JP2003019196A - Neurotization tube - Google Patents

Abstract

PROBLEM TO BE SOLVED: To reproduce cut nerves. SOLUTION: This neurotization tube includes a sponge made from a bioabsorbable polymer and tubular reinforcement made from another bioabsorbable polymer having a longer decomposition and absorption period than the sponge, with at least the inner surface of the tube being formed of the sponge.

Description

Detailed Description of the Invention

[0001]

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

[0002]

2. Description of the Related Art Regeneration of peripheral nerves 1
Since the publication of the silicone tube model by Lundberg et al. In 982, attempts have been made to extend the reproducible stump distance with silicone tubes. However, when a damaged nerve is repaired using a silicone tube, the silicone tube may circumscribe the nerve regenerated in the tube over time.

On the other hand, a technique relating to regeneration of nerve defect 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 such a support cannot provide sufficient strength.

Further, WO98 / 22155 discloses a tube made of a biodegradable and absorbable material and a collagen body having an inner cavity having a void penetrating the tube substantially parallel to the axis of the tube. , An artificial neural tube filled with a matrix gel containing laminin, etc. is disclosed. However, since the artificial neural tube is filled with collagen and matrix gel inside, Schwann cells cannot be seeded inside.

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

[0006]

The present invention provides the following nerve regeneration tube and a method for producing the same. Item 1. A nerve regeneration tube comprising a sponge composed of a bioabsorbable polymer and a tubular reinforcing material composed of a bioabsorbable polymer having a decomposition and absorption period longer than that of the sponge, at least the inner surface of which is a sponge. Item 2. Item 2. The tube according to Item 1, wherein the tubular reinforcing material has a shape of a braid, a knit, a woven fabric, a non-woven fabric, a punching sheet or a spiral mesh. Item 3. 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 comprising a cell adhesive factor. Item 5. Item 5. The tube according to any one of Items 1 to 4, further containing a growth factor. Item 6. Item 6. The tube according to any one of Items 1 to 5, wherein the Schwann cells are seeded on the inner surface of the tube. Item 7. The following steps (A) to (C): Step (A): Fixing a tubular reinforcing material composed of a bioabsorbable fiber on the outside of the tubular core, Step (B): Obtained reinforcing material The 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 tubular reinforcing material. Step (C): The freeze-dried product is removed from the tubular core, A method for producing a nerve regeneration tube having a sponge and a tubular reinforcing material, which comprises inverting as necessary. Item 8. The method further includes a step (B1) of immersing the tubular core body having the sponge and the reinforcing material obtained in the step (B) in a solution of a cell adhesive factor and / or a growth factor and freeze-drying the same.
Item 7 having an inner surface of a sponge coated with a cell adhesion factor and / or a growth factor, characterized in that the freeze-dried product obtained in the step (B1) is subjected to the step (C).
The method for producing a nerve regeneration tube according to [4]. Item 9. Item 9. A method for producing a nerve regeneration tube having Schwann cells on the inner surface, which further comprises a 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, both synthetic bioabsorbable polymer and natural bioabsorbable polymer can be used. Examples of the synthetic bioabsorbable polymer include aliphatic polyesters (polyglycolic acid, polylactic acid (D-form, L-form, DL-form), polycaprolactone, polyvalerolactone and copolymers thereof, for example, lactic acid-caprolactone copolymer. , Lactic acid-glycolic acid copolymer, glycolic acid-trimethylene carbonate copolymer, glycolic acid-trimethylene carbonate-dioxanone copolymer, glycolic acid-trimethylene carbonate-ε caprolactone copolymer, etc.), polyester ether (polyether) -1,4-dioxanone-2-one, poly-1,5-dioxepan-2-one, ethylene glycol-the aliphatic polyester copolymer, and the aliphatic polyester-polyester ether copolymer). To be Examples of natural bioabsorbable polymers include collagen, gelatin, hyaluronic acid, alginic acid and the like.

[0008] Examples of preferable synthetic bioabsorbable polymer constituting the sponge include polylactic acid, polyglycolic acid, polycaprolactone and copolymers thereof, and the like, as preferable synthetic bioabsorbable polymer constituting the reinforcing material. Examples thereof include polylactic acid, polyglycolic acid, polycaprolactone, and copolymers thereof.

