Cartilage repair membrane and its preparation method

Abstract

The present invention provides a cartilage repair membrane and a preparation method thereof. The cartilage repair membrane includes an umbilical cord layer, a first gel layer, and a second gel layer that are stacked in sequence. The umbilical cord layer is a sheet-like structure composed of umbilical cord tissue, and the umbilical cord tissue is formed from an umbilical cord from which vascular cells have been removed by mechanical means. Both the first gel layer and the second gel layer are formed by mixing and cross-linking chitosan, sodium alginate, and hydroxyapatite, and the concentration of hydroxyapatite in the first gel layer and the second gel layer is different. The cartilage repair membrane of the present invention has good biological activity, mechanical properties, porosity, and osteogenic properties, provides the best repair effect for the treatment of cartilage defects, and has significant effects in aspects such as antibacterial, hemostatic, promoting the adhesion, proliferation of osteoblasts, and the formation of mineralized bone matrix, can meet the requirements of clinical and tissue engineering to the greatest extent, and can improve the repair degree of articular cartilage injury.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly to a cartilage repair membrane and a preparation method thereof. Background Art

[0002] Osteochondral tissue covers the two ends of long bones to protect them from mechanical damage. Osteochondral tissue is a multi-layered tissue composed of cartilage, calcified cartilage and subchondral bone. The distribution of its water content, extracellular matrix (ECM), etc. in articular osteochondral tissue changes with the longitudinal gradient. As an interface tissue, osteochondral tissue exhibits different gradient characteristics in terms of biochemistry, biomechanics, structure and metabolism. Among them, from articular cartilage to subchondral bone, the mechanical properties of osteochondral tissue change gradiently with the longitudinal depth, and the change in mechanical behavior is most likely caused by the increase in the content of inorganic calcium salts. Osteochondral tissue is composed of extracellular matrix and chondrocytes, and has the characteristics of avascularity, no tension and weak regeneration potential. Therefore, once osteochondral tissue is damaged, it is difficult to repair itself, which leads to the occurrence of osteoarthritis and seriously affects the quality of life of patients.

[0003] At present, tissue-engineered cartilage transplantation repair is one of the treatment methods for osteochondral repair. Its basic principle is to combine biomaterial scaffolds, seed cells and growth factors in vitro and then implant them into bone defects. Through the degradation, absorption of biomaterials and the proliferation of autologous bone tissue, the complete repair of bone tissue in terms of morphology and function is finally achieved. Ideal bone tissue engineering materials require characteristics such as good biodegradability, biocompatibility, osteoinductivity, processability, promotion of cartilage formation and mechanical suitability. However, most of the currently common cartilage repair materials are constructed of a single component, and their repair efficiency is limited.

[0004] Chinese Patent with publication number CN102178981A discloses a preparation method of a cartilage repair scaffold material. Using animal dermal matrix as the raw material, a cartilage repair scaffold is obtained through processes such as decellularization, cross-linking, freeze-drying and surface modification with chondroitin sulfate. The above method uses a single material to prepare a single-phase scaffold, which cannot meet the complexity of the osteochondral matrix. Moreover, the single-phase cartilage scaffold has problems such as poor biocompatibility, poor mechanical properties, and inability to achieve a certain separation of different cell growth microenvironments.

[0005] Therefore, it is necessary to provide a novel cartilage repair membrane and a preparation method thereof to solve the above problems existing in the prior art. Summary of the Invention

[0006] The object of the present invention is to provide a cartilage repair membrane and a preparation method thereof, which have good biological activity, mechanical properties, porosity and osteogenic properties, provide the best repair effect for the treatment of cartilage defects, and solve the problems of poor biocompatibility, poor mechanical properties and inability to achieve a certain separation of different cell growth microenvironments in the single-phase cartilage scaffold in the prior art.

[0007] To achieve the above object, the cartilage repair membrane of the present invention comprises an umbilical cord layer, a first gel layer and a second gel layer which are sequentially laminated; the umbilical cord layer is a sheet-like structure composed of umbilical cord tissue, and the umbilical cord tissue is formed from an umbilical cord from which vascular cells have been removed by a mechanical method; both the first gel layer and the second gel layer are formed by mixing and crosslinking chitosan, sodium alginate and hydroxyapatite, and the concentrations of the hydroxyapatite in the first gel layer and the second gel layer are different.

[0008] The beneficial effects of the cartilage repair membrane of the present invention are as follows: The umbilical cord layer is a sheet structure composed of umbilical cord tissue, and the umbilical cord tissue is formed from an umbilical cord with vascular cells removed by mechanical means, that is, the cartilage repair membrane prepares a cartilage matrix material from the umbilical cord layer to serve as a three-dimensional porous structure scaffold for filling cartilage defects. Its main component is collagen, containing a large amount of collagen, and retaining a variety of natural active ingredients. It can recruit endogenous cells to the injury site, can simulate the microenvironment of the natural extracellular matrix to promote the proliferation and differentiation of the recruited cells, can achieve rapid vascularization and the repair and regeneration of the cartilage defect area, achieve the purpose of in-situ regeneration, and has the effects of inhibiting scar formation and osteogenesis; The first gel layer and the second gel layer are both formed by mixing and cross-linking chitosan, sodium alginate, and hydroxyapatite, and the concentrations of hydroxyapatite in the first gel layer and the second gel layer are different, so that the first gel layer and the second gel layer are a type of extremely hydrophilic hydrogel with a three-dimensional network structure, which is very similar to the extracellular matrix of natural cartilage. It can swell and retain a large amount of water, facilitating the loading of cells and factors, and having the ability of controlled release; At the same time, the multi-functional group characteristics of the hydrogel molecules make it easy to be functionalized, thereby improving its effectiveness as a scaffold and carrier. By sequentially laminating the first gel layer and the second gel layer on the umbilical cord layer, that is, setting two gel layers with gradients of different hydroxyapatite concentrations on the umbilical cord layer, it can provide the necessary mechanical support for the osteochondral tissue at each level in different longitudinal gradients, enabling the prepared cartilage repair membrane to better simulate the interface between bone and cartilage, and improving the mechanical strength, toughness, and stability of the cartilage repair membrane, which is suitable for the repair and regeneration of tissue-engineered cartilage. The present invention combines the components and performance characteristics of the raw materials to the greatest extent and compensates for each other's deficiencies, forming a composite multi-layer cartilage repair membrane for bionic construction of tissue-engineered cartilage gradient repair, having good biological activity, mechanical properties, porosity, and osteogenic properties, providing the best repair effect for cartilage defect treatment, and the cartilage repair membrane has significant effects in aspects such as antibacterial, hemostatic, promoting the adhesion, proliferation, and formation of mineralized bone matrix of osteoblasts, can meet the requirements of clinical and tissue engineering to the greatest extent, and can improve the repair degree of articular cartilage injury. It solves the problems in the prior art that single-phase cartilage scaffolds have poor biocompatibility, poor mechanical properties, and cannot achieve a certain separation of different cell growth microenvironments, etc.

[0009] Preferably, the concentration of hydroxyapatite in the first gel layer is less than that in the second gel layer. The beneficial effect is as follows: Osteochondral is a tissue with a multi-layer structure. In the distribution of articular osteochondral tissue, the content of mineral components, mainly hydroxyapatite, gradually increases along the longitudinal gradient direction and towards the interior of the osteochondral tissue. When the cartilage repair membrane is applied to the tissue defect, the second gel layer with a high hydroxyapatite concentration and the first gel layer with a low hydroxyapatite concentration are successively in contact with the tissue defect. In this way, the gradient distribution of hydroxyapatite in the articular osteochondral tissue is simulated, so that the double-layer hydroxyapatite gel layers with different contents can provide necessary mechanical support for osteochondral tissues at different longitudinal gradients and can better simulate the bone-cartilage interface.

[0010] Preferably, the concentration of hydroxyapatite in the first gel layer is 5 g / L to 7 g / L, and the concentration of hydroxyapatite in the second gel layer is 8 g / L to 10 g / L. The beneficial effect is as follows: The concentration of hydroxyapatite set in the first gel layer and the second gel layer is beneficial to maintaining the pore structure in the first gel layer and the second gel layer, and can also prevent the brittleness of the first gel layer and the second gel layer from being too high due to excessive hydroxyapatite concentration; moreover, the double-layer hydroxyapatite gel layers with different contents can provide necessary mechanical support for osteochondral tissues at different longitudinal gradients and can better simulate the bone-cartilage interface.

[0011] Preferably, the mass ratio of hydroxyapatite, chitosan and sodium alginate in the first gel layer is (1:2:2) to (1:4:8) ； The mass ratio of hydroxyapatite, chitosan and sodium alginate in the second gel layer is (1:1:1) to (1:2:4). The beneficial effect is as follows: This ratio enables the prepared first gel layer and second gel layer to maintain the morphology of a three-dimensional porous network structure and does not make the prepared cartilage repair membrane too brittle.

[0012] Preferably, the width of the cartilage repair membrane is 20 mm to 30 mm, and the length is 20 mm to 60 mm. The beneficial effect is as follows: Different specifications and sizes can meet different shaped wounds.

[0013] Preferably, the surface of the umbilical cord layer facing away from the first gel layer is a smooth surface, and the surface of the second gel layer facing away from the first gel layer is an indented surface, and the indented surface is provided with a convex structure and a concave structure. The beneficial effects are as follows: The cartilage repair membrane has a double-sided structure with an indented surface and a smooth surface. The double-sided structure can fit well with the part to be repaired, providing a relatively closed tissue growth environment, providing good space and time for bone regeneration, and promoting tissue repair and regeneration. Among them, the convex structure and the concave structure on the indented surface can effectively increase the degree of fit and the contact surface area between the cartilage repair membrane and the part to be repaired, improve the degree of fit between the cartilage repair membrane and the tissue defect, and improve the clinical use and treatment effect. Moreover, the cartilage repair membrane with this double-sided structure not only fits well with the bone tissue to be repaired, can selectively guide cell ingrowth, but also can be degraded synchronously with the reconstruction of the new tissue. It solves the problem in the prior art that there is a gap between the biofilm with smooth surfaces on both sides and the tissue defect, resulting in fibroblasts and epithelial cells with a faster growth and migration rate entering the bone defect area, forming a competitive inhibition with osteoblasts with a slower growth and migration rate, and having an adverse impact on the healing of bone tissue.

