Diaphragm repair material and method for manufacturing the same

Abstract

To provide a diaphragm repair material and a production method of the same, capable of suturing to a diaphragm to close a defective part, to repair a tissue on the defective part of the diaphragm, thereby suppressing recurrence of defective.SOLUTION: There is provided a diaphragm repair material for repairing a defective part of a diaphragm, the diaphragm repair material 1 comprises: a cell layer 2; first biocompatible long fiber nonwoven fabrics 3 each of which is arranged on one / the other of both sides of the cell layer 2; and a second biocompatible long fiber nonwoven fabric 4 arranged to contact one first biocompatible long fiber nonwoven fabric, the cell layer 2 including a stem cell. The first biocompatible long fiber nonwoven fabric 3 is a gelatin long fiber nonwoven fabric mainly formed of gelatin, and the second biocompatible long fiber nonwoven fabric 4 has a tension elastic modulus of 1.0 MPa or greater and 3.4 MPa or smaller.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a diaphragmatic repair material used to repair defects in the diaphragm, and a method for manufacturing the same. [Background technology]

[0002] Congenital diaphragmatic hernia is a condition in which there is a congenital defect in the diaphragm, causing some abdominal organs that should normally be in the abdomen to protrude into the chest cavity. If the defect is large, patch closure using artificial fabric may be performed. For example, PTFE sheets are widely used for patch closure of congenital diaphragmatic hernias (e.g., Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Camila Gonzalez Ruhrnschopf et al., Biological versus synthetic patch for the repair of congenital diaphragmatic hernia: 8-year experience at a tertiary center, Journal of Pediatric Surgery, Vol56, 2021, pp1957-1961 [Overview of the project] [Problems that the invention aims to solve]

[0004] However, while the diaphragm has elasticity, artificial fabrics such as PTFE sheets have poor elasticity, and especially when used in children, including newborns, they do not expand as the child grows, thus posing a risk of postoperative recurrence of diaphragmatic hernia.

[0005] To solve the aforementioned conventional problems, the present invention provides a diaphragm repair material and a method for manufacturing the same, which can repair the tissue of a diaphragm defect and improve the recurrence of the defect by suturing it to the diaphragm and closing the defect. [Means for solving the problem]

[0006] One or more embodiments of the present invention relate to a diaphragm repair material used to repair a defect in the diaphragm, wherein the diaphragm repair material comprises a cell layer, a first biocompatible long-fiber nonwoven fabric arranged on both sides of the cell layer, and a second biocompatible long-fiber nonwoven fabric arranged in contact with one side of the first biocompatible long-fiber nonwoven fabric, wherein the cell layer comprises stem cells, the first biocompatible long-fiber nonwoven fabric is a gelatin long-fiber nonwoven fabric mainly composed of gelatin, and the second biocompatible long-fiber nonwoven fabric has a tensile modulus of 1.0 MPa or more and 3.4 MPa or less.

[0007] One or more embodiments of the present invention relate to a method for manufacturing a diaphragm repair material, comprising the steps of: preparing a cell sheet; laminating a first biocompatible long-fiber nonwoven fabric onto one surface of the cell sheet to obtain a first laminate; laminating two first laminates so that the cell sheets are in contact with each other to obtain a second laminate; and laminating a second biocompatible long-fiber nonwoven fabric so that it is in contact with one side of the second laminate. [Effects of the Invention]

[0008] The present invention provides a diaphragm repair material and a method for manufacturing the same, which can repair the tissue in the defective portion of the diaphragm and improve the recurrence of the defect by suturing it to the diaphragm and closing the defective portion. [Brief explanation of the drawing]

[0009] [Figure 1] This is a schematic cross-sectional view of a diaphragm repair material according to one embodiment of the present invention. [Figure 2] This is a schematic cross-sectional view of a diaphragm repair material according to one embodiment of the present invention. [Figure 3] Schematic cross-sectional view of the diaphragm repair material according to one embodiment of the present invention. [Figure 4] In Example 1, it is a photograph showing the result of histological evaluation 1 of the diaphragm after the diaphragm repair experiment. [Figure 5] In Example 1 and Comparative Example 1, it is a photograph showing the result of histological evaluation 2 of the diaphragm after the diaphragm repair experiment. [Figure 6] In Example 1, it is a photograph showing the result of histological evaluation 3 of the diaphragm after the diaphragm repair experiment. [Figure 7] In Example 1, it is a photograph showing the result of histological evaluation 3 of the diaphragm after the diaphragm repair experiment.

Mode for Carrying Out the Invention

[0010] In order to solve the above-described problems, the inventors of the present invention repeated studies. As a result, as a diaphragm repair material, a cell layer containing stem cells, first biocompatible long fiber nonwoven fabrics are arranged on both sides of the cell layer, and a second biocompatible long fiber nonwoven fabric is arranged so as to contact the first biocompatible long fiber nonwoven fabric on one side. A laminate is used, a gelatin long fiber nonwoven fabric mainly composed of gelatin is used as the first biocompatible long fiber nonwoven fabric, and the tensile elastic modulus (also referred to as Young's modulus) of the second biocompatible long fiber nonwoven fabric is set to 1.0 MPa or more and 3.4 MPa or less. By doing so, the diaphragm repair material can be sutured to the diaphragm to close the defective part, repair the tissue of the defective part of the diaphragm, and improve the recurrence of the defect. Specifically, by setting the tensile elastic modulus of the second biocompatible long fiber nonwoven fabric to 1.0 MPa or more and 3.4 MPa or less, the suture property is good when suturing the defective part of the diaphragm with the diaphragm repair material, and it is easy to expand and contract in accordance with the expansion and contraction of the diaphragm after suturing. Further, by using the gelatin long fiber nonwoven fabric and the cell layer containing stem cells in combination, the bonding property between the diaphragm and the gelatin long fiber nonwoven fabric is enhanced, angiogenesis, striated muscle neogenesis, etc. progress, the tissue of the defective part of the diaphragm is repaired, and the recurrence of the defect can be suppressed.

[0011] <Cell layer> The cell layer may consist of one or more cell sheets and is not particularly limited, but it is preferable to include two or more cell sheets from the viewpoint of easily adjusting the number and distribution of cells. In one or more embodiments of the present invention, a cell sheet means a sheet-like structure formed by cells being joined together by intercellular bonds. The cells may be directly adhered to each other, or they may be adhered to each other via an intervening substance. The intervening substance is not particularly limited as long as it is a substance that can adhere cells to each other, but examples include the extracellular matrix. The intervening substance is not particularly limited, but it is preferable that it is cell-derived.

