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

Abstract

The present application relates to the fields of tissue engineering and medical biomaterials, and in particular, to a membrane tissue treatment composition, a membrane tissue treatment reagent, a tissue repair material, a preparation method therefor, and use thereof. The membrane tissue treatment composition comprises tea polyphenol and gallic acid in a molar ratio of about 1:(2-8).

Description

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

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

[0002] Oral repair membranes are widely used in the fields of periodontology, oral implantation and alveolar surgery. The oral repair membrane is a biocompatible material, which is placed between the oral soft tissue and the bone defect area by surgical operation to establish a biological barrier, so as to create a relatively closed bone regeneration environment, selectively block the migration of fibroblasts and epithelial cells with faster migration speed into the bone defect area, and at the same time, do not hinder the natural wound healing.

[0003] Oral repair membranes can be divided into collagen membranes, metal membranes, synthetic membranes and allogeneic bone membranes according to the source of the material, and can be divided into absorbable membranes and non-absorbable membranes according to whether the material can be degraded. Among them, the non-absorbable membrane needs a secondary surgery to remove it, and the non-collagen absorbable membrane does not need a secondary surgery, but has the side effect of inflammation caused by acidic degradation products, therefore, in comprehensive comparison, the collagen membrane is the most comprehensive product.

[0004] The common collagen membrane is a decellularized animal tissue membrane. The decellularized animal tissue membrane forms a natural double-layer collagen structure after treatment, and under electron microscopy, one layer of collagen fibers is arranged densely, and the other layer of collagen fibers is arranged relatively loosely. This unique double-layer structure has important significance in clinical application. The dense side provides a natural plane for the migration of epithelial cells, which is beneficial to rapid epithelialization, and the loose side completely retains the natural three-dimensional spatial structure of the skin, has a suitable pore size and porosity, which is beneficial to cell crawling and growth, provides a good scaffold for host cell growth and rapid vascularization, can regulate, guide and promote cell ingrowth, promote vascularization, and thus complete the repair and reconstruction of tissue defects.

[0005] At present, acellular animal tissue membranes from various organizations have been studied and applied. Commercialized tissue-engineered skin, cartilage and other products have officially entered clinical application, and the clinical application of tissue-engineered bone, tendon, skeletal muscle, cornea, mucosa, blood vessels, bladder, pancreas, genital organs, kidney, liver and the like has also begun to start and has achieved certain curative effect. Acellular animal tissue membranes can be used as the basis for constructing tissue engineering, which has greater advantages than synthetic materials. Acellular animal tissue membranes not only retain the natural three-dimensional structure and extracellular matrix, have no immunogenicity, biodegradability, sealing, non-toxicity and non-carcinogenicity, good biocompatibility and mechanical properties, but also retain basic fibroblast growth factor and are considered to be ideal scaffold materials for regeneration.

[0006] In the process of preparing acellular animal tissue membranes, commonly used decellularization methods include physical methods (freeze-thawing, pressurization, ultrasonic, etc.), chemical methods (acid, alkali, hypotonic and hypertonic solutions, non-ionic detergents, ionic detergents, zwitterionic detergents, metal ion chelating agents, etc.), enzyme methods (nucleic acid enzymes, trypsin, lipase, etc.) and combined use of the above methods. The mechanical properties of acellular animal tissue membranes obtained by different decellularization methods are different. Traditional decellularization methods are described in CN114191613A, CN104083803A and CN118236556A. How to improve the mechanical properties of acellular animal tissue membranes is the main goal of decellularization operation.

[0007] In view of this, the present application is proposed.

[0008] SUMMARY

[0009] 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. The technical solutions include the following:

[0010] 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).

[0011] In some embodiments of the present application, the membrane tissue treatment composition further comprises a high molecular polysaccharide compound, and the high molecular polysaccharide compound comprises one or more of ethyl cellulose and hydroxypropyl cellulose.

[0012] The molar ratio of the tea polyphenols, the gallic acid and the high molecular polysaccharide compound is about 1:(2-8):(3-5).

[0013] One or more embodiments of the present application also provide a membrane tissue treatment reagent, which comprises tea polyphenols, gallic acid and a solvent.

[0014] The molar ratio of the tea polyphenol and the gallic acid is about 1:(2-8).

[0015] In some embodiments of the present application, the total concentration of the tea polyphenol and the gallic acid is about 0.01M-1M.

[0016] In some embodiments of the present application, the membrane tissue treatment reagent further comprises a high molecular polysaccharide compound, the high molecular polysaccharide compound comprises one or more of ethyl cellulose and hydroxypropyl cellulose; the molar ratio of the tea polyphenol, the gallic acid and the high molecular polysaccharide compound is about 1:(2-8):(3-5).

[0017] In some embodiments of the present application, the total concentration of the tea polyphenol, the gallic acid and the high molecular polysaccharide compound is about 0.01M-1M.

[0018] In some embodiments of the present application, the solvent comprises a PBS buffer.

[0019] In some embodiments of the present application, the PBS buffer comprises about 0.1mol / L-0.3mol / L of disodium hydrogen phosphate and about 0.1mol / L-0.3mol / L of sodium dihydrogen phosphate, and the pH is about 6.5-7.5.

[0020] One or more embodiments of the present application further provide a preparation method of a tissue repair material, the preparation method comprising the step of decellularizing a biological membrane material by using the membrane tissue treatment reagent.

[0021] In some embodiments of the present application, the decellularization treatment condition comprises: being performed under a shaking condition, and the number of treatments is about 2-5 times, and each time is about 30min-90min.

[0022] In some embodiments of the present application, the preparation method comprises a plurality of decellularization treatment stages, and the biological membrane material is decellularized by using the membrane tissue treatment reagent in one of the decellularization treatment stages.

[0023] In some embodiments of the present application, the preparation method comprises a first decellularization treatment stage and a second decellularization treatment stage, and the biological membrane material is decellularized by using the membrane tissue treatment reagent in the second decellularization treatment stage.

[0024] In some embodiments of the present application, the step of the first decellularization treatment stage comprises: sequentially placing the biological membrane material in a high-osmotic solution and a low-osmotic solution for decellularization treatment.

