Effect of corneal collagen crosslinking on viscoelastic shear properties of the cornea

Abstract

The cornea is responsible for most of the refractive power in the eye and acts as a protective layer for internal contents of the eye. The cornea requires mechanical strength for maintaining its precise shape and for withstanding external and internal forces. Corneal collagen crosslinking (CXL) is a treatment option to improve corneal mechanical properties. The primary objective of this study was to characterize CXL effects on viscoelastic shear properties of the porcine cornea as a function of compressive strain. For this purpose, corneal buttons were prepared and divided into three groups: control group (n = 5), pseudo-crosslinked group (n = 5), and crosslinked group (n = 5). A rheometer was used to perform dynamics torsional shear experiments on corneal disks at different levels of compressive strain (0%-40%). Specifically, strain sweep experiments and frequency sweep tests were done in order to determine the range of linear viscoelasticity and frequency dependent shear properties, respectively. It was found that the shear properties of all samples were dependent on the shear strain magnitude, loading frequency, and compressive strain. With increasing the applied shear strain, all samples showed a nonlinear viscoelastic response. Furthermore, the shear modulus of samples increased with increasing the frequency of the applied shear strain and/or increasing the compressive strain. Finally, the CXL treatment significantly increased the shear storage and loss moduli when the compressive strain was varied from 0% to 30% (p < 0.05); larger shear moduli were observed at compressive 40% strain but the difference was not significant (P = 0.12).

Keywords: Collagen crosslinking treatment; Cornea; Porcine eyes; Shear properties; Torsional shear experiments.

Figure 1.

A schematic plot of the dynamic shear experimental setup. The corneal disks of 8 mm diameter were placed between the lower and upper plates of a rheometer in order to measure their dynamic shear properties at different levels of compressive strain. The strain sweep (with amplitude of 0.01% to 10%) and frequency sweep (with frequencies of 0.01 Hz to 2 Hz) torsional shear experiments were performed.

Figure 2.

The effect of CXL treatment on the variation of storage shear modulus G’ (a and b) and loss shear modulus G” (c and d) as a function of amplitude of the applied shear strain γ₀. The dependence of storage and loss shear moduli on the applied compressive strain ε is also shown. The strain sweep experiments were done at a frequency of 1 Hz. The symbols denote the average of experimental measurements and error bars indicate one standard deviation.

Figure 3.

The effect of CXL treatment on the variation of storage shear modulus G’ (a and b) and loss shear modulus G” (c and d) as a function of frequency. The dependence of storage and loss shear moduli on the applied compressive strain ε is also shown. The frequency sweep experiments were done within the range of linear viscoelastic at shear strain magnitude of γ₀ = 0.2%. The symbols denote the average of experimental measurements and error bars indicate one standard deviation.

Figure 4.

The normalized complex modulus of crosslinked and untreated samples as a function of the shear strain magnitude in strain sweep experiments. The experiments were done using compressive strain from 0% to 40% and at a frequency of 1 Hz. A characteristic curve, shown by the dashed line, independent of the compressive strain and whether the samples were crosslinked, is observed. The filled and unfilled symbols show the measurements obtained for the corneal disks in crosslinked and untreated groups, respectively. The range of linear viscoelasticity is also shown.

Figure 5.

The effect of corneal collagen crosslinking on the complex shear modulus ∣G*∣, averaged over the range of linear viscoelasticity, at different levels of compressive strain ε. The strain sweep experiments were done at frequency 1 Hz. No significant difference was observed between the shear response of pseudo-crosslinked and untreated samples; however, the crosslinking treatment significantly increased the magnitude of complex shear modulus when the compressive strain was less than 40% (no significant difference was observed at ε= 40%). The asterisk indicates significant differences between the two groups (p<0.05). The complex shear modulus of all samples increased significantly with increasing the compressive strain (no asterisk was shown for these groups so that the plot does not get crowded). The error bars indicate one standard deviation.

Figure 6.

A schematic showing that the CXL treatment creates additional crosslinks in corneal extracellular matrix increasing its structural integrity. The exact location of these crosslinks is not known but they possibly form between proteoglycans core proteins and collagen molecules, within collagen molecules, between glycosaminoglycan side chains and core proteins of proteoglycans, and between the proteoglycan core proteins [44].

Figure 7.

The effect of corneal collagen crosslinking on the complex shear modulus, obtained from torsional shear tests done at shear strain 0.2% and at frequency 0.01 Hz, as function of the applied compressive strain. No significant difference was observed between the shear response of pseudo-crosslinked and untreated samples; however, the crosslinking treatment significantly increased the magnitude of complex shear modulus when the compressive strain was less than 40% (no significant difference was observed at ε= 40%). The asterisk indicates significant differences between the two groups (p<0.05). The complex shear modulus of all samples increased significantly with increasing the compressive strain (no asterisk was shown for these groups so that the plot does not get crowded). The error bars indicate one standard deviation.