Optimizing Genipin Concentration for Corneal Collagen Cross-Linking: An ex vivo Study

<b><i>Purpose:</i></b> Studying genipin variable concentrations, treatment durations, and delivery methods as a substance to increase corneal stiffness by inducing corneal collagen cross-linking (CXL). <b><i>Materials and Methods:</i></b> 100 bovine corneas treated with different genipin concentrations (0.1, 0.5, and 1%) and treatment durations (15 min, 40 min, 2 h, and 3 days) through different delivery methods compared to 10 controls treated with riboflavin/UV. Histology examination, enzymatic digestion with collagenase and thermal differential scanning calorimetry were performed on the different samples. <b><i>Results:</i></b> Bovine corneas soaked in 0.5% genipin morphologically showed 4.7% CXL in comparison to 5.6% in controls (<i>p</i> &#x3c; 0.05). Corneas treated with topical 0.5% genipin, by a 140-µL drop applied hourly for 2 h, showed 7% corneal CXL. Corneas treated with topical genipin 0.5% for 30 min, 1 and 2 h showed 54 ± 6, 40 ± 7, and 39 ± 9% enzymatic degradation, respectively, in comparison to controls (74%). Corneas treated with 0.5% genipin for 1, 2, and 8 h showed higher thermal denaturation resistance (Td values of 64.9 ± 0.3, 64.7 ± 0.0 and 67.3 ± 0.9), respectively, in comparison to the control group (64.6 ± 0.5) (<i>p</i> &#x3c; 0.05). <b><i>Conclusions:</i></b> Genipin 0.5%, in a 140-µL drop applied hourly for 2 h, showed better potential to enhance corneal stiffness and stability through inducing CXL.