Transparent decellularized biological material as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological materials, and particularly discloses a transparent acellular biological material as well as a preparation method and application thereof. The transparent decellularized biological material provided by the invention is prepared from animal membrane tissues, and the thickness of the transparent decellularized biological material is 1-500 microns; the light transmittance is 89.10% to 95.05%, the breaking strength is 6.21 N to 50.95 N, and the sewing strength is 2.24 N to 25.60 N. The biological material disclosed by the invention is high in transparency and good in strength, can be used in the field of repairing biological patch materials with relatively high requirements on transparency degree, and is wide in application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a transparent decellularized biomaterial and a preparation method and 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 contains a variety of growth factors that play an important role in tissue repair and reconstruction. With the development of tissue engineering technology and in-depth research on the endogenous regeneration mechanism of biomaterials, it is found that when tissue is damaged, covering with allogeneic or xenogeneic cell matrix materials can promote the adhesion, proliferation and growth of cells at the wound on the surface of the material, thereby guiding tissue regeneration and promoting rapid recovery of wounds and blood vessels. Therefore, decellularized 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 pay attention to the efficacy of treatment while also considering the impact of treatment methods or equipment on nature and aesthetics. However, when using acellular matrix materials to treat and repair tissues or organs, the materials will be covered on the wound or tissue. The appearance of the material may greatly affect the aesthetics, especially when treating chronic skin wounds and gum repair. To reduce this impact, it is more appropriate to choose transparent repair materials. In addition, in some treatment scenarios such as corneal repair, whether the material is transparent or the degree of transparency will be a key technical indicator, which will have a great impact on the patient's life during treatment.

[0004] The transmittance and mechanical properties of decellularized matrix materials are sometimes also crucial for wound repair. The higher the transparency, the easier it is to observe the recovery of the wound through the decellularized matrix material. In addition, the transmittance and mechanics of decellularized matrix materials are also the key to the restoration of corneal transparency. High transparency and good mechanical properties are more conducive to the restoration of the cornea to a colorless and transparent state, achieving blindness elimination. Therefore, a decellularized matrix material with good mechanical properties, high transparency, degradation properties, 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, CN10322722775A discloses a method for preparing a dura mater biological patch, including separation of the small intestinal submucosa, virus inactivation and decellularization; CN116942913A discloses a decellularized biological material and 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 decellularized materials prepared by general traditional processes are white or slightly yellow membranes with poor transparency, which are not convenient for use in situations where transparency requirements are high or aesthetic requirements are high. Therefore, it is necessary to further study the preparation of decellularized matrix materials. Summary of the invention

[0006] One of the purposes of the present invention is to provide a decellularized biomaterial with high transparency and good strength, and a preparation method and application thereof.

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

[0008] A transparent decellularized biomaterial is prepared from animal membrane tissue. The transparent decellularized biomaterial has a thickness of 1-500 μm, a light transmittance of 89.10-95.05%, a breaking strength of 6.21-50.95 N, and a suture strength of 2.24-25.60 N.

[0009] Preferably, the thickness of the transparent decellularized biomaterial is 1-400 μm, more preferably 2-200 μm.

[0010] The present invention studies and obtains a novel transparent decellularized biomaterial, which has good physical properties and high transparency. When the decellularized matrix material is used 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 decellularized biomaterial of the present invention comprises the step of transparentizing the decellularized animal membrane tissue, wherein the transparentizing method is as follows: within 58 to 62 minutes, the animal membrane tissue is precooled from room temperature to -32 to -18°C and maintained for 28 to 32 minutes, then heated to -17 to -8°C within 88 to 92 minutes and maintained for 28 to 32 minutes, then heated to -2 to 2°C within 58 to 62 minutes and maintained for 178 to 182 minutes, then heated to 3 to 12°C within 58 to 62 minutes and maintained for 28 to 32 minutes, then heated to 13 to 22°C within 58 to 62 minutes and maintained for 28 to 32 minutes, finally heated to 18 to 27°C within 478 to 482 minutes and maintained for 28 to 32 minutes.

[0012] The present invention has found that when preparing decellularized biomaterials, decellularized animal membrane tissues are treated in a specific gradient dehydration manner to obtain biomaterials with good mechanical properties, high transparency, degradation properties, and good biocompatibility.

