Guided tissue regeneration cartilage repair material and preparation method thereof

Abstract

The invention discloses a guided tissue regeneration cartilage repair material and a preparation method thereof, and belongs to the technical field of medical materials. The tissue regeneration guided cartilage repair material is composed of three layers of different materials, namely a cartilage repair layer, a semi-permeable isolation layer and a subchondral bone repair layer from top to bottom in sequence, and the three layers of materials are bonded together through a biological adhesive to form a whole. Wherein the cartilage repair layer is composed of homogeneous or heterogeneous cartilage extracellular matrixes, is derived from natural cartilage tissues, is rich in collagen and glycosaminoglycan, has good biocompatibility, multiple growth factors and a vertical pore structure, and is beneficial to adhesion and growth of cells; the semi-permeable isolation layer is composed of a homogeneous or heterogeneous acellular dermal matrix, serves as a barrier, can prevent cells or blood vessels in subchondral bone tissue from growing into cartilage and affecting cartilage repair after being implanted, has permeability, and is beneficial for nutritional ingredients in tissue fluid below the cartilage to permeate into a cartilage area and nourish cartilage cells. The subchondral bone repair layer is a homogeneous or heterogeneous cancellous bone or a decalcified cancellous bone, so that bone tissue repair is facilitated, and clinical operation and fixation are facilitated. According to the three-layer cartilage repair material, in-vivo bone and cartilage repair environments are simulated respectively, bone and cartilage tissues are guided to be synchronously repaired, and the repair effect of articular cartilage defects is improved.

Description

Technical Field

[0001] The invention relates to a medical biomaterial, in particular to the field of cartilage damage repair, and mainly discloses a cartilage repair material for guiding tissue regeneration and a preparation method thereof. Background Art

[0002] Osteochondral defects are common diseases of the joints, often including defects in articular cartilage and subchondral bone. There are many causes of osteochondral damage, such as sports injuries, traffic accidents and various degenerative diseases. In addition, with the increase of age, the natural wear and tear of cartilage tissue can easily cause osteoarthritis, which is also the main cause of osteochondral damage. Although articular cartilage has its corresponding metabolic activities, due to its lack of nerves, blood vessels and lymphatic tissues, the nutrient supply is slow. Once the cartilage is damaged, it is difficult to self-recover and regenerate. As the cartilage damage progresses, if it is allowed to develop, the damage will further expand and even affect the subchondral bone. Therefore, the complete repair of articular cartilage and subchondral bone has always been a major unresolved problem in clinical practice.

[0003] Repairing osteochondral damage is one of the difficult problems in the medical field. On the one hand, cartilage damage involves not only articular cartilage, but also subchondral bone damage; on the other hand, the physiological environment in which articular cartilage is located is not easy to obtain nutrients, and is easily affected by the growth of subchondral bone cells and blood vessels. Therefore, according to the structure and physiological characteristics of the osteochondral damage site, simulating the structure and biochemical composition of natural bone and cartilage defects, providing an ideal environment for the repair of bone and cartilage respectively, guiding the repair of cartilage and bone to proceed simultaneously and promote each other, is the development direction of osteochondral repair materials.

[0004] In recent years, many multi-layer cartilage defect repair methods based on bionic structures have emerged, such as the patent with authorization announcement number CN 101574540B, which announced a tissue engineering bone / cartilage double-layer scaffold and its construction method and application; Zhu Min announced an integrated three-layer composite scaffold and preparation method for articular cartilage repair in 2017 (authorization announcement number CN107485731A); Zhang Mei announced an articular cartilage repair scaffold and its preparation method and application in 2019 (authorization announcement number CN110522536A); Cao Bin announced a cartilage or subchondral bone full-layer repair scaffold and its preparation method in 2021 (authorization announcement number CN110368529B). These patents have all imitated the cartilage tissue structure to a certain extent, but there are some materials that are not composed of natural bone and cartilage matrix materials, and some materials cannot provide a different environment for bone and cartilage repair, and cannot solve the problem of slow nutrient supply to articular cartilage tissue.

