A modified decellularized matrix

Abstract

The present application relates to the technical field of medical biomaterials, and particularly relates to a modified decellularized matrix. The present application can effectively reduce the phenomenon of low porosity after cross-linking by sequentially combining microcrystalline drying, immobilization and swelling treatment in the decellularization process, and can reduce the mechanical loss in the decellularization process, and can obtain high porosity while taking into account high mechanical properties. 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 decellularized matrix of the present application is thin and tough, and has high comfort after implantation, and the tensile strength is increased to more than 3 times that of the original material, and has extremely high application value in the medical field which requires mechanical properties, porosity, thickness and comfort.

Description

Technical Field

[0001] This invention relates to the field of medical biomaterials technology, and in particular to a modified decellularized matrix. Background Technology

[0002] Decellularized matrix refers to matrix materials that retain their bioactive components while removing cells from the extracellular matrix through a decellularization process, based on its composition and structure. It is a highly coordinated organic whole containing various signaling molecules, capable of inducing and promoting cell adhesion, proliferation, differentiation, and tissue formation, forming the basis of tissue repair. This material is the closest biological wound repair product to the physiological structure and function of body tissues. However, due to structural damage during the preparation process and the inherent structural characteristics of some materials, the mechanical properties of decellularized matrix materials are generally low, and they degrade rapidly after implantation, limiting their application in fields such as sports medicine. Modified decellularized matrix involves further processing of decellularized matrix materials, resulting in materials with higher advantages in mechanical properties and degradation time. However, modified materials are prone to reduced porosity. Studies have shown that porosity and cell proliferation rate are positively correlated within a certain range; therefore, modified decellularized matrix materials with high porosity have higher market value.

[0003] Due to the influence of their growth environment, aquatic animals exhibit significant diversity in the physicochemical properties of their extracellular matrix, with some animal tissues showing marked superior structural stability and mechanical properties compared to terrestrial animals. Patent CN118340945A discloses a method for preparing a decellularized matrix. This method uses gel polymers to adjust and modify the porosity of the decellularized matrix; some samples have a porosity of approximately 80%, indicating room for improvement. Furthermore, the modification process is unstable and prone to inconsistent cross-linking. Patent CN116236622A discloses a functionally graded biomimetic rotator cuff patch and its preparation method. This method uses aquatic-derived decellularized matrix material as the substrate, promoting the full impregnation of adhesive components into the substrate under room-temperature vacuum pressing conditions. However, this vacuum pressing method significantly damages the pore structure, hindering cell proliferation. Moreover, this method relies on physical adhesion to enhance mechanical properties, resulting in a thicker material with poor clinical comfort, and the adhesion carries a certain risk of detachment. Patent CN113476667A discloses a decellularized fish skin dermal matrix scaffold and its preparation method. However, the preparation method does not effectively protect the material structure, and there is still considerable room for improvement in its mechanical properties. Furthermore, the cross-linked decellularized matrix material prepared by this method exhibits certain cytotoxicity. In addition, the cross-linked material is prone to increased density and decreased porosity, which affects cell proliferation after implantation. Summary of the Invention

[0004] First, in order to solve the technical problem of the difficulty in achieving both high porosity and high mechanical properties, the present invention provides a modified decellularized matrix, the preparation method of which includes: using aquatic animal tissue as raw material, and obtaining it after decellularization and cross-linking treatment; the decellularization treatment includes microcrystal drying, immobilization and swelling treatment in sequence.

[0005] This invention effectively reduces the low porosity after cross-linking by sequentially combining microcrystal drying, immobilization, and swelling treatments during the decellularization process, while also reducing mechanical losses during decellularization, thus achieving high porosity while maintaining high mechanical properties.

[0006] In practice, the microcrystalline drying process involves freezing the material at a temperature below -40°C and then freeze-drying it.

[0007] Preferably, the microcrystalline drying involves quick-freezing the material at -40°C or below and then freeze-drying it until the moisture content is less than 15% (w / w).

