Preparation method of biological tissue material, prepared biological tissue material and application

Abstract

The invention provides a preparation method of a biological tissue material, which comprises the following steps: c, carrying out functional modification on a pretreated tissue, namely oxidizing groups on the pretreated tissue and grafting anti-inflammatory and anticoagulant functional groups. The invention also provides the biological tissue material prepared by the preparation method. The service life of the xenograft is prolonged by introducing anticoagulant functional molecules on the basis of non-aldehyde tissue fixation and antigen content reduction, the technical problem that an existing xenograft tissue material is high in antigen content is solved, and the problems of aldehyde group residue, depolymerization and the like caused by glutaraldehyde fixation can be effectively avoided; inflammation, thrombus and easy calcification caused by implantation are effectively improved through functional modification; and the technical problems of immune response of implanted biological tissue materials and the like are also solved.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a method for preparing a biological tissue material, the prepared biological tissue material and its application. Background Art

[0002] Xenogeneic tissue, derived from other animal species, can serve as a source of material for functional tissue grafts, such as heart valves, tendons, ligaments, and cartilage. However, xenogeneic tissue contains molecules called antigens that trigger an immune response upon implantation. The presence of antigens that trigger immune responses and ultimately lead to transplant rejection is a major obstacle to the use of xenogeneic tissue as a biomaterial. This graft-specific immune response can impair the biological performance of the graft and, in the worst case, lead to host death.

[0003] In order to more widely use xenogeneic tissues for clinical applications, it is necessary to remove antigenic components from the tissue while retaining the ideal composition, structure and function of the original tissue. Currently, many strategies have been used to remove or mask antigens in xenogeneic tissues so that they can be safely implanted in human patients. The most common strategy is to use chemical treatments, such as glutaraldehyde (GA), to cross-link proteins within xenogeneic tissues. The cross-linking process is designed to make the antigen epitopes unrecognizable to the human patient's immune system. However, studies have shown that glutaraldehyde cannot completely mask important antigens, including the Galalpha1,3-Galbeta1-4GlcNAc-R (α-gal) epitope. Glutaraldehyde-fixed tissues successfully overcome hyperacute or acute rejection reactions to xenogeneic heart valve replacements, but delayed immune recognition can lead to chronic immune degradation and calcification, thereby hindering valve performance.

[0004] On the other hand, it is possible to increase the lifespan of xenografts by eliminating antigenic proteins or inactivating antigenic determinants. A major approach involves decellularizing xenografts through various chemical and physical methods to reduce their antigenic content. Chemical treatments can involve tissue degradation or dissolution of cellular components and antigenic determinants, such as acid solutions, detergents, organic solvents, enzyme solutions, and hypotonic / hypertonic solutions. Cells can be disrupted and removed by osmotic pressure (e.g., hypotonic solutions) or by dissolving cell membranes (detergents and organic solvents). Physical treatments, such as freeze-thaw, sonication, and gamma irradiation or photooxidation (both gamma irradiation and photooxidation induce protein cross-linking within tissues), are used to disrupt cells within the tissue. Physical and chemical treatments are often combined. Ideally, reducing the antigenic content of xenografts should involve targeted removal of antigens that trigger immune responses without destroying the important structural molecules that impart mechanical properties to the tissue. However, this presents one of the current technical difficulties.

[0005] In addition to immune rejection caused by allogeneic grafts, which can impact graft longevity, clinical feedback indicates that bioprosthetic heart valves (BHVs) typically have a service life of less than 20 years. This is due to a high incidence of subclinical leaflet thrombosis (SLT, 15%-40%) and structural changes. Designing valve leaflet materials is crucial for addressing these issues. The high incidence of subclinical leaflet thrombosis and structural changes ultimately leads to symptoms such as leaflet dyskinesia, altered hemodynamics, increased risk of calcification, and even bioprosthetic heart valve failure. Currently commercial bioprosthetic heart valve leaflets are made from glutaraldehyde-cross-linked porcine or bovine pericardium. The presence of high levels of collagen in the decellularized tissue of these bioprosthetic heart valves may promote platelet adhesion and activation, as well as localized aggregation of coagulation factors, impairing the hemocompatibility of the leaflet tissue. Existing bioprosthetic heart valves are exposed to blood flow for a long time, and localized blood turbulence at the junction of the leaflet and the valve stent accelerates coagulation and thrombosis, leading to subclinical leaflet thrombosis. In addition, the aldehyde residues present in glutaraldehyde-fixed tissues will provide calcium attachment points, leading to calcification of bioprosthetic heart valves and shortening their service life. The toxicity of glutaraldehyde severely inhibits the endothelialization of valve leaflet materials, while effective endothelialization can improve the biocompatibility of valve leaflets in contact with blood.

[0006] Therefore, in order to solve the above-mentioned defects of bioartificial heart valves in the existing technology, the field urgently needs a method for preparing biological tissue materials that can improve material properties and a new biological tissue material prepared. When prepared into a bioartificial heart valve, it can significantly reduce the antigen content in the biological tissue material, improve the mechanical properties of the biomaterial, reduce the aldehyde content, improve the anticoagulant performance, reduce calcification, and ultimately increase biocompatibility, thereby extending the service life of the biological tissue material. Summary of the Invention

[0007] In view of the shortcomings of the prior art described above, the purpose of the present invention is to provide a method for preparing biological tissue materials that can improve material properties, so as to solve the problems in the prior art that after processing, the biological tissue materials have high antigen content, weak mechanical properties, high aldehyde content, weak anticoagulant properties, poor biocompatibility, and short service life.

[0008] To this end, the present invention provides a method for preparing a biological tissue material, which includes the following step c: functionally modifying the pretreated tissue, wherein the functional modification of the tissue is to oxidize the groups on the pretreated tissue and graft anti-inflammatory and anticoagulant functional groups.

[0009] As certain embodiments of the present invention, the pretreated tissue undergoes the following processing steps: a. tissue fixation, fixation to preserve cell-intrinsic substances and maintain the inherent morphology and structure of cells and tissues; b. tissue defatting to remove fat therein, and the tissue defatting includes physical defatting and / or chemical defatting.

[0010] As certain embodiments of the present invention, the tissue in step a is pericardial tissue, cartilage tissue, skin, or other membrane structure tissue. As certain embodiments of the present invention, the tissue in step a is pericardial tissue.

[0011] As certain embodiments of the present invention, the tissue fixation in step a is a tissue cross-linking treatment using ionic bonds and / or covalent bonds.

[0012] As certain embodiments of the present invention, the tissue defatting in step b is to treat the tissue fixed in step a with a betaine-type detergent and / or an alkaline alcohol reagent.

[0013] As certain embodiments of the present invention, the tissue functionalization modification is a combined treatment of oxidation and grafting.

[0014] As certain embodiments of the present invention, the ionic cross-linking treatment in step a is performed by soaking the tissue in a metal ion solution.

