Toward Measuring the Mechanical Stresses Exerted by Branching Embryonic Airway Epithelial Explants in 3D Matrices of Matrigel.
Toward Measuring the Mechanical Stresses Exerted by Branching Embryonic Airway Epithelial Explants in 3D Matrices of Matrigel.
复制标题
用于测量通过分支的胚胎气道上皮外植体所施加的机械应力。
DOI：
10.1007/s10439-022-02989-y
复制
发表时间：
2022-09
影响因子：
3.8
通讯作者：
Varner, Victor D.
中科院分区：
文献类型：
--
作者：
Patil, Lokesh S.;Varner, Victor D.
关键词：
动物体内的许多器官，包括肺、肾和乳腺，都含有上皮管的分支网络。这些结构是在发育过程中通过称为分支形态发生的过程产生的。先前的研究表明机械力直接影响这一过程，但分支胚胎上皮施加的机械应力的模式尚不清楚。部分原因是缺乏实验工具。传统的牵引力显微镜 (TFM) 测定依赖于使用具有明确机械性能的顺应性水凝胶。然而，分离的胚胎上皮外植体仅在重建基底膜蛋白或基质胶的三维（3D）基质中分支，基质胶是一种机械行为特征较差的生物材料，尤其是在大变形的情况下。在这里，为了计算分支上皮外植体产生的牵引应力，我们量化了重构基底膜蛋白凝胶在多轴机械载荷下的有限变形本构行为。然后，我们通过将荧光微球悬浮在周围凝胶内并跟踪其在培养过程中的运动，修改了分离胚胎气道上皮外植体离体培养的无间充质测定法。令人惊讶的是，在远离外植体的凝胶区域中，跟踪的珠子运动不为零，这表明基质内存在被动膨胀变形。为了计算准确的牵引应力，必须将这些膨胀变形从分支外植体产生的变形中分解出来。因此，我们跟踪了悬浮在无细胞基质中的珠子的运动，并量化了凝胶膨胀的时空模式。总而言之，这些被动膨胀数据可以与测量的凝胶机械特性相结合，以计算完整胚胎上皮外植体施加的牵引力。
Numerous organs in the bodies of animals, including the lung, kidney, and mammary gland, contain ramified networks of epithelial tubes. These structures arise during development via a process known as branching morphogenesis. Previous studies have shown that mechanical forces directly impact this process, but the patterns of mechanical stress exerted by branching embryonic epithelia are not well understood. This is, in part, owing to a lack of experimental tools. Traditional traction force microscopy (TFM) assays rely on the use of compliant hydrogels with well-defined mechanical properties. Isolated embryonic epithelial explants, however, have only been shown to branch in three-dimensional (3D) matrices of reconstituted basement membrane protein, or Matrigel, a biomaterial with poorly characterized mechanical behavior, especially in the regime of large deformations. Here, to compute the traction stresses generated by branching epithelial explants, we quantified the finite-deformation constitutive behavior of gels of reconstituted basement membrane protein subjected to multi-axial mechanical loads. We then modified the mesenchyme-free assay for the ex vivo culture of isolated embryonic airway epithelial explants by suspending fluorescent microspheres within the surrounding gel and tracking their motion during culture. Surprisingly, the tracked bead motion was non-zero in regions of the gel far away from the explants, suggestive of passive swelling deformations within the matrix. To compute accurate traction stresses, these swelling deformations must be decomposed from those generated by the branching explants. We thus tracked the motion of beads suspended within cell-free matrices and quantified spatiotemporal patterns of gel swelling. Taken together, these passive swelling data can be combined with the measured mechanical properties of the gel to compute the traction forces exerted by intact embryonic epithelial explants.
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