A decellularized matrix scaffold material and its preparation method and application

Abstract

The present invention relates to the field of biomaterial technology, and specifically discloses a decellularized matrix scaffold material and a preparation method and application thereof. The decellularized matrix scaffold material of the present invention is prepared from animal membrane tissue, and its light transmittance is 60%-88%, and its suture strength is 2.05N-3.5N. The present invention treats the decellularized animal membrane tissue with a dehydrating liquid and a structure regulator to prepare a decellularized matrix scaffold material with high transparency, good biological activity and ideal mechanical properties. The decellularized matrix scaffold material is suitable for use as a biological patch in animal membrane tissue repair, especially for corneal repair, which can promote corneal healing and provide vision for early corneal recovery.

Description

Technical Field

[0001] The present invention relates to the technical field of biomaterials, and in particular to a decellularized matrix scaffold material and a preparation method and application thereof. Background Art

[0002] Acellular matrix scaffolds refer to the extracellular matrix scaffolds of tissues and organs after the cells are removed by chemical, physical and biological methods. Because of low immunity and rich in various growth factors, and the transmission of physical, chemical and biological signals, they are conducive to cell proliferation, differentiation and angiogenesis. SIS material is a decellularized matrix scaffold of the submucosal layer of the small intestine of pigs after decellularization.

[0003] The ocular surface refers to the entire mucosal tissue bounded by the skin of the upper and lower eyelids in anatomy, namely the epithelial tissues such as the palpebral mucosal epithelium, palpebral conjunctiva, fornix conjunctiva, bulbar conjunctiva, limbus and cornea. Histologically, this epithelial surface is covered with two main areas, namely the cornea and conjunctiva. The integrity of the corneal epithelium is the basis for maintaining corneal transparency and normal visual function, and its stability depends on the continuous proliferation, differentiation and centripetal migration of limbal stem cells. Ocular surface diseases such as Stevens-Johnson syndrome, chemical, thermal, radiation damage, extensive microbial infections and genetic diseases can cause limbal stem cell deficiency or dysfunction, leading to ocular surface damage.

[0004] SIS and other acellular matrices can promote cell proliferation and differentiation as natural tissue engineering scaffolds and restore the function of the ocular surface. There are reports that SIS has been used to repair corneal damage in cats and dogs, with good results. However, the transparency of the SIS material itself is poor, and it is easy to cause vision loss in the early stage of implantation.

[0005] Existing patents disclose various methods for preparing scaffold materials for promoting the repair of corneal epithelium. For example, the Chinese publication number CN104288837B, the invention name of a decellularized dermal matrix and its preparation method and use, discloses a method for preparing a decellularized dermal matrix and a method for treating the decellularized dermal matrix before use. In the method, the collagen of the extracellular matrix is ​​arranged neatly, close to the arrangement of corneal fibers, but its transparency has not received attention. The Chinese publication number CN116171170A, the invention name of a fiber composite material, relates to a fiber composite material, which includes a non-fiber component with a water content of less than 10wt%; and a fiber component, a porous scaffold including multiple electrospun polymer fibers; and is applied in ophthalmology and wound care using electrospinning technology. The technology of electrospinning polymers in this scheme emphasizes mechanical properties and water content. As the part of its fiber scaffold, it is a polymer material, which does not have the biological activity of the decellularized matrix scaffold, and has a high immunogenicity. It only emphasizes its role as a substitute for amniotic membrane in ophthalmology, and does not pay attention to the transparent properties of corneal patches. The Chinese patent with publication number CN116850345A invented a method for preparing a transparent collagen membrane, which uses sodium hydroxide and acetic acid to perform three transparent treatments on the decellularized SIS membrane. This process will destroy the active factors of collagen, resulting in reduced activity, which is not conducive to promoting the repair of corneal epithelium. In summary, considering the visual function of the ocular surface repair material itself, it is necessary to obtain a decellularized matrix scaffold that increases the transparency of the material while maintaining the mechanical properties and biological activity of the material itself. Summary of the invention

[0006] One of the purposes of the present invention is to provide a decellularized matrix scaffold material with high light transmittance, good mechanical properties and good biological activity.

