A bone trauma repair bone paste and its preparation method

Abstract

The present invention relates to a bone trauma repair bone paste and a preparation method thereof, belonging to the technical field of biomedical material manufacturing. The bone paste of the present invention 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; the nano calcium phosphate biphasic ceramics are hydroxyapatite and β-tricalcium phosphate biphasic ceramics; the biomimetic mineralized collagen fibers are prepared by mineralizing freeze-dried fibers of type I collagen admixed with hydroxypropyl chitosan; the modified hyaluronic acid is hyaluronic acid-thiol and hyaluronic acid-hydrazide; the angiogenesis promoter is a polylactic acid sustained-release microsphere loaded with tanshinone and hydroxy polyethylene glycol biotin; the immunomodulatory additive is disodium glycyrrhizinate, lentinan and astragalus polysaccharide. The bone paste can effectively improve the bone induction ability, promote angiogenesis and reduce the risk of immunogenicity.

Description

Technical Field

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

[0002] Skeletal trauma is a common type of injury clinically, ranging from simple fractures to complex bone defects, which seriously affects the quality of life of patients. Fracture is the most common skeletal trauma. For simple fractures, traditional treatment methods such as plaster fixation and splint fixation limit 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 of bones, which seriously affects limb function.

[0003] Under such clinical needs, bone grafting has become one of the important means for treating complex skeletal traumas. Autologous bone grafting 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 skeletal trauma repair. It shows unique advantages and application prospects on the basis of overcoming the limitations of traditional treatment methods and materials. However, there are still the following problems in the process of using bone paste for skeletal trauma repair:

[0005] (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.

[0006] (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.

[0007] (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.

[0008] 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

[0009] 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:

[0010] 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.

[0011] 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, vacuum dry at 60°C to 80°C for 12h to 24h, and then rise 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.

[0012] 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, wherein 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 stirring, adding the mineralization solution to the collagen solution according to a 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.

[0013] In the above bone mud, the biodegradable polymer is polycaprolactone.

[0014] In the above bone mud, the mass ratio of the components of the modified hyaluronic acid is hyaluronic acid-thiol (HA-SH):hyaluronic acid-hydrazide (HA-Hy) = (2 - 3):(0.5 - 1).

[0015] 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 stirring conditions, adding the protein solution to the β-cyclodextrin aqueous solution according to a 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.

[0016] 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).

[0017] 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 polyethyleneglycol 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.

[0018] 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).

[0019] 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.

[0020] A bone paste for repairing bone trauma and its preparation method provided by the present invention have the following beneficial effects:

[0021] 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.

[0022] 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 properties, 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 conducive to cell growth in bone paste, promoting bone induction.

[0023] III. Biodegradable Polymer (Polycaprolactone) It has good biocompatibility, can slowly degrade 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 stability of the bone paste structure during the degradation process, working in synergy with nanoscale calcium phosphate biphasic ceramics and biomimetic mineralized collagen fibers to maintain the shape of the bone paste.

[0024] 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 the 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 micro-structure of the bone paste and promote cell activities within the bone paste.

[0025] V. Recombinant Human Bone Morphogenetic Protein-7 It is a strong bone induction factor that can stimulate the differentiation of mesenchymal stem cells 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.

[0026] VI. Tanshinone It promotes the proliferation and migration of vascular endothelial cells and induces angiogenesis; hydroxy polyethylene glycol biotin enhances biocompatibility and stability. When the two are used in combination, they can further improve the ability of blood vessel proliferation. Hydroxy polyethylene glycol biotin can improve the physical and chemical properties of the local microenvironment through its hydrophilicity, regulating the osmotic pressure, pH, 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 slowly degrade 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 the repair of large bone defects in bone paste, promoting the overall bone repair process.

[0027] 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.

[0028] In summary, the present invention uses nanoscale 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 to prepare bone paste 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 easy to implement, and it has good practical value. Specific Embodiments

[0029] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to these embodiments.

