A biofilm and its preparation method and application

Abstract

The present invention relates to the field of biotechnology, and specifically discloses a biofilm and a preparation method and application thereof. The present invention provides a biofilm, the preparation raw material of which is a decellularized animal membrane tissue, the biofilm has a light transmittance of 95.4%-97%, a suture strength of 2.4N-2.7N, and a water content of 5%-7%. The biofilm of the present invention has high transparency, good biocompatibility and excellent mechanical properties, and is absorbable. It can be used in the field of corneal repair or other biological patch materials with high transparency requirements, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to a biofilm and a preparation method and application thereof. Background Art

[0002] The cornea and sclera together form the outermost fibrous membrane of the eyeball. The cornea is also an important refractive matrix and the only way for external light to enter the eye and form an image on the retina. Any corneal lesions or corneal epithelial damage may lead to the destruction of the barrier function and integrity of the corneal epithelium, which may be accompanied by varying degrees of ocular surface inflammatory reactions. In severe cases, it may lead to corneal stromal lesions and even affect visual function. The commonly used treatment for corneal lesions or injuries is corneal transplantation for corneal repair, that is, using normal corneal tissue to replace the diseased or damaged corneal tissue, so as to restore the sight of the affected eye or control corneal lesions.

[0003] At present, corneal repair materials are mainly prepared from animals and allogeneic tissues, among which the most common ones are decellularized cornea and biological amniotic membrane. Decellularized cornea is mainly porcine cornea, but the raw material source of porcine cornea is small, expensive, and small in area, so its yield is low during the preparation process. In addition, since the decellularized cornea of ​​porcine cornea does not have the characteristics of corneal epithelium and endothelium, penetrating keratoplasty cannot be performed. Biological amniotic membrane has no blood vessels, nerves, and lymphatic vessels, has good toughness, is thin and translucent, and is similar to the tissue structure of human conjunctiva. Biological amniotic membrane can be used as a basement membrane for epithelial repair, promote epithelial healing, maintain normal epithelial cell phenotype, reduce inflammation, vascularization and scar hyperplasia, and the transplantation area is not limited. However, biological amniotic membrane still has certain disadvantages: such as low biomechanical properties, low suture strength, poor biocompatibility, few sources, and high price. Considering the above-mentioned shortcomings of decellularized cornea and biological amniotic membrane, new biological patch materials need to be developed for corneal repair.

[0004] Since the decellularized matrix material for corneal repair is very important for patients to achieve blindness relief, the light transmittance and mechanics of the decellularized matrix material are also the key to restore corneal transparency. High transparency and good mechanical properties are more conducive to the cornea to restore to a colorless and transparent state and achieve blindness relief. Therefore, a decellularized matrix material with good mechanical properties, high transparency, degradation performance and good biocompatibility is needed.

[0005] When preparing decellularized matrix materials, commonly used decellularization methods are divided into physical, chemical and biological methods, such as physical repeated freeze-thaw, physical swelling, chemical decellularization, enzymatic hydrolysis, etc. For example, Chinese patent CN116942913A discloses a decellularized biomaterial and its preparation method and application, including pre-treatment of the submucosal layer of the small intestine, swelling treatment, cold dissolution treatment, oxidase treatment and surfactant treatment, and the final freeze-drying step. The decellularized material prepared by the above-mentioned traditional process is a white or slightly yellow film with poor transparency, which is not convenient for use in situations where high transparency requirements are required, such as corneal repair, skin wound treatment and gum repair. Therefore, further research on the preparation of decellularized matrix materials is still needed. Summary of the invention

[0006] One of the purposes of the present invention is to provide a decellularized matrix material with good mechanical properties, high transparency, degradation properties and good biocompatibility.

[0007] In order to achieve this object, the technical solution of the present invention is as follows:

[0008] A biomembrane, the preparation material of which is decellularized animal membrane tissue, the biomembrane has a light transmittance of 95.4%-97%, a suture strength of 2.4N-2.7N, and a water content of 5%-7%.

[0009] The present invention provides a new biofilm material with high light transmittance, excellent mechanical properties and ideal biocompatibility. It is suitable for use in the field of biofilm repair with high transparency requirements, especially for repairing animal corneas, and provides a new method for the treatment of corneal diseases.

