A kind of hydroxyapatite microsphere with stable structure and easy injection and its preparation method

Abstract

The present invention discloses a structurally stable and easily injectable hydroxyapatite microsphere and a preparation method thereof, specifically relating to the field of biomedical materials technology. The preparation method comprises constructing a microfluidic reaction system, utilizing electroosmotic flow to precisely control the flow rate of the solution, mixing and adjusting the pH value, applying a pulsed electric field, reacting in a microfluidic chip, filtering through an ultrafiltration membrane, washing with supercritical carbon dioxide fluid extraction, freeze drying, and airflow milling, and finally mixing with a biodegradable polymer solution to prepare the microspheres by electrostatic spraying technology. The prepared hydroxyapatite microspheres are surface-grafted with arginine-glycine-aspartic acid polypeptides, with a particle size of 20-35 μm, a particle size distribution coefficient of variation of less than 8%, a compressive strength of 5-8 MPa, and a mass loss rate of less than 5% after immersion in a simulated physiological environment solution for 30 days. The microspheres have the advantages of stable structure, easy injection, and good biocompatibility, and have good application prospects in the fields of biomedicine and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and more particularly to structurally stable and easily injectable hydroxyapatite microspheres and a preparation method thereof. Background Art

[0002] In the field of biomedical materials, hydroxyapatite microspheres have become an ideal material for applications such as bone repair and drug delivery due to their excellent biocompatibility, bioactivity, and osteoconduction. However, traditional methods for preparing hydroxyapatite microspheres have many shortcomings.

[0003] Common preparation techniques such as hydrothermal, spray drying, sol-gel, and template methods often produce gases such as carbon dioxide during the reaction process, resulting in a loose spherical structure and poor shape retention of the microspheres. This makes it difficult for the microspheres to provide effective support when injected into the human body as bone repair materials, affecting the therapeutic effect. Moreover, these methods make it difficult to precisely control the reaction conditions, resulting in uneven particle size distribution of the products and large differences between batches, which is not conducive to large-scale industrial production.

[0004] While some existing preparation processes can improve the performance of microspheres to a certain extent, they still have shortcomings in improving the overall performance of microspheres, such as structural stability, injectability, and bioactivity. For example, the degradation rate of microspheres prepared by some processes is difficult to control in the body and may be too fast or too slow, affecting the continuity and safety of treatment. Some microspheres also have suboptimal surface properties and poor cell adhesion, which cannot effectively promote tissue repair and regeneration. Therefore, it is of great practical significance to develop a preparation method for hydroxyapatite microspheres that can be structurally stable, easily injectable, and have good bioactivity. Summary of the Invention

[0005] In order to overcome the above-mentioned defects of the prior art, the present invention provides a structurally stable and easily injectable hydroxyapatite microsphere and a preparation method thereof to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for preparing structurally stable and easily injectable hydroxyapatite microspheres, comprising the following steps:

[0007] (1) Constructing a microfluidic reaction system, injecting a phosphate aqueous solution and an alkaline regulator solution into a mixing area of ​​a microfluidic chip through independent microchannels; during the injection process, using electroosmosis to precisely control the flow rate of the phosphate aqueous solution to 10-20 μL / min and the flow rate of the alkaline regulator solution to 5-10 μL / min, so that the two are rapidly mixed in the mixing area, and the pH value of the phosphate aqueous solution is adjusted to 10.5-11.5, thereby obtaining a mixed solution 1;

[0008] (2) A calcium nitrate aqueous solution is injected into the microfluidic chip through another microchannel at a flow rate of 15-25 μL / min to mix with the mixed solution 1 to obtain a mixed solution 2; at the moment when the mixed solution 2 is formed, a pulse electric field is applied to the microfluidic chip, and then the pH value of the mixed solution 2 is adjusted to 11-12, and the reaction is carried out in the microfluidic chip to obtain a mixed solution 3;

[0009] (3) The mixed solution 3 is guided out of the microfluidic chip and filtered through an ultrafiltration membrane filtration device with a molecular weight cutoff of 1000-5000 Da to obtain a filter cake;

[0010] (4) The filter cake is placed in a supercritical carbon dioxide fluid extraction device, extracted and washed with anhydrous ethanol as an entrainer, then dried in a freeze dryer, and then pulverized using a jet mill to obtain a powder;

[0011] (5) The obtained powder is mixed with a biodegradable polymer solution with a mass fraction of 3%-8% to prepare a mixed aqueous solution with a mass fraction of 20%-40%, and hydroxyapatite microspheres are prepared by electrostatic spraying technology.