[0012] A cell sheet (cell layer) contains cells such as stem cells. Stem cells are cells that have the potential to differentiate into various specialized cell types. Stem cells are not particularly limited and include, for example, embryonic stem cells (ES cells), embryonic carcinoma cells (EC), embryonic germ stem cells (EG), induced pluripotent stem cells (iPS cells), adult stem cells, blastocyst-derived stem cells, germline-derived stem cells, teratoma-derived stem cells, oncostatin-independent stem cells (OISC), bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, amniotic membrane-derived mesenchymal stem cells, skin-derived mesenchymal stem cells, periosteum-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, dental pulp-derived stem cells, etc.

[0013] The stem cells described above may be used individually or in combination of two or more depending on the purpose. For example, from the viewpoint of increasing the engraftment rate in the diaphragmatic defect site, it is preferable that the stem cells include mesenchymal stem cells. Mesenchymal stem cells are not particularly limited to the tissue or organ of origin, and examples include bone marrow-derived mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, amniotic membrane-derived mesenchymal stem cells, skin-derived mesenchymal stem cells, periosteum-derived mesenchymal stem cells, umbilical cord-derived mesenchymal stem cells, umbilical cord blood-derived mesenchymal stem cells, dental pulp-derived stem cells, etc. Among these, it is more preferable to include one or more selected from the group consisting of bone marrow-derived stem cells and adipose-derived stem cells, and it is even more preferable to include adipose-derived stem cells.

[0014] From the viewpoint of suppressing immune rejection after suturing to the diaphragm, the cells are preferably allogeneic or autologous stem cells, and more preferably allogeneic or autologous mesenchymal stem cells.

[0015] The thickness of the cell layer is not particularly limited, but from the viewpoint of further improving angiogenesis and rhabdomyoogenesis, and further improving the repair of tissue in the defective area of ​​the diaphragm, it is preferably 10 μm to 200 μm, more preferably 20 μm to 170 μm, and even more preferably 30 μm to 140 μm.

[0016] <First biocompatible long-fiber nonwoven fabric> The first biocompatible long-fiber nonwoven fabric is a gelatin long-fiber nonwoven fabric mainly composed of gelatin. In one or more embodiments of the present invention, "main component" means a component that is present in an amount of 90% by mass or more. For example, "main component of gelatin" means that it contains 90% by mass or more of gelatin. The gelatin long-fiber nonwoven fabric may contain 95% by mass or more of gelatin, or may consist substantially of 100% by mass of gelatin. In addition to gelatin, the gelatin long-fiber nonwoven fabric may optionally contain other components in an amount of 10% by mass or less, or 5% by mass or less. The other components may be other biocompatible polymers, crosslinking agents, plasticizers, other additives, etc.

[0017] In one or more embodiments of the present invention, the diaphragm repair material is used in a swollen state. In one or more embodiments of the present invention, "swelling" means swelling the gelatin fiber nonwoven fabric with a liquid, such as water, saline solution, buffer solution, culture medium, etc., until it reaches a saturated state. The saturated state means a state in which the liquid is contained to the maximum extent possible, and the liquid content remains at a certain limit and does not increase further.

[0018] The type and part of the animal from which the collagen used as the raw material for the gelatin originates are not particularly limited. The collagen may be derived from, for example, vertebrates or fish. Collagen derived from various organs and tissues such as dermis, ligaments, tendons, bones, and cartilage can also be used as appropriate. The method for preparing gelatin from collagen is also not particularly limited and can be, for example, acid treatment, alkali treatment, and enzyme treatment. The molecular weight of the gelatin is also not particularly limited and can be appropriately selected and used. Furthermore, one type of gelatin may be used, or two or more types may be used in combination.

[0019] The gelatin is not particularly limited, but it has appropriate flexibility and hardness, and from the viewpoint of improving the handling properties of the gelatin long fiber nonwoven fabric, it is preferable that the gel strength is 100g or more and 400g or less, and more preferably 150g or more and 360g or less. In the present invention, the gel strength can be measured in accordance with JIS K 6503:2001. The gelatin may be a commercially available product.

[0020] The aforementioned other biocompatible polymers are not particularly limited, but for example, natural polymers and synthetic polymers can be used. Examples of natural polymers include proteins and polysaccharides. Examples of proteins include collagen, fibronectin, fibrinogen, laminin, and fibrin. Examples of polysaccharides include natural polymers such as chitosan, calcium alginate, heparan sulfate, chondroitin sulfate, hyaluronic acid, heparin, starch, gellan gum, agarose, guar gum, xanthan gum, carrageenan, pectin, locust bean gum, tamarind gum, and dieutan gum, or derivatives of natural polymers such as carboxymethylcellulose can be used. Examples of synthetic polymers include non-absorbent synthetic polymers such as polyethylene glycol, polypropylene glycol, polyethylene terephthalate, polyvinyl alcohol, thermoplastic elastomers, polypropylene, polyethylene, polystyrene, polymethyl methacrylate, polycarbonate, polydimethylsiloxane, cycloolefin polymers, and amorphous fluororesins, as well as bioabsorbable polymers such as polylactic acid, polyglycolic acid, polycaprolactone, and polydioxanone. The other biocompatible polymers mentioned above may be used individually or in combination of two or more.

[0021] Gelatin long fiber nonwoven fabric is suitable for use as a diaphragm repair material because it is primarily composed of gelatin, which is highly safe and has excellent bioabsorbability. In particular, when used in combination with a cell sheet composed of bone marrow-derived stem cells including mesenchymal stem cells or adipose-derived stem cells, a synergistic effect is likely to occur between the gelatin long fiber nonwoven fabric and the cell sheet, and differentiation into target cells is enhanced at the diaphragm defect site.

[0022] The gelatin long fibers preferably have an average fiber diameter of 2 μm to 400 μm, more preferably 5 μm to 300 μm, even more preferably 7 μm to 250 μm, and particularly preferably 10 μm to 150 μm. When the average fiber diameter of the gelatin long fibers is within the above range, stem cells can easily invade the gelatin long fiber nonwoven fabric, and the engraftment rate of stem cells in the diaphragmatic defect site is increased. In this specification, the "average fiber diameter of the gelatin long fibers" can be determined by measuring the diameter of 50 fibers arbitrarily selected from the gelatin long fiber nonwoven fabric after swelling and calculating the average value. The gelatin long fiber nonwoven fabric after swelling can be observed, for example, with a microscope (CKX53, manufactured by Olympus Corporation).

[0023] In the gelatin filament nonwoven fabric, the fiber intersections are at least partially welded. This allows the fibers in the gelatin filament nonwoven fabric to form a three-dimensional network structure, preventing the gelatin nonwoven fabric from collapsing even when swollen and increasing its strength in the swollen state. Furthermore, the formation of a three-dimensional network structure by the fibers in the gelatin filament nonwoven fabric makes it easier to mold into desired shapes and provides high molding stability. In the gelatin filament nonwoven fabric, the fiber intersections may be partially welded or all of them may be welded. The welding of the fiber intersections is not particularly limited, but can be achieved, for example, by depositing fibers that have not completely solidified during the manufacturing of the gelatin filament nonwoven fabric.

