Abstract
Introduction: Uncontrolled cell growth and scar tissue formation, known as fibrosis, are hallmarks of hypertrophic cardiomyopathy (HCM). Cardiac fibroblasts (CF), responsible for tissue fibrosis, are mechanosensitive cells, and increased extracellular matrix (ECM) stiffness may contribute to fibrotic pathways leading to disease progression. TGF- β signaling plays a pivotal role in mediating fibrosis by activating CF. State-of-the-art cell culture techniques allow the development of 3D cellular spheroids that can be mechanically tested and characterized. Cell spheroids exhibit low stiffness, thus undergo large deformations at small external forces, and current contact mechanics models fail to describe such deformation behavior. Here, we employ a hyperelastic model for large deformations to assess the mechanics of cell spheroids. As a case study, we compared the stiffness of HCM spheroids to that of control healthy ones, and tested the effect of TGF- β treatment on the mechanics of both of these groups.
Methods: Cell spheroids from primary human fibroblasts (ethical approval, Nr. 5/2018 Ethical Committee of the Province of Alto Adige/South Tyrol & Nr. 19337_bio Regional Ethical Committee for the clinical experimentation of Tuscany) were subjected to parallel-plate compression (MicroSquisher, CellScale) using a round tungsten cantilever and accompanying SquisherJoy V5.23 software. The fluid bath test chamber was filled with sterile PBS, and stage and optics were calibrated according to manufacturer’s instructions. Samples were compressed up to 50% apparent linear strain. F–δ data was fitted using linear least squares regression using the Tatara extended model (custom MATLAB code) with fully constrained contact points (F =0, δ =0).
Results: Our findings show the non-linear behavior of a cell spheroid compressed up to 50% strain. Transition from Hertzian behavior at small strains to a stiffer one at larger strainsis observed. Hertzian theory can be applied from 10% to 30% compressive strain depending on the displacement rate. However, at larger strains, where the force follows the third and fifth power of the displacement, the Tatara model was successfully applied. This model was used to extract the stiffness of different cell spheroids and results are presented here. HCM spheroids exhibit a nearly three-fold higher stiffness compared to control (healthy) ones. Additionally, stiffness of cell spheroids treated with TGF-β is approximately twice that of the untreated groups, in both HCM and control spheroids.
Discussion: Beyond the effect of HCM on stiffening of cell spheroids, our results show that TGF-β treatment noticeably influences spheroid stiffness. Possible mechanisms of this effect could be increased ECM production, crosslinking changes and enhanced cell contractility. HCM pathogenesis of cardiac fibrosis is yet to be fully understood and multiple mechanisms could contribute to tissue stiffening and disease progression, which can be studied with the models presented in a patient-specific manner.