A decellularized matrix material and a method of making the same

Abstract

The application relates to the field of biomaterials, and particularly discloses a kind of acellular matrix material and a preparation method thereof.The preparation method of the material comprises the following steps: extracting tissue material of a mammal, performing acellular treatment on the tissue material to obtain acellular tissue material, freeze-drying the tissue material, and then crushing the freeze-dried tissue material to obtain animal-derived acellular matrix; the animal-derived acellular matrix is resuspended in water, mixed with a modified epoxidized chitosan solution, and then transglutaminase is added to perform a reaction; the product obtained is freeze-dried to obtain the acellular matrix material.The method can improve the biocompatibility of the acellular matrix and reduce the immune rejection reaction by cross-linking the animal-derived acellular matrix with modified epoxidized chitosan; at the same time, since the cross-linking reaction is a biological cross-linking reaction under the action of transglutaminase, the biological activity factors contained in the acellular matrix are not easily destroyed during 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 traditional cosmetic medicine, commonly used fillers and restorative materials often have limitations. For example, synthetic materials may cause immune reactions or adverse reactions, and sometimes have difficulty integrating well with host tissue. To address these issues, decellularized extracellular matrix (dECM) has attracted widespread attention in recent years as a novel biomaterial.

[0003] dECM is a biomaterial extracted from animal tissue. It undergoes a special process to remove cellular components while retaining the natural extracellular matrix structure and its essential components, including growth factors, collagen, and other essential components. This makes dECM biocompatible, reducing immune responses while providing an ideal environment for supporting cell growth and repair. Compared to traditional synthetic materials, dECM demonstrates significant advantages and potential in the field of aesthetic medicine.

[0004] However, despite the numerous advantages displayed by dECM in aesthetic medical applications, its application in this field still faces numerous challenges. For example, due to its natural origin and preparation process, its stability and durability in certain applications are poor. Furthermore, despite treatment, decellularized matrices may still have potential biocompatibility issues, requiring further optimization and adjustment to improve compatibility with host tissues. Improving the biocompatibility and enhancing the safety of dECM have become important topics in current research and development. These efforts will help promote the widespread application of dECM in the field of aesthetic medicine, enhance its therapeutic efficacy and patient satisfaction, and thus meet the growing market demand. Summary of the Invention

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

[0006] This application provides the following technical solutions:

[0007] In one aspect, the present application provides a method for preparing a cell-free matrix material, comprising:

[0008] Extracting mammalian tissue material, decellularizing the tissue material to obtain decellularized tissue material, freeze-drying the tissue material, and then pulverizing it to obtain an animal-derived decellularized matrix;

[0009] 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, and 0.5-0.8% transglutaminase is added. The mixture is stirred at 33-35°C and 200-400 rpm for 18-24 hours. The resulting product is freeze-dried to obtain a decellularized matrix material.

[0010] 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 bioactive factors contained in the extracellular matrix, thereby affecting the biological activity and induction ability of the decellularized matrix.

[0011] This method, which crosslinks animal-derived acellular matrix with modified epoxidized chitosan, improves the biocompatibility of the acellular matrix and reduces immune rejection. Furthermore, because the crosslinking reaction is carried out using transglutaminase, the bioactive factors contained in the acellular matrix are not easily destroyed during the crosslinking process, and no chemical residues are left, making it safer than chemical crosslinking. During the crosslinking process, precise control of crosslinking conditions (such as pH, temperature, and time) ensures efficient crosslinking while effectively avoiding damage to the acellular matrix's function due to excessive crosslinking.

[0012] Furthermore, the mammalian tissue material includes any one of pig skin, cow skin, pig pericardium, bovine pericardium, pig integument and pig small intestinal submucosa.

[0013] 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.

[0014] The above technical solution limits the content of the animal-derived acellular matrix and modified epoxidized chitosan. The ratio of modified epoxidized chitosan to the acellular 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, affecting the material's in vivo application.

[0015] Compared to the direct cross-linking with natural chitosan, the present application adopts 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.

[0016] Furthermore, the modified epoxidized chitosan is obtained by composite-modifying epoxidized chitosan and bletilla striata polysaccharide in a mass ratio of 1-3:1.

[0017] Preferably, the composite modification method comprises:

[0018] 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%;

[0019] 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 the mixture is cross-linked at 60-70° C. for 5-6 hours.

