Polylactic acid composite porous microsphere with high dispersion and high hydroxyapatite encapsulation and preparation method thereof

Abstract

The invention relates to the technical field of tissue engineering, in particular to a polylactic acid composite porous microsphere with high dispersion and high hydroxyapatite encapsulation and a preparation method thereof.The preparation method comprises the following steps that an amphiphilic medium and hydroxyapatite are subjected to ball-milling compounding in an organic solvent I, the organic solvent I is removed through drying, and a compound is formed; dissolving the high polymer material and the compound in an organic solvent II to form an organic phase; dissolving a pore-foaming agent in water to form a water phase I; dissolving a surfactant in water to form a water phase II; dropwise adding the water phase I into the organic phase to form an emulsion; dropwise adding the emulsion into the water phase II, and uniformly mixing; stirring until the organic solvent is completely volatilized, forming dispersed microspheres in a water phase, filtering, washing and drying to obtain the composite porous microspheres. The prepared composite porous microspheres can be used as a scaffold for growth of adult cells, can also be used as a scaffold for attachment during growth and differentiation of stem cells, and can be applied to tissue engineering.

Description

Technical Field

[0001] The invention relates to the technical field of tissue engineering, and in particular to a polylactic acid composite porous microsphere with high dispersion and high encapsulation of hydroxyapatite and a preparation method thereof. Background Art

[0002] At present, the polyester materials used to prepare porous microspheres mainly include PLLA, PLGA, PLCL, etc. Polyester has attracted more and more attention due to its good properties, including excellent biocompatibility, biodegradability, non-toxicity, non-immunogenicity, morphological controllability and good mechanical strength. It has become one of the most commonly used biomaterials for preparing microspheres and is widely used in the pharmaceutical industry and medical devices. At present, many polyester-based medical devices have been approved by the US Food and Drug Administration (FDA) for drug delivery, microcarriers and tissue engineering.

[0003] However, the prepared porous microspheres also have various problems. The main reason is that the degradation products are acidic and the pH value is reduced, which can easily cause inflammation. The inorganic material hydroxyapatite is widely used in tissue engineering, especially in orthopedic research, because of its good biocompatibility. Hydroxyapatite releases weakly alkaline ions in aqueous solution, which can be used to neutralize the acidity of the degradation products of the above-mentioned polymer materials, and can also improve the biological activity of the material. However, hydroxyapatite is a hydrophilic material and is incompatible with polymer materials, so an amphiphilic medium is needed to improve the dispersibility and encapsulation rate of hydroxyapatite in the preparation process of composite porous microspheres.

[0004] Currently, there are few reports on polylactic acid and hydroxyapatite used together as carriers, and there are no reports at home and abroad on the method of using amphiphilic media to efficiently disperse and encapsulate hydroxyapatite. Summary of the invention

[0005] One of the purposes of the present invention is to provide a method for preparing highly dispersed and highly encapsulated hydroxyapatite polylactic acid composite porous microspheres, which has low cost and simple operation, and the prepared microspheres have controllable particle size and are easy to scale up and can be used as a scaffold for cell adhesion in tissue engineering.

[0006] The second purpose of the present invention is to provide a highly dispersed and highly encapsulated hydroxyapatite polylactic acid composite porous microsphere, which has uniform appearance size and rich pores. The particle size is 50-500μm and the pore size is 10-100μm. The interior of the microsphere is connected with the external environment and the water absorption rate is 500-5000%.

[0007] The solution adopted by the present invention to achieve one of the purposes is: a method for preparing polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite, comprising the following steps:

[0008] (1) ball-milling the amphiphilic medium and hydroxyapatite in an organic solvent I, and drying to remove the organic solvent I to form a composite;

[0009] (2) dissolving the polymer material and the composite simultaneously in an organic solvent II to form an organic phase;

[0010] (3) dissolving the porogen in water to form an aqueous phase I;

[0011] (4) dissolving the surfactant in water to form an aqueous phase II;

[0012] (5) adding the aqueous phase I dropwise to the organic phase, stirring and emulsifying to form an emulsion;

[0013] (6) Add the emulsion dropwise into the aqueous phase II under stirring and mix well;

[0014] (7) stirring at room temperature until the organic solvent is completely volatilized to form dispersed microspheres in the aqueous phase, and filtering, washing and drying to obtain polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite.

