A method for preparing decellularized dermal matrix

Abstract

This invention discloses a method for preparing acellular dermal matrix, belonging to the field of tissue engineering technology. The preparation method includes: selecting pelts from traceable animals aged one to three months, purifying them through steps such as soaking, fleshing, defatting, hair removal, liming, deliming, and softening in a tanning process to obtain acellular dermal matrix. The acellular dermal matrix obtained by this invention removes antigenic components such as cells, hair follicles, and extracellular proteins during the preparation process, minimizing its immunogenicity while preserving the collagen fiber structure in its extracellular matrix. Simultaneously, the preparation method of this invention moderately loosens the collagen fiber bundles. This process not only effectively alleviates the entanglement between fiber bundles but also successfully increases the exposure of hydrophilic groups, giving the acellular dermal matrix excellent hydrophilicity, which is more conducive to cell proliferation and migration.

Description

Technical Field

[0001] This invention belongs to the field of tissue engineering technology and relates to a method for preparing decellularized dermal matrix. Background Technology

[0002] The skin is the body's defensive barrier against external threats, and it is mainly composed of the epidermis, dermis, and subcutaneous tissue. Extensive or deep skin damage, especially damage involving the dermis, can lead to infection, shock, and poor wound elasticity, unevenness, and contractures after healing. Traditional treatments include autologous skin grafting, but this method can cause secondary harm to patients and prolong treatment time. To address these issues, scientists have developed a human skin substitute. The skin structure of mammals is similar to that of humans, making their skin an ideal source of replacement material.

[0003] Adenosine dermal matrix (ADM) is a dermal substitute obtained by processing fresh xenogeneic skin using bioengineering techniques to remove highly immunogenic cellular components while retaining extracellular matrix components. Since rejection in skin transplantation is primarily caused by cellular immunity, ADM, after removing highly immunogenic cellular components, exhibits very low antigenicity, allowing it to persist long-term in the host and eventually be absorbed and remodeled by normal tissue. As a human skin substitute that provides a scaffold structure for tissue cell regeneration, ADM can be used for wound coverage, filling of damaged tissue, promoting wound healing, guiding tissue regeneration, and accelerating cell proliferation. It features good tissue compatibility, strong guidance properties, and good tissue conformation, and is widely used in the field of human tissue wound repair.

[0004] Researchers have used methods such as repeated freeze-thaw cycles and trypsin digestion to prepare ADM (anti-dermal mesenchymal stem cells). However, these methods have not been widely adopted because they cannot completely remove dermal cellular components, leading to strong rejection reactions in the host after ADM transplantation. Furthermore, the preparation process is overly complex. Four common methods for ADM preparation are the Dispase II-Triton method, the hypertonic saline-SDS method, the hypertonic saline-NaOH ablation method, and the hypertonic saline-enzyme digestion method. Each method has its advantages and disadvantages. Generally speaking, most existing methods cannot simultaneously maintain low antigenicity and structural integrity. Removing antigenic components inevitably leads to structural damage, affecting fibroblast migration and collagen deposition, resulting in poor wound coverage or filling effects of ADM. Conversely, preserving structural integrity leads to incomplete decellularization and a certain degree of immunogenicity, thus limiting its application. Therefore, there is an urgent need to explore a novel ADM preparation method that can maintain low immunogenicity while ensuring structural integrity. Tanning, a traditional process for processing animal skin, involves multiple steps including soaking, liming, hair removal, softening, pickling, tanning, dyeing, and emulsion application. The mechanical action of a rotating drum promotes the uniform penetration and chemical reaction of various chemical materials. However, the primary purpose of tanning is to produce leather, not to prepare decellularized dermal matrix (ADM). This invention explores the potential applications of tanning in ADM preparation, aiming to find a novel, efficient, and practical preparation method that opens up broader prospects for the clinical application of ADM. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and disadvantages of the prior art. This invention uses a tanning method to process mammalian fur, removing antigenic components such as cells, hair follicles, and miscellaneous proteins, to obtain a decellularized dermal matrix with low immunogenicity and intact structure.

