Tissue repair material as well as preparation method and application thereof

Abstract

The invention relates to a preparation method of a tissue repair material. The preparation method comprises the steps of pretreatment, degreasing, virus inactivation, decellularization, material recovery, freeze-drying and sterilization. The invention also relates to the tissue repair material prepared by the method and application thereof. According to the preparation method of the tissue repair material provided by the invention, the stretching and elasticity of the tissue material can be better recovered on the basis of efficiently removing immunogen substances of the material, and a biological scaffold material with better biocompatibility and non-immunogen cell attachment, proliferation, differentiation and mechanical properties can be obtained. The tissue repair material obtained by the method disclosed by the invention has an excellent cartilage regeneration guiding effect.

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 functional failure is a major problem facing human medicine. Clinical treatment usually adopts surgical repair, tissue and organ transplantation, artificial prosthesis implantation, mechanical devices and other programs to enable patients to rebuild, restore or compensate for lost functions to varying degrees. Although artificial organs and transplantation surgery have benefited many patients, they have also revealed fatal weaknesses: the former is incompatible with the human body and cannot establish advanced human functions; the latter has limited donors and has problems such as immune rejection. The application of tissue engineering can solve this problem.

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

[0004] Common decellularization methods include physical methods (freeze-thaw, pressurization, ultrasound, etc.), chemical methods (acid, alkali, hypotonic and hypertonic solutions, non-ionic detergents, ionic detergents, zwitterionic detergents, metal ion chelators, etc.), enzyme methods (nuclease, trypsin, lipase, etc.) and the combination of the above methods. However, all decellularization methods will lead to 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 shock degreasing, ethanol solution virus inactivation, hypertonic and hypotonic solution decellularization, acid or alkali solution swelling treatment, rapid freeze drying and sterilization; the thickness of the obtained tissue repair material increases by 2-4 times, the material is loose and porous, the mechanical properties and degradability change little, the decellularization effect is better, it has high biosafety and biocompatibility, suitable thickness and loose and porous structural characteristics. However, the method still needs to be optimized to further reduce the 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] In view of the deficiencies of the above-mentioned product technologies, the present invention provides a method for preparing a tissue repair material, which can completely decellularize the obtained tissue repair material, without the residue of intact cell structure and cell fragments, and with low risk of DNA residue and reagent residue. On the basis of removing the immunogenic substances of the material, the present invention innovatively introduces reagents that can restore the mechanical properties of decellularized biomaterials, which can restore the good tensile properties and degradation properties of the material, etc., and has made great improvements to the industrialization of the product and the optimization of doctors' clinical operations.

[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 biomembrane 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 wash with water until there is no odor;

[0011] (4) Decellularization: The inactivated biofilm material is placed in a decellularization hypertonic solution and shaken for 30-90 min, then transferred to a decellularization hypotonic solution and shaken for 30-90 min, 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 for 6-15 times.

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

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

[0015] The inventor unexpectedly discovered that after the material is decellularized, the recovery solution of the present invention is used for treatment, which can 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 a better effect than single use. 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 the sodium hydroxide, potassium hydroxide, hydrochloric acid, and acetic acid be 0.01-1M; and the decellularized hypotonic solution is water.

[0020] Optionally, the a solution in step (5) is a 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, methyl cellulose, ethyl cellulose, and hemicellulose. The concentration of the polysaccharide polymer in the solution can range from 0.01-5M, preferably 0.1-2M.

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

[0022] The inventors found 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 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 for 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; preferably, the concentration of the high molecular weight polysaccharide solution is 0.3-1 M. 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 materials for guiding cartilage tissue regeneration;

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

[0030] The preparation method of the tissue repair material provided by the present invention can better restore the stretchability and elasticity of the tissue material on the basis of efficiently removing the immunogenic substances of the material, retain the microscopic and macroscopic structures and functional matrix proteins of the biological tissue, and obtain a biological scaffold material with better 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. 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 drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

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

[0033] Figure 2It is a microstructure comparison diagram of the products obtained by the methods of Example 2 and Comparative Example 2. Among them, 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 experiment results of the products obtained by the methods of Example 2 and Comparative Example 2.

