A double-layer osteochondral composite scaffold and its preparation method

Abstract

The application discloses a kind of osteochondral double-layer composite scaffold and its preparation method, wherein, osteochondral double-layer composite scaffold includes the three-dimensional porous ceramic scaffold of 3D printing integrated molding and the cartilage layer, hard bone layer being arranged on three-dimensional porous ceramic scaffold, in the application embodiment, using the above-mentioned one osteochondral double-layer composite scaffold and its preparation method, using the hard support structure of porous ceramic, respectively adding carrier in porous upper support layer, porous lower support layer and loading / bioactive substance filling, can realize bone cartilage integrated repair, can also solve the problem that flexible cartilage repair support is excessively compressed in the process of surgical implantation flexible cartilage repair support and leads to growth factor from carrier leakage, realize the slow release of bioactive substance after double-layer support is implanted, promote the differentiation of autologous bone marrow mesenchymal stem cells, and the through-hole structure in support interior, it is beneficial to the attachment of composite hydrogel and mesoporous bioactive glass, also beneficial to the adhesion and crawling of cell in support interior after implantation in vivo, promote new bone tissue.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a bone and cartilage double-layer composite scaffold and its preparation method. Background Technology

[0002] Cartilage tissue lacks blood vessels, nerves, and lymphatic vessels, resulting in limited self-repair capabilities. Once damaged, such as by sports injuries, trauma, or osteoarthritis, cartilage damage often fails to heal on its own, severely impacting joint function and the patient's quality of life.

[0003] Methods for cartilage repair mainly include microfracture, autologous chondrocyte transplantation, and allogeneic cartilage transplantation, but these methods all have certain limitations. While microfracture is relatively simple to perform, the resulting fibrocartilage differs significantly from normal hyaline cartilage in tissue structure and mechanical properties. Autologous chondrocyte transplantation carries risks such as donor site damage and potential phenotypic changes during cell expansion. Allogeneic cartilage transplantation, on the other hand, faces the risks of immune rejection and disease transmission.

[0004] Therefore, developing a cartilage repair material that can effectively promote cartilage regeneration and possesses good biocompatibility and mechanical properties is of significant clinical importance. Currently, many cartilage repair materials exist, including flexible ones. However, during implantation, doctors need to press the scaffold with their hands or instruments. This excessive compression of the carrier (the flexible scaffold softens upon contact with blood) can lead to excessive leakage of bioactive substances, affecting the cartilage repair and regeneration effect. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a bone-cartilage bilayer composite scaffold that can effectively promote cartilage regeneration and has good biocompatibility and mechanical properties, as well as its preparation method.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions.

[0007] This application provides a bone and cartilage double-layer composite scaffold, including a three-dimensional porous ceramic scaffold and a cartilage layer and a bone layer disposed on the three-dimensional porous ceramic scaffold;

[0008] The three-dimensional porous ceramic support is integrally formed by 3D printing and has a porous upper support layer and a porous lower support layer.

[0009] The cartilage layer includes a porous upper scaffold layer and a mesoporous bioactive glass tightly attached to the outer surface and the inner gap surface of the porous upper scaffold layer, as well as a first bioactive substance loaded by the mesoporous bioactive glass.

[0010] The bone layer includes a porous subscaffold layer, a lyophilized composite hydrogel infused in the internal gaps of the porous subscaffold layer, and a second bioactive substance loaded by the composite hydrogel.

[0011] The interlayer angle of the porous upper support layer is set to 60-90°, and the interlayer angle of the porous lower support layer is set to 30-60°.

[0012] Further specifying, in the above-mentioned osteochondral bilayer composite scaffold, the mesoporous bioactive glass is specifically configured as one or more of silicon-based mesoporous bioglass, silicon-phosphorus-based mesoporous bioglass, and silicon-calcium-phosphorus-based mesoporous bioglass.

[0013] And, or the first bioactive substance includes one or more of cartilage formation promoters, anti-inflammatory drugs, and analgesics.

[0014] Further specifying, in the above-mentioned osteochondral double-layer composite scaffold, the composite hydrogel comprises one or more of soybean protein-based solution, tocopherol acetate, glycerol, and lecithin;

[0015] And, or the second bioactive substance includes one or more of the following: bone formation promoters, bone resorption inhibitors, calcium metabolism regulators, anti-inflammatory drugs, and analgesics.

[0016] Further specifying, in the above-mentioned osteochondral double-layer composite scaffold, the cartilage layer has a multi-level pore size, wherein the first-level pore size is set to a range of 300-600μm, the second-level pore size is set to a range of 40-100μm, and the third-level pore size is set to a range of 20nm-40μm;

[0017] The primary pore size is formed by 3D printing. The secondary pore size is specifically composed of exposed internal granular nano-HA / β-TCP clusters after the MBG and nano-HA on the outer surface of the porous upper support layer are consumed. The tertiary pore size is specifically composed of nano-HA / β-TCP particles, surface-adsorbed mesoporous bioactive glass, and nano-needle-like hydroxyapatite carbonate clusters at the junction of the primary and secondary pore sizes.

[0018] Further specifying, in the above-mentioned osteochondral double-layer composite scaffold, the scleral layer has a multi-level pore size, wherein the first-level pore size is set to a range of 200-400μm, the second-level pore size is set to a range of 40-200μm, and the third-level pore size is set to a range of 100nm-40μm.

[0019] The primary pore size is formed by 3D printing, the secondary pore size is specifically composed of granular nano HA / β-TCP clusters and porous hydrogel, and the tertiary pore size is specifically composed of granular nano HA / β-TCP.

[0020] This application provides a preparation method for the osteochondral bilayer composite scaffold described in any of the above claims, comprising:

[0021] Slurry for preparing 3D printed preform scaffolds;

[0022] 3D printed preform support;

[0023] The green blank support is dried and sintered to form a three-dimensional porous ceramic support;

[0024] The porous subscaffold layer was impregnated under negative pressure into a soybean protein-based mixed solution loaded with a second bioactive substance. The impregnation was simultaneously ultrasonicated, and the porous subscaffold layer was then frozen and freeze-dried under vacuum after the impregnation and ultrasonication.

[0025] The porous scaffold layer is impregnated under negative pressure into a solution of mesoporous bioactive glass loaded with the first bioactive substance, and then vacuum dried.

[0026] Further specifying, in the above-mentioned preparation method, the slurry for the 3D printed preform scaffold is specifically set as an inorganic solid phase powder and a liquid phase adhesive mixed in a certain proportion.

[0027] Further specifying, in the above-mentioned preparation method, the inorganic solid raw material powder is specifically set as one or a mixture of two of nano-hydroxyapatite and nano-β-tricalcium phosphate with calcium stearate.

