Preparation method of injectable temperature-sensitive antibacterial adhesive high-activity bone repair hydrogel

Abstract

The invention discloses a preparation method of injectable temperature-sensitive antibacterial adhesive high-activity bone repair hydrogel, and belongs to the technical field of medical treatment. Comprising the following steps: synthesizing ZIF-8-coated BMP-2 nanoparticles, preparing PGO-SF, preparing an ALG-POL copolymer, and finally preparing the PGO-SF / ALG-POL / ZIF-8-coated BMP-2 composite bone repair gel. The bone repair hydrogel prepared by the invention has multiple functions such as heat sensitivity, adhesion, photo-thermal antibiosis, good fluidity and injection stability, can be injected in a non-surgical form, self-cures and adheres to a bone defect part, and releases Zn < 2 + > through degradation of ZIF-8 to reduce peripheral inflammatory response and slowly releases BMP-2 protein to play a bone repair role at the same time; the bone repair efficiency is effectively improved, and the method has a wide application prospect in the field of bone defect repair.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of medical treatment, in particular to a preparation method of an injectable temperature-sensitive antibacterial adhesive high-activity bone repair hydrogel. BACKGROUND

[0002] With the increase of population aging, the number of bone defects caused by various bone diseases is increasing year by year, and the demand for bone defect repair materials is also growing. Compared with traditional open implantation surgery, minimally invasive surgery has the advantages of reducing trauma, reducing the risk of infection and fast recovery, and can be used as an effective way to reduce the surgical risk of the elderly. The injectable hydrogel has similar physical properties to human soft tissue, adjustable physical and chemical properties, and can be used as a soft tissue scaffold and injected into the bone defect repair site through a minimally invasive method. The current hydrogel material has poor mechanical properties, insufficient bone activity, no tissue adhesion, and antibacterial properties. Bone morphogenetic protein (BMP) is the only growth factor that can induce ectopic bone formation in vivo, and is widely used in the field of bone repair due to its good bone induction. However, the bone induction of BMP protein is affected by the short blood circulation half-life and poor stability, making it difficult to be used alone. Therefore, the injectable hydrogel is used as a carrier for the loading of BMP protein to enhance the stability of the protein and endow the hydrogel with bone activity. The existing hydrogel technology mainly involves single network, no tissue adhesion and antibacterial properties, which is prone to protein burst release, hydrogel displacement and postoperative infection. SUMMARY

[0003] Therefore, the application provides a preparation method of an injectable temperature-sensitive antibacterial adhesive high-activity bone repair hydrogel, which encapsulates BMP-2 protein in zeolite imidazole acid salt framework-8 (ZIF-8) and further combines the multifunctional injectable hydrogel. The composite hydrogel has thermal sensitivity, adhesion, photothermal antibacterial properties, good fluidity and injection stability, and can be injected and adhered to the bone defect site in a non-surgical form with shape adaptability.

[0004] To achieve the above-mentioned purposes, the technical scheme adopted by the application is as follows:

[0005] A preparation method of an injectable temperature-sensitive antibacterial adhesive high-activity bone repair hydrogel, specifically comprising the following steps:

[0006] Step 1, BMP-2 and 2-MIM are dissolved to obtain a 0.08%-0.12% BMP-2 and 22%-28% 2-MIM solution, 13.38% zinc nitrate is added to the BMP-2, 2-MIM mixed solution under stirring, the solution is incubated at 30 DEG C for 10 minutes, the incubated solution is centrifuged at 13000 rpm for 10 minutes to obtain a milky white ZIF-8@BMP-2 nanoparticle, then the unreacted monomers are removed by washing twice with deionized water, and the ZIF-8@BMP-2 nanoparticle is obtained by freeze-drying;

[0007] Step 2, SF is dissolved in water to obtain an 8%-12% SF solution, 0.5% graphene oxide and 0.5-1.0% dopamine hydrochloride are added under ice bath conditions, 1 mol / L sodium hydroxide is used to adjust the pH value of the solution to 8.5, and the solution is reacted in the dark for 6h, then the solution is washed and centrifuged 3-5 times with deionized water, and PGO-SF is obtained by freeze-drying overnight;

