High-internal-phase Pickering emulsion 3D printing ink and preparation method thereof

Abstract

According to the 3D printing ink prepared from the high-internal-phase Pickering emulsion and the preparation method of the 3D printing ink, a continuous phase and a dispersed phase with the internal-phase volume ratio being 75%-85% are stirred and mixed at a high speed, and the 3D printing ink is obtained. Gelatin nanoparticles and hydroxyapatite are used as a stabilizer to form a double-particle stable liquid drop interface, so that the stability of the Pickering emulsion ink is improved. The animal gelatin is introduced into the continuous phase, the ink viscosity can be regulated and controlled, the printable time is prolonged, and the time requirement for gradient printing is met. The gradient biological scaffold is obtained by performing 3D printing on the Pickering emulsion ink, and the gradient biological scaffold has the advantages of good printability, relatively long printing time window, good formability, simple preparation process, mild conditions, low raw material consumption, wide source and low cost; the toxicity risk of the material is low, and the material has good stability and degradability; and the scaffold also has relatively high pore diameter and adjustable mechanical properties, can effectively promote proliferation and adhesion of cells to the interior of the scaffold, and has relatively high application and popularization values.

Description

Technical Field

[0001] The invention belongs to the field of biomedical engineering, and in particular relates to a high internal phase Pickering emulsion 3D printing ink and a preparation method thereof. Background Art

[0002] The repair of damaged osteochondral tissue is extremely challenging due to the limited supply of nutrients and progenitor cells caused by the avascularity of the joint microenvironment and the limited self-regeneration ability of articular cartilage. The method based on 3D printing scaffolds can be used to assist the regeneration of osteochondral tissue, making it easier to design scaffold biomimetic structures, and biomaterials customized according to the characteristics of osteochondral are used 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 technology (DIW) meets the needs of scaffold pore hierarchy through simple extrusion. With the use of suitable printing inks, the pore structure from microns to millimeters can be easily customized and the shape of the scaffold can be precisely controlled. The focus of 3D printing technology research is to develop printing inks with biocompatibility, mechanical strength, and shear thinning properties. In addition, the ink should also have printing adaptability (including shape fidelity, structural resolution, cell viability, etc.).

[0003] Multi-material 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 multi-material co-extrusion of polymer-nanoparticle composite inks and hydrogel bio-inks, which is considered promising for application in its current form to non-load-bearing bones. AbudurehemanMaihemuti proposed a 3D printed porous multilayer scaffold based on cold-water fish skin gelatin for osteoarticular 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 with nano-colloidal particles with interfacial wettability as stabilizers, which has the advantages of high stability and low cost. The stability of Pickering emulsion can reach several years, and the stabilizer content is only 1%, which is much lower than the amount of traditional surfactants (10%-40%), thus avoiding the toxic side effects of a large amount of surfactants. The solid particle material used as a stabilizer can use natural polymers as the matrix material. Pickering emulsion has natural shear thinning ability, and the emulsion template will form a multi-level microstructure, which can be used as 3D printing ink. The pore structure is multi-level, which 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 is applied to 3D printing gradient scaffolds to promote integrated repair of osteochondral defects.

[0005] In order to solve the above technical problems, a 3D printing ink with high viscosity, good printability, high mechanical strength, and longer printing time and a preparation method thereof are urgently needed in the art. Summary of the invention

[0006] In view of the above shortcomings, the present invention proposes a method for preparing 3D printing ink by compounding gelatin with Pickering emulsion. The 3D printing ink prepared by the present invention has better printability, longer printing time window, good formability, simple preparation process and mild conditions. The raw materials fish, bovine gelatin and hydroxyapatite are used in low amounts, widely available and low cost. The gradient bioscaffold prepared by 3D printing with the ink is safe and non-toxic, has a diverse pore structure, good stability and degradability, and the mechanical properties of the scaffold can be adjusted by a convenient post-printing cross-linking method, which can be used in bone tissue engineering to promote integrated bone and cartilage repair.

