FASEB J. Cell Migration Consortium
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bacabac, R. G.
Right arrow Articles by Klein-Nulend, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bacabac, R. G.
Right arrow Articles by Klein-Nulend, J.
(The FASEB Journal. 2006;20:858-864.)
© 2006 FASEB

Bone cell responses to high-frequency vibration stress: does the nucleus oscillate within the cytoplasm?

Rommel G. Bacabac*, Theo H. Smit{dagger}, Jack J. W. A. Van Loon*,{ddagger}, Behrouz Zandieh Doulabi*, Marco Helder* and Jenneke Klein-Nulend*,1

* Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam-Universiteit van Amsterdam and Vrije Universiteit, Amsterdam, The Netherlands;

{dagger} Department of Physics and Medical Technology, VU University Medical Center, Amsterdam, The Netherlands; and

{ddagger} Dutch Experiment Support Center, Vrije Universiteit, Amsterdam, The Netherlands

1Correspondence: ACTA-Universiteit van Amsterdam and Vrije Universiteit, Department Oral Cell Biology, Van der Boechorststraat 7, 1081-BT Amsterdam, The Netherlands. E-mail: j.kleinnulend{at}vumc.nl.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mechanosensing by cells directs changes in bone mass and structure in response to the challenges of mechanical loading. Low-amplitude, high-frequency loading stimulates bone growth by enhancing bone formation and inhibiting disuse osteoporosis. However, how bone cells sense vibration stress is unknown. Hence, we investigated bone cell responses to vibration stress at a wide frequency range (5–100 Hz). We used NO and prostaglandin E2 (PGE2) release, and COX-2 mRNA expression as parameters for bone cell response since these molecules regulate bone adaptation to mechanical loading. NO release positively correlated whereas PGE2 release negatively correlated to the maximum acceleration rate of the vibration stress. COX-2 mRNA expression increased in a frequency-dependent manner, which relates to increased NO release at high frequencies, confirming our previous results. The negatively correlated release of NO and PGE2 suggests that these signaling molecules play different roles in bone adaptation to high-frequency loading. The maximum acceleration rate is proportional to {omega}3 (frequency={omega}/2{pi}), which is commensurate with the Stokes-Einstein relation for modeling cell nucleus motion within the cytoplasm due to vibration stress. Correlations of NO and PGE2 with the maximum acceleration rate then relate to nucleus oscillations, providing a physical basis for cellular mechanosensing of high-frequency loading.—Bacabac, R. G., Smit, T. H., Van Loon, J. J. W. A., Doulabi, B. Z., Helder, M., Klein-Nulend, J. Bone cell responses to high-frequency vibration stress: does the nucleus oscillate within the cytoplasm?


Key Words: osteoblasts • mechanical loading • NO • prostaglandin • COX-2


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
BONE IS AN OBVIOUS BIOLOGICAL SYSTEM that exhibits the interplay of mechanical stress and adaptive response at both tissue and cellular levels (1 2 3) . Bones adapt their mass and structure in response to the demands of mechanical loading (4) . Furthermore, it has been suggested that the rate rather than the magnitude alone of the applied loading stimulus correlates to bone formation in vivo (5 ,6) . This suggests that both the frequency and amplitude of applied stresses are important for the osteogenic response of bone.

We recently found in another study that the NO production by MC3T3-E1 cells was linearly dependent on the rate of fluid shear stress, which depended on both the amplitude and frequency of stress (7) . In that study, however, the applied fluid shear stress was only up to a maximum of 9 Hz. NO and prostaglandin E2 production are essential for the induction of new bone formation in response to mechanical loading in vivo (8 9 10) . The constitutional, endothelial form of NOS (eNOS), one of the three NOS enzyme isoforms responsible for the synthesis of NO, is prominently expressed in osteocytes and up-regulated by mechanical loading (11) . Blocking of either cyclooxygenase-2 (COX-2), the key enzyme for mechanically induced PG production, or NOS could prevent mechanically induced bone formation (8 ,12 ,13) . Since osteoblasts respond to fluid shear stress as osteocytes, although to a lesser extent, osteoblasts could provide a practical model for osteocyte response to stress.