[0010] Preferably, the biofilm has a light transmittance of 95.40%-96.72%, a suture strength of 2.48N-2.67N, and a water content of 5.51%-6.50%.

[0011] The present invention also provides a method for preparing the above-mentioned biofilm, which comprises the step of subjecting the decellularized animal membrane tissue to transparent treatment, wherein the transparent treatment comprises:

[0012] (1) The first stage: the water content of the decellularized animal membrane tissue is reduced to 30-35.5% at a water reduction rate of 0.22% / min-0.45% / min;

[0013] (2) The second stage: The water content of the decellularized animal membrane tissue is reduced to 10.5-13% at a water reduction rate of 0.07% / min-0.15% / min.

[0014] Preferably, the transparency treatment includes:

[0015] (1) The first stage: the water content of the decellularized animal membrane tissue is reduced to 30-35.1% at a water reduction rate of 0.24% / min-0.43% / min;

[0016] (2) The second stage: The water content of the decellularized animal membrane tissue is reduced to 10.9~12.8% at a water reduction rate of 0.08% / min~0.13% / min.

[0017] Through research, the present invention has found that when the decellularized animal membrane tissue is dehydrated in a specific two-stage dehydration method, the transparency of the decellularized animal membrane tissue can be gradually improved, and finally a membrane with a transparency exceeding 95.4% is obtained. The mechanical strength and biocompatibility of the membrane are also very ideal, and the membrane is suitable for use as a biomembrane repair material in actual membrane tissue repair treatment.

[0018] Specifically, the present invention has found that the decellularized animal membrane tissue needs to be dehydrated to a specific water content at a specific rate, and then the dehydration rate is reduced to perform a specific secondary dehydration to ensure the transparency effect.

[0019] In the method of the present invention, the water content of the animal membrane tissue after decellularization is reduced by vacuum centrifugation.

[0020] Those skilled in the art can select the vacuum centrifugation conditions according to common knowledge in the art, as long as the moisture change limit in each stage of the present invention can be achieved, and the present invention does not impose any particular limitation on this.

[0021] As a specific implementation example, in the method of the present invention, the transparency processing includes:

[0022] (1) First stage: centrifuge for 120-180 minutes at a vacuum of 40-50 kPa, a temperature of 10-35°C, and a speed of 2000 r / min;

[0023] (2) Second stage: centrifuge for 180-240 minutes at a vacuum of 50-60 kPa, a temperature of 10-35°C, and a speed of 2000 r / min;

[0024] The vacuum value of the first stage is smaller than that of the second stage.

[0025] Preferably, the transparency treatment includes:

[0026] (1) First stage: centrifuge for 120 min at a vacuum of 50 kPa, a temperature of 25°C, and a speed of 2000 r / min;

[0027] (2) Second stage: centrifuge for 180 min at a vacuum of 60 kPa, a temperature of 25°C, and a rotation speed of 2000 r / min.

[0028] In the method of the present invention, the preparation method of the decellularized animal membrane tissue includes the steps of washing the animal membrane tissue, inactivating (killing bacteria, viruses and other microorganisms), removing the muscle layer and the serosal layer, secondary washing, defatting, decellularizing, and washing again.

[0029] The preparation of the decellularized animal membrane tissue of the present invention can be carried out by conventional methods in the art, as long as the pathogenic substances, fat and other cells in the animal tissue can be fully removed (for example, the residual fat content is less than or equal to 2% (dry basis), and the remaining cells should have no complete cell nucleus), and the present invention does not impose any special restrictions.

[0030] As a specific example, pathogens can be inactivated by soaking in peracetic acid (eg, at a concentration of 3-3.2%); degreasing can be performed by using a mixed solvent of methanol and chloroform (eg, at a volume ratio of 1:1); and decellularization can be performed by treating with acetic acid.

[0031] Preferably, the animal membrane tissue is small intestine, animal pericardium or animal bladder; and the animal membrane tissue is from cow or pig.

[0032] In the method of the present invention, a drying step is further included after the transparent treatment step; preferably, the drying method is freeze-drying.

