A programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties, and a preparation method and application thereof

Abstract

This application relates to a programmable 3D / 4D printable hydrogel mesoporous scaffold with adjustable mechanical and topological properties, its preparation method, and its application, belonging to the field of food biosynthesis technology. The preparation method of the hydrogel mesoporous scaffold of this application includes the following steps: first, obtaining a printable composite hydrogel ink through a one-pot delayed double crosslinking process; then, transferring the composite hydrogel ink to an extrusion 3D printer for printing to obtain a preliminary scaffold product with a three-dimensional porous structure; finally, heat treatment to obtain the scaffold. This hydrogel scaffold possesses excellent mechanical properties, a concentrated pore size distribution, adjustable porosity, and exhibits a customizable ordered hierarchical structure and high water retention capacity; it also exhibits environmental responsiveness, and the non-uniform swelling behavior of the hydrogel mesoporous scaffold can be programmed by adjusting the gelatin concentration, making it suitable as a smart scaffold for producing cultured meat.

Description

Technical Field

[0001] This application relates to the field of food biosynthesis technology, and in particular to a programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties, its preparation method and application. Background Technology

[0002] The development of cultured meat technology urgently requires intelligent, edible mesoporous scaffolds capable of mimicking the complex structure and texture of natural muscle to simulate the dynamic changes during tissue development, thereby producing cultured meat with the same taste and nutritional value as traditional meat. Although hydrogels are considered ideal scaffold materials due to their good biocompatibility and tunable physicochemical properties, their preparation methods still face key challenges in practical applications. On the one hand, it is difficult to synergistically balance the mechanical strength and topology of the scaffold through simple and scalable processes. On the other hand, constructing edible hydrogel scaffolds with programmable deformability also presents technical difficulties. These two requirements are difficult to meet simultaneously in edible hydrogels.

[0003] Existing research primarily focuses on preparing static, fixed-shape scaffolds, and most methods rely on a single cross-linking mechanism (e.g., ionic cross-linking of pure sodium alginate). The resulting network structure is fragile, leading to insufficient mechanical properties and poor shape retention in the printed scaffolds, making it difficult to meet the structural stability requirements of subsequent cell culture processes. To improve mechanical properties, some studies have attempted to introduce dual cross-linking strategies. However, these methods often suffer from problems such as imprecise timing control, complex processes, or insufficient synergistic effects in the cross-linking network. For example, if both cross-linking reactions occur simultaneously and rapidly, ink flowability decreases, affecting rheological behavior and shape accuracy during extrusion molding; if they are carried out in stages, they often involve cumbersome multi-step processes, limiting the feasibility of large-scale fabrication. This often results in difficulties in synergistically optimizing the balance between mechanical strength and pore topology, or between printability and long-term stability in the prepared scaffolds. Furthermore, conventional 3D-printed scaffolds cannot directionally induce muscle fiber alignment, thus failing to simulate the anisotropic mechanical properties of natural muscle and limiting the tissue maturity and function of cultured tissue.

[0004] Therefore, it is necessary to develop a simple and easy-to-operate preparation method to regulate different cross-linking reaction kinetics in the system to achieve a synergy of high mechanical properties and good topology, and to prepare edible hydrogel scaffolds with programmable deformability for dynamic cell culture in tissue engineering. Summary of the Invention

[0005] In view of this, this application provides a programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties, its preparation method, and its application. The hydrogel mesoporous scaffold prepared by ionic crosslinking and enzyme-catalyzed crosslinking in this application exhibits excellent mechanical properties, adjustable pore topology, and high water retention capacity. Furthermore, by adjusting the gelatin concentration, the non-uniform swelling behavior of the hydrogel mesoporous scaffold can be programmed, allowing the hydrogel structure with thickness gradients to undergo predictable shape changes in aqueous solution, demonstrating its potential as a smart scaffold for cell culture meat and effectively overcoming the shortcomings of the aforementioned prior art.

[0006] The first aspect of this application provides a method for fabricating a programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties, comprising the following steps:

[0007] (1) Dissolve gelatin and sodium alginate in a solvent and stir magnetically to form a homogeneous hydrogel solution;

[0008] (2) Add an enzyme cross-linking agent and a continuous calcium ion release cross-linking agent to the hydrogel solution at the same time, stir evenly, and obtain a mixed system solution;

[0009] (3) The mixed system solution is allowed to stand at 15~25℃ to carry out a pre-crosslinking reaction, and a printable composite hydrogel ink is obtained.

[0010] (4) The composite hydrogel ink is transferred into the barrel of an extrusion 3D printer and printed according to a preset three-dimensional digital model to obtain a scaffold initial product with a three-dimensional porous structure.

[0011] (5) The initial product of the scaffold is incubated to complete sufficient ionic crosslinking and enzymatic crosslinking, and then heat-treated to obtain a programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties.

[0012] Preferably, in step (1), the gelatin is fish-derived gelatin; the sodium alginate is sodium alginate with a viscosity of 5.0~7.0 mPa·s, an M / G ratio of 1:0.8~1.2, and a molecular weight of 18000~20000 Da; the solvent is deionized water;

[0013] In step (1), the mass ratio of gelatin to sodium alginate is (0~10):3. Specifically, the mass concentration ratio of gelatin to sodium alginate is one of the following five combinations: 0% gelatin to 3% sodium alginate, 2.5% gelatin to 3% sodium alginate, 5% gelatin to 3% sodium alginate, 7.5% gelatin to 3% sodium alginate, and 10% gelatin to 3% sodium alginate.

