Neurotrophic factor carrier, method for producing the same, and method for regenerating a nerve using the same

Abstract

The present invention relates to a neurotrophic factor carrier, particularly to a neurotrophic factor carrier wherein the neurotrophic factor is contained in a porous nerve conduit having micropores formed in microchannels, a method for preparing the same and a method for regenerating a nerve using the same, wherein the neurotrophic factor carrier prepared according to the present invention is applicable to in-vitro and in-vivo researches on nerves.

Claims

1. A method for preparing a neurotrophic factor carrier including a porous nerve conduit having a microchannel structure with micropores, for regeneration of a central nerve or a peripheral nerve comprising: i) preparing a polymer material for a porous nerve conduit having a microchannel structure with micropores, by dissolving a hydrophobic biocompatible polymer of poly(lactic-co-glycolic acid) (PLGA) or polycaprolactone (PCL) in a water-miscible organic solvent of tetraglycol; ii) preparing a porous nerve conduit from the polymer material, the nerve conduit having a microchannel structure with micropores, iii) immersing the porous nerve conduit in a heparin solution; and iv) immersing the porous nerve conduit in a neurotrophic factor solution, wherein step ii) comprises: inserting a plurality of water-soluble glass fibers into a container having upper and lower channels; injecting the polymer material of step i into the container in which the plurality of water-soluble glass fibers are inserted; infiltrating the polymer material between the water-soluble glass fibers by applying vacuum to the upper channel; separating the water-soluble glass fibers with the polymer material for the porous nerve conduit infiltrated from the container; and dissolving the water-soluble glass fibers by immersing the separated glass fibers with the polymer material in water to form the microchannel structure of the hydrophobic polymer, wherein in the step of dissolving the water-soluble glass fibers, the microchannels are formed as the hydrophobic biocompatible polymer is cured, and the micropores are formed in the microchannels as the water-miscible organic solvent is mixed with the water and released from the hydrophobic polymer, and wherein the polymer material for a nerve conduit is one in which the hydrophobic biocompatible polymer is dissolved in the water-miscible organic solvent at a concentration of 10-40 weight/volume % (w/v %).

2. The method for preparing a neurotrophic factor carrier of claim 1, wherein the neurotrophic factor is selected from a group comprising of NT-3 (neurotrophin-3), NT-4 (neurotrophin-4), BDNF (brain-derived neurotrophic factor), NGF (nerve growth factor), GDNF (glial-derived neurotrophic factor), CNTF (ciliary neurotrophic factor) and a mixture thereof.

3. The method for preparing a neurotrophic factor carrier of claim 2, wherein the neurotrophic factor is a wild-type or recombinant neurotrophic factor.

4. The method for preparing a neurotrophic factor carrier of claim 1, wherein the lower channel has a smaller diameter than the upper channel and the container is sloped with a discontinuous angle.

5. The method for preparing a neurotrophic factor carrier of claim 1, wherein the polymer material for a nerve conduit is in a solution state at room temperature.

6. The method for preparing a neurotrophic factor carrier of claim 1, which further comprises, after the step of dissolving the glass fibers: a step of cooling a nerve conduit formed after the glass fibers are dissolved with liquid nitrogen; and a step of shaping the cooled nerve conduit by cutting.

7. The method for preparing neurotrophic factor carrier of claim 1, wherein the container is formed of a transparent material so that the infiltration of the polymer material for a nerve conduit can be checked visually.

8. The method for preparing a neurotrophic factor carrier of claim 1, wherein the application of vacuum is repeated multiple times.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The above and other features and advantages will become more apparent to those of ordinary skill in the art by describing in detail exemplary embodiments with reference to the attached drawings, in which:

(2) FIG. 1 shows photographs illustrating a method for preparing a porous nerve conduit. A shows glass fibers, a glass capillary and a glass capillary into which glass fibers are inserted, B shows a silicone tube coupled with a 2-way valve and a Luer lock syringe, C shows a silicone tube coupled with a 2-way valve and a Luer lock syringe, and D shows application of vacuum into a glass tube using a syringe.

