Preparation method of animal-derived decellularized tissue scaffold material with high biocompatibility

Abstract

The invention discloses a preparation method of a high-biocompatibility animal-derived decellularized tissue scaffold material, and relates to the technical field of cell tissue preparation. The method comprises the following steps: washing tissues such as small intestines of pigs with normal saline containing double antibodies and lysozyme; oscillating by using a mixed solution of TritonX-100 and the like, treating by using a solution of nuclease and the like, digesting by using a mixed solution of multiple enzymes, and washing by using ultrapure water containing chitosan; then carrying out ultrasonic disinfection by using a disinfectant containing nano-silver, and sealing by using a solution containing fetal calf serum and the like; after freeze drying, genipin is used for cross-linking and loading growth factors and the like, and finally a dopamine solution is used for modification, so that the scaffold has better hydrophilicity and cell adhesiveness. The cell residue rate of the scaffold is lower than 3%, the nucleic acid residue is low, and the immunogenicity is effectively reduced; the composite material has the advantages that extracellular matrixes are reserved, the mechanical property is good, the controllable bioactive substance release function is achieved, the characteristics of antibiosis, antioxidation and the like are achieved, the biocompatibility is good, and the application prospect is wide.

Description

Technical Field

[0001] The invention relates to the technical field of cell tissue manufacturing, in particular to a method for preparing an animal-derived decellularized tissue scaffold material with high biocompatibility. Background Art

[0002] In the field of tissue engineering and regenerative medicine, finding suitable scaffold materials to repair and replace damaged tissues is a key issue. Animal-derived decellularized tissue scaffold materials have become a research hotspot due to their wide sources and similar structures to human tissues. However, there are many challenges in the preparation of existing animal-derived decellularized tissue scaffold materials.

[0003] Current decellularization methods often fail to completely remove cellular components. Residual cells can trigger an immune response, causing the graft to be recognized and attacked by the host immune system, affecting tissue repair. Traditional chemical reagent treatment, such as the use of detergents such as TritonX-100, can remove some cells, but it is not thorough enough to remove some stubborn cell components and nucleic acids, and may destroy the structure and function of the extracellular matrix, reducing the biocompatibility and mechanical properties of the scaffold material.

[0004] The extracellular matrix is ​​an important basis for the function of scaffold materials. It provides the necessary microenvironment for cell adhesion, proliferation and differentiation. However, in the decellularization process, existing technologies make it difficult to effectively remove cells while completely retaining the composition and structure of the extracellular matrix. For example, some enzymatic digestion methods may over-degrade proteins and polysaccharides in the extracellular matrix, resulting in a decrease in the mechanical properties of the scaffold material, which cannot provide sufficient support for cells and affects tissue regeneration and repair.

[0005] In addition, existing scaffold materials are also deficient in terms of bioactivity. Tissue repair and regeneration require appropriate bioactive signals to guide cell behavior, such as cell migration, proliferation and differentiation. However, scaffold materials obtained by traditional preparation methods lack these necessary bioactive substances, or the release of bioactive substances is difficult to control, and cannot continuously and effectively promote cell growth and tissue repair.

[0006] In terms of disinfection and preservation, existing methods also have defects. Some disinfection methods may damage the stent material and affect its performance. During the preservation process, the stent material is prone to denaturation and degradation, resulting in a decrease in its biological activity and mechanical properties, shortening the service life of the stent material and increasing the cost and risk of clinical application.

[0007] Therefore, it is of great practical significance to develop a method for preparing a highly biocompatible animal-derived decellularized tissue scaffold material that can efficiently remove cells, completely retain the extracellular matrix, have good biological activity, and is easy to disinfect and preserve. This can not only improve the therapeutic effects of tissue engineering and regenerative medicine, but also provide better treatment options for patients and promote the development of this field. Summary of the invention

[0008] 1. Technical issues to be resolved In view of the deficiencies of the prior art, the present invention provides a method for preparing a highly biocompatible animal-derived decellularized tissue scaffold material.

