Acellular matrix material and preparation method thereof

Abstract

The invention relates to the field of biological materials, and particularly discloses an acellular matrix material and a preparation method thereof. The preparation method of the material comprises the following steps: extracting a tissue material of a mammal, carrying out decellularization treatment on the tissue material to obtain a decellularized tissue material, freeze-drying the tissue material, and crushing to obtain an animal-derived decellularized matrix; resuspending an animal-derived acellular matrix in water, mixing the animal-derived acellular matrix with the modified epoxidized chitosan solution, adding transglutaminase for reaction, and freeze-drying the obtained product to obtain the acellular matrix material. According to the method, the animal-derived acellular matrix and the modified epoxidized chitosan are cross-linked, so that the biocompatibility of the acellular matrix can be improved, and immunological rejection can be reduced; meanwhile, as the cross-linking reaction is biological cross-linking under the action of transglutaminase, bioactive factors contained in the acellular matrix are not easy to damage in the cross-linking process, and the biological activity and safety are better.

Description

Technical Field

[0001] The present application relates to the field of biomedical materials, and more specifically, to a decellularized matrix material and a preparation method thereof. Background Art

[0002] In the traditional field of cosmetic medicine, commonly used filling materials and repair materials often have certain limitations. For example, synthetic materials may cause immune reactions or adverse reactions, and sometimes it is difficult to achieve good integration with host tissues. In order to solve these problems, in recent years, decellularized extracellular matrix (dECM) has attracted widespread attention as a new type of biomaterial.

[0003] dECM is a biomaterial extracted from animal tissues. It has been specially processed to remove cellular components, but retains the natural extracellular matrix structure and the growth factors, collagen and other important components it contains. This makes dECM have good biocompatibility, can reduce immune response, and provide an ideal environment for supporting cell growth and repair. Compared with traditional synthetic materials, dECM has shown significant advantages and potential in the field of aesthetic medicine.

[0004] However, although dECM has shown many advantages in medical and aesthetic applications, its application in the field of medical and aesthetic medicine still faces many challenges. For example, due to its natural origin and preparation process, its stability and durability in certain applications are poor. In addition, despite the treatment, the decellularized matrix may still have potential biocompatibility issues, which require further optimization and adjustment to improve compatibility with host tissues. How to improve the biocompatibility of dECM and improve its safety has become an important topic of current research and development. These efforts will help promote the widespread application of dECM in the field of aesthetic medicine, enhance its therapeutic effects and patient satisfaction, and thus meet the growing demand in the market. Summary of the invention

[0005] In order to improve the biocompatibility of a decellularized matrix, the present application provides a decellularized matrix material and a preparation method thereof.

[0006] This application provides the following technical solutions: In one aspect, the present application provides a method for preparing a decellularized matrix material, comprising: Extracting mammalian tissue materials, decellularizing the tissue materials to obtain decellularized tissue materials, freeze-drying the tissue materials, and then crushing them to obtain animal-derived decellularized matrix; The animal-derived decellularized matrix is ​​resuspended in water to obtain a decellularized powder suspension, which is mixed with a modified epoxidized chitosan solution, the pH of the mixed system is adjusted to 7.5-8.0, 0.5-0.8% transglutaminase is added, the mixture is stirred at 33-35°C and 200-400 rpm for 18-24 hours, and the obtained product is freeze-dried to obtain a decellularized matrix material.

[0007] The above technical solution first decellularizes the entire mammalian tissue material and then crushes it. On the one hand, it is beneficial to completely remove the cellular components in the tissue material, especially the cell nucleus and cell membrane fragments, thereby reducing immunogenic substances; on the other hand, it can avoid the destruction of the bioactive factors contained in the extracellular matrix, thereby affecting the biological activity and induction ability of the decellularized matrix.

[0008] This method can improve the biocompatibility of the decellularized matrix and reduce immune rejection by cross-linking the animal-derived decellularized matrix with modified epoxidized chitosan. At the same time, since the cross-linking reaction is a biological cross-linking under the action of transglutaminase, the bioactive factors contained in the decellularized matrix are not easily destroyed during the cross-linking process, and there will be no chemical residues, which is safer than chemical cross-linking. During the cross-linking process, the cross-linking conditions (such as pH, temperature, and time) are precisely controlled to ensure the efficient cross-linking reaction, while effectively avoiding damage to the function of the decellularized matrix due to excessive cross-linking.

