JP6920691B2 - Meniscus recycled base material - Google Patents

Description

The present invention relates to a meniscus regeneration base material capable of promoting the regeneration of the meniscus by filling the defective portion of the meniscus of the knee joint in the regeneration treatment of the meniscus.

The meniscus is a cartilage-like tissue within the knee joint. The structure of the knee joint will be described below with reference to FIGS. 1 and 2. FIG. 1 is a schematic cross-sectional view of the right knee joint in the sagittal plane, and FIG. 2 is a schematic cross-sectional view of the right knee joint in the cross section. As shown in FIG. 1, the knee joint has a meniscus 1 between the femur 4 and the tibia 5, and cartilage 3 is formed on the side where the femur 4 and the tibia 5 face each other. The anterior surface of the knee is the patella 6, and the lower part thereof is the subpatella fat pad (IPFP) 2. The knee joint is wrapped with a joint capsule 7, and the inside of the joint is filled with joint fluid 8. As shown in FIG. 2, the meniscus 1 is formed in pairs so as to oppose each other on the medial and lateral sides of the knee joint, and the anterior surface side and the posterior surface side of the knee joint are thickened.

Meniscal degeneration and injury is one of the most common pathologies with cartilage degeneration in osteoarthritis of the knee (OA). It has also been reported that excision of the meniscus reduces cartilage tissue and promotes knee osteoarthritis. Since the meniscus is a tissue containing many avascular regions, it has poor self-renewal ability and is difficult to self-repair. Therefore, in surgery, in addition to meniscal suture, additional treatments such as growth factors, synovial transplantation, and bone marrow stimulation have been performed for the purpose of promoting healing of the meniscus, but the regeneration of the meniscus was insufficient. ..

On the other hand, recent advances in cell engineering technology have made it possible to culture a large number of animal cells, including human cells, and research on so-called regenerative medicine that attempts to reconstruct human tissues and organs using these cells. Is progressing rapidly. In regenerative medicine, the point is whether cells can proliferate and differentiate to construct a three-dimensional biological tissue-like structure. For example, a substrate is transplanted into the patient's body and cells are removed from surrounding tissues or organs. A method of invading a substrate and proliferating and differentiating it to regenerate a tissue or an organ has been carried out.

As a base material for such regenerative medicine, for example, it has been proposed to use a non-woven fabric made of a bioabsorbable material as disclosed in Patent Document 1. When a non-woven fabric made of a bioabsorbable material is used as a base material for regenerative medicine or a suture reinforcing material, cells invade the voids and proliferate, and the tissue is regenerated at an early stage. Attempts have also been made to use such a tissue regeneration base material in the regeneration treatment of the meniscus.
However, in reality, there is a problem that the regeneration of the meniscus is not promoted as expected even if the conventional tissue regeneration base material is used.

Japanese Unexamined Patent Publication No. 05-07656

An object of the present invention is to provide a meniscus regeneration base material capable of promoting the regeneration of the meniscus by filling the defective portion of the meniscus of the knee joint in the regeneration treatment of the meniscus.

The present invention is a meniscus regenerating base material capable of promoting the regeneration of the meniscus by filling the defective portion of the meniscus of the knee joint in the regenerating treatment of the meniscus, and is made of a bioabsorbable material. A film layer, a layer made of a non-woven fabric having an average pore size of 5 to 20 μm made of a bioabsorbable material, and a layer made of a non-woven fabric having an average pore size of 22 to 50 μm made of a bioabsorbable material are compositely integrated, and at least one of them is integrated. It is a meniscus regenerated base material made of a laminated body whose surface is a film layer made of the bioabsorbable material.
The present invention will be described in detail below.

The present inventors investigated the reason why the regeneration was not sufficiently promoted when the meniscus was regenerated using the conventional tissue regeneration base material. It is known that the non-woven fabric constituting the conventional tissue regeneration substrate preferably has an average pore size of about 5 to 30 μm in order to ensure cell invasion. As a result of diligent studies, the present inventor has found that such a conventional tissue regeneration base material is excellent in terms of cell invasion, but a part of the invaded cells passes through the tissue regeneration base material to form a tissue. It was found that it may leak out of the recycled substrate. It was considered that the tissue regeneration was not promoted as expected because the invading cells leaked out of the tissue regeneration substrate and the concentration of the cell component that actually stayed in the affected area was insufficient. In addition, for the regeneration of normal meniscal tissue, it is also important for cells to form dense tissues and proliferate and differentiate by interaction between cells, but with conventional tissue regeneration substrates, it is important. There is also a problem that it is difficult to form a dense tissue because the cells are separated from each other. On the other hand, if the average pore size of the non-woven fabric is reduced in order to prevent the leakage of cells or to bring the cells closer to each other, the permeability of the cells is impaired, so that the tissue is regenerated. Is limited to a small part of the surface of the substrate and cannot regenerate a structure having sufficient thickness. Further, in the regeneration of the meniscus, there is a problem peculiar to the regeneration of the meniscus that the transplanted base material and the cartilage come into contact with each other due to the movement of the knee joint and the surface of the base material is worn by the friction.

