the graft should be large enough to accomodate polymer swelling during degradation, which is seen with some polymers.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 cutoff of approximately 50,000 has been found suitable to allow diffusional transport of nutrients and other molecules while preventing cells from entering the conduit. Angiogenesis in the graft will also provide nutrient transport to the regenerating axons. [1]

Biodegradable grafts also have the significant advantage that, as they degrade, they can be made to release growth or trophic factors trapped in or adsorbed to the polymer. With controlled release, compounds with short in vivo half-lives can be supplied slowly to the regenerating axons over the life of the graft. [1]

A logical alternative to controlled release of growth factors and /or waiting for Schwann cells normally migrate into graft lumens is to add Schwann cells directly into the lumen of the graft. The reason is because they secrete neurotrophic factors and express cell-adhesion molecules that enhance regeneration; they form an endoneurial sheath, which serves as a guide for axonal growth; they aid in clearing debris and create a suitable environment for nerve growth; they myelinate axons. Experiments has shown that Schwann cells stimulate axons to elongate faster and over longer distances than is possible on acellular matrices.[1]

Some Biomaterials Used for Nerve Tissue Engineering

Experiments on leeches, in the animal, in isolated ganglia and on individual neurons grown in 3-D collagen gels, have shown that repair after damage is specific so that neurons become reconnected with a high degree of precision to restore the function that was lost when the damage occured. Three-dimensional culture systems have different advantages. Collagen, which they used in this experiment to construct 3-D lattices, is a major constituent of the ECM in mammals and in leeches. It has been used as a substrate for cultured mammalian cells for many decades, and there is an extensive literature describing the effects of collagen substrates on the morphology, migration, adhesion, differentiation and growth of mammalian cells. Collagen lattices are now widely used to reconstruct extracellular matrices since the three dimensional network of collagen fibers allows 3D growth by cells that is more in vivo –like than growth on the two-dimensional surface of a culture dish.

Single ganglia, chains of ganglia and isolated lengths of connectives dissected from leech were maintained in 3D collagen gels, prepared from type I collagen isolated from rat tails, for up to four weeks without contraction of the gels. An early regenerative response was the migration of cells into the gel matrix from the cut ends of nerve roots and from connectives. First migrating cells are likely to be microglial cells which migrate toward the site of injury, and express laminin at the lesion site. Regenerative growth of axons into the gel from the cut ends was consistently seen within 2-3 days of culture. (Fig. 6) The collagen gel cultures are stable, nerve cord and target tissue can be aligned and held in position without the need to pin tissue, and exogenous substances such as growth factors or antisense molecules can be incorporated into the gel or added to the overlying medium without disrupting the regenerative fibers. [17]

Figue 6: Nerve cord repair within the gel. Successive stages days 0-7.

In another experiment using collagen, researchers developed a nerve guide made of collagen filaments to improve permeability of the material employed. The collagen filaments were made by highly purified type-I collagen. The filaments were sterilized using polyethylene glycol diglycidyl ether, as the crosslinking agent, which crosslinked Î-NH₂ groups of collagen molecules. The crosslinked collagen is resorbed more slowly compared to non-crosslinked one and resists bacterial collagenase digestion. Filaments were further sterilized by UV. (Fig. 7)

Figure 7: SEM of collagen filaments Figure 8: Collagen-filament graft

Arrows indicate myelinated axons

Under deep peurobarbital anaesthesia, the right scratic nerve of rat was exposed from the scratic notch to the popliteal region, and 20 mm segment of the tibial division of the nerve was removed. The proximal and distal nerve stumps were sutured to the collagen-filament nerve guide with two sutures using 10-0 monofilament nylon epineurinal sutures to bridge the nerve defect.

Myelinated axons of the rat sciatic nerve had regenerated 20 mm along collagen filaments without a tube or neurotrophic additives by 8 week postoperatively. There were as many regenerated myelinated axons in the collagen-filaments nerve guide as there were in the nerve autograft. (Fig. 8) It has been reported that increased permeability improves axonal regeneration. A nerve guide made of filaments without a tube has high permeability. It is opened to the surrounding extracellular matrix. [6]

Studies of synthetic polymers as materials for nerve conduits have also been performed. Polymer conduits were constructed with poly (DL-lactic-co-glycolic acid) (PLGA) foams fabricated by a solvent casting particulate leaching technique using NaCl as the leachable component. The tubular conduits were interposed into the right sciatic nerve defect of 20 Sprague Dawley rats. Functional evaluation was performed monthly. All conduits remained flexible, allowing mobility of the rat extremity without breakage of conduit. However, partial collapse of the conduits and elongation to 18 mm was noted, due to conduit degradation.

