JP4740482B2 - Nerve regeneration tube - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a nerve regeneration tube and a manufacturing method thereof.
[0002]
[Prior art and problems]
Since the announcement of the silicone tube model by Lundberg et al. In 1982 regarding peripheral nerve regeneration, attempts have been made to extend the reproducible stump distance using silicone tubes. However, when a damaged nerve is repaired using a silicone tube, the silicone tube may press the nerve regenerated in the tube over time, over time.
[0003]
On the other hand, a technique relating to regeneration of nerve defects using a polymer composed of a bioabsorbable polymer is also known. For example, Japanese Patent Laid-Open No. 2001-70436 discloses a technique using a collagen support such as a sponge, a tube, and a coil, but sufficient strength cannot be obtained with such a support.
[0004]
WO98 / 22155 is composed of a biodegradable absorbent material tube and a collagen body having a cavity penetrating the tube along the tube substantially parallel to the axis of the biodegradable absorbent material. An artificial neural tube filled with a matrix gel containing is disclosed. However, since the artificial neural tube is filled with a collagen body and a matrix gel, Schwann cells cannot be seeded inside.
[0005]
An object of the present invention is to provide a nerve regeneration tube capable of promptly regenerating nerves even in a long nerve defect portion and having no adverse effect on the regenerating nerves, and a method for producing the same.
[0006]
[Means for Solving the Problems]
The present invention provides the following nerve regeneration tube and a method for producing the same.
Item 1. A nerve regeneration tube including a sponge composed of a bioabsorbable polymer and a cylindrical reinforcing material composed of a bioabsorbable polymer having a longer decomposition and absorption period than the sponge, and at least an inner surface being a sponge.
Item 2. Item 2. The tube according to Item 1, wherein the tubular reinforcing material has a shape of braid, knitted fabric, woven fabric, nonwoven fabric, punched sheet, or spiral mesh.
Item 3. Item 3. The tube according to Item 2, wherein the cylindrical reinforcing material is an outer layer and the sponge is an inner layer.
Item 4. Item 4. The tube according to any one of Items 1 to 3, further comprising a cell adhesion factor.
Item 5. Item 5. The tube according to any one of Items 1 to 4, further comprising a growth factor.
Item 6. Item 6. The tube according to any one of Items 1 to 5, wherein Schwann cells are seeded on the inner surface of the tube.
Item 7. The following steps (A) to (C):
Step (A): fixing a cylindrical reinforcing material composed of bioabsorbable fibers on the outside of the cylindrical core,
Step (B): The obtained reinforcing material fixed core is immersed in a bioabsorbable polymer solution and then freeze-dried to form a sponge having a shorter decomposition and absorption period than the cylindrical reinforcing material.
Step (C): A method for producing a nerve regeneration tube having a sponge and a cylindrical reinforcing material, comprising: removing a freeze-dried product from a cylindrical core and inverting it if necessary.
Item 8. The method further includes a step (B1) of immersing the cylindrical core body having the sponge and the reinforcing material obtained in the step (B) in a cell adhesion factor and / or growth factor solution and freeze-drying, the step (B1) Item 8. The method for producing a nerve regeneration tube according to Item 7, which has the sponge inner surface coated with a cell adhesion factor and / or a growth factor, wherein the obtained lyophilized product is subjected to the step (C).
Item 9. Item 9. A method for producing a nerve regeneration tube having Schwann cells on the inner surface, further comprising the step (D) of seeding and culturing Schwann cells on the tube inner surface obtained in the step (C) of Item 7 or 8.
[0007]
DETAILED DESCRIPTION OF THE INVENTION
In the present invention, as the bioabsorbable polymer, either a synthetic bioabsorbable polymer or a natural bioabsorbable polymer can be used. Synthetic bioabsorbable polymers include aliphatic polyesters (polyglycolic acid, polylactic acid (D-form, L-form, DL-form), polycaprolactone, polyvalerolactone, and copolymers thereof, such as lactic acid-caprolactone copolymers. , Lactic acid-glycolic acid copolymer, glycolic acid-trimethylene carbonate copolymer, glycolic acid-trimethylene carbonate-dioxanone copolymer, glycolic acid-trimethylene carbonate-ε caprolactone copolymer), polyester ether (poly -1,4-dioxanon-2-one, poly-1,5-dioxepan-2-one, ethylene glycol-the aliphatic polyester copolymer, and a copolymer of the aliphatic polyester and polyester ether). It is done. Examples of natural bioabsorbable polymers include collagen, gelatin, hyaluronic acid, alginic acid and the like.
