Oxygen-enriched hydrogel for culturing vascular organs and preparation method of oxygen-enriched hydrogel

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to oxygen-enriched hydrogel for culturing vascular organs and a preparation method thereof.The preparation method comprises the steps that hyaluronic acid carboxyl and cysteine amino are covalently bound to obtain hyaluronic acid-cysteine conjugate HA-C, methacrylic anhydride and hydroxyl of an acellular matrix are subjected to an esterification reaction, and the hydrogel is obtained; the preparation method comprises the following steps: grafting methylacryloyl onto ECM (extracellular matrix) to obtain methylacryloyl modified acellular matrix ECM-MA; hA-C and ECM-MA are mixed and dissolved in a water phase, and a photoinitiator and PVP-coated calcium peroxide are added for cross-linking to form the oxygen-enriched hydrogel. The hydrogel is constructed by utilizing the sulfhydrylated hyaluronic acid and the double-bond functionalized acellular matrix by utilizing a click chemical reaction, and meanwhile, the calcium peroxide is added, so that the material has the function of releasing oxygen; the obtained hydrogel has biocompatibility, controllability and biological activity, and provides a high-performance material basis for biomedical application.

Description

TECHNICAL FIELD

[0001] The present application relates to an oxygen-enriched hydrogel for culturing vascular organoids and a preparation method thereof, and belongs to the technical field of biomedical materials. BACKGROUND

[0002] Vascular complications, including cardiovascular and cerebrovascular diseases, atherosclerosis, diabetic retinopathy, and nephropathy, are the main causes of death in the elderly and pose a serious challenge to public health. Therefore, research on vascular problems has become an important issue in addressing human health problems. However, the complex three-dimensional environment of blood vessels, the influence of cell crosstalk, and organ-specific structures make it extremely difficult to find a real in vitro model. In recent years, in vitro organoid models can effectively simulate the complex three-dimensional structure of in vivo organs. For example, 3D vascular organoids have become a powerful tool for studying vascular lesions and complications. Another outstanding advantage of vascular organoids is their ability to integrate with other types of organoids, thereby promoting the maturation and vascularization of other organoids, making them more similar to real organs. In the field of tissue engineering, it is expected to solve the problem of necrotic core formation in organoids due to limited oxygen and nutrient supply. Therefore, the development of 3D vascular organoids plays an important role in studying vascular complications and promoting the vascularization of organoids.

[0003] Currently, oxygen-enriched hydrogels reported in the literature are mainly used for wound repair. For example, Zhang et al. prepared a sodium alginate hydrogel dressing and added CaO2-containing polylactic acid microspheres and exosomes produced by bone marrow mesenchymal stem cells. The results showed that oxygen release can last for 7 days, effectively reducing the expression of hypoxia factors in fibroblasts and promoting the healing of diabetic wounds. Zou et al. synthesized an injectable oxygen-producing hydrogel using GelMA and loading CaO2, which can effectively alleviate the local anaerobic environment and eliminate periodontal pathogens. BO Chani et al. used chitosan and tannic acid as the main raw materials and added FeCl3 and MnO2 nanosheets to prepare a hydrogel dressing with antibacterial and oxygen release functions, which can effectively promote the deposition and maturation of collagen fibers, improve anti-inflammatory effects, and promote wound healing. Fu et al. prepared a phenylboronic acid-modified sodium alginate using a chemical synthesis method, then blended it with PVA to prepare an injectable hydrogel dressing with borate ester cross-linking. The addition of oxygen-releasing microspheres and active agents endows the hydrogel with ROS scavenging and oxygen release functions. Animal experiment results show that it can effectively promote the regeneration of acute and infected rat skin wound tissues. There is no report on the use of oxygen-enriched hydrogels for culturing vascular organoids and promoting their growth.

[0004] The material for culturing the vascular organ at present is mainly Matrigel derived from tumor tissue, which has problems of unknown exact composition, immunogenicity, high price, batch difference, and the like, and the morphology and internal microstructure of Matrigel are not conducive to the diffusion of nutrients and metabolic waste; and most of the synthetic hydrogels are natural or synthetic polymer materials, which have problems of poor mechanical properties, insufficient cell adhesion, and insufficient diffusion of oxygen and nutrients, and are rarely used for culturing the vascular organ, and the present application constructs a gel network by using click chemistry based on the porcine spleen acellular matrix and hyaluronic acid, constructs a hydrogel material, and uses the hydrogel material for culturing the vascular organ. SUMMARY

[0005] (One) technical problem to be solved

[0006] In order to solve the above problems of the prior art, the present application provides an oxygen-rich hydrogel material for culturing a vascular organ and a preparation method thereof, which can be used for culturing the vascular organ and promoting the growth of the vascular organ.

