Dynamic-covalent hybrid cross-linked hydrogel material as well as preparation method and application thereof

Abstract

The invention relates to a dynamic-covalent hybrid cross-linked hydrogel material as well as a preparation method and application thereof. The preparation method comprises the following steps: dissolving a component A-phenylboronic acid modified polymer derivative in a biocompatible medium to obtain a solution A; dissolving a component B, namely a macromolecular derivative containing a vicinal diol group, into a biocompatible medium to obtain a solution B; dissolving a component C-double bond group modified polymer derivative in a biocompatible medium to obtain a solution C; and mixing the solution A, the solution B and the solution C with certain concentrations for a certain time, and performing illumination under a photoinitiator for a certain time to obtain the dynamic-covalent hybrid cross-linked hydrogel material. Compared with single dynamic bond crosslinking or covalent bond crosslinking, the prepared hydrogel material has machinability and mechanical stability; the hydrogel material has plasticity, shear thinning, self-repairing property and the like in the early stage of crosslinking; enough mechanical strength and long-term stability are realized in the later stage of crosslinking.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomaterials, relates to a hydrogel material, and particularly relates to a dynamic-covalent hybrid cross-linked hydrogel material and a preparation method and application thereof. Background Art

[0002] As an ideal biomedical material, hydrogel materials are widely used in the fields of drug sustained release, tissue engineering and regenerative medicine, bioprinting, cell biology, etc. due to their excellent biocompatibility, suitable mechanical properties, and highly fitting biological tissue microenvironment. From the cross-linking method, the chemical cross-linking methods of hydrogel materials are divided into covalent bond cross-linking and dynamic covalent bond cross-linking. Covalent bond cross-linking reactions such as Michael addition reaction cross-linking, amide condensation reaction cross-linking, photoinitiated free radical polymerization cross-linking, etc. However, the cross-linking speed is difficult to control whether it is too fast or too slow, and the processability is poor. Among them, photoinitiated free radical polymerization cross-linking reaction has attracted extensive attention of researchers due to its advantages such as non-physical contact and spatio-temporal controllability during the gelation process. Another important chemical cross-linking method is dynamic covalent bond cross-linking reaction, such as phenylborate ester bond, Schiff base bond, disulfide bond, etc. Although it has excellent operability such as plasticity, shear thinning, and self-healing during the initial stage of cross-linking, it generally lacks sufficient mechanical strength and long-term stability during the later stage of cross-linking. Among them, the phenylborate ester bond has many advantages such as mild cross-linking conditions, strong reversibility of cross-linking bonds, and structural modifiability, and is a currently widely used dynamic covalent bond cross-linking method. However, neither phenylborate dynamic covalent cross-linking nor photoinitiated free radical polymerization covalent cross-linking can balance processability and mechanical stability. If the advantages of both dynamic covalent bond cross-linking and covalent bond cross-linking can be utilized, it is expected to prepare a hydrogel material with both processability and mechanical stability. Summary of the Invention

[0003] In order to overcome the deficiencies of single dynamic bond cross-linking and covalent bond cross-linking, the present invention proposes a dynamic-covalent hybrid cross-linked hydrogel material and a preparation method and application thereof.

[0004] The object of the present invention can be achieved by the following technical solutions:

[0005] The first object of the present invention is to provide a preparation method of a dynamic-covalent hybrid cross-linked hydrogel material.

[0006] The preparation method of the dynamic-covalent hybrid cross-linked hydrogel material of the present invention includes the following steps:

[0007] Dissolve component A - phenylboric acid-modified polymer derivative in a biocompatible medium to obtain solution A;

[0008] Dissolve component B - polymer derivative containing vicinal diol groups in a biocompatible medium to obtain solution B;

[0009] Dissolve the component C - double bond group - modified polymer derivative in a biocompatible medium to obtain solution C;

[0010] Mix a certain concentration of solution A, solution B, and solution C for a certain period of time and irradiate with light under a photoinitiator for a certain period of time to obtain the dynamic - covalent hybrid cross - linked hydrogel material.

[0011] In one embodiment of the present invention, the component A - phenylboronic acid - modified polymer derivative has a structure as shown in formula I:

[0012]

[0013] Among them, n≥2, that is, the average number of phenylboronic acid groups (the structure in parentheses in formula I) on a single P1 polymer chain is greater than or equal to 2;

[0014] R1, R2, R3, R4, R5 are independently selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl group, mercapto group, amino group, nitro group, cyano group, aldehyde group, ketone group, carboxyl group, ester group, amide group, aryl group, alkyl group, modified alkyl group, etc.

[0015] Furthermore, the alkyl group is a saturated or unsaturated aliphatic straight - chain or branched - chain alkyl group having 1 - 20 carbon atoms.

[0016] The modified alkyl group is such that any carbon atom of the alkyl group is replaced by one selected from - F, - Cl, - Br, - I, - OH, - SH, - NH2, - NO2, - CN, - CHO, - COOH, ester group, amide group, aryl group, etc. The modified alkyl group has 1 - 20 carbon atoms, and its carbon - carbon single bond can be arbitrarily replaced by a carbon - carbon double bond or a carbon - carbon triple bond.

[0017] In one embodiment of the present invention, preferably, in the structure of formula I, at least two of R1, R2, R3, R4, R5 are connected to each other to form a saturated or unsaturated alicyclic or heteroalicyclic ring with carbon atoms, or form an aromatic ring or heteroaromatic ring.

[0018] The alicyclic ring is a saturated or unsaturated 3 - 10 - membered monocyclic or polycyclic alicyclic ring.

[0019] The heteroalicyclic ring is a saturated or unsaturated 3 - 10 - membered monocyclic or polycyclic heteroalicyclic ring containing at least one heteroatom selected from O, S, N on the ring.

[0020] The aromatic ring is a 5 - 10 - membered aromatic monocyclic ring or aromatic fused bicyclic ring.

[0021] The heteroaromatic ring is a 5 - 10 - membered aromatic monocyclic ring or aromatic fused bicyclic ring containing at least one heteroatom selected from O, S, N on the ring.

[0022] Furthermore, the preferred structures of the alicyclic or heteroalicyclic rings include:

[0023] etc.

[0024] Furthermore, the preferred structures of the aromatic or heteroaromatic rings include:

[0025] etc.

[0026] In one embodiment of the present invention, preferably, in the structure of Formula I, P1 can be connected to any one or more of the groups R1, R2, R3, R4, and R5; or is connected to a saturated or unsaturated alicyclic or heteroalicyclic ring formed between R1, R2, R3, R4, and R5, or an aromatic or heteroaromatic ring formed.

[0027] The linking bond is selected from the linking bond P1-O- obtained from hydroxyl groups; or the linking bond P1-S- obtained from mercapto groups; or the linking bond P1-NH- obtained from amino groups; or the linking bond P1- obtained from alkyl groups; or the linking bond P1-COO- obtained from ester bonds; or the linking bond P1-CONH- obtained from amide bonds. One end of this linking bond is connected to P1, and the other end is connected to the benzene ring of the molecule shown in Formula I.

