Melanin-Assisted Femtosecond Laser Crosslinking Restores Biomechanical Strength In A Chondroitinase-Induced Ectatic Corneal Model

The purpose of this study was to evaluate whether plasma-assisted femtosecond laser–mediated collagen crosslinking (CxL) can restore biomechanical strength in a Chondroitinase-ABC–induced ectatic corneal model simulating keratoconus. Our group previously developed a melanin-enhanced, plasma–mediated corneal crosslinking method, in which femtosecond laser pulses generate reactive oxygen species. Here, we assessed its efficacy in weakened corneal tissue and compared its biomechanical performance with established crosslinking approaches, including glycation and UVA-riboflavin treatments.

This ex vivo experimental study was conducted in a university-based academic laboratory. All rabbit samples were harvested from a local vendor. Paired rabbit corneal buttons were subjected to controlled enzymatic degradation to induce ectasia, followed by collagen crosslinking treatments and mechanical characterization.

Corneal tissues from 6 rabbits were trephined (n=60 buttons total) and mechanically tested by spherical micro-indentation (Optics11 Life, 47.5 µm probe radius; 0.44 N/m cantilever stiffness). The baseline equilibrium modulus was measured for all samples, and a 3-hour 0.1 U/ml Chondroitinase-ABC digestion protocol was followed. Samples were then treated with melanin-assisted laser CxL, Advanced Glycation Product mediated CxL, UVA-riboflavin CxL, or PBS-only as blank controls. After overnight storage at 4°C to reach hydration equilibrium, repeat micro-indentation was performed to characterize the effect of digestion and CxL. Modulus changes were analyzed using a one-way ANOVA with Tukey post hoc comparisons.

Significant reduction of corneal stiffness was found in digested ectatic corneas when compared to blank controls (-11.56 ± 6.54 vs -2.90 ± 3.24 kPa; p < 0.05). For the samples receiving CxL, all three modalities significantly increased the equilibrium modulus of digested samples  (ANOVA; F = 17.3; p < 0.0001), with 5.59 ± 5.31 kPa stiffening by melanin-assisted laser-based CxL. The glycated samples stiffened 3.84 ± 9.13 kPa, and the UVA riboflavin-treated samples stiffened 12.34 ± 12.20 kPa. Compared with the non-digested controls, only UVA-riboflavin CxL resulted in significant stiffening over the baseline (*p<0.01), whereas melanin-laser treatment restored modulus toward baseline without additional stiffening.

The Chondroitinase digestion protocol produces an ectatic corneal model that simulates a keratoconus cornea with significant biomechanical weakening. Melanin-assisted femtosecond laser CxL effectively restores biomechanical strength in the ectatic cornea while ensuring the achieved stiffness is similar to that achieved with established crosslinking methods. These findings support laser-mediated, melanin-assisted crosslinking as a strategy for strengthening ectatic corneal tissue.