Bionic brain matrix hydrogel for constructing mature vascular organoid and preparation method and application of bionic brain matrix hydrogel

Abstract

The invention relates to bionic brain matrix hydrogel for constructing mature blood vessel organs and a preparation method and application of the bionic brain matrix hydrogel. The hydrogel comprises 1-10% of skeleton components and 0.02-1% of bioactive polypeptide, and the skeleton components comprise natural protein, phenolated natural polysaccharide and catalytic enzyme; the bioactive polypeptide comprises an adhesion sequence and a pore confinement sequence. Natural protein and natural polysaccharide are utilized to form a covalent / non-covalent combined composite network, the composite network presents a pore structure and mechanical characteristics of a brain extracellular matrix, and the stability of the whole structure can be endowed; a pore channel confinement sequence modified in the pore channel network endows the matrix with space-time dynamic adjustability in internal microstructure and mechanical properties; a cell adhesion sequence is modified in a pore channel network structure, so that adhesion anchoring and space growth regulation and control of vascular organs are further realized. The bionic brain matrix hydrogel and vascular organs cooperate to promote vascular reconstruction, regeneration and repair of cerebral infarction and other diseases.

Description

Technical Field

[0001] This invention relates to a biomimetic brain matrix hydrogel for constructing mature vascular organoids, its preparation method and application, belonging to the field of biomedical materials technology. Background Technology

[0002] Josef Penninger, a member of the European Academy of Sciences, and his team have cultivated blood vessel organoids (BVOs) by accumulating and developing human induced pluripotent stem cells (iPSCs) in vitro. These BVOs exhibit structure and function similar to normal vascular networks, demonstrating significant potential for clinical applications. On one hand, addressing the issue of insufficient vascularization in tissue regeneration, the microvascular network of BVOs can provide new blood supply support for cell replacement therapy and tissue regeneration. On the other hand, BVOs can be used to simulate vascular models in a diabetic environment, supporting research on the mechanisms of vascular diseases, drug screening, and exploration of angiogenesis and development. Currently, the three-dimensional growth of BVOs relies on a hydrogel matrix formed by Matrigel and collagen. However, Matrigel's unclear composition, batch-to-batch variability, high cost, and tumor origin limit the mass production and clinical application of BVOs.

[0003] Currently, synthetic hybrid hydrogels can highly mimic natural extracellular matrix materials in terms of structure and mechanical properties, and have a clear composition and good batch-to-batch stability, making them one of the important choices for three-dimensional culture of organoids. However, synthetic hybrid hydrogels still face many challenges in constructing vascular organoids and in vivo transplantation, which can be summarized into three main aspects: (1) Insufficient overall stability: Hydrogels are prone to excessive swelling or collapse, resulting in the loss of effective support for organoid growth, affecting the long-term culture and in vivo transplantation of organoids; (2) Poor structural and mechanical adaptability: The internal structure and mechanical characteristics of hydrogels are often difficult to adapt to the dynamic changes in vascular structure during the growth and development of vascular organoids, hindering the three-dimensional expansion of blood vessels; (3) Limited bioactivity: The bioactivity of existing synthetic hybrid hydrogels may not be able to fully meet the complex needs of vascular organoids for cell adhesion, migration and differentiation, thereby limiting the formation and functional improvement of vascular networks. Currently, only one patent application, CN117143359A, discloses a method for preparing hydrogel materials suitable for the growth of vascular organoids. This method utilizes methacrylic anhydride-modified gelatin (GelMA) hydrogel for vascular organoid culture. However, the single active ingredient and structure of this matrix cannot effectively promote the budding of vascular organoids and the maturation of blood vessel lumens, let alone deliver vascular organoids for brain vascular reconstruction. Therefore, designing and synthesizing gel matrix materials with overall structural stability, internal biodegradability, growth-promoting properties, and biocompatibility to achieve precise control over the growth and development of vascular organoids and promote their maturation is a key technical problem that urgently needs to be solved. Summary of the Invention

[0004] (1) Technical issues to be resolved

[0005] To address the aforementioned problems in the prior art, this invention provides a method for preparing and applying a biomimetic brain matrix hydrogel for constructing mature vascular organoids. Through the synergistic effect of its structural components, mechanical properties, and bioactivity characteristics, it achieves precise regulation of the growth and development of vascular organoids, thereby constructing more mature vascular organoids for application in brain vascular reconstruction.

[0006] (2) Technical solution

[0007] To achieve the above objectives, the main technical solutions adopted by the present invention include:

[0008] A biomimetic brain matrix hydrogel for constructing mature vascular organoids comprises a 1-10% by mass of a scaffold component and a 0.02-1% by mass of a bioactive polypeptide; the biomimetic brain matrix hydrogel has a stiffness of 50-1000 Pa and a stress relaxation half-decay time of 1-10 s; the biomimetic brain matrix hydrogel has a pore size of 10-100 μm.

[0009] Preferably, in the biomimetic brain matrix hydrogel described above, the scaffold component comprises natural protein, phenolic natural polysaccharide, and catalytic enzyme, and the mass ratio of natural protein, phenolic natural polysaccharide, and catalytic enzyme in the preparation solution is 1:0.1–10:0.001–0.1; wherein,

[0010] The natural protein is any one of silk fibroin, collagen, fibrin, and serum albumin; the phenolic natural polysaccharide is obtained by phenolic modification of natural polysaccharide as substrate, and the theoretical modification rate of phenolic groups accounts for 5-20% of the natural polysaccharide; the catalytic enzyme is any one of cytochrome P450 enzyme, laccase, catechol oxidase, and catalase (containing hydrogen peroxide).

[0011] Furthermore, the method for preparing the phenolic natural polysaccharide is as follows: tyramine hydrochloride is added to an aqueous solution of the natural polysaccharide, stirred and dissolved, the pH is adjusted to 6-8, and a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in an equimolar ratio is added. The reaction pH is maintained at 6-8, and the mixture is stirred overnight at room temperature. The mixture is then purified by dialyzing in pure water and freeze-dried to obtain the phenolic natural polysaccharide.

[0012] Furthermore, the natural polysaccharide is any one of hyaluronic acid, sodium alginate, carboxymethyl cellulose, and chondroitin sulfate; the molar ratio of tyramine hydrochloride to the natural polysaccharide is 1 to 20:1.

[0013] The molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to natural polysaccharide is 1 to 10:1.

[0014] In the biomimetic brain matrix hydrogel described above, preferably, the bioactive polypeptide comprises a mixture of adhesion sequences and pore-confining sequences, wherein the mass ratio of the adhesion sequences to the pore-confining sequences is 1:0.1 to 1; the adhesion sequences are used to provide active sites for the adhesion and growth of vascular organoids, and the pore-confining sequences are used to regulate the internal structure and mechanical properties of the gel to match the growth of vascular organoids.

