Artificial corneal endothelial graft based on natural degradable protein as well as preparation method and application of artificial corneal endothelial graft

Abstract

The invention discloses an artificial corneal endothelial graft based on natural degradable protein and a preparation method and application thereof, and relates to the technical field of medical implant materials. Currently, the artificial corneal endothelium graft for the corneal endothelium decompensation disease is a non-degradable artificially synthesized polymer material, the graft is prepared by taking a natural degradable material as a base material, a silk fibroin solution extracted from a high-salt solution is blended with glycerol in a certain proportion to obtain a material preparation stock solution, and the stock solution is prepared into the artificial corneal endothelium graft for the corneal endothelium decompensation disease. Coating a mold with the uniformly mixed blending solution in a gradient spin coating manner, and drying to obtain a silk fibroin film; the obtained silk fibroin film is subjected to girdling and then placed in a mold for hot-pressing steam fumigation, and the obtained silk fibroin film can serve as an artificial cornea endothelial graft, provides a donor source for an artificial cornea and has potential application value in cornea transplantation.

Description

Technical Field

[0001] The present invention relates to the technical field of medical implant materials, and in particular to an artificial corneal endothelial transplant based on a natural degradable protein material-silk protein, and a preparation method and application thereof. Background Art

[0002] Corneal disease is a clinical blinding eye disease that urgently needs to be solved in the world. Corneal transplantation is the only effective method to treat corneal blindness. However, with the aging of the population and the extension of life expectancy, the increase in the incidence of infectious diseases and the widespread use of corneal excimer refractive surgery, the donor resources that can be used for corneal transplantation are becoming increasingly scarce. Although there are more than 1 million patients with corneal disease blindness in my country, less than 5,000 allogeneic keratoplasties can be performed each year. Therefore, seeking artificial corneas that can be used for human transplantation, expanding the donor source, and meeting patient needs have become urgent issues that need to be addressed.

[0003] Among corneal blindness cases that require transplantation, a large proportion of patients suffer from corneal endothelial blindness caused by corneal endothelial decompensation. The corneal endothelium can be replaced by corneal endothelial transplantation, which is less traumatic and has been developed in recent years. This procedure ensures the original curvature of the cornea, has low surgical risks, and fast postoperative recovery. Among corneal endothelial transplantation procedures, Descemet's membrane endothelial keratoplasty (DMEK) is the most effective procedure, which only transplants the Descemet's membrane and endothelial tissue, making the anatomical structure after surgery more consistent with the physiological structure of the cornea. In the prior art, Chinese patent document CN109363801B discloses a corneal endothelial graft that can be used to treat endothelial blindness, which uses femtosecond laser technology to assist in the treatment of porcine corneas to obtain ultra-thin, uniformly thick porcine corneal endothelial grafts with Descemet's membrane.

[0004] However, in actual applications, the thin thickness and insufficient mechanical strength of corneal endothelial grafts make it difficult to implant, unfold and attach them, which seriously restricts the clinical application of Descemet's membrane endothelial corneal transplantation. In addition, animal-derived Descemet's membrane also faces practical problems such as immunogenicity. Currently, artificial non-degradable polymer grafts have high mechanical strength and operability, but long-term implantation can also cause clinical problems such as foreign body reactions that cannot be ignored. Therefore, it is worthwhile for doctors, researchers and technicians in this field to explore novel biodegradable grafts with good biocompatibility. Summary of the invention

[0005] The purpose of the present invention is to provide an artificial corneal endothelial transplant based on natural degradable protein and a preparation method thereof, so as to solve the clinical difficulty of decompensation caused by corneal endothelial cell lesions.