[0013] Preferably, the transparent treatment method is: within 60 minutes, the animal membrane tissue is precooled from room temperature to -20°C 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, finally heated to 25°C within 480 minutes and maintained for 30 minutes; or,

[0014] The transparent treatment method is: within 60 minutes, the animal membrane tissue is pre-cooled from room temperature to -30°C 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, finally heated to 20°C within 480 minutes and maintained for 30 minutes.

[0015] The present invention further comprises a drying step after the decellularized animal membrane tissue is transparentized, and the drying is preferably freeze-drying.

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

[0017] As a specific embodiment, the freeze-drying process is: pre-cooling from room temperature to -52 to -48°C within 58 to 62 minutes, maintaining for 0.7 to 0.9 minutes, and then under vacuum, then heating to -38 to -42°C within 28 to 32 minutes, maintaining for 0.45 to 0.55 minutes, then heating to -28 to -32°C within 28 to 32 minutes, maintaining for 0.45 to 0.55 minutes, then heating to -18 to -22°C within 28 to 32 minutes, maintaining for 0.45 to 0.55 minutes , then within 28-32 minutes, the temperature was raised to -8--12°C and maintained for 0.45-0.55 minutes, then within 18-22 minutes, the temperature was raised to -2-2°C and maintained for 0.45-0.55 minutes, then within 18-22 minutes, the temperature was raised to 8-12°C and maintained for 0.45-0.55 minutes, then within 8-12 minutes, the temperature was raised to 18-22°C and maintained for 0.45-0.55 minutes, and finally within 238-242 minutes, the temperature was raised to 23-27°C and maintained for 0.45-0.55 minutes;

[0018] Preferably, the freeze-drying process is: pre-cooling from room temperature to -50°C within 60 minutes and maintaining for 0.8 minutes, then under vacuum, then heating to -40°C within 30 minutes and maintaining for 0.5 minutes, then heating to -30°C within 30 minutes and maintaining for 0.5 minutes, then heating to -20°C within 30 minutes and maintaining for 0.5 minutes, then heating to -10°C within 30 minutes and maintaining for 0.5 minutes, then heating to 0°C within 20 minutes and maintaining for 0.5 minutes, then heating to 10°C within 20 minutes and maintaining for 0.5 minutes, then heating to 20°C within 10 minutes and maintaining for 0.5 minutes, and finally heating to 25°C within 240 minutes and maintaining for 0.5 minutes.

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

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

[0021] And / or, the animal membrane tissue is prepared from animal tissue, the animal tissue is animal small intestine, animal pericardium or animal bladder; the animal is cow or pig.

[0022] Preferably, the method for preparing the decellularized animal membrane tissue comprises:

[0023] (1) Pretreatment: After washing and inactivating the animal tissue, the muscle layer and serosal layer are removed;

[0024] (2) Antigen removal: The pretreated material is washed, defatted, 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 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 in the final product is less than 2% (dry basis), and the remaining cells should have no complete cell nucleus), and the present invention is not particularly limited.

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

[0027] In the present invention, when the transparent decellularized biomaterial is prepared from multiple layers of decellularized animal membrane tissue, the step of stacking and bonding the multiple layers of decellularized animal membrane tissue is also included before the transparent treatment; preferably, the bonding 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 and stacked and the air between the layers is discharged. More preferably, the pressure is 3 to 12N and the pressure is maintained for 0.5min to 8min. Further preferably, the pressure is 5 to 10N and the pressure is maintained for 1min to 5min.

[0028] When laminating multiple layers of decellularized animal membrane tissues, the present invention can specifically spread out the antigen-free membrane sheets, expel the air in the unlaminated parts, stack them layer by layer, expel the air in the unlaminated parts, and then apply pressure to laminate them.

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

[0030] The present invention further provides the use of the above-mentioned transparent acellular biomaterial or preparation method in the preparation of a biological patch. Preferably, the biological patch is a corneal repair patch, a skin repair patch, a peritoneal repair patch or a dura mater repair patch.

[0031] The present invention further provides a biological patch, which comprises the transparent decellularized biological material.

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

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

[0034] Figure 1 This is the transparent state of the transparent decellularized biomaterial prepared in Example 1.

