Enhanced cornea stroma lens and preparation method and application thereof
By compressing and cross-linking the corneal stromal lens, an enhanced corneal stromal lens was prepared, which solved the problems of insufficient biocompatibility, anti-edema, and anti-degradation properties of existing materials in full-thickness corneal stromal transplantation, and achieved higher mechanical stability and biocompatibility.
Patent Information
- Application Number
- CN202510911947.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-02
- Publication Date
- 2025-10-24
AI Technical Summary
Existing synthetic polymer corneal replacement materials have poor biocompatibility in full-thickness corneal stromal transplantation, and the corneal stromal lens removed during surgery has insufficient anti-edema and anti-degradation properties, which cannot meet the needs of full-thickness corneal stromal transplantation.
By compressing the corneal stromal lens to a compression ratio of 50-60% under sterile conditions and then crosslinking it with a crosslinking agent solution, enhanced corneal stromal lenses were prepared using mild crosslinking agents such as 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide to improve their density and biocompatibility.
Enhanced corneal stromal lenses exhibit excellent anti-edema, anti-degradation, and mechanical stability. They have a lower water absorption rate than natural corneal stromal lenses, a significantly reduced degradation rate, a higher elastic modulus than natural corneal stromal lenses, and good biocompatibility.
Smart Images
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of tissue engineering, and in particular to an enhanced corneal stroma lens and a preparation method and application thereof. BACKGROUND
[0002] Full-thickness corneal stroma injury is a serious eye trauma, which is affected by mechanisms such as edema reaction after aqueous humor infiltration into the corneal stroma and degradation by collagenase. After the aqueous humor in the anterior chamber enters the corneal stroma and the epithelium, acute corneal edema occurs, which can cause rapid vision loss and eventually lead to corneal blindness. When full-thickness corneal stroma injury involves full-thickness corneal stroma scarring, endothelial damage and corneal perforation, full-thickness corneal transplantation is required. For the material required for full-thickness corneal transplantation, in addition to donor corneas with healthy endothelium, pre-made corneal prosthesis materials can also be used. Currently, common pre-made corneal prosthesis materials include PMMA-based Boston Kpro and PHEMA-based AlphaCor KPro, which are synthetic polymer-based corneal replacement materials that have entered practice. These synthetic polymer-based corneal replacement materials generally exhibit high mechanical plasticity and toughness to resist aqueous swelling, meeting the needs of full-thickness corneal stroma replacement. However, these artificial materials have poor biocompatibility and biological integration compared to biological materials, leading to complications such as stroma dissolution.
[0003] Small incision lenticule extraction (SMILE) is one of the mainstream surgeries for corneal refractive surgery, and the corneal stroma lens removed during the surgery has high transparency, natural biological material properties, large quantity, and diversified thickness. With the popularization of SMILE surgery, a large number of removed corneal lenses are used as a good biological material to correct hyperopia, presbyopia, corneal ulcers, and dilated corneal diseases. However, the corneal stroma lens removed during the surgery has poor resistance to edema and degradation, making it unable to be applied to full-thickness corneal stroma transplantation.
[0004] Therefore, there is an urgent need to provide a preparation method of an enhanced corneal stroma lens with good anti-edema, anti-degradation and mechanical stability. SUMMARY
[0005] The present application aims to provide an enhanced corneal stroma lens with good anti-edema, anti-degradation and mechanical stability, and a preparation method and application thereof.
[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:
[0007] The present application provides a preparation method of an enhanced corneal stroma lens, comprising the following steps:
[0008] (1) compressing a corneal stromal lens in a sterile environment to obtain a compressed corneal stromal lens; the compression ratio of the compression is 50-60%; the compression ratio is the ratio of the thickness of the compressed corneal stromal lens to the thickness of the corneal stromal lens;
[0009] (2) mixing the compressed corneal stromal lens obtained in the step (1) with a crosslinking agent solution to perform crosslinking modification to obtain an enhanced corneal stromal lens; the crosslinking agent in the crosslinking agent solution comprises one or more of 4-(4, 6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide.
[0010] Preferably, the temperature of the environment for the compression in the step (1) is 20-25℃; the humidity of the environment for the compression is 40-55%.
[0011] Preferably, the time for the compression in the step (1) is 24-120h.
[0012] Preferably, the curvature of the compressed corneal stromal lens in the step (1) is 43-44°.
[0013] Preferably, the preparation method of the crosslinking agent solution in the step (2) comprises: dissolving 4-(4, 6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride in a hydrochloric acid solution to obtain the crosslinking agent solution; the pH value of the hydrochloric acid solution is 5.5-6.5; the ratio of the mass of the 4-(4, 6-dimethoxytriazin-2-yl)-4-methylmorpholinium chloride to the volume of the hydrochloric acid solution is (20-40) mg: 1 mL.
