Dynamic flow-driven in vitro skin model reveals mechanosensitive remodeling pathways

Mechanical forces regulate skin biology, yet few in vitro systems replicate pathological remodeling under physiological stress. We developed a three-dimensional co-culture model of HaCaT keratinocytes and NIH-3T3 fibroblasts embedded in collagen matrices, subjected to continuous unidirectional fluid flow to mimic physiological mechanical stress. Epidermal organization, fibroblast responses, and mechanosensitive signaling were assessed by histology, immunohistochemistry and western blotting. Fluid flow markedly enhanced keratinocyte stratification, with increased cytokeratin 14 and involucrin expression. A similar increase in keratinocyte thickness was observed in the co-culture system using TIG-121 fibroblasts. Ki-67 staining revealed elevated keratinocyte proliferation, particularly under co-culture conditions. Fibroblast density increased with flow without altering dermal thickness, accompanied by a trend toward α-SMA expression. ERK and p38 MAPK pathways were selectively activated in flow-stimulated co-cultures. This fluidic skin model recapitulates key cellular and molecular responses to mechanical stress and provides a platform for investigating mechanoresponsive pathways in the skin.