A tissue repair hemostatic membrane and its preparation method and application

Abstract

The present invention belongs to the field of medical material technology and provides a tissue repair hemostatic membrane and its preparation method and application. The method comprises: S1, taking non-human mammal skin tissue and pre-treating it to sample the dermis; the removed skin tissue is divided into the epidermis and dermis; S2, subjecting the material obtained in step S1 to freeze-thaw treatment to remove cells to obtain a matrix material; S3, applying a protective agent to the matrix material and freeze-drying it; S4, kneading or repeatedly folding it after freeze-drying to make it soft. The present invention uses animal skin tissue, which has a wide range of raw materials and low cost, to obtain the matrix material after simple treatment. The gel material is placed on the dermis and freeze-dried to obtain the tissue repair hemostatic membrane. The raw materials used are widely available and low cost. The material processing method is simple and low cost. The product has diverse uses, is easy to operate in clinical use, saves time and effort, and has good practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of medical materials, and in particular relates to a tissue repair hemostatic membrane and a preparation method and application thereof. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0003] Acellular matrix materials are biologically derived materials. They are derived from allogeneic or xenogeneic animal skin tissue. After the cellular components are removed through physical, chemical, or biological methods, the extracellular matrix (ECM) scaffolding structure remains. Removing the cellular components reduces immune rejection, while the remaining scaffolding structure guides host cells to grow within it, promoting defect repair and enhancing tissue toughness.

[0004] When using animal skin tissue to prepare acellular matrix materials, the following processes are often performed: mechanical hair removal followed by decellularization. The inventors have discovered that mechanical hair removal can result in incomplete hair removal, leaving hair roots. Post-hair removal tissue processing methods commonly include enzymatic and chemical methods. Enzymatic methods often damage the integrity of the skin tissue, while chemical methods can cause issues such as solvent residue. Summary of the Invention

[0005] To address the shortcomings of the prior art, the present invention provides an acellular matrix tissue repair hemostatic membrane, its preparation method, and its application. This invention utilizes animal skin tissue, a widely available, low-cost raw material, to obtain a matrix material after simple processing. The gel material is then applied to the dermis and freeze-dried to create a tissue repair hemostatic membrane, demonstrating its potential for practical application.

[0006] One of the purposes of the present invention is to provide a method for preparing a tissue repair hemostatic membrane.

[0007] A second object of the present invention is to provide a tissue repair hemostatic membrane prepared by the above method.

[0008] A third object of the present invention is to provide applications of the above-mentioned tissue repair hemostatic membrane.

[0009] To achieve the above object, the present invention relates to the following technical solutions:

[0010] A first aspect of the present invention provides a method for preparing a tissue repair hemostatic membrane, the method comprising:

[0011] S1. Pre-processing skin tissue from a non-human mammal and sampling the dermis; minimizing the risk of sampling the subdermal tissue; and separating the removed skin tissue into the epidermis and dermis. The pre-processing includes at least laser hair removal.

[0012] S2, subjecting the material obtained in step S1 to freeze-thaw treatment to remove cells to obtain a matrix material;

[0013] S3, applying a protective agent to the matrix material and freeze-drying;

[0014] S4. Take the freeze-dried membrane and rub or fold it repeatedly to make it soft.

[0015] The second aspect of the present invention provides a tissue repair hemostatic membrane prepared by the above method. The tissue repair hemostatic membrane has excellent adhesion and significant hemostatic effect, can better promote wound repair and healing, and is safe and non-toxic.

[0016] After freeze-drying, this product is divided into two sides. One side is a dense epidermal dermis layer that prevents adhesion and blocks collagen fiber ingrowth. The other side is a gel layer that promotes angiogenesis, builds a scaffold for new cells, and promotes defect repair. In clinical applications, the placement of the two sides should be differentiated according to actual conditions. For example, in the field of oral medicine, membrane repair products used in oral implants, alveolar surgery, and periodontology can isolate oral soft tissue and bone defects. When used, the epidermal dermis layer that blocks collagen fiber ingrowth is placed on the outside, and the gel layer that can build a scaffold for new cells is placed in the bone defect area. At this time, the repair membrane establishes a biological barrier, creating a relatively closed environment for bone regeneration. When used in other clinical fields, the orientation of the two sides should also be selected according to needs.