[0014] Preferably, the first gel layer is coated on the umbilical cord layer and fixed by pressing and freeze-drying with a mold; the second gel layer is coated on the first gel layer and fixed by pressing and freeze-drying with a mold; the mold includes a first surface and a second surface arranged oppositely, the first surface is an indented surface adapted to the indented surface, the indented surface is provided with a convex structure and a concave structure, and the second surface is a smooth surface adapted to the smooth surface. The beneficial effects are as follows: Using this mold can press and freeze-dry the cartilage repair membrane into a double-sided structure with different structures; that is, the indented surface provided with the convex structure and the concave structure makes one surface of the cartilage repair membrane pressed and freeze-dried have a convex structure and a concave structure, so as to effectively increase the degree of fit and the contact surface area between the cartilage repair membrane and the part to be repaired, improve the degree of fit between the cartilage repair membrane medical material and the tissue defect, and improve the clinical use and treatment effect. It solves the problem in the prior art that there is a gap between the biofilm with smooth and flat surfaces on both sides and the tissue defect, resulting in fibroblasts and epithelial cells with a faster growth and migration rate entering the bone defect area, forming a competitive inhibition with osteoblasts with a slower growth and migration rate, and having an adverse impact on the healing of bone tissue.

[0015] Preferably, the umbilical cord layer is a double-layer porous structure. The umbilical cord layer includes a dense layer and a loose layer. The pore size of the dense layer is smaller than that of the loose layer. The loose layer is fixed to the first gel layer, and the smooth surface is provided on the dense layer. The beneficial effects are as follows: The cartilage repair membrane is used to be placed on the surface of damaged bone tissue to create a relatively closed bone regeneration environment. Therefore, the dense layer with a small pore size facing the bone tissue can block fibroblasts and epithelial cells with a relatively fast migration speed from entering the bone defect area, playing a barrier role. Compared with the prior art where the two different structures in the double-layer membrane structure are simply fixed by pressing, in the present invention, the loose layer is adhered to the gel layer. Through freeze-thaw treatment, the gel layer is adsorbed into the micropores of the loose layer of the umbilical cord layer, enhancing the adhesion between the gel layer and the umbilical cord layer, that is, greatly improving the bonding force between the bonding interfaces, and solving the problem of insufficient bonding force and easy delamination between the membrane layers in the prior art.

[0016] Preferably, the pore size of the loose layer is 20 μm to 100 μm. The beneficial effects are as follows: The umbilical cord itself is a natural double-sided structure. The dense layer retains the natural collagen fiber dense structure of the umbilical cord and is used to face the bone tissue, which can effectively prevent fibroblasts and epithelial cells from growing into the bone defect area, support the formation of bone tissue, and prevent soft tissue from growing into the bone defect area. The collagen arrangement in the loose layer is relatively sparse and has a certain pore size, which can achieve close binding in some areas with the gel layer during the freeze-thaw process, realizing the preparation of a double-layer membrane that does not delaminate after rehydration. Therefore, when applied to bone tissue defects, it can effectively play the respective roles of the dense layer and the loose layer.

[0017] Preferably, the umbilical cord is an allogeneic umbilical cord. The beneficial effects are as follows: The allogeneic umbilical cord has a porous double-layer structure, with one layer having a dense arrangement of collagen fibers and the other layer having a relatively loose arrangement of collagen fibers. And it contains a variety of bioactive components, has low immunogenicity, and has unique biological characteristics of promoting cell adhesion and tissue regeneration and healing, which has important significance in clinical applications.

[0018] Further preferably, the umbilical cord tissue is of human origin. The beneficial effects are as follows: Using human-derived umbilical cord tissue as the basal layer breaks the limitation of preparing cartilage repair membranes with animal-derived raw materials, and can effectively avoid rejection reactions in patients; the umbilical cord contains basic fibroblast growth factor, insulin-like growth factor, platelet-derived growth factor, transforming growth factor, etc., and can be used as a sustained-release carrier of growth factors to promote cell proliferation and osteogenic activity; moreover, human-derived umbilical cord has low immunogenicity, and its natural active ingredients are mainly composed of collagen, glycoprotein, amino group, glucan, mucopolysaccharide, and growth factors. It also retains a variety of bioactive factors, has low immunogenicity and good biocompatibility. It can not only provide a bionic three-dimensional support carrier for cells, but also promote the migration, proliferation, and differentiation of host cells through various proteins and growth factors to promote tissue regeneration and repair. The umbilical cord layer is derived from human umbilical cord tissue with vascular cells removed by mechanical means, and the cell matrix is removed, and its main component is collagen. Human-derived umbilical cord tissue is often regarded as "medical waste", and its application cost is correspondingly low.

[0019] The preparation method of the cartilage repair membrane of the present invention includes the following steps:

[0020] Provide the original umbilical cord, remove the blood vessels in the original umbilical cord by mechanical means to form the umbilical cord tissue to be shaped, and trim the umbilical cord tissue to be shaped into a sheet-like structure of the umbilical cord layer;

[0021] Mix and crosslink sodium alginate solution, chitosan solution, and two different concentrations of hydroxyapatite dispersion liquids respectively to obtain two gel solutions with different hydroxyapatite concentrations;

[0022] Coat the two gel solutions with different hydroxyapatite concentrations on one side of the umbilical cord layer in sequence to prepare the first gel layer and the second gel layer, and then freeze-dry to obtain the cartilage repair membrane.

[0023] The beneficial effects of the method for preparing the cartilage repair membrane of the present invention are as follows: By providing an original umbilical cord, the blood vessels in the original umbilical cord are mechanically removed to form umbilical cord tissue to be shaped. The umbilical cord tissue to be shaped is trimmed into a sheet-like umbilical cord layer, that is, the cartilage repair membrane is prepared from the umbilical cord layer to obtain a cartilage matrix material, which serves as a three-dimensional porous structure scaffold for filling cartilage defects. Its main component is collagen, containing a large amount of collagen, and retaining a variety of natural active ingredients. It can recruit endogenous cells to the injury site, can simulate the microenvironment of the natural extracellular matrix to promote the proliferation and differentiation of the recruited cells, can achieve rapid vascularization and the repair and regeneration of the cartilage defect area, achieve the purpose of in-situ regeneration, and has the effects of inhibiting scar formation and osteogenesis; By separately mixing and cross-linking sodium alginate solution, chitosan solution and two different concentrations of hydroxyapatite dispersion liquids, two gel solutions with different hydroxyapatite concentrations are obtained; The two gel solutions with different hydroxyapatite concentrations are sequentially coated on one side of the umbilical cord layer to prepare a first gel layer and a second gel layer, so that the first gel layer and the second gel layer are a type of extremely hydrophilic hydrogel with a three-dimensional network structure, which is very similar to the extracellular matrix of natural cartilage. It can swell and retain a large amount of water, is convenient for loading cells and factors, and has the ability of controlled release; At the same time, the multi-functional group characteristics of the hydrogel molecules make it easy to be functionalized, thereby improving its efficiency as a scaffold and carrier. By sequentially coating the two gel solutions with different hydroxyapatite concentrations on one side of the umbilical cord layer to prepare a first gel layer and a second gel layer, that is, two gel layers with gradients of different hydroxyapatite concentrations are arranged on the umbilical cord layer, so that it can provide necessary mechanical support for the osteochondral tissue at each level in different longitudinal gradients, enabling the prepared cartilage repair membrane to better simulate the interface between bone and cartilage, and improving the mechanical strength, toughness and stability of the cartilage repair membrane, and being suitable for the repair and regeneration of tissue-engineered cartilage. The present invention maximally combines the components and performance characteristics of the raw materials and compensates for each other's deficiencies, forming a composite multi-layer cartilage repair membrane for bionic construction of tissue-engineered cartilage gradient repair, having good biological activity, mechanical properties, porosity and osteogenic properties, providing the best repair effect for cartilage defect treatment, and the cartilage repair membrane has remarkable effects in aspects such as antibacterial, hemostatic, promoting the adhesion, proliferation of osteoblasts and the formation of mineralized bone matrix, can meet the requirements of clinical and tissue engineering to the greatest extent, and can improve the repair degree of articular cartilage injury. It solves the problems in the prior art that single-phase cartilage scaffolds have poor biocompatibility, poor mechanical properties, and cannot achieve a certain separation of different cell growth microenvironments, etc.

[0024] Preferably, the step of sequentially coating the two gel solutions with different hydroxyapatite concentrations on one side of the umbilical cord layer to prepare a first gel layer and a second gel layer includes:

[0025] Coat the gel solution with a low hydroxyapatite concentration in the gel solution on one side of the umbilical cord layer, and obtain the first gel layer after pressing and freeze-drying with a mold;

[0026] Coat the gel solution with a high hydroxyapatite concentration in the gel solution on the side of the first gel layer facing away from the umbilical cord layer, and obtain the second gel layer after pressing and freeze-drying with a mold. The beneficial effects are as follows: Osteochondral is a tissue with a multi-layer structure. In the distribution of articular osteochondral tissue, the content of the mineral component, that is, mainly hydroxyapatite, gradually increases along the longitudinal gradient direction and towards the inside of the osteochondral tissue. When the cartilage repair membrane is applied to the tissue defect, the second gel layer with a high hydroxyapatite concentration and the first gel layer with a low hydroxyapatite concentration are successively in close contact with the tissue defect. In this way, the gradient distribution of hydroxyapatite in the articular osteochondral tissue is simulated, so that the double-layer gel layers with different contents of hydroxyapatite can provide the necessary mechanical support for the osteochondral tissue at different longitudinal gradients and can better simulate the bone-cartilage interface.

