Double-nanoparticle co-stabilized 3D-printing pickering emulsion ink, method of preparation and use

Abstract

The present application relates to a double-nanoparticle co-stable 3D printing Pickering emulsion ink, a preparation method and an application, wherein trace functional element hydroxyapatite (X-HA) and gelatin nanoparticles (GNPs) are used as stabilizers, a mixed solution composed of distilled water, gelatin molecules and a crosslinking agent is used as a continuous phase, n-hexane is used as a dispersed phase, a Pickering emulsion ink is obtained after homogenization emulsification, a porous scaffold material is obtained through a direct ink writing technology of 3D printing, the porous scaffold material is soaked in salt solutions with different concentrations to improve mechanical properties, the dispersed phase is removed, and a hierarchical interconnected porous scaffold is obtained through freeze-drying.The 3D printing Pickering emulsion ink constructed by the present application has excellent rheological properties, can stably flow through a printer nozzle and allow an extruded filament to be immediately deposited.The printed product is stable, has a variety of channel structures, has good biocompatibility, and can be applied to the field of promoting bone repair in bone tissue engineering.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering, specifically a dual-nanoparticle co-stabilized 3D printing Pickering emulsion ink, its preparation method, and its application. Background Technology

[0002] Bone tissue engineering scaffolds offer a novel approach to treating bone defects in clinical applications. Bioactive scaffolds, which possess interconnected porous structures and can match the load-bearing capacity of the host bone to provide mechanical support, have become a research hotspot in recent years. Natural polymer-based hydrogel porous scaffolds are soft, water-rich, and can mimic the natural microenvironment for cell survival, exhibiting good biocompatibility. However, they still face numerous challenges, including limited scaffold shape, difficulty in controlling the porous structure, and poor mechanical strength. 3D-printed bone tissue engineering scaffolds overcome the limitations of traditional scaffolds, meeting the needs for personalized scaffolds and diverse pore sizes. Direct ink writing (DIW) technology, with its rapid extrusion molding characteristics, is an important processing route in 3D printing. The challenge of this technology lies not only in designing printing inks with shear-thinning behavior but also in ensuring high printing accuracy and good biocompatibility.

[0003] In recent years, Pickering emulsions, which use interfacial wettable nanocolloidal particles as stabilizers, have been applied as inks in 3D printing. Their stability can last for several years, and the stabilizer content is only 1%, far lower than the amount of traditional surfactants (40%). Therefore, the side effects or toxicity caused by large amounts of surfactants are avoided. Pickering emulsion inks exhibit shear-thinning behavior, which helps them to be smoothly extruded from the 3D printer and quickly return to a gel state after printing. By adjusting the volume fraction of the dispersed phase in the Pickering emulsion ink, droplets of different sizes are obtained, and then the dispersed phase is removed to obtain a 3D printed scaffold with a hierarchical porous structure.

[0004] The Hofmeister effect is one of the most important properties of proteins. Utilizing this effect, a one-step soaking method can rapidly improve the mechanical properties of porous protein scaffolds. The hydrophilic anions in the protein's sequence have been shown to have a strong salting-out effect, and are typically arranged in an ordered manner: SO42-. 2- >HPO4 2- CH3COO - >C5H7O5COO -Therefore, leveraging the mechanical enhancement effect of the Hofmeister effect is of great significance for preparing natural protein-based bioscaffolds with excellent mechanical properties. Chinese patent CN113831554A discloses a protein hydrogel based on the Hofmeister effect and its preparation method. By examining the effects of different concentrations and types of salts in the Hofmeister effect on the formation and properties of bovine serum albumin hydrogels, it demonstrates the effectiveness of this method in altering the properties of protein hydrogels. Chinese patent CN110396205B discloses a Pickering high internal phase emulsion, a 3D-printed porous scaffold material, and its preparation method. The pore size, pore structure, and mechanical properties of the material are altered by adjusting the content of polymer PCL and the concentration of nano-hydroxyapatite particles. This method improves the mechanical properties of the material without producing substances harmful to organisms. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides a dual-nanoparticle co-stabilized 3D printing Pickering emulsion ink, its preparation method, and its applications. The ink preparation process is simple, has a short cycle time, and operates under mild conditions, exhibiting excellent printability. The raw materials, gelatin and hydroxyapatite, are used in low quantities, are widely available, and are inexpensive. The porous bioscaffolds prepared by 3D printing using this ink are safe and non-toxic, possess diverse pore structures, and exhibit good stability and biodegradability. A convenient "one-step salt immersion" method can be used to induce a salting-out effect, allowing for adjustment of the scaffold's pore size and mechanical properties. This ink can be used in bone tissue engineering to promote bone repair.

