WO2024011448A1 - 一种高粘附的人工角膜内皮片及其制备方法和应用 - Google Patents

一种高粘附的人工角膜内皮片及其制备方法和应用 Download PDF

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WO2024011448A1
WO2024011448A1 PCT/CN2022/105454 CN2022105454W WO2024011448A1 WO 2024011448 A1 WO2024011448 A1 WO 2024011448A1 CN 2022105454 W CN2022105454 W CN 2022105454W WO 2024011448 A1 WO2024011448 A1 WO 2024011448A1
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artificial
adhesion
corneal endothelial
endothelial sheet
increasing
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PCT/CN2022/105454
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English (en)
French (fr)
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史伟云
王婷
周庆军
张晓玉
王红卫
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山东第一医科大学附属眼科研究所(山东省眼科研究所、山东第一医科大学附属青岛眼科医院)
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Priority to CN202280005923.9A priority Critical patent/CN116348157B/zh
Priority to PCT/CN2022/105454 priority patent/WO2024011448A1/zh
Priority to US18/041,954 priority patent/US20240123120A1/en
Publication of WO2024011448A1 publication Critical patent/WO2024011448A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/14—Eye parts, e.g. lenses, corneal implants; Implanting instruments specially adapted therefor; Artificial eyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/16—Macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/20—Polysaccharides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
    • C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
    • C08F220/10—Esters
    • C08F220/26—Esters containing oxygen in addition to the carboxy oxygen
    • C08F220/32—Esters containing oxygen in addition to the carboxy oxygen containing epoxy radicals
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/26—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring from cyclic ethers and other compounds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
    • C08J7/00—Chemical treatment or coating of shaped articles made of macromolecular substances
    • C08J7/04—Coating
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea

Definitions

  • the invention relates to a corneal implant treated with increased adhesion and a method for increasing the adhesion of an artificial corneal endothelial sheet, and belongs to the field of medical artificial substitute materials.
  • the corneal endothelial cell layer is the innermost layer of the cornea. Its structural integrity and functional integrity are important factors in maintaining normal physiological metabolism of the cornea. Decreased endothelial cell density and endothelial pump dysfunction can lead to decompensation of the corneal endothelium, manifested as corneal edema and turbidity, which seriously affects vision, causes significant eye pain, and decreases the quality of life of patients. It is one of the more common complications after cataract surgery and other inner eye surgeries. First, it is also the end-stage manifestation of primary diseases such as corneal endothelial dystrophy. Initially, the only surgical treatment for corneal endothelial decompensation was penetrating keratoplasty (PKP).
  • PGP penetrating keratoplasty
  • Endothelial transplantation Endothelial keratoplasty, EK
  • EK Endothelial keratoplasty
  • the surgical procedure is corneal Descemet's membrane peeling endothelium.
  • Transplantation Dermathelial stripping endothelial keratoplasty, DSEK
  • DMEK Descemet's membrane endothelial keratoplasty
  • Some patients may suffer from corneal graft endothelial decompensation due to excessive loss of corneal endothelial cells during surgery. 35% of patients have corneal graft detachment after surgery and require one or more anterior chamber gas injections.
  • Artificial corneal endothelial patch refers to an artificial material that is attached to the surface of the posterior corneal stroma and acts as a barrier to isolate the aqueous humor. It is also called a corneal implant, corneal endothelial patch, etc.
  • the purpose of the present invention is to provide an artificial corneal endothelial sheet treated with increased adhesion and its preparation method and application.
  • the modified artificial endothelial sheet has better adhesion to the posterior corneal stroma.
  • the artificial corneal endothelial sheet provided by the inventor is transparent, extremely soft, foldable, has good compatibility with aqueous humor, and has no toxic or side effects.
  • One aspect of the present invention provides a highly adhesive artificial corneal endothelial sheet, which is characterized in that, after the artificial corneal endothelial sheet has been treated to increase adhesion, a material with an adhesion-increasing function is attached to its surface;
  • the adhesion treatment is through the epoxy-amine ring-opening reaction of the polyamino polymer and glycidyl methacrylate to obtain the polyamino polymer functionalized with methacrylate, and then photoinitiated free radical polymerization Reaction to perform surface modification of artificial endothelial sheets.
  • the adhesion-increasing treatment includes the following steps:
  • Step 1) Put the material with the function of increasing adhesion into distilled water and stir until dissolved to form a solution with a concentration of 0.08-0.14g/mL;
  • Step 2 Add 1.0-2.0 mL glycidyl methacrylate to the solution in step 1);
  • Step 3) Place the mixed solution of Step 2) in an environment of 40-70°C for 4-8 hours;
  • Step 4) Use a 12-14kDa cut-flow dialysis tube to dialyze in distilled water to remove unreacted glycidyl methacrylate and oligomers. The resulting sample is vacuum freeze-dried to obtain a solid white foam;
  • Step 5) Put 100.0-300.0 mg of the solid white foam in step 4) into 2.0-6.0 mL of distilled water, and obtain a mixed solution after complete dissolution;
  • Step 6 Soak the artificial endothelial sheet in the mixed solution of step 5), and add 6.0-10.0 ⁇ L of 10% DMPA solution;
  • Step 7) Place the mixed solution in Step 6) under an ultraviolet lamp with a wavelength of 365 nm and irradiate it for 20-60 minutes. During the irradiation period, the mixed solution is continuously stirred;
  • Step 8) Wash with distilled water to obtain an artificial endothelial sheet treated to increase adhesion.
  • the mass ratio of the material with the function of increasing adhesion, distilled water, and glycidyl methacrylate in step 1) and step 2) is (4-7): 50: (1-2).
  • the material with the function of increasing adhesion is a polyamino polymer, selected from one or more of gelatin, chitosan, and serum albumin.