[0007] (Two) technical scheme

[0008] In order to achieve the above purpose, the main technical scheme adopted by the present application includes:

[0009] In a first aspect, the present application provides an oxygen-rich hydrogel material for culturing a vascular organ, which comprises a gel matrix, and the gel matrix is a composite material produced by crosslinking hyaluronic acid-cysteine conjugate, methacryl-modified acellular matrix, and PVP-coated calcium peroxide under the action of an initiator. Further, the initiator is a photoinitiator, and the photoinitiator forms the oxygen-rich hydrogel material in a PBS solution under ultraviolet light irradiation. The product has adjustable mechanical properties and oxygen release function.

[0010] In a second aspect, the present application further provides a preparation method of the oxygen-rich hydrogel material for culturing the vascular organ, which comprises the following steps:

[0011] S1, dissolving hyaluronic acid and an active agent in a MES buffer solution, stirring and dissolving to form a solution I, and activating to form a solution I;

[0012] S2, dissolving L-cysteine in a MES buffer solution, stirring and dissolving to form a solution II; then adding the solution II into the solution I, and reacting at room temperature; after the reaction is completed, the solution is dialyzed, and then freeze-dried to obtain a product, which is denoted as HA-C;

[0013] S2, decellularized matrix modification: take the animal decellularized matrix and configure into a decellularized matrix hydrogel solution, add methacrylic anhydride at room temperature, adjust the pH to be alkaline, stir the reaction at low temperature, neutralize with acid after the reaction is completed, adjust the pH to physiological pH, and freeze-dry the solution after dialysis to obtain a white sponge product, denoted as PECM-MA;

[0014] S3, hydrogel preparation: take HA-C and PECM-MA mixed and dissolved in an aqueous phase, add a photoinitiator and PVP-coated calcium peroxide, and crosslink under light to obtain a hydrogel.

[0015] The order of steps S1 and S2 is not limited.

[0016] As described above, preferably, in step S1, the concentration of hyaluronic acid in the solution I is 0.001-0.02 g / mL; the concentration of L-cysteine in solution II is 1-10 mg / mL; the concentration of the MES buffer solution is 0.01-0.2 mol / L; and the pH value is 5-7.

[0017] As described above, preferably, in step S1, the active agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and the concentration of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in the solution I is independently 1-10 mg / mL, and the activation time is 20-60 min.

[0018] The reaction time at room temperature is 12-24 h, the dialysis uses a dialysis membrane with a molecular weight cut-off of 8000 Da, and the dialysis time is 2-4 d.

[0019] As described above, preferably, in step S2, the concentration of the decellularized matrix hydrogel is 3-20 mg / mL, and the methacrylic anhydride and the decellularized matrix hydrogel are slowly added at a volume ratio of 5-15 μL: 1 mL.

[0020] As described above, preferably, in step S2, the way to adjust the pH to be alkaline is to adjust with a sodium hydroxide solution or a potassium hydroxide solution to a pH value of 9-10; and after the reaction is completed, the hydrochloric acid is neutralized to a physiological pH value of 7.2-7.4.

[0021] As described above, preferably, in step S2, the low temperature is 2-8℃, and the reaction time is 6-24 h.

[0022] The preparation method described above, preferably, in step S2, the dialysis adopts a dialysis bag with a molecular weight cut-off of 8000 Da, the dialysis time is 2-4 days, and the dialysis is carried out at low temperature.

[0023] The preparation method described above, preferably, in step S3, the HA-C and the PECM-MA are mixed and dissolved in PBS, wherein the concentration of the PECM-MA is 0.01-0.05 g / mL, and the concentration of the HA-C is 0.01-0.05 g / mL.

[0024] The addition amount of the photoinitiator in the reaction system is 0.001-0.005 g / mL, and the addition amount of the PVP-coated calcium peroxide in the reaction system is 0.1-10 mg / mL.

[0025] The preparation method described above, preferably, the photoinitiator is LAP, and the light irradiation condition is ultraviolet light, and the ultraviolet light irradiation time is 20-60 s. The present application uses the photoinitiator to initiate the thiol (-SH) provided by the hyaluronic acid-cysteine conjugate and the carbon-carbon double bond (C=C) provided by the methyl methacryl-modified decellularized matrix to carry out a thiol-ene click reaction, so as to realize rapid cross-linking and solidification of the hydrogel.

