Neurotization tube

Description: TECHNICAL FIELD [Detailed description of the invention]

【0001】

[Technical field to which the invention belongs]

The present invention relates to a nerve regeneration tube and a method for producing the same.

【0002】

[Conventional technology and its problems]

Since the publication of the silicone tube model by Lundberg et al., Reported in 1982, regarding the regeneration of peripheral nerves, attempts have been made to extend the regenerative stump distance using silicone tubes. However, when the injured nerve is repaired using the silicone tube, the silicone tube may press the nerve regenerated in the tube all around over time.

【0003】

On the other hand, a technique relating to the regeneration of nerve defects using a polymer composed of a bioabsorbable polymer is also known. For example, Japanese Patent Application Laid-Open No. 2001-70436 discloses a technique using a collagen support such as a sponge, a tube, and a coil, but such a support cannot provide sufficient strength.

【0004】

In addition, WO98 / 22155 is composed of a tube made of a biodegradable and absorbent material and a collagen body having a gap in its lumen that penetrates the tube substantially parallel to the axis of the tube, and the gap is collagen, laminin, or the like. Disclosed is an artificial neural tube filled with a matrix gel containing. However, since the artificial neural tube is filled with collagen body and matrix gel, Schwann cells cannot be seeded inside.

【0005】

An object of the present invention is to provide a nerve regeneration tube capable of rapid nerve regeneration even in a long nerve defect portion and having no adverse effect on the regenerated nerve, and a method for producing the same.

【0006】

[Means for solving problems]

The present invention provides the following nerve regeneration tube and a method for producing the same. Item 1. A nerve regeneration tube containing a sponge composed of a bioabsorbable polymer and a tubular reinforcing material composed of a bioabsorbable polymer having a longer decomposition and absorption period than the sponge, and at least the inner surface is a sponge. .. Item 2. The tube according to Item 1, wherein the tubular reinforcing material has the shape of braid, knitted fabric, woven fabric, non-woven fabric, punching sheet or spiral mesh. Item 3. The tube according to Item 2, wherein the tubular reinforcing material is an outer layer and the sponge is an inner layer. Item 4. The tube according to any one of Items 1 to 3, further containing a cell adhesion factor. Item 5. The tube according to any one of Items 1 to 4, further containing a growth factor. 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 step (C): Step (A): Fixing a tubular reinforcing material composed of bioabsorbable fibers to the outside of the tubular core. Step (B): The obtained reinforcing material fixing 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 tubular reinforcing material. Step (C): A method for producing a nerve regeneration tube having a sponge and a tubular reinforcing material, which comprises removing the lyophilized product from the tubular core and inversion as needed. Item 8. The step (B1) further includes a step (B1) of immersing the tubular core having the sponge and the reinforcing material obtained in the step (B) in a solution of the cell adhesion factor and / or the growth factor and freeze-drying. Item 2. Production of the nerve regeneration tube according to Item 7, which has an inner surface of a sponge coated with a cell adhesion factor and / or a growth factor, which comprises subjecting the lyophilized product obtained in B1) to the step (C). Method. 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 inner surface of the tube obtained in the step (C) of Item 7 or 8.

【0007】

BEST MODE FOR CARRYING OUT 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 their copolymers, for example, lactic acid-caprolactone copolymer. , Lactic acid-glycolic acid copolymer, glycolic acid-trimethylone carbonate copolymer, glycolic acid-trimethylene carbonate-dioxanone copolymer, glycolic acid-trimethylene carbonate-ε caprolactone copolymer, etc.), polyester ether (poly) -1,4-dioxanone-2-one, poly-1,5-dioxepan-2-one, ethylene glycol-the aliphatic polyester copolymer and the copolymer of the aliphatic polyester and polyester ether) are mentioned. Be done. Examples of the natural bioabsorbable polymer include collagen, gelatin, hyaluronic acid, alginic acid and the like.