[0006] This invention also provides a method for preparing a 3D printing Pickering emulsion ink stabilized by two nanoparticles. The method uses hydroxyapatite, a trace functional element, and gelatin nanoparticles as stabilizers to form a stable droplet interface, thereby improving the stability of the Pickering emulsion ink. The addition of hydroxyapatite can adjust the ink viscosity to meet printability requirements. The porous biological scaffold obtained by 3D printing has a micro / nano morphology composed of two nanoparticles on its pore wall surface, which can endow the pore wall surface of the biological scaffold with functions such as promoting bone growth, promoting angiogenesis, and antibacterial activity, showing promising application prospects.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] (1) In the trace functional element-doped hydroxyapatite (X-HA) system, a Ca(OH)2 / H3PO4 solution was prepared according to a (Ca+X) / P ratio of 1.67. The specific preparation method is as follows: The compound containing the functional element was dissolved in water, Ca(OH)2 and polyethylene glycol-400 were added, and the mixture was stirred at room temperature for 2 hours until a uniform suspension was formed. H3PO4 solution was slowly added dropwise, and stirring was continued for 4 hours. After stirring, the lower layer of liquid aged at room temperature was washed twice alternately with distilled water and anhydrous ethanol, dried, and ground to obtain trace functional element-doped hydroxyapatite (X-HA). The trace functional element (X) can be any one of Cu, Mg, Zn, Co, Sr, Mn, or Se. Among them, the doping source of Cu, Mg, and Zn is the reaction between Cu, Mg, and Zn and nitrate ions (NO3). - ), hydroxide ions (OH-) - ), chloride ions (Cl) - ), sulfate ions (SO4) 2- ), carbonate ions (CO3) 2- The compound is composed of Co, Sr, and Mn, with Co, Sr, and Mn as doping sources and nitrate ions (NO3) as doping sources. - The compounds are composed of sodium selenite (Na2SeO3) as the Se doping source. In the compounds containing Cu, the molar ratio of Cu to Ca is (0.5-0.1):(0.95-0.9). In the compounds containing Mg, Zn, Co, Sr, Mn, and Se, the molar ratio of Mg, Zn, Co, Sr, Mn, and Se to Ca is (0.5-0.15):(0.95-0.85).

[0009] (2) Dissolve type B gelatin in distilled water and stir at 50°C to form a gelatin solution. Add acetone to form a precipitate. Redissolve the precipitate in distilled water and adjust the pH to 12.0. Add acetone again until a precipitate is formed. Then add 2.5-5% glutaraldehyde to the solution and continue the reaction at 50°C for 3-10 hours. Centrifuge the solution after the reaction at 10000g for 20-30 minutes to obtain gelatin nanoparticles (GNPs).

[0010] (3) A continuous phase consisting of distilled water, X-HA, GNPs, gelatin molecules, and a crosslinking agent is mixed with a dispersed phase containing n-hexane at a volume ratio of 1.5:1 to 1:4 at 10,000 to 15,000 rpm to form an O / W type Pickering emulsion ink for later use. The crosslinking agent in the continuous phase can be either genipin or 1-ethyl-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide, wherein 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, wherein the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is (1 to 2):1.

[0011] This dual-nanoparticle co-stabilized 3D printing Pickering emulsion ink can be used to fabricate porous biological scaffolds. The specific method is as follows:

[0012] (1) After setting the 3D printing model and printing parameters, the Pickering emulsion ink prepared above is poured into the syringe, and 3D printing is performed at room temperature. During the process of the printing ink being extruded from the needle, the Pickering emulsion ink undergoes a gel-sol-gel transformation until the printing is completed, and the finished product is obtained. The printing parameters are adjusted according to the following ranges: extrusion speed and printing speed are 7-15 mm / s, filament pitch 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℃, and the temperature of the substrate is controlled at 4-10℃.