[0009] (II) Technical solution A method for preparing a highly biocompatible animal-derived decellularized tissue scaffold material comprises the following steps: S1: Select the pig small intestinal submucosa, bovine pericardium or sheep acellular dermis tissue from healthy animals aged 3-6 months, and rinse them with normal saline containing 0.1%-0.5% penicillin and streptomycin and 0.01%-0.05% lysozyme at 4°C for 3-5 times, each time for 10-20 minutes; S2: Place the rinsed tissue in a mixed solution of 0.5%-2% TritonX-100, 0.05%-0.2% sodium deoxycholate, and 0.02%-0.08% sodium lauryl sulfate at 25°C-37°C and an oscillation speed of 80-120 r / min for 12-24 hours; S2.5: Place the tissue in a solution containing 0.02%-0.05% nuclease and 0.01%-0.03% proteinase K at 37°C for 4-8 hours; S3: Digest with a mixed solution containing 0.05%-0.2% trypsin, 0.01%-0.05% EDTA, and 0.005%-0.02% papain at 37°C for 2-4 hours. Preheat the solution to 37°C before digestion and shake gently every 30 minutes during the process. S4: Rinse the digested tissue with ultrapure water containing 0.005%-0.01% chitosan at 4°C for 5-8 times, each time for 15-30 minutes; S5: Soak the tissue in a mixed disinfectant containing 1%-3% hydrogen peroxide, 0.1%-0.5% peracetic acid and 0.05%-0.2% nanosilver particles, and disinfect at 25°C, 20-40kHz ultrasonic frequency, and 100-200W power for 2-4 hours; S6: Incubate with PBS solution (pH 7.2-7.4) containing 10%-20% fetal bovine serum and 0.01%-0.05% arginine at 37°C for 12-24 hours for blocking treatment, and change the solution every 4 hours; S7: freeze-dry the treated tissue, pre-freeze at -80°C for 6-12 hours, and then dry in a freeze dryer at -50°C--40°C and 0.01-0.1 mbar for 24-48 hours; S8: cross-linking the scaffold material using a solution of 0.1%-0.5% genipin and 0.02%-0.08% serine at 25°C for 12-24h; S9: Load the scaffold material with bioactive substances by immersing the scaffold in a solution containing 100-500 μg / mL growth factors including VEGF, bFGF, and 0.05%-0.2% hyaluronic acid at 4°C for 24-48 hours; S10: The surface of the loaded scaffold material was modified by treating it with Tris-HCl buffer containing 0.01%-0.05% dopamine at 25°C for 6-12 hours to make the scaffold surface have better hydrophilicity and cell adhesion.

[0010] Furthermore, in step S1, before flushing, the selected animal tissue is first three-dimensionally imaged using a laser scanning confocal microscope to record the initial cell distribution and structure of the tissue, and a pulse flushing method is used during flushing, with a pulse interval of 3-5 minutes.

[0011] Furthermore, in step S2, during the oscillation treatment, the mixed solution is subjected to component analysis and concentration detection every 3-6 hours, and the corresponding components are supplemented according to the detection results to maintain the solution concentration. At the same time, a nitrogen mixture containing 5%-10% carbon dioxide is introduced into the solution at a gas flow rate of 0.5-1L / min.

[0012] Furthermore, in step S2.5, during the treatment with nuclease and proteinase K solution, an alternating magnetic field is used to assist the treatment, with a magnetic field strength of 0.1-0.3T and a frequency of 10-20Hz to accelerate the degradation of nucleic acids and proteins.

[0013] Furthermore, in step S3, 0.001%-0.005% heparin is added to the mixed digestive enzyme solution to enhance the digestion effect on proteoglycans in the extracellular matrix.

