Low-swelling decellularized cornea, preparation method therefor and use thereof

Abstract

Disclosed in the present invention are a low-swelling decellularized cornea, a preparation method therefor and the use thereof. The low-swelling decellularized cornea is obtained by means of compounding a methacrylated compound into a decellularized cornea. The decellularized cornea has thermosensitivity and photopolymerization properties, and can exude a monomer polymer at 25°C or more, and by means of photopolymerization, can create stable bioadhesion on the surface of the implantation bed. The decellularized cornea has the characteristics of a rich source of materials and rapid repair of wounds without surgical sutures.

Description

A low-swelling acellular cornea and its preparation method and application Technical Field

[0001] The present invention belongs to the field of tissue engineering and regenerative medicine, and in particular relates to a low-swelling acellular cornea and a preparation method and application thereof. Background Art

[0002] Keratoplasty is the ultimate treatment for patients with corneal blindness. Corneal transplant procedures include penetrating keratoplasty, lamellar keratoplasty, and deep lamellar keratoplasty. Penetrating keratoplasty involves transplanting the entire cornea, including the corneal endothelium, replacing the damaged cornea with a healthy, intact cornea. This not only restores the integrity of the eye, controls corneal disease, but also significantly improves vision. Penetrating keratoplasty is widely used for a variety of indications, including infectious keratitis, ocular trauma, bullous keratopathy, secondary corneal transplantation, keratoconus, corneal tumors, corneal degeneration or dystrophy, Mooren's ulcer, and other corneal diseases. Currently, penetrating keratoplasty is the most commonly used corneal transplant procedure in China. While this procedure has a high success rate, the quality of the donor cornea required for penetrating keratoplasty is extremely high, requiring a healthy endothelium to prevent corneal edema and loss of transparency after transplantation. Due to the influence of ethics, morals and social customs in our country, there is a serious shortage of donor corneas, and the vast majority of corneal blind patients cannot receive immediate treatment.

[0003] While artificial corneas can be used for penetrating keratoplasty, they are primarily intended for surgical restoration of vision in patients with end-stage ocular surface conditions, such as those with severe chemical and thermal ocular burns, limbal stem cell deficiency, extensive corneal vascularization, and autoimmune diseases. Artificial corneas are typically made of synthetic polymer materials and are unable to bio-integrate with the recipient tissue to achieve tissue regeneration. Postoperative complications include retinal detachment, corneal calcification, glaucoma, corneal dissolution and implant expulsion, aqueous humor leakage, and severe cataracts.

[0004] In recent years, the launch of acellular keratin products has alleviated the shortage of corneal donors. However, due to the swellability of fresh corneal stroma in contact with water, acellular keratin cannot be directly exposed to aqueous humor. Therefore, its indication is limited to repairing corneal ulcers that do not involve the entire cornea.

[0005] In addition, implanting a corneal donor in a sutureless manner is clinically very desirable and can significantly reduce patient discomfort, infection risk, recovery time and treatment costs. A Chinese invention patent (application number 202310284669.7) discloses a suture-free composite artificial cornea and a preparation method thereof. The invention consists of a biological front membrane and a polymer back membrane, and the cornea is repaired without suture by inserting a fixed guide wire in the biological front membrane into the implant bed. Inserting a fixed guide wire into the cornea will cause secondary damage, and there is a risk of forming stromal scars and stimulating new blood vessels. In addition, the presence of a fixed guide wire also affects the transparency and beauty of the cornea. There is currently no transplant material that can replace the donor cornea for penetrating keratoplasty and can be used for sutureless repair.

[0006] Summary of the Invention

[0007] The purpose of this section is to summarize some aspects of the embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of this application to avoid obscuring the purpose of this section, the abstract and the title of the invention, and such simplifications or omissions should not be used to limit the scope of the present invention.

[0008] To address the deficiencies of the prior art, the first aspect of the present invention provides a decellularized cornea containing a solidified monomer polymer solution, wherein the monomer polymer solution comprises component 1 and component 2, wherein component 1 is a methacrylated derivative; and component 2 is selected from one or more of polyethylene glycol diacrylate (PEGDA) or ethylene glycol methyl ether methacrylate (PEGMA).

[0009] In the present invention, when the decellularized cornea is irradiated with visible light or ultraviolet light, the monomer polymers inside undergo polymerization to form a polymer network in the corneal stroma; the polymer network has no swelling property or low swelling property, thereby limiting the swelling of the decellularized cornea in the solution.

[0010] In the present invention, the monomer polymers in the decellularized cornea undergo polymerization reaction and form a polymer network in the corneal stroma. The transmittance in the visible light region is greater than 70%. After being immersed in physiological saline for 48 hours at room temperature, the water absorption rate is 10-35%.

[0011] In the present invention, the acellular cornea is selected from biological materials suitable for preparing artificial corneas, including but not limited to corneas from mammals such as humans, pigs, horses, and cows, acellular corneas, and transgenic corneas.

[0012] The recipient is an individual who receives the implanted acellular cornea, and can be a vertebrate, preferably a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples.

[0013] In the present invention, the monomer-polymer solution comprises component 1, which is selected from one or more methacrylic acid derivatives; and its concentration is 10-50% (w / v).

[0014] In a preferred embodiment, the methacrylated derivative comprises a methacrylated polysaccharide, protein or decellularized matrix.

[0015] In a more preferred embodiment, the methacryloylated derivative comprises methacryloylated gelatin and / or methacryloylated chondroitin sulfate.

