(227a) Local 3D Forces and Intracellular Calcium Dynamics Induced By Low-Intensity Ultrasound Stimulation of Cell-Hydrogel Constructs for Bone Repair

Mechanical forces play a key role in regulating bone lineage cell fate and function, bone maintenance, and fracture repair. Researchers have thus sought to encourage bone formation by controlling or delivering physical forces to cells and tissue engineering constructs through the use of external stimuli. Low-intensity pulsed ultrasound (LIPUS) is one such stimulation modality that non-invasively applies low-level acoustic radiation forces (ARFs) to cells and has shown promise in promoting osteogenic activity and fracture healing. In the collective effort to better understand the mechanisms by which cells respond to LIPUS-derived ARFs, the nature of the local ultrasound-induced forces that develop around hydrogel-encapsulated cells has not been known. Intracellular calcium ion (Ca²⁺)_i dynamics have also garnered attention as an immediate cellular response to acoustic forces and other mechanical stimuli, and are well-known to activate downstream signaling pathways leading to osteogenic differentiation and bone formation. However, it has not been previously determined how to quantify ultrasound-induced forces around cells in 3D hydrogels, nor whether ARF-induced (Ca²⁺)_i responses in osteogenic cells vary with stimulation parameters. To this end, we have developed a novel 3D force microscopy (3D-FM) technique that extends the principles of lower-dimensional traction force microscopy (TFM) to quantify these forces within cell-laden biomimetic hydrogels. We have also performed in vitro live-cell fluorescence imaging of (Ca²⁺)_i dynamics within pre-osteoblast cells exposed to ARF with different intensities and pulsation frequencies that correspond to clinically relevant ranges for bone repair, to see if these parameters can be used to modulate Ca²⁺ responses known to influence downstream osteogenic cell behavior.

Methods

For the 3D-FM experiment, primary bone marrow stromal cells (BMSCs) from green fluorescent reporter mice were encapsulated within 3 mg/mL type I collagen hydrogels along with 1.0-µm red fluorescent microspheres acting as fiducial markers of local hydrogel deformation. Z-stack images of a sub-volume in a cell+bead-laden gel were taken with an epifluorescence microscope (Leica DMi8) before, during, and after real-time stimulation with low intensity ultrasound, with the transducer immersed into the medium 1-2 mm above the gel. Ultrasound-induced displacements of the red beads around a green cell were tracked between the before- and during-ultrasound Z-stacks using the TrackMate plugin in ImageJ/Fiji. Custom MATLAB scripts were then designed and used to i) 3D-interpolate these measured displacements onto a regular meshgrid domain, and ii) reconstruct the associated ultrasound-induced force-density vector field using linear elastostatic theory and Fourier domain methods. For live-cell calcium imaging during ultrasound stimulation, MC3T3 pre-osteoblast cells were cultured on coverslip-bottomed dishes and incubated in Fluo-4 calcium indicator dye using the Fluo-4 AM kit from ThermoFisher. Imaging medium was then added to each dish before placement on the microscope and immersion of the ultrasound transducer. Images were captured every 5 seconds over the following timeline: 1-minute baseline period, 5- or 30-second bout of ultrasound, and 15-minute recovery period. In the first experiment, a 5-second bout of continuous-wave ultrasound was delivered at 30 or 300 mW/cm² intensity. In the second experiment, a 30-second bout of LIPUS was delivered with fixed 300 mW/cm² intensity and 50% duty cycle, and variable pulse repetition frequency of 0.1, 1, 10, 100, or 1000 Hz. Image sequences were analyzed with Fiji and MATLAB codes that calculated mean Ca²⁺ fluorescence per cell for all cells in view, visualized with color-mapped histograms of fluorescence intensity vs. time.

Results

A demonstration of the 3D-FM technique was carried out on a 220x160x100 µm volume of collagen hydrogel containing an isolated BMSC. The elastic parameters for the gel were estimated from separate bulk rheometry experiments, yielding an average Young’s modulus of 600 Pa and Poisson’s ratio of 0.5 by assuming the gels were incompressible. Bead displacements around the cell induced by 1000 mW/cm² continuous-wave ultrasound had a peak magnitude of 5 µm. The force-density reconstruction algorithm was designed to solve the general inverse 3D problem of linear elastostatics by regularized Fourier-domain Green’s function methods. Force reconstruction with appropriate regularization revealed prominent clusters of ultrasound-induced forces near the cell, with an approximate net force magnitude of 450 nN and vector components both parallel and transverse to the z-axis of ultrasound propagation. For pre-osteoblasts in 2D culture, (Ca²⁺)_i fluorescence data captured before, during, and after brief ultrasound stimulation showed that even 5 seconds of ultrasound could trigger noticeable increases in (Ca²⁺)_i in the majority of cells, beyond any spontaneous and asynchronous Ca²⁺ responses seen without ultrasound present. Moreover, peak (Ca²⁺)_i responses occurred later and were more pronounced with the higher 300 mW/cm² intensity, suggesting that the Ca²⁺ response is not binary but rather may be altered by acoustic intensity. For pre-osteoblasts in 2D culture exposed to different LIPUS pulse frequencies (PRFs), Ca²⁺ responses again occurred gradually, peaking 1-2 minutes following LIPUS stimulation. Interestingly, among the PRFs tested, 10 Hz appeared to cause slightly stronger Ca²⁺ responses across all cells in view relative to the pre-stimulation baseline. This suggests that Ca²⁺ responses to ARF in osteogenic cells may also depend on the temporal pattern in which the acoustic energy is delivered.

Implications

The 3D-FM technique developed in this work proved to be a useful tool for quantifying the direct microscopic physical effects of low-intensity ultrasound on cells delivered in biomimetic hydrogels for bone repair. Next, 3D-FM could be used to measure the local forces on cells in hydrogels with different stiffnesses exposed to different ultrasound intensities. The findings from the Ca²⁺ imaging experiments suggest that Ca²⁺ influx, which is known to be upstream of important osteogenic pathways, can be evoked by low-intensity ultrasound and may be sensitive to changes in acoustic intensity and pulse repetition frequency. Future studies will seek to evaluate whether Ca²⁺ responses in marrow-derived stem cells, particularly in 3D hydrogel culture, are also sensitive to the LIPUS temporal pattern. The ability to quantify the ultrasound-induced forces imparted to cells within tissue-mimetic constructs and manipulate (Ca²⁺)_i signaling via ARF could facilitate the design of optimized LIPUS regimens that enhance osteogenic activity and improve bone repair.