Preparation method of hypoallergenic acellular matrix implant

Abstract

The invention discloses a preparation method of a hypoallergenic acellular matrix implant, and belongs to the technical field of biomedical materials. The preparation method comprises the following steps: swelling animal soft tissues in an alkaline solution for a short time, quickly freezing and crushing; placing the crushed sample in a hypertonic-hypotonic solution system, and performing efficient and mild decellularization treatment through circulating oscillation; then, endotoxin and other sensitizers are specifically removed by adopting a chromatography technology in sequence, denatured protein is washed by utilizing a salt solution, and finally, purification is performed through ultrafiltration or dialysis. According to the method, cell debris, nucleic acid and endotoxin can be deeply removed through multi-step synergy and combination of hypertonic-hypotonic circulating decellularization and chromatographic purification, the immunogenicity of an implant is remarkably reduced, and the natural three-dimensional structure and biological activity of an extracellular matrix are reserved to the maximum extent. The implant prepared by the method has the characteristics of low DNA residual quantity, extremely low endotoxin content and excellent biocompatibility, and is suitable for soft tissue repair and regeneration.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a method for preparing a hypoallergenic decellularized matrix implant. Background Technology

[0002] With the rapid development of tissue engineering and regenerative medicine, decellularized matrix, as an ideal biological scaffold material, has shown great potential in clinical repair applications. These materials are derived from allogeneic or xenogeneic biological tissues (such as skin, submucosa of the small intestine, pericardium, dermis, etc.), and their cellular components are removed through physical, chemical, and biochemical methods, thereby minimizing the elimination of the main factors causing host immune rejection. The retained extracellular matrix framework is rich in functional components such as collagen, elastin, and glycosaminoglycans, which not only provide three-dimensional support for the migration, proliferation, and differentiation of host cells, but also release beneficial biological signals to guide endogenous tissue regeneration, achieving a repair effect of "remodeling" rather than "foreign body encapsulation."

[0003] However, despite the relatively mature decellularization technology, decellularized matrix implants prepared using existing technologies still face a key challenge in clinical applications: the risk of residual immunogenicity or sensitization. This mainly stems from the following aspects: 1. Residual Cellular Components and Incomplete Antigen Removal: While standard decellularization processes can remove most intact cells and nuclear material, they struggle to completely eliminate all subcellular structures, cell membrane fragments, and highly immunogenic epitopes. For example, the matrix from xenogeneic tissues such as pigs and cattle may contain residual α-Gal antigens (galactose-α-1,3-galactose), and pre-existing high titers of anti-α-Gal antibodies in the human body can trigger hyperacute or acute rejection reactions. Furthermore, residual DNA fragments in mitochondria and the cell nucleus also possess immunostimulatory properties.

[0004] 2. New Allergens Introduced by Cross-linking Agents: To enhance the mechanical strength, anti-degradation capacity, and reduce immunogenicity of decellularized matrix, many existing technologies employ chemical cross-linking (such as glutaraldehyde and carbodiimide). However, cross-linking agents such as glutaraldehyde are themselves cytotoxic, and their residues or stable compounds formed with the matrix may trigger long-term chronic inflammatory responses, calcification, and hinder cell ingrowth, essentially introducing new biocompatibility issues. Some individuals may experience allergic reactions to these chemical cross-linking agents or their byproducts.

[0005] 3. Endogenous immunogenic components of the matrix: In addition to cell-derived antigens, some components of the extracellular matrix itself, such as certain types of collagen fragments, specific domains of fibronectin, and elastin degradation products, may also exhibit certain immunogenicity or inflammatory stimulation potential, especially when initial degradation occurs after material implantation.

[0006] 4. Immunogenicity alterations caused by the manufacturing process: While drastic decellularization treatments (such as treatments with strong alkalis or strong oxidants) effectively remove antigens, they may excessively damage the natural microstructure, bioactive factors, and mechanical properties of the extracellular matrix, causing it to lose its ability to guide functional regeneration. This structural damage can sometimes expose new antigenic sites or produce degradation products with pro-inflammatory properties.

[0007] Currently, to obtain products with low allergenicity, the industry typically employs more stringent decellularization steps, sequential treatment with multiple detergents / enzymes, or methods combining specific solvents to remove lipid antigens. However, these improvements often involve trade-offs: the cost of improved biosafety may be a significant decrease in the bioactivity of the material, loss of mechanical properties, increased complexity in the preparation process, and increased costs.

[0008] Therefore, there is an urgent need in the clinical and commercial sectors for an innovative preparation method that can deeply eliminate various immunogenic substances (including residual cellular antigens, xenogeneic specific antigens, and residual cross-linking agents) while maximally preserving or optimizing the three-dimensional microstructure and mechanical integrity of the extracellular matrix, thereby truly producing high-performance decellularized matrix implants with "low allergenicity" or even "non-allergenicity". This invention addresses this technological bottleneck, aiming to provide an efficient, controllable, and balanced solution. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention provides a process for decellularizing, removing antigenic components from, and modifying the structure and function of biological tissues derived from animals or humans. The aim is to prepare a decellularized matrix implant with extremely low immunogenicity, excellent biocompatibility, and good biological activity, which can be applied to various surgical scenarios such as hernia repair, dura mater repair, breast reconstruction, skin wound repair, and ligament enhancement.

[0010] The purpose of this invention is to provide a method for preparing a hypoallergenic decellularized matrix implant, comprising the following steps performed in sequence: 1. Swelling: The pretreated animal soft tissue is immersed in a low-concentration alkaline solution for a short time to swell, making the tissue loose and easier for subsequent processing.

[0011] 2. Grinding: The swollen sample is rapidly frozen and then mechanically ground to increase the specific surface area and improve the decellularization efficiency.

[0012] 3. Decellularization: The pulverized sample is placed in a circulating system consisting of a hypertonic treatment solution and a hypotonic treatment solution for processing. The hypertonic solution promotes cell rupture, while the hypotonic solution further permeates and washes away cell debris. Within the circulating device, agitation promotes full contact and exchange of substances between the solution and the material, achieving efficient and gentle decellularization.

