Measuring Biomechanical Changes Of Collagen Cross-Linking With Rose Bengal And Green Light Using Atomic Force Microscopy

The standard protocol of riboflavin corneal crosslinking (CXL) is approved for corneas >400µm to manage corneal ectasia. Modifications to the standard protocol of CXL have been established for thin corneas (<400 um) in order to mitigate the risk of damage to the endothelium, but complications may still arise.  A potential alternative is using rose bengal (RB) which only penetrates ~100 µm into the cornea. This study investigated the changes in corneal biomechanics stiffness after rose bengal crosslinking (RB-CXL) using atomic force microscopy (AFM).

Experiements where performed between the Ophthalmic Biophysics Center, Department of Ophthalmology, Bascom Palmer Eye Institute. And the Biomedical Atomic Force Microscopy Laboratory Department of Biomedical Engineering. Both belong to the University of Miami.

21 pairs of ex vivo human corneas (<10 days post-mortem) were collected and de-epithelialized following the Dresden protocol. In each pair, OS  was left untreated and served as the control. OD cornea of each pair was soaked in the 0.1% RB solution for 30 minutes, and then irradiated with green light for a total energy of 5.4 J/cm² at two different light intensities: either 6 mW/cm² for 15 minutes (n=10) or 18 mW/cm² for 5 minutes (n=11). Using the AFM to measure the Young’s modulus of elasticity, measurements were taken at two depths: Bowman’s layer and 50 µm below the surface. A microkeratome with a 50 µm blade was used for the second measurement. OCT was performed initially and after each cut to verify corneal thickness.

At the 50 µm depth, the Young’s modulus of elasticity of corneas in the 18 mW/cm² irradiation group (25.7 ± 16.7 kPa) were significantly higher than the contralateral control corneas (10.4 ± 5.0 kPa), p<0.001. No differences were observed between the 18 mW/cm² and 6 mW/cm² groups or 6 mW/cm² group and the control at 50 µm, p≥0.05. There were no differences in Young’s modulus between any of the groups at Bowman’s layer, which was expected.

This study demonstrates that RB-CXL with high intensity green light irradiation can significantly increase corneal stiffness at a depth of 50 µm, specifically when irradiated at 18 mW/cm². The lack of significant differences between the 6 mW/cm² and 18 mW/cm² irradiation groups at Bowman’s layer indicates that deeper layers of the cornea are more affected by the cross-linking process. These findings highlight the potential of RB-CXL for thin corneas, where traditional riboflavin CXL protocols carry the risk of endothelial damage.