An integrated keratoprosthesis based on in-situ gradient solidification and a method of making the same

Abstract

This invention discloses an integrated artificial cornea and its preparation method, belonging to the field of biomedical engineering technology. The artificial cornea comprises a main corneal layer and a posterior corneal layer. The main corneal layer includes a central optical zone, a transitional junction zone, and a decellularized corneal annular zone. The main corneal layer uses a decellularized cornea as its overall framework. Polymerizable monomers are introduced into the decellularized corneal stroma and then cured using in-situ gradient curing technology. The integrated artificial cornea provided by this invention uses a decellularized cornea as its overall framework, eliminating the need for central perforation, resulting in better morphological stability. The optical zone constructed using gradient curing technology provides a favorable environment for cell ingrowth, promoting healing between the transplanted tissue and the corneal graft material.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, specifically relating to an integrated artificial cornea prepared based on in-situ gradient curing technology and its preparation method. Background Technology

[0002] Corneal transplantation has always been the most effective way to restore vision for patients blinded by corneal diseases. Currently, there are many methods for corneal transplantation. Early corneal transplants generally used allogeneic donors, but the number of corneal donations is far from meeting the demand for corneal transplantation, and the availability of donor corneal grafts is insufficient. Allogeneic corneal transplantation is also prone to inducing immune rejection. Therefore, researchers have gradually begun to develop various artificial corneas to address the urgent need for transplantation. Many products suitable for corneal transplantation have been developed, such as Boston-type artificial corneas, osteodental artificial corneas, AlphaCor artificial corneas, and MICOF artificial corneas. However, these products have not achieved the desired results due to issues such as material biocompatibility and harvesting methods.

[0003] Traditional non-biological artificial corneas are prepared using inorganic materials or non-bioactive polymers or hydrogels. Inorganic materials, such as alloys and hydroxyapatite, have poor biocompatibility, and some even possess biotoxicity or potential biotoxicity, easily inducing various complications such as glaucoma and anterior chamber leakage, thus failing to meet the clinical needs of corneal transplantation. While polymeric materials (such as PMMA and PHEMA) have higher biotoxicity and biocompatibility compared to inorganic materials, they lack bioactive substances, making it difficult to achieve good biological healing with the cornea after implantation and easily inducing complications.

[0004] In recent years, bioactive materials have attracted increasing attention and favor, and more and more bio-based corneal products have gained public acceptance. Examples include autologous dental graft-based artificial corneas and tibial scaffold-based artificial corneas. These corneas have good biocompatibility and high survival rates after transplantation. However, because the materials are taken from the patient's own dental grafts or tibia, the sampling process can cause some harm to the recipient. In addition, there are some decellularized porcine corneal products and composite products, but the corneal transmittance changes after decellularization, and direct application to corneal transplantation may affect postoperative visual recovery. Currently available decellularized composite materials cannot be directly used for full-thickness corneal transplantation.

[0005] To meet the needs of corneal transplant patients worldwide, there is an urgent need to develop high-performance, low-risk corneal products. Artificial corneas require sufficiently high biocompatibility and low inflammatory response to avoid high-risk complications. Furthermore, artificial corneas should also promote cell and nerve fiber regeneration and specifically inhibit angiogenesis to prevent immune responses. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing artificial corneal materials by providing a one-piece artificial corneal implant with high biocompatibility, high transparency, cell-inducing properties, and non-multi-component chimera structure, as well as its preparation method. Single decellularized materials, such as porcine decellularized corneal biomaterials, rapidly biodegrade after transplantation, and the endothelial and epithelial cells cannot regenerate in time, failing to meet the clinical requirements for long-term implantation and vision restoration. Currently, artificial corneas prepared from composite materials of decellularized materials and other materials are mostly chimera assemblies, as shown in patent documents CN202022891532.2 and CN201811116393.7; or they form a one-piece structure through transition zone bonding, as shown in patent documents CN201210508733.7 and CN201910154284.2. The artificial corneas created using the above methods lack the conditions to induce the growth of endothelial and endothelial cells on the surface of the central optical region. Only in the outer stromal region are there sites of integration with surrounding tissues. However, the rate of cell ingrowth in the decellularized matrix is ​​limited, and there is a risk of displacement and detachment after suture removal. Based on this background, the present invention provides an integrated artificial corneal implant with biological healing function, prepared by organically combining natural and synthetic materials through in-situ gradient curing technology.

