CA2066660C

CA2066660C - Method for achieving epithelialization of synthetic lenses

Info

Publication number: CA2066660C
Application number: CA002066660A
Authority: CA
Inventors: Cary Reich; Jeffrey Forsberg; Harold Levy; Jean Toner-Webb
Original assignee: Surmodics Inc
Current assignee: Surmodics Inc
Priority date: 1989-09-15
Filing date: 1990-09-05
Publication date: 2002-07-30
Anticipated expiration: 2010-09-05
Also published as: DE69029735T2; WO1991003990A1; ATE147613T1; EP0491860A1; KR920702975A; US6090995A; BR9007643A; CA2066660A1; JPH05501971A; EP0491860B1; EP0491860A4; DE69029735D1

Abstract

Synthetic surfaces such as surfaces of implantable prosthetic devices are modified to enhance their ability to support the growth, migration and attachment of epithelial cells. A surface modifier composition is covalently bound to the synthetic surface, and an epithelial cell-supporting coating is applied to the modified surface. The surface modifier composition may also include an epithelial cell-supporting material.
The invention is particularly suited towards the modification of synthetic epikeratophakia lenses.

Description

~06~66~
14f32-132 BB:520:14 MF'PHOD FOR ACHTLVTNG LPI'fHLLTALT~ATTON
OF SYN'fIjG'fTC L~NSL:S
Background of the Tnvention 1. 'Pechnical Field Tlae present invention relates to methods for modifying synthetic surfaces to support Lhe attachment, growth and migration of epithelial cells both in vitro and in vivo, as well as the modified surfaces themselves. More specifically, the invention relates to methods for modifying the tissue-contacting surfaces of synthetic, implantable prosthetic devices, especially contact lenses, to better support the attachment, growth and migration of epithelial cells.
The invention also relates to the prosthetic devices themselves.

2. Brief Description of the Background Art °fhere are a number of prosthetic devices which necessarily or desirably can be implanted either completely or partially beneath epithelial tissues. It is to be understood that reference to "epithelial°' tissues herein includes epidermal i.issue as well as other epithelial tissues. Implantation beneath the epithelium may be done for purposes of fixation of the device relative to other tissues and/or for cosmetic purposes. examples of implanted prostheses include dental prostheses such as artificial teeth and bridgework, hearing aids, dermal implants, vascular access devices, such as those associated with hyper-al.i.merntation, colostomy devices and prosthetic corneas.
While t3ie present invent ion will be described with reference to prosthetic corneas for subepithelial implantation, and with specific reference to an epikeratophakia lens, it will be readily understood that the invention is not so limited.
The permanent implantation in the eye of a synthetic epi.keratophakia lens has major advantages over operations such as radial keratotomy to correct severe vision prablems. Tmplanting the synthetic epikeratophakia lens does not involve compromising the anterior chamber, for example. In the implantation procedure, the epithelial layer is removed via a trephine and scrape, the wound is undermined and the lens is i:ucked into place. Re-epithelialization of the lens is expected to result in a perrnanent correction of vision for the patient. By "re-epithelializa~ion" it is meant not only the growth and migration (or °spread.i.ng°) of epithelial cells, but also the attachment and stabilization of these cells.
Re-epithelialization of the implant is important for a variety of reasons. For example, re-epithelialization is very important in order to ensure long term anchorage of an implant. The layer of new cells also acts as a barrier to prevent tear-barn and other materials from depositing on the lens surface.
Unfortunately, many materials which exhibit beneficial properties Waen formed into prosthetic devices (such as stability and lack of immune response) do not adequately support the growth, migration and attachment of epithelial cells.
'1'1ie methods and modified synthetic surfaces of the present invention also are useful for the in vitro growth of epithelial cells. Lpithelial cells grown in 2osssso the laboratory upon surfaces modified according to the present invention exhibit attachment, growth and migration quite s3.mi:Lar to the in vivo growth pattern of epithel.i.a1 cells.
Sur~unary of the :Invention In orre aspect, the present invention relates to a method for modifying a synthetic surface, comprising applying to a synthetic surface a surface modifying cornposilion comprising a polymer having pendant functional groups capable of being converted to nitrene groups and then converting the functional groups to nitrenes and thereby covalently binding the surface modifying composition to the synthetic surface. The surface modifying composition optionally includes a material which supports or enhances the attachment, growth and migration of epithelial cells.
In another aspect, the: present invention relates to a method for coating a synthetic surface with an epithelial cell--supporting coating, comprising applying to a syn ttretic surface a surface modifying composition comprising a polymer having pendant functional groups capable of being converted to nitrene groups, converting the functional groups to nitrene groups and thereby covalently binding the surface modifying compos.i.tiora to the polymeric surface to thereby rnod.ify the synthetic surface, and subsequently applying an epithelial cell-supporting coating onto the modified syntLietic surface. In a preferred embodiment, the epithelial cell supporting coating is provided as a plurality of layers to more closely resemble native tissues. The coating also may be crosslinked so that it is stabilized and resistant: to the action of.
proteases.

