Repeated application of riboflavin during corneal cross-linking does not improve the biomechanical stiffening effect ex vivo

To evaluate whether repeated application of riboflavin during corneal cross-linking (CXL) has an impact on the corneal biomechanical strength in ex-vivo porcine corneas. Laboratory investigation. Sixty-six porcine corneas with intact epithelium were divided into three groups and analyzed. All corneas were pre-soaked with an iso-osmolar solution of 0.1% riboflavin in a phosphate-buffered saline (PBS) solution (“riboflavin solution”). Then, the corneas in Groups 1 and 2 were irradiated with a standard epi-off CXL (S-CXL) UV-A irradiation protocol (3 mW/cm2 for 30 min); while the corneas in Group 3 were not irradiated and served as control. During irradiation, Group 1 (CXL-PBS-Ribo) received repeated riboflavin solution application while corneas in Group 2 (CXL-PBS) received only repeated iso-osmolar PBS solution. Immediately after the procedure, 5-mm wide corneal strips were prepared, and elastic modulus was calculated to characterize biomechanical properties. Significant differences in stress-strain extensiometry were found between two cross-linked groups with control group (P = 0.005 and 0.002, respectively). No significant difference was observed in the normalized stiffening effect between Groups 1 and 2 (P = 0.715). The repeated application of riboflavin solution during UV-A irradiation does not affect the corneal biomechanical properties achieved with standard epi-off CXL. Riboflavin application during CXL may be omitted without altering the biomechanical stiffening induced by the procedure.