A transparent acellular biological material and its preparation method and application

Abstract

The present invention relates to the technical field of biomaterials, and specifically discloses a transparent acellular biomaterial, a preparation method thereof and an application. The transparent acellular biomaterial provided by the present invention is prepared from animal membrane tissue, and the thickness of the transparent acellular biomaterial is 1-500 μm; the light transmittance is 89.10-95.05%, the breaking strength is 6.21-50.95 N, and the suture strength is 2.24-25.60 N. The biomaterial of the present invention has high transparency and good strength, and can be used in the field of biological patch materials with higher requirements for transparency, and has broad application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular, to a transparent acellular biomaterial, a preparation method thereof, and an application thereof. Background Art

[0002] Tissues are composed of cells and extracellular matrix (ECM). ECM can timely reflect changes in the microenvironment and plays an important role in cell migration, differentiation, and proliferation. It can not only act as a scaffold material but also contain various growth factors that play an important promoting role in tissue repair and reconstruction. With the development of tissue engineering technology and the in-depth study of the endogenous regeneration mechanism of biomaterials, it is found that when tissues are damaged, covering them with allogeneic or xenogeneic cell matrix materials can promote the adhesion, proliferation, and growth of cells at the wound site on the material surface, thereby guiding tissue regeneration and promoting the rapid recovery of wounds and blood vessels. Therefore, acellular matrix materials are widely used in the field of biological patches, such as hernia repair patches, pericardial patches, etc.

[0003] With the improvement of living and medical standards, people will also take into account the impact of treatment methods or instruments on nature and aesthetics while paying attention to the curative effect. However, when using acellular matrix materials to treat and repair tissues or organs, the materials will be covered on the wound or tissue site, and the appearance of the materials may greatly affect the aesthetic degree, especially in the treatment of chronic skin wounds and gum repair. To reduce this impact, it is more suitable to choose transparent repair materials. In addition, in some treatment scenarios such as corneal repair, whether the material is transparent or the transparency level will be a key technical indicator, which will have a great impact on the patient's life during the treatment.

[0004] The light transmittance, mechanical properties, and other properties of acellular matrix materials are sometimes also crucial for wound repair. The higher the transparency, the easier it is to observe the wound recovery situation through the acellular matrix material; in addition, the light transmittance and mechanics of acellular matrix materials are also the key to corneal transparency recovery. Higher transparency and good mechanical properties are more conducive to the cornea returning to a colorless and transparent state and achieving blindness recovery. Therefore, an acellular matrix material with good mechanical properties, high transparency, degradability, and good biocompatibility is needed.

[0005] When preparing acellular matrix materials, the commonly used decellularization methods are divided into physical, chemical and biological methods, such as physical repeated freezing and thawing, physical swelling, chemical decellularization, enzymatic hydrolysis, etc. For example, CN10322722775A discloses a method for preparing a dural biological repair patch, including separating the small intestinal submucosa, virus inactivation and decellularization; CN116942913A discloses an acellular biological material, its preparation method and application, including pretreatment of the small intestinal submucosa, swelling treatment, cold dissolution treatment, oxidase treatment and surfactant treatment, and finally freeze-drying. However, the acellular materials prepared by general traditional processes are white or slightly yellow membranes with poor transparency, which are not convenient for use in cases with higher transparency requirements or aesthetic requirements. Therefore, it is necessary to further study the preparation of acellular matrix materials. Summary of the Invention

[0006] One of the purposes of the present invention is to provide a highly transparent and strong acellular biological material, its preparation method and application.

[0007] To achieve this purpose, the technical solution of the present invention is as follows:

[0008] A transparent acellular biological material, which is obtained by preparing from animal membrane tissue. The thickness of the transparent acellular biological material is 1-500 μm; the light transmittance is 89.10-95.05%, the breaking strength is 6.21-50.95 N, and the suture strength is 2.24-25.60 N.

[0009] Preferably, the thickness of the transparent acellular biological material is 1-400 μm, and more preferably 2-200 μm.

[0010] The present invention has obtained a new type of transparent acellular biological material with good physical properties and high transparency. When using this acellular matrix material to treat and repair tissues or organs, the wound surface can be better observed, and the applicability is wider.

[0011] The preparation method of the transparent acellular biological material of the present invention includes the step of performing a transparency treatment on the decellularized animal membrane tissue. The method of the transparency treatment is as follows: within 58-62 min, cool the animal membrane tissue from room temperature to -32 to -18 °C for pre-cooling and maintain for 28-32 min, then heat it to -17 to -8 °C within 88-92 min and maintain for 28-32 min, then heat it to -2 to 2 °C within 58-62 min and maintain for 178-182 min, then heat it to 3-12 °C within 58-62 min and maintain for 28-32 min, continue to heat it to 13-22 °C within 58-62 min and maintain for 28-32 min, and finally heat it to 18-27 °C within 478-482 min and maintain for 28-32 min.

