[ Back to index of term-papers ]

Nerve Tissue Engineering

Nihal Engin VRANA

Definition of Tissue Engineering

            Tissue engineering is an interdisciplinary engineering branch.Primary aim of tissue engineering is the reconstruction of biological tissues by using knowledge stem from the life sciences and utilizing general engineering methodology.Definition quoted below is the first official definition declared in 1988 at U.S.A.  National Science Foundation workshop;

            “The application of principles and methods of engineering and life sciences toward fundamental understanding of structure-function relationships in normal and pathological mammalian tissues and the development of biological substitutes to restore, maintain or improve tissue function”

            Main promise of Today’s tissue engineering research is the possibility of creating completely functional living tissue that would replace failing organs. First approach for this end is the cell cultures. A 3-d scaffold which consists of  building materials like extracellular matrix or biodegradable polymer is prepared and seeded with living cells that are taken from a donor site.( with a small biopsy)Thus with aid of growth factors ex vivo proliferation of cells is meant to be achieved. Cells increase in number in the scaffold; they start to fill the scaffold’s pores and assume a 3-d shape. These cultured cells can be implanted to a part of the body and they can take up the place of a disrupted tissue (Of course cultures are prepared by considering the final destination of them. Hence selection of progenitor cells and growth factors are dependent on the desired tissue). Revascularization of the artificial tissue occurs in time; Meanwhile scaffold in which cells proliferated starts to disintegrate; leaving place for the growth of extracellular matrix and   finally(Also hopefully) introduced artificial tissue becomes an integral part of the body and functions as good as the natural one.

            A second approach is the development of engineered elements that would directly take over the function of the malfunctioning tissue, organ (Like cochlear implants) or that would facilitate the natural regeneration processes (Like nerve guidance conduits)

            Still another approach is the construction of novel tissues or improvement of inherent ones. Although this kind of research is quite rare and its aims seem so far away; with increasing elaboration of cellular mechanisms it will be possible to conduct such daring attempts to alter the structure of living organisms.

            Remaining part of this review will focus on nerve tissue engineering, its intentions, methods, shortcomings and recent advances.

Nerve Tissue Engineering

            The nervous system is divided into two structural components; central nervous system and peripheral nervous system. Together they regulate body activities, control body posture and movements, permit accumulation of experiences and control behavior. Central nervous system is composed of Brain and spinal cord; whereas PNS is the total of nerves and ganglia all around the body.

      Rehabilitation of nerve injuries is complicated with the fact that the mature nerve tissue do not replicate. So impairment of any part of nervous system results in both malfunctioning of itself and also the other parts of the body. Especially CNS damages have dramatic effects; since regeneration in CNS is near to none. In PNS axons are able to regenerate but they can cross only a certain distance, and in most cases; recovery of function is not complete or function is totally lost.

      Defects in nervous system can be due to accidents, damages inflicted during medical processes (Like removal of tumors), autoimmunesytem diseases (Like Multiple sclerosis) or diseases like Huntington’s or Parkinson’s. Location and gravity of impairment is the most important parameter; and each case needs different techniques. Some of these techniques are;

1) Nerve Guidance Conduits

      Mainly employed in peripheral nerve injuries; when gap between proximal and distal end is larger then the critical distance (Critical distance can be defined as the gap for which full recovery by natural nerve regeneration is expected)

      Several organic, inorganic materials can be used as conduit material. They are reviewed in a subsequent part of the report

2) Nerve grafts- Cell Transplantations

      Grafts can be used for peripheral nerves for large gaps due to injury. Generally autologous grafts; taken from the patients body; in most cases harvested from sensory nerves from sural portion. Cell transplants, Transplantation of cultured cells in a biomaterial scaffold, can be used in CNS damages due to  injury or disease(Such as ischemic hypoxia)Recently olfactory ensheathing cells are used for such transplants .Transplantation can be used for increasing the effectiveness of regeneration( Cultured Schwann cell plantation into nerve conduits).In some cases cells with a heightened secretion ability for a specific molecule can be transplanted into bodies that has lost the ability to produce that molecule(For instance Dopamine releasing cells transplanted to the brain’s of Parkinson patients)

3) Electronic stimulation-Electrodes

      Controlled stimulation of organs which has lost their innervations. Implantation of electrodes has been widely used for observation of nervous system and with developments in  the external control systems it is quite possible to design electrode based systems for paralyzed patients or patients with motor-neuron pathway problems(Such as amyhotrophic lateral sclerosis)

      Other than these some new methods are under development. Electronic brain implants is a hot research topic and some sound improvements has already been achieved in this area.

PERIPHERAL NERVE REPAIR

NERVE GRAFTS AND NERVE GUIDANCE CONDUITS

Peripheral nerves are composed of 3 layers; endoneurium, perineurium and epineurium. Each nerve contains several fascicles that have the same composition with the total nerve structure. Although nerves are generally designated as motor or sensory nerves; no nerve is entirely comprised from one type of nerves. Motor nerves are largely myelinated; whereas sensory nerves are unmyelinated. Between nerve fascicles; an areolar layer is present, which gives nerve ability to contract or expand due to motions of surrounding tissues. Another important property of nerves that must be considered is their tendency to intermingle; this forms nerve plexuses. This is the reason of relative difficulty of recovery of proximal (Close to ganglions) injuries. In proximal ends intercommunication is more extensive; thus retrieval of these communications is rather hard.

As described above, several events may lead to nerve damage. If left unrepaired, these defects may result in motor function problems, loss of sensation and neuropathies. So Practitioners of medical sciences have been trying to find ways to cure these problems.  The first documented nerve repair was done by Ferrar in 1608. In the second half of 19^th century Doctors start to use epineurial suturing. In this method, which is initially proposed by Hueter in 1873, proximal and distal ends of damaged nerve are bind to each other by using sutures. This method has received wide acceptance and it is used whenever ends are sufficiently close to each other. Main advantages of this method is it does not effect intraneural content; so fibrosis normally associated with the injury is minimal and it also introduces minimum amount of foreign material(Sutures)Thus inflammatory response is also minimal. But there are disadvantages too; first one is the tension problem; if the gap in the axon is large suturing would exert tremendous amount of tension on the cells which might be detrimental. To avoid this proximal and distal ends are mobilized in these procedures. Another problem is the misalignment of fascicles; this might affect the recovery of function.

As a remedy to this problem; Bora suggested alignment of fascicles through microscopial observation; so perineurium can be sutured instead of epineurium; which would give the surgeons a greater control over the relative orientation of two ends. But with this microsurgical method several problems arouse .Introduction of sutures into nerve is the biggest problem; this increased the intraneural fibrosis. Also general problems of nerve suturing techniques are still present; namely scar tissue formation and compression of axon due to the tension.

Today main method of bridging peripheral nerve gaps is nerve grafting. Grafts can be classified into three categories; autologous grafts(Nerve is taken from an uninjured and rarely used part of the patient’s body, and re-implanted into injured part; widely used donor nerves are sural nerve and sensory nerves of upper extremities), allografts  (Nerve taken from a different individual of the same species) and  xenografts (Nerve taken from a different species) Use of both Xenografts and allografts requires immunosuppression; and they cause amplified inflammatory response. Formation of tumors has been seen in allografts also. Since allografts and xenografts can’t reach the efficiency level of autologous grafts; they are not utilized frequently. Autologous perineural grafts are the golden standard of peripheral nerve surgery. In autologous grafts two choices are possible; fresh autografts and predegenerated ones. Several researches emphasized that predegenerated nerve grafts were better. This condition is attributed to the increased proliferation of Schwann cells due to Wallerian degeneration and related augmentation in the secretion of Nerve growth factors.

However; autografts have their own problems. First of this procedure requires an additional operation and as it can be easily guessed; donor sites are not very abundant in human body. Also morbidity of donor site is another problem. Attention should be given to the structural differences between the graft and the bridged nerve (Fascicles cannot be in proper orientation; so again they must be aligned). Moreover; grafts have a limited capacity and for gaps longer than a certain critical distance they are insufficient and may also cause necrosis in the site. There is no determined maximal length for grafts; but reports of recovery in 1.5 to7 cm are present. Taylor and Ham proposed revascularization of the grafts. But even this may not solve the problem. Another factor is the availability of grafts. Storage attempts have been unsuccessful. According to Gulati nerve grafts stored for a prolonged period of time tend to lose their regeneration supporting ability. Of course some difficulties can be overcome by new approaches. For alignment of fascicles several methods have been tried. Most popular method is the approximation of fascicle complementarity with respect to diameter or size. Labeling of motor and sensory molecules by using specific molecules was also attempted (Motor fascicles with acetylcholinesterase and sensory fascicles with carbonic anhydrase) but there is a significant time delay problem for labeling procedure which renders it useless. Another current method is the electrical stimulation of isolated fascicles. Fascicles are enveloped by a loop which isolates them from other fascicles; externally stimulated proximal nerve fascicle will produce an action potential which would be received by an External device which can send controlled stimulus to the distal end. Then, by analyzing the response of the distal fascicles; proximal and distal ends are tried to be matched. However, not all problems (Like availability) have immediate resolutions. Thus new methods have been developed.

