Contact guidance in human dermal fibroblasts is modulated by population pressure

Abstract

Morphogenesis is underpinned by orientated cell division, motility and growth. The substratum for migrating cells in vivo comprises either extracellular matrix or the surfaces of adjacent cells and both are believed to inform the dynamic behaviour of adherent cells through contact guidance. Collisions between migrating cells in vitro can induce the phenomena of contact inhibition of locomotion and division, suggesting that their sensitivity to substratum-derived cues may also be influenced by population density. In the present study dermal fibroblasts, which are known to be motile in culture and are fundamental to the organization of the extracellular matrix, were used to examine the influence of population pressure on the ability of substratum topography to induce contact guidance. The findings suggest that sensitivity to substratum-derived morphogenetic guidance cues, as revealed by alignment of cells to microtopography, is modulated by population pressure.

Fig. 1

Phase contrast photomicrographs illustrating human dermal fibroblast morphology in P1 primary culture. In low-density culture (A) cells are not orientated in relation to each other, do not tend to cluster and most often adopt a multipolar morphology. Cells emanating from explants of dermis are crowded together and migrate radially outwards across the culture substratum (B). Fibroblasts in confluent monolayers are similarly crowded and tend to exhibit a parallel alignment, often becoming stratified into separate cellular layers whose orientation is often mutually perpendicular (C). Scale bar on A = 50 µm, and B and C are at the same magnification.

Fig. 2

Line graphs illustrating the proportion of cells aligned to the underlying linear substratum microtopography within low-density and confluent cultures. Graphs A and B represent confluent cultures, C and D low-density cultures, and each displays alignment data for cells cultured on sets of grooves of increasing pitch and also increasing depth. Population pressure can be seen to exert maximum influence on alignment of cells on narrower grooves with depths between 380 and 1300 nm. On the narrowest grooves most cells in low-density cultures were insensitive to gratings 27 nm depth, but at confluence 25% of cells were aligned to the substratum.

Fig. 3

Phase contrast micrographs illustrating the effect of population density on alignment responses of fibroblasts. The micrographs are in pairs illustrating the same substratum but with analysis of alignment carried out on low-density and confluent cell cultures. A and B = 110 nm depth × 2 µm pitch, C and D = 780 nm depth × 25 µm pitch, E and F = 780 nm depth × 50 µm pitch. A, C, E, confluent cultures; B, D, F, low-density cultures. Scale bar on A = 100 µm, and the other images are at the same magnification.