Methacrylic anhydride modified sclera extracellular matrix repair material and curing method thereof

Abstract

The invention provides a methacrylic anhydride modified sclera extracellular matrix repair material and a curing method thereof, and belongs to the technical field of medical repair materials. The methacrylic anhydride modified sclera extracellular matrix repair material provided by the invention is prepared from the following raw materials: a methacrylic anhydride modified sclera extracellular matrix, methacrylated gelatin, a photoinitiator and a solvent. The sclera extracellular matrix modified by methacrylic anhydride is adopted, methacrylic anhydride contains methacrylic acid, double bonds can be introduced, the sclera extracellular matrix has the photo-initiation polymerization capacity, and in-situ gelling can be carried out at sclera and cornea damage positions under the action of a photoinitiator; the sclera extracellular matrix has good biocompatibility and histocompatibility, so that sclera matrix cells and corneal epithelial cells are adhered and proliferated, and the sutureless repair is realized; the methacrylated gelatin can provide a photo-crosslinking gelatin network under the action of a photoinitiator, so that cell adhesion and proliferation can be promoted, and repair is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of medical repair materials, and particularly relates to a methacrylic anhydride-modified scleral extracellular matrix repair material and a curing method thereof. Background Art

[0002] The sclera forms the outermost layer of the eyeball, primarily protecting it. Covered by the conjunctiva and fascia, the sclera is shielded from direct contact with the outside world. Furthermore, the sclera is primarily composed of densely interwoven collagen and elastic fibers, with relatively few cellular components and blood vessels. When scleral tissue is damaged, the disease progresses slowly, the tissue repair capacity is poor, and drug treatment is ineffective. Once the sclera is damaged by external factors such as infection, trauma, or ophthalmic surgery, necrosis becomes difficult to heal, potentially leading to scleral perforation.

[0003] Currently, acellular scleral lenses are the primary method for repairing scleral and corneal defects. However, this repair material requires extensive suturing, which not only increases surgical difficulty and requires advanced clinical suturing techniques, but also leads to various suture-related complications after surgery, such as suture loosening, suture exposure, and inflammatory reactions. Therefore, the development of a suture-free alternative material with high tissue adhesion is an urgent technical challenge in this field. Summary of the Invention

[0004] The present invention aims to provide a methacrylic anhydride-modified scleral extracellular matrix repair material and a curing method thereof. The methacrylic anhydride-modified scleral extracellular matrix repair material provided by the present invention does not require suturing and has high tissue adhesion performance.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a methacrylic anhydride-modified scleral extracellular matrix repair material. The raw materials include methacrylic anhydride-modified scleral extracellular matrix, methacrylated gelatin, a photoinitiator and a solvent.

[0007] Preferably, the method for preparing the methacrylic anhydride-modified scleral extracellular matrix comprises:

[0008] The scleral extracellular matrix, a buffer solution and methacrylic anhydride are mixed and grafted to obtain the scleral extracellular matrix modified with methacrylic anhydride.

[0009] Preferably, the ratio of the mass of the scleral extracellular matrix to the volume of methacrylic anhydride is (0.5-1.5) g: (0.1-0.3) mL.

[0010] Preferably, the grafting temperature is 45-55° C., and the grafting time is 2-4 hours.

[0011] Preferably, the mass ratio of the methacrylic anhydride-modified scleral extracellular matrix to the methacrylated gelatin is 1:2 to 2:1.

[0012] Preferably, the mass ratio of the methacrylic anhydride-modified scleral extracellular matrix to the methacrylated gelatin is 1:1.

[0013] Preferably, the photoinitiator is phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt.

[0014] Preferably, the total concentration of the methacrylic anhydride-modified scleral extracellular matrix and methacrylated gelatin in the scleral extracellular matrix repair material is 15-20% (w / v).

[0015] The present invention also provides a method for curing the scleral extracellular matrix repair material modified with methacrylic anhydride according to the above technical solution, wherein the curing reaction is ultraviolet light irradiation.

[0016] Preferably, the wavelength of the ultraviolet light is 365 nm, and the ultraviolet light irradiation time is 2 to 4 minutes.

[0017] The present invention provides a methacrylic anhydride-modified scleral extracellular matrix repair material, the raw materials of which include methacrylic anhydride-modified scleral extracellular matrix, methacrylated gelatin, a photoinitiator, and a solvent. The present invention uses methacrylic anhydride-modified scleral extracellular matrix. The methacrylic anhydride contains methacrylic acid, which can introduce double bonds, giving the scleral extracellular matrix the ability to photoinitiate polymerization. Under the action of the photoinitiator, the scleral extracellular matrix can form gel in situ at the site of scleral and corneal damage. The scleral extracellular matrix is a natural biomaterial with good biocompatibility and tissue compatibility, allowing scleral stromal cells and corneal epithelial cells to adhere and proliferate, achieving seamless repair. Under the action of the photoinitiator, the methacrylated gelatin can provide a photocrosslinked gelatin network. This flexible network forms a rigid polymer bond with the aforementioned methacrylic anhydride-modified scleral extracellular matrix, which can meet the mechanical requirements of ocular tissue. In addition, the methacrylated gelatin retains the natural RGD sequence of gelatin, which can further promote cell adhesion and proliferation, achieving further repair. Experimental results show that the methacrylic anhydride-modified scleral extracellular matrix repair material provided by the present invention has good adhesion performance and excellent repair effect after ultraviolet light irradiation. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a photo of the acellular sclera hydrogel of Application Example 1;

