WO2011004971A2

WO2011004971A2 - Nerve conduit for peripheral nerve regeneration

Info

Publication number: WO2011004971A2
Application number: PCT/KR2010/003781
Authority: WO
Inventors: Soon Jung Hwang; In Sook Kim; Sung June Kim; Tae Hyung Lee
Original assignee: Seoul National University Dental Hospital
Priority date: 2009-07-10
Filing date: 2010-06-11
Publication date: 2011-01-13
Also published as: WO2011004971A3; KR20110005501A; KR101126645B1

Abstract

A nerve conduit for peripheral nerve regeneration composed of a polymeric material, which is coated on its inner surface with collagen and nerve growth factor (NGF); is equipped with an electrical stimulation device; or has both the coating and the device is provided, the conduit being effective in peripheral nerve regeneration, due to high adhesiveness, sustained-release of nerve growth factor, and continuous electrical stimulation.

Description

NERVE CONDUIT FOR PERIPHERAL NERVE REGENERATION FIELD OF THE INVENTION The present invention relates to a nerve conduit for peripheral nerve regeneration, and more particularly to a conduit composed of a polymeric material, which is coated on its inner surface with collagen and nerve growth factor (NGF); is equipped with an electrical stimulation device; or has both the coating and the device.

BACKGROUND OF THE INVENTION

The nervous system is morphologically divided into two categories: the central nervous system (CNS) consisting of the brain and spinal cord; and the peripheral nervous system (PNS) consisting of the ganglion and nerve fiber. The PNS functions to transmit external stimuli to the CNS and also to transmit the response from the CNS to the organ. Peripheral nerve injuries are classified into following three types: neurapraxia, a temporary loss of function due to a temporary conduction block; axonotmesis, a condition with disruption of the neuronal axon, but with maintenance of the myelin sheath, wherein the axon can be regenerated along Schwann's tube; and neurotmesis, a condition with complete transection of the nerve trunk and with no potential of recovery.

Nerve injuries caused by traffic accidents, industrial accidents, sporting injuries and surgical excision belong to neurotmesis. Peripheral nerves, unlike other tissues, have limitations on autogenous regenerative capability and autologous tissue grafts and, accordingly, they are hardly recovered from such transection.

To solve such problem, there have been attempted a number of methods for peripheral nerve regeneration, e.g., autologous nerve grafts, tissue engineering approaches, artificial nerves and the like. Autologous nerve grafts involves grafting a piece of a nerve from another part of the body by using surgical anastomosis so as to bridge nerve gaps, however, it has a defect that the function recovery rate of transplanted nerves is as low as 20-50%, as well as a loss of function in area from which the nerve was removed. Further, as a tissue engineering approach, there was an approach which comprises reconnecting a transected nerve using a conduit prepared from a biocompatible polymer, and filling the conduit with in vitro cultured nerve cells or stem cells in conjunction with a nerve growth-promoting material, e.g., nerve growth factor (NGF) or brain-derived neurotrophic factor (BDNF). However, no better outcomes have been reported in animal tests until now, as compared with the autologous nerve grafts. Meanwhile, although artificial nerves have been intensively investigated, they have the problems of insufficient regeneration of nerve cells and nervous signal transduction from the proximal residing nerves, and difficulty in functionally associating them with the proximal residing nerves. In addition, an approach using biomaterials only without combined use of a cell therapy or a gene therapy is now at uppermost limit. Nevertheless, nerve regeneration has been recently attempted by employing various artificial nerves as substitutes for autologous nerves. A nerve conduit acts as a passage for reconnecting and regenerating severed nerve tissues. When the ends of a severed nerve are respectively connected to both ends of a nerve conduit, the nerve becomes generated within the conduit by the growth of nerve fibers at the ends of the severed nerve. Currently, there have been developed a wide variety of nerve conduits. For instance, Korean Patent Publication No. 10-2003-0087196 discloses a conduit comprised of any one material selected from bioresorbable polymers, collagen, alginate and bioresorbable ceramics, the conduit having a chitosan-coating inner layer. U.S. Patent No. 4,877,029 describes a conduit employing acryl copolymer, polyurethane isocyanate and other biocompatible semi-permeable polymers as major constituents. Additionally, U.S. Patent No. 5,026,381 discloses a conduit composed of collagen, and U.S. Patent No. 5,019,087 describes a conduit comprised of a matrix of collagen and laminin-containing material.