(3) FIG. 2 schematically shows a method for preparing a porous nerve conduit.

(4) FIG. 3A and FIG. 3B show channel formation in a container with a discontinuous (A) or continuous (B) slope.

(5) FIG. 4 shows transverse cross-sectional SEM images of a porous PLGA nerve conduit; scale bar=(left) 100 μm, (right) 10 μm.

(6) FIG. 5 shows magnified SEM images showing a microstructure at the transverse cross section of a porous nerve conduit; scale bar=(A, C) 10 μm, (B, D) 1 μm, =micropores inside microchannels.

(7) FIG. 6 shows longitudinal cross-sectional SEM images of a porous nerve conduit; scale bar=(A) 100 μm, (B) 10 μm, (C) 10 μm, (D) 1 μm, =micropores inside microchannels.

(8) FIG. 7 shows TG released from a porous nerve conduit and submerged in distilled water (DW); arrow: TG.

(9) FIG. 8 shows porous nerve conduits prepared with various diameters and lengths depending on applications.

(10) FIG. 9 shows 3D micro-CT images (sagittal plane) of a nerve conduit prepared according to an exemplary embodiment of the present invention.

(11) FIG. 10 schematically shows an expression vector for the recombinant neurotrophic factor BDNF.

(12) FIG. 11 shows an SDS-PAGE result for the recombinant neurotrophic factor BDNF.

(13) FIG. 12 schematically shows an expression vector for the recombinant neurotrophic factor NGF.

(14) FIG. 13 shows an SDS-PAGE result for the recombinant neurotrophic factor NGF.

(15) FIG. 14 shows the release behavior of a neurotrophic factor carrier containing the recombinant neurotrophic factor BDNF in vitro for 30 days.

(16) FIG. 15 shows the release behavior of a neurotrophic factor carrier containing the recombinant neurotrophic factor NGF in vitro for 30 days.

(17) FIG. 16 illustrates an in-vivo experiment procedure for confirming the nerve regeneration effect of a neurotrophic factor carrier according to the present invention in the sciatic nerve. A shows a PCL tube and a PLGA nerve conduit prepared for an in-vivo experiment, B shows an image of a nerve conduit inserted in the PCL tube, and C shows image of a 16-mm nerve conduit inserted after cutting the sciatic nerve of a rat.

(18) FIG. 17 shows the result of an in-vivo experiment procedure for confirming the nerve regeneration effect of a neurotrophic factor carrier according to the present invention in the sciatic nerve. A shows a tissue into which a hollow PCL tube with no nerve conduit inserted is transplanted (Hollow), B shows an autografted nerve (Autograft), C shows a tissue into which a nerve conduit not containing NGF is transplanted (Scaffold), and D shows a tissue into which a nerve conduit containing NGF is transplanted.

(19) FIG. 18 shows a procedure of inserting a nerve conduit in a complete spinal cord transection model.

(20) FIG. 19 shows the result of an in-vivo experiment procedure for confirming the nerve regeneration effect of a neurotrophic factor carrier according to the present invention in the spinal cord. A shows a tissue into which a nerve conduit is not transplanted, B shows a tissue into which a nerve conduit not containing NT-3 is transplanted (Scaffold), and C shows a tissue into which a nerve conduit containing NT-3 is transplanted (Scaffold+NT-3).

(21) In the following description, the same or similar elements are labeled with the same or similar reference numbers.

DETAILED DESCRIPTION

(22) The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

(23) The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “includes”, “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. In addition, a term such as a “unit”, a “module”, a “block” or like, when used in the specification, represents a unit that processes at least one function or operation, and the unit or the like may be implemented by hardware or software or a combination of hardware and software.

(24) Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and will not be interpreted in an idealized or overly formal sense unless expressly so defined herein.