[0005] Articular cartilage consists of three parts: the hyaline cartilage upper layer, the mineralized cartilage middle layer, and the subchondral bone lower layer. The hyaline cartilage is mainly composed of type II collagen, proteoglycans, and chondrocytes. It has a flexible and oriented collagen fiber structure, which supports and buffers pressure. The middle mineralized layer acts as a barrier to prevent the growth of cells and blood vessels in the subchondral bone layer. It also has a certain permeability to maintain the relative stability of the microenvironment between the layers. The subchondral bone is composed of cancellous bone. The cancellous bone trabeculae are rich in blood vessels and provide nutrients for themselves and the surrounding adjacent cartilage tissue. Cartilage is an avascular tissue, and its nutrition mainly comes from synovial fluid. Although the blood circulation of the subchondral bone is rich, it is limited by the separation of the interface, and the cartilage obtains very little nutrition from it. The present invention selects biomaterials suitable for the structural characteristics of each layer of articular cartilage according to the structure and physiological characteristics of articular cartilage, and prepares a cartilage repair material that can guide the regeneration of each layer of tissue, wherein the upper layer is a cartilage matrix layer with a longitudinal pore structure, which can provide a guiding scaffold for the growth of chondrocytes; the middle layer is a semi-permeable isolation layer, which can provide nutrient-rich tissue fluid for the cartilage layer while preventing the growth of blood vessels and non-chondrocytes; the lower layer is a subchondral bone repair layer, which provides a scaffold for guiding bone repair. The material is convenient for surgical operation and fixation, and can achieve the effect of full-layer repair. Summary of the invention

[0006] In view of the above-mentioned deficiencies existing in the prior art, the purpose of the present invention is to provide a cartilage repair material and a preparation method for articular cartilage repair, which has the function of guiding each layer of tissue to repair. On the one hand, it guides the cartilage layer and the subchondral bone tissue to repair separately to prevent the two from affecting each other, and at the same time solves the problem of insufficient nutrient source of the cartilage repair layer of the existing material.

[0007] The technical solution adopted by the present invention to achieve the above-mentioned purpose is: prepare subchondral bone repair material, semi-permeable isolation layer and cartilage repair material separately, and bond the above materials together from bottom to top for full-thickness implantation in articular cartilage and subchondral bone defects to facilitate full-thickness repair of cartilage defects.

[0008] A cartilage repair material for guiding tissue regeneration and a preparation method thereof, characterized in that the cartilage repair scaffold material is composed of three layers of materials from top to bottom: cartilage repair material, semi-permeable isolation membrane, and subchondral bone repair material. The three layers of materials are bonded together by an adhesive to obtain the cartilage repair scaffold material.

[0009] The cartilage repair material has a vertically oriented pore structure, which is conducive to the adhesion and growth of chondrocytes and provides an ideal growth microenvironment for cells. The preparation method includes washing, crushing, gradient centrifugation, and freeze drying. The specific steps are as follows: Step 1: Obtain homologous or heterologous articular cartilage tissue under sterile conditions, rinse with sterile buffer for 3 to 10 minutes, soak in 3% hydrogen peroxide solution with a volume ratio of 1:1 for 30 to 100 minutes until there is no blood, and wash with purified water at least 3 times; Step 2: adding purified water to the soft tissue at a material to water volume ratio of 1:1 to 3, and grinding in a grinder below 4°C for 1 minute each time, at least 20 times, to obtain a homogenate; Step 3: The homogenate is subjected to gradient centrifugation at 1000-2000 r / min for at least 30 min to obtain the supernatant, at 4000-6000 r / min for at least 30 min to obtain the supernatant, and at 8000-10000 r / min for at least 30 min to obtain the precipitate; Step 4: Add purified water to the obtained precipitate at a material to water volume ratio of 1:1 to 3, mix thoroughly and add to a cylindrical mold with a diameter of 5 to 7 mm, pre-freeze on an ice table below -30°C for 30 to 60 minutes, and after being completely frozen, enter a freeze dryer precooled to below -20°C for freeze drying for at least 24 hours;

