Mesoporous hydroxyapatite microspheres regulated and controlled by malic acid and preparation method of mesoporous hydroxyapatite microspheres

Abstract

The invention provides a malic acid regulated mesoporous hydroxyapatite microsphere and a preparation method thereof, and the preparation method comprises the following steps: sequentially dissolving a calcium source and a phosphorus source in deionized water, uniformly mixing, dropwise adding nitric acid or sodium hydroxide into a reaction solution, and adjusting the pH value of the solution to be clear; malic acid with different concentrations is added into the clarified reaction solution to serve as a morphology regulating agent, and then urea is added; transferring the prepared solution into a hydrothermal reaction kettle, and carrying out heat preservation reaction for 15 minutes to 12 hours at a set temperature; and after the reaction is completed, cooling the solution to room temperature, and centrifugally drying to obtain a hydroxyapatite powder sample. According to the method, the sphericity, the particle size, the surface micro-nano structure, the crystallinity and the like of the hydroxyapatite microspheres are adjusted by controlling the concentration of the malic acid serving as a morphological control agent and adjusting the reaction time and temperature. The hydroxyapatite microspheres prepared by the method are expected to be applied to the clinical application fields of drug controlled release systems, injectable facial wrinkle removal, hard tissue repair and the like.

Description

Technical Field

[0001] The invention relates to the technical field of inorganic biomaterials, and in particular to mesoporous hydroxyapatite microspheres regulated by malic acid and a preparation method thereof. Background Art

[0002] Hydroxyapatite (HAP) is the main inorganic component of human hard tissue and is widely used in biomedical fields such as bone implants, wound repair, regeneration filling, and drug carriers. The biocompatibility and osteoconductivity of HAP with bone tissue give it significant application advantages in bone defect repair. Designing HAP particles with specific morphology and structure according to the application requirements of different fields is crucial to improving its functionality and application performance.

[0003] The morphology and surface nanostructure of HAP particles have an important influence on their performance in biomedicine. Porous HAP microspheres can significantly enhance the adsorption capacity, interaction with proteins and cells, and biological activity of materials due to their high specific surface area and unique pore structure, and show strong application potential in drug loading and tissue engineering. Therefore, the development of HAP microspheres with adjustable pore structure and morphology is crucial to improve their application performance in the biomedical field.

[0004] At present, the methods for preparing calcium phosphate microspheres include sol-gel method, microemulsion method, spray drying method, template method, etc. The synthesis conditions of sol-gel method and microemulsion method are mild, but the yield is low and it is difficult to accurately control the morphology and surface nanostructure of microspheres; although the spray drying method has high production efficiency, it is difficult to control the morphology and size of microspheres, resulting in poor uniformity of the obtained particles. In contrast, the template method provides a relatively simple and effective means for regulating the morphology and surface structure of calcium phosphate particles. In particular, the soft template method, by self-assembling in the solution to form a template with a specific morphology, uses the interaction between organic molecules and specific sites on the surface of inorganic crystals to regulate nucleation and crystal growth, thereby achieving control of the morphology of HAP particles. However, compared with macromolecular template agents, the singleness of small molecules in adsorption mode and the limitation of the coverage range of crystal surface make them insufficient in stability in controlling crystal morphology. Therefore, how to combine the advantages of small molecule regulators to develop a stable and efficient hydrothermal synthesis method to achieve precise control of the morphology and structure of HAP microspheres is a problem that still needs to be solved. Summary of the invention

[0005] In view of the prior art. In order to achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present application provides a method for preparing mesoporous hydroxyapatite microspheres regulated by malic acid, comprising the following steps:

[0007] Step S1: dissolving a calcium source and a phosphorus source in deionized water in sequence, and mixing them evenly to prepare a reaction solution;

[0008] Step S2: adding nitric acid or sodium hydroxide dropwise to the reaction solution to adjust the pH of the solution to a clear state;

[0009] Step S3: adding malic acid of different concentrations as a morphology regulator to the clarified reaction solution, then adding urea, and continuously stirring for 5 to 15 minutes;

[0010] Step S4: transfer the prepared solution into a reaction kettle and keep the reaction at a set temperature for 15 minutes to 12 hours;

[0011] Step S5: After the reaction is completed, the solution is cooled to room temperature, the precipitate is separated by centrifugation, the precipitate is repeatedly washed with deionized water and ethanol, and then dried at 80° C. for 48 hours. After centrifugal drying, a hydroxyapatite powder sample with uniform morphology and high specific surface area is finally obtained.

