# Nanoscale Topographies for Corneal Endothelial Regeneration
## 1 Introduction  
The cornea is a transparent tissue located in the anterior segment of the eye. This vascular tissue allows light to enter the eye and accounts for most of its refractive power [1]. It is composed of five layers (Figure 1): the epithelium, the Bowman's layer, the stroma, the Descent's membrane and the endothelium. The corneal epithelium is the outermost layer and consists of a stratified sheet of epithelial cells residing on the Bowman's layer. The corneal stroma accounts for 80 to 90% of the total tissue volume and provides the cornea with mechanical strength. This layer is composed of highly structured collagen fibers and other extracellular matrix (ECM) proteins produced by the corneal keratocytes, which maintain the stromal homeostasis. The inner part of the cornea is composed of a monolayer of hexagonal endothelial cells that is in contact with the stroma through the [PERSON]'s membrane (Figures 1 and 2), the latter being characterized by well-defined architecture, porosity and collagen fibrils dimension [2, 3]. It is believed that a dual relationship exists between the [PERSON]'s membrane and the corneal endothelium, where the pathophysiology of both of these parts can equally affect or be affected by the state of the other [3]. These (human) corneal endothelial cells (hCECs) play a crucial role in maintaining the corneal transparency by actively pumping ions and other metabolites from the stroma to the aqueous humor of the eye, consequently regulating the corneal hydration level. Unlike vascular endothelial cells, hCECs derive from the neural crest during development. In adults, hCECs are retained in a non-dividing G1 phase [4] and, thus, cannot contribute to the regeneration of this layer through cell division [5]. Corneal dystrophies, damage after surgery or infections can cause a loss of hCECs. The pathological profile of corneal endothelial disease is characterized by cornea edema and opacification. Between 2012 and 2013, about 200,000 corneal transplantations were performed worldwide, with an estimation of 12.7 million patients awaiting transplantation [6]. Currently, the state-of-the-art therapy for corneal endothelial disease involves the selective replacement of the corneal endothelium with that of a donor [7]. However, only one donor cornea is available for every seventy patients in need [6]. The primary culture of hCECs is a promising alternative cell source to treat corneal endothelial disease [8], which would challenge the current one donor-one patient paradigm to make the therapy available for more patients. Despite hCECs being arrested in a non-proliferative state, it is  
Figure 1: Anatomy of the cornea with schematic representation of its five main layers. Epithelial cells are indicated in blue, stromal keratocytes in purple and endothelial cells in orange. Figure 2: Human corneal endothelium biopsy analysis. Brightfield image of the typical hexagonal morphological pattern exhibited by human corneal endothelial cells (hCECs) **(A)**; immunofluorescence analysis of native human corneal endothelium (**B** and **C**); cell nuclei were stained with Hoechst and are shown in blue. Functional marker Na\({}^{+}\)/K\({}^{+}\)-ATPase (**B**) and tight junction marker ZO-1 (**C**) are shown in green. Scale bars: **(A)**: 100 \(\upmu\)m; **(B)** and **(C)**: 50 \(\upmu\)m.  
possible to force their in vitro proliferation by culturing them with different basal media compositions, growth factors, such as fibroblast growth factor 2 or nerve growth factor, and other additives, such as ascorbic acid or calcium chloride [9]. Nonetheless, forcing hCECs to exit their quiescent state to enter a proliferative state by solely using biochemical factors is known to cause an undesired endothelial-to-mesenchymal transition resulting in a loss of function [10].