[0010] The semi-permeable isolation layer can prevent red blood cells from passing through or blood vessels from growing in after implantation, while allowing nutrients to pass through, thereby preventing cells from growing into the cartilage layer and causing cartilage hyperplasia. The preparation method includes cleaning, disinfection, skinning, epidermal removal, cell removal, cross-linking, and freeze-drying. The specific steps are as follows: Step 5: Obtain the same or different skin, rinse with purified water for 3 to 10 minutes, disinfect the surface with iodine, and then remove the hair; Step 6, using a drum-type skin extractor to extract a matrix with a thickness of 0.2 to 0.4 mm from the epidermis side; Step 7, adding 0.2-0.5% pancreatic enzyme solution at a volume ratio of solution to material of at least 2:1, and oscillating in a constant temperature shaking incubator for 3-6 hours to remove the epidermis, with a rotation speed of 120 r / min and a temperature of 35±5°C; Step 8, adding 0.2-0.5% sodium dodecyl sulfate (SDS) solution at a volume ratio of solution to material of at least 2:1, and shaking in a constant temperature shaking incubator for 2-4 hours to decellularize at a speed of 120 r / min and a temperature of 35±5°C; Step nine, adding 0.1-0.5% glutaraldehyde solution at a volume ratio of at least 1:1 to the material, soaking for 0.5-10.0 hours for cross-linking, and thoroughly washing after cross-linking; Step 10: freeze-dry the cross-linked dermal matrix for at least 20 hours at a pre-freezing temperature below -35°C, and cut into a circle with a diameter of 5.0 to 7.0 mm.

[0011] The subchondral bone repair layer has a certain strength, which not only plays an osteogenesis role, but also facilitates the operation and fixation of clinicians. The preparation method includes cleaning, molding, degreasing, decalcification, and freeze drying. The specific steps are as follows: Step 11, bone tissue is obtained under sterile conditions, soft tissue is removed with a scalpel, and purified water is sonicated at room temperature until there is no blood; Step 12: ultrasonic cleaning at least 10 times with purified water at 50-60°C, each time for no less than 30 minutes; Step 13, processing the cancellous bone material into a cylinder with a diameter of 5 to 7 mm and a height of 1 to 5 mm on a lathe; Step 14, adding chloroform and formaldehyde in a ratio of 1:1 to prepare a degreasing solution, placing the cancellous bone processed in the previous step into the degreasing solution and soaking it at room temperature for 2 to 10 hours, and then washing it thoroughly with purified water to obtain a cancellous bone matrix; Step 15: Add 0.2-0.5M hydrochloric acid or other acidic solution to the cancellous bone matrix in the previous step, soak and decalcify for 2-10 hours at room temperature, and obtain decalcified cancellous bone matrix after washing.

[0012] Step 16, take the above three materials of the same diameter, and bond them in the order of cartilage repair material, semi-permeable isolation membrane, and subchondral bone repair material from top to bottom using 5% sodium alginate solution or other adhesives, freeze-dry and package them after bonding, and sterilize them by irradiation with a sterilization dose of 10 to 25 kGy.

[0013] In step 4, growth factors that promote cartilage growth can be added to the purified water.

[0014] In the step sixteen, the adhesive may also be any one or more of hyaluronic acid, chitosan, gelatin, collagen, sodium alginate, chondroitin sulfate, and carboxymethyl cellulose.

