Non-invasive techniques to control protein structure and function are needed for tissue engineering applications. This study tested the hypothesis that non-thermal effects of ultrasound can produce changes in collagen microstructure that support directional cell migration. Type I rat tail collagen was polymerized for 15 minutes in the presence of an 8-MHz ultrasound standing wave field over a range of 0 to 30 W/cm2 spatial peak, temporal average intensity. Temperature-matched sham gels were manufactured in a heated water bath without exposure to ultrasound. To test for effects of acoustically-modified collagen on cellular behavior, fibronectin-null mouse embryonic fibroblasts or mouse skin explants were seeded on gel surfaces and cultured up to 7 days prior to imaging with phase or second harmonic generation (SHG) microscopy. Acellular acoustically-modified collagen gels were characterized by regions of radial fiber alignment, increased pore size, and denser fibers, with greater heterogeneity at higher intensities. Ultrasound-exposed gels supported rapid directional cell migration with accumulation of cells in the regions of highest SHG signal. Neither fiber alignment nor cellular migration was observed in temperature-matched sham gels. Results demonstrate that ultrasound exposure during collagen polymerization can result in functionally altered collagen microstructure, in part through a non-thermal mechanism.

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