Animal small intestinal submucosa acellular matrix anti-adhesion membrane and its preparation method and use

Abstract

The application discloses an animal small intestinal submucosa acellular matrix anti-adhesion membrane and a preparation method and application thereof, and belongs to the field of biological materials. The animal small intestinal submucosa acellular matrix anti-adhesion membrane is obtained by sterilizing one side of the animal small intestinal submucosa acellular matrix after laser treatment. The animal small intestinal submucosa acellular matrix anti-adhesion membrane has reduced thickness, improved degradation performance, can guide postoperative wound tissue regeneration, can prevent other tissues from adhering to a wound surface, and is particularly suitable for a case that the degradation rate of a sheet material needs to be further improved, and has a wide application prospect in preparation of anti-adhesion tissue repair materials.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomaterials, in particular to an animal small intestinal submucosa acellular matrix anti-adhesion membrane and a preparation method and use thereof. BACKGROUND

[0002] Tissue adhesion refers to a pathological state in which the contact surface of adjacent tissues or organs is connected together by fibrous or newly formed fibrous tissue in scar tissue. Adhesion is the most common surgical complication and can occur in various parts of the body, such as the chest cavity, abdominal cavity, uterine cavity, pericardium, tendon, joint, skin and subcutaneous tissue, etc. For example, 60% to 90% of gynecological and obstetrical patients will develop adhesion after pelvic surgery, and about 90% of patients will develop postoperative adhesion after abdominal surgery. Adhesion can cause serious complications and even endanger life.

[0003] Anti-adhesion materials are one of the main ways to prevent and treat tissue adhesion. In recent years, a large number of studies have shown that anti-adhesion materials can act as a barrier to effectively block adhesion and prevent adhesion-related adverse events. An ideal anti-adhesion material should have good biocompatibility, be able to effectively block external tissue invasion, and be able to promote postoperative tissue repair and easy degradation and absorption.

[0004] Anti-adhesion materials can be divided into film and liquid types. Film anti-adhesion materials are generally sponge-like or film-like and are mainly made of cellulose derivatives, chitosan and polylactic acid and other natural polymer materials. Such film materials have hard texture and poor adhesion during use. In addition, the local accumulation of acidic products during polylactic acid degradation can also easily produce mild aseptic inflammation. Liquid anti-adhesion materials are in the form of gel or solution and are mainly made of hyaluronic acid, chitosan or solution. Such gels or solutions are easy to flow and have unstable efficacy risks.

[0005] Bioactive materials such as small intestinal submucosa (SIS), pericardial tissue, dermal tissue and biological amniotic membrane are a kind of natural extracellular matrix membrane materials, which have good tissue compatibility, low immunogenicity and good flexibility after decellularization and other antigen-removing treatments, and are widely used in myocardial tissue, skin tissue, oral tissue, blood vessels and other soft tissue repair applications. Such materials can overcome the problems of poor flexibility and inflammation caused by degradation products of other anti-adhesion materials, and are one of the potential ideal anti-adhesion materials. The invention patent CN106983918B discloses an anti-adhesion film material prepared by small intestinal submucosa tissue, which can not only play a barrier role in anti-adhesion by smooth surface, but also guide tissue repair by porous structure of rough surface. However, on the one hand, the film material prepared by this method is composed of at least two layers of small intestinal submucosa material, which may have the risk of delamination during use; on the other hand, the degradation time of the film material prepared by this method is relatively long, and it may not be suitable for the case where the degradation rate is required to be fast. For example, the expert consensus on prevention and diagnosis of pelvic and abdominal adhesions after gynecological surgery (2020 edition) points out that the key period of postoperative pelvic and abdominal adhesion formation and mesothelial repair is 3-5 days, and if the degradation is too slow, it may cause chronic inflammation and foreign body reaction and other problems. SUMMARY

[0006] To solve the above problems, the purpose of the present application is to provide an animal small intestinal submucosa decellularized matrix anti-adhesion film and its preparation method and use, which has reduced thickness and improved degradation performance, and can guide postoperative wound tissue regeneration and prevent other tissues from adhering to the wound surface.

