The Resistance of Riboflavin/UV-A Corneal Cross-Linking to Enzymatic Digestion Is Oxygen-Independent.

Abstract

Corneal cross-linking (CXL) with riboflavin and UV-A induces several effects in the cornea, including biomechanical stiffening, generation of reactive oxygen species, and increased resistance to enzymatic digestion. Whereas the biomechanical stiffening effect is oxygen-dependent, little is known about the effect of oxygen on the resistance to enzymatic digestion. Here, we examined CXL-induced enzymatic resistance in the absence of oxygen. Ex vivo porcine corneas (n = 160) were assigned to 5 groups. Group 1 was the control group (abrasion and riboflavin application). Groups 2 and 3 received accelerated 10 and 15 J/cm2 high-fluence CXL protocols in the presence of oxygen (9'15″ @ 18 mW/cm2 and 8'20″ @ 30 mW/cm2, respectively), whereas groups 4 and 5 received accelerated 10 and 15 J/cm2 high-fluence CXL protocols in the absence of oxygen (oxygen content less than 0.1%). After CXL, corneas were digested in 0.3% collagenase A solution. Mean time until complete dissolution was determined. The mean times to digestion in groups 1 through 5 were 22.31 ± 1.97 hours, 30.78 ± 1.83 hours, 32.22 ± 2.22 hours, 31.38 ± 2.18 hours, and 31.69 ± 2.53 hours, respectively. Experimental CXL groups showed significantly higher (P < 0.001) resistance to digestion than nonirradiated controls. There was no significant difference in time to digestion across all experimental CXL groups, irrespective of fluence delivered or the absence of oxygen. The resistance to digestion in accelerated high-fluence riboflavin/UV-A CXL is oxygen-independent, which is of particular importance when developing future optimized CXL protocols for corneal ectasia and infectious keratitis.