[0008] During the microcrystal drying process, intracellular water rapidly condenses to form ice crystals, which are then freeze-dried. During the freeze-drying process, the ice crystals sublimate, resulting in a thickened and porous material.

[0009] In the specific implementation process, the immobilization is to place the material in a fixative (crosslinking agent) for crosslinking.

[0010] By using fixatives to initially fix the fibrous tissue of the material, the bonding between fibrin proteins can be improved, thereby effectively reducing the mechanical loss of the material during decellularization.

[0011] In the specific implementation process, the swelling treatment involves washing the material sequentially in a surfactant and a swelling reagent.

[0012] In specific implementation, 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).

[0013] In specific implementation, the fixative includes at least one of acrylates, polyisocyanates, polyols, polyamines, aziridines, epoxy compounds, aldehydes, organic peroxides, carbodiimides, and organosilicon crosslinking agents.

[0014] In the specific implementation process, the immobilization time is 0.1~6 h, 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, Tween20, and octyl glucoside; the swelling agent includes at least one of salt, alkali, acid, and polymer.

[0016] In specific implementation, the swelling reagents include, but are not limited to, salts, alkalis, acids, and polymers.

[0017] Preferably, the acids in the swelling reagent include, but are 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.

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

[0019] Preferably, the salts in the swelling reagent include, but are not limited to, NaCl, KCl, CaCl2, ZnCl2, MgCl2, and Ca2(NO3).

[0020] Preferably, the polymer in the swelling agent includes, but is not limited to, polydioxanone.

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

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

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

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

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

[0026] Reagent residues can be thoroughly removed by at least two precise cleanings, improving the biosafety of the material and further enhancing cell proliferation rate.

[0027] In specific implementation, 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, and calcium salt solution.

[0028] Preferably, the pH of the salt solution is 6.5 to 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 rinsing, and the second precision cleaning uses salt solution rinsing.

[0031] Performing two precision cleanings in the above order yields better results.

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

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

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

[0035] Preferably, the density of the aquatic animal tissue is 0.05–0.80 g / cm³. 3 Preferably, the concentration is 0.10–0.70 g / cm³. 3 More preferably 0.20–0.60 g / cm³ 3 .

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

[0037] Preferably, the pretreatment includes washing the raw material after removing residual meat, skin, and fat impurities.

[0038] In the specific implementation process, biological enzymes, acids, and other methods can also be introduced during the pretreatment process.

[0039] In the specific implementation process, the biological enzymes include, but are not limited to, cathepsins, papain, pepsin, trypsin, subtilisin, serine proteases, chymotrypsin, aspartate dehydrogenase, thiol proteases, triacylglycerol acyl hydrolases, etc.

[0040] In the specific implementation process, the acids used in the pretreatment process include, but are 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 specific implementation, the concentration of the biological enzyme is 0.01% to 0.1% (w / w), preferably 0.001% to 0.05% (w / w).

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

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

[0044] Preferably, the drying process is freeze-drying.

[0045] In the specific implementation process, the crosslinking agent in the crosslinking treatment includes at least one of acrylates, polyisocyanates, polyols, polyamines, aziridines, epoxy compounds, aldehydes, organic peroxides, carbodiimides, and organosilicon crosslinking agents.

[0046] In the specific implementation process, the concentration of the crosslinking agent in the crosslinking treatment is 0.1% to 10.0% (w / w), preferably 0.5% to 9.5% (w / w).

[0047] In the specific implementation process, the crosslinking treatment time is 0.1 to 96 hours, preferably 4 to 72 hours, and more preferably 24 to 72 hours.

[0048] In the specific implementation process, the temperature of the crosslinking treatment is 20℃~25℃.

[0049] Optionally, the crosslinking process is carried out in a buffer system.

