Department of Biomedical Engineering, Worcester Polytechnic Institute, Worcester, Massachusetts, United States of America.
PLoS One. 2011;6(8):e23272. doi: 10.1371/journal.pone.0023272. Epub 2011 Aug 15.
Cells have the ability to actively sense their mechanical environment and respond to both substrate stiffness and stretch by altering their adhesion, proliferation, locomotion, morphology, and synthetic profile. In order to elucidate the interrelated effects of different mechanical stimuli on cell phenotype in vitro, we have developed a method for culturing mammalian cells in a two-dimensional environment at a wide range of combined levels of substrate stiffness and dynamic stretch. Polyacrylamide gels were covalently bonded to flexible silicone culture plates and coated with monomeric collagen for cell adhesion. Substrate stiffness was adjusted from relatively soft (G' = 0.3 kPa) to stiff (G' = 50 kPa) by altering the ratio of acrylamide to bis-acrylamide, and the silicone membranes were stretched over circular loading posts by applying vacuum pressure to impart near-uniform stretch, as confirmed by strain field analysis. As a demonstration of the system, porcine aortic valve interstitial cells (VIC) and human mesenchymal stem cells (hMSC) were plated on soft and stiff substrates either statically cultured or exposed to 10% equibiaxial or pure uniaxial stretch at 1 Hz for 6 hours. In all cases, cell attachment and cell viability were high. On soft substrates, VICs cultured statically exhibit a small rounded morphology, significantly smaller than on stiff substrates (p<0.05). Following equibiaxial cyclic stretch, VICs spread to the extent of cells cultured on stiff substrates, but did not reorient in response to uniaxial stretch to the extent of cells stretched on stiff substrates. hMSCs exhibited a less pronounced response than VICs, likely due to a lower stiffness threshold for spreading on static gels. These preliminary data demonstrate that inhibition of spreading due to a lack of matrix stiffness surrounding a cell may be overcome by externally applied stretch suggesting similar mechanotransduction mechanisms for sensing stiffness and stretch.
细胞具有主动感知其机械环境的能力，并通过改变其黏附、增殖、迁移、形态和合成特性来响应基质硬度和拉伸的变化。为了阐明不同机械刺激对体外细胞表型的相互关联的影响，我们开发了一种在二维环境中培养哺乳动物细胞的方法，该方法可在广泛的基质硬度和动态拉伸组合水平下进行。聚丙稀酰胺凝胶通过共价键与柔性硅树脂培养板结合，并涂覆单体胶原以促进细胞黏附。通过改变丙烯酰胺与双丙烯酰胺的比例，可将基质硬度从相对柔软（G'=0.3 kPa）调整到坚硬（G'=50 kPa），并通过施加真空压力在圆形加载柱上拉伸硅树脂膜，以实现近乎均匀的拉伸，如应变场分析所证实。作为该系统的一个实例，猪主动脉瓣膜间质细胞（VIC）和人骨髓间充质干细胞（hMSC）被接种到柔软和坚硬的基质上，要么静态培养，要么在 1 Hz 下暴露于 10%的等双轴或纯单轴拉伸 6 小时。在所有情况下，细胞黏附率和细胞活力都很高。在柔软的基质上，静态培养的 VIC 呈小而圆的形态，明显小于坚硬基质上的细胞（p<0.05）。在等双轴循环拉伸后，VIC 扩展到与坚硬基质上培养的细胞相当的程度，但没有响应单轴拉伸而向坚硬基质上拉伸的细胞那样重新定向。hMSC 的反应不如 VIC 明显，这可能是由于在静态凝胶上扩展的细胞的刚度阈值较低。这些初步数据表明，由于细胞周围基质刚度的缺乏而导致的扩展抑制可能会被外部施加的拉伸所克服，这表明感知刚度和拉伸的机械转导机制相似。
