Acellular matrix membrane, preparation method, and use

Abstract

The present application provides an acellular matrix membrane, a preparation method, and use. The acellular matrix membrane comprises at least one of collagen, laminin, glycosaminoglycan, and keratin. The collagen comprises type I collagen, and the type I collagen has a triple-helix structure. The light transmittance of the acellular matrix membrane is 80% to 99%. The present application uses a conventional opaque animal-derived membrane material to prepare a transparent acellular matrix membrane by means of a special transparency treatment process. This membrane exhibits improved light transmittance while effectively retaining the microstructure of collagen, which is conducive to cell crawling and proliferation. In addition to having a relatively good biological repair function, it also facilitates real-time visual observation of a wound regeneration process in clinical practice.

Description

A decellularized matrix membrane, its preparation method and uses

[0001] This application claims priority to Chinese Patent Application No. 202510300648.9, filed on March 14, 2025, entitled "A decellularized matrix membrane, preparation method and use", the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of biomedical materials technology, and in particular to a decellularized matrix membrane, its preparation method, and its uses. Background Technology

[0003] When conventional animal-derived membrane materials are implanted or used to cover wounds, they fuse with the surrounding new tissue during the regeneration process. Currently approved implantable, regenerative, and repair-functional decellularized matrix membrane products are mostly non-transparent, limiting the visualization of the submembrane wound regeneration process or the addition of substances. Physicians cannot visually evaluate the submembrane wound regeneration without tearing open the membrane material, and using testing equipment would increase the cost of evaluation and introduce a certain degree of error. Summary of the Invention

[0004] The purpose of this application is to provide a decellularized matrix membrane, its preparation method, and its uses, so as to improve the light transmittance of the decellularized matrix membrane.

[0005] The first aspect of this application provides a decellularized matrix membrane comprising at least one of collagen, laminin, glycosaminoglycans and keratin, wherein the collagen comprises type I collagen having a triple helix structure, and the translucency of the decellularized matrix membrane is 80% to 99%.

[0006] In one embodiment of this application, the thickness of the decellularized matrix membrane is 0.1 mm to 0.5 mm.

[0007] In one embodiment of this application, the tensile strength of the decellularized matrix membrane is from 5 MPa to 25 MPa.

[0008] The second aspect of this application provides a method for preparing the decellularized matrix membrane described in the first aspect of this application, comprising:

[0009] 1) Pretreatment: Collect mammalian soft tissue materials, process them to obtain matrix materials. The mammalian soft tissue materials are selected from bovine pericardium, porcine pericardium, sheep gastric mucosa, porcine peritoneum, porcine small intestinal mucosa or dermal tissue.

[0010] 2) Degreasing: The matrix material is mixed with a degreasing agent and then treated to obtain the degreased material;

[0011] 3) Disinfection: Mix the degreased material with disinfectant and shake to obtain the disinfected material;

[0012] 4) Decellularization: The sterilized material is mixed with alkaline protease solution and shaken to obtain the decellularized material;

[0013] 5) Removal of α-Gal antigen: The removal is carried out by at least one of the following methods: repeated freeze-thaw washing, mechanical crushing washing, or washing with α-galactosidase solution, to obtain the material after removal of α-Gal antigen.

[0014] 6) Gradient dehydration: The material after α-Gal antigen removal is placed in dehydrating agent solutions of 25% to 35% (v / v), 45% to 55% (v / v), 65% to 75% (v / v), and 85% to 95% (v / v) at a mass ratio of 1:(10 to 50). The solutions are shaken for 0.5 to 8 hours at a shaking speed of 50 to 500 rpm and a shaking temperature of 20°C to 40°C. After removal, the material is rinsed with pure water for 5 to 40 minutes to obtain the dehydrated material. The dehydrating agent is selected from at least one of glycerol, ethanol, butylene glycol, propanol, and mannitol.

[0015] Alternatively, clearing treatment: Mix the material after removing the α-Gal antigen with a 50%–80% (v / v) clearing reagent solution at a mass ratio of 1:(10–50), shake for 12–72 hours, change the solution periodically, drain the clearing reagent after the treatment, place it in a 0.1–1 wt% sodium chloride solution, and shake and wash 3–15 times, 10–100 minutes each time, to obtain cleared material. The clearing reagent is selected from at least one of polyethylene glycol, methanol, phenylethanol, CUBIC tissue clearing reagent, and FDISCO ex vivo tissue clearing reagent.

[0016] 7) Pressing: The dehydrated or transparent material is laid flat on the pressing plate and pressed together with a pressing force of 10N to 800N to obtain the pressed material;

[0017] The pressure plate is made of stainless steel mesh plate, stainless steel plate, acrylic plate, polypropylene plastic plate, polyethylene plastic plate, acrylonitrile-butadiene-styrene copolymer plastic plate, rigid polyvinyl chloride plastic plate or polycarbonate plastic plate.

[0018] The moisture content of the pressed material is ≤15%;

[0019] 8) The pressed material is freeze-dried and sterilized to obtain a decellularized matrix membrane.

[0020] In one embodiment of this application, step 2) includes: mixing the matrix material with an 80% to 100% (v / v) degreasing agent solution at a mass ratio of 1:(2 to 100), shaking in a shaker for 6 to 72 hours, changing the solution periodically, draining off the degreasing agent solution, adding 0.1 to 1 wt% sodium chloride solution for washing, repeating 3 to 5 times to obtain the degreased material. The degreasing agent is selected from at least one of acetone, chloroform, methanol, diethyl ether, isopropanol, and ethanol.

