Osteochondral repair hydrogel with dynamic repair performance and preparation method thereof

Abstract

The invention discloses osteochondral repair hydrogel with dynamic repair performance and a preparation method thereof, the hydrogel has a double-layer structure, the upper layer is obtained by photocuring a solution containing silk fibroin, benzoyl peroxide, riboflavin and acylated chondroitin sulfate, and the lower layer is obtained by photocuring a solution containing silk fibroin, benzoyl peroxide, riboflavin and acylated chondroitin sulfate; the lower layer is obtained by carrying out ultrasonic treatment on a solution containing silk fibroin, benzoyl peroxide, riboflavin and amorphous calcium phosphate and then carrying out light curing. The upper layer of the hydrogel has good elasticity, the lower layer of the hydrogel has high hardness and good supporting performance, the two layers of the hydrogel have good interface bonding capacity, the oxygen release amount of the upper layer and the oxygen release amount of the lower layer can be adjusted by adjusting the content of benzoyl peroxide, and therefore the hydrogel can meet the oxygen condition needed by cartilage tissue and bone tissue repair at the same time; the hydrogel has antibacterial and anti-inflammatory functions, the mineralization process of the amorphous calcium phosphate component in the hydrogel is perfectly matched with the bone repair process, and dynamic bone repair is induced on a wound surface in the bone repair process.

Description

Technical Field

[0001] This invention belongs to the technical field of prosthetic materials or prosthetic covering materials, and particularly relates to a osteochondral repair hydrogel with dynamic repair properties and its preparation method. Background Technology

[0002] Osteochondrial defects are a common joint disease in clinical practice. Because cartilage tissue lacks blood vessels and nerve supply, its self-repair ability is extremely limited. Once damaged, it will continue to deteriorate, causing joint pain, swelling, and limited mobility, severely impacting the patient's quality of life. Currently, commonly used repair strategies include microfracture surgery, autologous chondrocyte transplantation, and autologous osteochondral transplantation. However, these methods all have significant limitations. Microfracture surgery produces fibrocartilage instead of normal hyaline cartilage. Autologous osteochondrocyte transplantation requires a second surgery, increasing surgical risks. Autologous osteochondral transplantation requires harvesting from healthy cartilage, which can cause further damage.

[0003] Hydrogels possess excellent biocompatibility, providing a suitable environment for cell growth and promoting cartilage and bone regeneration. Bilayer hydrogels mimic the layered structure of natural osteochondral, offering different growth conditions for cartilage and bone tissue respectively, thus enhancing repair efficiency: the upper layer imitates the softness and elasticity of cartilage, while the lower layer mimics the hardness and support of subchondral bone, contributing to a more natural repair effect. The mechanical properties of the bilayer hydrogel can be adjusted according to needs; the soft upper layer can withstand joint pressure, while the rigid lower layer provides support, thereby better adapting to the mechanical environment of the joint and reducing the risk of repair failure.

[0004] Oxygen-releasing hydrogels continuously release oxygen, improving the local hypoxic environment, promoting the survival, proliferation, and differentiation of chondrocytes and stem cells, and accelerating tissue regeneration. Oxygen is crucial for cell metabolism and energy synthesis. By providing oxygen, oxygen-releasing hydrogels enhance cellular metabolic activity, support chondrocytes in synthesizing matrix components such as collagen and proteoglycans, and promote cartilage repair. Hypoxic environments exacerbate inflammatory responses; oxygen-releasing hydrogels improve oxygen supply, reduce the release of inflammatory factors, and lower the inflammatory response, creating a more favorable microenvironment for tissue repair. The oxygen released by oxygen-releasing hydrogels stimulates angiogenesis, increases blood supply to the damaged area, provides nutrients and oxygen for subchondral bone repair, and promotes osteochondral integration. Osteochondrial injuries are often hypoxic, affecting cell survival and repair.

[0005] In view of this, the present invention provides a bone and cartilage repair hydrogel with dynamic repair properties and its preparation method. The bilayer oxygen-releasing silk fibroin-based hydrogel has significant advantages in terms of biomimetic structure, dual oxygen release, simultaneous repair of cartilage and bone, biocompatibility, anti-inflammatory properties, antibacterial properties, and mechanical properties, and can more effectively promote the repair of bone and cartilage defects. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to address the above-mentioned deficiencies in the prior art by providing a hydrogel with dynamic repair properties for osteochondral repair and its preparation method. The hydrogel has a bilayer structure and can adjust the mechanical properties and oxygen release properties of the upper and lower layers according to the different mechanical properties and O2 requirements of bone and cartilage tissues, thus meeting the needs of both tissues. It can also continuously provide amorphous calcium phosphate (ACP) during bone repair, inducing dynamic bone repair at the wound site. Furthermore, the mineralization process can be sustained for 90 days under the hydrogel coating, perfectly matching the bone repair process and realizing dynamic repair of bone and cartilage tissues.

[0007] The first aspect of this invention is to provide a osteochondral repair hydrogel with dynamic repair properties. The hydrogel has a bilayer structure. The upper layer is obtained by photocuring a solution containing silk fibroin, benzoyl peroxide (BPO), riboflavin, and acylated chondroitin sulfate. The lower layer is obtained by first ultrasonically treating a solution containing silk fibroin, benzoyl peroxide, riboflavin, and amorphous calcium phosphate, and then photocuring it. The upper hydrogel, by introducing the acylated chondroitin sulfate functional component, successfully constructs a biomaterial system with immunomodulatory activity. Acylated chondroitin sulfate, as a modified natural polysaccharide derivative, retains the original biocompatibility of chondroitin sulfate. Experiments by the applicant have demonstrated that it can effectively inhibit the expression of pro-inflammatory factors while upregulating the secretion level of anti-inflammatory factors, significantly improving the material's inhibitory efficiency against inflammatory responses. The lower hydrogel introduces an amorphous calcium phosphate component. Due to its high solubility and activity, amorphous calcium phosphate can more effectively release calcium ions (Ca) in the human body environment. 2+ ) and phosphate ions (PO4) 3- This provides raw materials for the mineralization of new bone tissue and eventually transforms into more stable hydroxyapatite. Furthermore, the amorphous calcium phosphate in the lower hydrogel gradually transforms into hydroxyapatite in about 90 days, exhibiting osteogenic induction ability and matching the bone regeneration process well, thus achieving dynamic repair.

[0008] According to the above scheme, the preparation method of the acylated chondroitin sulfate is as follows: chondroitin sulfate is added to deionized water and stirred thoroughly under ice bath conditions to fully dissolve the chondroitin sulfate and obtain a chondroitin sulfate solution. Under light-protected and fully stirred conditions, methacrylic anhydride (MA) is slowly added dropwise to the chondroitin sulfate solution, followed by an acylation reaction. After the reaction is completed, post-treatment is performed to obtain acylated chondroitin sulfate with an acylation rate of 40-60%.

