Self-assembled calcium phosphate microspheres with high specific surface area and preparation method thereof

Abstract

The invention discloses a self-assembled calcium phosphate microsphere with a high specific surface area and a preparation method thereof, and relates to the field of calcium phosphate biological materials, the self-assembled calcium phosphate microsphere is obtained by blending and compounding a self-assembled calcium phosphate microsphere doped with bismuth ions (Bi < 3 + >) and gallium ions (Ga < 3 + >) and polyurethane containing conjugated linoleate, and the characteristic of the high specific surface area of the calcium phosphate microsphere is retained. The calcium phosphate microspheres disclosed by the invention have excellent degradability, antibacterial property and osteogenesis promotion property, and are worthy of popularization and application in the fields of bone tissue repair, drug carriers and the like.

Description

Technical Field

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

[0002] Calcium phosphate is an inorganic compound that usually exists in a variety of mineral forms. For example, hydroxyapatite is one of the main components of bones and teeth and has good biocompatibility and bioactivity. Calcium phosphate 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 a type of calcium phosphate biomaterial. They are widely used in separation, catalysis, sensing, tissue engineering and drug release 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, degradability, easy operation and convenient drug loading. In the usual preparation process of calcium phosphate microspheres, calcium phosphate needs to be sintered after it is spheroidized. During the sintering process, the crystal structure of calcium phosphate becomes more stable, and the density increases, resulting in a decrease in internal voids, which affects the penetration of water and biological enzymes during the degradation 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 infected bone defects, and higher requirements are placed on the antibacterial properties of calcium phosphate microspheres.

[0004] Previous studies have used calcium phosphate biomaterials and polyurethane to improve the biocompatibility, degradability and mechanical properties of calcium phosphate biomaterials, and are expected to be used in cartilage tissue substitutes. However, the raw material MDI used in the preparation of the polyurethane has poor degradability, and the calcium phosphate biomaterial used in this study is hydroxyapatite, which has insufficient osteoinduction ability and unsatisfactory ability to promote bone growth. This is not conducive to the use of the polyurethane material in the field of bone repair, which has high requirements for degradability and osteogenic properties after being compounded with calcium phosphate microspheres.

[0005] Therefore, the present invention aims to provide a suitable modification method to improve the degradability and antibacterial properties of calcium phosphate microspheres; provide a raw material with good degradability for preparing polyurethane, and compound it with calcium phosphate microspheres, so as to obtain calcium phosphate biomaterials with excellent properties such as degradability, antibacterial properties, and osteogenic properties, and expand its scope of application to obtain self-assembled calcium phosphate microsphere materials with high specific surface area and considerable 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 high specific surface area and a preparation method thereof.