Using animal models, low magnitude (<10 µ{epsilon}), high-frequency (10–100 Hz) mechanical stimuli have been shown to be capable of stimulating bone growth by doubling bone formation rates and inhibiting disuse osteoporosis (14) . Thus, it would seem that higher frequencies are also stimulatory to bone cells. Much is unknown, however, about how high-frequency loading might permeate bone despite the presence of soft tissues. We showed earlier that external loading from exercise could involve frequencies reaching 9 Hz (7) . Frequencies experienced by cells beyond 10 Hz might occur at much lower amplitudes considering the damping effects of soft tissue. Regardless of frequency range, the applied rate of loading, which is dependent on both frequency and magnitude, seems to be a decisive factor in bone formation and maintenance.

Paradigms for understanding mechanosensing by cells include models for the mechanical properties of the cytoplasm as predominantly a continuum (15) or as composed of linked polymers that transfer forces through the cytoskeleton to the nucleus (16) . Cellular activation by mechanical loads in general or vibration in particular, leading to a biochemical cascade requires some form of cellular deformation as a mechanism for sensing forces. Although there is evidence that bone cells respond to dynamic loading, how bone cells might sense mechanical vibration is unknown. Therefore, we studied the response of bone cells to mechanical vibration over a wide frequency range (5–100 Hz) at different magnitudes. We tested whether vibration stress applied with varying frequencies and amplitudes affects NO and prostaglandin E2 (PGE2) production, and mRNA expression for COX-2 by MC3T3-E1 osteoblastic cells. Based on the results, we empirically derived a model to propose an alternative mechanism by which cells might sense high-frequency mechanical loading.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bone cell cultures
MC3T3-E1 cells (kindly provided by Dr. Kumegawa, Mekai University School of Dentistry, Sakado, Saitama, Japan; 17 ) were cultured up to near-confluency in 75 cm2 cell culture flasks (Nunc, Roskilde, Denmark) using {alpha}-modified Eagle’s medium ({alpha}-MEM; Life Technologies, Inc., Paisley, UK) supplemented with 10% FBS (FBS; Life Technologies, Inc.), ascorbate (50 µg/ml; Merck, Darmstadt, Germany), ß-glycerophosphate disodium salt hydrate (10 mM; Sigma, St. Louis, MO, USA), L-glutamine (300 µg/ml; Merck), gentamycine (50 µg/ml; Life Technologies, Inc.), fungizone (1.25 µg/ml; Life Technologies, Inc.), at 37°C with 5% CO2 in air. Cells were then harvested using 0.25% trypsin (Difco Laboratories, Detroit, MI, USA) and 0.1% EDTA (Sigma) in PBS and seeded at 0.7 x 105 cells per well in a 24-well plate, then incubated overnight in {alpha}-MEM with 10% FBS to promote cell attachment prior to vibration stress treatment as described below.

Application of vibration stress
For vibration stress treatment, the culture medium was changed to CO2-independent medium (Life Technologies, Inc., Grand Island, NY, USA) with 2% FBS and incubated for 5 min in the presence of mechanical vibration at varying frequencies and amplitudes (see Table 1 ). The applied frequency range (5–100 Hz) was based on various studies that reported the osteogenic benefits of dynamic mechanical loading (18 19 20 21) . The applied vibration stress amplitudes were chosen so that the maximum acceleration rate increased with increasing frequency whereas the maximum velocity did not. Vibration stress was implemented on attached cells by sinusoidal displacement of the 24-well plate along the cells’ plane of attachment using a voltage controlled linear actuator (Fig. 1 A). Fluid movement across the monolayer surface was prevented by ensuring that the wells were completely filled with fluid and by tightly sealing them. It was further required not to have air bubbles in the filled and tightly sealed wells. Conditioned medium was sampled after 5 min of vibration stress treatment to measure accumulated NO in medium produced by MC3T3-E1 cells.