[0014] Preferably, in step (1), the specific conditions for magnetic stirring are: mixing with magnetic stirring at a constant temperature of 60°C for 1 to 1.5 hours.

[0015] Preferably, in step (2), the enzyme cross-linking agent is transglutaminase; the sustained calcium ion release cross-linking agent is a mixture of CaCO3 and D-(+)-gluconic acid-δ-lactone, wherein the molar ratio of CaCO3 to D-(+)-gluconic acid-δ-lactone is 1:1.5~2.5. Specifically, the preparation method is a one-pot delayed double cross-linking method. More specifically, the one-pot delayed double cross-linking method involves simultaneously adding the enzyme cross-linking agent (transglutaminase) and the sustained calcium ion release cross-linking agent (CaCO3 and D-(+)-gluconic acid-δ-lactone) to a gelatin / sodium alginate mixed solution. The rate of transglutaminase cross-linking of gelatin is delayed by controlling the temperature (15~25℃), which has a synergistic effect with the sustained calcium ion release system, thereby providing the required strength and shape support for 3D printing. After printing, the printed sample continues to be incubated to ensure full cross-linking of transglutaminase and the continuous calcium ion release cross-linking agent, thereby further enhancing the mechanical strength and shape retention of the 3D printed sample.

[0016] It should be noted that the cross-linking temperature is set much lower than the optimal temperature for enzyme activity, which is a necessary condition for this method. If the optimal temperature for enzyme cross-linking is used, the cross-linking will be too fast, making it difficult to perform 3D printing.

[0017] The preparation method of this hydrogel scaffold is "one-pot delayed double cross-linking method".

[0018] The delay refers to using a temperature below the optimal enzyme activity to slow down the cross-linking rate of transglutaminase, and using a combination of CaCO3 and D-(+)-gluconic acid-δ-lactone to control the release rate of calcium ions for pre-cross-linking.

[0019] The one-pot method refers to the simultaneous addition of raw materials (gelatin, sodium alginate) and dual cross-linking agents (enzymes and calcium ion solution), allowing for 3D printing through simple pre-cross-linking.

[0020] The topology and response characteristics of the scaffold are designed by controlling the gelatin concentration.

[0021] Numerous patents already exist regarding methods such as using calcium ion slow release alone, enzyme cross-linking alone, or cross-linking using both enzyme cross-linking agents and calcium ion distribution through soaking. For example, Chinese patent CN110237301A discloses a gel preparation method using CaCO3 and glucono-δ-lactone (GDL) as the cross-linking system. Its core lies in utilizing the pH decrease caused by GDL hydrolysis to slowly release calcium ions. 2+This allows for uniform ionic crosslinking of sodium alginate, avoiding the problems of excessively rapid local gelation and uneven structure caused by the traditional direct addition of calcium chloride. Chinese patent CN113416326B discloses a method for preparing hydrogels by mixing gelatin solution and transglutaminase solution and then incubating, improving the mechanical properties of the hydrogel. Furthermore, Chinese patent CN117298337A discloses a method for crosslinking hydrogels by first immersing them in CaCl2 solution and then in a transglutaminase solution, improving the strength of the hydrogel scaffold. However, single enzyme crosslinking or calcium ion crosslinking results in hydrogels with limited overall mechanical strength and structural stability, making it difficult to meet the requirements of applications requiring high stress (such as tissue engineering scaffolds). Distributed immersion double crosslinking requires two independent post-processing steps, which is cumbersome and time-consuming. Moreover, repeated immersion of the formed sample in the post-processing solution can easily lead to swelling, deformation, or even structural collapse, making it difficult to maintain the precision of complex shapes and resulting in uneven crosslinking. To address the aforementioned shortcomings, the present invention proposes a one-pot delayed dual crosslinking method. By leveraging the synergistic effect of low-temperature delayed enzyme crosslinking and calcium ion slow release, the dual crosslinking mechanism is integrated into the one-pot ink. This solves the problems of insufficient strength of a single crosslinking network and uneven crosslinking during immersion, while also overcoming the drawbacks of cumbersome step-by-step processes and easily changeable structures. This provides a new solution for high-precision 3D printing of high-performance hydrogel scaffolds.

[0022] The amount of CaCO3 added is 0.6-0.8% of the total mass of the hydrogel solution, and the molar ratio of CaCO3 to D-(+)-glucono-δ-lactone is 1:1.5-2.5. This allows for the initiation of ionic cross-linking of sodium alginate through continuous calcium ion release, thereby enhancing the shape and strength required for 3D printing. The amount of transglutaminase added is 2-6 U / g, and the cross-linking rate between transglutaminase and gelatin is slowed down by low-temperature regulation, thus creating a synergistic effect with the cross-linking process of the sustained-release calcium ion system (CaCO3 / D-(+)-glucono-δ-lactone).

[0023] Preferably, in step (3), the temperature of the pre-crosslinking reaction is 15~25℃, and the time of the pre-crosslinking reaction is 15~20 minutes.

[0024] Preferably, in step (4), the printing process parameters are: printing temperature of 15~25℃, nozzle diameter of 0.8mm, extrusion speed of 10~20 mm / s, and barrel capacity of 100 ml; the three-dimensional digital model is a 20×20×20 mm cube model.

