Injectable bone repair material based on hydroxyapatite microspheres and preparation method thereof

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to an injectable bone repair material based on hydroxyapatite microspheres and a preparation method of the injectable bone repair material. The preparation method of the injectable bone repair material comprises the following steps: taking polycaprolactone (PCL4000) and glycerol, carrying out gradient heating, then adding hydroxyapatite microspheres, and stirring to obtain a mixture A; cooling the mixture A, adding carboxymethyl cellulose and collagen, and stirring to obtain a mixture B; and injecting the mixture B into an injection device to obtain the injectable bone repair material based on the hydroxyapatite microspheres. The injectable bone repair material prepared by the invention adopts the raw materials with good biocompatibility, realizes the integration of multiple functions of hemostasis, bone repair, absorbability and injectability, and is beneficial to clinical bone defect repair treatment according to different types and parts.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and in particular relates to an injectable bone repair material based on hydroxyapatite microspheres and a preparation method thereof. Background Art

[0002] Repairing severe bone defects caused by trauma, tumor resection, infection, or degenerative disease in clinical practice is a major surgical challenge. Although bone tissue has the ability to heal itself, defects exceeding a critical size still require bone graft support. Current mainstream repair methods include autologous bone, allogeneic bone, and artificial bone material transplantation, but all have significant limitations: autologous bone transplantation faces the problems of limited donor sites, secondary surgical trauma, and difficulty in filling large bone defects; allogeneic bone carries the risk of immune rejection and disease transmission; and metal prostheses are prone to loosening and fracture and are non-degradable. For this reason, artificial bone materials based on inorganic bone components such as hydroxyapatite (HA) have become an important research direction. Type I collagen, as a core component of the bone matrix, has excellent biocompatibility, osteogenic inductivity, and low antigenicity. However, when used alone, it degrades too quickly and lacks mechanical strength, requiring compounding with inorganic materials such as HA or tricalcium phosphate. Studies have shown that HA / collagen composite scaffolds can mimic the natural bone subfiber nanostructure (average pore size of 142.2μm), and animal experiments have shown that they can promote new bone deposition and angiogenesis; HA microspheres can not only act as temporary scaffolds, but also load antibiotics to construct a local drug delivery system, synergistically controlling infection and accelerating bone regeneration.

[0003] In recent years, injectable bone repair materials have garnered significant attention due to their minimally invasive advantages. While ceramics (such as HA and calcium phosphates) match natural bone composition and exhibit osteoconductivity, bulk HA is prone to fragmentation and loosening. Single polymer gels are prone to voiding due to degradation and are unable to support large defects. The location and morphology of bone defects place high demands on bone repair materials. Consequently, ceramic / polymer composites with biomimetic properties suitable for diverse bone defect scenarios, combining the structural strength of the inorganic phase with the formability of the organic phase, have become an ideal option for addressing various bone defect repair bottlenecks.

[0004] There is an urgent need for an injectable bone repair material that has the properties of hemostasis, active osteogenesis, applicability to different bone defects and excellent biocompatibility. Summary of the Invention

[0005] In response to the above technical deficiencies, the present application provides an injectable bone repair material based on hydroxyapatite microspheres and a preparation method thereof, which realizes the multifunctional integration of hemostasis, bone repair, absorbability and injectability.

[0006] To achieve the above objectives, the present application provides an injectable bone repair material based on hydroxyapatite microspheres, which is composed of hydroxyapatite microspheres, a water-soluble material, a polymer material and a natural polymer material.

[0007] Furthermore, the water-soluble material is at least one of glycerol and PEG600.

[0008] Furthermore, the polymer material is at least one of polycaprolactone, a blend of polycaprolactone and glycerol, type I collagen, and type II collagen.

[0009] Furthermore, the natural polymer material is at least one of chitosan, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, and polyvinyl alcohol.

[0010] Furthermore, the content of the hydroxyapatite microspheres is 20-80 wt%.

[0011] Furthermore, the particle size of the hydroxyapatite microspheres is 1-200 μm.

[0012] Furthermore, the polymer material content is 5 wt%.

[0013] Furthermore, the natural polymer material is 12-20 wt%.

[0014] A method for preparing an injectable bone repair material based on hydroxyapatite microspheres comprises the following steps: S1. PCL4000 and glycerol are heated in a gradient manner, and then hydroxyapatite microspheres are added and stirred to obtain a mixture A; S3, cooling the mixture A, adding purified water, carboxymethyl cellulose and collagen, and stirring to obtain a mixture B; S4. Pour the mixture B into an injection device, freeze it, and thaw it to obtain an absorbable and injectable bone repair material based on hydroxyapatite microspheres.