Because of collapse and elongation of PLGA conduits, another alternative polymer substrate PLLA was used. It was similarly designed and substituted for the in vivo trials. Conduits appeared to have maintained their stability and were more rigid. The results for PLLA were significantly improved over those for PLGA. Throughout all time periods, the PLLA conduits remained structurally intact and demonstrated tissue incorporation and vascularization. [7]

One of the studies aiming to promote tissue regeneration across large lesions of the brain and spinal cord, focuses on the porous hydrogel, as polymer scaffolds for neural tissue repair and regeneration. They developed the NeuroGel, a biocompatible hydrogel of poly [N-2 (hydroxypropyl) methacrylamide] (PHPMA) to provide a framework for tissue repair of the CNS. The swelling property of this polymer when in contact with host neural tissue determines its transport properties, which allow it to maintain both a chemical balance and physiologically suitable environment for the growth of host cells. Another important property of NeuroGel is its multimodal size distribution compromised of micropores (< 2 nm), mesopores (>2 nm, < 50 nm), and macropores (>50 nm < 300 mm). These pore dimensions allow a wide range of transport from small molecules to large molecules, cells, blood vessels. Other important feature is the viscoelastic characteristics of NeuroGel, which match those of the host tissue, and its low interfacial tension for biologic fluids and structural stability. Tissue formation is encouraged because of the large surface:volume ratio and the high fractional porosity of the hydrogel. Rats that received a severe spinal cord injury using a balloon catheter showed significant improvement following treatment with NeuroGel. (Fig. 9)

Figure 9: SEM of cross-section through a PHPMA hydrogel containing nerve cells.

In a recent study biohybrid hydrogel systems were studied. These combine polymer hydrogels with living cells. Charles Vacanti at the University of Massachusetts, together with his students, is trying to combine two techniques. The one is molding the polymer scaffold (any polymer) in the shape of tissue, and the other is to immerse a liquid hydrogel containing tissue precursor cells. As a result, the growing tissue will be in the same shape with the natural one. The polymer scaffold gives a strength and shape to the graft. The formation of a biohybrid hydrogel system relies on introducing developing cells into the reaction mixture prior polymerization. The liquid form of hydrogel-cell mixture is seeded into a polymer scaffold and when implanted into the body the hydrogel hardens, keeping the cells in place so that they are evenly distributed throughout the scaffold. (Figure 10) [19]

Figure 10

Polylactide foams also have been used as artificial nerve graft. Foams were prepared by freeze-drying polymer solutions in 1,4 dioxane or in 1,4 dioxane/water mixtures. Left sciatic nerve of a rat was cut. The polymer strip and 2 mm segment of both proximal and distal nerve stumps were enrolled with PLA film, which provides a better stability. (Fig. 11)

Figure 11: Nerve graft

After one month, the transplant and the two nerve stumps were connected to each other in many of the animals tested. PLA is a synthetic material, received FDA approval, which is known for biocompatibility and resorbability, combines lack of antigenicity, availability and versatility of the physiochemical properties. One disadvantage is its hydrophobicity, but it has been already overcome by immersing PLA foams into PVA (polyvinyl alcohol). [10] (Fig. 12)

Figure12: SEM of nerve cell seeded PLA

Numerous studies have highlighted the importance of ECM components like collagen, laminin, fibronectin, for appropriate cell migration during morphogenesis of the nervous system as well as for neurite outgrowth in vivo. As a result of this, surface coating of some polymers by these proteins or certain surface modifications has been done. [14]

In one study PLLA filaments fabricated by a melt extrusion process, were cut into 1 cm lengths, rinsed with phosphate buffered saline (PBS) (pH 7.2), and coated with laminin. Isolated DRG were cultured on laminin-coated PLLA filaments. As a result, coating the filaments with laminin greatly enhanced the attachment of DRG and subsequent neurite growth compared with uncoated ones. These filaments further can be made microporous and be loaded with neurotrophic factors, which are released as they degrade and thus acting as a neurotrophic reservoir. Another alternative is to seed activated Schwann cells onto the filaments. [11]

The goal of another study is to immobilize fibronectin on a serum-resistant surface in order to control neuronal attachment and neurite outgrowth in a ligand density manner. Fibronectin is a flexible heterogeneous glycoprotein composed of functionally specific domains. In this study they provide evidence that FN bioactivity is influenced by mode of presentation, i.e. cell attachment and neurite outgrowth on a range of FN surface concentrations was markedly different between adsorbtion and covalent immobilization.

