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, corneal implants; Implanting instruments specially adapted therefor; Artificial eyes
  • A61F2/145—Corneal inlays, onlays, or lenses for refractive correction
• • 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
  • 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
  • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified 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

• This invention relates to the field of ophthalmology, and more particularly is directed to corneal implants.
• the cornea is a complex layered structure comprising an outer layer of epithelial cells, Bowman's membrane posterior of the epithelial layer, the stroma posterior of the Bowman's membrane, Descemet's membrane posterior of the stroma, and the endo- thelium immediately posterior of Descemet's membrane.
• a number of surgical procedures involve implanting a lens structure into or onto one or more of these corneal components.
• the epithelial cell layer is removed and a corrective lens structure is placed and secured at the location where the cells are removed.
• a portion of the layer of epithelial cells is removed and a wedge-shaped annulus from Bowman's membrane and the underlying stroma is removed.
• An incision is then made from the posterior end of the resulting groove radiating outwardly in an annular zone to define a flap.
• a corrective lens structure is then attached by inserting the wing of the lens structure beneath the corneal flap and suturing it in place.
• a corrective lens structure can also be placed entirely within the stroma. This surgical procedure involves making an incision in the cornea to gain access to the stroma and also involves disrupting the stroma by placing a lens structure therein.
• corneal corrective surgeries are employed for the treatment of severe visual disturbances such as those associated with excessive or insufficient corneal curvature, traumatic injury to the cornea, for contact lens intolerant patients and the like. These procedures are generally known as keratoplasties.
• Hydrogels and like "porous" polymers block the free movement of proteins and other high molecular weight tissue fluid components and so compartmentalize the tissue anterior to a corneal implant from that beneath the implant. This has significant deleterious effects as will be hereinafter described. Hydrogels also lack the requisite mechanical properties necessary for ocular implants. For example, hydrogel ocular implants described in Australian Patent No 623137 shrink by about 22 % (that is shrinkage from initial volume) when placed in physiological saline (see page 16, lines 19 to 24 of Australian Patent No 623137). Clearly such materials would not be appropriate for implantation into the eye. Moreover, cells attach very poorly to hydrogel polymers with the result that epithelial recolonization of hydrogel implants would be impeded.
• tissue fluid components such as proteins and glycoproteins (for example, growth factors, peptide and protein hormones, and proteins associated with the transport of essential metals) and the like, across a corneal implant, that is, from epithelial cells to stromal cells and even to the endothelial layer and beyond, is essential for long term maintenance and viability of tissue anterior and posterior to a corneal implant.
• tissue fluid components such as proteins and glycoproteins (for example, growth factors, peptide and protein hormones, and proteins associated with the transport of essential metals) and the like
• corneal implants of the prior art prevent the passage of high molecular weight proteins and glycoproteins (such as those having a molecular weight up to or greater than 10,000 daltons and up to and in excess of 200,000 daltons or more), and adversely effect the formation of an epithelial layer over an anterior surface of a lens implant inserted within the epithelial layer of the cornea.
• tissue posterior to corneal implants suffers from stromal degeneration which is believed to result from the inability of high molecular weight proteinaceous components to pass from epithelium tissue to stromal tissue.
• This invention is particularly directed towards an implant to be placed beneath, within, or through corneal epithelial tissue, or within the corneal stroma or other tissue layers of the cornea.
• corneal onlays all such implants will be referred to hereafter as corneal onlays.
• a corneal onlay for use in surgical implantation into or onto the cornea of a mammal, said onlay having an optical axis region with optical characteristics which provide visual acuity therethrough, said onlay being comprised of a non-biodegradable non-hydrogel ocularly biocompatible material, and characterized in that said onlay has a porosity sufficient to allow passage therethrough of tissue fluid components having a molecular weight greater than 10,000 daltons, thereby providing for a flux of tissue fluid between cells anterior of the implanted onlay and cells posterior thereof, wherein the porosity of the optical axis region is such that it allows the flux of tissue fluid components whilst excluding ingrowth of ocular tissue, and wherein said onlay is adapted for epithelial recolonization.
• a method for correcting the optical properties of an eye or altering the appearance thereof comprising, surgically implanting into or onto the cornea a corneal onlay for use in surgical implantation into or onto the cornea of a mammal, said onlay having an optical axis region with optical characteristics which provide visual acuity therethrough, said onlay being comprised of a non-biodegrad ⁇ able non-hydrogel ocularly biocompatible material, and characterized in that said onlay has a porosity sufficient to allow passage therethrough of tissue fluid components having a molecular weight greater than 10,000 daltons, thereby providing for a flux of tissue fluid between cells anterior of the implanted onlay and cells posterior thereof, wherein the porosity of the optical axis region is such that it allows the flux of tissue fluid components whilst excluding ingrowth of ocular tissue, and wherein said onlay is adapted for epithelial recolonization.
