Journal of Therapeutic Ultrasound Full text Ultrasound stimulation…

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Ultrasound stimulation increases of MC3T3-E1 preosteoblast-like cells

* author: Amit Katiyar

Author Affiliations

1 Department of Engineering, University of Delaware, DE 19716, USA

2 Department of Biological University of Delaware, Newark, DE USA

3 Department of Mechanical and Aerospace George Washington University, DC 20052, USA


Mechanical of bone increases bone and fracture healing, at least in through increases in proliferation of and osteoprogenitor cells. Researchers previously performed in vitro of ultrasound-induced osteoblast proliferation but used fixed ultrasound and have reported widely and inconclusive results. Here we investigated the effects of the excitation of low-intensity pulsed ultrasound stimulation on proliferation of MC3T3-E1 cells in monolayer cultures.

We a custom-designed ultrasound exposure to vary the key ultrasound parameters—intensity, and excitation duration. MC3T3-E1 were seeded in 12-well culture plates. Unless specified, treated cells, in of three, were excited for 10 min with an interval of 24 h in between cell seeding. Proliferation of these cells were using BrdU and MTS assays 24 h the last LIPUS excitation.

All are presented as the mean ± standard The statistical significance was determined Student’s two-sample two-tailed t

Using discrete LIPUS ranging from 1 to 500 mW/cm 2 spatial average-temporal average), we that approximately 75 mW/cm 2 the greatest increase in osteoblast Ultrasound exposures at higher (approximately 465 mW/cm 2 ) significantly proliferation in MC3T3-E1 cells, that high-intensity pulsed may increase apoptosis or loss of in these cells.

Variation in frequency from 0.5 MHz to 5 MHz indicated osteoblast proliferation rate was not dependent. We found no difference in the in proliferation rate if LIPUS was for 30 min/day or 10 min/day, indicating a response.

This study that a short-term stimulation optimum intensity can enhance of preosteoblast-like bone cells plays an important role in formation and accelerated fracture also suggesting a possible treatment for reduced bone


Osteoblast; Proliferation; Mechanical stimulation; LIPUS

Bone fracture healing is a physiological process that involves initial inflammation, and hard-callus formation and finally repair and remodeling [1 ]. Every millions of fractures are reported to the World Health Organization, in approximately 9.0 million osteoporotic were reported worldwide, of them in America and Europe. with the state of the art clinical 5%–10% of bone fractures in the USA (nonunion) or take more usual time (delayed to heal [2 ]. The extended treatment require surgical intervention for bone-grafting and/or internal

Mechanical forces are required for homeostasis [3 ,4 ]. LIPUS is a nonthermal and source of mechanical energy intensity = 5–100 mW/cm 2 ) [5 ]. of low-intensity pulsed ultrasound has been approved for treatment of as well as nonunion fractures by the and Drug Administration. LIPUS in a significant reduction in the overall healing time in several models [6 ,7 ] and clinical trials [8 -11 ]. can produce micromechanical strains in which, in turn, can trigger cellular responses [12 ]. However, are not completely understood [13 -15 ]. Previous investigated the effects of LIPUS on cellular activities such as proliferation [16 ], cell differentiation [5 ], collagen synthesis, protein and synthesis, gene expression, and calcium levels [16 ].

To date, investigations of LIPUS of bones concentrated on the bone-forming the osteoblast [17 ,18 ]. In vitro studies of osteoblast proliferation, however, reported widely varying Doan et al. [19 ] found significant but distributed increase in human osteoblast cell proliferation at 5 mW/cm 2 spatial average 5% at 15 mW/cm 2 SA, 35% at 30 mW/cm 2 SA, and 18% at 50 mW/cm 2 SA) a near-field continuous ultrasound at 45 kHz.

