Fibrocartilaginous tissue reconstruction graft material and manufacturing method therefor

Abstract

The present invention relates to a fibrocartilaginous tissue reconstruction graft material for reconstructing fibrocartilaginous transitional zones, and a manufacturing method therefor. The fibrocartilaginous tissue reconstruction graft material has a cylindrical structure, and thus facilitates fastening to implantable tendons or ligaments and implantation into bone tunnels, and has a biphasic structure of calcified and non-calcified tissues, and thus can simulate the hierarchical structure of the tendon-bone interface (TBI) tissue.

Description

Graft material for reconstructing fibrocartilaginous tissue and method for manufacturing the same

[0001] The present invention provides a graft material for reconstructing fibrocartilage tissue for reconstructing a fibrocartilage transition area and a method for manufacturing the same.

[0002] This invention was conducted as a research (22C0601L1-11) supported by the Inter-Ministry Regenerative Medicine Technology Development Project with funding from the government (Ministry of Science and ICT, Ministry of Health and Welfare).

[0003] The tendon-bone interface (TBI) between the bone and the ligament is composed of a gradient transitional structure of bone, calcified fibrocartilage, non-calcified fibrocartilage, and ligament. This tendon-bone interface (TBI) acts as a buffer for the different mechanical properties between the bone and the ligament, and serves as a mediator for biological interactions.

[0004] In cases of ligament damage such as anterior cruciate ligament rupture, reconstruction surgery is currently being performed by drilling a tunnel into the bone tissue and inserting an autologous or allograft tendon to simply fix the ligament to the bone tissue.

[0005] However, when performing ligament reconstruction as described above, it is difficult to reconstruct the TBI hierarchical structure, and in most cases, scar tissue forms in the bone-ligament interstitial tissue. Scar tissue formation due to TBI causes excessive fibrous tissue formation, making it difficult to buffer physical energy between the two tissues and hindering biological regeneration. Ultimately, existing surgical methods have difficulty replicating TBI scar tissue formation and the TBI hierarchical structure, making them the primary biological cause of re-rupture after ligament reconstruction.

[0006] To overcome these shortcomings, there is a need to develop medical technologies that can mimic the hierarchical structure of TBI.

[0007] The purpose of the present invention is to provide a graft material for reconstructing fibrocartilage tissue capable of simulating the hierarchical structure between bone and ligament, and a method for manufacturing the same.

[0008] In order to achieve the above object, the present invention provides a graft for reconstructing fibrocartilage tissue between a bone and a ligament or tendon, comprising: a porous demineralized bone matrix support having a tube structure having a through hole through which a ligament or tendon fiber bundle for transplantation passes; a first hydrogel composition filled in the pores of a first region of the porous demineralized bone matrix support surrounding the through hole and containing an extracellular matrix derived from autologous or heterologous fibrocartilage; and a second hydrogel composition filled in the pores of a second region of the porous demineralized bone matrix support surrounding the first region and containing an extracellular matrix derived from autologous or heterologous fibrocartilage and a calcification-inducing substance.

[0009] In addition, the present invention provides a method for manufacturing a graft for reconstructing fibrous cartilage tissue between a bone and a ligament or tendon, comprising the steps of: manufacturing a porous demineralized bone matrix support having a tube structure having a through hole through which a ligament or tendon fiber bundle for transplantation passes; manufacturing a first hydrogel composition including an extracellular matrix derived from autologous or heterologous fibrocartilage, and a second hydrogel composition including an extracellular matrix derived from autologous or heterologous fibrocartilage and a calcification-inducing substance, respectively; And a step of filling the first and second hydrogel compositions into the porous demineralized bone matrix support, respectively, and the step of filling the first and second hydrogel compositions into the porous demineralized bone matrix support, respectively, is characterized in that the first hydrogel composition is filled into the pores of the first region of the porous demineralized bone matrix support surrounding the through hole and crosslinked, and the second hydrogel composition is filled into the pores of the second region of the porous demineralized bone matrix support surrounding the first region and crosslinked.

[0010] In addition, the present invention provides a method for manufacturing a graft material for reconstructing fibrocartilage tissue, comprising the steps of: manufacturing first and second porous demineralized bone matrix scaffolds having a tube structure having through holes having different diameters; manufacturing a first hydrogel composition comprising an extracellular matrix derived from autologous or heterologous fibrocartilage and a second hydrogel composition comprising an extracellular matrix derived from autologous or heterologous fibrocartilage and a calcification-inducing substance, respectively; filling the first hydrogel composition into a first tube structure formed of a porous demineralized bone matrix scaffold and crosslinking the first hydrogel composition, filling the second hydrogel composition into a second tube structure formed of a porous demineralized bone matrix scaffold and having an inner diameter and an outer diameter larger than those of the first tube structure, and crosslinking the second hydrogel composition; and assembling the first tube structure so as to be positioned inside the second tube structure.

[0011] In addition, the present invention provides a method for reconstructing fibrocartilage tissue between a bone and a ligament or tendon, comprising the steps of: forming a tibial tunnel penetrating the tibia; forming a femoral tunnel disposed parallel to the tibial tunnel and penetrating the femur; fixing a ligament or tendon for transplantation to the fibrocartilage tissue reconstruction graft described above; and positioning the tendon or ligament for transplantation fixed to the fibrocartilage tissue reconstruction graft in the tibial tunnel and the femoral tunnel.

[0012] The graft material for fibrocartilage tissue reconstruction according to the present invention has a cylindrical structure, which makes it easy to connect with a tendon or ligament for transplantation and to transplant it into a bone tunnel, and has a biphasic structure of calcified and non-calcified tissue, which can simulate the hierarchical structure of the trabecular bone-ligament interstitial tissue (TBI).

[0013] Figure 1 is a schematic diagram illustrating a method for manufacturing a graft material for reconstructing fibrocartilage tissue according to one embodiment of the present invention (1-3) and a method for reconstructing fibrocartilage tissue between a bone and a ligament or tendon (4-5).

[0014] FIG. 2 is a graph showing the results of evaluating the physicochemical properties of demineralized bone matrix in a graft for fibrocartilage tissue reconstruction according to one embodiment of the present invention, wherein (A) is a gross image, (B) is a scanning electron microscope (SEM) image, (C) is a graph showing the results of energy dispersive X-ray spectroscopy (EDS), (D) is a graph showing DNA content, (E) is a graph showing sulfated glycosaminoglycan (sGAG) content, and (F) is a graph showing collagen content.

[0015] FIG. 3 is a graph showing the results of evaluating the physicochemical properties of a meniscus-derived extracellular matrix (DMECM) hydrogel in a graft for fibrocartilage tissue reconstruction according to one embodiment of the present invention, in which (A) is a photograph (Gross image) and a scanning electron microscope (SEM) image, (B) is the result of SDS PAGE analysis, (C) is the dsDNA content, (D) is the collagen content, and (E) is the sulfated glycosaminoglycan (sGAG) content.

[0016] FIG. 4 is a graph showing the results of a concentration optimization experiment of a meniscus-derived extracellular matrix (DMECM) hydrogel in a graft for fibrous cartilage tissue reconstruction according to one embodiment of the present invention. (A) Graphs showing morphological changes according to concentration changes, (B) Graphs showing changes in elastic modulus according to temperature changes according to concentration changes, and (C) Graphs showing changes in storage modulus and loss modulus according to concentration changes.

[0017] FIG. 5 is a graph showing the results of a characteristic evaluation according to a method of filling a demineralized bone matrix support with a meniscus-derived extracellular matrix (DMECM) hydrogel in a graft for fibrocartilage tissue reconstruction according to an embodiment of the present invention. (A) is an image of the result of the stirring method after immersion, (B) is an image after dispensing according to hydrogel concentration, (C) is an image of the 3D printing method, (D) is a graph showing the collagen content according to hydrogel concentration, and (E) is a graph showing the sulfated glycosaminoglycan (sGAG) content according to hydrogel concentration.

[0018] FIG. 6 is a graph showing the results of evaluating the extracellular matrix content in a demineralized bone matrix support of a meniscus-derived extracellular matrix (DMECM) hydrogel in a graft for fibrocartilage tissue reconstruction according to one embodiment of the present invention. (A) is a photographic image and a scanning electron microscope (SEM) image according to the number of hydrogel fillings, (B) is a graph showing the collagen content according to the number of fillings, and (E) is a graph showing the sulfated glycosaminoglycan (sGAG) content according to the number of fillings.

[0019] FIG. 7 is a graph showing the results of in vitro fibrocartilage differentiation of a demineralized bone matrix scaffold according to the concentration of meniscus-derived extracellular matrix (DMECM) hydrogel in a graft for fibrocartilage tissue reconstruction according to one embodiment of the present invention. (A) is a histological analysis result image according to the concentration of DMECM hydrogel, (B) is a graph showing the collagen content according to the concentration of DMECM hydrogel, and (E) is a graph showing the sulfated glycosaminoglycan (sGAG) content according to the concentration of DMECM hydrogel.

[0020] FIG. 8 is a graph showing the results of evaluating the physicochemical properties of a fibrous cartilage tissue reconstruction graft (DMECM-DBM scaffold) according to a cross-linking method in a method for manufacturing a fibrous cartilage tissue reconstruction graft according to one embodiment of the present invention, where (A) is a graph showing compressive modulus, (B) is a graph showing swelling ratio, and (C) is a graph showing biodegradability.