• the corneal onlay may comprise the same visual acuity across its dimensions, as do conventional contact lenses. However, as visual acuity is only required for that portion of the onlay which covers the pupil, hereafter referred to as the optical axis region, it is preferred to provide a corneal onlay having an optical axis region having a porosity sufficient to allow passage therethrough of tissue fluid components having a molecular weight up to or greater than 10,000 daltons, but which excludes ingrowth of ocular tissue. In such a situation, the periphery of the onlay surrounding the optical axis region may define a skirt having a porosity sufficient to permit ingrowth of ocular tissue so as to facilitate anchorage of the onlay to the cornea.
• the porosity of the corneal onlay over the optical axis region of the eye allows the flow or flux of tissue fluid components comprising high molecular weight protein, as well as small molecular weight nutrients such as glucose and respiratory gases through the onlay.
• the porosity of the onlay allows for protein species having a molecular weight of several thousand daltons up to several hundred thousand daltons or more to freely pass through the corneal layers as hereinbefore described. It is the passage of high molecular weight proteinaceous components, such as growth factors, hormones, signalling proteins, molecular transmitters, and transport proteins such as transferrin which transport essential metal ions, which provide for the maintenance, integrity and health of tissues posterior and anterior to an implanted corneal onlay.
• the corneal onlay as herein described is capable of being surgically associated with, and preferably attached to, a living cornea so as to change its optical properties (such as correct vision deficiencies of the eye) or to change the appearance of the eye, such as coloration of the pupil.
• the porosity of the corneal onlay may be provided by virtue of the material from which the onlay is formed, that is, by the inherent porosity of the material.
• pores may be introduced into the material from which the onlay is formed by various procedures well known in the art such as those described in WO 90/07575, WO 91/07687, US Patent No 5,244,799, US Patent No 5,238,613, US Patent No 4,799,931 and US Patent No 5,213,721. Pores may be formed after production of the onlay shape (such as by polymer casting), or in bulk material prior to onlay shape formation. Such procedures may involve:
• Pores may be conveniently produced by using excimer lasers, such as a pulse type laser under microprocessor control as described, for example, in WO 91/07687;
• porosity may be provided by an inter-penetrating network of holes throughout the onlay material prepared such as by polymerization in the presence of an insoluble material. Subsequent removal of the insoluble material gives rise to interstices throughout the formed polymer material.
• the onlay must have a porosity sufficient to admit proteins of a molecular weight up to and greater than 10,000 daltons, such as from 10,000 to 1,000,000 daltons, but not sufficient to admit cells and thus tissue invasion into the optical axis region of the corneal onlay.
• porosity of the onlay is provided by pores
• the optical axis region comprises a plurality of pores, the number of which is not in any way limiting on the invention, but which is sufficient to provide flow of tissue components from the anterior to posterior regions of an implanted onlay.
• the number of pores provided in the onlay may range from 200 pores per square millimetre to 300,000 pores per square millimetre or more.
• the pores formed within the optical axis region are in the range of 5 nanometers to 15 x 10 3 nanometers in diameter. Pores of this size within the optical axis region would not cause refraction of visible light to an extent that would cause any problem with regard to vision correction. More preferably, the pores in the optical axis region have a diameter of from 15 to 300 nanometers, and even more preferably from 20 to 150 nanometers in diameter. It is to be understood that the term pore does not put any geometric limitation on the nature of the pores which may be of regular or irregular morphology. In this context the term "average width" may be preferable to the term "diameter". Within the above mentioned preferred diameters, it should be recognized that not all pores may be of the same diameter, but may vary within the range given.
• the onlay may have the same porosity as the optical axis region, that is, allowing passage through the onlay of small molecular weight nutrients, respiratory gases, and proteinaceous and other components of tissue fluid having a molecular weight up to and greater than 10,000 daltons (for example, up to 1,000,000 daltons or more) whilst being of a porosity which specifically excludes tissue ingrowth.
• this region of the onlay surrounding the periphery of the optical axis region which may be referred to as the skirt, may allow the ingrowth of cells of the cornea thereby assisting in anchorage of the onlay to the eye.