[0014] Preferably, the preparation method of the crosslinking agent solution in the step (2) comprises: dissolving N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide in a 2-morpholinoethanesulfonic acid buffer solution to obtain the crosslinking agent solution; the concentration of the 2-morpholinoethanesulfonic acid buffer solution is 5-10 mg / mL; the concentration of N-hydroxysuccinimide in the crosslinking agent solution is 10-20 mg / mL; the concentration of 1-ethyl-(3-dimethylaminopropyl) carbodiimide in the crosslinking agent solution is 20-30 mg / mL.
[0015] Preferably, the ratio of the number of the compressed corneal stromal lenses to the volume of the crosslinking agent solution is 1 piece: (1-2) mL.
[0016] Preferably, the temperature of the crosslinking modification in the step (2) is 20-30℃; the time of the crosslinking modification is 30 min-120 min.
[0017] The application further provides an enhanced corneal stromal lens prepared by the preparation method.
[0018] The present invention also provides the use of the enhanced corneal stroma lens described in the above technical solution as a full-thickness corneal stroma transplantation material.
[0019] The present invention provides a preparation method of an enhanced corneal stromal lens, comprising the following steps: compressing the corneal stromal lens in a sterile environment to obtain a compressed corneal stromal lens; the compression ratio of the compression is 50-60%; the compression ratio is the ratio of the thickness of the compressed corneal stromal lens to the thickness of the corneal stromal lens; mixing the compressed corneal stromal lens with a crosslinking agent solution, performing crosslinking modification, and obtaining the enhanced corneal stromal lens; the crosslinking agent in the crosslinking agent solution comprises one or more of 4-(4,6-dimethoxytriazine-2-yl)-4-methylmorpholine hydrochloride, N-hydroxysuccinimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide. The present invention compresses the corneal stromal lens under a sterile environment, thereby reducing the distance between fibers in the compressed corneal stromal lens, making the compressed corneal stromal lens more compact, and improving the mechanical properties and degradation resistance of the compressed corneal stromal lens. Furthermore, the compressed corneal stromal lens is relatively compact, making it difficult for water molecules to pass through, thereby improving its anti-edema ability and enabling the prepared enhanced corneal stromal lens to have better anti-edema properties. The present invention cross-links and modifies the compressed corneal stromal lens by mixing it with a cross-linking agent solution, thereby shaping the compressed corneal stromal lens to maintain a stable compact state, improving the mechanical properties and anti-edema stability of the enhanced corneal stromal lens. Furthermore, the cross-linking agent used in the present invention is relatively mild, enabling the enhanced corneal stromal lens to maintain good biocompatibility. The results of the examples show that the enhanced corneal stromal lens prepared by the present invention has a light transmittance similar to that of a natural corneal stromal lens; the water absorption rate of the enhanced corneal stromal lens is significantly lower than that of a natural corneal stromal lens, and it has excellent anti-edema properties; after being dissolved by collagenase, the degradation rate of the enhanced corneal stromal lens is significantly lower than that of a natural corneal stromal lens, and it has excellent anti-degradation properties; after being immersed in PBS solution, the thickness change rate of the enhanced corneal stromal lens is significantly lower than that of a natural corneal stromal lens, and the elastic modulus of the enhanced corneal stromal lens is significantly higher than that of a natural corneal stromal lens, and it has excellent mechanical stability and excellent biocompatibility. BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Figure 1 A physical picture of the mold base and mold sheet used in the embodiments of the present invention;
[0021] Figure 2 Physical pictures of the natural corneal stromal lens provided in Comparative Example 1 of the present invention and the enhanced corneal stromal lens prepared in Example 1;
[0022] Figure 3Light transmittance curves of the natural corneal stromal lens provided for Invention Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1;
[0023] Figure 4 SEM images of the natural corneal stromal lens provided for Invention Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1;
[0024] Figure 5 HE staining images of the natural corneal stromal lens provided for Invention Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1;
[0025] Figure 6 Transmission electron microscope images of the natural corneal stromal lens provided for Invention Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1;
[0026] Figure 7 Actual images of the natural corneal stromal lens provided for Invention Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1 before and after immersion in PBS solution for 48 h, respectively;
[0027] Figure 8 Water absorption rate-weight change curves of the natural corneal stromal lens provided for Invention Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1 after immersion in PBS solution for 48 h, respectively;
[0028] Figure 9 Water absorption rate-thickness change curves of the natural corneal stromal lens provided for Invention Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1 after immersion in PBS solution for 48 h, respectively;
[0029] Figure 10 Actual images of the natural corneal stromal lens provided for Invention Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1 before and after degradation;
[0030] Figure 11 Degradation rate curves of the natural corneal stromal lens provided for Invention Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1 after degradation;
[0031] Figure 12 TEM images of the natural corneal stromal lens provided for Invention Comparative Example 2 and the comparative corneal stromal lens prepared in Comparative Example 2 and the comparative corneal stromal lens after immersion in PBS solution;
[0032] Figure 13 Slit lamp microscope images of the natural corneal stromal lens provided for Invention Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1 4 weeks after corneal endothelial transplantation;
[0033] Figure 14The preoperative OCT results of the natural corneal stromal lens provided for Invention Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1 after corneal endothelial transplantation for 4 weeks;
[0034] Figure 15 The slit lamp microscope images of the natural corneal stromal lens provided for Invention Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1 after double-layer corneal transplantation for 4 weeks;
[0035] Figure 16 The fluorescence staining results of the natural corneal stromal lens provided for Invention Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1 after double-layer corneal transplantation for 4 weeks;
[0036] Figure 17 The preoperative OCT results of the natural corneal stromal lens provided for Invention Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1 after double-layer corneal transplantation for 4 weeks. DETAILED DESCRIPTION
[0037] The present application provides a preparation method of an enhanced corneal stromal lens, comprising the following steps:
[0038] (1) compressing a corneal stromal lens in a sterile environment to obtain a compressed corneal stromal lens; the compression ratio of the compression is 50-60%; the compression ratio is the ratio of the thickness of the compressed corneal stromal lens to the thickness of the corneal stromal lens;
[0039] (2) mixing the compressed corneal stromal lens obtained in step (1) with a crosslinking agent solution to perform crosslinking modification to obtain an enhanced corneal stromal lens; the crosslinking agent in the crosslinking agent solution comprises one or more of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbonyldiimidazole.
