Determination of the relationship between mechanical properties, ultrastructural changes and intrafibrillar bond formation in corneal UVA/riboflavin cross-linking treatment for keratoconus.

PurposeUVA/riboflavin induced corneal cross-linking is a common therapeutic approach used to prevent progression in keratoconus. However, there is limited understanding of how changes to the mechanical behavior in cross-linked corneas relate to changes in the corneal ultrastructure. The aim of this study was to determine these relationships and, by so doing, provide a reference against which future modifications to cross-linking approaches may be compared.MethodsPorcine corneas were treated following the “Dresden” protocol, the current gold standard for clinical treatment, consisting of dropwise application of 0.1% riboflavin in 20% dextran followed by 30 minutes of UVA irradiation. The effect of crosslinking was assessed using uniaxial tensile testing, transmission electron microscopy, and Fourier Transform Infrared Spectroscopy with results compared against corneas treated with each of the treatment solution components individuallyResults Data revealed that UVA/riboflavin cross-linked corneas displayed 28±17% increase in the material tangent modulus and altered collagen architecture within the first 300 m of stromal depth consisting of 5% increase in the thickness of collagen fibrils with no significant changes to interfibrillar spacing, resulting in an 8% to 12% decrease in number of fibrils per unit area. Fourier Transform Infrared Spectroscopy confirmed formation of interfibrillar bonds (p=0.012) induced by UVA mediated cross-linking.ConclusionsOur data support a model wherein collagen fibril diameter and structural density are fundamental parameters in defining tissue stiffening following UVA/riboflavin corneal cross-linking, and provide the key benchmarks against which modifications to the Dresden cross-linking protocol can be evaluated.