• the skirt may contain a plurality of pores of diameter of 20 microns or more, preferably 50 microns to 1,000 microns, more preferably 50 to 500 microns, and even more preferably 50 to 300 microns. Pores may be formed in the skirt in the same manner as that described above for the production of pores in the optical axis region of the onlay.
• Porosity in the skirt may be an inherent feature of the material from which the skirt is formed.
• the skirt may be formed of the same material as the optical axis region and may be integral therewith. In this situation, pores of differing diameter may be formed in the optical axis region and the skirt.
• the skirt may be formed in a distinct and separate manner to that of the optical axis region (for example, by polymerization in the presence of an insoluble agent such as glucose which is subsequently dissolved from a polymerized matrix to give a highly porous interpenetrating network), such that whilst formed of the same material as the optical axis region, the skirt has a much greater porosity thereby allowing tissue ingrowth.
• the skirt may be formed of a different material from the optical axis region, which material is of a higher porosity than the optical axis region so as to allow tissue ingrowth.
• the skirt may be processed in a manner to produce high porosity such as by preparing a coherent mass of melt drawn fibres having an interconnecting network of pores, such as fibres made of a polyolefin material, which is applied to the optical axis region.
• the skirt may, for example, be formed from a highly porous ceramic material which is fused to the optical axis region such as by solvent welding, heat fusion or use of adhesives. Highly porous ceramics which facilitate tissue ingrowth are well known (see, for example, J. Biomed. Mater. Res. Symp, No 4, pp 1-23, 1973).
• the corneal onlay may be prepared from any one of the known natural or synthetic polymeric materials with the requisite mechanical properties such as polymers and copolymers of the classes commonly known as acrylics, polyolefins, fluoropolymers, silicones, styrenics, vinyls, polyesters, polyurethanes, polycarbonates, cellulosics or proteins such as collagen based materials.
• Hydrogel polymeric compositions are specifically excluded due to their propensity of shrinkage, lack of porosity to high molecular weight species, and poor cellular attachment properties.
• the skirt may be formed of different polymeric or other materials than the optical axis region so as to allow tissue ingrowth, this being a function of greater porosity.
• the skirt region may be formed from one or more of the aforementioned polymers, ceramics or any other biocompatible non-biodegradable material having pores with a diameter greater than about 20 microns so as to allow tissue ingrowth.
• the corneal onlay of this invention may be produced according to standard techniques for the production of corneal onlays as are well known in the art Clearly the precise techniques used for the polymerization of polymers, copolymers, mixtures thereof, production of ceramics, and the joining of optical axis region to skirt where necessary, will depend upon the nature of the materials employed, with appropriate methodologies being adopted for respective materials according to conventional procedures.
• the corneal onlay may be shaped during or at the conclusion of its production process according to standard milling, polishing, and/or shaping procedures as are well known in the field for the production of corneal prostheses.
• the corneal onlay produced by polymerization is formed to shape either by moulding during polymerization, or by lathing of a preformed button.
• the optical axis region may be first formed to shape, for example, by either moulding or lathing of a preformed button, whereafter a skirt is attached to the periphery of the optical axis region by polymerization thereabouts.
• the optical axis region may be attached to a porous outer skirt, for example, by dipping the skirt in acetone and pressing the two together.
• an optically acceptable adhesive may be used of materials fused together by the application of heat such as generated by a pulse laser.
• a skirt may first be formed having an annular orifice, within which the optical axis is formed either by polymerization of fixation of a pre-formed button.
• the onlay or a portion thereof may be coloured with one or more pigments or dyes as are well known in the field. Coloured onlays, and particularly colouration around the pupil area, will result in change of eye coloration on implantation.
• the corneal onlay according to this invention is adapted for epithelial recolonization.
• the polymeric substance from which the onlay is formed does not inhibit cellular attachment or motility.
• the respective surfaces of the onlay may be modified as is hereinafter described to facilitate cellular attachment.
• the outer posterior and anterior surface of the corneal onlay may be modified by the application of a polymer having pendant groups capable of being converted to reactive functional groups which are thereafter capable of covalently coupling to the onlay surface.
• a surface modifying composition may comprise a poly amino acid, such as polylysine, where, for example, about 10 mol per cent of pendant groups are capable of being converted to nitrene functional groups.
• the modified surface may of itself, stimulate the adhesion of cells adjacent to the implanted onlay, such as epithelial cells, or cells of the stroma.
• the modified surface may be coated with one or more components which promote the growth of tissue adjacent to the implanted onlay.
