The Physical Microenvironment Affects Fibroblast Migration and Wound Healing

Cell migration depends on mechanotransduction—the conversion of mechanical cues from the microenvironment to internal biochemical cellular signals, such as by protein phosphorylation. Lipid signals are more difficult to study than proteins, but also play a key role in mechanotransduction pathways. In this study, we apply bioengineering approaches to manipulate physical cues of topography and stiffness in order to reprogram cells, assessing the effects of lipid signaling (PIP2) on the actin cytoskeleton. Primary rat neonatal fibroblasts were grown on polyacrylamide or polydimethylsiloxane (PDMS) with stiffness of 10, 100, or 400 kPa and glass (>1 GPa). Microstructures were fabricated from poly (ethylene glycol) dimethacrylate (PEGDMA) of different stiffness (100 x 15 x 15 µm3). To test the role of lipid signaling, PIP2 availability was reduced by neomycin (500 µM) scavenging, or the PIP2 level was increased by PI3K inhibition with wortmannin (1 µM). The localization of actin and lamellipodin (a PIP2 / actin binding protein) depends on stiffness and topography and was changed by drugs. The actin cytoskeleton had more prominent stress fibers extending to lamella edge with neomycin treatment, whereas the untreated and wortmannin treatment did not show that stress fibers extension. The lamellipodin was widely distributed along the cell membrane of the lamellipodia with neomycin treatment but was comparatively low with untreated or wortmannin treatment. Fibroblast migration is known to decelerate as the surface stiffens, and was measured here through a gap created by removal of a barrier. Migration velocity at all stiffnesses was much faster with reduction of PIP2 availability by neomycin treatment than in untreated cells. For example, when fibroblasts were grown on 10 kPa, the velocity nearly doubled with neomycin treatment (P<0.0002, n= 4). With wortmannin treatment, the velocity was lower than in the untreated cells (P <0.04, n=4). We conclude that primary rat neonatal fibroblasts respond to stiffness via lipid signaling to regulate cell migration through modulation of the architecture of the actin cytoskeleton. Neomycin was loaded into the microstructures and its release in culture also stimulated fibroblast migration. Therefore, the manipulation of the microenvironment and the drug delivery might be beneficial in improving therapeutics geared toward control of fibrosis.