Modified acellular matrix

Abstract

The invention relates to the technical field of medical biological materials, in particular to a modified acellular matrix. According to the method, microcrystal drying, immobilization and swelling treatment are combined in sequence in the decellularization treatment process, so that the phenomenon of low porosity after crosslinking can be effectively reduced, meanwhile, the mechanical loss in the decellularization process is reduced, and the high mechanical property is considered while the high porosity is obtained. According to the modified acellular matrix, the defect that the mechanical property of an acellular matrix material with the unit thickness is insufficient is remarkably overcome, and the modified acellular matrix has high porosity and high mechanical property at the same time. The acellular matrix is thin and tough, the comfort degree is high after implantation, the tensile strength is improved to more than three times that of an original material, and the acellular matrix has extremely high application value in the medical field with requirements for mechanical property, porosity, thickness and comfort degree.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of medical biomaterials, and in particular to a modified decellularized matrix. BACKGROUND

[0002] The decellularized matrix refers to a matrix material that is obtained by removing cells while retaining the biological activity components of the extracellular matrix components and structures through a decellularization process. The decellularized matrix is a highly coordinated organic whole composed of various signal molecules, can induce and promote cell adhesion, proliferation, differentiation and tissue formation, and is the basis of tissue repair of the body. The material is the most physiological structure and function of the biological type of wound repair product closest to the body tissue. However, due to the destruction of the structure during the preparation process and the structural characteristics of part of the material itself, the mechanical properties of the decellularized matrix material are generally low, and the material degrades rapidly after implantation, which limits its application in the field of sports medicine. The modified decellularized matrix is further processed on the basis of the decellularized matrix material, and the material prepared has higher advantages in mechanical properties, degradation time and other indicators. However, the modified material is prone to a decrease in porosity, and research shows that the porosity and cell proliferation rate are positively correlated within a certain range. Therefore, the modified decellularized matrix material with high porosity has higher market value.

[0003] Aquatic animals show significant diversity in the physicochemical properties of their extracellular matrix due to the influence of their growth environment. Some animal tissues have better structural stability and mechanical properties than terrestrial animals. Patent CN118340945A discloses a preparation method of a decellularized matrix. The method adjusts the porosity of the modified decellularized matrix by using gel polymers. The porosity of some samples is about 80%, which still has room for improvement. Moreover, the modification process is unstable and prone to inconsistent crosslinking degrees. Patent CN116236622A discloses a functional active gradient biomimetic shoulder sleeve patch and a preparation method thereof. The preparation method uses a decellularized matrix material from aquatic sources as a substrate and promotes the full infiltration of the adhesive components into the substrate under the condition of vacuum pressing at room temperature. However, the vacuum pressing method significantly damages the pore structure, which is not conducive to cell proliferation. Moreover, the method physically enhances the mechanical properties with the aid of adhesion, and the material is relatively thick, which results in poor clinical comfort and a certain risk of adhesion loss. Patent CN113476667A discloses a fish skin decellularized dermal matrix scaffold and a preparation method thereof. The preparation method does not effectively protect the material structure, and the mechanical properties still have a large room for improvement. Moreover, the crosslinked decellularized matrix material prepared by the method has certain cytotoxicity. In addition, the material is prone to an increase in density and a decrease in porosity after crosslinking, which affects cell proliferation after implantation. SUMMARY

[0004] Firstly, in order to solve the technical problem that high porosity and high mechanical property are difficult to be compatible, the application provides a modified decellularized matrix, and a preparation method thereof comprises: taking aquatic animal tissue as raw material, and preparing after decellularization treatment and crosslinking treatment; the decellularization treatment comprises sequentially performing microcrystal drying, immobilization and swelling treatment.

[0005] The application can effectively reduce the phenomenon of low porosity after crosslinking, and reduce the mechanical loss in the decellularization process, so as to obtain high porosity and high mechanical property.

[0006] In the specific implementation process, the microcrystal drying is freeze-drying after quick freezing at-40 DEG C or below.

[0007] Preferably, the microcrystal drying is freeze-drying to less than 15% (w / w) of moisture content after quick freezing at-40 DEG C or below.

[0008] In the microcrystal drying process, the intracellular water is rapidly condensed to form ice crystals, and then freeze-drying, and the ice crystals sublimate in the freeze-drying process, so that the material is thickened and porous.

