A corneal filler based on natural extracellular matrix and its preparation method and application

Abstract

The application discloses a kind of corneal fillers based on natural extracellular matrix and its preparation method and application.The corneal filler has the fiber mesh structure that decellularized corneal extracellular matrix microfibril and methacrylated derivative photocrosslinking network are interwoven and compounded;The corneal filler comprises 30%-70% (v / v) decellularized corneal extracellular matrix microfibril, 0.1%-0.5% (w / v) photo initiator and 5%-30% (w / v) methacrylated derivative.The filler provided by the application can realize the sutureless filling of corneal lesion, and has long-term stability, biomimetic structure and matrix regeneration capacity.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, specifically to a corneal filler based on natural extracellular matrix, its preparation method, and its application. Background Technology

[0002] The cornea is a crucial transparent tissue that maintains the structure and visual function of the eye. The corneal stroma is primarily composed of regularly arranged collagen fibers and proteoglycans. It is avascular and has extremely limited self-regenerative capacity; once structural defects occur, it is difficult for the cornea to restore its original thickness and optical integrity through self-repair. Clinically, for focal corneal stromal defects caused by infection, trauma, or degeneration, the ideal repair method is allogeneic corneal transplantation. However, there is a severe global shortage of corneal donors, and the surgery is highly invasive and expensive. Furthermore, the transplant suturing procedure easily causes complications such as deformation of the corneal optical interface, iatrogenic astigmatism, stimulation of local scar hyperplasia, and neovascularization. Especially for focal lesions less than 5-6 mm in diameter (corneal diameter approximately 10-12 mm), penetrating or lamellar keratoplasty often yields poor results due to overly dense sutures. For such defects, ideal repair materials have long been lacking clinically, and patients often face the risks of persistent refractive errors, poor epithelial repair, and scarring.

[0003] In recent years, injectable in-situ shaping hydrogels have provided new ideas for minimally invasive corneal repair and are often used as "corneal fillers" or "repair agents" in preclinical research. For example, patent document TW202229419A discloses a corneal occlusion and tissue adhesion material based on methacrylamide gelatin (GelMA) and its mixtures; patent document WO2025097846A1 proposes an in-situ corneal filler composed of methacrylic acid succinic acid diesterized starch and methacrylamide gelatin; and patent document CN118105542A introduces a photocurable biomaterial constructed by preparing collagen fibers through electrospinning and introducing photopolymerizable groups. However, existing corneal repair materials are mostly based on synthetic polymers or single-source natural polymers (such as gelatin and its derivatives), and their chemical composition is relatively simple, making it difficult to simulate the high complexity of the natural corneal extracellular matrix (ECM). Studies have shown that natural human corneal ECM contains more than 1,300 proteins, while existing materials generally lack this complex biochemical composition, making it difficult to effectively regulate the directional migration, orderly proliferation, and regular secretion of functional stroma cells. During routine repair processes, these materials exhibit insufficient regeneration capacity, difficulty in repairing larger corneal defects (greater than 3 mm), and excessively rapid degradation rates, limiting their clinical application potential.

[0004] Furthermore, patent document CN113336973B discloses a dual-network hydrogel constructed from pepsin-digested decellularized corneal stroma and modified hyaluronic acid. While this material exhibits good biocompatibility and healing-promoting ability in in vitro and short-term animal experiments, its long-term sustainability still faces significant challenges, primarily manifested in rapid in vivo degradation and limited repair efficacy for large corneal defects. In vivo studies show that, similar to most injectable hydrogels, this type of material often degrades within 2-3 months after implantation, leading to reduced stroma thickness and decreased epithelial adhesion stability in the repair area, making long-term structural repair difficult.

[0005] In summary, the development of in-situ corneal filling materials needs to move from simply "sealing defects" to "functional regeneration". Summary of the Invention

[0006] To address the aforementioned technical problems, this invention aims to provide a corneal tampon that efficiently fills corneal stromal defects, restores corneal refractive and mechanical characteristics, and promotes tissue regeneration, thereby meeting the clinical demand for corneal defect repair materials.

[0007] To achieve the above objectives, in a first aspect, the present invention provides a method for preparing decellularized corneal extracellular matrix microfibers, comprising breaking up a decellularized corneal homogenate to obtain decellularized corneal extracellular matrix microfibers, specifically including the following steps:

[0008] S1) The decellularized cornea is broken into a homogenate to obtain a suspension containing extracellular matrix microfibers of the decellularized cornea.

[0009] Preferably, the decellularized cornea is derived from mammals such as pigs, cattle, or humans, and undergoes decellularization to remove immunogenic components. This invention does not limit the method for preparing the decellularized cornea; those skilled in the art can purchase it commercially or prepare it using existing technologies, such as the method in patent CN117100913B.

[0010] Preferably, the stirring method is to use a homogenizer. More preferably, the stirring method is intermittent homogenization, i.e., running-pausing, repeating cycles. This invention does not limit the number of cycles; those skilled in the art can determine the number of cycles according to actual needs.

[0011] Preferably, the homogenization time for a single homogenization is 10-20 seconds, and the cumulative homogenization time is 1-3 minutes. More preferably, the homogenization time for a single homogenization is 10-20 seconds, and the cumulative homogenization time is 1-2 minutes.

[0012] Preferably, the rotation speed of the homogenizer is 10,000-25,000 rpm, more preferably 20,000-25,000 rpm.

[0013] Preferably, a cooling step is inserted during the homogenization process. The present invention does not impose any particular limitation on the specific method of inserting the cooling step; those skilled in the art can choose according to the actual situation. For example, a cooling step may be performed after each homogenization cycle, or a cooling step may be performed after several homogenization cycles.

[0014] Preferably, the cooling environment temperature is -40 to 0°C, the single cooling time is 3 to 5 minutes, and the cumulative cooling time is 3 to 10 minutes.

[0015] Preferably, the homogenization process is carried out in a low-temperature environment, wherein the low temperature is 0-25°C.

[0016] Preferably, the temperature of the decellularized cornea is maintained at 0~40°C by controlling the environment, time and interval of homogenization, the insertion method of cooling, and the temperature and time of the cooling environment; more preferably, the temperature of the decellularized cornea is maintained at 4~30°C; and even more preferably, the temperature of the decellularized cornea is maintained at 4~20°C.

[0017] Preferably, the above-mentioned method for preparing decellularized corneal extracellular matrix microfibers further includes:

[0018] S2) Centrifuge the suspension, and the precipitate is the decellularized corneal extracellular matrix microfiber.