[0007] The present application provides an animal small intestinal submucosa decellularized matrix anti-adhesion film, which is obtained by sterilizing one side of the animal small intestinal submucosa decellularized matrix after laser treatment; the parameters of the laser treatment are as follows: wavelength 10-400 nm, frequency ≥50 kHz, pulse width ≤1000 fs, power 0.1-30 w, filling processing line spacing 0.001-0.1 mm, and filling frequency 1-10 times.

[0008] Further, the parameters of the laser treatment are as follows: wavelength 350-360 nm, frequency 190-210 kHz, pulse width 290-310 fs, power 3-5 w, filling processing line spacing 0.005-0.02 mm, and filling frequency 4-7 times.

[0009] Further, the parameters of the laser treatment are as follows: wavelength is 355 nm, frequency is 200 kHz, pulse width is 300 fs, power is 4 w, filling processing line spacing is 0.01 mm, and filling number is 7 times; or, the parameters of the laser treatment are as follows: wavelength is 355 nm, frequency is 200 kHz, pulse width is 300 fs, filling processing line spacing is 0.01 mm, filling number is 7 times, filling is disordered, and power is randomly changed in the range of 0.1-30 w.

[0010] In the application, fs, i.e., femtosecond, 10 -15 s.

[0011] Further, the sterilization is gamma ray irradiation sterilization, and the dose of the irradiation sterilization is preferably 15-25 KGy.

[0012] Further, the preparation method of the animal small intestinal submucosa acellular matrix comprises the following steps: taking animal small intestinal submucosa tissue, virus inactivation, washing, antigen removal, washing, drying, and obtaining the animal small intestinal submucosa acellular matrix.

[0013] And / or, the animal is a mammal, preferably a pig or a cow.

[0014] Further, the washing before antigen removal comprises the following steps: washing with water and filtering dry water.

[0015] Further, the washing before antigen removal is washing to a conductivity of 10 muS / cm or less.

[0016] Further, the washing after antigen removal comprises the following steps: washing with water.

[0017] Further, the washing after antigen removal is washing to a conductivity of 10 muS / cm or less.

[0018] Further, the animal small intestinal submucosa tissue is obtained by removing the mucosa layer, serosa layer and muscle layer from the animal small intestinal tissue after cleaning.

[0019] The virus inactivation is achieved by treating the animal small intestinal submucosa tissue with a peracetic acid-ethanol solution; in the peracetic acid-ethanol solution, the volume percentage concentration of peracetic acid is 0.2-2%, the volume percentage concentration of ethanol is 10%-30%, the volume ratio of the peracetic acid-ethanol solution to the animal small intestinal submucosa tissue material is (2-15) ︰ 1, the treatment time is 2-4 hours, and the temperature is 10-40℃.

[0020] The deantigenization method is to sequentially use a methanol-chloroform mixture and a Triton X-100 solution to de-fat and de-cell the animal small intestinal submucosa tissue; in the methanol-chloroform mixture, the volume percentage of methanol is 30% to 70%, the volume percentage of chloroform is 30% to 70%, the volume ratio of the methanol-chloroform mixture to the small intestinal submucosa tissue material is (2 to 10) to 1, the de-fat time is 2 to 6 hours, and the temperature is 30 to 40℃; the mass percentage of the Triton X-100 solution is 0.1% to 5%, the volume ratio of the Triton X-100 solution to the small intestinal submucosa tissue material is (1 to 10) to 1, the de-cell time is 0.5 to 4 hours, and the temperature is 30 to 40℃.