[0014] Furthermore, in step S5, during the disinfection process, in addition to ultrasound assistance, a rotating platform is used to rotate the tissue at a uniform speed of 10-20 r / min, and after disinfection, the tissue is subjected to antioxidant treatment with a solution containing 0.02%-0.05% vitamin C for 1-2 h.

[0015] Furthermore, during the freeze-drying process of step S7, a gradient cooling method is adopted in the pre-freezing stage, firstly decreasing from room temperature to -20°C at a rate of 1-2°C / min, maintaining for 2-3h, and then decreasing to -80°C at a rate of 3-5°C / min. During the drying stage, the water vapor partial pressure in the drying chamber is detected and the vacuum degree is adjusted every 6-8h.

[0016] Furthermore, after the cross-linking treatment in step S8, the scaffold is protected with a solution containing 0.01%-0.03% trehalose for 6-12 hours, and then subjected to secondary freeze-drying at -60°C--50°C and 0.005-0.05 mbar for 12-24 hours.

[0017] Furthermore, after the surface modification in step S10, the scaffold was immersed in a solution containing 0.005%-0.02% chitosan quaternary ammonium salt for 3-6 hours, and then rinsed with deionized water for 3-5 times, each time for 5-10 minutes.

[0018] A highly biocompatible animal-derived decellularized tissue scaffold material obtained by applying the method for preparing the highly biocompatible animal-derived decellularized tissue scaffold material, wherein the cell residual rate of the scaffold material is less than 3%, the nucleic acid residual amount is less than 30 ng / mg dry weight, the porosity is 85%-98%, and the pore size is 150-600 μm.

[0019] 3. Beneficial technical effects Compared with the prior art, the present invention has the following beneficial effects: In terms of decellularization effect, the combined treatment of multiple reagents and innovative operating steps, such as pulsed flushing and alternating magnetic field assistance, can reduce the cell residue rate to less than 3% and the nucleic acid residue to less than 30ng / mg dry weight, effectively reducing immunogenicity, reducing the host's immune response, and improving the safety and applicability of the scaffold material.

[0020] In terms of retaining the extracellular matrix, by optimizing the reagent ratio and processing conditions, such as adding heparin to the digestive enzyme solution and strictly controlling the pH value, the composition and structure of the extracellular matrix are retained to the greatest extent, so that the scaffold material has good mechanical properties, can provide stable support for cells, and promote cell adhesion, proliferation and differentiation.

[0021] In terms of biological activity, by loading bioactive substances such as growth factors and hyaluronic acid, and using special loading and surface modification methods, the scaffold material has a controllable release function of bioactive substances, which can continuously and effectively provide cells with appropriate growth signals and promote tissue repair and regeneration. At the same time, surface modification also gives the scaffold better hydrophilicity and cell adhesion, further improving its biocompatibility.

[0022] In terms of disinfection and preservation, innovative disinfection methods and protective treatment steps, such as combined ultrasonic and rotating platform disinfection, the addition of antioxidants and trehalose protection, etc., can not only effectively kill bacteria and viruses, but also reduce damage to the stent material, improve its stability and service life, and reduce the cost and risk of clinical application.

[0023] In summary, the scaffold material prepared by the present invention has significantly improved biocompatibility, mechanical properties, biological activity and stability, providing a high-quality scaffold material for the field of tissue engineering and regenerative medicine, with broad application prospects and important clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the process for preparing highly biocompatible animal-derived decellularized tissue scaffold materials. DETAILED DESCRIPTION

[0025] refer to Figure 1 , the specific implementation methods of the present invention are as follows:

