Surgeons have used phakic IOLs and implantable collamer lenses (ICLs; Staar Surgical Company, Monrovia, CA) as alternatives to the popular excimer laser correction of refractive errors. The main concerns regarding phakic IOLs include the potential development of cataracts, pigmentary dispersion, and glaucoma.1 ICLs are placed behind the iris in the posterior chamber, anterior to the crystalline lens. Although deemed to be safer than LASIK in an FDA clinical trial,2 ICLs have nevertheless been associated with a loss of corneal endothelial cells.

Synthetic lenticules that are implanted within the corneal stroma (inlays) or underneath the epithelium (onlays) have been in development as alternatives to refractive surgery for the correction of refractive errors.3 Both modalities are designed with similar material properties, including adequate glucose transport, persistent clarity as well as a lack of inflammatory reaction, and an absence of neovascularization in the host cornea. Recent work has largely focused on onlays, which, because of their superficial location, may potentially offer reversible correction of refractive errors.

DESIGN
Manipulating the corneal onlay's shape modifies the shape of the air/cornea interface. This step in combination with the onlay's inherent refractive index produces a change in corneal power. Biologically, the lenticules become incorporated into the cornea by becoming attached to the epithelium and the underlying Bowman's membrane during the natural healing process without epithelial cell undergrowth. Clinically, the onlay should be removable to permit reversal of the treatment if necessary due to changes in the ocular optics.

Although the surgical procedure to implant onlays may be simple and the lenticule resembles a fairly uncomplicated contact lens, the design and development of onlays are demanding processes. The polymer(s) used in the device must produce a lenticule that is highly transparent, has low optical scatter, is biostable, and is permeable in addition to the aforementioned properties. The requirement for permeability to glucose and other nutrients is critical to providing an adequate nutritional flux through the device and thus maintaining a functional corneal epithelium anterior to the onlay. The surface topography and porosity may be important for normal corneal epithelial growth and stratification.4

Recently, Evans et al3 described a sophisticated, porous corneal onlay based on a perfluoropolyether (PFPE) copolymer, overcoated with a chemically bound, collagen I layer. PFPE is a transparent, isorefractive polymer that offers high chemical and thermal stability. Their clinical testing in the feline cornea demonstrated that porous PFPE lenticules support the growth of corneal epithelium in vivo when coated with collagen I. When the lenticules were implanted into cats, the epithelium completely covered a feline wound bed (sham) by days 3 to 9, and it covered the coated PFPE lenticule surface by days 5 to 11 in six of the seven implanted corneas. Overall, the implanted corneas were quiet, remained transparent, maintained multilayered epithelial cover (eight to 10 layers by 8 weeks), and supported a stable tear film during the observation period. There was no evidence of thinning (stromal melting) during this test period. The steady initial growth of epithelium over the lenticules was attributed to the presence of the covalently immobilized collagen I layer on the anterior surface of the lenticules.

OUR RESEARCH

Developing the Onlay
We recently developed a biosynthetic hybrid of the protein collagen and a synthetic N-isopropylacrylamide-based polymer that contains a laminin-derived bioactive peptide that comprises tyrosine-isoleucine-glycine-serine-arginine (YIGSR).5 This biosynthetic polymer can be molded to the same curvature and dimensions of a human cornea, has the optical clarity of a natural cornea, and is sufficiently robust to be sutured (Figure 1). When transplanted into the eyes of miniature swine (pigs with a body weight of 30 to 70kg at maturity) by means of a lamellar keratoplasty procedure, these implants allowed ingrowth and stratification of the epithelium. Additionally, healthy stromal cells and corneal nerves from the host grew through the implant, thereby effectively replacing the cells and functional nerves that were removed during the surgery. We obtained similar results from matrices comprising 1-ethyl-3.3'(dimethylaminopropyl)-carbodiimide and N-hydroxysuccinimide (EDC/NHS) crosslinked collagen. These materials have now been implanted in rabbits for 6 months as lamellar grafts6 without any adverse reactions.

The biosynthetic materials developed as corneal substitutes are also candidate materials as onlays, owing to the desired commonalities in the material's properties for both applications. The collagen-based matrices were fabricated into onlays (6mm in diameter, 70µm in thickness centrally, truncated at 30µm from the sloped edges) using contact lens molds.

