Placenta amnion decellularized material as well as preparation method and application thereof

Abstract

The invention relates to the technical field of tissue repair, in particular to a placenta amnion decellularized material as well as a preparation method and application thereof. The amnion is subjected to decellularization treatment by adopting a high-salt freeze-thaw combined composite enzymolysis method, and the result proves that the amnion decellularization material prepared by the invention is a high-activity bioremediation material, and the natural three-dimensional structure and bioactive components of an amnion extracellular matrix are reserved to the greatest extent; and the prepared filling material has better biocompatibility and tissue inductivity.

Description

Technical Field

[0001] The present invention relates to the technical field of tissue repair, and in particular to a placenta amniotic membrane decellularized material and a preparation method and application thereof. Background Art

[0002] Decellularized placental amniotic membrane is prepared through a decellularization process and exhibits excellent biocompatibility and low immunogenicity, making it a biomaterial with great application potential. The placental amniotic membrane itself is the innermost layer of the placenta and is rich in extracellular matrix components such as collagen, laminin, and fibronectin, as well as various growth factors such as epidermal growth factor and fibroblast growth factor. These components endow the amniotic membrane with excellent cell adhesion, proliferation, and differentiation-inducing abilities, promoting cell growth and tissue repair.

[0003] The goal of decellularization is to remove cellular components from the amniotic membrane while preserving the integrity and bioactivity of its extracellular matrix. Common methods include physical, chemical, and enzymatic methods, as well as combinations of these. Decellularized amniotic membrane material is virtually free of residual cellular components, yet retains the original extracellular matrix structure and bioactive components. For example, minimal residual DNA in decellularized amniotic membrane prevents the host from identifying xenogeneic cells as foreign, thereby reducing immune and inflammatory responses. Decellularized placental amniotic membrane has a wide range of applications in regenerative medicine. For example, in endometrial repair, decellularized amniotic membrane matrix can promote endometrial regeneration, increase endometrial thickness, enhance collagen deposition, enhance tissue regeneration and angiogenesis, and improve pregnancy outcomes, likely due to its excellent immune tolerance. In bone tissue engineering, decellularized amniotic membrane, when combined with other materials, exhibits mechanical and degradation properties comparable to cancellous bone, guiding bone tissue regeneration and promoting bone defect repair. Furthermore, decellularized amniotic membrane has been studied and applied in guided bone regeneration membranes and wound dressings, demonstrating broad application prospects.

[0004] Despite its promising prospects, the clinical application of placental amniotic membrane materials still faces challenges: there is a lack of standardized preparation methods, and the stability of active ingredients and retention of functional activity in large-scale production need further verification and development. Summary of the Invention

[0005] In response to the technical problems existing in the prior art, the present invention provides a placental amniotic membrane decellularized material and its preparation method and application. The present invention adopts high salt, freeze-thaw and combined composite enzymatic hydrolysis method to perform decellularization treatment from the amniotic membrane. The high salt pretreatment allows the cells in the amniotic membrane to absorb water, swell and break in advance, which is conducive to the subsequent use of freeze-thaw technology to remove cell fragments and residual cells. At the same time, for intracellular substances that are difficult to remove, a coenzyme enzymatic hydrolysis process is used, and a combination of trypsin and DNA enzyme is used to remove intracellular organelles and nucleic acids and other small molecules remaining in the amniotic membrane, thereby achieving efficient removal of cells in the amniotic membrane. The results confirm that the amniotic membrane decellularized material prepared by the present invention is a highly active biorepair material that retains the natural three-dimensional structure and bioactive components of the amniotic membrane extracellular matrix to the greatest extent, so that the prepared filling material has better biocompatibility and tissue induction.

[0006] Specifically, the present invention first provides a method for preparing a placenta amniotic membrane decellularized material, the method comprising the following steps: 1) Amniotic membrane washing; 2) Freeze-thaw treatment of amniotic membrane decellularized material; 3) Enzymatic treatment of amniotic membrane decellularized material.

[0007] Preferably, step 1) comprises mechanically separating the amniotic membrane from the placenta and washing it with a high salt washing solution.

[0008] Preferably, the high-salt cleaning solution in step 1) comprises 300-600 mmol / L NaCl, 5-10 mmol / L EDTA and 100-200 mmol / L Tris-HCl (pH 7.5-9.5).