[0026] (III) Beneficial effects

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

[0028] The oxygen-rich hydrogel material provided by the present application has stable appearance and shape, the storage modulus is greater than the loss modulus, the frequency does not affect the gel state, the vascular organ grows well, and the endothelial cell sprouting state is good. The hydrogel material formed by the addition reaction of thiol and double bond and the disulfide bond cross-linking between thiols has certain viscoelasticity, meanwhile, the presence of the decellularized matrix can enhance cell adhesion, the presence of the nanoparticles can slowly release oxygen, on the other hand, the components of the decellularized matrix are mainly collagen, and the three-dimensional structure of the formed hydrogel is beneficial to the diffusion of small molecules, thus, the problems of poor mechanical properties, insufficient cell adhesion and insufficient diffusion of oxygen and nutrients are solved to a certain extent.

[0029] The hydrogel combines the moisturizing property of hyaluronic acid and the biological activity of the decellularized matrix, has good biocompatibility, degradability and mechanical properties, the photo-crosslinking method can accurately control the gelation time and shape, and the oxygen released by the calcium peroxide can improve the local microenvironment, such as promoting cell survival in wound healing, and is suitable for the biomedical field such as vascular organ, tissue engineering scaffold, wound dressing, drug delivery carrier and the like.

[0030] The application provides a preparation method of the oxygen-enriched hydrogel material, which utilizes a click chemistry reaction, constructs a hydrogel by using thiolated hyaluronic acid and double bond functionalized decellularized matrix, and adds calcium peroxide to make the material have an oxygen releasing function; the whole reaction process is realized by chemical modification such as grafting of a functional group and crosslinking reaction, so that the natural biological material (hyaluronic acid, decellularized matrix) is converted into a composite hydrogel with specific functions, and the biological compatibility, controllability and biological activity are considered, thereby providing a high-performance material basis for biomedical applications. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 A gelation schematic diagram of the oxygen-enriched hydrogel material prepared in the examples of the application;

[0032] Figure 2 A modulus-amplitude test diagram of the gel;

[0033] Figure 3 A modulus-frequency test diagram of the gel;

[0034] Figure 4 A growth schematic diagram of the gel for culturing blood vessel-like devices for different time. DETAILED DESCRIPTION

[0035] The application provides an oxygen-enriched hydrogel material, which is prepared by first aminoacylating hyaluronic acid to obtain a hyaluronic acid-cysteine conjugate (HA-C), that is, hyaluronic acid grafted and modified by cysteine; methyl methacrylate anhydride is subjected to esterification reaction with hydroxyl groups of a decellularized matrix (ECM) to graft a methacryl group (containing a carbon-carbon double bond, C=C) to the ECM molecule to obtain a methacryl-modified decellularized matrix (ECM-MA); the hyaluronic acid-cysteine conjugate is mixed and dissolved with the ECM-MA, and a light initiator and PVP-coated calcium peroxide are added to crosslink to form the oxygen-enriched hydrogel.

[0036] The EDC used in the application is a carbodiimide type activator, which can be subjected to an activation reaction with carboxyl groups (-COOH) in a hyaluronic acid (HA) molecule to generate an unstable O-acylisourea intermediate; the NHS is used as an auxiliary reagent, which is subjected to a reaction with the intermediate to generate a more stable NHS ester (-COO-NHS), so that a hydrolysis byproduct such as direct reaction of the carboxyl group with the amino group is reduced.

[0037] The amino group (-NH2) in the L-cysteine molecule is used as a nucleophilic reagent, which is subjected to a substitution reaction with the NHS ester of the HA to form an amide bond (-CO-NH-), so that the cysteine is grafted to the HA molecular chain to occur an amidation reaction, and the HA-C is obtained. Unreacted EDC, NHS, cysteine and other small molecular impurities are removed by dialysis, and the solid-state HA-C is obtained by freeze-drying.