[0006] In a first aspect, the present invention provides a method for preparing a decellularized dermal matrix, wherein mammalian fur undergoes soaking, fleshing, defatting, hair removal, liming, deliming, and softening treatments in a tanning process, followed by rinsing, freeze-drying, and irradiation sterilization to obtain a decellularized dermal matrix.

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

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

[0009] The penetrant is fatty alcohol polyoxyethylene ether;

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

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

[0012] The degreasing process takes 60–90 minutes.

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

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

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

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

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

[0018] The surfactant is any one of Pintoline 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 immersion treatment time is 14 to 16 hours.

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

[0022] The glacial acetic acid solution was prepared by diluting glacial acetic acid and water at a volume ratio of 1:10.

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

[0024] The sodium bisulfite comprises 0.4% to 0.5% of the weight of mammalian fur.

[0025] The deashing process takes 50 minutes.

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

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

[0028] The softening process takes 60 minutes.

[0029] Furthermore, the rinsing process described in this invention includes alcohol rinsing and PBS rinsing;

[0030] The alcohol concentration for the alcohol rinsing is 50% to 70%, and the alcohol rinsing time is 10 to 30 minutes.

[0031] The pH of the PBS buffer used for PBS rinsing is 7.2–7.4, and the PBS rinsing time is 60–90 min.

[0032] Secondly, the present invention provides an acellular dermal matrix, which has low antigenicity, intact structure, well preserved basement membrane, and good hydrophilicity, thermal stability and mechanical properties.

[0033] Thirdly, this invention provides the application of mammalian fur prepared by tanning in the preparation of acellular dermal matrix. This invention purifies the acellular dermal matrix through steps such as soaking, fleshing, defatting, hair removal, liming, deliming, and softening in the tanning process. Because this invention minimizes the immunogenicity of the acellular dermal matrix while preserving the collagen fiber structure in its extracellular matrix, and simultaneously loosens the tangled collagen fiber bundles, exposing more hydrophilic groups, it exhibits excellent hydrophilicity, which is more conducive to cell proliferation and migration. This has promising application prospects in the fields of chronic wound healing, wound repair, and plastic surgery.

[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) Without destroying the collagen scaffold formed by the interweaving and entanglement of collagen microfibers, collagen fibers and collagen fiber bundles, the present invention removes antigenic components such as cells, hair follicles, miscellaneous proteins and fats through the steps of soaking, fleshing, defatting, hair removal, liming, deliming and softening in traditional leather making methods, thereby minimizing its immunogenicity, while preserving the complete basement membrane and the light and dark overlapping D periodic transverse striation structure, ensuring its structural integrity and high biological activity.

[0036] (2) Using the traditional lime hair removal method, the epidermis with high fat content can be removed, the hair is removed cleanly without leaving any hair roots, and it will not damage the basement membrane, preserving 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 decellularized dermal matrix prepared by the present invention has the deep penetration of surfactants and enzymes in the degreasing, softening and other steps. While removing immunogenic substances, the collagen fibers are loosened to a certain extent, and the hydrophilic groups on the chain are exposed to a greater extent. The water contact angle is maintained at about 50°, which has excellent hydrophilicity and is more conducive to cell adhesion, growth, proliferation and migration. It is also convenient to cover the wound to keep the wound moist and is more conducive to collagen deposition and granulation growth in the wound.

[0038] (4) In the process of ash soaking, alcohol soaking and cleaning, and irradiation disinfection and sterilization, the strong alkaline environment, alcohol and γ-rays cause the inactivation of viruses and fungi, which greatly reduces the risk of viruses.

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

[0040] (6) The method provided by the present invention has a simple process flow, requires inexpensive reagents, and has a high degree of automation. It is also suitable for the transformation of the traditional leather industry, and can be mass-produced without the need to lay out new production lines. Attached Figure Description

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

[0042] Figure 2 This is a SEM image of the decellularized dermal matrix.

