Measuring and Understanding Pericellular Stiffness

While tissue stiffness is thought to play a role in regulation of cellular behavior, for the most part, stiffness is measured at the bulk level. The bulk measurement masks microscale dynamics within the fibrous extracellular matrix (ECM) and is insensitive to changes as cells remodel their local ECM. In order to investigate, cell-ECM dynamics I have developed an automated active microrheology (AMR) system and used it to probe the ECM near both single, isolated cells and multi-cell angiogenic sprouts, quantifying the pericellular distribution of stiffness. Additionally, I developed a new technique to modify stiffness within the ECM, at a scale relevant to the pericellular distribution of stiffness. My work shows that both human fibroblasts and smooth muscle cells establish a complex heterogeneous pericellular stiffness landscape. As expected, cell contraction strain hardened the matrix, but surprisingly, cells must also be competent in ECM proteolysis, which is to say the matrix must be broken down for cell-mediated stiffening. My findings suggest pericellular stiffness distributions should be considered in the study of cell-ECM interactions. In collaboration with Professor Andrew Putnam, I measured the evolution of stiffness change within a capillary morphogenesis model over time. We applied both bulk rheology and AMR to measure stiffness at different length scales. This data highlighted that bulk rheology was dominated by the activity of supportive stromal cells but blinded to the stiffness heterogeneity found proximal to vessels via AMR. These findings underscore that characterizing ECM mechanics across length scales can provide a deeper understanding of the microenvironment’s role within these complex processes. Lastly, I developed and evaluated a method to modify stiffness within fibrin matrices at the micron-scale. This method allows for a patterning of stiffness at a spatial scale and magnitude similar to that observed by cell-mediated stiffening. By using ruthenium-catalyzed photo-crosslinking coupled with our laser scanning confocal microscope, we can selectively illuminate and thereby selectively crosslink regions of interest within the volume of a hydrogel. This results in a stiffness increase of up to 25X, with a steep stiffness gradient in the surrounding area. Selective crosslinking could be of great utility in creating more complex patterns of stiffness, which could be invaluable for the investigation of mechanotransduction within a natural 3D ECM context. Collectively, these works show that the mechanical topography surrounding cells within ECM is varied and must be considered in future study of mechanically driven hypotheses. Microrheology in combination with selective photo-crosslinking provides a new tool to better understand roles for tissue stiffness in cell regulation, and vice versa