Bio-ink for printing vascularized scaffolds and methods of making and using the same

Abstract

This invention provides a bio-ink for printing vascularized scaffolds, its preparation method, and its application. The bio-ink is either a substrate bio-ink or a sacrificial bio-ink. The substrate bio-ink comprises agarose, methacrylamide gelatin, methylcellulose, and LAP, wherein the concentration of agarose is 0.4%–0.6% wv, the concentration of methacrylamide gelatin is 5%–7% wv, the concentration of methylcellulose is 0.2%–0.4% wv, and the concentration of LAP is 0.25% wv. The sacrificial bio-ink comprises Pluronic acid and sodium alginate, wherein the concentration of Pluronic acid is 10%–30% wv, and the concentration of sodium alginate is 1%–3% wv. The substrate bio-ink has good mechanical properties and biocompatibility, while the sacrificial bio-ink has rapid molding and removal capabilities and good printability. It can be used for printing intraoperative bone repair vascularized scaffolds.

Description

Technical Field

[0001] This invention relates to the fields of tissue engineering and regenerative medicine, and in particular to a bio-ink for printing vascularized scaffolds, its preparation method, and its application. Background Technology

[0002] Skull defects can be caused by a variety of diseases, including trauma, tumors, and congenital bone abnormalities, and their treatment has always been considered a major challenge in the medical field. Traditional methods have limitations in the availability of grafts in practical applications and are prone to triggering strong immunogenic reactions. Tissue engineering technology offers new ideas and solutions for skull defect repair, but in vitro printing presents many problems, such as inconsistencies between external culture conditions and the human body's internal environment, mismatch between the implanted scaffold and the defect site, and potential complications like infection and rejection during surgery.

[0003] To address the challenges of in vitro printing, the following intraoperative printing strategy is currently employed: During surgery, a bioprinter is used to directly bioprint the target site on a living organism using cells and biomaterials. This technology allows for precise control of the printing process based on the patient's specific anatomy and lesion, ensuring a high degree of matching between the printed structure and surrounding tissue. Printing directly in the in vivo environment reduces the risk of infection during defect repair and provides a suitable developmental environment for the printed structure. Cells in the body can be naturally induced to migrate into the printed structure, promoting tissue regeneration. Vascularization, building upon intraoperative printing technology, further facilitates rapid bone defect regeneration. Vascularization plays a crucial role in the development, growth, and repair of tissues and organs, ensuring the supply of nutrients and oxygen, as well as the removal of metabolic waste. A biomimetic microchannel network is designed and constructed within the printed scaffold. These microchannels not only mimic the vascular system in natural bone tissue but also effectively enhance the transport efficiency of oxygen, nutrients, and metabolic waste, thereby accelerating and optimizing the bone regeneration process.

[0004] Intraoperatively printed vascularized scaffolds, as an emerging tissue engineering method, face a major obstacle in their development: the lack of suitable substrate bio-inks, sacrificial bio-inks, and corresponding printing processes. Bio-inks, defined as bioprintable media, include biomaterials, cells, and active factors. The growth of these living cells and factors depends on a "biomicroenvironment" composed of sufficient water, oxygen, nutrients, and a suitable pH. Currently, much of the development of bio-inks is geared towards in vitro printing. The environmental and time constraints of intraoperative printing mean that many bio-inks cannot meet the basic characteristics required for intraoperative printing. Intraoperative printing requires control of the printing and gelation temperatures of the bio-ink, a pH value that meets human requirements, and rapid prototyping to shorten surgical time. Achieving hollow vessels requires the preparation of sacrificial materials, and in vivo printed sacrificial materials need to simultaneously meet the requirements of rapid gelation under in vivo conditions and rapid, harmless removal. Most in vitro printed sacrificial materials are produced using heating or solvent washing methods, neither of which are suitable for intraoperative printing. Therefore, there is an urgent need to develop novel bio-inks and printing processes for both types of inks to meet the specific needs of intraoperative bioprinting. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a bio-ink for printing vascularized stents.

