Preparation process for modified hydroxyapatite and use thereof

Abstract

Provided are a preparation process for a modified hydroxyapatite and the use thereof. The preparation process comprises the following steps: S1. removing impurities from coral by using a sodium hypochlorite solution, followed by crushing to obtain coral particles; S2. subjecting the coral particles and a phosphate solution to a hydrothermal reaction at a pH value of 9-11 to obtain coralline hydroxyapatite; S3. mixing and reacting the coralline hydroxyapatite and an ion source to obtain an ion source-doped coralline hydroxyapatite; and S4. heating the ion source-doped coralline hydroxyapatite, and sintering and cooling same to obtain an ion source-doped coralline β-tricalcium phosphate, wherein the ion source comprises a strontium source. The modified hydroxyapatite prepared by the preparation process has a better degradation performance and mechanical adaptability, which improves the biological activity and osteoinductivity of coral artificial bones, and facilitates bone tissue ingrowth.

Description

Preparation process and application of modified hydroxyapatite Technical Field

[0001] The present invention relates to the field of biomedical materials, and in particular to a preparation process and application of modified hydroxyapatite. Background Art

[0002] At present, the repair of large bone defects caused by diseases or accidents is still a common clinical problem. Bone injury repair materials are generally divided into autologous bone, allogeneic bone and artificial synthetic bone. At present, autologous bone is the gold standard for clinical treatment of bone defects, but its donor source is limited and its clinical application is limited. In addition, allogeneic bone has problems such as immunogenicity and disease transmission. With the continuous development of regenerative medicine technology, artificial synthetic bone has emerged due to its advantages such as wide source, non-immunogenicity and strong designability, and has been widely researched and developed in recent decades.

[0003] Marine natural materials such as corals, shells, sea urchins, nacre, conch shells and fish bones have unique structures, and their main chemical components are calcium carbonate, and they are widely used in the field of bone repair material research and development. Among them, coral, as a natural porous aquatic mineralized structure, is mainly composed of inorganic calcium carbonate. Its microstructure and mechanical properties are similar to those of cancellous bone, which can meet the clinical needs of mechanical adaptation. It has the ability to guide the orderly growth of bone and vascular tissue, and is an ideal material for filling and repairing large bone tissue defects. However, coral is brittle and degrades too quickly in the acidic microenvironment of bone defects, which cannot meet the requirements of clinical use. Some researchers have solved the problem of coral degradation rate being too fast by transforming coral into coral hydroxyapatite with a hydroxyapatite structure on the surface. However, the slow degradation rate of hydroxyapatite results in poor mechanical matching with bone tissue, insufficient bone induction, and insufficient biological activity, which is not conducive to bone defect repair.

[0004] Therefore, there is an urgent need to develop a new generation of coral hydroxyapatite bone repair materials.

[0005] Summary of the Invention

[0006] The present invention aims to address at least one of the technical problems existing in the aforementioned prior art. To this end, the present invention proposes a process for preparing modified hydroxyapatite. The resulting modified hydroxyapatite exhibits improved degradation properties and mechanical adaptability, enhancing the bioactivity of coral artificial bone and effectively stimulating tissue regeneration at bone defect sites.

[0007] The present invention also provides a bone defect repairing material.

[0008] According to one aspect of the present invention, a process for preparing modified hydroxyapatite is proposed, comprising the following steps:

[0009] S1. Using sodium hypochlorite solution to remove impurities from the coral and then crushing it to obtain coral particles;

[0010] S2. hydrothermally reacting the coral particles with a phosphate solution at a pH of 9 to 11 to obtain coral hydroxyapatite;

[0011] S3. mixing the coralline hydroxyapatite and the ion source to obtain ion source-doped coralline hydroxyapatite;

[0012] S4. heating the ion source-doped coral hydroxyapatite, sintering it, and cooling it to obtain the ion source-doped coral β-tricalcium phosphate;

[0013] The ion source includes a strontium source.

[0014] The inventive concept of the present invention is:

[0015] This invention combines hydrothermal reaction and high-temperature sintering techniques to develop surface-doped coral β-tricalcium phosphate artificial bone with an ion source. Specifically, the process involves: 1) subjecting the coral surface to hydroxyapatite conversion treatment, reacting coral particles with a phosphate solution in an alkaline environment to produce coral hydroxyapatite with a hydroxyapatite surface structure; 2) subjecting the coral hydroxyapatite to a hydrothermal reaction with an ion source, causing the ions to replace calcium ions in the hydroxyapatite, producing ion-doped coral hydroxyapatite; and 3) sintering the ion-doped coral hydroxyapatite at high temperature and rapidly cooling it to retain the β-tricalcium phosphate phase. This forms an ion-rich apatite-like inorganic mineral structure on the coral surface that promotes bone regeneration, resulting in the ion-doped coral β-tricalcium phosphate artificial bone.

[0016] The embodiments according to the first aspect of the present invention have at least the following beneficial effects:

[0017] 1. Coral hydroxyapatite (CHA) with a hydroxyapatite structure on the surface has a composition similar to that of mineralized bone and a similar porous structure, and a structure similar to that of trabecular bone. It is the main form of coral currently used in clinical practice. However, hydroxyapatite degrades slowly, has poor compatibility with tissue mechanics, and has poor biological activity, resulting in limited bone induction. Compared with hydroxyapatite, β-tricalcium phosphate (β-TCP) is a ceramic material with better biodegradability. In addition, the present invention has found that strontium can regulate the bone formation process of bone metabolism and promote bone tissue regeneration by acting on macrophages and stem cells. In view of this, this patent combines hydrothermal reaction and sintering techniques to prepare surface-doped ion source coral β-tricalcium phosphate artificial bone. Specifically, the process involves: 1) reacting coral particles with a diammonium hydrogen phosphate solution to transform the coral surface into a hydroxyapatite structure, producing coral hydroxyapatite; 2) hydrothermally reacting the coral hydroxyapatite with a strontium source solution, causing strontium ions to replace calcium ions in the hydroxyapatite crystal structure, thereby preparing strontium-doped coral hydroxyapatite; and 3) sintering the strontium-doped coral hydroxyapatite at high temperature and rapidly cooling it to retain the β-tricalcium phosphate phase, thereby producing coral β-tricalcium phosphate artificial bone with a surface coral-doped strontium. Compared to hydroxyapatite, β-tricalcium phosphate exhibits superior degradation and mechanical compatibility. β-tricalcium phosphate has a chemical formula of Ca₃(PO₄)₂, a Ca / P ratio similar to that of normal bone tissue, and a low degree of crystallinity. After degradation, the local tissue pH decreases, creating a weakly acidic microenvironment that further accelerates the material's degradation process, ensuring rapid incorporation of new bone tissue into the material, matching the rate of new bone formation. On the other hand, the mechanical strength of β-tricalcium phosphate is similar to that of human cancellous bone and can be adjusted by modifying the material's physical properties, such as pore size and porosity. In summary, β-tricalcium phosphate has improved degradation resistance and mechanical adaptability, effectively promoting the integration of autologous bone tissue with the implant surface and inducing bone tissue regeneration. The artificial bone obtained in this invention has enhanced bioactivity and osteoinductive properties, effectively promoting tissue regeneration in bone defect areas, and has broad clinical application prospects.

