Membrane tissue treatment composition, membrane tissue treatment reagent, tissue repair material and preparation method and application thereof

Abstract

The invention relates to the field of medical biological materials in tissue engineering, in particular to a membrane tissue treatment composition, a membrane tissue treatment reagent, a tissue repair material and a preparation method and application of the membrane tissue treatment composition and the membrane tissue treatment reagent. The membrane tissue treatment composition includes tea polyphenol and gallic acid in a molar ratio of about 1: (2-8).

Description

Technical Field

[0001] The present application relates to the field of medical biomaterials for tissue engineering, and in particular to a membrane tissue processing composition, a membrane tissue processing reagent, a tissue repair material, and a preparation method and application thereof. Background Art

[0002] Oral repair membranes are widely used in oral medicine fields such as periodontology, oral implants and alveolar surgery. Oral repair membranes are a biocompatible material that is placed between oral soft tissue and bone defect areas through surgical procedures to establish a biological barrier, thereby creating a relatively closed bone regeneration environment, selectively blocking fibroblasts and epithelial cells with faster migration speeds from entering the bone defect area, while not hindering the natural healing of the wound.

[0003] Oral repair membranes can be divided into collagen membranes, metal membranes, synthetic membranes, and allogeneic periosteum based on the source of the material, and can be divided into absorbable membranes and non-absorbable membranes based on whether the material can be degraded. Among them, non-absorbable membranes require a second surgery to remove, and although non-collagen absorbable membranes do not require a second surgery, they have the side effect of acidic degradation products causing inflammation. Therefore, in a comprehensive comparison, collagen membranes are the products with the most comprehensive advantages.

[0004] Common collagen membranes are decellularized animal tissue membranes. After being processed, decellularized animal tissue membranes form a natural double-layer collagen structure. Under an electron microscope, one layer of collagen fibers is densely arranged, while the other layer is loosely arranged. This unique double-layer structure is of great significance in clinical applications. The dense side provides a natural plane for the migration of epithelial cells, which is conducive to rapid epithelialization. The loose side completely retains the natural three-dimensional spatial structure of the skin, has a suitable pore size and porosity, is conducive to the crawling and growth of cells, and provides a good scaffold for the growth and rapid vascularization of host cells. It can regulate, guide, and promote cell growth and promote vascularization, thereby completing the repair and reconstruction of tissue defects.

[0005] At present, decellularized animal tissue membranes from various tissues have been studied and applied. Commercialized tissue engineering skin, cartilage and other products have officially entered clinical application, and the clinical application of tissue engineering bone, tendon, skeletal muscle, cornea, mucosa, blood vessels, bladder, pancreas, genitals, kidneys, liver, etc. has also started and achieved certain therapeutic effects. Decellularized animal tissue membranes can be used as the construction basis of tissue engineering and have greater advantages than synthetic materials. Decellularized animal tissue membranes not only retain the natural three-dimensional structure and extracellular matrix, 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 renewable scaffold materials.

[0006] In the process of preparing decellularized animal tissue membranes, commonly used 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 (nucleases, trypsin, lipase, etc.) and the combined use of the above methods. The mechanical properties of decellularized animal tissue membranes obtained by different decellularization methods are different. Traditional decellularization methods are recorded in, for example, CN114191613A, CN104083803A, and CN118236556A. How to improve the mechanical properties of decellularized animal tissue membranes is the main goal of decellularization operations.

[0007] In view of this, this application is hereby filed. Summary of the invention

[0008] One or more embodiments of the present application provide a membrane tissue treatment composition, a membrane tissue treatment reagent, a tissue repair material, and a preparation method and application thereof, including the following technical solutions:

[0009] One or more embodiments of the present application provide a membrane tissue treatment composition, which comprises tea polyphenols and gallic acid in a molar ratio of about 1:(2-8).

[0010] In some embodiments of the present application, the membrane tissue treatment composition further comprises a high molecular weight polysaccharide compound, and the high molecular weight polysaccharide compound comprises one or more of ethyl cellulose and hydroxypropyl cellulose;

[0011] The molar ratio of the tea polyphenols, the gallic acid and the high molecular weight polysaccharide compound is about 1:(2-8):(3-5).

[0012] One or more embodiments of the present application further provide a membrane tissue treatment reagent, the membrane tissue treatment reagent comprising tea polyphenols, gallic acid and a solvent;

[0013] The molar ratio of the tea polyphenols to the gallic acid is about 1:(2-8).

[0014] In some embodiments of the present application, the total concentration of the tea polyphenols and the gallic acid is about 0.01M to 1M.

[0015] In some embodiments of the present application, the membrane tissue processing reagent also includes a high molecular weight polysaccharide compound, and the high molecular weight polysaccharide compound includes one or more of ethyl cellulose and hydroxypropyl cellulose; the molar ratio of the tea polyphenols, the gallic acid and the high molecular weight polysaccharide compound is about 1: (2-8): (3-5).

[0016] In some embodiments of the present application, the total concentration of the tea polyphenols, the gallic acid and the high molecular weight polysaccharide compound is about 0.01M to 1M.

[0017] In some embodiments of the present application, the solvent includes PBS buffer.

[0018] In some embodiments of the present application, the PBS buffer comprises about 0.1 mol / L to 0.3 mol / L of disodium hydrogen phosphate and about 0.1 mol / L to 0.3 mol / L of sodium dihydrogen phosphate, and the pH is about 6.5 to 7.5.

[0019] One or more embodiments of the present application also provide a method for preparing a tissue repair material, the method comprising the step of using the membrane tissue treatment reagent to perform a decellularization treatment on a biofilm material.

[0020] In some embodiments of the present application, the conditions for the decellularization treatment include: performing the treatment under shaking conditions, the number of treatments is about 2 to 5 times, and each treatment lasts about 30 minutes to 90 minutes.

[0021] In some embodiments of the present application, the preparation method includes multiple decellularization treatment stages, and in one of the decellularization treatment stages, the membrane tissue treatment reagent is used to perform decellularization treatment on the biofilm material.

