Highly dispersed and encapsulated hydroxyapatite polylactic acid composite porous microspheres and preparation method thereof

Abstract

The application relates to the technical field of tissue engineering, and particularly relates to a kind of high-dispersed high-encapsulated polylactic acid composite porous microspheres of hydroxyapatite and a preparation method, which comprises the following steps: ball-milling and compounding amphiphilic medium and hydroxyapatite in organic solvent I, removing the organic solvent I by drying to form a compound; dissolving the polymer material and the compound in organic solvent II to form an organic phase; dissolving a pore-forming agent in water to form water phase I; dissolving a surfactant in water to form water phase II; adding water phase I to the organic phase dropwise to form an emulsion; adding the emulsion to water phase II dropwise and mixing uniformly; stirring until the organic solvent is completely volatilized, and dispersed microspheres are formed in the water phase; after filtration, washing and drying, the composite porous microspheres are obtained. The composite porous microspheres prepared by the application can be used as a scaffold for growth of adult cells, and can also be used as a scaffold for growth and differentiation of stem cells, and are applied to tissue engineering.

Description

Technical Field

[0001] This invention relates to the technical field of tissue engineering, specifically to a polylactic acid composite porous microsphere with highly dispersed and highly encapsulated hydroxyapatite and its preparation method. Background Technology

[0002] Currently, the main polyester materials used for preparing porous microspheres include PLLA, PLGA, and PLCL. Due to their excellent properties, including superior biocompatibility, biodegradability, non-toxicity, non-immunogenicity, morphological controllability, and good mechanical strength, polyesters have attracted increasing attention and have become one of the most commonly used biomaterials for microsphere preparation, widely applied in the pharmaceutical industry and medical devices. Currently, many polyester-based medical devices have been approved by the U.S. Food and Drug Administration (FDA) for drug delivery, microcarriers, and tissue engineering.

[0003] However, the prepared porous microspheres also have various problems, mainly because the degradation products are acidic, leading to a decrease in pH and increased susceptibility to inflammation. Hydroxyapatite, an inorganic material, is widely used in tissue engineering, particularly in orthopedic research, due to its excellent biocompatibility. Hydroxyapatite releases weakly alkaline ions in aqueous solution, which can neutralize the acidity of the degradation products of the aforementioned polymeric materials and also improve the bioactivity of the material. However, hydroxyapatite is a hydrophilic material and incompatible with polymeric materials. Therefore, an amphiphilic medium is needed to improve the dispersibility and encapsulation efficiency of hydroxyapatite during the preparation of composite porous microspheres.

[0004] Currently, there are few reports on polylactic acid and hydroxyapatite used together as a carrier, and no methods for efficiently dispersing and encapsulating hydroxyapatite using amphiphilic media have been reported domestically or internationally. Summary of the Invention

[0005] One of the objectives of this invention is to provide a method for preparing polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite. This method is low in cost, simple to operate, and the particle size of the prepared microspheres is controllable. It is easy to scale up and can be used as a scaffold for cell adhesion in tissue engineering.

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

[0007] One of the solutions adopted to achieve the objective of this invention is: a method for preparing polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite, comprising the following steps:

[0008] (1) The amphiphilic medium and hydroxyapatite were ball-milled in organic solvent I and dried to remove organic solvent I, forming a complex.

[0009] (2) The polymer material and the complex are simultaneously dissolved in organic solvent II to form an organic phase;

[0010] (3) Dissolve the pore-forming agent in water to form aqueous phase I;

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

[0012] (5) Add the above aqueous phase I dropwise to the organic phase and stir to emulsify and form an emulsion;

[0013] (6) Add the emulsion dropwise to aqueous phase II while stirring, and mix thoroughly;

[0014] (7) Stir at room temperature until the organic solvent is completely evaporated, and dispersed microspheres are formed in the aqueous phase. After filtration, washing and drying, polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite are obtained.

[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 1000-200000 Da, 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 pore-forming agent is at least one of ammonium bicarbonate, sodium bicarbonate, F127, sodium oleate, and gelatin, and the final concentration of the pore-forming agent dissolved in 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 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 to organic phase at a rate of 1-10 s / 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 to aqueous phase II at a rate of 1-5s / drop while being stirred.

[0023] The solution adopted to achieve the second objective of this invention is: a highly dispersed and highly encapsulated polylactic acid composite porous microsphere of hydroxyapatite, prepared by the aforementioned preparation method.