[0015] As certain embodiments of the present invention, the covalent bond cross-linking treatment in step a is performed by immersing the substrate in a mixed solution of a carbonyl activation reagent and a coupling reagent.

[0016] In certain embodiments of the present invention, the ionic crosslinking in step a is performed by immersing the tissue in a 0.01 mM to 1 mM metal ion solution. In certain embodiments of the present invention, the tissue is immersed in a 0.01 mM to 1 mM metal ion solution at 10-15° C. for 0.5-2 h.

[0017] The concentration of the metal ion solution can be selected from 0.01mM, 0.02mM, 0.03mM, 0.04mM, 0.05mM, 0.06mM, 0.07mM, 0.08mM, 0.09mM, 0.1mM, 0.15mM, 0.2mM, 0.25mM, 0.3mM, 0.35mM, 0.4mM, 0.45mM, 0.5mM, 0.55mM, 0.6mM, 0.65mM, 0.7mM, 0.75mM, 0.8mM, 0.85mM, 0.9mM, 0.95mM, and 1mM.

[0018] The tissue is immersed in a 0.01 mM to 1 mM metal ion solution at a temperature selected from 10°C, 11°C, 12°C, 13°C, 14°C, and 15°C.

[0019] The tissue is immersed in the 0.01mM to 1mM metal ion solution for a time selected from 0.5h, 0.6h, 0.7h, 0.8h, 0.9h, 1h, 1.1h, 1.2h, 1.3h, 1.4h, 1.5h, 1.6h, 1.7h, 1.8h, 1.9h, and 2h.

[0020] In certain embodiments of the present invention, the covalent cross-linking treatment in step a is performed by immersing the sample in a mixed solution of 10-60 mM carbonyl activating reagent and 5-30 mM coupling reagent. In certain embodiments of the present invention, the covalent cross-linking treatment is performed by immersing the sample in a mixed solution of 10-60 mM carbonyl activating reagent and 5-30 mM coupling reagent for 4-12 hours.

[0021] The concentration of the carbonyl activating agent can be selected from 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 27mM, 28mM, 29mM, 30mM, 31mM, 32mM, 33mM, 34mM , 35mM, 36mM, 37mM, 38mM, 39mM, 40mM, 41mM, 42mM, 43mM, 44mM, 45mM, 46mM, 47mM , 48mM, 49mM, 50mM, 51mM, 52mM, 53mM, 54mM, 55mM, 56mM, 57mM, 58mM, 59mM, 60mM.

[0022] The concentration of the coupling reagent can be selected from 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 27mM, 28mM, 29mM, 30mM.

[0023] The covalent bond cross-linking treatment is performed by immersing the mixture in a mixed solution of 10-60 mM carbonyl activation reagent and 5-30 mM coupling reagent for a time selected from 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, and 12 h.

[0024] As certain embodiments of the present invention, the alkaline alcohol reagent treatment in step b is soaking in an alkaline alcohol reagent solution.

[0025] As certain embodiments of the present invention, the betaine-type detergent treatment in step b is a 0.01% to 3% w / w betaine-type detergent solution.

[0026] The betaine type detergent concentration can be selected from 0.01% w / w, 0.02% w / w, 0.03% w / w, 0.04% w / w, 0.05% w / w, 0.06% w / w, 0.07% w / w, 0.08% w / w, 0.09% w / w, 0.10% w / w, 0. 15% w / w, 0.20% w / w, 0.25% w / w, 0.30% w / w, 0.35% w / w, 0.40% w / w, 0.45% w / w, 0.50% w / w, 0.55% w / w, 0.60% w / w, 0.65% w / w, 0.70% w / w, 0.75% w / w, 0.80% w / w, 0.85% w / w, 0.90% w / w, 0.95% w / w, 1% w / w, 1.1% w / w, 1.2% w / w, 1.3% w / w, 1.4% w / w, 1.5% w / w, 1.6% w / w, 1.7% w / w, 1.8% w / w, 1.9% w / w, 2.0% w / w, 2.1% w / w, 2.2% w / w, 2.3% w / w, 2.4% w / w, 2.5% w / w, 2.6% w / w, 2.7% w / w, 2.8% w / w, 2.9% w / w, 3.0% w / w.

[0027] As certain embodiments of the present invention, the alkaline alcohol reagent treatment in step b is soaking in an alkaline alcohol solvent at 2-8° C. As certain embodiments of the present invention, the alkaline alcohol reagent treatment is soaking in an alkaline alcohol solvent at 2-8° C. for 2-12 hours.

[0028] The soaking temperature of the alkaline alcohol solvent is selected from 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, and 8°C.

[0029] The treatment time of the alkaline alcohol reagent is selected from 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, and 12h.

[0030] As certain embodiments of the present invention, the betaine-type detergent treatment in step b is soaking in a betaine-type detergent solvent at 15° C. As certain embodiments of the present invention, the betaine-type detergent treatment is soaking in a betaine-type detergent solvent at 15° C. for 2 hours.

[0031] As certain embodiments of the present invention, the oxidation treatment in step c is to immerse the tissue material in an oxidant. As certain embodiments of the present invention, the combined grafting treatment in step c is to use 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole to graft and introduce at least one of taurine, L-cysteic acid, N-(2-acetamido)-2-aminoethanesulfonic acid, 2-[(2-aminoethyl)amino]ethanesulfonic acid sodium salt, curcumin, salicylic acid, tea polyphenols, anthocyanidins, tannic acid, gallic acid, and propyl gallate.

[0032] In certain embodiments of the present invention, the oxidation treatment in step c is to immerse the tissue material in a 0.01-0.1 mM oxidant. In certain embodiments of the present invention, the oxidation treatment is to immerse the tissue material in a 0.01-0.1 mM oxidant at 5-30° C. for 10-60 minutes.

[0033] The concentration of the oxidant is selected from 0.01 mM, 0.02 mM, 0.03 mM, 0.04 mM, 0.05 mM, 0.06 mM, 0.07 mM, 0.08 mM, 0.09 mM, 0.1 mM.

[0034] The temperature of the oxidation treatment is selected from 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, and 30°C.

[0035] The oxidation treatment time is selected from 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, and 60min.

[0036] As certain embodiments of the present invention, the grafting combined treatment in step c is to soak for 12 to 24 hours at 10 to 25° C. using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole, and grafting and introducing at least one of 0.1 to 5% (w / w) taurine, L-cysteic acid, N-(2-acetamido)-2-aminoethanesulfonic acid, 2-[(2-aminoethyl)amino]ethanesulfonic acid sodium salt, curcumin, salicylic acid, tea polyphenols, anthocyanins, tannic acid, gallic acid, and propyl gallate.

[0037] The grafting combined treatment temperature is selected from 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, and 25°C.

[0038] The grafting combined treatment time is selected from 12h, 13h, 14h, 15h, 16h, 17h, 18h, 19h, 20h, 21h, 22h, 23h, and 24h.