[0009] Further, the mammalian tissue material includes any one of pig skin, cow skin, pig pericardium, bovine pericardium, pig integument and pig small intestinal submucosa.

[0010] Furthermore, the mass concentration of the animal-derived decellularized matrix in the decellularized powder suspension is 3.5-4.5%, the mass concentration of the modified epoxidized chitosan in the epoxidized chitosan solution is 1.5-2%, and the volume ratio of the decellularized powder suspension to the epoxidized chitosan solution in the mixed system is 4-5:1.

[0011] The above technical solution limits the content of animal-derived decellularized matrix and modified epoxidized chitosan. The content ratio of modified epoxidized chitosan to decellularized matrix affects the biocompatibility and immunogenicity of the material. An appropriate amount of chitosan can improve the biocompatibility of the material, but an excessive amount of chitosan may cause an immune response and affect the application of the material in the body.

[0012] Compared with the direct cross-linking of natural chitosan, the present application uses modified epoxidized chitosan to cross-link with the decellularized matrix. Since epoxidized chitosan contains multiple epoxy groups, it can form stable covalent bonds with the amino groups in the decellularized matrix, thereby improving the cross-linking efficiency and the stability of the material. In order to further enhance the biocompatibility of the material and improve the safety of use, in the optimized scheme of the present application, epoxidized chitosan and bletilla striata polysaccharide are compositely modified. Under the action of a cross-linking agent, the two undergo a cross-linking reaction to form a stable cross-linked network structure, which has almost no toxic effect on cells. Moreover, since both chitosan and bletilla striata polysaccharide have good biocompatibility, after cross-linking modification, the biocompatibility of the material is further enhanced, reducing the immune response to the organism, which is beneficial to the adhesion, proliferation and differentiation of cells.

[0013] Furthermore, the modified epoxidized chitosan is obtained by composite-modifying epoxidized chitosan and bletilla striata polysaccharide in a mass ratio of 1-3:1.

[0014] Preferably, the composite modification method comprises: Dissolving epoxidized chitosan in an acidic solution to obtain a chitosan solution with a concentration of 3-5 wt %, and dissolving bletilla striata polysaccharide in an acidic solution to obtain a bletilla striata polysaccharide solution with a concentration of 1-3 wt %; The chitosan solution and the bletilla striata polysaccharide solution are mixed, the pH value of the mixed solution is adjusted to 4-6, a cross-linking agent is added to the mixed solution, and then cross-linked at 60-70° C. for 5-6 hours.

[0015] Preferably, the cross-linking agent is 0.1-1% genipin. Genipin is the product of jasmine glycoside hydrolysis by β-glucosidase, and its chemical structure contains hydroxyl, carboxyl and a special olefinic carbon atom (C-3 position). Under acidic conditions, these reactive groups can react with the active groups on chitosan and bletilla striata polysaccharide during the cross-linking process by nucleophilic reaction. Genipin acts as a cross-linking bridge to connect the polysaccharide molecules to form a stable network structure.

[0016] Furthermore, the method for decellularizing tissue material includes: After pretreatment of the tissue material, a digestion treatment was performed in a trypsin solution; The resulting tissue material is then placed in a nuclease solution for secondary digestion; The obtained tissue material was then placed in a sodium N-lauroyl-L-glutamate solution for three deep treatments.

[0017] The above technical solution, through pretreatment of tissue materials and subsequent three treatments, helps to completely decellularize, remove residual cell debris, residual DNA and reagents, reduce immunogenic substances, and thus improve the safety of material use. In particular, three deep treatments with sodium N-lauroyl-L-glutamate can be inserted into the lipid bilayer of the cell membrane, destroy the lipid structure, dissociate and dissolve the cell membrane, and help remove proteins, organelles, etc. in the cell, further ensuring the complete removal of cells and improving the purity of the decellularized matrix.

[0018] Preferably, in the three deep treatments, the concentration of the sodium N-lauroyl-L-glutamate solution is 5-6%, the treatment temperature is 30-35° C., and the treatment time is 8-10 h.

[0019] Preferably, the concentration of the pancreatic enzyme solution is 0.15-0.25%, and the treatment time is 8-9 hours; the nuclease includes 0.01-0.02% ribonuclease and 0.02-0.04% deoxyribonuclease, and the treatment time is 4-5 hours.

[0020] Preferably, the particle size of the animal-derived decellularized matrix is ​​20-300 μm.

[0021] In a second aspect, the present application provides a decellularized matrix material prepared by the above method.