[0012] The use of a specific morphology regulator, an organic small molecule acid - malic acid, and controlling the concentration of the morphology regulator can regulate the particle size, pore size and specific surface area of ​​hydroxyapatite, and has a good spheroidizing effect.

[0013] Preferably, in step S1, calcium nitrate tetrahydrate and disodium hydrogen phosphate dodecahydrate are added sequentially into 50 mL of deionized water to make the calcium source concentration be 0.1 mol / L and the calcium-phosphorus molar ratio be 1.67, to prepare a reaction solution.

[0014] Preferably, in step S2, the pH value of the solution is adjusted with nitric acid or sodium hydroxide until the solution is clear and the pH value of the solution is measured to be 2-3.

[0015] Preferably, in step S3, the molar ratio of the urea to the calcium source is 0.56:0.1, and the malic acid is L-malic acid, and the molar ratio of the urea to the calcium source is (0.05-0.2):0.1.

[0016] Preferably, in step S3, the concentration of the added malic acid is 335 mg-1.34 g / L.

[0017] Preferably, in step S4, the set temperature is 120°C-180°C, and the particle size and pore size of the hydroxyapatite microspheres can be adjusted by adjusting the reaction time and temperature.

[0018] Preferably, as the reaction time increases, the particles are transformed from amorphous nanoparticles ACP to HAP particles, and the particle phase changes; at the same time, as the reaction time increases, the crystallinity of the HAP particles increases.

[0019] Preferably, an increase in malic acid concentration leads to a dense surface of hydroxyapatite particles, a reduced pore size and a corresponding decrease in the average particle size. Specifically, the average particle size decreases from 17.90±7.52 μm to 11.97±4.79 μm, and the pore size decreases from micrometer level to nanometer level. When the malic acid concentration is 100 mM, larger particles of about 40 μm appear.

[0020] Preferably, the hydroxyapatite powder obtained by the preparation method has uniform morphological characteristics and significant specific surface area, and can realize the preparation of hydroxyapatite microspheres with different morphological structures.

[0021] In a second aspect, the present application also provides mesoporous hydroxyapatite microspheres regulated by malic acid, which are obtained by the above-mentioned preparation method.

[0022] The beneficial effects of the present invention are:

[0023] The present invention proposes a method for preparing mesoporous hydroxyapatite microspheres regulated by malic acid, and synthesizes hydroxyapatite (HAP) particles with uniform morphology and high specific surface area by a hydrothermal method. The method aims to solve the problems of inaccurate morphology control, difficult particle size control, and low specific surface area in the existing HAP preparation technology. By using malic acid as a small molecule regulator, the present invention can effectively adjust the morphology, size and surface microstructure of HAP particles, thereby obtaining more uniform HAP microspheres. Compared with the prior art, the preparation method proposed by the present invention has the advantages of simple preparation process and high morphology controllability, and can achieve high crystallinity HAP particles without relying on complex equipment. It is suitable for the preparation of HAP materials that require high specific surface area and uniform morphology, and lays a foundation for further biomedical research and application.

[0024] The present invention uses malic acid as a small molecule regulator and achieves precise control of the morphology of HAP particles by adjusting its concentration. The obtained particles present a uniform spherical nanostructure and have a high specific surface area, which is conducive to further applications, such as drug loading, bone defect filling or preparation of composite biomaterials.

[0025] The present invention uses the regulating effect of malic acid to obtain particles with a porous surface structure, which greatly increases the specific surface area of ​​HAP. This high specific surface area not only improves the adsorption capacity of the material, but also provides it with broad application potential in the fields of drug loading and sustained release and bone defect filling.

[0026] The present invention adopts a hydrothermal synthesis process, which is simple to operate, has mild reaction conditions, does not require complex equipment, and is easy to achieve large-scale preparation. Compared with other complex template methods or spray drying methods, it can achieve precise control of the morphology of HAP particles while ensuring high crystallinity.

[0027] The present invention can synthesize HAP particles of different sizes and structures by controlling the ratio of malic acid to calcium source, so as to meet the specific requirements of materials in different application fields. In particular, in applications such as bone repair materials and drug carriers, the adjustability of particle size and morphology greatly improves the applicability of the material.

