Multifunctional hydrogel for anti-inflammatory and bone repair, preparation method and application thereof

Abstract

The application discloses a multifunctional hydrogel for anti-inflammation and bone repair, a preparation method and application, and the gel material is mainly composed of a temperature-sensitive hydrogel carrier material, a bone regeneration and repair growth factor and an anti-inflammatory bioactive nanomicelle, wherein the temperature-sensitive hydrogel carrier material is poloxamer P407; the bone regeneration and repair growth factor is human recombinant bone morphogenetic protein 9; and the anti-inflammatory bioactive nanomicelle is a functional amphiphilic polymer, which is composed of a hexachlorophosphazene mother nucleus, 1 hydrophilic polyethylene glycol chain and 5 hydrogen peroxide scavenging / responsive groups of phenylboronic acid ester derivatives connected in a 1:5 ratio through a step-by-step affinity substitution reaction, and the amphiphilic polymer can self-assemble into nanomicelles in an aqueous solution through intermolecular interaction. The multifunctional hydrogel is in a liquid state at low temperature, is injected locally in a gel state in the body, and can effectively regulate an inflammatory and oxidative stress microenvironment and promote the regeneration and repair of a defect bone when applied locally. The multifunctional hydrogel can be used for the local treatment of inflammatory bone defect diseases and has good biological safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, and particularly to a composition of a multifunctional hydrogel for anti-inflammatory and bone repair promotion, a preparation method thereof and application thereof in treating inflammatory bone defect diseases. BACKGROUND

[0002] For inflammatory bone defect diseases, persistent inflammatory response is the main reason for hindering bone tissue repair and regeneration. In the early stage of the disease, the inflammatory microenvironment recruits a large number of immune cells such as neutrophils, T cells, lymphocytes, etc. to the inflammation site, and secretes a large number of pro-inflammatory factors such as interleukin 6, interleukin 17, tumor necrosis factor alpha, etc. These inflammatory factors can activate macrophages to transform into osteoclasts while inhibiting the function of osteoblasts, leading to bone absorption and destruction [1-4]. In addition, the presence of inflammation causes mitochondrial damage in cells to produce excess reactive oxygen species, and the accumulation of reactive oxygen species further aggravates the inflammatory response by inducing oxidative stress damage, which eventually leads to further absorption and destruction of bone [5, 6]. Studies have shown that the recruitment, proliferation and osteogenic differentiation of endogenous stem cells play a crucial role in bone regeneration and repair [7, 8]. Under normal circumstances, stem cells around bone defects can differentiate into osteoblasts by receiving specific environmental signals, thereby achieving bone repair and regeneration [9-12]. However, the long-term inflammatory microenvironment greatly inhibits the cell activity of endogenous stem cells, severely hindering the regeneration and repair of bone tissue

[11] . Therefore, relieving the local inflammatory microenvironment is one of the main strategies for treating inflammatory bone defects and bone regeneration and repair.

[0003] In recent years, corticosteroids, non-steroidal anti-inflammatory drugs and antibiotics are often used to relieve local inflammatory response [13-15]. However, their long-term use is often accompanied by serious side effects such as osteoporosis, liver toxicity, gastrointestinal discomfort and antibiotic resistance, etc. In order to overcome these problems, in recent years, a variety of sustained-release systems based on biomaterials have been widely studied, which can control drug release to regulate local inflammation and immune response at the bone defect site, thereby relieving inflammation to effectively promote bone regeneration

[16] . However, in addition to the complex bone repair system, the drug delivery carrier itself or its degradation products may cause other side effects, such as some acidic products of ester degradation may exacerbate inflammation to some extent [17-20]. Therefore, it is still necessary to construct an effective and safe innovative strategy to regulate the pathological microenvironment of bone defects to promote new bone formation.