[0030] Example 1: A bone paste for repairing bone trauma, comprising the following raw materials in parts by mass: 35 parts of nanoscale 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.

[0031] 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 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, and 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 heat it to 950 °C at a heating rate of 5 °C / min, calcine it for 2.5 h, and pulverize it to obtain nanoscale calcium phosphate biphasic ceramics with a particle size below 200 nm.

[0032] Among them, the preparation method of the biomimetic mineralized collagen fiber includes the following steps: Prepare a collagen solution, which contains 2.5 wt% type I collagen freeze-dried fibers and 2.5 wt% hydroxypropyl chitosan, and the solvent is 0.8 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 1.5 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 to stir and react for 3 h, transfer the reaction solution to a dialysis bag, and dialyze in deionized water for 4 h; Lyophilize the dialyzed product to obtain the biomimetic mineralized collagen fiber.

[0033] 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 solution of β-cyclodextrin 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 solution of β-cyclodextrin according to the volume ratio of protein solution:aqueous solution of β-cyclodextrin = 1.2:23, continue to stir and react for 3 h, cool down to 8 °C, centrifuge at 7000 r / min for 25 min, take the precipitate, and lyophilize to obtain β-cyclodextrin-coated recombinant human bone morphogenetic protein-7.

[0034] 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 7%; Prepare a mixed solution containing 0.45 mg / mL of tanshinone and 0.15 mg / mL of hydroxy poly(ethylene glycol) biotin (HO-PEG-Biotin), and the solvent is phosphate buffer solution (PBS, pH 7.4); Prepare an aqueous solution of polyvinyl alcohol (PVA) with a mass concentration of 2.5%; 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 solution of polyvinyl alcohol according to the ratio of emulsion:aqueous solution of polyvinyl alcohol = 1:2.5, stir under reduced pressure at 32 °C and 4000 r / min for 3.5 h to fully volatilize ethyl acetate, and 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 lyophilize to obtain the angiogenesis promoter.

[0035] The above method for preparing bone mud for skeletal trauma repair comprises the following steps: By mass fraction, 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 65°C for melting and then mixed with the mixture, and the fluidity is adjusted with physiological saline to obtain bone mud.

[0036] Example 2: A bone mud for skeletal trauma repair, comprising the following raw materials by mass fraction: 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 mass ratio of the components 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 mass ratio of the components of the immunomodulatory additive is disodium glycyrrhizinate: lentinan: astragalus polysaccharide = 5:2:2.

[0037] Among them, the nanoscale calcium phosphate biphasic ceramics are hydroxyapatite and β-tricalcium phosphate biphasic ceramics; the method for preparing the nanoscale calcium phosphate biphasic ceramics comprises the following steps: By 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, and the reaction time is 2 h to generate a calcium phosphate precursor precipitate. The precipitate is centrifuged at 6000 r / min for 20 min, washed twice with deionized water, and then washed twice with absolute ethanol, dried in vacuum at 60°C for 12 h, and then heated to 900°C at a heating rate of 4°C / min and calcined for 2 h, and then pulverized to obtain nanoscale calcium phosphate biphasic ceramics with a particle size below 200 nm.

[0038] Among them, the preparation method of the biomimetic mineralized collagen fiber includes the following steps: Prepare a collagen solution, the collagen solution contains 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, according to the volume ratio of mineralization solution:collagen solution = 1:1.5, add the mineralization solution to the collagen solution, continue stirring and reacting for 2 h, transfer the reaction solution to a dialysis bag, and dialyze in deionized water for 3 h; Lyophilize the dialyzed product to obtain the biomimetic mineralized collagen fiber.