[0050] Optionally, the buffer system includes, but is not limited to, phosphate buffer, tris(hydroxymethyl)aminomethane buffer, barbiturate buffer, borate buffer, bicarbonate buffer system, glycine-sodium hydroxide buffer, etc.

[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 higher (preferably 35, 36, or 37 MPa or higher); and / or, the porosity is 91% or higher (preferably 92%, 93%, 94%, 95%, 96%, or 96.7% or higher); and / or, the thickness is 0.88 to 1.22 mm; and / or, the cell proliferation rate is 92% or higher (preferably 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, or 105% or higher).

[0054] Preferably, the residual DNA 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 ​​above 96% (more preferably above 97% or above, or above 97.86%).

[0056] No significant immune response was observed after implantation of the modified decellularized matrix of the present invention.

[0057] Compared with the prior art, the beneficial effects of the present invention are as follows: The modified decellularized matrix of this invention significantly improves upon the shortcomings of insufficient mechanical properties per unit thickness of decellularized matrix materials, achieving both high porosity and high mechanical performance. The resulting decellularized matrix is ​​thin yet tough, providing high comfort after implantation. Its tensile strength is more than three times that of the original material, and the high porosity facilitates cell migration and ingrowth. This modified decellularized matrix has extremely high application value in medical fields where mechanical properties, porosity, thickness, and comfort are critical (e.g., rotator cuff patches). Attached Figure Description

[0058] Figure 1 This is an electron micrograph of the modified decellularized matrix of Example 6 of the present invention. Detailed Implementation

[0059] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In the embodiments provided in this specification, where specific techniques or conditions are not specified, they are performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0060] As an example, this embodiment provides a modified decellularized matrix, the preparation method of which includes: using aquatic animal tissue as raw material, and obtaining it after decellularization and cross-linking treatment; the decellularization treatment includes microcrystal drying, immobilization and swelling treatment in sequence.

[0061] As an example, the microcrystalline drying is achieved by freeze-drying the material after quick-freezing it at a temperature below -40°C.

[0062] As an example, the microcrystalline drying involves placing the material at -40°C or below for quick freezing and then freeze-drying it until the moisture content is less than 15% (w / w).

[0063] As an example, the immobilization involves placing the material in a fixative (crosslinking agent) for crosslinking.

[0064] As an example, the swelling treatment involves washing the material sequentially in a surfactant and a swelling agent.

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

[0066] As an example, the fixative includes at least one of acrylates, polyisocyanates, polyols, polyamines, aziridines, epoxy compounds, aldehydes, organic peroxides, carbodiimides, and organosilicon crosslinking agents.

[0067] As an example, the immobilization time is 0.1 to 6 hours, preferably 0.1 to 4 hours.

[0068] As an example, the surfactant includes at least one of sodium cholate, sodium deoxycholate, sodium dodecyl sulfate, Triton X-100, Triton X-114, Tween 20, and octyl glucoside; the swelling agent includes at least one of salt, alkali, acid, and polymer.

[0069] As an example, the swelling agent includes, but is not limited to, salts, alkalis, acids, and polymers.

[0070] As an example, the acids in the swelling reagent include, but are 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 example, the alkali in the swelling reagent 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 example, the salts in the swelling reagent include, but are not limited to, NaCl, KCl, CaCl2, ZnCl2, MgCl2, and Ca2(NO3).

[0073] As an example, the polymer in the swelling agent includes, but is not limited to, polydioxanone.

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

[0075] As an example, the washing time in surfactant and / or swelling agent is 1 to 30 hours, preferably 1 to 10 hours.

[0076] As an example, the swelling treatment temperature is 2°C to 8°C.

[0077] As an example, the preparation method further includes: performing a precision cleaning after the decellularization treatment, and performing a second precision cleaning after the crosslinking treatment.

[0078] As one embodiment, the primary and / or secondary precision cleaning uses a salt solution or water for cleaning.

[0079] As an example, 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, and calcium salt solution.

[0080] As an example, the pH of the salt solution is 6.5 to 7.5.