[0016] In the present invention, the monomer polymer solution includes component 2, which is selected from one or more of polyethylene glycol diacrylate (PEGDA) or polyethylene glycol methyl ether methacrylate (PEGMA); and the concentration thereof is 0.001 g / ml-1 g / ml.

[0017] In a preferred embodiment, the monomer polymer solution is formed by mixing 10-30% (w / v) component 1 with 0.001g / ml-0.5g / ml component 2; preferably, it is formed by mixing 10-30% (w / v) component 1 with 0.05-0.2g / ml component 2; the volume ratio is 10:0-8:2, and the preferred volume ratio is 9:1-8:2.

[0018] In a more preferred embodiment, the monomer polymer solution comprises component 1: 10-30% (w / v) methacrylated gelatin and / or methacrylated chondroitin sulfate; component 2: 0.001 g / ml-0.5 g / ml polyethylene glycol diacrylate (PEGDA) or polyethylene glycol methyl ether methacrylate (PEGMA); the volume ratio of component 1 to component 2 is 10:0-8:2, preferably 9:1-8:2.

[0019] In a more preferred embodiment, the monomer polymer solution comprises 10-30% (w / v) methacrylated gelatin and 0.05-0.2 g / ml polyethylene glycol diacrylate (PEGDA) in a volume ratio of 9:1-8:2.

[0020] In a more preferred embodiment, the monomer polymer solution comprises 20% (w / v) methacrylated gelatin solution and 0.1 g / ml polyethylene glycol diacrylate (PEGDA) solution in a volume ratio of 9:1-8:2.

[0021] Furthermore, the monomer polymer solution contains a photoinitiator or a temperature initiator.

[0022] In a preferred embodiment, the photoinitiator is a blue light or ultraviolet light initiator, including 2,4,6-trimethylbenzoyl phosphate lithium salt (LAP); the temperature initiator includes azobisisobutyronitrile (AIBN).

[0023] In a preferred embodiment, the concentration of the photoinitiator is 0.1%-0.5%; the concentration of the temperature initiator is 0.1-0.2 mol / L.

[0024] In a preferred embodiment, the coagulated monomer polymer solution is maintained at a temperature of 4-25°C, preferably 4-20°C.

[0025] In a preferred embodiment, the liquefaction condition is that the surface temperature is greater than 25°C, preferably 35-38°C.

[0026] In a preferred embodiment, the wavelength range of visible light is 300-800 nm, and the energy of light is 100-500 mW / cm 2 , the illumination time is 30 seconds to 2 minutes; more preferably, the wavelength range is 365-405nm, and the illumination time is 30-60 seconds.

[0027] A second aspect of the present invention provides a method for preparing an acellular cornea, comprising:

[0028] The decellularized cornea is immersed in a monomer polymer solution, the decellularized cornea is taken out, and then the polymerization reaction of the monomer polymer in the decellularized cornea is activated.

[0029] In the present invention, the monomer-polymer solution contains the methacrylated derivative; the concentration thereof is 10-50% (w / v).

[0030] In a preferred embodiment, the methacrylated derivative comprises a methacrylated polysaccharide, protein or decellularized matrix.

[0031] In a more preferred embodiment, the methacryloylated derivative comprises methacryloylated gelatin and / or methacryloylated chondroitin sulfate.

[0032] In a preferred embodiment, the monomer polymer solution further comprises one or more selected from polyethylene glycol diacrylate (PEGDA) or polyethylene glycol methyl ether methacrylate (PEGMA); the concentration thereof is 0.001 g / ml-1 g / ml.

[0033] In a preferred embodiment, the monomer polymer solution comprises: the monomer polymer solution is formed by mixing 10-30% (w / v) component 1 with 0.001g / ml-0.5g / ml component 2; preferably, the monomer polymer solution is formed by mixing 10-30% (w / v) component 1 with 0.05-0.2g / ml component 2; the volume ratio is 10:0-8:2, and preferably the volume ratio is 9:1-8:2.

[0034] In a more preferred embodiment, the monomer polymer solution comprises component 1: 10-30% (w / v) methacrylated gelatin and / or methacrylated chondroitin sulfate; component 2: 0.001 g / ml-0.5 g / ml polyethylene glycol diacrylate (PEGDA) and / or polyethylene glycol methyl ether methacrylate (PEGMA); the volume ratio of component 1 to component 2 is 10:0-8:2, preferably 9:1-8:2.

[0035] In a more preferred embodiment, the monomer polymer solution comprises 10-30% (w / v) methacrylated gelatin and 0.05-0.2 g / ml polyethylene glycol diacrylate (PEGDA) in a volume ratio of 9:1-8:2.

[0036] In a more preferred embodiment, the monomer polymer solution comprises 20% (w / v) methacrylated gelatin solution and 0.1 g / ml polyethylene glycol diacrylate (PEGDA) solution in a volume ratio of 9:1-8:2.

[0037] In the present invention, the monomer-polymer solution may further contain anti-inflammatory agents such as voriconazole, methacryloyl chondroitin sulfate or methacryloyl derivatives, antibiotics, and / or substances for improving biocompatibility.

[0038] Furthermore, the monomer polymer solution contains a photoinitiator or a temperature initiator.

[0039] In a preferred embodiment, the photoinitiator is a blue light or ultraviolet light initiator, including 2,4,6-trimethylbenzoyl lithium phosphate (LAP); the temperature initiator includes azobisisobutyronitrile (AIBN).

[0040] In a preferred embodiment, the concentration of the photoinitiator is 0.1%-0.5%; the concentration of the thermal initiator is 0.1-0.2 mol / L. When light activation is used, the wavelength is 300nm-600nm, and the activation time is 30 seconds to 2 minutes; when temperature activation is used, the reaction time is 2 hours to 12 hours at a temperature above 60°C.

