Super-elastic hydrogel materials, super-elastic injectable scaffolds, and methods of making and using the same

Abstract

The present application relates to the technical field of biomedical materials, in particular to a super-elastic hydrogel material, a super-elastic injectable scaffold and a preparation method and application thereof. The super-elastic hydrogel material is obtained by cross-linking a base material with an initiator; the base material comprises GelMA and PEGDA with a molecular weight of 2000-20000; the injectable scaffold comprises a three-dimensional structure scaffold composed of super-elastic material, which can restore the original shape after being compressed and injected. The present application utilizes the excellent mechanical properties and shape memory of super-elastic material to realize solid injection and restore the original shape after injection, thereby providing a new idea and approach for large-size injectable scaffolds. The scaffold of the present application can be used in the fields of tissue engineering and regenerative medicine, minimally invasive injection transplantation, artificial skin, myocardial patch, in-vitro engineered tissue model construction, tissue physiological function remodeling and pathological process mechanism research, mechanical regulation and stress regulation mechanism research, drug development and drug screening, etc.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a superelastic hydrogel material, a superelastic injectable scaffold, its preparation method and application. Background Technology

[0002] Minimally invasive injection transplantation is a promising surgical intervention that avoids open surgery, offering advantages such as minimal surgical trauma, less patient discomfort, and a lower risk of postoperative infection. It reduces postoperative complications and provides a viable treatment option for critically ill and elderly patients. With the help of injectable biomaterials, therapeutic cells, cytokines, or pre-assembled functional tissues can be effectively delivered to damaged sites, providing better cell therapy or tissue regeneration effects. To meet the requirements of minimally invasive injection transplantation, biomaterials must be injected through needles or medical catheters with an inner diameter on the order of millimeters. Traditional injectable biomaterials employ a pre-injection, post-curing approach, which may face problems such as insufficient mechanical strength, uncontrollable gel shape, and material leakage into surrounding tissues. This has led to widespread attention on pre-formed injectable scaffolds. However, considering injectability, the size of pre-formed injectable scaffolds in existing technologies is typically small. For example, injectable scaffolds such as microrods, hydrogel microspheres, and microgels (also known as modular tissue engineering) have sizes ranging from hundreds of μm to several millimeters. They can be flexibly delivered to defect sites and freely assembled at the injection site, supporting the regeneration of damaged tissue.

[0003] Compared to the aforementioned small-sized injectable scaffolds, centimeter-sized injectable scaffolds offer greater cell loading capacity, enabling the formation of well-defined confined structures at defect sites and avoiding issues such as material leakage. Among these, gel scaffolds are the most widely studied system. The macroporous structure formed by freeze-drying the interior of gel improves the material's mechanical properties and injection capability, and its strength supports the injection process. However, the preparation of gel scaffolds requires freeze-drying, making cell-loaded fabrication impossible; cells can only be planted after the scaffold is manufactured, which may lead to uneven planting. Compared to gel, hydrogel materials have a modulus similar to that of natural tissues, providing a biomimetic microenvironment for cell growth, and their fabrication process allows for cell loading. However, hydrogels have poor mechanical properties and limited elastic deformation capacity. During deformation, the microstructure is damaged and densified, causing irreversible plastic deformation, making them unsuitable for the manufacture of injectable scaffolds, especially large-sized ones.

[0004] In summary, the fabrication of pre-formed hydrogel injectable scaffolds still faces significant challenges. There is a need to develop novel hydrogel materials with high elastic deformation capacity or superelastic properties to provide new pathways for the fabrication of injectable scaffolds ranging from millimeter to centimeter sizes, and to provide new carriers for the cell-borne fabrication and long-term cell culture of injectable scaffolds. Summary of the Invention

[0005] The purpose of this invention is to provide a superelastic hydrogel material, a superelastic injectable scaffold, its preparation method and application. By preparing a superelastic hydrogel material with excellent mechanical strength, high elastic deformation capacity, shape memory, injectability and biocompatibility, it can be pre-formed, compressed and injected, and can restore its original shape, providing a new and effective way for the manufacture of large-size injectable scaffolds.

[0006] To achieve the above objectives, in a first aspect, the present invention provides a superelastic hydrogel material, comprising a substrate material and an initiator, wherein the substrate material comprises a natural biomaterial and a synthetic biomaterial modified with photo-initiated crosslinking groups; the natural biomaterial modified with photo-initiated crosslinking groups comprises methacrylic anhydride gelatin (GelMA), and the synthetic biomaterial modified with photo-initiated crosslinking groups comprises polyethylene glycol dimethacrylate (PEGDA) with a crosslinking capacity of 2000 to 20000 Daltons.

[0007] This invention demonstrates that crosslinking GelMA and PEGDA together yields a stable hydrogel crosslinking network or an interpenetrating hydrogel crosslinking network. The material exhibits a pervasive porous microstructure and displays excellent mechanical behavior, superelasticity, and fatigue resistance, with controllable mechanical properties. This material can be compressed to 1%–99% of its volume, preferably 5%–50%, and then injected back to its original shape.

[0008] Furthermore, the molecular weight of the polyethylene glycol dimethacrylate (PEGDA) is 5000–20000 Daltons, for example, 5500, 6000, 7000, 8000, 9000, 10000, 12000, 15000, 18000 Daltons, etc. This invention selects PEGDA with a higher molecular weight, which significantly contributes to the material's excellent mechanical behavior, hyperelastic behavior, and fatigue resistance. Within the molecular weight range defined in this invention, PEGDA exhibits good flowability and plasticity, making it easy to print or mold, and the resulting structure possesses hyperelasticity. Excessively high molecular weight results in poor flowability, while excessively low molecular weight leads to brittleness and makes it impossible to obtain the hyperelastic hydrogel material described in this invention.

[0009] Furthermore, the grafting rate of the methacrylic anhydride gelatin is 30%–95%, and the concentration is 1%–20%; the concentration of the polyethylene glycol dimethacrylate is 0.5%–20%; typically, GelMA and PEGDA are dissolved in PBS buffer to prepare a stock solution. The volume ratio of the methacrylic anhydride gelatin to the polyethylene glycol dimethacrylate is 15:(1–8), for example, 15:1, 15:2, 15:3, 15:4, 15:6, 15:7, etc., preferably 15:(3–5).

[0010] Furthermore, the natural biomaterials also include one or more of alginate, hyaluronic acid, chitosan, carboxymethyl chitosan, and polysaccharides; the synthetic biomaterials also include one or more of polyvinyl alcohol and epoxy resin. This invention demonstrates that, while ensuring the simultaneous inclusion of GelMA and PEGDA crosslinking matrices, adding other photoinitiated crosslinking groups to modify the biomaterials can also yield high-performance superelastic hydrogel materials, while also allowing for flexible control of the material's mechanical properties.

[0011] Furthermore, the photoinitiating crosslinking group includes one or more of methacrylic anhydride, methacrylate, cinnamoyl, azobenzene, o-nitrobenzyl, and diacylpropionyl, preferably methacrylic anhydride and / or methacrylate; by grafting the photoinitiating crosslinking group, it is easy to crosslink under the action of the initiator to obtain a stable hydrogel crosslinking network or an interpenetrating hydrogel crosslinking network.

[0012] The initiator includes ultraviolet light initiators and visible light initiators; based on the initiation wavelength of the photocrosslinking reaction, an initiator with a matching absorption peak is selected and mixed with the aforementioned substrate material.

