JP7652382B2 - Regenerated meniscus base material - Google Patents

Description

The present invention relates to a meniscus regeneration substrate that is easy to handle and durable, and that can promote regeneration of the meniscus by wrapping and fixing the affected area of a damaged meniscus.

The meniscus is a cartilage-like tissue in the knee joint. The structure of the knee joint will be described below with reference to Figures 1 and 2. Figure 1 is a schematic cross-sectional view of a right knee joint in a sagittal plane, and Figure 2 is a schematic cross-sectional view of a right knee joint in a transverse plane. As shown in Figure 1, the knee joint has a meniscus 1 between the femur 4 and the tibia 5, and cartilage 3 is formed on each side where the femur 4 and the tibia 5 face each other. The patella 6 is located in the front of the knee, and below it is the infrapatellar fat pad (IPFP) 2. The knee joint is surrounded by a joint capsule 7, and the inside of the joint is filled with synovial fluid 8. As shown in Figure 2, a pair of menisci 1 are formed on the inside and outside of the knee joint, and the front and rear sides of the knee joint are thicker.

Degeneration and damage of the meniscus is one of the pathologies commonly seen along with cartilage degeneration in osteoarthritis (OA). It has also been reported that removal of the meniscus reduces cartilage tissue and advances osteoarthritis. As the meniscus is a tissue that contains many avascular areas, it has poor self-regenerative ability and self-repair is difficult. For this reason, in addition to meniscal suture surgery, additional treatments such as growth factors, synovial membrane transplants, and bone marrow stimulation have been performed to promote meniscal healing, but meniscal regeneration has been insufficient.

On the other hand, recent advances in cell engineering technology have made it possible to culture a variety of animal cells, including human cells, and research into so-called regenerative medicine, which uses these cells to reconstruct human tissues and organs, is progressing rapidly. In regenerative medicine, the key point is whether cells can proliferate and differentiate to build three-dimensional structures resembling living tissues. For example, one method is to transplant a substrate into the patient's body, and allow cells from the surrounding tissues or organs to invade the substrate, proliferate and differentiate, thereby regenerating the tissue or organ.

As a substrate for regenerative medicine, for example, it has been proposed to use a nonwoven fabric made of a bioabsorbable material, as disclosed in Patent Document 1. When a nonwoven fabric made of a bioabsorbable material is used as a substrate for regenerative medicine or as a suture reinforcement material, cells invade and grow in the voids, resulting in rapid tissue regeneration. Attempts have also been made to use such tissue regeneration substrates in meniscus regeneration treatment, and for example, tissue regeneration substrates with a structure in which a nonwoven fabric made of a bioabsorbable material is sandwiched between films made of a bioabsorbable material, and tissue regeneration substrates with multiple layers of nonwoven collagen laminated therein, are being considered for use in meniscus regeneration.

Japanese Patent Application Publication No. 05-076586

However, tissue regeneration substrates in which a nonwoven fabric made of a bioabsorbable material is sandwiched between films made of a bioabsorbable material have problems with low meniscus regeneration ability and are difficult to bend, making it difficult to perform transplant operations such as wrapping or wrapping the affected area of the meniscus. Furthermore, while tissue regeneration substrates in which collagen is laminated in multiple layers have a higher meniscus regeneration ability than tissue regeneration substrates in which a nonwoven fabric made of a bioabsorbable material is sandwiched between films made of a bioabsorbable material, this is still not sufficient, and there is also the problem that the strength is inferior to that of the meniscus when used in harsh environments exposed to high loads and friction.

The present invention aims to provide a meniscus regeneration substrate that is easy to handle and durable, and that can promote regeneration of the meniscus by wrapping and fixing the damaged area of the meniscus.

The present invention is a meniscus regeneration substrate that can promote the regeneration of the meniscus by wrapping and fixing so as to cover a damaged portion of the meniscus in a knee joint in meniscus regeneration treatment, and the meniscus regeneration substrate comprises a nonwoven fabric made of a bioabsorbable material and a film made of a bioabsorbable material integrated onto one side of the nonwoven fabric.
The present invention will be described in detail below.