(25) Preferred embodiments will now be described more fully hereinafter with reference to the accompanying drawings. However, they may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

Example 1

Porous Nerve Conduit Containing Nerve Growth Factor (NGF)

(26) 1-1: Preparation of Porous PLGA Nerve Conduit

(27) A 20% (w/v) PLGA-TG solution (polymer material) was prepared by mixing the hydrophobic polymer poly(lactic acid-co-glycolic acid) (PLGA) (lactic acid/glycolic acid mol %, 85:15) and the water-miscible solvent tetraglycol (TG) (density: 1.09 g/mL, Sigma-Aldrich, USA) at a weight/volume (w/v) ratio of 20% (w/v) and then dissolving at 60° C. for 18 hours.

(28) A glass capillary with an inner diameter of 1.6 mm and a length of 13 cm was heated at the center portion to form a bottleneck, thereby forming upper and lower channels sloped with a discontinuous angle. The lower channels were formed to have smaller diameters than the upper channel. Then, 7000-8500 strands of a water-soluble glass fiber (50P.sub.2O.sub.5-20CaO-30Na.sub.2O in mol % (1100° C., 800 rpm)) with diameters of 10-20 μm were cut to 5-6 cm and inserted densely into the upper channels of the glass tube along the axis direction (FIG. 1A and FIG. 2A).

(29) A pressure device prepared by connecting a Luer lock syringe equipped with a silicone tube of an inner diameter of 0.8 mm and a length of 15 cm, coupled with a 2-way valve, to the upper channels of the glass fiber-inserted glass tube (FIG. 1B and FIG. 1C).

(30) After immersing the lower channels of the glass tube in the 20% (w/v) PLGA-TG solution at room temperature, vacuum was repeatedly applied into the glass tube using a syringe such that the 20% (w/v) PLGA-TG solution was completely infiltrated into the void space between the glass fibers (FIG. 1D and FIG. 2C).

(31) The specific configuration of the glass tube (container) is shown in FIG. 3A. As shown in FIG. 3A, the diameter of the lower channels was decreased than that of the upper channels with a discontinuous angle. If the angle is continuous (FIG. 3B), it is difficult to maintain constant intervals between the glass fibers because the intervals between the glass fibers decrease gradually.

(32) If the nerve conduit is prepared in the state where the intervals between the glass fibers are not constant, the intervals between the microchannels of the nerve conduit will not be constant too. Then, the direction of nerve regeneration induced by the glass fibers will be different depending on the microchannel. As a result, it is difficult to induce nerve regeneration in the same direction.

(33) The PLGA-TG solution-infiltrated glass fibers were separated from the glass tube using a wire with a diameter of 1.5 mm and a length of 15 cm and, immediately thereafter, completely immersed in distilled water (DW) at 10-20° C. for at least 24 hours (FIG. 2D), so that the glass fibers were completely dissolved, and about 7,000-8,500 (7,777±716.2) microchannels of PLGA, with diameters of 10-20 μm (16.54±3.6 μm), were formed in the space where the glass fibers had been dissolved (FIG. 2E and FIG. 4). The microchannels were formed as the glass fibers were dissolved in the water at 10-20° C. and the hydrophobic polymer PLGA was cured at the same time. Also, micropores were formed inside the microchannels as the TG was mixed with the water and released from the hydrophobic polymer while the glass fibers infiltrated with the PLGA-TG solution were immersed in the DW (FIG. 4, FIG. 5 and FIG. 6). Because the TG released from the nerve conduit had a higher density than the DW, it was submerged like heat haze in the DW (FIG. 7).

(34) After the glass fibers and the TG were removed through the treatment with DW, the prepared porous microchannels formed of PLGA, i.e., the nerve conduit, was frozen in liquid nitrogen for about 30 seconds, cut to a desired size and then shaped into a desired shape (FIG. 8).

(35) 1-2: Investigation of Microstructure Inside Porous Nerve Conduit

(36) The microstructure formed in the microchannels inside the nerve conduit prepared in Example 1-1 was investigated by scanning electron microscopy (SEM) (FIG. 4, FIG. 5 and FIG. 6).