[0013] The ultraviolet photoinitiator includes one or more commercially available ultraviolet photoinitiators selected from 2-hydroxy-4-(2-hydroxyethoxy)-2-methylphenylacetone (i2959), phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide (i819), azodimethyl N-2-hydroxybutylpropionamide (VA-086), and lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP), with LAP being preferred;

[0014] The visible light initiator is selected from the eosin-Y system (composed of disodium eosin-Y, trihydroxyethylamine and vinylpyrrolidone) or composed of disodium eosin-Y, vinylpyrrolidone and dithiothreitol, with the eosin-Y system being preferred.

[0015] Furthermore, the superelastic hydrogel material also includes auxiliary materials and crosslinking agents; the auxiliary materials include bioactive hydrogel materials derived from the extracellular matrix and / or artificially designed materials that have a regulatory effect on cells. The crosslinking agent is the corresponding crosslinking agent of the auxiliary materials; if the auxiliary materials do not require additional crosslinking, then no crosslinking agent is needed.

[0016] The auxiliary material is selected from one or more of the following: agarose, matrix gum, decellularized matrix, collagen, collagen derivatives, hyaluronic acid, hyaluronic acid derivatives, cellulose, cellulose-derived materials, chitosan, chitosan-derived materials, proteoglycans, proteoglycan-derived materials, glycoproteins, glycoprotein-derived materials, laminin, fibronectin, fibroin, fibrin, fibrin derivatives, osteopontin, silk fibroin, silk fibroin derivatives, functionalized peptides, peptide hydrogels, amino acids, amino acid derivatives, and DNA hydrogels, preferably fibrinogen and type I collagen;

[0017] Preferably, the concentration of fibrinogen is 0.1 mg / mL to 20 mg / mL, and the concentration of type I collagen is 0.01 mg / mL to 8 mg / mL;

[0018] The cross-linking agent is thrombin (derived from bovine blood). The concentration of thrombin is 0.1 IU / mL to 100 IU / mL, and the cross-linking time is 1 to 20 minutes.

[0019] The superelastic hydrogel material has a through-hole microstructure with a porosity of 10% to 90%, an average pore diameter of 10 μm to 200 μm, and an average pore wall thickness of 2 μm to 20 μm.

[0020] The superelastic hydrogel material exhibits excellent mechanical behavior, with an elastic modulus ranging from 1 kPa to 200 kPa. The material demonstrates superelastic behavior and fatigue resistance, exhibiting a typical nonlinear elastic range during deformation. The fracture strain is as high as 50% to 95%. When subjected to cyclic deformation with a strain equal to half the fracture strain, the material exhibits fatigue failure after 10 to 3000 cycles.

[0021] The mechanical properties of the superelastic hydrogel material are tunable. By selecting the base material and / or auxiliary materials, the hydrogel material can be an elastomer or a viscoelastic body, exhibiting the characteristics of an elastomer, or possessing the stress relaxation and creep properties of a viscoelastic body.

[0022] In a second aspect, the present invention provides a method for preparing the superelastic hydrogel material according to any one of the above claims, comprising the following steps:

[0023] (1) Mix the substrate material and the initiator to obtain a precursor solution;

[0024] (2) The precursor solution is cross-linked by photoinitiation according to the pre-designed structure to obtain the desired three-dimensional structure.

[0025] The substrate material is preferably GelMA and PEGDA, wherein the grafting rate of GelMA is 30%–95% and the concentration is 1%–20%, and the molecular weight of PEGDA is 2000 Daltons–20000 Daltons and the concentration is 0.5%–20%. If the substrate material is subjected to ultraviolet light crosslinking, the initiator is preferably LAP with a concentration of 0.01 mg / mL–5 mg / mL, the wavelength of light used in the photocrosslinking reaction is 405 nm, and the light intensity is 1–100 mW / cm². 2 The required illumination time for the crosslinking reaction is 30 seconds to 10 minutes. For visible light crosslinking, the preferred initiator is the eosin-γ system, wherein the concentration of disodium eosin-γ is 1 μmol / L to 1 mmol / L, the volume fraction of trihydroxyethylamine is 0.01% to 1%, the concentration of vinylpyrrolidone is 1 nmol / L to 4 μmol / L, the visible light wavelength used for the photocrosslinking reaction is 780 nm to 400 nm, and the light intensity is 1 to 100 mW / cm². 2 The light exposure time required for the cross-linking reaction is 5 to 30 minutes.

[0026] Furthermore, the precursor solution comprises 8%–20% methacrylic anhydride gelatin, 1–10% polyethylene glycol dimethacrylate, and 0.01 mg / mL–5 mg / mL LAP;

[0027] Alternatively, the preparation method of superelastic hydrogel materials includes the following steps:

[0028] (1) Mix the substrate material, auxiliary material and initiator to obtain a precursor solution;

[0029] (2) The precursor solution is photo-initiated to crosslink according to a pre-designed structure to obtain the desired three-dimensional structure;

[0030] (3) Add a crosslinking agent to crosslink the auxiliary material to obtain a superelastic hydrogel material.

[0031] Furthermore, the precursor solution comprises 5-10% methacrylic anhydride gelatin, 1-5% polyethylene glycol dimethacrylate, 0.1-0.3 mg / mL type I collagen, 1-3 mg / mL fibrinogen, 0.02-0.06 mmol / mL eosin-γ disodium salt, 0.2-0.6% (v / v) trihydroxyethylamine, and 130-160 nmol / mL vinylpyrrolidone.

[0032] like Figure 1As shown, the precursor solution of the superelastic hydrogel material is composed of a base material and / or auxiliary materials and corresponding initiators and / or crosslinking agents. After crosslinking, the base material and / or auxiliary materials can obtain a stable hydrogel crosslinking network or an interpenetrating hydrogel crosslinking network. The material has a through-porous microstructure and exhibits excellent mechanical behavior, superelastic behavior and fatigue resistance on a macroscopic scale. Moreover, the mechanical properties can be controlled.

[0033] Preferably, the precursor solution further contains cells and / or cell clusters. The cells and / or cell clusters are selected from one or more of the following cell types and / or cell clusters obtained through pre-culture of one or more of the following cells:

[0034] Embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells, stem cells from various organs, progenitor cells from various organs, mesenchymal stem cells, cells derived from the induced differentiation of various stem cells, fibroblasts from various organs, epithelial cells from various organs, epidermal cells from various organs, endothelial cells from various organs, muscle cells from various organs, amniotic cells, cone cells, nerve cells, blood cells, erythrocytes, leukocytes, platelets, vascular cells, phagocytes, immune cells, lymphocytes, eosinophils, basophils, plasma cells, etc. Cells, mast cells, antigen-presenting cells, cells of the mononuclear phagocyte system, melanocytes, chondrocytes, bone-derived cells, smooth muscle cells, skeletal muscle cells, cardiomyocytes, secretory cells, adipocytes, ciliated cells, pancreatic cells, kidney cells, intestinal mucosal cells, hepatocytes, liver-derived stem cells or progenitor cells, hepatic macrophages, Kupffer cells, stellate cells, bile duct epithelial cells, hepatic sinusoidal endothelial cells and other cells of various tissue and organ origin, as well as various tumor cells, various cells used for immunotherapy, and various cells and cell lines that have undergone gene editing, viral packaging or modification.

[0035] The forming methods of the three-dimensional structure include: casting technology, lost foam technology, bio-3D printing technology, inkjet printing, fused deposition modeling, electrospinning, electrostatically driven printing, particle leaching, gas foaming technology, stereolithography technology, laser sintering technology, etc., with bio-3D printing technology being preferred.