[0013] (2) After printing, the finished product is immersed in a salt solution composed of hydrophilic ions in the Hofmeister sequence for 6–24 hours to obtain a 3D-printed porous biological scaffold. The selected salt solution is NH4+. + CS + K + Na + With SO4 2- HPO4 2- CH3COO - C5H7O5COO - The compounds consist of salt solutions with a concentration of 0.5–2 M.

[0014] Pickering emulsion inks are used for 3D printing, and 3D printed porous biological scaffolds with micron to millimeter-scale pore structures are formed by removing the dispersed phase. The scaffolds have good biocompatibility, adjustable mechanical properties and excellent stability. Moreover, the micro-nano morphology composed of dual nanoparticles on the pore surface endows the biological scaffold material with osteogenic properties and functions such as promoting angiogenesis and antibacterial activity. This is of great significance to the field of bone tissue engineering and promoting bone repair.

[0015] This invention uses natural polymer gelatin as a raw material to prepare nanoparticles, and introduces gelatin into the continuous phase using a very small amount (0.5 wt%) of gelatin nanoparticles (GNPs) and trace amounts of the functional element hydroxyapatite (X-HA) as stabilizers to prepare a 3D printing Pickering emulsion ink with excellent stability. The gelatin molecular chain is rich in hydroxyl, carboxyl, and amino groups, which can endow Pickering emulsion ink with gelation properties and facilitate its functional modification. However, gelatin molecules have poor mechanical strength without cross-linking, making it difficult to match the mechanical properties of environmental tissues. Therefore, utilizing the Hofmeister effect, salt immersion treatment of gelatin-based biological scaffolds can produce a strong salting-out effect, rapidly improving the mechanical properties of the scaffold.

[0016] The salt used in this invention is safe and non-toxic, and is a compound composed of kosmotropic ions in the Hofmeister sequence. Kosmotropic ions have been shown to have a strong salting-out effect, reducing the solubility of gelatin by affecting the hydrogen bonds between gelatin and surrounding water molecules, as well as the hydrophobic interactions between gelatin molecules. Regarding emulsion stability, the addition of salt ions interferes with the electrostatic repulsion between particles, thereby affecting the interfacial adsorption of protein nanoparticles and causing the emulsion to become metastable. During this process, droplet ripening and aggregation form large droplets, which ultimately serve as templates for forming macroporous structures. Therefore, through salting-out, the gelatin molecules in the continuous phase of the emulsion form a denser three-dimensional gel network, further enhancing the stability and mechanical properties of 3D-printed porous bioscaffolds, showing great promise for applications in bone tissue engineering.

[0017] Therefore, the 3D printing Pickering emulsion ink prepared using the present invention has the following advantages:

[0018] (1) The hydroxyapatite (X-HA) doped with trace functional elements prepared in this invention has excellent biocompatibility and osteogenic induction properties, and is an extremely important material for bone tissue engineering. In the process of HA synthesis, some Ca can be replaced by various trace functional elements to synthesize X-HA. The addition of trace functional elements can improve the defects of single HA such as insufficient mechanical strength and susceptibility to bacterial infection, and endow HA with more biological functions, including promoting osteoogenesis, angiogenesis, and exerting certain antibacterial effects.

[0019] (2) The 3D printing Pickering emulsion ink prepared in this invention uses GNPs and X-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 X-HA can adjust the ink viscosity, giving it good shear-thinning properties and meeting the printability requirements.

[0020] (3) The 3D printed macroporous biological scaffold prepared by the present invention is obtained by 3D printing the 3D printing Pickering emulsion ink at room temperature. The shape fidelity and structural stability of the scaffold can be precisely adjusted by changing the gelatin concentration, stabilizer dosage and printing parameters.