The synthetic bioabsorbable polymer that constitutes the sponge and the synthetic bioabsorbable polymer that has a longer decomposition and absorption period than the sponge that constitutes the reinforcing material may be the same or different.

"A 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 than the synthetic bioabsorbable polymer of the sponge. In addition to a polymer that is highly resistant to decomposition in vivo, the polymer itself is the same, or the polymer of the reinforcing material is more easily decomposed, but the tubular reinforcing material is better than the sponge. Since it is difficult to be decomposed, it means that the synthetic bioabsorbable fiber as a whole has a longer decomposition and absorption period than the synthetic bioabsorbable sponge.

Examples of constituent elements of the reinforcing material of the present invention include fibers such as monofilaments, multifilaments, and strings, sheets, and nonwoven fabrics. The diameter of the fiber is 10 to 20
The thickness is about 00 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 spirally knitted with filament yarn, and the like, and preferably braided braids. The thickness of the tubular reinforcement is 10-2000μ
m, preferably about 50 to 1000 μm.

The sponge of the present invention has a thickness of 0.1-5 mm
About 0.5 to 2 mm, and the sponge has a pore size of about 1 to 500 μm, preferably 10 to 200.
It is about μm. The reinforcing material may be inside the sponge to integrally form a nerve regeneration tube, and 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.

The thickness of the nerve regeneration tube of the present invention is 0.1.
Is about 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.

As a cell adhesion factor, collagen (I
Type, IV type, etc.), laminin, fibronectin and the like.

As a growth factor, nerve growth factor (NG
F), neurotrophic factors, neurite outgrowth factors and the like.

The cell adhesive 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.

The nerve regeneration tube of the present invention can be manufactured by a method including the following steps (A) to (C): Step (A): Synthetic bioabsorbable fiber is formed on the outside of the tubular core. Step (B): Immobilize the tubular reinforcing material, soaking the obtained reinforcing material-fixed core in a synthetic bioabsorbable polymer solution, and then freeze-drying it to obtain a sponge layer having a shorter decomposition and absorption period than the tubular reinforcing material. And step (C): The freeze-dried product is removed from the tubular core body and inverted as necessary.

In the step (A), the tubular reinforcing material may be externally fitted to the outer side of the tubular core body. In this case,
A nerve regeneration tube including a tubular reinforcing material in the outer layer is obtained (if the reinforcing material has an opening, the sponge penetrates into the opening). In addition, a detachable protrusion (for example, a radial protrusion, a donut-shaped brim, or the like) for fixing the tubular reinforcing material at a predetermined position of the tubular core (for example, a position corresponding to the length of the nerve regeneration tube). ), It may be fixed at a position away from the tubular 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 tubular core. In this way, by fixing the tubular reinforcing material at a position apart from the tubular core body, a nerve regeneration tube in which the reinforcing material and the sponge are integrated (the reinforcing material is surrounded by the sponge) is obtained.

As the cylindrical core, one in which the synthetic bioabsorbable polymer solution does not enter can be used.

In the step (B), the synthetic bioabsorbable polymer solution is preferably a solution which does not dissolve the polymer constituting the reinforcing material as much as possible. As the solvent of the solution,
Examples 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 fixed core is added to the synthetic bioabsorbable polymer solution. It is desirable to keep the body soaked for as short a time as possible, and to freeze-dry immediately after soaking.

The concentration of the synthetic bioabsorbable polymer solution is about 0.1 to 20% by weight, preferably about 1 to 10% by weight.

In step (C), when the freeze-dried product after immersion is removed from the tubular core body, the sponge exists outside the reinforcing material (the reinforcing material is adhered and fixed to the cylindrical core body) or outside. It may exist both inside (the reinforcing material is fixed at a position apart from the tubular core body). If the sponge is present as an outer layer of reinforcement, it is inverted to obtain a nerve regeneration tube with the sponge on the inner surface. When the reinforcing material has sponge layers on both sides, it is not 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 side, it is inverted and the inner sponge layer It is desirable to have a thicker nerve regeneration tube.

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

When no inversion is carried out, the reinforcing material is hollow first.
The synthetic bioabsorbable polymer solution is fixed to the inside of the tubular core material, and the tubular second tubular core material is further fixed to the center portion between the first tubular core material and the second tubular core material. If you pour and freeze-dry,
A nerve regeneration tube having a sponge on the inner surface can be obtained without inversion.