[0008] Step 3, sodium alginate and poloxamer are dissolved in water to obtain a 0.2-0.4% sodium alginate and 9-12% poloxamer mixed solution, 0.76% N-hydroxysuccinimide and 0.92% 1-ethyl-(3-dimethylaminopropyl) carbodiimide are added, and the solution is stirred and reacted at room temperature for 24h, then the solution is dialyzed against deionized water for 3 days using a dialysis membrane, and ALG-POL copolymer is obtained by freeze-drying;

[0009] Step 4, 7-9% PGO-SF, 5-8% ALG-POL, 0.5% hydrogen peroxide solution and 0.5% HRP solution are mixed uniformly, then 0.1 ZIF-8@QCT nanoparticles are added, and the mixture is uniformly mixed to obtain a PGO-SF / ALG-POL / ZIF-8@BMP-2 composite hydrogel.

[0010] Compared with the prior art, the present application has the following advantages:

[0011] 1, The injectable temperature-sensitive hydrogel of the present application is convenient for clinical operation, and the composite hydrogel can be injected into the bone defect site through a minimally invasive method, and the temperature-sensitive poloxamer gel realizes the sol-gel transition under the response of human body temperature.

[0012] 2, The composite gel modified by polydopamine has rich catechol and amino active groups, and provides good tissue adhesion, so that it can be stably adhered to the surface of bone tissue and reduce the adverse effects of material displacement on bone repair.

[0013] 3, The composite hydrogel has dual antibacterial function through the release of Zn 2+ and the photothermal effect of PGO, which can reduce the risk of post-implantation infection and improve the success rate of bone repair.

[0014] 4. After encapsulating BMP-2 in ZIF-8, the BMP-2 protein is doped in the double network hydrogel to realize long-acting slow release of the BMP-2 protein, continuously exert the bone induction effect, and effectively improve the efficiency of bone repair. BRIEF DESCRIPTION OF DRAWINGS

[0015] Figure 1 The graph of the cumulative release amount of the protein of the embodiment of the present application over time. DETAILED DESCRIPTION

[0016] The present application will be further described below in combination with the drawings and specific embodiments.

[0017] The present embodiment discloses a preparation method of an injectable composite hydrogel (PGO-SF / ALG-POL / ZIF-8@BMP-2). The composite hydrogel has thermal sensitivity, adhesion, photothermal antibacterial property, good fluidity and injection stability, can be injected in a non-surgical form and adhered to the bone defect site with shape self-adaptability. The basic components are polydopamine graphene oxide (PGO) modified silk fibroin (PGO-SF) and poloxamer grafted alginate (ALG-POL), wherein the PGO-SF and the ALG-POL form an injectable temperature-sensitive double network hydrogel (PGO-SF / ALG-POL) through covalent and physical crosslinking network, which can rapidly undergo sol-gel transition at a location close to physiological temperature and pH value after injection, and has shape self-adaptability.

[0018] The PGO-SF / ALG-POL / ZIF-8@BMP-2 composite hydrogel can stably adhere to the bone defect site, can release Zn 2+ , play an antibacterial and pro-osteogenesis / angiogenesis role, and act as a BMP-2 protein carrier, double-encapsulate the BMP-2 protein in the hydrogel, protect the biological activity of the BMP-2 protein, and realize sustained slow release. 2+ The PGO-SF / ALG-POL / ZIF-8@BMP-2 composite hydrogel can stably adhere to the bone defect site, can release Zn 2+ , play an antibacterial and pro-osteogenesis / angiogenesis role, and act as a BMP-2 protein carrier, double-encapsulate the BMP-2 protein in the hydrogel, protect the biological activity of the BMP-2 protein, and realize sustained slow release.