The present inventors have developed a nerve conduit composed of polymeric materials as a main component, (i) which is coated on its inner surface with collagen and nerve growth factor (NGF); (ii) which is equipped with an electrical stimulation device; or (iii) which has both the coating and the device, and confirmed that the conduits are very effective in the functional regeneration of peripheral nerves.

SUMMARY OF THE INVENTION

Therefore, it is an object of the present invention to provide a nerve conduit useful in the peripheral nerve regeneration.

In accordance with one aspect of the present invention, there is provided a nerve conduit composed of a polymeric material, which is coated on its inner surface with collagen and nerve growth factor.

Further, there is provided a nerve conduit composed of a polymeric material, which is equipped with an electrical stimulation device comprising a conduit-shaped electrode. BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, which respectively show: Fig. 1 : an exemplary in-vitro film for nerve regeneration according to the present invention;

Fig. 2: microscopic images showing cells attached to A) a film not coated with collagen; and B) a film coated with collagen;

Fig. 3: microscopic images showing hematoxylin-stained cells attached to A) a film not coated with collagen; and B) a film coated with collagen;

Fig. 4: a photograph showing that PC 12 cells cultured on a film for nerve regeneration grow with protruding neurites, when treated with nerve growth factor;

Fig. 5: graphs showing the amount of nerve growth factors released from a film coated with nerve growth factor (NGF) only or a film coated with collagen and nerve growth factor (Col+NGF), depending on the culture period;

Fig. 6: graphs showing the amounts of nerve growth factor released from a film coated with nerve growth factor (NGF) only or a film coated with collagen and nerve growth factor (Col+NGF), cultured in the presence of PC 12 cells, depending on the culture period;

Fig. 7: an exemplary in vivo nerve conduit according to the present invention;

Fig. 8: an exemplary in vivo nerve conduit according to the present invention, which is equipped with an electrical stimulation device;

Fig. 9: photographs showing the procedure of transecting a rat nerve and reconnecting the transected nerve by using the in vivo nerve conduit according to the present invention;

Fig. 10: a graph showing the sciatic function indexes (SFI) determined in the rats of which nerves are reconnected using various nerve conduits;

Fig. 11 : a graph showing the static sciatic index (SSI) determined in the rats of which nerves are reconnected using various nerve conduits;

Fig. 12: photographs showing the procedure for measuring the sciatic nerve-evoked action potential from a rat;

Fig. 13: a photograph showing the measurement of the evoked potential;

Fig. 14: histological appearance of regenerated nerve tissue immunostained PMP-22 (peripheral myelin protein-22) within nerve conduit; and

Fig. 15: fluorecent microphotographs of regenerated nerve tissue within nerve conduit. Regenerated nerve tissue was dual-stained with NeuN for axon and with PMP22 for Schwann cells, showing that axons were ensheathed by myelin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a nerve conduit composed of a polymeric material, which is coated on its inner surface with collagen and nerve growth factor (NGF).

In the nerve conduit of the present invention, the polymeric material is preferably a substance which induces neither tissue rejection nor complications such as inflammation, etc., and also it is preferably a substance which is capable of having an electrical stimulation device inside. Exemplary polymeric materials include paralene, SU-8, polynorbornene and polyimide, but not limited thereto. When a bioresorbable polymer is employed as a main component, it degrades naturally within the body and thus requires no additional surgery to remove it. When a non-bioresorbable polymer is employed as a main component, it may be removed by simple surgical procedure, or may be kept intact as long as it evokes no tissue rejection or inflammation.