[0009] Preferably, the high-salt cleaning solution in step 1) comprises 300 mmol / L NaCl, 5 mmol / L EDTA and 100 mmol / L Tris-HCl (pH 7.5).

[0010] Preferably, the high-salt cleaning solution in step 1) comprises 500 mmol / L NaCl, 8 mmol / L EDTA and 150 mmol / L Tris-HCl (pH 8.0).

[0011] Preferably, the high salt cleaning solution in step 1) comprises 600 mmol / L NaCl, 10 mmol / L EDTA and 200 mmol / L Tris-HCl pH 9.5.

[0012] Preferably, step 2) comprises evenly spreading the amniotic membrane cleaned in step 1) and then placing it in liquid nitrogen for freeze-thaw treatment. The specific process conditions include: freezing in liquid nitrogen for 5-10 minutes, quickly removing it, and thawing it in a 37°C water bath; and repeating the freezing and thawing process 3-5 times to destroy the cell structure.

[0013] Preferably, the freeze-thaw treatment process conditions in step 2) include: freezing in liquid nitrogen for 5 minutes, quickly taking out, and thawing in a 37°C water bath; and repeating the freezing and thawing process three times to destroy the cell structure.

[0014] Preferably, the freeze-thaw treatment process conditions in step 2) include: freezing in liquid nitrogen for 8 minutes, quickly taking out, and thawing in a 37°C water bath; and repeating the freezing and thawing process 4 times to destroy the cell structure.

[0015] Preferably, the freeze-thaw treatment process conditions in step 2) include: freezing in liquid nitrogen for 10 minutes, quickly taking out, and thawing in a 37°C water bath; and repeating the freezing and thawing process 5 times to destroy the cell structure.

[0016] Preferably, step 3) comprises soaking the frozen-thawed amniotic membrane in a 3-8% Triton X-100, 0.8-2% SDS and 150-300 mmol / L NaCl solution for 10-20 minutes each time, and repeating the treatment 3-5 times; then soaking the amniotic membrane in an aqueous solution containing 2000-4000 U / L trypsin and 1000-3000 U / L DNase for enzymatic hydrolysis, wherein the enzymatic hydrolysis conditions are 37°C and a shaker at 150-200 rpm for 48-96 hours; after enzymatic hydrolysis, washing is performed with a high salt washing solution.

[0017] Preferably, step 3) comprises soaking the frozen-thawed amniotic membrane in a 3% Triton X-100, 0.8% SDS and 150 mmol / L NaCl solution for 10 minutes each time, and repeating the treatment three times; then soaking the amniotic membrane in an aqueous solution containing 2000 U / L trypsin and 1000 U / L DNase for enzymatic hydrolysis, wherein the enzymatic hydrolysis conditions are 37°C and 150 rpm on a shaker for 96 hours; after enzymatic hydrolysis, washing is performed with a high salt washing solution.

[0018] Preferably, step 3) comprises soaking the frozen-thawed amniotic membrane in a 5% Triton X-100, 1% SDS and 200 mmol / L NaCl solution for 15 minutes each time, repeated 4 times; then soaking the amniotic membrane in an aqueous solution containing 3000 U / L trypsin and 2000 U / L DNase for enzymatic hydrolysis, wherein the enzymatic hydrolysis conditions are 37°C and 180 rpm on a shaker for 72 hours; after enzymatic hydrolysis, washing is performed with a high salt washing solution.

[0019] Preferably, step 3) comprises soaking the frozen-thawed amniotic membrane in an 8% Triton X-100, 2% SDS and 300 mmol / L NaCl solution for 20 minutes each time, and repeating the treatment 5 times; then soaking the amniotic membrane in an aqueous solution containing 4000 U / L trypsin and 3000 U / L DNase for enzymatic hydrolysis, wherein the enzymatic hydrolysis conditions are 37°C and a shaker at 200 rpm for 48 hours; after enzymatic hydrolysis, washing is performed with a high salt washing solution.

[0020] Furthermore, the present invention also provides a placenta amniotic membrane decellularized material obtained by the above preparation method.

