Role of boundary conditions in determining cell alignment in response to stretch

This study investigated why many cell types align perpendicular to cyclic stretch on 2D membranes but often align parallel to stretch in 3D gels, explicitly testing the “strain avoidance” hypothesis that attributes 3D parallel alignment to avoidance of larger transverse compaction strains. Primary adult rat cardiac fibroblasts were embedded in collagen gels and cultured under systematically varied boundary conditions that independently controlled transverse compaction and restraint during static and cyclic stretching. The results showed that, under static or low-frequency conditions, fibroblast alignment correlated with the presence or absence of restraining boundary conditions rather than with the magnitude/pattern of transverse compaction: gels restrained in the loading direction produced strong alignment parallel to the restraint even when compaction was isotropic, while biaxially restrained gels produced low/random alignment. Cyclic stretch could drive perpendicular alignment in 3D, but only at substantially higher frequencies than typically required in 2D stretching studies. A modified stress-fiber thermodynamics/kinetics model incorporating traction boundary conditions and reduced strain transmission in soft gels reproduced the experimental findings across 3D conditions and matched published 2D frequency-dependent alignment trends, providing a unified mechanistic explanation for the apparent 2D vs 3D discrepancy.