[0006] Another technical problem to be solved by the present invention is to provide a method for preparing the above-mentioned bio-ink for printing vascularized stents.

[0007] Another technical problem to be solved by the present invention is to provide the application of the above-mentioned bio-ink for printing vascularized stents.

[0008] The technical solution adopted in this invention is: A bio-ink for printing vascularized stents is a substrate bio-ink or a sacrificial bio-ink. The substrate bio-ink comprises agarose, methacrylamide gelatin, methylcellulose, and LAP (photoinitiator), wherein the concentration of agarose is 0.4%–0.6% wv, the concentration of methacrylamide gelatin is 5%–7% wv, the concentration of methylcellulose is 0.2%–0.4% wv, and the concentration of LAP is 0.25% wv. The sacrificial bio-ink comprises Pluronic acid and sodium alginate, wherein the concentration of Pluronic acid is 10%–30% wv and the concentration of sodium alginate is 1%–3% wv.

[0009] Preferably, in the above-mentioned bio-ink for printing vascularized stents, the substrate bio-ink has an agarose concentration of 0.5% wv, a methacrylamide gelatin concentration of 6% wv, a methylcellulose concentration of 0.2% wv, and a LAP concentration of 0.25% wv.

[0010] Preferably, in the above-mentioned bio-ink for printing vascularized stents, the sacrificial bio-ink has a Prönkel concentration of 30% wv and a sodium alginate concentration of 1% wv.

[0011] The above-mentioned method for preparing bio-ink for printing vascularized scaffolds includes the following specific steps for preparing the substrate bio-ink: (1) Dissolve agarose powder in double-distilled water and heat until the solution becomes clear and transparent; (2) When the solution cools to 50°C, add methylcellulose powder, continue heating and stirring at 50°C until fully dissolved to obtain a mixed solution, keep warm for later use; (3) Weigh out the methacrylated gelatin and add α-MEM medium to it. Heat and shake until the methacrylated gelatin is completely dissolved. (4) Add the prepared mixed solution to the methacrylamide gelatin solution and stir thoroughly at 45°C until homogeneous; (5) Add LAP solution and continue mixing in an environment protected from light and at a temperature of 40°C to obtain the final product.

[0012] The above-mentioned method for preparing bio-ink for printing vascularized stents includes the following specific steps for preparing sacrificial bio-ink: (1) Heat the double-distilled water to 50 degrees and add sodium alginate powder while stirring continuously until the sodium alginate powder is completely dissolved in the double-distilled water; (2) Keep the environment at 50°C, add Pluronic F-127 powder to the solution of dissolved sodium alginate, continue to stir thoroughly to ensure that the powder is evenly dispersed, and after stirring evenly, let it stand at 4°C for 24 hours to obtain the product.

[0013] The above-mentioned bio-ink for printing vascularized scaffolds is used in bone repair vascularized scaffolds.

[0014] Preferably, in the above application, the bone repair vascularization scaffold is an intraoperative bone repair vascularization scaffold.

[0015] Preferably, the specific molding and printing process for the above application is as follows: a spatially structured vascular network is established using SolidWorks. Based on the hierarchical and bifurcation characteristics of the vascular structure of the skull, a bifurcation biomimetic vascular structure is designed and printed. First, a layer of substrate bio-ink is printed. After the substrate gels, the vascular structure sacrificial bio-ink is printed. After cross-linking into a gel, another layer of substrate bio-ink is printed. Finally, the hollow vascular structure scaffold is obtained by rinsing with physiological saline. The scaffold is then irradiated with 405 nm blue light for a duration controlled between 10 and 30 seconds.

[0016] Preferably, in the above application, the bifurcated biomimetic blood vessel structure has a main vessel outer diameter of 0.4–0.85 mm, a branching angle of 80°–100°, and a bifurcated vessel outer diameter of <0.25 mm.

[0017] Preferably, in the above application, the printing parameters are: printing height of 0.1-0.5 mm, printing speed of 5-7 mm / s, extrusion layer height of 0.10-0.25 mm, extrusion ratio of 0.7; needle diameter of 0.16-0.33 mm, and needle length of 6.5-15 mm.

