Oxygen release type microsphere material for bone tissue engineering and preparation method thereof

Abstract

The invention discloses an oxygen release type microsphere material for bone tissue engineering and a preparation method of the oxygen release type microsphere material. A high-molecular polymer is used as a microsphere forming matrix, and inorganic peroxide is used as an oxygen release source; an emulsion solvent volatilization-pore-foaming agent method and an emulsion-freeze drying method are respectively adopted for oil-soluble polymers and water-soluble polymers to prepare the oxygen release type microspheres. Experimental results show that the oxygen release type microsphere material prepared by the method has an open and through porous structure, and oxygen can be continuously released within 25 days. In-vitro cell experiments prove that the oxygen release type microspheres can promote adhesion, proliferation and osteogenic differentiation of cells. The microsphere provided by the invention can be used for supplying oxygen when a cell-scaffold compound in the field of bone tissue engineering is implanted into a body and maintaining early survival of cells.

Description

Technical Field

[0001] The present invention relates to the technical field of bone tissue engineering, and in particular to an oxygen-releasing microsphere material for bone tissue engineering and a preparation method thereof. Background Art

[0002] The preparation, performance, tissue regeneration and defect repair of tissue engineering microspheres are the frontier and hot topics in the field of regenerative medicine today. Tissue defects that exceed the size that the body can repair on its own require external force to assist in clinical treatment.

[0003] Tissue engineering regenerative medicine is a new and promising approach to treat tissue defects. However, it has two major drawbacks: First, the stem cell source of seed cells is unstable, and autologous stem cells can be used to overcome this problem. Second, a large number of seed cells die in the early stage of implantation. After the seed cells cultured in vitro are loaded onto the scaffold and implanted into the defect site, the blood supply system of the defect site is severely damaged, and the seed cells can only rely on tissue fluid penetration or blood vessel growth to provide nutrients for survival. Studies have shown that the penetration depth of nutrients through tissue fluid is limited (200-300μm), which can only maintain the survival of cells on the surface of the scaffold, and it usually takes one to several weeks for new blood vessels to grow into the inside of the scaffold. Therefore, how to maintain the survival of seed cells in the early stage of implantation in the body, and at the same time quickly promote the vascularization of the implanted scaffold, provide new energy substances and discharge metabolites for cells before consuming stored energy substances, and ensure the subsequent survival and growth of cells, is the key link to achieve the success of tissue engineering technology in repairing defective tissues and regenerating new tissues.

[0004] Rapid vascularization has always been a hot topic in tissue engineering technology research. There are many related reports and significant research results have been achieved, which has a significant effect on improving cell survival. However, most cells have died in large numbers before vascularization. The problem of delayed vascularization leading to the death of implanted seed cells is still a bottleneck problem restricting the development of tissue engineering technology. Maintaining the early survival of implanted cells by releasing oxygen (O2) through the scaffold (cells use the O2 released by the scaffold for aerobic respiration and consume stored energy substances to maintain survival) is considered to be a potential effective method to maintain the early survival of implanted cells.

[0005] The decomposition and release of O2 by peroxides (such as calcium peroxide, hydrogen peroxide, organic peroxides, etc.) is a common way to obtain O2 sources in the field of materials. Magnesium peroxide and calcium peroxide are insoluble in water, but they can slowly decompose and release O2 in water, and can be used as a tissue engineering scaffold to provide a source of O2 to seed cells after implantation in the body. In addition, the magnesium and calcium ions produced by magnesium peroxide / calcium peroxide can induce osteogenic differentiation of mouse bone marrow mesenchymal stem cells and promote bone regeneration, thereby enhancing the bone repair ability of the bone tissue repair system. However, simple magnesium peroxide / calcium peroxide does not have cell loading capacity, and its use as a tissue engineering scaffold loaded with cells to implant in the body to repair bone defects is limited. Therefore, the use of suitable biopolymer materials to load magnesium peroxide / calcium peroxide to construct a suitable tissue engineering scaffold is expected to maintain the survival of seed cells before vascularization and improve the osteogenic ability of the scaffold, thereby improving the final bone defect repair effect.