[0033] A person skilled in the art may dry the transparent film by conventional methods in the art, preferably by freeze-drying. The specific freeze-drying conditions may adopt conventional procedures in the art, and the present invention does not impose any particular limitation on this.

[0034] As a specific example, the freeze-drying process of the present invention is:

[0035] Precool from room temperature to -40~-50℃ within 58~62min and maintain for 0.8-1min, then under vacuum, then heat to -35~-40℃ within 28~32min and maintain for 0.5-0.8min, then heat to -25~-30℃ within 28~32min and maintain for 0.5-0.8min, then heat to -18~-22℃ within 28~32min and maintain for 0.5-0.8min, then heat to -5~-10℃ within 28~32min and maintain for 0.5-0.8 min, then heating to -2~2°C within 18~22min and maintaining for 0.5-0.8min, then heating to 10~15°C within 18~22min and maintaining for 0.5-0.8min, then heating to 20~25°C within 8~12min and maintaining for 0.5-0.8min, finally heating to 25~35°C within 238~242min and maintaining for 0.5-0.8min; or, the freeze-drying process is: first freezing at -18~-22°C for 22-24h; then vacuum drying at a vacuum degree of 58-62 kpa and a temperature of -48~-52°C for 18-22h.

[0036] In the present invention, "room temperature" refers to 18±28°C.

[0037] The present invention also provides the use of the above-mentioned film or method in preparing a biological patch. Preferably, the biological patch is a corneal repair patch, a skin repair patch, a gum repair patch or an abdominal repair patch.

[0038] The present invention further provides a biological patch, which comprises the above-mentioned biological membrane or the biological membrane prepared by the above-mentioned method.

[0039] The beneficial effects of the present invention are at least:

[0040] The biofilm of the present invention has low moisture content, is easy to preserve, has high transparency, has good biocompatibility and excellent mechanical properties, is degradable, can be used in the field of corneal repair or other biological patch materials with high transparency requirements, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is the transparent state of the transparent decellularized biomaterial prepared in Example 1.

[0042] Figure 2 This is the transparent state of the transparent decellularized biomaterial prepared in Comparative Example 1.

[0043] Figure 3 The transmittance results of the decellularized materials prepared in various examples and comparative examples are shown.

[0044] Figure 4The suture strength results of the decellularized materials prepared in various examples and comparative examples are shown.

[0045] Figure 5 The water content results of the decellularized materials prepared in various examples and comparative examples are shown in FIG. DETAILED DESCRIPTION

[0046] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. It should be understood that the following examples are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0047] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available or prepared according to conventional methods in the art unless otherwise specified.

[0048] Example 1

[0049] This embodiment uses pig small intestine as a preparation material to provide a membrane (transparent decellularized biomaterial) and a preparation method thereof. The details are as follows:

[0050] 1. Preprocessing:

[0051] 1.1 Cleaning: Wash fresh pig small intestine repeatedly with purified water.

[0052] 1.2 Inactivation: Soak the cleaned pig small intestine in 3.2% peracetic acid solution for 60 minutes.

[0053] 1.3 Mechanical treatment: Clean the soaked materials and use mechanical methods to preliminarily remove the muscle layer and serosal layer of the materials.

[0054] 2. Antigen removal:

[0055] 2.1 Ultrasonic cleaning: Place the pretreated material in a container filled with purified water and clean it with ultrasound for 20 minutes (ultrasonic power 300W).

[0056] 2.2 Degreasing: Place the cleaned material in an oscillator and degrease it with methanol and chloroform in a volume ratio of 1:1.

[0057] 2.3 Decellularization: The defatted material was placed in acetic acid for 4 h for decellularization.

[0058] 2.4 Cleaning: Place the decellularized material in a container filled with purified water, clean it with ultrasound for 20 minutes (ultrasonic power 300W), and then clean it with water for injection to obtain the decellularized small intestine fresh film.

[0059] 3. Transparency:

[0060] The decellularized small intestinal matrix fresh film was squeezed until no water was dripped, placed in a vacuum centrifuge, and vacuum centrifuged according to the process described in Table 1 to obtain a single-layer transparent decellularized matrix membrane sheet.