[0026] Embodiment 1: Materials preparation: The small intestinal submucosal tissue of healthy pigs aged 3 months was selected. Prepare physiological saline containing 0.1% double antibody (penicillin-streptomycin) and 0.01% lysozyme; a mixed solution of 0.5% TritonX-100, 0.05% sodium deoxycholate and 0.02% sodium lauryl sulfate; a solution containing 0.02% nuclease and 0.01% proteinase K; a mixed solution containing 0.05% trypsin, 0.01% EDTA, 0.005% papain and 0.001% heparin; ultrapure water containing 0.005% chitosan; A disinfectant mixture of 1% hydrogen peroxide, 0.1% peracetic acid and 0.05% nanosilver particles (particle size 10-50nm); a PBS solution (pH 7.2) containing 10% fetal bovine serum and 0.01% arginine; a solution of 0.1% genipin and 0.02% serine; a solution containing 100μg / mL VEGF and 0.05% hyaluronic acid; a Tris-HCl buffer (pH=8.5) containing 0.01% dopamine; and a solution containing 0.005% chitosan quaternary ammonium salt.

[0027] Preparation steps: S1. Tissue washing: The porcine small intestinal submucosal tissue was pulsed with saline containing 0.1% double antibody and 0.01% lysozyme at 4°C, with a pulse interval of 3 minutes, and rinsed 3 times, each time for 10 minutes. Before rinsing, a laser scanning confocal microscope was used to record the initial cell distribution and structure of the tissue in three dimensions.

[0028] S2, Oscillation processing: The rinsed tissue was placed in the mixed solution and treated at 25°C and 80r / min shaking speed for 12h. During this period, the mixed solution was analyzed for components and concentrations every 3h, and the corresponding components were supplemented. At the same time, a nitrogen mixture containing 5% carbon dioxide was introduced at a gas flow rate of 0.5L / min.

[0029] S2.5. Nucleic acid and protein degradation treatment: The tissue was placed in a solution containing nuclease and proteinase K and treated at 37°C and 0.1T alternating magnetic field (frequency 10 Hz) for 4 h.

[0030] S3, digestion treatment: Digestion was performed at 37°C for 2 h with a mixed solution containing multiple enzymes and heparin. The solution was preheated to 37°C before digestion and gently shaken every 30 min during the process. The pH value of the solution was monitored in real time and controlled at 7.2.

[0031] S4, flushing treatment: The digested tissue was rinsed five times with ultrapure water containing 0.005% chitosan at 4°C for 15 min each time.

[0032] S5. Disinfection and antioxidant treatment: Soak the tissue in the mixed disinfectant and disinfect it for 2 hours at 25°C, 20kHz ultrasonic frequency, 100W power, and 10r / min rotation speed. The disinfectant is freshly prepared. After disinfection, the tissue is treated with an antioxidant solution containing 0.02% vitamin C for 1 hour.

[0033] S6. Closed processing: The cells were blocked by incubation with PBS solution containing 10% fetal bovine serum and 0.01% arginine at 37°C for 12 h, and the solution was replaced every 4 h.

[0034] S7. Freeze drying: First, the temperature was lowered from room temperature to -20°C at a rate of 1°C / min and maintained for 2 hours. Then, the temperature was lowered to -80°C at a rate of 3°C / min and pre-frozen for 6 hours. Then, the temperature was dried at -50°C and 0.01 mbar for 24 hours. During the drying stage, the vacuum degree was checked and adjusted every 6 hours.

[0035] S8, cross-linking and secondary freeze-drying: The scaffolds were cross-linked with a solution of 0.1% genipin and 0.02% serine at 25°C for 12 h. After cross-linking, the scaffolds were protected with a solution containing 0.01% trehalose for 6 h and then dried at -60°C and 0.005 mbar for 12 h.

[0036] S9. Bioactive substance loading: The scaffolds were immersed in a solution containing VEGF and hyaluronic acid and loaded at 4°C for 24 h.

[0037] S10, surface modification and antimicrobial treatment: The scaffolds were treated with Tris-HCl buffer containing 0.01% dopamine at 25°C for 6 h, and then immersed in a solution containing 0.005% chitosan quaternary ammonium salt for 3 h, and finally rinsed with deionized water three times, each time for 5 min.

