Preparation and application of a hydroxyapatite microsphere composite injection

Abstract

The present invention belongs to the technical field of material preparation, and specifically relates to the preparation and application of a hydroxyapatite microsphere composite injection. The present invention first provides a method for preparing hydroxyapatite composite microspheres, wherein the hydroxyapatite composite microspheres are modified hydroxyapatite microspheres with amino groups and carboxylic acid groups, and the method comprises preparing hydroxyapatite microspheres, preparing modified hydroxyapatite microspheres with amino groups, and preparing modified hydroxyapatite microspheres with amino groups and carboxylic acid groups. Without introducing a toxic crosslinking agent, the electrostatic adsorption between the groups is utilized to achieve uniform dispersion of the hydroxyapatite microspheres in an aqueous solution.

Description

Technical Field

[0001] The invention belongs to the technical field of material preparation, and specifically relates to the preparation and application of a hydroxyapatite microsphere composite injection. Background Art

[0002] Hydroxyapatite (HAp), also known as hydroxyapatite, is a natural apatite mineral with a molecular formula of Ca5(PO4)3(OH). 10 (PO4)6(OH)2 represents a crystal structure consisting of two molecules. HAp is the main inorganic component of the growing bones of the human body and other animals, and is widely used in the field of repair and regeneration biomaterials, pharmaceutical fields or medical fields.

[0003] Modern medical cosmetology or medical cosmetology is a new industry in the cosmetic surgery market that has emerged in recent years due to the vast market demand. It belongs to the category of modern medicine. The most common method of medical cosmetic surgery includes filling, which mainly uses fillers to fill the sunken areas of the face to modify the facial features, modify the facial contours, and achieve a full and three-dimensional face. Because this technology has the characteristics of fast effect and high safety, it is currently the first choice for those who need medical beauty. HAp has a high level of biocompatibility, is not prone to allergic reactions after injection, and has the effect of stimulating collagen regeneration after injection into the subcutaneous tissue, so it is used in the field of medical cosmetology.

[0004] The current mainstream hydroxyapatite injection products are basically a combination of sodium carboxymethylcellulose (CMC-Na) gel + hydroxyapatite microspheres, which use CMC-Na gel to disperse hydroxyapatite microspheres, increase the viscosity of the injection, improve the sedimentation of hydroxyapatite microspheres, and avoid the generation of large particle agglomeration. However, most hydroxyapatite microspheres are unevenly dispersed in the gel matrix, and may aggregate and migrate subcutaneously or in vivo after injection, causing patients to experience consequences such as foreign body sensation or granuloma at the injection site. For example, patents with publication numbers CN104853742A (20150819) and CN104703582A (20150610) disclose homogenizing hyaluronic acid, a biomacromolecule, with HAp microspheres to obtain a viscoelastic mixed injection gel. However, this technology is only a simple physical uniform mixing of hydroxyapatite microspheres and gel, and it is difficult to truly achieve uniform distribution of hydroxyapatite microspheres in the gel matrix. Although the patent with publication number CN115282339A (20221104) introduces photocross-linking technology on the basis of physical homogeneity, thereby combining macromolecules with HAp to reduce their aggregation, the introduced photoinitiators and photocross-linkers are still a major threat to the safety of injectable products.

[0005] In summary, it is necessary to propose new methods and strategies to alleviate or partially alleviate the shortcomings of existing technologies. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a hydroxyapatite composite microsphere and a preparation method and application thereof, and the specific technical scheme is as follows.