[0024] The gelatin long fiber nonwoven fabric is not particularly limited, but for example, from the viewpoint of handling, adhesion to the cell sheet, and enhancing interaction with the cell sheet after diaphragm repair, it is preferably 0.1 mm to 3.0 mm thick, more preferably 0.2 mm to 2.5 mm thick, and even more preferably 0.3 mm to 2.0 mm thick.

[0025] The gelatin long fiber nonwoven fabric is not particularly limited, but for example, from the viewpoint of improving handling, adhesion to cell sheets, and interaction with cell sheets after diaphragm repair, a basis weight of 50 g / m² is desirable. 2 More than 300g / m2 Preferably, it is less than 100 g / m². 2 More than 200g / m 2 The following, and more preferably 130 g / m² 2 More than 170g / m 2 The following applies: In this specification, the basis weight of gelatin nonwoven fabric can be measured in accordance with JIS L 1913:2010.

[0026] The gelatin long-fiber nonwoven fabric is not particularly limited, but for example, from the viewpoint of handling, adhesion to cell sheets, and interaction with cell sheets after diaphragm repair, it is preferable that the compressive deformation rate (hereinafter also simply referred to as "compressive deformation rate") at a compressive stress of 1.0 kPa is 1% or more and 40% or less, more preferably 5% or more and 35%, and even more preferably 10% or more and 30% or less. In this specification, the compressive deformation rate is calculated using the following formula (1) in the laminate after swelling with water to a saturated state, where the thickness under no load is (H1) and the thickness under a compressive stress of 1.0 kPa is (H2). The compression test is performed as described below. Compression deformation rate (%) = 100 - {(H2 / H1) × 100} ... (1)

[0027] In one or more embodiments of the present invention, the gelatin long fiber nonwoven fabric may be coated with cell adhesion factors, cell induction factors, cell growth factors, substances that provide nutrients and energy to cells, substances that inhibit or enhance cell function, etc. Examples of cell adhesion factors are not particularly limited, but include fibronectin. Coating the gelatin long fiber nonwoven fabric with cell adhesion factors strengthens adhesion to the cell sheet. Examples of substances that provide nutrients and energy to cells are not particularly limited, but include ATP, pyruvate, glutamine, etc. Furthermore, in one or more embodiments of the present invention, the gelatin long fiber nonwoven fabric may be immersed in a solution containing physiologically active substances such as cell induction factors and cell growth factors to incorporate these components. After suturing to the diaphragm, these physiologically active substances are gradually released from the gelatin long fiber nonwoven fabric, thereby promoting differentiation into target cells, etc.

[0028] In one or more embodiments of the present invention, the gelatin long fiber nonwoven fabric is not particularly limited, but from the viewpoint of suppressing the generation of impurities and preventing product contamination, it is preferable to produce it by extruding a spinning solution containing a biocompatible polymer such as gelatin into the air from a nozzle discharge port, injecting a pressurized fluid forward from a fluid injection port located behind the nozzle discharge port and not in contact with the nozzle discharge port, allowing the extruded spinning solution to form fibers along with the pressurized fluid, and then accumulating the resulting biocompatible long fibers to form a nonwoven fabric.

[0029] In one or more embodiments of the present invention, the gelatin long fiber nonwoven fabric is preferably crosslinked. This enhances morphological stability and water resistance. Crosslinking may be chemical crosslinking using compounds such as crosslinking agents, but from the viewpoint of biosafety, it is preferable to crosslink using a biosafe crosslinking agent or to crosslink without a crosslinking agent. Examples of crosslinking without a crosslinking agent include thermal crosslinking, electron beam crosslinking, radiation crosslinking such as gamma rays, and ultraviolet crosslinking. In the case of electron beam irradiation or radiation irradiation such as gamma rays, sterilization and crosslinking can be performed simultaneously. From the viewpoint of easily obtaining the desired crosslinking effect, thermal crosslinking is preferred, and thermal dehydration crosslinking is more preferred. Thermal dehydration crosslinking may be performed, for example, at 100°C to 200°C for 24 hours to 96 hours. Thermal dehydration crosslinking may also be performed, for example, under a vacuum of 1 kPa or less. The laminate may be dried before crosslinking. Drying may be done by air drying at room temperature or by vacuum freeze-drying.

[0030] In one or more embodiments of the present invention, the gelatin long fiber nonwoven fabric may be specifically prepared as described in International Publication No. 2018 / 235745 and used after swelling as necessary. Alternatively, as the gelatin long fiber nonwoven fabric, commercially available products such as the "Genocel®" sheet type manufactured by Nikke Medical Co., Ltd. may be used after being appropriately swollen.

[0031] <Second biocompatible long-fiber nonwoven fabric> The second biocompatible long-fiber nonwoven fabric has a tensile modulus of 1.0 MPa to 3.4 MPa. This ensures good sutureability when suturing the diaphragmatic repair material to the diaphragm to close the defect site, and after suturing the diaphragmatic repair material to the diaphragm to close the defect site, the diaphragmatic repair material easily follows the expansion and contraction of the diaphragm, reducing the risk of defect recurrence and complications such as thoracic deformity and scoliosis. The tensile modulus of the second biocompatible long-fiber nonwoven fabric is preferably 1.1 MPa to 3.3 MPa, more preferably 1.2 MPa to 3.2 MPa, even more preferably 1.2 MPa to 3.1 MPa, even more preferably 1.3 MPa to 3.0 MPa, and particularly preferably 1.4 MPa to 3.0 MPa. The tensile modulus of the second biocompatible long-fiber nonwoven fabric can be measured as described in the examples.

[0032] The second biocompatible long-fiber nonwoven fabric preferably has a tensile strength of 18 N or more. This allows for greater suture strength when suturing the diaphragmatic repair material to the diaphragm to close the defect, and also prevents tearing. The tensile strength of the second biocompatible long-fiber nonwoven fabric is more preferably 18 N to 150 N, even more preferably 20 N to 100 N, and even more preferably 25 N to 80 N. The tensile strength of the second biocompatible long-fiber nonwoven fabric can be measured as described in the examples.

[0033] The second biocompatible long-fiber nonwoven fabric preferably has an elongation rate (hereinafter also referred to as the 50% 50-cycle elongation rate) of 70% to 200% after 50 repeated stretch tests at a strain rate of 50%, more preferably 100% to 150%, and even more preferably 120% to 140%. The diaphragm generally expands to 1.5 times its size when breathing, and the respiratory rate of children is generally 30 to 45 breaths per minute, while that of adults is generally 15 to 20 breaths per minute. Therefore, if the 50% 50-cycle elongation rate of the second biocompatible long-fiber nonwoven fabric is within the above range, the diaphragm repair material will be able to more easily follow the expansion and contraction of the diaphragm of adults and children during breathing. The 50% 50-cycle elongation rate of the second biocompatible long-fiber nonwoven fabric can be measured as described in the examples.