[0040] The present application compresses a corneal stromal lens in a sterile environment to obtain a compressed corneal stromal lens.
[0041] The present application does not have special limitations on the source and size of the corneal stromal lens, and any clinical corneal stromal lens available to those skilled in the art can be used. In the examples of the present application, the corneal stromal lens is a clinical corneal stromal lens, i.e. a natural corneal stromal lens, and the size of the natural corneal stromal lens can be 6.5 mm in diameter and 120 μm in thickness.
[0042] The present application does not have specific limitations on the control method of the sterile environment, and any method for controlling the sterile environment known in the art can be used. The present application is compressed in a sterile environment, which can ensure the safety of the enhanced corneal stromal lens prepared.
[0043] In the present application, the compression ratio of the compression is 50-60%. As an embodiment of the present application, the compression ratio can be 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59% or 60%. In the present application, the compression ratio is the ratio of the thickness of the compressed corneal stromal lens to the thickness of the corneal stromal lens. The present application controls the compression ratio in the above range, which can make the compressed corneal stromal lens have excellent compactness, and improve the anti-degradation ability, mechanical properties and anti-edema of the compressed corneal stromal lens.
[0044] In the present application, the compression method is preferably that the corneal stromal lens is completely flattened on the mold sheet, and the mold base is completely matched with the mold sheet before compression.
[0045] In the present application, the surface curvature of the mold sheet is preferably 43-44°. The present application uses the above mold sheet, which can make the prepared enhanced corneal stromal lens conform to the natural corneal curvature, and better fit in application. In the embodiment of the present application, the physical diagram of the mold sheet is preferably as shown in Figure 1 (a).
[0046] In the present application, the contact surface of the mold base and the mold sheet is an inwardly concave curved surface, and the curvature of the inwardly concave is preferably 43-44°. The present application uses the above mold base, which can make the mold base and the mold sheet be completely matched, and the corneal stromal lens located between the mold base and the mold sheet be compressed, so that the compressed corneal stromal lens with uniform thickness is obtained. In the embodiment of the present application, the physical diagram of the mold base is preferably as shown in Figure 1 (b).
[0047] In the present application, the compression environment temperature is preferably 20-25℃. As an embodiment of the present application, the compression environment temperature can be 20℃, 21℃, 22℃, 23℃, 24℃ or 25℃. In the present application, the compression environment humidity is preferably 40-55%. As an embodiment of the present application, the compression environment humidity can be 40%, 45%, 50% or 55%. In the present application, the compression time is preferably 24-120h. As an embodiment of the present application, the compression time can be 24h, 36h, 48h, 72h, 84h, 96h, 108h or 120h. The present application performs compression in the above environment, which can prevent the shrinkage of the corneal stromal lens caused by unstable humidity.
[0048] In the present application, the curvature of the compressed corneal stromal lens is preferably 43-44°. The present application controls the curvature of the compressed corneal stromal lens in the above range, which can make the prepared enhanced corneal stromal lens conform to the natural corneal curvature.
[0049] After obtaining the compressed corneal stroma lens, the present application mixes the compressed corneal stroma lens with a crosslinking agent solution to perform crosslinking modification, thereby obtaining an enhanced corneal stroma lens.
[0050] In the present application, the crosslinking agent in the crosslinking agent solution includes one or more of 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride (abbreviated as DMTMM), N-hydroxysuccinimide (abbreviated as NHS), and 1-ethyl-(3-dimethylaminopropyl) carbodiimide (abbreviated as EDC), and is more preferably DMTMM or a mixture of NHS and EDC. The above crosslinking agent used in the present application is a mild crosslinking agent, which does not destroy the structure of the compressed corneal stroma lens and retains good biocompatibility of the compressed corneal stroma lens.
