A high internal phase Pickering emulsion 3D printing ink and its preparation method

Abstract

This invention proposes a method for preparing 3D printing ink using a high-internal-phase Pickering emulsion. The method involves high-speed mixing of a continuous phase (75%-85% internal phase volume ratio) with a dispersed phase. Gelatin nanoparticles and hydroxyapatite are used as stabilizers to form a stable two-particle droplet interface, improving the stability of the Pickering emulsion ink. The introduction of animal gelatin into the continuous phase allows for control of ink viscosity, extending printable time and meeting the time requirements for gradient printing. This invention enables the 3D printing of gradient biological scaffolds using Pickering emulsion ink, exhibiting excellent printability, a long printing time window, good formability, a simple and mild preparation process, low raw material consumption, wide availability, and low cost. The material also has low toxicity risk, good stability, and biodegradability; furthermore, it possesses high pore size and adjustable mechanical properties, effectively promoting cell proliferation and adhesion into the scaffold, demonstrating significant application and promotion value.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering, specifically relating to a high internal phase Pickering emulsion 3D printing ink and its preparation method. Background Technology

[0002] Repairing damaged osteochondral tissue is extremely challenging due to the avascular nature of the joint microenvironment, which limits the supply of nutrients and progenitor cells, and the limited self-regenerative capacity of articular cartilage. 3D-printed scaffolds can be used to assist in osteochondral tissue regeneration, facilitating the design of biomimetic scaffold structures and allowing for the use of customized biomaterials to repair defects. The main design strategies for osteochondral scaffolds include single-layer (homogeneous), double-layer, multi-layer (more than two layers), and gradient scaffolds. 3D printing direct ink writing (DIW) technology meets the need for hierarchical pore structures in scaffolds through simple extrusion. Using suitable printing inks, pore structures from micrometers to millimeters can be easily customized, and the scaffold shape can be precisely controlled. The focus of 3D printing technology research is on developing printing inks with biocompatibility, mechanical strength, and shear-thinning properties. Furthermore, the inks should also possess printability (including shape fidelity, structural resolution, and cell viability).

[0003] Multimaterial extrusion techniques generally exhibit low mechanical properties, and some studies have explored combining other materials (such as hydroxyapatite) with bio-inks to enhance the mechanical strength of 3D-printed scaffolds. Researchers have reported a combined 3D bioprinting strategy that enables the co-extrusion of polymer-nanoparticle composite inks and hydrogel bio-inks, a strategy considered promising for application in its current form to non-load-bearing bones. Abudureheman Maihemuti proposed a 3D-printed porous multilayer scaffold based on cold-water fish skin gelatin for articular cartilage regeneration, combining cold-water fish skin gelatin with sodium alginate to increase viscosity, printability, and mechanical strength. However, these scaffolds require cross-linking to remain stable at room temperature, and they are still not ideal materials for completely replacing bone tissue in vivo.

[0004] Pickering emulsion is an emulsion prepared using interfacially wettable nanocolloidal particles as stabilizers, offering advantages such as high stability and low cost. Pickering emulsions can maintain stability for several years, and the stabilizer content is only 1%, far lower than the amount of traditional surfactants (10%-40%), thus avoiding the toxic side effects of large amounts of surfactants. The solid particulate material used as the stabilizer can be a natural polymer as the matrix material. Pickering emulsion has natural shear-thinning ability, and the emulsion template forms a multi-level microstructure, which can be used as 3D printing ink. The multi-layered pore structure is more conducive to the repair of osteochondral defects. By changing the composition of the continuous phase of the emulsion, adjusting the ink viscosity, and extending the printing time, it can be applied to 3D printing gradient scaffolds to promote integrated osteochondral repair.

[0005] To address the aforementioned technical issues, a 3D printing ink with high viscosity, good printability, high mechanical strength, and longer printing time, along with its preparation method, is urgently needed in this field. Summary of the Invention

[0006] In view of the above shortcomings, this invention proposes a method for preparing 3D printing ink using gelatin compounded with Pickering emulsion. The 3D printing ink prepared by this invention has better printability, a longer printing time window, good formability, and a simple and mild preparation process. The raw materials—fish, bovine gelatin, and hydroxyapatite—are used in low amounts, are widely available, and are inexpensive. The gradient bioscaffolds prepared by 3D printing using this ink are safe and non-toxic, have diverse pore structures, good stability and biodegradability, and their mechanical properties can be adjusted using a convenient post-printing cross-linking method. They can be used in bone tissue engineering to promote integrated osteochondral repair.