[0009] In the specific implementation process, the immobilization is crosslinking the material in the fixing agent (crosslinking agent).

[0010] By preliminarily fixing the fiber organization of the material by using the fixing agent, the bonding between the fiber proteins can be improved, so that the mechanical loss of the material in the decellularization process can be effectively reduced.

[0011] In the specific implementation process, the swelling treatment is sequentially washing the material in the surfactant and the swelling agent.

[0012] In the specific implementation process, the concentration of the fixing agent is 0.01%~2% (w / w), preferably 0.01%~0.5% (w / w), and more preferably 0.01%~0.2% (w / w).

[0013] In the specific implementation process, the fixing agent comprises at least one of acrylate, polyisocyanate, polyol, polyamine, aziridine, epoxy compound, aldehyde, organic peroxide, carbodiimide and organosilicon crosslinking agent.

[0014] In the specific implementation process, the immobilization time is 0.1~6 h, and preferably 0.1~4 h.

[0015] In specific implementation, the surfactant includes at least one of sodium cholate, sodium deoxycholate, sodium dodecyl sulfate, Triton X-100, Triton X-114, Tween 20, octyl glucoside; and the swelling agent includes at least one of a salt, a base, an acid, and a polymer.

[0016] In specific implementation, the swelling agent includes but is not limited to a salt, a base, an acid, and a polymer.

[0017] Preferably, the acid in the swelling agent includes but is not limited to hydrochloric acid, formic acid, acetic acid, nitric acid, phosphoric acid, carbonic acid, hypochlorous acid, phthalic acid, isophthalic acid, oxalic acid, malic acid, citric acid.

[0018] Preferably, the base in the swelling agent includes but is not limited to sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, aluminum hydroxide, magnesium hydroxide, barium hydroxide, sodium carbonate, sodium bicarbonate.

[0019] Preferably, the salt in the swelling agent includes but is not limited to NaCl, KCl, CaCl2, ZnCl2, MgCl2, Ca2(NO3).

[0020] Preferably, the polymer in the swelling agent includes but is not limited to poly-p-dioxanone.

[0021] In specific implementation, the concentration of the swelling agent is 0.05-0.5% (w / w).

[0022] In specific implementation, the time for washing in the surfactant and / or the swelling agent is 1-30 h, preferably 1-10 h.

[0023] In specific implementation, the temperature of the swelling treatment is 2-8℃.

[0024] Preferably, the preparation method further includes: performing a first precision cleaning after the decellularization treatment, and performing a second precision cleaning after the crosslinking treatment.

[0025] Preferably, the first precision cleaning and / or the second precision cleaning uses a salt solution or water cleaning.

[0026] Through at least two precision cleanings, reagent residues can be more thoroughly removed, the biological safety of the material is improved, and the cell proliferation rate is further improved.

[0027] In specific implementation, the salt solution includes but is not limited to a phosphate buffer, a borate buffer, a bicarbonate buffer system, a glycine-sodium hydroxide buffer, a potassium salt solution, a sodium salt solution, a calcium salt solution.

[0028] Preferably, the pH of the salt solution is 6.5-7.5.

[0029] Preferably, the concentration of the salt solution is 0.1-0.3 mol / L.

[0030] Preferably, the first precision cleaning uses water cleaning, and the second precision cleaning uses salt solution cleaning.

[0031] The effect of the two precision cleanings performed in the above sequence is better.

[0032] In the specific implementation, the aquatic animal tissue is connective tissue of aquatic animals.

[0033] Specifically, the aquatic animals include fish, including but not limited to salmon, cod, carp, tilapia, grass carp, sea bass, tuna, black fish, sturgeon, bighead carp, blue carp, silver carp, crucian carp, etc.

[0034] Preferably, the aquatic animal tissue is swim bladder, skin, etc. of fish.

[0035] Preferably, the density of the aquatic animal tissue is 0.05-0.80 g / cm 3 , preferably 0.10-0.70 g / cm 3 , and more preferably 0.20-0.60 g / cm 3 .

[0036] In the specific implementation, pretreatment is performed before the decellularization treatment.

