Amniotic membrane acellular matrix for filling material and preparation method thereof

Abstract

The application relates to the technical field of tissue repair, in particular to an amniotic membrane acellular matrix for filling material and a preparation method. The amniotic membrane acellular matrix with a tissue repair function is prepared from amniotic membrane tissue, and the preparation method of the amniotic membrane acellular matrix adopts specific processes such as enzymolysis, so that the content of growth factors, collagen, glycosaminoglycan and other components in the amniotic membrane acellular matrix can be effectively improved, different types of tissue repair functions can be met, the amniotic membrane acellular matrix is suitable for being used as a personalized biological filling material, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of tissue repair, in particular to an amniotic membrane acellular matrix for filling material and a preparation method thereof. BACKGROUND

[0002] Tissue repair is the process by which the body restores damaged tissue, aiming to restore the structure and function of the tissue. Tissue repair generally includes the following stages: inflammatory response: after injury, local tissue will have an inflammatory response, manifested as hyperemia, plasma exudation and leukocyte migration, and local redness. Cell proliferation and migration: cells around the damaged tissue begin to proliferate and migrate to fill the damaged area. For example, the skin and subcutaneous tissue at the edge of the wound will move towards the center until the wound is closed. Granulation tissue formation: within 2-3 days after tissue injury, granulation tissue begins to appear, filling the wound or organizing foreign bodies. Granulation tissue is composed of newly formed capillaries, proliferating fibroblasts and loose extracellular matrix. Extracellular matrix synthesis and remodeling: fibroblasts begin to synthesize more extracellular matrix and accumulate extracellularly. Over time, the granulation tissue gradually matures into fibrous connective tissue and transforms into scar tissue.

[0003] Currently, tissue repair can be carried out in two ways:

[0004] 1. Regeneration: replacing damaged tissue with the same type of cells to complete repair. This approach is commonly used in tissues with high regenerative capacity, such as the superficial layer of the epidermis, mucosa or fetal skin.

[0005] 2. Fibrosis: involves the repair of fibrous connective tissue, particularly the formation of scar tissue. This is the main form of wound healing in adult skin.

[0006] During tissue repair, a variety of cellular and molecular mechanisms work together. For example, cell types such as fibroblasts, endothelial cells and macrophages, as well as growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF) and transforming growth factor-β (TGF-β), all play important roles in tissue repair.

[0007] Tissue repair is a complex and orderly process aimed at restoring the structure and function of damaged tissue. Although this process is effective in repairing damage in most cases, it can also lead to scar formation or other functional disorders in some cases. At present, there is no repair product that combines multiple functions in one, which is not suitable for the development of individualized repair programs and is not conducive to the development of the industry. SUMMARY

[0008] In view of the technical problems existing in the prior art, the application provides an amniotic membrane acellular matrix for filling material and a preparation method. The amniotic membrane acellular matrix having a tissue repair function is prepared from amniotic membrane tissue. The preparation method of the amniotic membrane acellular matrix adopts an enzymolysis process, so that the content of growth factors, collagen, glycosaminoglycans and other components in the amniotic membrane acellular matrix can be effectively increased, different types of tissue repair functions can be met, the amniotic membrane acellular matrix is suitable for being used as a personalized biological filling material, and the amniotic membrane acellular matrix has a wide application prospect.

[0009] Specifically, the application first provides a preparation method of an amniotic membrane acellular matrix for filling material, and the preparation method comprises the following steps:

[0010] 1) amniotic membrane pretreatment;

[0011] 2) amniotic membrane decontamination treatment;

[0012] 3) amniotic membrane acellular matrix preparation.

[0013] Preferably, step 1) comprises separating the amniotic membrane from a placenta, washing, and freeze-thawing to destroy cell structures.

[0014] Preferably, step 1) comprises mechanically separating the amniotic membrane from the placenta, washing with 0.9% physiological saline, and then performing 3-5 times of freeze-thawing treatment on the amniotic membrane to destroy cell structures.

[0015] Further preferably, step 1) comprises mechanically separating the amniotic membrane from the placenta, washing with 0.9% physiological saline, and then performing 3 times of freeze-thawing treatment on the amniotic membrane to destroy cell structures.

