The nanomorphology of cell surfaces of adhered osteoblasts

Abstract

The functionality of living cells is inherently linked to subunits with dimensions ranging from several micrometers down to the nanometer scale. The cell surface plays a particularly important role. Electric signaling, including information processing, takes place at the membrane, as well as adhesion and contact. For osteoblasts, adhesion and spreading are crucial processes with regard to bone implants. Here we present a comprehensive characterization of the 3D nanomorphology of living, as well as fixed, osteoblastic cells using scanning ion conductance microscopy (SICM), which is a nanoprobing method that largely avoids mechanical perturbations. Dynamic ruffles are observed, manifesting themselves in characteristic membrane protrusions. They contribute to the overall surface corrugation, which we systematically study by introducing the relative 3D excess area as a function of the projected adhesion area. A clear anticorrelation between the two parameters is found upon analysis of ca. 40 different cells on glass and on amine-covered surfaces. At the rim of lamellipodia, characteristic edge heights between 100 and 300 nm are observed. Power spectral densities of membrane fluctuations show frequency-dependent decay exponents with absolute values greater than 2 on living osteoblasts. We discuss the capability of apical membrane features and fluctuation dynamics in aiding the assessment of adhesion and migration properties on a single-cell basis.

Automatic Actin Filament Quantification and Cell Shape Modeling of Osteoblasts on Charged Ti Surfaces

“…This height is directly accessible by SICM, a scanning probe microscopy method, largely avoiding forces on cells. As we pointed out earlier [47], the cell edge height is a strongly varying quantity (between 100 nm and 1 µm) when measuring around the whole perimeter of the cell. Therefore, we took average values for each cell.…”

“…First, 4000 MG-63 cells/cm 2 were cultured on pristine glass cover slides (Thermo Fisher Scientific, Waltham, MA, USA) as well as plasma polymer (PPAAm)-coated cover slides for 1 and 3 h. After fixation with 4% PFA, cells were washed thrice with PBS before measuring their nanomorphology in PBS with a Scanning Ion Conductance Microscope (SICM) (NX-bio, Park Systems, Korea) [47]. The measurements were performed with borosilicate glass nanopipettes exhibiting opening diameters below 80 nm.…”

“…However, if we consider regions of peripheral ruffles, we find again a significant difference between 246 nm ± 17 nm on glass and 174 nm ± 10 nm on PPAAm. Note: This type of ruffle is a loosened and folded-back membrane and it can originate from membrane excess, which was more pronounced on glass [47]. The ruffles need to be excluded for this analysis, because they would lead to overestimation of the edge height in the case of glass.…”

Applications of scanning ion conductance microscope in biomedical fields

“…But the significant difference is that SICM can detect the small‐scale dynamics of the surface of living cells or the movement of the whole cell. At present, SICM has been used to study cells with different morphological characteristics such as cardiomyocytes, renal epithelial cells, 33 neuronal cells, 34 and osteoblasts 35 . It can even be combined with solid‐state nanopores to achieve precise imaging of the nuclear membrane surface 36 .…”

“…At present, SICM has been used to study cells with different morphological characteristics such as cardiomyocytes, renal epithelial cells, 33 neuronal cells, 34 and osteoblasts. 35 It can even be combined with solid-state nanopores to achieve precise imaging of the nuclear membrane surface. 36 Overall, SICM is working to explore faster ways to image smaller units of living cells.…”