[0007] A method for preparing hydroxyapatite composite microspheres, wherein the hydroxyapatite composite microspheres are modified hydroxyapatite microspheres with (electrostatically adsorbed) amino groups and carboxylic acid groups, and the preparation method is a solution reaction method, comprising the following steps:

[0008] S01: weighing a calcium salt and adding it to an aqueous solution containing a morphology-forming agent, stirring and dissolving the solution to obtain a calcium-containing reaction solution, wherein the morphology-forming agent includes citric acid, polyaspartic acid, sodium polyacrylate or polyethylene glycol;

[0009] S02: preparing a phosphate solution and adding it to the calcium-containing reaction solution prepared in S01, adjusting the pH of the reaction system so that the pH value is greater than or equal to 7.0, and the molar ratio of calcium to phosphate (calcium:phosphate) in the reaction system is 1.5 to 1.7:1 (preferably 1.68±0.2:1);

[0010] S03: placing the reaction system of S02 in a reaction kettle and reacting at 100-180° C. for 5-24 hours, centrifuging, and collecting the precipitate after centrifugation to obtain hydroxyapatite microspheres;

[0011] S04: adding the hydroxyapatite microspheres obtained in S03 to an acidic solution (e.g., acetic acid) and mixing them thoroughly, then adding a silane coupling agent, adjusting the pH value of the reaction system to be greater than or equal to 7.0, stirring, centrifuging after sufficient reaction, collecting the precipitate after centrifugation, and obtaining modified hydroxyapatite microspheres with amino groups;

[0012] S05: dissolving and dispersing the modified hydroxyapatite microspheres with amino groups obtained in S04 with purified water, then adding sodium carboxymethyl cellulose, adjusting the pH value of the reaction system to be greater than or equal to 8.0, stirring to fully react, and after the reaction is completed, dialyzing and freeze-drying to obtain the modified hydroxyapatite microspheres with amino groups and carboxylic acid groups.

[0013] Furthermore, the mass ratio of the calcium salt in S01 to the aqueous solution containing the morphology forming agent is 10:1 to 4:1.

[0014] Preferably, the mass ratio of the calcium salt solution to the aqueous solution containing the morphology forming agent in S01 includes 10:1, 9:1, 8:1, 7:1, 6:1, 5:1 or 4:1.

[0015] Furthermore, the calcium salt used in S01 includes calcium carbonate, calcium chloride, calcium nitrate or calcium bicarbonate; the phosphate used in S02 includes potassium phosphate, sodium phosphate, disodium hydrogen phosphate or dipotassium hydrogen phosphate.

[0016] Furthermore, the mass of hydroxyapatite microspheres in the S04 reaction system is 5-10 times the mass of the silane coupling agent.

[0017] Preferably, the mass of hydroxyapatite microspheres in the S04 reaction system is 5-6 times, 6-7 times, 7-8 times, 8-9 times or 9-10 times the mass of the silane coupling agent.

[0018] Furthermore, the silane coupling agent used in S04 includes γ-aminopropyltriethoxysilane, γ-aminopropyltrimethoxysilane, N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane, γ-methacryloxypropyltrimethoxysilane or γ-aminopropylmethyldiethoxysilane.

[0019] Furthermore, the mass ratio of the modified hydroxyapatite microspheres with amino groups to sodium carboxymethyl cellulose in S05 is 5:1 to 10:1.

[0020] Preferably, the mass ratio of the modified hydroxyapatite microspheres with amino groups to sodium carboxymethyl cellulose in S05 is 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

[0021] The hydroxyapatite composite microspheres prepared by the preparation method have surfaces connected with amino groups and carboxylic acid groups through electrostatic adsorption.

[0022] Furthermore, the particle size of the hydroxyapatite composite microspheres is in the range of 30-50 μm.

[0023] The hydroxyapatite composite microspheres are used in the preparation of medical injections, wherein the medical injections are aqueous dispersions and may further contain sodium carboxymethylcellulose.

[0024] The above preparation method is used in the preparation of hydroxyapatite microsphere composite injection, wherein the hydroxyapatite microsphere composite injection is an aqueous dispersant.

[0025] The above method prepares hydroxyapatite microspheres that can be uniformly dispersed in a solution.