[0012] Preferably, the phosphate aqueous solution is an aqueous solution of dipotassium hydrogen phosphate and potassium dihydrogen phosphate mixed in a molar ratio of 1:1-3:1, with a total concentration of 0.4-0.6 mol / L, and the alkaline regulator is a lithium hydroxide solution with a concentration of 0.5-1.5 mol / L.

[0013] Preferably, the electric field strength of the pulsed electric field is 50-100 V / cm, the pulse frequency is 10-20 Hz, and the duration is 5-10 s.

[0014] Preferably, during the reaction process of step (2), the reaction temperature is maintained at 45-55° C. using a temperature control module of the microfluidic chip, the reaction time is 20-28 hours, and the concentration of the calcium nitrate aqueous solution is 0.4-0.6 mol / L.

[0015] Preferably, in step (4), the extraction and washing are performed 2-4 times at a temperature of 40-50°C and a pressure of 10-15 MPa, with each extraction time being 30-60 minutes, and then dried in a freeze dryer at a temperature of -40-30°C and a vacuum degree of 10-30 Pa for 12-18 hours, and then pulverized using a jet mill for 1-3 hours to obtain a powder.

[0016] Preferably, the biodegradable polymer is a polylactic acid-glycolic acid copolymer, and the molar ratio of lactic acid to glycolic acid is 75:25-85:15; the process parameters of the electrostatic spray are: voltage of 10-15 kV, the distance between the nozzle and the receiving device is 15-25 cm, the solution flow rate is 5-10 mL / h, the ambient humidity is controlled at 30%-50%, and the ambient temperature is controlled at 20-25°C.

[0017] Preferably, the mixing area of ​​the microfluidic chip in step (1) adopts a T-shaped or Y-shaped structure, and the inner diameter of the microchannel is 50-100 μm.

[0018] Preferably, when the pulsed electric field is applied in step (2), the conductivity of the solution in the microfluidic chip is controlled at 1-3 mS / cm.

[0019] Preferably, the gas source for the jet mill in step (4) is nitrogen, and the gas pressure is 0.8-1.2 MPa.

[0020] A hydroxyapatite microsphere, prepared by the above-described preparation method, wherein a bioactive molecule is grafted onto the surface of the hydroxyapatite microsphere, wherein the bioactive molecule is an arginine-glycine-aspartic acid polypeptide, and the grafting rate is 0.5-2 μg / m²; the particle size of the microsphere is 20-35 μm, and the coefficient of variation of the particle size distribution is less than 8%; the compressive strength of the microsphere reaches 5-8 MPa, and after immersion in a simulated physiological environment solution for 30 days, the mass loss rate is less than 5%.

[0021] The technical effects and advantages of the present invention are as follows:

[0022] By utilizing microfluidics and electroosmosis to precisely control the flow rate of the solution, the phosphate aqueous solution and the alkaline regulator solution can be quickly and accurately mixed in the mixing area of ​​the microfluidic chip, adjusting the pH value to a specific range, ensuring the consistency of the reaction starting conditions and improving the purity and stability of the product.

[0023] By applying a pulsed electric field with specific parameters, combined with precisely controlled reaction temperature, time, and reactant concentration, the reaction process and the microstructure of the product can be changed, which helps to improve the crystallinity of hydroxyapatite microspheres, making their structure denser, thereby enhancing the compressive strength of the microspheres and improving their performance in practical applications.

[0024] By adopting a series of post-processing steps such as ultrafiltration membrane filtration, supercritical carbon dioxide fluid extraction and washing, freeze drying and air flow milling, impurities are effectively removed and the powder particle size is controlled, laying the foundation for the subsequent preparation of high-quality microspheres and further improving the purity and quality stability of the microspheres.

[0025] By using specific biodegradable polymers and controlling their proportions and electrostatic spraying process parameters, the prepared microspheres have good biodegradability, uniform particle size, stable structure, and are easy to inject. At the same time, arginine-glycine-aspartic acid polypeptides are grafted onto the surface of the microspheres to enhance biocompatibility, resulting in a low mass loss rate in a simulated physiological environment, allowing them to better function in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a flow chart of the preparation method of the present invention. DETAILED DESCRIPTION

[0027] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work shall fall within the scope of protection of the present invention.

