Preparation method of acellular dermal matrix

Abstract

The invention discloses a preparation method of an acellular dermal matrix, and belongs to the technical field of tissue engineering. The preparation method comprises the following steps: selecting traceable animal fur of one to three months old, and purifying through the steps of soaking, fleshing, degreasing, unhairing, liming, deliming, softening and the like in a tanning method to obtain the acellular dermal matrix. In the preparation process of the obtained acellular dermal matrix, antigen components such as cells, hair follicles and impure proteins are removed, the immunogenicity of the acellular dermal matrix is reduced to the maximum extent, and a collagenous fiber structure in an extracellular matrix of the acellular dermal matrix is reserved. Meanwhile, according to the preparation method, collagen fiber bundles are moderately released, and the process not only effectively relieves entanglement among the fiber bundles, but also successfully increases the exposure degree of hydrophilic groups, so that the acellular dermal matrix has excellent hydrophilicity and is more beneficial to proliferation and migration of cells.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue engineering and relates to a method for preparing an acellular dermal matrix. Background Art

[0002] The skin is the body's defense barrier against external threats, and it is mainly divided into the epidermis, dermis, and subcutaneous tissue. If the skin suffers extensive or deep damage, especially damage to the dermis, it may cause infection, shock, and problems such as poor elasticity, unevenness, and easy contracture of the wound surface after healing. Traditional treatments include autologous skin transplants, but this method may cause secondary damage to patients and prolong treatment time. To solve these problems, scientists have developed a human skin substitute. The skin structure of mammals is similar to that of humans, so their skin has become an ideal source of substitutes.

[0003] Fresh xenogeneic skin is processed by bioengineering technology to remove highly immunogenic cell components and retain extracellular matrix components. The resulting dermal substitute is called xenogeneic acellular dermal matrix (ADM). Since the rejection in skin transplantation is mainly cellular immunity, after removing the cell components with strong immunogenicity, ADM has only very low antigenicity and can remain in the host for a long time and eventually be absorbed and remodeled by normal tissues. As a human skin substitute that can provide a scaffold structure for the regeneration of tissue cells, ADM can cover wounds, fill defective tissues, promote wound repair, guide tissue regeneration, and accelerate the process of cell proliferation. It has the characteristics of good tissue compatibility, strong guidance, and good tissue conformity, and is widely used in the field of human tissue wound repair.

[0004] Researchers have used repeated freezing and thawing methods, trypsin digestion methods, etc. to prepare ADM, but these methods cannot completely remove the cell components in the dermis, resulting in a strong host rejection reaction after ADM transplantation, and the preparation process is too complicated, so it has not been widely used. There are four commonly used methods for the preparation of ADM, namely DispaseII-Triton method, hypertonic salt-SDS method, hypertonic salt-NaOH erosion method, and hypertonic salt-enzyme digestion method. These four methods have their own advantages and disadvantages. In general, most of the existing methods cannot maintain low antigenicity and structural integrity at the same time. The removal of antigenic components is bound to cause structural damage, thereby affecting the migration of fibroblasts and the deposition of collagen, resulting in poor coverage or filling effect of ADM on the wound surface. If the structural integrity is retained, it will lead to incomplete decellularization and a certain immunogenicity, thereby limiting its scope of application. Therefore, it is urgent to explore a new ADM preparation method that can maintain low immunogenicity while ensuring structural integrity. As a traditional process for processing animal skin, the tanning method involves multiple processes such as soaking, liming, depilation, softening, pickling, tanning, dyeing, and emulsion oiling, and promotes the uniform penetration of various chemical materials and the completion of chemical reactions through the mechanical action of a rotating drum. However, the main purpose of the tanning method is to make leather, not to prepare acellular dermal matrix. The present invention explores the potential application of the tanning method in the preparation of ADM, in order to find a new preparation method that is both efficient and practical, and to open up broader prospects for the clinical application of ADM. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies and disadvantages of the above-mentioned prior art. The present invention uses a tanning method to process mammalian fur, removes antigenic components such as cells, hair follicles, and miscellaneous proteins, and obtains a low-immunogenic, structurally intact acellular dermal matrix.

