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WO1994017851A1 - Hydrogels composites a deux couches pour protheses corneennes - Google Patents

Hydrogels composites a deux couches pour protheses corneennes Download PDF

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Publication number
WO1994017851A1
WO1994017851A1 PCT/US1994/001419 US9401419W WO9417851A1 WO 1994017851 A1 WO1994017851 A1 WO 1994017851A1 US 9401419 W US9401419 W US 9401419W WO 9417851 A1 WO9417851 A1 WO 9417851A1
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WIPO (PCT)
Prior art keywords
hydrogel
collagen
collagen material
keratoprosthesis
corneal
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PCT/US1994/001419
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English (en)
Inventor
Edward Perez
David Miller
Edward W. Merrill
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Massachusetts Institute Of Technology
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Publication of WO1994017851A1 publication Critical patent/WO1994017851A1/fr

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Classifications

    • 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 or corneal implants; Artificial eyes
    • A61F2/142—Cornea, e.g. artificial corneae, keratoprostheses or corneal implants for repair of defective corneal tissue
    • 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
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—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
    • 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 present invention is generally in the field of ocular materials and in particular is an improved hydrogel material for engineering of comeal surfaces.
  • the major anatomic divisions of the eye are the sclera, uveal tract, retina, vitreous, lens, anterior chamber, and the cornea.
  • the clear cornea and gray- white opaque sclera are fused together and completely enclose the other portions of the eye. Under the influence of intraocular pressure, the cornea and sclera form a semi-rigid spherical organ.
  • the cornea occupies the anterior pole of what is referred to as the globe. In adults, the cornea measures 12 mm in the horizontal meridian and 11 mm in the vertical meridian. The central one-third of the cornea is nearly spherical and measures 4 mm in diameter. The cornea is thinner (0.5 mm) in the center than in the periphery (1.0 mm). Histologically, the cornea is composed of five layers: epithelium, Bowman's membrane, stroma, Descemet's membrane, and endothelium. The most metabolically active layers are the limiting layers, as described by Zuraws i CA, McCarey BE, and Schmidt: Glucose Consumption in Cultured Comeal Cells.
  • Keratoprostheses are generally indicated in cases of chemical burns, ocular pemphigoid (severe dry eyes) , heavily vascularized corneas, and in cases of multiple graft failures.
  • a viable prosthetic alternative to comeal transplants is also needed for treatment of severely intractable cornea cases as well as for cases in underdeveloped countries where human comeal tissue is unavailable due to poor donor supply and storage conditions.
  • the appearance of AIDS and other tissue-borne diseases has caused increased scrutiny and mistrust of donor tissue even in developed countries.
  • the first issue is the optical function and the second is the anatomic barrier function of the cornea.
  • Use of synthetic materials in comeal surgery has existed, at least theoretically, since 1771 when Pellier de Quengsy conceived of implanting transparent material in the scarred cornea.
  • glass buttons were implanted into the cornea to increase the clarity of scarred corneas.
  • Modern devices have been designed as optically clear poly (methyl methacrylate) cylinders that penetrate the cornea and are anchored by a collar, as reported by Cardona H: Keratoprosthesis. Ophthalmology (1976) PP 284-299.
  • cornea including one or both of the limiting layers (epithelium or endothelium) is still addressed with materials such as poly (methyl methacrylate) or silicone membranes.
  • materials such as poly (methyl methacrylate) or silicone membranes.
  • These currently used keratoprostheses have relatively short lifetimes of between a few weeks and a few months as a result of no continuous epithelial boundary, tissue death around the implant (sterile necrosis) , secondary infection (endophthalmitis) , and peri-implant membrane formation.
  • Another approach to this problem attempts to synthesize a material whose intrinsic surface is one conducive to epithelial cell growth.
  • the synthesis of these materials utilizes crosslinking methods to form hydrogels from the collagen types (I, described by Geggel HS, Friend J, and Throft RA: Collagen Gels for Ocular Surface. 1985 Reports Invest. Qphthal. Vis. Sci. 26: 901-905, and IV, described by Thompson et al. Synthetic Collagen IV lenticules as a biomaterial for epikeratoplasty. 1990 ARVO Absts. Invest. Qphthal. Vis. Sci. 31(suppl), 301.
  • materials like these have been found to be susceptible to proteolysis in vivo .
  • Rao KP and Joseph KT Collagen Graft Copolymers and Their Biomedical Applications in Collagen, Vol 3, Nimni, M.E., ed. (CRC press, Boca Raton Florida 1988) pp 63-85, and Franzblau C et al: Cell Growth on Collagen-HEMA Hydrogels. id. pp 191-208, have synthesized collagen- HEMA copoly ers to grow epithelial cells. These materials were also found to be susceptible to proteolysis and were brittle.
  • Refractive errors are presently corrected with the use of eyeglasses or contact lenses.
  • Another major application of synthetics in corneal surgery is the rising interest in refractive surgery, as reviewed by Thompson KP: Current Status of Synthetic Epikeratoplasty. Refractive and Corneal Surgery 7:240- 248.
  • a refractive prosthesis could affect the curvature of the corneal surface and influence the cornea's ability to focus an image on the retina thereby circumventing the need for glasses.
  • epikeratophakic grafts Implanting material onto the cornea is receiving much attention as a means of correcting refractive errors. These superficial corneal augmentations have been termed epikeratophakic grafts. Originally, epikeratophakic grafts were made from donor corneal tissue. These were not optimal, however, because of difficulty in manufacture, requiring two to three weeks post-operatively to regain clarity, and susceptibility to normal tissue remodeling processes. Synthetic epikeratophakic grafts offer ease in manufacturing and stability in clarity and geometric form. However, in the effort to develop usable synthetic epikeratophakic grafts, investigators have identified difficulties with the synthetic materials as a result of the lack of normal regeneration of the epithelial cell layer on these materials. As a result, the development of a synthetic graft with a suitable surface for regeneration of an epithelial cell layer, which maintains the desired optical and structural properties, is required to make these devices clinically useful.