• such materials include fibronectm, laminin, chondroitan sulphate, collagen, cell attachment proteins, anti-gelatine factor, cold-insoluble globulin, chondronectin, epidermal growth factor, mussel adhesive protein, thrombospondin, vitronectin, and various proteoglycans, and/or derivatives of the above and mixtures thereof.
• Fibronectm, derivatives of fibronectm, epidermal growth factor, derivatives of epidermal growth factor and mixtures thereof are particularly useful.
• the corneal onlay itself without surface modification may be directly coated with one or more components which promote the growth of tissue adjacent to the implanted onlay and/or cell adhesion to the onlay.
• components may be any component or components which provide cell adhesion and/or promote cell growth such as growth factors and adhesion factors.
• Preferred materials are those detailed in connection with the application to a surface modified corneal onlay.
• the coating of components which promote the growth of tissue adjacent to the implanted onlay are covalently bonded to the onlay, without effecting the optical properties thereof.
• the onlay of this invention is adopted for epithelial recolonization.
• the corneal onlay according to the invention described herein is distinct and superior to prior art proposals in that it provides an effective flow of high molecular weight tissue fluid components to the anterior surface from the tissue beneath the implant which serves to support and to stimulate the coverage of the anterior surface of the implant with corneal epithelial cells through the processes of migration from the adjacent tissue and stratification, and to support the maintenance of the epithelial tissue that covers the implant, or indeed any other tissue which covers the implant.
• Transcorneal signalling is provided, for example, during the initial period after implantation, such as during coverage of the implant with epithelium, and furthermore provides for the porosity of the implant towards therapeutic compounds applied to the anterior surface of the implant.
• the corneal implant according to this invention may be inserted into the eye according to established keratectomy procedures as are well known in the field.
• FIGURE 1 is a histogram which depicts the percentage (%) of animals with corneal ulcers for specific membrane pore sizes.
• Column A represents the membrane materials Cuprophan / Gambrane.
• FIGURE 2 is a histogram which depicts the percentage (%) of animals with epithelial and anterior stromal thinning for specific membrane pore sizes.
• Column A represents the membrane materials Cuprophan / Gambrane.
• FIGURE 3 is a histogram which shows bovine corneal epithelial cell attachment to tissue culture polystyrene (TCPS) in fractionated serum after twenty four hours.
• the X-axis represents the medium.
• DD represents serum free culture medium and 20% (v/v) fibronectin and vitronectin-depleted serum
• “100k” represents serum-free culture medium, to which was added 20% (v/v) of Fraction 1, having serum factors of 1000 to 100,000 daltons in size
• "30k” represents serum-free culture medium, to which was added 20% (v/v) of serum factors of 1000 to 30,000 daltons in size
• “10k” represents serum-free culture medium, to which was added 20% (v/v) of serum factors of 1000 to 10,000 daltons in size
• “100+30+lOk” represents serum-free culture medium, to which was added 20% (v/v) of serum factors of 1000 to 100,000 daltons in size
• “DD” is serum-free culture medium + 20% (v/v) Fibronectm- and Vitronectin-depleted serum (not fractionated as to size)
• SFM is serum free culture medium
• Fn denotes that the tissue culture polystyrene surface v/as preco
• FIGURE 4 is a histogram which shows corneal epithelial cell outgrowth on tissue culture polystyrene (TCPS) in fractionated serum after six days culture.
• the X-axis represents the medium.
• the Y-axis represents the percentage cell number 24 hr/5 (control at day 6); cell outgrowth 6 days / 24 hr (wherein the hatched portion of the column represents the outgrowth, and the black squares represent the cell number).
• DD serum-free culture medium + 20% (v/v) Fibronectin- and Vitronectin-depleted serum (not fractionated as to size);
• 100k represents serum-free culture medium, to which was added 20% (v/v) of Fraction 1, having serum factors of 1000 to 100,000 daltons in size;
• 30k represents serum-free culture medium, to which was added 20% (v/v) of serum factors of 1000 to 30,000 daltons in size;
• 10k represents serum-free culture medium, to which was added 20% (v/v) of serum factors of 1000 to 10,000 daltons in size;
• SFM serum free culture medium; and Fn denotes that the tissue culture polystyrene surface was precoated with fibronectin.
• Example 1 Passage of Macromolecules Across the Cornea is Essential for Maintenance of Normal Corneal Structure/Integrity
• Membranes of differing pore size were implanted into the eyes of cats to determine the effects of facilitating or blocking the flux of high molecular weight tissue fluid components across the cornea, both posterior and interior of a corneal implant.