[0006] At present, in the field of tissue engineering, peroxide is usually embedded in polymers to prepare three-dimensional block tissue engineering scaffolds such as electrospun membranes, hydrogels, and porous scaffolds to achieve O2 release. However, three-dimensional block scaffolds are not injectable, and three-dimensional scaffolds are used for cell culture. Since cells can directly contact the loaded peroxide, they have greater cytotoxicity. In order to achieve that the loaded seed cells can obtain the O2 supply diffused by the decomposition of peroxide in the body, and avoid or be less affected by the toxicity of peroxide, it has become a difficult problem that needs to be solved in the field of oxygen supply tissue engineering technology. Summary of the invention

[0007] In order to solve the problems existing in the prior art, the purpose of the present invention is to overcome the shortcomings of the existing scaffold oxygen supply technology in tissue engineering and provide an oxygen-releasing microsphere material for bone tissue engineering and a preparation method thereof.

[0008] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is: an oxygen-releasing microsphere material for bone tissue engineering, characterized in that it is prepared by using an oxygen-releasing source and a high molecular polymer, the oxygen-releasing source is an inorganic peroxide, specifically one or more combinations of calcium peroxide, magnesium peroxide, and strontium peroxide, and the high molecular polymer is divided into an oil-soluble high molecular polymer and a water-soluble high molecular polymer.

[0009] Furthermore, the oil-soluble high molecular polymer used is one or more combinations of polylactic acid, polyglycolic acid, polylactide-glycolide, and polycaprolactone.

[0010] Furthermore, the water-soluble high molecular polymer used is one or more combinations of chitosan, carboxymethyl chitosan, gelatin, silk fibroin, sericin, sodium alginate, and sodium hyaluronate.

[0011] Furthermore, the composition ratio of the inorganic peroxide to the high molecular polymer is that the mass proportion of the inorganic peroxide is 0.1-7%.

[0012] Furthermore, the oxygen-releasing microspheres have a diameter of 50-500 μm, a pore size of 15-100 μm, and a porosity of 50-96%.

[0013] Furthermore, the oxygen-releasing microspheres have the ability to continuously and stably release oxygen in the body, the oxygen release time is 1-30 days, and the oxygen release amount is 0.1-1.2 mg / L.

[0014] Furthermore, the oxygen-releasing microsphere material can be used alone in tissue defect sites or loaded with animal cells for use in tissue defect sites.

[0015] A method for preparing an oxygen-releasing microsphere material for bone tissue engineering comprises an emulsion solvent volatilization-porogen method and an emulsion-freeze drying method.

[0016] The emulsion solvent volatilization-porogen method comprises the following steps:

[0017] (a) Preparation of porogen solution: Dissolve a suitable porogen in deionized water to prepare a solution with a concentration of 0.5-10% (w / v).

[0018] (b) Preparation of polymer solution: The oil-soluble polymer is dissolved in an organic solvent to prepare a solution with a concentration of 1 to 5% (w / v).

[0019] (c) Preparing a high molecular polymer solution containing an oxygen-releasing source: mixing the peroxide in a proportion of 0.1 to 10% (w / w) into the polymer solution.

[0020] (d) preparing an emulsion: mixing solution c and solution a in a volume ratio of 10:0.5 to 10:2.5, stirring and emulsifying at room temperature to obtain an emulsion.

[0021] (e) Dispersion liquid preparation: Polyvinyl alcohol is used as the dispersion liquid, and it is prepared with deionized water at a concentration of 0.5% to 2% (w / v)

[0022] (f) Forming of microspheres: Solution d is added to solution e at a volume ratio of 30:1 to 100:1 under stirring until the organic solvent is completely evaporated. The porogen is removed.

[0023] The emulsion-freeze drying method comprises the following steps:

[0024] (a) preparing an emulsion: adding sorbitan oleate to oily ether at a volume ratio of 0.1:10 to 0.5:10, and stirring until the sorbitan oleate is completely dissolved.

[0025] (b) Preparing a high molecular polymer solution: dissolving the high molecular polymer in deionized water and stirring until completely dissolved, with a concentration of 1 to 5% (w / v).

