A self-assembled calcium phosphate microsphere with a high specific surface area and a preparation method thereof

Abstract

The present invention discloses a self-assembled calcium phosphate microsphere with a high specific surface area and a preparation method thereof, relating to the field of calcium phosphate biomaterials. It is obtained by blending and compounding a self-assembled calcium phosphate microsphere doped with bismuth ions (Bi 3+ ‌) and gallium ions (Ga 3+ ‌) with a polyurethane containing conjugated linoleate, retaining the characteristics of the high specific surface area of the calcium phosphate microsphere. The calcium phosphate microsphere of the present invention has excellent degradability, antibacterial property, and osteogenic property, and is worthy of popularization and use in the fields of bone tissue repair and drug carriers, etc.

Description

Technical Field

[0001] The present invention relates to the field of calcium phosphate biomaterials, and particularly to a self-assembled calcium phosphate microsphere with a high specific surface area and a preparation method thereof. Background Art

[0002] Calcium phosphate is an inorganic compound, which usually exists in various mineral forms. For example, hydroxyapatite is one of the main components of bones and teeth, and has good biocompatibility and bioactivity, etc. Calcium phosphate-based biomaterials play an important role in medical fields such as bone tissue defect repair, drug and gene carriers, and medical imaging.

[0003] Calcium phosphate microspheres are one of the calcium phosphate-based biomaterials, and are widely used in separation, catalysis, sensing, tissue engineering, drug release, etc. due to their high specific surface area. In clinical applications, calcium phosphate microspheres used as bone tissue repair materials need to have good biocompatibility, bioactivity, anti-disintegration property, degradability, easy operation, and convenient drug loading, etc. In the usual preparation process of calcium phosphate microspheres, after the calcium phosphate forms spheres, it needs to be sintered. During the sintering process, the crystal structure of calcium phosphate becomes more stable, and at the same time, the density increases, resulting in a reduction in internal voids, which affects the penetration of water and bioenzymes during the degradation process of calcium phosphate, thereby further reducing the degradability. Therefore, the degradability of the obtained calcium phosphate microspheres does not meet the use requirements. In addition, calcium phosphate microspheres still face the risk of bacterial infection when treating infectious bone defects, and higher requirements are put forward for the antibacterial property of calcium phosphate microspheres.

[0004] Previously, there was a study using a composite of calcium phosphate-based biomaterials and polyurethane to improve the biocompatibility, degradability, and mechanical properties, etc. of calcium phosphate-based biomaterials, which was expected to be applied to cartilage tissue substitutes. However, the raw material MDI used in the preparation process of this polyurethane has poor degradability, and the calcium phosphate-based biomaterial used in this study is hydroxyapatite, with insufficient osteogenic induction ability and unsatisfactory bone growth promotion ability, which is not conducive to the use of this polyurethane material after being compounded with calcium phosphate microspheres in the bone repair field with high requirements for degradability and osteogenesis.

[0005] Therefore, the present invention aims to provide a suitable modification method to improve the degradability and antibacterial property of calcium phosphate microspheres; provide raw materials with better degradability for preparing polyurethane, and compound it with calcium phosphate microspheres, in order to obtain calcium phosphate-based biomaterials with excellent properties such as degradability, antibacterial property, and osteogenesis, and expand its scope of application, and obtain a self-assembled calcium phosphate microsphere material with good application prospects. Summary of the Invention

[0006] In order to solve the above technical problems, the present invention provides a self-assembled calcium phosphate microsphere with a high specific surface area and a preparation method thereof.

[0007] The object of the present invention can be achieved by the following technical solutions:

[0008] A self-assembled calcium phosphate microsphere with a high specific surface area, the preparation of which comprises the following steps:

[0009] Step S1: Add calcium chloride, bismuth nitrate, and gallium chloride into deionized water and stir for 20 - 25 min, then add potassium sodium tartrate tetrahydrate under stirring. After adding, continue stirring for 10 - 15 min, and then add sodium dihydrogen phosphate under stirring. After adding, continue stirring for 30 - 35 min. Then place it in a microwave chemical reactor and stir at 200 - 210 °C for 24 - 25 h, then let it stand at 150 - 160 °C for 3 - 3.5 h. After cooling to room temperature, perform centrifugal separation, wash it repeatedly with deionized water and ethanol 3 times each, and then dry it overnight at 60 - 80 °C to obtain the calcium phosphate microsphere product;