[0016] Further preferably, step 1) comprises mechanically separating the amniotic membrane from the placenta, washing with 0.9% physiological saline, and then performing 4 times of freeze-thawing treatment on the amniotic membrane to destroy cell structures.

[0017] Further preferably, step 1) comprises mechanically separating the amniotic membrane from the placenta, washing with 0.9% physiological saline, and then performing 5 times of freeze-thawing treatment on the amniotic membrane to destroy cell structures.

[0018] Preferably, step 2) comprises: soaking the freeze-thawed amniotic membrane in a 3-6% Triton X-100 solution, performing shaker treatment, and repeating the treatment; then continuing to perform shaker treatment by using a 4-6% deoxycholic acid sodium solution; then performing shaker treatment by using a 0.1-0.3% p-chlorobenzoic acid / 4-6% ethanol solution, and then washing.

[0019] Preferably, step 2) comprises: soaking the freeze-thaw treated amniotic membrane in a 3-6% Triton X-100 solution, 25-35°C, 150-200 rpm shaker for 2-6 hours, repeated for 3-6 times; followed by 4-6% sodium deoxycholate solution for 3-6 times, 2-4 hours each time; followed by 0.1-0.3% p-chlorobenzoic acid / 4-6% ethanol solution for 4-8 hours on a shaker, and finally rinsed with sterile PBS for 15-30 minutes, and then rinsed with sterile deionized water for 2-5 times, 15-30 minutes each time.

[0020] Further preferably, step 2) comprises: soaking the freeze-thaw treated amniotic membrane in a 3% Triton X-100 solution, 25°C, 150 rpm shaker for 2 hours, repeated for 3 times; followed by 4% sodium deoxycholate solution for 3 times, 2 hours each time; followed by 0.1% p-chlorobenzoic acid / 4% ethanol solution for 4 hours on a shaker, and finally rinsed with sterile PBS for 15 minutes, and then rinsed with sterile deionized water for 2 times, 15 minutes each time.

[0021] Further preferably, step 2) comprises: soaking the freeze-thaw treated amniotic membrane in a 5% Triton X-100 solution, 30°C, 180 rpm shaker for 4 hours, repeated for 4 times; followed by 5% sodium deoxycholate solution for 4 times, 3 hours each time; followed by 0.2% p-chlorobenzoic acid / 5% ethanol solution for 6 hours on a shaker, and finally rinsed with sterile PBS for 20 minutes, and then rinsed with sterile deionized water for 3 times, 20 minutes each time.

[0022] Further preferably, step 2) comprises: soaking the freeze-thaw treated amniotic membrane in a 6% Triton X-100 solution, 35°C, 200 rpm shaker for 6 hours, repeated for 6 times; followed by 6% sodium deoxycholate solution for 6 times, 4 hours each time; followed by 0.3% p-chlorobenzoic acid / 6% ethanol solution for 8 hours on a shaker, and finally rinsed with sterile PBS for 30 minutes, and then rinsed with sterile deionized water for 5 times, 30 minutes each time.

[0023] Preferably, step 3) comprises: soaking the rinsed amniotic membrane of step 2) in a solution containing 1-3 mg / mL of papain, 3-5 mg / mL of elastase and 0.01-0.03 N HCl, and treated on a shaker; neutralize the pH, incubate, and make the amniotic membrane decellularized matrix polymerize.

[0024] Preferably, step 3) comprises soaking the amniotic membrane after washing in step 2) in a solution containing 1-3 mg / mL of papain, 3-5 mg / mL of elastase and 0.01-0.03 N HCl on a 150-200 rpm shaker for 72-96 hours; the amniotic membrane decellularized matrix is polymerized by adding 1xPBS solution containing 0.1-0.3 N NaOH to neutralize the pH value to 7.0, and then incubated at 37°C.

[0025] Further preferably, step 3) comprises soaking the amniotic membrane after washing in step 2) in a solution containing 1 mg / mL of papain, 3 mg / mL of elastase and 0.01 N HCl on a 150 rpm shaker for 72 hours; the amniotic membrane decellularized matrix is polymerized by adding 1xPBS solution containing 0.1 N NaOH to neutralize the pH value to 7.0, and then incubated at 37°C.