[0007] The present invention also provides a method for preparing a high internal phase Pickering emulsion 3D printing ink, using gelatin nanoparticles and hydroxyapatite as stabilizers to form a double-particle stable droplet interface, thereby improving the stability of the Pickering emulsion ink. The addition of fish gelatin can adjust the ink viscosity and meet the printing time window requirements. The pore wall surface of the gradient bioscaffold obtained by 3D printing forms a micro-nano morphology composed of double nanoparticles, which can give the pore wall surface of the bioscaffold functions such as promoting osteogenesis and cartilage formation, and has good application prospects.

[0008] The present invention is achieved by the following technical means:

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

[0010] The continuous phase and the dispersed phase with an internal phase volume ratio of 75% to 85% are stirred and mixed at 10000 to 15000 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] Further, the hydroxyapatite (HA) is prepared by the following method:

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

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

[0016] Take 1.25g of type B gelatin (i.e. bovine gelatin) and dissolve it in 25mL of distilled water, stir it at a constant temperature of 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 value to 12.0, add acetone again until a precipitate is produced, then add 56mg of genipin to the solution and continue to react at a constant temperature of 50℃ for 3h, and the solution after the reaction is frozen and centrifuged at 10000g for 30min to obtain gelatin nanoparticles (GNPs).

[0017] Furthermore, the gelatin molecules are formed by mixing 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] The present invention also discloses an application of the high internal phase Pickering emulsion 3D printing ink in gradient biological scaffold 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 of the needle, the Pickering emulsion ink undergoes a gel-sol-gel transition. After the lower layer bracket is printed, the ink is replaced to continue printing the upper layer to obtain a finished product;

[0021] (2) After printing is completed, the finished product is immersed in a cross-linking agent for 6 to 24 hours to obtain a 3D printed gradient bioscaffold.

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

[0023] Furthermore, the cross-linking agent in step (2) is selected from any one of genipin or 1-ethyl-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide.

[0024] Furthermore, the genipin accounts for 1% to 5% of the mass of gelatin, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide accounts for 15% to 80% of the mass of gelatin; 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] The present invention uses 3-10 mg / mL of gelatin nanoparticles (GNPs) and 0-7 mg / mL of hydroxyapatite (HA) as stabilizers in the entire continuous phase to prepare a high internal phase Pickering emulsion 3D printing ink with excellent stability. The gelatin molecular chain is rich in hydroxyl, carboxyl and amino groups, which can give the Pickering emulsion ink gelation properties and help it to be functionally modified, but the mechanical strength of the gelatin molecule is poor without cross-linking and cannot match the mechanical properties of the environmental tissue. Therefore, after printing and then cross-linking, the gradient scaffold constructed based on the gelatin molecule is immersed, and the mechanical properties of the scaffold can be significantly improved.

[0026] The invention introduces fish gelatin and bovine gelatin into the continuous phase at the same time, which is beneficial to adjusting the ink viscosity, controlling the printing time and extending the printing window. Through research, it is found that when the mass ratio of fish gelatin to bovine gelatin reaches 1:1, the emulsion ink has better printability, good formability and strong shape fidelity, and has a good application prospect in bone tissue engineering.

[0027] The beneficial effects of the present invention are:

[0028] (1) The high internal phase Pickering emulsion 3D printing ink prepared by the present invention introduces animal gelatin into the continuous phase, which can adjust the ink viscosity, extend the printing time, and meet the time requirements for 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 by the present invention uses GNPs and HA as stabilizers to form a double-particle stable droplet interface, thereby improving the stability of the Pickering emulsion ink. Gelatin nanoparticles (GNPs) have good wettability and stability. Only a small amount of gelatin nanoparticles is required to form the Pickering emulsion. The addition of HA gives the ink good shear thinning properties, meeting the printability requirements.

[0030] (3) The 3D printed gradient bioscaffold prepared by the present invention is obtained by 3D printing at room temperature using the Pickering emulsion 3D printing ink. Changing the fish gelatin concentration, the amount of stabilizer and the printing parameters can accurately adjust the shape fidelity and structural stability of the scaffold.

[0031] (4) The 3D printed gradient bioscaffold prepared by the present invention uses 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, and can directly contact cells to promote osteogenic differentiation. Combined with 3D printing technology, the scaffold is given a pore structure from micrometer to millimeter level, and the purpose of personalized customization and rapid prototyping is achieved.