[0038] Embodiment 2:

[0039] Materials preparation: Pericardial tissue of healthy cows aged 4 months was selected. Prepare physiological saline containing 0.3% double antibody and 0.03% lysozyme; a mixed solution of 1.2% TritonX-100, 0.12% sodium deoxycholate and 0.05% sodium lauryl sulfate; a solution containing 0.035% nuclease and 0.02% proteinase K; a mixed solution containing 0.12% trypsin, 0.03% EDTA, 0.012% papain and 0.003% heparin; ultrapure water containing 0.008% chitosan; A mixed disinfectant containing 2% hydrogen peroxide, 0.3% peracetic acid and 0.12% nanosilver particles; a PBS solution (pH 7.3) containing 15% fetal bovine serum and 0.03% arginine; a solution of 0.3% genipin and 0.05% serine; a solution containing 300 μg / mL bFGF and 0.12% hyaluronic acid; a Tris-HCl buffer (pH = 8.5) containing 0.03% dopamine; and a solution containing 0.012% chitosan quaternary ammonium salt.

[0040] Preparation steps: S1. Tissue washing: The bovine pericardial tissue was pulse-washed with physiological saline containing 0.3% double antibody and 0.03% lysozyme at 4°C, with a pulse interval of 4 minutes, and washed 4 times, each time for 15 minutes. Initial imaging was also performed.

[0041] S2, Oscillation processing: The tissue was placed in the mixed solution for 18 h at 31°C and 100 r / min shaking speed. The components of the mixed solution were checked and replenished every 4 h, and a nitrogen mixed gas containing 7% carbon dioxide was introduced at a flow rate of 0.7 L / min.

[0042] S2.5. Nucleic acid and protein degradation treatment: The tissue was treated with a solution containing nuclease and proteinase K for 6 h at 37°C and 0.2 T alternating magnetic field (frequency 15 Hz).

[0043] S3, digestion treatment: Digest with a mixed solution containing multiple enzymes and heparin at 37°C for 3 h. Preheat the solution, control the pH at 7.3, and shake every 30 min.

[0044] S4, flushing treatment: The tissues were rinsed six times with ultrapure water containing 0.008% chitosan at 4°C, each time for 20 min.

[0045] S5. Disinfection and antioxidant treatment: Disinfect the tissue with a mixed disinfectant at 25°C, 30kHz ultrasonic frequency, 150W power, and 15r / min rotation speed for 3 hours. Prepare the disinfectant now and use a solution containing 0.035% vitamin C for antioxidant treatment for 1.5 hours after disinfection.

[0046] S6. Closed processing: The cells were blocked by incubation with PBS solution containing 15% fetal bovine serum and 0.03% arginine at 37°C for 18 h, and the solution was changed every 4 h.

[0047] S7. Freeze drying: Reduce the temperature to -20℃ at 1.5℃ / min and maintain for 2.5h. Reduce the temperature to -80℃ at 4℃ / min and prefreeze for 9h. Dry at -45℃ and 0.05mbar for 36h. Check and adjust the vacuum degree every 7h during drying.

[0048] S8, cross-linking and secondary freeze-drying: The cross-linking was carried out with a solution of 0.3% genipin and 0.05% serine at 25°C for 18 h. After cross-linking, the cross-linking was protected with a solution containing 0.02% trehalose for 9 h and dried at -55°C and 0.02 mbar for 18 h.

[0049] S9. Bioactive substance loading: The scaffolds were immersed in a solution containing bFGF and hyaluronic acid and loaded at 4°C for 36 h.

[0050] S10, surface modification and antimicrobial treatment: The samples were treated with Tris-HCl buffer containing 0.03% dopamine at 25°C for 9 h, immersed in a solution containing 0.012% chitosan quaternary ammonium salt for 4.5 h, and rinsed with deionized water 4 times, 7 min each time.