[0028] And, or the liquid phase adhesive is specifically configured as a mixture of polyvinyl alcohol, ammonium polyacrylate, tocopherol acetate, glycerol and deionized water of different molecular weights.

[0029] Further specifying, in the above-mentioned preparation method, the immersion height of the porous subscaffold layer in the soybean protein-based mixed solution is set to 0.2-0.5 times the height of the porous subscaffold layer;

[0030] And, or the immersion height of the porous upper scaffold layer in the solution of mesoporous bioactive glass is set to 0.5-1.0 times the height of the porous upper scaffold layer.

[0031] Further specifying, the above-mentioned preparation method further includes:

[0032] Electron beam irradiation sterilization was performed on the osteocartilage double-layer composite scaffold.

[0033] The sterilization dose range for electron beam irradiation sterilization is 10-30 KGy.

[0034] This invention has at least the following beneficial effects:

[0035] 1. A rigid scaffold structure with porous ceramic as the main body is adopted. The porous upper scaffold layer and porous lower scaffold layer are respectively filled with carriers and loaded / filled with bioactive substances, which can realize integrated bone and cartilage repair. At the same time, it can solve the problem of growth factors leaking from the carrier due to excessive compression of the flexible cartilage repair scaffold during surgical implantation. The bioactive substances are slowly released after implantation of the double-layer scaffold. The porous structure of the scaffold is conducive to the adhesion of composite hydrogel and mesoporous bioactive glass, and also facilitates the adhesion and crawling of cells inside the scaffold after implantation, promoting the formation of new bone tissue.

[0036] 2. The primary pore size of the cartilage layer facilitates the absorption of more nutrients and the recruitment and accommodation of more bone marrow mesenchymal stem cells (the role of a bioreactor). The secondary pore size is a hierarchical structure between the primary and tertiary pore sizes. The multi-level pore structure can well simulate the hierarchical structure of human bones. After the porous upper scaffold layer is treated with adsorption mesoporous bioactive glass and mineralization, it has a rougher and larger surface, which makes it easier for proteins and nutrients to be adsorbed. In the space of the primary pore size, nutrients are provided for the life activities of differentiation into chondrocytes and chondrocytes. At the same time, the primary bioactive substances attached to the mesoporous bioactive glass will promote the differentiation of stem cells into chondrocytes in the upper layer, and generate cartilage tissue in situ based on the upper layer.

[0037] 3. The primary pore size of the bone layer facilitates the absorption of more nutrients and the recruitment and accommodation of more bone marrow mesenchymal stem cells (the role of a bioreactor), providing growth space for new bone tissue. The porous hydrogel not only helps the bone layer recruit more bone marrow mesenchymal stem cells, but the ovogloss it contains can also slowly release bioactive substances that promote bone formation. The porous sponge will slowly degrade over time after implantation and will not hinder the ingrowth of new bone tissue. The secondary pore size composed of granular nano HA / β-TCP clusters degrades more slowly and will degrade after the composite hydrogel. Since the secondary pore size runs through the primary pore size, it is conducive to the ingrowth and crawling of blood vessels in the mid-to-late stages of new bone tissue. The tertiary pore size has a larger specific surface area, which increases the contact area between cells and materials and provides more nucleation sites for HA carbonate, which is conducive to the induction of new bone formation in vivo.

[0038] 4. Mesoporous bioactive glass containing the first bioactive substance is introduced into the porous scaffold layer by immersion in SBF (simulated body fluid) under negative pressure. Immersion in simulated body fluid causes mineralization on the outer surface of the mesoporous bioactive glass, forming nano-needle clusters of hydroxyapatite carbonate. In particular, a large number of nano-needle clusters of hydroxyapatite carbonate are generated at the interface between the scaffold and the mesoporous bioactive glass on the surface. This large number of nano-needle clusters of hydroxyapatite carbonate creates a bonding effect at the interface, making the mesoporous bioactive glass firmly attached to the scaffold surface. In addition, the microporous structure of the scaffold surface promotes the formation of this process and accelerates this bonding effect. The nano-needle clusters of hydroxyapatite on the scaffold surface have a larger specific surface area, which is more conducive to the adhesion of cartilage matrix. This makes the autologous cartilage tissue stabilized on the scaffold, which is conducive to the further generation of hyaline cartilage tissue.

[0039] 5. Optimize the electron beam irradiation sterilization process and adjust the sterilization dose range to 10-30KGy. The appropriate sterilization dose range can meet the sterilization requirements and ensure that the loaded bioactive substances are not deactivated, thus maintaining an effective load. Attached Figure Description

[0040] Figure 1 This is a schematic diagram of the structure of the osteochondral double-layer composite scaffold according to an embodiment of this application;

[0041] Figure 2 This is a schematic diagram of the structure of "cartilage layer 100" in the osteochondral double-layer composite scaffold of this application embodiment;

[0042] Figure 3 This is a schematic diagram of the structure of the "hard bone layer 200" in the osteochondral double-layer composite scaffold of this application embodiment;

[0043] Figure 4 This is a schematic diagram of the structure of the osteochondral double-layer composite scaffold according to an embodiment of this application;

[0044] Figure 5 This is an electron microscope scan of the osteochondral double-layer composite scaffold according to an embodiment of this application;

[0045] Figure 6 This is a structural mapping diagram of the osteochondral double-layer composite scaffold according to an embodiment of this application;

[0046] Figure 7 This is a schematic diagram of the structure of the osteochondral double-layer composite scaffold according to an embodiment of this application;

[0047] Figure 8 This is a test schematic diagram of the osteochondral double-layer composite scaffold according to an embodiment of this application.

[0048] Figure Labels

[0049] Cartilage layer - 100, bone layer - 200. Detailed Implementation

[0050] The technical solutions of the embodiments of this application will be clearly described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this application. All other embodiments obtained by those skilled in the art based on the embodiments of this application are within the scope of protection of this application.

[0051] The terms "first," "second," etc., used in the specification and claims of this application are used to distinguish similar objects and not to describe a specific order or sequence. It should be understood that such use of data can be interchanged where appropriate so that embodiments of this application can be implemented in orders other than those illustrated or described herein, and the objects distinguished by "first," "second," etc., are generally of the same class and the number of objects is not limited; for example, a first object can be one or more. Furthermore, in the specification and claims, "and / or" indicates at least one of the connected objects, and the character " / " generally indicates that the preceding and following objects are in an "or" relationship.

[0052] The following description, in conjunction with the accompanying drawings, details the osteochondral double-layer composite scaffold and its preparation method provided in this application through specific embodiments and application scenarios.

[0053] Example 1

[0054] like Figures 1 to 8 As shown, this application provides a bone and cartilage double-layer composite scaffold, including a three-dimensional porous ceramic scaffold and a cartilage layer 100 and a bone layer 200 disposed on the three-dimensional porous ceramic scaffold. The three-dimensional porous ceramic scaffold is integrally formed by 3D printing and has a porous upper scaffold layer and a porous lower scaffold layer.