[0025] Preferably, in step (5), the temperature of static incubation is 15~25℃ and the time of static incubation is 2.5~3.5 hours.

[0026] Preferably, in step (5), the heat treatment conditions are: heat treatment in a water bath at 75~90℃.

[0027] The second aspect of this application also provides a programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties, which is a programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties prepared by the above method.

[0028] The third aspect of this application also provides the application of the above-mentioned programmable 3D / 4D printed hydrogel mesoporous scaffolds with adjustable mechanical and topological properties in the preparation of cell culture meat products or tissue engineering products.

[0029] Compared with the prior art, this application has the following advantages:

[0030] 1. The programmable 3D / 4D printed hydrogel mesoporous scaffold of this application has adjustable mechanical and topological properties. It has an interpenetrating polymer network structure composed of synergistic ionic crosslinking and enzymatic crosslinking. The scaffold exhibits a regular honeycomb mesoporous topology with an average pore size distribution between 164 μm and 385 μm and a concentrated pore size distribution. The porosity of the scaffold is adjustable, and it exhibits a customizable ordered hierarchical structure, excellent mechanical properties, rheological properties and high water retention capacity.

[0031] 2. The programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties of this application is responsive. When printed as a non-uniform structure, it can generate non-uniform swelling by absorbing water, thereby undergoing a preset shape change. The non-uniform structure is a structure with a thickness gradient.

[0032] 3. This application achieves a synergistic improvement in mechanical properties and pore topology: Through a "one-pot delayed double cross-linking" strategy, this application cleverly coordinates the continuous ionic cross-linking of sodium alginate and the delayed enzymatic cross-linking of gelatin. These two networks interpenetrate and synergistically enhance each other, enabling the scaffold to achieve significantly enhanced mechanical strength (e.g., approximately 20-fold increase in tensile strength and approximately 4-fold increase in compressive modulus) while forming and maintaining a regular, interconnected honeycomb-like mesoporous structure. This effectively solves the contradiction between high mechanical strength and high porosity / large pore size in traditional hydrogels, providing cells with an ideal three-dimensional environment that combines mechanical support and nutrient transport capabilities.

[0033] 4. This application provides customizable mechanical and structural properties: By simply adjusting the gelatin concentration, this application enables precise and convenient control over the mechanical properties of the scaffold (such as storage modulus, compressive modulus, and tensile strength) and the pore topology (such as pore size, distribution, and porosity). This customizability allows this application to flexibly meet the specific physical performance requirements of different application scenarios (such as cultured meat and tissue engineering).

[0034] 5. The process of this application is simple, green and easy to promote: The "one-pot" preparation process adopted in this application is simple and does not require complicated multi-step reactions or post-processing. The materials used (gelatin and sodium alginate) are all edible and biocompatible natural polymers. The cross-linking process is mild and conforms to the concept of green manufacturing. It is very suitable for the large-scale preparation of cultured meat and tissue engineering scaffolds.

[0035] 6. This application endows the scaffold with intelligent responsiveness and programmable deformation capabilities: The scaffold fabricated in this application exhibits water responsiveness. By designing the spatial non-uniformity of the printed structure (such as thickness gradients) and utilizing the control of the swelling-stiffness balance relationship by gelatin concentration, the deformation behavior of the hydrogel in aqueous solution can be programmed. This provides an innovative material platform and scalable manufacturing method for developing intelligent scaffolds for dynamic cell culture. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in this application or the prior art, the drawings used in the description of this application or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0037] Figure 1 These are rheological diagrams of the hydrogel mesoporous scaffolds prepared in Examples 1, 2, 3, 4 and Comparative Example 1;

[0038] Figure 2 These are the compressive stress-strain curves and compressive modulus-hysteresis energy diagrams of the hydrogel mesoporous scaffolds prepared in Examples 1, 2, 3, 4 and Comparative Example 1;

[0039] Figure 3 These are tensile stress-strain curves and tensile modulus diagrams of the hydrogel mesoporous scaffolds prepared in Examples 1, 2, 3, 4 and Comparative Example 1;

[0040] Figure 4 These are scanning electron microscope images and pore size analysis diagrams of the hydrogel mesoporous scaffolds prepared in Examples 1, 2, 3, 4 and Comparative Example 1;

[0041] Figure 5 This is a porosity diagram of the hydrogel mesoporous scaffolds prepared in Examples 1, 2, 3, 4 and Comparative Example 1;

[0042] Figure 6 The diagram shows the water retention properties of the hydrogel mesoporous scaffolds prepared in Examples 1, 2, 3, 4 and Comparative Example 1.

[0043] Figure 7 It is a cubic lattice scaffold printed with the composite hydrogel ink prepared in Examples 2, 3 and 4;

[0044] Figure 8 This is a schematic diagram showing the shape change of the hydrogel strip with thickness gradient prepared in Example 5 after immersion in deionized water for 1.5 hours. Detailed Implementation

[0045] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0046] Unless otherwise specified, the experimental methods used in the embodiments of this application are all conventional methods.

[0047] In the following examples and comparative examples, unless otherwise specified, all raw materials can be obtained by commercial purchase or conventional methods.