[0013] 4. Chromatography: Chromatography is used to process the decellularized matrix to specifically adsorb and remove residual endotoxins, nucleic acid fragments, and other charged or specific molecular weight sensitizing impurities.

[0014] 5. Salt washing: Washing with a salt solution of a specific concentration to remove proteins and other hydrophobic impurities that may have been denatured during the previous treatment through salting out.

[0015] 6. Purification: Finally, small molecule impurities, salts, and unbound surfactants are removed by ultrafiltration or dialysis to obtain high-purity decellularized matrix implants.

[0016] Preferably, the alkaline solution in step 1 is a 0.01-0.2 M sodium hydroxide solution, potassium hydroxide solution, or sodium bicarbonate solution, which swells for 1-30 minutes.

[0017] Preferably, the hypertonic treatment solution in step 3 contains 0.1%-2% protease complex, 0.15%-5% nonionic surfactant and 0.5-4 M hypertonic solute.

[0018] Preferably, the hypertonic solute is at least one of sodium chloride, potassium chloride, sorbitol, and glucose.

[0019] Preferably, the protease complex is trypsin and / or lipase, and its solvent is PBS buffer or 0.9% biological agent. Salt water.

[0020] Preferably, the hypotonic treatment solution contains 10-30 mM HCl and 0.15%-5% nonionic surfactant.

[0021] Preferably, the nonionic surfactant is AEO-9 or Span-80, Triton X-100, or sodium deoxycholate. One less.

[0022] Preferably, the cyclic decellularization process in step 3 is carried out in a circulation device, specifically including: a) Hypertonic treatment stage: The pulverized sample and hypertonic treatment solution are mixed at a mass (g) to volume (ml) ratio of 1:(5-15), and circulated for 2-8 hours under oscillation conditions of 25-33℃ and 60-180 rpm; b) Separation and hypotonic treatment stage: Separate and recover the solid material after hypertonic treatment, mix it with hypotonic treatment liquid at a mass (g) to volume (ml) ratio of 1:(5-15), and circulate it for 2-8 hours under oscillation conditions of 25-33℃ and 60-180 rpm; c) Washing stage: After decellularization, the solid material is washed with PBS buffer. The mass (g) to volume (ml) ratio of the washing solution to the material is 1:10. The washing is carried out by shaking at 25-33℃ and 100-150 rpm for 20-40 minutes.

[0023] Preferably, the chromatography packing material is at least one of ion exchange chromatography medium, gel filtration medium, or desalination medium; the ion exchange chromatography medium is at least one of DEAE, Q, CM, or SP.

[0024] The beneficial effects of this invention are: 1. Significantly Reduced Immunogenicity and High Biocompatibility: This invention achieves multi-dimensional and in-depth removal of sensitizing substances through the synergistic effect of three core processes: "alkaline swelling pretreatment," "hyperosmolar-hypoosmolar cyclic decellularization," and "chromatographic purification." Alkaline swelling and cyclic oscillation treatment efficiently lyse and elute cellular components, significantly reducing DNA residue; while the dedicated chromatographic step specifically adsorbs and removes endotoxins and charged impurities that are difficult to remove with conventional washing. Implants prepared using this method have extremely low allergen residue, significantly reducing the risk of host immune rejection or inflammatory responses after implantation, demonstrating excellent biocompatibility.

[0025] 2. Effective preservation of the matrix's natural structure and active ingredients: This invention abandons the destructive methods of prolonged strong acid, strong alkali, or violent mechanical treatment in traditional processes, and adopts short-term, mild physicochemical conditions. The alkaline swelling time is short, mainly serving a loosening effect; cyclic decellularization is carried out under mild pH and concentration conditions, and dynamic oscillation replaces static soaking, ensuring decellularization efficiency while providing a good structural basis for cell attachment, proliferation, and tissue remodeling.

[0026] 3. Superior overall performance of the final product: Thanks to the gentle yet thorough preparation process described above, the resulting acellular matrix implant not only exhibits low immunogenicity but also possesses excellent mechanical properties, superior biocompatibility, and biodegradability. These characteristics enable it to demonstrate better repair efficacy and long-term safety than traditional acellular materials in various clinical applications such as hernia repair, soft tissue defect filling, and skin regeneration. Attached Figure Description

[0027] Figure 1 Sirius red staining diagrams of embodiments and comparative examples of the present invention; Figure 2 HE staining images of embodiments and comparative examples of the present invention; Figure 3 Electron micrographs of embodiments and comparative examples of the present invention; Figure 4 Electrophoresis diagrams of embodiments and comparative examples of the present invention; Detailed Implementation

[0028] To make the technical means, creative features, objectives and effects of this invention easier to understand, the invention will be further described below in conjunction with specific embodiments.

[0029] Example 1: Preparation of a low-sensitivity de-scraping matrix 1. Swelling: Immerse the pretreated animal soft tissue in a 0.2M sodium hydroxide solution for 5 minutes to swell, making the tissue loose and easier for subsequent processing.

[0030] 2. Grinding: The swollen sample is rapidly frozen and then mechanically ground to increase the specific surface area and improve the decellularization efficiency.

[0031] 3. Decellularization: Prepare the hypertonic treatment solution: a 2% (w / v) complex of trypsin and lipase, a 5% (w / v) AEO-9, and a 4 M sodium chloride solution; prepare the hypotonic treatment solution: a 30 mM HCl solution and a 5% (w / v) AEO-9. Place the hypertonic solution in container A, controlling the discharge using a level gauge. Mix the hypertonic solution and the solid in container B at a volume / mass ratio of approximately 15:1, and circulate at low speed for 8 hours at 150 rpm and 33°C. After completion, separate the solid and liquid components using a pressure pump and a filter between containers A and B. Adjust the level gauge in container C to release the hypotonic solution, allowing it to mix with the solid in container B at a volume / mass ratio of approximately 15:1, and circulate at low speed for 2 hours at 120 rpm and 28°C. After completion, solid and liquid separation was achieved through a pressurized pump and a filter between the AB cups. The decellularized material was then washed with PBS using a circulating pump. The ratio of washing solution to material (mass:volume) was approximately 10:1, and the process was carried out using a circulating shaking method at 100 rpm and 25°C for 30 minutes.