[0037] Preferably, the pretreatment includes cleaning after removing residual meat, epidermis, and fat impurities of the raw material.

[0038] In the specific implementation, biological enzymes, acid, etc. can be introduced in the pretreatment process.

[0039] In the specific implementation, the biological enzymes include but are not limited to cathepsin, papain, pepsin, trypsin, subtilisin, serine protease, chymotrypsin, aspartate dehydrogenase, sulfhydryl protease, triacylglycerol acylhydrolase, etc.

[0040] In the specific implementation, the acid in the pretreatment process includes but is not limited to hydrochloric acid, acetic acid, sulfuric acid, nitric acid, phosphoric acid, carbonic acid, hypochlorous acid, phthalic acid, isophthalic acid, oxalic acid, malic acid, citric acid, etc.

[0041] In the specific implementation, the concentration of the biological enzyme is 0.01%-0.1% (w / w), and preferably 0.001%-0.05% (w / w).

[0042] In a specific embodiment, the concentration of the acid is 0.01% to 0.2% (w / w), preferably 0.01% to 0.1% (w / w).

[0043] In a specific embodiment, the drying treatment is performed after the cross-linking treatment or the secondary precision cleaning.

[0044] Preferably, the drying treatment is a freeze-drying treatment.

[0045] In a specific embodiment, the cross-linking agent in the cross-linking treatment comprises at least one of acrylates, polyisocyanates, polyols, polyamines, aziridines, epoxides, aldehydes, organic peroxides, carbodiimides, and silicone cross-linking agents.

[0046] In a specific embodiment, the concentration of the cross-linking agent in the cross-linking treatment is 0.1% to 10.0% (w / w), preferably 0.5% to 9.5% (w / w).

[0047] In a specific embodiment, the time of the cross-linking treatment is 0.1 to 96 h, preferably 4 to 72 h, and more preferably 24 to 72 h.

[0048] In a specific embodiment, the temperature of the cross-linking treatment is 20°C to 25°C.

[0049] Optionally, the cross-linking treatment is performed in a buffer system.

[0050] Optionally, the buffer system includes, but is not limited to, a phosphate buffer, a tris buffer, a barbiturate buffer, a borate buffer, a bicarbonate buffer system, a glycine-sodium hydroxide buffer, and the like.

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

[0052] Optionally, 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 tensile strength of the decellularized matrix is 34 MPa or more (preferably 35, 36, 37 MPa or more); and / or, the porosity is 91% or more (preferably 92%, 93%, 94%, 95%, 96%, 96.7% or more); and / or, the thickness is 0.88 to 1.22 mm; and / or, the cell proliferation rate is 92% or more (preferably 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105% or more).

[0054] Preferably, the DNA residue in the modified decellularized matrix is less than 24.2 ng / mg (more preferably 23 ng / mg, 22 ng / mg, 21 ng / mg, 20 ng / mg, 19 ng / mg, 18 ng / mg, 16.3 ng / mg).

[0055] Preferably, the protein content of the modified decellularized matrix is more than 96% (more preferably more than 97%, more than 97.86%).

[0056] The modified decellularized matrix of the present application has no obvious immune response after implantation.

[0057] Compared with the prior art, the present application has the beneficial effects that: The modified decellularized matrix of the present application significantly improves the shortcomings of insufficient mechanical properties of unit thickness decellularized matrix material, and has high porosity while taking into account high mechanical properties. The prepared decellularized matrix is thin and tough, has high comfort after implantation, and the tensile strength is increased to more than 3 times that of the original material. The high porosity is beneficial to cell migration and ingrowth. The modified decellularized matrix of the present application has extremely high application value in the medical field (such as shoulder sleeve patch) which has requirements for mechanical properties, porosity, thickness and comfort. BRIEF DESCRIPTION OF DRAWINGS

[0058] Figure 1 Figure 6 is an electron microscope characterization diagram of the modified decellularized matrix of Example 6 of the present application. DETAILED DESCRIPTION

[0059] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme in the present application will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. In the examples provided in the present specification, the specific technology or conditions are not specified, which are according to the technology or conditions described in the literature in the art, or according to the product manual. The reagents or instruments used are not specified by the manufacturer, which are conventional products that can be purchased through regular channels.