[0028] The preparation method provided by the present invention adopts a specific morphology regulating agent, an organic small molecule acid, malic acid, and controls the concentration of the morphology regulating agent to regulate the particle size, pore size and specific surface area of ​​hydroxyapatite, and has a good sphering effect; by adjusting the reaction time and temperature, the particle size and pore size of hydroxyapatite microspheres can be adjusted; and the preparation of hydroxyapatite microspheres with different morphological structures can be achieved.

[0029] Other features and advantages of the present invention will be described in the following description, and partly become apparent from the description, or be understood by implementing the embodiments of the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures particularly pointed out in the written description, claims, and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0031] Figure 1A This is a scanning electron microscope image of the hydroxyapatite microspheres prepared in Example 1 of the present invention;

[0032] Figure 1B This is the X-ray diffraction pattern of the hydroxyapatite microspheres prepared in Example 1 of the present invention;

[0033] Figure 2A This is a scanning electron microscope image of hydroxyapatite microspheres prepared in Example 2 of the present invention;

[0034] Figure 2B This is the X-ray diffraction pattern of the hydroxyapatite microspheres prepared in Example 2 of the present invention;

[0035] Figure 2C This is a particle size distribution diagram of hydroxyapatite microspheres prepared in Example 2 of the present invention;

[0036] Figure 3A This is a scanning electron microscope image of the hydroxyapatite microspheres prepared in Example 3 of the present invention;

[0037] Figure 3BThis is the X-ray diffraction pattern of the hydroxyapatite microspheres prepared in Example 3 of the present invention;

[0038] Figure 3C This is a particle size distribution diagram of hydroxyapatite microspheres prepared in Example 3 of the present invention;

[0039] Figure 3D This is a Fourier transform infrared spectrum of hydroxyapatite microspheres prepared in Example 3 of the present invention;

[0040] Figure 4A This is a scanning electron microscope image of the hydroxyapatite microspheres prepared in Example 4 of the present invention;

[0041] Figure 4B This is a particle size distribution diagram of hydroxyapatite microspheres prepared in Example 4 of the present invention;

[0042] Figure 5A This is a scanning electron microscope image of the hydroxyapatite microspheres prepared in Example 5 of the present invention;

[0043] Figure 5B This is the X-ray diffraction pattern of the hydroxyapatite microspheres prepared in Example 5 of the present invention;

[0044] Figure 5C This is a particle size distribution diagram of hydroxyapatite microspheres prepared in Example 5 of the present invention;

[0045] Fig. 6A This is a scanning electron microscope image of the hydroxyapatite microspheres prepared in Example 6 of the present invention;

[0046] Figure 6B This is a particle size distribution diagram of hydroxyapatite microspheres prepared in Example 6 of the present invention;

[0047] Figure 7 This is a scanning electron microscope image of the hydroxyapatite microspheres prepared in Example 7 of the present invention;

[0048] Figure 8 This is a scanning electron microscope image of the hydroxyapatite microspheres prepared in Example 8 of the present invention;

[0049] Fig. 9 The present invention is a schematic diagram of the process for preparing hydroxyapatite microspheres regulated by malic acid. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] It should be noted that similar reference numerals and letters represent similar items in the following drawings, so once an item is defined in one drawing, it does not need to be further defined and explained in the subsequent drawings. At the same time, in the description of the present invention, the terms "first", "second", etc. are only used to distinguish the description and cannot be understood as indicating or implying relative importance.

[0052] The existing methods for preparing calcium phosphate microspheres include sol-gel method, microemulsion method, spray drying method, template method, etc. The sol-gel method and microemulsion method have mild synthesis conditions, but low yield and it is difficult to accurately control the morphology and surface nanostructure of the microspheres; although the spray drying method has high production efficiency, it is difficult to control the morphology and size of the microspheres, resulting in poor uniformity of the obtained particles. The present invention provides a technical solution for synthesizing mesoporous hydroxyapatite (HAP) microspheres with uniform morphology and high specific surface area by hydrothermal method using malic acid as a morphology regulator. The preparation method of mesoporous hydroxyapatite microspheres regulated by malic acid proposed in the present invention introduces malic acid in the hydrothermal reaction process, and utilizes its coordination effect with calcium ions to accurately regulate the nucleation and growth process of HAP particles, thereby achieving control of the particle morphology. Specifically, by adjusting the ratio of malic acid to calcium source, reaction temperature and time, spherical HAP particles with nanoporous structure can be obtained, and both the particle size and specific surface area can be regulated.