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

[0008] A decellularized matrix scaffold material is prepared from animal membrane tissue, has a light transmittance of 60%-88%, and a suture strength of 2.05N-3.5N.

[0009] The present invention provides a new acellular matrix scaffold material, which is prepared from animal membrane tissue, has high light transmittance and good mechanical properties while maintaining the biological activity of the animal membrane tissue itself, and is suitable for practical application in animal membrane tissue repair, especially for scenes with high requirements for membrane tissue transparency. For example, in corneal repair, the material of the present invention can provide vision for animals in the early stage of repair and promote repair, avoiding the drawbacks of traditional materials.

[0010] The preparation method of the decellularized matrix scaffold material of the present invention comprises the step of transparentizing the decellularized animal membrane tissue, wherein the transparentizing method comprises: firstly immersing the decellularized animal membrane tissue in a dehydrating liquid until the water content of the animal membrane tissue reaches 14%-18% (preferably 14.5%-17.5%, more preferably 14.8%-17.25%), and then mixing in a structure regulator for treatment;

[0011] The dehydration liquid comprises an ether organic solvent, an alcohol organic solvent and a ketone organic solvent; the volume ratio of the ether organic solvent, the alcohol organic solvent and the ketone organic solvent is (1-10): (1-3): (1-10);

[0012] The structure regulator is N,N-dimethylformamide (DMF), and the volume ratio of the dehydrating liquid to the structure regulator is (3-4.2):1.

[0013] After decellularization, general animal membrane tissue (such as SIS material) is observed by naked eyes as a milky white, translucent membrane with a light transmittance of about 28.7-31.2%, which is not suitable for use in animal membrane tissue repair with high transparency requirements. The present invention has found that when the decellularized animal membrane tissue prepared by conventional methods is first dehydrated with a specific dehydrating liquid and then a specific structure regulator is added, a decellularized matrix scaffold material with high transparency and good mechanical properties can be obtained on the basis of maintaining the original biological activity of the raw material, thereby meeting the needs of different application scenarios, and is particularly suitable for corneal repair, which can not only promote the speed of corneal repair but also provide vision in the early stage of repair.

[0014] Specifically, the present invention first uses a specific dehydrating liquid to specifically adjust the internal crystalline structure of the decellularized animal membrane tissue, and then adds DMF to the dehydrating liquid soaked with the decellularized animal membrane tissue, and further adjusts and stabilizes the crystalline structure with a specific ratio of dehydrating liquid and DMF, thereby ultimately achieving the effect of improving the transparency of the material without affecting the original biological activity and mechanical properties of the material.

[0015] When preparing the decellularized matrix scaffold material of the present invention, the volume ratio of the ether organic solvent, the alcohol organic solvent and the ketone organic solvent is preferably (1.5-3.5): (1-3): (1-9), so as to better improve the transparency of the material.

[0016] Preferably, the ether organic solvent includes one or more of propyl ether, ethyl ether, dipropyl ether, and diisopropyl ether, the alcohol organic solvent includes one or more of n-propanol, isopropanol, n-butanol, and isobutanol, and the ketone organic solvent includes acetone and / or ketone; more preferably, the ether organic solvent is propyl ether, the alcohol organic solvent is n-propanol, and the ketone organic solvent is acetone.

[0017] Preferably, when the decellularized animal membrane tissue is immersed in the present invention, the mass volume ratio of the decellularized animal membrane tissue to the dehydration liquid is 1:90-110 g / mL, preferably 1:100 g / mL.

[0018] When the decellularized animal membrane tissue is soaked in the dehydrating liquid, the dehydrating liquid should be completely immersed in the decellularized animal membrane tissue to ensure the efficiency and effect of adjusting the membrane structure performance. Soaking in the preferred ratio defined in the present invention is conducive to taking into account both the treatment effect and the cost.

[0019] When the acellular matrix scaffold material of the present invention is prepared, the time for treating with the structure regulator is 1-2 hours.

[0020] The structure regulator treatment step can be completed within 1-2 hours at the DMF addition ratio specified in the present invention, which can not only take into account the treatment effect, but also avoid the negative impact of long-term organic solvent treatment on material properties.