[0015] Furthermore, the adhesion sequence is any one or a mixture of two or more of SEQ ID NO.1: GRGDSPY, SEQ ID NO.2: HAVDIGGY, or SEQ ID NO.3: GYGYIGSR; the pore confinement sequence is any one or a mixture of two or more of SEQ ID NO.4: GYRDGPQGIWGQDRYG, SEQ ID NO.5: GYRDGPQGIAGQDRYG, or SEQ ID NO.6: GYRDGDQGIAGFDRYG.

[0016] Preferably, the N-terminus or C-terminus of the adhesion sequence and / or the pore confinement sequence is acetylated and capped.

[0017] The preparation method of the biomimetic brain matrix hydrogel as described above includes the following steps:

[0018] S1. Dissolve natural protein, phenolic natural polysaccharide and catalytic enzyme in aqueous solution, and mix under ice bath to obtain hydrogel framework preparation solution;

[0019] S2. Add bioactive peptides to the hydrogel framework preparation solution and mix well under ice bath to obtain the stock solution of biomimetic brain matrix hydrogel.

[0020] S3. Adjust the pH of the biomimetic brain matrix hydrogel stock solution to 6-8 and let it stand at 37°C for 0.5-2 hours. Under the action of the previously catalytic enzyme, further covalent cross-linking is carried out to obtain a hydrogel with a structure and mechanical modulus similar to that of the brain extracellular matrix.

[0021] In the preparation method described above, preferably, the mass concentration of the skeletal component in the biomimetic brain matrix hydrogel stock solution is 1-10%, which contains natural protein, phenolic natural polysaccharide and catalytic enzyme in a mass ratio of 1:0.1-10:0.01-0.1.

[0022] In the preparation method described above, preferably, the bioactive polypeptide in the biomimetic brain matrix hydrogel stock solution has a mass concentration of 0.02-1%, wherein the bioactive polypeptide contains an adhesion sequence and a pore confinement sequence, and the mass ratio of the two is 1:0.1-1.

[0023] Applications of biomimetic brain matrix hydrogels or biomimetic brain matrix hydrogels obtained by the above-described preparation methods in the construction of blood vessels or vascular organoids.

[0024] Application of the biomimetic brain matrix hydrogel or the biomimetic brain matrix hydrogel obtained by the above-described preparation method in the preparation of organ-on-a-chip.

[0025] The application of the biomimetic brain matrix hydrogel or the biomimetic brain matrix hydrogel obtained by the above-described preparation method in the preparation of vascular reconstruction and regeneration repair materials for diseases such as cerebral infarction, spinal cord injury, and myocardial infarction, in conjunction with vascular organoids.

[0026] (3) Beneficial effects

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

[0028] This invention provides a biomimetic brain matrix hydrogel for constructing mature vascular organoids. Compared with Matrigel and other synthetic hybrid matrices, the biomimetic brain matrix hydrogel provided by this invention utilizes a variety of natural proteins and natural polysaccharides to form a covalently / non-covalently combined composite network, resulting in an anti-swelling overall structure. Simultaneously, the confined sequences modified in the pore network endow the matrix with spatiotemporal dynamic tunability in its internal microstructure and mechanical properties. Furthermore, the cell adhesion sequences modified in the pore network structure further enable adhesion anchoring and spatial growth regulation of vascular organoids. The biomimetic brain matrix hydrogel possesses characteristics such as well-defined composition, overall anti-swelling properties, tunable mechanical properties, internal biodegradability, and biosafety, presenting broad clinical application prospects, especially showing potential in organ-on-a-chip and cerebral infarction repair.

[0029] The biomimetic brain matrix hydrogel provided by this invention simulates the pore structure and mechanical characteristics of the brain extracellular matrix through a natural protein / polysaccharide composite network. The design of pore confinement and adhesion sequences further replicates the physiological conditions of angiogenesis, forming a multi-scale biomimetic structure. Its main features are reflected in the following four aspects:

[0030] (1) In terms of overall structural stability, natural proteins and polysaccharides provide long-term matrix stability through covalent cross-linking (such as carbon-carbon bonds) and non-covalent assembly (hydrogen bonds, ionic bonds, and hydrophobic interactions). Covalent bonds and hydrophobic interactions effectively avoid the swelling and collapse problems that occur in traditional hydrogels (such as pure hyaluronic acid) in aqueous environments. Hydrogen bonds and electrostatic interactions endow the material with self-healing capabilities. These properties ensure that the pore structure provides stable support for vascular development during long-term culture, which helps to construct a stable vascular topology.

[0031] (2) In terms of structural and mechanical adaptability, by covalently modifying the pore-confined polypeptide sequence in the matrix network, vascular organoids can actively degrade and remodel the matrix interior by secreting matrix metalloproteinases (MMPs), thus realizing the dynamic adjustment of structure and mechanical properties (stiffness, stress relaxation), matching the migration and self-assembly process of vascular cells, and meeting the spatiotemporal dynamic needs from vascular sprouting to vascular maturation.

[0032] (3) In terms of growth regulation, covalently modified adhesion peptide sequences such as RGD, IKVAV and YIGSR in the matrix network can guide the adhesion and anchoring of endothelial cells / pericytes, and promote the formation of vascular lumen and branching morphology.

[0033] (4) In terms of biosafety, the preparation method provided by the present invention uses natural proteins and natural polysaccharides that are easy to obtain, with accurate composition, and can be produced in a standardized GMP manner; the natural substrate has low immunogenicity, and the components, such as hyaluronic acid, are important components of the brain extracellular matrix, playing an important role in organ development and angiogenesis, and have good biocompatibility.

[0034] The biomimetic brain matrix hydrogel provided by this invention solves key problems in the culture of vascular organoids, such as insufficient microenvironment simulation, poor structural stability, uncontrollable cell behavior, and low biosafety, and provides an ideal platform for the standardized construction and clinical translation of vascular organoids. Attached Figure Description

[0035] Figure 1 This is a comparison diagram of the storage modulus of the biomimetic brain matrix of the present invention, the natural brain extracellular matrix, and the traditional Matrigel-Collagen matrix.

[0036] Figure 2 This is a comparison diagram of the pore size between the biomimetic brain matrix of this invention and the traditional Matrigel-Collagen matrix;

[0037] Figure 3 This is a comparison of the stress relaxation half-decay time of the biomimetic brain matrix of this invention and the traditional Matrigel-Collagen matrix;

[0038] Figure 4 To assess the structural stability and biodegradability of the biomimetic brain matrix of this invention compared to the traditional Matrigel-Collagen matrix under culture conditions: (A) changes in swelling ratio, (B) changes in pore size;

[0039] Figure 5 The storage modulus changes of the biomimetic brain matrix of this invention and the traditional Matrigel-Collagen matrix under culture conditions;

[0040] Figure 6 This is a comparison of the sprouting of vascular organoids in the biomimetic brain matrix of this invention and the traditional Matrigel-Collagen matrix;

[0041] Figure 7 Live and dead cell staining of vascular organoids in the biomimetic brain matrix of this invention and the traditional Matrigel-Collagen matrix;

[0042] Figure 8 The results of immunofluorescence staining of vascular organoids in the biomimetic brain matrix and the traditional Matrigel-Collagen matrix of this invention, as well as the quantitative analysis data of the entire vascular network structure, are presented.