  • Hydrogel materials have received much attention in relation to their applicability as prosthetic corneal devices, as reported by Trinkhaus-Randall V et al: The Development of a Biopolymeric Keratoprosthetic Material. Invest. Qphthal. Vis. Sci. Vol. 29, No. 3 (1988) pp. 393-400; Sipehia R, et al. Towards an Artificial Cornea. Biomat.. Art Cell. Art Organs 18(5) pp. 643- 655 (1990) ; and Thompson KP: Current Status of Synthetic Epikeratoplasty. Refractive Corneal Sur ⁇ . Vol. 7 (1991) pp. 240-248, because of the properties of hydrogels, including clarity, flexibility, and ability to allow diffusive flow of nutrients.
  • a penetrating corneal prosthesis must be fully enclosed in the cornea with a contiguous epithelium; i.e., contiguous epithelium must develop between the implant and host via colonization of the implant surface by epithelial cells which migrate from the host cornea.
  • a hydrogel material must be constructed possessing a surface environment conducive to corneal epithelial cell growth in addition to maintaining other desirable characteristics of hydrogels. This has not yet been achieved.
  • a two-layer composite material composed of a thin- layer of corneal tissue or collagen and a hydrogel, preferably formed of an electron-beam crosslinkable polymer such as a synthetic polyethylene oxide (PEO) hydrogel, is described.
  • the material is designed to provide a suitable substrate for corneal epithelial cell growth while mainlining the desirable characteristics of hydrogels, i.e., clarity, flexibility and ability to allow diffusive flow of nutrients.
  • the gels are synthesized via electron irradiation induced crosslinking of an aqueous solution of PEO onto a thin layer of collagenous tissue substrate.
  • the collagenous tissue substrate, or stromal layer is glued to a preformed hydrogel by polymerization of a liquid polymer solution, for example a polyethylene oxide solution that is polymerized by electron beam irradiation.
  • a liquid polymer solution for example a polyethylene oxide solution that is polymerized by electron beam irradiation.
  • Figure 1 is a micrograph of a 15% PEO gel adhered onto corneal tissue using 10 Mrad of electron beam crosslinking.
  • Figure 2 is an SEM micrograph of a 10 ⁇ m layer of cornea adhered onto 15% PEO hydrogel (top view) .
  • Figure 3 is a micrograph of a cross-section of a composite lenticule grown in organ culture prepared by implantation of a composite hydrogel/stroma lenticule into a freshly enucleated rabbit eye which was grown in organ culture.
  • the top layer of cells are confluent basal epithelial cells, overlying stromal tissue, with the hydrogel appearing as a clear portion at the bottom of the micrograph.
  • On the surface of the basal epithelial cells are migrating epithelial cells.
  • a hydrogel composite whose ultimate use will be for the construction of an artificial cornea or epikeratoprostheses has been synthesized.
  • the design rationale is to construct a material possessing a surface environment conducive to epithelial cell growth in addition to possessing the proper optical, diffusive. and mechanical characteristics of the cornea.
  • EII electron-irradiation-induced crosslinking is used to synthesize a hydrogel network and simultaneously attach the polymeric network to a collagenous substrate.
  • Described herein is a material, and methods of use thereof as a synthetic lenticule or epikeratophakic graft, synthesized of a composite material formed of a synthetic hydrogel covalently attached to a thin collagen matrix.
  • a synthetic hydrogel covalently attached directly to the collagen (or stromal) matrix
  • the second where there is an intermediate material which is used to adhere the collagen matrix to the hydrogel.
  • polyethylene oxide (PEO) or hydroxyethylmethacrylate (HEMA) and excised corneal stromal tissue are used to synthesize the hydrogel and matrix surface.
  • a hydrogel is a water swollen polymer, between two weight percent and 60 weight percent per volume of gel. Hydrogels are typically 80 to 90% water, having indices of refraction close 1.3, which is similar to that of water and, therefore the cornea. Mechanically, the hydrogels should be able to support a breaking tensile stress of between 40,000 and 60,000 dynes/cm 2 . This is a relatively weak force.
  • the polymer should be completely transparent to visible light and not absorb in the range of 300 nm to 850 nm. Chemically, the hydrogels should remain stable and not degrade in vivo . Small amounts of degradation product can cause an inflammatory response.
  • hydrogel materials implanted into the center of the cornea have proven to be perfectly compatible and yield no deleterious effects, presumably in part because the cornea is not a vascularized organ and is therefore privileged, or isolated, from the immune system.
  • reactive functional groups on the surface of hydrogels including amines, carboxyls, and hydroxyls, have been associated with inflammation.
  • combining these materials with corneal stroma or collagen appears to make all of the hydrogels biocompatible within the corneal environment.
  • a number of polymers can be used to form the hydrogel, including hydroxyethylmethacrylate, polyethylene oxide, polyvinyl alcohol, polydioxolane, poly(acrylic acid) , poly(aerylamide) , and poly(N-vinyl pyrilidone) . These polymers can be crosslinked by electron beams. These materials are commercially available from Polysciences, Sigma Chemical Co., and Aldrich Chemical.
  • the hydrogel is formed of crosslinked polyethylene oxide or hydroxyethylmethacrylate.
  • Crosslinked PEO has previously been used in the synthesis of hydrogel materials for corneal implantation, as reported by Peiffer RL, Werblin TP, Fryczkowski AW: Pathology of Corneal Hydrogel Alloplastic Implants. Ophthalmology Aug 1985,92(9) p. 1294-1304. These materials have shown negligible tissue reaction and do not adsorb proteins, as reported by Merrill EW, Salzman EW: Polyethylene Oxide as a Biomaterial. Amer. Soc. for Art Int. Organs, Vol. 6, No. 2 April/June (1983) p. 60-64; Merrill EW, et al. : Nonadsorptive Hydrogels for Blood Contact. Progress in Artificial Organs, ISAO Press, Cleveland (1986) pp. 909-912.