[0017] Therefore, the third aspect of the present invention provides the use of the above-mentioned tissue repair hemostatic membrane in the preparation of medical materials; the medical materials have multiple functions such as sealing, anti-adhesion, and hemostasis.

[0018] Beneficial effects of the above technical solution:

[0019] The above technical solution uses animal skin tissue as raw material and successfully prepares an acellular matrix tissue repair hemostatic membrane with multiple functions such as sealing, anti-adhesion, and hemostasis. The raw materials used are widely available and low in cost. The material processing method is simple and low in cost, and it is easy to promote and apply industrially. The product has diverse uses, is easy to operate in clinical use, saves time and effort, and has good practical application value. DETAILED DESCRIPTION

[0020] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0021] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0022] As previously mentioned, the preparation of acellular matrix materials from animal skin tissue often involves mechanical hair removal followed by decellularization. The inventors have discovered that mechanical hair removal can result in incomplete hair removal, leaving hair roots. Post-hair removal tissue processing methods often include enzymatic and chemical methods. Enzymatic methods often damage the integrity of the skin tissue, while chemical methods can lead to residual solvents.

[0023] In view of this, in a typical embodiment of the present invention, a method for preparing a tissue repair hemostatic membrane is provided, the method comprising:

[0024] S1. Pre-processing skin tissue from a non-human mammal and sampling the dermis; minimizing the risk of sampling the subdermal tissue; wherein the pre-processing includes at least laser hair removal of the skin tissue;

[0025] S2, subjecting the material obtained in step S1 to freeze-thaw treatment to remove cells to obtain a matrix material;

[0026] S3, applying a protective agent to the matrix material and freeze-drying;

[0027] S4. Take the freeze-dried membrane and rub or fold it repeatedly to make it soft.

[0028] In step S1, the non-human mammal may be a cow, pig, or the like, and the selected animal is 9 months of age or younger. Since the skin of animals such as cows and pigs is densely covered with hair, laser hair removal can be performed repeatedly, for example, 1-12 times. The interval between each hair removal treatment is controlled to be at least one day or longer. In one embodiment of the present invention, the interval between hair removal treatments is one week.

[0029] Sampling is performed at least one day after the last hair removal. The sampling can be performed using a drum-type skin-sampling machine, a roller knife or other skin-sampling devices, which are not specifically limited here.

[0030] In a specific embodiment of the present invention, the sampling thickness can be controlled to be 0.1 mm-3 mm.

[0031] Furthermore, after the sampling is completed, the sample is rinsed with running water to remove residual impurities, transferred to PBS (pH 7.3) buffer, ultrasonically shaken for 10-20 minutes, and then soaked and rinsed with 10-20 times the amount of water for 0.5-2 hours; the soaking operation is repeated 2-10 times.

[0032] In step S2, the freeze-thaw treatment is specifically performed as follows: the membrane material is spread on a flat plate and placed in a -40 to -10°C environment for freezing, taken out after 6 to 24 hours, and ultrasonically shaken in a 30 to 40°C water bath for 10 to 60 minutes; after ultrasonic shaking, the membrane material is taken out and soaked in PBS buffer or pure water for 10 to 60 minutes; and the freeze-thaw operation at -40 to -10°C is repeated 2 to 10 times.

[0033] In step S3, the protective agent can be any one or more of sodium alginate, cellulose and its derivatives, chitosan and its derivatives, hyaluronic acid and its derivatives, gum arabic, xanthan gum, and lithium magnesium silicate. By adding the above protective agent, a three-dimensional scaffold structure is formed to protect the dermis during the freeze-thaw process, providing a scaffold for the host repair cells to grow into, ensuring the repair effect, and improving the performance of the repair hemostatic membrane finally prepared. At the same time, the protective agent enhances the adhesion of the product, so that when the repair membrane is used in certain parts, a suture-free operation can be performed to achieve a sealing effect, and the clinical operation is simple; moreover, the protective agent is a gel preparation that can carry drugs and has a sustained release effect, thereby achieving targeted drug delivery; the gel contains ions that can coagulate blood to form thrombi, which has a hemostatic effect.

