Preparation process of high-efficiency acellular dermal matrix

Abstract

The application discloses a preparation process of efficient acellular dermal matrix, and the acellular dermal matrix is prepared through the following steps: obtaining dermal tissue through pretreatment of animal fetal skin, and then performing specific acellular treatment and defatting treatment processes, and finally performing crosslinking, freeze-drying and sterilization. The acellular dermal matrix preparation process can effectively remove impurities and immunogenic substances, and at the same time, the activity of extracellular matrix is maximally protected. The obtained acellular dermal matrix product has complete triple-helix structure of collagen, low residues of DNA and alpha-Gal antigen, extremely low fat content, no cytotoxicity and high safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, and particularly relates to a preparation process of high-efficiency acellular dermal matrix. BACKGROUND

[0002] The acellular dermal matrix is a biological scaffold material prepared by removing the cellular components in the skin tissue through physical, chemical and biological methods, and retaining the extracellular matrix structure mainly composed of collagen. It is widely used in many medical fields such as chronic wound repair and tissue engineering materials due to its good biocompatibility and ability to induce tissue regeneration.

[0003] In the prior art, the preparation of the acellular dermal matrix mainly relies on the combined use of chemical detergents, acid-base treatment and enzyme treatment. For example, Chinese Patent Application CN109364298A discloses a preparation method of acellular dermal matrix material, which uses sodium dodecyl benzene sulfonate for defatting, sodium hydroxide for alkaline treatment to remove hair and inactivate viruses, and biochemical pancreatin for decellularization treatment, and finally uses crosslinking agent GPE for crosslinking modification. Although this method can achieve cell removal and virus inactivation to some extent, the use of strong surfactants and high-efficiency acid-base reagents can easily damage the fragile bioactive components in the extracellular matrix, resulting in damage to the matrix structure and loss of biological activity, and further affecting its ability to guide regeneration. Chinese Patent Application CN116019982A discloses an acellular dermal matrix and its preparation method and application. The animal fetal skin is first pretreated with purified water and 0.1-1wt% sodium chloride solution, then defatted with a defatting reagent (40-80wt% ethanol solution, 0.5-2wt% sodium hydroxide solution or 50-100wt% isopropanol solution), disinfected with a disinfectant, and then decellularized with a decellularization reagent (0.5-2wt% sodium hydroxide solution or 0.2-2wt% trypsin-EDTA solution), and finally treated with 0.2-5U / mL α-galactosidase solution to remove α-Gal antigen, and then sterilized. This method uses animal fetal skin as raw material to reduce the innate immunogenicity, and uses α-galactosidase to specifically remove the key α-Gal antigen in xenotransplantation, but the defatting reagent and decellularization reagent used in this method will inevitably damage the fine natural conformation and active components in the extracellular matrix during defatting and decellularization, especially the components such as elastin, fibronectin and glycosaminoglycans (such as chondroitin sulfate) which have important biological functions and have low content in fetal calf skin, are particularly damaged, resulting in that the biological activity and regeneration induction ability of the final product cannot reach the optimum. In addition, the current preparation method of acellular dermal matrix needs to be treated with enzymes, which has high production cost, and if the enzyme treatment process is not properly controlled, enzyme residues will be introduced, which will introduce the risk of immunogenicity.

[0004] Therefore, it is urgent to develop a new preparation method, which can maximize the protection of active ingredients such as elastin, fibronectin, laminin and chondroitin sulfate in extracellular matrix under the premise of efficient cell removal, low antigen residue and complete virus inactivation, maintain the natural three-dimensional structure and biological activity thereof, and ensure that the final product has good biocompatibility, mechanical properties and clinical safety. SUMMARY

[0005] In order to overcome the shortcomings of the prior art, the present application aims to provide a preparation process of efficient acellular dermal matrix.

