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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 cross-linking (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/cm2) for 2:50 minutes (fluence 1.53 J/cm2). CXL was performed as either a single (1×CXL) or a repeated (2×CXL) treatment. Un-irradiated corneas served as controls. In the 2×CXL 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 two-dimensional 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 1×CXL corneas and 2×CXL corneas. No significant difference (P = .70) was detected between the 1×CXL and 2×CXL groups. The stress remaining after relaxation was 355 ± 25.2 kPa in the control group, 457 ± 34.1 kPa in the 1×CXL group, and 463 ± 22.2 kPa in the 2×CXL 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. [J Refract Surg. 2017;33(1):56-60.].