[0021] Further, in the peracetic acid-ethanol solution, the volume percentage concentration of peracetic acid is 0.5%, the volume percentage concentration of ethanol is 20%, the volume ratio of the peracetic acid-ethanol solution to the animal small intestinal submucosa tissue material is 10 to 1, the treatment time is 2 hours, and the temperature is 25℃;

[0022] In the methanol-chloroform mixture, the volume percentage of methanol is 40%, the volume percentage of chloroform is 60%, the volume ratio of the methanol-chloroform mixture to the small intestinal submucosa tissue material is 8 to 1, the de-fat time is 3 hours, and the temperature is 37℃; the mass percentage of the Triton X-100 solution is 0.3%, the volume ratio of the Triton X-100 solution to the small intestinal submucosa tissue material is 5 to 1, the de-cell time is 2 hours, and the temperature is 37℃.

[0023] Further, the animal small intestinal submucosa de-cellular matrix anti-adhesion membrane has a layer number of ≥1.

[0024] Further, the animal small intestinal submucosa de-cellular matrix anti-adhesion membrane has a sheet structure, can include 1 to 8 layers, and has a thickness of 0.1 to 1 mm.

[0025] Further, the animal small intestinal submucosa de-cellular matrix anti-adhesion membrane includes a smooth surface and a rough surface, one side after laser treatment is the rough surface, and the other side without laser treatment is the smooth surface.

[0026] Further, the smooth surface is dense and has few pores, and the rough surface is loose and has many pores.

[0027] Further, the rough surface micropore is completely or partially filled with a surface.

[0028] Further, the rough surface micropore has a pore size of 10 to 500 μm, and a pore depth of no more than 60% of the material thickness.

[0029] Further, the animal small intestinal submucosa acellular matrix anti-adhesion membrane has a degradation time of 0.5-3 months.

[0030] Further, the animal small intestinal submucosa acellular matrix anti-adhesion membrane has a tensile strength greater than 5N and a tear strength greater than 0.8N.

[0031] The application further provides a preparation method of the animal small intestinal submucosa acellular matrix anti-adhesion membrane, which comprises the following steps: sterilizing the animal small intestinal submucosa acellular matrix after one side of the animal small intestinal submucosa acellular matrix is treated by laser, to obtain the animal small intestinal submucosa acellular matrix anti-adhesion membrane.

[0032] The application further provides a use of the animal small intestinal submucosa acellular matrix anti-adhesion membrane in preparing an anti-adhesion tissue repair material.

[0033] Compared with the prior art, the application has the following beneficial effects:

[0034] (1) The animal small intestinal submucosa acellular matrix anti-adhesion membrane is obtained by sterilizing the animal small intestinal submucosa acellular matrix after one side of the animal small intestinal submucosa acellular matrix is treated by laser under specific conditions. The animal small intestinal submucosa acellular matrix anti-adhesion membrane has a reduced thickness and improved degradation performance, can guide postoperative wound tissue regeneration and prevent other tissues from adhering to a wound surface, and is particularly suitable for a case where the degradation rate of a sheet material needs to be further improved, and has a wide application prospect in preparing an anti-adhesion tissue repair material.

[0035] (2) The animal small intestinal submucosa acellular matrix anti-adhesion membrane has a porous surface after being treated by laser, and has improved adhesion performance, which can improve the stability of the material in use.

[0036] (3) The animal small intestinal submucosa acellular matrix anti-adhesion membrane is a film material having a smooth surface and a rough surface structure. The rough surface has a porous structure and can support cell growth to guide wound repair, and the smooth surface is dense and smooth and can effectively prevent the invasion of external cells or tissues, thereby playing an anti-adhesion role.

[0037] (4) The application adopts advanced femtosecond laser processing technology, realizes non-contact processing of the material surface, introduces no pollutants, has very small thermal effect, and almost has no thermal damage to the material surface.

[0038] Obviously, according to the above content of the application, according to the ordinary technical knowledge and common means in the art, other various forms of modifications, replacements or changes can be made without departing from the above basic technical idea of the application.