As a result of further diligent studies, the present inventors have made a bioabsorbable material with respect to a layer made of a non-woven fabric having an average pore size of 22 to 50 μm (hereinafter, also referred to as “cell invasion layer”). A layer made of a non-woven fabric having an average pore diameter of 5 to 20 μm (hereinafter, also referred to as a “cell leakage prevention layer”) is combined, and a film layer made of a bioabsorbable material (hereinafter, simply “film”) is formed on at least one surface. By forming a laminate with "layers" (also referred to as "layers"), it is possible to prevent cells from leaking out of the regenerating base material of the crescent plate while maintaining excellent cell invasion, and to have a sufficient thickness. The present invention has been completed by finding that it is possible to regenerate the non-woven fabric of the non-woven fabric and prevent wear due to friction with the cartilage.

The meniscus regenerated base material of the present invention comprises a laminated body in which a film layer, a cell invasion layer, and a cell leakage prevention layer are compositely integrated.
The cell-invading layer serves as a scaffold for cell proliferation that invades from surrounding tissues after transplantation, and has a role of promoting the regeneration of the meniscus. The laminate may have only one cell invasion layer or two or more layers.

The cell invading layer is made of a non-woven fabric made of a bioabsorbable material.
Examples of the bioabsorbable material constituting the cell-invading layer include polyglycolide, polylactide, poly-ε-caprolactone, lactide-glycolide copolymer, glycolide-ε-caprolactone copolymer, and lactide-ε-caprolactone copolymer. Coalescence, polydioxanone, polycitrate, polyappleic acid, poly-α-cyanoacrylate, poly-β-hydroxyic acid, polytrimethylene oxalate, polytetramethylene oxalate, polyorthoester, polyorthocarbonate, polyethylene carbonate, poly-γ Synthetic polymers such as -benzyl-L-glutamate, poly-γ-methyl-L-glutamate, poly-L-alanine, polysaccharides such as starch, alginic acid, hyaluronic acid, chitin, pectinic acid and derivatives thereof, and gelatin. , Natural polymers such as proteins such as collagen, albumin and fibrin. These bioabsorbable materials may be used alone or in combination of two or more.

Among the above-mentioned bioabsorbable materials, polyglycolide is preferable. When polyglycolide is used, it is particularly excellent in cell invasion and can regenerate normal tissue. When polyglycolide is made into a fiber and immersed in physiological saline at 37 ° C., the period until the tensile strength becomes 1/2 of that before immersion is about 14 days. By having such degradability, the base material is gradually decomposed and absorbed at the time when the cells proliferate and the tissue is regenerated, and the tissue regenerated to the inside of the base material is constructed, and as a result, good quality regeneration is performed. It is thought that an organization will be built. Furthermore, since the cells of the inflammatory system disappear within a few days after implantation in the living body, it is possible to exert an excellent effect that it is difficult to cause tissue adhesion.

In the present specification, polyglycolide means a polymer of glycolide such as polyglycolic acid, but other bioabsorbable properties such as lactide, ε-caprolactone, p-dioxanone, etc., as long as the effects of the present invention are not impaired. It may be a copolymer with the component of. Further, it may be a mixture with other bioabsorbable materials such as polylactide as long as the effect of the present invention is not impaired.
When the polyglycolide is a copolymer with other bioabsorbable components such as lactide, ε-caprolactone and p-dioxanone, the preferable lower limit of the blending amount of the glycolide component in the copolymer is 60 mol%. .. By setting the blending amount of the glycolide component to 60 mol% or more, the excellent effect of the present invention of excellent cell invasion and regeneration of normal tissue can be particularly exhibited.
When a mixture of the above polyglycolide and another bioabsorbable material such as polylactide is used, the preferable lower limit of the amount of polyglycolide to be blended in the mixture is 50 mol%. By setting the blending amount of polyglycolide to 50 mol% or more, the excellent effect of the present invention of excellent cell invasion and regeneration of normal tissue can be particularly exhibited.

