A bone-like fiber directional ordered structure bioceramic, a preparation method and application thereof

Abstract

The application relates to a bone-like fiber directional ordered structure bioceramic and a preparation method and application thereof. The bone-like fiber directional ordered structure bioceramic comprises a directional arrangement hydroxyapatite fiber bundle matrix, calcium carbonate deposited on the surface and inside of the directional arrangement hydroxyapatite fiber bundle matrix, and a biocompatible polymer material uniformly distributed in the bioceramic; the biocompatible polymer material is sodium polyacrylate or sodium alginate; the mass percentage of the directional arrangement hydroxyapatite fiber bundle matrix is 90-95%, the mass percentage of the calcium carbonate is 3-5%, and the mass percentage of the biocompatible polymer material is 2-5% in the total mass of the bone-like fiber directional ordered structure bioceramic.

Description

Technical Field

[0001] This invention belongs to the field of bioceramic materials, specifically relating to a bone fiber-inspired oriented ordered structure bioceramic, its preparation method, and its application. Background Technology

[0002] Skeleton is the primary organ in living organisms, providing support and protection. Supporting the entire weight of the human body, skeletons require sufficient strength and toughness to resist external impacts, making them a typical high-strength, high-toughness natural material. Human cortical bone is mainly composed of two nanophases: collagen molecules and hydroxyapatite nanocrystals. One reason for the excellent comprehensive mechanical properties of cortical bone lies in its exquisite and complex multi-level ordered structure. At the micro- and nanoscale, hydroxyapatite nanocrystals are embedded within collagen fibers and arranged in a periodic, alternating pattern along the direction of the collagen fibers, forming mineralized collagen fibers. These mineralized collagen fibers further align in a specific direction to form more macroscopic bone units, bone plates, and other structures, ultimately forming bone—a tough yet lightweight, and to some extent self-repairing multifunctional material. The directional arrangement of mineralized collagen fibers effectively regulates the strength and toughness of bone, providing inspiration for the design of the structure and mechanical properties of artificial bone materials.

[0003] Bioceramics are a class of biomaterials with good biocompatibility and bioactivity, boasting advantages such as tunable composition and abundant sources. However, the inherent brittleness of bioceramics makes them unable to match the toughness of bone tissue. Furthermore, their excessively high modulus makes them prone to "stress shielding" after implantation, resulting in poor osseointegration and thus limiting their clinical application. Therefore, the research and preparation of bioceramics with good biocompatibility and mechanical properties matching those of natural bone tissue is of great practical significance. Summary of the Invention

[0004] To address the issues of high brittleness and high modulus in bioceramics, this invention provides a method for preparing a bone-fiber-inspired, oriented, ordered bioceramic structure and its application in bone tissue repair. This bone-fiber-inspired, oriented, ordered bioceramic structure possesses mechanical properties matching those of natural bone tissue, including flexural strength and elastic modulus, while also exhibiting good biocompatibility, showing broad prospects for clinical applications.

[0005] In a first aspect, the present invention provides a bone-fiber-inspired oriented ordered structure bioceramic, the bone-fiber-inspired oriented ordered structure bioceramic comprising: an oriented hydroxyapatite fiber bundle matrix, calcium carbonate deposited on the surface and inside the oriented hydroxyapatite fiber bundle matrix, and a biocompatible polymer material uniformly distributed within the bioceramic; the biocompatible polymer material is sodium polyacrylate or sodium alginate.

[0006] Based on the total mass of the bone-like fiber-oriented ordered structured bioceramic as 100%, the mass percentage of the oriented hydroxyapatite fiber bundle matrix is ​​90-95%, the mass percentage of calcium carbonate is 3-5%, and the mass percentage of biocompatible polymer material is 2-5%.

[0007] Preferably, the diameter of the hydroxyapatite fiber bundle is 450-500 μm and the aspect ratio is not less than 200, preferably 300-350.

[0008] Secondly, the present invention provides a method for preparing the above-mentioned bone-fiber-inspired oriented ordered structure bioceramic, comprising: injecting ultralong hydroxyapatite nanowire slurry into anhydrous ethanol via an injection self-assembly process to obtain hydroxyapatite fiber bundles with oriented nanowires; then, coating the surface of the hydroxyapatite fiber bundles with a biocompatible polymer material, sodium polyacrylate or sodium alginate, by impregnation, and oriented them into a mold, and then pressing and assembling them to obtain a hydroxyapatite matrix; then, circulating a mineralization solution in the hydroxyapatite matrix to mineralize it, so as to achieve the deposition of calcium carbonate on the surface and inside of the hydroxyapatite matrix; and finally, drying and pressing to obtain the bone-fiber-inspired oriented ordered structure bioceramic.

[0009] Preferably, the diameter of the ultralong hydroxyapatite nanowires is 20-30 nm and the length is 80-100 μm; the nanowire solid content of the ultralong hydroxyapatite nanowire slurry is 30-60 mg / mL, preferably 45 mg / mL.

[0010] Preferably, the parameters of the injection self-assembly process include: an injection rate of 1-4 mL / min, preferably 2 mL / min; a needle length of 100-200 mm, preferably 150 mm, an inner diameter of 0.72-1.05 mm, preferably 0.86 mm; and a needle movement speed of 2-6 cm / s, preferably 4.5 cm / s.

[0011] Preferably, the concentration of the sodium polyacrylate solution does not exceed 1.0 wt%, and more preferably 0.5 wt%.

[0012] Preferably, the mineralization solution is a mixed solution of calcium bicarbonate, polyacrylic acid, and magnesium chloride; wherein the concentration of calcium bicarbonate is 1.5-1.7 g / L, the degree of polymerization of polyacrylic acid is 25-30 and the concentration is 1-1.5 g / L, and the concentration of magnesium chloride is 2-2.5 g / L.

