Bone paste for repairing bone trauma and preparation method thereof

Abstract

The invention relates to bone paste for repairing bone wounds and a preparation method of the bone paste, and belongs to the technical field of biomedical material manufacturing. The bone paste is prepared from nanoscale calcium-phosphorus biphase ceramic, biomimetic mineralized collagen fiber, biodegradable polymer, modified hyaluronic acid, beta-cyclodextrin coated recombinant human bone morphogenetic protein-7, an angiogenesis accelerant and an immunoregulation additive according to a certain proportion. The nano-scale calcium-phosphorus dual-phase ceramic is hydroxyapatite and beta-tricalcium phosphate dual-phase ceramic; the biomimetic mineralized collagenous fiber is prepared by mixing type I collagen freeze-dried fiber with hydroxypropyl chitosan and performing mineralization; the modified hyaluronic acid is hyaluronic acid-sulfydryl and hyaluronic acid-hydrazide; the angiogenesis accelerant is a polylactic acid sustained release microsphere loaded with tanshinone and hydroxyl polyethylene glycol biotin; the immunoregulation additive is disodium glycyrrhizinate, lentinan and astragalus polysaccharide. The bone paste can effectively improve the bone induction capacity, promote vascularization generation and reduce the immunogenicity risk.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical material manufacturing, and particularly relates to a bone paste for repairing bone trauma and a preparation method thereof. Background Art

[0002] Bone trauma is a common type of injury clinically. From simple fractures to complex bone defects, it seriously affects the quality of life of patients. Fracture is the most common bone trauma. For simple fractures, traditional treatment methods such as plaster fixation and splint fixation restrict the movement of the fracture site through external fixation and utilize the body's own repair mechanism to achieve fracture healing. However, for complex traumas such as comminuted fractures and bone defects, these methods are difficult to achieve ideal effects. In the case of comminuted fractures, the number of fracture fragments is large and small, making it difficult to achieve accurate reduction and stable fixation through traditional fixation methods, which easily leads to complications such as delayed fracture healing and malunion. Bone defects are usually caused by trauma, tumor resection, infection, etc. When the bone defect range exceeds a certain critical value, the body's own repair ability is difficult to completely fill the defect area, resulting in long-term nonunion, which seriously affects limb function.

[0003] Under such clinical needs, bone transplantation has become one of the important means for treating complex bone traumas. Autologous bone transplantation has always been regarded as the "gold standard" for bone defect repair. It has good osteoconductivity, osteoinductivity, and biocompatibility, and there is no immune rejection reaction. However, the source of autologous bone is limited. Obtaining autologous bone will bring additional trauma and pain to patients and may also cause donor site complications. With the rapid development of materials science and biomedical engineering, bone substitute materials have gradually become a research hotspot, and bone paste is one of them. Bone paste is a bone substitute material with special morphology and properties, usually composed of one or more components such as bioactive ceramics, biodegradable polymers, growth factors, and cells.

[0004] The emergence of bone paste has brought new hope to the field of bone trauma repair. Based on overcoming the limitations of traditional treatment methods and materials, it demonstrates unique advantages and application prospects. However, there are still the following problems in the process of bone paste for bone trauma repair: (1) Limited osteoinductive ability: The types of growth factors or bioactive substances contained in some bone pastes are different, resulting in insufficient osteoinductive ability, which cannot fully stimulate the activity and proliferation of osteoblasts, affecting the speed and quality of new bone formation.

[0005] (2) Insufficient vascularization: Bone paste performs poorly in promoting angiogenesis. The growth of new bone tissue requires sufficient blood supply to provide nutrients and oxygen. Insufficient vascularization will limit the application of bone paste in the repair of large bone defects.

[0006] (3) Immunogenic risk: Although most of the components of bone paste have good biocompatibility, some biomaterials or added growth factors may still have potential immunogenicity, which may trigger an immune response in the body, affecting the treatment effect and the recovery of patients.

[0007] Therefore, it is necessary to continuously improve and innovate bone paste materials, enhance bone induction ability, promote angiogenesis, and improve biocompatibility to bring better treatment effects and quality of life to patients with bone trauma. Summary of the Invention

[0008] Aiming at the problems of limited bone induction ability, insufficient vascularization, and immunogenic risk existing in the existing bone paste for bone trauma repair, the present invention provides a bone paste for bone trauma repair and its preparation method. The bone paste is prepared by mixing nano calcium phosphate biphasic ceramics, biomimetic mineralized collagen fibers, biodegradable polymers, modified hyaluronic acid, β-cyclodextrin-coated recombinant human bone morphogenetic protein-7, angiogenesis promoters, and immunomodulatory additives in a certain proportion, which can effectively improve bone induction ability, promote angiogenesis, and reduce immunogenic risk. The specific technical solution is as follows: A bone paste for bone trauma repair, comprising the following raw materials in parts by mass: 30 to 40 parts of nano calcium phosphate biphasic ceramics, 15 to 20 parts of biomimetic mineralized collagen fibers, 20 to 30 parts of biodegradable polymers, 2 to 4 parts of modified hyaluronic acid, 0.5 to 1 part of β-cyclodextrin-coated recombinant human bone morphogenetic protein-7, 1 to 3 parts of angiogenesis promoters, and 0.5 to 1 part of immunomodulatory additives.

[0009] In the above bone paste, the nano calcium phosphate biphasic ceramics are hydroxyapatite and β-tricalcium phosphate biphasic ceramics; the preparation method of the nano calcium phosphate biphasic ceramics comprises the following steps: by mass ratio, calcium nitrate: diammonium hydrogen phosphate = (1.8 to 2.0): (1 to 1.2), dissolve calcium nitrate and diammonium hydrogen phosphate in 3 to 5 times the mass of deionized water respectively to obtain a calcium nitrate solution and a diammonium hydrogen phosphate solution. Under stirring, add the diammonium hydrogen phosphate solution to the calcium nitrate solution, and at the same time adjust the pH value to 10 to 11 with ammonia water, the reaction temperature is 60°C to 70°C, the reaction time is 2h to 3h, to generate a calcium phosphate precursor precipitate. Centrifuge and separate, wash the precipitate with deionized water and absolute ethanol 2 to 3 times respectively, dry in vacuum at 60°C to 80°C for 12h to 24h, and then raise the temperature to 900°C to 1000°C at a heating rate of 4°C / min to 6°C / min, calcine for 2h to 3h, and pulverize to obtain nano calcium phosphate biphasic ceramics with a particle size below 200nm.