[0006] In order to achieve the above object, the present invention provides a method for preparing an artificial corneal endothelial transplant based on natural degradable protein, comprising the following steps: S1: preparing protein solution by degradable protein material; S2: adding glycerol to the protein solution, mixing well, performing gradient spin coating, and obtaining a protein film after drying; S3: calculating the radius of curvature of the mold arc according to different corneal diopter requirements, and circularly cutting the protein film according to the radius of curvature to obtain a circular film, wherein the mold has a concave mold and a convex mold that are adapted to each other; S4: placing the obtained circular film in the center of the concave mold and the convex mold of the mold, performing heat pressing treatment, and then shaping to obtain a protein film with the physiological curvature of the cornea; S5: The protein film is subjected to water annealing treatment, and the obtained finished product is an artificial corneal endothelial transplant.

[0007] Preferably, the degradable protein material in the above preparation method can be selected from other degradable protein materials such as silk.

[0008] Preferably, the protein solution in the above preparation method is a silk fibroin solution, and its preparation process is as follows: (1) Prepare 2.12 g / L Na2CO3 solution, add silk to steam, remove and clean, and dry; (2) Prepare 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. (3) The silk fibroin solution was dialyzed in pure water at 4°C, the dialyzate was centrifuged at 10,000 rpm, and the supernatant was the silk fibroin solution.

[0009] Preferably, in 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 blended solution is added dropwise to the center of the culture dish, the mold is fixed in the center of the spin coater, and the solution is evenly coated on the bottom of the dish at a low speed of 100 r / min-300 r / min for 10-30 seconds; the speed is increased to 500 r / min-700 r / min, and the spin coating is continued for 10-30 seconds; the speed is increased to 800 r / min-1000 r / min again, and the spin coating is continued for 10-30 seconds.

[0010] Preferably, in S3 of the above preparation method, the calculation formula for 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 diopter, n is the relative refractive index of the air-corneal interface, and n=1.3375; this radius of curvature is used as the circular cutting diameter of the protein film.

[0011] Preferably, in S4 of the above preparation method, the hot pressing treatment is: pressing the concave mold and the convex mold up and down, and hot pressing at 60°C-80°C for not less than 4 hours; the shaping is natural cooling shaping.

[0012] Preferably, in S5 of the above preparation method, the water annealing treatment is carried out in a 60° C. water vapor environment for 15 min-60 min.

[0013] The present invention also provides an artificial corneal endothelial transplant sheet prepared by any one of the above preparation methods, the thickness of the finished artificial corneal endothelial transplant sheet is 10μm-100μm, the artificial corneal endothelial transplant sheet provided by the present invention can provide a donor source for artificial corneas, and has potential application value in the field of corneal transplantation.

[0014] The present invention has the following advantages: The present 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. The film has certain mechanical strength and flexibility, and can meet the needs of different surgical conditions.

[0015] The silk fibroin film provided by the present invention conforms to the physiological curvature of the cornea, can be closely attached to the posterior elastic layer of the cornea after implantation, prevent corneal edema, and replace the function of the corneal endothelium to a certain extent, providing a more convenient and efficient new option for clinical corneal endothelial transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the overall shape of the silk film obtained in the present invention.

[0017] Figure 2 The microscopic morphology of the silk film surface observed by SEM in the present invention.

[0018] Figure 3 The side morphology of the silk film observed by SEM in the present invention.

[0019] Figure 4 The tensile strength test results of the silk film of the present invention are shown in FIG.

[0020] Figure 5 This is an overall view of the transparency of the silk film in the present invention.

[0021] Figure 6 It is the transmittance of the silk film to visible light in the present invention.

[0022] Figure 7 The figure shows the degradation of the silk film in the present invention under phosphate buffer solution.

[0023] Figure 8The residual mass percentage of the silk fibroin membrane after in vitro degradation in the present invention.

[0024] Fig. 9 It is the hydrostatic pressure test result of the silk fibroin membrane in the present invention.

[0025] Fig.10 The results of culturing human corneal endothelial cells on the surface of silk membrane.

