Preparation method of acellular dermal matrix as well as product and application of acellular dermal matrix

Abstract

The invention relates to a preparation method of an acellular dermal matrix as well as a product and application of the acellular dermal matrix, the preparation method comprises the following steps: mixing skin tissues with an alkali solution, and removing hair, an epidermal layer and subcutaneous tissues to obtain a dermal layer; and mixing the corium layer with an alkali solution, and cleaning to obtain the corium layer. The method is easy and convenient to operate, the steps are greatly simplified, manpower and cost input is remarkably reduced, damage or residue of enzymes, surfactants and the like to the dermis structure is avoided, main components and the complete structure of the dermis layer are reserved to the maximum extent, and it is ensured that the obtained dermis matrix has good mechanical strength and biological performance.

Description

Technical Field

[0001] This invention belongs to the field of biomimetic materials technology, and relates to a method for preparing decellularized dermal matrix, its products, and applications. Background Technology

[0002] Traditional methods for preparing decellularized dermal matrix typically involve several cumbersome steps. First, hair on the donor skin surface must be removed mechanically, such as by hand shaving or using powered mechanical devices. Subsequently, to remove the epidermis, large equipment and techniques like shaving and slicing are usually required; the operation of this equipment relies heavily on experience and can easily lead to unevenness in the skin grafts. Furthermore, mechanical removal of subcutaneous tissues such as subcutaneous fat may be necessary, which is time-consuming and labor-intensive. After obtaining the dermis, subsequent decellularization typically relies on multiple steps using biochemical reagents such as enzymes (e.g., trypsin, nucleases) and surfactants.

[0003] The aforementioned traditional methods are complex, time-consuming, and labor-intensive, requiring a large amount of manpower and various reagents. The mechanical scraping step can also easily damage the dermal structure. Enzyme treatment is costly, and if not handled properly, it may affect the mechanical properties and biocompatibility of the dermal matrix. Furthermore, incomplete cleaning of surfactants can easily lead to cytotoxic risks.

[0004] Therefore, there is an urgent need to provide a simple method that requires less reagent to prepare decellularized dermal matrix. Summary of the Invention

[0005] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a method for preparing decellularized dermal matrix, its products, and applications.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing acellular dermal matrix, the method comprising:

[0008] (1) Mix the skin tissue with an alkaline solution to remove hair, epidermis and subcutaneous tissue to obtain the dermis;

[0009] (2) Mix the dermis with an alkaline solution and wash to obtain the product;

[0010] The concentration gradient of the alkaline solution in step (2) is 1-5.

[0011] This invention provides a method for preparing decellularized dermal matrix. By controlling the concentration of the alkali solution and the treatment temperature, efficient hair removal, epidermal removal, and subcutaneous tissue removal can be achieved. It utilizes the different tolerances of different structural layers of the skin to alkali, eliminating the need for complex mechanical scraping or enzymatic digestion steps. Simply by treating the skin with alkali under appropriate alkali concentration and temperature conditions, the hair, epidermis, and subcutaneous tissue can be separated from the dermis on their own. Then, they can be easily removed by gently scraping with a small handheld tool (such as a scraper or spatula) to obtain a complete dermis.

[0012] In the subsequent decellularization process of the dermis, only alkaline gradient treatment is used to achieve efficient exchange of antigenic substances in the dermis with the external environment, thereby achieving effects such as cell destruction, degreasing, nucleic acid removal, antigen removal, and virus inactivation.

[0013] Compared with traditional methods, the method of the present invention is simple to operate, the steps are greatly simplified, significantly reducing manpower and cost input, and avoiding damage or residue of the dermal structure by enzymes, surfactants, etc., preserving the main components and intact structure of the dermal layer to the greatest extent, and ensuring that the obtained dermal matrix has good mechanical strength and biological properties.

[0014] Preferably, the mass percentage of alkali in the alkaline solution in step (1) is 1-10%, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0015] Preferably, the ratio of skin tissue to alkaline solution is 1 g:(4-20) mL. The specific point values ​​in (4-20) can be selected from 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0016] Preferably, the mixing temperature in step (1) is 4-42°C, and the processing time for a single treatment is 0.5-48 h.

[0017] Temperatures can be selected from 4℃, 8℃, 10℃, 12℃, 15℃, 18℃, 20℃, 22℃, 25℃, 28℃, 30℃, 32℃, 35℃, 38℃, 40℃, 42℃, etc., and time can be selected from 0.5 h, 0.8 h, 1 h, 2 h, 5 h, 10 h, 15 h, 20 h, 25 h, 30 h, 35 h, 40 h, 45 h, 48 h, etc. Other specific point values ​​within the above ranges can also be selected, which will not be elaborated here.

