Cross-linked amniotic membrane material and preparation method and application thereof

Abstract

The present application belongs to the technical field of biomedical materials, and relates to a treatment material for eye diseases, in particular to a cross-linked amniotic membrane material and a preparation method and application thereof. A mechanical method is used to separate a chorion layer of a fetal membrane, and a sponge layer and an amniotic membrane layer are retained, denoted as an amniotic membrane / sponge layer, and the amniotic membrane / sponge layer is cleaned; the amniotic membrane / sponge layer is placed under a condition of 15-40 DEG C for drying, so that the water content of the dried amniotic membrane / sponge layer is 10-20%; and the dried amniotic membrane / sponge layer is placed in a low-concentration cross-linking agent solution for cross-linking, and the cross-linked amniotic membrane material is obtained. The cross-linked amniotic membrane material prepared by the present application not only has suitable mechanical properties, is beneficial to the operation of an ophthalmic surgery, but also has a relatively fast degradation speed, avoids causing a foreign body reaction, and can avoid the damage to active substances of the amniotic membrane. The preparation method of the present application is simple and can avoid the toxic effect of a residual cross-linking agent.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, and relates to therapeutic materials for eye diseases, specifically to a cross-linked amniotic membrane material and its preparation method and application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Amniotic membrane materials are currently widely used in the surgical treatment of various eye diseases, such as pterygium, glaucoma, and corneal injury. The amniotic membrane has a relatively dense basement membrane and compact layer, while the spongy layer has a loose, gel-like structure that easily separates from the main body of the amniotic membrane during processing. Therefore, the spongy layer generally needs to be removed during the preparation of amniotic membrane materials. However, amniotic membrane materials prepared using traditional methods are relatively thin, prone to curling, difficult to flatten during surgery, and prone to tearing during sutures. According to the inventors' research, to overcome this drawback, cross-linking technology can be used to prepare amniotic membrane products. Because the amniotic membrane itself has poor mechanical properties, a high concentration of cross-linking agent is often required to achieve a good improvement effect, thereby enhancing the mechanical properties of the amniotic membrane tissue. However, this method significantly prolongs the degradation time after cross-linking. For applications such as ophthalmology where the amniotic membrane is needed for temporary coverage and isolation, excessively long degradation times may cause foreign body reactions and hinder the healing of the tissue itself. Alternatively, amniotic membrane can be combined with other natural or synthetic materials (such as animal pericardium, acrylamide hydrogel, PCL fiber membrane, etc.) to improve its mechanical properties. However, this involves the introduction of multiple components such as adhesives, crosslinking agents, and other polymer materials, and the complex processing procedure severely affects the excellent biological properties of the amniotic membrane itself, and also presents the problem of prolonged degradation time. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a cross-linked amniotic membrane material, its preparation method, and its applications. The cross-linked amniotic membrane material prepared by this invention not only possesses suitable mechanical properties, facilitating ophthalmic surgery, but also exhibits a rapid degradation rate, avoiding foreign body reactions and preventing damage to the active substances in the amniotic membrane. The preparation method of this invention is simple and avoids the toxic effects of residual cross-linking agents.

[0005] To achieve the above objectives, the technical solution of the present invention is as follows:

[0006] On the one hand, a method for preparing a cross-linked amnion material includes the following steps:

[0007] The chorionic membrane layer of the fetal membranes was peeled off mechanically, leaving the spongy layer and the amniotic membrane layer, referred to as the amniotic membrane / sponge layer, and the amniotic membrane / sponge layer was cleaned.

[0008] The amniotic membrane / sponge layer was dried at 15–40°C to achieve a moisture content of 10–20% after drying.

[0009] The dried amniotic membrane / sponge layer is placed in a low-concentration crosslinking agent solution for crosslinking to obtain the product; the low-concentration crosslinking agent solution contains a crosslinking agent, and the mass concentration of the crosslinking agent is not higher than 0.5%.