By mixing a cell adhesion factor and / or a growth factor in a synthetic bioabsorbable polymer solution at a predetermined concentration, a nerve regeneration tube containing the factor in a sponge can be obtained.

By subjecting the tubular core body having the sponge and the reinforcing material obtained in the step (B) to a solution (preferably an aqueous solution) of a cell adhesive factor and / or a growth factor and freeze-drying the same. Also, a nerve regeneration tube having a sponge inner surface coated with a cell adhesive factor and / or a growth factor can be obtained. Alternatively, the growth factor may be incorporated into the crosslinked gelatin fine particles and then combined with the sponge.

The concentration of the cell adhesion factor is 0.01
An example is about 10%.

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 hydrous alcohol may be used.

By seeding Schwann cells on the inner surface of the tube and culturing, a nerve regeneration tube having a single layer of Schwann cells on the inner surface of the tube, preferably on the entire circumference of the lumen, can be obtained. In order to attach the Schwann cells to the entire circumference of the lumen, it is preferable to pipette while rotating (rolling) the tube to seed the Schwann cells throughout the lumen.

[0030]

EXAMPLES The present invention will now be described in more detail with reference to examples. Production Example 1 Polylactic acid fiber (40d) was braided using a braiding machine to obtain a tubular core material. Attach this to a stainless steel rod, lactate / ε-
After dipping in a dioxane solution (5% by weight) of a caprolactone copolymer (molar ratio 50/50), it was frozen at -40 ° C and then freeze-dried at 30 ° C for 24 hours. In this way
A composite 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.

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

The obtained nerve regeneration tube is shown in FIG. 1 (a magnified photograph of the surface of the braid reinforcement layer 50 times), FIG. 2 (a photograph of the sponge inner layer magnified 400 times), and FIG.
As shown in the enlarged photograph), 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 fiber (40d) was braided using a braiding machine to obtain a tubular core material. Attach this to a stainless steel rod, lactate / ε-
After dipping in a dioxane solution (5% by weight) of a caprolactone copolymer (molar ratio 25/75), it was frozen at -40 ° C and then lyophilized at 30 ° C for 24 hours. In this way
A composite 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. Production Example 3 A collagen tube (Type D of Experimental Example 4) in which the entire inner surface including Type IV collagen was coated was obtained in the same manner as in Production Example 1 except that Type IV collagen was used instead of Type I collagen. Production Example 4 Type I collagen and Type IV instead of Type I collagen
A collagen tube coated with Type I + Type IV collagen ((E) of Experimental Example 4) was obtained in the same manner as in Production Example 1 except that a mixture of collagen (1: 1 weight ratio) was used. Experimental Example 1 Ten sciatic nerves of 10 male Fischer 344 rats are temporarily dissected. On the right side is a tube (inner diameter 2 mm, length 4 mm) made of P (LA / CL) sponge inner layer and core material PLLA braid outer layer.
Using a ype I collagen-coated product, nerves were inserted into both ends by 2 mm, and these nerve stumps were brought into close contact with each other to perform nerve junction. Nerve junction was performed by suturing the nerve to a tube with 8.0 nylon thread. On the other hand, on the left side, the temporarily cut sciatic nerve was sutured with 8-0 nylon thread.

Bilateral sciatic nerves were excised at 8 weeks after the operation, and the number of central and peripheral axons and the density of axons were measured.

According to these results, nerve regeneration was shown to be good when a tube in which P (LA / CL) sponge was reinforced with a core material composed of a PLLA braid was used, which is easier than suturing a cut nerve. The same or better results have been obtained with various methods. Experimental Example 2 About 12 mm of both sciatic nerves of 10 male Fischer 344 rats
I removed it. The tube on the right is made of P (LA / CL) sponge inner layer and PLLA braid outer layer (inner diameter 2 mm, length 16 mm).
Using a ype I collagen-coated product, nerves were inserted by 2 mm at both ends so that the defect interval was 12 mm, and both ends of the tube and nerve fibers were sutured with 8.0 nylon thread for fixation. Type I on the left
Nerve fibers were fixed in the same manner except that Type IV collagen was used instead of collagen for coating.

The bilateral sciatic nerves were excised 8 weeks after the operation, and the number of central and peripheral axons and the density of axons were measured.