[0036] Thirdly, this invention provides a hyperelastic injectable scaffold, comprising a three-dimensional scaffold structured from a hyperelastic material. After being compressed and injected with a syringe, the scaffold recovers its original three-dimensional structure. Existing technologies employ a pre-injection and subsequent curing method, which faces numerous problems such as insufficient mechanical strength, uncontrollable gel shape, and material leakage into surrounding tissues. This invention selects a hyperelastic material to first fabricate a three-dimensional scaffold structure. Utilizing its hyperelasticity and fatigue resistance, it can be highly compressed before injection and recover its original shape, thus ensuring shape control and convenient injection. This invention's use of a hyperelastic material for solid injection breaks with conventional thinking on injectable scaffolds, providing a new approach and method for their development.

[0037] The hyperelastic injectable stent possesses elastic deformation capability, shape memory, and injectability. For example, the hyperelastic injectable stent can be compressed or stretched, with a deformation exceeding 50% of its size, and it fully recovers its shape after the external force is removed; this deformation process can be repeated multiple times. An example of the elastic deformation capability is a 10mm × 10mm circular hyperelastic injectable stent that can be compressed and deformed 30 times while maintaining its integrity. Another example is that the hyperelastic injectable stent can be delivered intact through a small opening with an inner diameter comparable to that of a clinically used medical catheter or syringe needle; after injection, the stent's shape and internal structure remain intact. An example of injectability is a 15mm × 15mm hyperelastic stent that can be inserted through an injection needle with an inner diameter of 1.5mm while maintaining its integrity.

[0038] Furthermore, the superelastic material includes any of the superelastic hydrogel materials described above or the superelastic hydrogel materials prepared by the methods described above. The resulting superelastic injectable scaffold has adjustable chemical composition, controllable mechanical properties, and its size and structure can be customized. It possesses excellent mechanical strength, elastic deformation capacity, shape memory, injectability, good biocompatibility, high structural stability, supports long-term cell culture, regulates cell assembly, and exhibits good cell function.

[0039] The macroscopic dimensions and structural design of the superelastic injectable scaffold are adjustable, with macroscopic dimensions ranging from 1mm to 50mm. The scaffold's shape can be blocky, sheet-like, spherical, tubular, or a combination of any shape. The internal structure can be solid, hollow, or filled with a custom pattern, a combination of any shape, or a custom gradient structure. Preferably, the scaffold's macroscopic dimensions are 10mm to 20mm, its shape is a square sheet, and its internal structure includes grid-like, radial, honeycomb, convex polygonal, concave polygonal, sinusoidal, Zigzag, or arc-shaped fillings. Figure 2 The diagram shows several possible support structures.

[0040] For cell-free scaffolds, the superelastic hydrogel materials prepared above are tested and / or injected according to the application needs (e.g., for tissue engineering and regenerative medicine research, minimally invasive injection transplantation, artificial skin, myocardial patches, etc.).

[0041] For scaffolds containing cells, the superelastic hydrogel material prepared above is cultured and / or tested in vitro, and then applied in vitro and / or injected in vivo as needed.

[0042] The in vitro culture time and method are determined by the application requirements and cell type, with a culture time ranging from 0 to 60 days. Culture methods can include static culture, shaker culture, microarray perfusion culture, or a combination of these methods. Applications include, but are not limited to, using the prepared scaffold for tissue engineering and regenerative medicine research, minimally invasive injection transplantation, artificial skin, myocardial patches, construction of in vitro engineered tissue models, study of tissue physiological function remodeling and pathogenesis mechanisms, study of mechanical and stress regulation mechanisms, drug development, and drug screening.

[0043] Fourthly, the present invention provides an application of any of the above-mentioned superelastic hydrogel materials or the above-mentioned superelastic injectable scaffolds, specifically applicable to (1) tissue engineering and regenerative medicine research; (2) minimally invasive injection transplantation; (3) artificial skin and myocardial patches; (4) cell culture and construction of in vitro engineered tissue models; (5) research on the mechanism of tissue physiological function remodeling and pathological process; (6) research on the mechanism of mechanical regulation and stress regulation; (7) drug development and drug screening, etc.

[0044] The beneficial effects of this invention are as follows:

[0045] The superelastic injectable scaffold provided by this invention uses a superelastic material to first fabricate a three-dimensional scaffold structure. Utilizing its excellent mechanical properties and shape memory, it enables solid injection and restores its original shape after injection, providing a new approach and method for large-size injectable scaffolds. Specific effects are as follows:

[0046] (1) The scaffold in this invention is made of a superelastic hydrogel material. The material has a through-hole microstructure with a porosity of 10% to 90%, an average pore diameter of 10 μm to 200 μm, and an average pore wall thickness of 2 μm to 20 μm. It exhibits excellent mechanical behavior, superelastic behavior, and fatigue resistance.

[0047] (2) The scaffold in this invention has excellent mechanical properties. Traditional hydrogel materials have insufficient mechanical strength and elastic deformation capacity, making it impossible to manufacture injectable scaffolds, especially centimeter-sized injectable scaffolds. The scaffold provided by this invention has high mechanical strength, good elastic deformation capacity, shape memory and injectability, and its mechanical properties are significantly better than those of traditional hydrogel scaffolds, which can meet the needs of applications such as minimally invasive injection transplantation and myocardial patching.

[0048] (3) The mechanical behavior of the scaffold in this invention is controllable. By selecting the base material and / or auxiliary materials, the hydrogel material can be an elastomer or a viscoelastic body, exhibiting the characteristics of an elastomer, or having the stress relaxation and creep characteristics of a viscoelastic body.

[0049] (4) The scaffold in this invention has good biocompatibility. The substrate materials and / or auxiliary materials used in this invention have good biocompatibility and bioactivity, and the scaffold is manufactured in a mild and controllable manner. The scaffold in this invention can provide a biomimetic microenvironment for cells, support the three-dimensional culture of various cell types, and support cell growth, proliferation, assembly, functional maintenance and enhancement.

[0050] (5) The biological activity of the scaffold in this invention is tunable. By selecting auxiliary materials, the chemical composition of the scaffold can be regulated and changed to better simulate the tissue-specific extracellular matrix, or to provide specific chemical stimuli for regulating cell growth and assembly, thereby meeting the needs of cell culture and application in different scenarios.

[0051] (6) The scaffold structure of this invention has high stability. The scaffold of this invention has a stable cross-linked network structure, excellent mechanical strength and anti-disturbance properties, and can be cultured for up to 1 month while maintaining structural integrity. The scaffold of this invention provides a good substrate for long-term cell culture and can better support cell assembly and functional remodeling in a three-dimensional environment.

[0052] (7) The scaffold of the present invention can maintain cell function and / or regulate cell assembly. The scaffold of the present invention can maintain cell function, and by regulating the chemical composition and mechanical behavior of the scaffold, the scaffold of the present invention can provide chemical and mechanical stimulation to cells, thereby improving cell function or regulating cell behavior.

[0053] (8) The macroscopic dimensions and structure of the scaffold in this invention can be designed independently. Its macroscopic dimensions range from 1mm to 50mm. The scaffold's shape can be block-shaped, sheet-like, spherical, tubular, or a combination of any shapes. The internal structure can be solid, hollow, or filled with custom patterns, combinations of any shapes, or a custom gradient structure. The scaffold can be manufactured using various advanced manufacturing technologies, such as casting technology, lost foam technology, bio-3D printing technology, inkjet printing, fused deposition modeling, electrospinning, electrostatically driven printing, particle leaching, gas foaming technology, stereolithography, and laser sintering technology.