[0041] In a preferred embodiment, the preparation process of the monomer polymer solution is:

[0042] a) preparing a 10-30% (w / v) methacryloyl derivative solution with a 0.1-0.5% LAP solution to prepare a component 1 solution;

[0043] b) preparing a 0.001 g / ml to 0.5 g / ml PEGDA solution to prepare a component 2 solution;

[0044] c) Mixing the component 1 solution and the component 2 solution in a volume ratio of 10:0-8:2.

[0045] In the present invention, during the soaking process, ultrasound, vacuuming or stirring can be used simultaneously to accelerate the soaking or immersion process and promote the monomer polymer to enter the stroma of the acellular cornea.

[0046] In a preferred embodiment, the immersion operation is to immerse the decellularized cornea in at least 10 volumes of the monomer and polymer solution and shake on a shaker for 18-48 hours.

[0047] Furthermore, the method of the present invention further comprises a cooling step, that is, immersing the decellularized cornea in the monomer polymer solution, taking out the decellularized cornea and cooling it to solidify the monomer polymer in the decellularized cornea.

[0048] In a preferred embodiment, the cooling method is to place the decellularized cornea in a 4°C environment for 15-30 minutes, and then store it in a 4-25°C environment; preferably, store it in a 4-20°C environment.

[0049] In the above-mentioned preparation method provided by the present invention, preferably all operations are carried out in a sterile ultra-clean environment.

[0050] Furthermore, the method of the present invention includes a post-coagulation treatment step, which involves removing the monomer and polymer solution from the surface of the decellularized cornea. This can be accomplished using conventional procedures, such as rinsing and wiping. For example, using the decellularized cornea as an example, removal can involve grasping the decellularized cornea with tweezers and wiping it on a dust-free absorbent paper surface to remove excess monomer and polymer solution. The method also includes sterilizing the product and cutting it into grafts of varying sizes.

[0051] In the present invention, the decellularized cornea includes but is not limited to biological tissues, decellularized scaffold materials, transgenic organs, transgenic tissues, cell products and their derivatives, and artificial bionic materials.

[0052] Furthermore, the method described in the present invention also includes a pre-treatment step for the decellularized cornea, including femtosecond cutting to obtain a corneal stroma sheet with a thickness of 100-400 μm and a diameter of 9 mm; scraping the epithelium and endothelium, decellularization process, and virus inactivation to obtain a decellularized cornea containing anterior elastic layer and corneal stroma.

[0053] The third aspect of the present invention provides acellular cornea obtained by the above preparation method.

[0054] In the present invention, the decellularized cornea contains and / or has a solidified monomer polymer solution attached to its surface. After being applied to the recipient cornea, the monomer polymer solution liquefies and partially seeps out of the decellularized cornea and then soaks into the space between the recipient cornea and the decellularized cornea, and into the recipient cornea. Under visible light or ultraviolet light irradiation, the monomer polymer undergoes a polymerization reaction to form an adhesion layer between the decellularized cornea and the recipient cornea.

[0055] In the present invention, the recipient refers to an individual who receives the implanted acellular cornea, and can be a vertebrate, preferably a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples.

[0056] In the present invention, the adhesive layer is composed of a polymeric network formed by a methacryloyl derivative (methacryloyl gelatin, methacryloyl chondroitin sulfate, etc.) between the recipient cornea and the stromal network of the decellularized cornea.

[0057] In a preferred embodiment, the coagulated monomer-polymer solution is maintained at a temperature of 4-25°C.

[0058] In a preferred embodiment, the liquefaction condition is that the surface temperature is greater than or equal to 25°C, preferably 35-38°C.

[0059] In a preferred embodiment, the wavelength range of visible light is 300-800 nm, and the energy of light is 100-500 mW / cm 2 , the illumination time is 30 seconds to 2 minutes; more preferably, the wavelength range is 365-405nm, and the illumination time is 30-60 seconds.

[0060] A fourth aspect of the present invention provides a method for preparing the above-mentioned decellularized cornea and / or acellular cornea, and its use in preparing corneal donors, corneal transplants, tissue replacement materials, tissue sealants, medical dressings, or drug carriers. Beneficial effects

[0061] The present invention utilizes the physical property of methacrylated gelatin (Gelma) that it solidifies at 4-20°C and quickly turns into a liquid state when the temperature rises to 35-38°C. This allows the Gelma, which has been immersed in the biomaterial matrix network, to quickly liquefy and ooze out after being placed on the body surface, and to form a polymer network through light activation, thereby achieving the effect of bonding the implant. When Gelma is mixed with other monomer polymers in a specific proportion, this temperature-sensitive physical property can be retained, while improving the degradation resistance, bonding strength, and swelling properties of the adhesive layer. The low-swelling decellularized cornea prepared by the present invention has temperature sensitivity and photopolymerization properties, and the material source is rich, and the wound can be quickly repaired without surgical suture. In particular, the decellularized cornea provided by the present invention can directly contact the aqueous humor, can be used to replace the corneal stroma, and can be suitable for penetrating corneal transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 Physical states of monomer-polymer solutions with different ratios.

[0063] Figure 2 Viscosity of monomer-polymer solutions with different ratios.

[0064] Figure 3. Cytocompatibility of monomers and polymers with different ratios after polymerization.

[0065] Figure 4 Monomer polymers were infused into the decellularized porcine corneal stroma.