[0006] In a first aspect, the present invention provides a method for preparing an acellular dermal matrix, wherein mammalian fur is subjected to a tanning process of soaking, fleshing, degreasing, depilating, liming, deliming, and softening, and then rinsed, freeze-dried, and irradiated to obtain an acellular dermal matrix.

[0007] Furthermore, the mammalian fur used in the present invention is salt-dried fur of 1 to 3 month old kid goats.

[0008] Further, in the degreasing treatment of the present invention, the degreasing agent is composed of 100 to 200 parts of soda ash, 10 to 20 parts of penetrant, 30 to 80 parts of surfactant and 9730 to 9800 parts of water, in parts by weight;

[0009] The penetrant is fatty alcohol polyoxyethylene ether;

[0010] The surfactant is any one of Peregal O, Triton X-100, and sodium dodecyl sulfate;

[0011] The mass ratio of the mammalian fur to the degreasing agent is 1:3-5;

[0012] The degreasing treatment time is 60 to 90 minutes.

[0013] Further, in the depilatory treatment of the present invention, the depilatory agent is composed of 500 parts of sodium sulfide, 4000-5000 parts of quicklime, 40-60 parts of ammonium sulfide, 10-20 parts of surfactant and 4950-5430 parts of water, in parts by weight;

[0014] The surfactant is any one of Peregal O, Triton X-100, and sodium dodecyl sulfate;

[0015] The mass ratio of the mammalian fur to the depilatory agent is 1:1-2;

[0016] The depilatory treatment time is 2.5 to 3.5 hours.

[0017] Further, in parts by weight, the liming solution in the liming treatment of the present invention is composed of 200-300 parts of sodium sulfide, 300-500 parts of calcium hydroxide, 2-5 parts of trypsin, 5-10 parts of surfactant and 9290-9390 parts of water;

[0018] The surfactant is any one of Peregal O, Triton X-100, and sodium dodecyl sulfate;

[0019] The mass ratio of the mammalian fur to the degreasing agent is 1:3;

[0020] The liming treatment time is 14 to 16 hours.

[0021] Furthermore, in parts by weight, the deliming liquid in the deliming treatment of the present invention comprises: mixing ammonium sulfate and sodium bisulfite to form a mixed liquid, and adjusting the pH of the mixed liquid to 7.5 to 8.0 using glacial acetic acid solution;

[0022] The glacial acetic acid solution is prepared by diluting glacial acetic acid and water in a volume ratio of 1:10;

[0023] The mass of the ammonium sulfate is 0.8% to 1% of the mass of the mammal's fur;

[0024] The mass of the sodium bisulfite is 0.4% to 0.5% of the mass of the mammal's fur;

[0025] The deashing treatment time is 50 minutes.

[0026] Further, in parts by weight, the softening liquid in the softening treatment of the present invention is composed of 40 to 60 parts of ammonium chloride, 40 to 60 parts of trypsin and 9890 to 9900 parts of water;

[0027] The mass ratio of the mammalian fur to the softening liquid is 1:1.5-2;

[0028] The softening treatment time is 60 minutes.

[0029] Furthermore, the rinsing of the present invention includes alcohol rinsing and PBS rinsing;

[0030] The alcohol concentration of the alcohol rinse is 50% to 70%, and the alcohol rinse time is 10 to 30 minutes;

[0031] The pH of the PBS buffer for rinsing with PBS is 7.2-7.4, and the PBS rinsing time is 60-90 minutes.

[0032] In a second aspect, the present invention provides an acellular dermal matrix having low antigenicity, an intact structure, a well-preserved basement membrane, and good hydrophilicity, thermal stability, and mechanical properties.

[0033] In a third aspect, the present invention provides the use of mammalian fur prepared by a tanning process in the preparation of an acellular dermal matrix. The present invention obtains an acellular dermal matrix after purification through the steps of soaking, fleshing, degreasing, depilating, liming, deliming, softening and the like in the tanning process. Because the present invention reduces the immunogenicity of the acellular dermal matrix to the maximum extent, and retains the collagen fiber structure in its extracellular matrix. At the same time, the tangled and intertwined collagen fiber bundles are appropriately loosened to expose more hydrophilic groups, so that it has excellent hydrophilicity, which is more conducive to cell proliferation and migration, and has good application prospects in the fields of chronic wound healing, wound repair, plastic surgery, etc.

