Customizable two-phase hydrogel stent as well as preparation method and application thereof

Abstract

The invention discloses a customizable biphasic hydrogel scaffold which comprises a hyaline cartilage layer and a subchondral bone layer, the hyaline cartilage layer comprises methacrylated I-type recombinant collagen and bone marrow mesenchymal stem cells, and the subchondral bone layer comprises methacrylated I-type recombinant collagen and bioactive glass loaded with resveratrol. The invention also provides a preparation method and application of the double-phase hydrogel scaffold. The biphase hydrogel stent has the advantages of being customizable, resistant to oxidation and inflammation, high in biocompatibility, capable of promoting wound healing and bone repair and the like.

Description

Technical Field

[0001] The present invention belongs to the field of bone repair, and in particular relates to a customizable biphasic hydrogel scaffold and a preparation method and application thereof. Background Art

[0002] Articular cartilage is the connective tissue covering the epiphyseal surfaces of the elbow and knee joints. It possesses excellent elasticity, compressive resistance, and lubricity, effectively reducing wear between bones and withstanding high-frequency dynamic stress. Articular cartilage damage is a common degenerative orthopedic condition, typically manifesting as joint pain and dysfunction. Because cartilage lacks blood vessels, lymphatic vessels, and nerves, it relies on synovial fluid and subchondral bone for nutrition, resulting in a low regenerative capacity.

[0003] Currently, the methods for treating articular cartilage damage are divided into three categories: conservative treatment, repair treatment and regenerative treatment.

[0004] However, current treatment methods have many disadvantages: (1) Conservative treatments, such as arthroscopic lavage, can provide short-term pain relief, but cannot prevent the progression of damage and carry the risk of damaging the surrounding healthy cartilage. (2) Restorative treatments include bone marrow stimulation, which stimulates the formation of blood clots in the subchondral bone, promotes the migration of mesenchymal stem cells to the cartilage area, and generates fibrocartilage. However, fibrocartilage cannot replace the biomechanical properties of natural cartilage. Although autologous and allogeneic transplants are effective, there are problems such as donor sources, tissue integration, and immune response. (3) Regenerative treatments, such as autologous chondrocyte implantation (ACI) and matrix-assisted chondrocyte implantation (MACI), can generate functional and stable cartilage by culturing chondrocytes in vitro and transplanting them into the defect site. However, the treatment cost is high, the recovery period is long, and the regeneration of the bone-cartilage interface and subchondral bone remains challenging.

[0005] Therefore, there is an urgent need to develop a multifunctional treatment method that can simulate the biomechanical properties of natural bone, and has anti-oxidation, anti-inflammatory, and cartilage regeneration promoting properties. It can effectively deal with the complex situation of articular cartilage defects, while having lower treatment costs, shorter recovery period, and can promote the regeneration of the bone cartilage interface and subchondral bone, thereby providing a more comprehensive and sustainable solution. Summary of the Invention

[0006] One object of the present invention is to provide a customizable biphasic hydrogel scaffold for the above technical problems, which can be customized to simulate the biomechanical properties of natural bones and have antioxidant, anti-inflammatory and healing-promoting effects.

[0007] Another object of the present invention is to provide a method for preparing the biphasic hydrogel scaffold.

[0008] Another object of the present invention is to provide applications of the biphasic hydrogel scaffold.

[0009] In order to achieve the above object of the invention, the present invention provides the following technical solutions:

[0010] In a first aspect, the present invention provides a customizable biphasic hydrogel scaffold, comprising a hyaline cartilage layer and a subchondral bone layer, wherein the hyaline cartilage layer comprises methacryloylated recombinant collagen type I (Col1MA) and bone marrow mesenchymal stem cells (BMSCs), and the subchondral bone layer comprises methacryloylated recombinant collagen type I (Col1MA) and resveratrol-loaded bioactive glass (MBG@RES).

[0011] Preferably, in the hyaline cartilage layer, the content of methacryloylation type I recombinant collagen is 5-15% by mass volume, and the content of bone marrow mesenchymal stem cells is 1-2×10 5 pieces / mL.

[0012] More preferably, in the hyaline cartilage layer, the methacryloylation type I recombinant collagen is 10%, the bone marrow mesenchymal stem cells are 1×10 5 pieces / mL.

[0013] Preferably, in the subchondral bone layer, the methacrylated type I recombinant collagen accounts for 7.5-15% and the resveratrol-loaded bioactive glass accounts for 1-2% by mass volume percentage.

[0014] More preferably, in the subchondral bone layer, by mass volume percentage, the methacrylated type I recombinant collagen accounts for 10% and the resveratrol-loaded bioactive glass accounts for 2%.

[0015] Preferably, the methacrylylated type I recombinant collagen is prepared by the following steps:

[0016] 2 g of type I collagen was dissolved in 100 mL of deionized water, the pH was adjusted to 7, 2 mL of methacrylic anhydride was added dropwise, and the mixture was reacted at room temperature for 24 h. The product was dialyzed in deionized water and centrifuged at 10,000 rpm for 15 minutes to remove impurities. The supernatant was lyophilized to obtain the methacryloyl-recombinant type I collagen (Col1MA).

[0017] Preferably, the resveratrol-loaded bioactive glass is prepared by the following steps:

[0018] 1 g of resveratrol was dissolved in 50 mL of deionized water to prepare a 2% resveratrol solution; 1 g of bioactive glass was added to the resveratrol solution and ultrasonically dispersed for 15 minutes, followed by stirring and mixing at 40°C. After the reaction, the glass was washed with deionized water to remove unbound resveratrol and freeze-dried to obtain the final product, resveratrol bioactive glass (MBG@RES).

[0019] More preferably, the bioactive glass is prepared by the following steps:

[0020] 0.7 g of hexadecyltrimethylammonium bromide (CTAB) was dissolved in 33 mL of deionized water, 10 mL of ethyl acetate was added, and after stirring for 30 minutes, 7 mL of 5 M ammonia water was added and stirring was continued for 15 minutes; 3.6 mL of tetraethyl orthosilicate and 0.36 mL of triethyl phosphate were slowly added dropwise to the above solution respectively; 2.28 g of calcium nitrate tetrahydrate was dissolved in 5 mL of deionized water and slowly added dropwise to the above solution, and stirred at room temperature for 4 hours; the reaction solution was collected and centrifuged at 5000 rpm for 5 minutes to obtain a white precipitate; the precipitate was washed three times with anhydrous ethanol and deionized water respectively, and dried at 60°C for 24 hours; the dried white powder was then calcined at 600°C at a heating rate of 1°C / min for 5 hours to obtain the bioactive glass.