[0020] Preferably, the crosslinking agent is 0.1-1% genipin. Genipin is the product of geniposide hydrolysis by β-glucosidase. Its chemical structure contains hydroxyl groups, carboxyl groups, and a special olefinic carbon atom (C-3 position). Under acidic conditions, these reactive groups can undergo nucleophilic reactions with the active groups on chitosan and bletilla striata polysaccharide during the crosslinking process. Genipin acts as a crosslinking bridge to connect the polysaccharide molecules, forming a stable network structure.

[0021] Furthermore, the method for decellularizing tissue materials includes:

[0022] After pretreatment of the tissue material, a digestion treatment was performed in a trypsin solution;

[0023] The resulting tissue material is then placed in a nuclease solution for secondary digestion;

[0024] The obtained tissue material was then placed in a sodium N-lauroyl-L-glutamate solution for three deep treatments.

[0025] The above technical solution, through pretreatment of tissue materials and subsequent three treatments, facilitates thorough decellularization, removing residual cell debris, DNA residue, and reagent residues, reducing immunogenic substances, and thus improving the safety of material use. In particular, the three-step deep treatment with sodium N-lauroyl-L-glutamate can insert into the lipid bilayer of the cell membrane, disrupting the lipid structure, causing cell membrane dissociation and dissolution, and facilitating the removal of intracellular proteins, organelles, etc., further ensuring the complete removal of cells and improving the purity of the decellularized matrix.

[0026] 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.

[0027] 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.

[0028] Preferably, the particle size of the animal-derived acellular matrix is ​​20-300 μm.

[0029] In a second aspect, the present application provides a decellularized matrix material prepared by the above method.

[0030] In summary, this application has the following beneficial effects:

[0031] 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 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.

[0032] 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, material stability and biocompatibility. DETAILED DESCRIPTION

[0033] The embodiments of the present invention will be described in detail below with reference to the 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. Specific conditions not specified in the examples are carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used without indicating the manufacturer are all conventional products that can be purchased commercially.

[0034] The following is a detailed description of the specific embodiments of the present invention. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0035] Preparation Example 1

[0036] This preparation example provides a modified epoxidized chitosan, and its preparation method is as follows:

[0037] (1) 80 g of epoxidized chitosan (purchased from Xi'an Qiyue Biological Company) was dispersed in 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 0.1 M phosphate solution to obtain a Bletilla striata polysaccharide solution with a concentration of 2 wt%;

[0038] (2) The chitosan solution and the bletilla striata polysaccharide solution were mixed, and the pH of the mixed solution was adjusted to 5. Genipin was added to the mixed solution in an amount of 0.5%. The obtained mixed solution was heated in a water bath at 65°C for 5 h. The obtained product was washed and dried to obtain modified epoxidized chitosan.

[0039] Preparation Example 2

[0040] This preparation example provides a modified epoxidized chitosan, and its preparation method is as follows:

[0041] (1) 80 g of epoxidized chitosan was dispersed in 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 0.1 M phosphate solution to obtain a Bletilla striata polysaccharide solution with a concentration of 3 wt%;

[0042] (2) The chitosan solution and the bletilla striata polysaccharide solution were mixed, and the pH of the mixed solution was adjusted to 4-6. Genipin was added to the mixed solution in an amount of 0.1%. The obtained mixed solution was heated in a water bath at 60°C for 6 hours. The obtained product was washed and dried to obtain modified epoxidized chitosan.

[0043] Preparation Example 3

[0044] This preparation example provides a modified epoxidized chitosan, and its preparation method is as follows:

[0045] (1) 80 g of epoxidized chitosan was dispersed in 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 0.1 M phosphate solution to obtain a Bletilla striata polysaccharide solution with a concentration of 1 wt%;

[0046] (2) The chitosan solution and the bletilla striata polysaccharide solution were mixed, and the pH of the mixed solution was adjusted to 4. Genipin was added to the mixed solution in an amount of 1%. The obtained mixed solution was heated in a water bath at 70°C for 5 h. The obtained product was washed and dried to obtain modified epoxidized chitosan.

[0047] Preparation Example 4

[0048] This preparation example provides a modified epoxidized chitosan, and its preparation method is the same as 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.

[0049] Comparative Preparation Example 5

[0050] This preparation example provides a modified epoxidized chitosan, and its preparation method is the same as 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.

[0051] Comparative Preparation Example 6

[0052] This preparation example provides a modified epoxidized chitosan, and its preparation method is the same as 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.