[0022] In summary, this application has the following beneficial effects: The present application can improve the biocompatibility of the decellularized matrix and reduce immune rejection reactions by cross-linking the animal-derived decellularized matrix with modified epoxidized chitosan; at the same time, since the cross-linking reaction is a biological cross-linking carried out under the action of transglutaminase, the bioactive factors contained in the decellularized matrix are not easily destroyed during the cross-linking process, and there will be no chemical residues, which is safer than chemical cross-linking.

[0023] Compared with the direct cross-linking of natural chitosan, the present application uses modified epoxidized chitosan to cross-link the decellularized matrix. Since epoxidized chitosan contains multiple epoxy groups, it can form stable covalent bonds with the amino groups in the decellularized matrix, thereby improving the cross-linking efficiency, material stability and biocompatibility. DETAILED DESCRIPTION

[0024] The embodiments of the present invention will be described in detail below in conjunction with examples. However, those skilled in the art will understand that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. The specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0025] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0026] Preparation Example 1 This preparation example provides a modified epoxidized chitosan, and the preparation method thereof is as follows: (1) 80 g of epoxidized chitosan (purchased from Xi'an Qiyue Biological Company) was dispersed in a 0.1 M phosphate buffer solution to obtain a chitosan solution with a concentration of 4 wt%; 40 g of Bletilla striata polysaccharide was dissolved in a 0.1 M phosphate solution to obtain a Bletilla striata polysaccharide solution with a concentration of 2 wt%; (2) The chitosan solution and the bletilla striata polysaccharide solution are mixed, and the pH value of the mixed solution is adjusted to 5. Genipin is added to the mixed solution in an amount of 0.5%. The mixed solution is heated in a water bath at 65° C. for 5 h. The obtained product is washed and dried to obtain modified epoxidized chitosan.

[0027] Preparation Example 2 This preparation example provides a modified epoxidized chitosan, and the preparation method thereof is as follows: (1) 80 g of epoxidized chitosan was dispersed in a 0.1 M phosphate buffer solution to obtain a chitosan solution with a concentration of 5 wt%; 40 g of bletilla striata polysaccharide was dissolved in a 0.1 M phosphate solution to obtain a bletilla striata polysaccharide solution with a concentration of 3 wt%; (2) The chitosan solution and the bletilla striata polysaccharide solution are mixed, and the pH value of the mixed solution is adjusted to 4-6. Genipin is added to the mixed solution in an amount of 0.1%. The mixed solution is heated in a water bath at 60° C. for 6 h. The obtained product is washed and dried to obtain modified epoxidized chitosan.

[0028] Preparation Example 3 This preparation example provides a modified epoxidized chitosan, and the preparation method thereof is as follows: (1) 80 g of epoxidized chitosan was dispersed in a 0.1 M phosphate buffer solution to obtain a chitosan solution with a concentration of 3 wt%; 40 g of bletilla striata polysaccharide was dissolved in a 0.1 M phosphate solution to obtain a bletilla striata polysaccharide solution with a concentration of 1 wt%; (2) The chitosan solution and the bletilla striata polysaccharide solution are mixed, and the pH value of the mixed solution is adjusted to 4. Genipin is added to the mixed solution in an amount of 1%. The mixed solution is heated in a water bath at 70° C. for 5 h. The obtained product is washed and dried to obtain modified epoxidized chitosan.

[0029] Preparation Example 4 This preparation example provides a modified epoxidized chitosan, and its preparation method is consistent with that of Preparation Example 1, except that: the mass ratio of epoxidized chitosan to the bletilla striata polysaccharide is 3:1, that is, the mass of epoxidized chitosan in step 1 is 120 g.

[0030] Comparative Preparation Example 5 This preparation example provides a modified epoxidized chitosan, and its preparation method is consistent with that of Preparation Example 1, except that the mass ratio of epoxidized chitosan to the bletilla striata polysaccharide is 1:1, that is, the mass of epoxidized chitosan in step 1 is 40 g.

[0031] Comparative Preparation Example 6 This preparation example provides a modified epoxidized chitosan, and its preparation method is consistent with that of Preparation Example 1, except that the mass ratio of epoxidized chitosan to the bletilla striata polysaccharide is 4:1, that is, the mass of epoxidized chitosan in step 1 is 160 g.

