Preparing method of nerve conduits

Abstract

The present disclosure relates to a method for preparing a nerve conduit, more particularly to a method for preparing a porous nerve conduit having micropores formed in microchannels and the nerve conduit prepared according to the present disclosure can be usefully used in in-vitro and in-vivo researches on nerves.

Claims

1. A method for preparing a nerve regeneration device, the method comprising: preparing a porous nerve conduit having a microchannel structure with micropores formed by: a step of inserting a plurality of water-soluble glass fibers into a container having upper and lower channels; a step of injecting a polymer material for a nerve conduit comprising a hydrophobic biocompatible polymer and a water-miscible organic solvent into the container in which the plurality of water-soluble glass fibers are inserted; a step of infiltrating the polymer material between the water-soluble glass fibers by applying vacuum to the upper channel; a step of separating the water-soluble glass fibers with the polymer material infiltrated from the container; and a step of forming a microchannel structure with micropores, comprising: immersing the separated water-soluble glass fibers with the polymer material in water; and dissolving the glass fibers, wherein the microchannel structure is formed in the space where the glass fibers were dissolved, and micropores are formed by mixing the water-miscible organic solvent with the water and releasing the water-miscible organic solvent from the polymer material, wherein the micropores are created in the microchannel structure where the water-miscible organic solvent was released from the polymer material, wherein the lower channel has a smaller diameter than the upper channel and the container is sloped with a discontinuous angle, 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 %), preparing a biocompatible polymer tube having micropores by: a step of immersing a forming tube in a mixture solution of a hydrophobic biocompatible polymer and a water-miscible organic solvent to form a thin coat on the forming tube, a step of curing the biocompatible polymer coat by immersing the coated forming tube in water, and a step of removing the biocompatible polymer tube from the forming tube, and inserting the nerve conduit having a microchannel structure with micropores into the biocompatible polymer tube having micropores to form the nerve regeneration device.

2. The method for preparing a porous nerve regeneration device of claim 1, wherein the porous nerve regeneration device is for regeneration of a central nerve or a peripheral nerve.

3. The method for preparing a porous nerve regeneration device of claim 1, wherein the polymer material for a nerve conduit is one in which a nanoparticle is further added in addition to the hydrophobic polymer and the water-miscible organic solvent.

4. The method for preparing a porous nerve regeneration device of claim 1, wherein the polymer material for a nerve conduit is in a solution state at room temperature.

5. The method for preparing a porous nerve regeneration device of claim 1, wherein the method for preparing a porous nerve conduit 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.

6. The method for preparing a porous nerve regeneration device 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.

7. The method for preparing a porous nerve regeneration device of claim 1, wherein the application of vacuum is repeated multiple times.

8. The method for preparing a porous nerve regeneration device of claim 1, wherein the hydrophobic biocompatible polymer is selected from a group comprising of polylactic acid (PLA), poly-L/D-lactide (PLDA), poly-L-lactic acid (PLLA), polyglycolic acid (PGA)), polydioxanone, polyhydroxybutyrate (PHB), polyhydroxyalkanoate (PHA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), a copolymer thereof and a mixture thereof; and the water-miscible organic solvent is selected from a group comprising of ethanol, isopropyl alcohol, N-methyl-2-pyrrolidone, 2-pyrrolidone, glycerol, propylene glycol, polyethylene glycol, tetraglycol, glycerol formal, ethyl acetate, ethyl lactate, diethyl carbonate, propylene carbonate, acetone, methyl ethyl ketone, dimethyl sulfoxide, dimethyl sulfone, tetrahydrofuran, tetrahydrofurfuryl alcohol, succinic acid diethyl ester, triethyl citrate, dibutyl sebacate, dimethylacetamide, lactic acid butyl ester, propylene glycol diacetate, diethylene glycol monoethyl ether and a mixture thereof.

9. The method for preparing a porous nerve regeneration device of claim 3, wherein the nanoparticle is a fluorescent nanoparticle.

