Introduction

There are various factors important in the design of nerve guidance conduits (NGCs) used in peripheral nerve injuries. These factors include biodegradability, conductivity, permeability and supporting neural cell adhesion, proliferation and migration. Since the structure of molecules and cell-ECM interactions occur within the nanoscale, the surface of NGCs implanted should mimic the nanophase topography of ECM. While there are many studies investigating the effects of nanophase topography on various cells types, i.e. endothelial cells, mesenchymal stem cells, fibroblasts, etc., studies focusing on the effects of nanophase (<100nm) topography on neural cell functions are limited. As a matter of fact, most studies in neural tissue engineering focus on submicron level (>100nm) surface topography. Therefore, the aim of the study was to investigate the effects of <100nm topographical structures on poly(lactic-co- glycolic) acid (PLGA) on neural cell functions.

Methods

Nanopit features possessing 30 and 80 nm diameters were formed on stainless steel by anodization, and these structures were transferred onto PLGA surfaces via replica molding method. To confirm whether nanophase topographies were correctly obtained, scanning electron microscopy (SEM) and atomic force microscopy (AFM) characterizations were conducted. Additionally, hydrophobicity of the surfaces were determined using sessile drop water contact angle measurements. For the neural cell viability (mouse neuroblastoma N2a cell line), MTT analysis was conducted. To understand neural cell spreading, cells were stained with DAPI, vinculin, and f-actin. Finally, neural cell activity was assessed with c-fos protein expression levels by Western-blotting.

Results and Discussion

It was confirmed that nanophase topographies were successfully transferred onto PLGA surfaces. Sessile drop water contact angle measurements showed that hydrophilicity increased on surfaces having nanophase topographical features. For the proliferation studies, cellular density was found to increase up to 2-folds for nanophase surfaces compared to

control ones on the 3rd and 5th days of culture. As a result of the neural cell spreading, it was found that more neurite extensions were formed on nanophase topographical surfaces (A and B) than the control (C, Fig. 1). Additionally, c-fos protein expression levels increased up to 2- fold for nanophase topographical surfaces than the control.

Conclusion

As a conclusion, the nanophase topographies successfully transferred onto PLGA surfaces, and these topographies were correlated with the increase in hydrophilicity. Additionally, it was observed that nanophase surface topography had favorable effects for cellular proliferation, cell spreading and cellular activity on neural cells. All of these results showed that fabrication of <100nm sized topographical features is a promising approach for NGC applications.

Acknowledgement

This work was financially supported by The Scientific and Technological Research Council of Turkey (Grant no: 217M952). The authors would like to thank Corbion Purac (Amsterdam, The Netherlands) for PURASORB® poly (lactic-co-glycolic acid) (w/w, 50:50), BIOMATEN-METU Center of Excellence in Biomaterials and Tissue Engineering (BIOMATEN), Middle East Technical University (METU) Central Laboratory.

Keywords: nanostructure, PLGA, neural cells