Matrix compliance regulates cellular and nuclear confinement of fibroblasts in tunable protein-based hydrogel microenvironments

Abstract The extracellular matrix (ECM) influences cellular behavior, fate, and various mechanisms underlying homeostasis, development, and disease. Biophysical and biochemical properties of the ECM are known to affect three-dimensional (3D) cellular behavior, and phenotype that regulate a wide range of pathological conditions. Tunable biomimetic hydrogels are extensively employed to regulate cellular dynamics in defined 3D microenvironments, enabling detailed investigation of cell-matrix interactions. This study aims to establish the relationship between varying hydrogel properties (adhesivity, degradability, porosity, and stiffness, hereby collectively referred to as ‘matrix compliance’) and fibroblast morphology at the cellular and nuclear levels. Poly(ethylene glycol diacrylate)-fibrinogen (PF)-based hydrogels were fabricated and their matrix properties were tuned using two different non-degradable co-monomers of varying molecular weights and concentrations. NIH3T3 mouse fibroblasts were cultured in 3D hydrogels for 10 days and their cellular and nuclear morphology was analyzed via confocal imaging. Cells in degradable, soft, porous, and adhesive hydrogels displayed higher degree of spreading, higher cell density, longer protrusions, and elongated and larger nuclei compared to those in less degradable, stiffer, less porous, and less adhesive hydrogels. Matrix compliance conjointly regulated cell density, protrusion length, and protrusion frequency (collectively referred to as ‘cellular confinement’) and nuclear volume (referred to as ‘nuclear confinement’). These results highlight the complex interplay and interdependence of these cellular and nuclear morphological features as regulated by engineered tunable matrices, providing guidance for future studies of 3D cell behavior in novel biomaterials. Statement of significance Biomimetic tunable hydrogels are commonly used to support, and control, three-dimensional (3D) cellular behavior to recapitulate various developmental processes and disease states. Specific biophysical and biochemical characteristics of the extracellular matrix (ECM) can be modeled using these hydrogels by controlling the compositions and crosslinking mechanisms. Using this approach, the behavior of cells in 3D hydrogel matrices can be correlated with the matrix properties, thereby providing mechanistic insights into cell-matrix interactions. This study assesses the variations in the morphological features of fibroblasts encapsulated in 3D protein-based hydrogel matrices with varying crosslinking mechanisms. Our results reveal the combinatorial role of matrix adhesivity, degradability, porosity, and stiffness in regulating cellular and nuclear confinement of fibroblasts in 3D microenvironments. Overall, this study elucidates how matrix characteristics influence cellular behavior, serving as a practical guide for researchers developing innovative biomaterial matrices for 3D cell culture applications.