[0021] Furthermore, the present invention also provides the use of the placenta amniotic membrane decellularized material obtained by the above preparation method in preparing tissue repair materials.

[0022] Preferably, the tissue repair includes but is not limited to skin, cartilage, and bone tissue.

[0023] Preferably, the tissue repair material further includes common pharmaceutical excipients.

[0024] The advantages of the present invention are as follows: The present invention uses a high-salt, freeze-thaw, and combined enzymatic hydrolysis method to decellularize the amniotic membrane. The high-salt pretreatment causes the cells in the amniotic membrane to absorb water, swell, and break in advance, which facilitates the subsequent freeze-thaw technology to remove cell fragments and residual cells. At the same time, for difficult-to-remove intracellular substances, a coenzyme enzymatic hydrolysis process is used, using a combination of trypsin and DNase to remove residual intracellular organelles and small molecules such as nucleic acids from the amniotic membrane, thereby achieving efficient removal of cells from the amniotic membrane. The results confirm that the amniotic membrane decellularized material prepared by the present invention is a highly active biorepair material that retains the natural three-dimensional structure and bioactive components of the amniotic membrane extracellular matrix to the greatest extent, making the prepared filling material have better biocompatibility and tissue induction. DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below with reference to specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0026] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative work are within the scope of protection of the present invention.

[0027] The present invention first provides a method for preparing a placenta amniotic membrane decellularized material, the method comprising the following steps: 1) Amniotic membrane washing; 2) Freeze-thaw treatment of amniotic membrane decellularized material; 3) Enzymatic treatment of amniotic membrane decellularized material.

[0028] Preferably, step 1) comprises mechanically separating the amniotic membrane from the placenta and washing it with a high salt washing solution.

[0029] Preferably, the high-salt cleaning solution in step 1) comprises 300-600 mmol / L NaCl, 5-10 mmol / L EDTA and 100-200 mmol / L Tris-HCl (pH 7.5-9.5).

[0030] Preferably, the high-salt cleaning solution in step 1) comprises 300 mmol / L NaCl, 5 mmol / L EDTA and 100 mmol / L Tris-HCl (pH 7.5).

[0031] Preferably, the high-salt cleaning solution in step 1) comprises 500 mmol / L NaCl, 8 mmol / L EDTA and 150 mmol / L Tris-HCl (pH 8.0).

[0032] Preferably, the high salt cleaning solution in step 1) comprises 600 mmol / L NaCl, 10 mmol / L EDTA and 200 mmol / L Tris-HCl pH 9.5.

[0033] Preferably, step 2) comprises evenly spreading the amniotic membrane cleaned in step 1) and then placing it in liquid nitrogen for freeze-thaw treatment. The specific process conditions include: freezing in liquid nitrogen for 5-10 minutes, quickly removing it, and thawing it in a 37°C water bath; and repeating the freezing and thawing process 3-5 times to destroy the cell structure.

[0034] Preferably, the freeze-thaw treatment process conditions in step 2) include: freezing in liquid nitrogen for 5 minutes, quickly taking out, and thawing in a 37°C water bath; and repeating the freezing and thawing process three times to destroy the cell structure.

[0035] Preferably, the freeze-thaw treatment process conditions in step 2) include: freezing in liquid nitrogen for 8 minutes, quickly taking out, and thawing in a 37°C water bath; and repeating the freezing and thawing process 4 times to destroy the cell structure.

[0036] Preferably, the freeze-thaw treatment process conditions in step 2) include: freezing in liquid nitrogen for 10 minutes, quickly taking out, and thawing in a 37°C water bath; and repeating the freezing and thawing process 5 times to destroy the cell structure.

[0037] Preferably, step 3) comprises soaking the frozen-thawed amniotic membrane in a 3-8% Triton X-100, 0.8-2% SDS and 150-300 mmol / L NaCl solution for 10-20 minutes each time, and repeating the treatment 3-5 times; then soaking the amniotic membrane in an aqueous solution containing 2000-4000 U / L trypsin and 1000-3000 U / L DNase for enzymatic hydrolysis, wherein the enzymatic hydrolysis conditions are 37°C and a shaker at 150-200 rpm for 48-96 hours; after enzymatic hydrolysis, washing is performed with a high salt washing solution.