[0034] Compared with the prior art, the technical solution provided by the present invention has at least the following beneficial effects or advantages:

[0035] (1) The present invention removes antigenic components such as cells, hair follicles, miscellaneous proteins, and fat through the steps of soaking, fleshing, degreasing, depilating, liming, deliming, and softening in the traditional leather making method without destroying the collagen scaffold formed by the interweaving and entanglement of collagen microfibers, collagen fibers, and collagen fiber bundles, thereby reducing their immunogenicity to the greatest extent, while retaining the intact basement membrane and the D-shaped periodic horizontal stripe structure with light and dark overlapping, thereby ensuring its structural integrity and high biological activity.

[0036] (2) The traditional lime hair removal method can remove the epidermis with high fat content, remove hair cleanly without leaving any hair roots, and will not cause damage to the basement membrane, retaining the complete structure. Without adding any exogenous means, it has excellent mechanical properties and thermal stability, which is convenient for daily storage, transportation and use. At the same time, it avoids the introduction of new materials and the occurrence of biological toxicity, and has higher safety.

[0037] (3) The acellular dermal matrix prepared by the present invention, due to the deep penetration of surfactants and enzymes in the steps of degreasing and softening, while removing immunogenic substances, the collagen fibers are loosened to a certain extent, and the hydrophilic groups on the chains are exposed to a greater extent. The water contact angle is maintained at about 50°, and the hydrophilicity is excellent, which is more conducive to the adhesion, growth, proliferation and migration of cells, is convenient for covering wounds and keeping wounds moist, and is more conducive to the collagen deposition and granulation growth of wounds.

[0038] (4) During the liming, alcohol immersion cleaning, and irradiation disinfection and sterilization processes, the strong alkaline environment, alcohol, and gamma rays inactivate viruses and fungi, greatly reducing the risk of viruses.

[0039] (5) The method of the present invention has wide applicability and is suitable for treating the fur of mammals such as pigs, cattle, and sheep.

[0040] (6) The method provided by the present invention has a simple process flow, low-cost reagents, a high degree of automation, and is suitable for the transformation of the traditional leather industry. It can be mass-produced without laying a new production line. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a histological photograph of acellular dermal matrix.

[0042] Figure 2 SEM image of acellular dermal matrix.

[0043] Figure 3 FT-IR image of acellular dermal matrix.

[0044] Figure 4 : is a photo of the water contact angle of the acellular dermal matrix. Among them, A is the acellular dermal matrix prepared in Example 1; B is the acellular dermal matrix prepared in Example 2; C is the acellular dermal matrix prepared in Example 3.

[0045] Figure 5 This is a comparison chart of the mechanical properties of acellular dermal matrix, where A is the tensile strength and B is the elongation at break.

[0046] Figure 6 The thermal stability diagram of acellular dermal matrix. DETAILED DESCRIPTION

[0047] The technical scheme of the present invention is described below in conjunction with the embodiments, but the present invention is not limited to the following embodiments. The experimental methods and detection methods described in each embodiment are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0048] Example 1

[0049] This embodiment provides a method for preparing an acellular dermal matrix.

[0050] s1. Select the salt-dried fur of a three-month-old kid, and trim the unevenly thick fur on the abdomen and limbs. Put the trimmed salt-dried fur of the kid into a drum, add clean water, the mass ratio of clean water to salt-dried fur is 1:3, and wash for 60 minutes. In order to ensure the washing effect, the washing process adopts a combination of steaming and running water washing, that is, after a certain period of steaming, it is switched to running water washing to remove impurities and residues on the fur. This process needs to be repeated three times until the water after washing becomes clear and transparent to ensure the cleanliness of the fur.

[0051] s2. Put the salt-dried fur of the goat in s1 into the drum again, add clean water, the mass ratio of clean water to salt-dried fur of the goat is 3:1, then add 0.2% of JFC penetrant (fatty alcohol polyoxyethylene ether) based on the mass of the salt-dried fur of the goat, set the drum to slow speed, continue for 6 hours, control the waste liquid and wash with water.

[0052] s3. Spread the dried salted skin of the kid goat in s2 flat and remove the fascia, minced meat and oil film attached to the skin.

