Abstract
Cell–cell communication plays a fundamental role in mediating corneal wound healing following injury or infection. Depending on the severity of the wound, regeneration of the cornea and the propensity for scar development are influenced by the acute resolution of the pro-fibrotic response mediated by closure of the wound via cellular and tissue contraction. Damage of the corneal epithelium, basement membrane, and anterior stroma following a superficial keratectomy is known to lead to significant provisional matrix deposition, including secretion of fibronectin and thrombospondin-1, as well as development of a corneal scar. In addition, corneal wounding has previously been shown to promote release of extracellular vesicles from the corneal epithelium, which, in addition to soluble factors, may play a role in promoting tissue regeneration. In this study, we report the development and characterization of a co-culture system of human corneal epithelial cells and corneal stromal fibroblasts cultured for 4 weeks to allow extracellular matrix deposition and tissue maturation. The secretion of provisional matrix components, as well as small and large extracellular vesicles, was apparent within the constructs, suggesting cell–cell communication between epithelial and stromal cell populations. Laminin-1β was highly expressed by the corneal epithelial layer with the presence of notable patches of basement membrane identified by transmission electron microscopy. Interestingly, we identified expression of collagen type III, fibronectin, and thrombospondin-1 along the epithelial–stromal interface similar to observations seen in vivo following a keratectomy, as well as expression of the myofibroblast marker, α-smooth muscle actin, within the stroma. Our results suggest that this corneal epithelial–stromal model may be useful in the study of the biochemical phenomena that occur during corneal wound healing.
Highlights
The cornea serves as a transparent barrier to protect the eye against environmental stresses
The human corneal fibroblasts (hCFs) were grown on a porous transwell membrane and stimulated to secrete and deposit an extracellular matrix (ECM), which occurred over 3 weeks in the presence of a stable Vitamin C (VitC) derivative that is known to stimulate collagen production [28]
The human corneal epithelial cells (hCECs) were seeded onto the monoculture construct, and the co-culture system was cultivated for an additional 7–10 days, with the hCECs submerged in serum-free epithelial media for 4 days and airlifted to an air–liquid interface for the final 3–6 days, to allow for the maturation of the epithelial layer [5]
Summary
The cornea serves as a transparent barrier to protect the eye against environmental stresses. The optical properties of the tissue require high transparency mediated by intricate organization of the stromal extracellular matrix (ECM). Prolonged or irregular wound healing may promote opacification of the cornea leading to fibrotic ECM deposition and vision loss. Over six million people are visually impaired due to corneal scarring, primarily a result of trachoma infections [1]. Corneal transplantation remains the only therapeutic option to recover vision loss due to corneal scarring. Physiologically relevant models to examine the pharmacological and toxicological effects of infection, chemical application, and disease are needed to develop novel therapeutics to prevent permanent corneal scarring
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