[0039] 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; According to a concentration of 0.5 mg / mL, add recombinant human bone morphogenetic protein-7 to phosphate buffer solution (PBS, pH 7.2) to prepare a protein solution; Under the stirring condition of 60 r / min, according to the volume ratio of protein solution:β-cyclodextrin aqueous solution = 1:20, add the protein solution to the β-cyclodextrin aqueous solution, continue stirring and reacting for 2 h, cool down to 5 °C, centrifuge at 6000 r / min for 20 min, take the precipitate, and lyophilize to obtain β-cyclodextrin-coated recombinant human bone morphogenetic protein-7.

[0040] 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, the mixed solution contains 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% aqueous solution of polyvinyl alcohol (PVA); According to the 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 ratio of emulsion:aqueous polyvinyl alcohol solution = 1:2, add the emulsion to the aqueous polyvinyl alcohol solution, and stir under negative pressure at 30 °C and 3000 r / min for 3 h to fully volatilize ethyl acetate, and 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 lyophilize to obtain the angiogenesis promoter.

[0041] The above method for preparing bone paste for bone trauma repair includes the following steps: By mass fraction, 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 60°C to melt and mixed with the mixture, and the fluidity is adjusted with physiological saline to obtain bone paste.

[0042] Example 3: A bone paste for bone trauma repair includes the following raw materials by mass fraction: 40 parts of nanoscale 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 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) = 3:1. 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 = 8:3:3.

[0043] 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 includes the following steps: By mass ratio, calcium nitrate: diammonium hydrogen phosphate = 2.0:1.2. Calcium nitrate and diammonium hydrogen phosphate are respectively dissolved in 5 times the mass of deionized water to obtain a calcium nitrate solution and a diammonium hydrogen phosphate solution. Under the stirring state of 300 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 11 with ammonia water. The reaction temperature is 70°C, and the reaction time is 3 h to generate a calcium phosphate precursor precipitate. Centrifugal separation is carried out at 8000 r / min for 30 min. The precipitate is washed 3 times with deionized water and then 3 times with absolute ethanol, dried in vacuum at 80°C for 24 h, and then heated to 1000°C at a heating rate of 6°C / min and calcined for 3 h, and then pulverized to obtain nanoscale calcium phosphate biphasic ceramics with a particle size of less than 200 nm.

[0044] 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 type I collagen fibers and 3 wt% hydroxypropyl chitosan, and the solvent is 1 mol / L acetic acid solution; Prepare a mineralizing 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 mineralizing solution to the collagen solution according to the volume ratio of mineralizing solution: collagen solution = 1:2, continue stirring and reacting for 4 h, transfer the reaction solution to a dialysis bag, and dialyze in deionized water for 5 h; Lyophilize the dialyzed product to obtain the biomimetic mineralized collagen fiber.

[0045] 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 stirring and reacting for 4 h, cool down to 10 °C, centrifuge at 8000 r / min for 30 min, take the precipitate, and lyophilize to obtain β-cyclodextrin-coated recombinant human bone morphogenetic protein-7.

[0046] 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, stir under negative pressure at 35 °C and 5000 r / min for 4 h to fully volatilize ethyl acetate, and solidify polylactic acid to form microspheres. Centrifuge at 8000 r / min for 30 min, take the precipitate, wash it repeatedly with deionized water 6 times, and lyophilize to obtain the angiogenesis promoter.

[0047] The preparation method of the above-mentioned bone trauma repair bone paste includes the following steps: By mass fraction, nanoscale calcium phosphate biphasic ceramic, biomimetic mineralized collagen fiber, 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 to melt and mixed with the mixture, and the fluidity is adjusted with physiological saline to obtain bone paste.

[0048] In the above-mentioned examples, the freeze-dried fiber of type I collagen is 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 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.

[0049] Comparative Example 1

[0050] In the preparation method of the biomimetic mineralized collagen fiber, hydroxypropyl chitosan is not added; other parameters and methods are the same as in Example 1.

[0051] Comparative Example 2

[0052] In the bone paste, modified hyaluronic acid is replaced with hyaluronic acid (the same manufacturer's hyaluronic acid model JPHA-1-10k); other parameters and methods are the same as in Example 1.