New trend in the peripheral nerve regeneration techniques is the development of Nerve guidance conduits. NGC would eliminate morbidity caused by autologous nerve graft; in addition to that NGC would give us the chance to regulate several factors that effect nerve regeneration; so we can manipulate the process of regeneration or determine the relative importance of these factors (Composition of extracellular matrix, neurotrophic factors, and architecture of the matrix)

             3 key components associated with peripheral nerve regeneration are a path for axonal regeneration (This is provided by the scaffold material), presence of Schwann cells; which are necessary for myelination process and also secrete several growth factors and extracellular matrix. Extracellular matrix both provides surface for attachment and also rear the cells forward by specific interactions between its components (Such as laminin) and cell surface proteins. A good artificial nerve conduit should either supply these elements or perfectly mimic their functions. (Such type of biomimetic materials are discussed later).Moreover several other factors should be taken into consideration while designing this kind of conduits. Ease and flexibility of fabrication(Whether new architectures is possible or not; such as  variable length and luminal diameter in tubular conduits without effecting properties such as tensile strength), biodegradation rate and mode(Enzymatic, or bulk degradation) Products of degradation (Cytotoxicity), selective permeability of conduit and porosity(For controlling entry of oxygen and nutrient molecules and to inhibit the infiltration of fibroblasts), hydrophilicity of inner surface(For enhanced cell adhesion) Revascularization(Which is important for recovery of natural way of getting nutrients and also for transfer of  factors synthesized by the body. Vascularization degree is also dependent on porosity, low antigenicity and required mechanical strength for the specific application (Prevention of long term compression)

            First guidance conduit ever used is a human branchial artery segment. It was used to link two ends of a canine sciatic nerve in 1891.Usage of conduits reduces the tension applied by the sutures; since nerve ends are placed into this tubular structure. Both autografts and conduits decrease the invasion the tissue by fibrous tissue. There is a diverse array of materials that can be used as nerve conduits. For instance Gluck used decalcified bone as a conduit in his experiments. Several organic and inorganic materials have followed these substances such as vein grafts, silicon tubes, fallopian tubes etc. Also ensheathed silicon tubes were used (for decreasing immunologic response). Several researchers have tried to improve the capabilities of these conduits. Wang utilized inverted jugular vein in order to expose regenerating axons to a collagen rich surface.2 other natural materials used are fibronectin and fibrin gels. Recent researches showed that fibronectin can be further stabilized by introduction of copper and it acts as a very good substrate for Schwann cell growth. 3-D fibrin gels also provide a good scaffold for tissue ingrowth. Another approach developed was the filling of the tubes; like dialyzed plasma or with extracellular matrix elements like collagen and laminin. Later on collagen tubes was also used. ECM proteins such as fibrin and collagen is preferable due to their adhesiveness for several cell types; which would improve their survival .Also collagen has a high angiogenetic potential; which is vital for revascularization. All this experiments showed improvement in nerve regeneration with the presence of the additives.

            We can divide nerve conduits into two classes; natural and synthetic ones. In addition to the ones mentioned above Natural Materials such as acellularized muscle, acellularized nerve generally improve biocompatibility and they also increase the migration of Schwann cells (A cell type found in peripheral nervous system. They also cause a decrease in toxicity due to the implantation. But amount and source of these materials may cause some complexities. Specific immune responses have been seen; Macrophage activity (Both resident and invading macrophages) was observed to persist around the implants; which may lead to prolonged inflammation. Also graft rejection is possible; this situation necessitates the usage of immunosuppressor drugs. (And this is highly undesirable in some cases). Also mechanical difficulties can hinder nerve generation. For instance; veins have a thin-walled structure and they may deform due to the external pressure exerted on them; since tube is practically empty. Also scar tissue formed in the tissue (both due to the injury and the implantation) may constrict the material; so nerve regeneration can be adversely effected. More severe problems can accompany the usage of muscle tissue; nerves can leave their path and this type of undirected regeneration would lead to neuroma. Also manufacturing of these materials is not easy; in terms of standardization of products; natural materials are inferior to synthetic ones.

            Main advantage of synthetic materials is the ease of fabrication; several manufacturing parameters can be easily controlled (Like porosity, water content, materials concentration); also they can be given any desired geometric shape and their mechanical strength can be calibrated. In synthetic materials most important class is the biodegradable polymers. These materials can provide necessary scaffold for regeneration and then can dissolve in biological environment. By playing with the fabrication parameters degradation period can be regulated. Nondegradable materials have desirable properties strengthwise; but their presence in the body as a foreign material increases the scar formation; which, in turn, may squeeze the nerves; hence impair their functions.(Secondary impairment).Another setback associated with these materials is their inflexibility(They may damage the surrounding tissues). Several materials that have been employed up to now are silicone rubber, acrylic polymers, polyethylene, elastomer hydrogel and porous stainless steel.

             First synthetic material used as a nerve conduit was Gelatin. Then polyurethane based nerve guides were tested; but these conduits’ degradation products were proved to be toxic to cells and also total degradation was not realized. Nevertheless they were shown to promote nerve regeneration in an 8 mm gap in rat sciatic nerve. (Usage of Sciatic nerve is very common in nerve regeneration procedures.) Following these experiments new biodegradable materials were tested as nerve guide conduits. Semi-crystalline poly-l-lactide and poly-e-caprolactone composite was shown to be quite good for nerve regeneration. But material fragments were found around the nerve 2 years after implantation. Other two suggested materials are PLGA (Poly DL lactic-co-glycolic acid with a ratio of 75:25 respectively).PLGA shows good distal end innervation and improvent in Sciatic Functional index is observed in 16 weeks and no inhibitory effects of degradation of co-polymer is observed. But mechanical stability of the material isn’t in the expected values. Elongation of conduits, even partial collapses was detected. A copolymer synthesized by using poly-dl-lactic acid and poly-e-caprolactone was also tested. PLLA followed the PLGA; which has superior mechanical capacities; it is ten times stronger than PLGA and it can hold its form even after 8 week of degradation. Tissue incorporation is high; and transverse blood vessels entering the tube was also seen(Indication of good vascularization ).PLLA is shown to be effective in nerve gaps of 10 mm.(Several papers suggest that  the 10 mm gap is the critical nerve defect)Poly D lactic acid was also used for 10 mm sciatic nerve defects. This material degraded completely within 1 year and reaction of body to this material was mild. Connection of larger gaps was investigated by using animals like cat. By using a poly glycolic acid/collage type 1 composite Kiyotani reported myelinated fibers in regeneration of a 25 mm sciatic nerve defect. Largest gap examined was 50 mm gaps in cats.

Several other materials are under consideration nowadays; like hyaff11 (which is a hyaluronic acid derivative; previously Seckel showed that usage of hyaluronic acid in nerve guides (He used polyurethane guides) increases conduction velocity, axon counts and facilitates myelination. Presence of hyaluronic acid decreases scar formation and improves the formation of fibrin matrix. Also it is hypothesized that hyaluronic acid creates open lattices in fibrin matrices; that create surface for tissue ingrowth. Studies showed that hyarulonic acid has a good biocompatibility and long biodegradation period. Low biodegradation rate can be an advantageous property; since we don’t want that guidance conduit lose its shape within the time-span of nerve regeneration. This would lead to migration of fibroblasts and macrophages into the tube early and can hamper axon growth. But there are some concerns about usage of hyarulonic acid based conduits; mainly the release of hyaluronic acid as degradation product. This acidic substance might be harmful for surrounding tissues. Lately electrically conductive polymers such as oxidized  polypyrrole is also shown to be positively effective on nerve regeneration. It is known that electrical fields have the ability to promote regeneration in tissues; so several researches have been done to manufacture materials with permanent charges or tempororary surface charges. However such materials can not be controlled after implantation; so their effect is rather random. Properties of polypyrrole allow external control; since in this material electrical cues are transferred by electron movement between different polymer chains. So by application of adjusted voltages electrical stimulation can be easily manipulated. This property of polypyrrole can be also handy as an interface between nerve tissue and the electronic prosthetic devices. Main drawback of polypyrrole is its nondegradibility. Recently an electrically-active and biodegradable material was suggested as a new option for conduit material. BECP is composed of short chains of polypyrrole which are held together by ester linkages. Ester linkages can be easily degraded by the body and polymer chains are small enough to be phagocysed by macrophages. Development of such materials is very promising; since they will give us the opportunity to merge chemical and electrical induction of growth.