[0018] Second, hydroxyapatite has poor degradation properties, which limits the role of strontium ions in regulating the immune system and promoting osteogenesis. The coralline hydroxyapatite of the present invention is sintered at high temperatures to form β-tricalcium phosphate, which has better degradation properties. In bone, particularly alveolar bone, where the pH of the microenvironment is low, β-tricalcium phosphate degrades relatively quickly, releasing more strontium ions during the inflammatory phase. This regulates the bone immune system, promotes the chemotaxis of macrophages toward an anti-inflammatory phenotype, stimulates the production of anti-inflammatory factors, facilitates the secretion of growth factors, and achieves excellent osteogenesis.

[0019] 3. The sodium hypochlorite in the present invention is mainly used to remove organic matter covering the surface of the coral, thereby avoiding the damage to the porous structure and mechanical properties of the coral caused by etching of the coral pores by dilute hydrochloric acid.

[0020] 4. Hydrothermal treatment of coralline hydroxyapatite with strontium. In the hexagonal crystal structure of hydroxyapatite, Ca 2+ Surrounded by phosphorus and oxygen tetrahedra. The strontium ion has a radius similar to that of the calcium ion. The properties of hydroxyapatite can be tuned by specifically modifying the calcium sites. Surface-doped strontium hydroxyapatite was prepared by hydrothermal reaction of strontium ions with coralline hydroxyapatite under hydrothermal conditions.

[0021] In some embodiments of the present invention, the ion source comprises a magnesium source.

[0022] In some embodiments of the present invention, the molar ratio of the strontium source to the magnesium source is 1:1-3.

[0023] In some preferred embodiments of the present invention, the molar ratio of the strontium source to the magnesium source is 1:1.

[0024] Magnesium is an essential functional element for bone homeostasis and metabolism. A lack of magnesium ions in the body can interfere with systemic bone metabolism, reduce the rate of bone resorption, disrupt the balance between osteogenesis and bone resorption, and lead to insufficient bone mass and density. Degradable metal materials such as magnesium and magnesium alloys exhibit excellent bone integration and bone formation after implantation. Furthermore, in vivo animal studies with osteoporosis have shown that magnesium ions have a positive effect on bone repair. As an osteogenic functional factor, magnesium ions mediate the polarization of macrophages during the inflammatory phase, causing them to polarize toward the pro-M2 type, secreting functional signaling factors that are beneficial to the proliferation, differentiation, and gene expression of osteoblast stem cells, thereby promoting bone regeneration at the defect site.

[0025] In some embodiments of the present invention, the strontium source includes at least one of strontium nitrate, strontium chloride, and strontium hypochlorite.

[0026] In some embodiments of the present invention, the magnesium source includes at least one of magnesium chloride and magnesium sulfate.

[0027] In some embodiments of the present invention, the phosphate comprises diammonium phosphate.

[0028] In some embodiments of the present invention, the concentration of the phosphate is 2 to 3 mol / L.

[0029] In some embodiments of the present invention, the mass concentration of the sodium hypochlorite is 10-20%.

[0030] In some embodiments of the present invention, in step S1, the impurity removal step includes rinsing and soaking with sodium hypochlorite solution for 3 to 4 days.

[0031] In some embodiments of the present invention, the size of the coral particles is 0.25 mm to 1 mm.

[0032] In some embodiments of the present invention, step S2 includes: hydrothermally reacting the coral particles and a phosphate solution at pH 10 to obtain coral hydroxyapatite.

[0033] The hydroxyapatite prepared by the present invention under alkaline conditions of pH 10 has good crystallinity and single components.

[0034] In some embodiments of the present invention, in step S2, the temperature of the hydrothermal reaction is 160-180° C., the time of the hydrothermal reaction is 20-24 h, and the pressure of the hydrothermal reaction is 0.3-0.5 MPa.

[0035] In some embodiments of the present invention, in step S3, the temperature of the mixing reaction is 160-180° C., the time of the mixing reaction is 20-24 h, and the pressure of the mixing reaction is 0.3-0.5 MPa.

[0036] In some embodiments of the present invention, in step S4, the sintering temperature is 1200-1400° C., and the sintering time is 2-3 hours.

[0037] The above-mentioned sintering temperature ensures that the reaction produces a single-phase β-tricalcium phosphate. The Ca / P ratio of β-tricalcium phosphate is similar to that of normal bone tissue, and it has a low degree of crystallinity. After degradation, the pH value of the local tissue decreases. This weakly acidic microenvironment further accelerates the degradation process of the material, ensuring that new bone tissue can quickly grow into the interior of the material and match the rate of new bone formation. On the other hand, the mechanical strength of β-tricalcium phosphate is similar to that of human cancellous bone, and it can be adjusted by changing the physical properties of the material, such as pore size and porosity. In short, β-tricalcium phosphate has better degradation performance and mechanical adaptability, can effectively promote the integration of autologous bone tissue with the implant surface, and has better bioactivity, inducing bone tissue regeneration.

[0038] In some embodiments of the present invention, in step S4, the heating rate is 7-10°C / min.

[0039] In some embodiments of the present invention, the concentration of the strontium source is 0.6-0.7 mol / L.

[0040] In some embodiments of the present invention, the cooling rate is 6-8°C / min.

[0041] In some embodiments of the present invention, the ion source further comprises at least one of manganese ions, copper ions, zinc ions and borate ions.

[0042] According to the second aspect of the present invention, a bone defect repair material is provided, wherein the raw materials for preparation include the modified hydroxyapatite prepared by the preparation process.

