A double-layer hydrogel bioactive scaffold and its preparation method and application

Abstract

The present invention discloses a double-layer hydrogel bioactive scaffold, its preparation method, and application. The preparation method of the double-layer hydrogel bioactive scaffold comprises the following steps: first, a first biohydrogel is deposited using a bioprinting process to form a cartilage layer; then, a second biohydrogel is deposited on the cartilage layer using a bioprinting process to form a subchondral bone layer, thereby obtaining a double-layer hydrogel bioactive scaffold; wherein, the pore size of the cartilage layer is 100-300 μm; and the pore size of the subchondral bone layer is 300-600 μm. The double-layer hydrogel bioactive scaffold obtained by the preparation method of the present invention not only has good biocompatibility, but also has a good homing effect on cells and growth factors in the cartilage and subchondral bone layers, thereby providing a foundation for optimal repair ability when repairing joints.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioactive scaffolds, and in particular relates to a double-layer hydrogel bioactive scaffold and a preparation method and application thereof. Background Art

[0002] With the aging of the population and the increase in sports injuries, the incidence of bone and joint diseases is rising, seriously affecting people's quality of life. Various degenerative diseases and traumatic injuries, as well as the natural wear and tear of cartilage tissue with aging, leading to osteoarthritis, are the main causes of osteochondral damage.

[0003] The current treatments for cartilage damage can be summarized into three categories: ① bone marrow stimulation techniques: drilling, microfracture, autologous matrix-induced cartilage regeneration, etc.; ② direct cartilage replacement: osteochondral transplantation, mosaic transplantation, etc.; ③ cell therapy: autologous chondrocyte transplantation, matrix-induced autologous chondrocyte transplantation, etc. These treatment methods have the following disadvantages: limited donor sources, easy lesions in the treatment area, large-area damage (>2cm 2 ) have limited repair effects. In recent years, tissue engineering has emerged as a new generation of cartilage repair technology, involving the construction of a cartilage repair scaffold in vitro and subsequent implantation at the site of the cartilage defect. Existing reports on bone-cartilage repair scaffolds primarily focus on constructing double-layer or multi-layer scaffolds that promote bone and cartilage growth. However, these scaffolds often suffer from issues with biocompatibility and poor cell growth on the scaffold material surface. Summary of the Invention

[0004] The purpose of the present invention is to construct a novel double-layer hydrogel bioactive scaffold and apply it to the repair of articular cartilage defects.

[0005] To achieve the above objectives, the technical solution of the present invention is implemented as follows: a method for preparing a double-layer hydrogel bioactive scaffold, the method specifically comprising: firstly depositing a first biohydrogel using a bioprinting process to form a cartilage layer; then depositing a second biohydrogel using a bioprinting process on the cartilage layer to form a subchondral bone layer, thereby obtaining a double-layer hydrogel bioactive scaffold;

[0006] The pore size of the cartilage layer is 100-300um; the pore size of the subchondral bone layer is 300-600um.

[0007] Preferably, the first bio-hydrogel and the second bio-hydrogel are both photocurable bio-inks.

[0008] Preferably, the first bio-hydrogel is made of methacrylated gelatin as a raw material; and the second bio-hydrogel is made of methacrylated alginate as a raw material.

[0009] Preferably, the first bio-hydrogel is prepared by mixing a methacrylated gelatin precursor solution with a photoinitiator I2959 and then photopolymerizing the mixture using 365 nm ultraviolet irradiation.

[0010] Preferably, the second hydrogel is prepared by mixing a methacrylated alginate precursor solution with a photoinitiator I2959 and then photopolymerizing the mixture under ultraviolet irradiation at 365 nm.

[0011] Preferably, the mass concentration of methacrylated gelatin in the methacrylated gelatin precursor solution is 8-12%; the mass concentration of methacrylated alginate in the methacrylated alginate precursor solution is 1-3%.

[0012] Preferably, the methacrylated gelatin is prepared by dissolving methacrylate monomer and gelatin in a CB buffer solution at 40-60° C. for 2-4 hours to obtain methacrylate anhydride gelatin after the methacrylate reacts with the amine group of the gelatin.

[0013] Preferably, the methacrylated alginate is obtained by combining 2-aminoethyl methacrylate hydrochloride with the carboxyl group of sodium alginate activated by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS).

