Skin repairing material as well as preparation method and application thereof

Abstract

The invention discloses a skin repair material as well as a preparation method and application thereof. The skin repair material is prepared from pig skin tissues subjected to virus inactivation and sterilization, glycerol, ethylenediamine tetraacetic acid, sodium chloride and water, and the skin repair material is prepared from the following components in percentage by weight: 20.0001 percent to 30.000 percent of the pig skin tissues subjected to virus inactivation and sterilization, 0.500 percent to 5.000 percent of the glycerol, 0.029 percent to 0.292 percent of the ethylenediamine tetraacetic acid, 0.200 percent to 0.600 percent of the sodium chloride and 64.108 percent to 79.271 percent of the water. Wherein the pig skin tissue is the skin tissue of a gene editing pig without an alpha-Gal antigen. The skin repair material disclosed by the invention is high in stability and low in immunogenicity, can be stored for a long time in an environment of-30 DEG C to 30 DEG C, and can be used after being unsealed.

Description

Technical Field

[0001] The present invention relates to the field of biological repair materials, and in particular to a skin repair material and a preparation method and application thereof. Background Art

[0002] There is a serious shortage of allogeneic skin tissue sources, which makes it difficult to meet the clinical needs of skin defect and trauma repair. Using xenogeneic skin materials is one of the fundamental ways to solve the shortage of dermis sources, among which pigs are a relatively ideal source of xenogeneic tissue donors. Pigs are similar to humans in genetics, physiology and anatomy, and are far from humans. The probability of transmitting human-pig co-morbid diseases is low. In addition, the low cost of pig farming and the short breeding cycle facilitate large-scale production.

[0003] However, using animal-derived materials such as pig skin to help human wound repair has potential immune risks, because there are a large number of α-Gal antigen epitopes in animal-derived tissues and natural anti-α-Gal antibodies in the human body. The combination of α-Gal antigen and anti-α-Gal antibody is an immune barrier that can lead to rejection and persistent chronic inflammation. Removing α-Gal antigens from animal-derived materials has become an important means to reduce adverse immune rejection reactions in the human body. [1-2] . In the past decade, the rapid development of gene editing technology has opened up a huge new field. Xenogeneic tissue and organ transplantation has developed rapidly, and to a large extent, problems such as immune rejection have been solved. Through gene editing, the α-1,3-galactosyltransferase gene (GTKO) in the pig genome can be knocked out, which can remove the hyperacute rejection reaction caused by the presence of the α-1,3-galactose (α-1,3-galactose) antigen epitope. Gene editing technology can also change other xenoantigens, coagulation regulatory factors, complement regulatory factors, cellular immune response factors, anti-apoptosis and anti-inflammatory factors, etc., which can effectively avoid or alleviate the problem of immunological incompatibility.

[0004] However, porcine endogenous retroviruses (PERVs) in the pig genome can infect human fibroblasts, T lymphocytes and B lymphocytes, and are a high-risk cross-infection virus between xenotransplantation species. The titer of PERVs in pigs can be reduced by screening pig breeds to obtain PERV-deficient pig strains. CRISPR / Cas9 gene editing technology can also inactivate some PERVs copies, greatly reducing the infection ability of PERVs. In recent years, as gene editing technology has become increasingly sophisticated, it has promoted the application of pig organs, tissues and cells in clinical xenotransplantation, and tissue and organ xenotransplantation research between pigs and non-human primates, and between pigs and humans has made rapid progress.

[0005] At the same time, in addition to the risk of xenoimmunity, xenotransplantation also has other biosafety risks. For example, there are many pathogenic microorganisms in pigs, such as cytomegalovirus (PCMV), hepatitis E virus (HEV), porcine lymphotropic herpesviruses (PLHVs), porcine circoviruses (PCVs), etc. These pathogens are pathogenic to pigs themselves, and can also bring the risk of disease infection to human recipients through tissue and organ transplantation. At present, these pathogenic microorganisms in pigs can be avoided through measures such as donor screening and sterile environment breeding. [3] However, this method requires large investment, low efficiency and high cost. Summary of the invention

[0006] The following is an overview of the subject matter of the detailed description of the present invention. This overview is not intended to limit the scope of protection of this application.