[0028] In the present invention, in the phenylboronic acid-modified polymer derivative, P1 is selected from natural polysaccharide substances and their modified or degraded products, or protein substances and their modified or degraded products.

[0029] The natural polysaccharide substances include hyaluronic acid, cellulose, alginic acid, dextran, agarose, heparin, chondroitin sulfate, chitosan, etc. The protein substances include gelatin, polypeptide, collagen, silk fibroin, elastin, recombinant protein, etc. Protein degradation products include gelatin or polypeptide, etc.

[0030] In one embodiment of the present invention, more preferably, Formula I can be selected from the structures of the following components A-1 to A-12:

[0031]

[0032] In components A-1 to A-12, n≥2, Col is collagen; GL is gelatin; HA is hyaluronic acid; CMC is cellulose; Alg is alginic acid; Dex is dextran; CTS is chitosan; Sil is silk fibroin; Ela is elastin; PGA is polyglutamic acid; PLL is polylysine.

[0033] The present invention also provides a first feasible preparation method of the phenylboronic acid-modified polymer derivative: Dissolve the carboxyl group-containing phenylboronic acid compound (PB-COOH, PB is phenylboronic acid) in deionized water, add the condensing agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and the activating agent N-hydroxysuccinimide (NHS), stir and react at room temperature for 1 h, dropwise add the water-soluble polymer solution containing amino groups, continue to stir and react at 40 °C for 24 h, add the reaction solution into a dialysis bag and dialyze with deionized water for 2-3 d, and then freeze-dry to obtain the phenylboronic acid-modified polymer derivative.

[0034] The present invention also provides a second feasible preparation method of the phenylboronic acid-modified polymer derivative: Dissolve the carboxyl group-containing phenylboronic acid compound (PB-COOH, PB is phenylboronic acid) in deionized water, add the condensing agent N,N'-dialkylcarbodiimide (DCC) and the catalyst 4-N,N-dimethylaminopyridine (DMAP), stir and react at room temperature for 1 h, dropwise add the water-soluble polymer solution containing hydroxyl groups, continue to stir and react at 40 °C for 24 h, add the reaction solution into a dialysis bag and dialyze with deionized water for 2-3 d, and then freeze-dry to obtain the phenylboronic acid-modified polymer derivative.

[0035] The present invention also provides a third feasible preparation method of the phenylboronic acid-modified polymer derivative: Dissolve the water-soluble polymer containing carboxyl groups in a 0.01 mol / L 2-(N-morpholino)ethanesulfonic acid MES buffer solution (pH = 5.2), add the phenylboronic acid compound containing amino groups (PB-NH2, PB is phenylboronic acid), and stir until completely dissolved. Dissolve 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride DMTMM in the MES buffer solution, and dropwise add it into the above reaction solution, react at 40 °C for 24 h, add the reaction solution into a dialysis bag and dialyze with deionized water for 2-3 d, and then freeze-dry to obtain the phenylboronic acid-modified polymer derivative.

[0036] The component B - the polymer derivative containing a vicinal diol group has a structure as shown in Formula II:

[0037]

[0038] Among them, n≥2, that is, the average number of vicinal diol groups (i.e., the structure in the brackets in Formula II) on a single P2 polymer chain is greater than or equal to 2.

[0039] In the present invention, in the polymer derivative containing a vicinal diol group, P2 includes natural polysaccharide substances and their modified or degraded products, or synthetic polymers containing vicinal diol groups, or vicinal diol-modified protein substances and their modified or degraded products.

[0040] The natural polysaccharide substances include hyaluronic acid, cellulose, alginic acid, dextran, agarose, heparin, chondroitin sulfate, chitosan, etc.

[0041] Further, it is preferably hyaluronic acid, cellulose, alginic acid, dextran, etc.

[0042] The synthetic polymer containing an adjacent diol group is preferably polyvinyl alcohol and its modified products.

[0043] Preparation method of the adjacent diol-modified proteinaceous substance: Dissolve D-(+)-glucono-δ-lactone and triethylamine in a small amount of DMSO, dropwise add the water-soluble polymer solution of the proteinaceous substance containing amino groups, continue to stir and react at room temperature for 48 h, then add the reaction solution into a dialysis bag and dialyze with deionized water for 2 - 3 d, and freeze-dry to obtain the adjacent diol-modified proteinaceous substance.

[0044] The proteinaceous substances include gelatin, polypeptide, collagen, fibroin, elastin, recombinant protein, etc.

[0045] The C-double bond group-modified polymer derivative of the component has a structure as shown in Formula III:

[0046]

[0047] Among them, n≥2, that is, the average number of double bond groups (i.e., the structure in the brackets in Formula III) on a single P3 polymer chain is greater than or equal to 2.

[0048] R’ is selected from hydrogen, alkyl, aryl, etc.

[0049] Further, the alkyl is a saturated or unsaturated aliphatic straight-chain or branched-chain alkyl having 1 - 5 carbon atoms. It is preferably methyl, ethyl, isopropyl, etc.

[0050] R is the connecting bond between the P3 polymer and the double bond group, and is selected from the connecting bond P3- obtained from alkyl groups; or the connecting bond P3-O- obtained from hydroxyl groups; or the connecting bond P3-NH- obtained from amino groups; or the connecting bond P3-COO- obtained from ester bonds; or the connecting bond P3-CONH- obtained from amide bonds. One end of this connecting bond is connected to P3, and the other end is connected to the double bond group of the molecule shown in Formula III.

[0051] In the present invention, in the double bond group-modified polymer derivative, P3 includes natural polysaccharide substances and their modified products or degradation products, or proteinaceous substances and their modified products or degradation products, or synthetic polymers and their modified products.

[0052] The natural polysaccharide substances include hyaluronic acid, cellulose, alginic acid, dextran, agarose, heparin, chondroitin sulfate, chitosan, etc. The protein substances include gelatin, polypeptide, collagen, fibroin, elastin, recombinant protein, etc.

[0053] The synthetic polymers include polyethylene glycol, polyethyleneimine, polyacrylic acid, polymethacrylic acid, polyacrylate, polymethacrylate, polyacrylamide, polymethacrylamide, polyvinyl alcohol, polyvinylpyrrolidone, etc.

[0054] In one embodiment of the present invention, further preferably, the formula III can be selected from the structures of the following components C-1 to C-5:

[0055]

[0056] Among components C-1 to C-5, n≥2. Component C-1 is acrylate-modified polyethylene glycol; component C-2 is methacrylate-modified hyaluronic acid; component C-3 is methacrylate-modified gelatin; component C-4 is methacrylate-modified chondroitin sulfate; component C-5 is acrylate-modified alginic acid.

[0057] The first feasible preparation method of the double bond group-modified polymer derivative: Dissolve the polymer containing hydroxyl groups in dichloromethane, add a small amount of triethylamine, and then dropwise add acryloyl chloride or methacryloyl chloride. After reacting for 10 h, pour the reaction solution into ethanol for reprecipitation. The obtained crude product is redissolved in deionized water, dialyzed for 2 - 3 d, and then freeze-dried to obtain the double bond group-modified polymer derivative.

