Cross-linked amniotic membrane material, and preparation method therefor and use thereof

Abstract

The present invention relates to the technical field of biomedical materials, relates to a material for treating eye diseases, and in particular to a cross-linked amniotic membrane material, and a preparation method therefor and a use thereof. The preparation method comprises: mechanically peeling off a chorion layer from a fetal membrane, preserving a spongy layer and an amniotic membrane layer and recording same as an amniotic membrane / spongy layer, and cleaning the amniotic membrane / spongy layer; drying the amniotic membrane / spongy layer at a temperature of 15-40°C to enable the water content of the dried amniotic membrane / spongy layer to be 10-20%; and putting the dried amniotic membrane / spongy layer in a low-concentration crosslinker solution for cross-linking. The cross-linked amniotic membrane material prepared in the present invention has suitable mechanical properties, facilitating the operation of ophthalmic surgery, and has a fast degradation rate, avoiding causing a foreign body reaction and also avoiding damage to active substances in an amniotic membrane. The preparation method of the present invention is simple and can avoid the toxic effect of crosslinker residues.

Description

A cross-linked amniotic membrane material and its preparation method and application

[0001] The present invention claims priority to the Chinese patent application filed with the Patent Office of China on August 10, 2023, with application number 202311007590.6 and invention name “A cross-linked amniotic membrane material, its preparation method and application”, the entire contents of which are incorporated by reference into the present invention. Technical Field

[0002] The present invention belongs to the technical field of biomedical materials, relates to therapeutic materials for eye diseases, and specifically relates to a cross-linked amniotic membrane material and a preparation method and application thereof. Background Art

[0003] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to those skilled in the art.

[0004] At present, amniotic membrane materials have been widely used in the surgical treatment of various eye diseases such as pterygium, glaucoma, and corneal damage. The structure of the basement membrane and the dense layer in the amniotic membrane is relatively dense, and the spongy layer has a loose structure and is a gel-like thin layer. It is easy to separate and fall off from the main body of the amniotic membrane during processing. Therefore, the process of preparing amniotic membrane materials generally requires the removal of the spongy layer. However, the amniotic membrane materials prepared by traditional methods are relatively thin and easy to curl. They are difficult to flatten during surgical operations and are easy to tear during sutures. According to the inventor's research, in order to overcome this shortcoming, cross-linking technology can be used to prepare amniotic membrane products. Because the mechanical properties of the amniotic membrane itself are poor, a higher concentration of cross-linking agent is often required to achieve a better improvement, thereby improving the mechanical properties of the amniotic membrane tissue; however, the degradation time after cross-linking of this method is greatly prolonged. For some application scenarios such as ophthalmology that require the amniotic membrane to play a temporary covering and isolation role, a degradation time that is too long may cause a foreign body reaction, which is not conducive to the healing of the own tissue. In addition, the amniotic membrane can also be compounded with other natural or synthetic materials (such as animal pericardium, acrylamide hydrogel, PCL fiber membrane, etc.) to improve its mechanical properties. However, the introduction of multiple components such as adhesives, cross-linking agents, and other polymer materials, and the complex processing process seriously affect the excellent biological properties of the amniotic membrane itself, and there is also the problem of prolonged degradation time.

[0005] Summary of the Invention

[0006] To address the deficiencies of the prior art, the present invention provides a cross-linked amniotic membrane material, its preparation method, and its application. The cross-linked amniotic membrane material prepared by the present invention not only exhibits suitable mechanical properties, facilitating ophthalmic surgical procedures, but also exhibits rapid degradation, avoiding foreign body reactions and damage to the active substances in the amniotic membrane. The preparation method of the present invention is simple and avoids the toxic effects of residual cross-linking agents.

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

[0008] In one aspect, a method for preparing a cross-linked amniotic membrane material comprises the following steps:

[0009] The chorionic membrane was mechanically peeled off, and the spongy layer and amniotic layer were retained, recorded as the amniotic / spongy layer, and the amniotic / spongy layer was cleaned;

[0010] The amniotic membrane / sponge layer is placed under 15-40° C. for drying, so that the moisture content of the dried amniotic membrane / sponge layer is 10-20%;

[0011] The dried amniotic membrane / sponge layer is placed in a low-concentration cross-linking agent solution for cross-linking, thereby obtaining the product; the low-concentration cross-linking agent solution contains a cross-linking agent, and the mass concentration of the cross-linking agent is not higher than 0.5%.

