Mechanical characterization of soft materials and hydrogels for mechanotransduction studies by nanoindentation

Biomimetic biomaterials, including hydrogels, are used for in vitro three-dimensional cell cultures with the overall goal to mimic the native extracellular matrix (ECM) of the cells and to investigate cellular mechanotransduction mechanisms induced by the physical microenvironment and stimuli surrounding the cells. Indeed, the mechanical properties of the surrounding environment influence the cellular behavior and functions and can trigger pathological processes. Therefore, characterizing the mechanical properties of biological tissues and proposed biomimetic substrates at a cellular length scale is gaining significant importance in mechanotransduction investigations. The general aim of this work was to support the design of gelatin-methacryloyl (GelMA) hydrogels developed for mimicking in vitro the human healthy and pathological lung tissue, and in particular to characterize by nanoindentation method the mechanical properties of human healthy and pathological lung tissue and GelMA hydrogels. In detail, the following samples were tested: a sample of human healthy lung tissue; a sample of human cancerous lung tissue; four batches of GelMA hydrogel, each one composed by twelve identical samples, prepared diluting bovine gelatin in PBS at 10% w/v and adding Methacrylic Anhydride (MA) to obtain the desired degree of methacrylation; two batches of 10% GelMA hydrogel mixed with human lung cancer epithelial cells (A549 cell line). GelMA hydrogel samples without and with cells were tested at day 1, day 2 and day 6 after production (analyzing four samples for each day of test) in order to understand if modifications in mechanical properties occur during time, also due to interaction between cells and substrate. All nanoindentation tests were performed in wet conditions (DMEM culture medium) at the physiological temperature of 37°C, for reproducing the conditions of the in vivo microenvironment. The analysis of the indentation curves, obtained in the sample regions where a linear elastic and isotropic response was assumed, allowed assessing the sample elastic response by calculating the effective Young’s Modulus (Eeff). Nanoindentation tests on human lung tissue showed high variability of the Eeff values, probably due to the heterogeneity of the tissue. The results obtained were Eeff = 186.08 ± 259.04 Pa for healthy lung tissue and 1.98 ± 6.41 kPa for cancerous one, respectively. As regards the GelMA samples, the Eeff values obtained vary from a few hundred of Pa to about 2 kPa for GelMA without cells and from approximately 600 Pa to about 1 kPa for GelMA with cells. These values are in the range of the Eeff values of the tested healthy and cancerous tissue and in agreement for the human healthy tissue with values found in literature (1-2 kPa for the healthy lung and around 16 kPa for the fibrotic lung). Further tests on GelMA samples obtained by modifying the w/v percentage and methacrylation will be performed for identifying the parameter combination most suitable to mimic the pathological lung tissue.