[0038] Preferably, step 3) comprises soaking the frozen-thawed amniotic membrane in a 3% Triton X-100, 0.8% SDS and 150 mmol / L NaCl solution for 10 minutes each time, and repeating the treatment three times; then soaking the amniotic membrane in an aqueous solution containing 2000 U / L trypsin and 1000 U / L DNase for enzymatic hydrolysis, wherein the enzymatic hydrolysis conditions are 37°C and 150 rpm on a shaker for 96 hours; after enzymatic hydrolysis, washing is performed with a high salt washing solution.

[0039] Preferably, step 3) comprises soaking the frozen-thawed amniotic membrane in a 5% Triton X-100, 1% SDS and 200 mmol / L NaCl solution for 15 minutes each time, repeated 4 times; then soaking the amniotic membrane in an aqueous solution containing 3000 U / L trypsin and 2000 U / L DNase for enzymatic hydrolysis, wherein the enzymatic hydrolysis conditions are 37°C and 180 rpm on a shaker for 72 hours; after enzymatic hydrolysis, washing is performed with a high salt washing solution.

[0040] Preferably, step 3) comprises soaking the frozen-thawed amniotic membrane in an 8% Triton X-100, 2% SDS and 300 mmol / L NaCl solution for 20 minutes each time, and repeating the treatment 5 times; then soaking the amniotic membrane in an aqueous solution containing 4000 U / L trypsin and 3000 U / L DNase for enzymatic hydrolysis, wherein the enzymatic hydrolysis conditions are 37°C and a shaker at 200 rpm for 48 hours; after enzymatic hydrolysis, washing is performed with a high salt washing solution.

[0041] Furthermore, the present invention also provides a placenta amniotic membrane decellularized material obtained by the above preparation method.

[0042] Furthermore, the present invention also provides the use of the placenta amniotic membrane decellularized material obtained by the above preparation method in preparing tissue repair materials.

[0043] Preferably, the tissue repair includes but is not limited to skin, cartilage, and bone tissue.

[0044] Preferably, the tissue repair material further includes common pharmaceutical excipients.

[0045] In the examples of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0046] Example 1 A method for preparing a placenta amniotic membrane decellularized material comprises the following steps: 1) Amniotic membrane cleaning The amniotic membrane was mechanically separated from the placenta and washed with a high-salt washing solution containing 300 mmol / L NaCl, 5 mmol / L EDTA, and 100 mmol / L Tris-HCl pH 7.5. 2) Freeze-thaw treatment of amniotic membrane decellularized material The amniotic membrane cleaned in step 1) was evenly spread out and then placed in liquid nitrogen for freeze-thaw treatment. The specific process conditions included: freezing in liquid nitrogen for 5 minutes, quickly removing, and thawing in a 37°C water bath; and repeating the freezing and thawing process three times to disrupt the cell structure. 3) Enzymatic treatment of amniotic membrane decellularized material The frozen-thawed amniotic membrane was immersed in a 3% Triton X-100, 0.8% SDS, and 150 mmol / L NaCl solution for 10 minutes each time, repeated three times; then, the amniotic membrane was immersed in an aqueous solution containing 2000 U / L trypsin and 1000 U / L DNase for enzymatic hydrolysis. The enzymatic hydrolysis conditions were 37°C and 150 rpm on a shaker for 96 hours; after enzymatic hydrolysis, the membrane was washed with a high-salt cleaning solution.

[0047] Example 2 A method for preparing a placenta amniotic membrane decellularized material comprises the following steps: 1) Amniotic membrane cleaning The amniotic membrane was mechanically separated from the placenta and washed with a high-salt washing solution containing 500 mmol / L NaCl, 8 mmol / L EDTA, and 150 mmol / L Tris-HCl pH 8.0. 2) Freeze-thaw treatment of amniotic membrane decellularized material The amniotic membrane cleaned in step 1) was evenly spread out and then placed in liquid nitrogen for freeze-thaw treatment. The specific process conditions included: freezing in liquid nitrogen for 8 minutes, quickly removing, and thawing in a 37°C water bath; and repeating the freezing and thawing process four times to disrupt the cell structure. 3) Enzymatic treatment of amniotic membrane decellularized material The frozen-thawed amniotic membrane was immersed in a 5% Triton X-100, 1% SDS, and 200 mmol / L NaCl solution for 15 minutes each time, repeated four times; then, the amniotic membrane was immersed in an aqueous solution containing 3000 U / L trypsin and 2000 U / L DNase for enzymatic hydrolysis. The enzymatic hydrolysis conditions were 37°C and 180 rpm on a shaker for 72 hours; after enzymatic hydrolysis, the membrane was washed with a high-salt cleaning solution.