Also other possibilities are being researched. Usage of cross-linked collagen filaments was also shown to be effective in bridging long gaps (20 mm). (Cross-linking slows down the absorption rate of the filaments; thus it is a way of adjusting the degradation time to needed regeneration period. Also resistance to the bacterial collagenase enzyme decreases occurrence of infection.) Usage of filaments instead of tubular structures has two advantages; first since it is not an enclosed system in filamentous conduits cells are free to interact with the surrounding ECM; this would also facilitate the revascularization and may enhance the regeneration. Second advantage is flexibility; for long gaps usage of tubular conduits might be dangerous; especially in the extremities. Movement of the surrounding tissues may exert large moments on the tube and this may lead to mechanical failure. Because of their superior flexibility filamentous conduits may avoid this calamity. Of course tissue response and intervention of regeneration by the wound repair system is the main concerns in using an open conduit. (Degree of fibrosis and effects of macrophage activities)In-vitro trials for synthetic filaments were also done. Although bare PLLA filaments were demonstrated to be poor substrates for cellular attachment; filaments coated with laminin gave good results. (All in cellular attachment, neurite growth and Schwann cell migration)These filaments can be used to manipulate the direction of growth within the tubular conduits. Also it is claimed that usage of this filaments only as NGC would reduce the tissue reaction due to the products of degradation of the PLLA; since less material would be used. Also a filamentous configuration would produce large surface area for cell attachment.

            Although practical applications of NGC systems is not widespread right now; experimental results are very encouraging; and with the proper optimization of several factors NGC can take place of the autografts in near future.

            There are several factors that should be considered while assessing the capabilities of NGCs. Obviously Empty nerve guides don’t provide a good alternative to autografts; but several improvement methods has been suggested. These methods generally incorporate a part of normal regeneration mechanism into the nerve conduit systems.

Implantation of cultured Schwann cells into nerve guides was shown to increase the regeneration rate and quality. SCs provide a substrate for axon migration in PNS. They have heterochromatin rich nucleus and an electron-dense cytoplasm. They are able to synthesize and secrete some neurotrophic factors like Nerve Growth Factor (Some other factor are also effective in promotion of nerve regeneration like Brain derived neurotrophic factor and ciliary neurotrophic factor).Release of these factors enhance regeneration. Schwann cells also provide ECM proteins laminin collagen and also express several cell adhesion proteins, namely gamma 1 integrin, N-cadherin and neural cell adhesion molecules. Axon-Schwann cell attachment is very important for controlled sprouting and elongation of axons. Following injury axonal segment distal to the lesion degenerates; this is called Wallerian degeneration. Soma of the neurons generally survives; if the lesion is not very close to it. In soma an increased metabolic activity and migration of nucleus to periphery are seen after the injury; which is named as chromotolysis. At the distal end Survived   Schwann cells proliferates and begin to phagocyte myelin sheaths with the aid of incoming macrophages. Mechanism is as follows; in the subsequent hours of the injury SCs begin to express MAC-2 which is a galactos specific lectin and can target galactolipid rich myelin sheath; so lectinophagocytosis happens. Afterwards Schwann cells form bands called band of Büngner which is a solid cord upon which axons can grow and axonal sprouting occurs. If this band is absent neurons grow in an uncontrolled manner and form a neuroma. In early phases of sprouting axons are unmyelinated; but soon after axons and Schwann cells stick to each other; and myelination takes place.

            So Schwann cells are an indispensable part of regeneration in PNS. Researchers have also attempted to enhance CNS regeneration by utilizing SCs; but with a limited success. SCs were tried both with biological conduits and synthetic ones. Possible natural matrices are vein, epineurium and acellularized muscle (Acellular allografts show decreased antigenicity; so acellularization is a viable method for obtaining bulk amount of natural scaffolds from non-self sources).As for synthetic materials; PLLA, poly-lactide-co-glycolide and polyglactin 9 are some examples for which studies have been carried out. Studies demonstrated that migration rate of Schwann cells is slower than that of axon sprouts. This means that after a given period of regeneration Schwann cells would lag behind and without the factors supplied by SC; regeneration would slow down or even halt. So for large gaps addition of Schwann cells is a good option for sustaining axon elongation.

However addition of SC cells does not guarantee a successful regeneration process. Some studies showed that SCs do not always have a positive effect on nerve regeneration. A recent experiment using epineurium or acellularized epineurium gave results in which addition of SC have no discernable advantage. This is in contrast with the body of publications about the role of SC cells within scaffolds; Most of the publications present an increased number of myelinated axons, increased number of axons in both middle and distal transverse sections with the addition of SC. It is hypothesized that rather than the amount of Schwann cells their condition is important. An additional factor is the amount of debris in the acellular grafts which can intervene with the activities of Schwann cells. It was demonstrated that the predegenerated acellular grafts are superior to fresh ones; and situation is interpreted in light of the wallerian degeneration; removal of myelin sheaths and other intraneural material opens up space for faster ingrowth. Good results observed in acellularized muscle grafts can be related to their ability to provide basal lamina for attachment of Schwann cells. So from this results this can be inferred; condition of the conduit must be thoroughly known; otherwise efforts for improvement can be proved to be futile.

To prevent this kind of interference related failures a novel approach is proposed. This is the production of artificial nerves. This approach uses biodegradable polymers and cultured Schwann cells. With using both membrane and fibrillous configurations of the polymer a three dimensional scaffold can be obtained. (A rolled membrane conduit filled with oriented fibrils)This scaffold is covered with a glue and then ECM protein Laminin is added. So a suitable matrix for adhesion and proliferation of SC is produced. Most important part of this procedure is the coculturing of polymer with cultured and purified Schwann cells (All contaminating fibroblasts is removed) this coculture results in a biodegradable matrix with a population of SC in it. These artificial nerves are shown to be very effective in promoting nerve regeneration in rabbits. These results can be surmised within the context of the argument in the paragraph below. That is established Schwann cells (This can be also judged as a condition) are more competent than directly added ones. Similar results have been attained with biodegradable polymer PLLA injected with Schwann cells in a matrix of collagen. It can be said that utilization of Schwann cells is highly advantageous provided that the circumstances are suitable for their activity. Another aspect of Schwann cell condition is the formation of the Büngner bands; axons grow on Büngner bands in natural regeneration. But in practice Schwann Cells are introduced into conduits as a suspension; which could delay the formation of these bands .So some groups claimed that usage of adherent monolayers of Schwann cells would be advantageous. But using conduits with a single lumen limits the number of Schwann cells that could attach to the inner surface of the tubule. Using a single lumen also limits the amount of substances that can be incorporated. This problem can be surmounted by using SC cells coated on polymer filaments; which can greatly increase the surface for formation of Büngner bands.

Storage and obtainment of Schwann cells are problematic aspects of this approach. If autologous Schwann cells are not available; another problem is the expected body response to transplanted allogenic Schwann cells; some harsh reaction can be expected for the relatively high number of Schwann cells. So, in spite of the fact that the Schwann cells are best available candidates for peripheral nerve regeneration, other possible substitutes have been investigated to circumvent these difficulties. Two possible nominees are olfactory ensheathing cells and nerve progenitor cells. Nerve progenitor cells has shown to be highly preservable in culture conditions. These cells can be obtained from hippocampus and they have the ability to differentiate or to proliferate without differentiation. Experiments in which progenitor cells cultured in collagen matrices are implanted within a biodegrable NGC(PGA); showed that progenitor cells co-implanted with nerve conduits have the capability of increasing axon regeneration and some of the cells are found to have differentiated to Schwann cells.

For promotion of recovery another possible method is the addition of nerve growth factors into the conduits. Nerve growth factors not only increase the rate of axon migration but also protect neurons from death when a lesion occurs. Furthermore NGF can determine the direction of nerve growth by acting as a chemoattractive. NGF’s protective properties can be used for increasing survival rate of Dorsal root Ganglions following an injury. Expression of NGF receptors located on the membrane of neurons is increased by the local high concentration of NGF’s. NGF also increases the gene expression related to the BDNF; thus it protects BDNF-response hormones. In addition to these NGFs can also act on Schwann cells; it is shown that the presence of NGF increases the rate of migration of SCs through interaction with low-Affinity receptors on the SCs.50 fold Up-regulation of expression of these receptor proteins in Schwann cells in the distal end when nerve is severed, is a good evidence of importance of NGF for migration. NGF can still act as chemotactic attractants in their bound form (to receptors)

Fibroblast growth factors were also proved to be helpful in nerve regeneration. Recently IGF (Insulin like growth factor) is suggested as a nerve regeneration promoter. In addition to these VSGF, a growth factor which increases the rate of vascularization may also be used for accelerated vascularization. Nerve growth factors mentioned earlier may be directly put into the lumen of conduits. Silicon tubes filled with growth factor containing saline solution was shown to increase regeneration. But like all growth factors nerve growth factors have very short half-lifes so they would be effective only for a short time span. Since Nerve regeneration is a long process; several controlled delivery systems have been proposed. One of these systems is heparin containing fibrin based growth matrix. (Matrix also contains heparin binding peptides for maximizing the amount of bound heparin)This system has been used for several controlled delivery problems and since it is well-characterized it is a plausible candidate for controlled release of NGF. But unfortunately NGF has a very low affinity for heparin. But even this low affinity system was shown too effective in slowing down the passive release of the factor. It was proved that this matrix reduces the speed of diffusion for several nerve growth factors. Tests with dorsal root ganglia containing matrices also proved that controlled delivery system is more efficient in axon regeneration promotion than untreated conduits. Free neutrotrophins were shown to be ineffective with respect to other procedures for encouragement neurite extension. In vivo analysis showed a concentration dependent effect of immobilized growth factors; with increasing concentrations efficiency of the factor increases. This type of delivery systems is called affinity delivery systems. Heparin is the agent in the system; which attracts molecules with a basic domain by electrostatic forces. (Heparin’s sulfated groups.)This interaction protects the growth factor from degradation and retards its release. In body degradation of fibrin matrix also contributes to the regulation of factor diffusion.