[0026] (c) Preparing a high molecular polymer solution containing an oxygen-releasing source: mixing the peroxide in a proportion of 0.1 to 10% (w / w) into the polymer solution.

[0027] (d) Forming microspheres: Add the solution of step d to the solution of step a at a volume ratio of 1:2 to 1:8, stir and emulsify for 1 to 3 minutes, quickly pour in petroleum ether at -10 to -75°C, remove the mobile liquid phase, obtain ice crystal particles and place them in a vacuum freeze dryer to dry until the water evaporates completely.

[0028] The present invention uses a high molecular polymer as a matrix, an inorganic peroxide as an oxygen release source, and uses a high molecular polymer to prepare microspheres to load seed cells and peroxides respectively. The microspheres carrying the seed cells are fully cultured and functionally differentiated in vitro, and after the cell inoculation is completed, they are mixed evenly with the peroxide-carrying microspheres in a certain proportion, and injected in vivo to supply oxygen to the tissues. Since the microspheres can be mixed evenly, the seed cells on the cell-carrying microspheres can obtain the O2 supply diffused from the peroxide-carrying microspheres to maintain survival. At the same time, the seed cells do not directly contact the peroxide, and the cells are less affected by the toxicity of the peroxide. The oxygen-releasing microsphere material provided by the present invention can be used for oxygen supply including but not limited to bone tissue engineering technology, protecting the early survival of implanted cells in vivo, and has important application prospects.

[0029] Compared with the prior art, the present invention has the following outstanding features and obvious advantages:

[0030] 1. The oxygen-releasing microsphere material for bone tissue engineering of the present invention has the ability to continuously and stably release oxygen in vivo and in vitro, the oxygen release time is 1-30 days, and the oxygen release amount is 0.1-1.2 mg / L.

[0031] 2. The oxygen-releasing microsphere material for bone tissue engineering of the present invention can be used alone to provide oxygen to tissue defect sites, or can be loaded with animal cells to provide oxygen thereto.

[0032] 3. The oxygen-releasing microsphere material for bone tissue engineering of the present invention can provide oxygen to the seeded cells and effectively avoid the toxic side effects of peroxide on the seed cells.

[0033] Other features and advantages of the present invention can be understood from the following drawings and detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is an electron microscope photograph of the sodium alginate-magnesium peroxide oxygen-releasing microspheres according to Example 1 of the present invention.

[0035] Figure 2 This is an oxygen release curve of an oxygen release test in a phosphate buffered saline (PBS) solution of sodium alginate-magnesium peroxide oxygen-releasing composite microspheres added with different concentrations of MgO2 after cross-linking and curing in Example 1 of the present invention.

[0036] Figure 3 This is the green fluorescence staining of the cell membrane of bone marrow mesenchymal stem cells (BMSCs) inoculated in sodium alginate-magnesium peroxide oxygen-releasing microspheres and cultured for 1, 7 and 14 days in Example 1 of the present invention.

[0037] Figure 4 This is a proliferation diagram of BMSCs inoculated with sodium alginate-magnesium peroxide oxygen-releasing microspheres and cultured for 1, 7, and 14 days in Example 1 of the present invention.

[0038] Figure 5 Example 1 of the present invention The gene expression of alkaline phosphatase (ALP) was measured after BMSCs were inoculated on sodium alginate-magnesium peroxide oxygen-releasing microspheres and cultured for 1, 7, and 14 days. DETAILED DESCRIPTION

[0039] In order to further illustrate and fully describe the technical solution of the present invention, the microsphere material capable of releasing oxygen and the preparation method thereof for use in the field of bone tissue engineering technology provided by the present invention are described below in conjunction with embodiments. The protection scope of the present invention is not limited by the following embodiments.

[0040] Example 1: Preparation, oxygen release and cell compatibility of sodium alginate-magnesium peroxide microspheres

[0041] In this embodiment, a method for preparing sodium alginate-magnesium peroxide oxygen-releasing microspheres comprises the following steps:

[0042] (a) Preparation of sodium alginate-magnesium peroxide solution: 2-10 g of sodium alginate is dissolved in deionized water and stirred until the sodium alginate is completely dissolved to obtain a sodium alginate solution with a concentration of 1-5% (w / v); then 0.2-1 g of magnesium peroxide is added to the sodium alginate solution and mechanically stirred at room temperature until the magnesium peroxide is evenly dispersed.

