Acellular matrix membrane as well as preparation method and application thereof

Abstract

The invention provides an acellular matrix membrane, a preparation method and application, the acellular matrix membrane comprises at least one of collagen, laminin, glycosaminoglycan and keratin, the collagen comprises type I collagen, the type I collagen is of a triple helix structure, and the light transmittance of the acellular matrix membrane is 80%-99%. The transparent acellular matrix membrane is prepared from a conventional opaque animal-derived membrane material through a special transparent treatment process, the light transmittance is improved, the microstructure of collagen is effectively reserved, cell crawling and proliferation are facilitated, and the acellular matrix membrane has a good bioremediation function and also has a good application prospect. And the real-time visual observation of the wound regeneration process in clinic is also facilitated.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biomedical materials, in particular to a decellularized matrix membrane, a preparation method and an application thereof. BACKGROUND

[0002] After the conventional animal-derived membrane material is implanted or covered on a wound, the membrane material will be fused with the surrounding newly generated tissue along with the regeneration process. The currently approved membrane products with regeneration and repair functions are mostly non-transparent products, which limits the visual observation of the regeneration process of the wound under the membrane or the added substances. Doctors cannot evaluate the regeneration of the wound under the membrane by naked eye without tearing the membrane material, and the use of detection equipment will increase the evaluation cost and there will be certain errors. SUMMARY

[0003] The present application aims to provide a decellularized matrix membrane, a preparation method and an application thereof to improve the light transmittance of the decellularized matrix membrane.

[0004] The first aspect of the present application provides a decellularized matrix membrane, which comprises at least one of collagen, laminin, glycosaminoglycan and keratin, the collagen comprises type I collagen, the type I collagen is a triple helix structure, and the light transmittance of the decellularized matrix membrane is 80% to 99%.

[0005] In an embodiment of the present application, the thickness of the decellularized matrix membrane is 0.1mm to 0.5mm.

[0006] In an embodiment of the present application, the tensile strength of the decellularized matrix membrane is 5MPa to 25MPa.

[0007] The second aspect of the present application provides a preparation method of the decellularized matrix membrane according to the first aspect of the present application, which comprises:

[0008] 1) Pretreatment: collecting mammalian soft tissue material, and obtaining a matrix material after treatment, the mammalian soft tissue material is selected from bovine pericardium, porcine pericardium, sheep gastric mucosa, porcine peritoneum, porcine small intestinal mucosa or dermal tissue;

[0009] 2) Defatting: mixing the matrix material with a defatting agent, and obtaining a defatted material after treatment;

[0010] 3) Disinfection: mixing the defatted material with a disinfectant, and obtaining a disinfected material after oscillation treatment;

[0011] 4) Decellularization: mixing the disinfected material with an alkaline protease solution, and obtaining a decellularized material after oscillation treatment;

[0012] 5) removing α-Gal antigen: at least one of repeated freeze-thaw washing, mechanical disruption washing, or washing with α-galactosidase solution is selected to obtain the material after removing α-Gal antigen;

[0013] 6) gradient dehydration: the material after removing α-Gal antigen is sequentially placed in 25% to 35% (v / v), 45% to 55% (v / v), 65% to 75% (v / v), and 85% to 95% (v / v) dehydrating agent solution at a mass ratio of 1:(10-50), oscillated for 0.5-8 h, with an oscillation speed of 50-500 rpm and an oscillation temperature of 20-40°C, and then washed with pure water for 5-40 min to obtain the material after dehydration; the dehydrating agent is at least one of glycerol, ethanol, butanediol, propanol, and mannitol;

[0014] Alternatively, transparent treatment: the material after removing α-Gal antigen is mixed with 50%-80% (v / v) transparent reagent solution at a mass ratio of 1:(10-50), oscillated for 12-72 h, with regular liquid change, and then drained of the transparent reagent and placed in 0.1-1 wt% sodium chloride solution, oscillated and washed for 3-15 times, each time for 10-100 min, to obtain the transparent material; the transparent reagent is at least one of polyethylene glycol, methanol, phenethyl alcohol, CUBIC tissue transparent reagent, and FDISCO ex vivo tissue transparent reagent;

[0015] 7) pressing: the material after dehydration or the transparent material is laid on a pressing plate for pressing, with a pressing force of 10-800 N, to obtain the material after pressing;

[0016] The pressing plate is a stainless steel mesh plate, a stainless steel plate, an acrylic plate, a polypropylene plastic plate, a polyethylene plastic plate, an acrylonitrile / butadiene / styrene copolymer plastic plate, a polyvinyl chloride hard plastic plate, or a polycarbonate plastic plate;

[0017] The water content of the material after pressing is ≤15%;

[0018] 8) the material after pressing is subjected to freeze-drying and sterilization to obtain the decellularized matrix membrane.