[0026] Fig.11 The results are the experimental results of implanting the silk membrane into the rabbit corneal endothelium. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Note: The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0029] Example 1 Preparation of silk fibroin film This embodiment provides a method for preparing a silk fibroin film, which is as follows: 1. Preparation of silk fibroin solution: 1. Add 2.12g Na2CO3 to 1L deionized water and boil. Pull a proper amount of silk loose, cut into pieces and add to the water. After boiling for 1 hour, remove the silk, filter and wash with deionized water. Repeat the above steps three times.

[0030] 2. Spread the washed silk flat and put it in an oven to dry at 37℃ for more than 24 hours until it is completely dry.

[0031] 3. Prepare 9.3 mol / L LiBr solution, with the ratio of silk to LiBr solution being 1 g:4 mL. Heat the LiBr solution to about 60°C while stirring, and slowly add the silk into the bottle until it is completely dissolved, stirring for about 2 hours.

[0032] Pour the completely dissolved silk solution into a dialysis bag and dialyze it in 4°C pure water, changing the water every 1, 4, 12, 24, and 48 hours.

[0033] 4. The filtered solution was divided into centrifuge tubes and centrifuged at 10,000 rpm for 15 minutes to precipitate impurities in the solution, and the supernatant was poured out. Repeat three times to obtain the final silk fibroin solution.

[0034] 5. Use the drying method to determine the concentration of the silk fibroin solution. Take a culture dish, weigh it, record it as W0; draw an appropriate amount of solution on the culture dish, weigh it, record it as W1; put the culture dish in an oven, dry it thoroughly, weigh it, record it as W2. The concentration C of the silk fibroin solution can be expressed by the following formula 1. Repeat three times, and take the average value as the concentration of the silk fibroin solution.

[0035] 𝐶 = (𝑊2 − 𝑊0) / (𝑊1 − 𝑊0) × 100% (Formula 1) 2. Preparation of silk fibroin film by gradient spin coating-drying method: 1. Dilute the silk fibroin solution prepared above with deionized water to a concentration (mass fraction) of 5%-10%.

[0036] 2. Add 1% to 5% by volume of glycerol to the 5% to 10% silk fibroin solution and mix well to obtain a mixed solution.

[0037] 3. Select a culture dish with a diameter of 35 mm and made of glass or polystyrene as the base, take 1 mL of the blended solution and drop it in 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 seconds to evenly coat the bottom of the dish; increase the speed to 500 r / min-700 r / min, and continue to spin coat for 10-30 seconds; increase the speed to 800 r / min-1000 r / min again, and continue to spin coat for 10-30 seconds.

[0038] 4. After spin coating, dry naturally for more than 24 hours to obtain an extremely thin, uniform, transparent and flexible silk fibroin film.

[0039] 3. Preparation of silk fibroin curvature film by hot pressing-water annealing method: 1. Using polytetrafluoroethylene or polystyrene as raw materials, design and prepare a mold for hot pressing silk film with corneal physiological curvature. The mold is divided into two parts, namely the concave mold and the convex mold. Its curvature conforms to the curvature of the corneal biological curvature of the experimental animal. The experimental case is New Zealand rabbit, whose corneal refractive power is about 46.5D. The curvature radius of the designed mold curvature is calculated according to the following formula:

[0040] r=(n-1) / D Where r is the radius of curvature of the arc surface (m), D is the corneal diopter, and n is the relative refractive index of the air-corneal interface, n = 1.3375. The calculated curvature radius r = 7.26 mm.

[0041] 2. Use a corneal trephine to cut the silk fibroin film prepared above into a circular film with a diameter of 6 mm to 8 mm.

[0042] 3. Place the circular film in the center of the concave and convex molds, press them tightly, heat press them at 60℃-80℃ for more than 4 hours, and then place them at room temperature for more than 12 hours, and cool them naturally to form a silk film with the physiological curvature of the cornea. Take the silk film out of the mold and perform water annealing treatment at 60℃ water vapor for 15min-60min to enhance its strength. The overall morphology of the obtained silk film is as follows: Figure 1 As shown, a is the front view and b is the side view.