[0039] The concentrations of taurine, L-cysteic acid, N-(2-acetamido)-2-aminoethanesulfonic acid, 2-[(2-aminoethyl)amino]ethanesulfonic acid sodium salt, curcumin, salicylic acid, tea polyphenols, anthocyanidins, tannic acid, gallic acid, and propyl gallate are selected from 0.1% w / w, 0.11% w / w, 0.12% w / w, 0.13% w / w, 0.14% w / w, 0.15% w / w, 0.16% w / w, 0.17% w / w, 0.18% w / w, 0.19% w / w, 0.2% w / w, 0. 25%w / w, 0.30%w / w, 0.35%w / w, 0.40%w / w, 0.45%w / w, 0.50%w / w, 0.55%w / w, 0.60%w / w, 0.65%w / w, 0.70%w / w, 0.75%w / w, 0.80% w / w, 0.85% w / w, 0.90% w / w, 0.95% w / w, 1% w / w, 1.5% w / w, 2.0% w / w, 3% w / w, 3.5% w / w, 4% w / w, 4.5% w / w, 5% w / w.

[0040] As certain embodiments of the present invention, the metal ions of the metal ion solution are selected from at least one of iron ions, aluminum ions, and zinc ions.

[0041] As certain embodiments of the present invention, the carbonyl activating reagent is selected from at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dimethylaminopropyl ethylcarboamide and the like carbonyl activating reagents.

[0042] As certain embodiments of the present invention, the coupling reagent is selected from at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide sulfonic acid sodium salt, 1-hydroxybenzotriazole, and benzyloxycarbonyl succinimide.

[0043] As certain embodiments of the present invention, the alkaline alcohol reagent is an alcohol reagent whose acidity and alkalinity are adjusted by an alkali-binding agent, wherein the alcohol reagent is selected from at least one of ethanol, isopropanol, propanol, butanol, octanol, pentanol, hexanol, heptanol, hemp alcohol, ethylene glycol, glycerol, propylene glycol, tert-butyl alcohol, or 1,3-butanediol, and the alkali-binding agent is selected from at least one of potassium carbonate, ammonium carbonate, or sodium carbonate;

[0044] As certain embodiments of the present invention, the betaine-type detergent is at least one of betaine salicylate, (3-([N,N-dimethyldimethyl(3-myristamidepropyl)ammonium]propane-1-sulfonate (ABS-14), and 3-[N,N-dimethyl(3-palmitamidopropyl)ammonium]-propanesulfonic acid inner salt (ABS-16)).

[0045] As certain embodiments of the present invention, the oxidizing agent is at least one selected from sodium periodate, potassium periodate, potassium dichromate, peracetic acid, and 2,2,6,6-tetramethylpiperidinyl oxide (TEMPO).

[0046] As certain embodiments of the present invention, the biological tissue material preparation method is as follows: after peeling, trimming and cleaning the biological tissue material, soaking it in a 0.05mM ferric chloride solution at 10°C for 2 hours, and then rinsing it with physiological saline; then soaking it in a 1% (w / w) 3-([N,N-dimethyldimethyl(3-tetradecylamidepropyl)ammonium]propane-1-sulfonate (ABS-14) solution at 15°C for 2 hours, then soaking it in an alkaline 1,3-butanediol solution at 2°C for 4 hours, and rinsing it with PBS solution and / or D-Hanks solution; then soaking it in a 0.1mM sodium periodate solution at 25°C for 30 minutes; and finally soaking it in a mixed solution of 0.5% (w / w) curcumin and containing 20mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10mM 1-hydroxybenzotriazole at 10°C for 24 hours; wherein, the reagents attached to the biological tissue material are cleaned by rinsing with physiological saline.

[0047] As certain embodiments of the present invention, the biological tissue material preparation method is as follows: after the biological tissue material is peeled, trimmed and cleaned, the biological tissue material is subjected to a 10°C, 0.01mM ferric chloride and 20mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 10mM The material is immersed in a mixed solution of sodium N-hydroxysuccinimide sulfonic acid for 4 hours, and then rinsed with physiological saline; then immersed in a 1% (w / w) 3-[N,N-dimethyl(3-palmitamidopropyl)ammonium]-propanesulfonic acid inner salt (ABS-16) solution at 15°C for 2 hours, and then immersed in an alkaline isopropanol solution at 2°C for 2 hours, and rinsed with PBS solution and / or D-Hanks solution; then immersed in a 0.1mM 2,2,6,6-tetramethylpiperidinoxide (TEMPO) solution at 25°C for 50 minutes; finally, immersed in a mixed solution of anthocyanin at 10°C, 0.1% (w / w) and containing 40mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 20mM 1-hydroxybenzotriazole for 24 hours; wherein, the reagent attached to the biological tissue material is rinsed with physiological saline to be cleaned.

[0048] As certain embodiments of the present invention, the biological tissue material preparation method is as follows: after peeling, trimming and cleaning the biological tissue material, soaking it in 0.1mM zinc chloride at 15°C for 2 hours and rinsing with physiological saline; then soaking it in 1% (w / w) 3-([N,N-dimethyldimethyl(3-tetradecylamidepropyl)ammonium]propane-1-sulfonate (ABS-14) at 15°C for 2 hours, and then soaking it in an alkaline propylene glycol solution at 8°C for 4 hours; rinsing it with PBS solution and / or D-Hanks solution; then soaking it in 0.01mM peracetic acid solution at 25°C for 30 minutes; and finally soaking it in a mixed solution of 0.5% (w / w) L-cysteic acid, 20mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10mM 1-hydroxybenzotriazole at 10°C for 24 hours; wherein, the reagents attached to the biological tissue material are cleaned by rinsing with physiological saline.

[0049] As certain embodiments of the present invention, the biological tissue material preparation method is as follows: after peeling, trimming and cleaning the biological tissue material, soaking it in a mixed solution of 0.05mM zinc chloride, 40mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 20mM N-hydroxysuccinimide sulfonic acid sodium salt at 10°C for 12 hours, rinsing with physiological saline; and then soaking it in a 1% (w / w) 3-[N,N-dimethyl(3-palmitamidopropyl)ammonium]-propanesulfonic acid inner salt (ABS-16) solution at 15°C. Soak for 2 hours, then soak in an alkaline tert-butyl alcohol solution at 5°C for 2 hours; rinse with PBS solution and / or D-Hanks solution; then soak in a 0.1mM sodium periodate solution at 25°C for 30 minutes; finally soak in a mixed solution of 0.5% (w / w) taurine and containing 20mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10mM 1-hydroxybenzotriazole at 10°C for 24 hours; wherein, the reagent attached to the biological tissue material is rinsed with physiological saline to be cleaned.

