A suture-free acellular biological amniotic membrane ocular surface composite material and use method thereof

Abstract

The present invention discloses a suture-free decellularized biological amniotic membrane ocular surface composite material and a method of use, which belongs to the field of biomaterials. The composite material includes an amniotic membrane matrix and a support supporting the amniotic membrane matrix, wherein the amniotic membrane matrix is ​​derived from human amniotic membrane tissue material, the support includes an animal cornea or a non-biological material cornea, and the support is prepared to match the shape of a corneal contact lens with the shape of a human cornea. In clinical use, the amniotic membrane composite material can be placed in the conjunctival sac and attached to the surface of the eyeball. The decellularized biological membrane matrix can effectively protect the wound surface of the cornea, bulbar conjunctiva or palpebral conjunctiva, prevent the wound surface from adhering to normal tissue, and inhibit the occurrence of clouding. There is no need to suture the wound surface with a dressing, which is convenient for ophthalmic clinical surgery and the application of later replacement of eye dressings. It has been experimentally verified that the suture-free decellularized biological amniotic membrane ocular surface composite material prepared by the present invention can significantly improve eye diseases, and provides a new method and direction for the effective treatment of clinical eye diseases.

Description

Technical Field

[0001] The invention relates to the field of biomaterials, and in particular to a suture-free acellular biological amniotic membrane ocular surface composite material and a use method thereof. Background Art

[0002] In ophthalmology, there are a large number of diseases with large-area ocular epithelial defects that need to be covered by biofilms, such as chemical injuries, thermal burns, and intractable ocular epithelial non-healing diseases such as Stevens-Johnson syndrome, which lead to chronic corneal inflammatory damage, regardless of large-scale epithelial defects or systemic epithelial deficiency; chronic and unhealed course. All of them can lead to corneal ulcers, scar changes, blepharoplasty and even blindness.

[0003] Since scholars introduced intact amniotic membrane into ophthalmic clinical treatment in the 1990s, basic and clinical research on amniotic membrane has been gradually carried out. Amniotic membrane has been proven to promote corneal epithelial repair and rebuild the normal structure of the ocular surface. It also has anti-inflammatory, anti-scarring and anti-angiogenic effects. In recent years, many excellent animal-derived membrane materials have emerged. They have the same low antigenicity and excellent tissue compatibility, and can also be used as wound biological dressings to provide a suitable healing environment for the wound.

[0004] Chinese patent CN102631707A discloses a biomaterial based on amniotic membrane and its preparation method and use. The patent fully introduces the material collection, preparation and clinical healing effect of amniotic membrane. It has obvious healing effect on superficial injuries of cornea and conjunctiva. There is no obvious inflammatory reaction in the postoperative wound, no immune response, significantly shortens the healing time, reduces the pain of patients, and has high safety. However, it needs to be sutured on the wound surface.

[0005] Chinese patent CN1985778A discloses a bio-artificial cornea and its preparation method, which is composed of a base material of an animal cornea that has been cross-linked and fixed with a fixative and treated to remove antigens. The advantages of this invention are that it is non-immunogenic, its physical properties are similar to those of a human cornea, it has a stable morphology, and good flexibility. It can be processed into different curvatures, does not swell in water, and is an ideal carrier or scaffold. However, the bio-artificial cornea in this patent must be clinically positioned to replace the human cornea.

[0006] Chinese patent CN100353835C discloses an amniotic membrane covering for tissue surface and a device that facilitates membrane fixation, specifically discloses: a biopolymer covering for tissue surface, which includes gauze, bandages, covers such as bandage contact lenses, compositions or coverings for protecting tissues, coverings that prevent adhesion, isolate bacteria, inhibit bacterial activity, or promote tissue healing or growth. This composition particularly emphasizes an example of an amniotic membrane covering for the ocular surface, using two rings of different sizes to fix the amniotic membrane by biting the two rings. This patent lacks a support, and the device has no curvature in the middle, and cannot fully fit the patient's cornea, and is only placed in the conjunctival sac. The inventors of this group have made similar devices and found that the device cannot move with the eyeball, the position of the amniotic membrane is always fixed, and as the eyeball moves, the amniotic membrane constantly rubs against the cornea, the cornea is severely stimulated, and the subject cannot last more than ten minutes after wearing it. If this device is worn on a patient with corneal injury, the new corneal epithelium will also be constantly wiped off by the amniotic membrane device, delaying corneal healing. If this device is to be put into practical use, more design details must be fully improved.