At 1 MHz pulsed ultrasound increased cell proliferation was in the same study but only at higher intensities (47% at 0.7 2 spatial average-pulse average and 37% at 1 W/cm 2 SAPA). Hayton et al. [20 ] approximately 10% rise of proliferation in osteoblast-like cells Saos-2 due to a excitation of standard LIPUS MHz frequency, 1 kHz PRF (pulse repetition 200 μs pulse duration and 30 mW/cm 2

In contrast, Suzuki et al. [5 ,21 ] showed there is no effect on cell for a near-field and 20-min standard exposure to rat osteoblast-like cells ROS Most recently, Kang et al. [22 ] the effects of 20 min a day stimulation by a low-intensity (1 MHz, 30 mW/cm2 continuous wave) in combination with vibratory strain (1 Hz, 10% strain) on cells in a 3D scaffold. The stimulation did not the cell proliferation over a of 10 days, but significantly up-regulated gene expressions—COL-I, OC, RUNX2, and accelerated differentiation.

It is clear LIPUS parameters for peak vary and the effects on osteoblast or cells are not always the same. is a need for a systematic study of the effects varying the parameters of such as intensity, frequency, and The objective of this study was to the effects of near-field LIPUS-induced stimulation on osteoblast cell in a monolayer culture and to understand its on key ultrasound parameters: intensity, and the excitation period.



The MC3T3-E1 cells 20–27), a preosteoblastic cell were cultured in α-minimal medium (Sigma Chemical, St. MO, USA) containing 10% fetal serum (Gibco, New York, NY, 100 units/ml penicillin G (Sigma) and 100 streptomycin (Sigma). Cells cultured in a humidified incubator at with 95% air and 5% CO 2 and subcultured every 72 h.


In previous studies, clinical devices have used to produce LIPUS [16 ,18 ,19 ,23 ,24 ]. to obtain better control on the parameters of US, we used a custom-designed exposure system. The arrangement of instruments for ultrasound exposure is in Figure 1 a. A programmable function (33250A, Agilent, Palo CA, USA) produced standard 200 μs pulses (sinusoidal waves) at 1 kHz

The transmit signal was amplified by a 55 dB laboratory RF power amplifier A-150; ENI, Rochester, NY, and then supplied to a single-element immersion transducer (part A306S, GE Panametrics, Waltham, MA, The transducer had an outside diameter of 16 mm and a frequency of 2.5 MHz. For frequency study, we used transducers different center frequencies.

1. Setup. (a) Customized ultrasound system. (b) Schematic representation of exposure setup.

The ultrasound and an XYZ positioning stage (Newport CA, USA) were sterilized 75% of ethanol and kept under light for at least 2 h before the Based on the diameter of the transducer area 2.01 cm 2 ), we found cell culture plates area 3.80 cm 2 ) appropriate for our MC3T3-E1 cells were on the bottom of the 12-well plate 1.5 mL of cell culture medium.

The transducer head was positioned over the culture well, touching the surface of the medium 1 b). In this configuration, the distance the transducer head and the bottom of the was approximately 4 ± 0.5 mm (determined from the XYZ as well as the cross-section area of the and the volume of the medium) and kept for all the experiments.

Note that the are in the near field, as in several investigations [19 ,21 ] and, therefore, are to a spatially nonuniform field. the setup has the advantage of direct by the immersed transducer unimpeded by an medium which would attenuate the signal.

Note several animal and clinical of therapeutic ultrasound involved stimulation by transducers in direct with the skin [8 ,9 ,11 ]. Li et al. [24 ] specifically the optimum intensity for far-field and found it to be comparable to the near-field quoted in the literature. For the stimulation here, the proper spatial are computed using the relations in the Appendix.

MC3T3-E1 cells at confluence (2.28 × 10 4 cells per ≈ 6 × 10 3 cells/cm 2 ) were seeded in cell culture plates. We verified that these remain in linear phase of up to approximately 4 × 10 4 cells per well. received their first US 24 h after seeding.

Unless mentioned, stimulation was given for 10 min and at an interval of 24 h. The control group the same experimental treatment the ultrasound powered off.

We that there is a possibility of transfer of mechanical energy of to the neighboring wells [25 ]. We investigated the ultrasound stimulation in neighboring and found it less than 1% to transferred directly. We note our setup for ultrasound stimulation has frequently used for studying its effects and release properties of bearing vesicles.