[0018] Preferably, the skin tissue includes human skin, sheepskin, cowhide, pigskin, donkey skin, horse skin, rabbit skin, or rat skin.

[0019] Preferably, the concentration of the alkaline solution each time is not higher than the concentration of the previous alkaline solution.

[0020] The concentration gradient of the alkaline solution can be selected as 2, 3, 4, or 5.

[0021] Preferably, the mass percentage of the alkaline solution in step (2) is 0.5-4%, for example, 0.5%, 0.8%, 1%, 1.2%, 1.5%, 1.8%, 2%, 2.2%, 2.5%, 2.8%, 3%, 3.2%, 3.5%, 3.8%, 4%, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0022] Preferably, the ratio of the dermis to the alkaline solution in step (2) is 1 g:(4-20) mL. The specific point values ​​in (4-20) can be selected from 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. Other specific point values ​​within the above range can be selected, and will not be described in detail here.

[0023] Preferably, the temperature of the treatment in step (2) is 4-45℃, and the time for a single treatment is 1-24 h.

[0024] Temperatures can be selected from 4℃, 8℃, 10℃, 12℃, 15℃, 18℃, 20℃, 22℃, 25℃, 28℃, 30℃, 32℃, 35℃, 38℃, 40℃, 42℃, 45℃, etc., and time can be selected from 1 h, 2 h, 5 h, 10 h, 12 h, 15 h, 18 h, 20 h, 22 h, 24 h, etc. Other specific point values ​​within the above ranges can also be selected, which will not be elaborated here.

[0025] Preferably, the alkali includes sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, and ammonia, and more preferably sodium hydroxide and / or potassium hydroxide.

[0026] Preferably, the cleaning specifically includes cleaning the dermis to neutrality, and the reagents used for cleaning include one or more of PBS buffer, deionized water, sodium chloride solution, or calcium chloride solution.

[0027] In a second aspect, the present invention provides an acellular dermal matrix prepared according to the method for preparing acellular dermal matrix as described in the first aspect.

[0028] Thirdly, the present invention provides an application of the decellularized dermal matrix prepared according to the second aspect in the preparation of biomimetic materials.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] Compared with traditional methods, the method of the present invention is simple to operate, the steps are greatly simplified, significantly reducing manpower and cost input, and avoiding damage or residue of the dermal structure by enzymes, surfactants, etc., preserving the main components and intact structure of the dermal layer to the greatest extent, and ensuring that the obtained dermal matrix has good mechanical strength and biological properties. Attached Figure Description

[0031] Figure 1 These are photographs of the decellularization process in Example 1. Image A shows the image before decellularization; A-1 is the subcutaneous tissue surface, and A-2 is the hair surface. Image B shows the image after treatment in sodium hydroxide at 37°C for 2 hours, after which hair, epidermis, subcutaneous fat, and other structural layers have been scraped off; B-1 is the scraped subcutaneous tissue surface, and B-2 is the scraped hair and epidermis surface. Image C shows both sides after alkali treatment. Image D shows both sides after washing with 1×PBS solution until neutral.

[0032] Figure 2 This is an image of a section stained with hematoxylin and eosin (HE) before decellularization.

[0033] Figure 3 This is an image of a decellularized section stained with hematoxylin and eosin (HE).

[0034] Figure 4 The images show the surface morphology and longitudinal section morphology of the decellularized bovine dermal matrix. A and B represent the microscopic morphology of the two sides of the decellularized bovine dermal matrix, while C and D represent the longitudinal section morphology of the decellularized bovine dermal matrix. Detailed Implementation

[0035] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0036] Example 1

[0037] This embodiment provides a method for preparing acellular dermal matrix, the method comprising:

[0038] (1) Obtain bovine skin from the slaughterhouse, rinse it with water to remove blood and impurities, cut it to an appropriate size, place it in a polypropylene bottle ①, and add 3% sodium hydroxide solution at a solid-liquid ratio of 1:6, then tighten the cap. Place the polypropylene bottle ① in a constant temperature shaker and shake for 8 hours (25℃, 150 rpm). Then remove the polypropylene bottle ①, replace it with a new 2% sodium hydroxide solution at the same ratio, and place it in a constant temperature water bath at 37℃ for 2 hours. Then remove the bovine skin from the polypropylene bottle ①, and use a hand scraper to easily scrape off the hair, epidermis, subcutaneous fat, and other accessory layers to obtain the complete dermis.

