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Endothelial actin and cell stiffness is modulated by substrate stiffness in 2D and 3D
Authors:Fitzroy J Byfield  Rashmeet K Reen  Tzu-Pin Shentu  Irena Levitan  Keith J Gooch
Institution:1. Institute of Health Service and Transfusion Medicine, Academy of Military Medical Sciences, Beijing 100039, P.R.China;2. Department of Blood Transfusion, The 988 hospital of PLA, Zhengzhou 450042, P.R.China;3. Institute of Biomechanics and Medical Engineering, School of Aerospace Engineering, Tsinghua University, Beijing 100084, P.R.China;4. National Centre for Nanoscience and Technology, Beijing 100190, P.R.China
Abstract:There is a growing appreciation of the profound effects that passive mechanical properties, especially the stiffness of the local environment, can have on cellular functions. Many experiments are conducted in a 2D geometry (i.e., cells grown on top of substrates of varying stiffness), which is a simplification of the 3D environment often experienced by cells in vivo. To determine how matrix dimensionality might modulate the effect of matrix stiffness on actin and cell stiffness, endothelial cells were cultured on top of and within substrates of various stiffnesses. Endothelial cells were cultured within compliant (1.0–1.5 mg/ml, 124±8 to 202±27 Pa) and stiff (3.0 mg/ml, 502±48 Pa) type-I collagen gels. Cells elongated and formed microvascular-like networks in both sets of gels as seen in previous studies. Cells in stiffer gels exhibited more pronounced stress fibers and ~1.5-fold greater staining for actin. As actin is a major determinant of a cell's mechanical properties, we hypothesized that cells in stiff gels will themselves be stiffer. To test this hypothesis, cells were isolated from the gels and their stiffness was assessed using micropipette aspiration. Cells isolated from relatively compliant gels were 1.9-fold more compliant than cells isolated from relatively stiff gels (p<0.05). Similarly, cells cultured on top of 1700 Pa polyacrylamide gels were 2.0-fold more compliant that those cultured on 9000 Pa (p<0.05). These data demonstrate that extracellular substrate stiffness regulates endothelial stiffness in both three- and two-dimensional environments, though the range of stiffnesses that cells respond to vary significantly in different environments.
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