r The present invention also relates to the modified synthet.i.c surfaces ner se. ~rhus, one aspect of the invention relates to a mocliFiQd synthetic surface for supporting the attachment, growth acrd migration of epithelial cells, comprising a synthe',;ic surface and a surface modifying composition covalently bound to the synthetic surface, wherein the surface modifying composition is capable of supporting epithelial cells.
In another aspect the invention provides a modified synthetic surface for supporting the growth and migration of epithelial cells, comprising a synthetic surface, a surface modifying composition covalently bound to the synthetic surface, and an epithelial cell supporting coating disposed on the modified surface.
'i'he invention also provides a treated prosthetic device for subepithelial implantation in a human or animal comprising a prosthetic device having a surface modifying composition covalently bound thereto, the surface modifying composition comprising a polymer having a plurality of pendant amino or carboxyl groups, and an epithelial cell supporting coating thereon.
In a particularly preferred embodiment, the invention provides a treated epikeratophakia lens comprising a synthetic lens, a surface modifying composition covalently bound to the lens, the surface modifying composition comprising a lysine polymer modified to contain pendant groups derived from N--hydroxy-succinimidyl-4-azidobenzoate or methyl 1-9-azidobenzoimidate, and an epithelial cell-supporting coating bound to the surface modifying composition and disposed on an exposed surface of the thus-treated lens. 't'he epithelial cell-supporting coating preferably is covalently bound to the surface modifying ~osso~o composition. 'fhe epithelial cell-supporting coating also may be crosslinked in situ.
Deta.i.led Description of the Preferred ~tnbodimenLs According to the present invention, a synthetic 5 surface which ordinarily is not well su3.ted to the binding of proteins is rendered more suitable fox , protein binding by the application of a surface modifier composition. 'This aspect of the invertti.on is applicable to a wide variety of synthetic surfaces.
'this spec.i.fication describes the invention in connection with hydroqels of, e.g., N-vinylpyrrolidone / methyl methacrylate copolymers commonly employed in implantable lenses, but is not so limited. ether hydrogels which may be modified according to the present invention incltade polymers of 2-hydroxyethylacrylate (e. g. polymacon), various copolymers of 2-hydroxyethylmethacrylate (e. g, haf:ilcon A and B, vifilcon A, tetrafilcon, dimefilcon, bufilcon, perfilcon, etc.), copolymers of N-vinylpyrrolidone (e. g. lidofilcon A and B, scafilcon A, surfilcon, vifilcon, filcon YA, etc.) and hydrogels containing collagen, far'example as described in U.S, patents 4,952,925 and 4,388,928 and in P.N.A.S., USA, 77 (No.
4), 206~t-2068 (1980).
The invention is also useful for providing modif.ieci surfaces on vascular graft implants. Such implants are fabricated, for example, from Dacron, polyu.reLhaaies, polypropylene, silicone, crosslinked collagens, collagen-plastic composites or phospho-lipid 3U paiymers.
Ilydrogels are preferred constituents of epikeratoptrakia lenses due to their permeability and, consequently, their ability to transport oxygen, glucose and other nutrients and metabolites. 'i'issue 20~~~~~