[0043] Figure 9 Immunofluorescence staining of local vascular network structures (central and leading edge regions) of vascular organoids in the biomimetic brain matrix of this invention and the traditional Matrigel-Collagen matrix;

[0044] Figure 10 This invention provides the expression and distribution of vascular markers at the angiogenesis front of vascular organoids in the biomimetic brain matrix and the traditional Matrigel-Collagen matrix.

[0045] Figure 11 To observe the cerebral cortical blood perfusion in mice after cerebral infarction treatment using laser speckle imaging;

[0046] Figure 12 Staining tissue sections to show angiogenesis in the damaged area after cerebral infarction treatment in mice;

[0047] Figure 13 This is a schematic diagram illustrating the application of biomimetic brain matrix hydrogels in the in vitro culture and in vivo delivery of vascular organoids.

[0048] Figure 14 To assess the recovery of motor function in mice after treatment for cerebral infarction using behavioral methods;

[0049] Figure 15 This invention presents a comparison of the budding and growth of vascular organoids in three different states of the biomimetic brain matrix: the original form, the form after removing adhesion sequences, and the form after removing pore confinement sequences. Detailed Implementation

[0050] This invention develops a biomimetic brain matrix hydrogel for constructing mature vascular organoids. On one hand, natural proteins and phenolic natural polysaccharides are cross-linked under enzymatic catalysis to form a covalently and non-covalently combined composite structure, mimicking the mechanical modulus of brain tissue to construct an anti-swelling biomimetic hydrogel that provides support for organoid growth. Simultaneously, bioactive polypeptide sequences are covalently anchored and embedded within the hydrogel's porous network, promoting cell adhesion and dynamically regulating the gel's internal structure and mechanical properties. This biomimetic brain matrix hydrogel design not only provides growth space and bio-induction for vascular organoid development but also achieves effective regulation during the maturation process of vascular organoids through the synergistic effect of both components.

[0051] On one hand, the present invention provides a biomimetic brain matrix hydrogel for constructing mature vascular organoids, characterized in that it comprises a skeleton component with a mass concentration of 1-10% and a bioactive polypeptide with a mass concentration of 0.02-1%; the biomimetic brain matrix hydrogel has the mechanical characteristics of the brain extracellular matrix, with a stiffness of 50-1000 Pa and a stress relaxation half-decay time of 1-10 s; the pore size of the biomimetic brain matrix hydrogel is 10-100 μm.

[0052] The skeletal components comprise natural protein, phenolic natural polysaccharide, and catalytic enzyme. The mass ratio of natural protein, phenolic natural polysaccharide, and catalytic enzyme in the preparation solution is 1:0.1–10:0.001–0.1. Further, the natural protein is any one of silk fibroin, collagen, fibrin, and serum albumin. The phenolic natural polysaccharide is obtained by phenolic modification of a natural polysaccharide substrate, with a theoretical phenolic modification rate of 5–20% of the natural polysaccharide. The catalytic enzyme is any one of cytochrome P450 enzyme, laccase, catechol oxidase, and catalase (containing hydrogen peroxide).

[0053] The bioactive polypeptide is a mixture of an adhesion sequence and a pore-confining sequence; the mass ratio of the adhesion sequence to the pore-confining sequence is 1:0.1 to 1; the adhesion sequence provides active sites for the adhesion and growth of vascular organoids; the pore-confining sequence regulates the internal structure and mechanical properties of the gel to match the growth requirements of vascular organoids; further, the adhesion sequence includes any one or a mixture of two or more of SEQ ID NO.1-SEQ ID NO.3; the pore-confining sequence includes any one or a mixture of two or more of SEQ ID NO.4-SEQ ID NO.6.

[0054] This invention provides a method for preparing a biomimetic brain matrix hydrogel for constructing mature vascular organoids, comprising the following steps:

[0055] S1. Dissolve natural protein, phenolic natural polysaccharide and catalytic enzyme in aqueous solution and mix under ice bath to obtain hydrogel framework preparation solution;

[0056] S2. Rapidly add the bioactive polypeptide sequence and mix under ice bath to obtain the stock solution of biomimetic brain matrix hydrogel;

[0057] S3. Adjust the pH of the biomimetic brain matrix hydrogel stock solution to 6-8 and let it stand at 37°C for 0.5-2 hours. Further covalent cross-linking is carried out under the action of the previously catalytic enzyme to obtain a hydrogel with a mechanical modulus similar to that of brain tissue.

[0058] Preferably, the mass concentration of the hydrogel framework component in the biomimetic brain matrix hydrogel stock solution is 1-10%, containing natural proteins, phenolic natural polysaccharides, and catalytic enzymes in a mass ratio of 1:0.1-10:0.01-0.1. This ratio ensures that the biomimetic brain matrix hydrogel has a stiffness of 50-1000 Pa, matching the mechanical support stiffness of biological tissues; a stress relaxation half-decay time of 1-10 s, promoting dynamic mechanical regulation of cells; and cell-scale pore size (10-100 μm), satisfying cell migration and ingrowth.

[0059] More preferably, the bioactive polypeptide in the biomimetic brain matrix hydrogel stock solution has a mass concentration of 0.02-1%, wherein the bioactive polypeptide contains adhesion sequences and pore-confining sequences, and the mass ratio of the two is 1:0.1-1. This ratio ensures that the biomimetic brain matrix hydrogel effectively promotes cell adhesion and assembly.

[0060] To better explain and facilitate understanding of this invention, the following detailed description, in conjunction with the accompanying drawings and specific embodiments, is provided. All materials used in this invention are commercially available products. For example, silk fibroin, collagen, fibrin, and serum albumin can be purchased from Sigma-Aldrich; the catalytic enzymes, including cytochrome P450 enzyme, laccase, catechol oxidase, and catalase, can be purchased from Sigma-Aldrich; 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide can be purchased from Beijing Innocare Technology Co., Ltd.; hyaluronic acid, sodium alginate, carboxymethyl cellulose, and chondroitin sulfate can be purchased from Shanghai Maclean Biotechnology Co., Ltd.; adhesion sequences and pore-confining sequences can be synthesized by Nanjing Genscript Biotech Co., Ltd.; Matrigel can be purchased from Corning Incorporated (USA); Collagen can be purchased from Stemcell (Canada); vascular organoid maturation culture medium can be purchased from Chongqing Jiukang Medical Research Institute Co., Ltd.; and hiPSC cells can be purchased from the National Model and Special Experimental Cell Resource Bank.

