Cell sensitivity to the elastic properties of the extracellular matrix : from single cell to collective response to 2D or 3D spatial modulations of the rigidity. – ModElast

We analyze how a spatial modulation of the elastic properties of the extracellular matrix influences (i) the distribution of several proteins involved in cell adhesion, (ii) the mechanical equilibrium of the cells, through the measurement of the intracellular stresses and the forces the cells exert on the extracellular matrix. Rigidity modulated extracellular matrices are obtained by photolithography (patented process). Cell forces are directly derived from the 3D deformations of the extracellular matrix due to cell pulling.

We have proposed an innovative, extremely simple mathematical approach that allows calculating the intracellular stresses in cellular assemblies of any size. Thanks to his work, making such mechanical analysis, that was previously reserved to experts in numerical analysis only, becomes within easy reach to a large community of biophysicists and biologists. Using it, we show analyzing endothelial tissues that, (i) the larger the cell assembly, the smaller the internal cellular stresses, (ii) internal cellular stresses propagate on micrometer range, limiting cell-cell interaction to the very close neighborhood of intracellular adhesions, and (iii) the observed larger range of interaction between cells (3 to 5 cells) occurs through cell/matrix interaction. In addition, we show that the distribution of intracellular stresses in isolated cells cannot be deduced from the observation of the contractile, fiber like, actin cytoskeleton that is observed on the basal membrane.
This work has been submitted for publication to a rank A peer reviewed journal.

We can now analyze how a gradient of elasticity in the extracellular matrix influences the intracellular stresses in single cell or in cellular assemblies, and correlate them to the biochemistry of cell adhesion, the organization of the cytoskeleton, or to calcium fluxes. To get a full snapshot, we must end setting up a mathematical approach that allows calculating the forces the cells transmit to extracellular matrices with non uniform elastic properties. Thus we will propose a package of easy handling tools and analysis methods that can be used for any cell type. In this project, we will use it with endothelial tissues, so to shed light on some remodeling and tissue repair mechanisms, and with epithelial and neural cellular assemblies to give some clues on potential mechanisms of invasion triggered by the physics of the extracellular matrix.

Intracellular stresses in patterned cell assemblies , Michel Moussus, Christelle der Loughian, David Fuard, Marie Courçon, Danielle Gulino Debrac, Hélène Delanoë-Ayari*, and Alice Nicolas* , submitted.
We propose a new method to calculate the internal stresses in a cellular assembly. We show that endothelial cells are all the more « stressed » that they are less numerous.

Nanoscale surface topography improves neuronal development in culture , Ghislain Bugnicourt, Jacques Brocard, Alice Nicolas, Catherine Villard*, submitted.
We observe that neurites grow more rapidly on surfaces with nanometric roughness and get more rapidly differentiated in axons than on flat surfaces. This article shows the influence of the physical environment on the growth and the axonal differentiation.

For the last tens years, experiments on isolated cells have shown that, in addition to chemical cues, adherent cells feel the mechanical properties of the extracellular matrix. This mechanical sensitivity was shown to profoundly influence cell proliferation, differentiation, motility or polarity, and consequently has opened great opportunities in tissue engineering. We recently developed a process, now protected by a patent, that allows the elaboration of polymerized hydrogel matrices exhibiting elastic properties that are modulated in the bulk of the material. We observed that these extracellular matrices could be used for cell localization and guidance. Although cell behavior on such matrices shares some common feature with those observed on chemically patterned, adhesive substrates, mechanisms at work differ, enlightening the chemical versus mechanical sensitivity of the cell to its environment. Thus modulating the elastic properties of the extracellular matrix in order to manipulate cell fate is a brand new strategy, which was formerly limited by the technological issue of getting a crosslinking modulation of gels at the micrometric scale. Applications are of importance: patterning cells for screening, organizing cell shape, guiding cells along routes for tissue engineering, designing 2D ou 3D niches for specific stem cell differentiation, elaborating biochips for predicting organ targeting of tumor cells, are among the potentialities of these materials with modulated elastic properties. In addition, this technology is very simple to handle, cheap and robust.
The aim of this proposal is to shed some light on the mechanisms that allow cell organization on such matrices, either in 2D or in 3D. We will explore the cases of sparse cells, as well as cell doublets for which immature intercellular adhesions compete with cell/matrix adhesions, and eventually conclude with tackling the mechanosensitivity of cell monolayers. A mechanical approach, based on the quantification of the stresses that are transmitted to the matrix by doublets or monolayers of endothelial, epithelial or myoblastic cells, will be developed, and then correlated with a biological study aiming at identifying cell-cell junction partners that contribute to the inhibition of cell sensitivity to rigidity that we observed with endothelial cells.
Two tools will be developed during the course of this project: (I) a 3D algorithm for quantifying forces exerted on substrates with modulated rigidity. Such algorithm will be inspired from a mathematical analysis of elastic inverse problems. (II) The design of a 3D biocompatible and non degradable hydrogel, for 3D confinement of cells. Composite hydrogels derived from alginate are good candidate to elaborate hydrogels with an approximately constant pore size and a variable elasticity. These experiments will support the extension of a model we formerly proposed, that describes cell/matrix adhesion mechanosensitivity at the scale of the adhesion site. The extended model will account for the coupling of the dynamics of the mechanosensitive adhesion site with the mechanosensitive response of the actin stress fibers. An innovative approach is proposed in this model to account for the mechanical interplay between cell/cell and cell/matrix adhesions. These fundamental approaches will offer a rational for the design of biochips or synthetic extracellular matrices for cell confinement, guidance or sorting.