[0019] In an embodiment of the present application, step 2) comprises: mixing the matrix material with 80%-100% (v / v) defatting agent solution at a mass ratio of 1:(2-100), oscillating for 6-72 h with a regular liquid change, and then draining the defatting agent and adding 0.1-1 wt% sodium chloride solution for washing, repeated for 3-5 times, to obtain the material after defatting; the defatting agent is at least one of acetone, chloroform, methanol, diethyl ether, isopropyl alcohol, and ethanol.

[0020] The third aspect of the present application provides a use of the decellularized matrix membrane of the first aspect of the present application in the preparation of a regenerative tissue repair membrane.

[0021] In an embodiment of the present application, the regenerative tissue repair membrane comprises a skin repair membrane.

[0022] In an embodiment of the present application, the regenerative tissue repair membrane comprises at least one of a bone repair post-wrapping membrane, a bone membrane 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, a soft tissue repair membrane, an abdominal wall defect repair membrane, a digestive tract repair membrane, a laminectomy isolation membrane, a dura mater repair membrane, a spinal cord repair membrane, and a pock filling membrane.

[0023] In an embodiment of the present application, the soft tissue repair membrane is selected from at least one of a tendon repair membrane, a skin repair membrane, a blood vessel repair membrane, and a nerve repair membrane.

[0024] Advantages of the present application:

[0025] The present application provides a decellularized matrix membrane comprising at least one of collagen, laminin, glycosaminoglycan, and keratin, the collagen comprising type I collagen, the type I collagen being a triple helix structure, and the decellularized matrix membrane having a light transmittance of 80% to 99%. The present application uses a conventional opaque animal-derived membrane material to prepare a decellularized matrix membrane with a transparent property through a special transparent treatment process, improves the light transmittance, effectively retains the microstructure of collagen, is conducive to cell crawling and proliferation, has good biological repair function, and is also conducive to real-time visual observation of the wound regeneration process in the clinic.

[0026] Of course, implementing any product or method of the present application does not necessarily require achieving all the advantages described above at the same time. BRIEF DESCRIPTION OF DRAWINGS

[0027] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the embodiment description will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other embodiments can also be obtained by those skilled in the art based on these drawings.

[0028] Figure 1 Photos of the decellularized matrix membrane prepared for Example 1 of the present application;

[0029] Figure 2 Photos of the decellularized matrix membrane prepared for Example 1 of the present application and the decellularized matrix membrane prepared for Comparative Example 1 placed at the skin defect site of a rabbit ((a) is a photo of the skin defect site of a rabbit; (b) is the decellularized matrix membrane prepared for Example 1, and (c) is the decellularized matrix membrane prepared for Comparative Example 1);

[0030] Figure 3SEM image of surface morphology of the decellularized matrix membrane prepared for Example 1 of the present application;

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

[0032] Figure 5 Column chart of cell survival rate after MTT cytotoxicity test of the decellularized matrix membrane prepared for Example 1 of the present application, blank group and positive control, negative control;

[0033] Figure 6 Light microscope image of surface morphology of the decellularized matrix membrane prepared for Example 1 of the present application after testing by direct contact method;

[0034] Figure 7 Light microscope image of surface morphology of the decellularized matrix membrane prepared for Example 1 of the present application after testing by direct contact method. DETAILED DESCRIPTION

[0035] The technical solutions in the present application will be described in detail below with reference to the embodiments of the present application and the accompanying drawings. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. All other embodiments obtained by those skilled in the art based on the present application are within the scope of protection of the present application.

[0036] The first aspect of the present application provides a decellularized matrix membrane, which comprises at least one of collagen, laminin, glycosaminoglycan and keratin, the collagen comprises collagen type I, the collagen type I is in a triple helix structure, and the light transmittance of the decellularized matrix membrane is 80% to 99%. For example, the light 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 formed by any two of them. The present application uses a conventional opaque animal-derived membrane material to prepare a decellularized matrix membrane with transparent properties through a special transparent treatment process, which improves the light transmittance and effectively preserves the microstructure of collagen, is conducive to cell crawling and proliferation, has good biological repair function, and is also conducive to real-time visual observation of the wound regeneration process in clinical use.

[0037] In an embodiment of the present 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 defined by any two of the above values. By controlling the thickness of the decellularized matrix membrane within the above range through the dehydration method and the pressing process, the light transmittance of the decellularized matrix membrane is improved, which helps to meet the needs of intraoperative and postoperative visualization of the clinician.

[0038] In an embodiment of the present application, the tensile strength of the decellularized matrix membrane is 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 defined by any two of the above values. The tensile strength of the decellularized matrix membrane within the range of the present application, the decellularized matrix membrane has better light transmittance and mechanical properties, which is conducive to the decellularized matrix membrane to withstand physiological pressure and mechanical pressure after being implanted into the body, maintain the structural integrity of the tissue repair area, such as the tendon part; at the same time, the decellularized matrix membrane can provide a long enough time for the growth of new tissues before biodegradation, which is conducive to the smooth progress of the regeneration process; the stable physical structure provides a good microenvironment for cells, supports cell adhesion, proliferation and differentiation, accelerates the tissue regeneration process, and improves the repair effect after being implanted into the body. The decellularized matrix membrane with the tensile strength within the range of the present application is conducive to meeting the repair needs of various biological tissues.