【0008】

Examples of preferred synthetic bioabsorbable polymers constituting the sponge include polylactic acid, polyglycolic acid, polycaprolactone and copolymers thereof, and preferred synthetic bioabsorbable polymers constituting the reinforcing material are poly. 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 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 in vivo than the synthetic bioabsorbable polymer of sponge. In addition to the case where the polymer has high decomposition resistance, the polymer itself is the same, or the polymer of the reinforcing material is more easily decomposed, but the tubular reinforcing material is less likely to be decomposed than the sponge. Therefore, as a whole, it means both cases where the synthetic bioabsorbable fiber has a longer decomposition and absorption period than the synthetic bioabsorbable sponge.

【0011】

Examples of the constituent elements of the reinforcing material of the present invention include monofilaments, multifilaments, fibers such as strings, sheets, and non-woven fabrics. The diameter of the fiber is about 10 to 2000 μm, preferably about 50 to 1000 μm. Examples of the tubular reinforcing material include braids, woven fabrics, knitted fabrics, non-woven fabrics, punching sheets, spiral meshes woven spirally with filament yarns, and the like, preferably braids. The thickness of the tubular reinforcing material is about 10 to 2000 μm, preferably about 50 to 1000 μm.

【0012】

The thickness of the sponge of the present invention is about 0.1 to 5 mm, preferably about 0.5 to 2 mm, and the pore size of the sponge is about 1 to 500 μm, preferably about 10 to 200 μm. The reinforcing material may be inside the sponge to integrally form a nerve regeneration tube, the sponge may be the inner layer, and the reinforcing material may be the outer layer. In this case, the inner layer of the sponge and the outer layer of the reinforcing material may be completely separated, or there may be a layer in which the sponge and the reinforcing material are mixed.

【0013】

The thickness of the nerve regeneration tube of the present invention is about 0.1 to 5 mm, preferably about 0.5 to 2 mm, and the inner diameter is about 0.1 to 5 mm, preferably about 0.5 to 3 mm.

【0014】

Examples of cell adhesion factors include collagen (type I, type IV, etc.), laminin, fibronectin, and the like.

【0015】

Growth factors include nerve growth factor (NGF), neurotrophic factors, neurite outgrowth factors 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 inside 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 tubular reinforcing material composed of synthetic bioabsorbable fibers to the outside of the tubular core. Step (B): The obtained reinforcing material fixing core is immersed in a synthetic bioabsorbable polymer solution and then freeze-dried to form a sponge layer having a shorter decomposition and absorption period than the tubular reinforcing material. Step (C): Remove the lyophilized product from the tubular core and invert it if necessary.

【0018】

In the step (A), the tubular reinforcing material may be externally fitted so as to be in close contact with the outside of the tubular core body, and in this case, a nerve regeneration tube containing the tubular reinforcing material in the outer layer is obtained (the reinforcing material is If it has an opening, the sponge will enter the opening). In addition, a removable protrusion (for example, a radial protrusion, a donut-shaped collar, etc.) for fixing the tubular reinforcing material at a predetermined position of the tubular core body (for example, a position corresponding to the length of the nerve regeneration tube), etc. ) May be provided so that the core body can be fixed at a position away from the tubular core body. In this case, the reinforcing material or the protrusion has an opening so that the synthetic bioabsorbable polymer solution penetrates into the gap between the reinforcing material and the tubular core body. By fixing the tubular reinforcing material at a position away from the tubular core body in this way, a nerve regeneration tube in which the reinforcing material and the sponge are integrated (the reinforcing material is surrounded by the sponge) can be obtained.

【0019】

As the tubular core body, one that does not allow the synthetic bioabsorbable polymer solution to penetrate inside can be used.

【0020】

In step (B), the synthetic bioabsorbable polymer solution is preferably 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 saturated solubility, or the reinforcing material fixing core is added to the synthetic bioabsorbable polymer solution. It is desirable to immerse the body for as short a time as possible and freeze-dry it immediately after immersing.