[0007] This invention also provides a method for preparing a high-internal-phase Pickering emulsion 3D printing ink. Gelatin nanoparticles and hydroxyapatite are used as stabilizers to form a stable dual-particle droplet interface, improving the stability of the Pickering emulsion ink. The addition of fish gelatin can adjust the ink viscosity to meet the printing time window requirements. The pore wall surface of the gradient biological scaffold obtained by 3D printing forms a micro-nano morphology composed of dual nanoparticles, which can endow the pore wall surface of the biological scaffold with functions such as promoting osteogenic and cartilage regeneration, showing good application prospects.

[0008] This invention is achieved through the following technical means:

[0009] This invention first discloses a method for preparing a high internal phase Pickering emulsion 3D printing ink, comprising the following steps:

[0010] A continuous phase with an internal phase volume ratio of 75%-85% and a dispersed phase are stirred and mixed at 10,000-15,000 rpm to obtain a high internal phase Pickering emulsion 3D printing ink; wherein:

[0011] The continuous phase is prepared by mixing distilled water, gelatin molecules, gelatin nanoparticles (GNPs), and hydroxyapatite (HA);

[0012] The dispersed phase is any one of n-hexane, benzene, toluene, p-xylene, and styrene.

[0013] Furthermore, the hydroxyapatite (HA) is prepared by the following method:

[0014] Dissolve 0.74 g of Ca(OH)2 in 200 mL of deionized water to make a suspension. Stir magnetically in a water bath for 10 min, then slowly add 120 mL of 85% phosphoric acid solution while stirring. Measure the pH of the mixed solution at the same time. After the phosphoric acid is added and the pH remains constant, age the mixed solution at room temperature for 24 h, then dry, sinter and grind to obtain hydroxyapatite (HA).

[0015] Furthermore, the gelatin nanoparticles (GNPs) are prepared by the following method:

[0016] Dissolve 1.25g of type B gelatin (i.e., bovine gelatin) in 25mL of distilled water and stir at 50℃ to form a gelatin solution. Add 25mL of acetone, remove the supernatant, redissolve the precipitate in 15mL of distilled water, and adjust the pH to 12.0. Add acetone again until a precipitate is formed. Then add 56mg of genipin to the solution and continue to react at 50℃ for 3h. Centrifuge the reacted solution at 10000g for 30min to obtain gelatin nanoparticles (GNPs).

[0017] Furthermore, the gelatin molecules are composed of a mixture of fish gelatin and bovine gelatin; the mass ratio of fish gelatin to bovine gelatin in the continuous phase is 1:3-4:0.

[0018] The present invention also discloses a high internal phase Pickering emulsion 3D printing ink prepared according to any of the above preparation methods.

[0019] This invention also discloses an application of the above-mentioned high internal phase Pickering emulsion 3D printing ink in gradient bioscaffold printing, comprising:

[0020] (1) After setting the 3D printing model and printing parameters, pour the high internal phase Pickering emulsion 3D printing ink into the syringe and perform 3D printing at room temperature. During the process of the printing ink being squeezed out from the needle, the Pickering emulsion ink undergoes a gel-sol-gel transformation. After the lower support is printed, replace the ink and continue printing the upper layer to obtain the finished product.

[0021] (2) After printing, the finished product is immersed in crosslinking agent for 6 to 24 hours to obtain 3D printed gradient biological scaffold.

[0022] Further, the printing parameters in step (1) include: extrusion speed and printing speed are both 7-15 mm / s, filament spacing is 0.8-2 mm, layer height is 0.5-1.2 mm, filament diameter is 0.6-1.2 mm, the temperature of the extrusion needle is controlled at 25-28℃ during printing, and the temperature of the base plate is controlled at 4-10℃.

[0023] Further, the crosslinking agent in step (2) is selected from either genipin or 1-ethyl-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide.

[0024] Further, the genipin accounts for 1% to 5% of the gelatin mass, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide accounts for 15% to 80% of the gelatin mass; in the 1-ethyl-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide, the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is (1 to 2):1.