[0026] The present invention can also provide a method for preparing hydroxyapatite microspheres that can be uniformly dispersed in a solution, wherein the hydroxyapatite composite microspheres are modified hydroxyapatite microspheres that electrostatically adsorb amino groups and carboxylic acid groups, and the preparation steps are as follows:

[0027] S01: weighing a calcium salt and adding it to an aqueous solution containing a morphology-forming agent, stirring and dissolving the solution to obtain a calcium-containing reaction solution, wherein the morphology-forming agent includes citric acid, polyaspartic acid, sodium polyacrylate or polyethylene glycol;

[0028] S02: preparing a phosphate solution and adding it to the calcium-containing reaction solution prepared in S01, adjusting the pH of the reaction system so that the pH value is greater than or equal to 7.0, and the molar ratio of calcium to phosphate (calcium:phosphate) in the reaction system is 1.5 to 1.7:1 (preferably 1.68±0.2:1);

[0029] S03: placing the reaction system of S02 in a reaction kettle and reacting at 100-180° C. for 5-24 hours, centrifuging, and collecting the precipitate after centrifugation to obtain hydroxyapatite microspheres;

[0030] S04: adding the hydroxyapatite microspheres obtained in S03 to an acidic solution (e.g., acetic acid) and mixing them thoroughly, then adding a silane coupling agent, adjusting the pH value of the reaction system to be greater than or equal to 7.0, stirring, centrifuging after sufficient reaction, collecting the precipitate after centrifugation, and obtaining modified hydroxyapatite microspheres with amino groups;

[0031] S05: dissolving and dispersing the modified hydroxyapatite microspheres with amino groups obtained in S04 with purified water, then adding sodium carboxymethyl cellulose, adjusting the pH value of the reaction system to be greater than or equal to 8.0, stirring to fully react, and after the reaction is completed, dialyzing and freeze-drying to obtain the modified hydroxyapatite microspheres with amino groups and carboxylic acid groups.

[0032] Beneficial technical effects

[0033] The present invention provides a modified hydroxyapatite microsphere with a double group, which is an amino group and a carboxylic acid group, and can also be called a hydroxyapatite microsphere coated with a macromolecular biomaterial. The modified hydroxyapatite microsphere with a double group forms a uniformly dispersed composite microsphere system, and maintains the uniform dispersion characteristics for a long time without sedimentation. In addition, the microsphere is preferably composed of carbonate hydroxyapatite, has good biocompatibility, and is therefore suitable for preparing medical injections. When the medical injection is injected into the body, due to the high dispersibility of the microspheres, the retention time of the microspheres after injection can be extended, thereby reducing the nodule phenomenon caused by particle migration and aggregation after filling.

[0034] The present invention also provides a method for preparing the modified hydroxyapatite microspheres with double groups, firstly using a morphology control agent to induce the conversion and crystallization of a phosphate solution containing calcium ions into a calcium phosphate mineral to form hydroxyapatite microspheres, and then using electrostatic adsorption to connect the modified hydroxyapatite with a macromolecular substance to form a composite microsphere system.

[0035] Finally, the present invention adopts a solution preparation method, most of the materials participate in the reaction in the form of liquid, and the reaction conditions are generally mild. Therefore, the preparation process is simple and convenient, suitable for industrial promotion, and the electrostatic adsorption between groups is utilized without introducing toxic cross-linking agents to achieve uniform dispersion of hydroxyapatite microspheres in an aqueous solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0037] Figure 1 A reaction mechanism diagram of one embodiment of the present invention;

[0038] Figure 2 A process flow chart of one embodiment of the present invention;

[0039] Figure 3 This is a particle size distribution diagram of modified hydroxyapatite with amino and carboxylic acid groups prepared in one embodiment of the present invention;

[0040] Figure 4 This is a scanning electron micrograph of modified hydroxyapatite with amino and carboxylic acid groups prepared in one of the embodiments of the present invention (scale bar: 30 μm);

[0041] Figure 5 This is a diagram showing the liquid dispersion effects of different samples tested in one embodiment of the present invention after 48 hours;

[0042] Figure 6 This is a diagram showing the liquid dispersion effects of different samples tested in one embodiment of the present invention after 5 days. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0044] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0045] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0046] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0047] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0048] In this specification, some embodiments may be disclosed in a format of being within a certain range. It should be understood that such description of "being within a certain range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the range The description of should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within this range, for example, 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0049] Example 1

[0050] This embodiment provides an example of a method for preparing hydroxyapatite composite microspheres.