Microencapsulation is a second alternative for controlled delivery of neurotrophic factors. Microencapsulation is based on biodegradable polymers. Controlled delivery of the substance is achieved by encapsulating the molecule in a sphere of polymer and by regulating the degradation rate of the polymer. Polymer with a designed half life in in-vivo conditions degrades in time and releases its contents into the tissue. Several biodegradable polymers such as PLGA and Polyphosphoester were tested with NGF for controlled delivery studies. Dynamics associated with PLGA was found as a slow release rate after an initial burst of diffusion. This is a desired pattern provided that the initial burst does not release more than necessary amount of molecule; which would cause a decrease in available growth factor for later stages of regeneration. Another problem of PLGA microspheres is the interaction of acidic by-products of degradation with the growth factors; this would either inhibit the release or totally inactivate the factor. In microencapsulation it is proposed that efficiency increases if factor are put into capsules with a buffer such as BSA. This might decrease incidents of irreversible inactivation of added proteins due to oxidation or aggregation.

 Degradation can occur in two ways; surface erosion and bulk degradation. In Polyphosphoester backbone bonds can be broken by hydrolysis in physiological conditions; a reaction which does not produce any interfering by-products. (Bulk degradation)By using this property a polymer named P(DAPG-EOP)  was developed; it has a backbone of oligomeric blocks of lactides and distributed phosphate bonds. Addition of phosphate bonds is a measure of regulation for these polymer systems. By this method degradation rate can be precisely manipulated.(By higher phosphate content faster degradation rate is attained) Stereoisomerism is a further factor that influence degradation rate. Relative percentage of degradation by surface erosion is also a key determinant; since surface erosion decreases the interaction between degradation products and encapsulated factor. In-vitro and in-vivo studies show that a prolonged effectiveness for nerve growth factor with employment of microspheres (Around 12 weeks) compared to free NGF (2 weeks) Conduit used with microspheres is also very important. Even though non-degradable materials such as silicon are undesirable generally; its inertness may provide a good environment for extended period of activity of microspheres. Biodegradable conduits’ porosity may cause the leakage of microspheres or in-flux of other molecules that might interfere with the activity of NGFs.

A third option for delivering nerve growth factor would be the transplantation of genetically engineered cells. These modified cells can secrete necessary growth factors in desired amounts without affecting the regeneration process. Experiments with modified fibroblasts and dermoblasts showed an enhancement in regeneration when such cells are present.

            One step further has been taken in activating the growth matrix. In gene incorporated matrices matrix contains engineered DNA molecules (Plasmids, cosmids etc whatever means are appropriate for the situation) with this matrices intention is to locally deliver several genes that may alter the healing process. Since microencapsulation techniques (sonication, exposure to organic solvents etc) may cause inactivation of some of the neurotrophic factors; hence decreasing the effectiveness of the matrix; controlled local gene delivery systems seems to be a logical solution. Another concern that stimulated researches for gene delivery systems is the systemic effects of cytokines and growth factors if they escape the local environment that they are put into. By adding DNA instead of protein this trouble can be avoided; since recombinant DNA is inactive in extracellular conditions and since its transformation ability is quite restricted it is highly improbable that it can affect other cells before being degraded. Of course it is a well-known fact that gene transfer is very hard in mammals; and generally transfer efficiency is quite low. Another obstacle is the complex expression patterns of eukaryotic cells; it hinders our ability to manipulate the cellular mechanisms. But there are some positive factors that may improve the effectiveness of gene delivery. During regeneration period related high mitotic rate may significantly increase gene delivery. Also by fixing the recombinant DNA into the matrix possible surface for transformation is also increased. Gene activated matrices was tried for cranial nerve injuries by Berry; they found that recombinant DNA was transported in retrograde fashion and they noted increase amount of gene products in the cell bodies. Also they demonstrated that plasmid cells enhanced rate of survival for retinal ganglion cells. In delivery systems molecular conjugates is a method for increasing the in vivo stability of the DNA. In Berry’s experiments it was shown that linking plasmid DNA with FGF2 increase the uptake of recombinant DNA. (Passage of DNA molecules through negatively charge cellular membranes is very difficult; due to their phosphate groups. So associating DNA with a cationic molecule or a molecule which is rapidly taken up by the cells is an eligible method for increasing transfer efficicacy) another possible approach would be the direct attachment of short signal peptides to plasmid vector. For controlled delivery both synthetic polymers and natural materials can be used. For example for fibroblast transformation collagen gels were used; degradation of collagen matrix by incoming fibroblast cells liberated plasmids and fibroblasts are transformed. Although this mechanism is not reasonable for nerve regeneration; other mechanism that might use secreted products during regeneration or direct controlled delivery systems via adjusted degradation rates of polymeric systems is possible. Atellocollagen was used for controlled delivery systems by Takahiro Ochiya^{; it} is a by-product of collagen degradation and has a very low antigenicity.

            Of course there are several concerns that cause medical practitioner to refrain form this kind of treatments; such as prospect of oncogenesis due to high expression of growth factors or effects of insertional mutagenesis due to the non-homologous recombination. Gene therapy is a very young branch of biological sciences and further studies both in vitro and in vivo will give us better evaluation of the feasibility of this kind of methods.

            For successful nerve regeneration interaction of cells with the extracellular matrix is very important. Migration of nerve cells to their right position in embryonic development can’t happen without the laminin tracks. Adhesion of cells to extracellular matrix proteins via membrane proteins (Like integrins) is an established fact. This fact can be put into use by using these proteins within biomaterials. When a biomaterial is introduced into the body; ECM molecules are non-specifically adsorbed on the material; so interaction of biomaterial and the cells would be through these ECM proteins. But for better control over the tissue growth it is desirable to be able to direct these interactions; so by modifying biomaterials through physical or chemical process such biologically active substances can be included. Moreover incorporation f some peptide sequences may also rise an opportunity to control in-vivo degradation; since these peptides renders biomaterial vulnerable to certain proteases. For example addition of VRN or APGL peptides to material makes it susceptible for degradation by plasmin and collagenase respectively. This type of materials called biomimetic materials augments cell adhesion and migration. (In broader terms; gene-activated matrices and conduits with NGFs are also biomimetic materials) Biomimetic materials provide a framework for specific manipulation of both rate and direction of new tissue formation and also cytoskeletal organization by carefully designing the spatial distribution and concentration of bio-active elements. In other words Biomimetic materials enhance tissue growth by acting like a natural ECM.

            ECM proteins (Insoluble extracellular matrix molecules includes

Laminin, fibronectin, and some forms of collagen) can be integrated in their complete structure; but this is shown to be ineffective and proteins can not elucidate their function fully. This is a result of random folding of proteins within the biomimetic materials. There are some specific peptide sequences within these proteins that interact with the surface proteins of the membranes or other ECM proteins. If random folding causes enclosure of this sequences with in the protein structure then protein can not interact. Since short recognition sequences have been determined for most of the ECM proteins; this short peptides can be used instead of the original protein. Although the activity of short peptides is lower than the normal protein due to the absence of stabilizing domains; steric availability compensates this loss. Another attractive property of short peptides is their ease of fabrication. Several examples to these short peptides are RGD, YIGSR, and IKVAV…

            There are several techniques for binding peptides to biomaterial surfaces; for example if material has NH2 moieties peptides can be bind through their C-Terminal carboxylic acid groups. If such groups are absent binding can be achieved by using photochemical methods. Another method used for peptide addition is the use of spacer molecules. Spacer molecules are the linkage between material network and the bioactive peptide and they increase the mobility of peptide sequences by moving them away from the bulk of the biomaterial. So peptides can more effectively interact with the regenerating cells with minimum steric hindrance. As spacers generally inert polymer chains (like PEG) or non-specific short peptides (PVELP) are used. Addition of spacers decreases the concentration of bioactive peptide necessary for sought biological outcomes.

