Tissue repair material and its preparation method and application

Abstract

The present invention relates to a method for preparing a tissue repair material, comprising the steps of pretreatment, defatting, virus inactivation, decellularization, material recovery, freeze-drying, and sterilization. It also relates to a tissue repair material prepared by this method and its application. The method for preparing a tissue repair material provided by the present invention can effectively remove immunogenic substances from the material while better restoring the tensile strength and elasticity of the tissue material, thereby obtaining a bioscaffold material with improved biocompatibility, non-immunogenic cell attachment, proliferation, differentiation, and mechanical properties. The tissue repair material obtained by the method of the present invention has excellent efficacy in guiding cartilage regeneration.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and more specifically relates to a tissue repair material and a preparation method and application thereof. Background Art

[0002] Tissue and organ damage or failure is a major challenge in human medicine. Clinical treatments typically employ surgical repair, tissue and organ transplantation, prosthetic implants, and mechanical devices to reconstruct, restore, or compensate for lost function to varying degrees. While artificial organs and transplants have benefited numerous patients, they also exhibit significant weaknesses: artificial organs are incompatible with the human body and cannot replicate advanced human functions; artificial organs have limited donors and are subject to immune rejection. Tissue engineering can address these challenges.

[0003] Currently, decellularized bioscaffolds derived from various tissues have been studied and applied. Commercialized tissue-engineered skin, cartilage, and other products have officially entered clinical use. The clinical application of tissue-engineered bone, tendon, skeletal muscle, cornea, mucosa, blood vessels, bladder, pancreas, genitals, kidney, liver, etc. has also begun and achieved certain therapeutic effects. Decellularized bioscaffolds can serve as the construction basis of tissue engineering and have greater advantages than synthetic materials. Decellularized matrices not only retain the three-dimensional structure and extracellular matrix of native tissues, but also have non-immunogenicity, biodegradability, sealing, non-toxicity, non-carcinogenicity, good biocompatibility and mechanical properties. They also retain basic fibroblast growth factor, etc., and are considered to be ideal scaffold materials for regeneration.

[0004] Common decellularization methods include physical methods (freeze-thaw, pressurization, ultrasound, etc.), chemical methods (acids, alkalis, hypotonic and hypertonic solutions, non-ionic detergents, ionic detergents, zwitterionic detergents, metal ion chelators, etc.), enzymatic methods (nucleases, trypsin, lipase, etc.), and combinations of these methods. However, all decellularization methods result in potential loss of surface structure and composition and structural damage. Minimizing unnecessary damage to the extracellular matrix (ECM) is the goal of the decellularized biomaterials field.

[0005] The invention patent (publication number CN104524634 B) discloses a method for preparing a tissue repair material, which is completed by pretreatment, organic solvent degreasing, ethanol solution virus inactivation, hypertonic and hypotonic solution decellularization, acid or alkali solution swelling treatment, rapid freeze-drying and sterilization. The resulting tissue repair material has a thickness increased by 2-4 times, the material is loose and porous, the mechanical properties and degradability change little, the decellularization effect is better, and it has high biosafety and biocompatibility, suitable thickness and loose and porous structural characteristics. However, this method still needs to be optimized to further reduce damage to the ECM under the premise of excellent decellularization effect, so that the material can achieve better mechanical properties. Summary of the Invention

[0006] To address the shortcomings of the aforementioned product technologies, the present invention provides a method for preparing a tissue repair material that is completely decellularized, leaving no intact cell structure or cell fragments, and minimizing the risk of residual DNA and reagents. By removing the immunogenic substances from the material, the present invention innovatively introduces reagents that restore the mechanical properties of the decellularized biomaterial, restoring the material's excellent tensile and degradation properties. This significantly improves the industrialization of the product and optimizes clinical procedures for physicians.