[0021] FIG. 9 is a graph showing the results of evaluating the cell affinity and fibrocartilage differentiation induction characteristics of a fibrocartilage tissue reconstruction graft (DMECM-DBM scaffold) according to a cross-linking method in a method for manufacturing a fibrocartilage tissue reconstruction graft according to an embodiment of the present invention, in which (A) is a graph showing cell proliferation capacity, (B) type 1 collagen (COL1), (C) type 2 collagen (COL2), (D) aggrecan (ACAN), and (E) changes in gene expression of SOX9.

[0022] FIG. 10 is a graph showing the results of evaluating the physicochemical properties of musculoskeletal tissue-derived extracellular vesicles (TEVs) in a graft for fibrocartilage tissue reconstruction according to one embodiment of the present invention, in which (A) and (B) are SEM images, (C) particle size distribution, and (D) Western blot analysis results.

[0023] Figure 11 shows the results of evaluating the calcification-inducing properties of musculoskeletal tissue-derived extracellular vesicles (TEVs) in a graft for fibrocartilage tissue reconstruction according to one embodiment of the present invention. (A) and (B) are the results of evaluating the calcification-inducing properties according to the concentration of the extracellular vesicles treated, and (C) and (D) are the results of evaluating the calcification-inducing properties according to the origin of the extracellular vesicles.

[0024] FIG. 12 shows the results of an evaluation of the in vitro calcification induction ability of a graft material for fibrocartilage tissue reconstruction (DMECM / TEVs-DBM scaffold) according to one embodiment of the present invention. (A) is a graph showing the results of scanning electron microscopy (SEM) and energy dispersive spectroscopy (EDS), (B) is a table showing the results of energy dispersive spectroscopy (EDS), and (C) is a graph showing cell gene expression (RT-PCR).

[0025] FIG. 13 shows the results of physicochemical evaluation according to the manufacturing method of a graft material for fibrocartilage tissue reconstruction (DMECM / TEVs-DBM scaffold) according to one embodiment of the present invention, (A) is an image of a single porous demineralized bone matrix scaffold and a method for manufacturing a graft material through a 3D printing method, (B) is an image of a double porous demineralized bone matrix scaffold and a method for manufacturing a graft material through an immersion method, (C) is a graph of collagen content and sulfated glycosaminoglycan (sGAG) content, and (D) is a graph of compressive modulus.

[0026] FIG. 14 shows the results of evaluating the effectiveness of tissue reconstruction in a rabbit bone-ligament injury model of a fibrocartilage tissue reconstruction graft (DMECM / TEVs-DBM scaffold) according to one embodiment of the present invention. (A) is a graph showing the results of Safranin-O histological staining, (B) is a graph showing the results of quantitative analysis of the histological effectiveness of tissue reconstruction according to the concentration of DMECM hydrogel, and (C) is a graph showing the results of maximum tensile strength measurement according to the concentration of DMECM hydrogel.

[0027] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. The present invention can be modified in various ways and can take various forms, and thus specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific disclosed form, but should be understood to include all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing each drawing, similar reference numerals are used to indicate similar components. In the attached drawings, the dimensions of structures are shown larger than actual size to ensure clarity of the present invention.

[0028] While terms like "first" and "second" may be used to describe various components, these components should not be limited by these terms. These terms are used solely to distinguish one component from another. For example, without departing from the scope of the present invention, a first component could be referred to as a "second component," and similarly, a second component could also be referred to as a "first component."

[0029] The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this application, it should be understood that the terms "comprise" or "have" indicate the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but do not preclude the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0030] Meanwhile, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning within the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0031]

[0032] The present invention provides a graft for reconstructing fibrocartilage tissue between a bone and a ligament or tendon, comprising: a porous demineralized bone matrix support having a tube structure having a through hole through which a ligament or tendon fiber bundle for transplantation passes; a first hydrogel composition filled in the pores of a first region of the porous demineralized bone matrix support surrounding the through hole and containing an extracellular matrix derived from autologous or heterologous fibrocartilage; and a second hydrogel composition filled in the pores of a second region of the porous demineralized bone matrix support surrounding the first region and containing an extracellular matrix derived from autologous or heterologous fibrocartilage and a calcification-inducing substance.

[0033] The above porous demineralized bone matrix support may include cancellous bone, compact bone or a combination thereof, and specifically, may include both cancellous bone and compact bone, but is not limited thereto.

[0034] The above porous demineralized bone matrix support may be in the form of a layered structure in which compact bone and cancellous bone are combined, and the compact bone and cancellous bone may be combined at an average thickness ratio of 1:2 to 5, but is not limited thereto.

[0035] According to the present invention, it can be confirmed that the compressive strength and tensile strength of a demineralized bone matrix support containing both cancellous bone and compact bone are significantly increased compared to a demineralized bone matrix support containing only cancellous bone.

[0036] The porous demineralized bone matrix support may have a tube structure having a through hole through which a graft ligament or tendon fiber bundle passes, and may include a first region surrounding the through hole, and a second region surrounding the first region.

[0037] Specifically, the first region is a portion that surrounds a through hole through which a graft ligament or tendon fiber bundle passes and can induce non-calcified fibrocartilaginous tissue by including a first hydrogel composition, and the second region surrounds the first region and can induce calcified fibrocartilaginous tissue by including a second hydrogel composition.

[0038] More specifically, the outer diameter of the first region may be on average 6.5 to 14 mm, 8 to 12.5 mm or 9.5 to 11 mm, the diameter of the through hole may be on average 6 to 13 mm, 7.5 to 11.5 mm or 9 to 10 mm, and the outer diameter of the second region may be on average 7 to 15 mm, 8.5 to 13.5 mm or 10 to 12 mm.

[0039] For example, the thicknesses of the first and second regions may be independently from each other an average of 0.2 to 3 mm, 0.3 to 2 mm, or 0.5 to 1 mm. The thickness refers to the difference between the outer diameter and the inner diameter of each region.

[0040] In addition, the porous demineralized bone matrix support may have a tube structure, and the length of the porous demineralized bone matrix support may be on average 20 to 60 mm, 25 to 55 mm, or 30 to 50 mm.

[0041] The graft material for fibrocartilage tissue reconstruction according to the present invention has the advantage of facilitating connection with a ligament or tendon for transplantation and being convenient for transplantation into a bone tunnel, as the porous demineralized bone matrix support has a tube (cylindrical) structure as described above.

[0042] According to one embodiment of the present invention, the biphasic structure of the porous demineralized bone matrix support can be achieved by filling the first hydrogel composition and the second hydrogel composition into the first region and the second region, respectively.

[0043] The first hydrogel composition is filled within the pores of the first region of the porous demineralized bone matrix support and may include an extracellular matrix derived from autologous or heterologous fibrocartilage.

[0044] The above autologous or xenogeneic fibrocartilage-derived extracellular matrix may be a xenogeneic fibrocartilage-derived extracellular matrix. For example, the fibrocartilage-derived extracellular matrix may be a meniscus tissue as a fibrocartilage composite tissue.

[0045] Specifically, the first hydrogel composition may comprise 0.5 to 10 w / v%, 0.5 to 7 w / v%, 1 to 5 w / v%, 3 to 4 w / v%, or 1 to 2 w / v% of an extracellular matrix derived from autologous or heterologous fibrocartilage.

[0046] According to one embodiment of the present invention, the first hydrogel composition may include an autologous or heterologous cartilage-derived extracellular matrix as described above, and may have a temperature sensitivity that allows it to exist in a liquid state at room temperature and to gel at a temperature of 37°C or higher, preferably 37°C to 45°C, thereby maintaining a specific shape.

[0047] The second hydrogel composition is filled within the pores of the second region of the porous demineralized bone matrix support and may include the autologous or heterologous cartilage-derived extracellular matrix and a calcification-inducing substance.

[0048] The above-mentioned calcification-inducing substance may include at least one selected from the group consisting of musculoskeletal tissue-derived extracellular vesicles (TEVs), bone morphogenetic protein-2 (BMP-2), and hydroxyapatite (HA). Specifically, the above-mentioned calcification-inducing substance may be musculoskeletal tissue-derived extracellular vesicles (TEVs). More specifically, the musculoskeletal tissue may be composed of cartilage tissue, bone tissue, and semicircular cartilage tissue.

[0049] The particle size of the above calcification-inducing material may be on average 10 to 1000 nm, 50 to 1000 nm, 100 to 1000 nm or 200 to 800 nm.

[0050] Specifically, the second hydrogel composition may comprise 0.5 to 10 w / v%, 0.5 to 7 w / v%, 1 to 5 w / v%, 3 to 4 w / v% or 1 to 2 w / v% of the autologous or heterologous cartilage-derived extracellular matrix, and may comprise 0.01 to 500 μg / ml, 1 to 500 μg / ml, 100 to 500 μg / ml or 10 to 100 μg / ml of the calcification-inducing substance.

[0051] The second hydrogel composition may include a 2-1 hydrogel composition filled in pores of a 2-1 region of the porous demineralized bone matrix support adjacent to the first region among the second regions, and containing the calcification-inducing substance at a first concentration; and a 2-2 hydrogel composition filled in pores of a 2-2 region of the porous demineralized bone matrix support surrounding the 2-1 region among the second regions, and containing the calcification-inducing substance at a second concentration higher than the first concentration.

[0052] According to one embodiment of the present invention, the second hydrogel composition may include an autologous or heterologous cartilage-derived extracellular matrix as described above, and may have thermosensitivity to exist in a liquid state at room temperature, gel at a temperature of 37°C or higher, preferably 37°C to 45°C, and maintain a specific shape, and may include a calcification-inducing substance as described above to increase mineral levels such as calcium and phosphorus, thereby inducing bone differentiation and calcification in vivo or in vitro.