[0054] (9) The chemical composition is adjustable, the mechanical properties, including viscoelastic characteristics and hyperelastic behavior, are controllable, the scaffold size and structure can be customized, and it has excellent mechanical strength, elastic deformation ability, shape memory, injectability, good biocompatibility, high structural stability, supports long-term cell culture, regulates cell assembly, and has good cell function. Attached Figure Description

[0055] Figure 1 This is a schematic diagram of the composition and cross-linking structure of the superelastic hydrogel material of the present invention.

[0056] Figure 2 Schematic diagram of injectable scaffolds with different macroscopic dimensions and structures using various advanced manufacturing technologies.

[0057] Figure 3 The image shows a physical photograph of the superelastic hydrogel material prepared in Example 1 of this invention. A is a top view of the cylindrical hydrogel sample; B is a side view of the cylindrical hydrogel sample.

[0058] Figure 4 The results of the cyclic compression test of the superelastic hydrogel material in Example 1 of this invention are shown. In Figure A, the results of the cyclic compression test of the hydrogel sample under increasing strain, with the strain increasing from 10% to 95%, are shown in small figures in Figure A, which are deformation photographs of the hydrogel sample with strains of 10%, 50%, and 90%, respectively. Figure B shows the stress-strain hysteresis curves of the sample under different cycles.

[0059] Figure 5 The image shows the shape memory characterization results of the annular stent in Embodiment 2 of the present invention. A is a macroscopic photograph of the stent during the 30th cycle of compression; B is a macroscopic photograph of the stent during the 30th cycle of stretching.

[0060] Figure 6 The images show the injection performance characteristics of the printed scaffold in Embodiment 3 of this invention. A is a macroscopic photograph of the printed scaffold; B shows the scaffold fully expanded after being injected with adhesive through a 1.5mm inner diameter dispensing needle.

[0061] Figure 7This is the result of using the scaffold for embryonic stem cell culture in Example 4 of the present invention. A represents the live / dead staining results of cells within the scaffold on day 0 and day 12 after printing; B represents the fold increase in cell proliferation within the scaffold on day 12 compared to day 0.

[0062] Figure 8 This is an example of the scaffold used in the in vitro liver model construction of Embodiment 5 of the present invention. A shows the results of cell viability and death staining, as well as cell morphology and arrangement, on days 0 and 14 after printing; B shows the immunofluorescence staining results of the liver-specific protein ALB on day 14.

[0063] Figure 9 In Embodiment 6 of the present invention, after injection, the stent spreads on the surface of the liver and freely adheres to the surface of the liver.

[0064] Figure 10 This is the expression of drug metabolism-related genes in cells within the scaffold after one week of rifampicin treatment in Example 7 of the present invention. Detailed Implementation

[0065] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention are described clearly and completely below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0066] Unless otherwise specified, the percentage sign "%" used in this invention refers to the mass percentage; however, the percentage of a solution, unless otherwise specified, refers to the number of grams of solute contained in 100 mL of solution.

[0067] Unless otherwise defined, all technical terms used in this invention have the same meaning as understood by one of ordinary skill in the art.

[0068] The term "superelasticity" used in this invention refers to the phenomenon that a material, under the action of external force, produces a strain much greater than its elastic limit strain, and the strain can automatically recover upon unloading.

[0069] In this invention, "injectable" means that the scaffold, engineered structure, and biomimetic tissue pre-cultured in vitro can pass through a small opening with an inner diameter comparable to that of a medical catheter or syringe, while maintaining structural integrity without breakage, damage, or loss of function.

[0070] In this invention, "photocrosslinking" refers to the process by which polymer compounds connect their chains to each other through photochemical reactions, forming an infusible and insoluble network structure.

[0071] In this invention, "initiator" refers to an initiator for photocrosslinking reaction, which is a type of compound that can absorb energy of a certain wavelength in the ultraviolet or visible light region, generate free radicals, cations, etc., thereby initiating the crosslinking and curing of monomers.

[0072] In this invention, the "crosslinking agent" of the auxiliary material refers to a substance that can initiate the crosslinking reaction of the auxiliary material.

[0073] In this invention, "precursor solution" refers to a material that has not undergone cross-linking reaction, is in a fluid state at room temperature, and becomes a gel state at low temperature due to physical cross-linking.

[0074] In this invention, "stress" refers to the internal force generated between different parts of an object when it deforms due to external factors (force, humidity, temperature field changes, etc.). The internal force per unit area is called stress. It is expressed as the ratio of internal force to cross-sectional area, and the unit is Pa. "Strain" refers to the ratio of the increase in length (positive when elongating) of a small line segment in a certain direction due to deformation to its original length.

[0075] In this invention, the needle model "G (gauge)" is the international number of the needle. The inner diameter of the 21G needle is 0.51mm and the outer diameter is 0.81mm.

[0076] The term "3D printing" as used in this invention refers to the precise deposition of three-dimensional materials using a method compatible with 3D printing, via an automatic or semi-automatic, computer-aided 3D forming apparatus (e.g., a 3D printer).

[0077] In the 3D printing process, the "suitable gel state" of the material refers to the viscosity of the material increased by temperature control of the printing equipment. Material in a suitable gel state has good forming effect. When extruded through the needle, the material filaments are stable and the surface of the filaments is flat and smooth.

[0078] In some embodiments, the three-dimensional structures of the present invention are engineered. The term "engineered" means that, according to a computer script, raw materials (e.g., biocompatible materials and / or precursor solutions) and their layers are placed to form a three-dimensional structure using a computer-aided device (e.g., a 3D printer). In further embodiments, the computer script is, for example, one or more computer programs, computer applications, or computer modules.

[0079] The embodiments disclosed herein include commercial methods. In some embodiments, the speed and scalability of the techniques and methods disclosed herein are used to design, construct, and operate industrial and / or commercial facilities for the production of scaffolds for implantation or drug screening, or for the generation of cell-based tools (for research and development, e.g., in vitro analysis). In further embodiments, scaffolds and arrays thereof are manufactured, stored, distributed, marketed, advertised, and sold, for example as platforms for cell culture, tissue engineering, large-scale cell culture, or cell arrays (e.g., microarrays or chips), tissue arrays (e.g., microarrays or chips), and kits for bioanalysis and high-throughput drug screening. In other embodiments, scaffolds and arrays thereof are manufactured and used for bioanalysis and / or drug screening as a service.

[0080] Example 1: Preparation and crosslinking method of superelastic hydrogel

[0081] 1. Preparation of substrate materials and initiators

[0082] 16% GelMA stock solution: The lyophilized GelMA material (Genenobio Biotechnology Co., Ltd., R08020102) was mixed with phosphate-buffered saline (PBS) (BI Biological Industries). The mass fraction of GelMA material in the mixed solution was 16%. After dispersing and stirring with a vortex stirrer, the solution was heated at 70°C until completely dissolved to obtain a clear GelMA solution. The solution was stored at 4°C in the dark for later use.

[0083] 15% PEGDA stock solution: Mix PEGDA powder (Carbon Technology, 80020110, molecular weight 6000 Daltons) with PBS. The mass fraction of PEGDA in the mixed solution is 15%. Use a vortex stirrer to disperse and stir until all PEGDA is dissolved to obtain a clear PEGDA solution. Store at 4°C in the dark for later use.