[0007] In one aspect, the present invention provides a method for preparing a tissue repair material, characterized in that it comprises the following steps:

[0008] (1) Pretreatment: Spread the membrane tissue from mammals on a flat plate, remove attached fat, connective tissue, or damaged tissue at the edge, and then wash with water until there is no blood to obtain the biofilm material;

[0009] (2) Degreasing: The obtained biofilm material is placed in an organic solvent and shaken for degreasing for 2-10 hours, then the liquid is changed, and the degreasing is repeated 2-4 times, and then washed with water until there is no odor;

[0010] (3) Virus inactivation: Place the defatted biofilm material in an inactivation solution for 1-3 hours and then wash with water until it is odorless;

[0011] (4) Decellularization: The inactivated biofilm material is placed in a decellularization hypertonic solution and shaken for 30-90 minutes, then transferred to a decellularization hypotonic solution and shaken for 30-90 minutes, and this cycle is repeated 2-5 times;

[0012] (5) Material recovery: The decellularized biofilm material is placed in a recovery solution and allowed to stand for 30-90 minutes. The recovery solution includes a and / or b solution. The a solution is a blue copper peptide solution. Preferably, the final concentration of the blue copper peptide in the a solution is 0.5-3 M. The b solution is a vitamin P solution. Preferably, the final concentration of the vitamin P in the b solution is 0.5-3 M. Preferably, after the material recovery is completed, it is rinsed with water 6-15 times.

[0013] (6) Freeze-drying: Flatten the recovered biofilm material, place it on a plate, and place it directly into the freeze dryer, or add water to the plate to cover the surface of the material before placing it into the freeze dryer; freeze-drying conditions are to cool the material to -80°C to -20°C at a rate of 5-12°C / min and maintain it at -20°C to -10°C for 10-16 hours;

[0014] (7) Sterilization: Sterilize by ethylene oxide or cobalt 60 irradiation to obtain the tissue repair material.

[0015] The inventors unexpectedly discovered that after decellularizing the material, the recovery solution of the present invention is used for treatment to better restore the mechanical properties of the material, which is better than other recovery solution formulas. During the processing, one type of solution can be used alone or multiple solutions can be used in a proportioned manner. Preferably, the combination of solution a and solution b can achieve better results than using either solution alone. The ratio of solution a to solution b can range from 0.1-10:1, preferably 0.1-5:1, and more preferably 0.5-3:1. In a specific embodiment, the ratio of solution a to solution b is 1:1.

[0016] Optionally, the mammal in step (1) is selected from any one of pigs, cattle, and sheep, and the membrane tissue is selected from any one of dermis, bladder matrix membrane, peritoneum, fascia, pericardium, heart valve, small intestine, and basement membrane. Preferably, the membrane tissue is peritoneal tissue. In a specific embodiment, the mammalian membrane tissue selected in the present invention is porcine peritoneal tissue. The material processing method of the present invention can better protect the porcine peritoneal structure, so that the final product has better safety and mechanical properties.

[0017] Optionally, the organic solvent in step (2) is selected from any one or more of ethanol, propanol, isopropanol, a mixture of methanol and chloroform, n-hexane, and ethyl acetate.

[0018] Optionally, the inactivation solution in step (3) includes one or more of sodium hydroxide, sodium chloride, potassium hydroxide, ammonia water, hydrochloric acid, nitric acid, phosphoric acid, acetic acid, ethanol, propanol, isopropanol, and methanol.

[0019] Optionally, the decellularized hypertonic solution in step (4) is a solution formed by adding one or more of sodium hydroxide, potassium hydroxide, hydrochloric acid, and acetic acid to a 0.5-5M sodium chloride solution to make the final concentration of one or more of sodium hydroxide, potassium hydroxide, hydrochloric acid, and acetic acid be 0.01-1M; the decellularized hypotonic solution is water.

[0020] Optionally, the solution a in step (5) is a solution of blue copper peptide dissolved in a polysaccharide polymer solution, wherein the polysaccharide polymer is selected from one or more of chitosan, sodium hyaluronate, sodium carboxymethyl cellulose, sodium alginate, methylcellulose, ethyl cellulose, and hemicellulose. The concentration of the polysaccharide polymer in the solution can range from 0.01 to 5M, preferably 0.1 to 2M.

[0021] Optionally, the solution b in step (5) is vitamin P dissolved in a collagen solution. The concentration of collagen in the solution can range from 0.01 to 5M, preferably from 0.1 to 2M.

[0022] The inventors have discovered that using a polysaccharide polymer solution to dissolve blue copper peptides and using a collagen solution to dissolve vitamin P can better exert the material recovery function of blue copper peptides and vitamin P compared to using other solvents.

