In situ measurement of native extracellular matrix strain

Cells directly interact with the extracellular matrix (ECM) in their microenvironment; however, the mechanical properties of the networks at this scale are not well defined. This work describes a method to quantify ECM network strain in situ after the application of a known load. Visualization of the ECM in the native 3D organization is facilitated using murine embryos and a novel decellularization method. During embryonic development, the ECM architecture is less dense making it easier to visualize and manipulate. Briefly, embryonic day (E)14.5 forelimbs were harvested and incubated in an acrylamide-based hydrogel mixture to maintain the 3D architecture of the ECM during decellularization. After decellularization, forelimbs were stained for fibrillin-2 and proteoglycans to visualize different networks. Samples were imaged, before and after the application of a static load, using confocal microscopy. A MATLAB-based fast iterative digital volume correlation algorithm was used to quantify network displacement fields by comparing the reference and compressed z-stacks. We observed that the amount of Green-Lagrange strain experienced by different proteins was dependent on whether the sub-region analyzed was located within cartilage or the adjacent connective tissue. The combination of these experimental and computational methods will enable the development of constitutive equations that describe the material behavior of ECM networks. In the future, this information has the potential to improve the fabrication of physiologically relevant scaffolds by establishing mechanical guidelines for microenvironments that support beneficial cell-ECM interactions.