Recent have indicated that in setup, reflections from the interface create a standing pattern giving rise to a varying acoustic field [20 ,21 ]. note that unlike in a of drug bearing particles, is a monolayer of cells with which is much smaller the wavelength. Therefore, the variation of between cells is negligibly and the current setup is adequate for our

Determination of cell proliferation


The BrdU ELISA Cell Proliferation Biotrak system, version 2, GE Healthcare Corp. Piscataway, NJ, USA) is on incorporation of 5-bromo-2′-deoxyuridine (BrdU) DNA synthesis in proliferating cells.

To the cell proliferation (24 h after US stimulation), BrdU labeling diluted with cell medium (0.4 ml of 1:1,000 v / v ) was to each well of 12-well and the cells were reincubated for 2 h in a incubator at 37°C with 95% air and 5% CO 2 . the labeling period, BrdU is in place of thymidine into the DNA of cells. The BrdU labeling was then removed from the and 0.4 mL of fixative solution (for fixation and DNA denaturation) supplied in the kit was to each well, and the cells incubated for an additional 30 min at room (RT).

The denaturation of the DNA is necessary to improve the of the incorporated BrdU for detection by the The fixative solution was then and 0.4 mL of 1:10 diluted blocking (also supplied in the kit to block the binding surface and prevent any binding of the antibodies) was added to well.

Following incubation at temperature for 30 min, the blocking was removed and 0.4 mL of 1:100 of diluted anti-BrdU (monoclonal antibody mouse cells conjugated to lyophilized, and stabilized) working was added. The peroxidase-labeled anti-BrdU is diluted with supplied dilution solution. Cells incubated in this solution at temperature for 90 min.

The peroxidase-labeled binds to the BrdU, which is in newly synthesized cellular The anti-BrdU working solution was removed, and the cells were with 1 ml of 1:10 diluted buffer solution (phosphate saline (PBS), 10× concentrate) times at room temperature. temperature-equilibrated 3,3′5,5-tetramethylbenzidine (TMB) solution (0.4 mL) in 15% ( v / v ) dimethyl (DMSO) was then added to well.

The immune complex after adding the peroxidase-labeled reacts with TMB substrate. approximately 10 min, a light color solution is obtained and the was then stopped by adding 100 μL of 2 M H 2 SO 4 to each well. The optical (absorbance) of 150 μL of resultant yellowish solution was read at 450 nm in a 96-well spectrophotometer.

The absorbance values directly to the amount of DNA synthesis and to the number of proliferating cells in

MTS assay

To corroborate the BrdU osteoblast cell number was determined using [3-(4,5-dimethylthiazol-2-yl)-5-(3-caroxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay (CellTiter 96 Aqueous, Madison, WI, USA). This is colorimetric based on the reduction of the MTS by the living cells to a formazan The absorbance of the formazan product is at 490 nm and the generation of this product is proportional to the cell mass.

In assay, 80 μL of the MTS solution was diluted 0.4 ml of cell culture medium and to each well. The cell plate was incubated 37°C for 2 h in a 5% CO 2 atmosphere. The absorbance was recorded at 490 nm a 96-well plate reader.

In study, each single was repeated at least three on three different passages of All data are presented as the mean ± error (SE). The statistical was determined using Student’s two-tailed t tests.

Values of p were considered to be statistically


MC3T3-E1 osteoblasts to LIPUS with increase in proliferation, and the details are provided in the subsections.

Intensity dependence of proliferation

To determine the peak response, the ultrasound intensity was varied over the range of 1 to 500 2 (SATA). The exposure time was set at 10 min and all parameters (frequency = 1.5 MHz, PRF = 1 pulse duration = 200 μs) were the same.

We varied the input signal to transducer by a factor of 2 and which increased the ultrasound by a factor of 4 and 6.25 respectively 4.64, 18.57, 74.27, and mW/cm 2 ). Figure 2 shows the of ultrasound excitation at different on osteoblast cell proliferation. The assay shows that the proliferation increased approximately 30%, 36%, and 49% for the four intensities, respectively.