  • the polymer solutions to be crosslinked range from 2 to 15 weight per volume.
  • the preferred thickness in combination with corneal tissue or collagen is in the range of between 50 and 100 microns.
  • EII crosslinking is used to crosslink a hydrogel network onto a collagenous matrix substrate. which serves as a substrate for cell growth.
  • EII crosslinking is described by Dole M: The Radiation Chemistry of Macro olecules. Academic Press, new York (1972) and Shultz, AR: Radiation and Crosslinking by Radiation, in Fettes EM (ed) , Chemical Reactions of Polymers. Interscience, New York 1964) , the teachings of which are incorporated herein, and described in more detail in the examples below.
  • Water is the only solvent used for electron beam crosslinking. An advantage of this method is that it can be used to polymerize and sterilize at the same time.
  • hydrogels can be synthesized. Either covalent chemical or physical bonds can be used to adhere the hydrogel to the corneal tissue or collagen. For example, endlinking of polymers with functionalized ends can be used.
  • a hydrogel can also be formed by polymerizing monomer in the presence of crosslinking agents, such as by polymerizing acrylamide and bis acrylamide in the presence of a free radical initiator like ammonium persulfate. Endlinking and polymerizing a network is performed in a non-polar solvent such as hexane.
  • the hydrogel is prepared in a mold shaped to provide an implant with the appropriate surface for placement in the eye and replacement of optical and physiological function, i.e., which maintains the desired geometry of the lens.
  • the mold will typically contain a layer of corneal stroma or collagen which the hydrogel is bonded to.
  • the hydrogel is ultimately placed on the inside of the eye, so that the epithelial cells adjacent to the implant abut the stroma or collagen and can grow over the surface of the implant.
  • the hydrogel will preferably be in a layer of between 50 and 100 microns.
  • the corneal tissue or collagen layer will preferably be in a layer of between 10 and 50 microns.
  • a thin layer of between approximately 10 ⁇ m and 50 microns of corneal tissue is grafted to the surface of a hydrogel to form a suitable surface for cell growth and to impart added mechanical stability to the device.
  • the tissue is preferably obtained fresh, frozen to -20°C cut on a microtome. After the tissue is placed in the mold, the tissue is lyophilized.
  • Corneal tissue can be obtained from the same or different species since xenogeneic tissue exchange has been shown to form viable corneal transplants.
  • Human corneal tissue is obtained from commercial eye banks. Rabbit eyes have also been used as a source of tissue, with up to thirty films being obtained from one rabbit cornea. Bovine eyes can also be used as donor material. As noted above, the material is preferably lyophilized, which further decreases the likelihood of a reaction against the material.
  • the bulk of the cornea is collagen I (70% of dry weight) with the balance being glycosaminoglycans and cellular elements, as reviewed by Maurice D: The Cornea and Sclera. in the Eye, Vol. IB, Davson H, editor. Florida, 1984, Academic Press, pp. 1-158.
  • Collagen type I has been proven to be a suitable substrate for epithelial growth in a variety of circumstances.
  • Collagen type I also contains peptide regions known to be involved in cell-substrate adhesion.
  • Collagen in modified forms is a good substrate for growth of many cell types, as reported by Geggel HS, Friend J, and Throft RA: Collagen Gels for Ocular Surface. Rep. Invest. Qphthal. Vis.
  • the first concern is the conservation of tissue architecture of collagen.
  • the bonding is only by passive adsorption. This is a simpler approach than by chemical bonding and does not attempt to preserve the higher organizational structure of collagen in vivo .
  • Collagen in corneal tissue is organized into a complex structure of fibrils oriented in parallel planes. The collagen in this tissue architecture is not easily manipulated in processing techniques unless it is solubilized, yet its soluble form no longer has the architecture it has in connective tissue.
  • cross-linked collagen type I In addition to corneal collagen, films of less than 20 microns thick cross-linked collagen type I have been used to develop cross-linking strategies and analytical techniques.
  • the electron beam is used to polymerize a polymer layer between the collagen matrix and the hydrogel which thereby covalently crosslinks the hydrogel to the collagen matrix.
  • a number of polymeric materials can be used to attach the hydrogel to the collagen matrix, including any of the materials described above with reference to formation of the hydrogel itself. These materials can be the same as or different from the material forming the hydrogel. Electron-beam irradiation has been used extensively to crosslink polymers onto hydrogels and also to attach polymers onto surfaces, as reported by, for example, Wilson JE: Radiation Chemistry of Monomers, Polymers, and Plastic. Marcel Dekker, Inc., New York, NY (1974) .
  • hydrogels can be synthesized which are covalently bound to fibrillar collagen.
  • This type of crosslinking is achieved by use of a Vandergraf generator, which generates up to a dose of 0.5 Mrads/second.
  • the higher the dosage the greater the amount of crosslinking.
  • the dosage and extent of crosslinking must be limited to avoid damage to the underlying corneal tissue or collagen substrate, which causes, for example, the tissue to become opaque.
  • This crosslinking mechanism is based upon chemical activation of polymer molecules that result in the formation of polymer-polymer crosslinks to form a hydrogel and polymer-collagen, as reported by Pietrucha K, Kroh J: Radiation Crosslinking of Poly(butyl acrylate) During Polymerization and Grafted Copolymerization with Cr(III) Crosslinked Collagen. 1986 Radiation Physics Che . 28(9) ,373-376; and Rao et al. Grafting of Vinyl Monomers onto Modified Collagen by Ceric Ion -Studies on the Grafting Sites. 1969 Leather Sci. 16, crosslinks to unite the collagen and hydrogel.
  • the adherence or bonding between the insoluble collagen substrate and the hydrogel network arise from collagen amino acids crosslinked to synthetic polymer, as expected based on Pietrucha K, Lobis M: Some Reactions of OH Radicals with Collagen and Tyrosine in Aqueous Solutions. 1990 Rad. Physics Chem. 36(2), 155-160.