[0012] The research of the present invention has found that when preparing acellular biomaterials, by treating the acellular animal membrane tissue in a specific gradient dehydration manner, a biomaterial with good mechanical properties, high transparency, degradability, and good biocompatibility can be obtained.

[0013] Preferably, the method of the transparency treatment is as follows: within 60 minutes, the animal membrane tissue is cooled from room temperature to -20°C for pre-cooling and maintained for 30 minutes, then heated to -10°C within 90 minutes and maintained for 30 minutes, then heated to 0°C within 60 minutes and maintained for 180 minutes, then heated to 10°C within 60 minutes and maintained for 30 minutes, then heated to 20°C within 60 minutes and maintained for 30 minutes, and finally heated to 25°C within 480 minutes and maintained for 30 minutes; or,

[0014] the method of the transparency treatment is as follows: within 60 minutes, the animal membrane tissue is cooled from room temperature to -30°C for pre-cooling and maintained for 30 minutes, then heated to -15°C within 90 minutes and maintained for 30 minutes, then heated to 0°C within 60 minutes and maintained for 180 minutes, then heated to 5°C within 60 minutes and maintained for 30 minutes, then heated to 15°C within 60 minutes and maintained for 30 minutes, and finally heated to 20°C within 480 minutes and maintained for 30 minutes.

[0015] After the transparency treatment of the acellular animal membrane tissue in the present invention, a drying step is further included, and preferably the drying is freeze-drying.

[0016] The method of freeze-drying in the present invention can adopt the conventional methods in the art and is not particularly limited herein.

[0017] As a specific embodiment, the process of freeze-drying is as follows: cool down from room temperature to -52 to -48 °C within 58 - 62 minutes and maintain for 0.7 - 0.9 minutes, then under vacuum, then heat up to -38 to -42 °C within 28 - 32 minutes and maintain for 0.45 - 0.55 minutes, then heat up to -28 to -32 °C within 28 - 32 minutes and maintain for 0.45 - 0.55 minutes, then heat up to -18 to -22 °C within 28 - 32 minutes and maintain for 0.45 - 0.55 minutes, then heat up to -8 to -12 °C within 28 - 32 minutes and maintain for 0.45 - 0.55 minutes, then heat up to -2 to 2 °C within 18 - 22 minutes and maintain for 0.45 - 0.55 minutes, then heat up to 8 to 12 °C within 18 - 22 minutes and maintain for 0.45 - 0.55 minutes, then heat up to 18 to 22 °C within 8 - 12 minutes and maintain for 0.45 - 0.55 minutes, and finally heat up to 23 to 27 °C within 238 - 242 minutes and maintain for 0.45 - 0.55 minutes;

[0018] Preferably, the process of freeze-drying is as follows: cool down from room temperature to -50 °C within 60 minutes and maintain for 0.8 minutes, then under vacuum, then heat up to -40 °C within 30 minutes and maintain for 0.5 minutes, then heat up to -30 °C within 30 minutes and maintain for 0.5 minutes, then heat up to -20 °C within 30 minutes and maintain for 0.5 minutes, then heat up to -10 °C within 30 minutes and maintain for 0.5 minutes, then heat up to 0 °C within 20 minutes and maintain for 0.5 minutes, then heat up to 10 °C within 20 minutes and maintain for 0.5 minutes, then heat up to 20 °C within 10 minutes and maintain for 0.5 minutes, and finally heat up to 25 °C within 240 minutes and maintain for 0.5 minutes.

[0019] In the present invention, "room temperature" refers to 18 ± 28 °C.

[0020] In the present invention, the transparent acellular biomaterial is prepared from one or more layers of acellular animal membrane tissues, preferably 1 - 10 layers;

[0021] And / or, the animal membrane tissue is prepared from animal tissues, and the animal tissues are animal small intestine, animal pericardium or animal bladder; the animal is cattle or pigs.

[0022] Preferably, the preparation method of the acellular animal membrane tissue includes:

[0023] (1) Pretreatment: After washing and inactivating the animal tissue, remove the muscular layer and serous layer;

[0024] (2) Antigen removal: The pretreated material is washed, degreased, decellularized, and washed again to obtain animal membrane tissue.

[0025] The preparation of the decellularized animal membrane tissue of the present invention can be carried out in a conventional manner in the art, as long as pathogenic substances, fats and other cells in the animal tissue can be sufficiently removed (for example, the residual fat content in the final product is less than 2% (dry basis), and the remaining cells should have no intact cell nuclei), and the present invention does not make special restrictions.

