# Nanoscale Topographies for Corneal Endothelial Regeneration
## 3 The Impact of Bio-Instructive Nanosubstrates for Culture on Corneal Endothelial Cells
In another study, monolayers of the human corneal endothelial cell line B4G12 could be successfully formed using blends of nanofibrous structures made of silk fibroin and poly(L-lactic acid-co-\(\epsilon\)-caprolactone) (P(LLA-CL)) [75]. The authors stated that the combination of natural and synthetic materials offered by silk(P(LLA-CL)) constructs could provide both biocompatibility and mechanical properties necessary for the culture of endothelial cells. This study evaluated different parameters such as cell adherence, proliferation and gene expression as well as light transmittance, identifying 25:75 as the optimal ratio of SF:P(LLA-CL) for the culture of corneal endothelial cells. [PERSON]'s group extensively studied bio-instructive substrates replicating features such as pillars and wells of various dimensions in different materials (Figure 5) [76; 77; 78; 79; 80; 81]. In particular, they were able to improve not only hCECs culture but also to recreate a disease  
Figure 4: Scanning electron microscope (SEM) images of the human [PERSON]’s membrane obtained by decellularization of the corneal endothelium. model such as [PERSON] endothelial dystrophy by using these topographies (Figure 5B), which is the main indication for endothelial keratoplasty (surgery) worldwide and for which currently no functional in vitro models are available [80]. Interestingly, the use of substrates with higher pillar density and with larger features (>10 \(\upmu\)m) was shown to prevent monolayer formation and induce [PERSON] endothelial dystrophy conditions (i.e., mimicking guttata), stressing the importance of sub-cellular features for physiological hCEC expansion and phenotype. Regarding the influence of particular patterns on hCECs expansion and phenotype, nanopillars and nanowells showed improved guidance compared to other nanopatterns [76; 77]. Pillars patterned on gel-methacrylate (GelMA) substrates induced enhanced ZO-1 expression and morphological parameters according to whether they were arranged in a square or hexagonal pattern [81]. The authors also showed that the expression of ZO-1 could be retained upon removal of the biophysical cues, i.e., when cells were subsequently cultured again on conventional tissue culture plastic, indicating a memory effect of cells after exposure to topographical cues [79]. Nanotopographies can also be used to generate carriers for tissue engineered corneal endothelium, therefore not only for ensuring hCECs expansion in vitro but also for main  
Figure 5: Examples of substrate topographies used for the culture of hCECs. Nanopillars (**E,F,H** in top image) and nanowells (**D,G,E,I** in top image) were demonstrated to better induce hCECs expansion and maintenance of their phenotype compared to other topographies (**A–C** in top image) (**A**), while patterns with larger dimensions can be used for modelling disease of hCECs such as [PERSON]’s endothelial dystrophy (**B**). Scale bars: 20 \(\upmu\)m unless specified. Reproduced from [77] (**A**) and [80] (**B**). taining the hCECs phenotype upon transplantation. In a study by [PERSON] and coworkers, nanoporous (pore size \(<\)200 nm diameter) silk fibroin films were explored as a carrier for rabbit corneal endothelial cells [82]. Although this study was not focused on comparing different nanofeatures but assessed the effect of sericin content in the films, it demonstrated that this nanomaterial supports cell attachment and morphology well, as well as expression of ZO-1 and Na+/K+-ATPase both at the mRNA and protein level.