[0012] When preparing the amniotic membrane material, the present invention increases the thickness of the amniotic membrane material by retaining the sponge layer, and then cross-linking is performed in a low concentration cross-linking agent solution, thereby improving the mechanical properties of the cross-linked amniotic membrane material and reducing the concentration of the cross-linking agent used. However, studies have found that when cross-linking is performed after retaining the sponge layer, the formed material will absorb water and swell, the thickness will greatly increase, and the transparency will deteriorate, which is not conducive to being used as a therapeutic material for ophthalmic surgery. The present invention controls the temperature for drying before cross-linking, and controls the water content of the amniotic membrane / sponge layer to control the tight connection between the sponge layer and the amniotic membrane layer, thereby avoiding the problems of water absorption and swelling, greatly increased thickness, and deteriorated transparency caused by cross-linking. The purpose of controlling the temperature during drying is to avoid damage to the active substances in the amniotic membrane.

[0013] In another aspect, a cross-linked amniotic membrane material is obtained by the above preparation method.

[0014] In a third aspect, a use of the above-mentioned cross-linked amniotic membrane material in the preparation of materials for surgical treatment of ophthalmic diseases.

[0015] In a fourth aspect, a method for surgical treatment of ophthalmic diseases is provided, wherein the cross-linked amniotic membrane material is used to repair, temporarily cover and / or isolate the diseased area.

[0016] The beneficial effects of the present invention are:

[0017] The present invention combines the amniotic membrane and the sponge layer together, and only requires a relatively low concentration of cross-linking agent for cross-linking to obtain an amniotic membrane sample with good mechanical properties. The amniotic membrane sample is soft and does not curl when clamped, is easy to flatten, and is convenient for surgical operation. It has a degradation rate close to that of natural amniotic membrane, while avoiding the damage to the active substances of the amniotic membrane caused by repeated washing after using high doses of cross-linking agents, as well as the toxic effects of residual cross-linking agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0019] FIG1 is an appearance diagram of the cross-linked amniotic membrane material prepared in Example 1 of the present invention when clamped;

[0020] FIG2 is an appearance diagram of the cross-linked amniotic membrane material prepared in Example 2 of the present invention when clamped;

[0021] FIG3 is an appearance diagram of the cross-linked amniotic membrane material prepared in Comparative Example 1 of the present invention when clamped;

[0022] FIG4 is an appearance diagram of the cross-linked amniotic membrane material prepared in Comparative Example 2 of the present invention when clamped;

[0023] FIG5 is an appearance diagram of the cross-linked amniotic membrane material prepared in Comparative Example 3 of the present invention when clamped;

[0024] FIG6 is an appearance diagram of the cross-linked amniotic membrane material prepared in Comparative Example 4 of the present invention when clamped;

[0025] FIG7 is an appearance diagram of the cross-linked amniotic membrane material prepared in Comparative Example 5 of the present invention when laid flat;

[0026] FIG8 is an appearance diagram of the cross-linked amniotic membrane material prepared in Comparative Example 5 of the present invention when clamped;

[0027] FIG9 is a bar graph showing the growth factor content of the cross-linked amniotic membrane materials prepared in Examples 1 and 2 of the present invention. DETAILED DESCRIPTION

[0028] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs.

[0029] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0030] In order to solve the problem that existing amniotic membrane materials cannot take into account mechanical properties, degradation time and biological properties, the present invention proposes a cross-linked amniotic membrane material and its preparation method and application.

[0031] A typical embodiment of the present invention provides a method for preparing a cross-linked amniotic membrane material, comprising the following steps:

[0032] The chorionic membrane was mechanically peeled off, and the spongy layer and amniotic layer were retained, recorded as the amniotic / spongy layer, and the amniotic / spongy layer was cleaned;

[0033] The amniotic membrane / sponge layer is placed under 15-40° C. for drying, so that the moisture content of the dried amniotic membrane / sponge layer is 10-20%;

[0034] The dried amniotic membrane / sponge layer is placed in a low-concentration cross-linking agent solution for cross-linking, thereby obtaining the product; the low-concentration cross-linking agent solution contains a cross-linking agent, and the mass concentration of the cross-linking agent is not higher than 0.5%.