[0039] The above mentioned subject matter of the present application is further explained in detail by way of specific embodiments in the form of examples. However, it should be understood that the scope of the above mentioned subject matter of the present application is not limited to the examples below. Any technology achieved based on the above mentioned subject matter of the present application falls within the scope of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 SEM photographs of the microstructure of the matt and glossy surface of the anti-blocking film of the present application.

[0041] Figure 2 Photograph of the anti-blocking film obtained in Test Example 1 with a power of 1 w and a filling processing line interval of 0.005 mm.

[0042] Figure 3 Photograph of the anti-blocking film obtained in Test Example 1 with a power of 4 w and a filling processing line interval of 0.005 mm.

[0043] Figure 4 Photograph of the anti-blocking film obtained in Test Example 1 with a power of 7 w and a filling processing line interval of 0.005 mm.

[0044] Figure 5 Photograph of the anti-blocking film obtained in Test Example 1 with a power of 4 w and a filling processing line interval of 0.01 mm.

[0045] Figure 6 Photograph of the anti-blocking film obtained in Test Example 1 with a power of 4 w and a filling processing line interval of 0.02 mm.

[0046] Figure 7 Photograph of the anti-blocking film obtained in Test Example 1 with a power of 7 w and a filling processing line interval of 0.02 mm.

[0047] Figure 8 HE staining diagram of the cross section of the anti-blocking film of the present application and statistical diagram of thickness change.

[0048] Figure 9 Comparison diagram of the tensile and tear properties of the anti-blocking film after laser surface treatment.

[0049] Figure 10 Comparison diagram of the degradation properties of the anti-blocking film after laser surface treatment.

[0050] Figure 11 Comparison diagram of the adhesion properties of the anti-blocking film after laser treatment.

[0051] Figure 12 Animal experiment results of the anti-blocking film of the present application. DETAILED DESCRIPTION

[0052] The reagents and devices used in the present application are known products, which are obtained by purchasing commercially available products.

[0053] Preparation of the small intestine submucosa acellular matrix anti-adhesion membrane of the animal in Example 1

[0054] The specific preparation method is as follows:

[0055] (1) Tissue collection and cleaning: pig small intestine tissue was taken, cleaned, and the mucosa layer, serosa layer, and muscle layer were removed to obtain small intestine submucosa tissue.

[0056] (2) Virus inactivation: the small intestine submucosa tissue was treated with a peracetic acid-ethanol solution, wherein the volume percentage concentration of peracetic acid was 0.5%, the volume percentage concentration of ethanol was 20%, the volume ratio of the peracetic acid-ethanol solution to the small intestine submucosa tissue material was 10:1, the inactivation time was 2 hours, and the temperature was 25°C.

[0057] (3) Washing: the small intestine was repeatedly washed with purified water 5 times, and the water amount was 20 L each time.

[0058] (4) Antigen removal: the small intestine submucosa tissue was subjected to lipid removal and decellularization treatment using a methanol-chloroform mixture and a Triton X-100 solution in sequence. First, the methanol-chloroform mixture used was composed of 40% methanol and 60% chloroform by volume, and the volume ratio of the methanol-chloroform mixture to the small intestine submucosa tissue material was 8:1, the lipid removal time was 3 hours, and the temperature was 37°C; then, a Triton X-100 solution with a mass percentage of 0.3% was prepared using a PBS solution (pH = 6-8), and the volume ratio of the Triton X-100 solution to the small intestine submucosa tissue material was 5:1, the decellularization time was 2 hours, and the temperature was 37°C.

[0059] (5) Washing: the small intestine submucosa tissue material was repeatedly washed with water in an ultrasonic cleaner 5 times, the water flow rate was 7 L / min, the volume ratio of the washing water to the small intestine submucosa tissue material was maintained at 20:1, the frequency of the ultrasonic cleaner was 40 kHz, and the power was 3000 W; after the washing was completed, the excess water was gently rolled off by hand, and the small intestine submucosa matrix material was obtained.