(37) FIG. 4 shows the transverse cross section of the nerve conduit, FIG. 5 shows magnified images showing the microstructure at the transverse cross section of the nerve conduit and FIG. 6 shows the longitudinal cross section of the nerve conduit. It can be seen that the microchannels were formed continuously inside the nerve conduit and micropores were formed in the microstructure.

(38) 1-3: 3D Micro-CT Imaging of Porous Nerve Conduit

(39) The 3D CT images of the nerve conduit of Example 1-1 are shown in FIG. 9. Intact microchannels inside the nerve conduit are observed as seen from FIG. 9.

(40) 1-4: Preparation of Heparin-Coated Porous Nerve Conduit

(41) The nerve conduit of Example 1-1 was washed with a 2 wt % NaCl aqueous solution 3 times, for 5 minutes each. After preparing a heparin solution by mixing heparin in a 2 wt % NaCl aqueous solution to a concentration of 1 mg/mL, the nerve conduit was immersed in the heparin solution at 4° C. for 3 hours. The heparin-coated nerve conduit was washed with a 2 wt % NaCl aqueous solution 3 times, for 5 minutes each. Then, the heparin-coated nerve conduit was washed with water 3 times, for 5 minutes each.

(42) 1-5: Coating of NGF on Heparin-Coated Porous Nerve Conduit

(43) After preparing an NGF solution by mixing NGF in distilled water to a concentration of 0.1-1000 μg/mL, the heparin-coated nerve conduit of Example 1-4 was immersed in the NGF solution at room temperature for 6 hours. Then, the nerve conduit was washed with PBS (phosphate-buffered saline, pH ˜7.4) 3 times, for 5 minutes each. The amount of NGF coated on the surface of the heparin-coated nerve conduit was about 1 mg.

Example 2

Recombinant Neurotrophic Factor

(44) 2-1: Large-Scale Production of Fluorescent BDNF (Brain-Derived Neurotrophic Factor)

(45) In order to construct a recombinant fluorescent BDNF expression vector, human BDNF (hBDNF) cDNA was amplified by PCR using a forward primer 5′-GACGGTACCGCACCCATGGCAGAAGG-3′ (SEQ ID NO 1) and a reverse primer 5′-AGAATTCTCACCGCCTCGGCTTGTC-3′ (SEQ ID NO 2). The PCR was performed by using 30 μL of a mixture containing 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 100 μg/mL gelatin, 0.2 mM dNTPs, 1.25 units of DNA polymerase (ELPiS Biotech, Korea) and 50 μmol of forward and reverse primers each. A cycle of annealing at 55° C. for 1 minute, extension at 72° C. for 2 minutes and denaturation at 94° C. for 1 minute was repeated 30 times. The amplification product was treated with BglII and KpnI restriction enzymes and then ligated into a pBAD-HisA vector (Invitrogen, USA) to obtain a pBAD-HisA-BDNF vector. GFP cDNA was amplified by PCR using a forward primer 5′-GGAATTCGTGAGCAAGGGCGAGGAG-3′ (SEQ ID NO 3) and a reverse primer 5′-TGAATTCTACTTGTACAGCTCGTC-3′ (SEQ ID NO 4). The amplification product was in-frame ligated into the EcoRI site of the pBAD-HisA-BDNF vector to obtain a pBAD-HisA-BDNF-GFP vector (FIG. 10).