  • the artificial corneal endothelial sheet is made of an acrylic material, selected from the group consisting of hydroxyethyl methacrylate/methyl methacrylate copolymer, polymethyl methacrylate, polyhydroxyethyl methacrylate, and acrylic acid.
  • acrylic material selected from the group consisting of hydroxyethyl methacrylate/methyl methacrylate copolymer, polymethyl methacrylate, polyhydroxyethyl methacrylate, and acrylic acid.
  • hydrogel and methacrylic hydrogel One or more of hydrogel and methacrylic hydrogel.
  • the light transmittance of the artificial corneal endothelium sheet is 79%-85% (400nm-800nm).
  • Another aspect of the present invention provides a method for increasing the adhesion of an artificial corneal endothelial sheet, which is characterized by comprising the following steps:
  • Step 1) Put the material with the function of increasing adhesion into distilled water and stir until dissolved to form a solution with a concentration of 0.08-0.14g/mL;
  • Step 2 Add 1.0-2.0 mL glycidyl methacrylate to the solution in step 1);
  • Step 3) Place the mixed solution in step 2) in an environment of 40-70°C for 4-8 hours;
  • Step 4) Use a 12-14kDa cut-flow dialysis tube to dialyze in distilled water to remove unreacted glycidyl methacrylate and oligomers. The resulting sample is vacuum freeze-dried to obtain a solid white foam;
  • Step 5) Put 100.0-300.0 mg of the solid white foam in step 4) into 2.0-6.0 mL of distilled water, and obtain a mixed solution after complete dissolution;
  • Step 6) Soak the artificial endothelial sheet in the mixed solution of step 5) for 1-2 hours, and add 6.0-10.0 ⁇ L of 10% DMPA solution;
  • Step 7) Place the mixed solution in Step 6) under an ultraviolet lamp with a wavelength of 365 nm and irradiate it for 20-60 minutes. During the irradiation period, the mixed solution is continuously stirred;
  • Step 8) Wash with distilled water to obtain an artificial endothelial sheet treated to increase adhesion.
  • the mass ratio of the material with the function of increasing adhesion, distilled water, and glycidyl methacrylate in step 1) and step 2) is (4-7): 50: (1-2).
  • the material with the function of increasing adhesion is a polyamino polymer, selected from one or more of gelatin, chitosan, and serum albumin;
  • the material of the artificial corneal endothelial sheet is Acrylate material, selected from the group consisting of hydroxyethyl methacrylate/methyl methacrylate copolymer, polymethyl methacrylate, polyhydroxyethyl methacrylate, acrylic hydrogel, and methacrylic hydrogel.
  • Acrylate material selected from the group consisting of hydroxyethyl methacrylate/methyl methacrylate copolymer, polymethyl methacrylate, polyhydroxyethyl methacrylate, acrylic hydrogel, and methacrylic hydrogel.
  • hydroxyethyl methacrylate/methyl methacrylate copolymer selected from the group consisting of hydroxyethyl methacrylate/methyl methacrylate copolymer, polymethyl methacrylate, polyhydroxyethyl methacrylate, acrylic hydrogel
  • Another aspect of the present invention provides the above-mentioned artificial corneal endothelial sheet and/or a method for increasing the adhesion of the artificial corneal endothelial sheet for use in alleviating or treating corneal endothelial damage, corneal endothelial cell dysfunction, and corneal endothelial cell dysfunction. Compensatory medical device applications.
  • Another aspect of the present invention provides the above-mentioned artificial corneal endothelial sheet and/or a method for increasing the adhesion of the artificial corneal endothelial sheet for use in alleviating or treating patients with corneal endothelial cell decompensation, abnormal corneal thickness, and corneal thickness abnormalities.
  • Application in medical devices such as decreased transparency, corneal edema, vision loss or dryness, eye dryness, and pain.
  • the medical device is a graft, a patch or a device box.
  • the artificial corneal endothelial sheet provided by the invention is a transparent, non-degradable optical material that uses polyamino polymers with small antigenicity, such as gelatin, chitosan, and serum albumin, and is combined with methyl
  • the epoxy-amine ring-opening reaction of glycidyl acrylate prepares methacrylate-modified polyamino polymers.
  • the modified product generates a layer of hydrogel on the surface of the acrylic artificial endothelial sheet through light-initiated free radical polymerization, which can It increases the adhesion between the artificial endothelial sheet and the posterior corneal stroma. It has the characteristics of strong adhesion, good biocompatibility, and no toxicity.
  • the artificial corneal endothelial sheet is not easy to fall off after implantation; the artificial endothelial sheet is extremely soft and easy to fold. It can be implanted into the anterior chamber through a small incision without the need to load cells. There is no need to consider cell destruction during the operation, which greatly reduces the difficulty of the operation, thereby reducing excessive loss of corneal endothelial cells and corneal graft detachment after surgery. complication.
  • the corneal implant provided by the present invention is expected to replace the traditional donor corneal endothelial graft and reduce the number of corneal transplants.
  • Figure 1 shows the appearance of the artificial corneal endothelial sheet in Example 1.
  • a and B show that the artificial corneal endothelial sheet is transparent and has curvature;
  • C is after the operation of experimental group 1 in Example 4, showing that the anterior chamber is filled with air bubbles and the artificial corneal endothelial sheet reign.
  • Figure 2 shows the light transmittance of the adhesion-increasing artificial corneal endothelial sheet prepared in Examples 1-3 and the normal rabbit cornea, indicating that their transparency is similar.
  • Figure 3 is a slit lamp and OCT observation picture of the rabbit cornea after operation in the blank group, the control group, the experimental group 1, the experimental group 2, and the experimental group 3 in Example 4, in which the experimental group 1, the experimental group 2, and the experimental group 3 represent respectively Animal experiment results corresponding to the corresponding modification methods in Examples 1-3.