[0060] As an embodiment, the present embodiment provides a modified decellularized matrix, and a preparation method thereof includes: using aquatic animal tissue as raw material, and preparing after decellularization treatment and crosslinking treatment; the decellularization treatment includes sequentially performing microcrystalline drying, immobilization and swelling treatment.

[0061] As an embodiment, the microcrystalline drying is freeze-drying after freezing the material at-40℃ or below.

[0062] As an embodiment, the microcrystalline drying is freeze-drying the material after being frozen at below -40℃ to a moisture content of less than 15% (w / w).

[0063] As an embodiment, the immobilization is cross-linking the material in a fixative (cross-linking agent).

[0064] As an embodiment, the swelling treatment is washing the material in a surfactant and a swelling agent in sequence.

[0065] As an embodiment, the concentration of the fixative is 0.01%~2% (w / w), preferably 0.01%~0.5% (w / w), and more preferably 0.01%~0.2% (w / w).

[0066] As an embodiment, the fixative includes at least one of acrylate, polyisocyanate, polyol, polyamine, aziridine, epoxide, aldehyde, organic peroxide, carbodiimide, and silicone cross-linking agent.

[0067] As an embodiment, the time of the immobilization is 0.1~6 h, preferably 0.1~4 h.

[0068] As an embodiment, the surfactant includes at least one of sodium cholate, sodium deoxycholate, sodium dodecyl sulfate, Triton X-100, Triton X-114, Tween20, and octyl glucoside; and the swelling agent includes at least one of salt, base, acid, and polymer.

[0069] As an embodiment, the swelling agent includes, but is not limited to, salt, base, acid, and polymer.

[0070] As an embodiment, the acid in the swelling agent includes, but is not limited to, hydrochloric acid, formic acid, acetic acid, nitric acid, phosphoric acid, carbonic acid, hypochlorous acid, phthalic acid, isophthalic acid, oxalic acid, malic acid, and citric acid.

[0071] As an embodiment, the base in the swelling agent includes, but is not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, aluminum hydroxide, magnesium hydroxide, barium hydroxide, sodium carbonate, and sodium bicarbonate.

[0072] As an embodiment, the salt in the swelling agent includes, but is not limited to, NaCl, KCl, CaCl2, ZnCl2, MgCl2, and Ca2(NO3).

[0073] As an embodiment, the polymer in the swelling agent includes, but is not limited to, poly-p-dioxanone.

[0074] As an embodiment, the concentration of the swelling agent is 0.05~0.5% (w / w).

[0075] As an embodiment, the time for washing in the surfactant and / or swelling agent is 1-30 h, preferably 1-10 h.

[0076] As an embodiment, the temperature of the swelling treatment is 2-8°C.

[0077] As an embodiment, the preparation method further comprises: performing a first precision cleaning after the decellularization treatment, and performing a second precision cleaning after the cross-linking treatment.

[0078] As an embodiment, the first precision cleaning and / or the second precision cleaning uses a salt solution or water cleaning.

[0079] As an embodiment, the salt solution includes but is not limited to phosphate buffer, borate buffer, bicarbonate buffer system, glycine-sodium hydroxide buffer, potassium salt solution, sodium salt solution, calcium salt solution.

[0080] As an embodiment, the pH of the salt solution is 6.5-7.5.

[0081] As an embodiment, the concentration of the salt solution is 0.1-0.3 mol / L.

[0082] As an embodiment, the first precision cleaning uses water cleaning, and the second precision cleaning uses salt solution cleaning.

[0083] As an embodiment, the aquatic animal tissue is aquatic animal connective tissue.

[0084] As an embodiment, the aquatic animal includes fish, including but not limited to salmon, cod, carp, tilapia, grass carp, sea bass, tuna, black fish, sturgeon, bighead carp, blue carp, silver carp, crucian carp, etc.

[0085] As an embodiment, the aquatic animal tissue is swim bladder, skin, etc. of fish.

[0086] As an embodiment, the density of the aquatic animal tissue is 0.05-0.80 g / cm 3 , preferably 0.10-0.70 g / cm 3 , more preferably 0.20-0.60 g / cm 3 .

[0087] As an embodiment, a pretreatment is performed before the decellularization treatment.

[0088] As an embodiment, the pretreatment includes cleaning after removing residual meat, epidermis and fat impurities of the raw material.

