Drug-loaded microsphere-modified porous calcium phosphate cement and its preparation method and repair material

Abstract

This invention relates to the field of bone repair technology, specifically to drug-loaded microsphere-modified porous calcium phosphate bone cement, its preparation method, and repair material. The solid phase of the porous calcium phosphate bone cement consists of a porous calcium phosphate bone cement matrix and drug-loaded microspheres dispersed therein. The porous calcium phosphate bone cement matrix has a three-dimensional interconnected pore structure, and the drug-loaded microspheres have a core-shell structure, with the outer shell being a biodegradable material and the core being a osteoinducing factor. This porous calcium phosphate bone cement utilizes porous CPC to provide space for bone integration, and the drug-loaded microspheres achieve long-term controlled release of the osteoinducing factor, achieving efficient bone defect repair through an integrated "bone conduction-bone induction-bone integration" process.

Description

Technical Field

[0001] This invention relates to the field of bone repair technology, and more specifically, to porous calcium phosphate bone cement modified with drug-loaded microspheres, its preparation method, and repair materials. Background Technology

[0002] Calcium phosphate cement (CPC) has become a commonly used material for filling and supporting bone defects in clinical practice due to its similar chemical composition to human bone tissue (mainly composed of calcium phosphate salts such as hydroxyapatite), good biocompatibility and biodegradability, and its ability to solidify and conform to the shape of the bone defect site through a hydration reaction. However, CPC itself only possesses "bone guiding" properties (guiding surrounding bone tissue to grow into the material) and lacks "bone induction" properties (actively inducing mesenchymal stem cells to differentiate into osteoblasts and promoting new bone formation). For complex bone defects with poor blood supply and large defect areas, CPC alone is often insufficient to achieve effective bone tissue regeneration and functional reconstruction, and it is necessary to combine it with osteogenic inducing factors to improve the repair effect.

[0003] Currently available osteoinductive CPC bone cements incorporate BMP-2 directly. After implantation, BMP-2 readily diffuses and dissolves through the cement pores, resulting in an initially high release (potentially exceeding physiological needs) followed by a rapid decline in effective concentration and a short release cycle. Furthermore, the initial high-concentration BMP-2 release may trigger local inflammatory responses and excessive bone proliferation.

[0004] The existing patent CN109771693A describes a composite artificial bone that requires sodium hydroxypolyphosphate, carbodiimide-modified CPC, and the addition of collagen, nano-bacterial cellulose, and PLGA / rhBMP-2 / PAV microspheres. It relies on the synergistic effect of multiple components to enhance performance. However, this composite artificial bone has a complex composition and is prone to clogging pores due to issues with the amount of additives used, thus affecting bone tissue ingrowth.

[0005] Existing calcium phosphate cement (CPC)-based composite bone repair materials have significant shortcomings: pure CPC has poor mechanical and degradation compatibility, poor osteoinduction, and its dense structure makes it difficult for bone tissue to ingrow into it; direct addition of osteoinducing factors such as BMP-2 is prone to burst release and inactivation, and carrier loading often encounters problems of poor compatibility and uncontrolled release; the composite system relies on a variety of CPC modifiers and exogenous organic additives, resulting in complex composition and complicated processes, and is also prone to clogging of pores due to additive issues.

[0006] In view of this, the present invention is proposed. Summary of the Invention

[0007] The purpose of this invention is to provide drug-loaded microsphere-modified porous calcium phosphate bone cement, its preparation method, and repair material. Embodiments of this invention provide a novel porous calcium phosphate bone cement that can simultaneously improve or solve the aforementioned problems. The porous calcium phosphate bone cement utilizes porous CPC to provide space for bone integration, and drug-loaded microspheres enable long-term controlled release of osteoinducing factors, achieving highly efficient bone defect repair through an integrated process of "osteoconduction-osteoinduction-osteointegration."

[0008] This invention is implemented as follows: In a first aspect, the present invention provides a porous calcium phosphate bone cement modified with drug-loaded microspheres, the raw material of which includes a solid phase component; the solid phase component is composed of a porous calcium phosphate bone cement matrix and drug-loaded microspheres dispersed therein; The porous calcium phosphate bone cement matrix has a three-dimensional interconnected pore structure. The drug-loaded microspheres have a core-shell structure, with the outer shell made of biodegradable material and the core being a bone-inducing factor.

[0009] In an optional embodiment, the drug-loaded microspheres satisfy at least one of the following conditions: (1) The biodegradable material includes at least one of polycaprolactone, polylactic acid, and polylactic acid-glycolic acid copolymer; (2) The bone-inducing factor includes at least one of bone morphogenetic protein, osteogenic growth peptide and transforming growth factor-β; (3) The amount of bone-inducing factor added is 1% to 20% of the mass of the biodegradable material.

[0010] In an optional embodiment, the raw materials forming the porous calcium phosphate bone cement further include liquid phase components; Preferably, the mass ratio of the solid phase component to the liquid phase component is (1.5:1) to (3:1). Preferably, the liquid phase component includes any one of sodium hydrogen phosphate solution, citric acid solution, and deionized water.

[0011] In an optional embodiment, the interconnected porosity of the porous calcium phosphate bone cement matrix is ​​not less than 40%, the total porosity of the porous calcium phosphate bone cement matrix is ​​not less than 70%, and the average pore size of the porous calcium phosphate bone cement matrix is ​​10~300μm. Preferably, the median particle size of the drug-loaded microspheres is 5~500 μm; Preferably, the mass of the drug-loaded microspheres accounts for 5% to 30% of the total mass of the solid phase components.

[0012] In an optional embodiment, the porous calcium phosphate bone cement meets at least one of the following requirements: (1) The porous calcium phosphate bone cement releases bone-inducing factors in phosphate buffer solution at pH 7.4 in vitro. The burst release of bone-inducing factors within 24 hours does not exceed 35% of the total load, and can continue to be released slowly over the next 6 to 8 weeks. (2) The compressive strength of the porous calcium phosphate bone cement is in the range of 2~20MPa.