[0032] 4. Chromatography: Activated GM1250 packing material is used for wet packing, allowing natural sedimentation to form a uniform column bed and avoiding air bubbles and layer breaks. Simultaneously, the collagen sample concentration is adjusted to 1–5 mg / mL and slowly added to the top of the column. The column outlet is opened, and after the sample has completely entered the column bed, 1–2 cm of equilibration buffer is added to the top of the column. Eluent is collected from the point where the sample has completely entered the column bed. After sample loading, the column bed is washed with equilibration buffer to elute any remaining unbound collagen. The wash solution is combined with the main peak flow-through solution to obtain a collagen solution free of residual endotoxins, nucleic acid fragments, and other charged or specific molecular weight sensitizing impurities.

[0033] 5. Salt washing: Wash the sample after chromatography in step (4) with 0.3M salt solution to remove denatured proteins and impurities, and collect the precipitate by centrifugation.

[0034] 6. Purification: Finally, small molecule impurities, salts, and unbound surfactants are removed by ultrafiltration or dialysis to obtain high-purity decellularized matrix implants.

[0035] Example 2 1. Swelling: Immerse the pretreated animal soft tissue in a 0.2M potassium hydroxide solution for 20 minutes to swell, making the tissue loose and easier for subsequent processing.

[0036] 2. Grinding: The swollen sample is rapidly frozen and then mechanically ground to increase the specific surface area and improve the decellularization efficiency.

[0037] 3. Decellularization: Prepare the hypertonic treatment solution: a mixture of trypsin and lipase at a mass / volume concentration of 1.2%, sodium deoxycholate at a mass / volume concentration of 0.15%, and a 0.5 M sodium chloride solution; prepare the hypotonic treatment solution: 30 mM HCl and sodium deoxycholate at a mass / volume concentration of 0.15%. Place the hypertonic solution in container A, and control the discharge using a level gauge. Mix the hypertonic solution and the solid in container B at a volume / mass ratio of approximately 5:1, and circulate at low speed for 2 hours. The rotation speed is 60 rpm, and the temperature is 33°C. After completion, separate the solid and liquid components using a pressure pump and a filter between containers A and B. Adjust the level gauge of container C to release the hypotonic solution, allowing the solution to mix with the solid in container B at a volume / mass ratio of approximately 5:1, and circulate at low speed for 2 hours. The rotation speed is 100 rpm, and the temperature is 33°C. After completion, solid and liquid separation was achieved through a pressurized pump and a filter between the AB cups. The decellularized material was then washed with PBS using a circulating pump. The ratio of washing solution to material (mass:volume) was approximately 10:1, and the process was carried out using a circulating shaking method at 100 rpm and 25°C for 30 minutes.

[0038] 4. Chromatography: Activated GM1250 packing material is used for wet packing, allowing natural sedimentation to form a uniform column bed and avoiding air bubbles and layer breaks. Simultaneously, the collagen sample concentration is adjusted to 1–5 mg / mL and slowly added to the top of the column. The column outlet is opened, and after the sample has completely entered the column bed, 1–2 cm of equilibration buffer is added to the top of the column. Eluent is collected from the point where the sample has completely entered the column bed. After sample loading, the column bed is washed with equilibration buffer to elute any remaining unbound collagen. The wash solution is combined with the main peak flow-through solution to obtain a collagen solution free of residual endotoxins, nucleic acid fragments, and other charged or specific molecular weight sensitizing impurities.

[0039] 5. Salt washing: The sample after chromatography in step (4) is washed with 0.3M salt solution to remove proteins and other hydrophobic impurities that may have been denatured in the previous treatment through salt precipitation.

[0040] 6. Purification: Finally, small molecule impurities, salts, and unbound surfactants are removed by ultrafiltration or dialysis to obtain high-purity decellularized matrix implants.

[0041] Example 3 1. Swelling: Immerse the pretreated animal soft tissue in a 0.2M sodium hydroxide solution for 5 minutes to swell, making the tissue loose and easier for subsequent processing.

[0042] 2. Grinding: The swollen sample is rapidly frozen and then mechanically ground to increase the specific surface area and improve the decellularization efficiency.

[0043] 3. Decellularization: Prepare the hypertonic treatment solution: a 2% (w / v) complex of trypsin and lipase, a 2.5% (w / v) Span-80, and a 4 M sodium chloride solution; prepare the hypotonic treatment solution: a 30 mM HCl solution and a 1% (w / v) Triton X-100. Place the hypertonic solution in container A, controlling the discharge using a level gauge. Mix the hypertonic solution and the solid in container B at a volume / mass ratio of approximately 15:1, and circulate at low speed for 8 hours at 150 rpm and 33°C. After completion, separate the solid and liquid components using a pressure pump and a filter between containers A and B. Adjust the level gauge in container C to release the hypotonic solution, allowing it to mix with the solid in container B at a volume / mass ratio of approximately 15:1, and circulate at low speed for 2 hours at 120 rpm and 28°C. After completion, solid and liquid separation was achieved through a pressurized pump and a filter between the AB cups. The decellularized material was then washed with PBS using a circulating pump. The ratio of washing solution to material (mass:volume) was approximately 10:1, and the process was carried out using a circulating shaking method at 100 rpm and 25°C for 30 minutes.