[0004] In addition to regulating the inflammatory microenvironment, the rational introduction of growth factors with bone regeneration / bone induction properties is also a major strategy for enhancing bone regeneration in the process of promoting the repair of bone defects. Currently, high concentrations of bone regeneration growth factors are introduced to achieve local bone regeneration and repair. However, the application of high concentrations of exogenous growth factors also has adverse effects. In order to achieve bone repair and regeneration, the addition of bone repair growth factors through covalent bonding with materials and other methods can also promote bone repair to some extent, but the effect is limited [21-23]. Therefore, it is still necessary to construct a delivery system with high growth factor loading capacity and local release effect and good tissue compatibility.

[0005] Based on the above results, from the two key points of inflammatory bone defect regeneration and repair, improving the local pathological microenvironment of bone defects and combining growth factors to enhance the osteogenic differentiation ability of local stem cells, the synergistic promotion of bone regeneration and repair is achieved. Therefore, the development of a system that integrates a nano-therapeutic strategy for effectively alleviating local inflammation and growth factors with bone regeneration / bone induction properties in a temperature-sensitive hydrogel, and has a simple synthesis method and good biological safety, is the focus of research in this field. SUMMARY

[0006] In view of the deficiencies of the prior art, the purpose of the present application is to provide an anti-inflammatory and bone repair multifunctional hydrogel, a preparation method and application. In addition, the present application also verifies the synergistic therapeutic effect of the multifunctional hydrogel in alleviating local inflammation and promoting bone regeneration at the defect site in the treatment of various inflammatory bone defect diseases.

[0007] To achieve the above purpose, the technical scheme adopted by the present application is an anti-inflammatory and bone repair multifunctional hydrogel, which comprises a temperature-sensitive gel carrier material, a bone regeneration and repair growth factor, and an anti-inflammatory bioactive nanomicelle. The concentration of the bone regeneration and repair growth factor in the gel is 0.2-5 μg / mL, the concentration of the anti-inflammatory bioactive nanomicelle in the gel is 1 mg / mL, and the particle size of the nanomicelle is about 30 nm.

[0008] The temperature-sensitive hydrogel is selected from poloxamer P407. The bone regeneration and repair growth factor is selected from human recombinant bone morphogenetic protein 9.

[0009] The chemical structure of the anti-inflammatory bioactive nanomicelle is

[0010]

[0011] Wherein:

[0012] R is a single-end amino polyethylene glycol R1 is a phenylboronic acid ester derivative containing an active group The total number of R and R1 substituents in hexachlorophosphazene is 6.

[0013] The application further provides a preparation method of the multifunctional hydrogel for anti-inflammatory and bone repair, comprising the following steps: first, preparing the anti-inflammatory bioactive nanodrug; under nitrogen protection, hexachlorophosphazene is gradually subjected to affinity substitution reaction with a single-end amino polyethylene glycol and a phenylboronic acid ester derivative containing an active group in an ultradry organic solvent, the time for each substitution reaction is 0.5-48 h, and the reaction temperature is-80-100 DEG C; after the reaction is completed, the reaction solution is concentrated by vacuumizing and rotary evaporation, and finally, is precipitated in diethyl ether, filtered and freeze-dried to obtain the anti-inflammatory bioactive nanodrug; then, poloxamer P407 powder is mixed with sterile water at 4 DEG C and magnetically stirred for 3 h until completely dissolved to obtain a poloxamer P407 hydrogel carrier material; human recombinant bone morphogenetic protein 9 and the anti-inflammatory bioactive nanodrug are added to the poloxamer P407 hydrogel at 4 DEG C, and magnetically stirred at 4 DEG C for 10 min, so that the anti-inflammatory bioactive nanodrug can self-assemble to form an anti-inflammatory bioactive nanomicelle, that is, the multifunctional hydrogel for anti-inflammatory and bone repair is obtained.