[0048] Example 1

[0049] This embodiment provides a method for preparing a 3D-printed gelatin / sodium alginate (Gel / SA) hydrogel mesoporous scaffold, the specific steps of which are as follows:

[0050] (1) 2.5 g gelatin and 3 g sodium alginate were dissolved in 100 ml of deionized water and mixed with magnetic stirring at 60°C for 1 hour to form a hydrogel solution with a concentration of 2.5% gelatin and 3% sodium alginate.

[0051] (2) Cool the hydrogel solution obtained in step (1) to room temperature. Then, add 0.7% CaCO3 and D-(+)-gluconic acid-δ-lactone (GDL) (the molar ratio of CaCO3 to GDL is 1:2), and continuously inject CaCO3 into the solution. 2+The process releases sodium alginate to initiate ionic cross-linking, thereby enhancing the shape and strength required for 3D printing. Simultaneously, 4 U / g of transglutaminase (TG) is added, and the cross-linking rate between TG and gelatin is slowed down by low-temperature regulation, thus creating a synergistic effect with the cross-linking process of the sustained-release calcium ion system (CaCO3 / GDL). After adding TG and CaCO3 / GDL, the mixture is stirred for approximately one minute until homogeneous. After standing for 15 minutes for pre-cross-linking, a composite hydrogel ink suitable for 3D printing is obtained.

[0052] (3) Load the composite hydrogel ink obtained in step (2) into the barrel of the FOODINI 3D printer. Set the printing parameters as follows: printing temperature 25℃, nozzle diameter 0.8 mm, extrusion speed 15 mm / s. Print according to the preset cubic three-dimensional model with a side length of 20 mm to obtain the initial product of the bracket.

[0053] (4) The printed scaffold was incubated at room temperature for 3 hours to complete the full ionic crosslinking of sodium alginate and the enzymatic crosslinking of gelatin. Subsequently, the hydrogel mesoporous scaffold was heated in an 85°C water bath to obtain the final hydrogel mesoporous scaffold (named 2.5Gel3SA).

[0054] Example 2

[0055] This embodiment provides a method for preparing a 3D-printed gelatin / sodium alginate (Gel / SA) hydrogel mesoporous scaffold, the specific steps of which are as follows:

[0056] (1) 5 g gelatin and 3 g sodium alginate were dissolved in 100 ml of deionized water and mixed with magnetic stirring at 60°C for 1 hour to form a hydrogel solution with a concentration of 5% gelatin and 3% sodium alginate.

[0057] (2) Cool the hydrogel solution obtained in step (1) to room temperature. Then, add 0.7% CaCO3 and D-(+)-gluconic acid-δ-lactone (GDL) (the molar ratio of CaCO3 to GDL is 1:2), and continuously inject CaCO3 into the solution. 2+ The process releases sodium alginate to initiate ionic cross-linking, thereby enhancing the shape and strength required for 3D printing. Simultaneously, 4 U / g of transglutaminase (TG) is added, and the cross-linking rate between TG and gelatin is slowed down by low-temperature regulation, thus creating a synergistic effect with the cross-linking process of the sustained-release calcium ion system (CaCO3 / GDL). After adding TG and CaCO3 / GDL, the mixture is stirred for approximately one minute until homogeneous. After standing for 15 minutes for pre-cross-linking, a composite hydrogel ink suitable for 3D printing is obtained.

[0058] (3) Load the composite hydrogel ink obtained in step (2) into the barrel of the FOODINI 3D printer. Set the printing parameters as follows: printing temperature 25℃, nozzle diameter 0.8 mm, extrusion speed 15 mm / s. Print according to the preset cubic three-dimensional model with a side length of 20 mm to obtain the initial product of the bracket.

[0059] (4) The printed scaffold was incubated at room temperature for 3 hours to complete the full ionic crosslinking of sodium alginate and the enzymatic crosslinking of gelatin. Then, the hydrogel mesoporous scaffold was heated in an 85°C water bath to obtain the final hydrogel mesoporous scaffold (named 5Gel3SA).

[0060] Compared to Example 1, the gelatin concentration in Example 2 was increased by 2 times.

[0061] Example 3

[0062] This embodiment provides a method for preparing a 3D-printed gelatin / sodium alginate (Gel / SA) hydrogel mesoporous scaffold, the specific steps of which are as follows:

[0063] (1) 7.5 g gelatin and 3 g sodium alginate were dissolved in 100 ml of deionized water and mixed with magnetic stirring at 60°C for 1 hour to form a hydrogel solution with a concentration of 7.5% gelatin and 3% sodium alginate.

[0064] (2) Cool the hydrogel solution obtained in step (1) to room temperature. Then, add 0.7% CaCO3 and D-(+)-gluconic acid-δ-lactone (GDL) (the molar ratio of CaCO3 to GDL is 1:2), and continuously inject CaCO3 into the solution. 2+ The process releases sodium alginate to initiate ionic cross-linking, thereby enhancing the shape and strength required for 3D printing. Simultaneously, 4 U / g of transglutaminase (TG) is added, and the cross-linking rate between TG and gelatin is slowed down by low-temperature regulation, thus creating a synergistic effect with the cross-linking process of the sustained-release calcium ion system (CaCO3 / GDL). After adding TG and CaCO3 / GDL, the mixture is stirred for approximately one minute until homogeneous. After standing for 15 minutes for pre-cross-linking, a composite hydrogel ink suitable for 3D printing is obtained.