[0007] The present application provides a skin repair material and its preparation method and application. The skin of gene-edited pigs without α-Gal antigen raised under normal conditions is processed by rapid cleaning, virus inactivation, sterilization and low-temperature cell inactivation technology to prepare a low-immunogenicity skin repair material that can be directly used for skin wound repair.

[0008] The first aspect of the present application provides a skin repair material, which comprises: virus-inactivated and sterilized pig skin tissue, glycerol, ethylenediaminetetraacetic acid, sodium chloride and water.

[0009] In an exemplary embodiment, the skin repair material consists of: virus-inactivated and sterilized pig skin tissue, glycerol, ethylenediaminetetraacetic acid, sodium chloride and water.

[0010] In an exemplary embodiment, the skin repair material is composed of the following by weight percentage: 20.000% to 30.000% of virus-inactivated and sterilized pig skin tissue, 0.500% to 5.000% of propylene glycol, 0.029% to 0.292% of ethylenediaminetetraacetic acid, 0.200% to 0.600% of sodium chloride and 64.108% to 79.271% of water; wherein the pig skin tissue is the skin tissue of gene-edited pigs without α-Gal antigen raised under normal environment.

[0011] In an exemplary embodiment, the skin repair material is composed of the following by weight percentage: 22.00% to 26.00% of virus-inactivated and sterilized pig skin tissue, 3.00% to 4.00% of glycerol, 0.10% to 0.15% of ethylenediaminetetraacetic acid, 0.40% to 0.5% of sodium chloride and 69.45% to 74.50% of water.

[0012] In an exemplary embodiment, the skin repair material is composed of the following by weight percentage: 25.00% of virus-inactivated and sterilized pig skin tissue, 4.00% of glycerol, 0.10% of ethylenediaminetetraacetic acid, 0.40% of sodium chloride and 70.50% of water.

[0013] In an exemplary embodiment, the skin repair material is composed of the following by weight percentage: 23.3% virus-inactivated and sterilized pig skin tissue, 3.3% glycerol, 0.08% ethylenediaminetetraacetic acid, 0.39% sodium chloride, and 72.93% water.

[0014] The second aspect of the present application provides a method for preparing the above-mentioned skin repair material, comprising the following steps:

[0015] 1) Collect skin tissue of gene-edited pigs without α-Gal antigen, remove hair, and layer;

[0016] 2) Use purified water to clean surface dirt;

[0017] 3) Virus inactivation and sterilization with peracetic acid solution;

[0018] 4) Rinse with sterile purified water to remove peracetic acid;

[0019] 5) Soaking in an aqueous solution containing glycerol, ethylenediaminetetraacetic acid, and sodium chloride;

[0020] 6) Sterile packaging;

[0021] 7) Low temperature inactivation;

[0022] 8) Store for future use.

[0023] In an exemplary embodiment, step 1) includes: gene editing is performed to knock out the α-1,3-galactosyltransferase gene in the pig genome, pig skin tissue is collected after slaughter, hair and subcutaneous fat are removed, the pig skin tissue is layered to obtain multiple layers of cortex, each layer having a thickness of 0.3 mm to 0.9 mm, and the cortex is cut into desired sizes.

[0024] In an exemplary embodiment, step 3) includes: soaking the cleaned pig skin tissue in a sodium chloride solution, and then adding peracetic acid until the final peracetic acid concentration reaches 0.05% to 0.15%, for example, 0.05%, 0.1%, 0.15%, etc. or any two of them as the range value between the endpoint values, but not limited to the listed values, other unlisted values ​​within the numerical range are also applicable; performing virus inactivation and sterilization.

[0025] In an exemplary embodiment, in step 3), the concentration of the sodium chloride solution is 2.0% to 5.0%, and 3 to 10 liters of sodium chloride solution are added per kilogram of pig skin tissue.

[0026] In an exemplary embodiment, in step 3), the time for virus inactivation and sterilization is 10 to 40 minutes, for example, 10 minutes, 20 minutes, 30 minutes, 40 minutes, etc., or a range value between any two of them as endpoint values, but is not limited to the listed values, and other unlisted values ​​within the numerical range are also applicable.

[0027] In an exemplary embodiment, step 4) comprises: rinsing the virus-inactivated and sterilized pig skin tissue with sterile purified water for 5-30 minutes.

