Optimizing genipin concentration for corneal collagen cross-linking: An ex vivo study

Purpose: Studying genipin variable concentrations, treatment durations, and delivery methods as a substance to increase corneal stiffness by inducing corneal collagen cross-linking (CXL). Materials and Methods: 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. Results: Bovine corneas soaked in 0.5% genipin morphologically showed 4.7% CXL in comparison to 5.6% in controls (p < 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) (p < 0.05). Conclusions: 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.