[0006] The present application is realized by the following technical solutions:

[0007] In a first aspect, the present application provides a preparation process of efficient acellular dermal matrix, comprising the following steps:

[0008] S1. Pretreatment: taking animal fetal skin, removing the hair on the surface of the animal fetal skin, soaking and treating with 0.05-0.2wt% sodium hypochlorite solution, then washing with water and filtering out the water; removing the upper epidermis layer and lower fat layer, retaining the middle dermis layer, washing and cutting into small pieces to obtain dermis tissue;

[0009] S2. Decellularization treatment: soaking the dermis tissue in 3-8wt% sodium chloride solution for decellularization treatment, and then washing with water;

[0010] S3. Defatting treatment: soaking the dermis tissue after decellularization treatment in step S2 in 0.08-0.4wt% sodium hydroxide solution, 2-5wt% sodium carbonate solution, 0.1-0.8wt% Triton X-100 solution and 50-80wt% isopropanol solution in sequence, and washing with water after each soaking treatment;

[0011] S4. Crosslinking: soaking the dermis matrix obtained after defatting treatment with a crosslinking agent solution, stirring for crosslinking reaction, and then washing with water;

[0012] S5. Freeze-drying and sterilization: freeze-drying the dermis matrix after crosslinking modification, and then irradiation sterilization to obtain the acellular dermis matrix.

[0013] Further, in step S1, the animal fetal skin is selected from bovine fetal skin. The fetal bovine skin is relatively thin, the collagen fibers are finer, the crosslinking is less, the structure is more uniform, and the complete hair follicle, gland and other structures have not yet been formed, so it is easier to remove cells and alpha-gal antigens, and obtain an acellular dermis matrix with low immunogenicity.

[0014] Further, in step S1, the soaking treatment time is 5-15 min; the water washing is purified water washing, and the washing times are at least 3 times. In step S1 of the present application, the animal fetal skin is soaked in sodium hypochlorite solution for treatment, which can effectively remove microorganisms and make the endotoxin of the final product lower.

[0015] Further, in step S1, the thickness of the intermediate dermis layer is 0.3-0.8 mm.

[0016] Further, in step S2, the decellularization treatment is carried out under stirring, the mass / volume ratio of the material to the liquid is 1 g:8-16 mL, the stirring speed is 100-300 rpm, and the treatment time is 6-18 h; the water washing is injection water washing, and the washing times are at least 3 times.

[0017] Further, in step S3, when using sodium hydroxide solution for soaking treatment, the mass / volume ratio of the material to the liquid is 1 g:5-15 mL, and the soaking time is 30-90 min; when using sodium carbonate solution for soaking treatment, the mass / volume ratio of the material to the liquid is 1 g:15-30 mL, and the soaking time is 2-4 h; when using Triton X-100 solution for soaking treatment, the mass / volume ratio of the material to the liquid is 1 g:10-30 mL, and the soaking time is 30-100 min; when using isopropyl alcohol for soaking treatment, the mass / volume ratio of the material to the liquid is 1 g:5-20 mL, and the soaking time is 6-12 h.

[0018] Further, in step S3, the defatting treatment is carried out as follows: after the dermis tissue treated by decellularization in step S2 is soaked in sodium hydroxide solution, it is washed with injection water until neutral; then it is soaked in sodium carbonate solution and washed with injection water until neutral; then it is soaked in Triton X-100 solution and washed with injection water at least 5 times; finally, it is soaked in isopropyl alcohol and washed with injection water at least 6 times.

[0019] Further, in step S4, the crosslinking agent solution is an aqueous solution of carbodiimide / N-hydroxysuccinimide composite crosslinking agent, wherein the concentration of carbodiimide is 30-70 mM, and the molar ratio of carbodiimide to N-hydroxysuccinimide is 1.5-3.5:1.

[0020] Further, in step S4, the stirring speed of the crosslinking reaction is 150-350 rpm, and the reaction time is 18-36 h; the water washing is injection water washing, and the washing times are at least 8 times.

[0021] Further, in step S5, the freeze-drying is performed by using a freeze vacuum dryer to freeze-dry the cross-linking modified dermal matrix. Preferably, the freeze-drying is performed under the following conditions: pre-freezing (-80 to -20℃, 4-8h), primary drying (-60 to -40℃, 20Pa, 28-48h) and secondary drying (25-35℃, 4-8h).