[0007] The purpose of the present invention can be achieved through the following technical solutions: A self-assembled calcium phosphate microsphere with a high specific surface area, the preparation of which comprises the following steps: Step S1, adding calcium chloride, bismuth nitrate and gallium chloride into deionized water and stirring for 20-25 minutes, then adding potassium sodium tartrate tetrahydrate under stirring, continuing stirring for 10-15 minutes after the addition, and then adding sodium dihydrogen phosphate under stirring, continuing stirring for 30-35 minutes after the addition, and then placing in a microwave chemical reactor and stirring at 200-210° C. for 24-25 hours, and then standing at 150-160° C. for 3-3.5 hours, cooling to room temperature, centrifuging, washing with deionized water and ethanol repeatedly for 3 times each, and drying at 60-80° C. overnight to obtain a calcium phosphate microsphere product; Further, the usage ratio of calcium chloride, bismuth nitrate, gallium chloride, deionized water, potassium sodium tartrate tetrahydrate, and sodium dihydrogen phosphate is 10-12mmol: 0.3-0.35mmol: 0.25-0.30mmol: 180-190mL: 10-12mmol: 6.3-6.5mmol; During the reaction of step S1, bismuth ions (Bi 3+ ) and gallium ions (Ga 3+ ‌) doped calcium phosphate microsphere products; Step S2, adding 1-(4-nitrophenyl)ethanol to cyclohexane, adding alkali solution under stirring, and then adding epichlorohydrin after 3-5 minutes, stirring and reacting at room temperature for 5-5.5 hours to obtain reaction product 1; in a protective gas atmosphere, adding conjugated linoleic acid to ethyl acetate, adding potassium hydroxide at 40-45° C., and then adding reaction product 1 under stirring, continuing to stir and react for 9-9.5 hours, then raising the temperature to 100-105° C., and continuing to stir for 1-1.5 hours to obtain reaction product 2; Further, 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; During the reaction of step S2, the alcoholic hydroxyl group in 1-(4-nitrophenyl)ethanol reacts with epichlorohydrin to obtain a reaction product 1 containing an epoxy group; the carboxyl group of conjugated linoleic acid reacts with the epoxy group of the reaction product 1 to obtain a reaction product 2 containing a nitro group, a hydroxyl group and a conjugated linoleic acid ester; 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, adding the mixed solution 2 dropwise to the mixed solution 1 in an ice water bath, and then heating to 45-50° C. and stirring for reaction for 8-8.5 hours to obtain a reaction product 3; Further, the amount ratio of reaction product 2, potassium carbonate, sodium hydroxide and dimethyl sulfoxide in mixed solution 1 is 57-58 g: 1.4-1.5 g: 4-4.2 g: 115-125 mL; the amount ratio of p-nitrobenzoyl chloride and dimethyl sulfoxide in mixed solution 2 is 19-20 g: 40-45 mL; the amount ratio of mixed solution 1 and mixed solution 2 is 120-130 mL: 45-50 mL; During the reaction of step S3, the hydroxyl group of the reaction product 2 reacts with p-nitrobenzoyl chloride to obtain a reaction product 3 containing a dinitro group and a conjugated linoleic acid ester; Step S4, adding the reaction product 3 to ethyl acetate, heating to 50-55° C., then adding sodium dithionite, reflux stirring and reacting for 1.5-1.7 hours to obtain a reaction product 4; in a protective gas atmosphere, adding the reaction product 4 and triphosgene to cyclohexane at 70-75° C., then heating to 110-120° C., reflux stirring and reacting for 4-4.5 hours to obtain a reaction product 5; Further, the usage ratio of reaction product 3, ethyl acetate, and sodium dithionite is 77-78 g: 200-220 mL: 18-18.5 g; the usage ratio of reaction product 4, triphosgene, and cyclohexane is 75-76 g: 30-31 g: 220-230 mL; During the reaction of step S4, the dinitro group of the reaction product 3 is selectively reduced to an amino group by sodium dithionite to obtain a reaction product 4; the amino group of the reaction product 4 reacts with triphosgene to generate chloroamide, which is then heated to dehydrochlorinate to generate a diisocyanate, namely, the reaction product 5; Step S5, in a protective gas atmosphere, mixing castor oil, 1,4-butanediol, distilled water, dibutyltin dilaurate, triethanolamine and silicone oil at 28-32° C. for 30-35 min, then adding reaction product 5, stirring at 800-900 r / min for 20-25 s, pouring into a mold, standing at 20-23° C. for foaming for 30-35 min, and curing at room temperature for 72-73 h after demolding to obtain reaction product 6; heating the calcium phosphate microsphere product and reaction product 6 at 170-175° C. for 20-30 min, drying at 70-80° C. for 24-25 h after cooling, and then grinding 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; Further, 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; In the reaction process of step S5, a polyurethane containing conjugated linoleic acid ester, i.e., reaction product 6, is prepared by a full water foaming method; the calcium phosphate microsphere product and reaction product 6 are mixed by heating to obtain a polyurethane containing conjugated linoleic acid ester and doped with bismuth ions (Bi 3+ ) and gallium ions (Ga 3+ ‌) of calcium phosphate microspheres.