[0013] Secondly, the present invention provides a method for preparing porous calcium phosphate bone cement modified with drug-loaded microspheres as described in the foregoing embodiments, comprising: solidifying solid phase components.

[0014] In an optional embodiment, the mixture further includes: curing the solid phase component and the liquid phase component after mixing; Preferably, the step of forming the solid phase component includes: physically blending porous CPC powder, which forms a porous calcium phosphate bone cement matrix, with drug-loaded microspheres.

[0015] In an optional embodiment, the step of forming the porous CPC powder includes: mixing a solid substrate and a coagulation accelerator and then freeze-drying the mixture; Preferably, the step of forming the porous CPC powder includes: mixing a solid substrate and a solution containing a coagulation promoter, then freezing at -75~-85°C for at least 30 minutes, followed by freeze-drying for 1~2 days, and then lightly crushing. Preferably, the steps for forming the drug-loaded microspheres include: mixing a biodegradable material, an organic solvent, and a bone-inducing factor to form an oil phase; mixing the oil phase with a polyvinyl alcohol solution to form an oil-in-water emulsion; then removing the solvent, followed by solidification, collection, washing, and drying.

[0016] In an optional embodiment, the conditions for forming the porous CPC powder satisfy at least one of the following requirements: (1) The solid substrate comprises one or more of α-tricalcium phosphate, β-tricalcium phosphate, tetracalcium phosphate and anhydrous calcium hydrogen phosphate; (2) The coagulation accelerator includes sodium hydrogen phosphate solution; (3) The molar concentration of the solution containing the coagulation promoter is 1~1.5 mol / L; (4) The mass-to-volume ratio of the solid substrate to the solution containing the coagulation accelerator is 1.4~2.5 g / ml; Preferably, the conditions for forming the drug-loaded microspheres satisfy at least one of the following requirements: (1) The mass-to-volume ratio of the biodegradable material to the organic solvent is 0.025~0.05 g / ml; (2) The organic solvent includes at least one of amide solvents, halogen-substituted C1-C3 alkanes and C3-C5 ketone solvents; (3) The mass-volume concentration of the polyvinyl alcohol solution is 0.002~0.02 g / ml; (4) The oil phase accounts for 10% to 20% of the volume of the polyvinyl alcohol solution.

[0017] Thirdly, the present invention provides a repair material for filling and supporting bone defects, which is prepared by porous calcium phosphate bone cement modified with drug-loaded microspheres as described in the foregoing embodiments.

[0018] The present invention has the following beneficial effects: (1) The solid phase composition of the porous calcium phosphate bone cement provided by the present invention includes only the porous calcium phosphate bone cement matrix and drug-loaded microspheres containing osteoinducing factors, without other CPC modifiers and exogenous organic additives, which simplifies the composition of porous calcium phosphate bone cement and avoids the problem of pore blockage caused by additives.

[0019] (2) The porous calcium phosphate bone cement matrix provided in the embodiments of the present invention provides an initial integration space for bone tissue ingrowth and promotes bone growth; when the drug-loaded microspheres degrade, they can further improve the pore structure of the calcium phosphate bone cement, form more interconnected pores, provide osteoblast migration, improve osteogenic performance, and accelerate bone healing; thus, it realizes efficient bone defect repair integrating "bone conduction-bone induction-bone integration".

[0020] (3) The porous calcium phosphate bone cement provided in this embodiment of the invention can continuously and slowly release bone-inducing factors through drug-loaded microspheres, avoiding the defects of easy inactivation and initial burst release of factors when added directly in the prior art, effectively promoting osteogenic differentiation and bone repair process, and improving the overall efficiency of bone defect repair. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The graphs show the release curves of bone-inducing factors in vitro in phosphate buffer for the porous calcium phosphate bone cement modified with drug-loaded microspheres provided in Examples 1-3 of this invention. Figure 2 The bar chart shows the compressive strength test results of the drug-loaded microsphere-modified porous calcium phosphate bone cement provided in Examples 1-3 of this invention. Figure 3 This is a diagram showing the cell adhesion results of different groups of samples cultured for 1 day, as provided in the experimental examples of this invention. Figure 4This is a diagram showing the cell adhesion results of different groups of samples cultured for 7 days, as provided in the experimental examples of this invention. Figure 5 The graph shows the alkaline phosphatase (ALP) activity results of different groups of samples cultured for 14 days, which are provided as experimental examples of the present invention. Figure 6 The results of Runx2 gene expression in different groups of samples provided for the experimental examples of this invention are shown in the figure. Figure 7 The results of OPN gene expression in different groups of samples provided in the experimental examples of this invention are shown in the figure. Figure 8 The figure shows the results of OCN gene expression in different groups of samples provided in the experimental examples of this invention. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0024] Analysis of existing calcium phosphate bone cements reveals the following problems: (1) insufficient space for bone integration in traditional dense CPCs, making it difficult to accommodate bone tissue ingrowth; (2) easy burst release and inactivation of bone-inducing factors directly mixed into CPCs; and (3) reliance on multiple CPC modifiers and organic additives leading to system complexity and pore blockage. Based on these findings, this invention provides a drug-loaded microsphere-modified porous calcium phosphate bone cement that simultaneously addresses the above three problems while achieving the overall goal of improving bone defect repair efficiency.

[0025] Specifically, the raw material of the drug-loaded microsphere-modified porous calcium phosphate bone cement includes a solid phase component; the solid phase component consists of a porous calcium phosphate bone cement matrix and drug-loaded microspheres dispersed therein.

[0026] As can be seen, the porous calcium phosphate bone cement modified with drug-loaded microspheres provided in this embodiment of the invention achieves efficient bone defect repair by: providing a three-dimensional interconnected bone integration space (osteoconduction) through the porous calcium phosphate bone cement matrix; providing long-term controlled release (osteoinduction) through drug-loaded microspheres containing osteoinducing factors, while avoiding initial burst release; and further optimizing the pore structure during the degradation process of the drug-loaded microspheres to promote osteoblast migration and bone tissue ingrowth.

