Spiral composite hydrogel stent as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of artificial meat tissue engineering, and particularly relates to a spiral composite hydrogel scaffold as well as a preparation method and application thereof. The spiral composite hydrogel scaffold provided by the invention adopts a bionic microstructure design, and can simulate fiber arrangement and a multi-scale hierarchical structure of natural beef tissues, so that ordered distribution of muscle cells and fat cells is guided in a microscopic space. By optimizing hydrogel components and a cross-linking mode, the mechanical strength and the elasticity of the stent are controllable in a relatively large range. A three-dimensional culture environment constructed by using the scaffold can promote differentiation and synchronous maturation of beef related muscle cells and fat cells, the time of a texturization process is greatly shortened, and the fibrosis degree and marbling effect of artificial meat are improved. Compared with a traditional two-dimensional culture method or a single scaffold method, the method can efficiently obtain textured artificial meat with a bionic structure and a texture close to that of natural beef, and is suitable for the fields of artificial meat manufacturing, functional food development, tissue engineering and the like.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering technology for artificial meat, specifically relating to a spiral composite hydrogel, its preparation method, and its application. Background Technology

[0002] Cultivated meat refers to alternative foods that achieve a high degree of similarity to natural meat in taste, texture, and nutritional composition by culturing animal-derived muscle cells and fat cells in vitro. In recent years, with population growth and the increasing prominence of issues such as food safety and carbon emissions, the cultivated meat industry has experienced rapid development. However, the core challenge currently hindering the industrialization of cultivated meat lies in how to efficiently and cost-effectively obtain high-density, well-structured beef tissue. Traditional two-dimensional culture and single-layer scaffold methods struggle to achieve large-scale expansion and co-differentiation of muscle and fat cells, resulting in low cell density, disordered arrangement, and loose tissue structure. To meet future industrialization needs, it is essential to develop novel, highly efficient three-dimensional bioreactors and cell scaffold systems capable of simultaneously supporting high-density cell seeding, stable adhesion, growth expansion, and directed differentiation, thereby achieving simultaneous improvements in the biomimetic structure, mechanical properties, and maturity of beef tissue.

[0003] Cell scaffolds are core components of three-dimensional cell culture systems, primarily providing cells with biomimetic spatial structures, mechanical support, and biochemical microenvironments. They are widely used in tissue engineering, organ-on-a-chip technology, and artificial meat manufacturing. Currently, common scaffold materials include natural polymers (such as gelatin, collagen, and alginate) and synthetic polymers (such as PEG and PVA). Among these, hydrogel-based cell scaffolds have become a research hotspot due to their high biocompatibility, biodegradability, and high designability. By adjusting the crosslinking density and component ratio of the hydrogel, precise control over the scaffold structure, porosity, and mechanical strength can be achieved. However, achieving highly biomimetic hydrogel scaffolds in artificial meat construction remains a significant challenge. For example, beef muscle fibers are highly parallel, and a suitable mechanical strength of approximately 15 kPa is needed to effectively promote muscle cell differentiation; while adipocytes tend to form isotropic arrangements in a soft matrix at around 3 kPa, thus achieving more efficient adipogenesis. Existing scaffolds cannot simultaneously achieve spatial orientation and mechanical optimization of different cell types in the same system. Therefore, there is an urgent need to develop hydrogel scaffolds that combine biomimetic microstructures and adjustable mechanical properties to promote the efficient maturation of artificial meat tissues. Summary of the Invention

[0004] The technical problems to be solved by this invention are: uneven distribution of cell scaffolds, lack of biomimetic structure, mismatch of mechanical properties, and excessively long culture cycle in artificial meat.

[0005] To address the aforementioned technical problems, this invention proposes a spiral composite hydrogel, its preparation method, and its application. This is achieved through the following technical solution: On the one hand, the purpose of this invention is to provide a spiral composite hydrogel scaffold, which adopts the following technical solution: A helical composite hydrogel scaffold is provided, comprising a plurality of circumferentially spaced helical blades intersecting at a central point; each helical blade consists of a front end and a rear end, which are connected; the front end is a two-dimensional helix, and the endpoints of the front ends are circumferentially distributed on a circle with a radius equal to the chord length of the helical blade; the helical composite hydrogel scaffold includes a biocompatible curable polymer and a photoinitiator.