[0022] Further, in step S5, the irradiation sterilization is performed by using a 25KGY cobalt-60 irradiation sterilization.

[0023] In a second aspect, the present application provides a decellularized dermal matrix prepared by the preparation method of the present application.

[0024] In a third aspect, the present application provides the use of the decellularized dermal matrix in chronic wound repair or tissue engineering material.

[0025] The present application has the following advantages:

[0026] The preparation method of the decellularized dermal matrix of the present application optimizes the decellularization and defatting processes. The decellularization is performed by using a specific concentration of sodium chloride solution to efficiently lyse cells and release cell components. The defatting is performed by using a low concentration of sodium hydroxide solution to preliminarily remove fat and change the properties of cell fragments, followed by using a sodium carbonate solution to further saponify fat and provide a mild alkaline environment, thereby effectively removing fat components by combining sodium hydroxide solution and sodium carbonate solution. Then, the decellularized dermal matrix is soaked in Triton X-100 solution to further remove fat and effectively remove cell membrane fragments, DNA and key α-Gal antigens. Finally, isopropyl alcohol treatment can completely remove residual lipids and assist in disinfection. The whole process is mild, and subsequent enzyme treatment is not required, which can effectively remove impurities and immunogenic substances, while maximizing the protection of active ingredients such as fragile elastin, fibronectin, laminin and chondroitin sulfate in the extracellular matrix. Subsequent moderate cross-linking by EDC / NHS effectively enhances the mechanical properties and anti-degradation ability of the matrix, prolongs the degradation time of the product in the body, and has low cytotoxicity. The decellularized dermal matrix prepared by the preparation process of the present application has complete triple helix structure of collagen, low residual DNA and α-Gal antigens, complete virus inactivation, extremely low fat content, extremely low endotoxin content, no cytotoxicity and high safety. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Circular dichroism spectrum of the decellularized dermal matrix prepared in Example 1.

[0028] Figure 2 Circular dichroism spectrum of the decellularized dermal matrix prepared in Comparative Example 3.

[0029] Figure 3 Circular dichroism spectrum of the acellular dermal matrix prepared for Comparative Example 5. DETAILED DESCRIPTION

[0030] The following examples are provided to better enable those skilled in the art to further understand and practice the application, and are not intended to limit the scope of the application in any way. Any product derived from the application or any modification of the application in combination with other prior art discloses the same or similar products as the application, and falls within the scope of the application.

[0031] The specific experimental steps or conditions are not indicated in the examples, which can be carried out according to the conventional experimental steps described in the literature in the art or the operation or conditions. The reagents or instruments used are not indicated by the manufacturer, which are conventional reagent products that can be obtained by purchase.

[0032] Unless otherwise specified, the room temperature or normal temperature in the examples of the application refers to 25±1℃.

[0033] Example 1

[0034] A preparation process of a high-efficiency acellular dermal matrix is as follows:

[0035] S1. Pretreatment: Take the fetal bovine skin, remove the hair on the surface of the fetal bovine skin with scissors, immerse in 0.1% sodium hypochlorite solution for 10 min, then wash with purified water for 3 times, and filter out the water; remove the upper epidermis layer and lower fat layer with a special skinning machine, only keep the middle dermis layer with a thickness of about 0.5 mm, wash with purified water, and cut into small pieces of about 0.5 cm×0.5 cm to obtain bovine dermis tissue;

[0036] S2. Decellularization treatment: immerse the bovine dermis tissue in 5% sodium chloride solution according to the solid-liquid mass volume ratio of 1 g: 12 mL, and carry out decellularization treatment under the condition of 150 rpm stirring for 12 h, then wash with water for injection for 3 times;

[0037] S3. Defatting treatment: immerse the dermis tissue after decellularization treatment in step S2 in 0.1% sodium hydroxide solution according to the solid-liquid mass volume ratio of 1 g: 12 mL for 60 min, and wash with water for injection until neutral; then immerse in 3% sodium carbonate solution according to the solid-liquid mass volume ratio of 1 g: 20 mL for 3 h, and wash with water for injection until neutral; then immerse in 0.5% Triton X-100 solution according to the solid-liquid mass volume ratio of 1 g: 20 mL for 60 min, and wash with water for injection for 5 times; finally, immerse in 70% isopropyl alcohol according to the solid-liquid mass volume ratio of 1 g: 10 mL for 8 h, and wash with water for injection for 6 times;

