A collagen scaffold material based on 3D printing technology and in-situ mineralization and a preparation method thereof

Abstract

The application discloses a kind of in-situ mineralization collagen support material based on 3D printing technology and preparation method thereof, comprising the following steps: S1: using organic solvent to dissolve collagen, configure into preset concentration of collagen solution;S2: using purified water respectively configure preset concentration of calcium ion solution and phosphate ion solution;S3: in collagen solution, respectively join calcium ion solution and phosphate ion solution, configure as two kinds of in-situ mineralization 3D printing ink containing different mineral ions;S4: be loaded into different printing cartridge and be printed, after printing, the scaffold is placed in drying dish and dried for a preset time;S5: configure preset concentration of crosslinking solution, the scaffold is soaked, the obtained scaffold is washed with purified water, and the in-situ mineralization collagen support material is obtained after drying.Belongs to the technical field of bone implant, the structure can solve the problem that the general strength of existing degradable artificial bone material is low and there is no suitable pore size and porosity to meet the bone cell crawl replacement.

Description

Technical Field

[0001] This invention relates to the field of bone implant technology. Background Technology

[0002] In clinical practice, bone grafting is usually performed to repair and reconstruct the shape and function of the defect. With the increasing aging of the population, the frequent occurrence of traffic accidents and congenital bone injuries, the clinical demand for ideal bone defect repair materials is becoming more and more urgent. Bone grafting has become the second most in-demand graft after blood transfusion, and the demand is increasing year by year.

[0003] Currently, autologous bone, allogeneic / xenogeneic bone, and artificial bone materials such as biodegradable metals, bone cement, and artificial polymer scaffolds are often used to implant into the defect site to promote bone repair.

[0004] Autologous bone grafting is currently the most widely used method in clinical practice, but it is a treatment that sacrifices healthy bone tissue, causing new damage to the donor site, and the source of autologous bone is limited.

[0005] Although allogeneic or xenograft bone transplantation overcomes the limitations of limited sources and secondary trauma associated with autologous bone transplantation, it still carries the risk of post-transplant immune rejection and the potential for disease transmission.

[0006] To overcome the various problems associated with autologous bone, allogeneic bone, and xenogeneic bone transplantation, people have been dedicated to developing artificial bone materials.

[0007] However, existing artificial bone materials still face significant technical bottlenecks in practical applications: Existing biodegradable artificial bone materials generally have low strength and cannot provide effective mechanical support for bone defects. Existing artificial bone scaffolds lack suitable pore size and porosity, failing to meet the needs of bone cell migration and replacement to achieve bone growth. In terms of simulating the composition and microstructure of natural bone, existing preparation methods, such as directly adding nano-hydroxyapatite particles, have obvious limitations. It is difficult to achieve a uniform distribution of minerals within the material, which makes it impossible to construct an ideal micro-bionic scaffold structure, thus limiting its role in promoting the activity of osteoblast mesenchymal stem cells.

[0008] Therefore, there is an urgent need to develop an in-situ mineralized collagen scaffold material based on 3D printing technology and its preparation method to solve the problems in the existing technology. Summary of the Invention

[0009] The purpose of this invention is to provide an in-situ mineralized collagen scaffold material based on 3D printing technology and its preparation method, which can solve the problems of existing biodegradable artificial bone materials generally having low strength and lacking suitable pore size and porosity to meet the needs of bone cell crawling replacement. Moreover, it has a simple structure and is easy to use, thus solving the problems mentioned in the background art.

[0010] To achieve the above objectives, the present invention provides the following technical solution: A method for preparing in-situ mineralized collagen scaffold material based on 3D printing technology, by simulating the composition and microstructure of natural bone, achieves uniform mineral distribution in the scaffold and improves mechanical properties, and has a significant promoting effect on the proliferation, migration, and osteogenic differentiation of osteoblast mesenchymal stem cells. The preparation method includes the following steps: S1: Prepare collagen solution: Dissolve collagen in an organic solvent to prepare a collagen solution of a preset concentration; S2: Prepare ion solutions: Dissolve calcium salt and phosphate source separately in purified water to prepare calcium ion solution and phosphate ion solution of preset concentrations; S3: 3D printing ink preparation: Calcium ion solution and phosphate ion solution are added to collagen solution to prepare two in-situ mineralization 3D printing inks containing different mineral ions. S4: Alternating double-layer printing: In-situ mineralization 3D printing ink containing different ions is loaded into different printing barrels for printing. During the printing process, the preset angle is adjusted every two layers to build a support structure of a specific shape and size. After printing, the support is placed in a desiccant to dry for a preset time. S5: In-situ crosslinking: Prepare a crosslinking solution of a preset concentration, immerse the scaffold in the crosslinking solution for a preset time, wash the obtained scaffold with purified water, and dry it by freeze drying to obtain in-situ mineralized collagen scaffold material.