[0050] As certain embodiments of the present invention, the biological tissue material preparation method is as follows: after peeling, trimming and cleaning the biological tissue material, soaking it in 0.1mM aluminum chloride at 15°C for 2 hours, rinsing with physiological saline; then soaking it in 1% (w / w) 3-[N,N-dimethyl(3-palmitamidopropyl)ammonio]-propanesulfonic acid inner salt (ABS-16) at 15°C for 2 hours, and then soaking it in alkaline ethanol solution at 5°C for 12 hours; washing it with PBS solution and / or D- The material was rinsed with Hanks solution; then immersed in a 0.01 mM peracetic acid solution at 25°C for 30 minutes; and finally immersed in a mixed solution of 0.5% (w / w) N-(2-acetamido)-2-aminoethanesulfonic acid, 20 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and 10 mM 1-hydroxybenzotriazole at 10°C for 24 hours; wherein, the reagent attached to the biological tissue material was rinsed with physiological saline to be cleaned.

[0051] As certain embodiments of the present invention, the biological tissue material preparation method is as follows: after peeling, trimming and cleaning the biological tissue material, soaking it in a mixed solution of 0.05mM aluminum chloride, 60mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 30mM N-hydroxysuccinimide sulfonic acid sodium salt at 10°C for 4 hours, rinsing with physiological saline; then soaking it in a 1% (w / w) 3-[N,N-dimethyl(3-palmitamidopropyl)ammonium]-propanesulfonic acid inner salt (ABS-16) solution at 15°C for 2 hours; and then rinsing it at 5°C. The biological tissue material is immersed in an alkaline 1,3-butanediol solution for 2 hours; rinsed with PBS solution and / or D-Hanks solution; then immersed in a 0.1 mM sodium periodate solution at 25°C for 30 minutes; finally immersed in a mixed solution of 0.5% (w / w) 2-[(2-aminoethyl)amino]ethanesulfonic acid sodium salt, 20 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mM 1-hydroxybenzotriazole at 10°C for 24 hours; wherein, the reagent attached to the biological tissue material is rinsed with physiological saline to be cleaned.

[0052] To achieve the above and other related objectives, the present invention also provides a biological tissue material prepared by the biological tissue material preparation method. In certain embodiments of the present invention, the biological tissue material is selected from pericardial tissue, cartilage tissue, skin, and other membranous tissues. In certain embodiments of the present invention, the biological tissue material is selected from pericardial tissue.

[0053] The present invention also provides an artificial heart valve, which is prepared using the biological tissue material.

[0054] As described above, the method for preparing a biological tissue material capable of improving material properties, the prepared biological tissue material, and its application of the present invention have the following beneficial effects:

[0055] The present invention increases the service life of xenografts by introducing molecules with anticoagulant function on the basis of non-aldehyde fixation of tissues and reduction of antigen content;

[0056] The present invention aims to solve at least one of the technical problems of existing xenotransplant tissue materials, effectively avoiding the problems of aldehyde residue and depolymerization caused by glutaraldehyde fixation, and effectively improving implantation-induced inflammation, thrombosis, and tissue calcification through functional modification.

[0057] Technical difficulties such as improving the immune response of implanted biological tissue materials can also be solved to a certain extent. The biological tissue material processing technology provided by the present invention, including tissue fixation, tissue degreasing, and tissue functionalization modification, can effectively improve or eliminate the immune response of implanted biological tissue materials.

[0058] The biological tissue material prepared by the present invention has less residual aldehyde groups and less retained antigens, thereby improving anticoagulant performance, reducing calcification, ultimately increasing biocompatibility, and improving the mechanical properties of the biological material. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 is a flow chart of the preparation method of the biological tissue material of the present invention;

[0060] Figure 2A Schematic diagram of the aldehyde group residues of the control sample 1 of Control Example 1 after glutaraldehyde fixation;

[0061] Figure 2B Schematic diagram of the aldehyde residues of control sample 2 of control example 2 after glutaraldehyde fixation;

[0062] Figure 2C Schematic diagram of the aldehyde residues of control sample 2 after glutaraldehyde fixation and glycine capping;

[0063] Figure 2D This is a schematic diagram of the aldehyde residues of Sample 4 of Example 4 after fixation with glutaraldehyde;

[0064] Figure 3A Schematic diagram of HE staining of control sample 2 prepared in control example 2 before subcutaneous implantation in rats;

[0065] Figure 3B Schematic diagram of HE staining of sample 4 prepared in Example 4 before subcutaneous implantation in rats;

[0066] Figure 3C This is a schematic diagram of HE staining of the control sample 2 prepared in Control Example 2 after being implanted subcutaneously in rats;

[0067] Figure 3D Schematic diagram of HE staining of sample 4 prepared in Example 4 after being implanted subcutaneously in rats;

[0068] Figure 4A This is a photo of the control sample 2 taken out 8 weeks after the control sample 2 was implanted subcutaneously in rats;

[0069] Figure 4B This is a photo of sample 4 taken out 8 weeks after sample 4 was subcutaneously implanted in rats.

[0070] Reference numerals:

[0071] Figure 3C The arrow in a shows obvious inflammatory cell infiltration in the tissue; the arrow in b shows abnormal overall structure of the pericardial tissue, with large areas of swelling, rupture and necrosis of collagen fibers in the tissue valves; the arrow in c shows a large area of ​​calcification in the tissue valves. DETAILED DESCRIPTION

[0072] The present invention is further described below with reference to specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention.

[0073] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0074] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0075] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0076] The following preparation examples 1 to 6 illustrate the biomaterial preparation method of the present invention with specific examples; and control samples 1 to 2 prepared in control examples 1 to 2 are used as controls.

[0077] Comparative Example 1:

[0078] Biological tissue materials were obtained from a local slaughterhouse. After peeling, trimming and cleaning, control sample 1, namely fresh bovine pericardial tissue, was obtained.

[0079] Comparative Example 2:

[0080] Biological tissue materials were obtained from a local slaughterhouse. After being peeled, trimmed, and cleaned, they were immersed in a 0.625% glutaraldehyde solution at room temperature for 24 hours, i.e., the biological tissue materials were prepared using a conventional glutaraldehyde treatment method to obtain control sample 2.

[0081] Preparation Example 1:

[0082] Biological tissue materials were obtained from a local slaughterhouse. After being peeled, trimmed, and cleaned, they were immersed in a 0.05 mM ferric chloride solution at 10°C for 2 h and rinsed several times with physiological saline; then immersed in a 1% (w / w) 3-([N,N-dimethyldimethyl(3-tetradecylamidepropyl)ammonium]propane-1-sulfonate (ABS-14) solution at 15°C for 2 h, immersed in an alkaline 1,3-butanediol solution at 2°C for 4 h, and rinsed several times with PBS solution and / or D-Hanks solution; then immersed in a 0.1 mM sodium periodate solution at 25°C for 30 min; finally, immersed in a mixed solution of 0.5% (w / w) curcumin and containing 20 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mM 1-hydroxybenzotriazole at 10°C for 24 h to obtain sample 1.