[0028] As attached Figure 1 The method for preparing a structurally stable and easily injectable hydroxyapatite microsphere comprises the following steps:

[0029] (1) Constructing a microfluidic reaction system, injecting a phosphate aqueous solution and an alkaline regulator solution into a mixing area of ​​a microfluidic chip through independent microchannels; during the injection process, using electroosmosis to precisely control the flow rate of the phosphate aqueous solution to 10-20 μL / min and the flow rate of the alkaline regulator solution to 5-10 μL / min, so that the two are rapidly mixed in the mixing area, and the pH value of the phosphate aqueous solution is adjusted to 10.5-11.5, thereby obtaining a mixed solution 1;

[0030] (2) A calcium nitrate aqueous solution is injected into the microfluidic chip through another microchannel at a flow rate of 15-25 μL / min to mix with the mixed solution 1 to obtain a mixed solution 2; at the moment when the mixed solution 2 is formed, a pulse electric field is applied to the microfluidic chip, and then the pH value of the mixed solution 2 is adjusted to 11-12, and the reaction is carried out in the microfluidic chip to obtain a mixed solution 3;

[0031] (3) The mixed solution 3 is guided out of the microfluidic chip and filtered through an ultrafiltration membrane filtration device with a molecular weight cutoff of 1000-5000 Da to obtain a filter cake;

[0032] (4) The filter cake is placed in a supercritical carbon dioxide fluid extraction device, extracted and washed with anhydrous ethanol as an entrainer, then dried in a freeze dryer, and then pulverized using a jet mill to obtain a powder;

[0033] (5) The obtained powder is mixed with a biodegradable polymer solution with a mass fraction of 3%-8% to prepare a mixed aqueous solution with a mass fraction of 20%-40%, and hydroxyapatite microspheres are prepared by electrostatic spraying technology.

[0034] Specifically, by constructing a microfluidic reaction system and using electroosmotic flow to precisely control the solution flow rate, a series of operations, including mixing, pH adjustment, reaction, filtration, extraction and washing, drying and pulverization, and electrostatic spraying, are performed within the microfluidic chip. Microfluidic technology enables precise microscale reaction control, and electroosmotic flow control ensures accurate mixing of the reactants. Subsequent steps involve separation, purification, and molding of the products, enabling precise control of various reaction process parameters. This helps improve the purity and consistency of the products, resulting in more stable quality and superior performance of the prepared hydroxyapatite microspheres.

[0035] Example 1

[0036] The first embodiment provides a method for preparing structurally stable and easily injectable hydroxyapatite microspheres, comprising the following steps:

[0037] A microfluidic reaction system was constructed. A phosphate aqueous solution with a total concentration of 0.4 mol / L and a lithium hydroxide solution with a molar ratio of dipotassium hydrogen phosphate to potassium dihydrogen phosphate of 1:1 and a concentration of 0.5 mol / L were injected into the T-shaped structure mixing area through independent microchannels, respectively. The flow rate of the phosphate aqueous solution was controlled to 10 μL / min, and the flow rate of the lithium hydroxide solution was controlled to 5 μL / min by electroosmosis. The pH value was adjusted to 10.5 to obtain a mixed solution 1.

[0038] A 0.4 mol / L calcium nitrate aqueous solution was injected into the microfluidic chip at a flow rate of 15 μL / min and mixed with mixed solution 1 to produce mixed solution 2. Immediately after the formation of mixed solution 2, a pulsed electric field with an electric field strength of 50 V / cm, a pulse frequency of 10 Hz, and a duration of 5 seconds was applied. The pH value was adjusted to 11, and the reaction temperature was maintained at 45°C using a temperature control module. The reaction was allowed to react for 20 hours to produce mixed solution 3.

[0039] The mixed solution 3 was filtered through an ultrafiltration membrane with a molecular weight cut-off of 1000 Da to obtain a filter cake.

[0040] The filter cake was placed in a supercritical carbon dioxide fluid extraction apparatus and extracted and washed twice with anhydrous ethanol as an entrainer at a temperature of 40°C and a pressure of 10 MPa, each time for 30 minutes. It was then dried in a freeze dryer at a temperature of -40°C and a vacuum of 10 Pa for 12 hours, and then pulverized in a jet mill using nitrogen as the gas source and a gas pressure of 0.8 MPa for 1 hour to obtain a powder.