[0032] (5) The present invention uses the natural biological cross-linking agent genipin to perform a cross-linking reaction on the gradient scaffold, which will not produce substances harmful to the organism. The slow cross-linking property of genipin is utilized to improve the stability and mechanical properties of the 3D printed gradient scaffold.

[0033] (6) The present invention uses Pickering emulsion ink for 3D printing to obtain a gradient bioscaffold, which has good printability, a long printing time window, good formability, a simple preparation process, mild conditions, low raw material usage, a wide source, and low cost; the material has a low toxicity risk, good stability and degradability; it also has a high pore size and adjustable mechanical properties, can effectively promote cell proliferation and adhesion to the inside of the scaffold, and has great application and promotion value. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0036] Figure 3 Actual pictures of 3D printed gradient bioscaffolds prepared in Example 2 and Example 3;

[0037] Figure 4 Live and dead staining images of cells in 3D printed scaffolds prepared in Examples 1, 2 and 3. DETAILED DESCRIPTION

[0038] The present invention will be further described below in conjunction with specific embodiments. The advantages and features of the present invention will become clearer with the description of the embodiments, but the embodiments are only exemplary and do not constitute any limitation to the scope of the present invention.

[0039] The materials involved in the following examples can all be obtained from commercial channels. The type B gelatin used in the present invention is all bovine gelatin, and the gelatin molecules used are a mixture of fish gelatin and bovine gelatin.

[0040] Example 1

[0041] 1.25 g of type B gelatin was dissolved in 25 mL of distilled water, stirred at a constant temperature of 50°C to form a gelatin solution, 25 mL of acetone was added, the supernatant was removed, the precipitate was redissolved in 15 mL of distilled water and the pH value was adjusted to 12.0, acetone was added dropwise again until a precipitate was produced, and then 56 mg of genipin (2%-5.5% of the mass of gelatin) was added to the solution and the reaction was continued at a constant temperature of 50°C for 3 h. The reacted solution was centrifuged at 10,000 g for 30 min to obtain gelatin nanoparticles (GNPs).

[0042] 2.5 mL of gelatin solution was prepared as the continuous phase, in which the concentration of GNPs was 10 mg / mL, the concentration of fish gelatin molecules was 125 mg / mL, and the concentration of bovine gelatin molecules was 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 to a cuboid with a length of 20 mm, a width of 20 mm, and a height of 5 mm; in the printing parameter setting, the extrusion speed and printing speed were 10 mm / s, the needle diameter was 0.84 mm, the wire spacing was 2 mm, and the layer height was 0.5 mm. The temperature of the extrusion needle during printing was 25°C, the base plate 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 bioscaffold. After printing, the scaffold was placed at 4°C for 15 hours and then immersed in a genipin solution for 24 hours for standby use.

[0044] Example 2

[0045] 1.25 g of type B gelatin was dissolved in 25 mL of distilled water, stirred at a constant temperature of 50°C to form a gelatin solution, 25 mL of acetone was added, the supernatant was removed, the precipitate was redissolved in 15 mL of distilled water and the pH value was adjusted to 12.0, acetone was added dropwise again until a precipitate was produced, and then 56 mg of genipin (2%-5.5% of the mass of gelatin) was added to the solution and the reaction was continued at a constant temperature of 50°C for 3 h. The reacted solution was centrifuged at 10,000 g for 30 min to obtain gelatin nanoparticles (GNPs).

[0046] Take 0.74g Ca(OH)2 and dissolve it in 200mL deionized water to make a suspension. Stir it magnetically in a water bath for 10min, slowly add 120mL 85% phosphoric acid solution and stir it. At the same time, measure the pH of the mixed solution. After the phosphoric acid is added and the pH remains unchanged, age the mixed solution at room temperature for 24h, dry it, sinter it and grind it to obtain hydroxyapatite (HA).

[0047] 2.5 mL of gelatin solution was prepared as the continuous phase, in which the concentration of GNPs was 3 mg / mL, the concentration of HA was 7 mg / mL, the concentration of fish gelatin molecules was 125 mg / mL, and the concentration of bovine gelatin molecules was 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 with dual nanoparticle co-stabilization.