[0083] Preparation Example 2:

[0084] Biological tissue materials were obtained from a local slaughterhouse. After being peeled, trimmed, and cleaned, they were immersed in a mixed solution of 0.01 mM ferric chloride, 20 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 10 mM sodium salt of N-hydroxysuccinimide sulfonate at 10°C for 4 h, rinsed several times with physiological saline, and then immersed in a 1% (w / w) solution of 3-[N,N-dimethyl(3-palmitamidopropyl)ammonio]-propanesulfonic acid inner salt (ABS-16) at 15°C for 2 h. Soak in an alkaline isopropanol solution at 2°C for 2 hours, rinse several times with PBS solution and / or D-Hanks solution; then soak in a 0.1mM 2,2,6,6-tetramethylpiperidinoxide (TEMPO) solution at 25°C for 50 minutes; finally soak in a mixed solution of 0.1% (w / w) anthocyanin and containing 40mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 20mM 1-hydroxybenzotriazole at 10°C for 24 hours to obtain sample 2.

[0085] Preparation Example 3:

[0086] Biological tissue materials were obtained from a local slaughterhouse. After being peeled, trimmed and cleaned, they were immersed in 0.1 mM zinc chloride at 15°C for 2 h and rinsed several times with physiological saline; then immersed in 1% (w / w) 3-([N,N-dimethyldimethyl(3-tetradecylamidepropyl)ammonium]propane-1-sulfonate (ABS-14) at 15°C for 2 h, and then immersed in an alkaline propylene glycol solution at 8°C for 4 h; rinsed several times with PBS solution and / or D-Hanks solution; then immersed in 0.01 mM peracetic acid solution at 25°C for 30 min; finally immersed in a mixed solution of 0.5% (w / w) L-cysteic acid, 20 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mM 1-hydroxybenzotriazole at 10°C for 24 h to obtain sample 3.

[0087] Preparation Example 4:

[0088] Biological tissue materials were obtained from a local slaughterhouse. After stripping, trimming, and washing, they were immersed in a mixed solution of 0.05 mM zinc chloride, 40 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 20 mM sodium N-hydroxysuccinimide sulfonate at 10°C for 12 h, rinsed several times with physiological saline, and then incubated at 15°C with 1% (w / w) 3-[N,N-dimethyl(3-palmitamidopropyl)ammonio]-propanesulfonic acid inner salt (ABS-16). The sample was immersed in an alkaline tert-butyl alcohol solution at 5°C for 2 hours, and then immersed in an alkaline tert-butyl alcohol solution at 5°C for 2 hours; rinsed several times with PBS solution and / or D-Hanks solution; then immersed in a 0.1mM sodium periodate solution at 25°C for 30 minutes; finally immersed in a mixed solution of 0.5% (w / w) taurine and containing 20mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10mM 1-hydroxybenzotriazole at 10°C for 24 hours to obtain sample 4.

[0089] Preparation Example 5:

[0090] Biological tissue materials were obtained from a local slaughterhouse. After being peeled, trimmed and cleaned, they were immersed in 0.1 mM aluminum chloride at 15°C for 2 h and rinsed several times with physiological saline; then immersed in 1% (w / w) 3-[N,N-dimethyl(3-palmitamidopropyl)ammonio]-propanesulfonic acid inner salt (ABS-16) at 15°C for 2 h, and then immersed in an alkaline ethanol solution at 5°C for 12 h; rinsed several times with PBS solution and / or D-Hanks solution; then immersed in 0.01 mM peracetic acid solution at 25°C for 30 min; finally immersed in a mixed solution of 0.5% (w / w) N-(2-acetamido)-2-aminoethanesulfonic acid containing 20 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mM 1-hydroxybenzotriazole at 10°C for 24 h to obtain sample 5.

[0091] Preparation Example 6:

[0092] Biological tissue materials were obtained from a local slaughterhouse. After being peeled, trimmed, and cleaned, they were immersed in a mixed solution of 0.05 mM aluminum chloride, 60 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 30 mM sodium N-hydroxysuccinimide sulfonate at 10°C for 4 h, rinsed several times with physiological saline, and then immersed in a 1% (w / w) 3-[N,N-dimethyl(3-palmitamidopropyl)ammonio]-propanesulfonic acid inner salt (ABS-16) solution at 15°C for 2 h. Soak in alkaline 1,3-butanediol solution at 5°C for 2 hours; rinse several times with PBS solution and / or D-Hanks solution; then soak in 0.1mM sodium periodate solution at 25°C for 30 minutes; finally soak in a mixed solution of 0.5% (w / w) 2-[(2-aminoethyl)amino]ethanesulfonic acid sodium salt and 20mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10mM 1-hydroxybenzotriazole at 10°C for 24 hours to obtain sample 6.

[0093] Example 7:

[0094] This example studies the comparison of mechanical properties of samples 1 to 6 prepared in preparation examples 1 to 6 and a control sample prepared in a control example.

[0095] 7.1 Mechanical properties testing

[0096] The mechanical properties of the samples were tested using a uniaxial tensile tester (BioTester, 100 N load cell; CellScale, Waterloo, Ontario, Canada).

[0097] Samples 1-6 were cut into rectangular shapes (10 mm x 50 mm, n = 6). The sample thickness (δ) was calculated as the average thickness measured at three random points using a thickness gauge. The two sides of the sample were then fixed to a fixture with a fixed initial distance of 25 mm (L 0). Uniaxial tensile testing was performed using a 100 N load cell at a constant tensile rate of 100 mm / min.

[0098] The test data are shown in Table 1.

[0099] Table 1 Mechanical properties test of samples prepared in control example and preparation examples 1 to 6

[0100] Group Maximum tensile load (N) Tensile strength (Mpa) Elongation at break (%) Control sample 2 66.02±11.89 13.75±2.38 45±12.2 Sample 1 119.93±9.5 25.52±1.25 59±15.1 Sample 2 116.79±8.4 27.16±1.45 55.48±8.3 Sample 3 102.06±9.78 18.56±1.38 61±15.5 Sample 4 125.74±5.86 25.15±0.25 59±4.5 Sample 5 100.78±10.2 21±1.38 59±9.8 Sample 6 116.6±6.8 25.91±2.31 56.2±6.5

[0101] As shown in the uniaxial tensile test data in Table 1, the experimental results of Samples 1-6 are significantly superior to those of the control sample. This also demonstrates that, after treating pericardial tissue using Preparation Examples 1-6, the mild preparation methods employed in Preparation Examples 1-6 significantly improve the mechanical properties of pericardial tissue compared to existing technology (Control Sample 2), while removing antigens while minimizing damage to the important structural molecules that impart mechanical properties to the tissue. Comprehensive mechanical property evaluations indicate that the significant increase in mechanical properties significantly improves the lifespan of pericardial tissue.