[0055] The cartilage layer 100 includes a porous upper scaffold layer, a mesoporous bioactive glass tightly attached to the outer surface and the inner interstitial surface of the porous upper scaffold layer, and a first bioactive substance loaded by the mesoporous bioactive glass.

[0056] The bone layer 200 includes a porous subscaffold layer and a lyophilized composite hydrogel perfused in the internal gaps of the porous subscaffold layer, as well as a second bioactive substance loaded by the composite hydrogel.

[0057] The interlayer angle of the porous upper support layer is set to 60-90°, and the interlayer angle of the porous lower support layer is set to 30-60°.

[0058] It is understandable that the interlayer angle is an execution parameter of 3D printing, that is, the angle between the bidirectional printing trajectories. Different interlayer angles correspond to different pore types. The interlayer angle of the porous upper scaffold layer is set to 60-90°, which has a higher porosity than the porous lower scaffold layer. It can recruit more bone marrow mesenchymal stem cells and accommodate more nutrients, which is beneficial to the differentiation and regeneration of cartilage.

[0059] In this embodiment, a bilayer composite osteocartilage scaffold is used, employing a rigid scaffold structure with porous ceramic as the main body. Carriers are added and loaded / filled with bioactive substances in the porous upper and lower scaffold layers, respectively. This enables integrated osteocartilage repair and solves the problem of growth factor leakage from the carrier caused by excessive compression of the flexible cartilage repair scaffold during surgical implantation. The scaffold allows for the slow release of bioactive substances after implantation. The porous structure of the scaffold facilitates the adhesion of composite hydrogels and mesoporous bioactive glass, and also promotes the adhesion and migration of cells inside the scaffold after implantation, thus promoting new bone tissue formation.

[0060] In a preferred embodiment, the three-dimensional porous ceramic scaffold is specifically configured as a three-dimensional porous calcium phosphate ceramic scaffold.

[0061] In a preferred embodiment, the three-dimensional porous ceramic scaffold may also be a three-dimensional porous calcium phosphate ceramic scaffold containing calcium oxide.

[0062] In a preferred embodiment, the mesoporous bioactive glass is specifically configured as one or more of silicon-based mesoporous bioglass, silicon-phosphorus-based mesoporous bioglass, and silicon-calcium-phosphorus-based mesoporous bioglass.

[0063] Understandably, mesoporous bioactive glass can promote the repair of subchondral bone, as well as the healing and regeneration of cartilage defects. As a carrier, mesoporous bioactive glass can carry bioactive substances that are beneficial to cartilage formation.

[0064] In a preferred embodiment, the shape of the mesoporous bioactive glass is set to one of the following: porous sheet, needle, rod, irregular granular, or regular spherical shape.

[0065] In a preferred embodiment, the mesoporous bioactive glass is configured to have a regular spherical shape, a particle size range of 1-100 μm, and a pore size range of 3 nm-10 μm.

[0066] In a preferred embodiment, the first bioactive substance includes one or more of a cartilage formation promoter, an anti-inflammatory drug, and an analgesic.

[0067] Understandably, the first bioactive substance loaded on mesoporous bioactive glass can promote the differentiation of bone marrow mesenchymal stem cells into chondrocytes and the secretion of extracellular matrix by chondrocytes.

[0068] In a preferred embodiment, the first bioactive substance includes growth factors such as glucosamine sulfate, chondroitin sulfate, diacerein, phentolamine, nanomedicine, Kartogenin and its derivatives, TGF-β (transforming growth factor-β), BMPs (bone morphogenetic proteins), IGF (insulin-like growth factor), and FGF (fibroblast growth factor); polysaccharides such as hyaluronic acid (HA) and chitosan, protein materials such as collagen and silk fibroin; and other exosomes and bioactive particles.

[0069] In a preferred embodiment, the composite hydrogel includes, but is not limited to, a soybean protein-based solution, tocopherol acetate, glycerol, and lecithin.

[0070] Understandably, the composite hydrogel is porous and sponge-like, which makes it easy to carry bioactive substances that promote osteogenic formation. In the early stages of implantation, it can recruit a large number of bone marrow mesenchymal stem cells and continuously release bioactive substances to promote osteogenic formation, which is beneficial to the repair of subchondral bone (hard bone layer).

[0071] Meanwhile, the lecithin (emulsifier) ​​and lecithin amphiphilic substances contained in the freeze-dried composite hydrogel can attract water and fat, and can also be used in conjunction with drug carriers (targeted drugs). Lecithin and lecithin particles loaded with bioactive substances can maintain the composite hydrogel in the bone layer 200, improve the bioavailability and stability of the drug, and are beneficial for the treatment of subchondral bone defects caused by bone tumors.

[0072] In a preferred embodiment, the second bioactive substance includes one or more of the following: bone formation promoters, bone resorption inhibitors, calcium metabolism regulators, anti-inflammatory drugs, and analgesics.

[0073] In a preferred embodiment, the second bioactive substance includes one or more of teriparatide, romosuzumab, bisphosphonates, denosumab, calcium, vitamin D, calcitonin, bioactive glass, and hydroxyapatite.

[0074] In a preferred embodiment, the radial cross-section of the three-dimensional porous ceramic support can be set as a circle, triangle, square, or other regular or irregular shape that can be achieved by 3D printing.

[0075] In a preferred embodiment, the equivalent diameter of the radial cross-section of the three-dimensional porous ceramic support is set to 3-28 mm, and the axial height of the three-dimensional porous ceramic support is set to 4-30 mm.

[0076] In a preferred embodiment, the axial height of the porous upper support layer is set to 0.5-10 mm, and the axial height of the porous lower support layer is set to 2.5-20 mm.

[0077] In a preferred embodiment, the cartilage layer 100 has a multi-level pore size, wherein the first-level pore size is set to a range of 300-600 μm, the second-level pore size is set to a range of 40-100 μm, and the third-level pore size is set to a range of 20 nm-40 μm.

[0078] The primary pore size is formed by 3D printing; the secondary pore size is specifically composed of exposed internal granular nano-HA / β-TCP (a composite bioceramic material composed of nano-sized hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP)) clusters after the MBG and nano-HA on the outer surface of the porous upper scaffold layer are consumed; the tertiary pore size is specifically composed of nano-HA / β-TCP particles, mesoporous bioactive glass adsorbed on the surface, and nano-needle-like hydroxyapatite carbonate clusters at the interface of the primary and secondary pore sizes.