[0010] In the above bone mud, the preparation method of the biomimetic mineralized collagen fiber comprises the following steps: preparing a collagen solution containing 2 wt% - 3 wt% type I collagen freeze-dried fibers and 2 wt% - 3 wt% hydroxypropyl chitosan, with the solvent being a 0.5 mol / L - 1 mol / L acetic acid solution; preparing a mineralization solution containing calcium ions and phosphate ions, where the calcium ion concentration is 1 mmol / L - 2 mmol / L, the phosphate ion concentration is 0.6 mmol / L - 1.2 mmol / L, and the pH value is adjusted to 7.2 - 7.4; under the stirring state, adding the mineralization solution to the collagen solution according to the volume ratio of mineralization solution:collagen solution = 1:(1.5 - 2), stirring and reacting for 2 h - 4 h, transferring the reaction solution to a dialysis bag, and dialyzing in deionized water for 3 h - 5 h; freeze-drying the dialyzed product to obtain the biomimetic mineralized collagen fiber.

[0011] In the above bone mud, the biodegradable polymer is polycaprolactone.

[0012] In the above bone mud, the component mass ratio of the modified hyaluronic acid is hyaluronic acid-thiol (HA-SH):hyaluronic acid-hydrazide (HA-Hy) = (2 - 3):(0.5 - 1).

[0013] In the above bone mud, the preparation method of β-cyclodextrin-coated recombinant human bone morphogenetic protein-7 comprises the following steps: preparing a 5 wt% - 6 wt% β-cyclodextrin aqueous solution at 50 °C - 60 °C; adding recombinant human bone morphogenetic protein-7 to a phosphate buffer solution (PBS, pH 7.2 - 7.4) according to a concentration of 0.5 mg / mL - 1 mg / mL to prepare a protein solution; under the stirring condition, adding the protein solution to the β-cyclodextrin aqueous solution according to the volume ratio of protein solution:β-cyclodextrin aqueous solution = (1 - 1.5):(20 - 25), stirring and reacting for 2 h - 4 h, centrifuging and separating, taking the precipitate, and freeze-drying to obtain β-cyclodextrin-coated recombinant human bone morphogenetic protein-7.

[0014] In the above bone mud, the angiogenesis promoter is a polylactic acid sustained-release microsphere loaded with tanshinone and hydroxy poly(ethylene glycol) biotin (HO-PEG-Biotin).

[0015] In the above bone paste, the preparation method of the angiogenesis promoter comprises the following steps: dissolving polylactic acid in ethyl acetate to prepare a polylactic acid solution with a mass concentration of 5% - 8%; preparing a mixed solution containing 0.4 mg / mL - 0.5 mg / mL of tanshinone and 0.1 mg / mL - 0.2 mg / mL of hydroxy polyethylene glycol biotin (HO-PEG-Biotin), with the solvent being phosphate buffer solution (PBS, pH 7.2 - 7.4); preparing an aqueous solution of polyvinyl alcohol (PVA) with a mass concentration of 2% - 3%; adding the mixed solution to the polylactic acid solution at a ratio of mixed solution: polylactic acid solution = 1: (3 - 5), and homogenizing at 10000 r / min - 15000 r / min to form a water-in-oil (W / O) type emulsion; then adding the emulsion to the aqueous solution of polyvinyl alcohol at a ratio of emulsion: aqueous solution of polyvinyl alcohol = 1: (2 - 3), and stirring under negative pressure at 30°C - 35°C for 3 h - 4 h to fully volatilize ethyl acetate, and solidifying polylactic acid to form microspheres, centrifuging and separating, taking the precipitate and washing it repeatedly with deionized water for 4 - 6 times, and freeze-drying to obtain the angiogenesis promoter.

[0016] In the above bone paste, the mass ratio of the components of the immunomodulatory additive is disodium glycyrrhizinate: lentinan: astragalus polysaccharide = (5 - 8): (2 - 3): (2 - 3).

[0017] The preparation method of the above bone paste for repairing bone trauma comprises the following steps: mixing nanoscale calcium phosphate biphasic ceramics, biomimetic mineralized collagen fibers, modified hyaluronic acid, β-cyclodextrin-coated recombinant human bone morphogenetic protein-7, angiogenesis promoter, and immunomodulatory additive by mass parts to obtain a mixture; heating and melting the biodegradable polymer, mixing it with the mixture, and adjusting the fluidity with physiological saline to obtain the bone paste.

[0018] A bone paste for repairing bone trauma and its preparation method provided by the present invention have the following beneficial effects: First, the nanoscale calcium phosphate biphasic ceramics are calcined at high temperature to endow them with appropriate crystallinity and mechanical properties. The nanoscale size greatly increases the specific surface area, enhances the bioactivity and osteoconductivity. Similar to the inorganic components of human bone tissue, after being implanted into the body, it serves as a template for new bone growth, guides osteoblasts to adhere and proliferate on its surface, promotes the deposition of calcium and phosphate ions, accelerates the formation of new bone, provides a solid basic scaffold for bone repair, and synergizes with other components to provide initial mechanical support for the overall bone paste.

[0019] II. Biomimetic Mineralized Collagen Fibers Type I collagen is the main organic component of natural bone, providing cell recognition sites and promoting cell adhesion; hydroxypropyl chitosan enhances the stability, antibacterial property, etc. of collagen fiber mineralization. After mineralization, the mechanical properties of the fibers are improved, and it mimics the natural bone structure, working in synergy with nanoscale calcium phosphate biphasic ceramics to jointly provide structural support and biological signals for new bone growth. At the same time, its biological activity is beneficial to the growth of cells in bone paste, promoting bone induction.

[0020] III. Biodegradable Polymer (Polycaprolactone) It has good biocompatibility, can be slowly degraded in the body, and the degradation products are non-toxic. It encapsulates other components, providing plasticity and a certain initial mechanical strength for bone paste. As it degrades, it creates space for new bone growth, and maintains the structural stability of bone paste during the degradation process, working in synergy with nanoscale calcium phosphate biphasic ceramics and biomimetic mineralized collagen fibers to maintain the shape of bone paste.

[0021] IV. Hyaluronic Acid It has good moisturizing and biocompatibility by itself. After modification with mercapto and hydrazide groups, its interaction with other components such as biomimetic mineralized collagen fibers and polycaprolactone is enhanced. It can form a hydrated environment inside bone paste, facilitating the diffusion of nutrients and metabolites, and can also regulate cell-cell interactions, working in synergy with other components to maintain the stability of the microstructure of bone paste and promote cell activities within bone paste.

