Acellular matrix and preparation method thereof

Abstract

The invention relates to the technical field of medical biological materials, in particular to an acellular matrix and a preparation method thereof. The preparation method comprises the steps of decellularization, stabilization, crosslinking and cleaning. The cross-linking comprises circulating standing cross-linking in a cross-linking agent solution and ultrasonic equilibrium in an equilibrium buffer solution system, and each time of completion of standing cross-linking and ultrasonic equilibrium is a circulating process; the concentration of the cross-linking agent is increased for next cross-linking after each circulation. The invention provides the preparation method of the acellular matrix material with higher mechanical property and cell proliferation rate, the prepared acellular matrix material is thin and tough, the problems of poor mechanical property, poor comfort and low cell proliferation rate in the prior art are solved, and the acellular matrix material has higher value in the field of medical application and is suitable for popularization and application. The method is especially suitable for the field of sports medicine and has wide application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of medical biomaterials, and in particular to a decellularized matrix and a preparation method thereof. Background Art

[0002] Acellular matrix materials refer to matrix materials that, based on the composition and structure of the extracellular matrix, remove cells through a decellularization process while retaining their bioactive components. They are highly coordinated organic entities composed of multiple signal molecules, and can induce and promote cell adhesion, proliferation, differentiation and tissue formation. They are the basis of body tissue repair and are the biological wound repair products that are closest to the physiological structure and function of body tissues. However, due to the destruction of the structure during the preparation process and the structural characteristics of some materials themselves, the overall mechanical properties of acellular matrix materials are relatively low, limiting their application in fields such as sports medicine.

[0003] Aquatic animal-derived decellularized matrix has received increasing attention in recent years due to its good biocompatibility and no risk of zoonotic virus transmission. Due to the influence of the growth environment, the physical and chemical properties of the extracellular matrix of aquatic animals show significant diversity, and some animal tissues are significantly better than terrestrial animals in terms of structural stability and mechanical properties. Patent CN116236622A discloses a functional active gradient bionic biological rotator cuff patch and its preparation method. The preparation method uses aquatic decellularized matrix material as a substrate, and promotes the adhesive component to fully infiltrate into the substrate under vacuum pressing conditions at room temperature. The vacuum pressing method used in this method will significantly damage the pore structure and is not conducive to cell proliferation; and this method is to enhance the mechanical properties through physical bonding, the material is thick, the clinical comfort is poor, and there is a certain risk of falling off during bonding. Patent CN113476667A discloses a fish skin decellularized dermal matrix scaffold and its preparation method. The preparation method does not effectively protect the material structure, and the mechanical properties of the prepared scaffold material still have a lot of room for improvement; and the cross-linked decellularized matrix material prepared by this method has certain cytotoxicity and a low cell proliferation rate. Patent CN111359016A discloses a low-antigenic fish skin tissue and a method for processing the same. The mechanical properties of the cross-linked material are low, and there are cross-linking agent residues in the material prepared by this method. The cross-linking agent is not thoroughly and effectively cleaned, and there are certain risks in clinical application. Summary of the invention

[0004] First, in order to solve the problems of insufficient mechanical properties, low cell proliferation rate and poor comfort of decellularized matrix materials prepared by the prior art, the present invention provides a decellularized matrix, and its preparation method includes: decellularization, stabilization, cross-linking and cleaning steps; the cross-linking includes cyclic static cross-linking in a cross-linking agent solution and ultrasonic balancing in an equilibrium buffer system, and each completion of cross-linking and ultrasonic balancing is a cycle process; after each cycle, the cross-linking agent concentration is increased for the next cross-linking.

