Substrate Stiffness Affects Human Keratinocyte Colony Formation.

Restoration of epidermal organization and function in response to a variety of pathophysiological insults is critically dependent on coordinated keratinocyte migration, proliferation, and stratification during the process of wound healing. These processes are mediated by the reconfiguration of both cell-cell (desmosomes, adherens junctions) and cell-matrix (focal adhesions, hemidesmosomes) junctions and the cytoskeletal filament networks that they serve to interconnect. In this study, we investigated the role of substrate elasticity (stiffness) on keratinocyte colony formation during the process of nascent epithelial sheet formation as triggered by the model of keratinocyte culture. Keratinocytes cultured on pepsin digested type I collagen coated (nominal = 1.2 kPa) polyacrylamide gels embedded with fluorescent microspheres exhibited (i) smaller spread contact areas, (ii) increased migration velocities, and (iii) increased rates of colony formation with more cells per colony than did keratinocytes cultured on (nominal = 24 kPa) polyacrylamide gels. As assessed by tracking of embedded microsphere displacements, keratinocytes cultured on substrates generated large local substrate deformations that appeared to recruit adjacent keratinocytes into joining an evolving colony. Together with the observed differences in keratinocyte kinematics and substrate deformations, we developed two analyses, termed distance rank (DR) and radius of cooperativity (RC), that help to objectively ascribe what we perceive as increasingly behavior of keratinocytes cultured on versus during the process of colony formation. We hypothesize that the differences in keratinocyte colony formation observed in our experiments could be due to cell-cell mechanical signaling generated via local substrate deformations that appear to be correlated with the increased expression of β4 integrin within keratinocytes positioned along the periphery of an evolving cell colony.