            Concentration of peptides should be carefully assessed before the procedures; otherwise undesired results may be obtained. For instance high peptide concentrations may enhance cell adhesion immensely; but this high adhesion profile also inhibits the migration ability of the cells and this is very unwanted outcome for nerve regeneration. Not only migration but also proliferation is dependent on the concentration of peptides. Thus optimal concentration of the peptide should be determined and treatments of the biomaterial should be done accordingly. We should also take into account the ligand-receptor affinity,

            ECM proteins have the ability to direct regenerating axons. This is defined as haptotaxis. Growth cone successively bind to the ligands at the extracellular matrix and this binding would generate a pull force which would move growth cone forward. As pointed out earlier Laminin is the most active ECM protein in nerve growth. In vitro tests employing laminin showed positive effects on both cell attachment and neurite outgrowth. It was later found out that 2 sequences YIGSR and IKVAV are responsible for membrane –protein interaction. Also another sequence was found which increases the specific attachment of astrocytes. (KHIFSDDSSE)Another possibility is the usage of ECM analogues. These materials can be produce by grafting GAG’s to a matrix composed of collagen type 1; a carefully designed matrix can result in a material that can successfully mimic ECM.

            Architecture of conduits (For the distribution of peptides or ECM proteins) can be accurately regulated. For PLA-PEG it was shown that nanometer scale patterning can be achieved by employing absorption affinity. In this method avidin modified surface strongly attracts and binds with biotinlyated peptides. It was shown that cells prefentially follow these paths; which would be very useful for directing nerves to their proper places. Physically patterning can be achieved by incorporation of peptides into gels by using a laser source. Another problem that should be overcome is the non-specific absorption of ECM molecules during processing. This can be avoided by using interpenetrating polymer network (IPN) which consists of a hydrophobic chain which limit the adhesion of the ECM molecules and a second chain with a high affinity for the ECM molecules. A second remedy would be immobilization of inert chains to the areas where the adsorption of ECM proteins is undesirable. In biomimetic materials main effort is to increase our control over the spatial distribution of the bioreactive molecules within the material.

            A second parameter that can greatly affect the dynamics of axon regeneration is the surface properties of the conduit. Silicon tubes that have either rough or smooth surfaces displayed different characteristics of axonal movement promotion. Experiments conducted by using silicon elastomers which were divided into two parts by polymer strips (One part had a rough surface and the other one smooth) showed that smooth surfaces facilitates axonal proliferation. In rough part tissue adhere to both sides of the polymer; whereas in smooth surface axonal growth was distinct. However these results can also be interpreted as an increase in adhesion capability of tissue on rough surfaces. A very interesting paper suggests an analogy between the movement of earthworm and neurite outgrowth. Their results showed that there is an optimum roughness in which ground worms moves faster. Considering the structure of extracellular matrix; this seem plausible also for axonal growth. Thus cell culture experiments should be done to determine this optimal value. Other parameters might be the presence of grooves(or striations) to which tissue may prefer to attach; their size with respect to the size of nerves; curvature of grooves and how can outgrowths can adapt to this directional changes. By using these topographic parameters we can enhance our ability to manipulate the directionality of regeneration and also may increase the rate of axonal regeneration. There are some experiments which aim to determine the behavior of astrocytes on different surfaces too. These experiments will be discussed in the related part of this review.

            So we can say that surface geometry of the inner surface of lumen is a very important factor in axonal regeneration. One of the requirements is the increased surface for better distribution of Schwann cells or microcapsules containing NGF or the necessary 3-d network of ECM matrix proteins or biologically active peptides. One way of increasing surface is increasing roughness; but as discussed in the previous part above a definite roughness movement of axons is inhibited. So a more straightforward way of increasing surface area is proposed. Multichannelled proteins contains more than 1 channel for axonal outgrowth. This configuration not only increases the surface for manipulation but also it can be beneficial for routing the nerve fascicles toward their original positions. Previously increase in surface area was achieved by insertion of filaments of collagen, polyamide etc. Although this method produce good results; orientation of filaments were disorganized and highly irreproducible; a possible solution to this problem would be the usage of aligned collagen fibers within conduits. Collagens aligned by using magnetic fields; would provide large surface for adhesion. Moreover aligned filaments would increase the directional stability of the regeneration. Hydrogels with guiding channels of differing diameters is another solution. Their fabrication is rather easy; and several parameters such as water content, porosity, average diameter size and number can be controlled. Two of the possible methods of manufacturing is fiber templating and low-pressure injection molding.

            In fiber templating extruded polymer fibers are embedded into another polymer gel and then dissolved; as a result they leave pores which have nearly the same diameter with the fibers. Procedure is defining for cross-linked pHema (poly 2-hydroxyethyl methacrylate) gel and polycaprolactone fibers. Dissolution of PCL fibers is accomplished by sonication of composite in acetone. PCL is used because it is insoluble in Hema while it is soluble in acetone. Thus it can be easily removed from composite and since interaction of acetone with hema is moderately small, acetone do not affect the structural integrity of the remaining gel. Channel diameters can be controlled by managing the diameter of PCL fibers during extrusion (by calibrating winder speed and piston speed).Then fiber bundles are formed and placed into a polymerization mold; into which hema solution is added. Following the polymerization reaction; sonication can be carried out. Products are either large or small pore size multichannels. Of course introduction of pores results in a decrease in strength of the gel and increase in porosity; but these changes do not significantly affect the capability of the gel. These gels are designed to act as scaffolds within empty conduits; such as PLLA conduits.

            Second method, injection molding, utilizes cold mold chambers. As a specific example; PLGA dissolved in glacial acetic acid is injected to cold mold. This induces a solid-liquid phase-separation and then polymer solidifies. Afterwards acetic acid is removed by lyophilization. In the end what is left is a hydrogel with longitudinal channels. Size of channels is determined by using stainless steel wires within the mold. These wires are hold in place in the molds but using a spring and a plate that would hold them taut. Design of injection mold and constant injection pressure are important. Mold must be designed so that polymer solution in the injection mold freezes later than the solution within the mold; constant pressure of injection minimizes the shrinkage which is inevitable during solidification. But by holding pressure constant shrinkage void can be seized in a reasonable range. Selection of solvent is very crucial in this method; since not all of the solvent can be removed. So solvent should be non-carcinogenic and it should not interfere with any molecules that is put in to enhance the nerve regeneration (Like nerve growth factors) Final material is a macroporous foam. It is highly anisotropic (which means dependence of material characteristics to the direction within the material) so its mechanical properties should be examined carefully to prevent unforeseen failures. Besides that test results of this type of multi channeled materials is highly encouraging; a 2.5 fold increase in Schwann cell adhesion is observed with macroporous PLGA conduits.

            Building up a 3-d scaffold within the conduits may have some drawbacks too; it is shown that filing up conduits with collagen can delay the revascularization and this can adversely effect Schwann cell proliferation. So these opposing needs should be carefully optimized. Additional problems have been reported. Some papers claimed that conduit thickness is an important factor in neuroma formation (Thinner conduits cause less neuroma formation) obviously using thinner conduits might be a big compromise (Strengthwise) .Hardness of implant should also be considered. Stiff implants may shear the surrounding tissue and can cause necrosis. So decreasing mechanical damage might be an extra advantage of porous conduits. Once all necessary data accumulates; it will become important to evaluate all advantages and disadvantages and propose optimal conduit; that would use a combination of the techniques described above.

Methods of assessing NGCs

            Evaluation of NGCs is generally based on their ability to promote nerve regeneration. Regeneration efficacy can be measured by comparing number of axons, degree of myelination(for motor nerves),axon growth rate, axonal diameter, revascularization level, number of axons per fascicle etc. between the NGC and the positive control(Generally autografts are used as positive controls; inverted isographs is the most common method. But comparison can also be done with respect to normal nerves).Histochemical methods are used for determination. For example toluidine blue is the dye that is used for determination of myelination. Transverse cross-sections are taken from mid-point and the distal end of nerve. Some other indexes are also in use; such as critical axon elongation which is the gap length for which reinnervation probability is 50%.

            Other means are also possible; like measuring the degree of reinnervation of body parts (Like gastrocnemius muscle) or degree of muscle atrophy due to denervation can also be used as indicators of the extent of regeneration. Atrophy is accompanied with weight loss and with reinnervation muscle gets some of its weight back. So this is an indirect method of measuring reinnervation. Moreover neurophysiological techniques can be employed. Velocity and amplitude of signal conducted through the regenerated nerve might be used to assess the degree of regeneration.

            Number of Schwann cells is also very important parameter. Schwann cells can be detected either by enzymatic monitoring methods (by employing specific antibodies such as p75) or by radiation based methods. For example incorporation of BrdU (Bromodeoxyuridine) into Schwann cells and subsequent monitoring is a widespread technique.

