Limits...
Mechanobiological modulation of cytoskeleton and calcium influx in osteoblastic cells by short-term focused acoustic radiation force.

Zhang S, Cheng J, Qin YX - PLoS ONE (2012)

Bottom Line: Cell viability was not affected.Application of pulsed ultrasound radiation generated only a minimal temperature rise of 0.1°C, and induced a streaming resulting fluid shear stress of 0.186 dyne/cm(2), suggesting that hyperthermia and acoustic streaming might not be the main causes of the observed cell responses.In conclusion, these data provide more insight in the interactions between acoustic mechanical stress and osteoblastic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Stony Brook University, Stony Brook, New York, United States of America.

ABSTRACT
Mechanotransduction has demonstrated potential for regulating tissue adaptation in vivo and cellular activities in vitro. It is well documented that ultrasound can produce a wide variety of biological effects in biological systems. For example, pulsed ultrasound can be used to noninvasively accelerate the rate of bone fracture healing. Although a wide range of studies has been performed, mechanism for this therapeutic effect on bone healing is currently unknown. To elucidate the mechanism of cellular response to mechanical stimuli induced by pulsed ultrasound radiation, we developed a method to apply focused acoustic radiation force (ARF) (duration, one minute) on osteoblastic MC3T3-E1 cells and observed cellular responses to ARF using a spinning disk confocal microscope. This study demonstrates that the focused ARF induced F-actin cytoskeletal rearrangement in MC3T3-E1 cells. In addition, these cells showed an increase in intracellular calcium concentration following the application of focused ARF. Furthermore, passive bending movement was noted in primary cilium that were treated with focused ARF. Cell viability was not affected. Application of pulsed ultrasound radiation generated only a minimal temperature rise of 0.1°C, and induced a streaming resulting fluid shear stress of 0.186 dyne/cm(2), suggesting that hyperthermia and acoustic streaming might not be the main causes of the observed cell responses. In conclusion, these data provide more insight in the interactions between acoustic mechanical stress and osteoblastic cells. This experimental system could serve as basis for further exploration of the mechanosensing mechanism of osteoblasts triggered by ultrasound.

Show MeSH

Related in: MedlinePlus

Schematic diagram of experimental setup for spatiotemporal measurements of the effects of acoustic radiation force on MC3T3-E1 cells.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.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3368843&req=5

pone-0038343-g001: Schematic diagram of experimental setup for spatiotemporal measurements of the effects of acoustic radiation force on MC3T3-E1 cells.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.

Mentions: A modified high intensity focused ultrasound system (Figure 1) was used for the mechanobiological studies. The excitation signal generated by a waveform generator (AFG3021, Tektronix Inc, Beaverton, OR), was attenuated 10 times (using an 860 Attenuator, Kay Elemetrics Corp, Lincoln Pk, NJ) before exposure to a radio-frequency power amplifier (E&I 2100L, Electronics & Innovation, Ltd., Rochester, NY), which in turn drives a single spherical and concave transducer (H-102C, Sonic Concepts, Bothell, WA). The transducer has an active outside diameter of 64.0 mm, a radius of curvature (distance to focus from inner surface) of 62.6 mm and a central 20-mm hole that houses a piece of plexiglass and a laser module (VLM-650-03-LPA, Quarton Inc, CA), focused by a plano-convex lens with a focus of 72 mm (NT32-850, Edmund Optics, NJ). A schematic diagram of the device is shown in Figure 1. The focal point of the ultrasound beam was positioned at the midpoint of cells cultured in Petri dishes through a three-dimensional support framework. The transducer assembly was attached to a polycarbonate coupling cone with a 10-mm diameter exit hole. The cone was filled with degassed water and the distal end was sealed with a silicon membrane (Figure 2A). Prior to the experiment, the transducer was perpendicularly positioned such that the distal end of the coupling cone was submerged in the culture medium and 5 mm above the MC3T3-E1 cell monolayer, which settled on the bottom of the culture well. The beam shape at the focal zone was determined by mapping the acoustic field using a capsule-type hydrophone (HGL-0200, ONDA Corp., Sunnyvale, CA) mounted on a three-way micropositioner. At the half-power points, the focal zone was approximately 4.0 mm axially and 0.4 mm transversely (Figure 2B). The laser guideline light was aligned to be coaxial and confocal with the transducer. The ultrasound focal point was located in the middle field of view of the microscope objective with guidance from the laser guideline light (Figure 2C). The focal area was 0.126 mm2, so the entire field of view of the microscope (200×200 µm at ×40, 400×400 µm at ×20) was radiated with ultrasound. Ultrasound was turn on for one minute at the third harmonic of the transducer, 3.3 MHz with pulse duration of 300 ms, pulse repetition frequency of 0.5 Hz, duty factor of 0.15 (300 ms on and 1700 ms off). The maximum acoustic output power in this study was 6 W, the spatial averaged intensity was calculated to be 4,800 W/cm2 and the peak negative pressure amplitude was 9.18 MPa. Efficiency was calculated to be 68.8% [29].