[0019] The mechanism of the injectable composite hydrogel disclosed in the present application is as follows: graphene oxide is doped in dopamine hydrochloride, and is modified on the surface of silk fibroin by the method of self-polymerization of dopamine hydrochloride oxidation under alkaline conditions. The poloxamer grafted sodium alginate component is prepared by condensation reaction and cross-linking between the primary amine molecules of sodium alginate and poloxamer activated by 1-ethyl-(3-dimethylaminopropyl) carbodiimide / N-hydroxysuccinimide. The ZIF-8@BMP-2 nanoparticles are prepared by doping BMP-2 protein, and the ZIF-8@BMP-2 nanoparticles are doped into the hydrogel system. The PGO-SF hydrogel component is prepared by enzyme catalytic cross-linking method, and the poloxamer grafted sodium alginate component is cross-linked by temperature-sensitive sol-gel transformation method. The mixed components are injected into the human bone tissue site, and the PGO-SF / ALG-POL / ZIF-8@BMP-2 composite hydrogel is synthesized by responding to the human body temperature.

[0020] The specific implementation method steps of the present application are as follows:

[0021] (1) Synthesis of ZIF-8@BMP-2 nanoparticles: dissolve BMP-2 (bone morphogenetic protein-2) and 2-methylimidazole (2-MIM) to obtain a 0.08%-0.12% (w / v) BMP-2 and 22-28% (w / v) 2-MIM solution, and add 13.38% (w / v) zinc nitrate to the above BMP-2 / 2-MIM mixed solution under vigorous stirring, and incubate the solution at 30°C for 10 minutes, centrifuge the incubated solution at 13000 rpm for 10 minutes to obtain white ZIF-8@BMP-2 nanoparticles, then wash twice with deionized water to remove unreacted monomers, and freeze-dry to obtain ZIF-8@BMP-2 nanoparticles.

[0022] (2) Preparation of PGO-SF: dissolve silk fibroin (SF) in water to obtain an 8%-12% (w / v) SF solution, and add 0.5% (w / v) graphene oxide and 0.5-1.0% (w / v) dopamine hydrochloride under ice bath conditions, respectively, adjust the pH value of the solution to 8.5 using 1 mol / L sodium hydroxide, avoid light reaction for 6h, wash and centrifuge the reaction product with deionized water for 3-5 times, and freeze-dry overnight to obtain PGO-SF.

[0023] (3) Preparation of ALG-POL copolymer: Sodium alginate and poloxamer were dissolved in water to obtain a mixed solution of 0.2-0.4% (w / v) sodium alginate and 9-12% (w / v) poloxamer, and 0.76% (w / v) N-hydroxysuccinimide (NHS) and 0.92% (w / v) 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) were added, and the reaction was stirred at room temperature for 24 h. After the reaction was completed, the deionized water was dialyzed for 3 days using a dialysis membrane (MWCF: 12-15k), and then freeze-dried to obtain the ALG-POL copolymer.

[0024] (4) Preparation of PGO-SF / ALG-POL / ZIF-8@BMP-2 composite hydrogel: 7-9% (w / v) PGO-SF, 5-8% (w / v) ALG-POL, 0.5% (w / v) hydrogen peroxide solution (500 mmol / L) and 0.5% (w / v) HRP solution (1000 U / mL) were mixed uniformly, and then 0.1 (w / v) ZIF-8@QCT nanoparticles were added, and the mixture was uniformly mixed to obtain the PGO-SF / ALG-POL / ZIF-8@BMP-2 composite hydrogel.

[0025] Example 1:

[0026] (1) Synthesis of ZIF-8@BMP-2 nanoparticles: BMP-2 and 2-methylimidazole (2-MIM) were dissolved to obtain a 0.08% (w / v) BMP-2 and 22% (w / v) 2-MIM solution. Under vigorous stirring, 13.38% (w / v) zinc nitrate was added to the above BMP-2 / 2-MIM mixed solution, and the solution was incubated at 30°C for 10 minutes. The incubated solution was centrifuged at 13000 rpm for 10 minutes to obtain white ZIF-8@BMP-2 nanoparticles, which were then washed twice with deionized water to remove unreacted monomers, and then freeze-dried to obtain ZIF-8@BMP-2 nanoparticles.

[0027] (2) Preparation of PGO-SF: Silk fibroin (SF) was dissolved in water to obtain an 8% (w / v) SF solution. Under ice bath conditions, 0.5% (w / v) graphene oxide and 0.5% (w / v) dopamine hydrochloride were added, and the pH value of the solution was adjusted to 8.5 using 1 mol / L sodium hydroxide. The reaction was carried out in the dark for 6 h. After the reaction was completed, the solution was washed and centrifuged 3-5 times with deionized water, and then freeze-dried overnight to obtain PGO-SF.

