ALP positive population and activity.

<div><p>a) ALP positive population signigicantly goes down to levels of control in M cultures. Application of LIPUS restores the ALP positive population in MU samples and significantly increases in GU samples relative to G samples.</p> <p>b) ALP Activity showed same pattern as ALP positive population, as ALP activity in M samples was significantly reduced and application of LIPUS restored ALP activity in differentiating cells. Application of LIPUS increases ALP activity in GU cultures. (*p<0.01, ** p< 0.001).</p></div

Collagen Content and Quantification.

<div><p>a) Sirius Red stain showing collagen clusters forming in extracellular matrix. Control samples show little or no collagen and Gravity (G) samples showed evenly distributed collagen clusters over the ECM. Simulated microgravity (M) samples showed little or no collagen clusters and much more comparable to control cultures. Application of LIPUS to simulated microgravity + LIPUS (MU) samples apparently increased collagen content, making them more comparable to G samples. Gravity + LIPUS (GU) samples showed denser collagen clusters and formation of thick collagen fibers (scale bar 500um).</p> <p>b) Sirius red quantification showed a significant decrease in collagen content in M cultures to the levels of control. Application of LIPUS restores collagen content in MU cultures, making them more comparable to G samples. LIPUS exposure significantly increases collagen in MU and GU samples (*p < 0.001).</p></div

LIPUS increases osteogenic genes expression and reduces osteoclastogenic gene expression in Hfob cells.

<div><p>a) RUNX2 showed no change in GU but was significantly reduced in M cultures. Application of LIPUS increased RUNX2 expression significantly relative to M cultures.</p> <p>b) OSX decreased significantly in M cultures and application of LIPUS increased OSX expression significantly in MU but didn’t show any significant changes in Gravity samples.</p> <p>c) RANKL expression showed decreases M; exposure of LIPUS further decreased RANKL expression in MU cultures contrary to LIPUS effect on hMSC (Figure 2c). LIPUS exposure show little change in RANKL expression in GU samples.</p></div

Matrix mineralization and Alizarin Red Quantification.

<div><p>a) No or little mineralization was apparent in Control samples. Gravity samples showed formation of mineralized clusters distributed evenly over the area of ECM. M samples showed no visible clusters and ECM morphology comparative to Controls. Application of LIPUS in MU samples induced mineralization as calcium clusters are apparent in ECM and morphology was more comparable to G samples. GU cultures showed denser calcium cultures and with higher frequency (scale bare 500um).</p> <p>b) Alizarin red quantification showed significant decrease of ECM calcification in M cultures almost to the level of the control group. LIPUS exposure restored mineralization of ECM. Application of LIPUS significantly increased calcification in MU cultures relative to M and GU cultures relative to G. (*p<0.001).</p></div

Calculated Dynamic Parameters for Different Acoustical Power.

<p>Notes. Values for velocity and τ represent mean ± SE. The radius of the focal region is 0.2 mm. Efficiency of power conversion is 68.8%. Fluid shear stress and radiation force were calculated according to both formulas (see text for details).</p

Schematic diagram of the transducer assembly. A

<p>: Spherical transducer with coupling cone and a centrally attached laser guideline light. The ellipsoidal acoustic focal zone covers approximately 4 mm axially and 0.4 mm transversely at the half-pressure points. <b>B</b>: Normalized pressure distribution through the culture dish for the single element on the radial plane at the distance of 62.75 mm of the transducer’s surface. <b>C</b>: The focal point of focused ultrasound guided by red laser light in the middle field of view of the microscope objective. Scale bar, 50 µm.</p

Primary cilium projections from the surface of an MC3T3-E1 osteoblast and its bending during radiation of pulsed ultrasound.

<p>The upper panel shows primary cilium stained with an antibody against acetylated α-tubulin (red) (arrowheads, A and D), cell body stained with CMFDA (green) (B and D) and nucleus stained with DAPI (blue) (C and D) in a MC3T3-E1 osteoblast. The lower panel shows the top views of bending movement of a primary cilium (arrowheads) extending from the surface of an MC3T3-E1 osteoblast before (E) and during (F and G) application of pulsed ultrasound from top to bottom. Scale bar, 10 µm.</p

Confocal scanning images from the live/dead assay, of the MC3T3-E1 osteoblast region with pulsed ultrasound applied in Petri dishes.

<p>Compared to controls (A), viability of cell cultures assessed 24 hours post-ultrasound treatment (B) showed no significant change. Scale bar, 20 µm.</p

Schematic diagram of experimental setup for spatiotemporal measurements of the effects of acoustic radiation force on MC3T3-E1 cells.

<p>The setup includes an inverted microscope and a high intensity focused ultrasound system. Movement of the spherical transducer is controlled by the three-dimensional support framework.</p

Measured average ultrasound-induced temperature rise vs. duration of different acoustical power and stimulation modes.

<p>Error bars (30-s intervals) show the standard deviation between the different measurements. Temperature was measured with a thermocouple probe immersed in the culture medium.</p