At the higher intensity of approximately 465 2. the proliferation decreased by approximately 6%, an inhibitory effect on osteoblast growth. Some cells also found detached the base of the cell culture after the excitation at this intensity.

Figure 2. Change in proliferation of MC3T3-E1 cells ultrasound intensity. Change in of ultrasound stimulated MC3T3-E1 (normalized with control) at ultrasound intensities (SATA) frequency = 1.5 MHz, PRF = 1 kHz, duration = 200 μs and exposure time = 10 Values significantly different control group have indicated by filled stars for p

The BrdU measurements were with an MTS assay. As shown in 2. the increase in cell proliferation was 16%, 25%, 36%, and 52% for the lower intensities respectively. stimulation at the higher intensity of 465 mW/cm 2 was detrimental to cell with a proliferation decrease of 21%.

These results by an independent assay are similar to obtained by the BrdU assay.

images of MC3T3-E1 preosteoblastic after two 10-min US excitations at interval and at different ultrasound are shown in Figure 3. These were taken at the central of the respective wells where ultrasound intensity was delivered. the increase in cell number due to US over control group is not visually distinct, Figure 3 that the cell count approximately 19%, 32%, and 53% for the four lower intensities, For the higher LIPUS intensity of 465 mW/cm 2. cells distinctly compressed and damaged.

Figure 3. images of MC3T3-E1 cells at the end of stimulation. Microscopic images of growth after two 10-min excitations at 24-h interval and at ultrasound intensities (a) control, (b) mW/cm 2. (c) 4.64 mW/cm 2. (d) mW/cm 2. (e) 74.27 mW/cm 2. and (f) mW/cm 2. Magnification × 20.

Frequency of proliferation in MC3T3-E1 cells

the optimum intensity was identified, of excitation frequency were over frequencies ranging 0.5 to 5 MHz at the optimal intensity (75 mW/cm 2 ) a 10-min exposure time. In experiment, we ensured that the duration (200 μs) and PRF (1 kHz) the same by changing the number of while changing the frequency. 4 shows that the ultrasound increased osteoblast cell at all three frequencies.

However, is no statistically significant difference in at different frequencies.

Figure 4. in proliferation of MC3T3-E1 cells ultrasound frequency. Change in of ultrasound stimulated MC3T3-E1 (normalized with control) at frequencies of 0.5, 1.5, and 5 MHz US intensity = 75 mW/cm 2 (SATA), PRF = 1 pulse duration = 200 μs, and exposure = 10 min. Values that are different from control have been indicated by for p

In several previous in vitro researchers have explored the of ultrasound application from a few to several hours [16 ,20 ,26 ,27 ]. To determine if proliferation was dependent on the excitation we varied it for 5, 10, 20, and 30 min. Figure 5 that the ultrasound stimulation cell proliferation for each period tested. However, 10 min and more exposure periods statistically significant increase the control group.

There was no statistically significant among the LIPUS excited at different exposure times of 10, 20, and 30

Figure 5. Change in proliferation of cells ultrasound exposure Change in proliferation of ultrasound-stimulated cells (normalized with at different ultrasound exposure of 5, 10, 20, and 30 min with ultrasound intensity = 75 2 (SATA), frequency = 1.5 MHz, PRF = 1 and pulse duration = 200 μs. Values different from control have been indicated by star for p 0.05.


stimuli play an important in the development and maintenance of healthy Increased mechanical loading on enhances bone formation and bone resorption to increase mass [28 ,29 ]. Bone cells the mechanical forces and produce signals to bring the changes in microenvironment. For example, mechanical generates microstrains and causes flow through lacunar and spaces of the bone.

The resulting shear stress can stimulate proliferation [30 ], contributing to the increase in mass. Ultrasound is a source of mechanical stimulation that can acoustic streaming (unidirectional in an ultrasonic pressure field), microstreaming (rapidly rotating fluid motion around bubbles), and cavitation (formation of gas bubbles in the tissues as the result of vibration) [31 ]. Because of the low intensity and low mechanical index (0.078 for the of 75 mW/cm 2 and 0.488 at 465 mW/cm 2 ) of the used, we do not expect any cavitation [15 ]. Although we did not try to detect cavitation in the setup, the excitation in the range of used here did not generate in water.