[0007] This invention creatively utilizes in-situ gradient curing technology in the central region of the decellularized cornea to obtain an optical region with a gradient effect. This optical region, due to its surface containing extracellular matrix components rather than a single polymer material, exhibits higher bioactivity, inducing endothelial and epithelial cell regeneration, and promoting healing between the body's cells and the corneal graft. Furthermore, the polymers in this optical region are interwoven and interlocked, without clearly defined boundaries, resulting in a stronger bond compared to constructing the optical region by drilling holes in the central region.

[0008] This invention includes the following technical solutions:

[0009] In a first aspect, the present invention provides an integrated artificial cornea, characterized in that the structure of the artificial cornea includes a corneal main layer and a posterior lamina layer, the corneal main layer includes a central optical zone, a transitional junction zone and a decellularized corneal annular zone, the corneal main layer uses a decellularized cornea as an integral framework, and polymerizable monomers are introduced into the decellularized corneal stroma and prepared by curing the monomers through in-situ gradient curing technology.

[0010] The in-situ gradient curing technology is achieved through a gradient-effect ultraviolet filter. The gradient-effect ultraviolet filter is an optical filter with a diameter of 8-10 mm. The center of the filter has a circular hole with a diameter of 3-4 mm. The periphery of the circular hole consists of a gradient light-transmitting zone and a completely light-blocking zone. The gradient light-transmitting zone has a width of 1-2 mm and is densely covered with micropores with a diameter of 10-20 μm. The density of the micropores gradually decreases from the inside to the outside, so that the ultraviolet light transmittance decreases uniformly until it is completely opaque. The completely light-blocking zone is completely opaque to ultraviolet light.

[0011] The artificial cornea obtained by curing with the above-mentioned gradient-effect ultraviolet filter has a central optical region with complete monomer polymerization and curing, resulting in a central optical region with a light transmittance of over 97%. The transition connection region is a composite region of incompletely polymerized and cured polymer material and decellularized cornea, with light transmittance decreasing from 97-100% to 40-50%, and it has pores. The decellularized corneal annular region is a decellularized corneal stroma region that has not undergone polymer polymerization and curing.

[0012] Secondly, the present invention provides a method for preparing an integrated artificial cornea, the method comprising the following steps:

[0013] (1) Introducing polymerizable monomers into the decellularized corneal stroma via gradient infiltration;

[0014] (2) Place the decellularized cornea, which has been permeated with polymerizable monomers, on a petri dish, cover the cornea with a gradient UV filter, irradiate with UV for 15-30 min, and then flip it over to irradiate the other side for 15-30 min.

[0015] (3) Soak the cornea in deionized water to remove residual monomers and photoinitiators from the corneal surface and obtain the corneal substrate;

[0016] (4) Place the corneal substrate in a petri dish, add 2-3 drops of hydroxyethyl methacrylate with a mass concentration of 100% containing photoinitiator to the surface of the substrate, allow it to penetrate for 5-10 minutes, then use a gradient UV filter with a gradient effect for gradient curing, and soak and wash with deionized water to obtain an integrated artificial cornea.

[0017] The gradient permeation method described in step (1) specifically involves preparing a polymerizable monomer solution containing a photoinitiator with a mass concentration gradient of 45-100%, and immersing the decellularized cornea in the solution sequentially from low to high concentration for 1-4 hours to fully remove the water from the decellularized corneal stroma.

[0018] Preferably, the mass concentration of the photoinitiator in the polymerizable monomer solution is 1-5%, more preferably, the mass concentration of the photoinitiator is 3%. The photoinitiator of the present invention is selected from one or more combinations of 2,4,6-(trimethylbenzoyl)diphenylphosphine oxide, 1-hydroxy-cyclohexyl-phenyl ketone, and 1-hydroxy-cyclohexyl-phenyl ketone.