[0089] As an embodiment, the pretreatment process can also introduce biological enzymes, acid and other methods for treatment.

[0090] As an embodiment, the biological enzymes include but are not limited to cathepsin, papain, pepsin, trypsin, subtilisin, serine protease, chymotrypsin, aspartate dehydrogenase, sulfhydryl protease, triacylglycerol acylhydrolase, etc.

[0091] As an embodiment, the acid in the pretreatment process includes but is not limited to hydrochloric acid, acetic acid, sulfuric acid, nitric acid, phosphoric acid, carbonic acid, hypochlorous acid, phthalic acid, isophthalic acid, oxalic acid, malic acid, citric acid, etc.

[0092] As an embodiment, the concentration of the biological enzyme is 0.01%~0.1% (w / w), preferably 0.001%~0.05% (w / w).

[0093] As an embodiment, the concentration of the acid is 0.01%~0.2% (w / w), preferably 0.01%~0.1% (w / w).

[0094] As an embodiment, after the cross-linking treatment or the secondary precision cleaning, a drying treatment is performed.

[0095] As an embodiment, the drying treatment is a freeze-drying treatment.

[0096] As an embodiment, the cross-linking agent in the cross-linking treatment includes at least one of acrylate, polyisocyanate, polyol, polyamine, aziridine, epoxide, aldehyde, organic peroxide, carbodiimide, and organosilicon cross-linking agent.

[0097] As an embodiment, the concentration of the cross-linking agent in the cross-linking treatment is 0.1%~10.0% (w / w), preferably 0.5%~9.5% (w / w).

[0098] As an embodiment, the time of the cross-linking treatment is 0.1~96 h, preferably 4~72 h, and more preferably 24~72 h.

[0099] As an embodiment, the temperature of the cross-linking treatment is 20℃~25℃.

[0100] As an embodiment, the cross-linking treatment is performed in a buffer system.

[0101] As an embodiment, the buffer system includes but is not limited to phosphate buffer, tris buffer, barbiturate buffer, borate buffer, bicarbonate buffer system, glycine-sodium hydroxide buffer, etc.

[0102] As an embodiment, the pH of the buffer system is 7.5-10.5, preferably 8.5-9.5.

[0103] As an embodiment, the concentration of the buffer system is 0.1-1.0 mol / L, preferably 0.1-0.5 mol / L.

[0104] As an embodiment, the tensile strength of the decellularized matrix is 34 MPa or more (preferably 35, 36, 37 MPa or more); and / or, the porosity is 91% or more (preferably 92%, 93%, 94%, 95%, 96%, 96.7% or more); and / or, the thickness is 0.88-1.22 mm; and / or, the cell proliferation rate is 92% or more (preferably 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105% or more).

[0105] As an embodiment, the DNA residue in the modified decellularized matrix is less than 24.2 ng / mg (more preferably 23 ng / mg, 22 ng / mg, 21 ng / mg, 20 ng / mg, 19 ng / mg, 18 ng / mg, 16.3 ng / mg).

[0106] As an embodiment, the protein content of the modified decellularized matrix is 96% or more (more preferably 97% or more, 97.86% or more).

[0107] The present application is described below with more specific embodiments.

[0108] Embodiments 1-10 This embodiment takes aquatic animal tissue-fish skin (density 0.40 g / cm 3 ) as an example, and provides a modified decellularized matrix, which is prepared as follows: (1) Impurity removal: remove residual meat, epidermis, fat and other impurities with a stainless steel spoon, an ultrasonic knife and other tools, select materials with a thickness of 0.30 mm, soak in 0.02% (w / w) pepsin for 1 h, and then wash with purified water for standby; (2) Decellularization: (a) microcrystalline drying: freeze the material at -40°C, and then freeze-dry to a water content of less than 15% (w / w) to obtain a loose and porous material; (b) immobilization: after freeze-drying of the material, immobilize it in a low-concentration fixing agent (crosslinking agent); (c) swelling treatment: wash the material in surfactants and swelling reagents at a temperature of 6°C, and the specific conditions are shown in Table 1.

[0109] (3) Cleaning 1: wash the reagent residues in the decellularized matrix material with purified water.