When the bioabsorbable material is polyglycolide, the preferable lower limit of the weight average molecular weight of the polyglycolide is 30,000, and the preferable upper limit is 400,000. If the weight average molecular weight of the polyglycolide is less than 30,000, the strength may be insufficient and a sufficient tissue reinforcing effect may not be obtained. If it exceeds 400,000, the decomposition rate in the living body becomes slow and a foreign body reaction occurs. Sometimes. The more preferable lower limit of the weight average molecular weight of the polyglycolide is 50,000, and the more preferable upper limit is 300,000.

When the bioabsorbable material is polyglycolide, melt flow rate may be used as an alternative index of the molecular weight of the polyglycolide. The preferred lower limit of the melt flow rate of the polyglycolide is 0.1 g / 10 minutes, and the preferred upper limit is 100 g / 10 minutes. Within this range, it becomes easy to produce a non-woven fabric made of polyglycolide. The more preferable lower limit of the melt flow rate of polyglycolide is 1 g / 10 minutes, and the more preferable upper limit is 50 g / 10 minutes.
The measurement condition of the melt flow rate means a value measured under the condition of a load of 4 kgf after holding the polyglycolide in a cylinder at 240 ° C. for 10 minutes to melt it.

The non-woven fabric constituting the cell invasion layer has an average pore diameter of 22 μm at the lower limit and 50 μm at the upper limit. When the cell invading layer satisfies such an average pore size, it is possible to provide a meniscus regeneration base material which is excellent in cell invasion and is suitable for the regeneration of a normal meniscus. Cells can easily invade the pores having a pore size of 22 to 50 μm in the non-woven fabric, and can proliferate and differentiate in the cell invading layer to form a meniscal tissue. On the other hand, if the average pore size is less than 22 μm, cells cannot invade efficiently, and if the average pore size exceeds 50 μm, cells can invade, but the distance between cells is too large. , Cells adhering to the cell invasion layer and cells invading from surrounding tissues cannot sufficiently proliferate and differentiate. The preferable lower limit of the average pore size of the nonwoven fabric constituting the cell invasion layer is 24 μm, the preferable upper limit is 40 μm, the more preferable lower limit is 25 μm, and the more preferable upper limit is 30 μm.
In the present specification, the average pore size of the non-woven fabric means the average pore size measured by the bubble point method.

The measurement of the pore size distribution of the non-woven fabric by the bubble point method will be described.
In the bubble point method, a liquid that well wets the membrane to be measured is absorbed in the pores of the membrane in advance, installed in an instrument as shown in FIG. 3, and air pressure is applied from the back side of the membrane to apply the membrane. This is a method in which the minimum pressure (bubble point) at which the generation of bubbles can be observed on the surface is measured, and the pore size distribution is estimated from the relational expression between the surface tension of the liquid and the bubble point (FIG. 4).
Specifically, after the wetting liquid (for example, a fluorine-based solvent, trade name: Polofil (trademark)) is absorbed in the non-woven fabric to be measured, an instrument as shown in FIG. 3 (for example, Porometer manufactured by Nippon Bell Co., Ltd.) After installing the test piece in 3G) so as to have a circular shape with a diameter of 25 mm, air pressure is applied from the back side of the non-woven fabric to measure the minimum pressure (bubble point) at which the generation of bubbles can be observed on the film surface.
In the formula for calculating the pore diameter shown in FIG. 4, γ represents the surface tension of the infiltrating liquid, θ represents the contact angle of the infiltrating liquid on the non-woven fabric material, and ΔP represents the bubble point pressure.

The average fiber diameter of the non-woven fabric constituting the cell invasion layer is not particularly limited, but the preferable lower limit is 10 μm and the preferable upper limit is 50 μm. When the average fiber diameter of the non-woven fabric is in this range, it becomes easy to adjust the average pore diameter to the above-specified range. The more preferable lower limit of the average fiber diameter of the non-woven fabric is 15 μm, and the more preferable upper limit is 40 μm.

The thickness of the cell invading layer is not particularly limited, but the preferable lower limit is 100 μm and the preferable upper limit is 3 mm. If the thickness of the cell invading layer is less than 100 μm, the meniscus having a sufficient thickness may not be regenerated, and if it exceeds 3 mm, the handleability may be poor. The more preferable lower limit of the thickness of the cell invasion layer is 150 μm, and the more preferable upper limit is 2.5 mm.