[0035] Figure 2 This is the transparent state of the transparent decellularized biomaterial prepared in Example 2.

[0036] Figure 3 This is the transparent state of the transparent decellularized biomaterial prepared in Example 3.

[0037] Figure 4 This is the transparent state of the transparent decellularized biomaterial prepared in Example 4.

[0038] Figure 5 This is the transparent state of the transparent decellularized biomaterial prepared in Example 5.

[0039] Figure 6 This is the transparent state of the transparent decellularized biomaterial prepared in Comparative Example 1.

[0040] Figure 7 This is the transparent state of the transparent decellularized biomaterial prepared in Comparative Example 2.

[0041] Figure 8 This is the transparent state of the transparent decellularized biomaterial prepared in Comparative Example 3.

[0042] Fig. 9 This is the transparent state of the opaque decellularized biomaterial prepared in Comparative Example 4.

[0043] Fig.10 The transmittance results of the decellularized materials prepared in various examples and comparative examples are shown.

[0044] Fig.11 The following are the breaking strength results of the decellularized materials prepared in various examples and comparative examples.

[0045] Fig.12 The suture strength results of the decellularized materials prepared in various examples and comparative examples are shown. DETAILED DESCRIPTION

[0046] The preferred embodiments of the present invention will be described in detail below in conjunction with examples. According to the content of the present invention, according to the common technical knowledge and customary means in this area, without departing from the basic technical idea of ​​the present invention, other various forms of modification, replacement or change can also be made. 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, without departing from the purpose, principle and spirit of the present invention, the changes, modifications, combinations, substitutions and simplifications made should all be equivalent replacement modes, and are all included in the protection scope 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 takes the bladder as an example to provide a transparent decellularized biomaterial and a preparation method thereof. The preparation process of the transparent decellularized biomaterial includes pretreatment, antigen removal, transparentization and freeze-drying steps. The details are as follows:

[0050] 1) Preprocessing:

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

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

[0053] 1.3 Manual processing: Clean the soaked materials and remove the muscular layer and serous layer of the materials manually.

[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: The cleaned material is placed in an oscillator and degreased with an organic solvent, wherein the organic solvent is methanol and chloroform in a volume ratio of 1:1.

[0057] 2.3 Decellularization: The defatted material was placed in an EDTA-trypsin mixed solution 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 injection water to obtain a decellularized bladder matrix fresh film.

[0059] 3) Transparency:

[0060] 3.1 Spreading the membrane: Spread a layer of acellular bladder matrix fresh film evenly and expel the air from the unattached part.

[0061] 3.2 Transparentization: Transparentization is achieved through gradient dehydration to obtain a single layer of transparent acellular bladder matrix. The transparentization process is shown in Table 1.

[0062] Table 1 Transparency parameters

[0063]

[0064] 4) Freeze drying: freeze drying the transparent decellularized matrix film under the freeze drying conditions in Table 2, and finally obtain transparent decellularized biomaterials such as Figure 1 As shown, the thickness is 3 μm. The remaining fat content is 2% (dry basis percentage), and the remaining cells have no complete nucleus.

[0065] Table 2 Freeze drying parameters

[0066]

[0067] Example 2

[0068] This embodiment takes the small intestinal submucosa (SIS) as an example and provides a transparent decellularized biomaterial and a preparation method thereof.

[0069] The preparation process of transparent decellularized biomaterials includes pretreatment, antigen removal, transparentization and freeze-drying steps. The details are as follows:

[0070] 1) Preprocessing:

[0071] 1.1 Cleaning: Wash fresh pig small intestine repeatedly with purified water.

[0072] 1.2 Inactivation: Soak the cleaned pig small intestine in 3% peracetic acid for 60 minutes.

[0073] 1.3 Mechanical treatment: Clean the soaked material and use mechanical methods to preliminarily remove the muscle layer and serosal layer of the material.

[0074] 2) Antigen removal:

[0075] 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).

[0076] 2.2 Degreasing: The cleaned material is placed in an oscillator and degreased with an organic solvent, wherein the organic solvent is methanol and chloroform in a volume ratio of 1:1.