[0036] Figure 5 The results of the cartilage defect repair animal experiment of the product obtained by the method of Example 2. A1, B1, C1: Gross photos, HE staining and Safranin-O staining results of the experimental group 1 month after sampling; A2, B2, C2: Gross photos, HE staining and Safranin-O staining results of the experimental group 6 months after sampling; A3, B3, C3: Gross photos, HE staining and Safranin-O staining results of the experimental group 12 months after sampling.

[0037] Figure 6 The results of the cartilage defect repair animal experiment of the product obtained by the method of comparative example 2 are shown in Figure 1. A1, B1, C1: Gross photos and Safranin-O staining results of the control group 1 month after sampling; A2, B2, C2: Gross photos and Safranin-O staining results of the control group 6 months after sampling; A3, B3, C3: Gross photos and Safranin-O staining results of the control group 12 months after sampling. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with 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, the technical terms and scientific terms in this specification have the same meaning as those commonly understood by those skilled in the art. Although similar or identical methods and materials as those described herein may be used in experiments or practical applications, the materials and methods are described herein below. In the event of a conflict, the present specification including the definitions therein shall prevail. In addition, the materials, methods and examples are provided for illustration only and are not intended to be limiting.

[0040] 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 unless otherwise specified.

[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 obtained biofilm material is placed in an organic solvent for degreasing by shaking for 6 hours, then the solution is changed, and the degreasing is repeated 3 times, and the biofilm material is washed with water until there is no odor. The organic solvent is propanol.

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

[0050] Step 4, decellularization: the inactivated biofilm material is placed in a decellular hypertonic solution and shaken for 40 minutes, then transferred to a decellular hypotonic solution and shaken for 60 minutes, and this cycle is repeated 3 times; the decellular hypertonic solution is 0.5M sodium chloride solution with 1M sodium hydroxide added; the decellular hypotonic solution is 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 material dissolved in sodium hyaluronate solution (sodium hyaluronate concentration 0.5M), with a final concentration of 0.8M; vitamin P dissolved in collagen solution (collagen concentration 1.0M), with a final concentration of 0.8M. After the material recovery was completed, it was rinsed with water 9 times.

[0052] Step 6, freeze drying: flatten the biofilm material obtained in step 5, 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 keeping it at -20°C for 16 hours. After freeze drying, it is cut and packaged.

[0053] Step 7: Sterilization: Use ethylene oxide for sterilization 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 obtained biofilm material is placed in an organic solvent for degreasing by shaking for 4 hours, then the solution is changed, and the degreasing is repeated 3 times, and the biofilm material is washed with water until there is no odor. The organic solvent is isopropanol.

[0057] Step 3: Virus inactivation: The obtained biofilm material is placed in an inactivation solution for 1 hour and then washed with water until there is no odor. The inactivation solution is 75% ethanol.

[0058] Step 4, decellularization: the inactivated biofilm material is placed in a decellular hypertonic solution and shaken for 30 minutes, then transferred to a decellular hypotonic solution and shaken for 60 minutes, and this cycle is repeated 3 times; the decellular hypertonic solution is 0.25M sodium hydroxide added to 3M sodium chloride solution; the decellular hypotonic solution is 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 material dissolved in chitosan solution (chitosan concentration 0.2M), with a final concentration of 0.8M; vitamin P dissolved in collagen solution (collagen concentration 0.3M), with a final concentration of 0.8M. After the material recovery was completed, it was rinsed with water 9 times.

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

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

[0062] Example 3

[0063] The preparation method of Example 2 is followed, except that the recovery solution is a blue copper peptide chitosan solution of the same volume as the recovery solution of Example 2, and other preparation methods remain the same.

[0064] Example 4

[0065] The preparation method of Example 2 is followed, except that the recovery solution is a vitamin P collagen solution of the same volume as the recovery solution of Example 2, and other preparation methods remain 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 tested for the mechanical properties of biomaterials.