[0008]
Examples of preferable synthetic bioabsorbable polymers constituting the sponge include polylactic acid, polyglycolic acid, polycaprolactone and copolymers thereof, and preferred synthetic bioabsorbable polymers constituting the reinforcing material include Examples include lactic acid, polyglycolic acid, polycaprolactone and copolymers thereof.
[0009]
The synthetic bioabsorbable polymer constituting the sponge and the synthetic bioabsorbable polymer having a longer decomposition and absorption period than the sponge constituting the reinforcing material may be the same or different.
[0010]
"Reinforcing material composed of a synthetic bioabsorbable polymer having a longer decomposition and absorption period than the sponge" means that the synthetic bioabsorbable polymer itself constituting the reinforcing material is more in vivo than the synthetic bioabsorbable polymer of the sponge. In addition to the case of the polymer having high decomposition resistance, the polymer itself is the same, or the polymer of the reinforcing material is more easily decomposed, but the cylindrical reinforcing material is less easily decomposed than the sponge. Therefore, it means both the case where the synthetic bioabsorbable fiber as a whole has a longer decomposition and absorption period than the synthetic bioabsorbable sponge.
[0011]
Examples of the component of the reinforcing material of the present invention include monofilaments, multifilaments, fibers such as strings, sheets, and nonwoven fabrics. The diameter of the fiber is about 10 to 2000 μm, preferably about 50 to 1000 μm. Examples of the cylindrical reinforcing material include braids, woven fabrics, knitted fabrics, non-woven fabrics, punching sheets, spiral meshes knitted spirally with filament yarns, and preferably braids. The thickness of the cylindrical reinforcing material is about 10 to 2000 μm, preferably about 50 to 1000 μm.
[0012]
The sponge of the present invention has a thickness of about 0.1 to 5 mm, preferably about 0.5 to 2 mm, and the pore diameter of the sponge is about 1 to 500 μm, preferably about 10 to 200 μm. The reinforcing material may be integrated into the nerve regeneration tube inside the sponge, and the sponge may be an inner layer and the reinforcing material may be an outer layer. In this case, the sponge inner layer and the reinforcing material outer layer may be completely separated, or there may be a layer in which the sponge and the reinforcing material are mixed.
[0013]
The nerve regeneration tube of the present invention has a thickness of about 0.1 to 5 mm, preferably about 0.5 to 2 mm, and an inner diameter of about 0.1 to 5 mm, preferably about 0.5 to 3 mm.
[0014]
Examples of the cell adhesion factor include collagen (type I, type IV, etc.), laminin, fibronectin and the like.
[0015]
Examples of growth factors include nerve growth factor (NGF), neurotrophic factor, neurite outgrowth factor and the like.
[0016]
The cell adhesion factor and the growth factor may be contained inside the sponge or may be coated on the surface of the sponge. The cell adhesion factor and the growth factor may be further contained in the reinforcing material, or may be coated on the surface of the reinforcing material.
[0017]
The nerve regeneration tube of the present invention can be produced by a method including the following steps (A) to (C):
Step (A): Fixing a cylindrical reinforcing material composed of synthetic bioabsorbable fibers on the outside of the cylindrical core,
Step (B): The obtained reinforcing material-fixing core is immersed in a synthetic bioabsorbable polymer solution and freeze-dried to form a sponge layer having a shorter decomposition and absorption period than the cylindrical reinforcing material, and the step (C) ): Remove the freeze-dried product from the cylindrical core and invert as necessary.