  • Figure 4 is a HE staining picture of the cornea in Example 4, where A is a HE staining picture of a normal cornea; B is a HE staining picture of the central cornea 2 weeks after implantation of the endothelial sheet treated with increased adhesion in Experimental Group 1 in Example 4 The figure shows that there is no obvious inflammatory reaction on the endothelial surface.
  • the present invention has no special restrictions on the source of the artificial corneal endothelial sheet, and any source of artificial corneal endothelial sheet well known in the art can be used.
  • the artificial corneal endothelial sheet is manufactured by the affiliated Ophthalmology Research Institute of Shandong First Medical University.
  • the material of the artificial corneal endothelial sheet is acrylic, which can be selected from hydroxyethyl methacrylate/methyl methacrylate copolymer, polymethyl methacrylate, polyhydroxyethyl methacrylate, acrylic hydrogel, One or more of methacrylic hydrogels.
  • Artificial corneal endothelial sheet diameter 5.0-7.0mm, thickness 25.0-70.0 ⁇ m, radius of curvature 6.0-9.0mm.
  • the light transmittance of the artificial corneal endothelium sheet gradually increases with the increase of light wavelength in the wavelength range of 300-800nm, and can reach 79%-85% in the wavelength range above 400nm.
  • Example 1 Preparation of gelatin-modified artificial corneal endothelial sheet with increased adhesion
  • step (4) Place the centrifuge tube containing the mixed solution obtained in step (4) in a 50°C water bath for reaction for 6 hours;
  • step (6) Vacuum freeze-dry the sample obtained in step (6) for 72 hours, and store the obtained solid white foam at 4°C until use again;
  • step (12) Add a magnetic rotor to the above mixed solution, and under the action of the magnetic stirrer, place the flask with the solution and artificial endothelial sheet in step (12) under a UV lamp with a wavelength of 365nm and irradiate it for 30 minutes;
  • the light transmittance of the gelatin-modified artificial endothelial sheet was tested using a UV spectrophotometer. The results are shown in Figure 2.
  • the light transmittance of the prepared gelatin-modified artificial corneal endothelial sheet was similar to that of the normal rabbit cornea, indicating that the transparency of the two is similar. .
  • Example 2 Preparation of chitosan-modified artificial corneal endothelial sheet with increased adhesion
  • step (4) Place the centrifuge tube containing the mixed solution obtained in step (4) in a 50°C water bath for reaction for 5 hours;
  • step (6) Vacuum freeze-dry the sample obtained in step (6) for 72 hours, and store the obtained solid white foam at 4°C until use again;
  • step (12) Add a magnetic rotor to the above mixed solution, and under the action of the magnetic stirrer, place the flask with the solution and artificial endothelial sheet in step (12) under a UV lamp with a wavelength of 365nm and irradiate it for 30 minutes;
  • the light transmittance of the chitosan-modified artificial endothelial sheet was tested using a UV spectrophotometer. The results are shown in Figure 2.
  • the light transmittance of the prepared chitosan-modified artificial corneal endothelial sheet was similar to that of the normal rabbit cornea, indicating that Both have similar transparency.
  • step (4) Place the centrifuge tube containing the mixed solution obtained in step (4) in a 50°C water bath for reaction for 8 hours;
  • step (6) Vacuum freeze-dry the sample obtained in step (6) for 72 hours, and store the obtained solid white foam at 4°C until use again;
  • step (12) Add a magnetic rotor to the above mixed solution, and under the action of the magnetic stirrer, place the flask with the solution and artificial endothelial sheet in step (12) under a UV lamp with a wavelength of 365nm and irradiate it for 30 minutes;
  • the light transmittance of the artificial endothelial sheet modified with serum albumin was tested using a UV spectrophotometer. The results are shown in Figure 2.
  • the light transmittance of the prepared artificial corneal endothelial sheet modified with serum albumin was similar to that of the normal rabbit cornea, indicating that the two Transparency is similar.
  • Blank group The endothelium in the central area of the rabbit cornea was removed, and no artificial corneal endothelium was implanted.
  • Control group The endothelium in the central area of the rabbit cornea was removed, and an artificial corneal endothelial sheet without any adhesion-increasing treatment was implanted (the diameter of the endothelial sheet was 6.5 mm, the thickness was 50.0 ⁇ m, and the radius of curvature was 7.32 mm).
  • Experimental group 1 The endothelium of the central area of the rabbit cornea was removed, and the gelatin-modified artificial corneal endothelial sheet of Example 1 was implanted (the diameter of the endothelial sheet was 6.5 mm, the thickness was 50.0 ⁇ m, and the radius of curvature was 7.32 mm).
  • Experimental group 2 The endothelium of the central area of the rabbit cornea was removed, and the chitosan-modified artificial corneal endothelial sheet of Example 2 (the diameter of the endothelial sheet was 6.5 mm, the thickness was 50.0 ⁇ m, and the radius of curvature was 7.32 mm) was implanted.
  • Experimental group 3 The endothelium of the central area of the rabbit cornea was removed, and the artificial corneal endothelial sheet modified with serum albumin in Example 3 (the diameter of the endothelial sheet was 6.5 mm, the thickness was 50.0 ⁇ m, and the radius of curvature was 7.32 mm) was implanted.
  • New Zealand white rabbits were anesthetized by intravenous injection of 25.0 mg/kg pentobarbital sodium into the ear margin.
  • the rabbits in the control group and the experimental group were injected with viscoelastic agent again to form the anterior chamber.
  • the control group was implanted with an artificial endothelial sheet without treatment to increase adhesion.
  • the experimental group 1 was implanted with the gelatin-modified artificial endothelium in Example 1.
  • the experimental group 2 was implanted with the chitosan-modified artificial endothelial sheet in Example 2, and the experimental group 3 was implanted with the serum albumin-modified artificial endothelial sheet in Example 3.