[0007] Although the technology of using amniotic membrane as a substrate to prepare biomaterials is already available, due to the existence of various problems as mentioned above, there is an urgent need for a new biomaterial that is safer, easier to operate, and does not damage the cornea for use in the treatment of clinical ophthalmic diseases. Summary of the invention

[0008] The purpose of the present invention is to provide a suture-free acellular biological amniotic membrane ocular surface composite material and a method of use to solve the problems existing in the above-mentioned prior art. The composite material has the characteristics of suture-free placement after eye surgery and convenient multiple painless and non-invasive dressing changes, and can also significantly improve eye diseases, providing a new method and direction for the effective treatment of clinical eye diseases.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] The present invention provides a suture-free acellular biological amniotic membrane ocular surface composite material, comprising an amniotic membrane matrix and a support for supporting the amniotic membrane matrix, wherein the amniotic membrane matrix is ​​derived from human amniotic membrane tissue material, the support comprises an animal cornea or a non-biological material cornea, the support is prepared into a corneal contact lens shape that matches the shape of a human cornea, and the amniotic membrane matrix is ​​attached to the concave surface or convex surface of the support having the shape of a corneal contact lens.

[0011] Preferably, the method for preparing the amniotic matrix comprises the steps of removing antigenic components from the human amniotic tissue material and retaining a low-antigenic matrix.

[0012] Preferably, the animal cornea includes a pig cornea, which is treated with a cross-linking agent and processed into a corneal contact lens shape using a mold to make the pig cornea fit the shape of a human cornea. That is, after the pig cornea is cross-linked, the appearance has a certain curvature, which perfectly matches the human cornea.

[0013] Preferably, the cross-linking agent comprises a non-aldehyde fixing agent, and the non-aldehyde fixing agent comprises genipin or diglycidyl ethyl ether;

[0014] The treatment is a soaking treatment at a low temperature of 0-4°C for 2-6 hours.

[0015] Preferably, the non-biological cornea material includes degradable non-biological material and non-degradable non-biological material.

[0016] Preferably, the degradable non-biological material includes at least one of collagen gelatin, cellulose, hyaluronic acid or sodium alginate, and the non-degradable non-biological material includes polyacrylamide polymer or silicone polymer.

[0017] The present invention also provides a method for preparing the suture-free acellular biological amniotic membrane ocular surface composite material, comprising any one of the following methods (1)-(2):

[0018] (1) The amniotic membrane matrix is ​​flattened on the concave surface of the support without tension, freeze-dried, sealed and irradiated to obtain the suture-free acellular biomembrane ocular surface composite material

[0019] (2) After a thin layer of hydrogel is evenly applied on the convex surface of the support, the amniotic membrane matrix is ​​flattened on the hydrogel without tension, freeze-dried, sealed and irradiated to obtain the suture-free acellular biological amniotic membrane ocular surface composite material.

[0020] Preferably, the hydrogel comprises the following components in the following amounts: 1-8% chitosan, 1-20% glycerol, 1-10% lactic acid and 1-30% gelatin, with the remainder being water. All components are expressed in mass percentage.

[0021] Preferably, the irradiation condition is: the irradiation dose is 15-40Gy.

[0022] The present invention also provides a method for using the suture-free decellularized biological amniotic membrane ocular surface composite material, wherein the suture-free decellularized biological amniotic membrane ocular surface composite material is selected according to the location of the ocular wound surface;

[0023] If the wound is on the cornea and bulbar conjunctiva, choose a suture-free acellular biological amniotic membrane ocular surface composite material with the amniotic membrane matrix attached to the concave surface, and place it in the conjunctival sac on the surface of the eyeball; if the wound is on the palpebral conjunctiva side, choose a suture-free acellular biological amniotic membrane ocular surface composite material biological membrane with the amniotic membrane matrix attached to the convex surface, and place it in the conjunctival sac on the surface of the eyeball.