The cell leakage prevention layer has a role of preventing the invading cells from leaking out of the meniscus regeneration base material. In addition, the cells proliferated in the cell invasion layer also invade the inside of the cell leakage prevention layer, and the cells form a dense tissue with each other to promote proliferation and differentiation, and promote the regeneration of a more normal meniscal tissue. It also has a role to play. The laminate may have only one layer for preventing cell leakage, or may have two or more layers.

The cell leakage prevention layer is made of a non-woven fabric made of a bioabsorbable material.
As the bioabsorbable material constituting the cell leakage prevention layer, the same bioabsorbable material as that used for the cell invasion layer can be used. The bioabsorbable material constituting the cell leakage prevention layer may be the same as or different from the bioabsorbable material constituting the cell invasion layer.

The non-woven fabric constituting the cell leakage prevention layer has an average pore diameter of 5 μm at the lower limit and 20 μm at the upper limit. When the cell leakage prevention layer satisfies such an average pore size, cell leakage can be prevented, and cells proliferated in the cell invasion layer can also invade the inside of the cell leakage prevention layer, so that the cells can enter each other. Can form a dense tissue. If the average pore size of the non-woven fabric constituting the cell leakage prevention layer exceeds 20 μm, the leakage of cells from the meniscus regeneration substrate cannot be sufficiently prevented. If the average pore size of the non-woven fabric constituting the cell leakage prevention layer is less than 5 μm, the cell invasion into the cell leakage prevention layer may be lowered, and the regeneration of the meniscus tissue may not be sufficiently promoted. The preferable lower limit of the average pore size of the non-woven fabric constituting the cell leakage prevention layer is 6 μm, the preferable upper limit is 18 μm, the more preferable lower limit is 7 μm, and the more preferable upper limit is 16 μm.

The average fiber diameter of the non-woven fabric constituting the cell leakage prevention layer is not particularly limited, but the preferable lower limit is 0.7 μm and the preferable upper limit is 7.0 μm. When the average fiber diameter of the non-woven fabric is in this range, it becomes easy to adjust the average pore diameter to the above-specified range. The more preferable lower limit of the average fiber diameter of the non-woven fabric is 0.9 μm, and the more preferable upper limit is 5.0 μm.

The thickness of the cell leakage prevention layer is not particularly limited, but a preferable lower limit is 10 μm and a preferable upper limit is 150 μm. If the thickness of the cell leakage prevention layer is less than 10 μm, it may not be possible to sufficiently prevent the leakage of cells from the meniscus regeneration substrate, and if it exceeds 150 μm, the area where cells cannot invade becomes large, which is normal. The meniscal tissue may not be regenerated. The more preferable lower limit of the thickness of the cell leakage prevention layer is 30 μm, and the more preferable upper limit is 100 μm.

The film layer is arranged on at least one surface of the laminate, further preventing cells that have invaded into the meniscus regenerated substrate of the present invention from leaking, and improving slipperiness. It has a role of preventing the surface of the base material from being worn by friction due to contact with cartilage after transplantation.
The film layer may be arranged on at least one surface of the laminate, but is preferably arranged on both surfaces.
In the present specification, the film means a thin film-like body having at least μm-order pores that can be observed with an optical microscope.

As the bioabsorbable material constituting the film layer, the same bioabsorbable material as that used for the cell invasion layer and the cell leakage prevention layer can be used. The bioabsorbable material constituting the film layer may be the same as or different from the bioabsorbable material constituting the cell invasion layer and the cell leakage prevention layer. Of these, a lactide-ε-caprolactone copolymer is preferable because it can exhibit high wear resistance.

The film layer may contain a lubricant for the purpose of further improving the slipperiness as long as the medical use is not impaired.

The thickness of the film layer is not particularly limited, but a preferable lower limit is 10 μm and a preferable upper limit is 500 μm. If the thickness of the film layer is less than 10 μm, it may not be possible to sufficiently prevent the surface of the base material from being worn due to friction due to contact with cartilage after transplantation. May be inferior. The more preferable lower limit of the thickness of the film layer is 25 μm, and the more preferable upper limit is 250 μm.