[0039] (2) The above-mentioned dermal layer was transferred to a clean polypropylene bottle ②, and 1% sodium hydroxide solution was added at a solid-liquid ratio of 1:6. The bottle cap was tightened, and the bottle was placed in a constant temperature shaker for 16 h (25℃, 50 rpm). Then, the polypropylene bottle ② was removed, and a new 0.5% sodium hydroxide solution was added at the same ratio. The bottle was then placed in a constant temperature shaker for 12 h (25℃, 50 rpm). Subsequently, the bovine dermal layer was removed from the polypropylene bottle ② and thoroughly washed with 1×PBS solution until neutral to obtain decellularized bovine dermal matrix.

[0040] See diagram of the decellularization process. Figure 1 .

[0041] Example 2

[0042] This embodiment provides a method for preparing acellular dermal matrix, the method comprising:

[0043] (1) Obtain bovine skin from the slaughterhouse, rinse it with water to remove blood and impurities, cut it to an appropriate size, and place it in a polypropylene bottle ①. Add 2% sodium hydroxide solution at a solid-liquid ratio of 1:5 and tighten the cap. Place the polypropylene bottle ① in a constant temperature shaker and shake for 16 h (25℃, 50 rpm). Then remove the polypropylene bottle ①, replace it with a new 2% sodium hydroxide solution at the same ratio, and place it in a constant temperature shaker and shake for 2 h (37℃, 150 rpm). Then remove the bovine skin from the polypropylene bottle ① and use a hand scraper to easily scrape off the hair, epidermis, subcutaneous fat, and other accessory layers to obtain the complete dermis.

[0044] (2) Transfer the above-mentioned dermal layer into a clean polypropylene bottle ②, add 2% sodium hydroxide solution at a solid-liquid ratio of 1:5, tighten the cap, and continue to shake in a constant temperature shaker for 24 h (25℃, 50 rpm). Then remove the polypropylene bottle ②, replace it with a new 1% sodium hydroxide solution at the same ratio, and shake in a constant temperature shaker for another 24 h (25℃, 50 rpm). Then remove the bovine dermal layer from the polypropylene bottle ②, wash it thoroughly with deionized water until neutral, and obtain decellularized bovine dermal matrix.

[0045] Example 3

[0046] This embodiment provides a method for preparing acellular dermal matrix, the method comprising:

[0047] (1) Obtain cow skin from the slaughterhouse, rinse it with water to remove blood and impurities, cut it to an appropriate size, place it in a polypropylene bottle ①, and add 4% potassium hydroxide solution at a solid-liquid ratio of 1:6. Tighten the bottle cap. Place the polypropylene bottle ① in a constant temperature shaker and shake for 12 h (10℃, 100 rpm). Then remove the polypropylene bottle ①, replace it with a new 3% potassium hydroxide solution at the same ratio, and place it in a constant temperature shaker and shake for 1.5 h (37℃, 150 rpm). Then remove the cow skin from the polypropylene bottle ① and use a hand scraper to easily scrape off the hair, epidermis, subcutaneous fat, and other accessory layers to obtain the complete dermis.

[0048] (2) The above-mentioned dermal layer was transferred to a clean polypropylene bottle ②, and 1.5% potassium hydroxide solution was added at a solid-liquid ratio of 1:5. The bottle cap was tightened, and the bottle was placed in a constant temperature shaker for 12 h (25℃, 150 rpm). Then, the polypropylene bottle ② was removed, and a new 1% potassium hydroxide solution was added at the same ratio. The bottle was then placed in a constant temperature shaker for 12 h (25℃, 150 rpm). The solution was then replaced with a new 0.5% potassium hydroxide solution at the same ratio. The bottle was then placed in a constant temperature shaker for 12 h (25℃, 150 rpm). The bovine dermal matrix was then removed from the polypropylene bottle ②, thoroughly washed with 0.75% CaCl2 solution and deionized water until neutral, and then freeze-dried to obtain the decellularized bovine dermal matrix.

[0049] Example 4

[0050] This embodiment provides a method for preparing acellular dermal matrix, the method comprising:

[0051] (1) Obtain cow skin from the slaughterhouse, rinse it with water to remove blood and impurities, cut it to an appropriate size, place it in a polypropylene bottle ①, add 1% sodium hydroxide solution at a solid-liquid ratio of 1:6, and tighten the bottle cap. Place the polypropylene bottle ① in a constant temperature shaker and shake for 36 h (37℃, 100 rpm). Then remove the cow skin from the polypropylene bottle, and use a hand scraper to easily scrape off the hair, epidermis, subcutaneous fat, and other accessory layers to obtain the complete dermis.

