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 signalling 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. Current analysis methods are often limited to Hertzian theory and its modifications, that only account for small deformations. However, spheroids exhibit low stiffness, thus undergoing large deformations at small external forces, and therefore current contact mechanics models fail to describe such 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.
DISCUSSION AND CONCLUSIONS Linear elastic continuum mechanics with some important modifications can be applied to the case of large deformations. Beyond the effect of HCM on stiffening of cell spheroids, our results show that also 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.
MATERIALS AND METHODS Four groups of cell spheroids, consisting of primary human fibroblasts cultured for two days (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 testing (MicroSquisher, CellScale) fitted with a round tungsten cantilever and accompanying SquisherJoy V5.23 software (CellScale, Ontario, Canada). The fluid bath test chamber was filled with sterile phosphate buffered saline (pH=7.4). Stage and optics were calibrated according to manufacturer’s instructions. Samples were compressed up to 50% apparent linear strain at different displacement rates. F–ε data was fitted using linear least squares regression on the Hertz and Tatara model and its extended version (custom MATLAB code) with fully constrained contact points (F=0, δ=0). During compression, images were captured via two digital cameras to determine lateral expansion of cell spheroids over a range of deformations. The effect of TGF-β on the collagen concentration was studied using the Zeiss LSM800 confocal microscope. The cell spheroids were stained with Picrosirius red solution.
RESULTS Our data shows the non-linear force response of a cell spheroid compressed up to 50% strain. Transition from softer behavior at small strains to a stiffer one at larger strains is 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 here presented. 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. This can be attributed to the increased collagen production in the treated spheroids. Another advantage of the Tatara model is its capability to compute the deformed shape of the spheroid. The compression modulus was calculated for the new Poisson’s ratio obtained from image analysis, showing 40% increase.