[0021] A third aspect of this application provides the use of the decellularized matrix membrane described in the first aspect of this application in the preparation of a regenerative tissue repair membrane.

[0022] In one embodiment of this application, the regenerative tissue repair membrane includes a skin repair membrane.

[0023] In one embodiment of this application, the regenerative tissue repair membrane includes at least one of the following: bone repair wrapping membrane, periosteum repair membrane, ligament repair membrane, ocular surface repair membrane, tympanic membrane repair membrane, oral cavity isolation membrane, soft tissue isolation membrane, soft tissue repair membrane, abdominal wall defect repair membrane, digestive tract repair membrane, laminectomy isolation membrane, dura mater repair membrane, spinal dura mater repair membrane, and acne scar filling membrane.

[0024] In one embodiment of this application, the soft tissue repair membrane is selected from at least one of tendon repair membranes, skin repair membranes, vascular repair membranes, and nerve repair membranes.

[0025] The beneficial effects of this application are:

[0026] This application provides a decellularized matrix membrane comprising at least one of collagen, laminin, glycosaminoglycans, and keratin. The collagen includes type I collagen, which has a triple helix structure. The translucency of the decellularized matrix membrane is 80% to 99%. This application uses conventional opaque animal-derived membrane materials and a special transparentizing process to prepare a transparent decellularized matrix membrane. The decellularized matrix membrane of this application has high translucency and effectively preserves the microstructure of collagen, which is conducive to cell migration and proliferation. It has good biological repair function and is also beneficial for real-time visualization of wound regeneration in clinical practice. Attached Figure Description

[0027] The accompanying drawings, which are provided to further illustrate this application and form part of this application, illustrate exemplary embodiments of this application and are used to explain this application, but do not constitute an undue limitation of this application.

[0028] Figure 1 is a photograph of the decellularized matrix membrane prepared in Example 1 of this application;

[0029] Figure 2 shows photographs of the decellularized matrix membrane prepared in Example 1 and the decellularized matrix membrane prepared in Comparative Example 1 placed on the skin defect site of a rabbit ((a) is a photograph of the skin defect site of a rabbit; (b) is a decellularized matrix membrane prepared in Example 1; (c) is a decellularized matrix membrane prepared in Comparative Example 1).

[0030] Figure 3 is a SEM image of the surface morphology of the decellularized matrix membrane prepared in Example 1 of this application;

[0031] Figure 4 is a SEM image of the triple helix structure of type I collagen in the decellularized matrix membrane prepared in Example 1 of this application;

[0032] Figure 5 is a bar chart showing the cell viability of the decellularized matrix membrane, blank group, positive control, and negative control prepared in Example 1 of this application after MTT cytotoxicity test.

[0033] Figure 6 is a light microscope image of the surface morphology of a decellularized matrix membrane prepared in Example 1 of this application after testing by the direct contact method at a location.

[0034] Figure 7 is a light microscope image of the surface morphology of another location of the decellularized matrix membrane prepared in Example 1 of this application after testing by the direct contact method. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are merely some embodiments of this application, and not all embodiments. All other embodiments obtained by those skilled in the art based on this application are within the scope of protection of this application.

[0036] The first aspect of this application provides a decellularized matrix membrane comprising at least one of collagen, laminin, glycosaminoglycans, and keratin. The collagen includes type I collagen, which has a triple helix structure. The transmittance of the decellularized matrix membrane is 80% to 99%. For example, the transmittance of the decellularized matrix membrane can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or a range of any two of these values. This application uses conventional opaque animal-derived membrane materials and a special transparentizing process to prepare a transparent decellularized matrix membrane. The decellularized matrix membrane of this application has high transmittance and effectively preserves the microstructure of collagen, which is beneficial for cell migration and proliferation. It possesses good biological repair function and is also conducive to real-time visualization of the wound regeneration process in clinical use.

[0037] In one embodiment of this application, the thickness of the decellularized matrix membrane is 0.1 mm to 0.5 mm. For example, the thickness of the decellularized matrix membrane can be 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, or a range of any two of these values. Controlling the thickness of the decellularized matrix membrane within the above range through dehydration methods and / or pressing processes is beneficial for improving the light transmittance of the decellularized matrix membrane, thus helping to meet the visualization needs of clinicians during and after surgery.

[0038] In one embodiment of this application, the tensile strength of the decellularized matrix membrane is from 5 MPa to 25 MPa. For example, the tensile strength of the decellularized matrix membrane can be 5 MPa, 6 MPa, 7 MPa, 8 MPa, 9 MPa, 10 MPa, 11 MPa, 12 MPa, 13 MPa, 14 MPa, 15 MPa, 16 MPa, 17 MPa, 18 MPa, 19 MPa, 21 MPa, 22 MPa, 23 MPa, 24 MPa, 25 MPa, or a range of any two of these values. The tensile strength of the decellularized matrix membrane falls within the scope of this application. While possessing good light transmittance, the decellularized matrix membrane also exhibits good mechanical properties, which is beneficial for withstanding physiological and mechanical stresses after implantation into the organism, maintaining the structural integrity of the tissue repair area, such as tendons. Simultaneously, the decellularized matrix membrane provides sufficient time for new tissue growth before biodegradation, facilitating the smooth regeneration process. Its stable physical structure provides a favorable microenvironment for cells, supporting cell adhesion, proliferation, and differentiation, accelerating tissue regeneration, and improving the repair effect after implantation. Decellularized matrix membranes with tensile strength within the scope of this application are beneficial for meeting the repair needs of various biological tissues.