In the nerve conduit of the present invention, collagen plays a role in facilitating adhesion of nerve cells, and is preferably employed in an amount of 0.0001 to 1 mg based on 100 mm² of the conduit. Further, nerve growth factor plays a role in shortening the nerve regeneration time by helping the growth of attached nerve cells, and is preferably employed in an amount of 0.01 to 1 mg based on 100 mm² of the conduit. Preferably, the inner surface of the present conduit is first coated with collagen and, then, with NGF, but the coating order is not limited thereto.

Meanwhile, the nerve conduit of the present invention preferably has pores for exchange of nutrients with surrounding environments. Preferably, the nerve conduit may have 1 to 1,000 pores based on 100 mm² of the conduit, the diameter of pores preferably ranging from 1 to 500 μm.

Furthermore, the nerve conduit of the present invention may be cultured in conjunction with nerve cells, before use. The inventive nerve conduit coated with collagen and nerve growth factor releases more nerve growth factor when cultured in conjunction with nerve cells. The nerve cells are preferably

PC 12 nerve cells, but not limited thereto. Additionally, in an embodiment of the present invention, there is provided a nerve conduit composed of a polymeric material, which is equipped with an electrical stimulation device comprising a conduit-shaped electrode. The electrical stimulation device provides stimulations by delivering constant currents to peripheral nerves, leading to enhanced nerve regeneration. Specifically, the electrical stimulation device comprises an inner stimulation part that generates and outputs a nerve stimulation signal with a wave-pattern corresponding to the control signal received from the outside, and a nerve electrode part that transmits the nerve stimulation signal output from the inner stimulation part to nerve cells in a mammalian body. In the nerve electrode part, a conduit-shaped electrode is comprised. The electrical stimulation is preferably biphasic electrical current stimulation. Among many forms of electrical stimulations, the biphasic electrical stimulation has the advantages that the biphasic current is a waveform flowing in a human body; that it can provide constant currents because the charge is balanced, thereby no charged proteins adhering to an electrode; and that it causes no toxicity to cells and tissues owing to no pH change. Clinically, the biphasic electrical stimulation is used in an artificial ear cochlear implant (for hearing loss), deep brain stimulator (for treatment of Parkinson's disease) and cardioverter difϊbrillators. In particular, the electrical stimulation according to the present invention preferably has a strength of 1 to 100 μA and a pulse width of 1 to 200 μs, and it is preferably applied in 1 to 200 times per second. In addition, the period for electrical stimulation may be varied depending upon the degree of nerve injury, but continuous application over two weeks is more preferable for rapid nerve regeneration, rather than temporary application. The inner surface of the nerve conduit may be further coated with collagen and nerve growth factor.

The present inventors prepared a film for in vitro nerve regeneration, which is composed of polyimide and coated thereon with collagen and/or nerve growth factor, and evaluated the cell adhesion of the film. As a result, it was found that cell adhesive force increased by coating with collagen and the growth of neurites increased by coating with nerve growth factor {see Figs. 2 to 4). Additionally, the effects of collagen coating or cell culture on the release of nerve growth factor were examined, and it was demonstrated that more nerve growth factor is sustainedly released by collagen coating and much more is released by culturing the film together with PC 12 cell line (see Figs. 5 and 6). These results suggest that it is preferable to coat the nerve conduit of the present invention with collagen and nerve growth factor, followed by culturing in the presence of cells.

Meanwhile, the present inventors rolled up the in vitro nerve regeneration film and clamped it with a silicon ring to prepare an in vivo nerve conduit. A transected nerve in a rat was bridged via the conduit and the values representing the nerve recovery were assessed. As indicated by sciatic function index (SFI), sciatic static index (SSI) and sciatic nerve-evoked action potential, a polyimide conduit coated with collagen and nerve growth factor was effective in nerve regeneration (see Figs. 8 and 11, and Table 1). Further, in histochemical analysis, many myelin sheaths present in normal nerves were significantly observed in a group coated with collagen and nerve growth factor (see Fig. 14). Also, a lot of myelin sheaths were observed in a group coated with collagen and applied with an electrical stimulation (see Fig. 14). The above results suggest that the conduits of the present invention are excellent in nerve regeneration owing to the collagen and nerve growth factor coatings and electrical stimulation, and is useful in peripheral nerve regeneration.