[0084] Eosin-Y initiator stock solution: Eosin-Y disodium salt (Sigma-Aldrich) powder was uniformly mixed with PBS and stirred until completely dissolved. The concentration of the eosin-Y disodium salt stock solution was 40 mmol / L. Trihydroxyethylamine solution (Sigma-Aldrich) was uniformly mixed with PBS to obtain a homogeneous solution. The volume fraction of the trihydroxyethylamine stock solution was 40%. Vinylpyrrolidone (Sigma-Aldrich) was uniformly mixed with PBS to obtain a homogeneous solution. The concentration of vinylpyrrolidone was 14.8 μmol / L. After preparation, the above initiator stock solutions were sterilized by filtration using a 22 μm filtration device (Millex) and stored at 4°C protected from light for later use.

[0085] 2. Preparation of auxiliary materials and crosslinking agents

[0086] 8 mg / mL type I collagen stock solution, purchased from Advanced BioMatrix, stored at 4°C for later use.

[0087] 20 mg / mL fibrinogen stock solution: Fibrinogen powder (Shanghai Yuanye Biotechnology Co., Ltd.) was mixed with PBS to a concentration of 20 mg / mL. The insoluble matter in the mixture was gently shaken several times to disperse it, and then dissolved overnight at 37°C to obtain a homogeneous fibrinogen stock solution. The prepared fibrinogen stock solution should be used immediately.

[0088] 1 IU / mL Thrombin Solution: Thrombin powder (Solepro Biotechnology Co., Ltd.) is fully dissolved in PBS to obtain a thrombin stock solution with a final concentration of 100 IU / mL. After aliquoting, it is stored at -20℃ for later use. Before use, the thrombin stock solution is thawed and diluted with PBS to a thrombin concentration of 1 IU / mL to obtain a fibrinogen cross-linking agent.

[0089] 3. Preparation of precursor solution

[0090] Before mixing, the aforementioned 16% GelMA stock solution, 15% PEGDA stock solution and eosin-γ initiator stock solution were incubated at 37°C, PBS was placed at room temperature, and type I collagen stock solution was placed on an ice plate for insulation.

[0091] GelMA stock solution, PEGDA stock solution, and fibrinogen stock solution were mixed uniformly at a volume ratio of 15:4:3 and placed at room temperature. This mixture is denoted as mixed solution A.

[0092] After the aforementioned mixed solution A was brought to room temperature, it was rapidly and thoroughly mixed with PBS and type I collagen stock solution to obtain a mixed solution B of substrate material and auxiliary material.

[0093] The aforementioned mixed solution B and the eosin-Y initiator mother liquor were uniformly mixed at a volume ratio of 988:1:10:1 to obtain the hydrogel precursor solution.

[0094] In the precursor solution, the final concentrations of each material component are: 8% GelMA, 2% PEGDA, 0.2 mg / mL L1 type collagen, 2 mg / mL fibrinogen, 0.04 mmol / mL eosin-γ disodium salt, 0.4% (v / v) trihydroxyethylamine, and 148 nmol / mL vinylpyrrolidone.

[0095] 4. Hydrogel crosslinking

[0096] After the aforementioned precursor solution was prepared, photocrosslinking was performed using a halogen tungsten lamp light source (Beijing Newbit Technology Co., Ltd.). The light source was equipped with a UV cutoff filter (UVCUT400), providing a light wavelength range of 780nm to 400nm and a light intensity of 12mW / cm². 2 The light exposure time required for the photocrosslinking reaction is 12 minutes.

[0097] After photocrosslinking, the sample surface was rinsed with PBS to remove some of the residual initiator. Subsequently, thrombin solution, a crosslinking agent, was added to crosslink the fibrinogen in the hydrogel for 3 minutes.

[0098] After the thrombin crosslinking is completed, the remaining solution is aspirated to obtain the superelastic hydrogel described in this invention. The appearance of the hydrogel is shown in the attached figure. Figure 3 .

[0099] 5. Characterization of the hyperelastic behavior of hydrogels

[0100] A block hydrogel with a diameter of 10 mm and a height of 1.5 mm was prepared according to the aforementioned steps. The deformation behavior of the hydrogel was characterized by dynamic mechanical testing using a fatigue testing machine (Electroforce 3200 BioDynamic, Bose Company). The hydrogel sample was placed in the center of the indenter of the testing machine, and cyclic compression tests were performed at a frequency of 1 Hz. Deformation amounts equivalent to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95% of the sample height were applied sequentially, with each deformation amount being cyclically compressed 5 times. During compression, the force (Newtons) and deformation (mm) were collected in real time. After the experiment, the stress and strain of the sample were calculated based on the obtained data, and the corresponding stress-strain curves were plotted. During compression, the hydrogel sample remained intact without fatigue failure, exhibiting excellent hyperelastic behavior. The data processing results of the cyclic compression tests are shown below. Figure 4 .

[0101] Example 2: Fabrication of a hyperelastic scaffold using a molding method

[0102] 1. Preparation of substrate materials and initiators

[0103] 16% GelMA stock solution: The lyophilized GelMA material was mixed with PBS, and the mass fraction of GelMA material in the mixed solution was 16%. After dispersing and stirring with a vortex stirrer, it was heated at 70°C until completely dissolved to obtain a clear GelMA solution, which was stored at 4°C in the dark for later use.

[0104] 15% PEGDA stock solution: PEGDA powder (molecular weight 6000 Daltons) is mixed with PBS, and the mass fraction of PEGDA in the mixed solution is 15%. The mixture is dispersed and stirred with a vortex stirrer until all PEGDA is dissolved to obtain a clear PEGDA solution. Store at 4°C in the dark for later use.

[0105] LAP initiator stock solution: LAP powder (Advanced BioMatrix) was uniformly dissolved in PBS to obtain a clear LAP stock solution with a LAP concentration of 17 mg / mL. The solution was filtered through a 22 μm filtration device and stored at 4°C protected from light. The shelf life of the initiator stock solution was 2 weeks.

[0106] 2. Preparation of superelastic hydrogel precursor solution

[0107] Before mixing, the aforementioned 16% GelMA stock solution, 15% PEGDA stock solution, LAP initiator stock solution and PBS were incubated at 37°C.

[0108] GelMA stock solution, PEGDA stock solution, and PBS were uniformly mixed at a volume ratio of 15:4:5, and then mixed with LAP initiator stock solution at a volume ratio of 1:0.02 to obtain a superelastic hydrogel precursor solution. In the superelastic hydrogel precursor solution, the final concentrations of each material component were: 10% GelMA, 2.5% PEGDA, and 0.34 mg / mL LAP.

[0109] 3. Mold manufacturing

[0110] The mold used in this embodiment was made of polydimethylsiloxane (PDMS) (Dow Corning). The PDMS base and curing agent were thoroughly mixed at a mass ratio of 10:1 and poured into a 60mm diameter cauldron. Air bubbles were removed by vacuum degassing, and the mixture was placed in a 70°C oven for curing for 65 minutes, at which point the PDMS was completely cured.

[0111] Demold the PDMS, clean it with deionized water, and cut it into square sheets of PDMS with a side length of 15mm. Then, use a punch to punch holes to obtain an annular groove mold with a height of 1mm. The inner diameter of the groove is 8mm and the outer diameter is 10mm.