[0026] As a specific embodiment, the present invention can inactivate pathogenic substances by soaking with peracetic acid (such as a concentration of 2.9-3.2%); can degrease with a mixed solvent of methanol and chloroform (such as a volume ratio of 1:1); can decellularize by using an EDTA-trypsin mixed solution or acetic acid treatment.

[0027] In the present invention, when the transparent decellularized biomaterial is prepared from multiple layers of decellularized animal membrane tissue, before the transparentization treatment, it further includes a step of superposing and laminating the multiple layers of decellularized animal membrane tissue; preferably, the lamination of the multiple layers of decellularized animal membrane tissue is achieved by applying pressure to the multiple layers of decellularized animal membrane tissue that are unfolded, overlapped, and the air between layers is discharged. More preferably, the pressure is 3-12 N, and the duration of the continuous pressure is 0.5 min-8 min. Further preferably, the pressure is 5-10 N, and the duration is 1 min-5 min.

[0028] When laminating multiple layers of decellularized animal membrane tissue in the present invention, specifically, the antigen-removed membrane sheets can be spread out, the air in the unlaminated part is discharged, and then they are stacked layer by layer and the air in the unlaminated part is discharged, and then pressure is applied for lamination.

[0029] The present invention also provides a preparation method of the above-mentioned transparent decellularized biomaterial, and the preparation method is as described above.

[0030] The present invention further provides the application of the above-mentioned transparent decellularized biomaterial or preparation method in the preparation of a biological patch. Preferably, the biological patch is a corneal repair patch, a skin repair patch, an abdominal cavity repair patch or a dura mater repair patch.

[0031] The present invention further provides a biological patch, which comprises the above-mentioned transparent decellularized biomaterial.

[0032] The beneficial effects of the present invention are at least as follows:

[0033] The present invention provides a transparent acellular biomaterial which is easy to preserve, highly transparent, has good biocompatibility, can well support cell adhesion, proliferation and differentiation; has excellent biomechanical properties, low immunogenicity, and will not cause obvious postoperative immune rejection reactions after allogeneic transplantation; and has degradability. This transparent acellular biomaterial can be used in the field of biological patch materials with high requirements for transparency, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Transparent state of the transparent acellular biomaterial prepared in Example 1.

[0035] Figure 2 Transparent state of the transparent acellular biomaterial prepared in Example 2.

[0036] Figure 3 Transparent state of the transparent acellular biomaterial prepared in Example 3.

[0037] Figure 4 Transparent state of the transparent acellular biomaterial prepared in Example 4.

[0038] Figure 5 Transparent state of the transparent acellular biomaterial prepared in Example 5.

[0039] Figure 6 Transparent state of the transparent acellular biomaterial prepared in Comparative Example 1.

[0040] Figure 7 Transparent state of the transparent acellular biomaterial prepared in Comparative Example 2.

[0041] Figure 8 Transparent state of the transparent acellular biomaterial prepared in Comparative Example 3.

[0042] Figure 9 Transparent state of the opaque acellular biomaterial prepared in Comparative Example 4.

[0043] Figure 10 Transmittance results of the acellular materials prepared in each example and comparative example.

[0044] Figure 11 Tensile strength results of the acellular materials prepared in each example and comparative example.

[0045] Figure 12 Suture strength results of the acellular materials prepared in each example and comparative example. DETAILED DESCRIPTION OF THE INVENTION

[0046] The preferred embodiments of the present invention will be described in detail below in conjunction with the embodiments. According to the content of the present invention, other various forms of modifications, substitutions or changes can be made without departing from the basic technical idea of the present invention according to the common general knowledge and conventional means in the art. It should be understood that the following embodiments are given only for the purpose of illustration and are not used to limit the scope of the present invention. Without departing from the purpose, principle and spirit of the present invention, the changes, modifications, combinations, substitutions and simplifications made by those skilled in the art should all be equivalent substitution methods and are included in the protection scope of the present invention.

[0047] Unless otherwise specified, the experimental methods used in the following embodiments are all conventional methods. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources or prepared by conventional methods in the art unless otherwise specified.

[0048] Example 1

[0049] In this example, taking the bladder as an example, a transparent acellular biomaterial and its preparation method are provided. The preparation process of the transparent acellular biomaterial includes pretreatment, antigen removal, transparency and freeze-drying steps. Specifically as follows:

[0050] 1) Pretreatment:

[0051] 1.1 Cleaning: Repeatedly clean the fresh porcine bladder with purified water.

[0052] 1.2 Inactivation: Immerse the cleaned bladder in 3% peracetic acid for 60 min.

[0053] 1.3 Manual treatment: Clean the soaked material and manually remove the muscular layer and serous layer of the material initially.