[0058] The second feasible preparation method of the double bond group-modified polymer derivative: Dissolve the water-soluble polymer containing hydroxyl groups or amino groups in deionized water, cool it to 0 - 4 °C, add acrylic anhydride or methacrylic anhydride, and then slowly dropwise add 5M NaOH. Stir and react for 24 h. Add the reaction solution into a dialysis bag and dialyze with deionized water for 2 - 3 d, and then freeze-dry to obtain the double bond group-modified polymer derivative.

[0059] The polymer containing hydroxyl groups can be selected from hyaluronic acid, cellulose, alginic acid, dextran, agarose, heparin, chondroitin sulfate.

[0060] The polymer containing amino groups can be selected from chitosan, gelatin, polypeptide, collagen, fibroin, elastin, recombinant protein.

[0061] In one embodiment of the present invention, the biocompatible medium is selected from distilled water, physiological saline, buffer solution or cell culture medium solution. Different media can be selected according to different applications.

[0062] In one embodiment of the present invention, solution A, solution B, and solution C are mixed evenly to form a hydrogel precursor solution. The certain concentration is 0.1% w / v - 60% w / v, preferably 1% w / v - 20% w / v; the molar ratio of phenylboronic acid groups to vicinal diol groups is 1:0.01 - 100, preferably 1:0.1 - 10; the proportion of component C in the total solid content is 0.1% w / v - 90% w / v, preferably 1% w / v - 50% w / v.

[0063] In one embodiment of the present invention, the mixing for a certain time is 0.001 - 24 h, preferably 0.1 - 120 min, and further preferably 1 - 30 min.

[0064] In one embodiment of the present invention, the photoinitiator is preferably I2959, LAP, and Eosin - Y / triethanolamine initiator.

[0065] In one embodiment of the present invention, the light irradiation for a certain time is 1 s - 30 min, preferably 5 s - 2 min.

[0066] In one embodiment of the present invention, in the preparation of the dynamic - covalent hybrid cross - linked hydrogel material, in addition to adding component A, component B, and component C, component D can also be added. Component D is a beneficial component for tissue regeneration and repair, including drugs, bioactive factors, or cells.

[0067] In the present invention, the drugs include lidocaine, insulin, local anesthetics, antibiotics, anti - inflammatory drugs, anti - cancer drugs, etc.; the bioactive factors include BMP - 2 to BMP - 9 (bone morphogenetic proteins), EGF (epidermal growth factor), TGFα, TGFβ (transforming growth factor), FGF (fibroblast growth factor), IGF - Ⅰ, IGF - Ⅱ (insulin - like growth factors), NGF (nerve growth factor), etc.

[0068] In one embodiment of the present invention, the drugs and bioactive factors can be loaded on carriers by using drug - controlled release technology. The drug carriers can be selected from microgels, PLGA microspheres, mesoporous silica nanospheres, metal - organic framework nanospheres, etc.

[0069] In one embodiment of the present invention, the cells include osteoblasts, chondrocytes, endothelial cells, myoblasts, fibroblasts, nerve cells, mesenchymal stem cells, embryonic stem cells, etc.

[0070] In the preparation process of the dynamic - covalent hybrid cross - linked hydrogel material of the present invention, component A, component B, and component C are essential components, and component D can be selectively added according to the actual application needs.

[0071] The second object of the present invention is to provide a product prepared by a method for preparing a dynamic-covalent hybrid crosslinked hydrogel material, that is, a dynamic-covalent hybrid crosslinked hydrogel material.

[0072] The third object of the present invention is to provide an application of the dynamic-covalent hybrid crosslinked hydrogel material.

[0073] The present invention provides an application of the dynamic-covalent hybrid crosslinked hydrogel material in the preparation of a skin wound repair material.

[0074] The present invention provides an application of the dynamic-covalent hybrid crosslinked hydrogel material in the preparation of a urethral wound repair material.

[0075] The present invention provides an application of the dynamic-covalent hybrid crosslinked hydrogel material in the preparation of a meniscus defect regeneration and repair material.

[0076] The present invention provides an application of the dynamic-covalent hybrid crosslinked hydrogel material in the preparation of a joint defect regeneration and repair material.

[0077] The present invention provides an application of the dynamic-covalent hybrid crosslinked hydrogel material in the preparation of a bone defect regeneration and repair material.

[0078] The present invention provides an application of the dynamic-covalent hybrid crosslinked hydrogel material in the preparation of a substrate material for three-dimensional cell culture.

[0079] The present invention provides an application of the dynamic-covalent hybrid crosslinked hydrogel material in 3D printing bioink.

[0080] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0081] (1) The present invention discloses a new method for a dynamic-covalent hybrid crosslinked hydrogel material. Compared with single dynamic bond crosslinking or covalent bond crosslinking, the prepared hydrogel material has both processability and mechanical stability: it has plasticity, shear thinning, self-healing property, etc. in the initial stage of crosslinking; it has sufficient mechanical strength and long-term stability in the later stage of crosslinking. (As Figure 3 shown)

[0082] (2) The hydrogel material disclosed by the present invention can be injected into the tissue defect site by a single-component syringe in the initial stage of crosslinking, and can provide physical effects such as a long-term wet healing environment, protective isolation, and mechanical strength support in the later stage of crosslinking.

[0083] (3) The hydrogel material disclosed by the present invention can load and slowly release beneficial components for promoting tissue regeneration and repair, and is an ideal tissue engineering scaffold material, which can be applied to the regeneration and repair of soft tissues, cartilage, bone and other defects. Description of the Drawings

[0084] Figure 1 For the crosslinking mechanism and macroscopic view of p-FPB-GL / PVA / CSMA hydrogel.

[0085] Figure 2 For the rheological curve of gel formation of p-FPB-GL / PVA / CSMA hydrogel.

[0086] Figure 3 For the compression performance test curve of p-FPB-GL / PVA / CSMA hydrogel.

[0087] Figure 4 For the three-dimensional cell culture diagram of p-FPB-GL / PVA / CSMA hydrogel.

[0088] Figure 5 For the skin defect repair diagram of p-FPB-GL / PVA / HAMA hydrogel.

[0089] Figure 6 For the meniscus defect repair diagram of p-FPB-GL / PVA / CSMA hydrogel. Specific implementation manners

[0090] The present invention will be described in more detail with the following examples. The present invention will be further described below in conjunction with the accompanying drawings and examples, but these examples are only descriptions of the best implementation manners of the present invention and do not limit the scope of the present invention in any way. Any other changes and modifications made by those skilled in the art without departing from the spirit and protection scope of the present invention are still included within the protection scope of the present invention.

[0091] Example 1: Synthesis of p-phenylboronic acid modified collagen polymer derivative (p-PB-Col)

[0092] Synthesis of p-PB-Col: Dissolve p-phenylboronic acid compound (p-PB, 50 mg) in deionized water, add condensing agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 0.1 g) and activator N-hydroxysuccinimide (NHS, 0.1 g), stir and react at room temperature for 1 h, then dropwise add collagen (Collagen, from rat tail, 1 g) polymer aqueous solution, continue to stir and react at 40 °C for 24 h, add the reaction solution into a dialysis bag (MWCO 3500) and dialyze with deionized water for 2 - 3 d, and then freeze-dry to obtain p-PB-Col polymer derivative (0.85 g, yield 85%). According to 1 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of phenylboronic acid group can be calculated to be 23%.