[0052] (2) The bovine dermis layer was transferred to a clean polypropylene bottle ②, and 2% sodium hydroxide solution was added at a solid-liquid ratio of 1:5. The bottle cap was tightened, and the bottle was placed in a constant temperature shaker for 8 h (25℃, 150 rpm). Then, the polypropylene bottle ② was removed, and a new 1% sodium hydroxide solution was added at the same ratio. The bottle was then placed in a constant temperature shaker for 8 h (25℃, 150 rpm). The solution was then replaced with a new 0.5% sodium hydroxide solution at the same ratio, and the bottle was placed in a constant temperature shaker for 16 h (25℃, 150 rpm). The bovine dermis was then removed from the polypropylene bottle ② and washed alternately with 1 M and 0.1 M NaCl solutions until neutral, to obtain acellular bovine dermis matrix.

[0053] Example 5

[0054] This embodiment provides a method for preparing acellular dermal matrix, the method comprising:

[0055] (1) Obtain cow skin from the slaughterhouse, rinse it with water to remove blood and impurities, cut it to an appropriate size, place it in a polypropylene bottle ①, add 8% sodium hydroxide solution at a solid-liquid ratio of 1:5, and tighten the bottle cap. Place the polypropylene bottle ① in a constant temperature shaker and shake for 1 h (42℃, 100 rpm). Then remove the cow skin from the polypropylene bottle and use a hand scraper to easily scrape off the hair, epidermis, subcutaneous fat, and other accessory layers to obtain the complete dermis.

[0056] (2) The bovine dermis layer was transferred to a clean polypropylene bottle ②, and 4% sodium hydroxide solution was added at a solid-liquid ratio of 1:5. The bottle cap was tightened, and the bottle was placed in a constant temperature shaker for 1 h (25℃, 150 rpm). Then, the polypropylene bottle ② was removed, and a new 2% sodium hydroxide solution was added at the same ratio. The bottle was then placed in a constant temperature shaker for 4 h (25℃, 150 rpm). The solution was then added at the same ratio, and a new 1% sodium hydroxide solution was added. The bottle was then placed in a constant temperature shaker for 8 h (25℃, 150 rpm). The bovine dermis was then removed from the polypropylene bottle ② and washed alternately with 1 M and 0.1 M NaCl solutions until neutral, to obtain the decellularized bovine dermis matrix.

[0057] Example 6

[0058] This embodiment provides a method for preparing acellular dermal matrix, which differs from Embodiment 1 only in that step (1) is as follows: “Obtain bovine skin from the slaughterhouse, rinse it with water to remove blood and impurities, cut it to an appropriate size and place it in a polypropylene bottle ①, then add 0.5% sodium hydroxide solution at a solid-liquid ratio of 1:6, and tighten the cap. Place the polypropylene bottle ① in a constant temperature shaker and shake for 8 h (25℃, 150 rpm). Then take out the polypropylene bottle ①, replace it with a new 0.5% sodium hydroxide solution at the same ratio, and place it in a constant temperature water bath at 37℃ for 2 h. Then take out the bovine skin from the polypropylene bottle ①, and it is almost impossible to scrape off the hair, epidermis, subcutaneous fat and other appendages by hand with a scraper”, and other operations remain unchanged.

[0059] Example 7

[0060] This embodiment provides a method for preparing acellular dermal matrix, which differs from Embodiment 1 only in that step (1) is as follows: “Obtain bovine skin from a slaughterhouse, rinse it with water to remove blood and impurities, cut it to an appropriate size and place it in a polypropylene bottle ①, then add 12% sodium hydroxide solution at a solid-liquid ratio of 1:6, and tighten the bottle cap. Place the polypropylene bottle ① in a constant temperature shaker and shake for 8 h (25℃, 150 rpm). Then take out the polypropylene bottle ①, replace it with a new 12% sodium hydroxide solution at the same ratio, and place it in a constant temperature water bath at 37℃ for 2 h. Check the bovine skin in the polypropylene bottle ①, and the bovine skin is completely dissolved.”

[0061] Test Example 1

[0062] The decellularized matrix prepared in Example 1 was subjected to HE staining and observation of its appearance morphology.

[0063] HE staining was performed, and the cellular components and histological structure of the matrix in bovine hide before and after decellularization were observed under a light microscope. The results are as follows: Figure 2 and Figure 3 As shown. Among them. Figure 2 Before decellularization, the bovine hide tissue contains a large number of skin appendages such as hair follicles, sweat glands, fibroblasts, and blood vessels, which are stained blue-purple by hematoxylin. Figure 3 After decellularization, the above-mentioned skin appendage structures disappeared, the collagen fiber bundles were arranged relatively densely, and no obvious disorder or destruction of the collagen fiber bundle structure was observed. The entire matrix was uniformly stained red with eosin.