[0035] In some embodiments, the donor of the fetal membrane is tested negative for HBsAg, HIV, HBV, BCV, and syphilis, thereby ensuring the safety of the prepared cross-linked amniotic membrane material from the raw material level.

[0036] In some embodiments, the amniotic membrane / sponge layer is washed with physiological saline, which can better remove blood cells and impurities on the amniotic membrane, thereby preventing the blood cells and impurities from affecting the prepared cross-linked amniotic membrane material.

[0037] In some embodiments, the drying time is 10 to 40 hours, which can better ensure that the water content of the amniotic membrane / sponge layer is 10 to 20%.

[0038] In some embodiments, the crosslinker used in the crosslinker solution is an epoxide crosslinker, a dialdehyde crosslinker, EDC / NHS, or genipin. The dialdehyde crosslinker can be glutaraldehyde, dialdehyde starch, dialdehyde hyaluronic acid, dialdehyde dextran, or dialdehyde carboxymethyl cellulose. The epoxide crosslinker can be 1,4-butanediol diglycidyl ether, epichlorohydrin, ethylene glycol diglycidyl ether, or polyglycerol polyglycidyl ether. Studies have shown that using epoxide crosslinkers, especially 1,4-butanediol diglycidyl ether, for crosslinking is more effective.

[0039] Different crosslinking systems have different suitable crosslinking pHs. For example, for EDC / NHS and glutaraldehyde, the suitable crosslinking pH is acidic, such as 3.5-6.0; for dialdehyde starch, the suitable crosslinking pH is neutral, and so on. In some embodiments, the pH of the crosslinker solution is 8-11. These conditions are more conducive to the crosslinking effect of 1,4-butanediol bisglycidyl ether.

[0040] In some embodiments, after cross-linking, the sample is washed with physiological saline to remove unreacted cross-linking agent.

[0041] In some embodiments, the cross-linked amniotic membrane material is sterilized by gamma irradiation after cross-linking.

[0042] Another embodiment of the present invention provides a cross-linked amniotic membrane material obtained by the above preparation method.

[0043] A third embodiment of the present invention provides a use of the cross-linked amniotic membrane material described above in preparing materials for surgical treatment of ophthalmic diseases.

[0044] A fourth embodiment of the present invention provides a method for surgical treatment of ophthalmic diseases, using the cross-linked amniotic membrane material to repair, temporarily cover and / or isolate the diseased area.

[0045] For example, the cross-linked amniotic membrane material is used to perform ophthalmic surgeries such as corneal reconstruction, conjunctival defect repair, corneal ulcer, and pterygium treatment.

[0046] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0047] Example 1

[0048] Under sterile conditions, fetal membranes were obtained by cesarean section from healthy pregnant women with negative HbsAg, HIV, HBV, BCV, and syphilis tests. The chorionic layer was mechanically stripped, and the sponge and amniotic layers were retained. The membranes were then washed with normal saline. The amniotic membrane / sponge layer was dried at 15°C for 10 hours to a moisture content of 20%, ensuring a tight fit. The amniotic membrane / sponge layer was then cross-linked in 0.05% 1,4-butanediol diglycidyl ether at a pH of 10. The cross-linked amniotic membrane / sponge layer was washed twice with normal saline for 1 hour each time. The washed amniotic membrane / sponge layer was spread on nitrocellulose filter paper and sterilized by gamma irradiation to obtain the cross-linked amniotic membrane material.

[0049] Example 2

[0050] Under sterile conditions, fetal membranes were obtained by cesarean section from healthy pregnant women with negative HbsAg, HIV, HBV, BCV, and syphilis tests. The chorionic layer was mechanically stripped, and the sponge and amniotic layers were retained. The membranes were then washed with normal saline. The amniotic membrane / sponge layer was dried at 30°C for 15 hours to a moisture content of 15%, ensuring a tight fit. The amniotic membrane / sponge layer was then cross-linked in 0.1% 1,4-butanediol diglycidyl ether at a pH of 8. The cross-linked amniotic membrane / sponge layer was washed three times with normal saline, each for 1 hour. The washed amniotic membrane / sponge layer was spread on nitrocellulose filter paper and sterilized by gamma irradiation to obtain the cross-linked amniotic membrane material.