[0110] (4) Cross-linking: Cross-linking using a cross-linking agent at a temperature of 20°C, the specific conditions are shown in Table 1.

[0111] (5) Washing 2: Using purified water to wash the reagent residues in the decellularized matrix material.

[0112] (6) Drying: After draining the material, freeze-drying treatment is performed to obtain a modified decellularized matrix.

[0113] Comparative Examples 1-2 The present comparative examples provide a modified decellularized matrix, and the preparation methods of Comparative Examples 1-2 are only different from Examples 1-2, respectively, in the following: Step (2) The decellularization process is first fixed, then the microcrystals are dried, and finally the swelling treatment is performed.

[0114] Comparative Examples 3-4 The present comparative examples provide a modified decellularized matrix, and the preparation methods of Comparative Examples 3-4 are only different from Examples 1-2, respectively, in the following: Step (2) The decellularization process is first fixed, then the microcrystals are dried, and finally the swelling treatment is performed.

[0115] Comparative Examples 5-6 The present comparative examples provide a modified decellularized matrix, and the preparation methods of Comparative Examples 5-6 are only different from Examples 1-2, respectively, in the following: Step (2) The decellularization process omits the microcrystal drying.

[0116] Comparative Examples 7-8 The present comparative examples provide a modified decellularized matrix, and the preparation methods of Comparative Examples 7-8 are only different from Examples 1-2, respectively, in the following: Step (2) The decellularization process omits the fixation.

[0117] Comparative Examples 9-10 The present comparative examples provide a modified decellularized matrix, and the preparation methods of Comparative Examples 9-10 are only different from Examples 1-2, respectively, in the following: Step (2) The decellularization process omits the swelling treatment.

[0118] Table 1

[0119] Test Example 1 The modified decellularized matrix prepared in the above examples and comparative examples is subjected to performance testing. The tensile strength is determined according to 5.3.3 tensile test in YY / T0606.5-2007 “Tissue Engineering Medical Products Part 5: Performance and Test of Matrix and Scaffolds” and GB / T 1040.3 “Test Methods for Tensile Properties of Plastics”. The size of the test sample is 54x10 mm, and the gauge length is 36 mm.

[0120] The porosity is tested as follows: the modified decellularized matrix is soaked in a container containing V1 volume of anhydrous ethanol, the gas in the material is removed by negative pressure suction, the volume V2 of the solution is recorded, the material is removed, and the volume V3 of the solution is recorded. The porosity is [(V1-V3) / (V2-V3)]x100%.

[0121] The thickness is tested as follows: five points are randomly selected at the center point and near the four corners of the product for thickness testing, and the thickness of each test point is recorded to calculate the average thickness.

[0122] Meanwhile, L929 cells are used to test the cell proliferation rate 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”. The test results are shown in Table 2.

[0123] Table 2

[0124] The results show that, compared with the comparative examples, the sequential combination of microcrystalline drying, immobilization and swelling during the decellularization process can effectively reduce the low porosity after crosslinking, and reduce the mechanical loss during decellularization, so that the modified decellularized matrix has high porosity, high cell proliferation rate and high mechanical properties.

[0125] Examples 11-12 The present example provides a modified decellularized matrix, and the preparation method thereof is only different from that of Example 2 in that: The first precision cleaning is performed before step (3) crosslinking, and the second precision cleaning is performed after step (3) crosslinking. The specific conditions of the two precision cleanings are shown in Table 3. The cell proliferation rate of the modified decellularized matrix prepared is detected by referring to the detection method of the above test example, and the results are shown in Table 3.

[0126] Table 3

[0127] Examples 13-14 The present example provides a modified decellularized matrix, and the preparation method thereof is only different from that of Example 2 in that: A first precision cleaning was performed before the cross-linking in step (3), and a second precision cleaning was performed after the cross-linking in step (3); the specific conditions of the two precision cleanings are shown in Table 4. The cell proliferation rate of the modified decellularized matrix prepared was detected according to the detection method of the above test example, and the results are shown in Table 4.

[0128] Table 4

[0129] It can be seen that the effect of first precision cleaning with water and then second precision cleaning with salt solution is better than that of first precision cleaning with salt solution and then second precision cleaning with water.

