Abstract
In this study we test the hypothesis that nonlinear optical (NLO) multiphoton photoactivation of riboflavin using a focused femtosecond (FS) laser light can be used to induce cross-linking (CXL) and mechanically stiffen collagen as a potential clinical therapy for the treatment of keratoconus and corneal ectasia. Riboflavin-soaked, compressed collagen hydrogels are cross-linked using a FS laser tuned to 760 nm and set to either 100 mW (NLO CXL I) or 150 mW (NLO CXL II) of laser power. FS pulses are focused into the hydrogel using a 0.75 NA objective lens, and the hydrogel is three-dimensionally scanned. Measurement of hydrogel stiffness by indentation testing show that the calculated elastic modulus (E) values are significantly increased over twofold following NLO CXL I and II compared with baseline values (P<0.05). Additionally, no significant differences are detected between NLO CXL and single photon, UVA CXL (P>0.05). This data suggests that NLO CXL has a comparable effect to conventional UVA CXL in mechanically stiffening collagen and may provide a safe and effective approach to localize CXL at different regions and depths within the cornea.
Highlights
The cornea is the outer, transparent part of the eye that provides two thirds of the refractive power for focusing light back to the retina
We have evaluated the effectiveness of nonlinear optical (NLO) CXL to mechanically stiffen compressed type I collagen hydrogels and compared the stiffening effect with that obtained using conventional ultraviolet A light (UVA) CXL
Photoactivation of riboflavin using either UVA or NLO at the two different powers showed significantly increased post-treatment E values compared to baseline (P < 0.05)
Summary
The cornea is the outer, transparent part of the eye that provides two thirds of the refractive power for focusing light back to the retina. The cornea is composed of five tissue layers: the epithelium, Bowman’s layer, stroma, Descemet’s membrane, and endothelium. Past studies by Kokott suggested the presence of both a circumferential band of collagen that constrains the corneal curvature at the limbus, and a preferred nasal-temporal/superior-inferior orientation of deeper collagen layers that aligns with the extra ocular muscles.[1] Studies using x-ray diffraction have confirmed these earlier findings and shown a preferential alignment of collagen fibers in the mid–to posterior central cornea and an apparent tangential orientation in the corneal periphery.[2] More recent studies using nonlinear optical microscopy to image second-harmonic-generated (SHG) signals from collagen have identified sutural or bow-string collagen fibers in the anterior stroma that insert into Bowman’s layer and extensively intertwine with orthogonally arranged collagen fibers.[3] The degree of collagen intertwining
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