The ability of five FARs (DAU, IMU, SMG, DMDM, OCT) to cross-link intact cadaveric rabbit cornea, a substrate for collagenous tissue, was assessed using an ex vivo TXL simulation setup. Cross-linking effects were measured using DSC, an assay method based on changes in thermal denaturation temperature (
Tm). Our results indicate that two out of the seven FARs studied, DAU and SMG, are effective collagen cross-linking agents for the cornea with the epithelium left intact (in the ex vivo simulation setup) at 1/2max allowed concentrations. Diazolidinyl urea was effective at pH 8.5, and SMG was effective at both pH 8.5 and 7.4. This was evidenced by shifts in the thermal denaturation temperature of corneal tissue (
Fig. 1). Sodium hydroxymethylglycinate pH 8.5 showed the greatest upward shift in
Tm (
ΔTm = 3.573 ± 0.578°C,
P < 0.05) followed by DAU at pH 8.5 (
ΔTm = 3.210 ± 0.742°C,
P < 0.05). Sodium hydroxymethylglycinate also showed effective cross-linking at pH 7.4 (
ΔTm = 2.281 ± 0.697°C,
P < 0.05). Some inconsistencies in the shifts in
Tm induced by SMG at pH 7.4, however, were noted, and a sample size of
n = 8 was required to reach statistical significance. A negative shift in
Tm on the order of ~0.5°C was observed for DAU at pH 7.4 and for IMU at both pH 8.5 and 7.4 (
Fig. 1), but the shift was significant only for IMU at pH 8.5 (
ΔTm = −0.69 ± 0.697°C,
P < 0.05). The lack of effect under these conditions may reflect issues related to epithelial permeability since both DAU and IMU are significantly larger than SMG. Furthermore, an increase in
Tm was observed for DMDM at both pH 8.5 and 7.4 (
ΔTm = 2.04 ± 0.225°C and 2.13 ± 0.273°C, respectively) and for OCT at pH 8.5 (
ΔTm = 1.10 ± 0.246°C). However, these observed increases in
Tm were not statistically significant using paired controls, which included the contralateral eye for each sample. Rabbit cornea cross-linked using UVA-riboflavin (CXL) showed an increase in
Tm comparable to values previously reported. In the present study, the
ΔTm following CXL was 1.73 ± 0.487°C. We previously reported a
ΔTi = 1.9°C,
19 which was similar to that reported by Spoerl et al.
34 at 2.5°C.
Ti values indicate the onset of thermal shrinkage (
Ts) temperature, and change in
Ti (
ΔTi) is another parameter widely used to measure extent of TXL. As previously reported, the CXL effect is relatively mild from a “thermal transition shifting” standpoint if one considers the potential magnitude of shifts in
Tm that may be induced using chemical agents. Lastly, it is worth noting that corneal tissue remained clear to visual inspection using either chemical or photochemical cross-linking treatment.