Figure 2. ECM microparticles amorphously packed in a hydrogel beyond a percolation threshold result in a composite material with mechanical properties that approach native tissue.

(A) Packing microparticles to high density in hydrogels increases cell-ECM contact and the volume ratio of microparticle to gel. As the volume ratio increases, the amount of void space between particles decreases until microparticles contact each other and form a new network. (B) The increase in microparticle volume ratio is visualized by taking 405 nm confocal microscopy z-stacks of DAPI stained tissue clay constructs. (C) Hydrogels were mechanically tested at 0.1%/sec to a deformation magnitude of 40%, and compressive modulus was calculated from the linear portion of the stress vs. strain curve. (D) The modulus of the increasing density tissue clays demonstrates an inflection point, f_c. The General Effective Medium (GEM) percolation model fit to the compressive modulus vs. density curve defines the percolation threshold at 0.57 volume ratio: the mathematical point where microparticles are predicted to interact and form a new network to propagate loading. (E, F) Tissue clay can be composed of a variety of hyaluronic acid concentrations and microparticle sizes, but for work presented here we utilized 1% HA hydrogels and 250 μm sized particles due to a balance of material consistency and strong mechanics. (*p<.05, **p<.001, +p<.05 as compared with hydrogel before centrifugation).