[0025] In some embodiments of the present application, the first decellularization stage satisfies one or more of the following conditions:

[0026] 1) the hypertonic solution comprises about 0.01M-1M sodium chloride solution with about 0.01M-1M base or acid 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;

[0027] 2) the hypotonic solution comprises water;

[0028] 3) the steps of the first decellularization stage are repeated about 2-5 times; and,

[0029] 4) performed under shaking conditions, each of the shaking in the hypertonic solution and the hypotonic solution is independently about 30min-90min.

[0030] In some embodiments of the present application, the biomembrane material is treated to remove attached fat, connective tissue and marginal damaged tissue, delipidation and viral inactivation.

[0031] In some embodiments of the present application, the delipidation satisfies one or more of the following conditions:

[0032] (I) the delipidation reagent used comprises one or more of methanol, chloroform, ethanol, propanol, isopropanol, n-hexane and ethyl acetate; and,

[0033] (II) performed under shaking conditions, the delipidation is about 2-4 times, each time about 2h-10h, and after each delipidation, fresh delipidation reagent is added for the next delipidation.

[0034] In some embodiments of the present application, the viral inactivation is performed by chemical method.

[0035] In some embodiments of the present application, the chemical method satisfies one or more of the following conditions:

[0036] I) the inactivation reagent used comprises one or more of acid, base and alcohol; optionally, the base comprises one or more of sodium hydroxide, sodium chloride, potassium hydroxide and ammonia; optionally, the acid comprises one or more of hydrochloric acid, nitric acid, phosphoric acid and acetic acid; and the alcohol comprises one or more of ethanol, propanol, isopropanol and methanol; and,

[0037] II) performed under static conditions, the viral inactivation is about 1h-3h.

[0038] In some embodiments of the present application, after the decellularization, the obtained tissue repair material is further subjected to freezing, slicing and sterilization.

[0039] In some embodiments of the present application, the freezing condition comprises: freeze-drying is performed at a rate of about 5℃ / min to about 12℃ / min to about -80℃ to about -20℃, and maintaining at about -20℃ to about -10℃ for about 10h to about 16h.

[0040] In some embodiments of the present application, the sectioning controls the thickness of the membrane to be about 0.1mm to about 1mm.

[0041] In some embodiments of the present application, the sterilization is performed by physical sterilization or chemical sterilization.

[0042] In some embodiments of the present application, the biological membrane material satisfies one or more of the following conditions:

[0043] (i) the biological membrane material is derived from dermis, bladder matrix membrane, peritoneum, fascia, pericardium, heart valve, small intestine, or basement membrane; and,

[0044] (ii) the biological membrane material is derived from pig, cow, or sheep.

[0045] One or more embodiments of the present application also provide a tissue repair material prepared by the preparation method of the tissue repair material.

[0046] One or more embodiments of the present application also provide a method for repairing bone defect, which comprises the step of repairing the bone defect area with the tissue repair material.

[0047] In some embodiments of the present application, the bone defect area is located in the oral cavity.

[0048] The details of one or more embodiments of the present application are set forth in the accompanying description, which makes apparent to those skilled in the art other features, purposes, and advantages of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0049] In order to more clearly illustrate the technical solutions in the embodiments of the present application, more completely understand the present application and its beneficial effects, the accompanying drawings needed to be used in the description of the embodiments will be briefly introduced. Obviously, the accompanying drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0050] Figure 1 is a tissue section observation figure of the tissue repair material prepared with the product of the first decellularization stage and the tissue repair material prepared with the product of the second decellularization stage in Example 1;

[0051] Fig. 2 is a microstructure observation 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;

[0052] Fig. 3 is a graph of the effectiveness animal test 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

[0053] The present application will be described in further detail with reference to the accompanying drawings, embodiments and examples. It is to be understood that these embodiments and examples are illustrative of the present application and are not restrictive of the scope of the present application, and the purpose of these embodiments and examples is to make the present disclosure more thorough and complete, and to fully convey the scope of the present application to those skilled in the art. It is also to be understood that the present application can be carried out in many different forms and should not be construed as limited to the embodiments and examples set forth herein. Rather, these embodiments and examples are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present application to those skilled in the art. Also, in the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced without one or more of these specific details.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the description herein is for the purpose of describing embodiments and examples only and is not intended to be limiting of the present application.

[0055] Terminology

[0056] Unless otherwise indicated or unless contradicted by context, the terms or phrases used herein have the following meanings:

[0057] The selection range of the terms "and / or", "or / and", "and / or" used in the present application includes any one of two or more relevant listed items, and also includes any and all combinations of the relevant listed items, which includes any two relevant listed items, any more relevant listed items, or all relevant listed items. It should be noted that when at least two conjunctions selected from "and / or", "or / and", "and / or" are combined to connect at least three items, it should be understood that in the present application, the technical solution undoubtedly includes the technical solution connected by "logical and", and also undoubtedly includes the technical solution connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For another example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C and D (i.e. the technical solution connected by "logical or"), and also includes any and all combinations of A, B, C and D, i.e. includes the combination of any two or any three of A, B, C and D, and also includes the four-item combination of A, B, C and D (i.e. the technical solution connected by "logical and").

[0058] In the present application, "multiple", "various", "multiple times", "multiple" and the like are used without specific limitation, which means greater than or equal to 2 in quantity. For example, "one or more" means one or greater than or equal to two.

[0059] As used herein, "combinations thereof", "any combination thereof", "any combination manner thereof" and the like include all suitable combination manners of any two or more listed items.

[0060] As used herein, "suitable", "suitable", "any suitable manner" and the like are subject to the implementation of the technical solutions of the present application, the solution of the technical problems of the present application, and the realization of the intended technical effects of the present application.

[0061] As used herein, "preferably", "better", "better", "preferably" only describe the implementation manner or embodiment with better effect, and it should be understood that it does not constitute a limitation on the protection scope of the present application.

[0062] In the present application, "further", "more further", "particularly" and the like are used for description purposes to represent differences in content, but should not be understood as a limitation on the protection scope of the present application.

[0063] In the present application, "optionally", "optional", "optional" means optional, i.e. selected from two parallel solutions of "yes" or "no". If there are multiple "optional" in a technical solution, and there is no specific description, and there is no contradictory relationship or mutual restriction, each "optional" is independent.