[0021] Preferably, the bone marrow mesenchymal stem cells are prepared by the following steps:

[0022] The rat limb bones were collected, and the muscles were removed in a PBS buffer containing 1% penicillin-streptomycin double antibody solution. Small incisions were made at both ends of the bones in α-MEM basal culture medium, and the bone marrow in the bones was flushed out. The bone marrow-culture medium mixture was collected and centrifuged at 1000 rpm for 5 minutes; the supernatant was discarded, and red blood cell lysis buffer was added to lyse for 10 minutes; PBS was added to terminate the reaction, and the mixture was centrifuged at 1000 rpm for 5 minutes; the supernatant was discarded, and the mixture was resuspended in α-MEM complete culture medium and cultured at 37°C and 5% CO2 for 48 hours. The cell medium was changed, and the cells were passaged when they grew to 80% confluence. After 2-3 generations of passages, the cells were washed 3 times with PBS when they grew to 80% confluence to obtain the bone marrow mesenchymal stem cells.

[0023] In a second aspect, the present invention further provides a method for preparing the biphasic hydrogel scaffold, comprising the following steps:

[0024] S1. Preparing the hyaline cartilage layer:

[0025] A photoinitiator solution was prepared by dissolving methacrylated type I recombinant collagen in the photoinitiator solution to obtain a methacrylated type I recombinant collagen solution, which served as the bio-ink for the first layer of the hydrogel scaffold. Cultured bone marrow mesenchymal stem cells were digested with 0.25% trypsin and resuspended, followed by centrifugation at 1000 rpm for 5 minutes. The supernatant was discarded, and the bone marrow mesenchymal stem cells were resuspended in the bio-ink, mixed evenly, and then 3D printed.

[0026] S2. preparing the subchondral bone layer:

[0027] A photoinitiator solution was prepared, and methacryloyl-type I recombinant collagen was dissolved in the above-mentioned photoinitiator solution to obtain a methacryloyl-type I recombinant collagen solution; the resveratrol-loaded bioactive glass was added and vortexed for 30 seconds to mix evenly, and the bio-ink for the second layer of the hydrogel scaffold was used. After the first layer was printed and formed, the second layer was continued to be printed.

[0028] Preferably, the curing condition for the first layer of 3D printing is a light intensity of 17mW / cm 2 , exposure time 20s, exposure time for base layer 24s; curing condition for printing the second layer is light intensity 18mW / cm 2 , exposure time 22s, base exposure time 27s.

[0029] In a third aspect, the present invention further provides the use of the biphasic hydrogel scaffold in the preparation of drugs that promote wound healing or bone repair or are anti-oxidative or anti-inflammatory.

[0030] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0031] (1) Performance of simulating the natural bone microenvironment: The internal and external structures of the scaffold can be designed to be gradient through 3D printing technology or other manufacturing methods to simulate the hardness gradient of natural bone, thereby providing corresponding mechanical support for bone tissue repair at different locations and degrees.

[0032] (2) Customizable scaffold materials: Cartilage tissue lacks blood vessels and other channels, has a high density of extracellular matrix, and has gradient heterogeneity. It is very suitable for using 3D printing technology to precisely customize scaffolds to repair (bone) cartilage defects.

[0033] (3) Design and preparation of composite materials: By combining methacryloyl-type I recombinant collagen (Col1MA) with bioactive glass (MBG), a composite material with structural stability and biocompatibility is formed. Methacryloyl-type I collagen enhances the stability and integration of the interface between the bone and cartilage layers. By modifying the composite material, the effective loading and controlled release of RES (resveratrol) are achieved, thereby enhancing the antioxidant and anti-inflammatory functions.

[0034] (4) Improved biocompatibility: The use of natural materials Col1MA and MBG to improve biocompatibility can significantly reduce the occurrence of local inflammation or allergic reactions compared to traditional synthetic materials. In addition, by adjusting the content of Col1MA and MBG, the scaffold can simulate the biomechanical properties of natural bone, improve the repair effect, and accelerate bone tissue regeneration.

[0035] (5) The addition of resveratrol improves the antioxidant capacity, can effectively scavenge free radicals, slow down cell damage, and protect cells from oxidative stress.

[0036] (6) Enhanced anti-inflammatory and pro-healing effects: The design of this material not only focuses on biomechanical properties but also integrates anti-inflammatory and pro-healing components. RES not only helps control the inflammatory response but also promotes the osteogenic differentiation of BMSCs, thereby promoting bone tissue regeneration and repair, meeting clinical needs.

[0037] (7) Multifunctional design: can be customized, anti-oxidant, anti-inflammatory and promote healing. The material design integrates multiple functions such as stability, anti-oxidant, anti-inflammatory and promotion of bone tissue regeneration to meet the complex needs of cartilage repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 An image of a hydrogel scaffold is shown.

[0039] Figure 2 The maximum compressive strength of the hydrogel is shown.

[0040] Figure 3 The drug loading efficiency of RES is shown.

[0041] Figure 4 The cumulative release rate of RES is shown.

[0042] Figure 5 The results of the cell compatibility evaluation are shown.

[0043] Figure 6 The results of MDA content determination are shown.

[0044] Figure 7 The results of anti-inflammatory experiments are shown. DETAILED DESCRIPTION

[0045] The present invention will be further described below with reference to specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0046] Unless otherwise specified, the reagents, instruments, etc. used in the embodiments of the present invention are conventional reagents and instruments available in the art.

[0047] 1. Preparation of methacryloyl-type I recombinant collagen (Col1MA):

[0048] Weigh 2 g of type I collagen and dissolve it in 100 mL of deionized water. Adjust the pH to 7, then add 2 mL of methacrylic anhydride dropwise. Allow to react at room temperature for 24 hours. The resulting methacryloyl-modified recombinant type I collagen (Col1MA) was dialyzed against deionized water using a 3500 Da dialysis bag. Impurities were removed by centrifugation at 10,000 rpm for 15 minutes. The supernatant was lyophilized and stored at -20°C until use.

