Myofibroblast persistence with real-time changes in boundary stiffness.

Myofibroblasts are critical for connective tissue remodeling and wound healing since they can close wound beds and shape tissues rapidly by generating high traction forces and secreting abundant extracellular matrix proteins and matrix metalloproteinases. However, their presence in excessive numbers is associated with fibrotic and calcific diseases and tissue thickening in engineered tissues. While activation of the myofibroblast phenotype has been studied extensively, whether myofibroblasts are "cleared" by phenotypic reversal or by apoptosis remains controversial. The goal of this work is to test the hypothesis that mechanical inhibition of myofibroblast force generation leads to de-differentiation or apoptosis depending upon the magnitude of the decrease in tension. To test this hypothesis, we cultured valvular interstitial cells (VICs) in fibrin micro-tissues suspended between flexible posts and dynamically altered the ability of the cells to generate tension by altering boundary stiffness via magnetic forces applied to posts. The flexible posts capped with magnetic beads enable the measurement and modulation of tension generated by the cells within the tissue. As expected, the cell-generated forces were elevated with dynamically increased boundary (post) stiffness, yet surprisingly, the forces continued to increase following dynamic reduction of boundary stiffness back to baseline levels. Increased apoptosis and reduced α-SMA staining were observed with complete freeing of the tissues from the posts but not upon removal of the magnet, resulting in a twofold decrease in post stiffness. Together, these data indicate that an increase in myofibroblast force generation, even if modest and temporary (1 day), can have lasting effects on myofibroblast persistence in tissues, and that a significant reduction in the ability of the cells to generate tension is required to trigger dedifferentiation and/or apoptosis. The ability to dedifferentiate myofibroblasts to a quiescent phenotype and to control the percentage of apoptosis would be of great benefit for therapeutic and tissue engineering applications. STATEMENT OF SIGNIFICANCE: Myofibroblasts play an important role in tissue remodeling and wound healing. However, excessive activation of this phenotype is associated with fibrotic diseases and scar formation. Being able to dedifferentiate these cells or controlling their clearance with apoptosis (programmed cell death) would be beneficial. It is known that releasing rigid tissue boundaries trigger apoptosis, while reducing the substrate stiffness can cause myofibroblast dedifferentiation. However, the mechanical tension was not quantified in any of the studies. Here we used micro-cantilever posts at tissue boundaries to measure tension and to regulate boundary stiffness in real time by pulling posts with magnets. We show that temporary stiffening of boundary causes irreversible myofibroblast activation and the magnitude of tension drop controls apoptosis.