[0010] This invention improves the mechanical properties of the cross-linked amniotic membrane material by retaining the sponge layer during preparation and then performing cross-linking in a low-concentration cross-linking agent solution, while simultaneously reducing the concentration of the cross-linking agent. However, research has found that retaining the sponge layer during cross-linking leads to water absorption and swelling, significantly increasing thickness and reducing transparency, making it unsuitable as a treatment material for ophthalmic surgery. This invention addresses this by controlling the drying temperature before cross-linking. By controlling the moisture content of the amniotic membrane / sponge layer, a tight bond between the sponge and amniotic membrane layers is maintained, thus avoiding the problems of water absorption and swelling, significant thickness increase, and reduced transparency caused by cross-linking. Temperature control during drying aims to prevent damage to the active substances in the amniotic membrane.

[0011] On the other hand, a cross-linked amniotic membrane material is obtained by the above preparation method.

[0012] Thirdly, the application of the aforementioned cross-linked amniotic membrane material in the preparation of materials for surgical treatment of ophthalmic diseases.

[0013] Fourthly, a surgical treatment method for ophthalmic diseases, using the aforementioned cross-linked amniotic membrane material to repair, temporarily cover, and / or isolate the diseased area.

[0014] The beneficial effects of this invention are as follows:

[0015] This invention combines the amniotic membrane and the sponge layer together, requiring only a low concentration of crosslinking agent to obtain an amniotic membrane sample with good mechanical properties. It is soft, does not curl when held, is easy to flatten, facilitates surgical operations, and has a degradation rate close to that of natural amniotic membrane. At the same time, it avoids the damage to the active substances of the amniotic membrane caused by repeated washing after using high doses of crosslinking agent, as well as the toxic effects of crosslinking agent residue. Attached Figure Description

[0016] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0017] Figure 1 This is an image showing the appearance of the cross-linked amniotic membrane material prepared in Example 1 of the present invention when it is clamped.

[0018] Figure 2 This is an image showing the appearance of the cross-linked amniotic membrane material prepared in Example 2 of the present invention when it is clamped.

[0019] Figure 3 This is an image showing the appearance of the cross-linked amniotic membrane material prepared in Comparative Example 1 of this invention when it is being held in place;

[0020] Figure 4 This is an image showing the appearance of the cross-linked amniotic membrane material prepared in Comparative Example 2 of this invention when it is being held in place;

[0021] Figure 5 This is an image showing the appearance of the cross-linked amniotic membrane material prepared in Comparative Example 3 of the present invention when it is clamped.

[0022] Figure 6 This is an image showing the appearance of the cross-linked amniotic membrane material prepared in Comparative Example 4 of this invention when it is being held in place.

[0023] Figure 7 This is an image of the cross-linked amniotic membrane material prepared in Comparative Example 5 of the present invention when laid flat.

[0024] Figure 8 This is an image showing the appearance of the cross-linked amniotic membrane material prepared in Comparative Example 5 of the present invention when it is clamped.

[0025] Figure 9 This is an image showing the appearance of the amnion when it is held in place by an uncrosslinked amnion.

[0026] Figure 10 The bar chart shows the growth factor content of the cross-linked amniotic membrane materials prepared in Examples 1 and 2 of this invention. Detailed Implementation

[0027] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0028] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0029] To address the issue that existing amniotic membrane materials cannot simultaneously achieve optimal mechanical properties, degradation time, and biological performance, this invention proposes a cross-linked amniotic membrane material, its preparation method, and its applications.

[0030] A typical embodiment of the present invention provides a method for preparing a cross-linked amniotic membrane material, comprising the following steps:

[0031] The chorionic membrane layer of the fetal membranes was peeled off mechanically, leaving the spongy layer and the amniotic membrane layer, referred to as the amniotic membrane / sponge layer, and the amniotic membrane / sponge layer was cleaned.

[0032] The amniotic membrane / sponge layer was dried at 15–40°C to achieve a moisture content of 10–20% after drying.

[0033] The dried amniotic membrane / sponge layer is placed in a low-concentration crosslinking agent solution for crosslinking to obtain the product; the low-concentration crosslinking agent solution contains a crosslinking agent, and the mass concentration of the crosslinking agent is not higher than 0.5%.

[0034] In some embodiments, the donors who obtained the amniotic membranes tested negative for HBsAg, HIV, HBV, BCV, and syphilis. This ensures the safety of the prepared cross-linked amniotic membrane material at the raw material level.

[0035] In some embodiments, the amnion / sponge layer is washed with physiological saline. This method can better remove blood cells and impurities on the amnion, avoiding the influence of blood cells and impurities on the prepared cross-linked amnion material.

