Preparation method of bionic artificial dense bone based on mineralized collagen fibers

Abstract

The invention discloses a mineralized collagen fiber-based bionic artificial dense bone preparation method, which specifically comprises: mixing mineralized collagen fibers and a polyvinyl alcohol solution according to a certain ratio, stirring to form a milk white thick liquid slurry as a mixed slurry, and freezing the mixed slurry to obtain a multilayer block structure, and forming the multi-layer block structure into the multi-layer bionic artificial dense bone structure under the conditions of proper temperature and pressure. According to the preparation method of the bionic artificial dense bone based on the mineralized collagen fibers, the problems that bionic bone materials prepared in the prior art are disordered and insufficient in mechanical property are solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of preparation methods of biomedical materials, and relates to a preparation method of a biomimetic artificial dense bone based on mineralized collagen fibers. Background Art

[0002] Bone structures generally exhibit multi-level ordered structural characteristics, mainly composed of collagen and hydroxyapatite, with a mineral content generally of 60-70%. The composition is relatively simple, but the bone structure shows excellent strength and toughness. However, for the preparation of biomimetic bone materials that meet a series of excellent properties such as multi-level order, excellent mechanical properties, and good biocompatibility, there are few reports in the current technology or literature on successful synthesis.

[0003] The existing process is to mechanically mix mineralized collagen fibers (or hydroxyapatite nanoparticles, etc.) into some polymers to prepare biomimetic bone materials, such as PCL, PLLA, PMMA. Whether it is a porous or dense structure, it is disordered, and there are problems of insufficient mechanical properties or inability to degrade. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method of a biomimetic artificial dense bone based on mineralized collagen fibers, which solves the problems in the existing technology that the prepared biomimetic bone materials are all disordered and have insufficient mechanical properties.

[0005] The technical solution adopted by the present invention is a preparation method of a biomimetic artificial dense bone based on mineralized collagen fibers, specifically: mixing mineralized collagen fibers and a polyvinyl alcohol solution in a certain proportion and stirring to form a milky white thick liquid slurry as a mixed slurry, then freezing the mixed slurry to obtain a multi-layer block structure, and then forming a multi-level biomimetic artificial dense bone structure under appropriate temperature and pressure conditions.

[0006] The present invention is further characterized in that

[0007] The process of preparing the polyvinyl alcohol solution is as follows:

[0008] Mix polyvinyl alcohol and deionized water in a ratio of 0.8-2 g: 50-100 mL, then heat to 85-95 °C, stir while heating until the polyvinyl alcohol is completely dissolved and the solution becomes clear and transparent.

[0009] The mineralized collagen fibers are freeze-dried mineralized collagen fibers or wet-state mineralized collagen fibers.

[0010] If freeze-dried mineralized collagen fibers are used for the mineralized collagen fibers, the mass ratio of the freeze-dried mineralized collagen fibers to polyvinyl alcohol is: 8-9.2 g: 0.8-2 g;

[0011] If wet mineralized collagen fibers are used, the mass ratio of wet mineralized collagen fibers to polyvinyl alcohol is: 45 g - 48 g : 0.8 - 2 g.

[0012] The mixed slurry is frozen in a gradient freezing manner to form a wedge-shaped multi-layer block structure, specifically:

[0013] A T-shaped copper plate is horizontally placed on the upper end of a metal tank filled with liquid nitrogen. The lower end of the T-shaped copper plate is inserted into the liquid nitrogen in the metal tank. The upper surface of the T-shaped copper plate is the operating table surface, and the temperature of the operating table surface is -30°C to -45°C. A PDMS wedge-shaped mold is prepared. The PDMS wedge-shaped mold is placed on the operating table surface. Then the mixed slurry is poured into the PDMS wedge-shaped mold. After dropping glutaraldehyde with a concentration of 50% into the mixed slurry and immediately stirring, it is poured into the PDMS wedge-shaped mold. It is frozen for 25 - 35 min to form a frozen block structure. Then the frozen block structure is freeze-dried at a pressure of 5 - 10 Pa and a temperature of -50°C for 48 - 72 h to form a stable multi-layer block structure. Then the multi-layer block structure is hot-pressed at a temperature of 80 - 90°C and a pressure of 500 - 1000 MPa for 10 min using a corresponding mold to form a multi-level bionic artificial dense bone structure with a corresponding shape.

[0014] The wedge angle of the PDMS wedge-shaped mold is set to 15 - 20 degrees.

[0015] The PDMS wedge-shaped mold is 20 - 40 mm away from the liquid nitrogen contact end of the T-shaped copper plate on the operating table surface.

[0016] The size of the PDMS wedge-shaped mold is height * width * slope length = 20 - 25 mm * 20 - 25 mm * 40 - 60 mm.

[0017] The dropping amount of glutaraldehyde with a concentration of 50% and the mass-volume ratio of polyvinyl alcohol is 0.1 - 0.2 mL : 0.8 - 2 g.

[0018] After mixing the mineralized collagen fibers and the polyvinyl alcohol solution, stir for 30 - 48 h until there are no visible lumps of agglomeration to obtain a mixed slurry.

[0019] The beneficial effects of the present invention are:

[0020] The mineralized collagen nanofibers doped with elements adopted in the present invention have good bone compatibility, osteoconductivity and certain osteogenic induction ability. They can not only achieve the combination with the original bone tissue after material implantation, but also recruit osteogenesis-related cells and promote their differentiation into the osteogenic direction, thus realizing bone regeneration. The use of polyvinyl alcohol also has excellent biocompatibility. The bionic artificial dense bone prepared by the method of the present invention has the advantages of light weight, high strength and high toughness. The bionic artificial dense bone prepared by the present invention has a high similarity in microstructure with natural bone cortical bone, meets the requirements of mineral content, density and strength, and also has good biocompatibility.