[0015] In the above process, PCL4000 gradually disentangles during the heating process, which can reduce the local supersaturation precipitation caused by molecular chain aggregation, promote the formation of a dynamic hydrogen bond network with glycerol, ensure the formation of an amorphous blend with glycerol, and enhance the stability of the matrix; cooling and adding collagen can maintain its natural fiber structure and retain biological activity, and cooling and adding carboxymethyl cellulose can avoid the entanglement of molecular chains caused by excessive swelling at high temperature, ensuring the uniform dispersion of carboxymethyl cellulose; the surface porosity and crystallinity of hydroxyapatite microspheres of different particle sizes ensure the injectability of bone repair materials.

[0016] Furthermore, the particle size of the hydroxyapatite microspheres is preferably 20-80 μm.

[0017] Furthermore, the gradient temperature rise is 25-40°C at a heating rate of 3-5°C / min, and 40-60°C at a heating rate of 1-3°C / min.

[0018] In summary, this application has the following beneficial effects: This application describes an injectable bone repair material based on hydroxyapatite microspheres. These microspheres are highly similar in composition to natural bone, exhibiting no immune rejection after implantation and excellent biocompatibility. Their surface absorbs growth factors, such as bone morphogenetic proteins (BMPs), creating a three-dimensional scaffold structure that promotes osteoconduction, guiding the attachment and growth of new bone cells. They also exert osteoinduction, promoting the differentiation of mesenchymal stem cells into osteoblasts, thereby accelerating bone defect repair. The porous structure within the microspheres accelerates fluid permeation and ion exchange, allowing for slow dissolution. They also serve as a carrier for drugs (such as antibiotics and growth factors) for targeted therapy and controlled release, with functional transformation occurring simultaneously with degradation. The PCL component in the system imparts excellent plasticity and controlled degradability. The collagen molecules self-assemble into a highly ordered structure, synergizing with the carboxymethyl cellulose network to create a hierarchical porous scaffold, further enhancing the material's integration with bone tissue.

[0019] The present invention discloses an injectable bone repair material based on hydroxyapatite microspheres. The injectable bone repair material is a composite of drug-loaded hydroxyapatite microspheres and degradation-regulating polymers, achieving the multifunctional integration of hemostasis, bone repair, controllable degradation and injectability. This absorbable composite injectable bone repair material innovatively integrates the degradable material polycaprolactone (PCL) with natural polymer materials, and collaborates with hydroxyapatite microspheres to construct a triple repair system, which significantly improves the treatment effect of bone defects. The metabolites of PCL are non-toxic and have no risk of immune rejection. Collagen simulates the natural extracellular matrix. The material has a strong structure that can reduce antigenicity and is easily absorbed by the human body. It can also promote cell proliferation and differentiation and activate platelet aggregation to accelerate hemostasis. The metabolic products of hydroxyapatite microspheres are physiologically essential ions. The calcium ions and phosphate ions released after hydroxyapatite degradation can be directly utilized by the human body to participate in the new bone mineralization process. There is no toxic residue. The calcium and phosphorus concentrations in the blood are precisely regulated by parathyroid hormone (PTH) and other factors. Excess ions can be excreted through the kidneys, avoiding the risk of hypercalcemia, providing patients with a safe and affordable repair solution, and simultaneously solving the problems of biocompatibility, functional activity and clinical applicability. The injectable bone repair material prepared in this application uses highly biocompatible hydroxyapatite microspheres and polycaprolactone and carboxymethyl cellulose as the main raw materials for bone defect repair, promoting bone tissue regeneration, reducing pain and discomfort in patients, and accelerating the repair of normal tissue. It is also injectable and applicable to a variety of bone defect scenarios, which is beneficial for clinical bone defect repair treatment according to different types and locations. DETAILED DESCRIPTION

[0020] The following will be combined with the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the embodiments described are only 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 efforts are within the scope of protection of the present invention.

[0021] The raw materials involved in the specific implementation of this application, among which hydroxyapatite microspheres are selected from Kunshan Overseas Chinese Technology New Materials Co., Ltd., specification HQ-HA-Q3; collagen is derived from type I collagen of bovine Achilles tendon, with a molecular weight of 200kDa; carboxymethyl cellulose has a molecular weight of 30kDa and a degree of substitution of 0.8.