[0019] Figure 2 This is a cross-sectional image of the acellular sclera hydrogel of Application Example 1 after H&E (hematoxylin-eosin) staining;

[0020] Figure 3 This is a picture of the methacrylic anhydride-modified scleral extracellular matrix repair material prepared in Examples 1 to 3;

[0021] Figure 4 This is a picture of the methacrylic anhydride-modified scleral extracellular matrix repair material prepared in Examples 4 to 6;

[0022] Figure 5 These are pictures of the methacrylic anhydride-modified scleral extracellular matrix repair materials prepared in Example 1 and Example 4;

[0023] Figure 6 This is a photograph of the methacrylic anhydride-modified scleral extracellular matrix repair material prepared in Example 1 being dropped onto a corneal defect and then irradiated with a 365nm ultraviolet lamp for 3 minutes;

[0024] Figure 7 Curves showing changes in viscosity of the scleral extracellular matrix repair materials prepared in Examples 1 to 3 and the repair material in Comparative Example 1 as a function of temperature;

[0025] Figure 8 This is the nuclear magnetic resonance spectrum of the scleral extracellular matrix modified with methacrylic anhydride in Example 1;

[0026] Figure 9 is the transmittance curve of the acellular sclera hydrogel and human cornea in Application Example 1;

[0027] Figure 10 Slit lamp gross photograph of intraoperative preparation for scleral perforation;

[0028] Figure 11 Slit lamp macroscopic image of a rabbit 4 weeks after scleral perforation repair using the methacrylic anhydride-modified scleral extracellular matrix repair material prepared in Application Example 1;

[0029] Figure 12 This is a slit lamp macroscopic image of a rabbit scleral perforation performed in Comparative Example 2;

[0030] Figure 13 This is a slit lamp macroscopic image of a rabbit 4 weeks after scleral perforation surgery in comparative example 2;

[0031] Figure 14 This is a slit lamp macroscopic image of a rabbit with lamellar corneal defect repaired using the methacrylic anhydride-modified scleral extracellular matrix repair material prepared in Application Example 1, taken 5 weeks after surgery.

[0032] Figure 15 This is an optical coherence tomography image of the methacrylic anhydride-modified scleral extracellular matrix repair material prepared in Application Example 1, taken 5 weeks after the lamellar corneal defect repair surgery in a rabbit;

[0033] Figure 16 This is a slit lamp macroscopic image of a rabbit with lamellar corneal defect in comparative example 3 5 weeks after surgery;

[0034] Figure 17 This is an optical coherence tomography image of the lamellar corneal defect in the rabbit of comparative example 3 5 weeks after surgery. DETAILED DESCRIPTION

[0035] The invention provides a methacrylic anhydride-modified scleral extracellular matrix repair material. The raw materials include methacrylic anhydride-modified scleral extracellular matrix, methacrylated gelatin, a photoinitiator and a solvent.

[0036] The present invention has no particular limitation on the sources of the raw materials, and commercially available products known to those skilled in the art may be used.

[0037] The raw materials for preparing the methacrylic anhydride-modified scleral extracellular matrix repair material of the present invention include methacrylic anhydride-modified scleral extracellular matrix. The present invention uses methacrylic anhydride-modified scleral extracellular matrix. Methacrylic anhydride contains methacrylic acid, which can introduce double bonds, giving the scleral extracellular matrix the ability to photoinitiate polymerization. Under the action of a photoinitiator, it can form an in situ gel at the site of scleral and corneal damage. Furthermore, the scleral extracellular matrix is a natural biomaterial with excellent biocompatibility and tissue compatibility, allowing scleral stromal cells and corneal epithelial cells to adhere and proliferate, achieving seamless repair.

[0038] In the present invention, the method for preparing the methacrylic anhydride-modified scleral extracellular matrix preferably comprises:

[0039] The scleral extracellular matrix, a buffer solution and methacrylic anhydride are mixed and grafted to obtain the scleral extracellular matrix modified with methacrylic anhydride.

[0040] In the present invention, the method for preparing the scleral extracellular matrix preferably comprises:

[0041] The acellular sclera sheet is dried, shredded, enzymatically hydrolyzed and alkali neutralized in sequence to obtain the sclera extracellular matrix.

[0042] In the present invention, the method for preparing the acellular sclera sheet preferably comprises sequentially subjecting the porcine sclera to a pretreatment and a decellularization treatment to obtain the acellular sclera sheet.

[0043] In the present invention, the porcine sclera is preferably a fresh eyeball obtained within 2 hours; the porcine sclera is preferably cleaned with sterilized double distilled water before use. The present invention does not specifically limit the operation of cleaning with sterilized double distilled water, and the operation well known to those skilled in the art can be used.