[0064] In the present application, the terms "first", "second", "third", "fourth" and the like in the "first aspect", "second aspect", "third aspect", "fourth aspect" and the like are used only for descriptive purposes, and cannot be construed as indicating or implying relative importance or quantity, nor can they be construed as implicitly indicating the importance or quantity of the technical features indicated. Moreover, "first", "second", "third", "fourth" and the like only serve the purpose of non-exhaustive enumeration and description, and should be understood as not constituting a closed limitation on the quantity.

[0065] In the present application, among the technical features described in an open manner, a closed technical solution consisting of the listed features is also included, as well as an open technical solution containing the listed features.

[0066] In the present application, with respect to a numerical interval (i.e. a numerical range), if no special instructions are given, the optional numerical distribution within the above-mentioned numerical interval is considered to be continuous, and includes the two numerical end points (i.e. the minimum value and the maximum value) of the numerical range, as well as every numerical value between the two numerical end points. If no special instructions are given, when the numerical interval only points to the integers within the numerical interval, including the two end point integers of the numerical range and every integer between the two end points, in this document, it is equivalent to directly listing every integer, for example, t is an integer selected from 1 to 10, which means that t is any integer selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe a feature or a characteristic, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed in this document should be understood to include any and all sub-ranges included therein.

[0067] In the present application, the temperature parameters, if not specifically limited, allow both constant temperature treatment and fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows fluctuations within the accuracy range controlled by the instrument. Fluctuations within a range such as ±5°C, ±4°C, ±3°C, ±2°C, ±1°C are allowed.

[0068] 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.

[0069] All the documents mentioned in the present application are cited as references in the present application as if each document is cited as a reference individually. Unless and to the extent that the cited documents conflict with the application purpose and / or technical solution of the present application, the cited documents are cited in the present application in their entirety, in their entirety purpose. When the present application refers to the cited documents, the definition of the relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited in the present application. When the present application refers to the cited documents, the examples, preferred modes of the relevant technical features cited are also cited in the present application as references, but are limited to the implementation of the present application. It should be understood that when the cited content conflicts with the description in the present application, the present application is for reference or is modified adaptively according to the description in the present application.

[0070] The main methods of decellularization of biological scaffolds are physical, chemical and enzymatic methods. No matter which decellularization method can destroy the structure and composition of ECM. Due to the tissue-specific factors such as cell density, matrix density and geometric factors including tissue thickness and shape, the best method of decellularization in tissues and organs is different. Due to the incomplete removal of cell residues, the decellularization process may cause some damage to the matrix. The effect of removing cells from the tissue depends on the source of the tissue and the decellularization method used. Each method affects the biochemical composition, tissue ultrastructure and mechanical properties of the remaining ECM, and then affects the body's response to the material, which is not conducive to the repair and regeneration of the tissue. In view of the deficiencies of the prior art, one of the purposes of the embodiments of the present application is to provide a preparation method of a tissue repair material, so that the obtained tissue repair material is completely decellularized, the material has antioxidant and anti-chronic inflammation effects, and has good biocompatibility. It is beneficial to the repair and regeneration of the tissue. On the basis of removing the immunogenic substances of the material, the anti-inflammatory and antioxidant reagents including tea polyphenols, gallic acid and ethyl cellulose are innovatively introduced, which can promote the healing of the tissue, induce bone regeneration, and enhance the mechanical properties. Greatly improve the clinical operation of the existing product and improve the effectiveness of the product.

[0071] Tea polyphenols can be used as a biological additive for food packaging materials due to their antioxidant and antimicrobial functions to reduce oxidation, 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.

[0072] Gallic acid (GA) is a natural polyphenol commonly found in plants, which has strong antioxidant and antibacterial effects. Ethyl cellulose is a high molecular polysaccharide compound. Using one or more of tea polyphenols, gallic acid and ethyl cellulose to configure a solution can increase the antioxidant and antibacterial effects of the decellularized biological material, and has good biocompatibility. It is beneficial to the repair and regeneration of the tissue.

[0073] The present inventors have also unexpectedly found that the use of tea polyphenols and gallic acid solution in combination has a synergistic effect, which is better than the effect of single use. In addition, ethyl cellulose is combined with tea polyphenols and gallic acid to form a complex solution system, which is superior to other similar substitutes.

[0074] After the membrane is treated by the above-mentioned reagent, a thickness cutting device is used in the drying process, so that the thickness uniformity of the product can be more accurately controlled, the product thickness meeting the needs of the clinical market can be prepared, and the industrial utilization rate of animal membrane, the production cost and the production capacity are greatly improved. The natural tissue fiber structure of the product after cutting is not damaged, the material is softer, it is more convenient for clinical use and improves the use effect.

[0075] In a first aspect, the present embodiment provides a membrane tissue treatment composition, which comprises tea polyphenols and gallic acid in a molar ratio of about 1:(2-8). For example, the molar ratio of tea polyphenols and gallic acid is 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, or 1:8.

[0076] In some examples, the membrane tissue treatment composition further comprises a high molecular polysaccharide compound, which comprises one or more of ethyl cellulose and hydroxypropyl cellulose; and the molar ratio of the tea polyphenols, the gallic acid and the high molecular polysaccharide compound is about 1:(2-8):(3-5). For example, the molar ratio of the tea polyphenols, the gallic acid and the high molecular polysaccharide compound is 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, or 1:8:5.

[0077] In a second aspect, the present application provides a membrane tissue processing reagent, which comprises tea polyphenols, gallic acid and a solvent; the molar ratio of the tea polyphenols and 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.

[0078] In some examples, the membrane tissue processing reagent further comprises a high molecular polysaccharide compound, which comprises one or more of ethyl cellulose and hydroxypropyl cellulose; the molar ratio of the tea polyphenols, the gallic acid and the high molecular 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 polysaccharide compound 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.

[0079] In some examples, the solvent comprises a PBS buffer. Optionally, the PBS buffer comprises about 0.1-0.3mol / L (for example, 0.1, 0.15, 0.2, 0.25, 0.3mol / L) of disodium hydrogen phosphate and about 0.1-0.3mol / L (for example, 0.1, 0.15, 0.2, 0.25, 0.3mol / L) of sodium dihydrogen phosphate, and the pH is about 6.5-7.5 (for example, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5).