[0093]

[0094] Example 2: Synthesis of p-Fluorophenylboronic Acid Modified Gelatin Polymer Derivative (p-FPB-GL)

[0095] Synthesis of p-FPB-GL: Dissolve p-fluorophenylboronic acid compound (p-FPB, 50 mg) in deionized water, add condensing agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 0.1 g) and activator N-hydroxysuccinimide (NHS, 0.1 g), stir and react at room temperature for 1 h, then dropwise add an aqueous solution of gelatin (Gelatin, derived from porcine skin, sigma V900863, 1 g) polymer. Continue to stir and react at 40 °C for 24 h. Then add the reaction solution into a dialysis bag (MWCO 3500) and dialyze with deionized water for 2 - 3 d, and freeze-dry to obtain the p-FPB-GL polymer derivative (0.88 g, yield 88%). According to 1 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of phenylboronic acid groups can be calculated to be 47%.

[0096]

[0097] Example 3: Synthesis of o-Phenylboronic Acid Modified Hyaluronic Acid Polymer Derivative (o-PB-HA)

[0098] Synthesis of o-PB-HA: Dissolve o-phenylboronic acid compound (o-FPB, 50 mg) in deionized water, add condensing agent N,N'-dicyclohexylcarbodiimide (DCC, 0.1 g) and catalyst 4-N,N-dimethylaminopyridine (DMAP, 20 mg / DMSO solution), stir and react at room temperature for 1 h, then dropwise add an aqueous solution of hyaluronic acid (Hyaluronic Acid, derived from biological fermentation, Freda, molecular weight 340 kDa, 1 g) polymer. Continue to stir and react at 40 °C for 24 h. Then add the reaction solution into a dialysis bag (MWCO 3500) and dialyze with deionized water for 2 - 3 d, and freeze-dry to obtain the o-PB-HA polymer derivative (0.74 g, yield 74%). According to 1 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of phenylboronic acid groups can be calculated to be 27%.

[0099]

[0100] Example 4: Synthesis of m-Chlorophenylboronic Acid Modified Alginate Polymer Derivative (m-CPB-Alg)

[0101] Synthesis of m-CPB-Alg: Dissolve the m-chlorophenylboronic acid compound (m-CPB, 50 mg) in deionized water, add the condensing agent N,N'-dialkylcarbodiimide (DCC, 0.1 g) and the catalyst 4-N,N-dimethylaminopyridine (DMAP, 20 mg / DMSO solution), stir and react at room temperature for 1 h, dropwise add the aqueous solution of alginate (Alginate, sigma180947, 1 g), continue to stir and react at 40 °C for 24 h, add the reaction solution into a dialysis bag (MWCO 3500) and dialyze with deionized water for 2 - 3 d, then freeze-dry to obtain the m-CPB-Alg polymer derivative (0.71 g, yield 71%). According to 1 the 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of the phenylboronic acid group can be calculated to be 22%.

[0102]

[0103] Example 5: Synthesis of p-isopropylphenylboronic acid modified chitosan polymer derivative (p-PPB-CTS)

[0104] Synthesis of p-PPB-CTS: Dissolve the p-isopropylphenylboronic acid compound (p-PPB, 50 mg) in deionized water, add the condensing agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 0.1 g) and the activator N-hydroxysuccinimide (NHS, 0.1 g), stir and react at room temperature for 1 h, dropwise add the aqueous solution of chitosan (Chitosan, sigma448869, 1 g), continue to stir and react at 40 °C for 24 h, add the reaction solution into a dialysis bag (MWCO 3500) and dialyze with deionized water for 2 - 3 d, then freeze-dry to obtain the p-PPB-CTS polymer derivative (0.65 g, yield 65%). According to 1 the 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of the phenylboronic acid group can be calculated to be 13%.

[0105]

[0106] Example 6: Synthesis of m-fluorochlorophenylboronic acid modified silk fibroin polymer derivative (m-FCPB-Sil)

[0107] Synthesis of m-FCPB-Sil: Dissolve the m-fluorochlorobenzeneboronic acid compound (m-FCPB-Sil, 50 mg) in deionized water, add the condensing agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 0.1 g) and the activating agent N-hydroxysuccinimide (NHS, 0.1 g). After stirring the reaction at room temperature for 1 h, gradually add dropwise the aqueous solution of silk fibroin (derived from silk, 1 g) polymer. Continue stirring the reaction at 40 °C for 24 h, then add the reaction solution into a dialysis bag (MWCO 3500) and dialyze it with deionized water for 2 - 3 d, and then freeze-dry to obtain the m-FCPB-Sil polymer derivative (0.78 g, yield 78%). According to 1 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of the benzeneboronic acid group can be calculated to be 34%.

[0108]

[0109] Example 7: Synthesis of p-methoxybenzeneboronic acid-modified elastin polymer derivative (p-OPB-Ela)

[0110] Synthesis of p-OPB-Ela: Dissolve the p-methoxybenzeneboronic acid compound (p-OPB-Ela, 50 mg) in deionized water, add the condensing agent 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl, 0.1 g) and the activating agent N-hydroxysuccinimide (NHS, 0.1 g). After stirring the reaction at room temperature for 1 h, gradually add dropwise the aqueous solution of elastin (derived from pig aorta, 1 g) polymer. Continue stirring the reaction at 40 °C for 24 h, then add the reaction solution into a dialysis bag (MWCO 3500) and dialyze it with deionized water for 2 - 3 d, and then freeze-dry to obtain the p-OPB-Ela polymer derivative (0.71 g, yield 71%). According to 1 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of the benzeneboronic acid group can be calculated to be 27%.

[0111]

[0112] Example 8: Synthesis of p-methylaminobenzeneboronic acid-modified polyglutamic acid polymer derivative (p-NPB-PGA)

[0113] Synthesis of p-NPB-PGA: Dissolve polyglutamic acid polymer (PGA, sigma P4886, 1 g / 10 mL deionized water) in 0.01 mol / L 2-(N-morpholino)ethanesulfonic acid MES buffer solution (pH = 5.2), and add p-methylaminophenylboronic acid compound (p-NPB-PGA, 50 mg). Stir until completely dissolved. Dissolve 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (DMTMM, 0.1 g) in MES buffer solution and add it dropwise to the above reaction solution. After reacting at 40 °C for 24 h, add the reaction solution to a dialysis bag (MWCO 3500) and dialyze it with deionized water for 2 - 3 d, then freeze-dry to obtain the p-NPB-PGA polymer derivative (0.86 g, yield 86%). According to 1 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of phenylboronic acid group can be calculated to be 38%.