[0065] (3) Load the composite hydrogel ink obtained in step (2) into the barrel of the FOODINI 3D printer. Set the printing parameters as follows: printing temperature 25℃, nozzle diameter 0.8 mm, extrusion speed 15 mm / s. Print according to the preset cubic three-dimensional model with a side length of 20 mm to obtain the initial product of the bracket.

[0066] (4) The printed scaffold was incubated at room temperature for 3 hours to complete the full ionic crosslinking of sodium alginate and the enzymatic crosslinking of gelatin. Then, the hydrogel mesoporous scaffold was heated in an 85°C water bath to obtain the final hydrogel mesoporous scaffold (named 7.5Gel3SA).

[0067] Compared to Example 1, the gelatin concentration in Example 3 was increased by 3 times.

[0068] Example 4

[0069] This embodiment provides a method for preparing a 3D-printed gelatin / sodium alginate (Gel / SA) hydrogel mesoporous scaffold, the specific steps of which are as follows:

[0070] (1) 10 g gelatin and 3 g sodium alginate were dissolved in 100 ml of deionized water and mixed with magnetic stirring at 60°C for 1 hour to form a hydrogel solution with a concentration of 10% gelatin and 3% sodium alginate.

[0071] (2) Cool the hydrogel solution obtained in step (1) to room temperature. Then, add 0.7% CaCO3 and D-(+)-gluconic acid-δ-lactone (GDL) (the molar ratio of CaCO3 to GDL is 1:2), and continuously inject CaCO3 into the solution. 2+ The process releases sodium alginate to initiate ionic cross-linking, thereby enhancing the shape and strength required for 3D printing. Simultaneously, 4 U / g of transglutaminase (TG) is added, and the cross-linking rate between TG and gelatin is slowed down by low-temperature regulation, thus creating a synergistic effect with the cross-linking process of the sustained-release calcium ion system (CaCO3 / GDL). After adding TG and CaCO3 / GDL, the mixture is stirred for approximately one minute until homogeneous. After standing for 15 minutes for pre-cross-linking, a composite hydrogel ink suitable for 3D printing is obtained.

[0072] (3) Load the composite hydrogel ink obtained in step (2) into the barrel of the FOODINI 3D printer. Set the printing parameters as follows: printing temperature 25℃, nozzle diameter 0.8 mm, extrusion speed 15 mm / s. Print according to the preset cubic three-dimensional model with a side length of 20 mm to obtain the initial product of the bracket.

[0073] (4) The printed scaffold was incubated at room temperature for 3 hours to complete the full ionic crosslinking of sodium alginate and the enzymatic crosslinking of gelatin. Then, the hydrogel mesoporous scaffold was heated in an 85°C water bath to obtain the final hydrogel mesoporous scaffold (named 10Gel3SA).

[0074] Compared to Example 1, the gelatin concentration in Example 4 was increased by 4 times.

[0075] Example 5

[0076] This embodiment aims to demonstrate the shape changes and programmability of the hydrogel mesoporous scaffold prepared in this application in aqueous solution. The specific steps are as follows:

[0077] (1) Four kinds of composite hydrogel inks were prepared according to the formulations and steps (1) to (2) of Examples 1 to 4 respectively.

[0078] (2) The obtained composite hydrogel ink was loaded into the barrel of the FOODINI 3D printer. The printing parameters were set as follows: printing temperature 25℃, nozzle diameter 0.8 mm, and extrusion speed 15 mm / s. The initial product of the support was obtained by printing a strip with a linear gradient from 50 mm long × 6 mm wide × 1.5 mm thick to 4 mm according to the preset three-dimensional model size.

[0079] (3) The initial product of the scaffold described in step (2) was incubated at room temperature for 3 h and then inactivated at 85°C to obtain the final hydrogel strips (named 2.5Gel3SA-4D, 5Gel3SA-4D, 7.5Gel3SA-4D, and 10Gel3SA-4D, respectively).

[0080] (4) After soaking the four groups of hydrogel strips in deionized water at room temperature for 1.5 h, observe and record their degree of bending.

[0081] Comparative Example 1

[0082] This comparative example provides a method for preparing a 3D-printed sodium alginate hydrogel scaffold, the specific steps of which are as follows:

[0083] (1) 3 g of sodium alginate was dissolved in 100 ml of deionized water and mixed with magnetic stirring at 60°C for 1 hour to form a 3% sodium alginate hydrogel solution.

[0084] (2) Cool the hydrogel solution obtained in step (1) to room temperature. Then, add 0.7% CaCO3 and D-(+)-gluconic acid-δ-lactone (GDL) (the molar ratio of CaCO3 to GDL is 1:2), and continuously inject CaCO3 into the solution. 2+ The release of sodium alginate initiates ionic crosslinking, thereby enhancing the shape and strength required for 3D printing. After adding CaCO3 / GDL, stir for about one minute until homogeneous, and allow to stand for 15 minutes for pre-crosslinking to obtain a hydrogel ink suitable for 3D printing.

[0085] (3) Load the hydrogel ink obtained in step (2) into the barrel of the FOODINI 3D printer. Set the printing parameters as follows: printing temperature 25℃, nozzle diameter 0.8 mm, extrusion speed 15 mm / s. Print according to the preset 3D model of a cube with a side length of 20 mm to obtain the hydrogel scaffold (named 3SA).

[0086] Compared to Example 1, no gelatin was added to the comparative example.