[0028] (3) Preparation of ALG-POL copolymer: Sodium alginate and poloxamer were dissolved in water to obtain a mixed solution of 0.28% (w / v) sodium alginate and 9% (w / v) poloxamer, and 0.76% (w / v) N-hydroxysuccinimide (NHS) and 0.92% (w / v) 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) were added, and the reaction was stirred at room temperature for 24 h. After the reaction was completed, the deionized water was dialyzed for 3 days using a dialysis membrane (MWCF: 12-15k), and freeze-drying was performed to obtain the ALG-POL copolymer.

[0029] (4) Preparation of PGO-SF / ALG-POL / ZIF-8@BMP-2 composite hydrogel: 8% (w / v) PGO-SF, 6% (w / v) ALG-POL, 0.5% (w / v) hydrogen peroxide solution (500 mmol / L) and 0.5% (w / v) HRP solution (1000 U / mL) were mixed uniformly, and then 0.1 (w / v) ZIF-8@QCT nanoparticles were added, and the mixture was uniformly mixed to obtain the PGO-SF / ALG-POL / ZIF-8@BMP-2 composite hydrogel.

[0030] Example 2:

[0031] (1) Synthesis of ZIF-8@BMP-2 nanoparticles: BMP-2 and 2-methylimidazole (2-MIM) were dissolved to obtain a 0.1% (w / v) BMP-2 and 25% (w / v) 2-MIM solution. Under vigorous stirring, 13.38% (w / v) zinc nitrate was added to the above BMP-2 / 2-MIM mixed solution, and the solution was incubated at 30°C for 10 minutes. The incubated solution was centrifuged at 13000 rpm for 10 minutes to obtain white ZIF-8@BMP-2 nanoparticles, which were then washed twice with deionized water to remove unreacted monomers, and freeze-dried to obtain ZIF-8@BMP-2 nanoparticles.

[0032] (2) Preparation of PGO-SF: Silk fibroin (SF) was dissolved in water to obtain an 8% (w / v) SF solution. Under ice bath conditions, 0.5% (w / v) graphene oxide and 0.6% (w / v) dopamine hydrochloride were added, and the pH value of the solution was adjusted to 8.5 using 1 mol / L sodium hydroxide. The reaction was carried out in the dark for 6 h. After the reaction was completed, the solution was washed and centrifuged 3-5 times with deionized water, and freeze-dried overnight to obtain PGO-SF.

[0033] (3) Preparation of ALG-POL copolymer: Sodium alginate and poloxamer were dissolved in water to obtain a mixed solution of 0.44% (w / v) sodium alginate and 9.5% (w / v) poloxamer, and 0.76% (w / v) N-hydroxysuccinimide (NHS) and 0.92% (w / v) 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) were added, and the reaction was stirred at room temperature for 24 h. After the reaction was completed, the deionized water was dialyzed for 3 days using a dialysis membrane (MWCF: 12-15k), and then freeze-dried to obtain the ALG-POL copolymer.

[0034] (4) Preparation of PGO-SF / ALG-POL / ZIF-8@BMP-2 composite hydrogel: 8.5% (w / v) PGO-SF, 5% (w / v) ALG-POL, 0.5% (w / v) hydrogen peroxide solution (500 mmol / L) and 0.5% (w / v) HRP solution (1000 U / mL) were mixed uniformly, and then 0.1 (w / v) ZIF-8@QCT nanoparticles were added, and the mixture was uniformly mixed to obtain the PGO-SF / ALG-POL / ZIF-8@BMP-2 composite hydrogel.

[0035] Example 3:

[0036] (1) Synthesis of ZIF-8@BMP-2 nanoparticles: BMP-2 and 2-methylimidazole (2-MIM) were dissolved to obtain a 0.08% (w / v) BMP-2 and 25% (w / v) 2-MIM solution. Under vigorous stirring, 13.38% (w / v) zinc nitrate was added to the above BMP-2 / 2-MIM mixed solution, and the solution was incubated at 30°C for 10 minutes. The incubated solution was centrifuged at 13000 rpm for 10 minutes to obtain white ZIF-8@BMP-2 nanoparticles, which were then washed twice with deionized water to remove unreacted monomers, and then freeze-dried to obtain ZIF-8@BMP-2 nanoparticles.

