# Nanoscale Topographies for Corneal Endothelial Regeneration
## 4 Conclusions and Future Directions  
Effective cell therapy and regenerative medicine approaches require hCECs to form confluent monolayers, while maintaining their original phenotype. To induce the formation of a functional, confluent hCECs monolayer, there is a limitation on the features and dimensions of topographies that can be used. In particular, topographical features (both lateral size and height), with dimensions comparable to or larger than the cellular size (i.e., \(\geq\)2-5 \(\upmu\)m), can hinder the formation of a continuous layer of cells and steer cells away from the corneal endothelium phenotype. For this reason, subcellular (nano-)topographical patterns have demonstrated better support for the physiological hCECs' phenotype over micro-topographical patterns. In light of the studies reviewed in this work, it is evident that nanotopographies are a potential technology to further support primary hCEC expansion and, therefore, increase the pool of cells suitable for corneal endothelial regenerative therapy, while larger micro-topographies could provide avenues for modelling hCEC disease.  
While nanofabrication techniques enabled the generation of improved substrates for hCECs culture, further improvements are required. For instance, imprinting nanotopographies on electrospun fiber meshes by fiber-level patterning results in hierarchically structured substrates, which could improve current protocols for hCECs regeneration [83]. Moreover, the combination of multiple nanotechnologies that allows the superimposition of porosity onto nanopatterns can also generate new strategies for recapitulating the native endothelial environment and facilitate nutrient, water, ion and metabolite transport of tissue engineered corneal endothelium. Creating controlled porosity on substrates is possible by various methods, such as photolithography and ion-track technology [84], the latter being already implemented to fabricate porous polycarbonate membranes, which are commercialized for cell culture applications [85]. On the other hand, self-assembly techniques such as water droplet-assisted methods [86] including breath figure [87] and phase separation methods [88] can still be considered as alternative to lithography techniques.  
The combination of biomaterials, fabrication techniques and specific patterns could impact on the overall biophysical and biological performances [89, 90, 91]. Biomechanical properties, biodegradability as well as ions and nutrient diffusion, for instance, are all important factors needing careful consideration when selecting suitable materials and fabrication techniques for translational applications. Moreover, major steps for the design of future studies also include the optimization of surface chemistry and substrate stiffness.  
Thus far, nanotopographies demonstrated efficacy towards inducing corneal endothelial regeneration; however, new (combinations) of existing nanotechnology offer the potential to provide additional tools for further advancing this field. The studies reported in this review can, therefore, provide basic insights to design novel studies for hCECs regeneration and eventually provide therapeutic solutions for corneal endothelium dysfunctions.  
Conceptualization: N.F.; methodology, writing--original draft preparation, resources: N.F., G.S. and P.C.; writing--review and editing: N.F., G.S., P.C., S.G., R.T., V.L.S.L., M.M.D. and R.M.M.A.N.; supervision, resources: S.G., R.T., V.L.S.L., M.M.D. and R.M.M.A.N.; project administration and funding acquisition: S.G. All authors have read and agreed to the published version of the manuscript.  
This research was part of BM3.02 EyeSciTe and performed under the framework of Chemelot InSciTe.  
**Institutional Review Board Statement:** Not applicable.