[0039] The second aspect of the present application provides a preparation method of the decellularized matrix membrane of the first aspect of the present application, which comprises:

[0040] 1) Pretreatment: collecting mammalian soft tissue material, and obtaining a matrix material after treatment, wherein the mammalian soft tissue material is selected from bovine pericardium, porcine pericardium, sheep gastric mucosa, porcine peritoneum, porcine small intestinal mucosa, or dermal tissue;

[0041] 2) Defatting: mixing the matrix material with a defatting agent, and obtaining a defatted material after treatment;

[0042] 3) Disinfection: mixing the defatted material with a disinfectant, and obtaining a disinfected material after oscillation treatment;

[0043] 4) Decellularization: mixing the disinfected material with an alkaline protease solution, and obtaining a decellularized material after oscillation treatment;

[0044] 5) removing α-Gal antigen: at least one of repeated freeze-thaw washing, mechanical disruption washing or washing with α-galactosidase solution is selected to obtain the material after removing α-Gal antigen;

[0045] 6) gradient dehydration: the material after removing α-Gal antigen is sequentially placed in 25% to 35% (v / v), 45% to 55% (v / v), 65% to 75% (v / v) and 85% to 95% (v / v) dehydrating agent solution according to the mass ratio of 1: (10-50), oscillated for 0.5-8h, the oscillation speed is 50-500rpm, the oscillation temperature is 20℃ to 40℃, after taking out, washed with pure water for 5min-40min to obtain the material after dehydration; the dehydrating agent is at least one of glycerol, ethanol, butanediol, propanol, mannitol; for example, the mass ratio of the material after removing α-Gal antigen and the dehydrating agent solution can be 1:10, 1:20, 1:30, 1:40, 1:50 or a range composed of any two of the above values; the oscillation time can be 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h or a range composed of any two of the above values; the oscillation speed can be 50rpm, 100rpm, 150rpm, 200rpm, 250rpm, 300rpm, 350rpm, 400rpm, 450rpm, 500rpm or a range composed of any two of the above values; the oscillation temperature can be 20℃, 22℃, 25℃, 28℃, 30℃, 32℃, 35℃, 38℃, 40℃ or a range composed of any two of the above values;

[0046] Alternatively, transparent treatment: the material after removing α-Gal antigen is mixed with 50%-80% (v / v) transparent reagent solution according to the mass ratio of 1: (10-50), so that the material after removing α-Gal antigen is completely immersed in the transparent reagent solution, oscillated for 12h-72h, the solution is replaced every 6-8h, after the end, the transparent reagent is drained, placed in 0.1-1wt% sodium chloride solution, oscillated for 3-15 times, each time for 10-100min, to obtain the transparent material, the transparent reagent is at least one of polyethylene glycol, methanol, phenethyl alcohol, CUBIC tissue transparent reagent, FDISCO ex vivo tissue transparent reagent; for example, the volume percentage of the transparent reagent solution can be 50% (v / v), 60% (v / v), 70% (v / v), 80% (v / v) or a range composed of any two of the above values; the mass ratio of the material after removing α-Gal antigen and the transparent reagent solution can be 1:10, 1:20, 1:30, 1:40, 1:50 or a range composed of any two of the above values.

[0047] 7) pressing: the dehydrated material or the transparentized material is laid on a pressing plate for pressing, the pressing force is 10N to 800N, the pressing time is 0.5h to 10h, and the pressed material is obtained; the pressing plate is a stainless steel mesh plate, a stainless steel plate, an acrylic plate, a polypropylene plastic plate, a polyethylene plastic plate, an acrylonitrile / butadiene / styrene copolymer plastic plate, a polyvinyl chloride hard plastic plate or a polycarbonate plastic plate; the water 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 a range formed by any two of them; the pressing time can be 0.5h, 1h, 2h, 4h, 6h, 8h, 10h or a range formed by any two of them; the water content of the pressed material can be 1%, 5%, 10%, 15% or a range formed by any two of them.

[0048] 8) freeze-drying and sterilizing the pressed material to obtain a decellularized matrix membrane.

[0049] In an embodiment of the present application, the pretreatment step can be: washing off the residues on the surface layer of the animal soft tissue with purified water, placing the animal soft tissue in a 0.1-1wt% sodium chloride solution, oscillating for 15-20h, and removing the muscle layer and epidermis layer by physical scraping to obtain a matrix material.