[0044] In the present invention, the pretreatment preferably involves removing the uveal membrane and accessory tissues of the porcine sclera, and more preferably, removing the uveal membrane on the inner surface of the porcine sclera, the uveal membrane on the outer surface of the porcine sclera, and the accessory muscle tissue. The present invention does not particularly limit the procedures for removing the uveal membrane on the inner surface of the porcine sclera, the uveal membrane on the outer surface of the porcine sclera, and the accessory muscle tissue, and the procedures well known to those skilled in the art can be used for removal.

[0045] After the pretreatment is completed, the present invention preferably slices and cleans the product obtained by the pretreatment in sequence.

[0046] The present invention has no special limitation on the slicing operation, and operations well known to those skilled in the art may be used.

[0047] In the present invention, the cleaning solution used for cleaning is preferably a mixture of TritonX-100 and PBS buffer; the mass concentration of TritonX-100 in the mixture is preferably 2-4%, more preferably 3%; the cleaning solution is preferably used after sterilization.

[0048] The present invention has no particular limitation on the PBS buffer, and any PBS buffer well known to those skilled in the art can be used.

[0049] As an embodiment, the concentration of the PBS buffer may be 0.02 mol / L; the pH of the PBS buffer may be 7.4.

[0050] The present invention has no particular limitation on the amount of the cleaning solution, as long as the product obtained by the pretreatment is cleaned.

[0051] In the present invention, the reagent used for the decellularization treatment is preferably a mixture of TritonX-100 and super nuclease; the mass concentration of the TritonX-100 in the mixture is preferably 1-3%, more preferably 2%; the concentration of the super nuclease in the mixture is preferably 2000 μ / mL.

[0052] The present invention has no particular limitation on the amount of the mixture of TritonX-100 and super nuclease, as long as the decellularization process is sufficiently performed. The present invention can remove scleral fibroblasts through decellularization, thereby reducing the rejection reaction of the scleral lens after living body transplantation surgery.

[0053] The present invention has no particular limitation on the temperature and time of the decellularization treatment, and the temperature and time well known to those skilled in the art can be used.

[0054] As an embodiment, the temperature of the decellularization treatment may be 37° C., and the time of the decellularization treatment may be 12 hours.

[0055] After the decellularization process is completed, the product obtained by the decellularization process is preferably washed; the washing agent used for the washing is preferably PBS buffer. The present invention has no particular limitation on the washing process, and the product can be washed thoroughly according to a process well known in the art.

[0056] The present invention has no particular limitation on the PBS buffer, and any PBS buffer well known to those skilled in the art can be used.

[0057] In the present invention, the drying is preferably performed in a freeze dryer. The present invention has no particular limitation on the type of freeze dryer, and any equipment familiar to those skilled in the art can be used. The present invention has no particular limitation on the drying time, and the drying can be performed until constant weight is achieved.

[0058] In the present invention, the shredding is preferably performed using sterile scissors on a clean bench. The present invention has no particular limitation on the specific shredding operation, and any operation well known to those skilled in the art may be used.

[0059] In the present invention, the enzymatic hydrolysis agents used are preferably pepsin and hydrochloric acid; the concentration of the hydrochloric acid is preferably 0.1N; and the ratio of the mass of the pepsin to the volume of the hydrochloric acid to the mass of the shredded product is preferably 3 mg:3 mL:30 mg. The temperature and time of the enzymatic hydrolysis are not particularly limited in the present invention, and procedures familiar to those skilled in the art can be used.

[0060] In the present invention, the base used for the alkali neutralization is preferably a NaOH solution; the concentration of the NaOH solution is preferably 0.5 to 1.5 N, more preferably 1.0 N. The amount of the NaOH solution used is not particularly limited in the present invention, as long as the pH value of the solution obtained after alkali neutralization is within the range of 7.35 to 7.45.

[0061] After the alkali neutralization is completed, the present invention preferably dries the product obtained by the alkali neutralization to obtain the scleral extracellular matrix.

[0062] In the present invention, the drying is preferably freeze drying. The present invention has no particular limitation on the freeze drying operation, and the process may be drying to a constant weight.

[0063] In the present invention, the buffer solution is preferably PBS buffer. The present invention has no particular limitation on the amount of the buffer solution, as long as the raw materials are completely dissolved.

[0064] In the present invention, the ratio of the mass of the scleral extracellular matrix to the volume of methacrylic anhydride is preferably (0.5-1.5) g: (0.1-0.3) mL, more preferably 1 g: 0.2 mL. Limiting the ratio of the mass of the scleral extracellular matrix to the volume of methacrylic anhydride within this range can further increase the grafting rate of methacrylic anhydride.

[0065] In the present invention, the mixing of the scleral extracellular matrix, the buffer solution and methacrylic anhydride is preferably performed by mixing the scleral extracellular matrix and the buffer solution and then adding methacrylic anhydride dropwise.

[0066] The present invention has no particular limitation on the operation of mixing the scleral extracellular matrix and the buffer solution, and the technical scheme for preparing the mixed material well known to those skilled in the art can be used.

[0067] The present invention has no particular limitation on the dropping rate, and any dropping rate well known to those skilled in the art may be used.

