An artificial corneal endothelial graft based on natural degradable protein, its preparation method and application

Abstract

This invention discloses an artificial corneal endothelial graft based on natural degradable proteins, its preparation method, and its application, relating to the field of medical implant materials technology. Currently, artificial corneal endothelial grafts for corneal endothelial decompensation are all non-degradable synthetic polymer materials. This invention uses a natural degradable material as the substrate to prepare the graft. A material preparation stock solution is obtained by mixing a silk fibroin solution extracted from a high-salt solution with a certain proportion of glycerol. The mixed solution is then spin-coated onto a mold using a gradient method, and dried to obtain a silk fibroin film. The obtained silk fibroin film is then circumcised and placed in a mold for hot-press steam fumigation. The resulting silk fibroin film can serve as an artificial corneal endothelial graft, providing a donor source for artificial corneas and possessing potential application value in corneal transplantation.

Description

Technical Field

[0001] This invention relates to the field of medical implant materials technology, specifically to an artificial corneal endothelial graft based on a natural biodegradable protein material—silk protein—and its preparation method and application. Background Technology

[0002] Corneal diseases are among the most pressing causes of blindness worldwide, and corneal transplantation is the only effective treatment for corneal blindness. However, with an aging population, increased life expectancy, rising incidence of infectious diseases, and the widespread use of excimer laser refractive surgery, donor resources for corneal transplantation are becoming increasingly scarce. Although there are over one million patients with corneal blindness in my country, fewer than 5,000 allogeneic corneal transplants are performed annually. Therefore, finding suitable artificial corneas for human transplantation, expanding the donor pool, and meeting patient needs have become urgent issues that need to be addressed.

[0003] In cases of corneal blindness requiring transplantation, a significant proportion of patients suffer from endothelial blindness due to corneal endothelial decompensation. This can be addressed through corneal endothelial transplantation, a less invasive procedure developed in recent years. This technique preserves the original curvature of the cornea, has low surgical risk, and allows for rapid postoperative recovery. Among corneal endothelial transplantations, Descemet's membrane endothelial transplantation (DMEK) is the most effective method, transplanting only the Descemet's membrane and endothelial tissue, resulting in a postoperative anatomical structure that better conforms to the physiological structure of the cornea. In the prior art, Chinese patent document CN109363801B discloses a corneal endothelial graft for treating endothelial blindness, which utilizes femtosecond laser technology to assist in the treatment of porcine cornea, obtaining an ultra-thin, uniformly thick porcine corneal endothelial graft with the Descemet's membrane.

[0004] However, in practical applications, corneal endothelial grafts, due to their thinness and insufficient mechanical strength, present significant challenges in surgical implantation, deployment, and attachment, severely limiting the clinical application of Descemet's membrane endothelial transplantation. Furthermore, animal-derived Descemet's membranes also face practical issues such as immunogenicity. Currently, grafts based on artificial non-degradable polymers offer high mechanical strength and manipulation; however, long-term implantation can cause significant clinical problems such as foreign body reactions. Therefore, exploring novel, biocompatible, and biodegradable biologically derived grafts warrants in-depth consideration by physicians, researchers, and technicians in this field. Summary of the Invention

[0005] The purpose of this invention is to provide an artificial corneal endothelial graft based on natural degradable proteins and its preparation method, in order to solve the clinical difficulties caused by corneal endothelial cell lesions.

[0006] To achieve the above objectives, the present invention provides a method for preparing an artificial corneal endothelial graft based on a natural degradable protein, comprising the following steps:

[0007] S1: Preparation of protein solutions using biodegradable protein materials;

[0008] S2: Add glycerol to the protein solution, mix well, perform gradient spin coating, and dry to obtain a protein film;

[0009] S3: Calculate the radius of curvature of the mold arc according to different corneal refractive power requirements, and circumferentially cut the protein film according to the radius of curvature to obtain a circular film. The mold has a concave mold and a convex mold that are compatible with each other.

[0010] S4: Place the obtained circular film in the center of the concave and convex molds of the mold, perform hot pressing, and then shape it to obtain a protein film with the physiological curvature of the cornea.

[0011] S5: The protein membrane is subjected to water annealing treatment, and the finished product is the artificial corneal endothelial graft.