[0022] In some embodiments of the present application, the preparation method includes a first decellularization treatment stage and a second decellularization treatment stage, and in the second decellularization treatment stage, the biofilm material is decellularized using the membrane tissue treatment reagent.

[0023] In some embodiments of the present application, the steps of the first decellularization stage include: placing the biofilm material in a hypertonic solution and a hypotonic solution in sequence for decellularization.

[0024] In some embodiments of the present application, the first decellularization treatment stage satisfies one or more of the following conditions:

[0025] 1) The hypertonic solution comprises a sodium chloride solution of about 0.01M to 1M to which a base or acid of about 0.01M to 1M is added; optionally, the base comprises one or more of sodium hydroxide and potassium hydroxide; optionally, the acid comprises one or more of hydrochloric acid and acetic acid;

[0026] 2) the hypotonic solution comprises water;

[0027] 3) the steps of the first decellularization stage are repeated about 2 to 5 times; and,

[0028] 4) The method is carried out under shaking conditions, wherein the shaking time in the hypertonic solution and the hypotonic solution is independently about 30 min to 90 min.

[0029] In some embodiments of the present application, the biofilm material is processed as follows: removal of attached fat, connective tissue and damaged tissue at the edges, defatting and virus inactivation.

[0030] In some embodiments of the present application, degreasing satisfies one or more of the following conditions:

[0031] (I) the degreasing agent used includes one or more of methanol, chloroform, ethanol, propanol, isopropanol, n-hexane and ethyl acetate; and,

[0032] (II) The degreasing process is carried out under shaking conditions for about 2 to 4 times, each time for about 2 hours to 10 hours. After each degreasing process, fresh degreasing reagent is replaced before the next degreasing process.

[0033] In some embodiments of the present application, virus inactivation is performed using chemical methods.

[0034] In some embodiments of the present application, the chemical method satisfies one or more of the following conditions:

[0035] Ⅰ) the inactivation reagent used includes one or more of an acid, a base and an alcohol; optionally, the base includes one or more of sodium hydroxide, sodium chloride, potassium hydroxide and ammonia water; optionally, the acid includes one or more of hydrochloric acid, nitric acid, phosphoric acid and acetic acid; the alcohol includes one or more of ethanol, propanol, isopropanol and methanol; and,

[0036] II) The virus is inactivated under static conditions for about 1 to 3 hours.

[0037] In some embodiments of the present application, after the decellularization process, the obtained tissue repair material is further frozen, sliced ​​and sterilized.

[0038] In some embodiments of the present application, the freezing conditions include: freeze-drying is cooling to about -80°C to -20°C at a rate of about 5°C / min to 12°C / min, and maintaining at about -20°C to -10°C for 10h to 16h.

[0039] In some embodiments of the present application, the thickness of the sliced ​​control membrane is about 0.1 mm to 1 mm.

[0040] In some embodiments of the present application, sterilization is carried out by physical sterilization or chemical sterilization.

[0041] In some embodiments of the present application, the biofilm material satisfies one or more of the following conditions:

[0042] (i) the biomembrane material is derived from dermis, bladder matrix membrane, peritoneum, fascia, pericardium, heart valve, small intestine or basement membrane; and,

[0043] (ii) The biofilm material is derived from pigs, cattle or sheep.

[0044] One or more embodiments of the present application also provide a tissue repair material, which is prepared by the tissue repair material preparation method.

[0045] One or more embodiments of the present application also provide a bone defect repair method, which includes the step of repairing the bone defect area using the tissue repair material.

[0046] In some embodiments of the present application, the bone defect area is located in the oral cavity.

[0047] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0049] Figure 1 Observation diagram of tissue sections of the tissue repair material prepared by using the product of the first decellularization stage and the tissue repair material prepared by using the product of the second decellularization stage in Example 1;

[0050] Figure 2 The microstructure observation diagram of the tissue repair material prepared by using the product of the first decellularization stage and the tissue repair material prepared by using the product of the second decellularization stage in Example 1;

[0051] Figure 3 This is an animal test diagram of the effectiveness of the tissue repair material prepared using the product of the first decellularization stage and the tissue repair material prepared using the product of the second decellularization stage in Example 1. DETAILED DESCRIPTION

[0052] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not used to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms, is not limited to the embodiments and examples described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the present application, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0053] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0054] the term

[0055] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0056] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0057] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0058] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0059] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0060] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0061] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0062] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0063] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0064] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0065] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0066] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0067] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0068] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0069] The methods for decellularization of biological scaffolds are mainly divided into physical methods, chemical methods and enzymatic methods. No matter which decellularization method is used, the structure and composition of ECM can be destroyed. Due to tissue-specific factors such as cell density, matrix density and geometric factors including tissue thickness and shape, the best methods for decellularization in tissues and organs are different. Because cell residues cannot be completely removed, the decellularization process may cause some damage to the matrix. The effect of removing cells from tissues depends on the source of the tissue and the decellularization method used. Each method will affect the biochemical composition, tissue ultrastructure and mechanical properties of the remaining ECM, thereby affecting the body's response to the material, which is not conducive to the repair and regeneration of tissues. In view of the shortcomings of the prior art, one of the purposes of the embodiments of the present application is to provide a method for preparing a tissue repair material, so that the obtained tissue repair material is completely decellularized, the material has antioxidant and anti-chronic inflammatory effects, and has good biocompatibility. It is conducive to the repair and regeneration of tissues. On the basis of removing the immunogenic substances of the material, the innovative introduction of anti-inflammatory and antioxidant reagents, including tea polyphenols, gallic acid and ethyl cellulose, can promote tissue healing, induce bone regeneration, and enhance mechanical properties. Greatly improve the clinical operation of existing products and improve the effectiveness of product use.

[0070] Tea polyphenols can be used as biological additives in food packaging materials due to their antioxidant and antimicrobial functions to reduce oxidation and prevent food spoilage and contamination by infectious pathogens. At the same time, tea polyphenols also play an important role in the treatment of chronic inflammation.