• dialysis membranes with a 1.4 nanometre pore size were implanted into the cornea of cats. These membranes, whilst allowing the passage of glucose, amino acids, water and respiratory gases, block the passage of components having a molecular weight greater than 10,000 daltons. Both the corneal epithelium and stroma of test animals lost integrity over the test period of four weeks. This experiment shows that tissue components having a molecular weight greater than 10,000 daltons, namely proteins and glycoproteins, are essential for corneal integrity.
• corneal integrity including slit lamp evaluation were conducted. Details of corneal transparency, vessel hyperaemia and penetration, surface charac ⁇ teristics, opacities, etc, were assessed.
• the dialysis membrane was rinsed and autoclaved in sterile isotonic saline after laser perforation.
• the cats were anaesthetised to a depth of stage 3 - plane 2.
• a diamond knife was used to make a 6 mm incision just inside the superior limbus.
• a corneal dissector with a shape edge was used to form a lamellar pocket of 14 mm to 15 mm diameter.
• Euthanasia of cats was performed with an intravenous overdose of sodium pentobarbitone at 140 mg kg of body weight.
• fixative was dropped on to both eyes (2.5 % glutaradehyde in 0.1 M sodium cacodylate trihydrate, 2 mM calcium chloride, pH adjusted to 7.2 with 1M HC1, at 25°C) at a sufficient rate to ensure that the anterior corneal surface remained moist Immediately after death 0.3 ml of fixative was injected into the anterior chamber of each eye to aid the fixation of the posterior corneal surface.
• Corneas were separated from the remainder of the eye and immersed in fixative for sixty hours at 4°C. Two 1 mm wide strips were cut from each cornea; one from periphery to centre along the superior-inferior axis; one from periphery to centre along the nasal-temporal axis.
• Tables 3 and 4 provide a summary of the study design and findings.
• Corneal neovascularization started at the site of incision and continued to penetrate into the stroma overlying the membrane in all cats. However, corneal neovascularization regressed in one eye (Cat 4) during the study. The neovascularization observed was a complication of the surgical procedure rather than vessel growth due to lack of nutrients.
• variable 20-30 in perforated region
• Example 2 Onlay Porosity Required for Maintenance of Epithelial and Stromal Health
• the details and characteristics of the membranes used in this study are set forth in Table 5.
• the membranes Prior to implantation, the membranes were trephined to a diameter of 12 mm, 10 mm or 8 mm.
• Example 6 Surgical procedure and follow up evaluation was carried out according to Example 1. The number of animals which were implanted with specific membranes are set forth in Table 6.
• Figure 1 shows a histogram which sets out the percentage of animals in each implanted group with corneal ulcers. Forty three percent of animals implanted with a membrane having a porosity of 25 nm showed corneal ulceration. Eighty percent of animals implanted with a membrane have a pore size of 15 nm exhibited corneal ulceration. Effectively the same result was observed for the cuprophan and gambrane membranes which have a pore size of approximately less than 1.5 nm. No ulceration was seen in animals implanted with membranes having pore sizes of 50 nm and 100 nm.
• Figure 2 shows a histogram which sets out the percentage of animals with epithelial and anterior stromal thinning following ocular implantations of membranes of varying pore size.
• Figure 2 clearly shows that as the pore size of the implanted membrane decreases, the number of animals with epithelial and anterior stromal thinning increases. All animals implanted with membranes having a pore size of 15 nm or less exhibited epithelial and anterior stromal thinning. Membranes having a pore size of 25 nm resulted in 75 % of animals showing epithelial and anterior stromal thinning. This figure fell to 31 % and 27 % respectively for membranes having a pore size of 50 nm and 100 nm.
• Figures 1 and 2 clearly show that the pore size of corneal onlays for the insertion into the eyes of animal should be greater than about 15 nm.
• Example 3 Corneal Epithelial Tissue Requires Trophic Factors of a Molecular Weight Greater Than 10.000 Daltons for Cell Attachment, Migration and Epithelial Outgrowth
• tissue fluid factors as exemplified by serum
• Corneal epithelial cells were maintained in a culture medium (serum-free) which provided for the requirements of the cells for glucose, nutrients, metabolites and small physiological molecules.
• the purpose of the experiment was to determine the requirement of these cells for tissue trophic factors, in order to permit certain cellular functions that would enable the colonisation of a corneal implant with corneal epithelium and for the maintenance of that epithelial tissue. This was demonstrated by examining the effect of the addition to the culture medium of serum, as an example of such tissue fluids, upon migratory and outgrowth activities of corneal epithelial cells.