[0026] Further preferably, step 3) comprises soaking the amniotic membrane after washing in step 2) in a solution containing 2 mg / mL of papain, 4 mg / mL of elastase and 0.02 N HCl on a 180 rpm shaker for 84 hours; the amniotic membrane decellularized matrix is polymerized by adding 1xPBS solution containing 0.2 N NaOH to neutralize the pH value to 7.0, and then incubated at 37°C.

[0027] Further preferably, step 3) comprises soaking the amniotic membrane after washing in step 2) in a solution containing 3 mg / mL of papain, 5 mg / mL of elastase and 0.03 N HCl on a 200 rpm shaker for 96 hours; the amniotic membrane decellularized matrix is polymerized by adding 1xPBS solution containing 0.3 N NaOH to neutralize the pH value to 7.0, and then incubated at 37°C.

[0028] Further preferably, the preparation method further comprises amniotic membrane decellularized matrix quality evaluation: analyzing the content of growth factors, collagen, glycosaminoglycans and other components in the amniotic membrane decellularized matrix prepared in the application by methods such as ELISA and SDS-PAGE.

[0029] Another aspect of the application also provides an amniotic membrane decellularized matrix prepared by the above preparation method.

[0030] Further, the application also provides the use of the amniotic membrane decellularized matrix prepared by the above preparation method in the preparation of tissue repair filling materials.

[0031] Preferably, the tissue includes but is not limited to skin, cartilage, bone tissue.

[0032] Further preferably, the tissue repair filling material further comprises a pharmaceutical excipient commonly used in pharmacy.

[0033] The advantages of the present application are as follows: the present application is prepared from amniotic membrane tissue to obtain an amniotic membrane acellular matrix with tissue repair function, and a special preparation process is used in the preparation method of the amniotic membrane acellular matrix, so that the content of growth factors, collagen, glycosaminoglycan and other components in the amniotic membrane acellular matrix can be effectively improved, different types of tissue repair functions can be met, it is suitable as a personalized biological filling material, and has a wide application prospect. DETAILED DESCRIPTION

[0034] The present application will be further described in detail below with specific examples, so that those skilled in the art can more clearly understand the present application.

[0035] The following examples are only used to illustrate the present application and are not intended to limit the scope of the present application. Based on the specific examples in the present application, all other examples obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0036] In the embodiments of the present application, all raw material components are commercially available products well known to those skilled in the art unless otherwise specified; in the embodiments of the present application, the technical means used are conventional means well known to those skilled in the art unless otherwise specified.

[0037] Papain: item number S27241, 10u / mg, source leaf biological. Elastase: item number S27996, 10u / mg, source leaf biological.

[0038] Example 1

[0039] An amniotic membrane acellular matrix for filling material, the preparation method comprises the following steps:

[0040] 1) Amniotic membrane pretreatment

[0041] The amniotic membrane is mechanically separated from the placenta and washed with 0.9% physiological saline, and then the amniotic membrane is subjected to 3 times of freeze-thaw treatment, the freeze-thaw conditions are-40℃ for 10 minutes and 37℃ for 10 minutes, to destroy the cell structure;

[0042] 2) Amniotic membrane decontamination treatment

[0043] The freeze-thaw treated amniotic membrane is soaked in 3wt% Triton X-100 solution, treated at 25℃, 150rpm shaking bed for 2h, and repeatedly treated for 3 times; then 4wt% sodium deoxycholate solution is used to continue the treatment on the shaking bed for 3 times, each for 2 hours; then 0.1wt% p-chlorobenzoic acid / 4wt% ethanol aqueous solution is used to treat on the shaking bed for 4 hours, finally washed with sterile PBS for 15 minutes, and then washed with sterile deionized water for 2 times, each for 15 minutes;

[0044] 3) Preparation of amniotic membrane acellular matrix

[0045] The amniotic membrane after rinsing in step 2) is soaked in a solution containing 1 mg / mL of papain, 3 mg / mL of elastase and 0.01 N HCl on a 150 rpm shaker for 72 hours; the pH value is neutralized by adding 0.1 N NaOH in 1xPBS solution to reach 7.0, and then the amniotic membrane is incubated at 37℃ to make the amniotic membrane acellular matrix polymerize.

[0046] Quality evaluation of amniotic membrane acellular matrix: the content of growth factors, collagen, glycosaminoglycans and other components in the amniotic membrane acellular matrix prepared in the application is analyzed by ELISA and SDS-PAGE methods.