In the laminated body, the film layer, the cell leakage prevention layer, and the cell invasion layer are compositely integrated.
If the above layers are not compositely integrated, a part or all of the above layers may be peeled off when the meniscus regenerated base material of the present invention is transplanted. If even a part of each of the above layers is peeled off, cell accumulation may occur in the space formed in the peeled portion, and normal tissue or organ may not be regenerated.
In the present specification, the fact that the above layers are compositely integrated means that when the meniscus regenerated base material of the present invention is transplanted, the above layers are not separated even if they are folded and transplanted. More quantitatively, the peel strength measured according to the peel strength test method specified in JIS L 1021-9 is preferably 0.1 N or more, and more preferably 0.3 N or more. ..

The laminate may have a plurality of layers as long as the film layer, the cell leakage prevention layer, and the cell invasion layer are compositely integrated and at least one surface is the film layer. Specifically, for example, a five-layer structure of a film layer / cell invasion layer / cell leakage prevention layer / cell invasion layer / film layer, or a film layer / cell invasion layer / film layer / cell leakage prevention layer / film layer / cell invasion. It may have a 10-layer structure of a layer / film layer / cell leakage prevention layer / film layer / cell invasion layer.

FIG. 5 shows a schematic view showing an example of the regenerated meniscus substrate of the present invention.
In the meniscus regeneration base material 10 of FIG. 5 (1), the film layer 11, the cell leakage prevention layer 12, the cell invasion layer 13, and the film layer 11 are laminated and integrated in this order.
In the meniscus regeneration base material 20 of FIG. 5 (2), the film layer 21, the cell invasion layer 23, the cell leakage prevention layer 22, the cell invasion layer 23, and the film layer 21 are laminated and integrated in this order.

The method for producing the regenerated meniscus substrate of the present invention is not particularly limited. For example, the film layer, the cell leakage prevention layer and the cell invasion layer are separately prepared and then bonded together using a medical adhesive. Alternatively, a method of dissolving a part of the surface of each layer with a solvent and then laminating the layers can be mentioned.
Further, for example, after preparing the cell invasion layer in advance, a thread made of a bioabsorbable material is discharged onto the cell invasion layer by a melt blow method to form the cell leakage prevention layer, and further, the above-mentioned laminate. It is also preferable to perform needle punching so that the needle enters from the cell leakage prevention layer side to compositely integrate the cell invasion layer and the cell leakage prevention layer, and then laminate a film layer on one or both surfaces. Is. According to such a method, the cell invasion layer and the cell leakage prevention layer can be compositely integrated without blocking the joint surface between the cell invasion layer and the cell leakage prevention layer.

An example of the method for producing the recycled meniscus substrate of the present invention will be described in more detail.
In the above production method, first, the above cell invasion layer is prepared.
The method for preparing the cell invasion layer is not particularly limited, and for example, an electrospinning deposition method, a melt blow method, a needle punch method, a spunbond method, a flash spinning method, a water flow confounding method, an airlaid method, a thermal bond method, and a resin bond. Conventionally known methods such as a method and a wet method can be used. Above all, the needle punch method is suitable for preparing the cell invading layer.

The surface of the cell invasion layer on the side where the cell leakage prevention layer is laminated may be subjected to a feathering treatment. By applying a feathering treatment to the surface of the cell invasion layer in advance, the adhesion to the cell leakage prevention layer is further improved.
Specific examples of the feathering treatment include a method of raising the surface of the cell invasion layer on the side where the cell leakage prevention layer is laminated by using a raising machine. When the needle punch method is used as the method for producing the cell invading layer, the surface of the obtained cell invading layer is already raised, so that the same effect as that of the feathering treatment can be obtained. Be done.

In the above manufacturing method, a thread made of a bioabsorbable material is then discharged onto the cell invasion layer by a melt blow method to form the cell leakage prevention layer.
The melt blow method is a melt spinning method in which a bioabsorbable material as a raw material is made into a non-woven fabric in one step. Specifically, the bioabsorbable material melted by the extruder was blown out from a die having a large number of caps in the width direction toward the fiber collection point in a thread shape by a high temperature and high speed air flow, and stretched into a fiber shape. The resin is accumulated on the conveyor. A non-woven fabric is formed because fibers are entangled and fused between discharges and accumulations. Specifically, for example, the cell invasion layer is arranged on a conveyor so as to be in front of the fiber collection point by the melt blow method, and the fibers are discharged onto the cell invasion layer while the cell invasion layer is moved by the conveyor. To form a cell leakage prevention layer.
At this time, the fiber diameter, density, thickness, etc. of the cell leakage prevention layer formed can be controlled by adjusting the polymer discharge amount of the melt blow method, the air air velocity near the discharge outlet, the speed of the conveyor, and the like.