View this table:
[in this window]
[in a new window]
  		Table 1. Data of applied vibration stressa


Figure 1
View larger version (44K):
[in this window]
[in a new window]
  		Figure 1. Vibration stress application. A) Cells were seeded onto the bottom surface of the 24-well plate (as described in Materials and Methods). Sinusoidal motion is along the x axis (arrow). B) Simplified model of a cell with an approximately rigid spherical nucleus compared to its viscoelastic cell body.

Nuclear oscillations induced by vibration stress
Bone cells were cultured as a monolayer onto the bottom surface of cylindrical wells using a 24-well plate configuration. Vibration stress was induced by oscillating the plate along the horizontal axis using a voltage controlled linear actuator at a wide range of frequencies and amplitudes. We take a minimalist approach to understand how the cells are possibly receiving stress by translational oscillation. We consider the nucleus to be spherical and embedded in the viscoelastic medium of the cell body. The source of mechanical stimulus is then attributed to the possible motion of the cellular nucleus (modeled to be a sphere; Fig. 1B ) inside the cell. Based on the Generalized Stokes-Einstein relation, we estimated the displacement of the nucleus in the cell xn({omega}), modeled as a rigid sphere compared to the cytoplasm, as proportional to the applied force F:

Formula 1(1)
where G({omega}) is here considered to be the elastic modulus of the cell cytoplasm (on the order of 100 Pa) (18) and the acting force is due to the mass of the nucleus {rho}nVn, with a volume Vn, and radius Rn, considering a relative acceleration of the nucleus with respect to the cell body. In this approximation, we neglect the time dependence of the modulus, so, G({omega}) -> Go. The nucleus is considered to be 4-fold more rigid than the cytoplasm (e.g., the elastic modulus of the nucleus is at the order of 400 Pa) (19) . The force acting on the nucleus is due to the acceleration it experiences due to the applied sinusoidal vibration, with amplitude xo and frequency = {omega}/(2{pi}):

Formula 2(2)
Thus, the absolute maximum force due to the nucleus Fmax, is related to the maximum acceleration of the entire plate:

Formula 3(3)
where xo{omega}2 is the maximum acceleration of the plate. The maximum velocity of the plate is related to nuclear velocity vn, which in turn is related to {omega}:

Formula 4(4)
where {rho}'n is the density difference between the cell nucleus and the surrounding cytoplasm. Here, we approximate the elastic modulus to be constant Go. The maximum rate of acceleration by the plate is related to the rate of acceleration ran by the nucleus at {omega}:

Formula 5(5)
Using this in vitro system, for a non-negligible density difference between the cell nucleus and the surrounding cytoplasm, we are able to mechanically stimulate cells by inducing body forces by vibration stress. Hence, by correlating the amount of released signaling molecules to the maximum velocity or rate of acceleration of the plate we characterized the effect of vibration stress to bone cell mechansosensitivity.

Nitric oxide and prostaglandin E2 determination
The conditioned medium was assayed for NO, which was measured as nitrite (NO2–) accumulation in the conditioned medium, using Griess reagent (1% sulfanilamide, 0.1% naphtylethelene-diamine-dihydrochloride, and 2.5 M H3PO4) (24) . The absorbance was measured at 540 nm. NO concentrations were determined using a standard curve derived from known concentrations of NaNO2 in nonconditioned culture medium. PGE2 was measured in conditioned medium by an enzyme immunoassay (EIA) system (Amersham, Buckinghamshire, UK) using an antibody raised against mouse PGE2. The detection limit was 16 pg/ml. Absorbance was measured at 450 nm.

Total DNA, RNA, and total protein determination
DNA, RNA and protein were isolated from the bone cell cultures using Trizol reagent according to the manufacturer’s instructions. The amount of protein was determined using a bicinchoninic acid (BCA) protein Assay Reagent Kit (Pierce, Rockford, IL, USA), the absorbance was read at 570 nm. The RNA and DNA content were determined by measuring absorbance in water at 260 nm using an Ultraspec III spectrophotometer (Amersham).