[0051] Step 1: Preparation of hydroxyapatite microspheres (HA-MP)

[0052] S01: Weigh a certain amount of calcium salt and dissolve it in an aqueous solution containing a morphology forming agent (citric acid, polyaspartic acid, sodium polyacrylate, polyethylene glycol, etc.), stir it to dissolve it, and form a calcium-containing reaction solution. The calcium salt can be selected from calcium carbonate, calcium chloride, calcium nitrate or calcium bicarbonate, etc. The mass ratio of the calcium salt to the aqueous solution containing the morphology forming agent is about 10:1 to 4:1.

[0053] S02: Weigh a certain amount of phosphate and dissolve it in purified water to prepare a phosphorus solution. The phosphate can be selected from potassium phosphate, sodium phosphate, disodium hydrogen phosphate or dipotassium hydrogen phosphate.

[0054] S03: Mix the calcium-containing reaction solution and the phosphorus solution to make the molar ratio of calcium to phosphate ion 1.5 to 1.7:1 (optimum 1.68±0.2:1), and adjust the pH value to make the pH value of the reaction system greater than or equal to 7.0.

[0055] S04: Stir the reaction solution until white precipitate appears in the reaction system, pour the reaction system into a reaction kettle, and react at 100-180° C. for 5-24 hours.

[0056] S05: After the reaction is completed, the reactor is taken out and cooled, and the white precipitate at the lower layer is collected by centrifugation and washed to obtain hydroxyapatite microspheres, i.e., the first reaction product.

[0057] Step 2: Preparation of the first modified hydroxyapatite microspheres (HA-MP-NH2)

[0058] S01: dissolving a silane coupling agent in a 95% ethanol aqueous solution, stirring at room temperature to dissolve it. The silane coupling agent can be selected from γ-aminopropyltriethoxysilane (A-1100), γ-aminopropyltrimethoxysilane (A-1110), N-(β-aminoethyl)-γ-aminopropyltrimethoxysilane (A-1120), γ-methacryloxypropyltrimethoxysilane (A-174) or γ-aminopropylmethyldiethoxysilane (A-2100) and the like.

[0059] S02: adding hydroxyapatite microsphere powder to an acid solution with a pH value of about 5.0-6.0, and fully homogenizing and mixing to obtain a hydroxyapatite microsphere solution. The acid solution can be selected from acetic acid, hydrochloric acid, and the like.

[0060] S03: Pour the hydroxyapatite microsphere solution into the silane coupling agent solution so that the mass of the hydroxyapatite microsphere is 5-10 times the mass of the silane coupling agent.

[0061] S04: Adjust the pH value of the reaction solution to about 7.0-9.0, and stir the reaction for 3-5 hours.

[0062] S05: Collect the white precipitate at the lower layer by centrifugation, wash and dry to obtain modified hydroxyapatite with amino groups, i.e., the second reaction product.

[0063] Step 3: Preparation of the second modified hydroxyapatite microspheres (HA-MP-NH2 / CMC)

[0064] S01: Dissolve the second reaction product in purified water and disperse it fully.

[0065] S02: Weigh a certain amount of sodium carboxymethyl cellulose (CMC-Na) and pour it into the aqueous solution of the second reaction product so that the mass ratio of the second reaction product to sodium carboxymethyl cellulose is 5:1-10:1, and mix them thoroughly.

[0066] S03: Adjusting the pH of the reaction solution so that the pH value of the reaction solution is approximately greater than or equal to 8.0.