[0102] 7.2 Thermal shrinkage and cross-linking degree detection

[0103] Thermal Shrinkage Detection Principle

[0104] Testing method: Use a laser cutting machine to cut each group of pericardium into 53.5mm long and 5mm wide; the inner rectangle is 43.5mm long and 3mm wide; the center distance is 48.5, and the circle radius is 1mm. Before the test, the test specimens are placed in physiological saline for heavy soaking and rinsing. The test conditions are a heating rate of 2℃ / min, and the reference standard (QBT 2713-2005 Leather Physical and Mechanical Test Shrinkage Temperature Determination Standard). Purpose: To evaluate the basis for the stability of pericardium cross-linking, test indicators: shrinkage deformation, tensile deformation temperature values. Detection of free amino content

[0105] The degree of cross-linking of pericardial tissue was determined using ninhydrin solution. For a sample size of 10, a 5 μmol / L glycine solution was used as a standard curve. 10 μL of tissue extract was mixed with 300 μL of deionized water, allowed to stand for 60 minutes, and then 3 mL of ninhydrin solution was added dropwise. The sample was heated at 100°C for 20 minutes. After cooling to room temperature, 15 mL of isopropanol was added dropwise. The absorbance at 570 nm was measured using a UV-visible spectrophotometer. The molar number of α-amino acids in the pericardial tissue was calculated using the standard curve. The degree of cross-linking was calculated using the following formula: Cross-linking degree (%) = (free amino acid content of uncross-linked pericardial tissue - free amino acid content of cross-linked pericardial tissue) / free amino acid content of uncross-linked pericardial tissue × 100%.

[0106] The test data are shown in Table 2.

[0107] Table 2 Thermal shrinkage and crosslinking degree test of samples prepared in the control example and preparation examples 1 to 6

[0108] Group Thermal shrinkage (℃) Crosslinking degree (%) Control sample 2 85.0±1.0 87.44±5 Sample 1 86.0±0.8 89.25±3.5 Sample 2 87.5±0.35 92.15±2.4 Sample 3 85.5±0.76 88.97±3.8 Sample 4 88.9±0.31 95.09±1.5 Sample 5 87.2±0.95 90.25±2.4 Sample 6 89.6±0.24 93.82±3.5

[0109] The thermal shrinkage and cross-linking degree test data show that samples 1-6 have higher thermal shrinkage and cross-linking degrees than the control sample. This is because samples 1-6 have been treated with non-aldehyde composite cross-linking, which increases the cross-linking degree and improves the thermodynamic properties of pericardial tissue.

[0110] Example 8

[0111] Detection of aldehyde content of sample 4, control sample 2 and control sample 1

[0112] Cut sample 4 and control samples 1-2 into 1cm×1cm pieces. Fix sample 4 and control samples 1-2 with glutaraldehyde. Block the glutaraldehyde-fixed control sample 2 with glycine. Then soak these four samples in the prepared red-sodium bisulfite stain and incubate at 37℃ for 0.5h, rinse with purified water 3 times, place them on a slide, and take photos. Figures 2A to 2D : Figure 2A Schematic diagram of the aldehyde group residues of the control sample 1 of Control Example 1 after glutaraldehyde fixation; Figure 2B Schematic diagram of the aldehyde residues of control sample 2 of control example 2 after glutaraldehyde fixation; Figure 2C Schematic diagram of the aldehyde residues of control sample 2 after glutaraldehyde fixation and glycine capping; Figure 2D Schematic diagram of the aldehyde residues of sample 4 in Example 4 after fixation with glutaraldehyde.

[0113] The amount of free aldehyde groups in Preparation Example 4 and Control Groups 1 to 2 was judged by the color depth. The darker the color, the higher the content of free aldehyde groups; the lighter the color, the less the content of free aldehyde groups. Figures 2A to 2D It can be seen that compared with Figure 2A Color, Figure 2B The darkest color, Figure 2C The color becomes noticeably lighter, Figure 2D No obvious color change occurs. Figure 2A The biological material in the figure is sample 1, that is, fresh bovine pericardial tissue, which appears light red. This is due to the electrostatic interaction between fibers. Figure 2B The control sample 2 was treated with glutaraldehyde but not capped, so its tissue contained a lot of aldehyde groups and was obviously dark in color. Figure 2CThe control sample 2 in the experiment was treated with glutaraldehyde and then capped with glycine. The aldehyde content in the tissue was less than that in the control sample 2, and the color was relatively Figure 2B shallow. Figure 2D Sample 4 in the test showed no aldehyde residues and almost no color change because the method of the present application was used. In summary, it can be seen that no aldehyde groups can be detected in the biological tissue material prepared by this method, which will bring about the following results: avoiding calcification and shortening of the service life of the bioartificial heart valve, reducing the toxicity of glutaraldehyde and causing a series of adverse physiological reactions. The biocompatibility of the leaflets is significantly improved, and the biocompatibility of the leaflets in contact with the blood is improved. In addition, due to the reduction of aldehyde residues, the calcium attachment points provided are also reduced; when prepared into an artificial valve, the service life can be increased. However, using conventional glutaraldehyde cross-linking treatment (Control Example 2 Group, Figure 2B ), and end-capping based on glutaraldehyde cross-linking treatment ( Figure 2C ), there are still aldehyde residues.

[0114] Example 9

[0115] The changes of control sample 2 and sample 4 before and after subcutaneous implantation in rats (ie, removed 8 weeks after subcutaneous implantation in rats) were compared by hematoxylin-eosin staining (HE) analysis of tissue components.

[0116] Hematoxylin stain is alkaline, which mainly stains the chromatin in the cell nucleus and the nucleic acids in the cytoplasm purple-blue; eosin is an acidic dye, which mainly stains the components in the cytoplasm and extracellular matrix red.

[0117] Figure 3A Schematic diagram of HE staining of control sample 2 prepared in control example 2 before subcutaneous implantation in rats; Figure 3B Schematic diagram of HE staining of sample 4 prepared in Example 4 before subcutaneous implantation in rats; Figure 3C This is a schematic diagram of HE staining of the control sample 2 prepared in Control Example 2 after being implanted subcutaneously in rats; Figure 3D Schematic diagram of HE staining of Sample 4 prepared in Example 4 after subcutaneous implantation in rats. The staining results show that the tissue fibers of Sample 4 are neatly arranged, without the presence of complete cellular tissue components, while the tissue fibers of Control Sample 2 are disorderly arranged, with a relatively large number of cellular tissue components.

[0118] from Figure 3C It can be seen that after implantation, the pericardial tissue of control sample 2 had an abnormal overall structure, with large areas of swelling, rupture, and necrosis of the collagen fibers of the tissue valve. Figure 3C The arrow in b shows a large area of ​​calcification in the tissue valve, as shown by the arrow in c. There is also obvious inflammatory cell infiltration in the tissue, as shown by the arrow in a.

[0119] from Figure 3D It can be seen that after implantation into rat skin, the overall structure of the tissue valve of sample 4 was basically normal, the collagen fibers of the tissue valve were arranged in a relatively neat manner, and no obvious swelling or breakage of the collagen fibers was observed. Figure 3D The middle arrows indicate collagen fibers. No significant inflammatory cell infiltration was observed in the tissue. There was also no significant calcification. This is because the low antigen content in Sample 4 improves tissue biocompatibility, avoids immune responses, and thus reduces inflammatory responses. This also reduces the calcification foci caused by tissue immune responses.