[0014] Specifically, the organic solvent is selected from dichloromethane, tetrahydrofuran or 1,4-dioxane; the molar ratio of hexachlorophosphazene to single-end amino polyethylene glycol is 1:1-3; the molar ratio of hexachlorophosphazene to the phenylboronic acid ester derivative containing an active group is 1:6; the concentration of the poloxamer P407 hydrogel is 19%; the human recombinant bone morphogenetic protein 9 storage solution is directly added into the hydrogel liquid as needed, and the final concentration is 0.2-5 mu g / mL; and the anti-inflammatory bioactive nanomicelle is directly added into the hydrogel liquid as needed, and the final concentration is 1 mg / mL.

[0015] The application further provides application of the multifunctional hydrogel for anti-inflammatory and bone repair in preparation of a medicine for treating inflammatory bone defect diseases.

[0016] The application takes mandibular bone defects and skull defects as models to verify the effect of the medicine of the application in treating related diseases.

[0017] The application has the following beneficial effects:

[0018] (1) The temperature-sensitive hydrogel carrier material can realize good liquid-solid phase conversion, that is, it is in a liquid state with good fluidity when injected at room temperature, and is in a gel solid state after being injected into local tissues, and can effectively serve as a carrier material to load drugs.

[0019] (2) The anti-inflammatory bioactive nanomicelle can effectively remove multiple active oxygen and inhibit secretion of inflammatory factors of inflammatory cells.

[0020] (3) The anti-inflammatory bioactive nanomicelles can have good biocompatibility, in vivo degradability, and degradation products have no toxic side effects on the body.

[0021] (4) The anti-inflammatory bioactive nanomicelles can promote the regeneration and repair of bone defects, and have no toxic side effects on the body.

[0022] (5) After local injection of the anti-inflammatory bioactive nanomicelles, the anti-inflammatory bioactive nanomicelles can respond to the inflammatory microenvironment of the defect site, thereby achieving a local anti-inflammatory effect. Compared with the control group without anti-inflammatory bioactive nanomicelles, the local secretion of inflammatory factors can be significantly reduced, which is beneficial to bone repair and regeneration.

[0023] (6) After local injection of the anti-inflammatory bioactive nanomicelles, human recombinant bone morphogenetic protein 9 enhances the local cell osteogenic differentiation capacity by slowly releasing from the gel, thereby promoting bone regeneration and repair. Compared with the control group without human recombinant bone morphogenetic protein 9, the bone defect repair and regeneration capacity can be significantly enhanced.

[0024] (7) The anti-inflammatory repair multifunctional hydrogel can quickly form a gel, slowly release anti-inflammatory bioactive nanomicelles and human recombinant bone morphogenetic protein 9 after local injection, thereby synergistically exerting the effects of regulating local inflammation and enhancing the local cell osteogenic differentiation capacity. The treatment effect of the multifunctional hydrogel on mandibular defects and skull defects is obviously superior to that of the control group treated with a single drug and the non-drug control group.

[0025] (8) The preparation method of the anti-inflammatory repair multifunctional hydrogel is relatively simple and low in cost, has good biological safety, and is easy to realize industrialization of the nanodrug combined gel material. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 TEM, size distribution and surface potential diagram of anti-inflammatory bioactive nanomicelles (PP5NMs);

[0027] Figure 2 Anti-inflammatory bioactive nanomicelles (PP5NMs) clear multiple ROS (A-C), inhibit macrophage secretion of inflammatory factors TNF-α, IL-6, IL-1β;

[0028] Figure 3 PXB hydrogel loaded with different concentrations of pro-osteogenic repair human recombinant bone morphogenetic protein 9 (BMP9) was locally injected into the mandibular defect site;

[0029] Figure 4Figure 1 shows the results of local injection of PXB hydrogel loaded with different concentrations of human recombinant bone morphogenetic protein 9 (BMP9) into the skull defect site;

[0030] Figure 5 Figure 5 shows the scanning electron microscopy (SEM) images of the powder of the product PXNB obtained in Example 5 after freeze-drying;

[0031] Figure 6 Figure 6 shows the in vitro erosion and quantitative analysis of PXNB in a 37°C environment;

[0032] Figure 7 Figure 7 shows the solid-liquid phase transition diagram (A) and the results of rheological testing (B) of PX, PXN, PXB and PXNB;