[0043] Figure 3 This is an FT-IR image of decellularized dermal matrix.

[0044] Figure 4 Photographs showing the water contact angle of decellularized dermal matrix. In the figures, A represents the decellularized dermal matrix prepared in Example 1; B represents the decellularized dermal matrix prepared in Example 2; and C represents the decellularized dermal matrix prepared in Example 3.

[0045] Figure 5 This is a comparison of the mechanical properties of decellularized dermal matrix. In the figure, A represents tensile strength; B represents elongation at break.

[0046] Figure 6 This is a graph showing the thermal stability of the decellularized dermal matrix. Detailed Implementation

[0047] The technical solution of the present invention will be described below with reference to embodiments. However, the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods and detection methods described in each embodiment are conventional methods; unless otherwise specified, the reagents and materials can be purchased commercially.

[0048] Example 1

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

[0050] s1. Select salt-dried pelts from three-month-old goats. Trim the pelts to remove uneven thickness from areas such as the abdomen and limbs. Place the trimmed salt-dried pelts in a tumbler, add water (water to pelt mass ratio 1:3), and wash for 60 minutes. To ensure cleaning effectiveness, a combination of simmering and running water rinsing is used. After a certain period of simmering, switch to running water rinsing to remove impurities and residues from the pelts. This process needs to be repeated three times until the rinsing water becomes clear and transparent, ensuring the pelts are clean.

[0051] s2. Put the dried goat fur from s1 back into the drum, add water, the mass ratio of water to dried goat fur is 3:1, then add 0.2% of JFC penetrant (fatty alcohol polyoxyethylene ether) by mass of dried goat fur, set the drum to slow speed and run for 6 hours, drain the waste liquid and wash with water.

[0052] s3. Lay the salt-dried goat skin from s2 flat and remove the fascia, bits of meat, and oil film attached to the skin.

[0053] s4. Place the salt-dried goat hides from s3 into a rotating drum, add a degreasing agent (mass ratio of salt-dried goat hides to degreasing agent 1:4), control the reaction temperature at 38℃, react for 60 minutes, and then drain the salt-dried goat hides for several hours until the waste liquid is completely dry. The degreasing agent contains 0.5% surfactant, 2% soda ash, 0.2% JFC penetrant, and 97.3% water, wherein the surfactant is Pingpingjia O.

[0054] s5. Place the dried goat pelt from s4 with the flesh side facing up, and evenly apply the depilatory agent. The mass ratio of the dried goat pelt to the depilatory paste is 1:1. After piling for 3.5 hours, manually push the hair away. The depilatory paste contains 5% sodium sulfide, 40% quicklime, 0.6% ammonium sulfate, 0.1% surfactant, and 54.3% water. The surfactant is Pingpingjia O.

[0055] s6. Wash the salt-dried goat hides from s5 several times with water until the water becomes clear. Place the salt-dried goat hides in a rotating drum, control the temperature at 22℃, add the liming solution (mass ratio of salt-dried goat hides to liming solution 1:3), rotate for 30 minutes, stop for 60 minutes, rotate again for 30 minutes, stop the drum overnight, and then rotate for 15 minutes the next day before washing with water. The total liming treatment time is 16 hours. The liming solution includes 2% flake sodium sulfide, 4% calcium hydroxide, 0.04% trypsin, 0.06% surfactant, and 93.9% water, wherein the surfactant is Pingpingjia O.

[0056] s7. Place the dried goat fur from s6 into a rotating drum for deliming treatment, controlling the temperature at 35℃. Add ammonium sulfate and sodium bisulfite, rotate for 10 minutes, then slowly drip in glacial acetic acid solution while continuing to rotate the drum until the pH stabilizes at 7.5–8.0. Stop adding glacial acetic acid and rotate for 40 minutes. The deliming solution is a mixed solution of 1% ammonium sulfate and 0.4% sodium bisulfite by weight of the dried goat fur, adjusted to pH 7.5–8.0 with glacial acetic acid solution. The glacial acetic acid solution is a mixture of glacial acetic acid and water at a volume ratio of 1:10.