[0080] In a third aspect, the present application provides a method for preparing a tissue repair material, comprising a step of decellularizing a biological membrane material using the membrane tissue processing reagent.

[0081] In some examples, the decellularization conditions comprise: being performed under shaking conditions, and being performed for about 2-5 times (e.g., 2, 3, 4, 5 times), each time for about 30-90 minutes (e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 minutes).

[0082] In some examples, the method for preparing a tissue repair material comprises a plurality of decellularization stages, and in one of the decellularization stages, the biological membrane material is decellularized using the membrane tissue processing reagent.

[0083] In some examples, the method for preparing a tissue repair material comprises a first decellularization stage and a second decellularization stage, and in the second decellularization stage, the biological membrane material is decellularized using the membrane tissue processing reagent.

[0084] In some examples, the step of the first decellularization stage comprises: sequentially placing the biological membrane material in a high-osmotic solution and a low-osmotic solution for decellularization. Optionally, the first decellularization stage satisfies one or more of the following conditions:

[0085] 1) the high-osmotic solution comprises about 0.01-1 M (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, 1 M) sodium chloride solution to which about 0.01-1 M (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, 1 M) of a base or an acid 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;

[0086] 2) the low-osmotic solution comprises water;

[0087] 3) the step of the first decellularization stage is repeated for about 2-5 times (e.g., 2, 3, 4, 5 times); and,

[0088] 4) being performed under shaking conditions, and being shaken in the high-osmotic solution and the low-osmotic solution for about 30-90 minutes (e.g., 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90 minutes) each time.

[0089] In some examples, the biomembrane material is treated to remove attached fat, connective tissue and marginal damaged tissue, delipidation and viral inactivation.

[0090] The delipidation step in the embodiments of the present application is not particularly limited. In some examples, the delipidation satisfies one or more of the following conditions:

[0091] (I) the delipidation reagent used includes one or more of methanol, chloroform, ethanol, propanol, isopropanol, n-hexane and ethyl acetate; and

[0092] (II) the delipidation is performed under shaking conditions, and the delipidation is performed for about 2 to 4 times (for example, 2, 3, 4 or 5 times), for about 2 to 10 hours (for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 hours) each time, and fresh delipidation reagent is replaced after each delipidation to perform the next delipidation.

[0093] The viral inactivation method in the embodiments of the present application is not particularly limited, and includes but is not limited to a chemical method. In some examples, the chemical method satisfies one or more of the following conditions:

[0094] (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; optionally, the acid includes one or more of hydrochloric acid, nitric acid, phosphoric acid and acetic acid; and the alcohol includes one or more of ethanol, propanol, isopropanol and methanol; and

[0095] (II) the viral inactivation is performed under static conditions, and the viral inactivation is performed for about 1 to 3 hours (for example, 1, 1.5, 2, 2.5 or 3 hours).

[0096] It can be understood that, after the decellularization treatment, the obtained tissue repair material is subjected to freezing, slicing and sterilization. The specific steps of freezing, slicing and sterilization in the embodiments of the present application are not particularly limited.

[0097] In some examples, the freezing conditions include: freeze-drying is performed at a rate of about 5 to 12 ℃ / min (for example, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5 or 12 ℃ / min) to about -80 to -20 ℃ (for example, -80, -75, -70, -65, -60, -55, -50, -45, -40, -35, -30, -25 or -20 ℃), and then kept at about -20 to -10 ℃ (for example, -20, -18, -16, -14, -12 or -10) for 10 to 16 hours (for example, 10, 11, 12, 13, 14, 15 or 16 hours).

[0098] In some examples, the slice controls the thickness of the membrane to be about 0.1mm-1mm, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1mm.

[0099] In some examples, the sterilization is by physical sterilization. In other examples, the sterilization is by chemical sterilization.

[0100] In some examples, the biological membrane material satisfies one or more of the following conditions:

[0101] (i) the biological membrane material is derived from dermis, urinary bladder matrix membrane, peritoneum, fascia, pericardium, heart valve, small intestine, or basement membrane; and,

[0102] (ii) the biological membrane material is derived from a pig, a cow, or a sheep.

[0103] In a fifth aspect, the present application provides a tissue repair material prepared by the method of preparing a tissue repair material according to the fourth aspect.

[0104] In a sixth aspect, the present application provides a method of repairing a bone defect, which comprises the step of repairing a bone defect region with the tissue repair material according to the fifth aspect.

[0105] In some examples, the bone defect region is located in the oral cavity.

[0106] The subject to be repaired according to the embodiments of the present application is not particularly limited and can be any animal that can have a bone defect and needs to be repaired. It can be a human or other non-human mammal. The term "mammal" in the present application mainly refers to warm-blooded vertebrate mammals, including but not limited to, for example, cats, dogs, rabbits, bears, foxes, wolves, monkeys, deer, mice (e.g., rats, mice), pigs, cows, sheep, horses, humans, etc. Preferably, the mammal is a primate, and more preferably, the mammal is a human.

[0107] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. The experimental methods in the following examples, if not specified, are preferred to refer to the instructions given in the present application, and can also be performed according to the experimental manuals or conventional conditions in the art, or according to the conditions suggested by the manufacturers, or according to the known experimental methods in the art.

[0108] In the following specific examples, the measurement parameters of the raw material components, if not specifically stated, can have slight deviations within the weighing accuracy range. For temperature and time parameters, acceptable deviations caused by instrument testing accuracy or operation accuracy are allowed.

[0109] Example 1

[0110] Step one, pretreatment: The bovine pericardium tissue was laid on a flat plate, and the attached fat, connective tissue and edge damaged tissue were removed. Then it was washed with water until no blood color was observed, and the biomembrane material was obtained.

[0111] Step two, defatting: The obtained biomembrane material was placed in an organic solvent for 6h of shaking defatting, and then the solution was changed. The defatting was repeated for 3 times, and the biomembrane material was washed with water until no odor was observed. The organic reagent was propanol.

[0112] Step three, virus inactivation: The obtained biomembrane material was placed in a chemical reagent for 2h of static inactivation, and then it was washed with water until no odor was observed. The chemical reagent was a sodium hydroxide solution with a sodium hydroxide content of 0.1M.