[0079] In this embodiment, a bone-cartilage double-layer composite scaffold is used. The primary pore size of the cartilage layer 100 facilitates the absorption of more nutrients and the recruitment and accommodation of more bone marrow mesenchymal stem cells (the role of a bioreactor). The secondary pore size is a hierarchical structure between the primary and tertiary pore sizes. The multi-level pore structure can well simulate the hierarchical structure of human bones. After being treated with adsorbed mesoporous bioactive glass and mineralization, the porous upper scaffold layer has a rougher and larger surface, which makes it easier for proteins and nutrients to be adsorbed. In the space of the primary pore size, nutrients are provided for the life activities of differentiating into chondrocytes and chondrocytes. At the same time, the first bioactive substance attached to the mesoporous bioactive glass will promote the differentiation of stem cells into chondrocytes in the upper layer and generate cartilage tissue in situ based on the upper layer.

[0080] In a preferred embodiment, the bone layer 200 has a multi-level pore size, with the first-level pore size ranging from 200 to 400 μm, the second-level pore size ranging from 40 to 200 μm, and the third-level pore size ranging from 100 nm to 40 μm.

[0081] The primary pore size is formed by 3D printing; the secondary pore size is specifically composed of granular nano HA / β-TCP clusters and porous hydrogel; and the tertiary pore size is specifically composed of granular nano HA / β-TCP.

[0082] In this embodiment, a bilayer composite scaffold of osteochondral is used. The primary pore size of the bone layer 200 facilitates the absorption of more nutrients and the recruitment and accommodation of more bone marrow mesenchymal stem cells (the role of a bioreactor), providing growth space for new bone tissue. The porous hydrogel not only helps the bone layer 200 recruit more bone marrow mesenchymal stem cells, but the ovogloss contained therein can also slowly release bioactive substances that promote bone formation. The porous sponge will slowly degrade over time after implantation and will not hinder the ingrowth of new bone tissue. The secondary pore size composed of granular nano-HA / β-TCP clusters degrades more slowly and will degrade after the composite hydrogel. Since the secondary pore size runs through the primary pore size, it is conducive to the ingrowth and migration of blood vessels in the mid-to-late stages of new bone tissue. The tertiary pore size has a larger specific surface area, increasing the contact area between cells and materials, providing more nucleation sites for HA carbonate, which is beneficial for inducing new bone formation in vivo.

[0083] Example 2

[0084] This application provides a preparation method for the osteochondral double-layer composite scaffold in the above embodiments, comprising:

[0085] Slurry for preparing 3D printed preform scaffolds;

[0086] 3D printed preform support;

[0087] The green blank support is dried and sintered to form a three-dimensional porous ceramic support;

[0088] The porous subscaffold layer was impregnated under negative pressure into a soybean protein-based mixed solution loaded with a second bioactive substance. The impregnation was simultaneously ultrasonicated, and the porous subscaffold layer was then frozen and freeze-dried under vacuum after the impregnation and ultrasonication.

[0089] The porous scaffold layer is impregnated under negative pressure into a solution of mesoporous bioactive glass loaded with the first bioactive substance, and then vacuum dried.

[0090] Understandably, after high-temperature sintering, the green blank support has an irregular porous structure at the nano and micron levels.

[0091] In a preferred embodiment, the slurry for the 3D printed preform scaffold is specifically configured as an inorganic solid phase powder and a liquid phase adhesive mechanically mixed in a certain proportion.

[0092] In a preferred embodiment, the inorganic solid raw material powder is specifically configured as a mixture of one or two of nano-hydroxyapatite and nano-β-tricalcium phosphate with calcium stearate.

[0093] In a preferred embodiment, the liquid phase adhesive is specifically configured as a mixture of polyvinyl alcohol, ammonium polyacrylate, tocopherol acetate, glycerol, and deionized water of different molecular weights.

[0094] In a preferred embodiment, the 3D printed preform scaffold includes:

[0095] Configure 3D printing parameters;

[0096] The model's bottom diameter is set to 5-30mm, the layer height to 0.25-0.5mm, the line spacing to 0.8-1.2mm, and the printing path to a Z-shape.

[0097] The height of the porous support layer is set to 5-20mm, and the pore structure is set to 0-60-120° filling.

[0098] The height of the porous upper support layer is set to 1-10mm, and the pore structure is set to 0-90° filling.

[0099] The printing output speed is set to 0.5-4.5 mm³ / s, the line feed speed is set to 1.0-4.8 mm / s, the syringe and table temperature is set to a constant 25℃, and the printing needle uses a 0.5 mm diameter.

[0100] In a preferred embodiment, drying the green blank support specifically involves:

[0101] Place the unfinished blank in a room temperature or 37°C oven and let it stand for 24 hours.

[0102] In a preferred embodiment, the process of sintering the green body support into a three-dimensional porous ceramic support specifically involves:

[0103] First, heat the blank support to 300-400℃ at a rate of 1℃ / min and hold it at that temperature for 2-3 hours;

[0104] The blank support is then heated to 1100℃-1250℃ at a rate of 3℃ / min and held at that temperature for 2-4 hours.

[0105] In a preferred embodiment, the immersion height of the porous subscaffold layer in the soybean protein-based mixed solution is set to 0.2-0.5 times the height of the porous subscaffold layer, the vacuum degree of the negative pressure impregnation is set to 0.01-0.07 MPa, the temperature of the composite hydrogel is set to 20-40℃, and the time for negative pressure impregnation and ultrasonic treatment is set to 30-300s.

[0106] In a preferred embodiment, the solvent in the solution of the mesoporous bioactive glass loaded with the first bioactive substance is deionized water, physiological saline, SBF (simulated body fluid) or PBS (buffer solution), the immersion height of the porous upper scaffold layer in the solution of the mesoporous bioactive glass is set to 0.5-1.0 times the height of the porous upper scaffold layer, the negative pressure immersion time is set to 4-24 hours, and the vacuum degree is set to 0.01MPa-0.03MPa.

[0107] In this embodiment, the above-mentioned preparation method is used to introduce mesoporous bioactive glass containing the first bioactive substance into a porous scaffold layer by immersing it in SBF (simulated body fluid) under negative pressure. Immersion in the simulated body fluid causes the outer surface of the mesoporous bioactive glass to mineralize and generate nano-needle clusters of hydroxyapatite carbonate. In particular, a large number of nano-needle clusters of hydroxyapatite carbonate are generated at the interface between the scaffold and the mesoporous bioactive glass on its surface. This large number of nano-needle clusters of hydroxyapatite carbonate will create a bonding effect at the interface, making the mesoporous bioactive glass firmly attached to the surface of the scaffold. In addition, the microporous structure of the scaffold surface promotes the formation of this process and accelerates this bonding effect. The nano-needle clusters of hydroxyapatite on the scaffold surface have a larger specific surface area, which is more conducive to the adhesion of cartilage matrix, so that the autologous cartilage tissue is stabilized on the scaffold, which is conducive to the further generation of hyaline cartilage tissue.