[0038] The decellularized matrix used in the application is preferably a pig spleen decellularized matrix (PECM), which mainly contains biological macromolecules such as collagen and glycosaminoglycan, and is rich in hydroxyl groups (-OH); the acyl groups (-COO-) in methacrylic anhydride (MA) undergo esterification with the hydroxyl groups of ECM under alkaline conditions (pH 9-10, adjusted by sodium hydroxide), and methacryl groups (containing carbon-carbon double bonds, C=C) are grafted onto the ECM molecules. Dilute hydrochloric acid is used to neutralize the excess base, adjust the pH to the physiological range (~ 7.4), dialysis (molecular weight cutoff 8000 Da) to remove unreacted MA and small molecular impurities, and freeze-drying to obtain white sponge-like PECM-MA. HA-C contains sulfhydryl groups (-SH), and PECM-MA contains carbon-carbon double bonds (C=C). Under the action of a photoinitiator, ultraviolet light excitation generates free radicals, which initiate the "thiol-ene click reaction" between the sulfhydryl groups and the double bonds, forming a covalent cross-linked network; PVP-coated calcium peroxide can slowly release oxygen, reducing the impact of internal hypoxia on cells, while PVP enhances the dispersibility and stability of calcium peroxide, ultimately obtaining an oxygen-rich hydrogel. In addition, the carbonyl oxygen of PVP chelates CaO2 decomposed by CaO2 to generate Ca 2+ , preventing the formation of Ca(OH)2 precipitate (avoiding local pH > 9.0), improving the rheological properties of the gel, and improving the storage modulus, and PVP shields the surface active oxygen (ROS) burst of CaO2, improving cell survival rate.

[0039] The material obtained by the application can be used for culturing vascular organoids, and the oxygen-releasing hydrogel for culturing vascular organoids.

[0040] The application also provides a preparation method of the oxygen-rich hydrogel material, which comprises the following steps:

[0041] S1, dissolving hyaluronic acid and an active agent in a MES buffer solution, stirring to dissolve, and activating for 20-60 min to form solution I;

[0042] L-cysteine is dissolved in the same concentration of MES buffer solution in a beaker, stirred to dissolve, and then solution II is added to solution I, and reacted at room temperature for 12-24 h. After the reaction is completed, the solution is dialyzed, and then freeze-dried to obtain a product, which is denoted as HA-C;

[0043] S2, modification of the decellularized matrix: take pig spleen decellularized matrix (other animal decellularized matrix can also be used) and configure it into a hydrogel solution, then add methacrylic anhydride under room temperature, adjust the pH to alkaline, stir in a refrigerator at 4℃ overnight, neutralize with acid after the reaction is completed, adjust the pH to physiological pH, dialyze, and then freeze-dry the solution to obtain a white sponge-like product, which is denoted as PECM-MA;

[0044] S3, hydrogel preparation: HA-C and PECM-MA mixed solution was taken, and light initiator and PVP-coated calcium peroxide were added, and finally cross-linked to form a hydrogel.

[0045] In one embodiment, preferably in step S1, the concentration of hyaluronic acid in solution I is 0.001-0.02 g / mL; the concentration of L-cysteine in solution II is 1-10 mg / mL; the concentration of MES buffer solution is 0.01-0.2 mol / L; and the pH is 5-7.

[0046] Preferably, the active agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and the concentration of EDC and NHS in solution I is 1-10 mg / mL.

[0047] In one embodiment, preferably in step S2, the concentration of the decellularized matrix hydrogel is 3-20 mg / mL, and methyl methacrylate anhydride is slowly added to the decellularized matrix hydrogel at a volume ratio of 5-15:1.

[0048] In one embodiment, preferably in step S2, the pH is adjusted to be alkaline using a sodium hydroxide solution or a potassium hydroxide solution, and the pH is 9-10; the acid is hydrochloric acid, and the physiological pH value is 7.2-7.4.

[0049] In one embodiment, preferably in step S2, the low temperature is 2-8°C, and the reaction time is 6-24 h.

[0050] In one embodiment, preferably in step S2, dialysis is performed using a dialysis bag with a molecular weight of 8000 Da, and the dialysis time is 2-4 d. The dialysis is performed at low temperature, preferably 2-8°C.

[0051] In one embodiment, preferably in step S3, the solution in the hydrogel uses a PBS solution, and PECM-MA is added to the hydrogel at a concentration of 0.01-0.05 g / mL; HA-C is added to the hydrogel at a concentration of 0.01-0.05 g / mL; the light initiator is added to the hydrogel at a concentration of 0.001-0.005 g / mL; and the concentration of PVP-coated calcium peroxide is 0.1-10 mg / mL.

[0052] In one embodiment, preferably the light initiator is LAP.

[0053] In one embodiment, preferably in step S3, the cross-linking to form a hydrogel is after ultraviolet light irradiation.