[0048] Example 3 A method for preparing a placenta amniotic membrane decellularized material comprises the following steps: 1) Amniotic membrane cleaning The amniotic membrane was mechanically separated from the placenta and washed with a high-salt washing solution containing 600 mmol / L NaCl, 10 mmol / L EDTA, and 200 mmol / L Tris-HCl pH 8.0. 2) Freeze-thaw treatment of amniotic membrane decellularized material The amniotic membrane cleaned in step 1) was evenly spread out and then placed in liquid nitrogen for freeze-thaw treatment. The specific process conditions included: freezing in liquid nitrogen for 10 minutes, quickly removing, and thawing in a 37°C water bath; and repeating the freezing and thawing process five times to disrupt the cell structure. 3) Enzymatic treatment of amniotic membrane decellularized material The frozen-thawed amniotic membrane was immersed in 8% Triton X-100, 2% SDS, and 300 mmol / L NaCl solution for 20 minutes each time, and the treatment was repeated 5 times; then the amniotic membrane was immersed in an aqueous solution containing 3000 U / L trypsin and 2000 U / L DNase for enzymatic hydrolysis. The enzymatic hydrolysis conditions were 37°C and 200 rpm on a shaker for 48 hours; after enzymatic hydrolysis, it was washed with a high salt cleaning solution.

[0049] Comparative Example The preparation method of Comparative Example 1 is the same as that of Example 1, except that in step 2), ultrasonic treatment is performed, and the process conditions are as follows: the frequency of the ultrasonic cleaner is 80 kHz, the power is 100 W, and the treatment time is 6 h; the remaining steps are the same as those of Example 1. The preparation steps of Comparative Example 2 are the same as those of Example 1, except that, in step 3), the product is treated with an organic solvent comprising ethanol and acetone in a mass ratio of 3:1, and the treatment conditions are soaking for 10 minutes followed by washing. The remaining steps are the same as those of the preparation method of Example 1. The preparation steps of Comparative Example 3 are the same as those of Example 1, except that 3000 U / L trypsin hydrolysis treatment is performed in step 3).

[0050] The preparation steps of Comparative Example 4 are the same as those of Example 1, except for the 3000 U / L DNA enzymatic hydrolysis treatment in step 3).

[0051] Verification test HE staining was used to analyze the decellularization effects of the above different methods. The blank group was the amniotic membrane that had not been treated by any method. The results are shown in Table 1.

[0052] Table 1. Relative cell number statistics Group Relative number of cells (%) Blank group 100 Example 1 5 Example 2 3 Example 3 2 Comparative Example 1 80 Comparative Example 2 75 Comparative Example 3 18 Comparative Example 4 21 Before decellularization, a large number of cells were present in the amniotic tissue. After treatment with the method described in any one of Examples 1-3 of the present invention, the number of cells in the amniotic membrane decreased significantly, and almost no relevant cell debris remained. However, in Comparative Examples 1-4, some cell fragments were present, and the decellularization effect was not obvious. The above experiments further confirmed that the decellularization method of the present invention using high salt, freeze-thaw and combined with a composite enzymatic hydrolysis method has a very superior technical effect. In particular, the high salt pretreatment causes the cells in the amniotic membrane to absorb water, swell and break in advance, which is conducive to the subsequent use of freeze-thaw technology to remove cell fragments and residual cells. At the same time, for intracellular substances that are difficult to remove, a coenzyme enzymatic hydrolysis process is used, and a combination of trypsin and DNase is used to remove small molecules such as intracellular organelles and nucleic acids remaining in the amniotic membrane, thereby achieving efficient removal of cells in the amniotic membrane.