[0013] Preferably, the parameters of the mineralization process include: a mineralization time of 10-18 days, preferably 14 days; more preferably, the mineralization process is divided into three stages according to the difference in mineralization liquid flow rate: the flow rate is 25 mL / min for the first 1-4 days; the flow rate is 50 mL / min for the 5th-7th days; and the flow rate is 10 mL / min for the 8th day to the last day of mineralization.

[0014] Thirdly, the present invention provides an application of the above-mentioned bone-fiber ordered structure bioceramic in the preparation of bone repair materials.

[0015] Beneficial effects

[0016] This invention utilizes a biomimetic strategy to prepare bioceramics with excellent biocompatibility and mechanical properties matching those of natural bone tissue, enabling better integration with bone tissue after implantation and enhancing bone repair. This bioceramic, with mechanical properties matching those of natural bone tissue, is prepared by constructing a hydroxyapatite matrix using a step-by-step assembly method with flexible, ultra-long hydroxyapatite nanowires, combined with a biomimetic mineralization method to mineralize and deposit calcium carbonate on the surface and inside of the matrix. The method is simple, low-cost, and can be mass-produced.

[0017] This invention is the first to prepare a bone-fiber oriented ordered bioceramic with excellent biocompatibility and mechanical properties that match bone tissue, thus promoting the development of bioceramic science and its clinical applications. Attached Figure Description

[0018] Figure 1 The images show the microstructure and crystal phase characterization of the ultralong hydroxyapatite nanowires prepared in Example 1; where (ab) is the SEM characterization image of the ultralong hydroxyapatite nanowires, (c) is the XRD characterization image of the ultralong hydroxyapatite nanowires, (d) is the TEM characterization image of the ultralong hydroxyapatite nanowires, and the inset is the selected area electron diffraction image of the ultralong hydroxyapatite nanowires.

[0019] Figure 2 This is a schematic diagram of the directional assembly of ultralong hydroxyapatite nanowires to construct hydroxyapatite fibers using the injection self-assembly method in Example 1; where (a) is an optical photograph of the ultralong hydroxyapatite nanowire slurry, (b) is an optical photograph of the hydroxyapatite fiber, and (cd) is a SEM image of the surface morphology of a single hydroxyapatite fiber at different magnifications.

[0020] Figure 3 The following are the assembly process and related optical photographs of the hydroxyapatite matrix in Example 1; wherein, (a) is the specific assembly process of the hydroxyapatite matrix, (b) is an optical photograph of the hydroxyapatite matrix, and (c) is an optical photograph of the ceramic material obtained after the hydroxyapatite matrix is ​​compacted under a pressure field.

[0021] Figure 4 The images shown are Micro-CT reconstructed images and SEM images of the cross-sectional morphology of the hydroxyapatite matrix along the fiber direction in Example 1; where (a) is a Micro-CT reconstructed image of the hydroxyapatite matrix and (b) is a SEM image of the cross-sectional morphology of the hydroxyapatite matrix along the fiber direction.

[0022] Figure 5 This is a schematic diagram showing the results of optimizing the preparation conditions of the hydroxyapatite matrix in Example 1; where (ab) represents the optimization of the pressure magnitude for the pressure field-assisted densification of the hydroxyapatite matrix, and (cd) represents the optimization of the sodium polyacrylate mass concentration for the pressure field-assisted densification of the hydroxyapatite matrix.

[0023] Figure 6 Examples 1 show the mineralization device and optical images of the bioceramic with a bone fiber-like oriented ordered structure; where (a) is an optical image of the mineralization device for the bioceramic with a bone fiber-like oriented ordered structure, and (b) is an optical image of the bioceramic with a bone fiber-like oriented ordered structure.

[0024] Figure 7 The images show the in vitro cytotoxicity test results for the unmineralized hydroxyapatite matrix, the bone fiber-like oriented ordered structure bioceramic obtained in Example 1, and the blank group; where (a) is a cell live / dead staining image for in vitro cytotoxicity test, and (b) is a quantitative statistical graph of cell survival rate for in vitro cytotoxicity test.

[0025] Figure 8 In vivo biocompatibility test diagram of unmineralized hydroxyapatite matrix and 14-day mineralized bone fiber-like oriented ordered structure bioceramic in rats;

[0026] Figure 9 SEM images of cross-sectional morphology along the fiber direction of unmineralized hydroxyapatite matrix and bioceramics with bone-like fiber-oriented ordered structure at different mineralization times; where (ab) is the cross-sectional morphology SEM image of unmineralized hydroxyapatite matrix, (cd) is the cross-sectional morphology SEM image of bioceramics with bone-like fiber-oriented ordered structure after 10 days of mineralization, (ef) is the cross-sectional morphology SEM image of bioceramics with bone-like fiber-oriented ordered structure after 14 days of mineralization, and (gh) is the cross-sectional morphology SEM image of bioceramics with bone-like fiber-oriented ordered structure after 18 days of mineralization.

[0027] Figure 10 , 11 Comparison of mechanical properties of unmineralized hydroxyapatite matrix and bioceramics with bone-fiber oriented ordered structure at different mineralization times; Figure 10 The flexural strength and flexural modulus of unmineralized hydroxyapatite matrix and bone fiber-like oriented ordered bioceramics mineralized for 10 days, 14 days, and 18 days were measured. Figure 11 Nanoindentation elastic modulus and nanoindentation hardness of unmineralized hydroxyapatite matrix and 14-day mineralized bone fiber-like oriented ordered structure bioceramics. Detailed Implementation

[0028] The present invention is further illustrated by the embodiments described below. It should be understood that the embodiments described below are for illustrative purposes only and are not intended to limit the present invention.