[0019] Preferably, the centrifugation is performed at 5000-20000 rpm for 2-20 minutes at 0-8℃. More preferably, it is performed at 15000-20000 rpm for 2-10 minutes at 0-4℃. This invention does not limit the number of centrifugation cycles; those skilled in the art can centrifuge all at once or in multiple cycles depending on the actual situation. Multiple centrifugation cycles are preferred.

[0020] In a second aspect, the present invention provides decellularized corneal extracellular matrix microfibers prepared by the above method.

[0021] The decellularized corneal extracellular matrix microfibers provided by this invention retain the protein and polysaccharide macromolecules in the cornea, including but not limited to type I collagen, core proteoglycans, corneal proteoglycans, laminin and fibronectin family glycoproteins.

[0022] In a third aspect, the present invention provides a corneal tampon containing the aforementioned decellularized corneal extracellular matrix microfibers.

[0023] Preferably, the corneal granulator comprises 30%-70% (v / v) decellularized corneal extracellular matrix microfibers, 0.1%-0.5% (w / v) photoinitiator, and 5%-30% (w / v) methacrylamide derivative. More preferably, the corneal granulator comprises 30-60% (v / v) decellularized corneal extracellular matrix microfibers, 10%-20% (w / v) methacrylamide derivative, and 0.2%-0.4% (w / v) photoinitiator.

[0024] Preferably, the methacrylamide derivative is selected from one or more of methacrylamide gelatin (GelMA), methacrylamide chondroitin sulfate (ChsMA), methacrylamide dextran (DexMA), and polyethylene glycol diacrylate (PEGDA).

[0025] Preferably, the photoinitiator is sodium phenyl-2,4,6-trimethylbenzoylphosphinic acid (NAP) or lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid (LAP) or a combination thereof.

[0026] In a fourth aspect, the present invention provides a method for preparing the corneal tampon in the third aspect, comprising the following steps: obtaining decellularized corneal extracellular matrix microfibers according to the decellularized corneal fragmentation method provided in the first aspect;

[0027] S3) Incubate a mixture of decellularized corneal extracellular matrix microfibers, photoinitiator, and methacrylamide derivative to obtain a corneal filler in the form of a gel precursor; or incubate a mixture of decellularized corneal extracellular matrix microfibers and methacrylamide derivative, and then add a photoinitiator to obtain a corneal filler in the form of a precursor.

[0028] During incubation, the methacrylamide derivative reduces protein denaturation of decellularized corneal extracellular matrix microfibrils through intermolecular hydrogen bonds and steric hindrance effects.

[0029] Preferably, the corneal tampon includes 30%-70% (v / v) decellularized corneal extracellular matrix microfibers, 0.1%-0.5% (w / v) photoinitiator and 5%-30% (w / v) methacrylamide derivative.

[0030] More preferably, the final concentration of decellularized corneal extracellular matrix microfibers is 30-60% (v / v), for example, 30%-60%, 30%-50%, 40%-60%, 40%-50%, or 45%-55%. If the content of decellularized corneal extracellular matrix microfibers is too low, it cannot provide sufficient biomimetic adhesion sites and structural support for corneal cells, weakening the effect of guiding tissue regeneration; if the solid content is too high, the viscosity of the gel precursor increases, making injection difficult, and uneven mixing with methacrylamide derivatives can lead to porosity, uneven mechanical properties, and even affect corneal transparency after cross-linking.

[0031] More preferably, the final concentration of the methacrylamide derivative is 10%-20% (w / v), for example 10%-15% or 15%-20%; and the final concentration of the photoinitiator is 0.2%-0.4% (w / v), for example 0.2%-0.3% or 0.3%-0.4%. If the concentration of the methacrylamide derivative is too low, it will not gel or the hydrogel network formed after cross-linking will be sparse and lack strength, making it prone to cracking and peeling under the physiological environment of the ocular surface; if the concentration is too high, it will be difficult to dissolve, and the formation of a suspension will reduce the light transmittance of the filler and affect corneal visual function.

[0032] Preferably, the methacrylamide derivative is selected from one or more of methacrylamide gelatin (GelMA), methacrylamide chondroitin sulfate (ChsMA), methacrylamide dextran (DexMA), and polyethylene glycol diacrylate (PEGDA).

[0033] Preferably, the photoinitiator is sodium phenyl-2,4,6-trimethylbenzoylphosphinic acid (NAP) or lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid (LAP) or a combination thereof.

[0034] This invention does not limit the solvent, and those skilled in the art can choose according to actual needs. Preferably, the solvent is phosphonate buffer (PBS) to maintain the system at an appropriate physiological pH value and ensure biocompatibility.

[0035] Preferably, the incubation is performed in a water bath at 60-80°C for 20-120 minutes, for example, incubation at 60°C for 40-80 minutes.

[0036] Preferably, the method for preparing the corneal tampon further includes a pretreatment step: drying the decellularized cornea until it is completely dehydrated.

[0037] Preferably, the preparation method of the above-mentioned corneal tampon further includes a post-processing step: centrifuging the incubated solution containing decellularized corneal extracellular matrix microfibers at 5000-10000 rpm for 5-10 minutes and discarding the bottom precipitate.

[0038] Preferably, the corneal tampon is stored at 0-6°C, more preferably 0-4°C.

[0039] This invention does not limit the specific operations of stirring, mixing, centrifuging, drying, sterilizing, filtering, resuspending, and storing the solvent in the above steps; those skilled in the art can choose according to the actual situation. The embodiments described in this invention are some embodiments of this invention, but not all embodiments.

[0040] In use, the corneal calorie is applied (e.g., injected) to the corneal defect. Under illumination with a light source of 365-405 nm wavelength, the corneal calorie undergoes in-situ cross-linking and curing to form a hydrogel filler that matches the shape of the defect.

[0041] The hydrogel filler obtained by this invention has a hydrogel network structure and can be used as a cell carrier or drug carrier to load corneal stromal cells, growth factors, antibacterial and anti-inflammatory drugs, etc.

[0042] The physicochemical properties of hydrogel fillers include an elastic modulus of 90-200 kPa, a swelling ratio of 5-80%, a porosity of 20-50%, and a light transmittance of 70-90%.

[0043] In a fifth aspect, the present invention provides the use of the above-mentioned decellularized corneal extracellular matrix microfibers and corneal tampon containing decellularized corneal extracellular matrix microfibers in the preparation of pharmaceuticals or medical devices for repairing corneal damage; or in the preparation of cell carriers or drug carriers for repairing corneal damage.