[0022] Example 1: A method for preparing an injectable bone repair material based on hydroxyapatite microspheres, comprising the following steps: S1. Mix 0.5 g of PCL4000, purified water, and 6.5 g of glycerol in a mass ratio of PCL4000 to glycerol of 1:2. Then, heat the mixture gradually (25-40°C at a heating rate of 4°C / min, 40-60°C at a heating rate of 2°C / min). Then, add hydroxyapatite microspheres with a particle size of 5 μm and stir at a speed of 500 rpm for 1 h to obtain mixture A. S2. Cooling mixture A to 40°C, adding 1 g of carboxymethyl cellulose and 0.5 g of type I collagen, and stirring (at a speed of 300 rpm for 1 h) to obtain mixture B; S3. Pour the mixture B into an injection device to obtain an injectable bone repair material based on hydroxyapatite microspheres.

[0023] Example 2: A method for preparing an injectable bone repair material based on hydroxyapatite microspheres, comprising the following steps: S1. Mix 0.5 g of PCL4000, purified water, and glycerol (a total of 6.5 g) at a PCL4000 to glycerol ratio of 1:6. The mixture was then heated at a gradient rate (25-40°C at a heating rate of 4°C / min and 40-60°C at a heating rate of 2°C / min). Hydroxyapatite microspheres with a particle size of 20 μm were then added and stirred at 500 rpm for 1 h to obtain mixture A. S2. Cooling mixture A to 40°C, adding 1 g of carboxymethyl cellulose and 0.5 g of type I collagen, and stirring (at a speed of 300 rpm for 1 h) to obtain mixture B; S4. Pour the mixture B into an injection device to obtain an injectable bone repair material based on hydroxyapatite microspheres.

[0024] Example 3: A method for preparing an absorbable and injectable bone repair material based on hydroxyapatite microspheres, comprising the following steps: S1. Mix 0.5 g of PCL4000, purified water, and glycerol (a total of 6.5 g) at a PCL4000 to glycerol ratio of 1:12. Then, heat the mixture gradually (25-40°C at a heating rate of 4°C / min, 40-60°C at a heating rate of 2°C / min). Then, add hydroxyapatite microspheres with a particle size of 20 μm and stir at a speed of 500 rpm for 1 h to obtain mixture A. S3. Cooling mixture A to 40° C., adding 1 g of carboxymethyl cellulose and 0.5 g of type I collagen, and stirring (at a speed of 300 rpm for 1 h) to obtain mixture B; S4. Pour the mixture B into an injection device to obtain an injectable bone repair material based on hydroxyapatite microspheres.

[0025] Control Example 1: This control example differs from Example 1 in that carboxymethyl cellulose is used instead of type I collagen.

[0026] Comparative Example 2: The difference between this comparative example and Example 2 is that the preparation method of an injectable bone repair material based on hydroxyapatite microspheres in this comparative example specifically includes the following steps: S1. Mix 0.5 g of PCL4000, purified water, and glycerol (a total of 6.5 g) at a PCL4000 to glycerol ratio of 1:6. Then, add hydroxyapatite microspheres with a particle size of 20 μm and stir (500 rpm for 1 h) to obtain mixture A. S2. Cooling mixture A to 40°C, adding 1 g of carboxymethyl cellulose and 0.5 g of type I collagen, and stirring (at a speed of 300 rpm for 1 h) to obtain mixture B; S4. Pour the mixture B into an injection device to obtain an injectable bone repair material based on hydroxyapatite microspheres.

[0027] Comparative Example 3: The difference between this comparative example and Example 2 is that the preparation method of an injectable bone repair material based on hydroxyapatite microspheres in this comparative example specifically includes the following steps: S1. Mix 0.5 g of PCL4000, purified water, and glycerol (a total of 6.5 g) at a PCL4000 to glycerol ratio of 1:12. Then, heat the mixture gradually (25-40°C at a heating rate of 4°C / min, 40-60°C at a heating rate of 2°C / min). Then, add hydroxyapatite microspheres with a particle size of 80 μm and stir at a speed of 500 rpm for 1 h to obtain mixture A. S3. Cooling mixture A to 40° C., adding 1 g of carboxymethyl cellulose and 0.5 g of type I collagen, and stirring (at a speed of 300 rpm for 1 h) to obtain mixture B; S4. Pour the mixture B into an injection device to obtain an injectable bone repair material based on hydroxyapatite microspheres.