[0029] First, this invention provides a bone-fiber-inspired oriented ordered structure bioceramic. The bone-fiber-inspired oriented ordered structure bioceramic comprises: an oriented hydroxyapatite fiber bundle matrix, calcium carbonate deposited on the surface and interior of the oriented hydroxyapatite fiber bundle matrix, and a biocompatible polymer material uniformly distributed within the bioceramic.

[0030] In some embodiments, the diameter of the hydroxyapatite fiber bundle can be 450-500 μm, and the aspect ratio can be not less than 200, preferably 300-350. The diameter of the hydroxyapatite fiber bundle determines its mechanical strength. If the fiber bundle diameter is too small, the fiber bundle will exhibit low mechanical strength and be prone to breakage. Conversely, if the hydroxyapatite fiber bundle diameter is too large, it will result in an excessively large needle diameter, leading to an increased slurry extrusion volume during the slurry extrusion process. This can cause insufficient contact between the ultralong hydroxyapatite nanowires and anhydrous ethanol in the slurry near the middle of the fiber, preventing proper directional assembly and also reducing the mechanical properties of the fiber bundle.

[0031] Furthermore, the biocompatible polymer primarily functions as a binder in bioceramics to maintain the structural stability of the hydroxyapatite matrix. In some embodiments, the biocompatible polymer material can be sodium polyacrylate or sodium alginate.

[0032] The bone-fiber-inspired oriented ordered bioceramic provided by this invention is a dense, blocky ceramic whose dimensions can be adjusted as needed, preferably with a length of not less than 20 mm, a width of not less than 5 mm, and a thickness of not less than 0.5 mm. In some embodiments, based on the total mass of the bone-fiber-inspired oriented ordered bioceramic as 100%, the mass percentage of the oriented hydroxyapatite fiber bundle matrix can be 90-95%, the mass percentage of calcium carbonate can be 3-5%, and the mass percentage of biocompatible polymer materials can be 2-5%. The composition of the bone-fiber-inspired oriented ordered bioceramic provided by this invention is mainly composed of bioactive hydroxyapatite. The purpose of mineralized calcium carbonate is mainly to further regulate the mechanical properties of the ceramic; a mineralization content within the range of 3-5% can effectively regulate the mechanical properties of the bioceramic. The role of biocompatible polymer materials such as sodium polyacrylate is mainly to enhance the bonding force between the hydroxyapatite fiber bundles, aiming to enhance the mechanical properties and structural stability of the hydroxyapatite matrix; its mass percentage mainly depends on the content of mineralized calcium carbonate.

[0033] The bioceramic with bone-like fiber oriented ordered structure provided by the present invention has a flexural strength of 130-185 MPa, a flexural modulus of 33.6-50.1 GPa, a nanoindentation elastic modulus of 17.8-26.9 GPa, and a nanoindentation hardness of 0.72-1.47 GPa, exhibiting good mechanical properties similar to those of dense human bone.

[0034] The following is an exemplary description of a method for preparing a bone-fiber-inspired oriented ordered bioceramic structure provided by the present invention, which may include the following steps.

[0035] (1) Preparation of ultra-long hydroxyapatite nanowire slurry. Ultra-long hydroxyapatite nanowire slurry can be synthesized by a conventional calcium oleate precursor solvothermal method: sodium hydroxide, calcium chloride and sodium dihydrogen phosphate dihydrate are added sequentially to a mixed solution of deionized water, oleic acid and methanol, and then the reaction system is subjected to a hydrothermal reaction to obtain ultra-long hydroxyapatite nanowire slurry.

[0036] As an example: Add 270 mL of deionized water and 210 mL of oleic acid (C 18 H 34O2) and 120 mL of methanol (CH4O) were mixed under mechanical stirring. Every 30 minutes, 300 mL of aqueous solution prepared with 21 g of sodium hydroxide (NaOH), 240 mL of aqueous solution prepared with 6.66 g of anhydrous calcium chloride (CaCl2), and 360 mL of aqueous solution prepared with 18.72 g of sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O) were slowly added to the above mixed solution. Then, the reaction system was transferred to a hydrothermal reactor made of polytetrafluoroethylene and kept at 180°C for 24 h to obtain ultralong hydroxyapatite nanowire slurry.

[0037] (2) Preparation of hydroxyapatite matrix. Ultra-long hydroxyapatite nanowire slurry was injected into anhydrous ethanol by injection self-assembly process to obtain hydroxyapatite fiber bundles with oriented nanowires; then, the surface of the hydroxyapatite fiber bundles was coated with biocompatible polymer sodium polyacrylate or sodium alginate by impregnation, and oriented into a mold, and then pressed and assembled to obtain hydroxyapatite matrix.

[0038] In some embodiments, the diameter of the ultralong hydroxyapatite nanowires can be 20–30 nm, and the length can be 80–100 μm; the nanowire solid content of the ultralong hydroxyapatite nanowire slurry can be 30–60 mg / mL, preferably 45 mg / mL. The solid content of the ultralong hydroxyapatite nanowire slurry determines the thickness and strength of the injected fibers. The slurry contains high levels of oleic acid, methanol, and water, which diffuse into the anhydrous ethanol during injection, and the remaining nanowires assemble into hydroxyapatite (HAP) fibers. Too low a solid content results in low HAP content and poor mechanical properties of the fibers; conversely, too high a nanowire proportion and low levels of oleic acid and other components lead to insufficient nanowire assembly, resulting in HAP fibers with poor mechanical properties. Controlling the nanowire solid content within the optimized range described above allows for the preparation of HAP fibers with certain mechanical properties and good handling.