[0014] The second technical solution of the present invention is achieved as follows: a double-layer hydrogel bioactive scaffold prepared by the above-mentioned preparation method, wherein the double-layer hydrogel bioactive scaffold has a porous structure.

[0015] The third technical solution of the present invention is achieved as follows: an application of the above-mentioned double-layer hydrogel bioactive scaffold in wound repair.

[0016] Compared with the prior art, in the process of preparing a double-layer hydrogel bioactive scaffold, the present invention adopts a first biohydrogel and a second biohydrogel to deposit respectively to form a cartilage layer and a subchondral bone layer, and sets the pore size of the cartilage layer to 100-300um, and the pore size of the subchondral bone layer to 300-600um, so that the obtained double-layer hydrogel bioactive scaffold has good biocompatibility and has an adsorption effect on wound exudate, thereby laying the foundation for providing repair ability when repairing the wound; in addition, the double-layer hydrogel bioactive scaffold obtained by the present invention has a porous structure, the pore size of the cartilage layer is 100-300um, and the pore size of the subchondral bone layer is 300-600um, which is conducive to the slow release of cells and their growth factors in the porous structure to the location of the joint defect, thereby achieving a long-term repair effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1Schematic diagram of a double-layer hydrogel bioactive scaffold obtained in an embodiment of the present invention;

[0018] Figure 2 Schematic diagram of the cartilage layer under the double-layer hydrogel bioactive scaffold obtained in an embodiment of the present invention, wherein: A is a schematic diagram of the subchondral bone layer under the scaffold; B is a schematic diagram of the cartilage layer under the scaffold;

[0019] Figure 3 Figure 1 is a schematic diagram of the process for preparing an animal experimental model in an embodiment of the present invention, wherein: A is a schematic diagram of creating a cartilage defect in the rat knee cartilage; B is a schematic diagram of a cartilage defect (3 mm in diameter, 2 mm in depth, a large and deep defect); C is a schematic diagram of implanting a sample into the defect site;

[0020] Figure 4 Schematic diagram of rat cartilage repair;

[0021] Figure 5 Schematic diagram of rat cartilage repair, wherein: A is a schematic diagram of HE detection of rat cartilage repair; B is a schematic diagram of aniline blue detection of rat cartilage repair;

[0022] Figure 6 Schematic diagram of cell growth on the surface of the material. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0024] In the description of the present invention, it should be clarified that the terms "vertical", "transverse", "longitudinal", "front", "rear", "left", "right", "up", "down", "horizontal", etc. indicating orientation or positional relationship are based on the orientation or positional relationship shown in the accompanying drawings, which are only for the convenience of describing the present invention, and do not mean that the device or element referred to must have a specific orientation or position, and therefore cannot be understood as a limitation on the present invention. In the description of the present invention, it should be noted that, unless otherwise clearly stipulated and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be directly connected or indirectly connected through an intermediate medium. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0025] An embodiment of the present invention provides a method for preparing a double-layer hydrogel bioactive scaffold, which specifically comprises: firstly depositing a first biohydrogel using a bioprinting process to form a cartilage layer; then depositing a second biohydrogel using a bioprinting process on the cartilage layer to form a subchondral bone layer, thereby obtaining a double-layer hydrogel bioactive scaffold;

[0026] The pore size of the cartilage layer is 100-300um; the pore size of the subchondral bone layer is 300-600um.

[0027] The first bio-hydrogel and the second bio-hydrogel are both photocurable bio-inks; the first bio-hydrogel is made of methacrylated gelatin as a raw material; the second bio-hydrogel is made of methacrylated alginate as a raw material; the first bio-hydrogel is made by mixing a methacrylated gelatin precursor solution with a photoinitiator I2959 and photopolymerizing it under 365nm ultraviolet irradiation; the second bio-hydrogel is made by mixing a methacrylated alginate precursor solution with a photoinitiator I2959 and photopolymerizing it under 365nm ultraviolet irradiation; the mass concentration of methacrylated gelatin in the methacrylated gelatin precursor solution is 8-12%; the mass concentration of methacrylated alginate in the methacrylated alginate precursor solution is 1-3%;

[0028] The methacrylated gelatin is prepared by dissolving methacrylate monomer and gelatin in a CB buffer solution at 40-60°C for 2-4 hours to obtain methacrylated gelatin after the methacrylate reacts with the amine groups of the gelatin. By adjusting the amount of methacrylate (MA) added to the reaction mixture, different degrees of methacryloyl substitution can be achieved in the methacrylated gelatin (GelMA), thereby producing methacrylated gelatin (GelMA) with different physical properties. Due to the introduction of methacryloyl groups into the gelatin molecular skeleton to replace the amino groups in the gelatin molecules, the methacrylated gelatin (GelMA) acquires a certain degree of photosensitivity. Under the irradiation of a photoinitiator and light of a certain wavelength, the methacrylate groups will cross-link to form a hydrogel with adjustable mechanical properties.