[0130] Test Example 2 In this test example, the DNA residual amount and protein content of the modified decellularized matrix prepared in the above examples were detected.

[0131] The detection method of the DNA residual amount is as follows: after the modified decellularized matrix is digested and dissolved with proteinase K, the DNA residual content in the material is determined according to YYT0606.25-2014 Tissue Engineering Medical Products Part 25: Determination of DNA Residual Amount of Animal-derived Biomaterials: Fluorescent Staining Method. The detection method of the protein content is as follows: it is determined according to the 2020 edition of the People's Republic of China Pharmacopoeia General Part 0704 "Nitrogen Determination Method Third Law Nitrogen Instrument Method".

[0132] The test results are shown in Table 5.

[0133] Table 5

[0134] The results show that the modified decellularized matrix material prepared by the application has a lower DNA residual amount and a higher protein content.

[0135] Test Example 3 In this test example, the serum immunoglobulin and complement component of the modified decellularized matrix prepared in the above examples were determined according to GB / T 16886.20-2015 "Medical Devices Biological Evaluation Part 20: Principles and Methods of Immunotoxicological Tests for Medical Devices" and YY / T 1465.2-2016 "Evaluation Methods for Immunogenicity of Medical Devices Part 2 Determination of Serum Immunoglobulin and Complement Component ELISA Method", and the specific method is as follows: Balb / c mice were selected and grouped according to the examples, a negative control group and a positive control group, 10 mice in each group, the negative control group (sham operation group) was operated, but no material was implanted; the positive control group was subcutaneously injected with a mixed emulsion of BSA (bovine serum albumin) and CFA (complete Freund's adjuvant) one week after the operation, the final concentration of BSA was 1.67 mg / mL, 0.12 mL per mouse, and the injection was performed once a week for a total of 3 times; the mice of the examples were subcutaneously implanted with a piece of sample 1 (1.5 cm 2 ) on the left side of the back of the mice. After 30 days, the peripheral blood of the mice was collected, and after being placed at 4℃ for 2 hours, it was centrifuged at 1000 g for 20 min, and the upper serum was collected. The content of immunoglobulin IgM and IgG was determined by an ELISA kit.

[0136] The test results are shown in Table 6.

[0137] Table 6

[0138] Note: The asterisk indicates that P < 0.01 compared with the negative control group.

[0139] The results show that there is a very significant difference in the serum IgM and IgG concentrations between the positive control group and the negative control group, and there is no significant difference in the serum IgM and IgG concentrations between all the example groups and the negative control group. Therefore, the acellular matrix material prepared in the application has no obvious immune response after being implanted into mice, and the immunological risk and biological safety risk are very low.

[0140] Test Example 4 This test example is to characterize the modified acellular matrix prepared in the above example 6 by scanning electron microscopy. The electron microscopy results are shown in Figure 1 The electron microscopy results show that the modified acellular matrix of the application has a higher porosity and more complete pores, most of the pore diameters are 100-200 microns, which is suitable for cell growth and migration, and has a good effect on promoting cell proliferation.

[0141] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the application, but not to limit them; although the application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the application.

Claims

1. A modified decellularized matrix, characterized in that, The preparation method comprises the following steps: taking aquatic animal tissue as raw material, and preparing after decellularization treatment and crosslinking treatment; the decellularization treatment comprises sequentially performing microcrystalline drying, immobilization and swelling treatment.

2. The modified decellularized matrix of claim 1, wherein, The microcrystalline drying is freeze-drying after quick freezing of the material at-40 ℃ or below; the immobilization is crosslinking of the material in a fixing agent; and the swelling treatment is sequentially washing the material in a surfactant and a swelling agent; Preferably, the time for washing in the surfactant and / or the swelling agent is 1-30 h, preferably 1-10 h; Preferably, the concentration of the fixing agent is 0.01%-2% (w / w), preferably 0.01%-0.5% (w / w), and more preferably 0.01%-0.2% (w / w); Preferably, the fixing agent comprises at least one of acrylate, polyisocyanate, polyol, polyamine, aziridine, epoxy compound, aldehyde, organic peroxide, carbodiimide and organosilicon crosslinking agent; Preferably, the time for immobilization is 0.1-6 h, preferably 0.1-4 h.