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

[0025] The method for preparing highly dispersed and highly encapsulated hydroxyapatite polylactic acid composite porous microspheres 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 highly dispersed and highly encapsulated hydroxyapatite prepared by the method of this invention can be used as scaffolds for cell adhesion in tissue engineering. The composite porous microspheres have uniform size and abundant pores, with a particle size of 50-500 μm and a pore size of 10-100 μm. This interconnects the interior of the microspheres with the external environment. The amphiphilic medium improves the dispersibility of hydroxyapatite and its encapsulation rate within the porous microspheres. Loading hydroxyapatite increases its hydrophilicity, with a water absorption rate of 500-5000%.

[0027] The polylactic acid composite porous microspheres with highly dispersed and highly encapsulated hydroxyapatite prepared by the preparation method of the present invention can be used as scaffolds for adult cell growth, and can also be used as scaffolds for stem cell growth and differentiation, and can be applied to tissue engineering. Attached Figure Description

[0028] Figure 1 The dispersion diagram (a) and DLS diagram (b) of hydroxyapatite dissolved in dichloromethane with and without an amphiphilic medium are shown.

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

[0030] Figure 3 Thermogravimetric analysis of two polylactic acid composite porous microspheres loaded with 10% hydroxyapatite. Detailed Implementation

[0031] To better understand the present invention, the following embodiments are further illustrations of the present invention, but the content of the present invention is not limited to the following embodiments.

[0032] A method for preparing highly dispersed and highly encapsulated hydroxyapatite polylactic acid composite porous microspheres, comprising:

[0033] The amphiphilic dispersant and hydroxyapatite were ball-milled in organic solvent I for 5-60 minutes to form a composite. After ball milling, the composite was placed in a vacuum drying oven for 5-60 minutes to evaporate the organic solvent and form a composite.

[0034] The polymer material and the complex are simultaneously dissolved in organic solvent II, so that the polymer material and the complex are dispersed in the organic solvent to form an organic phase;

[0035] The pore-forming agent is dissolved in water at room temperature to form aqueous phase I;

[0036] The surfactant is dissolved in water and stirred at room temperature to form aqueous phase II;

[0037] The aqueous phase I was added uniformly to the organic phase at a rate of 1-10 drops / second, and emulsified for 5-30 minutes under magnetic stirring at 500-1500 rpm to form an emulsion.

[0038] The emulsion was added uniformly to aqueous phase II at a rate of 1-5 drops / second while stirring, and stirred at a mechanical stirring rate of 100-500 rpm for 1-5 hours.

[0039] The mixture was continuously stirred at room temperature until the organic solvent completely evaporated, forming dispersed microspheres in the aqueous phase. The microspheres were collected by vacuum filtration, washed multiple times with deionized water, 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 to 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 selected from polylactic acid (PLLA), polylactic acid (PDLA), and racemic polylactic acid (PDLLA), and the molecular weight of the polymer material is 1000-200000 Da. The organic solvent II is at least one selected from 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 pore-forming agent is at least one of ammonium bicarbonate, sodium bicarbonate, F127, sodium oleate, and gelatin, and the final concentration of the pore-forming agent dissolved in aqueous phase I is 0.1-30% (w / v).

[0044] Furthermore, the surfactant is PVA, and the final concentration of the surfactant in 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] 200 mg of hydroxyapatite and 400 mg of soybean lecithin were weighed into two ball mill jars, and an appropriate amount of acetone was added to balance the mixture. The mixture was then ball milled for 30 min. After ball milling, the product was collected and dried in a vacuum drying oven at 37 °C for 30 min to obtain the complex soybean lecithin-encapsulated hydroxyapatite (SL@HAp). The complex containing an equal amount of hydroxyapatite and pure hydroxyapatite were added to an equal amount of dichloromethane, and the particle size distribution was observed using a Malvern laser particle size analyzer.