[0053] The graft material for fibrocartilage tissue reconstruction according to the present invention can induce hierarchical reconstruction of fibrocartilage tissue between bone and ligament by filling the first and second hydrogel compositions as described above into a porous demineralized bone matrix support, thereby having a dual structure of a first region exhibiting non-calcified properties and a second region exhibiting calcified properties.

[0054]

[0055] Figure 1 (1-3) is a schematic diagram illustrating a method for manufacturing a graft material for fibrocartilage tissue reconstruction according to one embodiment of the present invention.

[0056] Referring to (1-3) of FIG. 1, a method for manufacturing a graft for reconstructing fibrous cartilage tissue according to an embodiment of the present invention is a method for manufacturing a graft for reconstructing fibrous cartilage tissue between a bone and a ligament or tendon, comprising the steps of: manufacturing a porous demineralized bone matrix support having a tube structure having a through hole through which a ligament or tendon fiber bundle for transplantation passes; manufacturing a first hydrogel composition including an autologous or heterologous fibrocartilage-derived extracellular matrix, and a second hydrogel composition including an autologous or heterologous fibrocartilage-derived extracellular matrix and a calcification-inducing substance, respectively; And a step of filling the first and second hydrogel compositions into the porous demineralized bone matrix support, respectively, and the step of filling the first and second hydrogel compositions into the porous demineralized bone matrix support, respectively, may be characterized in that the first hydrogel composition is filled into the pores of the first region of the porous demineralized bone matrix support surrounding the through hole and crosslinked, and the second hydrogel composition is filled into the pores of the second region of the porous demineralized bone matrix support surrounding the first region and crosslinked.

[0057] The step of manufacturing the above porous demineralized bone matrix support may include the steps of obtaining a bone-cartilage composite tissue and removing minerals from the composite tissue; the step of removing cartilage tissue from the composite tissue from which the minerals have been removed to manufacture a porous demineralized bone matrix support; and the step of forming the porous demineralized bone matrix support into a tube shape having through holes.

[0058] The step of removing minerals from the above complex tissue can be performed by treating with an acidic solution to remove minerals or calcium, etc.

[0059] The step of removing the above cartilage tissue to produce a porous demineralized bone matrix scaffold can be performed by washing with a surfactant solution for more than 24 hours to remove cells and genetic material.

[0060] After removing the above cartilage tissue, a washing step may be further included to remove cells and foreign substances.

[0061] The porous demineralized bone matrix scaffold manufactured according to the above method may be composed of cancellous bone, compact bone or a combination thereof, and may preferably include both cancellous bone and compact bone, but is not limited thereto.

[0062] Specifically, the step of forming the porous demineralized bone matrix support into a tube shape having a through hole can manufacture a porous demineralized bone matrix support having an inner diameter of an average of 6 to 13 mm, 7.5 to 11.5 mm, or 9 to 10 mm and an outer diameter of an average of 7 to 15 mm, 8.5 to 13.5 mm, or 10 to 12 mm using a biopsy punch.

[0063] In addition, the step of forming the porous demineralized bone matrix support into a tube shape having a through hole can produce a porous demineralized bone matrix support having an average length of 20 to 60 mm, 25 to 55 mm, or 30 to 50 mm.

[0064] The first hydrogel composition can be prepared to include an extracellular matrix derived from autologous or heterologous fibrocartilage.

[0065] The above autologous or xenogeneic fibrocartilage-derived extracellular matrix may be a xenogeneic fibrocartilage-derived extracellular matrix. For example, the fibrocartilage-derived extracellular matrix may be a meniscus tissue as a fibrocartilage composite tissue.

[0066] Specifically, the first hydrogel composition can be prepared to include 0.5 to 10 w / v%, 0.5 to 7 w / v%, 1 to 5 w / v%, 3 to 4 w / v%, or 1 to 2 w / v% of an extracellular matrix derived from autologous or heterologous fibrocartilage.

[0067] According to one embodiment of the present invention, the first hydrogel composition may include an autologous or heterologous fibrocartilage-derived extracellular matrix as described above, and may have a temperature sensitivity that allows it to exist in a liquid state at room temperature and to gel at a temperature of 37°C or higher, preferably 37°C to 45°C, thereby maintaining a specific shape.

[0068] The second hydrogel composition can be prepared to include an extracellular matrix derived from autologous or heterologous fibrocartilage and a calcification-inducing substance.

[0069] The above-mentioned calcification-inducing substance may include at least one selected from the group consisting of musculoskeletal tissue-derived extracellular vesicles (TEVs), bone morphogenetic protein-2 (BMP-2), and hydroxyapatite (HA). Specifically, the above-mentioned calcification-inducing substance may be musculoskeletal tissue-derived extracellular vesicles (TEVs). More specifically, the musculoskeletal tissue may be composed of cartilage tissue, bone tissue, and semicircular cartilage tissue.

[0070] Specifically, the second hydrogel composition may comprise 0.5 to 10 w / v%, 0.5 to 7 w / v%, 1 to 5 w / v%, 3 to 4 w / v% or 1 to 2 w / v% of the autologous or heterologous cartilage-derived extracellular matrix, and may comprise 0.01 to 500 μg / ml, 1 to 500 μg / ml, 100 to 500 μg / ml or 10 to 100 μg / ml of the calcification-inducing substance.

[0071] More specifically, the second hydrogel composition can be used to prepare a 2-1 hydrogel composition comprising the calcification-inducing substance at a first concentration, and a 2-2 hydrogel composition comprising the calcification-inducing substance at a second concentration higher than the first concentration.

[0072] The above 2-1 hydrogel composition can be filled and crosslinked within the pores of the 2-1 region of the porous demineralized bone matrix support adjacent to the first region among the second regions, and the above 2-2 hydrogel composition can be filled and crosslinked within the pores of the 2-2 region of the porous demineralized bone matrix support surrounding the 2-1 region among the second regions.

[0073] The step of filling the first and second hydrogel compositions into the porous demineralized bone matrix support, respectively, may be performed by filling the first and second hydrogel compositions into the internal pores of the porous demineralized bone matrix support, respectively, through one method selected from the group consisting of immersion, stirring after immersion, dispensing, and 3D printing, and then crosslinking at a temperature of 37°C or higher to implement a biphasic structure.

[0074] The above immersion can be performed by immersing in the hydrogel composition at room temperature for 20 minutes to 2 hours.

[0075] The above-mentioned post-immersion stirring can be performed by incubating the hydrogel composition at a temperature of 37°C or higher while stirring at a speed of 100 to 300 rpm for 30 minutes to 2 hours at room temperature.

[0076] The above dispensing can be performed by dispensing the hydrogel composition into a porous demineralized bone matrix support, allowing it to penetrate or be absorbed into the support, and then incubating it at a temperature of 37°C or higher.

[0077] The above 3D printing can be performed at a speed of 5 to 15 mm / sec or 7 to 13 mm / sec at a pressure of less than 80 kPa and a temperature of 37°C or higher using a nozzle having an average diameter of 200 to 600 μm or 300 to 600 μm.

[0078] The method for manufacturing a graft for fibrocartilage tissue reconstruction according to the present invention can induce hierarchical reconstruction of fibrocartilage tissue between bone and ligament by filling the first and second hydrogel compositions as described above into a porous demineralized bone matrix support, thereby having a dual structure of a first region exhibiting non-calcified properties and a second region exhibiting calcified properties.

[0079]

[0080] According to one embodiment of the present invention, a method for manufacturing a graft for fibrocartilage tissue reconstruction may include: manufacturing first and second porous demineralized bone matrix supports having a tube structure having through holes having different diameters; manufacturing a first hydrogel composition including an extracellular matrix derived from autologous or heterologous fibrocartilage and a second hydrogel composition including an extracellular matrix derived from autologous or heterologous fibrocartilage and a calcification-inducing substance, respectively; filling the first hydrogel composition into a first tube structure formed of a porous demineralized bone matrix support and crosslinking the first hydrogel composition, and filling the second hydrogel composition into a second tube structure formed of a porous demineralized bone matrix support and having an inner diameter and an outer diameter larger than those of the first tube structure, and crosslinking the second hydrogel composition; and assembling the first tube structure so as to be positioned inside the second tube structure.

[0081] The step of manufacturing the first and second porous demineralized bone matrix scaffolds having a tube structure having through holes having different diameters may include the steps of obtaining a bone-cartilage composite tissue and removing minerals from the composite tissue; the step of removing cartilage tissue from the composite tissue from which the minerals have been removed to manufacture a porous demineralized bone matrix scaffold; and the step of forming the porous demineralized bone matrix scaffold into a tube shape having through holes.

[0082] The step of removing minerals from the above complex tissue can be performed by treating with an acidic solution to remove minerals or calcium, etc.

[0083] The step of removing the above cartilage tissue to produce a porous demineralized bone matrix scaffold can be performed by washing with a surfactant solution for more than 24 hours to remove cells and genetic material.

[0084] After removing the above cartilage tissue, a washing step may be further included to remove cells and foreign substances.

[0085] The porous demineralized bone matrix scaffold manufactured according to the above method may be composed of cancellous bone, compact bone or a combination thereof, and may preferably include both cancellous bone and compact bone, but is not limited thereto.

[0086] Specifically, the step of forming the porous demineralized bone matrix support into a tube shape having through holes may include a step of manufacturing a first porous demineralized bone matrix support having an inner diameter of an average of 6 to 13 mm, 7.5 to 11.5 mm, or 9 to 10 mm and an outer diameter of an average of 6.5 to 14 mm, 8 to 12.5 mm, or 9.5 to 11 mm; and a step of manufacturing a second porous demineralized bone matrix support having an inner diameter of an average of 6.5 to 14 mm, 8 to 12.5 mm, or 9.5 to 11 mm and an outer diameter of an average of 7 to 15 mm, 8.5 to 13.5 mm, or 10 to 12 mm.