[0051] Comparative Example 1

[0052] Under sterile conditions, fetal membranes were obtained by cesarean section from healthy pregnant women with negative HbsAg, HIV, HBV, BCV, and syphilis tests. The chorionic and spongy layers were mechanically removed and washed with normal saline. The membranes were then cross-linked in 0.1% 1,4-butanediol diglycidyl ether at a pH of 10. The cross-linked membranes were washed three times with normal saline for one hour each, then spread onto nitrocellulose filter paper and sterilized by gamma irradiation to obtain the cross-linked membrane material.

[0053] Comparative Example 2

[0054] Under sterile conditions, fetal membranes were obtained by cesarean section from healthy pregnant women with negative HbsAg, HIV, HBV, BCV, and syphilis tests. The chorionic and spongy layers were mechanically removed and washed with normal saline. The membranes were then cross-linked in 1% 1,4-butanediol diglycidyl ether at a pH of 10. The cross-linked membranes were washed three times with normal saline for one hour each, then spread onto nitrocellulose filter paper and sterilized by gamma irradiation to obtain the cross-linked membrane material.

[0055] Comparative Example 3

[0056] Under sterile conditions, fetal membranes were obtained by cesarean section from healthy pregnant women with negative HbsAg, HIV, HBV, BCV, and syphilis tests. The chorionic and spongy layers were mechanically removed and washed with saline. The membranes were then cross-linked in 2% 1,4-butanediol diglycidyl ether at a pH of 10. The cross-linked membranes were washed three times with saline for one hour each, then spread onto nitrocellulose filter paper and sterilized by gamma irradiation to obtain the cross-linked membrane material.

[0057] Comparative Example 4

[0058] Under sterile conditions, fetal membranes were obtained by cesarean section from healthy pregnant women with negative HbsAg, HIV, HBV, BCV, and syphilis tests. The chorionic and spongy layers were mechanically removed and washed with normal saline. The membranes were then cross-linked in 10% 1,4-butanediol diglycidyl ether at a pH of 10. The cross-linked membranes were washed three times with normal saline for one hour each, then spread onto nitrocellulose filter paper and sterilized by gamma irradiation to produce the cross-linked membrane material.

[0059] Comparative Example 5

[0060] Fetal membranes were obtained under sterile conditions from healthy pregnant women undergoing cesarean section and tested negative for HbsAg, HIV, HBV, BCV, and syphilis. The chorionic layer was mechanically removed, retaining the spongy and amniotic layers, and then washed with normal saline. The amniotic membrane / sponge layer was then cross-linked in 0.1% 1,4-butanediol diglycidyl ether at a pH of 10. The cross-linked amniotic membrane / sponge layer was washed three times with normal saline, each wash lasting one hour. The washed amniotic membrane / sponge layer was then spread on nitrocellulose filter paper and sterilized by gamma irradiation to obtain the cross-linked amniotic membrane material.

[0061] Performance testing:

[0062] 1. Sample appearance:

[0063] The appearance of the cross-linked amniotic membrane materials prepared in Examples 1 and 2 of the present invention and Comparative Examples 1 to 5 are shown in Figures 1 to 8. The cross-linked amniotic membrane materials prepared in Examples 1 and 2 have good mechanical properties, are not easily curled when clamped, are easy to flatten, and are easy to operate for ophthalmic surgery.

[0064] The cross-linked amniotic membrane materials prepared using only the amniotic membrane layer in Comparative Examples 1 to 4 require high concentration conditions to achieve the same performance as the cross-linked amniotic membrane materials prepared in Examples 1 to 2.

[0065] After soaking, the sample obtained in Comparative Example 5 had a certain separation between the amniotic membrane and the sponge layer, and swelled after absorbing water, with a greatly increased thickness and deteriorated transparency, which was not conducive to use in ophthalmic surgery.

