preparation-hydroxy-paam-hydrogels-for-decoupling-effects

Preparation of Hydroxy-PAAm Hydrogels for Decoupling the Effects of Mechanotransduction Cues

It is now well established that many cellular functions are regulated by interactions of cells with physicochemical and mechanical cues of their ...

This work describes a new orientation method for semicrystalline poly(3-hexylthiophene) (P3HT) thin films, coupling nanorubbing and subsequent crystallization. Using the stylus of an atomic force microscope, we align the polymer chains on P3HT surfaces with a spatial and geometrical control of the oriented domains (nanorubbing). These chain-aligned structures can be made permanent thanks to the crystallization process, which propagates the orientation obtained at the surface to the bulk.

Nanorubbing of polythiophene surfaces.

In attempting to reduce the size of functional devices, the thickness of polymer films has reached values even smaller than the diameter of the unperturbed molecule. However, despite enormous efforts for more than a decade, our understanding of the origin of some puzzling properties of such thin films is still not satisfactory and several peculiar observations remain mysterious. For example, under certain conditions, such films show negative expansion coefficients or show undesirable rupture although energetically they are expected to be stable. Here, we demonstrate that many of these extraordinary effects can be related to residual stresses within the film, resulting from the preparation of these films from solution by fast evaporation of the solvent. Consequently, depending on thermal history and ageing time, such films show significant changes even in the glassy state, which we quantify by dewetting experiments and corresponding theoretical studies. Identifying the relevance of frozen-in polymer conformations gives us a handle for manipulating and controlling properties of nanometric thin polymer films.

Residual stresses in thin polymer films cause rupture and dominate early stages of dewetting.

Molecular relaxations determine the viscoelastic properties of polymers, which, in turn, control macroscopic processes like dewetting. Here, we demonstrate experimentally that the onset of rim instabilities is correlated with the longest relaxation ("reptation") time of the dewetting polymer. Conversely, such experiments allow us to determine the reptation time of polystyrene in thin films as a function of molecular weight. Our approach opens up new possibilities for testing rheological properties of polymers confined in thin films.

Disentanglement time of polymers determines the onset of rim instabilities in dewetting.

Performing detailed studies of viscoelastic dewetting of thin polystyrene films on solid substrates, we demonstrate the existence of residual stress due to strongly out of equilibrium chain conformations and a reduced entanglement density resulting from film preparation by spin coating. The ratio of stress over elastic modulus was found to increase strongly with decreasing film thickness and increasing chain length. Full equilibration of chain conformations required long times comparable to bulk reptation times. However, for chains longer than about 3000 monomers, the residual stress relaxed faster, at a rate independent of chain length.

Relaxation of residual stress and reentanglement of polymers in spin-coated films.

We present a model describing the image formation in DIC (Differential Interference Contrast) mode microscopy, by including the actual refractive indexes and reflection coefficients of objects and substrates. We calculate the contrast of flat and level objects of nanometric thickness versus the bias retardation Gamma and the numerical aperture NA. We show that high contrasts, of the edge and of the inner object, can be achieved in DIC mode with special anti-reflective substrates and large NA values. The calculations agree with contrast measurements on nanometric steps of silica and explain also the extreme ability to detect single molecules (stretched DNA molecules).

Influence of surface reflective properties on differential interference contrast microscopy.

Circulating leukocyte sequestration in pulmonary capillaries is arguably the initiating event of lung injury in acute respiratory distress syndrome. We present a microfluidic investigation of the roles of actin organization and myosin II activity during the different stages of leukocyte trafficking through narrow capillaries (entry, transit and shape relaxation) using specific drugs (latrunculin A, jasplakinolide, and blebbistatin). The deformation rate during entry reveals that cell stiffness depends strongly on F-actin organization and hardly on myosin II activity, supporting a microfilament role in leukocyte sequestration. In the transit stage, cell friction is influenced by stiffness, demonstrating that the actin network is not completely broken after a forced entry into a capillary. Conversely, membrane unfolding was independent of leukocyte stiffness. The surface area of sequestered leukocytes increased by up to 160% in the absence of myosin II activity, showing the major role of molecular motors in microvilli wrinkling and zipping. Finally, cell shape relaxation was largely independent of both actin organization and myosin II activity, whereas a deformed state was required for normal trafficking through capillary segments.

Microfluidic investigation reveals distinct roles for actin cytoskeleton and myosin II activity in capillary leukocyte trafficking.

This article describes a simple and low-tech microfluidic method for single-cell experimentation, which permits cell selection without stress, cell manipulation with fine control, and passive self-exclusion of all undesired super-micronic particles. The method requires only conventional soft lithography microfabrication techniques and is applicable to any microfluidic single-cell circuitry. The principle relies on a bypass plugged in parallel with a single-cell assay device and collecting 97% of the flow rate. Cell selection into the single cell device is performed by moving the cell of interest back and forth in the vicinity of the junction between the bypass and the analysis circuitry. Cell navigation is finely controlled by hydrostatic pressure via centimetre-scale actuation of external macroscopic reservoirs connected to the device. We provide successful examples of biomechanical and biochemical assays on living human leukocytes passing through 4 mum wide capillaries. The blebbing process dynamics are monitored by conventional 24 fps videomicroscopy and subcellular cytoskeleton organization is imaged by on-chip immunostaining.

A simple microfluidic method to select, isolate, and manipulate single-cells in mechanical and biochemical assays.