[0054] 2) Antigen removal:

[0055] 2.1 Ultrasonic cleaning: Put the pretreated material into a container filled with purified water and clean it ultrasonically for 20 min (ultrasonic power 300 W).

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

[0057] 2.3 Decellularization: Place the degreased material in an EDTA-trypsin mixed solution for 4 h for decellularization.

[0058] 2.4 Cleaning: Put the decellularized material into a container filled with purified water, clean it ultrasonically for 20 min (ultrasonic power 300 W), and then clean it with injection water to obtain a fresh acellular bladder matrix membrane.

[0059] 3) Transparency:

[0060] 3.1 Diaphragm spreading: Evenly spread a layer of fresh acellular bladder matrix membrane, and expel the air in the non - adhered part.

[0061] 3.2 Transparency: Achieve transparency through gradient dehydration to obtain a single - layer transparent acellular bladder matrix. The transparency process is shown in Table 1.

[0062] Table 1 Transparency parameters

[0063]

[0064] 4) Freeze - drying: Perform freeze - drying on the transparent acellular matrix membrane using the freeze - drying conditions in Table 2, and finally obtain a transparent acellular biomaterial as Figure 1 shown, with a thickness of 3μm. Among them, the residual fat content is 2% (dry - basis ratio), and the remaining cells have no intact cell nuclei.

[0065] Table 2 Freeze - drying parameters

[0066]

[0067] Example 2

[0068] This example takes small intestinal submucosa (SIS) as an example to provide a transparent acellular biomaterial and its preparation method.

[0069] The preparation process of the transparent acellular biomaterial includes pre - treatment, antigen removal, transparency, and freeze - drying steps. Specifically as follows:

[0070] 1) Pre - treatment:

[0071] 1.1 Cleaning: Repeatedly clean fresh porcine small intestine with purified water.

[0072] 1.2 Inactivation: Immerse the cleaned porcine small intestine in 3% peracetic acid for 60 min.

[0073] 1.3 Mechanical treatment: Clean the soaked material and preliminarily remove the muscular layer and serosa layer of the material by mechanical means.

[0074] 2) Antigen removal:

[0075] 2.1 Ultrasonic cleaning: Put the pre - treated material into a container filled with purified water and perform ultrasonic cleaning for 20 min (ultrasonic power 300W).

[0076] 2.2 Degreasing: Place the cleaned material in an oscillator and degrease it with an organic solvent. The organic solvent is methanol and chloroform with a volume ratio of 1:1.

[0077] 2.3 Decellularization: The degreased material was placed in acetic acid for 4 h for decellularization.

[0078] 2.4 Cleaning: The decellularized material was put into a container filled with purified water and ultrasonically cleaned for 20 min (ultrasonic power 300 W), and then cleaned with water for injection to obtain a fresh decellularized small intestinal submucosa membrane.

[0079] 3) Clearing:

[0080] 3.1 Membrane spreading: A layer of fresh decellularized small intestinal submucosa membrane was evenly spread out to expel the air in the unbonded part.

[0081] 3.2 Clearing: Clearing was achieved through gradient dehydration to obtain a single-layer cleared decellularized matrix membrane. The clearing process is shown in Table 3.

[0082] Table 3 Clearing parameters

[0083]

[0084] 4) Freeze-drying: The cleared decellularized matrix membrane was freeze-dried under the freeze-drying conditions shown in Table 4, and finally a cleared decellularized biomaterial was obtained as Figure 2 shown, with a thickness of 2 μm. Among them, the residual fat content was 2% (dry basis ratio), and the remaining cells had no intact cell nuclei.

[0085] Table 4 Freeze-drying parameters

[0086]

[0087] Example 3

[0088] This example provides a method for preparing a cleared decellularized biomaterial, which is basically the same as the preparation method in Example 2, except that in step 3), the clearing treatment was carried out using the clearing parameters shown in Table 5.

[0089] Table 5 Clearing parameters

[0090]

[0091] Then, step 4) in Example 2 was continued, and the finally obtained cleared decellularized biomaterial was as Figure 3 shown, with a thickness of 3 μm.

[0092] Example 4

[0093] This example provides a method for preparing a cleared decellularized biomaterial, which is basically the same as the preparation method in Example 2, except that in step 3), 2-layer materials were prepared. The specific operation of step 3) is as follows:

[0094] 3.1 Membrane spreading: Evenly spread a layer of fresh acellular small intestine matrix membrane, expel the air from the unbonded part, then lay the second layer on top and expel the air from the unbonded part.

[0095] 3.2 Multilayer formation: Place the two spread membrane sheets into a press, evenly apply a pressure of 5 N for 1 minute to achieve interlayer bonding.