[0112] 4. Manufacturing of superelastic scaffolds using the casting method

[0113] The superelastic hydrogel precursor solution obtained in step 2 was poured into the mold manufactured in step 3, avoiding the formation of air bubbles within the material during the pouring process. Photocrosslinking was performed using a halogen tungsten lamp light source (Beijing Newbit Technology Co., Ltd.), equipped with an ultraviolet bandpass filter (405nm), providing 405nm ultraviolet light. The light intensity was 12mW / cm².2 The light exposure time required for the photocrosslinking reaction is 1 minute.

[0114] After crosslinking, the sample surface was rinsed three times with PBS to remove unreacted photoinitiator, and a ring scaffold was obtained after demolding.

[0115] 5. Characterization of scaffold shape memory

[0116] The scaffold obtained in step 4 is immersed in PBS. Using tweezers, the scaffold is held by both sides and slowly squeezed until the sides contact each other. Then the tweezers are released, allowing the scaffold to recover its shape. This completes one cycle. The cycle is repeated 30 times. The scaffold remains intact and does not break (e.g., Figure 5 (As shown in Figure A). Hold the support structure firmly on both sides with tweezers, slowly stretch and deform it, then release the tweezers to allow the support structure to return to its original shape. This completes one cycle. Repeat this cycle 30 times. The support structure should remain intact and not break (as shown in Figure A). Figure 5 (As shown in Figure B). The superelastic hydrogel scaffold has good deformation capacity and shape memory.

[0117] Example 3: Fabrication of a hyperelastic injectable scaffold via 3D printing

[0118] 1. Preparation of precursor solution

[0119] The preparation method of the precursor solution is described in Example 1.

[0120] 2. 3D printing

[0121] The 3D printing process in this embodiment uses the technology of Genofine Biotechnology Co., Ltd. The SR bio-3D printer is complete. Unless otherwise stated, all consumables used are printer-compatible consumables.

[0122] The precursor solution prepared in step 1 was loaded into a 5 mL light-shielded bio-ink tube and pre-cooled at 4°C for 5 minutes. A 21G flat-tipped injection needle was then attached to the tip of the bio-ink tube, which was then loaded onto a bio-3D printer. The temperatures of the ink tube, needle, and printing platform were controlled at 24°C, 22°C, and 12°C, respectively. When the precursor solution reached a suitable gel state, bio-3D printing was performed according to the pre-designed printing path. The pneumatic pump pressure was 0.14 MPa, the needle movement speed was 5 mm / s, and the structure was printed in a 35 mm diameter culture dish (cornering).

[0123] The printed material stack structure achieved good forming effect, complete shape, and neat filament edges. The overall dimensions of the structure are 15mm × 15mm. The repeating units are concave hexagons, and the structure is filled with 18 repeating units. The printing layer is 2 layers. A macroscopic photograph of the printed structure is shown below. Figure 6 As shown in Figure A.

[0124] 3. Crosslinking of the printed structure

[0125] After printing is completed, the printed structure is cross-linked to obtain a stable scaffold. The cross-linking process of the printed structure is the same as in Example 1 above. During cross-linking, the printed structure is placed on an ice plate.

[0126] 4. Characterization of stent injectability

[0127] The cross-linked scaffold was gently separated from the culture dish bottom and stained with a water-soluble pigment to make the morphology of the structure easier to distinguish during injection. Using PBS as the carrier solution, extrusion injection was performed using a 1.5mm inner diameter dispensing needle. The scaffold was observed to deform, fold, and compress successfully through the needle. After injection, the scaffold immediately spread out, maintaining structural integrity. The injection process and post-injection scaffold morphology are shown in the figure. Figure 6 As shown in Figure B. This injection result demonstrates that the centimeter-sized scaffold printed using the superelastic hydrogel material exhibits excellent injectability and shape memory.

[0128] Example 4: Superelastic injectable scaffold for embryonic stem cell culture

[0129] 1. Preparation of precursor solution

[0130] The preparation method of the precursor solution is described in Example 1. After the precursor solution is prepared, embryonic stem cells (Life Technologies) are uniformly mixed with the precursor solution, and the final cell suspension contains 1 × 10⁻⁶ cells. 6 per mL.

[0131] 2. Crosslinking of 3D Printing and Printed Structures

[0132] For cell 3D printing, the consumables required for printing need to be autoclaved before printing, and the interior of the bio-3D printer needs to be irradiated with ultraviolet light for 30 minutes to kill bacteria on the surface of the printer parts. Aseptic operation must be maintained during printing and cross-linking to avoid structural contamination. The 3D printing method of the scaffold is described in Example 3. The scaffold is printed in a 12-well plate (Corning), and the cross-linking method is the same as in Example 3. After cross-linking, a scaffold carrying embryonic stem cells is obtained.

[0133] 3. Scaffold culture and detection of cell viability and proliferation within the scaffold

[0134] Add 2 mL of culture medium to the scaffolds printed in each step 2 and place them in a carbon dioxide incubator (37°C, 5% CO2) for incubation. Replace the culture medium with fresh medium daily during the incubation process.

[0135] Cell viability assay: To assess cell viability in the scaffolds, live / dead cell staining was performed on days 0 and 12. The live / dead cell staining assay was performed using a Calcein-AM and propidium iodide (PI) double staining kit (Dojindo, C346). Calcein-AM and PI were dissolved in PBS to prepare the live / dead cell staining working solution, with a final concentration of 2 μmol / L for Calcein-AM and 4.5 μmol / L for PI. The scaffolds to be tested were transferred to glass-bottomed culture dishes (NEST), and the live / dead cell staining working solution was added to immerse the scaffolds. Staining was performed in the dark for 15 minutes. Subsequently, the cell viability in the scaffolds was observed and recorded using a laser scanning confocal microscope (LSCM, Nikon, Z2). The results are shown in the figure below. Figure 7 A. Quantitatively, cell viability was calculated using the formula: (Number of viable cells / Total number of cells) × 100%. On day 0, the cell viability within the scaffold was 90.05%, indicating that the 3D printing and cross-linking process caused minimal damage to the cells, maintaining high cell activity. On day 12, the cells within the scaffold showed good survival and significant proliferation, forming numerous cell clusters. Figure 7 (A)

[0136] Cell proliferation assay: On days 0 and 12, scaffold materials were degraded using collagenase (Solepro Biotechnology Co., Ltd., T1320), and cells and / or cell clusters were harvested by centrifugation. For cell clusters obtained on day 12, trypsin-EDTA (Solepro Biotechnology Co., Ltd.) was used for digestion for 3 minutes, followed by the addition of an equal volume of cell culture medium to terminate digestion, and cell harvesting by centrifugation. The cell samples were resuspended in culture medium, and the cell count was determined using a cell counter (Countstar) to calculate the number of cells harvested from each scaffold. The statistical results of cell proliferation are shown below. Figure 7 As shown in Figure B, after 12 days of culture, the embryonic stem cells in the scaffold proliferated to 4.66 times that of day 0.

[0137] Example 5: Hyperelastic injectable scaffold for in vitro liver model construction

[0138] 1. Preparation of precursor solution

[0139] The preparation method of the precursor solution is described in Example 1. After the precursor solution is prepared, liver progenitor cells (Life Technologies) are uniformly mixed with the precursor solution, and the final cell suspension contains 9 × 10⁻⁶ cells. 6 per mL.

[0140] 2. Crosslinking of 3D Printing and Printed Structures

[0141] The three-dimensional printing and cross-linking process of the precursor solution is the same as that described in Example 4.