[0054] Further, the ultraviolet light irradiation time is 20-60 s.

[0055] The hyaluronic acid used in the preparation method of the application is preferably used as the core raw material (matrix material) of the reaction to provide carboxyl (-COOH) sites, laying a foundation for subsequent grafting reaction with cysteine; the MES buffer solution is preferably used to provide a stable weakly acidic reaction environment, which is suitable for EDC / NHS-mediated carboxyl activation reaction, reduces the hydrolysis loss of EDC, and improves the reaction efficiency, and the working concentration thereof is preferably 0.01-0.2 mol / L, and the pH value thereof is preferably 5-7.

[0056] The 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) used in the application is used as a carboxyl activator, reacts with the carboxyl of HA to generate an unstable O-acyl isourea intermediate, and is an activated site for subsequent reaction with an amino group; N-hydroxysuccinimide (NHS) reacts with the carboxyl intermediate activated by EDC to generate a more stable NHS ester (-COO-NHS), reduces the hydrolysis rate of the intermediate, improves the specificity and efficiency of the reaction with the amino group, and reduces the side reaction; the concentration of EDC and NHS in the reaction is preferably 1-10 mg / mL.

[0057] The decellularized matrix used in the application is preferably a pig spleen decellularized matrix (PECM), which is a natural bioactive matrix and retains collagen, glycosaminoglycan, growth factors and other components in the tissue, provides biological signals required for cell adhesion and proliferation, and the concentration of the hydrogel prepared from the pig spleen decellularized matrix is preferably 3-20 mg / mL; and the concentration is most preferably 5-15 mg / mL.

[0058] The role of methacrylic anhydride is to provide a methacryl group (containing a carbon-carbon double bond, C=C) to graft the hydroxyl group (-OH) of PECM through esterification reaction, so that PECM obtains photo-crosslinking ability; methacrylic anhydride is slowly added to the decellularized matrix hydrogel at a volume ratio of 5-15:1. The pH value of the reaction system of the decellularized matrix modification is adjusted to an alkaline environment to promote the esterification reaction of methacrylic anhydride and the hydroxyl group in PECM, and the alkaline condition can enhance the nucleophilicity of the hydroxyl group, and the pH value is further preferably 9-10.

[0059] After the reaction is completed, acid is used for neutralization, mainly to neutralize the excess sodium hydroxide, adjust the pH value of the reaction system to the physiological range of 7.2-7.4, and avoid the damage of strong alkalinity to the biological activity of PECM.

[0060] The dialysis bag with a molecular weight cut-off of 8000 Da is preferably used in the preparation method of the application to remove unreacted methacrylic anhydride and small molecule byproducts, purify PECM-MA, and at the same time retain the macromolecular matrix components after modification. Stirring overnight is preferably carried out in a low-temperature environment to reduce the volatilization of methacrylic anhydride and the degradation of PECM, and to ensure that the reaction proceeds gently; low-temperature dialysis further stabilizes the product structure.

[0061] Further, the PECM-MA obtained by the freeze-drying treatment is white and spongy, and retains a porous structure, which is beneficial to subsequent swelling and loading of functional components (e.g., drugs and cells).

[0062] The preparation method provided by the application and the obtained hyaluronic acid-cysteine conjugate (HA-C) provide a thiol group (-SH) as a nucleophile for a cross-linking reaction to react with a carbon-carbon double bond of the PECM-MA to form a covalent network; and the methacryl-modified decellularized matrix (PECM-MA) is used to provide a carbon-carbon double bond (C=C) as an electrophile for a cross-linking reaction to react with the thiol group of the HA-C; at the same time, the hydrogel is endowed with the biological activity of a natural tissue; the photoinitiator generates free radicals under the excitation of ultraviolet light to initiate a "thiol-ene click reaction" between the thiol group (-SH) and the carbon-carbon double bond (C=C), so as to realize the rapid cross-linking and solidification of the hydrogel. The PVP-coated calcium peroxide, in which the calcium peroxide is used to slowly release oxygen (react with water to generate O2), improves the hypoxic environment inside the hydrogel and promotes cell survival, and is particularly suitable for tissue engineering; and the PVP (polyvinylpyrrolidone) is used as a coating material to enhance the dispersibility, stability and biocompatibility of the calcium peroxide and avoid direct reaction of the calcium peroxide with biomolecules.