[0096] 3.2 Transparency: Achieve transparency through gradient dehydration to obtain a two-layer transparent acellular matrix membrane sheet. The transparency process is shown in Table 3.

[0097] Then continue with step 4) in Example 2. The finally obtained transparent acellular biomaterial is as Figure 4 shown, with a thickness of 5 μm.

[0098] Example 5

[0099] This example provides a method for preparing a transparent acellular biomaterial, which is basically the same as the preparation method in Example 2, except that in step 3), a 10-layer material is prepared. The specific operation of step 3) is as follows:

[0100] 3.1 Membrane spreading: Evenly spread a layer of fresh acellular small intestine matrix membrane, expel the air from the unbonded part, then lay the second layer on top and expel the air from the unbonded part. Then repeat the operation until 10 layers are laid.

[0101] 3.2 Multilayer formation: Place the ten spread membrane sheets into a press, evenly apply a pressure of 10 N for 5 minutes to achieve interlayer bonding.

[0102] 3.2 Transparency: Achieve transparency through gradient dehydration to obtain a ten-layer transparent acellular matrix membrane sheet. The transparency process is shown in Table 3.

[0103] Then continue with step 4) in Example 2. The finally obtained transparent acellular biomaterial is as Figure 5 shown, with a thickness of 200 μm.

[0104] Comparative Example 1

[0105] This comparative example provides a method for preparing a transparent acellular biomaterial, which is basically the same as the preparation method in Example 2, except that in step 3), the transparency treatment is carried out using the transparency parameters shown in Table 6.

[0106] Table 6 Transparency parameters

[0107]

[0108] The obtained transparent acellular biomaterial is as Figure 6 shown, with a thickness of 5 μm.

[0109] Comparative Example 2

[0110] This comparative example provides a method for preparing a transparent decellularized biomaterial, which is basically the same as the preparation method of Example 2, except that in step 3), the transparency parameters shown in Table 7 are used for transparency treatment.

[0111] Table 7 Transparency parameters

[0112]

[0113]

[0114] The obtained transparent decellularized biomaterial is as Figure 7 shown, with a thickness of 4 μm.

[0115] Comparative Example 3

[0116] This comparative example provides a method for preparing a transparent decellularized biomaterial, which is basically the same as the preparation method of Example 2, except that in step 3), the transparency parameters shown in Table 8 are used for transparency treatment.

[0117] Table 8 Transparency parameters

[0118]

[0119] The obtained transparent decellularized biomaterial is as Figure 8 shown, with a thickness of 4 μm.

[0120] Method for preparing an opaque decellularized biomaterial in Comparative Example 4

[0121] This comparative example provides a method for preparing an opaque decellularized biomaterial, which is basically the same as the preparation method of Example 2, except that the transparency process in step 3) is removed and the freeze-drying process is directly carried out. The obtained opaque decellularized biomaterial is as Figure 9 shown, with a thickness of 6 μm.

[0122] Experimental Example

[0123] In this experimental example, the transparent decellularized matrix materials prepared in the above examples and comparative examples were detected: a transmittance tester (model: AT-172) was used for transmittance detection; a universal material testing machine was used for mechanical characterization, that is, the detection of breaking strength and suture strength.

[0124] The specific experimental methods for mechanical characterization are as follows: (1) Tensile strength: Tensile strength tests were carried out on specimens with a specification of 54×10 mm. Before the test, the samples were soaked in physiological saline for 5 min. The upper and lower ends of the samples were fixed on the fixture, and the moving speed of the gripper was set at 50 mm / min. The tensile machine was started until the sample broke, and the maximum tensile force borne by the sample at the time of breakage was recorded as the tensile strength. (2) Suture strength: Seam strength tests were carried out on specimens with a specification of 54×10 mm. Before the test, the samples were soaked in physiological saline for 5 min. At a point 2 mm from the midpoint of the short side of the sample towards the center of the sample, a nylon 11-0 suture was passed through and knotted into a ring. The length of the line segment was 5 cm after the ring was straightened. The suture was fixed to the upper part of the fixture, and the lower end of the sample was fixed to the lower part of the fixture. The moving speed of the fixture was set at 50 mm / min, and the tensile machine was started until the sample broke. The maximum tensile force borne by the sample at the time of breakage was recorded as the suture strength. The results are as Figures 10 to 12 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.