[0027] Preferably, the step of coating the gel solution with a low hydroxyapatite concentration in the gel solution on one side of the umbilical cord layer and obtaining the first gel layer after pressing and freeze-drying with a mold includes:

[0028] Cover the smooth surface of the mold with the side of the dense layer in the umbilical cord layer;

[0029] Uniformly coat 1 mL to 2 mL of the gel solution with a low hydroxyapatite concentration of 5 g / L to 7 g / L on the side of the loose layer in the umbilical cord layer;

[0030] Cover the indentation surface of another mold on the side of the umbilical cord layer coated with the gel solution with a low hydroxyapatite concentration;

[0031] Freeze the mold in the pressed state at a temperature of -5 °C to -20 °C for at least 4 h to obtain the first gel layer. The beneficial effects are as follows: After the pressing and freeze-drying treatment with the mold, the loose layer of the sheet umbilical cord layer is bonded to the first gel layer, and through subsequent freeze-thaw cycles and vacuum freeze-drying treatment, the bonding degree between the loose layer and the gel layer can be deeper, there is no obvious delamination, and the structural continuity is good.

[0032] Preferably, the step of coating the gel solution with a high hydroxyapatite concentration in the gel solution on the side of the first gel layer facing away from the umbilical cord layer and obtaining the second gel layer after pressing and freeze-drying with a mold includes:

[0033] Remove another mold covering the first gel layer;

[0034] Apply 1 mL to 2 mL of the gel solution with a high hydroxyapatite concentration of 8 g / L to 10 g / L evenly on the side of the first gel layer facing away from the umbilical cord layer within 3 min to 5 min.

[0035] Cover the indented surface of the other mold on the side of the first gel layer coated with the gel solution with a high hydroxyapatite concentration.

[0036] Freeze the mold in the pressed state at a temperature of -6°C to -20°C for 4 h to 8 h to obtain the second gel layer. The beneficial effect is that after the mold pressing and freeze-drying treatment, the cartilage repair membrane has a double-sided structure with an indented surface and a smooth surface. Usually, the flatness of the surface to be repaired is not good. The presence of the indented structure can effectively increase the fitting degree and the contact surface area between the cartilage repair membrane and the part to be repaired, which is beneficial to promoting tissue healing. Moreover, the second gel layer has an indented surface, so that the indented surface of the second gel layer with hydroxyapatite faces the bone defect, which can improve the survival rate of osteoblasts and the amount of osteogenic mineralization at the same time.

[0037] Preferably, the step of freeze-drying to obtain the cartilage repair membrane includes: performing 4 to 10 times of repeated freeze-thaw treatment on the preform containing the umbilical cord layer, the first gel layer, and the second gel layer; the parameters of the freeze-thaw treatment are: placing the preform at a temperature of 0°C to 20°C for 10 min to 60 min, and immediately placing the preform at a temperature of -6°C to -20°C for freezing for 10 min to 60 min when the surface of the second gel layer shows a dissolved state. The beneficial effect is that repeated freeze-thaw treatment is performed to enhance the bonding strength between the loose layer of the umbilical cord layer and the first gel layer, and between the first gel layer and the second gel layer. That is, through the freeze-thaw treatment, the first gel layer can be adsorbed into the micropores of the loose layer of the umbilical cord layer, which enhances the adhesion between the first gel layer and the umbilical cord layer, and at the same time enhances the adhesion between the first gel layer and the second gel layer. That is, the bonding force between the fitting interfaces is greatly improved, solving the problem of insufficient bonding force and easy delamination between the film layers in the prior art.

[0038] Preferably, the step of preparing the cartilage repair membrane by freeze-drying further includes: after subjecting the pre-product to repeated freeze-thaw treatment, gradually cooling it to -80 ± 5°C at a rate of 5°C / min to 10°C / min at a freezing temperature of -20 ± 5°C, and then performing vacuum freeze-drying at a temperature of -80 ± 5°C for 8 h to 20 h to obtain the cartilage repair membrane. The beneficial effects are as follows: during the vacuum freeze-drying process, by slowly cooling to -80 ± 5°C at a rate of 5°C / min to 10°C / min, it is possible to avoid the destruction of tissue structure caused by too rapid temperature drop, effectively retain the ultrastructure of the umbilical cord matrix, significantly improve the drying efficiency of the cartilage repair membrane, and obtain a relatively uniform pore structure, so that while maintaining a certain sealing effect, oxygen, blood, and bioactive substances can enter, promoting local cell growth and osteochondral regeneration.

[0039] Preferably, the step of separately mixing and cross-linking the sodium alginate solution, the chitosan solution, and two different concentrations of hydroxyapatite dispersion liquids to obtain gel solutions with two different hydroxyapatite concentrations includes:

[0040] Dissolve chitosan in an acetic acid aqueous solution with a concentration of 1 wt% to 2 wt% and stir well to obtain the chitosan solution with a concentration of 8 g / L to 20 g / L;

[0041] Dissolve sodium alginate in deionized water and stir well to obtain the sodium alginate solution with a concentration of 8 g / L to 40 g / L;

[0042] Dissolve and disperse hydroxyapatite in deionized water and stir well to obtain the hydroxyapatite dispersion liquids with concentrations of 5 g / L to 7 g / L and 8 g / L to 10 g / L respectively;

[0043] Sequentially add the chitosan solution, the hydroxyapatite dispersion liquid, and the cross-linking agent solution to the sodium alginate solution, and stir and cross-link well to obtain the gel solutions with two different hydroxyapatite concentrations. The beneficial effects are as follows: for the preparation of the gel solution, first prepare the sodium alginate solution, and then slowly add the chitosan solution to the sodium alginate solution. This addition sequence can avoid the problems of poor adhesion and connectivity between the internal pores of the material, thereby obtaining a porous cartilage repair membrane with an appropriate pore structure, good elasticity and mechanical properties, and a uniform pore structure distribution, without adhesion and uneven size distribution.

[0044] Preferably, the crosslinking agent is any one or two of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, genipin and formaldehyde. The beneficial effect is that it is beneficial to further improve the mechanical properties of the first gel layer and the second gel layer, that is, it can effectively improve the mechanical properties of the cartilage repair membrane.

[0045] Preferably, the crosslinking agent is a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Through EDC crosslinking, the cartilage repair membrane has the characteristics of non-toxicity and good biocompatibility, and the collagen-based scaffold crosslinked by EDC shows more excellent cell compatibility. During the crosslinking process, EDC and NHS do not enter the collagen matrix but are converted into water-soluble urea derivatives, which can be eliminated or washed away.

[0046] More preferably, the molar ratio of the EDC to the NHS is (4:1).

[0047] Preferably, the chitosan solution and the hydroxyapatite dispersion liquid are sequentially added to the sodium alginate solution to prepare a mixed gel liquid, and the mass ratio of the crosslinking agent solution to the mixed gel liquid is (20:1) to (40:3).

[0048] Preferably, the mass ratio of the hydroxyapatite, the chitosan to the sodium alginate in the gel solution with a low hydroxyapatite concentration is (1:2:2) to (1:4:8). ； The mass ratio of the hydroxyapatite, the chitosan to the sodium alginate in the gel solution with a high hydroxyapatite concentration is (1:1:1) to (1:2:4). The beneficial effect is that this ratio enables the prepared first gel layer and second gel layer to maintain the morphology of a three-dimensional porous network structure and does not make the cartilage repair membrane too brittle.

[0049] Preferably, the step of trimming the umbilical cord tissue to be shaped into a sheet-like umbilical cord layer includes: after washing the umbilical cord tissue to be shaped with a cleaning solution, trimming it into a sheet and washing it with deionized water 1 to 3 times to obtain the umbilical cord layer, and the cleaning temperature does not exceed 15°C, and the cleaning time does not exceed 5 minutes. The cleaning solution is any one or more of sterile physiological phosphate buffer solution, sterile sodium chloride injection solution and purified water. The beneficial effect is that the umbilical cord tissue is washed with a cleaning solution to wash away blood stains and destroy cells, and rinsed with deionized water to keep the umbilical cord layer moist, so as to facilitate adhesion to the gel layer. By keeping the cleaning temperature not exceeding 15°C and the cleaning time not exceeding 5 minutes, it is quickly processed in a low-temperature environment to retain the three-dimensional collagen structure and natural active ingredients in the umbilical cord to the greatest extent. Description of the Drawings

[0050] Figure 1 Schematic diagram of the electron microscope scan of the longitudinal section of the cartilage repair membrane of the present invention;

[0051] Figure 2 Schematic diagram of the mold of the first embodiment of the present invention;

[0052] Figure 3 Schematic diagram of the mold of the second embodiment of the present invention;

[0053] Figure 4 is Figure 3 Enlarged schematic diagram of the structure of the surface of the first layer body in the mold shown;

[0054] Figure 5 is Figure 3 Enlarged schematic diagram of the structure of the surface of the second layer body in the mold shown. Detailed implementation manners

[0055] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present invention. Unless otherwise defined, the technical terms or scientific terms used herein should have the ordinary meaning understood by those of ordinary skill in the art to which the present invention belongs. The words such as "including" used herein mean that the elements or objects appearing before the word cover the elements or objects listed after the word and their equivalents, without excluding other elements or objects.

[0056] In addition, for those not specified in the embodiments, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. All reagents or instruments not specified by the manufacturer are common commercially available products and can be purchased in the market.

[0057] To overcome the problems existing in the prior art, the embodiments of the present invention provide a cartilage repair membrane and its preparation method, which have good biological activity, mechanical properties, porosity and osteogenic properties, provide the best repair effect for the treatment of cartilage defects, and solve the problems of poor biocompatibility, poor mechanical properties, and inability to achieve a certain separation of different cell growth microenvironments in the prior art single-phase cartilage scaffolds.

[0058] In some embodiments of the present invention, the cartilage repair membrane comprises an umbilical cord layer, a first gel layer, and a second gel layer which are stacked in sequence; the umbilical cord layer is a sheet-like structure composed of umbilical cord tissue, and the umbilical cord tissue is formed from an umbilical cord from which vascular cells have been removed by mechanical means; both the first gel layer and the second gel layer are formed by mixing and crosslinking chitosan, sodium alginate, and hydroxyapatite, and the concentrations of hydroxyapatite in the first gel layer and the second gel layer are different.