[0010] Furthermore, the dosage ratio of calcium chloride, bismuth nitrate, gallium chloride, deionized water, potassium sodium tartrate tetrahydrate, and sodium dihydrogen phosphate is 10 - 12 mmol: 0.3 - 0.35 mmol: 0.25 - 0.30 mmol: 180 - 190 mL: 10 - 12 mmol: 6.3 - 6.5 mmol;

[0011] During the reaction process of step S1, in a microwave environment, self-assembly obtains a calcium phosphate microsphere product doped with bismuth ions (Bi 3+ ), and gallium ions (Ga 3+ ‌);

[0012] Step S2: Add 1-(4-nitrophenyl)ethanol into cyclohexane, add an alkali solution under stirring, add epichlorohydrin after 3 - 5 min, and stir and react at room temperature for 5 - 5.5 h to obtain reaction product 1; in an inert gas atmosphere, add conjugated linoleic acid into ethyl acetate, add potassium hydroxide at 40 - 45 °C, then add reaction product 1 under stirring, continue stirring and reacting for 9 - 9.5 h, then raise the temperature to 100 - 105 °C, and continue stirring for 1 - 1.5 h to obtain reaction product 2;

[0013] Furthermore, the dosage ratio of 1-(4-nitrophenyl)ethanol, cyclohexane, alkali solution, and epichlorohydrin is 17.5 - 18.5 g: 60 - 70 mL: 6 - 7 mL: 9.3 - 9.6 g, and the alkali solution is a sodium hydroxide solution with a mass fraction of 5 - 6%; the dosage ratio of conjugated linoleic acid, ethyl acetate, potassium hydroxide, and reaction product 1 is 29 - 29.5 g: 110 - 120 mL: 0.6 - 0.65 g: 27 - 27.5 g;

[0014] During the reaction process of step S2, the alcohol hydroxyl group in 1-(4-nitrophenyl)ethanol reacts with epichlorohydrin to obtain reaction product 1 containing an epoxy group; the carboxyl group of conjugated linoleic acid reacts with the epoxy group of reaction product 1 to obtain reaction product 2 containing a nitro group, a hydroxyl group and a conjugated linoleic acid ester;

[0015] Step S3: Stir and mix reaction product 2, potassium carbonate, sodium hydroxide and dimethyl sulfoxide to obtain mixture 1; mix p-nitrobenzoyl chloride and dimethyl sulfoxide to obtain mixture 2, and dropwise add mixture 2 to mixture 1 in an ice-water bath, then raise the temperature to 45 - 50 °C and stir and react for 8 - 8.5 h to obtain reaction product 3;

[0016] Furthermore, the dosage ratio of reaction product 2, potassium carbonate, sodium hydroxide and dimethyl sulfoxide in mixture 1 is 57 - 58 g : 1.4 - 1.5 g : 4 - 4.2 g : 115 - 125 mL; the dosage ratio of p-nitrobenzoyl chloride and dimethyl sulfoxide in mixture 2 is 19 - 20 g : 40 - 45 mL; the dosage ratio of mixture 1 and mixture 2 is 120 - 130 mL : 45 - 50 mL;

[0017] During the reaction process of step S3, the hydroxyl group of reaction product 2 reacts with p-nitrobenzoyl chloride to obtain reaction product 3 containing a dinitro group and a conjugated linoleic acid ester;

[0018] Step S4: Add reaction product 3 into ethyl acetate, raise the temperature to 50 - 55 °C, then add sodium dithionite, and reflux and stir and react for 1.5 - 1.7 h to obtain reaction product 4; in an atmosphere of protective gas, add reaction product 4 and triphosgene into cyclohexane at 70 - 75 °C, then raise the temperature to 110 - 120 °C, and reflux and stir and react for 4 - 4.5 h to obtain reaction product 5;

[0019] Furthermore, the dosage ratio of reaction product 3, ethyl acetate and sodium dithionite is 77 - 78 g : 200 - 220 mL : 18 - 18.5 g; the dosage ratio of reaction product 4, triphosgene and cyclohexane is 75 - 76 g : 30 - 31 g : 220 - 230 mL;

[0020] During the reaction process of step S4, the dinitro group of reaction product 3 is selectively reduced to an amino group by sodium dithionite to obtain reaction product 4; the amino group of reaction product 4 reacts with triphosgene to form chloroamide, and then dehydrochlorination occurs by heating to generate diisocyanate, which is reaction product 5;

[0021] Step S5: In an atmosphere of protective gas, mix castor oil, 1,4-butanediol, distilled water, dibutyltin dilaurate, triethanolamine, and silicone oil at 28 - 32°C for 30 - 35 min. Then add reaction product 5, stir at 800 - 900 r / min for 20 - 25 s, pour it into a mold, and let it stand and foam at 20 - 23°C for 30 - 35 min. After demolding, cure it at room temperature for 72 - 73 h to obtain reaction product 6; heat and blend the calcium phosphate microsphere product and reaction product 6 at 170 - 175°C for 20 - 30 min, cool it, dry it at 70 - 80°C for 24 - 25 h, and then grind it into a powder with a particle size of 0.5 - 1 mm to obtain self-assembled calcium phosphate microspheres with a high specific surface area.