[0034] The protective agent is applied to one side of the dermis. Specifically, the protective agent can be added using the following method: dissolve the protective agent into a gel (controllable content of 2-6%, w / w) and spread it on a flat plate, at a height lower than the skin tissue to be treated; with the epidermis side facing up, the dermis side is exposed to the gel; or, with the skin tissue spread on a flat plate, with the dermis side facing up, the gel protective agent is applied. After adding the protective agent, to further ensure its penetration into the dermis, the protective agent is allowed to stand for 10 minutes or more before freeze-drying.

[0035] The specific freeze-drying procedure includes: pre-freezing at -40 to -30°C for 1-4 hours; vacuuming after pre-freezing, and maintaining at -40 to -30°C for 1-3 hours; then heating to -30 to -25°C, heating time for 1-3 hours, and maintaining for 2-3 hours; then heating to -20°C, heating time for 2-3 hours, and maintaining for 2-3 hours; then heating to -15°C, heating time for 1-3 hours, and maintaining for 1-3 hours; then heating to -10°C, heating time for 1-3 hours, and maintaining for 1-3 hours; then heating to 0°C, heating time for 1-3 hours, and maintaining for 1-3 hours; then heating to 10°C, heating time for 1-3 hours, and maintaining for 1-3 hours; then heating to 20°C, heating time for 1-3 hours, and maintaining for 1-3 hours; then heating to 30°C, heating time for 2-3 hours, and maintaining for 2-3 hours.

[0036] Take the freeze-dried membrane and rub or fold it repeatedly to make it soft.

[0037] The method further includes the steps of cutting the hemostatic repair membrane after kneading or repeatedly folding it to soften it, marking the front and back of the membrane after cutting, packaging, and sterilizing it. For example, the hemostatic repair membrane can be cut into appropriate sizes and then packaged in blisters or aluminum-plastic bags. After packaging, sterilization can be performed using radiation sterilization or ethylene oxide sterilization, which will not be described in detail here.

[0038] In another specific embodiment of the present invention, a tissue repair hemostatic membrane prepared by the above method is provided. The tissue repair hemostatic membrane has excellent adhesion and significant hemostatic effect, can better promote wound repair and healing, and is safe and non-toxic.

[0039] After freeze-drying, this product separates into two sides. One side comprises a dense epidermal dermis layer, which prevents adhesion and inhibits fiber ingrowth. The other side, a gel layer, promotes angiogenesis, provides a scaffold for new cells, and promotes defect repair. In clinical applications, the placement of the two sides is determined based on practical needs. For example, in the field of stomatology, membrane repair products used in oral implantology, alveolar surgery, and periodontology can isolate oral soft tissue from bone defects. During use, the epidermal dermis layer, which prevents collagen fiber ingrowth, is placed on the outside, while the gel layer, which provides a scaffold for new cells, is placed within the bone defect area. This repair membrane then establishes a biological barrier, creating a relatively closed environment for bone regeneration. When used in other clinical areas, the orientation of the two sides should also be selected based on needs. Therefore, another specific embodiment of the present invention provides the use of the above-mentioned tissue repair hemostatic membrane in the preparation of medical materials; these medical materials have multiple functions, including sealing, preventing adhesion, and hemostasis.

[0040] The present invention is further explained by the following examples, but is not intended to limit the present invention. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention. The test methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions.