In the above-mentioned production method, next, needle punching is performed so that the needle enters from the above-mentioned cell leakage prevention layer side of the obtained laminate of the above-mentioned cell invasion layer and the above-mentioned cell leakage prevention layer, and the above-mentioned cell invasion layer and the above-mentioned cells are performed. The leak prevention layer is compositely integrated.
By the needle punch, the cell invasion layer and the cell leakage prevention layer can be reliably combined and integrated without blocking the pores of each layer on the bonding surface.

In the above production method, the film layer is then laminated on one or both surfaces of the obtained laminate of the cell invasion layer and the cell leakage prevention layer. As a method of laminating the film layer, for example, a method of applying a solvent capable of dissolving the film layer to the surface of the film layer to dissolve a part of the film layer, and then laminating the film layer on one or both surfaces of the laminated body. And so on.

According to the present invention, in the meniscus regeneration treatment, it is possible to provide a meniscus regeneration base material capable of promoting the regeneration of the meniscus by filling the defective portion of the meniscus of the knee joint.

It is a cross-sectional schematic diagram in the sagittal plane of the right knee joint. It is sectional drawing of the right knee joint in the cross section. It is a schematic diagram explaining the method of measuring the pore size distribution of a non-woven fabric by a bubble point method. It is a schematic diagram explaining the method of estimating the pore size distribution of a non-woven fabric from the data obtained by the bubble point method. It is a schematic diagram which shows an example of the meniscus regenerated base material of this invention. It is a micrograph image which transplanted the meniscus regenerated base material of Example 2 and stained the transplanted part with safranin O at the 12th week.

Hereinafter, embodiments of the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

(Example 1)
(1) Preparation of non-woven fabric for cell invasion layer A polyglycolide having a weight average molecular weight of 250,000 is used as a bioabsorbable material, and a cloth made of yarn obtained by spinning this is made into a non-woven fabric by a needle punching method. A non-woven fabric for a cell invasion layer having a diameter of about 20 μm and a thickness of about 300 μm was obtained.

The obtained non-woven fabric was absorbed with a fluorine-based solvent (trade name: Porofil (trademark)) as a wetting liquid, and then installed on a Porometer 3G manufactured by Nippon Bell Co., Ltd. so that the size of the test piece was a circle with a diameter of 25 mm. Then, air pressure was applied from the back side of the non-woven fabric to measure the minimum pressure (bubble point) at which the generation of bubbles could be observed on the film surface. A graph showing the pore size distribution of the non-woven fabric was obtained based on the obtained bubble points, and the average pore size was calculated from the graph and found to be 28 μm.

(2) Lamination of Cell Leakage Prevention Layer The obtained non-woven fabric for the cell invasion layer was placed on the conveyor so as to be in front of the fiber collection point by the melt blow method. While moving the conveyor, a thread made of polyglycolide was discharged onto the non-woven fabric for the cell invasion layer by the melt blow method, and a cell leakage prevention layer having an average fiber diameter of about 1 μm and a thickness of about 80 μm was laminated.
The melt blow method used polyglycolide having a weight average molecular weight of 250,000 as a raw material, and was carried out under the conditions of a polymer discharge rate of 0.2 kg / h and an air wind speed of 11 m / sec near the discharge outlet. The moving speed of the conveyor was set to a speed at which the density of the non-woven fabric obtained by the melt blow method was 10 g / m ^2.
A non-woven fabric composed of only the cell leakage prevention layer was separately produced under the same conditions, and the average pore size of the non-woven fabric was calculated by the bubble point method and found to be 12 μm.

(3) Composite integration of cell invasion layer and cell leakage prevention layer Needle punching is performed so that the needle enters from the cell leakage prevention layer side of the obtained laminate, and the cell invasion layer and cell leakage prevention layer are combined. It was integrated.

(4) Lamination of film layer L-lactide-ε-caprolactone copolymer (molar ratio 50:50, weight average molecular weight 200,000) was dissolved in dioxane to prepare a 4 wt% dioxane solution. The obtained solution was poured into a glass petri dish, air-dried and heat-treated to obtain a film having a thickness of about 100 μm.
A small amount of dioxane is applied to the surface of one surface of the obtained film to partially dissolve it, and the obtained laminate of the cell invasion layer and the cell leakage prevention layer is formed on the film on the cell leakage prevention layer side. The film was laminated so as to be in contact with each other, dried, and the film and the laminate were compositely integrated to obtain a recycled meniscus substrate.