[0067] S04: The reaction solution was further stirred at room temperature for 12-48 hours.

[0068] S05: After the reaction is completed, the alkali is removed by dialysis and freeze-dried to obtain modified hydroxyapatite with amino and carboxylic acid groups, which is the final product of the reaction.

[0069] The reaction mechanism diagram of the present invention is shown in Figure 1 .

[0070] Example 2

[0071] This embodiment provides an example of a specific preparation method of hydroxyapatite composite microspheres.

[0072] Step 1: Preparation of hydroxyapatite microspheres (HA-MP)

[0073] Dissolve 1.67g of calcium carbonate in 20mL of purified water containing 150μg / mL citric acid and stir magnetically at 100rpm. After it is fully dissolved, add 3.58g of sodium phosphate and stir to mix evenly. Use 0.1M NaOH to adjust the pH and control the pH value of the reaction solution to about 8.0 until a white precipitate appears in the reaction vessel. Further pour the precipitated reaction solution into the tetrafluoroethylene liner of the reactor, and then heat the entire reactor at 150℃ for 10h. After the reaction time is reached, take out the reactor and cool it to room temperature, collect the white precipitate in the liner by centrifugation at a rate of 2000rpm, and centrifuge and wash it 3 times with purified water and anhydrous ethanol respectively. Finally, collect the white precipitate and dry it at 60℃ for 12h to obtain hydroxyapatite microspheres, i.e., the first reaction product.

[0074] Step 2: Preparation of the first modified hydroxyapatite microspheres (HA-MP-NH2)

[0075] Weigh 0.2g of the first reaction product and dissolve it in 40mL of glacial acetic acid solution at pH 5.5. After using 20kHz ultrasound for 15min, use a homogenizer to homogenize at 10000rpm for 2min to fully dissolve the hydroxyapatite microspheres. Then pour the ethanol solution containing 0.02g of γ-aminopropyltrimethoxysilane (A-1110) prepared in advance into it, stir it sufficiently, adjust the pH value of the reaction solution to about 9.0, and further stir and react for 4 hours. The reaction product is centrifuged, the precipitate is collected and the precipitate is properly washed to obtain modified hydroxyapatite with amino groups, i.e., the second reaction product.

[0076] Step 3: Preparation of the second modified hydroxyapatite microspheres (HA-MP-NH2 / CMC)

[0077] Weigh 0.5g of the second reaction product and dissolve it in 40mL of purified water. Use a homogenizer and ultrasound to disperse it evenly. Add 20mL of aqueous solution containing 0.1g of sodium carboxymethyl cellulose (CMC-Na) and stir to mix it thoroughly. Adjust the pH value of the reaction solution to about 8.5, and stir the reaction at room temperature for 24 hours. After the reaction is completed, dialyze with distilled water for 24 hours (using a 3500Da dialysis bag), change the water 4 times during the process, and vacuum freeze-dry the reaction product for 48-72 hours to obtain modified hydroxyapatite with amino and carboxylic acid groups, i.e., the final product of the reaction.

[0078] It should be understood that the specific preparation method shown in this embodiment is only an example and not a limitation.

[0079] The process flow chart of this embodiment is shown in Figure 2 The particle size distribution diagram and scanning electron microscope image of the modified hydroxyapatite with amino and carboxylic acid groups prepared in this example are shown in Figure 3 and Figure 4 . Figure 3 It can be seen that the particle size range of the modified hydroxyapatite with amino and carboxylic acid groups prepared in this embodiment is about 10-60 μm, and the present invention further preferably selects microspheres with a particle size range of 30-50 μm for preparing medical injections. This is because when the particle size range of the microspheres is 30-50 μm, they can better avoid being phagocytosed by immune cells in the body.