[0022] V. Recombinant Human Bone Morphogenetic Protein - 7 It is a strong bone induction factor, which can stimulate mesenchymal stem cells to differentiate into osteoblasts and promote new bone formation. The hydrophobic cavity of β-cyclodextrin entraps the protein, protecting it from degradation by proteases in the body, achieving slow release and continuously exerting the bone induction effect. Working in synergy with nanoscale calcium phosphate biphasic ceramics and biomimetic mineralized collagen fibers, it greatly enhances the bone induction ability and accelerates bone repair.

[0023] VI. Tanshinone It promotes the proliferation and migration of vascular endothelial cells and induces angiogenesis; hydroxy polyethyleneglycol biotin enhances biocompatibility and stability. When the two are used in combination, it can further improve the vascular proliferation ability. Hydroxy polyethyleneglycol biotin can improve the physicochemical properties of the local microenvironment through its hydrophilicity, regulating the osmotic pressure, pH value, etc. of the microenvironment, creating more favorable conditions for tanshinone to exert its angiogenesis-inducing effect, and is also beneficial to the proliferation, migration and differentiation of vascular endothelial cells, indirectly promoting angiogenesis. Poly(lactic acid) microspheres degrade slowly and continuously release tanshinone, providing sufficient blood supply for bone tissue growth. Working in synergy with other components, it creates good blood circulation conditions for bone paste to repair large bone defects and promotes the overall bone repair process.

[0024] VII. Disodium Glycyrrhizinate It has anti-inflammatory effects and regulates immunity; lentinan and astragalus polysaccharide regulate the activity of immune cells and enhance the body's immune regulation ability. The three work in synergy to reduce the immunogenicity of bone paste, reduce immune rejection reactions, create an immune-friendly environment for bone repair, enable other components to play better roles, and promote bone tissue regeneration.

[0025] In summary, the present invention prepares bone mud by mixing nano calcium phosphate biphasic ceramics, biomimetic mineralized collagen fibers, biodegradable polymers, modified hyaluronic acid, β-cyclodextrin-coated recombinant human bone morphogenetic protein-7, angiogenesis promoters, and immunomodulatory additives in a certain proportion, which can effectively improve bone induction ability, promote angiogenesis, and reduce the risk of immunogenicity. The preparation methods of each component are simple and feasible, and it has good practical value. Detailed implementation manners

[0026] The present invention will be further described below in combination with specific implementation cases, but the present invention is not limited to these embodiments.

[0027] Example 1: A bone mud for repairing bone trauma, comprising the following raw materials in parts by mass: 35 parts of nano calcium phosphate biphasic ceramics, 18 parts of biomimetic mineralized collagen fibers, 25 parts of biodegradable polymers, 3 parts of modified hyaluronic acid, 0.7 part of β-cyclodextrin-coated recombinant human bone morphogenetic protein-7, 2 parts of angiogenesis promoters, and 0.8 part of immunomodulatory additives. The biodegradable polymer is polycaprolactone. The component mass ratio of the modified hyaluronic acid is hyaluronic acid-thiol (HA-SH): hyaluronic acid-hydrazide (HA-Hy) = 2.5:0.8. The angiogenesis promoter is a polylactic acid sustained-release microsphere loaded with tanshinone and hydroxy polyethylene glycol biotin (HO-PEG-Biotin). The component mass ratio of the immunomodulatory additive is disodium glycyrrhizinate: lentinan: astragalus polysaccharide = 7:2.8:2.5.

[0028] Among them, the nano calcium phosphate biphasic ceramics are hydroxyapatite and β-tricalcium phosphate biphasic ceramics; the preparation method of the nano calcium phosphate biphasic ceramics includes the following steps: according to the mass ratio, calcium nitrate: diammonium hydrogen phosphate = 1.9:1, dissolve calcium nitrate and diammonium hydrogen phosphate in 4 times the mass of deionized water respectively to obtain a calcium nitrate solution and a diammonium hydrogen phosphate solution. Under the stirring state of 200 r / min, add the diammonium hydrogen phosphate solution to the calcium nitrate solution, and at the same time adjust the pH value to 10.5 with ammonia water. The reaction temperature is 65 °C, the reaction time is 2.5 h, to generate a calcium phosphate precursor precipitate. Centrifuge at 7000 r / min for 25 min, wash the precipitate twice with deionized water, and then wash it three times with absolute ethanol. Dry it in vacuum at 70 °C for 18 h, and then raise the temperature to 950 °C at a heating rate of 5 °C / min, calcine for 2.5 h, and pulverize to obtain nano calcium phosphate biphasic ceramics with a size below 200 nm.

[0029] Among them, the preparation method of the biomimetic mineralized collagen fiber includes the following steps: Prepare a collagen solution containing 2.5 wt% freeze-dried type I collagen fibers and 2.5 wt% hydroxypropyl chitosan, with the solvent being 0.8 mol / L acetic acid solution; Prepare a mineralization solution containing calcium ions and phosphate ions using calcium chloride and sodium dihydrogen phosphate, where the calcium ion concentration is 1.5 mmol / L, the phosphate ion concentration is 1 mmol / L, and the pH value is adjusted to 7.4; Under the stirring state of 200 r / min, add the mineralization solution to the collagen solution according to the volume ratio of mineralization solution:collagen solution = 1:1.8, continue stirring and reacting for 3 h, transfer the reaction solution to a dialysis bag, and dialyze in deionized water for 4 h; Freeze-dry the dialyzed product to obtain the biomimetic mineralized collagen fiber.

[0030] Among them, the preparation method of β-cyclodextrin-coated recombinant human bone morphogenetic protein-7 includes the following steps: Prepare a 5.5 wt% aqueous β-cyclodextrin solution at 55 °C; Add recombinant human bone morphogenetic protein-7 to phosphate buffer solution (PBS, pH 7.4) at a concentration of 0.8 mg / mL to prepare a protein solution; Under the stirring condition of 70 r / min, add the protein solution to the aqueous β-cyclodextrin solution according to the volume ratio of protein solution:aqueous β-cyclodextrin solution = 1.2:23, continue stirring and reacting for 3 h, cool down to 8 °C, centrifuge at 7000 r / min for 25 min, take the precipitate, and freeze-dry to obtain β-cyclodextrin-coated recombinant human bone morphogenetic protein-7.