[0041] The obtained powder was mixed with a polylactic acid-glycolic acid copolymer solution with a mass fraction of 3% and a molar ratio of lactic acid to glycolic acid of 75:25 to prepare a 20% mass fraction mixed aqueous solution. Microspheres were prepared by electrostatic spraying under the conditions of a voltage of 10 kV, a distance of 15 cm between the nozzle and the receiving device, a solution flow rate of 5 mL / h, an ambient humidity of 30%, and an ambient temperature of 20°C.

[0042] Microsphere performance: The arginine-glycine-aspartic acid polypeptide grafting rate on the microsphere surface is 0.5μg / m², the particle size is 20μm, the particle size distribution variation coefficient is 7%, the compressive strength is 5MPa, and after immersion in a simulated physiological environment solution for 30 days, the mass loss rate is 4%.

[0043] Example 2

[0044] The second embodiment provides a method for preparing structurally stable and easily injectable hydroxyapatite microspheres, comprising the following steps:

[0045] A microfluidic reaction system was constructed. A phosphate aqueous solution with a molar ratio of dipotassium hydrogen phosphate to potassium dihydrogen phosphate of 2:1 and a total concentration of 0.5 mol / L and a lithium hydroxide solution with a concentration of 1 mol / L were injected into the Y-shaped structure mixing region through independent microchannels, respectively. The flow rate of the phosphate aqueous solution was controlled to 15 μL / min, and the flow rate of the lithium hydroxide solution was controlled to 8 μL / min by electroosmosis. The pH value was adjusted to 11 to obtain a mixed solution 1.

[0046] A 0.5 mol / L calcium nitrate aqueous solution was injected into the microfluidic chip at a flow rate of 20 μL / min and mixed with mixed solution 1 to produce mixed solution 2. Immediately after the formation of mixed solution 2, a pulsed electric field with an electric field strength of 75 V / cm, a pulse frequency of 15 Hz, and a duration of 8 seconds was applied. The pH was adjusted to 11.5, and the reaction temperature was maintained at 50°C using a temperature control module. The reaction was allowed to proceed for 24 hours to produce mixed solution 3.

[0047] The mixed solution 3 was filtered through an ultrafiltration membrane with a molecular weight cut-off of 3000 Da to obtain a filter cake.

[0048] The filter cake was placed in a supercritical carbon dioxide fluid extraction apparatus and extracted and washed three times with anhydrous ethanol as an entrainer at a temperature of 45°C and a pressure of 12 MPa, each time for 45 minutes. It was then dried in a freeze dryer at a temperature of -35°C and a vacuum of 20 Pa for 15 hours, and then pulverized in a jet mill using nitrogen as the gas source and a gas pressure of 1 MPa for 2 hours to obtain a powder.

[0049] The obtained powder was mixed with a polylactic acid-glycolic acid copolymer solution with a mass fraction of 5% and a molar ratio of lactic acid to glycolic acid of 80:20 to prepare a 30% mixed aqueous solution. Microspheres were prepared by electrostatic spraying under the conditions of a voltage of 12 kV, a distance of 20 cm between the nozzle and the receiving device, a solution flow rate of 7 mL / h, an ambient humidity of 40%, and an ambient temperature of 22°C.

[0050] Microsphere performance: The arginine-glycine-aspartic acid polypeptide grafting rate on the microsphere surface is 1.2μg / m², the particle size is 28μm, the coefficient of variation of the particle size distribution is 6%, the compressive strength is 6MPa, and after immersion in a simulated physiological environment solution for 30 days, the mass loss rate is 3%.

[0051] Example 3

[0052] The third embodiment provides a method for preparing structurally stable and easily injectable hydroxyapatite microspheres, comprising the following steps:

[0053] A microfluidic reaction system was constructed. A phosphate aqueous solution with a molar ratio of dipotassium hydrogen phosphate to potassium dihydrogen phosphate of 3:1 and a total concentration of 0.6 mol / L and a lithium hydroxide solution with a concentration of 1.5 mol / L were injected into the T-shaped structure mixing area through independent microchannels, respectively. The flow rate of the phosphate aqueous solution was controlled to 20 μL / min, and the flow rate of the lithium hydroxide solution was controlled to 10 μL / min by electroosmosis. The pH value was adjusted to 11.5 to obtain a mixed solution 1.

