Repeated Cross-linking After a Short Time Does Not Provide Any Additional Biomechanical Stiffness in the Mouse Cornea In Vivo

PURPOSE: To study whether repeated collagen crosslinking (CXL) performed in vivo in mice shows an additive effect on mechanical corneal stiffness. METHODS: In this experimental study, epithelium-off CXL was performed in a total of 18 eyes from male C57BL/6 mice, with 0.27%-riboflavin solution applied for 20 minutes, followed by ultraviolet-A (UVA) irradiation (365 nm, 9mW/cm(2)) for 2:50 minutes (fluence 1.53 J/cm(2)). CXL was performed as either a single (1xCXL) or a repeated (2xCXL) treatment. Un-irradiated corneas served as controls. In the 2xCXL group, the procedure was performed on day 1 and day 4 to ensure complete reepithelialization between sessions. Biomechanical analysis was performed on day 7. Corneas were harvested with a small scleral ring and mounted on a customized twodimensional flap holder. The biomechanical measurement consisted of three parts: (1) pre-conditioned during three cycles from 0.04 to 0.4 N, (2) stress relaxation during 120 seconds following 0.4 N force application, and (3) stress-strain curve until break. RESULTS: After the relaxation period of 120 seconds, highly significant differences (P <.001) were found between the controls and both 1xCXL corneas and 2xCXL corneas. No significant difference (P =.70) was detected between the 1xCXL and 2xCXL groups. The stress remaining after relaxation was 355 +/- 25.2 kPa in the control group, 457 +/- 34.1 kPa in the 1xCXL group, and 463 +/- 22.2 kPa in the 2xCXL group. No significant differences in the stress-strain curves were found between the conditions. CONCLUSIONS: Repeated CXL 3 days after the first procedure does not further increase corneal stiffness in mice in vivo.