[0071] Gallic acid (GA) is a natural polyphenol commonly found in plants and has strong antioxidant and antibacterial effects. Ethyl cellulose is a high molecular weight polysaccharide compound. The use of one or more of tea polyphenols, gallic acid and ethyl cellulose to prepare a solution can increase the antioxidant and antibacterial effects of decellularized biomaterials and have good biocompatibility. It is beneficial to the repair and regeneration of tissues.

[0072] The inventors of the present application also unexpectedly found that the combined use of tea polyphenols and gallic acid solution has a synergistic effect, which is better than using them alone. In addition, ethyl cellulose, tea polyphenols and gallic acid form a composite solution system, which is better than other similar substitutes.

[0073] The membranes treated with the above reagents can be used with thickness cutting equipment in the drying process to more accurately control the thickness uniformity of the product, and prepare products with thickness that meets the needs of the clinical market, greatly improving the industrial utilization rate of animal membranes, reducing production costs, and increasing production capacity. The natural tissue fiber structure of the product after cutting is not destroyed, the material is softer, and fits the surface of soft tissue, which is more convenient for clinical use and improves the use effect.

[0074] In a first aspect of an embodiment of the present application, a membrane tissue treatment composition is provided, wherein the membrane tissue treatment composition comprises tea polyphenols and gallic acid in a molar ratio of about 1: (2-8). The molar ratio of tea polyphenols to gallic acid is, for example, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5, 1:6, 1:6.5, 1:7, 1:7.5, 1:8.

[0075] In some examples, the membrane tissue processing composition further includes a high molecular weight polysaccharide compound, and the high molecular weight polysaccharide compound includes one or more of ethyl cellulose and hydroxypropyl cellulose; the molar ratio of the tea polyphenols, the gallic acid and the high molecular weight polysaccharide compound is about 1: (2-8): (3-5). The molar ratio of the tea polyphenols, the gallic acid and the high molecular weight polysaccharide compound is, for example, 1:2:3, 1:2.5:3, 1:3:3, 1:3.5:3, 1:4:3, 1:4.5:3, 1:5:3, 1:5.5:3, 1:6:3, 1:6.5:3, 1:7:3, 1:7.5:3, 1:8:3, 1:2:4, 1:2.5:4, 1:3:4, 1:3.5:4, 1:4:3 :4:4, 1:4.5:4, 1:5:4, 1:5.5:4, 1:6:4, 1:6.5:4, 1:7:4, 1:7.5:4, 1:8:4, 1:2:5, 1:2.5:5, 1:3:5, 1:3.5:5, 1:4:5, 1:4.5:5, 1:5:5, 1:5.5:5, 1:6:5, 1:6.5:5, 1:7:5, 1:7.5:5, 1:8:5.

[0076] In a second aspect of an embodiment of the present application, a membrane tissue treatment reagent is provided, the membrane tissue treatment reagent comprising tea polyphenols, gallic acid and a solvent; the molar ratio of the tea polyphenols to the gallic acid is about 1: (2-8), for example, 1: 2, 1: 2.5, 1: 3, 1: 3.5, 1: 4, 1: 4.5, 1: 5, 1: 5.5, 1: 6, 1: 6.5, 1: 7, 1: 7.5, 1: 8. Optionally, the total concentration of the tea polyphenols and the gallic acid is about 0.01M-1M, for example, 0.01, 0.02, 0.04, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1M.

[0077] In some examples, the membrane tissue treatment reagent further includes a high molecular weight polysaccharide compound, and the high molecular weight polysaccharide compound includes one or more of ethyl cellulose and hydroxypropyl cellulose; the molar ratio of the tea polyphenols, the gallic acid and the high molecular weight polysaccharide compound is about 1:(2-8):(3-5), for example, 1:2:3, 1:2.5:3, 1:3:3, 1:3.5:3, 1:4:3, 1:4.5:3, 1:5:3, 1:5.5:3, 1:6:3, 1:6.5:3, 1:7:3, 1:7. : 5:3, 1:8:3, 1:2:4, 1:2.5:4, 1:3:4, 1:3.5:4, 1:4:4, 1:4.5:4, 1:5:4, 1:5.5:4, 1:6:4, 1:6.5:4, 1:7:4, 1:7.5:4, 1:8:4, 1:2:5, 1:2.5:5, 1:3:5, 1:3.5:5, 1:4:5, 1:4.5:5, 1:5:5, 1:5.5:5, 1:6:5, 1:6.5:5, 1:7:5, 1:7.5:5, 1:8:5. Optionally, the total concentration of the tea polyphenols, the gallic acid and the high molecular weight polysaccharide compound is about 0.01M to 1M, for example, 0.01, 0.02, 0.04, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1M.

[0078] In some examples, the solvent includes a PBS buffer. Optionally, the PBS buffer includes about 0.1 mol / L to 0.3 mol / L (e.g., 0.1, 0.15, 0.2, 0.25, 0.3 mol / L) of disodium hydrogen phosphate and about 0.1 mol / L to 0.3 mol / L (e.g., 0.1, 0.15, 0.2, 0.25, 0.3 mol / L) of sodium dihydrogen phosphate, and a pH of about 6.5 to 7.5 (e.g., 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5).

[0079] A third aspect of the embodiments of the present application provides a method for preparing a tissue repair material, the method comprising the step of performing a decellularization treatment on a biomembrane material using the membrane tissue treatment reagent.

[0080] In some examples, the conditions for decellularization treatment include: performing the treatment under shaking conditions, the number of treatments is about 2 to 5 times (for example, 2, 3, 4, 5 times), and each treatment is about 30 minutes to 90 minutes (for example, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 minutes).

[0081] In some examples, the preparation method includes multiple decellularization treatment stages, and in one of the decellularization treatment stages, the biofilm material is decellularized using the membrane tissue treatment reagent.

[0082] In some examples, the preparation method includes a first decellularization treatment stage and a second decellularization treatment stage, and in the second decellularization treatment stage, the biofilm material is decellularized using the membrane tissue treatment reagent.