[0019] The polymerizable monomer described in this invention is a monomer containing acrylate functional groups. In a specific embodiment, the monomer containing acrylate functional groups is selected from one or more combinations of methyl methacrylate, ethyl methacrylate, propyl methacrylate, isopropyl methacrylate, n-butyl methacrylate, tert-butyl methacrylate, n-hexyl methacrylate, cyclohexyl methacrylate, octyl methacrylate, decyl methacrylate, undecyl methacrylate, dodecyl methacrylate, lauryl methacrylate, hydroxyethyl methacrylate (HEMA), hydroxyethyl acrylate, glycidyl methacrylate, polyethylene glycol methacrylate, and tetrahydrofuran methacrylate.

[0020] In the most preferred embodiment of the present invention, the polymerizable monomer is hydroxyethyl methacrylate (HEMA).

[0021] The decellularized cornea described in this invention is prepared by decellularizing xenogeneic corneas using conventional methods in the art. The xenogeneic corneas described in this invention are selected from the corneas of pigs, monkeys, rabbits, cats, dogs, cattle, and sheep. In the most preferred embodiment of this invention, the xenogeneic cornea is a pig cornea.

[0022] The decellularized porcine cornea was prepared by the following method:

[0023] S1: Take a fresh pig eyeball and carefully scrape off the epithelial cells with a scalpel. Remove the cornea, clean it, place the cornea at -80℃ to cool it down completely, and then take it out. Thaw it at room temperature and repeat the freeze-thaw cycle three times.

[0024] S2: Repeatedly freeze-thawed porcine corneas are placed in a decellularization solution (such as SDS, sodium deoxycholate, or a combination of two or more of Triton X-100) for 6-8 hours, with sonication for 15 minutes every 2 hours during this period. Then, they are treated with a super nuclease at 37°C for 6 hours, placed in the decellularization solution again to remove cellular components, and finally soaked in deionized water to wash away residual decellularization reagents in the cornea. Stress is applied to the cornea to remove some moisture and it is stored at -80°C.

[0025] In a specific embodiment of the present invention, the integrated artificial cornea is prepared by the following method:

[0026] A: Prepare aqueous solutions of hydroxyethyl methacrylate with mass concentrations of 45%, 60%, 75%, and 90%, and a 100% hydroxyethyl methacrylate solution. Add a 3% photoinitiator to the solutions and mix well. Place the decellularized cornea in the 45% hydroxyethyl methacrylate aqueous solution and shake for 1-4 hours. Remove and aspirate any residual solution from the surface. Then place the cornea in the 60% hydroxyethyl methacrylate aqueous solution and shake for 1-4 hours. Remove and aspirate any residual solution from the surface. Repeat the above steps, shaking the cornea in the 75% and 90% hydroxyethyl methacrylate aqueous solutions respectively, removing and aspirating any residual solution from the surface. Finally, shake the cornea in the 100% hydroxyethyl methacrylate aqueous solution and remove and aspirate any residual solution from the surface.

[0027] B: Place the decellularized cornea, which has been permeated with hydroxyethyl methacrylate monomer, in a petri dish, cover it with a UV filter with a gradient effect, and irradiate it with UV light for 20-30 minutes. Then flip it over and irradiate the other side for 15-20 minutes.

[0028] C: Soak the irradiated cornea with deionized water to remove residual monomers and photoinitiators from the corneal surface and obtain the corneal substrate;

[0029] D: Place the corneal substrate in a petri dish, add 2-3 drops of 100% hydroxyethyl methacrylate containing a photoinitiator to the surface of the corneal substrate, allow it to penetrate for 5-10 minutes, then cover it with a gradient UV filter, irradiate with UV light for 15-20 minutes, and then soak and wash with deionized water to obtain an integrated artificial cornea.

[0030] The integrated artificial cornea provided by this invention uses a decellularized cornea as its framework. The central optical zone is a light-transmitting area with a transmittance >97% formed by photopolymerization of monomers containing acrylate functional groups on the framework of the decellularized cornea. The area connecting to the central optical zone is a transition connection zone, which is formed by incomplete photopolymerization of polymerizable monomers on the framework of the decellularized cornea using a gradient-effect ultraviolet filter, resulting in a region with uniformly decreasing transmittance. The area connecting to the transition connection zone is the decellularized corneal annular zone, which is the area of ​​the decellularized cornea where monomer polymerization has not occurred.