[0053] s4. Put the salt-dried goat fur in s3 into a drum, add a degreasing agent, the mass ratio of the salt-dried goat fur to the degreasing agent is 1:4, the reaction temperature is controlled to 38°C, the reaction is carried out for 60 minutes, and the salt-dried goat fur is controlled to be water-controlled for several hours until the waste liquid is controlled dry. The degreasing agent contains 0.5% surfactant, 2% soda ash, 0.2% JFC penetrant and 97.3% water, wherein the surfactant is perchlorate O.

[0054] s5. Place the goat salt-dried fur in s4 with the flesh side facing upwards and evenly apply the depilatory agent. The mass ratio of the goat salt-dried fur to the depilatory paste is 1:1. After stacking for 3.5 hours, push the fur manually. The depilatory paste contains 5% sodium sulfide, 40% quicklime, 0.6% ammonium sulfate, 0.1% surfactant and 54.3% water, wherein the surfactant is perchlorate O.

[0055] s6. Wash the salt-dried fur of the kid goat in s5 several times until the water becomes clear. Put the salt-dried fur of the kid goat into a rotary drum, control the temperature to 22°C, add liming liquid, the mass ratio of the salt-dried fur of the kid goat to the liming liquid is 1:3, rotate for 30 minutes, stop for 60 minutes, rotate for another 30 minutes, stop the drum overnight, and wash with water after rotating for 15 minutes the next day. The total liming treatment time is 16 hours. The liming liquid includes 2% flaky sodium sulfide, 4% calcium hydroxide, 0.04% trypsin, 0.06% surfactant and 93.9% water, wherein the surfactant is perchlorate O.

[0056] s7. Put the salt-dried fur of the kid goat in s6 into a rotary drum for deashing treatment, control the temperature to 35°C, add ammonium sulfate and sodium bisulfite, rotate for 10 minutes, then slowly drip glacial acetic acid solution while starting the rotary drum until the pH stabilizes at 7.5-8.0, stop adding glacial acetic acid, and rotate for 40 minutes. The deashing liquid for deashing treatment is a mixed solution of 1% ammonium sulfate and 0.4% sodium bisulfite by weight of the kid goat salt-dried fur, and the pH of the mixed solution is adjusted to 7.5-8.0 by glacial acetic acid solution. The glacial acetic acid solution is a mixture of glacial acetic acid and water in a volume ratio of 1:10.

[0057] s8. Put the salt-dried goat fur in s7 into a drum, control the temperature to 37°C, add softening liquid, the mass ratio of softening liquid to salt-dried goat fur is 1:2, make the pH of the solution in the drum 7.5-8.2, and then rotate for 60 minutes until the grain surface is white, smooth, delicate, non-greasy, and the leather body feels loose. In parts by weight, the softening liquid is 0.5% trypsin solution, 0.6% ammonium chloride solution and 98.9% water.

[0058] s9. Put the salt-dried fur of the small goat in s8 into a drum, soak and clean it with 60% alcohol for 15 minutes, soak it in PBS buffer for 60 minutes, rinse it three times, rinse it thoroughly with physiological saline, put it into a freeze dryer for freeze drying, sterilize it by γ-ray irradiation, and obtain acellular dermal matrix, which is recorded as GADM-1. The PBS buffer contains 0.8% sodium chloride, 0.04% potassium chloride, 0.02% disodium hydrogen phosphate dodecahydrate, 0.01% potassium dihydrogen phosphate, 0.04% sodium bicarbonate and 99.09% water, and then a sterile sodium bicarbonate solution is used to adjust the pH to 7.2-7.4.

[0059] Example 2

[0060] This embodiment provides a method for preparing an acellular dermal matrix.

[0061] s1. Select the salt-dried fur of a one-month-old kid goat, and trim the unevenly thick fur on the abdomen and limbs. Put the trimmed salt-dried fur of the kid goat into a drum, add clean water, and the mass ratio of clean water to the salt-dried fur of the kid goat is 1:3, and wash it with water for 60 minutes. In order to ensure the washing effect, the washing process adopts a combination of steaming washing and running water washing, that is, after a certain period of steaming washing, switch to running water washing to remove impurities and residues on the salt-dried fur of the kid goat. This process needs to be repeated three times until the water after washing becomes clear and transparent to ensure the cleanliness of the fur.