[0053] Embodiment 1:

[0054] Reference Fig. 9 , a schematic diagram of a method for preparing hydroxyapatite microspheres regulated by malic acid, this embodiment provides a method for preparing mesoporous hydroxyapatite microspheres regulated by malic acid:

[0055] S1. Add 1.180 g Ca(NO3)2·4H2O and 1.074 g Na2HPO4·12H2O to 50 mL of deionized water in sequence so that the calcium-phosphorus molar ratio is 1.67, and continue stirring for a period of time, and the stirring time is 5-15 minutes.

[0056] S2. Gradually add nitric acid until the solution is clear. Add about 500 μL and measure the solution pH to be 2-3.

[0057] S3. Add 335.2 mg of malic acid (MA) to the solution to make the concentration of malic acid 50 mM. Then add 1.68 g of urea and continue stirring for a period of time to obtain the final reaction solution. The stirring time is 5-15 minutes.

[0058] S4. The final reaction solution was transferred into a hydrothermal reactor and placed in a forced air drying oven heated to 150° C. for reaction for 3 h.

[0059] S5. After the reaction is completed, the solution is cooled to room temperature. Centrifuged at 5000 r / min for 3 min, the precipitate is repeatedly washed with deionized water and ethanol, twice each. The washed precipitate is dried in an oven at 80°C for 48 hours to obtain hydroxyapatite microspheres.

[0060] The scanning electron microscope image of the hydroxyapatite microspheres prepared in this example is as follows: Figure 1A As shown in the figure, it can be seen that the particles collected after the reaction have a certain non-spherical cluster structure, in which the particles present a flower-like structure composed of flakes.

[0061] The X-ray diffraction pattern of the hydroxyapatite microspheres prepared in this example is as follows: Figure 1B As shown in the figure, it can be seen that the obtained malic acid-regulated hydroxyapatite microspheres (MA-HA) correspond to the standard diffraction characteristic peaks of pure hydroxyapatite, indicating that the phase component of the obtained microspheres is hydroxyapatite, and no other impurities are generated.

[0062] Embodiment 2:

[0063] Reference Fig. 9 , a schematic diagram of a method for preparing hydroxyapatite microspheres regulated by malic acid, this embodiment provides a method for preparing mesoporous hydroxyapatite microspheres regulated by malic acid:

[0064] S1. Add 1.180 g Ca(NO3)2·4H2O and 1.074 g Na2HPO4·12H2O to 50 mL of deionized water in sequence so that the calcium-phosphorus molar ratio is 1.67, and continue stirring for a period of time, and the stirring time is 5-15 minutes.

[0065] S2. Gradually add nitric acid until the solution is clear. Add about 500 μL and measure the pH value of the solution to 2-3.

[0066] S3. Add 670.4 mg of malic acid to the solution to make the concentration of malic acid 100 mM. Then add 1.68 g of urea and continue stirring for 5 to 15 minutes to obtain the final reaction solution.

[0067] S4. The final reaction solution was transferred into a hydrothermal reactor and placed in a forced air drying oven heated to 150° C. for reaction for 3 h.

[0068] S5. After the reaction is completed, the solution is cooled to room temperature. Centrifuged at 5000 r / min for 3 min, the precipitate is repeatedly washed with deionized water and ethanol, twice each. The washed precipitate is dried in an oven at 80°C for 48 hours to obtain hydroxyapatite microspheres.

[0069] The scanning electron microscope image of the hydroxyapatite microspheres prepared in this example is as follows: Figure 2A As shown in the figure, it can be seen that the particles collected after the reaction have a good spherical structure and nanopores appear on the surface of the particles. The particle size distribution diagram of the hydroxyapatite microspheres prepared in this example is shown in Figure 2C As shown in the figure, it can be seen that the particle size distribution range is 5-30 μm, mainly concentrated around 15 μm, and the average particle size is 15.58±4.65 μm. Compared with Example 1, it is shown that the increase in the concentration of the regulating agent is conducive to the formation of spherical particles.

[0070] The X-ray diffraction pattern of the hydroxyapatite microspheres prepared in this example is as follows: Figure 2B As shown in the figure, it can be seen that the obtained MA-HA corresponds to the standard diffraction characteristic peak of pure hydroxyapatite, indicating that the phase component of the prepared microspheres is hydroxyapatite, and no other impurities are generated.