[0066] Figure 5 Rheological properties of low swelling (composite) decellularized porcine cornea and ordinary decellularized porcine cornea as a function of temperature.

[0067] Figure 6 Rheological properties of low-swelling (composite) decellularized porcine cornea and ordinary decellularized porcine cornea before and after 405 nm light activation.

[0068] Figure 7 Comparison of tensile strength between low swelling (composite) decellularized porcine cornea and ordinary decellularized porcine cornea.

[0069] Figure 8 Changes in corneal transparency before and after corneal swelling.

[0070] Figure 9 Water absorption rate of cornea after swelling.

[0071] Figure 10 Light transmittance after corneal swelling.

[0072] Figure 11 Young's modulus of cornea before and after low swelling (composite type) treatment.

[0073] Figure 12 Mechanical properties of cornea before and after low swelling (composite) treatment.

[0074] Figure 13: Interface strength pattern diagram of low swelling (composite) decellularized porcine cornea test.

[0075] Figure 14 Comparison of interface strength between low swelling (composite) acellular porcine cornea and medical bioglue.

[0076] Figure 15 Low swelling (composite) acellular porcine cornea for sutureless repair of corneal stromal defects.

[0077] Figure 16. Gross photographs of low-swelling (composite) acellular porcine cornea before and after repair of corneal stromal defects, and comparison of AS-OCT and thickness scan results.

[0078] Figure 17: Low swelling (composite) acellular porcine cornea forms stable adhesion with natural cornea.

[0079] FIG18 is a histological staining photograph of the adhesive layer formed by low swelling (composite) decellularized porcine cornea.

[0080] Figure 19 shows the results of slit lamp, fluorescein staining, AS-OCT and thickness scanning in in vivo animal experiments.

[0081] Figure 20 Comparison of the transplanted effects of low-swelling (composite) acellular porcine cornea and donor cornea.

[0082] Figure 21 Comparative effect of low swelling (composite) decellularized porcine cornea and ordinary decellularized porcine cornea after transplantation. DETAILED DESCRIPTION

[0083] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work shall fall within the scope of protection of the present invention.

[0084] Although steps, materials or substances, and reaction conditions similar or equivalent to those described herein can be used in the practice of the present invention, the preferred steps, materials or substances, and reaction conditions are described herein.

[0085] When a numerical range is described herein, unless otherwise indicated, the range is intended to include its endpoints and all integers and fractions within the range, and all values ​​within the range can achieve the effects of the present invention.

[0086] Unless defined otherwise, all technical and scientific terms and abbreviations used herein have the same meaning as commonly understood by one of ordinary skill in the art in the field of the invention or in the field to which the terms are applied.

[0087] As used herein, words in the singular include the plural and vice versa. Thus, "a," "an," and "the" generally include the plural form of the respective terms. As used herein, "one embodiment" or "an embodiment" refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The appearance of "in one embodiment" in various places in this specification does not necessarily refer to the same embodiment, nor does it refer to separate or selective embodiments that are mutually exclusive with other embodiments.

[0088] As used herein, "comprising," "having," "including," or "containing" is inclusive or open-ended and does not exclude additional, unrecited materials or method steps.

[0089] As used herein, "none," "low," or "reduction" means producing a reduced or absent state, or reducing a detectable or observable amount. In some embodiments, such reduction or decrease or disappearance is measured using one of the assessment tools described herein. In some embodiments, none, reduction, or reduction represents a difference.

[0090] Swelling refers to the phenomenon that the volume of a polymer expands and increases in a solvent.

[0091] Swelling property refers to the ability of a substance to swell in a solvent, typically expressed as a percentage weight increase, swelling rate, or water absorption rate. As used herein, "low swelling" or "reduced swelling" refers to a state in which the phenomenon or tendency of a substance (cornea) to increase in volume upon contact with or immersion in a solvent (aqueous humor) is reduced or decreased, or the amount of observable or measurable swelling is reduced or decreased, or a difference in reduction or decrease occurs.

[0092] Decellularized cornea: The corneas of mammals such as humans, pigs, horses, and cows are treated with conventional decellularization methods (including repeated freezing and thawing, high and low osmotic pressure treatment, surfactant treatment, ultra-static pressure treatment, nuclease treatment, and phospholipase treatment) to cause the stromal cells and endothelial cells to fall off, thus obtaining decellularized corneas.

[0093] Acellular porcine cornea: prepared by virus inactivation and decellularization process, including the anterior Descemet's layer and corneal stroma.

[0094] As used herein, low swelling acellular cornea and composite acellular cornea have the same meaning, both referring to the acellular cornea disclosed in the present invention.

[0095] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or those recommended by the manufacturer. All reagents or instruments used, if the manufacturer is not specified, are commercially available conventional products.

[0096] Sources of some materials in the embodiments of the present invention

[0097] Fresh porcine cornea: fresh porcine cornea without denaturation purchased from the market.

[0098] SDS solution: sodium dodecyl sulfate solution, purchased from Beijing Solebow Technology Co., Ltd., product number S8010.

[0099] Nuclease: Deoxyribonuclease was purchased from Beijing Sino Biological Science and Technology Co., Ltd., catalog number SSNP01.

[0100] The photoinitiator LAP phenyl (2,4,6-trimethylbenzoyl) phosphate lithium salt was purchased from Shanghai Yuanye Biotechnology Co., Ltd., product number Y43995.

[0101] Methacrylated gelatin (GelMA): a gelatin derivative obtained by the reaction of gelatin and methacrylic anhydride, purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd., item numbers EFL-GM-90 and EFL-GM-70.