[0087] The following are the relevant performance tests of the hydrogel mesoporous scaffold involved in this application:

[0088] 1. Rheological testing of hydrogel scaffolds

[0089] Rheological measurements of the hydrogel mesoporous scaffold were performed using a rheometer (MARS40, Thermo Fisher Scientific, USA) equipped with a plate (35 mm in diameter, 1 mm gap). The oscillation frequency was 0.1–50 Hz, the strain was 0.5%, and the temperature was 25 °C.

[0090] The results are as follows Figure 1 As shown, the hydrogel mesoporous scaffold exhibits viscoelastic properties similar to solids, with its storage modulus (G') significantly greater than its loss modulus (G''), highlighting its excellent elastic performance. Furthermore, the G' and G'' of the gel / SA composite hydrogel mesoporous scaffold show minimal variation across the entire frequency range, indicating low frequency dependence and excellent mechanical strength and structure retention. This is primarily due to the interweaving of the "egg-box" structure formed by sodium alginate ion crosslinking and the network structure formed by gelatin covalent crosslinking, enhancing the interactions between hydrogel particles (including electrostatic interactions, hydrogen bonds, and van der Waals forces). With increasing gelatin concentration, the storage modulus of the hydrogel mesoporous scaffold increases, indicating that higher gelatin concentrations result in a more robust and compact protein-polysaccharide interaction network structure. The excellent mechanical strength and structure retention of the hydrogel mesoporous scaffold ensure the structural stability of the scaffold during cell culture, preventing scaffold structural damage caused by changes in the external environment, thus better maintaining the three-dimensional structure of cultured meat.

[0091] 2. Tissue and mechanical property testing of hydrogel scaffolds

[0092] (1) Organizational testing

[0093] The gel strength, tensile strength, hardness, and chewiness of the hydrogel mesoporous scaffold were tested using a texture analyzer (TA. XT Plus, Stable Micro Systems, UK) equipped with a P / 0.5 probe. The parameters were set as follows: single compression mode, pre-test velocity 1 mm / s, mid-test velocity 1 mm / s, post-test velocity 1 mm / s, strain 50%, trigger force 5.0 g.

[0094] The results are shown in Table 1: With increasing gelatin concentration, the gel strength, hardness, and chewiness of the Gel / SA composite hydrogel mesoporous scaffold significantly improved, and its fracture strength was nearly 20 times higher than that of the pure sodium alginate hydrogel mesoporous scaffold. This indicates that the one-pot delayed double crosslinking strategy resulted in a denser crosslinking network in the composite hydrogel mesoporous scaffold, and the density of the crosslinking network increased with increasing gelatin concentration, thereby greatly enhancing the composite hydrogel mesoporous scaffold's resistance to deformation and structural damage. Furthermore, this adjustable mechanical property allows the hydrogel mesoporous scaffold to be optimized according to different application scenarios and needs. For example, in cultured meat applications requiring high mechanical support, the mechanical strength of the hydrogel mesoporous scaffold can be enhanced by increasing the gelatin concentration, thereby better mimicking the mechanical environment of natural tissues and providing suitable support for cell adhesion, proliferation, and differentiation.

[0095] Table 1. Gel strength, tensile strength, hardness, and chewiness of hydrogel scaffolds at 50% strain.

[0096]

[0097] Different letters in the same column indicate significant differences (P<0.05).

[0098] (2) Compression test

[0099] The hydrogel mesoporous scaffold was placed at the center of the test plate of a texture analyzer (TA.XT Plus, Stable Micro Systems, UK). A single-compression mode was selected, using a P / 36R probe with a pre-compression velocity of 1 mm / s, a mid-compression velocity of 1 mm / s, and a post-compression velocity of 1 mm / s, and a trigger force of 5.0 g, compressing the hydrogel mesoporous scaffold to 30% of its thickness. The compressive modulus was determined by calculating the slope of the strain in the first 5% of the stress-strain curve. The hysteresis energy was determined by calculating the area of ​​the hysteresis loop formed during the loading and unloading processes of the stress-strain curve.

[0100] The results are as follows Figure 2As shown, the compressive modulus of the gel / SA composite hydrogel mesoporous scaffold increases significantly with increasing gelatin concentration, with the modulus of 10Gel3SA being approximately four times that of 3SA. This enhanced stiffness is crucial for preventing structural collapse during 3D printing. Furthermore, 3SA exhibits the lowest hysteresis energy, indicating poor energy dissipation and brittleness. In contrast, the composite hydrogel mesoporous scaffold demonstrates higher hysteresis energy, with 2.5Gel3SA showing the highest. This is primarily due to the double cross-linking, which makes the network structure of the composite hydrogel mesoporous scaffold more uniform and dense, resulting in superior overall energy absorption and dissipation performance.

[0101] (3) Tensile test

[0102] A rectangular hydrogel measuring 60 mm in length, 10 mm in width, and 5 mm in height was printed using a 3D printer (FOODINI) for tensile testing. The tensile test was conducted at a speed of 1 mm / s, and the Young's modulus was determined by calculating the slope of the first 5% of strain in the stress-strain curve.

[0103] The results are as follows Figure 3 As shown, the pure sodium alginate hydrogel scaffold (3SA) is brittle and cannot be effectively tensile tested. In contrast, the gel / SA composite hydrogel mesoporous scaffold exhibits a clear tensile stress-strain curve, and the Young's modulus increases significantly with increasing gelatin concentration, which further verifies the mechanical reinforcement effect of the double cross-linked network.

