Epithelial Cell-Like Elasticity Modulates Actin-Dependent E-Cadherin Adhesion Organization.

E-cadherin adhesions are essential for cell-to-cell cohesion and mechanical coupling between epithelial cells and reside in a microenvironment that comprises the adjoining epithelial cells. While E-cadherin has been shown to be a mechanosensor, it is unknown if E-cadherin adhesions can differentially sense stiffness within the range of that of epithelial cells. A survey of literature shows that epithelial cells' Young's moduli of elasticity lie predominantly in the sub-kPa to few-kPa range, with cancer cells often being softer than noncancerous ones. Here, we devised oriented E-cadherin-coated soft silicone substrates with sub-kPa or few-kPa elasticity but with similar viscous moduli and found that E-cadherin adhesions differentially organize depending on the magnitude of epithelial cell-like elasticity. Our results show that the actin cytoskeleton organizes E-cadherin adhesions in two ways─by supporting irregularly shaped adhesions at localized regions of high actin density and linear shaped adhesions at the end of linear actin bundles. Linearly shaped E-cadherin adhesions associated with radially oriented actin─but not irregularly shaped E-cadherin adhesions associated with circumferential actin foci─were much more numerous on 2.4 kPa E-cadherin substrates compared to 0.3 kPa E-cadherin substrates. However, the total amount of E-cadherin in both types of adhesions taken together was similar on the 0.3 and 2.4 kPa E-cadherin substrates across many cells. Our results show how the distribution of E-cadherin adhesions, supported by actin density and architecture, is modulated by epithelial cell-like elasticity and have significant implications for disease states like carcinomas characterized by altered epithelial cell elasticity.

Stiffness Measurement of Soft Silicone Substrates for Mechanobiology Studies Using a Widefield Fluorescence Microscope.

Soft tissues in the human body typically have stiffness in the kilopascal (kPa) range. Accordingly, silicone and hydrogel flexible substrates have been proven to be useful substrates for culturing cells in a physical microenvironment that partially mimics in vivo conditions. Here, we present a simple protocol for characterizing the Young's moduli of isotropic linear elastic substrates typically used for mechanobiology studies. The protocol consists of preparing a soft silicone substrate on a Petri dish or stiff silicone, coating the top surface of the silicone substrate with fluorescent beads, using a millimeter-scale sphere to indent the top surface (by gravity), imaging the fluorescent beads on the indented silicone surface using a fluorescence microscope, and analyzing the resultant images to calculate the Young's modulus of the silicone substrate. Coupling the substrate's top surface with a moduli extracellular matrix protein (in addition to the fluorescent beads) allows the silicone substrate to be readily used for cell plating and subsequent studies using traction force microscopy experiments. The use of stiff silicone, instead of a Petri dish, as the base of the soft silicone, enables the use of mechanobiology studies involving external stretch. A specific advantage of this protocol is that a widefield fluorescence microscope, which is commonly available in many labs, is the major equipment necessary for this procedure. We demonstrate this protocol by measuring the Young's modulus of soft silicone substrates of different elastic moduli.

Human Osteoblast-Derived Extracellular Matrix with High Homology to Bone Proteome Is Osteopromotive.

Efficient osteogenic differentiation of mesenchymal stromal cells (MSCs) is crucial to accelerate bone formation. In this context, the use of extracellular matrix (ECM) as natural 3D framework mimicking in vivo tissue architecture is of interest. The aim of this study was to generate a devitalized human osteogenic MSC-derived ECM and to investigate its impact on MSC osteogenic differentiation to improve MSC properties in bone regeneration. The devitalized ECM significantly enhanced MSC adhesion and proliferation. Osteogenic differentiation and mineralization of MSCs on the ECM were quicker than in standard conditions. The presence of ECM promoted in vivo bone formation by MSCs in a mouse model of ectopic calcification. We analyzed the ECM composition by mass spectrometry, detecting 846 proteins. Of these, 473 proteins were shared with the human bone proteome we previously described, demonstrating high homology to an in vivo microenvironment. Bioinformatic analysis of the 846 proteins showed involvement in adhesion and osteogenic differentiation, confirming the ECM composition as key modulator of MSC behavior. In addition to known ECM components, proteomic analysis revealed novel ECM functions, which could improve culture conditions. In summary, this study provides a simplified method to obtain an in vitro MSC-derived ECM that enhances osteogenic differentiation and could be applied as natural biomaterial to accelerate bone regeneration.