To investigate the changes in cell morphology that occurred from the new nanoneedle arrays, we cultured human mesenchymal stem cells (hMSCs) on flat silicon (as a control), nanopillars, two different blunt nanoneedles, and one set of sharp nanoneedles. We fixed the cells for immunostaining at four time-points (6, 12, 24, and 72 h after seeding). We then used image-based cell profiling to analyze 5,372 immunofluorescent microscopy images and extract single-cell morphological and protein localization features for over 100,000 cells. From this high-content image analysis, we were able to quantify pronounced, systematic morphological changes as a function of nanoneedle sharpness. In particular, decreasing the tip diameter reduced the spread area of both cells and nuclei, promoted cell body elongation, and decreased the protrusion ratio, which is the area of cell protrusions divided by the total cell area. In addition, nuclear solidity (a measure of nuclear perimeter tortuosity) visualized as slight scalloping in the in-plane nuclear membrane around the nanoneedles, decreased with increasing nanoneedle sharpness. Background-corrected and batch-normalized intensities of cytoskeletal proteins (F-actin, α-tubulin) were also influenced by changing tip diameter. Local cell density, determined by Voronoï tessellation, was also greater on nanoneedles than flat surfaces or nanopillars.
We next looked at the impact of nanoneedle tip diameter on gene expression. hMSCs were cultured for 6 and 24 h on flat silicon substrates or nanoneedles with varying tip diameters, before measuring the expression of a wide portfolio of genes, including focal adhesions proteins (PXN), nuclear lamins (LMNA, LMNB). Interestingly, LMNA expression was significantly influenced by both the presence of a nanostructured substrate and as a function of increasing nanoneedle tip diameter. LMNA codes for lamin A, a major structural component of the nuclear lamina, and our observation is consistent with previous studies showing a strong correlation between nuclear deformation and lamin expression. This finding was consistent with the reported increase in LMNA expression in cells on porous nanoneedles. We did not observe any significant changes in LMNB, the gene encoding lamin B.
Moreover, we were also able to significantly reduce the gene expression of PXN after 6 h of culture on the sharp nanoneedles, compared to the flat substrates (Figure 2c). This result was consistent with previous studies performed on porous nanoneedles, however, our non-degradable arrays enabled us to investigate gene expression beyond a 6 h time point. This analysis revealed a return to baseline expression levels after 24 h, which was expected given that PXN codes for the paxillin, a protein that is expressed at focal adhesions of during cell attachment. Indeed, immunostaining for paxillin showed a reduced intensity and reduced focal adhesion points for the hMSCs cultured on sharp nanoneedles at 24 h.
Finally, we used focused-ion-beam scanning electron microscopy (FIB-SEM) to image cross-sections through the cell-nanoneedle interface in order to visualize how biological membranes were perturbed by the nanotopography. For both the sharp nanoneedles and nanopillars, the plasma cell membrane was strongly perturbed by the vertical arrays, wrapping conformably around the silicon nanostructures after just 6 h. However, the depth of impingement of the plasma membrane was far greater on the sharp needles, moreover, the nuclear deformation was highly dependent upon tip diameter, with only the sharp nanoneedles able to perturb the nuclear membrane. The degree of cellular and nuclear membrane deformation increased with culture time, an important insight for the design of non-degradable nanoneedle arrays for long-term culture, intracellular delivery and sensing. We further investigated these structural changes for the 12 h timepoint by reconstructing consecutive FIB-SEM slices into a volumetric map, which allowed us to fully visualize the cell-nanoneedle interface in 3D. This reconstruction analysis showed that the impingement behavior was consistent across the entire cell and nuclear area.
