Preparation method of bioactive beta-tricalcium phosphate hollow microspheres

Abstract

The invention belongs to the field of medical materials. The preparation method of the bioactive beta-tricalcium phosphate hollow microspheres comprises the following steps: S1, preparing a K2HPO4 solution with the concentration of 0.25 M, and adjusting the pH value to 7; s2, Li-Ca-B glass microspheres are prepared, the Li-Ca-B glass microspheres and the phosphate solution in the step S1 are mixed, and calcium-deficient hydroxyapatite hollow microspheres with the calcium-phosphorus ratio Ca / P being 1.5 are obtained; s3, washing the calcium-deficient hydroxyapatite hollow microspheres, and drying the calcium-deficient hydroxyapatite hollow microspheres after washing; and S4, carrying out heat treatment on the calcium-deficient hydroxyapatite hollow microspheres to obtain the beta-tricalcium phosphate hollow microspheres. The beta-tricalcium phosphate hollow microspheres prepared by the method have hollow and spherical shell porous structures, are beneficial to ingrowth of tissue cells, are higher in degradation rate, and can improve the repair speed of bone tissues.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medical materials, and particularly relates to a preparation method of bioactive beta-tricalcium phosphate hollow microspheres. BACKGROUND

[0002] Calcium phosphate materials (such as hydroxyapatite, beta-tricalcium phosphate, etc.) are widely used in bone defect repair, drug delivery and regenerative medicine due to their high similarity to human bone composition, excellent biocompatibility and bone conductivity. Traditional bulk materials have problems such as low porosity, mismatched degradation rate and bone regeneration, and poor drug loading capacity. For example, the porosity of hydroxyapatite bulk prepared by traditional sintering method is usually less than 40%, and the average pore size is less than 10 μm, which is difficult to support vascular ingrowth (pore size > 100 μm) and cell migration. Current methods for preparing calcium phosphate hollow microspheres include template method, chemical pore preparation, spray drying, hydrothermal method and electrospinning method, etc.

[0003] The current preparation methods of calcium phosphate hollow microspheres have certain defects. The template method needs multiple conversion steps and is easy to leave template impurities, reducing biocompatibility. The pore size of the microspheres prepared by the spray drying method is limited, and the pore connectivity is poor. In the hydrothermal method, the temperature / pressure distribution in the reaction kettle is uneven, resulting in large particle size dispersion of the microspheres, which limits the clinical application. The prepared calcium phosphate microspheres have insufficient pore structure regulation. The internal structure of the calcium phosphate microspheres is mostly closed or small pores, which cannot meet the demand for large pores (> 100 μm), hindering tissue ingrowth. And the main products on the market are hydroxyapatite porous or hollow microspheres, which have extremely slow degradation rate (more than 5 years for complete degradation in vivo), and are easy to leave mechanical barriers, inhibiting the mineralization of new bone. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a preparation method of bioactive beta-tricalcium phosphate hollow microspheres. The beta-tricalcium phosphate hollow microspheres prepared by the present application have a hollow and porous shell structure, and a relatively fast degradation rate, which can promote bone tissue repair.

[0005] The technical solution adopted by the present application to solve the above problems is as follows:

[0006] A preparation method of bioactive beta-tricalcium phosphate hollow microspheres, comprising the following steps:

[0007] S1. Prepare a K2HPO4 solution with a concentration of 0.25 M and adjust the pH to 7;

[0008] S2. Prepare Li-Ca-B glass microspheres, mix the Li-Ca-B glass microspheres with the phosphate solution of step S1, control the temperature at 37℃, and react for 2-4 days under stirring to obtain calcium-deficient hydroxyapatite hollow microspheres with a calcium to phosphorus ratio Ca / P of 1.5;

[0009] S3. washing the calcium-deficient hydroxyapatite hollow microspheres obtained in step S2, and then drying the washed calcium-deficient hydroxyapatite hollow microspheres;

[0010] S4. heat-treating the calcium-deficient hydroxyapatite hollow microspheres obtained in step S3 to obtain β-tricalcium phosphate hollow microspheres.