[0009] According to the above scheme, the concentration of the chondroitin sulfate solution is 0.01-0.04 g / mL, and the mass-to-volume ratio of chondroitin sulfate to methacrylic anhydride is 0.2-0.3 g / mL.

[0010] According to the above scheme, the acylation reaction conditions are as follows: the system pH value is 8-9, and the reaction is carried out at room temperature (15-35℃) for 24-48 hours under conditions of thorough stirring and protection from light.

[0011] According to the above scheme, the upper layer of the hydrogel has a thickness of 1-3 mm, a pore size of 50-200 μm, and a maximum compressive stress of 50-80 kPa; the lower layer has a thickness of 2-6 mm, a pore size of 10-100 μm, and a maximum compressive stress of 450-650 kPa. Due to its porous and loose network structure, the upper layer of the bilayer hydrogel is prone to compressive deformation under stress, effectively dispersing and absorbing external stress, thus playing a buffering and protective role. The lower layer of the hydrogel has a relatively dense network structure, and its shape remains basically unchanged under the same load, providing reliable mechanical support for the entire material system.

[0012] A second aspect of this invention is to provide a method for preparing the above-mentioned osteochondral repair hydrogel with dynamic repair properties, the specific steps of which are as follows: 1) Preparation of silk fibroin solution: Silk fibroin fibers obtained by degumming and drying silk are dispersed in lithium bromide solution, heated to dissolve and stirred thoroughly, and then dialyzed. The dialyzed solution is filtered, centrifuged, and the supernatant is diluted to obtain silk fibroin solution. 2) Preparation of upper silk fibroin / chondroitin sulfate mixed solution: First, prepare a photoinitiator solution, then disperse acylated chondroitin sulfate in the photoinitiator solution to obtain a solution containing acylated chondroitin sulfate. Add benzoyl peroxide and riboflavin solution to the silk fibroin solution obtained in step 1) to obtain a silk fibroin solution containing benzoyl peroxide and riboflavin. Then add the solution containing acylated chondroitin sulfate to it, stir evenly in the dark, and obtain the upper silk fibroin / chondroitin sulfate mixed solution. 3) Preparation of osteocartilage repair hydrogel with dynamic repair properties: Add benzoyl peroxide and riboflavin solution to the silk fibroin solution obtained in step 1), and then add amorphous calcium phosphate to obtain a lower mixed solution. Then, perform ultrasonic treatment. After treatment, immediately place it under a UV lamp for the first photocuring. Before it is completely gelled, add the upper silk fibroin / chondroitin sulfate mixed solution prepared in step 2) to continue the second photocuring to obtain osteocartilage repair hydrogel with dynamic repair properties.

[0013] According to the above scheme, the silk degumming method in step 1) is as follows: add silk to deionized water, heat to boiling, add sodium carbonate, continue boiling for 30-60 minutes, then thoroughly wash the silk with deionized water, and repeat this process 2-3 times.

[0014] According to the above scheme, in step 1), the mass ratio of silk to deionized water is 1:25-100, and the concentration of sodium carbonate in deionized water is 0.05-0.1wt%.

[0015] According to the above scheme, the concentration of lithium bromide solution in step 1) is 9.3M (mol / L), and the concentration of silk fibroin fiber in lithium bromide solution is 0.1-0.3g / mL.

[0016] According to the above scheme, the heating temperature in step 1) is 40-60℃.

[0017] According to the above scheme, the concentration of the silk fibroin solution in step 1) is 2-8 wt%.

[0018] According to the above scheme, the photoinitiator solution in step 2) is obtained by dissolving the photoinitiator Irgacure 2959 in water, with a concentration of 0.1-0.5wt%.

[0019] According to the above scheme, the concentration of acylated chondroitin sulfate in the solution containing acylated chondroitin sulfate in step 2) is 1-5 wt%.

[0020] According to the above scheme, the concentration of the riboflavin solution in step 2) is 0.01-0.5 mmol / L.

[0021] According to the above scheme, in step 2), the mass of benzoyl peroxide is 0.3-3.0% of the mass of the silk fibroin solution, and the volume ratio of the silk fibroin solution to the riboflavin solution is 50-100:1.

[0022] According to the above scheme, the volume ratio of the silk fibroin solution containing benzoyl peroxide and riboflavin in step 2) to the solution containing acylated chondroitin sulfate is 0.25-4:1.

[0023] According to the above scheme, the mass of benzoyl peroxide in step 3) is 0.3-3.0% of the mass of the silk fibroin solution.

[0024] According to the above scheme, the concentration of the riboflavin solution in step 3) is 0.01-0.5 mmol / L, and the volume ratio of the silk fibroin solution to the riboflavin solution is 50-100:1.

[0025] According to the above scheme, the mass of the amorphous calcium phosphate in step 3) is 15-50% of the mass of silk fibroin protein in the silk fibroin solution.

[0026] According to the above scheme, the volume ratio of the lower mixed solution to the upper silk fibroin / chondroitin sulfate mixed solution in step 3) is 1-2:1.

[0027] According to the above scheme, the process conditions for ultrasonic treatment in step 3) are: ultrasonic power 100-200W, ultrasonic treatment time 3-30s.

[0028] According to the above scheme, the conditions for the first light curing in step 3) are: light curing reaction is carried out under ultraviolet lamp irradiation, the power of ultraviolet lamp is 100-900W, and the light curing reaction time is 1-2 minutes.

[0029] According to the above scheme, the conditions for the second light curing in step 3) are: light curing reaction is carried out under ultraviolet lamp irradiation, the power of ultraviolet lamp is 100-900W, and the light curing reaction time is 5-10 minutes.

[0030] A third aspect of the present invention is to provide the application of the above-mentioned osteochondral repair hydrogel with dynamic repair properties in osteochondral repair. The specific application method is as follows: the osteochondral repair hydrogel with dynamic repair properties is implanted between bone tissue and cartilage, with the upper layer attached to the cartilage tissue and the lower layer attached to the bone tissue.