[0043] In some embodiments of the present invention, the bone defect repair material includes an alveolar bone defect repair material. DETAILED DESCRIPTION

[0044] The following will clearly and completely describe the concept and technical effects of the present invention in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0045] Example 1

[0046] This embodiment prepares a modified hydroxyapatite material, and the specific preparation method is as follows:

[0047] A1. Soak the corals in deionized water at room temperature, ultrasonically clean them, and dry them. Then, rinse and soak them in 10% sodium hypochlorite solution for 4 days to remove organic residues, dead plants and animals, and sand from the surface or interior of the corals. Then, rinse and ultrasonicate them in distilled water and dry them for later use.

[0048] A2. Crush the coral and take 0.25mm to 1mm coral particles as raw materials;

[0049] A3. Add 60 ml of 2 mol / L diammonium hydrogen phosphate solution to 4 g of the coral treated in steps A1 and A2, adjust the pH of the solution to 10 with concentrated ammonia water, and then place the solution in a hydrothermal reactor at 180° C. for 24 h at a pressure of 0.5 MPa to obtain coral hydroxyapatite with a hydroxyapatite surface.

[0050] A4. Subsequently, 4 g of the coralline hydroxyapatite prepared in step A3 was added with 30 ml of a 0.33 mol / L strontium nitrate solution and 30 ml of a 0.33 mol / L magnesium nitrate solution, and the mixture was placed in a hydrothermal reactor at 180° C. and reacted for 24 h at a reaction pressure of 0.5 MPa to obtain strontium-magnesium-doped coralline hydroxyapatite.

[0051] A5. Then, the strontium-magnesium-doped coralline hydroxyapatite was sintered at 900°C with a heating rate of 7°C / min, kept at this temperature for 3 hours, and rapidly cooled to obtain a strontium-magnesium-doped coralline β-tricalcium phosphate artificial bone.

[0052] A6. Dry the strontium-magnesium-doped coral β-tricalcium phosphate artificial bone and store it in a dry place.

[0053] Example 2

[0054] In this example, a modified hydroxyapatite was prepared. The difference between this example and Example 1 is that the pH of the solution in step A5 is 9, and the other conditions are the same.

[0055] Example 3

[0056] In this embodiment, a modified hydroxyapatite is prepared. The difference between this embodiment and Example 1 is that in step A3, the strontium-magnesium doped coralline hydroxyapatite is sintered at a high temperature of 800° C., and the other conditions are the same.

[0057] Example 4

[0058] This embodiment prepares a modified hydroxyapatite material, and the specific preparation method is as follows:

[0059] A1. Soak the corals in deionized water at room temperature, ultrasonically clean them, and dry them. Then, rinse and soak them in 10% sodium hypochlorite solution for 4 days to remove organic residues, dead plants and animals, and sand from the surface or interior of the corals. Then, rinse and ultrasonicate them in distilled water and dry them for later use.

[0060] A2. Crush the coral and take 0.25mm to 1mm coral particles as raw materials;

[0061] A3. Add 60 ml of 2 mol / L diammonium hydrogen phosphate solution to 4 g of the coral treated in steps A1 and A2, adjust the pH of the solution to 10 with concentrated ammonia water, and then place the solution in a hydrothermal reactor at 180° C. for 24 h at a pressure of 0.5 MPa to obtain coral hydroxyapatite with a hydroxyapatite surface.

[0062] A4. Subsequently, 4 g of the coralline hydroxyapatite prepared in step A3 was added to 30 ml of a 0.33 mol / L strontium nitrate solution, and the mixture was placed in a hydrothermal reactor at 180° C. and reacted for 24 h at a reaction pressure of 0.5 MPa to obtain strontium-doped coralline hydroxyapatite.

[0063] A5. Then, the strontium-doped coralline hydroxyapatite was sintered at 900°C with a heating rate of 7°C / min, kept at this temperature for 3 hours, and rapidly cooled to obtain strontium-doped coralline β-tricalcium phosphate artificial bone.

[0064] A6. Dry the strontium-doped coral β-tricalcium phosphate artificial bone and store it in a dry place.

[0065] Comparative Example 1

[0066] In this comparative example, a modified hydroxyapatite material is prepared. The difference between this comparative example and Example 1 is that this comparative example does not include steps A5 to A6, and the other conditions are the same.

[0067] Comparative Example 2

[0068] In this comparative example, a modified hydroxyapatite material was prepared. The difference between this comparative example and Example 1 is that step A1 of this comparative example comprises: soaking and cleaning the coral in water to remove organic matter from the coral, drying the coral, rinsing the coral with a sodium hypochlorite solution, and then rinsing the coral with distilled water. After drying, the coral pores were etched with 3% dilute hydrochloric acid for 30 minutes to roughen the pore surface. All other conditions were the same.

[0069] Comparative Example 3

[0070] In this comparative example, a modified hydroxyapatite material is prepared. The difference between this comparative example and Example 1 is that this comparative example does not include the step of doping strontium and magnesium, and the other conditions are the same.

[0071] Comparative Example 4

[0072] In this comparative example, a modified hydroxyapatite material was prepared. The difference between this comparative example and Example 1 is that in step A2 of this comparative example, coralline hydroxyapatite was obtained by hydrothermal reaction at pH 8, and the other conditions were the same.

[0073] Test Example 1:

[0074] To verify the effects of the microenvironmental pH values ​​of Example 4 (strontium-doped coral β-tricalcium phosphate artificial bone) and Example 1 (strontium-magnesium-doped coral β-tricalcium phosphate artificial bone) on bone marrow mesenchymal stem cells (BMSCs), we used the following experimental steps:

[0075] 1) Strontium-doped coral β-tricalcium phosphate artificial bones and strontium-magnesium-doped coral β-tricalcium phosphate artificial bones were immersed in 37°C (20 mg / mL) SBF solution for 0.25, 24, 48, 72, 120, and 168 hours. At each time point, the extracts were obtained by membrane filtration. Three parallel groups were set up for each group.

[0076] 2) At the corresponding time point, the pH value of the material extract is tested using a pH microelectrode.

[0077] The test results showed that the pH values ​​of the strontium-doped coral β-tricalcium phosphate artificial bone at 0.25, 24, 48, 72, 120, and 168 hours were 7.35, 7.29, 7.39, 7.29, and 7.29, respectively. The pH values ​​of the strontium-magnesium-doped coral β-tricalcium phosphate artificial bone were 7.45, 7.59, 7.59, 7.49, and 7.52. The overall pH value of Example 1 was higher than that of Example 4. This is because the presence of magnesium ions increases the pH of the entire material system, thereby affecting the pH of the extract. The acidic products at the bone defect site can be neutralized by the alkaline degradation products of the magnesium-containing material, reducing the risk of local inflammation and infection.