[0044] 4. Chromatography: Activated GM1250 packing material is used for wet packing, allowing natural sedimentation to form a uniform column bed and avoiding air bubbles and layer breaks. Simultaneously, the collagen sample concentration is adjusted to 1–5 mg / mL and slowly added to the top of the column. The column outlet is opened, and after the sample has completely entered the column bed, 1–2 cm of equilibration buffer is added to the top of the column. Eluent is collected from the point where the sample has completely entered the column bed. After sample loading, the column bed is washed with equilibration buffer to elute any remaining unbound collagen. The wash solution is combined with the main peak flow-through solution to obtain a collagen solution free of residual endotoxins, nucleic acid fragments, and other charged or specific molecular weight sensitizing impurities.

[0045] 5. Salt washing: Wash the sample after chromatography in step (4) with 2M salt solution to remove denatured proteins and impurities, and collect the precipitate by centrifugation.

[0046] 6. Purification: Finally, small molecule impurities, salts, and unbound surfactants are removed by ultrafiltration or dialysis to obtain high-purity decellularized matrix implants.

[0047] Comparative Example 1 1. Swelling: Immerse the pretreated animal soft tissue in a 0.2M sodium hydroxide solution for 5 minutes to swell, making the tissue loose and easier for subsequent processing.

[0048] 2. Grinding: The swollen sample is rapidly frozen and then mechanically ground to increase the specific surface area and improve the decellularization efficiency.

[0049] 3. Decellularization: (1) Preparation of decellularization solution: a complex of trypsin and lipase with a mass / volume concentration of 2% and Span-80 with a mass / volume concentration of 2.5%, with the material and decellularization solution at a mass / volume ratio of approximately 15:1, soaked at 33℃ for 8 h. (2) Preparation of decellularization solution: Span-80 with a mass / volume concentration of 2.5%, with the material and decellularization solution at a mass / volume ratio of approximately 15:1, soaked at 28℃ for 2 h. (3) Soaking the decellularized material in PBS. The ratio of washing solution to material (mass:volume) is approximately 10:1, and the treatment is carried out at 25℃ for 30 min.

[0050] 4. Chromatography: Activated GM1250 packing material is used for wet packing, allowing natural sedimentation to form a uniform column bed and avoiding air bubbles and layer breaks. Simultaneously, the collagen sample concentration is adjusted to 1–5 mg / mL and slowly added to the top of the column. The column outlet is opened, and after the sample has completely entered the column bed, 1–2 cm of equilibration buffer is added to the top of the column. Eluent is collected from the point where the sample has completely entered the column bed. After sample loading, the column bed is washed with equilibration buffer to elute any remaining unbound collagen. The wash solution is combined with the main peak flow-through solution to obtain a collagen solution free of residual endotoxins, nucleic acid fragments, and other charged or specific molecular weight sensitizing impurities.

[0051] 5. Salt washing: Wash the sample after chromatography in step (4) with 2M salt solution to remove denatured proteins and impurities, and collect the precipitate by centrifugation.

[0052] 6. Purification: Finally, small molecule impurities, salts, and unbound surfactants are removed by ultrafiltration or dialysis to obtain high-purity decellularized matrix implants.

[0053] Comparative Example 2 A 0.3 mm thick porcine dermal tissue was subjected to enzymatic hydrolysis, elution, detergent treatment, and re-elution (repeated twice), a "four-step, eight-cycle" decellularization process to remove cellular components from the dermal tissue and prepare a decellularized dermal matrix. ① Enzymatic hydrolysis: 0.25% trypsin, hydrolyze at 37℃ for 4 hours; Step 2: use 2.5 U / ML neutral protease, hydrolyze at room temperature for 4 hours.

[0054] ② Washing: Each washing process requires 7 washes. The specific washing steps are as follows: a) Soak and rinse once with purified water for 5 minutes; b) Perform ultrasonic cleaning twice, 5 minutes each time; c) Wash twice with purified water using a shaker, 5 minutes each time; d) Wash twice with physiological saline on a shaker for 5 minutes each time.

[0055] ③ Detergent treatment: The porcine dermal matrix was immersed in a 2% ionic detergent SDS solution for both treatments, and then shaken at 37°C for 12 hours. This yielded acellular dermal matrix.

[0056] Comparative Example 3 1. Swelling: Immerse the pretreated animal soft tissue in a 0.2M sodium hydroxide solution for 5 minutes to swell, making the tissue loose and easier for subsequent processing.

[0057] 2. Grinding: The swollen sample is rapidly frozen and then mechanically ground to increase the specific surface area and improve the decellularization efficiency.

[0058] 3. Decellularization: Prepare the hypertonic treatment solution: a 2% (w / v) complex of trypsin and lipase, a 5% (w / v) AEO-9, and a 4 M sodium chloride solution; prepare the hypotonic treatment solution: a 30 mM HCl solution and a 5% (w / v) AEO-9. Place the hypertonic solution in container A, controlling the discharge using a level gauge. Mix the hypertonic solution and the solid in container B at a volume / mass ratio of approximately 15:1, and circulate at low speed for 8 hours at 150 rpm and 33°C. After completion, separate the solid and liquid components using a pressure pump and a filter between containers A and B. Adjust the level gauge in container C to release the hypotonic solution, allowing it to mix with the solid in container B at a volume / mass ratio of approximately 15:1, and circulate at low speed for 2 hours at 120 rpm and 28°C. After completion, solid and liquid separation was achieved through a pressurized pump and a filter between the AB cups. The decellularized material was then washed with PBS using a circulating pump. The ratio of washing solution to material (mass:volume) was approximately 10:1, and the process was carried out using a circulating shaking method at 100 rpm and 25°C for 30 minutes.

[0059] 4. Salt washing: Wash the sample from step (3) with 0.3M salt solution to remove denatured proteins and impurities, and collect the precipitate by centrifugation.

[0060] 5. Purification: Finally, small molecule impurities, salts, and unbound surfactants are removed by ultrafiltration or dialysis to obtain high-purity decellularized matrix implants.