[0044] The corneal tampon provided by this invention is used to repair corneal damage, including but not limited to ocular surface damage, corneal epithelial damage, corneal stromal damage, corneal ulcer, corneal perforation, and corneal laceration, as well as as an ocular surface dressing, auxiliary sealing and adhesive material in ocular surface transplantation surgery.

[0045] The components of the corneal tampon of this invention work synergistically to achieve a breakthrough effect of "structure-function integrated regeneration" tailored to the physiological environment and clinical needs of corneal repair, including but not limited to the following properties:

[0046] 1. The biomimetic structure has strong adaptability and combines efficient organization and regeneration capabilities.

[0047] The filler retains the natural decellularized corneal extracellular matrix components. The three-dimensional network structure of the filler is similar to that of the natural extracellular matrix, providing a biomimetic microenvironment for corneal cell adhesion and proliferation, promoting in-situ regeneration of corneal tissue, and achieving functional repair of the defect area, rather than simply physical filling. This effectively reduces the risk of postoperative corneal fibrosis and poor epithelial repair.

[0048] 2. Good biocompatibility and low immunogenicity

[0049] Decellularized corneal ECM removes immunogenic components through decellularization. Methacrylamide derivatives and photoinitiators are all made of non-toxic, biocompatible materials. The implanted filler will not cause significant inflammation or rejection.

[0050] 3. Minimally invasive procedure with strong clinical applicability

[0051] The filler is an injectable paste that can be injected into the corneal defect via a minimally invasive procedure. Under irradiation with a 365-405nm light source, it can complete in-situ cross-linking and curing within 60 seconds, quickly forming and precisely matching the shape of the defect. No sutures are required, reducing the difficulty of the surgery and medical costs. The filler is particularly suitable for focal corneal stromal defects with a diameter of less than 5-6 mm, filling the gap in the lack of ideal repair materials for such defects in clinical practice. Furthermore, the corneal curvature is stable after surgery, which can effectively improve the patient's refractive error.

[0052] 4. Multifunctionality and Clinical Expansion

[0053] This filler can be used not only for the direct repair of defects such as corneal ulcers, perforations, and lacerations, but its three-dimensional hydrogel network can also serve as a cell carrier or drug carrier, loading corneal stromal cells, growth factors, antibacterial and anti-inflammatory drugs, etc., to achieve a combined treatment of "filling + cell transplantation + drug sustained release", which is suitable for the repair of various corneal injuries such as infectious, traumatic, and degenerative diseases. Attached Figure Description

[0054] Figure 1 Image A shows electrophoresis diagrams of ECM microfibrils prepared by different extraction methods; Image B shows a quantitative comparison of protein concentrations.

[0055] Figure 2 The image shows a comparison of the ultrastructure of ECM microfibers extracted by different methods using transmission electron microscopy.

[0056] Figure 3 Image A shows protein electrophoresis diagrams prepared using different incubation methods; Image B shows the protein concentration quantification results.

[0057] Figure 4 A shows the WAXD characterization results; B shows the FTIR spectral analysis results.

[0058] Figure 5 The results of the analysis of the filler prepared by the present invention and the natural corneal protein components are shown, where A is PCA analysis; B is the number of proteins identified; C is the differential protein volcano plot; D is the ECM component clustering; and E is the core ECM component heatmap analysis.

[0059] Figure 6 The ultrastructures of different ECM-based fillers are shown.

[0060] Figure 7 Postoperative slit-lamp and fluorescein staining results of rabbit corneal defects repaired with different ECM-based fillers are shown.

[0061] Figure 8 This shows the postoperative OCT results of rabbit corneal defects repaired with different ECM-based fillers.

[0062] Figure 9 This study presents a comparison of corneal thickness topography after repair of rabbit corneal defects using different ECM-based fillers.

[0063] Figure 10 The H&E staining results of different ECM-based fillers repairing rabbit corneal defects are shown.

[0064] Figure 11 The results of slit-lamp and OCT examinations after surgery are shown in two patients from clinical trials of this invention. Detailed Implementation

[0065] Unless otherwise stated, all technical and scientific terms and abbreviations used herein have the meanings commonly understood by one of ordinary skill in the art or the field of application of such terms.

[0066] In this article, decellularized cornea refers to corneal tissue derived from mammals such as humans, pigs, horses, and cattle that has been treated with decellularization methods (including repeated freeze-thaw cycles, high and low osmotic pressure, surfactants, superhydrostatic pressure, nucleases, and phospholipases) to remove epithelial cells, stromal cells, and endothelial cells, while mainly preserving the natural extracellular matrix (ECM) scaffold of the cornea.

[0067] In this article, decellularized corneal extracellular matrix microfibers refer to injectable fluid / viscoelastic fluid materials formed by the disruption of the complex network structure composed of protein and polysaccharide macromolecules in the extracellular matrix. The types of macromolecules include, but are not limited to, glycosaminoglycans, proteoglycans, collagen, elastin, fibronectin, and laminin.

[0068] As used herein, when a proportion, equivalent, concentration, time, part by weight, or other value or parameter is expressed as a range, preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within the range, and all values ​​within the range are effective in achieving the effects of the invention.

[0069] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0070] Unless otherwise specified in the examples, the procedures should be performed under standard conditions or as recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available or conventionally obtainable products.

[0071] Example 1: Preparation of corneal tampon

[0072] 1. Decellularized corneal pretreatment: Take 5 decellularized porcine corneas, place them in a clean bench, and let them stand at room temperature for 10 hours until dry.

[0073] 2. Low-Temperature Homogenization: Measure 50 mL of pre-cooled phosphonate buffer (PBS, 4°C) and add it along with the dried cornea to the homogenizer container, placing it in an ice-water bath to maintain a low temperature. Set the homogenization speed to 20,000 rpm and use intermittent homogenization mode, i.e., pause after each 15-second run. Repeat the above cycle as needed; in this example, intermittent homogenization was repeated 8 times. Furthermore, after every two intermittent homogenization runs, the container was cooled by moving it to a -20°C freezer for 5 minutes. The temperature of the homogenate was measured with a handheld infrared thermometer during each pause, and it was never higher than 40°C. After completion, a 1 mL pipette tip was used to check for blockages in the homogenate.

[0074] 3. Preliminary centrifugation and separation: Transfer the homogenate to a 50 mL centrifuge tube and centrifuge at 10,000 rpm for 3 minutes at 4°C. Discard the supernatant and collect the precipitate.

[0075] 4. Resuspension of precipitate: Add 1.5 mL of PBS to the precipitate and gently pipette using a 1 mL pipette tip with a trimmed tip to fully resuspend the precipitate.