[0061] Example 1

[0062] The components and preparation method of biomimetic brain matrix hydrogels for constructing mature vascular organoids include the following steps:

[0063] S1. Prepare aqueous solutions of silk fibroin, phenolic hyaluronic acid, and cytochrome P450 enzyme at mass concentrations of 10%, 10%, and 1%, respectively. Then, mix these three solutions in an ice bath at a volume ratio of 1:1:0.1 to obtain a hydrogel framework preparation solution with a mass ratio of silk fibroin, phenolic hyaluronic acid, and cytochrome P450 enzyme of 1:1:0.01.

[0064] The preparation of a 10% silk fibroin aqueous solution can be carried out by dissolving 10g of silk fibroin in 100ml of deionized water, and the aqueous solution can be prepared in the same manner.

[0065] The preparation method of phenolic hyaluronic acid is as follows: (1) First, hyaluronic acid (molecular weight 10kD) is dissolved in deionized water to prepare a hyaluronic acid solution with a mass concentration of 10% (e.g., 10g of hyaluronic acid is dissolved in 100mL of deionized water); (2) Tyramine hydrochloride (0.87g, 5mmol) is added to 10mL of hyaluronic acid solution and stirred until it is completely dissolved to obtain a mixed solution; sodium hydroxide solution is added to adjust the pH of the mixed solution to 6-8; (3) A solid mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.48g, 2.5mmol) and N-hydroxysuccinimide (0.29g, 2.5mmol) is added at one time, and the reaction pH is maintained at 6-8 and stirred overnight at room temperature; (4) The hyaluronic acid is purified by dialysis in pure water for 3 days using a dialysis bag (molecular weight cutoff 7kD) and then freeze-dried to obtain phenolic hyaluronic acid. The nuclear magnetic resonance spectrum of phenolic hyaluronic acid was collected. The percentage of phenolic groups in all disaccharide repeating units was calculated by (phenolic hydrogen peak area ÷ 4) / (methyl hydrogen peak area of ​​disaccharide repeating unit ÷ 3) × 100%, and the actual phenolic modification rate of hyaluronic acid was found to be 10%.

[0066] S2. Prepare a mixed aqueous solution of bioactive peptide sequences with a total mass concentration of 2% (e.g., 2g of bioactive peptide dissolved in 100mL of deionized water), wherein the mass ratio of GRGDSPY (adhesion sequence) and GYRDGPQGIWGQDRYG (pore confinement sequence) is 1:1; then add the mixture in equal volume to the hydrogel framework preparation solution prepared in S1 and mix under ice bath to obtain the stock solution of biomimetic brain matrix hydrogel.

[0067] S3. Add sodium hydroxide solution to adjust the pH of the biomimetic brain matrix hydrogel stock solution to 6-8 and let it stand at 37°C for 0.5 h. During this process, the catalytic enzyme (cytochrome P450 enzyme) further covalently crosslinks to obtain a hydrogel with a mechanical modulus similar to that of brain tissue.

[0068] Example 2

[0069] The preparation method of biomimetic brain matrix hydrogel for constructing mature vascular organoids includes the following steps:

[0070] S1. Prepare aqueous solutions of collagen, phenolic sodium alginate, and laccase with mass concentrations of 1%, 10%, and 1%, respectively. Then, mix these three solutions in an ice bath at a volume ratio of 1:1:0.1 to obtain a hydrogel framework preparation solution with a mass ratio of collagen, phenolic sodium alginate, and laccase of 1:10:0.1.

[0071] The preparation method of phenolic sodium alginate is as follows: (1) First, sodium alginate (molecular weight 100kD) is dissolved in deionized water to prepare a sodium alginate solution with a mass concentration of 5%; (2) Tyramine hydrochloride (1.74g, 10mmol) is added to 10mL of sodium alginate solution and stirred until completely dissolved; sodium hydroxide solution is added to adjust the pH of the mixed solution to 6-8; (3) A solid mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.96g, 5mmol) and N-hydroxysuccinimide (0.58g, 5mmol) is added at one time, and the reaction pH is maintained at 6-8 and stirred overnight at room temperature; (4) The phenolic sodium alginate is purified by dialysis in pure water for 3 days using a dialysis bag (molecular weight cutoff 7kD) and then freeze-dried to obtain phenolic sodium alginate. The nuclear magnetic resonance spectrum of phenolized sodium alginate was collected. The percentage of phenolic groups in all monosaccharide repeating units was calculated by (phenolic hydrogen peak area ÷ 4) / (monosaccharide repeating unit hydrogen peak area ÷ 5) × 100%, and the actual phenolic modification rate of sodium alginate was found to be 8%.

[0072] S2. Prepare a mixed aqueous solution of bioactive peptide sequences with a mass concentration of 1.5%, wherein the mass ratio of GRGDSPY (adhesion sequence) and GYRDGPQGIAGQDRYG (pore confinement sequence) is 1:0.5; then add the mixture in equal volume to the hydrogel framework preparation solution prepared in S1 and mix under ice bath to obtain the stock solution of biomimetic brain matrix hydrogel.

[0073] S3. Add sodium hydroxide solution to adjust the pH of the biomimetic brain matrix hydrogel stock solution to 6-8 and let it stand for 1 hour. During this process, the catalytic enzyme (laccase) further covalently crosslinks to obtain a hydrogel with a mechanical modulus similar to that of brain tissue.

[0074] Example 3

[0075] A method for preparing a biomimetic brain matrix hydrogel for constructing mature vascular organoids includes the following steps:

[0076] S1. Prepare aqueous solutions of fibrin, phenolic carboxymethyl cellulose and catechol oxidase with mass concentrations of 10%, 1% and 0.5%, respectively. Then, mix the three solutions in an ice bath at a volume ratio of 1:1:0.1 to obtain a hydrogel framework preparation solution with a mass ratio of fibrin, phenolic carboxymethyl cellulose and catechol oxidase of 1:0.1:0.005.