[0031] The integrated artificial cornea provided by this invention has the following technical advantages:

[0032] (1) The artificial corneal scaffold provided by the present invention is derived from decellularized cornea of ​​animals, has good biocompatibility, is easy to integrate with tissues, is conducive to the healing of artificial cornea with the body, and solves problems such as corneal prolapse and aqueous humor leakage.

[0033] (2) When the central optical region is entirely composed of HEMA polymer, the body tissue cannot grow in sufficiently, which increases the probability of the artificial cornea melting. The artificial cornea provided by this invention uses decellularized cornea as its framework, which is not only more conducive to the overall stability of the cornea, but also provides a good proliferation environment for the growth of body cells in the decellularized corneal stroma.

[0034] (3) Due to incomplete photopolymerization, the transition connection area of ​​the artificial cornea provided by the present invention has a porous structure that allows cells to adhere, which is conducive to the growth, migration and differentiation of body cells in the porous structure, so that the artificial cornea and the recipient tissue have a tight connection and avoid the occurrence of corneal transplantation complications. Secondly, the transition connection area and the central optical zone interlock, which improves the mechanical strength of the connection and makes the overall morphological stability of the artificial cornea better. Attached Figure Description

[0035] Figure 1 Flowchart of integrated artificial cornea fabrication process.

[0036] Figure 2 Diagram of an integrated artificial cornea.

[0037] Figure 3 Structure diagram of an ultraviolet filter with gradient effect.

[0038] Figure 4 White light images of the cornea prepared in Example 1 and Comparative Example 2.

[0039] Figure 5 SEM image of the integrated artificial cornea prepared in Example 1.

[0040] Figure 6 Corneal tensile force data prepared in Example 1 and Comparative Example 1.

[0041] Figure 7 Image of an integrated artificial corneal slice prepared in Example 1. Detailed Implementation

[0042] 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.

[0043] Construction of UV filters with gradient effect

[0044] The structure of the ultraviolet filter is as follows: Figure 3As shown, the optical filter has a diameter of 8-10 mm. The center of the filter has a circular hole with a diameter of 3-4 mm, which allows ultraviolet light to pass through completely. The outer ring of the hole consists of a gradient light-transmitting zone and a completely opaque zone. The gradient light-transmitting zone has a width of 1-2 mm and is densely covered with micropores with a diameter of 10-20 μm. The density of the micropores gradually decreases from the inside to the outside, so that the ultraviolet light transmittance decreases uniformly from 100% to completely opaque. The completely opaque zone is made of conventional ultraviolet filter material and does not transmit ultraviolet light at all.

[0045] Preparation of decellularized porcine corneal stroma

[0046] S1: Take a fresh pig eyeball, carefully scrape off the epithelial cells with a scalpel, remove the cornea and clean it. Place the cornea at -80℃ to cool it down completely, then take it out and thaw it at room temperature. Repeat the freeze-thaw operation three times.

[0047] S2: Repeatedly freeze-thawed porcine corneas are placed in decellularization solutions (such as SDS, sodium deoxycholate, Triton X-100) for 6-8 hours, with sonication for 15 minutes every 2 hours. Then, they are treated with a super nuclease at 37°C for 6 hours, and placed in the decellularization solution again to fully remove cellular components. Finally, the decellularized corneal stroma is soaked in deionized water to fully wash away any residual decellularization reagents. Stress is applied to the corneal stroma to remove some moisture, and it is stored at -80°C to maintain its shape.

[0048] Preparation of integrated artificial cornea Example 1

[0049] Taking hydroxyethyl methacrylate (HEMA) as an example, it is constructed using in-situ polymer gradient curing technology, which is generally divided into three steps: introducing monomers, in-situ gradient curing, and removing monomers.

[0050] First, prepare aqueous solutions of hydroxyethyl methacrylate with mass concentrations of 45%, 60%, 75%, and 90%, and a hydroxyethyl methacrylate solution with a mass concentration of 100%. Then, add a 3% (by mass) photoinitiator (2,4,6-(trimethylbenzoyl)diphenylphosphine oxide).

