A method for preparing injectable diabetic osteochondral repair hydrogel containing V2CuS4 nanozymes based on hydrothermal method and its application.

Abstract

The application discloses a method for preparing active oxygen scavenging type diabetes osteochondral repair hydrogel based on a hydrothermal method and application thereof, and belongs to the technical field of biological medical materials and tissue engineering. The method comprises the following steps: synthesizing V2CuS4 nanoscale enzyme with peroxidase-like activity by a hydrothermal method and preparing injectable three-dimensional network hydrogel with photogelation characteristics. The application realizes efficient scavenging of active oxygen through cascade catalysis of glucose oxidase and V2CuS4 nanoscale enzyme, and the photogelation characteristics of methacrylamidated silk fibroin hydrogel can adapt to bone and cartilage defects with different areas and has excellent individual adaptability. In a simulated physiological environment, the hydrogel prepared by the application has high hydroxyl radical scavenging rate and good biocompatibility, and can significantly improve the oxidative stress regulation ability of the microenvironment of bone and cartilage defect repair, and provides a new functional material with mechanical support and biological activity for bone and cartilage tissue engineering.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and tissue engineering, and in particular to a method for preparing an injectable diabetic osteochondral repair hydrogel containing V2CuS4 nanozymes based on a hydrothermal method, and its application. Background Technology

[0002] In the pathological microenvironment of diabetic osteochondral defects, long-term hyperglycemia triggers a series of oxidative stress reactions. This process directly damages the mitochondrial function of osteoblasts, leading to a significant decrease in mitochondrial membrane potential (possibly below 48.7mV, far below the 120mV threshold required to maintain normal function), becoming the core pathological mechanism that hinders bone matrix mineralization.

[0003] Existing biomaterial systems for osteochondral repair mainly include bioactive materials and metal-based antioxidant materials. Bioactive materials, such as silicate bioactive glasses like the 45S5 series, while possessing excellent in vitro bone-inducing capabilities (hydroxyapatite deposition rate exceeding 3 μm / d), show an efficiency of less than 35% in scavenging reactive oxygen species (ROS) such as hydroxyl radicals in a simulated high-glucose environment (10 mM glucose) of diabetes, making it difficult to alleviate pathological-grade oxidative stress damage. Metal-based antioxidant materials, such as some metal-organic frameworks (MOFs), face another challenge. Although they can regulate ROS levels by simulating the Fenton reaction, the released metal ions (such as Fe)... 2+ and Cu 2+ The release of metal ions is often uncontrollable, and excessive release of metal ions can lead to abnormally high permeability of osteoblast lysosomal membranes (LMP value exceeding 60%), which in turn triggers apoptosis of up to 78% or more, severely inhibiting osteogenic activity.

[0004] The activity of nanozymes is generally regulated by synthesizing ternary sulfides (such as CuFeS2) via a high-temperature solvothermal method (>200℃). However, this method suffers from thermodynamic imbalance, resulting in high crystal defect density (transmission electron microscopy showing dense dislocations) and limited specific surface area (<80 μm²). 2 The activity of the nanozyme was significantly reduced (only 2.1 U / mg), significantly decreasing its peroxidase-like (POD) activity. This deficiency directly weakens the nanozyme's efficiency in scavenging reactive oxygen species, making it difficult to meet the intervention requirements for strong oxidative stress in the high-glucose microenvironment of diabetes. In the construction of the multi-stage reaction system, the excessive spatial separation between glucose oxidase (GOx) and the nanozyme (spacing > 50 nm) resulted in a high H2O2 diffusion loss rate of up to 78%, severely limiting the reaction efficiency. The physical mixing system, lacking spatial confinement regulation, could not achieve directional substrate transport, causing the catalytic kinetic constant to stagnate at 1.6 × 10³ M. -1 s -1 The following are difficult to match the rapid response requirements in the diabetic microenvironment.