[0062] s2. Put the salt-dried fur of the goat in s1 into the drum again, add clean water, the mass ratio of clean water to salt-dried fur of the goat is 3:1, then add 0.2% of the mass of JFC penetrant of the salt-dried fur of the goat, set the drum to slow speed, continue for 6 hours, control the waste liquid and wash with water.

[0063] s3. Spread the dried salted skin of the kid goat in s2 flat and remove the fascia, minced meat and oil film attached to the skin.

[0064] s4. Put the salt-dried goat fur in s3 into a drum, add a degreasing agent, the mass ratio of the salt-dried goat fur to the degreasing agent is 1:3, control the reaction temperature to 38°C, react for 90 minutes, and control the water content of the salt-dried goat fur for several hours until the waste liquid is dried. The degreasing agent contains 0.3% surfactant, 1.5% soda ash, 0.2% JFC penetrant and 98% water, wherein the surfactant is Triton X-100.

[0065] s5. Place the salt-dried goat fur in s4 with the flesh side facing upwards, and evenly apply depilatory paste, the mass ratio of the salt-dried goat fur to the depilatory paste is 1:1.5, and after stacking for 3.0 hours, push the fur manually. The depilatory paste contains 5% sodium sulfide, 45% quicklime, 0.4% ammonium sulfate, 0.1% surfactant and 49.50% water, wherein the surfactant is Triton X-100.

[0066] s6. Wash the salt-dried fur of the kid goat in s5 several times with water until the water becomes clear. Put the salt-dried fur of the kid goat into a rotary drum, control the temperature to 22°C, add liming liquid, the mass ratio of the salt-dried fur of the kid goat to the liming liquid is 1:3, rotate for 30 minutes, stop for 60 minutes, rotate for another 30 minutes, stop the drum overnight, and wash with water after rotating for 15 minutes the next day. The total liming treatment time is 16 hours. The liming liquid includes 2% flaky sodium sulfide, 5% calcium hydroxide, 0.02% trypsin, 0.08% surfactant and 92.9% water, wherein the surfactant is Triton X-100.

[0067] s7. Put the salt-dried fur of the kid goat in s6 into a rotary drum for deashing treatment, control the temperature to 35°C, add ammonium sulfate and sodium bisulfite, rotate for 10 minutes, then slowly drip glacial acetic acid solution while turning the drum until the pH stabilizes at 7.5-8.0, stop adding glacial acetic acid, and rotate for 40 minutes. The deashing liquid for deashing treatment is a mixed solution of 0.8% ammonium sulfate and 0.5% sodium bisulfite by weight of the kid goat salt-dried fur, and the pH of the mixed solution is adjusted to 7.5-8.0 by glacial acetic acid solution. The glacial acetic acid solution is a mixture of glacial acetic acid and water in a volume ratio of 1:10.

[0068] s8. Put the salt-dried goat fur in s7 into a drum, control the temperature to 37°C, add softening liquid, control the temperature to 37°C, add softening liquid, the mass ratio of softening liquid to salt-dried goat fur is 1:1.5, make the pH of the solution in the drum 7.5-8.2, and then rotate for 60 minutes until the grain surface is white, smooth, delicate, non-greasy, and the leather body has a loose feel. The softening liquid includes 0.5% trypsin solution, 0.4% ammonium chloride solution and 99.1% water.

[0069] s9. Put the salt-dried fur of the small goat in s8 into a drum, soak and clean it with 50% alcohol for 10 minutes, soak it in PBS buffer for 90 minutes, rinse it 3 times, rinse it thoroughly with physiological saline, put it into a freeze dryer for freeze drying, sterilize it by γ-ray irradiation, and obtain acellular dermal matrix, which is recorded as GADM-2. The PBS buffer contains 0.8% sodium chloride, 0.04% potassium chloride, 0.02% disodium hydrogen phosphate dodecahydrate, 0.01% potassium dihydrogen phosphate, 0.04% sodium bicarbonate and 99.09% water, and then a sterile sodium bicarbonate solution is used to adjust the pH to 7.2-7.4.

[0070] Example 3

[0071] This embodiment provides a method for preparing an acellular dermal matrix.