[0061] Table 1 Transparency process

[0062]

[0063] 4. Freeze drying:

[0064] The transparent acellular matrix film was freeze-dried using the freeze-drying conditions in Table 2, and finally a transparent acellular biomaterial was obtained, wherein the residual fat content was ≤2% (dry basis percentage), and the residual cells had no complete cell nucleus, as shown in the photo. Figure 1 As shown, the transparency is 95.63%, the suture strength is 2.48N, and the water content is 5.51%.

[0065] Table 2 Freeze drying process

[0066]

[0067] Example 2

[0068] This embodiment provides a transparent decellularized biomaterial and a preparation method thereof, which is substantially the same as the preparation method of Example 1, except that the transparent treatment is performed using the process described in Table 3 in step 3. Freeze drying is performed using the process described in Table 4 in step 4. The transparency of the finally obtained transparent decellularized biomaterial is 96.72%, the suture strength is 2.67N, and the water content is 5.60%.

[0069] Table 3 Transparency process

[0070]

[0071] Table 4 Freeze drying process

[0072]

[0073] Example 3

[0074] This embodiment provides a transparent decellularized biomaterial and a preparation method thereof, which is basically the same as the preparation method of Example 1, except that step 3 adopts the process described in Table 5 for transparent treatment. The freeze-drying method of step 4 is: a. Freeze the material at -20°C for 24 hours; b. Set the vacuum degree to 60 kpa, the temperature to -50°C, and vacuum dry for 20 hours. The transparency of the finally obtained transparent decellularized biomaterial is 95.85%, the suture strength is 2.60N, and the water content is 6.20%.

[0075] Table 5 Transparency process

[0076]

[0077] Example 4

[0078] This embodiment provides a transparent decellularized biomaterial and a preparation method thereof, which is substantially the same as the preparation method of Example 1, except that step 3 uses the process described in Table 6 for transparent treatment. Then, step 4 in Example 1 is continued, and the transparent decellularized biomaterial finally obtained has a transparency of 96.14%, a suture strength of 2.57N, and a water content of 6.50%.

[0079] Table 6 Transparency process

[0080]

[0081] Example 5

[0082] This embodiment provides a transparent decellularized biomaterial and a preparation method thereof, which is substantially the same as the preparation method of Example 1, except that step 3 uses the process described in Table 7 for transparent treatment. Then, step 4 in Example 1 is continued, and the transparent decellularized biomaterial finally obtained has a transparency of 95.40%, a suture strength of 2.51N, and a water content of 5.75%.

[0083] Table 7 Transparency process

[0084]

[0085] Example 6

[0086] This embodiment provides a transparent decellularized biomaterial and a preparation method thereof, which is substantially the same as the preparation method of Example 1, except that step 3 uses the process described in Table 8 for transparent treatment. Then, step 4 in Example 1 is continued, and the transparent decellularized biomaterial finally obtained has a transparency of 96.52%, a suture strength of 2.55N, and a water content of 5.81%.

[0087] Table 8 Transparency process

[0088]

[0089] Comparative Example 1

[0090] This comparative example provides a decellularized biomaterial and a preparation method thereof, which is substantially the same as the preparation method of Example 1, except that the transparentization process of step 3 is omitted and the subsequent freeze-drying process is directly performed. The photo of the decellularized biomaterial finally obtained is shown in Figure 2, transparency is 43.11%, suture strength is 1.51N, and water content is 22.70%.

[0091] The transparency and suture strength test results of this comparative example are significantly lower than those of the embodiments, indicating that the decellularized biomaterial that has not been subjected to the transparent step cannot achieve a transparent effect.

[0092] Comparative Example 2

[0093] This comparative example provides a decellularized biomaterial and a preparation method thereof, which is substantially the same as the preparation method of Example 1, except that step 3 adopts the process described in Table 9 for transparent treatment. Then, step 4 in Example 1 is continued, and the transparentized decellularized biomaterial finally obtained has a transparency of 82.27%, a suture strength of 1.99N, and a water content of 6.19%.

[0094] Table 9 Transparency process

[0095]

[0096] Compared with Example 1, the transparency and suture strength test results of this comparative example decreased, indicating that the moisture content in the first stage of the transparent process was not reduced enough (the moisture content was higher than 35.5%), which would eventually affect the transparency and suture strength of the transparent decellularized biomaterial.