[0038] S4. Crosslinking: After the obtained dermal matrix after defatting treatment was filtered dry, it was infiltrated with EDC / NHS solution (EDC concentration was 50 mM, EDC:NHS molar ratio was 2.5:1), crosslinking reaction was carried out at room temperature with 200 rpm stirring for 24 h, then it was washed with water for injection for 8 times;

[0039] S5. Freeze-drying and sterilization: the crosslinking modified dermal matrix was freeze-dried by using a freeze vacuum dryer, then it was sterilized by cobalt-60 25 KGY irradiation, to obtain the acellular dermal matrix.

[0040] Example 2

[0041] A preparation process of a high-efficiency acellular dermal matrix, the steps are as follows:

[0042] S1. Pretreatment: fetal bovine skin was taken, the hair on the surface of the fetal bovine skin was removed by scissors, then the fetal bovine skin was soaked in 0.05% sodium hypochlorite solution for 15 min, then it was washed with purified water for 3 times, and water was filtered out; the upper epidermis layer and the lower fat layer were removed by using a special skinning machine, only the middle dermis layer with a thickness of about 0.5 mm was reserved, after being washed with purified water, it was cut into small pieces with a size of about 0.5 cm x 0.5 cm, to obtain bovine dermis tissue;

[0043] S2. Decellularization treatment: the bovine dermis tissue was soaked in 5% sodium chloride solution according to the solid-liquid mass volume ratio of 1 g:16 mL, and decellularization treatment was carried out under the condition of 150 rpm stirring for 16 h, then it was washed with water for injection for 3 times;

[0044] S3. Defatting treatment: the dermis tissue after decellularization treatment in step S2 was soaked in 0.3% sodium hydroxide solution according to the solid-liquid mass volume ratio of 1 g:10 mL for 40 min, and it was washed with water for injection until it was neutral; then it was soaked in 2% sodium carbonate solution according to the solid-liquid mass volume ratio of 1 g:25 mL for 3.5 h, and it was washed with water for injection until it was neutral; then it was soaked in 0.2% Triton X-100 solution according to the solid-liquid mass volume ratio of 1 g:25 mL for 80 min, and it was washed with water for injection for 5 times; finally, it was soaked in 50% isopropyl alcohol according to the solid-liquid mass volume ratio of 1 g:10 mL for 10 h, and it was washed with water for injection for 6 times;

[0045] S4. Crosslinking: After the obtained dermal matrix after defatting treatment was filtered dry, it was infiltrated with EDC / NHS solution (EDC concentration was 50 mM, EDC:NHS molar ratio was 2.5:1), crosslinking reaction was carried out at room temperature with 200 rpm stirring for 24 h, then it was washed with water for injection for 8 times;

[0046] S5. Freeze-drying and sterilization: the crosslinking modified dermal matrix was freeze-dried by using a freeze vacuum dryer, then it was sterilized by cobalt-60 25 KGY irradiation, to obtain the acellular dermal matrix.

[0047] Example 3

[0048] A preparation process of efficient acellular dermal matrix, the steps are as follows:

[0049] S1. Pretreatment: take fetal bovine skin, remove the hair on the surface of the fetal bovine skin with scissors, soak in 0.2% sodium hypochlorite solution for 5 min, then wash with purified water for 3 times, filter out the water; remove the upper epidermis layer and lower fat layer with a special skinning machine, only keep the middle dermis layer with a thickness of about 0.5 mm, wash with purified water, then cut into small pieces of about 0.5 cm x 0.5 cm, obtain bovine dermis tissue;

[0050] S2. Acellular treatment: according to the solid-liquid mass volume ratio of 1g:10mL, the bovine dermis tissue is soaked in 8% sodium chloride solution, and the acellular treatment is carried out under the condition of 150rpm stirring for 8h, then washed with water for injection for 3 times;