[0015] Preferably, in step (1), the amphiphilic medium is at least one of phospholipids, soybean lecithin, cholesterol and glycolipids, and the mass ratio of the amphiphilic medium to hydroxyapatite is 1:1-10.

[0016] Preferably, in step (1), the organic solvent I is at least one of acetone, dichloromethane, ethanol, dimethyl sulfoxide, tetrahydrofuran and ethyl acetate.

[0017] Preferably, in step (2), the polymer material is at least one of PLLA, PDLA, and PDLLA, the molecular weight of the polymer material is 1000-200000Da, and the organic solvent II is at least one of acetone, dichloromethane, ethanol, dimethyl sulfoxide, tetrahydrofuran, and ethyl acetate.

[0018] Preferably, in step (2), the final concentration of the polymer material in the organic phase is 0.1%-30% (w / v), and the final concentration of hydroxyapatite in the organic phase is 0.1%-30% (w / v).

[0019] Preferably, in step (3), the porogen is at least one of ammonium bicarbonate, sodium bicarbonate, F127, sodium oleate and gelatin, and the final concentration of the porogen dissolved in the aqueous phase I is 0.1%-30% (w / v).

[0020] Preferably, in step (4), the surfactant is PVA, and the final concentration of the surfactant in the aqueous phase II is 0.01%-10% (w / v).

[0021] Preferably, in step (5), the volume ratio of aqueous phase I to organic phase is 1:1-10, and aqueous phase I is uniformly added dropwise to the organic phase at a rate of 1-10s / drop.

[0022] Preferably, in step (6), the volume ratio of aqueous phase II to organic phase is 5-20:1, and the emulsion is uniformly added dropwise to aqueous phase II at a rate of 1-5 s / drop under stirring.

[0023] The solution adopted by the present invention to achieve the second purpose is: a polylactic acid composite porous microsphere with high dispersion and high encapsulation of hydroxyapatite is prepared by the preparation method.

[0024] The present invention has the following advantages and beneficial effects:

[0025] The method for preparing the polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite of the present invention is simple and convenient to operate, low in cost, safe to use, and conducive to market promotion.

[0026] The polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite prepared by the preparation method of the present invention can be used as a scaffold for cell adhesion in tissue engineering. The composite porous microspheres have uniform appearance size and rich pores, with a particle size of 50-500 μm and a pore size of 10-100 μm, connecting the inside of the microspheres with the external environment, improving the dispersibility of hydroxyapatite and the encapsulation rate in the porous microspheres through the amphiphilic medium, and increasing its hydrophilicity by loading hydroxyapatite, and its water absorption rate is 500-5000%.

[0027] The polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite prepared by the preparation method of the present invention can be used as a scaffold for the growth of adult cells, and can also be used as a scaffold for the attachment of stem cells during growth and differentiation, and can be applied to tissue engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The dispersion diagram (a) and DLS diagram (b) of hydroxyapatite dissolved in dichloromethane with and without the addition of amphiphilic medium;

[0029] Figure 2 Schematic diagram of porous composite microspheres without adding amphiphilic medium (a) and with adding amphiphilic medium (b) of the present invention;

[0030] Figure 3 Thermogravimetric graphs of two polylactic acid composite porous microspheres loaded with 10% hydroxyapatite. DETAILED DESCRIPTION

[0031] For a better understanding of the present invention, the following examples are provided to further illustrate the present invention, but the present invention is not limited to the following examples.

