Riboflavin/UVA Collagen Cross-Linking-Induced Changes in Normal and Keratoconus Corneal Stroma

Purpose To determine the effect of Ultraviolet-A collagen cross-linking with hypo-osmolar and iso-osmolar riboflavin solutions on stromal collagen ultrastructure in normal and keratoconus ex vivo human corneas. Methods Using small-angle X-ray scattering, measurements of collagen D-periodicity, fibril diameter and interfibrillar spacing were made at 1 mm intervals across six normal post-mortem corneas (two above physiological hydration (swollen) and four below (unswollen)) and two post-transplant keratoconus corneal buttons (one swollen; one unswollen), before and after hypo-osmolar cross-linking. The same parameters were measured in three other unswollen normal corneas before and after iso-osmolar cross-linking and in three pairs of swollen normal corneas, in which only the left was cross-linked (with iso-osmolar riboflavin). Results Hypo-osmolar cross-linking resulted in an increase in corneal hydration in all corneas. In the keratoconus corneas and unswollen normal corneas, this was accompanied by an increase in collagen interfibrillar spacing (p<0.001); an increase in fibril diameter was also seen in two out of four unswollen normal corneas and one unswollen keratoconus cornea (p<0.001). Iso-osmolar cross-linking resulted in a decrease in tissue hydration in the swollen normal corneas only. Although there was no consistent treatment-induced change in hydration in the unswollen normal samples, iso-osmolar cross-linking of these corneas did result in a compaction of collagen fibrils and a reduced fibril diameter (p<0.001); these changes were not seen in the swollen normal corneas. Collagen D-periodicity was not affected by either treatment. Conclusion The observed structural changes following Ultraviolet-A cross-linking with hypo-osmolar or iso-osmolar riboflavin solutions are more likely a consequence of treatment-induced changes in tissue hydration rather than cross-linking

The effect of riboflavin/UVA collagen cross-linking therapy on the structure and hydrodynamic behaviour of the ungulate and rabbit corneal stroma. PLoS One 2013

Abstract Purpose: To determine the effect of Ultraviolet-A collagen cross-linking with hypo-osmolar and iso-osmolar riboflavin solutions on stromal collagen ultrastructure in normal and keratoconus ex vivo human corneas. Methods: Using small-angle X-ray scattering, measurements of collagen D-periodicity, fibril diameter and interfibrillar spacing were made at 1 mm intervals across six normal post-mortem corneas (two above physiological hydration (swollen) and four below (unswollen)) and two post-transplant keratoconus corneal buttons (one swollen; one unswollen), before and after hypo-osmolar cross-linking. The same parameters were measured in three other unswollen normal corneas before and after iso-osmolar cross-linking and in three pairs of swollen normal corneas, in which only the left was cross-linked (with isoosmolar riboflavin). Results: Hypo-osmolar cross-linking resulted in an increase in corneal hydration in all corneas. In the keratoconus corneas and unswollen normal corneas, this was accompanied by an increase in collagen interfibrillar spacing (p,0.001); an increase in fibril diameter was also seen in two out of four unswollen normal corneas and one unswollen keratoconus cornea (p,0.001). Iso-osmolar cross-linking resulted in a decrease in tissue hydration in the swollen normal corneas only. Although there was no consistent treatment-induced change in hydration in the unswollen normal samples, iso-osmolar cross-linking of these corneas did result in a compaction of collagen fibrils and a reduced fibril diameter (p,0.001); these changes were not seen in the swollen normal corneas. Collagen D-periodicity was not affected by either treatment. Conclusion: The observed structural changes following Ultraviolet-A cross-linking with hypo-osmolar or iso-osmolar riboflavin solutions are more likely a consequence of treatment-induced changes in tissue hydration rather than crosslinking

Changes in collagen fibril diameter following Riboflavin/UVA cross-linking.

<p>The contour maps show collagen fibril diameter in an unswollen normal (N1) and keratoconus (K1) cornea before and after hypo-osmolar riboflavin/UVA collagen cross-linking (Hypo-R CXL) and an unswollen normal cornea (N7) before and after iso-osmolar riboflavin/UVA collagen cross-linking (Iso-R CXL). The centre of each corneal button corresponds approximately with the centre of each map. The locations of the stellate scar (solid line) and the scar at Descemet's membrane level (broken line) (as recorded schematically by the operating surgeon) have been superimposed onto the contour maps of K1.</p

Tissue hydration and collagen parameters measured before (CXL-) and after (CXL+) riboflavin/UVA collagen cross-linking.

<p> <b>Average values (+/− SEM) of collagen parameters for all samples (excluding N10–N15) were calculated using >50 measurements recorded from the central 8 mm region of the same corneas before (CXL−) and after cross-linking (CXL+). Averaged data shown for samples N10–N15 (3 pairs of corneas in which the left of each pair (N10, N12 and N14) was cross-linked (CXL+) and the right (N11, N13 and N15) remained untreated (CXL−)) is based on a single measurements obtained from the centre of each cornea. Bold type is used to indicate pre and post treatment differences in collagen parameters at p<0.001.</b></p

Sample details and treatments.

<p> <b>Details of pre-treatment tissue hydration (recorded at the time of data collection), classification and UVA cross-linking treatment (hypo-osmolar/iso-osmolar riboflavin solution) are shown for each sample. Each cornea has been classified as being either ‘unswollen’ (at or below a physiological hydration of H = 3.2) or ‘swollen’ (above physiological hydration).</b></p><p>*<b>The pre-treatment hydration of samples N10, N12 and N14 was assumed to be the same as that of their untreated pair (N11, N13 and N15 respectively).</b></p

Changes in collagen interfibrillar spacing following Riboflavin/UVA cross-linking.

<p>The contour maps show collagen interfibrillar spacing (IFS) in an unswollen normal cornea (N3) and a slightly swollen keratoconus cornea (K2) before and after hypo-osmolar riboflavin/UVA cross-linking (Hypo-R CXL) and a normal unswollen cornea (N8) before and after iso-osmolar riboflavin/UVA cross-linking (Iso-R CXL). The centre of each corneal button corresponds approximately with the centre of each map.</p