[0068] In the present invention, the grafting temperature is preferably 45-55°C; the grafting time is preferably 2-4 hours, more preferably 3 hours. In the present invention, limiting the grafting temperature and time within the above ranges can further improve the grafting rate.

[0069] As an embodiment, the grafting temperature may be 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C or 54°C.

[0070] After the grafting is completed, the present invention preferably performs dialysis and freeze-drying on the grafted product in sequence to obtain methacrylic anhydride-modified scleral extracellular matrix.

[0071] In the present invention, the dialysis preferably uses pure water and PBS buffer in sequence.

[0072] In the present invention, the dialysis time using pure water is preferably 24 to 72 hours; the temperature of the dialysis using pure water is preferably 30 to 40°C, more preferably 35°C; and the pure water is preferably replaced every 0.5 and 1.5 hours during the dialysis using pure water. The present invention does not specifically limit the amount of pure water used, and an amount known to those skilled in the art can be used.

[0073] As an embodiment, the dialysis time using pure water can be 48 hours or 60 hours.

[0074] The present invention has no particular limitation on the amount of the PBS buffer, and any amount known to those skilled in the art may be used.

[0075] In the present invention, the temperature of the dialysis using PBS buffer is preferably 45-55°C, more preferably 50°C; when the dialysis using PBS buffer is performed, it is preferred to first dialyze overnight and then replace the PBS buffer every 3 hours.

[0076] The present invention has no particular limitation on the freeze-drying operation, and any operation well known to those skilled in the art may be used.

[0077] In the present invention, the grafting rate of the methacrylic anhydride-modified scleral extracellular matrix is ≥80%.

[0078] The raw materials used to prepare the methacrylic anhydride-modified scleral extracellular matrix repair material of the present invention include methacrylated gelatin. In the present invention, the methacrylated gelatin, under the action of a photoinitiator, can provide a photocrosslinked gelatin network. This flexible network, combined with the rigid polymer formed by the methacrylic anhydride-modified scleral extracellular matrix, can meet the mechanical requirements of ocular tissue. Furthermore, the methacrylated gelatin retains the natural RGD sequence of gelatin, which can further promote cell adhesion and proliferation, achieving further repair.

[0079] In the present invention, the mass ratio of the methacrylic anhydride-modified scleral extracellular matrix to the methacrylated gelatin is preferably 1:2 to 2:1, more preferably 1:1. Limiting the mass ratio of the methacrylic anhydride-modified scleral extracellular matrix to the methacrylated gelatin within the above range can enhance gel formation, thereby improving repair performance.

[0080] In the present invention, the total concentration of the methacrylic anhydride-modified scleral extracellular matrix and methacrylated gelatin in the scleral extracellular matrix repair material is preferably 15-20% (w / v), more preferably 20% (w / v). In the present invention, the total concentration of the methacrylic anhydride-modified scleral extracellular matrix and methacrylated gelatin in the scleral extracellular matrix repair material is 15-20% (w / v), that is, the ratio of the total mass of the methacrylic anhydride-modified scleral extracellular matrix and methacrylated gelatin to the volume of the solvent is (150-200) mg:1 mL; limiting the concentration within the above range can further improve adhesion performance.

[0081] The raw materials for preparing the methacrylic anhydride-modified scleral extracellular matrix repair material of the present invention include a photoinitiator; the photoinitiator is preferably phenyl (2,4,6-trimethylbenzoyl) lithium phosphate. The photoinitiator can initiate cross-linking and curing of the methacrylic anhydride-modified scleral extracellular matrix and methacrylated gelatin.

[0082] The raw materials for preparing the methacrylic anhydride-modified scleral extracellular matrix repair material of the present invention include a solvent; the solvent is preferably PBS buffer or sterile water for injection. The present invention uses a solvent to dissolve other raw materials.

[0083] In the present invention, the concentration of the phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt in the methacrylic anhydride-modified scleral extracellular matrix repair material is preferably 0.2-0.3% (w / v), more preferably 0.25% (w / v). In the present invention, the concentration of the phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt in the methacrylic anhydride-modified scleral extracellular matrix repair material is 0.2-0.3% (w / v), that is, the ratio of the mass of the phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt to the volume of the solvent is (2-3 mg): 1 mL.

[0084] The methacrylic anhydride-modified scleral extracellular matrix repair material provided by the present invention can achieve seamless repair of scleral and corneal defects during repair, and has excellent effects of promoting wound healing and tissue regeneration.

[0085] The raw materials of the invention are cheap and readily available, the cost is low, and it is conducive to large-scale production.

[0086] The present invention has no particular limitation on the preparation method of the methacrylic anhydride-modified scleral extracellular matrix repair material, as long as the raw materials are completely mixed.

[0087] The present invention also provides a method for curing the scleral extracellular matrix repair material modified with methacrylic anhydride according to the above technical solution, wherein the curing reaction is ultraviolet light irradiation.