[0031] Among them, the preparation method of the angiogenesis promoter includes the following steps: Dissolve polylactic acid in ethyl acetate to prepare a 7% polylactic acid solution by mass concentration; Prepare a mixed solution containing 0.45 mg / mL of tanshinone and 0.15 mg / mL of hydroxy polyethylene glycol biotin (HO-PEG-Biotin), with the solvent being phosphate buffer solution (PBS, pH 7.4); Prepare a 2.5% aqueous solution of polyvinyl alcohol (PVA) by mass concentration; Add the mixed solution to the polylactic acid solution according to the ratio of mixed solution:polylactic acid solution = 1:4, and homogenize at 12000 r / min to form a water-in-oil (W / O) type emulsion; Then add the emulsion to the aqueous PVA solution according to the ratio of emulsion:aqueous PVA solution = 1:2.5, stir under negative pressure at 32 °C and 4000 r / min for 3.5 h to fully volatilize ethyl acetate, and the polylactic acid solidifies to form microspheres. Centrifuge at 7000 r / min for 25 min, take the precipitate, wash it repeatedly with deionized water 5 times, and freeze-dry to obtain the angiogenesis promoter.

[0032] The above method for preparing bone paste for bone trauma repair comprises the following steps: According to mass parts, nanoscale calcium phosphate biphasic ceramics, biomimetic mineralized collagen fibers, modified hyaluronic acid, β-cyclodextrin-coated recombinant human bone morphogenetic protein-7, angiogenesis promoter, and immunomodulatory additive are uniformly mixed to obtain a mixture; polycaprolactone is heated to melt at 65 °C, mixed with the mixture, and the fluidity is adjusted with physiological saline to obtain bone paste.

[0033] Example 2: A bone paste for bone trauma repair, comprising the following raw materials in mass parts: 30 parts of nanoscale calcium phosphate biphasic ceramics, 15 parts of biomimetic mineralized collagen fibers, 20 parts of biodegradable polymer, 2 parts of modified hyaluronic acid, 0.5 part of β-cyclodextrin-coated recombinant human bone morphogenetic protein-7, 1 part of angiogenesis promoter, and 0.5 part of immunomodulatory additive. The biodegradable polymer is polycaprolactone. The component mass ratio of the modified hyaluronic acid is hyaluronic acid-thiol (HA-SH): hyaluronic acid-hydrazide (HA-Hy) = 2:0.5. The angiogenesis promoter is a polylactic acid sustained-release microsphere loaded with tanshinone and hydroxy polyethylene glycol biotin (HO-PEG-Biotin). The component mass ratio of the immunomodulatory additive is disodium glycyrrhizinate: lentinan: astragalus polysaccharide = 5:2:2.

[0034] Among them, the nanoscale calcium phosphate biphasic ceramics are hydroxyapatite and β-tricalcium phosphate biphasic ceramics; the preparation method of the nanoscale calcium phosphate biphasic ceramics comprises the following steps: According to the mass ratio, calcium nitrate: diammonium hydrogen phosphate = 1.8:1.1, calcium nitrate and diammonium hydrogen phosphate are respectively dissolved in 3 times the mass of deionized water to obtain a calcium nitrate solution and a diammonium hydrogen phosphate solution. Under the stirring state of 100 r / min, the diammonium hydrogen phosphate solution is added to the calcium nitrate solution, and at the same time, the pH value is adjusted to 10 with ammonia water. The reaction temperature is 60 °C, the reaction time is 2 h, a calcium phosphate precursor precipitate is generated, centrifuged and separated at 6000 r / min for 20 min, the precipitate is washed 2 times with deionized water and then 2 times with absolute ethanol, vacuum dried at 60 °C for 12 h, and then heated to 900 °C at a heating rate of 4 °C / min, calcined for 2 h, and pulverized to obtain nanoscale calcium phosphate biphasic ceramics with a particle size below 200 nm.

[0035] Among them, the preparation method of the biomimetic mineralized collagen fiber includes the following steps: Prepare a collagen solution containing 2 wt% type I collagen freeze-dried fibers and 2 wt% hydroxypropyl chitosan, and the solvent is 0.5 mol / L acetic acid solution; Prepare a mineralization solution containing calcium ions and phosphate ions with calcium chloride and sodium dihydrogen phosphate, where the calcium ion concentration is 1 mmol / L, the phosphate ion concentration is 0.6 mmol / L, and the pH value is adjusted to 7.2; Under the stirring state of 150 r / min, add the mineralization solution to the collagen solution according to the volume ratio of mineralization solution:collagen solution = 1:1.5, continue stirring and reacting for 2 h, transfer the reaction solution to a dialysis bag, and dialyze in deionized water for 3 h; Freeze-dry the dialyzed product to obtain the biomimetic mineralized collagen fiber.

[0036] Among them, the preparation method of β-cyclodextrin-coated recombinant human bone morphogenetic protein-7 includes the following steps: Prepare a 5 wt% β-cyclodextrin aqueous solution at 50 °C; Add recombinant human bone morphogenetic protein-7 to phosphate buffer solution (PBS, pH 7.2) at a concentration of 0.5 mg / mL to prepare a protein solution; Under the stirring condition of 60 r / min, add the protein solution to the β-cyclodextrin aqueous solution according to the volume ratio of protein solution:β-cyclodextrin aqueous solution = 1:20, continue stirring and reacting for 2 h, cool down to 5 °C, centrifuge at 6000 r / min for 20 min, take the precipitate, and freeze-dry to obtain β-cyclodextrin-coated recombinant human bone morphogenetic protein-7.

[0037] Among them, the preparation method of the angiogenesis promoter includes the following steps: Dissolve polylactic acid in ethyl acetate to prepare a polylactic acid solution with a mass concentration of 5%; Prepare a mixed solution containing 0.4 mg / mL of tanshinone and 0.1 mg / mL of hydroxy poly(ethylene glycol) biotin (HO-PEG-Biotin), and the solvent is phosphate buffer solution (PBS, pH 7.2); Prepare a 2 wt% polyvinyl alcohol (PVA) aqueous solution; According to the volume ratio of mixed solution:polylactic acid solution = 1:3, add the mixed solution to the polylactic acid solution, and homogenize at 10000 r / min to form a water-in-oil (W / O) type emulsion; Then, according to the volume ratio of emulsion:polyvinyl alcohol aqueous solution = 1:2, add the emulsion to the polyvinyl alcohol aqueous solution, stir under negative pressure at 30 °C and 3000 r / min for 3 h to fully volatilize ethyl acetate, and the polylactic acid solidifies to form microspheres. Centrifuge at 6000 r / min for 20 min, take the precipitate, wash it repeatedly with deionized water 4 times, and freeze-dry to obtain the angiogenesis promoter.