[0083] In some examples, the first decellularization step includes placing the biofilm material in a hypertonic solution and a hypotonic solution in sequence for decellularization. Optionally, the first decellularization step satisfies one or more of the following conditions:

[0084] 1) The hypertonic solution comprises about 0.01M to 1M (e.g., 0.01, 0.02, 0.04, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1M) sodium chloride solution added with about 0.01M to 1M (e.g., 0.01, 0.02, 0.04, 0.08, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1M) base or acid; optionally, the base comprises one or more of sodium hydroxide and potassium hydroxide; optionally, the acid comprises one or more of hydrochloric acid and acetic acid;

[0085] 2) the hypotonic solution comprises water;

[0086] 3) the steps of the first decellularization treatment stage are repeated about 2 to 5 times (for example, 2, 3, 4, 5 times); and,

[0087] 4) carrying out the step under shaking conditions, wherein the shaking time in the hypertonic solution and the hypotonic solution is independently about 30 min to 90 min (e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 min).

[0088] In some examples, the biofilm material is processed by removing attached fat, connective tissue and damaged marginal tissue, defatting and inactivating viruses.

[0089] The present application embodiment does not specifically limit the degreasing step. In some examples, the degreasing satisfies one or more of the following conditions:

[0090] (I) the degreasing agent used includes one or more of methanol, chloroform, ethanol, propanol, isopropanol, n-hexane and ethyl acetate; and,

[0091] (II) The degreasing treatment is carried out under shaking conditions for about 2 to 4 times (for example, 2, 3, 4, 5 times), each time for about 2 hours to 10 hours (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 hours), and after each degreasing treatment, fresh degreasing reagent is replaced before the next degreasing treatment.

[0092] The embodiments of the present application do not specifically limit the method of virus inactivation, including but not limited to chemical methods; in some examples, the chemical method satisfies one or more of the following conditions:

[0093] Ⅰ) the inactivation reagent used includes one or more of an acid, a base and an alcohol; optionally, the base includes one or more of sodium hydroxide, sodium chloride, potassium hydroxide and ammonia water; optionally, the acid includes one or more of hydrochloric acid, nitric acid, phosphoric acid and acetic acid; the alcohol includes one or more of ethanol, propanol, isopropanol and methanol; and,

[0094] II) The virus is inactivated under static conditions for a period of about 1 h to 3 h (e.g., 1, 1.5, 2, 2.5, or 3 h).

[0095] It is understandable that, in the embodiment of the present application, after the decellularization treatment, the obtained tissue repair material is further frozen, sliced ​​and sterilized. The embodiment of the present application does not particularly limit the specific steps of freezing, slicing and sterilization.

[0096] In some examples, the freezing conditions include: freeze-drying is cooled at a rate of about 5°C / min to 12°C / min (for example, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12°C / min) to about -80°C to -20°C (for example, -80, -75, -70, -65, -60, -55, -50, -45, -40, -35, -30, -25, -20°C), and maintained at about -20°C to -10°C (for example, -20, -18, -16, -14, -12, -10) for 10h to 16h (for example, 10, 11, 12, 13, 14, 15, 16h).

[0097] In some examples, the slice control membrane thickness is about 0.1 mm to 1 mm, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mm.

[0098] In some examples, the sterilization is performed by physical sterilization. In other examples, the sterilization is performed by chemical sterilization.

[0099] In some examples, the biofilm material satisfies one or more of the following conditions:

[0100] (i) the biomembrane material is derived from dermis, bladder matrix membrane, peritoneum, fascia, pericardium, heart valve, small intestine or basement membrane; and,

[0101] (ii) The biofilm material is derived from pigs, cattle or sheep.

[0102] A fifth aspect of the embodiments of the present application provides a tissue repair material prepared by the method for preparing the tissue repair material described in the fourth aspect.

[0103] A sixth aspect of the embodiments of the present application provides a method for repairing bone defects, comprising the step of repairing the bone defect area using the tissue repair material described in the fifth aspect.

[0104] In some examples, the bone defect is located in the oral cavity.

[0105] The present application embodiment does not specifically limit the subject to be repaired, and can be any animal that may have a bone defect and needs to be repaired. It can be a human or other non-human mammals. The term "mammal" in this application mainly refers to warm-blooded vertebrate mammals, including but not limited to: such as cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (such as rats, mice), pigs, cattle, sheep, horses, humans, etc., preferably primates, more preferably humans.

[0106] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0107] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0108] Example 1

[0109] Step 1: Pretreatment: Spread the bovine pericardium 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 the biofilm material.

[0110] Step 2: Degreasing: The obtained biofilm material is placed in an organic solvent and shaken for degreasing for 6 hours, then the liquid is changed, and the degreasing is repeated 3 times, and the material is washed with water until there is no odor. The organic solvent is propanol.

[0111] Step 3: Virus inactivation: The obtained biofilm material is placed in a chemical reagent for inactivation for 2 hours, and then washed with water until it is odorless. The chemical reagent is a sodium hydroxide solution with a sodium hydroxide content of 0.1 M.

[0112] Step 4: Decellularization:

[0113] The first decellularization stage: the virus-inactivated biofilm material is 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 is repeated three times; wherein, the decellularization hypertonic solution is an aqueous solution containing 0.5M sodium chloride and 1M sodium hydroxide, and the decellularization hypotonic solution is water.

[0114] In the second decellularization stage, PBS buffer solution (containing 0.2 mol / L disodium hydrogen phosphate and 0.2 mol / L sodium dihydrogen phosphate, pH 7) was used to dissolve tea polyphenols, gallic acid and ethyl cellulose, and the treatment reagent was prepared. The concentrations of tea polyphenols, gallic acid and ethyl cellulose in the treatment reagent were 0.02 M, 0.04 M and 0.06 M, respectively. The biofilm material was shaken in the treatment reagent for 50 minutes, and then replaced with fresh treatment reagent after the shaking. This cycle was repeated 3 times (i.e., after shaking treatment once, replaced with fresh treatment reagent for treatment for the second time, and replaced with fresh treatment reagent for treatment for the third time), and washed with water until there was no odor.