• the experiments were designed in a manner to remove the possibility that any stimulatory effect of serum factors could arise solely due to direct stimulation of cell attachment following adsorption of the serum factors onto the polymer surface.
• Two tissue and serum factors, fibronectin or vitronectin, can be found in a soluble form and can also adsorb onto surfaces, and in this insoluble form can serve to promote cellular attachment.
• the cell culture assay system used for the determination of the effect of tissue trophic factors was chosen to represent the corneal onlay material of the prior art, in being supportive of the processes of initial cell attachment.
• the corneal epithelial cells were cultured on the synthetic polymer surface called tissue culture polystyrene, which is an oxidized polystyrene surface that has been optimized for support of cellular attachment.
• tissue culture polystyrene which is an oxidized polystyrene surface that has been optimized for support of cellular attachment.
• this polymer surface resembles that of an example of a corneal onlay described in the prior art, insofar as cell adhesion is concerned.
• extracellular matrix components such as fibronectin, laminin or collagens may stimulate the cellular attachment activity of corneal epithelial cells.
• different extracellular matrix components were compared for their ability to support the initial attachment and outgrowth of corneal epithelial cells when the factors were adsorbed onto a culture surface, in order to determine an optimal surface for the stimulation of corneal epithelial cell migratory activity.
• the culture medium used was serum-free medium containing 20% (v/v) foetal calf serum depleted of both Fibronectin and Vitronectin, in order that the effectiveness of the different extracellular matrix components may be determined without any contribution from vitronectin or fibronectin adsorbed from the serum component of the culture medium.
• the cells in the epithelial island were then free to grow out over the surface, at a rate that could be determined in part by the extracellular matrix component that had been preadsorbed onto the polymer surface.
• this assay two different formats were possible. Cells may be seeded either within the region of the central chamber thereby effectively creating a circular island of cells, or alternatively outside the chamber and so leaving a central region of the surface devoid of cells. Using these assay formats, the extracellular matrix components were compared for their influence upon the outgrowth of epithelial cells.
• Bovine serum fibronectin at 10 ⁇ g ml. (from Sigma);
• Murine laminin at 50 ⁇ g ml. Purified from EHS sarcoma, provided from Collaborative Biomedical;
• Murine Collagen Type IV at 40 ⁇ g ml. Purified from EHS sarcoma, provided from Collaborative Biomedical;
• Bovine Collagen Type I at 1.5 mg/ml. (Purified from bovine calf skin, "Cellagen” from ICN).
• precoating concentrations were chosen to be levels that would offer maximal stimulation of cell adhesion.
• 80 ⁇ l of matrix solution were added at the predetermined concentrations to replicate tissue culture polystyrene wells (96-well culture trays) and incubated for one hour at 37°C.
• the matrix solutions were then removed and the surfaces 'blocked' with a solution of 1% (w/v) bovine serum albumin in serum-free culture medium and incubated for one hour at 37°C. This 'blocking' step ensures that all non-matrix supplied cell binding sites are effectively neutralized prior to cell seeding.
• the blocked tissue culture polystyrene surface was also included in each assay and, as a result of the treatment with albumin, served as an effective negative control.
• Cell Attachment Assay One hundred ⁇ l aliquots of 0.7 - 1.0 x 10 5 cells/ml for cultured cells, or 3.0 - 5.0 x 10 5 cells/ml for primary cells in 1 % (w/v) bovine serum albumin in serum-free culture medium, were added to each well and incubated for ninety minutes at 37°C. Cell adhesion was determined by methylene blue staining and absorbances read at 650 nm on a plate-reader. Collagen Type I was used as the reference substrate for each cell origin type and cell attachment set at 100 %, and so for the purposes of reporting, cell adhesion to all other matrices was expressed as a percentage of that on the Collagen Type I in the same experiment.
• the culture medium used was 20 % (v/v) feotal bovine serum, depleted of fibronectin and vitronectin, in 40% (v/v) Dulbecco's Minimal Essential Medium, 40 % (v/v) Ham's F12 medium.
• the precoatings were incubated for one hour at 37°C prior to use. During the culture period, the culture medium was changed every third day.
• fibronectin was also stimulatory of attachment of cells from each region, but for each cell origin the response of these primary isolates towards fibronectin was variable from experiment to experiment (mean response varying over a 2.3 to 2.8 fold range). Vitronectin, although stimulatory, was consistently much less effective than laminin, the coUagens or fibronectin in promoting cell attachment of each of these primary isolate cells. This is a surprising finding insofar as vitronectin is concerned.