[0120] The staining results show that the tissue fibers of Sample 4 are neatly arranged, with no intact cellular tissue components present. In contrast, the tissue fibers of Control Example 2 are chaotically arranged, with a relatively large number of cellular tissue components present. Therefore, the present method reduces residual cellular components, making it more effective than existing methods in eliminating antigenic proteins or inactivating antigenic determinants, thereby reducing the content of exogenous antigens. Furthermore, the tissue fibers are neatly arranged, demonstrating that the present method does not destroy the structure that imparts mechanical properties to the tissue.

[0121] HE staining analysis of pericardial tissue morphology and inflammatory infiltration before and after implantation revealed differences between control samples 2 and 4. The residual antigenic components in the biological tissue material prepared by this method were significantly lower than those in the control group. Alternatively, the immune response caused by the residual antigenic components in the biological tissue material prepared by this method was significantly lower than that in the control group, thus avoiding post-transplant immune rejection. Therefore, the post-implantation experimental results clearly demonstrate that the biocompatibility and immune rejection of tissue treated by this method were significantly improved.

[0122] At the same time, sample 4 prepared by this preparation method did not show a large area of ​​calcified tissue, which can avoid the obstruction of valve performance when preparing heart valves.

[0123] Example 10

[0124] Anticoagulant function test of sample 4 and control sample 2

[0125] Cut sample 4 and control sample 2 into 1 cm x 1 cm pieces and clean them. Sterilize surgical instruments before use. Subcutaneously implant one test sample and one control sample into the right back of the rats, and suture the skin incisions. After 8 weeks, weigh the animals and remove the implants. Rinse the implants with saline, flatten them, and photograph them. The experimental results are shown in Figure 4.

[0126] The tissue morphology in the photographs shows that Sample 4 is intact, with no obvious blood cell deposits. Control Sample 2 exhibits extensive blood cell deposits and tissue hyperplasia, and the tissue fibers in Control Sample 2 are disorganized compared to Sample 4. A comprehensive assessment of implant biocompatibility and anticoagulant properties, based on surface fiber, thrombus, and hyperplasia, indicates that Sample 4 exhibits excellent anticoagulant properties and, when fabricated into an artificial valve, could enhance the hemocompatibility of the valve leaflets.

[0127] Samples 1 to 3, 5 to 6 prepared in Examples 1 to 3, 5 and 6 were tested using the same test method as described in Examples 7 to 10, and the test results were similar to those of Sample 4, indicating that the samples prepared by the preparation method of the present application have good mechanical and thermodynamic properties, blood compatibility, and a significantly reduced aldehyde content.

[0128] In summary, the present invention effectively overcomes the various shortcomings of the prior art and has high industrial application value. The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concepts disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A method for preparing a biological tissue material, characterized in that: The biological tissue material preparation method comprises the following steps c: The pretreated tissue is functionally modified, wherein the tissue functional modification is oxidation of the groups on the pretreated tissue and grafting of anti-inflammatory and anticoagulant functional groups.

2. The method for preparing biological tissue material according to claim 1, characterized in that: The pre-treated tissue is processed through the following steps: a. Tissue fixation: fixation is to preserve the intrinsic substances of cells and maintain the intrinsic morphology and structure of cells and tissues; b. Tissue defatting to remove fat in the tissue, wherein the tissue defatting includes physical defatting and / or chemical defatting; Preferably, the tissue in step a is pericardial tissue, cartilage tissue, skin, or other membrane structure tissue; more preferably, the tissue in step a is pericardial tissue; Preferably, the tissue fixation in step a is to treat the tissue with ionic cross-linking and / or covalent cross-linking; the tissue defatting in step b is to treat the tissue fixed in step a with a betaine-type detergent and / or an alkaline alcohol reagent; Preferably, the ionic bond cross-linking treatment in step a is to immerse the tissue in a metal ion solution; The covalent bond cross-linking treatment in step a is soaking in a mixed solution of a carbonyl activation reagent and a coupling reagent; Preferably, the ionic crosslinking in step a is to immerse the tissue in a 0.01mM to 1mM metal ion solution; more preferably, the tissue is immersed in a 0.01mM to 1mM metal ion solution at 10 to 15° C. for 0.5 to 2h; Preferably, the covalent cross-linking treatment in step a is soaking in a mixed solution of 10-60 mM carbonyl activation reagent and 5-30 mM coupling reagent; more preferably, the covalent cross-linking treatment is soaking in a mixed solution of 10-60 mM carbonyl activation reagent and 5-30 mM coupling reagent for 4-12 hours.

3. The method for preparing a biological tissue material according to any one of claims 1 to 2, characterized in that: The alkaline alcohol reagent treatment in step b is soaking in an alkaline alcohol reagent solution; The betaine-type detergent treatment in step b is 0.01% to 3% w / w betaine-type detergent solution; Preferably, the alkaline alcohol reagent treatment in step b is soaking in an alkaline alcohol solvent at 2 to 8°C; more preferably, the alkaline alcohol reagent treatment is soaking in an alkaline alcohol solvent at 2 to 8°C for 2 to 12 hours; Preferably, the betaine-type detergent treatment in step b is soaking in a betaine-type detergent solvent at 15° C.; more preferably, the betaine-type detergent treatment is soaking in a betaine-type detergent solvent at 15° C. for 2 hours.

4. The method for preparing a biological tissue material according to any one of claims 2 to 3, characterized in that: The oxidation treatment of step c is to immerse the tissue material in an oxidant; the grafting combined treatment of step c is to use 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole to graft and introduce at least one of taurine, L-sulfoalanine, N-(2-acetamido)-2-aminoethanesulfonic acid, 2-[(2-aminoethyl)amino]ethanesulfonic acid sodium salt, curcumin, salicylic acid, tea polyphenols, anthocyanidins, tannic acid, gallic acid, and propyl gallate; Preferably, the oxidation treatment in step c is to immerse the tissue material in a 0.01-0.1 mM oxidant; more preferably, the oxidation treatment is to immerse the tissue material in a 0.01-0.1 mM oxidant at 5-30° C. for 10-60 min; Preferably, the grafting combined treatment in step c is to soak for 12 to 24 hours at 10 to 25° C. using 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1-hydroxybenzotriazole to graft and introduce 0.1 to 5% (w / w) of taurine, L-sulfoalanine, N-(2-acetamido)-2-aminoethanesulfonic acid, 2-[(2-aminoethyl)amino]ethanesulfonic acid sodium salt, curcumin, salicylic acid, tea polyphenols, anthocyanins, tannic acid, gallic acid, and at least one of propyl gallate.

5. The method for preparing a biological tissue material according to any one of claims 2 to 4, characterized in that: The metal ion of the metal ion solution is selected from at least one of iron ion, aluminum ion and zinc ion; The carbonyl activating agent is selected from at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, dimethylaminopropylethylcarbodiimide and the like carbonyl activating agents; The coupling reagent is selected from at least one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide sulfonic acid sodium salt, 1-hydroxybenzotriazole, and benzyloxycarbonyl succinimide.

