Neurons sense nanoscale roughness with nanometer sensitivity

Abstract

The interaction between cells and nanostructured materials is attracting increasing interest, because of the possibility to open up novel concepts for the design of smart nanobiomaterials with active biological functionalities. In this frame we investigated the response of human neuroblastoma cell line (SH-SY5Y) to gold surfaces with different levels of nanoroughness. To achieve a precise control of the nanoroughness with nanometer resolution, we exploited a wet chemistry approach based on spontaneous galvanic displacement reaction. We demonstrated that neurons sense and actively respond to the surface nanotopography, with a surprising sensitivity to variations of few nanometers. We showed that focal adhesion complexes, which allow cellular sensing, are strongly affected by nanostructured surfaces, leading to a marked decrease in cell adhesion. Moreover, cells adherent on nanorough surfaces exhibit loss of neuron polarity, Golgi apparatus fragmentation, nuclear condensation, and actin cytoskeleton that is not functionally organized. Apoptosis/necrosis assays established that nanoscale features induce cell death by necrosis, with a trend directly related to roughness values. Finally, by seeding SH-SY5Y cells onto micropatterned flat and nanorough gold surfaces, we demonstrated the possibility to realize substrates with cytophilic or cytophobic behavior, simply by fine-tuning their surface topography at nanometer scale. Specific and functional adhesion of cells occurred only onto flat gold stripes, with a clear self-alignment of neurons, delivering a simple and elegant approach for the design and development of biomaterials with precise nanostructure-triggered biological responses.

Fig. 1.

AFM analysis of gold surfaces with increasing level of nanoroughness. The surface morphology shifts from a flat gold film (A) to uniformly nanostructured surface with different level of nanoroughness (B–E). R_a indicates the mean surface roughness, calculated on 40 × 40 μm² regions. On the bottom of each substrate, representative line profiles are reported.

Fig. 2.

(A) SH-SY5Y cell adhesion onto different nanostructured gold surfaces with increasing values of nanoroughness (from 0 to 100 nm R_a), after 24 h of culture. (B) AnnexinV-FITC/PI in vivo staining of SH-SY5Y cells cultured for 24 h onto substrates with different nanoroughness. By this test, it was possible to distinguish and count between apoptotic cells (AnnexinV⁺/PI^-), necrotic cells (AnnexinV^-/PI⁺), and viable cells (AnnexinV^-/PI^-).

Fig. 3.

Confocal images of SH-SY5Y cultured onto (A) flat and (B) nanorough (R_a = 100 nm) surfaces. Cells were incubated with Phalloidin-TRITC conjugate for actin cytoskeleton staining and with Hoechst 33258 for nuclei staining.

Fig. 4.

Confocal images of SH-SY5Y cultured onto flat (A, B) and nanorough (C, D) surfaces. Cells were stained with antivinculin antibodies to mark focal adhesions (Green) and Hoechst 33258 (Blue) for nuclei labeling.

Fig. 5.

Representative confocal images of SH-SY5Y cultured onto flat (A, B) and nanorough (C, D) surfaces. Cells were labeled with anti-golgin 97 antibodies to stain Golgi network (Red) and Hoechst 33258 (Blue) for nuclei staining.

Fig. 6.

Representative confocal images showing the selective adhesion of SH-SY5Y cultured onto flat/nanorough micropatterned 100-μm stripes (A–C) and 50-μm stripes (D). Cells were stained with Phalloidin-TRITC conjugate and with Hoechst 33258 for cytoskeleton and nuclei imaging, respectively.