[0018] Preferably, in the above application, a printing needle with a diameter of 0.26 mm and a length of 15 mm is used. The printing temperature of the syringe is set to 37°C, the printing layer height is set to 0.15 mm, the extrusion ratio is 0.7, the printing speed is 6 mm / s, and the printing height is 0.4 mm. The printing is performed in the order of substrate bio-ink - sacrificial bio-ink - substrate bio-ink. After solidification, the substrate is rinsed with physiological saline at a low temperature (about 10°C). After each substrate layer is printed, it is photocrosslinked by irradiation with 405 nm blue light for 10 s.

[0019] The beneficial effects of this invention are: The aforementioned bio-inks for printing vascularized scaffolds provide a novel composition and precise ratio of substrate bio-inks and sacrificial bio-inks for intraoperative printing of vascularized structures. The substrate bio-ink possesses excellent mechanical properties and biocompatibility, while the sacrificial bio-ink exhibits rapid molding and removal capabilities and good printability. Furthermore, the design of vascularized structure models is provided, and the most suitable printing process parameters are selected for the sacrificial bio-ink to ensure the printing accuracy and quality of the model. These two bio-inks and printing processes have opened up a completely new path for intraoperative printing of vascularized scaffolds and brought new development opportunities to the fields of tissue engineering and regenerative medicine. Attached Figure Description

[0020] Figure 1 Scanning electron microscope image of the bio-ink substrate.

[0021] Figure 2 A statistical chart showing the swelling rate of 9 groups of substrate bio-inks.

[0022] Figure 3 The graph shows the degradation rate of bio-inks on nine substrates.

[0023] Figure 4 This is a flowchart of the vascularization structure formation process.

[0024] Figure 5 This is a diagram showing the results of an in vitro angiogenesis experiment.

[0025] Figure 6 This is a cross-linking process for bio-inks.

[0026] Figure 7 Experimental diagram for sacrificing the printability of bio-inks.

[0027] Figure 8 Front view and micrograph of the test results printed with bio-ink process parameters.

[0028] Figure 9 To print out the experimental process and results.

[0029] Figure 10 Printing images for animal experiments. Detailed Implementation

[0030] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0031] Example 1 Preparation of bio-inks for printing vascularized scaffolds First, add 10 ml of double-distilled water to an Erlenmeyer flask. Dissolve 0.05 g of agarose powder in the double-distilled water, then heat in a microwave oven until the solution becomes clear and transparent. Transfer the solution to a clean beaker and let it cool to 50°C. Add 0.04 g of methylcellulose powder and continue heating and stirring in a 50°C water bath until fully dissolved. Place this mixture in a water bath for later use. Next, select a light-protected centrifuge tube and accurately weigh 1.2 g of methacrylamide gelatin, then add 9 ml of α-MEM culture medium. Place the centrifuge tube in a water bath and heat and shake until the methacrylamide gelatin is completely dissolved. Add the previously prepared mixture to the methacrylamide gelatin solution and mix thoroughly in a 45°C water bath to ensure a homogeneous solution. After mixing thoroughly, add 1 ml of LAP solution and continue mixing in a light-protected environment at 40°C to complete the preparation of the substrate bio-ink. To strictly ensure the sterility of the bio-ink, the prepared bio-ink must undergo standardized processing. First, transfer the bio-ink into a sterile syringe and filter it through a 0.22-micron filter under a laminar flow hood for sterilization. Afterward, transfer the bio-ink to sterile and light-protected centrifuge tubes. Next, place the centrifuge tubes containing the bio-ink in a 37°C water bath to allow air bubbles to dissipate, and then store them for later use. Note that the entire process must be conducted in a light-protected environment.

[0032] Based on tests of gelation time and hydrogel formation at 37℃ for different ratios of bio-ink, it was found that when the agarose concentration ranged from 0.4% to 0.6% wv, the methacrylamide gelatin concentration ranged from 5% to 7% wv, the methylcellulose concentration ranged from 0.2% to 0.4% wv, and the LAP concentration ranged from 0.25% wt, the bio-ink exhibited the best gelation speed and printing effect at 37℃.