[0005] In current technological systems, materials such as bioactive glass and MOFs suffer from low bone integration rates due to the decoupling of their antioxidant and osteogenic functions and the toxicity of metal ions. Traditional ternary sulfide nanozymes have limited catalytic efficiency due to defects in high-temperature synthesis, and processes such as wet spinning are insufficient to achieve uniform enzyme-nanozyme composites. Traditional injectable hydrogels exhibit significant uncontrolled volume swelling (change rate >300%) in the high glucose-serum complex microenvironment of diabetes, and have a short reactive oxygen species scavenging half-life (<24h), resulting in insufficient mechanical stability and antioxidant durability. This leads to structural disintegration and rapid loss of active ingredients after implantation, making it impossible to maintain long-term regulation of the pathological microenvironment. Therefore, developing novel materials that combine structural stability with synergistic antioxidant and bone mineralization-promoting capabilities has significant clinical translational value. Summary of the Invention

[0006] To address the problem of poor antioxidant and osteogenic functions in materials used in diabetic bone regeneration technology, this invention provides a method for preparing an injectable diabetic osteocartilage repair hydrogel containing V2CuS4 nanozyme using a hydrothermal method, and its application. The invention innovatively develops an injectable hydrogel that combines reactive oxygen species scavenging (hydroxyl radical scavenging rate ≥95%) with mechanical stability by controlling the hydrothermal synthesis of V2CuS4 nanozyme and constructing a cascade reaction system through room temperature mixing. It also leverages the photogelation properties of methacrylamidized silk fibroin hydrogel.

[0007] The method for preparing reactive oxygen species scavenging diabetic osteochondral repair hydrogel based on hydrothermal method according to the present invention includes the following steps: (1) V2CuS4 nanozyme was obtained by mixing copper salt, vanadium salt and sulfur source and dissolving in water, followed by hydrothermal reaction, centrifugation, washing and drying.

[0008] (2) Glucose oxidase and V2CuS4 nanozyme are mixed evenly to obtain an enzyme mixture. Methacrylamide silk fibroin is added to the enzyme mixture to obtain a mixed solution. A photoinitiator is added to the mixed solution to obtain a composite material aqueous solution. The composite material aqueous solution is ultrasonically dispersed to form an injectable three-dimensional network hydrogel with photogelation properties.

[0009] Preferably, in step (1) of the present invention, the copper salt is one of copper chloride, copper nitrate, and copper sulfate; the vanadium salt is one of vanadium oxysulfate, sodium vanadate, and vanadium oxide; and the sulfur source is one of sodium thiosulfate, thiourea, and sulfur powder.

[0010] Preferably, in step (1) of the present invention, the Cu:V:S stoichiometric ratio of copper salt, vanadium salt and sulfur source is 1:2:4-8.

[0011] Preferably, the hydrothermal reaction vessel in step (1) of the present invention is a high-pressure reactor with a polytetrafluoroethylene liner; the hydrothermal reaction conditions are constant temperature treatment at 120-180℃ for 12-24 hours.

[0012] Preferably, the centrifugation parameters in step (1) of the present invention are 2000-6000 rpm and 10-30 minutes.

[0013] Preferably, in step (1) of the present invention, the washing is performed three times by alternating between anhydrous ethanol and deionized water.

[0014] Preferably, the vacuum drying conditions in step (1) of the present invention are 40-60℃ and -0.2 to -0.8MPa for 6-18h.

[0015] Preferably, the V2CuS4 nanozyme in step (1) of the present invention has a particle size of 20-50 nm and a specific surface area ≥80 m². 2 / g.

[0016] Preferably, the activity of glucose oxidase in step (2) of the present invention is ≥150U / mg.

[0017] Preferably, in step (2) of the present invention, the mass ratio of glucose oxidase to V2CuS4 nanozyme in the enzyme mixture is 1:1-5; the mass ratio of enzyme mixture to methacrylamide silk fibroin in the mixed solution is 1:80-100; and the mass percentage of mixed solution and photoinitiator in the aqueous solution of the composite material is 1:0.05-0.2%.

[0018] Preferably, the ultrasonic dispersion parameters in step (2) are 20-60 kHz and 30-70 W / cm². 2 The mixing time is 5-15 minutes, the mixing temperature is 15-30℃, and the resulting hydrogel can be gelled in 20-45 seconds under a 405nm UV lamp.

[0019] The hydrogel prepared by the method of the present invention can be applied to the bone and cartilage repair material for diabetes. Specifically, the hydrogel is loaded into the bone and cartilage repair scaffold material to form a diabetic bone repair material with active oxygen scavenging function.