[0034] The second biocompatible long-fiber nonwoven fabric preferably has an elongation rate (hereinafter also referred to as the 25% 100-cycle elongation rate) of 70% to 200% after 100 repeated stretch tests at a strain rate of 25%, more preferably 100% to 125%, and even more preferably 105% to 120%. When the 25% 100-cycle elongation rate of the second biocompatible long-fiber nonwoven fabric is within the above range, the diaphragm repair material is more likely to follow the expansion and contraction of the diaphragm during small breaths in newborns and children. The 25% 100-cycle elongation rate of the second biocompatible long-fiber nonwoven fabric can be measured as described in the examples.

[0035] The second biocompatible long-fiber nonwoven fabric can be any biocompatible long-fiber nonwoven fabric mainly composed of a biocompatible polymer, within the range that satisfies the tensile strength and tensile modulus described above. From the viewpoint of biodegradability and adhesion to gelatin, it is preferable that the biocompatible long-fiber nonwoven fabric mainly composed of one or more bioabsorbable polymers selected from the group consisting of polylactic acid, polyglycolic acid, polycaprolactone, and polydioxanone, and more preferably that it is a polyglycolic acid long-fiber nonwoven fabric mainly composed of polyglycolic acid.

[0036] The polylactic acid long fiber nonwoven fabric may contain 90% by mass or more of polylactic acid, may contain 95% by mass or more, or may be substantially composed of 100% by mass of polylactic acid. In addition to polylactic acid, the polylactic acid long fiber nonwoven fabric may contain other components of 10% by mass or less, or 5% by mass or less, as necessary. The other components may be other biocompatible polymers or the like described in the column of the first biocompatible long fiber nonwoven fabric.

[0037] The weight average molecular weight of polylactic acid is not particularly limited. For example, from the viewpoint of increasing the tensile strength and elastic modulus, it is preferably 7,000 or more and 130,000 or less, more preferably 7,500 or more and 100,000 or less, and even more preferably 8,000 or more and 80,000 or less. In this specification, the weight average molecular weight of polylactic acid is measured by gel permeation chromatography (GPC).

[0038] The basis weight of the polylactic acid long fiber nonwoven fabric is not particularly limited. For example, from the viewpoint of increasing the tensile strength and elastic modulus, it is preferably 60 g / m 2 or more and 2 300 g / m 2 or less, more preferably 70 g / m 2 or more and 2 200 g / m 2 or less, and even more preferably 80 g / m 2 or more and 2 180 g / m 2 or less. In this specification, the basis weight of the polylactic acid long fiber nonwoven fabric can be measured in accordance with JIS L 1913:2010.

[0039] The thickness of the polylactic acid long fiber nonwoven fabric is not particularly limited. For example, from the viewpoint of improving handling properties and sewing strength, it is preferably 0.1 mm or more and 3.0 mm or less, more preferably 0.2 mm or more and 1.0 mm or less, and even more preferably 0.3 mm or more and 0.8 mm or less.

[0040] The fineness of the single fiber of the polyglycolic acid long fiber is not particularly limited, but for example, from the viewpoint of biodegradability, it is preferably 10 dtex or more and 200 dtex or less, more preferably 20 dtex or more and 150 dtex or less, and even more preferably 40 dtex or more and 80 dtex or less.

[0041] The polyglycolic acid long fiber nonwoven fabric is not particularly limited, but for example, from the viewpoint of increasing tensile strength and elastic modulus, it is preferably a long fiber needle-punched nonwoven fabric obtained by needle-punching a knitted fabric composed of polyglycolic acid long fibers, and more preferably a long fiber needle-punched nonwoven fabric obtained by needle-punching a knitted fabric (also called a knit) composed of polyglycolic acid long fibers. The knit may be flat knit or circular knit.

[0042] Long-fiber needle-punched nonwoven fabrics can be manufactured using known methods and are not particularly limited, but for example, they can be manufactured as follows. (1) Knitted fabric is produced using a knitting machine such as a tubular knitting machine. (2) Lay the knitwear flat and stack the two pieces on top of each other to prevent wrinkles. (3) The two layers of knitted material are needle-punched using a needle punching machine to produce a nonwoven fabric. Needle punching should be continued until the fibers are intertwined and difficult to unravel. (4) Heat setting is performed to fix the expansion and contraction of the nonwoven fabric. Heat setting can be performed, for example, at a temperature of 40°C to 180°C for 1 minute to 60 minutes. (5) Heat press the material using a press machine to correct the thickness. Heat pressing can be performed as needed to obtain the desired thickness, for example, under conditions of a pressure of 5 MPa to 40 MPa and a temperature of 40°C to 180°C.

[0043] <Diaphragm repair material> The diaphragmatic repair material includes a cell layer, a first biocompatible long-fiber nonwoven fabric positioned on both sides of the cell layer, and a second biocompatible long-fiber nonwoven fabric positioned in contact with one side of the first biocompatible long-fiber nonwoven fabric. The cell layer may include two or more cell sheets.

[0044] Figure 1 is a schematic cross-sectional view (a cross-section perpendicular to the thickness direction) of a diaphragm repair material according to one embodiment of the present invention. The diaphragm repair material 1 of this embodiment includes a cell layer 2, a first biocompatible long-fiber nonwoven fabric 3 (3a, 3b) arranged on both sides of the cell layer 2, and a second biocompatible long-fiber nonwoven fabric 4 arranged in contact with the first biocompatible long-fiber nonwoven fabric 3b. The first biocompatible long-fiber nonwoven fabric 3 (3a, 3b) can be the gelatin long-fiber nonwoven fabric described above as appropriate. The second biocompatible long-fiber nonwoven fabric can preferably be the polyglycolic acid long-fiber nonwoven fabric described above. The cell layer 2 includes two layers of cell sheets, cell sheets 2a and 2b. The cell sheets can be the cell sheets containing stem cells described above as appropriate. In a cross-section perpendicular to the thickness direction of the diaphragm repair material 1, the cell sheets 2a and 2b and the first biocompatible long-fiber nonwoven fabrics 3a and 3b are the same size, while the size of the second biocompatible long-fiber nonwoven fabric 4 is smaller than the size of the first biocompatible long-fiber nonwoven fabric 3.

[0045] The first biocompatible long-fiber nonwoven fabrics 3a and 3b may be partially adhered to one surface of the cell sheets 2a and 2b, or they may be adhered to the entire surface. The cell sheets 2a and 2b may be partially adhered to each other or they may be adhered to the entire surface.