[0039] In some embodiments, the parameters of the injection self-assembly process may include: an injection rate of 1-4 mL / min, preferably 2 mL / min; a needle length of 100-200 mm, preferably 150 mm, an inner diameter of 0.72-1.05 mm, preferably 0.86 mm; and a needle movement speed of 2-6 cm / s, preferably 4.5 cm / s.

[0040] Excessive extrusion (injection) speed can cause the slurry to clump together, preventing the formation of uniformly sized hydroxyapatite fibers. Conversely, excessively slow extrusion speeds can lead to the slurry extrusion rate failing to keep pace with the needle movement speed, resulting in fiber breakage. The needle length should ideally be longer than 150mm to apply sufficient shear force to the nanowires during extrusion, promoting further orientation. An excessively large needle inner diameter can prevent proper orientation of nanowires near the fiber interior; an excessively small inner diameter can cause the extruded fiber bundle to curl at the extrusion nozzle. The needle movement speed must match the extrusion speed to facilitate the formation of hydroxyapatite fibers.

[0041] The slurry for ultralong hydroxyapatite nanowires contains oleic acid, methanol, and water. Oleic acid molecules are adsorbed onto the nanowires through the interaction of their carboxyl groups with calcium ions in the hydroxyapatite nanowires, while the alkyl chains on the oleic acid molecules are exposed in the slurry. During the injection of the slurry into anhydrous ethanol, excess methanol, water, and oleic acid diffuse into the anhydrous ethanol. Because the alkyl chains of the oleic acid molecules are incompatible with the polar solvent (ethanol), the ultralong HAP (hydroxyapatite) nanowires separate from the anhydrous ethanol. During solvent diffusion, the oleic acid molecules adsorbed on the surface of the ultralong hydroxyapatite nanowires regulate the self-assembly process of the nanowires through the "ethanol-oleic acid" interaction. Furthermore, due to the ultralong length of the hydroxyapatite nanowires, each nanowire tightly overlaps with the others in one direction, forming hydroxyapatite fibers. Ultimately, the ultralong hydroxyapatite nanowires are expelled from the ethanol, forming a bundle of oriented nanowires.

[0042] In some embodiments, the concentration of the sodium polyacrylate solution can be controlled to be no more than 1.0 wt%, preferably 0.5 wt%. The mass ratio of the hydroxyapatite fiber bundles to sodium polyacrylate or sodium alginate can be 90-95:2-5.

[0043] As an example: Ultra-long hydroxyapatite nanowire slurry was placed in a 60℃ oven for 30 minutes. Using a 20mL syringe, 20mL of the slurry was taken and injected into anhydrous ethanol at an injection rate of 2mL / min using a stainless steel flat-tipped needle with an inner diameter of 0.86mm and a length of 150mm. During the injection, the stainless steel needle maintained a moving speed of 4.5cm / s. Hydroxyapatite fibers were obtained, and then the obtained hydroxyapatite fibers were further... The fibers were cleaned by soaking in ethanol for 2 hours to remove excess oleic acid, resulting in hydroxyapatite fiber bundles. Next, a suitable amount of 4cm long fiber bundles were prepared, and a sodium polyacrylate solution with a mass concentration of 0.5wt% was prepared. The fiber bundles were soaked in the sodium polyacrylate solution for 5 minutes, removed and neatly arranged in a steel mold, and pressed into a hydroxyapatite matrix with a size of 40*10*5mm. Then, they were transferred to a -80℃ freezer for freezing and freeze-drying to obtain a macroscopic hydroxyapatite matrix with certain porosity.

[0044] This invention regulates the assembly process by adjusting the nanowire mass fraction of the ultralong hydroxyapatite slurry, the injection rate of the slurry, and the diameter and movement speed of the injection needle. This allows for further control over the macroscopic morphology and mechanical properties of the obtained hydroxyapatite fibers. This method offers advantages such as simple process and easily controllable conditions, enabling the directional assembly of one-dimensional nanowires from the nanoscale to the microscale, and can be used to construct multi-scale ordered structures.

[0045] The biocompatible polymer used in this invention does not affect the structure of the bioceramic. However, this polymer is rich in carboxyl groups, which can effectively chelate with the calcium ions of hydroxyapatite, significantly enhancing the bonding force between HAP fibers in the bioceramic, thereby improving the mechanical properties of the bioceramic. At the same time, it can also ensure that the HAP matrix is ​​not dispersed by the long-term flowing mineralization liquid during the mineralization process, so that the material obtained after mineralization can still maintain the fiber orientation and ordered structure of the HAP matrix.

[0046] (3) Preparation of bone fiber-like oriented ordered structure bioceramics. A mineralization solution at 25-40℃ (e.g., 40℃) is circulated in a hydroxyapatite matrix to achieve the deposition of calcium carbonate on the surface and inside the hydroxyapatite matrix; after drying and compaction, the bone fiber-like oriented ordered structure bioceramics are obtained.

[0047] In some embodiments, the mineralization solution may be a mixed solution of calcium bicarbonate, polyacrylic acid, and magnesium chloride; wherein the concentration of calcium bicarbonate is 1.5-1.7 g / L, the degree of polymerization of polyacrylic acid is 25-30 and the concentration is 1-1.5 g / L, and the concentration of magnesium chloride is 2-2.5 g / L.

[0048] As an example: Measure 1L of deionized water, weigh 5g of calcium carbonate (CaCO3) powder, and bubble carbon dioxide into the water while stirring at room temperature for 1 hour; then, at 4°C, stir and bubble carbon dioxide for 20 minutes; filter out excess calcium carbonate to obtain a clear saturated calcium bicarbonate (Ca(HCO3)2) solution; dissolve 1.2g of polyacrylic acid (C3H4O2) in 1L of saturated calcium bicarbonate solution. n A clear mineralized solution can be obtained by mixing 2.29 g of anhydrous magnesium chloride (MgCl2) powder with 15 min of anhydrous magnesium chloride (MgCl2) powder.