[0087] For example, the thicknesses of the first and second porous demineralized bone matrix supports may be independently from each other an average of 0.2 to 3 mm, 0.3 to 2 mm, or 0.5 to 1 mm. The thickness refers to the difference between the outer diameter and the inner diameter of each support.

[0088] The steps of preparing the first hydrogel composition including the autologous or heterologous fibrocartilage-derived extracellular matrix and the second hydrogel composition including the autologous or heterologous fibrocartilage-derived extracellular matrix and the calcification-inducing substance are substantially the same as those described in the method for preparing a graft material for fibrocartilage tissue reconstruction according to another embodiment of the present invention, and therefore, a detailed description thereof will be omitted.

[0089] The above first tube structure can be manufactured by filling the first hydrogel composition into a first porous demineralized bone matrix support and then crosslinking it.

[0090] The above second tube structure can be manufactured by filling the second hydrogel composition into a second porous demineralized bone matrix support and then crosslinking it.

[0091] The second tube structure may be crosslinked after filling the 2-1 hydrogel composition into the pores of the 2-1 region of the second tube structure adjacent to the first tube structure among the second tube structures, and the 2-2 hydrogel composition may be crosslinked after filling the pores of the 2-2 region of the second tube structure surrounding the 2-1 region of the second tube structure.

[0092] The first tube structure and the second tube structure can be manufactured by filling the first hydrogel composition or the second hydrogel composition into a porous demineralized bone matrix support, respectively, through one method selected from the group consisting of immersion, stirring after immersion, dispensing, and 3D printing, and then crosslinking at a temperature of 37°C or higher.

[0093] The above immersion, stirring after immersion, dispensing, and 3D printing are substantially the same as those described in the method for manufacturing a graft material for fibrocartilage tissue reconstruction according to another embodiment of the present invention, and therefore, redundant detailed descriptions thereof are omitted.

[0094]

[0095] Figure 1 (4-5) is a schematic diagram illustrating a method for reconstructing fibrocartilage tissue between a bone and a ligament or tendon using a graft material for reconstructing fibrocartilage tissue according to one embodiment of the present invention.

[0096] Referring to (4-5) of FIG. 1, a method for reconstructing fibrocartilage tissue between a bone and a ligament or tendon according to one embodiment of the present invention may include the steps of forming a tibial tunnel penetrating the tibia; forming a femoral tunnel penetrating the femur and arranged parallel to the tibial tunnel; fixing a ligament or tendon for transplantation to the fibrocartilage tissue reconstruction graft described above; and positioning the tendon or ligament for transplantation fixed to the fibrocartilage tissue reconstruction graft in the tibial tunnel and the femoral tunnel.

[0097] The step of forming the tibial tunnel and the step of forming the femoral tunnel may each form the tunnel using a reamer or an expander. Specifically, the tibial tunnel and the femoral tunnel may be independently formed to an average size of 20 to 60 mm, 25 to 55 mm, or 30 to 50 mm.

[0098] The step of fixing a ligament or tendon for transplantation to the above-mentioned graft for fibrocartilage tissue reconstruction can be performed by fixing a bundle of ligament or tendon fibers for transplantation to a through hole provided in a porous demineralized bone matrix support of the above-mentioned graft for fibrocartilage tissue reconstruction.

[0099] The step of positioning the graft tendon or ligament fixed to the graft material for fibrocartilage tissue reconstruction in the tibial tunnel and femoral tunnel can be performed by positioning the tendon or ligament so that it passes through the tibial tunnel and the femoral tunnel.

[0100] Hereinafter, to aid understanding of the present invention, examples and other embodiments will be described in detail. However, the following examples and other embodiments merely illustrate the content of the present invention and are not intended to limit the scope of the present invention. The examples and other embodiments of the present invention are provided to more fully explain the present invention to those of average skill in the art.

[0101]

[0102] <Example 1> Evaluation of physicochemical properties of demineralized bone matrix support

[0103] 1-1. Manufacturing of demineralized bone matrix (DBM) scaffold

[0104] Demineralized bone matrix (DBM) tissue can be obtained from animal tissues, including humans, and specifically, can be isolated from osteo-cartilage tissue obtained from cadavers, patients who have undergone artificial joint surgery, or pigs. To remove minerals and calcium from the harvested bone tissue, it was treated with a 5% nitric acid solution at 4℃ for 48 hours, and the nitric acid solution was replaced every 24 hours. After the demineralization process, the bone tissue was washed with a 15% Triton-X 100 solution at 4℃ for 72 hours to remove cells and genetic material, and the Triton-X 100 solution was replaced every 24 hours. After the decellularization process, the bone tissue was stirred for a total of 48 hours, twice for 24 hours each, to remove any Triton-X 100 remaining in the bone tissue. The demineralized and decellularized DBM can be cut and shaped to the desired shape and size. In addition, the above bone matrix is ​​a structure in which spongy bone and compact bone are combined. Spongy bone has high porosity and excellent tensile strength and elasticity, making it easy to form a support, while compact bone has relatively low porosity and is hard, providing good physical strength. Depending on the intended use of the support, only spongy bone or both spongy bone and compact bone can be used. Figure 2(A) is a gross image of a DBM scaffold manufactured by the method described above.

[0105]

[0106] 1-2. Microstructural Characterization of Demineralized Bone Matrix (DBM) Scaffolds

[0107] To observe the microstructure of the DBM scaffold, scanning electron microscope (SEM) analysis was performed. Microstructural observations using SEM were performed by photographing and analyzing native bone and DBM scaffolds, respectively (Fig. 2B). SEM image analysis results confirmed that the pore size and porosity of the DBM scaffold manufactured using the above process technology were well maintained with no significant difference compared to native bone.

[0108]

[0109] 1-3. Analysis of decalcification of DBM support

[0110] In Fig. 2C, the content of mineral elements such as calcium and phosphorus was analyzed through energy dispersive X-ray spectroscopy (EDS) to confirm the presence or absence of decalcification of the DBM scaffold manufactured by the above process. As a result, calcium was 65.5% in the native bone, and calcium in the demineralized bone matrix (DBM) scaffold was 0.08%, indicating a significant decrease in calcium, confirming that the demineralization process was successfully performed.

[0111]

[0112] 1-4. Biochemical Characterization of Demineralized Bone Matrix (DBM) Scaffolds

[0113] To confirm whether decellularization was successful, the dsDNA content of the decellularized DBM scaffold was analyzed using the Picogreen assay. As a result, as shown in Figure 2D, the dsDNA content of the DBM group was 33.26±3.4 ng / mg, which was below the dsDNA level of 50 ng / mg, which is the standard for decellularization, confirming successful decellularization. To analyze the biochemical properties of the DBM scaffold, the content of sGAG and collagen was analyzed. Figures 2E-F show the results of biochemical analysis quantifying the contents of sulfated glycosaminoglycan (sGAG) and collagen in the DBM scaffold. sGAG was analyzed using the DMMB assay (B1000, Biocolor), and collagen was analyzed using the Sirius Red assay (S1000, Biocolor). The sGAG content of the native bone group was 5.48±0.4 μg / mg, the sGAG content of the demineralized bone matrix group was 1.76±0.3 μg / mg, the collagen content of the native bone group was 2.65±0.6 μg / mg, and the collagen content of the DBM group was 1.62±0.4 μg / mg. Therefore, it was confirmed that sGAG and collagen, which are major extracellular matrix (ECM) components of bone tissue, were preserved even after the demineralization process.

[0114]

[0115] <Example 2> Evaluation of physicochemical properties of meniscus-derived extracellular matrix (DMECM) hydrogel

[0116] 2-1. Preparation of a thermosensitive hydrogel derived from meniscus-derived extracellular matrix (DMECM)

[0117] To fabricate a thermosensitive DMECM hydrogel using porcine meniscus tissue, meniscus tissue was first harvested from a porcine knee using a surgical blade (Fig. 3A, upper left). The meniscus tissue was then washed three times with distilled water and then freeze-dried and freeze-disintegrated to obtain a powder. The obtained meniscus tissue powder was treated with a hypotonic solution at room temperature for 4 hours, followed by decellularization in a surfactant solution containing sodium dodecyl sulfate (SDS) for 2 hours. This was followed by six washes with distilled water to remove the SDS. Finally, to remove genetic material present in the meniscus extracellular matrix powder, the solution was treated with a DNAse solution for 12 hours. This was followed by six additional washes with distilled water to complete the decellularization process. Next, an acidic solution (0.01 to 0.1 M HCl or 0.1 to 0.5 M acetic acid) containing pepsin (0.01 to 1% w / v) was prepared, mixed with meniscus tissue powder to dissolve it, and then the pH was neutralized to 7 to 8 with NaOH. Then, highly concentrated phosphate buffered saline (PBS) was added to adjust the concentration of physiological ions to prepare an aqueous DMECM Pre-hydrogel (Fig. 3A, bottom left).

[0118] To examine the microstructure of the meniscus tissue and components contained in DMECM, SEM analysis was performed. The meniscus tissue exhibited a structure in which large fibers were densely arranged (Fig. 3A, upper right), while in the DMECM group, the fiber structure was observed to be digested by the pepsin enzyme, resulting in a random arrangement of collagen fibers (Fig. 3A, lower right).