[0049] from Figure 1 As can be seen from the image, due to its strong hydrophilicity, hydroxyapatite immediately sinks to the bottom and forms a noticeable precipitate when added to hydrophobic dichloromethane. In contrast, hydroxyapatite encapsulated in soybean lecithin exhibits excellent dispersibility, not only dispersing uniformly in dichloromethane but also showing no sedimentation after standing for a period of time. Figure 1 b shows that hydroxyapatite, being a nanoparticle, is prone to agglomeration. When pure hydroxyapatite is dissolved in dichloromethane, two peaks appear at 142 nm and 521 nm. However, after being coated with soybean lecithin, the particle size distribution of hydroxyapatite in dichloromethane is significantly narrowed, with only one peak and a particle size of about 50 nm. This further confirms the good dispersibility and stability of hydroxyapatite coated with soybean lecithin in dichloromethane.

[0050] Example 2

[0051] 1.5 g of L-polylactic acid and 150 mg of hydroxyapatite were weighed and dissolved in 25 mL of chloromethane, and the mixture was magnetically stirred to form an organic phase. 350 mg of sodium bicarbonate was weighed and dissolved in 5 mL of water at room temperature to form aqueous phase I. 2.5 g of sodium bicarbonate was weighed and dissolved in 250 mL of deionized water, and the mixture was mechanically stirred at 350 rpm to form aqueous phase II. Aqueous phase I was added dropwise to the organic phase at a rate of 1 s / drop under magnetic stirring at 780 rpm, and emulsified for 10 min to form a primary emulsion. The primary emulsion was then added dropwise to aqueous phase II at a rate of 1 s / drop, and emulsified for 3 h. The mixture was then stirred overnight at room temperature to evaporate the organic solvent, forming dispersed microspheres in the aqueous phase. The microspheres were collected by vacuum filtration, washed repeatedly with deionized water, and then freeze-dried to obtain hydroxyapatite / polylactic acid composite porous microspheres (PH). 1.5 g of polylactic acid (PLA) and 450 mg of soybean lecithin-encapsulated hydroxyapatite complex were weighed and dissolved in 25 mL of chloromethane. The mixture was magnetically stirred to form an organic phase. Subsequent steps were the same as above to prepare highly dispersed and highly encapsulated hydroxyapatite-encapsulated PSH composite porous microspheres.

[0052] Scanning electron microscopy revealed that the hydroxyapatite / L-polylactic acid porous microspheres appeared as lumps or cakes, failing to form an ideal spherical structure. Furthermore, these microspheres themselves lacked a clear porous structure; only a very few microspheres exhibited pores, and their number was extremely limited, typically consisting of only 1-2 large pores. Figure 2 a). Soybean lecithin@hydroxyapatite / L-polylactic acid porous microspheres have a rich porous structure, connecting the interior of the microspheres with the external environment. Figure 2 b) indicates that the addition of soybean lecithin can make the microspheres more regular in morphology and more abundant and uniform in pore size.

[0053] Example 3

[0054] First, L-polylactic acid, soybean lecithin, and hydroxyapatite were heated from room temperature to 800℃ in a nitrogen atmosphere at a heating rate of 10℃ / min. It can be seen that hydroxyapatite hardly underwent thermal decomposition before 800℃. This is because the high thermal stability of hydroxyapatite allows it to maintain its structure and properties even at high temperatures. Soybean lecithin and L-polylactic acid, on the other hand, essentially completed thermal decomposition at approximately 550℃. Figure 3 a) Therefore, 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℃ in a nitrogen atmosphere at a heating rate of 10℃ / min. The final residual contents of the two types of microspheres were 4.3% and 8.3%, respectively, with encapsulation efficiencies of 42.5% and 83%, respectively. Figure 3 b). The above results fully demonstrate 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 mill jars, add an appropriate amount of acetone to balance the mixture, and ball mill for 30 min. After ball milling, collect the product and dry it in a vacuum drying oven at 37℃ for 30 min to obtain the composite. Weigh 1.5 g of polylactic acid and 450 mg of the composite and dissolve them in 25 mL of chloromethane, stirring magnetically to form the organic phase. Weigh 350 mg of sodium bicarbonate and dissolve it in 5 mL of water at room temperature to form aqueous phase I. Weigh 2.5 g of sodium bicarbonate and dissolve it in 250 mL of deionized water, stirring mechanically at 350 rpm to form aqueous phase II. Under magnetic stirring at 780 rpm, the above aqueous phase I was added dropwise to the organic phase at a rate of 1 s / drop, and emulsified for 10 min to form a primary emulsion. The above primary emulsion was then added dropwise to the aqueous phase II at a rate of 1 s / drop, and emulsified for 3 h. After emulsification, the mixture was stirred overnight at room temperature to evaporate the organic solvent, and dispersed microspheres were formed in the aqueous phase. The microspheres were collected by vacuum filtration, washed multiple times with deionized water, and then freeze-dried to obtain polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite.

