Figure 1.
Cross-section of cross-linked and non-cross-linked pig corneas.
Small-angle x-ray scattering images were obtained at 25 µm intervals (red circles) throughout the anterior 300 µm of riboflavin/UVA treated and untreated strips of pig cornea using a microfocus x-ray beam. Each graduation on the scale bar represents 150 µm.
Figure 2.
Enzymatic digestion rate of treated and untreated pig corneas.
Table 1.
Average collagen interfibrillar spacing, D-periodicity and order factor in treated and untreated pig corneas.
Table 2.
Average collagen interfibrillar spacing, D-periodicity and order factor in treated and untreated paired rabbit corneas.
Figure 3.
Collagen parameters in cross-linked and non-cross-linked pig corneas.
Average measurements of interfibrillar spacing (A) and fibril diameter (B) at 25 µm intervals throughout the anterior 300 µm of treated and untreated pig corneas. Significant differences between treatment groups are highlighted by asterices (p<0.05).
Figure 4.
Depth profile of collagen intermolecular spacing in treated and untreated sheep corneas.
Table 3.
Average collagen intermolecular spacing in treated and untreated pig corneas.
Figure 5.
Stromal thickness in pig corneas before and after treatment.
Controlled air drying of the most hydrated corneas ensured that all corneas were of a similar thickness at the start of the swelling study.
Figure 6.
Treated and untreated corneal buttons before and after in vitro swelling.
A black dot was drawn on the graph paper beneath each cornea for qualitative assessment of corneal transparency before (A) and after (B) 5 hours of swelling in saline solution.
Figure 7.
Stromal swelling rate in treated and untreated pig corneas.
Data shows the rapid stromal swelling phase (A) and the subsequent slower swelling period (B).
Table 4.
Maximum achievable hydration of treated and untreated pig corneas.
Figure 8.
Schematic showing possible cross-linking scenarios.
A simplified model showing three collagen fibrils, each with a coating (outer limit shown as a broken line) consisting mainly of proteoglycans which are attached to the fibril and form a porous network with fractal dimension (based on Fratzl and Daxer's theoretical model of collagen fibrils in the corneal stroma [48]). Coloured lines indicate the possible location(s) of riboflavin/UVA induced cross-links that are discussed in the text.
Figure 9.
Schematic showing likely collagen shrinkage during electron microscopy processing of cross-linked and non-cross-linked corneas.
(i) The theoretical structure of a coated collagen fibril (F) in the corneal stroma (as proposed by Fratzl and Daxer [48]). The coating (outer limit shown as a broken line), consists mainly of proteoglycans (P) which are attached to the fibril and form a porous network with fractal dimension. We propose that riboflavin/UVA induced cross-links are formed within the coating of the collagen fibril between proteoglycan core proteins and/or on the surface of the fibril within and between collagen molecules (M) (ii) and prevent the usual shrinkage associated with tissue dehydration during electron microscopy processing (iii and iv). Hence, when viewed by electron microscopy, collagen fibrils in riboflavin/UVA treated corneas (iv) may misleadingly appear larger in diameter than those in untreated corneas (iii).