[0051] In the present application, the preparation method of the crosslinking agent solution preferably includes: dissolving DMTMM in a hydrochloric acid solution to obtain a crosslinking agent solution.
[0052] In the present application, the pH value of the hydrochloric acid solution is preferably 5.0-6.5, and more preferably 5.5-6.5.
[0053] In the present application, the mass ratio of DMTMM to the volume of the hydrochloric acid solution is preferably (20-40) mg: 1 mL. As an embodiment of the present application, the mass ratio of DMTMM to the volume of the hydrochloric acid solution can be 20 mg: 1 mL, 25 mg: 1 mL, 30 mg: 1 mL, 35 mg: 1 mL, or 40 mg: 1 mL.
[0054] In the present application, the preparation method of the crosslinking agent solution preferably includes: dissolving NHS and EDC in a 2-morpholinoethanesulfonic acid (abbreviated as MES) buffer solution to obtain a crosslinking agent solution.
[0055] In the present application, the concentration of the MES buffer solution is preferably 5-10 mg / mL. As an embodiment of the present application, the concentration of the MES buffer solution can be 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, or 10 mg / mL.
[0056] In the present application, the concentration of NHS in the crosslinking agent solution is preferably 10-20 mg / mL, and more preferably 15 mg / mL; and the concentration of EDC in the crosslinking agent solution is preferably 20-30 mg / mL, and more preferably 25 mg / mL.
[0057] In the present invention, the ratio of the number of compressed corneal stromal lenses to the volume of the crosslinker solution is preferably 1 lens: (1-2) mL, more preferably 1 lens: (1.5-2) mL. By controlling the ratio of the number of compressed corneal stromal lenses to the volume of the crosslinker solution within this range, the compressed corneal stromal lenses can be fully crosslinked and modified.
[0058] In the present invention, the temperature of the cross-linking modification is preferably 20 to 30°C, more preferably 22 to 27°C. In the present invention, the time of the cross-linking modification is preferably 30 to 120 minutes. As an embodiment of the present invention, the time of the cross-linking modification can be 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes or 120 minutes. The present invention performs cross-linking modification at the above temperature and time, which can fully react the compressed corneal stromal lens with the cross-linking agent, fix the compressed corneal stromal lens, prevent the enhanced corneal stromal lens from swelling under the action of water, and improve the anti-edema stability of the enhanced corneal stromal lens.
[0059] In the present invention, the modified corneal stromal lens obtained by cross-linking modification is preferably cleaned, dried and plasma treated in sequence to obtain an enhanced corneal stromal lens.
[0060] In the present invention, the cleaning method preferably comprises: placing the modified corneal stromal lens in sterile injection water and performing shaking cleaning to obtain the cleaned modified corneal stromal lens. The present invention can remove the crosslinking agent that has not participated in the reaction by cleaning.
[0061] In the present invention, the volume of sterile water for injection used for each modified corneal stroma lens is preferably 20 to 40 mL, more preferably 30 to 40 mL.
[0062] In the present invention, the temperature of the shaking table cleaning is preferably 0-4°C; the number of shaking table cleanings is preferably 3-6 times. In the present invention, the time for each shaking table cleaning is preferably 60 minutes.
[0063] The present invention has no particular limitation on the drying method, and any conventional drying method may be used. In an embodiment of the present invention, the drying method is preferably natural drying; the temperature of the natural drying is preferably 20 to 30° C., more preferably 25° C.; and the humidity of the natural drying is preferably 40 to 55%, more preferably 50%.
[0064] In the present application, the power of the plasma treatment is preferably 1200W, and the irradiation sterilization energy of the plasma treatment is preferably 15kGy. The device for the plasma treatment is not particularly limited in the present application, and a conventional plasma treatment device can be used. In the embodiment of the present application, the plasma treatment is preferably tantec plasma treatment, and the model of the plasma treatment device is DK6640. The surface activity of the enhanced corneal stroma lens can be improved by the plasma treatment.
[0065] The enhanced corneal stroma lens obtained by the plasma treatment is preferably stored at low temperature in the present application. In the embodiment of the present application, the temperature of the low-temperature storage is preferably-20 to-80℃.
[0066] The present application also provides an enhanced corneal stroma lens prepared by the preparation method.
[0067] The present application also provides the use of the enhanced corneal stroma lens in full-thickness corneal stroma transplantation.
[0068] In the present application, the enhanced corneal stroma lens is preferably used in corneal endothelial transplantation surgery or in double-layer corneal transplantation surgery. The method of the corneal endothelial transplantation surgery is not particularly limited in the present application, and a conventional corneal endothelial transplantation surgery method can be used.
[0069] In the embodiment of the present application, the method of the double-layer corneal transplantation surgery can be as follows: the enhanced corneal stroma lens is used as an inner layer graft and is fixed to the corneal endothelial surface by intermittent suture with 2 needles, and the diameter is greater than 1-2mm larger than the defect. The natural corneal stroma lens is used as a surface layer graft and is fixed to the defect by intermittent suture, and the diameter is greater than 0.25mm larger than the defect.