[0071] Embodiment 3:

[0072] Reference Fig. 9 , a schematic diagram of a method for preparing hydroxyapatite microspheres regulated by malic acid, this embodiment provides a method for preparing mesoporous hydroxyapatite microspheres regulated by malic acid:

[0073] S1. Add 1.180 g Ca(NO3)2·4H2O and 1.074 g Na2HPO4·12H2O to 50 mL of deionized water in sequence so that the calcium-phosphorus molar ratio is 1.67, and continue stirring for 5 to 15 minutes.

[0074] S2. Gradually add nitric acid until the solution is clear. Add about 500 μL and measure the pH value of the solution to 2-3.

[0075] S3. Add 1340.9 mg of malic acid to the solution to make the malic acid concentration 200 mM. Then add 1.68 g of urea and continue stirring for 5 to 15 minutes to obtain the final reaction solution.

[0076] S4. The final reaction solution was transferred into a hydrothermal reactor and placed in a forced air drying oven heated to 150° C. for reaction for 3 h.

[0077] S5. After the reaction is completed, the solution is cooled to room temperature. Centrifuged at 5000 r / min for 3 min, the precipitate is repeatedly washed with deionized water and ethanol, twice each. The washed precipitate is dried in an oven at 80°C for 48 hours to obtain hydroxyapatite microspheres.

[0078] The scanning electron microscope image of the hydroxyapatite microspheres prepared in this example is as follows: Figure 3A As shown in the figure, it can be seen that the particles collected after the reaction have a good spherical structure and a nanoporous structure on the surface of the particles. The particle size distribution diagram of the hydroxyapatite microspheres prepared in this example is shown in Figure 3C As shown in the figure, it can be seen that the particle size distribution range is 0-25 μm, mainly concentrated around 11 μm, and the average particle size is 11.36±3.49 μm. Compared with Example 2, it is explained that the increase in the concentration of the regulating agent reduces the particle size.

[0079] The X-ray diffraction pattern of the hydroxyapatite microspheres prepared in this example is as follows: Figure 3B As shown in the figure, it can be seen that the obtained MA-HA corresponds to the standard diffraction characteristic peak of pure hydroxyapatite. The Fourier transform infrared spectrum of the hydroxyapatite microspheres prepared in this example is as follows Figure 3D As shown in the figure, it can be seen that the obtained MA-HA corresponds to the characteristic absorption peak of pure hydroxyapatite, indicating that the phase component of the prepared microspheres is hydroxyapatite, and no other impurities are generated.

[0080] Embodiment 4:

[0081] Reference Fig. 9 , a schematic flow chart of the preparation method of hydroxyapatite microspheres regulated by malic acid, in this embodiment, the reaction time in step S4 in embodiment 1 is changed to 5h, and the remaining operation steps are the same as those in embodiment 1.

[0082] The scanning electron microscope image of the hydroxyapatite microspheres prepared in this example is as follows: Figure 4A As shown in the figure, it can be seen that the particles collected after the reaction have a certain spherical structure and the surface morphology of the particles is uneven. The particle size distribution diagram of the hydroxyapatite microspheres prepared in this embodiment is shown in Figure 4BAs shown in the figure, it can be seen that the particle size distribution range is 5-35 μm, and the average particle size is 14.15±6.84 μm. Compared with Example 1, it is shown that the reaction time has an effect on particle growth.

[0083] Embodiment 5:

[0084] Reference Fig. 9 , a schematic diagram of the preparation method of hydroxyapatite microspheres regulated by malic acid, in this embodiment, the reaction time in step S4 in embodiment 2 is changed to 5h, and the remaining operation steps are the same as embodiment 2.

[0085] The scanning electron microscope image of the hydroxyapatite microspheres prepared in this example is as follows: Figure 5A As shown in the figure, it can be seen that the particles collected after the reaction have a good spherical structure and the surface morphology of the particles is uneven. The particle size distribution diagram of the hydroxyapatite microspheres prepared in this example is shown in Figure 5C As shown in the figure, it can be seen that the particle size distribution range is 5-45 μm, the average particle size is 17.90±7.52 μm, and the particle size distribution range is relatively large. Compared with Example 2, larger microsphere particles of about 40 μm appear, indicating that the reaction time has an effect on particle growth.

[0086] The X-ray diffraction pattern of the hydroxyapatite microspheres prepared in this example is as follows: Figure 5B As shown in the figure, it can be seen that the obtained MA-HA corresponds to the standard diffraction characteristic peak of pure hydroxyapatite, indicating that the phase component of the prepared microspheres is hydroxyapatite, and no other impurities are generated.