[0018]
In the step (A), the tubular reinforcement may be externally fitted so as to be in close contact with the outside of the tubular core. In this case, a nerve regeneration tube including the tubular reinforcement in the outer layer is obtained (the reinforcement is If there is an opening, the sponge enters the opening). In addition, a detachable protrusion (for example, a radial protrusion, a donut-shaped ridge, etc.) for fixing the cylindrical reinforcing material to a predetermined position (for example, a position corresponding to the length of the nerve regeneration tube) of the cylindrical core. ) May be fixed at a position away from the cylindrical core. In this case, the reinforcing material or the protrusion has an opening so that the synthetic bioabsorbable polymer solution enters the gap between the reinforcing material and the cylindrical core. In this way, by fixing the cylindrical reinforcing material at a position away from the cylindrical core, a nerve regeneration tube in which the reinforcing material and the sponge are integrated (the reinforcing material is surrounded by the sponge) is obtained.
[0019]
In addition, the cylindrical core can use what a synthetic | combination bioabsorbable polymer solution does not penetrate | invade inside.
[0020]
In the step (B), the synthetic bioabsorbable polymer solution is desirably a solution that does not dissolve the polymer constituting the reinforcing material as much as possible. Examples of the solvent for the solution include dioxane, chloroform, acetonitrile, methylene chloride and the like. When the solvent can dissolve the polymer constituting the reinforcing material, the concentration and temperature are adjusted so that the concentration of the solvent is close to the saturation solubility, or the reinforcing material fixing core is added to the synthetic bioabsorbable polymer solution. It is desirable to make the time for soaking the body as short as possible and freeze-dry immediately after soaking.
[0021]
The concentration of the synthetic bioabsorbable polymer solution is about 0.1 to 20% by weight, preferably about 1 to 10% by weight.
[0022]
In step (C), when the lyophilized product after immersion is removed from the cylindrical core, is the sponge present outside the reinforcing material (the reinforcing material is closely fixed to the cylindrical core), or both the outer and inner sides? (Reinforcing material may be fixed at a position away from the cylindrical core). When the sponge is present as the outer layer of the reinforcing material, it is inverted to obtain a nerve regeneration tube having the sponge on the inner surface. If there is a sponge layer on both sides of the reinforcing material, the inner surface is a sponge, so it is not always necessary to invert, but if the outer sponge layer of the reinforcing material is thicker than the inner, it is inverted and the inner sponge layer It is desirable to obtain a nerve regeneration tube that is thicker.
[0023]
Inversion can be performed by pushing the end of the tube into the tube while holding the end of the tube with a holder thinner than the inner diameter of the tube.
[0024]
When the reversal is not performed, the reinforcing material is fixed to the inside of the hollow first cylindrical core material, the cylindrical second cylindrical core material is further fixed to the center portion, and the first cylindrical core material and the second cylindrical material are fixed. If a synthetic bioabsorbable polymer solution is poured between the core materials and freeze-dried, a nerve regeneration tube having a sponge on the inner surface can be obtained without inversion.
[0025]
If a cell adhesion factor and / or a growth factor are blended in the synthetic bioabsorbable polymer solution at a predetermined concentration, a nerve regeneration tube in which the factor is contained in a sponge can be obtained.
[0026]
Cells can also be obtained by immersing the cylindrical core having the sponge and reinforcing material obtained in step (B) in a cell adhesion factor and / or growth factor solution (preferably an aqueous solution) and freeze-drying. A nerve regeneration tube having a sponge inner surface coated with an adhesion factor and / or a growth factor can be obtained. Alternatively, the growth factor may be included in the crosslinked gelatin fine particles and then combined with the sponge.
[0027]
An example of the concentration of the cell adhesion factor is about 0.01 to 10%.
[0028]
The cell adhesion factor and / or growth factor solution is usually an aqueous solution, but an aqueous solution such as hydrous alcohol may be used.
[0029]
When Schwann cells are seeded on the inner surface of the tube and cultured, a nerve regeneration tube can be obtained in which Schwann cells are formed in a single layer on the inner surface of the tube, preferably around the entire lumen. In order to adhere Schwann cells all around the lumen, it is preferable to seed the Schwann cells throughout the lumen by pipetting while rotating (rolling) the tube.
[0030]
【Example】
Hereinafter, the present invention will be described in more detail based on examples.