[0006] This invention provides a helical composite hydrogel scaffold. Based on GelMA material, this helical composite hydrogel scaffold is a three-dimensional scaffold with helical biomimetic microchannels fabricated using a molding method, and incorporates vortex flow driving to achieve rapid, high-density aggregation and uniform seeding of cells. Through scaffold-flow field coupling and optimized hydrogel formulation and cross-linking process, this invention achieves controllable local mechanical property gradients within the helical composite hydrogel scaffold, enabling muscle cells to exhibit parallel orientation in areas with a hardness of approximately 15 kPa, and adipocytes to form an isotropic distribution in areas with a softness of approximately 3 kPa, significantly improving the co-differentiation efficiency of the two cell types. A three-dimensional beef tissue culture system constructed using this scaffold achieves rapid maturation without prolonged cell expansion, forming highly biomimetic "marbled" artificial beef tissue in just 4 days. This invention overcomes the structural and mechanical limitations of existing hydrogel scaffolds, providing a novel technological path for the efficient, low-cost, and large-scale production of high-quality artificial meat.

[0007] In a preferred embodiment of the present invention, the angle between the tangent at the tip of the spiral blade and the tangent on the circumference of the tip of the blade is α, where 0° < α ≤ 90°; the starting point of the rear end of the spiral blade is located at an angle β between the tangent at the tip of the blade and the tangent on the circumference of the rear end, where 0° < β ≤ 90°; the distance from the starting point of the tip of the spiral blade to the ending point of the rear end is the chord length r of the spiral blade; the diameter of the helical composite hydrogel scaffold is 2r, where 100μm ≤ 2r ≤ 2cm.

[0008] In a preferred embodiment of the present invention, the number of helical blades is n, where 3 ≤ n ≤ 11; and the height of the helical blades is 0.5 mm to 10 mm.

[0009] In a preferred embodiment of the present invention, the curable polymer is selected from one or more of gelatin methacrylamide, plant fiber, calcium alginate, sodium alginate and calcium chloride; the photoinitiator is selected from one or more of vitamin B or vitamin C.

[0010] On the other hand, the present invention provides a method for preparing a spiral composite hydrogel scaffold, which adopts the following technical solution: The process includes the following steps: Step 1, designing a hydrogel 3D mold and photopolymerizing and printing the mold; Step 2, filling the mold with hydrogel material; Step 3, curing the hydrogel material.

[0011] On the other hand, this application provides an application of a spiral composite hydrogel scaffold in the manufacture of artificial meat.

[0012] On the other hand, a method for regulating cell tissue arrangement includes the following steps: Step 1, preparing a spiral composite hydrogel scaffold using the preparation method described above; Step 2, introducing a cell suspension vortex coupled with the spiral composite hydrogel scaffold.

[0013] On the other hand, a method for promoting the maturation of artificial meat includes the following steps: Step 1, preparing a spiral composite hydrogel scaffold using the preparation method described above; Step 2, placing the spiral composite hydrogel scaffold in a culture dish containing cell culture medium, and introducing a vortex of cell suspension to couple with the spiral composite hydrogel scaffold; Step 3, adding precursor cells to the culture dish to induce differentiation of the cultured cells.

[0014] In a preferred embodiment of the present invention, the precursor cells include one or both of adipocytes and muscle cells.

[0015] In another aspect, there is an artificial meat tissue constructed from a spiral composite hydrogel scaffold as described above; comprising one or both of anisotropically arranged muscle cells in the inner ring and isotropically arranged fat cells in the outer ring.

[0016] The beneficial effects of this invention compared to the prior art are: 1. The hydrogel scaffold of the present invention is simple to prepare, easy to operate, highly reproducible, and can be highly integrated and scaled up.

[0017] 2. The hydrogel scaffold designed in this invention can efficiently enrich cells and has an adjustable Young's modulus to adapt to the optimal mechanical strength for the differentiation of muscle cells and adipocytes.

[0018] 3. The hydrogel scaffold prepared by this invention can control the vortex flow field to local laminar and turbulent flow, thereby achieving anisotropic and isotropic biomimetic micro-arrangement of cells. Attached Figure Description

[0019] Figure 1 Flowchart of biomimetic design and fabrication of hydrogel scaffolds; Figure 1 a is a schematic diagram of the hydrogel scaffold structure; Figure 1 b is a flowchart of the hydrogel scaffold fabrication process; Figure 1 c represents the macroscopic and microscopic structure of Wagyu beef; Figure 1 d is a schematic diagram of the biomimetic design and flow field control of the hydrogel scaffold.