[0072] s1. Select the salt-dried fur of a month-old kid goat, and trim the uneven fur on the abdomen and limbs. Put the trimmed salt-dried fur into a drum, add clean water, the mass ratio of clean water to the salt-dried fur of the kid goat is 1:3, and wash for 60 minutes. In order to ensure the washing effect, the washing process adopts a combination of steaming and running water washing, that is, after a certain period of steaming, switch to running water washing to remove impurities and residues on the fur. This process needs to be repeated three times until the water after washing becomes clear and transparent to ensure the cleanliness of the fur.

[0073] s2. Put the salt-dried fur of the goat in s1 into the drum again, add clean water, the mass ratio of clean water to salt-dried fur of the goat is 3:1, then add 0.2% of the mass of JFC penetrant of the salt-dried fur of the goat, set the drum to slow speed, continue for 6 hours, control the waste liquid and wash with water.

[0074] s3. Spread the dried salted skin of the kid goat in s2 flat and remove the fascia, minced meat and oil film attached to the skin.

[0075] s4. Put the salt-dried goat fur in s3 into a drum, add a degreasing agent, the mass ratio of the salt-dried goat fur to the degreasing agent is 1:5, control the reaction temperature to 38°C, react for 60 minutes, and control the water content of the salt-dried goat fur for several hours until the waste liquid is dried. The degreasing agent contains 0.8% surfactant, 1% soda ash, 0.2% JFC penetrant and 98% water, wherein the surfactant is sodium dodecyl sulfate.

[0076] s5. Place the salt-dried goat fur in s4 with the flesh side facing upwards, and evenly apply depilatory paste, the mass ratio of the salt-dried goat fur to the depilatory paste being 1:2, and after stacking for 2.5 hours, push the fur manually. The depilatory paste contains 5% sodium sulfide, 40% quicklime, 0.6% ammonium sulfate, 0.1% surfactant and 54.3% water, wherein the surfactant is sodium dodecyl sulfate.

[0077] s6. Wash the salt-dried fur of the kid goat in s5 several times with water until the water becomes clear. Put the salt-dried fur of the kid goat into a rotary drum, control the temperature to 22°C, add liming liquid, the mass ratio of the salt-dried fur of the kid goat to the liming liquid is 1:3, rotate for 30 minutes, stop for 60 minutes, rotate for another 30 minutes, stop the drum overnight, and wash with water after rotating for 15 minutes the next day. The total liming treatment time is 14 hours. The liming liquid includes 3% flaky sodium sulfide, 3% calcium hydroxide, 0.06% trypsin, 0.05% surfactant and 93.89% water, wherein the surfactant is sodium dodecyl sulfate.

[0078] s7. Put the salt-dried fur of the kid goat in s6 into a rotary drum for deashing treatment, control the temperature to 35°C, add ammonium sulfate and sodium bisulfite, rotate for 10 minutes, then slowly drip glacial acetic acid solution while turning the drum until the pH stabilizes at 7.5-8.0, stop adding glacial acetic acid, and rotate for 40 minutes. The deashing liquid for deashing treatment is a mixed solution of 1% ammonium sulfate and 0.5% sodium bisulfite by weight of the kid goat salt-dried fur, and the pH of the mixed solution is adjusted to 7.5-8.0 by glacial acetic acid solution. The glacial acetic acid solution is a mixture of glacial acetic acid and water in a volume ratio of 1:10.

[0079] s8. Put the salt-dried goat fur in s7 into a drum, control the temperature to 37℃, add softening liquid, the mass ratio of softening liquid to salt-dried goat fur is 1:1.5, make the pH of the solution in the drum 7.5-8.2, and then rotate for 60 minutes until the grain surface is white, smooth, delicate, non-greasy, and the leather body feels loose. The softening liquid is 0.6% trypsin solution, 0.5% ammonium chloride solution and 98.90% water.

[0080] s9. Put the salt-dried fur of the small goat in s8 into a drum, soak and clean it with 70% alcohol for 30 minutes, soak it in PBS buffer for 90 minutes, rinse it 3 times, rinse it thoroughly with physiological saline, put it into a freeze dryer for freeze drying, sterilize it by γ-ray irradiation, and obtain acellular dermal matrix, which is recorded as GADM-3. The PBS buffer contains 0.8% sodium chloride, 0.04% potassium chloride, 0.02% disodium hydrogen phosphate dodecahydrate, 0.01% potassium dihydrogen phosphate, 0.04% sodium bicarbonate and 99.09% water, and then a sterile sodium bicarbonate solution is used to adjust the pH to 7.2-7.4.