10. A method for preparing a nerve regeneration device, the method comprising: preparing a porous nerve conduit having a microchannel structure with micropores formed by: inserting a plurality of water-soluble glass fibers into a container having upper and lower channels; injecting a polymer material for a nerve conduit comprising a hydrophobic biocompatible polymer and a water-miscible organic solvent into the container in which the plurality of water-soluble glass fibers are inserted; infiltrating the polymer material between the glass fibers by applying vacuum to the upper channel; separating the water-soluble glass fibers with the polymer material infiltrated from the container; forming a microchannel structure by dissolving the glass fibers; and forming micropores in the microchannel structure by separating the water-miscible organic solvent from the hydrophobic biocompatible polymer, wherein the lower channel has a smaller diameter than the upper channel and the container is sloped with a discontinuous angle, 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 %), preparing a biocompatible polymer tube having micropores by: a step of immersing a forming tube in a mixture solution of a hydrophobic biocompatible polymer and a water-miscible organic solvent to form a thin coat on the forming tube, a step of curing the biocompatible polymer coat by immersing the coated forming tube in water, and a step of removing the biocompatible polymer tube from the forming tube, and inserting the nerve conduit having a microchannel structure with micropores into the biocompatible polymer tube having micropores to form the nerve regeneration device.

11. The method for preparing a porous nerve regeneration device of claim 10, wherein the porous nerve regeneration device is for regeneration of a central nerve or a peripheral nerve.

12. The method for preparing a porous nerve regeneration device of claim 10, wherein the polymer material for a nerve conduit is one in which a nanoparticle is further added in addition to the hydrophobic polymer and the water-miscible organic solvent.

13. The method for preparing a porous nerve regeneration device of claim 10, wherein the polymer material for a nerve conduit is in a solution state at room temperature.

14. The method for preparing a porous nerve regeneration device of claim 10, wherein the method for preparing a porous nerve conduit further comprising: cooling a nerve conduit formed after the glass fibers are dissolved with liquid nitrogen; and shaping the cooled nerve conduit by cutting.

15. The method for preparing a porous nerve regeneration device of claim 10, 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.

16. The method for preparing a porous nerve regeneration device of claim 10, wherein the application of vacuum is repeated multiple times.

17. The method for preparing a porous nerve regeneration device of claim 10, wherein the hydrophobic biocompatible polymer is selected from a group comprising of polylactic acid (PLA), poly-L/D-lactide (PLDA), poly-L-lactic acid (PLLA), polyglycolic acid (PGA)), polydioxanone, polyhydroxybutyrate (PHB), polyhydroxyalkanoate (PHA), poly(lactic-co-glycolic acid) (PLGA), polycaprolactone (PCL), a copolymer thereof and a mixture thereof; and the water-miscible organic solvent is selected from a group comprising of ethanol, isopropyl alcohol, N-methyl-2-pyrrolidone, 2-pyrrolidone, glycerol, propylene glycol, polyethylene glycol, tetraglycol, glycerol formal, ethyl acetate, ethyl lactate, diethyl carbonate, propylene carbonate, acetone, methyl ethyl ketone, dimethyl sulfoxide, dimethyl sulfone, tetrahydrofuran, tetrahydrofurfuryl alcohol, succinic acid diethyl ester, triethyl citrate, dibutyl sebacate, dimethylacetamide, lactic acid butyl ester, propylene glycol diacetate, diethylene glycol monoethyl ether and a mixture thereof.

18. The method for preparing a porous nerve regeneration device of claim 12, wherein the nanoparticle is a fluorescent nanoparticle.

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 channel.

(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 channel.

(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 disclosure.

(11) FIG. 10 shows transverse cross-sectional SEM images of a porous PCL nerve conduit; scale bar=(left) 500 μm, (center) 10 μm (right) 10 μm. A shows a transverse cross-sectional image, B shows a magnified transverse cross-sectional SEM images showing a microstructure and C shows a longitudinal cross-sectional image.

(12) FIG. 11 schematically shows a method for preparing a nerve conduit containing a fluorescent nanoparticle according to an exemplary embodiment of the present disclosure.

(13) FIG. 12 shows photographs illustrating a method for preparing a porous nerve conduit containing a fluorescent nanoparticle. 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, D shows application of vacuum into a glass tube using a syringe, E shows a green fluorescent silica particle stock solution, F shows a mixture solution of a green fluorescent silica particle and 20% (w/v) PLGA-TG, and G shows a mixture solution of a green fluorescent silica particle and 20% (w/v) PLGA-TG mixture solution with light blocked for preservation of fluorescence.

(14) FIG. 13 shows fluorescence microscopic images of a nerve conduit not containing a fluorescent nanoparticle (A) and a nerve conduit containing a fluorescent nanoparticle (B).

(15) FIG. 14 shows fluorescence microscopic images of a nerve conduit containing a fluorescent nanoparticle kept at the same location under an in-vitro environment for 60 days.

(16) FIG. 15 shows the change in fluorescence intensity of a fluorescent nanoparticle for 60 days.

(17) FIG. 16 shows transverse cross-sectional SEM images of a biocompatible polymer tube with micropores formed in which a nerve conduit according to the present disclosure is inserted (A) and magnified images showing parts of the tube (B, C).