[0032] Example 1 This embodiment provides a decellularized matrix material, and the preparation method thereof comprises the following steps: 1. Take pig skin, remove the epidermis and fat layer, and wash it clean; 2. Place the obtained pig skin in a 2-4% sodium hydroxide solution and shake for 2 hours to remove residual fat; 3. Place the obtained pig skin in a 0.2% pancreatic enzyme solution and soak it at 36°C for 8 hours with stirring for a digestion treatment to degrade the protein in the extracellular matrix; 4. Wash the digested material and place it in a nuclease solution (containing 0.01% ribonuclease and 0.02% deoxyribonuclease), stir and soak for 4 hours at 36°C for secondary digestion to degrade the nucleic acid molecules in the extracellular matrix; 5. Wash the digested material and place it in a 6% sodium N-lauroyl-L-glutamate solution at 35°C for 8 hours. After three deep treatments, wash the tissue material in hot water for three times and freeze-dry it. 6. The decellularized tissue material is crushed, and the portion with a particle size of 60-80 μm is taken as the animal-derived decellularized matrix; 7. Resuspend the animal-derived decellularized matrix in water to obtain a decellularized powder suspension with a mass concentration of 3.5%, and mix it with a modified epoxidized chitosan solution (mass concentration of 1.5%, purchased from Xi'an Qiyue Biological Company) at a volume ratio of 4:1. Adjust the pH of the mixed system to 7.5-8.0, add 0.8% transglutaminase, place at 33-35°C and stir at 300 rpm for 20 hours, and freeze-dry the resulting product to obtain a decellularized matrix material.

[0033] Example 2 This embodiment provides a decellularized matrix material, and the preparation method thereof comprises the following steps: 1. Take pig skin, remove the epidermis and fat layer, and wash it clean; 2. Place the obtained pig skin in a 2-4% sodium hydroxide solution and shake for 12 hours to remove residual fat; 3. Place the obtained pig skin into 0.15% pancreatic enzyme solution and soak it at 36°C for 9 hours with stirring for a digestion treatment to degrade the protein in the extracellular matrix; 4. Wash the digested material and place it in a nuclease solution (containing 0.02% ribonuclease and 0.04% deoxyribonuclease), stir and soak for 5 hours at 36°C for secondary digestion to degrade the nucleic acid molecules in the extracellular matrix; 5. Wash the digested material and place it in a 5% sodium N-lauroyl-L-glutamate solution at 30°C for 10 hours. After three deep treatments, wash the tissue material in hot water for three times and freeze-dry it. 6. The decellularized tissue material is crushed, and the portion with a particle size of 60-80 μm is taken as the animal-derived decellularized matrix; 7. The animal-derived decellularized matrix was resuspended in water to obtain a decellularized powder suspension with a mass concentration of 4.5%, and the suspension was mixed with a modified epoxidized chitosan solution (mass concentration of 2%, purchased from Xi'an Qiyue Biological Co., Ltd.) at a volume ratio of 5:1. The pH of the mixed system was adjusted to 7.5-8.0, and 0.5% transglutaminase was added. The mixture was stirred at 35°C and 200 rpm for 24 hours. The obtained product was freeze-dried to obtain the decellularized matrix material.

[0034] Example 3 This embodiment provides a decellularized matrix material, and its preparation method is basically the same as that of embodiment 1, except for step (6): The animal-derived decellularized matrix was resuspended in water to obtain a decellularized powder suspension with a mass concentration of 4.5%, which was mixed with a modified epoxidized chitosan solution (mass concentration of 2%, purchased from Xi'an Qiyue Biological Company) at a volume ratio of 5:1, the pH of the mixed system was adjusted to 7.5-8.0, and 0.6% transglutaminase was added. The mixture was stirred at 400 rpm at 33°C for 18 hours, and the resulting product was freeze-dried to obtain a decellularized matrix material.

[0035] Embodiment 4-9 This embodiment provides a decellularized matrix material, and its preparation method is basically the same as that of Example 1, except that the modified epoxidized chitosan in step (6) is different: Example 4: Select the modified epoxidized chitosan provided in Preparation Example 1; Example 5: Select the modified epoxidized chitosan provided in Preparation Example 2; Example 6: Select the modified epoxidized chitosan provided in Preparation Example 3; Example 7: Select the modified epoxidized chitosan provided in Preparation Example 4; Example 8: Selecting the modified epoxidized chitosan provided in Comparative Preparation Example 5; Example 9: The modified epoxidized chitosan provided in Comparative Preparation Example 6 was selected.

[0036] Comparative Example 1 This comparative example provides a decellularized matrix material, which differs from Example 1 in that: there is no step (6), and its essence is an animal-derived decellularized matrix that is crushed after three treatments.