[0060] (6) Drying: the small intestine submucosa matrix material was laid flat in one layer, and a vacuum oven (60°C, 4h) was used for drying and dehydration treatment to obtain the decellularized matrix material.

[0061] (7) The decellularized matrix material was scanned and processed on one side using three specific parameters of ultraviolet femtosecond laser to form a rough surface, and three kinds of membrane pieces were prepared.

[0062] The three ultraviolet femtosecond laser processing parameters are as follows:

[0063] Sequential processing A: wavelength 355 nm, frequency 200 khz, pulse width 300 fs, power 4 w, filling processing line spacing 0.01 mm, filling 4 times; the processing surface microstructure is as shown in Figure 1 and the sample is named as "sequential processing A" film;

[0064] Sequential processing B: wavelength 355 nm, frequency 200 khz, pulse width 300 fs, power 4 w, filling processing line spacing 0.01 mm, filling 7 times; the processing surface microstructure is as shown in Figure 1 and the sample is named as "sequential processing B" film;

[0065] Disordered processing: wavelength 355 nm, frequency 200 khz, pulse width 300 fs, filling processing line spacing 0.01 mm, filling 7 times, disordered filling, power randomly changes in the range of 0.1-30 w during processing; the processing surface microstructure is as shown in Figure 1 and the sample is named as "disordered processing" film.

[0066] (8) Sterilization: the film is irradiated and sterilized by Co-60 (15 KGy) to obtain an animal small intestinal submucosa decellularized matrix anti-adhesion film, which is named as "sequential processing A" anti-adhesion film, "sequential processing B" anti-adhesion film and "disordered processing" anti-adhesion film, respectively.

[0067] Preparation of non-processed animal small intestinal submucosa decellularized matrix anti-adhesion film in Comparative Example 1

[0068] The sample is prepared according to the preparation method in Reference Example 1, and the only difference is that the decellularized matrix film is not processed by laser in step (7) (and the sample is named as "non-processed" film), to obtain a non-processed animal small intestinal submucosa decellularized matrix anti-adhesion film, which is named as "non-processed" anti-adhesion film.

[0069] The beneficial effects of the present application are demonstrated by the following test examples.

[0070] Test Example 1 Optimization of ultraviolet femtosecond laser processing parameters of anti-adhesion film

[0071] The sample is prepared according to the preparation method in Reference Example 1, and the only difference is that the decellularized matrix material is scanned and processed by ultraviolet femtosecond laser with different parameters on one side in step (7), to obtain an animal small intestinal submucosa decellularized matrix anti-adhesion film. The specific parameters of the ultraviolet femtosecond laser are set as follows: wavelength 355 nm, frequency 200 khz, pulse width 300 fs, filling times 7, adjusting power (1 w / 4 w / 7 w) and filling processing line spacing (0.005 mm / 0.01 mm / 0.02 mm).

[0072] Observe whether the processing traces of the animal small intestinal submucosa decellularized matrix anti-adhesion membrane prepared under different parameters are obvious and whether there are damages. As shown in Table 1, under the parameters of wavelength 355 nm, frequency 200 khz, pulse width 300 fs, power 4 w, filling processing line spacing 0.01 mm, and filling 7 times, the animal small intestinal submucosa decellularized matrix anti-adhesion membrane prepared has no damage and the processing traces are the most clear and obvious.

[0073] Table 1 Influence of different power and filling processing line spacing on ultraviolet femtosecond laser processing anti-adhesion membrane

[0074]

[0075] SEM test results of the anti-adhesion membrane of Test Example 2

[0076] 1. Test method

[0077] Spray gold on the disordered processing, ordered processing A, and ordered processing B anti-adhesion membrane samples for 5 min, and observe and take photos of the processing area (400X), non-processing area (400X), and the junction (40X) by SEM.