            Mechanical strength of the conduits is very important. Porous conduits with hollow lumens can be manufactured by utilizing several methods. Porous meshes can be rolled and sealed by suturing, gluing or welding; each of them affecting the strength of the final material. Usually in-vitro analysis are performed to determine some characteristics of guidance conduits; such as modulus of elasticity, ultimate tensile strength, ultimate tensile strain etc. For example experiments with acellularized nerve showed that acellularization leads to significant decreases both in tensile strength and also tensile strain capacity. Material stiffness (Young’s modulus) seems to remain same for both specimens. One further step was taken in this experiment in which both specimens are coapted with nylon sutures. Since conduits are sutured to nerve ends; their mechanical behavior when sutures are present should be examined. Also this is an indicator of suture-holding ability of the conduit. Again a decrease in tensile strength was observed. But Young’s modulus was same for both specimens and their values were significantly higher than that of non-coapted ones. This situation brings us to a factor; composite structure of suture and conduit. Since nylon is a stiff material we can expect an increase in the total stiffness of the structure. What we must carefully consider is the orientation of the materials within the composite; like in the case of springs. If two springs are in parallel their stiffness is added; so total stiffness of the system increase. However when springs are in series total of reciprocals of their stiffness is the stiffness of the system; so there might be decrease in stiffness in such composite systems. In our case; sutures are parallel to conduits; so they increase the stiffness.

            Recovery of function should be assessed too. For sciatic nerve experiments; this is done by using walking track analysis. And data obtained from this analysis is evaluated by using SFI (sciatic function index).Before denervation operation rats are trained to walk down a darkened area. Then after a period of time following the operation (around 16 weeks to allow enough time for nerve regeneration) hind legs of rats are dipped into a dye; and stains they left are evaluated; parameters are; 

            Print Length function which is the difference between experimental and normal print length, toe spread function(Between 1^st and 5^th toes) again difference between experimental and normal values and intermedian toe spread function(between 2^nd and 4^th  toes;);this is also difference between experimental and normal value. By using these parameters SFI is defined as follows:

SFI= -38.3PLF +109.5TSF +13.3 ITF -8.8

            This empirical formula proves to be a good indicator of functional recovery. SFI values that approach to 0 are indicative of good recovery.

Central Nervous System Injuries

            CNS injuries have very dramatic results and body, to a large extend, is unable to recover from them. Mature nerve cells can not divide; so dead cells can not be replaced by the body. But complications in CNS goes beyond this obstacle. As discussed in the previous part; peripheral nerves have a high capacity of axon regeneration; but in CNS this is not the case. This is due to the different behavior of helper cells in PNS and CNS. In PNS Schwann cells provide necessary conditions for regeneration; whereas in CNS microglial cells act in a way that inhibit the regeneration of lost axons. Glial scar formation and release of inhibitory molecules (such as Nogo) hinders the recovery. Moreover retrograde cell death of neuron somas (necrosis or apoptosis) also occurs following the injury and even if cells are not dead gene expression patterns do not support the regeneration.

            Other than injuries neurodegenerative diseases are also affecting many people around the world. These diseases generally render patients crippled and eventually cause death. Huntington’s disease, Multiple Sclerosis and Parkinson’s disease are the most frequently encountered ones. Each of these diseases affect the CNS in different manner and their effects are very drastic.

            Of course problems associated with CNS injuries are not as simple and straightforward as in the peripheral injuries. For example; in spinal cord injury several functions of the body can be lost; like cardiovascular system control, bowel control, bladder control. Loss of body functions can be accompanied by the loss of intrinsic control of neuron excitability in several pathways; which may result in hyperactivity in some motor or sensory neurons. Consequences would be overwhelming pain, prolonged muscle spasms and so on. In addition to these; secondary problems may arise; like high infection percentage of urinary tract following SCI; which was a fatal condition it the past. So in treatment of Spinal cord injuries priority has been given to diminish the effects of such problems; which is generally accomplished by pharmaceutical methods. This is why improvement in this area is slow and number of clinical trials is low. For example Sound results of human studies of cell transplantation aside from dopaminergic cell transplantation in Parkinson’s diseases have not been published yet.

            But there are several attempts to develop solutions to CNS injuries and diseases. This attempts range form direct cell transplantations to totally artificial remedies (Brain-computer interface). Although satisfactory results haven’t been seen yet; improvements have been definitely achieved.

            Up to 1980’s it was believed that any damage to CNS would be permanent and there was no hope for recovery. Even though some sprouting of axons occurs; this axons can’t find their proper routes and may cause spasticity or pain rather than retrieval of functions. This problem was attributed to the incapability of CNS neurons to sustain its regenerative abilities. But in 1980 it was shown that; CNS neurons have considerable regeneration potential and retardation of regeneration is due to the hostile environment of the CNS. This was demonstrated by grafting peripheral nerves to injury sites. At the end of these experiments considerable regeneration was observed. Several experiments supported the fact that the composition of CNS following the injury was the one of the main reasons that prevent proper recovery. Histological explanation is as follows; after the injury axons try to regrow by transporting cytoskeletal elements with organelles anterogradely that would provide a tugging force; which promotes axon regeneration. But if the environmental concentration of some molecules are very high; this process is stopped. For instance high concentration of Calcium ions in the surroundings may lead to an influx of the ions; which may disrupt the structure of neurofilaments.

            Later on; investigations demonstrated discrepancies between axonal elongation during embryonic growth and regeneration following the repair. Without going into much detail; it can be said that relative contributions of cytoskeletal elements in these two phases are distinctly different and dissimilarities between extracellular and intracellular conditions of these two periods (Like age dependent decrease in cAMP concentration; or distribution of laminin in extracellular matrix) trigger two different mechanisms. In comparison regenerative process is the inferior one. In addition to these; some differences between the availability and involvement of cytoskeletal elements in peripheral nerves and CNS nerves is also noted as a factor for low regeneration capability of CNS.

            An encouraging result of animal studies (Lampreys, frogs) is the relative specificity of CNS regeneration. This property is attributed to the activities of membrane bound ligands called ephrins and guidance molecule netrin which can both act as a chemoattractive and a chemorepulsive. Although underlying mechanisms have not been fully elucidated yet; comprehensive studies may provide the tools necessary for influencing these interactions.

            Information about the inhibitory factors are more readily available. Sources of these factors are oligodendrocytes and CNS myelin (Since oligodendrocytes are the cells responsible for myelination of nerves in CNS; this two sources are interconnected) Most important ones of these molecules are Nogo, MAG, OMGP and tenascin. First three molecules use the same membrane-bound receptor to confer their effects (Ng-R) Moreover chondroitin sulfate proteoglycans which are deposited by astrocytes have also inhibitory effects on axon elongation. Besides extracellular effectors some intracellular proteins were shown to be important in nerve regeneration; such as Gap-43 and Cap-23.

 These findings stimulated the investigations for agents that might improve the conditions or inhibit the molecules that are responsible for suspension of regeneration. Treatments with neurotrophic factors, anti-apoptotic agents or molecules that would interfere with the actions of inhibitory molecules (Injection of antibodies or competitive antagonists) have been devised and all of these methods provided better results. As mentioned before transplantation of fetal tissue or cultured cells are the most widely used techniques. Utilization of peripheral nerve grafts for connecting two sides of the lesions is amenable too. Increasing the axonal regeneration only is not enough for recovery; since glial scar formed following the injury create a physical barrier that hinder the axonal growth. So grafts can be helpful in bridging the lesions. (Environment provided by transplants is permissive for axon growth; so regenerating axons can cross the lesion region).  But as in the case of the peripheral nerves; even with these techniques functional recovery is insufficient. Also even in the situations where axons can cross the lesion; grow into the host region at the distal end is minimal. So techniques that combine transplantation and the application of chemical factors are in development. Of course recipients of the trophic or other factors are also important(Whether cells at the site of the injury or cells distal to the injury are stimulated)For spinal cord injuries direct injection of neurotrophic factors; such as neurotrophin 3 or transplantation of modified fibroblast that secrete these factors were confirmed to have positive effects.

Aforementioned cultured cells can be selected from a variety of cell types; such as olfactory ensheating cells (which is a special type of glial cells), Schwann cells, stem cells etc…Although it is possible to culture pluripotent stem cells; researches have shown that environment provided by spinal cord generally is in favor of differentiation of cells into glial cells. Thus for incorporation of neuronal cells; cell lines that have already committed to be neurons must be implanted. But implantation of pluripotent stem cells may have another advantage; some of these cells may differentiate into oligodendrocytes. So if excessive demyelination of cells in the injury site is at hand; this might be a remedy. Furthermore; for cleaning up the degenerative remains of injury activated macrophages (Activation is done by put macrophages in touch with regenerating peripheral nerves) can be transplanted; subsequent removal of regeneration inhibitory factor might be beneficial for axonal growth. All transplanted cells have different properties and help regeneration in a different manner.Schwann cells can act as substrate for regeneration; whereas fetal tissue merely provides a uninhibitory medium for growth. On the other hand olfactory ensheathing cells may be the solution to regrow into host tissue; since their main role in normal physiological conditions is supporting of growth of new axons. So one should consider several factors before embarking on transplantation; like degree of demyelination, length of the lesion, degree of cell death, number of severed connections etc. Also availability should be considered; for example harvesting olfactory ensheathing cells can damage smell sensation. Stem cells are probably the best option to solve availability problem. It is shown that Stem cells have a very high plasticity; in other words they have the ability to dedifferentiate or transdifferentiate. This would allow us to use stem cells from bone marrow or other resources in nerve injury treatment. Moreover several stem cells have been identified in adult human brain which can be used for nerve regeneration.