[0059] Specifically, since the umbilical cord layer is a sheet-like structure composed of umbilical cord tissue, and the umbilical cord tissue is formed from an umbilical cord from which vascular cells have been removed by mechanical means, that is, the cartilage repair membrane prepares a cartilage matrix material from the umbilical cord layer to serve as a three-dimensional porous structure scaffold for filling cartilage defects. Its main component is collagen, which contains a large amount of collagen and retains a variety of natural active ingredients. It can recruit endogenous cells to the injury site, can simulate the microenvironment of the natural extracellular matrix to promote the proliferation and differentiation of the recruited cells, can achieve rapid vascularization and the repair and regeneration of the cartilage defect area, achieve the purpose of in-situ regeneration, and has the effects of inhibiting scar formation and osteogenesis; since both the first gel layer and the second gel layer are formed by mixing and crosslinking chitosan, sodium alginate, and hydroxyapatite, and the concentrations of hydroxyapatite in the first gel layer and the second gel layer are different, the first gel layer and the second gel layer are a type of extremely hydrophilic hydrogel with a three-dimensional network structure, which is very similar to the extracellular matrix of natural cartilage. It can swell and retain a large amount of water, is convenient for loading cells and factors, and has the ability of controlled release; at the same time, the multi-functional group characteristics of the hydrogel molecules make it easy to be functionalized, thereby improving its efficiency as a scaffold and carrier. By stacking the first gel layer and the second gel layer on the umbilical cord layer in sequence, that is, by setting two gel layers with gradients of different hydroxyapatite concentrations on the umbilical cord layer, it can provide the necessary mechanical support for the osteochondral tissue at each level in different longitudinal gradients, so that the prepared cartilage repair membrane can better simulate the interface between bone and cartilage, and moreover, improve the mechanical strength, toughness, and stability of the cartilage repair membrane, and is suitable for the repair and regeneration of tissue-engineered cartilage. The present invention combines the components and performance characteristics of the raw materials to the greatest extent and compensates for each other's deficiencies, forming a composite multi-layer cartilage repair membrane for bionic construction of gradient repair of cartilage tissue engineering, which has good biological activity, mechanical properties, porosity, and osteogenic properties, provides the best repair effect for the treatment of cartilage defects, and the cartilage repair membrane has significant effects in aspects such as antibacterial, hemostatic, promoting the adhesion, proliferation, and formation of mineralized bone matrix of osteoblasts, can meet the requirements of clinical and tissue engineering to the greatest extent, and can improve the repair degree of articular cartilage injury. It solves the problems in the prior art that single-phase cartilage scaffolds have poor biocompatibility, poor mechanical properties, and cannot achieve a certain separation of different cell growth microenvironments, etc.

[0060] In some embodiments of the present invention, the concentration of hydroxyapatite in the first gel layer is less than the concentration of hydroxyapatite in the second gel layer. Osteochondral tissue is a multi-layered tissue. In the distribution of articular osteochondral tissue, the content of the mineral component, which is mainly hydroxyapatite, gradually increases along the longitudinal gradient direction and towards the interior of the osteochondral tissue. When the cartilage repair membrane is applied to the tissue defect, the second gel layer with a high hydroxyapatite concentration and the first gel layer with a low hydroxyapatite concentration are successively in contact with the tissue defect, thereby simulating the gradient distribution of hydroxyapatite in the articular osteochondral tissue, so that the double-layer hydroxyapatite gel layers with different contents can provide necessary mechanical support for osteochondral tissues at different longitudinal gradients and can better simulate the bone-cartilage interface.

[0061] In some embodiments of the present invention, the concentration of hydroxyapatite in the first gel layer is 5 g / L to 7 g / L, and the concentration of hydroxyapatite in the second gel layer is 8 g / L to 10 g / L. The concentration of hydroxyapatite set in the first gel layer and the second gel layer is beneficial to maintaining the pore structure in the first gel layer and the second gel layer, and can also prevent the brittleness of the first gel layer and the second gel layer from being too high due to excessive hydroxyapatite concentration; moreover, the double-layer hydroxyapatite gel layers with different contents can provide necessary mechanical support for osteochondral tissues at different longitudinal gradients and can better simulate the bone-cartilage interface.

[0062] In some embodiments of the present invention, the mass ratio of hydroxyapatite, chitosan and sodium alginate in the first gel layer is (1:2:2) to (1:4:8). ； The mass ratio of hydroxyapatite, chitosan and sodium alginate in the second gel layer is (1:1:1) to (1:2:4). This ratio enables the prepared first gel layer and second gel layer to maintain the morphology of a three-dimensional porous network structure and does not make the prepared cartilage repair membrane too brittle.

[0063] In some embodiments of the present invention, the width of the cartilage repair membrane is 20 mm to 30 mm, and the length is 20 mm to 60 mm. Different specifications and sizes can meet different shaped wounds.

[0064] In some embodiments of the present invention, the surface of the umbilical cord layer facing away from the first gel layer is a smooth surface, and the surface of the second gel layer facing away from the first gel layer is an indented surface, and the indented surface is provided with a convex structure and a concave structure. Thus, the cartilage repair membrane has a double-sided structure with an indented surface and a smooth surface. The double-sided structure can fit well with the part to be repaired, providing a relatively closed tissue growth environment, providing good space and time for bone regeneration, and promoting tissue repair and regeneration. Among them, the convex structure and the concave structure on the indented surface can effectively increase the degree of fitting and the contact surface area between the cartilage repair membrane and the part to be repaired, improve the fitting degree of the cartilage repair membrane and the tissue defect, and improve the clinical use and treatment effect. Moreover, the double-sided cartilage repair membrane not only fits well with the bone tissue to be repaired, can selectively guide cell ingrowth, but also can be degraded synchronously with the reconstruction of the new tissue. This solves the problem in the prior art that there is a gap between the biofilm with smooth surfaces on both sides and the tissue defect, resulting in fibroblasts and epithelial cells with a faster growth and migration rate entering the bone defect area, forming a competitive inhibition with osteoblasts with a slower growth and migration rate, and having an adverse impact on the healing of bone tissue.

[0065] In some embodiments of the present invention, the first gel layer is coated on the umbilical cord layer and fixed by freeze-drying under pressure with a mold, and the second gel layer is coated on the first gel layer and fixed by freeze-drying under pressure with a mold; the mold includes a first surface and a second surface arranged opposite to each other, the first surface is an indented surface adapted to the indented surface, the indented surface is provided with a convex structure and a concave structure, and the second surface is a smooth surface adapted to the smooth surface. Thus, using this mold can press and freeze-dry the cartilage repair membrane into a double-sided structure with different structures; that is, the indented surface provided with the convex structure and the concave structure makes one surface of the cartilage repair membrane pressed and freeze-dried have a convex structure and a concave structure, thereby effectively increasing the degree of fitting and the contact surface area between the cartilage repair membrane and the part to be repaired, improving the fitting degree of the cartilage repair membrane medical material and the tissue defect, and improving the clinical use and treatment effect. This solves the problem in the prior art that there is a gap between the biofilm with smooth and flat surfaces on both sides and the tissue defect, resulting in fibroblasts and epithelial cells with a faster growth and migration rate entering the bone defect area, forming a competitive inhibition with osteoblasts with a slower growth and migration rate, and having an adverse impact on the healing of bone tissue.

[0066] In some embodiments of the present invention, the umbilical cord layer is a double-layer porous structure. The umbilical cord layer includes a dense layer and a loose layer. The pore size of the dense layer is smaller than that of the loose layer. The loose layer is fixed to the first gel layer, and the smooth surface is provided on the dense layer. The cartilage repair membrane is used to be placed on the surface of damaged bone tissue to create a relatively closed bone regeneration environment. Therefore, the dense layer with a small pore size facing the bone tissue can block fibroblasts and epithelial cells with a relatively fast migration speed from entering the bone defect area to play a barrier role. Compared with the prior art where the two different structures in the double-layer membrane structure are simply fixed by pressing, in the present invention, the loose layer is adhered to the gel layer, and through freeze-thaw treatment, the gel layer is adsorbed into the micropores of the loose layer of the umbilical cord layer, which enhances the adhesion between the gel layer and the umbilical cord layer, that is, greatly improves the bonding force between the bonding interfaces, and solves the problem of insufficient bonding force and easy delamination between the membrane layers in the prior art.

[0067] In some embodiments of the present invention, the pore size of the loose layer is 20 μm to 100 μm. The umbilical cord itself is a natural double-sided structure. The dense layer retains the natural collagen fiber dense structure of the umbilical cord and is used to face the bone tissue, which can effectively prevent fibroblasts and epithelial cells from growing into the bone defect area, support the formation of bone tissue, and prevent soft tissue from growing into the bone defect area. The collagen arrangement of the loose layer is relatively sparse and has a certain pore size, which can achieve partial area tight bonding with the gel layer during the freeze-thaw process, realizing the preparation of a double-layer membrane that does not delaminate after rehydration. Therefore, when applied to bone tissue defects, it can effectively play the respective roles of the dense layer and the loose layer.

[0068] In some embodiments of the present invention, the umbilical cord is an allogeneic umbilical cord. The allogeneic umbilical cord has a porous double-layer structure, with one layer having a dense arrangement of collagen fibers and the other layer having a relatively loose arrangement of collagen fibers. And it contains a variety of bioactive components, has low immunogenicity, and has unique biological properties of promoting cell adhesion and tissue regeneration and healing, which has important significance in clinical applications.

[0069] In some embodiments of the present invention, the umbilical cord tissue is derived from the human body. Selecting human-derived umbilical cord tissue as the basal layer breaks the limitation of preparing cartilage repair membranes with animal-derived raw materials and can effectively avoid rejection reactions in patients. The umbilical cord contains basic fibroblast growth factor, insulin-like growth factor, platelet-derived growth factor, transforming growth factor, etc., and can serve as a sustained-release carrier for growth factors to promote cell proliferation and osteogenic activity. Moreover, human-derived umbilical cord has low immunogenicity, and its natural active components are mainly composed of collagen, glycoprotein, amino group, glucan, mucopolysaccharide, and growth factors. It also retains a variety of bioactive factors, has low immunogenicity and good biocompatibility. It can not only provide a bionic three-dimensional support carrier for cells, but also promote the migration, proliferation, and differentiation of host cells through various proteins and growth factors to promote tissue regeneration and repair. The umbilical cord layer is derived from human umbilical cord tissue with vascular cells removed by mechanical means, and the cell matrix is removed, and its main component is collagen. Human-derived umbilical cord tissue is often regarded as "medical waste", and its application cost is correspondingly low.