[0020] In a simulated diabetic microenvironment (10 mM glucose concentration, 100-500 μM H2O2 concentration), the hydrogel of this invention exhibits a scavenging rate of ≥95% for hydroxyl radicals and a scavenging rate of ≥85% for superoxide anions.

[0021] This invention involves dissolving degummed silkworm cocoons using an Ajisawa system or a LiBr system to obtain silk fibroin; amidating the silk fibroin with methacrylic anhydride or glycidyl methacrylate; and introducing photoinitiating functional groups onto the silk fibroin molecular chain using photoinitiator 2959 [2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone] or LAP [lithium phenyl(2,4,6-trimethylbenzoyl)phosphate] to achieve photoinitiated gelation of the methacrylamide-amylated silk fibroin. Other methods may also be used to introduce photoinitiating functional groups onto the silk fibroin molecular chain, which will not be specifically described here.

[0022] Mechanism of this invention: This invention repairs diabetic osteochondrocytes by integrating silk fibroin-based photoinitiated hydrogels and GOx-V2CuS4-mediated cascade reactions. The hydrogels prepared by this invention can decompose glucose and scavenge reactive oxygen species. The GOx-V2CuS4-mediated cascade reactions continuously provide the microbial support required for tissue repair, significantly inhibiting the accumulation of advanced glycation end products (AGEs) in diabetes. By efficiently scavenging reactive oxygen species, it significantly improves the oxidative stress microenvironment. This invention enhances bone mineralization capacity while synergistically resisting oxidation, which is beneficial for bone matrix regeneration and mineralization.

[0023] The beneficial effects of this invention are: This invention overcomes the limitations of traditional single antioxidants or metal nanoparticles. The V2CuS4 nanozyme, constructed via a hydrothermal method, is uniformly dispersed and possesses both catalase-like activity and stability. It can stably and long-term scavenge reactive oxygen species generated during glucose degradation and in cascade reactions, significantly improving the microenvironment of oxidative stress-induced diabetic bone defects. The cascade reaction system formed in this invention overcomes the shortcomings of traditional antioxidant materials, such as low scavenging efficiency and short action period.

[0024] This invention employs a room-temperature mixing and photogelation approach, avoiding the damage to enzyme activity caused by high-temperature treatment. Simultaneously, it utilizes the photogelation properties of methacrylamide-modified silk fibroin to achieve in-situ molding of injectable gels with excellent biocompatibility. Compared to traditional chemically cross-linked hydrogels, this process significantly improves the retention rate of bioactive components and precisely matches the filling of osteochondral defects with individual and temporal variations. Attached Figure Description

[0025] Figure 1 This is a SEM morphology analysis of the V2CuS4 nanozyme synthesized in Example 1 of this invention.

[0026] Figure 2 This is the XRD pattern of the V2CuS4 nanozyme synthesized in Example 1 of this invention.

[0027] Figure 3This is the verification of the peroxidase activity of V2CuS4 nanozymes based on the TMB colorimetric reaction in Example 1 of the present invention.

[0028] Figure 4 This is a schematic diagram of the photogelation properties of the V2CuS4-GOx-methacrylamide silk fibroin hydrogel cascade reaction system prepared in Example 1 of this invention.

[0029] Figure 5 This is a biocompatibility verification of the V2CuS4-GOx-methacrylamide silk fibroin hydrogel prepared in Example 1 of this invention.

[0030] Figure 6 This is a schematic diagram illustrating the application mode of the hydrogel of the present invention in osteocartilage. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited to the content described.

[0032] Copper salts include copper nitrate (Cu(NO3)2·3H2O), copper chloride (CuCl2), and copper sulfate (Cu2SO4); vanadium salts include vanadium oxysulfate (VOSO4·2H2O), sodium vanadate (Na3VO4), and vanadium oxide (V2O5); sulfur sources include thiourea (CH4N2S); anhydrous calcium chloride (CaCl2); and methacrylic anhydride. All reagents were purchased from Aladdin China and have a purity ≥99.0%.

[0033] Sodium thiosulfate (Na2S2O3, purity ≥99.0%); anhydrous ethanol, hydrogen peroxide (H2O2, concentration 3wt%), 3,3',5,5'-tetramethylbenzidine (TMB), glucose oxidase (GOx, activity ≥100U / mg) 3 The reagents included: 1) 94% pure methacrylic anhydride (94% pure) containing 0.2% topanol stabilizer; 2) 99.0% pure methacrylic anhydride; and 3) methacrylic anhydride (94% pure) containing 0.2% topanol stabilizer. All reagents were purchased from Ron.