[0039] The second aspect of this application provides a method for preparing the decellularized matrix membrane described in the first aspect of this application, comprising:

[0040] 1) Pretreatment: Collect mammalian soft tissue materials, process them to obtain matrix materials, wherein the mammalian soft tissue materials are selected from bovine pericardium, porcine pericardium, sheep gastric mucosa, porcine peritoneum, porcine small intestinal mucosa or dermal tissue;

[0041] 2) Degreasing: The matrix material is mixed with a degreasing agent and then treated to obtain the degreased material;

[0042] 3) Disinfection: Mix the degreased material with disinfectant and shake to obtain the disinfected material;

[0043] 4) Decellularization: The sterilized material is mixed with alkaline protease solution and shaken to obtain the decellularized material;

[0044] 5) Removal of α-Gal antigen: The removal is carried out by at least one of the following methods: repeated freeze-thaw washing, mechanical crushing washing, or washing with α-galactosidase solution, to obtain the material after removal of α-Gal antigen.

[0045] 6) Gradient dehydration: The material after α-Gal antigen removal is placed sequentially in dehydrating agent solutions of 25% to 35% (v / v), 45% to 55% (v / v), 65% to 75% (v / v), and 85% to 95% (v / v) at a mass ratio of 1:(10-50). The mixture is shaken for 0.5-8 hours at a shaking speed of 50-500 rpm and a shaking temperature of 20°C to 40°C. After removal, it is rinsed with pure water for 5-40 minutes to obtain the dehydrated material. The dehydrating agent is selected from at least one of glycerol, ethanol, butylene glycol, propanol, and mannitol. For example, the mass ratio of the material after α-Gal antigen removal to the dehydrating agent solution can be 1:10, 1:20, 1:10, 1:20, 1:20, 1:3 ... The oscillation ratios can be 1:30, 1:40, 1:50, or any two of these ratios; the oscillation time can be 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, or any two of these values; the oscillation speed can be 50rpm, 100rpm, 150rpm, 200rpm, 250rpm, 300rpm, 350rpm, 400rpm, 450rpm, 500rpm, or any two of these values; the oscillation temperature can be 20℃, 22℃, 25℃, 28℃, 30℃, 32℃, 35℃, 38℃, 40℃, or any two of these values.

[0046] Alternatively, a clearing treatment can be performed: The material after α-Gal antigen removal is mixed with a 50%–80% (v / v) clearing reagent solution at a mass ratio of 1:(10–50), ensuring the material is completely submerged. The mixture is shaken for 12–72 hours, with the solution changed every 6–8 hours. After the treatment, the clearing reagent solution is drained off, and the material is placed in a 0.1–1 wt% sodium chloride solution and shaken and washed 3–15 times, each time for 10–100 minutes, to obtain the cleared material. The clearing reagent is selected from polyethylene glycol and methanol. At least one of phenylethanol, CUBIC tissue clearing reagent, and FDISCO ex vivo tissue clearing reagent; for example, the volume percentage of the clearing reagent solution may be 50% (v / v), 60% (v / v), 70% (v / v), 80% (v / v), or a range of any two of these values; the mass ratio of the material after removal of α-Gal antigen to the clearing reagent solution may be 1:10, 1:20, 1:30, 1:40, 1:50, or a range of any two of these ratios.

[0047] 7) Pressing: The dehydrated or transparent material is laid flat on a pressing plate and pressed together. The pressing force is 10N to 800N, and the pressing time is 0.5h to 10h to obtain the pressed material. The pressing plate can be a stainless steel mesh plate, stainless steel plate, acrylic plate, polypropylene plastic plate, polyethylene plastic plate, acrylonitrile-butadiene-styrene copolymer plastic plate, rigid polyvinyl chloride plastic plate, or polycarbonate plastic plate. The moisture content of the pressed material is ≤15%. For example, the pressing force can be 10N, 50N, 100N, 200N, 300N, 400N, 500N, 600N, 700N, 800N, or any two of these values. The pressing time can be 0.5h, 1h, 2h, 4h, 6h, 8h, 10h, or any two of these values. The moisture content of the pressed material can be 1%, 5%, 10%, 15%, or any two of these values.

[0048] 8) The pressed material is freeze-dried and sterilized to obtain a decellularized matrix membrane.

[0049] In one embodiment of this application, the pretreatment step may be: washing away the residue on the surface of the animal soft tissue with purified water, placing the animal soft tissue in a 0.1-1 wt% sodium chloride solution, shaking for 15-20 hours, and removing the muscle layer and epidermis by physical scraping to obtain the matrix material.

[0050] The degreasing step can be as follows: mix the matrix material with an 80%–100% (v / v) degreasing agent solution at a mass ratio of 1:(2–100), shake in a shaker for 6–72 hours, change the solution every 6–8 hours, drain off the degreasing agent solution, add 0.1–1 wt% sodium chloride solution for washing, repeat 3–5 times to obtain the degreased material. The degreasing agent is selected from at least one of acetone, chloroform, methanol, diethyl ether, isopropanol, and ethanol. For example, the volume percentage of the degreasing agent in the degreasing agent solution can be 80% (v / v), 85% (v / v), 90% (v / v), 95% (v / v), 100% (v / v), or any range of two such values. The mass ratio of the matrix material to the degreasing agent solution can be 1:2, 1:10, 1:20, 1:40, 1:60, 1:80, 1:100, or any range of two such values. The degreasing method provided in this application, which can be repeated multiple times, helps remove lipid components in tissues that hinder light penetration, facilitating subsequent clearing treatment and making the treated tissue easier for the clearing agent solution to penetrate, thereby improving the transparency of the decellularized matrix membrane. This application does not have any particular limitations on the number of times or the duration of washing with 0.1–1 wt% sodium chloride solution, as long as the objective of this application is achieved. For example, the number of washes with 0.1–1 wt% sodium chloride solution can be 3–5 times, and the washing time for each wash can be 15–30 minutes.