RNA isolation and reverse transcription
Total RNA from the cells was isolated using Trizol reagent with one modification; 5 µg of glycogen (Roche Diagnostics, Mannheim, Germany) was added to RNA and isopropanol solution prior to the centrifuge step to increase RNA yield. Total RNA concentration was quantified spectrophotometrically. cDNA synthesis was performed using 0.5–1 µg total RNA in a 20 µl reaction mix consisting of 5 units of Transcriptor Reverse Transcriptase according to the manufacturer’s instructions (Roche Diagnostics) with 0.08 A260 units random primers (Roche Diagnostics), 1 mM of each dNTP (Invitrogen, San Diego, CA, USA), and Transcriptor RT reaction buffer. cDNA was diluted five times and stored at –80°C prior to real-time polymerase chain reaction (PCR).

Real-time PCR
Real-time PCR reactions were performed using the SYBRGreen reaction kit according to the manufacturer’s instructions (Roche Diagnostics) in a LightCycler (Roche Diagnostics). cDNA (2 µl each) was diluted to a vol of 20 µl with PCR mix (Light Cycler DNA Master Fast start plus Kit, Roche Diagnostics) containing a final concentration of 0.2 pmol of primers. Relative housekeeping gene expression (18S; whose expression was not subjected to time and or treatment-related variations) and relative target gene expression (COX-2) were determined. Primers (Invitrogen) used for real-time PCR listed in Table 2 were designed using Clone manager suite software program version 6 (Scientific & Educational Software, Cary, NC, USA); the amplified PCR fragment had an extension over at least one exon border except for 18S, whose gene is encoded only by one exon. Values of relative target gene expression were normalized for relative 18S housekeeping gene expression.


View this table:
[in this window]
[in a new window]
  		Table 2. Primers used for real time PCR

Real-time PCR data analyses
With the Light Cycler software (version 2), the crossing points were assessed and plotted vs. the serial dilution of known concentrations of the standards derived from each gene. PCR efficiency (E) was obtained by the formula: E=10–1/slope and the data were used if and only if the PCR efficiency was calculated between 1.85 and 2.0.

Statistics
Data were pooled from the results of at least five experiments for each vibration stress regime. The effects of vibration stress regimes were analyzed with the nonparametric Wilcoxon signed rank sum test of the S-Plus 2000 package (release 1). Differences between total DNA, RNA, and protein expressions for different vibration stress regimes were tested using one-way ANOVA. The relation between the release of NO or PGE2 against the peak rate acceleration by the vibration stress, and against each other, was characterized by linear regression. Significant differences were considered at a P value < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Application of vibration stress for 5 min to the MC3T3-E1 cells did not result in visible changes in cell shape or alignment of the cells to any orientation (data not shown). No cells were removed by any of the vibration stress regimes, as assessed by visually inspecting the cultures before and after vibration stress treatment, and by measuring the total amount of DNA, RNA, and protein (Table 3 ).


View this table:
[in this window]
[in a new window]
  		Table 3. Total DNA, RNA, and proteina

The rapid response to vibration stress by bone cells was measured as the accumulation of NO released in the medium after 5 min of treatment with the different vibration stress regimes (Table 1) . NO production in rapid response to treatment with vibration stress linearly correlated with the applied maximum acceleration rate, (Fig. 2 A; P<0.05, r=0.95). However, this response did not correlate linearly to the applied maximum velocity (Fig. 2 B). The highest response was due to the 100 Hz regime (see Table 1 ), which was significantly larger than the response to 5 Hz and 60 Hz regimes (Fig. 2A, B , P<0.03).


Figure 2
View larger version (15K):
[in this window]
[in a new window]
  		Figure 2. Effect of vibration stress on NO production by bone cells. A) Bone cells respond in positive correlation to the applied maximum acceleration rate (max. acc. rate) of vibration stress immediately after 5 min (P<0.05). B) The response to mechanical vibration does not correlate linearly to the applied maximum velocity (max. velocity). The response to vibration stress of 100 Hz was significantly larger than the response to 5 Hz and 60 Hz (*P<0.03). See Table 1 for the applied vibration stress regimes. Values are mean total amount ± SE.