Over the past decade, investigators have attempted to establish the pathophysiological mechanisms by which non-penetrating injuries damage the brain. Several studies have implicated either membrane poration or ion channel dysfunction pursuant to neuronal cell death as the primary mechanism of injury. We hypothesized that traumatic stimulation of integrins may be an important etiological contributor to mild Traumatic Brain Injury. In order to study the effects of forces at the cellular level, we utilized two hierarchical, in vitro systems to mimic traumatic injury to rat cortical neurons: a high velocity stretcher and a magnetic tweezer system. In one system, we controlled focal adhesion formation in neurons cultured on a stretchable substrate loaded with an abrupt, one dimensional strain. With the second system, we used magnetic tweezers to directly simulate the abrupt injury forces endured by a focal adhesion on the neurite. Both systems revealed variations in the rate and nature of neuronal injury as a function of focal adhesion density and direct integrin stimulation without membrane poration. Pharmacological inhibition of calpains did not mitigate the injury yet the inhibition of Rho-kinase immediately after injury reduced axonal injury. These data suggest that integrin-mediated activation of Rho may be a contributor to the diffuse axonal injury reported in mild Traumatic Brain Injury.

A possible role for integrin signaling in diffuse axonal injury.

Growing evidence suggests that cytoplasmic actin filaments are essential factors in the modulation of nuclear shape and function. However, the mechanistic understanding of the internal orchestration between cell and nuclear shape is still lacking. Here we show that orientation and deformation of the nucleus are regulated by lateral compressive forces driven by tension in central actomyosin fibres. By using a combination of micro-manipulation tools, our study reveals that tension in central stress fibres is gradually generated by anisotropic force contraction dipoles, which expand as the cell elongates and spreads. Our findings indicate that large-scale cell shape changes induce a drastic condensation of chromatin and dramatically affect cell proliferation. On the basis of these findings, we propose a simple mechanical model that quantitatively accounts for our experimental data and provides a conceptual framework for the mechanistic coordination between cell and nuclear shape.

Spatial coordination between cell and nuclear shape within micropatterned endothelial cells.

Physico-chemical and biochemical factors in the local cellular microenvironment are known to impact on multiple aspects of cell behaviour through specific signal pathways. These mechanotransduction cues can couple each other to regulate cell fate, and it remains unclear whether mechanotransduction in different contexts shares common mechanisms. Undoubtedly, a challenge will involve the further characterization of such cooperative mechanisms, as well as clearly defining the individual role of each mechanical and biochemical parameter. To control these mechanotransduction cues in an independent manner, we developed a simple and efficient strategy to immobilize any desired nature of proteins on polyacrylamide hydrogels and independently control various parameters of the cell microenvironment, such as matrix stiffness, cell-binding ligand density and confined adhesiveness. This novel platform is validated by conducting single-cell experiments and opens a broad avenue for studying complex interplays involved in mechanotransduction with a facile and versatile approach.

A simple route to functionalize polyacrylamide hydrogels for the independent tuning of mechanotransduction cues.

This protocol describes a simple method to deposit protein micropatterns over a wide range of culture substrate stiffness (three orders of magnitude) by using two complementary polymeric substrates. In the first part, we introduce a novel polyacrylamide hydrogel, called hydroxy-polyacrylamide (PAAm), that permits to surmount the intrinsically nonadhesive properties of polyacrylamide with minimal requirements in cost or expertize. We present a protocol for tuning easily the rigidity of "soft" hydroxy-PAAm hydrogels between ~0.5 and 50 kPa and a micropatterning method to locally deposit protein micropatterns on these hydrogels. In a second part, we describe a protocol for tuning the rigidity of "stiff" silicone elastomers between ~100 and 1000 kPa and printing efficiently proteins from the extracellular matrix. Finally, we investigate the effect of the matrix rigidity on the nucleus of primary endothelial cells by tuning the rigidity of both polymeric substrates. We envision that the complementarity of these two polymeric substrates, combined with an efficient microprinting technique, can be further developed in the future as a powerful mechanobiology platform to investigate in vitro the effect of mechanotransduction cues on cellular functions, gene expression, and stem cell differentiation.

Article	Year
Nanorubbing of polythiophene surfaces. Journal of the American Chemical Society\| PubMed ID: 15926818	2005
Residual stresses in thin polymer films cause rupture and dominate early stages of dewetting. Nature materials\| PubMed ID: 16184173	2005
Disentanglement time of polymers determines the onset of rim instabilities in dewetting. Physical review letters\| PubMed ID: 16712174	2006
Relaxation of residual stress and reentanglement of polymers in spin-coated films. Physical review letters\| PubMed ID: 17678296	2007
Influence of surface reflective properties on differential interference contrast microscopy. Optics express\| PubMed ID: 18542552	2008
Microfluidic investigation reveals distinct roles for actin cytoskeleton and myosin II activity in capillary leukocyte trafficking. Biophysical journal\| PubMed ID: 19450501	2009
A simple microfluidic method to select, isolate, and manipulate single-cells in mechanical and biochemical assays. Lab on a chip\| PubMed ID: 20480111	2010
A possible role for integrin signaling in diffuse axonal injury. PloS one\| PubMed ID: 21799943	2011
Spatial coordination between cell and nuclear shape within micropatterned endothelial cells. Nature communications\| PubMed ID: 22334074	2012
A simple route to functionalize polyacrylamide hydrogels for the independent tuning of mechanotransduction cues. Lab on a chip\| PubMed ID: 23334710	2013
Micropatterning hydroxy-PAAm hydrogels and Sylgard 184 silicone elastomers with tunable elastic moduli. Methods in cell biology\| PubMed ID: 24560501	2014

Sylvain Gabriele

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