• TCPS . tissue culture polystyrene
• NS foetal calf serum (not depleted of fibronectin or vitronectin)
• Fn fibronectin
• DD foetal calf serum, depleted of both fibronectin and vitronectin
• TCPS tissue culture polystyrene
• NS foetal calf serum (not depleted of fibronectin or vitronectin)
• Fn fibronectin
• DD foetal calf serum, depleted of both fibronectin and vitronectin
• both fibronectin and collagen I are stimulatory of both the initial cell attachment of corneal epithelial cells to polymer surfaces and also the outgrowth and migration of the cells from cell islands.
• the fibronectin-coated polymer surface was included as being representative of a surface containing a biological adhesive factor.
• Foetal bovine serum was depleted of the cell-adhesive molecules vitronectin (Vitronectin) and fibronectin (Fibronectin) by the methods we have previously described. This depleted Fibronectin and Vitronectin-depleted serum was then passed through a series of ultra-filtration membranes, in order to separate the serum components into fractions containing components of defined ranges of molecular weights. Three different non-ionic Amicon ultrafiltration membranes were used:
• the membranes were cleaned prior to use as per the manufacturer's recommendations.
• the membranes were each pretreated with a solution of 1 % (w/v) bovine serum albumin in PBS, followed by a wash with sterile PBS, in order to reduce nonspecific binding of proteins onto the membrane surface and consequental loss of serum components, during the subsequent filtration step.
• the various membranes were held in an ultrafiltration assembly (Amicon) and the Fibronectin and Vitronectin-depleted serum or fraction thereof was stirred with a magnetic stirrer and filtered at an air pressure of 400 kPa.
• the fractionation of the serum was performed by the following method: The Fibronectin and Vitronectin-depleted serum was diluted with two volumes of sterile phosphate-buffered saline (PBS) and put through a series of sequential filtrations. All of the Fibronectin and Vitronectin-depleted and diluted serum was firsdy passed through a 43 mm diameter XM100 membrane and the filtrate collected. The retentate from this membrane was discarded. Two aliquots of the filtrate from the XM100 membrane were taken and passed through either a PM10 or a PM3025 mm diameter membrane. The filtrates from these two membranes separately collected and the retentates of these membranes were discarded.
• PBS sterile phosphate-buffered saline
• Fraction 1 The filtrate of the XM100 membrane that was subsequently retained upon the UM2 membrane - this preparation would contain components in the 1,000 - 100,000 daltons range.
• Fibronectin and Vitronectin-depleted serum and depleted serum fractions prepared as described above, were added to the culture medium (1:1 Dulbecco's Modified Essential Medium/Ham's F12 serum-free culture medium) at a concentration of 20 % (v/v) in order to study the effects of the various fractions upon the attachment and growth of bovine corneal epithelial cells and the assays were conducted over a culture period of six days.
• Culture wells were set up with sterile stainless steel "fences" which were contained within the wells and centrally located. Aliquots (100 ⁇ l) of a cell suspension of corneal epithelial cells (prepared from bovine corneal tissue) were each seeded into stainless steel "fence" enclosure at a density of 5 x 10 5 cells/ml. This cell loading was sufficient to give a confluent monolayer twenty four hours after seeding the cells into the culture area defined by the lumen of the fence, in a culture medium that contained intact serum. In these experiments the fences were removed from the culture wells after twenty four hours culture, then the culture medium volume adjusted and the culture allowed to continue.
• Each test sample of Fibronectin and Vitronectin-depleted serum fraction was assayed in triplicate wells, with one well being for quantitation of the area of the culture containing cells after a twenty four hour culture period, and two wells being used for measuring the area after seven days culture.
• the cells were fixed with formol-saline and stored in sterile PBS at 4°C until required.
• the cell density was determined by staining with Methylene Blue after Oliver et al., and the amount of bound stain measured colourimetrically at 655 nm on a plate-reader.
• Methylene blue staining was used to determine both cell outgrowth areas (by image analysis using a Quantimet 570 image analyser, detecting cells by the increased colour following uptake of the blue stain), and to measure the extent of cell proliferation (determined colourimetrically at 655 nm, on an immunoassay plate reader).