[0051] Embodiment 3:

[0052] Materials preparation: Acellular dermis tissues of 6-month-old healthy sheep were selected. Prepare physiological saline containing 0.5% double antibody and 0.05% lysozyme; a mixed solution of 2% TritonX-100, 0.2% sodium deoxycholate and 0.08% sodium lauryl sulfate; a solution containing 0.05% nuclease and 0.03% proteinase K; a mixed solution containing 0.2% trypsin, 0.05% EDTA, 0.02% papain and 0.005% heparin; ultrapure water containing 0.01% chitosan; and 3% lysozyme. A disinfectant containing 0.5% hydrogen peroxide, 0.5% peracetic acid and 0.2% nanosilver particles; a PBS solution (pH 7.4) containing 20% ​​fetal bovine serum and 0.05% arginine; a solution of 0.5% genipin and 0.08% serine; a solution containing 500 μg / mL VEGF and 0.2% hyaluronic acid; a Tris-HCl buffer (pH = 8.5) containing 0.05% dopamine; and a solution containing 0.02% chitosan quaternary ammonium salt.

[0053] Preparation steps: S1. Tissue washing: The sheep acellular dermis tissue was pulse-washed with normal saline containing 0.5% double antibody and 0.05% lysozyme at 4°C, with a pulse interval of 5 minutes, and washed 5 times, each time for 20 minutes, for initial imaging.

[0054] S2, Oscillation processing: The tissue was placed in the mixed solution for 24 hours at 37°C and 120 r / min shaking speed. The components of the mixed solution were checked and replenished every 6 hours, and a nitrogen mixed gas containing 10% carbon dioxide was introduced at a flow rate of 1 L / min.

[0055] S2.5. Nucleic acid and protein degradation treatment: The tissue was treated with a solution containing nuclease and proteinase K at 37°C and 0.3T alternating magnetic field (frequency 20 Hz) for 8 h.

[0056] S3, digestion treatment: Digest with a mixed solution containing multiple enzymes and heparin at 37°C for 4 hours. Preheat the solution, control the pH at 7.4, and shake every 30 minutes.

[0057] S4, flushing treatment: The tissues were rinsed eight times with ultrapure water containing 0.01% chitosan at 4°C, each time for 30 min.

[0058] S5. Disinfection and antioxidant treatment: Disinfect the tissue with a mixed disinfectant for 4 h at 25°C, 40 kHz ultrasonic frequency, 200 W power, and 20 r / min rotation speed. Prepare the disinfectant freshly and use a solution containing 0.05% vitamin C for antioxidant treatment for 2 h after disinfection.

[0059] S6. Closed processing: The cells were blocked by incubation with PBS solution containing 20% ​​fetal bovine serum and 0.05% arginine at 37°C for 24 h, and the solution was changed every 4 h.

[0060] S7. Freeze drying: Reduce the temperature to -20℃ at 2℃ / min and keep it for 3h. Then reduce the temperature to -80℃ at 5℃ / min and pre-freeze it for 12h. Dry it at -40℃ and 0.1mbar for 48h. During drying, check and adjust the vacuum degree every 8h.

[0061] S8, cross-linking and secondary freeze-drying: The cross-linking was carried out with a solution of 0.5% genipin and 0.08% serine at 25°C for 24 h. After cross-linking, the cells were protected with a solution containing 0.03% trehalose for 12 h and dried at -50°C and 0.05 mbar for 24 h.

[0062] S9. Bioactive substance loading: The scaffolds were immersed in a solution containing VEGF and hyaluronic acid and loaded at 4°C for 48 h.

[0063] S10, surface modification and antimicrobial treatment: The samples were treated with Tris-HCl buffer containing 0.05% dopamine at 25°C for 12 h, immersed in a solution containing 0.02% chitosan quaternary ammonium salt for 6 h, and rinsed with deionized water 5 times, 10 min each time.