[0033] Figure 8 Figure 8 shows the BSA release curve and the PP5NM release curve in PXNB in a 37°C environment in vitro;

[0034] Figure 9 Figure 9 shows the fluorescence images and quantitative statistical results of the mandibular and skull in vivo after local injection of PXN gel loaded with Cy5-PP5NMs;

[0035] Figure 10 Figure 10 shows the levels of H2O2, IL-6 and TNF-a in the local bone tissue homogenate after treatment of mandibular defects with PX loaded with different drugs;

[0036] Figure 11 Figure 11 shows the results of local bone regeneration and repair after treatment of mandibular defects with PX loaded with different drugs;

[0037] Figure 12 Figure 12 shows the results of local bone regeneration and repair after treatment of skull defects with PX loaded with different drugs. DETAILED DESCRIPTION

[0038] The present application will now be described in further detail with reference to the specific embodiments. It should be understood that the embodiments of the present application are only used to explain the present application and not to limit the present application. Various substitutions and modifications can be made to the embodiments according to the ordinary technical knowledge and common practices in the art without departing from the technical idea of the present application, and all such substitutions and modifications should be included in the scope of the present application.

[0039] Example 1

[0040] Under nitrogen protection, 0.1 mmol hexachlorophosphazene was dissolved in 10 mL super dry tetrahydrofuran, 2.4 mmol triethylamine was added, and then 0.1 mmol mono-amino polyethylene glycol (molecular weight 2000) was reacted at -80°C for 6 h, followed by 0.6 mmol glycine-modified 4-hydroxymethylphenylboronic acid ester at 75°C for 48 h. After the reaction was completed, the generated salt was removed by vacuum filtration, the reaction solution was concentrated to 5 mL by rotary evaporation, and finally precipitated in 45 mL ice ethyl ether at -40°C. After filtration, the product, i.e. the amphiphilic polymer of hexachlorophosphazene modified with one polyethylene glycol and five glycine-modified 4-hydroxymethylphenylboronic acid esters, was obtained, and after freeze-drying, the anti-inflammatory bioactive nanomedicine PP5 was obtained.

[0041] Example 2

[0042] At 4°C, 190 mg of poloxamer P407 was mixed with 1 mL of sterile water, and magnetically stirred for 3 h until completely dissolved to obtain the liquid phase PX hydrogel.

[0043] Example 3

[0044] At 4°C, 190 mg of poloxamer P407 was mixed with 1 mL of sterile water, and magnetically stirred for 3 h until completely dissolved, and then 1 mg of anti-inflammatory bioactive nanomedicine was added and magnetically stirred for 10 min to obtain PXN containing 1 mg / mL of anti-inflammatory bioactive nanomicelles.

[0045] Example 4

[0046] At 4°C, 190 mg of poloxamer P407 was mixed with 1 mL of sterile water, and magnetically stirred for 3 h until completely dissolved, and then 0.2-5 μg of human recombinant bone morphogenetic protein 9 was added and magnetically stirred for 10 min to obtain PXB containing 0.2-5 μg / mL of human recombinant bone morphogenetic protein 9.

[0047] Example 5

[0048] At 4°C, 190 mg of poloxamer P407 was mixed with 1 mL of sterile water, and magnetically stirred for 3 h until completely dissolved, and then 1 mg of anti-inflammatory bioactive nanomedicine and 1 μg of human recombinant bone morphogenetic protein 9 were added and magnetically stirred for 10 min to obtain the multifunctional hydrogel PXNB containing 1 mg / mL of anti-inflammatory bioactive nanomicelles and 1 μg / mL of human recombinant bone morphogenetic protein 9.