[0054] A 0.6 mol / L calcium nitrate aqueous solution was injected into the microfluidic chip at a flow rate of 25 μL / min and mixed with mixed solution 1 to produce mixed solution 2. Immediately after the formation of mixed solution 2, a pulsed electric field with an electric field strength of 100 V / cm, a pulse frequency of 20 Hz, and a duration of 10 s was applied. The pH was adjusted to 12, and the reaction temperature was maintained at 55°C using a temperature control module. The reaction was allowed to proceed for 28 hours to produce mixed solution 3.

[0055] The mixed solution 3 was filtered through an ultrafiltration membrane with a molecular weight cut-off of 5000 Da to obtain a filter cake.

[0056] The filter cake was placed in a supercritical carbon dioxide fluid extraction apparatus and extracted and washed four times with anhydrous ethanol as an entrainer at a temperature of 50°C and a pressure of 15 MPa, each time for 60 minutes. It was then dried in a freeze dryer at a temperature of -30°C and a vacuum of 30 Pa for 18 hours, and then pulverized in a jet mill using nitrogen as the gas source and a gas pressure of 1.2 MPa for 3 hours to obtain a powder.

[0057] The obtained powder was mixed with a polylactic acid-glycolic acid copolymer solution with a mass fraction of 8% and a molar ratio of lactic acid to glycolic acid of 85:15 to prepare a mixed aqueous solution with a mass fraction of 40%. Microspheres were prepared by electrostatic spraying under the conditions of a voltage of 15 kV, a distance of 25 cm between the nozzle and the receiving device, a solution flow rate of 10 mL / h, an ambient humidity of 50%, and an ambient temperature of 25°C.

[0058] Microsphere performance: The arginine-glycine-aspartic acid polypeptide grafting rate on the microsphere surface is 2μg / m², the particle size is 35μm, the coefficient of variation of the particle size distribution is 5%, the compressive strength is 8MPa, and after immersion in a simulated physiological environment solution for 30 days, the mass loss rate is 2%.

[0059] Example 4

[0060] The fourth embodiment provides a method for preparing structurally stable and easily injectable hydroxyapatite microspheres, comprising the following steps:

[0061] A microfluidic reaction system was constructed. A phosphate aqueous solution with a molar ratio of dipotassium hydrogen phosphate to potassium dihydrogen phosphate of 1.5:1 and a total concentration of 0.45 mol / L and a lithium hydroxide solution with a concentration of 0.8 mol / L were injected into the Y-shaped structure mixing region through independent microchannels, respectively. The flow rates of the phosphate aqueous solution and the lithium hydroxide solution were controlled by electroosmotic flow to 12 μL / min and 6 μL / min, and the pH value was adjusted to 10.8 to obtain a mixed solution 1.

[0062] A 0.45 mol / L calcium nitrate aqueous solution was injected into the microfluidic chip at a flow rate of 18 μL / min and mixed with mixed solution 1 to produce mixed solution 2. Immediately after the formation of mixed solution 2, a pulsed electric field with a strength of 60 V / cm, a pulse frequency of 12 Hz, and a duration of 6 seconds was applied. The pH was adjusted to 11.2, and the reaction temperature was maintained at 48°C using a temperature control module. The reaction was allowed to proceed for 22 hours to produce mixed solution 3.

[0063] The mixed solution 3 was filtered through an ultrafiltration membrane with a molecular weight cut-off of 2000 Da to obtain a filter cake.

[0064] The filter cake was placed in a supercritical carbon dioxide fluid extraction apparatus and extracted and washed three times with anhydrous ethanol as an entrainer at a temperature of 42°C and a pressure of 11 MPa, each time for 40 minutes. It was then dried in a freeze dryer at a temperature of -38°C and a vacuum of 15 Pa for 13 hours, and then pulverized in a jet mill using nitrogen as the gas source and a gas pressure of 0.9 MPa for 1.5 hours to obtain a powder.

[0065] The obtained powder was mixed with a polylactic acid-glycolic acid copolymer solution with a mass fraction of 4% and a molar ratio of lactic acid to glycolic acid of 78:22 to prepare a mixed aqueous solution with a mass fraction of 25%. Microspheres were prepared by electrostatic spraying under the conditions of voltage 11 kV, distance between the nozzle and the receiving device 18 cm, solution flow rate 6 mL / h, ambient humidity 35%, and ambient temperature 21°C.

[0066] Microsphere performance: The arginine-glycine-aspartic acid polypeptide grafting rate on the microsphere surface is 0.8μg / m², the particle size is 23μm, the coefficient of variation of the particle size distribution is 7%, the compressive strength is 5.5MPa, and after immersion in a simulated physiological environment solution for 30 days, the mass loss rate is 3.5%.