[0041] Example 1

[0042] Preparation of matrix materials

[0043] Select a livestock farm with qualified epidemic prevention qualifications and select three-month-old pigs of either sex. Secure the pig's forelimbs and hind limbs appropriately, exposing the abdomen and back. First, roughly shave with a razor, then use a laser hair removal device. Select a commercially available laser hair removal device. Set the instrument parameters according to the device's manual. Repeat the laser hair removal procedure one week later. Take samples one week later. Use a drum skin harvester, roller blade, or other skin harvesting device to harvest skin down to the dermis. Samples should be approximately 0.2 mm thick. After sampling, rinse with running water to remove impurities, transfer to PBS (pH 7.3) buffer, sonicate for 15 minutes, and then soak and rinse in 10 times the volume of purified water for 1 hour. Repeat this purified water soaking cycle twice. After soaking, remove the membrane and freeze-thaw. Spread the membrane flat on a flat plate and freeze at -20°C. After 6 hours, remove the membrane and sonicate in a 37°C water bath for 30 minutes. After sonication, remove the membrane and soak and rinse in purified water for 1 hour. Repeat the freeze-thaw process at -20°C and rinse with purified water twice. Then store in physiological saline.

[0044] Gel preparation

[0045] Take lithium magnesium silicate, add it into purified water at a content of 2% (w / w), and disperse and dissolve it into a transparent gel under strong stirring.

[0046] Preparation of repair hemostatic membrane

[0047] The matrix material was spread evenly on a flat plate, with the epidermis and dermis in contact with the plate and the other side facing up. Lithium magnesium silicate gel was evenly applied to the surface. The sample was allowed to stand for 1 hour before freeze-drying. The freeze-drying procedure was as follows: pre-freeze at -30°C for 3 hours. After pre-freezing, vacuum was applied and the sample was held at -30°C for 3 hours. The sample was then heated to -25°C for 3 hours and held for 3 hours. The sample was then heated to -20°C for 3 hours and held for 3 hours. The sample was then heated to -15°C for 3 hours and held for 3 hours. The sample was then heated to -10°C for 3 hours and held for 3 hours. The sample was then heated to 0°C for 3 hours and held for 3 hours. The sample was then heated to 10°C for 3 hours and held for 3 hours. The sample was then heated to 20°C for 3 hours and held for 3 hours. The sample was then heated to 30°C for 3 hours and held for 3 hours.

[0048] Kneading of the diaphragm

[0049] Take the freeze-dried membrane and rub it to make it soft. The rubbing can be repeated.

[0050] After completion, the samples are cut into appropriate sizes, and each film is marked on a corner to distinguish the front and back. The film is then packaged in blisters or aluminum-plastic bags. After packaging, the film is sterilized, either by radiation or ethylene oxide.

[0051] Example 2

[0052] Preparation of matrix materials

[0053] Select a livestock farm with qualified epidemic prevention qualifications and select six-month-old cattle of either sex. Secure the cattle's forelimbs and hind limbs appropriately, exposing the abdomen and back. First, roughly shave with a razor, then use a laser hair removal device. Choose a commercially available laser hair removal device. Set the instrument parameters according to the device's manual. Repeat the laser hair removal procedure two weeks later, repeating the procedure twice. One week later, sample the skin using a drum skin remover, roller blade, or other skin removal device, down to the dermis. Samples should be approximately 0.6 mm thick. After sampling, rinse with running water to remove impurities, transfer to PBS (pH 7.3) buffer, and sonicate for 30 minutes. Continue soaking and rinsing in 10 times the volume of PBS buffer for 0.5 hours. Repeat this soaking process five times in purified water. After soaking, remove the membrane and freeze-thaw. Spread the membrane flat on a plate and freeze at -10°C. After 12 hours, remove the membrane and sonicate in a 37°C water bath for 15 minutes. After sonication, remove the membrane and soak in PBS buffer for 30 minutes. Repeat the freeze-thaw cycle at -10°C and the subsequent PBS rinses eight times. After the final PBS rinse, remove the tube and soak in purified water. Stir at low speed for 1 hour, changing the water five times. After the final water change, remove the tube and store in purified water.

[0054] Gel preparation

[0055] Take sodium alginate, add it into purified water at a content of 3% (w / w), and disperse and dissolve it into a transparent gel under stirring.