[0115] Step 5, freeze-drying: flatten the biofilm material obtained in step 4, stick it on a plate, and directly put it into a freeze dryer for freeze drying. The freeze drying is carried out 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.

[0116] Step 6: Cutting: The freeze-dried biofilm is processed using a cutting device. The film thickness is selected to be 0.4mm for precise cutting to achieve uniform thickness of single-specification materials.

[0117] Step 7: Sterilization: Use ethylene oxide to perform chemical sterilization to obtain tissue repair materials.

[0118] Example 2

[0119] 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 the biofilm material.

[0120] Step 2: Degreasing: The obtained biofilm material is placed in an organic solvent and shaken for degreasing for 4 hours, then the liquid is changed, and the degreasing is repeated 3 times, and the material is washed with water until there is no odor. The organic solvent is ethanol.

[0121] Step 3: Virus inactivation: The obtained biofilm material is placed in a chemical reagent for inactivation for 1 hour, and then washed with water until there is no odor. The chemical reagent is a sodium hydroxide solution with a sodium hydroxide content of 0.1 M.

[0122] Step 4: Decellularization:

[0123] The first decellularization stage: the virus-inactivated biofilm material is 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 is repeated three times; wherein the decellularization hypertonic solution is an aqueous solution containing 0.5M sodium chloride and 1M sodium hydroxide, and the decellularization hypotonic solution is water.

[0124] Second decellularization stage: Use PBS buffer solution (containing 0.2 mol / L disodium hydrogen phosphate and 0.2 mol / L sodium dihydrogen phosphate, pH 7) to dissolve tea polyphenols, gallic acid and ethyl cellulose, and prepare treatment reagents. The concentrations of tea polyphenols, gallic acid and ethyl cellulose in the treatment reagents are 0.02 M, 0.04 M and 0.1 M, respectively. Shake the biofilm material in the treatment reagent for 50 minutes, replace it with fresh treatment reagent after shaking, repeat this cycle 3 times, and wash with water until there is no odor.

[0125] Step 5, freeze drying: flatten the biofilm material obtained in step 4, stick it on a plate, and put it directly 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.

[0126] Step 6: Cutting: The freeze-dried biofilm is processed using a cutting device. The film thickness is selected to be 0.3mm for precise cutting to achieve uniform thickness of single-specification materials.

[0127] Step 7: Sterilization: Use irradiation sterilization to obtain tissue repair materials.

[0128] Example 3

[0129] This embodiment is a variation of the embodiment 1, and the variation relative to the embodiment 1 is as follows:

[0130] Step 2: Degreasing: The obtained biofilm material is placed in an organic solvent and shaken for degreasing for 2 hours, then the liquid is changed, and the degreasing is repeated 4 times, and the material is washed with water until there is no odor. The organic solvent is propanol.

[0131] Step 3: Virus inactivation: Place the obtained biofilm material in hydrochloric acid for 1 hour for inactivation, and then wash with water until there is no odor.

[0132] Step 4: Decellularization:

[0133] The first decellularization stage: the virus-inactivated biofilm material is placed in a decellularization hypertonic solution and shaken for 50 minutes, then transferred to a decellularization hypotonic solution and shaken for 30 minutes, and this cycle is repeated 5 times; wherein the decellularization hypertonic solution is an aqueous solution containing 2M sodium chloride and 0.01M sodium hydroxide, and the decellularization hypotonic solution is water.

[0134] Second decellularization stage: Use PBS buffer solution (containing 0.1 mol / L disodium hydrogen phosphate and 0.3 mol / L sodium dihydrogen phosphate, pH 6.5) to dissolve tea polyphenols, gallic acid and ethyl cellulose, and prepare treatment reagents. The molar concentration ratio of tea polyphenols, gallic acid and ethyl cellulose in the treatment reagents is 1:2:5, and the total concentration of tea polyphenols, gallic acid and ethyl cellulose is 0.01 M. Shake the biofilm material in the treatment reagent for 30 minutes, replace it with fresh treatment reagent after shaking, repeat this cycle 5 times, and wash with water until there is no odor.

[0135] Step 5, freeze-drying: flatten the biofilm material obtained in step 4, stick it on a plate, and directly put it into a freeze dryer for freeze drying. The freeze drying is performed by cooling to -20°C at a rate of 5°C / min and keeping it at -10°C for 10 hours. After freeze drying, it is cut and packaged.

[0136] Step 6: Cutting: The freeze-dried biofilm is processed using a cutting device. The film thickness is selected to be 0.1mm for precise cutting to achieve uniform thickness of single-specification materials.

[0137] The rest is the same as in Example 1.

[0138] Example 4

[0139] This embodiment is a variation of the embodiment 1, and the variation relative to the embodiment 1 is as follows:

[0140] Step 2: Degreasing: The obtained biofilm material was placed in ethyl acetate and degreased by shaking for 10 hours, then the liquid was changed, and the degreasing was repeated 4 times, and then washed with water until there was no odor.

[0141] Step 3: Virus inactivation: The obtained biofilm material was placed in propanol for 3 hours for inactivation, and then washed with water until there was no odor.

[0142] Step 4: Decellularization:

[0143] The first decellularization stage: the virus-inactivated biofilm material is placed in a decellularization hypertonic solution and shaken for 90 minutes, then transferred to a decellularization hypotonic solution and shaken for 90 minutes, and this cycle is repeated twice; wherein, the decellularization hypertonic solution is an aqueous solution containing 5M sodium chloride and 0.5M sodium hydroxide, and the decellularization hypotonic solution is water.