Clinical Testing of the Onlay
We implanted the onlays subepithelially within the corneas of miniature swine. One eye of each of six animals received the implant, and the contralateral corneas (untouched) served as controls. Performed preoperatively and 3 weeks postoperatively, clinical examinations of the anesthetized animals included a slit-lamp examination, tonometry, aesthesiometry, in vivo confocal microscopy, and corneal topography. All corneas with onlays were optically clear by slit-lamp examination, with no vascular infiltration and no changes in IOP. Topographical analysis showed the expected change of approximately 70µm in the thickness of the central corneal elevation (Figure 2). At 3 weeks, aesthesiometry demonstrated that the implanted eyes were sensitive to touch, a finding consistent with good nerve integration. Implanted corneas were recovered 3 weeks postoperatively and processed for histology. The onlays were fully biocompatible and showed smooth host-implant integration. The epithelium adhered well to all lenticules (Figure 3), and it expressed Keratin 3 and E-cadherin at levels comparable to the untreated controls. Staining for alpha6 integrin, which mediates the adhesion of epithelial cells to the basement membrane, showed localization in the basal epithelial cells in both operated and unoperated eyes. Type VII collagen staining for anchoring fibers within the basement membrane complex, however, showed weaker staining in treated corneas. Histology also demonstrated conclusively that the implants contained neurofilament-positive nerves (Figure 4), a finding that is consistent with the observed recovery of corneal sensitivity at 3 weeks.

CONCLUSION
Short-term results with the collagen-coated onlays and the fully collagen-based onlays show promise for correcting refractive errors. The long-term biocompatibility and refractive-power stability of these new materials as well as mechanical shaping will be critical to surgeons' acceptance of onlays as an alternative to laser-based refractive surgery.

The authors received research funding from National Sciences and Engineering Research Councils of Canada strategic project grant, No. 246418, and Ocular Sciences Inc. They state that they hold no direct financial interest in the products and company mentioned herein.

Cecilia Becerril, MSc, is Projects Manager at University of Ottawa Eye Institute.
Donna Bueckert, MSc, is Senior Research Assistant at University of Ottawa Eye Institute.
David Carlsson, PhD, is Researcher Emeritus for the National Research Council of Canada-ICPET in Ottawa.
May Griffith, PhD, is Associate Professor at University of Ottawa Eye Institute and the Department of Cellular and Molecular Medicine, University of Ottawa, and she is Senior Scientist at the Ottawa Health Research Institute.
William Hodge, MD, PhD, is Associate Professor at University of Ottawa Eye Institute.
W. Bruce Jackson, MD, is Professor and Chairman, Department of Ophthalmology, University of Ottawa, and Director General of the University of Ottawa Eye Institute. Dr. Jackson may be reached at (613) 737-8759; bjackson@ottawahospital.on.ca.
Fengfu Li, PhD, is Associate Scientist at Ottawa Health Research Institute.
Yuwen Liu, PhD, is a visiting postdoctoral fellow for the National Research Council of Canada-ICPET in Ottawa.
Rejean Munger, PhD, is Assistant Professor at University of Ottawa Eye Institute and is Scientist at Ottawa Health Research Institute.
1. Pineda-Fernandez A, Jaramillo J, Vargas J, et al. Phakic posterior chamber intraocular lens for high myopia. J Cataract Refract Surg. 2004;30:2277-2283.
2. Sanders DR, Doney K, Poco M; ICL in Treatment of Myopia Study Group. United States Food and Drug Administration clinical trial of the implantable collamer lens (ICL) for moderate to high myopia: three-year follow-up. Ophthalmology. 2004;111:1683-1692.
3. Evans MDM, Xie RZ, Fabbri M, et al. Progress in the development of a synthetic corneal onlay. Invest Ophthalmol Vis Sci. 2002;43:3196-3201.
4. Evans MDM, Farland GA, Taylor S, Walboomers XF. The response of healing corneal epithelium to grooved polymer surfaces. Biomaterials. 2005;26:1703-1711 (e-publication 2004, available at: http://www.sciencedirect.com).
5. Li F, Carlsson D, Lohmann C, et al. Cellular and nerve regeneration within a bio-synthetic extracellular matrix for corneal transplantation. Proc Natl Acad Sci USA. 2003;100:15346-15351.
6. Gan L, Fagerholm P, Liu Y, et al. Artificial cornea—rapport of human corneal cells and animal tests. Ophthalmic Res. 2004;36(suppl 1):203.