[0051] Alternatively, the matrix material is mixed with an 80%–100% (v / v) degreasing agent solution at a mass ratio of 1:(2–100), shaken for 50–200 min, and then the degreasing agent solution is drained off. The mixture is then washed in a 0.1–1 wt% sodium chloride solution 3–15 times, each time for 10–100 min, to obtain the degreased material. The degreasing agent is selected from at least one of acetone, chloroform, methanol, diethyl ether, isopropanol, and ethanol. For example, the volume percentage of the degreasing agent solution can be 80% (v / v), 85% (v / v), 90% (v / v), 95% (v / v), 100% (v / v), or any range of two such values. The mass ratio of the matrix material to the degreasing agent solution can be 1:2, 1:10, 1:20, 1:40, 1:60, 1:80, 1:100, or any range of two such values.

[0052] The disinfection process can be as follows: Mix the degreased material with the disinfectant solution at a material-to-liquid mass ratio of 1:(2-100), shake for 60-240 minutes, drain the disinfectant solution after the process, and place the mixture in a 0.1-1 wt% sodium chloride solution and shake and wash 3-15 times, each time for 10-100 minutes, to obtain the disinfected material; wherein, the disinfectant solution is selected from at least one of 0.1-0.2% benzalkonium chloride solution, a mixed solution of 0.05 wt% peracetic acid and 40 wt% ethanol, or a 75-95 wt% ethanol solution, hydrogen peroxide, and peracetic acid;

[0053] The decellularization step can be as follows: Mix the sterilized material with 0.01-0.3 wt% alkaline protease solution at a material-to-liquid mass ratio of 1:(2-100), shake for 60-300 min, drain the alkaline protease solution, and place the mixture in 0.1-1 wt% sodium chloride solution for shaking and washing 3-15 times, 10-100 min each time, to obtain the decellularized material; the alkaline protease solution is selected from at least one of neutral protease solution, DNase solution, and trypsin solution;

[0054] The steps for removing α-Gal antigen can be: selected from at least one of repeated freeze-thaw washing, mechanical disruption washing, or washing with α-galactosidase solution to obtain material after α-Gal antigen removal; for example, the step of washing with α-galactosidase solution to remove α-Gal antigen is as follows: mix the decellularized material with 0.25-5 U / mL α-galactosidase solution at a material-to-solution mass ratio of 1:(2-100), shake at 25-37°C for 12-48 h, discard the α-galactosidase solution, add 0.05M-0.15M PBS solution, and shake and wash 3-15 times, 10-100 min each time, to obtain material after α-Gal antigen removal;

[0055] The freeze-drying sterilization process can be as follows: the pressed material is pre-frozen in a -20℃ freezer for 30 to 180 minutes, and then vacuum freeze-dried at a temperature of -60℃ to -30℃ and a vacuum degree of 0.05 mbar to 2 mbar to obtain the freeze-dried material; the freeze-dried material is then sterilized by irradiation sterilization or ethylene oxide sterilization to obtain a decellularized matrix membrane. For example, the pre-freezing time can be 30 min, 50 min, 80 min, 1000 min, 120 min, 150 min, 180 min, or any two of these values; the freeze-drying temperature can be -60℃, -50℃, -40℃, -30℃, or any two of these values; and the vacuum level can be 0.05 mbar, 0.1 mbar, 0.2 mbar, 0.5 mbar, 0.8 mbar, 1 mbar, 1.2 mbar, 1.5 mbar, 1.8 mbar, 2 mbar, or any two of these values.

[0056] In this application, oscillation is a conventional operation in the field, and there are no particular limitations on it, as long as it can achieve the purpose of this application. For example, oscillation can be performed using an oscillator.

[0057] In this application, sodium chloride solution, degreasing agent, disinfectant solution, alkaline protease solution, α-galactosidase solution, dehydrating agent, and clearing agent can be commercially available conventional substances, and there are no particular restrictions on them, as long as they can achieve the purpose of this application.

[0058] A third aspect of this application provides the use of the decellularized matrix membrane described in the first aspect of this application in the preparation of a regenerative tissue repair membrane.

[0059] In one embodiment of this application, the regenerative tissue repair membrane includes a skin repair membrane.

[0060] In one embodiment of this application, the regenerative tissue repair membrane includes at least one of the following: a bone repair wrapping membrane, a periosteal repair membrane, a ligament repair membrane, an ocular surface repair membrane, a tympanic membrane repair membrane, an oral cavity isolation membrane, a soft tissue isolation membrane, an abdominal wall defect repair membrane, a digestive tract repair membrane, a laminectomy isolation membrane, a dura mater repair membrane, a spinal dura mater repair membrane, and an acne scar filling membrane. This application prepares a transparent decellularized matrix membrane using a transparentization process, giving it not only good biocompatibility and mechanical properties but also excellent light transmittance. This facilitates real-time visualization of the wound regeneration process in clinical settings, thus enhancing its application in the preparation of regenerative tissue repair membranes. For ocular surface repair products, this helps address the current limitation on the source of donated human tissue materials.

