Gel electrophoretic analysis of corneal collagen after photodynamic cross-linking treatment.

PURPOSE: Photodynamic collagen cross-linking by using ultraviolet A (UVA) irradiation and the photosensitizer riboflavin has been recently introduced as a new possible treatment of progressive keratoconus. This is the first study, to our knowledge, investigating biochemical aspects of the new procedure. Its aim was to analyze the possible changes in the electrophoretic pattern of corneal collagen type I after collagen cross-linking treatment.

METHODS

Twenty fresh postmortem porcine corneas were cross-linked; another 20 porcine corneas treated with physiologic saline were used as controls. After removal of the central 10 mm of the epithelium, the corneas were treated with the photosensitizer riboflavin and UVA irradiation for 30 minutes by using a double UVA diode (370 nm, 3 mW/cm). For biochemical analysis, the central 10-mm corneal buttons were trephined, tissue was homogenized, and collagen type I was extracted. Subsequently, the collagen extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

RESULTS

In the controls, the typical collagen pattern of normal cornea was found with 1 gamma trimer band, 2 beta dimer bands, and 2 alpha monomer bands. In the cross-linked samples, there was an additional intense polymer band in the stacking gel that was resistant to mercaptoethanol, heat, and pepsin treatment. Its molecular size was estimated to be at least 1000 kDa.

CONCLUSIONS

In the cross-linked corneas, a strong band of high-molecular-weight collagen polymers was shown as the biochemical correlate of the cross-linking effect, showing the efficiency of the new cross-linking procedure. This polymer band complies well with the morphologic correlate of an increased fiber diameter after cross-linking treatment. Its chemical stability supports hopes of a long-term effect of the new treatment.