[0008] Beneficial effects of the invention: The invention discloses a self-assembled calcium phosphate microsphere with a high specific surface area, which is composed of self-assembled calcium phosphate microspheres doped with bismuth ions (Bi 3+ ) and gallium ions (Ga 3+ ‌) calcium phosphate microspheres are blended with polyurethane containing conjugated linoleic acid esters, retaining the high specific surface area characteristics of calcium phosphate microspheres. 3+ ) and gallium ions (Ga 3+ ‌) after co-doping, it not only gives the calcium phosphate microspheres good antibacterial properties; it can also change the morphology of calcium phosphate and cause lattice distortion, thereby improving the biodegradability of the calcium phosphate microspheres; it can also make the surface of the calcium phosphate microspheres positively charged, making it easier to load negatively charged drugs through charge interaction. After the conjugated linoleic acid is formed into a conjugated linoleic acid ester through the reaction, it is introduced into the reaction product 5, which not only makes the reaction product 5, i.e., the diisocyanate used in the preparation process of the reaction product 6, i.e., the polyurethane, biodegradable and antibacterial, but also makes up for the deficiency that the antibacterial property of conjugated linoleic acid is easily affected by the adverse effects of pH value, thereby improving the biodegradability and antibacterial properties of the prepared polyurethane. In addition, conjugated linoleic acid has a promoting effect on human bone growth, helps maintain bone density, and prevents bone diseases such as osteoporosis. When it is combined with bismuth ions (Bi 3+ ) and gallium ions (Ga 3+ ‌) of calcium phosphate microspheres to further improve the biodegradability of calcium phosphate microspheres and combine with bismuth ions (Bi 3+ ) and gallium ions (Ga 3+ ‌) can synergistically enhance antibacterial properties and also promote bone growth. DETAILED DESCRIPTION

[0009] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0010] Example 1

[0011] A self-assembled calcium phosphate microsphere with a high specific surface area, the preparation of which comprises the following steps: Step S1, adding calcium chloride, bismuth nitrate and gallium chloride to deionized water and stirring for 20 minutes, then adding potassium sodium tartrate tetrahydrate under stirring, continuing to stir for 10 minutes after the addition, and then adding sodium dihydrogen phosphate under stirring, continuing to stir for 30 minutes after the addition, and then placing in a microwave chemical reactor (supplier: Shandong Hengmei Electronic Technology Co., Ltd., model: HM-WH4) and stirring at 200°C for 24 hours, and then standing at 150°C for 3 hours, cooling to room temperature, centrifuging, washing repeatedly with deionized water and ethanol for 3 times each, and drying at 60°C overnight to obtain a calcium phosphate microsphere product; the amount ratio of calcium chloride, bismuth nitrate, gallium chloride, deionized water, potassium sodium tartrate tetrahydrate, and sodium dihydrogen phosphate is 10mmol: 0.3mmol: 0.25mmol: 180mL: 0.01mol: 6.3mmol; Step S2, adding 1-(4-nitrophenyl)ethanol to cyclohexane, adding alkali solution under stirring, and then adding epichlorohydrin after 3 minutes, stirring and reacting at room temperature for 5 hours to obtain reaction product 1; in a nitrogen atmosphere, adding conjugated linoleic acid to ethyl acetate, adding potassium hydroxide at 40°C, and then adding reaction product 1 under stirring, continuing to stir and react for 9 hours, then heating to 100°C, and continuing to stir for 1 hour to obtain reaction product 2; the amount ratio of 1-(4-nitrophenyl)ethanol, cyclohexane, alkali solution, and epichlorohydrin is 17.5g:60mL:6mL:9.3g, and the alkali solution is a sodium hydroxide solution with a mass fraction of 5%; the amount ratio of conjugated linoleic acid, ethyl acetate, potassium hydroxide, and reaction product 1 is 29g:110mL:0.6g:27g; 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, dropping the mixed solution 2 into the mixed solution 1 in an ice-water bath, then heating to 45° C. and stirring for reaction for 8 hours, filtering, washing, distilling under reduced pressure, and drying to obtain a reaction product 3; the amount ratio of the reaction product 2, potassium carbonate, sodium hydroxide and dimethyl sulfoxide in the mixed solution 1 is 57g:1.4g:4g:115mL; the amount ratio of p-nitrobenzoyl chloride and dimethyl sulfoxide in the mixed solution 2 is 19g:40mL; the amount ratio of the mixed solution 1 to the mixed solution 2 is 120mL:45mL; Step S4, adding the reaction product 3 to ethyl acetate, heating to 50°C, adding sodium dithionite, reflux stirring and reacting for 1.5 hours to obtain a reaction product 4; in a nitrogen atmosphere, adding the reaction product 4 and triphosgene to cyclohexane at 70°C, heating to 110°C, reflux stirring and reacting for 4 hours to obtain a reaction product 5; the amount ratio of the reaction product 3, ethyl acetate, and sodium dithionite is 77g:200mL:18g; the amount ratio of the reaction product 4, triphosgene, and cyclohexane is 75g:30g:220mL; 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 minutes, and then the reaction product 5 was added, stirred at 800r / min for 20s, poured into a mold, and allowed to stand at 20°C for foaming for 30 minutes. After demolding, the reaction product 6 was cured at room temperature for 72 hours to obtain the calcium phosphate microsphere product and the reaction product 6 at 170°C. Mix for 20 minutes, cool and dry at 70°C for 24 hours, and then grind 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 ratio of castor oil, 1,4-butanediol, distilled water, dibutyltin dilaurate, triethanolamine, silicone oil, and reaction product 5 is 95g:5g:2g:18mg:9mg:0.5g:60g; the dosage ratio of calcium phosphate microsphere product and reaction product 6 is 90g:10g.

