Nonlinear optical collagen cross-linking and mechanical stiffening: a possible photodynamic therapeutic approach to treating corneal ectasia

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.