[0020] Figure 2 Schematic diagram of eddy current-induced tissue cell arrangement in a hydrogel scaffold; Figure 2 a is a picture of the actual hydrogel scaffold; Figure 2 b shows the morphological pattern of cell aggregation within the hydrogel scaffold; Figure 2 c is a finite element numerical simulation diagram of the eddy current field of the hydrogel scaffold; Figure 2 d represents two flow field modes of the hydrogel scaffold, namely laminar flow and turbulent flow; Figure 2 e is a schematic diagram of the layered arrangement of muscle and fat cells.

[0021] Figure 3 Artificial meat prepared using hydrogel scaffolds; Figure 3 a represents the macroscopic and microscopic structure of Australian sirloin beef, including Masson staining and H&E staining. Figure 3 b represents artificial meat prepared using a hydrogel scaffold. Purple fluorescence represents MHC protein, a marker of muscle cell maturation, while green fluorescence represents lipid droplets from adipocytes.

[0022] Figure 4 Examples of large-scale artificial meat assembly and performance comparison charts; Figure 4 a shows the assembly process and a picture of the artificial meat; Figure 4 b shows the physical properties of the artificial meat and a comparison with Australian sirloin steak. Detailed Implementation

[0023] The following will describe the embodiments of the present invention and the appendices thereto. Figures 1-4 The technical solutions in the embodiments of the present invention will be described in detail below.

[0024] Example 1: Preparation of artificial meat using a spiral composite hydrogel scaffold (1) Preparation of spiral composite hydrogel scaffold like Figure 1 As shown in b, a negative mold of the spiral composite hydrogel scaffold was first designed using CINEMA 4D software and printed using a DLP printer. A hydrogel solution mixed with vitamin B (riboflavin) and vitamin C was poured into the negative mold. After vacuuming to remove air bubbles, the scaffold was cured by UV exposure and then demolded to obtain the spiral composite hydrogel scaffold, as shown in the diagram. Figure 1 As shown in a. The support was placed in the center of the culture dish and bonded and fixed using uncured hydrogel through ultraviolet exposure.

[0025] Figure 2 Image a shows a physical image of a spiral composite hydrogel scaffold, which consists of two concentric rings.

[0026] The inner circle is a circular region with a radius equal to the chord length of the helical blade. The angle between the tangent at the tip of the helical blade and the tangent on the circumference of the circle containing the tip of the blade is 45°. A 10 wt% GelMA (gelatin methacrylamide) solution containing 3 mM riboflavin and 0.3 mM vitamin C is infused into this region. After photoinitiated polymerization, a hydrogel with a Young's modulus of approximately 13 kPa is formed to simulate the microenvironment of muscle tissue.

[0027] The outer ring is a circular region formed by the axial arrangement of the rear ends of the helical blades. The starting point of the rear end of the helical blade is located at an angle β (90°) between the tangent at the end of the front end of the blade and the circumferential tangent at the end of the rear end. After the inner ring gels, a 10 wt% GelMA solution containing 1 mM vitamin B and 0.1 mM vitamin C is infused into this region. After polymerization, a hydrogel with a Young's modulus of approximately 3 kPa is obtained to simulate the microenvironment of adipose tissue.

[0028] (2) Cultivation of biomimetic artificial meat The muscle precursor cells (C2C12) and adipose precursor cells (3T3-L1) used in this embodiment were purchased from ATCC.

[0029] like Figure 1 As shown in Figure d, a peristaltic pump drives the culture medium to form a vortex flow field in a circular culture dish. First, C2C12 cells are added to the culture system. Under the influence of the vortex, the cells aggregate to the inner region of the scaffold and achieve anisotropic alignment. Subsequently, 3T3-L1 cells are added, and under the influence of the vortex, they aggregate to the outer region of the scaffold and achieve isotropic alignment.

[0030] After cell seeding, a co-culture protocol was used to simultaneously induce the differentiation of C2C12 and 3T3-L1 cells to accelerate the maturation of artificial meat.

[0031] Flow field control, such as Figure 2 As shown in Figure c, the arrows in the figure indicate the direction of cell inflow.

[0032] The blue area in the figure represents the local turbulent flow field formed between the spiral blades of the scaffold when the flow rate is 0~1.0 mm / s, which promotes the anisotropic arrangement of muscle precursor cells and their differentiation into muscle tissue.

[0033] The orange area in the figure represents the laminar flow field formed by the culture medium on the outer ring of the scaffold when the flow rate is 1.0~2.0 mm / s, which promotes the isotropic arrangement of adipocyte precursor cells and their differentiation into adipose tissue.

[0034] Figure 2 b shows a comparison of the microstructure before and after the aggregation of muscle precursor cells.

[0035] Figure 2d shows two flow field modes of the spiral composite hydrogel scaffold: laminar flow and turbulent flow.