[0102] PEGDA solution: polyethylene glycol diacrylate solution, purchased from Sigma, product number 455008.

[0103] PBS buffer: phosphate buffered saline, purchased from Beijing Solebow Technology Co., Ltd., catalog number P1020.

[0104] Rabbits were purchased from Xilingjiao Breeding and Propagation Center in Jinan.

[0105] Example 1 Physical properties of methacrylated gelatin and its mixture

[0106] PEGDA and PEGMA polymers have low swelling properties and, after forming a polymeric network in decellularized cornea (DPC), can limit the swelling of DPC in aqueous solutions. However, cells have difficulty adhering to the surfaces of PEGDA and PEGMA polymers. Therefore, methacryloyl derivatives such as methacryloyl gelatin / chondroitin sulfate, which have good biocompatibility, can be mixed with PEGDA and PEGMA in specific ratios to improve the cell adhesion of PEGDA and PEGMA.

[0107] After mixing 20% ​​GelMA (methacrylated gelatin) with a 0.1g / ml PEGMA solution (average mass fraction of 700) in varying proportions, the physical state, viscosity, transmittance, water absorption, and cell adhesion properties were analyzed. The results are shown in Table 1, Figures 1, and 2. When the 20% GelMA ratio was greater than 20%, the gel was solid at 4-20°C and liquid at 37°C. As the GelMA ratio increased, viscosity increased, transmittance decreased, and water absorption increased. When the 20% GelMA ratio was greater than 80%, the gel exhibited good cell adhesion.

[0108] Table 1

[0109] Example 2 Cytocompatibility of mixtures of PEGDA and methacryloyl gelatin in different proportions

[0110] A 0.1 g / ml PEGDA solution was thoroughly mixed with a 20% methacryloylated gelatin solution in ratios of 9:1, 8:2, 5:5, 2:8, and 1:9, respectively. One ml of the mixture was added to each well of a 6-well cell culture plate at 37°C and solidified by light curing. Corneal epithelial cells were seeded into the 6-well plate, and cell culture medium was added. The cells were cultured in a CO2 incubator. After 72 hours, cell attachment and growth were observed under a microscope. As shown in Figure 3, corneal epithelial cells adhered to the gel surface when the PEGDA and methacryloylated gelatin were mixed in ratios of 2:8 and 1:9. Gels prepared with other mixtures did not adhere to the gel surface.

[0111] Example 3 Preparation of low swelling (composite) acellular porcine cornea

[0112] (1) Preparation of decellularized porcine cornea: fresh porcine cornea was scraped to remove the epithelium and endothelium, immersed in 0.5% SDS solution, treated with 500 U / ml nuclease for 2 hours, and then washed with sufficient balanced salt buffer for 6-8 times.

[0113] (2) Preparation of methacrylated compound solution: 20% methacrylated gelatin solution and 0.1 g / ml PEGDA (PEGDA with an average molecular weight of 700) solution were prepared with 0.3% LAP solution. The methacrylated gelatin solution and PEGDA solution were mixed at a volume ratio of 8:2 to 10:0.

[0114] (3) Composite methacrylated gelatin: The decellularized porcine cornea was immersed in a methacrylated compound solution and then placed in a 37°C constant temperature shaker with agitation at 120 rpm for 24 hours. Immunofluorescence analysis showed that after 24 hours, the monomer polymer was completely immersed in the decellularized porcine corneal stroma (Figure 4).

[0115] (4) Preparation of low-swelling decellularized porcine cornea: The decellularized porcine cornea was removed, the surface solution was removed, and the gelatin derivative was cooled at 4-25°C (e.g., 4°C, 10°C, 15°C, 20°C, 25°C) to solidify.

[0116] Example 4 Preparation of acellular porcine cornea

[0117] (1) Preparation of decellularized porcine cornea: fresh porcine cornea was scraped to remove the epithelium and endothelium, immersed in 0.5% SDS solution, treated with 500 U / ml nuclease for 2 hours, and then washed with sufficient balanced salt buffer for 6-8 times.

[0118] (2) Preparation of voriconazole-methacrylated gelatin mixture: 30% methacrylated gelatin solution was prepared with 0.5% LAP solution. 5 mg of voriconazole powder was weighed and dissolved in 10 ml of dimethyl sulfoxide solution to prepare a 500 μg / ml stock solution. The methacrylated gelatin solution and the voriconazole solution were mixed at a volume ratio of 9:1. (3) Preparation of voriconazole-composite decellularized porcine cornea: The decellularized porcine cornea was immersed in the voriconazole-methacrylated gelatin mixture, and then placed in a 37°C constant temperature shaker with shaking at 120 rpm for 24 hours. The decellularized porcine cornea was removed, the surface solution was removed, and the cornea was cooled to 4°C.

[0119] Test Example 1 Rheological properties, storage modulus, and loss modulus

[0120] The rheological properties of the low swelling decellularized porcine cornea and the ordinary decellularized porcine cornea prepared in Example 3 under different temperature conditions were tested by a rheometer. The results are shown in Figure 5. Under conditions below 20°C, the rheological properties of the low swelling decellularized porcine cornea remain stable. When the temperature is above 20°C, the storage modulus of the low swelling decellularized porcine cornea begins to decrease, reaches the lowest point at around 27°C, and then begins to rise. This is related to the fact that the methacrylated gelatin in the low swelling decellularized porcine cornea changes from solidification to liquid exudation from the decellularized porcine cornea when the temperature is above 20°C. The rheological properties of the ordinary decellularized porcine cornea remain stable within the observation range of 0-40°C, and no significant changes occur. This proves that the low swelling decellularized porcine cornea is more temperature sensitive than the ordinary decellularized porcine cornea.