[0070] The enhanced corneal stroma lens provided by the present application has the characteristics of anti-edema, anti-degradation and strong mechanical stability, and can be used as a full-thickness corneal stroma transplantation material.
[0071] The technical solutions in the present application will be described clearly and completely in combination with the embodiments in the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.
[0072] Embodiment 1
[0073] A preparation method of an enhanced corneal stroma lens, comprising the following steps:
[0074] (1) The clinical corneal stroma lens (diameter 6.5 mm, thickness 120 μm) was placed on the mold piece with a curvature of 43-44° under sterile conditions to completely flatten, and the mold base was completely matched with the mold piece. After 72 h of compression in a sterile environment at a temperature of 25°C and a humidity of 50%, the thickness of the corneal stroma lens was measured and controlled in real time by an electronic corneal thickness measuring instrument (547-301, Mitutoyo, Japan), and a compressed corneal stroma lens with a compression ratio of 50% was obtained.
[0075] (2) The crosslinking agent DMTMM was dissolved in a hydrochloric acid solution with a pH of 6.0 (crosslinking agent mass: volume ratio of 30 mg: 1 mL), and a piece of the compressed corneal stroma lens obtained in step (1) was placed in 1.5 mL of the crosslinking agent solution for crosslinking modification at 25°C for 60 min. The modified corneal stroma lens obtained by crosslinking modification was washed with sterile water on a shaking table at a temperature of 4°C, with each washing lasting 60 min, and the lens was washed for 3 times, with 30 mL of water used for each washing. After natural drying of the washed modified corneal stroma lens, plasma treatment (DK6640, 1200W) was performed, and the lens was stored at -80°C after 15 kGy irradiation sterilization for later use, to obtain an enhanced corneal stroma lens.
[0076] Example 2
[0077] A method for preparing an enhanced corneal stroma lens, comprising the following steps:
[0078] (1) The clinical corneal stroma lens (diameter 6.5 mm, thickness 120 μm) was placed on the mold piece with a curvature of 43-44° under sterile conditions to completely flatten, and the mold base was completely matched with the mold piece. After 72 h of compression in a sterile environment at a temperature of 25°C and a humidity of 50%, the thickness of the corneal stroma lens was measured and controlled in real time by an electronic corneal thickness measuring instrument (547-301, Mitutoyo, Japan), and a compressed corneal stroma lens with a compression ratio of 50% was obtained.
[0079] (2) 300 mg of NHS was dissolved in 20 mL of a MES solution with a concentration of 5 mg / mL, and 500 mg of EDC was added after mixing at room temperature. A piece of the compressed corneal stroma lens obtained in step (1) was placed in 1.5 mL of the crosslinking agent solution for crosslinking modification at 25°C for 30 min. The modified corneal stroma lens obtained by crosslinking modification was washed with sterile water on a shaking table at a temperature of 4°C, with each washing lasting 60 min, and the lens was washed for 3 times, with 30 mL of water used for each washing. After natural drying of the washed modified corneal stroma lens, plasma treatment (DK6640, 1200W) was performed, and the lens was stored at -80°C after 15 kgy irradiation sterilization for later use, to obtain an enhanced corneal stroma lens.
[0080] Example 3
[0081] A preparation method of an enhanced corneal stromal lens, comprising the following steps:
[0082] (1) placing a clinical corneal stromal lens (6.5 mm in diameter and 120 μm in thickness) on a mold piece with a curvature of 43-44° under sterile conditions to completely flatten, completely matching the mold base with the mold piece, and then compressing for 72 h under sterile conditions at a temperature of 25°C and a humidity of 50%, and measuring and controlling the thickness of the corneal stromal lens in real time by using an electronic corneal thickness measuring instrument (547-301, Mitutoyo, Japan) to obtain a compressed corneal stromal lens with a compression ratio of 60%;
[0083] (2) dissolving a crosslinking agent DMTMM in a hydrochloric acid solution with a pH of 6.5 (the mass: volume ratio of the crosslinking agent is 30 mg: 1 mL), placing a piece of the compressed corneal stromal lens obtained in the step (1) into 1.5 mL of the crosslinking agent solution, and performing crosslinking modification at 25°C for 30 min; cleaning the modified corneal stromal lens obtained by crosslinking modification on a shaking table at a temperature of 4°C for 60 min each time for 3 times, and the water amount for cleaning each piece is 30 mL; drying the cleaned modified corneal stromal lens, and then performing plasma treatment (DK6640, 1200 W), and then performing irradiation sterilization at 15 kGy and storing at -80°C for standby use to obtain an enhanced corneal stromal lens.
[0084] Comparative Example 1
[0085] A natural corneal stromal lens, which is the same as the clinical corneal stromal lens used in the step (1) of the embodiment 1.