[0049] In some embodiments, the parameters of the mineralization process include: a mineralization time of 10-18 days, preferably 14 days; more preferably, the mineralization process is divided into three stages according to the difference in mineralization solution flow rate: days 1-4, flow rate of 25 mL / min; days 5-7, flow rate of 50 mL / min; and day 8 to the last day of mineralization, flow rate of 10 mL / min. Adjusting the flow rate of the mineralization solution can promote the nucleation and growth of calcium carbonate.

[0050] In some embodiments, the pressure for compaction is 105–270 MPa, preferably 215 MPa.

[0051] As an example: The hydroxyapatite matrix was placed in a mineralization mold and sealed. At 40°C, a peristaltic pump was used to continuously flow the mineralization solution inside the hydroxyapatite matrix. 250 mL of mineralization solution was prepared for each hydroxyapatite matrix and the solution was changed daily. The mineralization time was 14 days. The mineralization process was divided into three stages according to the difference in mineralization solution flow rate: the flow rate was 25 mL / min from day 1 to day 4, 50 mL / min from day 5 to day 7, and 10 mL / min from day 8 to day 14. After mineralization, the sample was taken out and dried at room temperature in a fume hood for 24 hours. Then, it was compacted under a pressure of 215 MPa to complete the preparation of the bone fiber ordered oriented structure bioceramic.

[0052] This invention employs a peristaltic pump-assisted calcium carbonate mineralization deposition method. The prepared mineralization solution is used, with different flow rates set at different time periods, allowing the solution to circulate and peristalt within the HAP matrix for 10-18 days. Saturated calcium bicarbonate solution can be thermally decomposed at approximately 40°C to obtain calcium carbonate. Furthermore, the polyacrylic acid added to the mineralization solution contains abundant carboxyl groups, which can adsorb calcium ions in the solution through electrostatic interactions, forming nucleation sites for calcium carbonate. In the initial stage of mineralization, the obtained calcium carbonate is an amorphous phase. The magnesium ions from the added magnesium chloride further promote the transformation of amorphous calcium carbonate into aragonite-phase calcium carbonate. Aragonite-phase calcium carbonate is a type of calcium carbonate sheet with a large aspect ratio and superior mechanical properties. Therefore, through the preparation process provided by this invention, after mineralization, calcium carbonate can be deposited within the HAP matrix, resulting in a denser bulk and significantly improved mechanical properties of the bioceramic (especially flexural modulus).

[0053] This invention utilizes an injection self-assembly method of "multi-scale hierarchical assembly" to achieve the directional assembly of ultralong hydroxyapatite nanowires to prepare hydroxyapatite fibers. These fibers are then further assembled into centimeter-sized hydroxyapatite matrices. Subsequently, peristaltic pump-assisted mineralization of calcium carbonate (depositing calcium carbonate on the template surface and interior through biomimetic mineralization) and pressure field densification are applied to this matrix to achieve the preparation of bone-fiber-inspired directional ordered bioceramics. Using ultralong hydroxyapatite nanowires, a biocompatible biomaterial, as the assembly unit, this invention proposes a method of "directional assembly-biomimetic mineralization-pressure-assisted densification" to prepare bone-fiber-inspired directional ordered bioceramics, which is expected to achieve mechanical properties matching those of natural bone tissue.

[0054] Compared to traditional hydroxyapatite ceramics prepared by high-temperature sintering, the bioceramics prepared in this invention exhibit higher flexural strength and lower flexural modulus, closely resembling the mechanical properties of human bone tissue. This allows for better matching with bone tissue, overcoming the problem of traditional bioceramics being difficult to match with bone tissue due to their high brittleness and modulus. These superior mechanical properties are attributed to the oriented and ordered fiber structure design and mineralization reinforcement of the bioceramics. During fracture, the material does not fracture instantaneously but layer by layer. The gradual fracture of HAP fibers effectively increases energy dissipation and enhances the material's toughness.

[0055] The bone-fiber ordered structure bioceramic obtained by the preparation method provided by this invention has both good biocompatibility and mechanical properties matching natural bone tissue. It can be used to prepare bone repair materials and is expected to improve its in vivo bone integration capacity to further enhance the bone repair effect.

[0056] The following examples further illustrate the present invention in detail. It should also be understood that the following examples are only for further explanation of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-essential improvements and adjustments made by those skilled in the art based on the above description of the present invention fall within the scope of protection of the present invention. The specific process parameters, etc., in the following examples are merely examples within a suitable range; that is, those skilled in the art can make appropriate selections within the appropriate range based on the description herein, and are not intended to be limited to the specific values ​​in the examples below.

[0057] Example 1

[0058] (1) Preparation of ultralong hydroxyapatite nanowire slurry. 270 mL of deionized water, 210 mL of oleic acid and 120 mL of methanol were mixed under mechanical stirring. Every 30 minutes, sodium hydroxide aqueous solution (21 g / 300 mL), anhydrous calcium chloride aqueous solution (6.66 g / 240 mL) and sodium dihydrogen phosphate dihydrate aqueous solution (18.72 g / 360 mL) were slowly added to the mixed solution in sequence. Then, the reaction system was transferred to four 500 mL polytetrafluoroethylene hydrothermal reactors, and the whole system was transferred to an oven and kept at 180 °C for 24 h. When the temperature dropped to room temperature, the white precipitate was collected, and the pale yellow supernatant in the reactor was collected at the same time. The two were mixed to prepare an ultralong hydroxyapatite nanowire slurry with a nanowire solid content of 45 mg / mL.