Problem of regeneration inhibitory factors can be solved with other methods; genetically modified cells(hybridomas) that has the ability to produce and secrete Nogo antibodies promotes axonal regeneration when grafted to injured spinal cord; introduction of antibodies by injection was also proved to be effective method for boosting the recovery. However most important point that should be remembered while using antibodies that all three known inhibitory proteins act on the same receptor and eliminating only one of them might not be sufficient for adequate recovery. Removal of chondroitin sulfate deposits can be done by injection of enzymes (Chondroitinase ABC) Moreover treatments that would affect the inhibitory cells (Microglial cells or astrocytes) should also be developed too.

 A third approach would be the coaxing of the injured cells to act like their embryonic counterparts; which would facilitate the axonal growth. So for mimicking the embryonic conditions several factors should be supplied. Prevention of effects of inhibitory molecules can be achieved by terminating intracellular pathways that result in the stoppage of regeneration. Decreasing the intracellular concentration of Rho protein or increasing cyclic Amp might be suitable methods. It is said that neurotrophins perform their actions by increasing cAMP concentrations; thus other molecules that have the ability to increase cAMP concentration can be used instead of neurotrophic factors. However other known effect of neurotrophic factor is that they are capable of saving injured neuronal cells from atrophy. Atrophy can be impeded by using antiapoptotic proteins; but these proteins do not increase regeneration. So all trophic molecules should be carefully evaluated before deciding on what is the optimum therapy. For example clinical trials on animals illustrated that transplantation of modified Schwann cells concomitant with neurotrophin-3 and Nogo antibodies was better than separate utilization of each method.

Lastly regenerating axon must re-enter into the correct location; otherwise functional recovery is impossible. Cease of growth and synaptogenesis must be controlled too. For guidance several chemoattractive and chemorepulsive molecules are present; so techniques that manipulate the concentrations of these molecules can be an answer to problem of guidance.

            Usage of biomaterials has become important in this aspect. If scaffolds are provided for transplanted cells; better results can be expected. A scaffold would offer a chance to manipulate the direction of axon growth; which is essential. As in the case of the peripheral repair Hydrogels are the most pronounced candidates for such usages. Things  expected from the Hydrogels are mechanical stability(Ability to withstand forces act on them in normal motions of spinal cord; in other words mechanical characters similar to that of host tissue) Providing a surface for cell adhesion and subsequent growth; a porous structure for promoting angiogenesis and transport of nutrients. Hydrogels can be used to supply continuity between the severed ends and at the same time it would provide the surface for modification. Also scaffolds may provide ECM-like environment which is very important in nerve regeneration in PNS. But it is nearly absent in the CNS; thus incorporation of ECM proteins or short active peptide sequences. For example peptides like RGD fragment can be immobilized to increase cell adhesion; experiments with poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) shows an increase in regeneration after surface treatment with RGD.PHPMA is a biocompatible hydrogel and it is a patented product (Neurogel) which is shown to be effective in CNS injuries in animal studies. (Both acute and chronic spinal cord injuries and brain damages) Addition of RGD increase the integration of the hydrogel with the tissue. Also; since many neurotrophicfactors can affect the regeneration controlled release of neurotrophic factors is important. This is possible by methods such as microencapsulation, heparin based systems within these conduits. Inner surface of conduits can be designed in away that promotes guidance of axons in a controlled manner. Another factor is the interface between the hydrogel and the nerves; which should not be inhibitory; otherwise growth of regenerating nerve into the host tissue may stop.

            In acute injuries; it is important to restrict the degree of damage following the injury. Studies illustrate that all nerve damage does not occur at the time of the injury. Inflammatory response and the content of dead cells turn the injury into a hostile site for nerve cells. To decrease this extra damage; several drugs are designed. Methylprednisolone is one of these drugs; which enhances spinal cord recovery; provided that it is applied within 8 hours following the injury. This time-dependent hostility of the environment is harmful for transplants as well. So 2-4 weeks of waiting period following the injury is recommended before cell transplantation. Lastly all resolutions described should be modified when they are to be used for chronic injuries. Chronic injuries generally tend to be more resistant to rehabilitation; since scar formation, cyst cavity and the necrosis of the tissue is not prevented immediately after the injury. So different strategies must be seeked out; like extended periods of trophic factor treatments preceding transplantations may increase the efficiency of recovery.

            Another branch of Central Nervous system repair is the cell transplantation techniques used in neurodegenerative diseases. In this area most success has been realized in Parkinson’s disease. Parkinson Disease is characterized by the degeneration of dopamine producing neurons in the substantia nigra. Symptoms of this disease are tremors, stiffness of the body, difficulties in motor functions and depression. Transplantation of foetal tissue to degenerated area has been successful in several cases; but public opinion against the usage of foetal tissues stimulated researches that investigate the potential utilization of stem cells. Stem cells are shown to be neuroprotective (they protect dopaminergic neurons from degeneration) Also genetically modified stem cells which secrete GDNF (Glial derived neurotrophic factor) showed increased neuroprotective abilities. Other methods used in therapy of Parkinson’s disease is the direct injection of dopamine. But of course controlled release of missing factor is more desirable. In past osmotic pumps were used for delivery of chemical factors; but elevated inflammatory response was observed. So, instead, polymer -based techniques for controlled release is in use for delivering dopamine now; of course by taking the conditions of the CNS into account. In addition to these; there are some efforts to transform degenerative cells in vivo (Vectors containing dopamine gene)

            Multiple Sclerosis is a debilitating disease; in which autoimmune responses lead to the degradation of myelin sheaths. This slows down the nerve conduction; hence cause problems in motor functions. In later phases of the disease nerves are also damaged and severe problems are seen in all nervous system functions. Up to now no remedies for this disease have come up; but using cell transplants for remyelination is under examination. Main problems is the extent of demyelination; it is very hard for donor cells to remyelinate such huge distances. Another problem is the diffuse characteristic of the disease; it virtually upsets all body. Use of stem cells in correspondence with immunosuppressor drugs might lessen the effects of the disease.

            Amyotrophic lateral Sclerosis is a disease that results in the loss of motor pathways; degeneration of both motor neurons and spinal cord. Thus a cell replacement therapy is needed for this disease. However; as mentioned earlier, stem cell transplants are shown to increase the repair process rather than replacing the lost neurons. But in the early stages of the disease; transplantation of stem cells simultaneously with growth factor may protect the motor neurons and can enhance the condition of the patient.

            Other current studies to defeat these challenging diseases are transplantation of genetically modified cell lines, direct injection of missing molecules or gene transfer techniques and lastly electrical stimulation methods in which a brain-computer interface is established and this interface circumvents the damaged area and give control over the lost functions .But all this methods had limited success so far.

ELECTRONIC IMPLANTS

            In some cases, regeneration or transplantation are not viable options. For example control of a limb prosthesis can not be gained in natural ways. Moreover in some circumstances nervous pathways are disrupted to a degree that rejuvenation is impossible; such as genetically inherited deafness or blindness. Answer to these problems are implantable devices that can assume the role of a damaged part of the nervous system. Till now several devices have been designed and applied successfully (Like cochlear implants) Advances in this area is rather fast and exciting. Usage of these devices not only has some medical importance but also they can be used as analyzing tools. Recording of action potentials or other data using electrodes is a well-known method of monitoring nervous system activity. At the extreme end of this branch of nerve tissue engineering stands the researches conducted for development of hybrid computers. Several studies are underway to incorporate nerve cells into the computers.

            In ordinary procedures; Electrodes are used either for detecting nerve signals or for stimulating them. To achieve these tasks they must be in close contact with the nerves. So several properties of electrodes must be considered while designing such devices. Biocompatibility, mechanical strength, flexibility and conductivity are some of these parameters. Shape of the electrodes and their orientation with respect to the nerves are also important. As electrode material most appropriate choice is platinum. It is highly inert; released platinum ions in negligible even for long periods. Iridium is also in-use; its passivating oxide-layer strongly increases its electrical conductivity. Also glassy carbons were used as electrodes because of their high biocompatibility. For insulation several common polymers like Teflon or silicon rubber can be used. But there are some problems related with this kind of implants; for example if an electrode array is not properly fixed on a nerve; it can break loose and might damage the nerve fibers. Or if the insulation of individual electrodes is not sufficient; sensitivity of electrodes would decrease. (Functional impairment of the implant; which would render the presence of the implant futile)Another problem is the chronic inflammation related to prolonged presence of such implants. Shape and composition of the substrate on which the electrodes are placed should be carefully selected too. For instance silicone can be used; but its hardness might damage the surrounding tissues. Aside from their mechanical damage capacity; the most important problem with electrodes is the disruption of nerve cells due to high amount of electrical discharge; it was shown that frequent electrical stimulation damages the neurons.