[0063] In the application, the substances are synergistically combined through the logic of "activation-grafting-crosslinking", the small molecule reagents EDC, NHS and methacrylic anhydride are responsible for the introduction of functional groups, the natural biomaterials HA and decellularized matrix provide the matrix skeleton and biological activity, the post-processing steps of dialysis and freeze-drying ensure the purity and stability of the product, and finally the cross-linking reaction and the functional additive PVP-coated calcium peroxide endow the hydrogel with adjustable physical properties and biological functions, so that the oxygen-rich hydrogel is obtained.

[0064] In order to better explain the application and facilitate understanding, the application is described in detail below by means of specific embodiments in combination with the drawings. The reagents used in the embodiments of the application can be commercially available products, such as hyaluronic acid, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, MES buffer solution, L-cysteine and LAP photoinitiator, which can be purchased from Shanghai McLean Biochemical Reagent Co., Ltd.; the PVP-coated calcium peroxide can be purchased from Beijing Zhongke Keyou Technology Co., Ltd.; and the pig spleen decellularized matrix is from the Chongqing Organ Intelligent Biological Manufacturing Engineering Research Center.

[0065] Example 1

[0066] S1, in a 100 mL beaker, 0.5 g of hyaluronic acid, 0.4 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), 0.25 g of N-hydroxysuccinimide (NHS) and 50 mL of MES buffer solution with a concentration of 0.02 M (pH = 5.5) were added, stirred and dissolved to form solution I and transferred to a 100 mL two-necked flask, and stirring was continued for 45 min of activation; in another 100 mL beaker, 0.2 g of L-cysteine and 20 mL of MES buffer solution with a concentration of 0.02 M (pH = 5.5) were added, stirred and dissolved to form solution II; finally, solution II was transferred to the two-necked flask, and reaction was carried out under stirring at 25°C for 24 h; then the reacted solution was collected and dialyzed in water with a dialysis membrane with a molecular weight cut-off of 8000 KDa for 3 days, and then the product was obtained after freeze-drying, and was named as HA-C-1;

[0067] S2, the pig spleen decellularized matrix was prepared into a hydrogel solution with a concentration of 15 mg / mL using PBS buffer solution, then 15 mL of the hydrogel solution was taken and 20 mL of deionized water was added and stirred uniformly, then 225 μL of methacrylic anhydride was slowly dropped at room temperature, then the pH was adjusted to 9-10 using sodium hydroxide solution, and stirring was carried out in a refrigerator at 4°C overnight, after the reaction was completed, neutralization was carried out using dilute hydrochloric acid, the pH was adjusted to a physiological pH value of 7.2-7.4, finally, dialysis was carried out in a dialysis bag with a molecular weight cut-off of 8000 Da at 4°C for 4 days, and finally the solution was freeze-dried to obtain a white sponge-like product (named as PECM-MA-1).

[0068] Example 2

[0069] S1, in a 100 mL beaker, 0.5 g of hyaluronic acid, 0.4 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, 0.25 g of N-hydroxysuccinimide and 50 mL of MES buffer solution with a concentration of 0.02 M (pH = 5.5) were added, stirred and dissolved to form solution I and transferred to a 100 mL two-necked flask, and stirring was continued for 45 min of activation; in another 100 mL beaker, 0.4 g of L-cysteine and 20 mL of MES buffer solution with a concentration of 0.02 M (pH = 5.5) were added, stirred and dissolved to form solution II; finally, solution II was transferred to the two-necked flask, and reaction was carried out under stirring at 25°C for 24 h; then the reacted solution was collected and dialyzed in water with a dialysis membrane with a molecular weight cut-off of 8000 KDa for 3 days, and then the product was obtained after freeze-drying, and was named as HA-C-2;

[0070] S2, take pig spleen decellularized matrix and prepare hydrogel solution with PBS buffer to a concentration of 15 mg / mL, then take 15 mL of the hydrogel solution and add 20 mL of deionized water to stir evenly, then slowly drop 225 μL of methacrylic anhydride at room temperature, then adjust the pH to 9-10 with sodium hydroxide solution, stir overnight in a 4°C refrigerator, after the reaction is completed, neutralize with dilute hydrochloric acid, adjust the pH to the physiological pH range of 7.2-7.4, finally dialyze the solution at 4°C for 4 days with a dialysis bag with a molecular weight cut-off of 8000 Da, and finally freeze-dry the solution to obtain a white sponge-like product (named PECM-MA-1).