6. The method for preparing a biological tissue material according to any one of claims 3 to 5, characterized in that: The alkaline alcohol reagent is a base-binding agent used to adjust the acidity and alkalinity of the alcohol reagent, the alcohol reagent is selected from at least one of ethanol, isopropanol, propanol, butanol, octanol, pentanol, hexanol, heptanol, hemp alcohol, ethylene glycol, glycerol, propylene glycol, tert-butyl alcohol, or 1,3-butanediol, and the base-binding agent is selected from at least one of potassium carbonate, ammonium carbonate, or sodium carbonate; The betaine-type detergent is at least one of betaine salicylate, (3-([N,N-dimethyldimethyl(3-myristoylamidepropyl)ammonium]propane-1-sulfonate (ABS-14), and 3-[N,N-dimethyl(3-palmitamidopropyl)ammonium]-propanesulfonic acid inner salt (ABS-16)).

7. The method for preparing a biological tissue material according to any one of claims 4 to 6, characterized in that: The oxidant is selected from at least one of sodium periodate, potassium periodate, potassium dichromate, peracetic acid, and 2,2,6,6-tetramethylpiperidinyl oxide (TEMPO).

8. The method for preparing a biological tissue material according to any one of claims 1 to 7, characterized in that: The biological tissue material preparation method is selected from any one of the following: After the biological tissue material is peeled, trimmed and cleaned, it is immersed in a 0.05mM ferric chloride solution at 10°C for 2 hours, and then rinsed with physiological saline; then immersed in a 1% (w / w) 3-([N,N-dimethyldimethyl (3-tetradecanoylpropyl) ammonium] propane-1-sulfonate (ABS-14) solution at 15°C for 2 hours, and then immersed in an alkaline 1,3-butanediol solution at 2°C for 4 hours, and rinsed with a PBS solution and / or a D-Hanks solution; then immersed in a 0.1mM sodium periodate solution at 25°C for 30 minutes; finally immersed in a mixed solution of 0.5% (w / w) curcumin and containing 20mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 10mM 1-hydroxybenzotriazole at 10°C for 24 hours; or After the biological tissue materials were peeled, trimmed and washed, they were heated at 10°C in the presence of 0.01 mM ferric chloride, 20 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 10 mM Soak in a mixed solution of sodium salt of N-hydroxysuccinimide sulfonate for 4 hours, then rinse with physiological saline; then soak in a 1% (w / w) 3-[N,N-dimethyl (3-palmitamidopropyl) ammonium]-propane sulfonic acid inner salt (ABS-16) solution at 15°C for 2 hours, then soak in an alkaline isopropanol solution at 2°C for 2 hours, rinse with PBS solution and / or D-Hanks solution; then soak in a 0.1mM 2,2,6,6-tetramethylpiperidin oxide (TEMPO) solution at 25°C for 50 minutes; finally soak in a mixed solution of anthocyanidins at 10°C, 0.1% (w / w) and containing 40mM 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and 20mM 1-hydroxybenzotriazole for 24 hours; or After the biological tissue material is peeled, trimmed and cleaned, it is immersed in 0.1mM zinc chloride at 15°C for 2 hours and rinsed with physiological saline; then immersed in 1% (w / w) 3-([N,N-dimethyldimethyl(3-tetradecanoylpropyl)ammonium]propane-1-sulfonate (ABS-14) at 15°C for 2 hours, and then immersed in an alkaline propylene glycol solution at 8°C for 4 hours; rinsed with PBS solution and / or D-Hanks solution; then immersed in 0.01mM peracetic acid solution at 25°C for 30 minutes; finally immersed in a mixed solution of 0.5% (w / w) L-sulfoalanine and containing 20mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10mM 1-hydroxybenzotriazole at 10°C for 24 hours; or After the biological tissue materials were peeled, trimmed and cleaned, they were immersed in a mixed solution of 0.05 mM zinc chloride, 40 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 20 mM sodium salt of N-hydroxysuccinimide sulfonate at 10°C for 12 h, rinsed with physiological saline; then, they were immersed in a 1% (w / w) 3-[N,N-dimethyl(3-palmitamidopropyl)ammonio]-propanesulfonic acid inner salt (ABS-16) solution at 15°C. Soak in an alkaline tert-butyl alcohol solution at 5°C for 2 hours, then soak in an alkaline tert-butyl alcohol solution at 5°C for 2 hours; rinse with PBS solution and / or D-Hanks solution; then soak in a 0.1 mM sodium periodate solution at 25°C for 30 minutes; finally soak in a mixed solution of 0.5% (w / w) taurine and containing 20 mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mM 1-hydroxybenzotriazole at 10°C for 24 hours; or After the biological tissue material is peeled, trimmed and cleaned, it is immersed in 0.1mM aluminum chloride at 15°C for 2 hours and rinsed with physiological saline; then immersed in 1% (w / w) 3-[N,N-dimethyl(3-palmitamidopropyl)ammonio]-propanesulfonic acid inner salt (ABS-16) at 15°C for 2 hours, and then immersed in an alkaline ethanol solution at 5°C for 12 hours; rinsed with PBS solution and / or D-Hanks solution; then immersed in 0.01mM peracetic acid solution at 25°C for 30 minutes; finally immersed in a mixed solution of 0.5% (w / w) N-(2-acetamido)-2-aminoethanesulfonic acid and containing 20mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10mM 1-hydroxybenzotriazole at 10°C for 24 hours; or After peeling, trimming and cleaning, the biological tissue materials were immersed in a mixed solution of 0.05 mM aluminum chloride, 60 mM 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 30 mM sodium salt of N-hydroxysuccinimide sulfonate at 10°C for 4 h, rinsed with physiological saline; then immersed in a 1% (w / w) 3-[N,N-dimethyl(3-palmitamidopropyl)ammonio]-propanesulfonic acid inner salt (ABS-16) solution at 15°C for 2 h; and then immersed in a mixed solution of 1% (w / w) 3-[N,N-dimethyl(3-palmitamidopropyl)ammonio]-propanesulfonic acid inner salt (ABS-16) at 5 ℃ alkaline 1,3-butanediol solution for 2 hours; rinse with PBS solution and / or D-Hanks solution; then soak in 25℃, 0.1mM sodium periodate solution for 30 minutes; finally soak in 10℃, 0.5% (w / w) 2-[(2-aminoethyl)amino]ethanesulfonic acid sodium salt and a mixed solution containing 20mM 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10mM 1-hydroxybenzotriazole for 24 hours; The reagents attached to the biological tissue material are rinsed with physiological saline to be cleaned.

9. The biological tissue material prepared according to the biological tissue material preparation method according to any one of claims 1 to 8; preferably, the biological tissue material is selected from pericardial tissue, cartilage tissue, skin, and other membrane structure tissues; more preferably, the biological tissue material is selected from pericardial tissue.

10. An artificial heart valve prepared using the biological tissue material according to claim 9.

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