[0043] Experimental Example 1 Observation of the surface morphology of silk film The obtained silk film was observed by scanning electron microscope (SEM), and the microscopic morphology of the silk film surface under different magnifications was obtained. Figure 2 As shown, Figure 2 a and b in the figure are microscopic images under different backgrounds. The side morphology of the silk film is shown in Figure 3 As shown, Figure 3 Figures a, b, and c are the side morphologies of the prepared silk film at different positions on the edge and middle. SEM observations show that the surface of the silk film is flat and uniform. High-power microscope (5000X) observations show that the surface has a rough structure, which may be conducive to cell attachment. Observation of the side of the silk film shows that the film thickness is average. By controlling the content of the silk solution and the spin coating speed, the film thickness ranges from 10μm to 100μm. Under the premise of meeting the implantation conditions of animal experiments, it can be adjusted according to the actual needs of the surgery.

[0044] Experimental Example 2 Mechanical Test The tensile strength of the silk film was measured using a Shimadzu universal mechanical testing machine. The silk film was cut into samples with a length of 20 mm and a width of 5 mm. Both ends of the sample were fixed on a glass slide with 502 glue, and then the glass slide was fixed on the fixture of the mechanical testing machine. The stretching speed was set to 1 cm / min, and the silk film was slowly stretched until it broke. The stress-strain curve was recorded, and the tensile strength and elongation at break were calculated. Among them, the tensile strength (Ts) was calculated according to the following formula:

[0045] Ts=F / S F is the maximum tensile force (N) when the membrane breaks, S is the cross-sectional area of ​​the membrane (m 2 ).

[0046] The elongation at break (E) is calculated according to the following formula: E=[(L1- L0 ) / L0 ]x100% L0 is the original length of the membrane (mm), and L1 is the length of the membrane when it breaks (mm).

[0047] The test samples were divided into 6 groups, namely: 1. Silk film without glycerol, not treated with water vapor; 2. Silk film without glycerol, treated with water vapor annealing at 60℃-80℃ for 15min-60min; 3. Silk film with 3% glycerol, not treated with water vapor; 4. Silk film with 3% glycerol, treated with water vapor annealing at 60℃-80℃ for 15min-60min; 5. Silk film with 5% glycerol, not treated with water vapor; 6. Silk film with 5% glycerol, treated with water vapor annealing at 60℃-80 for 15min-60min. Three parallel samples were set in each group, and the test results were averaged.

[0048] The tensile strength test results of silk fibroin membranes under different experimental conditions are shown in Figure 2. Figure 4 As shown, a is the stress-strain curve of silk films of different groups; b is the effect of water vapor fumigation on the tensile modulus of silk film; c is the effect of glycerol content on the maximum elongation of silk film; wherein Gn means that the silk film contains n% glycerol, and W means that the silk film was treated with 60℃ water vapor fumigation for 15min.

[0049] It can be seen that the stress-strain curve of the pure silk film belongs to a typical brittle fracture, and its strain is less than 1.6%. The addition of glycerol can act as a small molecule plasticizer, significantly increasing the flexibility of the silk film, and the tensile strain is increased to more than 60%. Water vapor fumigation treatment can improve the tensile strength of the silk film. The tensile strength of each group is higher than the tensile strength of the human cornea of ​​3.8MPa, which can meet the needs of clinical surgery.

[0050] Experimental Example 3 Transparency Test The transparency of the silk film was measured using a visible light spectrophotometer. The silk film was cut into a size suitable for the cuvette (40 mm long and 12 mm wide), the light source of the visible light spectrophotometer was preheated for 20 minutes to reach a stable state, a blank sample (i.e., a cuvette without a silk film) was placed in the light path, and the reference transmittance was recorded and set to 100%. The silk film was attached to the inner wall of the cuvette to ensure that the light beam can pass through the sample vertically. The scanning range was set to the visible light region (400-800 nm), and the wavelength was stepped by 100 nm, and the transparency data at each wavelength was scanned and recorded.

