Patent Application: US-60679603-A

Abstract:
a membrane for corneal implant or keratoprosthesis comprising a biological polymer and a polyacrylamide is described . the mixture of both polymers produces a hydrogel that becomes a transparent film or membrane upon drying . the resulting device and tissue engineered implants are useful for biomedical applications of the cornea , such as tissue repair and transplantation .

Description:
the invention is in the field of opthalmology . in embodiments , the invention relates to a membrane which serves as the basis of a substitute for corneal implant or graft ( i . e ., penetrating keratoplasty ), keratoprosthesis and to a replacement of amniotic membrane used as corneal implant and support for cultured epithelial and precursor cells . in embodiments , the substitute , i . e . the membrane or corneal implant of the invention , can be transplanted onto the cornea to assist cornea wound healing after eye surgery , cornea injury and disorders , or excimer laser surgery . in an aspect , the invention relates to a corneal implant or keratoprosthesis comprising a membrane of the invention , which is produced by the combination of biological polymer , such as collagen , and a polyacrylamide , e . g . a polyalkylacrylamide such as poly ( n - isopropylacrylamide ) ( pnipaam ). the use of such synthetic polymers is advantageous as it provides good transparency and strength to the membrane , contributing to its use as a corneal implant . prior to the studies described herein , pnipaam and its derivatives have not been used in any cornea or ocular device . in an embodiment , the biological polymer is collagen , in a further embodiment , type i telocollagen , a soluble form of non - pepsinized collagen that may for example be purified from rat tail tendon ( rtt ). other sources of collagen , including mammalian sources , such as bovine , porcine , and human telocollagen or atelocollagen ( pepsin or pronase digested telocollagen ) may also be used . collagen may also be from a recombinant source . in the examples described below , the synthetic polymer pnipaam homopolymer has been used . “ corneal implant ” as used herein refers to any material or device which may be applied to or comes in contact with the cornea of a subject . in an embodiment the subject is a mammal , in a further embodiment , a human . such an implant may be used for repair or replacement of a cornea or a portion thereof , or as a cornea wound dressing , due to defects for example caused by corneal injury or disease . in a preferred embodiment , the implant is substantially transparent . the pnipaam family of polymers is one of the few synthetic materials which support cell in - growth and growth of polymer encapsulated cells ( vernon et al 1999 ; stille et al 1999 ). pnipaam has been extensively studied in vitro for cell culture use that demonstrates its non cytotoxicity ( takezawa et al 1990 ). takezawa et al . ( 1992 ) have combined collagen with pnipaam as cell culture substrate in order to form spheroids , producing only a thin ( 2 μm ) layer of collagen - pnipaam . moreover , u . s . pat . no . 6 , 030 , 634 ( wu et al . ; feb . 29 , 2000 ) describes the combination of pnipaam with gelatin ( denaturated collagen ) which results in a polymer gel having the properties of an interpenetrating polymer network structure with a shrinking temperature of 35 ° c ., having surgical application for the repair of damaged tissue , but not replacement or substitution . further , corneal applications are not contemplated nor mentioned . an improved shrinking rate for a drug delivery system using such network structure is also described . furthermore , activated pnipaam has also been conjugated to protein a , various enzymes , biotin , phospholipids , rgd peptide sequences , and other interactive molecules such as monoclonal antibodies ( as described in u . s . pat . no . 4 , 780 , 409 ; oct . 25 , 1988 ; monji and hoffman , 1987 ). the polymer gel compositions of the present invention comprises a matrix , such as an interpenetrating pnipaam - collagen hydrogel network which leads to strengthen the final collagen - based product while keeping its transparency . in embodiments , the membrane of the invention may be made with different ratios of collagen to pnipaam . in an embodiment , the collagen to pnipaam ratio is in the range of about 0 . 2 : 1 . 0 to 1 . 0 : 0 . 2 ( w / w ). in an embodiment , the collagen to pnipaam ratio is about 0 . 3 : 1 . 