[0031] This invention, based on the differences in mechanical properties and O2 requirements between bone and cartilage tissues, designs a bilayered repair hydrogel. The layered structure simulates natural bone and cartilage tissue. The upper layer (silk fibroin + acylated chondroitin sulfate) mimics the softness and elasticity of cartilage. Silk fibroin and chondroitin sulfate each possess unique advantages in cartilage repair; their combined use exerts a synergistic effect, significantly enhancing the repair outcome. Silk fibroin provides mechanical support and a cell growth scaffold, while chondroitin sulfate provides bioactivity and anti-inflammatory effects. Together, they simulate the microenvironment of natural cartilage, promoting cartilage regeneration and functional recovery. The lower layer (silk fibroin + amorphous calcium phosphate) simulates the hardness and support of subchondral bone. Silk fibroin and amorphous calcium phosphate each possess unique advantages in subchondral bone repair; their combined use exerts a synergistic effect, significantly enhancing the repair outcome. Silk fibroin provides mechanical support and a cell growth scaffold, while acylated calcium phosphate provides calcium and phosphorus elements and bioactivity. Together, they simulate the microenvironment of the natural bone matrix, promoting bone tissue regeneration and functional recovery.

[0032] This invention utilizes benzoyl peroxide not only as an oxygen-releasing agent (because benzoyl peroxide reacts with water to generate oxygen), but also as a cross-linking agent for silk fibroin gel formation. In the repair of osteochondral defects, infected bone defects are often accompanied by the risk of bacterial infection. Benzoyl peroxide, as a peroxide with antibacterial properties, can also impart significant antibacterial properties to the material, meeting the needs of osteochondral repair. Furthermore, the oxygen release behavior of the hydrogel can be effectively controlled by varying the amount of benzoyl peroxide added. Hydrogels with lower benzoyl peroxide content release less oxygen, better meeting the low-oxygen environment required for cartilage tissue repair, while hydrogels with higher benzoyl peroxide content release more oxygen, better meeting the oxygen requirements for bone tissue repair. Therefore, by precisely controlling the benzoyl peroxide content, suitable hydrogel materials can be designed for the oxygen requirements of different tissues, supporting differentiated regeneration of bone and cartilage.

[0033] The beneficial effects of this invention are as follows: 1. The hydrogel provided by this invention has a double-layer structure. The upper hydrogel is soft and has good elasticity, while the lower hydrogel has high hardness and good support. The two hydrogels have good interfacial bonding ability. Moreover, the oxygen release of the upper and lower hydrogels can be adjusted by adjusting the content of benzoyl peroxide to simultaneously meet the oxygen conditions required for cartilage and bone tissue repair. In addition, the hydrogel has antibacterial and anti-inflammatory functions, which can prevent implant-related infections and bacterial infections of infected osteochondral defects. The amorphous calcium phosphate component in the hydrogel can maintain its mineralization process for about 90 days under the encapsulation of the hydrogel, perfectly matching the bone repair process and realizing the continuous supply of amorphous calcium phosphate to induce dynamic bone repair at the wound site during the bone repair process.

[0034] 2. The preparation method provided by this invention is fast and simple, and can achieve mass production. Attached Figure Description

[0035] Figure 1 This is a comparison chart of the antibacterial test results of Control, silk fibroin solution, and SF in Comparative Example 1 of this invention; Figure 2 Comparison of oxygen release from hydrogels with different BPO contents prepared in Comparative Example 3; Figure 3 SF prepared for Comparative Example 1, CSMA prepared for Comparative Example 2, and SF prepared for Example 1 20 / CSMA 80 SF 50 / CSMA 50 SF 80 / CSMA 20 The stress-strain curve; Figure 4 SF prepared for Comparative Example 1, CSMA prepared for Comparative Example 2, and SF prepared for Example 1 20 / CSMA 80 SF 50 / CSMA 50 SF 80 / CSMA 20 SEM comparison images of freeze-dried products; Figure 5 The SF prepared in Comparative Example 1, the CSMA prepared in Comparative Example 2, and the hydrogel SF prepared in Example 1 50 / CSMA 50 Anti-inflammatory performance test chart; Figure 6Stress-strain curves of SF prepared in Comparative Example 1 and SF / 5%ACP, SF / 10%ACP, SF / 15%ACP, SF / 20%ACP, and SF / 25%ACP prepared in Example 2; Figure 7 SEM images of SF prepared in Comparative Example 1 and SF / 15% ACP prepared in Example 2 after freeze-drying; Figure 8 Comparison of alkaline phosphatase staining after 7 and 14 days of culture of SF prepared in Comparative Example 1 and SF / 15% ACP prepared in Example 2, respectively. Figure 9 This is a comparison image of the solution before and after curing of the two-layer gel in Example 3, observed using a 45° tilt method. Figure 10 A cross-sectional SEM image of the osteochondral repair hydrogel with dynamic repair properties prepared in Example 3; Figure 11 Photographs of the osteochondral repair hydrogel with dynamic repair properties prepared in Example 3 after being immersed in PBS buffer at 37°C for 24 h and 48 h; Figure 12 The image shows a Live / Dead immunofluorescence staining of mouse bone marrow mesenchymal stem cells cultured with the osteochondral repair hydrogel with dynamic repair properties prepared in Example 3. Figure 13 XRD comparison images of the osteochondral repair hydrogel with dynamic repair properties prepared in Example 3 after 90 days of static storage; Figure 14 The image shows a comparison of SEM images of the osteochondral repair hydrogel with dynamic repair properties prepared in Example 3 during a 90-day static period. Detailed Implementation

[0036] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0037] The preparation method of amorphous calcium phosphate used in the embodiments and comparative examples of this invention is as follows: At room temperature, 58.443g of sodium chloride, 3.675g of calcium chloride dihydrate, 0.373g of potassium chloride, 1.016g of magnesium chloride hexahydrate, and 1.199g of anhydrous disodium hydrogen phosphate were added to 1000mL of deionized water. After complete dissolution, 0.840g of sodium bicarbonate was added. The mixture was stirred at room temperature for 30min, and a white precipitate was formed. The reaction solution was then filtered, and the filter residue was washed with anhydrous ethanol to remove excess water, yielding amorphous calcium phosphate powder, which was stored in a vacuum.

[0038] Comparative Example 1 A pure silk fibroin hydrogel (SF) is prepared as follows: S1. Preparation of silk fibroin solution: Take 100g of silkworm silk, add 5000mL of deionized water, heat to boiling, add Na2CO3, and continue boiling for 30min. Then, thoroughly wash the silkworm silk with deionized water. Repeat this degumming process 3 times. The concentrations of Na2CO3 added in the deionized water during the 3 degumming processes are 0.1wt%, 0.1wt%, and 0.05wt%, respectively. Place the degummed silkworm silk in a 60℃ oven and dry for 48 hours, constantly loosening it during the process. After drying, obtain silk fibroin fiber. Take 5g of silk fibroin fiber, cut it into small pieces, and add it to 25mL of LiBr solution (9.3mol / L). Place it in a 60℃ water bath to dissolve for 30min, then stir for 30min. Take out the solution, cool it, and transfer it to a dialysis bag. Dialyze it in a 4℃ refrigerator for 72h. Filter and centrifuge the dialysis solution, and dilute the supernatant to obtain a silk fibroin solution with a concentration of 4wt%. Place it in a 4℃ refrigerator for later use. S2. Take 5 mL of the silk fibroin solution prepared in step S1, add 0.075 g of benzoyl peroxide and 50 μL of riboflavin solution (0.1 mmol / L) to obtain a silk fibroin mixture. Place it under a UV lamp with a power of 600 W and photocuring for 10 min to obtain pure silk fibroin hydrogel.