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

[0049] Example 3

[0050] 1.25 g of type B gelatin was dissolved in 25 mL of distilled water, stirred at a constant temperature of 50°C to form a gelatin solution, 25 mL of acetone was added, the supernatant was removed, the precipitate was redissolved in 15 mL of distilled water and the pH value was adjusted to 12.0, acetone was added dropwise again until a precipitate was produced, and then 56 mg of genipin (2%-5.5% of the mass of gelatin) was added to the solution and the reaction was continued at a constant temperature of 50°C for 3 h. The reacted solution was centrifuged at 10,000 g for 30 min to obtain gelatin nanoparticles (GNPs).

[0051] 2.5 mL of gelatin solution was prepared as the continuous phase, in which the concentration of GNPs was 10 mg / mL, the concentration of fish gelatin molecules was 125 mg / mL, and the concentration of bovine gelatin molecules was 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 to a cuboid with a length of 20 mm, a width of 20 mm, and a height of 2 mm; in the printing parameter setting, the extrusion speed and printing speed were 10 mm / s, the needle diameter was 0.84 mm, the wire spacing was 1.8 mm, and the layer height was 0.5 mm. The temperature of the extrusion needle during printing was 25°C, the base plate temperature was 4°C, the Pickering emulsion ink was poured into the syringe, and 3D printing was continued at room temperature to obtain the upper 3D printed porous bioscaffold. After printing, the gradient scaffold was placed at 4°C for 15 hours and then immersed in the genipin solution for 24 hours for standby use.

[0053] Comparative Example 1

[0054] 1.25 g of type B gelatin was dissolved in 25 mL of distilled water, stirred at a constant temperature of 50°C to form a gelatin solution, 25 mL of acetone was added, the supernatant was removed, the precipitate was redissolved in 15 mL of distilled water and the pH value was adjusted to 12.0, acetone was added dropwise again until a precipitate was produced, and then 56 mg of genipin (2%-5.5% of the mass of gelatin) was added to the solution and the reaction was continued at a constant temperature of 50°C for 3 h. The reacted solution was centrifuged at 10,000 g for 30 min to obtain gelatin nanoparticles (GNPs).

[0055] 2.5 mL of gelatin solution was prepared as the continuous phase, in which the concentration of GNPs was 10 mg / mL and the concentration of bovine gelatin molecules was 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 to a cuboid with a length of 20 mm, a width of 20 mm, and a height of 5 mm; in the printing parameter setting, the extrusion speed and printing speed were 10 mm / s, the needle diameter was 0.84 mm, the wire spacing was 2 mm, and the layer height was 0.5 mm. The temperature of the extrusion needle during printing was 25°C, the base plate 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 bioscaffold. After printing, the scaffold was placed at 4°C for 15 hours and then immersed in a genipin solution for 24 hours for standby use.

[0057] Comparative Example 2

[0058] 1.25 g of type B gelatin was dissolved in 25 mL of distilled water, stirred at a constant temperature of 50°C to form a gelatin solution, 25 mL of acetone was added, the supernatant was removed, the precipitate was redissolved in 15 mL of distilled water and the pH value was adjusted to 12.0, acetone was added dropwise again until a precipitate was produced, and then 56 mg of genipin (2%-5.5% of the mass of gelatin) was added to the solution and the reaction was continued at a constant temperature of 50°C for 3 h. The reacted solution was centrifuged at 10,000 g for 30 min to obtain gelatin nanoparticles (GNPs).

[0059] 2.5 mL of gelatin solution was prepared as the continuous phase, in which the GNPs concentration was 10 mg / mL, the fish gelatin molecule concentration was 187.5 mg / mL, and the bovine gelatin molecule concentration was 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 to a cuboid with a length of 20 mm, a width of 20 mm, and a height of 5 mm; in the printing parameter setting, the extrusion speed and printing speed were 10 mm / s, the needle diameter was 0.84 mm, the wire spacing was 2 mm, and the layer height was 0.5 mm. The temperature of the extrusion needle during printing was 25°C, the base plate 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 bioscaffold. After printing, the scaffold was placed at 4°C for 15 hours and then immersed in a genipin solution for 24 hours for standby use.

