Pig-derived decellularized patch as well as preparation method and application thereof

Abstract

The invention relates to the technical field of tissue repair materials, and particularly discloses a porcine-derived decellularized patch as well as a preparation method and application thereof. The preparation method of the porcine-derived decellularized patch specifically comprises the following steps: sequentially carrying out washing and repeated freezing and thawing treatment, trypsin digestive juice treatment, SDS-containing isopropanol solution treatment, TritonX-100-containing isopropanol solution treatment, 100wt% isopropanol solution treatment and freeze drying on a porcine-derived patch sample, so as to obtain the porcine-derived decellularized patch. The trypsin digestive juice is a solution containing 0.1 wt%-0.5 wt% of trypsin and 0.1 wt%-0.5 wt% of EDTA (Ethylene Diamine Tetraacetic Acid). Through the preparation method, the pig-derived decellularized patch prepared by the application has relatively low content of DNA and has excellent mechanical properties.

Description

Technical Field

[0001] The present application relates to the technical field of tissue repair materials, and in particular to a porcine decellularized patch and a preparation method and application thereof. Background Art

[0002] A massive rotator cuff tear is a severe rotator cuff injury caused by acute trauma, degenerative changes or multiple factors. The surgery is difficult and the prognosis is poor. The re-tear rate after repair is as high as 40%. Therefore, it is a difficult and hot issue in the current research field of sports medicine.

[0003] Traditional repair methods include directly suturing the torn tendon or using autologous or allogeneic grafts. However, these methods also face risks such as donor site complications, immune rejection, and pathogen transmission. As an emerging treatment method, acellular matrix technology provides a new option for the repair of giant rotator cuff tears. It has good cell affinity, biocompatibility, certain mechanical properties, and the ability to induce cell growth and proliferation. It has become one of the research hotspots in the field of tissue repair and biomedical materials in recent years.

[0004] The main sources of acellular matrix patches include human and animal extracellular matrices. Human patches have good repair effects, but their limited supply is the main challenge in practical applications. Animal-derived acellular extracellular matrix patches have the advantages of wide availability and low price. Compared with other animals, porcine dermis-derived extracellular matrix patches are more similar to human ECM in composition and function, and therefore show broad prospects in the field of rotator cuff tear repair. However, due to differences in tissue sources, processing methods, and sterilization methods, their mechanical load strength and bioactivity will be affected. In addition, there is currently a lack of large-scale randomized controlled trials to evaluate the safety and effectiveness of porcine dermal grafts in repairing giant rotator cuff tears. Therefore, it is necessary to develop a technically mature decellularization scheme so that the patch has both excellent mechanical properties and bioactivity. Summary of the invention

[0005] In order to solve the above technical problems, the present application provides a porcine decellularized patch and a preparation method and application thereof.

[0006] In first aspect, the present application provides a method for preparing a porcine acellular patch for repairing rotator cuff tears, characterized in that the method specifically comprises the following steps: subjecting the porcine patch sample to sequential washing and repeated freeze-thaw treatment, trypsin digestion solution treatment, isopropanol solution treatment containing SDS, isopropanol solution treatment containing TritonX-100, 100wt% isopropanol solution treatment, and freeze-drying; the trypsin digestion solution is a solution containing 0.1wt%-0.5wt% trypsin and 0.1wt%-0.5wt% EDTA.

[0007] The porcine patch samples were washed in sequence to remove excess impurities on the surface, and then repeatedly frozen and thawed, which helped to destroy the cell membrane structure, promote cell lysis and release of contents, thereby helping to remove cell components in the tissue. Trypsin is an enzyme that can selectively degrade proteins. It can dissolve the connection between cells and degrade proteins in the cytoplasm to further remove cell components; EDTA is a chelating agent that can bind metal ions and help destroy certain metal-dependent adhesion molecules (such as calcium ions) in the extracellular matrix, further promoting cell shedding; the concentration of trypsin and EDTA is between 0.1wt%-0.5wt%, ensuring sufficient protein degradation and metal chelation while avoiding excessive damage to the ECM structure. It was then treated in an isopropanol solution containing SDS. Sodium dodecyl sulfate (SDS) is an anionic surfactant that is mainly used to dissolve lipid bilayers, destroy cell membranes and organelle membranes, and completely release the contents of cells. Then, the tissue was treated with an isopropanol solution containing TritonX-100. TritonX-100 is a non-ionic surfactant that can gently destroy the lipid components of the cell membrane. It has a relatively mild effect and replenishes the cell membrane lipids that may remain after the SDS action, ensuring complete decellularization while protecting the structural integrity of the tissue as much as possible. The 100wt% isopropanol solution treatment mainly dehydrates the tissue, causing it to lose moisture and prepare for freeze-drying. At the same time, isopropanol can further remove any residual lipid substances.