[0059] (2) Preparation of hydroxyapatite matrix. Ultra-long hydroxyapatite nanowire slurry was injected into anhydrous ethanol using a 150 mm long syringe with an inner diameter of 0.86 mm at a syringe movement speed of 4.5 cm / s and an injection rate of 2 mL / min to obtain hydroxyapatite fibers. Then, a 0.5 wt% sodium polyacrylate aqueous solution was uniformly coated on the fiber surface, and the fibers were further pressed and assembled into a three-dimensional hydroxyapatite matrix, denoted as Unmineralized.

[0060] (3) Preparation of bioceramics with bone fiber-like oriented ordered structure. A hydroxyapatite matrix was subjected to a 14-day biomimetic mineralization process using a mixed solution of calcium bicarbonate, polyacrylic acid, and magnesium chloride. The mineralization process was divided into three stages according to the difference in mineralization solution flow rate: 25 mL / min for days 1-4, 50 mL / min for days 5-7, and 10 mL / min for days 8-14. After mineralization, the sample was taken out and dried at room temperature in a fume hood for 24 h. Then, it was compacted under a pressure of 215 MPa to prepare a bioceramic with bone fiber-like oriented ordered structure, denoted as Mineralized-14d.

[0061] Figure 1The images show the microstructure and crystal phase characterization of the ultralong hydroxyapatite nanowires prepared in Example 1. (ab) are SEM images of the ultralong hydroxyapatite nanowires, (c) are XRD images, and (d) are TEM images. The inset is a selected area electron diffraction (SED) image of the ultralong hydroxyapatite nanowires. As can be seen from the images, the hydroxyapatite nanowires exhibit a typical one-dimensional nanowire structure with a large aspect ratio. They are uniform in size (d = 20–30 nm) and have good crystallinity, growing along the c-axis of the hydroxyapatite nanowire.

[0062] Figure 2 This image shows a schematic diagram of the directional assembly of ultralong hydroxyapatite nanowires to construct hydroxyapatite fibers using the injection self-assembly method in Example 1. (a) is an optical photograph of the ultralong hydroxyapatite nanowire slurry, (b) is an optical photograph of the hydroxyapatite fiber, and (cd) are SEM images of the surface morphology of a single hydroxyapatite fiber at different magnifications. As can be seen from the images, the hydroxyapatite fiber diameter is approximately 450-500 μm. Uniformly sized hydroxyapatite fibers can be prepared controllably and efficiently using the injection self-assembly method. Furthermore, the ultralong hydroxyapatite nanowires are tightly arranged together with a specific orientation, demonstrating that the injection self-assembly method can achieve directional assembly of nanowires, proving the scientific validity and reliability of this method.

[0063] Figure 3 The figures show the assembly process and related optical photographs of the hydroxyapatite matrix in Example 1; (a) shows the specific assembly process of the hydroxyapatite matrix, (b) is an optical photograph of the hydroxyapatite matrix, and (c) is an optical photograph of the ceramic material obtained after the hydroxyapatite matrix is ​​compacted under pressure. As can be seen from the figures, the hydroxyapatite fibers, coated with the biocompatible polymer sodium polyacrylate, are neatly arranged within the mold. The hydroxyapatite matrix is ​​constructed through low-pressure pre-compression, where the sodium polyacrylate concentration is 0.5 wt%. The template contains certain pores, which is beneficial for further mineralization.

[0064] Figure 4 The images show the Micro-CT reconstructed image and the SEM image of the cross-sectional morphology along the fiber direction of the hydroxyapatite matrix in Example 1; where (a) is the Micro-CT reconstructed image of the hydroxyapatite matrix, and (b) is the SEM image of the cross-sectional morphology along the fiber direction of the hydroxyapatite matrix. As can be seen from the images, the hydroxyapatite matrix exhibits a clear fiber-oriented structure and contains a certain amount of porosity, which facilitates subsequent mineralization.

[0065] Figure 5This diagram illustrates the results of optimizing the preparation conditions of the hydroxyapatite matrix in Example 1. (ab) represents the optimized pressure for densifying the hydroxyapatite matrix under pressure field assistance, and (cd) represents the optimized sodium polyacrylate mass concentration for densifying the hydroxyapatite matrix under pressure field assistance. The flexural properties of the hydroxyapatite matrices prepared under different pressure conditions and sodium polyacrylate concentrations were tested using a universal testing machine. The results showed that the optimized hydroxyapatite matrix had a flexural strength of 144.1 MPa and a flexural modulus of 31.8 GPa. The preparation parameters of 215 MPa pressure and 0.5 wt% sodium polyacrylate concentration were selected as the final preparation conditions for the bone-fiber-inspired oriented ordered bioceramic structure.

[0066] Figure 6 Examples 1 show the mineralization apparatus and optical images of the bioceramic with a bone fiber-like oriented ordered structure. (a) is an optical image of the mineralization apparatus for the bioceramic with a bone fiber-like oriented ordered structure, and (b) is an optical image of the bioceramic with a bone fiber-like oriented ordered structure.

[0067] In vitro cytotoxicity test of Mineralized-14d bioceramics:

[0068] The in vitro cytotoxicity of the unmineralized hydroxyapatite matrix and the bone-fiber-inspired oriented ordered structure bioceramic obtained in Example 1 was investigated using the Transwell assay, with a blank control group as a reference. The in vitro cytotoxicity of the materials was comprehensively evaluated using cell viability / dead staining and quantitative statistical analysis of cell viability. The basic procedure for the Transwell assay for in vitro cytotoxicity testing is as follows: Rabbit bone marrow mesenchymal stem cells (rBMSCs) were used to evaluate the in vitro cytotoxicity of the unmineralized and mineralized scaffolds, with the scaffold-free group serving as a blank control group; 1×10⁻⁶ cells were added to the matrix... 5 Culture medium for 100 cells was added to each well of a 24-well plate, and the scaffold was placed in a Transwell chamber. After co-culturing with the cells in an incubator (37°C, 5% CO2) for 24 hours, cell viability was quantitatively assessed using the CCK-8 cell viability assay. In addition, the Calcein-AM / PI double staining kit was used to stain the live and dead cells in the well plate. The staining reagent was diluted with PBS at a ratio of PBS:AM:PI = 1000:2:3. After adding the live and dead staining reagent and incubating at 37°C for 25 min, the distribution of green fluorescent live cells and red fluorescent dead cells in the well plate was observed using a fluorescence microscope.