【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】

When the lyophilized product after immersion is removed from the tubular core in step (C), is the sponge present on the outside of the reinforcing material (the reinforcing material is firmly fixed to the tubular core), or both outside and inside? It may be present (fixing the reinforcing material at a position away from the tubular core). If 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. When there are sponge layers on both sides of the reinforcing material, it is not always necessary to invert because the inner surface is a sponge, but when the outer sponge layer of the reinforcing material is thicker than the inner surface, it is inverted and the inner sponge layer is inverted. It is desirable to obtain a thicker nerve regeneration tube.

【0023】

Inversion can be performed by pushing the end of the tube into the tube while holding one end of the tube with a holder thinner than the inner diameter of the tube.

【0024】

When not reversing, the reinforcing material is fixed inside the hollow first tubular core material, and the tubular second tubular core material is further fixed in the center, and the first tubular core material and the second cylinder are fixed. By pouring a synthetic bioabsorbable polymer solution between the core materials and freeze-drying, 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 is blended in a 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】

The cells can also be obtained by immersing the tubular core having the sponge and the reinforcing material obtained in step (B) in a solution of cell adhesion factor and / or growth factor (preferably an aqueous solution) and freeze-drying the cells. A nerve regeneration tube having an inner surface of a sponge 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 compounded with the sponge.

【0027】

The concentration of the cell adhesion factor is exemplified by about 0.01 to 10%.

【0028】

As the solution of the cell adhesion factor and / or the growth factor, an aqueous solution is usually used, but an aqueous solution such as a hydroalcohol may be used.

【0029】

By seeding and culturing Schwann cells on the inner surface of the tube, it is possible to obtain a nerve regeneration tube in which Schwann cells are present in one layer on the inner surface of the tube, preferably all around the lumen. In order to adhere Schwann cells to the entire circumference of the lumen, it is preferable to seed the Schwann cells over the entire lumen by pipetting while rotating (rolling) the tube.

【0030】

[Example]

Hereinafter, the present invention will be described in more detail based on examples. Manufacturing example 1 Polylactic acid fiber (40d) was braided using a braiding machine to form a tubular core material. This is fitted into a stainless steel rod, immersed in a dioxane solution (5% by weight) of a lactic acid / ε-caprolactone copolymer (molar ratio 50/50), frozen at -40 ° C, and then frozen at 30 ° C 24. Freeze-dried for hours. In this way, a complex having a lactic acid / ε-caprolactone copolymer sponge in the inner layer and a reinforcing material composed of a polylactic acid braid in the outer layer ((A) of Experimental Example 4) was obtained.

【0031】

A 0.1% solution of collagen (Type I, porcine tendon-derived atelocollagen) was sufficiently permeated into the sponge complex, frozen at -100 ° C, and then freeze-dried at 30 ° C for 24 hours. A collagen tube ((C) in Experimental Example 4) was obtained in which Type I collagen was coated on the entire surface including the inner surface.

【0032】

The obtained nerve regeneration tube is shown in Fig. 1 (50 times magnified photograph of the braid reinforcement 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). As described above, the inner layer was a sponge, the outer layer was a reinforcing material, and the sponge layer and the reinforcing material layer were separated. Manufacturing example 2 Polylactic acid fiber (40d) was braided using a braiding machine to form a tubular core material. This is fitted on a stainless steel rod, immersed in a dioxane solution (5% by weight) of a lactic acid / ε-caprolactone copolymer (molar ratio 25/75), frozen at -40 ° C, and then frozen at 30 ° C 24. Freeze-dried for hours. In this way, a complex having a lactic acid / ε-caprolactone copolymer sponge in the inner layer and a reinforcing material composed of a polylactic acid braid in the outer layer ((B) of Experimental Example 4) was obtained. Manufacturing example 3 A collagen tube ((D) in Experimental Example 4) was obtained in which Type IV collagen was coated on the entire surface including the inner surface in the same manner as in Production Example 1 except that Type IV collagen was used instead of Type I collagen. Manufacturing example 4 Type I collagen and Type instead of Type I collagen A collagen tube ((E) in Experimental Example 4) coated with Type I + Type IV collagen was obtained in the same manner as in Production Example 1 except that a mixture of IV collagen (1: 1 weight ratio) was used. Experimental example 1 Temporarily dissect both sciatic nerves of 10 male Fischer 344 rats. On the right side, a tube (inner diameter 2 mm, length 4 mm) made of P (LA / CL) sponge inner layer and core material PLLA braid outer layer coated with Type I collagen is used, and nerves are inserted at both ends by 2 mm. These nerve stumps were brought into close contact with each other to perform nerve junctions. Nerve junctions were performed by suturing the nerve to the tube with 8.0 nylon thread. On the left side, the temporarily dissected sciatic nerve was sutured with 8-0 nylon thread.