[0036] In some embodiments, the drying time is 10–40 hours. This condition better ensures that the moisture content of the amniotic membrane / sponge layer is 10–20%.

[0037] In some embodiments, the crosslinking agent used in the crosslinking agent solution is an epoxide crosslinking agent, a dialdehyde crosslinking agent, EDC / NHS, or genipin. The dialdehyde crosslinking agent can be glutaraldehyde, dialdehyde starch, dialdehyde hyaluronic acid, dialdehyde dextran, or dialdehyde carboxymethyl cellulose. The epoxide crosslinking agent can be 1,4-butanediol diglycidyl ether, epichlorohydrin, ethylene glycol diglycidyl ether, or polyglycerol polyglycidyl ether. Studies have shown that using epoxide crosslinking agents, especially 1,4-butanediol diglycidyl ether, yields better crosslinking results.

[0038] The suitable crosslinking pH varies depending on the crosslinking system. 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 crosslinking agent solution is 8–11. Under these conditions, 1,4-butanediol diglycidyl ether is more conducive to exerting its crosslinking effect.

[0039] The volume of low-concentration crosslinking agent solution added per square centimeter of amnion / sponge layer is 5–15 mL, preferably 9–11 mL. This ensures more thorough crosslinking.

[0040] In this invention, the dried amniotic membrane / sponge layer is placed in a low-concentration crosslinking agent solution for crosslinking, with the goal of achieving sufficient crosslinking. Different crosslinking times are used for different crosslinking systems; for example, EDC / NHS crosslinking time is 12h–30h; dialdehyde starch crosslinking time is 10min–60min; glutaraldehyde crosslinking time is 12h–36h; and so on. In some embodiments, the crosslinking time is 60h–80h. This condition is more conducive to the sufficient crosslinking of 1,4-butanediol diglycidyl ether.

[0041] In some embodiments, the crosslinking agent is washed with physiological saline to remove unreacted crosslinking agents.

[0042] In some embodiments, the cross-linked amniotic membrane material is sterilized by gamma irradiation.

[0043] Another embodiment of the present invention provides a cross-linked amniotic membrane material obtained by the above preparation method.

[0044] A third embodiment of the present invention provides the application of the above-mentioned cross-linked amniotic membrane material in the preparation of materials for surgical treatment of ophthalmic diseases.

[0045] A fourth embodiment of the present invention provides a method for surgically treating ophthalmic diseases, which uses the above-mentioned cross-linked amniotic membrane material to repair, temporarily cover and / or isolate the diseased area.

[0046] For example, the cross-linked amniotic membrane material is used for ophthalmic surgeries such as corneal reconstruction, conjunctival defect repair, corneal ulcer treatment, and pterygium treatment.

[0047] To enable those skilled in the art to better 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.

[0048] Example 1

[0049] Fetal membranes were obtained under aseptic conditions from healthy mothers who tested negative for HBsAg, HIV, HBV, BCV, and syphilis via cesarean section. The chorionic villus layer was bluntly detached, retaining the spongy layer and amnion layer, and washed with physiological saline for 1 hour. The amnion / spongy layer was dried at 15°C for 10 hours, and the moisture content was determined to be 20% by loss on drying, ensuring a tight bond between the two layers. At room temperature, the amnion / spongy layer was cross-linked in a 0.05% (w / w) aqueous solution of 1,4-butanediol diglycidyl ether, with a cross-linking agent solution volume to amnion / spongy layer area ratio of 10 mL: 1 cm². 2The pH of the crosslinking system was adjusted to 10, and the crosslinking time was 72 hours. The crosslinked amnion / sponge layer was washed twice with physiological saline, 1 hour each time. The washed amnion / sponge layer was then laid on nitrocellulose filter paper, sealed in a PAPE bag, and sterilized by Co-60 irradiation to obtain the crosslinked amnion material.