[0024] The present invention discloses the following technical effects:

[0025] The present invention is composed of an amniotic membrane matrix and a support with a certain supporting function (including an animal cornea or a non-biological material cornea). Specifically, the animal-derived support is a pig cornea, the corneal epithelial layer and the endothelial layer are removed, the antigen is removed, and after cross-linking, the cornea is formed in a mold similar to a corneal contact lens, and then the amniotic membrane matrix is ​​composited into a finished product, and experimental verification is carried out. Specifically, when the wound surface is on the cornea or bulbar conjunctiva side, the amniotic membrane matrix is ​​selected on the concave surface of the composite material, the composite material moves with the eyeball, and the amniotic membrane has no obvious displacement relative to the corneal wound surface, which can effectively protect the repair of the corneal epithelium; when the wound surface is located on the upper and lower eyelid conjunctiva side, the amniotic membrane matrix is ​​selected on the convex surface of the composite material, and an additional layer of hydrogel layer is added between the amniotic membrane matrix and the decellularized biomaterial (such as the pig cornea). When the amniotic membrane composite material is implanted in the patient's conjunctival sac, the hydrogel layer absorbs water and gels. When the composite material moves with the eyeball, the amniotic membrane matrix can slide on the hydrogel surface to a limited extent, thereby ensuring that the position of the amniotic membrane matrix relative to the palpebral conjunctiva wound surface remains unchanged, effectively protecting the growth of the epithelium on the palpebral conjunctiva wound surface. Specifically, in clinical use, the amniotic membrane composite material can be placed in the conjunctival sac and attached to the surface of the eyeball. The decellularized biofilm matrix can effectively protect the corneal, bulbar conjunctiva or palpebral conjunctiva wound surface, prevent the wound surface from adhering to normal tissue, and inhibit the occurrence of clouding. There is no need to suture the wound surface with a dressing, which is convenient for ophthalmic clinical surgery and the application of later replacement of eye dressings. It has been experimentally verified that the suture-free decellularized bio-amniotic membrane ocular surface composite material prepared by the present invention has the characteristics of suture-free placement after eye surgery and convenient multiple painless and non-traumatic dressing replacements, and can also significantly improve eye diseases, providing new methods and directions for the effective treatment of clinical eye diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0027] Figure 1 The convex surface (A) and concave surface (B) of the suture-free acellular bio-amniotic membrane ocular surface composite material prepared with genipin as a biological cross-linking agent;

[0028] Figure 2 The results of animal experiments using Example 2 as an example; A: a diagram of the constructed corneal epithelial defect; B: a diagram of the composite material product prepared by the present invention wearing the dressing; C: a diagram of the appearance 7 days after surgery; D: a diagram of the corneal epithelial recovery 7 days after surgery;

[0029] Figure 3 The convex surface (A) and concave surface (B) of the acellular bio-amniotic membrane ocular surface composite material prepared using non-aldehyde epoxide as a bio-crosslinking agent;

[0030] Figure 4 The results of animal experiments using Example 4 as an example; A: a diagram of the constructed corneal epithelial defect; B: a diagram of the composite material product prepared by the present invention wearing the dressing; C: a diagram of the appearance 7 days after surgery; D: a diagram of the corneal epithelial recovery 7 days after surgery;

[0031] Figure 5 The convex surface (A) and the concave surface (B) of the suture-free acellular biological amniotic membrane ocular surface composite material prepared in Example 5;

[0032] Figure 6 The results of animal experiments using Example 5 as an example; A: the state of the eyeball after wearing for 1 week; B: the recovery of corneal cells after wearing for 1 week; C: the state of the eyeball after wearing for 2 weeks; D: the recovery of corneal cells after wearing for 2 weeks; E: the state of the control eyeball; F: the cell changes of the control cornea. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0034] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0035] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0036] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0037] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0038] The present invention provides a suture-free acellular biological amniotic membrane ocular surface composite material, comprising an amniotic membrane matrix and a support for supporting the amniotic membrane matrix, wherein the amniotic membrane matrix is ​​derived from human amniotic membrane tissue material, the support comprises an animal cornea or a non-biological material cornea, and the support is prepared into a corneal contact lens shape that matches the shape of a human cornea. By preparing the support into a corneal contact lens shape, the composite material of the amniotic membrane matrix attached to the support can be placed in the conjunctival sac and move with the eyeball without friction.