[0051] The test samples were divided into 6 groups, namely: 1. Silk film without glycerin, not treated with water vapor; 2. Silk film without glycerin, treated with 60℃ water vapor fumigation for 15 minutes; 3. Silk film with 3% glycerin, not treated with water vapor; 4. Silk film with 3% glycerin, treated with 60℃ water vapor fumigation for 15 minutes; 5. Silk film with 5% glycerin, not treated with water vapor; Silk film with 5% glycerin, treated with 60℃ water vapor fumigation for 15 minutes. Three parallel samples were set in each group, and the test results were averaged.

[0052] The overall transparency of silk membranes in different groups is shown in Figure 5 As shown, the transmittance in the visible light range (wavelength 400nm-800nm) was measured, and the results are shown in Figure 6 As shown. It can be seen that in the visible light range (wavelength of 400nm-800nm), the transmittance of pure silk film can reach more than 85%. Adding glycerol can further improve the transparency of silk film, the transmittance is more than 90%, and the transmittance at a wavelength of 800nm ​​is close to 100%, which is similar to the natural cornea. Figure 5 , 6 Gn in the figure means that the silk film contains n% glycerol, and W means that the silk film has been treated with 60°C water vapor fumigation for 15 min.

[0053] Experimental Example 4 In vitro degradation test The silk membrane was placed in an in vitro environment (phosphate buffer solution) and degraded for a total of 42 days. The membrane was weighed every 7 days to calculate the remaining weight percentage. Figure 7 The results of the residual mass percentage of each stage of in vitro degradation are shown in Figure 8 It can be seen that after 42 days of degradation in vitro, the shape and structure of the silk film remained basically intact, and the remaining weight percentage was more than 90%.

[0054] Experimental Example 5 Hydrostatic Pressure Test The hydrostatic pressure test of the silk membrane was carried out. The experimental apparatus was designed to be a measuring cylinder with a 15mm diameter hole at the bottom and a flange with a 15mm diameter center hole. The edge of the measuring cylinder and the flange can be fixed and tightened by screws. The silk membrane was fixed in the center of the flange and the measuring cylinder. Deionized water was added to the measuring cylinder. The pressure on the silk membrane was the water pressure of the water column.

[0055] The results of the hydrostatic pressure test of silk fibroin membrane are shown in Fig. 9 As shown, it is known that the normal intraocular pressure range is 10-21 mmHg. Calculated as 21 mmHg = 285 mmH2O, the silk membrane can withstand the maximum intraocular pressure without rupture or leakage, and lasts for 75 days.

[0056] Experimental Example 6 In vitro cell test The silk film prepared above was cut into a size suitable for the culture dish, spread flat on the bottom of the culture dish, and sterilized using ultraviolet light for 30 minutes on both the front and back sides.

[0057] Human corneal endothelial cell line B4G12 was revived from liquid nitrogen, added with preheated culture medium, and placed in a 37°C, 5% CO2 incubator. Cells were subcultured to the logarithmic growth phase at a 1:3 subculture ratio. The sterilized silk membrane was placed in a 6-well plate and 1-2 × 10 cells were seeded in each well. 4 Cells were placed on the membrane surface, and DMEM medium containing 10% FBS and 1% double antibody (penicillin-streptomycin) was added. At the same time, corneal endothelial cells were cultured in a well plate without silk membrane as a control group. The well plate was placed in an incubator and cultured for 7 days, with the culture medium replaced every 1-2 days.

[0058] On the 1st, 3rd and 7th days, the cell morphology and growth on the silk membrane were observed using an inverted microscope and images were recorded. The results are shown in Fig.10 a in.