[0049] Example 6

[0050] Under the protection of nitrogen, 0.1 mmol hexachlorophosphazene was dissolved in 10 mL super dry dichloromethane, 2.4 mmol triethylamine was added, and then 0.1 mmol mono-amine polyethylene glycol (molecular weight 2000) was reacted at -80°C for 0.5 h, followed by reaction with 0.6 mmol glycine 4-hydroxymethyl phenyl borate at 75°C for 48 h. After the reaction was completed, the generated salt was removed by vacuum filtration, the reaction solution was concentrated to 5 mL by rotary evaporation, and finally precipitated in 45 mL ice ethyl ether at -40°C. After filtration, the product, i.e. one polyethylene glycol and five glycine 4-hydroxymethyl phenyl borate modified hexachlorophosphazene amphiphilic polymer, was obtained. After freeze-drying, the anti-inflammatory bioactive nanomedicine PP5 was obtained.

[0051] At 4°C, 190 mg of poloxamer P407 was mixed with 1 mL of sterile water, and magnetically stirred for 3 h until completely dissolved. Then, 1 mg of anti-inflammatory bioactive nanomedicine and 0.2-5 μg of human recombinant bone morphogenetic protein 9 were added and magnetically stirred for 10 min to obtain the multifunctional hydrogel PXNB containing 1 mg / mL of anti-inflammatory bioactive nanomicelles and 0.2-5 μg / mL of human recombinant bone morphogenetic protein 9.

[0052] Example 7

[0053] Under the protection of nitrogen, 0.1 mmol hexachlorophosphazene was dissolved in 10 mL super dry 1,4-dioxane, 2.4 mmol triethylamine was added, and then 0.3 mmol mono-amine polyethylene glycol (molecular weight 2000) was reacted at -80°C for 6 h, followed by reaction with 0.6 mmol glycine 4-hydroxymethyl phenyl borate at 75°C for 48 h. After the reaction was completed, the generated salt was removed by vacuum filtration, the reaction solution was concentrated to 5 mL by rotary evaporation, and finally precipitated in 45 mL ice ethyl ether at -40°C. After filtration, the product, i.e. one polyethylene glycol and five glycine 4-hydroxymethyl phenyl borate modified hexachlorophosphazene amphiphilic polymer, was obtained. After freeze-drying, the anti-inflammatory bioactive nanomedicine PP5 was obtained.

[0054] At 4°C, 190 mg of poloxamer P407 was mixed with 1 mL of sterile water, and magnetically stirred for 3 h until completely dissolved. Then, 1 mg of anti-inflammatory bioactive nanomedicine and 0.2-5 μg of human recombinant bone morphogenetic protein 9 were added and magnetically stirred for 10 min to obtain the multifunctional hydrogel PXNB containing 1 mg / mL of anti-inflammatory bioactive nanomicelles and 0.2-5 μg / mL of human recombinant bone morphogenetic protein 9.

[0055] Figure 1The amphiphilic polymer synthesized in Example 1 is one polyethylene glycol and five glycine-modified 4-hydroxymethylphenylboronic acid ester products, which self-assemble into anti-inflammatory bioactive nanomicelles (PP5 NMs) in aqueous solution. Transmission electron microscopy scanning shows that PP5 NMs are spherical with clear outlines, with an average particle size of 29 ± 4 nm and a surface potential of 3.9 ± 0.7 mV.

[0056] Figure 2 The anti-inflammatory bioactive nanomicelles PP5 NMs can effectively scavenge multiple reactive oxygen species in vitro and inhibit the secretion of inflammatory factors by macrophages. This shows that PP5 NMs can effectively scavenge reactive oxygen species and also have good anti-inflammatory effects.

[0057] Figure 3 After the PXB hydrogel loaded with different concentrations of human recombinant bone morphogenetic protein 9 (BMP9) for bone repair was locally injected into the mandibular defect site, it had a good effect on promoting bone repair and regeneration in a concentration-dependent manner.

[0058] Figure 4 After the PXB hydrogel loaded with different concentrations of human recombinant bone morphogenetic protein 9 (BMP9) for bone repair was locally injected into the mandibular defect site, it had a good effect on promoting bone repair and regeneration in a concentration-dependent manner.