[0125] According to Figure 10 it can be seen that compared with the opaque acellular material, the transmittance of the 1-layer, 2-layer, and 10-layer transparent acellular biomaterials prepared by the method provided by the present invention is significantly improved. The transmittance of the 1-layer transparent material can reach 95.05%, the transmittance of the 2-layer transparent material can reach 92.12%, and the transmittance of the 10-layer transparent material can reach 89.10%. There is no significant difference in the transparency of the transparent acellular biomaterials in each example; according to Figure 11 it can be seen that compared with the opaque acellular material, the tensile strength of the 1-layer transparent acellular biomaterial prepared by the method provided by the present invention is significantly enhanced. The tensile strength of the 1-layer transparent material can reach 6.21 N, the tensile strength of the 2-layer can reach 12.38 N, and the tensile strength of the 10-layer can reach 50.95 N. There is a significant difference between the 1-layer, 2-layer, and 10-layer transparent materials. The more layers there are, the stronger the tensile strength. The tensile strength of Comparative Example 4 is 2.87 N; according to Figure 12 it can be seen that compared with the opaque acellular material, the suture strength of the 1-layer transparent acellular biomaterial prepared by the method provided by the present invention is significantly enhanced. The suture strength of the 1-layer transparent material can reach 2.24 N, the suture strength of the 2-layer can reach 6.03 N, and the suture strength of the 10-layer can reach 25.60. There is a significant difference between the 1-layer, 2-layer, and 10-layer transparent materials. The more layers there are, the stronger the suture strength. The suture strength of Comparative Example 4 is 1.43 N. The corresponding transparent acellular biomaterial can be selected according to the usage range and usage site of the product and the requirements for mechanical characterization.

[0126] In summary, the transparent decellularized biomaterial of the present invention can be a single layer or 2-10 stacked layers. The thickness of the transparent decellularized biomaterial is 2-200 μm, the light transmittance is 89.10-95.05%, the breaking strength is 6.21-50.95 N, and the suture strength is 2.24-25.60 N. The transparent decellularized biomaterial has excellent mechanical properties, good biocompatibility, and is easy to store.

[0127] Although the present invention has been described in detail with general descriptions and specific embodiments above, on the basis of the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

Claims

1. A transparent acellular biological material, characterized in that, Obtained by preparing from animal membrane tissue, the thickness of the transparent acellular biomaterial is 1 - 500 μm; the light transmittance is 89.10 - 95.05%, the breaking strength is 6.21 - 50.95 N, and the suture strength is 2.24 - 25.60 N; The preparation method of the transparent acellular biomaterial includes the step of performing a transparency treatment on the acellular animal membrane tissue. The method of the transparency treatment is as follows: within 58 - 62 min, cool the animal membrane tissue from room temperature to -32 - -18 °C for pre-cooling and maintain it for 28 - 32 min, then heat it to -17 - -8 °C within 88 - 92 min and maintain it for 28 - 32 min, then heat it to -2 - 2 °C within 58 - 62 min and maintain it for 178 - 182 min, then heat it to 3 - 12 °C within 58 - 62 min and maintain it for 28 - 32 min, continue to heat it to 13 - 22 °C within 58 - 62 min and maintain it for 28 - 32 min, and finally heat it to 18 - 27 °C within 478 - 482 min and maintain it for 28 - 32 min; After the transparency treatment of the acellular animal membrane tissue, it further includes a drying step, and the drying is freeze-drying; the process of the freeze-drying is as follows: within 58 - 62 min, cool it from room temperature to -52 - -48 °C for pre-cooling and maintain it for 0.7 - 0.9 min, then under vacuum, then heat it to -38 - -42 °C within 28 - 32 min and maintain it for 0.45 - 0.55 min, then heat it to -28 - -32 °C within 28 - 32 min and maintain it for 0.45 - 0.55 min, then heat it to -18 - -22 °C within 28 - 32 min and maintain it for 0.45 - 0.55 min, then heat it to -8 - -12 °C within 28 - 32 min and maintain it for 0.45 - 0.55 min, then heat it to -2 - 2 °C within 18 - 22 min and maintain it for 0.45 - 0.55 min, then heat it to 8 - 12 °C within 18 - 22 min and maintain it for 0.45 - 0.55 min, then heat it to 18 - 22 °C within 8 - 12 min and maintain it for 0.45 - 0.55 min, and finally heat it to 23 - 27 °C within 238 - 242 min and maintain it for 0.45 - 0.55 min; The animal membrane tissue is prepared from animal tissue, and the animal tissue is animal small intestine, animal pericardium or animal bladder.

2. The transparent acellular biological material according to claim 1, wherein The thickness of the transparent acellular biomaterial is 1 - 400 μm.

3. The transparent acellular biological material according to claim 2, wherein The thickness of the transparent acellular biomaterial is 2 - 200 μm.