[0046] The first biocompatible long-fiber nonwoven fabrics 3a and 3b may have the same basis weight, thickness, average fiber diameter of the long fibers, etc., or they may be different.

[0047] Cell sheets 2a and 2b may be composed of the same type of cells, or they may be composed of different types of cells. Furthermore, cell sheets 2a and 2b may each be composed of one type of cell, or they may each be composed of two or more types of cells.

[0048] The diaphragm repair material 1 may further include, as necessary, other cell sheets in addition to cell sheets 2a and 2b, and it is preferable that the other cell sheets be positioned between cell sheets 2a and 2b. There may be one other cell sheet or two or more.

[0049] The diaphragm repair material 1 may further include, as necessary, other gelatin long fiber nonwoven fabrics in addition to the first biocompatible long fiber nonwoven fabrics 3a and 3b, and the other gelatin long fiber nonwoven fabrics may be placed between the cell sheets.

[0050] Figure 2 is a schematic cross-sectional view (a cross-section perpendicular to the thickness direction) of a diaphragm repair material according to one embodiment of the present invention. The diaphragm repair material 11 of this embodiment has the same configuration as the diaphragm repair material 1, except that the size of the second biocompatible long-fiber nonwoven fabric 14 is the same as the size of the first biocompatible long-fiber nonwoven fabric 3 in a cross-section perpendicular to the thickness direction.

[0051] Figure 3 is a schematic cross-sectional view (a cross-section perpendicular to the thickness direction) of a diaphragm repair material according to one embodiment of the present invention. The diaphragm repair material 21 of this embodiment has the same configuration as the diaphragm repair material 1, except that the size of the second biocompatible long-fiber nonwoven fabric 24 is larger than the size of the first biocompatible long-fiber nonwoven fabric 3 in a cross-section perpendicular to the thickness direction.

[0052] In the cross-section perpendicular to the thickness direction of the diaphragm repair material shown in Figures 1 to 3, the sizes of cell sheet 2a, cell sheet 2b, the first biocompatible long-fiber nonwoven fabric 3a, and the first biocompatible long-fiber nonwoven fabric 3b are the same, however the sizes of cell sheet 2a, cell sheet 2b and the first biocompatible long-fiber nonwoven fabric 3a and the first biocompatible long-fiber nonwoven fabric 3b may differ. Furthermore, the sizes of cell sheet 2a and cell sheet 2b may also differ. Furthermore, the sizes of the first biocompatible long-fiber nonwoven fabric 3a and the first biocompatible long-fiber nonwoven fabric 3b may also differ. For example, from the viewpoint of further improving angiogenesis and rhabdomyoogenesis, and further improving the repair of tissue in the defective portion of the diaphragm, it is preferable that the sizes of cell sheet 2a, cell sheet 2b, the first biocompatible long-fiber nonwoven fabric 3a, and the first biocompatible long-fiber nonwoven fabric 3b are the same in the cross-section perpendicular to the thickness direction of the diaphragm repair material.

[0053] In one or more embodiments of the present invention, the diaphragm repair material may be directly bonded to the gelatin long fiber nonwoven fabric and the cell sheet, or they may be bonded via an intervening substance, such as an extracellular matrix. Furthermore, in one or more embodiments of the present invention, the cell sheets may be directly bonded to each other, or they may be bonded via an intervening substance, such as an extracellular matrix.

[0054] <Method for manufacturing diaphragm repair material> In one or more embodiments of the present invention, the preparation of the diaphragm repair material includes the following steps, although this is not particularly limited. (1) Steps to prepare the cell sheet; (2) A step of laminating a cell sheet and a gelatin long fiber nonwoven fabric layer to obtain a first laminate; (3) A step of stacking two first laminates so that the cell sheets are in contact with each other to obtain a second laminate; (4) A step of laminating the second biocompatible long-fiber nonwoven fabric so as to be in contact with the first biocompatible long-fiber nonwoven fabric on one side of the second laminate.

[0055] In one or more embodiments of the present invention, a cell sheet can be obtained, for example, by adhering cells to a known culture method. The cell sheet can be removed from a culture vessel such as a culture dish by a known method. The culture vessel such as a culture dish is not particularly limited, but it is preferable to use a culture vessel that makes it easy to remove the cell sheet. As such a culture vessel, commercially available products such as temperature-responsive culture substrates manufactured by Cellseed Co., Ltd. may be used.

[0056] During cell culture, the culture medium is not particularly limited, and any medium containing the components necessary for cell survival and proliferation can be used as appropriate, depending on the type of cell. The medium may also contain serum, antibiotics, and growth factors. For example, bovine serum, fetal bovine serum, horse serum, human serum, etc. can be used as appropriate. For antibiotics, penicillin, streptomycin, gentamicin, amphotericin, ampicillin, minocycline, kanamycin, etc. can be used as appropriate. For growth factors, cell growth factors, differentiation-inducing factors, cell adhesion factors, etc. can be used as appropriate.

[0057] The number of cells to seed during cell culture should be determined appropriately depending on the type of cell, etc., but for example, 0.10 x 10 cells relative to the contact area of ​​the culture vessel. 5 Cells / cm 2 The above 0.60x10 5 Cells / cm 2 It may also be less than or equal to 0.20x10 5 Cells / cm 2 The above 0.50x10 5 Cells / cm 2 The following is also acceptable: 0.35x10 5 Cells / cm 2 More than 0.45x10 5 Cells / cm 2 The following is even more preferable:

[0058] Cell culture may be carried out at temperatures between 27°C and 40°C, or between 31°C and 37°C. The carbon dioxide concentration may be between 2% and 10%.

[0059] The culture time can be determined appropriately depending on the cell type, cell number, etc., but for example, it may be continued for 2 to 8 days, 3 to 7 days, or 4 to 6 days. The culture medium may be changed every 2 to 4 days.

[0060] A gelatin fiber nonwoven fabric, swollen with culture medium, is laminated onto one surface of a cell sheet detached from a culture vessel, without any culture medium present. By allowing it to stand for approximately 2 to 10 minutes at room temperature (20°C to 25°C), the gelatin fiber nonwoven fabric and the cell sheet are bonded together to obtain a first laminate. The gelatin fiber nonwoven fabric can be cut to a predetermined size to match the size of the cell sheet before use.

[0061] Two first laminates are prepared, and the two first laminates are stacked so that the cell sheets are in contact with each other. For example, by culturing them in the absence of culture medium, in an atmosphere of 2% to 10% carbon dioxide, at a temperature of 27°C to 40°C, preferably 35°C to 40°C, for about 60 minutes to 120 minutes, the cell sheets can be adhered to each other to obtain a second laminate.