[0028] In an exemplary embodiment, step 5) includes: soaking the pig skin tissue rinsed with sterile purified water in an aqueous solution containing glycerol, ethylenediaminetetraacetic acid, and sodium chloride for 30-90 minutes, wherein the osmotic pressure of the aqueous solution is 280-650mOsm / L (milliosmole per liter), and 3 to 10 liters of the aqueous solution are added per kilogram of pig skin tissue.

[0029] In an exemplary embodiment, step 6) includes: aseptically packaging the pig skin tissue, the packaged pig skin tissue, by weight percentage, is composed of: 20.000% to 30.000% of virus-inactivated and sterilized pig skin tissue, 0.500% to 5.000% of glycerol, 0.029% to 0.292% of ethylenediaminetetraacetic acid, 0.200% to 0.600% of sodium chloride and 64.108% to 79.271% of water.

[0030] In an exemplary embodiment, step 7) includes: placing the encapsulated pig skin tissue in a low-temperature environment below -20°C for cell freezing and inactivation.

[0031] In an exemplary embodiment, step 8) includes: after low-temperature freezing and inactivation, long-term storage at -30°C to 30°C is performed and the product can be used immediately after opening.

[0032] The third aspect of the present application provides a skin repair material prepared by the above method.

[0033] The fourth aspect of the present application provides an application of a skin repair material in the preparation of a skin defect repair material.

[0034] In an exemplary embodiment, the skin defects include: skin defects caused by skin trauma, burns, donor site wounds, diabetic foot and other chronic wounds.

[0035] Compared with the prior art in this field, this application has the following technical effects:

[0036] The skin repair material in the present application has high stability and low immunogenicity, can be stored for a long time at an environment of -30°C to 30°C, and can be used immediately after opening; there are no live pig skin cells in the pig skin tissue after the frozen cell-inactivated treatment, which further reduces the immunogenicity and improves the safety of the product.

[0037] In the preparation method of the present application, the pig skin tissue is first immersed in a sodium chloride solution containing 2.0% to 5.0%, and then 0.05% to 0.15% peracetic acid is added to perform virus inactivation and sterilization treatment, which can not only play a role in virus inactivation and sterilization, but also reduce the side effects of peracetic acid treatment. After immersion, the product can be stored for a long time at -30°C to 30°C.

[0038] The low immunogenicity skin repair material disclosed in the present application can be directly transplanted into the wound surface of human skin defects to help repair the wound surface.

[0039] In the experiment of transplanting the skin repair material into the full-thickness skin defect wound of rats, the skin repair material can grow attached to the wound after transplantation, and the surface of the skin repair material in direct contact with the skin defect wound (inner surface) has rat cells growing in and new blood vessels forming. Part of the skin tissue of the skin repair material is embedded in the wound surface for symbiosis, and is absorbed and utilized to participate in the formation of new rat skin. The epithelialization of the rat skin defect wound occurs in the middle layer of the transplanted skin, and the pig skin surface (outer surface) that is not in direct contact with the skin defect wound falls off automatically. There is no significant difference in the repair effect of the skin repair material and the rat allogeneic skin graft.

[0040] The low-immunogenicity skin repair material prepared by the method of the present application is transplanted into the wound surface of heterologous skin. Even without decellularization treatment, it can be embedded in the tissue and coexist, and participate in the regeneration of new skin through tissue remodeling.

[0041] Other features and advantages of the present application will be described in the following description, and partly become apparent from the description, or be understood by implementing the present application. Other advantages of the present application can be realized and obtained by the schemes described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] The accompanying drawings are used to provide an understanding of the technical solution of the present application and constitute a part of the specification. Together with the embodiments of the present application, they are used to explain the technical solution of the present application and do not constitute a limitation on the technical solution of the present application.