[0039] The antibacterial properties of the hydrogel prepared in this comparative example were tested, with the silk fibroin solution prepared in step S1 as a control. A blank control group was also set up. Staphylococcus aureus and Escherichia coli were used as bacterial models to evaluate the antibacterial effect of the hydrogel. The specific experimental steps are as follows: (1) Preparation of liquid culture medium: Dissolve 1g tryptone, 0.5g yeast extract and 1g sodium chloride in 100mL deionized water, add sodium hydroxide solution to adjust the pH to 7.0-7.5, and then put it in an autoclave for sterilization. (2) Preparation of solid culture medium: Dissolve 2g tryptone, 1g yeast extract and 2g sodium chloride in 250mL deionized water, add sodium hydroxide solution to adjust the pH to 7.0-7.5, then add 5g agar, and then put it in an autoclave for sterilization. (3) First, add 5 mL of liquid culture medium to the test tube, then add 500 μL of the original bacterial solution. Seal the test tube and place it in a constant temperature incubator at 37℃. After incubating with shaking at 150 rpm for 18 h, place the sterilized hydrogel sample in the test tube. After the bacteria have been in contact with the sample for 18 h, take 100 μL of the bacterial solution and dilute it 10 times until the dilution factor is 10. 5Take 100 μL of the bacterial culture and place it in the center of a cooled solid agar plate. Spread the mixture evenly from top to bottom using a spreader. Invert the plate and incubate at 37°C for 18 hours. Quantify the colony count using an automated colony counter. Repeat each experiment three times. Figure 1 The figure shows a comparison of the antibacterial test results of the Control, silk fibroin solution, and SF groups. The silk fibroin solution group had a relatively large number of Staphylococcus aureus and Escherichia coli colonies, indicating that its antibacterial ability was limited. However, after adding BPO, the number of colonies of both bacteria was significantly reduced, with the inhibitory effect on Staphylococcus aureus being particularly obvious. Almost no obvious colony growth was observed on the culture plate. This shows that the addition of BPO effectively enhanced the antibacterial properties of SF hydrogel and showed a stronger inhibitory effect on Gram-positive bacteria.

[0040] Comparative Example 2 A pure acylated chondroitin sulfate hydrogel (CSMA) is prepared by the following method: S1. Add 4.0 g of chondroitin sulfate to 100 mL of deionized water and stir thoroughly in an ice bath until the chondroitin sulfate is fully dissolved to obtain a chondroitin sulfate solution. Under light-protected and well-stirred conditions, slowly add 16 mL of methacrylic anhydride (MA) to the above solution. Then adjust the pH value to 8-9 with NaOH solution (10 mol / L). React for 24 h under well-stirred and light-protected conditions. After the reaction is completed, add the resulting milky white reaction solution to 500 mL of anhydrous ethanol and freeze at -20 °C for 12 h. Then centrifuge (5000 rpm, 10 min) to collect the precipitate. Dissolve the precipitate and transfer it to a dialysis bag (3500 Da) for dialysis. After dialysis for 48 h, freeze-dry the dialyzed solution under vacuum to obtain acylated chondroitin sulfate (acylation rate 58%). S2. Preparation of pure acylated chondroitin sulfate hydrogel: First, prepare a 0.135wt% photoinitiator Irgacure 2959 solution. Take 0.4g of the acylated chondroitin sulfate prepared in step S1 and disperse it in 10mL of the photoinitiator Irgacure 2959 solution. Then, place it under a UV lamp for photocrosslinking (UV lamp power 600W, photocrosslinking time 10min) to obtain pure acylated chondroitin sulfate hydrogel.

[0041] Comparative Example 3 The effect of BPO dosage on oxygen release time and oxygen release amount was investigated. The dosage of benzoyl peroxide in Comparative Example 1 was adjusted to 0.8% (0.04 g), 1.5% (0.075 g), and 3% (0.15 g) of the silk fibroin solution, respectively. The resulting hydrogels were named SF / BPO. 0.8% SF / BPO 1.5%SF / BPO 3% .

[0042] Under an atmospheric pressure of 101 kPa and a temperature of 37°C, the three hydrogels prepared in this embodiment were made into cylindrical rods with a diameter of 12 mm and a height of 6 mm. These rods were then immersed in 20 mL of deionized water and sealed to prevent gas exchange with the outside environment. The dissolved oxygen content in the water was measured using a dissolved oxygen meter over 1-8 days. Three parallel samples were set up for each experimental group, and the average value was calculated.

[0043] like Figure 2 The figure shows a comparison of oxygen release rates of hydrogels with different BPO contents prepared in this comparative example, where SF / BPO... 3% It can release oxygen for about 7 days. The daily release rates from day 1 to day 7 are 0.57 mg / L, 0.47 mg / L, 0.36 mg / L, 0.25 mg / L, 0.2 mg / L, 0.14 mg / L, and 0.05 mg / L, respectively. On day 8, there is virtually no oxygen release. SF / BPO 1.5% It can release oxygen for about 5 days, releasing 0.25 mg / L, 0.24 mg / L, 0.13 mg / L, 0.08 mg / L, and 0.06 mg / L per day from day 1 to day 5, respectively. On day 6, there is virtually no oxygen release. SF / BPO 0.8% It can release oxygen for approximately 5 days, with daily releases of 0.14 mg / L, 0.13 mg / L, 0.07 mg / L, 0.05 mg / L, and 0.04 mg / L respectively from day 1 to day 5. On day 6, there is virtually no oxygen release. The oxygen release behavior of the hydrogel can be effectively controlled by adjusting the BPO content. Hydrogels with lower BPO content release less oxygen, better meeting the low-oxygen environment required for cartilage tissue repair, while hydrogels with higher BPO content release more oxygen, better meeting the oxygen requirements for bone tissue repair. Therefore, by precisely controlling the BPO content, suitable hydrogel materials can be designed to meet the oxygen needs of different tissues, supporting differentiated regeneration of bone and cartilage.