[0062] A diaphragm repair material can be obtained by laminating a second biocompatible long-fiber nonwoven fabric so that it is in contact with one side of the first biocompatible long-fiber nonwoven fabric of the second laminate. If necessary, after laminating the second biocompatible long-fiber nonwoven fabric so that it is in contact with one side of the first biocompatible long-fiber nonwoven fabric of the second laminate, the second laminate and the second biocompatible long-fiber nonwoven fabric may be integrated using sutures or a biocompatible adhesive (such as fibrin glue) to obtain a diaphragm repair material, or the second laminate and the second biocompatible long-fiber nonwoven fabric may be integrated by suturing them together with absorbable sutures (also called absorbable threads) to obtain a diaphragm repair material.

[0063] In one or more embodiments of the present invention, in the case of a disease having a defect in the diaphragm, such as a congenital diaphragmatic hernia, the diaphragmatic repair material can be positioned so that the first biocompatible long-fiber nonwoven fabric is closest to the tissue, and the diaphragmatic repair material and the diaphragm can be sutured together to close the defect. [Examples]

[0064] The present invention will be described in more detail below using examples. However, the present invention is not limited to the following examples.

[0065] (Manufacturing Example 1) Using polyglycolic acid (hereinafter also referred to as PGA) filament (multifilament yarn, 10 filaments, single fiber fineness 4.6 dtex, total fineness 46 dtex), knitted on a tubular knitting machine (weight 12 g / m). 2 A 0.25mm thick knit was produced. The resulting knit was laid flat, and three layers were stacked to prevent wrinkles. Needle punching was then performed using a needle punching machine until the fibers were intertwined and difficult to unravel, thus producing a nonwoven fabric. Next, heat setting was performed to fix the expansion and contraction of the nonwoven fabric. Then, heat pressing was performed using a press machine to correct the thickness, resulting in a basis weight of 70g / m². 2 A 0.3mm thick PGA long-fiber needle-punched nonwoven fabric was produced.

[0066] (Manufacturing example 2) Except for using five layers of knit fabric, the manufacturing process was the same as in Example 1, with a weight of 120g / m². 2 A 0.4mm thick PGA long-fiber needle-punched nonwoven fabric was produced.

[0067] (Manufacturing Example 3) Except for using eight layers of knit fabric, the manufacturing process was the same as in Example 1, with a weight of 180g / m². 2 A 0.6mm thick PGA long-fiber needle-punched nonwoven fabric was produced.

[0068] (Reference example 1) Non-absorbable hernia, chest wall, and abdominal wall prosthetic material made from polytetrafluoroethylene (hereinafter also referred to as PTFE) (weight 360g / m²) 2It uses a 1.0mm thick fabric manufactured by WLGore&Associates GK (Japan Gore G.K.), product name "Dual Mesh".

[0069] (Reference example 2) Absorbable tissue reinforcement material made from PGA (basis weight 35g / m²) 2 A sheet with a thickness of 0.15 mm, manufactured by Gunze Medical Japan Co., Ltd., product name "NeoVeil (registered trademark)", 015G sheet type was used.

[0070] Tensile and stretch tests were performed on the PGA long-fiber needle-punched nonwoven fabrics of Manufacturing Examples 1-3, the PTFE dual mesh of Reference Example 1, and the structural reinforcement material of Reference Example 2, as described below. Tensile strength, tensile modulus, 50% elongation at 50 cycles, and 25% elongation at 100 cycles were measured. The results are shown in Table 1 below.

[0071] (Tensile test) Using a test specimen measuring 1 cm in width and 8 cm in length, with a grip width of 2 cm and a gripping distance of 4 cm, the specimen was pulled at a tensile speed of 100 mm / min using an Autograph (Shimadzu Corporation, model number "AGS-X, 10N-10kN") until fracture, and a tensile stress-strain curve was obtained. The tensile strength at fracture was used as the tensile strength, and the tensile modulus was determined from the obtained tensile stress-strain curve by linear regression between strains of 0.05 and 0.25%, according to ISO 527-1, as shown in formula (2) below. Three samples were taken from each of the longitudinal and transverse directions of the test specimen, and the average of the total six measurements was calculated. E=(σ2−σ1) / (ε2−ε1)…(2) However, in equation (2), E represents the tensile modulus, σ1 represents the tensile stress measured at strain ε1 = 0.05%, and σ2 represents the tensile stress measured at strain ε2 = 0.25%.

[0072] (Stretch Test 1) Using a test specimen measuring 1 cm in width and 8 cm in length, with a grip width of 2 cm and a gripping distance of 4 cm, the elongation rate was measured after 50 reciprocating movements of pulling the specimen 2 cm back and forth at a tensile speed of 100 mm / min using an Autograph (Shimadzu Corporation, model number "AGS-X, 10N-10kN"). The test specimens were sampled three times in both the longitudinal and transverse directions, and the average of the six measurements was calculated.

[0073] (Stretch Test 2) Using a test specimen measuring 1 cm in width and 8 cm in length, with a grip width of 2 cm and a gripping distance of 4 cm, the elongation rate was measured after 100 reciprocating movements of pulling the specimen 1 cm back and forth at a tensile speed of 100 mm / min using an Autograph (Shimadzu Corporation, model number "AGS-X, 10N-10kN"). The test specimens were sampled three times in both the longitudinal and transverse directions, and the average of the six measurements was calculated.

[0074] [Table 1]

[0075] As can be seen from the data in Table 1, the PGA long-fiber needle-punched nonwoven fabrics of manufacturing examples 1 to 3 have a tensile modulus of elasticity of 1.0 MPa to 3.4 MPa, exhibiting good sutureability when suturing to the diaphragm. Furthermore, after suturing to the diaphragm and closing the defect, they readily follow the expansion and contraction of the diaphragm. This suggests that using the PGA long-fiber needle-punched nonwoven fabric in combination with fiber sheets and gelatin long-fiber nonwoven fabric as a diaphragm repair material may repair the tissue in the diaphragm defect and improve the recurrence of the defect.

[0076] As can be seen from the data in Table 1, the PGA long-fiber needle-punched nonwoven fabrics of manufacturing examples 1-3 have a tensile strength of 18N or higher, do not tear when sutured to the diaphragm, and can further increase suture strength.

[0077] On the other hand, the PTFE dual mesh in Reference Example 1, which is used as an artificial fabric, has a tensile modulus of elasticity exceeding 3.4 MPa, making it difficult to follow the growth and expansion of the diaphragm after it has been sutured to close the defect. The tissue reinforcement material in Reference Example 2 has a tensile modulus of elasticity of less than 1.0 MPa, making it difficult to suture to the diaphragm, and its low tensile strength makes it prone to tearing when sutured to the diaphragm.