[0069] Figure 7The figures show in vitro cytotoxicity test results for the unmineralized hydroxyapatite matrix, the bone fiber-like oriented ordered structure bioceramic obtained in Example 1, and the blank control group; where (a) is a cell live / dead staining image for in vitro cytotoxicity testing, and (b) is a quantitative statistical graph of cell viability for in vitro cytotoxicity testing. As can be seen from the figures, neither the unmineralized hydroxyapatite matrix nor the bone fiber-like oriented ordered structure bioceramic obtained in Example 1 showed significant cytotoxicity.

[0070] In vivo biocompatibility testing of Mineralized-14d bioceramics:

[0071] Unmineralized hydroxyapatite matrix and 14-day mineralized bone-fiber oriented ordered bioceramic were implanted subcutaneously into male SD rats. The scaffold size was 5*5*1mm. Subcutaneous implantation experiments were conducted in rats for 7 and 14 days. The in vivo biocompatibility of the bioceramic was evaluated by H&E staining of tissue sections.

[0072] Figure 8 This image shows the in vivo biocompatibility test results of unmineralized hydroxyapatite matrix and 14-day mineralized bone-fiber oriented ordered structure bioceramics in rats. H&E staining of tissue sections showed that neither the implanted unmineralized hydroxyapatite matrix nor the 14-day mineralized bone-fiber oriented ordered structure bioceramics exhibited significant inflammation 14 days after implantation. These results indicate that the bone-fiber oriented ordered structure bioceramics possesses good in vivo biocompatibility.

[0073] Example 2

[0074] The preparation process of bioceramics in Example 2 is the same as that in Example 1, with the main difference being that the mineralization time is 10 days and the mineralization process is divided into three stages according to the difference in the flow rate of the mineralizing solution: the flow rate is 25 mL / min for days 1 to 4, 50 mL / min for days 5 to 7, and 10 mL / min for days 8 to 10; the prepared bioceramics with a bone fiber oriented ordered structure are denoted as Mineralized-10d.

[0075] Example 3

[0076] The preparation process of bioceramics in Example 3 is the same as that in Example 1, with the main difference being that the mineralization time is 18 days and the mineralization process is divided into three stages according to the difference in the flow rate of the mineralizing solution: the flow rate is 25 mL / min for days 1 to 4, 50 mL / min for days 5 to 7, and 10 mL / min for days 8 to 18; the prepared bioceramics with a bone fiber oriented ordered structure are denoted as Mineralized-18d.

[0077] Comparative Example 1

[0078] Unmineralized hydroxyapatite bioceramics were prepared as a control using a stepwise assembly technique:

[0079] (1) Preparation of ultralong hydroxyapatite nanowire slurry. 270 mL of deionized water, 210 mL of oleic acid and 120 mL of methanol were mixed under mechanical stirring. Every 30 minutes, sodium hydroxide solution (21 g / 300 mL), anhydrous calcium chloride solution (6.66 g / 240 mL) and sodium dihydrogen phosphate dihydrate aqueous solution (18.72 g / 360 mL) were slowly added to the mixed solution in sequence. Then, the reaction system was transferred to four 500 mL polytetrafluoroethylene hydrothermal reactors, and the whole system was transferred to an oven and kept at 180 °C for 24 h. When the temperature dropped to room temperature, the white precipitate was collected, and the pale yellow supernatant in the reactor was collected at the same time. The two were mixed to prepare a hydroxyapatite nanowire slurry with a nanowire solid content of 45 mg / mL.

[0080] (2) Preparation of hydroxyapatite matrix. Ultra-long hydroxyapatite nanowire slurry was injected into anhydrous ethanol using a 150 mm long syringe with an inner diameter of 0.86 mm at a syringe movement speed of 4.5 cm / s and an injection rate of 2 mL / min to obtain hydroxyapatite fibers. Then, a 0.5 wt% sodium polyacrylate aqueous solution was uniformly coated on the fiber surface, and the fibers were further pressed and assembled into a three-dimensional hydroxyapatite matrix to prepare unmineralized hydroxyapatite bioceramics, denoted as Unmineralized.

[0081] Figure 9 The images show SEM images of the cross-sectional morphology along the fiber direction of the unmineralized hydroxyapatite matrix and the bone-fiber-inspired oriented ordered bioceramics at different mineralization times. Specifically, (ab) is the cross-sectional SEM image of the unmineralized hydroxyapatite matrix, (cd) is the cross-sectional SEM image of the bone-fiber-inspired oriented ordered bioceramics after 10 days of mineralization, (ef) is the cross-sectional SEM image of the bone-fiber-inspired oriented ordered bioceramics after 14 days of mineralization, and (gh) is the cross-sectional SEM image of the bone-fiber-inspired oriented ordered bioceramics after 18 days of mineralization. As can be seen from the images, the ultralong hydroxyapatite nanowires inside the mineralized bone-fiber-inspired oriented ordered bioceramics still maintain a highly ordered and oriented structure, and the internal structure is more dense compared to the unmineralized hydroxyapatite matrix.