After extensive research, the inventors discovered that laminating a film made of a bioabsorbable material onto one side of a nonwoven fabric made of a bioabsorbable material and integrating the resulting composite resulted in a material that exhibited higher meniscus regeneration performance than conventional tissue regeneration substrates, and thus completed the present invention.

The meniscus regeneration substrate of the present invention has a nonwoven fabric (hereinafter, also simply referred to as nonwoven fabric) made of a bioabsorbable material.
The nonwoven fabric serves as a scaffold for the proliferation of cells that have invaded from outside the meniscus and cells within the meniscus. In addition, since the nonwoven fabric is made of a bioabsorbable material, it is gradually absorbed into the body as the meniscus regenerates, eliminating the need for a second surgery to remove the fabric, and providing a high level of safety.

The bioabsorbable material is not particularly limited, and examples thereof include synthetic polymers such as polyglycolide, polylactide, poly-ε-caprolactone, lactide-glycolide copolymer, glycolide-ε-caprolactone copolymer, lactide-ε-caprolactone copolymer, polydioxanone, polycitric acid, polymalic acid, poly-α-cyanoacrylate, poly-β-hydroxy acid, polytrimethylene oxalate, polytetramethylene oxalate, polyorthoester, polyorthocarbonate, polyethylene carbonate, poly-γ-benzyl-L-glutamate, poly-γ-methyl-L-glutamate, and poly-L-alanine, and natural polymers such as polysaccharides such as starch, alginic acid, hyaluronic acid, chitin, pectinic acid, and derivatives thereof, and proteins such as gelatin, collagen, albumin, and fibrin. These bioabsorbable materials may be used alone, or two or more types may be used in combination. Of these, polyglycolide is preferred because it makes it easier to collect cells from the edges of the meniscus.

The preferred lower limit of the weight average molecular weight of the polyglycolide is 30,000, and the preferred upper limit is 400,000. If the weight average molecular weight of the polyglycolide is 30,000 or more, the strength can be further increased, and if it is 400,000 or less, the foreign body reaction can be further suppressed. The more preferred lower limit of the weight average molecular weight of the polyglycolide is 50,000, and the more preferred upper limit is 300,000.

The melt flow rate may be used as an alternative index of the molecular weight of the polyglycolide. The melt flow rate of the polyglycolide has a preferred lower limit of 0.1 g/10 min and a preferred upper limit of 100 g/10 min. Within this range, it is easy to prepare a nonwoven fabric made of polyglycolide. The melt flow rate of the polyglycolide is more preferably 1 g/10 min and more preferably 50 g/10 min.
The melt flow rate is measured under the condition that polyglycolide is melted by holding it in a cylinder at 240° C. for 10 minutes, and then the value is measured under the condition of a load of 4 kgf.

The nonwoven fabric preferably has an average fiber diameter of 0.9 μm or more and 7.0 μm or less.
When the average fiber diameter of the nonwoven fabric is within the above range, it is possible to facilitate cell penetration and also to increase the strength of the nonwoven fabric. The average fiber diameter of the nonwoven fabric is more preferably 1.5 μm or more, more preferably 3.0 μm or less, and even more preferably 2.0 μm or less.
In this specification, the average fiber diameter of a nonwoven fabric refers to the average value of fiber diameters measured at 10 random locations by cutting out a portion from the center of the fabric and observing the portion under an electron microscope.

The nonwoven fabric preferably has a basis weight of 3 g/m2 or ^more and 20 g/ ^m2 or less.
By setting the average fiber diameter and basis weight of the nonwoven fabric within the above range, it is possible to make it easier for cells to penetrate and also to increase the strength. The basis weight of the nonwoven fabric is preferably 5 g/ ^m2 or more and 15 g/ ^m2 or less.

The thickness of the nonwoven fabric is not particularly limited, but the preferred lower limit is 50 μm and the preferred upper limit is 200 μm. By making the thickness of the nonwoven fabric within the above range, the strength of the obtained meniscus regeneration substrate can be further increased and the meniscus regeneration ability can be improved. A more preferred lower limit of the thickness of the nonwoven fabric is 70 μm and a more preferred upper limit is 130 μm.