[0049] 2. Preparation of bioactive glass (MBG):

[0050] Weigh 0.7 g of cetyltrimethylammonium bromide (CTAB) and dissolve it in 33 mL of deionized water, add 10 mL of ethyl acetate, and stir for 30 minutes. Then add 7 mL of ammonia water (5M) and continue stirring for 15 minutes. Slowly add 3.6 mL of tetraethyl orthosilicate (TEOS) and 0.36 mL of triethyl phosphate to the above solution. Dissolve 2.28 g of calcium nitrate tetrahydrate in 5 mL of deionized water and slowly add it to the above solution. Stir vigorously at room temperature for 4 hours. Collect the reaction solution and centrifuge at 5000 rpm for 5 minutes to obtain a white precipitate. Wash the precipitate with anhydrous ethanol and deionized water three times each, and dry at 60°C for 24 hours. Then calcine the dried white powder at 600°C with a temperature rise rate of 1°C / min for 5 hours to obtain MBG.

[0051] 3. Preparation of resveratrol-loaded bioactive glass (MBG@RES):

[0052] Dissolve 1 g of resveratrol (RES) in 50 mL of deionized water or a suitable solvent to prepare a 2% RES solution. Add 1 g of bioactive glass to the RES solution and ultrasonically disperse for 15 min, then stir the mixture at 40°C to promote the interaction between RES and the surface of the bioactive glass. After the reaction is complete, wash with deionized water to remove unbound RES. Freeze-dry the modified bioactive glass (MBG@RES) for 48 h to obtain the final product.

[0053] 4. Extraction and culture of bone marrow mesenchymal stem cells (BMSCs)

[0054] Take 3-4 week old SD rats, immerse them in 75% alcohol after dislocation and disinfect for 5 min, then transfer the rats to a clean bench, remove the rat limbs using a high-temperature high-pressure sterilization instrument, remove the muscles in PBS buffer (containing 1% penicillin-streptomycin double-antibiotic solution), cut a small opening at both ends of the bone in α-MEM basic medium, and use a 1 mL syringe to flush out the bone marrow. Collect the bone marrow-medium mixture and centrifuge at 1000 rpm for 5 min. Discard the supernatant, add red blood cell lysis solution and lyse for 10 min. Add PBS to terminate the reaction and centrifuge at 1000 rpm for 5 min. Discard the supernatant, resuspend in α-MEM complete medium, and culture in a 37°C constant temperature carbon dioxide incubator (containing 5% CO2) for 48 h. Replace the medium when the cells are 80% confluent, and passage the cells when they are 80% confluent. Wash the cells with PBS 3 times, digest the cells with trypsin, add α-MEM complete medium when the cells are rounded, collect the cell suspension, and centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells in bio-ink.

[0055] Example 1:

[0056] First, prepare the photoinitiator solution: take 1 mg of LAP (phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt, photoinitiator) and 0.6 mg of lemon yellow (light blocker) and add them to 1 mL of deionized water, vortex for 30 seconds to mix them thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in the solution. As the bio-ink on the upper layer of the hydrogel scaffold, the cultured BMSCs cells were digested and resuspended with 0.25% trypsin and centrifuged (1000 rpm, 5 min). Discard the supernatant and resuspend the cells with the upper bio-ink at a density of 1×10 5 The cell suspension of 100 cells / mL was quickly mixed with the bio-ink in the upper layer to prepare the first layer of bio-ink.

[0057] Next, 0.075 g of Col1MA was weighed and dissolved in the above solution, and then 0.01 g of MBG@RES was added to prepare the second layer of bio-ink.

[0058] Then, the prepared first layer of bio-ink was poured into the groove of the 3D printer, and the printing model and its size were selected for printing (light intensity: 17mW / cm 2 , exposure time: 20s, number of base layers: 1 layer, base exposure time: 24s), and then continue to print the second layer on this layer of hydrogel after molding (light intensity: 18mW / cm 2 , exposure time: 22s, number of base layers: 1 layer, base exposure time: 27s).

[0059] Finally, the printed hydrogel scaffold was washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.

[0060] Example 2:

[0061] First, prepare the photoinitiator solution: take 1 mg of LAP (photoinitiator) and 0.6 mg of lemon yellow (light blocker) and add them to 1 mL of deionized water, vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in the solution as the bio-ink on the upper layer of the hydrogel scaffold. After the cultured BMSCs cells are digested with 0.25% trypsin and resuspended, centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells with the upper layer of bio-ink at a density of 1×10 5 The cell suspension of 100 cells / mL was quickly mixed with the bio-ink in the upper layer to prepare the first layer of bio-ink.

[0062] Next, 0.075 g of Col1MA was weighed and dissolved in the above solution, and then 0.015 g of MBG@RES was added to prepare the second layer of bio-ink.

[0063] Then, the prepared first layer of bio-ink was poured into the groove of the 3D printer, and the printing model and its size were selected for printing (light intensity: 17mW / cm 2 , exposure time: 20s, number of base layers: 1 layer, base exposure time: 24s), and then continue to print the second layer on this layer of hydrogel after molding (light intensity: 18mW / cm 2 , exposure time: 22s, number of base layers: 1 layer, base exposure time: 27s).

[0064] Finally, the printed hydrogel scaffold was washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.

[0065] Example 3:

[0066] First, prepare the photoinitiator solution: take 1 mg of LAP (photoinitiator) and 0.6 mg of lemon yellow (light blocker) and add them to 1 mL of deionized water, vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in the solution as the bio-ink on the upper layer of the hydrogel scaffold. After the cultured BMSCs cells are digested with 0.25% trypsin and resuspended, centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells with the upper layer of bio-ink at a density of 1×10 5 The cell suspension of 100 cells / mL was quickly mixed with the bio-ink in the upper layer to prepare the first layer of bio-ink.