[0071] Example 3

[0072] S1, in a 100 mL beaker, add 0.5 g of hyaluronic acid, 0.4 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, 0.25 g of N-hydroxysuccinimide and 50 mL of 0.02 M concentration MES buffer solution (pH = 5.5), stir to dissolve to form solution I and transfer to a 100 mL two-neck flask, continue to stir and activate for 45 min; in another 100 mL beaker, add 0.2 g of L-cysteine and 20 mL of 0.02 M concentration MES buffer solution (pH = 5.5), stir to dissolve to form solution II; finally, transfer solution II to the two-neck flask and react under stirring at 25°C for 24 h; then collect the reacted solution and dialyze in water for 3 days with a dialysis membrane with a molecular weight cut-off of 8000 KDa, then freeze-dry to obtain the product, named HA-C-1;

[0073] S2, take pig spleen decellularized matrix and prepare hydrogel solution with PBS buffer to a concentration of 15 mg / mL, then take 15 mL of the hydrogel solution and add 20 mL of deionized water to stir evenly, then slowly drop 225 μL of methacrylic anhydride at room temperature, then adjust the pH to 9-10 with sodium hydroxide solution, stir overnight in a 4°C refrigerator, after the reaction is completed, neutralize with dilute hydrochloric acid, adjust the pH to the physiological pH range of 7.2-7.4, finally dialyze the solution at 4°C for 4 days with a dialysis bag with a molecular weight cut-off of 8000 Da, and finally freeze-dry the solution to obtain a white sponge-like product (named PECM-MA-1).

[0074] Example 4

[0075] In a 100 mL beaker, add 0.5 g of hyaluronic acid, 0.4 g of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, 0.25 g of N-hydroxysuccinimide and 50 mL of MES buffer solution with a concentration of 0.02 M (pH = 5.5), stir to dissolve to form solution I and transfer to a 100 mL two-necked flask, continue to stir and activate for 45 min; in another 100 mL beaker, add 0.4 g of L-cysteine and 20 mL of MES buffer solution with a concentration of 0.02 M (pH = 5.5), stir to dissolve to form solution II; finally, transfer solution 2 to the two-necked flask and react under stirring at 25°C for 24 h; then collect the reacted solution and dialyze for 3 days, then freeze-dry to obtain the product, named HA-C-2; take the porcine spleen decellularized matrix and configure it into a hydrogel solution with a concentration of 15 mg / mL, then take 15 mL of the hydrogel solution and add 20 mL of deionized water to stir uniformly, then slowly drop 450 μL of methacrylic anhydride at room temperature, then adjust the pH to between 9 and 10 with sodium hydroxide solution, stir overnight in a 4°C refrigerator, neutralize with dilute hydrochloric acid after the reaction is completed, adjust the pH to physiological pH, and finally dialyze in water with a molecular weight cut-off of 8000 Da at 4°C for 4 days, and then freeze-dry the solution to obtain a white sponge-like product, named PECM-MA-2.

[0076] Example 5

[0077] Take a certain amount of HA-C-2 and PECM-MA-2 and dissolve them in PBS solution to configure HA-C-2 and PECM-MA-2 with concentrations of 0.02 g / mL and 1 mL of volume, then add a photoinitiator LAP with a concentration of 0.0025 g / mL, then add PVP-coated calcium peroxide (CPO) nanoparticles with a concentration of 1 mg / mL, stir uniformly, and then irradiate with 405 nm ultraviolet light for 30 s to form a gel.

[0078] The gel sample test data in Example 5 are as follows:

[0079] Figure 1 The schematic diagram of the hydrogel after gelation is shown in the figure, and the results show a stable colloidal appearance;

[0080] The rheological test of the hydrogel after gelation is tested using a rheometer, model: TA Discovery HR-1, USA, and the results are as follows: Figure 2 and Figure 3 The schematic diagrams of the modulus and deformation, and the modulus and frequency, respectively, show that within the low strain and test frequency range, the storage modulus (G') of the gel > the loss modulus (G''), which meets the colloidal characteristics.

[0081] Example 6

[0082] Hydrogel preparation: Take a certain amount of HA-C-1 and PECM-MA-1 mixed and dissolved in PBS solution, and prepare HA-C-2 and PECM-MA-2 with concentrations of 0.02 g / mL respectively, take 1 mL of volume, then add the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) with a concentration of 0.0025 g / mL, then add PVP-coated calcium peroxide (CPO) with a concentration of 1 mg / mL, stir uniformly, then irradiate with 405 nm ultraviolet light for 30 s to form a gel.