[0021] The bionic artificial dense bone prepared by the present invention can be slowly degraded and has very good compatibility. The present invention is a bionic of an ordered structure, which can also promote the regeneration and development of bones. Moreover, the bionic artificial dense bone prepared by the present invention has the characteristics of being arbitrarily cold-worked and shaped and secondary processed, which is also more convenient to use than other materials. Description of the Drawings

[0022] Figure 1 It is a schematic diagram of the mixed slurry prepared in Example 1 of the preparation method of the bionic artificial dense bone based on mineralized collagen fibers of the present invention;

[0023] Figure 2 It is a macroscopical view of a porous wedge obtained by bidirectional freezing of the mixed slurry in Example 1 of the preparation method of the bionic artificial dense bone based on mineralized collagen fibers of the present invention;

[0024] Figure 3 It is a macroscopical morphology diagram of a parallel porous block obtained by bidirectional freezing of the mixed slurry in Example 1 of the preparation method of the bionic artificial dense bone based on mineralized collagen fibers of the present invention;

[0025] Figure 4 It is a microscopical morphology diagram of a parallel porous block obtained by bidirectional freezing of the mixed slurry in Example 1 of the preparation method of the bionic artificial dense bone based on mineralized collagen fibers of the present invention;

[0026] Figure 5 It is a diagram of the gradual evolution process of the multi-level ordered structure in Example 1 of the preparation method of the bionic artificial dense bone based on mineralized collagen fibers of the present invention;

[0027] Figure 6 It is a mineral content diagram of the bionic mineralized collagen fibers and the bionic artificial dense bone in Example 1 of the preparation method of the bionic artificial dense bone based on mineralized collagen fibers of the present invention;

[0028] Figure 7 It is a Raman spectrum diagram of the mineralized collagen fibers and the bionic artificial dense bone in Example 1 of the preparation method of the bionic artificial dense bone based on mineralized collagen fibers of the present invention;

[0029] Figure 8 is the X-ray diffraction pattern of the biomimetic artificial dense bone prepared in Example 1 of the present invention;

[0030] Figure 9 is the bending stress-strain curve diagram of the biomimetic artificial dense bone prepared in Example 1 of the present invention obtained through a three-point bending test;

[0031] Figure 10 is the statistical result diagram of the bending strength and bending modulus of the biomimetic artificial dense bone prepared in Example 1 of the present invention;

[0032] Figure 11 is the fracture toughness test diagram of the biomimetic artificial dense bone prepared in Example 1 of the present invention through a single-edge notched beam specimen;

[0033] Figure 12 is the test diagram of the cell compatibility test of the biomimetic artificial dense bone prepared in Example 1 of the present invention;

[0034] Figure 13 is the result diagram of the CCK-8 experiment of the biomimetic artificial dense bone prepared in Example 1 of the present invention;

[0035] Figure 14 is the schematic diagram of the secondary processing of the biomimetic artificial dense bone prepared in Example 1 of the present invention.

[0036] Figure 15 is the morphological diagram of the freeze-dried biomimetic bone material prepared by adding mineralized collagen fibers to PCL in the prior art;

[0037] Figure 16 is Figure 15 the microscopic morphological diagram of the biomimetic bone material prepared therein. Specific Embodiments

[0038] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0039] The preparation method of the biomimetic artificial dense bone based on mineralized collagen fibers of the present invention is specifically as follows: Mix mineralized collagen fibers and a polyvinyl alcohol solution in a certain proportion and stir to form a milky white thick liquid slurry as a mixed slurry, then freeze the mixed slurry to obtain a multi-layer block structure, and then form a multi-level biomimetic artificial dense bone structure under appropriate temperature and pressure conditions.

[0040] The preparation process of the polyvinyl alcohol solution is as follows:

[0041] Mix polyvinyl alcohol and deionized water at 0.8 - 2 g: 50 - 100 mL, and then heat to 85 - 95 °C, stirring while heating until the polyvinyl alcohol is completely dissolved and the solution becomes clear and transparent. The mineralized collagen fibers are freeze-dried mineralized collagen fibers or wet mineralized collagen fibers.

[0042] If freeze-dried mineralized collagen fibers are used for the mineralized collagen fibers, the mass ratio of the freeze-dried mineralized collagen fibers to polyvinyl alcohol is: 8 - 9.2 g : 0.8 - 2 g;

[0043] If wet mineralized collagen fibers are used, the mass ratio of the wet mineralized collagen fibers to polyvinyl alcohol is: 45 g - 48 g : 0.8 - 2 g.

[0044] The mixed slurry is frozen by a gradient freezing method to form a wedge-shaped multi-layer block structure. Specifically:

[0045] A T-shaped copper plate is horizontally placed on the upper end of a metal tank filled with liquid nitrogen. The lower end of the T-shaped copper plate is inserted into the liquid nitrogen in the metal tank. The upper surface of the T-shaped copper plate is the operation table surface, and the temperature of the operation table surface is -30°C to -45°C. A PDMS wedge-shaped mold is prepared and placed on the operation table surface. Then the mixed slurry is poured into the PDMS wedge-shaped mold. After dropping glutaraldehyde with a concentration of 50% into the mixed slurry and immediately stirring, it is poured into the PDMS wedge-shaped mold. It is frozen for 25 - 35 min to form a frozen block structure, and then the frozen block structure is freeze-dried at a pressure of 5 - 10 Pa and a temperature of -50°C for 48 - 72 h to form a stable multi-layer block structure. Then the multi-layer block structure is hot-pressed for 10 min at a temperature of 80 - 90°C and a pressure of 500 - 1000 MPa using a corresponding mold to form a multi-level bionic artificial dense bone structure with a corresponding shape.