[0027] The porous calcium phosphate bone cement matrix has a three-dimensional interconnected pore structure with a connected porosity of not less than 40%, such as 40%, 45%, 50%, 55%, 60%, etc., preferably any value between 40% and 60%. Its total porosity is not less than 70%, such as 70%, 75%, 80%, 85%, etc., preferably any value between 70% and 80%. Its average pore size is 10~300μm; for example, 10μm, 50μm, 100μm, 150μm, 200μm, 250μm, 300μm, or any value between 10 and 300μm.

[0028] It should be noted that: interconnected porosity refers to the percentage of the volume of pores in bone cement that are interconnected and communicate with the external surface of the material, relative to the total volume of the material (including pores and solid matrix).

[0029] Furthermore, the median particle size of the drug-loaded microspheres is 5~500μm, for example, 5μm, 50μm, 100μm, 150μm, 200μm, 300μm, 400μm, 500μm or any value between 5 and 500μm.

[0030] The drug-loaded microspheres comprise 5% to 30% of the total mass of the solid phase components, for example, any value between 5%, 10%, 15%, 20%, 25%, 30%, or 5% to 30%. The drug-loaded microspheres have a core-shell structure. The outer shell is formed from a biodegradable material, including, but not limited to, at least one of polycaprolactone, polylactic acid, and polylactic-glycolic acid copolymer. The core is a bone-inducing factor, including, but not limited to, at least one of bone morphogenetic proteins, osteogenic growth peptides, and transforming growth factor-β.

[0031] Furthermore, the raw materials forming the porous calcium phosphate bone cement also include a liquid phase component; the liquid phase component includes any one of sodium hydrogen phosphate solution, citric acid solution, and deionized water. Specifically, the mass ratio of the solid phase component to the liquid phase component is (1.5:1) to (3:1); for example, any value between (1.5:1) and (3:1), such as 1.5:1, 2:1, 2.5:1, 3:1, etc.

[0032] As described above, this invention achieves highly efficient bone defect repair through an integrated approach of "bone conduction-bone induction-bone integration" by using a porous calcium phosphate bone cement matrix combined with improved drug-loaded microspheres. Common sense dictates that increasing porosity or microsphere content often leads to decreased compressive strength and exacerbated drug burst release (due to more and more permeable diffusion channels), and the combination of both exacerbates these issues. In other words, conventionally, this invention employs a connected porosity of not less than 40%, a total porosity of not less than 70%, and drug-loaded microspheres comprising 5% to 30% of the total mass of the solid phase components. This means that the compressive strength of the porous calcium phosphate bone cement formed by combining the porous calcium phosphate bone cement matrix with improved drug-loaded microspheres should be reduced, and drug burst release should be significant. However, the porous calcium phosphate bone cement provided in this embodiment has a compressive strength range of 2 to 20 MPa, which meets the needs of various bone defect filling and support methods. Meanwhile, it releases bone-inducing factors in phosphate buffer solution at pH 7.4 in vitro. The burst release of bone-inducing factors within 24 hours does not exceed 35% of the total load, and it can continue to be released slowly over the following 6 to 8 weeks. This indicates that there is no obvious burst release, which shows that the porous calcium phosphate bone cement provided by the embodiments of the present invention has excellent compressive strength, and the drug can be released slowly rather than burst, which shows that the present invention has unexpected technical effects.

[0033] Secondly, the present invention provides a method for preparing the drug-loaded microsphere-modified porous calcium phosphate bone cement described in the foregoing embodiments, comprising: S1. Preparation of drug-loaded microspheres; A biodegradable material, an organic solvent, and a bone-inducing factor are mixed to form an oil phase. This oil phase is then mixed with a polyvinyl alcohol solution to form an oil-in-water emulsion. The solvent is removed, followed by solidification, collection, washing, and drying. Specifically, the biodegradable material is dissolved in an organic solvent at a mass-to-volume ratio of 0.025–0.05 g / ml to form a homogeneous solution. The bone-inducing factor is then added to this solution, and the mixture is magnetically stirred until homogeneous, forming the oil phase. The oil phase is added dropwise at a volume ratio of 10%–20% to a polyvinyl alcohol aqueous solution at a mass-to-volume ratio of 0.002–0.02 g / ml. The mixture is vortexed for at least 1 minute to form an oil-in-water emulsion (O / W). The organic solvent is then evaporated by magnetic stirring at 30–40°C. After solidification, the mixture is collected, washed, and dried to obtain drug-loaded microspheres. The amount of bone-inducing factor added is 1%–20% of the biodegradable material.

[0034] The organic solvent includes at least one of amide solvents, halogen-substituted C1-C3 alkanes, and C3-C5 ketone solvents; for example, including but not limited to one or more of dichloromethane, acetone, or N,N-dimethylformamide.

[0035] S2. Prepare porous CPC powder; The solid substrate and coagulation accelerator are mixed and then freeze-dried. Specifically, the solid substrate and the solution containing the coagulation accelerator are mixed and stirred at 37°C for no more than 8 minutes, then frozen at -80°C for no less than 30 minutes, followed by freeze-drying for 1 to 2 days, and then lightly crushed to obtain porous CPC powder.

[0036] The specific conditions for freeze drying are as follows: vacuum degree of 20 Pa, cold trap temperature of -40℃; pre-freeze at -40℃ for 3 hours, then slowly raise to 0℃ for a first drying of 6~12 hours, and finally slowly raise to 37℃ for a second drying of 15~33 hours.

[0037] The solid substrate comprises one or more of α-tricalcium phosphate, β-tricalcium phosphate, tetracalcium phosphate, and anhydrous calcium hydrogen phosphate; the setting accelerator comprises sodium hydrogen phosphate; the molar concentration of the solution containing the setting accelerator is 1~1.5 mol / L; and the mass-to-volume ratio of the solid substrate to the solution containing the setting accelerator is 1.4~2.5 g / ml. These conditions are more conducive to the formation of porous calcium phosphate bone cement matrix.

[0038] S3. Preparation of solid phase components; Porous CPC powder and drug-loaded microspheres are physically blended to form a solid phase.