culture plates are other synthetic surfaces which are enhanced by the methods of the present invention.
'1'ha surface modifier composition can be based on virtual:ly arty polymer having a plurality of pendant groups. Preferred polymers include a plurality of pendant amino and/or carboxyl groups and are exemplified by poly(amino acids such as poly(lysine).
Other polyamines can be used, e.g. polyethyleneimine, as can other compounds with high amine content. In the alternative, bio-compatible compounds with high carboxyl content may be used.
The molecular weight or chain length of the polymer employed in the surface modifier composition is not critical to the invention. For example, poly-L-and poly-D-lysines of 90,000 to 990,000 daltons have been used successfully in the surface modification method of. i:he invention . Polymers of lower (or higher ) molecular weight also are useful.
In order to provide a highly stable modified surface, the surface modifier composition is covalently bound to the synthetic surface, t~ovalent binding is accomplished via the use of appropriate coupling agents. In general, in surface modifier compositions based on polymers having a plurality of pendant groups, the pendant groups first are converted into functional groups capable of forming highly reactive radicals.
The polymers then are covalently bound to the synthetic surface by converting the groups to their corresponding highly reactive functional groups, preferably via photolysis. The highly reactive functional groups then covalently couple with the synthetic surface.
Advantageously, the synthetic surface does not have to be derivatized or otherwise specially treated prior to the application of tire surface modifier a composition. Pre-treatment of a hydrogel surface with a methyl alcohol solution (which causes a swelling of tt~e cok~olyrner) does enhance b.i.nding, however, and thus ~t.s recommended.
One preferred binding rnetarod is to covalently couple to the synthetic surface a surface modifier composition based on a poly(lysine} which has been modified so that about 10 rnol percent of the pendant amino groups have been modified by a functional group containing a moiety capable of being converted into a nitrene or other highly reactive group. Nitrene groups are highly reactive with the synthetic surface and are formed, for example, by the photolysis of an azido (-N3) group.
A portion of the pendant amino groups of a poly(lysine) polymer can be derivatized by reacting the lysine polymer with N-hydroxysuccinimidyl-9-azidobenzoate ("HSAB"), a polyfunctional compound which contains an amine-reactive group as well as an azido group. t)pon incubation of the hydrogel lens with the ttSAB-derivatized poly(lysine), and photolysis with UV
light (typically in the 265-275 nm .range), the poly(lysine) chains are covalently bound to the hydrogel. Crosslinking among polymer chains also occurs. Methyl 1-4-azidobenzoimidate (MABI) is another compound useful for modifying the lysine polymer.
Those skilled in the art will be able to select other aL~proC~r:iate polyfunctional coupling agents.
The present invention also provides for the inclusion of a variety of other anaterials in the surface modifier compositions. If desired, the compositions can contain medicaments and/or other Irlilterial.s which promote wound healing. ror example, an antibiotic material can be dispersed in thre surface 2a~~~~0 a modifying composition. Suitable antibiotics include gentamicin, neomycin, bacitracin and the like. In addition, other antirnicrobial agents, anriviral agents, anti.-inflammatory agents, anti-protease agents, hormones, vitamins, analges.i.cs, ahelat.ir~g agents, mitogen.i.c agents (including growth factors) and the like may be incorporated in the surface modifying composition.
Preferred materials for incorporation into the surface modifier compositions are biological maU:erials which are known to support the attachment, growth and migration of epithelial cells. 'these materials are referred to as "epithelial cell-supporting" materials herein. Advantageously, materials to be incorporated within ttx~e surface modifying compositions do not need to be modified or derivatized. Useful native, underivatized materials include (but are not limited to) collagen types I, III, IV and/or others, fibronectin, laminin, chondroitin sulfate and virtually any other protein or other desired material desired to be covalently attached to the synthetic surface. If desired, these materials may be altered, derivatized or crosslinked prior to being combined with the HSA13-rnockified poly(lysine) and applied to the hydrogel.
Upon photolysis, the included material is crosslinked by some of the nitren,e groups attached to the poly(lysine), whereas other nitrene groups attached to the poly(lysine) covalently bind to the lens surface.
Thus Lhe lens (or other synthetic surface) is now coated with a covalently-attached layer of a surface modifier composition.
A derivatized poly(lysine) molecule is prepared by .i.ncubation of the native poly{lysine) with the bifunctional crosslinker HSAB under appropriate r ,.
..
conc~ii:ions. Any urrreacteci crosslinker is removed by ultrafill:ration or other non-destructive methods suctr as dialysis. In addition to paly(lysine), other polymers capable of binding to a bifunctianal crossli.nker containing a secondary group capable of forrninc3 a highly-reactive radical upon exposure to light may be used in this process.
Lxam~le I
Aar HSA13-derivatized poly(lysine) is prepared according to the procedure described in detail in Example V. HSAB is available from Pierce Chemical Company, Itockford, IL, USA. A hydrogel lens prepared from err N-vinylpyrrolidone, methyl methacrylate copolymer is placed in a chamber anterior side up and incubated with the 1-iSAB-derivatized poly(lysine) solution (2.O to 10.0 mg/ml, preferably 5.0 mg/ml) (which is hereinafter referred to as "HSAB-plys"), with or without 10-20~ MeCH added to swell the hydrogel during coating. The lens is then irradiated with UV
light for 4 to l0 minutes per coat fox 5 to 10 coats.
The lens is then extracted in aqueous solutions of plain water, saline or 0.05 M glacia2 acetic acid to remove unbound IISAB-plys.
The coating is visualized on a test lens from the lot by a novel Coomassien° staining/destaining process that visualizes only covalently bound I~ISAB-plys on the hydroge:l lens . '1'he coated lens and a control (uncaated) lens are submerged in a stain composed of 0.1 to 0.25 ~ (w/v) Coomassie Brilliant Blue It (Sigma B-0630), 7 to 10 ~k glacial acetic acid and 25 ~
methanol in water (see Laemmli, U.K., Nature 227, 680 (170). Alternately, a stain composed of 0.1 to 0:25 (w/v) Coomassie Brilliant Blue R, 0 to 10 ~ glacial ~ :': .. ', ,,. . , , acetic acid, 45 ~ methanol and 45 ~ acetone (balance water, methanol and/or acetone) rnay be used. Wtrile the Laee~unl..i. process employs acetic acid to f.i.x the proteins) of interest to an acrylamide gel, the use of acetic acid is not requ.i.red in this process as tire poly(lysine) i.s covalently bound to the synthetic surface .
'file lenses are incubated .i.n the stain for 2U to 30 minutes. The lenses are extracted with three 2U-minute 1U extract.i.orrs (or until the control lens is completely clear) of destaining solution composed of 0 to 10 (w/v) glacial acetic acid, 45 ~ methanol and 45 ~
acetone (balance water, mettranal and/or acetic acid) to remove the unbound Coomassie stain. ~i'he acetone advantageously swells the hydrogel to aid the release of unbound stain. Under these staining/destaining conditions urxbound iFSAB-plys (or plys alone) is removed from tire lens and only lenses to which the poly(lysine) is covalently bound retain the stain.
'1'ile5e lenses, having surfaces modified with HSAF3-plys alone, are capable of binding epithelial cells, brrt the cells do not seem to spread well. Thus it is desirable to bind collagen o~ other epithelial cell-srrpporting materials to this poly(lysine) i.n order to support epithelial growth. This may be done in one step, by incorporating an epithelial cell-supporting material within the surface modifier composition, or in several steps, by providing an epithelial cell-srapporti.ng coating over the surface modifier composition, Variations in the destaining solutipn mentioned above are possible. In general, destaining solutions contain ing 1U-45 ~ acetone, 25-45 ~ methanol, 10-25 ~
glyme (dirnethoxyethane), balance water and/or glacial 11 ~ooss~o acetic acid (FIOAc) are useful for removing unbound Coomassie-type stain while swcalling the hydrogel (or other polymer). Specific examples are set forth below wherein all amounts are percent (w/v):
Acetone MeOIi IIzO HOAc Olyme 1) 45 45 0-10 0-10 2) 1U 45 35 - 10 3) 25 - 50 - 25 4) 10 25 55 - 1U
The acetone component of the destaining solution appears to function as a solvent which softens the poly(methyl methacrylate) component of the hydrogel to aid in the release of unbound stain. Other solvent s WhlCh function in a similar manner may be employed in lieu of or in combination with acetone. Of course, the choice of particular solvents will be based on the composition of the synthetic surface to be treated in accordance with the invention.
Lxample II
In a one step method, ~ISAB-plys and unmodified collagen may be simultaneously covalently bound to, the hydrogel surface in the following manner. A hydrogel leas is incubated with a solution containing both IISAB-plys and combinations of collagen I, III and IV
(optionally along with filaronectin, laminin, chondroitin sulfate or any other desired material) in a range from 100:1 to 1:100 ratio by weight HSAB-plys:collagen (or other ratios allowing some of the IiSAB moieties on the poly ( lysine ) to be used for coupling to the hydrogel lens, and Borne to be used for coupling to the collagen). Multiple coats (5-10) are coupled via irradiation onto the lens. 'Phe protein coating may be visualised by staining as described above. Alternate specific stains may be used to -.