[0076] 5. Centrifugation and separation again: Transfer the resuspension to a 2.5 mL centrifuge tube and centrifuge at 12,000 rpm for 5 minutes at 4°C. Discard the supernatant to obtain concentrated corneal extracellular matrix (ECM) microfiber precipitate (approximately 800 µL).

[0077] 6. Preparation of composite slurry: Add 800 µL of a 40% (w / v) methacrylamide gelatin (GelMA) solution containing 0.6% (w / v) LAP photoinitiator to the above ECM precipitate. Mix thoroughly by blowing with a pipette tip for 3 minutes.

[0078] 7. Incubation: Incubate the mixture in a 60°C water bath for 80 minutes, then centrifuge at 10,000 rpm for 5 minutes, discard the bottom precipitate to improve the homogeneity and transparency of the solution, and obtain the corneal filler.

[0079] 8. Finished product filling and storage: Fill the composite gel precursor into a sterile, light-protected syringe and store it at 4°C for later use.

[0080] Examples 2-21: Preparation of corneal tampon

[0081] Following the method of Example 1, the mixing ratio of ECM microfiber deposits, photoinitiators, and methacrylamide derivatives was adjusted, while keeping the other steps unchanged, to prepare different corneal fillers, as shown in Table 1:

[0082] Table 1

[0083] Example 1 50% 20% GelMA 0.3% LAP Example 2 25% 20% GelMA 0.3% LAP Example 3 30% 20% GelMA 0.3% LAP Example 4 70% 20% GelMA 0.3% LAP Example 5 75% 20% GelMA 0.3% LAP Example 6 50% 20% GelMA 0.05% LAP Example 7 50% 20% GelMA 0.1% LAP Example 8 50% 20% GelMA 0.5% LAP Example 9 50% 20% GelMA 0.6% LAP Example 10 50% 20% GelMA 0.3% NAP Example 11 50% 2% GelMA 0.3% LAP Example 12 50% 10% GelMA 0.3% LAP Example 13 50% 30% GelMA 0.3% LAP Example 14 50% 35% GelMA 0.3% LAP Example 15 30% 5% ChsMA 0.1% LAP Example 16 50% 10% ChsMA 0.3% LAP Example 17 50% 10% DexMA 0.3% LAP Example 18 30% 5% GelMA 0.1% LAP Example 19 70% 15% GelMA 0.2% LAP Example 20 50% 30% GelMA 0.4% LAP Example 21 70% 30% GelMA 0.5% LAP

[0084] Examples 22-25: Preparation of corneal tampon

[0085] Following the method of Example 1, the incubation steps were adjusted while the other steps and components remained unchanged, resulting in the preparation of different corneal ferrules, as shown in Table 2.

[0086] Table 2

[0087] Example 1 60℃, 80 minutes Example 22 60℃, 120 minutes Example 23 80℃, 40 minutes Example 24 40℃, 120 minutes Example 25 100℃, 30 minutes

[0088] Test Example 1: Testing and Comparison of Physicochemical Properties of Fillers

[0089] To systematically evaluate the physicochemical properties of different corneal keratin fillers, the elastic modulus, swelling properties, porosity, and in vitro degradation rate of each sample were quantitatively measured. All tests were performed under conditions simulating the physiological environment of the ocular surface, using phosphonate buffered saline (PBS, pH 7.4) at 37°C, with six replicates for each sample group.

[0090] Detection method:

[0091] Elastic modulus: A uniaxial tensile test was conducted on the light-cured dumbbell-shaped sample (8 mm in diameter and 2 mm in thickness) using a universal testing machine at a rate of 0.5 mm / min, and the stress-strain curve was recorded.

[0092] Swelling rate: Weigh the lyophilized sample (W0), incubate in PBS at 37℃ until swelling equilibrium is reached, remove excess liquid from the surface, and weigh again (W). s ), swelling ratio = (W s -W0) / W0×100%.

[0093] Porosity: The porosity of the sample was determined using the mercury intrusion porosimetry method.

[0094] In vitro enzymatic degradation rate: The photocured sample was weighed (W1) and incubated in PBS containing 2 U / mL collagenase at 37°C. After 72 hours, it was weighed (W2). Degradation rate = (W1-W2) / W1×100%.

[0095] Transmittance: After photocuring, the sample was made into a standard thin film with a thickness of 500 μm, and the transmittance was measured at a wavelength of 550 nm using a UV-Vis spectrophotometer.

[0096] Implant retention rate: A rabbit corneal lamellar stromal defect model with a diameter of 4 mm was established, filler was implanted and light-cured, and the implant was observed to be intact and retained in situ by slit lamp and OCT 7 days after the operation. The percentage of eyes with intact implants was counted.

[0097] Healing rate: Three days post-surgery, sodium fluorescein staining was performed, and the defect area was photographed under a slit lamp. The percentage of corneal epithelial healing area was calculated using ImageJ software, which is the 3-day epithelial healing rate.

[0098] The test results of the core physicochemical properties of each filler are shown in Table 3:

[0099] Table 3

[0100] Example 1 135.4±4.8 24.7±5.2 40.8±1.5 22.4±0.9 82.5±1.5 97.7±1.3 93.3±1.5 Example 2 92.3±5.0 18.5±4.7 48.6±1.4 35.8±1.4 88.2±1.4 55.2±1.2 76.3±1.1 Example 3 102.4±5.2 22.3±4.5 42.7±1.6 28.7±1.6 85.3±1.5 97.5±1.5 86.7±1.4 Example 4 178.7±7.9 27.7±1.4 34.3±1.9 17.9±0.8 72.6±5.9 99.0±0.2 98.9±0.8 Example 5 195.3±6.0 30.2±3.2 33.5±1.3 15.1±1.1 58.2±1.4 82.1±1.3 81.5±1.2 Example 6 54.9±0.5 34.0±1.8 50.8±2.4 42.4±1.0 88.5±7.2 37.7±9.6 63.3±8.9 Example 7 133.4±4.2 25.4±2.2 42.8±1.4 23.2±0.4 84.1±1.4 94.7±1.8 96.3±1.0 Example 8 137.6±3.7 26.4±3.2 40.9±0.7 22.4±0.6 86.4±0.7 95.6±0.8 98.6±0.5 Example 9 140.8±3.9 24.7±1.2 40.8±2.4 20.4±1.0 81.3±7.1 87.7±9.6 82.4±8.7 Example 10 134.7±3.3 25.7±1.2 41.8±2.4 22.4±1.0 85.7±7.1 94.7±3.6 97.3±1.9 Example 11 / / / / / / / Example 12 102.4±4.0 21.5±3.6 38.6±1.5 21.3±1.5 87.1±1.3 97.5±1.5 96.7±1.4 Example 13 176.0±7.6 25.8±0.9 34.7±1.9 16.8±0.7 82.5±5.1 99.1±0.7 84.0±7.5 Example 14 196.3±9.6 27.3±0.7 31.6±1.6 14.0±0.6 68.4±6.7 98.7±0.8 69.3±6.8 Example 15 108.7±6.1 6.3±3.5 32.1±1.5 7.5±1.2 82.1±1.5 85.3±1.5 89.2±1.6 Example 16 198.6±4.7 5.9±5.0 22.2±1.4 4.1±1.1 81.3±1.5 93.8±2.5 90.5±1.4 Example 17 125.3±4.9 26.2±5.1 49.5±1.5 23.6±1.0 75.7±1.6 93.5±3.6 91.1±1.5 Example 18 13.0±2.6 65.3±1.9 58.0±4.1 77.0±2.1 92.4±8.7 21.4±6.4 98.9±0.8 Example 19 155.6±5.9 26.1±3.7 35.2±1.6 18.8±1.3 75.3±1.6 98.2±1.4 98.1±1.6 Example 20 172.8±5.5 25.3±4.1 33.5±1.6 17.5±1.4 82.8±1.5 98.1±1.6 86.2±1.5 Example 21 205.2±5.0 27.8±4.8 22.3±1.6 12.2±1.6 72.5±1.5 94.1±1.4 85.3±1.4 Example 22 153.2±5.8 25.5±3.6 36.1±1.5 17.1±1.2 79.6±1.6 95.0±1.5 97.8±1.5 Example 23 138.2±5.4 21.3±3.8 40.3±1.6 24.6±1.3 85.9±1.5 94.6±1.4 89.6±1.4 Example 24 104.2±5.2 14.4±4.0 45.8±1.5 34.8±1.4 86.6±1.6 53.9±1.3 68.1±4.3 Example 25 106.5±6.0 17.9±3.5 44.8±1.4 36.5±1.1 74.9±1.7 55.3±1.6 73.2±1.6

[0101] Based on the above results, it can be seen that when the concentration of methacrylamide derivative (GelMA) is 2% (w / v), a stable hydrogel cannot be formed, and the relevant physicochemical indicators cannot be effectively measured; a concentration in the range of 5%-30% (w / v) can achieve stable gel formation and possess basic repair properties; among them, the 10%-20% (w / v) range has the best comprehensive performance, with suitable elastic modulus, low swelling rate, reasonable porosity, controllable degradation rate, high light transmittance, high graft retention rate, and rapid epithelial healing ability. With a formulation range of 30%-70% (v / v) ECM microfibers and 0.1%-0.5% (w / v) photoinitiator, the corneal ECM microfiber filler of this invention exhibits excellent comprehensive performance within the patent-protected formulation range: it possesses good mechanical rigidity, low swelling, high porosity, and anti-enzymatic ability, and can stably maintain structural integrity; it has high light transmittance, and its optical performance is close to that of natural cornea; the material and tissue interface are tightly bonded, resulting in excellent graft retention rate; it can significantly promote rapid epithelial healing 3 days after surgery; and it has outstanding biocompatibility and repair-promoting ability, fully meeting the clinical needs of minimally invasive corneal filling.

[0102] Comparative Example 1: Dry Grinding Method

[0103] 1. Grinding: Cut 5 fresh decellularized porcine corneas into small fragments using sterile scissors and allow them to dry at room temperature. Then, transfer the corneal fragments to a grinding tube, add an appropriate amount of grinding beads, and grind at a frequency of 30 times / second for 3 hours until the corneal fragments are ground into a uniform powder.

[0104] 2. Weighing: Remove the grinding beads and weigh 100 mg of decellularized corneal powder.

[0105] 3. Mixing: Add 40% (w / v) GelMA solution (pre-dissolved in buffer containing 0.6% LAP) to the centrifuge tube containing corneal powder at a 1:1 volume ratio. Mix thoroughly by pipetting for 3 minutes.

[0106] Comparative Example 2: Enzymatic hydrolysis

[0107] 1. Enzymatic digestion: Five decellularized corneas were cut into four equal parts, air-dried, and then mixed in a mass-to-volume ratio of 30 mg decellularized cornea: 3 mg pepsin: 1 mL hydrochloric acid solution (0.1 mol / L). The mixture was placed on a magnetic stirrer and continuously stirred and digested at 4°C for approximately 12-18 hours.

[0108] 2. Termination: While continuously stirring, slowly add 1N NaOH solution dropwise to the digestive fluid to adjust the pH of the mixture to neutral (pH 7.0-7.4) to terminate the pepsin digestion reaction.

[0109] 3. Mixing: Add 40% (w / v) GelMA solution (pre-dissolved in buffer containing 0.6% LAP) to the centrifuge tube containing the digestion solution at a 1:1 volume ratio. Use a pipette tip to mix thoroughly.

[0110] Comparative Example 3: Traditional Gel Method

[0111] 1. Solution preparation: Accurately weigh 20.0 g of methacrylamide gelatin powder, dissolve it in 100 mL of pre-cooled phosphonate buffer, and stir at 40°C for 1 hour to ensure complete dissolution, thus preparing a 20% (w / v) GelMA stock solution.

[0112] 2. Photoinitiator addition: Add LAP photoinitiator to the above GelMA solution to achieve a final concentration of 0.3% (w / v), and stir thoroughly under light-protected conditions to dissolve and obtain a homogeneous gel precursor solution.

[0113] Test Example 2: The Effect of Different Extraction Methods on ECM Proteins

[0114] To evaluate the effects of different methods on the integrity and extraction efficiency of proteins in decellularized corneal ECM microfibrils, the ECM microfibrils prepared in Example 1 (steps 1-5), Comparative Example 1 (steps 1-2), and Comparative Example 2 (steps 1-2) were subjected to SDS-PAGE electrophoresis, Coomassie brilliant blue staining, and BCA protein quantification. Specifically, proteins were extracted from the ECM microfibrils prepared in each group using RIPA lysis buffer, followed by SDS-PAGE electrophoresis, Coomassie brilliant blue staining, and observation of protein band distribution. Simultaneously, protein concentration was determined using a BCA protein quantification kit.