[0037] Comparative Example 2 This comparative example provides a decellularized matrix material, which differs from Example 1 in that natural unmodified chitosan is selected in step (6).

[0038] Comparative Example 3 This comparative example provides a decellularized matrix material, which differs from Example 4 in that the mass concentration of transglutaminase in step (6) is 0.3%.

[0039] Comparative Example 4 This comparative example provides a decellularized matrix material, which differs from Example 4 in that the mass concentration of transglutaminase in step (6) is 1.0%.

[0040] Comparative Example 5 This comparative example provides a decellularized matrix material, which differs from Example 1 in that the tissue material is processed differently: the pretreated pig skin is crushed to 60-80 μm, and the resulting powder is subjected to a primary digestion treatment, a secondary digestion treatment, and a tertiary deep treatment in sequence, and the processing method is consistent with Example 1.

[0041] Comparative Example 6 This comparative example provides a decellularized matrix material, which differs from Example 1 in that the treatment solution used in the third deep treatment of the tissue material is a Triton X-100 / ethanol solution.

[0042] 1. Biological performance evaluation The biological performance of the decellularized matrix materials provided in some embodiments and comparative examples was evaluated according to the GB / T16886 standard method. The evaluation items included relative cell proliferation rate, sensitization reaction and intradermal irritation test. The results are shown in Tables 1 and 2: Table 1. Biological performance evaluation results of the examples

[0043] Table 2. Biological performance evaluation results of comparative examples

[0044] As can be seen from Table 1 and Table 2, the relative cell proliferation rates of the acellular matrix materials provided in the examples and comparative examples of the present application are both greater than 70%, indicating that they have no potential cytotoxicity and no allergic reaction or intradermal irritation reaction.

[0045] Compared with Example 1 and Example 2, Example 1 is only a decellularized tissue material, and Example 2 is a cross-linked product of a decellularized tissue material and natural chitosan. Although the relative proliferation rate of cells in Example 2 is higher than that in Example 1 to a certain extent, it is still lower than that in Example 1 after cross-linking with epoxidized chitosan. This shows that the embodiment of the present application uses epoxidized chitosan and decellularized tissue materials for biological cross-linking, which can improve the biocompatibility of decellularized tissue materials and reduce rejection reactions.

[0046] Compared with Examples 4-9, Example 1 uses epoxidized chitosan, and Examples 4-9 use epoxidized chitosan modified by blending epoxidized chitosan and Bletilla striata polysaccharide. The relative cell proliferation rate of Examples 4-9 is significantly better than that of Example 1, indicating that the blending modification of Bletilla striata polysaccharide can improve the biocompatibility of the material and facilitate cell adhesion and proliferation. Furthermore, in Examples 5 and 6, if too much or too little Bletilla striata polysaccharide is added during the modification process, the relative cell proliferation rate of the material will be affected to a certain extent.

[0047] Compared with Comparative Examples 3 and 4, Example 4 uses epoxidized chitosan modified by blending epoxidized chitosan and Bletilla striata polysaccharide for cross-linking. However, the amount of transglutaminase added will significantly affect the relative cell proliferation rate of the material. When the amount of transglutaminase added is too small, the cross-linking is incomplete; when the amount of transglutaminase added is too much, it is easy to affect the bioactive factors contained in the decellularized matrix, thereby reducing the biocompatibility.

[0048] Compared with Comparative Examples 5 and 6, the treatment method of tissue materials in Example 1 will also affect the properties of the matrix material. In Comparative Example 5, the first pulverization and then digestion treatment may damage the bioactive factors in the decellularized matrix. In Comparative Example 6, Triton X-100 / ethanol solution is used in the third deep treatment process, the decellularization process is not thorough, and the amount of residual cells is large, which leads to a decrease in the relative cell growth rate of the material.

