Fibrin hydrogels are used as a model system for studying cell-ECM biophysical interactions. Bulk mechanical stiffness of these hydrogels has been correlated to mechanotransduction and downstream signaling. However, stiffness values proximal to cells can vary by orders of magnitude at the length scale of microns. Patterning of matrix stiffness at this spatial scale can be useful in studying such interactions. Here we present and evaluate a technique to selectively stiffen defined regions within a fibrin hydrogel. Laser scanning illumination activates ruthenium-catalyzed crosslinking of fibrin tyrosine residues, resulting in tunable stiffness changes spanning distances as small as a few microns and a localized compaction of the material. As probed by active microrheology, stiffness increases by as much as 25X, similar to previously observed stiffness changes around single cells in 3D culture. In summary, our method allows for selective modification of fibrin stiffness at the micron scale with the potential to create complex patterns, which could be valuable for the investigation of mechanotransduction in a biologically meaningful way.
Bundles of stiff filaments are ubiquitous in the living world, found both in the cytoskeleton and in the extracellular medium. These bundles are typically held together by smaller cross-linking molecules. We demonstrate, analytically, numerically, and experimentally, that such bundles can be kinked, that is, have localized regions of high curvature that are long-lived metastable states. We propose three possible mechanisms of kink stabilization: a difference in trapped length of the filament segments between two cross-links, a dislocation where the endpoint of a filament occurs within the bundle, and the braiding of the filaments in the bundle. At a high concentration of cross-links, the last two effects lead to the topologically protected kinked states. Finally, we explore, numerically and analytically, the transition of the metastable kinked state to the stable straight bundle.
Understanding force propagation through the fibrous extracellular matrix can elucidate how cells interact mechanically with their surrounding tissue. Presumably, due to elastic nonlinearities of the constituent filaments and their random connection topology, force propagation in fiber networks is quite complex, and the basic problem of force propagation in structurally heterogeneous networks remains unsolved. We report on a new technique to detect displacements through such networks in response to a localized force, using a fibrin hydrogel as an example. By studying the displacements of fibers surrounding a two-micron bead that is driven sinusoidally by optical tweezers, we develop maps of displacements in the network. Fiber movement is measured by fluorescence intensity fluctuations recorded by a laser scanning confocal microscope. We find that the Fourier magnitude of these intensity fluctuations at the drive frequency identifies fibers that are mechanically coupled to the driven bead. By examining the phase relation between the drive and the displacements, we show that the fiber displacements are, indeed, due to elastic couplings within the network. Both the Fourier magnitude and phase depend on the direction of the drive force, such that displacements typically propagate farther, but not exclusively, along the drive direction. This technique may be used to characterize the local mechanical response in 3-D tissue cultures, and to address fundamental questions about force propagation within fiber networks.
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