[0043] (b) Preparation of oil phase solution: Add 0.5 mL of sorbitan oleate to 40 mL of oily ether and stir until the sorbitan oleate is completely dissolved.

[0044] (c) Emulsion preparation: The solution of step b is added to the solution of step b, and mechanical stirring is performed for emulsification at room temperature to obtain a milky white solution.

[0045] (d) Preparation of microspheres: The solution in step c was quickly poured into petroleum ether precooled at -25°C, and mechanically stirred for 5-10 min in a -16°C freezer until completely solidified. The flowable upper liquid phase was removed, and the ice crystal particles were placed in a freezer for 72 h. Samples were collected and sealed for storage.

[0046] In this embodiment, an oxygen release experimental method of sodium alginate-magnesium peroxide oxygen-releasing microspheres comprises the following steps:

[0047] Another 0.1g of the sodium alginate-magnesium peroxide oxygen-releasing microspheres prepared in step (d) above was soaked in anhydrous ethanol for 1h, and then the microspheres were added to 500mL of a 15% calcium chloride solution and gently stirred for 2h. The sodium alginate-magnesium peroxide oxygen-releasing microspheres were sieved with a 100-mesh sieve, and then the microspheres were placed in 100mL of 1×PBS solution and shaken in a constant temperature shaker at 37°C for oxygen release test. Five parallel control groups were set for each concentration of MgO2, and the solution was changed every day and the oxygen release was measured.

[0048] In this embodiment, a cell compatibility test method of sodium alginate-magnesium peroxide oxygen-releasing microspheres includes the following steps:

[0049] The third generation of BMSCs were inoculated on sodium alginate-magnesium peroxide oxygen-releasing microspheres. Before inoculation, all microsphere samples were sterilized with 75% ethanol solution for at least 24 hours. After sterilization, the microspheres were washed three times with sterile 0.1M PBS solution and cell culture medium, and then immersed in complete cell culture medium for 24 hours. After precipitation and separation, 30 mg of microspheres were added to a T-25 cell culture flask containing 10 mL of cell culture medium and allowed to stand for 10 minutes. Then, 1 cm3 containing 4*10 6 The cell culture medium of each BMSCs was gently dripped onto the microspheres. After static culture for 5 hours, the culture system was gently blown 3-5 times with a pipette. Then, the culture bottle was shaken at 60 rpm for 10 hours, and 20 mL of cell culture medium was added for long-term culture. The distribution of cell growth in the microspheres was observed by laser confocal microscopy of the Dio-stained microsphere / cell complex. Cell proliferation was analyzed by DNA quantitative analysis of cell number after the cells were cultured on the microspheres for 1, 3, 5, and 7 days. The operation steps of DNA quantitative analysis of cell number are briefly described as follows: After the microsphere / cell complex was repeatedly frozen and thawed and ultrasonically broken, the supernatant was collected by centrifugation at 10,000 rpm. Then, the cell number was quantitatively obtained by fluorescence observation using Hoechst 33258 dye assay.

[0050] After the cells were cultured on the microspheres for 7 days, osteogenic differentiation was initiated. The operation process is briefly described as follows: After 7 days of microsphere culture, the culture medium of the system was replaced with osteogenic differentiation cell culture medium for continued culture. The induction culture medium was changed every two days, and the microsphere / cell complex was cultured in the adipogenic differentiation cell culture medium system for 1, 7, and 14 days before the osteogenic differentiation ALP test was performed.

[0051] Figure 1 As shown, the sodium alginate-magnesium peroxide oxygen-releasing microspheres prepared by the method of this embodiment have an open and continuous porous structure with an average pore size of 35±6.8 μm and a particle size that can be controlled within a range of 180-320 μm.

[0052] Figure 2 As shown, the oxygen release amount of sodium alginate-magnesium peroxide oxygen-releasing microspheres with different MgO2 concentrations.