The late response to vibration stress by bone cells was measured as the accumulation of PGE2 released in the medium after 5 min of treatment with the different vibration stress regimes (Table 1) . PGE2 was assayed in the conditioned medium after 30 min of postincubation at 37°C without vibration stress. The PGE2 released by bone cells in response to vibration stress negatively correlated with the applied peak acceleration rate (Fig. 3 A, P<0.006). However, the response to mechanical vibration did not correlate linearly to the applied maximum velocity (Fig. 3B ). The highest response to vibration stress was due to the 5 Hz regime (see Table 1 ), which was significantly larger than the response to 100 Hz (Fig. 3 , P<0.003).


Figure 3
View larger version (13K):
[in this window]
[in a new window]
  		Figure 3. Effect of vibration stress on PGE2 production by bone cells. A) Bone cells respond in negative correlation to the applied maximum acceleration rate (max. acc. rate) of vibration stress (P<0.006). B) The response to mechanical vibration does not correlate linearly to the applied maximum velocity (max. velocity). The response to vibration stress of 5 Hz was significantly larger than the response to 100 Hz (*P<0.003). PGE2 was assayed from medium harvested after 30 min of postincubation subsequent to 5 min of vibration stress. See Table 1 for the applied vibration stress regimes. Values are mean total amount ± SE.

To investigate whether the production of NO and PGE2 was related, the measured release of these signaling molecules to corresponding increasing frequencies of the vibration stress were correlated by linear regression. The NO and PGE2 released by bone cells in response to vibration stress at varying frequencies, were found to be negatively correlated (Fig. 4 , P<0.013, r = 0.99).


Figure 4
View larger version (13K):
[in this window]
[in a new window]
  		Figure 4. Negatively correlated release of NO and PGE2 by bone cells in response to vibration stress. Values are mean total amount ± SE.

To investigate the long-term effect of vibration stress on bone cells, the mRNA expression for COX-2 was measured in relation to PGE2 release, at 30 min subsequent to 5 min vibration stress. The bone cells were harvested for COX-2 mRNA expression after 2.5 h of postincubation subsequent to 5 min of vibration stress. The mRNA expression for COX-2 was found to be up-regulated the most in response to 100 Hz vibration stress, which was significantly higher than the response to all the other regimes (Fig. 5 ). The mRNA expression for COX-2 in response to 100 Hz was 2-fold higher than the response to 5 and 30 Hz. The mRNA expression for COX-2 in response to 60 Hz vibration was found to be 1.5-fold higher than the response to the 30 Hz regime (Fig. 5 , P<0.047).


Figure 5
View larger version (37K):
[in this window]
[in a new window]
  		Figure 5. Vibration stress up-regulates mRNA for COX-2 expression by bone cells. mRNA expression for COX-2, was 2-fold regulated in response to 100 Hz vibration stress (b, higher than the response to 5 Hz, P < 0.028, higher than 30 Hz, P < 0.011, and higher than 60 Hz, P < 0.05). mRNA expression for COX-2, in response to 60 Hz vibration was 1.5-fold higher than the response to 30 Hz (aP<0.047). Cells were harvested for measuring mRNA expression for COX-2 after 2 h of postincubation subsequent to 5 min of vibration stress. Values are relative to housekeeping gene expression (18S). Values are mean total amount ± SE.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The membrane-bound soluble enzyme called endothelial cell NOS (ecNOS or NOS III, one of three members of the NOS family) is involved in the NO response of bone cells to mechanical stress (25 , 26) . NO production is an essential step for mechanical loading-induced bone formation as observed in rats in vivo (8) . We have found earlier that mRNA expression for ecNOS in bone cells (osteocytes isolated from chicken calvaria) is up-regulated in response to fluid shear stress (27) . Prostaglandins are generated by the release of arachidonic acid from phospholipids in the cell membrane, followed by conversion of arachidonic acid into prostaglandin G2 and subsequently H2. Prostaglandin H2 is further isomerized to the biological active prostanoids, prostaglandin E2 (PGE2). The in vivo acute prostaglandin production by loading stimulated bone cells seems to be more important than the sustained prostaglandin release (13 , 28) . Hence, NO and PGE2 production are meaningful parameters for measuring bone cell activation. Furthermore, our previous studies showed that both NO and PGE2 production by bone cells increase until a plateau is reached before 1 h and that the highest response occurs within 5 min of mechanical stimulation (7 , 29) . Thus we investigated the rapid response to vibration stress.