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

[0078] 2.4 Cleaning: Place the decellularized material into a container filled with purified water, clean it with ultrasound for 20 minutes (ultrasonic power 300W), and then clean it with injection water to obtain a decellularized small intestinal matrix fresh film.

[0079] 3) Transparency:

[0080] 3.1 Spreading the membrane: Spread a layer of decellularized intestinal matrix film evenly and expel the air from the unattached part.

[0081] 3.2 Transparentization: Transparentization is achieved through gradient dehydration to obtain a single-layer transparent acellular matrix membrane. The transparentization process is shown in Table 3.

[0082] Table 3 Transparency parameters

[0083]

[0084] 4) Freeze drying: freeze drying the transparent decellularized matrix membrane using the freeze drying conditions in Table 4, and finally obtain transparent decellularized biomaterials such as Figure 2 As shown, the thickness is 2 μm. The remaining fat content is 2% (dry basis percentage), and the remaining cells have no complete nucleus.

[0085] Table 4 Freeze drying parameters

[0086]

[0087] Example 3

[0088] This embodiment provides a method for preparing a transparent decellularized biomaterial, which is substantially the same as the preparation method in Example 2, except that in step 3), the transparentization parameters shown in Table 5 are used for transparentization treatment.

[0089] Table 5 Transparency parameters

[0090]

[0091] Then, continue with step 4) in Example 2 to finally obtain a transparent decellularized biomaterial such as Figure 3 As shown, the thickness is 3 μm.

[0092] Example 4

[0093] This embodiment provides a method for preparing a transparent decellularized biomaterial, which is substantially the same as the preparation method of embodiment 2, except that a two-layer material is prepared in step 3). The specific operation of step 3) is:

[0094] 3.1 Spreading the membrane: Spread a layer of decellularized small intestinal matrix fresh film evenly, expel the air from the unattached part, and then lay the second layer and expel the air from the unattached part.

[0095] 3.2 Multilayering: Place the two layers of spread membrane into the press and apply 5N pressure evenly for 1 minute to achieve interlayer bonding.

[0096] 3.2 Transparentization: Transparentization is achieved through gradient dehydration to obtain a two-layer transparent acellular matrix membrane. The transparentization process is shown in Table 3.

[0097] Then, continue with step 4) in Example 2 to finally obtain a transparent decellularized biomaterial such as Figure 4 As shown, the thickness is 5 μm.

[0098] Example 5

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

[0100] 3.1 Spreading the membrane: Spread a layer of decellularized intestinal matrix fresh film evenly, expel the air from the unattached part, then stack the second layer, expel the air from the unattached part, and repeat the operation until 10 layers are stacked.

[0101] 3.2 Multilayering: Place the 10 layers of spread membrane into the press and apply 10N pressure evenly for 5 minutes to achieve interlayer bonding.

[0102] 3.2 Transparentization: Transparentization was achieved through gradient dehydration to obtain a 10-layer transparent acellular matrix membrane. The transparentization process is shown in Table 3.

[0103] Then, continue with step 4) in Example 2 to finally obtain a transparent decellularized biomaterial such as Figure 5 As shown, the thickness is 200 μm.

[0104] Comparative Example 1

[0105] This comparative example provides a method for preparing a transparent decellularized biomaterial, which is substantially the same as the preparation method in Example 2, except that in step 3), the transparentization parameters shown in Table 6 are used for transparentization treatment.

[0106] Table 6 Transparency parameters

[0107]

[0108] The obtained transparent decellularized biomaterial is Figure 6 As shown, the thickness is 5 μm.

[0109] Comparative Example 2

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

[0111] Table 7 Transparency parameters

[0112]

[0113]

[0114] The obtained transparent decellularized biomaterial is Figure 7 As shown, the thickness is 4 μm.

[0115] Comparative Example 3

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

[0117] Table 8 Transparency parameters

[0118]

[0119] The obtained transparent decellularized biomaterial is Figure 8 As shown, the thickness is 4 μm.

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

[0121] This comparative example provides a method for preparing an opaque decellularized biomaterial, which is substantially the same as the preparation method of Example 2, except that the transparentization step of step 3) is removed and the freeze-drying step is directly performed. The obtained opaque decellularized biomaterial is as follows Fig. 9 As shown, the thickness is 6 μm.

