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Purpose  To investigate the viscoelastic changes of the human cornea induced by riboflavin/UV-A cross-linking using Atomic Force Microscopy (AFM) at the nano level.    Methods  Seven eye bank donor corneas were investigated, after gently removing the epithelium, using a commercial AFM in the force spectroscopy mode. Silicon cantilevers with tip radius of 10 nm and spring elastic constants between 26- and 86-N/m were used to probe the viscoelastic properties of the anterior stroma up to 3 µm indentation depth. Five specimens were tested before and after riboflavin/UV-A cross-linking; the other two specimens were chemically cross-linked using glutaraldehyde 2.5% solution and used as controls. The Young’s modulus ( E ) and the hysteresis ( H ) of the corneal stroma were quantified as a function of the application load and scan rate.    Results  The Young’s modulus increased by a mean of 1.1-1.5 times after riboflavin/UV-A cross-linking (P<0.05). A higher increase of  E , by a mean of 1.5-2.6 times, was found in chemically cross-linked specimens using glutaraldehyde 2.5% (P<0.05). The hysteresis decreased, by a mean of 0.9-1.5 times, in all specimens after riboflavin/UV-A cross-linking (P<0.05). A substantial decrease of  H , ranging between 2.6 and 3.5 times with respect to baseline values, was observed in glutaraldehyde-treated corneas (P<0.05).    Conclusions  The present study provides the first evidence that riboflavin/UV-A cross-linking induces changes of the viscoelastic properties of the cornea at the scale of stromal molecular interactions.

Understanding of the Viscoelastic Response of the Human Corneal Stroma Induced by Riboflavin/UV-A Cross-Linking at the Nano Level