Micro-tensile rheology of fibrous gels quantifies strain-dependent anisotropy

Semiflexible fiber gels such as collagen and fibrin have unique nonlinear mechanical properties that play an important role in tissue morphogenesis, wound healing, and cancer metastasis. Optical tweezers microrheology has greatly contributed to the understanding of the mechanics of fibrous gels at the microscale, including its heterogeneity and anisotropy. However, the explicit relationship between micromechanical properties and gel deformation has been largely overlooked. We introduce a unique gel-stretching apparatus and employ it to study the relationship between microscale strain and stiffening in fibrin and collagen gels, focusing on the development of anisotropy in the gel. We find that gels stretched by as much as 15 % stiffen significantly both in parallel and perpendicular to the stretching axis, and that the parallel axis is 2-3 times stiffer than the transverse axis. We also measure the stiffening and anisotropy along bands of aligned fibers created by aggregates of cancer cells, and find similar effects as in gels stretched with the tensile apparatus. Our results illustrate that the extracellular microenvironment is highly sensitive to deformation, with implications for tissue homeostasis and pathology. STATEMENT OF SIGNIFICANCE: The inherent fibrous architecture of the extracellular matrix (ECM) gives rise to unique strain-stiffening mechanics. The micromechanics of fibrous networks has been studied extensively, but the deformations involved in its stiffening at the microscale were not quantified. Here we introduce an apparatus that enables measuring the deformations in the gel as it is being stretched while simultaneously using optical tweezers to measure its microscale anisotropic stiffness. We reveal that fibrin and collagen both stiffen dramatically already at ∼10 % deformation, accompanied by the emergence of significant, yet moderate anisotropy. We measure similar stiffening and anisotropy in the matrix remodeled by the tensile apparatus to those found between cancer cell aggregates. Our results emphasize that small strains are enough to introduce substantial stiffening and anisotropy. These have been shown to result in directional cell migration and enhanced force propagation, and possibly control processes like morphogenesis and cancer metastasis.