[0061] Performance Testing and Results Analysis I. DNA Residual Amount (Reagent Kit Operation): The method utilizes a fluorescent dye to specifically bind to double-stranded DNA, forming a complex that generates a strong fluorescent signal when excited at a wavelength of 480 nm. This complex can be detected using a fluorescent microplate reader at a wavelength of 520 nm. Within a certain range of DNA concentration and in the case of excess dye, the fluorescence intensity is directly proportional to the DNA concentration. The amount of residual DNA in the sample can be calculated based on the fluorescence intensity of the sample.

[0062] reagents (1) 1 mol / L Tris solution (pH 7.5), adjust the pH to 7.5 with hydrochloric acid.

[0063] (2) 0.5 mol / L disodium ethylenediaminetetraacetate solution (pH 7.5), adjust the pH to 7.5 with 10 mol / L sodium hydroxide solution.

[0064] (3) TE buffer (pH 7.5): Measure 1.0 ml of 1 mol / L Tris solution (pH 7.5) and 0.2 ml of 0.5 mol / L disodium ethylenediaminetetraacetate solution (pH 7.5), and add sterile water for injection to 100 ml.

[0065] (4) Double-stranded DNA fluorescent dye, prepared according to the reagent instructions.

[0066] (5) DNA standard: Dissolve an appropriate amount of DNA standard in TE buffer to prepare 50 μg / ml DNA standard, and store at -20℃.

[0067] The concentration of DNA standards is calculated using the following formula: DNA concentration (μg / ml) = 50 × A260 Preparation of DNA standard solutions: DNA standards were prepared into standard solutions with concentrations of 0, 1.25 ng / ml, 2.5 ng / ml, 5.0 ng / ml, 10 ng / ml, 20 ng / ml, 40 ng / ml, and 80 ng / ml using TE buffer.

[0068] Assay Method: Accurately measure 400 μl each of the DNA standard solution and the test solution into 1.5 ml centrifuge tubes. Add 400 μl of freshly prepared double-stranded DNA fluorescent dye to each tube, mix well, and incubate at room temperature in the dark for 5 minutes. Transfer 250 μl of the above reaction solution to a 96-well black ELISA plate, making 3 replicates. Measure the fluorescence intensity using a fluorescence microplate reader at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. Using the fluorescence intensity measured with TE buffer as the background, measure and record the fluorescence value of each well. Perform a linear regression between the concentration of the standard solution and its corresponding fluorescence intensity to obtain the linear regression equation (the correlation coefficient should be no less than 0.99). Substitute the fluorescence intensity of the test solution into the linear regression equation to calculate the residual DNA content in the test sample.

[0069] II. α-Gal clearance rate: (1) Preparation of control standard for the experimental system: SP2 / 0 cells were cultured in RPMI 1640 complete medium until they reached good growth status, and the cell concentration was adjusted to 1×10⁻⁶. 7 Cells / mL. Resuspend 1×10⁻⁶ cells / mL in 1% BSA. 6 10 SP2 / 0 cells were prepared, mixed thoroughly, and then serially diluted by adding 1% BSA solution to both the original cell solution and the cell dilution buffer, resulting in four concentrations (including a cell-free control group). The volume ratio of the original cell solution in each of the remaining groups decreased progressively with each serial dilution. Three replicates were prepared for each concentration.

[0070] (2) Sample preparation: Weigh three portions of the same batch of α-Gal antigen removal samples before and after treatment, add 1% BSA solution to prepare a sample suspension of a certain concentration, and dry the wetted samples in a 37℃ drying oven. Then, accurately weigh and record the test samples, cut them into appropriate sizes with sterile ophthalmic scissors, and homogenize them at low temperature until all samples are pulverized (particle size less than 100μm) to form a uniform tissue homogenate. Perform serial dilution with 1% BSA solution to a total of four concentrations.

[0071] (3) Sample reaction with antibody I: Take the sample and M86 antibody (M86 antibody was diluted 200 times with antibody dilution buffer) at a volume ratio of 1:4, and incubate at 37°C with gentle shaking for 2 hours, then transfer to 4°C and incubate overnight. Centrifuge the next day and collect the supernatant for later use.

[0072] (4) Indirect method for detecting the remaining M86 antibody in the supernatant: Prepare an α-Gal-BSA solution with a concentration of 2 μg / mL. Add 100 μL of α-Gal antigen solution to the corresponding well of a 96-well plate, incubate overnight at 4°C, wash the plate 3 times with washing buffer, and then blot dry. Add 200 μL of 1% (w / v) BSA to each well for blocking, incubate at 37°C with shaking for 2 h, and then wash the plate 3 times again. Add the supernatant from the above (3) reaction to the corresponding antigen-coated 96-well plate, 100 μL / well, incubate at 37°C with shaking for 2 h, wash the plate 3 times with washing buffer, add HRP-labeled anti-mouse secondary antibody, incubate at 37°C with shaking for 1 h, and then wash the plate 3 times with washing buffer. After color development, read the OD value of each well at a wavelength of 450 nm using a microplate reader.

[0073] III. Mechanical Strength: Each group of samples was freeze-dried under the same conditions, and the samples were cut into rectangles with a length of 1.5 cm and a width of 1 cm. The samples were placed on a tensile testing machine, clamped and fixed at both ends, and tensile tests were performed at a tensile speed of 100 mm / min. The tensile strength at break was recorded.

[0074] IV. Fat content: (1) Acid hydrolysis: Weigh 5g of sample (accurate to 0.001g), place it in an Erlenmeyer flask, add 50ml of 2mol / L hydrochloric acid solution and a few fine glass beads, cover with a watch glass, heat on a hot plate until it just boils (you can hear the glass clinking), maintain for 1 hour, and rotate and shake once every 10 minutes. Remove the Erlenmeyer flask, add 150ml of hot water, mix well, filter, wash the Erlenmeyer flask and watch glass with hot water, and filter the hot water together. Wash the precipitate with hot water until neutral (test with blue litmus paper; the paper does not change color when neutral). Place the precipitate and filter paper on a large watch glass, and dry in a drying oven at 100℃±5℃ for 1 hour, then cool.