[0039] S4. Preparation of porous calcium phosphate bone cement modified with drug-loaded microspheres; The solid and liquid components are mixed and then cured. Specifically, the solid and liquid components are mixed at a mass ratio of 1.5:1 to 3:1 to obtain an injectable slurry, which cures within 10 to 30 minutes. At this point, the porous CPC powder composite forms a large porous calcium phosphate bone cement matrix with a three-dimensional interconnected pore structure.

[0040] The drug-loaded microsphere-modified porous calcium phosphate bone cement provided in this invention can be used for filling and supporting bone defects. Therefore, this invention also provides a repair material for filling and supporting bone defects, which is prepared by drug-loaded microsphere-modified porous calcium phosphate bone cement.

[0041] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0042] Example 1 This embodiment provides a method for preparing drug-loaded microsphere-modified porous calcium phosphate bone cement, including: S1. Preparation of drug-loaded microspheres; Polylactic acid-glycolic acid copolymer (PLGA) was dissolved in dichloromethane at a mass-to-volume ratio of 0.025 g / ml to form a homogeneous solution. Bone morphogenetic protein (BMP-2, at a mass of 5% of PLGA) was added to this solution and magnetically stirred until homogeneous, forming the oil phase. The oil phase was then slowly added dropwise to a polyvinyl alcohol (PVA) aqueous solution at a mass-to-volume ratio of 10% (v / v) and vortexed for 1 min to form an oil-in-water emulsion (O / W). Subsequently, the organic solvent was evaporated by magnetic stirring at 30°C. After solidification, the emulsion was collected, washed, and dried to obtain PLGA drug-loaded microspheres.

[0043] SEM particle size statistics (n≥300) show that: D50≈5μm, D10 / D90≈3~8μm.

[0044] S2, Preparation of porous CPC powder; α-Tricalcium phosphate (α-TCP) and sodium hydrogen phosphate (Na2HPO4) solution (molar concentration 1.5 mol / L) were mixed at 1.4 g / ml (substrate mass / solution volume) and stirred for 4 min at 37 °C; subsequently... Freeze at 80℃ for 30 min, freeze-dry for 1 day. The freeze-drying conditions are as follows: vacuum degree of 20 Pa, cold trap temperature of -40℃; first, pre-freeze at -40℃ for 3 h, then slowly rise to 0℃ for a first drying of 6 h, and finally slowly rise to 37℃ for a second drying of 15 h; the freeze-dried block is lightly crushed to obtain porous calcium phosphate bone cement (CPC) powder, i.e., porous CPC powder.

[0045] S3, Preparation of composite bone cement; The porous CPC powder obtained in step (2) was blended with the drug-loaded microspheres obtained in step (1) (microspheres accounted for 5% of the solid phase composition by mass) to obtain the solid phase composition. Na2HPO4 solution was added at a solid-liquid ratio of 1.5:1 and mixed to obtain an injectable slurry. The extrusion force was tested with an 18G needle and an advance rate of 12 mm / min: maximum extrusion force ≈ 30 N, stable section ≈ 22 N; Vicat method determination: initial coagulation ≈ 10 min, final coagulation ≈ 15 min (n≥5). Mercury intrusion method determination: interconnected porosity ≈ 40%, total porosity ≈ 70%; SEM statistical average pore size 10~150 μm; microCT analysis confirmed three-dimensional connectivity.

[0046] In vitro release (PBS, pH 7.40±0.05, 37℃, 50 rpm, equal volume sampling and replenishment): 24h burst release ≈ 12.6%, cumulative release ≈ 28.5% on day 7, followed by slow release, with cumulative release reaching ≈ 85.2% by week 8.

[0047] Compressive strength (ISO5833, n≥5, mean ±SD): ≈16.2MPa.

[0048] It should be noted that the test method for in vitro release is as follows: Pharmacopoeia of the People's Republic of China: 2025 Edition, Part IV, 9013 Guidelines for Sustained-Release, Controlled-Release and Delayed-Release Preparations.

[0049] The test method for compressive strength is ISO 5833:2002.

[0050] Example 2 This embodiment provides a method for preparing drug-loaded microsphere-modified porous calcium phosphate bone cement, including: S1. Preparation of drug-loaded microspheres; Polylactic acid (PLA) was dissolved in dichloromethane at a mass-to-volume ratio of 0.0375 g / ml. Osteogenic growth peptide (OGP, added at 10% of the mass of PLA) was added and magnetically stirred until homogeneous, forming the oil phase. The oil phase was then added dropwise to a PVA aqueous solution at a mass-to-volume ratio of 0.011 g / ml at a volume ratio of 15% (v / v), and vortexed for 2 min to form an O / W emulsion. The solvent was evaporated by magnetic stirring at 35 °C, and the emulsion was collected, washed, and dried to obtain PLA drug-loaded microspheres.

[0051] SEM particle size statistics (n≥300): D50≈27.5μm, D10 / D90≈20~35μm.

[0052] S2, Preparation of porous CPC powder; β-tricalcium phosphate (β-TCP) and tetracalcium phosphate (TTCP) were mixed at a mass ratio of 1:1 as a base material, and stirred at 37℃ for 6 min with Na2HPO4 solution (molar concentration of 1.5 mol / L) at a concentration of 1.95 g / ml. The mixture was then frozen at -80℃ for 45 min and freeze-dried for 1.5 days under the following conditions: vacuum degree of 20 Pa, cold trap temperature of -40℃; pre-freezing at -40℃ for 3 h, followed by slow drying at 0℃ for 9 h, and finally slow drying at 37℃ for 24 h. The freeze-dried block was lightly crushed to obtain porous calcium phosphate bone cement (CPC) powder. S3. Preparation of composite bone cement; The porous CPC powder obtained in step (2) was blended with the drug-loaded microspheres obtained in step (1) (microspheres accounted for 15% of the solid phase by mass) to obtain the solid phase. Citric acid solution was added at a solid-liquid ratio of 2.0:1 and mixed thoroughly to prepare an injectable slurry. Extrusion force was tested with an 18G needle at 12 mm / min: maximum extrusion force ≈ 25 N, stable section ≈ 18 N; Vicat method determination: initial coagulation ≈ 20 min, final coagulation ≈ 25 min (n ≥ 5). Mercury intrusion method determination: interconnected porosity ≈ 50%, total porosity ≈ 75%; SEM statistical average pore size 50~200 μm; microCT confirmed three-dimensional connectivity.