[0011] By adopting the above technical solution, the overall function of simulating the composition and microstructure of natural bone is realized. By separating the ion source and combining double-layer printing, the minerals inside the scaffold are evenly distributed and the mechanical properties are improved, ultimately promoting the proliferation, migration and differentiation of osteoblast mesenchymal stem cells.

[0012] As a further aspect of the present invention: in S1, the organic solvent is hexafluoroisopropanol, with a preset concentration of 25%.

[0013] By adopting the above technical solution, the overall function of providing a stable dissolution environment for collagen is achieved. Through specific organic solvent and concentration ratio, the optimal matrix rheological properties are ensured for subsequent ion binding and printing processes.

[0014] As a further aspect of the present invention: in S2, the calcium salt is anhydrous calcium chloride, and the phosphoric acid source is a concentrated phosphoric acid solution.

[0015] By adopting the above technical solution, the function of providing an ion source with high solubility and high ionization degree is realized. Through the combination of specific calcium salt and phosphate source, the formation of low-solubility byproducts is prevented in the ink preparation stage, ensuring the smooth progress of the in-situ mineralization reaction.

[0016] As a further aspect of the present invention: in step S3, calcium ion solution and phosphate ion solution are added to collagen solution respectively, diluted to a stable solution of 200mM, and NaOH solution is added to adjust the pH of the solution to 7.4.

[0017] By adopting the above technical solution, the overall function of precisely controlling ion balance and ink state is realized. Through specific concentration dilution and pH adjustment, the mixed ink presents a stable milky white state without precipitation, ensuring the continuity of the printing process.

[0018] As a further aspect of the present invention: in S4, the printing parameters include a line spacing of 0.4 cm and an interlayer rotation angle of 90°.

[0019] By adopting the above technical solution, the function of constructing a regular interlaced channel structure is realized. By coordinating specific line spacing and rotation angle, the micro-layered structure of natural bone is imitated, providing the most suitable physical pores for cell crawling and nutrient transport.

[0020] As a further aspect of the present invention: in S4, the in-situ mineralization 3D printing inks containing different ions all use 27G needles, wherein the printing pressure of the phosphorus-containing in-situ mineralization 3D printing ink is 220kPa and the moving speed is 8mm / s, and the printing pressure of the calcium-containing in-situ mineralization 3D printing ink is 300kPa and the moving speed is 10mm / s.

[0021] By adopting the above technical solution, the function of customizing extrusion kinetics for different ionic inks is realized. By matching specific needle specifications, pressure and speed parameters, it is ensured that both differentiated inks can be accurately deposited and maintain line shape.

[0022] As a further aspect of the present invention: in step S4, the support is placed in a desiccant and dried for 48 hours.

[0023] By adopting the above technical solution, the function of removing solvent without damaging the microstructure is realized. Through static drying for a specific period of time, the geometric dimensions and shape of the printed bracket are kept absolutely stable before cross-linking.

[0024] As a further aspect of the present invention: in S5, the crosslinking solution is prepared by mixing 0.02 mmol / L N-hydroxysuccinimide ethanol solution and 0.02 mmol / L 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride ethanol solution in a ratio of 5:3.

[0025] By adopting the above technical solution, the function of establishing a stable covalent network between collagen fibers is realized. By using a specific concentration of two-component crosslinking agent, the structural integrity and mechanical toughness of the scaffold are significantly enhanced without introducing impurities.

[0026] As a further aspect of the present invention: in step S5, the stent is immersed in a crosslinking solution for 2 hours for crosslinking.

[0027] By adopting the above technical solution, the overall function of precisely controlling the degree of cross-linking reaction is realized. By limiting the soaking time, the scaffold can be ensured to achieve a complete structural enhancement effect, while avoiding scaffold brittleness or loss of bioactivity caused by excessive cross-linking.

[0028] This invention also discloses an in-situ mineralized collagen scaffold material based on 3D printing technology. The material is prepared according to the above-mentioned method for preparing an in-situ mineralized collagen scaffold material based on 3D printing technology. The in-situ mineralized collagen scaffold material is characterized by the in-situ mineralization of the material by introducing an ion solution to construct a micro-bionic scaffold structure with a uniform distribution of minerals inside. Furthermore, the fiber is precisely arranged through a double-layer alternating printing technology to form a scaffold structure with a specific shape and size.