[0036] Figure 2 e shows the layered distribution of two types of cells within the scaffold: the inner layer consists of anisotropically arranged muscle cells, and the outer layer consists of isotropically arranged fat cells. The right side shows a microscopic schematic diagram of the cell arrangement of the two tissues under the corresponding flow fields.

[0037] The cultured meat produced in this embodiment has an appearance and texture similar to animal meat. The above method effectively shortens the cultivation time and reduces the amount of culture medium used, potentially significantly reducing the cultivation cost of cultured meat.

[0038] Example 2: Spiral composite hydrogel scaffold for the preparation of artificial meat (1) Preparation of spiral composite hydrogel scaffold like Figure 1 Figure b shows the flowchart for the preparation of the hydrogel scaffold. First, a negative mold of the spiral composite hydrogel scaffold was designed using CINEMA 4D software and printed using a DLP printer. A hydrogel mixture containing vitamin C was poured into the negative mold, air bubbles were removed by vacuuming, and the scaffold was cured by UV exposure before demolding to obtain the hydrogel scaffold. The scaffold was placed in the center of a culture dish, and the uncured hydrogel was bonded and fixed using UV exposure.

[0039] The spiral composite hydrogel scaffold consists of two concentric rings: Inner circle: A circular region with the helical blade chord length as its radius, where the angle between the tip of the helical blade and the tangent of the circumference is 45°. A 2.0 wt% sodium alginate solution is infused into this region, and polymerization is initiated with a 100 mM calcium chloride solution to form a hydrogel with a Young's modulus of approximately 13 kPa, used to simulate the microenvironment of muscle tissue.

[0040] The outer ring is a circular region formed by the axial arrangement of the rear ends of the spiral blades, with the angle between the end point of the blade's rear end and the tangent of the circumference being 90°. After the inner ring gel forms, a 1.0 wt% sodium alginate solution is poured into this region, and polymerization is initiated with a 50 mM calcium chloride solution to obtain a hydrogel with a Young's modulus of approximately 3 kPa, used to simulate the microenvironment of adipose tissue.

[0041] (2) Cultivation of biomimetic artificial meat The same cultivation steps as in Example 1 for biomimetic artificial meat.

[0042] Example 3: Spiral composite hydrogel scaffold for the preparation of artificial meat (1) Preparation of spiral composite hydrogel scaffold The same preparation steps as in Example 2 for the spiral composite hydrogel scaffold.

[0043] (2) Cultivation of biomimetic artificial meat A peristaltic pump drives the culture medium to create a vortex flow field within a circular culture dish. First, muscle precursor cells (C2C12 cells) are added to the culture system. Under the influence of the vortex, the cells aggregate in the inner 45° region of the scaffold, achieving a parallel alignment. Subsequently, adipocyte precursor cells (3T3-L1 cells) are added, and under the influence of the vortex, they aggregate in the outer region of the scaffold, achieving an isotropic alignment.

[0044] After cell seeding, a co-culture protocol was used to simultaneously induce the differentiation of C2C12 and 3T3-L1 cells to accelerate the maturation of the artificial meat. The prepared artificial meat has an appearance and texture similar to animal meat. Figure 1 c shows the macroscopic and microscopic structures of marbled beef. This method significantly shortens the culture time and reduces the amount of culture medium used, potentially leading to a substantial reduction in culture costs.

[0045] Example 4: Microstructural observation of artificial meat prepared by a spiral composite hydrogel scaffold The microstructure of the artificial meat tissue prepared using the method of Example 1 is as follows: Figure 3 As shown. Figure 3 Image a shows a H&E stained micrograph of an Australian sirloin steak, used for comparative observation of its microstructure. Figure 3 b shows a microscopic image of the obtained artificial meat, where purple fluorescence represents the labeling signal of MHC protein, a marker of muscle cell maturation, and green fluorescence represents the labeling signal of lipid droplets in adipocytes. Figure 3 Upon further magnification of the right side of area b, the distribution details of purple and green fluorescence can be observed. As seen in the image, the outer green fluorescent region (fat ring) of the artificial meat tissue is similar in structure to the fat ring of Australian sirloin steak; the inner purple fluorescent region (muscle tissue cells) is similar in structure to the muscle tissue of Australian sirloin steak.

[0046] Furthermore, the distribution of extracellular matrix collagen can be observed through Masson staining. In this embodiment, the artificial meat sample was labeled with MHC protein primary antibody and 594nm wavelength secondary antibody, and stained with lipid droplet staining agent to obtain the aforementioned fluorescence signal.