[0078] 2. Experimental results

[0079] As Figure 1 are SEM images of the processing area, non-processing area, and processing junction after laser processing, and are anti-adhesion membrane materials prepared by using disordered processing parameters, ordered parameter A, and ordered parameter B, respectively. The results show that laser can be used for surface pattern control and roughening of the anti-adhesion membrane material, increase the surface porosity, and significantly increase the surface roughness. It is shown that the anti-adhesion membrane prepared by the embodiment of the present application after laser processing can be used for the preparation of rough surfaces in contact with the wound surface.

[0080] HE staining results of the anti-adhesion membrane of Test Example 3

[0081] 1. Test method

[0082] According to the method of Embodiment 1, the processing area of one side of the decellularized matrix material is scanned and processed using ultraviolet femtosecond laser with three specific parameters to form a rough surface, and the non-processing area is not treated by laser. The disordered processing, ordered processing A, and ordered processing B anti-adhesion membrane samples are embedded in wax, then the material slices are deparaffinized to water and stained with hematoxylin for 5-10 min, washed with water, differentiated with hydrochloric acid alcohol, returned to blue with ammonia water, and stained with eosin for 3-5 min, then dehydrated with anhydrous ethanol, and finally made transparent by xylene, and the transparent slices are sealed with gum. After the gum is dried, the biological membrane material is observed and photographed (100x) using a microscope, and the thickness change is counted.

[0083] 2. Experimental results

[0084] As Figure 8 The HE image results (processing, non-processing area respectively on the left and right sides of the dotted line, the processing surface upward) in a) showed that the surface of the biological membrane material after laser processing had no thermal damage, and the thickness was reduced; and with the increase of the filling times, the laser processing showed obvious thinning effect. The thickness change statistics of the laser processing anti-adhesion membrane are as shown in Figure 8 b), the thinning rate of the membrane material processed by disorder processing and ordered processing B parameters reached about 50%. The above results showed that laser processing could perform thinning treatment on biological membrane materials to regulate the thickness of the membrane.

[0085] Test Example 4 Mechanical properties of anti-adhesion membrane

[0086] 1. Test method

[0087] Tensile strength test: non-processing, disorder processing, ordered processing A, ordered processing B anti-adhesion membrane samples with a size of 40x40mm were used for testing. The test sample was soaked in physiological saline for 5-10min before testing, and then fixed on the clamps of the tensile testing machine, and the tensile rate was set to 50mm / min for testing. The maximum tensile force at the time of membrane fracture was recorded as the tensile strength, repeated 3 times, and the average value was calculated as the final result.

[0088] Tear strength test: anti-adhesion membrane with a size of 10x25mm was used for testing, and the test was carried out according to the detection method specified in YY / T1794-2021.

[0089] 2. Experimental results

[0090] Test item Non-processed Disordered processing Ordered processing A Ordered processing B Tensile strength / N 5.54 5.46 5.50 5.43 Tear strength / N 0.94 0.85 0.89 0.86

[0091] As shown in the above table and Figure 9 a-b) are respectively the tensile strength and tear strength test results of each sample. Compared with the non-processing sample, the tensile strength and tear strength of each sample changed little. It is shown that compared with the non-processing anti-adhesion membrane material, the mechanical properties of each sample after laser processing have no obvious change, and the laser processing has almost no effect on the mechanical properties of the biological membrane material, which can be used to fit various shaped defect tissues and avoid rupture during treatment.

[0092] Test Example 5 Degradation performance

[0093] 1. Test method

[0094] The non-processed, disorderly-processed, orderly-processed A and orderly-processed B anti-adhesion film samples with a size of 40x40 mm were used for the degradation performance test. The samples were placed in a collagenase type I solution (4 mL, 4 mg / mL) and reacted at 37°C for 6 h, then the samples were taken out and washed twice with purified water by shaking, and dried and placed in a constant weight volumetric flask for weighing. The test was repeated three times, and the average value was calculated as the final result.