[0061] In one embodiment of this application, the soft tissue repair membrane is selected from at least one of tendon repair membranes, skin repair membranes, vascular repair membranes, and nerve repair membranes.

[0062] Example

[0063] The embodiments and comparative examples provided below illustrate the implementation of this application in more detail. Various tests and evaluations were conducted according to the methods described below. Furthermore, unless otherwise specified, "parts" and "%" are quality standards.

[0064] Test methods and equipment:

[0065] Thickness test:

[0066] The cut decellularized matrix membrane was 3cm × 4cm. The thickness was measured using a precision thickness gauge (model: 32CH0F1030). The thickness was measured at five points: the four corners of the rectangular decellularized matrix membrane and the center of the rectangular decellularized matrix membrane. The average value of these measurements was recorded as the thickness of the decellularized matrix membrane.

[0067] Light transmittance test:

[0068] Refer to Method A, Section 7.1 of the national standard GB / T 2410-2008 "Determination of transmittance and haze of transparent plastics": Haze meter method to detect the transmittance of samples prepared in the examples and comparative examples.

[0069] Tensile strength test:

[0070] The test was conducted according to the national standard GB / T 1040.3-2006 "Test of tensile properties of plastics - Part 3: Test conditions for films and sheets".

[0071] Using the barbell-shaped specimen mold (5B) as described in the national standard GB / T 1040.2-2022 "Tests of tensile properties of plastics - Part 2: Test conditions for molded and extruded plastics", the sample was cut, the average thickness of the sample was measured, the tensile rate was 5 mm / min, a 20 kg sensor was used for testing, and the tensile strength was calculated according to the formula F / A, where F is the corresponding load force (N) and A is the cross-sectional area of ​​the sample.

[0072] SEM image:

[0073] Characterization was performed using a scanning electron microscope (SEM, model SU8010, Hitachi). The samples prepared in the examples or comparative examples were cut and then adhered to the sample stage with conductive adhesive. The samples were then sputter-coated with gold at magnifications of 20,000x and 50,000x, respectively. The surface morphology of the decellularized matrix membrane was observed under the electron microscope.

[0074] Biocompatibility testing:

[0075] (1) MTT cytotoxicity assay: The cytotoxicity assay assesses the potential cytotoxicity of the sample prepared in Example 1 to L-929 cells (mouse fibroblasts) using a quantitative method.

[0076] The test sample was cultured in MEM complete medium containing 10% fetal bovine serum (FBS) at a depth of 6 cm. 2 Extraction was performed at a ratio of / mL (double-sided area, GB / T 16886.12-2017) in an incubator at 37℃±1℃ for 72±2h. Negative control (high-density polyvinyl alcohol film, product model PVA-1788) and positive control (polyurethane film, 0.25% ZDBC, product model Aquatex WPU-400) were used. According to the recommendations in Table 1 of GB / T 16886.12, the negative and positive controls were prepared at a thickness of 3cm. 2 / mL and 6cm 2 Extraction was performed under the same conditions at a ratio of / mL. Specifically, 100%, 50%, and 25% extracts were obtained by adding MEM complete culture medium to the extract of the sample prepared in Example 1.

[0077] L-929 cells were seeded in 96-well plates at a density of 1×10⁶ cells / well. 4 L-929 cells were cultured at 37℃±1℃ for approximately 24 hours until a nearly confluent cell layer formed. The cells were then exposed to either the respective group's extract or MEM complete medium. The group exposed to MEM complete medium was designated as the control group. After incubating L-929 cells in either the extract or MEM complete medium for 24 hours, the extract or MEM complete medium was removed, and thiazolyl blue (MTT) solution was added to each well. The cell culture plates were incubated at 37℃±1℃ for 2 hours, after which the MTT solution was removed and 100 μL of isopropanol was added. The culture plates were placed on a microplate reader, and absorbance was read at 570 nm as the detection wavelength and 650 nm as the reference wavelength. Cytotoxicity was assessed by measuring cell viability, which was determined by the percentage of OD570 (mean OD570) of each group relative to the OD value of the control group at 570 nm. A higher mean OD570 indicated a higher cell viability in that group.

[0078] (2) Direct contact method test: L929 cells were injected at a concentration of 1×10⁻⁶. 4Cells were seeded at a density of [number] cells / mL in 96-well plates and incubated in a CO2 incubator at 37°C until they nearly converged. The culture medium in the plates was discarded, and fresh culture medium was added to each well. The decellularized matrix membrane prepared in Example 1 was carefully placed on the cell layer at the center of each well, after being trimmed to ensure that the membrane covered approximately one-tenth of the cell layer surface. After 24 hours, the wells were observed under an optical microscope (CKX53) to monitor cell adhesion to the decellularized matrix membrane, as shown in Figures 6 and 7. A higher cell count in the decellularized matrix membrane indicates better biocompatibility.

[0079] (3) In vivo animal characterization: Two SD rats (aged 8-10 weeks) were anesthetized and their skin was prepared. Two drills were prepared on the back of each rat. The circular wounds were divided into two groups. One group had the decellularized matrix membrane prepared in Example 1 placed on it as the decellularized matrix membrane group, while the other group had no material placed on it as the blank group.