[0021] (4) The 3D-printed macroporous bioscaffold prepared in this invention utilizes a Pickering emulsion template to form a multi-level interconnected pore structure within the scaffold, which further promotes cell proliferation and adhesion. X-HA, after lyophilization, is deposited on the surface of the internal pores, allowing direct contact with cells and promoting osteogenic differentiation. By doping with different functional elements, it can meet different application requirements, such as promoting angiogenesis and antibacterial effects. Combined with 3D printing technology, the scaffold is endowed with pore structures ranging from micrometers to millimeters, achieving personalized customization and rapid prototyping.

[0022] (5) The present invention uses genipin, a natural biological crosslinking agent, to crosslink the continuous phase gelatin. This will not produce substances that are harmful to organisms. By utilizing the slow crosslinking properties of genipin, the viscosity of Pickering emulsion ink during the printing process is not affected, and the stability and mechanical properties of the 3D printed scaffold are improved.

[0023] (6) The 3D-printed porous biological scaffold prepared by this invention uses a "one-step salt soaking" method to induce a strong salting-out effect, which can change the scaffold pore size, form a macroporous structure scaffold, and rapidly improve its mechanical properties. Depending on the type of salt, salt concentration, and soaking time, it can produce multi-level pore structures and different gradients of mechanical strength, making it better suited for practical applications.

[0024] (7) This invention uses Pickering emulsion ink to 3D print porous biological scaffolds. The operation steps are simple, the cycle is short, the conditions are mild, the raw material consumption is low, the source is wide, and the cost is low. 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. It has great application and promotion value. Attached Figure Description

[0025] Figure 1 Images of the 3D-printed porous biological scaffolds prepared in Examples 1 and 2.

[0026] Figure 2 Shear thinning curves (G) of the 3D printing Pickering emulsion inks prepared in Examples 1 and 3 x and H y (Representing gelatin nanoparticles and hydroxyapatite doped with trace amounts of functional element and their concentrations, respectively).

[0027] Figure 3 Scanning electron microscope image of the interior of the 3D printed porous biological scaffold prepared in Example 1 and mechanical property curves comparing the scaffold without soaking and soaked in different salts or for different times. Detailed Implementation

[0028] 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.

[0029] Example 1

[0030] 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 250μL of glutaraldehyde solution (25% aqueous solution) to the solution and continue to react at 50℃ for 3 hours. Centrifuge the reacted solution at 10000g for 30 minutes to obtain gelatin nanoparticles (GNPs).

[0031] Take 1.570g of Zn(NO3)2·6H2O, dissolve it in 200mL of distilled water, then add 3.520g of Ca(OH)2 solution, stir until a uniform suspension is formed, add 1mL of polyethylene glycol-400 during the process, and then continue to add 100mL of 0.3M H3PO4 solution while stirring. After stirring, age for 24h, wash the settled liquid twice with anhydrous ethanol and distilled water, dry and grind to obtain zinc-doped hydroxyapatite (Zn-HA).

[0032] A 4 mL gelatin solution was prepared as the continuous phase, with Zn-HA concentration of 5 mg / mL, GNPs concentration of 5 mg / mL, gelatin molecule concentration of 177 mg / mL, and genipin concentration of 1.78 mg / mL. 6 mL of n-hexane was added as the dispersed phase. The two phases were stirred at 13500 rpm for 30 s to obtain the dual nanoparticle co-stabilized 3D printing Pickering emulsion ink.

[0033] The printing model was set as a cylinder with a diameter of 17 mm and a height of 7 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 1.7 mm, and layer height of 0.7 mm. The extrusion needle temperature was 25°C, and the substrate temperature was 4°C. The 3D printing 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 left at room temperature for 24 hours and then immersed in a 1.5 M ammonium sulfate solution for 15 hours for later use.

[0034] like Figure 3 As shown, the scanning electron microscope image of the interior of the 3D printed porous biological scaffold prepared in Example 1 and the mechanical property curves comparing the scaffold without soaking and soaked in different salts or for different times are shown.

[0035] Example 2

[0036] 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 250μL of glutaraldehyde solution (25% aqueous solution) to the solution and continue to react at 50℃ for 3 hours. Centrifuge the reacted solution at 10000g for 30 minutes to obtain gelatin nanoparticles (GNPs).