distinguish collagen or other materials from the poly(lysi.ne) staining; however, Coomassie stain can also distinguish the collagerx/po7.y(lysine) coating from a poly(lysine) coating using the procedure described in Lxample z. As further evidence of the covalent binding of collagen and paly(lysine) to the hydrogel surfaces, autoclaving these coated lenses results in retention of the collagen/poly(lysine) coating as visual.i.xed by the staining procedure. However, cell culture results an such autoclaved lenses are negative, i.e. cells do not adhere or spread on these lenses. Thus, even when the collagen is denatured by autoclaving, it is still covalently bound to the lrydrogel surface.
Lxample TII
In a two step method, HSAm-plys and collagen (or Other moleCilles ) rnay be bound to the surface of the hydrogel. rirst, IiSAB-plys is covalently bound to the surface by incubation with the lrydrogel in the presence of UV light, as discussed above. Secondly, collagen, and/or other molecules containing carboxyl groups are incubated with the poly(lysine) coated lens in the presence of a crosslinke.r such as z-ethyl-3-(3-dimethylaminopropyl)carbodiirnide (~~EDC~~, available from Pierce Chemical Company, Rockford, I1,, USA), or other ' carbodiimides, which crosslinks the collagen to the bound poly(lysine), and 'thus to the hydrogel lens. If other. derivatized polymers are used, instead of poly(lysine), and covalently bound to the hydrogel surface, then other crosslinkers are chosen which can crosslirrk functional groups on the polymer to materials which will support epithdlial growth, or other molecules as desired. Thus both homobifunctional and heterobifunctional crosslinkers may be used where ~