[0037] (2) Preparation of PGO-SF: Silk fibroin (SF) was dissolved in water to obtain a 6.5% (w / v) SF solution. Under ice bath conditions, 0.5% (w / v) graphene oxide and 0.5% (w / v) dopamine hydrochloride were added, and the pH value of the solution was adjusted to 8.5 using 1 mol / L sodium hydroxide. The reaction was carried out in the dark for 6 h. After the reaction was completed, the solution was washed and centrifuged 3-5 times with deionized water, and then freeze-dried overnight to obtain PGO-SF.

[0038] (3) Preparation of ALG-POL copolymer: Sodium alginate and poloxamer were dissolved in water to obtain a mixed solution of 0.35% (w / v) sodium alginate and 9% (w / v) poloxamer, and 0.76% (w / v) N-hydroxysuccinimide (NHS) and 0.92% (w / v) 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) were added, and the reaction was stirred at room temperature for 24 h. After the reaction was completed, the deionized water was dialyzed for 3 days using a dialysis membrane (MWCF: 12-15k), and freeze-drying was performed to obtain the ALG-POL copolymer.

[0039] (4) Preparation of PGO-SF / ALG-POL / ZIF-8@BMP-2 composite hydrogel: 7.5% (w / v) PGO-SF, 8% (w / v) ALG-POL, 0.5% (w / v) hydrogen peroxide solution (500 mmol / L), and 0.5% (w / v) HRP solution (1000 U / mL) were mixed uniformly, and then 0.1 (w / v) ZIF-8@QCT nanoparticles were added, and the mixture was uniformly mixed to obtain the PGO-SF / ALG-POL / ZIF-8@BMP-2 composite hydrogel.

[0040] Example 4:

[0041] (1) Synthesis of ZIF-8@BMP-2 nanoparticles: BMP-2 and 2-methylimidazole (2-MIM) were dissolved to obtain a 0.12% (w / v) BMP-2 and 28% (w / v) 2-MIM solution. Under vigorous stirring, 13.38% (w / v) zinc nitrate was added to the above BMP-2 / 2-MIM mixed solution, and the solution was incubated at 30°C for 10 minutes. The incubated solution was centrifuged at 13000 rpm for 10 minutes to obtain white ZIF-8@BMP-2 nanoparticles, which were then washed twice with deionized water to remove unreacted monomers, and freeze-dried to obtain ZIF-8@BMP-2 nanoparticles.

[0042] (2) Preparation of PGO-SF: Silk fibroin (SF) was dissolved in water to obtain a 10% (w / v) SF solution. Under ice bath conditions, 0.5% (w / v) graphene oxide and 1.0% (w / v) dopamine hydrochloride were added, and the pH value of the solution was adjusted to 8.5 using 1 mol / L sodium hydroxide. The reaction was carried out in the dark for 6 h. After the reaction was completed, the solution was washed and centrifuged 3-5 times with deionized water, and freeze-dried overnight to obtain PGO-SF.

[0043] (3) Preparation of ALG-POL copolymer: Sodium alginate and poloxamer were dissolved in water to obtain a mixed solution of 0.35% (w / v) sodium alginate and 10% (w / v) poloxamer, and 0.76% (w / v) N-hydroxysuccinimide (NHS) and 0.92% (w / v) 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) were added, and the reaction was stirred at room temperature for 24 h. After the reaction was completed, the deionized water was dialyzed for 3 days using a dialysis membrane (MWCF: 12-15k), and freeze-drying was performed to obtain the ALG-POL copolymer.

[0044] (4) Preparation of PGO-SF / ALG-POL / ZIF-8@BMP-2 composite hydrogel: 8% (w / v) PGO-SF, 7.5% (w / v) ALG-POL, 0.5% (w / v) hydrogen peroxide solution (500 mmol / L), and 0.5% (w / v) HRP solution (1000 U / mL) were mixed uniformly, and then 0.1 (w / v) ZIF-8@QCT nanoparticles were added, and the mixture was uniformly mixed to obtain the PGO-SF / ALG-POL / ZIF-8@BMP-2 composite hydrogel.