[0080] Comparative Example 1

[0081] Weigh 0.5g of the first reaction product in Example 2 and dissolve it in 40mL of purified water. Use a homogenizer and ultrasound to disperse it evenly, then add 20mL of an aqueous solution containing 0.1g of CMC-Na and stir to mix it thoroughly. Adjust the pH of the reaction solution to 8.5 and stir the reaction at room temperature for 24h. After the reaction is completed, dialyze with distilled water for 24h (using a 3500Da dialysis bag), change the water 4 times, and vacuum freeze-dry the reaction product for 48-72h to obtain modified hydroxyapatite microspheres with only carboxylic acid groups.

[0082] Comparative Example 2

[0083] 0.2 g of the first reaction product in Example 2 was weighed and dissolved in 40 mL of glacial acetic acid solution at pH 5.5. After using 20 kHz ultrasound for 15 min, the hydroxyapatite microspheres were fully dissolved by homogenizing with a homogenizer at 10000 rpm for 2 min. Then, an ethanol solution containing 0.02 g of γ-aminopropyltrimethoxysilane (A-1110) prepared in advance was poured into it, and after sufficient stirring, the pH value of the reaction solution was adjusted to about 9.0, and further stirred and reacted for 4 hours. The reaction product was centrifuged, the precipitate was collected and the precipitate was properly washed to obtain modified hydroxyapatite microspheres with only amino groups.

[0084] Comparative Example 3

[0085] 1.67g of calcium carbonate was dissolved in 20mL of purified water containing 150μg / mL citric acid and magnetically stirred at 100rpm. After it was fully dissolved, 3.58g of sodium phosphate was added and stirred to mix evenly. The pH was adjusted with 0.1M NaOH to control the pH value of the reaction solution to be about 8.0 until a white precipitate appeared in the reaction vessel. The precipitated reaction solution was further poured into the tetrafluoroethylene liner of the reactor, and then the entire reactor was heated at 150℃ for 10h. After the reaction time was reached, the reactor was taken out and cooled to room temperature, and the white precipitate in the liner was collected by centrifugation at a rate of 2000rpm, and purified water and anhydrous ethanol were centrifuged and washed 3 times each. Finally, the white precipitate was collected and dried at 60℃ for 12h to obtain unmodified hydroxyapatite microspheres.

[0086] Example 4

[0087] Weigh the samples prepared in Example 2 and Comparative Examples 1-3, dissolve them in 5 mL of aqueous solution, and observe the sedimentation of the samples in the liquid within 48 h and 5 days, as shown in Tables 1 and Figure 5 , Figure 6 . Figure 6It is shown that after 5 days, after the dispersion test with comparative example 1, the sample of Example 2 is still homogeneously dispersed in the solution containing CMC-Na, while the sample of comparative example 1 has some white precipitates gathered at the bottom of the test tube. This shows that after the hydroxyapatite microspheres with two group modifications prepared by the preferred method of the present invention are mixed with sodium carboxymethyl cellulose, the microsphere aggregation and precipitation phenomenon of the solution can be effectively reduced. Therefore, after being prepared as an injection, the nodular swelling and bulging phenomenon of the hydroxyapatite injection at the injection site can be effectively improved.

[0088] Table 1 Dispersion test of different sample solutions

[0089]

[0090] Conclusion: Unmodified hydroxyapatite microspheres and hydroxyapatite microspheres modified with only a single group began to precipitate after 6 hours, while the modified hydroxyapatite microspheres with amino and carboxylic acid groups prepared by the present invention only showed a small amount of precipitation after 5 days, indicating that such microspheres meet the requirements for further preparation as medical injections.

[0091] Example 5

[0092] This embodiment provides another example of a specific preparation method of hydroxyapatite composite microspheres.