3. The modified decellularized matrix of claim 2, wherein, The surfactant comprises at least one of sodium cholate, sodium deoxycholate, sodium dodecyl sulfate, Triton X-100, Triton X-114, Tween 20 and octyl glucoside.

4. The modified decellularized matrix of claim 2, wherein, The swelling agent comprises at least one of salt, base, acid and polymer; Preferably, the acid in the swelling agent comprises, but is not limited to, hydrochloric acid, formic acid, acetic acid, nitric acid, phosphoric acid, carbonic acid, hypochlorous acid, phthalic acid, isophthalic acid, oxalic acid, malic acid and citric acid; Preferably, the base in the swelling agent comprises, but is not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, aluminum hydroxide, magnesium hydroxide, barium hydroxide, sodium carbonate and sodium bicarbonate; Preferably, the salt in the swelling agent comprises, but is not limited to, NaCl, KCl, CaCl2, ZnCl2, MgCl2 and Ca2(NO3); Preferably, the polymer in the swelling agent comprises, but is not limited to, poly-p-dioxanone; Preferably, the concentration of the swelling agent is 0.05-0.5% (w / w); Preferably, the temperature for the swelling treatment is 2-8 ℃.

5. The modified decellularized matrix of claim 1, wherein, The preparation method further comprises: performing primary precision cleaning after the decellularization treatment and secondary precision cleaning after the crosslinking treatment.

6. The modified decellularized matrix of claim 5, wherein, The primary precision cleaning and / or the secondary precision cleaning is performed by using a salt solution or water; Preferably, the salt solution comprises, but is not limited to, phosphate buffer, borate buffer, bicarbonate buffer system, glycine-sodium hydroxide buffer, potassium salt solution, sodium salt solution and calcium salt solution; Preferably, the pH of the salt solution is 6.5-7.5; Preferably, the concentration of the salt solution is 0.1-0.3 mol / L; Preferably, the primary precision cleaning is performed by using water, and the secondary precision cleaning is performed by using a salt solution.

7. The modified decellularized matrix of any one of claims 1-6, wherein, The aquatic animal tissue is connective tissue of aquatic animals; Preferably, the aquatic animals comprise fish, including, but not limited to, salmon, cod, carp, tilapia, grass carp, sea bass, tuna, black fish, sturgeon, bighead carp, blue carp, silver carp or crucian carp. Preferably, the aquatic animal tissue is swim bladder or skin of fish. Preferably, the density of the aquatic animal tissue 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 .

8. The modified decellularized matrix of any one of claims 1-7, wherein, After the cross-linking treatment or the secondary precision cleaning, a drying treatment is performed, preferably, the drying treatment is a freeze-drying treatment.

9. The modified decellularized matrix of any one of claims 1-8, wherein, The cross-linking agent in the cross-linking treatment includes at least one of acrylate, polyisocyanate, polyol, polyamine, aziridine, epoxy compound, aldehyde, organic peroxide, carbodiimide, and organosilicon cross-linking agent; Preferably, the concentration of the cross-linking agent in the cross-linking treatment is 0.1% to 10.0% (w / w), preferably 0.5% to 9.5% (w / w); Preferably, the time of the cross-linking treatment is 0.1 to 96 h, preferably 4 to 72 h, and more preferably 24 to 72 h; Preferably, the temperature of the cross-linking treatment is 20℃ to 25℃; Optionally, the cross-linking treatment is performed in a buffer system; Optionally, the buffer system includes, but is not limited to, phosphate buffer, tris buffer, barbiturate buffer, borate buffer, bicarbonate buffer system, glycine-sodium hydroxide buffer; Optionally, the pH of the buffer system is 7.5 to 10.5, preferably 8.5 to 9.5; Optionally, the concentration of the buffer system is 0.1 to 1.0 mol / L, preferably 0.1 to 0.5 mol / L.

10. The modified decellularized matrix of any one of claims 1-9, wherein, The tensile strength of the decellularized matrix is 34 MPa or more; and / or, the porosity is 91% or more; and / or, the thickness is 0.88 to 1.22 mm; and / or, the cell proliferation rate is 92% or more; and / or, the DNA residue in the modified decellularized matrix is less than 24.2 ng / mg; and / or, the protein content of the modified decellularized matrix is 96% or more.

Images