0 w / w , which is the preferred ratio for corneal implant design . collagen can be replaced or mixed with other biological polymers , including proteins such as gelatin , fibrin - fibrinogen , glycosaminoglycans , elastin , and glycoproteins or peptides such as adhesive peptide sequences , cytokines , chemokines or growth factors . the membranes can be layered together , in a further embodiment laminated together , to form a composite material in which thickness can be a controlled parameter . in addition , a multilayered membrane may result in the guidance of the stroma regeneration within the implant . as such , in an embodiment , a corneal implant of the invention may comprise a plurality of the membranes of the invention . the layered membranes in such a structure may be heterogeneous , i . e . having different polymeric compositions and ratios of components . in addition , a mixture of crosslinked and non - crosslinked membranes may be used . the membranes in such a layered structure may be crosslinked to one another using for example the crosslinking agents and methods described herein . a variety of agents or compounds ( crosslinking , plasticizer , etc .) can be introduced during manufacturing of the membrane before and / or after the formation of the membrane . depending on the step in which the agent or compound is added , the physicochemical and biological properties of the membrane may vary . in embodiments , the membrane of the invention may be crosslinked with different agents . in an embodiment , edc and nhs are the crosslinking agents . pnipaam can be modified physically to provide different biophysical and biological properties for different ophthalmic applications ( e . g ., wound dressings ) and mixed with collagen . in a further embodiment , collagen membranes can be crosslinked chemically either during the procedure or afterwards . in an embodiment , the membrane is transparent and its thickness range is between about 20 μm and about 400 μm , depending upon hydration . in a further embodiment , the thickness of the membrane is in the range of about 50 μm to about 100 μm . different thicknesses can be produced , as well as different front and back curvature for cornea replacement . in an embodiment , the membrane comprises an elastic modulus of less than about 10 mpa , a tensile strength at break of less than 6 mpa , an elongation at break of less than 80 % and a tensile energy to break of less than 2 mj . in an embodiment , the membrane of the invention is strong enough to support suturing stresses . its physical properties such as strength and elasticity can be modified by crosslinking ( e . g . with carbodiimide - type crosslinking agents such as edc ), but other crosslinking agents ( e . g ., nhs ) and plasticizers such as glycerol can be used . the physical properties of the membrane may be modified for example as a function of rehydration , or via the presence of lipids and / or proteins . physical modification of pnipaam can lead to different properties that may enhance biological and biophysical characteristics for corneal implants or membrane substitutes . the membrane of the invention provides a device which remains robust and optically clear in the eye for extended periods of time . in the embodiments of the invention , there is cell ingrowth into the polymeric membrane , and epithelialization over the membrane with no hyperplasia . the membrane may induce the deposition of organized extracellular matrix proteins resembling the cornea stroma with close reorganisation of the newly deposited collagen and stromal cells . the membrane of the invention may further comprise / have associated with it various compounds e . g . drugs , biological materials ( e . g . peptides / proteins , lipids , etc . ), crosslinkers , plasticizers , cytokines , etc . to fulfill or further contribute to an aspect of the desired functionality of the corneal implant in any particular situation . such agents or compounds may be introduced during the making of the membranes or after their formation . the invention further relates to a method of preparing a corneal implant of the invention , comprising combining a biological polymer and a polyacrylamide ( or providing such a mixture ), and allowing the mixture to dry to form a membrane for use as a corneal implant . in embodiments , the biological polymer is collagen provided as a collagen solution of about 1 - 6 mg / ml , in a further embodiment , about 3 . 0 - 3 . 5 mg / ml ( e . g . in 0 . 