[0088] In the present invention, the wavelength of the ultraviolet light is preferably 365 nm; the ultraviolet light irradiation time is preferably 2 to 4 minutes, more preferably 3 minutes. The methacrylic anhydride-modified scleral extracellular matrix repair material of the present invention can form a gel in situ after ultraviolet light irradiation, and can produce an acellular scleral hydrogel with good adhesion and rapid gelation. The hydrogel has the characteristics of high transparency, good adhesion, rapid in situ gelation, and high biocompatibility. It can promote tissue repair and wound healing of the sclera and cornea, repair corneal stromal defects, and serve as a substitute material for corneal donors.

[0089] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. The embodiments of the present invention and all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0090] The concentration of the PBS buffer used in the examples and comparative examples was 0.02 mol / L, and the pH was 7.4.

[0091] Example 1

[0092] The raw materials of a methacrylic anhydride-modified scleral extracellular matrix repair material are methacrylic anhydride-modified scleral extracellular matrix, methacrylated gelatin, a photoinitiator and a solvent;

[0093] The mass ratio of the methacrylic anhydride-modified scleral extracellular matrix to the methacrylated gelatin is 1:1;

[0094] The total concentration of the methacrylic anhydride-modified scleral extracellular matrix and methacrylated gelatin is 20% (w / v);

[0095] The photoinitiator is phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt, with a concentration of 0.25% (w / v);

[0096] The solvent is PBS buffer;

[0097] The preparation method of the methacrylic anhydride-modified scleral extracellular matrix repair material is as follows: 2.5 mg of a photoinitiator is dissolved in 1 mL of a solvent, and then a total mass of 200 mg of methacrylic anhydride-modified scleral extracellular matrix and methacrylated gelatin are added to obtain the methacrylic anhydride-modified scleral extracellular matrix repair material;

[0098] The preparation method of the methacrylic anhydride-modified scleral extracellular matrix is as follows:

[0099] (1) Take fresh pig eye sclera within 2 hours, then remove the inner surface pigment membrane, outer surface pigment membrane and attached muscle tissue of the pig eye sclera, and then slice it with a blade, and then place it in a mixture of TritonX-100 and PBS buffer for washing, then place it in a mixture of TritonX-100 and super nuclease, and perform decellularization treatment on a shaker at 37°C for 12 hours, and finally wash it with PBS buffer to obtain a decellularized sclera sheet; wherein the mass concentration of TritonX-100 in the mixture of TritonX-100 and PBS buffer is 2%; the mass concentration of TritonX-100 in the mixture of TritonX-100 and super nuclease is 2%; the concentration of super nuclease in the mixture of TritonX-100 and super nuclease is 2000μ / mL;

[0100] (2) The acellular sclera obtained in step (1) is placed in a freeze dryer for drying, and then cut into pieces with sterile scissors on a clean bench, followed by enzymatic hydrolysis with pepsin and hydrochloric acid, and then alkaline neutralization with 1.0N NaOH solution until the solution pH value is 7.4, and then freeze-dried to obtain a scleral extracellular matrix; wherein the concentration of hydrochloric acid is 0.1N; and the ratio of the mass of pepsin, the volume of hydrochloric acid, and the mass of the product obtained by cutting into pieces is 3 mg:3 mL:30 mg;

[0101] (3) Weigh 1 g of scleral extracellular matrix into a 50 mL spherical bottle, add 10 mL of PBS buffer to dissolve it, the dissolution temperature is 50 ° C, after complete dissolution, add 0.2 mL of methacrylic anhydride to the beaker, and graft at 50 ° C for 3 hours. The solution is visibly turbid, and then dialyzed with pure water for 48 hours, wherein the pure water is replaced once every 0.5 hours and 1.5 hours, the dialysis temperature is 35 ° C, and a white precipitate is generated in the solution. Then dialyzed with PBS buffer at 50 ° C overnight, and the PBS buffer was replaced every 3 hours on the second day, and replaced 7 times. A white precipitate is generated, followed by freeze-drying to obtain 0.9 g of methacrylic anhydride-modified scleral extracellular matrix with a grafting rate of 80%.

[0102] Application Example 1

[0103] 200 μL of the methacrylic anhydride-modified scleral extracellular matrix repair material prepared in Example 1 was placed in a culture dish and irradiated with a 365 nm ultraviolet lamp for 3 minutes to obtain an acellular scleral hydrogel.

[0104] Figure 1 This is a photograph of the acellular sclera hydrogel of Application Example 1.

[0105] from Figure 1 It can be seen that the acellular sclera hydrogel is easy to form and has high transparency.

[0106] The slices of the decellularized scleral hydrogel of Application Example 1 after H&E (hematoxylin-eosin) staining are shown in FIG. Figure 2 As shown, the specific method is:

[0107] 1. Fixation: The acellular sclera hydrogel of Application Example 1 was placed in a 10 wt% formaldehyde solution for 24 h;

[0108] 2. Dehydration: Place the fixed material in an embedding box, rinse with running water for 30 minutes, and then dehydrate in an alcohol solution;

[0109] 3. Transparency: Place the dehydrated material in xylene to replace the alcohol;

[0110] 4. Wax dipping and embedding: Place the material in melted paraffin, wait for the paraffin to completely cover the material, and then cool it down to embed;

[0111] 5. Slicing, spreading, and baking: Place the embedded material in a microtome and cut it into 3 μm thick slices. Flatten the slices in water and attach them to a glass slide. Dry the attached slides in a 60°C constant temperature oven.