[0053] In vitro release (PBS, pH 7.40±0.05, 37℃, 50 rpm, equal volume sampling and replenishment): 24h burst release ≈ 18.4%, cumulative release ≈ 35.1% on day 7, followed by slow release, reaching a cumulative release of ≈ 90.1% by week 8.

[0054] Compressive strength (ISO5833, n≥5, mean ±SD): ≈9.4MPa.

[0055] Example 3 This embodiment provides a method for preparing drug-loaded microsphere-modified porous calcium phosphate bone cement, including: S1. Preparation of drug-loaded microspheres; Polycaprolactone was dissolved in dichloromethane at a mass-volume ratio of 0.05 g / ml, and transforming growth factor-β (TGF-β, added at 15% of the mass of polycaprolactone) was added. The mixture was magnetically stirred until homogeneous and used as the oil phase. The oil phase was added dropwise to a PVA aqueous solution at a mass-volume ratio of 0.02 g / ml at a volume ratio of 20% (v / v), and vortexed for 3 min to form an O / W emulsion. The solvent was evaporated by magnetic stirring at 40 °C, and the emulsion was collected, washed, and dried to obtain polycaprolactone drug-loaded microspheres.

[0056] SEM particle size statistics (n≥300): D50≈50μm, D10 / D90≈40~60μm.

[0057] S2, Preparation of porous CPC powder; Anhydrous calcium dicalcium phosphate (DCPA) and tetracalcium phosphate (TTCP) were mixed at a mass ratio of 1:1 as a base material. This mixture was then stirred at 37°C for 7.5 min with a Na₂HPO₄ solution (1.5 mol / L) at a concentration of 2.5 g / ml. The mixture was then frozen at -80°C for 60 min and freeze-dried for 2 days under the following conditions: vacuum degree of 20 Pa, cold trap temperature of -40°C; pre-freezing at -40°C for 3 h, followed by a slow rise to 0°C for a first drying of 12 h, and finally a slow rise to 37°C for a second drying of 33 h. The freeze-dried block was then lightly crushed to obtain porous calcium phosphate bone cement (CPC) powder. S3. Preparation of composite bone cement; The porous CPC powder obtained in step (2) was blended with the drug-loaded microspheres obtained in step (1) (microspheres accounted for 30% of the solid phase by mass) to obtain the solid phase. Deionized water was added at a solid-liquid ratio of 3.0:1 and mixed thoroughly to obtain an injectable slurry. Extrusion force was tested with an 18G needle at 12mm / min: maximum extrusion force ≈20N, stable section ≈15N; Vicat method determination: initial setting ≈25min, final setting ≈30min (n≥5). Mercury intrusion method determination: interconnected porosity ≈60%, total porosity ≈80%; SEM statistical average pore size 150~300μm; microCT confirmed three-dimensional connectivity.

[0058] In vitro release (PBS, pH 7.40±0.05, 37℃, 50 rpm, equal volume sampling and replenishment): 24h burst release ≈ 30.1%, cumulative release ≈ 58.2% on day 7, followed by slow release, with cumulative release reaching ≈ 95.7% by week 8.

[0059] Compressive strength (ISO5833, n≥5, mean ±SD): ≈2.1MPa.

[0060] In summary, the release curves of bone-inducing factors in vitro in phosphate buffer for the porous calcium phosphate bone cement prepared in Examples 1-3 are shown below. Figure 1 See the bar chart for the compressive strength test results. Figure 2 Wherein, 5% represents the porous calcium phosphate bone cement with microspheres accounting for 5% of the solid phase mass fraction provided in Example 1; 15% represents the porous calcium phosphate bone cement with microspheres accounting for 15% of the solid phase mass fraction provided in Example 2; and 30% represents the porous calcium phosphate bone cement with microspheres accounting for 30% of the solid phase mass fraction provided in Example 3.

[0061] Comparative Example 1 This comparative example provides a method for preparing calcium phosphate bone cement, which is the same as the method provided in Example 1, except that the loading method of the bone-inducing factor is changed. The specific process is as follows: Using the porous CPC powder prepared in S2 of Example 1 as the solid substrate, bone morphogenetic protein (BMP-2) was dissolved in sodium hydrogen phosphate (Na2HPO4) solution (molar concentration of 1.5 mol / L) to prepare bone induction factor working solution, so that the nominal content of BMP-2 in the final cured bone cement is the same as that in Example 1.

[0062] Porous calcium phosphate bone cement matrix and BMP-2 working fluid were mixed at a solid-liquid ratio of 1.5:1 and stirred for 4 minutes to prepare injectable grout. The Vicat method was used to determine the initial setting time: ≈9 minutes, and the final setting time: ≈14 minutes (n≥5).

[0063] In vitro release (PBS, pH 7.40±0.05, 37℃, 50 rpm, equal volume sampling and replenishment): 24h burst release ≈ 55.2%, 7d cumulative release ≈ 73%, by week 3 the release was almost complete, showing severe burst release and short-term rapid release behavior.

[0064] Compressive strength (ISO5833, n≥5, mean ±SD): ≈17.3MPa.

[0065] Compared with Example 1, under the same conditions of porous CPC powder and nominal BMP-2 feed amount, the direct blending of osteoinductive factor in this comparative example resulted in a significant increase in the initial burst release and a shortened effective release time in the later stage, making it difficult to meet the requirements of low burst release and long-lasting sustained release.

