Substratum surface topography alters cell shape and regulates fibronectin mRNA level, mRNA stability, secretion and assembly in human fibroblasts

Abstract

The regulation of cell shape, fibronectin mRNA level, secretion and assembly by substratum surface topography was investigated in early passage human gingival fibroblasts cultured on titanium-coated smooth or V-shaped grooved substrata produced by micromachining. Cells on grooved surfaces were significantly elongated and orientated along the grooves of the substratum, while cell height, measured using confocal scanning laser microscopy, was approximately 1.5-fold greater than that of cells on smooth surfaces. Northern hybridization analysis revealed that on a per cell basis the grooved surface increased the amounts of fibronectin mRNA/cell approximately 3.5-fold at 16 hours, approximately 1.9-fold at 40 hours and approximately 2.2-fold at 90 hours, while the mRNA levels of the house-keeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPD) were constant. The amounts of secreted fibronectin on the grooved surface were increased approximately 2-fold for all time points. The stability of fibronectin mRNA was also altered by substratum surface topography. The half-life of fibronectin mRNA on smooth surfaces was estimated to be approximately 5 hours, but on the grooved surfaces the half-life of fibronectin mRNA showed a two-phase response: a rapid 60% reduction in the first half-life (t1/2 approximately 2 hours) and a 2.4-fold increase in the second half-life (t1/2 approximately 12 hours) relative to that observed on the smooth surface. The GAPD mRNA half-lives were essentially unaffected by the surface topography of the substrata. The grooved surface was also found to alter the amount of fibronectin assembled into the extracellular matrix, producing a approximately 2-fold increase in the cultures at all time points. It thus appears that substratum surface topography alters cell shape and modulates fibronectin at the transcriptional and post-transcriptional levels, as well as the amount of fibronectin assembled into extracellular matrix. Micromachining, which has the ability to precisely control surface topography over a wide range of dimensions and shapes, appears to be a useful technique in investigating the relationship between cell shape and function.