[0053] Example 1

[0054] This embodiment provides a decellularized matrix material, the preparation method of which includes the following steps:

[0055] 1. Take the pig skin, remove the epidermis and fat layer, and wash it clean;

[0056] 2. Place the obtained pig skin in 2-4% sodium hydroxide solution and shake for 2 hours to remove residual fat;

[0057] 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 process to degrade the proteins in the extracellular matrix;

[0058] 4. Wash the digested material and place it in a nuclease solution (containing 0.01% ribonuclease and 0.02% deoxyribonuclease) at 36°C for 4 hours with stirring for secondary digestion to degrade nucleic acid molecules in the extracellular matrix.

[0059] 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, the tissue material was washed three times in hot water and freeze-dried.

[0060] 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;

[0061] 7. Resuspend the animal-derived decellularized matrix in water to obtain a 3.5% decellularized powder suspension, which is then mixed with a modified epoxidized chitosan solution (1.5% mass concentration, purchased from Xi'an Qiyue Biological Co., Ltd.) at a volume ratio of 4:1. Adjust the pH of the mixture to 7.5-8.0, add 0.8% transglutaminase, and stir at 300 rpm for 20 hours at 33-35°C. The resulting product is freeze-dried to obtain the decellularized matrix material.

[0062] Example 2

[0063] This embodiment provides a decellularized matrix material, the preparation method of which includes the following steps:

[0064] 1. Take the pig skin, remove the epidermis and fat layer, and wash it clean;

[0065] 2. Place the obtained pig skin in 2-4% sodium hydroxide solution and shake for 12 hours to remove residual fat;

[0066] 3. Place the obtained pig skin in a 0.15% pancreatic enzyme solution and soak it at 36°C for 9 hours with stirring for a digestion process to degrade the proteins in the extracellular matrix;

[0067] 4. Wash the digested material and place it in a nuclease solution (containing 0.02% ribonuclease and 0.04% deoxyribonuclease) at 36°C for 5 hours with stirring for secondary digestion to degrade nucleic acid molecules in the extracellular matrix.

[0068] 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 three times and freeze-dry.

[0069] 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;

[0070] 7. Resuspend the animal-derived decellularized matrix in water to obtain a 4.5% decellularized powder suspension, which is then mixed with a modified epoxidized chitosan solution (2% mass concentration, purchased from Xi'an Qiyue Biological Co., Ltd.) at a volume ratio of 5:1. Adjust the pH of the mixture to 7.5-8.0, add 0.5% transglutaminase, and stir at 35°C and 200 rpm for 24 hours. Freeze-dry the resulting product to obtain the decellularized matrix material.

[0071] Example 3

[0072] This embodiment provides a decellularized matrix material, and its preparation method is basically the same as that of Example 1, except for step (6):

[0073] The animal-derived decellularized matrix was resuspended in water to obtain a decellularized powder suspension with a mass concentration of 4.5%. The suspension was then 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 0.6% transglutaminase was added. The mixture was stirred at 400 rpm at 33°C for 18 hours. The resulting product was freeze-dried to obtain the decellularized matrix material.

[0074] Examples 4-9

[0075] 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:

[0076] Example 4: The modified epoxidized chitosan provided in Preparation Example 1 was selected;

[0077] Example 5: The modified epoxidized chitosan provided in Preparation Example 2 was selected;

[0078] Example 6: The modified epoxidized chitosan provided in Preparation Example 3 was selected;

[0079] Example 7: The modified epoxidized chitosan provided in Preparation Example 4 was selected;

[0080] Example 8: The modified epoxidized chitosan provided in Comparative Preparation Example 5 was selected;

[0081] Example 9: The modified epoxidized chitosan provided in Comparative Preparation Example 6 was selected.

[0082] Comparative Example 1

[0083] 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 has been crushed after three treatments.

[0084] Comparative Example 2

[0085] This comparative example provides a decellularized matrix material, which differs from Example 1 in that natural unmodified chitosan is selected in step (6).

[0086] Comparative Example 3

[0087] 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%.

[0088] Comparative Example 4

[0089] 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%.

[0090] Comparative Example 5

[0091] 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 the same manner as in Example 1.

[0092] Comparative Example 6

[0093] 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 Triton X-100 / ethanol solution.

[0094] (1) Biological performance evaluation

[0095] The biological performance of the acellular matrix materials provided in some examples 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:

[0096] Table 1. Biological performance evaluation results of the examples

[0097]

[0098] Table 2. Biological performance evaluation results of comparative examples

[0099]

[0100] 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.

[0101] Compared with Comparative Example 1 and Comparative Example 2, Comparative Example 1 is only acellular tissue material, and Comparative Example 2 is a crosslinked product of acellular tissue material and natural chitosan. Although the relative cell growth rate of Comparative Example 2 is increased to a certain extent compared with Comparative Example 1, it is still lower than that of the material crosslinked by epoxidized chitosan in Example 1. Therefore, it is proved that the bio-compatibility of the acellular tissue material can be improved and the rejection reaction can be reduced by using epoxidized chitosan to bio-crosslink the acellular tissue material in the application.