[0046] The wedge angle of the PDMS wedge-shaped mold is set to 15 - 20 degrees.

[0047] The PDMS wedge-shaped mold is placed on the operation table surface 20 - 40 mm away from the liquid nitrogen contact end of the T-shaped copper plate.

[0048] The size of the PDMS wedge-shaped mold is height * width * slope length = 20 - 25 mm * 20 - 25 mm * 40 - 60 mm.

[0049] The dropping amount of glutaraldehyde with a concentration of 50% and the mass-volume ratio of polyvinyl alcohol is 0.1 - 0.2 mL : 0.8 - 2 g.

[0050] After mixing the mineralized collagen fibers and the polyvinyl alcohol solution, stir for 30 - 48 h until there are no visible lumps of agglomeration to obtain a mixed slurry.

[0051] In the examples of the present invention, the raw material sources are as follows: polyvinyl alcohol (PVA, analytical amount 5000 daltons, Shanghai Aladdin Biochemical Technology Co., Ltd.), anhydrous calcium chloride (CaCl 2 , analytical pure), sodium hydroxide (NaOH, analytical pure) and ammonia water (NH 4OH (analytical grade) was purchased from Sinopharm Chemical Reagent Co., Ltd.; type I collagen sponge (telopeptide - removed collagen from bovine tail skin, MW 300,000, bovine tail, Hebei Kaolishen Co., Ltd.); phosphoric acid (H 3 PO 4 , analytical grade, Shanghai Titan Scientific Co., Ltd.); Tris - HCl (C 4 H 11 NO 3 ·HCl, analytical grade, Beijing Baidi Biotechnology Co., Ltd.).

[0052] The mineralized collagen fibers used in Example 1 and Example 2 of the present invention were prepared according to the invention patent named "A Mineralized Collagen Nanofiber Doped with Active Elements and Its Preparation Method" (publication number: CN113913961B, publication date: May 16, 2023).

[0053] Example 1

[0054] Dissolve 2 g of PVA in 50 mL of deionized water. The oil bath temperature is 90 degrees Celsius until it is completely dissolved until the solution is transparent and clear to obtain a PVA aqueous solution. Add 45 g of wet mineralized collagen fibers to the PVA aqueous solution and stir for 36 h until there are no visible lumps to the naked eye and the solution shows an obvious milky white viscous mixed slurry as Figure 1 shown. This mixed slurry is the pre - freezing precursor solution. Let it stand for 30 min before use and there will be no precipitation effect. Before freezing, add 0.2 mL of 50% glutaraldehyde and stir quickly to ensure that PVA can be cross - linked before freezing to obtain the cross - linked slurry;

[0055] Immerse one end of the T - type platform into liquid nitrogen to ensure a continuous cooling process. When the operating table surface temperature of the T - type copper plate is - 35 degrees Celsius, place the PDMS wedge - shaped mold on the operating table surface to ensure that the bottom of the PDMS wedge - shaped mold is in full contact with the surface of the T - type copper plate. Pour the cross - linked slurry into the PDMS wedge - shaped mold for freezing operation and wait for 35 min for cooling and solidification to obtain a block structure containing ice crystals. Among them, the size of the PDMS wedge - shaped mold is 20 * 20 * 45 mm; freeze - dry the block structure at a pressure of 5 Pa and a temperature of - 50 degrees Celsius to obtain the final parallel porous block structure as Figure 2 shown, which is the macroscopic morphology of the wedge - shaped parallel porous block structure; as Figure 3 shown, which is the microscopic morphology of the wedge - shaped parallel porous block structure; as can be seen from Figure 2-3 , it presents a porous structure arranged in parallel with each other.

[0056] Pressure forming is carried out using a circular or cuboid pressure mold. The pressure parameters are set to 900 MPa, the temperature is 85 °C, a stable pressure is applied for 10 min, and the pressure direction is perpendicular to the parallel plane of the parallel porous block structure. The multi-level ordered lamellar bone structure presents a milky white morphology and can be cut into different shapes to match the defect area, such as Figure 4 The block structure after pressure forming is shown, and it can be scaled up for production to obtain a larger block structure.

[0057] The formed structure obtained by the above pressure forming can be arbitrarily shaped and reprocessed. During the application of bone defect repair in animals, it is reprocessed into a columnar structure with a diameter of 3 mm, a height of 4 mm, and a central pore diameter of 0.8 mm to obtain bionic artificial dense bone. The bionic artificial dense bone is sterilized using cobalt-60 rays and stored aseptically.

[0058] Such as Figure 5 shown, is the gradual evolution process of the multi-level ordered structure in this embodiment, Figure 5 From left to right are the transmission electron microscope morphologies of the bionic mineralized collagen fibers used. It can be seen that they present a disordered distribution morphology, the transmission electron microscope morphology after the bidirectional freezing process makes the mineralized collagen fibers arranged neatly and parallel (the scale bar is 50 nm), the scanning electron microscope image of the neatly parallel arranged mineralized collagen fibers (the scale bar is 100 nm), the scanning electron microscope image of the parallel lamellar structure (the scale bar is 200 microns), and the morphology image after pressure forming. Compared with the real bone morphology, they are basically the same.

[0059] Figure 6 This is the mineral content of the bionic mineralized collagen fibers and the bionic artificial dense bone structure measured by a thermogravimetric analyzer in this embodiment. It can be seen that the mineral content of the bionic artificial dense bone reaches 70.3%;

[0060] Figure 7 This is the Raman spectrum of the mineralized collagen fibers and the bionic artificial dense bone in this embodiment. It can be seen from the figure that the internal crystal structures of the mineralized collagen fibers and the bionic artificial dense bone are the same and consistent with the crystal structure of bone.

[0061] Figure 8 This is the X-ray diffraction pattern of the bionic artificial dense bone prepared in this embodiment. It can be seen from the figure that the internal crystal structure of the bionic artificial dense bone is consistent with the crystal structure of bone.