[0045] Example 5:

[0046] (1) Synthesis of ZIF-8@BMP-2 nanoparticles: BMP-2 and 2-methylimidazole (2-MIM) were dissolved to obtain a 0.12% (w / v) BMP-2 and 24% (w / v) 2-MIM solution. Under vigorous stirring, 13.38% (w / v) zinc nitrate was added to the above BMP-2 / 2-MIM mixed solution, and the solution was incubated at 30°C for 10 minutes. The incubated solution was centrifuged at 13000 rpm for 10 minutes to obtain white ZIF-8@BMP-2 nanoparticles, which were then washed twice with deionized water to remove unreacted monomers, and freeze-dried to obtain ZIF-8@BMP-2 nanoparticles.

[0047] (2) Preparation of PGO-SF: Silk fibroin (SF) was dissolved in water to obtain an 11% (w / v) SF solution. Under ice bath conditions, 0.5% (w / v) graphene oxide and 0.8% (w / v) dopamine hydrochloride were added, and the pH value of the solution was adjusted to 8.5 using 1 mol / L sodium hydroxide. The reaction was carried out in the dark for 6 h. After the reaction was completed, the solution was washed and centrifuged 3-5 times with deionized water, and freeze-dried overnight to obtain PGO-SF.

[0048] (3) Preparation of ALG-POL copolymer: sodium alginate and poloxamer were dissolved in water to obtain a mixed solution of 0.34% (w / v) sodium alginate and 10.5% (w / v) poloxamer, 0.76% (w / v) N-hydroxysuccinimide (NHS) and 0.92% (w / v) 1-ethyl-(3-dimethylaminopropyl) carbodiimide (EDC) were added, and the reaction was stirred at room temperature for 24 h. After the reaction was completed, the deionized water was dialyzed for 3 days using a dialysis membrane (MWCF: 12-15k), and then freeze-dried to obtain the ALG-POL copolymer.

[0049] (4) Preparation of PGO-SF / ALG-POL / ZIF-8@BMP-2 composite hydrogel: 7.5% (w / v) PGO-SF, 6.8% (w / v) ALG-POL, 0.5% (w / v) hydrogen peroxide solution (500 mmol / L) and 0.5% (w / v) HRP solution (1000 U / mL) were mixed uniformly, and then 0.1 (w / v) ZIF-8@QCT nanoparticles were added. After mixing uniformly, the PGO-SF / ALG-POL / ZIF-8@BMP-2 composite hydrogel was obtained.

[0050] The performance tests of the composite hydrogels with different parameter ratios in the five examples are as follows:

[0051] (1) Verification of sol-gel transition time at 37°C: the five kinds of hydrogels described in the examples were placed in different vials, and the vials were inverted every 15 s in a 37°C water bath. The flowability of the liquid in the vial was observed, and the time when the solution stopped flowing was recorded as the sol-gel conversion time.

[0052] (2) Verification of the adhesion performance of injectable hydrogel: a clean and smooth steel plate was selected to contact the left and right sides of the hydrogel, which was fixed on the base with adhesive tape. The release liner of the hydrogel was removed, and a 5*5 cm test sample was attached to the steel plate and fixed. The test sample was peeled off at 90°C using a hydrogel strength tester, and the adhesion strength was determined by the force required at the adhesion interface.

[0053] (3) Verification of the photothermal performance of the injectable hydrogel: a 5*5 cm test hydrogel sample was placed under a stable sunlight (1Kw.m -2 ) for 30 min, and an infrared thermal imager was used to capture the surface temperature of the hydrogel. The photothermal conversion ability of the material surface was verified by the temperature.

[0054] (4) Verification of the antibacterial effect of the injectable hydrogel: a sterile cotton swab was used to dip a solution with a concentration of 5*10 5 cfu / ml-5*10 6The test bacteria suspension of cfu / ml was evenly smeared on the surface of the nutrient agar medium plate for 3 times. Cover the plate and dry it at room temperature for 5 min. The 5*5mm water gel disc with a thickness of less than 4mm was sterilized by ultraviolet and placed in the bacteria plate, and placed in a 38℃ incubator for 18h. The diameter of the inhibition ring was measured with a caliper, and the inhibition ring greater than 7mm was determined to have antibacterial effect.