[0022] Further, the dosage ratio of castor oil, 1,4-butanediol, distilled water, dibutyltin dilaurate, triethanolamine, silicone oil, and reaction product 5 is 95 - 105 g : 5 - 10 g : 2 - 2.4 g : 18 - 20 mg : 9 - 10 mg : 0.5 - 0.7 g : 60 - 65 g; the dosage ratio of the calcium phosphate microsphere product and reaction product 6 is 90 - 95 g : 5 - 10 g.

[0023] During the reaction in step S5, a polyurethane containing conjugated linoleic acid ester, i.e., reaction product 6, is prepared by the all-water foaming method; the calcium phosphate microsphere product and reaction product 6 are heated and blended to obtain calcium phosphate microspheres doped with bismuth ions (Bi 3+ ), and gallium ions (Ga 3+ ‌) and compounded with a polyurethane containing conjugated linoleic acid ester.

[0024] Beneficial effects of the present invention: The present invention discloses self-assembled calcium phosphate microspheres with a high specific surface area, which are obtained by blending and compounding self-assembled calcium phosphate microspheres doped with bismuth ions (Bi 3+ ), and gallium ions (Ga 3+ ‌) with a polyurethane containing conjugated linoleic acid ester, retaining the characteristics of the high specific surface area of the calcium phosphate microspheres. Bismuth ions (Bi 3+ ), and gallium ions (Ga 3+After co-doping, it not only endows the calcium phosphate microspheres with good antibacterial properties, but also can change the morphology of calcium phosphate and cause lattice distortion, thereby improving the degradability of the calcium phosphate microspheres. Moreover, it can make the surface of the calcium phosphate microspheres positively charged, which is more convenient for loading negatively charged drugs through charge interaction. After forming conjugated linoleic acid ester through reaction and introducing it into reaction product 5, it not only makes reaction product 5, i.e., diisocyanate used in the preparation process of reaction product 6, i.e., polyurethane, have degradability and antibacterial properties, but also makes up for the deficiency that the antibacterial property of conjugated linoleic acid is easily affected by pH value, thereby improving the degradability and antibacterial properties of the prepared polyurethane. And conjugated linoleic acid has a promoting effect on human bone growth, helps maintain bone density, and prevents bone diseases such as osteoporosis. After its combination with calcium phosphate microspheres doped with bismuth ions (Bi 3+ ), and gallium ions (Ga 3+ ), it further improves the degradability of the calcium phosphate microspheres. When combined with bismuth ions (Bi 3+ ), and gallium ions (Ga 3+ ), it synergistically improves the antibacterial property and also improves the bone growth promoting property. Specific Embodiments

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts belong to the scope of protection of the present invention.

[0026] Example 1

[0027] A self-assembled calcium phosphate microsphere with a high specific surface area, the preparation of which includes the following steps:

[0028] Step S1: Add calcium chloride, bismuth nitrate, and gallium chloride to deionized water and stir for 20 min. Then, while stirring, add potassium sodium tartrate tetrahydrate. After adding, continue stirring for 10 min. Then, while stirring, add sodium dihydrogen phosphate. After adding, continue stirring for 30 min. Then, place it in a microwave chemical reactor (supplier: Shandong Hengmei Electronic Technology Co., Ltd., model: HM-WH4) and stir at 200 °C for 24 h. Then, let it stand at 150 °C for 3 h. After cooling to room temperature, centrifuge and wash with deionized water and ethanol repeatedly 3 times each, and then dry overnight at 60 °C to obtain the calcium phosphate microsphere product. The dosage ratio of calcium chloride, bismuth nitrate, gallium chloride, deionized water, potassium sodium tartrate tetrahydrate, and sodium dihydrogen phosphate is 10 mmol: 0.3 mmol: 0.25 mmol: 180 mL: 0.01 mol: 6.3 mmol;