(46) For expression of a recombinant fluorescent BDNF, competent cells were prepared using a CaCl.sub.2 buffer and the pBAD-HisA-BDNF-GFP vector, a recombinant fluorescent BDNF expression vector, was introduced into Escherichia coli TOP10 cells by applying a heat shock (42° C.). The Escherichia coli TOP10 cells into which the pBAD-HisA-BDNF-GFP vector was introduced were incubated overnight in an LB (Luria-Bertani) medium containing ampicillin at 37° C. When the absorbance (A600) of the medium reached 0.6, expression was induced using 0.25% (w/v) L-arabinose. 3 hours later, the culture was centrifuged and the bacteria were pelletized, lysed and then sonicated. After centrifuging at refrigerator temperature and 6,000 rpm for 30 minutes, the resulting supernatant was transferred to a fresh tube. The crude protein from the sonicated bacterial supernatant was purified to a purity of 95% or higher by affinity chromatography (ELPiS Biotech, Korea) and then analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (FIG. 11).

(47) 2-2: Large-Scale Production of Fluorescent NGF (Nerve Growth Factor)

(48) In order to construct a recombinant fluorescent NGF expression vector, human NGF (hNGF) cDNA was amplified by PCR using a forward primer 5′-GGTACCAGCAGCAGCCATCCGAT-3′ (SEQ ID NO 5) and a reverse primer 5′-GAATTCGCCGCACGACGCACCG-3′ (SEQ ID NO 6). The PCR was performed in the same manner as in Example 2-1. The amplification product was treated with KpnI and EcoRI restriction enzymes and then ligated into a pBAD-HisA vector to obtain a pBAD-HisA-NGF vector. RFP cDNA was amplified by PCR using a forward primer 5′-AAGCTTAATTAATTAAGTTTGTGCCCCAGTT-3′ (SEQ ID NO 7) and a reverse primer 5′-AAGCTTAATTAAGTTTGTGCCCCAGTTTGC-3′ (SEQ ID NO 8). The amplification product was in-frame ligated into the HindIII site of the pBAD-HisA-NGF vector to obtain a pBAD-HisA-NGF-RFP vector (FIG. 12).

(49) The recombinant fluorescent NGF was expressed in E. coli into which the recombinant fluorescent NGF expression vector pBAD-HisA-NGF-RFP was introduced in the same manner as in Example 2-1, which was then purified to a purity of 95% or higher and analyzed by SDS-PAGE (FIG. 13).

(50) 2-3: Large-Scale Production of Fluorescent GDNF (Glial-Derived Neurotrophic Factor)

(51) In order to construct a recombinant fluorescent GDNF expression vector, human GDNF (hGDNF) cDNA was amplified by PCR using a forward primer 5′-GGTACCAGTCCGGATAAACAAATGGCA-3′ (SEQ ID NO 9) and a reverse primer 5′-GAATTCAATACAACCACAACGTTTTGCG-3′ (SEQ ID NO 10). The PCR was performed in the same manner as in Example 2-1. The amplification product was treated with KpnI and EcoRI restriction enzymes and then ligated into a pBAD-HisA vector to obtain a pBAD-HisA-GDNF vector. GFP cDNA was amplified by PCR using a forward primer 5′-AAGCTTAATTAATTAAGTTTGTGCCCCAGTT-3′ (SEQ ID NO 3) and a reverse primer 5′-AAGCTTAATTAAGTTTGTGCCCCAGTTTGC-3′ (SEQ ID NO 4). The amplification product was in-frame ligated into the EcoRI site of the pBAD-HisA-GDNF vector to obtain a pBAD-HisA-GDNF-GFP vector.

(52) The recombinant fluorescent GDNF was expressed in E. coli into which the recombinant fluorescent GDNF expression vector pBAD-HisA-GDNF-GFP was introduced in the same manner as in Example 2-1, which was then purified to a purity of 95% or higher and analyzed by SDS-PAGE.

