Abstract
Diabetes mellitus is a disease caused by innate or acquired insulin deficiency, resulting in altered glucose metabolism and high blood glucose levels. Chronic hyperglycemia is linked to development of several ocular pathologies affecting the anterior segment, including diabetic corneal neuropathy and keratopathy, neovascular glaucoma, edema, and cataracts leading to significant visual defects. Due to increasing disease prevalence, related medical care costs, and visual impairment resulting from diabetes, a need has arisen to devise alternative systems to study molecular mechanisms involved in disease onset and progression. In our current study, we applied a novel 3D in vitro model of the human cornea comprising of epithelial, stromal, and neuronal components cultured in silk scaffolds to study the pathological effects of hyperglycemia on development of diabetic corneal neuropathy. Specifically, exposure to sustained levels of high glucose, ranging from 35 mM to 45 mM, were applied to determine concentration-dependent effects on nerve morphology, length and density of axons, and expression of metabolic enzymes involved in glucose metabolism. By comparing these metrics to in vivo studies, we have developed a functional 3D in vitro model for diabetic corneal neuropathy as a means to investigate corneal pathophysiology resulting from prolonged exposure to hyperglycemia.
Highlights
The polyol pathway has been linked to diabetic complications in the neural retina and lens through the production of excess reactive oxygen species (ROS) and reduced glutathione availability, causing osmotic damage[11]
We found that hECs and Human corneal stromal stem cell (hCSSC) in control constructs appeared dense and attached on silk films, having typical shape exemplified by extended, polygonal Human corneal epithelial cell (hCEC) and elongated structure for hCSSCs
We report having developed a functional model to mimic diabetic corneal neuropathy in vitro using silk scaffolds as a mechanically tunable material comparable to collagen-based approaches and corneal elasticity found within the human cornea[21,38]
Summary
The polyol pathway has been linked to diabetic complications in the neural retina and lens through the production of excess reactive oxygen species (ROS) and reduced glutathione availability, causing osmotic damage[11]. Given disease pervasiveness and a dearth of appropriate systems, though, a clinical need has arisen to establish accurate tissue models for diabetic research Some such approaches employing tissue engineered in vitro corneal models include a corneal equivalent for drug permeation studies[20], and an innervated model to examine nerve-target cell interactions[21]. The results of our study demonstrate a significant improvement upon past methods for visualizing diabetic neuropathy in vitro and present a novel approach to study diabetes-associated complications within the peripheral nervous system Development of this in vitro tissue model to mimic the effects of hyperglycemia on corneal innervation suggests further potential application of this bioengineered system for broader studies of chronic nerve function and dysfunction. Error bars represent standard error. *p < 0.05 as determined by a one-way ANOVA
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