[0082] Figure 10 , 11 Comparison of mechanical properties of unmineralized hydroxyapatite matrix and bioceramics with bone-fiber oriented ordered structure at different mineralization times; Figure 10 The flexural strength and flexural modulus of unmineralized hydroxyapatite matrix and bone fiber-like oriented ordered bioceramics mineralized for 10 days, 14 days, and 18 days were measured. Figure 11The figures show the nanoindentation elastic modulus and nanoindentation hardness of the unmineralized hydroxyapatite matrix and the bone-fiber-inspired oriented ordered bioceramic after 14 days of mineralization. As can be seen from the figures, the flexural strength of the bone-fiber-inspired oriented ordered bioceramic does not change significantly with increasing mineralization time, but its flexural modulus shows a trend of first gradually increasing and then stabilizing. Furthermore, compared to the unmineralized hydroxyapatite matrix, the 14-day mineralized bone-fiber-inspired oriented ordered bioceramic exhibits significantly improved elastic modulus and hardness. Bending test results show that the bone-fiber-inspired oriented ordered bioceramic possesses good flexural strength and flexural modulus, and nanoindentation test results also indicate that the bone-fiber-inspired oriented ordered bioceramic has a higher elastic modulus and nanoindentation hardness compared to the unmineralized hydroxyapatite matrix.

[0083] Table 1 below shows the performance parameters of the bioceramics prepared in this invention:

[0084]

[0085] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A bone-like fibrous oriented ordered structure bioceramic, characterized in that, The bone-like fiber directional ordered structure bioceramics comprises: a directional arranged hydroxyapatite fiber bundle matrix, calcium carbonate deposited on the surface and inside of the directional arranged hydroxyapatite fiber bundle matrix, and a biocompatible polymer material uniformly distributed in the bioceramics; the biocompatible polymer material is sodium polyacrylate or sodium alginate; The mass percentage of the directional arranged hydroxyapatite fiber bundle matrix is 90-95%, the mass percentage of the calcium carbonate is 3-5%, and the mass percentage of the biocompatible polymer material is 2-5%, based on 100% of the total mass of the bone-like fiber directional ordered structure bioceramics. The preparation method of the bone-like fiber directional ordered structure bioceramics comprises: injecting a super-long hydroxyapatite nanowire slurry into anhydrous ethanol through an injection self-assembly process to obtain a nanowire directional arranged hydroxyapatite fiber bundle; then, coating a biocompatible polymer material sodium polyacrylate or sodium alginate on the surface of the hydroxyapatite fiber bundle by immersion and directional arrangement into a mold, and molding by compression assembly to obtain a hydroxyapatite matrix; then, circulating the mineralization solution in the hydroxyapatite matrix to perform mineralization, so as to realize the deposition of calcium carbonate on the surface and inside of the hydroxyapatite matrix; and drying and compression compaction to obtain the bone-like fiber directional ordered structure bioceramics. The super-long hydroxyapatite nanowire slurry contains oleic acid, methanol and water, and the nanowire solid content of the super-long hydroxyapatite nanowire slurry is 30-60 mg / mL; the parameters of the injection self-assembly process include: an injection rate of 1-4 mL / min, a needle tube length of 100-200 mm, an inner diameter of 0.72-1.05 mm, and a needle tube moving rate of 2-6 cm / s. The mineralization solution is a mixed solution of calcium bicarbonate-polyacrylic acid-magnesium chloride; wherein the concentration of calcium bicarbonate is 1.5-1.7 g / L, the polymerization degree of polyacrylic acid is 25-30, the concentration is 1-1.5 g / L, and the concentration of magnesium chloride is 2-2.5 g / L.

2. The bone-imitative fiber directed ordered structure bioceramic according to claim 1, characterized in that, The diameter of the hydroxyapatite fiber bundle is 450-500 μm, and the aspect ratio is not less than 200.

3. The bone-imitative fiber directed ordered structure bioceramic according to claim 2, characterized in that, The aspect ratio of the hydroxyapatite fiber bundle is 300-350.

4. The bone-imitative fiber directed ordered structure bioceramic according to claim 1, characterized in that, In the preparation method, the diameter of the nanowire in the super-long hydroxyapatite nanowire slurry is 20-30 nm, and the length is 80-100 μm; and the nanowire solid content of the super-long hydroxyapatite nanowire slurry is 45 mg / mL.

5. The bone-imitative fiber directed ordered structure bioceramic according to claim 1, characterized in that, The parameters of the injection self-assembly process include: an injection rate of 2 mL / min, a needle tube length of 150 mm, an inner diameter of 0.86 mm, and a needle tube moving rate of 4.5 cm / s.

6. The bone-imitative fiber directed ordered structure bioceramic according to claim 1, characterized in that, In the preparation method, the concentration of the sodium polyacrylate solution used for immersion is not more than 1.0 wt%.

7. The bone-imitative fiber directed ordered structure bioceramic according to claim 6, characterized in that, The concentration of the sodium polyacrylate solution used for immersion is 0.5 wt%.

8. The bone-imitative fiber-directionally ordered structure bioceramic according to claim 1, characterized in that, The mineralization time is 10-18 days.

9. The bone-imitative fiber-oriented ordered structure bioceramic according to claim 8, characterized in that, The mineralization time is 14 days.

10. The bone-imitative fiber-directionally ordered structure bioceramic according to claim 1, characterized in that, The mineralization process was divided into three stages according to the flow rate of the mineralization solution: 25 mL / min from day 1 to day 4; 50 mL / min from day 5 to day 7; and 10 mL / min from day 8 to the last day of mineralization.

11. Use of the bone-like fiber-oriented ordered structure bioceramic according to claim 1 or 2 for the preparation of a bone repair material.

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