[0075] (2) Extraction: Place the filter paper tube into the drawer of the Soxhlet extractor and connect it to the receiving flask that has been dried to a stable weight. Add petroleum ether to the flask up to half its volume above the condenser tube of the extractor, and then heat in a water bath to continuously reflux the petroleum ether for extraction (6-8 times / h), generally for 6-10 hours. Collect a drop of extract with a ground glass rod. If there is no oil residue on the ground glass rod, the extraction is complete.

[0076] (3) Weighing: Remove the receiving bottle, recover the petroleum ether, and when only 1-2 ml of solvent remains in the receiving bottle, evaporate it to dryness in a water bath, then dry it in an oven at 100℃±5℃ for 1 h, cool it in a desiccator for 0.5 h, and then weigh it. Repeat the above operation until the weighing result is constant (i.e., the difference between two weighings does not exceed 2 mg).

[0077] (4) Results analysis: Fat content in the sample: X = (m １ -m ｏ ) / m ２ ×100 X: Fat content in the sample, in grams per 100 grams (g / 100g) m １ The content of fat in the receiving flask after constant weight, expressed in grams (g). m ｏ Mass of the receiving bottle, in grams (g). m ２ Mass of the sample, in grams (g). 100: Conversion factor.

[0078] V. Electrophoretic purity test: (1) Solution preparation Preparation of collagenase digestion solution: Dissolve collagenase in 20 mmol / L sodium dihydrogen phosphate solution (pH 7.4, containing 0.1 mmol / L calcium chloride) to prepare a collagenase digestion solution containing appropriate activity units.

[0079] Sample buffer preparation: Measure 2.5 mL of 0.5 mol / L Tris-HCl, 2.0 mL of glycerol, 4.0 mL of 10% SDS, 0.5 mL of 0.1% bromophenol blue, and bring the volume to 10 mL with ultrapure water.

[0080] Electrophoresis buffer preparation: Weigh 15.1 g Tris, 72 g g glycine, and 5.0 g SDS, dissolve them in ultrapure water, and bring the volume to 1000 mL. Dilute 5 times before use.

[0081] Preparation of Coomassie Brilliant Blue staining solution: Weigh 1 g of Coomassie Brilliant Blue R250, add 200 mL of methanol, 50 mL of glacial acetic acid, and 250 mL of water, and mix well.

[0082] Preparation of Coomassie Brilliant Blue decolorizing solution: Mix 400 mL of methanol, 100 mL of glacial acetic acid and 500 mL of water.

[0083] Separating gel concentration: 7%.

[0084] Concentration of stacking gel: 4%.

[0085] BSA solution preparation: Dilute BSA to a gradient concentration using ultrapure water.

[0086] (2) Analysis of contaminating proteins in the sample Collagen reference standard: Dissolve the collagen reference standard in 3% acetic acid or 0.01 mol / L hydrochloric acid to make the collagen concentration 1 mg / mL.

[0087] Sample a: Dissolve the sample in 3% acetic acid or 0.01 mol / L hydrochloric acid to make the collagen concentration 1 mg / mL.

[0088] Sample b: Dissolve the sample with collagenase digestion solution to make the collagen concentration 1 mg / mL, and incubate in a 37 °C water bath for 4 h.

[0089] Sample c: Take ultrapure water and add collagenase digestion solution to make the concentration of collagenase the same as that in sample b.

[0090] Sample loading and analysis: Mix the above sample with an equal volume of sample buffer (if the solution turns slightly yellow, adjust to blue with sodium hydroxide), then place in hot water at 90℃~100℃ for 1 min~2 min before performing SDS-PAGE electrophoresis. The loading volume for both the sample and BSA (using the staining limit) is 20 μL. After loading, perform electrophoresis, staining, and destaining. The destaining film can then be scanned and analyzed using an image analysis system.

[0091] (3) Calculation 1) When BC=0, Collagen purity (%) = (10000 - BSA limit) / 10000 × 100% 2) When BC≠0, Collagen purity (%) = A - (BC) × 100% Where A = (sum of optical densities of all bands in sample a) / (sum of optical densities of all bands in sample a) × 100%; B = (Sum of optical densities of all bands in sample b) / (Sum of optical densities of all bands in sample a) × 100%; C = (Sum of optical densities of all bands in sample c) / (Sum of optical densities of all bands in sample a) × 100%.

[0092] In the formula: The sum of the optical densities of all bands in sample a, % The sum of the optical densities of all bands in sample b, % The sum of the optical densities of all bands in sample c, %.

[0093] VI. Endotoxin Test: Take 1 ml of sample and add it to a dilution tube. Seal the tube with sealing film and heat it in a water bath for about 5-10 minutes until the sample is completely in a solution state. Remove the tube, vortex mix, and use the dynamic colorimetric method with a sensitivity of 50-0.005 EU / ml of Limulus amebocyte lysate (LAL) reagent for detection. Preheat the tube to 37±1℃ before adding the sample. Set the program (add and shake to mix for 1 minute), wavelength 405 nm, OD value: 0.1, and reaction time: 60-90 minutes.

[0094] VII. Sirius Red Staining Experiment: (1) The tissue was fixed in 10% formalin fixative (Cat# 60535ES) and routinely dehydrated and embedded.

[0095] (2) The sections are 3~8 μm thick and dewaxed to water using conventional xylene or environmentally friendly wax dewaxing and clearing solution (Cat# 60722ES).

[0096] (3) Stain with hematoxylin staining solution (Cat# 60502ES) for 3~10 min, then wash with water for 30~120 s.

[0097] (4) Stain with Sirius red staining solution for 10-60 min, or stain tissues that are easier to stain for 10-20 min.

[0098] (5) Rinse briefly with running water to remove the staining solution from the surface of the slide.