[0025] This invention utilizes 3-10 mg / mL gelatin nanoparticles (GNPs) and 0-7 mg / mL hydroxyapatite (HA) as stabilizers throughout the continuous phase to prepare a highly stable high-internal-phase Pickering emulsion 3D printing ink. The gelatin molecular chain is rich in hydroxyl, carboxyl, and amino groups, which can impart gelation properties to the Pickering emulsion ink and facilitate its functionalization modification. However, gelatin molecules exhibit poor mechanical strength without cross-linking, failing to match the mechanical properties of environmental tissues. Therefore, cross-linking after printing, followed by immersion treatment of the gradient scaffold constructed based on gelatin molecules, significantly improves the mechanical properties of the scaffold.

[0026] This invention introduces both fish gelatin and bovine gelatin into the continuous phase, which helps to adjust ink viscosity, control printing time, and extend the printing window. Research has shown that when the mass ratio of fish gelatin to bovine gelatin reaches 1:1, the emulsion ink exhibits better printability, good formability, and strong shape fidelity, demonstrating promising applications in bone tissue engineering.

[0027] The beneficial effects of this invention are as follows:

[0028] (1) The high internal phase Pickering emulsion 3D printing ink prepared by this invention introduces animal gelatin into the continuous phase, which can control the ink viscosity, extend the printable time, and meet the time requirements of gradient printing. The emulsion ink has good stability, excellent extrudability, good formability, and shape fidelity.

[0029] (2) The high internal phase Pickering emulsion 3D printing ink prepared in this invention uses GNPs and HA as stabilizers to form a stable droplet interface with two particles, thereby improving the stability of the Pickering emulsion ink. Gelatin nanoparticles (GNPs) have good wettability and stability. The formation of the Pickering emulsion only requires a small amount of gelatin nanoparticles. The addition of HA gives the ink good shear-thinning properties, meeting the printability requirements.

[0030] (3) The 3D printed gradient biological scaffold prepared by the present invention is obtained by 3D printing the Pickering emulsion 3D printing ink at room temperature. The shape fidelity and structural stability of the scaffold can be precisely adjusted by changing the fish gelatin concentration, stabilizer dosage and printing parameters.

[0031] (4) The 3D-printed gradient biological scaffold prepared in this invention utilizes a Pickering emulsion template to form a multi-level interconnected pore structure inside the scaffold, which is more conducive to cell proliferation and adhesion. HA is deposited on the surface of the internal pores after freeze-drying, allowing direct contact with cells and promoting osteogenic differentiation. Combined with 3D printing technology, the scaffold is endowed with pore structures ranging from micrometers to millimeters, achieving personalized customization and rapid prototyping.

[0032] (5) The present invention uses genipin, a natural biological cross-linking agent, to cross-link the gradient scaffold, which will not produce substances harmful to organisms. By utilizing the slow cross-linking properties of genipin, the stability and mechanical properties of the 3D printed gradient scaffold are improved.

[0033] (6) This invention uses Pickering emulsion ink to 3D print gradient biological scaffolds, which have good printability, long printing time window, good formability, simple preparation process, mild conditions, low raw material consumption, wide availability, and low cost; the material has low toxicity risk, good stability and degradability; it also has high pore size and adjustable mechanical properties, which can effectively promote cell proliferation and adhesion into the scaffold, and has great application and promotion value. Attached Figure Description

[0034] Figure 1 The gel points of the Pickering emulsion 3D printing inks prepared in Example 1, Comparative Example 1, and Comparative Example 2;

[0035] Figure 2 Temperature window of Pickering emulsion 3D printing ink prepared in Example 1;

[0036] Figure 3 Images of the 3D-printed gradient biological scaffolds prepared in Examples 2 and 3;

[0037] Figure 4 Cell live / dead staining images of 3D printed scaffolds prepared in Examples 1, 2, and 3. Detailed Implementation

[0038] The present invention will be further described below with reference to specific embodiments. The advantages and features of the present invention will become clearer as the embodiments are described. However, the embodiments are merely exemplary and do not constitute any limitation on the scope of the present invention.

[0039] All materials used in the following embodiments are commercially available. The type B gelatin used in this invention is bovine gelatin, and the gelatin molecules used are a mixture of fish gelatin and bovine gelatin.

[0040] Example 1

[0041] Dissolve 1.25g of type B gelatin in 25mL of distilled water and stir at 50℃ to form a gelatin solution. Add 25mL of acetone, remove the supernatant, redissolve the precipitate in 15mL of distilled water and adjust the pH to 12.0. Add acetone again until a precipitate is formed. Then add 56mg of genipin (2%-5.5% of the gelatin mass) to the solution and continue to react at 50℃ for 3h. Centrifuge the reacted solution at 10000g for 30min to obtain gelatin nanoparticles (GNPs).