[0012] Preferably, the degradable protein material in the above preparation method can be selected from other degradable protein materials such as silk.

[0013] Preferably, the protein solution in the above preparation method is a silk fibroin solution, and its preparation process is as follows:

[0014] (1) Prepare a 2.12 g / L Na2CO3 solution, add silkworm silk and boil it, then take it out, wash it clean and dry it;

[0015] (2) Prepare a 9.3 mol / L LiBr solution, heat the LiBr solution to 60°C, add silk and stir until completely dissolved to obtain a silk fibroin solution. The ratio of silk to LiBr solution is 1 g: 4 mL.

[0016] (3) Dialyze the silk fibroin solution in pure water at 4°C. Centrifuge the obtained dialysate at 10,000 rpm and collect the supernatant as the silk fibroin solution.

[0017] Preferably, in step S2 of the above preparation method, the mass fraction of the protein solution is adjusted to 5%-10%, and then glycerol with a volume fraction of 1%-5% is added and mixed. The gradient spin coating is as follows: 1 mL of the mixture is dropped into the center of the culture dish, the mold is fixed in the center of the spin coater, and the mixture is spin coated at a low speed of 100 r / min-300 r / min for 10-30 s to make the solution evenly coated on the bottom of the dish; the speed is increased to 500 r / min-700 r / min, and spin coated for another 10-30 s; the speed is increased again to 800 r / min-1000 r / min, and spin coated for another 10-30 s.

[0018] Preferably, in step S3 of the above preparation method, the formula for calculating the radius of curvature r is: r=(n-1) / D; where r is the radius of curvature of the arc surface, D is the corneal refractive power, n is the relative refractive index of the air-corneal interface, and n=1.3375; this radius of curvature is used as the circumferential diameter of the protein film.

[0019] Preferably, in step S4 of the above preparation method, the hot pressing treatment is as follows: pressing the die and the punch together at 60℃-80℃ for no less than 4 hours; the shaping is done by natural cooling.

[0020] Preferably, in step S5 of the above preparation method, the water annealing treatment is performed at 60°C in a steam environment for 15-60 minutes.

[0021] The present invention also provides an artificial corneal endothelial graft prepared by any of the above preparation methods. The thickness of the finished artificial corneal endothelial graft is 10μm-100μm. The artificial corneal endothelial graft provided by the present invention can provide a donor source for artificial cornea and has potential application value in the field of corneal transplantation.

[0022] The present invention has the following advantages:

[0023] This invention prepares a uniform, transparent, and soft silk fibroin film. While ensuring the overall uniformity of the film, the film thickness can be controlled by adjusting experimental conditions, and it also has certain mechanical strength and flexibility, which can meet the needs of different surgical conditions.

[0024] The silk fibroin film provided by this invention conforms to the physiological curvature of the cornea and can adhere tightly to the elastic layer after implantation into the cornea, preventing corneal edema and, to a certain extent, replacing the function of the corneal endothelium, providing a more convenient and efficient new option for corneal endothelial transplantation in clinical practice. Attached Figure Description

[0025] Figure 1 This refers to the overall morphology of the silk fibroin membrane obtained in this invention.

[0026] Figure 2 This is the surface microstructure of the silk fibroin membrane observed by SEM in this invention.

[0027] Figure 3 This is the side morphology of the silk fibroin membrane observed by SEM in this invention.

[0028] Figure 4 The results show the tensile mechanical strength of the silk fibroin membrane in this invention.

[0029] Figure 5 This is an overall view showing the transparency of the silk fibroin film in this invention.

[0030] Figure 6This shows the transmittance of the silk fibroin film to visible light in this invention.

[0031] Figure 7 This shows the degradation of the silk fibroin membrane in the phosphate buffer solution of the present invention.

[0032] Figure 8 This shows the percentage of the remaining mass of the silk fibroin membrane after in vitro degradation in this invention.

[0033] Figure 9 The results are the hydrostatic pressure test results of the silk fibroin membrane in this invention.

[0034] Figure 10 The results show the culture of human corneal endothelial cells on the surface of a silk fibroin membrane.