[0053] Comparative Example 3

[0054] 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.

[0055] Comparative Example 4

[0056] In the bone paste, recombinant human bone morphogenetic protein-7 is not coated with β-cyclodextrin, and other parameters and methods are the same as those in Example 1.

[0057] Comparative Example 5

[0058] In the preparation method of the angiogenesis promoter, the addition amounts of tanshinone and hydroxy poly(ethylene glycol) biotin are interchanged; other parameters and methods are the same as those in Example 1.

[0059] Comparative Example 6

[0060] In the preparation method of the angiogenesis promoter, hydroxy poly(ethylene glycol) biotin is not added; other parameters and methods are the same as those in Example 1.

[0061] Comparative Example 7

[0062] In the immunomodulatory additive, lentinan is replaced with disodium glycyrrhizinate; other parameters and methods are the same as those in Example 1.

[0063] Comparative Example 8

[0064] In the immunomodulatory additive, astragalus polysaccharide is replaced with disodium glycyrrhizinate; other parameters and methods are the same as those in Example 1.

[0065] Bone paste specimen preparation: For the convenience of in vitro detection, nano-scale calcium phosphate biphasic ceramics and biodegradable polymers are not added to the bone paste specimen, and the angiogenesis promoter does not prepare polylactic acid sustained-release microspheres, and tanshinone and hydroxy poly(ethylene glycol) biotin are directly added. The active ingredient powder contained in every 1 g of the whole-component bone paste is diluted with 2 mL of PBS to obtain the bone paste specimen. The active ingredient 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.

[0066] 1. Detection of bone induction ability

[0067] Detection item: Alkaline phosphatase (ALP) activity.

[0068] 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.

[0069] Detection method:

[0070] 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% CO2 until the logarithmic growth phase.

[0071] Bone mud sample: According to the dosages of biomimetic mineralized collagen fibers, modified hyaluronic acid, β-cyclodextrin-coated recombinant human bone morphogenetic protein-7, angiogenesis promoter, and immunomodulatory additive used in 1 g of whole-component bone mud, prepare the bone mud active substance (i.e., without nano-scale calcium phosphate biphasic ceramics and biodegradable polymers), and dilute it with 5 mL of PBS to obtain the bone mud sample.

[0072] Co-culture: Seed osteoblasts in the logarithmic growth phase into a 24-well plate at a density of 5×10 4 cells / mL, add 1 mL of culture medium to each well. After the cells adhere to the wall, add 100 μL of the bone mud sample of different samples respectively, and set 3 replicates for each sample.

[0073] Activity detection: On the 3rd, 7th, and 14th days of culture respectively, discard the culture medium in the wells, wash the cells 3 times with PBS, add 0.25% trypsin to digest the cells, collect the cell suspension, centrifuge at 1000 r / min for 5 min, and discard the supernatant. Add cell lysate, lyse on ice bath for 30 min, centrifuge at 12000 r / min for 15 min, and take the supernatant. According to the usage method of the ALP detection kit, add the p-nitrophenyl phosphate disodium substrate working solution, incubate at 37 °C for 15 min, then add the stop solution, and measure the absorbance at a wavelength of 405 nm on an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the ALP activity according to the standard curve. The detection results are shown in Table 1 below.

[0074] 2. Detection of angiogenesis ability

[0075] Detection item: In vitro angiogenesis experiment (tube formation experiment).

[0076] Detection principle: Human umbilical vein endothelial cells (HUVECs) can form tube-like structures similar to blood vessels under appropriate induction conditions. By observing and quantifying these structures, the angiogenesis promotion ability of the material can be evaluated.

[0077] Detection method:

[0078] Matrigel plating: Place a 96-well plate on ice, add 50 μL of Matrigel to each well, and quickly place it in a 37 °C constant temperature incubator for 30 min to solidify the Matrigel.