[0005] The present invention finds that by performing gradient crosslinking in a crosslinking agent with gradually increasing concentrations, the bonding between microfibril proteins can be effectively improved, while avoiding excessive crosslinking of the material surface due to excessive crosslinking agent concentration, which results in smaller micropores in the matrix tissue and hinders the crosslinking liquid from continuing to penetrate into the material, ultimately affecting the overall crosslinking degree and uniformity of the material, thereby achieving reinforcement of the material's mechanical properties and digestion resistance. The prepared decellularized matrix material is thin and tough, and has very high tensile strength and porosity, which can effectively solve the problems of insufficient mechanical properties and poor comfort of decellularized matrix materials in the prior art. At the same time, the cell proliferation rate of the decellularized matrix of the present invention is also high.

[0006] Preferably, the raw materials used in the preparation method of the decellularized matrix are aquatic animal-derived materials, preferably aquatic animal tissues, more preferably fish connective tissues, mollusk cartilage tissues or bone tissues, and more preferably fish skin or mollusk cartilage tissues.

[0007] Preferably, the density of the aquatic animal source material is 0.05 to 0.80 g / cm 3 , preferably 0.10 to 0.70 g / cm 3 , more preferably 0.20 to 0.60 g / cm 3 .

[0008] When selecting the above-mentioned aquatic animal-derived materials with higher tissue fiber density, better mechanical properties and comfort can be obtained by combining with a gradient cross-linking reaction.

[0009] In some embodiments, the initial concentration of the cross-linking agent solution is 0.05% to 0.25%, and the cross-linking agent concentration is increased by 1% to 5% after each cycle; preferably, the cross-linking agent concentration is increased by 1.5% to 4%.

[0010] In some embodiments, the number of cycles is 2 or more; preferably 3 or more.

[0011] In the specific implementation process, the appropriate cross-linking agent concentration gradient and cycle times can be selected according to different requirements of the decellularized matrix materials.

[0012] In a specific implementation process, the cross-linking is carried out at room temperature (preferably 20-25° C.).

[0013] In some embodiments, the crosslinking includes first sonicating in an equilibrium buffer system, then cyclically standing crosslinking in a crosslinker solution and sonicating in an equilibrium buffer system; the concentration of the crosslinker solution in the first cycle is 0.05% to 0.25%, the concentration of the crosslinker solution in the second cycle is 2% to 3%, and the concentration of the crosslinker solution in the third cycle is 5% to 6%.

[0014] In some embodiments, as the concentration of the crosslinking agent solution increases, the time for crosslinking in the crosslinking agent solution decreases gradually.

[0015] In some embodiments, the static cross-linking time in the first cycle is 15 to 18 hours, the static cross-linking time in the second cycle is 1 to 3 hours, and the static cross-linking time in the third cycle is 0.5 to 1.5 hours.

[0016] In some embodiments, the time of sonication in the equilibration buffer in each cycle is 1 to 3 hours.

[0017] In some embodiments, the cross-linking agent is at least one of epoxy compounds, aldehydes, organic peroxides, and carbodiimides; preferably, the cross-linking agent includes at least one of glycerol diglycidyl ether, glutaraldehyde, and acetic anhydride.

[0018] In some embodiments, the equilibration buffer comprises at least one of a phosphate buffer, a tris buffer, a barbiturate buffer, a borate buffer, a bicarbonate buffer system, and a glycine-sodium hydroxide buffer; preferably a phosphate buffer and a glycine-sodium hydroxide buffer.

[0019] In some embodiments, the pH of the equilibration buffer is 7.5-10.5 (preferably pH 7.0-9.0), and the concentration of the equilibration buffer is 0.01-1 mol / L (preferably 0.05-0.5 mol / L, more preferably 0.05-0.2 mol / L).

[0020] In some embodiments, the power of the ultrasound is 60-120W, preferably 80-100W.

[0021] Further preferably, the washing comprises performing gradient washing in a salt solution with gradually decreasing concentration.

[0022] In order to further solve the problem that the residual preparations in the decellularized matrix prepared by the prior art are difficult to remove and have high immune and biosafety risks, the present invention further finds that by washing with a gradient salt solution, the molecular diffusion effect of the concentration difference can be utilized to replace the residual reagents in the cross-linked matrix, thereby achieving effective removal of the residual reagents, reducing immune and biosafety risks, and at the same time preventing the problem of decreased mechanical properties of the decellularized matrix caused by the traditional cleaning process.