[0097] Comparative Example 3

[0098] This comparative example provides a decellularized biomaterial and a preparation method thereof, which is substantially the same as the preparation method of Example 1, except that step 3 adopts the process described in Table 10 for transparent treatment. Then, step 4 in Example 1 is continued, and the finally obtained transparentized decellularized biomaterial has a transparency of 85.42%, a suture strength of 2.13N, and a water content of 6.52%.

[0099] Table 10 Transparency Process

[0100]

[0101] Compared with Example 1, the transparency and suture strength test results of this comparative example decreased, indicating that the moisture content in the first stage of the transparent process decreased too low (the moisture content was less than 30%), which would eventually affect the transparency and suture strength of the transparent decellularized biomaterial.

[0102] Comparative Example 4

[0103] This comparative example provides a decellularized biomaterial and a preparation method thereof, which is substantially the same as the preparation method of Example 1, except that step 3 adopts the process described in Table 11 for transparent treatment. Then, step 4 in Example 1 is continued, and the transparentized decellularized biomaterial finally obtained has a transparency of 79.83%, a suture strength of 2.08N, and a water content of 10.15%.

[0104] Table 11 Transparency Process

[0105]

[0106] Compared with Example 1, the transparency and suture strength of this comparative example decreased, indicating that the moisture content in the second stage of the transparent process was not reduced enough (the moisture content was higher than 13%), which would eventually affect the transparency and suture strength of the transparent decellularized biomaterial.

[0107] Comparative Example 5

[0108] This comparative example provides a decellularized biomaterial and a preparation method thereof, which is substantially the same as the preparation method of Example 1, except that step 3 adopts the process described in Table 12 for transparent treatment. Then, step 4 in Example 1 is continued, and the finally obtained transparentized decellularized biomaterial has a transparency of 86.71%, a suture strength of 2.11N, and a water content of 5.63%.

[0109] Table 12 Transparency Process

[0110]

[0111] Compared with Example 1, the transparency and suture strength of this comparative example decreased, indicating that the moisture content in the second stage of the transparent process decreased too low (the moisture content was lower than 10.5%), which would eventually affect the transparency and suture strength of the transparent decellularized biomaterial.

[0112] Comparative Example 6

[0113] This comparative example provides a decellularized biomaterial and a preparation method thereof, which is substantially the same as the preparation method of Example 1, except that step 3 adopts the process described in Table 13 for transparent treatment. Then, step 4 in Example 1 is continued, and the transparentized decellularized biomaterial finally obtained has a transparency of 86.92%, a suture strength of 2.05N, and a water content of 5.42%.

[0114] Table 13 Transparency Process

[0115]

[0116] Compared with Example 1, the transparency and suture strength of this comparative example decreased, indicating that the rate of water reduction in the first stage of the transparent process was too fast (higher than 0.45% / min), which would eventually affect the transparency and suture strength of the transparent decellularized biomaterial.

[0117] Comparative Example 7

[0118] This comparative example provides a decellularized biomaterial and a preparation method thereof, which is substantially the same as the preparation method of Example 1, except that step 3 adopts the process described in Table 14 for transparent treatment. Then, step 4 in Example 1 is continued, and the transparentized decellularized biomaterial finally obtained has a transparency of 74.66%, a suture strength of 1.96N, and a water content of 12.64%.

[0119] Table 14 Transparency Process

[0120]

[0121] Compared with Example 1, the transparency and suture strength of this comparative example decreased, indicating that the rate of water reduction in the first stage of the transparent process was too slow (less than 0.22% / min), which would eventually affect the transparency and suture strength of the transparent decellularized biomaterial.

[0122] Comparative Example 8

[0123] This comparative example provides a decellularized biomaterial and a preparation method thereof, which is substantially the same as the preparation method of Example 1, except that step 3 adopts the process described in Table 15 for transparent treatment. Then, step 4 in Example 1 is continued, and the transparentized decellularized biomaterial finally obtained has a transparency of 85.39%, a suture strength of 1.91N, and a water content of 5.57%.