[0038] The above method for preparing bone mud for bone trauma repair includes the following steps: By mass fraction, uniformly mix nano calcium phosphate biphasic ceramics, biomimetic mineralized collagen fibers, modified hyaluronic acid, β-cyclodextrin-coated recombinant human bone morphogenetic protein-7, angiogenesis promoter, and immune regulation additive to obtain a mixture; heat polycaprolactone to melt at 60 °C, mix it with the mixture, and adjust the fluidity with physiological saline to obtain bone mud.

[0039] Example 3: A bone mud for bone trauma repair, comprising the following raw materials by mass fraction: 40 parts of nano calcium phosphate biphasic ceramics, 20 parts of biomimetic mineralized collagen fibers, 30 parts of biodegradable polymer, 4 parts of modified hyaluronic acid, 1 part of β-cyclodextrin-coated recombinant human bone morphogenetic protein-7, 3 parts of angiogenesis promoter, and 1 part of immune regulation additive. The biodegradable polymer is polycaprolactone. The mass ratio of the components of the modified hyaluronic acid is hyaluronic acid-thiol (HA-SH): hyaluronic acid-hydrazide (HA-Hy) = 3:1. The angiogenesis promoter is a polylactic acid sustained-release microsphere loaded with tanshinone and hydroxy polyethylene glycol biotin (HO-PEG-Biotin). The mass ratio of the components of the immune regulation additive is disodium glycyrrhizinate: lentinan: astragalus polysaccharide = 8:3:3.

[0040] Among them, the nano calcium phosphate biphasic ceramics are hydroxyapatite and β-tricalcium phosphate biphasic ceramics; the preparation method of the nano calcium phosphate biphasic ceramics includes the following steps: By mass ratio, calcium nitrate: diammonium hydrogen phosphate = 2.0:1.2, dissolve calcium nitrate and diammonium hydrogen phosphate in 5 times the mass of deionized water respectively to obtain a calcium nitrate solution and a diammonium hydrogen phosphate solution. Under the stirring state of 300 r / min, add the diammonium hydrogen phosphate solution to the calcium nitrate solution, and at the same time adjust the pH value to 11 with ammonia water. The reaction temperature is 70 °C, and the reaction time is 3 h to generate a calcium phosphate precursor precipitate. Centrifuge at 8000 r / min for 30 min, wash the precipitate 3 times with deionized water, and then wash it 3 times with absolute ethanol. Dry it in vacuum at 80 °C for 24 h, and then heat it to 1000 °C at a heating rate of 6 °C / min, calcine it for 3 h, and pulverize it to obtain nano calcium phosphate biphasic ceramics with a particle size below 200 nm.

[0041] Among them, the preparation method of the biomimetic mineralized collagen fiber includes the following steps: Prepare a collagen solution, the collagen solution contains 3 wt% freeze-dried fibers of type I collagen and 3 wt% hydroxypropyl chitosan, and the solvent is 1 mol / L acetic acid solution; Prepare a mineralization solution containing calcium ions and phosphate ions with calcium chloride and sodium dihydrogen phosphate, where the calcium ion concentration is 2 mmol / L, the phosphate ion concentration is 1.2 mmol / L, and the pH value is adjusted to 7.3; Under the stirring state of 250 r / min, add the mineralization solution to the collagen solution according to the volume ratio of mineralization solution:collagen solution = 1:2, continue to stir and react for 4 h, transfer the reaction solution to a dialysis bag, and dialyze in deionized water for 5 h; Freeze-dry the dialyzed product to obtain the biomimetic mineralized collagen fiber.

[0042] Among them, the preparation method of β-cyclodextrin-coated recombinant human bone morphogenetic protein-7 includes the following steps: Prepare a 6 wt% aqueous solution of β-cyclodextrin at 60 °C; Add recombinant human bone morphogenetic protein-7 to phosphate buffer solution (PBS, pH 7.3) at a concentration of 1 mg / mL to prepare a protein solution; Under the stirring condition of 80 r / min, add the protein solution to the aqueous solution of β-cyclodextrin according to the volume ratio of protein solution:aqueous solution of β-cyclodextrin = 1.5:25, continue to stir and react for 4 h, cool down to 10 °C, centrifuge at 8000 r / min for 30 min, take the precipitate, and freeze-dry to obtain β-cyclodextrin-coated recombinant human bone morphogenetic protein-7.

[0043] Among them, the preparation method of the angiogenesis promoter includes the following steps: Dissolve polylactic acid in ethyl acetate to prepare a polylactic acid solution with a mass concentration of 8%; Prepare a mixed solution, the mixed solution contains 0.5 mg / mL of tanshinone and 0.2 mg / mL of hydroxy poly(ethylene glycol) biotin (HO-PEG-Biotin), and the solvent is phosphate buffer solution (PBS, pH 7.3); Prepare an aqueous solution of polyvinyl alcohol (PVA) with a mass concentration of 3%; According to the volume ratio of mixed solution:polylactic acid solution = 1:5, add the mixed solution to the polylactic acid solution, and homogenize at 15000 r / min to form a water-in-oil (W / O) type emulsion; Then, according to the volume ratio of emulsion:aqueous solution of polyvinyl alcohol = 1:3, add the emulsion to the aqueous solution of polyvinyl alcohol, and stir under reduced pressure at 35 °C and 5000 r / min for 4 h to fully volatilize ethyl acetate, and polylactic acid solidifies to form microspheres, centrifuge at 8000 r / min for 30 min, take the precipitate, wash it repeatedly with deionized water 6 times, and freeze-dry to obtain the angiogenesis promoter.

[0044] The preparation method of the above-mentioned bone trauma repair bone paste includes the following steps: by mass parts, nanoscale calcium phosphate biphasic ceramics, biomimetic mineralized collagen fibers, modified hyaluronic acid, β-cyclodextrin-coated recombinant human bone morphogenetic protein-7, angiogenesis promoter, and immunomodulatory additive are uniformly mixed to obtain a mixture; polycaprolactone is heated to 70°C and melted, mixed with the mixture, and the fluidity is adjusted with physiological saline to obtain bone paste.

