Surface ultrastructure of collagen fibrils and their association with proteoglycans in human cornea and sclera by atomic force microscopy and energy-filtering transmission electron microscopy

Purpose. We aimed to investigate the possible association of proteoglycans with D-periodic collagen fibrils in the human cornea and sclera, using ener gy-filtering transmission electron microscopy (EF-TEM) and atomic force mic roscopy (AFM). Methods. Human cornea and sclera were digested with keratana se to eliminate keratan sulfate proteoglycans (KSPGs). For EF-TEM observati on, surface proteoglycans were detected by cupromeronic blue (CB) staining. For AFM observation, cornea and sclera were treated with sodium hydroxide before and after keratanase digestion, and the surface topology of collagen fibrils was analyzed. Results. With CB staining, numerous CB-positive shor t filaments of surface proteoglycans (proteoglycan filaments) were observed in the interfibrillar spaces of cornea and sclera associated with collagen fibrils. AFM imaging showed that the depth and periodicity of D-periodic c ollagen fibrils in keratanase-treated corneal collagens were deeper and mor e regular than in untreated ones. Moreover, the depth and periodicity of ke ratanase-untreated corneal collagens were shallow and irregular in comparis on with keratanase-untreated scleral collagens. On the other hand, there wa s no difference in depth or regularity between keratanase-treated and-untre ated scleral colla.-en fibrils. Using AFM imaging, additional thin grooves Sub-bands were detected on the surface of keratanase-treated corneal collag en fibrils. The grooves were not detected in keratanase-untreated collagen fibrils nor in scleral collagen fibrils with or without keratanase digestio n. Comparing densitometry waves, the grooves of D-periodic corneal collagen sub-bands corresponded to a and c bands. Conclusion. Using AFM and EF-TEM to study corneal and scleral collagen fibrils and their association with pr oteoglycans, we conclude that KSPG is found in ample amounts in the human c ornea in comparison with sclera. Moreover, we topologically detected KSPG a ttached to a and c bands of collagen fibrils.