  • the corneas of both the experimental group and the control group completely returned to transparency within 1 week after surgery, while the blank group had significant corneal edema and turbidity, proving that the artificial corneal endothelium sheet has the effect of With the barrier function of blocking aqueous humor, after increasing the adhesion treatment, the artificial endothelial patch adheres well to the posterior corneal stroma, reducing the risk of endothelial patch falling off after surgery.
  • anterior segment OCT (see Table 1, Figure 3), the central corneal thickness was measured on the 1st, 7th, and 14th day after surgery.
  • Corneal OCT showed that on the first day after surgery, all rabbits in the control group had artificial endothelial patches falling off (100%). The endothelial patches usually fell off and were located in the lower anterior chamber.
  • the proportion of artificial corneal endothelial patches falling off in the experimental group was as follows.
  • Experimental group 1 was 20 %, the experimental group 2 was 20%, the experimental group 3 was 40%, the corneal thickness of both the experimental group and the control group returned to normal thickness within 1 week after surgery, the average corneal thickness of experimental group 1 was (223 ⁇ 16) ⁇ m, The average corneal thickness of experimental group 2 was (278 ⁇ 44) ⁇ m, the average corneal thickness of experimental group 3 was (242 ⁇ 19) ⁇ m, the average corneal thickness of the control group was (227 ⁇ 13) ⁇ m, and the cornea of the blank group 1 week after surgery The average thickness is about (1643 ⁇ 50) ⁇ m. Moreover, the experimental group and the control group could maintain corneal transparency for 14 days after surgery. It is proved that the artificial corneal endothelial sheet treated with increased adhesion adheres well to the corneal stroma and can act as a barrier to aqueous humor.
  • a is compared with the blank group, P ⁇ 0.05; compared with the experimental group and the control group, there is no statistical difference between the two.
  • A is the HE staining picture of the normal rabbit cornea
  • B is the HE staining picture of the central cornea 14 days after endothelial sheet implantation in Experimental Group 1 of Example 4, indicating that there is no obvious inflammatory reaction on the endothelial surface. , the implant has good biocompatibility.

Abstract