[0029] By adopting the above technical solution, the material has been successfully implanted as a high-quality bone repair carrier. It integrates a microscopic biomimetic mineralization structure with a macroscopically precisely arranged fiber morphology, possesses specific shape and size and excellent mechanical strength, and directly meets the needs of bone defect repair.

[0030] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention successfully constructs a micro-bionic scaffold structure by introducing calcium ion and phosphate ion solution for in-situ mineralization, so that minerals are evenly distributed inside the scaffold, effectively simulating the composition and microstructure of natural bone, and overcoming the defect of single composition of traditional artificial bone materials.

[0031] 2. This invention uses a double-layer alternating printing technology, combined with specific line spacing and rotation angle, to achieve precise fiber arrangement and construct a scaffold structure with specific shape, size and suitable pore size and porosity, which meets the personalized needs of bone repair and the physical conditions for cell crawling replacement.

[0032] 3. This invention uses in-situ cross-linking treatment with a specific cross-linking system to form a stable network inside the scaffold, which significantly improves the overall mechanical properties and toughness, provides an excellent microenvironment for osteogenic mesenchymal stem cells, and significantly promotes cell proliferation, migration and osteogenic differentiation, thereby achieving high-quality bone regeneration.

[0033] Other features and advantages of the present invention will be disclosed in detail in the following detailed description and accompanying drawings. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the steps involved in the preparation of an in-situ mineralized collagen scaffold material based on 3D printing technology and its preparation method, as described in an embodiment of the present invention. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] This approach starts by biomimetic construction of natural bone structures and uses 3D printing technology to create in-situ mineralized collagen scaffolds to improve the quality of bone regeneration and repair.

[0037] In this embodiment of the invention, an in-situ mineralized collagen scaffold material based on 3D printing technology and its preparation method are described below. Figure 1 As shown, the process includes the following: calcium and phosphate ion solutions are added to collagen ink, respectively, to prepare two types of collagen-based bio-inks containing different mineral ions, and then in-situ mineralized collagen scaffolds are prepared by alternating double-layer 3D printing.

[0038] in: 1. Prepare collagen solution: Dissolve collagen in hexafluoroisopropanol to prepare a collagen solution with a concentration of 15%~45%; 2. Prepare ion solutions: Dissolve anhydrous calcium chloride in purified water to prepare a 0.2~1.0M calcium ion solution; dilute phosphoric acid in purified water to prepare a 0.1~0.5M phosphate ion solution. 3. 3D printing ink preparation: Add calcium ion solution or phosphate ion solution to collagen solution to prepare stable in-situ mineralization 3D printing ink. The concentration of calcium ion and phosphate ion should be in a specific ratio to form a complex ionic compound with a specific ratio. The ion source should have high solubility and high ionization degree, and should not react to form byproducts with low solubility and low ionization degree. If calcium ion solution and phosphate ion solution are added to the same collagen solution at the same time, the high concentration of calcium and phosphate ions will instantly exceed the solubility product and react violently inside the barrel in advance to generate a large number of large-sized hydroxyapatite precipitates. This causes the ink to lose its fluidity and shear thinning properties, and completely clogs the printing needle.

[0039] Therefore, collagen ink containing pure calcium ions and collagen ink containing pure phosphate ions should be prepared separately.

[0040] In addition, in calcium- or phosphorus-containing inks alone, the system is in a metastable state due to the lack of corresponding reactive anions / cations. At this time, ions combine with charged groups such as carboxyl and amino groups on the collagen molecular chain through electrostatic interaction to form a milky white and stable colloidal solution without precipitation. This state retains ionic activity and meets the rheological requirements of 3D printing.

[0041] Meanwhile, the specific ratio of calcium ion to phosphate ion concentrations includes the following: In order to ultimately form hydroxyapatite crystals Ca10(PO4)6(OH)2 with the same height as natural bone inside the scaffold, the molar ratio of calcium ions to phosphate ions needs to be calculated based on stoichiometry.

[0042] By extracting the molar ratio of calcium to phosphorus in hydroxyapatite, which is 10:6, and simplifying it to 1.67:1, the total molar amount of calcium ions in calcium-containing ink and the total molar amount of phosphate ions in phosphorus-containing ink are strictly set to a specific ratio of 1.67:1.

