Abstract
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 1Hz 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.
Highlights
Proper spatiotemporal distributions of dynamic physical cues are necessary to guide the development, maintenance, and healing of tissues
Leung et al [3] first described the sensitivity of cells to dynamic stretch in vitro by demonstrating a change in protein production in equibiaxially cycled smooth muscle cells, and subsequent studies have demonstrated that mechanical stretching induces a wide range of cellular responses including cytoskeletal remodeling, synthesis of numerous extracellular matrix proteins, and altered expression of a multitude of genes [4,5]
Our preliminary data confirm our hypothesis that the application of cyclic stretch to cells cultured on soft hydrogels induces responses indicative of culture on stiff substrates; most strikingly, valvular interstitial cells exhibited a rounded morphology on soft static substrates but spread to the same extent as those cultured on stiff substrates upon application of cyclic equibixal stretch
Summary
Proper spatiotemporal distributions of dynamic physical cues are necessary to guide the development, maintenance, and healing of tissues. Cells such as fibroblasts, endothelial cells, and muscle cells actively sense both the external loading applied to them (outside-in signaling) and the stiffness of their surroundings (insideout signaling). Endothelial cells, and muscle cells actively sense both the external loading applied to them (outside-in signaling) and the stiffness of their surroundings (insideout signaling) They respond to these stimuli with changes in adhesion, proliferation, locomotion, morphology, and synthetic profile (reviewed in [1,2]). A better understanding of how complex combinations of mechanical stimuli regulate cell behavior is critical for the rational engineering of tissues in vitro and for guiding proper regeneration in vivo. As cells are not able to appreciably deform the relatively stiff silicone substrates used in standard cell-stretch systems (Young’s modulus
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