[0012] Example 2

[0013] A self-assembled calcium phosphate microsphere with a high specific surface area, the preparation of which comprises the following steps: Step S1, adding calcium chloride, bismuth nitrate and gallium chloride to deionized water and stirring for 22 minutes, then adding potassium sodium tartrate tetrahydrate under stirring, continuing to stir for 12 minutes after the addition, and then adding sodium dihydrogen phosphate under stirring, continuing to stir for 32 minutes after the addition, and then placing in a microwave chemical reactor (supplier: Shandong Hengmei Electronic Technology Co., Ltd., model: HM-WH4) and stirring at 200°C for 24 hours, and then standing at 150°C for 3 hours, cooling to room temperature, centrifuging, washing repeatedly with deionized water and ethanol for 3 times each, and drying at 60°C overnight to obtain a calcium phosphate microsphere product; the amount ratio of calcium chloride, bismuth nitrate, gallium chloride, deionized water, potassium sodium tartrate tetrahydrate, and sodium dihydrogen phosphate is 11mmol:0.32mmol:0.27mmol:185mL:11mmol:6.4mmol; Step S2, adding 1-(4-nitrophenyl)ethanol to cyclohexane, adding alkali solution under stirring, and then adding epichlorohydrin after 4 minutes, stirring and reacting at room temperature for 5.2 hours to obtain reaction product 1; in a nitrogen atmosphere, adding conjugated linoleic acid to ethyl acetate, adding potassium hydroxide at 42°C, and then adding reaction product 1 under stirring, continuing to stir and react for 9.3 hours, then heating to 103°C, and continuing to stir for 1.2 hours to obtain reaction product 2; the amount ratio of 1-(4-nitrophenyl)ethanol, cyclohexane, alkali solution, and epichlorohydrin is 18.0g:65mL:6.5mL:9.4g, and the alkali solution is a sodium hydroxide solution with a mass fraction of 5%; the amount ratio of conjugated linoleic acid, ethyl acetate, potassium hydroxide, and reaction product 1 is 29.3g:115mL:0.63g:27.2g; 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, dropping the mixed solution 2 into the mixed solution 1 in an ice-water bath, and then heating to 47° C. and stirring for reaction for 8.3 hours, filtering, washing, distilling under reduced pressure, and drying to obtain a reaction product 3; the amount ratio of the reaction product 2, potassium carbonate, sodium hydroxide and dimethyl sulfoxide in the mixed solution 1 is 57.5 g:1.4 g:4.1 g:120 mL; the amount ratio of p-nitrobenzoyl chloride and dimethyl sulfoxide in the mixed solution 2 is 19.5 g:43 mL; the amount ratio of the mixed solution 1 to the mixed solution 2 is 125 mL:47 mL; Step S4, adding the reaction product 3 to ethyl acetate, heating to 53°C, adding sodium dithionite, reflux stirring and reacting for 1.6 hours to obtain a reaction product 4; in a nitrogen atmosphere, adding the reaction product 4 and triphosgene to cyclohexane at 72°C, heating to 115°C, reflux stirring and reacting for 4.2 hours to obtain a reaction product 5; the amount ratio of the reaction product 3, ethyl acetate, and sodium dithionite is 77.5g:210mL:18.3g; the amount ratio of the reaction product 4, triphosgene, and cyclohexane is 75.5g:30.5g:225mL; 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 minutes, and then the reaction product 5 was added, stirred at 800 r / min for 22 seconds, poured into a mold, and allowed to stand at 22°C for foaming for 32 minutes. After demolding, the mixture was cured at room temperature for 72.5 hours to obtain a reaction product 6; the calcium phosphate microsphere product and the reaction product 6 were heated and blended at 172°C. 25min, cooled and dried at 75℃ for 24h, and then ground into a powder with a particle size of 0.6mm to obtain self-assembled calcium phosphate microspheres with 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 100g:7g:2.2g:19mg:9.5mg:0.6g:63g; the dosage ratio of calcium phosphate microsphere product and reaction product 6 is 93g:7g.