[0051] S3. Defatting treatment: the dermis tissue after acellular treatment in step S2 is soaked in 0.1% sodium hydroxide solution according to the solid-liquid mass volume ratio of 1g:15mL for 80min, washed with water for injection until neutral; then soaked in 5% sodium carbonate solution according to the solid-liquid mass volume ratio of 1g:15mL for 2h, washed with water for injection until neutral; then soaked in 0.8% Triton X-100 solution according to the solid-liquid mass volume ratio of 1g:15mL for 40min, washed with water for injection for 5 times; finally, soaked in 70% isopropyl alcohol according to the solid-liquid mass volume ratio of 1g:10mL for 10h, washed with water for injection for 6 times;

[0052] S4. Crosslinking: the dermis matrix obtained after defatting treatment is dried, then soaked in EDC / NHS solution (EDC concentration is 70mM, EDC:NHS molar ratio is 3:1), crosslinking reaction is carried out at room temperature under the condition of 200rpm stirring for 30h, then washed with water for injection for 8 times;

[0053] S5. Freeze-drying and sterilization: the crosslinking modified dermis matrix is freeze-dried by using a freeze vacuum dryer, then sterilized by cobalt-6025KGY irradiation, to obtain an acellular dermis matrix.

[0054] Comparative example 1

[0055] The difference from example 1 is that the bovine dermis tissue obtained in step S1 pretreatment is first defatted, then acellular treatment is carried out, and after crosslinking, freeze-drying and sterilization, an acellular dermis matrix is prepared. The process conditions of each step are the same as in the example.

[0056] Comparative example 2

[0057] The difference from example 1 is that 2wt% sodium chloride solution is used for decellularization treatment in step S2; the remaining steps and process conditions are the same as those of example 1, and a decellularized dermal matrix is prepared.

[0058] Comparative example 3

[0059] The difference from example 1 is that 0.5wt% sodium hydroxide solution is used for decellularization treatment in step S2; the remaining steps and process conditions are the same as those of example 1, and a decellularized dermal matrix is prepared.

[0060] Comparative example 4

[0061] The difference from example 1 is that 0.1wt% hydrochloric acid solution is used for decellularization treatment in step S2; the remaining steps and process conditions are the same as those of example 1, and a decellularized dermal matrix is prepared.

[0062] Comparative example 5

[0063] The difference from example 1 is that the concentration of sodium hydroxide solution in step S3 is 0.5wt%; the remaining steps and process conditions are the same as those of example 1, and a decellularized dermal matrix is prepared.

[0064] Comparative example 6

[0065] The difference from example 1 is that the 0.5% Triton X-100 solution soaking treatment operation in step S3 is omitted; the remaining steps and process conditions are the same as those of example 1, and a decellularized dermal matrix is prepared.

[0066] I. Performance test:

[0067] 1. Characterization of collagen secondary structure in decellularized dermal matrix by circular dichroism spectroscopy

[0068] Literature reports that the circular dichroism of collagen has a maximum negative absorption peak near 200nm and a maximum positive absorption peak at 220nm, and the ratio Rpn of the peak values of the positive and negative absorption peaks is about 0.12, indicating that the collagen has a complete triple helix structure. As an example, the circular dichroism spectrum of example 1 is provided (as shown in Figure 1 The detection result is: the positive absorption peak is at 221nm, the CD value is 12.6527, the negative absorption peak is at 197.5nm, the CD value is -106.3, and the calculated Rpn is 0.119, indicating that the sample has a complete triple helix structure.

[0069] 2. DNA and α-Gal antigen residual detection

[0070] Referring to the method for determining the residual amount of exogenous DNA in the 2020 edition of the People's Republic of China Pharmacopoeia, the residual amount of DNA in the decellularized dermal matrix is detected.

[0071] The α-Gal antigen clearance rate in the acellular dermal matrix was detected according to YY / T 1561-2017 Tissue Engineering Medical Device Animal-derived Scaffold Material Residue.

[0072] 3. Total fat content detection

[0073] The total fat content in the acellular dermal matrix was detected according to GB 5009.168-2016 Food Safety National Standard Determination of Fat Acid Content in Food.