[0059] On the 1st, 3rd, and 7th days, samples were taken and cells were fixed with 4% paraformaldehyde for 10 min, then stained with 1 μg / mL DAPI for 10 min at room temperature, and washed three times with PBS. Five fields of view were randomly selected under a fluorescence microscope, and the number of cell nuclei per unit area was counted to calculate the growth density. The results are shown in Fig.10 b in.

[0060] The cell proliferation activity was detected using the CCK-8 method. On days 1, 2, 3, 4, and 5, samples were taken, 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance at 450 nm was measured using an ELISA reader, and the cell proliferation curve was plotted. The results are shown in Fig.10 c in.

[0061] After 7 days of culture, the expression of functional proteins in corneal endothelial cells was detected by immunofluorescence staining. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton X-100 for 10 minutes, and blocked with 5% BSA at room temperature for 30 minutes. Specific primary antibodies against corneal endothelial functional proteins (ZO-1, Na⁺ / K⁺-ATPase) were added and incubated overnight at 4°C. Fluorescently labeled secondary antibodies were added and incubated at room temperature in the dark for 1 hour. The expression and distribution of proteins were observed using a confocal microscope. The results are shown in Fig.10 In d.

[0062] After 7 days of culture, the expression of functional proteins in corneal endothelial cells was detected by Western Blot (WB). The cells were lysed with RIPA lysis buffer, the lysate was collected and centrifuged, the total protein was extracted, and the protein concentration was determined using a BCA kit. Equal amounts of protein samples were loaded, separated by electrophoresis, and transferred to a PVDF membrane. Incubated with a primary antibody specific for the functional protein, followed by incubation with an HRP-labeled secondary antibody. ECL luminescent reagent was used for color development, protein bands were recorded, and protein expression levels were analyzed. The results are shown in Fig.10 The e in.

[0063] The results of human corneal endothelial cells cultured on the surface of silk membrane are shown in Fig.10 As shown, a is the morphological observation of corneal endothelial cells on the surface of the scaffold; b is the growth density of corneal endothelial cells; c is the proliferation activity of corneal endothelial cells; d is the immunofluorescence staining of corneal endothelial cell functional protein expression; e is the WB detection and quantitative analysis results of corneal endothelial cell functional protein expression. It can be seen that after culturing the human corneal endothelial cell line B4G12 on the surface of the silk film for 7 days, the cell morphology, cell density, cell proliferation activity, and cell function 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 crawling slice, but there was no statistical difference. The composite membrane had good biocompatibility.

[0064] Experimental Example 7 Animal Experiment Test Healthy, adult New Zealand rabbits weighing 2-3 kg were selected as experimental animals. Silk curvature membranes with diameters of 6 mm and 8 mm were prepared according to the materials and methods described above. Before surgery, the silk membranes were sterilized using ethylene oxide.

[0065] New Zealand rabbits were anesthetized by intravenous injection of 30 mg / kg sodium pentobarbital, and the rabbits were fixed on the operating table to ensure adequate exposure of the surgical eye. A small incision of about 2 mm was made around 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 torn off with corneal forceps. The silk film was rolled into a roll, and the silk film was sent into the anterior chamber through a small incision. The silk film was adjusted to fully unfold and attached to the position of the Descemet's membrane. Air bubbles were injected into the anterior chamber to help the silk film fix to the position of the Descemet's membrane of the cornea. In the control group, only the Descemet's membrane was torn off, and no material was implanted.

[0066] One day, one week, one month, two months, and three months after surgery, anterior segment photography was used to record corneal transparency and postoperative recovery, and to observe whether there was corneal edema, turbidity, 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 evaluate the degree of corneal edema.

[0067] One week and two weeks after surgery, the morphology, distribution and density of corneal endothelial cells on the silk membrane were observed using a corneal confocal microscope.