[0033] Nine groups of bio-inks with different contents were prepared using an orthogonal method, as shown in Table 1. Swelling and degradation characterization experiments were conducted by grouping to select the appropriate ratio. The swelling experiment method was as follows: The hydrogel prepared with the bio-ink was taken, and the initial mass was recorded. The sample was completely immersed in pre-prepared α-MEM medium and incubated at 37℃ in a CO2 incubator. Samples were removed at 3, 6, 12, 24, and 48 hours, and excess liquid was gently blotted with filter paper until no liquid seepage occurred. The weight was then recorded. The degradation experiment method was as follows: The hydrogel prepared with the bio-ink was frozen at -80℃ for 12 hours using a vacuum freeze dryer, followed by 12 hours of vacuum drying. The dry weight was recorded. The sample was completely immersed in PBS solution and incubated at 37℃ in a CO2 incubator. Samples were removed at 7, 14, and 21 days, freeze-dried again, and weighed. The degradation weight was recorded.

[0034] The microstructure of the substrate bio-ink gel was observed using SEM, and the scanning electron microscope images are shown below. Figure 1 As shown in the figure, the substrate bio-ink gel has a dense porous structure, satisfying both high mechanical strength and biocompatibility. The swelling degradation rate is statistically shown in the figure below. Figure 2 , Figure 3 As shown, the gel has a suitable swelling rate and a high degradation rate, which meets the design requirements.

[0035] Table 1

[0036] The following steps are required to prepare the sacrificial bio-ink: First, add 10 ml of double-distilled water to a clean beaker. Heat the beaker to 50°C in a water bath, then add 0.1 g of sodium alginate powder and stir continuously until the sodium alginate powder is completely dissolved in the double-distilled water. Next, maintaining the beaker at 50°C in the water bath, add 3 g of Pluronic F-127 powder to the dissolved sodium alginate solution and continue stirring thoroughly to ensure uniform powder dispersion. After thorough mixing, transfer the beaker to a refrigerator at 4°C and let it stand for 24 hours. This completes the preparation of the sacrificial bio-ink. Similarly, to ensure sterility, perform the same filtration and sterilization process as the base bio-ink. Transfer the sterilized bio-ink to a sterile centrifuge tube and store it at 4°C until use.

[0037] Sacrificial inks are used to print precise vascular structures. Based on tests of gel time and removal time in 10°C saline solution for sacrificial bio-inks with different formulations, it was found that when the Pluronic acid concentration ranged from 10% to 30% wv and the sodium alginate concentration ranged from 1% to 3% wv, the bio-ink exhibited faster gelation and removal rates at 37°C. To obtain the optimal formulation, nine groups of bio-inks with different concentrations were prepared, as shown in Table 2. Some printability experiments are as follows: Figure 7 As shown, the printability of bio-inks was evaluated by processing the print results using image analysis software to obtain parameters such as print spread and aspect ratio. The results showed that the bio-ink exhibited the best printability when the Prönkel concentration was 30% wv and the sodium alginate concentration was 1% wv.

[0038] Table 2

[0039] Print using the above-mentioned bio-ink: (1) Construction of the composite molding structure model of the support A spatially structured vascular network was established using SolidWorks, based on the hierarchical and bifurcation characteristics of cranial vascular structures, with an average curvature of 0.008-0.08 rad. Considering the limitations of in-situ printing difficulty, extrusion printing precision, and methods for removing sacrificial materials in vivo, a bifurcation biomimetic vascular structure was designed. The outer diameter of the main vessel is approximately 0.4–0.85 mm, the branching angle ranges from approximately 80° to 100°, and the outer diameter of the bifurcation vessels is <0.25 mm. The structural fabrication flowchart is shown below. Figure 4 As shown, first, a layer of substrate bio-ink is printed. After the substrate gels, sacrificial bio-ink for vascular structures is printed. This is then cross-linked into a gel, and another layer of substrate bio-ink is printed. Finally, the hollow vascular scaffold is obtained by rinsing with physiological saline at a low temperature (approximately 10°C). In vitro printing experiments verify the results. Figure 5 As shown, the sacrificial ink staining process was used for a clearer demonstration, revealing a clear hollow blood vessel structure, proving the feasibility of the design method.