一种经增加粘附性处理的角膜植入物及增加人工角膜内皮片粘附性的方法，通过多氨基高分子聚合物与甲基丙烯酸缩水甘油酯的环氧-胺开环反应得到甲基丙烯酸酯功能化修饰的多氨基高分子聚合物，其进一步通过光引发自由基聚合反应，可提高人工角膜内皮片的粘附性和生物相容性，具有高透氧、高粘附的优势。多氨基高分子聚合物为明胶、壳聚糖和血清白蛋白。所述的角膜植入物用于角膜内皮细胞失代偿患者的治疗，具有隔离房水的屏障作用，消除角膜水肿，降低术后发生角膜植片脱落的风险。

Description

一种高粘附的人工角膜内皮片及其制备方法和应用 技术领域
本发明涉及一种经增加粘附性处理的角膜植入物及增加人工角膜内皮片粘附性的方法，属于医用人工替代材料领域。
背景技术
角膜内皮细胞层是角膜的最内层，其结构完整和功能健全是维持角膜正常生理代谢的重要因素。内皮细胞密度降低和内皮泵功能失调会导致角膜内皮失代偿，表现为角膜水肿混浊，严重影响视力，患者眼疼明显，生活质量下降，是白内障手术等内眼手术后较常见的并发症之一，也是角膜内皮营养不良等原发疾病的终末期表现。最初对于角膜内皮失代偿的治疗，只能采取穿透性角膜移植术(Penetrating keratoplasty，PKP)手术治疗。随着手术技术的发展，内皮移植术(Endothelial keratoplasty,EK)的占比逐渐上升，即保留患者自身的角膜基质部分，而仅移植病变的内皮细胞层，术式为角膜后弹力层剥除内皮移植术(Descemet stripping endothelial keratoplasty，DSEK)或角膜后弹力层内皮移植术(Descemet’s membrane endothelial keratoplasty，DMEK)，但不仅需要新鲜角膜供体，并且手术操作难度高、对供体质量要求也较高，部分患者可能会因术中角膜内皮细胞丢失过多而再次发生角膜植片内皮失代偿，有35％患者术后发生角膜植片脱落而需要进行一次或多次前房注气术。
发明内容
人工角膜内皮片，是指贴附于角膜后基质表面，起隔离房水屏障作用的人工材料，也称为角膜植入物、角膜内皮贴片等。
基于大量的临床研究，本发明的目的是提供一种经增加粘附性处理的人工角膜内皮片及其制备方法和应用，经修饰后的人工内皮片与角膜后基质有更好的粘附性。发明人提供的人工角膜内皮片透明、极软、可折叠，与房水之间的有良好的相容性，且无任何毒副作用。
本发明的一方面，提供了一种高粘附的人工角膜内皮片，其特征在于，所述人工 角膜内皮片经增加粘附性处理后表面附着有增加粘附性功能的材料；所述增加粘附性处理是通过多氨基高分子聚合物与甲基丙烯酸缩水甘油酯的环氧-胺开环反应得到甲基丙烯酸酯功能化修饰的多氨基高分子聚合物，并通过光引发自由基聚合反应进行人工内皮片的表面修饰。
优选地，所述增加粘附性处理包括以下步骤：
步骤1)将所述有增加粘附性功能的材料放入蒸馏水中，搅拌至溶解，形成浓度为0.08-0.14g/mL的溶液；
步骤2)向步骤1)的溶液中加入1.0-2.0mL甲基丙烯酸缩水甘油酯；
步骤3)将步骤2)的混合溶液放置于40-70℃环境下反应4-8h；
步骤4)使用12-14kDa断流透析管在蒸馏水中透析，除去未反应的甲基丙烯酸缩水甘油酯和寡聚物，所得样品真空冻干后得到固体白色泡沫；
步骤5)将100.0-300.0mg步骤4)的固体白色泡沫放入2.0-6.0mL蒸馏水中，完全溶解后获得混合液；
步骤6)将人工内皮片置于步骤5)的混合液中浸泡，加入6.0-10.0μL 10％DMPA溶液；
步骤7)将步骤6)的混合液置于波长为365nm的紫外灯下照射20-60min，照射期间不断搅拌混合液；
步骤8)用蒸馏水洗涤后得到经增加粘附性处理的人工内皮片。
优选地，步骤1)和步骤2)所述有增加粘附性功能的材料、蒸馏水、甲基丙烯酸缩水甘油酯的质量比为(4-7)：50：(1-2)。
优选地，所述有增加粘附性功能的材料为多氨基的高分子聚合物，选自明胶、壳聚糖、血清白蛋白中的一种或多种。
优选地，所述人工角膜内皮片的材质为丙烯酸酯类材料，选自甲基丙烯酸羟乙酯/甲基丙烯酸甲酯共聚物、聚甲基丙烯酸甲酯、聚甲基丙烯酸羟乙酯、丙烯酸水凝胶、甲基丙烯酸水凝胶的一种或多种。
优选地，所述人工角膜内皮片的透光率为79％-85％(400nm-800nm)。
本发明的另一方面，提供了一种增加人工角膜内皮片粘附性的方法，其特征在于，包括以下步骤：
步骤1)将所述有增加粘附性功能的材料放入蒸馏水中，搅拌至溶解，形成浓度为0.08-0.14g/mL的溶液；
步骤2)向步骤1)的溶液中加入1.0-2.0mL甲基丙烯酸缩水甘油酯；
步骤3)将步骤2)中的混合溶液放置于40-70℃环境下反应4-8h；
步骤4)使用12-14kDa断流透析管在蒸馏水中透析，除去未反应的甲基丙烯酸缩水甘油酯和寡聚物，所得样品真空冻干后得到固体白色泡沫；
步骤5)将100.0-300.0mg步骤4)的固体白色泡沫放入2.0-6.0mL蒸馏水中，完全溶解后获得混合液；
步骤6)将人工内皮片置于步骤5)的混合液中浸泡1-2h，加入6.0-10.0μL10％DMPA溶液；
步骤7)将步骤6)的混合液置于波长为365nm的紫外灯下照射20-60min，照射期间不断搅拌混合液；
步骤8)用蒸馏水洗涤后得到经增加粘附性处理的人工内皮片。
优选地，步骤1)和步骤2)所述有增加粘附性功能的材料、蒸馏水、甲基丙烯酸缩水甘油酯的质量比为(4-7)：50：(1-2)。
优选地，所述有增加粘附性功能的材料为多氨基的高分子聚合物，选自明胶、壳聚糖、血清白蛋白中的一种或多种；所述人工角膜内皮片的材质为丙烯酸酯类材料，选自甲基丙烯酸羟乙酯/甲基丙烯酸甲酯共聚物、聚甲基丙烯酸甲酯、聚甲基丙烯酸羟乙酯、丙烯酸水凝胶、甲基丙烯酸水凝胶的一种或多种。