[0039] Furthermore, the preparation method of the amniotic matrix includes the steps of removing antigenic components from human amniotic tissue materials and retaining low antigenic matrix. The antigenic components that may produce rejection reactions in human amniotic tissue are removed, and the active substances and amniotic membrane that promote wound healing are retained, and the function of guiding autologous tissue growth is retained. Furthermore, physical methods such as deep cryogenics, mechanical scraping, biochemical enzymatic digestion, cross-linking methods, etc. can be used to remove antigenic components such as cells in amniotic tissue and retain low antigenic matrix.

[0040] Furthermore, the animal cornea includes a porcine cornea, which is treated with a cross-linking agent and processed into a corneal contact lens shape using a mold to make the porcine cornea fit the human cornea shape. After the treated porcine cornea is rehydrated, the corneal contact lens shape can still be maintained. This shape can ensure that after the suture-free decellularized biological amniotic membrane ocular surface composite material of the amniotic membrane matrix covers the patient's cornea, there is no air between the amniotic membrane matrix and the cornea, presenting a weak negative pressure state, ensuring that the suture-free decellularized biological amniotic membrane ocular surface composite material of the amniotic membrane matrix moves with the eyeball. The above-mentioned adaptation to the human cornea is distinguished by the base curve (BC) (the central arc of the inner curved surface of the lens. The base curve is longer, the curved surface of the lens is flatter; the base curve is shorter, and the curved surface of the lens is more curved). The present invention sets the following specifications (corneal curvature): 8.2, 8.2, 8.3, 8.5, 8.6, 8.7, 8.8, 8.9, a total of 8. Most of the base curves of the eyeball are between 8.4-8.6.

[0041] Furthermore, the cross-linking agent includes a non-aldehyde fixing agent, and the non-aldehyde fixing agent includes genipin or diglycidyl ethyl ether;

[0042] The treatment is performed by soaking at a low temperature of 0-4°C for 2-6 hours.

[0043] Furthermore, the non-biological cornea material includes degradable non-biological materials and non-degradable non-biological materials. Furthermore, the degradable non-biological material includes a mixture of one or more of collagen gelatin, cellulose, hyaluronic acid or sodium alginate, and the non-degradable non-biological material includes hydrogel materials such as polyacrylamide polymers or silicone polymers.

[0044] The present invention also provides a method for preparing a suture-free acellular biological amniotic membrane ocular surface composite material, comprising any one of the following methods (1)-(2):

[0045] (1) The amniotic membrane matrix is ​​flattened on the concave surface of the support without tension, freeze-dried, sealed and irradiated to produce a suture-free acellular biomembrane ocular surface composite material

[0046] (2) After a thin layer of hydrogel is evenly applied on the convex surface of the support, the amniotic membrane matrix is ​​flattened on the hydrogel without tension, freeze-dried, sealed and irradiated to obtain a suture-free acellular biomembrane ocular surface composite material.

[0047] Furthermore, the hydrogel comprises the following components in percentage by mass: 1-8% of deacetylated chitosan, 1-20% of glycerol, 1-10% of lactic acid and 1-30% of gelatin, with the remainder being water.

[0048] Furthermore, the irradiation conditions are: the irradiation dose is 15-40Gy.

[0049] The present invention also provides a method for using a suture-free acellular biological amniotic membrane ocular surface composite material, wherein the suture-free acellular biological amniotic membrane ocular surface composite material is selected according to the location of the ocular wound surface;

[0050] If the wound is on the cornea and bulbar conjunctiva, choose a suture-free acellular biological amniotic membrane ocular surface composite material with the amniotic membrane matrix attached to the concave surface, and insert it into the conjunctival sac on the surface of the eyeball; if the wound is on the palpebral conjunctiva side, choose a suture-free acellular biological amniotic membrane ocular surface composite material biomembrane with the amniotic membrane matrix attached to the convex surface, and insert it into the conjunctival sac on the surface of the eyeball.

