[Enzymatic evidence of the depth dependence of stiffening on riboflavin/UVA treated corneas].

PURPOSE: It has been shown that the treatment of keratoconus with riboflavin/ultraviolet A (UVA) causes significant stiffening of the cornea due to cross-linking. The aim of this study was to evaluate how deep the mechanical stabilization after collagen cross-linking could be shown biochemically.
METHOD: Ten out of 20 enucleated porcine eyes were treated with riboflavin as a photosensitizer and UVA (370 nm, 3 mW/cm2, 30 min). The other 10 eyes served as controls. With a Microkeratom device, two flaps with a thickness of 200 microm and a diameter of 8 mm were cut off from each eye and put in a collagenase solution (NaCl plus collagenase A, 1:1). The surfaces of the flaps were measured digitally every day to characterize the dissolving behavior.
RESULTS: The resistance (regarding corneal collagen against enzymatic digestion) of the treated superficial flaps was considerably higher (p=0.001) compared to those that were cut secondarily and to the control flaps. But even the flaps from deeper layers showed a significant increase in resistance (p=0.02) compared with the untreated flaps. The half-life of the surfaces of the treated superficial flaps was 220 h; of those cut secondarily, it was 80 h. Both untreated flaps had a half-life of 50 h.
CONCLUSIONS: The biochemical study showed that the treatment of the cornea with riboflavin/UVA leads to significant collagen cross-linking not only in the anterior slice of 200 microm but also in the following 200 microm. This locally limited cross-linking effect may be explained by the absorption behavior for UVA of the riboflavin-treated cornea; 65% of UVA irradiation is absorbed in the first 200 microm and only 25-30% in the next 200 microm. Therefore, deeper-lying structures and especially the endothelium are not affected.