[0080] The area S of the two wounds on the back of each rat was measured separately. Two weeks and four weeks after surgery, the healed area of ​​the two wounds on the back of both rats was measured, and the average value was recorded as S1. The postoperative healing rate was calculated as S1 / S × 100%. A higher postoperative healing rate indicates better repair effect, thus indicating better biocompatibility.

[0081] (4) Visual functional characterization: The decellularized matrix membranes prepared in Example 1 and Comparative Example 1 were placed on the skin defects of animals (rabbits) and photographs were taken, as shown in Figure 2.

[0082] DNA residual content analysis test:

[0083] DNA residue analysis: DNA residue was determined according to YY / T 1876-2023 "Determination of DNA Residue in Animal-Derived Biological Materials for Tissue Engineering Medical Products: Fluorescent Staining Method" using the DNeasy Blood & Tissue Kit (QIAGEN). The DNA residue level should not exceed 50 ng / mg.

[0084] Moisture content test of the material after pressing:

[0085] Three samples were cut from the pressed material, each weighing approximately 1g. The weight of each sample was recorded as m1. The samples were then dried in a desiccator at 105℃. The samples were removed every 1 hour, cooled to room temperature, and weighed and recorded. The mass of each sample was recorded until the mass change between two consecutive weighings was less than 0.3mg. The weight at this point was recorded as m2. The moisture content of the pressed material was calculated as (m1-m2) / m1. The average value of the three samples was taken as the final value.

[0086] Example 1

[0087] 1) Pretreatment: Rinse the dirt and residue of the obtained sheep gastric mucosa with purified water, place it in 0.9wt% sodium chloride solution, shake for 18 hours, take it out, scrape off the surface of the sheep stomach to obtain white or milky white sheep stomach tissue, drain off excess water until there is no obvious dripping water, and obtain matrix material for use.

[0088] 2) Degreasing: The obtained matrix material was mixed with 80% (v / v) isopropanol solution (solvent is purified water) at a mass ratio of 1:100, shaken for 180 min, and after the mixture was dehydrated, 0.9 wt% sodium chloride solution was added, and the mixture was washed 3 times for 15 min each time to obtain the degreased material.

[0089] 3) Disinfection: Mix the degreased material with a mixed solution of 0.05wt% peracetic acid and 40wt% ethanol (solvent is purified water) at a material-to-liquid mass ratio of 1:100, shake for 120 min, drain off the disinfectant, add 0.9wt% sodium chloride solution, shake and wash 3 times, 20 min each time, to obtain the disinfected material.

[0090] 4) Decellularization: Mix the sterilized material with 0.05wt% trypsin solution (solvent is PBS) at a material-to-solution mass ratio of 1:100, shake for 180 min, drain the trypsin solution, add 0.9wt% sodium chloride solution, shake and wash 3 times, 20 min each time, to obtain the decellularized material;

[0091] 5) Removal of α-Gal antigen: The decellularized material was mixed with 4.5 U / mL α-galactosidase solution (solvent is PBS) at a material-to-solvent mass ratio of 1:10. The mixture was shaken at 37°C for 30 h. The α-galactosidase solution was discarded. Then 0.1 M PBS solution was added and the mixture was shaken and washed 3 times for 15 min each time to obtain the material after removal of α-Gal antigen.

[0092] 6) Gradient dehydration: Step 1: After removing the α-Gal antigen, the material was placed in a 30% (v / v) glycerol solution (solvent is purified water) at a mass ratio of 1:20 for dehydration. The shaking speed was 300 rpm, the shaking temperature was 30℃, and the shaking time was 150 min. This concentration gradient dehydration was carried out for 2.5 h, followed by rinsing with purified water for 10 min.

[0093] Step 2: Dehydrate the product by shaking in a 50% (v / v) glycerol solution at a mass ratio of 1:20 for 4 hours at a shaking speed of 300 rpm and a shaking temperature of 30°C for 240 minutes. Rinse with purified water for 10 minutes.

[0094] Step 3: Dehydrate the product by shaking in a 70% (v / v) glycerol solution at a mass ratio of 1:20 for 4 hours at a shaking speed of 300 rpm and a shaking temperature of 30°C for 240 minutes. Rinse with purified water for 15 minutes.

[0095] Step 4: Dehydrate the material by shaking in a 90% (v / v) glycerol solution at a mass ratio of 1:20 for 2.5 hours. The shaking speed is 300 rpm, the shaking temperature is 30℃, and the shaking time is 150 min. Rinse with purified water for 20 min to obtain the dehydrated material.

[0096] 7) Pressing: The dehydrated material is laid flat on a stainless steel mesh plate and pressed together. The pressing force is 400N and the pressing time is 4h to obtain the pressed material.

[0097] 8) Freeze-drying: Spread the pressed material flat on a stainless steel plate, making sure there are no air bubbles. Place another stainless steel plate on top of the material and stack 8 layers (no more than 10 layers). Place it in a -20℃ freezer for 100 minutes and then transfer it to a freeze dryer. The freeze-drying temperature is -45℃, the vacuum degree is 1mbar, and the freeze-drying time is 12 hours to obtain the freeze-dried material.

[0098] 9) Sterilization: The packaged freeze-dried material was sterilized by irradiation at 25 kGy for 20 h to obtain a decellularized matrix membrane.

[0099] Examples 2 to 3

[0100] Except for adjusting the relevant parameters according to Table 1, the rest is the same as in Example 1.