[0050] The defatting step can be: mixing the matrix material with a 80%-100%(v / v) defatting agent solution at a mass ratio of 1:(2-100), oscillating for 6h-72h, replacing the solution every 6-8h, adding a 0.1-1wt% sodium chloride solution after draining off the defatting agent, and repeating the process 3-5 times to obtain a defatted material; the defatting agent is selected from at least one of acetone, chloroform, methanol, diethyl ether, isopropyl alcohol and ethanol; for example, the volume mass percentage of the defatting agent in the defatting agent solution can be 80%(v / v), 85%(v / v), 90%(v / v), 95%(v / v), 100%(v / v) or a range formed by any two of them; the mass ratio of the matrix material to the defatting agent solution can be 1:2, 1:10, 1:20, 1:40, 1:60, 1:80, 1:100 or a range formed by any two of them. The defatting method provided by the present application, and repeated multiple times, helps to remove the lipid components in the tissue that hinder the penetration of light, facilitates the subsequent transparentization process, and makes the treated tissue more easily penetrated by the transparentization reagent solution, thereby improving the transparency of the decellularized matrix membrane.

[0051] or, the matrix material is mixed with a 80%~100%(v / v) solution of a defatting agent at a mass ratio of 1:(2~100), oscillated for 50~200 min, and then the defatting agent is drained off, and the defatted material is obtained by washing in a 0.1~1 wt% sodium chloride solution for 3~15 times, each time for 10~100 min; wherein the defatting agent is at least one selected from acetone, chloroform, methanol, diethyl ether, isopropyl alcohol, and ethanol; for example, the volume mass percentage of the defatting agent solution can be 80%(v / v), 85%(v / v), 90%(v / v), 95%(v / v), 100%(v / v), or a range defined by any two of the above values; and the mass ratio of the matrix material to the defatting agent solution can be 1:2, 1:10, 1:20, 1:40, 1:60, 1:80, 1:100, or a range defined by any two of the above values.

[0052] The sterilization step can be: the defatted material is mixed with a sterilization agent solution at a solid-liquid mass ratio of 1:(2~100), oscillated for 60~240 min, and then the sterilization agent is drained off, and the sterilized material is obtained by oscillating washing in a 0.1~1 wt% sodium chloride solution for 3~15 times, each time for 10~100 min; wherein the sterilization agent solution is at least one selected from 0.1~0.2% new Jieer solution, a mixed solution of 0.05 wt% peroxyacetic acid and 40 wt% ethanol, or 75~95 wt% ethanol solution, hydrogen peroxide, and peroxyacetic acid;

[0053] The decellularization step can be: the defatted material is mixed with a 0.01~0.3 wt% alkaline protease solution at a solid-liquid mass ratio of 1:(2~100), oscillated for 60~300 min, and then the alkaline protease solution is drained off, and the decellularized material is obtained by oscillating washing in a 0.1~1 wt% sodium chloride solution for 3~15 times, each time for 10~100 min; wherein the alkaline protease solution is at least one selected from neutral protease solution, DNAase solution, and trypsin solution;

[0054] The step of removing α-Gal antigen can be at least one selected from repeated freeze-thaw washing, mechanical disruption washing, or washing with an α-galactosidase solution, to obtain the material after removing α-Gal antigen; for example, the step of removing α-Gal antigen by washing with an α-galactosidase solution is: the decellularized material is mixed with a 0.25~5 U / mL α-galactosidase solution at a solid-liquid mass ratio of 1:(2~100), oscillated at 25~37°C for 12~48 h, the α-galactosidase solution is discarded, and then a 0.05 M~0.15 M PBS solution is added, and the resulting mixture is oscillated and washed for 3~15 times, each time for 10~100 min, to obtain the material after removing α-Gal;

[0055] The freeze-drying sterilization step can be: placing the pressed material into a-20℃ refrigerator for pre-freezing for 30min to 180min, and then performing vacuum freeze-drying, the freeze-drying temperature is-60℃ to-30℃, the vacuum degree is 0.05mbar to 2mbar, to obtain the freeze-dried material; the freeze-dried material is sterilized by irradiation sterilization or ethylene oxide sterilization to obtain the decellularized matrix membrane. For example, the pre-freezing time can be 30min, 50min, 80min, 1000min, 120min, 150min, 180min, or a range formed by any two of the above values; the freeze-drying temperature can be-60℃, -50℃, -40℃, -30℃, or a range formed by any two of the above values; the vacuum degree can be 0.05mbar, 0.1mbar, 0.2mbar, 0.5mbar, 0.8mbar, 1mbar, 1.2mbar, 1.5mbar, 1.8mbar, 2mbar, or a range formed by any two of the above values.

[0056] In the present application, oscillation is a conventional operation in the art, and the present application does not have a particular limitation, as long as the purpose of the present application can be achieved. For example, oscillation can be performed by an oscillator.

[0057] In the present application, the sodium chloride solution, the defatting agent solution, the disinfectant solution, the alkaline protease solution, the alpha-galactosidase solution, the dehydrating agent solution, and the transparentizing reagent can be conventional commercial materials, and are not particularly limited, as long as the purpose of the present application can be achieved.