[0045] In the above-mentioned examples, the freeze-dried fibers of type I collagen are from Guangzhou Beijia Health Medical Technology Co., Ltd. Hydroxypropyl chitosan is from Shaanxi Qionghua Biotechnology Co., Ltd. with a purity of 99%. β-cyclodextrin is hydroxypropyl β-cyclodextrin from Shaanxi Lantai Bioengineering Co., Ltd. with a purity of 99%. Recombinant human bone morphogenetic protein-7 is from Wuhan AmyJet Scientific Co., Ltd., model CYT-333. Polylactic acid is from Guangzhou Chuling Biomedical Technology Co., Ltd., model jymz005. Tanshinone is from Xi'an Tongze Biotechnology Co., Ltd. containing 20% tanshinone IIA. Hydroxy poly(ethylene glycol) biotin (HO-PEG-Biotin) is from Beijing Jingpi Technology Co., Ltd., model JP0135-2k. Polyvinyl alcohol (PVA) is from Guangzhou Zhonggao Chemical Industry Co., Ltd., model 217. Polycaprolactone is from Xi'an Ryan Biotechnology Co., Ltd., model rn0086. Hyaluronic acid-thiol (HA-SH) is from Beijing Jingpi Technology Co., Ltd., model JPHA-3-10k. Hyaluronic acid-hydrazide (HA-Hy) is from Beijing Jingpi Technology Co., Ltd., model JPHA-5-10k. Disodium glycyrrhizinate is from Hubei Yamaide Biomedical Co., Ltd., pharmaceutical excipient grade. Lentinan is from Shanxi Shangnuoda Biotechnology Co., Ltd., first grade product. Astragalus polysaccharide is from Xi'an Wanfang Biotechnology Co., Ltd., model H-3.

[0046] Comparative Example 1 In the preparation method of the biomimetic mineralized collagen fibers, hydroxypropyl chitosan is not added; other parameters and methods are the same as in Example 1.

[0047] Comparative Example 2 In the bone paste, modified hyaluronic acid is replaced with hyaluronic acid (hyaluronic acid model JPHA-1-10k from the same manufacturer); other parameters and methods are the same as in Example 1.

[0048] Comparative Example 3 In the bone paste, all of the modified hyaluronic acid is replaced with hyaluronic acid-thiol; other parameters and methods are the same as in Example 1.

[0049] Comparative Example 4 In the bone paste, for recombinant human bone morphogenetic protein-7, β-cyclodextrin coating is not used, and other parameters and methods are the same as in Example 1.

[0050] Comparative Example 5 In the preparation method of the angiogenesis promoter, the addition amounts of tanshinone and hydroxy polyethyleneglycol biotin are interchanged; other parameters and methods are the same as those in Example 1.

[0051] Comparative Example 6 In the preparation method of the angiogenesis promoter, hydroxy polyethyleneglycol biotin is not added; other parameters and methods are the same as those in Example 1.

[0052] Comparative Example 7 In the immunomodulatory additive, lentinan is replaced with disodium glycyrrhizinate; other parameters and methods are the same as those in Example 1.

[0053] Comparative Example 8 In the immunomodulatory additive, astragalus polysaccharide is replaced with disodium glycyrrhizinate; other parameters and methods are the same as those in Example 1.

[0054] Preparation of bone mud specimen: For convenient in vitro detection, nano-scale calcium phosphate biphasic ceramics and biodegradable polymers are not added to the bone mud specimen, and the angiogenesis promoter does not prepare polylactic acid sustained-release microspheres, and tanshinone and hydroxy polyethyleneglycol biotin are directly added. The active substance powder contained in every 1 g of the whole-component bone mud is diluted with 2 mL of PBS to obtain the bone mud specimen. The active substance powder does not contain nano-scale calcium phosphate biphasic ceramics and biodegradable polymers, and the angiogenesis promoter does not prepare polylactic acid sustained-release microspheres.

[0055] 1. Detection of bone induction ability Detection item: Alkaline phosphatase (ALP) activity.

[0056] Detection principle: Under the action of the bone induction material, the ALP activity secreted by osteoblasts will change. ALP can catalyze the hydrolysis of disodium p-nitrophenyl phosphate to generate yellow p-nitrophenol. By detecting the absorbance of p-nitrophenol at 405 nm, the ALP activity is calculated, thereby reflecting the bone induction ability.

[0057] Detection method: Cell culture: Osteoblasts (MC3T3-E1) are cultured in α-MEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody in a constant temperature incubator at 37 °C and 5% CO 2 until the logarithmic growth phase.

[0058] Bone mud specimen: According to the dosages of the biomimetic mineralized collagen fiber, modified hyaluronic acid, β-cyclodextrin-coated recombinant human bone morphogenetic protein-7, angiogenesis promoter, and immunomodulatory additive used in every 1 g of the whole-component bone mud, the bone mud active substance (i.e., without nano-scale calcium phosphate biphasic ceramics and biodegradable polymers) is prepared and diluted with 5 mL of PBS to obtain the bone mud specimen.

[0059] Co - culture: Osteoblasts in the logarithmic growth phase were inoculated into 24 - well plates at a density of 5×10 4 cells / mL, and 1 mL of medium was added to each well. After the cells adhered to the wall, 100 μL of bone mud samples of different samples were added respectively, and 3 replicate wells were set for each sample.

[0060] Activity detection: On the 3rd, 7th, and 14th days of culture, the culture medium in the wells was discarded, the cells were washed 3 times with PBS, 0.25% trypsin was added to digest the cells, the cell suspension was collected, centrifuged at 1000 r / min for 5 min, and the supernatant was discarded. Cell lysis buffer was added, and the cells were lysed on ice bath for 30 min, then centrifuged at 12000 r / min for 15 min, and the supernatant was taken. According to the usage method of the ALP detection kit, p - nitrophenyl phosphate disodium substrate working solution was added, incubated at 37℃ for 15 min, then the termination solution was added, and the absorbance at a wavelength of 405 nm was measured on an enzyme - linked immunosorbent assay (ELISA) reader. The ALP activity was calculated according to the standard curve. The detection results are shown in Table 1 below.

[0061] 2. Detection of angiogenesis ability Detection item: In vitro angiogenesis experiment (tube formation experiment).

[0062] Detection principle: Human umbilical vein endothelial cells (HUVECs) can form lumen - like structures similar to blood vessels under appropriate induction conditions. By observing and quantifying these structures, the ability of the material to promote angiogenesis can be evaluated.

[0063] Detection method: Matrigel plating: Place the 96 - well plate on ice, add 50 μL of Matrigel to each well, and quickly place it in a 37℃ constant - temperature incubator for 30 min to solidify the Matrigel.