[0077] The preparation method of phenolic carboxymethyl cellulose is as follows: (1) First, carboxymethyl cellulose (molecular weight 90kD) is dissolved in deionized water to prepare a solution with a mass concentration of 5%; (2) Tyramine hydrochloride (3.48g, 20mmol) is added to 10mL of carboxymethyl cellulose solution and stirred to dissolve it completely; sodium hydroxide solution is added to adjust the pH of the mixed solution to 6-8; (3) A solid mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.84g, 20mmol) and N-hydroxysuccinimide (2.302g, 20mmol) is added at one time, and the reaction pH is maintained at 6-8 and stirred overnight at room temperature; (4) The phenolic carboxymethyl cellulose is obtained by dialysis in pure water for 3 days using a dialysis bag (molecular weight cutoff 7kD) and freeze-drying. The nuclear magnetic resonance spectrum of phenolic carboxymethyl cellulose was collected. The percentage of phenolic groups in all monosaccharide repeating units was calculated by (phenolic hydrogen peak area ÷ 4) / (methylene unit hydrogen peak area ÷ 2) × 100%, and the actual phenolic modification rate of carboxymethyl cellulose was found to be 6%.

[0078] S2. Prepare a mixed aqueous solution of bioactive peptide sequences with a mass concentration of 0.6%, wherein the mass ratio of HAVDIGGY (adhesion sequence) and GYRDGPQGIWGQDRYG (pore confinement sequence) is 1:0.5; then add the mixture in equal volume to the hydrogel framework preparation solution prepared in S1 and mix under ice bath to obtain the stock solution of biomimetic brain matrix hydrogel.

[0079] S3. Add sodium hydroxide solution to adjust the pH of the biomimetic brain matrix hydrogel stock solution to 6-8 and let it stand for 1 hour. Under the action of catalytic enzyme (catechol oxidase), further covalent cross-linking is carried out to obtain a hydrogel with a mechanical modulus similar to that of brain tissue.

[0080] Example 4

[0081] The preparation method of biomimetic brain matrix hydrogel for constructing mature vascular organoids includes the following steps:

[0082] S1. Prepare aqueous solutions of serum albumin, phenolic chondroitin sulfate, and catechol oxidase with mass concentrations of 10%, 5%, and 0.5%, respectively. Then, mix these three solutions in an ice bath at a volume ratio of 1:1:0.1 to obtain a hydrogel framework preparation solution with a mass ratio of serum albumin, phenolic chondroitin sulfate, and catechol oxidase of 1:0.5:0.005.

[0083] The preparation method of phenolic chondroitin sulfate is as follows: (1) First, chondroitin sulfate (molecular weight 10kD) is dissolved in deionized water to prepare a solution with a mass concentration of 10%; (2) Tyramine hydrochloride (1.74g, 10mmol) is added to 10mL of chondroitin sulfate solution and stirred to dissolve it completely; sodium hydroxide solution is added to adjust the pH of the mixed solution to 6-8; (3) A solid mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.92g, 10mmol) and N-hydroxysuccinimide (1.16g, 10mmol) is added at one time, and the reaction pH is maintained at 6-8 and stirred overnight at room temperature; (4) The phenolic chondroitin sulfate is obtained by dialysis in pure water for 3 days using a dialysis bag (molecular weight cutoff 7kD) and freeze-drying. The nuclear magnetic resonance spectrum of phenolic chondroitin sulfate was collected. The percentage of phenolic groups in all disaccharide repeating units was calculated by (phenolic hydrogen peak area ÷ 4) / (methyl hydrogen peak area of ​​disaccharide repeating unit ÷ 3) × 100%, and the actual phenolic modification rate of chondroitin sulfate was found to be 5%.

[0084] S2. Prepare a mixed aqueous solution of bioactive peptide sequences with a mass concentration of 0.4%, wherein the mass ratio of GYGYIGSR (adhesion sequence) and GYRDGDQGIAGFDRYG (pore confinement sequence) is 1:1; then add the mixture in equal volume to the hydrogel framework preparation solution prepared in S1 and mix under ice bath to obtain the stock solution of biomimetic brain matrix hydrogel.

[0085] S3. Add sodium hydroxide solution to adjust the pH of the biomimetic brain matrix hydrogel stock solution to 6-8 and let it stand for 2 hours. Under the action of catalytic enzyme (catechol oxidase), further covalent cross-linking is carried out to obtain a hydrogel with a mechanical modulus similar to that of brain tissue.

[0086] Example 5

[0087] The preparation method of biomimetic brain matrix hydrogel for constructing mature vascular organoids includes the following steps:

[0088] S1. Prepare aqueous solutions of silk fibroin, phenolic hyaluronic acid, catalase, and hydrogen peroxide with mass concentrations of 10%, 10%, 0.1%, and 3%, respectively. Then, mix these four solutions in an ice bath at a volume ratio of 1:1:0.1:0.1 to obtain a hydrogel framework preparation solution with a mass ratio of 1:1:0.001:0.03 for silk fibroin, phenolic hyaluronic acid, catalase, and hydrogen peroxide.

[0089] The preparation method of phenolic hyaluronic acid is as follows: (1) First, hyaluronic acid (molecular weight 10kD) is dissolved in deionized water to prepare a solution with a mass concentration of 10%; (2) Tyramine hydrochloride (0.87g, 5mmol) is added to 10mL of hyaluronic acid solution and stirred to dissolve it completely; sodium hydroxide solution is added to adjust the pH of the mixed solution to 6-8; (3) A solid mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.48g, 2.5mmol) and N-hydroxysuccinimide (0.29g, 2.5mmol) is added at one time, and the reaction pH is maintained at 6-8 and stirred overnight at room temperature; (4) The phenolic hyaluronic acid is obtained by dialysis in pure water for 3 days using a dialysis bag (molecular weight cutoff 7kD) and freeze-drying. The nuclear magnetic resonance spectrum of phenolic hyaluronic acid was collected. The percentage of phenolic groups in all disaccharide repeating units was calculated by (phenolic hydrogen peak area ÷ 4) / (methyl hydrogen peak area of ​​disaccharide repeating unit ÷ 3) × 100%, and the actual phenolic modification rate of hyaluronic acid was found to be 10%.

[0090] S2. Prepare a mixed aqueous solution of bioactive peptide sequences with a mass concentration of 1%, wherein the mass ratio of GRGDSPY (adhesion sequence) and GYRDGPQGIWGQDRYG (pore confinement sequence) is 1:1; then add the mixture in equal volume to the hydrogel framework preparation solution prepared in S1 and mix under ice bath to obtain the stock solution of biomimetic brain matrix hydrogel.

[0091] S3. Add sodium hydroxide solution to adjust the pH of the biomimetic brain matrix hydrogel stock solution to 6-8 and let it stand at 37°C for 2 hours. Under the action of catalytic enzymes, further covalent cross-linking is carried out to obtain a hydrogel with a mechanical modulus similar to that of brain tissue.

[0092] Comparative Example 1

[0093] Preparation of Matrigel-Collagen matrix

[0094] First, prepare the neutralization solution: thoroughly mix 11.6 μL of 1 mol / L NaOH, 163.4 μL of ultrapure water, and 75 μL of 10×DPBS. Then, add 500 μL of 3 mg / mL Collagen (type I collagen) solution to the neutralization solution, followed by 250 μL of Matrigel, and gently mix to avoid generating bubbles. All steps are performed on ice to prevent premature gelation. The final mixture is incubated at 37°C to form a gel, yielding the conventional vascular organoid culture medium (Matrigel-Collagen).