[0029] Step S2: Add 1-(4-nitrophenyl)ethanol into cyclohexane, add lye under stirring, add epichlorohydrin after 3 min, and stir and react at room temperature for 5 h to obtain reaction product 1; in a nitrogen atmosphere, add conjugated linoleic acid into ethyl acetate, add potassium hydroxide at 40 °C, then add reaction product 1 under stirring, continue to stir and react for 9 h, then raise the temperature to 100 °C and continue to stir for 1 h to obtain reaction product 2; the dosage ratio of 1-(4-nitrophenyl)ethanol, cyclohexane, lye, and epichlorohydrin is 17.5 g: 60 mL: 6 mL: 9.3 g, and the lye is a sodium hydroxide solution with a mass fraction of 5%; the dosage ratio of conjugated linoleic acid, ethyl acetate, potassium hydroxide, and reaction product 1 is 29 g: 110 mL: 0.6 g: 27 g;

[0030] Step S3: Stir and mix reaction product 2, potassium carbonate, sodium hydroxide, and dimethyl sulfoxide to obtain mixture 1; mix p-nitrobenzoyl chloride and dimethyl sulfoxide to obtain mixture 2, and add mixture 2 dropwise to mixture 1 in an ice-water bath, then raise the temperature to 45 °C and stir and react for 8 h, filter, wash with water, distill under reduced pressure, and dry to obtain reaction product 3; the dosage ratio of reaction product 2, potassium carbonate, sodium hydroxide, and dimethyl sulfoxide in mixture 1 is 57 g: 1.4 g: 4 g: 115 mL; the dosage ratio of p-nitrobenzoyl chloride and dimethyl sulfoxide in mixture 2 is 19 g: 40 mL; the dosage ratio of mixture 1 and mixture 2 is 120 mL: 45 mL;

[0031] Step S4: Add reaction product 3 into ethyl acetate, raise the temperature to 50 °C, then add sodium dithionite, and reflux and stir and react for 1.5 h to obtain reaction product 4; in a nitrogen atmosphere, add reaction product 4 and triphosgene into cyclohexane at 70 °C, then raise the temperature to 110 °C, and reflux and stir and react for 4 h to obtain reaction product 5; the dosage ratio of reaction product 3, ethyl acetate, and sodium dithionite is 77 g: 200 mL: 18 g; the dosage ratio of reaction product 4, triphosgene, and cyclohexane is 75 g: 30 g: 220 mL;

[0032] Step S5: In a nitrogen atmosphere, castor oil, 1,4-butanediol, distilled water, dibutyltin dilaurate, triethanolamine, and silicone oil were mixed at 30 °C for 30 min. Then, reaction product 5 was added, and after stirring at 800 r / min for 20 s, it was poured into a mold and left to foam at 20 °C for 30 min. After demolding, it was cured at room temperature for 72 h to obtain reaction product 6. The calcium phosphate microsphere product and reaction product 6 were heated and blended at 170 °C for 20 min, cooled, dried at 70 °C for 24 h, and then ground into a powder with a particle size of 0.5 mm to obtain self-assembled calcium phosphate microspheres with a high specific surface area. The dosage ratios of castor oil, 1,4-butanediol, distilled water, dibutyltin dilaurate, triethanolamine, silicone oil, and reaction product 5 were 95 g:5 g:2 g:18 mg:9 mg:0.5 g:60 g; the dosage ratios of the calcium phosphate microsphere product and reaction product 6 were 90 g:10 g.

[0033] Example 2

[0034] A kind of self-assembled calcium phosphate microsphere with a high specific surface area, and its preparation includes the following steps:

[0035] Step S1: Calcium chloride, bismuth nitrate, and gallium chloride were added to deionized water and stirred for 22 min. Then, potassium sodium tartrate tetrahydrate was added under stirring. After addition, stirring was continued for 12 min. Then, sodium dihydrogen phosphate was added under stirring. After addition, stirring was continued for 32 min. Then, it was placed in a microwave chemical reactor (supplier: Shandong Hengmei Electronic Technology Co., Ltd., model: HM-WH4) and stirred at 200 °C for 24 h. Then, it was left to stand at 150 °C for 3 h. After cooling to room temperature, it was centrifuged and washed repeatedly with deionized water and ethanol three times each, and then dried overnight at 60 °C to obtain the calcium phosphate microsphere product. The dosage ratios of calcium chloride, bismuth nitrate, gallium chloride, deionized water, potassium sodium tartrate tetrahydrate, and sodium dihydrogen phosphate were 11 mmol:0.32 mmol:0.27 mmol:185 mL:11 mmol:6.4 mmol;