[0023] In some embodiments, the cleaning method includes but is not limited to at least one of a high-speed stirring reflux method, a jet reflux ultrasound method, an ultrasonic oscillation method, and a jet ultrasound method.

[0024] Preferably, the rotation speed of the high-speed stirring is 200 to 2000 rpm, preferably 600 to 1600 rpm, and more preferably 800 to 1200 rpm.

[0025] Preferably, the cleaning times are 2 or more; more preferably, 3 or more.

[0026] Preferably, the concentration of the salt solution for initial cleaning is 0.5-1.5 mol / L, and the concentration of the salt solution is reduced by 0.2-0.6 mol / L after each cleaning; preferably, the concentration of the salt solution is reduced by 0.2-0.5 mol / L.

[0027] Preferably, the last cleaning is performed in pure water (preferably for 1 to 10 hours or 2 to 8 hours).

[0028] Preferably, the concentration of the salt solution in the first cleaning is 0.8-1 mol / L, the concentration of the salt solution in the second cleaning is 0.5-0.6 mol / L, and the concentration of the salt solution in the third cleaning is 0.1-0.3 mol / L.

[0029] Preferably, the pH of the salt solution is 6 to 8, preferably 6 to 7.6, and more preferably 7.2 to 7.6.

[0030] Preferably, the salt solution includes at least one of a phosphate buffer, a borate buffer, a bicarbonate buffer system, a glycine-sodium hydroxide buffer, a potassium salt solution, a sodium salt solution, and a calcium salt solution; more preferably, the salt solution includes at least one of a phosphate buffer, a potassium chloride solution, and a sodium chloride solution.

[0031] More preferably, the washing comprises gradient washing in a salt solution with gradually decreasing concentration, wherein the concentration of the salt solution in the first washing is 0.8-1 mol / L, and the temperature of the first washing is room temperature (e.g., 20-30°C); the concentration of the salt solution in the second washing is 0.5-0.6 mol / L, and the temperature of the second washing is 2-8°C; the concentration of the salt solution in the third washing is 0.1-0.3 mol / L, and the temperature of the third washing is 2-8°C; and the last washing is washing in pure water (preferably the temperature of the last washing is 2-8°C).

[0032] Preferably, the cleaning time for each time is 1 to 20 hours, preferably 1 to 8 hours, more preferably 6 to 8 hours.

[0033] Furthermore, the present invention provides a decellularized matrix obtained by the preparation method of any one of the above embodiments or a combination thereof.

[0034] Preferably, the tensile strength of the decellularized matrix is ​​10 MPa to 60 MPa (e.g., 10 MPa, 20 MPa, 25 MPa, 30 MPa, 31 MPa, 32 MPa, 33 MPa, 34 MPa, 35 MPa, 36 MPa, 37 MPa, 38 MPa, 39 MPa, 40 MPa, 45 MPa, 50 MPa, 55 MPa, 60 MPa).

[0035] Preferably, the porosity of the decellularized matrix is ​​greater than 90% (eg, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%).

[0036] Preferably, the cell proliferation rate of the decellularized matrix is ​​greater than 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 105%, 108%, 110%, 111%).

[0037] Preferably, the protein content of the decellularized matrix is ​​above 75%, preferably above 85%, further preferably above 95%, and most preferably above 96%.

[0038] Preferably, the in vitro complete degradation time of the decellularized matrix in a 5 mg / mL collagenase solution is 5 to 90 days, preferably 10 to 60 days, and more preferably 10 to 30 days.

[0039] Preferably, the acellular matrix of the present invention has no significant immune response after implantation.

[0040] The residual amount of DNA in the decellularized matrix of the present invention is lower than 30 ng / mg, lower than 25 ng / mg, lower than 20 ng / mg, or lower than 15 ng / mg.

[0041] In the specific implementation process, the preparation method further includes: performing an impurity removal operation before decellularization.