[0061] Test example

[0062] The samples prepared in the embodiment 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 ink state at different addition ratios of fish and cow gelatin was studied, and it was found that the gelling time of pure cow gelatin ink with a mass concentration of 20% was extremely short and could not be extruded for 3D printing; the ink with fish gelatin added to 15% and cow gelatin added to 5% had the best extrudability, but the formability was extremely poor and was easily affected by high temperature, and the ink was unstable; the ink with a mass concentration of 10% for both fish and cow gelatin had the best gelling 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] The ink was squeezed out under different temperature conditions, and the state of the filaments was observed to obtain the optimal printing time period. The ink viscosity was optimal when the fish gelatin and bovine gelatin were added in an amount of 10% each in the continuous phase, and the printing time was extended. The emulsion ink had good stability and formability, and the stent had good shape fidelity after printing.

[0067] according to Figure 3 It can be seen that: the actual image of the 3D printed gradient bioscaffold prepared in Example 2 and Example 3

[0068] By adjusting the printing parameters and the different ratios of the two types of particles, gradient bioscaffolds can be customized with pores ranging from micrometers to millimeters and a stable structure.

[0069] according to Figure 4 It can be seen that: the live and dead staining images of the 3D printed scaffold cells prepared in Example 1, Example 2 and Example 3

[0070] After co-culturing rat bone marrow mesenchymal stem cells (BMSC) with 3D printed scaffolds for 72 hours, it was observed that most cells showed green fluorescence, and the number of dead cells with red fluorescence was very small, proving that the scaffold has the ability to promote cell proliferation. The multi-level interconnected pore structure inside the scaffold provides more space for cell adhesion. HA is deposited on the surface of the internal pores to promote the osteogenic differentiation of BMSCs.

Claims

1. A method for preparing a high internal phase Pickering emulsion 3D printing ink, comprising: The continuous phase and the dispersed phase with an internal phase volume ratio of 75% to 85% are stirred and mixed at 10000 to 15000 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.

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

3. The preparation method according to claim 1, wherein: The gelatin nanoparticles are prepared by the following method: Take 1.25g of bovine gelatin and dissolve it in 25mL of distilled water. Stir it at a constant temperature of 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 value to 12.

0. Add acetone again until a precipitate is produced. Then add 56mg of genipin to the solution and continue to react at a constant temperature of 50℃ for 3h. The solution after the reaction is frozen and centrifuged at 10000g for 30min to obtain gelatin nanoparticles.

4. The preparation method according to claim 1, wherein: The gelatin molecules are made from 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.

5. The preparation method according to claim 1, wherein: The mass ratio of the gelatin nanoparticles to hydroxyapatite is 10:0-1:

9.

6. A high internal phase Pickering emulsion 3D printing ink prepared according to the preparation method according to any one of claims 1 to 5.

7. An application of the high internal phase Pickering emulsion 3D printing ink according to claim 6 in gradient biological scaffold 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 of the needle, the Pickering emulsion ink undergoes a gel-sol-gel transition. After the lower layer bracket is printed, the ink is replaced to continue printing the upper layer to obtain a finished product; (2) After printing is completed, the finished product is immersed in a cross-linking agent for 6 to 24 hours to obtain a 3D printed gradient bioscaffold.

8. The use according to claim 7, wherein: The printing parameters in step (1) include: The extrusion speed and printing speed are both 7-15mm / s, the wire spacing is 0.8-2mm, the layer height is 0.5-1.2mm, and the wire diameter is 0.6-1.2mm. During printing, the temperature of the extrusion needle is controlled at 25-28℃, and the base plate temperature is controlled at 4-10℃; The cross-linking agent in step (2) is selected from: Any one of genipin or 1-ethyl-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide.

9. The use according to claim 8, wherein: The genipin accounts for 1% to 5% of the gelatin mass, and the 1-ethyl-(3-dimethylaminopropyl)carbonyl diamine-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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