[0066] 2. In vitro degradation time:

[0067] The cross-linked amniotic membrane materials obtained in Examples 1 and 2 and Comparative Examples 1 to 4, as well as fresh amniotic membrane, were cut into 10 mm x 10 mm sheets and placed in a conical flask. 100 ml of a 1% pepsin hydrochloric acid solution (c(HCl) = 0.1 mol / L) preheated to 37°C was added, and the mixture was shaken in a 37°C shaking incubator. The digestion time was recorded. The results are shown in Table 1.

[0068] Table 1 Digestion time of each sample

[0069] The results in Table 1 show that the cross-linked amniotic membrane materials prepared in Examples 1-2 exhibited degradation times similar to those of fresh amniotic membrane. The sample obtained in Comparative Example 1 exhibited degradation times similar to those of fresh amniotic membrane at a cross-linker concentration of 0.1%, but exhibited poor mechanical properties and operability. The cross-linked amniotic membrane materials prepared in Comparative Examples 2-4 exhibited prolonged degradation times, or even prolonged periods of inactivity, making them unsuitable for use in ophthalmic surgery.

[0070] 3. Cytokine content:

[0071] The human basic fibroblast growth factor (bFGF) detection kit (name: QuantiCyto Human bFGF ELISA kit, manufacturer: Xinbosheng Biotechnology Co., Ltd.), the human epidermal growth factor (EGF) detection kit (name: QuantiCyto Human EGF ELISA kit, manufacturer: Xinbosheng Biotechnology Co., Ltd.), and the human hepatocyte growth factor (HGF) detection kit (name: QuantiCyto Human HGF ELISA Kit, manufacturer: Xinbosheng Biotechnology Co., Ltd.) were used to detect the bFGF, EGF, and HGF contents in the samples.

[0072] 20 mg of the cross-linked amniotic membrane material prepared in Examples 1-2 and fresh amniotic membrane were freeze-dried and minced, added with 1 ml of lysis buffer, homogenized with a homogenizer for 2 minutes, centrifuged at 13,000 rpm at 4°C for 15 minutes, and the supernatant was collected for detection according to the method specified in the instructions of each growth factor detection kit.

[0073] The results are shown in FIG9 , which indicates that the active factor content of the cross-linked amniotic membrane material obtained in Examples 1 and 2 is about 90% of that of the fresh amniotic membrane, and that the bioactive substances of the amniotic membrane can be well preserved.

[0074] Example 3

[0075] Under sterile conditions, fetal membranes were obtained by cesarean section from healthy pregnant women with negative HbsAg, HIV, HBV, BCV, and syphilis tests. The chorionic layer was mechanically stripped, and the sponge and amniotic layers were retained and washed with normal saline. The amniotic membrane / sponge layer was dried at 20°C for 20 hours to a water content of 15%, so that the two layers were tightly fitted. The amniotic membrane / sponge layer was placed in 0.5% dialdehyde starch for cross-linking, and the pH of the cross-linking system was 7. The cross-linked amniotic membrane / sponge layer was washed twice with normal saline, each time for 1 hour. The washed amniotic membrane / sponge layer was spread on nitrocellulose filter paper and sterilized by gamma irradiation to obtain the cross-linked amniotic membrane material.

[0076] Performance tests of sample appearance, in vitro degradation time, and cytokine content showed that the cross-linked amniotic membrane material prepared in this example had good mechanical properties, was not easy to curl when clamped, was easy to flatten, had a suitable degradation time, could better preserve the bioactive substances in the amniotic membrane, and was beneficial for ophthalmic surgery.

[0077] Example 4

[0078] Under sterile conditions, the fetal membranes were obtained by cesarean section from healthy pregnant women with negative HbsAg, HIV, HBV, BCV and syphilis tests. The chorionic layer was mechanically peeled off, and the sponge layer and amniotic layer were retained and washed with normal saline. The amniotic membrane / sponge layer was dried at 40°C for 10 hours to a water content of 18%, so that the two were tightly fitted. The amniotic membrane / sponge layer was placed in 0.1% glutaraldehyde for cross-linking, and the pH of the cross-linking system was 4. The cross-linked amniotic membrane / sponge layer was washed twice with normal saline, each time for 1 hour. The washed amniotic membrane / sponge layer was spread on nitrocellulose filter paper and sterilized by gamma ray irradiation to obtain the cross-linked amniotic membrane material.