[0124] Table 15 Transparency Process

[0125]

[0126] Compared with Example 1, the transparency and suture strength of this comparative example decreased, indicating that the rate of water reduction in the second stage of the transparent process was too fast (higher than 0.15% / min), which would eventually affect the transparency and suture strength of the transparent decellularized biomaterial.

[0127] Comparative Example 9

[0128] This comparative example provides a decellularized biomaterial and a preparation method thereof, which is substantially the same as the preparation method of Example 1, except that step 3 adopts the process described in Table 16 for transparent treatment. Then, step 4 in Example 1 is continued, and the transparentized decellularized biomaterial finally obtained has a transparency of 72.54%, a suture strength of 1.89N, and a water content of 14.21%.

[0129] Table 16 Transparency Process

[0130]

[0131] Compared with Example 1, the transparency and suture strength of this comparative example decreased, indicating that the rate of water reduction in the second stage of the transparent process was too slow (less than 0.07% / min), which would eventually affect the transparency and suture strength of the transparent decellularized biomaterial.

[0132] The test methods for transparency (i.e., light transmittance), suture strength, and moisture content of the specific embodiments of the present invention are as follows:

[0133] 1. Use a transmittance tester (model: AT-172) to test the transmittance of each sample; test the transmittance of the left, middle and right areas of each sample and take the average value.

[0134] 2. Use a universal material testing machine for mechanical characterization, that is, the detection of suture strength; specific experimental method: Use a specimen with a specification of 54×10 mm for seam strength test. Soak the sample in saline for 5 min before testing. Pass nylon 11-0 suture through and tie it into a ring at a point 2 mm from the midpoint of the short side of the sample to the center of the sample. Ensure that the length of the line segment is greater than 5 cm after the ring is straightened. Fix the suture to the upper part of the fixture and the lower end of the sample to the lower part of the fixture. Set the fixture movement speed to 50 mm / min, start the tensile machine until the sample breaks, and record the maximum tensile force that the sample withstands when it breaks, which is the suture strength. Perform three parallel experiments and take the average value of the results.

[0135] 3. Dry and test the moisture content at each stage according to the second method of moisture determination method 0832 of Part IV of the 2020 edition of the Pharmacopoeia of the People's Republic of China. Perform three parallel experiments and take the average value of the results.

[0136] The light transmittance results of each embodiment and comparative example are as follows: Figure 3 As shown in the figure, if the lowercase letters on the data are the same, it means that the difference between the data is not significant, if they are different, it means that the difference between the data is significant. The transparent decellularized biomaterial prepared according to the method provided by the present invention has significantly improved transmittance compared with the material of Comparative Example 1 without transparent treatment, and there is no significant difference in the transmittance of the transparent decellularized materials of each embodiment.

[0137] The suture strength results of each embodiment and comparative example are as follows: Figure 4As shown in the figure, if the lowercase letters on the data are the same, it means that the difference between the data is not significant, if they are different, it means that the difference between the data is significant. It can be seen that the suture strength of the transparent decellularized biomaterial prepared according to the method provided by the present invention is significantly enhanced compared with the material of Comparative Example 1 without transparent treatment, and there is no significant difference in the suture strength of the transparent decellularized materials of each embodiment.

[0138] The water content results of each embodiment and comparative example are as follows Figure 5 As shown, if the lowercase letters on the data in the figure are the same, it means that the difference between the data is not significant. If they are different, it means that the difference between the data is significant. The water content of the transparent decellularized biomaterial prepared according to the method provided by the present invention is significantly lower than that of the material of Comparative Example 1 without transparent treatment. There is no significant difference in the water content of the final transparent decellularized materials of each embodiment. The transparent decellularized biomaterial of the present invention has low water content and is easy to store.

[0139] Based on the results of the embodiments and comparative examples, the decellularized biomaterial prepared by the method of embodiment 2 has the best effect.

[0140] Experimental example

[0141] In this experimental example, the transparent decellularized materials prepared in various embodiments of the present invention were evaluated for biocompatibility according to the GB / T 16886 "Biological Evaluation of Medical Devices" series of standards. The specific items included: cytotoxicity test, skin sensitization test, intradermal reaction test, subcutaneous implantation test, pyrogen test, and subchronic systemic toxicity test.