[0037] Dissolve 1.570 g of Zn(NO3)2·6H2O in 200 mL of distilled water, then add 3.520 g of Ca(OH)2 solution and stir until a uniform suspension is formed. During this process, add 1 mL of polyethylene glycol-400, and then continue to add 100 mL of 0.3 M H3PO4 solution while stirring. After stirring, age for 24 h. Wash the settled liquid twice with anhydrous ethanol and distilled water, dry and grind to obtain copper-doped hydroxyapatite (Zn-HA).

[0038] A 4 mL gelatin solution was prepared as the continuous phase, with Zn-HA concentration of 5 mg / mL, GNPs concentration of 5 mg / mL, gelatin molecule concentration of 177 mg / mL, and genipin concentration of 1.78 mg / mL. 6 mL of n-hexane was added as the dispersed phase. The two phases were stirred at 13500 rpm for 30 s to obtain the dual nanoparticle co-stabilized 3D printing Pickering emulsion ink.

[0039] The printing model was set as a cuboid with a length and width of 20 mm and a height of 6 mm. The printing parameters were set as follows: extrusion speed and printing speed of 10 mm / s, needle diameter of 0.7 mm, filament spacing of 1.6 mm, and layer height of 0.6 mm. The extrusion needle temperature was 25°C, and the substrate temperature was 4°C. The 3D printing 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 left at room temperature for 24 hours and then immersed in a 1.5 M ammonium sulfate solution for 15 hours for later use.

[0040] The 3D-printed porous biological scaffolds prepared in Examples 1 and 2 are shown below. Figure 1 As shown.

[0041] Example 3

[0042] 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 250μL of glutaraldehyde solution (25% aqueous solution) to the solution and continue to react at 50℃ for 3 hours. Centrifuge the reacted solution at 10000g for 30 minutes to obtain gelatin nanoparticles (GNPs).

[0043] Take 1.570g of Zn(NO3)2·6H2O, dissolve it in 200mL of distilled water, then add 3.520g of Ca(OH)2 solution, stir until a uniform suspension is formed, add 1mL of polyethylene glycol-400 during the process, and then continue to add 100mL of 0.3M H3PO4 solution while stirring. After stirring, age for 24h, wash the settled liquid twice with anhydrous ethanol and distilled water, dry and grind to obtain zinc-doped hydroxyapatite (Zn-HA).

[0044] A 4 mL gelatin solution was prepared as the continuous phase, with Zn-HA concentration of 2.5 mg / mL, GNPs concentration of 2.5 mg / mL, gelatin molecule concentration of 177 mg / mL, and genipin concentration of 1.78 mg / mL. 6 mL of n-hexane was added as the dispersed phase. The two phases were stirred at 13500 rpm for 30 s to obtain the dual nanoparticle co-stabilized 3D printing Pickering emulsion ink.

[0045] The printing model was set as a cylinder with a diameter of 17 mm and a height of 7 mm. The printing parameters were set as follows: extrusion speed and printing speed of 12 mm / s, needle diameter of 0.84 mm, filament spacing of 1.7 mm, and layer height of 0.7 mm. The extrusion needle temperature was 25°C, and the substrate temperature was 4°C. The 3D printing 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 left at room temperature for 24 hours and then immersed in a 1.5 M sodium citrate solution for 15 hours for later use.

[0046] The shear thinning curves of the 3D printing Pickering emulsion inks prepared in Examples 1 and 3 are shown in the figure. Figure 2 As shown, (G) x and H y (Representing gelatin nanoparticles and hydroxyapatite doped with trace amounts of functional element and their concentrations, respectively).

[0047] Example 4

[0048] 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 250μL of glutaraldehyde solution (25% aqueous solution) to the solution and continue to react at 50℃ for 3 hours. Centrifuge the reacted solution at 10000g for 30 minutes to obtain gelatin nanoparticles (GNPs).

[0049] Dissolve 0.426 g of CuCl2·2H2O in 200 mL of distilled water, then add 3.520 g of Ca(OH)2 solution and stir until a uniform suspension is formed. During this process, add 1 mL of polyethylene glycol-400, and then continue to add 100 mL of 0.3 M H3PO4 solution while stirring. After stirring, age for 24 h. Wash the settled liquid twice with anhydrous ethanol and distilled water, dry and grind to obtain zinc-doped hydroxyapatite (Cu-HA).