(53) 2-4: Large-Scale Production of Fluorescent CNTF (Ciliary Neurotrophic Factor)

(54) In order to construct a recombinant fluorescent CNTF expression vector, human CNTF (hCNTF) cDNA was amplified by PCR using a forward primer 5′-GGTACCGCATTTACCGAACATAGTCCG-3′ (SEQ ID NO 11) and a reverse primer 5′-GAATTCCATTTTTTTGTTGTTGGCAATATAATGG-3′ (SEQ ID NO 12). The PCR was performed in the same manner as in Example 2-1. The amplification product was treated with KpnI and EcoRI restriction enzymes and then ligated into a pBAD-HisA vector to obtain a pBAD-HisA-CNTF vector. GFP cDNA was amplified by PCR using a forward primer 5′-AAGCTTAATTAATTAAGTTTGTGCCCCAGTT-3′ (SEQ ID NO 3) and a reverse primer 5′-AAGCTTAATTAAGTTTGTGCCCCAGTTTGC-3′ (SEQ ID NO 4). The amplification product was in-frame ligated into the EcoRI site of the pBAD-HisA-CNTF vector to obtain a pBAD-HisA-CNTF-GFP vector.

(55) The recombinant fluorescent CNTF was expressed in E. coli into which the recombinant fluorescent CNTF expression vector pBAD-HisA-CNTF-GFP was introduced in the same manner as in Example 2-1, which was then purified to a purity of 95% or higher and analyzed by SDS-PAGE.

Example 3

Monitoring of Neurotrophic Factor Release Behavior

(56) Nerve conduits with the recombinant neurotrophic factors BDNF and NGF of Example 2 coated on the surface were prepared according to the method of Example 1-5.

(57) Then, the release behavior of the neurotrophic factors in vitro was investigated by measuring the release of the neurotrophic factors from the nerve conduits in PBS buffer. The fluorescence-labeled recombinant neurotrophic factors were quantitated by using a fluorescence microscope and the release amount was analyzed by collecting solutions at predetermined time. The release amount was calculated as a percentage of the initial coating amount 1 mg in the carrier.

(58) As a result, it was confirmed that the recombinant neurotrophic factors BDNF and NGF were released in a sustained manner for over 30 days (FIG. 14 and FIG. 15). In particular, after 3 days, they showed release by diffusion through the microstructure of the nerve conduits. This long-term release behavior for over 30 days is considered effective for nerve regeneration.

(59) The daily release amount was about 3% for BDNF and about 1% for NGF per day. Considering that the initial amount of the neurotrophic factor loaded in the nerve conduit was 1 mg, the neurotrophic factor carrier exhibited a release amount of 10-30 μg per day, which is considered enough for nerve regeneration.

(60) Accordingly, it was confirmed that a large amount of the neurotrophic factor can be coated by adsorbing the protein onto the porous nerve conduit having the microstructure with a large surface area.

Example 4

Confirmation of Nerve Regeneration Effect in Peripheral Nerve Injury Model

(61) A nerve conduit with NGF, as one of NTF, coated on the surface thereof was prepared by the method of Example 1-5.

(62) Then, a polycaprolactone (PCL) tube for inserting the nerve conduit was prepared. The PCL tube was prepared by the following method. A glass tube with an outer diameter of 1.6-1.7 mm was immersed in a 15% (w/v) PCL-TG solution so as to form a thin PCL-TG coat on the surface of the glass tube. Then, the PCL-TG-coated glass tube was immersed in DW, so that the PCL polymer was contacted with the water and then cured and micropores were formed in the hydrophobic polymer as the TG was mixed with the DW and released from the hydrophobic polymer. After removing the glass tube by pushing or pulling with forceps, followed by freezing in liquid nitrogen for 30 seconds and cutting to a length of 18 mm, a PCL tube was completed. The nerve conduit containing NGF was inserted into the PCL tube with a diameter of 1.6-1.7 mm and a length of 18 mm (FIGS. 16A and 16B).