[0011] According to the technical scheme, although β-tricalcium phosphate cannot be directly precipitated from an aqueous solution, it can promote the precipitation of the apatite layer in an ionic aqueous solution due to its low interface energy relative to apatite. The calcium-deficient hydroxyapatite can be converted into β-tricalcium phosphate by heat treatment. The β-tricalcium phosphate hollow microspheres can be obtained only when the phosphate concentration is 0.25 M and the pH is 7. The β-tricalcium phosphate hollow microspheres cannot be obtained under other concentrations and pH values, and only powders can be directly collected. The β-tricalcium phosphate hollow microspheres prepared by the preparation method have a hollow and porous shell structure, a faster degradation rate, and are beneficial to the repair of bone tissue.

[0012] Further, the pH adjusting agent used for adjusting the pH of the K2HPO4 solution is hydrochloric acid.

[0013] Further, the composition of the Li-Ca-B glass microspheres is CaO: 15wt%, Li2O: 11wt%, and B2O3: 74wt%.

[0014] Further, the mass of the Li-Ca-B glass microspheres to the volume of the phosphate solution is 1g of Li-Ca-B glass microspheres to 200mL of phosphate solution.

[0015] According to the technical scheme, the Li-Ca-B glass microspheres and the phosphate solution can fully react under this mixing ratio, promoting the formation of the hollow and porous structure of the β-tricalcium phosphate hollow microspheres.

[0016] Further, the stirring speed of the mixture of the Li-Ca-B glass microspheres and the phosphate solution is 50-100r / min.

[0017] Further, the washing in step S3 is washing with deionized water and anhydrous ethanol in sequence.

[0018] Preferably, the washing in step S3 is washing with deionized water for three times and then washing with anhydrous ethanol for three times.

[0019] Further, the drying treatment includes first drying and second drying. The temperature of the first drying is 25°C, and the time is ≥12h. The temperature of the second drying is 90°C, and the time is ≥12h.

[0020] Through the above technical scheme, the first drying can make the washed calcium-deficient hydroxyapatite hollow microspheres better dispersed and not easy to agglomerate, and the second drying can make the residual liquid after washing fully volatilize, so as to ensure that the calcium-deficient hydroxyapatite hollow microspheres are fully dried.

[0021] Further, the temperature of the heat treatment in step S4 is 800℃, and the time of the heat treatment is 5h.

[0022] Through the above technical scheme, 800℃ calcination for 5h can ensure that the calcium-deficient hydroxyapatite is completely converted into β-tricalcium phosphate. Lower than 800℃ cannot make the calcium-deficient hydroxyapatite change, and higher than 800℃ will make the outermost shell structure of the β-tricalcium phosphate hollow microspheres dense, almost without pore structure, which is not conducive to the growth of tissue cells and the repair of bone tissue.

[0023] Further, the particle size of the Li-Ca-B glass microspheres is 150-250μm.

[0024] Further, the specific surface area of the β-tricalcium phosphate hollow microspheres in step S4 is 1.5±0.2(m 2 / g), and the average pore size is 7.7±0.2nm.

[0025] Further, the preparation method of the Li-Ca-B glass microspheres comprises the following steps:

[0026] A1. Heating CaCO3, LiCO3 and H3BO3 to a molten state;

[0027] A2. Spreading the mixture in the molten state obtained in step A1 on a stainless steel plate to quench, to obtain Li-Ca-B glass;

[0028] A3. Crushing the Li-Ca-B glass, sieving, to obtain Li-Ca-B glass particles;

[0029] A4. Passing the Li-Ca-B glass particles through a vertical furnace spheroidizing device to obtain Li-Ca-B glass microspheres.

[0030] Further, the temperature of the heating in step A1 is 1200℃, and the time of the heating is 45min.

[0031] Further, the mesh number of the sieving of the Li-Ca-B glass is 60 mesh and 100 mesh. The Li-Ca-B glass is first sieved through a 60 mesh sieve, and then sieved through a 100 mesh sieve.

[0032] By the technical scheme, the Li-Ca-B glass particles with a particle size less than 250 μm can be obtained after passing through the 60-mesh sieve, and the Li-Ca-B glass particles with a particle size of 150-250 μm can be obtained by passing the retained particles through the 100-mesh sieve.