FN was immobilized onto an end-group-activated polyethylene oxide (PEO)- containing copolymer, Pluronic^TM F108. PEO containing surface coatings are believed to effectively “buffer” proteins from the denaturing effects of the underlying hydrophobic material. Cell attachment can vary dramatically depending on the nature of the surface on which FN is deposited. This effect results from surface-induced conformational effects that either can expose or mask bioactive domains of FN-molecule. In observations it was seen that attachment values on F108-FN increases over the range of FN doses. Fibronectin is chosen because it contains several cell-adhesive domains, such as the RGD sequence and the CS-I domain, which are reported to participate selectively in neuronal attachment and neurite outgrowth. In this study it was shown that adsorbtion of FN to the surface, when compared with covalently immobilizing FN, shows poor neurite attachment, which is also related to the exposure of cell-adhesive domains in right conformation. (Fig. 13) [14]

Figure 13: A neurite extension on Fibronectin immobilized surfaces

In one study fibrin, a natural biomaterial of nerve regeneration, is modified by covalently incorporating exogenous peptides, containing heparin-binding domains, to enhance its ability to promote nerve regeneration. Cell adhesion is mediated by different types of cell-surface receptors and ligands in the ECM or on the surface of other cells. One class of adhesion domains are the heparin-binding domains, which possess the ability to bind heparin and other sulfated glycosaminoglycans, typically components of cell surface and ECM proteoglycans. Heparin binding domains are found in laminin, fibronectin, fibrinogen and some other proteins. The domains have been found to enhance neurite extension both from PNS and CNS.

Synthesized peptides containing heparin-binding domains are added to the coagulation mixtures and cross-linked to the fibrin gel during polymerization. For the test, DRGs of chicken embryos were dissected. Fibrin gels containing the peptides were put into wells containing DRG fractions and gels polymerize around the ganglia so that they are 3-D embedded within the gel. The heparin-binding domain has been shown to promote neurite extension. Furthermore, the neurite outgrowth induced by this domain could be inhibited by addition of heparin to the cell culture media, digestion with heparitinase, or inhibition of cell proteoglycan synthesis suggesting that the proteoglycan interacting with this domain was located on the cell surface. (Fig.14) [12]

Figure 14: Neurite extension on fibrin gels

Another study related with structural modifications is synthesis of PHPMA-RGD hydrogel. Previously described NeuroGel was functionalized by covalently grafting to the polymer backbone the synthetic peptide, which includes the cell adhesive sequence RGD.

RGD containing pseudo-peptide was covalently incorporated to the hydrogel. The monomer (HPMA), crosslinking agent MbisAA, polymerization initiator (AIBN), RGD peptides, TMAEM (a cationic agent used to neutralize the (-) charges of RGD) are used in synthesis of PHPMA-RGD hydrogel. The structure of PHPMA-RGD hydrogel displayed an interconnected porous structure, with viscoelastic properties similar to those of the neural tissue, and conductivity properties due to a peptide group. The polymer hydrogel provided a structural, 3-D continuity across the defect, facilitating the migration and reorganization of local wound-repair cells, as well as tissue development within the lesion. Angiogenesis and axonal growth also occurred within the microstructure of the tissue network. [13]

Chemically defined surfaces can be used to directly examine substratum-guided neuronal outgrowth in culture. Generally, substrates are coated with two different chemical components: one component with an adhesive property for cells, and the other with a non-adhesive property. In this study, glass cover slips are patterned with an extracellular matrix protein, laminin. A synthetic peptide P20, derived from a neurite-outgrowth-promoting domain of the B2 chain of mouse laminin (Fig. 15) was used for the surface modification.