[0067] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for preparing structurally stable and easily injectable hydroxyapatite microspheres, characterized in that: The following steps are involved: (1) Constructing a microfluidic reaction system, injecting a phosphate aqueous solution and an alkaline regulator solution into a mixing area of ​​a microfluidic chip through independent microchannels; during the injection process, using electroosmosis to precisely control the flow rate of the phosphate aqueous solution to 10-20 μL / min and the flow rate of the alkaline regulator solution to 5-10 μL / min, so that the two are rapidly mixed in the mixing area, and the pH value of the phosphate aqueous solution is adjusted to 10.5-11.5, thereby obtaining a mixed solution 1; (2) injecting calcium nitrate aqueous solution into the microfluidic chip through another microchannel at a flow rate of 15-25 μL / min to meet and mix with mixed solution 1 to obtain mixed solution 2; at the moment when mixed solution 2 is formed, applying a pulsed electric field to the microfluidic chip, wherein the electric field strength of the pulsed electric field is 50-100 V / cm, the pulse frequency is 10-20 Hz, and the duration is 5-10 s, and then adjusting the pH value of mixed solution 2 to 11-12, and reacting in the microfluidic chip to obtain mixed solution 3; (3) The mixed solution 3 is guided out of the microfluidic chip and filtered through an ultrafiltration membrane filtration device with a molecular weight cutoff of 1000-5000 Da to obtain a filter cake; (4) The filter cake is placed in a supercritical carbon dioxide fluid extraction device, extracted and washed with anhydrous ethanol as an entrainer, then dried in a freeze dryer, and then pulverized using a jet mill to obtain a powder; (5) mixing the obtained powder with a biodegradable polymer solution having a mass fraction of 3% to 8% to prepare a mixed aqueous solution having a mass fraction of 20% to 40%, and preparing hydroxyapatite microspheres by electrostatic spraying technology; During the reaction of step (2), the temperature control module of the microfluidic chip is used to maintain the reaction temperature at 45-55° C., the reaction time is 20-28 hours, and the concentration of the calcium nitrate aqueous solution is 0.4-0.6 mol / L.

2. The method for preparing structurally stable and injectable hydroxyapatite microspheres according to claim 1, characterized in that: The phosphate aqueous solution is an aqueous solution of dipotassium hydrogen phosphate and potassium dihydrogen phosphate mixed in a molar ratio of 1:1-3:1, with a total concentration of 0.4-0.6 mol / L; the alkaline regulator is a lithium hydroxide solution with a concentration of 0.5-1.5 mol / L.

3. The method for preparing structurally stable and injectable hydroxyapatite microspheres according to claim 1, characterized in that: In the step (4), extraction and washing are performed 2-4 times at a temperature of 40-50°C and a pressure of 10-15 MPa, with each extraction time being 30-60 minutes, and then drying is performed in a freeze dryer at a temperature of -40-30°C and a vacuum degree of 10-30 Pa for 12-18 hours, and then pulverizing is performed using a jet mill for 1-3 hours to obtain a powder.

4. The method for preparing structurally stable and injectable hydroxyapatite microspheres according to claim 1, characterized in that: The biodegradable polymer is a polylactic acid-glycolic acid copolymer, and the molar ratio of lactic acid to glycolic acid is 75:25-85:

15. The process parameters of the electrostatic spray are: voltage of 10-15kV, distance between the nozzle and the receiving device of 15-25cm, solution flow rate of 5-10mL / h, ambient humidity controlled at 30%-50%, and ambient temperature controlled at 20-25℃.

5. The method for preparing structurally stable and injectable hydroxyapatite microspheres according to claim 1, characterized in that: In the step (1), the mixing area of ​​the microfluidic chip adopts a T-shaped or Y-shaped structure, and the inner diameter of the microchannel is 50-100 μm.

6. The method for preparing structurally stable and injectable hydroxyapatite microspheres according to claim 1, characterized in that: In step (2), when the pulsed electric field is applied, the conductivity of the solution in the microfluidic chip is controlled at 1-3 mS / cm.

7. The method for preparing structurally stable and injectable hydroxyapatite microspheres according to claim 1, characterized in that: In step (4), the gas source for the jet mill is nitrogen, and the gas pressure is 0.8-1.2 MPa.

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