[0036] Step S2: 1-(4-Nitrophenyl)ethanol was added to cyclohexane, and an alkali solution was added under stirring. After 4 min, epichlorohydrin was added, and the mixture was stirred at room temperature for 5.2 h to obtain reaction product 1. In a nitrogen atmosphere, conjugated linoleic acid was added to ethyl acetate, potassium hydroxide was added at 42 °C, and then reaction product 1 was added under stirring. After continuing to stir and react for 9.3 h, the temperature was raised to 103 °C, and stirring was continued for 1.2 h to obtain reaction product 2. The dosage ratios of 1-(4-nitrophenyl)ethanol, cyclohexane, alkali solution, and epichlorohydrin were 18.0 g:65 mL:6.5 mL:9.4 g, and the alkali solution was a 5% sodium hydroxide solution by mass; the dosage ratios of conjugated linoleic acid, ethyl acetate, potassium hydroxide, and reaction product 1 were 29.3 g:115 mL:0.63 g:27.2 g;

[0037] Step S3: Stir and mix reaction product 2, potassium carbonate, sodium hydroxide, and dimethyl sulfoxide to obtain mixture 1; mix p-nitrobenzoyl chloride and dimethyl sulfoxide to obtain mixture 2. Dropwise add mixture 2 to mixture 1 in an ice-water bath, then raise the temperature to 47°C and stir and react for 8.3 h. Filter, wash with water, distill under reduced pressure, and dry to obtain reaction product 3. The dosage ratios of reaction product 2, potassium carbonate, sodium hydroxide, and dimethyl sulfoxide in mixture 1 are 57.5 g: 1.4 g: 4.1 g: 120 mL; the dosage ratios of p-nitrobenzoyl chloride and dimethyl sulfoxide in mixture 2 are 19.5 g: 43 mL; the dosage ratio of mixture 1 to mixture 2 is 125 mL: 47 mL;

[0038] Step S4: Add reaction product 3 to ethyl acetate, raise the temperature to 53°C, then add sodium dithionite, and reflux and stir and react for 1.6 h to obtain reaction product 4; in a nitrogen atmosphere, add reaction product 4 and triphosgene to cyclohexane at 72°C, then raise the temperature to 115°C, and reflux and stir and react for 4.2 h to obtain reaction product 5. The dosage ratios of reaction product 3, ethyl acetate, and sodium dithionite are 77.5 g: 210 mL: 18.3 g; the dosage ratios of reaction product 4, triphosgene, and cyclohexane are 75.5 g: 30.5 g: 225 mL;

[0039] Step S5: In a nitrogen atmosphere, mix castor oil, 1,4-butanediol, distilled water, dibutyltin dilaurate, triethanolamine, and silicone oil at 30°C for 30 min, then add reaction product 5. Stir at 800 r / min for 22 s, then pour into a mold, and let stand and foam at 22°C for 32 min. After demolding, cure at room temperature for 72.5 h to obtain reaction product 6; heat and blend calcium phosphate microsphere product and reaction product 6 at 172°C for 25 min, cool, dry at 75°C for 24 h, and then grind into a powder with a particle size of 0.6 mm to obtain self-assembled calcium phosphate microspheres with a high specific surface area. The dosage ratios of castor oil, 1,4-butanediol, distilled water, dibutyltin dilaurate, triethanolamine, silicone oil, and reaction product 5 are 100 g: 7 g: 2.2 g: 19 mg: 9.5 mg: 0.6 g: 63 g; the dosage ratio of calcium phosphate microsphere product to reaction product 6 is 93 g: 7 g.

[0040] Example 3

[0041] A self-assembled calcium phosphate microsphere with a high specific surface area, and its preparation includes the following steps:

[0042] Step S1: Calcium chloride, bismuth nitrate, and gallium chloride are added to deionized water and stirred for 25 min. Then, potassium sodium tartrate tetrahydrate is added under stirring. After addition, stirring is continued for 15 min. Then, sodium dihydrogen phosphate is added under stirring. After addition, stirring is continued for 35 min. Then, it is placed in a microwave chemical reactor (supplier: Shandong Hengmei Electronic Technology Co., Ltd., model: HM-WH4) and stirred at 210 °C for 25 h. Then, it is left standing at 160 °C for 3.5 h. After cooling to room temperature, it is centrifuged and washed repeatedly with deionized water and ethanol three times each, and then dried overnight at 80 °C to obtain calcium phosphate microsphere products. The dosage ratio of calcium chloride, bismuth nitrate, gallium chloride, deionized water, potassium sodium tartrate tetrahydrate, and sodium dihydrogen phosphate is 12 mmol: 0.35 mmol: 0.30 mmol: 190 mL: 12 mmol: 6.5 mmol;