本发明的另一方面，提供了上述人工角膜内皮片和/或如增加人工角膜内皮片粘附性的方法在制备用于缓解或治疗角膜内皮损伤、角膜内皮细胞功能紊乱、角膜内皮细胞功能失代偿的的医疗器械中的应用。
本发明的另一方面，提供了上述人工角膜内皮片和/或如增加人工角膜内皮片粘附性的方法在制备用于缓解或治疗患有角膜内皮细胞功能失代偿患者角膜厚度异常、角膜透明度下降、角膜水肿、视力下降或丧失、眼睛干涩、疼痛的医疗器械中的应用。
优选地，所述医疗器械为植片、贴片或器械盒。
有益效果
本发明提供的人工角膜内皮片，是一种透明的、不降解的光学材料，使用具有较小抗原性的多氨基高分子聚合物，如明胶、壳聚糖、血清白蛋白，通过与甲基丙烯酸缩水甘油酯的环氧-胺开环反应制备甲基丙烯酸酯修饰多氨基高分子聚合物，修饰产物通过光引发自由基聚合反应在丙烯酸酯类人工内皮片表面生成一层水凝胶，能够增加 人工内皮片与角膜后基质的粘附，具有粘附性强、生物相容性好、无任何毒性的特点，植入后人工角膜内皮片不易脱落；该人工内皮片极软且易折叠，可在小切口的情况下植入前房，且无需负载细胞，在手术过程中无需考虑细胞破坏的问题，大大降低了手术难度，从而减少术后角膜内皮细胞丢失过多、角膜植片脱落等并发症。
本发明提供的角膜植入物有望替代传统的供体角膜内皮植片，减少角膜移植的数量。
附图说明
图1为实施例1人工角膜内皮片外观图，其中A、B可见人工角膜内皮片透明、有曲率；C为实施例4实验组1手术结束后，显示前房内充满气泡，人工角膜内皮片在位。
图2为实施例1-3制备的增加粘附性处理的人工角膜内皮片与正常兔角膜的透光率，说明它们透明度相似。
图3为实施例4中空白组、对照组、实验组1、实验组2、实验组3术后兔角膜的裂隙灯及OCT观察图，其中实验组1、实验组2、实验组3分别代表实施例1-3相应修饰方法对应的动物实验结果。
图4为实施例4角膜HE染色图，其中A为正常角膜的HE染色图；B为实施例4中实验组1增加粘附性处理的内皮片植入术后2周，中央角膜的HE染色图，说明内皮面未见明显炎症反应。
具体实施方式
本发明进一步由解释本发明的以下实施例进行示例性说明，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。除非另有说明，本文所使用的技术和科学术语为本发明所属领域的普通技术人员通常所理解的含义。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
本发明对人工角膜内皮片的来源没有特殊限制，采用本领域所熟知的人工角膜内皮片来源即可。在本发明实施例中，所述人工角膜内皮片由山东第一医科大学附属眼科研究所自制。
人工角膜内皮片的相关参数：
1.人工角膜内皮片材料为丙烯酸酯类，可以选自甲基丙烯酸羟乙酯/甲基丙烯酸甲酯共聚物、聚甲基丙烯酸甲酯、聚甲基丙烯酸羟乙酯、丙烯酸水凝胶、甲基丙烯酸水凝胶中的一种或多种。
2.人工角膜内皮片：直径为5.0-7.0mm，厚度为25.0-70.0μm，曲率半径为6.0-9.0mm。
3.透光率：人工角膜内皮片透光率在300-800nm波长范围内均随着光波波长的增加而逐渐升高，在400nm以上波长范围内透光率可达到79％-85％。
实施例1：制备增加粘附性的明胶修饰的人工角膜内皮片
(1)向离心管中加入5.0g A型猪皮明胶；
(2)向步骤(1)的离心管中加入45.0mL蒸馏水；
(3)将上述混合物在50℃下搅拌至完全溶解，形成均匀明胶溶液；
(4)向步骤(3)的离心管中加入1.0mL甲基丙烯酸缩水甘油酯；
(5)将步骤(4)中所得到的盛有混合溶液的离心管放置于50℃水浴锅中反应6h；
(6)使用12-14kDa的断流透析管在大量蒸馏水中透析5天，除去未反应的甲基丙烯酸缩水甘油酯和寡聚物；
(7)将步骤(6)所得样品真空冻干72小时，得到的固体白色泡沫保存在4℃环境下，待再次使用；
(8)向离心管中加入200.0mg步骤(7)的固体白色泡沫；
(9)向步骤(8)的离心管中加入4.0mL蒸馏水；
(10)将上述离心管放置于50℃水浴锅中10h，直至完全溶解；
(11)将人工内皮片置于上述溶液，于烧瓶中浸泡1h；
(12)向上述混合溶液中加入8.0μL 10％DMPA溶液；
(13)向上述混合溶液中加入磁转子，在磁力搅拌器的作用下，将步骤(12)中带有溶液和人工内皮片的烧瓶置于波长为365nm的紫外灯下照射30min；
(14)用蒸馏水洗涤至少6次，得到经明胶化修饰的人工内皮片。
用紫外分光光度计测试经明胶修饰的人工内皮片的透光率，结果如图2所示，制 备的经明胶修饰的人工角膜内皮片与正常兔角膜的透光率相似，说明两者透明度相似。
实施例2：制备增加粘附性的壳聚糖修饰的人工角膜内皮片
(1)向离心管中加入4.5g壳聚糖；
(2)向步骤(1)的离心管中加入50.0mL蒸馏水；
(3)将上述混合物在50℃下搅拌至完全溶解，形成均匀壳聚糖溶液；
(4)向步骤(3)的离心管中加入1.0mL甲基丙烯酸缩水甘油酯；
(5)将步骤(4)中所得到的盛有混合溶液的离心管放置于50℃水浴锅中反应5h；
(6)使用12-14kDa的断流透析管在大量蒸馏水中透析5天，除去未反应的甲基丙烯酸缩水甘油酯和寡聚物；
(7)将步骤(6)所得样品真空冻干72小时，得到的固体白色泡沫保存在4℃环境下，待再次使用；
(8)向离心管中加入200.0mg步骤(7)的固体白色泡沫；
(9)向步骤(8)的离心管中加入4.5mL蒸馏水；
(10)将上述离心管放置于50℃水浴锅中10h，直至完全溶解；
(11)将人工内皮片置于上述溶液，于烧瓶中浸泡1h；
(12)向上述混合溶液中加入8.0μL 10％DMPA溶液；
(13)向上述混合溶液中加入磁转子，在磁力搅拌器的作用下，将步骤(12)中带有溶液和人工内皮片的烧瓶置于波长为365nm的紫外灯下照射30min；