Production Example 1
Polylactic acid fibers (40d) were stringed using a braiding machine to obtain a cylindrical core material. This was put on a stainless steel rod, immersed in a dioxane solution (5% by weight) of lactic acid / ε-caprolactone copolymer (molar ratio 50/50), frozen at −40 ° C. and then frozen at 30 ° C. for 24 hours. Dried. In this way, a composite material (Experimental Example 4 (A)) having a reinforcing material composed of a lactic acid / ε-caprolactone copolymer sponge in the inner layer and a polylactic acid braid in the outer layer was obtained.
[0031]
A 0.1% solution of collagen (Type I, porcine tendon-derived atelocollagen) was sufficiently infiltrated into the sponge complex, frozen at −100 ° C. and then freeze-dried at 30 ° C. for 24 hours. A collagen tube ((C) of Experimental Example 4) in which Type I collagen was coated on the entire surface including the inner surface was obtained.
[0032]
The obtained nerve regeneration tube is shown in FIG. 1 (50 times magnified photograph of the braid reinforcing material layer surface), FIG. 2 (400 times magnified photograph of the sponge inner layer), and FIG. 3 (50 times magnified photograph of the cross section of the tube). Thus, the inner layer was a sponge, the outer layer was a reinforcing material, and the sponge layer and the reinforcing material layer were separated.
Production Example 2
Polylactic acid fibers (40d) were stringed using a braiding machine to obtain a cylindrical core material. This was put on a stainless steel rod, immersed in a dioxane solution (5% by weight) of lactic acid / ε-caprolactone copolymer (molar ratio 25/75), frozen at −40 ° C. and then frozen at 30 ° C. for 24 hours. Dried. In this way, a composite material (Experimental Example 4 (B)) having a lactic acid / ε-caprolactone copolymer sponge in the inner layer and a reinforcing material made of polylactic acid braid in the outer layer was obtained.
Production Example 3
In the same manner as in Production Example 1 except that Type IV collagen was used in place of Type I collagen, a collagen tube (Type (D) of Experimental Example 4) in which the entire surface including the inner surface was coated with Type IV collagen was obtained.
Production Example 4
A collagen tube (Experimental Example 4) coated with Type I + Type IV collagen as a whole in the same manner as in Production Example 1 except that a mixture of Type I collagen and Type IV collagen (1: 1 weight ratio) was used instead of Type I collagen. (E)) was obtained.
Experimental example 1
Ten sciatic nerves of 10 Fischer 344 rats are temporarily dissected. The right side is a tube made of P (LA / CL) sponge inner layer and core material PLLA braid outer layer (inner diameter 2mm, length 4mm) coated with Type I collagen, and nerves are inserted 2mm at both ends. These nerve stumps were brought into close contact with each other to perform nerve junction. Nerve junctions were performed by stitching the nerve to the tube with 8.0 nylon yarn. On the other hand, the sciatic nerve that was temporarily cut off was sutured with 8-0 nylon thread on the left side.
[0033]
Eight weeks after the operation, bilateral sciatic nerves were removed, and the number of axons and the axon density at the center and the periphery of the transplanted part were measured.
[0034]
According to these results, nerve regeneration was better when using a tube reinforced with P (LA / CL) sponge with a core made of PLLA braid, which is a simpler method than suturing the cut nerve. Equivalent or better results were obtained.
Experimental example 2
About 12 mm of both sciatic nerves of 10 male Fischer 344 rats were excised. On the right side, a tube made of P (LA / CL) sponge inner layer and PLLA braid outer layer (inner diameter 2mm, length 16mm) coated with Type I collagen, nerves are inserted 2mm at both ends, and the defect interval The length of the tube was 12 mm, and both ends of the tube and nerve fiber were sutured with 8.0 nylon thread and fixed. On the left side, nerve fibers were fixed in the same manner except that coating was performed using Type IV collagen instead of Type I collagen.
[0035]
Eight weeks after the operation, bilateral sciatic nerves were removed, and the number of axons and the axon density at the center and the periphery of the transplanted part were measured.
[0036]
According to these results, nerve stumps showed good results for both Type I collagen and Type IV collagen.