[0051] In the above scheme, when the wound is on the corneal and conjunctival side, the amniotic membrane matrix is ​​selected on the concave surface of the composite material of the amniotic membrane matrix. The composite material of the amniotic membrane matrix moves with the eyeball, and the amniotic membrane has no obvious displacement relative to the corneal wound, thereby effectively protecting the repair of the corneal epithelium; when the wound is on the upper and lower eyelid mucosal side, the amniotic membrane matrix is ​​selected on the convex surface of the composite material of the amniotic membrane matrix, and an additional hydrogel layer is added between the amniotic membrane matrix and the decellularized biomaterial (such as pig cornea). When the composite material of the amniotic membrane matrix is ​​implanted into the patient's conjunctival sac, the hydrogel layer absorbs water and gels. When the composite material of the amniotic membrane matrix moves with the eyeball, the amniotic membrane matrix can slide on the hydrogel surface to a limited extent, thereby ensuring that the position of the amniotic membrane matrix relative to the conjunctival wound remains unchanged, thereby effectively protecting the growth of the epithelium on the conjunctival wound.

[0052] In order to further explain the above technical solution clearly, the following is an illustration by way of embodiments.

[0053] Example 1 A method for preparing a suture-free acellular biological amniotic membrane ocular surface composite material

[0054] (1) Amniotic membrane

[0055] The preparation was carried out by mechanical deep cryo-cell removal method, and the specific operation was as follows:

[0056] Under sterile conditions, the amniotic membrane facing the fetus is bluntly separated from the chorion, the blood clots are washed away, and after soaking in Chlorhexidine for 3-5 minutes, the epithelial basement membrane is spread upward on the nitrocellulose film, cut into appropriate sizes, and placed in a mixture of RPMI-1640 and glycerol with a volume ratio of 1:1 in a -80℃ refrigerator for 24 hours. After freeze-drying in a freeze dryer, the amniotic epithelial basement membrane is obtained for standby use. It can be stored for standby use or the amniotic epithelial cells can be removed by enzymatic digestion to make acellular amniotic matrix material.

[0057] (2) Animal membrane materials

[0058] We purchased commercially available fresh pig eyeballs that had passed quarantine, trimmed the pig corneas flat, and used a corneal lamellar knife to mechanically peel off the corneal epithelium and endothelium, retaining a pig corneal lamellar layer with a thickness of 450nm. We then soaked the pig corneal lamellar layer in a non-aldehyde fixative (genipin) at a low temperature of 4°C for 3 hours, and pressed it on an imitation eyeball base to create a corneal contact lens appearance for later use.

[0059] (3) Preparation of hydrogel

[0060] Select 1% deacetylated chitosan (deacetylated 80), 2% glycerol, 3% lactic acid, and 2% gelatin. Add them into a 50°C water bath pot and mix with water, stir evenly, and form a gel after cooling for later use.

[0061] (4) Preparation of amniotic membrane decellularized ocular surface covering device

[0062] The rehydrated amniotic epithelial basement membrane was flattened without tension toward the concave surface of the porcine corneal lamella, or a thin layer of hydrogel was evenly applied to the convex surface of the porcine corneal lamella, and then the rehydrated amniotic epithelial basement membrane was flattened without tension toward the hydrogel surface, placed in a freeze dryer for 3 hours, taken out, trimmed flat, placed in a sterile bag, and sealed. The finished product was irradiated with 15Gy.

[0063] Example 2 A method for preparing a suture-free acellular biological amniotic membrane ocular surface composite material

[0064] (1) Amniotic membrane processing

[0065] The preparation was carried out by mechanical deep cryo-cell removal method, and the specific operation was as follows:

[0066] Under sterile conditions, the amniotic membrane facing the fetus is bluntly separated from the chorionic membrane, the blood clots are washed away, and after soaking in chlorhexidine for 3-5 minutes, the epithelial basement membrane is spread upward on the nitrocellulose film, cut into appropriate sizes, and placed in a mixture of RPMI-1640 and glycerol with a volume ratio of 1:1 in a -80℃ refrigerator for 36 hours. After freeze-drying in a freeze dryer, the amniotic epithelial basement membrane is obtained for standby use. It can be stored for standby use or the amniotic epithelial cells can be removed by enzymatic digestion to make acellular amniotic matrix material.