  • Solution characteristics and irradiation conditions affect the degree of polymer-collagen crosslinking. These factors can be theoretically considered by examining the major reactions, the radical generation reactions and the crosslinking reactions.
  • the radiolysis of water is a fast reaction which creates a concentration of hydroxyl radicals, [OH], that are consumed as fast as they are created.
  • OH hydroxyl radicals
  • These hydroxyl radicals, OH " participate in the creation of macroradicals on polymers in solution. Scission that takes place is not considered since it is first order whereas recombination of macroradicals is second order (bimolecular) .
  • Factors which increase the concentration of macroradical should also increase the amount of crosslinking. Dose rate (Mrad/sec) directly affects the concentration of hydroxyl radicals.
  • Polymer concentration affects the concentration of hydroxyl radicals. Polymer concentration affects the amount of collagen-polymer crosslinking by the increased availability of polymer radicals. Since the amount of collagen is fixed in a given area, only an increase in dose rate affects the concentration of collagen radicals. Since the amount of collagen is fixed in a given area, only an increase in dose rate affects the concentration of collagen radicals. The relative proportion of collagen-polymer to polymer-polymer crosslinks is governed by the ratio of rate constants k pC and k pP and the polymer concentration. Collagen concentration is essentially fixed per a certain area. Formation of and Implantation of Composites These considerations aside, the collagen or corneal tissue substrate is placed in a mold of the appropriate desired shape.
  • corneal tissue layer substrates are prepared by slicing tissue using a cryostat or microtome to a thickness between 10 and 80 microns, at a plane tangential to the surface of the cornea.
  • the layers are placed in a pre-cooled (-20°C) glass mold in the shape of the final product.
  • An aliquot of the polymer solution is placed onto the substrate, then exposed to an electron bean in a dose sufficient to crosslink the solution into a gel so that its final dimensions will be stable.
  • the mold determines the basic shape and dimensions of the final product, this is then shaped in the same manner as other types of keratoprostheses or contact lens, trimming off material as necessary to achieve the desired curve in the cornea.
  • the implant is then placed in a patient's eye using standard techniques for implantation of keratoprosthesis.
  • the eye is topically anesthetized with 4% proparacaine HC1.
  • the cornea is locally anesthetized with 4% proparacaine by subconjunctival injections, and the eye is immobilized with a lid speculum, which simultaneously keeps the eyelids open and the eye fixed, as necessary.
  • a sterile drape is placed around the eye.
  • Absolute ethanol is applied to the central five mm of the cornea to remove the central epithelial layer.
  • the epithelium is further debrided from the central cornea using the blunt side of a scalpel blade.
  • the debrided surface is then irrigated using sterile balanced salt solution (BSS) to remove any other epithelial debris.
  • BSS sterile balanced salt solution
  • a superficial circular incision is made in the central cornea to a depth of 0.1 mm.
  • a lamellar keratotectomy is performed on the central core of the cornea.
  • a circumferential incision is then made using a rounded scalpel blade.
  • the implant is tucked into the annular incision that has been created.
  • the wound edges are sealed either with sutures, or, more preferably, by air-drying for three to five minutes.
  • a pressure patch is then applied to keep the eyelid from moving over the eye surface for at least two days, to allow healing and epithelialization of the implant surface.
  • Example l Synthesis and characterization of PEO hydrogels prepared by EII crosslinking of polyethylene oxide (PEO) to a substrate of fibrillar collagen. Light microscopy and scanning electron microscopy were used to visualize the gel/collagen interface. The materials were characterized by several chemical and spectroscopic techniques to elucidate the nature of the bonding of PEO to a substrate of collagen. Bulk proteinaceous material was removed with oxalic acid hydrolysis so the gel/collagen interface could be exposed. Electron spectroscopy for chemical analysis (ESCA) was used to determine the presence of nitrogen from amino acids on the surface. Fluorescamine amino- acid labelling was used to confirm that the nitrogen present was in amino-acid form. Attenuated Total Reflection Infrared (ATR-IR) spectroscopy was used to determine IR spectral changes indicative of bound amino acids.
  • EII crosslinking of polyethylene oxide (PEO) to a substrate of fibrillar collagen.
  • Light microscopy and scanning electron microscopy were
  • Collagenous substrates were either dried films of bovine collagen I (100 ⁇ m) or bovine corneal tissue (10 ⁇ m) .
  • Collagen films were made by air-drying a 10 mg/ml solution of icrofibrillar collagen (Avitene, MedChem Products, Woburn, MA) placed in a 25 mm polypropylene dish. Later the films were vacuum dried for 24 hours. Sheets of corneal tissue were cut from the anterior half of the adult bovine cornea on a cryostat (Tissue Tek II) set at -22°C.
  • the collagen substrates, from either method, were supported on glass coverslips during electron beam irradiation.
  • PEO hydrogels grafted onto corneal tissue were prepared into 6 ⁇ m sections and stained with hematoxylin and eosin for light microscopy.
  • the samples prepared for SEM were fixed in glutaraldehyde and stained with Os0 4 .
  • the samples were dried in a critical point dryer (AUTOSAMDRI model 814) and visualized in a dual stage scanning electron microscope (model ISI-DS130) .
  • the gel networks with collagen hydrolytically removed were buffered with 2 volumes of 0.2 M sodium borate buffer.
  • One volume of fluorescamine (15 mg/100 ml acetone) was added to the mixture, giving an approximately 20-fold excess versus protein, if 0.6 ⁇ g/cm 2 of protein binds to the hydrogel surface, as reported by Udenfriend S, et al.: Fluorescamine: A Reagent for Assay of Amino Acids, Peptides, Proteins, and Primary Amines. Science Vol. 178, Nov. 24, 1972. Fluorescence of the mixture was measured in a fluorescence spectrophotometer (Perkins Elmer Model 650- 10M) . The instrumentation is designed quantify fluorescence for solutions. The fluorescence of gels suspended in a solution can be tested but the determination of the gel surface amine concentration is not readily ascertained.