[0051] a. Introduction of monomers: Place the decellularized cornea in a 45% aqueous solution of hydroxyethyl methacrylate containing a photoinitiator and shake thoroughly for 2 hours. Remove the cornea and aspirate any residual solution from the surface. Then place it in a 60% aqueous solution of hydroxyethyl methacrylate containing a photoinitiator and shake thoroughly for 4 hours. Repeat this process until the concentration of the hydroxyethyl methacrylate solution is 100%. Shake thoroughly to remove water from the decellularized matrix and allow the hydroxyethyl methacrylate to fully penetrate into the decellularized corneal matrix.

[0052] b. Gradient curing: Wipe away the residual monomers on the surface of the decellularized cornea obtained in step a, place it in a petri dish, cover it with a gradient UV filter, irradiate with UV for 20 minutes, then flip it over, cover it with the filter again, and continue irradiation for 15 minutes.

[0053] c. Monomer removal: Soak the gradient-cured decellularized corneal matrix in deionized water and agitate it to fully remove unreacted monomers and initiators from the corneal matrix, obtaining a corneal main layer with a central gradient-cured zone;

[0054] d. Constructing the posterior plate layer: Place the above-mentioned corneal substrate layer on a petri dish, wipe off any residual moisture, add 2 drops of 100% hydroxyethyl methacrylate containing a photoinitiator, allow it to remain on the surface of the posterior plate layer of the substrate for 5-10 minutes to penetrate, then use a cotton ball to absorb the solution, and add 2 drops of the above solution again. Cover the top with a UV filter with a gradient effect, and irradiate under UV light for 15-20 minutes to allow the monomers to fully polymerize and obtain the water-resistant area of ​​the posterior plate layer. Finally, thoroughly soak and wash with deionized water to obtain an integrated artificial cornea.

[0055] Comparative Example 1

[0056] Prepare aqueous solutions of hydroxyethyl methacrylate with mass concentrations of 45%, 60%, 75%, and 90%, and a hydroxyethyl methacrylate solution with a mass concentration of 100%. Add 3% (by mass) of photoinitiator (2,4,6-(trimethylbenzoyl)diphenylphosphine oxide).

[0057] a. Introduction of monomers: Place the decellularized cornea in a 45% aqueous solution of hydroxyethyl methacrylate containing a photoinitiator and shake thoroughly for 2 hours. Remove the cornea and aspirate any residual solution from the surface. Then place it in a 60% aqueous solution of hydroxyethyl methacrylate containing a photoinitiator and shake thoroughly for 4 hours. Repeat this process until the concentration of the hydroxyethyl methacrylate solution is 100%. Shake thoroughly to remove water from the decellularized matrix and allow the hydroxyethyl methacrylate to fully penetrate into the decellularized corneal matrix.

[0058] b. Gradient curing: Wipe off the residual monomers on the surface of the decellularized cornea obtained in step a, place it in a petri dish, make a central hole with a diameter of 3-4 mm in the center of the decellularized cornea according to the design requirements, cover it with the gradient effect ultraviolet filter described in Example 1, irradiate with ultraviolet light for 20 min, flip it over, cover it with the filter and continue irradiation for 15 min.

[0059] c. Monomer removal: Soak the gradient-cured decellularized corneal matrix in deionized water and agitate it to fully remove unreacted monomers and initiators from the corneal matrix and obtain the main corneal layer;

[0060] d. Constructing the posterior plate layer: Place the above-mentioned corneal substrate layer on a petri dish, wipe off any residual moisture, add 2 drops of 100% hydroxyethyl methacrylate containing a photoinitiator, allow it to remain on the surface of the posterior plate layer of the substrate for 5-10 minutes to penetrate, then use a cotton ball to absorb the solution, and add 2 drops of the above solution again. Cover the top with a UV filter with a gradient effect, and irradiate under UV light for 15-20 minutes to allow the monomers to fully polymerize and obtain the water-resistant area of ​​the posterior plate layer. Finally, thoroughly soak and wash with deionized water to obtain an integrated artificial cornea.

[0061] Comparative Example 2

[0062] Provide 100% hydroxyethyl methacrylate by mass, and add 3% by mass of photoinitiator (2,4,6-(trimethylbenzoyl)diphenylphosphine oxide).