[0033] Further, the temperature of the vertical furnace spheroidizing device is 1200 ℃.

[0034] By the technical scheme, the loss rate of the Li-Ca-B glass microspheres can be reduced.

[0035] The present application has the following beneficial effects:

[0036] In terms of structure, the prepared β-tricalcium phosphate hollow microspheres have a hollow and porous shell structure, and the average pore size of the shell is 7.7±0.2, which is beneficial for the growth of tissue cells. As the β-tricalcium phosphate hollow microspheres degrade, the hollow structure inside them gradually opens, allowing cells and tissues to penetrate into larger pores, promoting the repair of bone tissue. In terms of degradation rate, compared with hydroxyapatite, the prepared β-tricalcium phosphate hollow microspheres have a faster degradation rate. As the β-tricalcium phosphate degrades, Ca 2+ , PO4 3- can promote bone tissue growth and improve the repair speed of bone tissue. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is the XRD pattern of the β-tricalcium phosphate hollow microspheres.

[0038] Figure 2 (a) is the scanning electron microscope image of the β-tricalcium phosphate hollow microspheres after being cut open.

[0039] Figure 2 (b) is the scanning electron microscope image of the outer surface of the β-tricalcium phosphate hollow microspheres.

[0040] Figure 2 (c) is the scanning electron microscope image of the inner surface of the β-tricalcium phosphate hollow microspheres.

[0041] Figure 3 (A) is the optical image of the H&E stained section of the rat skull defect after implanting the microspheres of Example 1 for 8 weeks.

[0042] Figure 3 (B) is the optical image of the H&E stained section of the rat skull defect after implanting the microspheres of Comparative Example 1 for 8 weeks.

[0043] Figure 4 (C) is the optical image of the H&E stained section of the rat subcutaneous defect after implanting the microspheres of Example 1 for 8 weeks.

[0044] Figure 4 (D) is the optical image of H&E staining section of rat subcutaneous defect after 8 weeks of implantation of microspheres of Comparative Example 1.

[0045] Explanation of reference signs:

[0046] 1, host bone; 2, new bone; 3, multinucleated cells; 4, blood vessels. DETAILED DESCRIPTION

[0047] In order to make the technical problems, technical solutions and technical advantages of the present application clearer, specific examples will be described in detail below, but the protection scope of the present application is not limited to the following specific examples, the described examples are only a part of the embodiments of the present application, but not all the embodiments, and even more not the limitation of the present application. Based on the examples in the present application, all other examples obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0048] Unless otherwise defined, all the professional terms used in the following have the same meaning as generally understood by those skilled in the art. The professional terms used in the present application are only for the purpose of describing specific examples, and are not intended to limit the protection scope of the present application.

[0049] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present application can be purchased from the market or can be prepared by existing methods.

[0050] In the quantitative test of the following examples, three repeated experiments were set, and the data were the average value or average value ± standard deviation of three repeated experiments.

[0051] Example 1

[0052] Preparation of Li-Ca-B glass microspheres:

[0053] 26.77 g of CaCO3, 26.29 g of Li2CO3, 132.10 g of H3BO3 were heated to 1200 °C in a platinum-gold crucible for 45 min; the obtained molten mixture was spread on a stainless steel plate to quench, to obtain a Li-Ca-B glass with a composition of CaO: 15 wt%, Li2O: 11 wt%, B2O3: 74 wt%; the Li-Ca-B glass was then crushed, first through a 60-mesh screen to obtain Li-Ca-B glass particles with a particle size of less than 250 μm, and then through a 100-mesh screen while retaining the Li-Ca-B glass particles that could not pass through the 100-mesh screen to obtain Li-Ca-B glass particles with a particle size of 150-250 μm; the temperature of the vertical furnace spheroidizing device was adjusted to 1200 °C, the glass particles were placed in a vibrator above the vertical furnace spheroidizing device, and the Li-Ca-B glass particles were uniformly discharged by vibration, so that the Li-Ca-B glass particles passed through the center of the heating pipe of the vertical furnace spheroidizing device, to obtain Li-Ca-B glass microspheres with a particle size of 150-250 μm.