[0144] Second decellularization stage: Use PBS buffer solution (containing 0.3 mol / L disodium hydrogen phosphate and 0.1 mol / L sodium dihydrogen phosphate, pH 7.5) to dissolve tea polyphenols, gallic acid and ethyl cellulose, and prepare treatment reagents. The molar concentration ratio of tea polyphenols, gallic acid and ethyl cellulose in the treatment reagents is 1:8:3, and the total concentration of tea polyphenols, gallic acid and ethyl cellulose is 1 M. The biofilm material is shaken in the treatment reagent for 50 minutes, and the fresh treatment reagent is replaced. This cycle is repeated 3 times, and the treatment reagent is washed with water until it is odorless.

[0145] Step 5, freeze-drying: flatten the biofilm material obtained in step 4, stick it on a plate, and directly put it into a freeze dryer for freeze drying. The freeze drying is carried out by cooling to -16°C at a rate of 12°C / min and keeping it at -15°C for 14 hours. After freeze drying, it is cut and packaged.

[0146] Step 6: Cutting: The freeze-dried biofilm is processed using a cutting device. The film thickness is selected to be 1mm for precise cutting to achieve uniform thickness of single-specification materials.

[0147] The rest is the same as in Example 1.

[0148] Example 5

[0149] This embodiment is a variation of Embodiment 1, and the variations relative to Embodiment 1 include:

[0150] In the second decellularization stage of step 4, no ethyl cellulose was added, that is, PBS buffer solution (containing 0.2 mol / L disodium hydrogen phosphate and 0.2 mol / L sodium dihydrogen phosphate, pH 7) was used to dissolve tea polyphenols and gallic acid, and the treatment reagent was prepared. The concentrations of tea polyphenols and gallic acid in the treatment reagent were 0.04 M and 0.08 M, respectively. The biofilm material was shaken in the solution for 50 minutes, and fresh treatment reagent was replaced after the shaking was completed. This cycle was repeated 3 times, and then washed with water until there was no odor.

[0151] The rest is the same as in Example 1.

[0152] Example 6

[0153] This embodiment is a variation of Embodiment 1, and the variations relative to Embodiment 1 include:

[0154] In the second decellularization stage of step 4, ethyl cellulose is replaced by hydroxypropyl cellulose. That is, PBS buffer solution (containing 0.2 mol / L disodium hydrogen phosphate and 0.2 mol / L sodium dihydrogen phosphate, pH 7) is used to dissolve tea polyphenols, gallic acid, and hydroxypropyl cellulose to prepare the treatment reagent. The concentrations of tea polyphenols, gallic acid, and hydroxypropyl cellulose in the treatment reagent are 0.02M, 0.04M, and 0.06M, respectively. The biofilm material is shaken in the treatment reagent for 50 minutes, and fresh treatment reagent is replaced after the shaking is completed. This cycle is repeated 3 times, and then washed with water until there is no odor.

[0155] The rest is the same as in Example 1.

[0156] Example 7

[0157] This embodiment is a variation of Embodiment 1, and the variations relative to Embodiment 1 include:

[0158] In the second decellularization stage of step 4, PBS buffer solution (containing 0.2 mol / L disodium hydrogen phosphate and 0.2 mol / L sodium dihydrogen phosphate, pH 7) was used to dissolve tea polyphenols, gallic acid and ethyl cellulose, and a treatment reagent was prepared. The concentrations of tea polyphenols, gallic acid and ethyl cellulose in the treatment reagent were 0.02 M, 0.04 M and 0.04 M, respectively. The biofilm material was shaken in the treatment reagent for 50 min, and a fresh treatment reagent was replaced. This cycle was repeated 3 times, and the biofilm material was washed with water until it was odorless.

[0159] The rest is the same as in Example 1.

[0160] Example 8

[0161] This embodiment is a variation of Embodiment 1, and the variations relative to Embodiment 1 include:

[0162] In the second decellularization stage of step 4, PBS buffer solution (containing 0.2 mol / L disodium hydrogen phosphate and 0.2 mol / L sodium dihydrogen phosphate, pH 7) was used to dissolve tea polyphenols, gallic acid and ethyl cellulose, and a treatment reagent was prepared. The concentrations of tea polyphenols, gallic acid and ethyl cellulose in the treatment reagent were 0.02 M, 0.04 M and 0.12 M, respectively. The biofilm material was shaken in the treatment reagent for 50 min, replaced with a fresh treatment reagent, and the cycle was repeated 3 times, and then washed with water until there was no odor.

[0163] The rest is the same as in Example 1.

[0164] Example 9

[0165] This embodiment is a variation of Embodiment 1, and the variations relative to Embodiment 1 include:

[0166] In the second decellularization stage of step 4, the molar concentration ratio of tea polyphenols, gallic acid and ethyl cellulose is 1:2:3, and the total concentration is 1.2M.

[0167] The rest is the same as in Example 1.

[0168] Example 10

[0169] This embodiment is a variation of Embodiment 1, and the variations relative to Embodiment 1 include:

[0170] In the second decellularization stage of step 4, the molar concentration ratio of tea polyphenols, gallic acid and ethyl cellulose is 1:2:3, and the total concentration is 0.009M.

[0171] The rest is the same as in Example 1.

[0172] Comparative Example 1

[0173] This comparative example is a comparative example of Example 1, and the only difference from Example 1 is that in the second decellularization stage of step 4, PBS buffer solution is used to dissolve tea polyphenols and prepare the treatment reagent, and the concentration of tea polyphenols in the treatment reagent is 0.06 M. The rest is the same as Example 1.

[0174] Comparative Example 2

[0175] This comparative example is a comparative example of Example 1, and the only difference from Example 1 is that in the second decellularization stage of step 4, PBS buffer solution is used to dissolve gallic acid and prepare the treatment reagent, and the concentration of gallic acid in the treatment reagent is 0.06 M. The rest is the same as Example 1.

[0176] Comparative Example 3

[0177] This comparative example is a comparative example of Example 1, and the only difference from Example 1 is that:

[0178] In step 4, tea polyphenols are replaced by tannic acid. That is, PBS buffer solution is used to dissolve tannic acid, gallic acid and ethyl cellulose to prepare a treatment reagent, wherein the concentrations of tannic acid, gallic acid and ethyl cellulose in the treatment reagent are 0.02 M, 0.04 M and 0.06 M, respectively. The rest are the same as in Example 1.