[0057] s8. Place the salt-dried goat hides from s7 into a rotating drum, control the temperature at 37℃, add softening solution (the mass ratio of softening solution to salt-dried goat hides is 1:2), ensuring the pH of the solution inside the drum is 7.5–8.2, then rotate for 60 minutes until the grains are white, smooth, fine, and free of greasiness, and the hide feels loose. The softening solution, by weight, consists of 0.5% trypsin solution, 0.6% ammonium chloride solution, and 98.9% water.

[0058] s9. The salt-dried goat fur from s8 was placed in a drum and soaked in 60% alcohol for 15 minutes, then soaked in PBS buffer for 60 minutes. After rinsing three times, it was thoroughly rinsed with physiological saline, freeze-dried, and sterilized by gamma irradiation to obtain decellularized dermal matrix, designated GADM-1. The PBS buffer contained 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. The pH was then adjusted to 7.2–7.4 using sterile sodium bicarbonate solution.

[0059] Example 2

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

[0061] s1. Select salt-dried pelts from one-month-old goats. Trim the pelts to remove uneven thickness from areas such as the abdomen and limbs. Place the trimmed pelts in a tumbler, add water (water to pelt weight ratio 1:3), and wash for 60 minutes. To ensure cleaning effectiveness, a combination of simmering and running water rinsing is used. After a certain period of simmering, switch to running water to remove impurities and residues from the pelts. This process is repeated three times until the rinsing water becomes clear and transparent, ensuring the pelts are clean.

[0062] s2. Put the dried goat fur from s1 back into the drum, add water, the mass ratio of water to dried goat fur is 3:1, then add 0.2% of JFC penetrant by mass of dried goat fur, set the drum to slow speed and run for 6 hours, drain the waste liquid and wash with water.

[0063] s3. Lay the salt-dried goat skin from s2 flat and remove the fascia, bits of meat, and oil film attached to the skin.

[0064] s4. Place the salt-dried goat hides from s3 into a rotating drum, add a degreasing agent (mass ratio of salt-dried goat hides to degreasing agent 1:3), control the reaction temperature at 38℃, react for 90 minutes, and then drain the salt-dried goat hides for several hours until the waste liquid is completely dry. The degreasing agent contains 0.3% surfactant, 1.5% soda ash, 0.2% JFC penetrant, and 98% water. The surfactant is Triton X-100.

[0065] s5. Place the dried goat pelt from s4 with the flesh side facing up, and evenly apply the depilatory paste. The mass ratio of the dried goat pelt to the depilatory paste is 1:1.5. After piling for 3.0 hours, manually push off the hair. The depilatory paste contains 5% sodium sulfide, 45% quicklime, 0.4% ammonium sulfate, 0.1% surfactant, and 49.50% water. The surfactant is Triton X-100.

[0066] s6. Wash the salt-dried goat hides from s5 several times with water until the water becomes clear. Place the salt-dried goat hides in a rotating drum, control the temperature at 22℃, add the liming solution (mass ratio of salt-dried goat hides to liming solution 1:3), rotate for 30 minutes, stop for 60 minutes, rotate again for 30 minutes, stop the drum overnight, and then rotate for 15 minutes the next day before washing with water. The total liming treatment time is 16 hours. The liming solution includes 2% flake sodium sulfide, 5% calcium hydroxide, 0.02% trypsin, 0.08% surfactant, and 92.9% water, wherein the surfactant is Triton X-100.