[0119]

[0120] 2-2. Biochemical Characterization of Thermosensitive DMECM Hydrogel

[0121] To analyze the differences in the protein cargo profiles of the meniscus tissue and DMECM hydrogel, SDS-PAGE analysis was performed (Fig. 3B). As a result, there was no significant difference in the type and size of bands between the native meniscus tissue group and the DMECM group, confirming that the constituent proteins were well maintained during the decellularization and hydrogel manufacturing processes. Next, to evaluate the biochemical content of DMECM, quantitative analysis of dsDNA, collagen, and sGAG was performed. When the dsDNA content of DMECM was analyzed using the Picogreen assay, the dsDNA content of the DMECM group was 20.5±13.2 ng / mg, confirming that the decellularization process was successfully performed. The collagen and sGAG contents of DMECM were 828.4±14.3 μg / mg and 19.7±1.7 μg / mg, respectively, which were slightly reduced compared to native tissue, but it was confirmed that the major ECM components were well maintained at a significant level.

[0122]

[0123] <Example 3> Optimization of DBM loading conditions for temperature-sensitive DMECM hydrogel

[0124] 3-1. Concentration optimization of thermosensitive DMECM hydrogel

[0125] To load the temperature-sensitive DMECM hydrogel into the DBM support, an optimization study on the concentration conditions of the DMECM hydrogel was conducted.

[0126] Figure 4A is a gross image of the thermosensitive DMECM hydrogel prepared above according to concentration. It was confirmed that the DMECM hydrogel prepared at 0.1% did not gel at 37°C and remained in an aqueous solution state. Meanwhile, the DMECM hydrogel group of 1% to 5% showed gelation at 37°C.

[0127] To investigate the rheological properties of the thermosensitive DMECM hydrogel according to temperature change, the shear rate was 1 s -1 The gelation rheology was measured while maintaining the temperature of the analytical device at 4℃ to 45℃ (Fig. 4B). As a result, it was confirmed that the elastic modulus of the DMECM hydrogel significantly increased at temperatures above 20℃ due to thermal gelation. In addition, the storage modulus of all groups was higher than the loss modulus, confirming that they possessed the rheological properties of hydrogels (Fig. 4C).

[0128] In conclusion, it was confirmed that DMECM hydrogel is thermosensitive, allowing for free administration of a desired amount at room temperature, and that it maintains specific viscoelasticity and shape according to concentration at 37°C.

[0129]

[0130] <Example 4> Evaluation of the characteristics of a temperature-sensitive DMECM hydrogel according to DBM loading conditions

[0131] 4-1 Development of a DBM-loading Method for Temperature-Sensitive DMECM Hydrogel

[0132] The method for loading the temperature-sensitive DMECM hydrogel onto the DBM scaffold manufactured above may be one of immersion, stirring after immersion, dispensing or seeding, or printing.

[0133] First, by the immersion / stirring method, the DBM scaffold manufactured above was immersed in a DMECM pre-hydrogel aqueous solution having a concentration of 1 to 4% (w / v), stirred at 200 rpm for 30 minutes to 2 hours at room temperature, and then incubated at 37°C to load the DMECM hydrogel into the DBM scaffold (Fig. 5A).

[0134] Second, the dispensing method was to dispense the DMECM aqueous solution onto the DBM scaffold manufactured above using a micropipette or syringe, and allow it to penetrate or be absorbed into the DBM scaffold, followed by incubation at 37°C to load the DMECM hydrogel into the DBM scaffold. Figure 5B is a photograph showing the difference in the degree of absorption into the DBM scaffold depending on the DMECM concentration. It was confirmed that the 1 to 4% DMECM pre-hydrogel aqueous solution was absorbed into the DBM scaffold within approximately 2 to 5 seconds after dispensing.

[0135] Third, using a 3D printing method, DMECM pre-hydrogel is used as a bioink to dispense it in the desired shape at the desired coordinates on the DBM scaffold, and then absorbed into the DBM scaffold. Then, the temperature of the lower plate of the 3D printer is set to 37℃ to gel and mount the DMECM hydrogel (Fig. 5C). In order to create a structure of the desired shape using a 3D printer, the printing conditions were set as follows. Considering the temperature sensitivity, viscosity, and printability of DMECM-bioink, the nozzle size was set to 200 μm to 600 μm or less, the air pressure was less than 80 KPa, and the printing speed was 7-13 mm / sec. The temperature of the lower plate of the 3D printer was set to 37℃. As a result, it was confirmed that the desired amount could be injected into the demineralized bone matrix (DBM) scaffold at the desired location using a 3D printer.

[0136]

[0137] 4-2 Evaluation of biochemical properties of DBM scaffolds according to DBM loading method in temperature-sensitive DMECM hydrogels

[0138] To analyze the biochemical properties of the DMECM+DBM scaffold according to the loading method of the temperature-sensitive DMECM hydrogel, the content analysis of collagen and sGAG was performed (Fig. 5 DE).

[0139] Quantitative analysis of collagen and sGAG showed that the collagen and sGAG contents significantly increased in all groups as the concentration of DMECM increased. In the low-concentration DMECM hydrogel group of 1 to 2%, no significant difference was observed in the biochemical contents according to the loading method. However, in the case of high-concentration DMECM hydrogel loading of 3 to 4%, it was confirmed that the immersion loading method significantly increased the collagen and sGAG contents compared to the dispensing and printing methods.

[0140]

[0141] 4-3 Evaluation of biochemical properties of DBM scaffolds according to the number of loadings of temperature-sensitive DMECM hydrogels

[0142] To evaluate the differences in physicochemical properties according to the number of dispensings of the thermosensitive DMECM hydrogel, gross images were taken and SEM analysis of the scaffold cross-section was performed (Fig. 6A). The test was performed by manufacturing a DBM scaffold with a diameter of 5 mm and a height of 5 mm, preparing a 2% DMECM pre-hydrogel aqueous solution, dispensing 200 μl of each onto the DBM scaffold, and incubating at 37°C. This process was repeated 0 to 6 times.

[0143] As a result, it was confirmed that the expression color of the DMECM hydrogel labeled with a yellow dye became darker as the number of times the temperature-sensitive hydrogel was loaded increased in the gross image. SEM image analysis confirmed that the pore structure within the DBM scaffold gradually became smaller and the porosity also significantly decreased, confirming that the physical properties of the DMECM-DBM scaffold can be controlled depending on the number of times of dispensing.

[0144] Next, to analyze the biochemical properties of the DMECM-DBM scaffold according to the number of dispensing of the thermosensitive hydrogel, collagen and sGAG content analysis was performed (Fig. 6 BC). As a result of collagen measurement, it was confirmed that collagen significantly increased in all groups loaded with hydrogel compared to the DBM only group (1.73±0.3 μg / mg) (1st time: 15.03±4.4 μg / mg, 2nd time: 18.6±5.1 μg / mg, 3rd time: 20.8±4.6 μg / mg, 4th time: 22.4±4.6 μg / mg, 5th time: 23.6±4.2 μg / mg, 6th time: 24.9±5.01 μg / mg). Similar to the collagen results, the sGAG measurement results also confirmed that the sGAG content significantly increased in all groups loaded with DMECM hydrogel compared to the DBM only group (1.76±0.3 μg / mg) (1st time: 13.5±1.7 μg / mg, 2nd time: 15.7±1.5 μg / mg, 3rd time: 18.8±0.5 μg / mg, 4th time: 22.3±1.01 μg / mg, 5th time: 23.8±0.5 μg / mg, 6th time: 24.4±0.5 μg / mg). Therefore, it was confirmed that the biochemical content of the DMECM-DBM scaffold could be controlled depending on the loading conditions.

[0145]

[0146] 4-4 Evaluation of in vitro fibrocartilage differentiation potential of DMECM-DBM scaffold according to the concentration of thermosensitive DMECM hydrogel

[0147] Next, the difference in the in vitro fibrocartilage differentiation ability of DMECM-DBM scaffolds was evaluated according to the loading concentration of the thermosensitive DMECM hydrogel. The test was conducted by producing DMECM-DBM scaffolds loaded with DMECM hydrogel at concentrations of 1%, 2%, 3%, and 4%, freeze-drying them, sterilizing them with EO gas, and then incubating them with 4x10 human bone marrow-derived mesenchymal stem cells (MSCs). 6After seeding with Cells / scaffold, they were cultured for 14 days at 37℃, 5% CO2, and histological and biochemical analyses were performed (Fig. 7).

[0148] As a result, it was confirmed that alcian blue staining, which specifically expresses sGAG in blue, and type 2 collagen (COL2) staining were expressed more strongly as the concentration of the hydrogel increased (Fig. 7A).

[0149] Next, to analyze the biochemical properties of the DBM+DMECM scaffold, collagen and sGAG content analysis was performed (Fig. 7B-C). As a result of collagen measurement, it was confirmed that collagen significantly increased according to the DMECM hydrogel loading concentration compared to the DBM only group (9.58±2.7 μg / mg), but there was no statistically significant difference between the 3% and 4% hydrogel loading groups (1%: 29.08±6.8 μg / mg, 2%: 46.9±9.3 μg / mg, 3%: 59.7±12.7 μg / mg, 4%: 68.6±10.4 μg / mg).

[0150] In the sGAG measurement results, it was confirmed that the sGAG content significantly increased depending on the loading concentration of the hydrogel compared to the DBM only group (4.54±1.8 μg / mg), but there was no statistically significant difference between the groups loaded with 3% and 4% hydrogel (1%: 16.2±5.5 μg / mg, 2%: 26.6±6.8 μg / mg, 3%: 34.1±6.6 μg / mg, 4%: 37.7±5.2 μg / mg).