[0104] 3. Microstructure and physical property characterization of hydrogel mesoporous scaffolds

[0105] (1) Scanning electron microscope

[0106] After freeze-drying, the hydrogel mesoporous scaffold was sliced ​​into thin sections and sputter-coated with gold. The microstructure of the hydrogel cross-section was observed using an intelligent tungsten filament scanning electron microscope (Axia, Thermo Fisher Scientific, USA). The SEM images were processed using ImageJ software to analyze the pore size of the hydrogel mesoporous scaffold.

[0107] The results are as follows Figure 4As shown, the microstructure of pure sodium alginate hydrogel scaffold (3SA) is relatively loose, with irregular pore shapes. In contrast, the composite hydrogel mesoporous scaffold (2.5Gel 3SA + 10Gel 3SA) gradually develops a more regular pore structure with increasing gelatin content, exhibiting a honeycomb-like porous structure. This porous structure is crucial for nutrient transport and the removal of metabolic waste, processes essential for cell survival and function in cultured meat production. Further statistical analysis of pore size revealed that 3SA has a wide and uneven pore size distribution, with an average pore size of 131.87 ± 80.19 μm. After adding gelatin, the average pore size of 2.5Gel 3SA significantly increased to 385.42 ± 72.21 μm. With increasing gelatin concentration, the average pore size of the composite hydrogel mesoporous scaffold gradually decreased, and the pore size distribution became more concentrated. This indicates that gelatin content has a regulatory effect on pore size and uniformity.

[0108] (2) Porosity measurement

[0109] The porosity of the hydrogel mesoporous scaffold was measured using the liquid displacement method. The lyophilized hydrogel mesoporous scaffold was weighed and recorded as M0. It was then completely immersed in anhydrous ethanol for 1 hour for adsorption. After removal, the anhydrous ethanol on the hydrogel surface was wiped off with filter paper and weighed again, recorded as M1. The porosity of the hydrogel mesoporous scaffold was calculated using the following formula:

[0110] ;

[0111] ρ is the density of anhydrous ethanol; V is the volume of the lyophilized hydrogel mesoporous scaffold before it was immersed in anhydrous ethanol.

[0112] The results are as follows Figure 5 As shown, 3SA exhibits the highest porosity, exceeding 90%. However, as the gelatin concentration increases from 2.5% to 10%, the porosity of the composite hydrogel mesoporous scaffold decreases, but remains at a high level. This is because the interwoven network formed by gelatin and sodium alginate transforms the hydrogel mesoporous scaffold from a loose porous structure into a compact, regular pore structure. Furthermore, adjusting the gelatin content can impart different topological properties to the hydrogel mesoporous scaffold. Therefore, by simply adjusting the gelatin concentration, the topological structure of the hydrogel mesoporous scaffold can be modulated, thereby adapting to the different cell requirements for porosity and pore structure.

[0113] (3) Water-holding capacity measurement

[0114] A certain mass of hydrogel mesoporous scaffold was centrifuged at 10000 g for 10 min at 4℃ and the water was removed. The formula for calculating the water-holding capacity of the hydrogel mesoporous scaffold is as follows:

[0115] ;

[0116] M0 is the mass of the empty centrifuge tube; M1 is the total mass of the centrifuge tube and hydrogel mesoporous scaffold before centrifugation; M2 is the total mass of the centrifuge tube and hydrogel mesoporous scaffold after centrifugation.

[0117] The results are as follows Figure 6 As shown, the water-holding capacity of pure sodium alginate hydrogel mesoporous scaffolds is less than 30%. With the gelatin content increasing from 2.5% to 10%, the water-holding capacity of the Gel / SA composite hydrogel mesoporous scaffold shows an upward trend, with the water-holding capacity of 10Gel / 3SA approaching 100%. This indicates that the tight network structure formed by the double cross-linking can more effectively "capture" water. Furthermore, gelatin is an amphiphilic polymer, and its complex amino acid composition allows it to bind water molecules more firmly through multiple mechanisms (such as hydrogen bonding), further enhancing the water-retention capacity of the hydrogel mesoporous scaffold. The high water-holding capacity of the hydrogel mesoporous scaffold can provide a stable and moist growth microenvironment for cells during cell culture, thereby promoting cell proliferation, differentiation, and metabolism.

[0118] (4) 3D printing of cubic lattice supports

[0119] Using a Foodini 3D printer, a cubic lattice support with a complex spatial structure was printed at the following printing parameters: temperature 25°C, nozzle diameter 0.8 mm, and extrusion speed 15 mm / s.

[0120] The results are as follows Figure 7 As shown, under the same printing conditions, both the pure sodium alginate hydrogel scaffold (3SA) and the low gelatin concentration composite scaffold (2.5Gel3SA) experienced structural collapse and could not maintain the preset three-dimensional morphology; while the groups with a gelatin concentration of not less than 5% (including 5Gel3SA, 7.5Gel3SA, and 10Gel3SA) were successfully printed. This result confirms the key role of the "one-pot delayed double crosslinking strategy" of this invention in realizing the molding of complex structures. The collapse of 3SA is attributed to the low mechanical strength and poor structural recovery of its single ionic crosslinking network; the printing failure of 2.5Gel3SA indicates that when the gelatin concentration is insufficient, it is impossible to form a sufficiently strong enzymatic covalent crosslinking network, making it difficult for it to effectively synergize with the initial ionic network to form a more stable double crosslinking network structure, and therefore still lacks the anti-collapse ability required to support complex models. When the gelatin concentration is greater than or equal to 5%, it can effectively increase the crosslinking points of gelatin and sodium alginate, enhance their electrostatic interaction and hydrogen bonding, thereby giving the hydrogel mesoporous scaffold good mechanical support ability.