[0023] Optionally, the recovery solution in step (5) includes solutions a and b.

[0024] Optionally, before step (6), the biofilm material is soaked in a high-molecular-weight polysaccharide solution for 30-60 minutes, then the biofilm is rinsed with water 6-15 times before the freeze-drying operation is performed; the high-molecular-weight polysaccharide is selected from one or more of chitosan, sodium hyaluronate, sodium carboxymethylcellulose, sodium alginate, methylcellulose, ethylcellulose, and hemicellulose; preferably, the concentration of the high-molecular-weight polysaccharide solution is 0.3-1M. Performing this specific step before freeze-drying can further enhance the protective effect on the ECM structure of the material.

[0025] On the other hand, the present invention also provides a tissue repair material, characterized in that it is prepared using the above-mentioned preparation method.

[0026] In another aspect, the present invention further provides the use of the above-mentioned tissue repair material in any one or more of the following:

[0027] (1) Preparation of materials for soft tissue repair;

[0028] (2) Preparation of guided cartilage tissue regeneration materials;

[0029] (3) Prepare materials to isolate the tissues surrounding the bone tissue repair area and prevent tissue adhesion.

[0030] The method for preparing a tissue repair material provided by this invention effectively removes immunogenic substances from the material, while better restoring the material's tensile strength and elasticity, preserving the micro- and macrostructure of biological tissues and their functional matrix proteins. This results in a bioscaffold material with improved biocompatibility, non-immunogenic cell attachment, proliferation, differentiation, and mechanical properties. The tissue repair material obtained by this method exhibits excellent efficacy in guiding cartilage regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0032] Figure 1 The following is a comparison of histological staining of the products obtained by the methods of Example 2 and Comparative Example 2.

[0033] Figure 2The microstructure comparison diagram of the products obtained by the methods of Example 2 and Comparative Example 2. A is the product obtained by the method of Comparative Example 2, and B is the product obtained by the method of Example 2.

[0034] Figure 3 This is a comparison chart of the animal experimental histological degradation performance results of the products obtained by the methods of Example 2 and Comparative Example 2.

[0035] Figure 4 This is a comparison chart of the jaw repair animal test results of the products obtained by the methods of Example 2 and Comparative Example 2.

[0036] Figure 5 Figures show the results of animal experiments on cartilage defect repair using the product obtained by the method of Example 2. A1, B1, C1: Gross images, HE staining, and Safranin-O staining of samples taken 1 month after sampling from the experimental group; A2, B2, C2: Gross images, HE staining, and Safranin-O staining of samples taken 6 months after sampling from the experimental group; A3, B3, C3: Gross images, HE staining, and Safranin-O staining of samples taken 12 months after sampling from the experimental group.

[0037] Figure 6 Results of animal experiments on cartilage defect repair using the product obtained using the method of Comparative Example 2. A1, B1, C1: Gross images and Safranin-O staining results of samples taken from the control group at 1 month; A2, B2, C2: Gross images and Safranin-O staining results of samples taken from the control group at 6 months; A3, B3, C3: Gross images and Safranin-O staining results of samples taken from the control group at 12 months. DETAILED DESCRIPTION

[0038] The present invention is further described below with reference to the examples. It should be understood that the examples are only used to further illustrate and explain the present invention and are not intended to limit the present invention.

[0039] Unless otherwise defined, technical and scientific terms used in this specification have the same meanings as commonly understood by those skilled in the art. Although methods and materials similar or identical to those described herein can be used in experiments or applications, the materials and methods are described herein below. In the event of conflict, the present specification, including definitions, will control. The materials, methods, and examples are provided for illustrative purposes only and are not intended to be limiting.

[0040] Unless otherwise specified, the test methods, detection methods and conventional experimental reagent preparation methods used in the embodiments of the present invention are all in accordance with conventional operations in the art.

[0041] The membrane tissue used in the examples was purchased from the Red Maple Breeding Professional Cooperative Company in Lintong District, Xi'an.

[0042] Blue copper peptide was purchased from Xi'an Tianguangyuan Biotechnology Co., Ltd. The sequence is Gly-His-Lys-Cu2+ .

[0043] Vitamin P was purchased from Shanxi Qianren Biotechnology Co., Ltd.