[0101] Example 4

[0102] Except for steps 1) pretreatment, 3) disinfection, 4) decellularization, 5) removal of α-Gal antigen, 8) lyophilization, and 9) sterilization, which are the same as in Example 1, the other steps are performed as follows to obtain a decellularized matrix membrane:

[0103] 2) Degreasing: Mix the obtained matrix material with 100% (v / v) acetone solution at a mass ratio of 1:50, shake in a shaker for 12 hours, change the solution every 6 hours, drain the acetone solution after the end, add 0.9wt% sodium chloride solution, wash 3 times, 15 minutes each time, to obtain matrix material that has been degreased once.

[0104] Then, the matrix material that has been degreased once is mixed with 100% (v / v) acetone solution at a mass ratio of 1:50. The mixture is shaken for 24 hours, and the solution is changed every 6 hours. After the mixture is shaken, the acetone solution is drained off, and 0.9wt% sodium chloride solution is added. The mixture is washed 5 times for 30 minutes each time to obtain the matrix material that has been degreased twice.

[0105] Then, the twice-degreased matrix material was mixed with 100% (v / v) acetone solution at a mass ratio of 1:50. The mixture was shaken for 48 hours, with the solution changed every 12 hours. After the mixture was shaken, the acetone solution was drained off, and 0.9 wt% sodium chloride solution was added. The mixture was washed 5 times, 30 minutes each time.

[0106] 6) Clearing treatment: Mix the material after removing α-Gal antigen with 80% (v / v) polyethylene glycol solution at a mass ratio of 1:20, so that the material after removing α-Gal antigen is completely immersed in the clearing reagent solution, shake for 48 hours, change the solution every 6 hours, drain the clearing reagent solution after the treatment, place it in 0.9wt% sodium chloride solution, shake and wash 10 times, 30 minutes each time, to obtain the cleared material;

[0107] 7) Pressing: The transparent material is laid flat on a stainless steel mesh plate and pressed together. The pressing force is 400N and the pressing time is 4h to obtain the pressed material.

[0108] Comparative Example 1

[0109] Compared with Example 1, the gradient dehydration and pressing process was not performed, but the rest of the preparation method was the same as that of Example 1, and the decellularized matrix membrane prepared in Comparative Example 1 was obtained.

[0110] The preparation and performance parameters of each embodiment and comparative example are shown in Table 1.

[0111] Table 1 Note: In Table 1, " / " indicates that there are no relevant parameters.

[0112] Table 2

[0113] The transmittance of the decellularized matrix membrane affects the real-time visualization of its role in the clinical wound regeneration process. The transmittance is influenced by the compression force applied during the preparation process. As seen in Examples 1 to 4 and Comparative Example 1, when the compression force applied during the preparation process is within the range specified in this application, the transmittance of the decellularized matrix membrane is relatively high, indicating that the decellularized matrix membrane prepared in these examples provides good real-time visualization of the clinical wound regeneration process.

[0114] In this application, the residual DNA content in all embodiments and comparative examples is less than 10 ng / mg.

[0115] The thickness of the decellularized matrix membrane is affected by the pressing force during the pressing process in its preparation. As can be seen from Examples 1 to 4 and Comparative Example 1, when the pressing force during the preparation of the decellularized matrix membrane is within the range of this application, the thickness of the decellularized matrix membrane is within the range of this application, and the light transmittance of the decellularized matrix membrane is high, it indicates that the decellularized matrix membrane prepared in these examples has a good real-time visualization effect in the clinical wound regeneration process.

[0116] The tensile strength of the decellularized matrix membrane is affected by the pressing force during the preparation process. As can be seen from Examples 1 to 4 and Comparative Example 1, when the pressing force during the preparation process of the decellularized matrix membrane is within the range of this application, the tensile strength of the decellularized matrix membrane is within the range of this application, and the light transmittance of the decellularized matrix membrane is high. This indicates that the decellularized matrix membrane prepared in these examples, while taking into account the clinical real-time visualization observation effect, is beneficial for promoting the repair of biological tissues.

[0117] As shown in Table 2, the decellularized matrix membrane group prepared by Example 1 had a higher healing rate at 2 and 4 weeks postoperatively, compared with the blank group. This indicates that the decellularized matrix membrane prepared by Example 1 has good biocompatibility and can effectively promote the repair of skin defects.

[0118] As shown in Figure 1, the decellularized matrix membrane prepared in Example 1 is transparent, which is beneficial for improving light transmittance and thus facilitates real-time visual observation of the wound regeneration process in clinical practice.

[0119] As shown in Figure 2, (a) is a photograph of the skin defect site of a rabbit. Compared with the decellularized matrix membrane prepared in Comparative Example 1 in Figure (c), the decellularized matrix membrane prepared in Example 1 in Figure (b) still clearly shows the biological tissue under the membrane after it is placed on the skin defect site of a rabbit. This indicates that the decellularized matrix membrane prepared in Example 1 has a better real-time visualization effect in the clinical wound regeneration process.

[0120] As shown in Figures 3 and 4, the decellularized matrix membrane prepared in Example 1 has a densely arranged fibrous structure and a clearly visible triple helix structure of type I collagen, which is beneficial for cell crawling and proliferation.

[0121] As shown in Figure 5, the 100%, 50%, and 25% extracts of the decellularized matrix membrane prepared in Example 1 showed good cell proliferation-promoting effects, indicating that the decellularized matrix membrane prepared in Example 1 has good biocompatibility.