[0112] 6. Dewaxing and dehydration: Dewax the slides in xylene, dehydrate them in alcohol, and then place them in distilled water;

[0113] 7. Staining: Stain the sections that have been soaked in distilled water in a hematoxylin aqueous solution for 10 minutes. Place them in 1 wt% hydrochloric acid for 3 seconds, then place them in tap water for 5 minutes to return to blue. Dehydrate them in 80%, 90%, 95%, and 100% alcohol by volume for 10 minutes each, and then stain them in eosin staining solution for 5 minutes.

[0114] 8. Dehydration and transparency: Place the dyed material in pure alcohol for dehydration;

[0115] 9. Sealing and reading the slides: drip resin on the slides, seal the slides with coverslips and then read the slides.

[0116] Depend on Figure 2 It can be seen that the acellular sclera hydrogel has uniform texture and consistent density.

[0117] Example 2

[0118] On the basis of Example 1, the mass ratio of the methacrylic anhydride-modified scleral extracellular matrix and the methacrylated gelatin was changed to 1:2, while other conditions remained unchanged.

[0119] Example 3

[0120] On the basis of Example 1, the mass ratio of the methacrylic anhydride-modified scleral extracellular matrix and the methacrylated gelatin was changed to 2:1, while other conditions remained unchanged.

[0121] Example 4

[0122] The raw materials of a methacrylic anhydride-modified scleral extracellular matrix repair material are methacrylic anhydride-modified scleral extracellular matrix, methacrylated gelatin, a photoinitiator and a solvent;

[0123] The mass ratio of the methacrylic anhydride-modified scleral extracellular matrix to the methacrylated gelatin is 1:1;

[0124] The total concentration of the methacrylic anhydride-modified scleral extracellular matrix and methacrylated gelatin is 10% (w / v);

[0125] The photoinitiator is phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt, with a concentration of 0.25% (w / v);

[0126] The solvent is PBS buffer;

[0127] The preparation method of the methacrylic anhydride-modified scleral extracellular matrix repair material is as follows: 2.5 mg of photoinitiator is dissolved in 1 mL of solvent, and then a total mass of 100 mg of methacrylic anhydride-modified scleral extracellular matrix and methacrylated gelatin are added to obtain the methacrylic anhydride-modified scleral extracellular matrix repair material. Other conditions are the same as in Example 1.

[0128] Example 5

[0129] Based on Example 4, the mass ratio of the methacrylic anhydride-modified scleral extracellular matrix to the methacrylated gelatin was changed to 1:2, while other conditions remained unchanged.

[0130] Example 6

[0131] Based on Example 4, the mass ratio of the methacrylic anhydride-modified scleral extracellular matrix to the methacrylated gelatin was changed to 2:1, while other conditions remained unchanged.

[0132] The pictures of the scleral extracellular matrix repair materials modified with methacrylic anhydride prepared in Examples 1 to 3 are as follows: Figure 3 As shown; the pictures of the scleral extracellular matrix repair materials modified with methacrylic anhydride prepared in Examples 4 to 6 are shown Figure 4 As shown; the pictures of the scleral extracellular matrix repair materials modified with methacrylic anhydride prepared in Example 1 and Example 4 are shown Figure 5 shown.

[0133] from Figures 3-5 It can be seen that the total concentration of methacrylic anhydride-modified scleral extracellular matrix and methacryloyl gelatin is 20%, which is easy to form a gel state, but not easy to form a gel state at a concentration of 10%. At a concentration of 20%, the mass ratios of methacrylic anhydride-modified scleral extracellular matrix and methacryloyl gelatin are 1:2 and 1:1, which are easier to form a gel state than 2:1. In order to ensure the maximum content of methacrylic anhydride-modified scleral extracellular matrix, the mass ratio of 1:1 is the optimal embodiment.

[0134] The adhesion test of the methacrylic anhydride-modified scleral extracellular matrix repair material prepared in Example 1 was performed, and the specific steps were as follows:

[0135] 1. Preparation of lamellar corneal defect: Select fresh pig eyeballs and remove the lamellar corneal stroma from the center of the cornea using a 6.0 mm trephine drill.

[0136] 2. 50 μL of the methacrylic anhydride-modified scleral extracellular matrix repair material prepared in Example 1 was evenly dropped onto the corneal defect, and then irradiated with a 365 nm ultraviolet lamp for 3 minutes. Figure 6 shown.

[0137] from Figure 6 It can be seen that the scleral extracellular matrix repair material prepared in Example 1 gels quickly after being exposed to ultraviolet light.

[0138] Comparative Example 1

[0139] On the basis of Example 1, the scleral extracellular matrix modified with methacrylic anhydride was omitted, the concentration of methacrylated gelatin was 20% (w / v), and other conditions remained unchanged to obtain a repair material.

[0140] The viscosity curves of the scleral extracellular matrix repair materials prepared in Examples 1 to 3 and the repair material in Comparative Example 1 as a function of temperature are shown in FIG. Figure 7 As shown in the figure, 20% GelMA is the curve of Comparative Example 1; 20% EMMA:GelMA=1:1 is the curve of Example 1; 20% EMMA:GelMA=2:1 is the curve of Example 3; 20% EMMA:GelMA=1:2 is the curve of Example 2.