[0179] Comparative Example 4

[0180] This comparative example is a comparative example of Example 1, and the only difference from Example 1 is that:

[0181] In the second decellularization stage of step 4, gallic acid is replaced by epigallocatechin gallate. That is, tea polyphenols, epigallocatechin gallate and ethyl cellulose are dissolved in PBS buffer solution to prepare a treatment reagent, wherein the concentrations of tea polyphenols, epigallocatechin gallate and ethyl cellulose in the treatment reagent are 0.02 M, 0.04 M and 0.06 M, respectively. The rest are the same as in Example 1.

[0182] Comparative Example 5

[0183] This comparative example is a comparative example of Example 1, and the only difference from Example 1 is that:

[0184] In the second decellularization stage of step 4, the molar ratio of tea polyphenols to gallic acid is 1:1. Specifically, PBS buffer solution is used to dissolve tea polyphenols, gallic acid and ethyl cellulose to prepare a treatment reagent, in which the concentrations of tea polyphenols, gallic acid and ethyl cellulose are 0.03 M, 0.03 M and 0.06 M, respectively. The rest are the same as in Example 1.

[0185] Comparative Example 6

[0186] This comparative example is a comparative example of Example 1, and the only difference from Example 1 is that:

[0187] In the second decellularization stage of step 4, the molar ratio of tea polyphenols to gallic acid is 1:9. Specifically, PBS buffer solution is used to dissolve tea polyphenols, gallic acid and ethyl cellulose to prepare a treatment reagent, in which the concentrations of tea polyphenols, gallic acid and ethyl cellulose are 0.01 M, 0.09 M and 0.06 M, respectively. The rest are the same as in Example 1.

[0188] Performance Testing

[0189] Experiment 1: Histological sections

[0190] Take the tissue repair material of Example 1 with a size of at least 1.0×1.0 cm and use conventional HE staining technology to detect it. The results are as follows Figure 1 shown. Figure 1 , the first row of three pictures show tissue repair materials prepared using the products of the first decellularization stage, and the second row of three pictures show tissue repair materials prepared using the products of the second decellularization stage.

[0191] Experiment 2: Microstructure Observation

[0192] Take the tissue repair material of Example 1 after complete rehydration for 5 minutes, cut it into pieces of about 1 mm × 5 mm, and observe it under a transmission electron microscope after treatment and staining. Figure 2 , Figure 2The middle left picture shows a tissue repair material prepared using the product of the first decellularization stage, and the right picture shows a tissue repair material prepared using the product of the second decellularization stage. The results show that the natural collagen fiber structure of the tissue repair material of Example 1 is intact, with obvious and clear collagen fiber arrangement structure, and periodic horizontal stripes / layered arrangement structure can be seen along the long axis of the collagen fibers.

[0193] Experiment 3: Mechanical properties testing

[0194] Conduct suture tear force testing of tissue repair materials in accordance with the provisions of YY 0500-2020;

[0195] The tensile strength and elongation at break of tissue repair materials were determined in accordance with the first part of GB / T 3923.1-2013 (strip method).

[0196] The results show that the mechanical properties of each embodiment are stronger. The use of the decellularization treatment reagent of the present application can enhance the toughness of the tissue repair material. The effect of the combined use of tea polyphenols, gallic acid and ethyl cellulose is better than that of the three ingredients used alone, and the mechanical properties are more advantageous.

[0197] Table 1. Suture tearing force

[0198]

[0199] In Table 1, "after decellularization" refers to the tissue repair material prepared using the product of the first decellularization stage, and "after solution treatment" refers to the tissue repair material prepared using the product of the second decellularization stage.

[0200] Table 2. Tensile strength at break

[0201]

[0202] In Table 2, "after decellularization" refers to the tissue repair material prepared using the product of the first decellularization stage, and "after solution treatment" refers to the tissue repair material prepared using the product of the second decellularization stage.

[0203] Table 3. Elongation at break

[0204]

[0205]

[0206] In Table 3, "after decellularization" refers to the tissue repair material prepared using the product of the first decellularization stage, and "after solution treatment" refers to the tissue repair material prepared using the product of the second decellularization stage.

[0207] Experiment 4: In vitro degradation assay

[0208] The tissue repair material samples of each example and comparative example were cut into uniform pieces, added into 10 mL of 0.1 M (pH 7.4) PBS buffer containing 50 CDU / mL of type I collagenase, and shaken at 37°C.

[0209] Results Figure 3 The results show that the tissue repair material of Example 1 has better degradation performance and longer in vitro degradation time. In clinical use, the barrier time is more advantageous and can effectively promote tissue healing, giving full play to the natural advantages of tissue repair materials. Figure 3 The second row of the accompanying drawings (tissue repair material prepared using the product of the second decellularization stage); Figure 3 The first row of figures shows tissue repair materials prepared using the products of the first decellularization stage.

[0210] Table 4. In vitro degradation

[0211]

[0212]

[0213] In Table 4, "after decellularization" refers to the tissue repair material prepared using the product of the first decellularization stage, and "after solution treatment" refers to the tissue repair material prepared using the product of the second decellularization stage.

[0214] Experiment 5: Animal testing

[0215] The tissue repair material of Example 1 (ie, the tissue repair material prepared using the product of the second decellularization stage) was tested for effectiveness in canine extraction socket filling to verify the material's degradation properties and healing properties for soft tissue.

[0216] The histological results of animal experiments show that: 2 weeks after implantation of the tissue repair material of Example 1, early vascularization occurred and early new bone formation occurred under the collagen membrane. 8 weeks after implantation, the tissue repair material gradually degraded. 12-16 weeks after implantation, new bone continued to form and the epithelium had covered the defect area.

[0217] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0218] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. A membrane tissue treatment composition, characterized in that: The membrane tissue treatment composition comprises tea polyphenols and gallic acid in a molar ratio of about 1:(2-8).