[0067] s7. Place the dried goat fur from s6 into a rotating drum for deliming treatment, controlling the temperature at 35℃. Add ammonium sulfate and sodium bisulfite, rotate for 10 minutes, then slowly drip in glacial acetic acid solution while continuing to rotate the drum until the pH stabilizes at 7.5–8.0. Stop adding glacial acetic acid and rotate for 40 minutes. The deliming solution is a mixed solution of 0.8% ammonium sulfate and 0.5% sodium bisulfite by weight of the dried goat fur, adjusted to pH 7.5–8.0 with glacial acetic acid solution. The glacial acetic acid solution is a mixture of glacial acetic acid and water at a volume ratio of 1:10.

[0068] s8. Place the salt-dried goat hides from s7 into a rotating drum, maintain the temperature at 37℃, add softening solution, and maintain the temperature at 37℃. The mass ratio of softening solution to salt-dried goat hides is 1:1.5, ensuring the pH of the solution inside the drum is 7.5–8.2. Then rotate for 60 minutes, until the grains are white, smooth, fine, and free of greasiness, and the hide feels loose. The softening solution consists of 0.5% trypsin solution, 0.4% ammonium chloride solution, and 99.1% water.

[0069] s9. The salt-dried goat fur from s8 was placed in a drum, soaked and washed with 50% alcohol for 10 minutes, then soaked in PBS buffer for 90 minutes, rinsed three times, thoroughly rinsed with physiological saline, and freeze-dried in a freeze dryer. After sterilization by gamma irradiation, the decellularized dermal matrix was obtained, designated GADM-2. The PBS buffer contained 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. The pH was then adjusted to 7.2–7.4 using sterile sodium bicarbonate solution.

[0070] Example 3

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

[0072] s1. Select salt-dried pelts from young goats of a certain age. Trim the pelts to remove uneven thickness from areas such as the abdomen and limbs. Place the trimmed pelts in a tumbler, add water (water to pelt weight ratio 1:3), and wash for 60 minutes. To ensure cleaning effectiveness, a combination of simmering and running water rinsing is used. After a certain period of simmering, switch to running water to remove impurities and residues from the pelts. This process is repeated three times until the rinsing water becomes clear and transparent, ensuring the pelts are clean.

[0073] s2. Put the dried goat fur from s1 back into the drum, add water, the mass ratio of water to dried goat fur is 3:1, then add 0.2% of JFC penetrant by mass of dried goat fur, set the drum to slow speed and run for 6 hours, drain the waste liquid and wash with water.

[0074] s3. Lay the salt-dried goat skin from s2 flat and remove the fascia, bits of meat, and oil film attached to the skin.

[0075] s4. Place the salt-dried goat hides from s3 into a rotating drum, add a degreasing agent (mass ratio of salt-dried goat hides to degreasing agent 1:5), control the reaction temperature at 38℃, react for 60 minutes, and then drain the salt-dried goat hides for several hours until the waste liquid is completely dry. 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 dried goat pelts from s4 with the flesh side facing up, and evenly apply the depilatory paste. The mass ratio of the dried goat pelts to the depilatory paste is 1:2. After piling for 2.5 hours, manually push the hair away. The depilatory paste contains 5% sodium sulfide, 40% quicklime, 0.6% ammonium sulfate, 0.1% surfactant, and 54.3% water. The surfactant is sodium dodecyl sulfate.

[0077] s6. Wash the salt-dried goat hides from s5 several times with water until the water runs clear. Place the salt-dried goat hides in a rotating drum, control the temperature at 22℃, add the liming solution (mass ratio of salt-dried goat hides to liming solution 1:3), rotate for 30 minutes, stop for 60 minutes, rotate again for 30 minutes, stop the drum overnight, and then rotate for 15 minutes the next day before washing with water. The total liming treatment time is 14 hours. The liming solution contains 3% flake sodium sulfide, 3% calcium hydroxide, 0.06% trypsin, 0.05% surfactant, and 93.89% water, wherein the surfactant is sodium dodecyl sulfate.