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

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

[0060] In an embodiment of the present application, the regenerative tissue repair membrane includes at least one of a bone repair post-wrapping membrane, a bone membrane 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 cord repair membrane, and a pock filling membrane. The present application prepares a decellularized matrix membrane with a transparent property through a transparent processing process, so that it not only has good biocompatibility and mechanical properties, but also has excellent light transmittance, which is conducive to real-time visual observation of the wound regeneration process in clinic, thereby facilitating its use in the preparation of a regenerative tissue repair membrane, and for the ocular surface repair product, it is conducive to solving the current situation of limited source of human donated tissue materials.

[0061] In one embodiment of the present application, the soft tissue repair membrane is selected from at least one of a tendon repair membrane, a skin repair membrane, a blood vessel repair membrane, and a nerve repair membrane.

[0062] Examples

[0063] Hereinafter, examples and comparative examples are cited to more specifically explain the embodiments of the present application. Various tests and evaluations were performed according to the following methods. In addition, unless otherwise specified, "parts" and "%" are on a mass basis.

[0064] Test method and equipment:

[0065] Thickness test:

[0066] The thickness of the decellularized matrix membrane after cutting was 3 cm x 4 cm; the thickness was measured using a precision thickness gauge (model: 32CH0F1030), and the average value of the thickness of the decellularized matrix membrane was recorded.

[0067] Transmittance test:

[0068] The transmittance of the samples prepared in the examples and comparative examples was detected according to the method A: haze meter method in the national standard GB / T 2410-2008 "Determination of transmittance and haze of transparent plastics".

[0069] Tensile strength test:

[0070] The test was performed according to the determination method in the national standard GB / T 1040.3-2006 "Test methods for tensile properties of plastics, part 3: test conditions for films and sheets".

[0071] The cutting sample was prepared using the dumbbell-shaped sample mold (5B) described in the national standard GB / T 1040.2-2022 "Test methods for tensile properties of plastics, part 2: test conditions for molded and extruded plastics", the average thickness of the sample was measured, the tensile rate was 5 mm / min, a 20 kg sensor was used for detection, and the tensile strength was calculated according to the formula: F / A, F is the corresponding load force (N), and A is the cross-sectional area of the sample.

[0072] SEM imaging:

[0073] The samples prepared in the examples or comparative examples were characterized by scanning electron microscopy (SEM, model: Hitachi su8010), the samples were cut to enable them to be adhered to the sample stage by conductive glue, then surface gold plating was performed, the magnification was 20000x and 50000x, respectively, and the surface morphology of the decellularized matrix membrane was observed under the electron microscope.

[0074] Biocompatibility test

[0075] (1) MTT cytotoxicity test: The cytotoxicity test evaluates the potential cytotoxicity of the sample prepared in Example 1 on L-929 cells (mouse fibroblasts) by a quantitative method.

[0076] The test sample was immersed in a 6 cm 2 / mL (double-sided area, GB / T 16886.12-2017) ratio in a 37℃±1℃ incubator for 72±2h, with a negative control (high-density polyvinyl alcohol film, product model PVA-1788) and a positive control (polyurethane film, 0.25% ZDBC, product model Aquatex WPU-400). According to the recommendations in Table 1 of GB / T 16886.12, the negative control and positive control were immersed in the same conditions at a ratio of 3 cm 2 / mL and 6 cm 2 / mL, respectively, according to the thickness. Among them, 100% immersion liquid, 50% immersion liquid, and 25% immersion liquid were obtained by adding MEM complete medium to the immersion liquid of the sample prepared in Example 1.

[0077] L-929 cells were seeded in a 96-well plate at a density of 1×10 4 cells / well and incubated in a 37℃±1℃ incubator for about 24h, until a nearly confluent cell layer was formed, and then exposed to each group of immersion liquid or MEM complete medium. The group exposed to MEM complete medium is recorded as the blank group. After incubating L-929 cells with each group of immersion liquid or MEM complete medium for 24h, the immersion liquid or MEM complete medium of each group was removed and MTT solution was added to each well. After incubating the cell culture plate in a 37℃±1℃ incubator for 2h, the MTT solution was removed and 100μL of isopropanol was added. The plate was placed in a microplate reader, and the absorbance value was read at a detection wavelength of 570nm and a reference wavelength of 650nm. Cell survival rate was detected by measuring cytotoxicity, and cell survival rate was determined by the percentage of OD570 (OD570 average) of each group relative to the OD value at 570nm of the blank group. The higher the OD570 average, the higher the cell survival rate of the group.

[0078] (2) Direct contact method test: L929 cells were seeded in a 96-well plate at a density of 1×10 4The cells were seeded in 96-well plates at a density of 1 x 104 / mL and cultured in a CO2incubator at 37°C until the cells grew to near confluence. The culture medium in the well plate was discarded, fresh culture medium was added to each well, and the acellular matrix membrane prepared in Example 1 was carefully placed on the cell layer in the central part of each well, and was cut to ensure that the acellular matrix membrane covered about one-tenth of the surface of the cell layer. After 24 h, the well plate was observed under an optical microscope (CKX53) to observe the cell adhesion to the acellular matrix membrane. The more cells on the acellular matrix membrane, the better the biocompatibility.