[0067] (2) Animal membrane material processing

[0068] Fresh pig eyeballs that have passed quarantine were purchased from the market. The pig cornea was trimmed flat, and the corneal epithelium and endothelium were mechanically peeled off with a corneal lamellar knife, retaining the pig corneal lamellar layer with a thickness of 450nm. The pig corneal lamellar layer was soaked in a non-aldehyde fixative (genipin) at a low temperature of 0℃ for 5 hours, and pressed on an imitation eyeball base to have a corneal contact lens appearance (the corneal curvature was set to 8.4) for later use.

[0069] (3) Preparation of hydrogel

[0070] Select 6% deacetylated chitosan (deacetylated 80), 12% glycerol, 5% lactic acid, and 20% gelatin, add them into a 55°C water bath in sequence, stir, and form a gel after cooling.

[0071] (4) Preparation of amniotic membrane decellularized ocular surface covering device

[0072] The rehydrated amniotic epithelial basement membrane was flattened without tension toward the concave surface of the porcine corneal lamella, or a thin layer of hydrogel was evenly applied to the convex surface of the porcine corneal lamella, and then the rehydrated amniotic epithelial basement membrane was flattened without tension toward the hydrogel surface, placed in a freeze dryer for 4 hours, taken out, trimmed flat, placed in a sterile bag, and sealed. The finished product was irradiated with 25Gy.

[0073] Example 3 Preparation method of a suture-free acellular biological amniotic membrane ocular surface composite material

[0074] (1) Amniotic membrane processing

[0075] The preparation was carried out by mechanical deep cryo-cell removal method, and the specific operation was as follows:

[0076] Under sterile conditions, the amniotic membrane facing the fetus is bluntly separated from the chorionic membrane, the blood clots are washed away, and after soaking in Chlorhexidine for 3-5 minutes, the epithelial basement membrane is spread upward on the nitrocellulose film, cut into appropriate sizes, and placed in a mixture of RPMI-1640 and glycerol with a volume ratio of 1:1 in a -80℃ refrigerator for 48 hours. After freeze-drying in a freeze dryer, the amniotic epithelial basement membrane is obtained for standby use. It can be stored for standby use or the amniotic epithelial cells can be removed by enzymatic digestion to make acellular amniotic matrix material.

[0077] (2) Animal membrane material processing

[0078] We purchased commercially available fresh pig eyeballs that had passed quarantine, trimmed the pig corneas flat, and used a corneal lamellar knife to mechanically peel off the corneal epithelium and endothelium, retaining a pig corneal lamellar layer with a thickness of 450nm. We then soaked the pig corneal lamellar layer in a non-aldehyde fixative (diglycidyl ethyl ether) at 4°C for 6 hours, and pressed it onto an imitation eyeball base to create a contact lens-like appearance for later use.

[0079] (3) Preparation of hydrogel

[0080] Choose 8% deacetylated chitosan (deacetylated 80), 20% glycerol, 10% lactic acid, and 30% gelatin. Add them into a 60°C water bath in sequence and stir. After cooling, form a gel for later use.

[0081] (4) Preparation of amniotic membrane decellularized ocular surface covering device

[0082] The rehydrated amniotic epithelial basement membrane was flattened without tension toward the concave surface of the porcine corneal lamella, or a thin layer of hydrogel was evenly applied to the convex surface of the porcine corneal lamella, and then the rehydrated amniotic epithelial basement membrane was flattened without tension toward the hydrogel surface, placed in a freeze dryer for 4 hours, taken out, trimmed flat, placed in a sterile bag, and sealed. The finished product was irradiated with 40Gy.