[0056] Preparation of repair hemostatic membrane

[0057] The matrix material was spread evenly on a flat plate, with the epidermis and dermis in contact with the plate and the other side facing up. Sodium alginate gel was evenly applied to the surface. The sample was allowed to stand for 2 hours before freeze-drying. The freeze-drying procedure was as follows: pre-freeze at -40°C for 1 hour. After pre-freezing, vacuum was applied and the sample was held at -40°C for 2 hours. The sample was then heated to -30°C for 2 hours and held for 2 hours. The sample was then heated to -20°C for 2 hours and held for 2 hours. The sample was then heated to -15°C for 2 hours and held for 2 hours. The sample was then heated to -10°C for 2 hours and held for 2 hours. The sample was then heated to 0°C for 2 hours and held for 2 hours. The sample was then heated to 10°C for 2 hours and held for 2 hours. The sample was then heated to 20°C for 2 hours and held for 2 hours. The sample was then heated to 30°C for 2 hours and held for 2 hours.

[0058] Kneading of the diaphragm

[0059] Take the freeze-dried membrane and rub it to make it soft. This rubbing can be repeated.

[0060] After completion, the samples are cut into appropriate sizes, and each film is marked on a corner to distinguish the front and back. The film is then packaged in blisters or aluminum-plastic bags. After packaging, sterilization is carried out, which can be done by radiation or ethylene oxide.

[0061] Example 3

[0062] Preparation of matrix materials

[0063] Select a livestock farm with qualified epidemic prevention qualifications and select one-month-old pigs of either sex. Secure the pig's forelimbs and hind limbs appropriately, exposing the abdomen and back. First, roughly remove the hair with a razor, then use a laser hair removal device. Choose a common brand of laser hair removal device. Set the parameters for use according to the device's manual. Repeat laser hair removal one week later. Take samples one week later. Use a drum skin harvester or other skin harvesting device to harvest the skin down to the dermis. The thickness should be approximately 1mm. After sampling, rinse with running water to remove impurities, transfer to PBS (pH 7.3) buffer, and sonicate for 60 minutes. Continue soaking and rinsing with 10 times the amount of PBS buffer for 1 hour. Repeat the purified water soaking process 10 times. After soaking, remove the membrane and perform freeze-thaw treatment. Spread the membrane flat on a flat plate and freeze at -40°C. After 24 hours, remove the membrane and sonicate in a 37°C water bath for 60 minutes. After sonication, remove the membrane and soak and rinse in PBS buffer for 10 minutes. Repeat the freeze-thaw cycle at -40°C and the subsequent rinsing with PBS buffer 10 times. After the final PBS rinse, remove the tube and soak in purified water. Stir at low speed for 10 minutes, changing the water six times. After the final water change, remove the tube and store in PBS buffer.

[0064] Gel preparation

[0065] Take carboxymethyl chitosan, add it into purified water at a content of 6% (w / w), and disperse and dissolve it into a transparent gel under stirring.

[0066] Preparation of repair hemostatic membrane

[0067] Spread the carboxymethyl chitosan gel flat on a flat plate. Place the matrix material evenly on the plate, with the epidermis and dermis facing up and the other side in contact with the gel. Let it sit for 5 hours before freeze-drying. During freeze-drying, peel off the membrane, place the gel side up, and spread it flat on the plate. The freeze-drying procedure is as follows: Prefreeze at -40°C for 4 hours. After pre-freezing, vacuum the temperature and keep it at -30℃ for 3h; then heat it to -25℃ for 2.5h and keep it for 2.5h; then heat it to -20℃ for 2h and keep it for 2h; then heat it to -15℃ for 1.5h and keep it for 1.5h; then heat it to -10℃ for 1h and keep it for 1h; then heat it to 0℃ for 1h and keep it for 1h; then heat it to 10℃ for 1h and keep it for 1h; then heat it to 20℃ for 1h and keep it for 1h; then heat it to 30℃ for 3h and keep it for 3h.

[0068] Repeated folding of the diaphragm

[0069] Take the freeze-dried membrane and fold it repeatedly to make it soft.