            There are several electrode designs which use different geometries and different materials. One of the earliest electrode models is the cuff electrode. In this model electrodes embedded on a suitable substrate are placed on a nerve and it surrounds the nerve like a cuff. Thus nerve tissue is not damaged by the implantation. Disadvantage of cuff electrodes is their low accessibility into the nerve fascicles. In other words; this type of electrode arrays can only stimulate or read superficial potentials; which might not be enough in various cases; like when a specific motor pathway within a nerve is wanted to be stimulated. Novel designs like half-cuff electrodes or interdigitating electrodes are proposed for increasing the level  of interface between nerves and the electrodes; but even with this approaches capacity of cuff electrodes for accurate stimulation is limited. To solve this problem penetrating electrodes are utilized. This electrode arrays are composed of electrodes with different heights which are put on a substrate. These electrodes can penetrate into the nerve and can selectively stimulate specific fascicles or record their activity. Of course most obvious problem is the harm inflicted upon the nerve; extend of this damage and its targets can not be predicted precisely. Still another electrode design is the sieve-shaped one. For implantation of this electrodes; nerve is severed and electrode is placed between proximal and distal ends. During regeneration nerve axons elongate within the holes on the electrode; so an intimate contact between nerve and the electrode is established; which would increase the efficacy of recording. Damaging the nerves is the major setback of this method; other problem is the loss of some fibers when they can not pass through the holes. In humans; another type of electrode have been tried too. This electrode is composed of a glass tube; in which several gold wires are present. This tube also contains neurotrophic factors which promote the growth of axons into the tube. So by providing a direct contact between the electrodes and nerves; recordings can be taken. Of course recording techniques are not restricted to direct cell contact. Methods like electroencephalogram measure the brain waves by electrodes placed on the cranium. Several studies are underway to find methods to utilize the EEG signals; i.e. to use EEG signals for controlling parts of the body via computer interfaces. Promising results have been demonstrated with rats; which learned to control the movement of a robotic arm without using their limbs(Neuronal action is recorded by electrodes) Glass tube electrodes was tested with paralyzed human patients and one of the patients learned to communicate through an computer; with a moderate rate. While more sophisticated applications, like control muscles of a paralyzed patient, needs intricate techniques (Like real-time corrections on the movement; or simultaneous control of several elements) If developed properly; this methods might be a new hope for paralyzed patients.

            Among electronic implants; cochlear implants are the most widely used ones. Cochlear implants are used for stimulation of auditory nerves when damages hinder the conduction of processed sound through the normal route. Cochlear implants are composed of an external part which contains microphone, power supply, processor and a transmitter and an internal part which has a receiver and electrodes which stimulates the auditory nerve. In which sound waves detected by the microphone are converted into electrical signals. These signals are transmitted to internal part of the implant and through electrodes nerve stimulation occurs. Main design restriction is the number of electrodes in this system. Obviously increase in electrode number would increase the quality of hearing sensation; but if number of electrodes increases interference between stimulations would increase too. Other problems are possibility of damaging of healthy cells during implantation. But, even though it isn’t perfect, cochlear implants have definitely increased the quality of hearing for many people and it is under continuous development. Other type of hearing aid devices is brain stem implants; which are used when auditory nerve is damaged also.

            For visual implants task is more complicated; since processing of visual stimuli is highly complex. For overcoming blindness two types of implants are under development. Retinal implants which take the place of photoreceptor cells; stimulate the optic nerve in a controlled manner and cortical implants which directly stimulate the appropriate parts of brain upon incoming visual stimuli. Cortical implants are used when optic nerve is lost. There are two types of retinal implants; epiretinal and subretinal. In the case of subretinal implants; implant takes the role of photoreceptor cells and stimulates other cells in the normal pathway (Horizontal cells, bipolar cells) whereas in epiretinal implants; electrodes directly stimulates the retinal ganglion cells which are connected to optic nerve. Clinical trials of these devices are in process now. Other application areas for electrons in nervous system is pain relieving systems, stimulation of phrenic nerve in the case of ventilatory insufficiency, bladder control…

            Main research topics in electronic implants are data processing, optimization of machine neural tissue interface, mechanisms of adaptation and learning and how can they be used for providing a better interface. Although they are very interesting, these are beyond the scope of this review.

Suggestions:

            So far; primary aim of nerve tissue engineering is the improvement of the nerve regeneration. Techniques discussed in this review all have promising results. Now the next problem that should be solved in the field is the controlled reinnervation of the target organs.

            In all experiments with nerve guidance conduits, regardless of the improvement method used (Nerve growth factors, Schwann cells), distal end innervation is not sufficient. At midline sections NGC’s axon number, fiber density etc. values is comparable with normal  nerve(And even higher in some cases)But at the distal end reinnervation is more random, myelinated fiber number is low; also total axon number is lower than that of the proximal end.

            In my opinion; this is due to the mode of current approaches. NGC filled with anything that is known to have positive effect on nerve regeneration, aims an increase in mass regeneration. Even though this is a desired goal; it is not enough for the real aim; that is functional recovery. I think for functional recovery more intricate methods should be employed. For this end my first suggestion would be the organized stimulation of the denervated muscles. It is known that the refining of neuromuscular junction is activity dependent and following denervation muscles goes into atrophy. For preventing atrophy generally transcutanous stimulation or training of the muscles is used. But stimulation by implanted electrodes may have several advantages over this technique. First, by knowing the mode of movement of the muscle tissue in question; some muscle fibers can be selected. Electrical stimulation should protect this fiber from atrophy and it may also give them an advantage over other fibers in competition for reinnervation; like increased expression of membrane bound recognition proteins. By this method randomness of reinnervation can be decreased; also degree of functional recovery can be improved. Electrode for this kind of controlled stimulation is available; such as the BION design of MIT (It is originally designed for external control of muscles of paralyzed patients) Of course this would complicate the surgical application and also insertion of permanent implants is not desirable. But for massive damages, when functional recovery is not possible with conventional methods, this kind of invasive techniques can be rectified. Another possible method is the injection or controlled delivery of factors to the muscles; which may simulate the condition of innervated state and prevent the switching of gene expression following the injury.For this end controlled release of neurotransmitters can be employed. This way muscles would be more receptive to the incoming axons.

            Decrease in the number of axons in the distal end can be viewed as a limitation in the capacity of Schwann cells at helping regeneration. For this I suggest the usage of composite tubes; i.e. two tubes which are welded together. These two tubes can be treated differently; like one can contain Microcapsules of a specific nerve growth factor; where as other may contain another factor immobilized which increases Schwann cell activity. If with in-vitro analysis persistence of growth and Schwann cell activity with respect to the length of gap can be modeled; these data can be used to predict the optimal lengths of two tubes; other than growth factors inner geometry of these tubes can be different; such as first part can be as smooth as possible to gain maximum fast regeneration and the second part can be rougher to selectively eliminate some axons while promoting others(Controlled reinnervation)Molecules that are known to activate Schwann Cells can be immobilized at the latter part; for attaining a second burst of growth or genetically modified Schwann cells which are sensitive to a certain substance can be used for more precise control. Of course using such tubes might cause problems in strength and welding material should be carefully selected; differences in composition may cause differential movement of two parts and bends or more dangerously breaks can occur. For decreasing the compression and distributing stress more equally a biodegrable outer coating can be used. Of course degradation rate of two materials should be optimized.

            Axon elongation is due to the polymerization of actins and microtubules. Microtubules provides the elongation and is effective at selection of growth directing and actins confer their effect through adhesion to membrane proteins. So, F-actin polymer is important in crawl-like motion of the growing axon. Gene delivery methods that might increase the polymerization of these molecules can increase regeneration. Vectors that contain tubulin gene (Monomer of microtubules) with a specific inducer at promoter region that can only be supplied externally can be used for increasing the tubulin concentration within regenerating neurons.   Positive effect of electrical stimulation might be the promotion of polymerization of these molecules. If stimulation’s mode of action can be quantified; controlled stimulation can be useful when branching of the axon cones is needed. Also some groups suggest that the elongation of the axons during growing should be utilized too. If piezoelectric conduits are used; change of their length with the applied electric field can be transmitted to the axons. This may stimulate mode of elongation which reacts to the tension. Of course this type of therapy should be highly regulated; since if tension exceeds a certain amount neurons would be damaged and recovery would become harder.

            Different Growth factors have different mode of actions; and they might be beneficial in different ways. So regulation of their concentration gradient with respect to time within the conduit can be beneficial. This can be done by using several microspheres manufactured from several polymers with distinct biodegradation rate.

            Schwann cells can synthesize NGF; but control of release is not possible unless Schwann cells are modified. But there might be regulatory pathways that balance the myelination and NGF release. So if Schwann cells are supported with other cells for secretion; regeneration can go on for a longer time. For This end co-transplantation of Schwann cells with genetically modified dermoblasts or fibroblast (Which secrete NGF with a pre-determined rate) might be effective.