[0083] The gel was used to culture vascular organoids, and the results were as follows Figure 4 As shown, the vascular organoids developed well and formed many strip-shaped microvessels. Specifically, the human-derived induced pluripotent stem cells were photographed after being cultured into a ball in the well plate (1d), then they were placed in the hydrogel for culture, and were photographed regularly (3, 5, 7, 9d) until budding, and the budding density and length of the newly formed blood vessels were observed, Figure 4 The growth of the vascular organoids in the gel in Example 5 is shown in the schematic diagram, and it can be seen from the diagram that the vascular organoids grow well, and the endothelial cell budding state is good.

[0084] The above is only a preferred embodiment of the present application, and is not intended to limit the present application in other forms. Any person skilled in the art can modify or change the above disclosed technical content to equivalent embodiments. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present application, which does not deviate from the technical solution content of the present application, still belongs to the protection scope of the technical solution of the present application.

Claims

1. An oxygen-rich hydrogel for culturing vascular organoids, characterized in that, It comprises a gel matrix which is a composite material produced by cross-linking of hyaluronic acid-cysteine conjugate, methylacryloyl modified decellularized matrix and PVP coated calcium peroxide under the action of initiator.

2. A method for preparing an oxygen-rich hydrogel for culturing vascular organoids, characterized by, It comprises the following steps: S1, dissolve hyaluronic acid and active agent in MES buffer solution, after stirring and dissolving, activate to form solution I; Dissolve L-cysteine in MES buffer solution, stir and dissolve to form solution II; Then add solution II to solution I, react at room temperature, after the reaction is completed, dialyze the solution, then freeze-dry to obtain the product, denoted as HA-C; S2, decellularized matrix modification: take animal decellularized matrix and configure into decellularized matrix hydrogel solution, add methacrylic anhydride under room temperature conditions, adjust pH to alkaline, stir and react under low temperature, after the reaction is completed, neutralize with acid, adjust pH to physiological pH, after dialysis, freeze-dry the solution to obtain white sponge-like product, denoted as PECM-MA; The order of steps S1 and S2 is not limited; S3, hydrogel preparation: dissolve HA-C and PECM-MA in aqueous phase, add photoinitiator and PVP coated calcium peroxide, cross-link under light to obtain hydrogel.

3. The production method according to claim 2, wherein In step S1, the concentration of hyaluronic acid in solution I is 0.001-0.02 g / mL; the concentration of L-cysteine is 1-10 mg / mL; the concentration of MES buffer solution is 0.01-0.2 mol / L; the pH value is 5-7.

4. The production method according to claim 2, wherein In step S1, the active agent is 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide, and the concentration of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide in solution I is independently 1-10 mg / mL, and the activation time is 20-60 min; The reaction time at room temperature is 12-24 h, the dialysis adopts a dialysis membrane with a molecular weight cut-off of 8000 Da, and the dialysis time is 2-4 d.

5. The production method according to claim 2, wherein In step S2, the concentration of decellularized matrix hydrogel is 3-20 mg / mL, and methacrylic anhydride is slowly added to the decellularized matrix hydrogel at a volume ratio of 5-15 μL:1 mL.

6. The production method according to claim 2, wherein The decellularized matrix is pig spleen decellularized matrix; the method for adjusting pH to alkaline is adjusting with sodium hydroxide solution or potassium hydroxide solution to pH 9-10; after the reaction is completed, neutralize with hydrochloric acid, and the physiological pH value is 7.2-7.

4.

7. The production method according to claim 2, wherein In step S2, the low temperature is 2-8℃, and the reaction time is 6-24 h.

8. The production method according to claim 2, wherein In step S2, the dialysis adopts a dialysis bag with a molecular weight cut-off of 8000 Da, and the dialysis time is 2-4 d, and the dialysis is carried out at low temperature.

9. The production method according to claim 2, wherein In step S3, dissolve HA-C and PECM-MA in PBS solution, wherein the concentration of PECM-MA is 0.01-0.05 g / mL, and the concentration of HA-C is 0.01-0.05 g / mL; The amount of the photoinitiator added in the reaction system is 0.001-0.005 g / mL, and the amount of the PVP-coated calcium peroxide added in the reaction system is 0.1-10 mg / mL.

10. The production method according to claim 2, wherein The photoinitiator is LAP, the light irradiation condition is ultraviolet light, and the irradiation time is 20-60 s.

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