[0067] Next, 0.075 g of Col1MA was weighed and dissolved in the above solution, and then 0.02 g of MBG@RES was added to prepare the second layer of bio-ink.

[0068] Then, the prepared first layer of bio-ink was poured into the groove of the 3D printer, and the printing model and its size were selected for printing (light intensity: 17mW / cm 2 , exposure time: 20s, number of base layers: 1 layer, base exposure time: 24s), and then continue to print the second layer on this layer of hydrogel after molding (light intensity: 18mW / cm 2 , exposure time: 22s, number of base layers: 1 layer, base exposure time: 27s).

[0069] Finally, the printed hydrogel scaffold was washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.

[0070] Example 4:

[0071] First, prepare the photoinitiator solution: take 1 mg of LAP (photoinitiator) and 0.6 mg of lemon yellow (light blocker) and add them to 1 mL of deionized water, vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in the solution as the bio-ink on the upper layer of the hydrogel scaffold. After the cultured BMSCs cells are digested with 0.25% trypsin and resuspended, centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells with the upper layer of bio-ink at a density of 1×10 5 The cell suspension of 100 cells / mL was quickly mixed with the bio-ink in the upper layer to prepare the first layer of bio-ink.

[0072] Next, 0.1 g of Col1MA was weighed and dissolved in the above solution, and then 0.01 g of MBG@RES was added to prepare the second layer of bio-ink.

[0073] Then, the prepared first layer of bio-ink was poured into the groove of the 3D printer, and the printing model and its size were selected for printing (light intensity: 17mW / cm 2 , exposure time: 20s, number of base layers: 1 layer, base exposure time: 24s), and then continue to print the second layer on this layer of hydrogel after molding (light intensity: 18mW / cm 2 , exposure time: 22s, number of base layers: 1 layer, base exposure time: 27s).

[0074] Finally, the printed hydrogel scaffold was washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.

[0075] Example 5:

[0076] First, prepare the photoinitiator solution: take 1 mg of LAP (photoinitiator) and 0.6 mg of lemon yellow (light blocker) and add them to 1 mL of deionized water, vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in the solution as the bio-ink on the upper layer of the hydrogel scaffold. After the cultured BMSCs cells are digested with 0.25% trypsin and resuspended, centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells with the upper layer of bio-ink at a density of 1×10 5 The cell suspension of 100 cells / mL was quickly mixed with the bio-ink in the upper layer to prepare the first layer of bio-ink.

[0077] Next, 0.1 g of Col1MA was weighed and dissolved in the above solution, and then 0.015 g of MBG@RES was added to prepare the second layer of bio-ink.

[0078] Then, the prepared first layer of bio-ink was poured into the groove of the 3D printer, and the printing model and its size were selected for printing (light intensity: 17mW / cm2 , exposure time: 20s, number of base layers: 1 layer, base exposure time: 24s), and then continue to print the second layer on this layer of hydrogel after molding (light intensity: 18mW / cm 2 , exposure time: 22s, number of base layers: 1 layer, base exposure time: 27s).

[0079] Finally, the printed hydrogel scaffold was washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.

[0080] Example 6:

[0081] First, prepare the photoinitiator solution: take 1 mg of LAP (photoinitiator) and 0.6 mg of lemon yellow (light blocker) and add them to 1 mL of deionized water, vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in the solution as the bio-ink on the upper layer of the hydrogel scaffold. After the cultured BMSCs cells are digested with 0.25% trypsin and resuspended, centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells with the upper layer of bio-ink at a density of 1×10 5 The cell suspension of 100 cells / mL was quickly mixed with the bio-ink in the upper layer to prepare the first layer of bio-ink.

[0082] Next, 0.1 g of Col1MA was weighed and dissolved in the above solution, and then 0.02 g of MBG@RES was added to prepare the second layer of bio-ink.

[0083] Then, the prepared first layer of bio-ink was poured into the groove of the 3D printer, and the printing model and its size were selected for printing (light intensity: 17mW / cm 2 , exposure time: 20s, number of base layers: 1 layer, base exposure time: 24s), and then continue to print the second layer on this layer of hydrogel after molding (light intensity: 18mW / cm 2 , exposure time: 22s, number of base layers: 1 layer, base exposure time: 27s).

[0084] Finally, the printed hydrogel scaffold was washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.

[0085] Example 7:

[0086] First, prepare the photo-initiation solution: take 1 mg of LAP (photo-initiator) and 0.6 mg of lemon yellow (light blocker) into 1 mL of deionized water, vortex for 30 s to mix thoroughly. Then, take 0.1 g of Col1MA and dissolve it in the solution as the upper layer of the bio-ink of the hydrogel scaffold. After resuspension of the cultured BMSCs cells by 0.25% trypsin, centrifugation (1000 rpm, 5 min) is performed. Discard the supernatant and resuspend the cells with the upper layer of the bio-ink at a density of 1 x 10 5 The first layer of bio-ink is prepared by rapidly mixing the cell suspension at a density of 1 x 10

[0087] Next, take 0.15 g of Col1MA and dissolve it in the above solution, and then add 0.01 g of MBG@RES to prepare the second layer of bio-ink.

[0088] Then, pour the prepared first layer of bio-ink into the groove of the 3D printer, and select the printing model and its size for printing (light intensity: 17 mW / cm 2 , exposure time: 20 s, base layer number: 1 layer, base layer exposure time: 24 s). After molding, the second layer is printed on the layer of hydrogel (light intensity: 18 mW / cm 2 , exposure time: 22 s, base layer number: 1 layer, base layer exposure time: 27 s).

[0089] Finally, the printed hydrogel scaffold is washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.

[0090] Example 8:

[0091] First, prepare the photo-initiation solution: take 1 mg of LAP (photo-initiator) and 0.6 mg of lemon yellow (light blocker) into 1 mL of deionized water, vortex for 30 s to mix thoroughly. Then, take 0.1 g of Col1MA and dissolve it in the solution as the upper layer of the bio-ink of the hydrogel scaffold. After resuspension of the cultured BMSCs cells by 0.25% trypsin, centrifugation (1000 rpm, 5 min) is performed. Discard the supernatant and resuspend the cells with the upper layer of the bio-ink at a density of 1 x 10 5 The first layer of bio-ink is prepared by rapidly mixing the cell suspension at a density of 1 x 10

[0092] Next, take 0.15 g of Col1MA and dissolve it in the above solution, and then add 0.015 g of MBG@RES to prepare the second layer of bio-ink.