[0028] Comparative Example 4: A method for preparing an absorbable and injectable bone repair material based on hydroxyapatite microspheres, comprising the following steps: S1. Mix 6.5 g of PCL4000, purified water, and glycerol in a 1:1 mass ratio. Then, heat the mixture gradually (25-40°C at a heating rate of 4°C / min, 40-60°C at a heating rate of 2°C / min). Then, add 20 μm hydroxyapatite microspheres and stir at 500 rpm for 1 h to obtain mixture A. S3. Cooling mixture A to 40° C., adding 1 g of carboxymethyl cellulose and 0.5 g of type I collagen, and stirring (at a speed of 300 rpm for 1 h) to obtain mixture B; S4. Pour mixture B into the injection device to obtain an injectable bone repair material based on hydroxyapatite microspheres.

[0029] Performance test: Physical property test and in vitro degradation test were performed on the injectable bone repair material samples prepared in Examples 1-3 and Comparative Examples 1-4: Smearability test: Spread the sample evenly on the sample to see if there are any obstacles in spreading and verify the ease of use. The spreadability is divided into three levels: Level 1: no resistance when spread and uniform film formation; Level 2: can be spread with light pressure and basic coverage; Level 3: difficult to spread and uneven coverage.

[0030] Adhesion test: A universal material testing machine was used to measure the bond strength (mPa) of the injectable bone repair material. A sample of the injectable bone repair material (80 × 10 × 2 mm) was overlapped with a cortical bone block (10 mm × 20 mm × 50 mm). A tensile force was applied unilaterally at a rate of 2 mm / min until the sample separated. The peak stress before failure was recorded.

[0031] Anti-washing test: The injectable bone repair materials of the above-mentioned embodiments and control examples were respectively made into 10×10×10 mm cubes, and washed with a large amount of flowing physiological saline to observe whether the injectable bone repair materials were loose and fell off.

[0032] Degradation rate test: Refer to the in vitro degradation test method described in YY / T1806.1-2021 for testing, and set the experimental time to 6 weeks. The test results are shown in Table 1: Table 1

[0033] It can be seen from Table 1 that the spreadability, adhesion, injectability, impact resistance and degradation rate of Examples 1-3 are relatively good, especially the injectable bone repair material prepared in Example 2 has the highest degradation rate; Compared with Example 1, Control Example 1 uses carboxymethyl cellulose instead of type I collagen. From the test results, it can be seen that the adhesion and degradation rate of the injectable bone repair material prepared in Control Example 1 are lower than those in Example 1, and the effect is not as good as that in Example 1, indicating that there is a synergistic effect between the materials in the examples of the present application; Compared with Example 2, the preparation conditions of Control Example 2 are changed, and the gradient heating step is missing. The experimental results show that the injectable bone repair material prepared in Control Example 2 has poor spreadability, poor adhesion, poor impact resistance, and unsatisfactory degradation rate. The test effect is not as good as that in Example 2, indicating that the preparation method of the injectable bone repair material in the examples of the present application can improve the effect of the injectable bone repair material; Compared with Example 2, the size of the hydroxyapatite microspheres in Control Example 3 is increased, and the test results are It shows that the injectable bone repair material prepared in Control Example 3 is not as good as Example 2 in terms of spreadability, adhesion, plasticity, impact resistance and degradation rate, indicating that the preparation method of the injectable bone repair material in the embodiment of the present application can improve the effect of the injectable bone repair material; the content of hydroxyapatite microspheres in Control Example 4 is increased compared with Example 3. The test results show that the degradation rate of the injectable bone repair material prepared in Control Example 4 is not as good as that in Example 3, and the spreadability, adhesion and plasticity are not good. The injectable bone repair material prepared in Control Example 4 is not as effective as that in Example 3, indicating that the dosage range of each material in the embodiment of the present application is the optimal dosage range; the degradation rate of the embodiment of the present application is high within 6 weeks, but it is not completely degraded, indicating that during bone regeneration, the injectable bone repair material prepared in the present application can be slowly degraded, and will not be unable to cover the entire repair process due to excessive degradation, or will not be unable to release bone repair-promoting substances due to excessive degradation, thereby achieving controllable degradation.

[0034] Cytotoxicity test: In the leaching test, prepare the sample extract (24 h). 100 μl per well, density 5×10 3 A 100 μl / μl osteoblast suspension was added to a 96-well plate and incubated at 37°C for 24 hours. The culture medium was then replaced with 100 μl of the sample extract and incubated for 24 and 48 hours, respectively. CCK-8 solution was then added to each well for 2 hours. Finally, the OD value at 450 nm was measured using a microplate reader to determine cell viability. The results are shown in Table 2. Table 2

[0035] As can be seen from Table 2, the cell survival rates of the examples of the present application and the control examples are relatively high, and both the examples of the present application and the control examples have good biocompatibility.