[0121] Subsequently, the low-swelling decellularized porcine cornea and conventional decellularized porcine cornea were illuminated with a 405nm light source. The results showed that the storage modulus and loss modulus of the low-swelling decellularized porcine cornea changed significantly, with the difference between the storage modulus and loss modulus increasing (Figure 6). In contrast, the rheological properties of the conventional decellularized porcine cornea did not change significantly with illumination. This demonstrates that the low-swelling decellularized porcine cornea is more photosensitized than the conventional decellularized porcine cornea.

[0122] Finally, the mechanical properties of low-swelling decellularized porcine cornea and ordinary decellularized porcine cornea were tested by tensile tests. The results are shown in Figure 7. The average tensile strength of the low-swelling decellularized porcine cornea is 7.52N, which is higher than that of natural human cornea (5.20N) and porcine cornea (5.03N), showing good mechanical properties. This is due to the polymerization of polymers within the stroma of the low-swelling decellularized porcine cornea, which increases the cross-linking strength of the cornea. However, due to the decellularization process, the internal collagen fibers of the decellularized porcine cornea are damaged, and the average tensile strength is 3.36N, which is lower than that of the low-swelling decellularized porcine cornea and the natural cornea.

[0123] Test Example 2 Mechanical Properties, Anti-Swelling, and Light Transmittance

[0124] Decellularized porcine corneas were prepared according to the procedures of Example 3, using a 9:1 volume ratio of methacrylated gelatin solution to PEGDA solution. The resulting low-swelling decellularized porcine cornea was irradiated with 365 nm UV light for 2 minutes to solidify. The corneas were then immersed in a balanced salt solution (normal saline) along with untreated, standard decellularized porcine corneas and natural corneas (fresh porcine corneas) at room temperature for 48 hours. The corneas were then photographed and compared for changes in water absorption and transparency before and after immersion.

[0125] The results showed that the low-swelling decellularized porcine cornea exhibited low swelling properties. After immersion in saline for 48 hours, the cornea remained transparent and showed no edema. In contrast, the conventional decellularized porcine cornea exhibited significant edema and decreased transparency (Figure 8). The water absorption rate of the low-swelling decellularized porcine cornea was 25±4%, compared to 324±12% for the conventional decellularized porcine cornea and 322±70% for the natural cornea (Figure 9). Spectrophotometric analysis revealed that the light transmittance of the low-swelling decellularized porcine cornea was 77±1.0%, compared to 26±1.3% for the conventional decellularized porcine cornea and 43±0.2% for the natural cornea (Figure 10).

[0126] Low-swelling decellularized porcine cornea exhibited a higher Young's modulus than conventional decellularized corneal stroma, demonstrating superior mechanical properties (Figures 11 and 12). The increased mechanical properties and swelling resistance of the cornea demonstrate that the monomeric polymers penetrated into the corneal stroma fibers, undergoing photostimulation and polymerization, forming a new polymeric network within the corneal stroma.

[0127] Test Example 3 Adhesion and interface strength

[0128] Currently, commonly used bioadhesives in clinical practice primarily include fibrin glue, α-cyanoacrylate glue, and PEG-based bioglue. The clinical application of PEG-based bioglue is significantly limited. Fibrin glue is a natural biomaterial with good biocompatibility, but its bonding strength is relatively weak. Furthermore, its raw material is derived from allogeneic tissue, posing a risk of viral transmission. α-cyanoacrylate glue offers high bonding strength, but it also has significant disadvantages. α-cyanoacrylate glue generates polymerization heat during use, causing thermal damage to the bonded tissue. Furthermore, the cured material is brittle and mechanically incompatible with soft tissue, resulting in a foreign body reaction that hinders wound healing. PEG-based bioglue is prone to swelling and can cause wound dehiscence. Furthermore, PEG gelation hinders cell adhesion, hindering wound repair. Compared to traditionally used representative bioadhesives, low-swelling decellularized porcine corneas achieve bioadhesion to recipient tissue without the use of bioglue, offering improved operability, biocompatibility, and the potential to promote wound healing.

[0129] The adhesion strength of the low swelling decellularized porcine cornea prepared in Example 3 and the traditional bioadhesive on the natural cornea surface was tested (Figure 13). The results showed that the low swelling decellularized porcine cornea could form a 91.1 J / m 2 The interfacial strength of fibrin glue (porcine fibrin adhesive Guangzhou Beixiu Biotechnology Co., Ltd.) is 22.3J / m 2 The interfacial strength of PEG glue (absorbable dura mater sealing medical glue, Cyxes Biotechnology Co., Ltd.) is 25.1 J / m 2 The interfacial strength of α-cyanoacrylate glue (α-cyanoacrylate fast medical glue Guangzhou Baiyun Medical Glue Co., Ltd.) is 38.6 J / m 2 The interfacial strength of low-swelling acellular porcine cornea on the natural corneal surface was significantly higher than that of conventional bioadhesives (Figure 14).

[0130] Application Example 1 In vitro experiment - in situ curing

[0131] (1) A circular corneal stromal defect with a diameter of 6 mm and a depth of 500 μm was created on the surface of the pig cornea using a trephine drill and a lamellar knife.

[0132] (2) A low-swelling acellular porcine cornea of ​​equal size prepared according to the method of Example 3 (wherein the volume ratio of methacryloylated gelatin solution to PEGDA solution was 8:2) was placed on the corneal defect. After the ambient temperature was raised to 37°C, the cornea was irradiated with 405 nm visible light for 60 seconds (Figure 15).