[0072] The suture tear force, tensile strength and elongation at break of the biomaterial after decellularization and material recovery were measured according to the method of the first part of GB / T 3923.1-2013 (strip method). The results showed that the mechanical properties of the biomaterial treated by the method of the present invention were significantly different from those of the comparative example.

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

[0083] Table 4 In vitro degradation assay

[0084]

[0085] Experimental Example 3 Detection of immunogenic substances

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

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

[0088] (2) DNA residue detection method: The detection shall be carried out in accordance with the requirements of YY / T 0606.25-2014 Tissue Engineering Medical Products Part 25: Determination of DNA Residue 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) Determination of α-Gal antigen clearance rate: α-Gal antigen clearance rate was tested using 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 techniques were 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 process of using the material of the present invention to restore, 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 material of the present invention is used for recovery, and the results show that an obvious and clear collagen fiber arrangement structure is visible, and a periodic horizontal stripe / layered arrangement structure is visible 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, the histological results of animal experiments show 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 Bone Repair

[0100] The samples prepared in Example 2 and Comparative Example 2 were tested for effectiveness in canine tooth extraction socket filling to evaluate the soft tissue healing and bone tissue regeneration capabilities. Figure 4 As shown in the figure, the histological results of the animal experiment show that the biomaterial of Example 2 is implanted for 2 weeks, and early vascularization occurs, and early new bone formation occurs under the collagen membrane. After 12-16 weeks of implantation, new bone continues to form, and the epithelium has covered the defect area. In contrast, the membrane material of the biomaterial of Comparative Example 2 has a less obvious early vascularization effect, and new bone formation is slower.

[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, from the histological results of animal experiments, it can be seen 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 Safranin-O metachromasia is weak, 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 there are cartilage pits, indicating that hyaline cartilage has been formed. When the sample was taken at 12m, the tide line gradually moved up, approaching normal cartilage, and the Safranin-O metachromasia was 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 showed that some fibrous tissue was generated ( Figure 6 As shown in the figure, the defect was filled with cartilage-like tissue when the sample was taken at 12 minutes. Safranin-O staining showed that the 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 specific method, scheme and material described, because these all can change.It should also be understood that the terminology used herein is only for the purpose of describing specific embodiment scheme, rather than being intended to limit the scope of the present invention, and the scope of the present invention is only limited to 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 biomembrane material; (2) Degreasing: The obtained biofilm material is placed in an organic solvent for degreasing by shaking for 2-10 hours, and then the liquid is changed, and the degreasing is repeated 2-4 times, and the biofilm material is washed with water until there is no odor; preferably, the organic solvent is selected from any one or more of ethanol, propanol, isopropanol, a mixture of methanol and chloroform, n-hexane, and ethyl acetate; (3) Virus inactivation: Place the defatted biofilm material in an inactivation solution for 1-3 hours and wash with water until there is no odor; (4) Decellularization: The inactivated biofilm material is placed in a decellularization hypertonic solution and shaken for 30-90 min, then transferred to a decellularization hypotonic solution and shaken for 30-90 min, and this cycle is repeated 2-5 times; (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 for 6-15 times. (6) Freeze drying: Flatten the recovered biofilm material, stick it on a plate, and directly put it into a freeze dryer, or add water to the plate to cover the surface of the material and then put it into the freeze dryer; the freeze drying conditions are to cool down to -80°C to -20°C at a rate of 5-12°C / min, and keep it at -20°C to -10°C for 10-16h; (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 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.

4. 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 one or more of sodium hydroxide, potassium hydroxide, hydrochloric acid, and acetic acid be 0.01-1M; the decellularized hypotonic solution is water.

5. 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.

6. 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.

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

8. 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 for 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; preferably, the concentration of the high molecular weight polysaccharide solution is 0.3-1M.

9. A tissue repair material, characterized in that: It is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the tissue repair material according to claim 9 in any one or more of the following: (1) Preparation of materials for soft tissue repair; (2) Preparation of materials for guiding cartilage tissue regeneration; (3) Prepare materials to isolate the tissues surrounding the bone repair area and prevent tissue adhesion.

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