[0108] In a preferred embodiment, the solution preparation method of the mesoporous bioactive glass loaded with the first bioactive substance is as follows:

[0109] First, a mixed solution of the first bioactive substance with a concentration of 15-45%wt is prepared. The first bioactive substance is uniformly dispersed into the solution by ultrasonication for 30-300 seconds. Then, mesoporous bioactive glass is immersed in the mixed solution under negative pressure (vacuum degree of 0.04MPa-0.08MPa) overnight. After vacuum drying, the particles are collected.

[0110] In a preferred embodiment, the porous support layer is further subjected to negative pressure impregnation followed by wiping off excess solution with a paper towel before vacuum drying.

[0111] In a preferred embodiment, the negative pressure impregnation and freeze-drying processes of the porous lower support layer and the porous upper support layer can be performed once or repeatedly.

[0112] In a preferred embodiment, it further includes:

[0113] Electron beam irradiation sterilizes the osteocartilage double-layer composite scaffold.

[0114] The sterilization dose range for electron beam irradiation sterilization is 10-30 KGy.

[0115] In this embodiment, the above-mentioned preparation method is used to optimize the electron beam irradiation sterilization process and adjust the sterilization dose range to 10-30 KGy. The appropriate sterilization dose range can meet the sterilization requirements and ensure that the loaded bioactive substances are not inactivated, thus maintaining an effective loading amount.

[0116] Comparative Example 1

[0117] This comparative example provides a method for preparing a bilayer calcium phosphate ceramic scaffold containing calcium oxide, comprising:

[0118] Step S1: Prepare the 3D printing slurry in the laboratory, specifically as follows:

[0119] (1) To prepare a liquid phase binder, weigh 8g of PVA (polyvinyl alcohol) powder and put it into a beaker. Add an appropriate size magnetic stir bar and 100mL of deionized water. Heat the mixture to 90-95℃ and stir magnetically for about 2 hours (until it becomes clear). Then lower the temperature to 60℃ and keep the speed unchanged. Add 0.1g of ammonium polyacrylate and 0.1g of tocopherol acetate and continue stirring for 1 hour. After cooling to room temperature, add 0.4ml of glycerol and stir evenly to finally obtain a colorless, uniform, and transparent solution.

[0120] (2) Prepare solid-phase mixed powder: Weigh 6g of nano hydroxyapatite powder, 4g of β-TCP (β-tricalcium phosphate) powder, and 0.5g of calcium stearate fine powder, and mechanically stir and mix evenly to obtain solid-phase mixed powder.

[0121] (3) Prepare 3D printing paste. Weigh 8g of solid mixed powder and add it slowly to the solution binder in multiple portions. Stir mechanically until uniform. After the solid mixed powder is completely added and stirred evenly, perform ultrasonic debubbling treatment to obtain a uniform printing paste with good extrudability and formability. Carefully transfer the prepared printing paste into the printing barrel to ensure that there are no air bubbles in the barrel and that it is filled evenly.

[0122] Step S2: Create a printable model, specifically as follows:

[0123] Import the STL format cylindrical printing model into the 3D printer, adjust the bottom diameter of the mold to 7mm, the layer height to 0.5mm, the line spacing to 1.2mm, and the printing path to Z-shape. Copy the model, keeping the bottom diameters of the two models equal. Adjust the height of the perforated lower support layer to 8mm and the infill path to 0-60-120°. Adjust the height of the perforated upper support layer to 2mm and the infill path to 0-90°. Then, superimpose the two models to obtain the final printed model.

[0124] Step S3: Set the printing parameters, specifically:

[0125] The output speed is set to 2.5 mm³ / s, the line feed speed to 2.0 mm / s, the cylinder and table temperature to a constant 25°C, the printing needle diameter to 0.5 mm, the printer extrusion method to be an electrically driven screw propeller, the worktable to make a combined motion along the xy axis, and the print head to move along the z axis, printing layer by layer, and finally completing the printing according to the specified model to obtain the blank support.

[0126] Step S4, drying the support, specifically:

[0127] Place the freshly printed blank holder in a constant temperature drying oven at 37℃ and dry for 12 hours;

[0128] Step S5, sintering of the support, specifically:

[0129] The completely dried support was placed in a corundum crucible, which was then placed in a high-temperature muffle furnace. The program was set to first raise the temperature to 300℃ at a rate of 1℃ / min and hold for 2 hours, then raise the temperature to 1200℃ at a rate of 3℃ / min and hold for 2 hours. After that, the temperature was lowered to room temperature. After sintering, a three-dimensional porous calcium phosphate ceramic support containing calcium oxide was finally obtained.

[0130] To evaluate the structure of the prepared three-dimensional porous calcium phosphate ceramic scaffold containing calcium oxide, the general appearance of the scaffold was assessed visually, and the surface and internal structure of the scaffold were evaluated using field emission scanning electron microscopy (FET). At the same time, the elemental distribution on the scaffold surface was evaluated using an energy dispersive spectrometer attached to the FET.

[0131] like Figure 4 As shown, Part A is an external image of the three-dimensional porous calcium phosphate ceramic scaffold. The scaffold is cylindrical in shape, with both the upper and lower layers exhibiting a three-dimensional porous structure. Part B is a SEM image of the porous upper scaffold layer (using a scanning electron microscope). The images obtained using microscope (SEM) show that the porous upper scaffold layer exhibits a 90-degree rectangular pore structure with uniform fiber arrangement, regular pore size, and relatively large pore size. Based on the structural characteristics of the porous upper scaffold layer, the larger pore size here is the first-order pore size, with a pore size range of 300-600 μm. Part C is the SEM image of the porous lower scaffold layer, which exhibits a 60-degree pore structure with uniform fiber arrangement, regular pore size, and relatively large pore size. Based on the structural characteristics of the porous lower scaffold layer, the larger pore size here is the first-order pore size, with a pore size range of 200-400 μm. Part D is the SEM image of the side stacking structure of the three-dimensional porous ceramic scaffold, that is, the sides of the three-dimensional porous ceramic scaffold also have interconnected pore structures, with a pore size range of 200-450 μm.

[0132] The primary pore structure in the porous lower scaffold layer and porous upper scaffold layer facilitates the absorption of more nutrients and the recruitment and accommodation of more bone marrow mesenchymal stem cells (the role of a bioreactor).

[0133] like Figure 5 As shown, it is a high-magnification SEM image of the surface and interior of the three-dimensional porous ceramic scaffold fibers. Parts A and B are SEM images of the surface of the three-dimensional porous ceramic scaffold fibers; parts C and D are SEM images of the internal fibers of the three-dimensional porous ceramic scaffold.