[0078] 2. Magnesium ions can increase the biological behaviors of BMSCs such as proliferation, adhesion and activity.

[0079] The control group (strontium-doped coral β-tricalcium phosphate artificial bone) and the experimental group (strontium-magnesium-doped coral β-tricalcium phosphate artificial bone) were immersed in α-MEM complete medium for 24 hours to obtain cell conditioned medium, and then the following experiments were carried out:

[0080] 2.1 Value-added test:

[0081] (1) 2×10 4 Cells were seeded into 96-well plates at a density of 10 cells / well, with four replicates per group. 200 μL of the extract was added to each well. The plates were then incubated in a 37°C, 5% CO2 incubator for 1, 3, 5, and 7 days, with the medium changed every 2 days. Four replicates were set per group.

[0082] (2) On days 1, 3, 5, and 7, the culture medium was aspirated, washed with PBS, and 200 μL of α-MEM complete culture medium containing 10% CCK-8 solution was added to each well. The cells were incubated in a cell culture incubator in the dark for 2 h.

[0083] (3) After 2 h of incubation, aspirate 100 μL of the working solution from each well and transfer it to a new 96-well plate, taking care to avoid creating bubbles. Subsequently, the absorbance was measured at 450 nm using a microplate reader.

[0084] 2.2 Cell adhesion assay

[0085] (1) 2×10 4 Cells were seeded into 96-well plates at a density of 100 cells / well, and 200 μL of the extract was added to each well. The plates were then placed in a 37°C, 5% CO2 cell culture incubator for 24 hours, with three replicates set up for each group.

[0086] (2) After culturing for 24 h, the culture medium was aspirated, the cells were washed three times with PBS, and 4% paraformaldehyde was added to fix the cells for 15 min.

[0087] (3) After fixation, the plates were washed three times with PBS, and then rhodamine-labeled phalloidin solution was added to the wells for staining in the dark for 1 hour.

[0088] (4) Discard the dye, wash three times with PBS, and stain with DAPI solution for 15 minutes.

[0089] (5) The cells were then washed three times with PBS to remove the dye, and the adhesion status of the cells was observed under a laser confocal scanning microscope.

[0090] 2.3 Live / Dead staining experiment

[0091] To verify the cytotoxicity of the sample extracts, this study conducted a Live / Dead staining experiment on BMSCs stem cells. The steps are as follows:

[0092] (1) 2×10 4 Cells were seeded into 90-well plates at a density of 100 cells / well, and 200 μL of the extract was added to each well. The plates were then placed in a 37°C, 5% CO2 cell culture incubator for 24 hours, with three replicates set up for each group.

[0093] (2) After 24 h of culture, the cells were washed three times with PBS, and Calcein-AM / PI staining solution was added to the wells. The cells were incubated in a 37°C cell culture incubator in the dark for 5 min.

[0094] (3) The plate was washed with PBS to remove the dye, and then observed and photographed under a laser confocal scanning microscope.

[0095] The test results showed that as the culture time increased, the OD absorbance values ​​of Example 4 (strontium-doped coral β-tricalcium phosphate) and Example 1 (strontium-magnesium-doped coral β-tricalcium phosphate) showed different trends. Overall, compared with Example 4, the cells in Example 1 increased in number and were non-cytotoxic. On the first day, Examples 1 and 4 showed consistent growth effects and were non-cytotoxic. Starting from the third day, the growth rate of Example 1 increased and was non-cytotoxic, while Example 4 began to show cytotoxicity. On the seventh day, the cell growth effect of Example 1 further expanded, approximately 0.5 times that of the Example 1, and was non-cytotoxic. However, the growth effect of Comparative Example 3 was further suppressed, showing significant cytotoxicity. Furthermore, the adhesion morphology of BMSCs in the extracts of each group and the Live / Dead staining results showed that compared with Example 4, the BMSCs in Example 1 were able to maintain normal cell morphology after 24 hours of cell culture, with better morphology and tentacles, better surface extension, and cell survival reaching over 95%, with no obvious dead cells. However, the cell survival rate of the cells in the example was only 85% after 24 hours of culture. This is because strontium and magnesium ions, as important synthetic elements in the human body, actively participate in the cell life cycle. By regulating favorable signaling pathways such as cell proliferation and adhesion, they phosphorylate cells, continuously upregulating downstream signaling substances that are beneficial to cell osteogenesis, and thus enhancing the cell osteogenesis effect.

[0096] The reason is that magnesium ions are released from the material, and the hydroxide ions produced by the material cause the pH value in the microenvironment to increase, thereby increasing the pH of the acidic environment in the bone defect area, thereby improving the pH of the cell microenvironment. This moderate alkalinity increases the vitality and activity of bone marrow mesenchymal stem cells and promotes the polarization of cells toward the anti-inflammatory M2 type, further promoting the production of anti-inflammatory factors and enhancing the ability to form new bone. However, it is known that an alkaline pH value greater than 8.5 will have adverse effects and lead to tissue necrosis.

[0097] 3. Magnesium ions reduce inflammatory cytokines and increase anti-inflammatory factors

[0098] To verify the effect of magnesium ions, the following test steps were used to conduct the experiment:

[0099] 1) Strontium-doped coralline β-tricalcium phosphate artificial bone and strontium-magnesium-doped coralline β-tricalcium phosphate artificial bone were immersed in DMEM medium containing 10% FBS and 1% streptomycin-penicillin for 24 hours to obtain the corresponding conditioned medium. The extraction conditions were set in an incubator at 37°C and 5% CO2. The following experiments were then conducted:

[0100] 2) In a 48-well plate, 4 × 10 4 M1 macrophages were inoculated at a density of 100 cells / well, and the macrophages were induced by adding 20 ng / ml IFN-γ and 100 ng / ml LPS to the culture medium to induce RAW264.7 macrophages for 12 hours and then continuing to culture with complete culture medium for 24 hours.

[0101] 4) The conditioned medium obtained in step 1 is used to replace the medium in step 2) to culture M1 macrophages, and the medium is changed every two days.

[0102] 5) After 3 and 7 days of culture, RT-qPCR was used to evaluate the effect of magnesium ions on M1 macrophage polarization. Specific polarization genes tested included tumor necrosis factor-α (TNF-α), interleukin-6 (IL6), interleukin-10 (IL10), and arginase.