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

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

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

[0084] As an example, the aquatic animals include fish, including but not limited to salmon, cod, carp, tilapia, grass carp, bass, tuna, snakehead, sturgeon, bighead carp, grass carp, silver carp, crucian carp, etc.

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

[0086] As an example, the density of the aquatic animal tissue is 0.05–0.80 g / cm³. 3 Preferably, the concentration is 0.10–0.70 g / cm³. 3 More preferably 0.20–0.60 g / cm³ 3 .

[0087] As an example, a pretreatment is performed before the decellularization process.

[0088] As one embodiment, the pretreatment includes washing the raw material after removing residual meat, skin, and fat impurities.

[0089] As an example, biological enzymes, acids, or other methods can also be introduced during the pretreatment process.

[0090] As an example, the biological enzymes include, but are not limited to, cathepsins, papain, pepsin, trypsin, subtilisin, serine proteases, chymotrypsin, aspartate dehydrogenase, thiol proteases, triacylglycerol acyl hydrolases, etc.

[0091] As an example, the acids used in the pretreatment process include, but are 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 example, the concentration of the biological enzyme is 0.01% to 0.1% (w / w), preferably 0.001% to 0.05% (w / w).

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

[0094] As an example, the product undergoes drying after crosslinking treatment or secondary precision cleaning.

[0095] As an example, the drying process is freeze-drying.

[0096] As an example, the crosslinking agent in the crosslinking treatment includes at least one of acrylates, polyisocyanates, polyols, polyamines, aziridines, epoxy compounds, aldehydes, organic peroxides, carbodiimides, and organosilicon crosslinking agents.

[0097] As an example, the concentration of the crosslinking agent in the crosslinking treatment is 0.1% to 10.0% (w / w), preferably 0.5% to 9.5% (w / w).

[0098] As an example, the crosslinking treatment time is 0.1 to 96 h, preferably 4 to 72 h, and more preferably 24 to 72 h.

[0099] As an example, the crosslinking treatment temperature is 20°C to 25°C.

[0100] As an example, the crosslinking process is carried out in a buffer system.

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

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

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

[0104] As an example, the tensile strength of the decellularized matrix is ​​34 MPa or more (preferably 35, 36, or 37 MPa or more); and / or, the porosity is 91% or more (preferably 92%, 93%, 94%, 95%, 96%, or 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%, or 105% or more).

[0105] As an example, the residual DNA 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 example, the protein content of the modified decellularized matrix is ​​96% or more (more preferably 97% or more, 97.86% or more).

[0107] The invention is illustrated below with more specific embodiments.

[0108] Examples 1-10 This embodiment uses aquatic animal tissue—fish skin (density 0.40 g / cm³). 3 For example, a modified decellularized matrix is ​​provided, and its preparation method is as follows: (1) Removal of impurities: Use stainless steel spoons, ultrasonic scalpels and other tools to remove residual meat, skin, fat and other impurities. Select materials with a thickness of 0.30 mm, soak them in 0.02% (w / w) pepsin for 1 h, and wash them with purified water before use. (2) Decellularization: (a) Microcrystal drying: The material is rapidly frozen at -40°C and then freeze-dried until the moisture content is less than 15% (w / w) to obtain a loose and porous material; (b) Immobilization: After the material is freeze-dried, it is immobilized in a low-concentration fixative (crosslinking agent); (c) Swelling treatment: The material is washed in surfactant and swelling reagent at 6°C in sequence, and the specific conditions are shown in Table 1.

[0109] (3) Cleaning 1: Use purified water to clean the decellularized matrix material of reagent residues.

[0110] (4) Crosslinking: Crosslinking is performed using a crosslinking agent at a temperature of 20°C, and the specific conditions are shown in Table 1.

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

[0112] (6) Drying: After draining the material, freeze-dry it to obtain the modified decellularized matrix.