[0021] The method for preparing the decellularized animal membrane tissue of the present invention comprises: washing; removing the muscle layer and the serosal layer; defatting; decellularizing;

[0022] Alternatively, it includes the steps of cleaning the animal membrane tissue, inactivating with disinfectants (killing bacteria, viruses and other microorganisms), removing the muscle layer and the serosal layer, ultrasonic cleaning, defatting, decellularizing, and cleaning again.

[0023] The method for preparing the decellularized animal membrane tissue of the present invention can be prepared by methods known 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 2% (dry basis), and the remaining cells should have no complete cell nucleus). The present invention does not impose any restrictions on this.

[0024] As a specific embodiment, the method for preparing the decellularized animal membrane tissue (decellularized animal small intestinal submucosa) of the present invention comprises:

[0025] 1. Clean and organize.

[0026] 2. Remove the muscle layer and serosa layer, and store in normal saline at 4°C overnight (12 hours).

[0027] 3. Degreasing: Immerse the drained material in a mixture of surfactant and ethanol for full degreasing, with the ratio of surfactant to ethanol being 1:1. The surfactant can be SDS, Tirton-100 or chloroform.

[0028] 4. Decellularization: Use 0.25% trypsin solution to decellularize overnight at 4°C.

[0029] 5. Rinse with PBS buffer (pH 5-10) until the liquid is colorless.

[0030] The animal membrane tissue of the present invention is from the small intestine or amniotic membrane. Preferably, the animal membrane tissue is from cattle or pigs.

[0031] When preparing the acellular matrix scaffold material of the present invention, after completing the transparent treatment, a drying step is also included. Preferably, the drying is vacuum freeze drying.

[0032] Those skilled in the art may use conventional methods in the art to dry the material after the transparency treatment, and the present invention is not limited to this.

[0033] As a specific embodiment, the present invention can be dried by vacuum freezing. Specifically, the decellularized matrix scaffold material can be expanded as much as possible to expand the surface area, and the vacuum pump can be started to a vacuum degree of ≤10Pa. The pre-freezing temperature of freeze drying is -40°C, the time is 30-120min, the sublimation temperature is -40-0°C, and the heating rate is 0.1°C / min.

[0034] The present invention also provides a method for preparing a decellularized matrix scaffold material, and the preparation method is as described above.

[0035] The method of the present invention has simple preparation steps, and the prepared decellularized matrix scaffold material exhibits good cell compatibility. When implanted into a rabbit model with shallow corneal damage, compared with biological amniotic membrane, the decellularized matrix scaffold material of the present invention has high corneal transparency in the early stage, has less effect on the visual function of the animal, and also exhibits the effect of promoting corneal healing in the later stage of implantation of the decellularized matrix scaffold material, thereby achieving an ideal application effect.

[0036] The present invention further provides an application of a decellularized matrix scaffold material in preparing a biological patch. Preferably, the biological patch is a corneal repair patch, a skin repair patch or an abdominal repair patch.

[0037] The present invention further provides a biological patch, which includes the above-mentioned acellular matrix scaffold material. Preferably, the biological patch is a corneal repair patch, a skin repair patch or an abdominal repair patch.

[0038] The beneficial effects of the present invention are at least:

[0039] The present invention provides a new acellular matrix scaffold material, which has high suture strength and high transparency while maintaining the biological activity of the material, and is particularly suitable for use in the repair of animal membrane tissues with high transparency requirements. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 These are representative photos of the cornea at different weeks after surgery under the slit lamp in Experimental Example 2.

[0041] Figure 2 Representative photos of HE staining in Experimental Example 2. DETAILED DESCRIPTION

[0042] 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.

[0043] 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.

[0044] The present invention provides a method for preparing a cell-free matrix scaffold material, comprising:

[0045] Preprocessing stage:

[0046] 1) Cleaning and sorting: Take fresh pig small intestines half an hour after slaughter, rinse the contents of the small intestines with water, turn the small intestines over, add salt and rub them, then rinse them repeatedly with water for 3 times, and cut the rinsed small intestines into short sections.

[0047] 2) Use a tongue depressor to scrape off the muscle layer and serosa layer, and store in normal saline at 4°C overnight.