[0113] Step four, decellularization:

[0114] The first decellularization stage: the biomembrane material after virus inactivation was placed in a high-osmotic decellularization solution for 40min of shaking, and then it was transferred into a low-osmotic decellularization solution for 60min of shaking. The above process was repeated for 3 times. The high-osmotic decellularization solution was an aqueous solution containing 0.5M sodium chloride and 1M sodium hydroxide, and the low-osmotic decellularization solution was water.

[0115] The second decellularization stage used a PBS buffer solution (containing 0.2mol / L disodium hydrogen phosphate and 0.2mol / L sodium dihydrogen phosphate, pH7) to dissolve tea polyphenols, gallic acid and ethyl cellulose, and prepare a treatment reagent. The concentrations of tea polyphenols, gallic acid and ethyl cellulose in the treatment reagent were 0.02M, 0.04M and 0.06M, respectively. The biomembrane material was shaken in the treatment reagent for 50min, and then it was replaced with fresh treatment reagent. The above process was repeated for 3 times (i.e. after the first shaking treatment, the biomembrane material was replaced with fresh treatment reagent for the second time, and then it was replaced with fresh treatment reagent for the third time). The biomembrane material was washed with water until no odor was observed.

[0116] Step five, freeze-drying: the biomembrane material obtained in step four was flattened and attached to a flat plate, and then it was directly placed in a freeze-dryer for freeze-drying. The freeze-drying was performed at a rate of 8℃ / min to-80℃, and the freeze-drying was maintained at-20℃ for 16h. After the completion of freeze-drying, the biomembrane material was cut and packaged.

[0117] Step six, cutting: the biomembrane sheet after freeze-drying was treated using a cutting device. The biomembrane sheet was precisely cut to a thickness of 0.4mm, so as to realize the uniformity of the thickness of the single-specification biomembrane material.

[0118] Step seven, sterilization: chemical sterilization was performed using ethylene oxide, and the tissue repair material was obtained.

[0119] Example 2

[0120] Step one, pretreatment: the porcine peritoneal tissue is laid on a flat plate, and the attached fat, connective tissue and edge damaged tissue are removed, and then washed with water until no blood color is obtained, to obtain a biological membrane material.

[0121] Step two, degreasing: the obtained biological membrane material is placed in an organic solvent for 4h, and then the solution is changed, and the degreasing is repeated for 3 times, and then washed with water until no odor is obtained, and the organic reagent is ethanol.

[0122] Step three, virus inactivation: the obtained biological membrane material is placed in a chemical reagent for 1h, and then washed with water until no odor is obtained, and the chemical reagent is a sodium hydroxide solution with a sodium hydroxide content of 0.1M.

[0123] Step four, decellularization:

[0124] The first decellularization stage: the virus-inactivated biological membrane material is placed in a decellularization high-osmotic solution for 30min, and then transferred into a decellularization low-osmotic solution for 60min, and the cycle is repeated for 3 times; wherein the decellularization high-osmotic solution is an aqueous solution containing 0.5M sodium chloride and 1M sodium hydroxide, and the decellularization low-osmotic solution is water.

[0125] The second decellularization stage: PBS buffer solution (containing 0.2mol / L disodium hydrogen phosphate and 0.2mol / L sodium dihydrogen phosphate, pH7) is used to dissolve tea polyphenol, gallic acid and ethyl cellulose to prepare a treatment reagent, and the concentrations of tea polyphenol, gallic acid and ethyl cellulose in the treatment reagent are 0.02M, 0.04M and 0.1M respectively. The biological membrane material is shaken in the treatment reagent for 50min, and then replaced with fresh treatment reagent, and the cycle is repeated for 3 times, and then washed with water until no odor is obtained.

[0126] Step five, freeze-drying: the biological membrane material obtained in step four is flattened and attached to a flat plate, and then directly placed into a freeze-dryer. The freeze-drying is performed at a rate of 8℃ / min to-80℃, and then kept at-20℃ for 16h. After freeze-drying, cutting and packaging are performed.

[0127] Step six, cutting: the freeze-dried biological membrane sheet is treated using a cutting device, and the thickness of the membrane sheet is selected to be 0.3mm for precise thickness cutting, so as to realize uniform thickness of single-specification material.

[0128] Step seven, sterilization: irradiation sterilization is adopted to obtain a tissue repair material.

[0129] Example 3

[0130] This example is a variation of example 1, and the difference from example 1 is that:

[0131] Step two, defatting: the obtained biological membrane material is placed in an organic solvent for 2 hours of defatting, and the liquid is changed after defatting, and the defatting is repeated 4 times, and the water is washed until there is no odor. The organic reagent is propyl alcohol.

[0132] Step three, virus inactivation: the obtained biological membrane material is placed in hydrochloric acid for 1 hour of inactivation, and the water is washed until there is no odor.

[0133] Step four, decellularization:

[0134] The first decellularization stage: the virus-inactivated biological membrane material is placed in a high-osmotic decellularization solution for 50 minutes of oscillation, and then transferred into a low-osmotic decellularization solution for 30 minutes of oscillation, and the cycle is repeated 5 times. The high-osmotic decellularization solution is an aqueous solution containing 2M sodium chloride and 0.01M sodium hydroxide, and the low-osmotic decellularization solution is water.

[0135] The second decellularization stage: PBS buffer solution (containing 0.1 mol / L disodium hydrogen phosphate and 0.3 mol / L sodium dihydrogen phosphate, pH 6.5) is used to dissolve tea polyphenols, gallic acid and ethyl cellulose to prepare a treatment reagent. The molar concentration ratio of tea polyphenols, gallic acid and ethyl cellulose in the treatment reagent is 1:2:5, and the total concentration of tea polyphenols, gallic acid and ethyl cellulose is 0.01M. The biological membrane material is oscillated in the treatment reagent for 30 minutes, and then fresh treatment reagent is replaced, and the cycle is repeated 5 times. The water is washed until there is no odor.