'~

appropriate. Multiple coats (4-5) are covalently bound to the surface in this fashion. Extensive extrac;tion with so line or a solution of 100 to 50U pg/ml gentamicin sulfate in saline is performed to :remove any non-covalently hound materials and reagents.
The EUC crosslinking may be done at a low pll, as is standard, or at physiological pH using N-hydroxysulfosuccinimide ("Sulfo-NHS" also available from Pierce Chemical Corrrpany) as a co-reactant in order to gently couple sensitive molecules or materials, such as laminin or basement membrane extract, which may be desired in the coating. Laminin may be added during the neutral pH EDC crosslinking. After an incubation of the lens with the EUC/collagen/low pTi mixture, the pd! may be raised before the addition of laminin or other sensitive molecules to be bound.
The above lenses, covalently coated with poly(lysine) and collagen (and/or other ep3.thelial cell-sutaporting materials) may be furthex crosslinked with glut:araldehyde alone, or glutaraldehyde followed by sodium cyanoborohydride, to stabilize the lenses to collagerrase activity and to provide a more desirable coating for epithelial migration. The coated lenses rnay be crosslinked with a low concentration glutaraldehyde solution ( 0. 2~ ) , and/or with a higPr.
concentration solution (up to 2.0~), or other concentrations, in a sodium phosphate/sodium chloride buffer. 'fhe lenses are extensively extracted and then treated wittr'a borate/glycine buffer to neutralize any unreacted glutaraldehyde. Tire unbound materials are removed by extensive extraction in aqueous solutions of saline or gentamicin sulfate in saline as described above. 'tire stability of the coating to collagenase digestion on such lenses is greater than that of control. lenses without glutaraldehyde crosslinking, as visualized by the stain/destaining method. In cell culture and in animal studies (rabbits and cats) ttrese lenses perform well, indicating that post or i.ntermecti.ate crosslin)cing with concentrations ranging from 0.2~ to 2.0~ glutaraldehyde does not deter epi.tliel.ial cell growth, a.nd may in fact enhance the growth of epithelial cells. 'the further treatment of these lenses with sodium cyanoborohydride to further stabilize the crosslinks forrned by the glutaraldehyde (to prevent possible reversal with time) also does not interfere with epithelial cell growth.
Additionally, the above lenses which have poly(lysine) surface modifier and collagen layers (and optionally also include fibronectin, laminin, or other desired materials), followed by glutaraldehyde treatment, may now have additional coats of collagen I, III and/or IV, laminin, fibronectin, or any combination of these or other appropriate molecules to support 2U epithelial growth, bound to the lens by the methods described above, ~i'he additional coats will present a more native surface to the spreading epithelial cells.
If desired, these extra coats may also be followed by a crossl.inking step {with glutaraldehyde for example) which further crosslinks all coats. Hydrogel lenses treated as above have given 100$ cell confluence in 1-2 days in cell culture. When implanted in rabbits, the lenses are essentially completely re-epithelialized in 4-7 days.
The following examples are intended to illustrate further the practice of the invention and are not intended to limit its scope in any way.