4. The transparent acellular biological material according to claim 1, wherein The method of the transparency treatment is as follows: within 60 min, the animal membrane tissue is cooled from room temperature to -20°C for pre-cooling and maintained for 30 min, then heated to -10°C within 90 min and maintained for 30 min, then heated to 0°C within 60 min and maintained for 180 min, then heated to 10°C within 60 min and maintained for 30 min, then continuously heated to 20°C within 60 min and maintained for 30 min, and finally heated to 25°C within 480 min and maintained for 30 min; or, The method of the transparency treatment is as follows: within 60 min, the animal membrane tissue is cooled from room temperature to -30°C for pre-cooling and maintained for 30 min, then heated to -15°C within 90 min and maintained for 30 min, then heated to 0°C within 60 min and maintained for 180 min, then heated to 5°C within 60 min and maintained for 30 min, then continuously heated to 15°C within 60 min and maintained for 30 min, and finally heated to 20°C within 480 min and maintained for 30 min.

5. The transparent acellular biological material according to claim 1, characterized in that, The process of freeze-drying is as follows: cooled from room temperature to -50°C within 60 min and maintained for 0.8 min, then under vacuum, then heated to -40°C within 30 min and maintained for 0.5 min, then heated to -30°C within 30 min and maintained for 0.5 min, then heated to -20°C within 30 min and maintained for 0.5 min, then heated to -10°C within 30 min and maintained for 0.5 min, then heated to 0°C within 20 min and maintained for 0.5 min, then heated to 10°C within 20 min and maintained for 0.5 min, then heated to 20°C within 10 min and maintained for 0.5 min, and finally heated to 25°C within 240 min and maintained for 0.5 min.

6. The transparent acellular biological material according to any one of claims 1-5, characterized in that, The transparent acellular biomaterial is prepared from one or more layers of acellular animal membrane tissues.

7. The transparent acellular biological material according to claim 6, characterized in that, The transparent acellular biomaterial is prepared from 1 - 10 layers of acellular animal membrane tissues.

8. The transparent acellular biological material according to claim 6, wherein The preparation method of the acellular animal membrane tissue includes: (1) Pretreatment: After cleaning and inactivating the animal tissue, the muscular layer and serous layer are removed. (2) Antigen removal: The pretreated material is washed, degreased, decellularized, and washed again to obtain the animal membrane tissue.

9. The transparent acellular biological material according to claim 1, wherein The animal is cattle or swine.

10. The transparent acellular biological material according to claim 6, wherein When the transparent acellular biomaterial is prepared from multiple layers of acellular animal membrane tissues, before the transparency treatment, it further includes a step of superposing and laminating the multiple layers of acellular animal membrane tissues.

11. The transparent acellular biological material according to claim 10, characterized in that, The lamination of the multiple layers of acellular animal membrane tissues is achieved by applying pressure to the acellular animal membrane tissues that are unfolded, overlapped, and laid out in multiple layers to discharge the air between the layers.

12. The transparent acellular biological material according to claim 11, wherein The pressure is 3 - 12 N, and the duration of the continuous pressure is 0.5 min - 8 min.

13. The transparent acellular biological material according to claim 12, wherein The pressure is 5 - 10 N, and the duration is 1 min - 5 min.

14. A method for preparing the transparent acellular biological material according to claim 1, characterized in that, The preparation method includes the step of performing transparency treatment on the acellularized animal membrane tissue. The method of the transparency treatment is as follows: within 58 - 62 minutes, cool the animal membrane tissue from room temperature to -32 - -18°C for pre-cooling and maintain it for 28 - 32 minutes, then heat it to -17 - -8°C within 88 - 92 minutes and maintain it for 28 - 32 minutes, then heat it to -2 - 2°C within 58 - 62 minutes and maintain it for 178 - 182 minutes, then heat it to 3 - 12°C within 58 - 62 minutes and maintain it for 28 - 32 minutes, continue to heat it to 13 - 22°C within 58 - 62 minutes and maintain it for 28 - 32 minutes, and finally heat it to 18 - 27°C within 478 - 482 minutes and maintain it for 28 - 32 minutes; After the transparency treatment on the acellularized animal membrane tissue, it further includes a drying step, and the drying is freeze-drying. The process of the freeze-drying is as follows: within 58 - 62 minutes, cool it from room temperature to -52 - -48°C for pre-cooling and maintain it for 0.7 - 0.9 minutes, then under vacuum, then heat it to -38 - -42°C within 28 - 32 minutes and maintain it for 0.45 - 0.55 minutes, then heat it to -28 - -32°C within 28 - 32 minutes and maintain it for 0.45 - 0.55 minutes, then heat it to -18 - -22°C within 28 - 32 minutes and maintain it for 0.45 - 0.55 minutes, then heat it to -8 - -12°C within 28 - 32 minutes and maintain it for 0.45 - 0.55 minutes, then heat it to -2 - 2°C within 18 - 22 minutes and maintain it for 0.45 - 0.55 minutes, then heat it to 8 - 12°C within 18 - 22 minutes and maintain it for 0.45 - 0.55 minutes, then heat it to 18 - 22°C within 8 - 12 minutes and maintain it for 0.45 - 0.55 minutes, and finally heat it to 23 - 27°C within 238 - 242 minutes and maintain it for 0.45 - 0.55 minutes; The animal membrane tissue is prepared from animal tissue, and the animal tissue is animal small intestine, animal pericardium or animal bladder.