[0070] After completion, the samples are cut into appropriate sizes, and each film is marked on a corner to distinguish the front and back. The film is then packaged in blisters or aluminum-plastic bags. After packaging, sterilization is carried out, which can be done by radiation or ethylene oxide.

[0071] Comparative Examples 1 to 3

[0072] In the preparation of Examples 1 to 3, except for applying and soaking the gel, the samples obtained by completing the remaining operations are Comparative Examples 1 to 3, respectively.

[0073] Adhesion test:

[0074] Cut two samples from the Examples / Comparative Examples into 2 cm x 4 cm pieces. Attach the two membranes, gel-side to gel-side, or dermis-side to dermis-side, for a length of 2 cm. Moisten the membranes with purified water. Allow the membranes to stand for 5 minutes. Then, clamp the two ends of the bonded samples in the chucks of a tensile testing machine (do not clamp the bonded area). Stretch the samples up and down at a speed of 5 mm / min until the two samples separate. Record the highest tensile force.

[0075] Table 1 Comparison of the maximum tensile strength values ​​of the embodiments and comparative examples

[0076]

[0077] After adding the gelling agent, the adhesion force of the membrane is much greater than that of the sample without gelling agent.

[0078] Hemostasis test:

[0079] Get rat and be divided into 3 groups at random, be respectively embodiment group, comparative example group and blank control group, 3 in every group, give pentobarbital solution intraperitoneal injection anesthesia, carry out the disinfection of surgical area with iodine tincture after abdomen skin preparation.A longitudinal incision is made in the middle of abdomen, fully exposes the right lobe of liver, excises the tip of the lower edge of the right lobe of liver, and in time covers with diaphragm after excision, observes hemostasis situation, records the time of complete hemostasis, and blank control group does not perform active hemostasis.In the test process, embodiment sample can adhere to wound surface in time.Hemostatic effect is known, and embodiment sample hemostatic effect is better than comparative example sample.

[0080] Table 2 Comparison of hemostatic effects between the examples and comparative examples

[0081]

[0082] Oral mucosal defect repair test:

[0083] New Zealand white rabbits of either sex were selected for the experiment and divided into an embodiment group, a comparative group and a blank control group, with 3 rabbits in each group. A mucosal defect with a diameter of 1 cm was prepared on the hard palate above the rabbit incisors, and samples from the embodiment group and the comparative group were implanted in the defect site. The blank control group was only given conventional gauze for hemostasis without any other treatment. The wound healing was observed after 28 days. The wounds of the embodiment group and the comparative group were completely healed, while the wound of the blank control group still had a large defect. After the experimental rabbits were killed, the repaired wounds were removed and the wound thickness was measured. The thickness of the embodiment group was greater than that of the comparative group. The repair hemostatic membrane after applying the gel can better promote wound repair.

[0084] Cytotoxicity assay:

[0085] Take Examples 1-3 and Comparative Examples 1-3 and conduct cytotoxicity tests according to GB / T 16886.5-2017 Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test. The test method is as follows: Examples 1-3 and Comparative Examples 1-3 are tested at 1.25 cm 2 Extraction medium was added at a ratio of 1:1 / ml. Extraction medium: serum-containing MEM medium, extraction temperature: 37°C-31°C, extraction time: 24h-32h. Extraction solution was prepared according to the method specified in GB / T 16886.5-2017, and cytotoxicity was evaluated quantitatively.

[0086] Table 3 Cytotoxicity results of various examples and comparative examples

[0087]

[0088] Cytotoxicity grade 1 indicates no cytotoxicity; cytotoxicity grade 2 indicates mild cytotoxicity; cytotoxicity grade 3 indicates moderate cytotoxicity; and cytotoxicity grade 4 indicates severe cytotoxicity.

[0089] From the above results, it can be seen that the cytotoxicity results of the embodiment and the comparative example are all level 1, which meets the clinical requirements.