[0042] A 2.5 mL gelatin solution was prepared as the continuous phase, with a GNP concentration of 10 mg / mL, a fish gelatin molecular concentration of 125 mg / mL, and a bovine gelatin molecular concentration of 125 mg / mL. 7.5 mL of n-hexane was added as the dispersed phase. The two phases were stirred at 13500 rpm for 30 s to obtain a high internal phase Pickering emulsion 3D printing ink.

[0043] The printing model was set as a cuboid with a length of 20 mm, a width of 20 mm, and a height of 5 mm. The printing parameters were set as follows: extrusion speed and printing speed of 10 mm / s, needle diameter of 0.84 mm, filament spacing of 2 mm, and layer height of 0.5 mm. The extrusion needle temperature was 25°C, and the substrate temperature was 4°C. The Pickering emulsion ink was poured into a syringe, and 3D printing was performed at room temperature to obtain a 3D-printed porous biological scaffold. After printing, the scaffold was placed at 4°C for 15 hours and then immersed in genipin solution for 24 hours for later use.

[0044] Example 2

[0045] Dissolve 1.25g of type B gelatin in 25mL of distilled water and stir at 50℃ to form a gelatin solution. Add 25mL of acetone, remove the supernatant, redissolve the precipitate in 15mL of distilled water and adjust the pH to 12.0. Add acetone again until a precipitate is formed. Then add 56mg of genipin (2%-5.5% of the gelatin mass) to the solution and continue to react at 50℃ for 3h. Centrifuge the reacted solution at 10000g for 30min to obtain gelatin nanoparticles (GNPs).

[0046] 0.74 g of Ca(OH)₂ was dissolved in 200 mL of deionized water to prepare a suspension. The suspension was magnetically stirred in a water bath for 10 min. 120 mL of 85% phosphoric acid solution was slowly added dropwise while stirring. The pH of the mixed solution was measured at the same time. After the phosphoric acid was added and the pH remained unchanged, the mixed solution was aged at room temperature for 24 h, dried, sintered and ground to obtain hydroxyapatite (HA).

[0047] A 2.5 mL gelatin solution was prepared as the continuous phase, with GNPs concentration of 3 mg / mL, HA concentration of 7 mg / mL, fish gelatin molecular concentration of 125 mg / mL, and bovine gelatin molecular concentration of 125 mg / mL. 7.5 mL of n-hexane was added as the dispersed phase. The two phases were stirred at 13500 rpm for 30 s to obtain a highly internally stable Pickering emulsion 3D printing ink with dual nanoparticle co-stability.

[0048] The printing model was set as a cuboid with a length of 20mm, a width of 20mm, and a height of 4mm. In the printing parameter settings, the extrusion speed and printing speed were 13mm / s, the needle diameter was 0.84mm, the filament spacing was 2mm, and the layer height was 0.5mm. During printing, the temperature of the extrusion needle was 25℃, and the temperature of the substrate was 4℃. The Pickering emulsion ink was poured into the syringe, and 3D printing was performed at room temperature to obtain the lower layer of 3D printed porous biological scaffold for later use.

[0049] Example 3

[0050] Dissolve 1.25g of type B gelatin in 25mL of distilled water and stir at 50℃ to form a gelatin solution. Add 25mL of acetone, remove the supernatant, redissolve the precipitate in 15mL of distilled water and adjust the pH to 12.0. Add acetone again until a precipitate is formed. Then add 56mg of genipin (2%-5.5% of the gelatin mass) to the solution and continue to react at 50℃ for 3h. Centrifuge the reacted solution at 10000g for 30min to obtain gelatin nanoparticles (GNPs).

[0051] A 2.5 mL gelatin solution was prepared as the continuous phase, with a GNP concentration of 10 mg / mL, a fish gelatin molecular concentration of 125 mg / mL, and a bovine gelatin molecular concentration of 125 mg / mL. 7.5 mL of n-hexane was added as the dispersed phase. The two phases were stirred at 13500 rpm for 30 s to obtain a high internal phase Pickering emulsion 3D printing ink.

[0052] The printing model was set as a cuboid with a length of 20 mm, a width of 20 mm, and a height of 2 mm. The printing parameters were set as follows: extrusion speed and printing speed of 10 mm / s, needle diameter of 0.84 mm, filament spacing of 1.8 mm, and layer height of 0.5 mm. The extrusion needle temperature was 25°C, and the substrate temperature was 4°C. The Pickering emulsion ink was poured into a syringe, and 3D printing continued at room temperature to obtain the upper 3D printed porous biological scaffold. After printing, the gradient scaffold was placed at 4°C for 15 hours and then immersed in genipin solution for 24 hours for later use.