[0134] Depend on Figure 5 It can be seen that the three-dimensional porous ceramic scaffold after high-temperature sintering exhibits uniform micropores on its inner and outer surfaces. The micropores are formed by the mixed stacking of larger nHA particles (nano-hydroxyapatite particles) and β-TCP particles (β-tricalcium phosphate particles) with a particle size range of 40μm to 100μm, while the micropores are formed by the mixed stacking of smaller nHA particles and β-TCP particles with a particle size range of 100nm to 40μm.

[0135] like Figure 6 As shown, it is specifically a mapping diagram of a selected region on the surface of a three-dimensional porous ceramic scaffold fiber, formed by... Figure 6 It can be seen that the three-dimensional porous ceramic scaffold prepared under the specified process (3D printing, sintering process) has a uniform distribution of each element (calcium, phosphorus, oxygen), which is consistent with the expectation. This indicates that 3D printing technology can be used to prepare a double-layer scaffold with a uniform composition distribution.

[0136] Comparative Example 2

[0137] This comparative example provides a method for preparing a double-layer composite scaffold for osteochondral bone, which differs from Comparative Example 1 above in that it further includes modification treatment of the porous lower scaffold layer and the porous upper scaffold layer.

[0138] The modification treatment of the porous substructure includes:

[0139] (1) Impregnation under negative pressure into a composite hydrogel solution loaded with a second bioactive substance

[0140] Weigh 10g of soy protein powder and dissolve it in 100mL of deionized water. Stir magnetically until the soy protein is completely dissolved. Adjust the pH of the solution to a slightly acidic level (e.g., pH 5.5–6.5) using 0.1M HCl. Add 0.1M calcium chloride solution (mix in equal volumes) to the mixture, then add 1mL of glycerol, 1g of tocopherol acetate, and 3g of lecithin (encapsulated with teriparatide). Place the mixture in a 37°C water bath, stir magnetically, and sonicate for 60 seconds to ensure that calcium ions are fully in contact with the protein.

[0141] The porous scaffold layer in Comparative Example 1 was immersed in the mixed sol liquid, and the immersion height was controlled to be 0.5 times the height of the porous scaffold layer. The system was then transferred to a vacuum drying oven (37°C, vacuum degree of negative pressure impregnation of 0.07MPa) for 2 hours. After 2 hours, obvious cross-linking phenomenon was observed.

[0142] (2) Freeze drying

[0143] After the negative pressure impregnation is completed, the porous sub-scaffold layer that has been gelled is removed. Excess solution is wiped off with a paper towel, and the surface is gently rinsed with deionized water to remove excess calcium ions. The porous sub-scaffold layer that has been gelled into hydrogel is placed in a -25°C freezer overnight, and then vacuum freeze-dried (-40°C, vacuum degree 90-300) to obtain a composite sponge-like porous sub-scaffold layer.

[0144] Modification treatment of the porous support layer includes:

[0145] (1) Preparation of mesoporous bioactive glass

[0146] Mesoporous bioactive glass can be prepared using, but is not limited to, 80SMBG (mesoporous bioactive glass with a silica content of 80 mol%), 77SMBG, 70SMBG, and 60SMBG. Mesoporous bioactive glass can be prepared by methods such as sol-gel method, solvent evaporation self-assembly method, or spray drying of bioactive glass precursor solution.

[0147] The specific preparation process for 80 SMBG using the sol-gel method is as follows:

[0148] At room temperature, 240g of ethanol was used as a solvent, and 16g of polyether P123 (Mn=5800), 26.8g of tetraethyl orthosilicate (silicon source), 2.92g of triethyl phosphate (phosphorus source), and 5.60g of calcium nitrate tetrahydrate (calcium source) were added sequentially. The mixture was stirred for 24 hours, and the solution was poured into a glass petri dish and placed in a ventilated cabinet. After the ethanol had mostly evaporated and the sol had turned into a wet gel, it was placed in a forced-air drying oven and dried at 60°C for 48 hours until it became a relatively dry gel sheet.

[0149] Grind the gel sheet into smaller fragments using an agate mortar and pestle, and evenly disperse them into a corundum crucible. Place the corundum crucible in a muffle furnace, set the calcination program, and start from room temperature. Increase the temperature to 650°C at a rate of 1°C / min and hold for 2 hours, then reduce to room temperature.

[0150] The calcined dry gel sheet was obtained. The sintered dry gel sheet, small and large zirconia ball milling mills were placed in a planetary ball mill jar at a mass ratio of 1:1:2, and the mass of the symmetrical ball mill jar and its contents was balanced. After grinding for 10 hours, the ball mill jar was removed, allowed to stand for 20 minutes, and the ball milling mills and 80 SMBG were separated using a standard sieve.

[0151] (2) Mesoporous bioactive glass support

[0152] At room temperature, 100 mL of deionized water was added to a beaker. 5 g of 80SMBG and 1 g of chondroitin sulfate were then added to the deionized water. The chondroitin sulfate and 80SMBG microspheres were uniformly dispersed in the solution by sonication for 180 seconds. The beaker and mixture were then transferred to a vacuum freeze dryer (vacuum degree adjusted to 0.08 MPa) and soaked under negative pressure overnight. After vacuum drying, 80SMBG microspheres loaded with chondroitin sulfate were obtained.

[0153] (3) Negative pressure impregnation of a mixed solution of mesoporous bioactive glass and the first bioactive substance

[0154] At room temperature, weigh 100 mL of freshly prepared simulated body fluid into a beaker, add a magnetic stir bar, weigh 3 g of chondroitin sulfate-loaded 80SMBG into the beaker, stir evenly, and ultrasonically disperse for 180 seconds.

[0155] Pour the mixture into a glass petri dish, immerse the porous upper scaffold layer of the three-dimensional porous ceramic scaffold in the solution, and control the immersion height to be 0.7 times that of the porous upper scaffold layer. Place the three-dimensional porous ceramic scaffold and the mixture in a vacuum drying oven (without drying, control the negative pressure to 0.01 MPa) for 4 hours, then turn off the vacuum, adjust the temperature to 37℃, and let the system stand for 20 hours. On the second day, take out the three-dimensional porous ceramic scaffold immersed in the mixture solution and place it in a 37℃ oven to dry, and obtain a three-dimensional porous ceramic scaffold with modified porous upper scaffold layer.

[0156] The three-dimensional porous ceramic scaffold is packaged in double or triple sealed plastic bags and then sterilized by irradiation with gamma rays or electron beams at a dose of 25 kGy.

[0157] like Figure 1 As shown, the osteochondral double-layer composite scaffold is a three-dimensional porous nHA / β-TCP ceramic scaffold containing calcium oxide, such as... Figure 2 As shown, the cartilage layer 100 includes a porous upper scaffold layer with an interlayer angle of 90°, mesoporous bioactive glass particles tightly attached to the outer surface and inner pore surface of the porous upper scaffold layer and covered with nanoneedle-like HA, and a first bioactive substance loaded thereon, such as... Figure 3 As shown, the bone layer 200 includes a porous subscaffold layer with an interlayer angle of 60°, a composite hydrogel (lyophilized) perfused in the pores of the porous subscaffold layer, and a second bioactive substance loaded on the composite hydrogel.