[0054] Preparation of β-tricalcium phosphate hollow microspheres:

[0055] A K2HPO4 solution with a concentration of 0.25 M was prepared, and a hydrochloric acid solution with a concentration of 0.1 M was added to adjust the pH to 7; the above-prepared Li-Ca-B glass microspheres were mixed with the phosphate solution, and the amount of the phosphate solution was controlled to be 200 mL per 1 g of the Li-Ca-B glass microspheres; the temperature was controlled to be 37 °C, and the reaction was carried out under stirring at 60 r / min for 4 days, to obtain calcium-deficient hydroxyapatite hollow microspheres with a calcium-phosphorus ratio Ca / P of 1.5; the calcium-deficient hydroxyapatite hollow microspheres were washed with deionized water, and the mass / volume ratio of the calcium-deficient hydroxyapatite hollow microspheres to the deionized water was 1:50 mL; after washing, the liquid was poured out after the solid settled to the bottom, and the washing was repeated three times; then, the calcium-deficient hydroxyapatite hollow microspheres were washed with anhydrous ethanol, and the mass / volume ratio of the calcium-deficient hydroxyapatite hollow microspheres to the anhydrous ethanol was 1:30 mL, and the washing was repeated three times; after washing, the first drying was carried out at a temperature of 25 °C for 12 h; then, the second drying was carried out at a temperature of 90 °C for 12 h; after drying, the calcium-deficient hydroxyapatite hollow microspheres were heated at a temperature of 800 °C for 5 h, to obtain β-tricalcium phosphate hollow microspheres.

[0056] Comparative Example 1

[0057] The difference from the examples is that the obtained calcium-deficient hydroxyapatite hollow microspheres with a calcium-phosphorus ratio Ca / P of 1.5 were not subjected to heat treatment.

[0058] The specific components and preparation conditions of the examples and the comparative example are shown in Table 1.

[0059] Table 1

[0060]

[0061] Experimental detection

[0062] The β-tricalcium phosphate hollow microspheres were characterized by quantitative X-ray diffraction (XRD), inductively coupled plasma optical emission spectrometry (ICP-OES), scanning electron microscopy (SEM), and H&E staining section optical images, including skull defect H&E staining section optical images and subcutaneous defect H&E staining section optical images, and the results are shown in Tables 2-3, Figures 1-4

[0063] Skull defect H&E staining section optical image experimental method: 3-month-old male SD rats were selected. Using aseptic instruments and aseptic techniques, two 4.6 mm diameter defects were made on the skull. The microspheres of Example 1 and Comparative Example 1 were respectively implanted in the two defects at 10 mg each, and only one kind of microsphere was implanted in each defect. After 8 weeks, the rats were sacrificed by inhaling CO2, and the skull defect site and surrounding bone and soft tissue were collected to observe the formation of new bone (2) and the residual microspheres, wherein the new bone (2) generation was evaluated by the percentage of new bone (2) in the defect after 8 weeks, and the experiment was repeated 6 times.

[0064] Subcutaneous defect H&E staining section optical image experimental method: 3-month-old male SD rats were selected, and each rat had four implantation sites, two below the shoulder and two above the hind leg. A surgical scissors was used to make a cut about 2 cm long on the skin tissue perpendicular to the spine. A skin retractor was gently inserted into the incision to form a pocket between the skin tissue and the skeletal muscle. The microspheres of Example 1 were implanted in two defects at 20 mg each, and the microspheres of Comparative Example 2 were implanted in the other two defects at 20 mg each. Then, the skin was closed with wound clips. All rats received the same postoperative care and monitoring as the skull implantation. After 8 weeks of implantation, the animals were sacrificed, and the subcutaneous defect site and surrounding soft tissue were collected for subsequent evaluation.