[0049] 1. Cell Proliferation Assay The cell matrix materials provided in Example 1, Example 4, Comparative Example 1 and Comparative Example 2 were made into thin sheets and placed at the bottom of a cell culture plate. Mouse L929 fibroblasts were seeded on the surface of the cell matrix materials at a seeding density of 1*10 4 The inoculated cell culture plate was placed in a CO2 cell culture incubator for cell culture. After 1 day, 3 days, and 7 days, the cell supernatant of each group was discarded, 50 μL of MTT solution was added to each well, incubated at 37°C for 3 hours, the supernatant was aspirated, 400 μL of DMSO solution was added to each well, and after shaking for 10 minutes, 200 μL of supernatant was aspirated from each well and transferred to a 96-well plate. The optical density (OD) value at 570 nm was measured by an enzyme-labeled instrument. The results are shown in Table 3: Table 3. Optical density measurement results

[0050] As can be seen from Table 3, the OD values ​​of the decellularized matrix materials provided in Examples 1 and 3 on the 3rd day were significantly higher than those of Comparative Examples 1 and 2, which indicates that the surface recellularization effect of the decellularized matrix materials obtained after cross-linking with epoxidized chitosan is better, and more conducive to cell adhesion and proliferation. Although the OD value of the decellularized matrix material provided in Example 4 was comparable to that of Example 1 on the 3rd day, the OD value on the 7th day was significantly higher than that of Example 1, indicating that the cross-linking of epoxidized chitosan after blending and modification with Bletilla striata polysaccharide can further improve the biocompatibility of the material.

[0051] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A method for preparing a cell-free matrix material, characterized in that: It includes: Extracting mammalian tissue material, performing decellularization on the tissue material to obtain decellularized tissue material, freeze-drying the tissue material, and then crushing it to obtain an animal-derived decellularized matrix; The animal-derived decellularized matrix is ​​resuspended in water to obtain a decellularized powder suspension, which is mixed with a modified epoxidized chitosan solution, the pH of the mixed system is adjusted to 7.5-8.0, 0.5-0.8% transglutaminase is added, and the mixture is stirred at 33-35° C. and 200-400 rpm for 18-24 hours. The obtained product is freeze-dried to obtain a decellularized matrix material.

2. The method for preparing the acellular matrix material according to claim 1, characterized in that: The mass concentration of the animal-derived decellularized matrix in the decellularized powder suspension is 3.5-4.5%, the mass concentration of the modified epoxidized chitosan in the epoxidized chitosan solution is 1.5-2%, and the volume ratio of the decellularized powder suspension to the epoxidized chitosan solution in the mixed system is 4-5:1; The mammalian tissue material includes any one of pig skin, cow skin, pig pericardium, bovine pericardium, pig integument, and pig small intestinal submucosa.

3. The method for preparing the decellularized matrix material according to claim 1, characterized in that: The modified epoxidized chitosan is obtained by compositely modifying epoxidized chitosan and bletilla striata polysaccharide in a mass ratio of 1-3:

1.

4. The method for preparing the decellularized matrix material according to claim 3, characterized in that: The composite modification method comprises: Dissolving epoxidized chitosan in an acidic solution to obtain a chitosan solution with a concentration of 3-5 wt %, and dissolving bletilla striata polysaccharide in an acidic solution to obtain a bletilla striata polysaccharide solution with a concentration of 1-3 wt %; The chitosan solution and the bletilla striata polysaccharide solution are mixed, the pH of the mixed solution is adjusted to 4-6, a cross-linking agent is added to the mixed solution, and then cross-linked at 60-70° C. for 5-6 hours.

5. The method for preparing the decellularized matrix material according to claim 4, characterized in that: The cross-linking agent is 0.1-1% genipin, and the mass ratio of the epoxidized chitosan to the bletilla striata polysaccharide is 2-3:

1.

6. The method for preparing the decellularized matrix material according to claim 1, characterized in that: The method for decellularizing the tissue material comprises: After pre-treating the tissue material, digestion is performed once in a pancreatic enzyme solution; The resulting tissue material is then placed in a nuclease solution for secondary digestion; The obtained tissue material was then placed in a sodium N-lauroyl-L-glutamate solution for three deep treatments.

7. The method for preparing the decellularized matrix material according to claim 6, characterized in that: In the three deep treatments, the concentration of the sodium N-lauroyl-L-glutamate solution is 5-6%, the treatment temperature is 30-35° C., and the treatment time is 8-10 hours.

8. The method for preparing the decellularized matrix material according to claim 6, characterized in that: The concentration of the pancreatic enzyme solution is 0.15-0.25%, and the treatment time is 8-9 hours; the nuclease includes 0.01-0.02% ribonuclease and 0.02-0.04% deoxyribonuclease, and the treatment time is 4-5 hours.

9. The method for preparing the decellularized matrix material according to claim 1, characterized in that: The particle size of the animal-derived decellularized matrix is ​​20-300 μm.

10. A decellularized matrix material, characterized in that: The compound is prepared by the preparation method provided by claims 1-9.

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