The following Examples and Experimental Examples are given for the purpose of illustration only, and are not intended to limit the scope of the invention.

Example 1: Preparation of in vitro films for nerve regeneration

<!-!> Preparation of polyimide film A film having 15mm^χ 10mm in size was prepared from liquid polyimide resin, by spin coating as used in semiconductor processing, and holes 300 μm in diameter were formed on the surface of the film at intervals of lmm. Then, the film was dipped in 100% ethanol, and treated with a UV-light for sterilization in a clean bench for 24 hrs to prepare a polyimide film.

<l-2> Preparation of a collagen-coated polyimide film

On the surface of the film prepared in Example 1-1, 400 μL of a mixture of collagen (3 mg/mL, collagen type I, Nitta gelatin, Japan) and 70% ethanol by 1:1 (v/v) was sprayed, followed by drying it in a clean bench for a day to prepare a film coated with collagen.

<l-3> Preparation of a nerve growth factor (NGF)-coated polyimide film

On the surface of the film prepared in Example 1-1, 30 μL of a mixture of nerve growth factor (10 μg/mL; R&D systems) and PBS by 2: 1 (v/v) was sprayed, followed by drying it in a clean bench for a day to prepare a film coated with nerve growth factor.

<l-4> Preparation of polyimide film which is coated with collagen and nerve growth factor (TSfGF)

On the surface of the film prepared in Example 1-2, 30 μL of a mixture of nerve growth factor (10 μg/mL; R&D systems) and PBS by 2:1 (v/v) was sprayed, followed by drying it in a clean bench for a day to prepare a film coated with collagen and nerve growth factor in consecutive order. The film for nerve regeneration is depicted in Fig. 1. Experimental Example 1: Cell adhesion assay

In order to evaluate the cell adhesion of the inventive film for nerve regeneration, PC 12 cells (Accession No. KCLB 21721; Korean Cell Line Bank) cultured in RPMI medium (10% FBS, 1% penicillin/streptomycin) were labeled with DiI I (1,1' -dioctadecyl-3,3,3'₅3'-tetramethylindocarbocyanine perchlorate) and were inoculated on the nerve regeneration films prepared in Examples 1-1 and 1-2 at a concentration of I x IO⁶ cells/film, respectively. After 24 hours of incubation, each film was supplemented with nerve growth factor (20 ng), and incubated for 7 days by replacing the medium with a fresh one every three days. After incubation, each film's surface was observed by a microscope (Olympus BX51) at ^χ40, * 100, ^χ200 and *400 magnifications. As a result, there were few cells adhered on the film not coated with collagen (Example 1-1) as shown in Fig. 2A, while there were lots of cells adhered on the film coated with collagen (Example 1-2) as shown in Fig. 2B.

Meanwhile, in order to confirm the cell adhesion, the films were stained with hematoxylin, followed by microscopic observation at ^χ40, * 100, *200 and ^χ400 magnifications. As shown in Figs. 3A and 3B, it was confirmed that a large number of cells were attached on the collagen-coated film (Example 1-2).

In addition, it was observed that PC 12 cells grew from round shapes (10 μm diameter) into neurite-protruding shapes by treatment of nerve growth factor (see Fig. 4).

As described above, it was confirmed that collagen would enhance cell adhesion and that nerve growth factor would induce the neurite outgrowth of PC 12 cells.

Experimental Example 2: Measurement of release of nerve growth factor by collagen coating and cell culture

<2-l> Effect of collagen on release of nerve growth factor

In order to examine the effect of collagen-coating on the release of nerve growth factor, a film coated with nerve growth factor (500 ng) only as a control group and a film coated with both collagen and nerve growth factor as an experimental group were prepared. Then, the films were incubated in RPMI medium, and the resulting culture media were collected every two or three days and analyzed for the released amount of nerve growth factor by ELISA method. The measurement results are shown in Fig. 5. As shown in Fig. 5, the film coated with both collagen and nerve growth factor (col+NGF) sustainedly released nerve growth factor at an amount of about 1.5 to 2.5 folds higher than the film coated with nerve growth factor (NGF) only each day. Thus, it was confirmed that collagen-coating has a significant influence on the sustained- release of nerve growth factor.