(14)用蒸馏水洗涤至少6次，得到经明胶化修饰的人工内皮片。
用紫外分光光度计测试经壳聚糖修饰的人工内皮片的透光率，结果如图2所示，制备的经壳聚糖修饰的人工角膜内皮片与正常兔角膜的透光率相似，说明两者透明度相似。
实施例3：制备增加粘附性的血清白蛋白修饰的人工角膜内皮片
(1)向离心管中加入6.0g血清白蛋白；
(2)向步骤(1)的离心管中加入40.0mL蒸馏水；
(3)将上述混合物在50℃下搅拌至完全溶解，形成均匀白蛋白溶液；
(4)向步骤(3)的离心管中加入1.5mL甲基丙烯酸缩水甘油酯；
(5)将步骤(4)中所得到的盛有混合溶液的离心管放置于50℃水浴锅中反应8h；
(6)使用12-14kDa的断流透析管在大量蒸馏水中透析5天，除去未反应的甲基丙烯酸缩水甘油酯和寡聚物；
(7)将步骤(6)所得样品真空冻干72小时，得到的固体白色泡沫保存在4℃环境下，待再次使用；
(8)向离心管中加入250.0mg步骤(7)的固体白色泡沫；
(9)向步骤(8)的离心管中加入4.0mL蒸馏水；
(10)将上述离心管放置于50℃水浴锅中10h，直至完全溶解；
(11)将人工内皮片置于上述溶液，于烧瓶中浸泡2h；
(12)向上述混合溶液中加入10.0μL 10％DMPA溶液；
(13)向上述混合溶液中加入磁转子，在磁力搅拌器的作用下，将步骤(12)中带有溶液和人工内皮片的烧瓶置于波长为365nm的紫外灯下照射30min；
(14)用蒸馏水洗涤至少6次，得到经明胶化修饰的人工内皮片。
用紫外分光光度计测试经血清白蛋白修饰的人工内皮片的透光率，结果如图2所示，制备的经血清白蛋白修饰的人工角膜内皮片与正常兔角膜的透光率相似，说明两者透明度相似。
实施例4：动物实验
1.材料与方法
1.1实验动物
新西兰白兔25只，体重3.0-3.5Kg，雄兔。
1.2分组
将25只新西兰大白兔随机分为5组，每组5只。
(1)空白组：剥除兔角膜中央区内皮，不植入人工角膜内皮片。
(2)对照组：剥除兔角膜中央区内皮，植入无增加粘附性处理的人工角膜内皮片(内皮片直径为6.5mm，厚度为50.0μm，曲率半径为7.32mm)。
(3)实验组1：剥除兔角膜中央区内皮，植入实施例1经明胶修饰的人工角膜内 皮片(内皮片直径为6.5mm，厚度为50.0μm，曲率半径为7.32mm)。
(4)实验组2：剥除兔角膜中央区内皮，植入实施例2经壳聚糖修饰的人工角膜内皮片(内皮片直径为6.5mm，厚度为50.0μm，曲率半径为7.32mm)。
(5)实验组3：剥除兔角膜中央区内皮，植入实施例3经血清白蛋白修饰的人工角膜内皮片(内皮片直径为6.5mm，厚度为50.0μm，曲率半径为7.32mm)。
1.3角膜内皮失代偿动物模型的建立
(1)25.0mg/kg戊巴比妥钠耳缘静脉注射麻醉新西兰白兔。
(2)将0.5％盐酸丙美卡因滴在兔角膜表面局部镇痛。
(3)使用0.9％氯化钠注射液冲洗结膜囊，使用棉签充分清洁眼表。
(4)使用6.5mm的环钻于兔角膜上皮面中央做印记。
(5)使用15°穿刺刀于12点位角膜缘做穿刺口，注入0.02mg/mL卡米可林注射液缩瞳，注入粘弹剂形成前房。
(6)使用1ml注射器针头及晶体调位钩去除角膜中央标记的内皮。
(7)使用0.9％氯化钠注射液冲洗前房，置换粘弹剂。
(8)依次向前房内注入4.0mg/mL庆大霉素注射液及肝素钠注射液，减轻前房炎症反应。
(9)空白组兔子缝合切口，使用0.9％氯化钠注射液形成前房。
(10)对照组及实验组兔子再次注入粘弹剂形成前房，对照组植入未经增加粘附性处理的人工内皮片，实验组1植入实施例1中的经明胶修饰的人工内皮片，实验组2植入实施例2中的经壳聚糖修饰的人工内皮片，实验组3植入实施例3中的经血清白蛋白修饰的人工内皮片。
(11)将人工角膜内皮片折叠，并使用无齿镊将其置入前房，使用调位钩将角膜植入物固定于中央刮除区内皮。
(12)使用0.9％氯化钠注射液再次冲洗前房，置换粘弹剂。
(13)使用10-0缝线缝合切口，并使用胰岛素针头进行前房注气。
2.结果(见表1)
2.1人工角膜内皮片的脱落率
术后第1天，对照组5只兔子术后均发生角膜植片脱落，发生率为100％，实验组1中1只兔子术后角膜植片脱落，发生率为20％，实验组2中1只兔子术后角膜植片 脱落，发生率为20％，实验组3中2只兔子术后角膜植片脱落，发生率为40％。
2.2角膜水肿情况
使用眼科检查用裂隙灯(图3)，在术后第1天、第7天、第14天拍摄眼部大体照片。眼部大体照显示，术后第1天，对照组兔子均发生人工内皮片脱落(100％)，需术后再次进行前房注气，实验组人工角膜内皮片脱落的比例如下，实验组1为20％，实验组2为20％，实验组3为40％，实验组与对照组均在术后1周内角膜完全恢复透明，而空白组角膜水肿混浊显著，证明人工角膜内皮片起到了阻挡房水的屏障作用，经增加粘附性处理后，人工内皮片与角膜后基质贴附良好，减小术后内皮片脱落的风险。
2.3人工角膜内皮片的贴附情况
使用眼前节OCT(见表1、图3)，在术后第1天、第7天、第14天进行角膜中央厚度测量。角膜OCT显示，术后第1天，对照组兔子均发生人工内皮片脱落(100％)，内皮片通常脱落后位于下方前房，实验组人工角膜内皮片脱落的比例如下，实验组1为20％，实验组2为20％，实验组3为40％，，实验组与对照组均在术后1周内角膜厚度恢复至正常厚度，实验组1角膜厚度平均为(223±16)μm，实验组2角膜厚度平均为(278±44)μm，实验组3角膜厚度平均为(242±19)μm，对照组角膜厚度平均为(227±13)μm，而空白组在术后1周角膜厚度平均为(1643±50)μm左右。并且，实验组与对照组在术后14天内可一直维持角膜透明。证明增加粘附性处理后的人工角膜内皮片与角膜基质贴附良好，能够起到阻挡房水的屏障作用。