[0044] Example 1 Preparation of silk fibroin / chondroitin sulfate hydrogel: S1. Take 5 mL of silk fibroin solution (prepared using the method in step S1 of Comparative Example 1), add 0.075 g of benzoyl peroxide and 50 μL of riboflavin solution (0.1 mmol / L) to obtain a silk fibroin mixture; S2. Prepare a 0.135wt% photoinitiator Irgacure 2959 solution. Take 0.02g of acylated chondroitin sulfate (prepared using the method in step S1 of Comparative Example 2) and disperse it in 5mL of photoinitiator Irgacure 2959 solution to obtain an acylated chondroitin sulfate solution. S3. Add the acylated chondroitin sulfate solution obtained in step S2 to the silk fibroin mixture obtained in S1, and mix them at volume ratios of 20:80, 50:50, and 80:20 respectively. Stir evenly in the dark to obtain three kinds of silk fibroin / chondroitin sulfate mixed solutions. Irradiate under a 600W UV lamp for 10 minutes to cure the reaction. Name the resulting silk fibroin / chondroitin sulfate hydrogels as SF. 20 / CSMA 80 SF 50 / CSMA 50 SF 80 / CSMA 20 .

[0045] like Figure 3 The images show SF prepared in Comparative Example 1, CSMA prepared in Comparative Example 2, and SF prepared in this embodiment. 20 / CSMA 80 SF 50 / CSMA 50 SF 80 / CSMA 20 The stress-strain curves show that CSMA and SF... 20 / CSMA 80 SF 50 / CSMA 50 SF 80 / CSMA 20 The maximum stresses of chondroitin sulfate and silk fibroin were 52.0 kPa, 55.2 kPa, 78.1 kPa, 72.1 kPa, and 69.6 kPa, respectively, and the maximum compressive strains were 59.3%, 67.2%, 86.6%, 81.2%, and 80.0%, respectively. This indicates that blending chondroitin sulfate and silk fibroin in an appropriate ratio enhances the mechanical properties of the hydrogel. 50 / CSMA 50 It has the best mechanical properties, meeting the needs of cartilage repair.

[0046] like Figure 4 The images show SF prepared in Comparative Example 1, CSMA prepared in Comparative Example 2, and SF prepared in this embodiment. 20 / CSMA 80 SF 50 / CSMA 50 SF 80 / CSMA 20SEM images of the hydrogels after freeze-drying at -80℃ for 24 hours in a freeze dryer, arranged in descending order of silk fibroin content, show that the hydrogels exhibit a loose and porous structure. Furthermore, as the proportion of silk fibroin components in the composite material gradually changes, the overall microstructure of the hydrogels also undergoes significant changes: its internal structure gradually transitions from the three-dimensional porous network unique to SF to a dense sheet-like structure dominated by acylated chondroitin sulfate, specifically manifested as increased pore size, clearer pore walls, and enhanced interpore connectivity, resulting in a more loose and porous morphology overall.

[0047] Testing the hydrogel SF prepared in this embodiment 50 / CSMA 50 The anti-inflammatory properties were assessed, with SF prepared in Comparative Example 1 and CSMA prepared in Comparative Example 2 used as control samples. The experimental methods are as follows: (1) Preheat PBS buffer and complete culture medium in a 37°C water bath. After observing that the confluence of mouse macrophages reaches 80%, pass the cells. Remove the culture medium, wash with PBS salt 2-3 times, add PBS buffer and let stand for about 5 min to digest, add 2 mL of complete culture medium, use a pipette to blow the bottom of the bottle until the cells are completely detached and suspended, collect the digestion liquid and transfer it to a centrifuge tube and centrifuge at 1000 r / min for 10 min. After centrifugation, remove the supernatant, add complete culture medium to resuspend the cells, and pass the cells at a cell ratio of 1:4. (2) Induction of polarization: In order to simulate a high-inflammatory environment, lipopolysaccharide (LPS) was used to polarize mouse macrophages. The old culture medium was removed, and the cells were washed three times with PBS. The cells were stimulated for 24 hours with complete culture medium containing LPS (1 μg / mL) to stimulate the macrophages from the M0 phenotype to the M1 phenotype. (3) Cell seeding: After sterilizing the hydrogel with 75% alcohol for 15 min, wash it three times with PBS buffer on a clean bench, and finally soak it in PBS buffer overnight. Then, seed induced polarized macrophages onto the surface of the hydrogel material (1×10⁶ cells / year). 5 (cells / well), and then place it in a CO2 incubator for 2-3 hours. After the cells adhere to the surface of the material, add complete culture medium, and then change the complete culture medium every 48 hours thereafter. (4) Immunofluorescence staining: Induced polarized macrophages were seeded into hydrogel material and cultured for 3 days. The culture medium was discarded and the cells were washed with PBS 2-3 times. 4% paraformaldehyde was added for fixation for 20 min. After washing with PBS buffer 2-3 times, 0.2% Triton X-100 solution was added and the cell membrane was permeabilized at room temperature for 5 min. The waste liquid was discarded and the cells were washed with PBS buffer 2-3 times. After washing with PBS buffer 3 times, 2% BSA / PBS solution was used for blocking for 30 min. Three types of hydrogels were stained with different immunofluorescence methods: For the first group, primary antibody CD206 (1:200) was added, and the mixture was incubated overnight at 4°C. The waste solution was discarded, and the gels were washed 2-3 times with PBS for 5 minutes each time. Then, secondary antibody Coralite® 488 (1:500) was added, and the gels were incubated in the dark for 1 hour. The waste solution was discarded, and the gels were washed 3 times with PBS buffer. Then, 5 μg / mL DAPI solution was added, and the gels were incubated in the dark for 10 minutes. The waste solution was removed, and the gels were washed 2-3 times with PBS buffer. Finally, 1-2 drops of anti-fluorescence quencher were added for mounting. For the second group, primary antibody CD86 (1:100) was added, and the gels were incubated overnight at 4°C. The waste solution was discarded, and the gels were washed 2-3 times with PBS for 5 minutes each time. Then, 5 μg / mL DAPI solution was added, and the gels were incubated in the dark for 10 minutes. The waste solution was removed, and the gels were washed 2-3 times with PBS buffer. Finally, 1-2 drops of anti-fluorescence quencher were added for mounting.