[0113] Comparative Examples 1-2 This comparative example provides a modified decellularized matrix. The preparation methods of Comparative Examples 1 and 2 differ from those of Examples 1 and 2, respectively, only in that: Step (2) The decellularization process involves immobilization, followed by microcrystal drying and finally swelling treatment.

[0114] Comparative Examples 3-4 This comparative example provides a modified decellularized matrix. The preparation methods of comparative examples 3 and 4 differ from those of examples 1 and 2, respectively, only in that: Step (2) Decellularization process: After drying the microcrystals, they are first swollen and then immobilized.

[0115] Comparative Examples 5-6 This comparative example provides a modified decellularized matrix. The preparation methods of comparative examples 5 and 6 differ from those of examples 1 and 2, respectively, only in that: Step (2) The decellularization process omits microcrystal drying.

[0116] Comparative Examples 7-8 This comparative example provides a modified decellularized matrix. The preparation methods of comparative examples 7 and 8 differ from those of examples 1 and 2, respectively, in that: Step (2) The decellularization process omits immobilization.

[0117] Comparative Examples 9-10 This comparative example provides a modified decellularized matrix. The preparation methods of comparative examples 9 and 10 differ from those of examples 1 and 2, respectively, only in that: Step (2) The swelling treatment is omitted in the decellularization process.

[0118] Table 1

[0119] Experimental Example 1 This experimental example tests the performance of the modified decellularized matrices prepared in the above examples and comparative examples. The tensile strength was determined according to YY / T0606.5-2007 "Tissue-engineered Medical Products Part 5: Performance and Testing of Matrices and Scaffolds" 5.3.3 Tensile Test and GB / T 1040.3 "Plastics - Tensile Properties Test". The test specimen size was 54 × 10 mm, and the gauge length was 36 mm.

[0120] The porosity test method is as follows: Immerse the modified decellularized matrix in anhydrous ethanol containing V1 volume, remove the gas inside the material using a negative pressure suction method, and record the solution volume V2. Remove the material and record the solution volume V3. Porosity = [(V1-V3) / (V2-V3)] × 100%.

[0121] The thickness test method is as follows: randomly select 5 points near the center point and four corners of the product for thickness measurement, record the thickness of each test point, and calculate the average thickness.

[0122] Simultaneously, L929 cells were used to test the cell proliferation rate of different materials according to the MTT assay recommended in GB / T 16886.5-2017 "Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Tests". The test results are shown in Table 2.

[0123] Table 2

[0124] The results showed that, compared with the control group, sequentially combining microcrystal drying, immobilization and swelling treatments during the decellularization process could effectively reduce the low porosity after cross-linking, while also reducing mechanical losses during the decellularization process. This resulted in the modified decellularized matrix possessing both high porosity and high cell proliferation rate, as well as high mechanical properties.

[0125] Examples 11-12 This embodiment provides a modified decellularized matrix, the preparation method of which differs from that of Example 2 only in that: A first precision cleaning was performed before crosslinking in step (3), and a second precision cleaning was performed after crosslinking in step (3); the specific conditions for the two precision cleanings are shown in Table 3. The cell proliferation rate of the prepared modified decellularized matrix was detected according to the detection method of the above experimental example, and the results are shown in Table 3.

[0126] Table 3

[0127] Examples 13-14 This embodiment provides a modified decellularized matrix, the preparation method of which differs from that of Example 2 only in that: A precision cleaning was performed before crosslinking in step (3), and a second precision cleaning was performed after crosslinking in step (3); the specific conditions for the two precision cleanings are shown in Table 4. The cell proliferation rate of the prepared modified decellularized matrix was detected according to the detection method of the above experimental example, and the results are shown in Table 4.

[0128] Table 4

[0129] It is evident that performing a first precision cleaning with water followed by a second precision cleaning with a salt solution is more effective than performing a first precision cleaning with a salt solution followed by a second precision cleaning with water.

[0130] Experimental Example 2 This experimental example tested the residual DNA and protein content of the modified decellularized matrix prepared in the above examples.