[0079] Performance tests of sample appearance, in vitro degradation time, and cytokine content showed that the cross-linked amniotic membrane material prepared in this example had good mechanical properties, was not easy to curl when clamped, was easy to flatten, had a suitable degradation time, could better preserve the bioactive substances in the amniotic membrane, and was beneficial for ophthalmic surgery.

[0080] Example 5

[0081] Under sterile conditions, fetal membranes were obtained by cesarean section from healthy pregnant women with negative HbsAg, HIV, HBV, BCV, and syphilis tests. The chorionic layer was mechanically stripped, and the sponge and amniotic layers were retained and washed with normal saline. The amniotic membrane / sponge layer was dried at 25°C for 15 hours to a water content of 15%, so that the two layers were tightly fitted. The amniotic membrane / sponge layer was placed in a 0.15% EDC / 0.2% NHS solution for cross-linking, and the pH of the cross-linking system was 5.5. The cross-linked amniotic membrane / sponge layer was washed three times with normal saline, each time for 1 hour. The washed amniotic membrane / sponge layer was spread on nitrocellulose filter paper and sterilized by gamma irradiation to obtain the cross-linked amniotic membrane material.

[0082] Performance tests of sample appearance, in vitro degradation time, and cytokine content showed that the cross-linked amniotic membrane material prepared in this example had good mechanical properties, was not easily curled when clamped, was easy to flatten, had a suitable degradation time, could well preserve the bioactive substances in the amniotic membrane, and was beneficial for ophthalmic surgery.

[0083] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A method for preparing a cross-linked amniotic membrane material, characterized in that: The steps include: The chorionic layer of the fetal membrane was mechanically peeled off, and the sponge layer and the amniotic layer were retained, recorded as the amniotic / sponge layer, and the amniotic / sponge layer was cleaned; The amniotic membrane / sponge layer is placed at 15-40° C. for drying, so that the moisture content of the dried amniotic membrane / sponge layer is 10-20%; The dried amniotic membrane / sponge layer is placed in a low-concentration cross-linking agent solution for cross-linking to obtain the product; the low-concentration cross-linking agent solution contains a cross-linking agent, and the mass concentration of the cross-linking agent is not higher than 0.5%.

2. The method for preparing the cross-linked amniotic membrane material according to claim 1, characterized in that: The results of HBsAg, HIV, HBV, BCV and syphilis tests of the donor of the fetal membrane were all negative.

3. The method for preparing the cross-linked amniotic membrane material according to claim 1, characterized in that: The amniotic membrane / sponge layer was cleaned with saline.

4. The method for preparing the cross-linked amniotic membrane material according to claim 1, characterized in that: The drying time is 10 to 40 hours.

5. The method for preparing the cross-linked amniotic membrane material according to claim 1, characterized in that: The crosslinking agent used in the crosslinking agent solution is an epoxide crosslinking agent, a dialdehyde crosslinking agent, EDC / NHS or genipin; preferably, the dialdehyde crosslinking agent is glutaraldehyde, dialdehyde starch, dialdehyde hyaluronic acid, dialdehyde dextran or dialdehyde carboxymethyl cellulose; preferably, the epoxide crosslinking agent is 1,4-butanediol diglycidyl ether, epichlorohydrin, ethylene glycol diglycidyl ether or polyglycerol polyglycidyl ether; preferably, the crosslinking agent used in the crosslinking agent solution is an epoxide crosslinking agent, and more preferably 1,4-butanediol diglycidyl ether.

6. The method for preparing the cross-linked amniotic membrane material according to claim 1, characterized in that: The pH of the crosslinking agent solution is 8-11.

7. The method for preparing the cross-linked amniotic membrane material according to claim 1, characterized in that: After cross-linking, the membranes were washed with saline.

8. The method for preparing the cross-linked amniotic membrane material according to claim 1, characterized in that: The cross-linked amniotic membrane material is sterilized by γ-ray irradiation.

9. A cross-linked amniotic membrane material, characterized in that: Obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the cross-linked amniotic membrane material according to claim 9 in preparing materials for surgical treatment of ophthalmic diseases.

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