[0029] The methacrylated alginate is obtained by combining 2-aminoethyl methacrylate hydrochloride with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS) to activate the carboxyl groups of sodium alginate; by adjusting the amount of methacrylated alginate (AEMA) in the reaction mixture, different degrees of methacrylated alginate can be obtained, and finally methacrylated alginate (AEMA) with different physical and mechanical properties can be obtained. The degree of methacrylated alginate can be determined based on the percentage of carboxyl groups replaced by methacrylate groups in the alginate, and the percentage of carboxyl groups replaced by methacrylate groups can be determined using 1 Determined by HNMR spectroscopy.

[0030] The biomedical materials of this embodiment are as follows:

[0031] Gelatin is widely available, exhibits excellent biocompatibility, hydrophilicity, biodegradability, and strong plasticity, making it a commonly used hydrogel material for cartilage regeneration. However, gelatin itself has poor mechanical properties and is unstable when heated. These properties can be enhanced through modification or compounding with other materials. Currently, gelatin is commonly methacrylated and then cross-linked using UV light to enhance its biomechanical strength. Alternatively, gelatin can be cross-linked with glutaraldehyde to enhance its mechanical strength. However, this significantly reduces gelatin's viscosity and elasticity, and its toxicity significantly affects cell growth. Because gelatin is rich in amino, carboxyl, and amide groups, current research focuses on grafting methacrylates and photopolymerizing them to enhance gelatin's mechanical strength, effectively avoiding the toxic effects of chemical cross-linkers on cells. Chen et al. generated a cartilage extracellular matrix / methacrylated gelatin exosome-loaded scaffold using a photocurable 3D printing process. Implanted in vivo, the scaffold effectively repaired chondrocyte mitochondrial dysfunction, strengthened cartilage, and significantly promoted cartilage regeneration in rabbit articular cartilage defects. Zhang et al. used photocurable 3D printing technology to fabricate a 3D polyethylene glycol diacrylate (PEGDA) mesh scaffold. The PEGDA mesh scaffold served as the mechanical support for the cartilage scaffold. Cells were then encapsulated with methacrylated gelatin (GelMA) / alginate composite hydrogel, and the methacrylated gelatin (GelMA) / alginate composite hydrogel with cells was then filled into the PEGDA mesh scaffold to prepare a cartilage scaffold with good biocompatibility, strong mechanical properties, cell adhesion, and biodegradability. Gao et al. fabricated an integrated cross-linked double-layer hydrogel scaffold of methacrylated gelatin (GelMA) and GelMA-hydroxyapatite (HAp) to evaluate the effect of the spacing of 3D printed filaments on osteochondral regeneration in a rabbit model. The porous scaffold with moderate spacing had better cartilage regeneration in the cartilage cavity and formation of subchondral bone. Therefore, control of the rheological properties of methacrylated gelatin (GelMA) hydrogels can be used to improve 3D printing, and the integrated bilayer hybrid scaffolds produced by continuous 3D printing are expected to serve as biomaterials for regenerating articular cartilage.