[0151] Therefore, it was confirmed that the induction of stem cell and artificial fibrocartilage tissue differentiation was promoted depending on the concentration of DMECM hydrogel, and it was confirmed that the concentration of DMECM hydrogel suitable for fibrocartilage differentiation was 1% to 4%.

[0152]

[0153] <Example 5> Evaluation of physicochemical properties of DMECM-DBM supports according to crosslinking methods

[0154] 5-1. Evaluation of mechanical properties (compressive strength) of DMECM-DBM scaffolds according to crosslinking method

[0155] The DMECM-DBM scaffold loaded with the above-mentioned temperature-sensitive DMECM hydrogel can undergo an additional physical / chemical cross-linking process when physicochemical properties such as mechanical strength, swelling degree, and biodegradability need to be controlled. More specifically, gamma-irradiation or energy beam (E-Beam) irradiation is possible as physical cross-linking, glutaraldehyde or EDC / NHS is possible as chemical cross-linking, and photocross-linking under UV or blue-ray irradiation through mixing the DMECM hydrogel with an appropriate photoinitiator is possible. In this example, representative cross-linking methods among the above methods were performed, such as glutaraldehyde (0.01%), gamma-ray irradiation (25 kGy), and cross-linking through mixing the DMECM hydrogel with a riboflavin photoinitiator and blue-ray irradiation, and the physicochemical property analyses thereof were compared, but the present invention is not limited thereto.

[0156] To analyze the mechanical properties of the DMECM-DBM scaffold according to the cross-linking method, the compressive strength was analyzed using a universal property tester (Fig. 8A). As a result of the analysis, the compressive strength of the chemical cross-linking group using glutaraldehyde (GA: Glutaraldehyde, Sigma, USA) was the highest among all groups, followed by gamma-ray irradiation, photo-crosslinking, and freeze-drying (GA: 147.5±6.7 kPa, gamma-ray irradiation: 139.8±8.6 kPa, photo-crosslinking group: 125.6±7.4 kPa, freeze-drying group: 83.2±5.8 kPa, DBM only: 72.5±5.1 kPa).

[0157]

[0158] 5-2. Evaluation of swelling degree of DMECM-DBM scaffold according to crosslinking method

[0159] In order to measure the swelling degree of the DMECM-DBM scaffold according to the crosslinking method, the freeze-dried scaffold was immersed in triple-distilled water at room temperature, and the weight change of the swollen scaffold was measured after 24 hours to measure the swelling degree of the manufactured DMECM-DBM scaffold (Fig. 8B).

[0160] The swelling degree is the weight of the swollen scaffold (W) as shown in Equation (2). s ) and the weight of the dried scaffold (W d ) Weight of the dried scaffold (W d ) and expressed as a percentage.

[0161] [Formula 2] Swelling (%) = W s - W d / W d x100

[0162] As a result of the analysis, it was confirmed that the swelling degree of the DMECM-DBM scaffold using gamma irradiation and photo-crosslinking crosslinking methods was 661.3% and 650.1%, respectively, which were significantly higher than that of other crosslinking groups (GA: 580.8%, Freeze drying: 575.9%, DBM only: 573.6%).

[0163]

[0164] 5-3. Evaluation of the decomposition rate of DMECM-DBM scaffolds according to cross-linking method

[0165] Since the required degradation period for medical biomaterials varies depending on the anatomical location where they are implanted or the desired performance, the controllability of the degradation period is very important.

[0166] To determine whether the degradation rate can be controlled depending on the cross-linking method of the DMECM-DBM scaffold, 10 mL of PBS was added to the DMECM-DBM scaffold in vitro, and the degradation behavior was evaluated from 0 to 10 weeks. The DMECM-DBM scaffold was freeze-dried, and the degradation rate was expressed as a percentage of the dry weight of the initial scaffold. The X-axis of the graph represents the period from 0 to 10 weeks in weeks, and the Y-axis represents the weight of the remaining scaffold as a percentage (%) (Fig. 8C).

[0167] As a result of the analysis, the decomposition rate of the freeze drying group without any cross-linking treatment was the fastest among all groups, and the initial residual amount after 10 weeks of hydrolysis was 58.4±1.3%.

[0168] On the other hand, the residual amount of chemical crosslinking groups through glutaraldehyde was the highest among all groups, followed by photo-crosslinking and X-ray gamma-ray irradiation (GA group: 84.7±1.7%, photo-crosslinking group: 62.7±2.1%, X-ray gamma-ray irradiation group: 60.2±2.9%). Therefore, the DMECM-DBM scaffold can be decomposed by using one or more of the above crosslinking methods depending on the desired degradation period.

[0169]

[0170] <Example 6> Evaluation of cell affinity and fibrocartilage differentiation induction properties of DMECM-DBM scaffolds according to cross-linking method

[0171] 6-1. Cell affinity evaluation of DMECM-DBM scaffold

[0172] To determine the cell proliferation ability of DMECM-DBM scaffolds according to the cross-linking method, 1Х10 per scaffold 6 After seeding MSCs, they were cultured with cell culture medium at 37℃, and cell proliferation was analyzed using WST assay on days 1, 3, and 7 (Fig. 9A). As a result, at all time points of 1, 3, and 7, the WST absorbance of each cross-linking group showed similar or higher values ​​than that of the DBM only scaffold, confirming their cell compatibility. In particular, on days 3 and 7 of proliferation, the gamma-irradiation and photocross-linking treatment group showed higher WST absorbance values ​​than the other groups, confirming that they induced the proliferation of MSCs.

[0173]

[0174] 6-2. Evaluation of the fibrocartilage differentiation induction properties of the DMECM-DBM scaffold

[0175] To evaluate the effect of DMECM-DBM scaffold on MSC differentiation according to the cross-linking method, 1Х10 per scaffold 6 RT-PCR analysis was performed 3 days after inoculation of MSCs.

[0176] As a result, as shown in Figures 9B-E, in the case of type 1 collagen (COL1), there was no significant difference from the DBM only group, but type 2 collagen (COL2), aggrecan (ACAN), and SOX9 showed higher gene expression in all groups cross-linked after DMECM loading than in the DBM only group, and the expression of genes related to fibrocartilage differentiation was observed to be higher in the order of photocross-linking, gamma-ray irradiation, and glutaraldehyde cross-linking. Therefore, it was confirmed that the biological efficacy for fibrocartilage differentiation was observed in all groups even after the cross-linking process.

[0177]

[0178] <Example 7> Evaluation of physicochemical properties of extracellular vesicles (TEVs) derived from musculoskeletal tissues

[0179] 7-1. Isolation of tissue-derived extracellular vesicles (TEVs) derived from musculoskeletal tissue

[0180] Musculoskeletal tissues, including bone, cartilage, and meniscus, were harvested from porcine knee joints using a surgical blade. Each harvested musculoskeletal tissue was lyophilized, aliquoted into 1 g portions, and then solubilized through treatment with enzymes containing collagenase or pepsin or chemical solvents containing urea. More specifically, 0.1 mg / ml type 2 collagenase or 1 mg / ml pepsin enzymes were suspended in acidic solvents such as 0.5 M acetic acid or 0.1 M HCl, or solubilized with a 0.5 urea solution. The lyophilized musculoskeletal tissues were solubilized in one or a combination of the above solvents for 24 hours. The tissue suspension prepared through the above-mentioned solubilization process was centrifuged in a high-speed centrifuge at 500 x g for 10 minutes, 2,500 x g for 20 minutes, and 10,000 x g for 30 minutes to remove cells and cell-derived genetic materials, and then sequentially concentrated using a 0.45 μm filter tube, a 0.2 μm filter tube, and a 3 kD filter tube. The concentrate was mixed with distilled water and centrifuged in an ultra-high-speed centrifuge at 100,000 x g for 1 to 5 hours to wash away water-soluble foreign substances and collect tissue-derived extracellular vesicles. Alternatively, the concentrate could be mixed with a polymer compound such as dextran or PEG, and centrifuged at 1,500 x g for 10 minutes to collect tissue-derived extracellular vesicles. The separated extracellular vesicle suspension was aliquoted into the amount of target protein through protein quantitative analysis such as the Bradford assay and stored in an ultra-low temperature freezer at -80°C.

[0181]

[0182] 7-2. Physicochemical Characteristics of Tissue-derived Extracellular Vesicles (TEVs)

[0183] Figure 10A is a scanning electron microscope (SEM) image of meniscus tissue and TEVs dissolved by enzyme treatment. The left image shows the microfibrous structure of ECM and the attachment of TEVs to the fibrous structure, and the right image shows the morphology of TEVs separated and purified by the above method.

[0184] Figure 10B shows SEM images of TEVs derived from meniscus, bone, and cartilage according to the three musculoskeletal tissue origins, showing that the TEVs have similar morphology and size.

[0185] Figure 10C shows the results of analyzing the particle size distribution of TEVs derived from each tissue, and it was confirmed that TEVs of all groups showed a particle size distribution between approximately 100 and 1000 nm.

[0186] In Figure 10D, the expression of CD81, CD63, and CD9, known as extracellular vesicle surface antigen markers, was confirmed through Western blot, confirming that the TEVs purified from each musculoskeletal tissue were extracellular vesicles.

[0187] Therefore, through the above physical and biological characteristic analysis, it was confirmed that TEVs isolated and purified from musculoskeletal tissue possess the characteristics of extracellular vesicles.