[0034] Photoinitiator 2959 [2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone] and photoinitiator LAP [lithium phenyl (2,4,6-trimethylbenzoyl)phosphate]; 14 kDa dialysis bags were purchased from Solarbio, USA. Sulfur powder (GSB0417732004) was purchased from the China Nonferrous Metals Research Institute, with a purity ≥99.0%.

[0035] The preparation of methacrylamide-modified silk fibroin is as follows: 2g of degummed silk fibroin, 8mL of water, and 350mM of methacrylic anhydride (0.5395g) were mixed evenly and heated to 60℃. The mixture was stirred at 300rpm for 3h and then dialyzed at 25℃ for 72h using a 14kDa dialysis bag to remove unreacted methacrylic anhydride, thus obtaining methacrylamidized silk fibroin. Example 1

[0036] A method for preparing reactive oxygen species scavenging diabetic bone repair hydrogel based on hydrothermal method, the specific steps of which are as follows: (1) Dissolve copper nitrate (Cu(NO3)2·3H2O), vanadium oxysulfate (VOSO4·2H2O) and thiourea (CH4N2S) in 30mL of deionized water at a Cu:V:S stoichiometric ratio of 1:2:4, stir until completely dissolved, and form a dark gray-blue precipitate.

[0037] (2) Transfer the dark gray-blue precipitate from (1) to a polytetrafluoroethylene-lined high-pressure reactor (volume 100 mL, filling rate 40%) and react at a constant temperature of 120℃ for 12 h.

[0038] (3) After the reaction in (2) is completed, the product is naturally cooled to room temperature. The product is separated by centrifugation at 3000 rpm for 10 minutes. The product is washed three times with anhydrous ethanol and deionized water (10 minutes each time).

[0039] (4) The product washed and separated in (3) was dried at 40°C and under vacuum of -0.2MPa for 12h to obtain V2CuS4 nanozyme powder.

[0040] (5) Mix glucose oxidase and V2CuS4 nanozyme at a mass ratio of 1:1 to obtain an enzyme mixture. Add methacrylamide silk fibroin at a mass ratio of 1:80 to obtain a mixed solution. Add photoinitiator at a mass percentage of 1:0.05% to obtain an aqueous solution of composite material. Stir magnetically at 15°C for 12 hours until completely dissolved to obtain an 8wt% aqueous solution of composite material.

[0041] (6) The aqueous solution of the composite material obtained in (5) is heated at 25°C with a frequency of 20kHz and a flow rate of 30W / cm. 2 Power density ultrasonic dispersion for 10 minutes forms an injectable three-dimensional network hydrogel with photoinitiated gelation properties, with a gelation time of 30 seconds. Example 2

[0042] A method for preparing reactive oxygen species scavenging diabetic bone repair hydrogel based on hydrothermal method, the specific steps of which are as follows: (1) Dissolve copper nitrate (Cu(NO3)2·3H2O), sodium vanadate (Na3VO4) and thiourea (CH4N2S) in 20 mL of deionized water at a Cu:V:S stoichiometric ratio of 1:2:6, stir until completely dissolved, and form a dark gray-blue precipitate.

[0043] (2) Transfer the dark brown precipitate in (1) to a polytetrafluoroethylene-lined high-pressure reactor (volume 100 mL, filling rate 40%) and react at a constant temperature of 180℃ for 24 h.

[0044] (3) After the reaction in (2) is completed, the product is naturally cooled to room temperature, and the product is separated by centrifugation at 6000 rpm for 20 minutes. The product is washed three times with anhydrous ethanol and deionized water (10 minutes each time).

[0045] (4) The product washed and separated in (3) was dried at 60 °C and -0.8 MPa vacuum for 18 h to obtain V2CuS4 nanozyme powder.

[0046] (5) Mix glucose oxidase and V2CuS4 nanozyme at a mass ratio of 1:5 to obtain an enzyme mixture. Add methacrylamide silk fibroin at a mass ratio of 1:100 to obtain a mixed solution. Add photoinitiator at a mass percentage of 1:0.2% to obtain an aqueous solution of composite material. Stir magnetically at 20°C for 12 hours until completely dissolved to obtain a 12wt% aqueous solution of composite material.

