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In Situ Measurement of Native Extracellular Matrix Strain

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Abstract

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.

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Acknowledgements

This work was supported by the National Institutes of Health [R21 AR069248, R01 AR071359 and DP2 AT009833 to S.C.]. The authors would like to thank Dr. Robert Mecham for providing the fibrillin-2 antibody. We would also like to acknowledge Michael Drakopoulos and Benjamin Sather for their technical assistance and support.

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Authors and Affiliations

  1. Weldon School of Biomedical Engineering, Purdue University, 206 South Martin Jischke Drive, West Lafayette, IN, 47907, USA

    A. Acuna, S. H. Sofronici, C. J. Goergen & S. Calve

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  1. A. Acuna
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  2. S. H. Sofronici
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  3. C. J. Goergen
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  4. S. Calve
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Correspondence to S. Calve.

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Appendix

Appendix

Fig. 7
figure 7

Displacement fields for representative ECM sub-stacks from sample B. ux, uy, and uz were plotted for FBN2 and WGA in the loose connective tissue (top) and the cartilage (bottom) regions with color indicating magnitude and directionality of displacement

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Fig. 8
figure 8

Comparison of Green-Lagrange strain components across independent samples. a. Significant differences between normal strains (Exx, Eyy, Ezz) across proteins and regions for sample A (left) and sample B (right) identified via a two-way ANOVA post-hoc Tukey test comparison are shown (*0.0021 < p ≤ 0.032; **0.0002 < p ≤ 0.0021) (n = 3; bars = S.D.), revealing similar trends in ECM strain profiles. b. Shear strain components (Exy, Exz, Eyz) for sample A (left) and Sample B (right) (n = 3; bars = S.D.)

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Fig. 9
figure 9

Gaussian filtered FIDVC displacement components at 3 representative positions (indicated by different colors) in sample B, including their corresponding polynomial fit. Negative z-displacements are in the direction of the applied load

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Table 1 FIDVC absolute displacement error
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Acuna, A., Sofronici, S.H., Goergen, C.J. et al. In Situ Measurement of Native Extracellular Matrix Strain. Exp Mech 59, 1307–1321 (2019). https://doi.org/10.1007/s11340-019-00499-y

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