[0032] A method for preparing polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite, comprising:

[0033] The amphiphilic medium and hydroxyapatite are compounded in an organic solvent I by ball milling for 5-60 minutes, and after the ball milling, the mixture is placed in a vacuum drying oven for 5-60 minutes to volatilize the organic solvent, thereby forming a composite;

[0034] Dissolving the polymer material and the composite in the organic solvent II at the same time, so that the polymer material and the composite are dispersed in the organic solvent to form an organic phase;

[0035] The porogen is dissolved in water at room temperature to form an aqueous phase I;

[0036] Dissolve the surfactant in water and stir to dissolve at room temperature to form aqueous phase II;

[0037] The aqueous phase I is uniformly added dropwise to the organic phase at a rate of 1-10s / drop, and emulsified for 5-30min at a magnetic stirring speed of 500-1500rpm to form an emulsion;

[0038] The emulsion was uniformly added dropwise to the aqueous phase II at a rate of 1-5 s / drop under stirring, and stirred for 1-5 h at a mechanical stirring rate of 100-500 rpm;

[0039] The mixture was stirred continuously at room temperature until the organic solvent was completely volatilized to form dispersed microspheres in the aqueous phase. The microspheres were collected by suction filtration, washed with deionized water for multiple times, and then freeze-dried to obtain polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite.

[0040] Furthermore, the amphiphilic medium is at least one of phospholipids, soybean lecithin, cholesterol, and glycolipids, the mass ratio of the amphiphilic medium to hydroxyapatite is 1:1-1:10, and the organic solvent I is at least one of acetone, dichloromethane, ethanol, dimethyl sulfoxide, tetrahydrofuran, and ethyl acetate.

[0041] Furthermore, the polymer material is at least one of left-handed polylactic acid (PLLA), right-handed polylactic acid (PDLA), and racemic polylactic acid (PDLLA), and the molecular weight of the polymer material is 1000-200000Da. The organic solvent II is at least one of acetone, dichloromethane, ethanol, dimethyl sulfoxide, tetrahydrofuran, and ethyl acetate.

[0042] Furthermore, the final concentration of the polymer material in the organic phase is 0.1-30% (w / v), and the final concentration of the hydroxyapatite in the organic phase is 0.1-30% (w / v).

[0043] Furthermore, the porogen is at least one of ammonium bicarbonate, sodium bicarbonate, F127, sodium oleate, and gelatin, and the final concentration of the porogen dissolved in the aqueous phase I is 0.1-30% (w / v).

[0044] Furthermore, the surfactant is PVA, and the final concentration of the surfactant in the aqueous phase II is 0.01-10% (w / v).

[0045] Furthermore, the volume ratio of the aqueous phase I to the organic phase is 1:1 to 1:10.

[0046] Furthermore, the volume ratio of the aqueous phase II to the organic phase is 5:1 to 20:1.

[0047] Example 1

[0048] Weigh 200 mg of hydroxyapatite and 400 mg of soybean lecithin into two ball mill jars, add appropriate amount of acetone to balance, and ball mill for 30 minutes. After the ball milling is completed, the product is collected and placed in a 37°C vacuum drying oven for drying for 30 minutes to obtain a composite soybean lecithin-encapsulated hydroxyapatite (SL@HAp). The composite containing an equal amount of hydroxyapatite and pure hydroxyapatite are added to an equal amount of dichloromethane, and the particle size distribution is observed by a Malvern laser particle size analyzer.

[0049] from Figure 1 a It can be seen that due to its strong hydrophilicity, when hydroxyapatite is added to hydrophobic dichloromethane, it will immediately sink to the bottom and form an obvious precipitate. In contrast, hydroxyapatite coated with soybean lecithin exhibits excellent dispersion performance. It can not only be evenly dispersed in dichloromethane, but also does not sink to the bottom after standing for a period of time. Figure 1 b It can be seen that since hydroxyapatite is a nanoparticle that is very easy to agglomerate, after pure hydroxyapatite is dissolved in dichloromethane, two peaks at 142nm and 521nm appear. However, the particle size distribution of hydroxyapatite after being coated with soybean lecithin in dichloromethane is significantly narrowed, with only one peak appearing, and the particle size is about 50nm, which further confirms the good dispersibility and stability of hydroxyapatite coated with soybean lecithin in dichloromethane.