[0093] Then, pour the prepared first layer of bio-ink into the groove of the 3D printer, and select the printing model and its size for printing (light intensity: 17 mW / cm2 , exposure time: 20 s, base layer number: 1 layer, base layer exposure time: 24 s), after the molding, the second layer was continued to be printed on the layer of hydrogel (light intensity: 18 mW / cm 2 , exposure time: 22 s, base layer number: 1 layer, base layer exposure time: 27 s).

[0094] Finally, the printed hydrogel scaffold was washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.

[0095] Example 9:

[0096] First, the photoinitiating solution was prepared: 1 mg of LAP (photoinitiator) and 0.6 mg of lemon yellow (light blocker) were taken and added to 1 mL of deionized water, and vortexed for 30 s to mix thoroughly. Subsequently, 0.1 g of Col1MA was dissolved in the solution as the upper layer of the bio-ink of the hydrogel scaffold, and the cultured BMSCs cells were resuspended after being digested with 0.25% trypsin and centrifuged (1000 rpm, 5 min). The supernatant was discarded, and the cells were resuspended with the upper layer of bio-ink at a density of 1 x 10 5 The first layer of bio-ink was prepared by rapidly mixing the cell suspension at a density of 1 x 10

[0097] Then, 0.15 g of Col1MA was weighed and dissolved in the above solution, and 0.02 g of MBG@RES was added to prepare the second layer of bio-ink.

[0098] Then, the prepared first layer of bio-ink was poured into the groove of the 3D printer, and the printing model and its size were selected for printing (light intensity: 17 mW / cm 2 , exposure time: 20 s, base layer number: 1 layer, base layer exposure time: 24 s), after the molding, the second layer was continued to be printed on the layer of hydrogel (light intensity: 18 mW / cm 2 , exposure time: 22 s, base layer number: 1 layer, base layer exposure time: 27 s).

[0099] Finally, the printed hydrogel scaffold was washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.

[0100] Comparative Example 1:

[0101] First, prepare the photoinitiator solution: take 1 mg of LAP (photoinitiator) and 0.6 mg of lemon yellow (light blocker) and add them to 1 mL of deionized water, vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in the solution as the bio-ink on the upper layer of the hydrogel scaffold. After the cultured BMSCs cells are digested with 0.25% trypsin and resuspended, centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells with the upper layer of bio-ink at a density of 1×10 5 The cell suspension of 100 cells / mL was quickly mixed with the bio-ink in the upper layer to prepare the first layer of bio-ink.

[0102] Next, 0.1 g of Col1MA was weighed and dissolved in the above solution to prepare the second layer of bio-ink.

[0103] Then, the prepared first layer of bio-ink was poured into the groove of the 3D printer, and the printing model and its size were selected for printing (light intensity: 17mW / cm 2 , exposure time: 20s, number of base layers: 1 layer, base exposure time: 24s), and then continue to print the second layer on this layer of hydrogel after molding (light intensity: 18mW / cm 2 , exposure time: 22s, number of base layers: 1 layer, base exposure time: 27s).

[0104] Finally, the printed hydrogel scaffold was washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.

[0105] Comparative Example 2:

[0106] First, prepare a photoinitiator solution: 1 mg of LAP (photoinitiator) and 0.6 mg of tartrazine (light blocker) are added to 1 mL of deionized water and vortexed for 30 seconds to thoroughly mix. Subsequently, 0.1 g of Col1MA is weighed and dissolved in this solution to prepare the first layer of bio-ink.

[0107] Next, 0.1 g of Col1MA was weighed and dissolved in the above solution, and then 0.02 g of MBG@RES was added to prepare the second layer of bio-ink.

[0108] Then, the prepared first layer of bio-ink was poured into the groove of the 3D printer, and the printing model and its size were selected for printing (light intensity: 17mW / cm 2 , exposure time: 20s, number of base layers: 1 layer, base exposure time: 24s), and then continue to print the second layer on this layer of hydrogel after molding (light intensity: 18mW / cm 2 , exposure time: 22s, number of base layers: 1 layer, base exposure time: 27s).

[0109] Finally, the printed hydrogel scaffold was washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.

[0110] Comparative Example 3:

[0111] First, prepare the photoinitiator solution: take 1 mg of LAP (photoinitiator) and 0.6 mg of lemon yellow (light blocker) and add them to 1 mL of deionized water, vortex for 30 seconds to mix thoroughly. Then, weigh 0.1 g of Col1MA and dissolve it in the solution as the bio-ink on the upper layer of the hydrogel scaffold. After the cultured BMSCs cells are digested with 0.25% trypsin and resuspended, centrifuge (1000 rpm, 5 min). Discard the supernatant and resuspend the cells with the upper layer of bio-ink at a density of 1×10 5 The cell suspension of 100 cells / mL was quickly mixed with the bio-ink in the upper layer to prepare the first layer of bio-ink.

[0112] Next, 0.1 g of Col1MA was weighed and dissolved in the above solution, and then 0.02 g of RES was added to prepare the second layer of bio-ink.

[0113] Then, the prepared first layer of bio-ink was poured into the groove of the 3D printer, and the printing model and its size were selected for printing (light intensity: 17mW / cm 2 , exposure time: 20s, number of base layers: 1 layer, base exposure time: 24s), and then continue to print the second layer on this layer of hydrogel after molding (light intensity: 18mW / cm 2 , exposure time: 22s, number of base layers: 1 layer, base exposure time: 27s).

[0114] Finally, the printed hydrogel scaffold was washed 2-3 times with sterile PBS solution to obtain the final hydrogel scaffold.

[0115] Test results

[0116] 1. Appearance of hydrogel scaffold

[0117] Figure 1 An image of the hydrogel scaffold soaked in PBS is shown for Example 1. The hydrogel scaffolds of Examples 2-9 had similar appearances.