[0047] Example 2

[0048] An amniotic membrane acellular matrix for filling material, the preparation method comprising the following steps:

[0049] 1) Pretreatment of amniotic membrane

[0050] The amniotic membrane is mechanically separated from the placenta and washed with 0.9% physiological saline, and then the amniotic membrane is subjected to 4 freeze-thaw treatments, with freeze-thaw conditions of -40℃ for 10 minutes and 37℃ for 10 minutes to destroy the cell structure;

[0051] 2) Decontamination of amniotic membrane

[0052] The freeze-thaw treated amniotic membrane is soaked in a 5% Triton X-100 solution and treated on a 30℃ 180 rpm shaker for 4h, with repeated treatment for 4 times; then a 5% sodium deoxycholate solution is used to continue the treatment on the shaker for 4 times, 3 hours each time; then a 0.2% p-chlorobenzoic acid / 5% ethanol aqueous solution is used to treat on the shaker for 6 hours, and finally the amniotic membrane is rinsed with sterile PBS for 20 minutes and then with sterile deionized water for 3 times, 20 minutes each time;

[0053] 3) Preparation of amniotic membrane acellular matrix

[0054] The amniotic membrane after rinsing in step 2) is soaked in a solution containing 1 mg / mL of papain, 3 mg / mL of elastase and 0.01 N HCl on a 150 rpm shaker for 72 hours; the pH value is neutralized by adding 0.1 N NaOH in 1xPBS solution to reach 7.0, and then the amniotic membrane is incubated at 37℃ to make the amniotic membrane acellular matrix polymerize.

[0055] Quality evaluation of amniotic membrane acellular matrix: the content of growth factors, collagen, glycosaminoglycans and other components in the amniotic membrane acellular matrix prepared in the application is analyzed by ELISA and SDS-PAGE methods.

[0056] Example 3

[0057] A decellularized amniotic membrane for filling material, the preparation method comprising the following steps:

[0058] 1) Amniotic membrane pretreatment

[0059] The amniotic membrane is mechanically separated from the placenta and washed with 0.9% normal saline, and then the amniotic membrane is subjected to 5 freeze-thaw treatments, with freeze-thaw conditions of -40°C for 10 minutes and 37°C for 10 minutes, to destroy the cell structure;

[0060] 2) Amniotic membrane decontamination

[0061] The freeze-thaw treated amniotic membrane is soaked in a 6% Triton X-100 solution, treated on a 35°C 200 rpm shaker for 6 hours, and repeatedly treated 6 times; then a 6% sodium deoxycholate solution is used to continue the treatment on the shaker for 6 times, 4 hours each time; then a 0.3% p-chlorobenzoic acid / 6% ethanol aqueous solution is used to treat on the shaker for 8 hours, and finally the amniotic membrane is washed with sterile PBS for 30 minutes and sterile deionized water for 5 times, 30 minutes each time;

[0062] 3) Preparation of amniotic membrane decellularized matrix

[0063] The amniotic membrane after washing in step 2) is soaked in a solution containing 3 mg / mL papain, 5 mg / mL elastase and 0.03N HCl on a 200 rpm shaker for 96 hours; the pH value is neutralized by adding 1×PBS solution containing 0.3N NaOH to reach 7.0, and then the amniotic membrane decellularized matrix is polymerized by incubation at 37°C.

[0064] Quality evaluation of amniotic membrane decellularized matrix: the content of growth factors, collagen, glycosaminoglycans and other components in the amniotic membrane decellularized matrix prepared in the application is analyzed by ELISA and SDS-PAGE methods.

[0065] Comparative example

[0066] The preparation method steps of Comparative Example 1 are the same as those of Example 1, except that in step 3) the enzymatic hydrolysis only uses 1 mg / mL papain treatment, which is the same as the method step;

[0067] The preparation method steps of Comparative Example 2 are the same as those of Example 1, except that in step 3) the enzymatic hydrolysis only uses 3 mg / mL elastase treatment;

[0068] The preparation method steps of Comparative Example 3 are the same as those of Example 1, except that in step 3) no enzymatic hydrolysis treatment is performed.

