Impact of substrate curvature on fibroblasts' architecture and function

Abstract

In biological systems, form and function are inherently correlated. Despite this key link between cells and the topography of the environment in which they naturally reside, it has not been since only a few years ago that three-dimensional (3D) topographical features have started to be considered when performing cellular experiments in vitro to understand physiological and pathological mechanisms underlying cell-cell and cell-environment interactions. One of these key features involved in the 3D architecture of natural tissues is curvature, which is found in most organs of our body, such as the small intestine, which has a particular topography based on a set of invaginations and protrusions. Recently, curvature has been implied as a cue directing several cellular processes and having an impact on cell behavior, but almost all the studies performed to the date have been carried out with substrates having a higher stiffness than physiological tissues, and they were fabricated with quite complex methods. Moreover, most studies have focused on nanofeatures or cell-scale topographies, leaving macroscale curvatures such as those of the rete ridges in skin or of the crypts and villi in the intestine largely unexplored. This is why in this project, taking as a reference the curvatures of the small intestine, soft hydrogel scaffolds have been fabricated by means of light-based 3D printing, a relatively simple and fast technique. The hydrogels contained convex, concave and no curvatures with different sizes in order to obtain distinct curvature ranges. During scaffolds functionalization, collagen reached all heights for convex curvatures, whereas on invaginations collagen did not retrieve the deepest ones, thus limiting their depth to 150 μm. Afterwards, NIH-3T3 fibroblasts have been seeded on these scaffolds and immunostainings for different cell markers have been performed in order to assess their expression levels and evaluate possible changes due to the different curvatures. The expression of YAP, laminin, α-SMA and vimentin has been found to differ significatively with changes in substrate curvature. Altogether, these results reveal that macroscale curvatures resembling the intestinal villi and crypts affect different cell processes including cytoskeleton organization, transcription factors localization and extracellular matrix (ECM) protein secretion. This project has been ideated as a preliminary study to observe whether curvature-dependent changes are taking place, and therefore check if it would be worth extending the research to other cell types with higher complexity.