[0062] Figure 9 This is the bending stress-strain curve diagram obtained by the three-point bending test of the bionic artificial dense bone in this embodiment, Figure 10 This is the statistical result diagram of the bending strength and bending modulus of the bionic artificial dense bone in this embodiment.

[0063] Figure 11This is a diagram showing the fracture toughness test of the bionic artificial dense bone in this embodiment using a single-edge notched beam specimen. The multi-level bionic artificial dense bone structure prepared in this embodiment has a relatively high KIC = 1.77 ± 0.01 MPa·m1 / 2 and KJC = 2.14 ± 0.041 MPa·m1 / 2. This indicates that the multi-level bionic artificial dense bone structure has excellent fracture toughness and the property of resisting crack propagation.

[0064] Figure 12 For the cytocompatibility test of the bionic artificial dense bone in this embodiment, the laser confocal microscope shows that rat bone marrow mesenchymal stem cells have good biocompatibility on the surface of the bionic artificial bone, with cell growth, migration, and a stretched state. Figure 13 This is a diagram of the CCK-8 experimental results. The CCK-8 results show that the cell proliferation and growth state is good.

[0065] During the animal experiment, a bone defect model with a diameter of 3 mm and a depth of 4 mm was established at the distal end of the humerus. The above-mentioned secondarily processed columnar structure was embedded in the defect, and samples were extracted and analyzed after 8 weeks and 12 weeks of repair. It can be found that the final repair effect is good, and new bone tissue has grown into the material, indicating that the bionic artificial bone has good biocompatibility.

[0066] Figure 14 The specimen in this embodiment was processed to form a columnar structure with a diameter of 3 mm and a height of 4 mm, and a 0.8-mm hole was drilled in the middle for animal experiments. Samples were taken at 8 weeks and 12 weeks respectively. A good repair effect was obtained at 12 weeks, and no inflammatory reaction occurred.

[0067] Figure 15 This is the morphology of the bionic bone material preparation method in the prior art after adding mineralized collagen fibers to PCL and freeze-drying. It has a porous structure, including PLLA, etc., and the macroscopic and microscopic morphologies are consistent.

[0068] Figure 16 For Figure 15 the microscopic morphology, the framework is mineralized collagen fibers, and the particles inside are aggregates of mineralized collagen fibers, with physical doping and a disordered state.

[0069] The specific preparation steps of the wet mineralized collagen fibers used in Example 1 of this embodiment are as follows:

[0070] (1) Dissolve type I collagen sponge in phosphoric acid aqueous solution. Add 0.5 g of collagen sponge to a phosphoric acid solution with a concentration of 6 mM, stir for 12 h, and maintain the stirring temperature at 37 degrees Celsius until the final solution is clear and transparent to obtain a phosphoric acid / collagen solution.

[0071] (2) Prepare a 10 mM calcium hydroxide solution: Add 10 mmol of calcium hydroxide to 2 L of ice water at 4 °C and stir to dissolve to obtain a calcium hydroxide solution;

[0072] (3) Prepare a Tris-HCl buffer solution: Dissolve 1 mM of Tris-HCL in 100 mL of water and adjust the pH value of the reaction cell between 8 and 10 with 0.5 mol / L ammonia water and HCl solution to obtain a reaction cell solution;

[0073] (4) Slowly add the phosphoric acid / collagen solution and the calcium hydroxide solution to the reaction cell solution at a dropping rate of 500 mL / h, maintain the pH between 8 - 10, the reaction temperature at 37 °C, seal the tube mouth, and stir and mineralize fully for 24 h to finally end the mineralization reaction.

[0074] (5) Wash and centrifuge the reacted liquid for standby. The washing process is achieved through multiple low-speed centrifugation processes. First, centrifuge at 2500 rpm, then measure the pH of the upper clear liquid; then add deionized water and pipette to mix evenly, and perform the same centrifugation speed and operation 3 - 5 times until the pH of the surface liquid = 7. Finally, centrifuge at 16500 rpm and remove the upper layer of water. The finally obtained mineralized collagen fibers are in the morphology as shown at the far left. To prepare mineralized collagen fibers doped with elements, only other cations or anions need to be incorporated, but the ion introduction amount needs to be less than 7%. The total molar ratio of cations to anions is 5:3. Figure 5 As shown at the far left. To prepare mineralized collagen fibers doped with elements, only other cations or anions need to be incorporated, but the ion introduction amount needs to be less than 7%. The total molar ratio of cations to anions is 5:3.

[0075] Example 2

[0076] On the basis of Example 1, when the pressure forming parameter is set to 500 MPa, the forming strength is about 50 MPa; when the pressure parameter is set to 800 MPa, the forming strength is about 70 - 80 MPa; when the pressure parameter is set to 1000 MPa, the forming strength is about 95 MPa.

[0077] Example 3

[0078] Dissolve 2 g of PVA in 50 mL of deionized water. The oil bath temperature is 90 °C until it is completely dissolved until the solution is transparent and clear to obtain a PVA aqueous solution. Add 8 g of freeze-dried mineralized collagen fibers to the PVA aqueous solution and stir to dissolve for 36 h until there are no visible lumps to the naked eye and the solution shows an obvious milky white viscous mixed slurry as shown. This mixed slurry is a frozen precursor solution. Let it stand for 30 min before use and there will be no precipitation effect. Before freezing, add 0.2 mL of 50% glutaraldehyde and stir quickly to ensure that PVA can be cross-linked before freezing to obtain a cross-linked slurry; Figure 1 As shown. This mixed slurry is a frozen precursor solution. Let it stand for 30 min before use and there will be no precipitation effect. Before freezing, add 0.2 mL of 50% glutaraldehyde and stir quickly to ensure that PVA can be cross-linked before freezing to obtain a cross-linked slurry;