[0048] like Figure 5 The image shows the anti-inflammatory properties of three hydrogels. Immunofluorescence staining was performed on the surface markers of M1 macrophages (CD86) and M2 anti-inflammatory macrophages (CD206) on SF6. 50 / CSMA 50 In the CSMA group and the SF group, a clear green fluorescence signal of CD206 was observed, while the red fluorescence signal of CD86 was extremely low, indicating that CSMA and SF... 50 / CSMA 50 Both hydrogels effectively inhibited the expression of pro-inflammatory factors while upregulating the secretion levels of anti-inflammatory factors, demonstrating a regulatory role in the immune microenvironment that facilitates the transformation from a pro-inflammatory to an anti-inflammatory phenotype. They also promoted macrophage polarization towards the M2 anti-inflammatory phenotype, exhibiting a good anti-inflammatory effect. In the SF group, the CD86 red fluorescence signal was significantly enhanced, indicating that SF did not show a significant anti-inflammatory regulatory effect. In contrast, CSMA and SF... 50 / CSMA 50 Macrophages in the group showed a significant polarization towards the M2 anti-inflammatory phenotype. This result further confirms that the introduction of CSMA significantly enhances the material's efficiency in inhibiting inflammatory responses and is a key component endowing the hydrogel with anti-inflammatory function. It can effectively regulate immune cell phenotypes and create favorable conditions for tissue repair in the inflammatory microenvironment.

[0049] Example 2 Preparation of silk fibroin / amorphous calcium phosphate hydrogel: Take 5 portions of silk fibroin solution (prepared using the method in step S1 of Comparative Example 1), 5 mL for each portion, add 0.075 g of benzoyl peroxide and 50 μL of riboflavin solution (0.1 mmol / L) to each portion, and then add amorphous calcium phosphate hydrogels accounting for 5% (0.01 g), 10% (0.02 g), 15% (0.03 g), 20% (0.04 g), and 25% (0.05 g) of the silk fibroin protein, respectively. Five silk fibroin / amorphous calcium phosphate mixed solutions were obtained by ultrasonic treatment at a power of 100W for 6 seconds. After treatment, the solutions were immediately placed under a 600W UV lamp for 10 minutes to cure the gels, resulting in five silk fibroin / amorphous calcium phosphate hydrogels, named SF / 5%ACP, SF / 10%ACP, SF / 15%ACP, SF / 20%ACP, and SF / 25%ACP, respectively.

[0050] Figure 6 The stress-strain curves for SF prepared in Comparative Example 1 and the SF / 5%ACP, SF / 10%ACP, SF / 15%ACP, SF / 20%ACP, and SF / 25%ACP prepared in this example are shown. The curves reveal that the maximum compressive stresses for SF, SF / 5%ACP, SF / 10%ACP, SF / 15%ACP, SF / 20%ACP, and SF / 25%ACP are 550.1 kPa, 553.3 kPa, and 559.0 kPa, respectively. a. The maximum compressive strains were 74.0%, 74.1%, 85.2%, 86.1%, 86.3%, and 88.5% at 584.8 kPa, 484.5 kPa, and 477.6 kPa, respectively. Among them, SF / 15% ACP showed the best mechanical properties, indicating that the mechanical properties of the hydrogel first increased and then decreased with the increase of the proportion of amorphous calcium phosphate. When the amount of amorphous calcium phosphate added was 15%, the mechanical properties of the hydrogel were the best, meeting the needs of subchondral bone repair.

[0051] like Figure 7 The images shown are SEM images of SF prepared in Comparative Example 1 and SF / 15% ACP prepared in this example, after being freeze-dried at -80°C for 24 hours. A1 and A2 are SEM images of SF at different magnifications after freeze-drying, and B1 and B2 are SEM images of SF / 15% ACP at different magnifications after freeze-drying. The comparison shows that the hydrogel without amorphous calcium phosphate exhibits a clearly visible loose porous network structure with relatively large pore sizes after freeze-drying. The hydrogel obtained after introducing amorphous calcium phosphate has a denser microstructure and significantly smaller pore sizes after freeze-drying. This is mainly due to the CaO content in the amorphous calcium phosphate. 2+This allows hydrogen bonds to form between silk fibroin molecular chains, effectively increasing the network cross-linking density.

[0052] The ability of SF / 15% ACP prepared in this example to promote the differentiation of bone marrow mesenchymal stem cells (BMSCs) into osteoblasts was tested using alkaline phosphatase, with SF prepared in Comparative Example 1 used as a control. Alkaline phosphatase (ALP) is a key monophosphate hydrolase that widely participates in and regulates the osteogenic differentiation and biomineralization process of cells. It is an important biomarker for assessing bone tissue formation and repair activity, and its activity level can directly reflect early osteogenic capacity. The specific experimental methods are as follows: (1) Differentiation of bone marrow mesenchymal stem cells: SF and SF / 15% ACP hydrogels were sterilized with 75% alcohol for 15 min, washed three times with PBS buffer on a clean bench, and then soaked in PBS buffer overnight. BMSCs cultured to passage 3 were then seeded onto the surface of the hydrogel material (1×10⁻⁶ cells / year). 5 (cells / wells), and then placed in CO2 2 After 2-3 hours in the incubator, once the cells have adhered to the surface of the hydrogel, add complete culture medium. After 24 hours, replace the complete culture medium with osteogenic induction medium. Subsequently, change the medium with osteogenic induction medium every 48 hours. The osteogenic induction medium is prepared as follows: add 10% fetal bovine serum (FBS) and 1% streptomycin-penicillin to DMEM / F12 culture medium to obtain complete culture medium, and then add 0.2% vitamin C solution (142mM), 0.1% dexamethasone solution (10mM), and 1% β-glycerophosphate disodium salt to obtain osteogenic induction medium. (2) Alkaline phosphatase (ALP) staining: 66 μL of nitrotetrazole blue (NBT) solution and 33 μL of p-toluidine blue (BCIP) solution were dissolved in 10 mL of alkaline phosphatase staining buffer and mixed evenly to obtain BCNP / NBT staining working solution. SF and SF / 15% ACP hydrogels were cultured for 7 and 14 days respectively, and the culture medium was discarded. Then, the samples were washed 3 times with PBS buffer, fixed with 4% paraformaldehyde for 20 min, and the waste liquid was discarded. After washing 3 times with PBS buffer, 1 mL of BCNP / NBT staining working solution was added to each sample and reacted at room temperature in the dark for 1 h. After discarding the waste liquid, the samples were washed 3 times with PBS buffer and finally placed under an optical microscope to observe the degree of differentiation.

[0053] like Figure 8The image shows a comparison of alkaline phosphatase (ALP) staining in SF and SF / 15% ACP hydrogels after 7 and 14 days of culture, respectively. The Control sample in the image represents the control sample without osteogenic induction medium; no obvious blue-purple precipitate was observed on its surface, indicating background staining. After 7 and 14 days of culture with osteogenic induction medium, the SF / 15% ACP hydrogel showed a more significant and widespread blue-purple precipitate than the SF hydrogel, indicating higher ALP activity on its surface. SF / 15% ACP may continuously release calcium and phosphate ions through its active component ACP, creating a microenvironment more conducive to the differentiation of bone marrow mesenchymal stem cells into osteoblasts.