[0087] Embodiment 6:

[0088] Reference Fig. 9 , a schematic flow chart of the preparation method of hydroxyapatite microspheres regulated by malic acid, in this embodiment, the reaction time in step S4 in embodiment 3 is changed to 5h, and the remaining operation steps are the same as those in embodiment 3.

[0089] The scanning electron microscope image of the hydroxyapatite microspheres prepared in this example is as follows: Fig. 6A As shown in the figure, it can be seen that the particles collected after the reaction have a good spherical structure and the surface morphology of the particles is relatively uniform. The particle size distribution diagram of the hydroxyapatite microspheres prepared in this example is shown in Figure 6B As shown, it can be seen from the figure that the particle size distribution range is 5-25μm, and the average particle size is 11.97±4.79μm.

[0090] Embodiment 7:

[0091] Reference Fig. 9, a schematic flow chart of a method for preparing hydroxyapatite microspheres regulated by malic acid, in this embodiment, the reaction time in step S4 in embodiment 2 is changed to 30 min, and the remaining operation steps are the same as those in embodiment 2.

[0092] The scanning electron microscope image of the hydroxyapatite microspheres prepared in this example is as follows: Figure 7 As shown in the figure, it can be seen that a good spherical structure appeared after 30 minutes of reaction, and the particle surface showed micron pores.

[0093] Embodiment 8:

[0094] Reference Fig. 9 , a schematic diagram of the preparation process of hydroxyapatite microspheres regulated by malic acid, in this embodiment, the reaction time in step S4 in embodiment 2 is changed to 1h, and the remaining operation steps are the same as those in embodiment 2.

[0095] The scanning electron microscope image of the hydroxyapatite microspheres prepared in this example is as follows: Figure 8 As shown in the figure, it can be seen that microsphere particles with relatively dense surface appear after 1 hour of reaction. Compared with Example 7, it is shown that as the reaction time increases, the hydroxyapatite crystals grow more densely. In summary, the present invention uses a reasonable and easy hydrothermal process and malic acid, a small molecule regulator, to achieve effective regulation of the morphology and structure of hydroxyapatite particles, providing a new solution for the preparation of high-performance biomedical materials.

[0096] It is understandable that, in the present invention, as shown in Examples 1 to 3, with the increase of malic acid concentration (such as from 50 mM to 200 mM), the morphology of hydroxyapatite microspheres changes from a cluster structure to a more uniform spherical structure, and nanopores appear on the particle surface. This shows that malic acid, as a morphology regulator, can effectively affect the crystal morphology and size of hydroxyapatite.

[0097] In the present invention, Examples 4 to 8 show that the change in reaction time has a significant effect on the morphology and size of hydroxyapatite microspheres. For example, compared with Example 1 (3 hours), Example 4 (5 hours reaction time) has uneven particle surface morphology, indicating that a longer reaction time may cause the particles to grow into a spherical structure. Compared with Example 8 (1 hour), Example 7 (30 minutes reaction time) has poor crystallinity, while Example 8 has increased crystallinity, indicating that the extension of reaction time helps to improve the crystallinity of hydroxyapatite.

[0098] It can be understood that the effect of reaction conditions on the size of hydroxyapatite microspheres is as follows: in Example 3, compared with Example 2, a higher malic acid concentration (200 mM vs. 100 mM) resulted in a decrease in particle size, indicating that a high concentration of malic acid may promote the formation of smaller particles. In Example 5 (reaction time 5 hours), compared with Example 2 (reaction time 3 hours), Example 8 (reaction time 1 hour), and Example 7 (reaction time 30 minutes), the average particle size first decreased and then increased, and microsphere particles of larger size (about 40 μm) appeared, indicating that as the reaction time increases, hydroxyapatite crystals may dissolve and recrystallize, and a longer reaction time leads to an increase in the size of some particles.

[0099] The results of the above examples are shown in Table 1:

[0100] Table 1. Summary of parameters and result data of various examples

[0101]

[0102]

[0103] In practical applications, all embodiments of the present invention mention the process of centrifugation and washing with deionized water and ethanol, which helps to remove unreacted precursors and by-products, thereby obtaining pure hydroxyapatite microspheres. In the embodiments, the step of drying in an oven at 80°C for 48 hours is mentioned, which is crucial for obtaining dry and morphologically stable hydroxyapatite microspheres.