[0057] Scanning electron microscopy revealed that the average particle size of the soybean lecithin@hydroxyapatite / polylactic acid porous microspheres was 304±32.6 μm. They exhibited a rich porous structure that connected the interior of the microspheres with the external environment; the average diameter of the pores 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 into two ball mill jars, add an appropriate amount of ethanol to balance the mixture, and ball mill for 60 min. After ball milling, collect the product and dry it in a vacuum drying oven at 37℃ for 30 min to obtain the composite. Weigh 2 g of polylactic acid and 600 mg of the composite and dissolve them in 20 mL of chloromethane, stirring magnetically to form the organic phase. Weigh 400 mg of sodium bicarbonate and dissolve it in 5 mL of water at room temperature to form aqueous phase I. Weigh 2 g of sodium bicarbonate and dissolve it in 300 mL of deionized water, stirring mechanically at 400 rpm to form aqueous phase II. Under magnetic stirring at 750 rpm, the above aqueous phase I was added dropwise to the organic phase at a rate of 1 s / drop, and emulsified for 15 min to form a primary emulsion. The above primary emulsion was then added dropwise to the aqueous phase II at a rate of 1 s / drop, and emulsified for 3 h. After emulsification, the mixture was stirred overnight at room temperature to evaporate the organic solvent, and dispersed microspheres were formed in the aqueous phase. The microspheres were collected by vacuum filtration, washed multiple times with deionized water, and then freeze-dried to obtain polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite.

[0060] Scanning electron microscopy revealed that the average particle size of the soybean lecithin@hydroxyapatite / polylactic acid porous microspheres was 280±23.5 μm. They exhibited a rich porous structure that connected the interior of the microspheres with the external environment; the average diameter of the pores was 41.2±6.0 μm. The water absorption rate was 3865.3%.

[0061] Example 6

[0062] 100 mg of hydroxyapatite and 300 mg of soybean lecithin were weighed into two ball mill jars, and an appropriate amount of acetone was added to balance the mixture. The mixture was ball milled for 15 min. After milling, the product was collected and dried in a vacuum drying oven at 37℃ for 30 min to obtain the composite. 1 g of polylactic acid and 400 mg of the composite were weighed and dissolved in 20 mL of chloromethane, and the solution was magnetically stirred to form the organic phase. 300 mg of sodium bicarbonate was weighed and dissolved in 5 mL of water at room temperature to form aqueous phase I. 3 g of sodium bicarbonate was weighed and dissolved in 300 mL of deionized water, and the solution was mechanically stirred at 250 rpm to form aqueous phase II. Under magnetic stirring at 800 rpm, the above aqueous phase I was added dropwise to the organic phase at a rate of 1 s / drop, and emulsified for 5 min to form a primary emulsion. The above primary emulsion was then added dropwise to the aqueous phase II at a rate of 1 s / drop, and emulsified for 3 h. After emulsification, the mixture was stirred overnight at room temperature to evaporate the organic solvent, and dispersed microspheres were formed in the aqueous phase. The microspheres were collected by vacuum filtration, washed multiple times with deionized water, and then freeze-dried to obtain polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite.

[0063] Scanning electron microscopy revealed that the average particle size of the soybean lecithin@hydroxyapatite / polylactic acid porous microspheres was 350±32.2 μm. They exhibited a rich porous structure that connected the interior of the microspheres with the external environment; the average diameter of the pores was 31.2±6.0 μm. The water absorption rate was 2804.6%.

[0064] Example 7

[0065] 100 mg of hydroxyapatite and 500 mg of soybean lecithin were weighed into two ball mill jars, and an appropriate amount of ethanol was added to balance the mixture. The mixture was ball-milled for 90 min. After milling, the product was collected and dried in a vacuum drying oven at 37 °C for 30 min to obtain the composite. 2 g of polylactic acid and 600 mg of the composite were weighed and dissolved in 30 mL of chloromethane, and the solution was magnetically stirred to form the organic phase. 350 mg of sodium bicarbonate was weighed and dissolved in 5 mL of water at room temperature to form aqueous phase I. 2.5 g of sodium bicarbonate was weighed and dissolved in 300 mL of deionized water, and the solution was mechanically stirred at 450 rpm to form aqueous phase II. Under magnetic stirring at 720 rpm, the above aqueous phase I was added dropwise to the organic phase at a rate of 1 s / drop, and emulsified for 10 min to form a primary emulsion. The above primary emulsion was then added dropwise to the aqueous phase II at a rate of 1 s / drop, and emulsified for 3 h. After emulsification, the mixture was stirred overnight at room temperature to evaporate the organic solvent, and dispersed microspheres were formed in the aqueous phase. The microspheres were collected by vacuum filtration, washed multiple times with deionized water, and then freeze-dried to obtain polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite.