[0093] Step 1: Preparation of hydroxyapatite microspheres (HA-MP)

[0094] Dissolve 1.67g of calcium chloride in 20mL of purified water containing 150μg / mL polyaspartic acid, and stir magnetically at 100rpm. After it is fully dissolved, add 3.58g of potassium phosphate and stir to mix evenly. Use 0.1M NaOH to adjust the pH and control the pH value of the reaction solution to about 8.0 until a white precipitate appears in the reaction vessel. Further pour the precipitated reaction solution into the tetrafluoroethylene liner of the reactor, and then place the entire reactor at a high temperature of 160℃ and heat for 8h. After the reaction time is reached, take out the reactor and cool it to room temperature, collect the white precipitate in the liner by centrifugation at a rate of 2000rpm, and centrifuge and wash it 3 times with purified water and anhydrous ethanol respectively, finally collect the white precipitate, dry it at 65℃ for 12h, and obtain hydroxyapatite microspheres, i.e., the first reaction product.

[0095] Step 2: Preparation of the first modified hydroxyapatite microspheres (HA-MP-NH2)

[0096] Weigh 0.2g of the first reaction product and dissolve it in 40mL of hydrochloric acid solution with pH 5.5. After using 40kHz ultrasound for 10min, use a homogenizer to homogenize at 10000rpm for 2min to fully dissolve the hydroxyapatite microspheres. Then pour the ethanol solution containing 0.02g of γ-aminopropyltriethoxysilane (A-1100) prepared in advance into it, stir it sufficiently, adjust the pH value of the reaction solution to about 9.0, and further stir and react for 4 hours. The reaction product is centrifuged, the precipitate is collected and the precipitate is properly washed to obtain modified hydroxyapatite with amino groups, i.e., the second reaction product.

[0097] Step 3: Preparation of the second modified hydroxyapatite microspheres (HA-MP-NH2 / CMC)

[0098] Weigh 0.6g of the second reaction product and dissolve it in 40mL of purified water. Use a homogenizer and ultrasound to disperse it evenly. Add 20mL of an aqueous solution containing 0.1g of sodium carboxymethyl cellulose (CMC-Na) and stir to mix it thoroughly. Adjust the pH value of the reaction solution to about 8.5, and stir the reaction at room temperature for 24 hours. After the reaction is completed, dialyze with distilled water for 24 hours (using a 3500Da dialysis bag), change the water 4 times during the process, and vacuum freeze-dry the reaction product for 48-72 hours to obtain modified hydroxyapatite with amino and carboxylic acid groups, i.e., the final product of the reaction.

[0099] It should be understood that the specific preparation method shown in this embodiment is only an example and not a limitation.

[0100] Example 6

[0101] This embodiment provides another example of a specific preparation method of hydroxyapatite composite microspheres.

[0102] Step 1: Preparation of hydroxyapatite microspheres (HA-MP)

[0103] Dissolve 1.67g of calcium bicarbonate in 20mL of purified water containing 150μg / mL polyethylene glycol and stir magnetically at 100rpm. After it is fully dissolved, add 3.58g of potassium dihydrogen phosphate and stir to mix evenly. Use 0.1MNaOH to adjust the pH and control the pH value of the reaction solution to about 8.0 until a white precipitate appears in the reaction vessel. Further pour the precipitated reaction solution into the tetrafluoroethylene liner of the reactor, and then heat the entire reactor at 120℃ for 15h. After the reaction time is reached, take out the reactor and cool it to room temperature, collect the white precipitate in the liner by centrifugation at a rate of 2000rpm, and centrifuge and wash it 3 times with purified water and anhydrous ethanol respectively. Finally, collect the white precipitate and dry it at 60℃ for 12h to obtain hydroxyapatite microspheres, i.e., the first reaction product.

[0104] Step 2: Preparation of the first modified hydroxyapatite microspheres (HA-MP-NH2)

[0105] Weigh 0.2g of the first reaction product and dissolve it in 40mL of glacial acetic acid solution at pH 5.5. After using 40kHz ultrasound for 10min, use a homogenizer to homogenize at 10000rpm for 2min to fully dissolve the hydroxyapatite microspheres. Then pour the ethanol solution containing 0.02g of γ-aminopropylmethyldiethoxysilane (A-2100) prepared in advance into it, stir it sufficiently, adjust the pH value of the reaction solution to about 9.0, and further stir and react for 4 hours. The reaction product is centrifuged, the precipitate is collected and the precipitate is properly washed to obtain modified hydroxyapatite with amino groups, i.e., the second reaction product.