<2-2> Effect of cells on release of nerve growth factor

In order to examine the effect of cells on the release of nerve growth factor, a film coated with nerve growth factor (500 ng) only and incubated together with PC 12 cells (a control group) and a film coated with both collagen and nerve growth factor and incubated together with PC 12 cells (an experimental group) were analyzed for the released amount of nerve growth factor, in accordance with the method described in Experimental Example 2- 1. Specifically, each film was sterilized and then incubated in a plate (incubation plate) in which PC 12 cells were attached at a concentration of 2^χ lO⁵. The incubated solutions were collected every two or three days for measurement of the released amount of nerve growth factor by ELISA method. The measurement results are shown in Fig. 6. As shown in Fig. 6, more nerve growth factor was sustainedly released from the film coated with both collagen and nerve growth factor (col+NGF), when co-cultured with cells. In addition, when compared with the film not co-cultured with cells as shown in Fig. 5, it was found that about two folds of nerve growth factor was released each day.

Considering the results of Figs. 5 and 6, the film, which is coated with both collagen and nerve growth factor, is suitable for nerve regeneration, probably mediated with sustained release of nerve growth factor.

Example 2: Preparation of in vivo conduits for nerve regeneration The film coated with collagen as described in Example 1-2 and the film coated with collagen and nerve growth factor as described in Example 1-4 (wherein the concentration of nerve growth factor is adjusted to 300 ng) were rolled up, respectively, and were clamped with silicon rings of 3 mm inside diameter to prepare in vivo nerve conduits for animal experiments, respectively. An exemplary shape of the conduits is depicted in Fig. 7. In Fig. 7, 174 indicates a conduit electrode, and 172, a nerve electrode. The conduit was attached to an electrical stimulation device, which can provide continuous electrical stimulations and can be transplanted into an experimental animal, and the electrical stimulation device comprised an electrical stimulation chip and a battery. The electrical stimulation device is depicted in Fig. 8. In Fig. 8, 150 indicates an inner stimulation part; 152, a silicon rubber; and 170, a nerve electrode part. Experimental Example 3: Animal experiment

<3-l> Reconnection of transected nerves in rat using a nerve conduit

Thirty (30) SD rats (220-250 g) of 7 week age were divided into five groups as follows:

Group I (control): a nerve conduit composed of polyimide;

Group II (col+NGF): a nerve conduit composed of polyimide, which is coated with collagen and nerve growth factor;

Group III (col+NGF+EC): a nerve conduit composed of polyimide, which is coated with collagen and nerve growth factor, and is equipped with an electrical stimulation device;

Group IV (EC): a nerve conduit composed of polyimide, which is equipped with an electrical stimulation device; and

Group V (col+EC) : a nerve conduit composed of polyimide, which is coated with collagen, and is equipped with an electrical stimulation device

After each rat was intraperitoneally anesthetized, the right sciatic nerve thereof was exposed and transected to create 10 mm gap. Subsequently, the ends of a transected nerve were ligated with the nerve conduit of each group, and then the incision was sutured. On 2 and 4 weeks after the operation, the sciatic functional index (SFI) and sciatic static index (SSI) were assessed, and on 4 weeks, the action potential was assessed and staining was performed. The process for anatomy of rat nerve is depicted in Fig. 9. <3-2> Measurement of SFI

For functional evaluation of sciatic nerves, the motor function of rat was assessed by using the sciatic function index (SFI) on postoperative 2^nd and 4^th weeks (Bain JR et al., Plast. Reconstr. Sur., 1989, Jan. 83(1): 129-138). Specifically, the rats' paws were painted with ink and the rats were allowed to walk along a dark straight corridor several times for footprints assessment. The SFI were calculated by following formula I using the experimental results.