[0043] Figure 1 A schematic diagram of the production process of the skin repair material for this application;

[0044] Figure 2 This is a hematoxylin-eosin staining micrograph of a cross section of the skin repair material prepared in Example 3 of the present application;

[0045] Figure 3 The growth of the transplanted skin in the full-thickness skin defect repair experiment of Sprague Dawley rats in Example 3 of the present application;

[0046] Figures 4 to 5 This is a pathological analysis diagram of wound repair after skin repair material transplantation and suture in Example 3 of the present application. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical scheme and advantages of the present application clearer, the embodiments of the present application will be described in detail below. Examples of these preferred embodiments are illustrated in the accompanying drawings. The embodiments of the present invention shown in the accompanying drawings and described according to the accompanying drawings are merely exemplary, and the present application is not limited to these embodiments.

[0048] In the present application, unless otherwise specified, all preparation raw materials / components are commercially available products well known to those skilled in the art.

[0049] Example 1: Collection of pig skin tissue and sterilization with peracetic acid

[0050] Six-month-old Bama pigs (Sus scrofa scrofa 'Bama miniaturepig') weighing about 45 kg were selected. After gene editing, the α-1,3-galactosyltransferase gene (GTKO) was knocked out and the human complement regulatory protein (hCD46) gene was transferred in. After the Bama pigs were slaughtered and bled, the hair was shaved, the full-thickness skin tissue of the trunk was peeled off manually, the subcutaneous fat was removed, and the dirt on the epidermis was scraped off. The pig skin tissue was layered by machine. The thickness of the Bama pig skin is 2mm to 3mm, which can be divided into multiple layers for use, and the thickness of each layer is 0.3mm to 0.9mm.

[0051] The obtained pig skin tissue was washed twice with purified water, each time for 20 minutes;

[0052] The cleaned pig skin tissue was first immersed in a 5% sodium chloride solution, 4 liters of sodium chloride solution was added per kilogram of pig skin tissue, and the skin tissue was divided into 4 groups; then peracetic acid was added to each group until the final peracetic acid concentration reached 0.025%, 0.05%, 0.10%, and 0.20%, and each group was sterilized for 0 minutes, 30 minutes, 60 minutes, and 120 minutes, respectively; three pig skin tissue samples were taken from each group for microbial quantity detection, and the obtained microbial quantity results are shown in Table 1 below.

[0053] Table 1: Number of microorganisms per 100 cm2 of pig skin tissue (CFU) (mean ± error, N = 3)

[0054]

[0055] Meanwhile, the contaminated pig skin tissue was first immersed in a 2% sodium chloride solution, and 5 liters of sodium chloride solution was added per kilogram of pig skin tissue; then peracetic acid was added until the final peracetic acid concentration reached 0.075%, and sterilization was performed for 0 minutes, 2 minutes, 5 minutes, 15 minutes, 30 minutes or 60 minutes. Three pig skin tissue samples were taken from each group for microbial quantity detection, and the obtained microbial quantity results are shown in Table 2 below.

[0056] Table 2: Number of microorganisms per 100 cm2 of pig skin tissue (CFU) (mean error, N=3)

[0057] Processing time (minutes) Microbial count (CFU) 0 minutes 117665±9192 2 minutes 3.0±0.6 5 minutes 1.0±0.6 15 minutes 0.1±0.6 30 minutes 0.3±0.3 60 minutes 0.3±0.3

[0058] Conclusion: Treatment of pig skin tissue in sodium chloride solution with low concentration peracetic acid solution can effectively inactivate microorganisms.

[0059] Example 2: Peracetic acid virus inactivation treatment

[0060] In this embodiment, the indicator virus is selected in accordance with the indicator viruses recommended in the "Guidelines for Technical Review of Registration of Animal-derived Medical Devices" (revised in 2017) and the pharmaceutical industry standard of the People's Republic of China YY / T 0771.3-2009 "Animal-derived Medical Devices Part 3: Confirmation of Removal and Inactivation of Viruses and Transmissible Spongiform Encephalopathy (TSE) Factors". Four representative viruses were selected: vesicular stomatitis virus (VSV), pseudorabies virus (PRV), poliovirus sabinI type (PV1) and porcine parvovirus (PPV).