[0142] 3. Scaffold culture and cell viability assay

[0143] The scaffold containing liver progenitor cells obtained from the printing process was cultured for 14 days, and the scaffold was cultured in the same manner as described in Example 4.

[0144] Cell viability assay: Cell viability in the scaffold was measured on days 0 and 14. The assay method was the same as described in Example 4. The results are shown in [Figure 4]. Figure 8 A. On day 0, the cell survival rate was 87.65%, indicating that the 3D printing and cross-linking process caused minimal damage to the cells, maintaining high cell viability. On day 14, the cells within the scaffold showed good survival, significant proliferation, and spreading and assembly within the scaffold.

[0145] 4. Liver-specific function tests

[0146] On day 14 of culture, immunofluorescence staining was used to detect the expression of the liver-specific protein ALB in the cells to assess the liver-specific function of the cells in the scaffold.

[0147] On day 14, the liver progenitor cell-loaded scaffold was transferred to a glass-bottomed culture dish, washed with PBS, and fixed with 2.5% glutaraldehyde solution (Sigma Aldrich, G5882) at room temperature for 4 hours.

[0148] Remove the remaining glutaraldehyde solution, wash the scaffold three times with PBS, and permeabilize the scaffold on ice for 20 minutes with 0.3% Triton-X100 (Solebio Biotechnology Co., Ltd., T8200); remove the remaining Triton-X100 solution, wash the scaffold three times with PBS, add a solution of 10% bovine serum albumin (Yeasen Biotech Company, 9048-46-8), and block the scaffold at room temperature for 1 hour; remove the remaining solution, and wash the scaffold three times with PBS.

[0149] Add diluted ALB primary antibody (Abcam, ab83465) (containing 0.3% Triton-X100 and 1% bovine serum albumin), and incubate overnight at 4°C; aspirate the remaining primary antibody solution, wash the rack three times with PBS, and add the corresponding secondary antibody Alexa. 488 (Abcam, ab150113) was incubated overnight at 4°C in the dark. The remaining secondary antibody solution was removed, and the scaffold was washed three times with PBS. Heochst staining solution (Solepro Biotechnology Co., Ltd.) was added, and the scaffold was incubated at room temperature in the dark for 30 minutes. Then, the protein expression of the cells in the scaffold was observed and photographed using a laser scanning confocal microscope.

[0150] The results of immunofluorescence staining are shown in Figure 8 B. The hepatic progenitor cells in the scaffold highly express ALB, indicating that the cells have albumin secretion function. This demonstrates that the scaffold can provide a biomimetic microenvironment for the cells, maintaining the survival of hepatic progenitor cells and liver-specific functions.

[0151] Example 6: Hyperelastic injectable scaffold for injection transplantation

[0152] 1. Stent manufacturing

[0153] The preparation of the precursor solution, 3D printing, and scaffold cross-linking processes are the same as in Example 3 above. To meet the requirements of injection implantation, the scaffold manufacturing process requires aseptic operation.

[0154] 2. Superelastic injectable scaffolds for injection transplantation

[0155] The scaffold prepared in step 1 was gently separated from the bottom of the culture dish and immersed in PBS buffer, which served as a carrier solution for the injection process. An injection transplantation experiment was performed using a disposable sterile syringe (BDI, 301942) equipped with a 1.5 mm inner diameter needle. The scaffold immersed in PBS was drawn into the syringe and injected onto the surface of fresh pig liver. The results of the injection experiment are shown below. Figure 9 .

[0156] During the experiment, it was observed that the scaffold had good injectability and could pass completely through the injection needle. At the same time, the superelastic hydrogel material has a hydrophilic surface, which can freely conform to the liver surface, and the soft, deformable scaffold can fit the liver surface well.

[0157] Example 7: Hyperelastic Injectable Stent for Drug Testing

[0158] 1. In vitro liver model fabrication and culture

[0159] The manufacturing and culture process of the in vitro liver model is the same as that in Example 5 above.

[0160] 2. Stents are used in drug testing.

[0161] Drug administration to samples: Scaffolds cultured for 7 days were used for drug testing. The scaffolds were divided into two groups: the experimental group was given 25 μmol / L rifampin, and the control group was given 0.1% dimethyl sulfoxide (Sigma Aldrich). The scaffolds were cultured for another 7 days.

[0162] Gene expression level detection: On day 7 after drug administration, the transcriptional levels of drug metabolism-related genes in the experimental and control groups were detected using qPCR. The results are shown below. Figure 10 The specific procedure for qPCR is as follows:

[0163] RNA Extraction: Harvest the scaffold on day 7. Hydrolyze the material using collagenase to harvest cells from the scaffold. Centrifuge and remove the supernatant. Add Trizol (Gibco) and mix thoroughly by pipetting. Incubate at room temperature for 10 minutes, then transfer to a 1.5 mL EP tube. Add 200 μL of chloroform, shake rapidly for 30 seconds, incubate at room temperature for 5 minutes, and then centrifuge at 12000 g for 10 minutes at 4°C. Remove the supernatant, add an equal volume of isopropanol, and centrifuge at 12000 g for 10 minutes at 4°C. Discard the supernatant, wash the precipitate with 75% anhydrous ethanol, and air-dry to obtain RNA. Dissolve the RNA in enzyme-free sterile water (Solebio Biotechnology Co., Ltd.).

[0164] RNA reverse transcription: using PrimeScript TM II. The 1st strand cDNA Synthesis Kit (Tiangen Biotech Co., Ltd.) was used, and the procedure was strictly followed according to the kit instructions. The primer was Oligo dT Primer. Reverse transcription was performed using a PCR instrument (ABI, SimpliAmp™ thermal cycler). The reverse transcription PCR program was: 42℃, 50 minutes, 95℃, 5 minutes, 4℃ incubation.

[0165] Quantitative real-time PCR (qPCR) procedure: Using the Maxima SYBR Green qPCR Master Mix (ThermoScientific, K0251) kit, follow the instructions exactly as per the kit's instructions. After adding the reaction solution as required, place the reaction plate in a qPCR instrument for detection. The reaction program is as follows: 95℃ for 10 min, 95℃ for 15 s, 60℃ for 30 s, 40 cycles, 72℃ for 30 s, 72℃ for 10 min.

[0166] After the reaction was completed, the amplification curve and the dissolution curve were confirmed, and the data were analyzed using the ΔΔCt method.

[0167] qPCR results showed that after 7 days of drug induction, the expression levels of drug metabolism-related genes (CYP1A2, CYP3A4) in hepatocytes in the scaffold were significantly upregulated, indicating that the hepatocytes in the scaffold had the ability to metabolize drugs.

[0168] Example 8

[0169] A superelastic injectable scaffold, which differs from Example 3 in that the molecular weight of PEGDA is 12,000 Daltons. Everything else is the same as in Example 3 and will not be repeated here.

[0170] Example 9

[0171] A superelastic injectable scaffold, which differs from Example 3 in that the molecular weight of PEGDA is 20,000 Daltons. Everything else is the same as in Example 3 and will not be repeated here.

[0172] Example 10

[0173] A superelastic injectable scaffold, which differs from Example 3 in that the molecular weight of PEGDA is 5000 Daltons. Everything else is the same as in Example 3 and will not be repeated here.

[0174] Example 11

[0175] A superelastic injectable scaffold, which differs from Example 3 in that the molecular weight of PEGDA is 2000 Daltons. Everything else is the same as in Example 3 and will not be repeated here.