[0053] Figure 3 , 4 As shown, the cell proliferation and alkaline phosphatase results of BMSCs seeded on sodium alginate-magnesium peroxide oxygen-releasing microspheres.

[0054] Example 2: Preparation and oxygen release of sodium alginate-calcium peroxide microspheres

[0055] In this embodiment, a method for preparing sodium alginate-calcium peroxide oxygen-releasing microspheres comprises the following steps:

[0056] (a) Preparation of sodium alginate-calcium peroxide solution: 2-10 g of sodium alginate is dissolved in deionized water and stirred until the sodium alginate is completely dissolved to obtain a sodium alginate solution with a concentration of 1-5% (w / v); then 0.2-1 g of calcium peroxide is added to the sodium alginate solution and mechanically stirred at room temperature until the calcium peroxide is evenly dispersed.

[0057] (b) Preparation of oil phase solution: Add 0.5 mL of sorbitan oleate to 40 mL of oily ether and stir until the sorbitan oleate is completely dissolved.

[0058] (c) Emulsion preparation: The solution of step b is added to the solution of step b, and mechanical stirring is performed for emulsification at room temperature to obtain a milky white solution.

[0059] (d) Preparation of microspheres: The solution in step c was quickly poured into petroleum ether precooled at -25°C, and mechanically stirred for 5-10 min in a -16°C freezer until completely solidified. The flowable upper liquid phase was removed, and the ice crystal particles were placed in a freezer for 72 h. Samples were collected and sealed for storage.

[0060] In this embodiment, an oxygen release experimental method of sodium alginate-calcium peroxide oxygen-releasing microspheres comprises the following steps:

[0061] Take 0.1g of the sodium alginate-calcium peroxide oxygen-releasing microspheres prepared in step (d) above and soak them in anhydrous ethanol for 1h, then add the microspheres to 500mL of 15% calcium chloride solution and stir gently for 2h. Sieve the sodium alginate-calcium peroxide oxygen-releasing microspheres through a sieve with a mesh of 100 mesh, then place the microspheres in 100mL of 1×PBS solution and shake in a constant temperature shaker at 37°C for oxygen release test. Set 5 parallel control groups for each concentration of CaO2, change the solution every day and measure the oxygen release.

[0062] Example 3: Preparation and oxygen release of chitosan-calcium peroxide microspheres

[0063] In this embodiment, a method for preparing chitosan-calcium peroxide oxygen-releasing microspheres comprises the following steps:

[0064] (a) Preparation of chitosan-calcium peroxide solution: 2-10 g of chitosan is dissolved in deionized water and stirred until the chitosan is completely dissolved to obtain a chitosan solution with a concentration of 1-5% (w / v); then 0.2-1 g of calcium peroxide is added to the chitosan solution and mechanically stirred at room temperature until the calcium peroxide is evenly dispersed.

[0065] (b) Preparation of oil phase solution: Add 0.5 mL of sorbitan oleate to 40 mL of oily ether and stir until the sorbitan oleate is completely dissolved.

[0066] (c) Emulsion preparation: The solution of step b is added to the solution of step b, and mechanical stirring is performed for emulsification at room temperature to obtain a milky white solution.

[0067] (d) Preparation of microspheres: The solution in step c was quickly poured into petroleum ether precooled at -25°C, and mechanically stirred for 5-10 min in a -16°C freezer until completely solidified. The flowable upper liquid phase was removed, and the ice crystal particles were placed in a freezer for 72 h. Samples were collected and sealed for storage.

[0068] In this embodiment, an oxygen release experimental method of chitosan-calcium peroxide oxygen-releasing microspheres comprises the following steps:

[0069] Take 0.1g of the chitosan-calcium peroxide oxygen-releasing microspheres prepared in step (d) above and soak them in anhydrous ethanol for 1h, then add the microspheres to 500mL of a sodium hydroxide solution with a mass concentration of 3mol / L and stir gently for 2h. Sieve the chitosan-calcium peroxide oxygen-releasing microspheres through a sieve with a mesh size of 100 mesh, wash with deionized water, and place the microspheres in 100mL of 1×PBS solution and shake in a constant temperature shaker at 37°C for oxygen release test. Set 5 parallel control groups for each concentration of CaO2, change the solution every day and measure the oxygen release.

