Substrate stiffness modulates integrin α5 expression and ECM-associated gene expression in fibroblasts

Biomaterials, like polydimethylsiloxane (PDMS), are soft, biocompatible, and tuneable, which makes them useful to delineate specific substrate factors that regulate the complex landscape of cell-substrate interactions. We used a commercial formulation of PDMS to fabricate substrates with moduli 40 kPa, 300 kPa, and 1.5 MPa, and cultured HMF3S fibroblasts on them. Gene expression analysis was performed by RT-PCR and Western blotting. Cellular and nuclear morphologies were analyzed using confocal imaging, and MMP-2 and MMP-9 activities were determined with gelatin zymography. Results, comparing mechanotransduction on PDMS substrates with control petridishes, show that substrate stiffness modulates cell morphologies and proliferations. Cell nuclei were rounded on compliant substrates and correlated with increased tubulin expression. Proliferations were higher on stiffer substrates with cell cycle arrest on softer substrates. Integrin alpha5 expression decreased on lower stiffness substrates, and correlated with inefficient TGF-beta; activation. Changes to the activated state of the fibroblast on higher stiffness substrates were linked to altered TGF-beta; secretion. Collagen I, collagen III, and MMP-2 expression levels were lower on compliant PDMS substrates as compared to stiffer ones; there was little MMP-9 activity on substrates. These results demonstrate critical feedback mechanisms between substrate stiffness and ECM regulation by fibroblasts which is highly relevant in diseases like tissue fibrosis.

Wrinkling Analysis of Double Nanofilms Embedded in Compliant Substrates Based on Nonlocal Elasticity Theory

In this paper, an analytical approach for wrinkling analysis of double nanofilms embedded in compliant substrates is presented. The governing differential equations for wrinkling of double nanofilms are derived based on Eringen’s nonlocal elasticity theory and the classical plate theory (CLPT). Substrates are regarded as elastic bodies of finite thickness and the normal pressure of substrates on the films is assumed to be linear with the lateral deformation of the films. Solutions for critical wrinkling load and wave number are computed in terms of the nonlocal parameter, the elastic properties and thickness of double nanofilms, the elastic properties and thickness of the substrates, and wrinkle modes. The parametric study shows that the dimensionless critical wave number and the dimensionless critical load decrease gradually with the increase of the nonlocal parameter. Four typical wrinkling states are studied: in-phase wrinkling, out-of-phase wrinkling, single-film wrinkling and asymmetric wrinkling. In-phase wrinkling has the highest chance to occur among four wrinkle modes. In addition, the results show that the dimensionless critical load and wave number are much smaller when the films become much stiffer and thinner than the substrates.

Compliant Substrates Enhance Macrophage Cytokine Release and NLRP3 Inflammasome Formation During Their Pro-Inflammatory Response

Immune cells process a myriad of biochemical signals but their function and behavior are also determined by mechanical cues. Macrophages are no exception to this. Being present in all types of tissues, macrophages are exposed to environments of varying stiffness, which can be further altered under pathological conditions. While it is becoming increasingly clear that macrophages are mechanosensitive, it remains poorly understood how mechanical cues modulate their inflammatory response. Here we report that substrate stiffness influences the expression of pro-inflammatory genes and the formation of the NLRP3 inflammasome, leading to changes in the secreted protein levels of the cytokines IL-1β and IL-6. Using polyacrylamide hydrogels of tunable elastic moduli between 0.2 and 33.1 kPa, we found that bone marrow-derived macrophages adopted a less spread and rounder morphology on compliant compared to stiff substrates. Upon LPS priming, the expression levels of the gene encoding for TNF-α were higher on more compliant hydrogels. When additionally stimulating macrophages with the ionophore nigericin, we observed an enhanced formation of the NLRP3 inflammasome, increased levels of cell death, and higher secreted protein levels of IL-1β and IL-6 on compliant substrates. The upregulation of inflammasome formation on compliant substrates was not primarily attributed to the decreased cell spreading, since spatially confining cells on micropatterns led to a reduction of inflammasome-positive cells compared to well-spread cells. Finally, interfering with actomyosin contractility diminished the differences in inflammasome formation between compliant and stiff substrates. In summary, we show that substrate stiffness modulates the pro-inflammatory response of macrophages, that the NLRP3 inflammasome is one of the components affected by macrophage mechanosensing, and a role for actomyosin contractility in this mechanosensory response. Thus, our results contribute to a better understanding of how microenvironment stiffness affects macrophage behavior, which might be relevant in diseases where tissue stiffness is altered and might potentially provide a basis for new strategies to modulate inflammatory responses.

Determination by Relaxation Tests of the Mechanical Properties of Soft Polyacrylamide Gels Made for Mechanobiology Studies

Following the general aim of recapitulating the native mechanical properties of tissues and organs in vitro, the field of materials science and engineering has benefited from recent progress in developing compliant substrates with similar physical and chemical properties. In particular, in the field of mechanobiology, soft hydrogels can now reproduce the precise range of stiffnesses of healthy and pathological tissues to study the mechanisms behind cell response to mechanics. However, it was shown that biological tissues are not only elastic but also relax at different timescales. Cells can indeed perceive and actually need this dissipation because it is a critical signal integrated with other signals to define adhesion, spreading and even more complicated functions. The mechanical definition of hydrogels used in mechanobiology is however commonly limited to the elastic stiffness (Young’s modulus) and this value is known to depend greatly on the measurement conditions that are rarely reported. Here, we report that a simple relaxation test performed under well defined conditions can provide all the necessary information to characterize soft materials mechanically, by fitting the dissipation behavior with a generalized Maxwell model (GMM). The method was validated using soft polyacrylamide hydrogels and proved to be very useful to unveil precise mechanical properties of gels that cells can sense and offer a set of characteristic values that can be compared with what is typically reported from microindentation tests.