  • the samples were prepared by air drying the materials overnight and then vacuum drying for six hours.
  • the material were studied in a Perkins Elmer IR spectrophotometer (Model 1430) utilizing a ZnSe crystal for multiple internal reflectance. The range of study was from 4000 cm" 1 to 700 cm" 1 .
  • the objective of the ESCA and fluorescamine labelling was to verify that stable covalent bonding between the gel and collagen had occurred during EII crosslinking and to characterize the nature of the bonds formed.
  • Aqueous irradiation forms carbon radicals on the amino acid which recombine with other carbon radicals produced on the polymeric network to form PEO collagen crosslinks.
  • the carbon-carbon bonds are very stable but peptide hydrolyzable with acidic media. Only the carbon-carbon bonds remain intact following hydrolysis of these peptide bonds, leaving single amino acids bound to the PEO network via carbon-carbon bonds.
  • Control surfaces were prepared by carrying out the same hydrolysis reaction in the presence of corneal collagen which was simply placed in contact with the gel, but which had not been crosslinked.
  • ESCA was used to investigate the presence of residual amino acids on the surface.
  • Survey scans of materials included those for PEO gels alone and with grafted collagen removed. The survey scan showed the presence of carbon, oxygen, sodium, chloride and nitrogen. The ratio of carbon to oxygen is roughly 2:1, indicative of the high concentration of PEO on the surface. Nitrogen is assumed to arise from the presence of amino acids bound to the surface of the hydrogels. PEO crosslinked without collagen was also exposed to the hydrolysis. After washing, these gels do not exhibit the presence of nitrogen on the surface.
  • Fluorescamine labelling was used to assay for nitrogen present in amino acid form. Primary amine- fluorescamine conjugates fluoresce at 475 nm with excitation at 390 nm. Fluorescence was noted for those collagen grafted gels which were treated hydrolytically. PEO gels not crosslinked to collagen did not fluoresce.
  • the collagen material gave an IR spectrum with characteristic peaks at 1640 cm' 1 and 1540 cm-1 due to Amide I and Amide II bands. This compares identically with published data for collagen, Weetal (1975) .
  • the non-crosslinked linear PEO shows characteristic absorption peaks of C-H vibrations and the distinctive peak at 1080 cm "1 due to the C-0 ether linkages, Vandeberg JT (editor) : An Infrared Spectroscopy Atlas for the Coating Industry. Federation of the Society for Coating Technologies, Philadelphia, PA (1980) .
  • Electron beam crosslinked PEO shows the appearance of carbonyl and carboxyl functionalities at 3080 cm “1 and 1535 cm “1 , Dennison KA: Radiation Crosslinked Poly(ethylene oxide) Hydrogel Membranes. PhD Thesis, MIT (1986) .
  • IR spectra elicited for collagen on PEO gel confirms the presence of collagen on the surface of the gel. After the hydrolytic removal of collagen from the surface of the gels, the IR spectra regressed to a form similar to that of non-grafted PEO gels also treated hydrolytically. The peak at 1085-1075 cm “1 identifies the major presence of PEO on the surface.
  • the significant differences in the infra-red spectrum of pure crosslinked PEO with gels which had collagen hydrolyticaily removed are the increased absorption at the Amide I, Amide II regions, and below 1000 cm" 1 , Silverstein, et al., "Spectrometric Identification of Organic Compound", 4th Ed. John Wiley and Sons, Inc. New York (1981) . There is a higher absorption for gels grafted onto collagen with 10 Mrad doses than those for 7.5 Mrad doses indicating a dose response behavior.
  • the IR spectra also identified the presence of amino-acids with accentuation of bands at 1640 cm “1 and 1540 cm” 1 .
  • the IR spectra identified a higher number of amino-acids covalently bound as a function of crosslinking dose.
  • the stability of covalent bonding further ensures that the thin-layer tissue will remain on the synthetic material truly imparting the corneal tissue biological activity, as reviewed by Brightbill FS, Corneal Surgery: Theory, Tissue and Technique. The C.V. Mosby Company St. Louis, Missouri (1986) , and strength to the implant.
  • collagen can be covalently linked to PEO hydrogels by Ell-crosslinking, and that the bonds are stable toward acid hydrolysis. Furthermore, the collagen retains features of its native architecture and may be a more suitable substrate for corneal epithelial cell growth than other formulations of collagen.
  • Example 2 Synthesis and Physical characterization of hydrogel bonded to collagen by EII.
  • the simultaneous synthesis and chemical attachment of hydrogels onto a collagenous matrix is performed using an electron beam source, a collagen substrate layered onto the bottom of a container (Fluoroware, H22 Series), and an aqueous solution of 5.25% polymer covering the collagen substrate.
  • the assembly (container, collagen, and solution) are irradiated with doses of 10 Mrads at a rate of up to 100 Mrad/sec.
  • the resulting hydrogels grossly appear to be optically clear, firmly gelled, and adherent to the collagen.
  • Substrates of corneal tissue 70% collagen (pre- extracted) and 100% collagen (extracted) are made into sheets from freshly enucleated bovine (calf) corneas.
  • the collagenous substrate is placed in a freezing microtome, and sheets of tissue, 10 mm in diameter, can be cut into thicknesses varying from 20 microns or larger.
  • the semi-rigid collagen sheets are placed into 25.4 mm diameter air-tight polypropylene dishes. After lyophilization, substrates are kept in a vacuum at 0°C until use.
  • Aqueous solutions of polyethylene oxide (PEO) or polyvinyl alcohol (PVA) are used in the synthesis since hydrogels made of these polymers are well-characterized (see Table 2) .
  • the polymer solutions are placed over the collagen sheets in polypropylene dishes which are now ready for electron beam irradiation.
  • the high voltage source of electron irradiation is supplied by a Van de Graft generator.
  • the irradiation area is 1 cm 2 .
  • This machine is capable of delivering dose rates of up to 10 Mrad/sec to samples conveyed along a belt at 1 cm/sec. Gelling and crosslinking is virtually instantaneous in these systems. After irradiation, the samples are ready for further analysis.