[0158] To evaluate the microstructure of the outer surface of the cartilage layer 100 of the modified osteochondral double-layer composite scaffold, field emission scanning electron microscopy was used to observe the cartilage layer 100 of the osteochondral double-layer composite scaffold.

[0159] like Figure 7 As shown, the surface of the cartilage layer 100 is covered by spherical objects with nano-needle-like clusters. Based on the preparation method and the mineralization process of 80SMBG in simulated body fluid, it can be concluded that the spherical objects on the surface of the cartilage layer 100 are 80SMBG microspheres, and the nano-needle-like clusters on the surface of the microspheres are nano-HA.

[0160] according to Figure 7 According to statistics from ImageJ software (an open-source image processing and analysis software), the porous structure on the surface of the osteochondral double-layer composite scaffold is composed of alternating nanometer and micrometer scales, with a pore size ranging from 20 nm to 40 μm. Figure 5 The observation results show that the mixed stacking of larger nHA particles and β-TCP particles constitutes the secondary pore size of cartilage layer 100, with a pore size range of 40μm-100μm. The pores presented by the mixed stacking of smaller nHA particles and β-TCP particles, together with the nano- and micro-scale porous structures on the surface of the osteochondral double-layer composite scaffold, constitute the tertiary pore size structure of cartilage layer 100, with a tertiary pore size range of 20nm-40μm.

[0161] After being adsorbed with MBG and mineralized, the cartilage layer 100 has a mineralized outer surface of mesoporous bioactive glass to form nanoneedle-shaped HA carbonate, which has a rougher and larger surface (tertiary pore size), making it easier for proteins and nutrients to be adsorbed. In the space of the primary pore size, nutrients are provided for the life activities of chondrocyte differentiation and chondrocyte life. In addition, the primary bioactive substances loaded in MBG will promote stem cells to differentiate into chondrocytes in the upper layer, and differentiate and proliferate in situ on the porous upper scaffold layer as a base to generate chondrocyte extracellular matrix, and then generate cartilage tissue. The secondary pore size structure of the cartilage layer 100 is a hierarchical structure between the primary and tertiary pore size structures. These three hierarchical structures well simulate the hierarchical structure of human bones.

[0162] Field emission scanning electron microscopy was used to evaluate the structure of the scleroderma layer 200 of the osteochondral double-layer composite scaffold. It was found that the pore size of the composite hydrogel permeating the interconnected channels of the scleroderma layer 200 ranged from 50 to 200 μm. Figure 5Characterization results show that the secondary porous structure of the bone layer 200 is mainly composed of a porous structure of composite hydrogel with a mixed stack of larger nHA particles and β-TCP particles that runs through the bone layer 200. The pore size ranges from 40 to 200 μm. The composite hydrogel can not only help recruit more bone marrow mesenchymal stem cells in the porous scaffold layer, but also contains ovulin, which can slowly release bioactive substances that promote bone formation. The porous sponge will slowly degrade over time and will not hinder the ingrowth of new bone tissue. The secondary pores composed of larger granular nano-HA / β-TCP clusters degrade more slowly and will degrade after the porous sponge. The secondary pores it provides run through the primary pores, which is conducive to the ingrowth and creep of blood vessels in the mid-to-late stages of new bone tissue.

[0163] Similarly, combining Figure 5 It is known that the tertiary pore size of the bone layer 200 ranges from 100nm to 40μm, and it is mainly composed of a mixture of smaller nHA particles and β-TCP particles. This provides a larger surface area, increases the contact area between cells and materials, provides more nucleation sites for HA carbonate, and induces the formation of new bone in vivo.

[0164] To evaluate the ability of the modified osteocartilage bilayer composite scaffold with modified bone layer 200 to adsorb simulated plasma, the osteocartilage bilayer composite scaffolds with unmodified bone layer 200 (Comparative Example 1) and modified bone layer 200 (Comparative Example 2) were placed in shallow simulated plasma. The mass of the scaffolds before immersion and after 3 seconds of immersion was recorded, and the scaffolds' ability to automatically adsorb simulated plasma was calculated. The conclusions are shown in the table below:

[0165]

[0166] Wherein, the fluid adsorption rate of the stent / % = (m stent wet weight - m stent dry weight) / m stent dry weight.

[0167] like Figure 8As shown, Part A shows the left side of the unmodified osteocartilage double-layer composite scaffold immersed in simulated body fluid, and the right side shows the right side of the modified osteocartilage double-layer composite scaffold immersed in simulated body fluid. Part B shows the frontal view of the two scaffolds removed from simulated plasma. It can be seen that the unmodified scaffold has limited ability to absorb simulated plasma in a short time, which is not as good as the modified scaffold (simulated plasma immediately covers the entire scaffold, which is visible to the naked eye and spreads rapidly to the upper layer). Part C shows the inverted view of the two scaffolds removed from simulated plasma. It can be seen that the porous foam structure formed by 200 oz hydrogel soaked in the bone layer and freeze-dried is more conducive to the adsorption and in-situ storage of simulated plasma. The porous hydrogel material after freeze-drying is mainly soy-based protein. The degradation rate of soy-based protein in vivo is about 15 days. It will be degraded and absorbed later. The degradation rate is faster than that of the main scaffold structure material, so the soy-based protein will not occupy the three-dimensional interconnected pore structure for a long time and will not hinder the crawling and growth of blood vessels and new bone tissue in the primary and secondary pore structures.

[0168] Combination Figure 8 As shown in the table above, the modified osteochondral bilayer composite scaffold has a stronger ability to adsorb simulated plasma, reaching 49.12±6.78%. This is very beneficial for recruiting bone marrow mesenchymal stem cells (BMSCs) from the subchondral bone in the early stage after the sample is implanted into the human body. At the same time, it can also promote the influx of BMSCs into the cartilage layer 100. Under the action of the bioactive substances that promote cartilage formation by slow release of MBG, the patient's own BMSCs are induced to differentiate into chondrocytes, which further secrete extracellular matrix of chondrocytes and finally generate cartilage tissue.