[0079] One end of the T-shaped platform is immersed in liquid nitrogen to ensure a continuous cooling process. When the operating table surface temperature of the T-shaped copper plate is -35 °C, place the PDMS wedge-shaped mold on the operating table surface to ensure that the bottom of the PDMS wedge-shaped mold is in full contact with the surface of the T-shaped copper plate. Pour the cross-linked slurry into the PDMS wedge-shaped mold for freezing operation, and wait for 35 minutes for cooling and solidification to obtain a block structure containing ice crystals. Among them, the size of the PDMS wedge-shaped mold is 20*20*45 mm; freeze-dry the block structure at a pressure of 5 Pa and a temperature of -50 °C to form a porous scaffold with a stable porous structure and a density of 0.35 g / cm 3 , dry the obtained porous structure in a drying oven at 25 °C for 24 hours to ensure the removal of residual trace glutaraldehyde, and obtain a porous structure with a mineral content of 64%. After sterilization with cobalt-60, it can be directly used for bone defect filling, and the porosity is 85%.

[0080] Set the pressure parameter of the obtained porous structure to 1000 MPa for the porous structure, the temperature is 85 °C, apply a stable pressure for 10 minutes for pressure forming, and the pressure direction is perpendicular to the parallel plane of the porous structure. The multi-level ordered lamellar bone structure presents a milky white morphology and can be cut into different shapes to match the defect area.

[0081] The formed structure obtained by the above pressure forming can be arbitrarily shaped and secondary processed into a columnar structure for shape-matching bone defect repair applications. After sterilization with cobalt-60, it can be directly used for bone defect filling. It can be processed into various structures such as skull locks and skull plugs for bone defect filling. The density of the multi-level artificial dense bone structure with 64% mineral content is 2.01 g / cm 3 , and the porosity is less than 0.5%.

[0082] The specific preparation steps of the freeze-dried mineralized collagen fibers used in this example are as follows:

[0083] (1) Dissolve type I collagen sponge in an ultrapure aqueous phosphoric acid solution, where the concentration of the phosphoric acid solution is 6 mM, and 0.2 g of type I collagen sponge is dissolved in the phosphoric acid aqueous solution. During the stirring process, maintain the temperature of the system between 35 °C and 38 °C, and finally obtain a transparent liquid;

[0084] (2) Prepare an ultrapure aqueous calcium chloride solution, add 7% molar relative percentage of MgCl2 to the CaCl2 solution and stir and dissolve evenly (i.e., the molar ratio of magnesium ions to calcium ions is 7:93), and the total concentration is 10 mM.

[0085] Prepare a 20 mM NaOH deionized aqueous solution and add it to the solution containing Mg ions;

[0086] (3) Dissolve Tris-HCl with a concentration of 5 mM in 50 mL of water as a buffer solution. Adjust the pH value of the reaction kettle liquid between 8 and 10 using 0.5 mol / L ammonia water and HCl solution to obtain a reaction solution;

[0087] (4) Control the flow rate of the solutions prepared in (1) and (2) to be maintained at 450 ml / h, titrate them into the reaction solution, and maintain the pH between 8 and 10. After titration, control the reaction temperature at 37 °C, and stir well for 24 h under light avoidance;

[0088] (5) Transfer the system in (4) to a centrifuge tube for low-speed centrifugal washing and high-speed centrifugation to retain the precipitate, and finally obtain the washed precipitate at 17,000 revolutions per minute;

[0089] (6) Place the washed precipitate at a cold well temperature of -50 °C, with a vacuum of 5 Pa, and the freeze-drying duration is more than 50 h. After drying is completed, magnesium-doped nano-mineralized collagen is obtained. In the prepared strontium-doped mineralized collagen nanofibers, the stoichiometric ratio of calcium ions and magnesium ions to the stoichiometric ratio of the phosphate groups is 1.56:1; the obtained Mg-doped mineralized collagen fibers are white powders, and after sterilization with cobalt-60, they can be used as bone powder to fill bone defects.

[0090] Example 4

[0091] The preparation method of the biomimetic artificial compact bone based on mineralized collagen fibers in the present invention is specifically as follows:

[0092] Prepare a polyvinyl alcohol solution: Mix polyvinyl alcohol and deionized water in a ratio of 0.8 - 2 g: 50 - 100 mL, then heat to 85 - 95 °C, and stir while heating until the polyvinyl alcohol is completely dissolved and the solution becomes clear and transparent;

[0093] Mix the freeze-dried mineralized collagen fibers and the polyvinyl alcohol solution in a certain proportion and stir for 30 - 48 h until there are no visible lumps of aggregation to obtain a mixed slurry; among them, the mass ratio of the freeze-dried mineralized collagen fibers to polyvinyl alcohol is: 8 - 9.2 g: 0.8 - 2 g;

[0094] The mixed slurry is frozen by gradient freezing to form a wedge-shaped multi-layer block structure. Specifically: a T-shaped copper plate is horizontally placed on the upper end of a metal tank filled with liquid nitrogen, the lower end of the T-shaped copper plate is inserted into the liquid nitrogen in the metal tank, the upper surface of the T-shaped copper plate is the operating table surface, and the temperature of the operating table surface is -30°C to -45°C. Prepare a PDMS wedge-shaped mold, place the PDMS wedge-shaped mold on the operating table surface, at a position 20 - 40 mm away from the liquid nitrogen contact end of the T-shaped copper plate on the operating table surface. Then pour the mixed slurry into the PDMS wedge-shaped mold, add glutaraldehyde with a concentration of 50% dropwise to the mixed slurry and immediately stir, then pour it into the PDMS wedge-shaped mold, freeze for 25 - 35 min to form a frozen block structure. Then freeze-dry the frozen block structure at a pressure of 5 - 10 Pa and a temperature of -50°C for 48 - 72 h to form a stable multi-layer block structure. Then use the corresponding mold to hot-press the multi-layer block structure at a temperature of 80 - 90°C and a pressure of 500 - 1000 MPa for 10 min to form a multi-level bionic artificial dense bone structure with the corresponding shape; among them, the wedge angle is set to 15 - 20 degrees, and the size of the PDMS wedge-shaped mold is height * width * slope length = 20 - 25 mm * 20 - 25 mm * 40 - 60 mm; the dropping amount of glutaraldehyde with a concentration of 50% and the mass-volume ratio of polyvinyl alcohol is 0.1 - 0.2 mL: 0.8 - 2 g;