[0008] This application designs and compares different decellularization methods to select the decellularization method with the best comprehensive performance; by subjecting the porcine patch samples to the above steps in sequence, a porcine decellularized patch is prepared. The finished material has a lower DNA content and excellent mechanical properties.

[0009] Preferably, the specific steps of the repeated freeze-thaw treatment are: immersing the sample in liquid nitrogen for 3-10 minutes, and then thawing it in pure water at 37° C. for 5-15 minutes; repeating the above steps 2-4 times in total.

[0010] Preferably, the specific steps of the trypsin digestion solution are: immersing the patch sample in an aqueous solution containing 0.1wt%-0.5wt% trypsin and 0.1wt%-0.5wt% EDTA at 35-40°C and 150-250rpm for 4-8h; and ultrasonically washing with deionized water at 30-40°C for 2-4 times.

[0011] Preferably, the specific steps of the trypsin-EDTA solution treatment are: immersing the patch sample in an aqueous solution containing 0.2wt%-0.4wt% trypsin and 0.2wt%-0.4wt% EDTA at 35-40°C and 150-250rpm for 4-8h; and ultrasonically washing with deionized water at 30-40°C for 2-4 times.

[0012] In some specific embodiments, the concentration of the trypsin can be 0.1-0.2wt%, 0.1-0.3wt%, 0.1-0.4wt%, 0.2-0.3wt%, 0.2-0.4wt%, 0.2-0.5wt%, 0.3-0.4wt%, 0.3-0.5wt%, 0.4-0.5wt%; the concentration of the EDTA can be 0.1-0.2wt%, 0.1-0.3wt%, 0.1-0.4wt%, 0.2-0.3wt%, 0.2-0.4wt%, 0.2-0.5wt%, 0.3-0.4wt%, 0.3-0.5wt%, 0.4-0.5wt%.

[0013] In a specific embodiment, the concentration of the trypsin can also be 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%; the concentration of the EDTA can also be 0.1wt%, 0.2wt%, 0.3wt%, 0.4wt%, 0.5wt%.

[0014] Through experimental analysis, it can be known that the present application uses the above-mentioned concentration of trypsin and EDTA aqueous solution to treat the sample, which effectively improves the performance of the porcine decellularized patch.

[0015] Preferably, the specific steps of the SDS-containing isopropanol solution treatment are: immersing the patch in an aqueous solution containing 0.05wt%-0.2wt% SDS and 60wt%-80wt% isopropanol at 35-40°C for 4-8h; and ultrasonically washing with deionized water at 30-40°C for 4 to 6 times.

[0016] Preferably, the specific steps of the SDS-containing isopropanol solution treatment are: immersing the patch in an aqueous solution containing 0.10wt%-0.15wt% SDS and 65wt%-75wt% isopropanol at 35-40°C for 4-8h; and ultrasonically washing with deionized water at 30-40°C for 4 to 6 times.

[0017] Preferably, the specific steps of treating with the isopropanol solution containing TritonX-100 are: immersing the patch in an aqueous solution containing 0.5wt%-1.5wt% TritonX-100 and 60wt%-80wt% isopropanol at 35-40°C for 10-14h, and replacing the solution every 2-6h; and ultrasonically washing the patch with deionized water at 30-40°C for 4 to 6 times.

[0018] Preferably, the specific steps of the 100wt% isopropanol solution treatment are: immersing the patch in a 100wt% isopropanol solution, degreasing at 35-40°C and 200-400rpm for 10-14h, replacing the solution every 2-6h; and ultrasonically washing with deionized water at 30-40°C for 5-7 times.