[0050] Example 2

[0051] Fetal membranes were obtained under aseptic conditions from healthy mothers who tested negative for HBsAg, HIV, HBV, BCV, and syphilis via cesarean section. The chorionic villus layer was bluntly detached, retaining the spongy layer and amnion layer, and washed with physiological saline for 1 hour. The amnion / spongy layer was dried at 30°C for 15 hours, and the moisture content was determined to be 15% by loss on drying, ensuring a tight bond between the two layers. At room temperature, the amnion / spongy layer was cross-linked in a 0.1% (w / w) aqueous solution of 1,4-butanediol diglycidyl ether, with a cross-linking agent solution volume to amnion / spongy layer area ratio of 10 mL: 1 cm². 2 The pH of the crosslinking system was adjusted to 8, and the crosslinking time was 60 hours. The crosslinked amnion / sponge layer was washed three times with physiological saline, one hour each time. The washed amnion / sponge layer was then laid on nitrocellulose filter paper, sealed in a PAPE bag, and sterilized by Co-60 irradiation to obtain the crosslinked amnion material.

[0052] Comparative Example 1

[0053] Amniotic membranes were obtained under aseptic conditions from healthy mothers who tested negative for HBsAg, HIV, HBV, BCV, and syphilis via cesarean section. The chorionic membrane layer was bluntly removed, and the spongy layer was scraped off with a cytometer. The membranes were then washed with physiological saline for 1 hour. At room temperature, the amniotic membranes were cross-linked in a 0.1% (w / w) aqueous solution of 1,4-butanediol diglycidyl ether, with a cross-linking agent solution volume to amniotic membrane area ratio of 10 mL: 1 cm². 2 The pH of the crosslinking system was adjusted to 10, and the crosslinking time was 72 hours. The crosslinked amniotic membrane was washed three times with physiological saline for 1 hour each time, laid on nitrocellulose filter paper, sealed in PAPE bags, and sterilized by Co-60 irradiation to obtain the crosslinked amniotic membrane material.

[0054] Comparative Example 2

[0055] Amniotic membranes were obtained under aseptic conditions from healthy mothers who tested negative for HBsAg, HIV, HBV, BCV, and syphilis via cesarean section. The chorionic villus layer was bluntly removed, and the spongy layer was scraped off with a cytometer. The membranes were then washed with physiological saline for 1 hour. At room temperature, the amniotic membranes were cross-linked in a 1% (w / w) aqueous solution of 1,4-butanediol diglycidyl ether, with a cross-linking agent solution volume to amniotic membrane area ratio of 10 mL: 1 cm². 2The pH of the crosslinking system was adjusted to 10, and the crosslinking time was 72 hours. The crosslinked amniotic membrane was washed three times with physiological saline for 1 hour each time, laid on nitrocellulose filter paper, sealed in PAPE bags, and sterilized by Co-60 irradiation to obtain the crosslinked amniotic membrane material.

[0056] Comparative Example 3

[0057] Amniotic membranes were obtained under aseptic conditions from healthy mothers who tested negative for HBsAg, HIV, HBV, BCV, and syphilis via cesarean section. The chorionic villus layer was bluntly removed, and the spongy layer was scraped off with a cytometer. The membranes were then washed with physiological saline for 1 hour. At room temperature, the amniotic membranes were cross-linked in a 2% (w / w) aqueous solution of 1,4-butanediol diglycidyl ether. The volume ratio of the cross-linking agent solution to the area of ​​the amniotic membrane was 10 mL: 1 cm². 2 The pH of the crosslinking system was adjusted to 10, and the crosslinking time was 72 hours. The crosslinked amniotic membrane was washed three times with physiological saline for 1 hour each time, laid on nitrocellulose filter paper, sealed in PAPE bags, and sterilized by Co-60 irradiation to obtain the crosslinked amniotic membrane material.

[0058] Comparative Example 4

[0059] Amniotic membranes were obtained under aseptic conditions from healthy mothers who tested negative for HBsAg, HIV, HBV, BCV, and syphilis via cesarean section. The chorionic villi were bluntly removed, and the spongy layer was scraped off with a cytometer. The membranes were then washed with physiological saline for 1 hour. At room temperature, the amniotic membranes were cross-linked in a 10% (w / w) aqueous solution of 1,4-butanediol diglycidyl ether. The volume ratio of the cross-linking agent solution to the area of ​​the amniotic membrane was 10 mL: 1 cm². 2 The pH of the crosslinking system was adjusted to 10, and the crosslinking time was 72 hours. The crosslinked amniotic membrane was washed three times with physiological saline for 1 hour each time, laid on nitrocellulose filter paper, sealed in PAPE bags, and sterilized by Co-60 irradiation to obtain the crosslinked amniotic membrane material.