[0063] a. Introducing monomers: Place decellularized cornea in the above-mentioned hydroxyethyl methacrylate containing photoinitiator and shake thoroughly for 4 hours;

[0064] b. Gradient curing: Wipe away the residual monomers on the surface of the decellularized cornea obtained in step a, place it in a petri dish, cover it with a gradient UV filter, irradiate with UV for 20 minutes, then flip it over, cover it with the filter again, and continue irradiation for 15 minutes.

[0065] c. Monomer removal: Soak the gradient-cured decellularized corneal matrix in deionized water and agitate it to fully remove unreacted monomers and initiators from the corneal matrix, obtaining a corneal main layer with a central gradient-cured zone;

[0066] d. Constructing the posterior plate layer: Place the above-mentioned corneal substrate layer on a petri dish, wipe off any residual moisture, add 2 drops of 100% hydroxyethyl methacrylate containing a photoinitiator, allow it to remain on the surface of the posterior plate layer of the substrate for 5-10 minutes to penetrate, then use a cotton ball to absorb the solution, and add 2 drops of the above solution again. Cover the top with a UV filter with a gradient effect, and irradiate under UV light for 15-20 minutes to allow the monomers to fully polymerize and obtain the water-resistant area of ​​the posterior plate layer. Finally, thoroughly soak and wash with deionized water to obtain an integrated artificial cornea.

[0067] Figure 4 To compare the white light images of the cornea before curing in Comparative Example 2 and Example 1, it can be seen from the images that, compared with Example 1, Comparative Example 1 did not use a gradient concentration of monomers when introducing the monomers but directly used 100% monomers, which resulted in excessively rapid dehydration and uneven shrinkage in the decellularized corneal stroma, making it impossible to proceed with the next step of in-situ curing.

[0068] Effect verification

[0069] I. Scanning electron microscope (SEM) images of an integrated artificial cornea

[0070] The integrated artificial cornea prepared in Example 1 of this invention was used as a sample. After processing, it was observed using a scanning electron microscope. The scanning electron microscope image of the transition connection region in the sample of Example 1 is shown below. Figure 5 As shown, the relatively loose and porous central area is the decellularized corneal stroma, while the surrounding relatively dense area is the solidified area. The image reveals that the in-situ gradient-solidified porcine corneal decellularized stroma forms a gradient-shaped bonding zone with the unsolidified area. After corneal implantation, the decellularized stroma is gradually replaced by corneal stromal cells. This bonding zone provides a higher bonding area between the cells and the optical zone, thereby improving the bonding strength between the artificial cornea and the eyeball in the post-operative period.

[0071] II. Tensile Strength of Integrated Artificial Cornea

[0072] The integrated artificial corneas prepared in Example 1 and Comparative Example 1 of this invention were used as test samples. Before testing, the samples were prepared from the center into strips 15 mm long, 7 mm wide, and approximately 1 mm thick, retaining the central optical zone and transition connection zone to determine the tensile strength of the bonding zone. The testing equipment was an electronic single-fiber tensile testing machine, model YG005A, with a clamping distance of 6 mm and a testing speed of 100 mm / min. The tensile distance and tensile force during the stretching process were recorded until the sample was completely broken. The tensile strength was calculated using the maximum tensile force applied during the stretching process.

[0073] Test results as follows Figure 6 As shown in the figure, the fracture strength of Comparative Example 1 is much lower than that of Example 1, indicating that the integrated artificial cornea (Example 1) prepared by the in-situ gradient curing technology provided by this invention has higher mechanical strength than the centrally perforated combined artificial cornea (Comparative Example 1). Asterisks in the figure indicate statistically significant differences, and *** indicates significant differences (p < 0.001).