[0044] Collagen was purchased from Guangzhou Oubedi Biotechnology Co., Ltd.

[0045] Collagen tripeptide was purchased from Jiangsu Fushengde Bioengineering Co., Ltd.

[0046] Example 1 Preparation of tissue repair materials

[0047] Step 1: Pretreatment: Spread the pig peritoneal tissue on a flat plate, remove attached fat, connective tissue and damaged tissue at the edge, and then wash with water until there is no blood to obtain a biofilm material;

[0048] Step 2: Degreasing: The biofilm material was placed in an organic solvent and shaken for 6 hours, then the solution was changed and degreasing was repeated three times, followed by washing with water until odorless. The organic solvent was propanol.

[0049] Step 3: Virus inactivation: The obtained biofilm material was placed in an inactivation solution and allowed to stand for 2 hours for inactivation, and then washed with water until odorless. The inactivation solution was a sodium hydroxide solution (concentration 0.2M).

[0050] Step 4: Decellularization: The inactivated biofilm material was placed in a decellularization hypertonic solution and shaken for 40 minutes, then transferred to a decellularization hypotonic solution and shaken for 60 minutes, and this cycle was repeated three times; the decellularization hypertonic solution was 0.5M sodium chloride solution with 1M sodium hydroxide added; the decellularization hypotonic solution was water.

[0051] Step 5. Material Recovery: The decellularized biofilm material was placed in a recovery solution for 50 minutes. The recovery solution was a 1:1 mixture of the following two solutions: blue copper peptide dissolved in sodium hyaluronate solution (sodium hyaluronate concentration 0.5M), to a final concentration of 0.8M; and vitamin P dissolved in collagen solution (collagen concentration 1.0M), to a final concentration of 0.8M. After the material recovery was complete, it was rinsed with water nine times.

[0052] Step 6: Freeze-drying: Flatten the biofilm material obtained in step 5 and stick it on a plate. Add water to the plate to cover the surface of the material and then put it into a freeze dryer. Freeze-drying is performed by cooling to -80°C at a rate of 8°C / min and maintaining it at -20°C for 16 hours. After freeze-drying, cut and package it.

[0053] Step 7: Sterilization: Sterilize with ethylene oxide to obtain tissue repair material.

[0054] Example 2 Preparation of tissue repair materials

[0055] Step 1: Pretreatment: Spread the pig peritoneal tissue on a flat plate, remove attached fat, connective tissue and damaged tissue at the edge, and then wash with water until there is no blood to obtain a biofilm material;

[0056] Step 2: Degreasing: The biofilm material was placed in an organic solvent and shaken for 4 hours, then the solution was changed and degreasing was repeated 3 times, followed by washing with water until odorless. The organic solvent was isopropyl alcohol.

[0057] Step 3: Virus inactivation: The obtained biofilm material was placed in an inactivation solution containing 75% ethanol for 1 hour and then washed with water until odorless.

[0058] Step 4: Decellularization: The inactivated biofilm material was placed in a decellularization hypertonic solution and shaken for 30 minutes, then transferred to a decellularization hypotonic solution and shaken for 60 minutes, and this cycle was repeated three times; the decellularization hypertonic solution was 3M sodium chloride solution with 0.25M sodium hydroxide added; the decellularization hypotonic solution was water.

[0059] Step 5. Material Recovery: The decellularized biofilm material was placed in a recovery solution for 50 minutes. The recovery solution was a 1:1 mixture of the following two solutions: blue copper peptide dissolved in chitosan solution (chitosan concentration 0.2M), to a final concentration of 0.8M; and vitamin P dissolved in collagen solution (collagen concentration 0.3M), to a final concentration of 0.8M. After recovery, the material was rinsed with water nine times.

[0060] Step 6. Freeze-drying: Sodium hyaluronate is used as the base reagent, and sodium carboxymethyl cellulose is added to a final concentration of 0.4M high-molecular-weight polysaccharide. The biofilm material is soaked for 40 minutes and rinsed with water 8 times. The biofilm material is flattened, attached to a plate, and directly placed in a freeze dryer. Freeze-drying is performed by cooling to -20°C at a rate of 5°C / min and maintaining at -10°C for 16 hours. After freeze-drying, it is cut and packaged.