[0050] Example 2

[0051] Weigh 1.5g of L-polylactic acid and 150mg of hydroxyapatite, dissolve in 25mL of chloroform, and stir magnetically to form an organic phase. Weigh 350mg of sodium bicarbonate and dissolve in 5mL of water, dissolve at room temperature to form aqueous phase I. Weigh 2.5g and dissolve in 250mL of deionized water, and dissolve by mechanical stirring at a rate of 350rpm to form aqueous phase II. Under a magnetic stirring speed of 780rpm, the above aqueous phase I is dripped into the organic phase at a rate of 1s / drop, emulsified for 10min to form a primary emulsion, and the above primary emulsion is dripped into the aqueous phase II at a rate of 1s / drop. After emulsification for 3h, continue stirring at room temperature overnight to volatilize the organic solvent, form dispersed microspheres in the aqueous phase, collect the microspheres by suction filtration, and wash them with deionized water several times and freeze-dry to obtain hydroxyapatite / polylactic acid composite porous microspheres (PH);. 1.5 g of L-polylactic acid and 450 mg of soybean lecithin-encapsulated hydroxyapatite complex were weighed and dissolved in 25 mL of methyl chloride, and magnetic stirring was performed to form an organic phase. The subsequent steps were the same as above to prepare highly dispersed and highly encapsulated hydroxyapatite L-polylactic acid composite porous microspheres (PSH).

[0052] Scanning electron microscope observation showed that the hydroxyapatite / L-polylactic acid porous microspheres were blocky or cake-shaped, and did not form an ideal spherical structure. Moreover, these microspheres themselves did not have obvious pore structures. Only a few microspheres had pores, and the number was very limited, usually only 1-2 large pores ( Figure 2 a). The appearance of soybean lecithin @ hydroxyapatite / L-polylactic acid porous microspheres has a rich pore structure, connecting the inside of the microspheres with the external environment ( Figure 2 b), indicating that the addition of soybean lecithin can make the microspheres more regular in shape and more uniform in pore size.

[0053] Example 3

[0054] First, poly(L-lactic acid), soybean lecithin, and hydroxyapatite were heated from room temperature to 800°C at a heating rate of 10°C / min in a nitrogen atmosphere. It can be seen that hydroxyapatite hardly undergoes thermal decomposition before 800°C. This is because the high thermal stability of hydroxyapatite enables it to maintain its structure and properties under high temperature conditions, while soybean lecithin and poly(L-lactic acid) have basically completed thermal decomposition at about 550°C ( Figure 3 a), so the approximate content of hydroxyapatite in the composite material can be obtained by thermogravimetric analysis. Hydroxyapatite / L-polylactic acid porous microspheres loaded with 10% hydroxyapatite and soybean lecithin@hydroxyapatite / L-polylactic acid porous microspheres were heated from room temperature to 800°C at a rate of 10°C / min in a nitrogen atmosphere. It can be seen that the final residual amounts of the two microspheres were 4.3% and 8.3%, respectively, and the encapsulation rates were 42.5% and 83%, respectively ( Figure 3 b). The above results fully prove that soybean lecithin can effectively improve the encapsulation rate of hydroxyapatite.