[0095] 2. Experimental results

[0096] Figure 10 The degradation performance test results of each sample showed that the degradation performance of each sample was improved after laser processing compared with the non-processed anti-adhesion film material. The degradation performance of the anti-adhesion film material prepared by disorderly processing and orderly processing B parameters was improved by 2-3 times.

[0097] Test Example 6: Adhesion performance of the anti-adhesion film

[0098] 1. Test method

[0099] The non-processed, disorderly-processed, orderly-processed A and orderly-processed B anti-adhesion film samples with a size of 40x40 mm were used for the adhesion strength test. The hairy surface of one end of the sample was attached to one end of the biological tissue with a length of 30 mm, and then the other end of the biological film material and the biological tissue was fixed on the clamps of the tensile testing machine with a length of 5 mm. The constant tensile speed of the tensile testing machine was set to 50 mm / min, and the tensile testing machine was started. The maximum tensile force when the sample was detached from the biological tissue was recorded as the adhesion strength. The test was repeated three times, and the average value was calculated as the final result.

[0100] 2. Experimental results

[0101] Figure 11 The adhesion strength results of the hairy surface of each sample showed that the hairy surface formed by laser processing could improve the adhesion performance of the film material to the biological tissue. The adhesion strength of the anti-adhesion film prepared by disorderly processing and orderly processing B parameters was increased by 30%-40%, which showed that it had significant adhesion performance on the surface of the wet tissue.

[0102] During the operation, appropriate adhesion is beneficial to the positioning of the film material, improves the success rate of the operation, and good adhesion performance can ensure that the material is tightly attached or wrapped around the wound during the movement of the patient after the operation, avoiding falling off and failure. The above results showed that the surface adhesion performance of the anti-adhesion film prepared by disorderly processing and orderly processing B parameters was significantly improved, which was beneficial to improving the treatment effect.

[0103] Test Example 7: Animal experiment of the anti-adhesion film

[0104] 1. Test method

[0105] A 4cm longitudinal incision was made in the lower left abdomen of the experimental rabbits (above the uterus) to access the abdominal cavity and locate the left uterus. The left uterus was repeatedly rubbed with a blunt scalpel until bleeding occurred, thus creating a model of intrauterine adhesions. The adhesion models were treated with three methods: no membrane material (positive control), membrane material covered with an untreated membrane (unprocessed), and a single-sided laser-treated membrane (ordered processing B). Absorbable sutures were used to fix the membrane material in the treated areas of the rabbits in all three groups. After moistening the abdominal cavity with physiological saline, the incisions in the abdominal wall muscle and skin layers were sutured continuously layer by layer. The degradation and adhesion of the samples were observed and photographed at each sampling time.

[0106] 2. Experimental Results

[0107] As shown in Table 2 below, compared with the untreated positive control, no significant adhesions were observed in the areas treated with the decellularized submucosal matrix of the small intestine, and the laser-treated single-sided membrane (ordered processing B) was completely degraded within 28 days. Figure 12 As shown, a) is an image of the intrauterine adhesion model covered by the sample on day 0, and b) and c) are the results of laser-treated single-sided membrane (ordered processing B) and positive control in rabbits after 28 days of treatment, respectively. The results show that the wound healed well after treatment with ordered processing B and no adhesion occurred, indicating that the anti-adhesion membrane material prepared by laser processing with ordered processing B has good degradation properties and anti-adhesion effect. After treatment, no inflammation was observed in the treated area and surrounding tissues by visual inspection, showing good tissue compatibility of the degradation products, suggesting its potential for in vivo anti-adhesion treatment.

[0108] Table 2 Evaluation of Animal Experiment Results

[0109]

Claims

1. A decellularized matrix anti-adhesion membrane for the submucosa of animal small intestine, characterized in that, It is obtained by sterilizing the decellularized matrix layer of the submucosa of animal small intestine after laser treatment; the parameters of the laser treatment are as follows: wavelength is 355 nm, frequency is 200 kHz, pulse width is 300 fs, power is 4 W, filling line spacing is 0.01 mm, and filling times are 7 times. The method for preparing the decellularized submucosal matrix of the animal small intestine includes the following steps: taking the submucosal tissue of the animal small intestine, inactivating the virus, washing, removing the antigen, washing again, and drying to obtain the decellularized submucosal matrix of the animal small intestine; the animal is a mammal.