[0053] Comparative Example 1

[0054] Dissolve 1.25g of type B gelatin in 25mL of distilled water and stir at 50℃ to form a gelatin solution. Add 25mL of acetone, remove the supernatant, redissolve the precipitate in 15mL of distilled water and adjust the pH to 12.0. Add acetone again until a precipitate is formed. Then add 56mg of genipin (2%-5.5% of the gelatin mass) to the solution and continue to react at 50℃ for 3h. Centrifuge the reacted solution at 10000g for 30min to obtain gelatin nanoparticles (GNPs).

[0055] A 2.5 mL gelatin solution was prepared as the continuous phase, with a GNP concentration of 10 mg / mL and a bovine gelatin molecule concentration of 250 mg / mL; 7.5 mL of n-hexane was added as the dispersed phase; the two phases were stirred at 13500 rpm for 30 s to obtain a high internal phase Pickering emulsion 3D printing ink.

[0056] The printing model was set as a cuboid with a length of 20 mm, a width of 20 mm, and a height of 5 mm. The printing parameters were set as follows: extrusion speed and printing speed of 10 mm / s, needle diameter of 0.84 mm, filament spacing of 2 mm, and layer height of 0.5 mm. The extrusion needle temperature was 25°C, and the substrate temperature was 4°C. The Pickering emulsion ink was poured into a syringe, and 3D printing was performed at room temperature to obtain a 3D-printed porous biological scaffold. After printing, the scaffold was placed at 4°C for 15 hours and then immersed in genipin solution for 24 hours for later use.

[0057] Comparative Example 2

[0058] Dissolve 1.25g of type B gelatin in 25mL of distilled water and stir at 50℃ to form a gelatin solution. Add 25mL of acetone, remove the supernatant, redissolve the precipitate in 15mL of distilled water and adjust the pH to 12.0. Add acetone again until a precipitate is formed. Then add 56mg of genipin (2%-5.5% of the gelatin mass) to the solution and continue to react at 50℃ for 3h. Centrifuge the reacted solution at 10000g for 30min to obtain gelatin nanoparticles (GNPs).

[0059] A 2.5 mL gelatin solution was prepared as the continuous phase, with a GNP concentration of 10 mg / mL, a fish gelatin molecular concentration of 187.5 mg / mL, and a bovine gelatin molecular concentration of 62.5 mg / mL. 7.5 mL of n-hexane was added as the dispersed phase. The two phases were stirred at 13500 rpm for 30 s to obtain a high internal phase Pickering emulsion 3D printing ink.

[0060] The printing model was set as a cuboid with a length of 20 mm, a width of 20 mm, and a height of 5 mm. The printing parameters were set as follows: extrusion speed and printing speed of 10 mm / s, needle diameter of 0.84 mm, filament spacing of 2 mm, and layer height of 0.5 mm. The extrusion needle temperature was 25°C, and the substrate temperature was 4°C. The Pickering emulsion ink was poured into a syringe, and 3D printing was performed at room temperature to obtain a 3D-printed porous biological scaffold. After printing, the scaffold was placed at 4°C for 15 hours and then immersed in genipin solution for 24 hours for later use.

[0061] Test case

[0062] The samples prepared in the embodiments of the present invention were measured.

[0063] according to Figure 1 It can be seen that the gel points of the Pickering emulsion 3D printing inks prepared in Example 1, Comparative Example 1, and Comparative Example 2 are...

[0064] The study investigated the ink state under different addition ratios of fish and bovine gelatin. It was observed that the pure bovine gelatin ink with a mass concentration of 20% had an extremely short gelation time and could not be extruded for 3D printing. The ink with 15% fish gelatin and 5% bovine gelatin had the best extrudability, but its formability was extremely poor and it was easily affected by high temperatures, making the ink unstable. The ink with a mass concentration of 10% fish and 10% bovine gelatin had the best gelation time and the best printability.

[0065] according to Figure 2 It can be seen that the temperature window of the Pickering emulsion 3D printing ink prepared in Example 1 is...

[0066] By extruding ink under different temperature conditions and observing the state of the filaments, the optimal printing time was determined. Ink with 10% fish gelatin and 10% bovine gelatin added to the continuous phase exhibited the best viscosity and extended printing time. The emulsion ink demonstrated good stability and formability, resulting in a printed formstock with excellent shape fidelity.