[0064] Scanning electron microscopy was used to observe the surface morphology and longitudinal section morphology of the decellularized bovine dermal matrix. The results are shown in the figure. Figure 4 A and B show the surface morphology of two sides of the decellularized bovine dermal matrix, while C and D show the longitudinal section morphology of the decellularized bovine dermal matrix at different magnifications. Figure 4 In the middle, A and B: one side of the decellularized bovine dermal matrix has a porous structure, and the other side has a rough lamellar structure. This appearance is conducive to cell adhesion and ingrowth. C and D: the longitudinal section shows densely arranged fibers, which can provide good mechanical support for the matrix. After decellularization and heat treatment, the fiber structure is still intact, the surface is relatively smooth, and there are no obvious signs of breakage or degradation.

[0065] Test Example 2

[0066] DNA content test.

[0067] The DNA content in the matrix of bovine hide before and after decellularization was determined according to YY / T 1876-2023, "Determination of DNA Residue in Animal-Derived Biological Materials: Fluorescent Staining Method".

[0068] Before decellularization, the DNA content in bovine skin was (3695.25±179.21) ng / mg; after decellularization, the DNA content in the bovine dermal matrix of Examples 1-6 was (9.44±3.53), (12.37±2.94), (7.88±1.67), (28.36±5.21), (15.11±3.22), and (186.29±17.41) ng / mg, respectively. The residual DNA content after decellularization in Examples 1-5 met the requirements for clinical application, while the residual DNA content after decellularization in Example 6 did not meet the requirements for clinical application.

[0069] Test Example 3

[0070] Fat content test.

[0071] The fat content in the matrix of bovine hide before and after decellularization was determined in accordance with GB 5009.6-2016, the national standard for food safety, "Determination of fat in food".

[0072] Before decellularization, the fat content in bovine skin was (5.31±0.38)%; after decellularization, the fat content in the bovine dermal matrix of Examples 1-6 was (0.52±0.09)%, (0.67±0.19)%, (0.38±0.12)%, (0.81±0.31)%, (0.94±0.25)%, and (2.85±0.54)%, respectively. The fat content after alkaline treatment in Examples 1-5 met the requirements for clinical application, while the fat content in the decellularized matrix of Example 6 posed a certain risk to clinical application.

[0073] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for preparing acellular dermal matrix, characterized in that, The preparation method includes: (1) Mix the skin tissue with an alkaline solution to remove hair, epidermis and subcutaneous tissue to obtain the dermis; (2) Mix the dermis with an alkaline solution, wash, and you will get the product; The concentration gradient of the alkaline solution in step (2) is 1-5.

2. The method for preparing decellularized dermal matrix according to claim 1, characterized in that, The alkali content in the alkaline solution in step (1) is 1-10% by mass; Preferably, the ratio of skin tissue to alkaline solution is 1 g:(4-20) mL.

3. The method for preparing decellularized dermal matrix according to claim 1, characterized in that, The mixing temperature in step (1) is 4-42℃, and the processing time for a single treatment is 0.5-48 h.

4. The method for preparing decellularized dermal matrix according to claim 1, characterized in that, The skin tissue includes human skin, sheepskin, cowhide, pigskin, donkey skin, horse skin, rabbit skin, or rat skin.

5. The method for preparing decellularized dermal matrix according to claim 1, characterized in that, The concentration of the alkaline solution each time should not be higher than the concentration of the previous alkaline solution.

6. The method for preparing decellularized dermal matrix according to claim 1, characterized in that, The alkaline solution in step (2) has a mass percentage of 0.5-4%; Preferably, the ratio of the dermis to the alkaline solution in step (2) is 1 g:(4-20) mL.

7. The method for preparing decellularized dermal matrix according to claim 1, characterized in that, The temperature of the treatment in step (2) is 4-45℃, and the time for a single treatment is 1-24 h.

8. The method for preparing decellularized dermal matrix according to claim 1, characterized in that, The alkali includes sodium hydroxide, potassium hydroxide, sodium carbonate, potassium carbonate, and ammonia, preferably sodium hydroxide and / or potassium hydroxide; Preferably, the cleaning specifically includes cleaning the dermis to neutrality, and the reagents used for cleaning include one or more of PBS buffer, deionized water, sodium chloride solution, and calcium chloride solution.

9. The decellularized dermal matrix prepared by the method according to any one of claims 1-8.

10. The application of the decellularized dermal matrix prepared according to claim 9 in the preparation of biomimetic materials.

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