[0131] The method for detecting residual DNA is as follows: After the modified decellularized matrix is ​​digested and dissolved using 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 Biological Materials: Fluorescent Staining Method. The method for detecting protein content is as follows: The protein content is determined according to the 2020 edition of the Pharmacopoeia of the People's Republic of China, Part IV, General Chapter 0704, "Nitrogen Determination Method, Third Method for Nitrogen Analyzer".

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

[0133] Table 5

[0134] The results show that the modified decellularized matrix material prepared in this invention has low DNA residue and high protein content.

[0135] Experimental Example 3 In this experimental example, the serum immunoglobulins and complement components of the modified decellularized matrix prepared in the above examples were determined according to GB / T 16886.20-2015 "Biological Evaluation of Medical Devices Part 20: Principles and Methods for Immunotoxicological Testing of Medical Devices" and YY / T 1465.2-2016 "Methods for Immunogenicity Evaluation of Medical Devices Part 2: Determination of Serum Immunoglobulins and Complement Components by ELISA". The specific methods are as follows: Balb / c mice were selected and grouped into three groups (n=10 per group) according to the example, negative control group, and positive control group. The negative control group (sham-operated group) underwent surgery but no material was implanted. The positive control group received subcutaneous injections of an equal volume of a mixed emulsion of BSA (bovine serum albumin) and CFA (complete Freund's adjuvant) starting one week post-surgery. The final BSA concentration was 1.67 mg / mL, with 0.12 mL injected per mouse, once a week for a total of three weeks. In the example mice, a 1.5 cm sample was implanted subcutaneously on the left side of the mouse's back. 2 Thirty days later, peripheral blood was collected from mice, incubated at 4°C for 2 hours, centrifuged at 1000 g for 20 min, and the supernatant serum was collected. The levels of immunoglobulins IgM and IgG were measured using an ELISA kit.

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

[0137] Table 6

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

[0139] The results showed that there were highly significant differences in serum IgM and IgG concentrations between the positive control group and the negative control group. There were no significant differences in serum IgM and IgG concentrations between the positive control group and the negative control group in all the example groups. Therefore, the decellularized matrix material prepared in this invention did not cause a significant immune response after being implanted into mice, and the immune risk and biosafety risk were extremely low.

[0140] Test Example 4 This experimental example demonstrates the scanning electron microscopy (SEM) characterization of the modified decellularized matrix prepared in Example 6 above. The SEM results are as follows: Figure 1 As shown in the figure, electron microscopy results show that the modified decellularized matrix of the present invention has high porosity and relatively intact pores, with most pores having a diameter of 100-200 micrometers, 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 embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A modified decellularized matrix, characterized in that, The preparation method includes: using aquatic animal tissue as raw material, and obtaining it after decellularization and cross-linking treatment; the decellularization treatment includes sequentially performing microcrystal drying, immobilization and swelling treatment; the microcrystal drying is to freeze-dry the material after quick-freezing at -40°C or below; the immobilization is to cross-link the material in a fixative, and to initially fix the fibrous tissue of the material by using a fixative; the swelling treatment is to wash the material sequentially in a surfactant and a swelling reagent.

2. The modified decellularized matrix according to claim 1, characterized in that, The washing time in surfactants and / or swelling agents is 1 to 30 hours.

3. The modified decellularized matrix according to claim 2, characterized in that, The washing time in surfactants and / or swelling agents is 1 to 10 hours.

4. The modified decellularized matrix according to claim 1, characterized in that, The concentration of the fixative is 0.01%~2% (w / w).

5. The modified decellularized matrix according to claim 4, characterized in that, The concentration of the fixative is 0.01%~0.5% (w / w).

6. The modified decellularized matrix according to claim 4, characterized in that, The concentration of the fixative is 0.01%~0.2% (w / w).