[0115] Electrophoresis results showed that in Example 1, the protein bands were clear and intact, with no obvious degradation or diffusion; in Comparative Example 1, the bands were blurred and uneven, with some characteristic components lost; in Comparative Example 2, the bands were significantly lighter and more diffused, with collagen characteristic bands almost disappearing. Figure 1 (A). BCA quantification results showed that the protein concentration in Example 1 was significantly higher than that in Comparative Example 2, and it could effectively extract ECM protein ( Figure 1 (See section B). The above results confirm that the low-temperature intermittent homogenization method used in this invention has high protein extraction efficiency, providing a key raw material basis for the excellent regeneration ability of corneal dermal fillers.

[0116] Test Example 3: The Effect of Different Extraction Methods on ECM Fiber Structure

[0117] To further compare the effects of different preparation methods on the natural ultrastructure of decellularized corneal ECM microfibers, transmission electron microscopy (TEM) was used to observe the samples from Example 1 (steps 1-5), Comparative Example 1 (steps 1-2), and Comparative Example 2 (steps 1-2). Specifically, the ECM microfiber suspensions prepared in Example 1, Comparative Example 1, and Comparative Example 2 were dropped onto a copper grid, negatively stained with phosphotungstic acid, and observed under a TEM to assess the degree of damage to the ultrastructure of the extracellular matrix caused by different preparation methods. The microfibers in Example 1 clearly showed natural collagen fibers with uniform diameter and intact structure; in Comparative Example 1, the fiber bundles were severely broken and twisted, and the fiber integrity was severely damaged; in Comparative Example 2, the natural collagen fiber structure completely disappeared, leaving only amorphous degradation fragments. The above results indicate that the low-temperature intermittent homogenization method used in this invention can effectively protect the natural structure of corneal ECM microfibers during the extraction process, while traditional dry grinding and enzymatic hydrolysis methods both cause structural damage or degradation of collagen fibers. Figure 2 ).

[0118] Test Example 4: Effect of heat incubation on ECM protein content and abundance

[0119] To evaluate the effects of different incubation protocols on the integrity and extraction efficiency of decellularized corneal ECM microfibrils, the corneal tampones prepared in Examples 1, 22, 23, 24, and 25 were subjected to SDS-PAGE electrophoresis and Coomassie brilliant blue staining. The protein content and component integrity were comprehensively evaluated using BCA protein quantification. Figure 3 ).

[0120] The results showed that the electrophoretic bands in Examples 1, 22, and 23 were clear and intact, with no significant degradation of collagen characteristic bands, while retaining medium and low molecular weight characteristic bands such as proteoglycans and glycoproteins. Figure 3 (A); BCA quantification results showed that the protein concentrations in the three groups (Example 1, Example 22, and Example 23) were 2.08±0.09 μg / μL, 3.15±0.12 μg / μL, and 1.60±0.32 μg / μL, respectively. The protein concentrations in Example 24 and Example 25 groups were significantly lower (0.30±0.04 μg / μL and 0.40±0.06 μg / μL, respectively), with noticeably lighter and more diffuse electrophoretic bands and weakened collagen characteristic band signals. Figure 3 (B). The above results indicate that a suitable incubation method (e.g., 60-80℃) can effectively reduce the loss of ECM proteins.

[0121] Test Example 5: Methacrylated derivatives provide protection for ECM during heat incubation

[0122] To verify the protective effect of methacrylamide gelatin (GelMA) and methacrylamide chondroitin sulfate (ChsMA) on ECM microfibers during incubation and the structural characteristics of the composite system, wide-angle X-ray scattering (WAXD) characterization was performed on 50% pure ECM microfibers (prepared according to steps 1-6 of Example 1, without heat bath), the GelMA-ECM composite system (Example 1, after 60°C heat bath), and the ChsMA-ECM composite system (Example 15, after 60°C heat bath). Figure 4 (A). WAXD analysis confirmed that the characteristic scattering signal of ECM microfibers was significantly weakened after a heat bath; while the composite system still retained a clear scattering signal under the same thermal conditions. These results indicate that both GelMA and ChsMA can effectively inhibit the thermal denaturation and structural damage of ECM proteins, providing a key structural basis for the excellent mechanical properties and long-term stability of the materials.

[0123] To further confirm the interaction and structural protection effect between GelMA and ECM proteins at the molecular level, Fourier transform infrared (FTIR) spectroscopy analysis was performed on pure ECM, pure GelMA, and the ECM-GelMA complex system. Figure 4(See Figure B). The results showed that compared with the characteristic peak of amide I in pure GelMA (1630 cm⁻¹), the amide I band of the ECM-GelMA composite system exhibited a significant red shift, moving to 1640 cm⁻¹. Simultaneously, the hydroxyl / amino stretching vibration peak at 3289 cm⁻¹ in the composite system was significantly enhanced and shifted to lower wavenumbers, indicating the formation of numerous stable intermolecular hydrogen bonds between GelMA and ECM microfibrils. Furthermore, the characteristic glycosyl peak of pure ECM at 1084 cm⁻¹ was significantly weakened in the composite system, suggesting that the two were not simply physically mixed, but rather formed a tight intermolecular interaction. These results confirm that GelMA can provide effective molecular-level protection for ECM during heat incubation, inhibiting ECM protein denaturation and maintaining its native conformation.

[0124] Test Example 6: Proteomic Comparison of Corneal Foils

[0125] To evaluate the similarity of the protein composition of the filler prepared in this invention to that of the natural corneal matrix, proteomics technology was used for comparative analysis. The filler from Example 1 was used as the experimental group, and the natural porcine corneal matrix and the filler prepared by enzymatic hydrolysis in Comparative Example 2 were used as control groups. Specifically, the filler from Example 1, the natural porcine corneal matrix, and the filler prepared by enzymatic hydrolysis were digested with trypsin, and then subjected to proteomics analysis by liquid chromatography-tandem mass spectrometry to identify the types and quantities of proteins. Principal component analysis (PCA), differential protein volcano plots, and core ECM component clustering were used to compare the similarity with that of the natural cornea. The results of principal component analysis (PCA) showed that the sample clusters of Example 1 and the natural cornea highly overlapped, while they were significantly separated from the samples of Comparative Example 2, indicating that the ECM microfibers prepared in Example 1 have extremely high similarity to the natural cornea in terms of overall protein expression profile, and good intra-group reproducibility. Figure 5 (A). The total number of proteins identified in Example 1 (369-505) was significantly higher than that in Comparative Example 2 (114-133). Figure 5 (B)

[0126] Compared to natural cornea, the filler in Example 1 showed significant downregulation of 182 proteins and significant upregulation of 253 proteins. The downregulated proteins were mainly enriched in various intracellular functional proteins; the significant loss of these intracellular proteins is a direct result of the material's decellularization process, which removed inherent cellular components. The upregulated proteins were primarily extracellular matrix-related proteins. Figure 5 (C) Cluster analysis showed that the extracellular matrix protein composition of the filler was highly similar to that of the natural cornea, retaining core extracellular matrix components, including collagen, proteoglycans, glycoproteins, and growth factors. Figure 5 (D). These components play crucial biological roles in maintaining corneal transparency, regulating the orderly arrangement of collagen fibers, and promoting corneal wound healing.