[0086] Comparative Example 2
[0087] A preparation method of a comparative corneal stromal lens, comprising the following steps:
[0088] (1) placing a clinical corneal stromal lens (6.5 mm in diameter and 120 μm in thickness) on a mold piece with a curvature of 43-44° under sterile conditions to completely flatten, completely matching the mold base with the mold piece, and then compressing for 72 h under sterile conditions at a temperature of 25°C and a humidity of 50%, and measuring and controlling the thickness of the corneal stromal lens in real time by using an electronic corneal thickness measuring instrument (547-301, Mitutoyo, Japan) to obtain a compressed corneal stromal lens with a compression ratio of 50%;
[0089] (2) drying the compressed corneal stromal lens obtained in the step (1) to obtain a comparative corneal stromal lens.
[0090] Test Example
[0091] (1) The actual pictures of the natural corneal stromal lens provided in Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1 are as follows: Figure 2 As shown. Figure 2 In the figure, Figure (a) is a physical picture of the natural corneal stromal lens provided in Comparative Example 1; Figure (b) is a physical picture of the enhanced corneal stromal lens prepared in Example 1. Figure 2 It can be seen that the enhanced corneal stromal lens prepared in the present invention has a transparency similar to that of a natural corneal stromal lens.
[0092] (2) The light transmittance of the natural corneal stromal lens provided in Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1 is as follows: Figure 3 As shown. Figure 3 It can be seen that the enhanced corneal stromal lens prepared in the present invention has a light transmittance similar to that of a natural corneal stromal lens.
[0093] (3) The SEM image of the natural corneal stroma lens provided in Comparative Example 1 is as follows Figure 4 (a) shows; the SEM image of the enhanced corneal stromal lens prepared in Example 1 is shown in FIG. Figure 4 (b) is shown. Figure 4 It can be seen that compared with the natural corneal stromal lens, the surface of the enhanced corneal stromal lens prepared in the present invention is flatter.
[0094] (4) HE staining of the natural corneal stroma lens provided in Comparative Example 1 is as follows Figure 5 (a) shows the HE staining of the enhanced corneal stromal lens prepared in Example 1. Figure 5 (b) is shown. Figure 5 It can be seen that the enhanced corneal stromal lens prepared in the present invention has a denser structure than the natural corneal stromal lens.
[0095] (5) The transmission electron microscope image of the natural corneal stroma lens provided in Comparative Example 1 is as follows Figure 6 (a) shows; the transmission electron microscope image of the enhanced corneal stromal lens prepared in Example 1 is as shown Figure 6 (b) is shown. Figure 6 It can be seen that the fiber spacing of the enhanced corneal stromal lens prepared by the present invention is significantly smaller than that of the natural corneal stromal lens.
[0096] (6) The natural corneal stromal lens provided in Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1 were soaked in PBS solution for 48 hours. Figure 7 As shown in (a), the photo after immersion for 48 hours is as follows Figure 7 (b) As shown; the physical picture of the enhanced corneal stromal lens prepared in Example 1 is as shown Figure 7 As shown in (c), the actual picture after immersion for 48 hours is as followsFigure 7 (d) shown.
[0097] The water absorption rate-weight change curve of the natural corneal stromal lens provided by Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1 after being soaked in PBS solution for 48h is shown in Figure 8
[0098] The water absorption rate-thickness change curve of the natural corneal stromal lens provided by Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1 after being soaked in PBS solution for 48h is shown in Figure 9
[0099] It can be seen that, compared with the natural corneal stromal lens, the enhanced corneal stromal lens prepared by the present application has higher resistance to edema. Figures 7-9
[0100] (7) The natural corneal stromal lens provided by Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1 were respectively dissolved by 5U / mL collagenase for 6h. The pre-degradation physical picture of the natural corneal stromal lens provided by Comparative Example 1 is shown in Figure 10 (a), and the post-degradation physical picture is shown in Figure 10 (b); the pre-degradation physical picture of the enhanced corneal stromal lens prepared in Example 1 is shown in Figure 10 (c), and the post-degradation physical picture is shown in Figure 10 (d).
[0101] The degradation rate curve of the natural corneal stromal lens provided by Comparative Example 1 and the enhanced corneal stromal lens prepared in Example 1 after being dissolved by 5U / mL collagenase for 6h is shown in Figure 11
[0102] It can be seen that, after being dissolved by 5U / mL collagenase, compared with the natural corneal stromal lens, the enhanced corneal stromal lens prepared by the present application has higher resistance to degradation. Figures 10-11
[0103] (8) The TEM image of the clinical corneal stromal lens (i.e. natural corneal stromal lens) used in Comparative Example 2 is shown in Figure 12 (a); the TEM image of the comparative corneal stromal lens prepared in Comparative Example 2 is shown in Figure 12 (b); the TEM image of the comparative corneal stromal lens prepared in Comparative Example 2 after being soaked in PBS solution for 5min is shown in Figure 12 (c).