[0060] Comparative Example 5

[0061] Fetal membranes were obtained under aseptic conditions from healthy mothers who tested negative for HBsAg, HIV, HBV, BCV, and syphilis via cesarean section. The chorionic villus layer was bluntly detached, while the spongy layer and amnion layer were preserved. The membranes were then washed with physiological saline for 1 hour. At room temperature, the amnion / spongy layer was cross-linked in a 0.1% (w / w) aqueous solution of 1,4-butanediol diglycidyl ether. The ratio of the cross-linking agent solution volume to the area of ​​the amnion / spongy layer was 10 mL: 1 cm². 2 The pH of the crosslinking system was adjusted to 10, and the crosslinking time was 72 hours. The crosslinked amnion / sponge layer was washed three times with physiological saline, one hour each time. The washed amnion / sponge layer was then laid on nitrocellulose filter paper, sealed in a PAPE bag, and sterilized by Co-60 irradiation to obtain the crosslinked amnion material.

[0062] Performance testing:

[0063] 1. Sample appearance:

[0064] The appearance of the cross-linked amniotic membrane materials prepared in Examples 1 and 2 and Comparative Examples 1 to 5 of this invention is as follows: Figures 1-8 As shown, the uncrosslinked amnion appears as follows: Figure 9 As shown. The cross-linked amniotic membrane materials prepared in Examples 1 and 2 have good mechanical properties, are not easily curled when clamped, and are easy to flatten, making them easy to operate in ophthalmic surgery.

[0065] The cross-linked amniotic membrane materials prepared using only the amniotic membrane layer in Comparative Examples 1-4 require high concentration conditions to achieve the same performance as the cross-linked amniotic membrane materials prepared in Examples 1-2.

[0066] The amniotic membrane and spongy layer of the sample obtained in Comparative Example 5 separated to some extent after soaking. The sample absorbed water and swelled, with a significant increase in thickness and a decrease in transparency, which is not conducive to ophthalmic surgery.

[0067] 2. In vitro degradation time:

[0068] The cross-linked amniotic membrane materials obtained in Examples 1 and 2 and Comparative Examples 1-4, as well as fresh amniotic membranes, were cut into 10 mm × 10 mm pieces and placed in Erlenmeyer flasks. 100 ml of 1% pepsin hydrochloric acid solution (c(HCl) = 0.1 mol / L) preheated to 37 °C was added, and the flasks were shaken in a 37 °C shaking incubator. The digestion time was recorded. The results are shown in Table 1.

[0069] Table 1. Complete digestion and degradation time for each sample

[0070] sample Digestion time fresh amnion 4.5h Cross-linked amnion material prepared in Example 1 7.5h Cross-linked amniotic membrane material prepared in Example 2 13h Cross-linked amnion material prepared in Comparative Example 1 12h Cross-linked amniotic membrane material prepared in Comparative Example 2 57h Cross-linked amniotic membrane material prepared in Comparative Example 3 More than 1 week without degradation Cross-linked amnion material prepared in Comparative Example 4 More than 1 week without degradation

[0071] Table 1 shows that the degradation time of the cross-linked amniotic membrane materials prepared in Examples 1-2 is close to that of fresh amniotic membrane. The degradation time of the sample obtained in Comparative Example 1 at a cross-linking agent concentration of 0.1% is close to that of fresh amniotic membrane, but its mechanical properties and operability are poor. The cross-linked amniotic membrane materials prepared in Comparative Examples 2-4 all have long degradation times or even remain undegraded for a long time, which is not conducive to their use in ophthalmic surgery.

[0072] 3. Cytokine levels:

[0073] The levels of bFGF, EGF, and HGF in the samples were detected using the following kits: Human Basic Fibroblast Growth Factor (bFGF) ELISA kit (QutiCyto HumanbFGF ELISA kit, manufacturer: Xinbosheng Biotechnology Co., Ltd.), Human Epidermal Growth Factor (EGF) ELISA kit (QutiCyto Human EGF ELISA kit, manufacturer: Xinbosheng Biotechnology Co., Ltd.), and Human Hepatocyte Growth Factor (HGF) ELISA kit (QutiCyto Human HGF ELISA Kit, manufacturer: Xinbosheng Biotechnology Co., Ltd.).