[0078] (Example 1) <Collection and primary culture of adipocytes> Rats were used as the animals. Under anesthesia, a midline incision was made in the lower abdomen, and adipose tissue (0.5-1.0 g) was excised. The excised adipose tissue was transferred to αMEM and finely chopped. Then, the adipose tissue was transferred to HBSS containing 0.1% collagenase and swirled at approximately 100 rpm at 37°C for 60-90 minutes until no visible solid adipose tissue remained. The enzyme-treated cell solution was transferred to a centrifuge tube. After centrifugation at 1200 rpm for 10 minutes, the supernatant was removed. The cells accumulated in the lower layer were suspended in fresh culture medium and seeded in a 10 cm culture dish. The culture dish was incubated under conditions of 5% CO2 and 37°C for 5 to 7 days until confluence was reached, during primary culture. During the primary culture period, the entire culture medium was changed every two days, and cells that did not adhere to the culture dish (hematologic cells, dead cells, etc.) were removed. Cells that adhered to and spread in a culture dish and proliferated were treated as adipocyte-derived cells. <Cell sheet preparation> Adipose-derived cells (P6-7) that had reached confluence after primary culture and subculturing were treated with trypsin and harvested from the culture dish. After centrifugation at 1300 rpm for 10 minutes, the supernatant was removed and the cells were resuspended in fresh medium. Cell counts were performed on the cell suspension, resulting in 5 × 10⁶ cells. 6 A cell suspension was prepared in fresh medium to a cell / mL concentration of 5.0 × 10⁶ cells. 6 Adipose-derived stem cells (ASCs) were labeled using the Cellview® Claretch Far-Infrared Fluorescent Cell Linker Mini Kit for general membrane labeling (Sigma-Aldrich), and then seeded in a 6 cm temperature-responsive culture dish. 3 mL of fresh medium was added, and the culture was incubated at 5% CO2 and 37°C for 10 days until overconfluence was reached. During this time, the medium was completely replaced every two days. <Fabrication of diaphragm repair material> After adipose-derived cells became overconfluent in a 6 cm temperature-responsive culture dish, the culture medium was removed, and the temperature at the bottom of the culture dish was lowered to 20-25°C. The culture dish was left to stand for 20 minutes, and the adipose-derived cells were detached from the culture dish and released into a sheet. The diameter of the cell sheet at this time was approximately 35 mm. A gelatin long-fiber nonwoven fabric (Genocel®, sheet type, manufactured by Nikke Medical Co., Ltd.), swollen with culture medium, was laminated onto one surface of the obtained cell sheet to adhere the gelatin long-fiber nonwoven fabric to the cell sheet, and the first laminate of gelatin long-fiber nonwoven fabric and cell sheet was recovered. The average fiber diameter of the gelatin long-fiber nonwoven fabric when swollen was 47-50 μm, the diameter was approximately 35 mm, and the thickness was approximately 450 μm. Two of the recovered first laminates were laminated so that the surfaces of the cell sheets overlapped. The laminate was placed in a 10 cm culture dish and incubated for 60 minutes at 5% CO2 and 37°C without any culture medium, so that the cell sheets would adhere to each other, to obtain a second laminate. To the second laminate obtained above, 10-12 mL of fresh medium was added, and the cells were cultured for 2-3 days without changing the medium. After 3 days of culture, the thickness of the cell layer sandwiched between gelatin long fiber nonwoven fabric immediately before transplantation was approximately 200 μm. Subsequently, the PGA long-fiber needle-punched nonwoven fabric (approximately 20 mm in diameter) from Production Example 2 was laminated so as to be in contact with the first biocompatible long-fiber nonwoven fabric on one side of the second laminate, and the two layers were sutured together with 3 stitches using 5-0 monofilament absorbable sutures to obtain a diaphragmatic repair material having the structure shown in Figure 1. Note that the absorbable sutures are not shown in Figure 1. <Diaphragm repair experiment> 15-20 week old male F344 / NSlc rats were used. A triple-component anesthetic was administered intraperitoneally, followed by endotracheal intubation. Under mechanical ventilation, a laparotomy was performed in the left upper abdomen, and a portion of the left diaphragm was resected (allowing for visualization of the lung) to create a diaphragmatic hernia model. The diaphragmatic repair material obtained above was placed in the diaphragmatic defect of the rat with the gelatin long fiber nonwoven fabric 3b side facing the nearest tissue. The diaphragmatic repair material and the diaphragm were sutured together with 3 stitches using 5-0 monofilament nonabsorbable sutures, and the abdomen was closed.

[0079] (Comparative Example 1) A laminate was obtained by stacking two gelatin long-fiber nonwoven fabrics swollen with the same culture medium as used in Example 1. Next, the laminate was placed in a 10 cm culture dish and incubated for 90 minutes at 5% CO2 and 37°C without any culture medium. Then, the PGA long-fiber needle-punched nonwoven fabric (approximately 20 mm in diameter) from Production Example 2 was laminated so as to be in contact with one of the first biocompatible long-fiber nonwoven fabrics, and the layers were sutured together with 3 stitches using 5-0 monofilament absorbable sutures to obtain a diaphragm repair material. A diaphragm repair experiment was conducted in the same manner as in Example 1, except that the diaphragm repair material was used.

[0080] (Comparative Example 2) A laminate was obtained by stacking two gelatin long-fiber nonwoven fabrics swollen with the same culture medium as used in Example 1. Next, the laminate was placed in a 10 cm culture dish and incubated for 90 minutes at 5% CO2 and 37°C without any culture medium. Then, a tissue reinforcement material (approximately 30 mm in diameter) made of PGA from Reference Example 2 was stacked so as to be in contact with one side of the first biocompatible long-fiber nonwoven fabric, and the two layers were sutured together with 3 stitches using 5-0 monofilament absorbable sutures to obtain a diaphragm repair material. When attempting to suture the diaphragm repair material to the diaphragm using 5-0 monofilament nonabsorbable sutures, after placing the diaphragm repair material in the diaphragm defect of a rat with the gelatin long-fiber nonwoven fabric side not in contact with the PGA long-fiber needle-punched nonwoven fabric being closest to the tissue, the diaphragm repair material tore, and suturing was not possible.

[0081] (Diaphragmatic histological evaluation 1) In Example 1, two weeks after the diaphragm repair experiment, the animals were anesthetized and the diaphragms were removed. 5 mm paraffin sections of the diaphragm were prepared, stained with hematoxylin and eosin (HE), and the area around the diaphragm defect was observed. In addition, 5 mm paraffin sections of the diaphragm were prepared, and the nuclei were stained with DAPI before observing adipose-derived stem cells. The results are shown in Figure 4.

[0082] In Figure 4, A is a cross-sectional image of the diaphragmatic defect, B and C are DAPI-stained images of the area near the diaphragmatic defect, and D is a magnified view of the area enclosed by a rectangle in C. In Figures B to D, adipose-derived stem cells are stained red. From Figure 4, it can be seen that the diaphragmatic repair material 1 has closed the diaphragmatic defect 50, and adipose-derived stem cells (one example is indicated by an arrow) have gathered towards the diaphragmatic defect 50, suggesting the possibility of regeneration.