SFI = -38.3 x (EPL-NPL)/NPL + 109.5 x (ETS-NTS) + 13.3 * (EIT- NIT)ZNIT - 8.8

EPL: experimental paw length;

NPL: unoperated normal paw length;

ETS: distance between the first and fifth toes of operated experimental foot;

NTS: distance between the first and fifth toes of unoperated experimental foot;

EIT: distance between the second and fourth toes of operated experimental foot;

NIT: distance between the second and fourth toes of unoperated experimental foot;

A SFI value of 0 is a status of perfect normality, and -100 is a status of perfect transection. The SFI values^■ measured after foot-printing are shown in Fig. 10.

Group II using the conduit coated with both collagen and nerve growth factor (col+NGF) showed no difference in SFI value on 2 weeks, but showed some recovered SFI value on 4 weeks, compared with Group I (control). For Group III (col+NGF+EC) using the conduit which is coated with both collagen and nerve growth factor and is equipped with an electrical stimulation device, SFI values on 2 and 4 weeks were similar to those of Group II (col+NGF). In addition, Group IV (EC) using the conduit which is equipped with an electrical stimulation device and the Group V (col+EC) using the conduit which is coated with collagen and is equipped with an electrical stimulation showed similar SFI values to those of Group II (col+NGF) on 2 and 4 weeks, respectively. Therefore, it is more effective for recovery from nerve injury to use a conduit coated with both collagen and nerve growth factor or to use a conduit in conjunction with electrical stimulation. <3-3> Measurement of SSI

For recovery evaluation of sciatic nerves, the static sciatic index (SSI) was measured on postoperative 2^nd and 4^th weeks (Bain JR et al., Plast. Reconstr.

Sur., 1989, Jan. 83(1): 129-138). Rats' paws were photographed with a camera, and the SSI values were calculated by the following formula using the obtained parameters each.

SSI = 108.44 x (OTS-NTS)/NTS + 31.85 x (OITS-NITS)/NITS-5.49

OTS: operated side toe spread; NTS: non-operated side toe spread;

OITS: operated side intermediate toe spread; and

NITS: non-operated side intermediate toe spread. The measurement results are shown in Fig. 11. As shown in Fig. 11, the SSI values, a nerve recovery index acquired when the animal is on a static position, were much higher in Group II (col+NGF) using the conduit coated with collagen and nerve growth factor than Group I (control) on both 2^nd and 4^th weeks. Group III (col+NGF+EC), using the conduit coated with collagen and nerve growth factor and being applied with an electrical stimulation, showed higher SSI values than Group I, but no synergic effect of the electrical stimulation and the coating was observed. Further, the index became higher than Group I when electrical stimulation was applied to an uncoated conduit (Group IV), and was much higher in Group V, wherein the conduit was coated with collagen and the electrical stimulation was applied. Therefore, it is effective for recovery from nerve injury to use a conduit coated with collagen and nerve growth factor or to use a conduit in conjunction with electrical stimulation. <3-4> Measurement of sciatic nerve-evoked action potential

In order to examine the degree of regeneration of sciatic nerve, the simulating currents were applied to the proximal nerves via bipolar stimulating electrodes and the evoked action potential was recorded on the distal nerves via recording electrodes, by using an electrical measuring apparatus (AD Instruments; stimulation & recording system and PowerLab version 5.01). The measured signals were filtered using a PC to obtain desired stimulation signals only. The measurement processes are shown in Fig. 12. In Fig. 12(a), the evoked potentials in normal nerves were measured to monitor the expressions of the threshold and action potential; and in Fig. 12(b), the evoked potentials were measured after the transection of normal nerves. Since only artifacts are observed in transected nerves, the action potential waveforms can be extracted from the artifacts by comparing the waveforms in the transected nerves with those of (a). The evoked potentials can be obtained from waveforms (a) and (b) and, thus it was examined under the same condition whether the evoked potentials are generated in nerves at the site of surgery. The method for measuring the evoked potentials using an electrical measurement apparatus is shown in Fig. 13, and the results are shown in Table 1 below.

N.D: Not detected.