[0048] 3) Degreasing: rinse with deionized water, filter with gauze, immerse in a mixture of chloroform and ethanol for thorough degreasing. The volume ratio of chloroform to ethanol is 1:1. Change the mixture every 2 hours on average and stir every half an hour.

[0049] 4) Decellularization: Rinse with deionized water 20 times, rinse repeatedly until there is no smell. Then put it in 0.25% trypsin solution and decellularize it at 4℃ overnight.

[0050] 5) Rinse with PBS buffer (pH 7.4) 20 times until the liquid becomes colorless.

[0051] Transparency stage:

[0052] 6) Preparation of dehydration solution: Take an ether organic solvent, an alcohol organic solvent and a ketone organic solvent, stir them thoroughly in a certain volume ratio to make a solution, and let them stand for 20 minutes to allow them to be fully mixed.

[0053] 7) Take the SIS material prepared in step 5), drain the water, and soak it in a dehydrating liquid until the SIS material reaches a specific moisture content. The mass volume ratio of the SIS material to the dehydrating liquid is 1:100 g / mL.

[0054] 8) Add structure regulator DMF (N,N-dimethylformamide) in proportion and continue soaking for 1~2 hours.

[0055] Drying stage:

[0056] 9) Freeze-dry the transparent SIS patch: unfold the decellularized matrix membrane as much as possible to expand the surface area, start the vacuum pump to a vacuum degree of ≤10Pa, the pre-freezing temperature for freeze drying is -40°C, the time is 30 minutes, the sublimation temperature is -10°C, and the heating rate is 0.1°C / min.

[0057] In the present invention, a UV-visible spectrophotometer is used to measure the light transmittance of the material at the same wavelength (380nm), and pure water is used for light transmittance blank correction. The light transmittance of the freeze-dried SIS material is directly tested, and the transparency results of the four corners of the square material are selected to calculate the average value to obtain the overall light transmittance of the material.

[0058] Examples 1-14

[0059] In each embodiment of the present invention, the acellular matrix scaffold material was prepared according to the above method, and the transmittance was tested. The specific implementation conditions and transmittance test results of the transparent stage of each embodiment are shown in Table 1.

[0060] Table 1

[0061]

[0062] Comparative Examples 1-6

[0063] The comparative examples of the present invention were prepared according to the above method for decellularized matrix scaffold materials, and the transmittance was detected, wherein the method of comparative example 1 was basically the same as that of Example 6, the method of comparative example 2 was basically the same as that of Example 11, the method of comparative example 3 was basically the same as that of Example 7, and the method of comparative example 4 was basically the same as that of Example 8, except that the volume ratio of ether, alcohol and ketone organic solvents was changed in each comparative example, as shown in Table 2 for details.

[0064] Table 2

[0065]

[0066] Comparative Example 7

[0067] This comparative example provides a decellularized matrix scaffold material, and the transmittance is tested. The specific preparation method is basically the same as the method in Example 2, except that in step 7), the SIS material is immersed in a dehydrating liquid until the water content of the SIS material is 13%. The transmittance of the final product is 51%.

[0068] Comparative Example 8

[0069] This comparative example provides a decellularized matrix scaffold material, and the transmittance is tested. The specific preparation method is basically the same as the method in Example 5, except that in step 8), the volume ratio of dehydration liquid to DMF is 4.6:1. The transmittance of the final product is 53%.

[0070] Comparative Example 9

[0071] This comparative example provides a decellularized matrix scaffold material, and the transmittance is tested. The specific preparation method is basically the same as the method in Example 12, except that acetic acid is used instead of propanol in the dehydration solution. The transmittance of the final product is 41%.

[0072] Comparative Example 10

[0073] This comparative example provides a decellularized matrix scaffold material, and the light transmittance is tested. The specific preparation method is basically the same as the method of Example 13, except that trichloroethylene is used to replace the propyl ether in the dehydrating liquid. The final product has a light transmittance of 54%.

[0074] Comparative Example 11

[0075] This comparative example provides a decellularized matrix scaffold material, and the transmittance is tested. The specific preparation method is basically the same as the method of Example 13, except that chloroform is used to replace acetone in the dehydration solution. The transmittance of the final product is 49%.