[0066] Scanning electron microscopy revealed that the average particle size of the soybean lecithin@hydroxyapatite / polylactic acid porous microspheres was 280±22.5 μm. They exhibited a rich porous structure, connecting the interior of the microspheres to the external environment. The average diameter of the porous structure was 36.2±5.4 μm. The water absorption rate was 3001.3%.

[0067] Example 8

[0068] 200 mg of hydroxyapatite and 400 mg of soybean lecithin were weighed into two ball mill jars, and an appropriate amount of acetone was added to balance the mixture. The mixture was ball milled for 45 min. After milling, the product was collected and dried in a vacuum drying oven at 37℃ for 30 min to obtain the composite. 1.5 g of polylactic acid and 600 mg of the composite were weighed and dissolved in 20 mL of chloromethane, and magnetically stirred to form the organic phase. 350 mg of sodium bicarbonate was weighed and dissolved in 5 mL of water at room temperature to form aqueous phase I. 2 g of sodium bicarbonate was weighed and dissolved in 200 mL of deionized water, and mechanically stirred at 300 rpm to form aqueous phase II. Under magnetic stirring at 700 rpm, the above aqueous phase I was added dropwise to the organic phase at a rate of 1 s / drop, and emulsified for 15 min to form a primary emulsion. The above primary emulsion was then added dropwise to the aqueous phase II at a rate of 1 s / drop, and emulsified for 3 h. After emulsification, the mixture was stirred overnight at room temperature to evaporate the organic solvent, and dispersed microspheres were formed in the aqueous phase. The microspheres were collected by vacuum filtration, washed multiple times with deionized water, and then freeze-dried to obtain polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite.

[0069] Scanning electron microscopy revealed that the average particle size of the soybean lecithin@hydroxyapatite / polylactic acid porous microspheres was 380±15.1 μm. They exhibited a rich porous structure that connected the interior of the microspheres with the external environment; the average diameter of the pores was 35.8±5.4 μm. The water absorption rate was 3158.9%.

[0070] The above description is merely a preferred embodiment of the present invention, and should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for preparing polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite, characterized in that, Includes the following steps: (1) The amphiphilic medium and hydroxyapatite were ball-milled in organic solvent I and dried to remove organic solvent I, forming a complex; (2) The polymer material and the complex are simultaneously dissolved in organic solvent II to form an organic phase; (3) Dissolve the pore-forming agent in water to form aqueous phase I; (4) Dissolve the surfactant in water to form aqueous phase II; (5) Add the above aqueous phase I dropwise to the organic phase and stir to emulsify and form an emulsion; (6) Add the emulsion dropwise to aqueous phase II while stirring, and mix thoroughly; (7) Stir at room temperature until the organic solvent is completely evaporated, and dispersed microspheres are formed in the aqueous phase. After filtration, washing and drying, polylactic acid composite porous microspheres with high dispersion and high encapsulation of hydroxyapatite are obtained. 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. In step (2), the polymer material is at least one of PLLA, PDLA, and PDLLA, and the molecular weight of the polymer material is 1000-200000 Da.

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

3. The method for preparing polylactic acid composite porous microspheres with highly dispersed and highly encapsulated hydroxyapatite according to claim 1, characterized in that: In step (2), organic solvent II is at least one of acetone, dichloromethane, ethanol, dimethyl sulfoxide, tetrahydrofuran, and ethyl acetate.

4. The method for preparing polylactic acid composite porous microspheres with highly dispersed and highly encapsulated hydroxyapatite according to claim 1, characterized in that: 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).

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

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

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

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

9. A polylactic acid composite porous microsphere with highly dispersed and highly encapsulated hydroxyapatite, characterized in that: It is prepared by any one of the preparation methods according to claims 1-8.

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