[0136] Step five, freeze-drying: the biological membrane material obtained in step four is flattened and attached to a flat plate, and then directly placed in a freeze-drying machine for freeze-drying. The freeze-drying is performed at a rate of 5℃ / min to -20℃, and the freeze-drying is maintained at -10℃ for 10 hours. After the freeze-drying is completed, cutting and packaging are performed.

[0137] Step six, cutting: the freeze-dried biological membrane sheet is treated using a cutting device. The thickness of the membrane sheet is selected to be 0.1mm for precise thickness cutting, so as to realize uniform thickness of single-specification material.

[0138] The rest is the same as example 1.

[0139] Example 4

[0140] This example is a variation of example 1, and the difference from example 1 is that:

[0141] Step two, defatting: the obtained biological membrane material is placed in ethyl acetate for 10 hours of defatting, and the liquid is changed after defatting, and the defatting is repeated 4 times, and the water is washed until there is no odor.

[0142] Step three, virus inactivation: the obtained biological membrane material is placed in propyl alcohol for 3 hours of inactivation, and the water is washed until there is no odor.

[0143] Step four, decellularization:

[0144] The first decellularization stage: the virus-inactivated biological membrane material is placed in a decellularization high-osmotic solution for 90 minutes of shaking, then transferred into a decellularization low-osmotic solution for 90 minutes of shaking, and the cycle is repeated twice; wherein the decellularization high-osmotic solution is an aqueous solution containing 5M sodium chloride and 0.5M sodium hydroxide, and the decellularization low-osmotic solution is water.

[0145] The second decellularization stage: tea polyphenols, gallic acid and ethyl cellulose are dissolved in a PBS buffer solution (containing 0.3 mol / L disodium hydrogen phosphate and 0.1 mol / L sodium dihydrogen phosphate, pH 7.5) to prepare a treatment reagent, the molar concentration ratio of tea polyphenols, gallic acid and ethyl cellulose in the treatment reagent is 1:8:3, and the total concentration of tea polyphenols, gallic acid and ethyl cellulose is 1M. The biological membrane material is shaken in the treatment reagent for 50 minutes, the fresh treatment reagent is replaced, and the cycle is repeated for 3 times, and then washed with water until no odor is generated.

[0146] Step five, freeze-drying: the biological membrane material obtained in step four is flattened and attached to a flat plate, and then directly placed in a freeze dryer for freeze-drying. The freeze-drying is performed at a rate of 12℃ / min to -16℃, and the freeze-drying is maintained at -15℃ for 14 hours. After the freeze-drying is completed, the biological membrane material is cut and packaged.

[0147] Step six, cutting: the freeze-dried biological membrane sheet is treated using a cutting device. The thickness of the membrane sheet is selected to be 1mm for precise thickness cutting, so as to realize uniform thickness of single-specification material.

[0148] The rest is the same as example 1.

[0149] Example 5

[0150] This example is a variation of example 1, and the changes relative to example 1 include:

[0151] In the second decellularization stage of step four, no ethyl cellulose is added, i.e. tea polyphenols and gallic acid are dissolved in a PBS buffer solution (containing 0.2 mol / L disodium hydrogen phosphate and 0.2 mol / L sodium dihydrogen phosphate, pH 7) to prepare a treatment reagent, and the concentrations of tea polyphenols and gallic acid in the treatment reagent are 0.04M and 0.08M respectively. The biological membrane material is shaken in the solution for 50 minutes, the fresh treatment reagent is replaced after the shaking is completed, and the cycle is repeated for 3 times, and then washed with water until no odor is generated.

[0152] The rest is the same as example 1.

[0153] Example 6

[0154] This example is a variation of example 1, and the changes relative to example 1 include:

[0155] In the second decellularization stage of Step 4, the ethyl cellulose is replaced with hydroxypropyl cellulose. That is, the tea polyphenol, gallic acid, and hydroxypropyl cellulose are dissolved in a PBS buffer solution (containing 0.2 mol / L of disodium hydrogen phosphate and 0.2 mol / L of sodium dihydrogen phosphate, pH 7) to prepare a treatment reagent, and the concentrations of the tea polyphenol, gallic acid, and hydroxypropyl cellulose in the treatment reagent are 0.02 M, 0.04 M, and 0.06 M, respectively. The biomembrane material is shaken in the treatment reagent for 50 minutes, and then fresh treatment reagent is replaced, and the above operation is repeated three times, and then the biomembrane material is washed with water until no odor is generated.

[0156] The rest is the same as in Example 1.

[0157] Example 7

[0158] This example is a variation of Example 1, and the changes relative to Example 1 include:

[0159] In the second decellularization stage of Step 4, the ethyl cellulose is replaced with hydroxypropyl cellulose. That is, the tea polyphenol, gallic acid, and hydroxypropyl cellulose are dissolved in a PBS buffer solution (containing 0.2 mol / L of disodium hydrogen phosphate and 0.2 mol / L of sodium dihydrogen phosphate, pH 7) to prepare a treatment reagent, and the concentrations of the tea polyphenol, gallic acid, and hydroxypropyl cellulose in the treatment reagent are 0.02 M, 0.04 M, and 0.06 M, respectively. The biomembrane material is shaken in the treatment reagent for 50 minutes, and then fresh treatment reagent is replaced, and the above operation is repeated three times, and then the biomembrane material is washed with water until no odor is generated.

[0160] The rest is the same as in Example 1.

[0161] Example 8

[0162] This example is a variation of Example 1, and the changes relative to Example 1 include:

[0163] In the second decellularization stage of Step 4, the ethyl cellulose is replaced with hydroxypropyl cellulose. That is, the tea polyphenol, gallic acid, and hydroxypropyl cellulose are dissolved in a PBS buffer solution (containing 0.2 mol / L of disodium hydrogen phosphate and 0.2 mol / L of sodium dihydrogen phosphate, pH 7) to prepare a treatment reagent, and the concentrations of the tea polyphenol, gallic acid, and hydroxypropyl cellulose in the treatment reagent are 0.02 M, 0.04 M, and 0.06 M, respectively. The biomembrane material is shaken in the treatment reagent for 50 minutes, and then fresh treatment reagent is replaced, and the above operation is repeated three times, and then the biomembrane material is washed with water until no odor is generated.

[0164] The rest is the same as in Example 1.

