This work provides a comprehensive view on how multiple mechanical cues of the substrate influence fundamental cellular processes, including spreading, proliferation, migration and differentiation, towards 3D tissue constructs.

Tissue engineers have explored a set of materials cues that can allow control of cell viability and guide cell fate and functions. Although the effect of substrate stiffness on cell fate has been extensively studied and established, the role of substrate stress relaxation, the ability of a substrate to dissipate cell-induced forces, is only emerging. Recently, several studies have demonstrated that substrate stress relaxation is an important mechanical cue for cell spreading, proliferation and differentiation in vitro. In this mini-review, we highlight the influence of substrate stress relaxation on cell behavior and function as well as provide future perspectives. Firstly, we describe the methods used for characterizing the stress relaxation/creep responses of hydrogels along with the molecular origin of viscoelastic properties. Then, we highlight the most recent studies elucidating the stress relaxation effect on cellular behavior using physically cross-linked hydrogels. Finally, we report on an emerging alternative design of tunable viscoelastic hydrogels: chemically cross-linked (reversible linkages) adaptable hydrogels that have been used as stable 3D cell culture platforms for a few years in the era of hydrogel systems.

The mechanical milieu of the extracellular matrix (ECM) plays a key role in modulating the cellular responses. The native ECM exhibits viscoelasticity with stress relaxation behavior. Here, we reported the preparation of degradation-mediated stress relaxing semi-interpenetrating (semi-IPN) polymeric networks of hydroxyethyl cellulose in the crosslinked gelatin-polyethylene glycol (PEG) architecture, leveraging a newly developed synthesis protocol which successively includes one-pot gelation under physiological conditions, freeze-drying and a post-curing process. Fourier transform infrared (FTIR) confirmed the formation of the semi-IPN blend mixture. A surface morphology analysis revealed an open pore porous structure with a compact skin on the surface. The hydrogel showed a high water-absorption ability (720.00 ± 32.0%) indicating the ability of retaining a hydrophilic nature even after covalent crosslinking with functionalized PEG. Detailed mechanical properties such as tensile, compressive, cyclic compression and stress relaxation tests were conducted at different intervals over 28 days of hydrolytic degradation. Overall, the collective mechanical properties of the hydrogel resembled the mechanics of cartilage tissue. The rate of stress relaxation gradually increased with an increasing swelling ratio. Hydrolytic degradation led to a marked increase in the percentage dissipation energy and stress relaxation response, indicating the degradation-dependent viscoelasticity of the hydrogel. Strikingly, the hydrogel maintained the structural stability even after degrading two-thirds of its initial mass after a month-long hydrolytic degradation. This study demonstrates that this semi-IPN G-PEG-HEC hydrogel possesses bright prospects as a potential scaffolding material in tissue engineering.

One of the main hurdles to improving scaffolds for regenerative medicine is the development of non-invasive methods to monitor cell proliferation within three-dimensional environments. Recently, an electrical impedance-based approach has been identified as promising for three-dimensional proliferation assays. A low-cost impedance-based solution, easily integrable with multi-well plates, is here presented. Sensors were developed using biocompatible carbon-based ink on foldable polyimide substrates by means of a novel aerosol jet printing technique. The setup was tested to monitor the proliferation of human mesenchymal stromal cells into previously validated gelatin-chitosan hybrid hydrogel scaffolds. Reliability of the methodology was assessed comparing variations of the electrical impedance parameters with the outcomes of enzymatic proliferation assay. Results obtained showed a magnitude increase and a phase angle decrease at 4 kHz (maximum of 2.5 kΩ and −9 degrees) and an exponential increase of the modeled resistance and capacitance components due to the cell proliferation (maximum of 1.5 kΩ and 200 nF). A statistically significant relationship with enzymatic assay outcomes could be detected for both phase angle and electric model parameters. Overall, these findings support the potentiality of this non-invasive approach for continuous monitoring of scaffold-based cultures, being also promising in the perspective of optimizing the scaffold-culture system.