[0019] In a second aspect, the present application provides a porcine decellularized patch prepared using the above-mentioned preparation method.

[0020] In a third aspect, the present application provides the use of the above-mentioned porcine decellularized patch in rotator cuff tear repair materials.

[0021] In summary, the technical solution of this application has the following effects: The present application designs and compares different decellularization methods to select the decellularization method with the best comprehensive performance; finally, a porcine decellularized patch is prepared by sequentially washing and repeatedly freezing and thawing the porcine patch samples, treating them with 0.1wt%-0.5wt% trypsin, 0.1wt%-0.5wt% EDTA solution, an isopropanol solution containing SDS, an isopropanol solution containing TritonX-100, a 100wt% isopropanol solution, and freeze-drying. The finished material has a low DNA content and excellent mechanical properties. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the HE staining result of the porcine decellularized patch prepared in Example 1. DETAILED DESCRIPTION

[0023] The present application is further described in detail below in conjunction with examples, comparative examples and performance testing experiments. These examples should not be construed as limiting the scope of protection claimed in the present application. Example

[0024] Examples 1-9 Examples 1-9 respectively provide a porcine decellularized patch and a preparation method thereof.

[0025] The difference between the above embodiments is that the process parameters of each step in the method for preparing the porcine decellularized patch are specifically shown in Table 1.

[0026] The method for preparing the porcine decellularized patch in the above embodiment specifically comprises the following steps: Washing and repeated freezing and thawing treatment: remove the meat and fat on the lower side of the pig skin; cut the pig skin into 2×5cm patches with a thickness of 1mm; wash with deionized water at 35°C for 3 times; immerse the sample in liquid nitrogen for 5min, and then thaw in pure water at 37°C for 10min; repeat the above steps for a total of 3 times of freezing and thawing.

[0027] Trypsin-EDTA solution treatment: The patch sample was immersed in an aqueous solution containing 0.1wt%-0.5wt% trypsin and 0.1wt%-0.5wt% EDTA at 37°C and 200rpm for 4-8h; and ultrasonically washed with deionized water at 37°C for 3 times.

[0028] Treatment with isopropanol solution containing SDS: immerse the patch in an aqueous solution containing 0.05wt%-0.2wt% SDS and 60wt%-80wt% isopropanol at 37°C for 4-8h; and wash the patch with deionized water at 37°C for 3 times by ultrasonic washing.

[0029] Treatment with isopropanol solution containing TritonX-100: immerse the patch in an aqueous solution containing 0.5wt%-1.5wt% TritonX-100 and 60wt%-80wt% isopropanol at 37°C for 10-14h, changing the solution every 4h; ultrasonically wash the patch with deionized water at 37°C for 3 times.

[0030] Treatment with 100% isopropanol solution: Immerse the patch in 100wt% isopropanol solution, degrease at 37°C and 300rpm for 10-14h, and replace the solution every 2-6h; and wash with deionized water ultrasonically at 37°C for 6 times.

[0031] Freeze drying: Freeze the patch sample at -80℃ for 16 hours and then freeze dry it in a vacuum freeze dryer for 60 hours.

[0032] Storage: Store the dried patch in a -20℃ refrigerator until use.

[0033] Table 1 Specific process parameters of each step in Examples 1-9 Comparative Example Comparative Example 1 Comparative Example 1 provides a porcine decellularized patch and a preparation method thereof.

[0034] The preparation method of the porcine decellularized patch in Comparative Example 1 specifically includes the following steps: Washing and repeated freezing and thawing treatment: remove the meat and fat on the lower side of the pig skin; cut the pig skin into 2×5cm patches with a thickness of 1mm; wash with deionized water at 35°C for 3 times; immerse the sample in liquid nitrogen for 5min, and then thaw in pure water at 37°C for 10min; repeat the above steps for a total of 3 times of freezing and thawing.

[0035] Treatment with isopropanol solution containing SDS: The patch was immersed in an aqueous solution containing 0.12 wt % SDS and 70 wt % isopropanol at 37° C. for 6 h; and ultrasonically washed with deionized water at 37° C. for 3 times.

[0036] Trypsin-EDTA solution treatment: The patch samples were immersed in an aqueous solution containing 0.3 wt% trypsin and 0.3 wt% EDTA at 37°C and 200 rpm for 6 h; and ultrasonically washed with deionized water at 37°C for 3 times.