[0079] Cell preparation: Culture HUVECs in ECM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody at 37 °C and 5% CO2 until the logarithmic growth phase, digest with 0.25% trypsin, and prepare a cell suspension of 2×10 4 cells / well.

[0080] Co-culture: Add 100 μL of the cell suspension to the solidified Matrigel in each well, and then add 50 μL of the bone mud sample of different samples respectively. Set 3 replicates for each sample.

[0081] Observation and analysis: After incubating the 96-well plate in an incubator at 37 °C and 5% CO2 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 vascular-like structures. The test results are shown in Table 1 below.

[0082] 3. Immunogenicity detection

[0083] Test items: Release amounts of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6).

[0084] 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, the immunogenicity of the material can be evaluated.

[0085] Detection method:

[0086] Cell culture: Culture RAW264.7 macrophages in DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody at 37 °C and 5% CO2 until the logarithmic growth phase.

[0087] Co-culture: Inoculate macrophages in the logarithmic growth phase into a 24-well plate at a density of 3×10 4 cells / mL, add 1 mL of medium to each well. After the cells adhere, add 100 μL of bone mud samples of different samples respectively, and set 3 replicates for each sample.

[0088] Factor detection: After incubating the 24-well plate in an incubator at 37 °C and 5% CO2 for 24 h, collect the supernatant. According to the use method of the ELISA kit, add standard products, samples, enzyme-labeled antibodies, substrates, etc. in sequence for reaction, measure the absorbance at a wavelength of 450 nm on an enzyme-labeled instrument, and calculate the release amounts of TNF-α and IL-6 according to the standard curve. The test results are shown in Table 1 below.

[0089] Table 1 Test data results of each example and comparative example

[0090]

[0091] Note: " / " indicates that the item was not detected.

[0092] From the above test 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.

[0093] 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 bionic 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 intercellular 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 bionic mineralized collagen fibers, and macrophages secrete more TNF-α and IL-6, leading to an increase in immunogenicity.

[0094] 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 characteristics 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 the interaction between cells, 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.

[0095] From the comparison of the results of Comparative Example 3, it can be seen that all hyaluronic acid-thiol is used. This disrupts the ratio balance between 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. It changes the ratio of functional groups of hyaluronic acid, affects its regulation of angiogenesis-related signaling pathways, and has a certain impact on the formation of vascular-like structures. The ratio imbalance leads to a change in the immune regulation ability and an enhanced macrophage response.

[0096] 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 is reduced, and the induction effect on osteoblasts is weakened.

[0097] 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, resulting in a corresponding decrease in ALP activity.

[0098] Comparing the results 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 indirectly affect the osteoinductive microenvironment, resulting in a decrease in ALP activity.

[0099] Comparing the results of Comparative Example 7, it can be seen that lentinan is replaced by disodium glycyrrhizinate. Lentinan and disodium glycyrrhizinate have different roles in immunomodulation. Lentinan can regulate the immune activity of macrophages and keep them in a moderate immune response state. After being replaced by disodium glycyrrhizinate, the immunomodulation is imbalanced, the immunogenicity is significantly increased, and the overall performance of bone paste is affected, resulting in a decrease in osteoinductive and angiogenesis abilities.

[0100] Comparing the results 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 indicators worse.