[0076] Comparative Example 12

[0077] This comparative example provides a decellularized matrix scaffold material, and the light transmittance is tested. The specific preparation method is basically the same as the method of Example 14, except that DMSO is used instead of DMF. The light transmittance of the final product is 52%.

[0078] Experimental Example 1

[0079] This experimental example further tests the cytotoxicity of the decellularized matrix scaffold material prepared in the above examples and comparative examples, and specifically uses mouse fibroblast L929 cells for detection according to the national standard GB / T 16886.5-2017 "Biological Evaluation of Medical Devices Part 5: In Vitro Cytotoxicity Test". The test results are shown in Table 3.

[0080] Table 3

[0081]

[0082] Experimental Example 2

[0083] This experimental example further tests the repair effect of the decellularized matrix scaffold material (transparentized SIS material) prepared in Example 12 on the corneal epithelium of rabbits. Specifically, 6 male New Zealand rabbits weighing 1.8-2.2 kg were selected, and the shallow corneal structure was drilled using a circular negative pressure trephine. The left eye was used as the test group and the right eye was used as the control group for the experiment. The control group was implanted with the biological amniotic membrane of the marketed product (Jiangxi Ruiji Bioengineering Technology Co., Ltd.; National Medical Device Registration No. 20173460958), and the test group was implanted with the decellularized matrix scaffold material prepared by the present invention.

[0084] The corneal recovery of all rabbits was observed from 1 week to 8 weeks after surgery. At 8 weeks after surgery, 2 rabbits (T5 and T6) were randomly selected, the repaired parts were taken, embedded in paraffin, and fixed, and then HE staining was performed to observe the corneal defect repair effect.

[0085] All rabbits were rated according to the rating criteria in Table 4, and the average values ​​of each group at each period were calculated. The results are shown in Table 5. Representative photos of the cornea at each week after surgery under slit lamp are shown in Figure 1 The HE staining results are shown in Table 6 and representative staining photos are shown in Figure 2 .

[0086] Table 4 Corneal transparency rating standards

[0087]

[0088] Table 5 Mean corneal transparency test results from 1 week to 8 weeks after surgery

[0089]

[0090] Table 6 Experimental results 8 weeks after surgery

[0091]

[0092] From the above results, it can be seen that the transparency of the patch of the animals in the test group in the early recovery stage of 1-8 weeks was higher than that of the biological amniotic membrane product, and the iris texture was clearer under the slit lamp, providing the animals with early vision in corneal recovery. The application effects of other embodiments of the present invention are similar to those of embodiment 12.

[0093] Experimental Example 3

[0094] This experimental example tests the suture strength of the acellular matrix scaffold material prepared in each embodiment and comparative example, and the specific method is as follows:

[0095] Use a universal material testing machine to test the seam strength. Soak the sample in saline for 5 min before testing. Pass nylon 11-0 suture thread through the upper 2mm of the sample and tie it into a ring. Make sure that the length of the line segment is greater than 5cm after the ring is straightened. Fix the suture thread 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 55mm / 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. If the sample slips out of the fixture during the test, another sample should be taken and re-measured. The results are shown in Table 7.

[0096] Table 7

[0097]

[0098] 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 decellularized matrix scaffold material prepared from animal membrane tissue, characterized in that: The light transmittance is 60%-88%, and the suture strength is 2.05N-3.5N; The preparation method of the decellularized matrix scaffold material comprises the step of transparentizing the decellularized animal membrane tissue, wherein the transparentizing method comprises: firstly immersing the decellularized animal membrane tissue in a dehydrating liquid until the water content of the animal membrane tissue reaches 14%-18%, and then mixing in a structure regulator for treatment; The dehydrating liquid comprises an ether organic solvent, an alcohol organic solvent and a ketone organic solvent; the volume ratio of the ether organic solvent, the alcohol organic solvent and the ketone organic solvent is (1-10):(1-3):(1-10); the ether organic solvent comprises one or more of propyl ether, ethyl ether, dipropyl ether and diisopropyl ether; the alcohol organic solvent comprises one or more of n-propanol, isopropanol, n-butanol and isobutanol; the ketone organic solvent comprises acetone and / or ketone; The structure regulator is N,N-dimethylformamide, and the volume ratio of the dehydration liquid to the structure regulator is (3-4.2):

1.