  • polymer-collagen crosslinking Important factors in polymer-collagen crosslinking are polymer concentration, polymer morphology, irradiation dose, dose rate, and depth of penetration. By varying the parameters stated above, polymer-collagen interfacial crosslinks can be varied. Interfacial Analysis
  • the collagen is removed by enzymatic and oxalic acid degradation, using the method of Pietrucha (1986) . This preserves the gel structure while still hydrolyzing the protein into amino acids.
  • Collagen is first cleaved by collagenase, then lysed into peptides by non-specific protease and finally hydrolyzed into amino acids (AA) by oxalic acid.
  • Proteolytic degradation removes all AAs except those which are chemically bonded onto polymer chains of the hydrogel.
  • Non-extracted amino-acids (from collagen) are considered covalently linked onto the hydrogel surface and are detectable by surface analytical techniques and amino acid labeling techniques.
  • the amino acids which are most highly reactive to participation in crosslinking are shown in Table 3.
  • Interface topology is studied with screening methods such as scanning electron microscopy (SEM) .
  • SEM scanning electron microscopy
  • the architecture of hydrogels in the hydrated state is preserved by certain processing methodologies or use of environmental scanning electron microscopy.
  • SEM- identified interface topology will verify any auxiliary mode of attachment such as mechanical intercalation of hydrogel into the collagen matrix. This may show up as roughness in the hydrogel interface.
  • a quantitative surface analysis technique is electron spectroscopy for chemical analysis (ESCA) .
  • Immobilized AAs analyzed in ESCA provide binding energy signals of nitrogen in addition to polymeric carbon and oxygen from PEO.
  • the nitrogen (N1S) peaks from ESCA can allow quantification of the amount of elemental nitrogen on the top 50 angstroms of the surface. This is an indirect measure of the quantity of surface bound AAs.
  • ESCA can analyze surface areas from 5 mm to 150 micrometers to a depth of 1-20 atomic layers.
  • the precision of elemental analysis varies from 10-20%. Elemental detection limits are from 0.1-1.0%.
  • the quantity of bound AAs and its relationship to polymer concentration, irradiation dose, dose rate, and irradiation depth of penetration is to be ascertained.
  • An additional method of studying interface amino acids bound to synthetic polymer is to label these amino acids with fluorescent or colored labelling molecules.
  • Amino acid labelling reagents have been used classically to label the amino termini of peptides being sequenced. Some of these labelling reagents are: ninhydrin, fluorescamine, dansyl chloride, and orthophthaldiol aldehyde (OPT) , which can be obtained from Sigma Chemical Co., St. Louis, MO.
  • the labelling protocols which use these reagents can detect nanogram to picogram quantities of the amino acids.
  • the particular absorbance characteristic of labelled amino acids allow the identification and quantification of particular amino acids.
  • Polymer/amino acid conjugates can be acid hydrolyzed from the hydrogel network and spectrophotometrica1ly quantified.
  • separation of the AAs can be performed with high pressure liquid chromatography. This technique can be used to find attachment sites of polymer to AAs.
  • Standards for such analyses can be made by electron beam irradiation of AA homopolymers within polymeric solutions. Standard amino acid homopolymers linked to polymer chains are hydrolyzed into single AAs. The networks and bound amino acids are washed to remove any residual amino acids not bound. The polymeric network is then hydrolyzed to free amino acid/monomer dimers. These dimers can be labelled with colored or fluorescent derivatives and separated through HPLC. Comparison of labelled elution fractions of samples against standard elution profiles derived from homopolymeric amino acids allows identification and quantification of modified AAs. Some irradiation modification of amino acids is expected but the majority of radical site generation will be on the alpha carbon of the peptide chain.
  • Light microscopy is used to analyze the histologic appearance of the collagen in the composite material. Immunofluorescence techniques are highly sensitive in antigenically determining biological changes. Antibodies to specific portions of the collagen type I molecule are commercially available. To perform immunofluorescent analyses, composites are sectioned and stained with the appropriate antibodies. Changes in the staining properties are used as a indirect measure of changes in the biological character of fibrillar collagen.
  • An in vitro enzymatic degradation assay can allow determination of the process induced changes which alter the proteolytic susceptibility of the collagen in the composite material.
  • Unmodified collagen is not susceptible to non-specific proteases and mildly sensitive to mammalian collagenase.
  • Test materials are incubated in a particular enzyme for 20 hours and the amount of hydrolysis is compared to that of corneal collagen. Differences in degradation rate between hydrogel/collagen composites and cornea can signal changes induced by the process of composite synthesis.
  • Attenuated total reflection Fourier transform infra-red spectroscopy utilizes infrared absorption to observe the chemical functionalities at a surface.
  • Example 3 Analysis of biological surface activity and growth of corneal epithelial cells onto composite. Epithelium formation was examined following surgical implantation of the composite hydrogen through organ culture of rabbit cornea. The ability of the hydrogel to support the outgrowth of epithelial cells in a physiologic fashion, i.e., from the remaining corneoscleral rim, was demonstrated by placing the entire cornea into culture.
  • Tissue explants were also studied to evaluate the migration and growth potential of corneal epithelial cells on the lenticule. Tissue explants provide cells which closely resemble normal cell populations in the body. In the assay, corneal tissue is placed epithelial side down on the lenticule, so that donor corneal epithelium is in direct contact with the adherent corneal stromal layer of the lenticule. Epithelial cells migrate off the tissue explant and onto the lenticule surface. Subsequently, the cells multiply and form an epithelium. Physiologically, corneal epithelial cell migration is important in normal healing.
  • the regrowth of the epithelial layer is typically the limiting factor in healing, and the return of function is dependent upon the regeneration of a normal epithelium.
  • Adhesion of stratified epithelium to the underlying stroma is ultimately dependent upon the generation of numerous subcellular components in the basal layer, including hemidesmosomes, the basal lamina, and anchoring fibrils. Sipehia, et al., Bio at.. Art Cells. Art Org. 18(5), 643-655 (1990).