[0083] Example 4 A method for preparing a suture-free acellular biological amniotic membrane ocular surface composite material

[0084] (1) Amniotic membrane processing

[0085] The amniotic epithelial cells were removed by enzymatic digestion to prepare acellular amniotic matrix material. The specific operation was as follows:

[0086] Under sterile conditions, the amniotic membrane facing the fetus is bluntly separated from the chorion, and blood clots are rinsed off. After soaking in chlorhexidine for 3-5 minutes, a surfactant solution is used in combination with a protease solution to control the final concentration of the surfactant to 0.1% and the final concentration of the protease to 0.1%. The cells in the amniotic membrane are ultrasonically digested to obtain a decellularized amniotic membrane matrix material for later use.

[0087] (2) Animal membrane material processing

[0088] Same as "(2) Animal membrane material treatment" in Example 2.

[0089] (3) Preparation of hydrogel

[0090] Same as “(3) Preparation of hydrogel” in Example 2.

[0091] (4) Preparation of amniotic membrane decellularized ocular surface covering device

[0092] Same as "(4) preparing an amniotic membrane decellularized ocular surface covering device" in Example 2.

[0093] Example 5

[0094] (1) Same as (1) of Example 2.

[0095] (2) The porcine corneal lamellae in Example 2 were replaced with commercially available qualified soft contact lenses (Bausch and Lomb daily disposable cosmetic lenses).

[0096] (3) The preparation method of the gel is the same as that of Example 2.

[0097] (4) For the purchased qualified soft contact lenses, apply the prepared gel evenly on the convex surface under sterile conditions, and then cover the surface of the gel with the decellularized amniotic membrane matrix material. Then, place the decellularized amniotic membrane matrix material toward the metal eyeball base, and place an appropriate weight on the composite material. After pressing for 34-48 hours, put it into a sealed bag, freeze-dry it together, and then put it into a sealed bag for 15-40Gy irradiation. In ophthalmology clinics, the composite material can be directly placed on the surface of the eyeball.

[0098] Example 6

[0099] (1) Amniotic membrane processing

[0100] The amniotic epithelial cells were removed by enzymatic digestion to prepare acellular amniotic matrix material. The specific operation was as follows:

[0101] Under sterile conditions, the amniotic membrane facing the fetus is bluntly separated from the chorion, and blood clots are rinsed off. After soaking in chlorhexidine for 3-5 minutes, a surfactant solution is used in combination with a protease solution to control the final concentration of the surfactant to 0.1% and the final concentration of the protease to 0.1%. The cells in the amniotic membrane are ultrasonically digested to obtain a decellularized amniotic membrane matrix material for later use.

[0102] (2) Replace the gel prepared in Example 2 with commercially available qualified medical polyacetamide gel.

[0103] (3) Purchase a qualified hard contact lens (Luxida RGP or Menicon RGP) on the market, remove the middle part of the hard contact lens under sterile conditions to form an appropriate contact lens ring, evenly apply the purchased medical polyacetamide gel on the concave or convex surface of the contact lens ring, cover the surface of the gel with the above wound protection membrane biomaterial, and then place the wound protection membrane biomaterial toward the metal eyeball base, place an appropriate weight on the composite material, press for 34-48 hours, put it into a sealed bag, freeze-dry it together, and then put it into a sealed bag for 15-40Gy irradiation. In ophthalmology clinics, the composite material can be placed directly on the surface of the eyeball.

[0104] Animal Experimentation:

[0105] 1. Experimental methods

[0106] (1) Anesthesia: According to the weight of the experimental animal (New Zealand white rabbit), 0.5 mL / kg of sulphuric acid was injected intramuscularly in the neck and 1 mL / kg of 2% phenobarbital was slowly pushed intravenously. After the combined anesthesia, the hair around the left eye was removed, and the rabbit was placed in the right side decubitus position. The periocular area was disinfected with 5% iodine and the surface of the eyeball was disinfected with 0.5% iodine. A sterile drape was laid, and only the left eye was exposed.

[0107] (2) Creating corneal pupil epithelial defect: Use a surgical blade to remove the corneal pupil epithelium and a small amount of lamellar layer of the left eye, drip 5% sodium fluorescein saline on the cornea, rinse the wound with saline three times, illuminate the left eye with a purple light pen, and take a picture;

[0108] (3) Placement of the suture-free acellular bio-amniotic membrane ocular surface composite material of the present invention: Rinse the left eye with eye drops containing antibiotics, and place the suture-free acellular bio-amniotic membrane ocular surface composite material of suitable size on the surface of the left eyeball. After observing that the material can move with the eyeball, the operation is completed.