[0043] During alternating double-layer printing, the ion diffusion at the interface between the two layers follows this ratio, ensuring that the complex ionic compounds generated by in-situ mineralization precisely correspond to the crystal phase ratio of hydroxyapatite. 4. Alternating double-layer printing: 3D printing inks containing different ions are loaded into different printing barrels for printing. During the printing process, double-layer rotation is used, that is, the angle is adjusted once every two layers are printed. After printing, the support is placed in a desiccant to dry for 24h~72h. In this process, in-situ mineralization does not occur inside the barrel, but rather at the interface between the layers during the extrusion of the alternating double-layer printing.

[0044] When the first layer of calcium-containing ink and the second layer of phosphorus-containing ink are stacked alternately, calcium ions and phosphate ions diffuse across the interface through the concentration gradient at the contact surface of the two layers of lines.

[0045] On the surface where collagen molecular chains are enriched with negative charges, the calcium and phosphorus ions that diffuse and meet reach a local supersaturation state, spontaneously nucleate and grow, thus completing the mineralization process while the scaffold is being formed.

[0046] 5. In-situ crosslinking: Prepare a crosslinking solution by mixing 0.01~0.05mM N-hydroxysuccinimide ethanol solution and 0.01~0.05mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride ethanol solution in a ratio of 5:1~5:3. Immerse the scaffold in the crosslinking solution for 2~8 hours. Wash the obtained scaffold with purified water and freeze dry.

[0047] Example 1 S1: Dissolve 250mg of collagen in 1ml of hexafluoroisopropanol. After dissolving for 1 hour, stir with a magnetic stirrer for 5 hours to ensure that the collagen is uniformly dissolved into a 25% collagen solution. S2: Dissolve the anhydrous calcium chloride and concentrated phosphoric acid solutions separately in deionized water; S3: Add to collagen solution and dilute to a stable 200mM solution. Continue stirring for 24 hours. When the solution turns milky white and there is no precipitate, add NaOH solution to adjust the pH of the solution to 7.4. Finally, stir the solution for 30 minutes to obtain stable in-situ mineralized 3D printing ink.

[0048] S4: Set the printing parameters to line spacing 0.4cm, interlayer rotation angle 90°, air cut-off speed 100mm / s, and trailing distance 5mm; The phosphorus-containing in-situ mineralization 3D printing ink uses a 27G needle, a printing pressure of 220kPa, and a moving speed of 8mm / s. The calcium-containing in-situ mineralization 3D printing ink uses a 27G needle, a printing pressure of 300kPa, and a moving speed of 10mm / s. The printed support was dried in a desiccator for 48 hours.

[0049] S5: Prepare 0.02 mmol / L N-hydroxysuccinimide ethanol solution and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride ethanol solution respectively, and prepare crosslinking agent in a 5:3 ratio. Immerse the scaffold in the crosslinking solution for 2 hours, wash the scaffold with purified water, and freeze dry to obtain a stable, continuous, regularly shaped in-situ mineralized collagen scaffold with certain strength and toughness.

[0050] Example 2 The technical features that distinguish this embodiment from Embodiment 1 are as follows: S1: Dissolve 450 mg of collagen in 1 ml of hexafluoroisopropanol and stir magnetically for 6 hours to prepare a 45% collagen solution.

[0051] S2: Dissolve anhydrous calcium chloride in deionized water to prepare a 1.0M calcium ion solution, and dilute phosphoric acid in deionized water to prepare a 0.5M phosphate ion solution.

[0052] S3: Add the above calcium ion solution and phosphate ion solution to two equal parts of the above collagen solution, respectively. Calculate the amount to be added according to the calcium-to-phosphorus molar ratio of 1.67:1. Dilute and stir until a stable solution is reached. Add NaOH solution to adjust the pH to 7.4.

[0053] S4: Use double-layer alternating printing with parameters set as follows: line spacing 0.4cm, interlayer rotation angle 90°, printing pressure of phosphorus ink 250kPa, moving speed 9mm / s, printing pressure of calcium ink 320kPa, moving speed 11mm / s, drying time 48h.

[0054] S5: Prepare a 0.05mM crosslinking solution at a ratio of 5:1, crosslink for 8 hours, wash, and then freeze dry.

[0055] The high-concentration collagen scaffold produced has a denser fiber network, making it more suitable for repairing bone defects in weight-bearing areas compared to existing technologies.

[0056] Example 3 The technical features that distinguish this embodiment from Embodiment 1 are as follows: S1: Prepare collagen solution Consistent with the original example, 250 mg of collagen was dissolved in 1 ml of hexafluoroisopropanol and stirred to form a 25% collagen solution, which was then divided into two equal portions for later use.