[0070] In some embodiments of the present invention, the method for preparing the cartilage repair membrane includes the following steps:

[0071] Providing an original umbilical cord, removing the blood vessels in the original umbilical cord by mechanical means to form an umbilical cord tissue to be shaped, and trimming the umbilical cord tissue to be shaped into a sheet-like umbilical cord layer;

[0072] Mixing and crosslinking sodium alginate solution, chitosan solution, and two different concentrations of hydroxyapatite dispersion liquids respectively to obtain two gel solutions with different hydroxyapatite concentrations;

[0073] Coating the two gel solutions with different hydroxyapatite concentrations on one side of the umbilical cord layer in sequence to prepare a first gel layer and a second gel layer, and then freeze-drying to prepare the cartilage repair membrane.

[0074] Specifically, by providing the original umbilical cord, removing the blood vessels in the original umbilical cord mechanically to form umbilical cord tissue to be shaped, trimming the umbilical cord tissue to be shaped into a sheet-like umbilical cord layer, that is, the cartilage repair membrane is prepared from the umbilical cord layer to obtain a cartilage matrix material, which is used as a three-dimensional porous structure scaffold for filling cartilage defects. Its main component is collagen, containing a large amount of collagen, and retaining a variety of natural active ingredients. It can recruit endogenous cells to the injury site, can simulate the microenvironment of the natural extracellular matrix to promote the proliferation and differentiation of the recruited cells, can achieve rapid vascularization and the repair and regeneration of the cartilage defect area, achieve the purpose of in-situ regeneration, and has the effects of inhibiting scar formation and osteogenesis; by mixing and cross-linking sodium alginate solution, chitosan solution and two different concentrations of hydroxyapatite dispersion liquids respectively, two gel solutions with different hydroxyapatite concentrations are obtained; the two gel solutions with different hydroxyapatite concentrations are sequentially coated on one side of the umbilical cord layer to prepare a first gel layer and a second gel layer, so that the first gel layer and the second gel layer are a class of extremely hydrophilic hydrogels with a three-dimensional network structure, which is very similar to the extracellular matrix of natural cartilage. It can swell and retain a large amount of water, is convenient for loading cells and factors, and has the ability of controlled release; at the same time, the multi-functional group characteristics of the hydrogel molecules make it easy to be functionalized, thereby improving its efficiency as a scaffold and carrier. By sequentially coating the two gel solutions with different hydroxyapatite concentrations on one side of the umbilical cord layer to prepare a first gel layer and a second gel layer, that is, two gel layers with gradients of different hydroxyapatite concentrations are arranged on the umbilical cord layer, so that the necessary mechanical support can be provided for the osteochondral tissue at each level in different longitudinal gradients, making the prepared cartilage repair membrane better simulate the interface between bone and cartilage, and improving the mechanical strength, toughness and stability of the cartilage repair membrane, which is suitable for tissue engineering cartilage repair and regeneration. The present invention combines the components and performance characteristics of the raw materials to the greatest extent and compensates for each other's deficiencies, forming a composite multi-layer cartilage repair membrane for bionic construction of gradient repair of cartilage tissue engineering, with good biological activity, mechanical properties, porosity and osteogenic properties, providing the best repair effect for cartilage defect treatment, and the cartilage repair membrane has significant effects in aspects such as antibacterial, hemostatic, promoting the adhesion, proliferation of osteoblasts and the formation of mineralized bone matrix, can meet the requirements of clinical and tissue engineering to the greatest extent, and can improve the repair degree of articular cartilage injury. It solves the problems in the prior art that single-phase cartilage scaffolds have poor biocompatibility, poor mechanical properties, and cannot achieve a certain separation of different cell growth microenvironments, etc.

[0075] In some embodiments of the present invention, the step of sequentially coating the two gel solutions with different hydroxyapatite concentrations on one side of the umbilical cord layer to prepare a first gel layer and a second gel layer includes:

[0076] Coat the gel solution with a low hydroxyapatite concentration in the gel solution on one side of the umbilical cord layer, and prepare the first gel layer after pressing and freeze-drying through a mold.

[0077] Coat the gel solution with a high hydroxyapatite concentration in the gel solution on the side of the first gel layer facing away from the umbilical cord layer, and prepare the second gel layer after pressing and freeze-drying through a mold. Osteochondral is a tissue with a multi-layer structure. In the distribution of articular osteochondral tissue, the content of mineral components, that is, mainly hydroxyapatite, gradually increases along the longitudinal gradient direction and towards the inside of the osteochondral tissue. When the cartilage repair membrane is applied to the tissue defect, the second gel layer with a high hydroxyapatite concentration and the first gel layer with a low hydroxyapatite concentration are successively in close contact with the tissue defect. In this way, the gradient distribution of hydroxyapatite in the articular osteochondral tissue is simulated, so that the double-layer gel layers with different contents of hydroxyapatite can provide necessary mechanical support for the osteochondral tissue at different longitudinal gradients and can better simulate the bone-cartilage interface.

[0078] In some embodiments of the present invention, the step of coating the gel solution with a low hydroxyapatite concentration in the gel solution on one side of the umbilical cord layer and preparing the first gel layer after pressing and freeze-drying through a mold includes:

[0079] Cover the smooth surface of the mold with the side of the dense layer in the umbilical cord layer;

[0080] Uniformly coat 1 mL to 2 mL of the gel solution with a low hydroxyapatite concentration of 5 g / L to 7 g / L on the side of the loose layer in the umbilical cord layer;

[0081] Cover the indentation surface of another mold on the side of the umbilical cord layer coated with the gel solution with a low hydroxyapatite concentration;

[0082] Freeze the mold in the pressed state at a temperature of -5°C to -20°C for at least 4 h to prepare the first gel layer. After the pressing and freeze-drying treatment through the mold, the loose layer of the sheet umbilical cord layer is bonded to the first gel layer, and then through subsequent freeze-thaw cycles and vacuum freeze-drying treatment, the bonding degree between the loose layer and the gel layer can be deeper, there is no obvious delamination, and the structural continuity is good.

[0083] In some embodiments of the present invention, the step of coating the gel solution with a high hydroxyapatite concentration in the gel solution on the side of the first gel layer facing away from the umbilical cord layer and preparing the second gel layer after pressing and freeze-drying through a mold includes:

[0084] Remove another mold covering the first gel layer;

[0085] Uniformly coat 1 mL to 2 mL of the gel solution with the high hydroxyapatite concentration of 8 g / L to 10 g / L on the side of the first gel layer facing away from the umbilical cord layer within 3 min to 5 min;

[0086] Cover the indented surface of the other mold on the side of the first gel layer coated with the gel solution with the high hydroxyapatite concentration;

[0087] Freeze the mold in the pressed state at a temperature of -6°C to -20°C for 4 h to 8 h to obtain the second gel layer. After freeze-drying treatment by pressing with the mold, the cartilage repair membrane has a double-sided structure with an indented surface and a smooth surface. Usually, the flatness of the surface to be repaired is not good. The presence of the indented structure can effectively increase the degree of fitting and the contact surface area between the cartilage repair membrane and the part to be repaired, which is beneficial to promoting tissue healing; moreover, the second gel layer has an indented surface, so that the indented surface of the second gel layer with hydroxyapatite faces the bone defect, which can simultaneously improve the survival rate of osteoblasts and the amount of osteogenic mineralization.

[0088] In some embodiments of the present invention, the step of freeze-drying to obtain the cartilage repair membrane includes: performing 4 to 10 times of repeated freeze-thaw treatment on the preform including the umbilical cord layer, the first gel layer and the second gel layer; the parameters of the freeze-thaw treatment are: placing the preform at a temperature of 0°C to 20°C for 10 min to 60 min, and immediately placing the preform at a temperature of -6°C to -20°C for freezing for 10 min to 60 min when the surface of the second gel layer shows a dissolved state. Performing repeated freeze-thaw treatment to enhance the bonding strength between the loose layer of the umbilical cord layer and the first gel layer, and between the first gel layer and the second gel layer. That is, through the freeze-thaw treatment, the first gel layer can be adsorbed into the micropores of the loose layer of the umbilical cord layer, enhancing the adhesion between the first gel layer and the umbilical cord layer, and at the same time enhancing the adhesion between the first gel layer and the second gel layer, that is, greatly improving the bonding force between the fitting interfaces, and solving the problem of insufficient bonding force and easy delamination between the film layers in the prior art.

[0089] In some embodiments of the present invention, the step of freeze-drying to obtain the cartilage repair membrane further includes: after subjecting the pre-product to repeated freeze-thaw treatment, gradually cooling it to -80 ± 5°C at a rate of 5°C / min to 10°C / min at a freezing temperature of -20 ± 5°C, and then performing vacuum freeze-drying at a temperature of -80 ± 5°C for 8h to 20h to obtain the cartilage repair membrane. The beneficial effects are as follows: during the vacuum freeze-drying process, by slowly cooling to -80 ± 5°C at a rate of 5°C / min to 10°C / min, it is possible to avoid the destruction of tissue structure caused by too rapid temperature drop, effectively retain the ultrastructure of the umbilical cord matrix, significantly improve the drying efficiency of the cartilage repair membrane, and obtain a relatively uniform pore structure, so that while maintaining a certain sealing effect, oxygen, blood, and bioactive substances can enter, promoting local cell growth and osteochondral regeneration.