[0055] Example 4

[0056] Weigh 150 mg of hydroxyapatite and 300 mg of soybean lecithin into two ball mills, add appropriate amount of acetone to balance, and ball mill for 30 minutes in a ball mill. After the ball milling is completed, collect the product and put it into a vacuum drying oven at 37°C for 30 minutes to obtain a composite. Weigh 1.5 g of polylactic acid, dissolve 450 mg of the composite in 25 mL of chloromethane, and stir magnetically to form an organic phase. Weigh 350 mg of sodium bicarbonate and dissolve it in 5 mL of water, dissolve at room temperature to form aqueous phase I. Weigh 2.5 g and dissolve it in 250 mL of deionized water, stir mechanically at a rate of 350 rpm to dissolve to form aqueous phase II. Under magnetic stirring at a speed of 780 rpm, the above-mentioned aqueous phase I was dripped into the organic phase at a rate of 1 s / drop, and emulsified for 10 minutes to form a primary emulsion. The above-mentioned primary emulsion was dripped into the aqueous phase II at a rate of 1 s / drop. After emulsification for 3 hours, stirring was continued at room temperature overnight to volatilize the organic solvent to form dispersed microspheres in the aqueous phase. The microspheres were collected by suction filtration, washed with deionized water several times, and then freeze-dried to obtain polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite.

[0057] Scanning electron microscope observation showed that the average particle size of soybean lecithin @ hydroxyapatite / polylactic acid porous microspheres was 304 ± 32.6 μm, and the appearance had a rich pore structure, connecting the inside of the microspheres with the external environment, and the average diameter of the pore structure was 33.4 ± 5.4 μm. The water absorption rate was 3243.7%.

[0058] Example 5

[0059] Weigh 300 mg of hydroxyapatite and 300 mg of soybean lecithin in two ball mills, add an appropriate amount of ethanol to balance, and ball mill in a ball mill for 60 minutes. After the ball milling is completed, collect the product and put it in a vacuum drying oven at 37°C to dry for 30 minutes to obtain a complex. Weigh 2 g of polylactic acid, dissolve 600 mg of the complex in 20 mL of chloromethane, and stir magnetically to form an organic phase. Weigh 400 mg of sodium bicarbonate and dissolve it in 5 mL of water, dissolve at room temperature to form aqueous phase I. Weigh 2 g of solution in 300 mL of deionized water, stir mechanically at a rate of 400 rpm to dissolve to form aqueous phase II. Under magnetic stirring at a speed of 750 rpm, the above-mentioned aqueous phase I was dripped into the organic phase at a rate of 1 s / drop, and emulsified for 15 minutes to form a primary emulsion. The above-mentioned primary emulsion was dripped into the aqueous phase II at a rate of 1 s / drop. After emulsification for 3 hours, stirring was continued at room temperature overnight to volatilize the organic solvent to form dispersed microspheres in the aqueous phase. The microspheres were collected by suction filtration, washed with deionized water several times, and then freeze-dried to obtain polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite.

[0060] Scanning electron microscope observation showed that the average particle size of soybean lecithin @ hydroxyapatite / polylactic acid porous microspheres was 280 ± 23.5 μm, and the appearance had a rich pore structure, connecting the inside of the microspheres with the external environment, and the average diameter of the pore structure was 41.2 ± 6.0 μm. The water absorption rate was 3865.3%.

[0061] Example 6

[0062] Weigh 100 mg of hydroxyapatite and 300 mg of soybean lecithin in two ball mills, add appropriate amount of acetone to balance, and ball mill in a ball mill for 15 minutes. After the ball milling is completed, collect the product and put it in a vacuum drying oven at 37°C to dry for 30 minutes to obtain a composite. Weigh 1 g of polylactic acid, dissolve 400 mg of the composite in 20 mL of chloromethane, and stir magnetically to form an organic phase. Weigh 300 mg of sodium bicarbonate and dissolve it in 5 mL of water, dissolve at room temperature to form aqueous phase I. Weigh 3 g and dissolve it in 300 mL of deionized water, stir mechanically at a rate of 250 rpm to dissolve to form aqueous phase II. Under magnetic stirring at a speed of 800 rpm, the above-mentioned aqueous phase I was dripped into the organic phase at a rate of 1 s / drop, and emulsified for 5 minutes to form a primary emulsion. The above-mentioned primary emulsion was dripped into the aqueous phase II at a rate of 1 s / drop. After emulsification for 3 hours, stirring was continued at room temperature overnight to volatilize the organic solvent to form dispersed microspheres in the aqueous phase. The microspheres were collected by suction filtration, washed with deionized water several times, and then freeze-dried to obtain polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite.