15 2060~~0 I~xarngle TV
Coupling call.ac;en directly ro a h~yrdrogel surface which coni:ai.ns carboxyl groups The hydrogel in this Lxample is a polymer consist.i.ng of vinyl pyrrolidone and methyl me~hacrylate, containing methacrylic acid as a source of carboxyl. functional groups. Collagen is type I, calf skin, 2.5 mg/ml as supplied in acidic solutions.
The hydrogel was incubated with collagen and EDC far 1 hr. at room temperature in a pII 4 sodium phosphate buffer, and subseduentiy rinsed. As visualized by protein staining, the hydrogel acquired a thin protein coating.
Example V
Preparation of poly-L-lysine derivatized with a he-Cerobifunctional crosslinkina reagent HSAB
Poly-L-lysine 0190,000 daltons), 500 mg, is dissolved into 95 ml of 0.5 M triethanolamine, 0.2 M
NaCl bu fer, pII Q.3-Q.4. A 10$ molar ratio of HSAa to total available amino groups is dissolved in a small .
volume of DMF (3 ml) in the dark. The HSAB in DMF' is then added, while stirring, to the poly-L-lysine and incubated at 4°C in the dark for 2 haurs, or until the process of binding is complete as deCerrnined by IiPLC
using a size exclusian column. The HSAB-derivatized poly-L-lysine (HSAB-plys) is exchanged into water via ultrafiltration for several changes, is sterile filtered and stored in the dark at 4° C until use.

~xam~ale VT
Coupl.i.ng IISAB-pal -y T, l.ysin~ to a nan-function~lized l~dr~el surface by exposure to UV l3aht '1'lie hydrogel (vinyl pyrrolidone, rnethylmethacrylate copolymer with no carboxyl or amine functional groups) lenses are incubated with a 5 mg/ml solution of HSAB-plys (from example V) and photolyzed with UV ligl:-t for 10 minutes. The solution is exchanged, and the process repeated for a total of 10 times to obtain 10 coats of poly-L-lysine covalently bound to the lens surface and to itself.
'fhe lenses are rinsed extensively and put into cell culture, or implanted into rabbits. Cell culture results show isolated patches of cells which show up to 40~ attactunent to the surface by day 2-5. These results imply that although cell attachment may be achieved, cell spreading is not achieved on this surface. Rabbit implants were stable, but epithelialization of the lens surface did not occur.
Example VTI
Coupling HSAB-poly-L-lysine and collag~en simultaneously to a non-functia_n_al_ized~
hydroael surface b~sLexpasure to UV liah~
tiydrogel lenses were incubated with so~,ulions containing molar ratios of 10:1, 30:1, and 100:1 collagen IV : HSAB-plys, with collagen concentration of 2 mg/ml. Z'en coats were applied using 10 minute exposures to UV light. Lenses with such coatings support epithelial growth in cell culture, with d5-90~
coverage by day 1, and 100 by day 4. Rabbit implants show epithelial growth up to the trephine cut by day 2, and at best, epithelial coverage up to 355 by day 6, followed by complete retreat of the epithelium from the lens by day Q.