[0043] Step S2: 1-(4-Nitrophenyl)ethanol is added to cyclohexane, and an alkali solution is added under stirring. After 5 min, epichlorohydrin is added, and the mixture is stirred at room temperature for 5.5 h to obtain reaction product 1. In a nitrogen atmosphere, conjugated linoleic acid is added to ethyl acetate, potassium hydroxide is added at 45 °C, and then reaction product 1 is added under stirring. Stirring is continued for 9.5 h, and then the temperature is raised to 105 °C and stirring is continued for 1.5 h to obtain reaction product 2. The dosage ratio of 1-(4-nitrophenyl)ethanol, cyclohexane, alkali solution, and epichlorohydrin is 18.5 g: 70 mL: 7 mL: 9.6 g, and the alkali solution is a 5% sodium hydroxide solution by mass fraction. The dosage ratio of conjugated linoleic acid, ethyl acetate, potassium hydroxide, and reaction product 1 is 29.5 g: 120 mL: 0.65 g: 27.5 g;

[0044] Step S3: Reaction product 2, potassium carbonate, sodium hydroxide, and dimethyl sulfoxide are stirred and mixed to obtain mixture 1. p-Nitrobenzoyl chloride and dimethyl sulfoxide are mixed to obtain mixture 2. Mixture 2 is added dropwise to mixture 1 in an ice-water bath, and then the temperature is raised to 50 °C and stirring is continued for 8.5 h. It is filtered, washed with water, distilled under reduced pressure, and dried to obtain reaction product 3. The dosage ratio of reaction product 2, potassium carbonate, sodium hydroxide, and dimethyl sulfoxide in mixture 1 is 58 g: 1.5 g: 4.2 g: 125 mL. The dosage ratio of p-nitrobenzoyl chloride and dimethyl sulfoxide in mixture 2 is 20 g: 45 mL. The dosage ratio of mixture 1 to mixture 2 is 130 mL: 50 mL;

[0045] Step S4: Add the reaction product 3 into ethyl acetate, heat up to 55 °C, then add sodium dithionite, and reflux and stir for 1.7 h to obtain reaction product 4; in a nitrogen atmosphere, add reaction product 4 and triphosgene into cyclohexane at 75 °C, then heat up to 120 °C, reflux and stir for 4.5 h to obtain reaction product 5; the dosage ratio of reaction product 3, ethyl acetate and sodium dithionite is 78 g: 220 mL: 18.5 g; the dosage ratio of reaction product 4, triphosgene and cyclohexane is 76 g: 31 g: 230 mL;

[0046] Step S5: In a nitrogen atmosphere, mix castor oil, 1,4-butanediol, distilled water, dibutyltin dilaurate, triethanolamine and silicone oil at 30 °C for 35 min, then add reaction product 5, stir at 900 r / min for 25 s, pour into a mold, stand and foam at 23 °C for 35 min, demold and cure at room temperature for 73 h to obtain reaction product 6; heat and blend the calcium phosphate microsphere product and reaction product 6 at 175 °C for 30 min, cool and dry at 80 °C for 25 h, then grind into powder with a particle size of 0.7 mm to obtain self-assembled calcium phosphate microspheres with a high specific surface area; the dosage ratio of castor oil, 1,4-butanediol, distilled water, dibutyltin dilaurate, triethanolamine, silicone oil and reaction product 5 is 105 g: 10 g: 2.4 g: 20 mg: 10 mg: 0.7 g: 65 g; the dosage ratio of the calcium phosphate microsphere product and reaction product 6 is 95 g: 5 g.

[0047] Comparative Example 1

[0048] Compared with Example 3, replace conjugated linoleic acid with sorbic acid, and the rest is exactly the same as Example 3 to prepare self-assembled calcium phosphate microspheres with a high specific surface area.

[0049] Comparative Example 2

[0050] Compared with Example 3, replace bismuth nitrate with iron nitrate, and the rest is exactly the same as Example 3 to prepare self-assembled calcium phosphate microspheres with a high specific surface area.