[0070] Example 4: Preparation and oxygen release of lactide-caprolactone-calcium peroxide microspheres

[0071] In this embodiment, a method for preparing lactide-caprolactone-calcium peroxide oxygen-releasing microspheres comprises the following steps:

[0072] (a) Preparation of porogen solution: 0.5-10 g of gelatin solid is dissolved in 100 mL of deionized water and stirred at 40-60° C. until completely dissolved to prepare a solution with a concentration of 0.5-10% (w / v).

[0073] (b) Preparation of polymer solution: 0.1-0.5 g of lactide or caprolactone is dissolved in 10 mL of chloroform to prepare a solution with a concentration of 1-5% (w / v).

[0074] (c) Preparing a high molecular polymer solution containing an oxygen-releasing source: mixing calcium peroxide in a ratio of 0.1 to 10% (w / w) into the lactide-caprolactone solution.

[0075] (d) preparing an emulsion: mixing solution c and solution a in a volume ratio of 10:0.5 to 10:2.5, stirring and emulsifying at room temperature to obtain an emulsion.

[0076] (e) Dispersion preparation: Weigh 0.5-2 g of polyvinyl alcohol solid into 100 mL of deionized water, stir at 90° C. until completely dissolved, and prepare a solution with a concentration of 0.5% to 2% (w / v).

[0077] (f) Forming of microspheres: Solution d is added to solution e at a volume ratio of 30:1 to 100:1 under stirring until the organic solvent is completely evaporated. The porogen is removed.

[0078] In this embodiment, an oxygen release experimental method of lactide-caprolactone-calcium peroxide oxygen-releasing microspheres includes the following steps:

[0079] Another 0.1 g of the lactide-caprolactone-calcium peroxide oxygen-releasing microspheres prepared in step (f) above was placed in 100 mL of 1×PBS solution and shaken in a constant temperature shaker at 37°C for oxygen release test. Five parallel control groups were set for each concentration of CaO2, and the solution was changed every day and the oxygen release was measured.

[0080] Example 5: Preparation and oxygen release of lactide-calcium peroxide microspheres

[0081] In this embodiment, a method for preparing lactide-calcium peroxide oxygen-releasing microspheres comprises the following steps:

[0082] (a) Preparation of porogen solution: 0.5-10 g of gelatin solid is dissolved in 100 mL of deionized water and stirred at 40-60° C. until completely dissolved to prepare a solution with a concentration of 0.5-10% (w / v).

[0083] (b) Preparation of polymer solution: 0.1-0.5 g of lactide is dissolved in 10 mL of chloroform to prepare a solution with a concentration of 1-5% (w / v).

[0084] (c) Preparing a high molecular polymer solution containing an oxygen-releasing source: mixing calcium peroxide in a ratio of 0.1 to 10% (w / w) into the lactide solution.

[0085] (d) preparing an emulsion: mixing solution c and solution a in a volume ratio of 10:0.5 to 10:2.5, stirring and emulsifying at room temperature to obtain an emulsion.

[0086] (e) Dispersion preparation: Weigh 0.5-2 g of polyvinyl alcohol solid into 100 mL of deionized water, stir at 90° C. until completely dissolved, and prepare a solution with a concentration of 0.5% to 2% (w / v).

[0087] (f) Forming of microspheres: Solution d is added to solution e at a volume ratio of 30:1 to 100:1 under stirring until the organic solvent is completely evaporated. The porogen is removed.

[0088] In this embodiment, an oxygen release experimental method of lactide-calcium peroxide oxygen-releasing microspheres comprises the following steps:

[0089] Another 0.1 g of the lactide-calcium peroxide oxygen-releasing microspheres prepared in step (f) above was placed in 100 mL of 1×PBS solution and shaken in a constant temperature shaker at 37°C for oxygen release test. Five parallel control groups were set for each concentration of CaO2, and the solution was changed every day and the oxygen release was measured.