[0064] Cell preparation: Culture HUVECs in ECM medium containing 10% fetal bovine serum and 1% penicillin - streptomycin double - antibody at 37℃ and 5% CO 2 until the logarithmic growth phase, digest with 0.25% trypsin, and make a cell suspension of 2×10 4 cells / well.

[0065] Co - culture: Add 100 μL of cell suspension to the solidified Matrigel in each well, and then add 50 μL of bone mud samples of different samples respectively. 3 replicate wells are set for each sample.

[0066] Observation and analysis: Place the 96 - well plate in an incubator at 37℃ and 5% CO 2 for 6 h, observe under an inverted microscope and use ImageJ software to analyze the total length, number of nodes, and number of branches of the vessel - like structures. The detection results are shown in Table 1 below.

[0067] 3. Immunogenicity detection Detection items: Release amounts of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6).

[0068] Detection principle: After macrophages contact immunogenic substances, they secrete inflammatory factors such as TNF-α and IL-6. By quantitatively detecting the release amounts of these factors through ELISA method, the immunogenicity of the material can be evaluated.

[0069] Detection method: Cell culture: RAW264.7 macrophages were cultured in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody at 37°C and 5% CO 2 until the logarithmic growth phase.

[0070] Co-culture: Macrophages in the logarithmic growth phase were inoculated into 24-well plates at a density of 3×10 4 cells / mL, 1 mL of medium was added to each well. After the cells adhered, 100 μL of bone mud specimens of different samples were added respectively, and 3 replicates were set for each sample.

[0071] Factor detection: After incubating the 24-well plates in an incubator at 37°C and 5% CO 2 for 24 h, the supernatant was collected. According to the usage method of the ELISA kit, standards, samples, enzyme-labeled antibodies, substrates, etc. were added in sequence for reaction, and the absorbance at a wavelength of 450 nm was measured on an enzyme-labeling instrument. The release amounts of TNF-α and IL-6 were calculated according to the standard curve. The detection results are shown in Table 1 below.

[0072] Table 1 Detection data results of each example and comparative example

[0073] Note: " / " indicates that this item was not detected.

[0074] From the above detection results, it can be seen that the bone mud active substances in Examples 1 to 3 have strong bone induction promotion ability, strong angiogenesis promotion ability, good biocompatibility, and lower immunogenicity risk.

[0075] From the comparison of the results of Comparative Example 1, it can be seen that hydroxypropyl chitosan is not added. Hydroxypropyl chitosan can interact with type I collagen, optimize the structure of biomimetic mineralized collagen fibers, and promote the adhesion, proliferation and differentiation of osteoblasts. The absence of it will disrupt this structure and function, reduce the activity of osteoblasts, and decrease the secretion of ALP, resulting in the ALP activity being lower than that of Example 1 on the 3rd, 7th, and 14th days. Hydroxypropyl chitosan has a positive effect on the construction of the extracellular matrix and cell - to - cell signal transduction. After its absence, the migration and lumen formation of endothelial cells are affected, and the total length, number of nodes and number of branches of the vascular - like structure are reduced. The absence of hydroxypropyl chitosan changes the biocompatibility of biomimetic mineralized collagen fibers, and macrophages secrete more TNF - α and IL - 6, resulting in an increase in immunogenicity.

[0076] From the comparison of the results of Comparative Example 2, it can be seen that hyaluronic acid is used to replace modified hyaluronic acid. Modified hyaluronic acid is modified by thiol and hydrazide, which enhances its binding ability with other components and its regulatory effect on cells. Ordinary hyaluronic acid lacks these properties and cannot efficiently promote the function of osteoblasts, resulting in a decrease in ALP activity. Modified hyaluronic acid can better regulate the release of cytokines and cell - to - cell interactions, and promote angiogenesis. Ordinary hyaluronic acid is less capable in this regard, resulting in the vascular - like structure parameters being inferior to those of Example 1. The special structure of modified hyaluronic acid can further reduce immunogenicity.

[0077] From the comparison of the results of Comparative Example 3, it can be seen that all hyaluronic acid - thiol is used. This disrupts the proportion balance of HA - SH and HA - Hy in modified hyaluronic acid, affects its synergistic effect with other components, weakens the induction effect on osteoblasts, and slightly reduces the ALP activity. Changing the proportion of functional groups of hyaluronic acid affects its regulation of angiogenesis - related signaling pathways, and the formation of the vascular - like structure is affected to a certain extent. The proportion imbalance leads to a change in immunomodulatory ability and an enhanced macrophage response.

[0078] From the comparison of the results of Comparative Example 4, it can be seen that recombinant human bone morphogenetic protein - 7 is not coated with β - cyclodextrin. β - cyclodextrin coating can protect the activity of recombinant human bone morphogenetic protein - 7, prevent its degradation and inactivation. When not coated, the protein has poor stability in the in vitro environment, its activity decreases, and the induction effect on osteoblasts weakens.

[0079] From the comparison of the results of Comparative Example 5, it can be seen that the tanshinone component in the angiogenesis promoter decreases, and the hydroxy polyethylene glycol biotin component increases. The component ratio changes, the synergistic effect becomes poor, the angiogenesis promoter cannot fully exert its efficacy, the total length, number of nodes and number of branches of the vascular - like structure are significantly reduced, and the bone induction ability is also affected to a certain extent due to insufficient angiogenesis, and the ALP activity decreases accordingly.

[0080] From the result comparison of Comparative Example 6, it can be seen that hydroxy polyethylene glycol biotin is not added. Hydroxy polyethylene glycol biotin can promote angiogenesis-related signal transduction by interacting with cell surface receptors and other biomolecules. The absence of it will hinder this process, reduce the angiogenesis ability, worsen the parameters of vascular-like structures, and at the same time indirectly affect the osteoinductive microenvironment, resulting in a decrease in ALP activity.

[0081] From the result comparison of Comparative Example 7, it can be seen that lentinan is replaced by disodium glycyrrhizinate. Lentinan and disodium glycyrrhizinate have different effects in immunomodulation. Lentinan can regulate the immune activity of macrophages and make them in a moderate immune response state. After being replaced by disodium glycyrrhizinate, the immunomodulation is imbalanced, the immunogenicity is significantly increased, and at the same time, the overall performance of bone paste is affected, resulting in a decrease in osteoinductive and angiogenesis abilities.

[0082] From the result comparison of Comparative Example 8, it can be seen that astragalus polysaccharide is replaced by disodium glycyrrhizinate. Astragalus polysaccharide has immunomodulatory and anti-inflammatory effects and can cooperate with other components to maintain immune balance. After being replaced by disodium glycyrrhizinate, the immunomodulatory function is disordered, the immunogenicity is increased, and it also has a negative impact on osteoinductive and angiogenesis abilities, making the relevant detection indexes worse.

