Mechanical characterization of GelMA hydrogels for tissue engineering applications using Nanoindentation

Living cells are constantly exposed to mechanical stimuli arising from the surrounding environment. The ability of a cell to transform stimuli into a biological response, known as mechanotransduction, is crucial in guiding cell behavior. Therefore, the mechanical properties of the extracellular matrix (ECM) determine, synergistically with other stimuli, cell fate, homeostasis, and disease. In this context, gelatin methacryloyl (GelMA) hydrogels have attracted significant attention in the field of tissue engineering and regenerative medicine due to their ability to mimic the native ECM, their excellent biocompatibility, and their tunable physicochemical properties. The main purpose of this study was to mechanically characterize various compositions of GelMA hydrogel, differing in methacrylation degree and GelMA concentration, at the cellular scale. Accordingly, stress-relaxation nanoindentation tests in phosphate-buffered saline and at 37°C (physiological conditions) were carried out. The elastic properties were calculated fitting the experimental curves through the Hertz model. The GelMA samples analyzed in this study showed that the elastic modulus (E) values range from approximately 0.5 kPa to 57 kPa. This family of materials is characterized by an intricate polymeric structure able to absorb high quantity of liquid. This leads to the hypothesis that their mechanical response is governed by viscoelastic and poroelastic mechanism. Viscoelasticity is associated with the micro-scale rearrangement of biopolymer networks, whereas poroelasticity involves the fluid flow within a porous elastic solid. In fact, in order to investigate the predominant relaxation mechanism of the GelMA samples, stress-relaxation curves were analyzed with three different models: viscoelastic, poroelastic and poroviscoelastic. The viscoelastic model revealed that all samples predominantly exhibit an elastic solid-like behavior. Meanwhile, the poroelastic analysis allowed to calculate the intrinsic permeability, a structural parameter directly related to pore size. This value ranges from 10-19 m2 to 10-17 m2, with higher values associated to softer samples, indicating larger pore sizes. Finally, the poroviscoelastic model revealed that the relaxation behavior is predominantly viscoelastic. A comprehensive micromechanical characterization of GelMA hydrogels was carried out through nanoindentation, aiming to support tissue engineering studies and to better understand how the production processes and the element stoichiometry influence the mechanical properties of the analyzed hydrogels.