[0079] (3) In vivo animal experiment characterization: Two SD rats (8-10 weeks old) were taken, anesthetized, and skinned, and a φ = 1.5 cm drill was used to prepare two φ = 1.5 cm circular wounds on the back of each rat. An acellular matrix membrane prepared in Example 1 was placed on one of the wounds as an acellular matrix membrane group, and no material was placed on the other wound as a blank group.

[0080] The area S of each of the two wounds on the back of each rat was measured, and the area of the two wounds on the back of the two rats was measured at two weeks and four weeks after the operation, respectively. The average value was taken as S1, and the healing rate after the operation was calculated as S1 / S x 100%. The lower the healing rate after the operation, the better the repair effect, and thus the better the biocompatibility.

[0081] (4) Visual function characterization: The acellular matrix membrane prepared in Example 1 or Comparative Example 1 was placed on the skin defect site of an animal (rabbit), and a photograph was taken.

[0082] DNA residual amount analysis test:

[0083] DNA residual amount analysis: According to YY / T 1876-2023 "Tissue Engineering Medical Products Animal-derived Biomaterial DNA Residual Amount Determination Method: Fluorescent Staining Method", the DNeasy Blood & Tissue Kit (QIAGEN) kit was used for determination. The DNA residual amount should be not more than 50 ng / mg.

[0084] Example 1

[0085] 1) Pretreatment: The dirt residue of the obtained sheep gastric mucosa was washed clean with purified water, placed in a 0.9 wt% sodium chloride solution, and shaken for 18 h. The sheep stomach surface layer was scraped clean, and a white or milky white sheep stomach tissue was obtained. The excess water was drained until no obvious dripping was observed, and the matrix material was obtained for use;

[0086] 2) Defatting: The obtained substrate material was mixed with 50% (v / v) isopropanol solution (solvent: purified water) at a mass ratio of 1:100, and oscillated for 180 min. After the end, the isopropanol solution was drained, 0.9 wt% sodium chloride solution was added, and the material was washed for 3 times, each for 15 min, to obtain the defatted material;

[0087] 3) Disinfecting: The obtained defatted material was mixed with a mixed solution of 0.05 wt% peracetic acid and 40 wt% ethanol (solvent: purified water) at a solid-liquid mass ratio of 1:100, and oscillated for 120 min. The disinfectant was drained, 0.9 wt% sodium chloride solution was added, and the material was washed for 3 times, each for 20 min, to obtain the disinfected material;

[0088] 4) Decellularization: The obtained disinfected material was mixed with a 0.05 wt% trypsin solution (solvent: PBS) at a solid-liquid mass ratio of 1:100, and oscillated for 180 min. The trypsin solution was drained, 0.9 wt% sodium chloride solution was added, and the material was washed for 3 times, each for 20 min, to obtain the decellularized material;

[0089] 5) Removal of α-Gal antigen: The obtained decellularized material was mixed with a 4.5 U / mL α-galactosidase solution (solvent: PBS) at a solid-liquid mass ratio of 1:10, and oscillated at 37°C for 30 h. The α-galactosidase solution was discarded, 0.1 M PBS solution was added, and the material was washed for 3 times, each for 15 min, to obtain the material after removal of the α-Gal antigen;

[0090] 6) Gradient dehydration: Step 1: The material after removal of the α-Gal antigen was placed in a 30% (v / v) glycerol solution (solvent: purified water) at a mass ratio of 1:20 for dehydration, with an oscillation speed of 300 rpm, an oscillation temperature of 30°C, and an oscillation time of 150 min. The concentration gradient dehydration was performed for 2.5 h, and the material was washed with purified water for 10 min;

[0091] Step 2: The material was placed in a 50% (v / v) glycerol solution for dehydration at a mass ratio of 1:20, with an oscillation speed of 300 rpm, an oscillation temperature of 30°C, and an oscillation time of 240 min. The material was washed with purified water for 10 min,

[0092] Step 3: The material was placed in a 70% (v / v) glycerol solution for dehydration at a mass ratio of 1:20, with an oscillation speed of 300 rpm, an oscillation temperature of 30°C, and an oscillation time of 240 min. The material was washed with purified water for 15 min,

[0093] Step 4: Dehydration in 90% (v / v) glycerol solution for 2.5 h at a mass ratio of 1:20, with a shaking speed of 300 rpm, a shaking temperature of 30 °C, a shaking time of 150 min, and a rinsing time of 20 min with purified water, and a water content of the pressed material of ≤ 15%;

[0094] 7) Pressing: The dehydrated material was laid on a stainless steel mesh plate for pressing, with a pressing force of 400 N and a pressing time of 4 h, to obtain the pressed material;

[0095] 8) Freeze-drying: The pre-frozen pressed material was laid on a stainless steel plate without air bubbles, and 8 layers (not more than 10 layers) were stacked on top of each other, and then placed in a -20 °C refrigerator for 100 min and transferred to a freeze dryer, with a freeze-drying temperature of -45 °C and a vacuum degree of 1 mbar;

[0096] 9) Sterilization: The packaged treated sheep stomach tissue was sterilized at 25 kGy for 20 h to obtain the decellularized matrix membrane.