[0042] In the specific implementation process, the impurity removal operation includes removing residual meat, skin and fat impurities, and draining after washing.

[0043] In a specific implementation process, the decellularization step includes: using chemical reagents to remove residual cells in the material.

[0044] Preferably, the chemical reagent includes but is not limited to a surfactant (including but not limited to sodium cholate, sodium deoxycholate, sodium dodecyl sulfate, Triton X-100, Triton X-114, Tween20, octyl glucoside), an acid or base (including but not limited to hydrochloric acid, acetic acid, sulfuric acid, carbonic acid, sodium hydroxide, potassium hydroxide, calcium hydroxide), a biological enzyme (including but not limited to tissue protease, papain, pepsin, trypsin, subtilisin), and a deodorizing and degreasing agent (including but not limited to methanol, ethanol, isopropanol, sodium hydroxide, potassium hydroxide, and peracetic acid).

[0045] Preferably, the concentration of the chemical reagent is 0.01-1.0%, preferably 0.01-0.50%.

[0046] Preferably, the decellularization reaction time is 1 to 30 h, more preferably the reaction time is 1 to 20 h.

[0047] Preferably, the decellularization reaction temperature is 4-10°C.

[0048] More preferably, the decellularization step comprises: removing residual cells of the material by oscillating washing with 0.1-1% TritonX-114, oscillating washing with 0.05-0.2% acetic acid, oscillating washing with 0.01-0.05% papain, and oscillating washing with 0.05-0.2% potassium hydroxide in sequence at a reaction temperature of 4-10°C.

[0049] In a specific implementation process, the stabilization step includes: stabilizing the material in a buffer system.

[0050] Preferably, the buffer system includes, but is not limited to, one or more of phosphate buffer, tris(hydroxymethylaminomethane) buffer, barbital buffer, borate buffer, bicarbonate buffer system, and glycine-sodium hydroxide buffer, preferably phosphate buffer and glycine-sodium hydroxide buffer.

[0051] Preferably, the pH of the buffer system is 7.5 to 10.5, preferably 8.5 to 9.5.

[0052] Preferably, the concentration of the buffer system is 0.1 to 1.0 mol / L, preferably 0.1 to 0.5 mol / L.

[0053] Preferably, the stabilization reaction time is 2 to 10 h, preferably 2 to 4 h.

[0054] Preferably, the stabilization reaction temperature is 4-10°C.

[0055] More preferably, the stabilization step comprises: oscillating and washing in a 0.1-0.5 mol / L pH 8.5-9.5 glycine-sodium hydroxide solution at a reaction temperature of 4-10°C for stabilization.

[0056] In a specific implementation process, the preparation method further includes: freeze-drying after the washing step.

[0057] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a decellularized matrix material with high mechanical properties and cell proliferation rate and a preparation method thereof. The prepared decellularized matrix is ​​thin and tough, overcoming the problems of poor mechanical properties, poor comfort and low cell proliferation rate in the prior art. It has high value in the field of medical applications, is particularly suitable for the field of sports medicine, and has broad application prospects. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The cross-linking agent concentration % in the following embodiments represents the final concentration of the cross-linking agent in the solution. For example, a cross-linking agent concentration of 1% means that 1g of cross-linking agent is contained in every 100g of water.

[0059] If no specific techniques or conditions are specified in the examples, all are carried out according to conventional methods or the techniques or conditions described in the literature in the field, or according to the product instructions. If no manufacturer is specified for the reagents and instruments used, all are conventional products that can be purchased through regular channels.