[0057] S2: Prepare ion stock solution Anhydrous calcium chloride was dissolved in purified water to prepare a 1.0 M calcium ion stock solution; phosphoric acid was diluted with purified water to prepare a 0.5 M phosphate ion stock solution.

[0058] S3: Configure 3D printing ink Since the main inorganic component of natural bone is hydroxyapatite Ca10(PO4)6(OH)2, in order to achieve perfect in-situ biomimetic mineralization, it is necessary to ensure that the molar ratio of free calcium ions to phosphate ions at the printing contact interface strictly follows the stoichiometric ratio of hydroxyapatite, that is, the calcium-phosphorus molar ratio is 1.67:1.

[0059] Based on this logic, the final concentrations of the two inks are precisely converted and prepared as follows: S31: Preparation of phosphorus-containing in-situ mineralization 3D printing ink: Take the above 0.5M phosphate ion stock solution, calculate the volume ratio and add it to one of the 25% collagen solutions, and then add purified water for dilution.

[0060] By precisely calculating the amount of water added, the final concentration of phosphate ions in the mixed ink system was precisely achieved to 200 mM. Stirring continued until the solution became a milky white color without any precipitate.

[0061] S32: Preparation of calcium-containing in-situ mineralization 3D printing ink: Take the above 1.0M calcium ion stock solution and add it to another 25% collagen solution, then add purified water for dilution.

[0062] To ensure that the calcium-to-phosphorus ratio at the interface reaches 1.67:1 during alternating printing, the amount of water added is precisely calculated so that the final concentration of calcium ions in the mixed ink system reaches 334mM. The calculation logic is: 200mM × 1.67 ≈ 334mM. Stirring continues until the solution becomes a milky white color without precipitate.

[0063] S33: pH adjustment: Add NaOH solution to the two inks with the final concentration determined above, and adjust the pH of both to 7.4. Finally, stir for 30 minutes to obtain two stable in-situ mineralization 3D printing inks with specific concentration ratios.

[0064] S4: Alternating double-layer printing The final concentrations of the above-mentioned calcium-containing in-situ mineralization 3D printing ink (334 mM) and phosphorus-containing in-situ mineralization 3D printing ink (200 mM) were respectively loaded into different printing cartridges.

[0065] Due to the slightly higher ion concentration and viscosity of calcium-containing inks, differentiated printing parameters were set: phosphorus-containing inks used a 27G needle with a printing pressure of 220kPa and a movement speed of 8mm / s; calcium-containing inks used a 27G needle with a printing pressure of 300kPa and a movement speed of 10mm / s.

[0066] The line spacing was 0.4 cm, and the interlayer rotation angle was 90°. The printed support was dried in a desiccator for 48 hours.

[0067] S5: In-situ crosslinking A crosslinking agent was prepared by mixing 0.02 mmol / L N-hydroxysuccinimide ethanol solution and 0.02 mmol / L 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride ethanol solution in a 5:3 ratio. After crosslinking for 2 hours, the mixture was washed and freeze-dried.

[0068] Comparative Example 1 The technical features that distinguish this comparative example from the embodiments are as follows: Anhydrous calcium chloride and concentrated phosphoric acid solution were added simultaneously to the same 25% collagen solution, so that the system directly contained calcium ions and phosphate ions. After stirring for 24 hours, a large amount of white flocculent precipitate was formed in the solution, and uniform ink could not be obtained.

[0069] Furthermore, forcibly loading the suspension containing a large amount of sediment into the printing cylinder for printing resulted in severe needle blockage when a pressure of 220 kPa was applied. The extrusion was intermittent, unable to form continuous lines, and even more unable to construct a support structure with a specific shape and size.

[0070] Comparative Example 2 The technical features that distinguish this comparative example from the embodiments are as follows: Two types of 3D printing inks containing calcium and phosphorus were used, exactly the same as in Example 1. However, in step S4, instead of adjusting the angle every two layers, the traditional 0° layer-by-layer orthogonal stacking printing was used, that is, the line direction of each layer was completely consistent and they were directly stacked. Other drying and crosslinking parameters remained completely consistent with those in Example 1.

[0071] Microstructural observation of the support in Comparative Example 2 revealed that although in-situ mineralization occurred, the lack of 90° interlayer rotation resulted in pores that were mainly interconnected in a single direction. The pore sizes exhibited severe polarization, with large and small pore regions clearly distinct, and a uniform pore network that could not be formed.