[0051] Comparative Example 3

[0052] Compared with Example 3, replace gallium chloride with iron chloride, and the rest is exactly the same as Example 3 to prepare self-assembled calcium phosphate microspheres with a high specific surface area.

[0053] Next, further effect detection is carried out on the calcium phosphate microspheres prepared by the present invention, and the detection results are as follows.

[0054] In vitro degradation rate: The calcium phosphate microspheres prepared by the present invention were immersed in 0.5 mol / L tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution at a ratio of 0.02 g / mL, and then placed in a constant temperature shaker (37 °C, 60 r / min) for an in vitro degradation experiment for 28 days. Equal amounts of fresh 0.5 mol / L tris (hydroxymethyl) aminomethane-hydrochloric acid buffer solution were replaced every 7 days. At 28 days, the calcium phosphate microspheres were washed three times with deionized water, and then the dry weight of the microspheres was weighed and the mass loss rate was calculated to evaluate the degradation rate;

[0055] In vitro antibacterial test: 0.01 g of the calcium phosphate microspheres prepared by the present invention was added to 15 mL of LB agar medium, and the medium doped with calcium phosphate microspheres was obtained by high-temperature sterilization. Under sterile conditions, 200 μL of the bacterial stock solution of Escherichia coli and Staphylococcus aureus at a certain concentration was added to the culture dish, and then the medium doped with calcium phosphate microspheres cooled to 45 °C was poured in. The mixture was placed flat on the table, shaken well, condensed into a plate, and then placed in an incubator at 37 °C for 24 h. Bacterial counting was carried out and the antibacterial rate of bacteria was calculated. The medium without the powder was used as the control sample. The antibacterial rate = (A0 - A) / A0 × 100%, where A0 is the number of recovered bacteria in the control sample and A is the number of recovered bacteria in the calcium phosphate microsphere sample;

[0056] ALP activity: The quantitative detection results of alkaline phosphatase (ALP) activity of bone marrow mesenchymal stem cells (BMSCs) after osteogenic induction culture with the calcium phosphate microspheres prepared by the present invention for 3 days and 7 days were used to evaluate the osteogenic promotion performance;

[0057] The results are recorded in Table 1;

[0058]

[0059] According to the data in Table 1, the calcium phosphate microspheres of the present invention have excellent degradability, antibacterial property and osteogenic promotion property. By comparing Example 3 with Comparative Example 1, it can be seen that when using conjugated linoleic acid instead of sorbic acid, the antibacterial rate is higher, and sorbic acid does not show osteogenic promotion property. Therefore, the use of conjugated linoleic acid makes the calcium phosphate microspheres have higher antibacterial property and osteogenic promotion property. By comparing Example 3 with Comparative Example 2, it can be seen that the co-doping of bismuth ions (Bi 3+ ‌) and gallium ions (Ga 3+ ‌) is more conducive to the improvement of the antibacterial rate compared with the co-doping of iron ions (Fe 3+ ‌) and gallium ions (Ga 3+ ‌), and the specific effect of the co-doping of bismuth ions (Bi 3+ ‌) and gallium ions (Ga 3+ ‌) on the calcium phosphate microsphere crystals is more conducive to improving the degradability of the calcium phosphate microspheres. By comparing Example 3 with Comparative Example 3, it can be seen that the co-doping of bismuth ions (Bi 3+ ‌) and gallium ions (Ga 3+‌) The co-doping shows a higher antibacterial rate of calcium phosphate microspheres compared to the co-doping of bismuth ions (Bi 3+ ) and iron ions (Fe 3+ ). Moreover, the specific effect of the co-doping of bismuth ions (Bi 3+ ) and gallium ions (Ga 3+ ) on the crystals of calcium phosphate microspheres also endows the calcium phosphate microspheres with better degradability.

[0060] The above content is only an example and illustration of the concept of the present invention. Those skilled in the art of the present technology can make various modifications or supplements to the described specific embodiments or use similar methods for substitution. As long as they do not deviate from the concept of the invention or exceed the scope defined by this claims, they shall fall within the protection scope of the present invention.

