The micromechanical force balance established between the cytoskeleton and ECM regulate cell fate in three dimensions

A recent goal of tissue engineering has been to develop tissue-equivalent constructs to aid in wound repair and regeneration. Cell remodeling processes are driven largely by the context of the local microenvironment. Fibrillar 3D extracellular matrix model systems offer a novel context by which to study basic cell-ECM dynamic interactions. In this work, a 3D fibrillar collagen matrix experimental system, in conjunction with a predictive model, was utilized to study individual fibroblast interactions with the localized surrounding ECM. Individual fibroblasts were monitored in real time using confocal reflection microscopy to quantify changes in local the ECM microstructure. In addition, a GFP-utrophin probe for F-actin was concurrently employed during live-cell imaging to monitor instantaneous cell surface area and actin stress fiber formation. Observed fibroblast behavior was monitored in response to variation in external ECM stiffness and quantified parameters were applied to a cell-ECM force balance model. The force balance model incorporated experimental measures of cell surface area, local matrix strain, contractile force, and matrix modulus. To validate the force balance model in terms of individual fibroblast contractile force, pharmacologic agents we employed to perturb internal cell stiffness. The role of cytoskeleton filament structure vs. function was documented using Cytochalasin D to study the role of actin structure, Blebbistatin to study myosin contractile function independent of actin structure, and Nocodazole to study microtubule function. By isolating cytoskeleton structure and function we simultaneously documented changes in cell surface area and local matrix strain imparted by an individual fibroblast. These measurements were used to predict contractile force using a single cell-ECM force balance equation previously proposed. Finally, the predicted force trends were corroborated with levels of myosin light chain phosphorylation, a known biochemical indicator of contractile force. Findings indicate that cells alter internal contractile force through modulation of the cytoskeleton, in addition to cell surface area, to match the stiffness of the external ECM. Such findings applied to a predictive model system are important because they provide, for the first time, a verified model by which important parameters governing 3D cell behavior are related. Finally, this predictive model system can be utilized as a means to strategically advance design parameters for tissue engineering applications