According to these results, the nerve stump is Type I
Both collagen and Type IV collagen showed similarly good results. Experimental Example 3 Using 10 Japanese white rabbits, about 20 mm in the common sural nerve on both sides
Created a defect. Next, 60 mm of the sural nerve on the same side was collected, 20 mm in length × 3 cable grafts were prepared, and the polarity was reversed under a surgical microscope and transplanted into the common sural nerve defect. Of these, 5 limbs and 10 limbs were left with the sutured part as it was, and closed as a control. With respect to the remaining 5 limbs, a bioabsorbable tube having a length of 6 mm and a lumen of 2 mm was longitudinally divided at the central and peripheral nerve junctions, and the whole circumference was wrapped and sutured again to form a lumen. An electrophysiological, functional and morphological search was performed 6 months after the operation on these two groups. 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 above complex Was cut in the axial direction to obtain a plate-shaped composite body, which was cut into a disk shape. The obtained disc-shaped complex was treated with ethanol, and Schwann cells collected from the rat dorsal root ganglia and cultured were treated with sponge-like CL of the complex.
It was seeded on the / PLLA copolymer or on the collagen coating layer covering the surface thereof, and its adhesion was examined by scanning electron microscopy and histology. For identification of Schwann cells
Immunostaining with S-100 antibody was performed. The result of immunostaining with the S-100 antibody using the complex (C) is shown in FIG.

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

[0038]

EFFECTS 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,
Moreover, it is not necessary to take it out after the operation since it is made of only the bioabsorbable material.

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

[Brief description of drawings]

FIG. 1 is a drawing-substitute photograph (magnified 50 times) showing the surface of a braid reinforcing material layer.

FIG. 2 is a drawing-substitute photograph (400 times magnification) showing an inner layer of a sponge.

FIG. 3 is a drawing-substitute photograph (magnified 50 times) showing a cross section of a tube.

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

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Seiji Takamatsu             6-4-5 Kamishio, Tennoji-ku, Osaka City, Osaka Prefecture             Zanne Yuhigaoka No.502 F-term (reference) 4C081 AB18 BA12 BA16 CA161                       CA162 CD122 CD132 DA03                       DA05 DA06 DB03 DC03                 4C097 AA14 BB04 CC02 DD02 DD05                       DD12 EE08 EE19 FF02 FF17                       MM04

Claims (9)

[Claims] 1. A nerve comprising a sponge composed of a bioabsorbable polymer and a tubular reinforcing material composed of a bioabsorbable polymer having a decomposition and absorption period longer than that of the sponge, at least the inner surface of which is a sponge. Play tube. 2. The tube according to claim 1, wherein the tubular reinforcing material has a shape of a braid, a knit, a woven fabric, a non-woven fabric, a punching sheet or a spiral mesh. 3. The tube according to claim 2, wherein the reinforcing material is an outer layer and the sponge is an inner layer. 4. The method according to claim 1, which further comprises a cell adhesion factor.
The tube according to any one of 1. 5. The tube according to claim 1, further comprising a growth factor. 6. The tube according to claim 1, wherein the Schwann cells are seeded on the inner surface of the tube. 7. The following steps (A) to (C): Step (A): Fixing a tubular reinforcing material composed of a bioabsorbable fiber on the outside of the tubular core body, Step (B): The obtained reinforcing material-immobilized core is immersed in a bioabsorbable polymer solution and freeze-dried to form a sponge having a shorter decomposition and absorption period than the tubular reinforcing material. Step (C): The freeze-dried material is tubular. A method for producing a nerve regeneration tube having a sponge and a tubular reinforcing material, which comprises removing from a core body and inverting as necessary. 8. The method further comprises a step (B1) of immersing the tubular core body having the sponge and the reinforcing material obtained in the step (B) in a solution of a cell adhesive factor and / or a growth factor and freeze-drying. The nerve regeneration according to claim 7, which has an inner surface of a sponge coated with a cell adhesion factor and / or a growth factor, characterized by subjecting the lyophilized product obtained in the step (B1) to the step (C). Tube manufacturing method. 9. A method for producing a nerve regeneration tube having Schwann cells on the inner surface thereof, which further comprises a step (D) of seeding and culturing Schwann cells on the inner surface of the tube obtained in the step (C) of claim 7 or 8. Method.