[0103] The test results showed that compared with Example 4, the expression of pro-inflammatory genes TNF-α and IL6 was significantly downregulated in Example 17d. Regarding the expression of anti-inflammatory genes, the expression of Arginase and IL10 in the experimental group was significantly stronger than that in Example 4. These results demonstrate that strontium-magnesium-doped coral β-tricalcium phosphate artificial bone can inhibit the expression of pro-inflammatory genes and promote the expression of anti-inflammatory genes. This indicates that strontium-magnesium-doped coral β-tricalcium phosphate artificial bone promotes the polarization of macrophages from M1 to M2.

[0104] It is generally believed that the stages of bone repair are divided into the early inflammatory stage, the early granulation stage, the mid-stage new bone formation stage, and the late bone remodeling stage. The process of magnesium ions promoting bone defect repair is primarily in the early verification stage, by regulating the expression of anti-inflammatory factors in immune cells such as macrophages, promoting macrophage polarization toward the M2 type (anti-inflammatory phenotype).

[0105] Test Example 2

[0106] Supplementary information on degradation performance: In order to verify that strontium-magnesium-doped coralline β-tricalcium phosphate has better degradation performance, this test example will conduct degradation performance tests on Comparative Example 1 and Example 1 (strontium-magnesium-doped coralline β-tricalcium phosphate artificial bone) in the following steps:

[0107] (1) Place 10 g of coral hydroxyapatite bone and strontium-magnesium-doped coral β-tricalcium phosphate samples in 50 mL centrifuge tubes. Add 40 mL of Tris-HCl solution (pH 7.4) to each tube. Place the tube in a 37°C incubator at 60 rpm. Prepare the Tris-HCl solution by weighing 13.25 g of Tris using a balance and dissolving it in 500 mL of deionized water. Adjust the pH of the solution to 7.4 with concentrated hydrochloric acid, and then dilute to 1000 mL.

[0108] (2) The samples were taken out after immersion for 1 day, 3 days, 1 week, 3 weeks, 5 weeks, 8 weeks, and 12 weeks, rinsed with deionized water three times, and then placed in a 50°C oven to dry for 24 hours. After drying, the samples were weighed using a balance and recorded.

[0109] (3) At each weighing time point, fresh Tris-HCl solution was replaced, and the degradation solution after 1 day, 3 days, 1 week, and 2 weeks of degradation was collected for subsequent analysis of the degradation product ion components.

[0110] (4) The concentrations of Ca, P, Sr and other ions in the degradation solution were determined using an inductively coupled plasma emission spectrometer.

[0111] Test results: Both Comparative Example 1 and Example 1 (strontium-magnesium-doped coral β-tricalcium phosphate) slowly degraded in a Tris-HCl solution, with weight losses of 10±1% and 15±1%, respectively, after 10 weeks. This indicates that the degradation rate of the materials can be increased by doping with strontium and magnesium and converting them into β-tricalcium phosphate. Statistical analysis of the cumulative concentrations of Ca, P, Sr, and Mg in the degradation solution, Tris-HCl, at days 1, 3, 7, and 21 revealed that the concentrations of Ca and P in Example 1 were higher than those in the control group. Furthermore, the release rate of strontium and magnesium ions from the strontium-magnesium-doped coral β-tricalcium phosphate was higher in the first 7 days than after day 7, while no strontium or magnesium ion release was observed in the coral hydroxyapatite bone group.

[0112] Therefore, the above experimental results show that the degradation performance of β-tricalcium phosphate is higher than that of comparative example 1, resulting in the degradation rate of the material in the leaching solution being higher than that of hydroxyapatite. As the test time increases, the structure of β-tricalcium phosphate is further destroyed, more Ca and P ions are released, and the release rate of strontium ions and magnesium ions is further increased.

[0113] Test Example 3

[0114] To verify that strontium and magnesium ions have a good osteogenic effect on mesenchymal stem cells (BMSCs), proliferation assay, live / dead assay and Sirius red staining were used to evaluate the effect.

[0115] The test groups were: Comparative Example 3 (coral β-tricalcium phosphate not doped with strontium-magnesium) and Example 1 (coral β-tricalcium phosphate doped with strontium-magnesium). The extracts were prepared by sterilizing the samples of Comparative Example 3 (coral β-tricalcium phosphate not doped with strontium-magnesium) and Example 1 (coral β-tricalcium phosphate doped with strontium-magnesium) in an autoclave for 20 minutes, followed by immersion of the sterilized samples (5 g) in 50 ml of serum-containing α-MEM complete medium (10% FBS + 5% double antibody + remaining medium) for 24 hours to obtain sample extracts. The test methods and results are analyzed as follows:

[0116] 1. Effects of strontium and magnesium ions on the biocompatibility of BMSCs:

[0117] 1.1 Value-added test

[0118] (1) 2×10 4 Cells were seeded into 96-well plates at a density of 10 cells / well, with four replicates per group. 200 μL of the extract was added to each well. The plates were then placed in a 37°C, 5% CO2 cell culture incubator for 1, 3, 5, and 7 days, with the medium changed every 2 days.

[0119] (2) On days 1, 3, 5, and 7, the culture medium was aspirated, washed with PBS, and 200 μL of α-MEM complete culture medium containing 10% CCK-8 solution was added to each well. The cells were incubated in a cell culture incubator in the dark for 2 h.

[0120] (3) After 2 h of incubation, aspirate 100 μL of the working solution from each well and transfer it to a new 96-well plate, taking care to avoid creating bubbles. Subsequently, the absorbance was measured at 450 nm using a microplate reader.

[0121] 1.2 Cell adhesion assay

[0122] (1) 2×10 4Cells were seeded into 96-well plates at a density of 100 cells / well, and 200 μL of the extract was added to each well. The plates were then placed in a 37°C, 5% CO2 cell culture incubator for 24 hours, with three replicates set up for each group.

[0123] (2) After culturing for 24 h, the culture medium was aspirated, the cells were washed three times with PBS, and 4% paraformaldehyde was added to fix the cells for 15 min.

[0124] (3) After fixation, the plates were washed three times with PBS, and then rhodamine-labeled phalloidin solution was added to the wells for staining in the dark for 1 hour.

[0125] (4) Discard the dye, wash three times with PBS, and stain with DAPI solution for 15 minutes.

[0126] (5) The cells were then washed three times with PBS to remove the dye, and the adhesion status of the cells was observed under a laser confocal scanning microscope.

[0127] 1.3 Live / Dead staining experiment

[0128] To verify the cytotoxicity of the sample extracts, this study conducted a Live / Dead staining experiment on BMSCs stem cells. The steps are as follows:

[0129] (1) 2×10 4 Cells were seeded into 90-well plates at a density of 100 cells / well, and 200 μL of the extract was added to each well. The plates were then placed in a 37°C, 5% CO2 cell culture incubator for 24 hours, with three replicates set up for each group.