2. The decellularized matrix anti-adhesion membrane for the submucosa of the animal small intestine according to claim 1, characterized in that, The sterilization is performed by gamma ray irradiation.

3. The decellularized matrix anti-adhesion membrane for the submucosa of the animal small intestine according to claim 2, characterized in that, The irradiation sterilization dose is 15~25 KGy.

4. The decellularized matrix anti-adhesion membrane for the submucosa of the animal small intestine according to claim 1, characterized in that, The mammal in question is either a pig or a cow.

5. The decellularized matrix anti-adhesion membrane for the submucosa of the animal small intestine according to claim 1, characterized in that, The animal small intestinal submucosal tissue is obtained by cleaning the animal small intestinal tissue and removing the mucosal layer, serosa layer and muscle layer; The virus inactivation method involves treating the submucosal tissue of the animal small intestine with a peracetic acid-ethanol solution; the peracetic acid-ethanol solution has a volume percentage concentration of 0.2-2% and an ethanol volume percentage concentration of 10-30%, the volume ratio of the peracetic acid-ethanol solution to the submucosal tissue of the animal small intestine is (2-15):1, the treatment time is 2-4 hours, and the temperature is 10-40℃. The method for removing the antigen involves sequentially using a methanol-chloroform mixture and a Triton X-100 solution to degrease and decellularize the submucosal tissue of the animal's small intestine. In the methanol-chloroform mixture, the volume percentages of methanol and chloroform are 30%–70% and 30%–70% respectively, with a volume ratio of (2–10):1 between the methanol-chloroform mixture and the submucosal tissue material. The degreasing time is 2–6 hours, and the temperature is 30–40°C. The mass percentage of the Triton X-100 solution is 0.1%–5%, with a volume ratio of (1–10):1 between the Triton X-100 solution and the submucosal tissue material. The decellularization time is 0.5–4 hours, and the temperature is 30–40°C.

6. The decellularized matrix anti-adhesion membrane for the submucosa of the animal small intestine according to claim 5, characterized in that, In the peracetic acid-ethanol solution, the volume percentage concentration of peracetic acid is 0.5%, the volume percentage concentration of ethanol is 20%, the volume ratio of peracetic acid-ethanol solution to animal small intestinal submucosa tissue material is 10:1, the treatment time is 2 hours, and the temperature is 25°C. In the methanol-chloroform mixture, the volume percentage of methanol is 40% and the volume percentage of chloroform is 60%. The volume ratio of the methanol-chloroform mixture to the submucosal tissue material of the small intestine is 8:

1. The defatting time is 3 hours and the temperature is 37°C. The mass percentage of the Triton X-100 solution is 0.3%. The volume ratio of the Triton X-100 solution to the submucosal tissue material of the small intestine is 5:

1. The decellularization time is 2 hours and the temperature is 37°C.

7. The decellularized matrix anti-adhesion membrane for the submucosa of the animal small intestine according to any one of claims 1 to 6, characterized in that, The number of layers of the decellularized matrix anti-adhesion membrane in the submucosa of the animal small intestine is ≥1.

8. The method for preparing the decellularized matrix anti-adhesion membrane of the animal small intestinal submucosa according to any one of claims 1 to 7, characterized in that, The method includes the following steps: treating one side of the decellularized matrix of the animal small intestinal submucosa with laser and then sterilizing it to obtain an anti-adhesion membrane of the decellularized matrix of the animal small intestinal submucosa.

9. The use of the decellularized matrix anti-adhesion membrane of the animal small intestinal submucosa according to any one of claims 1 to 7 in the preparation of anti-adhesion tissue repair materials.

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