7. The modified decellularized matrix according to claim 1, characterized in that, The fixative includes at least one of acrylates, polyisocyanates, polyols, polyamines, aziridines, epoxy compounds, aldehydes, organic peroxides, carbodiimides, and organosilicon crosslinking agents.

8. The modified decellularized matrix according to claim 1, characterized in that, The immobilization time is 0.1 to 6 hours.

9. The modified decellularized matrix according to claim 8, characterized in that, The immobilization time is 0.1 to 4 hours.

10. The modified decellularized matrix according to claim 1, characterized in that, The surfactant includes at least one of sodium cholate, sodium deoxycholate, sodium dodecyl sulfate, Triton X-100, Triton X-114, Tween20, and octyl glucoside.

11. The modified decellularized matrix according to claim 1, characterized in that, The swelling agent includes at least one of salt, alkali, acid, and polymer.

12. The modified decellularized matrix according to claim 11, characterized in that, The acids in the swelling reagent include hydrochloric acid, formic acid, acetic acid, nitric acid, phosphoric acid, carbonic acid, hypochlorous acid, phthalic acid, isophthalic acid, oxalic acid, malic acid, or citric acid.

13. The modified decellularized matrix according to claim 11, characterized in that, The alkali in the swelling reagent includes sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, aluminum hydroxide, magnesium hydroxide, barium hydroxide, sodium carbonate, or sodium bicarbonate.

14. The modified decellularized matrix according to claim 11, characterized in that, The salts in the swelling reagent include NaCl, KCl, CaCl2, ZnCl2, MgCl2, and Ca(NO3)2.

15. The modified decellularized matrix according to claim 11, characterized in that, The polymer in the swelling agent includes polydioxanone.

16. The modified decellularized matrix according to claim 11, characterized in that, The concentration of the swelling agent is 0.05~0.5% (w / w).

17. The modified decellularized matrix according to claim 11, characterized in that, The swelling treatment temperature is 2℃~8℃.

18. The modified decellularized matrix according to claim 1, characterized in that, The preparation method further includes: performing a precision cleaning after the decellularization treatment, and performing a second precision cleaning after the crosslinking treatment.

19. The modified decellularized matrix according to claim 18, characterized in that, The primary and / or secondary precision cleaning uses a salt solution or water.

20. The modified decellularized matrix according to claim 19, characterized in that, The salt solution includes phosphate buffer, borate buffer, bicarbonate buffer system, glycine-sodium hydroxide buffer, potassium salt solution, sodium salt solution or calcium salt solution.

21. The modified decellularized matrix according to claim 19, characterized in that, The pH of the salt solution is 6.5 to 7.

5.

22. The modified decellularized matrix according to claim 19, characterized in that, The concentration of the salt solution is 0.1~0.3 mol / L.

23. The modified decellularized matrix according to claim 19, characterized in that, The first precision cleaning uses water rinsing, and the second precision cleaning uses salt solution rinsing.

24. The modified decellularized matrix according to any one of claims 1 to 23, characterized in that, The aquatic animal tissue is the connective tissue of aquatic animals.

25. The modified decellularized matrix according to claim 24, characterized in that, The aquatic animals include fish, including salmon, cod, carp, tilapia, grass carp, bass, tuna, snakehead, sturgeon, bighead carp, grass carp, silver carp, or crucian carp.

26. The modified decellularized matrix according to claim 24, characterized in that, The aquatic animal tissue is the swim bladder or skin of a fish.

27. The modified decellularized matrix according to claim 24, characterized in that, The density of the aquatic animal tissue is 0.05–0.80 g / cm³. 3 .

28. The modified decellularized matrix according to claim 27, characterized in that, The density of the aquatic animal tissue is 0.10–0.70 g / cm³. 3 .

29. The modified decellularized matrix according to claim 27, characterized in that, The density of the aquatic animal tissue is 0.20–0.60 g / cm³. 3 .