[0078] s7. Place the dried goat fur from s6 into a rotating drum for deliming treatment, controlling the temperature at 35℃. Add ammonium sulfate and sodium bisulfite, rotate for 10 minutes, then slowly drip in glacial acetic acid solution while continuing to rotate the drum until the pH stabilizes at 7.5–8.0. Stop adding glacial acetic acid and rotate for 40 minutes. The deliming solution is a mixed solution of 1% ammonium sulfate and 0.5% sodium bisulfite by weight of the dried goat fur, adjusted to pH 7.5–8.0 with glacial acetic acid solution. The glacial acetic acid solution is a mixture of glacial acetic acid and water at a volume ratio of 1:10.

[0079] s8. Place the salt-dried goat hides from s7 into a rotating drum, control the temperature at 37℃, add softening solution (the mass ratio of softening solution to salt-dried goat hides is 1:1.5), ensuring the pH of the solution inside the drum is 7.5–8.2, then rotate for 60 minutes until the grains are white, smooth, fine, and free of greasiness, and the hide feels loose. The softening solution consists of 0.6% trypsin solution, 0.5% ammonium chloride solution, and 98.90% water.

[0080] s9. The salt-dried goat fur from s8 was placed in a drum and soaked in 70% alcohol for 30 minutes, then soaked in PBS buffer for 90 minutes. After rinsing three times, it was thoroughly rinsed with physiological saline, freeze-dried, and sterilized by gamma irradiation to obtain decellularized dermal matrix, designated GADM-3. The PBS buffer contained 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. The pH was then adjusted to 7.2–7.4 using sterile sodium bicarbonate solution.

[0081] Example 4

[0082] This embodiment provides the test results of the prepared decellularized dermal matrix.

[0083] Figure 1 Histological photographs of the decellularized dermal matrix obtained in Examples 1-3. Figure 1As shown, histological evaluation was performed using hematoxylin and eosin (HE), Masson's 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, numerous fluorescently stained nuclei were observed, indicating abundant cellular material. In contrast, purified GADM showed very few to no nuclei or nuclear debris. Masson's 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 adipose tissue content. A comprehensive analysis of the HE, Masson's, and Oil Red O staining results showed that, unlike the original goat salt-dried fur, purified GADM underwent complete removal of the epidermis. Furthermore, cells, hair follicles, sweat glands, fat, miscellaneous proteins, and other components were effectively eliminated. The preparation method provided by this invention removes non-collagenous components from goat skin while retaining collagen fibers that represent the most basic structural framework. Furthermore, the network structure and collagen fiber bundles are loosened to a certain extent; this loosening can promote cell proliferation, collagen deposition, and tissue healing when GADM is used as a skin substitute.

[0084] Figure 2 SEM images of the decellularized dermal matrix obtained in Examples 1-3. Figure 2 As shown, the prepared GADM surface is dense and exhibits a fully preserved uneven basement membrane and a clearly visible pore structure. The cross-section of the GADM is a three-dimensional network structure of loosely interwoven and stacked collagen fibers and fiber bundles, with D-cycles of approximately 65 nm visible 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 bands of GADM-1, GADM-2, and GADM-3 appear at 3445 cm⁻¹, respectively. -1 3452cm -1 and 3431cm -1 At this point, the stretching vibration of the NH bond is observed. The amide B band peaks of GADM-1, GADM-2, and GADM-3 are located at 3076 cm⁻¹. -1 3075cm -1 and 3079cm -1 At this location, a peak corresponding to the CH stretching vibration was observed. The amide I band peak representing the C=O stretching vibration appeared at 1662 cm⁻¹ in GADM-1, GADM-2, and GADM-3, respectively. -1 1660cm-1 and 1652cm -1 The amide II band peaks are located at 1557 cm⁻¹ for GADM-1, GADM-2, and GADM-3, respectively. -1 1554cm -1 and 1550cm -1 The presence of this peak indicates coupling between the NH bending vibration and the CN stretching vibration. Finally, the amide III characteristic peaks of GADM-1, GADM-2, and GADM-3 are concentrated at 1241 cm⁻¹. -1 1241cm -1 and 1240cm -1 The values ​​represent the CN stretching vibration of the amide bond, the NH bending vibration of the main chain, and the rocking vibration of the CH2 group in proline. These results indicate that the characteristic absorption peaks of the amide bands are present in GADM-1, GADM-2, and GADM-3 with minimal differences, suggesting that the decellularized dermal matrices prepared in Examples 1-3 have essentially similar secondary structures.