[0032] Alginate (Alg) is a substance extracted from seaweed such as brown algae, nori, Japanese kelp, ascomycetes and macrospora. Alg is a linear copolymer containing (1,4)-D-mannuronic acid and L-guluronic acid residues, which reacts with divalent cations (such as Ca 2+Cross-linked algae hydrogels have recently been used as cell delivery scaffolds for tissue engineering applications. The structure of the guluronic acid blocks allows a wide range of divalent cations to bind to the algae chains. This forms ionic bridges between the guluronic acid blocks of adjacent polymer chains, resulting in an egg-box-like structure. Alg hydrogels are a relatively common type of MSC-exos scaffold. For example, Shafei et al. prepared an Alg-exosome-loaded scaffold for skin wound repair. Compared to Alg scaffolds alone, the exosome-loaded scaffold significantly enhanced skin wound closure, collagen synthesis, and angiogenesis in the wound area. Therefore, it is considered an ideal bioactive composite dressing for treating skin injuries. In another study, an Alg scaffold loaded with exosomes isolated from human umbilical cord-derived MSCs was developed for the treatment of pain caused by nerve injury. Three-dimensional cell-printed alginate / PCL scaffolds containing TGF-β showed higher levels of ECM formation. Kesti et al. developed a cartilage-specific bioink blend of alginate and gellan gum extracellular matrix particles that was found to outperform native articular cartilage. Maxter et al. combined alginate and nanofibrillated cellulose, whose rapid cross-linking ability and shear-thinning properties made the scaffolds manufactured by 3D printing more stable. The PEG / alginate hydrogel composite created by 3D printing showed higher fracture resistance and higher cell viability than natural cartilage. Daly et al. produced a composite material for cartilage tissue engineering that included alginate, agarose, methacrylated gelatin (GelMA) and bio-ink and found that it was better than natural cartilage. Due to its high biocompatibility, biodegradability, non-antigenicity and high water absorption, Alg is considered to be a functional material suitable for biomedical applications.

[0033] The following are specific embodiments

[0034] Example 1

[0035] The double-layer hydrogel bioactive scaffold provided in Example 1 of the present invention is prepared by the following method:

[0036] A first biohydrogel made from methacrylated gelatin is first deposited using a bioprinting process to form a cartilage layer. A second biohydrogel made from methacrylated alginate is then deposited on the cartilage layer using a bioprinting process to form a subchondral bone layer. The pore size of the cartilage layer is 200 μm. The pore size of the subchondral bone layer is 500 μm, thus obtaining a double-layer hydrogel bioactive scaffold.

[0037] Among them, the first bio-hydrogel is prepared by mixing a methacrylated gelatin precursor solution with a mass concentration of 10% with a photoinitiator I2959 and photopolymerizing it under 365nm ultraviolet irradiation; the second bio-hydrogel is prepared by mixing a methacrylated alginate precursor solution with a mass concentration of 2% with a photoinitiator I2959 and photopolymerizing it under 365nm ultraviolet irradiation; the methacrylated gelatin is prepared by dissolving methacrylate monomer and gelatin in CB buffer at 50°C for 3h to obtain methacrylated gelatin after the methacrylate reacts with the amine group of gelatin; the methacrylated alginate is obtained by combining 2-aminoethyl methacrylate hydrochloride with the carboxyl group of sodium alginate activated by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS).

[0038] Example 2

[0039] The double-layer hydrogel bioactive scaffold provided in Example 2 of the present invention is prepared by the following method:

[0040] A first biohydrogel made from methacrylated gelatin is first deposited using a bioprinting process to form a cartilage layer. A second biohydrogel made from methacrylated alginate is then deposited on the cartilage layer using a bioprinting process to form a subchondral bone layer. The pore size of the cartilage layer is 100 μm. The pore size of the subchondral bone layer is 300 μm, thus obtaining a double-layer hydrogel bioactive scaffold.

[0041] Among them, the first bio-hydrogel is prepared by mixing a methacrylated gelatin precursor solution with a mass concentration of 8% with a photoinitiator I2959 and photopolymerizing it under 365nm ultraviolet irradiation; the second bio-hydrogel is prepared by mixing a methacrylated alginate precursor solution with a mass concentration of 1% with a photoinitiator I2959 and photopolymerizing it under 365nm ultraviolet irradiation; the methacrylated gelatin is prepared by dissolving methacrylate monomer and gelatin in CB buffer at 40°C for 2h to obtain methacrylated gelatin after the methacrylate reacts with the amine group of gelatin; the methacrylated alginate is obtained by combining 2-aminoethyl methacrylate hydrochloride with the carboxyl group of sodium alginate activated by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS).

[0042] Example 3

[0043] The double-layer hydrogel bioactive scaffold provided in Example 3 of the present invention is prepared by the following method:

[0044] A first biohydrogel made from methacrylated gelatin is first deposited using a bioprinting process to form a cartilage layer. A second biohydrogel made from methacrylated alginate is then deposited on the cartilage layer using a bioprinting process to form a subchondral bone layer. The pore size of the cartilage layer is 300 μm. The pore size of the subchondral bone layer is 600 μm, thereby obtaining a double-layer hydrogel bioactive scaffold.