[0114]

[0115] Example 9: Synthesis of diol-modified gelatin polymer derivative (diol-GL)

[0116] Synthesis of diol-GL: Dissolve D-(+)-glucono-δ-lactone (0.5 g) and triethylamine (0.2 mL) in 5 mL DMSO, and add it dropwise to the aqueous solution of gelatin (Gelatin, derived from pig skin, sigma V900863, 1 g / 10 mL deionized water) polymer. Continue to stir and react at 40 °C for 48 h. Then add the reaction solution to a dialysis bag and dialyze it with deionized water for 2 - 3 d, and freeze-dry to obtain the diol-GL polymer derivative (0.82 g, yield 82%). According to 1 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of vicinal diol group can be calculated to be 23%.

[0117]

[0118] Example 10: Synthesis of diol-modified polylysine polymer derivative (diol-PLL)

[0119] Synthesis of diol-PLL: Dissolve D-(+)-glucono-δ-lactone (0.5 g) and triethylamine (0.2 mL) in 5 mL DMSO, and add it dropwise to the aqueous solution of polylysine (PLL, sigma P9404, 1 g / 10 mL deionized water) polymer. Continue to stir and react at 40 °C for 48 h. Then add the reaction solution to a dialysis bag and dialyze it with deionized water for 2 - 3 d, and freeze-dry to obtain the diol-PLL polymer derivative (0.87 g, yield 87%). According to 1 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of vicinal diol group can be calculated to be 34%.

[0120]

[0121] Example XI: Synthesis of Double-Bond Modified Polyethylene Glycol Polymer Derivative (PEGDA)

[0122] Synthesis of PEGDA: Dissolve polyethylene glycol (PEG, sigma P2139, 1 g) polymer in 50 mL of dichloromethane, add 1 mL of triethylamine, and then dropwise add 5 mL of acryloyl chloride in dichloromethane solution. After reacting for 10 h, pour the reaction solution into ethanol for reprecipitation. The obtained crude product is redissolved in deionized water and freeze-dried to obtain PEGDA polymer derivative (0.82 g, yield 82%). According to 1 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of double-bond groups can be calculated to be 94%.

[0123]

[0124] Example XII: Synthesis of Double-Bond Modified Hyaluronic Acid Polymer Derivative (HAMA)

[0125] Synthesis of HAMA: Dissolve hyaluronic acid (HA, from biologic fermentation, Freda, molecular weight 340 kDa, 1 g) polymer in 50 mL of deionized water, cool to 0 - 4 °C, add 2 mL of methacrylic anhydride, and then slowly dropwise add 5 M NaOH. Stir and react for 24 h. Add the reaction solution into a dialysis bag and dialyze with deionized water for 2 - 3 d. Freeze-dry to obtain HAMA polymer derivative (0.88 g, yield 88%). According to 1 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of double-bond groups can be calculated to be 57%.

[0126]

[0127] Example XIII: Synthesis of Double-Bond Modified Chondroitin Sulfate Polymer Derivative (CSMA)

[0128] Synthesis of CSMA: Dissolve chondroitin sulfate (CS, from bovine cartilage, sigma C6737, 1 g) polymer in 50 mL of deionized water, cool to 0 - 4 °C, add 2 mL of methacrylic anhydride, and then slowly dropwise add 5 M NaOH. Stir and react for 24 h. Add the reaction solution into a dialysis bag and dialyze with deionized water for 2 - 3 d. Freeze-dry to obtain CSMA polymer derivative (0.83 g, yield 83%). According to 1 1H NMR nuclear magnetic resonance hydrogen spectrum, the grafting rate of double-bond groups can be calculated to be 44%.

[0129]

[0130] Example 14: Preparation of p-FPB-GL / PVA / HAMA Dynamic-Covalent Hybrid Crosslinked Hydrogel

[0131] According to the method of the present invention, component A - phenylboronic acid modified gelatin derivative p-FPB-GL (obtained in Example 2, 0.1 g) was dissolved in 1 mL of D-PBS (pH = 7.4), component B - polyvinyl alcohol (PVA, sigma360627, 20 mg) was dissolved in 1 mL of D-PBS (pH = 7.4), component C - double bond modified hyaluronic acid (obtained in Example 12, HAMA, 10 mg) and photoinitiator LAP (2 mg) were selected. Components A / B / C were mixed evenly in equal proportions (A / B / C in equimolar ratio), and left for 5 min until the phenylborate crosslinking was complete. Then, after irradiation with a 365 nm light source (20 mW / cm 2 ) for 1 min, the p-FPB-GL / PVA / HAMA dynamic-covalent hybrid crosslinked hydrogel could be obtained.

[0132] Example 15: Preparation of p-FPB-GL / PVA / CSMA Dynamic-Covalent Hybrid Crosslinked Hydrogel

[0133] According to the method of the present invention, component A - phenylboronic acid modified gelatin derivative p-FPB-GL (obtained in Example 2, 0.1 g) was dissolved in 1 mL of D-PBS (pH = 7.4), component B - polyvinyl alcohol (PVA, 20 mg) was dissolved in 1 mL of D-PBS (pH = 7.4), component C - double bond modified chondroitin sulfate (obtained in Example 13, CSMA, 10 mg) and photoinitiator LAP (2 mg) were selected. Components A / B / C were mixed evenly in equal proportions, and left for 5 min until the phenylborate crosslinking was complete. Then, after irradiation with a 365 nm light source (20 mW / cm 2 ) for 1 min, the p-FPB-GL / PVA / CSMA dynamic-covalent hybrid crosslinked hydrogel (as shown in Figure 1 ) could be obtained.

[0134] Example 16: Preparation of p-PB-Col / diol-GL / GelMA Dynamic-Covalent Hybrid Crosslinked Hydrogel

[0135] According to the method of the present invention, component A - phenylboronic acid - modified collagen derivative p - PB - Col (obtained in Example 1, 0.1 g) is dissolved in 1 mL of D - PBS (pH = 7.4), component B - vicinal diol - modified gelatin derivative (obtained in Example 9, diol - GL, 0.1 g) is dissolved in 1 mL of D - PBS (pH = 7.4), component C - double - bond - modified gelatin (GelMA, sourced from pigskin, double - bond labeling rate ~ 50%, 50 mg) and photoinitiator LAP (2 mg) are selected. Components A / B / C are mixed evenly in equal proportions, and left for 5 min until the phenylborate cross - linking is complete. Then, after irradiation with a 365 nm light source (20 mW / cm 2 ) for 1 min, the p - PB - Col / diol - GL / GelMA dynamic - covalent hybrid cross - linked hydrogel can be obtained.

[0136] Example 17: Preparation of p - PPB - CTS / PVA / PEGDA dynamic - covalent hybrid cross - linked hydrogel

[0137] According to the method of the present invention, component A - phenylboronic acid - modified chitosan derivative p - PPB - CTS (obtained in Example 5, 0.1 g) is dissolved in 1 mL of D - PBS (pH = 7.4), component B - polyvinyl alcohol (PVA, sigma360627, 20 mg) is dissolved in 1 mL of D - PBS (pH = 7.4), component C - double - bond - modified polyethylene glycol (PEGDA, Beijing Jiankai, molecular weight 10 kDa, 50 mg) and photoinitiator LAP (2 mg) are selected. Components A / B / C are mixed evenly in equal proportions, and left for 5 min until the phenylborate cross - linking is complete. Then, after irradiation with a 365 nm light source (20 mW / cm 2 ) for 1 min, the p - PPB - CTS / PVA / PEGDA dynamic - covalent hybrid cross - linked hydrogel can be obtained.