[0067] according to Figure 3 As can be seen from the actual images of the 3D-printed gradient biological scaffolds prepared in Examples 2 and 3:

[0068] By adjusting the printing parameters and different ratios of the two types of particles, gradient biological scaffolds can be customized to have channels ranging from micrometers to millimeters, resulting in a stable structure.

[0069] according to Figure 4 It can be seen that the cell liveness and death staining images of the 3D printed scaffolds prepared in Examples 1, 2 and 3 are as follows:

[0070] After co-culturing rat bone marrow mesenchymal stem cells (BMSCs) with a 3D-printed scaffold for 72 hours, most cells exhibited green fluorescence, while very few dead cells showed red fluorescence, demonstrating the scaffold's ability to promote cell proliferation. The multi-level interconnected pore structure within the scaffold provides more space for cell adhesion. HA deposition on the surface of the internal pores promotes osteogenic differentiation of BMSCs.

Claims

1. A method for preparing a high internal phase Pickering emulsion 3D printing ink, comprising: A continuous phase with an internal phase volume ratio of 75%-85% and a dispersed phase are stirred and mixed at 10,000-15,000 rpm to obtain a high internal phase Pickering emulsion 3D printing ink; wherein: The continuous phase is prepared by mixing distilled water, gelatin molecules, gelatin nanoparticles and hydroxyapatite. The dispersed phase is any one of n-hexane, benzene, toluene, p-xylene, and styrene; characterized in that: The gelatin molecules are a mixture of fish gelatin and bovine gelatin; The mass ratio of fish gelatin to bovine gelatin in the continuous phase is 1:3-4:0; The mass ratio of the gelatin nanoparticles to hydroxyapatite is 10:0-1:

9.

2. The preparation method according to claim 1, wherein: The hydroxyapatite is prepared by the following method: Dissolve 0.74g Ca(OH)2 in 200mL of deionized water to make a suspension. Stir magnetically in a water bath for 10min. Slowly add 120mL of 85% phosphoric acid solution while stirring. Measure the pH of the mixed solution at the same time. After the phosphoric acid is added and the pH remains unchanged, age the mixed solution at room temperature for 24h. Dry, sinter and grind to obtain hydroxyapatite.

3. The preparation method according to claim 1, wherein: The gelatin nanoparticles were prepared by the following method: Dissolve 1.25g of bovine gelatin in 25mL of distilled water and stir at 50℃ to form a gelatin solution. Add 25mL of acetone, remove the supernatant, redissolve the precipitate in 15mL of distilled water, and adjust the pH to 12.

0. Add acetone again until a precipitate is formed. Then add 56mg of genipin to the solution and continue to react at 50℃ for 3h. Centrifuge the reacted solution at 10000g for 30min to obtain gelatin nanoparticles.

4. A high internal phase Pickering emulsion 3D printing ink prepared by the preparation method according to any one of claims 1 to 3.

5. An application of the high internal phase Pickering emulsion 3D printing ink according to claim 4 in gradient bioscaffold printing, comprising: (1) After setting the 3D printing model and printing parameters, pour the high internal phase Pickering emulsion 3D printing ink into the syringe and perform 3D printing at room temperature. During the process of the printing ink being squeezed out from the needle, the Pickering emulsion ink undergoes a gel-sol-gel transformation. After the lower support is printed, replace the ink and continue printing the upper layer to obtain the finished product. (2) After printing, the finished product is immersed in crosslinking agent for 6 to 24 hours to obtain 3D printed gradient biological scaffold.

6. The application according to claim 5, wherein: The printing parameters in step (1) include: The extrusion speed and printing speed are both 7~15mm / s, the filament pitch is 0.8~2mm, the layer height is 0.5~1.2mm, and the filament diameter is 0.6~1.2mm. During printing, the temperature of the extrusion needle is controlled at 25~28℃, and the temperature of the substrate is controlled at 4~10℃. The crosslinking agent in step (2) is selected from: Genipin or 1-ethyl-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide.

7. The application according to claim 6, wherein: The genipin accounts for 1% to 5% of the gelatin mass, and 1-ethyl-(3-dimethylaminopropyl)carbamate-N-hydroxysuccinimide accounts for 15% to 80% of the gelatin mass. In the 1-ethyl-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide, the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is (1~2):1.

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