[0083] (Diaphragmatic histological evaluation 2) In both Example 1 and Comparative Example 1, 5 mm paraffin sections of the diaphragm were prepared two weeks after the diaphragm repair experiment and stained with HE. The results are shown in Figure 5.

[0084] In Figure 5, A and B are the HE staining results of tissue sections from Comparative Example 1, and C and D are the HE staining results of tissue sections from Example 1. In Figure 5, by comparing A and B with C and D, it can be seen that in Comparative Example 1, which uses a diaphragm repair material that does not contain stem cells, the gelatin long fiber nonwoven fabric 10 is coarse, while in Example 1, which uses a diaphragm repair material that contains stem cells, the gelatin long fiber nonwoven fabric 10 is dense. Furthermore, it can be seen that Example 1, which uses a diaphragm repair material that contains stem cells, has improved binding to the diaphragm 30.

[0085] (Diaphragmatic histological evaluation 3) Angiogenesis was evaluated using an anti-CD31 antibody. In both Example 1 and Comparative Example 1, 5 mm paraffin sections of the diaphragm were prepared two weeks after the diaphragm repair experiment. Vascular endothelial cells were immunohistochemically stained with CD31 antibody, and the nuclei were stained with DAPI. The results are shown in Figure 6.

[0086] In Figure 6, A is a fluorescent staining image of a tissue section from Comparative Example 1, and B is a fluorescent staining image of a tissue section from Example 1. In Figure 6, blue indicates cells stained with DAPI, red indicates CD31-stained CD31-positive cells (thick arrows), i.e., angiogenesis, and adipose-derived stem cells (thin arrows) are stained light blue. In Figure 6B, there are many red CD-positive cells, confirming angiogenesis.

[0087] (Diaphragmatic histological evaluation 4) Rhabdomyolysis was evaluated using rhabdomyolysis markers. In both Example 1 and Comparative Example 1, 5 mm paraffin sections of the diaphragm were prepared 2 weeks after the diaphragm repair experiment, and the rhabdomyolysis was immunohistochemically stained with anti-desmin antibody. The nuclei were also stained with DAPI. In Example 1, 5 mm paraffin sections of the diaphragm were prepared 4 weeks after the diaphragm repair experiment, and the rhabdomyolysis was immunohistochemically stained with anti-myoglobin antibody. The nuclei were also stained with DAPI. Desmin is a marker for immature rhabdomyolysis, and myoglobin is a marker for mature rhabdomyolysis. The results are shown in Figure 7.

[0088] In Figure 7, A is a fluorescently stained photograph of a tissue section from Comparative Example 1, B is a fluorescently stained photograph of a tissue section from Example 1 two weeks after the diaphragm repair experiment, and C is a fluorescently stained photograph of a tissue section from Example 1 four weeks after the diaphragm repair experiment. In Figures 7A and 7B, blue indicates cells stained with DAPI, green indicates desmin-positive cells, adipose-derived stem cells (thin arrows) are stained light blue, and red indicates αSMA (α-smooth muscle actin). In Figure 7C, blue indicates cells stained with DAPI, green indicates myoglobin-positive cells, and red indicates αSMA (α-smooth muscle actin).

[0089] In Example 1, as shown in Figure 7B, two weeks after the diaphragm repair experiment, areas stained green and areas positive for desmin, a marker of immature striated muscle, were observed within the gelatin long fiber nonwoven fabric 10. As shown in Figure 7C, four weeks after the diaphragm repair experiment, areas positive for myoglobin, a marker of striated muscle, were observed. These findings suggest that new muscle tissue is being generated.

[0090] As described above, by using the diaphragmatic repair material of the present invention, it is possible to close the diaphragmatic defect with good suture properties, and tissue repair can be promoted in the diaphragmatic defect, thereby improving the recurrence of the defect. [Explanation of Symbols]

[0091] 1 Diaphragm repair material 2 cell layers 2a, 2b Cell Sheets 3, 3a, 3b, 10 First biocompatible long-fiber nonwoven fabric (gelatin long-fiber nonwoven fabric) 4.20 Second Biocompatible Long Fiber Nonwoven Fabric 30 Diaphragm 50 Diaphragmatic defect

Claims

1. A diaphragmatic repair material used to repair defects in the diaphragm, The diaphragm repair material includes a cell layer, a first biocompatible long-fiber nonwoven fabric positioned on both sides of the cell layer, and a second biocompatible long-fiber nonwoven fabric positioned in contact with one side of the first biocompatible long-fiber nonwoven fabric. The aforementioned cell layer includes stem cells, The first biocompatible long-fiber nonwoven fabric is a gelatin long-fiber nonwoven fabric with gelatin as its main component. The second biocompatible long-fiber nonwoven fabric is a diaphragm repair material characterized by having a tensile modulus of elasticity of 1.0 MPa or more and 3.4 MPa or less.

2. The diaphragm repair material according to claim 1, wherein the second biocompatible long-fiber nonwoven fabric has a tensile strength of 18 N or more.

3. The diaphragm repair material according to claim 1, wherein the second biocompatible long-fiber nonwoven fabric is a polyglycolic acid long-fiber nonwoven fabric mainly composed of polyglycolic acid.

4. The diaphragm repair material according to claim 1, wherein the cell layer includes adipose tissue-derived stem cells.

5. The diaphragm repair material according to claim 1, wherein the cell layer comprises two or more cell sheets.

6. The diaphragm repair material according to claim 1, wherein the second biocompatible long-fiber nonwoven fabric has an elongation rate of 100% or more and 120% or less after being subjected to a stretch test 100 times at a strain rate of 25%.

7. The diaphragm repair material according to claim 1, wherein the second biocompatible long-fiber nonwoven fabric has an elongation rate of 100% or more and 140% or less after being subjected to a repeated stretch test 50 times at a strain rate of 50%.

8. A method for manufacturing a diaphragm repair material according to any one of claims 1 to 7, The process of preparing the cell sheet, A step of obtaining a first laminate by laminating a first biocompatible long-fiber nonwoven fabric onto one surface of a cell sheet, A step of stacking two first laminates so that the cell sheets are in contact with each other to obtain a second laminate, and A method for manufacturing a diaphragm repair material, comprising the step of laminating a second biocompatible long-fiber nonwoven fabric so as to be in contact with one side of a second laminate of a first biocompatible long-fiber nonwoven fabric.

9. The method for producing a diaphragm repair material according to claim 8, wherein the second biocompatible long-fiber nonwoven fabric is obtained by needle punching a woven or knitted fabric composed of biocompatible long fibers.

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