Claims

1. A bone trauma repair bone paste, characterized in that, It comprises raw materials in the following 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 promoter, and 0.5 to 1 part of immunomodulatory additive; The nano calcium phosphate biphasic ceramics are hydroxyapatite and β-tricalcium phosphate biphasic ceramics; The preparation method of the biomimetic mineralized collagen fibers comprises the following steps: preparing a collagen solution containing 2wt% to 3wt% of type I collagen freeze-dried fibers and 2wt% to 3wt% of hydroxypropyl chitosan, with the solvent being 0.5mol / L to 1mol / L acetic acid solution; preparing a mineralization solution containing calcium ions and phosphate ions, where 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 mineralization solution to the collagen solution according to a volume ratio of mineralization solution:collagen solution = 1:(1.5 to 2), stirring and reacting for 2h to 4h, transferring the reaction solution to a dialysis bag, and dialyzing in deionized water for 3h to 5h; freeze-drying the dialyzed product to obtain biomimetic mineralized collagen fibers; The mass ratio of the components of the modified hyaluronic acid is hyaluronic acid-thiol:hyaluronic acid-hydrazide = (2 to 3):(0.5 to 1); The angiogenesis promoter is a polylactic acid sustained-release microsphere loaded with tanshinone and hydroxy polyethylene glycol biotin; The mass ratio of the components of the immunomodulatory additive is disodium glycyrrhizinate:lentinan:astragalus polysaccharide = (5 to 8):(2 to 3):(2 to 3).

2. The bone mud for repairing bone trauma according to claim 1, wherein 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: according to the mass ratio, calcium nitrate:diammonium hydrogen phosphate = (1.8 to 2.0):(1 to 1.2), dissolving 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, adding the diammonium hydrogen phosphate solution to the calcium nitrate solution, and simultaneously adjusting 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, generating a calcium phosphate precursor precipitate, centrifuging and separating, washing the precipitate with deionized water and absolute ethanol 2 to 3 times respectively, drying in vacuum at 60°C to 80°C for 12h to 24h, and then heating to 900°C to 1000°C at a heating rate of 4°C / min to 6°C / min, calcining for 2h to 3h, and pulverizing to obtain nano calcium phosphate biphasic ceramics with a particle size below 200nm.

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

4. A bone trauma repair bone paste 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 an aqueous β-cyclodextrin solution at 50 °C to 60 °C with a concentration of 5 wt% to 6 wt%; preparing a protein solution by adding recombinant human bone morphogenetic protein-7 into a phosphate buffer solution at a concentration of 0.5 mg / mL to 1 mg / mL; under stirring conditions, adding the protein solution into the aqueous β-cyclodextrin solution according to a volume ratio of protein solution:aqueous β-cyclodextrin solution = (1 to 1.5):(20 to 25), stirring and reacting for 2 h to 4 h, centrifuging and separating, taking the precipitate, and freeze-drying to obtain β-cyclodextrin-coated recombinant human bone morphogenetic protein-7.

5. A bone trauma repair bone paste according to claim 1, 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 containing 0.4 mg / mL to 0.5 mg / mL of tanshinone and 0.1 mg / mL to 0.2 mg / mL of hydroxy poly(ethylene glycol) biotin, with the solvent being a phosphate buffer solution; preparing an aqueous polyvinyl alcohol solution with a mass concentration of 2% to 3%; adding the mixed solution into the polylactic acid solution according to a ratio of mixed solution:polylactic acid solution = 1:(3 to 5), and homogenizing at 10000 r / min to 15000 r / min to form a water-in-oil emulsion; then adding the emulsion into the aqueous polyvinyl alcohol solution according to a ratio of emulsion:aqueous polyvinyl alcohol solution = 1:(2 to 3), and performing suction stirring at 30 °C to 35 °C for 3 h to 4 h to fully volatilize ethyl acetate, and solidifying polylactic acid to form microspheres, centrifuging and separating, taking the precipitate, washing it repeatedly with deionized water for 4 to 6 times, and freeze-drying to obtain the angiogenesis promoter.

6. The preparation method of a bone trauma repair bone paste according to claim 1, characterized in that, Comprising the following steps: uniformly mixing nanoscale calcium phosphate biphasic ceramics, biomimetic mineralized collagen fibers, modified hyaluronic acid, β-cyclodextrin-coated recombinant human bone morphogenetic protein-7, the angiogenesis promoter, and an immune regulation additive by mass parts to obtain a mixture; heating and melting a biodegradable polymer, mixing it with the mixture, and adjusting the fluidity with physiological saline to obtain bone paste.

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