[0074] Take 20 mg each of the cross-linked amniotic membrane material prepared in Examples 1 and 2 and fresh amniotic membrane after freeze-drying, cut them into small pieces, add 1 ml of lysis buffer, homogenize with a homogenizer for 2 min, centrifuge at 13000 rpm at 4℃ for 15 min, take the supernatant, and perform the detection according to the method specified in the instructions of each growth factor detection kit.

[0075] The results are as follows Figure 10 As shown, Figure 10 This indicates that the content of active factors in the cross-linked amniotic membrane materials obtained in Examples 1 and 2 is about 90% of that in fresh amniotic membrane, which can better preserve the bioactive substances of the amniotic membrane.

[0076] 4. Animal experiments:

[0077] The cross-linked amniotic membranes prepared in Examples 1 and 2, along with the uncross-linked amniotic membranes, were used for temporary covering surgery for superficial corneal defects in rabbits. Compared to the uncross-linked amniotic membrane, the cross-linked amniotic membranes prepared in Examples 1 and 2 did not curl up easily, were easy to flatten, and had good operability, thus shortening the surgical operation time. The degradation time of each sample was shown in Table 2 after macroscopic observation and pathological tissue section observation.

[0078] Table 2 Degradation time of each sample

[0079] sample Degradation time Uncrosslinked amnion 7 days Cross-linked amnion material prepared in Example 1 8 days Cross-linked amniotic membrane material prepared in Example 2 10 days

[0080] Example 3

[0081] Fetal membranes were obtained under aseptic conditions from healthy mothers who tested negative for HBsAg, HIV, HBV, BCV, and syphilis via cesarean section. The chorionic villus layer was bluntly detached, retaining the spongy layer and amnion layer, and washed with physiological saline for 1 hour. The amnion / spongy layer was dried at 20°C for 20 hours, and the moisture content was determined to be 15% by loss on drying, ensuring a tight bond between the two layers. At room temperature, the amnion / spongy layer was cross-linked in a 0.5% (w / w) dialdehyde starch aqueous solution, with a cross-linking agent solution volume to amnion / spongy layer area ratio of 10 mL: 1 cm². 2The pH of the crosslinking system was adjusted to 7, and the crosslinking time was 30 min. The crosslinked amnion / sponge layer was washed twice with physiological saline, 1 h each time. The washed amnion / sponge layer was laid on nitrocellulose filter paper, sealed in PAPE bags, and sterilized by Co-60 irradiation to obtain the crosslinked amnion material.

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

[0083] Example 4

[0084] Fetal membranes were obtained under aseptic conditions from healthy mothers who tested negative for HBsAg, HIV, HBV, BCV, and syphilis via cesarean section. The chorionic villus layer was bluntly detached, retaining the spongy layer and amnion layer, and washed with physiological saline for 1 hour. The amnion / spongy layer was dried at 40°C for 10 hours, and the moisture content was determined to be 18% by loss on drying, ensuring a tight bond between the two layers. At room temperature, the amnion / spongy layer was cross-linked in a 0.1% (w / w) glutaraldehyde aqueous solution, with a cross-linking agent solution volume to amnion / spongy layer area ratio of 10 mL: 1 cm². 2 The pH of the crosslinking system was adjusted to 4, and the crosslinking time was 20 hours. The crosslinked amnion / sponge layer was washed twice with physiological saline, 1 hour each time. The washed amnion / sponge layer was then laid on nitrocellulose filter paper, sealed in a PAPE bag, and sterilized by Co-60 irradiation to obtain the crosslinked amnion material.

[0085] Performance tests, including sample appearance, in vitro degradation time, and cytokine content, showed that the cross-linked amniotic membrane material prepared in this embodiment has good mechanical properties, is not easily curled when clamped, is easy to flatten, has a suitable degradation time, and can better preserve the bioactive substances of the amniotic membrane, which is beneficial for ophthalmic surgery.