[0061] Step 7: Sterilization: Sterilize with cobalt 60 irradiation to obtain tissue repair material.

[0062] Example 3

[0063] The preparation method of Example 2 was followed, except that the recovery solution was a blue copper peptide chitosan solution of equal volume to the recovery solution of Example 2, and other preparation methods remained consistent.

[0064] Example 4

[0065] The preparation method of Example 2 was followed, except that the recovery solution was a vitamin P collagen solution of the same volume as that of the recovery solution of Example 2, and other preparation methods remained the same.

[0066] Comparative Example 1

[0067] The preparation method of Example 2 is followed, except that collagen tripeptide is used to replace blue copper peptide, and vitamin E is used to replace vitamin P.

[0068] Comparative Example 2

[0069] The tissue repair material was prepared by the method of Example 1 of CN104524634A.

[0070] Experimental Example 1 Mechanical Properties Test

[0071] The products obtained in Example 2, Example 3, Example 4 and Comparative Examples 1 and 2 were subjected to mechanical property testing of biomaterials.

[0072] Suture tear force, tensile strength, and elongation at break of biomaterials after decellularization and recovery were measured using the strip method, part 1 of GB / T 3923.1-2013. The results showed that the mechanical properties of biomaterials treated with the present method were significantly different from those of the control material.

[0073] Table 1 Suture tearing force

[0074]

[0075] Table 2 Tensile strength at break

[0076]

[0077]

[0078] Table 3 Elongation at break

[0079]

[0080] Experimental Example 2 In vitro degradation detection

[0081] The products obtained in Example 2, Example 3, Example 4, Comparative Example 1 and Comparative Example 2 were subjected to in vitro degradation testing.

[0082] After decellularization and material recovery, the sample was cut into uniform fragments and added to 10 mL of 0.1 M (pH 7.4) PBS buffer containing 50 CDU / mL type I collagenase. The membrane was shaken at 37°C, and the in vitro degradation of the membrane was observed and recorded. The results showed that the biomaterials of the present invention had better degradation performance and longer in vitro degradation time than the comparative examples. During clinical use, the barrier time was more advantageous, which can effectively promote tissue healing and give full play to the natural advantages of decellularized biomaterials.

[0083] Table 4 In vitro degradation test

[0084]

[0085] Experimental Example 3: Detection of Immunogenic Substances

[0086] To verify the safety of the samples, immunogenic substances were detected on the untreated biofilm tissues and the samples prepared in Examples 1 and 2.

[0087] (1) Cell residue detection method: biological materials and skin tissues were fixed with 10% formaldehyde, collected, dehydrated, paraffin-embedded, sliced, and stained with HE and Masson staining. The cell nucleus residue and matrix fiber structure were observed under a microscope.

[0088] (2) DNA residue detection method: The test shall be conducted in accordance with the requirements of YY / T 0606.25-2014 Tissue Engineering Medical Products Part 25: Determination of DNA Residues in Animal-Derived Biomaterials: Fluorescence Staining Method.

[0089] (3) Fat content detection method: Determine the fat content in accordance with the Soxhlet extraction method in the first method of GB 5009.6-2016 National Food Safety Standard Determination of Fat in Food.

[0090] (4) α-Gal antigen clearance rate determination: α-Gal antigen clearance rate detection was performed according to the inhibition ELISA test method specified in the industry standard YY / T 1465.5-2016 "Medical device immunogenicity evaluation method Part 5: Determination of α-Gal antigen clearance rate in animal-derived medical devices using M86 antibody".

[0091] Table 5 Immunogenicity test data

[0092]

[0093] Experimental Example 4 Histological Sections

[0094] Six tissues of at least 1.0×1.0 cm in size were taken and numbered A1-A3 and B1-B3, wherein A1-A3 were prepared according to the method of Comparative Example 2 and B1-B3 were prepared according to the method of Example 2. Conventional HE staining technique was used to perform histological staining on the above samples. Figure 1 As shown, the results show that the natural extracellular matrix structure of the biomaterials A1-A3 is damaged to a certain extent, and the collagen fibers are split to a certain extent; after the recovery process using the materials of the present invention, the results clearly show that the collagen fiber structure is repaired to a large extent, and the tissue structure is close to the natural extracellular matrix.

