(637e) Characterization of Length-Scale Dependent Rheology Using Bi-Disperse Multiple Particle Tracking during Cell-Material Interactions

hMSCs re-engineer their microenvironments to enable basic cellular processes, including motility and differentiation, during wound healing and tissue regeneration. This re-engineering is inherently length-scale dependent and the structure and rheological properties are important factors in retaining native function of hMSCs. During hMSC-mediated scaffold remodeling single cross-links break on the nanometer scale, cellular extensions pull material and degrade paths through the scaffold to enable motility on the micrometer scale and bulk scaffold degradation occurs on macroscopic scales. We measure length-scale dependent rheology during cell-mediated remodeling of the pericellular region using bi-disperse MPT. Traditionally, MPT uses a single particle size to characterize rheological properties. But in complex systems, MPT measurements with a single size particle can characterize distinct properties that are linked to the materials length-scale dependent structure. By varying probe size, MPT can measure material properties associated with different length-scales. Bi-disperse MPT measures length-scale dependent rheology by tracking a bi-disperse population of particles. 0.5 and 2 micron particles are embedded in the same sample and the Brownian motion of these particle populations are tracked separately using a brightness-based squared radius of gyration. The Brownian motion of each particle size is then related to rheological properties using the Generalized Stokes-Einstein Relation. We measure hMSC-mediated scaffold degradation in a well-defined poly(ethylene glycol) (PEG)-peptide hydrogel scaffold. A 4-arm star PEG-norbornene is cross-linked with a matrix metalloproteinase-degradable peptide sequence after exposure to UV light. An adhesion ligand, CGRDS, is also included in the hydrogel to enable cellular adhesion to the network during motility. We measure that cells preferentially re-engineer their microenvironment across length-scales using enzymatic secretions (irreversible degradation) and cytoskeletal tension (reversible degradation). Using bi-disperse MPT measurements we also identify the area where the cell is applying force by tracking probe particles and extending their trajectories to points of intersection. From these measurements, we identify that 2 micron particles are stuck in a loose gel network that has been partially degraded and is being reversibly remodeled by the hMSC. At the same time 0.5 micron particles are measuring irreversible scaffold degradation due to cell-secreted enzymes and are able to diffuse through the larger network structure cells are pulling on. By characterizing evolving length-scale dependent rheology, new materials can be designed which better mimic native tissue and instruct cell behavior.