[0045] Among them, the first biological hydrogel is prepared by mixing a methacrylated gelatin precursor solution with a mass concentration of 12% with a photoinitiator I2959 and photopolymerizing it under 365nm ultraviolet irradiation; the second bio-hydrogel is prepared by mixing a methacrylated alginate precursor solution with a mass concentration of 3% with a photoinitiator I2959 and photopolymerizing it under 365nm ultraviolet irradiation; the methacrylated gelatin is prepared by dissolving methacrylate monomer and gelatin in a CB buffer solution at 60°C for 4h to obtain methacrylated gelatin after the methacrylate reacts with the amine group of gelatin; the methacrylated alginate is obtained by combining 2-aminoethyl methacrylate hydrochloride with the carboxyl group of sodium alginate activated by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS).

[0046] Example 4

[0047] The double-layer hydrogel bioactive scaffold provided in Example 4 of the present invention is prepared by the following method:

[0048] A first biohydrogel made from methacrylated gelatin is first deposited using a bioprinting process to form a cartilage layer. A second biohydrogel made from methacrylated alginate is then deposited on the cartilage layer using a bioprinting process to form a subchondral bone layer. The pore size of the cartilage layer is 200 μm. The pore size of the subchondral bone layer is 500 μm, thus obtaining a double-layer hydrogel bioactive scaffold.

[0049] Among them, the first bio-hydrogel is prepared by mixing a methacrylated gelatin precursor solution with a mass concentration of 12% with a photoinitiator I2959 and photopolymerizing it under 365nm ultraviolet irradiation; the second bio-hydrogel is prepared by mixing a methacrylated alginate precursor solution with a mass concentration of 1% with a photoinitiator I2959 and photopolymerizing it under 365nm ultraviolet irradiation; the methacrylated gelatin is prepared by dissolving methacrylate monomer and gelatin in CB buffer at 40°C for 4h to obtain methacrylate anhydride gelatin after the methacrylate reacts with the amine group of gelatin; the methacrylated alginate is obtained by combining 2-aminoethyl methacrylate hydrochloride with the carboxyl group of sodium alginate activated by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS).

[0050] Example 5

[0051] The double-layer hydrogel bioactive scaffold provided in Example 5 of the present invention is prepared by the following method:

[0052] A first biohydrogel made from methacrylated gelatin is first deposited using a bioprinting process to form a cartilage layer. A second biohydrogel made from methacrylated alginate is then deposited on the cartilage layer using a bioprinting process to form a subchondral bone layer. The pore size of the cartilage layer is 200 μm. The pore size of the subchondral bone layer is 500 μm, thus obtaining a double-layer hydrogel bioactive scaffold.

[0053] Among them, the first bio-hydrogel is prepared by mixing a methacrylated gelatin precursor solution with a mass concentration of 8% with a photoinitiator I2959 and photopolymerizing it under 365nm ultraviolet irradiation; the second bio-hydrogel is prepared by mixing a methacrylated alginate precursor solution with a mass concentration of 3% with a photoinitiator I2959 and photopolymerizing it under 365nm ultraviolet irradiation; the methacrylated gelatin is prepared by dissolving methacrylate monomer and gelatin in a CB buffer solution at 60°C for 2h to obtain methacrylated gelatin after the methacrylate reacts with the amine group of gelatin; the methacrylated alginate is obtained by combining 2-aminoethyl methacrylate hydrochloride with the carboxyl group of sodium alginate activated by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS).

[0054] In order to verify the performance of the double-layer hydrogel bioactive scaffold obtained in the embodiment of the present invention, the double-layer hydrogel bioactive scaffold obtained in Example 1 was characterized. The specific characterization results are as follows:

[0055] Table 1 Mechanical test results of the double-layer hydrogel bioactive scaffold obtained in Example 1

[0056]

[0057]

[0058] The data in Table 1 above indicate that the mechanical test of the double-layer hydrogel bioactive scaffold obtained in the present invention reaches 405 kPa, which meets the requirements for cartilage implantation.

[0059] Biocompatibility testing, such as Figure 2 and 3 As shown, cells can grow and reproduce well on the surface of the scaffold material and have good biocompatibility.

[0060] CCK-8 was used to test the growth of the material, and electron microscopy was used to observe the growth of cells on the material surface. A degradation test was conducted in rats, as well as in vivo biocompatibility testing.