Claims

1. A bone mud for repairing bone trauma, characterized in that: The invention comprises the following raw materials in parts by weight: 30 to 40 parts of nano-scale calcium-phosphorus dual-phase ceramics, 15 to 20 parts of biomimetic mineralized collagen fibers, 20 to 30 parts of biodegradable polymers, 2 to 4 parts of modified hyaluronic acid, 0.5 to 1 part of beta-cyclodextrin-coated recombinant human bone morphogenetic protein-7, 1 to 3 parts of angiogenesis promoters and 0.5 to 1 part of immunomodulatory additives.

2. The bone mud for repairing bone trauma according to claim 1, characterized in that: The nano-scale calcium-phosphorus dual-phase ceramic is a dual-phase ceramic of hydroxyapatite and β-tricalcium phosphate; the preparation method of the nano-scale calcium-phosphorus dual-phase ceramic comprises the following steps: according to the mass ratio of calcium nitrate: diammonium hydrogen phosphate = (1.8-2.0): (1-1.2), calcium nitrate and diammonium hydrogen phosphate are respectively dissolved in 3-5 times the mass of deionized water to obtain a calcium nitrate solution and a diammonium hydrogen phosphate solution; under stirring, the diammonium hydrogen phosphate solution is added to the calcium nitrate solution, and ammonia is added to the solution. The pH value is adjusted to 10-11 with water, the reaction temperature is 60-70°C, the reaction time is 2h-3h, a calcium-phosphorus precursor precipitate is generated, the precipitate is centrifuged, the precipitate is washed 2-3 times with deionized water and anhydrous ethanol respectively, vacuum dried at 60-80°C for 12h-24h, then heated to 900-1000°C at a heating rate of 4-6°C / min / min, calcined for 2h-3h, and crushed to obtain a nano-scale calcium-phosphorus dual-phase ceramic with a size of less than 200nm.

3. The bone mud for repairing bone trauma according to claim 1, characterized in that: The preparation method of the biomimetic mineralized collagen fiber comprises the following steps: preparing a collagen solution, wherein the collagen solution contains 2wt% to 3wt% type I collagen freeze-dried fibers, 2wt% to 3wt% hydroxypropyl chitosan, and the solvent is 0.5mol / L to 1mol / L acetic acid solution; preparing a mineralizing solution containing calcium ions and phosphate ions, wherein the calcium ion concentration is 1mmol / L to 2mmol / L, the phosphate ion concentration is 0.6mmol / L to 1.2mmol / L, and the pH value is adjusted to 7.2 to 7.4; under stirring, adding the mineralizing solution to the collagen solution at a volume ratio of the mineralizing solution: the collagen solution = 1: (1.5 to 2), stirring for reaction for 2h to 4h, transferring the obtained reaction solution to a dialysis bag, and dialyzing in deionized water for 3h to 5h; and freeze-drying the dialyzed product to obtain the biomimetic mineralized collagen fiber.

4. The bone mud for repairing bone trauma according to claim 1, characterized in that: The biodegradable polymer is polycaprolactone.

5. The bone mud for repairing bone trauma according to claim 1, characterized in that: The mass ratio of the modified hyaluronic acid components is hyaluronic acid-thiol: hyaluronic acid-hydrazide = (2-3): (0.5-1).

6. The bone mud for repairing bone trauma according to claim 1, characterized in that: The preparation method of β-cyclodextrin-coated recombinant human bone morphogenetic protein-7 comprises the following steps: preparing a 50°C-60°C, 5wt%-6wt% β-cyclodextrin aqueous solution; adding recombinant human bone morphogenetic protein-7 to a phosphate buffer solution at a concentration of 0.5mg / mL-1mg / mL to prepare a protein solution; under stirring conditions, adding the protein solution to the β-cyclodextrin aqueous solution at a volume ratio of protein solution: β-cyclodextrin aqueous solution = (1-1.5): (20-25), stirring for reaction for 2h-4h, centrifuging, taking a precipitate, and freeze-drying to obtain the β-cyclodextrin-coated recombinant human bone morphogenetic protein-7.

7. The bone mud for repairing bone trauma according to claim 1, characterized in that: The angiogenesis promoter is polylactic acid sustained-release microspheres loaded with tanshinone and hydroxypolyethylene glycol biotin.

8. A bone mud for repairing bone trauma according to claim 1 or 7, characterized in that: The preparation method of the angiogenesis promoter comprises the following steps: dissolving polylactic acid in ethyl acetate to prepare a polylactic acid solution with a mass concentration of 5% to 8%; preparing a mixed solution, wherein the mixed solution contains 0.4 mg / mL to 0.5 mg / mL of tanshinone and 0.1 mg / mL to 0.2 mg / mL of hydroxypolyethylene glycol biotin, and the solvent is a phosphate buffer solution; preparing a polyvinyl alcohol aqueous solution with a mass concentration of 2% to 3%; adding the mixed solution to the polylactic acid solution at a ratio of mixed solution to polylactic acid solution = 1: (3 to 5), and homogenizing at 10000 r / min to 15000 r / min to form an oil-in-water type emulsion; then adding the emulsion to the polyvinyl alcohol aqueous solution at a ratio of emulsion to polyvinyl alcohol aqueous solution = 1: (2 to 3), stirring under negative pressure at 30° C. to 35° C. for 3 h to 4 h, so that the ethyl acetate is fully volatilized, the polylactic acid is solidified to form microspheres, centrifugation is performed, and the precipitate is repeatedly washed with deionized water for 4 to 6 times, and freeze-dried to obtain the angiogenesis promoter.

9. The bone cement for repairing bone trauma according to claim 1, characterized in that: The mass ratio of the components of the immunomodulatory additive is disodium glycyrrhizinate: lentinan: astragalus polysaccharide = (5-8): (2-3): (2-3).

10. The method for preparing bone mud for repairing bone trauma according to claim 1, characterized in that: The method comprises the following steps: uniformly mixing nanometer-scale calcium-phosphorus biphasic ceramics, biomimetic mineralized collagen fibers, modified hyaluronic acid, beta-cyclodextrin-coated recombinant human bone morphogenetic protein-7, angiogenesis promoters, and immunomodulatory additives according to mass fractions to obtain a mixture; heating and melting a biodegradable polymer, mixing the mixture with the mixture, and adjusting fluidity with physiological saline to obtain bone mud.

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