[0047] Example 5: Performance characterization of artificial meat prepared using a spiral composite hydrogel scaffold Using the method in Example 1, n composite hydrogel scaffolds were prepared and assembled into large artificial meat tissues simulating the structure of marbled beef, such as... Figure 4 As shown in figure a. Upon magnification, the assembled large piece of artificial meat appears as follows: Figure 4 On the right side of a, the yellow part represents adipose tissue cells, and the red part represents muscle tissue cells.

[0048] Texture analysis was performed on the assembled artificial meat tissue, and it was compared with Australian sirloin steak. The results showed ( Figure 4 (b) The texture properties of artificial meat are similar to those of Australian sirloin steak in terms of hardness, chewiness, elasticity, and viscosity.

[0049] The texture analysis method used in this embodiment is texture profile analysis (TPA).

[0050] The above embodiments are merely illustrative of the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solutions based on the technical concept proposed in this invention shall fall within the scope of protection of this invention.

Claims

1. A helical composite hydrogel scaffold, characterized in that, The helical composite hydrogel scaffold is composed of a plurality of circumferentially spaced spiral leaves, and the plurality of spiral leaves intersect at a center point; the spiral leaf is composed of a leaf front end and a leaf rear end, the leaf front end and the leaf rear end are connected, the leaf front end is a two-dimensional spiral line, and the terminal point of the leaf front end is distributed circumferentially on the circumference with the chord length of the spiral leaf as the radius; the helical composite hydrogel scaffold comprises a biocompatible curable polymer and a photoinitiator.

2. The helical composite hydrogel scaffold of claim 1, wherein, The helical composite hydrogel scaffold is composed of a plurality of circumferentially spaced spiral leaves, and the plurality of spiral leaves intersect at a center point; the spiral leaf is composed of a leaf front end and a leaf rear end, the leaf front end and the leaf rear end are connected, the leaf front end is a two-dimensional spiral line, and the terminal point of the leaf front end is distributed circumferentially on the circumference with the chord length of the spiral leaf as the radius; the helical composite hydrogel scaffold comprises a biocompatible curable polymer and a photoinitiator.

3. The helical composite hydrogel scaffold of claim 1, wherein, The helical composite hydrogel scaffold is composed of a plurality of circumferentially spaced spiral leaves, and the plurality of spiral leaves intersect at a center point; the spiral leaf is composed of a leaf front end and a leaf rear end, the leaf front end and the leaf rear end are connected, the leaf front end is a two-dimensional spiral line, and the terminal point of the leaf front end is distributed circumferentially on the circumference with the chord length of the spiral leaf as the radius; the helical composite hydrogel scaffold comprises a biocompatible curable polymer and a photoinitiator.

4. The helical composite hydrogel scaffold of claim 1, wherein, The number of spiral leaves is n, and 3≤n≤11; the height of the spiral leaf is 0.5mm~10mm.

5. A method for preparing a spiral composite hydrogel scaffold as described in any one of claims 1 to 4, characterized in that, The curable polymer is selected from one or more of gelatin methacrylamide, plant fiber, calcium alginate, sodium alginate and calcium chloride; the photoinitiator is selected from one or more of vitamin B and vitamin C. The method comprises the following steps: step 1, designing a hydrogel 3D mold and light curing the mold; step 2, filling the hydrogel material into the mold; step 3, curing the hydrogel material.

7. A method of regulating cell tissue arrangement, characterized by, 6. The helical composite hydrogel scaffold according to any one of claims 1-4 for use in the manufacture of artificial meat.

8. A method of facilitating the maturation of artificial meat, characterized by, The method comprises the following steps: step 1, preparing the helical composite hydrogel scaffold using the preparation method of claim 5; step 2, introducing a cell suspension vortex to couple with the helical composite hydrogel scaffold.

9. The method of claim 8, wherein the step of applying the vacuum is performed for a period of time of about 1 to about 5 minutes. The method comprises the following steps: step 1, preparing the helical composite hydrogel scaffold using the preparation method of claim 5; step 2, placing the helical composite hydrogel scaffold in a culture dish containing cell culture medium, introducing a cell suspension vortex to couple with the helical composite hydrogel scaffold; step three, adding precursor cells to the culture dish to differentiate the cultured cells.

10. An artificial meat tissue, characterized in that, The precursor cells include one or both of adipocytes and muscle cells. The artificial meat tissue is constructed from the helical composite hydrogel scaffold according to any one of claims 1-4; and includes one or both of an inner ring of anisotropic muscle cells and an outer ring of isotropic adipocytes.

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