[0086] Figure 4 The surface contact angles (θ) of GADM-1, GADM-2, and GADM-3 are shown, which are 48.4° ( Figure 4 A in the middle), 56.2° ( Figure 4 (B) and 50.2° Figure 4 In the C) process, the intertwined collagen microfibers, collagen fibers and collagen fiber bundles were loosened to a certain extent during the purification process, exposing the hydrophilic groups that were blocked by the adjacent D cycle. The richer hydrophilic groups resulted in better hydrophilicity. The results showed that the decellularized dermal matrix prepared in Examples 1 to 3 had little difference in water contact angle and stable hydrophilicity.

[0087] like Figure 5 As shown in Figure A, 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 were approximately 19.73%, 20.81%, and 21.32%, respectively. This indicates that the decellularized dermal matrix prepared in Examples 1-3 still possesses strong tensile strength and high elongation at break after various purification treatments.

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

[0089] The embodiments described above are some, but not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. A method for preparing acellular dermal matrix, characterized in that, After the tanning process, mammalian fur undergoes soaking, fleshing, degreasing, hair removal, liming, deliming, and softening, followed by rinsing, freeze-drying, and irradiation sterilization to obtain decellularized dermal matrix. By weight, the degreasing agent in the degreasing treatment consists of 100-200 parts of soda ash, 10-20 parts of penetrant, 30-80 parts of surfactant and 9730-9800 parts of water; The penetrant is fatty alcohol polyoxyethylene ether; By weight, the hair removal agent in the hair removal treatment consists of 500 parts sodium sulfide, 4000-5000 parts quicklime, 40-60 parts ammonium sulfide, 10-20 parts surfactant and 4950-5430 parts water; By weight, the leaching solution in the leaching treatment consists of 200-300 parts sodium sulfide, 300-500 parts calcium hydroxide, 2-5 parts trypsin, 5-10 parts surfactant and 9290-9390 parts water; The deashing solution in the deashing process includes: mixing ammonium sulfate and sodium bisulfite to prepare a mixed solution, and adjusting the pH of the mixed solution to 7.5~8.0 using glacial acetic acid solution; By weight, the softening solution in the softening treatment consists of 40-60 parts ammonium chloride, 40-60 parts trypsin, and 9890-9900 parts water.

2. The preparation method according to claim 1, characterized in that, The mammalian fur is salt-dried fur from a young goat; The goats mentioned are 1 to 3 months old.

3. The preparation method according to claim 1, characterized in that, The surfactant is any one of Pintoli 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 process takes 60-90 minutes.

4. The preparation method according to claim 1, characterized in that, The mass ratio of the mammalian fur to the depilatory agent is 1:1~2; The hair removal treatment time is 2.5~3.5 hours.

5. The preparation method according to claim 1, characterized in that, The mass ratio of the mammalian fur to the liming solution is 1:3; The immersion treatment time is 14-16 hours.

6. The preparation method according to claim 1, characterized in that, The glacial acetic acid solution was prepared by diluting glacial acetic acid and water at a volume ratio of 1:

10. The mass of the ammonium sulfate is 0.8% to 1% of the mass of mammalian fur; The sodium bisulfite is present in an amount of 0.4% to 0.5% of the weight of mammalian fur. The deashing process takes 50 minutes.

7. The preparation method according to claim 1, characterized in that, The mass ratio of the mammalian fur to the softening solution is 1:1.5~2; The softening process takes 60 minutes.

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

9. The decellularized dermal matrix prepared by any one of claims 1 to 8.

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