[0188]

[0189] <Example 8> Evaluation of the calcification-inducing properties of extracellular vesicles (TEVs) derived from musculoskeletal tissues

[0190] 8-1. Evaluation of calcification induction ability according to TEV treatment concentration

[0191] Figure 11A shows that MSCs are 1x10 6This is the result of evaluating the calcification differentiation inducing ability of TEVs by adding Meniscus TEVs at a concentration of 0.1 to 500 μg / mL to a 6 cm diameter culture dish seeded with cells and culturing them for 7 days at 37°C in 5% CO2 conditions, and then performing an Alizarin red staining test. For comparison, the control group was tested without adding Meniscus TEVs. As a result, it was confirmed that as the concentration of Meniscus TEVs increased, calcification progressed and Alizarin red staining was expressed more strongly.

[0192] To quantitatively confirm this, the Alizarin red-stained images were semi-quantified using ImageJ software to compare the expression levels between groups (Fig. 11B). As a result, the Alizarin red-stained area tended to significantly increase as the concentration of TEVs increased up to 100 μg / mL, but there was no statistical significance among the high-concentration groups of 100, 200, and 500 μg / mL.

[0193]

[0194] 8-2. Evaluation of the calcification-inducing ability of musculoskeletal tissue-derived extracellular vesicles (TEVs)

[0195] To analyze the differences in the calcification-inducing capacity of TEVs according to their musculoskeletal tissue origin, an evaluation was performed using the Alizarin red staining test method (Fig. 11C). BMP-2 (100 μg / ml), which induces osteogenic differentiation and calcification, was used as a positive control.

[0196] As a result, it was confirmed that the group to which three types of TEVs (derived from meniscus, bone, and cartilage) were added at a concentration of 100 μg / mL showed Alizarin red staining at a level that was statistically significantly higher than that of the negative control group and similar to that of the positive control group, BMP-2 group.

[0197] Therefore, we confirmed that extracellular vesicles (TEVs) derived from musculoskeletal tissues, including bone, cartilage, and meniscus, can induce calcification.

[0198]

[0199] <Example 9> Evaluation of the in vitro calcification-inducing properties of DMECM / TEVs-DBM scaffolds

[0200] 9-1. Analysis of calcification of DMECM / TEVs-DBM scaffolds according to TEV concentration

[0201] To determine the in vitro calcification induction ability of DMECM / TEVs-DBM scaffolds according to the concentration of TEVs, TEVs were suspended in 2% DMECM Pre-hydrogel at a concentration of 10 μg / mL to 100 μg / mL, dispensed onto DBM scaffolds, and incubated at 37°C to produce DMECM / TEVs-DBM scaffolds. Subsequently, 1X10 6 Human bone marrow-derived mesenchymal stem cells (MSCs) were seeded on the scaffold and cultured with cell culture medium at 37°C for 4 weeks in vitro. Afterwards, mineral elements such as calcium and phosphorus deposited on the scaffold surface were analyzed using energy-dispersive X-ray spectroscopy (EDS) to confirm the degree of induction of the calcification layer according to the concentration of TEVs. The DBM scaffold group, which was prepared by adding BMP-2 (100 μg / ml) and hydroxyapatite (HA, 100 μg / ml), which are known to induce calcification, to the DMECM Pre-hydrogel instead of TEVs, was used as a positive control group for analysis (Fig. 12 AB).

[0202] The analysis results showed that as the concentration of TEVs increased, the levels of minerals such as calcium and phosphorus on the surface of the DMECM / TEVs-DBM scaffold increased. In particular, the calcium and phosphorus content composition ratio of the 100 μg / mL TEVs-added group was higher than that of the BMP-2-added group, confirming that TEVs significantly induced calcification.

[0203] Next, RT-PCR analysis was performed to confirm the degree of osteogenic differentiation of stem cells cultured in the DMECM / TEVs-DBM scaffold (Fig. 12C). As a result, it was confirmed that as the concentration of TEVs increased, the expression of RUNX-2, ALP, COL1A1, and OCN genes increased, similar to the positive control groups BMP-2 and HA. Therefore, it was confirmed that the addition of TEVs can induce osteogenic differentiation and calcification of human bone marrow-derived mesenchymal stem cells in the DMECM / TEVs-DBM scaffold.

[0204]

[0205] <Example 10> Evaluation of physicochemical properties of DMECM / TEVs-DBM supports according to loading method

[0206] 10-1. Fabrication of DMECM / TEVs-DBM scaffolds

[0207] There are two methods for loading DMECM and TEVs as a double layer on the DBM support fabricated above: a bioprinting method and an immersion method.

[0208] First, the bioprinting method is a method in which a DBM scaffold is manufactured into a single cylindrical shape using a biopsy punch, as shown in the photograph of Fig. 13A, and then a first agent containing DMECM and a second agent combining DMECM and TEVs are printed on the DBM scaffold, respectively. For reference, the scaffold manufactured in this example was manufactured in a size smaller than the actual patient application standard for use in in vitro and in vivo animal testing.

[0209] For the first printing, a cylindrical shape with an inner diameter of 2 mm, an outer diameter of 4 mm, and a height of 5 mm was 3D modeled, and then 2% DMECM hydrogel was used to print using a 3D printer with a nozzle size of 300 μm to 600 μm or less, an air pressure of less than 80 Kpa, and a printing speed of 7-13 mm / sec, and the temperature of the lower plate of the 3D printer was set to 37℃.

[0210] For the second printing, a cylindrical shape with an inner diameter of 4 mm, an outer diameter of 8 mm, and a height of 5 mm was 3D modeled, and the printing area was determined by excluding the first printing area. Then, 2% of DMECM hydrogel and 100 μg / mL of TEVs were mixed and loaded, and printing was performed under the same conditions.

[0211] Next, in the immersion method, the DBM scaffold was manufactured in a double cylindrical shape as shown in the photo of Fig. 13B, and then the DMECM / TEVs-DBM scaffold was manufactured by immersing / stirring each scaffold in the first and second agents to combine them and then crosslinking them.

[0212] As a result, it was confirmed that the first and second agents were successfully loaded onto the DBM scaffold in both methods, and the calcified fibrocartilage zone (outer, blue) and the non-calcified fibrocartilage zone (inner, yellow) could be reproduced (Fig. 13 AB).

[0213]

[0214] 10-2. Physical and chemical properties of DMECM / TEVs-DBM supports according to the loading method

[0215] Next, to confirm the differences in biochemical content and physical properties of the DMECM / TEVs-DBM scaffolds according to the two loading methods, quantitative analysis of collagen and sGAG was performed and compressive strength was measured using a universal physical property tester (Fig. 13 CD).

[0216] Quantitative analysis of collagen and sGAG showed that the double-layer loading method of the first and second agents using the immersion method of the double cylindrical DBM support showed slightly higher biochemical content values ​​than the printing method, but there was no statistical significance between the two groups.

[0217] In the compressive strength analysis results, similar to the biochemical content test results above, the support manufactured by immersion method showed a slightly higher compressive strength value than the support manufactured by printing method, but there was no statistically significant difference between the two groups.

[0218]

[0219] <Example 11> Evaluation of the Tissue Regeneration Effectiveness of DMECM / TEVs-DBM Scaffolds in a Rabbit Bone-Ligament Injury Model

[0220] 11-1. Histological Evaluation after DMECM / TEVs-DBM Scaffold Transplantation in a Rabbit Bone-Ligamentation Injury Model

[0221] To determine whether the composition of the DMECM / TEVs-DBM scaffold affects the effectiveness of fibrocartilage tissue regeneration, an analysis was performed using a rabbit anterior cruciate ligament reconstruction model (Fig. 14).

[0222] The definitions of the groups used in the test are as follows:

[0223] 1. Defect: Group without any transplantation after bone-ligament damage

[0224] 2. DBM only: DBM only scaffold transplant group

[0225] 3. 1% or 2% DMECM-DBM: DMECM-DBM scaffold implant group with 1% or 2% DMECM loaded on both the medial and lateral sides and only non-calcified fibrocartilage layer

[0226] 4. 1% or 2% DMECM / 100 μg / mL TEVs-DBM: DMECM / TEVs-DBM scaffold implant group with a non-calcified fibrocartilage layer loaded with 1% or 2% DMECM on the inner side of the DBM and a calcified fibrocartilage layer loaded with TEVs at a concentration of 100 μg / mL combined with 1% or 2% DMECM on the outer side.

[0227] 5. 2% DMECM / BMP-2 or HA-DBM: DBM scaffold implantation group with a non-calcified fibrocartilage layer loaded with 2% DMECM on the inner side of the DBM, and BMP-2 or HA loaded at a concentration of 100 μg / mL instead of TEVs on the outer calcified fibrocartilage layer of the DBM scaffold.

[0228]

[0229] The above groups were transplanted into a rabbit anterior cruciate ligament reconstruction model, and the effectiveness of bone-ligament tissue reconstruction was confirmed by Safranin-O staining after 8 weeks (Fig. 14 A). As a result, it was confirmed that the group containing 2% DMECM and BMP-2 together showed the most predominant red Safranin-O staining indicating sGAG deposition. Next, the group containing 2% DMECM and 100 μg / mL TEVs together, the 2% DMECM group, and the HA group showed predominant Safranin-O staining in that order.

[0230] Next, the effectiveness of tissue reconstruction was quantitatively analyzed using a histological scoring system for anterior cruciate ligament regeneration (Fig. 14B). As a result, it was confirmed that the group loaded with 2% DMECM (medial non-calcified fibrocartilage layer) and 2% DMECM + TEVs or BMP-2 or HA (lateral calcified fibrocartilage layer) showed significantly higher histological scores than the other groups.

[0231] Additionally, biomechanical validity evaluation was performed by measuring the maximum tensile strength of rabbit bone-ligament tissue samples harvested from the above animal model (Fig. 14C).