[0064] Comparative Example: Material preparation; The pericardial tissue of healthy 4-month-old cows was selected. Prepare physiological saline containing 0.1% double antibody; 1% TritonX-100 solution; 0.1% trypsin solution; ultrapure water; disinfectant containing 2% hydrogen peroxide; PBS solution containing 10% fetal bovine serum; 0.5% glutaraldehyde solution; and 200 μg / mL bFGF solution.

[0065] Preparation steps: 1. Rinse the bovine pericardium tissue with 0.1% double antibody-containing saline at 4°C for 3 times, each time for 15 minutes.

[0066] 2. Place the rinsed tissue in 1% TritonX-100 solution and shake at 37°C for 24 hours.

[0067] 3. Digest with a solution containing 0.1% trypsin at 37°C for 4 hours.

[0068] 4. Rinse the tissue five times with ultrapure water at 4°C, 20 min each time.

[0069] 5. Soak the tissue in a disinfectant containing 2% hydrogen peroxide and disinfect it at 25°C for 3 hours.

[0070] 6. Block the cells by incubating with PBS solution containing 10% fetal bovine serum at 37°C for 12 hours.

[0071] 7. Prefreeze the treated tissue at -80°C for 12 h, and then freeze-dry at -50°C, 0.05 mbar for 48 h.

[0072] 8. Cross-link with 0.5% glutaraldehyde solution at 25°C for 24 hours.

[0073] 9. Soak the scaffold in a solution containing 200 μg / mL bFGF and load it at 4°C for 24 hours.

[0074] Performance Test: The performance of the scaffold materials prepared in Examples 1-3 and the comparative example was tested, and the results were as follows: It can be seen from the test results that the scaffold material prepared in the embodiment is significantly better than the comparative example in terms of cell residual rate, nucleic acid residual amount, porosity, pore size, cell adhesion rate, antibacterial rate and release time of bioactive substances, which fully demonstrates the effectiveness and superiority of the preparation method of the present invention.

[0075] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a highly biocompatible animal-derived decellularized tissue scaffold material, characterized in that: The following steps are involved: S1: Select the pig small intestinal submucosa, bovine pericardium or sheep acellular dermis tissue from healthy animals aged 3-6 months, and rinse them with normal saline containing 0.1%-0.5% penicillin and streptomycin and 0.01%-0.05% lysozyme at 4°C for 3-5 times, each time for 10-20 minutes; S2: Place the rinsed tissue in a mixed solution of 0.5%-2% TritonX-100, 0.05%-0.2% sodium deoxycholate, and 0.02%-0.08% sodium lauryl sulfate at 25°C-37°C and an oscillation speed of 80-120 r / min for 12-24 hours; S2.5: Place the tissue in a solution containing 0.02%-0.05% nuclease and 0.01%-0.03% proteinase K at 37°C for 4-8 hours; S3: Digest with a mixed solution containing 0.05%-0.2% trypsin, 0.01%-0.05% EDTA, and 0.005%-0.02% papain at 37°C for 2-4 hours. Preheat the solution to 37°C before digestion and shake gently every 30 minutes during the process. S4: Rinse the digested tissue with ultrapure water containing 0.005%-0.01% chitosan at 4°C for 5-8 times, each time for 15-30 minutes; S5: Soak the tissue in a mixed disinfectant containing 1%-3% hydrogen peroxide, 0.1%-0.5% peracetic acid and 0.05%-0.2% nanosilver particles, and disinfect at 25°C, 20-40kHz ultrasonic frequency, and 100-200W power for 2-4 hours; S6: Incubate with PBS solution containing 10%-20% fetal bovine serum and 0.01%-0.05% arginine at 37°C for 12-24 hours for blocking treatment, and replace the solution every 4 hours; S7: freeze-dry the treated tissue, pre-freeze at -80°C for 6-12 hours, and then dry in a freeze dryer at -50°C--40°C and 0.01-0.1 mbar for 24-48 hours; S8: cross-linking the scaffold material using a solution of 0.1%-0.5% genipin and 0.02%-0.08% serine at 25°C for 12-24h; S9: Load the scaffold material with bioactive substances by immersing the scaffold in a solution containing 100-500 μg / mL growth factors including VEGF, bFGF, and 0.05%-0.2% hyaluronic acid at 4°C for 24-48 hours; S10: The surface of the loaded scaffold material was modified by treating it with Tris-HCl buffer containing 0.01%-0.05% dopamine at 25°C for 6-12 hours to make the scaffold surface have better hydrophilicity and cell adhesion.