~

17 ~o6s6~o example Vala Crosslinkin~ oared hydroael lenses with cr:Lutara.ldehvde Lenses were coated with UV liclht as in example Vat with 15s1 collagen IV to IiSAB-plys. The coated lapses were Llien incubated for two 45 minute treatments with solutions of 0.2~ glutaraldehyde in a 0.5 M sod~.r,rrn phosphate, 0.15 M sodium chloride, pH 7.4 buffer.
Lenses were rinsed with water for injection and incubated with a 0.05 M sodium borate, 0.025 M glycine solution for three incubations of 20 minutes each, followed by extensive rinsing in aqueous solutions.
These lenses support epithelial growth in cell culture, with 90~ covdrage by day l, and 100~s by day 2. Rabbit implants show a maximum coverage of the lenses of 40$
by day 3 today Q, followed by regression to 0~ by day 14. A cat implant showed a stable maximum coverage of 70~ after 5 weeks, followed by a 3 day regression to 50~ and extrusion.
2O Example IX
Addition of 1~ Chondroitin Sulfate to the coating of hydroael lenses Lenses were coated us.Ing UV light, similarly to example VIT, with l0 coats for 5 minutes UV each and a 15a1 cell-supporting material . I~SAB-plys solution, with tl~e cell-supporting material consisting of a solution of 2.0 mg/ml collagen and a 0.02 mg/ml chondroitin sulfabe. Lenses were treated with glutaraldehyde as in Example VTIL: Rabbit implant results are similar to those of Example VITI with'a maximum of 40~ coverage by day 4, and regression to 20~
by day 9.

~O~fi660 ExamJale X
Addition of collagen coats via carbodiirnide coux~l..r.nct to the col lanen : r~oly-.~si.ne cc~a t;ed lenses Tenses were prepared sirna.;l,arly t;o Li~nse In example VII using collagen IV:HSAI3-p7.ys in a x5:1 molar ratio, for 9-10 coats. These lenses were then incubated with 2.0 rng/ml collagen :IV and 19.2 mg/ml EpC under acidic conditions for ~ coats of 20 minutes each. Lenses had either no further additions, or had additions of 1$ by weight of chondroitin sulfate (CS), fibranectin (Fn), or chondroitin sulfate and fibranectin. The lenses were treated with glutaraldehyde as in example VIII.
Results for lenses with the following EDC casts are seen in Table I. The expressed percentages refer to re-epitheiializaLion.
T~B~,E x Col IV: Cell culture, 80$ by day 2, 90$ by day -6, healthy cells.

- Rabbit implant, 85$ by day 7, 100$ by day 8 through 9, with regression to 20$

by day 1~.

Col LV

+ 1$ CS: Cell culture, 80$ by day 2, healthy -cells.

Col IV

-t- 1$ >'n: Rabbit implant, 100$ by day 4, 90$ lay -day 8, with regression to 10$ by day 1~.

Col IV

+ 1$ I'n i~ 1$ Cs: Cell culture, 80$ by day 2, 95-98$ by -day 6 with some rounded cells.

- Cat implant, 100$ epithelial coverage, stable out to 30 weeks at last observation:

2~~~s~o 1~
Example XI
All lenses were coated in the following mariner (steps 1-7):

1. 5 IiSAB-poly-L-lysine UV coats with 10~ MeOH, irradiated for 9 minutes each.

2. 10 coats Col I:HSAB-poly-L-lysine, 12:1 ratio by weight, at 1 mg/ml collagen and irradiated as in step 1.

3 . 4 coats EDC/NiiS-sulfo* pit 7 , ~ with collagen IV at 2 mg/ml and laminin**.

4. 0.2~ glutaraldehyde overnight under the conditions in example VIII.

5. 2.0$ glutaraldehyde for 45 minutes under the same conditions.

6. 2 coats EDC/NiiS-sulfo pit 7.9 with collagen IV, laminin, and 0.2~ chondroitin sulfate.

~. one of the following differences or additions to the treatment:

'Type 1: No further treatment.

'Pype 2: Poly-D-lysine was used in steps I and 2.

Type 3: EDC/NHS-sulfo coats of underivatized poly-lysine were added in between the first three coats of step 3.

'Type 4: A 2.0~ glutaraldehyde crosslinking step after all coats (after step 6 above) under the same conditions as in Example VITT.

