Carbon Nanotubes Substrates Alleviate Pro-Calcific Evolution in Porcine Valve Interstitial Cells

Figures

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  Figure 1. Evaluation of pVICs when developed on glass control and CNT substrates. (a) Scanning electron micrographs of CNT substrates at different magnifications pointing out carpet uniformity (left image) and CNT morphology (middle and right images). Scale bars: 50 µm, 2 µm, and 200 nm, respectively. (b) Low magnification electron micrographs of pVICs grown on a glass control coverslip (left) and a CNT substrate (right). Scale bars: 100 µm. (c) High magnification electron micrograph of a valve interstitial cell growth interfaced with a CNT-decorated substrate revealing the intimate contact between the cell and the filamentous nanotubes. Scale bar: 2 µm. (d) Immunofluorescence images of cells grown on glass (left column) and CNTs (right column) after 12 h (top row) and 72 h (bottom row) from seeding. Cell nuclei were stained in blue using DAPI, while cytoskeletal actin was pointed out in red using phalloidin. Scale bars: 100 µm. In (e), histograms comparing the cell densities calculated from the two different substrates at 12 (top) and 72 h (bottom) time points are shown. No differences were visible in terms of cell densities between controls and CNTs after 12 h (light green and light grey…

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  Figure 2. Morphological characterization of VICs grown on control glass and CNTs substrates. (a) Based on cell shape and elongation, we identified three distinct cell morphologies within our cultures: a spindle-like shape, associated with smooth muscles cells (SMc, orange), intermediate geometries ranging from unciform to sagittate shapes, associated with fibroblasts (Fib, blue), and large and cuspated shapes, associated with myofibroblasts (myFib, brown). (b) Percentages of the three different cell morphologies were evaluated in cultures developed for 72 h on both glass and CNTs. (c) Box plot pointing out cell dimensions after 12 h from seeding on glass controls (light green) and CNTs (light grey), and after 72 h (dark green and grey, respectively). (d) Degree of circularity (DOC) of cells developed on glass controls and CNTs for 12 and 72 h. (e) Number of cusps characterizing cells grown on glass controls and CNTs after 12 and 72 h. (f) Two representative fields of α-SMA staining (in green, bottom row) of cells developed for 72 h on glass and CNTs (actin filaments stained in red, top row). The generally lower and grainer fluorescence signal characterizing CNT samples should be noted.…

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  Figure 3. Evaluation of differences in cell parameters based on cell morphology. In (a) representative immunofluorescence images of cells grown on glass (top row) and CNTs (bottom row) after 72 h. Actin cytoskeleton was marked in red with phalloidin (left column), focal adhesions were highlighted in green with a staining again vinculin (middle column), and cell nuclei were made visible in blue by DAPI (right column). Scale bars: 100 µm. (b) Box plot summarizing cell areas distributions after 72 h from seeding on glass controls (in light orange SMc, in light blue Fib, and in light brown myFib), and on CNT substrates (in dark orange SMc, in dark blue Fib, and in dark brown myFib). (c) Box plot showing focal adhesion densities for the different cell morphologies when interfaced with glass and CNTs for 72 h. (d) Box plot summarizing the amount of stress fibers, normalized to the actin content, for the different cell morphologies when interfaced with glass and CNTs for 72 h. (e) Two representative fluorescent images of actin content (in light grey) of cells grown interfaced to glass controls (above) and CNTs (below) where stress fibers were highlighted for every cell with different false…

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  Figure 4. Characterization of the interaction between substrates and the inferior portion of the cell membrane. (a) AFM images of pVICs plasma membrane patches adherent to a flat glass substrate (left) and to a CNT carpet (right). (b) AFM image of the membrane lying on glass and corresponding to the region highlighted in the left image in (a). The two topographic profiles below correspond to a line scan on the uncovered glass substrate (top profile) and on the membrane patch (bottom profile). (c) AFM image of the membrane lying on CNTs and corresponding to the region highlighted in the right image in (a). The two topographic profiles below correspond to a line scan on the CNT carpet (top profile) and on the membrane patch (bottom profile). (d) Scanning electron micrographs of pVICs membrane patches adherent to a flat glass substrate (left) and to a CNT carpet (right). The two red arrows in the high magnification inset of the right image feature two carbon nanotubes interacting with the plasma membrane. Scale bars: 20 µm and 2 µm, respectively.