[0040] (2) Intraoperative deposition process of bio-ink The printing process of bio-inks directly affects the performance of the scaffold, and thus its functionality. Therefore, this study investigated the effects of the concentration of each component on the printability of bio-inks, including extrudability, print fidelity, and print accuracy.

[0041] Based on the design model, the two bio-inks employ different cross-linking methods. The bio-ink mixed with cells requires sufficient mechanical strength for support while maintaining a certain degree of biocompatibility, thus employing a dual cross-linking method. The cross-linking conditions for the bio-inks are as follows: Figure 6As shown, the printing process begins with temperature cross-linking. Since both the printing needle and the target area are maintained at the human physiological temperature of 37°C, the ink enters a pre-cross-linked state, ensuring uniform cell distribution and effectively preventing cell sedimentation and aggregation. While temperature cross-linking, as a physical method, causes minimal damage to cells, the mechanical strength of the scaffold after cross-linking still falls short of expectations. Therefore, after printing, photocross-linking is further employed, using 405 nm blue light for irradiation for 10 to 30 seconds. This cross-linking technology significantly enhances the structural stability of the printed scaffold while improving printing resolution and accuracy. Sacrificial bio-inks require rapid gelation and degelation with high printing precision; therefore, temperature-based single cross-linking is used, also involving pre-cross-linking during the printing process and rapid gelation after printing.

[0042] (3) Printing parameters The printing parameters were adjusted and optimized based on the designed printing model. First, the temperature of the Luer syringe was adjusted to physiological temperature, and the prepared bio-ink was loaded into the Luer syringe of the robotic arm system. The vascular model was sliced ​​using Simplify3D software. Various printing parameters, including printing layer height, printing speed, extrusion layer height, and extrusion ratio, were set in the slicing software. Experiments were conducted to optimize the printing parameters. After printing a double-layer orthogonal network structure, the structural accuracy and fidelity were quantitatively analyzed, and the width and height of the printed filaments and pore area were measured. After printing, the printed model was photographed using a microscope, and the Pr value and printing error were measured to evaluate the printing effect. The results are as follows: Figure 8 As shown in the figure. The optimal printing parameters for the bio-ink were obtained through the above experiments: printing height of 0.1–0.3 mm, printing speed of 5–7 mm / s, extrusion layer height of 0.10–0.25 mm, extrusion ratio of 0.7; needle diameter of 0.16–0.33 mm, and needle length of 6.5–15 mm.

[0043] Example 2 Preparation of vascular stents containing vascular growth factor (VEGF) 1. Preparation of substrate bio-ink: 1) Preparation of Agarose: Weigh 0.10g of agarose powder and place it in a thoroughly cleaned 20ml Erlenmeyer flask. Add 10ml of double-distilled water. Then, tightly wrap the mouth of the Erlenmeyer flask with aluminum foil and heat it in a microwave oven. When the solution begins to boil slightly, quickly remove the flask and gently shake it a few times. Then return it to the microwave oven and continue heating until the solution becomes clear and transparent. Transfer the clear agarose solution to a clean beaker and place the beaker in a water bath set to 50℃. Weigh 0.06g of methylcellulose powder. When the agarose solution cools to 50℃, add hyaluronic acid powder while continuously stirring with a glass rod until the powder is completely dissolved. Finally, place the beaker in the water bath to allow any air bubbles to escape naturally.

