Substrate pre-stretch reprograms macrophage behavior through surface topographical remodeling.

Mechanical regulation of macrophages is increasingly recognized, but whether a mechanically preconditioned surface remains biologically instructive after loading has ceased is unclear. Here, we developed a custom uniaxial pre-stretch device to generate polydimethylsiloxane (PDMS) membranes with defined tensile histories and used this system to examine how substrate mechanical regulates macrophage behavior. Atomic force microscopy showed that pre-stretch reduced membrane step height and surface roughness without significantly changing elastic modulus or water contact angle, indicating selective remodeling of surface topography. RAW264.7 macrophages cultured on pre-stretched membranes exhibited increased cell and nuclear spreading, reduced roundness, and decreased F-actin intensity, accompanied by enhanced cell-substrate adhesion and increased migratory activity. Pre-stretched surfaces also promoted phagocytosis and reactive oxygen species generation without affecting proliferation. In addition, macrophages on pre-stretched membranes showed elevated expression of TNFα and IL-1β and increased TNF-α secretion, whereas Arg1 and IL-10 were not enhanced, indicating a shift toward a pro-inflammatory phenotype. These findings demonstrate that pre-stretched PDMS membranes function as mechanically encoded interfaces that direct macrophage morphology, motility, functional activation, and inflammatory polarization. More broadly, this study identifies substrate mechanical as a previously underappreciated regulator of macrophage mechanoimmunology and provides a useful platform for biomaterials research, disease modeling, and drug screening.