[0059] Figure 5 After the product PXNB obtained in Example 5 was freeze-dried, the powder was observed by electron microscopy scanning to show that the gel had a honeycomb network structure. Cy5-labeled anti-inflammatory bioactive nanomaterials (Cy5-PP5 NMs) and FITC-labeled fetal bovine serum protein (FITC-BSA) (simulating human recombinant bone morphogenetic protein 9) were used to prepare PXNB, and immunofluorescence detection confirmed that the anti-inflammatory bioactive nanomicelles and small molecule proteins could be uniformly distributed in the gel.

[0060] Figure 6 This is the slow erosion of 2 mL of PXNB in the presence of 2 mL of normal saline at 37°C.

[0061] Figure 7 This is the liquid-solid phase transition of PX, PXN, PXB, and PXNB at 4°C and 37°C, which confirms that they can form a gel at 37°C. Rheological detection further confirms that the gel material can change from a liquid phase to a solid phase in a low-temperature environment to a high-temperature environment, and it is in a stable gel solid phase state at 37°C.

[0062] Figure 8For the preparation of PXNB with Cy5-labeled anti-inflammatory bioactive nanomaterial (Cy5-PP5NMs) and fetal bovine serum protein (BSA), 2 mL of PXNB coexists with 2 mL of normal saline at 37℃ environment, and the gel continuously and slowly releases Cy5-PP5NMs and BSA.

[0063] Figure 9 For the preparation of PXN with Cy5-labeled anti-inflammatory bioactive nanomaterial (Cy5-PP5NMs), after the construction of the mandibular and skull defect model, 25 μL of PXN is injected into the defect site, and the Cy5 fluorescence intensity is detected by the in vivo imaging instrument to confirm that the hydrogel is slowly degraded locally.

[0064] Figure 10 After the local injection of PXN, PXB and PXNB hydrogels into the mandibular defect site, the group containing the anti-inflammatory bioactive nanomicelle group can significantly reduce the content level of local tissue oxidative stress indicators H2O2 and inflammatory factors IL-6 and TNF-α, and there is a statistical difference, which shows that the anti-inflammatory bioactive nanomicelle has a better inhibitory effect on inflammation.

[0065] Figure 11 After the local injection of PXN, PXB and PXNB hydrogels into the mandibular defect site, the PXNB multifunctional hydrogel has a better effect of promoting bone repair and regeneration compared with other groups, which confirms that its anti-inflammatory and pro-osteogenic synergistic treatment effect is the best.

[0066] Figure 12 After the local injection of PXN, PXB and PXNB hydrogels into the skull defect site, the PXNB multifunctional hydrogel has a better effect of promoting bone repair and regeneration compared with other groups, which confirms that its anti-inflammatory and pro-osteogenic synergistic treatment effect is the best.

[0067] That is to say, the anti-inflammatory and pro-bone repair multifunctional hydrogel can promote bone regeneration and repair in inflammatory bone defect diseases by exerting a synergistic effect, mainly through the synergistic effect of removing local reactive oxygen species and inflammatory factor secretion by the anti-inflammatory bioactive nanomicelle and enhancing the pro-osteogenic differentiation ability of local stem cells by human recombinant bone morphogenetic protein 9, so as to achieve a treatment effect. The treatment effect of mandibular defect and skull defect is better than that of simple anti-inflammatory treatment or the addition of pro-osteogenic growth factor treatment. In addition, the gel is used as a carrier, the drug is slowly released from the gel to continuously exert the drug effect, which effectively avoids the burst release of the drug, and at the same time ensures that a low concentration of drug dosage can exert a better treatment effect, and the biological safety is better.