[0047] (6) The aqueous solution of the composite material obtained in (5) is subjected to a frequency of 60 kHz and a pressure of 70 W / cm at 20 °C. 2 Power density ultrasonic dispersion for 5 minutes forms an injectable three-dimensional network hydrogel with photo-initiated gelation properties, with a gelation time of 20 seconds. Example 3

[0048] A method for preparing reactive oxygen species scavenging diabetic bone repair hydrogel based on hydrothermal method, the specific steps of which are as follows: (1) Dissolve copper nitrate (Cu(NO3)2·3H2O), vanadium oxide (V2O5) and thiourea (CH4N2S) in 30mL of deionized water at a Cu:V:S stoichiometric ratio of 1:2:8, stir until completely dissolved, and form a dark gray-blue precipitate.

[0049] (2) Transfer the dark brown precipitate from (1) to a polytetrafluoroethylene-lined high-pressure reactor (volume 100 mL, filling rate 40%) and react at a constant temperature of 160℃ for 18 h.

[0050] (3) After the reaction in (2) is completed, the product is naturally cooled to room temperature. The product is separated by centrifugation at 4000 rpm for 30 minutes. The product is washed three times with anhydrous ethanol and deionized water (10 minutes each time).

[0051] (4) The product washed and separated in (3) was dried at 50°C and -0.5MPa vacuum for 12 h to obtain V2CuS4 nanozyme powder.

[0052] (5) Mix glucose oxidase and V2CuS4 nanozyme at a mass ratio of 1:3 to obtain an enzyme mixture. Add methacrylamide silk fibroin at a mass ratio of 1:90 to obtain a mixed solution. Add photoinitiator at a mass percentage of 1:0.1% to obtain an aqueous solution of composite material. Stir magnetically at 25°C for 12 hours until completely dissolved to obtain an 11wt% aqueous solution of composite material.

[0053] (6) The composite material dispersion obtained in (5) is subjected to a temperature of 15°C and a frequency of 40kHz and a concentration of 50W / cm. 2 Power density ultrasonic dispersion for 15 minutes forms an injectable three-dimensional network hydrogel with photo-initiated gelation properties, with a gelation time of 45 seconds. Example 4

[0054] A method for preparing reactive oxygen species scavenging diabetic bone repair hydrogel based on hydrothermal method, the specific steps of which are as follows: (1) Dissolve copper chloride (CuCl2), vanadium oxysulfate (VOSO4·2H2O) and sodium thiosulfate (Na2S2O3) in 20 mL of deionized water at a Cu:V:S stoichiometric ratio of 2:1:4, stir until completely dissolved, and form a dark gray-blue precipitate.

[0055] (2) Transfer the dark brown precipitate in (1) to a polytetrafluoroethylene-lined high-pressure reactor (volume 100 mL, filling rate 40%) and react at a constant temperature of 140℃ for 18 h.

[0056] (3) After the reaction in (2) is completed, the product is naturally cooled to room temperature. The product is separated by centrifugation at 2000 rpm for 20 minutes. The product is washed three times with anhydrous ethanol and deionized water (10 minutes each time).

[0057] (4) The product washed and separated in (3) was dried at 50°C and -0.5MPa vacuum for 6 hours to obtain V2CuS4 nanozyme powder.

[0058] (5) Mix glucose oxidase and V2CuS4 nanozyme at a mass ratio of 1:3 to obtain an enzyme mixture. Add methacrylamide silk fibroin at a mass ratio of 1:100 to obtain a mixed solution. Add photoinitiator at a mass percentage of 1:0.05% to obtain an aqueous solution of composite material. Stir magnetically at 30°C for 12 hours until completely dissolved to obtain a 10wt% aqueous solution of composite material.

[0059] (6) The composite material dispersion obtained in (5) is subjected to a temperature of 30°C and a frequency of 40kHz and a concentration of 50W / cm. 2 Power density ultrasonic dispersion for 10 minutes forms an injectable three-dimensional network hydrogel with photo-initiated gelation properties, with a gelation time of 40 seconds.