[0054] Example 3 A hydrogel for osteochondral repair with dynamic repair properties has a bilayer structure. The upper layer is obtained by photocuring a mixture of silk fibroin solution and a solution containing acylated chondroitin sulfate. The lower layer is obtained by ultrasonic treatment followed by photocuring of a mixture of silk fibroin solution and amorphous calcium phosphate. The specific preparation method is as follows: S1. Preparation of silk fibroin / chondroitin sulfate mixed solution: First, prepare a 0.135wt% photoinitiator Irgacure 2959 solution. Take 0.02g of acylated chondroitin sulfate (prepared using the method in step S1 of Comparative Example 2) and disperse it in 5mL of photoinitiator Irgacure 2959 solution to obtain an acylated chondroitin sulfate solution. Take 5mL of silk fibroin solution (prepared using the method in step S1 of Comparative Example 1), add 0.075g of benzoyl peroxide and 50μL of riboflavin solution (0.1mmol / L), and then add the aforementioned acylated chondroitin sulfate solution. Stir evenly in the dark to obtain a silk fibroin / chondroitin sulfate mixed solution, i.e., the upper mixed solution. S2. Preparation of osteochondral repair hydrogel with dynamic repair properties: Take 5 mL of silk fibroin solution (prepared using the method in step S1 of Comparative Example 1), add 0.075 g of benzoyl peroxide and 50 μL of riboflavin solution (0.1 mmol / L), and then add 0.03 g of amorphous calcium phosphate to obtain a lower mixed solution. Then take 600 μL of the lower mixed solution for ultrasonic treatment at a power of 100 W for 6 s. After treatment, immediately place it under a UV lamp with a power of 600 W. When the photocuring reaction is 1 min (not fully cured at this time), add 300 μL of the silk fibroin / chondroitin sulfate mixed solution prepared in step S1, and then continue photocuring for 10 min to obtain a bilayer structure osteochondral repair hydrogel with dynamic repair properties.

[0055] like Figure 9The figures shown are comparative images of the solutions before and after curing using a 45° tilt method. (a) shows the upper mixed solution after curing with UV light for 10 minutes (b), where the liquid level did not change when the bottle was tilted. (c) shows the lower mixed solution after ultrasonic treatment for 6 seconds and UV curing for 10 minutes (d), where the liquid level did not change when the bottle was tilted. In this embodiment, the lower mixed solution was first ultrasonically treated for 6 seconds and then photocured for 1 minute. The upper mixed solution was then added and photocured for 10 minutes to obtain the product. The liquid level did not change when the bottle was tilted (see figure (e)). This demonstrates the successful preparation of a bilayered osteochondral repair hydrogel with dynamic repair properties.

[0056] like Figure 10 The image shown is a cross-sectional SEM image of the osteochondral repair hydrogel with dynamic repair properties prepared in this embodiment after being freeze-dried at -80℃ for 24 hours. It can be seen that the upper layer is about 2 mm thick and the lower layer is about 4 mm thick, and there is a clear interface between the upper and lower hydrogel layers. The lower hydrogel used for bone repair has a denser pore size (pore size 10-100 μm) and provides mechanical support, while the upper hydrogel used for cartilage repair has a looser pore size (pore size 50-200 μm) and facilitates the transport of nutrients during the repair process of cartilage and bone.

[0057] To evaluate the interfacial bonding performance between the upper and lower layers of the bilayer hydrogel prepared in this embodiment, the osteochondral repair hydrogel with dynamic repair properties prepared in this embodiment was immersed in PBS buffer at 37°C to simulate its behavior in a real body fluid environment. Observations were made at 24h and 48h of immersion, and the test results are shown in the figure below. Figure 11 As shown, during the immersion process, the upper hydrogel swelled moderately due to its hydrophilic properties, resulting in a uniform expansion of its morphology. In contrast, the lower hydrogel, with its higher cross-linking density, maintained a stable overall morphology, without significant volume changes or structural loosening. Even under continuous immersion conditions lasting up to 48 hours, no interfacial delamination, displacement, or detachment was observed between the two hydrogel layers; their contact interface remained clear and intact, without any obvious gaps. These results clearly demonstrate that the bilayer hydrogels form a stable, continuous, and tightly bonded interfacial integrated structure through effective physical or chemical interactions, exhibiting excellent bonding strength and structural integrity in an aqueous environment, thus meeting the requirements for interlayer stability in bone / cartilage tissue repair.

[0058] The biocompatibility of the osteochondral repair hydrogel with dynamic repair properties prepared in this embodiment was tested using the following specific test methods: (1) Cell seeding: After sterilizing the hydrogel with 75% alcohol for 15 min, it was washed three times with PBS buffer on a clean bench, and finally soaked in PBS buffer overnight. Then, mouse bone marrow mesenchymal stem cells were seeded onto the surface of the hydrogel material (1×10⁻⁶). 5 (cells / well), and then place it in a CO2 incubator for 2-3 hours. After the cells adhere to the surface of the material, add complete culture medium, and then change the complete culture medium every 48 hours thereafter. (2) Preparation of live / dead cell staining solution: Add 0.5 μL of Calcein AM and 2 μL of propidium iodide (PI) to 1 mL of PBS buffer to obtain the live / dead cell staining working solution. Prepare fresh before use. (3) Live / dead cell staining: To observe cell viability, the hydrogel was cultured for 1, 3, and 7 days, and the culture medium was discarded. The cells were then washed three times with PBS buffer. 1 mL of live / dead cell staining reagent was added to each well, and the cells were then transferred to a cell culture incubator and incubated in the dark for 15 minutes. Finally, the cells were observed using a laser confocal microscope. Figure 12 The image shows a Live / Dead immunofluorescence staining image of cells cultured in hydrogel and mouse bone marrow mesenchymal stem cells. The results show that bone marrow mesenchymal stem cells grew well on both the upper and lower hydrogel layers, indicating that the bilayer hydrogel has excellent biocompatibility.