[0130] (2) After 24 h of culture, the cells were washed three times with PBS, and Calcein-AM / PI staining solution was added to the wells. The cells were incubated in a 37°C cell culture incubator in the dark for 5 min.

[0131] (3) The plate was washed with PBS to remove the dye, and then observed and photographed under a laser confocal scanning microscope.

[0132] The test results showed that with increasing culture time, the OD absorbance values ​​of Comparative Example 3 (undoped strontium-magnesium coral β-tricalcium phosphate) and Example 1 (strontium-magnesium-doped coral β-tricalcium phosphate) exhibited different trends. Compared with Comparative Example 3, Example 1 demonstrated superior cell proliferation. On day 1, Example 1 showed no significant cytotoxicity, while Comparative Example 3 began to exhibit cytotoxicity. Starting on day 3, the proliferation rate of Example 1 increased, with no cytotoxicity, while the cytotoxicity of Comparative Example 3 further intensified. On day 7, the cell proliferation of Example 1 further expanded, reaching approximately double that of Comparative Example 3, without cytotoxicity, while the proliferation of Comparative Example 3 was further inhibited, exhibiting significant cytotoxicity. Furthermore, the adhesion morphology of BMSCs in the extracts of each group and Live / Dead staining results showed that, compared with Comparative Example 3, the BMSCs in Example 1 maintained normal cell morphology after 24 hours of cell culture, with improved morphology and antennae, better surface extension, and a cell survival rate exceeding 95%, with no obvious dead cells. However, the survival rate of cells in Comparative Example 3 after 24 hours of culture was only 75%. The above results show that compared with Comparative Example 3, Example 1 is more conducive to cell proliferation and non-toxic. This is because strontium ions and magnesium ions, as important synthetic elements in the human body, actively participate in the cell life cycle activities. By regulating favorable signaling pathways such as cell proliferation, adhesion, and differentiation, they phosphorylate cells and continuously upregulate downstream signaling substances that are beneficial to cell osteogenesis, thereby enhancing biological behaviors such as cell biological proliferation, adhesion, and activity.

[0133] 2. Effects of strontium and magnesium ions on osteogenic differentiation of BMSCs:

[0134] Collagen secretion and calcium nodule formation are important markers of early and late osteogenic differentiation, respectively. This study used Sirius red staining and Alizarin red staining to test the osteogenic differentiation properties of the sample extracts. The experimental steps and results are as follows:

[0135] 2.1 Sirius red staining experiment:

[0136] (1) 4×10 4 BMSCs were seeded in a 48-well plate at a density of 10 cells / well, with 3 replicate wells in each group, and cultured in a 37°C incubator for 24 h.

[0137] (2) Mix the osteogenic induction components: 3 mM β-glycerophosphate, 50 μg / mL vitamin C, and 100 nM dexamethasone with the extract. Then, continue culturing the cells in the osteogenic induction medium prepared with the extract, changing the medium every 2 days.

[0138] (3) After 7 days of osteogenic induction culture, the culture medium was aspirated and the cells were washed three times with PBS. The cells were then fixed with 4% paraformaldehyde for 15 min and stained with Sirius red solution. The cells were then stained in the dark at 37°C for 1 h.

[0139] (4) Discard the staining solution, wash the plate three times with 0.1 M acetic acid, and then take images using an inverted microscope.

[0140] (5) Prepare an alkaline eluent by mixing equal volumes of 0.2 M sodium hydroxide solution and methanol. Add 200 μL of the eluent to each well and transfer the plate to a new 96-well plate. Then measure the absorbance at a wavelength of 540 nm to quantitatively detect the secretion of collagen.

[0141] 2.1 Alizarin red staining experiment:

[0142] (1) BMSCs were cultured at 4×10 4 The cells were seeded in a 48-well plate at a density of 100 cells / well, with 3 replicate wells in each group, and cultured in a 37°C incubator for 24 h.

[0143] (2) Mix the osteogenic induction components: 3mM β-glycerophosphate, 50μg / mL vitamin C and 100nM dexamethasone with the extract. Then continue to culture the cells using the osteogenic induction medium prepared with the extract, and change the medium every 2 days.

[0144] (3) After 21 days of osteogenic induction culture, the culture medium was aspirated and the cells were washed three times with PBS. The cells were then fixed with 4% paraformaldehyde for 15 min and finally stained with Alizarin Red solution at 37°C in the dark for 30 min.

[0145] (4) Discard the staining solution, wash the plate three times with ultrapure water, and then take images using an inverted optical microscope.

[0146] (5) Then, phosphate buffer (pH = 7) containing 10% cetylpyridinium chloride was prepared, 200 μL of eluent was added to each well and the cells were transferred to a new 96-well plate. The absorbance value was measured at 565 nm to quantitatively detect the number of calcium nodules.

[0147] Analysis of results: Differentiation is an important stage for stem cells to form characteristic tissues. Early, mid- and late osteogenic differentiation markers need to be detected to evaluate the osteogenic differentiation ability of BMSCs. This study used Sirius red staining and Alizarin red staining to detect the effects of different sample extracts on the osteogenic differentiation of BMSCs. The results of Sirius red staining showed that compared with the control group, the collagen secretion of the experimental group increased, basically covering the entire well plate area. The quantitative results showed that the collagen secreted by Example 1 was about 3 times that of Example 3. At the same time, the results of the Alizarin red staining test showed that the control group and the experimental group promoted the formation of calcium nodules to a certain extent, and the calcium nodules formed in Example 1 were the most, basically covering more than 85% of the well plate area. Quantification of calcium nodules showed that the calcium nodules secreted by Example 1 were about 4 times that of Example 3. The possible reason is that strontium ions and magnesium ions regulate the polarization process of macrophages during the inflammatory period, causing macrophages to polarize from M1 (pro-inflammatory phenotype) to M2 (anti-inflammatory phenotype), secreting large amounts of IL-4, IL-10 and IL-1ra factors, thereby acting on osteoblasts, further upregulating the expression of genes such as Col-Ⅰ and OCN of osteoblasts, and promoting the osteogenic differentiation process.

[0148] 3. In vivo testing:

[0149] Repairing alveolar bone defects has always been a challenge in clinical treatment. This study used in vivo animal experiments to evaluate the bone-forming abilities of Comparative Example 3 (undoped strontium-magnesium coralline β-tricalcium phosphate) and Examples 1 and 2 (strontium-magnesium-doped coralline β-tricalcium phosphate).