[0014] Example 3

[0015] A self-assembled calcium phosphate microsphere with a high specific surface area, the preparation of which comprises the following steps: Step S1, adding calcium chloride, bismuth nitrate and gallium chloride to deionized water and stirring for 25 minutes, then adding potassium sodium tartrate tetrahydrate under stirring, continuing to stir for 15 minutes after the addition, and then adding sodium dihydrogen phosphate under stirring, continuing to stir for 35 minutes after the addition, and then placing in a microwave chemical reactor (supplier: Shandong Hengmei Electronic Technology Co., Ltd., model: HM-WH4) and stirring at 210°C for 25 hours, and then standing at 160°C for 3.5 hours, cooling to room temperature, centrifuging, washing repeatedly with deionized water and ethanol for 3 times each, and drying at 80°C overnight to obtain a calcium phosphate microsphere product; the amount ratio of calcium chloride, bismuth nitrate, gallium chloride, deionized water, potassium sodium tartrate tetrahydrate, and sodium dihydrogen phosphate is 12mmol:0.35mmol:0.30mmol:190mL:12mmol:6.5mmol; Step S2, adding 1-(4-nitrophenyl)ethanol to cyclohexane, adding alkali solution under stirring, and then adding epichlorohydrin after 5 minutes, stirring and reacting at room temperature for 5.5 hours to obtain reaction product 1; in a nitrogen atmosphere, adding conjugated linoleic acid to ethyl acetate, adding potassium hydroxide at 45°C, and then adding reaction product 1 under stirring, continuing to stir and react for 9.5 hours, then heating to 105°C, and continuing to stir for 1.5 hours to obtain reaction product 2; the amount ratio of 1-(4-nitrophenyl)ethanol, cyclohexane, alkali solution, and epichlorohydrin is 18.5g:70mL:7mL:9.6g, and the alkali solution is a sodium hydroxide solution with a mass fraction of 5%; the amount ratio of conjugated linoleic acid, ethyl acetate, potassium hydroxide, and reaction product 1 is 29.5g:120mL:0.65g:27.5g; 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, adding the mixed solution 2 dropwise to the mixed solution 1 in an ice-water bath, and then heating to 50° C. and stirring for reaction for 8.5 hours, filtering, washing, distilling under reduced pressure, and drying to obtain a reaction product 3; the amount ratio of the reaction product 2, potassium carbonate, sodium hydroxide and dimethyl sulfoxide in the mixed solution 1 is 58g:1.5g:4.2g:125mL; the amount ratio of p-nitrobenzoyl chloride and dimethyl sulfoxide in the mixed solution 2 is 20g:45mL; the amount ratio of the mixed solution 1 to the mixed solution 2 is 130mL:50mL; Step S4, adding the reaction product 3 to ethyl acetate, heating to 55°C, adding sodium dithionite, reflux stirring and reacting for 1.7h to obtain a reaction product 4; in a nitrogen atmosphere, adding the reaction product 4 and triphosgene to cyclohexane at 75°C, heating to 120°C, reflux stirring and reacting for 4.5h to obtain a reaction product 5; the amount ratio of the reaction product 3, ethyl acetate, and sodium dithionite is 78g:220mL:18.5g; the amount ratio of the reaction product 4, triphosgene, and cyclohexane is 76g:31g:230mL; 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 35 minutes, and then the reaction product 5 was added, stirred at 900 r / min for 25 seconds, poured into a mold, and allowed to stand at 23°C for foaming for 35 minutes. After demolding, the mixture was cured at room temperature for 73 hours to obtain a reaction product 6; the calcium phosphate microsphere product and the reaction product 6 were heated and blended at 175°C for 3 hours. 0min, cooled and dried at 80℃ for 25h, and then ground into a powder with a particle size of 0.7mm to obtain self-assembled calcium phosphate microspheres with 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 105g:10g:2.4g:20mg:10mg:0.7g:65g; the dosage ratio of calcium phosphate microsphere product and reaction product 6 is 95g:5g.