[0035] Figure 11 This is the experimental result of implanting silk fibroin membrane into the endothelial layer of rabbit cornea in this invention. Detailed Implementation

[0036] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0037] Note: Unless otherwise specified, the experimental methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0038] Example 1: Preparation of silk fibroin film

[0039] This embodiment provides a method for preparing a silk fibroin film, as detailed below:

[0040] I. Preparation of silk fibroin solution:

[0041] 1. Boil 2.12g of Na2CO3 in 1L of deionized water. Loosen and cut an appropriate amount of silk into small pieces and add them to the water. After boiling for 1 hour, remove the silk, filter it, and rinse it thoroughly with deionized water. Repeat the above steps three times.

[0042] 2. Lay the washed silk flat and place it in an oven at 37℃ for more than 24 hours until it is completely dry.

[0043] 3. Prepare a 9.3 mol / L LiBr solution with a silkworm silk to LiBr solution ratio of 1 g: 4 mL. Heat the LiBr solution to about 60°C while stirring, and slowly add the silkworm silk into the bottle until it is completely dissolved. Stir for about 2 hours.

[0044] Pour the completely dissolved silk fibroin solution into a dialysis bag and dialyze it in pure water at 4°C. Change the water every 1, 4, 12, 24, and 48 hours.

[0045] 4. Dispense the filtered solution into centrifuge tubes and centrifuge at 10,000 rpm for 15 minutes to precipitate impurities. Discard the supernatant. Repeat three times to obtain the final silk fibroin solution.

[0046] 5. Determine the concentration of silk fibroin solution using the drying method. Take a petri dish, weigh it, and record the weight as W0; take an appropriate amount of solution onto the petri dish, weigh it, and record the weight as W1; place the petri dish in an oven to dry thoroughly, weigh it again, and record the weight as W2. The concentration C of the silk fibroin solution can then be expressed by the following formula 1. Repeat this process three times, and take the average value as the concentration of the silk fibroin solution.

[0047] 𝐶 = (𝑊2−𝑊0) / (𝑊1−𝑊0) × 100% (Equation 1)

[0048] II. Preparation of silk fibroin films using gradient spin-coating-drying method:

[0049] 1. Dilute the silk fibroin solution prepared above with deionized water to a concentration (mass fraction) of 5%-10%.

[0050] 2. Add 1%-5% glycerol by volume to a 5%-10% silk fibroin solution, mix well, and obtain a blend.

[0051] 3. Select a 35 mm diameter culture dish made of glass or polystyrene as the substrate. Add 1 mL of the blend solution to the center of the culture dish. Fix the mold in the center of the spin coater and spin coat at a low speed of 100 r / min-300 r / min for 10-30 s to evenly coat the bottom of the dish. Increase the speed to 500 r / min-700 r / min and continue spin coating for 10-30 s. Increase the speed again to 800 r / min-1000 r / min and continue spin coating for 10-30 s.

[0052] 4. After spin coating, allow it to air dry for more than 24 hours to obtain an extremely thin, uniform, transparent, and flexible silk fibroin film.

[0053] III. Preparation of silk fibroin curvature membranes by hot pressing-water annealing method:

[0054] 1. Using polytetrafluoroethylene (PTFE) or polystyrene as raw materials, design and fabricate a mold with the physiological curvature of the cornea for hot-pressing silk fibroin films. The mold consists of two parts, a concave mold and a convex mold, whose curvature conforms to the biological curvature of the cornea in experimental animals. In this case, a New Zealand rabbit was used, with a corneal refractive power of approximately 46.5D. The radius of curvature of the mold is calculated using the following formula:

[0055] r=(n-1) / D

[0056] Where r is the radius of curvature of the curved surface (m), D is the corneal refractive power, and n is the relative refractive index of the air-corneal interface, n=1.3375. The calculated radius of curvature r=7.26mm.

[0057] 2. Use a corneal trephine drill to circumferentially cut the prepared silk fibroin film into circular films with a diameter of 6mm-8mm.

[0058] 3. Place the circular film in the center of the concave and convex molds, press them tightly together, and hot-press at 60℃-80℃ for at least 4 hours. Then, place it at room temperature for at least 12 hours to allow it to cool and set naturally, resulting in a silk fibroin film with the physiological curvature of the cornea. Remove the silk fibroin film from the mold and perform water annealing in a 60℃ steam environment for 15-60 minutes to enhance its strength. The overall morphology of the resulting silk fibroin film is as follows: Figure 1 As shown, 'a' represents the front view and 'b' represents the side view.