Claims

1. An oxygen-releasing microsphere material for bone tissue engineering, characterized in that: It is prepared by using an oxygen-releasing source and a high molecular polymer. The oxygen-releasing source is an inorganic peroxide, specifically one or more combinations of calcium peroxide, magnesium peroxide, and strontium peroxide. The high molecular polymer is divided into an oil-soluble high molecular polymer and a water-soluble high molecular polymer.

2. The oxygen-releasing microsphere material for bone tissue engineering according to claim 1, characterized in that: The oil-soluble high molecular polymer used is one or more combinations of polylactic acid, polyglycolic acid, polylactide-glycolide and polycaprolactone.

3. The oxygen-releasing microsphere material for bone tissue engineering according to claim 1, characterized in that: The water-soluble high molecular polymer used is one or more combinations of chitosan, carboxymethyl chitosan, gelatin, silk fibroin, sericin, sodium alginate and sodium hyaluronate.

4. The oxygen-releasing microsphere material for bone tissue engineering according to claim 1, characterized in that: The composition ratio of the inorganic peroxide to the high molecular polymer is that the mass proportion of the inorganic peroxide is 0.1-7%.

5. The oxygen-releasing microsphere material for bone tissue engineering according to claim 1, characterized in that: The oxygen-releasing microspheres have a diameter of 50-500 μm, a pore size of 15-100 μm, and a porosity of 50-96%.

6. The oxygen-releasing microsphere material for tissue engineering according to claim 1, characterized in that: Oxygen-releasing microspheres have the ability to continuously and stably release oxygen in the body. The oxygen release time is 1-30 days and the oxygen release amount is 0.1-1.2 mg / L.

7. The oxygen-releasing microsphere material for bone tissue engineering according to claim 1, characterized in that: The oxygen-releasing microsphere material can be used alone in tissue defect sites or loaded with animal cells for use in tissue defect sites.

8. A method for preparing oxygen-releasing microsphere materials for bone tissue engineering, characterized in that: It includes emulsion solvent evaporation-porogen method and emulsion-freeze drying method.

9. The emulsion solvent volatilization-porogen method according to claim 8, characterized in that: The steps include: (a) Preparation of porogen solution: dissolving a suitable porogen in deionized water to prepare a solution with a concentration of 0.5 to 10% (w / v); (b) Preparation of a polymer solution: the oil-soluble polymer is dissolved in an organic solvent to prepare a solution with a concentration of 1 to 5% (w / v); (c) preparing a high molecular polymer solution containing an oxygen-releasing source: mixing the peroxide in a ratio of 0.1 to 10% (w / w) into the polymer solution; (d) preparing an emulsion: mixing solution c and solution a in a volume ratio of 10:0.5 to 10:2.5, stirring and emulsifying at room temperature to obtain an emulsion; (e) Dispersion liquid preparation: polyvinyl alcohol is used as the dispersion liquid, and deionized water is used to prepare the dispersion liquid, with a concentration of 0.5% to 2% (w / v); (f) Forming of microspheres: Solution d is added to solution e at a volume ratio of 30:1 to 100:1 under stirring, and the mixture is stirred until the organic solvent is completely evaporated to remove the porogen.

10. The emulsion-freeze drying method according to claim 8, characterized in that: The steps include: (a) preparing an emulsion: adding sorbitan oleate to oily ether at a volume ratio of 0.1:10 to 0.5:10, and stirring until the sorbitan oleate is completely dissolved; (b) preparing a polymer solution: dissolving the polymer in deionized water and stirring until completely dissolved, with a concentration of 1 to 5% (w / v); (c) preparing a high molecular polymer solution containing an oxygen-releasing source: mixing the peroxide in a ratio of 0.1 to 10% (w / w) into the polymer solution; (d) Forming microspheres: Add the solution of step d to the solution of step a at a volume ratio of 1:2 to 1:8, stir and emulsify for 1 to 3 minutes, quickly pour in petroleum ether at -10 to -75°C, remove the mobile liquid phase, obtain ice crystal particles and place them in a vacuum freeze dryer to dry until the water evaporates completely.

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