15. The preparation method of the transparent acellular biological material according to claim 14, wherein, The method of the transparency treatment is as follows: within 60 minutes, cool the animal membrane tissue from room temperature to -20°C for pre-cooling and maintain it for 30 minutes, then heat it to -10°C within 90 minutes and maintain it for 30 minutes, then heat it to 0°C within 60 minutes and maintain it for 180 minutes, then heat it to 10°C within 60 minutes and maintain it for 30 minutes, continue to heat it to 20°C within 60 minutes and maintain it for 30 minutes, and finally heat it to 25°C within 480 minutes and maintain it for 30 minutes; or, The method of the transparency treatment is as follows: within 60 minutes, cool the animal membrane tissue from room temperature to -30°C for pre-cooling and maintain it for 30 minutes, then heat it to -15°C within 90 minutes and maintain it for 30 minutes, then heat it to 0°C within 60 minutes and maintain it for 180 minutes, then heat it to 5°C within 60 minutes and maintain it for 30 minutes, continue to heat it to 15°C within 60 minutes and maintain it for 30 minutes, and finally heat it to 20°C within 480 minutes and maintain it for 30 minutes.

16. The preparation method of the transparent decellularized biological material according to claim 14, characterized in that, The process of freeze-drying is as follows: cool from room temperature to -50°C for pre-cooling within 60 minutes and maintain it for 0.8 minutes, then under vacuum, then heat it to -40°C within 30 minutes and maintain it for 0.5 minutes, then heat it to -30°C within 30 minutes and maintain it for 0.5 minutes, then heat it to -20°C within 30 minutes and maintain it for 0.5 minutes, then heat it to -10°C within 30 minutes and maintain it for 0.5 minutes, then heat it to 0°C within 20 minutes and maintain it for 0.5 minutes, then heat it to 10°C within 20 minutes and maintain it for 0.5 minutes, then heat it to 20°C within 10 minutes and maintain it for 0.5 minutes, and finally heat it to 25°C within 240 minutes and maintain it for 0.5 minutes.

17. The method for preparing a transparent decellularized biomaterial according to any one of claims 14-16, characterized in that, The transparent acellular biological material is prepared from one or more layers of acellularized animal membrane tissues.

18. The preparation method of the transparent decellularized biological material according to claim 17, wherein, The transparent acellular biological material is prepared from 1 to 10 layers of acellularized animal membrane tissues.

19. The preparation method of the transparent acellular biological material according to claim 17, characterized in that, The preparation method of the acellularized animal membrane tissue includes: (1) Pretreatment: After cleaning and inactivating the animal tissue, remove the muscular layer and serous layer. (2) Antigen removal: Clean, degrease, decellularize, and clean the pretreated material again to obtain the animal membrane tissue.

20. The preparation method of the transparent decellularized biological material according to claim 14, characterized in that, The animal is cattle or pigs.

21. The preparation method of the transparent decellularized biological material according to claim 17, wherein, When the transparent acellular biological material is prepared from multiple layers of acellularized animal membrane tissues, before the transparency treatment, it further includes a step of laminating and fitting the multiple layers of acellularized animal membrane tissues.

22. The preparation method of the transparent decellularized biological material according to claim 21, characterized in that, The lamination and fitting of the multiple layers of acellularized animal membrane tissues are achieved by applying pressure to the acellularized animal membrane tissues that are unfolded and laid out in multiple layers and the air between the layers is discharged.

23. The preparation method of the transparent decellularized biological material according to claim 22, characterized in that, The pressure is 3 to 12 N, and the duration of the continuous pressure is 0.5 minutes to 8 minutes.

24. The preparation method of the transparent acellular biological material according to claim 23, wherein, The pressure is 5 to 10 N, and the duration is 1 minute to 5 minutes.

25. Use of the transparent acellular biological material according to any one of claims 1 - 13 in the preparation of a biological patch.

26. The application according to claim 25, wherein, The biological patch is a corneal repair patch, a skin repair patch, an abdominal cavity repair patch, or a dura mater repair patch.

27. A biological patch, characterized in that, It includes the transparent acellular biological material according to any one of claims 1 - 13.

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