[0044] 2) Preparation of Methacrylated Gelatin: Take a clean brown centrifuge tube and weigh 0.7g of methacrylated gelatin into it. Add 5ml of α-MEM solution to the centrifuge tube. Place the centrifuge tube in a 5℃ water bath to dissolve. During the dissolution process, remove the centrifuge tube and gently shake it several times as needed. After the methacrylated gelatin is completely dissolved, add 5ml of a pre-mixed solution of agarose and hyaluronic acid to the brown centrifuge tube. Then, pipette 350μl of 3% LAP solution and add it to the centrifuge tube. Shake the centrifuge tube in a 45℃ water bath to mix thoroughly, then let it stand for a period of time to allow air bubbles to escape naturally. Load 10ml of pre-prepared bio-ink into a sterile syringe. Filter the solution through a 0.22μm filter into a sterile brown centrifuge tube, seal the centrifuge tube opening with sealing film, and place it in a 37℃ water bath to remove air bubbles before use.

[0045] 3) Add growth factors to prepare rabbit vascular vascular growth factor. Resuspend the growth factor in sterile pre-made bio-ink at a concentration of 2 ng / ml. Use a pipette to carefully pipette repeatedly to obtain bio-ink containing mixed vascular vascular growth factor.

[0046] 2. Preparation of sacrificial bio-ink: 1) To prepare sodium alginate, add 10 ml of double-distilled water to a clean beaker, heat it to 50 degrees Celsius in a water bath, add 0.1 g of sodium alginate and stir continuously with a stirring tool until the sodium alginate powder is completely dissolved in the double-distilled water.

[0047] 2) Preparation of Pluronic: Add 3g of Pluronic F-127 powder to the solution in a 50℃ water bath and continue stirring thoroughly to ensure uniform dispersion of the powder. After stirring evenly, transfer the beaker to a 4℃ refrigerator and let it stand for 24 hours. Load the bio-ink into a sterile syringe, filter the solution through a 0.22μm filter into a sterile centrifuge tube, seal the centrifuge tube opening with sealing film, and place it in a 4℃ refrigerator to remove air bubbles for later use.

[0048] 3. Intraoperative bioprinting: To simulate the intraoperative printing environment, a 12×12mm defect model was constructed using a rat head model. A circular defect with a diameter of 10mm and a depth of 2mm was then built on the defect model. To simulate the real environment of the target area during intraoperative printing, a heating plate was placed at the bottom of the defect model to maintain the physiological temperature during the printing process.

[0049] First, bio-ink containing angiogenic factors was loaded into a sterile Luer syringe of the bioprinter. A 0.26mm printing needle with a 15mm needle length was used, with an extrusion ratio of 0.7. The ink was extruded evenly until the defect surface was completely covered. After printing, the area was irradiated with 405nm blue light for 10 seconds for photocrosslinking. Next, a sterile Luer syringe containing sacrificial bio-ink was used. A 0.26mm printing needle with a 15mm needle length was used, with an extrusion ratio of 0.7, a printing speed of 6mm / s, and a layer height of 0.15mm to print the vascular structure. After solidification, the first step was repeated. After complete solidification, the area was rinsed with 10℃ saline to obtain a hollow vascular stent. The experimental printing process and results are as follows: Figure 9 As shown.

[0050] Skull defect modeling was performed using New Zealand white rabbits weighing approximately 2 kg. After intraperitoneal anesthesia, the hair in the skull area was removed, and the surgical area was disinfected with povidone-iodine solution. Then, a 12 mm incision was made along the midline of the skull. The skin was separated to expose and peel off the periosteum. Using a 5 mm diameter skull drill, the skull surface was gradually worn down until a complete circular defect was created, preserving a small portion of the skull bone. Forceps were then used to test for looseness, and the skull bone was removed along the periphery of the circular defect. The area was immediately rinsed with saline solution to ensure no bone fragments remained and the dura mater was undamaged, indicating successful modeling. A hollow vascular stent was printed intraoperatively using the same parameters and procedures as in the simulation experiment. The printed result is shown in the image below. Figure 10 As shown in the image. The entire experimental procedure took approximately 30 minutes. The rabbits were in good condition post-surgery. The hollow structure of the scaffold can be seen in the image, demonstrating that rapid gelation and harmless removal under in vivo conditions are feasible.