[0063] Scanning electron microscope observation showed that the average particle size of soybean lecithin @ hydroxyapatite / polylactic acid porous microspheres was 350 ± 32.2 μm, and the appearance had a rich pore structure, connecting the inside of the microspheres with the external environment, and the average diameter of the pore structure was 31.2 ± 6.0 μm. The water absorption rate was 2804.6%.

[0064] Example 7

[0065] Weigh 100 mg of hydroxyapatite and 500 mg of soybean lecithin in two ball mills, add an appropriate amount of ethanol to balance, and ball mill in a ball mill for 90 minutes. After the ball milling is completed, collect the product and put it in a vacuum drying oven at 37°C to dry for 30 minutes to obtain a complex. Weigh 2 g of polylactic acid, dissolve 600 mg of the complex in 30 mL of chloromethane, and stir magnetically to form an organic phase. Weigh 350 mg of sodium bicarbonate and dissolve it in 5 mL of water, dissolve at room temperature to form aqueous phase I. Weigh 2.5 g and dissolve it in 300 mL of deionized water, stir mechanically at a rate of 450 rpm to dissolve to form aqueous phase II. Under magnetic stirring at a speed of 720 rpm, the above-mentioned aqueous phase I was dripped into the organic phase at a rate of 1 s / drop, and emulsified for 10 minutes to form a primary emulsion. The above-mentioned primary emulsion was dripped into the aqueous phase II at a rate of 1 s / drop. After emulsification for 3 hours, stirring was continued at room temperature overnight to volatilize the organic solvent to form dispersed microspheres in the aqueous phase. The microspheres were collected by suction filtration, washed with deionized water several times, and then freeze-dried to obtain polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite.

[0066] Scanning electron microscope observation shows that the average particle size of soybean lecithin @ hydroxyapatite / polylactic acid porous microspheres is 280 ± 22.5 μm, and the appearance has a rich pore structure, which connects the inside of the microspheres with the external environment. The average diameter of the pore structure is 36.2 ± 5.4 μm. The water absorption rate is 3001.3%.

[0067] Example 8

[0068] Weigh 200 mg of hydroxyapatite and 400 mg of soybean lecithin in two ball mills, add appropriate amount of acetone to balance, and ball mill in a ball mill for 45 minutes. After the ball milling is completed, collect the product and put it in a vacuum drying oven at 37°C to dry for 30 minutes to obtain a composite. Weigh 1.5 g of polylactic acid, dissolve 600 mg of the composite in 20 mL of chloromethane, and stir magnetically to form an organic phase. Weigh 350 mg of sodium bicarbonate and dissolve it in 5 mL of water, dissolve at room temperature to form aqueous phase I. Weigh 2 g and dissolve it in 200 mL of deionized water, stir mechanically at a rate of 300 rpm to dissolve to form aqueous phase II. Under magnetic stirring at a speed of 700 rpm, the above-mentioned aqueous phase I was dripped into the organic phase at a rate of 1 s / drop, and emulsified for 15 minutes to form a primary emulsion. The above-mentioned primary emulsion was dripped into the aqueous phase II at a rate of 1 s / drop. After emulsification for 3 hours, stirring was continued at room temperature overnight to volatilize the organic solvent to form dispersed microspheres in the aqueous phase. The microspheres were collected by suction filtration, washed with deionized water several times, and then freeze-dried to obtain polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite.

[0069] Scanning electron microscope observation showed that the average particle size of soybean lecithin @ hydroxyapatite / polylactic acid porous microspheres was 380 ± 15.1 μm, and the appearance had a rich pore structure, connecting the inside of the microspheres with the external environment, and the average diameter of the pore structure was 35.8 ± 5.4 μm. The water absorption rate was 3158.9%.