[0053] To further demonstrate the superior tissue compatibility of the amniotic membrane decellularized material of the present invention, the amniotic membrane decellularized material prepared in Example 1 was co-cultured with HFF-1 fibroblasts. After 24 hours of culture, the proliferation of the HFF-1 fibroblasts in the amniotic membrane decellularized material was analyzed by MTT assay, and collagen secretion by the HFF-1 fibroblasts in the amniotic membrane decellularized material was analyzed by ELISA kits. These kits are commonly available in the art, and detailed methods can be adjusted based on the kit instructions. The results are shown in Tables 2 and 3.

[0054] Table 2. Fibroblast proliferation analysis Group OD value Example 1 Amniotic membrane decellularized material 0.189±0.024 fibroblast HFF-1 0.986±0.054 Example 1: Amniotic membrane decellularized material + fibroblast HFF-1 0.869±0.032 According to the results in Table 2, the amniotic membrane decellularized material of the present invention has good tissue compatibility, can effectively adsorb fibroblasts, and promote their proliferation activity, which is beneficial for the amniotic membrane decellularized material to exert its repair effect.

[0055] Table 3. Type I and Type III collagen secretion Group Type I collagen (ng / mL) Type III collagen (ng / mL) Example 1 Amniotic membrane decellularized material 23.54±3.65 0.25±0.08 fibroblast HFF-1 734.58±15.24 10.26±0.21 Example 1: Amniotic membrane decellularized material + fibroblast HFF-1 689.76±20.38 9.57±0.92 The results in Table 3 demonstrate that the decellularized amniotic membrane material of the present invention efficiently promotes the secretion of type I and type III collagen by fibroblasts, with comparable results to those achieved by the HFF-1 fibroblast group. These results demonstrate that the decellularized amniotic membrane material is a highly active bioremediation material. The preparation method of the present invention maximizes the preservation of the natural three-dimensional structure and bioactive components of the amniotic extracellular matrix, resulting in a filler material with enhanced biocompatibility and tissue induction.

[0056] It is important to note that the above embodiments are intended only to further illustrate and describe the technical solutions of the present invention and are not intended to further limit the technical solutions of the present invention. The methods of the present invention are merely preferred implementations and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A method for preparing a cell-free placental amniotic membrane material, characterized in that: The method comprises the following steps: 1) Amniotic membrane washing; 2) Freeze-thaw treatment of amniotic membrane decellularized material; 3) Enzymatic treatment of amniotic membrane decellularized material.

2. The method according to claim 1, wherein Step 1) involves mechanically separating the amniotic membrane from the placenta and washing it with a high-salt washing solution.

3. The method according to claim 2, wherein The high salt cleaning solution in step 1) includes 300-600 mmol / L NaCl, 5-10 mmol / L EDTA, and 100-200 mmol / L Tris-HCl (pH 7.5-9.5).

4. The method according to claim 1, wherein Step 2) includes evenly spreading the amniotic membrane cleaned in step 1) and then placing it in liquid nitrogen for freeze-thaw treatment. The specific process conditions include: freezing in liquid nitrogen for 5-10 minutes, quickly removing it, and thawing it in a 37°C water bath; and repeating the freezing and thawing process 3-5 times to destroy the cell structure.

5. The method according to claim 1, wherein Step 3) includes soaking the frozen-thawed amniotic membrane in a freeze-thaw equilibrium buffer solution for equilibrium treatment; then soaking the amniotic membrane in a composite enzymatic hydrolysis aqueous solution for enzymatic hydrolysis treatment; after enzymatic hydrolysis, washing with a high salt washing solution.

6. The method according to claim 5, wherein The freeze-thaw equilibration buffer in step 3) includes 3-8% Triton X-100, 0.8-2% SDS, and 150-300 mmol / L NaCl solution.

7. The method according to claim 5, wherein The complex enzymatic hydrolysis aqueous solution in step 3) includes an aqueous solution of 2000-4000 U / L of trypsin and 1000-3000 U / L of DNase.

8. The method according to claim 5, wherein The enzymatic hydrolysis conditions in step 3) are 37° C. and 150-200 rpm shaking for 48-96 hours.

9. The placenta amniotic membrane decellularized material prepared by the method according to any one of claims 1 to 8.

10. Use of the placental amniotic membrane decellularized material prepared by the method according to any one of claims 1 to 8 in the preparation of tissue repair materials.