[0095] Example 6

[0096] The biomimetic brain matrix hydrogel products prepared in Examples 1, 3, and 5, and the Matrigel-Collagen matrix of Comparative Example 1 were selected for mechanical and structural characterization. Hereinafter, the product of Example 1 will be referred to as "Bionic Brain Matrix A", the product of Example 5 as "Bionic Brain Matrix B", the product of Example 3 as "Bionic Brain Matrix C", and the Matrigel-Collagen matrix as "Matrigel-Collagen".

[0097] Rheological measurements of the hydrogel matrix were performed using a TA DHR-1 rheometer at 37°C. First, a cylindrical matrix was carefully placed on the Peltier temperature-controlled chassis of the rheometer. Then, the parallel plate clamp was slowly and gently lowered until it was in complete contact with the top surface of the matrix. To prevent moisture evaporation during the test, silicone oil was applied to the edges of the clamp for sealing. The stiffness of the matrix was evaluated by oscillation tests within its linear viscoelastic range. Specifically, dynamic frequency sweeps were performed from 0.1 to 10 Hz with a strain of 1% to obtain the mechanical response data of the matrix at different frequencies. The rheological characterization results are as follows: Figure 1 As shown, the storage moduli of the biomimetic hydrogel are 68.9±5.5 Pa (biomimetic brain matrix A), 282.0±4.0 Pa (biomimetic brain matrix B), and 426.3±21.7 Pa (biomimetic brain matrix C), all matching the mechanical characteristics of soft tissue (between 50 and 1000 Pa). Among them, the stiffness of biomimetic brain matrix B is closest to that of the brain ECM (the storage modulus of the brain ECM is 241.2±29.4 Pa). In contrast, the storage modulus of the traditional vascular organoid culture medium (Matrigel-Coll agen) is 101.0±13.6 Pa, which is lower than that of the brain ECM.

[0098] To assess the microstructure of the hydrogel matrix, detailed characterization was performed using cryo-electron microscopy (Cryo-SEM), and the results are as follows: Figure 2 As shown, the biomimetic brain matrix exhibits different pore structures with pore sizes of 42.8±8.3 μm (biomimetic brain matrix A), 19.5±4.2 μm (biomimetic brain matrix B), and 15.1±3.9 μm (biomimetic brain matrix C). In contrast, the Matrigel-Collagen matrix has a pore size of only 8.9±1.7 μm. This indicates that the pore size of the biomimetic brain matrix hydrogel is more conducive to cell migration and attachment.

[0099] Meanwhile, the dynamic mechanical properties of the biomimetic brain matrix hydrogel were evaluated through stress relaxation characterization, and the results are as follows: Figure 3 As shown, biomimetic brain matrix hydrogels all exhibit rapid mechanical relaxation properties, characterized by a low stress relaxation half-decay time (τ). 1 / 2The time intervals were 10.4±7.0 s (bionic brain matrix A), 1.5±0.7 s (bionic brain matrix B), and 0.82±0.16 s (bionic brain matrix C). This ensures the dynamic mechanical regulation of the bionic brain matrix hydrogel.

[0100] Example 7

[0101] The structural stability and degradability of the biomimetic brain matrix hydrogel (hereinafter referred to as "bionic brain matrix") prepared in Example 5 and the conventional Matrigel-collagen matrix (hereinafter referred to as "Matrigel-Collagen") prepared in Comparative Example 1 were further compared and studied.

[0102] according to Figure 4 As shown in Figure A, the overall volume of both the biomimetic brain matrix hydrogel and the Matrigel-Collagen matrix did not change significantly after immersion in vascular organoid maturation culture medium for 5 days. However, microstructural analysis based on Cryo-SEM (…) Figure 4 B) revealed different dynamic changes in the internal structure of the two matrices: compared with day 0, the biomimetic brain matrix hydrogel showed a significant increase in pore size on day 5; in contrast, the Matrigel-Collagen matrix did not show significant changes in pore size after 5 days of culture.

[0103] Changes in pore size are further reflected in the mechanical properties of the matrix (results as follows). Figure 5 As shown in the diagram: with prolonged immersion time in the vascular organoid maturation medium, the stiffness of the biomimetic brain matrix hydrogel gradually decreased; in contrast, the stiffness of the traditional Matrigel-Collogen matrix did not show a significant change. This indicates that the biomimetic brain matrix hydrogel possesses both a stable overall structure and a degradable internal structure, the latter characterized by a gradual increase in pore size during degradation, accompanied by a decrease in stiffness. This unique combination of structure and mechanical properties may be beneficial for regulating the growth and functional maturation of vascular organoids. Therefore, the biomimetic brain matrix hydrogel prepared in Example 5 exhibits potential advantages in both structure and mechanical properties compared to the traditional Matrigel-Collogen matrix, suggesting its potential as an alternative matrix material for vascular organoid culture.

[0104] Example 8

[0105] The study compared the performance differences of the biomimetic brain matrix hydrogel (hereinafter referred to as "bionic brain matrix") prepared in Example 5 and the conventional Matrigel-collagen matrix (hereinafter referred to as "Matrigel-Collagen") prepared in Comparative Example 1 in the in vitro culture of vascular organoids.

[0106] First, hiPSC cell aggregates were subjected to mesodermal-induced differentiation and angiogenesis-induced differentiation sequentially. On day 6, 500 μL of prepared biomimetic brain matrix hydrogel was pre-added to each well of a 12-well plate and incubated at 37°C for 2 hours to form a gel. Then, the initially induced vascular organoids were washed with DPBS, mixed thoroughly with 500 μL of the prepared biomimetic hydrogel, and added to the pre-added wells, with each well containing 40-60 vascular organoids. After incubation at 37°C for 2 hours until the hydrogel was completely formed, 2 mL of vascular maturation culture medium was added to each well for three-dimensional induction culture. Vascular organoids were then cultured using the same method as above using a conventional Matrigel-collagen matrix.

[0107] The growth status of vascular organoids in different matrices was observed under a microscope. Statistical analysis was performed on the germination efficiency and number of vascular organoids on the first day after coating, as well as the germination length at different times after coating (e.g., ...). Figure 6 (As shown in the figure). The results showed that the budding efficiency, budding length, and budding rate of vascular organoids in the biomimetic brain matrix hydrogel were comparable to those of vascular organoids cultured in Matrigel-collagen matrix. This indicates that the biomimetic brain matrix hydrogel possesses the basic activity for culturing vascular organoids.