Experimental example 3
Using 10 Japanese white rabbits, a defect of about 20 mm was created in the bilateral total sural nerve. Next, 60 mm of the ipsilateral sural nerve was sampled, a 20 mm long x 3 cable graft was prepared, and transplanted to the total sural nerve deficient part with the polarity reversed under a surgical microscope. Of these, 5 wings and 10 limbs were left as sutures, and were closed for control. The remaining 5 limbs and 10 limbs were sutured so that the entire circumference was wrapped with a bioabsorbable tube having a length of 6 mm and a lumen of 2 mm at the central and peripheral nerve junctions, and the entire circumference was rolled up again. Electrophysiological, functional and morphological searches were performed on these two groups at 6 months after surgery.
Experimental Example 4
The following composites (A) to (E) obtained in Production Examples 1 to 4 were used.
(A): CL: PLLA (50:50), collagen coating (-)
(B): CL: PLLA (75:25), collagen coating (-)
(C): CL: PLLA (50:50), Type I collagen coating (+)
(D): CL: PLLA (50:50), Type IV collagen coating (+)
(E): CL: PLLA (50:50), Type I + Type IV collagen coating (+)
The composite was cut in the axial direction to obtain a flat composite, which was cut into a disk. The obtained disc-like complex was treated with ethanol, and then Schwann cells collected from the rat dorsal root ganglion and cultured on the sponge-like CL / PLLA copolymer of the complex or a collagen coating covering the surface It was seeded on the layer and its adhesion was examined by scanning electron microscope and histology. For identification of Schwann cells, immunostaining with S-100 antibody was performed. FIG. 4 shows the result of immunostaining with S-100 antibody using the complex (C).
[0037]
As a result, in all the complexes (A) to (E), Schwann cell adhesion was observed on the CL / PLLA copolymer sponge in a more uniform manner.
[0038]
【The invention's effect】
According to the method of the present invention, nerve regeneration equivalent to or higher than that in the case of using a silicone tube can be performed, and since it is made of only a bioabsorbable material, it is not necessary to remove it after the operation.
[0039]
Although cadaveric nerve transplantation has been practiced in recent years, there is a risk of viral infection because it is necessary to suppress immunosuppression, and it is difficult to become a general technique, but the present invention does not have such a problem.
[Brief description of the drawings]
FIG. 1 is a drawing-substituting photograph (enlarged 50 times) showing the surface of a braid reinforcing material layer.
FIG. 2 is a drawing-substituting photograph (400 magnification) showing the sponge inner layer.
FIG. 3 is a drawing-substituting photograph (enlarged 50 times) showing a cross section of the tube.
4 is a drawing-substituting photograph showing the result of immunostaining with S-100 antibody of Experimental Example 4. FIG.

Claims (8)

A nerve regeneration tube comprising a sponge composed of a lactic acid-caprolactone copolymer and a braided reinforcing material composed of polylactic acid fibers , and at least the inner surface being a sponge. The tube according to claim 1, wherein the reinforcing material is an outer layer and the sponge is an inner layer. The tube according to claim 1 or 2, further comprising a cell adhesion factor. Furthermore, the tube in any one of Claims 1-3 containing a growth factor. The tube according to any one of claims 1 to 4, wherein Schwann cells are seeded on the inner surface of the tube. The following steps (A) to (C):
Step (A): fixing a braided reinforcing material composed of polylactic acid fibers on the outside of the cylindrical core,
Step (B): The obtained reinforcing material fixed core is immersed in a lactic acid-caprolactone copolymer solution and then freeze-dried to form a sponge having a shorter decomposition and absorption period than the braided reinforcing material.
Step (C): lyophilisate was removed from the cylindrical core body, characterized in that it comprises the inverted if necessary, have a sponge and braided reinforcement nerve regeneration tube least the inner surface is sponge Manufacturing method. The method further includes a step (B1) of immersing the cylindrical core body having the sponge and the reinforcing material obtained in the step (B) in a cell adhesion factor and / or growth factor solution and freeze-drying, the step (B1) The method for producing a nerve regeneration tube according to claim 6, which has an inner surface of a sponge coated with a cell adhesion factor and / or a growth factor, wherein the obtained freeze-dried product is subjected to the step (C). A method for producing a nerve regeneration tube having Schwann cells on the inner surface, further comprising a step (D) of seeding and culturing Schwann cells on the tube inner surface obtained in the step (C) of claim 6 or 7.

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