to .tenses coated as i.n _exarn~ple VIII, with various other treatments as indicated below ('Types 1-7 ~, ~osssso 'type 5: A 2.0~ glutaraldehyde step as in 'Pype 4 followed by sodium cyanoborohydride treatment .
'fype 6: Sodium cyanoborohydride treatrssent alone after all coats (after step 6 above).
't'ype 7: Lenses were etched before coating to give a rough coating surface.
* I:DC/Nt3S-sulfo was 19.2 rng/ml SDC, 9.6 mg/ml NtiS-sulfo at neutral ptl in sodium phosphate buffer.
** Laminin was provided by adding 13 ~ag/ml laminin to the collagen mixture.
*** Sodium cyanoborohydride was provided by adding 50 mM sodium cyanoborohydride in 0.5 M sodium acetate, pH 4.4.
't'he results obtained are as follows:
Types l through 6 lenses were implanted in rabbits. 'fhe best lens from Type 5 achieved 9~~ ~
coverage by day Fi and maintained 75 $ coverage as of day 62. The best lens from Type 4 achieved 80 to (35 ~
coverage by day 7 and maintained 70 sk coverage as of day 29. Z'he best lens from Type 3 achieved 70 to 75 ~k coverage by day 6 and maintained 60 ~ covarage as of day 22. Lenses from Types 1, 2 and 6 achieved maximum epithelial cell coverage of around 70 ~S and regressed to 15 $ or less by day 40.
Although the present invention has been described in connection with certain preferred embodiments and specific Examples, it is not so limited. Variations within the scope of the appended claims will be readily apparent to those skilled in the art.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed is defined as follows:

1. A method for modifying a synthetic surface, the method comprising the steps of:
a) applying to the synthetic surface a surface modifying composition comprising (i) polymer molecules having pendant functional groups capable of being converted by photolysis to highly reactive groups, and (ii) molecules of another material lacking pendant functional groups capable of being converted by photolysis to highly reactive groups, b) photolysing the composition in order to convert the functional groups to highly reactive groups and thereby simultaneously covalently bind the polymer molecules by reaction of the highly reactive groups with the other material and the synthetic surface, wherein the surface is the surface of a device selected from the group consisting of a prosthetic device to be implanted into the body, a tissue culture plate, and a device for supporting in vitro epithelial cell growth.

2. A method according to claim 1 wherein the polymer molecules are selected from the group consisting of polymers comprising a plurality of pendant amino or carboxyl groups or bath.

3. A method according to claim 2 wherein the polymer molecules are selected from the group consisting of poly(amino acid)s.

4. A method according to claim 1 wherein the other material supports the growth, migration and attachment of cells.

5. A method according to claim 1 wherein the other material is a biological material selected from the group consisting of antibiotics, antimicrobial agents, antiviral agents, anti-inflammatory agents, anti-protease agents, hormones, vitamins, analgesics, chelating agents, mitogenic agents.

6. A method according to claim 1 wherein the surface is fabricated from a material selected from the group consisting of Dacron, polyurethanes, polypropylene, silicone, crosslinked collagens, collagen-plastic composites and phospho-lipid polymers.

7. A method according to claim 1 wherein the surface comprises a hydrogel.

8. A modified synthetic surface formed by a process that comprises the steps of a) providing a synthetic surface, b) applying to the synthetic surface a surface modifying composition comprising (i) polymer molecules having pendant functional groups capable of being converted by photolysis to highly reactive groups, and (ii) molecules of another material lacking pendant functional groups capable of being converted by photolysis to highly reactive groups, and c) photolysing the composition in order to convert the functional groups to highly reactive groups and thereby simultaneously covalently bind the polymer molecules by reaction of the highly reactive groups with the other material and the synthetic surface, wherein the surface is the surface of a device selected from the group consisting of the surface of a prosthetic device to be implanted into the body, the surface of a tissue culture plate, and the surface of a device for supporting in vitro epithelial cell growth.

9. A surface according to claim 8 wherein the polymer molecules are selected from the group consisting of polymers comprising a plurality of pendant amino or carboxyl groups or both.

10. A surface according to claim 9 wherein the polymer molecules are selected from the group consisting of poly (amino acid)s.

11. A surface according to claim 8 wherein the other material supports the growth, migration and attachment of cells.

12. A surface according to claim 8 wherein the other material is a biological material selected from the group consisting of antibiotics, antimicrobial agents, antiviral agents, anti-inflammatory agents, anti-protease agents, hormones, vitamins, analgesics, chelating agents, mitogenic agents.

13. A surface according to claim 8 wherein the surface is fabricated from a material selected from the group consisting of Dacron, polyurethanes, polypropylene, silicone, crosslinked collagens, collagen-plastic composites and phospho-lipid polymers.

14. A surface according to claim 8 wherein the surface comprises a hydrogel.

15. A prosthetic device comprising a modified synthetic surface according to claim 8.