[0016] Comparative Example 1 Compared with Example 3, conjugated linoleic acid was replaced with sorbic acid, and the rest was exactly the same as Example 3, to prepare self-assembled calcium phosphate microspheres with high specific surface area.

[0017] Comparative Example 2 Compared with Example 3, bismuth nitrate was replaced by ferric nitrate, and the rest was exactly the same as Example 3, to obtain self-assembled calcium phosphate microspheres with high specific surface area.

[0018] Comparative Example 3 Compared with Example 3, gallium chloride was replaced by ferric chloride, and the rest was exactly the same as Example 3, to obtain self-assembled calcium phosphate microspheres with a high specific surface area.

[0019] The calcium phosphate microspheres prepared by the present invention were further tested for their effects, and the test results are as follows.

[0020] In vitro degradation rate: The calcium phosphate microspheres prepared by the present invention were immersed in 0.5 mol / L tris(hydroxymethylaminomethane)-hydrochloric acid buffer 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. An equal amount of new 0.5 mol / L tris(hydroxymethylaminomethane)-hydrochloric acid buffer was replaced every 7 days. At 28 days, the calcium phosphate microspheres were washed three times with deionized water, and the dry weight of the microspheres was weighed and the mass loss rate was calculated to evaluate the degradation rate; 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 sterilized at high temperature to obtain a medium containing calcium phosphate microspheres. Under sterile conditions, 200 μL of a certain concentration of bacterial stock solution of Escherichia coli and Staphylococcus aureus was added to the culture dish, and then the culture medium containing calcium phosphate microspheres cooled to 45°C was poured in, and the plate was placed flat on the table, shaken, condensed into a flat plate, and then placed in an incubator at 37°C for 24 hours, and the bacteria were counted and the antibacterial rate of the bacteria was calculated. The culture medium without powder was used as the control sample, and the antibacterial rate = (A0-A) / A0×100%, A0 is the number of bacteria recovered from the control sample, and A is the number of bacteria recovered from the calcium phosphate microsphere sample; ALP activity: The alkaline phosphatase (ALP) activity of bone marrow mesenchymal stem cells (BMSCs) was quantitatively detected after 3 days and 7 days of osteogenic induction culture using the calcium phosphate microspheres prepared by the present invention to evaluate the osteogenic performance; The results are recorded in Table 1;

[0021] According to the data in Table 1, the calcium phosphate microspheres of the present invention have excellent degradability, antibacterial and osteogenic properties. Compared with Comparative Example 1, Example 3 shows that the antibacterial rate of conjugated linoleic acid is higher than that of sorbic acid, and sorbic acid does not show osteogenic properties. Therefore, the use of conjugated linoleic acid makes the calcium phosphate microspheres have higher antibacterial and osteogenic properties. Compared with Comparative Example 2, Example 3 shows that bismuth ions (Bi 3+ ) and gallium ions (Ga 3+ ‌) co-doping compared to iron ions (Fe 3+ ) and gallium ions (Ga 3+ ‌) co-doping is more conducive to improving the antibacterial rate, and bismuth ions (Bi 3+ ) and gallium ions (Ga 3+ ‌) co-doping has a specific effect on calcium phosphate microsphere crystals, which is more conducive to improving the degradability of calcium phosphate microspheres. 3+ ) and gallium ions (Ga 3+ ‌) co-doping compared to bismuth ions (Bi 3+ ) and iron ions (Fe 3+ ) co-doped, the antibacterial rate of calcium phosphate microspheres is higher, and bismuth ions (Bi 3+ ) and gallium ions (Ga 3+ ‌) The specific effect of co-doping on calcium phosphate microsphere crystals also makes the calcium phosphate microspheres more degradable.

[0022] The above contents are merely examples and explanations of the concept of the present invention. The technicians in this technical field may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all 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 dosage ratio of the 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. Self-assembled calcium phosphate microspheres with high specific surface area are prepared by the method for preparing self-assembled calcium phosphate microspheres with high specific surface area according to any one of claims 1 to 9.

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