(63) After removing the sciatic nerve (length 16 mm) of a 12-week-old female Sprague-Dawley rat at 5 mm below the hip joint, the nerve conduit containing NGF (Scaffold+NGF) or a hollow PCL tube with no nerve conduit inserted (Hollow) or a nerve conduit not containing NGF (Scaffold) as controls was transplanted into the damaged area (FIG. 16C). In order to prevent the nerve conduit from being separated from the nerve, the both ends of the nerve conduit were sutured to the cut nerve terminals using a suture (10-0:0.02-0.029 mm thick nylon suture). As another control group, autografting was conducted after removing the sciatic nerve (length 16 mm) of a 12-week-old female Sprague-Dawley rat at 5 mm below the hip joint. The autografting was conducted by inverting the distal and proximal parts of the cut nerve and suturing with a 10-0 suture (FIG. 16D).

(64) Then, immunostaining was conducted to check the growth of the sciatic nerve. 2 weeks after the transplantation, the sciatic nerve containing the 18-mm long graft was taken out and fixed in 4% paraformaldehyde. Then, after treating with 30% sucrose for 3 days, the tissue was sliced to 16-μm thick sections. Mouse Tuj1 monoclonal antibody was used for staining of the neuronal axons and rabbit S100 polyclonal antibody was used for staining of the Schwann cells. The tissue sections were observed with a confocal microscope and the result is shown in FIG. 17. FIG. 17 shows merged images of mouse Tuj1 monoclonal antibody staining and rabbit S100 polyclonal antibody staining. FIG. 17A shows the tissue into which the hollow PCL tube with no nerve conduit inserted was transplanted (Hollow), FIG. 17B shows the autografted nerve (Autograft), FIG. 17C shows the tissue into which the nerve conduit not containing NGF was transplanted (Scaffold) and FIG. 17D shows the tissue into which the nerve conduit containing NGF was transplanted. More regenerated axons were observed in the tissue into which the nerve conduit containing NGF was transplanted.

Example 5

Confirmation of Nerve Regeneration Effect in Central Nerve Injury (Transection) Model

(65) A nerve conduit with NT-3, as one of NTF, coated on the surface thereof was prepared by the method of Example 1-5.

(66) A central nerve injury model was prepared using a 12-week-old female Sprague-Dawley rat and the nerve conduit was transplanted (FIG. 18). First, laminectomy was conducted on the thoracic vertebrae 9-10 for transplantation of the nerve conduit (FIG. 18A). Then, after cutting open the dura mater of the spinal cord and completely removing the 5-mm long spinal cord (FIG. 18B), the nerve conduit was transplanted into the corresponding area (FIGS. 18C, 18D and 18E). After the nerve conduit transplantation, the dura mater was sutured using a suture (10-0:0.02-0.029 mm thick nylon suture) (FIG. 18F). As a control group, the nerve conduit not containing NT-3 (Scaffold) was transplanted into a spinal cord transection model.

(67) Then, immunostaining was conducted to check the growth of the central nerve. 16 weeks after the transplantation, the central nerve containing the 5-mm long graft was taken out and fixed in 4% paraformaldehyde. Then, after treating with 30% sucrose for 3 days, the tissue was sliced to 16-μm thick sections. Mouse Tuj1 monoclonal antibody was used for staining of the neuronal axons and the result of confocal microscopic observation is shown in FIG. 19. FIG. 19A shows the tissue into which the nerve conduit was not transplanted, FIG. 19B shows the tissue into which the nerve conduit not containing NT-3 was transplanted (Scaffold) and FIG. 19C shows the tissue into which the nerve conduit containing NT-3 was transplanted (Scaffold+NT-3). More regenerated axons were observed in the tissue into which the nerve conduit containing NT-3 was transplanted.

(68) While the present disclosure has been described with reference to the embodiments illustrated in the figures, the embodiments are merely examples, and it will be understood by those skilled in the art that various changes in form and other embodiments equivalent thereto can be performed. Therefore, the technical scope of the disclosure is defined by the technical idea of the appended claims.

(69) The drawings and the forgoing description gave examples of the present invention. The scope of the present invention, however, is by no means limited by these specific examples. Numerous variations, whether explicitly given in the specification or not, such as differences in structure, dimension, and use of material, are possible. The scope of the invention is at least as broad as given by the following claims.