[0108] On day 5 of gel-coated culture, cell viability in the matrix was assessed. Live and dead cells were stained with Calcein-AM (green) / PI (red) and fluorescence statistics were performed (e.g., ...). Figure 7 (As shown in the image). Statistical results indicate that vascular organoids maintained high survival rates in both biomimetic brain matrix hydrogels and conventional Matrigel-Collagen matrixes. This result demonstrates the excellent biocompatibility of biomimetic brain matrix hydrogels.

[0109] To further evaluate the effects of the two matrices on vascular organoid development, immunofluorescence staining of vascularization-related indicators was performed on the vascular organoids 5 days after coating. Specifically, CD31 antibody was used to label vascular endothelial cells, and phalloidin was used to label the cytoskeleton (…). Figure 8 Subsequently, AngioTool software was used to quantitatively analyze the vascular network structure in the immunofluorescence images, thereby obtaining key parameters such as vessel diameter, vessel density, total vessel length, and number of vessel branches in the vascular organoids. The results are as follows: Figure 8 As shown, compared to vascular organoids in the Matrigel-Collagen matrix, vascular organoids in the biomimetic brain matrix hydrogel exhibit larger vessel diameters, higher vessel density, longer total vessel length, and richer vascular branching. Furthermore, a detailed comparative analysis of the local vascular network structure of the vascular organoids in the two matrices was conducted. Figure 9As shown, in the central region of the vascular organoids, the biomimetic brain matrix hydrogel group exhibited a richer network structure and larger vessel diameter, while the Matrigel-Collagen matrix group showed a simpler network structure. Particularly at the angiogenesis tip of the vascular organoids, the biomimetic brain matrix group displayed a large number of tip-like cells, resulting in thicker and rounder vessel tips, while the Matrigel-Collagen matrix group exhibited relatively fewer tip-like cells, making its vessel tips appear thinner and softer. Therefore, the vascular cell composition at the outer edge of the vascular organoids was further investigated. Figure 10 As shown, by co-staining vascular organoids with CD31 (an endothelial cell marker) and α-SMA (a smooth muscle cell marker), it was found that in the angiogenesis front region of the biomimetic brain matrix group vascular organoids, endothelial cells were surrounded by smooth muscle cells, while no endothelial cells were observed at the vascular tip position in the Matrigel group.

[0110] The high expression of CD31 and increased vessel diameter in the biomimetic brain matrix hydrogel group indicate increased vascular maturity. The richer network structure, higher vessel density, and increased branching index suggest improved vascular connectivity. The presence of tip-cell-like cells at the vessel tips, as well as the co-localization of endothelial cells and smooth muscle cells, indicate active angiogenesis and vascular remodeling. These results suggest that the biomimetic brain matrix hydrogel provides a more favorable microenvironment for the development and maturation of vascular organoids.

[0111] Example 9

[0112] Biomimetic brain matrix hydrogels in conjunction with vascular organoids for the repair and treatment of cerebral infarction

[0113] This experiment used 8-12 week old, 22-24g C57BL / 6 male mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) as experimental subjects. After anesthesia with 1.0% inhaled isoflurane, focal cerebral infarction was induced in the cerebral cortex of the mice using photochemical embolization. Five days after modeling, the mice were randomly divided into four groups (n=6): PBS group (5μL PBS), BVOs group (5μL vascular organoids, approximately 10), Gel group (5μL biomimetic brain matrix hydrogel; the specific preparation method of the biomimetic brain matrix hydrogel is described in Example 5), and BVOs+Gel group (5μL biomimetic brain matrix hydrogel loaded with vascular organoids; the specific preparation method of the biomimetic brain matrix hydrogel is described in Example 5, approximately 10 vascular organoids). The corresponding substances were injected into the stroke cavity of each group using a microsyringe. To suppress immune rejection, cyclosporine was injected into the transplanted mice daily from the day before intracranial transplantation until the day of tissue collection. On day 14 post-modeling, laser speckle imaging was used to assess blood perfusion in the cerebral cortex of mice in each group. Figure 11As shown, the PBS group exhibited a clear infarct area with no blood flow perfusion, indicating that the infarct model was successfully established. Compared with the PBS group, the blood flow perfusion in the infarct area of ​​both the BVOs and Gel groups showed a certain degree of recovery, suggesting that the use of vascular organoids or biomimetic brain matrix hydrogels alone may have a certain therapeutic effect. In particular, the BVOs+Gel group showed a significant reduction in the infarct area and a more significant recovery in blood flow perfusion. Statistical analysis showed that the decrease in blood flow perfusion level in the infarct area relative to the contralateral normal brain region in the BVOs+Gel group was significantly lower than that in other groups.

[0114] Brain tissue was sectioned and immunofluorescence staining was used to further verify angiogenesis (CD31). + ) situation. Result as follows Figure 12 As shown, almost no angiogenesis was observed in the infarct area of ​​the PBS group, a small amount of angiogenesis was observed in the BVOs and Gel groups, while the BVOs+Gel group showed significant angiogenesis, as evidenced by significantly higher levels of key indicators such as vascular area, vascular density, total vascular length, and number of vascular branches compared to other groups. These results confirm that biomimetic brain matrix hydrogels promote the development and maturation of vascular organoids, thereby achieving in vivo vascular reconstruction. Figure 13 As shown.

[0115] To assess the impact of the aforementioned angiogenesis on the recovery of behavioral function in mice after cerebral infarction, several behavioral tests were performed on day 14 post-modeling. The results showed ( Figure 14 The PBS group (without treatment intervention) showed the most impaired motor function, while other treatment groups showed significant signs of functional recovery compared to the PBS group. In grid walking and rotating bar tests, the BVOs+Gel combined treatment group showed the most significant improvement. Figure 14 (A, B), although their performance still did not reach the level of the normal control group. To provide a more comprehensive functional analysis, weight-based grip strength and open field tests were also performed. Results showed that the BVOs+Gel group significantly outperformed all other treatment groups in grip strength and motor activity, with recovery levels approaching those of normal mice (e.g., A, B). Figure 14 (C, D). The above experimental results indicate that biomimetic brain matrix hydrogels, in conjunction with vascular organoids, can improve functional recovery after cerebral infarction by promoting vascular remodeling in the infarct area.

[0116] Comparative Example 2

[0117] Biomimetic hydrogels (without adhesive sequences) for in vitro culture of vascular organoids

[0118] Referring to Example 5, the GRGDSPY (adhesion sequence) was removed, and hydrogels were prepared using the same method. HiPSC cell aggregates were sequentially subjected to mesodermal-induced differentiation and angiogenesis-induced differentiation. On day 6, 500 μL of the prepared hydrogel was added to each well of a 12-well plate as a base, and the plate was incubated at 37°C for 2 hours to allow gelation. Then, the initially obtained vascular organoids were washed with DPBS, and 40-60 organoids per well were mixed with 500 μL of the prepared hydrogel and added to the plate. After incubation at 37°C for 2 hours until the hydrogel was completely gelled, 2 mL of vascular maturation culture medium was added to each well for three-dimensional induction culture. Light microscopy and immunofluorescence staining images showed that the budding and growth of the vascular organoids were significantly inhibited (e.g., ...). Figure 15 (The non-adhesive sequence set in the middle).