[0127] Core ECM protein expression heatmap ( Figure 5 Further, it was confirmed that key functional components in the decellularized corneal stroma were successfully detected in Example 1, including type I collagen (COL1A1 / COL1A2), core proteoglycan (DCN), corneal proteoglycan (KERA), laminin (LAMA3 / LAMB3 / LAMC2), and fibrin family glycoproteins (EFEMP1 / EFEMP2), etc., and the expression abundance of most components was higher than that of the natural corneal stroma, indicating that this method can effectively preserve and enrich these key functional proteins.

[0128] Test Example 7: Comparison of the ultrastructure of corneal tampon

[0129] To reveal the effects of different treatment methods on the internal ultrastructure of fillers, transmission electron microscopy (TEM) was used to analyze the microstructure of different fillers. The method is as follows: After photocuring, the prepared fillers were fixed with 2.5% glutaraldehyde and 1% osmium tetroxide, dehydrated with graded ethanol, embedded in epoxy resin, and ultrathin sections were prepared. These sections were then stained with uranium acetate and lead citrate using TEM and observed under a transmission electron microscope. Experimental results (…) Figure 6 The results show that the filler in Example 1 exhibits a natural fiber structure under a transmission electron microscope. Figure 6 (Indicated by the white arrows) The hydrogel network and ECM fibers are interwoven and uniformly mixed. Comparative Example 1 filler shows aggregated regions formed by ECM fragments (…). Figure 6 (As indicated by the black arrow), but no ECM fiber bundles were observed. The filler prepared by the enzymatic hydrolysis method in Comparative Example 2 exhibited a highly uniform polymer network structure with no fibrous components. The filler prepared by the conventional gelation method in Comparative Example 3 showed a dense and uniform amorphous polymer network with no fibrous structure. These results demonstrate that the filler prepared by the method of this invention can better maintain the natural corneal ECM structure, which corroborates the results of Test Example 3.

[0130] Test Example 8: Long-term efficacy evaluation of corneal defect repair

[0131] To systematically compare the effects of different fillers on in vivo repair performance, this study established a rabbit corneal lamellar defect model for an 8-week evaluation of repair effects. Twenty-four healthy New Zealand white rabbits were randomly divided into four groups (six eyes per group), and repaired using fillers from Example 1, Comparative Example 1 (dry abrasion method), Comparative Example 2 (enzymatic digestion method), and Comparative Example 3 (20% GelMA), respectively. A standard lamellar defect with a diameter of 4 mm and a depth of 200 μm was created in the center of the rabbit cornea. After injecting each filler into the defect cavity, in-situ photocuring was achieved by irradiation with blue light at a wavelength of 405 nm and a power density of 10 mW / cm² for 60 seconds. Corneal transparency, epithelial integrity, and gross healing were observed at 1 week and 8 weeks post-operation using a slit-lamp microscope. Optical coherence tomography (OCT) was used to assess the structural repair, optical density, and central corneal thickness of the defect area.

[0132] Slit-lamp observation 1 week after surgery ( Figure 7 The results showed that the corneal epithelium in Example 1, Comparative Example 1, and Comparative Example 2 was basically healed, with a smooth wound surface and no obvious signs of infection or acute rejection. Comparative Example 3 showed slower epithelial healing, with a wound repair rate of <60%. Eight weeks post-operation, the corneal transparency in Example 1 was significantly better than in Comparative Example 1, with an intact and smooth epithelial layer and no obvious scarring or neovascularization. In Comparative Examples 2 and 3, obvious depressions (such as...) were observed in the original defect area. Figure 7 (As indicated by the white arrow).

[0133] OCT results 1 week after surgery ( Figure 8 This confirmed that all four groups of fillers successfully filled the defect cavities, but the optical properties of the materials showed differences between groups: the optical density of the filler in Example 1 was closest to that of the surrounding natural corneal stroma, and the interface fused naturally; the material area in Comparative Example 1 showed a relatively higher optical density, corresponding to its lower optical transparency; Comparative Examples 2 and 3 showed extremely low optical density, with the area appearing as a homogeneous transparent gel, exhibiting high contrast with the surrounding corneal tissue. Eight weeks post-surgery, OCT images of Example 1 showed that the filler outline was still clearly discernible, and new collagen deposition was visible in the material-host interface area, suggesting that the material was guiding stroma regeneration; OCT images of Comparative Examples 2 and 3 showed that their transparent hydrogel had almost completely disappeared, indicating that the material had essentially degraded within 8 weeks.

[0134] Quantitative data from central corneal thickness measurements confirmed the above morphological observations. Figure 9 Eight weeks post-surgery, the central corneal thickness in Example 1 group did not decrease significantly compared to one week post-surgery; while the corneal thickness reduction rates in Comparative Examples 2 and 3 were as high as 50% and 63%, respectively. This is consistent with the result that the material degrades rapidly and cannot provide long-term mechanical support, leading to collapse of the repair area.

[0135] These results indicate that the filler of Example 1 exhibits excellent wound healing and structural stability in vivo, which is beneficial for restoring corneal thickness and transparency. In contrast, the dry powder filler of Comparative Example 1 showed difficulty in rapidly restoring transparency after implantation. The enzymatically treated group (Comparative Example 2) and Comparative Example 3 degraded rapidly in vivo and could not effectively restore corneal thickness.

[0136] Test Example 9: Histological staining results of corneal defect repair

[0137] To further evaluate the long-term repair effects and degradation behavior of different fillers at the microscopic level, experimental animals in each group were euthanized 8 weeks post-surgery to obtain corneal tissue samples after repair. After fixation and paraffin embedding, serial sections were prepared and stained with hematoxylin and eosin. Histological sections ( Figure 10 The results showed that the corneal defect area in Example 1 was completely repaired, with a full contour and a thickness roughly equivalent to the surrounding normal corneal stroma. No obvious signs of inflammatory cell aggregation or material degradation were observed. The repair results in Comparative Example 1 were generally similar to those in Example 1. The filler remained stable within the defect area, but there was aggregated particle accumulation, which caused optical scattering and affected corneal transparency. The repair area thickness in Comparative Examples 2 and 3 was significantly reduced, forming obvious depressions compared to the surrounding cornea, indicating rapid material degradation. These results demonstrate that Example 1 exhibits excellent structural stability and anti-degradation ability in vivo, effectively maintaining the morphology and thickness of the repaired area. In contrast, the material in Comparative Example 2, treated with pepsin hydrolysis, and the synthetic gel in Comparative Example 3 both showed excessively rapid degradation rates.