[0106] Step 3: Preparation of the second modified hydroxyapatite microspheres (HA-MP-NH2 / CMC)

[0107] Weigh 0.5g of the second reaction product and dissolve it in 40mL of purified water. Use a homogenizer and ultrasound to disperse it evenly. Add 20mL of aqueous solution containing 0.1g of sodium carboxymethyl cellulose (CMC-Na) and stir to mix it thoroughly. Adjust the pH value of the reaction solution to about 8.5, and stir the reaction at room temperature for 24 hours. After the reaction is completed, dialyze with distilled water for 24 hours (using a 3500Da dialysis bag), change the water 4 times during the process, and vacuum freeze-dry the reaction product for 48-72 hours to obtain modified hydroxyapatite with amino and carboxylic acid groups, i.e., the final product of the reaction.

[0108] It should be understood that the specific preparation method shown in this embodiment is only an example and not a limitation.

[0109] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A method for preparing modified hydroxyapatite composite microspheres with double groups, characterized in that: The modified hydroxyapatite composite microspheres with double groups are modified hydroxyapatite microspheres with amino groups and carboxylic acid groups. The preparation method is a solution reaction method, comprising the following steps: S01: weighing a calcium salt and adding it to an aqueous solution containing a morphology-forming agent, stirring and dissolving the solution to obtain a calcium-containing reaction solution, wherein the morphology-forming agent includes citric acid, polyaspartic acid, sodium polyacrylate or polyethylene glycol; S02: preparing a phosphate solution and mixing it with the calcium-containing reaction solution prepared in S01, adjusting the pH value of the reaction system so that the pH value is greater than 7.0, and the molar ratio of calcium to phosphate in the reaction system is 1.5-1.7:1; S03: placing the reaction system of S02 in a reaction kettle and reacting at 100-180° C. for 5-24 hours, centrifuging, and collecting the precipitate after centrifugation to obtain hydroxyapatite microspheres; S04: adding the hydroxyapatite microspheres obtained in S03 to the acidic solution and mixing them thoroughly, then adding a silane coupling agent, wherein the mass of the hydroxyapatite microspheres is 10 times the mass of the silane coupling agent, adjusting the pH value of the reaction system to 9.0, stirring for 4 hours, centrifuging after sufficient reaction, collecting the precipitated material after centrifugation, and obtaining modified hydroxyapatite microspheres with amino groups; the silane coupling agent is γ-aminopropyltrimethoxysilane; S05: dissolving and dispersing the modified hydroxyapatite microspheres with amino groups obtained in S04 with purified water, and then adding sodium carboxymethyl cellulose, wherein the mass ratio of the modified hydroxyapatite microspheres with amino groups to sodium carboxymethyl cellulose is 5:1; adjusting the pH value of the reaction system to 8.5, stirring the reaction at room temperature for 24 hours, and after the reaction is completed, dialyzing and freeze-drying to obtain the modified hydroxyapatite microspheres with double groups; the particle size range of the modified hydroxyapatite microspheres with double groups is 30-50 μm.

2. The preparation method according to claim 1, characterized in that The calcium salt used in S01 includes calcium carbonate, calcium chloride, calcium nitrate or calcium bicarbonate; the phosphate used in S02 includes potassium phosphate, sodium phosphate, disodium hydrogen phosphate or dipotassium hydrogen phosphate.

3. Application of the preparation method according to claim 1 or 2 in the preparation of hydroxyapatite microsphere composite injection, characterized in that: The hydroxyapatite microsphere composite injection is an aqueous dispersant.

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