[0068] The results of the silk membrane implantation experiment on the rabbit corneal endothelium are as follows Fig.11 As shown, a is corneal anterior segment photography; b is central corneal thickness observed by OCT; c is corneal endothelial cells observed by confocal microscopy; d is the measurement results of central corneal thickness in each treatment group. Animal experiments observed for 3 months found that after the silk membrane was implanted into the corneal endothelium, the cornea gradually became transparent. The cornea of ​​the control group was turbid. After the silk membrane was implanted, it was closely attached to the posterior corneal stroma. The central corneal thickness (CCT) measured by OCT gradually became thinner, close to the CCT of normal rabbits. The silk membrane has good biocompatibility. After implantation, the peripheral corneal endothelium maintained a regular hexagonal shape, and no inflammatory cells were found.

[0069] In summary, the present invention provides a method for preparing a silk film based on silk protein. The prepared silk film has a tensile strength higher than that of a human cornea, a transmittance in the visible light range similar to that of a natural cornea, and has the characteristics of high pressure resistance and degradation resistance. It also has good biocompatibility and can be used as an artificial corneal endothelial transplant to provide a donor source for artificial corneas, and has potential application value in corneal transplantation.

[0070] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A method for preparing an artificial corneal endothelial transplant based on natural degradable protein, characterized in that: The following steps are included: S1: preparing protein solution by degradable protein material; S2: adding glycerol to the protein solution, mixing well, performing gradient spin coating, and obtaining a protein film after drying; S3: calculating the radius of curvature of the mold arc according to different corneal diopter requirements, and circularly cutting the protein film according to the radius of curvature to obtain a circular film, wherein the mold has a concave mold and a convex mold that are adapted to each other; S4: placing the obtained circular film in the center of the concave mold and the convex mold of the mold, performing heat pressing treatment, and then shaping to obtain a protein film with the physiological curvature of the cornea; S5: performing water annealing treatment on the protein film to obtain an artificial corneal endothelial transplant.

2. The preparation method according to claim 1, characterized in that: The degradable protein material can be selected from silk.

3. The preparation method according to claim 2, characterized in that: The protein solution is a silk fibroin solution, and its preparation process is as follows: Prepare 2.12g / L Na2CO3 solution, add silk to cook, remove and clean, and dry; 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, wherein the ratio of silk to LiBr solution is 1 g:4 mL; The silk fibroin solution was dialyzed in 4° C. pure water, the dialyzate obtained was centrifuged at 10,000 rpm, and the collected supernatant was the silk fibroin solution.

4. The preparation method according to claim 1, characterized in that: In the S2, 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 blended solution is added dropwise to the center of the culture dish, the mold is fixed in the center of the spin coater, and the spin coating is performed at a low speed of 100 r / min-300 r / min for 10-30 seconds to evenly coat the bottom of the dish with the solution; the rotation speed is increased to 500 r / min-700 r / min, and the spin coating is continued for 10-30 seconds; the rotation speed is increased to 800 r / min-1000 r / min again, and the spin coating is continued for 10-30 seconds.

5. The preparation method according to claim 1, characterized in that: In S3, the calculation formula of the curvature radius r is: r=(n-1) / D; wherein r is the curvature radius of the arc surface, D is the corneal diopter, n is the relative refractive index of the air-corneal interface, and n=1.3375; the curvature radius is used as the protein film ring cutting diameter.

6. The preparation method according to claim 1, characterized in that: The hot pressing treatment in S4 is: pressing the concave mold and the convex mold up and down, and hot pressing at 60° C.-80° C. for at least 4 hours; the shaping is natural cooling shaping.

7. The preparation method according to claim 1, characterized in that: The water annealing treatment in S5 is carried out in a 60° C. water vapor environment for 15 min to 60 min.

8. The preparation method according to claim 1, characterized in that: The finished product has a thickness of 10 μm-100 μm.

9. An artificial corneal endothelial transplant prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the artificial corneal endothelial transplant sheet as claimed in claim 9 in the field of corneal transplantation.

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