[0138] Example 18: Preparation of m - FCPB - Sil / HA / AlgMA dynamic - covalent hybrid cross - linked hydrogel

[0139] According to the method of the present invention, component A - phenylboronic acid - modified silk fibroin derivative m - FCPB - Sil (prepared in Example 6, 0.1 g) is dissolved in 1 mL of D - PBS (pH = 7.4), component B - hyaluronic acid (HA, sourced from biologic fermentation, Freda, molecular weight 340 kDa, 50 mg) is dissolved in 1 mL of D - PBS (pH = 7.4), component C - double - bond - modified alginic acid (AlgMA, 20 mg) and photoinitiator LAP (2 mg) are selected. Components A / B / C are mixed evenly in equal proportions, and left for 5 min until the phenylborate cross - linking is complete. Then, after irradiation with a 365 nm light source (20 mW / cm 2) After irradiating for 1 min, the m-FCPB-Sil / HA / AlgMA dynamic-covalent hybrid cross-linked hydrogel can be obtained.

[0140] Example XIX: Preparation of p-OPB-Ela / CSMA Dynamic-Covalent Hybrid Cross-Linked Hydrogel

[0141] According to the method of the present invention, component A - phenylboronic acid-modified elastin derivative p-OPB-Ela (obtained in Example VII, 0.1 g) is dissolved in 1 mL of D-PBS (pH = 7.4), and component B / C - double bond-modified chondroitin sulfate (CS, from bovine cartilage, sigma C6737, 20 mg) and photoinitiator LAP (2 mg) are dissolved in 1 mL of D-PBS (pH = 7.4). Components A / B / C are mixed evenly in equal proportions and left for 5 min until the phenylborate cross-linking is complete. Then, it is irradiated with a 365 nm light source (20 mW / cm 2 ) After irradiating for 1 min, the p-OPB-Ela / CSMA dynamic-covalent hybrid cross-linked hydrogel can be obtained.

[0142] Example XX: Preparation of p-NPB-PGA / diol-PLL / GelMA Dynamic-Covalent Hybrid Cross-Linked Hydrogel

[0143] According to the method of the present invention, component A - phenylboronic acid-modified polyglutamic acid derivative p-NPB-PGA (obtained in Example VIII, 0.1 g) is dissolved in 1 mL of D-PBS (pH = 7.4), component B - vicinal diol-modified polylysine derivative (obtained in Example X, diol-PLL, 0.1 g) is dissolved in 1 mL of D-PBS (pH = 7.4), component C - double bond-modified gelatin (GelMA, from porcine skin, double bond labeling rate ~ 50%, 50 mg) and photoinitiator LAP are mixed evenly in equal proportions and left for 5 min until the phenylborate cross-linking is complete. Then, it is irradiated with a 365 nm light source (20 mW / cm 2 ) After irradiating for 1 min, the p-NPB-PGA / diol-PLL / GelMA dynamic-covalent hybrid cross-linked hydrogel can be obtained.

[0144] Example XXI: Preparation of Dynamic-Covalent Hybrid Cross-Linked Hydrogel Material

[0145] According to the method of the present invention, the operation is carried out at 37 °C, and polymer aqueous solutions with different concentrations are respectively prepared, with the composition A:B = 1:1 (w / w), as shown in Table 1.

[0146] Table 1

[0147]

[0148]

[0149] Component C includes polyethylene glycol PEGDA, hyaluronic acid HAMA, gelatin GelMA, chondroitin sulfate CSMA, and alginic acid AlgMA, and the added concentration ranges from 0.1% to 10% w / v.

[0150] According to the method of the present invention, hydrogel precursor solutions with corresponding concentrations of the above different components (Component A, Component B, and Component C) are respectively prepared, mixed evenly in equal proportions, and left standing for 5 minutes to obtain hydrogel materials with different chemical compositions. Different hydrogel materials have different biological effects, and the composition of the hydrogel material can be selected specifically according to different applications.

[0151] In addition, beneficial component D for tissue regeneration and repair, including drugs, bioactive factors, or cells, can also be added to the above hydrogel. Among them, drugs include lidocaine, insulin, local anesthetics, antibiotics, anti-inflammatory drugs, anti-cancer drugs, etc.; bioactive factors include BMP-2 to BMP-9 (bone morphogenetic protein), EGF (epidermal growth factor), TGFα, TGFβ (transforming growth factor), FGF (fibroblast growth factor), IGF-I, IGF-II (insulin-like growth factor), NGF (nerve growth factor), etc.; cells include osteoblasts, chondrocytes, endothelial cells, myoblasts, fibroblasts, nerve cells, mesenchymal stem cells, embryonic stem cells, etc.

[0152] Example 22: Rheological test of dynamic-covalent hybrid crosslinked hydrogel

[0153] Rheological analysis was carried out using a HAAKE MARS rheometer on a test platform at 37°C. Taking the p-FPB-GL / PVA / CSMA dynamic-covalent hybrid crosslinked hydrogel in Example 15 as an example, when the two components are mixed, the hydrogel exhibits viscoelastic characteristics. After a certain time of light irradiation (365 nm, 20 mW / cm 2 ), G' gradually exceeds G", the elasticity of the hydrogel gradually increases, realizing the transformation from a plastic gel to an elastic gel, and the modulus at complete gelation can reach about 300 - 500 Pa (as Figure 2 shown).

[0154] Example 23: Mechanical test of dynamic-covalent hybrid crosslinked hydrogel

[0155] The compression test was carried out using a GT-TCS-2000 tensile testing machine. The test sample was a cylindrical specimen with a diameter of 10 mm and a height of 2 mm, and the test speed was 1 mm / min. Taking the p-FPB-GL / PVA / CSMA dynamic-covalent hybrid cross-linked hydrogel in Example 15 as an example, the compressive strength of the hydrogel material was tested. The experimental results showed that the single dynamic cross-linked hydrogel material (p-FPB-GL / PVA) had a low compressive strength (elastic modulus 10.5 ± 1.8 kPa), and the hydrogel material combined by dynamic bonds and covalent bonds (p-FPB-GL / PVA / CSMA) significantly improved its compressive strength (elastic modulus 27.4 ± 1.8 kPa). Adding PLGA microspheres loaded with growth factors (PLGA microspheres prepared by the conventional emulsification method for TGFβ) could slightly increase the compressive strength (elastic modulus 34.7 ± 2.4 kPa). The above results indicate that dynamic-covalent hybrid cross-linking can improve the mechanical strength and stability (as Figure 3 shown).