[0069] Analysis of experimental results

[0070] The content of growth factors, collagen, glycosaminoglycans and other components in the amniotic membrane decellularized matrix prepared in the application is analyzed by ELISA and SDS-PAGE and the like. The specific evaluation and analysis method can be referred to the kit instruction. The results are shown as follows:

[0071] Table 1 Growth factor content

[0072]

[0073]

[0074] As shown in Table 1: single enzymolysis cannot effectively improve the growth factor content in the amniotic membrane decellularized matrix, which lies in that single enzymolysis cannot effectively destroy the amniotic membrane decellularization, thereby leading to the released growth factor in the matrix being significantly lower than that of double enzymolysis. The results further confirm that the use of double enzymolysis can synergistically increase the growth factor content in the amniotic membrane decellularized matrix, and the growth factor content in the amniotic membrane decellularized matrix can be further improved by increasing the enzyme amount or prolonging the enzymolysis time, showing a dose-dependent phenomenon. The above results confirm that the amniotic membrane decellularized matrix prepared in the application has certain tissue repair potential.

[0075] Table 2 Collagen content

[0076] Group Collagen Type I (ng / mL) Collagen Type III (ng / mL) Example 1 452.32±10.28 3.41±0.13 Example 2 512.69±15.22 4.13±0.25 Example 3 556.38±12.55 5.26±0.31 Comparative Example 1 180.65±6.52 1.78±0.16 Comparative Example 2 171.26±4.34 1.56±0.17 Comparative Example 3 20.35±2.57 0.23±0.15

[0077] The results are shown in Table 2, and the type I and III collagen in the amniotic membrane decellularized matrix is detected by ELISA kit. It is found that the amniotic membrane decellularized matrix prepared by any one of the methods of embodiments 1-3 has higher collagen secretion, and the difference compared with the comparative examples 1-3 is extremely significant. It is further confirmed that the preparation method of the amniotic membrane decellularized matrix in the application can effectively meet the individualized biological filling material requirements of different tissue repair (such as skin, cartilage, bone tissue and the like).

[0078] Table 3 Glycosaminoglycan content

[0079]

[0080]

[0081] The glycosaminoglycan analysis results are shown in Table 3. The combination of papain and elastase can simultaneously improve the glycosaminoglycan content in the amniotic membrane decellularized matrix, which is helpful for the application of the amniotic membrane decellularized matrix in cartilage or bone tissue injury repair. The glycosaminoglycan in the amniotic membrane decellularized matrix is used for tissue repair to improve the injury healing ability.

[0082] It is necessary to point out here that the above embodiments are only for further illustrating and describing the technical solutions of the present application, and are not for further limiting the technical solutions of the present application. The method of the present application is only a preferred embodiment, and is not used for limiting the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a decellularized amniotic membrane matrix as a filling material, characterized in that, The preparation method includes the following steps: 1) Amniotic membrane pretreatment: This includes separating the amniotic membrane from the placenta, washing, and freezing and thawing to destroy its cellular structure; 2) Amniotic membrane decontamination treatment: This includes immersing the freeze-thawed amniotic membrane in a 3-6 wt% Triton X-100 solution, shaking it, and repeating the treatment; then continuing the shaking treatment with a 4-6 wt% sodium deoxycholate solution; finally, shaking it with a 0.1-0.3 wt% p-chlorobenzoic acid / 4-6 wt% ethanol solution, followed by rinsing. 3) Preparation of decellularized amniotic membrane matrix: This includes immersing the amniotic membrane after rinsing in step 2) in a solution containing 1-3 mg / mL papain, 3-5 mg / mL elastase and 0.01-0.03 N HCl, treating it on a shaker; neutralizing the pH, incubating, and allowing the decellularized amniotic membrane matrix to polymerize.

2. The preparation method according to claim 1, characterized in that, The preparation method also includes quality assessment of the decellularized amniotic membrane matrix.

3. The preparation method according to claim 2, characterized in that, The contents of growth factors, collagen, and glycosaminoglycans were analyzed using ELISA and SDS-PAGE methods.

4. The decellularized amniotic matrix obtained by the preparation method according to any one of claims 1-3.

5. The use of the decellularized amniotic membrane matrix obtained by the preparation method according to any one of claims 1-3 in the preparation of tissue repair filling materials.

6. The use as described in claim 5, characterized in that, The tissues include skin, cartilage, and / or bone tissue.

7. The use as described in any one of claims 5-6, characterized in that, Tissue repair filling materials also include pharmaceutical excipients.

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