[0059] Experimental Example 1: Observation of the surface morphology of silk fibroin membranes

[0060] The obtained silk fibroin membrane was observed by scanning electron microscopy (SEM), and the microstructure of the silk fibroin membrane surface under different magnifications was obtained as follows: Figure 2 As shown, Figure 2 In the figures, a and b represent the microscopic conditions under different backgrounds, and the morphology of the silk fibroin membrane side surface is as follows: Figure 3 As shown, where Figure 3 a, b, and c in the figures represent the lateral morphology of the prepared silk fibroin film at different positions along its edge and center. SEM observation revealed that the silk fibroin film surface was smooth and uniform, while high-magnification (5000X) observation showed a rough surface structure, which may be beneficial for cell adhesion. Observation of the sides of the silk fibroin film showed that the film thickness was uniform. By controlling the silk fibroin solution content and spin-coating speed, the film thickness varied from 10 μm to 100 μm, which can be adjusted according to the actual needs of surgery, provided that the implantation conditions for animal experiments are met.

[0061] Experiment Example 2: Mechanical Testing

[0062] The tensile mechanical strength of silk fibroin film was determined using a Shimadzu universal testing machine. The silk fibroin film was cut into samples 20 mm long and 5 mm wide. Both ends of the samples were fixed to glass slides using 502 glue, and the slides were then fixed to the fixtures of the testing machine. The tensile speed was set to 1 cm / min, and the silk fibroin film was slowly stretched until it broke. The stress-strain curves were recorded, and the tensile strength and elongation at break were calculated. The tensile strength (Ts) was calculated using the following formula:

[0063] Ts=F / S

[0064] F is the maximum tensile force (N) that the membrane can withstand when it ruptures, and S is the cross-sectional area of ​​the membrane (m²). 2 ).

[0065] Elongation at break (E) is calculated according to the following formula:

[0066] E=[(L1- L0 ) / L0 ]x100%

[0067] L0 is the original length of the membrane (mm), and L1 is the length of the membrane when it breaks (mm).

[0068] The test samples were divided into 6 groups: 1. Glycerin-free silk fibroin membrane, without steam treatment; 2. Glycerin-free silk fibroin membrane, treated with steam annealing at 60℃-80℃ for 15-60 minutes; 3. Silk fibroin membrane containing 3% glycerol, without steam treatment; 4. Silk fibroin membrane containing 3% glycerol, treated with steam annealing at 60℃-80℃ for 15-60 minutes; 5. Silk fibroin membrane containing 5% glycerol, without steam treatment; 6. Silk fibroin membrane containing 5% glycerol, treated with steam annealing at 60℃-80℃ for 15-60 minutes. Three parallel samples were set up for each group, and the average value of the test results was taken.

[0069] The tensile mechanical strength of silk fibroin membranes under different experimental conditions is as follows: Figure 4 As shown, a represents the stress-strain curves of different groups of silk fibroin membranes; b represents the effect of steam fumigation on the tensile modulus of silk fibroin membranes; c represents the effect of glycerol content on the maximum elongation of silk fibroin membranes; where Gn represents the presence of n% glycerol in the silk fibroin membrane, and W represents the silk fibroin membrane being treated with steam fumigation at 60℃ for 15 min.

[0070] It can be seen that the stress-strain curve of the pure silk fibroin membrane is a typical brittle fracture, with a strain of less than 1.6%. The addition of glycerol acts as a small-molecule plasticizer, significantly increasing the flexibility of the silk fibroin membrane and raising the tensile strain to over 60%. Steam fumigation treatment can improve the tensile strength of the silk fibroin membrane; the tensile strength of each group is higher than the tensile strength of human cornea (3.8 MPa), which meets the requirements of clinical surgery.

[0071] Experiment Example 3: Transparency Test

[0072] The transparency of the silk fibroin film was measured using a visible light spectrophotometer. The silk fibroin film was cut to a size suitable for the cuvette (40 mm long, 12 mm wide). The visible light spectrophotometer was preheated for 20 minutes to achieve stability. A blank sample (i.e., a cuvette without the silk fibroin film) was placed in the light path, and the baseline transmittance was recorded as 100%. The silk fibroin film was then attached to the inner wall of the cuvette, ensuring the light beam passed perpendicularly through the sample. The scanning range was set to the visible light region (400-800 nm), with wavelength steps of 100 nm, and the transparency data at each wavelength was recorded.