[0061] The pig skin tissue with a thickness of 0.3 mm to 1.0 mm in Example 1 was selected for virus inactivation experiment, and the steps were as follows:

[0062] 1) Cut the cleaned pig skin tissue sample into 2 cm × 3 cm pieces and put them into the virus solution for infection;

[0063] 2) Control group: The pig skin tissue sample after being exposed in step 1) was extracted with 10 times the sample volume of culture medium as a zero-point control sample and frozen at -70°C for testing;

[0064] 3) Treatment group: Add 4 times the weight of the sample to the pig skin tissue sample in step 1) with a virus inactivation solution (containing 5% sodium chloride and 0.10% peracetic acid), and pour out the virus inactivation solution after the prescribed time treatment; add a corresponding volume of 0.9% sodium chloride solution to rinse for 10-20 minutes, and then use 10 times the sample volume of culture medium for extraction as an inactivated sample, and freeze it at -70°C for inspection;

[0065] 4) Stop solution control: dilute the virus solution with culture medium as the stop solution control and freeze at -70°C for testing.

[0066] The virus activity titration test in pig skin tissue samples adopts 96-well plate cytopathic method (culture plate is placed in 37 ℃ and 5% CO2 incubator, and 8 replicate wells are set for each dilution), and the titer value of residual indicator virus at each sampling time point is determined.

[0067] The virus reduction factor was calculated based on the virus titer test value and the sample volume before and after virus inactivation / removal. The results are shown in Tables 3 to 6 below.

[0068] Table 3: Inactivation effect of VSV virus in samples treated with 0.10% peracetic acid solution for different time periods

[0069]

[0070]

[0071] Note: -0.5LgTCID 50 This is the detection limit of this test method.

[0072] Table 4: Effect of 0.10% peracetic acid solution treatment on PRV virus inactivation in samples at different time periods

[0073]

[0074] Note: -0.5LgTCID 50 This is the detection limit of this test method.

[0075] Table 5: Inactivation effect of PV1 virus in samples treated with 0.10% peracetic acid solution for different time periods

[0076]

[0077] Note: -0.5LgTCID 50 This is the detection limit of this test method.

[0078] Table 6: Effect of 0.10% peracetic acid solution treatment on PPV virus inactivation in samples at different time periods

[0079]

[0080]

[0081] Note: -0.5LgTCID 50 This is the detection limit of this test method.

[0082] The results showed that the virus could be effectively inactivated by treating pig skin tissue with a low concentration of peracetic acid solution in a sodium chloride solution.

[0083] Example 3: Animal experiment of low immunogenic skin repair material

[0084] 1) Preparation of low immunogenic skin repair materials:

[0085] The skin tissue of 6-month-old gene-edited Bama pigs was selected, and the hair and subcutaneous fat were removed. After scraping off the dirt on the epidermis, the pig skin tissue was divided into multiple layers by a machine, and the thickness of each layer was 0.7mm to 1.0 mm;

[0086] The pig skin tissue was washed with purified water three times, 10 minutes each time;

[0087] The cleaned pig skin tissue is first immersed in a 5% sodium chloride solution, with 4 liters of sodium chloride solution added per kilogram of pig skin tissue; then peracetic acid is added until the final peracetic acid concentration reaches 0.075% and treated for 20 minutes;

[0088] After discarding the peracetic acid solution, rinse once with sterile purified water for 30 minutes each time;

[0089] Soak the washed pig skin tissue in an aqueous solution containing glycerol, ethylenediaminetetraacetic acid, and sodium chloride for 30-90 minutes, adding 4 liters of the aqueous solution per kilogram of pig skin tissue. The contents of the components in the aqueous solution are as follows by weight percentage: 4.0% glycerol, 0.117% ethylenediaminetetraacetic acid, and 0.500% sodium chloride, and the osmotic pressure of the aqueous solution is 650mOsm / L;

[0090] The processed pig skin tissue was packaged in a sterile aluminum foil bag, and the packaged pig skin tissue was placed in a -25°C freezer to freeze overnight to inactivate cells;

[0091] After freezing and inactivation, the product is stored in a refrigerator at 2°C to 10°C to obtain a skin repair material with low immunogenicity.

[0092] The composition of the skin repair material was measured, and the results showed that its ingredients and composition, by weight percentage, were: 23.3% pig skin tissue, 3.3% glycerol, 0.08% ethylenediaminetetraacetic acid, 0.39% sodium chloride, and 72.93% water.