• the culture media combinations were: i) serum-free culture medium, to which was added 20 % (v/v) of Fraction 3, having serum factors of 1000 to 10,000 daltons in size; ii) serum-free culture medium, to which was added 20 % (v/v) of Fraction 2 having serum factors of 1000 to 30,000 daltons in size; iii) serum-free culture medium, to which was added 20 % (v/v) of Fraction 1, having serum factors of 1000 to 100,000 daltons in size; iv) serum-free culture medium, to which was added 20 % (v/v) of a combination of
• Fractions 1, 2 and 3 having serum factors of 1000 to 100,000 daltons in size; v) serum-free culture medium + 20 % (v/v) Fibronectin - and Vitronectin-depleted serum (not fractionated as to size), vi) serum-free medium alone.
• tissue culture polystyrene As described above, two culture surfaces were used: firstly, tissue culture polystyrene, and secondly, tissue culture polystyrene that had been precoated with bovine serum Fibronectin at 10 ⁇ g/ml and incubated for one hour at 37°C prior to cell loading.
• Fibronectin and Vitronectin-depleted serum By separating Fibronectin and Vitronectin-depleted serum into fractions containing known molecular weight ranges, it was possible to assess the effects of serum components, within specific molecular weight ranges, upon corneal epithelial cell attachment, migration and outgrowth.
• Figure 3 shows the effect of the different fractions prepared from Fibronectin and Vitronectin-depleted serum upon the attachment of corneal epithelial cells during the first twenty four hour of culture.
• the number of cells present on the surfaces in the different treatments were close to being the same for each treatment after twenty four hour of culture.
• the different treatments could therefore be compared for the rates of cell outgrowth and increase in cell number beyond the initial twenty four hour culture period, having a similar population of cells in the assay at the twenty four hour time point.
• Figure 4 shows the effects of these various fractions upon the area of the culture surface covered with cells during a six day culture period, expressed as the cell outgrowth index (calculated as described above). Figure 4 also compares these results in a manner that relates the increase in cell culture area over the six day culture period (expressed as a ratio of that at twenty four hours) to the cell attachment data (at twenty four hours).
• Figure 4 makes an interesting comparison between the different serum fractions. Similar levels of cell attachment after twenty four hours culture were seen with the treatments having either serum-free medium alone, or serum-free medium containing 20 % (v/v) Fraction 3 (having serum factors of 1000 to 10,000 daltons in size) or 20 % (v/v) of Fraction 2 (having serum factors of 1000 to 30,000 daltons in size) or 20 % (v/v) of Fraction 1 (having serum factors of 1000 to 100,000 daltons in size) or the recombination of Fractions 1, 2 and 3. Notwithstanding the initial cell attachment being equivalent for these different treatments, there was a marked difference in the increase in cell area.
• the experimental format used in these experiments is representative of corneal onlays previously described, providing for both cellular access to nutrients and other small molecules and also providing for a surface chemistry that supports corneal epithelial cell attachment.
• tissue trophic factors as exemplified by factor(s) in serum, that are required for optimal epithelial cell coverage of a synthetic polymer surface such as the tissue culture polystyrene surface.
• factor (s) are greater than 1000 daltons in size and are also retained on filters that exclude molecules of 10,000 and greater.
• the activity, or a portion thereof, can be identified as falling within the molecular weight range of 10,000 to 75,000 daltons (determined from being excluded by the Amicon PM10 membrane filter and included by the Amicon XM100 membrane filter).
• These serum factors are not the adhesion factors Fibronectin or Vitronectin.
• Tissue factors such as serum stimulate epithelial cell outgrowth from epithelial islands, and this effect is not dependent upon the serum adhesive proteins fibronectin and vitronectin.
• this activity has been shown to be greater than 10,000 daltons in size.
• the comeal onlays according to this invention have a porosity sufficient to allow passage therethrough of tissue fluid components having a molecular weight greater than 10,000 daltons.
• Corneal onlays may be prepared from a polymer comprising crosslinked macrocycles that are made up of units of the following formula
• A is alkylene or alkenylene each having from 3 to 10 carbon atoms and each of which may be substimted by one or more R 3 radicals, each of R 1 and R 2 , independently of die other, is hydrogen or lower alkyl, and R 3 is lower alkyl, fluorinated lower all yl or a siloxane radical.
• R 3 is lower alkyl, fluorinated lower all yl or a siloxane radical.
• onlays are prepared from copolymers of the formula I as described in EP-A-538,188, which is incorporated herein by reference in its entirety. Specifically, 100 mmol of tricyclo[ 1.1.1.0 1,3 ]pentane and 150 mmol of ethyl acrylate are copolymerized for two days in diethyl ether at room temperature, excluding oxygen. A sheet with a glass transition temperature of 100°C is obtained. The sheet is compression molded to give corneal onlays. Pores are formed in the onlay as described as above.

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