[0122] Experimental example

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

[0124] The specific experimental methods for mechanical characterization are as follows: (1) Breaking strength: A specimen with a specification of 54×10mm was used for the tensile strength test. Before the test, the sample was soaked in physiological saline for 5 minutes. The upper and lower ends of the sample were fixed on the clamp, and the clamp movement speed was set to 50mm / 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 breaking strength. (2) Suture strength: A specimen with a specification of 54×10mm was used for the seam strength test. Before the test, the sample was soaked in physiological saline for 5 minutes. Nylon 11-0 suture thread was passed through the midpoint of the short side of the sample 2mm away from the center of the sample and knotted into a ring. After the ring is straightened, the length of the line segment is 5cm. The suture thread is fixed to the upper part of the clamp, and the lower end of the sample is fixed to the lower part of the clamp. Set the clamp movement speed to 50mm / 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. The results are as follows Figures 10 to 12 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.

[0125] according to Fig.10 It can be seen that the 1-layer, 2-layer, and 10-layer transparent decellularized biomaterials prepared according to the method provided by the present invention have significantly improved transmittance compared with the opaque decellularized material. 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 decellularized biomaterials in each embodiment; according to Fig.11 It can be seen that the breaking strength of one layer of transparent decellularized biomaterial prepared according to the method provided by the present invention is significantly enhanced compared with that of the opaque decellularized material. The breaking strength of one layer of transparent material can reach 6.21N, the breaking strength of two layers can reach 12.38N, and the breaking strength of ten layers can reach 50.95N. There are significant differences between the transparent materials with one layer, two layers, and ten layers. The more layers, the stronger the breaking strength. The breaking strength of comparative example 4 is 2.87N. Fig.12 It can be seen that the suture strength of one layer of transparent decellularized biomaterial prepared by the method provided by the present invention is significantly enhanced compared with that of opaque decellularized material. The suture strength of one layer of transparent material can reach 2.24N, the suture strength of two layers can reach 6.03N, and the breaking strength of ten layers can reach 25.60. There are significant differences between one layer, two layers, and ten layers of transparent materials. The more layers, the stronger the suture strength. The suture strength of comparative example 4 is 1.43N. The corresponding transparent decellularized biomaterial can be selected according to the product use range, use site, and mechanical characterization requirements.

[0126] In summary, the transparent decellularized biomaterial of the present invention can be a single layer or 2-10 layers stacked together, the transparent decellularized biomaterial has a thickness of 2-200 μm, a transmittance of 89.10-95.05%, a breaking strength of 6.21-50.95 N, and a suture strength of 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 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 transparent decellularized biomaterial, characterized in that: The transparent decellularized biological material is prepared from animal membrane tissue, and has a thickness of 1-500 μm, a light transmittance of 89.10-95.05%, a breaking strength of 6.21-50.95N, and a suture strength of 2.24-25.60N.

2. The transparent decellularized biomaterial according to claim 1, characterized in that: The thickness of the transparent decellularized biomaterial is 1-400 μm, preferably 2-200 μm.

3. The transparent decellularized biomaterial according to claim 1 or 2, characterized in that: The preparation method of the transparent decellularized biomaterial comprises the step of transparentizing the decellularized animal membrane tissue, wherein the transparentizing method is as follows: within 58 to 62 minutes, the animal membrane tissue is precooled from room temperature to -32 to -18°C and maintained for 28 to 32 minutes, then heated to -17 to -8°C within 88 to 92 minutes and maintained for 28 to 32 minutes, then heated to -2 to 2°C within 58 to 62 minutes and maintained for 178 to 182 minutes, then heated to 3 to 12°C within 58 to 62 minutes and maintained for 28 to 32 minutes, then heated to 13 to 22°C within 58 to 62 minutes and maintained for 28 to 32 minutes, finally heated to 18 to 27°C within 478 to 482 minutes and maintained for 28 to 32 minutes; Preferably, the transparent treatment method is: within 60 minutes, the animal membrane tissue is precooled from room temperature to -20°C 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, finally heated to 25°C within 480 minutes and maintained for 30 minutes; or, The transparent treatment method is: within 60 minutes, the animal membrane tissue is pre-cooled from room temperature to -30°C 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, finally heated to 20°C within 480 minutes and maintained for 30 minutes.