[0118] 2. Swelling rate test:

[0119] Record the initial weight of the printed hydrogel, which is recorded as W0. Place the prepared hydrogel sample in a 24-well plate and add PBS buffer using a pipette to completely immerse the hydrogel sample. Remove the sample after 8 hours, carefully wipe off the excess water on the outside of the hydrogel with weighing paper, and then place it on a plastic culture dish and weigh it. Record the weight of the hydrogel and record it as W. t .

[0120] The swelling ratio is calculated according to the following formula:

[0121]

[0122] Table 1. Swelling ratio

[0123] Grouping Swelling rate Example 1 97.3±1.7％ Example 2 84.2±1.3％ Example 3 72.9±0.9％ Example 4 132.1±2.2％ Example 5 125.3±2.3％ Example 6 116.4±1.5％ Example 7 192.5±1.6％ Example 8 181.1±0.8％ Example 9 164.6±1.6％ Comparative Example 1 187.3±1.5％ Comparative Example 2 110.9±0.8％ Comparative Example 3 180.5±0.9％

[0124] The results show that: Too high a swelling rate may cause the material to expand in volume after absorbing water in the body, compressing the surrounding tissues or organs, and affecting the stability and biocompatibility after implantation. In addition, if the swelling rate of the material is too high, it may cause the material to soften, reduce its mechanical strength, and affect its supporting function in bone repair. As can be seen from Table 2, as the content of Col1MA increases, the swelling rate gradually increases. After adding MBG@RES, the swelling rate of the hydrogel decreases significantly. This is because MBG releases Ca in the hydrogel. 2+ MBG can interact with negatively charged groups (such as carboxyl or phosphate groups) in the hydrogel matrix to form ionic crosslinks. Ionic crosslinking increases the crosslink density between polymer chains, making the hydrogel more compact when absorbing water, thereby limiting the swelling of the hydrogel. However, excessive MBG may also partially block the pores in the hydrogel, thereby reducing the swelling rate.

[0125] 3. Compression strength

[0126] Test method: Place the sample on the sample stage, take the printed hydrogel scaffold, and adjust the sample stage height so that both the upper and lower surfaces are in contact with the fixture. Compress at a constant rate of 0.05 mm / min. Calculate the Young's modulus using the following formula:

[0127]

[0128] Where F is the compressive force on the stent, A is the cross-sectional area, L0 is the initial height of the stent before compression, and ΔL is the change in stent height.

[0129] Figure 2 The maximum compressive strength of each specimen is shown.

[0130] Result description:

[0131] The ideal compressive strength of hydrogel should be 1MPa to 2MPa, which can provide biomechanical properties similar to cartilage, and can withstand the dynamic load of joints while maintaining a certain flexibility, simulating the elasticity and durability of cartilage. Figure 2As shown in the figure, the mechanical strength of the scaffold gradually increases with increasing Col1MA content. Furthermore, MBG@RES also plays an important role in improving the mechanical strength of the scaffold, as clearly seen in all Examples and Comparative Example 2. This is likely because the addition of MBG@RES makes the three-dimensional network structure inside the sponge denser and more stable, helping the hydrogel scaffold resist large deformations.

[0132] 4. Resveratrol loading

[0133] Test method: Dissolve 1 mg of RES in 1 mL of anhydrous ethanol to prepare a 1 mg / mL solution. Then, dilute the solution with pure water to 500, 250, 125, 62.5, and 36.25 μg / mL. Measure the absorbance at 305 nm using a UV spectrophotometer to create a standard curve. After loading RES, centrifuge the supernatant and measure its absorbance at 305 nm using a UV spectrophotometer to calculate the drug content in the supernatant.

[0134] Calculation formula: Drug loading (%) = (total mass of loaded drug / total mass of MBG) × 100%

[0135] Figure 3 The drug loading efficiency of RES is shown.

[0136] Result description:

[0137] By measuring the absorbance of the supernatant of MBG after loading with RES, the drug loading capacity of RES was calculated to be 20.50±0.23%.

[0138] 5. Resveratrol Release

[0139] Test method: The printed hydrogel was placed in 1 mL of sterile PBS solution and incubated in a constant temperature shaking incubator at 37°C and 100 rpm. Samples were taken at different time points (1, 3, 6, 24, 48, 72, 96, 120, 144, 168, 192, 216, and 240 hours), and 1 mL of the culture medium was removed and replaced with an equal amount of fresh PBS. The sample was centrifuged at 4000 rpm for 5 minutes. The absorbance of resveratrol in PBS solution was measured using UV spectrophotometry at an excitation wavelength of 305 nm.

[0140] Calculation formula: Cumulative release rate (%) = (cumulative release amount / dose) × 100%

[0141] Figure 4 The cumulative release rate of RES is shown.

[0142] Results show that RES was loaded into amino-modified mesoporous bioactive glass (MBG@RES) and dispersed in Col1MA hydrogel. In Example 6, drug release exhibited two distinct phases: rapid release within the first five days, with approximately 31.03% of the drug released. Subsequently, the release rate gradually slowed, reaching a cumulative release of 57.61% on day 14 and nearly 92.54% on day 46. Hydrogels from other examples also exhibited similar drug release patterns.

[0143] The initial rapid release may be due to the fact that the drug molecules are loosely adsorbed on the surface of the carrier or close to the surface layer, making it easy to diffuse into the external environment. In addition, the hydrogel material swells rapidly when it encounters liquid, which also accelerates the initial release of the drug. Over time, the drug is slowly released from deep inside the carrier, and the drug release rate gradually slows down. This may be because the mesoporous structure of the carrier limits the diffusion of the drug, and the network structure of the hydrogel also plays a role in delaying drug release. This release behavior is very suitable for long-term drug delivery applications. Early rapid release can provide sufficient drug concentration to quickly exert a therapeutic effect, while the slow release in the later stage can maintain the long-term effect of the drug. In Comparative Example 3, RES showed a burst release phenomenon, reaching 95.61% at 14 days. This shows that the composite material of MBG@RES and Col1MA hydrogel has good sustained-release properties, which is particularly suitable for treatment scenarios that require long-term and stable drug supply.