[0142] The results show that: the transparent decellularized material of the present invention has no cytotoxic reaction; the transparent decellularized material has no skin sensitization reaction; the final scores of polar extraction and non-polar extraction of the transparent material are both less than 1.0; no abnormal signs were observed in the test animals at each time point during the implantation period of the transparent decellularized material; the transparent decellularized material has no pyrogenic reaction; the transparent decellularized material has no subchronic systemic toxicity; and the TK gene mutation test of the transparent decellularized material is negative. In summary, the transparent decellularized biomaterial prepared by the present invention has good biocompatibility.

[0143] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A biofilm, characterized in that The raw material is decellularized animal membrane tissue, the biofilm has a light transmittance of 95.4%-97%, a suture strength of 2.4N-2.7N, and a water content of 5%-7%; The method for preparing the biofilm includes the step of subjecting the decellularized animal membrane tissue to a transparent treatment, wherein the transparent treatment includes: (1) The first stage: the water content of the decellularized animal membrane tissue is reduced to 30-35.5% at a water reduction rate of 0.22% / min to 0.45% / min; (2) The second stage: the water content of the decellularized animal membrane tissue is reduced to 10.5-13% at a water reduction rate of 0.07% / min-0.15% / min.

2. A method for preparing the biofilm according to claim 1, characterized in that: The method comprises the step of subjecting the decellularized animal membrane tissue to transparent treatment, wherein the transparent treatment comprises: (1) The first stage: the water content of the decellularized animal membrane tissue is reduced to 30-35.5% at a water reduction rate of 0.22% / min to 0.45% / min; (2) The second stage: the water content of the decellularized animal membrane tissue is reduced to 10.5-13% at a water reduction rate of 0.07% / min-0.15% / min.

3. The method according to claim 2, characterized in that The transparency processing includes: (1) The first stage: the water content of the decellularized animal membrane tissue is reduced to 30-35.1% at a water reduction rate of 0.24% / min to 0.43% / min; (2) The second stage: the water content of the decellularized animal membrane tissue is reduced to 10.9-12.8% at a water reduction rate of 0.08% / min-0.13% / min.

4. The method according to claim 2 or 3, characterized in that: The water content of animal membrane tissue after decellularization can be reduced by vacuum centrifugation.

5. The method according to claim 4, characterized in that The transparency processing includes: (1) Centrifuge for 120-180 minutes at a vacuum degree of 40-50 kPa, a temperature of 10-35°C, and a speed of 2000 r / min; (2) centrifugation for 180-240 minutes at a vacuum degree of 50-60 kPa, a temperature of 10-35° C., and a rotation speed of 2000 r / min; The vacuum value of the first stage is smaller than that of the second stage.

6. The method according to claim 5, characterized in that The transparency processing includes: (1) Centrifuge for 120 min at a vacuum of 50 kPa, a temperature of 25°C, and a speed of 2000 r / min; (2) Centrifuge for 180 min at a vacuum of 60 kPa, a temperature of 25°C, and a rotation speed of 2000 r / min.

7. The method according to any one of claims 2-3, 5-6, characterized in that: The method for preparing the decellularized animal membrane tissue comprises the steps of washing the animal membrane tissue, inactivating the animal membrane tissue, removing the muscle layer and the serosal layer, washing the animal membrane tissue for the second time, defatting, decellularizing, and washing the animal membrane tissue again.

8. The method according to claim 4, characterized in that The method for preparing the decellularized animal membrane tissue comprises the steps of washing the animal membrane tissue, inactivating the animal membrane tissue, removing the muscle layer and the serosal layer, washing the animal membrane tissue for the second time, defatting, decellularizing, and washing the animal membrane tissue again.

9. The method according to claim 7, characterized in that: The animal membrane tissue is small intestine, animal pericardium or animal bladder; the animal membrane tissue is from cow or pig.

10. The method according to claim 8, characterized in that The animal membrane tissue is small intestine, animal pericardium or animal bladder; the animal membrane tissue is from cow or pig.

11. The method according to any one of claims 2-3, 5-6, 8-10, characterized in that: A drying step is also included after the transparent treatment step.