【0033】

Eight weeks after the operation, bilateral sciatic nerves were removed, and the number of axons and axon density at the center and periphery of the transplant site were measured.

【0034】

According to these results, nerve regeneration showed good results with a tube of P (LA / CL) sponge reinforced with a core made of PLLA braid, a simpler method than suturing a severed nerve. Equivalent or better results were obtained. Experimental example 2 Bisciatic nerves of 10 male Fischer 344 rats were resected approximately 12 mm. On the right side, a tube (inner diameter 2 mm, length 16 mm) made of P (LA / CL) sponge inner layer and PLLA braid outer layer coated with Type I collagen is used, and nerves are inserted at both ends by 2 mm, and the defect interval is Was fixed to 12 mm by suturing both ends of the tube and nerve fibers with 8.0 nylon thread. On the left side, nerve fibers were fixed in the same manner except that they were coated with 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 axon density at the center and periphery of the transplant site were measured.

【0036】

According to these results, both Type I collagen and Type IV collagen showed good results for nerve stumps. Experimental example 3 Using 10 Japanese white rabbits, a defect of about 20 mm was created in the total sural nerve on both sides. Next, 60 mm of the sural nerve on the ipsilateral side was sampled, and 3 cable grafts with a length of 20 mm were prepared and transplanted to the common peroneal nerve defect by reversing the polarity under a surgical microscope. Of these, 5 birds and 10 limbs were closed and controlled with the sutures left as they were. For the remaining 5 birds and 10 limbs, a bioabsorbable tube with a length of 6 mm and a lumen of 2 mm was vertically divided at the central and peripheral nerve junctions, and the entire circumference was involved and sutured so as to become a lumen again. Electrophysiological, functional and morphological searches were performed on these two groups 6 months after surgery. Experimental example 4 The following complexes (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 above complex was cut in the axial direction to obtain a flat plate-shaped complex, which was cut into a disk shape. After the obtained disk-shaped complex was treated with ethanol, Schwann cells collected from the dorsal root ganglion of the rat and cultured were subjected to a collagen coating on the sponge-like CL / PLLA copolymer of the complex or covering the surface thereof. It was seeded on the layer and its adhesiveness was examined by scanning electron microscopy and histology. In addition, immunostaining with S-100 antibody was performed to identify Schwann cells. Figure 4 shows the results of immunostaining with the S-100 antibody using the complex (C).

【0037】

As a result, in all the complexes (A) to (E), Schwann cell adhesion was further observed on the CL / PLLA copolymer sponge.

【0038】

[Effect of the invention]

According to the method of the present invention, nerve regeneration equal to or higher than that when a silicone tube is used can be performed, and since it is composed of only a bioabsorbable material, it does not need to be taken out after surgery.

【0039】

Nerve transplantation of corpses has been carried out in recent years, but there is a risk of immunosuppression and viral infection, and it is difficult to use a general method, but the present invention does not have such a problem.

[Simple explanation of drawings]

[Figure 1]

It is a drawing substitute photograph (magnification 50 times) showing the surface of the braid reinforcement layer.

[Figure 2]

It is a drawing substitute photograph (400 times magnification) showing the inner layer of the sponge.

[Fig. 3]

It is a drawing substitute photograph (magnification 50 times) showing the cross section of the tube.

[Fig. 4]

It is a drawing-substituting photograph showing the result of immunostaining with the S-100 antibody of Experimental Example 4.