Kural Mehmet H, Billiar Kristen L
Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, MA, USA.
Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, MA, USA.
Acta Biomater. 2016 Mar 1;32:223-230. doi: 10.1016/j.actbio.2015.12.031. Epub 2015 Dec 19.
Myofibroblasts are critical for connective tissue remodeling and wound healing since they can close wound beds and shape tissues rapidly by generating high traction forces and secreting abundant extracellular matrix proteins and matrix metalloproteinases. However, their presence in excessive numbers is associated with fibrotic and calcific diseases and tissue thickening in engineered tissues. While activation of the myofibroblast phenotype has been studied extensively, whether myofibroblasts are "cleared" by phenotypic reversal or by apoptosis remains controversial. The goal of this work is to test the hypothesis that mechanical inhibition of myofibroblast force generation leads to de-differentiation or apoptosis depending upon the magnitude of the decrease in tension. To test this hypothesis, we cultured valvular interstitial cells (VICs) in fibrin micro-tissues suspended between flexible posts and dynamically altered the ability of the cells to generate tension by altering boundary stiffness via magnetic forces applied to posts. The flexible posts capped with magnetic beads enable the measurement and modulation of tension generated by the cells within the tissue. As expected, the cell-generated forces were elevated with dynamically increased boundary (post) stiffness, yet surprisingly, the forces continued to increase following dynamic reduction of boundary stiffness back to baseline levels. Increased apoptosis and reduced α-SMA staining were observed with complete freeing of the tissues from the posts but not upon removal of the magnet, resulting in a twofold decrease in post stiffness. Together, these data indicate that an increase in myofibroblast force generation, even if modest and temporary (1 day), can have lasting effects on myofibroblast persistence in tissues, and that a significant reduction in the ability of the cells to generate tension is required to trigger dedifferentiation and/or apoptosis. The ability to dedifferentiate myofibroblasts to a quiescent phenotype and to control the percentage of apoptosis would be of great benefit for therapeutic and tissue engineering applications.
Myofibroblasts play an important role in tissue remodeling and wound healing. However, excessive activation of this phenotype is associated with fibrotic diseases and scar formation. Being able to dedifferentiate these cells or controlling their clearance with apoptosis (programmed cell death) would be beneficial. It is known that releasing rigid tissue boundaries trigger apoptosis, while reducing the substrate stiffness can cause myofibroblast dedifferentiation. However, the mechanical tension was not quantified in any of the studies. Here we used micro-cantilever posts at tissue boundaries to measure tension and to regulate boundary stiffness in real time by pulling posts with magnets. We show that temporary stiffening of boundary causes irreversible myofibroblast activation and the magnitude of tension drop controls apoptosis.
肌成纤维细胞对于结缔组织重塑和伤口愈合至关重要，因为它们能够通过产生高牵引力以及分泌大量细胞外基质蛋白和基质金属蛋白酶来迅速封闭伤口床并塑造组织。然而，它们数量过多与纤维化和钙化疾病以及工程组织中的组织增厚相关。虽然对肌成纤维细胞表型的激活已进行了广泛研究，但肌成纤维细胞是通过表型逆转还是通过凋亡被“清除”仍存在争议。这项工作的目标是检验以下假设：对肌成纤维细胞力产生的机械抑制会导致去分化或凋亡，这取决于张力降低的幅度。为了检验这一假设，我们将瓣膜间质细胞（VICs）培养在悬浮于柔性柱之间的纤维蛋白微组织中，并通过施加于柱上的磁力改变边界刚度来动态改变细胞产生张力的能力。带有磁珠的柔性柱能够测量和调节组织内细胞产生的张力。正如预期的那样，随着边界（柱）刚度动态增加，细胞产生的力升高，但令人惊讶的是，在边界刚度动态降低回到基线水平后，力仍继续增加。当组织完全从柱上释放时观察到凋亡增加和α - SMA染色减少，但在移除磁铁时未观察到，此时柱刚度降低了两倍。总之，这些数据表明，肌成纤维细胞力产生的增加，即使是适度且暂时的（1天），也可能对肌成纤维细胞在组织中的持续存在产生持久影响，并且细胞产生张力的能力显著降低是触发去分化和/或凋亡所必需的。将肌成纤维细胞去分化为静止表型以及控制凋亡百分比的能力对于治疗和组织工程应用将非常有益。
肌成纤维细胞在组织重塑和伤口愈合中起重要作用。然而，这种表型的过度激活与纤维化疾病和瘢痕形成相关。能够使这些细胞去分化或通过凋亡（程序性细胞死亡）控制其清除将是有益的。已知释放刚性组织边界会触发凋亡，而降低底物刚度会导致肌成纤维细胞去分化。然而，在任何研究中都未对机械张力进行量化。在这里，我们在组织边界使用微悬臂柱来测量张力，并通过用磁铁拉动柱实时调节边界刚度。我们表明，边界的暂时硬化会导致肌成纤维细胞不可逆激活，并且张力下降的幅度控制凋亡。