[0051] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A bio-ink for printing vascularized stents, characterized in that: The substrate bio-ink or sacrificial bio-ink comprises agarose, methacrylamide gelatin, methylcellulose, and LAP, wherein the concentration of agarose is 0.4%–0.6% wv, the concentration of methacrylamide gelatin is 5%–7% wv, the concentration of methylcellulose is 0.2%–0.4% wv, and the concentration of LAP is 0.25% wv; the sacrificial bio-ink comprises Pluronic acid and sodium alginate, wherein the concentration of Pluronic acid is 10%–30% wv and the concentration of sodium alginate is 1%–3% wv.

2. The bio-ink for printing vascularized stents according to claim 1, characterized in that: The substrate bio-ink contains 0.5% wv agarose, 6% wv methacrylamide gelatin, 0.2% wv methyl cellulose, and 0.25% wv LAP.

3. The bio-ink for printing vascularized stents according to claim 1, characterized in that: The sacrificial bio-ink contains 30% wv of Prönkel and 1% wv of sodium alginate.

4. The method for preparing the bio-ink for printing vascularized stents according to claim 1 or 2, characterized in that: The specific steps for preparing the substrate bio-ink are as follows: (1) Dissolve agarose powder in double-distilled water and heat until the solution becomes clear and transparent; (2) When the solution cools to 50°C, add methylcellulose powder, continue heating and stirring at 50°C until fully dissolved to obtain a mixed solution, keep warm for later use; (3) Weigh out the methacrylated gelatin and add α-MEM medium to it. Heat and shake until the methacrylated gelatin is completely dissolved. (4) Add the prepared mixed solution to the methacrylamide gelatin solution and stir thoroughly at 45°C until homogeneous; (5) Add LAP solution and continue mixing in an environment protected from light and at a temperature of 40°C to obtain the final product.

5. The method for preparing the bio-ink for printing vascularized stents according to claim 1 or 3, characterized in that: The specific steps for preparing sacrificial bio-ink are as follows: (1) Heat the double-distilled water to 50 degrees and add sodium alginate powder while stirring continuously until the sodium alginate powder is completely dissolved in the double-distilled water; (2) Keep the environment at 50°C, add Pluronic F-127 powder to the solution of dissolved sodium alginate, continue to stir thoroughly to ensure that the powder is evenly dispersed, and after stirring evenly, let it stand at 4°C for 24 hours to obtain the product.

6. The use of the bio-ink for printing vascularized scaffolds as described in any one of claims 1-3 in bone repair vascularized scaffolds.

7. The application according to claim 6, characterized in that: The specific molding and printing process steps are as follows: A spatially structured vascular network is established using SolidWorks. Based on the hierarchical and bifurcation characteristics of the vascular structure of the skull, a bifurcation biomimetic vascular structure is designed and printed. First, a layer of base bio-ink is printed. After the base gels, the vascular structure sacrificial bio-ink is printed. After cross-linking into a gel, another layer of base bio-ink is printed. Finally, the hollow vascular structure scaffold is obtained by rinsing with physiological saline. The scaffold is then irradiated with 405 nm blue light for 10–30 seconds.

8. The application according to claim 7, characterized in that: The bifurcated biomimetic blood vessel structure has a main vessel outer diameter of 0.4–0.85 mm, a branching angle of 80°–100°, and a bifurcated vessel outer diameter of <0.25 mm.

9. The application according to claim 7, characterized in that: The printing parameters are as follows: printing height is 0.1-0.5mm, printing speed is 5-7mm / s, extrusion layer height is 0.10-0.25mm, extrusion ratio is 0.7; needle diameter is 0.16-0.33mm, and needle length is 6.5-15mm.

10. The application according to claim 7 or 9, characterized in that: Using a printing needle with a diameter of 0.26 mm and a length of 15 mm, the printing temperature of the syringe was set to 37℃, the printing layer height was set to 0.15 mm, the extrusion ratio was 0.7, the printing speed was 6 mm / s, and the printing height was 0.4 mm. The printing was performed in the order of substrate bio-ink - sacrificial bio-ink - substrate bio-ink. After solidification, the substrate was rinsed with low-temperature saline. After each substrate layer was printed, it was photocrosslinked by irradiation with 405 nm blue light for 10 seconds.

Images