2n acetic acid in water ), and the polyacrylamide is pnipaam provided in a solution of about 2 - 10 %, in a further embodiment , about 4 % in water . in an embodiment , the solution of the biological polymer and the polyacrylamide may be combined in a ratio of about 1 : 1 . the polymer mixture may be poured into a suitable dish ( e . g . a plastic culture [ e . g . non - treated ] dish ) or mold ( e . g . a lens or cornea mold ) for drying . drying may be allowed to proceed until for example a constant weight ( e . g . about 1 - 10 % water residue , in a further embodiment about 7 % water residue ) is reached . drying may be performed under sterile conditions , under a laminar flow hood . drying may be performed at room temperature ( 15 - 22 ° c . ), for example for a period of about 2 - 4 days . the preparation may further involve a crosslinking step , using for example a crosslinking agent such as edc and / or nhs . the resulting membrane is typically rehydrated with a suitable solution prior to its use for cornea implantation . the invention further relates to the use of a membrane of the invention as a corneal implant , e . g . for application to the cornea for repair or replacement of damaged or otherwise inadequate cornea , thus for the treatment of a corneal defect , disorder , injury or disease . the invention further relates to a method of treating a condition characterized by a corneal defect ( e . g . corneal disorder , injury or disease ), said method comprising administering to a subject in need thereof a corneal implant of the invention . the invention further relates to a commercial package comprising a membrane of the invention together with instructions for its use as a corneal implant , e . g . for application to the cornea for repair or replacement of damaged or otherwise inadequate cornea , thus for the treatment of a corneal defect , disorder , injury or disease . although various embodiments of the invention are disclosed herein , many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in this art . such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way . numeric ranges are inclusive of the numbers defining the range . in the claims , the word “ comprising ” is used as an open - ended term , substantially equivalent to the phrase “ including , but not limited to ”. the following examples are illustrative of various aspects of the invention , and do not limit the broad aspects of the invention as disclosed herein . throughout this application , various references are referred to describe more fully the state of the art to which this invention pertains . the disclosures of these references are hereby incorporated by reference into the present disclosure . a sterile rtt collagen solution of 3 . 0 - 3 . 5 mg / ml ( w / v ) in acetic acid ( 0 . 02n in water ) is made , and kept at 4 ° c . a 4 % ( w / v ) solution of pnipaam homopolymer is made in water ( ddh2o ) and sterilized by autoclaving or by filtering ( 0 . 24tm ). the 4 % solution of pnipaam is diluted to a 1 % solution with sterile ddh2o . the 0 . 30 - 0 . 35 % solution of collagen and the 1 % solution of pnipaam are mixed ( 1 : 1 vol / vol ) in a sterile test tube at 4 ° c . by pumping with a pipette until well dispersed . cold mixing will avoid any premature gelification or fibrillogenesis of the collagen during this procedure . collagen - pnipaam is then poured into a plastic dish ( non - treated culture dish ) or a mold ( e . g ., lens or cornea mold ) and left air - drying under sterile conditions in a laminar flow hood for at least 2 - 3 days at room temperature . after drying to constant weight (˜ 7 % water residue ) at room temperature , under laminar flow at relatively constant humidity , the formed membrane is removed from the mold after soaking for a short period ( hours ) in a sterile buffered solution ( e . g ., hank &# 39 ; s balanced salt solution or hbss ) at room temperature . the sterile membrane is ready to be used for cornea implantation or for a transplantable membrane support for epithelial cells . conversely , rtt collagen solution that is not mixed with pnipaam as reported above , and only diluted in water or buffer at 1 : 1 ratio , fails to become a transplantable and suturable collagen membranes after drying . a sterile fibrinogen ( fraction i ; type i - s from bovine plasma ) solution ( 3 mg / ml in hbss ) is mixed with a 1 % pnipaam solution as reported in example 1 . during mixing , thrombin ( 50 u / ml purchased from parke davis ) is added at a final concentration of 0 . 