Adhesive peptide and polymer density modulate 3D cell traction forces within synthetic hydrogels

Cell-extracellular matrix forces provide pivotal signals regulating diverse physiological and pathological pro-
cesses. Although mechanobiology has been widely studied in two-dimensional configurations, limited research
has been conducted in three-dimensional (3D) systems due to the complex nature of mechanics and cellular
behaviors. In this study, we established a platform integrating a well-defined synthetic hydrogel system (PEG-
4MAL) with 3D traction force microscopy (TFM) methodologies to evaluate deformation and force responses
within synthetic microenvironments, providing insights that are not tractable using biological matrices because
of the interdependence of biochemical and biophysical properties and complex mechanics. We dissected the
contributions of adhesive peptide density and polymer density, which determines hydrogel stiffness, to 3D force
generation for fibroblasts. A critical threshold of adhesive peptide density at a constant matrix elasticity is
required for cells to generate 3D forces. Furthermore, matrix displacements and strains decreased with matrix
stiffness whereas stresses, and tractions increased with matrix stiffness until reaching constant values at higher
stiffness values. Finally, Rho-kinase-dependent contractility and vinculin expression are required to generate
significant 3D forces in both collagen and synthetic hydrogels. This research establishes a tunable platform for
the study of mechanobiology and provides new insights into how cells sense and transmit forces in 3D.