  • the implant under study was a composite of 2% polyethylene oxide hydrogel, 150 ⁇ m in thickness, with a 50 ⁇ m layer of native corneal tissue covalently attached. It was sterilized with 70% ethanol prior to implantation.
  • a peripheral lamellar dissection was performed at the bottom of the trephine cut with an angled lamellar dissector extending to the limbus, as described by McDonald, et al., Kaufman, 823-845 (1988).
  • the implant previously cut to a diameter of approximately 10 mm
  • the implant was placed on the denuded cornea and the edges were tucked below the recipient lip so that they lay in the peripheral lamellar keratotomy created by the lamellar dissector. Due to the large area of lenticule lying within the circular keratotomy, no sutures were required to mechanically secure the lenticule.
  • the cornea was periodically moistened with sterilized saline solution containing polymyxin B.
  • the cornea was then carefully excised, including a scleral rim of 2 mm, and the explant was brought into culture by. transfer into a 6 well plate (Costar) containing sufficient media to cover the tissue.
  • the media was prepared from keratinocyte serum-free media (Gibco) supplemented with epidermal growth factor (2.5 ⁇ g) , BPE (25 mg) , L-glutamine, and gentamicin (pento 20 ⁇ g) .
  • the plates were then incubated at 37°C and 5% C0 2 for 5 to 7 days with a media change after three days.
  • the eyes were immersed in 10% paraformaldehyde for fixation.
  • the corneas were imbedded in methacrylate and three ⁇ m cuts were made by microtome for histologic examination.
  • the sections were stained with hematoxylin-eosin (H&E) and toluidine blue for light microscopy and evaluation of cell morphology and differentiation.
  • H&E hematoxylin-eosin
  • the lenticules were immersed in half-strength Kamovsky's glutaraldehyde-formaldehyde fixative, post-fixed in 2% osmium tetroxide, stained en- bloc with uranyl acetate, dehydrated and embedded according to standard technique.
  • Semi-thin sections were cut and stained with para-phenylenediamine (PPDA) for phase contrast microscopy.
  • PPDA para-phenylenediamine
  • Organ Culture Histologic examination revealed migration of epithelial cells onto the hydrogel within a seven-day period. A confluent layer of epithelial cells was observed over the implant over time with scattered areas of two cell layered stratification.
  • TEM Transmission electron microscopy
  • the goal of this project is to evaluate epithelial cell interactions with a synthetic material designed for keratoprostheses.
  • Composite hydrogels are hybrid materials made of corneal stromal tissue and a synthetic polyethylene oxide hydrogel base.
  • the hydrogel is coated with a 40 ⁇ m layer of corneal stromal tissue bound to the surface of a hydrogel.
  • Epithelial wound closure was evaluated on these materials using a two chamber cell culture system meant to mimic the clinical situation.
  • the hydrogels are suspended between a humidified air chamber (5.5% C0 2 ) and a serum free media chamber. Epithelial cell migration and proliferation onto the material is observed daily via phase contrast microscopy. Samples are processed by immunohistochemistry to determine production of basement membrane proteins.
  • Epithelial cells from corneal tissue explants readily adhere and grow to confluence over the composite hydrogel surface. Three millimeter diameter wound areas are epithelialized within four days after an initial two day lag period. Histological results show a multilayered epithelium 2 to 3 cell layers thick. By week 8, immunohistochemistry demonstrates the deposition of a continuous basal layer of laminin.
  • Composite hydrogels of stroma and synthetic hydrogel support epithelial cell attachment and growth to confluence.
  • Laminin deposition and multilayer stratification demonstrates the ability of the materials to support regeneration of epithelial characteristics.

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  • Health & Medical Sciences (AREA)
  • Transplantation (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Veterinary Medicine (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Ophthalmology & Optometry (AREA)
  • Vascular Medicine (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Chemical & Material Sciences (AREA)
  • Dermatology (AREA)
  • Medicinal Chemistry (AREA)
  • Epidemiology (AREA)
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Abstract

Matière composite à deux couches composée d'une couche mince de collagène ou de tissu cornéen, et d'un hydrogel, formé de préférence d'un polymère réticulable par faisceau électronique tel qu'un hydrogel synthétique en oxyde de polyéthylène (PEO). La matière est conçue pour servir de substrat approprié pour la croissance de cellules épithéliales cornéennes tout en conservant les caractéristiques désirables des hydrogels, à savoir la transparence, la souplesse et l'aptitude à permettre un écoulement par diffusion de substances nutritives. Selon un mode préféré de réalisation représenté dans les exemples, la synthèse des gels s'effectue par la réticulation provoquée par irradiation électronique d'une solution aqueuse de PEO, sur une couche mince d'un substrat en tissu collagénique. Selon un autre mode préféré de réalisation, les gels sont couplés chimiquement au substrat en tissu collagénique par l'intermédiaire d'un second polymère.