[0036] In vivo animal experiments were conducted to determine hemostatic and bone repair properties: A 10 mm diameter circular defect was created in the rabbit iliac bone using a drill. The injectable bone repair materials prepared in Examples 1-3 and Comparative Examples 1-4 were applied to the defect wound. The effectiveness of hemostasis was recorded, and the hemostasis time of the injectable bone repair materials was measured. The results are shown in Table 3. Table 3

[0037] It can be seen from Table 3 that the hemostasis time of the examples of the present application is better than that of the control example, especially Example 2 has the fastest hemostasis speed and the shortest hemostasis time.

[0038] Bone repair performance: The injectable bone repair materials prepared in Examples 1-3 and Control Examples 1-4 were sterilized and then placed in 48-well culture plates. 20 μL of the injectable bone repair materials were seeded on each well at a density of 5×10 6 / mL mesenchymal stem cell suspension was placed in a 37°C, 5% CO2 incubator for two hours, and then 500μL of culture medium was added and cultured for 3 days, 7 days, and 14 days respectively. The culture medium was changed every 3 days. The absorbance at 405nm was measured using a microplate reader, and the alkaline phosphatase activity (U / mg) was calculated. The average value of 3 measurements was taken for each group. The results are shown in Table 4: Table 4

[0039] It can be seen from Table 4 that the alkaline phosphatase activity of cells in the injectable bone repair materials prepared in the examples gradually increased, especially the alkaline phosphatase activity in Example 2 was the highest, indicating that the injectable bone repair materials prepared in the examples of the present application are beneficial to the differentiation of osteoblasts and promote bone repair.

[0040] In summary, the injectable bone repair materials prepared in the embodiments of the present application have excellent effects, especially the injectable bone repair material prepared in Example 2 of the present application has the best effect, and Example 2 is the best embodiment of the present application.

[0041] The above content is merely an example and explanation of the concept of the present application. Technicians in this technical field may make various modifications or additions to the described specific embodiments or replace them in a similar manner. As long as they do not deviate from the concept of the invention or exceed the scope defined by the claims, they should all fall within the scope of protection of the present application.

Claims

1. An injectable bone repair material based on hydroxyapatite microspheres, characterized in that: The injectable bone repair material consists of hydroxyapatite microspheres, water-soluble materials, polymer materials and natural high molecular materials.

2. The injectable bone repair material based on hydroxyapatite microspheres according to claim 1, characterized in that: The water-soluble material is at least one of glycerol and PEG600.

3. The injectable bone repair material based on hydroxyapatite microspheres according to claim 1, characterized in that: The solvent is one or more of pure water, water for injection, saline solution, buffer solution or at least one of hexafluoroisopropanol, chloroform, dimethylformamide, tetrahydrofuran, chloroform or acetone.

4. The injectable bone repair material based on hydroxyapatite microspheres according to claim 1, characterized in that: The polymer material is at least one of polycaprolactone, a blend of polycaprolactone and glycerol, type I collagen, and type II collagen.

5. The injectable bone repair material based on hydroxyapatite microspheres according to claim 1, characterized in that: The natural polymer material is at least one of chitosan, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose and polyvinyl alcohol.

6. The injectable bone repair material based on hydroxyapatite microspheres according to claim 1, characterized in that: The content of the hydroxyapatite microspheres is 20-80 wt %.

7. The injectable bone repair material based on hydroxyapatite microspheres according to claim 1, characterized in that: The particle size of the hydroxyapatite microspheres is 30-200 μm.

8. The injectable bone repair material based on hydroxyapatite microspheres according to claim 1, characterized in that: The polymer material content is 5 wt %.

9. A method for preparing an injectable bone repair material based on hydroxyapatite microspheres according to any one of claims 1 to 8, characterized in that: The following steps are involved: S1. PCL4000 and glycerol are heated in a gradient manner, and then hydroxyapatite microspheres are added and stirred to obtain a mixture A; S3, cooling the mixture A, adding carboxymethyl cellulose and collagen, and stirring to obtain a mixture B; S4. Pour the mixture B into an injection device to obtain an injectable bone repair material based on hydroxyapatite microspheres.

10. The method for preparing an injectable bone repair material based on hydroxyapatite microspheres according to claim 9, characterized in that: The particle size of the hydroxyapatite microspheres is 30-200 μm; the gradient heating is performed at a heating rate of 3-5° C. / min from 25° C. to 40° C. and a heating rate of 1-3° C. / min from 40° C. to 60° C.

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