[0099] (6) Dehydrate with 95% ethanol for 1 min, dehydrate with anhydrous ethanol twice for 1 min each time, clear with xylene or environmentally friendly wax-removing and clearing solution (Cat# 60722ES) twice for 1 min each time, seal with neutral resin (Cat# 36313ES), and examine under a microscope.

[0100] 8. HE staining: 1. Procedure for HE staining of cell smears 1.1. Fixation: After adding tissue fixative to the well plate and fixing for 15 min, aspirate the fixative from the wells and wash with ultrapure water 3-4 times.

[0101] 1.2. Membrane breaking: Add membrane breaking solution to the well plate and break the membrane for 15 minutes. Aspirate the membrane breaking solution from the well and wash with ultrapure water 3-4 times.

[0102] 1.3. Pretreatment: Add an appropriate amount of high-definition constant pretreatment solution to the well plate and treat for 1 min.

[0103] 1.4. Hematoxylin staining: Add an appropriate amount of hematoxylin staining solution to the well plate and stain for 3-5 minutes. Aspirate the staining solution from the wells and wash with tap water 3-4 times. Differentiate with differentiation solution for 1-2 seconds, wash with tap water 3-4 times, and then use blueing solution to blue for 1-2 seconds. Wash with tap water 3-4 times.

[0104] 1.5. Eosin staining: Pretreatment with anhydrous ethanol for 1 min, aspirate the ethanol, add eosin staining solution for 2-3 min, aspirate the staining solution, and wash 3-4 times with anhydrous ethanol. 1.6. Mounting: After drying, take a clean glass slide, write a number on it, drop a drop of ultra-clean quick-drying mounting medium in the middle of the slide, take out the slide, place the side with the cells facing down, and mount it with neutral resin.

[0105] 2. HE staining procedure for sections 2.1. Dewaxing paraffin sections to water: Immerse the sections in environmentally friendly dewaxing solution I for 15 min, environmentally friendly dewaxing solution II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% alcohol for 5 min, then rinse with tap water.

[0106] Frozen sections were thawed and fixed: Frozen sections were taken out of the -20℃ freezer and brought to room temperature. They were fixed with acetone for 1 minute and then washed with water.

[0107] 2.2. Pretreatment: The sections were immersed in high-resolution constant staining pretreatment solution for 1 min.

[0108] 2.3. Hematoxylin staining: Immerse the sections in hematoxylin staining solution for 3 min, rinse with tap water, differentiate with differentiation solution for 3-5 s, rinse with tap water, blue with blueing solution for 3-6 s, and rinse with running water.

[0109] 2.4. Eosin staining: Dehydrate the sections in 95% alcohol for 1 min, then stain them in eosin staining solution for 15 s.

[0110] 2.5. Dehydration and mounting: The sections were sequentially immersed in anhydrous ethanol I for 1 min - anhydrous ethanol II for 1 min - anhydrous ethanol III for 1 min - n-butanol I for 1 min - n-butanol II for 1 min - xylene I for 1 min - xylene II for 1 min, and then cleared and mounted with neutral resin.

[0111] 2.6. Microscopic examination, image acquisition and analysis.

[0112] IX. Masson staining: 1. Dewaxing paraffin sections to water: Immerse the sections in environmentally friendly dewaxing and clearing solution I for 15 min, environmentally friendly dewaxing and clearing solution II for 15 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, and 75% alcohol for 5 min, then rinse with tap water.

[0113] Frozen sections were rewarmed and fixed: Frozen sections were taken out of the -20℃ freezer and brought to room temperature. They were fixed with tissue fixative for 15 minutes and then rinsed with running water.

[0114] 2. Immerse the slices in Masson A solution and soak in a 65°C oven for 30 minutes, then rinse with running tap water.

[0115] 3. Simultaneously preheat Masson D solution and Masson F solution in a 55°C water bath. 4. Immerse the slices in a staining solution containing an equal mixture of Masson B and Masson C solutions for 1 minute, rinse with tap water, differentiate with differentiation solution for a few seconds, and rinse with tap water.

[0116] 5. Immerse the sections in Masson D solution for 6 min, then rinse with tap water.

[0117] 6. Immerse in Masson E solution for 1 min.

[0118] 7. Do not rinse with water. Drain slightly and stain directly with Masson F solution for 2-30 seconds.

[0119] 8. Rinse the sections with 1% acetic acid for differentiation, and dehydrate them in two tanks of anhydrous ethanol.

[0120] 9. Clearing and mounting: Place the sections in anhydrous ethanol for 5 minutes, xylene for 5 minutes to clear, and then mount with neutral resin.

[0121] 10. Microscopic examination, image acquisition and analysis.

[0122] 10. Electron microscopy: 1. Equipment Information: Field Emission Scanning Electron Microscope, Model: JSM-IT800 2. Operating Procedures (1) Take a solid sample and attach it to a metal frustum with conductive adhesive. Use clean air to blow away any excess sample that is not adhered. Place the metal frustum with the sample in an automated ion sputtering instrument and spray gold (gold target) for 30 seconds. After the gold spraying is completed, store the sample in a desiccator.

[0123] (2) Place the sample in the field emission scanning electron microscope, adjust the instrument to a vacuum mode of 10⁻³-10⁻⁷ Pa, and scan the sample using a voltage of 10-20 keV. Observe the sample in the range of 40-5000x.

[0124] XI. Identification of Active Ingredients: 1. Preparation of test solution: Dissolve each group of samples in 3% acetic acid or 0.01 mol / L hydrochloric acid to achieve a collagen concentration of 1 mg / mL. Take 0.5 mL of the extruded liquid and dilute it 1-fold with test buffer (2×). Mix well and heat in a water bath at 90–100 ℃ for 3–5 min, then cool to room temperature. Perform the same procedure for the collagen control solution.

[0125] 2. Prepare the separating gel and stacking gel of SDS-PAGE using laboratory methods.