[0109] This experiment was conducted using the suture-free acellular biological amniotic membrane ocular surface composite materials prepared in Example 2, Example 4 and Example 5 as examples, and the experimental results are shown in Table 1 below:

[0110] Table 1 Animal experiment results of Example 2 and Example 4

[0111]

[0112] Table 2 Animal experiment results of Example 5

[0113]

[0114] It can be seen from the above results that the suture-free acellular biological amniotic membrane ocular surface composite material provided by the present invention can effectively protect the corneal epithelial defect area and prevent infection after being worn.

[0115] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A suture-free acellular biological amniotic membrane ocular surface composite material, characterized in that: The invention comprises an amniotic membrane matrix and a support for supporting the amniotic membrane matrix, wherein the amniotic membrane matrix is ​​derived from human amniotic membrane tissue material, the support comprises an animal cornea or a non-biological material cornea, the support is prepared to match the shape of a corneal contact lens with the shape of a human cornea, and the amniotic membrane matrix is ​​attached to the convex surface of the support having the shape of a corneal contact lens; The preparation method of the suture-free decellularized biological amniotic membrane ocular surface composite material comprises the following steps: after a thin layer of hydrogel is evenly applied on the convex surface of the support, the amniotic membrane matrix is ​​flattened on the hydrogel without tension, freeze-dried, sealed and irradiated to obtain the suture-free decellularized biological amniotic membrane ocular surface composite material; The hydrogel comprises the following components in the following amounts: 1-8% of deacetylated chitosan, 1-20% of glycerol, 1-10% of lactic acid and 1-30% of gelatin, and the rest is water.

2. The suture-free acellular biological amniotic membrane ocular surface composite material according to claim 1, characterized in that: The method for preparing the amniotic matrix comprises the steps of removing the antigenic components in the human amniotic tissue material and retaining the low-antigenic matrix.

3. The suture-free acellular biological amniotic membrane ocular surface composite material according to claim 1, characterized in that: The animal cornea comprises a pig cornea, which is treated with a cross-linking agent and then processed into a corneal contact lens shape using a mold so that the pig cornea is adapted to the shape of a human cornea.

4. The suture-free acellular biological amniotic membrane ocular surface composite material according to claim 3, characterized in that: The cross-linking agent includes a non-aldehyde fixing agent, and the non-aldehyde fixing agent includes genipin or diglycidyl ethyl ether; The treatment is a soaking treatment at a low temperature of 0-4°C for 2-6 hours.

5. The suture-free acellular biological amniotic membrane ocular surface composite material according to claim 1, characterized in that: The non-biological cornea material includes degradable non-biological material and non-degradable non-biological material.

6. The suture-free acellular biological amniotic membrane ocular surface composite material according to claim 5, characterized in that: The degradable non-biological material includes at least one of collagen gelatin, cellulose, hyaluronic acid or sodium alginate, and the non-degradable non-biological material includes polyacrylamide polymer or silicone polymer.

7. A method for preparing the suture-free acellular biological amniotic membrane ocular surface composite material according to any one of claims 1 to 6, characterized in that: The following steps are involved: After a thin layer of hydrogel is evenly applied on the convex surface of the support, the amniotic membrane matrix is ​​flattened on the hydrogel without tension, freeze-dried, sealed and irradiated to obtain the suture-free acellular biological amniotic membrane ocular surface composite material; The hydrogel comprises the following components in the following amounts: 1-8% of deacetylated chitosan, 1-20% of glycerol, 1-10% of lactic acid and 1-30% of gelatin, and the rest is water.

8. The preparation method according to claim 7, characterized in that: The irradiation conditions are: the irradiation dose is 15-40Gy.

9. Use of the suture-free acellular biological amniotic membrane ocular surface composite material according to any one of claims 1 to 6 in the preparation of an eye dressing.

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