Composition Matters: Collagen vs Polyacrylamide Modulates Distinct Trabecular Meshwork Cell Traction

Cells actively sense not only substrate stiffness but also matrix architecture and composition. To understand how these factors play a role in mechanobiology while minimizing stiffness as a dominant variable, we cultured normal high-flow human trabecular meshwork (TM) cells on a fibrous type I collagen gel and a collagen-coated polyacrylamide (PAM) gel (nominal stiffness ∼4.7 kPa for PAM; collagen fiber stiffness characterized by atomic force microscopy (AFM)) but with distinct microstructures, including a fibrous, elastic/viscoelastic type I collagen gel and an amorphous, linearly/non-linearly elastic polyacrylamide (PAM) gel. Using live 3D traction force microscopy and a traction-release (trypsin detachment) assay, we quantified cell-induced displacement fields and computed traction force distributions, strain, divergence (∇·u), and curl (∇ × u) on both gels. Across both gels, TM cells on collagen generated multi-fold higher tractions than on PAM throughout detachment; at 20 min, the median traction was ∼2.66 kPa (0.61-6.27 kPa) on collagen compared to ∼0.27 kPa (0.23-0.47 kPa) on PAM (∼9.8×). Median strain at 20 min was ∼1.31% (0.16-2.86) on collagen compared to ∼0.10% (0.076-0.163) on PAM (∼13×). Curl was consistently higher on collagen, i.e., 12 min medians: 4.24 compared to 0.27; ∼15×, whereas PAM remained near ∼0.3. Divergence stayed near zero on both substrates without a consistent negative shift on collagen. On collagen, traction tended to co-vary with weak divergence and higher curl over time, consistent with long-range force transmission and fiber recruitment, whereas on PAM, deformations were confined to the immediate adhesion footprint with minimal net divergence or rotation. Live confocal imaging and SEM corroborated distinct morphologies as elongated, fiber-aligned cells embedded within the 3D-like collagen network versus compact, uniformly spread cells on the planar PAM surface. Our data show that, with nominal stiffness approximately matched, switching from a collagen-coated 2D hydrogel to a fibrillar collagen matrix changes how TM cells generate, pattern, and transmit forces. These differences should be interpreted as the aggregate effect of the full matrix context rather than microstructure alone.