The method for producing the nonwoven fabric is not particularly limited, and any conventional method such as electrospinning deposition, melt-blowing, needle punching, spunbonding, flash spinning, hydroentanglement, airlaid, thermal bonding, resin bonding, or wet methods can be used.

The meniscus regeneration substrate of the present invention has a film made of a bioabsorbable material (hereinafter, also simply referred to as a film) on one side of the nonwoven fabric made of the above-mentioned bioabsorbable material.
The film facilitates the movement of bones in contact with the meniscus, such as the femur and tibia; in other words, it acts as a substitute for the cartilage of the damaged area of the meniscus until the area regenerates.
The film has a smooth surface, so it is unlikely to impede the movement of the bone in contact with the meniscus. In addition, since the film is made of a bioabsorbable material, it is gradually absorbed into the body as the meniscus regenerates, and eventually disappears, making it highly safe.

The bioabsorbable material constituting the film is not particularly limited, and the same bioabsorbable material as the nonwoven fabric can be used, but it is preferable that the bioabsorbable material constituting the film has a slower decomposition rate than the bioabsorbable material constituting the nonwoven fabric.
By forming the film from a bioabsorbable material that decomposes more slowly than the nonwoven fabric, it is possible to impart durability sufficient to withstand friction with the cartilage until the meniscus regenerates. Such a bioabsorbable material is appropriately determined depending on the bioabsorbable material constituting the nonwoven fabric, and when the bioabsorbable material constituting the nonwoven fabric is polyglycolide, it is preferably a lactide-ε-caprolactone copolymer.

The lactide-ε-caprolactone copolymer preferably has a molar ratio of lactide-derived structural units to ε-caprolactone-derived structural units of 45:55 or more and 55:45 or less. When the molar ratio of lactide-derived structural units to ε-caprolactone-derived structural units is within the above range, a film having high durability and an appropriate decomposition rate can be obtained. It is more preferable that the molar ratio of lactide-derived structural units to ε-caprolactone-derived structural units is 48:52 or more and 52:48 or less.

The preferred lower limit of the weight average molecular weight of the lactide-ε-caprolactone copolymer is 150,000, and the preferred upper limit is 600,000. If the weight average molecular weight of the polyglycolide is 300,000 or more, the strength can be further increased. The more preferred lower limit of the weight average molecular weight of the polyglycolide is 200,000, and the more preferred upper limit is 400,000.

The method for producing the film is not particularly limited, and any conventional method such as melt extrusion molding can be used.

The meniscus regenerating substrate of the present invention has the above-mentioned film integrated on one surface of the above-mentioned nonwoven fabric.
By laminating the film onto only one side of the nonwoven fabric, the side not laminated with the film exposes the nonwoven fabric, which has numerous voids, making it easier for cells to invade and proliferate from inside the meniscus and around the meniscus, thereby improving the meniscus regeneration ability compared to conventional tissue regeneration substrates.
In addition, the meniscus regeneration substrate of the present invention is easy to handle, such as wrapping or wrapping, because the film is laminated only on one side of the nonwoven fabric. Furthermore, since the film is included, the substrate is highly durable against friction with cartilage. Here, the term "composite integration" means that the nonwoven fabric and the film are firmly bonded together to such an extent that they do not easily separate. The nonwoven fabric and the film may be laminated with a plurality of nonwoven fabrics or films for the purpose of adjusting the thickness, etc.

The method for producing the regenerated meniscus substrate of the present invention is not particularly limited, and it can be produced, for example, by a method in which a film produced by the above method is layered on a nonwoven fabric produced by the above method, and vacuum-heated with a weight placed on top.

The meniscus regeneration substrate of the present invention is used for meniscus regeneration treatment.
Specifically, the regeneration of the meniscus can be promoted by wrapping the meniscus regeneration base material around the affected area of the damaged meniscus and fixing it by a method such as sewing it up. In particular, the present invention has a superior meniscus regeneration ability compared to conventional meniscus regeneration base materials, and therefore can reduce the risk of re-rupture of the regenerated meniscus.

According to the present invention, regeneration of the meniscus can be promoted by wrapping and fixing the damaged area of the meniscus, and a meniscus regeneration base material with excellent handling properties and durability can be provided.