[0133] (3) Comparison of the repaired pig eyeball with the preoperative one showed successful closure of the stromal defect under slit lamp observation. Anterior segment optical coherence tomography (AS-OCT) and thickness scans confirmed that the stromal defect was filled and the corneal curvature was restored (Figure 16).

[0134] (4) The entire eyeball can be lifted by pinching the composite decellularized porcine cornea with forceps, which proves that the composite decellularized porcine cornea forms a strong adhesion with the natural cornea and can still maintain stable adhesion after 48 hours (Figure 17).

[0135] (5) The cornea after bonding was histologically sectioned and stained. The H&E staining results showed that there was a polymerization reaction between the composite decellularized porcine cornea and the recipient cornea, forming an adhesive layer ( FIG. 18 ).

[0136] Application Example 2 In Vivo Experiment - In Situ Curing

[0137] (1) A circular corneal stromal defect with a diameter of 6 mm and a depth of 200 μm was created on the rabbit corneal surface using a trephine drill and a lamellar knife.

[0138] (2) In situ curing: A low-swelling decellularized porcine cornea of ​​the same size and thickness prepared according to the method of Example 3 (wherein the volume ratio of methacrylated gelatin solution to PEGDA solution was 8:2) was placed on the corneal defect and then irradiated with 405 nm visible light for 60 seconds.

[0139] (3) Postoperative examinations were performed every two weeks. Two weeks after surgery, the graft was stable without displacement or peeling. Epithelial defect staining showed that the corneal epithelium was fully repaired within 2 weeks. OCT and thickness map scans showed that the corneal defect was successfully repaired and the matrix was regenerated (Figure 19).

[0140] Application Example 3 In Vivo Experiment - Suturing

[0141] A penetrating defect with a diameter of 3 mm was created in the center of the rabbit cornea. Penetrating keratoplasty was then performed, and a low-swelling decellularized porcine cornea prepared by the method of Example 3 (wherein the methacrylated gelatin solution and the PEGDA solution were in a volume ratio of 9:1) and a donor cornea with a healthy endothelium (an allogeneic cornea obtained from another rabbit's eyeball) were implanted, and the corneal perforation was repaired by breakpoint suture (Figure 20). After the low-swelling decellularized porcine cornea was implanted as a graft, it was stable 5 days after surgery, no edema occurred, and the corneal transparency was good; 14 days after surgery, the corneal epithelium was repaired, the surgical irritation inflammation subsided, the graft did not edema, and no immune rejection occurred; 18 days after surgery, the cornea was stable, the graft was fused with the graft bed, the anterior chamber was stable, and no aqueous humor leakage occurred. 28 days after surgery, the repair effect of the decellularized porcine cornea was comparable to that of the donor cornea, with no corneal edema, good corneal transparency, a stable anterior chamber, and biological fusion of the graft with the graft bed.

[0142] Application Example 4 In Vivo Experiment - Suturing

[0143] A penetrating defect with a diameter of 6 mm was created in the center of the rabbit cornea. Penetrating keratoplasty was then performed, and the low-swelling decellularized porcine cornea (previously cut into 6 mm diameter grafts with a corneal trephine) prepared by the method of Example 3 (wherein the methacrylated gelatin solution and the PEGDA solution were in a volume ratio of 9:1) and the ordinary decellularized porcine cornea were implanted by breakpoint suture (Figure 21). After the low-swelling decellularized porcine cornea was implanted as a graft, the cornea remained transparent, the anterior chamber was stable, and there was no inflammatory stimulation 28 days after the operation. The transparency of the ordinary decellularized porcine cornea was completely lost, turning white, completely covering the pupil area, and the eye lost its visual function. Optical coherence scanning showed that the low-swelling decellularized porcine cornea did not swell, was comparable in thickness to the recipient cornea, and underwent biological fusion with the recipient cornea. The ordinary decellularized porcine cornea was severely edematous and its thickness was significantly higher than that of the recipient cornea.

[0144] The embodiments of the present invention are described in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only intended to help understand the method of the present invention and its core concept. At the same time, changes or modifications made by those skilled in the art based on the concept of the present invention, the specific implementation methods of the present invention, and the scope of application are all within the scope of protection of the present invention. In summary, the contents of this specification should not be understood as limiting the present invention.

Claims

1. A decellularized cornea, characterized in that: The decellularized cornea contains a solidified monomer polymer solution, which includes component 1 and component 2, wherein component 1 is a methacrylated derivative; component 2 is selected from one or more of polyethylene glycol diacrylate (PEGDA) or ethylene glycol methyl ether methacrylate (PEGMA).

2. The acellular cornea according to claim 1, characterized in that: When the decellular cornea is irradiated with visible light or ultraviolet light, the monomer polymers inside undergo a polymerization reaction to form a polymer network in the corneal stroma; the polymer network has no swelling property or low swelling property, thereby limiting the swelling of the decellular cornea in the solution.

3. The acellular cornea according to claim 2, characterized in that: The light transmittance of the decellularized cornea in the visible light region is greater than 70%. Under normal temperature conditions, after being immersed in physiological saline for 48 hours, its water absorption rate is 10-35%.

4. The acellular cornea according to claim 1 or 2, characterized in that: Component 1 is 10-50% (w / v) of methacrylic derivative; component 2 is 0.001g / ml-1g / ml polyethylene glycol diacrylate or polyethylene glycol methyl ether methacrylate or a mixture thereof; the monomer polymer solution is prepared by mixing component 1 and component 2, and the volume ratio of component 1 solution to component 2 solution is 10:0-8:2; wherein the methacrylic derivative includes methacrylic gelatin or methacrylic chondroitin sulfate or a mixture thereof.

