Matrix permissiveness regulates 3D confinement of fibroblasts in biosynthetic hydrogel microenvironments

The extracellular matrix (ECM) is a major regulator of 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 investigate cell-matrix interactions in defined 3D microenvironments. However, the dynamics of these interactions in complex coupled multiparametric microenvironments has been relatively less studied. This study aims to provide a framework for correlating cellular and nuclear behavior as a function of specific matrix properties (adhesivity, degradability, porosity, and stiffness) in a biosynthetic hydrogel system with varying crosslinking mechanisms. Poly(ethylene glycol diacrylate)-fibrinogen (PF)-based hydrogels were fabricated with varying crosslinking profiles, resulting in a series of hydrogels with varying matrix properties. NIH3T3 mouse fibroblasts were cultured in 3D and their matrix-associated morphological responses were studied over time. Matrix adhesivity and degradability (collectively termed 'matrix permissiveness') were found to be the most influential parameters regulating cell and nuclear behavior. Cells in permissive matrices displayed high viability, high cell density, increased spreading and protrusivity, and large elongated nuclei. Cells in restrictive matrices displayed reduced viability and lower cell density, rounded morphology, lower spreading, and smaller rounded nuclei. Cell confinement and nuclear confinement determined from various morphological features were correlated to matrix permissiveness. Overall, this study provides insights into regulation of cellular and nuclear behavior through modulation of matrix properties which could be used for future applications in various disease contexts. STATEMENT OF SIGNIFICANCE: Biomimetic engineered hydrogels are commonly used to support three-dimensional (3D) tissue-level behavior to recapitulate various developmental processes and disease states. Specific biophysical and biochemical cues in the engineered microenvironment can be used to control cellular behavior, morphology, and function, thereby providing mechanistic insights into cell-matrix interactions. This study assesses the variations in the cellular and nuclear features of fibroblasts encapsulated in 3D hydrogel matrices with varying crosslinking mechanisms. Our results reveal the combinatorial role of matrix adhesivity and degradability in regulating cellular and nuclear confinement of fibroblasts in 3D restrictive microenvironments. Overall, these matrix guiding principles can be implemented in the future to design tunable biomimetic matrices to modulate cell state, behavior, and function.