[0073] The test samples were divided into 6 groups: 1. Glycerin-free silk fibroin membrane, untreated with steam; 2. Glycerin-free silk fibroin membrane, treated with 60℃ steam for 15 minutes; 3. Silk fibroin membrane containing 3% glycerin, untreated with steam; 4. Silk fibroin membrane containing 3% glycerin, treated with 60℃ steam for 15 minutes; 5. Silk fibroin membrane containing 5% glycerin, untreated; and 6. Silk fibroin membrane containing 5% glycerin, treated with 60℃ steam for 15 minutes. Three parallel samples were set up for each group, and the average value of the test results was taken.

[0074] See the overall view of the transparency of silk fibroin membranes in different groups. Figure 5 As shown, its transmittance in the visible light range (wavelength 400nm-800nm) was measured, and the results are shown in [the table / reference]. Figure 6 As shown, within the visible light range (wavelength 400nm-800nm), the transmittance of pure silk fibroin film can reach over 85%. Adding glycerin further improves the transparency of the silk fibroin film, with a transmittance exceeding 90%, and approaching 100% transmittance at 800nm, similar to that of the natural cornea. Figure 5 , 6 In the text, Gn indicates that the silk fibroin membrane contains n% glycerol, and W indicates that the silk fibroin membrane has been treated with 60°C steam fumigation for 15 minutes.

[0075] Experimental Example 4: In vitro degradation test

[0076] The silk fibroin membrane was placed in an in vitro environment (phosphate buffer solution) for degradation over 42 days. Weighing was performed every 7 days to calculate the percentage of remaining weight. The overall morphology of the silk fibroin membrane after in vitro degradation is shown below. Figure 7 As shown, the percentage of residual mass after in vitro degradation at each stage is as follows: Figure 8 As shown, after 42 days of in vitro degradation, the shape and structure of the silk fibroin membrane remained largely intact, with a remaining weight percentage of over 90%.

[0077] Experiment Example 5: Hydrostatic Pressure Test

[0078] A hydrostatic pressure test was conducted on the silk fibroin membrane. The experimental apparatus consisted of a graduated cylinder with a 15mm diameter hole at the bottom and a flange with a 15mm diameter central hole. The edges of the graduated cylinder and the flange could be secured with screws. The silk fibroin membrane was fixed in the center of the flange and the graduated cylinder. Deionized water was added to the graduated cylinder, and the pressure exerted on the silk fibroin membrane was the water pressure of the water column.

[0079] The hydrostatic pressure test results of the silk fibroin membrane are shown below. Figure 9 As shown, the normal intraocular pressure range is known to be 10-21 mmHg. Based on the calculation that 21 mmHg = 285 mmH2O, the silk fibroin membrane can withstand the pressure of the maximum intraocular pressure without rupture or leakage, and this lasted for 75 days.

[0080] Experimental Example 6: In vitro cell experiments

[0081] The silk fibroin film prepared above was cut to a size suitable for the petri dish, laid flat on the bottom of the petri dish, and sterilized with ultraviolet light on both sides for 30 minutes.

[0082] Human corneal endothelial cell line B4G12 was resuscitated in liquid nitrogen, added to preheated culture medium, and incubated at 37°C in a 5% CO2 incubator. Cells were passaged to the logarithmic growth phase at a 1:3 passage ratio. Sterilized silk fibroin membranes were placed in 6-well plates, with 1-2 × 10⁶ cells per well. 4 Each cell was placed on the membrane surface and indwelled in DMEM medium containing 10% FBS and 1% penicillin-streptomycin. Simultaneously, corneal endothelial cells were cultured in wells without the silk fibroin membrane as a control group. The wells were placed in an incubator and cultured for 7 days, with the medium changed every 1-2 days.

[0083] On days 1, 3, and 7, the cell morphology and growth on the silk fibroin membrane were observed using an inverted microscope, and images were recorded. The results are shown in [Table missing]. Figure 10 'a' in 'a'.