[0095] Experimental Example 5 Microstructure Observation

[0096] The biomaterials prepared in Example 2 and Comparative Example 2 were cut into pieces of about 1 mm × 5 mm in size, and then treated and stained and observed using a transmission electron microscope. Figure 2 As shown, the results show that the natural collagen fiber structure of the biomaterial of Comparative Example 2 is destroyed, the unique triple helix structure is incomplete, and the collagen fibers are loosely arranged; the process after the restoration of the material of the present invention is used, and the results show that an obvious and clear collagen fiber arrangement structure can be seen, and a periodic horizontal stripe / layered arrangement structure can be seen along the long axis direction of the collagen fibers, and the collagen fiber structure is effectively restored.

[0097] Experimental Example 6: Degradation Performance Animal Experiment

[0098] The samples prepared in Example 2 and Comparative Example 2 were subjected to a subcutaneous implantation test in rats to verify the degradation performance of the materials. Figure 3 As shown in the histological results of animal experiments, it can be seen that the biomaterial of Comparative Example 2 is completely degraded after 6-8 months of implantation, and the biomaterial of Example 2 is completely degraded after 8-10 months of implantation. The use of material recovery reagents can effectively prolong the barrier effect time of its biomatrix.

[0099] Experimental Example 7: Effectiveness Experiment of Jaw Repair

[0100] The samples prepared in Example 2 and Comparative Example 2 were tested for effectiveness in filling canine extraction sockets to evaluate the soft tissue healing and bone tissue regeneration capabilities. Figure 4 As shown in the histological results of animal experiments, the biomaterial of Example 2 showed early vascularization and early bone formation beneath the collagen membrane after 2 weeks of implantation. New bone formation continued after 12-16 weeks of implantation, and the epithelium completely covered the defect area. In contrast, the membrane of the biomaterial of Comparative Example 2 showed less obvious early vascularization and slower new bone formation.

[0101] Experimental Example 8: Effectiveness Experiment of Cartilage Defect Repair

[0102] The samples prepared in Example 2 and Comparative Example 2 were subjected to a rabbit cartilage repair effectiveness test to evaluate the regenerative capacity of cartilage repair. Figure 5 and Figure 6As shown in the figure, the histological results of the animal experiments show that the new tissue of the biomaterial of Example 2 heals well with the surrounding tissue 1m, 6m and 12m after surgery. At 1m, incompletely degraded material can be seen, and the tissue has weak Safranin-O metachromasia, indicating that mature cartilage tissue has not yet formed. At 6m, the material is completely degraded, the cartilage tide line is obvious, the Safranin-O metachromasia is obvious, and cartilage pits are present, indicating that hyaline cartilage has formed. When the sample is taken at 12m, the tide line gradually moves upward, approaching normal cartilage, and the Safranin-O metachromasia is similar to that of normal cartilage. In contrast, the histological sections of the biomaterial of Comparative Example 2 taken at 1m and 6m after surgery show that some fibrous tissue has been generated ( Figure 6 By the time of sampling at 12 minutes, the defect was filled with cartilage-like tissue. Safranin-O staining showed that metachromasia was not obvious at each sampling time, and no hyaline cartilage repair was formed.

[0103] It should be understood that the present invention disclosed is not limited only to the particular methods, schemes and materials described, as these can vary. It should also be understood that the terms used herein are merely for the purpose of describing specific embodiments, rather than being intended to limit the scope of the present invention, which is limited only by the appended claims.

[0104] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein.Such equivalents are also intended to be encompassed by the appended claims.