[0015] Beneficial effects of the present invention: The cartilage repair material for guiding tissue regeneration prepared by the present invention has the following characteristics: (1) According to the repair characteristics of cartilage and bone tissue, a cartilage matrix material that is beneficial to cartilage repair and a bone matrix material that is beneficial to bone repair are respectively used to construct, which is convenient for guiding the repair of bone and cartilage. (2) The application of a semi-permeable isolation membrane can prevent blood vessels and cells in bone tissue from growing into the cartilage area, which affects the repair of cartilage, while not affecting the penetration of nutrients from bone tissue into cartilage tissue, thereby promoting cartilage repair; (3) The cartilage repair layer has a longitudinal pore structure, which is beneficial to the longitudinal growth of chondrocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments of the present application. Obviously, the drawings described below are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0017] Attached Figure 1 Appearance photos of materials prepared for the examples of this application Attached Figure 2 This is a scanning electron microscope photo of the cartilage repair layer in the embodiment of the present application, showing a longitudinal pore structure Attached Figure 3 Scanning electron microscope photos of the front and back sides of the semi-permeable isolation membrane prepared in the examples of the present application, showing gaps or pores of varying degrees Attached Figure 4 Appearance photo of the subchondral bone repair material prepared in the embodiment of this application Attached Figure 5 The photo shows a two-layer cartilage repair material without a semi-permeable isolation membrane with blood dripped onto the subchondral bone repair layer, and the photo was taken after standing for 1 hour. It can be seen that the entire layer of the control group was stained red.

[0018] Attached Figure 6 The three-layer cartilage repair material prepared in the embodiment of the present application was dripped with blood on the subchondral bone repair layer, and the photo was taken after standing for 1 hour. It can be seen that the subchondral bone repair material side is all dyed red, and the cartilage scaffold material side is white. Implementation

[0019] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Embodiment 1

[0020] A cartilage repair material for guiding tissue regeneration and a preparation method thereof, which consists of three layers of materials from top to bottom: a cartilage repair material, a semi-permeable isolation membrane, and a subchondral bone repair material. The three layers of material are bonded together by a biological adhesive to form a whole for implantation in a full-thickness repair of an articular cartilage defect area.

[0021] The preparation method of the cartilage matrix of the cartilage repair layer includes washing, crushing, gradient centrifugation, and freeze drying; the preparation method of the semi-permeable isolation membrane includes washing, disinfection, skinning, epidermal removal, cell removal, cross-linking, and freeze drying; the preparation method of the subchondral bone repair material includes washing, molding, and defatting. The specific steps are as follows: Step 1: Obtain the same type of articular cartilage tissue under sterile conditions, rinse with sterile buffer for 3 minutes, soak in a 3% hydrogen peroxide solution with a volume ratio of 1:1 for 30 minutes until there is no blood, and wash with purified water for 3 times; Step 2: Add purified water to the soft tissue in a material to water volume ratio of 1:1, and grind it in a grinder below 4°C for 1 minute each time, 20 times, to obtain a homogenate; Step 3: The homogenate was subjected to gradient centrifugation at 2000 r / min for 30 minutes, and the supernatant was taken after 30 minutes; at 6000 r / min for 30 minutes, the supernatant was taken after 30 minutes; and at 10000 r / min for 30 minutes, the precipitate was taken; Step 4: Add purified water to the obtained precipitate at a material to water volume ratio of 1:1, mix thoroughly and add to a 6 mm diameter cylindrical mold, place on a -35°C ice table for pre-freezing for 30 minutes, and after being completely frozen, place in a freeze dryer pre-cooled to below -20°C for freeze drying for 24 hours;