Figure 15: Laminin domain

Glass cover slips were cleaned by immersion in a 20% (V/V) sulfuric acid solution overnight to remove organic residues from the surfaces, and then rinsed with deionized water. Glasses are immersed into aminosaline solution to obtain an amine-derivatized glass surface. Chemically modified surface is exposed to UV using a photo-mask (patterned chrome deposited on a quartz plate), which protects the aminosaline film surface from the exposure while the uncovered surface regions are irradiated. The irradiated regions showed a hydrophilic character, so those sites are denatured. Before attaching the synthetic peptide to the surface crosslinker is used (sulfo-GMBS). This crosslinker possesses an amine-reactive site at one end and a sulfhydryl-reactive site at the other. (Fig. 16)

Figure 16: Couplin of synthetic peptide to an amine modified glass surface

At the end the cystein-labeled peptide is added and bound to the crosslinker. (Fig. 17)

Figure 17: The patterning procedure

When hippocampal neurons were grown on substrates having alternating stripes of synthetic peptide and irradiated DETA surfaces, their morphology reflected the geometric pattern of the surface. (Fig. 18)

Figure 18: Extension of neurons on patterned surface

Many studies exist related with developing micropatterned surfaces. Those pre-formed pathways of differentiated adhesiveness are crucial in guiding neuronal growth to distant targets in vivo.

The use of biomaterials for tissue regeneration, such as the promotion of nerve healing, is a promising field of research. By developing materials with specific adhesive characteristics, it is possible to control and enhance the level of cell migration within the material. This may lead to more effective forms of therapy in many areas of tissue engineering, such as promoting better integration at the tissue-biomaterial interface, promoting cell infiltration, and controlling morphogenesis in tissue regeneration. [16]

REFERENCES:

1- Heath CA, Rutkowski GE. The development of bioartificial nerve grafts for peripheral-nerve regeneration. TIBTECH April (1998) Vol 16, 163-168.

2- Fitch MT, Silver J. Glial cell extracellular matrix : boundaries for axon growth in development and regeneration. Cell Tissue Res. (1997) 290: 379-384

3- http://www.nih.gov/news/stemcell/chapter8.pdf

4- http://biomed.brown.edu/Courses/BI108/BI108_2001_Groups/Nerve_Regeneration/Default.html

5- Fuchs JK, Nasseri BA, MD, Vacanti JP, MD. Tissue Engineering: A 21st Century Solution to surgical Reconstruction. Ann Thorac Surg 2001; 72 : 577-91.

6- Yoshii S, Oka M. Collagen filaments as a scaffold for nerve regeneration. J Biomed Mater Res 56 : 400-405, 2001.

7- Evans GRD, MD, F.A.C.S. Challenges to Nerve Regeneration. Semin. Surg.Oncol. 19: 312-318, 2000

8- Woerly S. Restorative surgery of the central nervous system by means of tissue using NeuroGel implants. Neurosurg Rev (2000) 23: 59-77.

9- Borkenhagen M, Clemence JF, Sigrist H, Aebischer P. Three-dimensional extracellular matrix engineering in the nervous system. J Biomed Mater Res (1998) 40: 392-400.

10- Maquet V, Martin D, Malgrange B, Franzen R, Schoenen J, Moonen G, Jerome R. Peripheral Nerve Regeneration Using bioresorbable macroporous polylactide scaffolds. J Biomed Mater Res(2000) 52: 639-651.

11- Rangappa N, Romero A, Nelson KD, Eberhart RC, Smith GM. Laminin-coated poly( L-lactide) filaments induce robust neurite growth while providing directional orientation. J Biomed Mater Res (2000) 51: 625-634.

12- Sakıyama SE, Schense JC, Hubbell JA. Incorporation of heparin-binding peptides into fibrin gels enhances neurite extension: an example of designer matrices in tissue engineering. FASEB J (1999) 13 : 2214-2224

13- S. Worley, E. Pinet, L. de Robertis, D Van Diep, M. Bousmina. Spinal cord repair with PHPMA hydrogel containing RGD peptides (NeuroGelTM ). Biomaterials 22(2001) 1095-1111

14- Biran R, Webb K, Noble MD, Tresco PA. Surfactant-immobilised fibronectin enhances bioactivity and regulates sensory neurite outgrowth. J Biomed Mater Res (2001) 55:1-12.

15- Matsuzawa M, Liesi P, Knoll W. Chemically modifying glass surfaces to study substratum-guided neurite outgrowth in culture. J Neurosci Meth 69 (1996) 189-196.

16- Schense JC, Hubbell JA.Three-dimensional migration of neurites is mediated by adhesion site density and affinity. J Biol. Chem Vol 275, Issue 10; 6813-6818: (2000)

17- Blackshaw SE, Arkison S, Cameron C, Davies JA. Promotion of regeneration and axon growth following injury in an invertebrate nervous system by the use of three-dimensional collagen gels. Proc. R. Soc.Lond. B (1997) 264, 657-661.

18- http://guide.stanford.edu/People/sabelman/99nerve.pdf

19- http://www.techreview.com/magazine/may01/patents4.asp