Concentration independent modulation of local micromechanics in a fibrin gel.
Concentration independent modulation of local micromechanics in a fibrin gel.
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DOI：
10.1371/journal.pone.0020201
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Botvinick EL
中科院分区：
文献类型：
--
作者：
Kotlarchyk MA;Shreim SG;Alvarez-Elizondo MB;Estrada LC;Singh R;Valdevit L;Kniazeva E;Gratton E;Putnam AJ;Botvinick EL
在3D细胞培养中，调节细胞外基质(ECM)机制的方法依赖于增加蛋白质或交联分子的浓度，但无法控制重要参数，如孔大小、配体密度和分子扩散系数。或者，通过机械加载ECM，可以独立于蛋白质浓度来调节ECM的硬度。我们开发了一种新的装置，用于在自然衍生的ECM中产生刚度梯度，其中刚度通过诱导应变进行调节，而局部机械性能则由基于主动微观流变学(AMR)的激光镊子直接确定。在直径35 mm的皮氏培养皿内聚合的水凝胶基质通过嵌入的圆柱形柱子的精确旋转而非均匀地施加应变，并且表现出与位置相关的硬度，几乎不调节局部网格几何形状。在这里，我们介绍了在纤维蛋白水凝胶的背景下的设备。首先，AMR被用来直接测量纤维蛋白浓度增加的无张力水凝胶中的局部微观力学。然后在我们的设备中映射僵硬的变化，其中纤维蛋白浓度保持不变。荧光共聚焦成像和轨道粒子跟踪分别用于在微米和纳米水平上量化纤维蛋白的结构变化。在我们的外加载荷下，微观流变学测量的微机械应变硬化没有伴随着ECM微观结构的变化，而用共聚焦显微镜测量的结果是这样的。然而，超分辨率轨道跟踪揭示了纤维蛋白纤维的纳米结构伸直、延长和运动减少。此外，我们发现在我们的装置内培养的主动脉平滑肌细胞对诱导的机械梯度在形态上是敏感的。我们的结果证明了一种强大的细胞培养工具，可以用于研究自然衍生的3D ECM组织中细胞生理学的机械效应。
Methods for tuning extracellular matrix (ECM) mechanics in 3D cell culture that rely on increasing the concentration of either protein or cross-linking molecules fail to control important parameters such as pore size, ligand density, and molecular diffusivity. Alternatively, ECM stiffness can be modulated independently from protein concentration by mechanically loading the ECM. We have developed a novel device for generating stiffness gradients in naturally derived ECMs, where stiffness is tuned by inducing strain, while local mechanical properties are directly determined by laser tweezers based active microrheology (AMR). Hydrogel substrates polymerized within 35 mm diameter Petri dishes are strained non-uniformly by the precise rotation of an embedded cylindrical post, and exhibit a position-dependent stiffness with little to no modulation of local mesh geometry. Here we present the device in the context of fibrin hydrogels. First AMR is used to directly measure local micromechanics in unstrained hydrogels of increasing fibrin concentration. Changes in stiffness are then mapped within our device, where fibrin concentration is held constant. Fluorescence confocal imaging and orbital particle tracking are used to quantify structural changes in fibrin on the micro and nano levels respectively. The micromechanical strain stiffening measured by microrheology is not accompanied by ECM microstructural changes under our applied loads, as measured by confocal microscopy. However, super-resolution orbital tracking reveals nanostructural straightening, lengthening, and reduced movement of fibrin fibers. Furthermore, we show that aortic smooth muscle cells cultured within our device are morphologically sensitive to the induced mechanical gradient. Our results demonstrate a powerful cell culture tool that can be used in the study of mechanical effects on cellular physiology in naturally derived 3D ECM tissues.
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影响因子：
3.9
作者：
Hellriegel, Christian;Gratton, Enrico
通讯作者：
Gratton, Enrico
影响因子：
41.2
作者：
Janmey, Paul A.;Mccormick, Margaret E.;Mackintosh, Fred C.
通讯作者：
Mackintosh, Fred C.
影响因子：
3.4
作者：
Hudson, Nathan E.;Houser, John R.;Falvo, Michael R.
通讯作者：
Falvo, Michael R.
影响因子：
3.4
作者：
Guthold, M;Liu, W;Superfine, R
通讯作者：
Superfine, R
DOI：
10.2217/nnm.10.12
发表时间：
2010-04
期刊：
Nanomedicine (London, England)
影响因子：
--
作者：
Geckil H;Xu F;Zhang X;Moon S;Demirci U
通讯作者：
Demirci U