Claims

1. A method for preparing self-assembled calcium phosphate microspheres with high specific surface area, characterized in that: The steps include: Step S1, adding calcium chloride, bismuth nitrate and gallium chloride into deionized water, mixing with potassium sodium tartrate tetrahydrate and sodium dihydrogen phosphate, and reacting in a microwave chemical reactor to obtain a calcium phosphate microsphere product; Step S2, adding 1-(4-nitrophenyl)ethanol to cyclohexane, adding alkali solution, and then adding epichlorohydrin, stirring to react to obtain reaction product 1; in a protective gas atmosphere, adding conjugated linoleic acid to ethyl acetate, adding potassium hydroxide, and then adding reaction product 1, stirring to react to obtain reaction product 2; Step S3, stirring and mixing the reaction product 2, potassium carbonate, sodium hydroxide and dimethyl sulfoxide to obtain a mixed solution 1; mixing p-nitrobenzoyl chloride and dimethyl sulfoxide to obtain a mixed solution 2, and adding the mixed solution 2 dropwise to the mixed solution 1 in an ice water bath, stirring and reacting to obtain a reaction product 3; Step S4, adding the reaction product 3 to ethyl acetate, and then adding sodium dithionite, reflux stirring and reacting to obtain the reaction product 4; in a protective gas atmosphere, adding the reaction product 4 and triphosgene to cyclohexane, reflux stirring and reacting to obtain the reaction product 5; Step S5, in a protective gas atmosphere, castor oil, 1,4-butanediol, distilled water, dibutyltin dilaurate, triethanolamine and silicone oil are mixed, and then reaction product 5 is added, stirred and allowed to stand for foaming to obtain reaction product 6; the calcium phosphate microsphere product and reaction product 6 are heated, blended and ground to obtain self-assembled calcium phosphate microspheres with a high specific surface area.

2. The method for preparing a self-assembled calcium phosphate microsphere with a high specific surface area according to claim 1, characterized in that: In step S1, the usage ratio of calcium chloride, bismuth nitrate, gallium chloride, deionized water, potassium sodium tartrate tetrahydrate, and sodium dihydrogen phosphate is 10-12 mmol: 0.3-0.35 mmol: 0.25-0.30 mmol: 180-190 mL: 10-12 mmol: 6.3-6.5 mmol.

3. The method for preparing a self-assembled calcium phosphate microsphere with a high specific surface area according to claim 1, characterized in that: In step S2, the dosage ratio of 1-(4-nitrophenyl)ethanol, cyclohexane, alkali solution and epichlorohydrin is 17.5-18.5 g: 60-70 mL: 6-7 mL: 9.3-9.6 g, and the alkali solution is a sodium hydroxide solution with a mass fraction of 5-6%.

4. The method for preparing a self-assembled calcium phosphate microsphere with a high specific surface area according to claim 1, characterized in that: In step S2, the usage ratio of conjugated linoleic acid, ethyl acetate, potassium hydroxide and reaction product 1 is 29-29.5 g: 110-120 mL: 0.6-0.65 g: 27-27.5 g.

5. The method for preparing a self-assembled calcium phosphate microsphere with a high specific surface area according to claim 1, characterized in that: In step S3, the usage ratio of reaction product 2, potassium carbonate, sodium hydroxide and dimethyl sulfoxide in the mixed solution 1 is 57-58 g: 1.4-1.5 g: 4-4.2 g: 115-125 mL.

6. The method for preparing self-assembled calcium phosphate microspheres with high specific surface area according to claim 1, characterized in that: In step S3, the usage ratio of p-nitrobenzoyl chloride and dimethyl sulfoxide in mixed solution 2 is 19-20 g:40-45 mL; the usage ratio of mixed solution 1 and mixed solution 2 is 120-130 mL:45-50 mL.

7. The method for preparing self-assembled calcium phosphate microspheres with high specific surface area according to claim 1, characterized in that: In step S4, the usage ratio of reaction product 3, ethyl acetate and sodium dithionite is 77-78 g: 200-220 mL: 18-18.5 g.

8. The method for preparing self-assembled calcium phosphate microspheres with high specific surface area according to claim 1, characterized in that: In step S4, the usage ratio of reaction product 4, triphosgene and cyclohexane is 75-76 g: 30-31 g: 220-230 mL.

9. The method for preparing self-assembled calcium phosphate microspheres with high specific surface area according to claim 1, characterized in that: In step S5, the usage ratio of castor oil, 1,4-butanediol, distilled water, dibutyltin dilaurate, triethanolamine, silicone oil, and reaction product 5 is 95-105g: 5-10g: 2-2.4g: 18-20mg: 9-10mg: 0.5-0.7g: 60-65g; the usage ratio of calcium phosphate microsphere product and reaction product 6 is 90-95g: 5-10g.

10. A self-assembled calcium phosphate microsphere with a high specific surface area, prepared by the method for preparing a self-assembled calcium phosphate microsphere with a high specific surface area according to any one of claims 1 to 9.

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