[0232] As a result, similar to the histological results above, it was confirmed that the group loaded with DMECM (inner non-calcified fibrocartilage layer) and 2% DMECM + TEVs or BMP-2 or HA (outer calcified fibrocartilage layer) showed significantly higher maximum tensile strength values ​​than the other groups.

[0233]

[0234] The foregoing description of the present invention is for illustrative purposes only. Those skilled in the art will readily appreciate that modifications to other specific embodiments can be made without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0235] The scope of the present invention is indicated by the claims set forth below, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. In a graft material for reconstructing fibrocartilaginous tissue between bone and ligament or tendon, A porous demineralized bone matrix scaffold having a tubular structure having through holes through which graft ligaments or tendon fiber bundles pass; A first hydrogel composition filled in the pores of the first region of the porous demineralized bone matrix support surrounding the through hole, and including an extracellular matrix derived from autologous or heterologous fibrocartilage; and A graft for reconstructing fibrocartilage tissue, comprising a second hydrogel composition filled in the pores of a second region of the porous demineralized bone matrix support surrounding the first region, and containing an extracellular matrix derived from autologous or heterologous fibrocartilage and a calcification-inducing substance.

2. In paragraph 1, The second hydrogel composition is, A 2-1 hydrogel composition filled in the pores of the 2-1 region of the porous demineralized bone matrix support adjacent to the 1st region among the 2nd regions, and containing the calcification-inducing material at a first concentration; and A graft for reconstructing fibrocartilage tissue, comprising a 2-2 hydrogel composition filled in the pores of the 2-2 region of the porous demineralized bone matrix support surrounding the 2-1 region among the 2 regions, and containing the calcification-inducing substance at a second concentration higher than the first concentration.

3. In paragraph 1, The outer diameter of the first region is on average 6.5 to 14 mm, The diameter of the above through hole is 6 to 13 mm on average, A graft for fibrocartilage tissue reconstruction, characterized in that the outer diameter of the second region is 7 to 15 mm on average.

4. In paragraph 1, The above porous demineralized bone matrix support is, A graft material for reconstructing fibrocartilaginous tissue, comprising cancellous bone, compact bone or a combination thereof.

5. In paragraph 1, The first and second hydrogel compositions, A graft material for reconstructing fibrocartilage tissue, characterized in that it exists in a liquid state at room temperature and gels at 37°C or higher.

6. In paragraph 1, A graft for fibrocartilage tissue reconstruction, characterized in that the above autologous or heterologous fibrocartilage-derived extracellular matrix is ​​heterologous meniscus cartilage tissue.

7. In paragraph 1, A graft for reconstructing fibrocartilage tissue, wherein the calcification-inducing substance comprises at least one selected from the group consisting of musculoskeletal tissue-derived extracellular vesicles (TEVs), bone morphogenetic protein-2 (BMP-2), and hydroxyapatite (HA).

8. In paragraph 7, A graft for reconstruction of fibrocartilage tissue, characterized in that the above musculoskeletal tissue is composed of cartilage tissue, bone tissue, and semicircular cartilage tissue.

9. A method for manufacturing a graft for reconstructing fibrous cartilage tissue between a bone and a ligament or tendon, A step of manufacturing a porous demineralized bone matrix scaffold having a tubular structure having through holes through which ligament or tendon fiber bundles for transplantation pass; A step of preparing a first hydrogel composition comprising an autologous or heterologous cartilage-derived extracellular matrix, and a second hydrogel composition comprising an autologous or heterologous fibrocartilage-derived extracellular matrix and a calcification-inducing substance, respectively; and Comprising a step of filling the first and second hydrogel compositions into the porous demineralized bone matrix support, respectively; The step of filling the first and second hydrogel compositions into the porous demineralized bone matrix support, respectively, is: The first hydrogel composition is filled into the pores of the first region of the porous demineralized bone matrix support surrounding the through hole and crosslinked, A method for producing a graft material for reconstructing fibrocartilage tissue, characterized in that the second hydrogel composition is filled into the pores of the second region of the porous demineralized bone matrix support surrounding the first region and crosslinked.

10. A step of manufacturing first and second porous demineralized bone matrix supports having a tube structure having through holes with different diameter sizes; A step of preparing a first hydrogel composition comprising an extracellular matrix derived from autologous or heterologous fibrocartilage and a second hydrogel composition comprising an extracellular matrix derived from autologous or heterologous fibrocartilage and a calcification-inducing substance, respectively; A step of filling the first hydrogel composition into a first tube structure formed of a porous demineralized bone matrix support and then crosslinking, and filling the second hydrogel composition into a second tube structure formed of a porous demineralized bone matrix support and having an inner diameter and an outer diameter larger than the first tube structure and then crosslinking; and A method for manufacturing a graft material for reconstructing fibrocartilage tissue, comprising the step of assembling the first tube structure so as to be positioned inside the second tube structure.

11. In paragraph 9, The step of filling the first and second hydrogel compositions into the porous demineralized bone matrix support, respectively, is: A method for producing a graft material for reconstructing fibrocartilage tissue, characterized in that the first and second hydrogel compositions are filled into the internal pores of a porous demineralized bone matrix support by one method selected from the group consisting of immersion, stirring after immersion, dispensing, and 3D printing, and then crosslinking is performed at a temperature of 37°C or higher.

12. In paragraph 10, The above first tube structure and the second tube structure, A method for producing a graft for reconstructing fibrocartilage tissue, characterized in that the first hydrogel composition or the second hydrogel composition is filled into a porous demineralized bone matrix support by one method selected from the group consisting of immersion, stirring after immersion, dispensing, and 3D printing, and then crosslinked at a temperature of 37°C or higher.

13. In paragraph 11 or 12 A method for manufacturing a graft material for reconstructing fibrocartilage tissue, characterized in that the above 3D printing is performed at a speed of 5 to 15 mm / sec at a temperature of 37°C or higher and a pneumatic pressure of less than 80 kPa using a nozzle having an average diameter of 200 to 600 μm.

14. In paragraph 9 or 10, The step of manufacturing the above porous demineralized bone matrix support is: A step of obtaining bone-cartilage composite tissue and removing minerals from the composite tissue; A step of manufacturing a porous demineralized bone matrix support by removing cartilage tissue from the composite tissue from which the above minerals have been removed; and It comprises a step of forming the porous demineralized bone matrix support into a tube shape having a through hole, The step of forming the above porous demineralized bone matrix support into a tube shape having a through hole is as follows: A method for producing a graft material for reconstructing fibrocartilage tissue, characterized by producing a porous demineralized bone matrix scaffold having an average inner diameter of 6 to 13 mm and an average outer diameter of 7 to 15 mm.

15. In paragraph 14, The step of forming the above porous demineralized bone matrix support into a tube shape having a through hole is as follows: A step of manufacturing a first porous demineralized bone matrix support having an inner diameter of 6 to 13 mm on average and an outer diameter of 6.5 to 14 mm on average; and A method for producing a graft material for reconstructing fibrocartilage tissue, characterized by comprising the step of producing a second porous demineralized bone matrix support having an inner diameter of 6.5 to 14 mm on average and an outer diameter of 7 to 15 mm on average.

16. In paragraph 9 or 10, A method for producing a graft material for reconstructing fibrocartilage tissue, wherein the first hydrogel composition comprises 0.5 to 10 w / v% of an extracellular matrix derived from autologous or heterologous fibrocartilage.

17. In paragraph 9 or 10, A method for producing a graft material for reconstructing fibrocartilage tissue, wherein the second hydrogel composition comprises 0.5 to 10 w / v% of an extracellular matrix derived from autologous or heterologous fibrocartilage and 50 to 500 μg / ml of a calcification-inducing substance.

18. In paragraphs 9 and 10, The step of preparing the second hydrogel composition is: A second-first hydrogel composition comprising the above-mentioned calcification-inducing substance at a first concentration is prepared, A method for producing a graft material for reconstructing fibrocartilage tissue, characterized in that a second-second hydrogel composition is produced that includes the calcification-inducing substance at a second concentration higher than the first concentration.

19. In paragraph 18, The above 2-1 hydrogel composition is filled into the pores of the 2-1 region of the porous demineralized bone matrix support adjacent to the first region among the second regions, A method for producing a graft material for reconstructing fibrocartilage tissue, characterized in that the 2-2 hydrogel composition is filled into the pores of the 2-2 region of the porous demineralized bone matrix support surrounding the 2-1 region among the 2 regions.

20. In paragraph 9 or 10, A method for producing a graft material for reconstructing fibrocartilage tissue, characterized in that the autologous or heterologous fibrocartilage-derived extracellular matrix is ​​heterologous meniscus cartilage tissue.

21. In paragraph 9 or 10, A method for producing a graft for reconstructing fibrocartilage tissue, wherein the calcification-inducing substance comprises at least one selected from the group consisting of musculoskeletal tissue-derived extracellular vesicles (TEVs), bone morphogenetic protein-2 (BMP-2), and hydroxyapatite (HA).

22. Step of forming a tibial tunnel penetrating the tibia; A step of forming a femoral tunnel that is arranged parallel to the tibial tunnel and penetrates the femur; A step of fixing a ligament or tendon for transplantation to a graft material for fibrocartilage tissue reconstruction according to any one of claims 1 to 8; and A method for reconstructing fibrocartilage tissue between a bone and a ligament or tendon, comprising the step of positioning a graft tendon or ligament fixed to the graft material for reconstructing the fibrocartilage tissue in the tibial tunnel and the femoral tunnel.

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