Biomechanical changes after repeated collagen cross-linking on human corneas assessed in vitro using scanning acoustic microscopy.

Purpose: To explore the biomechanical changes induced by repeated cross-linking using scanning acoustic microscopy (SAM).

Methods: Thirty human corneas were divided into three groups. In group A, five corneas were cross-linked once. In group B, five corneas were cross-linked twice, 24 hours apart. In group C, five corneas were cross-linked three times, 24 hours apart. The contralateral controls in all groups had similar treatment but without UV-A. The speed of sound, which is directly proportional to the square root of the tissue's elastic modulus, was assessed using SAM.

Results: In group A, the speed of sound of the treated corneas was 1677.38 ± 10.70 ms(-1) anteriorly and 1603.90 ± 9.82 ms(-1) posteriorly, while it was 1595.23 ± 9.66 ms(-1) anteriorly and 1577.13 ± 8.16 ms(-1) posteriorly in the controls. In group B, the speed of sound of the treated corneas was 1746.33 ± 23.37 ms(-1) anteriorly and 1631.60 ± 18.92 ms(-1) posteriorly, while it was 1637.57 ± 22.15 ms(-1) anteriorly and 1612.30 ± 22.23 ms(-1) posteriorly in the controls. In group C, the speed of sound of the treated corneas was 1717.97 ± 18.92 ms(-1) anteriorly and 1616.62 ± 17.58 ms(-1) posteriorly, while it was 1628.69 ± 9.37 ms(-1) anteriorly and 1597.68 ± 11.97 ms(-1) posteriorly in the controls. The speed of sound in the anterior (200 × 200 μm) region between the cross-linked and control corneas in groups A, B, and C was increased by a factor of 1.051 (P = 0.005), 1.066 (P = 0.010), and 1.055 (P = 0.005) respectively. However, there was no significant difference among the cross-linked corneas in all groups (P = 0.067).

Conclusions: A significant increase in speed of sound was found in all treated groups compared with the control group; however, the difference among the treated groups is not significant, suggesting no further cross-links are induced when collagen cross-linking treatment is repeated.