12. The method according to claim 4, characterized in that A drying step is also included after the transparent treatment step.

13. The method according to claim 7, characterized in that A drying step is also included after the transparent treatment step.

14. The method according to claim 11, characterized in that The drying method is freeze drying.

15. The method according to claim 12 or 13, characterized in that The drying method is freeze drying.

16. The method according to claim 14, characterized in that The freeze-drying process is as follows: pre-cooling from room temperature to -40--50°C within 58-62 minutes and maintaining for 0.8-1 minute, then heating to -35--40°C within 28-32 minutes and maintaining for 0.5-0.8 minutes, then heating to -25--30°C within 28-32 minutes and maintaining for 0.5-0.8 minutes, then heating to -18--22°C within 28-32 minutes and maintaining for 0.5-0.8 minutes, then heating to -18--22°C within 28-32 minutes and maintaining for 0.5-0.8 minutes, then heating to -25--30 ...25--30°C within 28-32 minutes and maintaining for 0.5-0.8 minutes, then heating to -25--30°C within 28-32 minutes and maintaining for 0.5-0.8 minutes, then heating to -25--30°C within 28-32 minutes and maintaining for 0.5-0.8 minutes, then heating The temperature is raised to -5 to -10°C within 8 to 32 minutes and maintained for 0.5 to 0.8 minutes. Then, the temperature is raised to -2 to 2°C within 18 to 22 minutes and maintained for 0.5 to 0.8 minutes. Then, the temperature is raised to 10 to 15°C within 18 to 22 minutes and maintained for 0.5 to 0.8 minutes. Then, the temperature is raised to 20 to 25°C within 8 to 12 minutes and maintained for 0.5 to 0.8 minutes. Finally, the temperature is raised to 25 to 35°C within 238 to 242 minutes and maintained for 0.5 to 0.8 minutes. Alternatively, the freeze-drying process is: first freezing at -18 to -22°C for 22-24 hours; then vacuum drying at a vacuum degree of 58-62 kPa and a temperature of -48 to -52°C for 18-22 hours.

17. The method according to claim 15, characterized in that The freeze-drying process is as follows: pre-cooling from room temperature to -40--50°C within 58-62 minutes and maintaining for 0.8-1 minute, then heating to -35--40°C within 28-32 minutes and maintaining for 0.5-0.8 minutes, then heating to -25--30°C within 28-32 minutes and maintaining for 0.5-0.8 minutes, then heating to -18--22°C within 28-32 minutes and maintaining for 0.5-0.8 minutes, then heating to -18--22°C within 28-32 minutes and maintaining for 0.5-0.8 minutes, then heating to -25--30 ...25--30°C within 28-32 minutes and maintaining for 0.5-0.8 minutes, then heating to -25--30°C within 28-32 minutes and maintaining for 0.5-0.8 minutes, then heating to -25--30°C within 28-32 minutes and maintaining for 0.5-0.8 minutes, then heating The temperature is raised to -5 to -10°C within 8 to 32 minutes and maintained for 0.5 to 0.8 minutes. Then, the temperature is raised to -2 to 2°C within 18 to 22 minutes and maintained for 0.5 to 0.8 minutes. Then, the temperature is raised to 10 to 15°C within 18 to 22 minutes and maintained for 0.5 to 0.8 minutes. Then, the temperature is raised to 20 to 25°C within 8 to 12 minutes and maintained for 0.5 to 0.8 minutes. Finally, the temperature is raised to 25 to 35°C within 238 to 242 minutes and maintained for 0.5 to 0.8 minutes. Alternatively, the freeze-drying process is: first freezing at -18 to -22°C for 22-24 hours; then vacuum drying at a vacuum degree of 58-62 kPa and a temperature of -48 to -52°C for 18-22 hours.

18. Use of the biofilm according to claim 1 or the method according to any one of claims 2 to 17 in preparing a biological patch.

19. The method according to claim 18, characterized in that The biological patch is a cornea repair patch, a skin repair patch, a gum repair patch or an abdominal cavity repair patch.

20. A biological patch, characterized in that: The invention comprises the biofilm according to claim 1 or the biofilm prepared by the method according to any one of claims 2 to 17.

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