[0169] Example 3

[0170] This embodiment provides a method for preparing a bilayer composite scaffold for osteochondral bone, which differs from Comparative Example 2 in the method of preparing mesoporous bioactive glass, specifically by using a spray-drying method to prepare 80SMBG of bioactive glass precursor solution, including:

[0171] (1) Preparation of spray-dried precursor solution

[0172] Preparation of a 15% wt precursor solution: At room temperature, take 106 mL of deionized water, add 0.1 M HNO3 to adjust the pH of the aqueous solution to 2, add 10.0 g of polyether P123 (Mn = 5800), stir overnight until the solution becomes clear, add 6.7 g of tetraethyl orthosilicate (silicon source) to the mixed solution and stir magnetically until the solution is transparent and clear, then add 0.73 g of triethyl phosphate (phosphorus source) to the above clear solution and stir for 30 min, finally add 1.4 g of calcium nitrate tetrahydrate (calcium source) and stir for 20 min to obtain a clear precursor solution for later use;

[0173] (2) Preparation of mesoporous bioactive glass microspheres by spray drying

[0174] A spray dryer (nozzle diameter can be selected from 1 to 10 mm), preferably 1.5 mm, is used.

[0175] The parameters for preparing 80SMBG microspheres by spray drying precursor solution were as follows: the instrument circulation rate was 100%, the inlet temperature was 220℃, the precursor solution concentration was 15wt%, the air inlet flow rate was 1052L / h, and the feed rate was 3mL / min.

[0176] 80 SMBG microspheres were collected in a spray dryer; at this point, they were not yet biologically active and had not been calcined.

[0177] The collected microspheres were uniformly dispersed in an alumina crucible, which was then placed in a muffle furnace. A calcination program was set, and the temperature was increased from room temperature to 650°C at a rate of 1°C / min and held for 2 hours. The temperature was then lowered to room temperature, and the sample was collected.

[0178] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element. Furthermore, it should be noted that the scope of the methods and apparatuses in the embodiments of this application is not limited to performing functions in the order shown or discussed, but may also include performing functions substantially simultaneously or in the reverse order, depending on the functions involved. For example, the described methods may be performed in a different order than described, and various steps may be added, omitted, or combined. Additionally, features described with reference to certain examples may be combined in other examples.

[0179] The embodiments of this application have been described above with reference to the accompanying drawings. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. An osteochondral bilayer composite scaffold, characterized in that, The three-dimensional porous ceramic scaffold is a three-dimensional porous calcium phosphate ceramic scaffold or a three-dimensional porous calcium phosphate ceramic scaffold containing calcium oxide. The three-dimensional porous ceramic scaffold is integrally formed by 3D printing and has a porous upper scaffold layer and a porous lower scaffold layer. The cartilage layer includes a porous upper scaffold layer, mesoporous bioactive glass closely attached to the outer surface and internal gap surface of the porous upper scaffold layer, and a first bioactive substance loaded by the mesoporous bioactive glass. The hard bone layer includes a porous lower scaffold layer, a composite hydrogel infused in the internal gap of the porous lower scaffold layer in a freeze-dried state, and a second bioactive substance loaded by the composite hydrogel. The interlayer angle of the porous upper scaffold layer is set to 60-90°, and the interlayer angle of the porous lower scaffold layer is set to 30-60°.

2. The osteochondral double-layer composite scaffold according to claim 1, wherein, The mesoporous bioactive glass is specifically a silicon-based mesoporous bioglass. The first bioactive substance includes one or more of a cartilage formation promoter, an anti-inflammatory drug, and an analgesic.

3. The osteochondral bilayer composite scaffold of claim 2, wherein, The silicon-based mesoporous bioglass includes one or more of a silicon-phosphorus-based mesoporous bioglass and a silicon-calcium-phosphorus-based mesoporous bioglass.

4. The osteochondral double-layer composite scaffold according to claim 1 or 2, and a preparation method thereof, characterized in that, The composite hydrogel includes one or more of a soy protein-based solution, tocopherol acetate, glycerol, and lecithin. The second bioactive substance includes one or more of a bone formation promoter, a bone resorption inhibitor, a calcium metabolism regulator, an anti-inflammatory drug, and an analgesic.

5. The osteochondral bilayer composite scaffold of claim 1, wherein, The cartilage layer has multiple pore sizes, the first pore size is set to 300-600μm, the second pore size is set to 40-100μm, and the third pore size is set to 20nm-40μm. The first pore size is formed by 3D printing, the second pore size is specifically the mesoporous bioactive glass MBG and nano-hydroxyapatite HA consumed on the outer surface of the porous upper scaffold layer, and the third pore size is specifically the exposed internal granular nano-HA / β-TCP cluster.

6. The osteochondral bilayer composite scaffold of claim 1, wherein, The hard bone layer has multiple pore sizes, the first pore size is set to 200-400μm, the second pore size is set to 40-200μm, and the third pore size is set to 100nm-40μm. The first pore size is formed by 3D printing, the second pore size is specifically the granular nano-hydroxyapatite HA / β-TCP cluster and the porous hydrogel, and the third pore size is specifically the granular nano-HA / β-TCP.

7. A method of manufacture, characterized by, The bone-cartilage double-layer composite scaffold of any one of claims 1-6 comprises: A slurry for preparing a three-dimensional printed embryonic scaffold; Three-dimensional printing of the embryonic scaffold; Drying and sintering the embryonic scaffold to form a three-dimensional porous ceramic scaffold; The porous lower scaffold layer is immersed in a soy protein-based mixed solution loaded with a second bioactive substance under negative pressure, and the immersion is accompanied by ultrasonic treatment, and after the immersion and ultrasonic treatment of the porous lower scaffold layer, the porous lower scaffold layer is subjected to freezing, vacuum freezing and freeze-drying. The porous upper scaffold layer is immersed in a solution of mesoporous bioactive glass loaded with a first bioactive substance under negative pressure, and after the immersion, the porous upper scaffold layer is subjected to vacuum drying.

8. The preparation method according to claim 7, characterized in that, The slurry of the three-dimensional printed embryonic scaffold is specifically configured as a mixture of inorganic solid-phase powder and liquid-phase adhesive in a certain proportion.

9. A method of manufacture according to claim 8, wherein, The inorganic solid-phase raw material powder is specifically configured as a mixed powder of one or both of nano-hydroxyapatite and nano-beta-tricalcium phosphate and calcium stearate; and / or the liquid-phase adhesive is specifically configured as a mixture of polyvinyl alcohol, ammonium polyacrylate, tocopherol acetate, glycerol and deionized water with different molecular weights.

10. The preparation method according to claim 7, characterized in that, The immersion height of the porous lower scaffold layer in the soy protein-based mixed solution is set to 0.2-0.5 times the height of the porous lower scaffold layer. and / or the immersion height of the porous upper scaffold layer in the solution of mesoporous bioactive glass is set to 0.5-1.0 times the height of the porous upper scaffold layer.

11. The preparation method according to claim 7, characterized in that, Further comprising: electron beam irradiation sterilization of the osteochondral double-layer composite scaffold; wherein the sterilization dose of the electron beam irradiation sterilization ranges from 10-30 KGy.

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