[0141] from Figure 7 It can be seen that as the temperature increases, the viscosity of Examples 1 to 3 and Comparative Example 1 gradually decreases; when the mass ratio of methacrylic anhydride-modified scleral extracellular matrix to methacrylic gelatin is 1:1, the viscosity is relatively high while ensuring the content of the extracellular matrix, indicating that the methacrylic anhydride-modified scleral extracellular matrix repair material provided by the present invention has high tissue adhesion performance.

[0142] The grafting rate of methacrylic anhydride in the scleral extracellular matrix in Example 1 was detected by nuclear magnetic resonance. The results are as follows: Figure 8 As shown, Figure 8 This is the nuclear magnetic resonance spectrum of the scleral extracellular matrix modified with methacrylic anhydride in Example 1.

[0143] from Figure 8 It can be seen that the grafting rate is high, which is conducive to rapid gelation with methacrylated gelatin.

[0144] Figure 9 The transmittance curve of the decellularized sclera hydrogel material and human cornea of Application Example 1 is as follows: the decellularized sclera hydrogel material of Application Example 1 is moistened with PBS buffer and placed in a cuvette, which is then fixed in the sample compartment of a fluorescence spectrophotometer. The light absorbance of the sample in the wavelength range of 300 to 800 nm is detected to obtain the transmittance value, and a curve is drawn using Graphpad 8.0 as shown in FIG. Figure 9 shown.

[0145] Depend on Figure 9 It can be seen that the acellular sclera hydrogel material of Application Example 1 has high transparency, which is similar to that of human cornea, and can be used as an alternative material for corneal repair.

[0146] Application Example 2

[0147] The methacrylic anhydride-modified scleral extracellular matrix repair material prepared in Example 1 was used to repair a 3.0 mm rabbit scleral perforation. The steps for repairing scleral perforation in rabbits were as follows:

[0148] 1) Animal Model Preparation: Healthy New Zealand white rabbits were anesthetized, and the bulbar conjunctiva was incised at the superior corneal limbus to expose the scleral stroma. The full-thickness sclera with a diameter of 3.0 mm was drilled out using a trephine to create a scleral perforation model.

[0149] 2) Defect repair: 50 μL of the methacrylic anhydride-modified scleral extracellular matrix repair material prepared in Example 1 was dropped onto the perforation, and the perforation was irradiated with a 365 nm ultraviolet lamp for 3 minutes to allow the methacrylic anhydride-modified scleral extracellular matrix repair material to gel in situ at the perforation.

[0150] 3) Suturing the conjunctiva: Suture the bulbar conjunctiva with 10-0 nylon suture, apply tobramycin-dexamethasone eye ointment to the conjunctival sac, and return the New Zealand rabbit to the breeding room;

[0151] 4) Postoperative care: For 4 weeks after surgery, apply tobramycin-dexamethasone eye ointment every morning and evening to the New Zealand white rabbits.

[0152] Slit lamp macrophotograph of the scleral perforation during surgery Figure 10 As shown. Figure 10 It can be seen that the scleral stroma is fully peeled off and the pigment membrane is removed.

[0153] Four weeks after surgery, the repair effect of the scleral extracellular matrix repair material modified with methacrylic anhydride prepared in Example 1 was observed using a slit lamp. Figure 11 As shown, Figure 11 This is a slit lamp macroscopic image of a rabbit 4 weeks after the acellular sclera hydrogel was used to repair scleral perforation using the methacrylic anhydride-modified sclera extracellular matrix repair material prepared in Application Example 1.

[0154] Depend on Figure 11 It can be seen that 4 weeks after the operation, the repaired rabbit scleral perforation had a good pigment membrane at the perforation site, with no obvious pigment membrane prolapse, and no obvious congestion or rejection reaction of the corresponding bulbar conjunctiva.

[0155] Comparative Example 2

[0156] No repair is performed based on Application Example 2, and other operations remain unchanged.

[0157] Comparative Example 2: Slit lamp macroscopic images of scleral perforation in rabbits Figure 12 As shown. Figure 12 It can be seen that the entire scleral stroma is defective and the pigment membrane is prolapsed.

[0158] Figure 13 This is a slit lamp macroscopic image of a rabbit scleral perforation surgery performed in comparative example 2 4 weeks after surgery. Figure 13 It can be seen that 4 weeks after surgery, the upper conjunctiva was obviously congested with a large number of new blood vessels, the scleral perforation was obviously bulging, the pigment membrane was embedded in the perforation and partially protruded from the conjunctiva; Figure 11 By comparison, it can be seen that the healing effect of untreated scleral perforation is worse than that of the scleral perforation repaired with the scleral extracellular matrix repair material prepared in Example 1.