Mechanobiological modulation of cytoskeleton and calcium influx in osteoblastic cells by short-term focused acoustic radiation force.

Zhang S, Cheng J, Qin YX - PLoS ONE (2012)

Schematic diagram of experimental setup for spatiotemporal measurements of the effects of acoustic radiation force on MC3T3-E1 cells.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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368843&req=5

pone-0038343-g001: Schematic diagram of experimental setup for spatiotemporal measurements of the effects of acoustic radiation force on MC3T3-E1 cells.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.
Mentions: A modified high intensity focused ultrasound system (Figure 1) was used for the mechanobiological studies. The excitation signal generated by a waveform generator (AFG3021, Tektronix Inc, Beaverton, OR), was attenuated 10 times (using an 860 Attenuator, Kay Elemetrics Corp, Lincoln Pk, NJ) before exposure to a radio-frequency power amplifier (E&I 2100L, Electronics & Innovation, Ltd., Rochester, NY), which in turn drives a single spherical and concave transducer (H-102C, Sonic Concepts, Bothell, WA). The transducer has an active outside diameter of 64.0 mm, a radius of curvature (distance to focus from inner surface) of 62.6 mm and a central 20-mm hole that houses a piece of plexiglass and a laser module (VLM-650-03-LPA, Quarton Inc, CA), focused by a plano-convex lens with a focus of 72 mm (NT32-850, Edmund Optics, NJ). A schematic diagram of the device is shown in Figure 1. The focal point of the ultrasound beam was positioned at the midpoint of cells cultured in Petri dishes through a three-dimensional support framework. The transducer assembly was attached to a polycarbonate coupling cone with a 10-mm diameter exit hole. The cone was filled with degassed water and the distal end was sealed with a silicon membrane (Figure 2A). Prior to the experiment, the transducer was perpendicularly positioned such that the distal end of the coupling cone was submerged in the culture medium and 5 mm above the MC3T3-E1 cell monolayer, which settled on the bottom of the culture well. The beam shape at the focal zone was determined by mapping the acoustic field using a capsule-type hydrophone (HGL-0200, ONDA Corp., Sunnyvale, CA) mounted on a three-way micropositioner. At the half-power points, the focal zone was approximately 4.0 mm axially and 0.4 mm transversely (Figure 2B). The laser guideline light was aligned to be coaxial and confocal with the transducer. The ultrasound focal point was located in the middle field of view of the microscope objective with guidance from the laser guideline light (Figure 2C). The focal area was 0.126 mm2, so the entire field of view of the microscope (200×200 µm at ×40, 400×400 µm at ×20) was radiated with ultrasound. Ultrasound was turn on for one minute at the third harmonic of the transducer, 3.3 MHz with pulse duration of 300 ms, pulse repetition frequency of 0.5 Hz, duty factor of 0.15 (300 ms on and 1700 ms off). The maximum acoustic output power in this study was 6 W, the spatial averaged intensity was calculated to be 4,800 W/cm2 and the peak negative pressure amplitude was 9.18 MPa. Efficiency was calculated to be 68.8% [29].

Bottom Line: Cell viability was not affected.Application of pulsed ultrasound radiation generated only a minimal temperature rise of 0.1°C, and induced a streaming resulting fluid shear stress of 0.186 dyne/cm(2), suggesting that hyperthermia and acoustic streaming might not be the main causes of the observed cell responses.In conclusion, these data provide more insight in the interactions between acoustic mechanical stress and osteoblastic cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Stony Brook University, Stony Brook, New York, United States of America.

ABSTRACT
Mechanotransduction has demonstrated potential for regulating tissue adaptation in vivo and cellular activities in vitro. It is well documented that ultrasound can produce a wide variety of biological effects in biological systems. For example, pulsed ultrasound can be used to noninvasively accelerate the rate of bone fracture healing. Although a wide range of studies has been performed, mechanism for this therapeutic effect on bone healing is currently unknown. To elucidate the mechanism of cellular response to mechanical stimuli induced by pulsed ultrasound radiation, we developed a method to apply focused acoustic radiation force (ARF) (duration, one minute) on osteoblastic MC3T3-E1 cells and observed cellular responses to ARF using a spinning disk confocal microscope. This study demonstrates that the focused ARF induced F-actin cytoskeletal rearrangement in MC3T3-E1 cells. In addition, these cells showed an increase in intracellular calcium concentration following the application of focused ARF. Furthermore, passive bending movement was noted in primary cilium that were treated with focused ARF. Cell viability was not affected. Application of pulsed ultrasound radiation generated only a minimal temperature rise of 0.1°C, and induced a streaming resulting fluid shear stress of 0.186 dyne/cm(2), suggesting that hyperthermia and acoustic streaming might not be the main causes of the observed cell responses. In conclusion, these data provide more insight in the interactions between acoustic mechanical stress and osteoblastic cells. This experimental system could serve as basis for further exploration of the mechanosensing mechanism of osteoblasts triggered by ultrasound.

Show MeSH
Related in: MedlinePlus