[0072] Compression tests showed that Comparative Example 2 had extremely low mechanical strength in the direction perpendicular to the lines, and obvious interlayer slippage and collapse occurred; cell proliferation experiments showed that, due to the lack of suitable interlacing pores, osteoblastic mesenchymal stem cells mainly aggregated on the surface, and the depth of migration into the scaffold was reduced by more than 60% compared with Example 1.

[0073] This invention provides an in-situ mineralized collagen scaffold material based on 3D printing technology and its preparation method, which can solve the problems that existing biodegradable artificial bone materials generally have low strength and lack suitable pore size and porosity to meet the needs of bone cell crawling replacement.

[0074] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0075] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing in-situ mineralized collagen scaffold material based on 3D printing technology, characterized in that, The preparation method includes the following steps: S1: Prepare collagen solution: Dissolve collagen in an organic solvent to prepare a collagen solution of a preset concentration; S2: Prepare ion solutions: Dissolve calcium salt and phosphate source separately in purified water to prepare calcium ion solution and phosphate ion solution of preset concentrations; S3: 3D printing ink preparation: Calcium ion solution and phosphate ion solution are added to collagen solution to prepare two in-situ mineralization 3D printing inks containing different mineral ions. S4: Alternating double-layer printing: In-situ mineralization 3D printing ink containing different ions is loaded into different printing barrels for printing. During the printing process, the preset angle is adjusted every two layers to build a support structure of a specific shape and size. After printing, the support is placed in a desiccant to dry for a preset time. S5: In-situ crosslinking: Prepare a crosslinking solution of a preset concentration, immerse the scaffold in the crosslinking solution for a preset time, wash the obtained scaffold with purified water, and dry it by freeze drying to obtain in-situ mineralized collagen scaffold material.

2. The method for preparing an in-situ mineralized collagen scaffold material based on 3D printing technology according to claim 1, characterized in that, In S1, the organic solvent is hexafluoroisopropanol, with a preset concentration of 25%.

3. The method for preparing an in-situ mineralized collagen scaffold material based on 3D printing technology according to claim 1, characterized in that, In S2, the calcium salt is anhydrous calcium chloride, and the phosphoric acid source is a concentrated phosphoric acid solution.

4. The method for preparing an in-situ mineralized collagen scaffold material based on 3D printing technology according to claim 1, characterized in that, In step S3, calcium ion solution and phosphate ion solution are added to collagen solution respectively, diluted to a stable solution of 200mM, and NaOH solution is added to adjust the pH of the solution to 7.

4.

5. The method for preparing an in-situ mineralized collagen scaffold material based on 3D printing technology according to claim 1, characterized in that, In S4, the printing parameters include a line spacing of 0.4 cm and an interlayer rotation angle of 90°.

6. The method for preparing an in-situ mineralized collagen scaffold material based on 3D printing technology according to claim 1, characterized in that, In S4, the in-situ mineralization 3D printing inks containing different ions all use 27G needles. The printing pressure of the phosphorus-containing in-situ mineralization 3D printing ink is 220 kPa and the moving speed is 8 mm / s. The printing pressure of the calcium-containing in-situ mineralization 3D printing ink is 300 kPa and the moving speed is 10 mm / s.

7. The method for preparing an in-situ mineralized collagen scaffold material based on 3D printing technology according to claim 1, characterized in that, In step S4, the support is placed in a desiccator and dried for 48 hours.

8. The method for preparing an in-situ mineralized collagen scaffold material based on 3D printing technology according to claim 1, characterized in that, In step S5, the crosslinking solution is prepared by mixing 0.02 mmol / L N-hydroxysuccinimide ethanol solution and 0.02 mmol / L 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride ethanol solution in a ratio of 5:

3.

9. The method for preparing an in-situ mineralized collagen scaffold material based on 3D printing technology according to claim 1, characterized in that, In step S5, the stent is immersed in a crosslinking solution for 2 hours for crosslinking.

10. An in-situ mineralized collagen scaffold material based on 3D printing technology, prepared and obtained according to the method for preparing an in-situ mineralized collagen scaffold material based on 3D printing technology according to claim 1, characterized in that, The in-situ mineralized collagen scaffold material is constructed by introducing an ion solution for in-situ mineralization to create a micro-bionic scaffold structure with a uniform distribution of minerals inside. Furthermore, the fiber arrangement is achieved through a double-layer alternating printing technology, forming a scaffold structure with a specific shape and size.

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