[0060] Embodiment 1~5

[0061] In this example, aquatic animal tissue - fish skin (density 0.35 g / cm 3 ) as an example, a decellularized matrix is ​​provided, and the preparation method thereof is as follows: (1) Removal of impurities: remove impurities such as residual meat, skin, fat, etc., wash and drain; (2) Decellularization: At a reaction temperature of 6°C, the samples were washed with 0.5% Triton X-114 for 1 h, 0.1% acetic acid for 2 h, 0.01% papain for 4 h, and 0.1% potassium hydroxide for 2 h to remove residual cells. (3) Stabilization: Wash and stabilize in 0.3 mol / L glycine-sodium hydroxide solution at pH 9.0 at 6°C for 4 h. (4) Cross-linking: The material was gradient cross-linked using a cross-linking agent at a reaction temperature of 20°C. Ultrasonic balance treatment was combined during the cross-linking process, and the ultrasonic power was 80 W. The cross-linking steps were as follows: ultrasonic treatment in pH8.5 0.1 mol / L equilibrium buffer for 2 h; static cross-linking in gradient 1 concentration cross-linking agent for 16 h, and ultrasonic treatment in pH8.5 0.1 mol / L equilibrium buffer for 2 h; static cross-linking in gradient 2 concentration cross-linking agent for 2 h, and ultrasonic treatment in pH8.5 0.1 mol / L equilibrium buffer for 2 h; static cross-linking in gradient 3 concentration cross-linking agent for 1 h, and ultrasonic treatment in pH8.5 0.1 mol / L equilibrium buffer for 2 h. The specific cross-linking parameters of different embodiments are shown in Table 1; (5) Cleaning: After ultrasonic cleaning in pure water at 25°C for 24 h, the material was drained and freeze-dried to obtain a decellularized matrix.

[0062] Comparative Example 1

[0063] This comparative example provides a method for preparing a decellularized matrix, the steps of which are different from those of Example 1 only in that ultrasonic treatment in the equilibration buffer is omitted in the cross-linking step, and the specific cross-linking parameters are shown in Table 1.

[0064] Comparative Example 2

[0065] This comparative example provides a method for preparing a decellularized matrix, and the steps are only different from those of Example 1 in that a fixed crosslinking agent concentration is used for crosslinking in the crosslinking step.

[0066] Comparative Example 3

[0067] This comparative example provides a method for preparing a cell-free matrix, and the steps are different from those of Example 1 only in that the step of static cross-linking in a cross-linking agent solution is omitted in the cross-linking step.

[0068] Test Example 1

[0069] The decellularized matrices prepared in the above examples and comparative examples were subjected to performance tests. The tensile strength was measured in accordance with 5.3.3 tensile test of YY / T0606.5-2007 "Tissue Engineering Medical Products Part 5: Performance and Test of Matrix and Scaffold" and GB / T 1040.3 "Plastic Tensile Properties Test", with the test specimen size of 54×10 mm and a gauge length of 36 mm.

[0070] The porosity test method is as follows: immerse the decellularized matrix in anhydrous ethanol with a volume of V1, remove the gas in the material by negative pressure suction, record the volume of the solution V2, remove the material, and record the volume of the solution V3. Porosity = [(V1-V3) / (V2-V3)] × 100%.

[0071] At the same time, L929 cells were used to test the cell proliferation rates of different materials according to the MTT method recommended in GB / T 16886.5-2017 “Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test”.

[0072] The test results are shown in Table 1.

[0073] Table 1

[0074] The results show that compared with the comparative example, the preparation method of the embodiment can effectively improve the tensile strength and porosity of the decellularized matrix material, improve the mechanical properties and comfort, and also have a higher cell proliferation rate. Moreover, in Examples 1 to 5, selecting different cross-linking agents and buffer systems can effectively improve the tensile strength and porosity of the material.

[0075] Embodiments 6 to 11

[0076] This embodiment provides a method for preparing a decellularized matrix, and the steps are different from those of Example 4 only in that the washing steps are: washing at 200 rpm for 6 h in a salt solution of concentration 1 at 25°C; washing at 200 rpm for 8 h in a salt solution of concentration 2 at 6°C; washing at 200 rpm for 8 h in a salt solution of concentration 3 at 6°C; and finally washing at 200 rpm for 6 h in purified water at 6°C. The treatment conditions are shown in Table 2.

[0077] Test Example 2

[0078] According to the test method of Experimental Example 1, the tensile strength, porosity and cell proliferation rate of the decellularized matrices prepared in Examples 6 to 11 were tested.

