The transduction of biophysical cues from the extracellular space to intracellular machinery is collectively referred to as “mechanotransduction.” Recently, we developed high-aspect ratio, porous silicon nanoneedles (nN) for in vitro and in vivo manipulation of cell behaviour.  Of particular interest, nN physically deform both the cell membrane and the nucleus; however, the mechanisms by which these interactions occur, and the resulting effects on cell behaviour, in regards to the mechanotransduction machinery remain unexplored

In the present study, we have investigated the biophysical interactions of either human mesenchymal stem cells (hMSCs) or human umbilical vein endothelial cells (HUVECs) with nN.  Samples were prepared as previously described and were used as culture substrates for the two cell types over 6- and 48-hour periods.  Endpoint experiments included classical molecular biology techniques, such as quantitative polymerase chain reaction (qPCR), immunocytochemistry, and Western blot.  Super-resolution microscopy has also been employed to visualise cell-nN interactions.

Striking changes in cell behaviours were observed for hMSCs and HUVECs cultured on nN, as compared to flat silicon wafer controls.  These include the organization of the actin cytoskeleton, formation of focal adhesions, activation of mechanotransduction machinery, and expression of key nuclear envelope components.  Furthermore, cell shape descriptors and cell-nN interactions were correlated with the localisation of the mechanoresponsive co-factors, YAP and TAZ, indicating that whole cell-level changes in response to nN are biologically functional.  We have also developed “rescue” experiments to prove the role of nN features in regulating cell behaviours.  Our data indicate that the presence of nN disrupts the organization of the actin cytoskeleton and the morphology of the nucleus, and that focal adhesion formation and classical mechanotransduction pathways are compromised; these alterations coincide with cytosolic localisation of YAP/TAZ, indicating an inability to generate cytoskeletal tension.  When the nN degrade after 48 hours, these behaviours return to levels observed for cells cultured on flat substrates, indicating that the presence of the nN is responsible for the effects.  Finally, we are conducting biological rescue experiments to investigate how nN-induced changes in nuclear envelope morphology and composition impact YAP/TAZ and other classical signal transduction pathways. 

This ongoing work employs a novel cell culture platform for investigating and elucidating unconventional mechanotransduction cues, and offers insight for how cell-material interactions can be leveraged to regulate cell fate and function.

ACKNOWLEDGEMENTS: SW Crowder is funded by the Whitaker International Program, Institute of International Education, USA and Horizon 2020 Marie Curie Action, European Commission.