[0159] Application Example 3

[0160] The methacrylic anhydride-modified scleral extracellular matrix repair material prepared in Example 1 was used to repair a 6.0 mm rabbit lamellar corneal stromal defect, and lamellar corneal transplantation was performed in the rabbit in vivo, as follows:

[0161] 1) Animal Model Preparation: Healthy New Zealand white rabbits were anesthetized, and a corneal stromal defect model was prepared by drilling a 6.0 mm diameter, 200 μm thick corneal stroma in the center of the cornea using a trephine drill.

[0162] 2) Defect repair: 50 μL of the methacrylic anhydride-modified scleral extracellular matrix repair material prepared in Example 1 was dropped onto the corneal stroma defect, and the perforation was irradiated with a 365 nm ultraviolet lamp for 3 minutes to allow the methacrylic anhydride-modified scleral extracellular matrix repair material to gel in situ at the defect area;

[0163] 3) Rinse: Rinse the cornea with normal saline, put on a corneal bandage lens, and return the New Zealand rabbit to the breeding room;

[0164] 4) Postoperative care: Apply tobramycin-dexamethasone eye ointment every morning and evening for 5 weeks after surgery.

[0165] Figure 14 This is a slit lamp macroscopic image of a rabbit with lamellar corneal defect repaired using the methacrylic anhydride-modified scleral extracellular matrix repair material prepared in Application Example 1, taken 5 weeks after surgery. Figure 15 This is an optical coherence tomography image of the methacrylic anhydride-modified scleral extracellular matrix repair material prepared in Application Example 1 5 weeks after the lamellar corneal defect repair surgery in rabbits.

[0166] Depend on Figure 14It can be seen that 5 weeks after the operation, it was found that the scleral extracellular matrix repair material of the repaired rabbit cornea remained relatively transparent and the central pupil area was clear 5 weeks after the lamellar keratoplasty in the rabbit.

[0167] Depend on Figure 15 It can be seen that the scleral extracellular matrix repair material adheres well to the corneal stroma after ultraviolet light irradiation and has good peripheral apposition.

[0168] Comparative Example 3

[0169] No repair is performed based on Application Example 3, and other operations remain unchanged.

[0170] Figure 16 This is a slit lamp macroscopic image of a rabbit with lamellar corneal defect in comparative example 3 5 weeks after surgery; Figure 17 This is an optical coherence tomography image of the lamellar corneal defect in the rabbit of comparative example 3 5 weeks after surgery.

[0171] from Figure 16 It can be seen that 5 weeks after surgery, corneal stroma scarring was formed at the corneal defect site and corneal transparency decreased; Figure 14 By comparison, the postoperative healing effect of the untreated corneal stromal defect case was worse than that of case 3.

[0172] from Figure 17 It can be seen that the corneal defect becomes thinner and the corneal stroma density increases, indicating scar formation; Figure 15 By comparison, the recovery was worse than that in application example 3.

[0173] It can be seen from the above examples and comparative examples that the methacrylic anhydride-modified scleral extracellular matrix repair material provided by the present invention does not require suturing and has high tissue adhesion performance.

[0174] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A methacrylic anhydride-modified scleral extracellular matrix repair material, the raw materials comprising methacrylic anhydride-modified scleral extracellular matrix, methacrylated gelatin, a photoinitiator and a solvent.

2. The methacrylic anhydride-modified scleral extracellular matrix repair material according to claim 1, characterized in that: The preparation method of the methacrylic anhydride-modified scleral extracellular matrix comprises: The scleral extracellular matrix, a buffer solution and methacrylic anhydride are mixed and grafted to obtain the scleral extracellular matrix modified with methacrylic anhydride.

3. The methacrylic anhydride modified scleral extracellular matrix repair material according to claim 2, characterized in that: The ratio of the mass of the scleral extracellular matrix to the volume of methacrylic anhydride is (0.5-1.5) g: (0.1-0.3) mL.

4. The methacrylic anhydride modified scleral extracellular matrix repair material according to claim 2, characterized in that: The grafting temperature is 45-55° C., and the grafting time is 2-4 hours.

5. The methacrylic anhydride modified scleral extracellular matrix repair material according to claim 1, characterized in that: The mass ratio of the methacrylic anhydride-modified scleral extracellular matrix to the methacrylated gelatin is 1:2 to 2:

1.

6. The methacrylic anhydride-modified scleral extracellular matrix repair material according to claim 1 or 5, characterized in that: The mass ratio of the methacrylic anhydride-modified scleral extracellular matrix to the methacrylated gelatin is 1:

1.

7. The methacrylic anhydride modified scleral extracellular matrix repair material according to claim 1, characterized in that: The photoinitiator is phenyl (2,4,6-trimethylbenzoyl) lithium phosphate.

8. The methacrylic anhydride modified scleral extracellular matrix repair material according to claim 1, characterized in that: The total concentration of the methacrylic anhydride-modified scleral extracellular matrix and the methacrylated gelatin in the scleral extracellular matrix repair material is 15-20% (w / v).

9. The curing method of the methacrylic anhydride-modified scleral extracellular matrix repair material according to any one of claims 1 to 8, wherein the curing reaction is ultraviolet light irradiation.

10. The curing method according to claim 9, characterized in that: The wavelength of the ultraviolet light is 365 nm, and the ultraviolet light irradiation time is 2 to 4 minutes.

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