[0095] Then freeze the mixed slurry to obtain a multi-layer block structure, and then form a multi-level bionic artificial dense bone structure under appropriate temperature and pressure conditions.

[0096] Example 5

[0097] The preparation method of the bionic artificial dense bone based on mineralized collagen fibers of the present invention is specifically as follows:

[0098] Prepare a polyvinyl alcohol solution: Mix polyvinyl alcohol and deionized water according to 0.8 g: 50 mL, and then heat to 85°C, stirring while heating until the polyvinyl alcohol is completely dissolved and the solution becomes clear and transparent;

[0099] Mix the freeze-dried mineralized collagen fibers and the polyvinyl alcohol solution in a certain proportion and stir for 30 h until there are no visible lumps agglomerated by the naked eye to obtain a mixed slurry; among them, the mass ratio of the freeze-dried mineralized collagen fibers to polyvinyl alcohol is: 8 g: 0.8 g;

[0100] The mixed slurry is frozen using a gradient freezing method to form a wedge-shaped multi-layer block structure. Specifically: A T-shaped copper plate is horizontally placed on the upper end of a metal tank filled with liquid nitrogen. The lower end of the T-shaped copper plate is inserted into the liquid nitrogen in the metal tank. The upper surface of the T-shaped copper plate is the operating table surface, and the temperature of the operating table surface is -30°C. A PDMS wedge-shaped mold is prepared and placed on the operating table surface. The PDMS wedge-shaped mold is 20 mm away from the liquid nitrogen contact end of the T-shaped copper plate on the operating table surface. Then, the mixed slurry is poured into the PDMS wedge-shaped mold. After dropping glutaraldehyde with a concentration of 50% into the mixed slurry and immediately stirring, it is poured into the PDMS wedge-shaped mold. It is frozen for 25 min to form a frozen block structure. Then, the frozen block structure is freeze-dried at a pressure of 5 Pa and a temperature of -50°C for 48 h to form a stable multi-layer block structure. Then, the multi-layer block structure is hot-pressed at a temperature of 80°C and a pressure of 500 MPa for 10 min using a corresponding mold to form a multi-level bionic artificial dense bone structure with a corresponding shape; among them, the wedge angle is set to 15 degrees, and the size of the PDMS wedge-shaped mold is height * width * slope length = 20 mm * 20 mm * 40 mm; the dropping amount of glutaraldehyde with a concentration of 50% and the mass-volume ratio of polyvinyl alcohol is 0.1 mL:0.8 g;

[0101] The mixed slurry is frozen again to obtain a multi-layer block structure, and then the multi-layer block structure is formed into a multi-level bionic artificial dense bone structure under appropriate temperature and pressure conditions.

[0102] Example 6

[0103] The preparation method of the bionic artificial dense bone based on mineralized collagen fibers of the present invention is specifically as follows:

[0104] Prepare a polyvinyl alcohol solution: Mix polyvinyl alcohol and deionized water at a ratio of 2 g:100 mL, and then heat to 95°C, stirring while heating until the polyvinyl alcohol is completely dissolved and the solution becomes clear and transparent;

[0105] The freeze-dried mineralized collagen fibers and the polyvinyl alcohol solution are mixed and stirred in a certain proportion for 48 h until there are no visible lumps agglomerated by the naked eye to obtain a mixed slurry; among them, the mass ratio of the freeze-dried mineralized collagen fibers to polyvinyl alcohol is: 9.2 g:2 g;

[0106] The mixed slurry is frozen by gradient freezing to form a wedge-shaped multi-layer block structure. Specifically: a T-shaped copper plate is horizontally placed on the upper end of a metal tank filled with liquid nitrogen, the lower end of the T-shaped copper plate is inserted into the liquid nitrogen in the metal tank, the upper surface of the T-shaped copper plate is the operation table surface, and the temperature of the operation table surface is -45°C. Prepare a PDMS wedge-shaped mold, place the PDMS wedge-shaped mold on the operation table surface, and the PDMS wedge-shaped mold is 40 mm away from the liquid nitrogen contact end of the T-shaped copper plate on the operation table surface. Then pour the mixed slurry into the PDMS wedge-shaped mold, add glutaraldehyde with a concentration of 50% dropwise to the mixed slurry, immediately stir it, and then pour it into the PDMS wedge-shaped mold. Freeze for 35 min to form a frozen block structure, and then freeze-dry the frozen block structure at a pressure of 10 Pa and a temperature of -50°C for 72 h to form a stable multi-layer block structure. Then hot-press the multi-layer block structure with a corresponding mold at a temperature of 90°C and a pressure of 1000 MPa for 10 min to form a multi-level bionic artificial dense bone structure with a corresponding shape; among them, the wedge angle is set to 20 degrees, and the size of the PDMS wedge-shaped mold is height * width * slope length = 25 mm * 25 mm * 60 mm; the dropping amount of glutaraldehyde with a concentration of 50% and the mass-volume ratio of polyvinyl alcohol is 0.2 mL: 2 g;

[0107] Then freeze the mixed slurry to obtain a multi-layer block structure, and then form a multi-level bionic artificial dense bone structure under appropriate temperature and pressure conditions.