[0081] Example 4

[0082] This example provides the test results of the prepared acellular dermal matrix.

[0083] Figure 1 The following are histological photographs of the acellular dermal matrix prepared in Examples 1 to 3. Figure 1As shown, histological evaluation was performed using hematoxylin and eosin (HE), Masson trichrome, and Oil Red O staining to assess the presence of cell nuclei, collagen fibers, and fat, respectively. HE staining identified a significant reduction in cellular components after purification. In unpurified tissue, many fluorescently stained nuclei were observed, indicating that cellular material was abundant. In contrast, purified GADM showed very few to no nuclei or nuclear fragments. Masson trichrome staining showed a significant reduction in myofibrils (red) after purification. Unpurified tissue contained a large number of fluorescently stained myofibrils, while the purification process resulted in the almost complete elimination of these structures from GADM. Oil Red O staining confirmed a significant reduction in adipose tissue content. Many fluorescently stained adipose structures were present in unpurified tissue, while purified GADM showed minimal to undetectable fat content. A comprehensive analysis of the results of HE, Masson, and Oil Red O staining showed that, unlike the original kid salt-dried fur, purified GADM underwent complete removal of the epidermis. In addition, cells, hair follicles, sweat glands, fat, miscellaneous proteins, and other components were effectively eliminated. The preparation method provided by the present invention removes non-collagen components from goat skin while retaining collagen fibers representing the most basic structural support. In addition, the network structure and collagen fiber bundles also have a certain degree of looseness, which can promote cell proliferation, collagen deposition and tissue healing when GADM is used as a skin substitute.

[0084] Figure 2 The following are SEM images of the acellular dermal matrix prepared in Examples 1 to 3. Figure 2 As shown, the surface of the prepared GADM is dense and shows a completely preserved concave-convex basement membrane and a clearly visible pore structure. The cross section of GADM is a three-dimensional network structure of loose collagen fibers and fiber bundles interwoven and stacked, and D-loops of about 65 nm can be seen on the fiber bundles.

[0085] Figure 3 The FT-IR spectra of GADM-1, GADM-2, and GADM-3 are shown. The IR absorption peaks corresponding to the amide A band of GADM-1, GADM-2, and GADM-3 appear at 3445 cm -1 、3452cm -1 and 3431cm -1 The amide B bands of GADM-1, GADM-2, and GADM-3 are at 3076 cm -1 、3075cm -1 and 3079cm -1 The amide I band peaks representing C=O stretching vibration appeared at 1662 cm-1 for GADM-1, GADM-2, and GADM-3, respectively. -1 、1660cm-1 and 1652cm -1 The bands of amide II are located at 1557 cm-1 for GADM-1, GADM-2 and GADM-3 respectively. -1 、1554cm -1 and 1550cm -1 Finally, the characteristic peaks of amide III of GADM-1, GADM-2 and GADM-3 are concentrated at 1241 cm -1 、1241cm -1 and 1240cm -1 The results show that the characteristic absorption peaks of the amide bands exist in GADM-1, GADM-2 and GADM-3, with very little difference, indicating that the basic secondary structures of the acellular dermal matrices prepared in Examples 1 to 3 are similar.

[0086] Figure 4 The surface contact angles (θ) of GADM-1, GADM-2, and GADM-3 are shown to be 48.4° ( Figure 4 A in), 56.2°( Figure 4 B) and 50.2°( Figure 4 C), since the intertwined and tangled collagen microfibrils, collagen fibers and collagen fiber bundles are loosened to a certain extent during the purification process, the hydrophilic groups shielded by the adjacent D periods are exposed, and the richer hydrophilic groups lead to better hydrophilicity. The results show that the water contact angles of the acellular dermal matrices prepared in Examples 1 to 3 are not much different, and the hydrophilicity is stable.

[0087] like Figure 5 As shown in A in FIG, the tensile strengths of GADM-1, GADM-2, and GADM-3 are approximately 20.26 MPa, 19.83 MPa, and 21.73 MPa, respectively. Figure 5 As shown in B, the elongation at break of GADM-1, GADM-2 and GADM-3 are about 19.73%, 20.81% and 21.32%, respectively, indicating that the acellular dermal matrix prepared in Examples 1 to 3 still has strong tensile strength and high elongation at break after various purification treatments.