[0165] Example 9

[0166] This example is a variation of Example 1, and the changes relative to Example 1 include:

[0167] In the second decellularization stage of Step 4, the molar concentration ratio of tea polyphenol, gallic acid, and ethyl cellulose is 1:2:3, and the total concentration is 1.2 M.

[0168] The rest is the same as Example 1.

[0169] Example 10

[0170] This example is a variation of Example 1, and the changes relative to Example 1 include:

[0171] In the second decellularization stage of Step 4, the molar concentration ratio of tea polyphenol, gallic acid, and ethyl cellulose is 1:2:3, and the total concentration is 0.009 M.

[0172] The rest is the same as Example 1.

[0173] Comparative Example 1

[0174] This comparative example is a comparative example of Example 1, and the only difference relative to Example 1 is that in the second decellularization stage of Step 4, PBS buffer solution is used to dissolve tea polyphenol to prepare the treatment reagent, and the concentration of tea polyphenol in the treatment reagent is 0.06 M. The rest is the same as Example 1.

[0175] Comparative Example 2

[0176] This comparative example is a comparative example of Example 1, and the only difference relative to Example 1 is that in the second decellularization stage of Step 4, PBS buffer solution is used to dissolve gallic acid to 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.

[0177] Comparative Example 3

[0178] This comparative example is a comparative example of Example 1, and the only difference relative to Example 1 is that:

[0179] In Step 4, tea polyphenol is replaced by tannic acid. That is, PBS buffer solution is used to dissolve tannic acid, gallic acid, and ethyl cellulose to prepare the treatment reagent, and 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 is the same as Example 1.

[0180] Comparative Example 4

[0181] This comparative example is a comparative example of Example 1, and the only difference relative to Example 1 is that:

[0182] In the second decellularization stage of Step 4, gallic acid is replaced by epigallocatechin gallate. That is, a PBS buffer solution is used to dissolve tea polyphenols, epigallocatechin gallate and ethyl cellulose to prepare a treatment reagent, and the concentrations of tea polyphenols, epigallocatechin gallate and ethyl cellulose in the treatment reagent are 0.02M, 0.04M and 0.06M, respectively. The rest is the same as in Example 1.

[0183] Comparative Example 5

[0184] This comparative example is a comparative example of Example 1, and the only difference from Example 1 is that:

[0185] In the second decellularization stage of Step 4, the molar ratio of tea polyphenols to gallic acid is 1:1. Specifically, a PBS buffer solution is used to dissolve tea polyphenols, gallic acid and ethyl cellulose to prepare a treatment reagent, and the concentrations of tea polyphenols, gallic acid and ethyl cellulose in the treatment reagent are 0.03M, 0.03M and 0.06M, respectively. The rest is the same as in Example 1.

[0186] Comparative Example 6

[0187] This comparative example is a comparative example of Example 1, and the only difference from Example 1 is that:

[0188] In the second decellularization stage of Step 4, the molar ratio of tea polyphenols to gallic acid is 1:9. Specifically, a PBS buffer solution is used to dissolve tea polyphenols, gallic acid and ethyl cellulose to prepare a treatment reagent, and the concentrations of tea polyphenols, gallic acid and ethyl cellulose in the treatment reagent are 0.01M, 0.09M and 0.06M, respectively. The rest is the same as in Example 1.

[0189] Performance test

[0190] Experiment 1: Histological section

[0191] At least 1.0 x 1.0 cm of the tissue repair material of Example 1 was taken and detected by using a conventional HE staining technique. The results are shown in Figure 1. In Figure 1, the first row of three images is the tissue repair material prepared by using the product of the first decellularization stage, and the second row of three images is the tissue repair material prepared by using the product of the second decellularization stage.

[0192] Experiment 2: Microstructure observation

[0193] Take the fully rehydrated tissue repair material after 5 min of Example 1, cut into about 1 mm x 5 mm size, after treatment and staining, observed by transmission electron microscope. The results are shown in Figure 2, the left figure of Figure 2 is the tissue repair material prepared by the product of the first decellularization stage, and the right figure is the tissue repair material prepared by the product of the second decellularization stage, the results show that the tissue repair material of Example 1 has complete natural collagen fiber structure, and obvious and clear collagen fiber arrangement structure can be seen along the long axis direction of the collagen fiber.

[0194] Experiment 3: mechanical property detection

[0195] The suture tear force detection of the tissue repair material was carried out according to YY 0500-2020.

[0196] The tensile breaking strength and elongation at break of the tissue repair material were determined according to GB / T 3923.1-2013 Part 1 (strip method).

[0197] The results show that the mechanical properties of each example are stronger, and the use of the decellularization reagent of the application can enhance the toughness of the tissue repair material, and the effect of the combination of tea polyphenol, gallic acid and ethyl cellulose is better than that of the three components alone, and the mechanical properties are more advantageous.

[0198] Table 1, suture tear force

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

[0200] Table 2, tensile breaking strength

[0201] In Table 2, "after decellularization" refers to the tissue repair material prepared by the product of the first decellularization stage, and "after solution treatment" refers to the tissue repair material prepared by the product of the second decellularization stage.

[0202] Table 3, elongation at break

[0203] In Table 3, "after decellularization" refers to the tissue repair material prepared by the product of the first decellularization stage, and "after solution treatment" refers to the tissue repair material prepared by the product of the second decellularization stage.

[0204] Experiment 4: in vitro degradation detection

[0205] The tissue repair material samples of each example and comparative example were cut into uniform pieces and added to 10 mL of 0.1 M (pH 7.4) PBS buffer containing 50 CDU / mL collagenase type I, and shaken at 37°C.

[0206] The results are shown in Figure 3, which shows that the tissue repair material of Example 1 has more excellent degradation performance and a longer in vitro degradation time, and has an advantage in the barrier time during clinical use, can effectively promote the healing of the tissue, and plays the natural advantage of the tissue repair material. See the second row of the figure in Figure 3 (tissue repair material prepared using the product of the second decellularization stage); the first row of the figure in Figure 3 is the tissue repair material prepared using the product of the first decellularization stage.

[0207] Table 4, in vitro degradation

[0208] 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.