[0090] In some embodiments of the present invention, the step of separately mixing and cross-linking the sodium alginate solution, the chitosan solution, and two different concentrations of hydroxyapatite dispersion liquids to obtain gel solutions with two different hydroxyapatite concentrations includes:

[0091] Dissolve chitosan in an acetic acid aqueous solution of 1wt% to 2wt% and stir well to obtain the chitosan solution with a concentration of 8g / L to 20g / L;

[0092] Dissolve sodium alginate in deionized water and stir well to obtain the sodium alginate solution with a concentration of 8g / L to 40g / L;

[0093] Dissolve and disperse hydroxyapatite in deionized water and stir well to obtain the hydroxyapatite dispersion liquids with concentrations of 5g / L to 7g / L and 8g / L to 10g / L respectively;

[0094] Add the chitosan solution, the hydroxyapatite dispersion liquid, and the cross-linking agent solution to the sodium alginate solution in sequence, and stir and cross-link well to obtain the gel solutions with two different hydroxyapatite concentrations. For the preparation of the gel solution, it is selected to first prepare the sodium alginate solution, and then slowly drop the chitosan solution into the sodium alginate solution. This addition sequence can avoid the problems of poor adhesion and connectivity of the pores inside the material, thereby obtaining a porous cartilage repair membrane with a suitable pore structure, good elasticity and mechanical properties, and the pore structure is evenly distributed, without adhesion and no problem of uneven size distribution.

[0095] In some embodiments of the present invention, the crosslinking agent is any one or two of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, genipin, and formaldehyde. This is beneficial to further improve the mechanical properties of the first gel layer and the second gel layer, that is, it can effectively improve the mechanical properties of the cartilage repair membrane.

[0096] In some embodiments of the present invention, the crosslinking agent is a mixed solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Through EDC crosslinking, the cartilage repair membrane has the characteristics of being non-toxic and having good biocompatibility. Moreover, the collagen-based scaffold crosslinked by EDC exhibits better cytocompatibility. During the crosslinking process, EDC and NHS do not enter the collagen matrix but are converted into water-soluble urea derivatives, which can be removed or washed away.

[0097] In some embodiments of the present invention, the molar ratio of the EDC to the NHS is (4:1).

[0098] In some embodiments of the present invention, after the sodium alginate solution, the chitosan solution, and the hydroxyapatite dispersion liquid are sequentially added, a mixed gel liquid is obtained. The mass ratio of the crosslinking agent solution to the mixed gel liquid is (20:1) to (40:3).

[0099] In some embodiments of the present invention, the mass ratio of the hydroxyapatite, the chitosan, and the sodium alginate in the gel solution with a low hydroxyapatite concentration is (1:2:2) to (1:4:8). ； The mass ratio of the hydroxyapatite, the chitosan, and the sodium alginate in the gel solution with a high hydroxyapatite concentration is (1:1:1) to (1:2:4). This ratio enables the prepared first gel layer and second gel layer to maintain the morphology of a three-dimensional porous network structure and does not make the cartilage repair membrane too brittle.

[0100] In some embodiments of the present invention, the step of trimming the umbilical cord tissue to be shaped into a sheet-like umbilical cord layer includes: after washing the umbilical cord tissue to be shaped with a cleaning solution, trimming it into a sheet and washing it with deionized water 1 to 3 times to obtain the umbilical cord layer. The cleaning temperature does not exceed 15°C, and the cleaning time does not exceed 5 minutes. The cleaning solution is any one or more of a sterile physiological phosphate buffer solution, a sterile sodium chloride injection solution, and purified water. The beneficial effects are as follows: The umbilical cord tissue is washed with a cleaning solution to remove blood stains and destroy cells, and rinsed with deionized water to keep the umbilical cord layer moist for easy adhesion to the gel layer. By setting the cleaning temperature not to exceed 15°C and the cleaning time not to exceed 5 minutes, rapid treatment is carried out in a low-temperature environment to retain the three-dimensional collagen structure and natural active ingredients in the umbilical cord to the greatest extent.

[0101] Example 1, the preparation method of the cartilage repair membrane comprises the following steps:

[0102] S1. Prepare the umbilical cord membrane:

[0103] S11. By mass ratio, add the raw umbilical cord and the cleaning solution at a liquid-to-material ratio of at least 1:6 to clean the raw umbilical cord twice.

[0104] S12. Select annular drills with different inner diameters according to the diameter of the blood vessels to remove the blood vessels in the raw umbilical cord to obtain the umbilical cord tissue to be shaped; then cut it along the axial direction of the umbilical cord into flat sheets to trim the umbilical cord tissue to be shaped into a sheet-like structure of the umbilical cord membrane.

[0105] S13. Soak and clean it once with deionized water for standby.

[0106] S2. Prepare the gel solution:

[0107] S21. Weigh a certain amount of chitosan and dissolve it in 1wt% acetic acid aqueous solution, and stir well for 3h to obtain a 50mL chitosan solution with a mass concentration of 20g / L.

[0108] S22. Weigh a certain amount of sodium alginate and dissolve it in deionized water, and stir well for 3h to obtain a 50mL sodium alginate solution with a mass concentration of 30g / L.

[0109] S23. Dissolve and disperse hydroxyapatite in deionized water, and stir and mix well to prepare 50mL hydroxyapatite dispersion liquids with concentrations of 5g / L and 10g / L respectively.

[0110] S24. Under the condition of continuous stirring, according to the volume ratio of the chitosan solution, the hydroxyapatite dispersion liquid and the sodium alginate solution of 0.8:1:1, add one portion of the chitosan solution and the hydroxyapatite dispersion liquid with a concentration of 5g / L to one portion of the sodium alginate solution to prepare the first mixed gel liquid; under the condition of continuous stirring, according to the volume ratio of the chitosan solution, the hydroxyapatite dispersion liquid and the sodium alginate solution of 0.8:1:1, add another portion of the chitosan solution and the hydroxyapatite dispersion liquid with a concentration of 10g / L to another portion of the sodium alginate solution to prepare the second mixed gel liquid; then add the mixed cross-linking agent solution of EDC and NHS to the first mixed gel liquid and the second mixed gel liquid respectively, and the mass ratio of the first mixed gel liquid and the second mixed gel liquid to the mixed cross-linking agent of EDC and NHS is both 25:1, stir and mix well to obtain the gel solution loaded with 5g / L hydroxyapatite and the gel solution loaded with 10g / L hydroxyapatite.

[0111] S3. Preparation of cartilage repair membrane:

[0112] S31. Attach the umbilical cord membrane with a side length of 20 mm to the smooth side of the mold as the umbilical cord layer; uniformly coat 2 mL of a gel solution loaded with 5 g / L hydroxyapatite on the umbilical cord layer, then cover with another mold, and the indented surface of this mold faces the side of the umbilical cord layer coated with the gel solution loaded with 5 g / L hydroxyapatite. Place the mold in a -20°C environment and freeze for 4 h to form a first gel coating, which is the first gel layer; remove the mold covering the first gel layer, then uniformly coat 2 mL of a gel solution loaded with 10 g / L hydroxyapatite on the first gel layer, and cover with this mold, and the indented surface of this mold faces the side of the first gel layer coated with the gel solution loaded with 10 g / L hydroxyapatite. Place the mold in a -20°C environment and freeze for 4 h to form a second gel coating, which is the second gel layer. In this way, a preform containing the umbilical cord layer, the first gel layer, and the second gel layer is obtained;

[0113] S32. After the coating is completed, subject the preform in the pressed state together with the mold to repeated freeze-thaw treatment to enhance the bonding force between the coating layers, i.e., the first gel layer and the second gel layer, and the loose surface of the umbilical cord layer; and the parameters of the repeated freeze-thaw treatment are: place at 4°C for 60 min, and when it is observed that the surface of the coating layer, i.e., the second gel layer, is slightly dissolved, immediately place it at -20°C and freeze for 60 min, repeat 4 times;

[0114] S33. Place the preform in the pressed state together with the mold at -20°C and gradually cool it to -80°C at a rate of -10°C / min, then vacuum freeze-dry for 12 h, and after sterilization treatment, a cartilage repair membrane is prepared.

[0115] Specifically, the original umbilical cord in step S11 belongs to extra-embryonic tissue, has a wide source, and has no immune rejection reaction as a cartilage scaffold; the umbilical cord tissue contains rich glucosamine, Wharton's jelly, collagen, and a variety of active factors, and has a similar composition and biological function to cartilage matrix, which can provide a biomimetic three-dimensional support carrier for cells, and can also promote the migration, proliferation, differentiation, etc. of host cells through various proteins and growth factors interacting with the host.

[0116] Specifically, the processing environmental temperature of the original umbilical cord in step S11 does not exceed 15°C, that is, rapid processing in a low-temperature environment to retain the three-dimensional collagen structure and natural active ingredients in the umbilical cord to the greatest extent.

[0117] Specifically, the umbilical cord membrane in the form of a sheet structure in step S12 has different specifications and dimensions, and the dimensions are any one of 20mm×20mm, 20mm×30mm, 20mm×40mm, 20mm×50mm, 25mm×25mm, 30mm×40mm, 30mm×50mm, and 30mm×60mm.

[0118] Specifically, sodium alginate and chitosan are non-cytotoxic and have antibacterial activity. The gel layer scaffold prepared by freeze-drying with their participation has a microporous structure and certain permeability, which can improve the mechanical strength of the cartilage repair membrane scaffold.

[0119] Specifically, by adding the chitosan solution to the sodium alginate solution in the ratio and order in step S24, a porous cartilage repair membrane with a smoother surface and appropriate pore structure can be obtained. The reason for this phenomenon is that the chitosan dissolves more completely and the mixture is more uniform, and the low-proportion concentration solution is not too sticky, so that the formation of ice crystals is increased after freezing, thus increasing the pore structure formed in the cartilage repair membrane and increasing the pore size.

[0120] Specifically, the stability of the acellular matrix umbilical cord layer scaffold alone is very poor. Coating with a gel solution containing hydroxyapatite, sodium alginate, and chitosan to form a gel layer can improve the stability and mechanical properties of the umbilical cord layer.

[0121] Specifically, in step S31, two gel solutions with different concentrations of hydroxyapatite are coated on the umbilical cord layer to form two gel layers with a gradient, so as to provide the necessary mechanical support for each layer of cartilage tissue and better simulate the bone-cartilage interface.

[0122] Specifically, slow cooling in step S33 can avoid the rapid decline of temperature from damaging the tissue structure, effectively retain the ultrastructure of the umbilical cord matrix, significantly improve the drying efficiency of the cartilage repair membrane, and obtain a relatively uniform pore structure, so that while maintaining a certain sealing effect, oxygen, blood, and bioactive substances can enter, promoting local cell growth and bone regeneration.