[0022] Step 5: Obtain the same skin, rinse with purified water for 5 minutes, disinfect with iodine, and then shave the hair; Step 6, using a drum-type skin extractor to extract a matrix with a thickness of 0.4 mm from the epidermis side; Step 7: Add 0.5% pancreatic enzyme solution at a volume ratio of 2:1 between solution and material, and oscillate in a constant temperature shaking incubator for 3 hours to remove the epidermis, at a speed of 120 r / min and a temperature of 35°C; Step 8, adding 0.5% sodium dodecyl sulfate (SDS) solution at a volume ratio of solution to material of 2:1 and shaking in a constant temperature shaking incubator for 4 hours to decellularize at a temperature of 35°C; Step nine, adding 0.2% glutaraldehyde solution at a volume ratio of 1:1 between the solution and the material, soaking for 0.5h for cross-linking, and thoroughly washing after cross-linking; Step 10, freeze-drying the cross-linked dermal matrix for 24 hours at a pre-freezing temperature of -36°C, and cutting into a circle with a diameter of 6 mm after freeze-drying.

[0023] Step 11, obtaining the same cancellous bone under sterile conditions, removing the soft tissue with a scalpel, and ultrasonically cleaning with room temperature purified water until there is no blood; Step 12: ultrasonically clean the mixture with purified water at 50°C for 10 times, each time for 40 minutes; Step 13, processing the cleaned cancellous bone into a cylinder with a diameter of 6 mm and a height of 2 mm on a lathe; Step 14, the processed cancellous bone cylinder is placed in a 1:1 chloroform and formaldehyde mixed degreasing solution for 4 hours, and then thoroughly washed with purified water to obtain a cancellous bone matrix;

[0024] Step 15, the three materials with a diameter of 6 mm are bonded together in the order of cartilage matrix material, the same type of decellularized dermal matrix, and cancellous bone matrix from top to bottom using 0.8% carboxymethyl cellulose, freeze-dried and packaged after bonding, and sterilized by 15 kGy irradiation. Embodiment 2

[0025] A cartilage repair material for guiding tissue regeneration and a preparation method thereof, which consists of three layers of materials from top to bottom: a cartilage repair material, a semi-permeable isolation membrane, and a subchondral bone repair material. The three layers of material are bonded together by a biological adhesive to form a whole for implantation in a full-thickness repair of an articular cartilage defect area.

[0026] The preparation method of the cartilage matrix of the cartilage repair layer includes washing, crushing, gradient centrifugation, and freeze drying; the preparation method of the semi-permeable isolation membrane includes washing, disinfection, skinning, epidermal removal, cell removal, cross-linking, and freeze drying; the preparation method of the subchondral bone repair material includes washing, molding, defatting, decalcification, and freeze drying. The specific steps are as follows: Step 1: Obtain porcine articular cartilage tissue under sterile conditions, rinse with sterile buffer for 3 minutes, soak in a 3% hydrogen peroxide solution with a volume ratio of 1:1 for 30 minutes until there is no blood, and wash with purified water for 3 times; Step 2: Add purified water to the soft tissue in a material to water volume ratio of 1:1, and grind it in a grinder below 4°C for 1 minute each time, 20 times, to obtain a homogenate; Step 3: The homogenate was subjected to gradient centrifugation at 2000 rpm for 40 min, the supernatant was taken after 40 min, 5000 rpm for 40 min, and the precipitate was taken after 60 min at 9000 rpm. Step 4: Add purified water to the obtained precipitate at a material to water volume ratio of 1:1, mix thoroughly and add to a 6 mm diameter cylindrical mold, place on a -35°C ice table for pre-freezing for 40 minutes, and after being completely frozen, place in a freeze dryer pre-cooled to below -20°C for freeze drying for at least 24 hours;

[0027] Step 5, obtaining qualified pig skin, rinsing with purified water for 10 minutes, disinfecting with iodine tincture and shaving the hair; Step 6, using a drum-type skin extractor to extract a matrix with a thickness of 0.4 mm from the epidermis side; Step 7: Add 0.5% pancreatic enzyme solution at a volume ratio of 2:1 between solution and material, and oscillate in a constant temperature shaking incubator for 6 hours to remove the epidermis, with a rotation speed of 120 r / min and a temperature of 35±2°C; Step 8, adding 0.5% sodium dodecyl sulfate (SDS) solution at a volume ratio of solution to material of 2:1 and shaking in a constant temperature shaking incubator for 4 hours to decellularize at a temperature of 35±2°C; Step nine, adding 0.2% glutaraldehyde solution at a volume ratio of 1:1 between solution and material and soaking for 1 hour for cross-linking, and thoroughly washing after cross-linking; Step 10: freeze-dry the cross-linked dermal matrix for 24 hours at -35°C, and cut into a circle with a diameter of 6 mm.