5. The acellular cornea according to claim 1 or 2, characterized in that: The monomer polymer solution also includes a photoinitiator or a temperature initiator; the concentration of the photoinitiator is 0.1%-0.5%, and the concentration of the temperature initiator is 0.1-0.2 mol / L; The photoinitiator is a blue light or ultraviolet light initiator, including 2,4,6-trimethylbenzoyl lithium phosphate; the temperature initiator includes azobisisobutyronitrile.

6. A method for preparing acellular cornea, comprising: The decellular cornea is immersed in a monomer polymer solution, the decellular cornea is taken out, and then the polymerization reaction of the monomer polymer in the decellular cornea is activated.

7. The preparation method according to claim 6, characterized in that: include: The decellular cornea is immersed in a monomer polymer solution, the decellular cornea is taken out and then cooled to solidify the monomer polymer in the decellular cornea, and then the polymerization reaction of the monomer polymer in the decellular cornea is activated; The monomer polymer solution includes component 1: a methacryloyl derivative at a concentration of 10-50% (w / v).

8. The preparation method according to claim 6 or 7, characterized in that: The monomer polymer solution also includes component 2: polyethylene glycol diacrylate (PEGDA) or polyethylene glycol methyl ether methacrylate (PEGMA) or a mixture thereof at a concentration of 0.001 g / ml-1 g / ml; the volume ratio of component 1 to component 2 is 10:0-8:

2.

9. The preparation method according to claim 6 or 7, characterized in that: The monomer polymer solution comprises component 1: 10-30% (w / v) methacryloyl gelatin and / or methacryloyl chondroitin sulfate; component 2: 0.001 g / ml-0.5 g / ml polyethylene glycol diacrylate (PEGDA) and / or polyethylene glycol methyl ether methacrylate (PEGMA); the volume ratio of component 1 to component 2 is 10:0-8:

2.

10. The preparation method according to claim 6 or 7, characterized in that: The monomer polymer solution contains a photoinitiator or a temperature initiator; the photoinitiator is a blue light or ultraviolet light initiator, including 2,4,6-trimethylbenzoyl lithium phosphate (LAP); the temperature initiator includes azobisisobutyronitrile (AIBN).

11. The preparation method according to claim 10, characterized in that: The concentration of the photoinitiator is 0.1%-0.5%; the concentration of the temperature initiator is 0.1-0.2 mol / L; when light activation is adopted, the wavelength is 300nm-600nm, and the activation time is 30 seconds to 2 minutes; when temperature activation is adopted, above 60°C, the reaction time is 2 hours to 12 hours.

12. The preparation method according to claim 6 or 7, characterized in that: The preparation process of monomer polymer solution is as follows: a) preparing a 10-50% (w / v) methacryloyl derivative solution with a 0.1-0.5% LAP solution to prepare Obtain component 1 solution; b) preparing a solution of component 2 by adding 0.001 g / ml to 1 g / ml of polyethylene glycol diacrylate or polyethylene glycol methyl ether methacrylate or a mixture thereof; c) Mix the component 1 solution and the component 2 solution in a volume ratio of 10:0-8:

2.

13. The method according to claim 12, characterized in that: The step a) is to prepare a solution of component 1 by using a 0.1-0.5% LAP solution to prepare a 10-30% (w / v) methacryloyl gelatin or methacryloyl chondroitin sulfate solution; The step b) is to prepare a solution of component 2 by preparing 0.001 g / ml-0.5 g / ml of polyethylene glycol diacrylate or polyethylene glycol methyl ether methacrylate. In the step c), the component 1 solution and the component 2 solution are mixed in a volume ratio of 9:1-8:

2.

14. The preparation method according to claim 6 or 7, characterized in that: The soaking operation is to soak the decellularized cornea in at least 10 volumes of the monomer polymer solution for 18-48 hours.

15. The preparation method according to claim 7, characterized in that: The cooling method is to place the decellularized cornea in a 4° C. environment for 15-30 minutes.

16. Acellular cornea obtained by the preparation method according to any one of claims 6 to 15.

17. The acellular cornea according to claim 16, characterized in that: The decellularized cornea contains a solidified monomer polymer solution inside and / or adheres to its surface. After being applied to the recipient cornea, the monomer polymer solution liquefies and partially seeps out of the decellularized cornea and then soaks into the space between the recipient cornea and the decellularized cornea, and into the recipient cornea. Under visible light or ultraviolet light, the monomer polymer undergoes a polymerization reaction to form an adhesion layer between the decellularized cornea and the recipient cornea.

18. The acellular cornea according to claim 17, characterized in that: The adhesion layer consists of a polymeric network formed by the methacryloyl derivative between the recipient cornea and the stromal network of the decellularized cornea.

19. The acellular cornea according to claim 17, characterized in that: The condition for maintaining the solidified monomer polymer solution is 4-25°C; the condition for liquefaction is that the surface temperature of the recipient cornea is greater than 25°C; The wavelength of the visible light is in the range of 300-800nm, and the energy of the light is in the range of 100-500mW / cm 2 , the illumination time is 30 seconds to 2 minutes.

20. Use of the decellularized cornea according to any one of claims 1 to 5, or the preparation method according to any one of claims 6 to 15, or the decellularized cornea according to any one of claims 16 to 19 in preparing a corneal donor, a corneal transplant, a tissue substitute material, a tissue sealant, a medical dressing, or a drug carrier.

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