表1 植入修饰与未修饰人工角膜内皮片的处理及结果
Figure PCTCN2022105454-appb-000001
Figure PCTCN2022105454-appb-000002
a为与空白组比较，P＜0.05；实验组与对照组比较，两者无统计学差异。
2.4人工角膜内皮片的生物相容性
在术后第14天，将新西兰兔安乐死，取兔角膜行组织病理学检查，HE染色。如图4所示，其中A为正常兔角膜的HE染色图；B为实施例4实验组1的内皮片植入术后14天，中央角膜的HE染色图，说明内皮面未见明显炎症反应，植入物具有较好的生物相容性。
尽管本发明的具体实施方式己经得到详细的描述，本领域技术人员将会理解。根据己经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (12)

  1. 一种高粘附的人工角膜内皮片，其特征在于，所述人工角膜内皮片经增加粘附性处理后表面附着有增加粘附性功能的材料；所述增加粘附性处理是通过多氨基高分子聚合物与甲基丙烯酸缩水甘油酯的环氧-胺开环反应得到甲基丙烯酸酯功能化修饰的多氨基高分子聚合物，并通过光引发自由基聚合反应进行人工内皮片的表面修饰。
  2. 如权利要求1所述的高粘附的人工角膜内皮片，其特征在于，所述增加粘附性处理包括以下步骤：
    步骤1)将所述有增加粘附性功能的材料放入蒸馏水中，搅拌至溶解，形成浓度为0.08-0.14g/mL的溶液；
    步骤2)向步骤1)的溶液中加入1.0-2.0mL甲基丙烯酸缩水甘油酯；
    步骤3)将步骤2)的混合溶液放置于40-70℃环境下反应4-8h；
    步骤4)使用12-14kDa断流透析管在蒸馏水中透析，除去未反应的甲基丙烯酸缩水甘油酯和寡聚物，所得样品真空冻干后得到固体白色泡沫；
    步骤5)将100.0-300.0mg步骤4)的固体白色泡沫放入2.0-6.0mL蒸馏水中，完全溶解后获得混合液；
    步骤6)将人工内皮片置于步骤5)的混合液中浸泡，加入6.0-10.0μL 10％DMPA溶液；
    步骤7)将步骤6)的混合液置于波长为365nm的紫外灯下照射20-60min，照射期间不断搅拌混合液；
    步骤8)用蒸馏水洗涤后得到经增加粘附性处理的人工内皮片。
  3. 如权利要求2所述的高粘附的人工角膜内皮片，其特征在于，步骤1)和步骤2)所述有增加粘附性功能的材料、蒸馏水、甲基丙烯酸缩水甘油酯的质量比为(4-7)：50：(1-2)。
  4. 如权利要求1-3任一所述的高粘附的人工角膜内皮片，其特征在于，所述有增加粘附性功能的材料为多氨基的高分子聚合物，选自明胶、壳聚糖、血清白蛋白中的一种或多种。
  5. 如权利要求1-4任一所述的高粘附的人工角膜内皮片，其特征在于，所述人工角膜内皮片的材质为丙烯酸酯类材料，选自甲基丙烯酸羟乙酯/甲基丙烯酸甲酯共聚物、聚甲基丙烯酸甲酯、聚甲基丙烯酸羟乙酯、丙烯酸水凝胶、甲基丙烯酸水凝胶的一种 或多种。
  6. 如权利要求1-5任一所述的高粘附的人工角膜内皮片，其特征在于，所述人工角膜内皮片的透光率为79％-85％(400nm-800nm)。
  7. 一种增加人工角膜内皮片粘附性的方法，其特征在于，包括以下步骤：
    步骤1)将所述有增加粘附性功能的材料放入蒸馏水中，搅拌至溶解，形成浓度为0.08-0.14g/mL的溶液；
    步骤2)向步骤1)的溶液中加入1.0-2.0mL甲基丙烯酸缩水甘油酯；
    步骤3)将步骤2)中的混合溶液放置于40-70℃环境下反应4-8h；
    步骤4)使用12-14kDa断流透析管在蒸馏水中透析，除去未反应的甲基丙烯酸缩水甘油酯和寡聚物，所得样品真空冻干后得到固体白色泡沫；
    步骤5)将100.0-300.0mg步骤4)的固体白色泡沫放入2.0-6.0mL蒸馏水中，完全溶解后获得混合液；
    步骤6)将人工内皮片置于步骤5)的混合液中浸泡1-2h，加入6.0-10.0μL 10％DMPA溶液；
    步骤7)将步骤6)的混合液置于波长为365nm的紫外灯下照射20-60min，照射期间不断搅拌混合液；
    步骤8)用蒸馏水洗涤后得到经增加粘附性处理的人工内皮片。
  8. 如权利要求7所述的增加人工角膜内皮片粘附性的方法，其特征在于，步骤1)和步骤2)所述有增加粘附性功能的材料、蒸馏水、甲基丙烯酸缩水甘油酯的质量比为4-7：50：1-2。
  9. 如权利要求7-8所述的增加人工角膜内皮片粘附性的方法，其特征在于，所述有增加粘附性功能的材料为多氨基的高分子聚合物，选自明胶、壳聚糖、血清白蛋白中的一种或多种；所述人工角膜内皮片的材质为丙烯酸酯类材料，选自甲基丙烯酸羟乙酯/甲基丙烯酸甲酯共聚物、聚甲基丙烯酸甲酯、聚甲基丙烯酸羟乙酯、丙烯酸水凝胶、甲基丙烯酸水凝胶的一种或多种。
  10. 如权利要求1-6任一所述的人工角膜内皮片或如权利要求7-9所述的增加人工角膜内皮片粘附性的方法在制备用于缓解或治疗角膜内皮损伤、角膜内皮细胞功能紊乱、角膜内皮细胞功能失代偿的的医疗器械中的应用。
  11. 如权利要求1-6任一所述的人工角膜内皮片或如权利要求7-9所述的增加 人工角膜内皮片粘附性的方法在制备用于缓解或治疗患有角膜内皮细胞功能失代偿患者角膜厚度异常、角膜透明度下降、角膜水肿、视力下降或丧失、眼睛干涩、疼痛的医疗器械中的应用。
  12. 如权利要求10-11所述的应用，其特征在于，所述医疗器械为植片、贴片或器械盒。
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