30. The modified decellularized matrix according to any one of claims 1 to 23, characterized in that, After cross-linking treatment or secondary precision cleaning, the product is dried.

31. The modified decellularized matrix according to any one of claims 30, characterized in that, The drying process is freeze-drying.

32. The modified decellularized matrix according to any one of claims 1 to 23, characterized in that, The crosslinking agent in the crosslinking treatment includes at least one of acrylates, polyisocyanates, polyols, polyamines, aziridines, epoxy compounds, aldehydes, organic peroxides, carbodiimides, and organosilicon crosslinking agents.

33. The modified decellularized matrix according to claim 32, characterized in that, The concentration of the crosslinking agent in the crosslinking treatment is 0.1% to 10.0% (w / w).

34. The modified decellularized matrix according to claim 32, characterized in that, The concentration of the crosslinking agent in the crosslinking treatment is 0.5% to 9.5% (w / w).

35. The modified decellularized matrix according to claim 32, characterized in that, The crosslinking treatment time is 0.1 to 96 h.

36. The modified decellularized matrix according to claim 35, characterized in that, The crosslinking treatment time is 4~72h.

37. The modified decellularized matrix according to claim 35, characterized in that, The crosslinking treatment time is 24–72 h.

38. The modified decellularized matrix according to claim 32, characterized in that, The crosslinking treatment temperature is 20℃~25℃.

39. The modified decellularized matrix according to claim 32, characterized in that, The crosslinking process is carried out in a buffer system.

40. The modified decellularized matrix according to claim 39, characterized in that, The buffer system includes phosphate buffer, tris(hydroxymethyl)aminomethane buffer, barbiturate buffer, borate buffer, bicarbonate buffer system, or glycine-sodium hydroxide buffer.

41. The modified decellularized matrix according to claim 40, characterized in that, The pH of the buffer system is 7.5–10.

5.

42. The modified decellularized matrix according to claim 40, characterized in that, The pH of the buffer system is 8.5 to 9.

5.

43. The modified decellularized matrix according to claim 40, characterized in that, The concentration of the buffer system is 0.1–1.0 mol / L.

44. The modified decellularized matrix according to claim 40, characterized in that, The concentration of the buffer system is 0.1–0.5 mol / L.

45. The modified decellularized matrix according to any one of claims 1-23, 25-29, 31, and 33-44, characterized in that, The decellularized matrix has a tensile strength of ≥34 MPa; and / or a porosity of ≥91%; and / or a thickness of 0.88~1.22 mm; and / or a cell proliferation rate of ≥92%; and / or a DNA residue of ≥24.2 ng / mg in the modified decellularized matrix; and / or a protein content of ≥96% (w / w) in the modified decellularized matrix.

46. ​​The modified decellularized matrix according to claim 24, characterized in that, The decellularized matrix has a tensile strength of ≥34 MPa; and / or a porosity of ≥91%; and / or a thickness of 0.88~1.22 mm; and / or a cell proliferation rate of ≥92%; and / or a DNA residue of ≥24.2 ng / mg in the modified decellularized matrix; and / or a protein content of ≥96% (w / w) in the modified decellularized matrix.

47. The modified decellularized matrix according to claim 30, characterized in that, The decellularized matrix has a tensile strength of ≥34 MPa; and / or a porosity of ≥91%; and / or a thickness of 0.88~1.22 mm; and / or a cell proliferation rate of ≥92%; and / or a DNA residue of ≥24.2 ng / mg in the modified decellularized matrix; and / or a protein content of ≥96% (w / w) in the modified decellularized matrix.

48. The modified decellularized matrix according to claim 32, characterized in that, The decellularized matrix has a tensile strength of ≥34 MPa; and / or a porosity of ≥91%; and / or a thickness of 0.88~1.22 mm; and / or a cell proliferation rate of ≥92%; and / or a DNA residue of ≥24.2 ng / mg in the modified decellularized matrix; and / or a protein content of ≥96% (w / w) in the modified decellularized matrix.

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