The Resistance To Enzymatic Digestion Of High-Fluence Corneal Cross-Linking With Riboflavin/Uv-A Is Oxygen-Independent

Corneal cross-linking (CXL) with riboflavin and UV-A induces several effects in the cornea, including 1) biomechanical stiffening, 2) increased resistance to enzymatic digestion and 3) generation of reactive oxygen species (ROS).

For the biomechanical effect, oxygen is an essential and rate-limiting reagent: high-fluence protocols use up oxygen faster than it can be replenished, impairing the strengthening effect. 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.

Laboratory investigation. ELZA Institute, Dietikon, Switzerland and Dept. of Ophthalmology, University of Antwerp, Antwerp, Belgium

Ex vivo porcine corneas (n=129) were assigned to 5 groups. Group 1 was the control group (abrasion and riboflavin application). Groups 2 and 3 received an accelerated 10 J/cm² high-fluence CXL protocol (9′15″ @ 18 mW/cm²), and Groups 4 and 5 received an accelerated 15 J/cm² high-fluence protocol (8′20″ @ 30 mW/cm²). Groups 3 and 5 underwent CXL in a nitrogen-rich, anaerobic environment (oxygen content less than 0.1%). CXL procedures were performed using commercially available riboflavin (Ribo-Ker, EMAGine SA, Zug, Switzerland) and a commercially available CXL device (C-eye, EMAGine AG, Zug, Switzerland). All 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 h, 30.61 ± 1.79 h, 31.38 ± 2.29 h, 32.52 ± 2.56 h, and 31.71 ± s.57 h, respectively. All groups had significantly higher (p<0.001) digestion resistance than the non-irradiated riboflavin control group (Group 1). However, there was no significant difference in the mean time to digestion across all the experimental groups, irrespective of fluence delivered or the presence of oxygen.

High-fluence accelerated riboflavin/UV-A CXL protocols (10 J/cm² and 15 J/cm²) render ex vivo porcine corneas significantly more resistant to enzymatic digestion when compared to non-irradiated control corneas. This effect seems to be oxygen-independent.