Role of the cytoskeleton in the reaction of fibroblasts to multiple grooved substrata

Abstract

The role of the cytoskeleton and cell attachments in the alignment of baby hamster kidney fibroblasts to ridge and groove substratum topography was investigated using confocal scanning microscopy. This was carried out with normal cells and cells treated with the cytoskeleton modifiers cytochalasin D, colcemid, and taxol. Actin was localised with fluorescent phalloidin. Tubulin, vinculin, and intracellular adhesion molecule-1 were visualised by indirect immunofluorescence. The spreading, elongation, and orientation of the cells after 24 h of culture in these conditions were measured on grooves of 5, 10, and 25 microns width and 0.5, 1, 2, and 5 microns depth. We have also observed events over the first 30 min of cell attachment. Five minutes after cell attachment, F-actin condensations were seen close to the intersection of groove wall and ridge top, that is, at a topographic discontinuity. The condensations were often at right angles to the groove edge and showed a periodicity of 0.6 microns. Vinculin arrangement at the early stages of cell spreading was similar to that of actin. Organisation of the microtubule system followed later, becoming obvious at about 30 min after cell plating. The Curtis and Clark theory (that cells react to topography primarily at lines of discontinuity in the substratum by actin nucleation) is supported by these results. The use of cytoskeletal poisons did not entirely abolish cell reaction to grooves. Colcemid increased cell spreading and reduced cell orientation and elongation. Cytochalasin D reduced cell spreading, orientation, and elongation. Taxol reduced cell elongation but did not affect cell spreading and orientation. We conclude that the aggregation of actin along groove/ridge boundaries is a primary driving event in determining fibroblast orientation on microgrooved substrata.

Nanopattern-induced changes in morphology and motility of smooth muscle cells

“…Cell alignment and elongation of different cell types have been studied on micro- [15,17,18,21,25,[34][35][36][37][38][39][40] or nanoscale gratings [22]. Cell alignment, F-actin alignment and elongation are usually observed on the edges of micro-ridges.…”

“…Response of cells to topographical cues and the concept of contact guidance have been known for decades [8,9]. Various topographical features such as grooves, ridges, stops, pores, wells and nodes in micro-or nanoscale [10][11][12] have been presented to a wide variety of cells: fibroblasts [13][14][15][16][17], BHK cells [18], neuronal cells [19], macrophages [20,21], epithelial cells [22], endothelial cells, and smooth muscle cells (SMC) [23][24][25][26]. Topography can influence cellular responses from initial attachment and migration to differentiation and production of new tissue [10,12,27].…”

Biology on a Chip: Microfabrication for Studying the Behavior of Cultured Cells

“…Thus, the microtubule system appeared to be the principal but not the sole cytoskeletal substratum-response mechanism affecting topographic guidance of human gingival fibroblasts. 80 Wojciak-Stothard et al 81 investigated the role of the cytoskeleton proteins in the spreading, elongation, and orientation of baby hamster kidney (BHK) fibroblasts to ridge and groove substratum topography and derived a different conclusion. They observed F-actin condensations close to the intersection of the wall groove and ridge top, 5 minutes after cell attachment.…”

The Effects of Colloidal Nanotopography on Initial Fibroblast Adhesion and Morphology

“…Materials must fit criteria specific to the tissue under reconstruction including the mechanical properties, chemistry, and physical architecture of an implant relative to the native tissue where it will be situated. With respect to the tissue-implant interface, in vitro investigations indicate that microtopography can be utilized to control cell behavior, including that of fibroblasts, via initial adhesive interactions [12]- [15]. Furthermore, the effects of nanopatterned surfaces on cell adhesion and behavior have emerged in-line with developments in electronics and materials fabrication techniques [16]- [23], compounding the importance of substrate topography on micro-and nanometric levels in fundamental cell behavior [24], [25].…”