[0150] The specific test plan is as follows:

[0151] Sixteen 8-week-old male SPF Sprague-Dawley rats were anesthetized with isopropyl fluoride. The rats were then secured to an operating table with their abdomen facing upward. The mandibles were pulled upward and posteriorly with appropriate instruments to fully expose the maxilla. The rats were randomly divided into groups: 4-SH1, 4-SH2, 4-SH3, and 4-SH4 in the 4-week control group, and 4-SH-Sr-Mg 1, 4-SH-Sr-Mg 2, 4-SH-Sr-Mg 3, and 4-SH-Sr-Mg 4 in the 8-week control group, and 8-SH1, 8-SH2, 8-SH3, and 8-SH4 in the experimental group.

[0152] 2) An 8 mm sagittal incision was made at the mesial alveolar ridge of the bilateral first molars, and blunt dissection was performed to expose the maxillary bone surface of the rat. After the bilateral first molars were extracted using vascular forceps, a 5 × 3 × 4 mm sac was prepared on the bilateral first molars and the mesial of the first molars using a high-speed drill with a 1 mm diameter ball drill. 3 During this process, 0.9% sterile saline was slowly and continuously injected into the bone defect area using a 50mL syringe to cool the heat generated during ball milling and flush out ball residue. At the same time, gauze was used to absorb excess liquid to prevent excessive liquid from interfering with the experimental operation. After the alveolar bone defect was prepared, the control group and experimental group materials were respectively filled into the defect site.

[0153] 3) Finally, the periosteum and mucosa were sutured in layers using 4-0 surgical sutures and a suture needle. The wound was cleaned and disinfected with iodine. After the rats were anesthetized, they were placed in their cages. After surgery, each rat received an intramuscular injection of 1 mL of 80,000 U of gentamicin sulfate into the buttocks to prevent infection. This injection was repeated regularly for three consecutive days, and the rats' condition was continuously observed.

[0154] 4) After 4 and 8 weeks, the osteogenic ability of the material was evaluated by Micro-CT imaging analysis and HE and Masson tissue section staining tests, respectively.

[0155] 3.1 Micro-CT imaging analysis

[0156] Four and eight weeks after alveolar bone defect repair surgery in rats, rats in each group were killed by intraperitoneal injection of an overdose of isopropyl fluoride. The maxilla and surrounding soft tissues were then carefully excised, and the specimens were fixed in 4% paraformaldehyde for 72 hours before micro-CT examination. Three-dimensional reconstruction was performed using the image processing software provided by the Micro-CT, and the microstructure of the new bone was analyzed. Bone density (BV / TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N) were calculated using the auxiliary software CTAn. Qualitative results showed that at four weeks, the alveolar ridge height of the experimental group was significantly greater than that of the control group. New bone formation was the most effective in both frontal and top views of the bone defect area, essentially covering 70±5% of the defect area. In contrast, the bone formation in Comparative Example 1 was approximately 60% of that in Example 1, with relatively little new bone formation. The bone formation in Example 2 was approximately 90% of that in Example 1, with less new bone formation. The bone formation in Example 3 was approximately 88% of that in Example 1, with less new bone formation. Furthermore, at four weeks, quantitative data showed that Example 1 exhibited 30% greater bone density, trabecular thickness, and trabecular number than Comparative Example 1, while trabecular separation was 20% less. Micro-CT test results further confirmed the superior osteogenesis of strontium-magnesium-doped coralline β-tricalcium phosphate. After eight weeks, the osteogenesis effect was further enhanced.

[0157] 3.2 Tissue section staining test:

[0158] To investigate the status of newly formed bone within the bone defect area during the repair process of the scaffolds in the control and experimental groups, HE and Masson staining were performed on the specimens. Prior to staining, the animal specimens were decalcified and sectioned. The specific steps are as follows:

[0159] (1) After Micro-CT, 8-week-old animals were selected and immersed in a 10% EDTA composite decalcification solution for decalcification.

[0160] (2) The decalcified specimens were dehydrated and transparentized.

[0161] (3) Embed the dehydrated and transparent specimens in paraffin.

[0162] (4) Fix the embedded specimen onto the slice holder for slicing.

[0163] (5) Dry the slices and store them at room temperature.

[0164] 3.2.1 HE staining:

[0165] Staining steps:

[0166] (1) Dewax with xylene for 25 min.

[0167] (2) The samples were treated with anhydrous ethanol, 95% ethanol, 80% ethanol, 60% ethanol, 40% ethanol, and deionized water washing procedures for rehydration.

[0168] (3) The specimen was stained with hematoxylin staining solution for 7 minutes, washed with deionized water for 5 minutes, differentiated with 1% hydrochloric acid ethanol for 2 seconds, and finally rinsed with deionized water for 10 minutes to make the sample blue.

[0169] (4) The specimens treated with hematoxylin staining solution were stained with eosin staining solution for 40 seconds, and then dehydrated. The concentration gradient of dehydration treatment was selected as follows: 80% ethanol, 95% ethanol, anhydrous ethanol, and finally transparentized with xylene for 10 minutes.

[0170] (5) Use neutral gum to seal the slices. After sealing, let the slices dry naturally at room temperature and fix them. Then observe and photograph them under a microscope.

[0171] At 8 weeks after surgery, new trabeculae were formed inside the bone defects in the control group, but the trabeculae were blocked by residual materials and no continuous new bone tissue was formed; while in the experimental group, more new trabeculae were generated inside the bone defects, and the new trabeculae were connected into continuous new bone tissue. The residual materials were all wrapped by the new bone tissue, which could be gradually degraded and replaced by new bone tissue in the subsequent bone repair process, eventually forming dense bone tissue.

[0172] 3.2.2 Masson staining:

[0173] Staining steps:

[0174] (1) Dewax with xylene for 25 min.

[0175] (2) The samples were treated with anhydrous ethanol, 95% ethanol, 80% ethanol, 60% ethanol, 40% ethanol, and deionized water washing procedures for rehydration.

[0176] (3) The specimens were stained with Weigert iron hematoxylin staining solution for 5 min, washed with deionized water for 2 min, differentiated with 1% hydrochloric acid ethanol for 2 s, and finally rinsed with deionized water for 5 min.

[0177] (4) The specimens after acid Pinceau staining were stained with eosin solution for 3 minutes, then treated with citric acid solution for 30 seconds, stained with Orange G for 6 minutes, then treated with citric acid solution for 30 seconds, and then stained with brilliant green for 5 minutes. Then, the specimens were rinsed with deionized water for 5 minutes and air-dried.

