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.

中文翻译：

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原生细胞外基质应变的原位测量

细胞直接与微环境中的细胞外基质 (ECM) 相互作用；然而，这种规模的网络的机械性能并没有得到很好的定义。这项工作描述了一种在应用已知负载后原位量化 ECM 网络应变的方法。使用鼠胚胎和一种新的去细胞化方法促进了原生 3D 组织中 ECM 的可视化。在胚胎发育过程中，ECM 架构的密度较低，因此更易于可视化和操作。简而言之，收获胚胎日 (E)14.5 前肢并在基于丙烯酰胺的水凝胶混合物中孵育，以在脱细胞过程中保持 ECM 的 3D 结构。去细胞化后，前肢被染色为 fibrillin-2 和蛋白多糖以可视化不同的网络。样品被成像，在施加静态载荷之前和之后，使用共聚焦显微镜。基于 MATLAB 的快速迭代数字体积相关算法用于通过比较参考和压缩 z 堆栈来量化网络位移场。我们观察到不同蛋白质所经历的格林拉格朗日应变的量取决于所分析的子区域是位于软骨内还是位于相邻的结缔组织内。这些实验和计算方法的结合将有助于开发描述 ECM 网络材料行为的本构方程。将来，这些信息有可能通过为支持有益细胞-ECM 相互作用的微环境建立机械指南来改进生理相关支架的制造。