[0093] 2) Determination of DNA content in pig skin tissue:

[0094] The low immunogenic skin repair material sample obtained in 1) was soaked in physiological saline for 30 minutes, 100-150 mg of the sample was taken, and after complete digestion with proteinase K at 62°C, the DNA content in the sample was detected in accordance with standard YY / T0606.25-2014 "Tissue Engineering Medical Products, Part 25, Determination of Residual DNA in Animal-Derived Biomaterials: Fluorescence Staining Method" and Invitrogen's Quanti-iT PicoGreen dsDNA kit.

[0095] The results showed that the DNA content in the skin repair material sample was 199.5±24.4 ng / mg (dry weight), which was only slightly less than 10% of the DNA content in fresh pig skin tissue without any treatment (220.1±25.8 ng / mg dry weight), indicating that there was no decellularization in the sample. Figure 2 As shown, the original basic natural structure of the gene-edited pig skin tissue was retained.

[0096] 3) Repair of full-thickness skin defects in Sprague Dawley rats:

[0097] Twelve 6-week-old SD rats were selected, and a 15 mm × 15 mm full-thickness skin defect was made on the back of the rats. The skin repair material that had been cleaned, virus-inactivated and sterilized in 1) was used to cover the defect. The transplanted skin graft was sutured at the four corners of the wound, and a wound dressing was applied externally to protect the wound.

[0098] Twelve rats were randomly divided into three groups. One group of animals was sacrificed 5 days, 12 days and 20 days after surgery to observe the progress of wound healing and to make pathological sections to evaluate the growth of the transplanted skin. The results are as follows: Figures 3 to 5 shown.

[0099] Figure 3 In the experiment, after the skin graft was sutured, it was initially tightly attached to the wound. After 5 days, the wound contracted and the edge of the skin graft began to separate from the wound by 1-2 mm, but the middle part still grew together. After 12 days, the separation between the edge of the skin graft and the wound became more obvious, and the middle part of the wound still grew together. After 20 days, the skin graft fell off completely and the wound was basically completely epithelialized.

[0100] Figures 4 to 5 In the experiment, the skin graft of the skin repair material was sutured to the full-thickness skin defect wound, but did not fully participate in the regeneration of the skin. Instead, an interesting stratification phenomenon occurred. The inner layer of the dermis where the skin graft was in direct contact with the wound had host cells growing in and blood vessels forming, and this part was eventually absorbed and utilized to participate in the formation of new skin tissue; there was a severe inflammatory transition zone between the inner and outer layers; the wound repaired epithelialized from the outside to the inside along the inflammatory transition zone, the inner skin tissue was embedded in the symbiosis, and the outer tissue of the skin graft (including the epidermis and part of the dermis) was separated and fell off.

[0101] As the host cells grow in and vascularize, the original dermis structure disappears and a remodeling zone without obvious structural features is formed. The remodeling zone gradually remodels into new dermis tissue. After the skin defect wound is completely epithelialized, the dermis remodeling is far from complete.

[0102] Conclusion: The low-immunogenic porcine skin prepared by the method of the present application can be transplanted into a xenogeneic skin wound and can be embedded in the tissue for symbiosis even without decellularization, and participate in the regeneration of new skin through tissue remodeling.

[0103] Although the embodiments of the present application have been shown and described above, it can be understood that the above embodiments are exemplary and cannot be understood as limitations on the present application. Ordinary technicians in this field can change, modify, replace and modify the above embodiments within the scope of the present application.

[0104] References:

[0105] [1] Ke Linnan, Fang Yu, Shan Yongqiang, et al. α-Gal antigen and immunogenicity risk control of animal-derived medical devices[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(25): 4051-4056.

[0106] KeL N,Fang Y,ShanYQ,et al.Alpha-Galantigenandimmunityriskcontrolofanimal-derivedmedicaldevices[J].ChinJTissueEngRes,2014,18(25):4051-4056.

[0107] [2]GaliliU.AvoidingDetrimentalHuman ImmuneResponse AgainstMammalianExtracellular MatrixImplants[J].TissueEngineering Part BReviews,2014,21(2):231-241.

[0108] [3]Denner J.Why was PERVnot transmittedduringpreclinicalandclinicalxenotransplantationtrials and after inoculationof animals?[J].Retrovirology,2018,15(1):28.