[0061] Cartilage defect repair test in rats, e.g. Figure 4 As shown,

[0062] In addition, it should be noted that the hydrogel in the embodiment of the present invention is prepared by mixing a precursor solution of methacrylated gelatin (GelMA) or methacrylated alginate (AEMA) with a photoinitiator I2959, which is then injected into the printer loading tank and photopolymerized using 365 nm ultraviolet irradiation during the printing process (e.g., Figure 1 The printed shape was set to a cube (length and width = 25 mm, height = 10.0 mm), and further compression testing was performed after printing.

[0063] Based on factors such as hydrogel loading strength tested in previous studies and the fact that cells proliferate and differentiate more easily in low-viscosity precursor solutions, GelMA (G10, 10% w / v) and ALMA (A2, 2% w / v) were ultimately selected as the precursor solutions for preparing the double-layer hydrogel scaffolds. The prepared double-layer hydrogel scaffolds were then subjected to mechanical strength testing. Sample gelation was tested using a dynamic mechanical analyzer (DMA) at a load of 10% strain / minute, and the elastic modulus was calculated within the strain range from 10% to 20%.

[0064] In addition, this embodiment also provides the application of the above-obtained double-layer hydrogel bioactive scaffold in the repair of articular cartilage defects. The specific application steps are as follows:

[0065] The cartilage defect was filled with a 3D bioprinted double-layer hydrogel bioactive scaffold. After 12 weeks of repair, a good repair effect was achieved. Tissue sections showed that the joint defect was well repaired. HE staining, toluidine blue staining, and safranin O staining showed that chondrocytes were arranged in an orderly manner, type I collagen was arranged in an orderly manner, and the articular cartilage tissue was well repaired. For details, see Figure 5 and Figure 6 shown.

[0066] In summary, in the process of preparing a double-layer hydrogel bioactive scaffold of the present invention, the first biohydrogel and the second biohydrogel are respectively deposited to form a cartilage layer and a subchondral bone layer, and the pore size of the cartilage layer is set to 100-300um, and the pore size of the subchondral bone layer is set to 300-600um, so that the obtained double-layer hydrogel bioactive scaffold has good biocompatibility, has a homing effect on cells and their growth factors, and thus lays the foundation for providing repair ability during cartilage repair; in addition, the double-layer hydrogel bioactive scaffold obtained by the present invention has a porous structure, the pore size of the cartilage layer is 100-300um, and the pore size of the subchondral bone layer is 300-600um, which is conducive to the slow release of cells and their growth factors in the porous structure to the articular cartilage defect position, thereby achieving a long-term repair effect.

[0067] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for preparing a double-layer hydrogel bioactive scaffold, characterized in that: The method specifically comprises the following steps: firstly, a first biohydrogel is deposited using a bioprinting process to form a cartilage layer; then, a second biohydrogel is deposited on the cartilage layer using a bioprinting process to form a subchondral bone layer, thereby obtaining a double-layer hydrogel bioactive scaffold; Among them, the pore size of the cartilage layer is 100-300um; the pore size of the subchondral bone layer is 300-600um; the first biohydrogel and the second biohydrogel are both photocured bio-inks; the first biohydrogel is made of methacrylated gelatin as raw material; the second biohydrogel is made of methacrylated alginate as raw material; the first biohydrogel is made by mixing a methacrylated gelatin precursor solution with a photoinitiator I2959 and then photopolymerizing it under 365nm ultraviolet irradiation; the second biohydrogel is made by mixing a methacrylated alginate precursor solution with a photoinitiator I2959 and then photopolymerizing it under 365nm ultraviolet irradiation; the mass concentration of methacrylated gelatin in the methacrylated gelatin precursor solution is 8-12%; the mass concentration of methacrylated alginate in the methacrylated alginate precursor solution is 1-3%.

2. The method for preparing a double-layer hydrogel bioactive scaffold according to claim 1, characterized in that: The methacrylated gelatin is prepared by dissolving methacrylate monomer and gelatin in a CB buffer solution at 40-60° C. for 2-4 hours to obtain methacrylate anhydride gelatin after the methacrylate reacts with the amine group of the gelatin.

3. The method for preparing a double-layer hydrogel bioactive scaffold according to claim 1, characterized in that: The methacrylated alginate is obtained by combining 2-aminoethyl methacrylate hydrochloride with the carboxyl group of sodium alginate activated by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) / N-hydroxysuccinimide (NHS).

4. A double-layer hydrogel bioactive scaffold prepared by the preparation method according to any one of claims 1 to 3, characterized in that: The double-layer hydrogel bioactive scaffold has a porous structure.

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