How cells, when exposed to changing biomechanical and biochemical conditions, modify their extracellular matrix environment

Abstract

Abstract Background: Fibrosis, a condition characterized by excessive accumulation of extracellular matrix (ECM) components, is a major contributor to organ failure in chronic diseases such as kidney and liver disease. ECM stiffness is known to play a critical role in modulating cellular behavior, particularly through mechanotransduction pathways mediated by integrins. Understanding how cells, specifically fibroblasts, respond to changes in matrix stiffness is crucial for elucidating the mechanisms underlying fibrosis and developing potential therapeutic strategies. Objectives: This study aimed to investigate the impact of matrix stiffness on fibroblast morphology, adhesion characteristics, and ECM deposition. The specific objectives were to determine how matrix stiffness influences cell shape, focal adhesion dynamics, and the production of ECM components. Methods: Telomerase immortalized foreskin fibroblasts (Tiffs) were cultured on polyacrylamide gels with defined stiffnesses of 1.5 kPa (soft) and 28 kPa (stiff), coated with fibronectin. Cell morphology was assessed through measurements of cell area, aspect ratio, and roundness. Focal adhesion characteristics were quantified by counting the number of adhesions per cell. ECM deposition was evaluated by measuring the thickness of the ECM and fluorescence intensity as a proxy for ECM protein content. High-resolution 11 confocal microscopy and ImageJ Fiji software were used for imaging and analysis. Statistical analyses were performed using unpaired t-tests by GraphPad Prism. Results: Fibroblasts cultured on stiffer matrices exhibited a more elongated morphology, with a significantly higher aspect ratio and lower roundness compared to those on softer matrices. The number of focal adhesions was significantly reduced on stiffer matrices, indicating that stiffer environments promote fewer but potentially more stable adhesions. ECM thickness was significantly greater on stiffer matrices, suggesting enhanced ECM deposition in response to increased stiffness. However, no statistically significant difference in fluorescence intensity was observed between the two stiffness conditions, though a trend toward higher intensity on stiffer matrices was noted. Conclusions: The findings demonstrate that matrix stiffness significantly influences fibroblast morphology, adhesion dynamics, and ECM deposition, underscoring the importance of mechanical signals in regulating cellular behavior. These results have important implications for understanding the pathogenesis of fibrosis and suggest that targeting matrix stiffness could be a viable therapeutic approach. The study's limitations, including the use of a single cell type, 2D culture system, and limited ECM components, highlight the need for future research to explore these phenomena in more complex and physiologically relevant models.