[0123] Figure 1 It is a schematic diagram of the electron microscope scan of the longitudinal section of the cartilage repair membrane of the present invention.

[0124] As Figure 1As shown, the cartilage repair membrane has an obvious bilayer structure. Since only the concentration of hydroxyapatite is different between the first gel layer and the second gel layer, the first gel layer and the second gel layer are not significantly stratified. The umbilical cord layer has a dense structure and small pore sizes. It can be seen that the umbilical cord layer retains the natural dense structure of collagen fibers in the umbilical cord, which can effectively prevent fibroblasts and epithelial cells from growing into the osteochondral defect area and prevent soft tissues from growing into the bone defect area. Moreover, the various natural active ingredients retained by the umbilical cord layer can improve the wound healing ability and at the same time reduce the scars generated after surgery; the first gel layer and the second gel layer have a loose structure, have an appropriate porosity and can withstand a certain external force. The pore sizes of the gel layers are relatively larger than those of the umbilical cord layer, and have a certain density and roughness, which are conducive to cell adhesion, proliferation and differentiation. When applied to tissue defects, it can effectively guide bone tissue regeneration.

[0125] The active factors and components of the cartilage repair membrane prepared in Example 1 were tested, and the contents of various factors in the umbilical cord layer of the cartilage repair membrane of the present invention and in the fresh original umbilical cord were determined. The data of various factors in the umbilical cord layer of the cartilage repair membrane and in the fresh original umbilical cord are shown in Table 1.

[0126] Table 1

[0127]

[0128] It can be analyzed from Table 1 that the contents of various factors in the umbilical cord layer and in the fresh original umbilical cord used as raw materials are not much different, indicating that the preparation method of the cartilage repair membrane of the present invention can retain various natural active factor components in the original umbilical cord. Compared with simple collagen membranes or acellular matrix products, it has unique biological characteristics that can promote healing.

[0129] The porosity of the cartilage repair membrane prepared in Example 1 was tested. The porosity of the cartilage repair membrane was measured by the anhydrous ethanol liquid method. A total of 3 groups of samples were tested, and the test results are shown in Table 2. The specific steps for porosity testing are as follows: Weigh the dry weight of the sample and record it as M1. Subsequently, soak the cartilage repair membrane completely in anhydrous ethanol for 30 minutes. Take out the cartilage repair membrane that has been saturatedly adsorbed, quickly weigh it after blotting the excess liquid on the surface with filter paper, and record it as M2. Then place the cartilage repair membrane in a graduated cylinder containing a fixed volume of anhydrous ethanol, and record the increment V of the volume in the graduated cylinder at this time. The porosity (P) of the cartilage repair membrane is calculated by the following formula: P=(M2 - M1) / ρV, where ρ = 0.8 g / cm 3 , which is the density of anhydrous ethanol.

[0130] Table 2

[0131]

[0132] Table 2 shows that the cartilage repair membrane has a good porosity, indicating that the cartilage repair membrane of the present invention is beneficial to the three-dimensional growth, proliferation and differentiation of cells during the cartilage repair process.

[0133] The mechanical properties of the cartilage repair membrane prepared in Example 1 were tested. Specifically, a universal testing machine was used to conduct a compression mechanical experiment. The sample size was measured before each test, and the compression speed was set at 1 mm / min. Then, stress and strain curves were plotted to evaluate the mechanical properties of the scaffold. A total of 3 groups of samples were tested, and the test results are shown in Table 3.

[0134] Table 3

[0135]

[0136] Table 3 shows that the cartilage repair membrane has excellent mechanical properties, indicating that the cartilage repair membrane of the present invention can meet the mechanical requirements for cartilage defect repair as much as possible when implanted into the cartilage defect site of a patient.

[0137] Figure 2 Schematic diagram of the mold of the first embodiment of the present invention.

[0138] In some embodiments of the present invention, the mold is an integrally formed structure. The mold includes a first surface and a second surface arranged opposite to each other. Referring to Figure 2 , the first surface is the indentation surface 1, and the indentation surface 1 is provided with a convex structure (not marked in the figure) and a concave structure (not marked in the figure). The second surface (not marked in the figure) is a smooth surface.

[0139] Figure 3 Schematic diagram of the mold of the second embodiment of the present invention.

[0140] In some embodiments of the present invention, referring to Figure 3 , the mold includes a first layer body 11 and a second layer body 12. The first layer body 11 and the second layer body 12 are overlapped and fixed up and down. The opposite surfaces of the first layer body 11 and the second layer body 12 are the first surface 111 and the second surface (not marked in the figure) respectively. Both the first layer body 11 and the second layer body 12 are woven from 316L stainless steel wires. The structure is simple, the manufacturing process is easy, the manufacturing cost is low, and the stainless steel material itself has corrosion resistance and weldability, making the mold have good stability, plasticity and high mechanical strength.

[0141] Figure 4 For Figure 3 Schematic diagram of the enlarged structure of the surface of the first layer body in the mold shown; Figure 5 For Figure 3 Schematic diagram of the enlarged structure of the surface of the second layer body in the mold shown.

[0142] In some embodiments of the present invention, with reference to Figure 4 and Figure 5 , the first layer body 11 is woven in a twill weave pattern, such that the first surface 111 of the first layer body 11 is an indentation surface, and the indentation surface is provided with the regularly arranged raised structures 101 and the recessed structures 102. The twill weave refers to different wire diameters and different mesh counts for the warp wires and the weft wires, characterized by sparser warp and denser weft, thicker warp and thinner weft. The warp wires are in the length direction, and the weft wires are in the width direction. The length direction is the direction indicated by A as shown in Figure 4 , and the width direction is the direction perpendicular to A. The specific structure after the twill weave is as shown in Figure 5 . The weaving method is a conventional setting in the art and will not be elaborated herein. The second layer body 12 is woven in a twill weave pattern, such that the second surface (not marked in the figure) of the second layer body 12 is a smooth surface, with a relatively flat surface and dense and fine pore diameters. The twill weave refers to a weaving method in which each warp wire crosses over and under every two weft wires, and each weft wire crosses over and under every two warp wires. The specific structure after the twill weave is as shown in Figure 5 . The first layer body 11 woven in the twill weave pattern has good acid resistance, alkali resistance, and high temperature resistance, and strong compressive strength and wear resistance, etc.; the second layer body 12 woven in the twill weave pattern has characteristics such as a flat and smooth surface, strong corrosion resistance, and durability. The first layer body 11 is woven in the twill weave pattern, such that the surface of the first layer body 11 can effectively form the raised structures 101 and the recessed structures 102, and the second layer body 12 is woven in the twill weave pattern, such that the surface of the second layer body 12 is smooth and flat.

[0143] In some embodiments of the present invention, the warp wire diameter of the stainless steel wire for weaving the first layer body is 0.2 mm to 0.28 mm, the weft wire diameter is 0.14 mm to 0.2 mm, the pore diameter of the first layer body is 60 mesh - 100 mesh, and the thickness of the first layer body is 1.2 mm to 3.4 mm. This enables the first surface of the mold to better form the raised structures and the recessed structures, so that the raised structures and the recessed structures on the cartilage repair membrane after pressing and freeze-drying can effectively increase the degree of fit and the contact surface area between the membrane material and the part to be repaired.

[0144] In some specific embodiments of the present invention, the warp wire diameter of the stainless steel wire for weaving the first layer body is 0.25 mm, the weft wire diameter is 0.18 mm, the pore diameter of the first layer body is 80 mesh, and the thickness of the first layer body is 2 mm.

[0145] In some embodiments of the present invention, the wire diameter of the stainless-steel wire for weaving the second layer body is 0.04 mm to 0.045 mm, the pore diameter of the second layer body is 250 mesh - 300 mesh, and the thickness of the second layer body is 0.3 mm to 0.7 mm. This makes the second surface of the second layer body relatively flat, with dense and fine pore diameters.

[0146] In some embodiments of the present invention, the pore diameter of the second layer body is 0.0446 mm to 0.0556 mm.

[0147] In some specific embodiments of the present invention, the second layer body of the mold is made by plain weaving of stainless-steel wire with a diameter of 0.042 mm, the pore diameter of the second layer body is 0.05 mm, and the thickness of the second layer body is 0.5 mm. Weaving with a thinner stainless-steel wire makes the pore diameters on the second surface of the mold dense and fine, and the surface smooth, enabling a relatively flat morphological structure to be pressed out for the cartilage repair membrane material.

[0148] In some embodiments of the present invention, the raised structures and the sunken structures are arranged alternately, and the distance between adjacent raised structures in the warp direction is 0.14 mm to 0.2 mm, and the distance between adjacent raised structures in the weft direction is 0.2 mm to 0.28 mm. This makes the formed cartilage repair membrane have the raised structures and the sunken structures, so that it can closely fit the tissue defect area, block the entry of external soft tissue cells, play a role in isolating the growth of soft tissue into the bone defect area, and achieve a good treatment effect.

[0149] In some specific embodiments of the present invention, the raised structures and the sunken structures are arranged alternately, and the distance between adjacent raised structures in the warp direction is 0.15 mm, and the distance between adjacent raised structures in the weft direction is 0.26 mm.

[0150] In some embodiments of the present invention, the weft wire diameter of the stainless-steel wire for weaving the first layer body is the first wire diameter, the warp wire diameter of the stainless-steel wire for weaving the first layer body is the second wire diameter, the raised structures and the sunken structures are arranged alternately, and the distance between adjacent raised structures in the warp direction is equal to the first wire diameter, and the distance between adjacent raised structures in the weft direction is equal to the second wire diameter.

[0151] In some embodiments of the present invention, the warp wire diameter of the stainless-steel wire for weaving the first layer body is the second wire diameter, the raised structures and the sunken structures are arranged alternately, and the height difference between the raised structures and the sunken structures is equal to the second wire diameter.

[0152] In some embodiments of the present invention, the raised structures and the sunken structures are arranged alternately, and the height difference between the raised structures and the sunken structures is 0.2 mm to 0.28 mm.