Utmost care has taken to avoid the formation of in the medium. In any event, different effects caused by the ultrasound cause fluid flow in the space [12 ] and result in deformation and to osteoblasts. Thus, osteoblasts respond to LIPUS in part due to the mechanisms that are present in of shear forces from flow.

Using BrdU and MTS we found enhanced proliferation at LIPUS intensities with effect at approximately 75 mW/cm 2. optimum LIPUS intensity is of the order reported previously in Reher et al. [23 ] found the optimum to be 100 mW/cm 2 SAPA for osteoblastic lines with a 200-μs at 1 MHz frequency, whereas intensities than approximately 750 mW/cm 2 led to the of collagen and noncollagenous proteins.

Li et al. [24 ] an optimum intensity of 600 mW/cm 2 or 120 mW/cm 2 (SATA) for osteoblast at 100 Hz PRF, 1:4 duty cycle (2 ms period), 1 MHz US frequency, 15 min exposure and 24 cm exposure distance. We also that the US exposure at higher (approximately 465 mW/cm 2 SAPA) detrimental to osteoblasts. In a far-field exposure study, Li et al. [24 ] also complete inhibition of cell at 480 mW/cm 2 SAPA. High-intensity US have been shown to bone formation in animal as well [32 ].

In an attempt to determine the stimulation frequency, we investigated different frequencies: 0.5, and 5 MHz but found no statistically significant in osteoblast cell proliferation them. We also examined the of US stimulation to yield peak proliferation. At the optimum US intensity, we that longer stimulation of 30 min a day was not different from a shorter of 10 min a day, indicating a habituation It has been shown that mass increases if loading is in intermittent bouts, as bone and become less sensitive to mechanical stimulation [33 ].

How ultrasound alters cell remains uncertain. Studies rat bone marrow stromal primary osteoblasts, or intact have shown that markers are increased with mW/cm 2 LIPUS and that increase corresponds to the increases in adhesion kinase (FAK), activation, and MAP kinases [34 -37 ]. When the α 5 β 1 were blocked in primary LIPUS failed to increase PI3 and β-catenin activity, suggesting integrins could be the primary molecule for LIPUS [37 ]. However, studies in mechanotransduction in bone that other signaling could be sensitive to LIPUS to proliferation.

Release of ATP and the resultant signaling increases proliferation, differentiation, and can induce cell in numerous cells types [38 ]. We shown that ATP is released osteoblasts in response to fluid [39 ] and cyclic hydrostatic pressure [40 ] and activation of purinergic receptors is for mechanically induced bone [41 ]. This release of ATP is mediated activation of mechanosensitive and voltage-sensitive channels in osteoblasts [39 ] that also be responsive to LIPUS. For cells, intracellular and extracellular stores and their transport these stores can play an role in their response to stimuli such as LIPUS. calcium channels (VSCCs) been reported to be the key regulators of calcium signaling in osteoblasts [39 ]. In studies, we plan to investigate roles of ultrasound-induced calcium in enhanced osteoblast cell


This study that the application of near-field stimulation is a viable method to osteoblast cell proliferation in culture. It also supports the that US-induced increased proliferation plays an important in bone formation and accelerated healing. Our findings indicate the to better define the optimum of key ultrasound parameters for the maximum in clinical applications.

We have suggested potential mechanisms of enhancement of osteoblast proliferation to be in future research.


6. Setup to measure ultrasound (a) Experimental setup for ultrasound measurement using hydrophone. (b) representation of the concentric annular and the innermost circular region in beam for intensity measurement.

r is the radial coordinate vector on surface S . ρ is the medium density, and c is the of the sound in medium. p m is half of the peak-to-peak pressure at any position r . The average ultrasound intensity, I PA ( r ) and the ultrasound intensity, I TA ( r ) were as the following:

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