[0108] Example 7

[0109] The preparation method of the bionic artificial dense bone based on mineralized collagen fibers of the present invention is specifically as follows:

[0110] Prepare a polyvinyl alcohol solution: mix polyvinyl alcohol and deionized water at a ratio of 1 g: 80 mL, and then heat to 90°C, stirring while heating until the polyvinyl alcohol is completely dissolved and the solution becomes clear and transparent;

[0111] Mix and stir the wet mineralized collagen fibers and the polyvinyl alcohol solution in a certain proportion, and stir for 40 h until there are no visible lumps agglomerated by the naked eye to obtain a mixed slurry; among them, the mass ratio of the wet mineralized collagen fibers to the polyvinyl alcohol is: 46 g: 1 g;

[0112] The mixed slurry is frozen using a gradient freezing method to form a wedge-shaped multi-layer block structure. Specifically: A T-shaped copper plate is horizontally placed on the upper end of a metal tank filled with liquid nitrogen. The lower end of the T-shaped copper plate is inserted into the liquid nitrogen in the metal tank. The upper surface of the T-shaped copper plate is the operating table surface, and the temperature of the operating table surface is -40°C. Prepare a PDMS wedge-shaped mold and place the PDMS wedge-shaped mold on the operating table surface, 30 mm away from the liquid nitrogen contact end of the T-shaped copper plate on the operating table surface. Then pour the mixed slurry into the PDMS wedge-shaped mold. After dropping glutaraldehyde with a concentration of 50% into the mixed slurry and immediately stirring, pour it into the PDMS wedge-shaped mold. Freeze for 30 min to form a frozen block structure. Then freeze-dry the frozen block structure at a pressure of 8 Pa and a temperature of -50°C for 48 - 72 h to form a stable multi-layer block structure. Then hot-press the multi-layer block structure using a corresponding mold at a temperature of 85°C and a pressure of 800 MPa for 10 min to form a multi-level bionic artificial dense bone structure with a corresponding shape; among them, the wedge angle is set to 18 degrees, and the size of the PDMS wedge-shaped mold is height * width * slope length = 23 mm * 23 mm * 50 mm; the dropping amount of glutaraldehyde with a concentration of 50% and the mass-volume ratio of polyvinyl alcohol is 0.1 mL:1 g;

[0113] Freeze the mixed slurry again to obtain a multi-layer block structure, and then form a multi-level bionic artificial dense bone structure under appropriate temperature and pressure conditions.

[0114] Example 8

[0115] The preparation method of the bionic artificial dense bone based on mineralized collagen fibers of the present invention is specifically as follows:

[0116] Prepare a polyvinyl alcohol solution: Mix polyvinyl alcohol and deionized water at a ratio of 2 g:100 mL, and then heat to 95°C, stirring while heating until the polyvinyl alcohol is completely dissolved and the solution becomes clear and transparent;

[0117] Mix and stir the wet mineralized collagen fibers and the polyvinyl alcohol solution in a certain proportion for 40 h until there are no visible lumps of aggregation to obtain a mixed slurry; among them, the mass ratio of the wet mineralized collagen fibers to polyvinyl alcohol is: 48 g:2 g;

[0118] The mixed slurry is frozen using a gradient freezing method to form a wedge-shaped multi-layer block structure. Specifically: A T-shaped copper plate is horizontally placed on the upper end of a metal tank filled with liquid nitrogen. The lower end of the T-shaped copper plate is inserted into the liquid nitrogen in the metal tank. The upper surface of the T-shaped copper plate is the operating table surface, and the temperature of the operating table surface is -45°C. Prepare a PDMS wedge-shaped mold and place the PDMS wedge-shaped mold on the operating table surface, 40 mm away from the liquid nitrogen contact end of the T-shaped copper plate on the operating table surface. Then pour the mixed slurry into the PDMS wedge-shaped mold. After dropping glutaraldehyde with a concentration of 50% into the mixed slurry and immediately stirring, pour it into the PDMS wedge-shaped mold. Freeze for 35 minutes to form a frozen block structure. Then freeze-dry the frozen block structure at a pressure of 10 Pa and a temperature of -50°C for 72 hours to form a stable multi-layer block structure. Then hot-press the multi-layer block structure using a corresponding mold at a temperature of 90°C and a pressure of 1000 MPa for 10 minutes to form a multi-level bionic artificial dense bone structure with a corresponding shape; among them, the wedge angle is set to 20 degrees, and the size of the PDMS wedge-shaped mold is height * width * slope length = 25 mm * 25 mm * 60 mm; the dropping amount of glutaraldehyde with a concentration of 50% and the mass-volume ratio of polyvinyl alcohol is 0.2 mL: 2 g;

[0119] Then freeze the mixed slurry to obtain a multi-layer block structure, and then form a multi-level bionic artificial dense bone structure under appropriate temperature and pressure conditions.