[0084] On days 1, 3, and 7, samples were collected, cells were fixed with 4% paraformaldehyde for 10 min, stained with 1 μg / mL DAPI at room temperature for 10 min, and washed three times with PBS. Five fields of view were randomly selected under a fluorescence microscope to count the number of cell nuclei per unit area and calculate the growth density. The results are shown below. Figure 10 b in the text.

[0085] Cell proliferation activity was detected using the CCK-8 assay. Samples were collected on days 1, 2, 3, 4, and 5, and 10 μL of CCK-8 solution was added to each well. The cells were incubated at 37°C for 2 hours, and the absorbance at 450 nm was measured using a microplate reader. Cell proliferation curves were plotted. Results are shown below. Figure 10 c in the text.

[0086] After 7 days of culture, the expression of functional proteins in corneal endothelial cells was detected by immunofluorescence staining. Cells were fixed with 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% BSA at room temperature for 30 min. Specific primary antibodies against corneal endothelial functional proteins (ZO-1, Na⁺ / K⁺-ATPase) were added, and the cells were incubated overnight at 4°C. Fluorescently labeled secondary antibodies were added, and the cells were incubated at room temperature in the dark for 1 hour. Protein expression and distribution were observed using confocal microscopy. The results are shown in [Figure number missing]. Figure 10 d in the text.

[0087] After 7 days of culture, the expression of functional proteins in corneal endothelial cells was detected by Western blotting (WB). Cells were lysed with RIPA lysis buffer, the lysate was collected and centrifuged, total protein was extracted, and protein concentration was determined using a BCA kit. Equal volumes of protein samples were loaded, separated by electrophoresis, and transferred to a PVDF membrane. The cells were incubated with a functional protein-specific primary antibody, followed by incubation with an HRP-labeled secondary antibody. Protein bands were recorded using ECL luminescence reagent, and protein expression levels were analyzed. Results are shown in the table below. Figure 10 The 'e' in the middle.

[0088] The results of human corneal endothelial cell culture on the surface of silk fibroin membrane are shown in the figure. Figure 10 As shown, a) shows the morphological observation of corneal endothelial cells on the scaffold surface; b) shows the growth density of corneal endothelial cells; c) shows the proliferation activity of corneal endothelial cells; d) shows the immunofluorescence staining of corneal endothelial cell functional protein expression; and e) shows the results of Western blot detection and quantitative analysis of corneal endothelial cell functional protein expression. It can be seen that after culturing the human corneal endothelial cell line B4G12 on the silk fibroin membrane surface for 7 days, cell morphology, cell density, cell proliferation activity, and cell functional expression were observed. It was found that the cells grew well on the membrane surface, and the cell density was higher than that of the scaffold, but there was no statistically significant difference, indicating good biocompatibility of the composite membrane.

[0089] Experimental Example 7: Animal Experiment Testing

[0090] Healthy, adult New Zealand rabbits weighing 2-3 kg were selected as experimental animals. Silk fibroin curvature membranes with diameters of 6 mm and 8 mm were prepared according to the methods described above. The silk fibroin membranes were sterilized with ethylene oxide before surgery.

[0091] New Zealand rabbits were anesthetized by intravenous injection of 30 mg / kg sodium pentobarbital. The rabbits were then secured on the operating table, ensuring adequate exposure of the operated eye. A small incision of approximately 2 mm was made around the periphery of the cornea using a microscalpel. The anterior chamber of the cornea was filled with balanced salt solution, and the Descemet's membrane (including corneal endothelial cells) was gently removed using corneal forceps. A silk fibroin film was rolled into a tube and inserted into the anterior chamber through the small incision. The film was adjusted to fully unfold and adhere to the Descemet's membrane. Air bubbles were introduced into the anterior chamber to help fix the silk fibroin film in place. In the control group, only the Descemet's membrane was removed; no material was implanted.

[0092] At 1 day, 1 week, 1 month, 2 months, and 3 months post-surgery, corneal transparency and postoperative recovery were recorded using an anterior segment camera to observe for corneal edema, opacity, or other lesions. Optical coherence tomography (OCT) was used to scan the cornea, record central corneal thickness, compare changes in corneal thickness before and after surgery, and assess the degree of corneal edema.

[0093] One and two weeks post-surgery, the morphology, distribution, and density of corneal endothelial cells on the silk fibroin membrane were observed using a corneal confocal microscope.