[0065] In combination Figure 1 The broad peaks appearing in the XRD pattern of the Li-Ca-B glass microspheres mixed with the phosphate solution and converted may indicate that the material is poorly crystallized, or composed of nanoscale crystals, or both, which are typical characteristics of calcium-deficient hydroxyapatite, indicating that the Li-Ca-B glass microspheres have been converted into calcium-deficient hydroxyapatite after mixing and reacting with the phosphate solution. After heat treatment, the peak intensity of the microspheres increased significantly, which may be due to the increase in crystal fraction or crystal size. According to the XRD pattern after heat treatment combined with Figure 2 ​(a), which shows that the microspheres are β-tricalcium phosphate hollow microspheres. The microspheres are dissolved in nitric acid, and Ca / P is determined by inductively coupled plasma optical emission spectrometry (ICP-OES), which is consistent with the result of the XRD quantitative analysis.

[0066] Combining Figure 2 (a), it can be seen that the β-tricalcium phosphate hollow microspheres prepared in the application are hollow structures. Combining Figure 2 (b), it can be seen that the outer surface of the β-tricalcium phosphate hollow microspheres has a loose and porous structure. Combining Figure 2 (c), it can be seen that the inner surface of the β-tricalcium phosphate hollow microspheres also has a loose and porous structure, which shows that the shell of the β-tricalcium phosphate hollow microspheres is a porous structure. The ratio of the inner diameter to the outer diameter of the β-tricalcium phosphate hollow microspheres is 30% to 40%, the specific surface area is 1.5 ± 0.2 (m 2 / g), and the average pore size is 7.7 ± 0.2 nm. It shows that the β-tricalcium phosphate hollow microspheres prepared in the application have sufficient pores and can meet the requirements of macropores (> 100 μm), so as to facilitate the growth of tissue cells into the interior of the β-tricalcium phosphate hollow microspheres through the pores and promote the repair of bone tissue.

[0067] The formation of new bone (2) and the residual microspheres after 8 weeks of implantation of the rat skull defects of Example 1 and Comparative Example 1 are shown in Table 2.

[0068] Table 2

[0069] New bone (%) Residual microspheres (%) Example 1 30±3 26±4 Comparative Example 1 16±4 34±4

[0070] By comparing the test results of Example 1 and Comparative Example 1, it can be seen that the β-tricalcium phosphate hollow microspheres prepared in the application have a small amount of residual, which shows that the degradation speed is faster than that of the microspheres of Comparative Example 1, and can promote the growth of new bone (2). Combining Figure 3 (A), the two sides in the section are host bone (1), and the middle of the section is the defect, the β-tricalcium phosphate hollow microspheres have good biocompatibility in the rat body, and gradually degrade, and within 8 weeks, the growth of new bone (2) is obviously induced, and combining Figure 3 (B), the growth of new bone (2) in the section of Example 1 is significantly better than that of Comparative Example 1, which shows that the β-tricalcium phosphate hollow microspheres prepared in the application can promote the repair of bone tissue more than ordinary calcium-deficient hydroxyapatite. And with the degradation of the β-tricalcium phosphate hollow microspheres, there is obvious new bone (2) in the hollow of the β-tricalcium phosphate hollow microspheres, which shows that a larger pore structure can promote the growth of tissue.

[0071] The formation of new bone (2) and the residual microspheres after 8 weeks of implantation of the rat subcutaneous defects of Example 1 and Comparative Example 1 are shown in Table 3.

[0072] Table 3

[0073] Vessels (%) Foreign body giant cells (%) Example 1 1.16±0.32 0.98±0.35 Comparative Example 1 0.42±0.26 0.44±0.21

[0074] In combination Figure 4 , the nuclei of the cells in the slice are blue, the cytoplasmic connective tissue and extracellular matrix are purple or red, and the red blood cells are bright red, among which the multinucleated cells (3) are indicated by green arrows, and the blood vessels (4) are indicated by yellow arrows. The microspheres of Example 1 and Comparative Example 1 are surrounded by connective tissue, some large multinucleated cells (3), i.e. foreign body giant cells, and blood vessels (4). The biomaterials will trigger an immune response after being implanted into the human body, among which the foreign body giant cells play a key role in the foreign body reaction and continuously exist at the interface between the biomaterial and the tissue, and the foreign body giant cells have multiple functions, which can not only wrap the foreign body by forming a fibrous layer to promote the degradation of the degradable biomaterial, but also participate in activities such as immune regulation, tissue degradation and formation of blood vessels (4). In combination Figure 4 (C), the number of blood vessels (4) and foreign body giant cells in the tissue slice of Example 1 is obviously more than Figure 4 (D), which proves that the effect of Example 1 in promoting the generation of new bone (2) is better, and these results further indicate that the β-tricalcium phosphate hollow microspheres have the characteristics of promoting the generation of blood vessels (4).

