<p>Mechanical forces on cells and tissues are known to play key roles in regulating cell fate, function, and tissue repair. In bone tissue engineering, mechanical stimulation of cell-hydrogel constructs with low-intensity ultrasound has become a promising therapy for improving the pace and extent of bone regeneration in challenging defects, though its physical and biological mechanisms are not fully understood. In particular, the local ultrasound-induced forces that are imparted to fully encapsulated cells have not been directly quantified. Here, we have developed, validated, and applied a novel 3D force microscopy technique (3D-FM) that extends established principles of unconstrained, regularized, Fourier domain traction force microscopy to reconstruct forces within ultrasound-displaced 3D cell-hydrogel constructs. Validation tests with simulated data demonstrated that the algorithm is capable of reconstructing simple and complex force-density fields from simulated displacements and is robust against corruption with noise. 3D-FM was then used to estimate the ultrasound-induced forces around a bone marrow stromal cell within a soft collagen hydrogel. Localized forces near the cell had magnitudes comparable to other reported cell-scale forces (~ 100 nN), with components both parallel and perpendicular to the direction of ultrasound propagation. This work demonstrates that 3D-FM can elucidate the microscopic physical effects of low-intensity ultrasound on cells in soft matrices used in bone regeneration applications, which can provide valuable insight into the relationship between applied physical forces and cellular responses.</p>