[0088] Dynamic thermogravimetric analysis (TGA) was used to characterize the weight change of collagen during heating and to evaluate the thermal stability of GADM-1, GADM-2, and GADM-3 in various temperature ranges. Figure 6As shown, at all weight loss stages, the weight loss rates of GADM-1, GADM-2 and GADM-3 were similar with minimal differences, indicating that the thermal stabilities of the acellular dermal matrices prepared in Examples 1 to 3 were similar.

[0089] The embodiments described above are part of the embodiments of the present invention, but not all of them. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but only represents selected embodiments of the present invention. All other embodiments obtained without creative work and related deductions and substitutions made by ordinary technicians in the field under the conditions of the concept of the present invention belong to the scope of protection of the present invention.

Claims

1. A method for preparing an acellular dermal matrix, characterized in that: The mammalian fur is treated by soaking, fleshing, degreasing, dehairing, liming, deliming and softening in the tanning process, and then rinsed, freeze-dried and irradiated to obtain the acellular dermal matrix.

2. The preparation method according to claim 1, characterized in that: The mammal fur is salt-dried fur of a kid goat; The kid is 1 to 3 months old.

3. The preparation method according to claim 1, characterized in that: In the degreasing treatment, the degreasing agent is composed of 100 to 200 parts of soda ash, 10 to 20 parts of penetrant, 30 to 80 parts of surfactant and 9730 to 9800 parts of water in parts by weight; The penetrant is fatty alcohol polyoxyethylene ether; The surfactant is any one of Peregal O, Triton X-100, and sodium dodecyl sulfate; The mass ratio of the mammalian fur to the degreasing agent is 1:3-5; The degreasing treatment time is 60 to 90 minutes.

4. The preparation method according to claim 1, characterized in that: In parts by weight, the depilatory agent in the depilatory treatment is composed of 500 parts of sodium sulfide, 4000-5000 parts of quicklime, 40-60 parts of ammonium sulfide, 10-20 parts of surfactant and 4950-5430 parts of water; The surfactant is any one of Peregal O, Triton X-100, and sodium dodecyl sulfate; The mass ratio of the mammalian fur to the depilatory agent is 1:1-2; The depilatory treatment time is 2.5 to 3.5 hours.

5. The preparation method according to claim 1, characterized in that: In parts by weight, the liming solution in the liming treatment consists of 200-300 parts of sodium sulfide, 300-500 parts of calcium hydroxide, 2-5 parts of trypsin, 5-10 parts of surfactant and 9290-9390 parts of water; The surfactant is any one of Peregal O, Triton X-100, and sodium dodecyl sulfate; The mass ratio of the mammalian fur to the degreasing agent is 1:3; The liming treatment time is 14 to 16 hours.

6. The preparation method according to claim 1, characterized in that: The deliming liquid in the deliming treatment comprises: mixing ammonium sulfate and sodium bisulfite to form a mixed liquid, and using glacial acetic acid solution to adjust the pH of the mixed liquid to 7.5-8.0; The glacial acetic acid solution is prepared by diluting glacial acetic acid and water in a volume ratio of 1:10; The mass of the ammonium sulfate is 0.8% to 1% of the mass of the mammal's fur; The mass of the sodium bisulfite is 0.4% to 0.5% of the mass of the mammal's fur; The deashing treatment time is 50 minutes.

7. The preparation method according to claim 1, characterized in that: In parts by weight, the softening liquid in the softening treatment consists of 40 to 60 parts of ammonium chloride, 40 to 60 parts of trypsin and 9890 to 9900 parts of water; The mass ratio of the mammalian fur to the softening liquid is 1:1.5-2; The softening treatment time is 60 minutes.

8. The preparation method according to claim 1, characterized in that: The rinsing includes alcohol rinsing and PBS rinsing; The alcohol concentration of the alcohol rinse is 50% to 70%, and the alcohol rinse time is 10 to 30 minutes; The pH of the PBS buffer for rinsing with PBS is 7.2-7.4, and the PBS rinsing time is 60-90 minutes.

9. Acellular dermal matrix obtained by the preparation method according to any one of claims 1 to 8.

10. The use of mammalian fur prepared by tanning method in the preparation of acellular dermal matrix.

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