[0079] The test results are shown in Table 2.

[0080] Table 2

[0081] The results show that, compared with Example 4, Examples 6 to 11 can further improve the tensile strength and cell proliferation rate of the decellularized matrix by washing with a salt solution, and can further improve the tensile strength and cell proliferation rate of the decellularized matrix by washing with a salt solution of gradient concentration, and reduce the loss of mechanical properties of the material during the washing process.

[0082] Test Example 3

[0083] In this test example, the residual DNA and protein content of the decellularized matrix prepared in the above example were detected.

[0084] The method for detecting the residual DNA is as follows: after the decellularized matrix is ​​digested and dissolved with proteinase K, the residual DNA content in the material is determined according to YYT0606.25-2014 Tissue Engineering Medical Products Part 25: Determination of Residual DNA in Animal-Derived Biomaterials: Fluorescence Staining Method.

[0085] The method for detecting protein content is as follows: Determine according to the General Rules 0704 of Part IV of the 2020 edition of the Pharmacopoeia of the People's Republic of China "Nitrogen Determination Method - Third Method - Nitrogen Analyzer Method".

[0086] The test results are shown in Table 3.

[0087] Table 3

[0088] The results show that the acellular matrix material prepared by the present invention has low DNA residue.

[0089] Test Example 4

[0090] In this test example, the serum immunoglobulin and complement components of the acellular matrix prepared in the above example were determined according to GB / T 16886.20-2015 "Biological Evaluation of Medical Devices Part 20: Principles and Methods of Immunotoxicology Tests for Medical Devices" and YY / T 1465.2-2016 "Medical Device Immunogenicity Evaluation Methods Part 2 ELISA Method for Determination of Serum Immunoglobulin and Complement Components". The specific method is as follows: Balb / c mice were selected and divided into groups according to the embodiment, negative control group and positive control group, with 10 mice in each group. The negative control group (sham operation group) underwent surgery but no material was implanted. The positive control group began to be subcutaneously injected with a mixed emulsion of equal volumes of BSA (bovine serum albumin) and CFA (complete Freund's adjuvant) one week after surgery. The final concentration of BSA was 1.67 mg / mL, and each mouse was injected with 0.12 mL once a week for a total of 3 times. The mice in the embodiment were implanted with 1 piece of the sample (1.5 cm) subcutaneously on the left side of the back of the mouse. 2). After 30 days, peripheral blood was collected from mice, placed at 4°C for 2 hours, and then centrifuged at 1000 g for 20 min. The upper serum was collected and the contents of immunoglobulins IgM and IgG were determined using ELISA kits.

[0091] The test results are shown in Table 4.

[0092] Table 4

[0093] Note: "**" indicates P < 0.01 compared with the negative control group.

[0094] The results showed that there were extremely significant differences in serum IgM and IgG concentrations between the positive control group and the negative control group, and there were no significant differences in serum IgM and IgG concentrations between all embodiment groups and the negative control group. Therefore, the acellular matrix material prepared by the present invention did not have an obvious immune response after implantation into mice, and the immune risk and biosafety risk were extremely low.

[0095] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A decellularized matrix, characterized in that: The preparation method comprises: decellularization, stabilization, cross-linking and cleaning steps; the cross-linking comprises cyclic static cross-linking in a cross-linking agent solution and ultrasonic balancing in an equilibrium buffer system, and each completion of cross-linking and ultrasonic balancing is a cycle process; after each cycle, the cross-linking agent concentration is increased to carry out the next cross-linking.

2. The acellular matrix according to claim 1, characterized in that The initial concentration of the crosslinking agent solution is 0.05% to 0.25%, and the concentration of the crosslinking agent is increased by 1% to 5% after each cycle; preferably, the concentration of the crosslinking agent is increased by 1.5% to 4%; And / or, the number of cycles is 2 or more; preferably 3 or more.