(18) FIG. 17 illustrates an in-vivo experiment procedure for confirming the nerve regeneration effect of a nerve conduit according to the present disclosure. 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.

(19) FIG. 18 shows a result of immunohistochemical staining of an animal of a peripheral nerve injury model autografted with or without (control) transplantation of a nerve conduit observed by confocal microscopy. The images are merged images of mouse Tuj1 monoclonal antibody staining and rabbit S100 polyclonal antibody staining.

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

(21) FIG. 20 shows a result of immunohistochemical staining of an animal of a complete spinal cord transection model 2 weeks after transplantation with or without (control) transplantation of a nerve conduit.

(22) FIG. 21 shows a result of immunohistochemical staining of an animal of a complete spinal cord transection model 16 weeks spinal cord transection model 16 weeks after transplantation with or without (control) transplantation of a nerve conduit.

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

DETAILED DESCRIPTION

(24) 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.

(25) 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.

(26) 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.

(27) 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

(28) 1-1: Preparation of Porous PLGA Nerve Conduit

(29) 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.

(30) 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).

(31) A pressure device was 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).

(32) 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).

(33) 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.

(34) 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.

(35) 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 glass fibers infiltrated with the PLGA-TG solution were as the TG was mixed with the water while they 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).

(36) 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).

(37) 1-2: Investigation of Microstructure Inside Porous PLGA Nerve Conduit

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

(39) 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.

(40) 1-3:3D Micro-CT Imaging of Porous Nerve Conduit

(41) 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.

(42) 1-4: Preparation of Porous PCL Nerve Conduit

(43) A porous nerve conduit was prepared in the same manner as in Example 1-1 except that the polymer material was prepared using polycaprolactone (PCL) as the hydrophobic biocompatible polymer material instead of the PLGA. As the polymer material, an 18% (w/v) PCL-TG solution was prepared by mixing PCL and TG at a weight/volume (w/v) ratio of 18% (w/v) and then dissolving at 90° C. for 18-24 hours. Then, a nerve conduit was prepared in the same manner as in Example 1-1 (FIG. 10).

Example 2: Porous Nerve Conduit Containing Fluorescent Nanoparticle

(44) 2-1: Preparation of Porous Nerve Conduit Containing Fluorescent Nanoparticle

(45) 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.

(46) After transferring a 50 mg/mL stock solution of a green fluorescent silica nanoparticle (particle size: 500 nm, Sicastar®-greenF, micromod Partikeltechnologie, Germany) (FIG. 12E) of an amount corresponding to 1/50 of the volume of the 20% (w/v) PLGA-TG solution (e.g., when the volume of the 20% (w/v) PLGA-TG solution is 5 mL, the volume of the green fluorescent silica particle stock solution is 100 μL) to a 1.5-mL tube, centrifugation was performed at a speed of 8,000 rpm or lower for 30 seconds. Then, distilled water was removed from the green fluorescent silica particle stock solution suspended in the distilled water by removing the supernatant. After the removal of the distilled water, the green fluorescent silica particle was resuspended in a small amount (100 μL or less) of a TG stock solution and then completely mixed with the 20% (w/v) PLGA-TG solution (working concentration: 1 mg/mL) (FIG. 12F). The prepared mixture solution of the green fluorescent silica particle and the 20% (w/v) PLGA-TG was blocked from light for preservation fluorescence (FIG. 12G).

(47) 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. 12A).

(48) A pressure device was 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. 12B and FIG. 12C).

(49) After immersing the lower channels of the glass tube in the mixture solution of the green fluorescent silica particle and the 20% (w/v) PLGA-TG at room temperature, vacuum was repeatedly applied into the glass tube using a syringe such that the mixture solution was completely infiltrated into the void space between the glass fibers (FIG. 11C and FIG. 12D).

(50) 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. 11D), so that the glass fibers were completely dissolved, and about 7,000-8,500 (7,777±716.2) microchannels of the PLGA containing the green fluorescent silica particle, with diameters of 10-20 μm (16.54±3.6 μm), were formed in the space where the glass fibers had been dissolved (FIG. 11E). The microchannels were formed as the glass fibers were dissolved in the water at 10-20° C. and the PLGA containing the green fluorescent silica particle was cured at the same time. And, the infiltrated glass fibers were completely dissolved by the DW while they were immersed in the DW. The microchannels were formed as the hydrophobic polymer PLGA was contacted with the DW in the space formed as the glass fibers were dissolved and then cured. Also, micropores were formed inside the microchannels as the water-miscible solvent was mixed with the water and released from the microchannels. Because the TG released from the nerve conduit had a higher density than the DW, it was submerged like heat haze in the DW.