[0138] Test Example 10: Clinical Trial

[0139] This example is an investigator-initiated clinical study approved through ethical review, preliminarily evaluating the efficacy of a keratin sealant in the repair of defects after debridement for fungal corneal ulcers. Five patients with unilateral fungal corneal ulcers, approximately 5 mm in diameter and reaching the middle corneal stroma, were included. After infection control following topical antifungal treatment, debridement was performed to create a corneal tissue defect, which was then filled with the corneal protectant from Example 1 (single-dose administration, 2 μL). Postoperative follow-up showed ( Figure 11 The filler surface rapidly epithelialized, and fluorescein staining was negative on the 3rd postoperative day. The patient's vision improved, and the corneal curvature remained stable. By the 56th postoperative day, slit-lamp and OCT observations confirmed that the filler thickness had not decreased significantly, maintaining good structural support and corneal transparency, with no ulcer recurrence or significant complications observed.

[0140] The results show that the natural corneal ECM-based filler of the present invention is feasible and has preliminary effectiveness in the repair of corneal defects after infection control. It can support epithelial healing and maintain long-term morphological stability, providing a new potential strategy for minimally invasive repair of infected corneal ulcers.

[0141] In summary, the unique advantages of this invention include, but are not limited to:

[0142] This invention utilizes low-temperature intermittent cyclic cooling homogenization to achieve microfibrillation and high dispersion of corneal end-cell membranes (ECMs) while protecting the natural fiber conformation and activity. Subsequently, collagen / polysaccharide methacryloyl derivatives (GelMA, ChsMA, DexMA, etc.) are added for thermal incubation, allowing the derivatives to penetrate into the ECM fiber interstices. Through intermolecular interactions and mechanical interlocking, a dynamic protective layer is formed, isolating thermal shock and inhibiting collagen denaturation, thereby better preserving the ECM conformation and promoting regeneration activity. After photocuring, this filler constructs a stable interpenetrating network, simultaneously improving mechanical strength, light transmittance, and in vivo stability.

[0143] The specific embodiments described above do not constitute a limitation on the scope of protection of this invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can be made based on the inventive principles and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this invention should be included within the scope of protection of this invention.

Claims

1. A corneal calender, characterized in that, Includes 30%-70% v / v decellularized corneal extracellular matrix microfibers, 0.1%-0.5% w / v photoinitiator, and 5%-30% w / v methacrylamide derivatives; The method for preparing the decellularized corneal extracellular matrix microfibers includes the following steps: S1) The decellularized cornea is broken into a homogenate to obtain a suspension containing extracellular matrix microfibers of the decellularized cornea; the homogenate is an intermittent homogenate, the homogenization process is carried out at a low temperature and a cooling step is inserted during the homogenization process to keep the temperature of the decellularized cornea at 0-40℃. The homogenization time for a single homogenization is 10-20 seconds, and the cumulative homogenization time is 1-3 minutes. The homogenization speed is 10,000-25,000 rpm; Cooling conditions are -40 to 0℃, single cooling time is 3-5 minutes, and cumulative cooling time is 3-10 minutes; The low temperature range is 0-25℃; S2) Centrifuge the suspension, and the precipitate is decellularized corneal extracellular matrix microfibers; The centrifugation is carried out at 0-8℃ and 5000-20000 rpm for 2-20 minutes. The methacrylamide derivative is selected from one or more of methacrylamide gelatin, methacrylamide chondroitin sulfate, and methacrylamide dextran and polyethylene glycol diacrylate. The photoinitiator is one or a combination of two of sodium phenyl-2,4,6-trimethylbenzoylphosphinic acid and lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid.

2. The corneal dermal filler as described in claim 1, characterized in that, The decellularized corneas are derived from pigs, cattle, or humans.

3. The corneal dermal filler as described in claim 1, characterized in that, The decellularized corneal extracellular matrix microfibrils retain type I collagen, core proteoglycans, corneal proteoglycans, laminin, and fibronectin family glycoproteins from the decellularized cornea.

4. The corneal dermal filler as described in claim 1, characterized in that, The corneal tampon contains 30%-60% v / v decellularized corneal extracellular matrix microfibers, 10%-20% w / v methacrylamide derivatives, and 0.2%-0.4% w / v photoinitiator.

5. The corneal dermal filler as described in claim 1, characterized in that, After the corneal filler is irradiated with a 365-405nm light source and cross-linked and cured in situ, the resulting hydrogel has a three-dimensional network structure formed by the cross-linking of decellularized corneal extracellular matrix microfibers and methacrylamide derivatives.

6. The method for preparing the corneal tampon as described in any one of claims 1-5, characterized in that, Includes the following steps: A precursor corneal tampon is prepared by incubating a mixture of decellularized corneal extracellular matrix microfibers, a photoinitiator, and a methacrylated derivative; or by incubating a mixture of decellularized corneal extracellular matrix microfibers and a methacrylated derivative, and then adding a photoinitiator to the mixture to prepare a precursor corneal tampon.

7. The method for preparing the corneal dermal filler according to claim 6, characterized in that, The incubation is carried out in a water bath at 60-80℃ for 20-120 minutes.

8. The method for preparing the corneal dermal filler according to claim 6, characterized in that, Precursor-form corneal tampon should be stored at 0-6℃.

9. The method for preparing the corneal tampon according to claim 6, characterized in that, It also includes a post-processing step: centrifuge the incubated mixture at 5000-10000 rpm for 5-10 minutes and discard the bottom precipitate.

10. The method for preparing the corneal tampon according to claim 6, characterized in that, The solvent for the mixture is a phosphonate buffer.

11. The use of the corneal tampon according to any one of claims 1-5 in the preparation of a pharmaceutical or medical device for repairing corneal damage.

12. The use of the corneal tampon according to any one of claims 1-5 in the preparation of a cell carrier or drug carrier for repairing corneal damage.

13. The application according to claim 11 or 12, characterized in that, The corneal injury refers to ocular surface injury, corneal epithelial injury, corneal stromal injury, corneal ulcer, corneal perforation, or corneal laceration.

14. The application according to claim 11, characterized in that, The medical device is an ocular dressing, auxiliary sealing or adhesive material.

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