[0086] Example 5

[0087] Fetal membranes were obtained under aseptic conditions from healthy mothers who tested negative for HBsAg, HIV, HBV, BCV, and syphilis via cesarean section. The chorionic villus layer was bluntly detached, preserving the spongy layer and amnion layer. The membranes were then washed with physiological saline for 1 hour. The amnion / spongy layer was dried at 25°C for 15 hours, and the moisture content was found to be 15%, ensuring a tight bond between the two layers. At room temperature, the amnion / spongy layer was cross-linked in a 0.15% (w / w) EDC / 0.2% (w / w) NHS aqueous solution, with a cross-linking agent solution volume to amnion / spongy layer area ratio of 10 mL: 1 cm². 2The pH of the crosslinking system was adjusted to 5.5, and the crosslinking time was 24 hours. The crosslinked amnion / sponge layer was washed three times with physiological saline, one hour each time. The washed amnion / sponge layer was then laid on nitrocellulose filter paper, sealed in a PAPE bag, and sterilized by Co-60 irradiation to obtain the crosslinked amnion material.

[0088] Performance tests, including sample appearance, in vitro degradation time, and cytokine content, showed that the cross-linked amniotic membrane material prepared in this embodiment has good mechanical properties, is not easily curled when clamped, is easy to flatten, has a suitable degradation time, and can better preserve the bioactive substances of the amniotic membrane, which is beneficial for ophthalmic surgery.

[0089] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a cross-linked amnion material, characterized in that, Includes the following steps: The chorionic membrane layer of the fetal membranes was peeled off mechanically, leaving the spongy layer and the amniotic membrane layer, referred to as the amniotic membrane / sponge layer, and the amniotic membrane / sponge layer was cleaned. The amniotic membrane / sponge layer was dried at 15~40℃ to achieve a moisture content of 10~20% after drying. The dried amniotic membrane / sponge layer is placed in a low-concentration crosslinking agent solution for crosslinking to obtain the product; the low-concentration crosslinking agent solution contains a crosslinking agent, and the mass concentration of the crosslinking agent is not higher than 0.5%.

2. The method for preparing the cross-linked amniotic membrane material as described in claim 1, characterized in that, The donors who obtained the fetal membranes all tested negative for HBsAg, HIV, HBV, BCV, and syphilis.

3. The method for preparing the cross-linked amniotic membrane material as described in claim 1, characterized in that, The amnion / sponge layer was cleaned with physiological saline.

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

5. The method for preparing the cross-linked amniotic membrane material as described in claim 1, characterized in that, The crosslinking agents used in the crosslinking agent solution are epoxide crosslinking agents, dialdehyde crosslinking agents, EDC / NHS, or genipin.

6. The method for preparing the cross-linked amniotic membrane material as described in claim 5, characterized in that, The dialdehyde crosslinking agent is glutaraldehyde, dialdehyde starch, dialdehyde hyaluronic acid, dialdehyde dextran, or dialdehyde carboxymethyl cellulose.

7. The method for preparing the cross-linked amniotic membrane material as described in claim 5, characterized in that, The epoxide crosslinking agent is 1,4-butanediol diglycidyl ether, epichlorohydrin, ethylene glycol diglycidyl ether, or polyglycerol polyglycidyl ether.

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

9. The method for preparing the cross-linked amniotic membrane material as described in claim 1, characterized in that, The volume of low-concentration crosslinking agent solution added per square centimeter of amnion / sponge layer is 5~15 mL.

10. The method for preparing the cross-linked amniotic membrane material as described in claim 9, characterized in that, The volume of low-concentration crosslinking agent solution added per square centimeter of amnion / sponge layer is 9~11 mL.

11. The method for preparing the cross-linked amniotic membrane material as described in claim 1, characterized in that, The volume of low-concentration cross-linking agent solution added per square centimeter of amnion / sponge layer is such that the cross-linking time is 60-80 hours.

12. The method for preparing the cross-linked amniotic membrane material as described in claim 1, characterized in that, After cross-linking, the sample was washed with physiological saline.

13. The method for preparing the cross-linked amniotic membrane material as described in claim 1, characterized in that, The cross-linked amniotic membrane material was sterilized by gamma irradiation.

14. A cross-linked amnion material, characterized in that, Obtained by the preparation method according to any one of claims 1 to 13.

15. The use of the cross-linked amniotic membrane material of claim 14 in the preparation of materials for surgical treatment of ophthalmic diseases.

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