[0037] Treatment with isopropanol solution containing TritonX-100: The patch was immersed in an aqueous solution containing 1.0wt% TritonX-100 and 70wt% isopropanol at 37°C for 12h, and the solution was replaced every 4h; ultrasonic washing was performed with deionized water at 37°C for 3 times.

[0038] Treatment with 100% isopropanol solution: The patch was immersed in 100wt% isopropanol solution and degreased at 37°C and 300rpm for 12h, with the solution replaced every 4h; and ultrasonically washed with deionized water at 37°C for 6 times.

[0039] Freeze drying: Freeze the patch sample at -80℃ for 16 hours and then freeze dry it in a vacuum freeze dryer for 60 hours.

[0040] Storage: Store the dried patch in a -20℃ refrigerator until use.

[0041] Comparative Example 2 Comparative Example 2 provides a porcine decellularized patch and a preparation method thereof.

[0042] The preparation method of the porcine decellularized patch in Comparative Example 2 specifically includes the following steps: Washing and repeated freezing and thawing treatment: remove the meat and fat on the lower side of the pig skin; cut the pig skin into 2×5cm patches with a thickness of 1mm; wash with deionized water at 35°C for 3 times; immerse the sample in liquid nitrogen for 5min, and then thaw in pure water at 37°C for 10min; repeat the above steps for a total of 3 times of freezing and thawing.

[0043] Trypsin-EDTA solution treatment: The patch samples were immersed in an aqueous solution containing 0.3 wt% trypsin and 0.3 wt% EDTA at 37°C and 200 rpm for 6 h; and ultrasonically washed with deionized water at 37°C for 3 times.

[0044] SDS aqueous solution treatment: The patch was immersed in an aqueous solution containing 0.12 wt% SDS at 37°C for 6 h; and ultrasonically washed with deionized water at 37°C for 3 times.

[0045] Treatment with TritonX-100 aqueous solution: The patch was immersed in an aqueous solution containing 1.0wt% TritonX-100 at 37°C for 12h, and the solution was replaced every 4h; and ultrasonically washed with deionized water at 37°C for 3 times.

[0046] Treatment with 100% isopropanol solution: The patch was immersed in 100wt% isopropanol solution and degreased at 37°C and 300rpm for 12h, with the solution replaced every 2-6h; and ultrasonically washed with deionized water at 37°C for 6 times.

[0047] Freeze drying: Freeze the patch sample at -80℃ for 16 hours and then freeze dry it in a vacuum freeze dryer for 60 hours.

[0048] Storage: Store the dried patch in a -20℃ refrigerator until use.

[0049] Comparative Examples 3-4 Comparative Examples 3-4 respectively provide a porcine decellularized patch and a preparation method thereof.

[0050] The difference between the comparative example and Example 1 is that the process parameters of each step in the preparation method of the porcine decellularized patch are specifically shown in Table 2.

[0051] Table 2 Specific process parameters of each step in comparative examples 3-4 The remaining process parameters in the comparative example and embodiment 1 are the same.

[0052] Performance testing 1. HE staining results: The results are as follows Figure 1 As shown; the results show that the porcine decellularized patch prepared by the technical solution of the present application has no residual cells after HE staining and the collagen structure is intact.

[0053] (2) DNA content: The test shall be conducted in accordance with the method of YY / T 1876-2023 “Determination of DNA Residue in Animal-Derived Biomaterials for Tissue Engineering Medical Products: Fluorescence Staining Method”.

[0054] (3) Tensile strength: The test method is based on the preparation of samples using Type 3 test specimens in accordance with GB / T 528-2009.

[0055] (4) Suture strength: According to the method specified in YY / T 1814-2022.

[0056] Test results: as shown in Table 3.

[0057] Table 3 Performance test results of porcine decellularized patches in Examples and Comparative Examples Combined with the test results in Table 3, it can be seen that the present application prepares a porcine decellularized patch by subjecting the porcine patch sample to repeated freeze-thaw treatment, trypsin-EDTA solution treatment, SDS-containing isopropanol solution treatment, TritonX-100-containing isopropanol solution treatment, and 100wt% isopropanol solution treatment, and then freeze-drying. The finished material has a low DNA content and excellent mechanical properties.