2. The method for preparing a highly biocompatible animal-derived decellularized tissue scaffold material according to claim 1, characterized in that: In step S1, before flushing, the selected animal tissue is first three-dimensionally imaged using a laser scanning confocal microscope to record the initial cell distribution and structure of the tissue, and a pulse flushing method is used during flushing, with a pulse interval of 3-5 minutes.

3. The method for preparing a highly biocompatible animal-derived decellularized tissue scaffold material according to claim 1, characterized in that: In step S2, during the oscillation treatment, the mixed solution is subjected to component analysis and concentration detection every 3-6 hours, and the corresponding components are supplemented according to the detection results to maintain the solution concentration. At the same time, a nitrogen mixture containing 5%-10% carbon dioxide is introduced into the solution at a gas flow rate of 0.5-1L / min.

4. The method for preparing a highly biocompatible animal-derived decellularized tissue scaffold material according to claim 1, characterized in that: In step S2.5, during the treatment with nuclease and proteinase K solution, an alternating magnetic field is used to assist the treatment, with a magnetic field strength of 0.1-0.3T and a frequency of 10-20Hz to accelerate the degradation of nucleic acids and proteins.

5. The method for preparing a highly biocompatible animal-derived decellularized tissue scaffold material according to claim 1, characterized in that: In step S3, 0.001%-0.005% heparin is added to the mixed digestive enzyme solution to enhance the digestion effect of proteoglycans in the extracellular matrix.

6. The method for preparing a highly biocompatible animal-derived decellularized tissue scaffold material according to claim 1, characterized in that: In step S5, during the disinfection process, in addition to ultrasound assistance, a rotating platform is used to rotate the tissue at a uniform speed of 10-20 r / min, and after disinfection, the tissue is subjected to antioxidant treatment with a solution containing 0.02%-0.05% vitamin C for 1-2 h.

7. The method for preparing a highly biocompatible animal-derived decellularized tissue scaffold material according to claim 1, characterized in that: During the freeze-drying process of step S7, a gradient cooling method is adopted in the pre-freezing stage, firstly decreasing from room temperature to -20°C at a rate of 1-2°C / min, maintaining for 2-3h, and then decreasing to -80°C at a rate of 3-5°C / min. During the drying stage, the water vapor partial pressure in the drying chamber is detected and the vacuum degree is adjusted every 6-8h.

8. The method for preparing a highly biocompatible animal-derived decellularized tissue scaffold material according to claim 1, characterized in that: After the cross-linking treatment in step S8, the scaffold is protected with a solution containing 0.01%-0.03% trehalose for 6-12 hours, and then subjected to secondary freeze-drying at -60°C--50°C and 0.005-0.05 mbar for 12-24 hours.

9. The method for preparing a highly biocompatible animal-derived decellularized tissue scaffold material according to claim 1, characterized in that: After the surface modification in step S10, the scaffold was immersed in a solution containing 0.005%-0.02% chitosan quaternary ammonium salt for 3-6 h, and then rinsed with deionized water for 3-5 times, each time for 5-10 min.

10. A highly biocompatible animal-derived decellularized tissue scaffold material obtained by using the method for preparing a highly biocompatible animal-derived decellularized tissue scaffold material according to any one of claims 1 to 9, characterized in that: The cell residual rate of the scaffold material is less than 3%, the nucleic acid residual is less than 30ng / mg dry weight, the porosity is 85%-98%, and the pore size is 150-600μm.

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