[0066] Comparative Example 2 This comparative example provides a method for preparing calcium phosphate bone cement, which is the same as the method provided in Example 1, except that the calcium phosphate bone cement powder used is non-porous. The specific operation is as follows: α-Tricalcium phosphate (α-TCP) and drug-loaded microspheres (prepared from S1 in Example 1) were blended at a mass ratio of 95:5 to obtain a solid phase. Na2HPO4 solution was added at a solid-liquid ratio of 1.5:1, and the mixture was manually stirred for 4 min to obtain an injectable slurry. Extrusion force was tested using an 18G needle at an advance rate of 12 mm / min: maximum extrusion force ≈ 32 N, stable section ≈ 24 N; Vicat method determination: initial setting ≈ 11 min, final setting ≈ 16 min (n ≥ 5).

[0067] In vitro release (PBS, pH 7.40±0.05, 37℃, 50 rpm, equal volume sampling and replenishment): 24h burst release ≈ 10.2%; 7d cumulative release ≈ 23.8%; 8 weeks cumulative release ≈ 69.1%.

[0068] Compressive strength (ISO5833, n≥5, mean ±SD): ≈35.6MPa.

[0069] Compared with the drug-loaded microsphere-modified porous calcium phosphate bone cement of Example 1, although the initial burst release of this comparative example was slightly lower, the overall porosity and interconnected porosity were significantly reduced, the cumulative release in the later stage was lower, and the dense structure could not provide enough space for bone tissue ingrowth, which was not conducive to bone integration.

[0070] Comparative Example 3 This comparative example provides a method for preparing calcium phosphate bone cement, which is the same as the method provided in Example 1, except that the microspheres account for 40% of the solid phase by mass. The specific operation is as follows: The porous CPC powder prepared in S2 of Example 1 was blended with drug-loaded microspheres (prepared in S1 of Example 1) at a mass ratio of 60:40 to obtain a solid phase. Deionized water was added at a solid-liquid ratio of 3.0:1 and mixed thoroughly to obtain an injectable slurry. Extrusion force was tested with an 18G needle at 12 mm / min: maximum extrusion force ≈ 18 N, stable section ≈ 13 N; Vicat method determination: initial setting ≈ 27 min, final setting ≈ 32 min (n ≥ 5).

[0071] In vitro release (PBS, pH 7.40±0.05, 37℃, 50 rpm, equal volume sampling and replenishment): 24h burst release ≈ 38.5%; 7d cumulative release ≈ 72.4%; 8-week cumulative release close to 100%.

[0072] Compressive strength (ISO5833, n≥5, mean ±SD): ≈0.8MPa The results showed that when the mass fraction of drug-loaded microspheres in the solid phase increased to 40%, the compressive strength of the solidified body was significantly lower than 2 MPa, which could not meet the basic requirements for filling and supporting bone defects. At the same time, the 24-hour burst release was significantly higher than 35%, and the drug release behavior was difficult to control. This further proves that the porous calcium phosphate bone cement modified with drug-loaded microspheres provided in the embodiments of the present invention ensures its sustained release and compressive strength.

[0073] Comparative Example 4 This comparative example provides a method for preparing drug-loaded porous calcium phosphate bone cement. This method is the same as that provided in Example 1, except that the drug-loaded microspheres are replaced with BMP-2-loaded porous amorphous calcium carbonate nanoparticles. The specific steps are as follows: S1, BMP-2-loaded porous nano-amorphous calcium carbonate 1.11 g of calcium chloride was dissolved in 100 ml of deionized water and stirred until homogeneous to obtain a 0.1 mol / L calcium chloride solution. 0.5 g of BMP-2 was added to the calcium chloride solution. 0.074 g of sodium carbonate and 0.107 g of disodium hydrogen phosphate dodecahydrate were dissolved in 20 ml of deionized water and stirred until homogeneous to obtain a mixed solution. 20 ml of this mixed solution was added dropwise to 100 ml of the calcium chloride solution. The concentration of sodium carbonate was 3.7 g / L, and the concentration of disodium hydrogen phosphate dodecahydrate was 5.35 g / L. The resulting solution was stirred, allowed to stand for 12 hours to precipitate, and then centrifuged at 7500 rpm. The obtained product was then subjected to… Drug-loaded porous amorphous calcium carbonate nanoparticles were obtained by freeze-drying at 20℃ for 30h, with a pore size of 120~320nm. S2, Preparation of porous CPC powder; Porous CPC powder was prepared according to the S2 preparation method in Example 1.

[0074] S3, Preparation of composite bone cement; The porous CPC powder obtained in step (2) was mixed with the BMP-2-loaded porous nano-amorphous calcium carbonate (5% by mass of solid phase) obtained in step (1) to obtain the solid phase composition. Na2HPO4 solution was added at a solid-liquid ratio of 1.5:1 and mixed thoroughly to obtain an injectable slurry. Extrusion force was tested using an 18G needle at an advance rate of 12 mm / min: maximum extrusion force ≈ 31 N, stable section ≈ 22 N; Vicat method determination: initial setting ≈ 11 min, final setting ≈ 16 min (n ≥ 5).

[0075] In vitro release (PBS, pH 7.40±0.05, 37℃, 50 rpm, equal volume sampling and replenishment): The burst release was as high as 42.6% in 24 hours, and the cumulative release exceeded 76% in 7 days. By the third week, the release was almost complete, showing a serious burst release phenomenon and failing to achieve long-term sustained release.

[0076] Compressive strength (ISO5833, n≥5, mean ±SD): ≈14.5MPa.

[0077] This comparative example demonstrates that even when using the same porous CPC powder as in the embodiments of the present invention, simply replacing the drug delivery system with a porous nano-amorphous calcium carbonate drug delivery system results in a severe burst release of bone-inducing factor (BMP-2) of up to 42.6% within 24 hours and over 76% within 7 days, completely losing its long-term sustained-release function. This clearly shows that porous nano-amorphous calcium carbonate cannot effectively load BMP-2, and its technical principle is fundamentally different from the polymer-shell-core microsphere system innovated in the embodiments of the present invention to solve the problem of long-term controlled release of bone-inducing factor, and it cannot be replaced.

[0078] Comparative Example 5 This comparative example aims to investigate whether simply stacking the core functional component (drug-loaded microspheres) of the embodiments of the present invention into a multi-component complex system can achieve better or equivalent performance. The specific operation is as follows: S1. Preparation of drug-loaded microspheres PLGA-loaded BMP-2 microspheres were prepared according to method S1 of Example 1.