[0104] In summary, by adjusting the malic acid concentration and reaction time, the morphology, size and crystallinity of hydroxyapatite microspheres can be finely controlled. These factors are crucial to the final application performance of the material, such as in the fields of biomedicine, drug-loaded sustained release and bone defect filling. Through these embodiments, the synthesis conditions can be optimized to obtain hydroxyapatite microspheres with specific properties.

[0105] To achieve precise control of malic acid concentration and reaction time in the experiment, the following methods can be adopted:

[0106] 1. Use accurate measuring equipment: In the experiment, weigh the appropriate amount of malic acid powder directly on the analytical balance according to the amount of malic acid required for the experiment. Ensure the accuracy of the weighing process and avoid the influence of wind and vibration of the balance.

[0107] 2. Temperature control: Reaction temperature has a significant impact on the rate of chemical reactions. In experiments, high-throughput reaction devices with independent temperature control and monitoring functions can be used to accurately control the temperature of the reaction, thereby controlling the reaction time. High-throughput reaction devices often have the characteristics of automation, which can automatically adjust the temperature, reduce manual intervention, and improve the repeatability and reliability of the experiment.

[0108] 3. Time control: Use a timer or a device with time control function to accurately control the reaction time. For example, in some experiments, it may be necessary to take samples or add reagents at a specific time point. At this time, a timer can be used to ensure the accuracy of the operation.

[0109] 4. Monitoring of color changes: In the experiment, the precipitated calcium phosphate crystals in the solution can be removed by adjusting the pH value.

[0110] This is because calcium phosphate dissolves under acidic conditions and precipitates under alkaline conditions.

[0111] In summary, in the method for preparing mesoporous hydroxyapatite microspheres, malic acid is used as a morphology regulator: that is, in the embodiment, the morphology and size of the mesoporous hydroxyapatite microspheres are regulated by adding malic acid of different concentrations. This method is not common in the prior art. The addition of malic acid not only affects the morphology of the microspheres, but also affects their surface structure and pore size; at the same time, there is a synergistic effect of urea, that is, urea is added at the same time as malic acid, and the molar ratio of urea to calcium source is 0.56:0.1. This synergistic effect may have an important influence on the formation mechanism of the microspheres. This is rare in previous studies; at the same time, the temperature and time of the hydrothermal reaction are precisely controlled in the examples, such as reacting at 150°C for 5 hours. The optimization of such conditions helps to obtain the ideal microsphere morphology and size; it also mentions the use of a centrifugal speed of 5000 rpm and repeated washing with deionized water and ethanol. This step helps to remove impurities and improve the purity and surface properties of the microspheres. In the drying process, the examples mention drying in an oven at 80°C for 48 hours. Such precise drying conditions help to control the final morphology and physical properties of the microspheres.

[0112] Compared with other prior arts, such as a porous hydroxyapatite microsphere material and its preparation method, it is mentioned that sodium citrate is used as a template to regulate the growth of hydroxyapatite crystals, and a microsphere morphology with a surface porous structure is obtained. It is different from the use of malic acid as a regulator in this embodiment. Malic acid has a unique structure of two carboxyl groups and one hydroxyl group. The presence of the double carboxyl group enables it to provide a more stable binding mode in the process of regulating the nucleation and growth of HAP crystals, which helps to form a uniform, pore-controlled mesoporous structure. At the same time, the hydroxyl group in malic acid may also affect the surface chemical properties of HAP and improve its biocompatibility. Malic acid is relatively hydrophilic and easy to disperse under hydrothermal conditions, providing a more uniform regulation effect.

[0113] In addition, malic acid exists naturally in many fruits and is a common intermediate product in human metabolism. It has good biocompatibility and safety, and is suitable for the development of biomedical materials. L-malic acid is a naturally occurring form that does not produce harmful byproducts in the human metabolic pathway, making it more suitable for the application of biomedical materials.