[0060] Performance testing: 1. Morphological characteristics of V2CuS4 nanozymes The V2CuS4 nanozyme prepared in Example 1 was subjected to SEM at an accelerating voltage of 5-20 kV and a resolution of <5 nm, as shown in the figure. Figure 1 As shown in AB, the nanozymes exhibit uniform, spherical particle shapes. EDS-Mapping results indicate that the vanadium, copper, and sulfur elements in the nanozymes are relatively evenly distributed on the surface. Figure 1 C), X-ray diffraction (XRD) was used to characterize the crystal structure of the V2CuS4 nanozyme, using Cu-Kα rays (λ=1.5406 Å), with a scan range of 10°–80° (2θ) and a step size of 0.02°. The results showed that the V2CuS4 nanozyme was in high agreement with the theoretical simulation parameters. Figure 2 This confirms the effectiveness of the synthesis method. Figure 1 The proportions of Cu, V, and S in the hydrogel, as indicated by the amount of substance added, may differ from the actual surface atomic distribution. This is because the sensitivity to light elements such as S is relatively low, resulting in smaller observed values, which is reasonable. 2. In vitro hydrogen peroxide decomposition experiment of V2CuS4 nanozyme The V2CuS4 nanozyme prepared in Example 1 exhibited significant peroxidase-like activity under physiological conditions, such as... Figure 3 As shown, a 200 μL reaction system (16 mM TMB (3,3',5,5'-tetramethylbenzidine), 1 M H₂O₂, 0.2 M acetate buffer (pH 4.0), 300 μg / mL V₂CuS₄ nanozyme) was constructed during the assay, and the enzyme activity of the V₂CuS₄ nanozyme was calculated according to the following formula:

[0061] in : Rate of change of absorbance : Reaction volume (mL) : Molar extinction coefficient of TMB oxidation products (39,000 M⁻¹cm⁻¹). : Cuvette optical path length (cm) Enzyme mass (mg).

[0062] 3. Rheological experiments on injectable diabetic osteochondral repair hydrogel containing V2CuS4 nanozyme like Figure 4 As shown, the rheological tests were conducted at 18-30℃ with shear rates ranging from 0.1 to 100 s⁻¹. -1 The viscosity changes were recorded, and the injectability was evaluated (the viscosity was considered extrudable when it dropped to <10 Pa·s). The viscosity with both storage modulus and loss modulus at gelation was <10 Pa·s, which verified the photogelation properties and extrudability of the hydrogel prepared in Example 1.

[0063] 4. Hemolysis experiment of injectable diabetic osteochondral repair hydrogel containing V2CuS4 nanozyme The hydrogel prepared in Example 1 showed a hemolysis rate of <1.5% after co-culturing with red blood cells for 3 hours (ISO 10993-4 standard threshold ≤5%). Figure 5 The CCK-8 assay further showed that bone marrow mesenchymal stem cells (BMSCs) cultured in the composite hydrogel for 7 days had a survival rate of ≥90%, and alkaline phosphatase (ALP) activity increased by 1.5-fold, confirming its combination of biocompatibility and osteogenic induction function. The calculation method for CCK-8 cell viability is as follows:

[0064] The ALP activity assay was performed as follows: cells were lysed, pNPP substrate (p-nitrophenyl phosphate) was added, the rate of yellow product formation was monitored at 405 nm, and the amount of enzyme required to hydrolyze 1 μmol of pNPP per minute was used as the activity unit.

[0065] This invention utilizes a hydrothermal method to construct V2CuS4 nanozyme ( Figure 1 The SEM morphology and EDS-mapping shown indicate uniformly shaped and elementally distributed flower-shaped particles, and the X-ray diffraction (XRD) pattern (…) Figure 2 The effectiveness of the synthesis method was confirmed in the in vitro hydrogen peroxide decomposition experiment. Figure 3The V2CuS4 nanozyme exhibited significant peroxidase-like activity under physiological conditions. A photogelation cascade system composed of the V2CuS4 nanozyme, glucose oxidase (GOx), and methacrylamide-modified silk fibroin hydrogel achieved efficient degradation of glucose and oxidatively active species through enzymatic cascade reactions in a simulated diabetic microenvironment. Rheological tests verified the photogelation properties and extrudability of the hydrogel. Figure 4 Hemolysis test () Figure 5 The study confirmed that the hemolysis rate of the material was <1.4% after 3 hours of co-culture with erythrocytes (ISO 10993-4 standard threshold ≤5%). Further CCK-8 assays showed that bone marrow mesenchymal stem cells (BMSCs) had a survival rate ≥90% after 7 days of culture in the composite hydrogel, and alkaline phosphatase (ALP) activity increased by 1.5 times, demonstrating its combination of biocompatibility and osteogenic induction function.