PCT/US1994/001419 1993-02-08 1994-02-08 Hydrogels composites a deux couches pour protheses corneennes WO1994017851A1 (fr)

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996006582A1 (fr) * 1994-09-01 1996-03-07 University Of Miami Seringue utilisee pour realiser une keratoplastie par injection controlee de gel
WO1996006883A1 (fr) * 1994-09-01 1996-03-07 University Of Miami Implant sous forme de gel d'oxyde de polyethylene injectable et son procede de production
WO2003009789A1 (fr) * 2001-07-23 2003-02-06 Fos Holding S.A. Dispositif permettant la separation de la couche epitheliale de la surface de la cornee d'un oeil
WO2007067697A2 (fr) * 2005-12-07 2007-06-14 Zimmer, Inc. Procedes de liaison ou de modification d’hydrogels par irradiation
WO2007071169A1 (fr) * 2005-12-20 2007-06-28 Zhiguang (Gz) Biotech Co., Ltd Cornee biologique artificielle et procede pour la produire
US7335230B2 (en) 2000-11-24 2008-02-26 Universite De Montreal Connective tissue substitutes, method of preparation and uses thereof
US7476398B1 (en) * 2002-06-28 2009-01-13 Universite Laval Corneal implant and uses thereof
US7674289B2 (en) 2005-07-29 2010-03-09 Grandhope Biotech Co., Ltd. Biological artificial ligament and method of making
US7828844B2 (en) 2002-09-13 2010-11-09 Forsight Labs, Llc Inserting lenses into corneal epithelial pockets to improve vision
CN101658445B (zh) * 2008-08-29 2011-08-17 四川大学 一体式人工角膜及其制备方法
US8100970B2 (en) 2005-12-20 2012-01-24 Grandhope Biotech Co., Ltd. Biological surgical patch and method of making
US8197500B2 (en) 2005-08-04 2012-06-12 Grandhope Biotech Co., Ltd. Biological membrane-carrying aneurysm clip
US8292799B2 (en) 2005-07-29 2012-10-23 Grandhope Biotech Co., Ltd. Biological artificial blood vessel and method of making
US8366770B2 (en) 2005-12-20 2013-02-05 Grandhope Biotech Co. Ltd. Biological artificial nerve guide and method of making
CN105412974A (zh) * 2015-11-27 2016-03-23 广州市朴道联信生物科技有限公司 一种双层结构角膜修复材料的制备方法

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US5196027A (en) * 1990-05-02 1993-03-23 Thompson Keith P Apparatus and process for application and adjustable reprofiling of synthetic lenticules for vision correction

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US5067961A (en) * 1988-02-18 1991-11-26 Autogenesis Technologies, Inc. Non-biodegradable two phase corneal implant and method for preparing same
US5196027A (en) * 1990-05-02 1993-03-23 Thompson Keith P Apparatus and process for application and adjustable reprofiling of synthetic lenticules for vision correction

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5645583A (en) * 1990-07-12 1997-07-08 University Of Miami Injectable polyethylene oxide gel implant and method for production
US5681869A (en) * 1990-07-12 1997-10-28 University Of Miami Injectable polyethylene oxide gel implant and method for production
US5547468A (en) * 1990-07-12 1996-08-20 University Of Miami Instruments for use in performing gel injection adjustable keratoplasty
US5634943A (en) * 1990-07-12 1997-06-03 University Of Miami Injectable polyethylene oxide gel implant and method for production
AU690327B2 (en) * 1994-09-01 1998-04-23 University Of Miami Injectable polyethylene oxide gel implant and method for production
WO1996006883A1 (fr) * 1994-09-01 1996-03-07 University Of Miami Implant sous forme de gel d'oxyde de polyethylene injectable et son procede de production
WO1996006582A1 (fr) * 1994-09-01 1996-03-07 University Of Miami Seringue utilisee pour realiser une keratoplastie par injection controlee de gel
US7335230B2 (en) 2000-11-24 2008-02-26 Universite De Montreal Connective tissue substitutes, method of preparation and uses thereof
US7347872B2 (en) 2000-11-24 2008-03-25 Universite De Montreal Connective tissue substitutes, method of preparation and uses thereof
WO2003009789A1 (fr) * 2001-07-23 2003-02-06 Fos Holding S.A. Dispositif permettant la separation de la couche epitheliale de la surface de la cornee d'un oeil
US7476398B1 (en) * 2002-06-28 2009-01-13 Universite Laval Corneal implant and uses thereof
US7828844B2 (en) 2002-09-13 2010-11-09 Forsight Labs, Llc Inserting lenses into corneal epithelial pockets to improve vision
US7674289B2 (en) 2005-07-29 2010-03-09 Grandhope Biotech Co., Ltd. Biological artificial ligament and method of making
US8292799B2 (en) 2005-07-29 2012-10-23 Grandhope Biotech Co., Ltd. Biological artificial blood vessel and method of making
US7824447B2 (en) 2005-07-29 2010-11-02 Grandhope Biotech Co. Ltd. Biological artificial ligament and method of making
US8197500B2 (en) 2005-08-04 2012-06-12 Grandhope Biotech Co., Ltd. Biological membrane-carrying aneurysm clip
WO2007067697A3 (fr) * 2005-12-07 2008-10-02 Zimmer Inc Procedes de liaison ou de modification d’hydrogels par irradiation
US8262730B2 (en) 2005-12-07 2012-09-11 Zimmer, Inc. Methods of bonding or modifying hydrogels using irradiation
WO2007067697A2 (fr) * 2005-12-07 2007-06-14 Zimmer, Inc. Procedes de liaison ou de modification d’hydrogels par irradiation
CN1985778B (zh) * 2005-12-20 2010-10-13 广东冠昊生物科技股份有限公司 生物型人工角膜
JP2009519792A (ja) * 2005-12-20 2009-05-21 ズヒグアング（ジージェット） バイオテク シーオー．，エルテーデー． 生体人工角膜および作製方法
US8100970B2 (en) 2005-12-20 2012-01-24 Grandhope Biotech Co., Ltd. Biological surgical patch and method of making
WO2007071169A1 (fr) * 2005-12-20 2007-06-28 Zhiguang (Gz) Biotech Co., Ltd Cornee biologique artificielle et procede pour la produire
AU2006329154B2 (en) * 2005-12-20 2012-12-13 Grandhope (Gd) Biotech Co., Ltd. Biological artificial cornea and method of making
US8366770B2 (en) 2005-12-20 2013-02-05 Grandhope Biotech Co. Ltd. Biological artificial nerve guide and method of making
AU2006329149B2 (en) * 2005-12-20 2013-03-14 Summit (Gd) Biotech Co., Ltd. Biological surgical patch and method of making
CN101658445B (zh) * 2008-08-29 2011-08-17 四川大学 一体式人工角膜及其制备方法
CN105412974A (zh) * 2015-11-27 2016-03-23 广州市朴道联信生物科技有限公司 一种双层结构角膜修复材料的制备方法

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