FIG. 2 is a schematic sagittal cross-sectional view of the right knee joint. FIG. 2 is a schematic cross-sectional view of a right knee joint. 1 is a graph showing the results of tensile strength measurements. 1 is a graph showing the results of measuring threading strength. 1 shows photographs showing the results of quantitative evaluation of cell viability. 1 is a graph showing the results of quantitative evaluation of cell viability. 13 is a micrograph showing the condition of a meniscus defect site at 8 weeks in the evaluation of meniscus regeneration. 13 is a micrograph showing the condition of a meniscus defect site at 12 weeks in the evaluation of meniscus regeneration. 1 is a graph showing the results of Ishida score in the evaluation of meniscus regeneration. 13 shows micrographs showing the state of femoral cartilage at 8 weeks in the evaluation of femoral cartilage damage. 13 shows micrographs showing the state of femoral cartilage at 12 weeks in the evaluation of femoral cartilage damage. 1 is a graph showing the results of OARSI score in the evaluation of femoral cartilage damage.

The following examples further illustrate aspects of the present invention, but the present invention is not limited to these examples.

Example 1
Three sheets of a film made of lactide-ε-caprolactone copolymer (thickness: 100 μm, molar ratio of lactide-derived structural units to ε-caprolactone-derived structural units: 50:50) were layered on one side of a bioabsorbable nonwoven fabric (Neoveil ^Nano (NV-1010-D15G), manufactured by Gunze Co., Ltd., average fiber diameter: 3 μm, basis weight: 10 g/m 2 , thickness: 80 μm) to form a laminate. The laminate was placed in a vacuum dryer (DP41, manufactured by Yamato Scientific Co., Ltd.), sandwiched between silicone sheets, a plate-shaped weight was placed on top, and a load of 3 kPa was applied. The laminate was heated and pressed at 160°C for 10 minutes under vacuum to obtain a meniscus regeneration substrate.

(Comparative Example 1)
Gunze's SEAMDURASD-0909C (a three-layer structure in which a nonwoven fabric made of polyglycolide with a thickness of 80 μm is sandwiched between two films made of lactide-ε-caprolactone copolymer with a thickness of 100 μm) was used as the meniscus regeneration substrate.

(Comparative Example 2)
Bio-Gide (a two-layered collagen nonwoven fabric structure, thickness: 600 μm) manufactured by Geistlich was used as a meniscus regeneration substrate.

<Evaluation>
The meniscus regeneration base materials obtained in the examples and comparative examples were evaluated as follows.

(Measurement of tensile strength)
The obtained meniscus regeneration substrate was cut to 50 x 10 mm to prepare a measurement sample. The measurement sample was attached to a chuck, and the tensile strength was measured using an autograph (manufactured by Shimadzu Corporation) under the conditions of a chuck distance of 20 mm and a test speed of 50 mm/min. Next, measurement samples were prepared by immersing in physiological saline for 2 weeks, 4 weeks, and 8 weeks, and the tensile strength of each sample was measured in the same manner as above. The results are shown in Figure 3. Note that for Comparative Example 2, the meniscus regeneration substrate was completely decomposed at 4 weeks and 8 weeks, so it was not possible to measure.

(Measurement of thread strength)
The obtained meniscus regeneration substrate was cut to 22 mm x 22 mm, and a hole was drilled at a position 3 mm from the end of a pair of opposing sides, and a thread (2-0 nylon thread, diameter 0.3 mm) was passed through each of them. The thread was tied at both ends to form a ring, which was used as a measurement sample. For the obtained measurement sample, each thread was clamped with a chuck and the tensile strength (threading strength) of the thread was measured using an autograph (manufactured by Shimadzu Corporation) under conditions of a chuck distance of 40 mm and a test speed of 100 mm/min. Next, measurement samples were prepared by immersing in physiological saline for 2 weeks, 4 weeks, and 8 weeks, and the threading strength of each sample was measured in the same manner as above. The results are shown in Figure 4. Note that, for Comparative Example 2, the meniscus regeneration substrate was completely decomposed at 4 weeks and 8 weeks, so it was not possible to measure.