03 : 1 v / v ( thrombin : fibrin ), and incubated for 10 - 15 min at 37 ° c . to allow gelification ( hydrogel ) in a culture well . this procedure strengthens the fibrin gel , probably by aiding the development of a filamentous network as shown on fig1 c . a sterile rtt collagen viscous solution and a 1 % ( w / v ) solution of pnipaam are made as described in example 1 . the 0 . 30 - 0 . 35 % solution of collagen and the 1 % solution of pnipaam are mixed ( 1 : 1 vol / vol ) with a 10 % stock solution of 1 -( 3 - dimethylaminopropyl )- 3 - ethyl carbodiimide hydrochloride ( edc ) in water to give a final edc concentration of 0 . 5 ; 0 . 1 ; or 0 . 05 % ( w / v ) in a sterile test tube at 40 ° c . by pumping with a pipette until well dispersed . the collagen - pnipaam - edc mixture is then poured into a plastic dish ( non - treated culture dish ) or a mold ( e . g ., lens or cornea molds ) and left air - drying under sterile conditions in a laminar flow hood for at least 2 - 3 days at room temperature . after drying to constant weight (˜ 7 % water residue ), an interpenetrating network of pnipaam in cross - linked collagen is formed . each membrane is removed from the mold after soaking in a sterile buffered solution ( e . g ., hbss ) at room temperature . among the three different concentrations of edc tested , the one with the lowest concentration gave the most reliable procedure to toughen the membrane . the sterile membranes can be soaked in hbss containing glycine ( 5 % in hbss ) to remove residual unreactive crosslinking agent . membranes are further rinsed in hbbs ( at least 3 times ). thus , they are ready to be used for cornea implantation or for a transplantable membrane support for epithelial cells . membranes from examples 1 and 3 are used to form a thicker membrane that are produced by sticking one or several membranes together . for this procedure , hydrated membranes are bound to each other using a solution of 0 . 5 % edc solution . the resulting membranes of various thicknesses are left to dry , then rinsed as described in example 1 . one rehydrated membrane is left bonded to the bottom of the dish or mold the other membrane to be glued to the first one is released from its dish or mold the edc solution ( 10 μl of 10 % stock edc solution per cm 2 of membrane ) is poured over the unreleased membrane . edc plays the role of a glue between the 2 membranes . at once , the second membrane ( released one ) is laid down onto the solution and stuck to the unreleased membrane , combined membranes are dried , later rehydrated , and finally rinsed as described in example 1 . different combinations can be made such as one crosslinked membrane sandwiched between two uncrosslinked membranes , or other alternatives . a variety of agents or compounds ( e . g ., crosslinking , plasticizer , drugs , cytokines ) can be introduced during the making of the membranes from examples i to iv . compounds can be introduced either during the mixing of both collagen and pnipaam or after the formation of a membrane . the latter can be dried , thereby , the agents can be introduced during the rehydration process . otherwise , the agents can be introduced on the rehydrated membrane . the physicochemical and biological properties may vary ( table i ). the rehydrated implants from examples 1 and 3 to 5 are strong enough to support surgical manipulation , suture thread and needle ( table i ). they are relatively flexible . the presence of proteins and lipids ( e . g ., albumax ®) is likely to strengthen the membranes . similar properties are also observed in the presence of glycerol combined at the time of mixing collagen and pnippaam solutions . the membrane thickness can vary from 20 to 400 μm after hydration ( fig1 ). table i : shearing characteristics of the resulting membranes produced in different experimental conditions . shearing forces are roughly determined by handling the specimen between 2 forceps in opposite directions . the membranes result from the mixture of collagen and pnipaam . collagen can be prepared by making a solution either in acetic acid at ph 4 . 0 ( coll ( ac . ac .)) or in water at ph 3 . 