[0126] 3. Sample loading: Add 10 μl of the test solution and 10 μl of the standard solution to the sample loading well.

[0127] 4. After electrophoresis, a gel imaging system was used to collect and photograph the samples.

[0128] Table 1 Performance Test Table of Examples and Comparative Examples Using a hyperosmolar-hypoosmolar cyclic decellularization method, the amount of residual DNA was significantly lower than that of conventional methods (Comparative Examples 1 and 2), the α-Gal clearance rate was significantly higher than that of conventional methods (Comparative Examples 1 and 2), and the fat content was significantly lower than that of conventional methods (Comparative Examples 1 and 2). This indicates that hyperosmolar-hypoosmolar cyclic decellularization can effectively reduce sensitization reactions.

[0129] The greater the structural integrity, the greater the mechanical strength. Using the decellularization method designed in this invention, the mechanical strength can reach approximately 50 N. This indicates that the sample structure remains relatively intact.

[0130] After chromatographic treatment, the endotoxin level in the samples could be reduced to below 0.5 EU / mL, with a minimum of 0.008 EU / mL. Comparative examples 1, 2, and 3, which were not treated with chromatography, showed higher endotoxin levels than the examples. This demonstrates that chromatographic treatment can effectively reduce and remove impurities such as endotoxins.

[0131] Using the decellularization method and chromatography and purification methods designed in this invention, the purity of Examples 1-3 is all >99.5%.

[0132] HE and Sirius red staining showed that in Examples 1-3 and Comparative Example 3, which used a hyperosmolar-hypoosmolar decellularization method, there were no obvious cell nuclei, and the fiber structure was relatively orderly. Comparative Examples 1 and 2 showed obvious residual cell nuclei and broken fiber structures. Scanning electron microscopy images showed that, compared to Examples 1-3, Comparative Example 3 clearly showed the three-dimensional structure of the active ingredient collagen fiber bundles, Comparative Example 1 showed fiber structure breakage, and Comparative Example 2 showed that the three-dimensional structure of the active ingredient collagen fiber bundles was destroyed.

[0133] The decellularization method designed in this invention was used in Examples 1-3. The active ingredient in Comparative Example 3 was clearly visible on electrophoresis. Comparative Examples 1 and 2 had active ingredients, but the characteristic bands were significantly degraded or broken.

[0134] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or basic characteristics. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0135] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for the preparation of a low sensitizing decellularized matrix implant, characterized in that, The method comprises the following steps: (1) Swelling: the pretreated animal soft tissue is immersed in an alkaline solution for swelling; (2) Pulverization: the swollen sample is rapidly frozen and then pulverized; (3) Decellularization: the pulverized sample obtained in step (2) is placed in a high-osmotic-low-osmotic circulating decellularization solution system for circulating decellularization treatment; (4) Chromatography: the decellularized sample is treated with a chromatographic filler to remove allergens and impurities; (5) Salt washing: the sample after chromatography in step (4) is washed with a salt solution to remove denatured proteins and impurities, and the precipitate is collected by centrifugation; (6) Purification: the sample after salt washing is purified by ultrafiltration or dialysis to further remove impurities, thereby obtaining the low-allergenicity decellularized matrix implant.

2. The production method according to claim 1, characterized by, The alkaline solution in step 1 is at least one of sodium hydroxide solution, potassium hydroxide solution, and sodium bicarbonate solution, and the concentration of the alkaline solution is 0.01-0.2 M, and the swelling time is 1-30 min.

3. The preparation method according to claim 1, characterized in that, In step (3), the high-osmotic-low-osmotic circulating decellularization solution system comprises a high-osmotic treatment solution and a low-osmotic treatment solution; the high-osmotic treatment solution comprises a protease complex with a mass-volume concentration of 0.1%-2%, a non-ionic surfactant with a mass-volume concentration of 0.15%-5%, and a high-osmotic solute with a concentration of 0.5-4 M; and the low-osmotic treatment solution comprises HCl with a concentration of 10-30 mM and a non-ionic surfactant with a mass-volume concentration of 0.15%-5%.

4. The production method according to claim 3, characterized by, The non-ionic surfactant is at least one of AEO-9 or Span-80, Triton X-100, and sodium deoxycholate.

5. The preparation method according to claim 3, characterized in that, The high-osmotic solute is at least one of sodium chloride, potassium chloride, sorbitol, and glucose.

6. The preparation method according to claim 3, characterized in that, The protease complex is trypsin and / or lipase, and the solvent is PBS buffer or 0.9% physiological saline.

7. The preparation method according to claim 1, characterized in that, The circulating decellularization treatment in step (3) is carried out in a circulating device, specifically comprising: a) high-osmotic treatment stage: the pulverized sample is mixed with the high-osmotic treatment solution at a mass(g):volume(ml) ratio of 1:(5-15), and is treated under the conditions of 25-33℃ and 60-180 rpm oscillation for 2-8 hours; b) separation and low-osmotic treatment stage: the solid material after high-osmotic treatment is separated and recovered, mixed with the low-osmotic treatment solution at a mass(g):volume(ml) ratio of 1:(5-15), and treated under the conditions of 25-33℃ and 60-180 rpm oscillation for 2-8 hours; c) washing stage: after decellularization treatment, the solid material is washed with PBS buffer, and the mass(g):volume(ml) ratio of the washing liquid to the material is 1:10, and the oscillation washing is carried out under the conditions of 25-33℃ and 100-150 rpm for 20-40 minutes.

8. The method of claim 1, wherein, In step (4), the chromatographic filler is at least one of ion exchange chromatography medium, gel filtration medium, or desalting medium; and the ion exchange chromatography medium is at least one of DEAE, Q, CM, or SP.

9. The method of claim 1, wherein, In step (5), the salt solution is a sodium chloride solution having a final concentration of 0.1 to 2 M.

10. A low sensitizing decellularized matrix implant, characterized in that, made by the method of any one of claims 1 to 9.

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