[0102] Compared with Example 1 and Example 4-9, Example 1 uses epoxidized chitosan, and Example 4-9 uses epoxidized chitosan modified by blending with bletilla striata polysaccharide. The relative cell growth rate of Example 4-9 is obviously better than that of Example 1, which proves that the bio-compatibility of the material can be improved and the adhesion and proliferation of cells can be facilitated by modifying the epoxidized chitosan by blending with bletilla striata polysaccharide. Further, in Example 5 and Example 6, the addition amount of bletilla striata polysaccharide is too much or too little in the modification process, which will affect the relative cell growth rate of the material to a certain extent.

[0103] Compared with Comparative Example 3 and Comparative Example 4, both of which use epoxidized chitosan modified by blending with bletilla striata polysaccharide for crosslinking, the addition amount of transglutaminase will significantly affect the relative cell growth rate of the material. When the addition amount of transglutaminase is small, the crosslinking is not complete. When the addition amount of transglutaminase is too much, the biological active factors contained in the acellular matrix will be affected, thereby reducing the bio-compatibility.

[0104] Compared with Comparative Example 5 and Comparative Example 6, the treatment method of the tissue material will also affect the properties of the final matrix material. In Comparative Example 5, the tissue material is first crushed and then digested, which may damage the biological active factors in the acellular matrix. In Comparative Example 6, Triton X-100 / ethanol solution is used in the third deep treatment process, and the acellular process is not complete, so the cell residue amount is large, thereby reducing the relative cell growth rate of the material.

[0105] 1. Cell proliferation experiment

[0106] The cell matrix materials provided by Example 1, Example 4, Comparative Example 1 and Comparative Example 2 were made into thin slices and placed at the bottom of a cell culture plate. Mouse L929 fibroblasts were inoculated on the surface of the cell matrix material, and the inoculation density was 1*10 4The inoculated cell culture plates were placed in a CO2 cell culture incubator for cell culture. After 1, 3, and 7 days, the cell supernatant of each group was discarded, 50 μL of MTT solution was added to each well, and the cells were incubated at 37°C for 3 hours. The supernatant was aspirated, and 400 μL of DMSO solution was added to each well. 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 using a microplate reader. The results are shown in Table 3:

[0107] Table 3. Optical density measurement results

[0108]

[0109] As shown in Table 3, the OD values ​​of the decellularized matrix materials provided in Examples 1 and 3 were significantly higher than those of Comparative Examples 1 and 2 on day 3, indicating that the surface recellularization of the decellularized matrix materials obtained after cross-linking with epoxidized chitosan was more effective, which was 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 day 3, its OD value on day 7 was significantly higher than that of Example 1, indicating that cross-linking with epoxidized chitosan after blending and modification with Bletilla striata polysaccharide further improved the biocompatibility of the material.

[0110] 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 non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A method for preparing acellular matrix material, characterized in that: It includes: (1) extracting mammalian tissue material, decellularizing the tissue material to obtain a decellularized tissue material, freeze-drying the tissue material, and then pulverizing the tissue material to obtain an animal-derived decellularized matrix; (2) resuspending the animal-derived decellularized matrix in water to obtain a decellularized powder suspension, mixing the suspension with a modified epoxidized chitosan solution, adjusting the pH of the mixture to 7.5-8.0, adding 0.5-0.8% transglutaminase, and stirring at 33-35° C. and 200-400 rpm for 18-24 hours. The resulting product is freeze-dried to obtain a decellularized matrix material; The modified epoxidized chitosan is obtained by composite-modifying epoxidized chitosan and bletilla striata polysaccharide in a mass ratio of 1-3:1, and the composite-modification method includes: 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, and the pH value of the mixed solution is adjusted to 4-6. 0.1-1% genipin is added to the mixed solution, and then cross-linked at 60-70° C. for 5-6 hours.

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 acellular matrix material according to claim 1, wherein: The mass ratio of the epoxidized chitosan to the bletilla striata polysaccharide is 2-3:

1.

4. The method for preparing the acellular matrix material according to claim 1, wherein: 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.

5. The method for preparing the acellular matrix material according to claim 4, 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.

6. The method for preparing the acellular matrix material according to claim 4, 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.

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

8. A decellularized matrix material, characterized in that: The compound is prepared by the preparation method provided by any one of claims 1 to 7.

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