Claims

1. A method for preparing a tissue repair material, characterized in that: The following steps are involved: (1) Pretreatment: Spread the membrane tissue from mammals on a flat plate, remove attached fat, connective tissue, or damaged tissue at the edge, and then wash with water until there is no blood to obtain the biofilm material; (2) Degreasing: The obtained biofilm material is placed in an organic solvent and shaken for degreasing for 2-10 hours, then the liquid is changed, and the degreasing is repeated 2-4 times, and then washed with water until there is no odor; (3) Virus inactivation: Place the defatted biofilm material in the inactivation solution for 1-3 hours and wash with water until it is odorless; (4) Decellularization: Place the inactivated biofilm material in a decellularization hypertonic solution and shake for 30-90 minutes, then transfer it to a decellularization hypotonic solution and shake for 30-90 minutes, and repeat this cycle 2-5 times; (5) Material recovery: The decellularized biofilm material is placed in a recovery solution and allowed to stand for 30-90 min. The recovery solution includes solution a and / or solution b, wherein solution a is a blue copper peptide solution and solution b is a vitamin P solution. (6) Freeze-drying: Flatten the recovered biofilm material, place it on a plate, and place it directly into the freeze dryer, or add water to the plate to cover the surface of the material before placing it into the freeze dryer; freeze-drying conditions are to cool the material to -80°C~-20°C at a rate of 5-12°C / min and maintain it at -20°C~-10°C for 10-16 hours; (7) Sterilization: Sterilize by ethylene oxide or cobalt 60 irradiation to obtain the tissue repair material.

2. The method for preparing the tissue repair material according to claim 1, characterized in that: In step (1), the mammal is selected from any one of pigs, cattle, and sheep, and the membrane tissue is selected from any one of dermis, bladder matrix membrane, peritoneum, fascia, pericardium, heart valve, small intestine, and basement membrane.

3. The method for preparing the tissue repair material according to claim 1, characterized in that: The organic solvent in step (2) is selected from any one or more of ethanol, propanol, isopropanol, a mixture of methanol and chloroform, n-hexane, and ethyl acetate.

4. The method for preparing the tissue repair material according to claim 1, characterized in that: The inactivation solution in step (3) includes one or more of sodium hydroxide, sodium chloride, potassium hydroxide, ammonia water, hydrochloric acid, nitric acid, phosphoric acid, acetic acid, ethanol, propanol, isopropanol, and methanol.

5. The method for preparing the tissue repair material according to claim 1, characterized in that: The decellularized hypertonic solution in step (4) is a solution formed by adding one or more of sodium hydroxide, potassium hydroxide, hydrochloric acid, and acetic acid to a 0.5-5M sodium chloride solution to make the final concentration of the one or more of sodium hydroxide, potassium hydroxide, hydrochloric acid, and acetic acid be 0.01-1M; the decellularized hypotonic solution is water.

6. The method for preparing the tissue repair material according to claim 1, characterized in that: The recovery solution in step (5) includes solutions a and b.

7. The method for preparing the tissue repair material according to claim 1, characterized in that: The solution a in step (5) is blue copper peptide dissolved in a polysaccharide polymer solution, and the polysaccharide polymer is selected from one or more of chitosan, sodium hyaluronate, sodium carboxymethyl cellulose, sodium alginate, methyl cellulose, ethyl cellulose, and hemicellulose.

8. The method for preparing the tissue repair material according to claim 1, characterized in that: The solution b in step (5) is vitamin P dissolved in collagen solution.

9. The method for preparing the tissue repair material according to claim 1, characterized in that: The final concentration of the blue copper peptide in the solution a described in step (5) is 0.5-3M.

10. The method for preparing the tissue repair material according to claim 1, characterized in that: The final concentration of vitamin P in the solution b in step (5) is 0.5-3M.

11. The method for preparing the tissue repair material according to claim 1, characterized in that: After the material is restored in step (5), rinse with water 6-15 times.

12. The method for preparing the tissue repair material according to claim 1, characterized in that: Before performing step (6), the biofilm material is soaked in a high molecular weight polysaccharide solution for 30-60 minutes, then the biofilm is rinsed with water 6-15 times, and then the freeze-drying operation is performed; the high molecular weight polysaccharide is selected from one or more of chitosan, sodium hyaluronate, sodium carboxymethyl cellulose, sodium alginate, methyl cellulose, ethyl cellulose, and hemicellulose.

13. The method for preparing the tissue repair material according to claim 12, characterized in that: The concentration of the high molecular weight polysaccharide solution is 0.3-1M.

14. A tissue repair material, characterized in that: It is prepared using the preparation method according to any one of claims 1 to 13.

15. Use of the tissue repair material according to claim 14 in any one or more of the following: (1) Preparation of materials for soft tissue repair; (2) Preparation of guided cartilage tissue regeneration materials; (3) Prepare materials to isolate the surrounding tissues of the bone tissue repair area and prevent tissue adhesion.

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