[0050] A 4 mL gelatin solution was prepared as the continuous phase, with Cu-HA concentration of 5 mg / mL, GNPs concentration of 5 mg / mL, gelatin molecule concentration of 177 mg / mL, and genipin concentration of 1.78 mg / mL. 6 mL of n-hexane was added as the dispersed phase. The two phases were stirred at 13500 rpm for 30 s to obtain the dual nanoparticle co-stabilized 3D printing Pickering emulsion ink.

[0051] The printing model was set as a cuboid with a length and width of 20 mm and a height of 7 mm. The printing parameters were set as follows: extrusion speed and printing speed of 12 mm / s, needle diameter of 0.84 mm, filament spacing of 1.7 mm, and layer height of 0.7 mm. The extrusion needle temperature was 25°C, and the substrate temperature was 4°C. The 3D printing 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 left at room temperature for 24 hours and then immersed in a 1.5 M ammonium sulfate solution for 15 hours for later use.

[0052] Example 5

[0053] 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 250μL of glutaraldehyde solution (25% aqueous solution) to the solution and continue to react at 50℃ for 3 hours. Centrifuge the reacted solution at 10000g for 30 minutes to obtain gelatin nanoparticles (GNPs).

[0054] Dissolve 0.426 g of CuCl2·2H2O in 200 mL of distilled water, then add 3.520 g of Ca(OH)2 solution and stir until a uniform suspension is formed. During this process, add 1 mL of polyethylene glycol-400, and then continue to add 100 mL of 0.3 M H3PO4 solution while stirring. After stirring, age for 24 h. Wash the settled liquid twice with anhydrous ethanol and distilled water, dry and grind to obtain zinc-doped hydroxyapatite (Cu-HA).

[0055] A 4 mL gelatin solution was prepared as the continuous phase, with Cu-HA concentration of 2.5 mg / mL, GNPs concentration of 2.5 mg / mL, gelatin molecule concentration of 177 mg / mL, and genipin concentration of 1.78 mg / mL. 6 mL of n-hexane was added as the dispersed phase. The two phases were stirred at 13500 rpm for 30 s to obtain the dual nanoparticle co-stabilized 3D printing Pickering emulsion ink.

[0056] The printing model was set as a cylinder with a diameter of 16 mm and a height of 6 mm. The printing parameters were set as follows: extrusion speed and printing speed of 10 mm / s, needle diameter of 0.7 mm, filament spacing of 1.6 mm, and layer height of 0.6 mm. The extrusion needle temperature was 25°C, and the substrate temperature was 4°C. The 3D printing 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 left at room temperature for 24 hours and then immersed in a 1.5 M ammonium sulfate solution for 24 hours for later use.

Claims

1. A method for preparing a dual-nanoparticle co-stabilized 3D printing Pickering emulsion ink, characterized in that, Includes the following steps: (1) A continuous phase is prepared consisting of distilled water, hydroxyapatite X-HA doped with trace amounts of functional elements, gelatin nanoparticles GNPs, gelatin molecules and a crosslinking agent; the hydroxyapatite doped with trace amounts of functional elements and the gelatin nanoparticles are used together as Pickering emulsion stabilizers; the concentration ranges of the hydroxyapatite doped with trace amounts of functional elements, gelatin nanoparticles and gelatin molecules are 2 ~ 8 mg / mL, 2 ~ 8 mg / mL and 177 ~ 334 mg / mL, respectively. (2) The continuous phase and the dispersed phase are mixed at a volume ratio of 1.5:1 to 1:4 under the condition of 10000 ~ 15000 rpm to form O / W type Pickering emulsion ink.