[0144] 6. In vitro degradation test

[0145] Test method: The printed hydrogel scaffold was freeze-dried and weighed, which was recorded as W0. Then, the freeze-dried hydrogel sample was placed in a 24-well plate, and a fixed volume of PBS buffer was added with a pipette to completely immerse the hydrogel sample. The plate was then placed in a 37°C constant temperature shaker. The degradation solution was replaced every 2 days. On the 14th day, the sample was removed, the excess degradation solution was discarded, the sample was freeze-dried, and the weight of the hydrogel was recorded, which was recorded as W. t The degradation rate is calculated according to the following formula:

[0146]

[0147] Table 2. Degradation rate

[0148] Grouping Degradation rate Example 1 64.1±4.6％ Example 2 61.5±5.8％ Example 3 54.4±2.6％ Example 4 47.4±1.8％ Example 5 41.3±5.3％ Example 6 30.2±3.5％ Example 7 42.6±4.7％ Example 8 28.2±3.8％ Comparative Example 1 75.2±1.9％ Comparative Example 2 69.3±6.1％ Comparative Example 3 71.1±3.3％

[0149] The results show that the ideal hydrogel degradation rate should be 20%-40% at 14 days and gradually degrade to near completeness within 4-6 weeks. Through this design, the hydrogel scaffold can effectively support the initial repair of cartilage, and then provide sufficient space and environment to allow new cartilage tissue to occupy the position of the scaffold, and finally complete the repair process. The addition of Col1MA has a significant anti-degradation effect. As the amount of addition increases, the anti-degradation effect is enhanced. At the same time, the content of MBG@RES also has a great influence on the degradation rate. This may be because a higher content of MBG@RES can form a denser and more stable three-dimensional network structure, which increases the cross-linking degree and stability of the hydrogel. A higher cross-linking density is usually associated with a slower degradation rate because a tighter molecular network will limit the penetration of water molecules, thereby delaying the degradation of the material.

[0150] 7. Cytocompatibility evaluation

[0151] Test method: The cultured BMSCs cells were digested with 0.25% trypsin and resuspended at a density of 1×10 5 The cell suspension of 100 μL / mL was quickly mixed with the sterilized hydrogel, printed in a 3D printer, placed in a 24-well plate, and continued to be cultured with α-MEM complete medium. Each group has at least 3 wells. CCK8 was used to quantitatively analyze the cell survival rate. After 24 hours of culture, the well plate was removed, 300 μL of CCK8 working solution was added to each well, and the 3D printed scaffold was broken up as much as possible by blowing with a pipette tip. After incubation in a 37°C constant temperature carbon dioxide incubator (containing 5% CO2) for 1 to 2 hours, the absorbance (OD) was measured at a wavelength of 450 nm using a microplate reader, and the cell survival rate was calculated according to the formula:

[0152]

[0153] Figure 5 Cytocompatibility evaluation is shown.

[0154] The results show that the hydrogel scaffolds did not cause toxicity to cells, and the cell viability was higher than 90%, and even exceeded 100% (Examples 6-8), indicating that the prepared hydrogel scaffolds had no obvious toxicity to cells and could promote cell proliferation to a certain extent.

[0155] 8. Determination of MDA (malondialdehyde) content:

[0156] Test method:

[0157] BMSCs were cultured at a rate of 2×10 4 / well were inoculated into 24-well plates, and four groups were set up: Control (control), Example 6, Comparative Example 1 and Comparative Example 3. After the cells adhered to the wall, the corresponding materials were added into the groups for co-culture. After 12 hours, the culture medium was replaced, and the other four groups except the Control group were treated with 100μM H2O2 for 4 hours to induce oxidative stress response. Collect the cells into a centrifuge tube, discard the supernatant after centrifugation; add 1mL of extract for every 4 million cells, and ultrasonically break the bacteria or cells (power 20%, ultrasonic 3s, interval 10s, repeat 30 times); 8000g, 4℃ centrifugation for 10min, take the supernatant, and put on ice. Subsequently, the test was performed using the MDA kit (McLean).

[0158] Calculation formula: MDA content (nmol / 10 4 cells)=[ΔA×V 反总 / (ε×d)×10 9 ] / (400×V 样 / V 样总 )=0.0645×ΔA

[0159] In the above formula, V 反总 : Total volume of reaction system, 8×10 -4 L; ε: molar extinction coefficient of malondialdehyde, 155×10 3 L / mol / cm; d: cuvette optical path, 1 cm; V 样 : Sample volume added, 0.2mL; V 样总 : volume of extraction solution added; 400: total number of cells, 4 million.

[0160] Result description: From Figure 6 It can be seen that the MDA levels of Example 6 and the Control group are comparable, while Comparative Examples 1 and 3 both have higher MDA levels, indicating that the hydrogel scaffolds of Example 6 can effectively reduce the level of lipid peroxide MDA. This may be because the silicon, calcium, and phosphorus ions in the bioactive glass can promote antioxidant enzymes (such as superoxide dismutase, catalase, etc.) and resveratrol to effectively neutralize free radicals in the body when interacting with body fluids or cells, thereby slowing down cell oxidative damage. The two work synergistically to inhibit oxidative stress and provide a stable microenvironment for bone tissue. The hydrogel scaffolds of other examples also showed similar effects in reducing lipid peroxide MDA levels as in Example 6.

[0161] 8. Anti-inflammatory evaluation:

[0162] Test method: After anesthetizing the rat, use a scalpel or micro drill to remove the surface cartilage layer, remove the cartilage layer and penetrate into the subchondral bone. A defect of 2-3 mm in diameter is usually created. At this time, the wound usually bleeds to a certain extent. After stopping the bleeding with hemostatic cotton, a hydrogel scaffold is placed and the wound is closed with 7-0 absorbable sutures. Bone tissue samples are taken 10 days after surgery, washed in pre-cooled PBS (0.02mol / L, pH 7.0-7.2) to remove blood, and weighed for later use; 1.0g of tissue blocks are transferred to a glass homogenizer and 5mL of pre-cooled PBS is added for thorough grinding. This process must be performed on ice; the resulting homogenate is repeatedly frozen and thawed twice, and the prepared homogenate is centrifuged at 5000×g for 5 minutes. The supernatant is collected and tested with an ELISA kit.