[0074] 4. In vitro cytotoxicity detection

[0075] The cytotoxicity of the acellular dermal matrix was detected according to GB / T 16886.5-2017 Medical Devices Biological Evaluation: Part 5: In Vitro Cytotoxicity Test. The cell survival rate quantitative detection was carried out using 100% sample test solution.

[0076] The detection results are shown in Tables 1 and 2:

[0077] Table 1. Detection results of each performance of the acellular dermal matrix prepared in Examples 1-3

[0078]

[0079] Table 2. Detection results of each performance of the acellular dermal matrix prepared in Comparative Examples 1-6

[0080]

[0081] The above results show that the preparation process of the acellular dermal matrix of the application can ensure more thorough removal of impurities and immunogenic substances, while maximizing the protection of the activity of the extracellular matrix; the obtained acellular dermal matrix product has complete triple helix structure of collagen, low residual DNA and α-Gal antigen, extremely low fat content, no cytotoxicity, and high safety.

[0082] Comparative Example 1 was first subjected to defatting treatment, and then subjected to decellularization treatment, which could not achieve good removal of impurities and immunogenic substances, and the DNA and total fat residues increased significantly, and the α-Gal antigen clearance rate decreased.

[0083] In Comparative Example 2, 2wt% sodium chloride solution was used for decellularization treatment in step S2, and the concentration of the sodium chloride solution was too low, the decellularization efficiency was reduced, the effect of subsequent defatting treatment was affected, and finally the DNA and fat content in the product were high, and the α-Gal antigen clearance rate decreased.

[0084] The impurities and immunogenic substances can be well removed, but the triple helix structure of collagen in the final product is changed, as shown in Figure 2 The basic substance (sodium hydroxide) is treated for too long, resulting in structure damage, and the positive peak disappears on the spectrum, indicating that the triple helix structure is destroyed.

[0085] In step S2 of Comparative Example 4, 0.1wt% hydrochloric acid solution is used for decellularization treatment, and the decellularization efficiency is reduced. The DNA and fat content in the final product is high, the clearance rate of α-Gal antigen is reduced, the cytotoxicity is increased, and the safety is greatly reduced.

[0086] In step S3 of Comparative Example 5, the concentration of sodium hydroxide solution is too high, although the total fat removal effect is better, but the detection result of circular dichroism spectrum shows that the positive peak disappears, as shown in Figure 3 , indicating that the triple helix structure of collagen disappears, that is, the biological activity disappears, indicating that the high concentration of sodium hydroxide solution can destroy the biological activity of the decellularized dermal matrix.

[0087] In step S3 of Comparative Example 6, the 0.5% Triton X-100 solution soaking treatment operation is omitted, and the impurities and immunogenic substances cannot be well removed. The fat content of the product is obviously increased, and the clearance rate of α-Gal antigen is reduced.

[0088] II. Detection results of two-step virus inactivation of Example 1

[0089]

[0090] The above results show that the preparation process of the decellularized dermal matrix of the application can effectively inactivate viruses and has high safety.

[0091] The preparation method of the decellularized dermal matrix of the application optimizes the decellularization and defatting processes. First, a specific concentration of sodium chloride solution is used for decellularization treatment, and then a specific concentration of sodium hydroxide solution, sodium carbonate solution, Triton X-100 solution and isopropanol are used for soaking treatment in sequence. This can effectively remove impurities and immunogenic substances, while maximizing the protection of active ingredients in the extracellular matrix. Subsequently, EDC / NHS is used for moderate crosslinking, which effectively enhances the mechanical properties and anti-degradation ability of the matrix, prolongs the degradation time of the product in the body, and has low cytotoxicity. The decellularized dermal matrix product prepared by the preparation process of the application has complete triple helix structure of collagen, low residual DNA and α-Gal antigen, complete virus inactivation, extremely low fat content, extremely low endotoxin content, no cytotoxicity, and high safety.

[0092] The above embodiments are merely exemplary but not limiting on the implementation. Based on the above description, those skilled in the art can make further changes or modifications to the embodiments in different forms. Here, it is not necessary or possible to enumerate all the embodiments. The obvious changes or modifications derived from the above description are still within the scope of the present application.