2. The membrane tissue treatment composition according to claim 1, characterized in that: The membrane tissue treatment composition further comprises a high molecular weight polysaccharide compound, wherein the high molecular weight polysaccharide compound comprises one or more of ethyl cellulose and hydroxypropyl cellulose; The molar ratio of the tea polyphenols, the gallic acid and the high molecular weight polysaccharide compound is about 1:(2-8):(3-5).

3. A membrane tissue processing reagent, characterized in that: The membrane tissue treatment reagent comprises tea polyphenols, gallic acid and a solvent; The molar ratio of the tea polyphenols to the gallic acid is about 1:(2-8); Optionally, the total concentration of the tea polyphenols and the gallic acid is about 0.01M to 1M.

4. The membrane tissue processing reagent according to claim 3, characterized in that: The membrane tissue treatment reagent also includes a high molecular weight polysaccharide compound, and the high molecular weight polysaccharide compound includes one or more of ethyl cellulose and hydroxypropyl cellulose; The molar ratio of the tea polyphenols, the gallic acid and the high molecular weight polysaccharide compound is about 1:(2-8):(3-5); Optionally, the total concentration of the tea polyphenols, the gallic acid and the high molecular weight polysaccharide compound is about 0.01M to 1M.

5. The membrane tissue processing reagent according to any one of claims 3 to 4, characterized in that: The solvent includes PBS buffer; Optionally, the PBS buffer comprises about 0.1 mol / L to 0.3 mol / L of disodium hydrogen phosphate and about 0.1 mol / L to 0.3 mol / L of sodium dihydrogen phosphate, and the pH is about 6.5 to 7.

5.

6. A method for preparing a tissue repair material, characterized in that: The preparation method comprises the step of using the membrane tissue treatment reagent described in any one of claims 3 to 5 to perform decellularization treatment on the biofilm material.

7. The method for preparing a tissue repair material according to claim 6, characterized in that: The conditions for the decellularization treatment include: performing the treatment under shaking conditions, the number of treatments being about 2 to 5 times, and each treatment lasting about 30 minutes to 90 minutes.

8. The method for preparing a tissue repair material according to claim 6, characterized in that: The preparation method comprises a plurality of decellularization treatment stages, in one of which the membrane tissue treatment reagent is used to perform decellularization treatment on the biofilm material.

9. The method for preparing a tissue repair material according to claim 8, characterized in that: The preparation method comprises a first decellularization treatment stage and a second decellularization treatment stage, and in the second decellularization treatment stage, the membrane tissue treatment reagent is used to perform decellularization treatment on the biofilm material.

10. The method for preparing a tissue repair material according to claim 7, characterized in that: The steps of the first decellularization treatment stage include: placing the biofilm material in a hypertonic solution and a hypotonic solution in sequence for decellularization treatment; Optionally, the first decellularization treatment stage satisfies one or more of the following conditions: 1) The hypertonic solution comprises a sodium chloride solution of about 0.01M to 1M to which a base or acid of about 0.01M to 1M is added; optionally, the base comprises one or more of sodium hydroxide and potassium hydroxide; optionally, the acid comprises one or more of hydrochloric acid and acetic acid; 2) the hypotonic solution comprises water; 3) the steps of the first decellularization stage are repeated about 2 to 5 times; and, 4) The method is carried out under shaking conditions, wherein the shaking time in the hypertonic solution and the hypotonic solution is independently about 30 min to 90 min.

11. The method for preparing a tissue repair material according to any one of claims 6 to 10, characterized in that: The biofilm material is processed as follows: removing attached fat, connective tissue and damaged tissue at the edge, defatting and inactivating viruses.

12. The method for preparing a tissue repair material according to claim 11, characterized in that: Degreasing meets one or more of the following conditions: (I) the degreasing agent used includes one or more of methanol, chloroform, ethanol, propanol, isopropanol, n-hexane and ethyl acetate; and, (II) The degreasing process is carried out under shaking conditions for about 2 to 4 times, each time for about 2 hours to 10 hours. After each degreasing process, fresh degreasing reagent is replaced before the next degreasing process.

13. The method for preparing a tissue repair material according to claim 11, characterized in that: Virus inactivation uses chemical methods; Optionally, the chemical method satisfies one or more of the following conditions: Ⅰ) the inactivation reagent used includes one or more of an acid, a base and an alcohol; optionally, the base includes one or more of sodium hydroxide, sodium chloride, potassium hydroxide and ammonia water; optionally, the acid includes one or more of hydrochloric acid, nitric acid, phosphoric acid and acetic acid; the alcohol includes one or more of ethanol, propanol, isopropanol and methanol; and, II) The virus is inactivated under static conditions for about 1 to 3 hours.

14. The method for preparing a tissue repair material according to any one of claims 6 to 10 and 12 to 13, characterized in that: After the decellularization process, the obtained tissue repair material is also frozen, sliced, and sterilized; Optionally, the freezing conditions include: freeze drying by cooling to about -80°C to -20°C at a rate of about 5°C / min to 12°C / min, and maintaining at about -20°C to -10°C for 10h to 16h; Optionally, the slice controls the film thickness to be about 0.1 mm to 1 mm; Optionally, sterilization is performed by physical sterilization or chemical sterilization.

15. The method for preparing a tissue repair material according to any one of claims 6 to 10 and 12 to 13, characterized in that: The biofilm material meets one or more of the following conditions: (i) the biomembrane material is derived from dermis, bladder matrix membrane, peritoneum, fascia, pericardium, heart valve, small intestine or basement membrane; and, (ii) The biofilm material is derived from pigs, cattle or sheep.

16. A tissue repair material, characterized in that: The method for preparing a tissue repair material according to any one of claims 6 to 15 is used.

17. A method for repairing bone defects, characterized in that: The repair method comprises the step of repairing the bone defect area using the tissue repair material according to claim 16.

18. The bone defect repairing method according to claim 17, characterized in that: The bone defect area is located in the oral cavity.

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