2. The preparation method of the dual-nanoparticle co-stabilized 3D printing Pickering emulsion ink according to claim 1, characterized in that, The hydroxyapatite X-HA doped with trace functional element X is synthesized by liquid-phase coprecipitation through a Ca(OH)2 / H3PO4 system. The Ca(OH)2 / H3PO4 solution is prepared according to a molar ratio of (Ca+X) / P = 1.67, and the trace functional element X replaces part of the Ca in the system. The preparation method is as follows: a system is prepared using distilled water containing functional element compounds. Each 0.1 to 0.5 L of the system contains 0.2 to 0.5 M Ca(OH)2 solution. The mixture is stirred until a uniform suspension is formed. During this process, 0.25 to 0.1 mL of polyethylene glycol-400 is added, and 0.2 to 0.5 M H3PO4 solution is added dropwise while stirring. After stirring, the mixture is aged for 24 h, dried, and ground to obtain hydroxyapatite doped with trace amounts of functional elements.

3. The method for preparing the dual-nanoparticle co-stabilized 3D printing Pickering emulsion ink according to claim 2, characterized in that, The aforementioned trace functional element X is derived from inorganic compounds containing Cu, Mg, Zn, Co, Sr, Mn, and Se; The doping sources for Cu, Mg, and Zn are their reactions with nitrate ions (NO3). - OH- ions - Chloride ions Cl - Sulfate ions SO4 2- carbonate ions CO3 2- The compound is composed of Co, Sr, and Mn dopants, which are derived from the interaction of nitrate ions with NO3. - The compound is composed of sodium selenite (Na2SeO3); the Se doping source is sodium selenite. In compounds containing Cu, the molar ratio of Cu to Ca is 0.5 ~ 0.1: 0.95 ~ 0.

9. In compounds containing Mg, Zn, Co, Sr, Mn, and Se, the molar ratio of Mg, Zn, Co, Sr, Mn, and Se to Ca is 0.5 ~ 0.15: 0.95 ~ 0.

85.

4. The preparation method of the dual-nanoparticle co-stabilized 3D printing Pickering emulsion ink according to claim 1, characterized in that, The gelatin nanoparticles were prepared using a two-step solvent removal method. The preparation method was as follows: Type B gelatin was fully dissolved in distilled water, and precipitate was obtained by acetone. The precipitate was redissolved in distilled water and the pH was adjusted to 12.

0. Acetone was added dropwise again until precipitate was formed. Then, 2.5-5% glutaraldehyde was added to the solution and the reaction was continued at 50 °C for 3-10 hours. The reaction solution was centrifuged at 10000 g for 20-30 minutes to obtain gelatin nanoparticles.

5. The method for preparing the dual-nanoparticle co-stabilized 3D printing Pickering emulsion ink according to claim 1, characterized in that, The crosslinking agent in the continuous phase is either genipin or 1-ethyl-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide, wherein genipin accounts for 1% to 5% of the sum of the mass of gelatin nanoparticles (GNPs) and gelatin molecules, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide accounts for 15% to 80% of the mass of gelatin, wherein the mass ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 1 to 2:

1.

6. A dual-nanoparticle co-stabilized 3D printing Pickering emulsion ink, characterized in that, Obtained by the preparation method according to any one of claims 1 to 5.

7. The application of the dual nanoparticle co-stabilized 3D printing Pickering emulsion ink according to claim 6, characterized in that, Materials used to prepare porous biological scaffolds.

8. A method for preparing a porous biological scaffold, characterized in that, Includes the following steps: (1) After setting the 3D printing model and printing parameters, pour the dual nanoparticle co-stabilized 3D printing Pickering emulsion ink as described in claim 6 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 until the printing is completed and the finished product is obtained. (2) After printing, the finished product is placed in a salt solution composed of hydrophilic ions and soaked for 6 to 24 hours to obtain a 3D printed porous biological scaffold.

9. The method for preparing a porous biological scaffold according to claim 8, characterized in that, In step (1), the printing parameters are adjusted according to the following ranges: extrusion speed and printing speed are 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 °C, and the temperature of the substrate is controlled at 4 ~ 10 °C. The salt solution selected in step (2) is NH4 + CS + K + Na + With SO4 2- HPO4 2- CH3COO - C5H7O5COO - The compounds consist of salt solutions with a concentration of 0.5 to 2 M and a soaking time of 6 to 24 h.

10. A porous biological scaffold, characterized in that, Obtained by the preparation method described in claim 8.

Images