[0153] In some embodiments of the present invention, the length of the mold is 6 cm to 10 cm, the width is 4 cm to 8 cm, and the thickness is 1.5 mm to 4.1 mm. The mold is lightweight and will not crush the cartilage repair membrane during the preparation of the cartilage repair membrane. While ensuring the pressing effect, it saves materials and reduces the input cost.

[0154] In some specific embodiments of the present invention, the mold is a thin plate structure of 8 cm × 6 cm, which can not only meet the required size for the pressing and freeze-drying treatment of the existing cartilage repair membrane, but also will not cause the cartilage repair membrane material to be deformed by pressing, and also saves the production materials of the freeze-drying mold.

[0155] In some embodiments of the present invention, the first layer and the second layer are welded together by using micro-spot resistance welding or seamless butt welding. This makes the mold have no protruding solder joints and burrs, and the first layer and the second layer are closely combined without gaps, and there will be no delamination and peeling phenomena, having high stability.

[0156] In some embodiments of the present invention, both the first layer and the second layer include 4 to 8 layers of stainless steel wire mesh. If the number of layers is too many, the air permeability and permeability are poor, which will affect the freeze-drying effect of the cartilage repair membrane. If the number of layers is too few, the overall weight is light, which will affect the pressing effect of the mold on the cartilage repair membrane.

[0157] In some specific embodiments of the present invention, the preparation steps of the mold include:

[0158] Step 1, the mold is woven from 316L stainless steel wires into two different structures of stainless steel wire meshes by the dense weave method and the twill weave method respectively, and five pieces are made respectively;

[0159] Step 2, the five stainless steel wire meshes woven by the dense weave method are laminated and pressed in a flat surface manner and then vacuum sintered to obtain the first layer, and the other five stainless steel wire meshes woven by the twill weave method are laminated and pressed in a flat surface manner and then vacuum sintered to obtain the second layer;

[0160] Step 3, the first layer and the second layer are welded by seamless butt welding technology to obtain the mold.

[0161] In some embodiments of the present invention, the 316L stainless steel wire surface is subjected to electrolytic polishing treatment, so that it will not rust and deform during use, cleaning, etc.

[0162] In some embodiments of the present invention, the vacuum sintering process is simple to operate, can improve the purity of the cemented carbide to make it have high pressure resistance and good plasticity, and is convenient for subsequent processing, welding and assembly.

[0163] Although the embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations are all within the scope and spirit of the present invention as described in the claims. Moreover, the present invention as described herein may have other embodiments and can be implemented or realized in various ways.

Claims

1. A cartilage repair membrane, characterized in that, It includes an umbilical cord layer, a first gel layer, and a second gel layer which are stacked in sequence: The umbilical cord layer is a sheet-like structure composed of umbilical cord tissue, and the umbilical cord tissue is formed from an umbilical cord from which vascular cells have been removed by mechanical means; Both the first gel layer and the second gel layer are formed by mixing and cross-linking chitosan, sodium alginate, and hydroxyapatite, and the concentrations of hydroxyapatite in the first gel layer and the second gel layer are different; The concentration of hydroxyapatite in the first gel layer is 5 g / L to 7 g / L, and the concentration of hydroxyapatite in the second gel layer is 8 g / L to 10 g / L; The mass ratio of hydroxyapatite, chitosan, and sodium alginate in the first gel layer is (1:2:2) to (1:4:8); the mass ratio of hydroxyapatite, chitosan, and sodium alginate in the second gel layer is (1:1:1) to (1:2:4).

2. The cartilage repair membrane according to claim 1, wherein The width of the cartilage repair membrane is 20 mm to 30 mm, and the length is 20 mm to 60 mm.

3. The cartilage repair membrane according to claim 1, wherein The side of the umbilical cord layer facing away from the first gel layer is a smooth surface, and the side of the second gel layer facing away from the first gel layer is an indentation surface, and the indentation surface is provided with a convex structure and a concave structure.

4. The cartilage repair membrane according to claim 3, characterized in that, The first gel layer is coated on the umbilical cord layer and fixed by pressing and freeze-drying with a mold, and the second gel layer is coated on the first gel layer and fixed by pressing and freeze-drying with a mold; the mold includes a first surface and a second surface arranged opposite to each other, the first surface is an indentation surface adapted to the indentation surface, and the indentation surface is provided with a convex structure and a concave structure, and the second surface is a smooth surface adapted to the smooth surface.

5. The cartilage repair membrane according to claim 3, characterized in that, The umbilical cord layer is a double-layer porous structure, the umbilical cord layer includes a dense layer and a loose layer, the pore size of the dense layer is smaller than that of the loose layer, the loose layer is fixed to the first gel layer, and the dense layer is provided with the smooth surface.

6. A method for preparing a cartilage repair membrane, which is used to prepare the cartilage repair membrane according to any one of claims 1-5, characterized in that, It includes the following steps: Provide an original umbilical cord, remove the blood vessels in the original umbilical cord by mechanical means to form umbilical cord tissue to be shaped, and trim the umbilical cord tissue to be shaped into an umbilical cord layer in a sheet-like structure; Mix and cross-link a sodium alginate solution, a chitosan solution, and two dispersions of hydroxyapatite with different concentrations respectively to obtain two gel solutions with different hydroxyapatite concentrations; Coat the two gel solutions with different hydroxyapatite concentrations on one side of the umbilical cord layer in sequence, and after preparing the first gel layer and the second gel layer, then freeze-dry to obtain the cartilage repair membrane.

7. The preparation method of the cartilage repair membrane according to claim 6, wherein, The step of coating the two gel solutions with different hydroxyapatite concentrations on one side of the umbilical cord layer in sequence to prepare the first gel layer and the second gel layer includes: Coat the gel solution with a low hydroxyapatite concentration on one side of the umbilical cord layer, and after pressing and freeze-drying with a mold, obtain the first gel layer; Coat the gel solution with a high hydroxyapatite concentration on the side of the first gel layer facing away from the umbilical cord layer, and after pressing and freeze-drying with a mold, obtain the second gel layer.

8. The preparation method of the cartilage repair membrane according to claim 6, wherein, The step of coating the gel solution with a low hydroxyapatite concentration in the gel solution on one side of the umbilical cord layer and preparing the first gel layer after pressing and freeze-drying with a mold includes: Cover one side of the dense layer in the umbilical cord layer on the smooth surface of the mold; Uniformly coat 1 mL to 2 mL of the gel solution with a low hydroxyapatite concentration of 5 g / L to 7 g / L on one side of the loose layer in the umbilical cord layer; Cover the indentation surface of another mold on the side of the umbilical cord layer coated with the gel solution with a low hydroxyapatite concentration; Freeze the mold in the pressed state at a temperature of -5°C to -20°C for at least 4 h to prepare the first gel layer.

9. The preparation method of the cartilage repair membrane according to claim 8, wherein, The step of coating the gel solution with a high hydroxyapatite concentration in the gel solution on the side of the first gel layer facing away from the umbilical cord layer and preparing the second gel layer after pressing and freeze-drying with a mold includes: Remove another mold covering the first gel layer; Uniformly coat 1 mL to 2 mL of the gel solution with a high hydroxyapatite concentration of 8 g / L to 10 g / L on the side of the first gel layer facing away from the umbilical cord layer within 3 min to 5 min; Cover the indentation surface of another mold on the side of the first gel layer coated with the gel solution with a high hydroxyapatite concentration; Freeze the mold in the pressed state at a temperature of -6°C to -20°C for 4 h to 8 h to prepare the second gel layer.

10. The preparation method of the cartilage repair membrane according to claim 6 or 9, characterized in that, The step of freeze-drying to prepare the cartilage repair membrane includes: Perform 4 to 10 times of repeated freeze-thaw treatment on the preform including the umbilical cord layer, the first gel layer and the second gel layer; the parameters of the freeze-thaw treatment are: place the preform at a temperature of 0°C to 20°C for 10 min to 60 min, and immediately place the preform at a temperature of -6°C to -20°C for freezing for 10 min to 60 min when the surface of the second gel layer shows a dissolved state.

11. The preparation method of the cartilage repair membrane according to claim 10, wherein, The step of freeze-drying to prepare the cartilage repair membrane further includes: Gradually cool the preform after repeated freeze-thaw treatment from -20 ± 5°C at a rate of 5°C / min to 10°C / min to -80 ± 5°C, and then perform vacuum freeze-drying at -80 ± 5°C for 8 h to 20 h to prepare the cartilage repair membrane.

12. The preparation method of the cartilage repair membrane according to claim 6, characterized in that, The step of separately mixing and crosslinking the sodium alginate solution, the chitosan solution and two hydroxyapatite dispersions with different concentrations to obtain two gel solutions with different hydroxyapatite concentrations includes: Dissolve chitosan in an acetic acid aqueous solution of 1 wt% to 2 wt% and stir well to prepare the chitosan solution with a concentration of 8 g / L to 20 g / L; Dissolve sodium alginate in deionized water and stir well to prepare the sodium alginate solution with a concentration of 8 g / L to 40 g / L; Dissolve and disperse hydroxyapatite in deionized water and stir well to prepare the hydroxyapatite dispersions with concentrations of 5 g / L to 7 g / L and 8 g / L to 10 g / L respectively; The chitosan solution, the hydroxyapatite dispersion and the crosslinking agent solution are successively added to the sodium alginate solution, and crosslinked by sufficient stirring to obtain the gel solutions with two different hydroxyapatite concentrations.

13. The preparation method of the cartilage repair membrane according to claim 12, wherein The crosslinking agent is any one or two of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, N-hydroxysuccinimide, genipin and formaldehyde.

14. The preparation method of the cartilage repair membrane according to claim 6, wherein The step of trimming the umbilical cord tissue to be shaped into a sheet-like umbilical cord layer includes: After the umbilical cord tissue to be shaped is cleaned with a cleaning solution, it is trimmed into a sheet and washed with deionized water 1 to 3 times to obtain the umbilical cord layer. The cleaning temperature does not exceed 15°C, and the cleaning time does not exceed 5 minutes. The cleaning solution is any one or more of sterile physiological phosphate buffer solution, sterile sodium chloride injection solution and purified water.

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