3D force microscopy for volumetric quantification of ultrasound-induced loading: applications for bone repair.

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.