[0094] The experimental results of implanting silk fibroin membrane into the rabbit corneal endothelium are as follows: Figure 11 As shown, a) is anterior segment corneal photography; b) is central corneal thickness observed by OCT; c) is endothelial cell observation by corneal confocal microscopy; and d) is the central corneal thickness measurement results for each treatment group. Animal experiments observed for 3 months revealed that after the silk fibroin membrane was implanted into the corneal endothelium, the cornea gradually regained transparency. The control group showed corneal opacity. After implantation, the silk fibroin membrane adhered tightly to the posterior corneal stroma, and OCT measurements showed that the central corneal thickness (CCT) gradually thinned, approaching that of normal rabbits. The silk fibroin membrane exhibited good biocompatibility; after implantation, the peripheral corneal endothelium maintained a regular hexagonal morphology, and no inflammatory cells were observed.

[0095] In summary, this invention provides a method for preparing silk fibroin membranes based on silk fibroin. The prepared silk fibroin membrane has higher tensile strength than human cornea, and its light transmittance in the visible light range is similar to that of natural cornea. It also has high compressive strength and resistance to degradation, as well as good biocompatibility. It can be used as an artificial corneal endothelial graft to provide a donor source for artificial corneas and has potential application value in corneal transplantation.

[0096] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for producing an artificial corneal endothelial graft based on a naturally degradable protein, characterized by, The preparation method comprises the following steps: S1: preparing a protein solution by using a degradable protein material, wherein the degradable protein material is silk, and the protein solution is a silk fibroin solution; S2: adding glycerol into the protein solution, mixing, and then performing gradient spin coating, and obtaining a protein film after drying, wherein the mass fraction of the protein solution is adjusted to 5%-10%, and then 1%-5% glycerol by volume is added and mixed; the gradient spin coating is performed as follows: 1 mL of the blended solution is dropped in the center of a culture dish, a mold is fixed in the center of a spin coater, spin coating is performed at a low speed of 100 r / min-300 r / min for 10-30 s to uniformly coat the solution on the bottom of the dish, the speed is increased to 500 r / min-700 r / min, and spin coating is continuously performed for 10-30 s, and then the speed is increased to 800 r / min-1000 r / min, and spin coating is continuously performed for 10-30 s; S3: calculating the radius of curvature of the mold according to different corneal refractive power requirements, and cutting the protein film into a circular film according to the radius of curvature, wherein the mold comprises a concave mold and a convex mold which are matched with each other; S4: placing the obtained circular film in the center of the concave mold and the convex mold, performing hot pressing treatment, and then performing shaping to obtain a protein film with physiological curvature of the cornea; wherein the hot pressing treatment is performed as follows: the concave mold and the convex mold are pressed tightly, and hot pressing is performed at 60-80 ℃ for at least 4 h; and the shaping is performed by natural cooling; S5: performing water annealing treatment on the protein film, and obtaining an artificial corneal endothelial graft; wherein the water annealing treatment is performed in a 60 ℃ water vapor environment for 15-60 min; and the thickness of the product is 10-100 μm.

2. The production method according to claim 1, characterized by, The preparation process of the silk fibroin solution is as follows: 2.12 g / L Na2CO3 solution is prepared, and silk is cooked in the solution, and then taken out, washed and dried; 9.3 mol / L LiBr solution is prepared, the LiBr solution is heated to 60 ℃, and silk is added and stirred until completely dissolved to obtain a silk fibroin solution, wherein the ratio of the silk to the LiBr solution is 1 g:4 mL; The silk fibroin solution is dialyzed in 4 ℃ pure water, the obtained dialysate is centrifuged at 10000 rpm, and the collected supernatant is the silk fibroin solution.

3. The method of claim 1, wherein, In S3, the calculation formula of the radius of curvature r is: r=(n-1) / D; wherein r is the radius of curvature of the arc surface, D is the corneal refractive power, and n is the relative refractive index of the air-corneal interface, n=1.3375; and the radius of curvature is used as the cutting diameter of the protein film.

4. An artificial corneal endothelial graft prepared by the preparation method according to any one of claims 1-3.

5. Application of the artificial corneal endothelial graft according to claim 4 in the field of corneal transplantation.

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