[0066] The application modes of the hydrogel for osteocartilage described in this invention are as follows: Figure 6 As shown; in a simulated diabetic microenvironment (glucose concentration 10mM, H2O2 concentration 100-500μM, Science Advances, 2021, 7, 35), the hydrogel prepared in this invention was tested using a kit (purchased from Nanjing Jiancheng) and found to have a scavenging rate of ≥95% for hydroxyl radicals and a scavenging rate of ≥85% for superoxide anions. This demonstrates that the hydrogel prepared in this invention has both good structural suitability and biological benefits.

[0067] In summary, the hydrogel obtained by this invention has good free radical scavenging efficacy, injectability, and biocompatibility, and also has efficient antioxidant and mineralization-promoting functions, making it applicable to osteochondral repair materials for diabetic patients.

[0068] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A method for preparing a hydrogel for repairing osteochondral bone in diabetic patients with reactive oxygen species scavenging using a hydrothermal method, characterized in that, Includes the following steps: (1) Copper salt, vanadium salt and sulfur source were mixed and dissolved in water, and after hydrothermal reaction, centrifuged, washed and dried to obtain V2CuS4 nanozyme; (2) Glucose oxidase and V2CuS4 nanozyme are mixed evenly to obtain an enzyme mixture. Methacrylamide silk fibroin is added to the enzyme mixture to obtain a mixed solution. A photoinitiator is added to the mixed solution to obtain a composite material aqueous solution. The composite material aqueous solution is ultrasonically dispersed to form an injectable three-dimensional network hydrogel with photogelation properties. The Cu:V:S stoichiometric ratio in the copper salt, vanadium salt and sulfur source mentioned in step (1) is 1:2:4; The V2CuS4 nanozyme mentioned in step (1) has a particle size of 20-50 nm and a specific surface area ≥80 m². 2 / g; In step (2), the mass ratio of glucose oxidase to V2CuS4 nanozyme in the enzyme mixture is 1:1-5; the mass ratio of enzyme mixture to methacrylamide silk fibroin in the mixed solution is 1:80-100; and the mass percentage of mixed solution and photoinitiator in the aqueous solution of the composite material is 1:0.05-0.2%.

2. The method for preparing reactive oxygen species scavenging diabetic osteochondral repair hydrogel based on hydrothermal method according to claim 1, characterized in that, The copper salt mentioned in step (1) is one of copper chloride, copper nitrate, and copper sulfate; the vanadium salt is one of vanadium oxysulfate, sodium vanadate, and vanadium oxide; and the sulfur source is one of sodium thiosulfate, thiourea, and sulfur powder.

3. The method for preparing reactive oxygen species scavenging diabetic osteochondral repair hydrogel based on hydrothermal method according to claim 1, characterized in that, The hydrothermal reaction conditions described in step (1) are constant temperature treatment at 120-180℃ for 12-24 hours.

4. The method for preparing reactive oxygen species scavenging diabetic osteochondral repair hydrogel based on hydrothermal method according to claim 1, characterized in that, In step (1), the centrifugation parameters are 2000-6000 rpm and 10-30 minutes; the washing is performed three times by alternating between anhydrous ethanol and deionized water.

5. The method for preparing reactive oxygen species scavenging diabetic osteochondral repair hydrogel based on hydrothermal method according to claim 1, characterized in that, The activity of glucose oxidase in step (2) is ≥150 U / mg.

6. The method for preparing reactive oxygen species scavenging diabetic osteochondral repair hydrogel based on hydrothermal method according to claim 1, characterized in that, The ultrasonic dispersion conditions described in step (2) are: 20-60 kHz, 30-70 W / cm². 2 The ultrasonic dispersion time is 5-15 minutes, and the mixing temperature is 15-30℃.

7. The application of the reactive oxygen species scavenging diabetic osteocartilage repair hydrogel prepared by the method according to any one of claims 1 to 6 in the preparation of diabetic osteocartilage repair materials.

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