[0114] The present application selects a higher temperature and a short reaction time. These parameters have a significant impact on the addition of malic acid and its performance in the reaction: higher temperatures usually increase the reaction rate because high temperatures increase the movement speed and collision frequency of molecules, thereby accelerating the reaction process. In the present application, the reaction conditions of higher temperature and shorter time are selected to achieve the desired reaction conversion rate in a shorter time, thereby improving production efficiency. Therefore, at a higher temperature, even if the reaction time is shorter, it is possible to achieve a reaction efficiency similar to or better than that of a low temperature and long reaction. The thermal decomposition temperature of malic acid is 140°C, and the temperature used in the present application is 150°C. The principle of how to control the effective effect of malic acid is as follows: ① The hydrothermal reaction is carried out under a high pressure environment, which will inhibit the thermal decomposition of malic acid to a certain extent. Therefore, the selection of 150°C, which is slightly higher than the decomposition temperature of malic acid, not only ensures the reaction speed, but also ensures the effectiveness of malic acid. ② Malic acid has a large number of hydroxyl groups, good hydrophilicity, and good dispersibility, which provides good material dispersion conditions for rapid reaction. At higher temperatures, the growth rate of crystals may be accelerated, but smaller crystals may also be formed or the integrity of the crystals may be affected due to the shorter reaction time. The surface of the hydroxyapatite particles regulated by malic acid in this embodiment has dense flake stacking and has a more excellent surface micro-nano structure. The mesoporous surface makes it more conducive to drug loading and sustained release; the micron-level size makes it more conducive to applications such as bone defect filling.

[0115] The present invention adopts a reaction system, and a macroporous spherical structure appears at 30 minutes, and a mesoporous microsphere structure with good crystallinity and uniform morphology is obtained at 5 hours. The present invention provides a simple, time-saving and efficient method for preparing mesoporous hydroxyapatite microspheres with good crystallinity and uniform morphology, which can be applied to different biomedical fields.

[0116] In general, the present invention demonstrates a certain degree of creativity in the selection of morphology regulators, precise control of reaction conditions, and refinement of post-processing processes. These factors work together to help obtain mesoporous hydroxyapatite microspheres with specific morphologies and sizes, thus playing an important role in biomedicine and other fields.

[0117] It should be noted that, regarding the system in the above embodiment, the specific manner in which each module performs operations has been described in detail in the embodiment of the method, and will not be elaborated here.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

[0119] The above is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

Claims

1. A method for preparing mesoporous hydroxyapatite microspheres regulated by malic acid, characterized in that: The following steps are involved: Step S1: dissolving a calcium source and a phosphorus source in deionized water in sequence, and mixing them evenly to prepare a reaction solution; Step S2: adding nitric acid or sodium hydroxide dropwise to the reaction solution to adjust the pH of the solution to a clear state; Step S3: adding malic acid of different concentrations as a morphology regulator to the clarified reaction solution, and then adding urea; Step S4: transfer the prepared solution into a reaction kettle and keep the reaction at a set temperature for 15 minutes to 12 hours; Step S5: After the reaction is completed, the solution is cooled to room temperature and centrifuged and dried to obtain a hydroxyapatite powder sample.

2. The method for preparing mesoporous hydroxyapatite microspheres regulated by malic acid according to claim 1, characterized in that: In the step S1, calcium nitrate tetrahydrate and disodium hydrogen phosphate dodecahydrate are added to 50 mL of deionized water in sequence to make the calcium source concentration 0.1 mol / L and the calcium-phosphorus molar ratio 1.67, thereby preparing a reaction solution.

3. The method for preparing mesoporous hydroxyapatite microspheres regulated by malic acid according to claim 1, characterized in that: In step S3, the molar ratio of urea to the calcium source is 0.56:0.1, and the malic acid is L-malic acid, and the molar ratio of L-malic acid to the calcium source is (0.05-0.2):0.

1.

4. The method for preparing mesoporous hydroxyapatite microspheres regulated by malic acid according to claim 3, characterized in that: The increase in malic acid concentration leads to a dense surface of hydroxyapatite particles, a decrease in pore size, and a corresponding decrease in the average particle size.

5. The method for preparing mesoporous hydroxyapatite microspheres regulated by malic acid according to claim 1, characterized in that: In step S4, the set temperature is 120°C-180°C.

6. The method for preparing mesoporous hydroxyapatite microspheres regulated by malic acid according to claim 1, characterized in that: As the reaction time increases, the particles transform from amorphous nanoparticles ACP to HAP particles, and the particle phase changes; at the same time, as the reaction time increases, the crystallinity of HAP particles increases.

7. The method for preparing mesoporous hydroxyapatite microspheres regulated by malic acid according to claim 1, characterized in that: In step S2, the pH value of the solution is adjusted with nitric acid or sodium hydroxide until the solution is clear and the pH value of the solution is measured to be 2-3.

8. A mesoporous hydroxyapatite microsphere regulated by malic acid, characterized in that: Obtained by the preparation method described in any one of claims 1 to 7.

9. A hydroxyapatite powder obtained by the preparation method according to any one of claims 1 to 3, characterized in that: Has uniform morphological characteristics.

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