3. The acellular matrix according to claim 1, characterized in that The cross-linking comprises first ultrasonication in an equilibrium buffer system, then cyclically static cross-linking in a cross-linking agent solution and ultrasonic equilibrium in an equilibrium buffer system; the concentration of the cross-linking agent solution in the first cycle is 0.05% to 0.25%, the concentration of the cross-linking agent solution in the second cycle is 2% to 3%, and the concentration of the cross-linking agent solution in the third cycle is 5% to 6%.

4. The decellularized matrix according to claim 2, characterized in that As the concentration of the crosslinking agent solution increases, the time for crosslinking in the crosslinking agent solution decreases gradually.

5. The decellularized matrix according to claim 3, characterized in that The static cross-linking time in the first cycle is 15 to 18 hours, the static cross-linking time in the second cycle is 1 to 3 hours, and the static cross-linking time in the third cycle is 0.5 to 1.5 hours.

6. The acellular matrix according to any one of claims 1 to 5, characterized in that The cross-linking agent is at least one of epoxy compounds, aldehydes, organic peroxides, and carbodiimides; And / or, the equilibration buffer comprises at least one of a phosphate buffer, a tris(hydroxymethylaminomethane) buffer, a barbiturate buffer, a borate buffer, a bicarbonate buffer system, and a glycine-sodium hydroxide buffer; Preferably, the pH of the equilibration buffer is 7.5-10.5, and the concentration of the equilibration buffer is 0.01-1 mol / L.

7. The acellular matrix according to any one of claims 1 to 6, characterized in that The washing comprises performing gradient washing in salt solutions with gradually decreasing concentrations.

8. The decellularized matrix according to claim 7, characterized in that The concentration of the salt solution for initial cleaning is 0.5-1.5 mol / L, and the concentration of the salt solution is reduced by 0.2-0.6 mol / L after each cleaning; preferably, the concentration of the salt solution is reduced by 0.2-0.5 mol / L; Preferably, the cleaning is performed by oscillation cleaning or ultrasonic cleaning; Preferably, the number of cleanings is 2 or more times; more preferably 3 or more times; Preferably, the last wash is in pure water; Preferably, the concentration of the salt solution in the first cleaning is 0.8-1 mol / L, the concentration of the salt solution in the second cleaning is 0.5-0.6 mol / L, and the concentration of the salt solution in the third cleaning is 0.1-0.3 mol / L; Preferably, the pH of the salt solution is 6 to 8, preferably 6 to 7.6, more preferably 7.2 to 7.6; Preferably, the salt solution includes at least one of a phosphate buffer, a borate buffer, a bicarbonate buffer system, a glycine-sodium hydroxide buffer, a potassium salt solution, a sodium salt solution, and a calcium salt solution; More preferably, the cleaning comprises gradient cleaning in a salt solution with gradually decreasing concentration, wherein the concentration of the salt solution in the first cleaning is 0.8-1 mol / L, and the temperature of the first cleaning is room temperature; the concentration of the salt solution in the second cleaning is 0.5-0.6 mol / L, and the temperature of the second cleaning is 2-8°C; the concentration of the salt solution in the third cleaning is 0.1-0.3 mol / L, and the temperature of the third cleaning is 2-8°C; and the last cleaning is cleaning in pure water.

9. The decellularized matrix according to claim 1, characterized in that: The raw materials used in the preparation method are aquatic animal source materials, preferably aquatic animal tissues, more preferably connective tissues of fish, cartilage tissues or bone tissues of molluscs, and more preferably skin of fish or cartilage tissues of molluscs.

10. The acellular matrix according to any one of claims 1 to 9, characterized in that The tensile strength of the decellularized matrix is ​​10 MPa to 60 MPa; and / or the porosity of the decellularized matrix is ​​greater than 90%; and / or the cell proliferation rate of the decellularized matrix is ​​greater than 90%; and / or the protein content of the decellularized matrix is ​​greater than 75%; and / or the residual amount of DNA in the decellularized matrix is ​​less than 30 ng / mg.

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