The corneal specimens were divided in three groups; the study group (
n = 4) underwent transepithelial corneal cross-linking using a novel 0.1% riboflavin solution (Nanotech; Sooft Italia SpA, Montegiorgio, Italia) and UV-A irradiation of the cornea with a 10 mW/cm
2 device for 9 minutes (370 ± 8 nm; Vega, CSO, Scandicci, Italy), according to the manufacturer's instructions. Each corneoscleral tissue was placed in an artificial anterior chamber (AAC) (Coronet; Network Medical Products, Ltd., Ripon, UK), pressurized with the AAC filled with 0.9% sodium chloride using a 5-mm syringe. The hypotonic solution consisted of 0.1% (phosphate-free) riboflavin that was kept in solution by 2-hydroxypropyl-β-cyclodextrin nanoparticles; EDTA and trometamol were added as enhancers (
Table 1). The solution was instilled over the corneal epithelial surface every 20 to 30 seconds for 20 minutes using a silicone ring. Before UV-A irradiation, the epithelium was gently washed with balanced physiological solution. An irradiation area of 8.0-mm diameter was used in all cases; no riboflavin drop was administered over the epithelial surface during UV-A irradiation. Immediately after treatment, the epithelium was removed using an Amoils brush (Innovative Excimer Solutions, Inc., Toronto, ON, Canada), and the corneal specimens were kept in 20% dextran solution overnight.
A positive control group (
n = 4) underwent conventional corneal cross-linking; each corneal tissue was deepithelized using the Amoils brush and then was immersed in 20% dextran-enriched 0.1% riboflavin solution (Ricrolin; Sooft Italia SpA) for 30 minutes. Before UV-A irradiation, the specimens were gently washed with balanced physiological solution in order to remove the excess of riboflavin. Specimens were irradiated with a 3 mW/cm
2 UV-A device for 30 minutes (370 ± 8 nm; Vega, CSO). An irradiation area of 8.0-mm diameter was used in all cases; no riboflavin drop was administered over the stromal surface during UV-A irradiation. After treatment, the corneal specimens were kept in 20% dextran solution overnight. The entire procedure is schematized in
Figure 1. Untreated sclerocorneal tissues were used as negative controls (
n = 4). Data from control specimens were collected in previous studies.
15,17