[0070] The above is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and changes can be made without departing from the principle of the present invention, and these improvements and changes are also regarded as the protection scope of the present invention.

Claims

1. A method for preparing highly dispersed and highly encapsulated hydroxyapatite polylactic acid composite porous microspheres, characterized in that: The following steps are involved: (1) ball-milling the amphiphilic medium and hydroxyapatite in an organic solvent I, and drying to remove the organic solvent I to form a composite; (2) dissolving the polymer material and the composite simultaneously in an organic solvent II to form an organic phase; (3) dissolving the porogen in water to form an aqueous phase I; (4) dissolving the surfactant in water to form an aqueous phase II; (5) adding the aqueous phase I dropwise to the organic phase, stirring and emulsifying to form an emulsion; (6) Add the emulsion dropwise into the aqueous phase II under stirring and mix well; (7) stirring at room temperature until the organic solvent is completely volatilized to form dispersed microspheres in the aqueous phase, and filtering, washing and drying to obtain polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite.

2. The method for preparing highly dispersed and highly encapsulated hydroxyapatite polylactic acid composite porous microspheres according to claim 1, characterized in that: In the step (1), the amphiphilic medium is at least one of phospholipids, soybean lecithin, cholesterol, and glycolipids, and the mass ratio of the amphiphilic medium to hydroxyapatite is 1:1-10.

3. The method for preparing highly dispersed and highly encapsulated hydroxyapatite polylactic acid composite porous microspheres according to claim 1, characterized in that: In the step (1), the organic solvent I is at least one of acetone, dichloromethane, ethanol, dimethyl sulfoxide, tetrahydrofuran, and ethyl acetate.

4. The method for preparing highly dispersed and highly encapsulated hydroxyapatite polylactic acid composite porous microspheres according to claim 1, characterized in that: In the step (2), the polymer material is at least one of PLLA, PDLA, and PDLLA, the molecular weight of the polymer material is 1000-200000Da, and the organic solvent II is at least one of acetone, dichloromethane, ethanol, dimethyl sulfoxide, tetrahydrofuran, and ethyl acetate.

5. The method for preparing highly dispersed and highly encapsulated hydroxyapatite polylactic acid composite porous microspheres according to claim 1, characterized in that: In the step (2), the final concentration of the polymer material in the organic phase is 0.1%-30% (w / v), and the final concentration of hydroxyapatite in the organic phase is 0.1%-30% (w / v).

6. The method for preparing highly dispersed and highly encapsulated hydroxyapatite polylactic acid composite porous microspheres according to claim 1, characterized in that: In the step (3), the porogen is at least one of ammonium bicarbonate, sodium bicarbonate, F127, sodium oleate, and gelatin, and the final concentration of the porogen dissolved in the aqueous phase I is 0.1%-30% (w / v).

7. The method for preparing highly dispersed and highly encapsulated hydroxyapatite polylactic acid composite porous microspheres according to claim 1, characterized in that: In the step (4), the surfactant is PVA, and the final concentration of the surfactant in the aqueous phase II is 0.01%-10% (w / v).

8. The method for preparing highly dispersed and highly encapsulated hydroxyapatite polylactic acid composite porous microspheres according to claim 1, characterized in that: In the step (5), the volume ratio of the aqueous phase I to the organic phase is 1:1-10, and the aqueous phase I is uniformly added dropwise to the organic phase at a rate of 1-10s / drop.

9. The method for preparing highly dispersed and highly encapsulated hydroxyapatite polylactic acid composite porous microspheres according to claim 1, characterized in that: In the step (6), the volume ratio of aqueous phase II to organic phase is 5-20:1, and the emulsion is uniformly added dropwise into aqueous phase II at a rate of 1-5s / drop under stirring.

10. A polylactic acid composite porous microsphere with high dispersion and high encapsulation of hydroxyapatite, characterized in that: The method is prepared according to any one of claims 1 to 9.

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