[0075] The above describes the preferred embodiments of the present application in detail, but the present application is not limited to the specific details in the above-described embodiments, and within the technical concept range of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection range of the present application.

[0076] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, various possible combinations are not described again in the present application.

[0077] In addition, various different embodiments of the present application can also be combined in any manner, as long as it does not deviate from the idea of the present application, and it should also be considered as disclosed by the present application.

Claims

1. A method for preparing biologically active β-tricalcium phosphate hollow microspheres, characterized by, The method comprises the following steps: S1. preparing a K2HPO4 solution with a concentration of 0.25M and adjusting the pH to 7; S2. preparing Li-Ca-B glass microspheres, mixing the Li-Ca-B glass microspheres with the phosphate solution of step S1, controlling the temperature to be 37℃, and reacting for 2-4 days under stirring to obtain calcium-deficient hydroxyapatite hollow microspheres with a calcium-phosphorus ratio Ca / P of 1.5; S3. washing the calcium-deficient hydroxyapatite hollow microspheres obtained in step S2 and performing drying treatment after washing; S4. performing heat treatment on the calcium-deficient hydroxyapatite hollow microspheres obtained in step S3 to obtain β-tricalcium phosphate hollow microspheres.

2. The method of claim 1, wherein the bioactive β-tricalcium phosphate hollow microspheres are prepared by the steps of: The Li-Ca-B glass microspheres have a composition of CaO: 15wt%, Li2O: 11wt%, and B2O3: 74wt%. ​ 3. The method for preparing bioactive β-tricalcium phosphate hollow microspheres according to claim 1, characterized in that, The mass of the Li-Ca-B glass microspheres and the volume of the phosphate solution are in a ratio of 1g of the Li-Ca-B glass microspheres to 200mL of the phosphate solution.

4. The method for preparing bioactive β-tricalcium phosphate hollow microspheres according to claim 1, characterized in that, The calcium-deficient hydroxyapatite hollow microspheres in step S3 are washed with deionized water and anhydrous ethanol in sequence.

5. The method for preparing bioactive β-tricalcium phosphate hollow microspheres according to claim 1, characterized in that, The drying treatment comprises first drying and second drying, the first drying is performed at a temperature of 25℃ for ≥12h, and the second drying is performed at a temperature of 90℃ for ≥12h.

6. The method for preparing bioactive β-tricalcium phosphate hollow microspheres according to claim 1, characterized in that, The heat treatment in step S4 is performed at a temperature of 800℃ for 5h.

7. The method for preparing bioactive β-tricalcium phosphate hollow microspheres according to claim 1, characterized in that, The Li-Ca-B glass microspheres have a particle size of 150-250μm.

8. The method for preparing bioactive β-tricalcium phosphate hollow microspheres according to claim 1, characterized in that, The specific surface area of the β-tricalcium phosphate hollow microspheres in step S4 is 1.5 ± 0.2 (m 2 / g), and the average pore size is 7.7 ± 0.2 nm.

9. The method of producing biologically active β-tricalcium phosphate hollow microspheres according to any one of claims 1 to 8, characterized by, The method for preparing the Li-Ca-B glass microspheres comprises the following steps: A1. heating CaCO3, LiCO3, and H3BO3 to a molten state; A2. quenching the molten mixture obtained in step A1 on a stainless steel plate to obtain Li-Ca-B glass; A3. crushing the Li-Ca-B glass, sieving, and obtaining Li-Ca-B glass particles; A4. passing the Li-Ca-B glass particles through a vertical furnace spheroidization device to obtain Li-Ca-B glass microspheres.

10. The method of claim 9, wherein the bioactive β-tricalcium phosphate hollow microspheres are prepared by the steps of: The heating in step A1 is performed at a temperature of 1200℃ for 45min. ​

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