0 with hcl ( coll ( hcl )). a crosslinking agent such as edc is introduced either during the mixture or after the formation of a membrane . the latter fragilizes the membrane . glycerol can be used as a plasticizer . albumax ( a lipid rich bovine serum albumin , purchased from gibco / brl ) can be added during rehydration with hank &# 39 ; s balanced salt solution ( hbss ). other components can be introduced at different periods to induce different properties . composite collagen - pnipaam membranes from example 1 were uncrosslinked ( 1m ) or crosslinked by a carbodiimide derivative , the 1 -( 3 - dimethylaminopropyl )- 3 - ethyl carbodiimide ( 1mes ); other membranes were layered by chemically binding 2 sheets of the collagen - pnipaam composite which were uncrosslinked ( 2m ) or crosslinked ( 2mes ). chemical binding was performed with the same carbodiimide . biomechanical tests were performed on membranes using an instron apparatus ( canton , mass ., usa ). strips of membranes were placed between the two grips of the apparatus and uniaxial tension was applied . elongation of the membranes were recorded and analyzed ( table ii ). table ii . biomechanical assessments of uncrosslinked and crosslinked membranes . means and standard errors of means are presented . these results were from 2 separate experiments of 3 samples each . carbodiimide crosslinking of collagen - pnipaam improved the strength of the membranes , particularly with the one - layer membrane , which exhibited an associated increased elasticity and toughness . the two - layered membranes were very elastic , probably due to a sliding effect within the 2 membranes . although the strength of the two - layered membranes was weaker than the one - layer membrane , the overall toughness was sustained . the biomechanical properties of the membranes are similar to those reported for the human cornea ( zheng et al ., 2001 ; wang et al ., 2001 ). human stromal cells or keratocytes were seeded at 5 × 10 5 cells per cm 2 onto the collagen - pnipaam membranes from example 1 . they were then grown in the presence of culture medium and serum for 7 days . stromal cells form a cell layer on the surface of the membrane as seen on histological section ( see fig1 ). in other experiments , pnipaam was dissolved in the culture medium . human endothelial cells were grown in the presence of the polymer for 5 days at different concentrations ( 0 ; 0 . 06 ; 0 . 12 ; 0 . 25 and 1 . 0 mg / ml ). endothelial cell numbers were determined by spectrofluorometry after staining cells with a dna - specific dye ( hoechst 33342 ). cell growth was not impaired by increasing concentration of pnipaam ( no significant difference by anova test ) ( see fig2 ). collagen - pnipaam membranes from example 1 were implanted into the corneas of 15 rabbits . controls included sham operations and use of human amniotic membrane . fig3 a & amp ; b show rabbit corneas at 3 days after surgery and fluorescein staining of the wound , respectively . by 3 days post - operative , re - epithelialization had occurred and fluorescein staining was minimal with the collagen implants . conversely , 3 out of 9 rabbits with amniotic implants did not epithelialize by 3 days ( fig3 c & amp ; d ). after 6 days a complete epithelialization was observed by direct observation and there was no fluorescein staining . this was comparable to observations made on corneas covered with amniotic membranes , which are now being used clinically to treat corneal epithelial damage due to disease or injury . biopsies were taken at 12 days and show a complete epithelialization of the surface of the collagen - pnipaam membrane that was in place and non degraded ( fig4 a ). around the implant the formation of an oriented stroma was observed , that is organized like the nearby stroma of the cornea . there was an absence of immune and inflammatory reaction . furthermore , histological section of the cornea with the amniotic membrane showed a partial degradation of the membrane and a hyperplasia of the epithelial cell layer at the nearby of the cornea pocket ( fig4 b ). in addition , regenerating nerve axons were observed at the edges of the polymeric membrane underlying the wound at 11 days post - surgery and nerves had penetrated the periphery of the polymers by 28 days . by 3 months post - surgery , there was nerve re - growth into the polymeric membrane ( fig4 c ). moreover , fine neurites are observed within the polymer - epithelial interface . nerve regrowth into the membrane and overlying epithelium are consistent with previous observations of cornea nerve regeneration in rabbits ( tervo et al ., 1994 ) and demonstrate the feasibility of transplantable artificial corneas that promote nerve in - 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