Lamellar and dispersed fibrous collagen networks are organized and maintained via endogenous crosslinks along the superior-inferior and nasal-temporal directions in the stromal regions of corneal tissues. Collagen organization contributes to corneal transparency, tissue integrity, and the surface topography. Ultrastructural changes to the lamellar arrangement of collagen occur in diseases, such as keratoconus and ectasia post refractive surgery, resulting in impaired biomechanical properties, changes to the surface curvature, and irregular astigmatism. Collagen crosslinking with UV-A/riboflavin is used clinically to increase the structural integrity and halt corneal thinning; however it can cause complications in certain cases. Earlier studies suggest that crosslinking mediated by advanced glycation end products (AGE), associated with ageing, may increase corneal stiffness and prevent corneal thinning. The specific links between corneal properties and microstructural network features are however not well established. We used collagenase and non-enzymatic crosslinking using methylglyoxal (MGO) to investigate the effects of collagen content, organization, and crosslinking densities in an ex-vivo goat cornea model. We estimated the collagen contents using a biochemical assay, performed uniaxial mechanical tests, and used histology to quantify the underlying fiber tortuosity in untreated (control) and collagenase/MGO treated groups. We fit the experimental stress-strain data using an exponential strain energy function (SEF) that uses a generalized structure tensor to describe collagen fiber organization in tissues. Our results show that fiber tortuosity increased with collagenase treatment time. AGE-mediated non-enzymatic crosslinking using MGO caused a dramatic increase in the elastic modulus of tissues without significant changes to the fiber tortuosity or overall collagen content. Finally, we obtained scaling relationships linking tissue modulus to collagen volume fraction that may be useful clinically. Changes in fiber tortuosity with collagenase treatment suggest that collagen fiber organization and composition play a key role in regulating mechanobiological properties of the cornea.
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