[0163] Figure 7 Evaluation of anti-inflammatory effects is shown.

[0164] Result description: From Figure 7 It can be seen that compared with the saline group and the comparative example 1 group, the hydrogel scaffold loaded with Example 6 can effectively reduce the expression levels of inflammatory factors TNF-α, IL-6, and IL-1β, providing a stable microenvironment for bone tissue. The hydrogel scaffolds of other examples also showed similar effects as Example 6 in reducing the expression levels of inflammatory factors TNF-α, IL-6, and IL-1β.

Claims

1. A customizable biphasic hydrogel scaffold, characterized in that: The biphasic hydrogel scaffold includes a hyaline cartilage layer and a subchondral bone layer. The hyaline cartilage layer includes methacryloyl-type I recombinant collagen and bone marrow mesenchymal stem cells. The subchondral bone layer includes methacryloyl-type I recombinant collagen and resveratrol-loaded bioactive glass.

2. The biphasic hydrogel scaffold according to claim 1, characterized in that In the hyaline cartilage layer, the methacryloylation type I recombinant collagen is 5-15% by mass volume percentage, and the bone marrow mesenchymal stem cells are 1-2×10 5 pieces / mL.

3. The biphasic hydrogel scaffold according to claim 1, characterized in that In the subchondral bone layer, the content of methacrylated type I recombinant collagen is 7.5-15% by mass volume, and the content of resveratrol-loaded bioactive glass is 1-2%.

4. The biphasic hydrogel scaffold according to any one of claims 1 to 3, characterized in that The methacryloyl-type I recombinant collagen is prepared by the following steps: 2 g of type I collagen was dissolved in 100 mL of deionized water, the pH was adjusted to 7, 2 mL of methacrylic anhydride was added dropwise, and the mixture was reacted at room temperature for 24 h. The product was dialyzed in deionized water and centrifuged at 10,000 rpm for 15 minutes to remove impurities. The supernatant was lyophilized to obtain the methacrylated type I recombinant collagen.

5. The biphasic hydrogel scaffold according to any one of claims 1 to 3, characterized in that The resveratrol-loaded bioactive glass is prepared by the following steps: 1 g of resveratrol was dissolved in 50 mL of deionized water to prepare a 2% resveratrol solution; 1 g of bioactive glass was added to the resveratrol solution, ultrasonically dispersed for 15 minutes, and then stirred and mixed at 40°C. After the reaction was completed, the glass was washed with deionized water to remove unbound resveratrol and freeze-dried to obtain the final product.

6. The biphasic hydrogel scaffold according to claim 5, characterized in that: The bioactive glass is prepared by the following steps: 0.7 g of hexadecyltrimethylammonium bromide was dissolved in 33 mL of deionized water, 10 mL of ethyl acetate was added, and the mixture was stirred for 30 minutes. After that, 7 mL of 5 M ammonia water was added and the stirring was continued for 15 minutes. 3.6 mL of tetraethyl orthosilicate and 0.36 mL of triethyl phosphate were slowly added dropwise to the above solution respectively. 2.28 g of calcium nitrate tetrahydrate was dissolved in 5 mL of deionized water and slowly added dropwise to the above solution, and stirred at room temperature for 4 hours. The reaction solution was collected and centrifuged at 5000 rpm for 5 minutes to obtain a white precipitate. The precipitate was washed three times with anhydrous ethanol and deionized water respectively, and dried at 60° C. for 24 hours. The dried white powder was then calcined at 600° C. at a heating rate of 1° C. / min for 5 hours to obtain the bioactive glass.

7. The biphasic hydrogel scaffold according to any one of claims 1 to 3, characterized in that The bone marrow mesenchymal stem cells are prepared by the following steps: The rat limb bones were collected, and the muscles were removed in a PBS buffer containing 1% penicillin-streptomycin double antibody solution. Small incisions were made at both ends of the bones in α-MEM basal culture medium, and the bone marrow in the bones was flushed out. The bone marrow-culture medium mixture was collected and centrifuged at 1000 rpm for 5 minutes; the supernatant was discarded, and red blood cell lysis buffer was added to lyse for 10 minutes; PBS was added to terminate the reaction, and the mixture was centrifuged at 1000 rpm for 5 minutes; the supernatant was discarded, and the mixture was resuspended in α-MEM complete culture medium and cultured at 37°C and 5% CO2 for 48 hours. The cell medium was changed, and the cells were passaged when they grew to 80% confluence. After 2-3 generations of passages, the cells were washed 3 times with PBS when they grew to 80% confluence to obtain the bone marrow mesenchymal stem cells.

8. A method for preparing a biphasic hydrogel scaffold according to any one of claims 1 to 7, characterized in that The following steps are involved: S1. Preparing the hyaline cartilage layer: A photoinitiator solution was prepared by dissolving methacrylated type I recombinant collagen in the photoinitiator solution to obtain a methacrylated type I recombinant collagen solution, which served as the bio-ink for the first layer of the hydrogel scaffold. Cultured bone marrow mesenchymal stem cells were digested with 0.25% trypsin and resuspended, followed by centrifugation at 1000 rpm for 5 minutes. The supernatant was discarded, and the bone marrow mesenchymal stem cells were resuspended in the bio-ink, mixed evenly, and then 3D printed. S2. preparing the subchondral bone layer: A photoinitiator solution was prepared, and methacryloyl-type I recombinant collagen was dissolved in the above-mentioned photoinitiator solution to obtain a methacryloyl-type I recombinant collagen solution; the resveratrol-loaded bioactive glass was added and vortexed for 30 seconds to mix evenly, and the bio-ink for the second layer of the hydrogel scaffold was used. After the first layer was printed and formed, the second layer was continued to be printed.

9. The method according to claim 8, characterized in that The curing condition for the first layer of 3D printing is a light intensity of 17mW / cm 2 , exposure time 20s, exposure time for base layer 24s; the curing condition for printing the second layer is light intensity 18mW / cm 2 , exposure time 22s, base exposure time 27s.

10. Use of the biphasic hydrogel scaffold according to any one of claims 1 to 7 in the preparation of a drug for promoting wound healing or bone repair or anti-oxidation or anti-inflammatory effects.

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