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Claims

1. A multifunctional hydrogel with anti-inflammatory and bone-repairing properties, characterized in that: The product comprises a temperature-sensitive gel carrier material, a bone regeneration and repair growth factor, and anti-inflammatory bioactive nanomicelles. The concentration of the bone regeneration and repair growth factor in the gel is 0.2–5 μg / mL, and the concentration of the anti-inflammatory bioactive nanomicelles in the gel is 1 mg / mL. The temperature-sensitive gel carrier material is selected from poloxamer P407, and the concentration of the poloxamer P407 hydrogel is 19%. The bone regeneration and repair growth factor is selected from recombinant human bone morphogenetic protein 9. The chemical structure of the anti-inflammatory bioactive nanomicelles is as follows: Wherein: R is monoamino polyethylene glycol with a molecular weight of 2000, and R1 is glycined 4-hydroxymethylphenylboronic acid ester. In hexachlorophosphazene, the number of substituents of R and R1 are 1 and 5, respectively.

2. The multifunctional hydrogel with anti-inflammatory and bone-repairing properties according to claim 1, characterized in that: The anti-inflammatory bioactive nanomedicine was prepared by the following method: First, anti-inflammatory bioactive nanomedicine was prepared by stepwise affinity substitution reactions of hexachlorophosphazene with mono-amino-terminated polyethylene glycol and glycinated 4-hydroxymethylphenylboronic acid ester in ultra-dry organic solvents under nitrogen protection. The reaction time for each step was 0.5-48 h, and the reaction temperature was -80-100 ℃. After the reaction was completed, the reaction solution was concentrated by vacuum evaporation and rotary evaporation. Finally, the solution was precipitated in ice-cold ether, filtered, and freeze-dried to obtain the anti-inflammatory bioactive nanomedicine. Then, poloxamer P407 powder was mixed with sterile water at 4°C and magnetically stirred until completely dissolved to obtain poloxamer P407 hydrogel carrier material; human recombinant bone morphogenetic protein 9 and anti-inflammatory bioactive nanomedicine were added to poloxamer P407 hydrogel at 4°C and magnetically stirred at 4°C. In the solution, the anti-inflammatory bioactive nanomedicine can self-assemble into nanomicelles, thus obtaining a multifunctional hydrogel with anti-inflammatory and bone repair properties.

3. A method for preparing the anti-inflammatory and bone-repairing multifunctional hydrogel of claim 1, characterized in that, Includes the following steps: First, anti-inflammatory bioactive nanomedicines were prepared by stepwise affinity substitution reactions of hexachlorophosphazene with mono-amino-terminated polyethylene glycol and glycinated 4-hydroxymethylphenylboronic acid ester in ultra-dry organic solvents under nitrogen protection. The reaction time for each step ranged from 0.5 to 48 h, and the reaction temperature ranged from -80 to 100 °C. After the reaction was completed, the reaction solution was concentrated by vacuum evaporation and rotary evaporation. Finally, the solution was precipitated in ice-cold ether, filtered, and freeze-dried to obtain the anti-inflammatory bioactive nanomedicines. Then, poloxamer P407 powder was mixed with sterile water at 4°C and magnetically stirred until completely dissolved to obtain poloxamer P407 hydrogel carrier material; human recombinant bone morphogenetic protein 9 and anti-inflammatory bioactive nanomedicine were added to poloxamer P407 hydrogel at 4°C and magnetically stirred at 4°C. In the solution, the anti-inflammatory bioactive nanomedicine can self-assemble into nanomicelles, thus obtaining a multifunctional hydrogel with anti-inflammatory and bone repair properties.

4. The method for preparing a multifunctional hydrogel with anti-inflammatory and bone-repairing properties according to claim 3, characterized in that: The organic solvent is selected from dichloromethane, tetrahydrofuran, or 1,4-dioxane.

5. The method for preparing a multifunctional hydrogel with anti-inflammatory and bone-repairing properties according to claim 3, characterized in that: The molar ratio of hexachlorophosphazene to mono-amino polyethylene glycol is 1:1~3, and the molar ratio of hexachlorophosphazene to glycine-modified 4-hydroxymethylphenylboronic acid ester is 1:

6.

6. The use of the anti-inflammatory and bone-repairing multifunctional hydrogel of claim 1 or 2 in the preparation of a drug for treating inflammatory bone defects.

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