# Nanoscale Topographies for Corneal Endothelial Regeneration
## 3 The Impact of Bio-Instructive Nanosubstrates for Culture on Corneal Endothelial Cells
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Fabrication Method** & **Feature Dimension (\(\upmu\)m)** & **Cell Type** & **Reference** \\ \hline \multirow{3}{*}{Electron beam lithography and solvent casting} & Width: 20 nm-1 \(\upmu\)m & Rat dermal fibroblasts & [60] \\  & Depth: 5-350 nm & & \\ \hline \multirow{6}{*}{Electron beam lithography and hot embossing} & Diameter: 35-120 nm & Primary human fibroblasts & [61] \\  & Diameter: 120 nm & Human osteoprogenitor & \\  & Interval: 300 nm & and mesenchymal & [62] \\  & Height: 100 nm & stem cells & \\ \cline{2-4}  & Diameter: 120 nm & Human mesenchymal & \\  & Interval: 300 nm & stem cells & [63] \\ \cline{2-4}  & Depth: 100 nm & & \\ \cline{2-4}  & Diameter: 35-120 nm & Human fibroblasts and & \\  & Interval: 100-300 nm & rat epileton & [64] \\ \hline \multirow{3}{*}{Photolithography and soft lithography} & Diameter: 1 \(\upmu\)m & & \\  & Interval: 600 nm & Mouse 3T3 fibroblasts & [65] \\  & Height: 800 nm & & \\ \hline \multirow{3}{*}{Interference lithography and deep reactive ion etching} & Pitch: 230 nm & & \\  & Height: 50-600 nm & Human foreskin fibroblasts & [66] \\ \hline \multirow{3}{*}{Interference lithography and nanomiprint lithography} & Width: 200 nm & & \\  & Interval: 700 nm & & \\ \hline \multirow{3}{*}{Nanoimprint lithography} & Pitch: 420-800 nm & & \\  & Height: 0-350 nm & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Examples of fabrication methods to fabricate sub-micron features used in cell studies. and partially in descemetorhexis, without endothelial keratoplasty (DWEK), where the complete removal of a compromised ECM environment can also induce self-regeneration of a functional endothelium even without implantation of a new graft [70]. Similar to the hexagonal lattice of the [PERSON]'s membrane, collagen nanofibers constituting the native ECM (Figure 4) are also considered for delivering essential biophysical cues that control corneal endothelial cell phenotype. To mimic the nanoffibrillary contact guidance of native Descemet's membranes, instructive cell substrates can be produced by electrospinning using different materials. The [PERSON] group proposed synthetic blends composed of poly(glycerol sebacate) and poly(e-caprolactone) (PGS/PCL) as nanofibrous scaffolds for the culture of corneal endothelial cells [71; 72]. The scaffolds had elastic moduli in the same order of magnitude of native corneas. In these studies, a weight ratio of 4:1 parts of PGS with respect to PCL showed the best performances in terms of reaching confluence and exhibiting typical hCEC morphology. The scaffolds were made of aligned fibers with diameters between 200 and 500 nm. Such dimensions were reported to improve cell response when compared to fibers with thickness in the micrometer range [73], confirming the efficacy of nanotopographies for hCECs. While the aligned fibers of these scaffolds induced immortalized hCECs (HCEC-12) to align along their main axis, the authors reported the eventual formation of a confluent cell monolayer with typical hexagonal pattern. Cyto- and immune-compatibility was assessed by studying the interaction of the scaffolds with peripheral blood mononuclear cells and polymorphonuclear leukocytes. Similarly, [PERSON] and coworkers also investigated the cytocompatibility of electrospun scaffolds made of poly(methyl-methacrylate) (PMMA), poly(lactic-co-glycolic acid) (PLGA) and PCL [74]. Generally, PMMA electrospun meshes exhibited high cytotoxicity, while both PCL and PLGA showed good levels of biocompatibility. Regarding the topographical features, although keeping equal fabrication parameters for the different materials, differences in fiber diameter and interstitial spaces were noticed. Cells preserved their normal morphology only on smaller fibers with smaller interstitial space, which was obtained with PLGA although perfect cellular monolayers could not be generated with any of the materials tested.