[0156] The results of other mechanical tests are shown in Table 2:

[0157] Table 2

[0158] Hydrogel composition Elastic modulus (kPa) p-PB-Col / diol-GL 6.7±2.6 p-PB-Col / diol-GL / GelMA 21.5±3.1 p-PPB-CTS / PVA 8.7±2.2 p-PPB-CTS / PVA / PEGDA 32.3±3.5 m-FCPB-Sil / HA 7.7±1.3 m-FCPB-Sil / HA / AlgMA 28.6±2.8 p-OPB-Ela / CS 7.9±2.1 p-OPB-Ela / CSMA 37.8±4.3 p-NPB-PGA / diol-PLL 5.4±2.2 p-NPB-PGA / diol-PLL / GelMA 20.8±2.8

[0159] Example 24: Three-dimensional cell culture experiment of dynamic-covalent hybrid cross-linked hydrogel

[0160] In this experiment, taking the three-dimensional cell culture experiment of the p-FPB-GL / PVA / HAMA dynamic-covalent hybrid cross-linked hydrogel in Example 14 as an example, a hydrogel precursor solution with a certain concentration was prepared using cell culture medium DMEM (p-FPB-GL: 10% w / v; PVA: 2% w / v; HAMA: 1% w / v; mixed at 1:1:1). The chondrocytes were digested with trypsin and centrifuged to prepare a cell suspension. Then the cell suspension was fully mixed with the above hydrogel precursor solution and added to a confocal culture dish, left for 5 min and irradiated with light for 1 min (365 nm, 20 mW / cm 2 ) until it was completely gelled. Then, 1 mL of DMEM containing 10% FBS was added to each well and continued to be cultured for a certain period of time (1 d, 4 d, 7 d, 14 d) in an environment of 37 °C and 5% CO2. As Figure 4 shown, the live / dead cell results showed that chondrocytes were more conducive to cell spreading, proliferation, differentiation, etc. in the three-dimensional culture matrix of the dynamic-covalent hybrid cross-linked hydrogel and could maintain long-term three-dimensional culture.

[0161] Example 25: Application of dynamic-covalent hybrid cross-linked hydrogel in skin defect repair

[0162] In this experiment, taking the application of the p-FPB-GL / PVA / HAMA dynamic-covalent hybrid cross-linked hydrogel in skin defect repair in Example XIV as an example, a hydrogel precursor solution with a certain concentration was prepared (p-FPB-GL: 10% w / v; PVA: 2% w / v; HAMA: 1% w / v; mixed in a ratio of 1:1:1). The experiment used a zebra pig model, and a 3 cm × 3 cm square defect model was created on the pig skin, which was divided into 3 groups: ① blank group; ② hydrogel group (p-FPB-GL / PVA / HAMA). The hydrogel precursor solution was filled and covered the skin defect site, left for 5 min and irradiated for 1 min (365 nm, 20 mW / cm 2 ) until it completely gelled. The wound healing and tissue regeneration were observed one month after the operation. As Figure 5 shown, the moist healing conditions provided by the hydrogel material are beneficial to promoting tissue regeneration.

[0163] Example XXVI: Application of dynamic-covalent hybrid cross-linked hydrogel in meniscus defect repair

[0164] In this experiment, taking the application of the p-FPB-GL / PVA / CSMA dynamic-covalent hybrid cross-linked hydrogel in meniscus defect repair in Example XV as an example, a hydrogel precursor solution with a certain concentration was prepared (p-FPB-GL: 10% w / v; PVA: 2% w / v; CSMA: 1% w / v; mixed in a ratio of 1:1:1). The experiment used a New Zealand rabbit model, and a cylindrical defect model with a diameter of 5 mm was created on the rabbit meniscus, which was divided into 3 groups: ① blank group; ② hydrogel group (p-FPB-GL / PVA / CSMA); ③ hydrogel + growth factor group (p-FPB-GL / PVA / CSMA + TGFβ), where TGFβ with a concentration of 10 ng / mL was added during the preparation of the hydrogel precursor solution. The hydrogel precursor solution was filled and covered the meniscus defect site, left for 5 min and irradiated for 1 min (365 nm, 20 mW / cm 2 ) until it completely gelled. The regeneration and repair of the meniscus defect were observed at one month and three months after the operation respectively. The experimental results confirmed that the hydrogel material can stimulate the homing of endogenous stem cells to promote the regeneration of the meniscus defect, and the added TGFβ growth factor can accelerate the regeneration and repair speed, successfully realizing the regeneration and repair of the meniscus defect (as Figure 6 shown).

[0165] Example XXVII: Application of dynamic-covalent hybrid cross-linked hydrogel in maxillofacial bone defect repair

[0166] In this experiment, taking the application of the m-FCPB-Sil / HA / AlgMA dynamic-covalent hybrid cross-linked hydrogel in the repair of maxillofacial bone defects in Example 18 as an example, a hydrogel precursor solution with a certain concentration was prepared (m-FCPB-Sil: 10% w / v; HA: 2% w / v; AlgMA: 1% w / v; mixed in a ratio of 1:1:1). The experiment used a New Zealand rabbit model, and a cylindrical defect model with a diameter of 5 mm was created on the maxillofacial bone of the rabbit, which was divided into 3 groups: ① blank group; ② hydrogel group (m-FCPB-Sil / HA / AlgMA); ③ hydrogel + growth factor group (m-FCPB-Sil / HA / AlgMA + BMP-2), where BMP-2 at a concentration of 10 ng / mL was added during the preparation of the hydrogel precursor solution. The hydrogel precursor solution was filled and covered the maxillofacial bone defect site, left for 5 min and irradiated with light for 1 min (365 nm, 20 mW / cm 2 ) until it was completely gelled. The regeneration and repair of the maxillofacial bone defect were observed at 1 month and 3 months after the operation. The experimental results confirmed that the hydrogel material could stimulate the homing of endogenous stem cells to promote the regeneration of maxillofacial bone defects, and the added BMP-2 growth factor could accelerate the regeneration and repair speed, successfully achieving the regeneration and repair of maxillofacial bone defects.

[0167] The above description of the embodiments is to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and the improvements and modifications made by those skilled in the art without departing from the scope of the present invention according to the disclosure of the present invention should be within the protection scope of the present invention.

Claims

1. A method for preparing a dynamic-covalent hybrid crosslinked hydrogel material, characterized in that, It includes the following steps: Dissolve component A - phenylboronic acid - modified polymer derivative in a biocompatible medium to obtain solution A; Dissolve component B - polymer derivative containing a vicinal diol group in a biocompatible medium to obtain solution B; Dissolve component C - double - bond - modified polymer derivative in a biocompatible medium to obtain solution C; Mix a certain concentration of solution A, solution B, and solution C for a certain time and irradiate under a photoinitiator for a certain time to obtain the dynamic - covalent hybrid cross - linked hydrogel material; The component A - phenylboronic acid - modified polymer derivative has a structure as shown in formula I: Wherein, n≥2; P1 is a protein or a protein - modified substance or a protein degradation product. The protein is selected from collagen, silk fibroin, elastin, recombinant protein; the protein degradation product includes gelatin or polypeptide; R1, R2, R3, R4, R5 are independently selected from hydrogen, fluorine, chlorine, bromine, iodine, hydroxyl group, mercapto group, amino group, nitro group, cyano group, aldehyde group, ketone group, carboxyl group, ester group, amide group, aryl group, alkyl group, modified alkyl group.