Claims

1. A skin repair material, characterized in that: The skin repair material is composed of the following by weight percentage: 20.000% to 30.000% of virus-inactivated and sterilized pig skin tissue, 0.500% to 5.000% of propylene glycol, 0.029% to 0.292% of ethylenediaminetetraacetic acid, 0.200% to 0.600% of sodium chloride and 64.108% to 79.271% of water; wherein the pig skin tissue is the skin tissue of a gene-edited pig without α-Gal antigen.

2. The skin repair material according to claim 1, characterized in that: The skin repair material is composed of the following by weight percentage: 22.00% to 26.00% of virus-inactivated and sterilized pig skin tissue, 3.00% to 4.00% of glycerol, 0.10% to 0.15% of ethylenediaminetetraacetic acid, 0.40% to 0.5% of sodium chloride and 69.45% to 74.50% of water.

3. The skin repair material according to claim 2, characterized in that: The skin repair material is composed of the following by weight percentage: 25.00% of virus-inactivated and sterilized pig skin tissue, 4.00% of glycerol, 0.10% of ethylenediaminetetraacetic acid, 0.40% of sodium chloride and 70.50% of water.

4. A method for preparing the skin repair material according to any one of claims 1 to 3, comprising the following steps: 1) Collecting the skin tissue of gene-edited pigs without α-Gal antigen, removing the hair and layering; 2) Use purified water to clean surface dirt; 3) Virus inactivation and sterilization with peracetic acid solution; 4) Rinse with sterile purified water to remove peracetic acid; 5) Soaking in an aqueous solution containing glycerol, ethylenediaminetetraacetic acid, and sodium chloride; 6) Sterile packaging; 7) Low temperature inactivation; 8) Store for future use.

5. The method according to claim 4, characterized in that Step 1) comprises: a pig whose α-1,3-galactosyltransferase gene is knocked out in the pig genome by gene editing, pig skin tissue is collected after slaughter, hair and subcutaneous fat are removed, the pig skin tissue is layered to obtain multiple layers of cortex, each layer having a thickness of 0.3 mm to 0.9 mm, and the cortex is cut into a desired size.

6. The method according to claim 4 or 5, characterized in that: Step 3) comprises: soaking the cleaned pig skin tissue in a sodium chloride solution, and then adding peracetic acid until the final peracetic acid concentration reaches 0.05% to 0.15%, to inactivate the virus and sterilize; Optionally, the concentration of the sodium chloride solution is 2.0% to 5.0%, and 3 to 10 liters of sodium chloride solution are added per kilogram of the pig skin tissue; Optionally, the time for virus inactivation and sterilization is 10 to 40 minutes.

7. The method according to claim 4 or 5, characterized in that: Step 4) comprises: washing the virus-inactivated and sterilized pig skin tissue with sterile purified water for 5-30 minutes.

8. The method according to claim 4 or 5, characterized in that: Step 5) comprises: soaking the pig skin tissue rinsed with sterile purified water in an aqueous solution containing glycerol, ethylenediaminetetraacetic acid, and sodium chloride for 30-90 minutes, wherein the osmotic pressure of the aqueous solution is 280-650mOsm / L, and 3-10 liters of the aqueous solution are added per kilogram of the pig skin tissue.

9. The method according to claim 4 or 5, characterized in that: Step 6) comprises: aseptically encapsulating the pig skin tissue, wherein the encapsulated pig skin tissue is composed of the following by weight percentage: 20.000% to 30.000% of virus-inactivated and sterilized pig skin tissue, 0.500% to 5.000% of glycerol, 0.029% to 0.292% of ethylenediaminetetraacetic acid, 0.200% to 0.600% of sodium chloride and 64.108% to 79.271% of water; and / or Step 7) includes: placing the encapsulated pig skin tissue in a low temperature environment below -20°C for cell freezing inactivation; and / or Step 8) comprises: after low-temperature freezing and inactivation, long-term storage at -30°C to 30°C is performed and the mixture can be used immediately after opening.

10. A skin repair material prepared by the method according to any one of claims 4 to 9.

11. Use of the skin repair material according to any one of claims 1 to 3 and 10 in preparing a skin defect repair material.

12. The use according to claim 11, characterized in that: The skin defects include: skin wounds, burns, donor site wounds, diabetic foot and other chronic wounds.

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