4. The transparent decellularized biomaterial according to claim 3, characterized in that: After the decellularized animal membrane tissue is transparentized, a drying step is further included, and the drying is preferably freeze-drying.

5. The transparent decellularized biomaterial according to claim 4, characterized in that: The freeze-drying process is as follows: pre-cooling from room temperature to -52--48°C within 58-62 minutes and maintaining for 0.7-0.9 minutes, then under vacuum, then heating to -38--42°C within 28-32 minutes and maintaining for 0.45-0.55 minutes, then heating to -28--32°C within 28-32 minutes and maintaining for 0.45-0.55 minutes, then heating to -18--22°C within 28-32 minutes and maintaining for 0.45-0.55 minutes, then heating to -18--22°C within 28-32 minutes and maintaining for 0.45-0.55 minutes, then heating to -28--32°C within 28-32 minutes and maintaining for 0.45-0.55 minutes, then heating to -18--22°C within 28-32 minutes and maintaining for 0.45-0.55 minutes, then heating to -28--32°C within 28-32 minutes and maintaining for 0.45-0.55 minutes, then heating to -2 ... The temperature was raised to -8--12°C within 1-32 minutes and maintained for 0.45-0.55 minutes, then raised to -2-2°C within 18-22 minutes and maintained for 0.45-0.55 minutes, then raised to 8-12°C within 18-22 minutes and maintained for 0.45-0.55 minutes, then raised to 18-22°C within 8-12 minutes and maintained for 0.45-0.55 minutes, and finally raised to 23-27°C within 238-242 minutes and maintained for 0.45-0.55 minutes; Preferably, the freeze-drying process is: pre-cooling from room temperature to -50°C within 60 minutes and maintaining for 0.8 minutes, then under vacuum, then heating to -40°C within 30 minutes and maintaining for 0.5 minutes, then heating to -30°C within 30 minutes and maintaining for 0.5 minutes, then heating to -20°C within 30 minutes and maintaining for 0.5 minutes, then heating to -10°C within 30 minutes and maintaining for 0.5 minutes, then heating to 0°C within 20 minutes and maintaining for 0.5 minutes, then heating to 10°C within 20 minutes and maintaining for 0.5 minutes, then heating to 20°C within 10 minutes and maintaining for 0.5 minutes, and finally heating to 25°C within 240 minutes and maintaining for 0.5 minutes.

6. The transparent decellularized biomaterial according to any one of claims 1 to 5, characterized in that: The transparent decellularized biomaterial is prepared from one or more layers of decellularized animal membrane tissue, preferably 1-10 layers; And / or, the animal membrane tissue is prepared from animal tissue, the animal tissue is animal small intestine, animal pericardium or animal bladder; the animal is cattle or pig; Preferably, the method for preparing the decellularized animal membrane tissue comprises: (1) Pretreatment: After washing and inactivating the animal tissue, the muscle layer and serosal layer are removed; (2) Antigen removal: The pretreated material is washed, defatted, decellularized, and washed again to obtain animal membrane tissue.

7. The transparent decellularized biomaterial according to claim 6, characterized in that: When the transparent decellularized biomaterial is prepared from multiple layers of decellularized animal membrane tissue, the step of stacking and bonding the multiple layers of decellularized animal membrane tissue is also included before the transparent treatment; preferably, the bonding 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 and stacked and the air between the layers is discharged. More preferably, the pressure is 3 to 12N and the pressure is maintained for 0.5min to 8min. Further preferably, the pressure is 5 to 10N and the pressure is maintained for 1min to 5min.

8. A method for preparing a transparent decellularized biomaterial according to any one of claims 1 to 7, characterized in that: The preparation method is as described in any one of claims 3 to 8.

9. Use of the transparent decellularized biomaterial according to any one of claims 1 to 7 or the preparation method according to claim 8 in preparing a biological patch, preferably, the biological patch is a corneal repair patch, a skin repair patch, a peritoneal repair patch or a dura mater repair patch.

10. A biological patch, characterized in that: The transparent decellularized biomaterial comprises the transparent decellularized biomaterial according to any one of claims 1 to 7.

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