[0104] It can be seen that, compared with the natural corneal stromal lens, the enhanced corneal stromal lens prepared by the present application has higher resistance to degradation. Figure 12 It can be seen that compared with the natural corneal stroma lens, the fiber spacing of the compression corneal stroma lens is reduced, and the stroma fibers are dense; and after the compression corneal stroma lens is soaked in the PBS solution, the fiber spacing returns to the compression before, which is similar to the natural corneal stroma lens. This shows that the compression corneal stroma lens has not been cross-linked and modified, and cannot have a stable structure, and cannot have a stable anti-edema ability.
[0105] At the same time, since the compression corneal stroma lens has not been cross-linked and modified, it cannot have a stable structure, and therefore does not have stable anti-degradation and mechanical properties.
[0106] Application Example 1
[0107] The enhanced corneal stroma lens prepared in Example 1 is used as a full-layer corneal stroma transplantation material for corneal endothelial transplantation surgery.
[0108] The surgical process is as follows: 5.0 mm of the corneal endothelium in the center of the cornea is removed, and the 5.0 mm enhanced corneal stroma lens prepared in Example 1 is implanted into the corneal endothelial defect.
[0109] Comparative Application Example 1
[0110] The natural corneal stroma lens provided in Comparative Example 1 is used as a full-layer corneal stroma transplantation material for corneal endothelial transplantation surgery.
[0111] The surgical process is as follows: 5.0 mm of the corneal endothelium in the center of the cornea is removed, and the 5.0 mm natural corneal stroma lens provided in Comparative Example 1 is implanted into the corneal endothelial defect.
[0112] Test Example 2
[0113] (1) After the corneal endothelial transplantation of Application Example 1 and Comparative Application Example 1, slit lamp microscopy and anterior segment OCT are used for observation. The natural corneal stroma lens provided in Comparative Example 1 and the enhanced corneal stroma lens prepared in Example 1 are applied to the corneal endothelial transplantation surgery, and the postoperative slit lamp microscopy is as shown in Figure 13 ; the anterior segment OCT result 4 weeks after the corneal endothelial transplantation is as shown in Figure 14 . In Figure 13 and Figure 14 , (a) represents the photo of the natural corneal stroma lens provided in Comparative Example 1 after the corneal endothelial transplantation; (b) represents the photo of the enhanced corneal stroma lens prepared in Example 1 after the corneal endothelial transplantation.
[0114] It can be seen from Figure 13 that 4 weeks after the corneal endothelial transplantation of the natural corneal stroma lens provided in Comparative Example 1, the slit lamp photograph shows that the cornea is obviously edematous, thickened, and the transparency is decreased; 4 weeks after the corneal endothelial transplantation of the enhanced corneal stroma lens prepared in Example 1, the slit lamp photograph shows that the cornea is transparent, and there is no obvious edema.
[0115] From Figure 14 It can be seen that the natural corneal stroma lens provided by Comparative Example 1 provided the corneal endothelial transplantation postoperative 4 weeks of anterior segment OCT results, the cornea can be seen obvious edema, thickness increased; the preparation of the enhanced corneal stroma lens of Example 1 corneal endothelial transplantation postoperative 4 weeks of anterior segment OCT results, the graft and the bed can be seen good adhesion, corneal edema, thickness no obvious change.
[0116] Application Example 2
[0117] The enhanced corneal stroma lens prepared in Example 1 was used as a full layer corneal stroma transplantation material for double layer corneal transplantation surgery;
[0118] Surgical process: a 3.0mm full layer corneal stroma defect was made in the center of the cornea. The 4.0mm enhanced corneal lens prepared in Example 1 was sutured to the inner layer corneal stroma in the defect, and then the 3.25mm natural stroma lens provided by Comparative Example 1 was sutured to the surface layer corneal stroma defect.
[0119] Comparative Application Example 2
[0120] The enhanced corneal stroma lens prepared in Comparative Example 1 was used as a full layer corneal stroma transplantation material for double layer corneal transplantation surgery;
[0121] Surgical process: the 4.0mm natural corneal stroma lens provided by Comparative Example 1 was sutured to the inner layer corneal stroma in the defect, and then the 3.25mm natural stroma lens provided by Comparative Example 1 was sutured to the surface layer corneal stroma defect.
[0122] Test Example 3
[0123] After double layer corneal transplantation of Application Example 2 and Comparative Application Example 2, slit lamp microscope and anterior segment OCT were used for observation. The slit lamp microscope images of the natural corneal stroma lens provided by Comparative Example 1 and the enhanced corneal stroma lens prepared in Example 1 after double layer corneal transplantation for 4 weeks are shown in Figure 15 ; the corneal fluorescence staining results of the natural corneal stroma lens provided by Comparative Example 1 and the enhanced corneal stroma lens prepared in Example 1 after double layer corneal transplantation for 4 weeks are shown in Figure 16 ; the anterior segment OCT results of the natural corneal stroma lens provided by Comparative Example 1 and the enhanced corneal stroma lens prepared in Example 1 after double layer corneal transplantation for 4 weeks are shown in Figure 17 . In Figures 15-17 , (a) represents the photograph of the natural corneal stroma lens provided by Comparative Example 1 after double layer corneal transplantation; (b) represents the photograph of the enhanced corneal stroma lens prepared in Example 1 after double layer corneal transplantation.