[0209] Experiment 5: animal experiment

[0210] The sample of the tissue repair material of Example 1 (i.e., the tissue repair material prepared using the product of the second decellularization stage) was subjected to an effectiveness test for dog tooth socket filling to verify the degradation performance and the healing performance for soft tissue of the material.

[0211] From the histological results of the animal experiment, it can be seen that the tissue repair material of Example 1 was implanted for 2 weeks, early vascularization, and early new bone formation was formed under the collagen membrane. The tissue repair material was gradually degraded after being implanted for 8 weeks, and new bone formation was continuously formed after being implanted for 12-16 weeks, and the epithelium covered the defect area.

[0212] Each of the technical features of the above-described embodiments and examples can be combined in any suitable manner. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments and examples are described, however, as long as the combinations of the technical features do not contradict each other, they should be considered within the scope of the present disclosure.

[0213] The above-described embodiments only express several implementation manners of the present application, facilitate specific and detailed understanding of the technical solutions of the present application, but cannot be understood as a limitation on the patent protection scope of the present application. It should be noted that, for ordinary skilled persons in the art, several modifications and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. In addition, it should be understood that, after reading the above teaching content of the present application, the skilled person in the art can make various modifications or modifications to the present application, and the equivalent forms obtained are also within the protection scope of the present application. It should also be understood that, on the basis of the technical solutions provided by the present application, the skilled person in the art obtains the technical solutions through logical analysis, reasoning or limited experiments, and all of them are within the protection scope of the appended claims of the present application. Therefore, the protection scope of the patent of the present application should be based on the content of the appended 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 film tissue treatment composition according to claim 1, characterized in that, The membrane tissue treatment composition further comprises a high molecular polysaccharide compound, which comprises one or more of ethyl cellulose and hydroxypropyl cellulose. The molar ratio of the tea polyphenols, the gallic acid and the high molecular polysaccharide compound is about 1:(2-8):(3-5).

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

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

5. The membrane tissue treatment reagent according to any one of claims 3 to 4, characterized by, The solvent comprises a PBS buffer. Optionally, the PBS buffer comprises about 0.1mol / L-0.3mol / L disodium hydrogen phosphate and about 0.1mol / L-0.3mol / L sodium dihydrogen phosphate, with a pH of about 6.5-7.

5.

6. A method of preparing a tissue repair material, characterized by, The preparation method comprises a step of decellularizing the biomembrane material using the membrane tissue treatment reagent according to any one of claims 3-5.

7. The method of claim 6, wherein the tissue repair material is prepared by the steps of: The decellularization is performed under oscillation, and the number of times of treatment is about 2-5, with each time being about 30min-90min.

8. The method of claim 6, wherein the tissue repair material is prepared by the steps of: The preparation method comprises a plurality of decellularization stages, and the biomembrane material is decellularized using the membrane tissue treatment reagent in one of the decellularization stages.

9. The method of claim 8, wherein the tissue repair material is prepared by the steps of: The preparation method comprises a first decellularization stage and a second decellularization stage, and the biomembrane material is decellularized using the membrane tissue treatment reagent in the second decellularization stage.

10. The method of claim 7, wherein the tissue repair material is prepared by the steps of: The steps of the first decellularization stage comprise sequentially placing the biomembrane material in a high-osmotic solution and a low-osmotic solution for decellularization. Optionally, the first decellularization stage satisfies one or more of the following conditions: 1) the high-osmotic solution comprises about 0.01M-1M sodium chloride solution added with about 0.01M-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; 2) the low-osmotic solution comprises water; 3) the steps of the first decellularization stage are repeated about 2-5 times; and 4) the decellularization is performed under oscillation, and the oscillation time in the high-osmotic solution and the low-osmotic solution is independently about 30min-90min.

11. The method of producing a tissue repair material according to any one of claims 6 to 10, characterized in that, The biomembrane material is treated to remove attached fat, connective tissue and marginally damaged tissue, defat and inactivate viruses.

12. The method of claim 11, wherein the tissue repair material is prepared by the steps of: The defatting satisfies one or more of the following conditions: (I) the defatting reagent includes one or more of methanol, chloroform, ethanol, propanol, isopropanol, n-hexane and ethyl acetate; and (II) the defatting is performed under shaking condition, about 2 to 4 times, each time for about 2 to 10 hours, and fresh defatting reagent is used for each time of defatting.

13. The method of claim 11, wherein the tissue repair material is prepared by the steps of: The virus inactivation is performed by chemical method. Optionally, the chemical method satisfies one or more of the following conditions: (I) the inactivation reagent includes one or more of acid, base and alcohol; optionally, the base includes one or more of sodium hydroxide, sodium chloride, potassium hydroxide and ammonia; optionally, the acid includes one or more of hydrochloric acid, nitric acid, phosphoric acid and acetic acid; and the alcohol includes one or more of ethanol, propanol, isopropanol and methanol; and (II) the virus inactivation is performed under static condition, and the virus inactivation time is about 1 to 3 hours.

14. The method of producing a tissue repair material according to any one of claims 6 to 10 and 12 to 13, characterized in that, After the decellularization, the obtained tissue repair material is further subjected to freezing, slicing and sterilization. Optionally, the freezing condition includes: the freeze-drying is performed at a rate of about 5 to 12℃ / min to about -80 to -20℃, and the temperature is kept at about -20 to -10℃ for about 10 to 16 hours. Optionally, the slicing controls the thickness of the film to be about 0.1 to 1mm. Optionally, the sterilization is performed by physical sterilization method or chemical sterilization method.

15. The method of producing a tissue repair material according to any one of claims 6 to 10 and 12 to 13, characterized in that, The biological film material satisfies one or more of the following conditions: (I) the biological film material is derived from dermis, bladder matrix film, peritoneum, fascia, pericardium, heart valve, small intestine or basement membrane; and (II) the biological film material is derived from pig, cow or sheep.

16. A tissue repair material, characterized by, The tissue repair material is prepared by the method of any one of claims 6 to 15.

17. A method of repairing a bone defect, comprising: providing a bone graft material according to claim 1 ; and implanting the bone graft material into a bone defect. The repair method includes the step of repairing the bone defect area by using the tissue repair material of claim 16.

18. The method of repairing a bone defect of claim 17, wherein, The bone defect area is located in oral cavity.

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