Regional quantification of glycosaminoglycans and their association with collagen fibril ultrastructure in the sclera following enzymatic degradation

Sclera biomechanics play an important role in clear vision. Understanding the biomechanical, composition and ultrastructural topography of the sclera may help provide better insight into eye health. Some prior research has investigated the ultrastructural and biomechanical properties of the sclera in relation to regional variations. However, the complex association between regional variations in collagen fibril morphology and elasticity with proteoglycan types and quantities has not been investigated. This study aimed to explore regional variations in scleral collagen fibril topography and proteoglycan quantities and to investigate their role in the mechanical properties of the sclera. Atomic force microscopy (AFM), collagen autofluorescence, glycosaminoglycans (GAGs) quantification, and Coomassie blue staining techniques were used to assess alterations of the porcine sclera following treatment with amylase (Amy) and chondroitinase ABC (ChABC). Collagen fibril diameters were found to vary among the regions of the scleral stroma, with the largest diameters (268 ± 23 nm) in the anterior region and smallest diameters (148 ± 14 nm) in the posterior region. Collagen fibril stiffness and diameters were reduced following incubation with these enzymes. GAGs were depleted from the enzymatically treated tissues with the greater depletion in the posterior region. GAG depletion was inversely correlated (Pearson's r = −0.75 and −0.85 for the amylase and ChABC treated groups) with collagen fibril diameter and elastic modulus. In summary, we show the direct link between GAGs and collagen fibril properties at the nano-scale from the anterior to the posterior region of the porcine sclera.