(Assessment of cell viability quantification)
The obtained meniscus regeneration scaffolds were cut into 1 x 1 cm pieces, soaked in culture solution, and then ATDC-5 was sprayed onto each scaffold and incubated at 37°C. After 1, 2, and 4 weeks, viable cells were evaluated using a laser microscope with a LIVE/DEAD assay reagent.
The stained photographs are shown in FIG. 5, and a graph quantifying the results of the viable cell count is shown in FIG.
5 and 6 show that Example 1 is superior to Comparative Examples 1 and 2 in terms of cell proliferation performance.

(Evaluation of meniscus regeneration)
Rabbits (Japanese white rabbits, 6 months old, male) were used as experimental animals, and cylindrical defects of Φ2 mm were created in the anterior segment of the medial meniscus. The meniscus regeneration substrate was transplanted by wrapping it around the circumference of the meniscus defect and then sutured. Transplantation was performed on a total of four rabbits, including a rabbit without a meniscus regeneration substrate transplanted as a control, two rabbits transplanted with the meniscus regeneration substrate of Example 1, and two rabbits transplanted with the meniscus regeneration substrates of Comparative Example 1 and Comparative Example 2.
The animals were sacrificed 8 and 12 weeks after surgery, and the menisci were removed. Then, the resected portion of the meniscus was stained with Safranin O to prepare a microscope sample, and a microscope photograph was taken. The photograph at the 8th week is shown in FIG. 7, and the photograph at the 12th week is shown in FIG. 8. The tissue specimens were scored to calculate the Ishida score. The calculated Ishida score is shown in FIG. 9. The Ishida score is an evaluation method for meniscus tissue repair.
7 and 8, it can be seen that the defect site is filling up in Example 1, and the surface of the defect site is stained, which indicates that cells are invading from the surface. On the other hand, in Comparative Examples 1 and 2, the defect is hardly filled up, and the surface is hardly stained, which indicates that there is little cell invasion. This can also be confirmed from the Ishida score results in FIG. 9.

(Evaluation of femoral cartilage damage)
The femur of a rabbit sacrificed for the evaluation of meniscus regeneration was taken out. A microscope sample was prepared by staining with Safranin O for the cartilage in contact with the meniscus of the taken out femur, and a microscope photograph was taken. The photograph at 8 weeks is shown in FIG. 10, and the photograph at 12 weeks is shown in FIG. 11. In addition, the OARSI score was calculated by scoring the tissue specimen. The calculated OARSI score is shown in FIG. 12. The OARSI score is a scoring used to evaluate cartilage degeneration.
10 and 11, it can be seen that the stained area is large in Example 1, indicating that the chondrocytes are not damaged. On the other hand, in Comparative Examples 1 and 2, although the wear is less than that in the case where the meniscus regeneration substrate was not implanted, the stained area is small, indicating that the cells are damaged. This can also be confirmed from the OARSI score result in FIG. 12.

According to the present invention, regeneration of the meniscus can be promoted by wrapping and fixing the damaged area of the meniscus, and a meniscus regeneration base material with excellent handling properties and durability can be provided.

1 Meniscus 2 Infrapatellar fat pad 3 Cartilage 4 Femur 5 Tibia 6 Patella 7 Joint capsule 8 Synovial fluid

Claims (3)

A meniscus regeneration base material for promoting the regeneration of the meniscus by wrapping and fixing a damaged portion of the meniscus of a knee joint in a meniscus regeneration treatment,
A film made of a bioabsorbable material is integrated on one side of a nonwoven fabric made of a bioabsorbable material, and the nonwoven fabric is exposed on the other side.
Meniscus regeneration base material. The bioabsorbable material constituting the film decomposes slower than the bioabsorbable material constituting the nonwoven fabric.
The meniscus regeneration substrate according to claim 1 . A meniscus regeneration base material for promoting the regeneration of the meniscus by wrapping and fixing a damaged portion of the meniscus of a knee joint in a meniscus regeneration treatment,
A film made of lactide-ε-caprolactone copolymer is integrated on one side of a nonwoven fabric made of polyglycolide, and the nonwoven fabric is exposed on the other side.
Meniscus regeneration base material.

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