[0079] S2, Preparation of porous CPC powder; Step 1, Prepare porous β TCP powder.

[0080] 4.77 g of anhydrous calcium chloride was added to 100 ml of deionized water, resulting in a concentration of 0.43 mol / L. The mixture was stirred at 85 °C for 1 h to obtain a calcium chloride aqueous solution. 42.12 g of sodium dihydrogen phosphate monohydrate was weighed and added to 600 ml of deionized water, resulting in a concentration of 0.51 mol / L. The mixture was stirred at room temperature for 1 h to obtain a sodium dihydrogen phosphate monohydrate aqueous solution. The calcium chloride aqueous solution and the sodium dihydrogen phosphate monohydrate solution were mixed at a volume ratio of 1:6 and stirred at room temperature for 2 h to obtain a mixed solution. The mixed solution was poured into an atomizer, which directly sprayed the solution into liquid nitrogen. The gas-liquid ratio in the atomizer was 1:100, the pressure was set to 0.08 MPa, and the distance between the atomizer and the liquid nitrogen surface was 10 cm. This yielded solidified β-formaldehyde resin. TCP powder was freeze-dried in a vacuum environment of 1 Pa under the following conditions: The water in the sample was sublimated at 20℃ for 36 hours, resulting in porous β-type material. TCP powder.

[0081] Step 2, Preparation of porous magnesium particles: 1.5g of magnesium-aluminum alloy powder was completely immersed in 20ml of anhydrous ethanol. After ultrasonic cleaning and dispersion, the powder was placed in an oven and dried at 50℃ for 6h. The cleaned and dried magnesium-aluminum alloy powder was then dissolved in 6ml of 15mol / L hydrofluoric acid aqueous solution in an ultrasonic bath. After repeated washing with deionized water and anhydrous ethanol three times, the powder was dried at 50℃ for 6h to obtain porous magnesium particles.

[0082] S3, Preparation of composite bone cement; The porous β obtained in step (2) TCP powder and porous magnesium particles were mixed with PLGA microspheres loaded with BMP-2 obtained in step (1) (accounting for 5% of the solid phase composition by mass) to obtain the solid phase composition. Na2HPO4 solution was added at a solid-liquid ratio of 1.5:1 and mixed well to obtain an injectable slurry. Extrusion force was tested with an 18G needle and an advance rate of 12mm / min: maximum extrusion force ≈32N, stable section ≈23N; Vicat method determination: initial setting ≈11min, final setting ≈17min (n≥5). Mercury intrusion method determination: total porosity ≈16%.

[0083] In vitro release (PBS, pH 7.40±0.05, 37℃, 50 rpm, equal volume sampling and replenishment): 24-hour burst release: 7.2%, 7-day cumulative release: approximately 28%, followed by slow release, with the cumulative release at week 8 being only ≈68.7%.

[0084] Compressive strength (ISO5833, n≥5, mean ±SD): ≈32.4 MPa.

[0085] This comparative example demonstrates that a low-porosity matrix framework severely restricts the early release and long-term delivery efficiency of osteoinductive factors. Even when using the same drug-loaded microspheres as in the embodiments of this invention, the drug diffusion channels are severely blocked due to the physical barrier formed by the low porosity (≈16%) of the matrix, resulting in a large amount of osteoinductive factors being isolated inside the material and unable to be utilized, leading to incomplete release (the cumulative release amount after 8 weeks stagnated at ≈68.7%).

[0086] Test case To comprehensively evaluate the osteogenic capacity of the material of this invention and to verify the intrinsic relationship between its release behavior and biological function, the following in vitro cell experiments were conducted.

[0087] (1) Experimental grouping and sample preparation: A total of 5 groups of materials were set up for testing: 1) Experimental Group A: Drug-loaded microsphere modified porous calcium phosphate bone cement prepared in Example 1 of this invention.

[0088] 2) Control group B: The sample prepared in comparative example 4.

[0089] 3) Control group C: Blank porous CPC (i.e., the porous CPC powder prepared in S2 of Example 1 is directly cured without any drug-loaded components).

[0090] 4) Control group D: Drug-loaded microspheres only (PLGA / BMP-2 microspheres prepared by method S1 in Example 1, directly plated).

[0091] 5) Negative control group: ordinary cell culture plate (TCPS).

[0092] All block materials were made into discs with a diameter of 10 mm and a thickness of 2 mm. After being sterilized with ethylene oxide, they were pre-soaked in complete culture medium for 24 hours before cell inoculation.

[0093] (2) Cell culture: The mouse pre-osteoblast cell line MC3T3-E1 was used. Cells were spaced at 1 × 10⁶ cells per well. 4 Inoculate the samples at a density of 100 individuals onto the surface of each material or into culture plates using α-MEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and incubate at 37°C and 5% CO2. Change the medium every 2 days.

[0094] (3) Detection indicators and results: (A) Cell adhesion and proliferation (CCK-8 assay): Detected after 1 and 7 days of culture. See results below. Figure 3 and Figure 4 ,according to Figure 3 It can be seen that after 1 day of culture, the experimental group A showed the highest number of adherent cells compared to the control groups B and C, with no significant difference, indicating that the porous matrix of this invention has good cell compatibility. According to Figure 4 It can be seen that by day 7 of culture, the cell proliferation rate of experimental group A was significantly higher than that of all other material groups (p<0.05), showing the optimal cell growth microenvironment.

[0095] (B) Alkaline phosphatase (ALP) activity: After 14 days of culture, the activity was detected using the p-nitrophenyl phosphate (pNPP) method. See results below. Figure 5 ,according to Figure 5 It was found that after 14 days of culture, experimental group A showed the highest ALP activity, significantly better than all control groups. This demonstrates the excellent bone-inducing ability of the material of this invention. (C) Osteogenesis-related gene expression (qRT-PCR): After 14 days of culture, total RNA was extracted from cells, and the expression levels of core osteogenic genes Runx2, osteopontin (OPN), and osteocalcin (OCN) were detected. See results below. Figures 6-8 ,according to Figures 6-8It can be seen that the expression levels of these three genes in experimental group A were significantly upregulated, and their expression levels were 2.5 to 4 times higher than those in other material groups.