[0124] From Figure 15It can be seen that 4 weeks after the double-layer corneal transplantation, the transparency of the natural corneal stroma lens provided in Comparative Example 1 decreased significantly, and the double-layer corneal grafts were edematous and thickened; the double-layer corneal grafts prepared by the enhanced corneal stroma lens prepared in Example 1 were transparent without obvious edema.
[0125] from Figure 16 It can be seen that 4 weeks after the double-layer keratoplasty, the corneal epithelium of the natural corneal stroma lens provided in Comparative Example 1 had not healed and was partially stained; 4 weeks after the double-layer keratoplasty, the corneal epithelium of the enhanced corneal stroma lens prepared in Example 1 had healed well and had no obvious staining.
[0126] from Figure 17 It can be seen that 4 weeks after the double-layer keratoplasty, the natural corneal stromal lens provided in Comparative Example 1 had obvious edema in the inner corneal graft and poor fit with the defective corneal graft bed; the surface corneal graft had obvious edema and increased density; 4 weeks after the double-layer keratoplasty, the enhanced corneal stromal lens prepared in Example 1 had good fit between the double cornea and the defective graft bed, and the enhanced graft had no obvious edema.
[0127] The above results demonstrate that the enhanced corneal stromal lens prepared by the present invention not only has similar light transmittance to a natural corneal stromal lens, but also exhibits enhanced resistance to edema and degradation, higher mechanical stability, and excellent biocompatibility. This approach addresses the issue of corneal stromal lenses removed during SMILE surgery being unable to be used as materials for full-thickness corneal stromal transplants, thereby increasing resource utilization.
[0128] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.
Claims
1. A method of making an enhanced corneal stromal lens, characterized by, The preparation method comprises the following steps: (1) compressing a corneal stromal lens in a sterile environment to obtain a compressed corneal stromal lens; the compression ratio of the compression is 50-60%; the compression ratio is the ratio of the thickness of the compressed corneal stromal lens to the thickness of the corneal stromal lens; (2) mixing the compressed corneal stromal lens obtained in the step (1) with a crosslinking agent solution to perform crosslinking modification, thereby obtaining a reinforced corneal stromal lens; the crosslinking agent in the crosslinking agent solution comprises one or more of 4-(4, 6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride, N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide.
2. The method of making an enhanced corneal on-lens according to claim 1, wherein, The temperature of the environment for the compression in the step (1) is 20-25℃; the humidity of the environment for the compression is 40-55%.
3. The method of making an enhanced corneal on-lens according to claim 1 or 2, wherein, The time for the compression in the step (1) is 24-120h.
4. The method for preparing an enhanced corneal stromal lens according to claim 1, wherein: The curvature of the compressed corneal stromal lens in the step (1) is 43-44°.
5. The method of claim 1, wherein the step of forming the enhanced corneal stromal lens comprises forming the enhanced corneal stromal lens to have a diameter of about 8.0 mm to about 10.0 mm. The preparation method of the crosslinking agent solution in the step (2) comprises: dissolving 4-(4, 6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride in a hydrochloric acid solution to obtain a crosslinking agent solution; the pH value of the hydrochloric acid solution is 5.5-6.5; the ratio of the mass of the 4-(4, 6-dimethoxytriazin-2-yl)-4-methylmorpholinium hydrochloride to the volume of the hydrochloric acid solution is (20-40) mg: 1 mL.
6. The method of claim 1, wherein the step of forming the enhanced corneal stromal lens comprises forming the enhanced corneal stromal lens to have a diameter of about 8.0 mm to about 10.0 mm. The preparation method of the crosslinking agent solution in the step (2) comprises: dissolving N-hydroxysuccinimide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide in a 2-morpholinoethanesulfonic acid buffer solution to obtain a crosslinking agent solution; the concentration of the 2-morpholinoethanesulfonic acid buffer solution is 5-10 mg / mL; the concentration of N-hydroxysuccinimide in the crosslinking agent solution is 10-20 mg / mL; the concentration of 1-ethyl-(3-dimethylaminopropyl) carbodiimide in the crosslinking agent solution is 20-30 mg / mL.
7. The method of making an enhanced corneal on-lens according to claim 5 or 6, wherein, The ratio of the number of the compressed corneal stromal lenses to the volume of the crosslinking agent solution is 1 piece: (1-2) mL.
8. The method of claim 1, wherein the step of forming the enhanced corneal stromal lens comprises forming the enhanced corneal stromal lens to have a diameter of about 8.0 mm to about 10.0 mm. The temperature of the crosslinking modification in the step (2) is 20-30℃; the time of the crosslinking modification is 30 min-120 min.
9. The reinforced corneal stromal lens prepared by the preparation method in any one of claims 1-8.
10. The use of the reinforced corneal stromal lens in claim 9 as a full-layer corneal stromal grafting material.