As shown in Table 1, the evoked potential was not detected in Group I (control), which indicates that there was no nerve recovered. In contrast,

Group II (col+NGF), using the conduit coated with collagen and nerve growth factor, showed high action potentials of 1.23 mA in average, indicating that nerve regeneration in Group II is better than that in Group I (control). Group III (col+NGF+EC), using the conduit coated with collagen and received electrical stimulation, showed the action potential of 2.23 mA, which is higher than Group I (control) but not than Group II (col+NGF) using the conduit coated with collagen and nerve growth factor. In addition, the action potential was observed in just one of six rats in Group IV (EC) received only electrical stimulation, but it was better than Group I (control) wherein no action potential was detected. Further, Group V (col+EC) showed the action potential of 1.50 mA, which was much higher than that of Group IV. Therefore, it is effective for recovery from nerve injury to use a conduit coated with collagen and nerve growth factor or to use a conduit in conjunction with electrical stimulation.

<3-5> Histological evaluation After 15 mm sciatic nerves containing an experimental site were obtained from the experimental and control groups by exfoliation, the nerves were fixed in 10% paraformaldehyde, dehydrated, and embedded in paraffin. Subsequently, the nerves were cross-sectioned across the center of reconstructed site to prepare 2 μm-thick sections. After paraffin was removed using xylene, the sections were dehydrated using a series of ethanol, and were sequentially subjected to a reaction with hydrogen peroxide, proteinase K and serum. In order to stain myelin sheath encasing the axons of the peripheral nervous system, peripheral myelin protein 22 (PMP-22; santa cruz) was immunostained, thereby evaluating myelin formation. In addition, a shape of myelin around neurites was observed by fluorescence staining of PMP-22 (red) representing myelin sheath and Neu N (green) representing neurites.

The results are shown in Figs. 14 and 15. As shown in Fig. 14 displaying the immunostained PMP-22, few peripheral myelin proteins were observed in Group I, while more peripheral myelin proteins were observed in Group II (col+NGF) using the conduit coated with collagen and nerve growth factor. The amount of peripheral myelin proteins observed in Group III (col+NGF+EC) was less than that of Group II (col+NGF). In Group IV (EC) and Group V (col+EC), a lot of peripheral myelin proteins were observed.

Meanwhile, as shown in Fig. 15 representing the result of the fluorescence-staining, a small amount of the red-colored peripheral myelin proteins were observed in all Groups, but the neurites showing a green color were highly observed in Group II (col+NGF) and Group V (col+EC). While the invention has been described with respect to the above specific embodiments, it should be recognized that various modifications and changes may be made to the invention by those skilled in the art which also fall within the scope of the invention as defined by the appended claims.

Claims

What is claimed is:

1. A nerve conduit composed of a polymeric material, which is coated on its inner surface with collagen and nerve growth factor (NGF).

2. The nerve conduit of claim 1, wherein the polymeric material is selected from the group consisting of paralene, SU-8, polynorbornene and polyimide.

3. The nerve conduit of claim 1, wherein the collagen is employed in an amount of 0.0001 to 1 mg based on 100 mm of the conduit.

4. The nerve conduit of claim 1, wherein the nerve growth factor is coated in an amount of 0.01 to 1 mg based on 100 mm² of the conduit.

5. The nerve conduit of claim 1, wherein the inner surface is first coated with collagen and, then, with NGF.

6. The nerve conduit of claim 1, which has 1 to 1000 of pores with a pore diameter of 1 to 500 μm, based on 100 mm² of the conduit.

7. A nerve conduit composed of a polymeric material, which is equipped with an electrical stimulation device comprising a conduit-shaped electrode.

8. The nerve conduit of claim 7, wherein the electrical stimulation is biphasic electrical current stimulation.

9. The nerve conduit of claim 7, wherein the electrical stimulation has a strength of 1 to 100 μA.

10. The nerve conduit of claim 7, wherein the electrical stimulation has a pulse width of 1 to 200 μs.

11. The nerve conduit of claim 7, wherein the inner surface of the conduit is coated with collagen and nerve growth factor.