[0058] In Comparative Example 1, after the sample was treated with trypsin-EDTA solution, the sample was first treated with an isopropanol solution containing TritonX-100, and then with an isopropanol solution containing SDS. In Comparative Example 2, the sample was treated with an aqueous solution containing SDS and an aqueous solution containing TritonX-100 instead of an isopropanol solution containing SDS and an isopropanol solution containing TritonX-100, and the performance of the prepared porcine decellularized patch was poor.

[0059] By comparing the test results of Examples 1-5 with Comparative Examples 3-4, the present application selected 0.1wt%-0.5wt% trypsin and 0.1wt%-0.5wt% EDTA solution to treat the patch samples, which effectively improved the performance of the porcine decellularized patch.

[0060] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing a porcine acellular patch for repairing rotator cuff tears, characterized in that: Specifically, the method comprises the following steps: washing and repeatedly freezing and thawing the porcine patch sample, treating it with trypsin digestion solution, treating it with isopropanol solution containing SDS, treating it with isopropanol solution containing TritonX-100, treating it with 100wt% isopropanol solution, and freeze-drying it to obtain the product; the trypsin digestion solution is a solution containing 0.1wt%-0.5wt% trypsin and 0.1wt%-0.5wt% EDTA.

2. The method for preparing the porcine decellularized patch according to claim 1, characterized in that: The specific steps of the repeated freeze-thaw treatment are: immersing the sample in liquid nitrogen for 3-10 minutes, and then thawing it in pure water at 37° C. for 5-15 minutes; repeating the above steps 2-4 times in total.

3. The method for preparing the porcine decellularized patch according to claim 1, characterized in that: The specific steps of the trypsin digestion solution treatment are: immersing the patch sample in an aqueous solution containing 0.2wt%-0.4wt% trypsin and 0.2wt%-0.4wt% EDTA at 35-40°C and 150-250rpm for 4-8h; and ultrasonically washing with deionized water at 30-40°C for 2-4 times.

4. The method for preparing the porcine decellularized patch according to claim 3, characterized in that: The patch sample was immersed in an aqueous solution containing 0.25wt%-0.35wt% trypsin and 0.25wt%-0.35wt% EDTA at 35-40°C and 150-250rpm for 4-8h; and ultrasonically washed with deionized water at 30-40°C for 2-4 times.

5. The method for preparing the porcine decellularized patch according to claim 1, characterized in that: The specific steps of the SDS-containing isopropanol solution treatment are: immersing the patch in an aqueous solution containing 0.05wt%-0.2wt% SDS and 60wt%-80wt% isopropanol at 35-40°C for 4-8h; and ultrasonically washing with deionized water at 30-40°C for 4 to 6 times.

6. The method for preparing the porcine decellularized patch according to claim 5, characterized in that: The specific steps of the SDS-containing isopropanol solution treatment are: immersing the patch in an aqueous solution containing 0.10wt%-0.15wt% SDS and 65wt%-75wt% isopropanol at 35-40°C for 4-8h; and ultrasonically washing with deionized water at 30-40°C for 4 to 6 times.

7. The method for preparing the porcine decellularized patch according to claim 1, characterized in that: The specific steps of treating with the isopropanol solution containing TritonX-100 are: immersing the patch in an aqueous solution containing 0.5wt%-1.5wt% TritonX-100 and 60wt%-80wt% isopropanol at 35-40°C for 10-14h, and replacing the solution every 2-6h; and ultrasonically washing the patch with deionized water at 30-40°C for 4 to 6 times.

8. The method for preparing the porcine decellularized patch according to claim 1, characterized in that: The specific steps of the 100wt% isopropanol solution treatment are: immersing the patch in the 100wt% isopropanol solution, degreasing for 10-14h at 35-40°C and 200-400rpm, replacing the solution every 2-6h; and ultrasonically washing with deionized water at 30-40°C for 5-7 times.

9. A porcine decellularized patch, characterized in that: The method is prepared by any one of claims 1 to 8.

10. Use of the porcine decellularized patch according to claim 9 in rotator cuff tear repair materials.

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