(Comparative Example 1)
Polyglycolide having a weight average molecular weight of 250,000 is used as a bioabsorbable material, and a cloth made of yarn obtained by spinning this is made into a non-woven fabric by a needle punching method. The average fiber diameter is about 20 μm and the thickness is about 380 μm. A non-woven fabric for the cell invasion layer was obtained.
The average pore size of the obtained non-woven fabric for the cell invasion layer was calculated by the bubble point method and found to be 28 μm.

A 4 wt% dioxane solution was prepared by dissolving an L-lactide-ε-caprolactone copolymer (molar ratio 50:50, weight average molecular weight 200,000) in dioxane. The obtained solution was poured into a glass petri dish, air-dried and heat-treated to obtain a film having a thickness of about 100 μm.
A small amount of dioxane is applied to the surface of one surface of the obtained film to partially dissolve it, and the obtained non-woven fabric for the cell invasion layer is laminated on it and dried to combine the film and the non-woven fabric. Then, a meniscus regenerated base material was obtained.

(Comparative Example 2)
The Teflon (registered trademark) sheet was placed on the conveyor so as to be in front of the fiber collection point by the melt blow method. While moving the conveyor, a thread made of polyglycolide was discharged onto a Teflon (registered trademark) sheet by a melt blow method to obtain a cell leakage prevention layer having an average fiber diameter of about 1 μm and a thickness of about 380 μm.
The melt blow method used polyglycolide having a weight average molecular weight of 250,000 as a raw material, and was carried out under the conditions of a polymer discharge rate of 0.2 kg / h and an air wind speed of 11 m / sec near the discharge outlet. The moving speed of the conveyor was set to a speed at which the density of the non-woven fabric obtained by the melt blow method was 10 g / m ^2.
The average pore size of the obtained non-woven fabric for the cell leakage prevention layer was calculated by the bubble point method and found to be 12 μm.

A 4 wt% dioxane solution was prepared by dissolving an L-lactide-ε-caprolactone copolymer (molar ratio 50:50, weight average molecular weight 200,000) in dioxane. The obtained solution was poured into a glass petri dish, air-dried and heat-treated to obtain a film having a thickness of about 100 μm.
A small amount of dioxane is applied to the surface of one surface of the obtained film to partially dissolve it, and the obtained non-woven fabric of the cell leakage prevention layer is laminated on the surface and dried to compositely integrate the film and the non-woven fabric. Then, a meniscus regenerated base material was obtained.

(Comparative Example 3)
A meniscus regenerated base material was obtained in the same manner as in Example 1 except that the film layers were not laminated.

<Evaluation by animal experiments>
A rabbit was prepared as an experimental animal, and the anterior to intermediate phalanx (the part surrounded by the dotted line in FIG. 2) of the medial meniscus of the right knee joint was excised. The meniscus regenerated base material obtained in Examples and Comparative Examples was transplanted to the meniscus removal portion and sutured.
Eight weeks after the operation, the patient was sacrificed to death, and the right knee joint was observed. In Example 1, it was confirmed that the meniscal-like tissue was regenerated to the extent that the removed portion was completely filled. On the other hand, in Comparative Examples 1 to 3, regeneration of the meniscal-like tissue was not observed.

(Example 2)
(1) Preparation of non-woven fabric for cell invasion layer A polyglycolide having a weight average molecular weight of 250,000 is used as a bioabsorbable material, and a cloth made of yarn obtained by spinning this is made into a non-woven fabric by a needle punching method. A non-woven fabric for a cell invasion layer having a diameter of about 20 μm and a thickness of about 300 μm was obtained.

The obtained non-woven fabric was absorbed with a fluorine-based solvent (trade name: Porofil (trademark)) as a wetting liquid, and then installed on a Porometer 3G manufactured by Nippon Bell Co., Ltd. so that the size of the test piece was a circle with a diameter of 25 mm. Then, air pressure was applied from the back side of the non-woven fabric to measure the minimum pressure (bubble point) at which the generation of bubbles could be observed on the film surface. A graph showing the pore size distribution of the non-woven fabric was obtained based on the obtained bubble points, and the average pore size was calculated from the graph and found to be 28 μm.

(2) Preparation of Film for Film Layer An L-lactide-ε-caprolactone copolymer (molar ratio 50:50, weight average molecular weight 200,000) was dissolved in dioxane to prepare a 4 wt% dioxane solution. The obtained solution was poured into a glass petri dish, air-dried and heat-treated to obtain a film for a film layer having a thickness of about 100 μm.