[0028] Step 11, obtaining cancellous bones of pig limbs under sterile conditions, removing soft tissues with a scalpel, and ultrasonically cleaning with purified water until there is no blood; Step 12: ultrasonically clean the mixture with purified water at 55°C for 10 times, each time for 50 minutes; Step 13, processing the cleaned porcine cancellous bone into a cylinder with a diameter of 6 mm and a height of 3 mm; Step 14, the processed cancellous bone cylinder is placed in a 1:1 chloroform and formaldehyde mixed degreasing solution for 4 hours, and then thoroughly washed with purified water to obtain a cancellous bone matrix; Step 15, adding the above cancellous bone matrix to a 0.5M hydrochloric acid solution for decalcification for 4 hours, and washing thoroughly with purified water to obtain a decalcified cancellous bone matrix; Step 16, the three materials with a diameter of 6 mm are bonded together in the order of cartilage matrix material, decellularized dermal matrix semipermeable membrane, and decalcified cancellous bone matrix from top to bottom using 2% sodium alginate solution, and after bonding, they are freeze-dried and packaged, and sterilized by 20 kGy irradiation.

[0029] Comparative Example 1 The experimental group in this comparative example is a three-layer cartilage repair material, and its preparation method is the same as that described in Example 1. The control group is a two-layer cartilage repair material without a semi-permeable isolation membrane, and its preparation method does not add a semi-permeable isolation membrane, and other steps are the same as those in Example 1.

[0030] Blood was dripped on the subchondral bone repair material side of the experimental group and the control group respectively to observe the blood penetration and record whether red blood cells could reach the side of the cartilage scaffold material.

[0031] After 1 hour, the subchondral bone repair material side of the experimental group was completely stained red, and the cartilage scaffold material side was white; the entire layer of the control group was stained red.

[0032] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0033] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A cartilage repair material for guiding tissue regeneration and a preparation method thereof, characterized in that: The guided tissue regeneration cartilage repair material comprises, from top to bottom, a cartilage repair layer composed of acellular cartilage matrix, a semi-permeable isolation layer composed of acellular dermal matrix, and a subchondral bone repair layer composed of cancellous bone matrix or / and decalcified cancellous bone matrix. The three layers of material are bonded together as a whole by biological adhesive or / and physical bonding.

2. A cartilage repair material for guiding tissue regeneration and a preparation method thereof according to claim 1, characterized in that: The decellularized cartilage matrix of the cartilage repair layer is an extracellular matrix derived from homologous or heterologous cartilage and has a longitudinal pore structure.

3. The guided tissue regeneration cartilage repair material and preparation method thereof according to claim 1, characterized in that: The acellular dermal matrix of the semi-permeable isolation layer is derived from the same skin or different skin.

4. The guided tissue regeneration cartilage repair material and preparation method thereof according to claim 1, characterized in that: The cancellous bone matrix or decalcified cancellous bone matrix of the subchondral bone repair layer is derived from allogenic bone or xenogenic bone.

5. A cartilage repair material for guiding tissue regeneration and a preparation method thereof according to claims 2-3, characterized in that: The same species refers to humans, and the different species refers to animals, including monkeys, pigs, cattle, sheep, horses, dogs, and mice.