[0097] Examples 2 to 3

[0098] The relevant parameters were adjusted according to Table 1, and the rest was the same as in Example 1.

[0099] Example 4

[0100] In addition to the pre-treatment of step 1), sterilization of step 3), decellularization of step 4), removal of the a-Gal antigen of step 5), freeze-drying of step 8), sterilization of step 9), and Example 1, the other steps were performed according to the following steps to obtain the decellularized matrix membrane:

[0101] 2) Delipidation: The obtained matrix material was mixed with 100% (v / v) acetone solution at a mass ratio of 1:50, and the shaker was oscillated for 12 h, with liquid change every 6 h. After the end, the acetone solution was drained, 0.9 wt% sodium chloride solution was added, and the washing was performed 3 times for 15 min each time;

[0102] Then the sheep stomach tissue was mixed with 100% (v / v) acetone solution at a mass ratio of 1:50, and the shaker was oscillated for 24 h, with liquid change every 6 h. After the end, the acetone solution was drained, 0.9 wt% sodium chloride solution was added, and the washing was performed 5 times for 30 min each time;

[0103] Then the sheep stomach tissue was mixed with 100% (v / v) acetone solution at a mass ratio of 1:50, and the shaker was oscillated for 48 h, with liquid change every 12 h. After the end, the acetone solution was drained, 0.9 wt% sodium chloride solution was added, and the washing was performed 5 times for 30 min each time.

[0104] 6) transparent treatment: the material after removing α-Gal antigen was mixed with 80% (v / v) polyethylene glycol according to the mass ratio of 1:20, so that the material after removing α-Gal antigen was completely immersed in the transparent reagent solution, and was oscillated for 48 h, and the solution was replaced every 6 h, to obtain a transparent material;

[0105] 7) pressing: the transparent material was laid on a stainless steel mesh plate for pressing, the pressing force was 400 N, and the pressing time was 4 h, to obtain a pressed material.

[0106] Comparative Example 1

[0107] Comparative Example 1

[0108] The preparation parameters and performance parameters of each example and comparative example are shown in Table 1.

[0109] Table 1

[0110] Pressing force (N) Thickness (mm) Transmittance (%) Tensile strength (MPa) Example 1 400 0.36 95.17 15.78 Example 2 10 0.48 81.97 24.63 Example 3 800 0.1 98.92 5.35 Example 4 400 0.40 94.66 18.13 Comparative Example 1 / 0.62 12.98 27.38

[0111] Table 2

[0112]

[0113] The light transmittance of the decellularized matrix membrane will affect the real-time visual observation effect of the decellularized matrix membrane in the clinical wound regeneration process. The light transmittance of the decellularized matrix membrane will be affected by the pressing force of the pressing process in the preparation of the decellularized matrix membrane. As can be seen from Example 1 to Example 4 and Comparative Example 1, when the pressing force of the pressing process in the preparation of the decellularized matrix membrane is within the range of the present application, the light transmittance of the decellularized matrix membrane is higher, which indicates that the real-time visual observation effect of the decellularized matrix membrane prepared in the examples in the clinical wound regeneration process is better.

[0114] In the present application, the DNA residual amount of all examples and comparative examples is less than 10 ng / mg.

[0115] The thickness of the decellularized matrix membrane will be affected by the pressing force of the pressing process in the preparation of the decellularized matrix membrane. As can be seen from Example 1 to Example 4 and Comparative Example 1, when the pressing force of the pressing process in the preparation of the decellularized matrix membrane is within the range of the present application, the thickness of the decellularized matrix membrane is within the range of the present application, and the light transmittance of the decellularized matrix membrane is higher, which indicates that the real-time visual observation effect of the decellularized matrix membrane prepared in the examples in the clinical wound regeneration process is better.

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

[0117] Through in vivo animal experiment characterization, as can be seen from Table 2, compared with the blank group, the decellularized matrix membrane group obtained by the decellularized matrix membrane prepared in Example 1 has a higher healing rate at 2 weeks and 4 weeks after the operation, which indicates that the decellularized matrix membrane prepared in Example 1 has good biocompatibility and can better promote the repair of skin defects.

[0118] As shown in Figure 1 , the decellularized matrix membrane prepared in Example 1 is transparent, which is conducive to improving the light transmittance, thereby facilitating real-time visual observation of the wound regeneration process in clinical practice.