[0122] As shown in Figures 6 and 7, after 24 hours of direct contact testing, the decellularized matrix membrane prepared in Example 1 had a large number of cells, indicating that the cells tend to adhere to the decellularized matrix membrane, thus demonstrating that the decellularized matrix membrane prepared in Example 1 has good biocompatibility.

[0123] It should be noted that, in this document, the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0124] The various embodiments in this specification are described in a related manner. The same or similar parts between the various embodiments can be referred to each other. Each embodiment focuses on describing the differences from other embodiments.

[0125] The above description is only a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application.

Claims

1. A decellularized matrix membrane comprising at least one of collagen, laminin, glycosaminoglycans, and keratin; The collagen includes type I collagen, which has a triple helix structure, and the translucency of the decellularized matrix membrane is 80% to 99%.

2. The decellularized matrix membrane according to claim 1, wherein, The thickness of the decellularized matrix membrane is 0.1 mm to 0.5 mm.

3. The decellularized matrix membrane according to claim 1, wherein, The tensile strength of the decellularized matrix membrane is 5 MPa to 25 MPa.

4. A method for preparing a decellularized matrix membrane according to any one of claims 1 to 3, comprising: 1) Pretreatment: Collect mammalian soft tissue materials, process them to obtain matrix materials, wherein the mammalian soft tissue materials are selected from bovine pericardium, porcine pericardium, sheep gastric mucosa, porcine peritoneum, porcine small intestinal mucosa or dermal tissue; 2) Degreasing: The matrix material is mixed with a degreasing agent and treated to obtain a degreased material; 3) Disinfection: The degreased material is mixed with a disinfectant and subjected to shaking treatment to obtain the disinfected material; 4) Decellularization: The sterilized material is mixed with an alkaline protease solution and subjected to shaking treatment to obtain the decellularized material; 5) Removal of α-Gal antigen: The removal is carried out by at least one of the following methods: repeated freeze-thaw washing, mechanical crushing washing, or washing with α-galactosidase solution, to obtain the material after removal of α-Gal antigen. 6) Gradient dehydration: The material after α-Gal antigen removal is placed in dehydrating agent solutions of 25% to 35% (v / v), 45% to 55% (v / v), 65% to 75% (v / v), and 85% to 95% (v / v) at a mass ratio of 1:(10-50). The solutions are shaken for 0.5 to 8 hours at a shaking speed of 50-500 rpm and a shaking temperature of 20°C to 40°C. After removal, the material is rinsed with pure water for 5 to 40 minutes to obtain the dehydrated material. The dehydrating agent is selected from at least one of glycerol, ethanol, butylene glycol, propanol, and mannitol. Alternatively, a clearing treatment can be performed: the material after α-Gal antigen removal is mixed with a 50%–80% (v / v) clearing reagent solution at a mass ratio of 1:(10–50), shaken for 12–72 hours, and the solution is changed periodically. After the treatment, the clearing reagent solution is drained off, and the mixture is placed in a 0.1–1 wt% sodium chloride solution and shaken and washed 3–15 times, each time for 10–100 minutes, to obtain a cleared material. The clearing reagent is selected from at least one of polyethylene glycol, methanol, phenylethanol, CUBIC tissue clearing reagent, and FDISCO ex vivo tissue clearing reagent. 7) Pressing: The dehydrated material or the transparent material is laid flat on the pressing plate and pressed together with a pressing force of 10N to 800N to obtain the pressed material; The pressure plate is a stainless steel mesh plate, stainless steel plate, acrylic plate, polypropylene plastic plate, polyethylene plastic plate, acrylonitrile-butadiene-styrene copolymer plastic plate, rigid polyvinyl chloride plastic plate, or polycarbonate plastic plate. The moisture content of the pressed material is ≤15%; 8) The compressed material is freeze-dried and sterilized to obtain the decellularized matrix membrane.

5. The method for preparing the decellularized matrix membrane according to claim 4, wherein, Step 2) includes: mixing the matrix material with an 80% to 100% (v / v) degreasing agent solution at a mass ratio of 1:(2 to 100), shaking in a shaker for 6 to 72 hours, changing the solution periodically, draining off the degreasing agent solution, adding 0.1 to 1 wt% sodium chloride solution for washing, repeating 3 to 5 times to obtain the degreased material. The degreasing agent is selected from at least one of acetone, chloroform, methanol, diethyl ether, isopropanol, and ethanol.

6. Use of a decellularized matrix membrane according to any one of claims 1 to 3 in the preparation of a regenerative tissue repair membrane.

7. The use of the decellularized matrix membrane according to claim 6 in the preparation of regenerative tissue repair membranes, wherein, The regenerative tissue repair membrane includes a skin repair membrane.

8. The use of the decellularized matrix membrane according to claim 6 in the preparation of regenerative tissue repair membranes, wherein, The regenerative tissue repair membrane includes at least one of the following: bone repair wrapping membrane, periosteum repair membrane, ligament repair membrane, ocular surface repair membrane, tympanic membrane repair membrane, oral cavity isolation membrane, soft tissue isolation membrane, soft tissue repair membrane, abdominal wall defect repair membrane, digestive tract repair membrane, laminectomy isolation membrane, dura mater repair membrane, spinal dura mater repair membrane, and acne scar filling membrane.

9. The use of the decellularized matrix membrane according to claim 8 in the preparation of regenerative tissue repair membranes, wherein, The soft tissue repair membrane is selected from at least one of tendon repair membranes, skin repair membranes, blood vessel repair membranes, and nerve repair membranes.

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