[0059] To test the transformation process of amorphous calcium phosphate to hydroxyapatite (HAP) in the osteochondral repair hydrogel with dynamic repair properties prepared in this embodiment, the lower layer hydrogel prepared in this embodiment was placed in PBS solution at 37°C and allowed to stand for 90 days. Samples were taken at 0, 10, 20, 30, 60, and 90 days. The hydrogel was washed twice with deionized water and then freeze-dried at -80°C for 24 hours. XRD and SEM were then performed to observe the changes. Figure 13 The image shows a comparison of XRD patterns of the lower hydrogel after 90 days of standing. As time increases, the intensity of the characteristic peak of HAP in the hydrogel also increases, indicating improved crystallinity. Figure 14 SEM comparison can observe the changes in mineral morphology on the hydrogel network during the crystallization process. In the early stage, spherical ACP nanoparticles are distributed in the hydrogel network. As the crystallization process progresses, these spherical ACP nanoparticles gradually transform into a sheet-like structure, which can continuously provide ACP to induce dynamic bone repair in the wound during the bone repair process. Moreover, the mineralization process can be sustained for 90 days under hydrogel encapsulation, perfectly matching the bone repair process.

[0060] The above description is only a preferred embodiment of the present invention. The scope of protection of the present invention is not limited to the above embodiments. Any equivalent modifications or changes made by those skilled in the art based on the content disclosed in the present invention should be included within the scope of protection set forth in the claims.

Claims

1. A hydrogel for repairing osteochondral bone with dynamic repair properties, characterized in that, The hydrogel has a bilayer structure. The upper layer is obtained by photocuring a solution containing silk fibroin, benzoyl peroxide, riboflavin and acylated chondroitin sulfate. The lower layer is obtained by first ultrasonically treating a solution containing silk fibroin, benzoyl peroxide, riboflavin and amorphous calcium phosphate, and then photocuring it.

2. The osteochondral repair hydrogel with dynamic repair properties according to claim 1, characterized in that, The preparation method of the acylated chondroitin sulfate is as follows: chondroitin sulfate is added to deionized water and stirred thoroughly under ice bath conditions to fully dissolve the chondroitin sulfate and obtain a chondroitin sulfate solution. Under light-protected and fully stirred conditions, methacrylic anhydride is slowly added dropwise to the chondroitin sulfate solution, followed by an acylation reaction. After the reaction is completed, post-treatment is performed to obtain acylated chondroitin sulfate with an acylation rate of 40-60%.

3. The osteochondral repair hydrogel with dynamic repair properties according to claim 2, characterized in that, The concentration of the chondroitin sulfate solution is 0.01-0.04 g / mL, and the mass-to-volume ratio of chondroitin sulfate to methacrylic anhydride is 0.2-0.3 g / mL. The acylation reaction conditions are: the system pH is 8-9, and the reaction is carried out at room temperature for 24-48 h under conditions of thorough stirring and protection from light.

4. The osteochondral repair hydrogel with dynamic repair properties according to claim 1, characterized in that, The upper layer of the hydrogel has a thickness of 1-3 mm, a pore size of 50-200 μm, and a maximum compressive stress of 50-80 kPa; the lower layer has a thickness of 2-6 mm, a pore size of 10-100 μm, and a maximum compressive stress of 450-650 kPa.

5. A method for preparing a osteochondral repair hydrogel with dynamic repair properties according to any one of claims 1-4, characterized in that, The specific steps are as follows: 1) Preparation of silk fibroin solution: Silk fibroin fibers obtained by degumming and drying silk are dispersed in lithium bromide solution, heated to dissolve and stirred thoroughly, and then dialyzed. The dialyzed solution is filtered, centrifuged, and the supernatant is diluted to obtain silk fibroin solution. 2) Preparation of upper silk fibroin / chondroitin sulfate mixed solution: First, prepare a photoinitiator solution, then disperse acylated chondroitin sulfate in the photoinitiator solution to obtain a solution containing acylated chondroitin sulfate. Add benzoyl peroxide and riboflavin solution to the silk fibroin solution obtained in step 1) to obtain a silk fibroin solution containing benzoyl peroxide and riboflavin. Then add the solution containing acylated chondroitin sulfate to it, stir evenly in the dark, and obtain the upper silk fibroin / chondroitin sulfate mixed solution. 3) Preparation of osteocartilage repair hydrogel with dynamic repair properties: Add benzoyl peroxide and riboflavin solution to the silk fibroin solution obtained in step 1), and then add amorphous calcium phosphate to obtain a lower mixed solution. Then, perform ultrasonic treatment. After treatment, immediately place it under a UV lamp for the first photocuring. Before it is completely gelled, add the upper silk fibroin / chondroitin sulfate mixed solution prepared in step 2) to continue the second photocuring to obtain osteocartilage repair hydrogel with dynamic repair properties.

6. The method for preparing the osteochondral repair hydrogel with dynamic repair properties according to claim 5, characterized in that, Step 2) The photoinitiator solution is obtained by dissolving the photoinitiator Irgacure 2959 in water, with a concentration of 0.1-0.5 wt%; the concentration of acylated chondroitin sulfate in the acylated chondroitin sulfate solution is 1-5 wt%; and the concentration of the riboflavin solution is 0.01-0.5 mmol / L.

7. The method for preparing the osteochondral repair hydrogel with dynamic repair properties according to claim 5, characterized in that, Step 2) The mass of benzoyl peroxide is 0.3-3.0% of the mass of the silk fibroin solution, and the volume ratio of the silk fibroin solution to the riboflavin solution is 50-100:1; the volume ratio of the silk fibroin solution containing benzoyl peroxide and riboflavin to the solution containing acylated chondroitin sulfate is 0.25-4:

1.

8. The method for preparing the osteochondral repair hydrogel with dynamic repair properties according to claim 5, characterized in that, Step 3) The mass of benzoyl peroxide is 0.3-3.0% of the mass of the silk fibroin solution; Step 3) The concentration of the riboflavin solution is 0.01-0.5 mmol / L, and the volume ratio of the silk fibroin solution to the riboflavin solution is 50-100:1; The mass of the amorphous calcium phosphate is 15-50% of the silk fibroin protein mass in the silk fibroin solution; The volume ratio of the lower mixed solution to the upper silk fibroin / chondroitin sulfate mixed solution is 1-2:

1.

9. The method for preparing the osteochondral repair hydrogel with dynamic repair properties according to claim 5, characterized in that, Step 3) The ultrasonic treatment process conditions are as follows: ultrasonic power 100-200W, ultrasonic treatment time 3-30s; the first photocuring conditions are: photocuring reaction under ultraviolet lamp irradiation, ultraviolet lamp power 100-900W, photocuring reaction time 1-2 minutes; the second photocuring conditions are: photocuring reaction under ultraviolet lamp irradiation, ultraviolet lamp power 100-900W, photocuring reaction time 5-10 minutes.

10. The application of the osteochondral repair hydrogel with dynamic repair properties according to any one of claims 1-4 in osteochondral repair, characterized in that, The specific application method is as follows: the osteochondral repair hydrogel with dynamic repair properties is implanted between bone tissue and cartilage, with the upper layer attached to the cartilage tissue and the lower layer attached to the bone tissue.

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