(3) Preparation of non-woven fabric for cell leakage prevention layer Polyglycolide was used as a bioabsorbable material, and a non-woven fabric was prepared by a melt blow method using a general-purpose compact extruder having a screw diameter of 20 mm. Nitrogen gas was purged in the hopper, spinning was performed under hot air, and the discharge amount and the speed of the belt conveyor were adjusted to obtain a non-woven fabric for a cell leakage prevention layer having an average fiber diameter of about 1 μm and a thickness of about 80 μm. ..
The melt blow method used polyglycolide having a weight average molecular weight of 250,000 as a raw material, and was carried out under the conditions of a polymer discharge rate of 0.2 kg / h and an air wind speed of 11 m / sec near the discharge outlet. The moving speed of the conveyor was set to a speed at which the density of the non-woven fabric obtained by the melt blow method was 10 g / m ^2.
A non-woven fabric composed of only the cell leakage prevention layer was separately produced under the same conditions, and the average pore size of the non-woven fabric was calculated by the bubble point method and found to be 12 μm.

(4) Manufacture of a meniscus regenerated base material composed of a 10-layer structure laminate For a film layer For a cell invasion layer obtained by partially dissolving it by applying a small amount of dioxane to the surface of one surface of the film. Non-woven fabric was laminated. Next, a small amount of dioxane was applied to the surface of the other surface of the film for the film layer on which the non-woven fabric for the cell invasion layer was laminated to partially dissolve it, and the obtained non-woven fabric for the cell leakage prevention layer was laminated on the surface and dried. Obtained a laminate. By repeating substantially the same steps as described above, 10 layers of film layer / cell invasion layer / film layer / cell leakage prevention layer / film layer / cell invasion layer / film layer / cell leakage prevention layer / film layer / cell invasion layer A regenerated half-moon plate base material composed of a laminated body having a structure was obtained.

<Evaluation by animal experiments>
A rabbit was prepared as an experimental animal, and a part of the anterior to middle phalanx (the part surrounded by the dotted line in FIG. 2) of the medial meniscus of the right knee joint was excised. The meniscus regenerated base material obtained in Example 2 was transplanted to the meniscus removal portion and sutured.
In addition, as a comparison target, an experiment in which the regenerated meniscus base material was sutured without transplantation to the meniscus removal part, and an experiment in which a scalpel was inserted as a sham control and the wound was closed as it was without removing the meniscus were also performed.

Twelve weeks after the operation, the patient was sacrificed to death, the medial meniscus was removed, and the transplanted portion (the portion from the anterior segment to the middle segment of the medial meniscus in the comparative control) was removed. A micrograph image of the obtained specimen stained with safranin O is shown in FIG.
From FIG. 6, 12 weeks after the operation, regeneration of cartilage tissue (the portion stained in red in the stained image) was observed in the portion to which the meniscal regeneration substrate was transplanted, which was similar to that of the sham control.

According to the present invention, in the meniscus regeneration treatment, it is possible to provide a meniscus regeneration base material capable of promoting the regeneration of the meniscus by filling the defective portion of the meniscus of the knee joint.

1 Meniscus 2 Subpatellar fat pad 3 Cartilage 4 Femur 5 Tibia 6 Patella 7 Joint capsule 8 Joint fluid 10 Meniscus regeneration base material 11 Film layer 12 Cell leakage prevention layer 13 Cell invasion layer 20 Meniscus regeneration base material 21 Film layer 22 Cell leakage prevention layer 23 Cell invasion layer

Claims (1)

In the meniscus regeneration treatment, it is a meniscus regeneration base material that can promote the regeneration of the meniscus by filling the defective portion of the meniscus of the knee joint.
A composite of a film layer made of a bioabsorbable material, a layer made of a non-woven fabric having an average pore size of 5 to 20 μm made of a bioabsorbable material, and a layer made of a non-woven fabric made of a bioabsorbable material having an average pore size of 22 to 50 μm. integrated, Ri is at least one surface Do a laminate is a film layer made of the bioabsorbable material,
The film layer made of the bioabsorbable material is made of a lactide-ε-caprolactone copolymer.
The layer made of the bioabsorbable material and made of a non-woven fabric having an average pore size of 5 to 20 μm and the layer made of the bioabsorbable material and made of a non-woven fabric having an average pore size of 22 to 50 μm are made of polyglycolide. A recycled base material for meniscus.

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