[0119] Comparative Example 3

[0120] Biomimetic hydrogels (poreless confined sequences) for in vitro culture of vascular organoids

[0121] Referring to Example 5, the GYRDGPQGIWGQDRYG (pore-confined sequence) was removed, and hydrogels were prepared using the same method. HiPSC cell aggregates were sequentially subjected to mesodermal-induced differentiation and angiogenesis-induced differentiation. On day 6, 500 μL of the prepared hydrogel was added to each well of a 12-well plate as a base, and the plate was incubated at 37°C for 2 hours to allow gelation. Then, the initially obtained vascular organoids were washed with DPBS, and 40-60 organoids per well were mixed with 500 μL of the prepared hydrogel and added to the plate. After incubation at 37°C for 2 hours until the hydrogel was completely gelled, 2 mL of angiogenesis maturation medium was added to each well for three-dimensional induction culture. Light microscopy and immunohistographs showed that the budding and growth of the vascular organoids were significantly inhibited (e.g., Figure 15 (The unconfined sequence group in the middle).

[0122] Through the above comparative examples, compared to traditional Matrigel-Collagen three-dimensional culture media, the biomimetic brain matrix hydrogel provided by this invention, with its framework composed of covalent / non-covalently assembled natural molecules and anchored functional sequences (adhesion sequences and pore-confining sequences), exhibits a stable overall structure, degradable internal pore space, and excellent bioactivity and biocompatibility. Combined with its suitable mechanical characteristics, the biomimetic brain matrix hydrogel can more effectively promote the growth and maturation of vascular organoids. Furthermore, in terms of in vivo promotion of vascular regeneration, the biomimetic brain matrix hydrogel, in conjunction with vascular organoids, can effectively promote angiogenesis in the cerebral infarction area.

[0123] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other way. Any person skilled in the art can make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A biomimetic brain matrix hydrogel for constructing mature vascular organoids, characterized in that, It comprises a skeleton component with a mass concentration of 1-10% and a bioactive polypeptide with a mass concentration of 0.02-1%; the biomimetic brain matrix hydrogel has a stiffness of 50-1000 Pa and a stress relaxation half-decay time of 1-10 s; the biomimetic brain matrix hydrogel has a pore size of 10-100 μm.

2. The biomimetic brain matrix hydrogel as described in claim 1, characterized in that, The scaffold component comprises natural protein, phenolic natural polysaccharide, and catalytic enzyme, wherein the mass ratio of the natural protein, phenolic natural polysaccharide, and catalytic enzyme is 1:0.1–10:0.001–0.1; wherein, The natural protein is any one of silk fibroin, collagen, fibrin, and serum albumin; the phenolic natural polysaccharide is obtained by phenolic modification of natural polysaccharide as substrate, and the theoretical modification rate of phenolic groups accounts for 5-20% of the natural polysaccharide; the catalytic enzyme is any one of cytochrome P450 enzyme, laccase, catechol oxidase, and catalase.

3. The biomimetic brain matrix hydrogel as described in claim 2, characterized in that, The method for preparing the phenolic natural polysaccharide is as follows: tyramine hydrochloride is added to an aqueous solution of the natural polysaccharide, stirred and dissolved, the pH is adjusted to 6-8, and a mixture of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in an equimolar ratio is added. The reaction pH is maintained at 6-8, and the mixture is stirred overnight at room temperature. The polysaccharide is then purified by dialyzing in pure water and freeze-dried to obtain the phenolic natural polysaccharide.

4. The biomimetic brain matrix hydrogel as described in claim 3, characterized in that, The natural polysaccharide is any one of hyaluronic acid, sodium alginate, carboxymethyl cellulose, and chondroitin sulfate; the molar ratio of tyramine hydrochloride to natural polysaccharide is 1 to 20:1; the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to natural polysaccharide is 1 to 10:

1.

5. The biomimetic brain matrix hydrogel as described in claim 1, characterized in that, The bioactive polypeptide comprises a mixture of adhesion sequences and pore-confining sequences; the mass ratio of the adhesion sequences to the pore-confining sequences is 1:0.1 to 1; the adhesion sequences provide active sites for the adhesion and growth of vascular organoids; the pore-confining sequences regulate the internal structure and mechanical properties of the gel to match the growth requirements of vascular organoids; wherein the adhesion sequences comprise any one or a mixture of two or more of SEQ ID NO.1-SEQ ID NO.3; and the pore-confining sequences comprise any one or a mixture of two or more of SEQ ID NO.4-SEQ ID NO.

6.

6. A method for preparing the biomimetic brain matrix hydrogel according to any one of claims 1-5, characterized in that, It includes the following steps: S1. Dissolve natural protein, phenolic natural polysaccharide and catalytic enzyme in aqueous solution, and mix under ice bath to obtain hydrogel framework preparation solution; S2. Add bioactive polypeptide sequences to the hydrogel framework preparation solution and mix well under ice bath to obtain the stock solution of biomimetic brain matrix hydrogel. S3. Adjust the pH of the biomimetic brain matrix hydrogel stock solution to 6-8 and let it stand at 37°C for 0.5-2 hours to obtain a hydrogel with a structure and mechanical modulus similar to that of the brain extracellular matrix.

7. The preparation method according to claim 6, characterized in that, The biomimetic brain matrix hydrogel stock solution has a hydrogel framework component with a mass concentration of 1-10%, containing natural proteins, phenolic natural polysaccharides, and catalytic enzymes in a mass ratio of 1:0.1-10:0.001-0.1; the bioactive polypeptide has a mass concentration of 0.02-1%, containing adhesion sequences and pore-confined sequences in a mass ratio of 1:0.1-1.

8. The application of the biomimetic brain matrix hydrogel according to any one of claims 1-5 or the biomimetic brain matrix hydrogel obtained by the preparation method according to claim 6 or 7 in the construction of blood vessels or vascular organoids.

9. The application of the biomimetic brain matrix hydrogel according to any one of claims 1-5 or the biomimetic brain matrix hydrogel obtained by the preparation method according to claim 6 or 7 in the preparation of organ-on-a-chip.

10. The application of the biomimetic brain matrix hydrogel according to any one of claims 1-5 or the biomimetic brain matrix hydrogel obtained by the preparation method according to claim 6 or 7, in conjunction with vascular organoids, in the preparation of vascular reconstruction and regenerative repair materials for cerebral infarction, spinal cord injury, and myocardial infarction.

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