Claims

1. A process for the preparation of high efficiency acellular dermal matrix, characterized in that, The method comprises the following steps: S1. Pretreatment: taking animal fetal skin, removing the hair on the surface of the animal fetal skin, soaking and treating with 0.05-0.2wt% sodium hypochlorite solution, then washing with water and filtering out the water; removing the upper epidermis layer and the lower fat layer, retaining the middle dermis layer, washing and cutting into small pieces to obtain dermis tissue; S2. Decellularization treatment: soaking the dermis tissue in 3-8wt% sodium chloride solution for decellularization treatment, and then washing with water; S3. Defatting treatment: soaking the dermis tissue after decellularization treatment in step S2 in 0.08-0.4wt% sodium hydroxide solution, 2-5wt% sodium carbonate solution, 0.1-0.8wt% Triton X-100 solution and 50-80wt% isopropanol solution in sequence, and washing with water after each soaking treatment; S4. Crosslinking: after the defatting treatment, the dermis matrix obtained is filtered dry, soaked with a crosslinking agent solution, stirred for crosslinking reaction, and then washed with water; S5. Freeze-drying and sterilization: the dermis matrix after crosslinking modification is freeze-dried and then irradiation sterilized to obtain a decellularized dermis matrix.

2. The manufacturing process according to claim 1, characterized in that, In step S1, the animal fetal skin is selected from bovine fetal skin; the soaking treatment time is 5-15min; and the washing with water is washing with purified water, and the washing is performed at least 3 times.

3. The manufacturing process of claim 1, wherein, In step S2, the decellularization treatment is performed under stirring, the mass / volume ratio of the material to the liquid is 1g:8-16mL, the stirring speed is 100-300rpm, and the treatment time is 6-18h; the washing with water is washing with water for injection, and the washing is performed at least 3 times.

4. The manufacturing process of claim 1, wherein, In step S3, when the sodium hydroxide solution is used for soaking treatment, the mass / volume ratio of the material to the liquid is 1g:5-15mL, and the soaking time is 30-90min; when the sodium carbonate solution is used for soaking treatment, the mass / volume ratio of the material to the liquid is 1g:15-30mL, and the soaking time is 2-4h; when the Triton X-100 solution is used for soaking treatment, the mass / volume ratio of the material to the liquid is 1g:10-30mL, and the soaking time is 30-100min; when isopropanol is used for soaking treatment, the mass / volume ratio of the material to the liquid is 1g:5-20mL, and the soaking time is 6-12h.

5. The manufacturing process of claim 4, wherein, In step S3, the defatting treatment is performed as follows: after the dermis tissue after decellularization treatment in step S2 is soaked with the sodium hydroxide solution, the dermis tissue is washed with water for injection until neutral; then the dermis tissue is soaked with the sodium carbonate solution and washed with water for injection until neutral; then the dermis tissue is soaked with the Triton X-100 solution and washed with water for injection at least 5 times; finally, the dermis tissue is soaked with isopropanol and washed with water for injection at least 6 times.

6. The manufacturing process of claim 1, wherein, In step S4, the crosslinking agent solution is an aqueous solution of a carbodiimide / N-hydroxysuccinimide composite crosslinking agent, wherein the concentration of carbodiimide is 30-70mM, and the molar ratio of carbodiimide to N-hydroxysuccinimide is 1.5-3.5:

1.

7. The manufacturing process of claim 1, wherein, In step S4, the stirring speed for the crosslinking reaction is 150-350rpm, the reaction time is 18-36h, and the washing with water is washing with water for injection, and the washing is performed at least 8 times.

8. The manufacturing process of claim 1, wherein, In step S5, the freeze-drying is performed by using a freeze vacuum drier to freeze-dry the cross-linked and modified dermal matrix; and the irradiation sterilization is performed by using a cobalt-60 25KGY irradiation sterilization.

9. An acellular dermal matrix, characterized in that, obtained by the preparation process of any one of claims 1-8.

10. Use of the acellular dermal matrix of claim 9 in the preparation of a tissue engineering material.

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