2. The acellular matrix scaffold material according to claim 1, characterized in that: The volume ratio of the ether organic solvent, the alcohol organic solvent and the ketone organic solvent is (1.5-3.5):(1-3):(1-9).

3. The acellular matrix scaffold material according to claim 1 or 2, characterized in that: When the decellularized animal membrane tissue is immersed, the mass volume ratio of the decellularized animal membrane tissue to the dehydration liquid is 1: (90-110) g / mL.

4. The acellular matrix scaffold material according to claim 1 or 2, characterized in that: The treatment time with the structure regulator is 1-2 hours.

5. The acellular matrix scaffold material according to claim 3, characterized in that: The treatment time with the structure regulator is 1-2 hours.

6. The acellular matrix scaffold material according to any one of claims 1, 2 and 5, characterized in that: The method for preparing the decellularized animal membrane tissue comprises: washing; removing the muscle layer and the serous membrane layer; defatting; and decellularizing.

7. The acellular matrix scaffold material according to claim 3, characterized in that: The method for preparing the decellularized animal membrane tissue comprises: washing; removing the muscle layer and the serous membrane layer; defatting; and decellularizing.

8. The acellular matrix scaffold material according to claim 4, characterized in that: The method for preparing the decellularized animal membrane tissue comprises: washing; removing the muscle layer and the serous membrane layer; defatting; and decellularizing.

9. The acellular matrix scaffold material according to any one of claims 1, 2, 5, 7, and 8, characterized in that: The animal membrane tissue is from the small intestine or amnion.

10. The acellular matrix scaffold material according to claim 3, characterized in that: The animal membrane tissue is from the small intestine or amnion.

11. The acellular matrix scaffold material according to claim 4, characterized in that: The animal membrane tissue is from the small intestine or amnion.

12. The acellular matrix scaffold material according to claim 6, characterized in that: The animal membrane tissue is from the small intestine or amnion.

13. The acellular matrix scaffold material according to claim 9, characterized in that: The animal membrane tissue is from cattle or pigs.

14. The acellular matrix scaffold material according to any one of claims 10 to 12, characterized in that: The animal membrane tissue is from cattle or pigs.

15. The acellular matrix scaffold material according to any one of claims 1, 2, 5, 7, 8, 10-13, characterized in that: When the acellular matrix scaffold material is prepared, after the transparent treatment is completed, a drying step is also included.

16. The acellular matrix scaffold material according to claim 3, characterized in that: When the acellular matrix scaffold material is prepared, after the transparent treatment is completed, a drying step is also included.

17. The acellular matrix scaffold material according to claim 4, characterized in that: When the acellular matrix scaffold material is prepared, after the transparent treatment is completed, a drying step is also included.

18. The acellular matrix scaffold material according to claim 6, characterized in that: When the acellular matrix scaffold material is prepared, after the transparent treatment is completed, a drying step is also included.

19. The acellular matrix scaffold material according to claim 9, characterized in that: When the acellular matrix scaffold material is prepared, after the transparent treatment is completed, a drying step is also included.

20. The acellular matrix scaffold material according to claim 14, characterized in that: When the acellular matrix scaffold material is prepared, after the transparent treatment is completed, a drying step is also included.

21. The acellular matrix scaffold material according to claim 15, characterized in that: The drying is vacuum freeze drying.

22. The acellular matrix scaffold material according to any one of claims 16 to 20, characterized in that: The drying is vacuum freeze drying.

23. A method for preparing a decellularized matrix scaffold material, characterized in that: The preparation method is as described in any one of claims 1 to 22.

24. Use of the acellular matrix scaffold material according to any one of claims 1 to 22 in preparing a biological patch.

25. The use according to claim 24, characterized in that The biological patch is a cornea repair patch, a skin repair patch or an abdominal cavity repair patch.

26. A biological patch, characterized in that: The invention comprises the acellular matrix scaffold material according to any one of claims 1 to 22.

27. The biological patch according to claim 26, characterized in that: The biological patch is a cornea repair patch, a skin repair patch or an abdominal cavity repair patch.

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