[0176] The materials from Examples 8-11 were prepared into block hydrogels with a diameter of 10 mm and a height of 1.5 mm. The deformation behavior of the hydrogels was characterized by dynamic mechanical testing using a fatigue testing machine (Electroforce 3200 BioDynamic, Bose Company). The hydrogel specimen was placed in the center of the indenter of the testing machine, and cyclic compression tests were performed at a frequency of 1 Hz. Deformation amounts equivalent to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, and 95% of the specimen height were applied sequentially, with each deformation amount being cyclically compressed 5 times. The experimental results showed that the hydrogel specimens remained intact without fatigue failure even at deformation amounts up to 80%, exhibiting excellent superelastic behavior. Moreover, the compression deformation amount for molecular weights in the 5000-12000 Dalton range could reach 90%, with the samples remaining intact and rebounding to their original sample size after stress release. When the molecular weight is in the range of 2000-5000 Daltons or 12000-20000 Daltons, the material exhibits superelasticity, but with a slight reduction. Even with a deformation of up to 80%, the hydrogel sample can remain intact without fatigue failure.

[0177] The materials from Examples 8-11 were 3D printed into 15mm × 15mm × 1mm three-dimensional structures. Extrusion injection was performed using a medical catheter with an inner diameter of 2mm. It was observed that the three-dimensional structure could deform, fold, and compress, and was successfully injected through the catheter. After injection, the scaffold immediately unfolded and restored to the previously printed structure, and the gel microfilaments in the three-dimensional structure remained intact, demonstrating unique injectability.

[0178] Example 12

[0179] A superelastic injectable scaffold, compared to Example 3, differs in that the final concentrations of each material component in the precursor solution are: 7.5% GelMA, 2.5% PEGDA, 0.5 mg / mL matrix gel, 0.2 mg / mL decellularized matrix, 100 μmol / mL eosin-γ disodium salt, 0.8% (v / v) trihydroxyethylamine, and 500 nmol / mL vinylpyrrolidone. All other aspects are the same as in Example 3 and will not be repeated here.

[0180] Example 13

[0181] A superelastic injectable scaffold, compared with Example 3, differs in that the final concentrations of each material component in the precursor solution are: 5% GelMA, 5% PEGDA, 1 mg / mL hyaluronic acid, 0.1 mg / mL laminin, 500 μmol / mL eosin-γ disodium salt, 0.5% (v / v) trihydroxyethylamine, and 500 nmol / mL vinylpyrrolidone.

[0182] Example 14

[0183] A superelastic injectable scaffold, compared with Example 3, differs in that the final concentrations of each material component in the precursor solution are: 8% GelMA, 4.5% PEGDA, 0.1 mg / mL collagen, 0.5 mg / mL silk fibroin, 600 μmol / mL eosin-γ disodium salt, 0.6% (v / v) trihydroxyethylamine, and 600 nmol / mL vinylpyrrolidone.

[0184] Experimental results show that when the molecular weight of PEGDA is appropriate, within the concentration range defined by this invention, the resulting hydrogel materials all exhibit good superelasticity and can recover their original shape after injection extrusion. Therefore, they can be used to prepare injectable scaffolds.

[0185] The above description represents only preferred embodiments and is provided as an example only, not as a limitation on the combination of features necessary for carrying out the invention. The provided headings are not intended to limit the various embodiments of the invention. Terms such as “comprising,” “including,” and “including” are not intended to be limiting. Furthermore, unless otherwise stated, the plural form is included when not modified by a numeral, and “or” or “or” means “and / or.” Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0186] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention 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 of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a superelastic hydrogel material via solid injection in the preparation of artificial skin, myocardial patches, and in vitro engineered tissue models, characterized in that, The superelastic hydrogel material includes a substrate material and an initiator. The substrate material includes natural biomaterials modified with photo-initiated crosslinking groups and synthetic biomaterials modified with photo-initiated crosslinking groups. The natural biomaterials modified with photo-initiated crosslinking groups include methacrylic anhydride gelatin. The synthetic biomaterials modified with photo-initiated crosslinking groups include polyethylene glycol dimethacrylate with a crosslinking capacity of 2000 to 20000 Daltons. The preparation method of the superelastic hydrogel material includes the following steps: (1) Mix the substrate material and the initiator to obtain a precursor solution; (2) The precursor solution is photo-initiated to crosslink according to a pre-designed structure to obtain the desired three-dimensional structure; The superelastic hydrogel material recovers to its original three-dimensional structure after being compressed and injected with a syringe.

2. The application of a superelastic hydrogel material via solid injection in the preparation of artificial skin, myocardial patches, and in vitro engineered tissue models, characterized in that, The superelastic hydrogel material comprises a substrate material, an initiator, auxiliary materials, and a crosslinking agent. The substrate material comprises natural biomaterials modified with photo-initiated crosslinking groups and synthetic biomaterials modified with photo-initiated crosslinking groups. The natural biomaterials modified with photo-initiated crosslinking groups include methacrylic anhydride gelatin. The synthetic biomaterials modified with photo-initiated crosslinking groups include polyethylene glycol dimethacrylate with a crosslinking capacity of 2000–20000 Daltons. The preparation method of the superelastic hydrogel material includes the following steps: (1) Mix the substrate material, auxiliary material and initiator to obtain a precursor solution; (2) The precursor solution is photo-initiated to crosslink according to a pre-designed structure to obtain the desired three-dimensional structure; (3) Add a crosslinking agent to crosslink the auxiliary material to obtain a superelastic hydrogel material; The superelastic hydrogel material recovers to its original three-dimensional structure after being compressed and injected with a syringe.

3. The application according to claim 1 or 2, characterized in that, The molecular weight of the polyethylene glycol dimethacrylate is 5,000 to 20,000 Daltons.

4. The application according to claim 1 or 2, characterized in that, The initiator includes ultraviolet photoinitiators or visible photoinitiators; The ultraviolet photoinitiator includes one or more of 2-hydroxy-4-(2-hydroxyethoxy)-2-methylphenylacetone, phenylbis(2,4,6-trimethylbenzoyl)phosphine, azodimethyl N-2-hydroxybutylpropionamide, and lithium phenyl(2,4,6-trimethylbenzoyl)phosphate. The visible light initiator is selected from the eosin-γ system.

5. The application according to claim 4, characterized in that, The eosin-Y system is composed of eosin-Y disodium salt, vinylpyrrolidone, and dithiothreitol.

6. The application according to claim 1, characterized in that, The precursor solution comprises 8%–20% methacrylic anhydride gelatin, 1%–10% polyethylene glycol dimethacrylate, and 0.01 mg / mL–5 mg / mL lithium phenyl (2,4,6-trimethylbenzoyl) phosphate.

7. The application according to claim 2, characterized in that, The auxiliary materials are type I collagen and fibrinogen. The precursor solution includes 5%–10% methacrylic anhydride gelatin, 1%–5% polyethylene glycol dimethacrylate, 0.1–0.3 mg / mL type I collagen, 1–3 mg / mL fibrinogen, 1 μmol / L–1 mmol / L eosin-γ disodium salt, 0.01%–1% (v / v) trihydroxyethylamine, and 1 nmol / L–4 μmol / L vinylpyrrolidone.

8. The application according to claim 6 or 7, characterized in that, Cells were also added to the precursor solution.

9. The application according to claim 6 or 7, characterized in that, Cell clusters were also added to the precursor solution.

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