[0055] (5) Verify the protein release effect of the injectable hydrogel: ELISA protein determination kit (96 kit) is used to determine the protein release kinetics curve. The sample to be tested is completely immersed in 1ml PBS buffer solution, and is placed in a constant temperature shaking incubator at 37℃ and 80ppm. Simulate the in vitro release environment, take out the sample at the preset time point and place it in the same dose of PBS, and freeze the PBS protein release liquid at each time point in the-20℃ refrigerator. After all the release liquids at all time points are collected, they are uniformly detected. First, a standard curve is established by using the standard protein solution provided by the ELISA kit, and the specific reference is Figure 1 , and then the release concentration of rhBMP-2 in the sample is calculated based on the curve.

[0056] The performance of each of the 5 examples is shown in Table 1:

[0057] Table 1

[0058]

[0059] The verification conclusion of the example is:

[0060] Through the performance test related to the example, the sol-gel transition time of the injectable gel is appropriately increased by increasing the content of the poloxamer component to optimize the sol-gel transition time of the injectable gel; the bone tissue adhesion performance of the injectable gel is increased by increasing the content of the polydopamine component to optimize the bone tissue adhesion performance; the photothermal performance of the injectable gel is increased by increasing the content of the polydopamine-doped graphene oxide component to optimize the photothermal conversion performance of the injectable hydrogel; the photothermal antibacterial and Zn 2+ release antibacterial performance is verified by the inhibition ring experiment; the BMP-2 protein release performance of the injectable gel is optimized by increasing the content of the ZIF-8@BMP-2 nanoparticles in combination with the composite gel component.

[0061] The present application is not limited to the above examples, and more other equivalent examples can be included without departing from the concept of the present application, and the scope of the present application is determined by the scope of the appended claims.

Claims

1. A method for preparing an injectable, temperature-sensitive, antibacterial, adhesive, highly active bone repair hydrogel, characterized in that, Specifically, the following steps are included: Step 1: Dissolve BMP-2 and 2-MIM to obtain a 0.08%-0.12% w / v BMP-2 and 22%-28% w / v 2-MIM solution. Add 13.38% w / v zinc nitrate to the BMP-2 and 2-MIM mixed solution with stirring. Incubate the solution at 30°C for 10 minutes. Centrifuge the incubated solution at 13000 rpm for 10 minutes to obtain milky white ZIF-8@BMP-2 nanoparticles. Wash twice with deionized water to remove unreacted monomers. After freeze-drying, obtain ZIF-8@BMP-2 nanoparticles. Step 2: Dissolve SF in water to obtain an 8%-12% w / v SF solution. Add 0.5% w / v graphene oxide and 0.5-1.0% w / v dopamine hydrochloride under ice bath conditions. Adjust the pH of the solution to 8.5 with 1 mol / L sodium hydroxide. React in the dark for 6 hours. After the reaction is completed, wash with deionized water and centrifuge 3-5 times. Freeze-dry overnight to obtain PGO-SF. Step 3: Dissolve sodium alginate and poloxamer in water to obtain a mixed solution of 0.2-0.4% w / v sodium alginate and 9-12% w / v poloxamer. Add 0.76% w / v N-hydroxysuccinimide and 0.92% w / v 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC). Stir and react at room temperature for 24 h. After the reaction is completed, dialyze deionized water through a dialysis membrane for 3 days and freeze-dry to obtain ALG-POL copolymer. Step 4: Mix 7-9% w / v PGO-SF, 5-8% w / v ALG-POL, 0.5% w / v hydrogen peroxide solution and 0.5% w / v HRP solution evenly, then add 0.1 w / v ZIF-8@QCT nanoparticles and mix evenly to obtain PGO-SF / ALG-POL / ZIF-8@BMP-2 composite hydrogel.

2. The method for preparing an injectable, thermosensitive, antibacterial, adhesive, highly active bone repair hydrogel according to claim 1, characterized in that, In step 4, the hydrogen peroxide solution is 500 mmol / L and the HRP solution is 1000 U / mL.

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