[0120] Example 9

[0121] The preparation method of the bionic artificial dense bone based on mineralized collagen fibers of the present invention is specifically as follows:

[0122] Prepare a polyvinyl alcohol solution: Mix polyvinyl alcohol and deionized water at a ratio of 0.8 g: 50 mL, and then heat to 85 - 95°C, stirring while heating until the polyvinyl alcohol is completely dissolved and the solution becomes clear and transparent;

[0123] Mix and stir the wet mineralized collagen fibers and the polyvinyl alcohol solution in a certain proportion and stir for 30 h until there are no visible lumps agglomerated by the naked eye to obtain a mixed slurry; among them, the mass ratio of the wet mineralized collagen fibers to the polyvinyl alcohol is: 47 g: 1 g;

[0124] The mixed slurry is frozen using a gradient freezing method to form a wedge-shaped multi-layer block structure. Specifically: a T-shaped copper plate is horizontally placed on the upper end of a metal tank filled with liquid nitrogen, the lower end of the T-shaped copper plate is inserted into the liquid nitrogen in the metal tank, the upper surface of the T-shaped copper plate is the operating table surface, and the temperature of the operating table surface is -40°C. A PDMS wedge-shaped mold is prepared and placed on the operating table surface. The PDMS wedge-shaped mold is 40 mm away from the liquid nitrogen contact end of the T-shaped copper plate on the operating table surface. Then the mixed slurry is poured into the PDMS wedge-shaped mold. After dropping glutaraldehyde with a concentration of 50% into the mixed slurry and immediately stirring, it is poured into the PDMS wedge-shaped mold. It is frozen for 25 minutes to form a frozen block structure. Then the frozen block structure is freeze-dried at a pressure of 5 Pa and a temperature of -50°C for 48 hours to form a stable multi-layer block structure. Then the multi-layer block structure is hot-pressed at a temperature of 80°C and a pressure of 500 MPa for 10 minutes using a corresponding mold to form a multi-level bionic artificial dense bone structure with a corresponding shape; among them, the wedge angle is set to 15 degrees, and the size of the PDMS wedge-shaped mold is height * width * slope length = 20 mm * 20 mm * 40 mm; the dropping amount of glutaraldehyde with a concentration of 50% and the mass-volume ratio of polyvinyl alcohol is 0.1 mL:0.8 g;

[0125] Then the mixed slurry is frozen to obtain a multi-layer block structure, and then the multi-layer block structure is formed into a multi-level bionic artificial dense bone structure under appropriate temperature and pressure conditions.

Claims

1. A method for preparing bionic artificial dense bone based on mineralized collagen fibers, characterized in that: Specifically, the mineralized collagen fibers and polyvinyl alcohol solution are mixed in a certain proportion and stirred to form a milky white viscous liquid slurry as a mixed slurry, and then the mixed slurry is frozen to obtain a multi-layer block structure, and then the multi-layer block structure is formed into a multi-level bionic artificial dense bone structure under appropriate temperature and pressure conditions.

2. The method for preparing bionic artificial dense bone based on mineralized collagen fibers according to claim 1, characterized in that: The polyvinyl alcohol solution mixing process is as follows: Mix polyvinyl alcohol and deionized water at a ratio of 0.8-2 g: 50-100 mL, and heat to 85-95°C while stirring until the polyvinyl alcohol is completely dissolved and the solution becomes clear and transparent.

3. The method for preparing bionic artificial dense bone based on mineralized collagen fibers according to claim 2, characterized in that: The mineralized collagen fibers are freeze-dried mineralized collagen fibers or wet mineralized collagen fibers.

4. The method for preparing bionic artificial dense bone based on mineralized collagen fiber according to claim 3, characterized in that: If the mineralized collagen fibers are freeze-dried mineralized collagen fibers, the mass ratio of the freeze-dried mineralized collagen fibers to the polyvinyl alcohol is: 8-9.2 g: 0.8-2 g; If wet mineralized collagen fibers are used, the mass ratio of wet mineralized collagen fibers to polyvinyl alcohol is: 45g-48g:0.8-2g.

5. The method for preparing bionic artificial dense bone based on mineralized collagen fibers according to claim 4, characterized in that: The mixed slurry is frozen by a gradient freezing method to form a wedge-shaped multilayer block structure, specifically: A T-shaped copper plate is placed horizontally on the upper end of a metal tank filled with liquid nitrogen, and the lower end of the T-shaped copper plate is inserted into the liquid nitrogen in the metal tank. The upper end surface of the T-shaped copper plate is an operating table, and the temperature of the operating table is -30°C to -45°C. A PDMS wedge-shaped mold is prepared, and the PDMS wedge-shaped mold is placed on the operating table. Then, the mixed slurry is poured into the PDMS wedge-shaped mold, and 50% glutaraldehyde is added to the mixed slurry and immediately stirred and poured into the PDMS wedge-shaped mold. The mixture is frozen for 25-35 minutes to form a frozen block structure, and then the frozen block structure is freeze-dried at a pressure of 5-10Pa and a temperature of -50°C for 48-72 hours to form a stable multi-layer block structure. Then, the multi-layer block structure is hot-pressed for 10 minutes at a temperature of 80-90°C and a pressure of 500-1000MPa using a corresponding mold to form a multi-layer bionic artificial dense bone structure of a corresponding shape.

6. The method for preparing bionic artificial dense bone based on mineralized collagen fibers according to claim 5, characterized in that: The wedge angle of the PDMS wedge mold is set to 15-20 degrees.

7. The method for preparing bionic artificial dense bone based on mineralized collagen fibers according to claim 6, characterized in that: The PDMS wedge-shaped mold is placed on the operating table 20-40 mm from the end where the T-shaped copper plate contacts the liquid nitrogen.

8. The method for preparing bionic artificial dense bone based on mineralized collagen fibers according to claim 7, characterized in that: The dimensions of the PDMS wedge-shaped mold are height*width*slope length of 20-25mm*20-25mm*40-60mm.

9. The method for preparing bionic artificial dense bone based on mineralized collagen fibers according to claim 5, characterized in that: The mass volume ratio of the dropwise addition amount of 50% glutaraldehyde to polyvinyl alcohol is 0.1-0.2 mL: 0.8-2 g.

10. The method for preparing bionic artificial dense bone based on mineralized collagen fibers according to claim 9, characterized in that: After the mineralized collagen fibers and the polyvinyl alcohol solution are mixed, they are stirred for 30-48 hours until there are no visible agglomerations, thereby obtaining a mixed slurry.

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