[0074] III. Observation of Slices

[0075] The integrated artificial cornea prepared in Example 1 of this invention was subjected to section staining and observation. Figure 7 This is an optical microscope image of a frozen section of the sample from Example 1 stained with hematoxylin and eosin (HE) in the transition region. The relatively loose area on the left is the decellularized matrix area, and the dense area on the right is the solidified area. The image shows that the decellularized porcine corneal matrix, solidified in situ through a gradient process, forms a gradient-shaped junction with the unsolidified area.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An integrated keratoprosthesis, comprising: The structure of the artificial cornea comprises a corneal main layer and a posterior plate layer, the corneal main layer comprises a central optical zone, a transition connection zone and a decellularized corneal annular zone, the corneal main layer takes the decellularized cornea as an integral framework, a polymerizable monomer is introduced into the decellularized cornea, and the monomer is cured by an in-situ gradient curing technology to obtain the corneal main layer; The integrated artificial cornea is prepared by the following method: A: prepare hydroxyethyl methacrylate aqueous solutions with mass concentrations of 45%, 60%, 75% and 90%, and hydroxyethyl methacrylate with a mass concentration of 100%, add a photoinitiator with a mass concentration of 3% to the solutions, shake the decellularized cornea in the hydroxyethyl methacrylate aqueous solution with a mass concentration of 45% for 1-4 h, take out and remove the residual solution on the surface, shake the decellularized cornea in the hydroxyethyl methacrylate aqueous solution with a mass concentration of 60% for 1-4 h, take out and remove the residual solution on the surface, repeat the above operation in the hydroxyethyl methacrylate aqueous solutions with mass concentrations of 75% and 90%, take out and remove the residual solution on the surface, and finally shake the decellularized cornea in the hydroxyethyl methacrylate with a mass concentration of 100%, and take out and remove the residual solution on the surface; B: place the decellularized cornea infiltrated with the hydroxyethyl methacrylate monomer on a petri dish, cover the top with a UV filter with a gradient effect, and irradiate for 20-30 min, and then turn over and irradiate the other side for 15-20 min; C: soak the irradiated cornea in deionized water to remove the residual monomer and photoinitiator on the surface of the cornea, and obtain the corneal main layer; D: place the corneal main layer on a petri dish, add 2-3 drops of hydroxyethyl methacrylate with a mass concentration of 100% containing a photoinitiator on the surface of the corneal main layer, penetrate for 5-10 min, cover the top with a UV filter with a gradient effect, irradiate for 15-20 min, and then soak and wash the integrated artificial cornea in deionized water; The UV filter with a gradient effect is an optical filter with a diameter of 8-10 mm, the center of the filter is a circular hole with a diameter of 3-4 mm, the outer circle of the hole is a gradient light transmission zone and a complete light shielding zone in sequence, the gradient light transmission zone has a width of 1-2 mm, is densely covered with micropores with diameters of 10-20 μm, and has a gradually decreasing density from the inside to the outside, so that the UV light transmittance uniformly decreases to complete non-transmittance of UV light; and the complete light shielding zone completely does not transmit UV light.

2. A preparation method of the integrated artificial cornea according to claim 1, the method comprising the following steps: A: Prepare hydroxyethyl methacrylate aqueous solution with mass concentration of 45%, 60%, 75%, 90%, and hydroxyethyl methacrylate with mass concentration of 100%, add photoinitiator with mass concentration of 3% to the solution, mix well, place the decellularized cornea in the hydroxyethyl methacrylate aqueous solution with mass concentration of 45%, shake for 1-4 h, take out and remove the residual solution on the surface, then place the cornea in the hydroxyethyl methacrylate aqueous solution with mass concentration of 60%, shake for 1-4 h, take out and remove the residual solution on the surface, repeat the above operation in the hydroxyethyl methacrylate aqueous solution with mass concentration of 75% and 90%, take out and remove the residual solution on the surface, and finally shake in the hydroxyethyl methacrylate with mass concentration of 100%, take out and remove the residual solution on the surface; B: Place the decellularized cornea permeated with hydroxyethyl methacrylate monomer on a surface dish, cover the top with a UV filter with gradient effect, and irradiate with UV light for 20-30 min, flip and irradiate the other side for 15-20 min; C: Soak the irradiated cornea with deionized water to remove the residual monomer and photoinitiator on the surface of the cornea, and obtain the corneal stroma layer; D: Place the corneal stroma layer on a surface dish, add 2-3 drops of hydroxyethyl methacrylate with mass concentration of 100% containing photoinitiator on the surface of the corneal stroma layer, permeate for 5-10 min, cover the top with a UV filter with gradient effect, and irradiate with UV light for 15-20 min, then soak and wash with deionized water to obtain an integrated artificial cornea.

3. The preparation method according to claim 2, characterized in that, The decellularized cornea is prepared by following the conventional decellularization method in the art, and the xenogeneic cornea is selected from the cornea of pig, monkey, rabbit, cat, dog, cow, and sheep.

Images