6. The guided tissue regeneration cartilage repair material and preparation method thereof according to claim 1, characterized in that: The preparation method of the decellularized cartilage matrix is ​​as follows: (1) Obtain articular cartilage under sterile conditions, rinse with sterile buffer for 3 to 10 minutes, and soak in 1 to 3% hydrogen peroxide solution for 30 to 100 minutes until there is no blood; (2) Add purified water to the articular cartilage prepared in step (1) at a material to water volume ratio of 1:1 to 3, and grind in a grinder at a temperature below 4°C; (3) The homogenate obtained in step (2) is subjected to gradient centrifugation at 1000-2000 rpm for at least 30 min, and the supernatant is collected; the supernatant is collected by centrifugation at 4000-6000 rpm for at least 30 min; the precipitate is collected by centrifugation at 8000-10000 rpm for at least 30 min; (4) Add purified water to the precipitate in step (3) at a material to water volume ratio of 1:1 to 3, mix thoroughly and add to a cylindrical mold with an inner diameter of 5 to 7 mm, place on an ice table below -30°C for pre-freezing for 30 to 60 min, and freeze-dry after freezing.

7. The guided tissue regeneration cartilage repair material and preparation method thereof according to claim 1, characterized in that: The preparation method of the acellular dermal matrix is ​​as follows: (1) Obtain homologous or heterologous skin tissue, rinse with purified water for 3 to 10 minutes, and remove the hair after surface disinfection; (2) The thickness of the matrix obtained from the epidermis side of the skin extractor is 0.2-0.4 mm; (3) Place the matrix in step (2) into a 0.2-0.5% pancreatic enzyme solution and shake it in a constant temperature shaking incubator at 35±5°C for 3-6 hours to remove the epidermis and obtain the dermis; (4) placing the dermis obtained in step (3) in a 0.2-0.5% sodium dodecyl sulfate (SDS) solution and shaking in a constant temperature shaking incubator at 35±5°C for 2-4 hours to perform decellularization; (5) soaking the decellularized dermis obtained in step (4) in a 0.1-0.5% glutaraldehyde solution for crosslinking; (6) After washing, freeze-dry and cut into circles with a diameter of 5 to 7 mm.

8. The guided tissue regeneration cartilage repair material and preparation method thereof according to claim 1, characterized in that: The preparation methods of the cancellous bone matrix and the decalcified cancellous bone matrix are as follows: (1) Obtain cancellous bone tissue under sterile conditions, remove soft tissue, and ultrasonically clean with purified water at room temperature until there is no blood; (2) ultrasonically cleaning the cancellous bone in step (1) in purified water at 50 to 60° C. for at least 10 times, each time for no less than 30 minutes; (3) processing the cancellous bone in step (2) into a cylindrical shape with a diameter of 5 to 7 mm and a height of 1 to 5 mm; (4) degreasing the cancellous bone obtained in step (3) in a 1:1 mixed degreasing solution of chloroform and formaldehyde for 2 to 10 hours, and washing with purified water to obtain a cancellous bone matrix; (5) Decalcified cancellous bone matrix is ​​obtained by placing the cancellous bone matrix in step (4) in a 0.2-0.5 M hydrochloric acid solution for 2-10 hours and then thoroughly washing with purified water.

9. The guided tissue regeneration cartilage repair material and preparation method thereof according to claim 1, characterized in that: The bonding method of the three layers of materials is as follows: (1) Decellularized cartilage matrix, decellularized dermal matrix, cancellous bone matrix or decalcified cancellous bone matrix of the same diameter are bonded together using an adhesive in the order of cartilage repair layer, semi-permeable isolation layer and subchondral bone repair layer from top to bottom.

10. (2) The adhesive in step (1) is any one or more of hyaluronic acid, chitosan, gelatin, collagen, sodium alginate, chondroitin sulfate, and carboxymethyl cellulose aqueous solution.

11. (3) After bonding, freeze-dry and package, and sterilize by irradiation.

12. (4) The radiation sterilization dose in step (3) is 10 to 25 kGy.

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