(51) After the glass fibers and the TG were removed through the treatment with DW, the prepared porous microchannels formed of the green fluorescent silica particle and the 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.

(52) 2-2: Fluorescence Emission from Nerve Conduit

(53) Fluorescence emission from the nerve conduit containing a fluorescent nanoparticle prepared in Example 2-1 was observed using a fluorescence microscope. As seen from FIG. 13, the nerve conduit containing a fluorescent nanoparticle (Example 2-1) exhibited green fluorescence whereas the nerve conduit not containing a fluorescent nanoparticle (Example 1-1) did not exhibit green fluorescence.

(54) When the nerve conduit containing a fluorescent nanoparticle was imaged at the same location under an in-vitro environment for 60 days at a magnification of ×10, the fluorescence emission from the nerve conduit decreased with time as the fluorescent nanoparticle was degraded (FIG. 14). The fluorescence intensity of the fluorescent nanoparticle decreased greatly initially. 60 days later, the intensity was decreased to 5.10% at a magnification of ×5 and to 5.16% at a magnification of ×10 (FIG. 15). In FIG. 14 and FIG. 15, the “PDMS device” is a device used to fix the nerve conduit for the in-vitro experiment. A method for fixing the nerve conduit to the PDMS device is well known in the related art. To describe briefly, a mold of a desired shape is prepared first using a 3D printer, etc. and a PDMS solution is poured into the mold and then hardened.

Example 3: Nerve Regeneration after Transplantation of Nerve Conduit in Peripheral Nerve Injury Model

(55) A nerve conduit (diameter 1.6 mm, length 16 mm) was prepared by the method of Example 1-1. 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. FIG. 16 shows the prepared PCL tube having micropores formed.

(56) The nerve conduit prepared by the method of Example 1-1 was inserted to the polycaprolactone (PCL) tube with a diameter of 1.6-1.7 mm and a length of 18 mm (FIGS. 17, A and B). 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 was transplanted into the damaged area (FIG. 17C). 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) (FIG. 17D).

(57) As a 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.

(58) 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. 18. FIG. 18 shows merged images of mouse Tuj1 monoclonal antibody staining and rabbit S100 polyclonal antibody staining. As seen from FIG. 18, the growth of axons and Schwann cells along the channels at the proximal part of the nerve conduit was confirmed both in the autografted animal (control) and the animal in which the nerve conduit of the present disclosure was inserted. However, the animal in which the nerve conduit of the present disclosure was inserted showed higher peripheral nerve regeneration efficiency than the control group.

Example 4: Nerve Regeneration after Transplantation of Nerve Conduit in Central Nerve Injury (Transection) Model

(59) A nerve conduit (diameter 2.2 mm, length 5 mm) was prepared by the method of Example 1-1.

(60) A central nerve injury model was prepared using a 12-week-old female Sprague-Dawley rat and the nerve conduit was transplanted (FIG. 19). First, laminectomy for transplanting the nerve conduit was performed on the ninth and tenth thoracic vertebrae (FIG. 19A). Then, after cutting open the dura mater of spinal cord and completely removing 5 mm of the spinal cord (FIG. 19B), the nerve conduit was transplanted at the spinal cord-removed part (FIG. 19C, 19D, 19E). After the transplantation of the nerve conduit, the dura mater was sutured using a suture (10-0:0.02-0.029-mm thick nylon suture) (FIG. 19F). A spinal cord transection model in which the nerve conduit was not transplanted was used as a control group.

(61) Then, immunostaining was conducted to check the growth of the central nerve. 2 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 of the tissue sections. The tissue sections were observed with a confocal microscope and the result is shown in FIG. 20 and FIG. 21.

(62) FIG. 20 shows a result of immunohistochemical staining of the animal of a complete spinal cord transection model 2 weeks after the transplantation with or without (control) transplantation of the nerve conduit. From FIG. 20, axons (arrows) regenerating across the damaged area inside the transplanted nerve conduit are observed. In contrast, axon regeneration was not observed in the control group in which the nerve conduit was not transplanted.

(63) FIG. 21 shows a result of immunohistochemical staining of the animal of a complete spinal cord transection model 16 weeks after the transplantation autografting with or without (control) transplantation of the nerve conduit. From FIG. 21, axons regenerating across the damaged area inside the transplanted nerve conduit are observed. In contrast, axon regeneration was not observed in the control group in which the nerve conduit was not transplanted.

(64) 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.

(65) 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.