[0178] (5) The sections were dried naturally at room temperature and fixed, and then observed and photographed under a microscope.

[0179] The experimental results showed that 8 weeks after surgery, more discontinuous new bone tissue was formed inside the alveolar bone defect area of ​​the control group material; while continuous new bone tissue was formed in the bone defect area of ​​the experimental group material, and there were more osteoblasts around the new bone tissue.

[0180] The results of Masson and HE staining showed that the period of new bone formation in strontium- and magnesium-doped materials was earlier, the rate of new bone formation was faster, and they had better bone defect repair effects.

[0181] Test Example 4

[0182] Macrophages have strong plasticity, and changes in the microenvironment will prompt macrophages to transform between M1 and M2. In order to verify the effects of strontium ions and magnesium ions on macrophages RAW264.7, this test example put the materials of Comparative Example 3 (coral β-tricalcium phosphate not doped with strontium-magnesium) and Example 1 (coral β-tricalcium phosphate doped with strontium-magnesium) into complete culture medium and extracted them for 24 hours, and then used the extract to culture M1 macrophages. After 3 and 7 days of culture, this experiment used RT-qPCR technology to evaluate the polarization state of macrophages. The experimental results showed that compared with the control group, the expression of pro-inflammatory related genes TNF-α and IL6 in the experimental group were significantly downregulated at 3d and 7d, while the anti-inflammatory related genes Arginase and IL10 were significantly upregulated. It can be seen from this that strontium-magnesium-doped coral β-tricalcium phosphate can promote the polarization of M1 macrophages to M2 macrophages, and then regulate the directional differentiation of osteoblasts, which is beneficial for the repair of bone defects and the treatment of bone diseases such as osteoporosis.

[0183] Test Example 5

[0184] In order to verify that strontium-doped coral β-tricalcium phosphate can maintain the original porous structure of coral that is similar to human bone tissue, this test example will perform microscopic morphology analysis on Comparative Example 2 (strontium-magnesium-doped coral β-tricalcium phosphate etched with dilute hydrochloric acid) and Example 1 (strontium-magnesium-doped coral β-tricalcium phosphate). First, 2g of the two groups of material samples were placed on the SEM sample stage, affixed with conductive glue, and then sprayed with gold for 300s before electron microscopy. Subsequently, the samples were subjected to microscopic morphology analysis using a scanning electron microscope. The results showed that the strontium-magnesium-doped coral β-tricalcium phosphate in Example 1 retained the original structural characteristics of the coral, presenting a porous network with a pore size of 200-400μm and a porosity of 40%-60%. The physical structure, crystal arrangement, and bone density under the electron microscope were very similar to those of human bones. Dilute hydrochloric acid can undergo a relatively violent redox reaction with calcium carbonate, the main component of coral, resulting in the appearance of many corrosion holes on the surface of the strontium-magnesium coral β-tricalcium phosphate (Comparative Example 2) etched by dilute hydrochloric acid. Most of the pores are below 100 μm, which is not within the normal tissue pore size range and the pore size is enlarged.

[0185] Test Example 6

[0186] To verify the effect of pH on coral artificial bone, Comparative Example 4, Example 1 (strontium-magnesium-doped coral β-tricalcium phosphate at pH 10), and Example 2 (strontium-magnesium-doped coral β-tricalcium phosphate at pH 9) were selected as research materials. X-ray diffraction (XRD) and infrared absorption spectroscopy (IR) were used to observe and analyze the crystal phase and composition of the materials. The results revealed significant differences in the composition and crystallinity of Example 1. The crystals produced by the material in Example 1 exhibited superior crystallinity, with clear and sharp diffraction peaks, complete crystal growth, and a largely single phase. In contrast, the diffraction peaks of the sample in Comparative Example 1 weakened, the crystallinity deteriorated, and single crystals could not form. Compared to Example 1, the material in Example 2 failed to form a largely single-phase product, namely, an amorphous phosphate; and compared to Example 1, the material in Example 3 failed to form a largely single-phase product, namely, a non-static phosphate.

[0187] While the above description is in conjunction with the embodiments of the present invention, the present invention is not limited to the aforementioned embodiments. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A preparation process of modified hydroxyapatite, characterized in that, It includes the following steps: S1. After removing impurities from the coral with sodium hypochlorite solution, the coral is crushed to obtain coral particles; S2. The coral particles and phosphate solution are subjected to hydrothermal reaction at pH 9-11 to obtain coral hydroxyapatite; S3. The coral hydroxyapatite and ion source are mixed and reacted to obtain ion source-doped coral hydroxyapatite; S4. The ion source-doped coral hydroxyapatite is heated, sintered, and cooled to obtain ion source-doped coral β-tricalcium phosphate; The ion source includes a strontium source.

2. The preparation process of the modified hydroxyapatite according to claim 1, characterized in that, The ion source includes a magnesium source; preferably, the molar ratio of the strontium source to the magnesium source is 1:1-3.

3. The preparation process of the modified hydroxyapatite according to claim 1, characterized in that, Step S2 includes: The coral particles and phosphate solution are subjected to hydrothermal reaction at pH 10 to obtain coral hydroxyapatite.

4. The preparation process of the modified hydroxyapatite according to claim 1, characterized in that, In step S2, the temperature of the hydrothermal reaction is 160-180 °C, the time of the hydrothermal reaction is 20-24 h, and the pressure of the hydrothermal reaction is: 0.3-0.5 MPa.

5. The preparation process of the modified hydroxyapatite according to claim 1, characterized in that, In step S3, the temperature of the mixing reaction is 160-180 °C, the time of the mixing reaction is 20-24 h, and the pressure of the mixing reaction is: 0.3-0.5 MPa.

6. The preparation process of the modified hydroxyapatite according to claim 1, characterized in that, In step S4, the temperature of the sintering is 800-1000 °C, and the time of the sintering is 2-3 h.

7. The preparation process of the modified hydroxyapatite according to claim 1, characterized in that, The cooling rate is 6-8 °C / min.

8. The preparation process of the modified hydroxyapatite according to claim 1, characterized in that, The ion source further includes at least one of manganese ions, barium ions, copper ions, zinc ions, and borate ions.

9. A bone defect repair material, characterized in that, The preparation raw material includes the modified hydroxyapatite prepared by the preparation process according to any one of claims 1-8.

10. The bone defect repair material according to claim 9, characterized in that, The bone defect repair material includes an alveolar bone defect repair material.

Images