[0090] It should be noted that the above examples are only intended to illustrate the technical solutions of the present invention and are not intended to limit the same. Although the present invention has been described in detail with reference to the examples given, those skilled in the art may modify or replace the technical solutions of the present invention as needed without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. Application of tissue repair hemostatic membranes in the preparation of medical materials; the medical materials have multiple functions of sealing, anti-adhesion, and hemostasis; The tissue repair hemostatic membrane is prepared by: S1. Obtain skin tissue from a non-human mammal and sample it down to the dermis after pretreatment; the removed skin tissue is divided into the epidermis and dermis; wherein, The pretreatment at least includes using laser to remove hair from the skin tissue; the sampling thickness is controlled to be 0.1mm-3mm; S2, subjecting the material obtained in step S1 to freeze-thaw treatment to remove cells to obtain a matrix material; S3, applying a protective agent to the matrix material and freeze-drying; S4, kneading or repeated folding after freeze drying; In step S3, the protective agent is any one or more of sodium alginate, cellulose and its derivatives, chitosan and its derivatives, hyaluronic acid and its derivatives, gum arabic, xanthan gum, and lithium magnesium silicate; The protective agent is applied to one side of the dermis layer. Specifically, the protective agent is added by the following method: Dissolve the protective agent into a gel and spread it on a flat plate, with the height lower than the skin tissue to be treated; place the epidermis side upwards and the dermis side in contact with the gel; Alternatively, lay the skin tissue flat on a flat plate with the dermis facing up and apply a gel-like protective agent; After adding the protective agent, ensure that it is left to stand for 10 minutes or more before freeze-drying; When the tissue repair hemostatic membrane is used, the oral soft tissue and the bone defect are separated, the epidermis is placed on the outside, and the dermis with the protective agent applied is placed in the bone defect area.

2. The use according to claim 1, characterized in that In step S1, the non-human mammal is a cow or a pig, and the selected animal is 9 months old or younger. The laser hair removal treatment is repeated 1-12 times; the time interval between each hair removal treatment is controlled to be at least one day or more.

3. The use according to claim 2, characterized in that In the step S1, sampling is performed at least one day after the last hair removal, and the sampling is performed using a drum-type skin remover or a roller knife.

4. The use according to claim 3, characterized in that In step S1, after sampling is completed, the sample is rinsed with running water to remove residual impurities, transferred to PBS buffer, ultrasonically shaken for 10-20 minutes, and then soaked and rinsed with 10-20 times the amount of water for 0.5-2 hours; the soaking operation is repeated 2-10 times.

5. The use according to claim 1, characterized in that In step S2, the freeze-thaw treatment is specifically performed as follows: the membrane material is spread on a flat plate and placed in a -40 to -10°C environment for freezing, taken out after 6 to 24 hours, and ultrasonically shaken in a 30 to 40°C water bath for 10 to 60 minutes; after ultrasonic shaking, the membrane material is taken out and soaked in PBS buffer or pure water for 10 to 60 minutes; and the freeze-thaw operation at -40 to -10°C is repeated 2 to 10 times.

6. The use according to claim 1, wherein The specific freeze-drying procedure includes: pre-freezing at -40 to -30°C for 1-4 hours; vacuuming after pre-freezing, and maintaining at -40 to -30°C for 1-3 hours; then heating to -30 to -25°C, heating time for 1-3 hours, and maintaining for 2-3 hours; then heating to -20°C, heating time for 2-3 hours, and maintaining for 2-3 hours; then heating to -15°C, heating time for 1-3 hours, and maintaining for 1-3 hours; then heating to -10°C, heating time for 1-3 hours, and maintaining for 1-3 hours; then heating to 0°C, heating time for 1-3 hours, and maintaining for 1-3 hours; then heating to 10°C, heating time for 1-3 hours, and maintaining for 1-3 hours; then heating to 20°C, heating time for 1-3 hours, and maintaining for 1-3 hours; then heating to 30°C, heating time for 2-3 hours, and maintaining for 2-3 hours.

7. The use according to claim 1, wherein The method further comprises the steps of cutting the softened hemostatic repair membrane, marking the front and back sides thereof, packaging and sterilizing the membrane.

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