[0121] (5) Observation of shape changes of 4D hydrogel strips in aqueous solution

[0122] Four sets of 3D-printed hydrogel strips with thickness gradients were completely immersed in deionized water (temperature 25℃) and their shape changes were observed after 1.5 h.

[0123] The results are as follows Figure 8 As shown: After soaking, the hydrogel strips bend in the direction of their greater thickness, and the degree of bending gradually increases with the increase of gelatin concentration from 2.5% to 7.5%. However, when the gelatin concentration increases to 10%, the degree of bending of the hydrogel strips decreases. This is mainly because the thinner end of the hydrogel strip absorbs water and swells faster, generating internal stress that causes it to bend towards the thicker end. At lower concentrations (2.5%), the hydrogel network structure is relatively loose, with strong expansion capacity but low stiffness, thus lacking sufficient support to form significant bending. As the gelatin concentration increases, the network structure becomes denser, the cross-linking density increases, and the stiffness improves, but at this point, the swelling capacity effect still dominates, causing unevenness in the swelling process and increasing the degree of bending of the hydrogel strips. However, when the gelatin concentration further increases to 10%, the network structure becomes too dense, the swelling capacity weakens, and the stiffness increases significantly. At this point, the stiffness effect dominates, thus inhibiting deformation, and therefore the degree of bending of the hydrogel strips decreases. This phenomenon demonstrates that the swelling-stiffness balance can be precisely programmed by adjusting the gelatin concentration, thereby pre-setting the final shape of the 4D hydrogel deformation. Furthermore, employing a one-pot delayed double cross-linking strategy, pre-cross-linking is performed by delaying the cross-linking rate of the enzyme at low temperatures, followed by 3D printing, thus achieving controllable topology and 4D responsiveness. Therefore, by designing the degree and direction of curvature of the hydrogel mesoporous scaffold, muscle fibers can be induced to grow and align along specific directions, thereby improving the texture and mouthfeel of cultured meat.

[0124] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. A method for fabricating a programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties, characterized in that, Includes the following steps: (1) Dissolve gelatin and sodium alginate in a solvent and stir magnetically to form a homogeneous hydrogel solution; (2) Add an enzyme cross-linking agent and a continuous calcium ion release cross-linking agent to the hydrogel solution at the same time, stir evenly, and obtain a mixed system solution; (3) The mixed system solution is allowed to stand at 15~25℃ to carry out a pre-crosslinking reaction, and a printable composite hydrogel ink is obtained. (4) The composite hydrogel ink is transferred into the barrel of an extrusion 3D printer and printed according to a preset three-dimensional digital model to obtain a scaffold initial product with a three-dimensional porous structure. (5) The initial product of the scaffold is incubated to complete sufficient ionic crosslinking and enzymatic crosslinking, and then heat-treated to obtain a programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties.

2. The method for preparing a programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties according to claim 1, characterized in that, In step (1), the gelatin is fish-derived gelatin; the sodium alginate is sodium alginate with a viscosity of 5.0~7.0 mPa·s, an M / G ratio of 1:0.8~1.2, and a molecular weight of 18000~20000 Da; the solvent is deionized water; In step (1), the mass ratio of gelatin to sodium alginate is (0~10):

3.

3. The method for preparing a programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties according to claim 1, characterized in that, In step (1), the specific conditions for magnetic stirring are: mixing with magnetic stirring at a constant temperature of 60°C for 1 to 1.5 hours.

4. The method for preparing a programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties according to claim 1, characterized in that, In step (2), the enzyme cross-linking agent is transglutaminase; the continuous calcium ion release cross-linking agent is a mixture of CaCO3 and D-(+)-gluconic acid-δ-lactone, and the molar ratio of CaCO3 to D-(+)-gluconic acid-δ-lactone is 1:1.5~2.

5.

5. The method for preparing a programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties according to claim 1, characterized in that, In step (3), the temperature of the pre-crosslinking reaction is 15~25℃, and the time of the pre-crosslinking reaction is 15~20 minutes.

6. The method for preparing a programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties according to claim 1, characterized in that, In step (4), the printing process parameters are: printing temperature of 15~25℃, nozzle diameter of 0.8 mm, extrusion speed of 10~20 mm / s, and barrel capacity of 100 ml; the three-dimensional digital model is a 20×20×20 mm cube model.

7. The method for preparing a programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties according to claim 1, characterized in that, In step (5), the temperature for static incubation is 15~25℃, and the time for static incubation is 2.5~3.5 hours.

8. The method for preparing a programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties according to claim 1, characterized in that, In step (5), the heat treatment conditions are: heat treatment in a water bath at 75~90℃.

9. A programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties, characterized in that, Programmable 3D / 4D printed hydrogel mesoporous scaffolds with adjustable mechanical and topological properties are obtained by the method described in any one of claims 1 to 8.

10. The application of the programmable 3D / 4D printed hydrogel mesoporous scaffold with adjustable mechanical and topological properties as described in claim 9 in the preparation of cell culture meat products or tissue engineering products.

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