2. The preparation method of a dynamic - covalent hybrid cross - linked hydrogel material according to claim 1, wherein In formula I, the alkyl group is a saturated or unsaturated aliphatic straight - chain or branched - chain alkyl group having 1 - 20 carbon atoms; The modified alkyl group is an alkyl group in which any carbon atom is substituted by one selected from - F, - Cl, - Br, - I, - OH, - SH, - NH2, - NO2, - CN, - CHO, - COOH, ester group, amide group, aryl group; The modified alkyl group has 1 - 20 carbon atoms, and its carbon - carbon single bond can be arbitrarily replaced by a carbon - carbon double bond or a carbon - carbon triple bond; in the structure shown in formula I, at least two of R1, R2, R3, R4, R5 are connected to each other to form a saturated or unsaturated alicyclic or heteroalicyclic ring with carbon atoms, or form an aromatic ring or heteroaromatic ring; The alicyclic ring is a saturated or unsaturated 3 - 10 - membered monocyclic or polycyclic alicyclic ring, and the heteroalicyclic ring is a saturated or unsaturated 3 - 10 - membered monocyclic or polycyclic heteroalicyclic ring containing at least one heteroatom selected from O, S, N on the ring; The aromatic ring is a 5 - 10 - membered aromatic monocyclic or aromatic fused bicyclic ring, and the heteroaromatic ring is a 5 - 10 - membered aromatic monocyclic or aromatic fused bicyclic ring containing at least one heteroatom selected from O, S, N on the ring; In the structure of formula I, P1 is connected to any one or more groups of R1, R2, R3, R4, R5; or is connected to the saturated or unsaturated alicyclic or heteroalicyclic ring formed between R1, R2, R3, R4, R5, or forms an aromatic ring or heteroaromatic ring; The connecting bond is selected from the connecting bond P1 - O - obtained from hydroxyl groups; or the connecting bond P1 - S - obtained from mercapto groups; or the connecting bond P1 - NH - obtained from amino groups; or the connecting bond P1 - obtained from alkyl groups; or the connecting bond P1 - COO - obtained from ester bonds; or the connecting bond P1 - CONH - obtained from amide bonds. One end of this connecting bond is connected to P1, and the other end is connected to the benzene ring of the molecule shown in formula I.

3. The preparation method of a dynamic-covalent hybrid cross-linked hydrogel material according to claim 1, wherein, The formula I can be selected from the structures of the following components A - 1 to A - 12: Among components A-1 to A-12, n≥2, Col is collagen; GL is gelatin; HA is hyaluronic acid; CMC is cellulose; Alg is alginic acid; Dex is dextran; CTS is chitosan; Sil is silk fibroin; Ela is elastin; PGA is polyglutamic acid; PLL is polylysine.

4. The preparation method of a dynamic-covalent hybrid crosslinked hydrogel material according to claim 1, wherein The component B - a polymer derivative containing a vicinal diol group, with the structure shown in formula II: Wherein, n≥2; In the polymer derivative containing a vicinal diol group, P2 includes natural polysaccharide substances and their modified or degraded products, or synthetic polymers containing vicinal diol groups, or vicinal diol-modified protein substances and their modified or degraded products.

5. The preparation method of a dynamic-covalent hybrid cross-linked hydrogel material according to claim 1, characterized in that, The component C - a polymer derivative modified with a double bond group, with the structure shown in formula III: Wherein, n≥2; R’ is selected from hydrogen, alkyl, aryl; The alkyl is a saturated or unsaturated aliphatic straight-chain or branched-chain alkyl having 1-5 carbon atoms; R is a connecting bond between P3 polymer and the double bond group, selected from the connecting bond P3- obtained from alkyl groups; or the connecting bond P3-O- obtained from hydroxyl groups; or the connecting bond P3-NH- obtained from amine groups; or the connecting bond P3-COO- obtained from ester bonds; or the connecting bond P3-CONH- obtained from amide bonds. One end of this connecting bond is connected to P3, and the other end is connected to the double bond group of the molecule shown in formula III; P3 includes natural polysaccharide substances and their modified or degraded products, or protein substances and their modified or degraded products, or synthetic polymers and their modified products.

6. The preparation method of a dynamic-covalent hybrid cross-linked hydrogel material according to claim 5, characterized in that, The formula III is selected from the structures of the following components C-1 to C-5: Among components C-1 to C-5, n≥2, component C-1 is acrylate-modified polyethylene glycol; component C-2 is methacrylate-modified hyaluronic acid; component C-3 is methacrylate-modified gelatin; component C-4 is methacrylate-modified chondroitin sulfate; component C-5 is acrylate-modified alginic acid.

7. The preparation method of a dynamic-covalent hybrid cross-linked hydrogel material according to claim 1, characterized in that, The biocompatible medium is selected from distilled water, physiological saline, buffer solution or cell culture medium solution; Solution A, solution B and solution C are mixed evenly to form a hydrogel precursor solution. The certain concentration is 0.1% w / v - 60% w / v, the molar ratio of phenylboronic acid group to vicinal diol group is 1:0.01 - 100, and the proportion of component C in the total solid content is 0.1% w / v - 90% w / v; The mixing for a certain time is 0.001 - 24h; The photoinitiator is I2959, LAP or Eosin-Y / triethanolamine initiator; The light irradiation for a certain time is 1s - 30min.

8. The preparation method of a dynamic-covalent hybrid cross-linked hydrogel material according to claim 1, characterized in that, In the preparation of the dynamic-covalent hybrid cross-linked hydrogel material, in addition to adding component A, component B and component C, component D is also added. The component D is a beneficial component for tissue regeneration and repair, including drugs, bioactive factors or cells; The drugs include lidocaine, insulin, local anesthetics, antibiotics, anti-inflammatory drugs, anti-cancer drugs; The bioactive factors include BMP-2 to BMP-9, EGF, TGFα, TGFβ, FGF, IGF-Ⅰ, IGF-Ⅱ, NGF; The cells include osteoblasts, chondrocytes, endothelial cells, myoblasts, fibroblasts, nerve cells, mesenchymal stem cells, and embryonic stem cells.

9. A dynamic-covalent hybrid cross-linked hydrogel material prepared by the preparation method according to any one of claims 1-8.

10. The application of the dynamic-covalent hybrid cross-linked hydrogel material according to claim 9, selected from one of the following applications: The application of the dynamic-covalent hybrid cross-linked hydrogel material in the preparation of a skin wound repair material, The application of the dynamic-covalent hybrid cross-linked hydrogel material in the preparation of a urethral wound repair material, The application of the dynamic-covalent hybrid cross-linked hydrogel material in the preparation of a meniscus defect regeneration and repair material, The application of the dynamic-covalent hybrid cross-linked hydrogel material in the preparation of a joint defect regeneration and repair material, The application of the dynamic-covalent hybrid cross-linked hydrogel material in the preparation of a bone defect regeneration and repair material, The application of the dynamic-covalent hybrid cross-linked hydrogel material in the preparation of a substrate material for three-dimensional cell culture, The application of the dynamic-covalent hybrid cross-linked hydrogel material in 3D printing bioink.

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