Robust detection and visualization of cytoskeletal structures in fibrillar scaffolds from 3-dimensional confocal image

Abstract

Figure 1: Modes of F-actin organization in fiber-attaching cells. (a) Longitudinally oriented stress fibers (arrows) in a loosely attached cell, at 4h after scaffold seeding. In this cell, F-actin containing ruffles are also visible (arrowheads). (b) Concave actin bundles (CAB) in a cell cultivated in the scaffold for 10 days (arrowheads). Circular distribution of F-actin in CAB is shown in optical cross-sections across the supporting fiber (arrows). F-actin was stained by FITC-phalloidin (green), and the nuclei with DAPI (blue).

Actin grips: Circular actin-rich cytoskeletal structures that mediate the wrapping of polymeric microfibers by endothelial cells

“…Two-dimensional PCL films are well known as poorly adhesive substrates for cell cultivation, including endothelial and smooth muscle cells [ 19 – 22 ]. However, HUVECs could easily attach to SMFs prepared from PCL by electrospinning in micro-fibrillar form [ 12 , 13 ]. Using scanning electron microscopy (SEM), we identified cells attached directly to individual SMFs, or more seldom simultaneously to multiple fibers of smaller diameters ( Fig.…”

“…Digital unwrapping of the F-actin bundles from the SMFs was performed using either a Matlab program that projects the space around a cylinder on a plane [ 15 ] or, to section this space with planes parallel to cylinders’ axis, our original method [ 12 ]. In addition, to determine the properties of the scaffold inductive of cell attachment, we compared fiber diameters at cell-attachment site with overall distribution of fiber diameters in the scaffold.…”

“…In addition, to determine the properties of the scaffold inductive of cell attachment, we compared fiber diameters at cell-attachment site with overall distribution of fiber diameters in the scaffold. In brief, segmented 3D images of cell-seeded scaffolds stained with PKH26, extracted from digitized confocal images, were fitted with 1-μm long template cylinders of pre-determined diameters, as described in detail in our method paper [ 12 ]. Normalized histograms of these distributions were compared for dissimilarity by the Q-Q plot method [ 29 ].…”

“…Recently we [ 12 ] and others [ 13 ] have also found that ECs incubated with polymeric scaffold microfibers (SMFs) within their own size range re-organize their actin cytoskeleton in bands oriented transversally to cylinder’s axis. In the current study, we further address the mechanisms facilitating this interaction, specifically the occurrence and maintenance of a tubular morphology in this cellular system, by focusing on the organization of F-actin as a dynamic cytoskeletal component.…”

“…In fact, the PCL films required a chemical modification by macromolecular covalent modification [ 19 , 20 ] or alkalinization [ 21 , 22 ], to become an optimal culture support comparable with tissue culture polystyrene (TCPS) for HUVECs [ 19 – 21 ] and for human ECFCs [ 22 ], respectively. However, the behavior of EC-lineage cells on PCL scaffolds only recently started to be explored [ 12 , 13 ].…”