[0096] This demonstrates that the embodiments of the present invention utilize porous CPC to provide a space for bone integration, enabling long-term controlled release of osteoinducing factors via drug-loaded microspheres, thereby transforming them into efficient and continuous osteogenic signals, achieving efficient bone defect repair through the integrated process of "bone conduction-bone induction-bone integration".

[0097] In summary, the porous calcium phosphate bone cement matrix provided in this embodiment of the invention provides an initial three-dimensional interconnected pore structure, promoting bone tissue ingrowth and integration; the drug-loaded microspheres achieve long-term controlled release of osteoinducing factors, avoiding initial burst release and the easy inactivation of factors due to direct addition in existing technologies; the microsphere degradation process further optimizes the pore structure, forming more interconnected pores, promoting osteoblast migration and bone healing, effectively promoting osteogenic differentiation and bone repair processes, and improving the overall efficiency of bone defect repair.

[0098] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A porous calcium phosphate bone cement modified with drug-loaded microspheres, characterized in that, Its raw materials include a solid phase component; the solid phase component consists of a porous calcium phosphate bone cement matrix and drug-loaded microspheres dispersed therein; The porous calcium phosphate bone cement matrix has a three-dimensional interconnected pore structure, and the drug-loaded microspheres have a core-shell structure. The outer shell is made of a biodegradable material, and the core is a bone-inducing factor.

2. The porous calcium phosphate bone cement modified with drug-loaded microspheres according to claim 1, characterized in that, The drug-loaded microspheres satisfy at least one of the following conditions: (1) The biodegradable material includes at least one of polycaprolactone, polylactic acid, and polylactic acid-glycolic acid copolymer; (2) The bone-inducing factor includes at least one of bone morphogenetic protein, osteogenic growth peptide and transforming growth factor-β; (3) The amount of bone-inducing factor added is 1% to 20% of the mass of the biodegradable material.

3. The porous calcium phosphate bone cement modified with drug-loaded microspheres according to claim 1, characterized in that, The raw materials used to form the porous calcium phosphate bone cement also include liquid phase components; Preferably, the mass ratio of the solid phase component to the liquid phase component is (1.5:1) to (3:1). Preferably, the liquid phase component includes any one of sodium hydrogen phosphate solution, citric acid solution, and deionized water.

4. The porous calcium phosphate bone cement modified with drug-loaded microspheres according to claim 1, characterized in that, The interconnected porosity of the porous calcium phosphate bone cement matrix is ​​not less than 40%, the total porosity of the porous calcium phosphate bone cement matrix is ​​not less than 70%, and the average pore size of the porous calcium phosphate bone cement matrix is ​​10~300μm. Preferably, the median particle size of the drug-loaded microspheres is 5~500 μm; Preferably, the mass of the drug-loaded microspheres accounts for 5% to 30% of the total mass of the solid phase components.

5. The porous calcium phosphate bone cement modified with drug-loaded microspheres according to claim 1, characterized in that, The porous calcium phosphate bone cement meets at least one of the following requirements: (1) The porous calcium phosphate bone cement releases bone-inducing factors in phosphate buffer solution at pH 7.4 in vitro. The burst release of bone-inducing factors within 24 hours does not exceed 35% of the total load, and can continue to be released slowly over the next 6 to 8 weeks. (2) The compressive strength of the porous calcium phosphate bone cement is in the range of 2~20MPa.

6. A method for preparing porous calcium phosphate bone cement modified with drug-loaded microspheres as described in claim 1, characterized in that, include: Solidified solid phase components.

7. The preparation method according to claim 6, characterized in that, Also includes: The solid and liquid components are mixed and then cured. Preferably, the step of forming the solid phase component includes: physically blending porous CPC powder, which forms a porous calcium phosphate bone cement matrix, with drug-loaded microspheres.

8. The preparation method according to claim 7, characterized in that, The steps for forming the porous CPC powder include: mixing a solid substrate and a coagulation accelerator and then freeze-drying the mixture; Preferably, the step of forming the porous CPC powder includes: mixing a solid substrate and a solution containing a coagulation promoter, then freezing at -75~-85°C for at least 30 minutes, followed by freeze-drying for 1~2 days, and then lightly crushing. Preferably, the steps for forming the drug-loaded microspheres include: mixing a biodegradable material, an organic solvent, and a bone-inducing factor to form an oil phase; mixing the oil phase with a polyvinyl alcohol solution to form an oil-in-water emulsion; then removing the solvent, followed by solidification, collection, washing, and drying.

9. The preparation method according to claim 8, characterized in that, The conditions for forming the porous CPC powder satisfy at least one of the following requirements: (1) The solid substrate comprises one or more of α-tricalcium phosphate, β-tricalcium phosphate, tetracalcium phosphate and anhydrous calcium hydrogen phosphate; (2) The coagulation accelerator includes sodium hydrogen phosphate solution; (3) The molar concentration of the solution containing the coagulation promoter is 1~1.5 mol / L; (4) The mass-to-volume ratio of the solid substrate to the solution containing the coagulation accelerator is 1.4~2.5 g / ml; Preferably, the conditions for forming the drug-loaded microspheres satisfy at least one of the following requirements: (1) The mass-to-volume ratio of the biodegradable material to the organic solvent is 0.025~0.05 g / ml; (2) The organic solvent includes at least one of amide solvents, halogen-substituted C1-C3 alkanes and C3-C5 ketone solvents; (3) The mass-volume concentration of the polyvinyl alcohol solution is 0.002~0.02 g / ml; (4) The oil phase accounts for 10% to 20% of the volume of the polyvinyl alcohol solution.

10. A repair material for filling and supporting bone defects, characterized in that, It is prepared by using porous calcium phosphate bone cement modified with drug-loaded microspheres as described in claim 1.

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