[0119] As shown in Figure 2 , (a) is a photo of the skin defect site of a rabbit, and compared with the decellularized matrix membrane prepared in Comparative Example 1 in (c), the decellularized matrix membrane prepared in Example 1 in (b) can still be clearly seen after being placed on the skin defect site of a rabbit, which indicates that the decellularized matrix membrane prepared in Example 1 has a good real-time visual observation effect in the clinical wound regeneration process.

[0120] As shown in Figure 3 and Figure 4 , the fibrous structure of the decellularized matrix membrane prepared in Example 1 is dense, and the type I collagen maintains a clear triple helix structure, which is conducive to cell crawling and proliferation.

[0121] As shown in Figure 5 , the 100% extract, 50% extract and 25% extract of the decellularized matrix membrane prepared in Example 1 have good effects in promoting cell proliferation, which indicates that the decellularized matrix membrane prepared in Example 1 has good biocompatibility.

[0122] As shown in Figure 6 and Figure 7 Test method and equipment: Pressing force (N) Thickness (mm) Transmittance (%) Tensile strength (MPa) Example 1 Example 2 Example 3 Example 4 Comparative Example 1 Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 , after 24 hours of testing by the direct contact method, there are more cells on the decellularized matrix membrane prepared in Example 1, which indicates that the cells tend to adhere to the decellularized matrix membrane, and further indicates that the decellularized matrix membrane prepared in Example 1 has good biocompatibility.

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

[0124] Each of the embodiments in the present specification is described in a related manner, and the same or similar parts between the embodiments can be referred to each other. Each of the embodiments focuses on the difference from other embodiments.

[0125] The above only describes the preferred embodiments of the present application, and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A decellularized matrix membrane, comprising at least one of collagen, laminin, glycosaminoglycan and keratin; the collagen comprises type I collagen, which is a triple helix structure, and the decellularized matrix membrane has a light transmittance of 80% to 99%.

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

3. The decellularized matrix membrane of claim 1, wherein, The tensile strength of the decellularized matrix membrane is 5MPa to 25MPa. 4.A method for preparing the decellularized matrix membrane according to any one of claims 1 to 3, comprising: 1) pretreatment: collecting mammalian soft tissue material selected from bovine pericardium, porcine pericardium, sheep gastric mucosa, porcine peritoneum, porcine small intestinal mucosa or dermal tissue, and processing to obtain a matrix material; 2) defatting: mixing the matrix material with a defatting agent, and processing to obtain a defatted material; 3) disinfection: mixing the defatted material with a disinfectant, and processing by oscillation to obtain a disinfected material; 4) decellularization: mixing the disinfected material with an alkaline protease solution, and processing by oscillation to obtain a decellularized material; 5) removing α-Gal antigen: at least one method selected from repeated freeze-thaw washing, mechanical crushing washing or washing with an α-galactosidase solution, to obtain a material after removing α-Gal antigen; 6) gradient dehydration: placing the material after removing α-Gal antigen in 25% to 35% (v / v), 45% to 55% (v / v), 65% to 75% (v / v) and 85% to 95% (v / v) dehydrating agent solutions in sequence according to a mass ratio of 1:(10-50), oscillating for 0.5-8h at an oscillation speed of 50-500rpm and an oscillation temperature of 20℃ to 40℃, and then washing with pure water for 5min to 40min after taking out, to obtain a dehydrated material; the dehydrating agent is at least one selected from glycerol, ethanol, butanediol, propanol and mannitol; or, transparent treatment: mixing the material after removing α-Gal antigen with 50% to 80% (v / v) transparent reagent solution according to a mass ratio of 1:(10-50), oscillating for 12h to 72h, changing the solution regularly, draining the transparent reagent after finishing, and placing in 0.1-1wt% sodium chloride solution, oscillating for 3-15 times, each time for 10-100min, to obtain a transparent material; the transparent reagent is at least one selected from polyethylene glycol, methanol, phenethyl alcohol, CUBIC tissue transparent reagent and FDISCO ex vivo tissue transparent reagent; 7) pressing: placing the dehydrated material or the transparent material on a pressing plate for pressing, with a pressing force of 10N to 800N, to obtain a pressed material; the pressing plate is a stainless steel mesh plate, a stainless steel plate, an acrylic plate, a polypropylene plastic plate, a polyethylene plastic plate, an acrylonitrile / butadiene / styrene copolymer plastic plate, a polyvinyl chloride hard plastic plate or a polycarbonate plastic plate; the water content of the pressed material is ≤15%; 8) freeze-drying and sterilization of the pressed material to obtain the decellularized matrix membrane.

5. The method of producing a 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, adding 0.1 to 1 wt% sodium chloride solution, and washing. Repeating this process 3 to 5 times yields 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. Use of the decellularized matrix membrane according to claim 6 for the preparation of a regenerative tissue repair membrane, wherein, The regenerative tissue repair membrane includes a skin repair membrane.

8. Use of the decellularized matrix membrane according to claim 6 for the preparation of a regenerative tissue repair membrane, 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. Use of the decellularized matrix membrane according to claim 8 for the preparation of a regenerative tissue repair membrane, 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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