Cell Activities on Viscoelastic Substrates Show an Elastic Energy Threshold and Correlate with the Linear Elastic Energy Loss in the Strain‐Softening Region

Abstract

Energy‐sensing in viscoelastic substrates has recently been shown to be an important regulator of cellular activities, modulating mechanical transmission and transduction processes. Here, this study fine‐tunes the elastic energy of viscoelastic hydrogels with different physical and chemical compositions and shows that this has an impact on cell response in 2D cell cultures. This study shows that there is a threshold value for elastic energy (≈0.15 J m−3) above which cell adhesion is impaired. When hydrogels leave the linear stress–strain range, they show softening (plastic) behavior typical of soft tissues. This study identifies a correlation between the theoretical linear elastic energy loss in the strain‐softening region and the number of cells adhering to the substrate. This also has implications for the formation of vinculin‐rich anchorage points and the ability of cells to remodel the substrate through traction forces. Overall, the results reported in this study support that the relationship between cell activities and energy‐sensing in viscoelastic substrates is an important aspect to consider in the development of reliable ex vivo models of human tissues that mimic both normal and pathological conditions.

Controlled Quenching of Agarose Defines Hydrogels with Tunable Structural, Bulk Mechanical, Surface Nanomechanical, and Cell Response in 2D Cultures

“…The physical/chemical characteristics of the agarose are the following: total content of agaropectins (in terms of ashes) = 0.6% w/w; gelling temperature = 34 °C; rotational viscosity at 60 °C = 14 mPa s; total methylation = 7.4%. [21] Synthesis of Atto Rho101 NHS Ester-Labeled Agarose: 300 mg of agarose were dissolved in 15 mL of deionized water and the resulting mixture was stirred at ≈95 °C for 15 min to promote agarose solubilization. Then, the temperature was cooled down to 60 °C, and 125 μL of Atto Rho101 NHS ester (Sigma, USA) dissolved in DMSO at 2 mg mL −1 was added to the solution.…”

“…[10] Nevertheless, the residual methylation pattern has been shown to play a key role in modulating the onset of softening, which has fundamental implications for the mechanical sensing of the agarose substrates by the cells. [21] Standard protocols for the preparation of agarose hydrogels involve dissolving the agarose at high temperature and then rapidly cooling it to room temperature. However, this uncontrolled quenching could have an important influence on the final hydrogel structure, given the known particular temperatureassisted gelation of the biopolymer.…”

Ionic Strength Impacts the Physical Properties of Agarose Hydrogels

“…We have investigated the effects of ionic strength, I , generated by different alkali metal salts on the mechanical properties of hydrogels composed of a commercial agarose sample with low agaropectin content [ 22 ]. Agarose powder was dispersed in the presence of two monovalent salts, namely LiCl and NaCl, to vary the final ionic strength in the range 0–1 M. After autoclaving the mixtures at 121 °C and immediately quenching at room temperature, the agarose hydrogels were equilibrated at 37 °C for another 24 h [ 13 ] and analyzed by stress sweep experiments at 1 Hz.…”

“…The dependence of the elastic modulus G′ for strain values tending towards zero indicates a decrease in the elastic response for both LiCl- and NaCl-supplemented hydrogels for a total ionic strength of up to 0.75 M. A further increase in ionic strength does not show a variation in the case of LiCl, while an increase in G′ is observed for NaCl. The data of the long stress sweep were analyzed with respect to the stress-strain relationship and modelled according to Equation (1) to provide information about where b is a fitting parameter, the applied stress and the shear modulus for strain [ 14 , 22 ]. Figure 2 shows the trend of as a function of ionic strength when both monovalent salts are considered.…”

“…It is interesting to note that agarose-based hydrogels show different mesh size, ranging in the order of hundreds of nanometers, depending on its concentration [ 20 , 21 ]. Furthermore, the degree and pattern of residual methylation has been shown to play a role in the mechanical response of hydrogels from agaroses of different origins, especially when the nonlinear stress-strain region is considered [ 22 ].…”