NO and PGE2 production by bone cells linearly correlated with opposing signs, with the maximum applied acceleration rate, which is third order or cubic in frequency dimension (see Table 1 ). This suggests that the bone cell response to vibration stress treatment is highly dependent on the applied frequency of loading. The response, however, did not linearly correlate with the applied maximum velocity, which is a joint effect of the applied amplitude and frequency, both at first order. The most likely effect of the vibration stress might be the movement of the cell nucleus through the cell body induced by the acceleration of the plate. NO release and PGE2 release correlated with the rate of acceleration, which is proportional to the rate of the force on the nucleus. The rate of force is proportional to the rate of stress experienced by the cell through nuclear motion directly. This supports our earlier finding that bone cell response is linear to the applied fluid shear stress rate (7) . However, in this study we find for the first time correlations that suggest nuclear oscillation as the physical mechanism responsible for vibration stress response by bone cells. In our model for the mechanical effect of vibration stress, the acceleration rate corresponds to the rate of the force acting on the cell nucleus. Since stress is the amount of force about a cross-sectional area, the consistent signature for bone cell response to mechanical loading seems to be linear stress rate dependence. Fluid shear stress, which is contact stress primarily acting on the cell membrane, might be linearly rate dependent only for low frequencies (<10 Hz) since fluid flow might attenuate at high frequencies (>10 Hz). Thus, our model for the motion of the cell nucleus for vibration stress at high frequencies involving body forces explains the different way how bone cells might sense loading at high frequencies (>10 Hz) or at high impact loading.

The anticorrelation of NO and PGE2 release is closely linked to the rate of stress experienced by the bone cells at a wide range of frequencies induced by vibration. These opposing trends of molecular release suggest different roles for NO and PGE2 in regulating the mechanical adaptation of bone. It has been shown in vitro that osteoclasts migrate away from NO (30 , 31) . PGE2 increases mRNA levels of osteoprotegerin ligand (OPG-L)/osteoclast differentiation factor (ODF) from osteoblastic lineage cells. OPG-L/ODF stimulates osteoclast differentiation and activity, further inhibiting osteoclast apoptosis (30) . NO and PGE2 regulate the activity of osteoclasts (32) . Since at higher frequencies MC3T3-E1 tends to increase NO release but decrease PGE2 release, this suggests an acute tendency for bone cells to oppose the presence of osteoclasts at high-frequency loading. Since materials of higher density are expected to favor force transfer at high frequencies, in denser bone regions osteoclasts are driven away by increased NO levels and are not recruited due to decreased PGE2 levels. With fewer osteoclasts, denser bone is not degraded but maintained. It is possible that, in vivo, osteoblasts enhance their rejection of osteoclasts when stimulated with high frequencies. This might in turn stimulate osteoblast activity in the absence of osteoclasts. Indirectly, osteoclasts are possibly recruited onto regions not experiencing high-frequency loading where increased PGE2 levels might occur. It is possible that in bone, regions that are less dense (hence not responsive to high frequencies) are more prone to being degraded by osteoclasts. Furthermore, since the release of signaling molecules correlates strongly to {omega}3, the absence of high strains in normal daily activities does not necessarily correspond to bone loss. High frequencies in the spectrum of loading are expected during movements of high impact activity, as in exercise and sports, which have been shown to be beneficial for bone health (33 34 35) . It would seem that the loss in strain amplitude can be compensated for by the application of higher frequency loading.

Our results might also provide further insight on the effects of a broad band of frequencies on bone formation (6 , 36) . Our model for predicting the motion of the nucleus through the cell body during vibration stress application might relate to the reported osteogenic benefits of vibration-related stimulation. For example, the use of low-intensity ultrasound has been shown to stimulate a higher occurrence of spinal fusion compared to cases without low-intensity ultrasound application (37) . Much is unknown about the mechanisms responsible for the transfer of forces in bone as imparted by loading of minute magnitudes at high frequencies. It is possible that high-frequency vibration attenuate through soft tissue surrounding the bone mineralized matrix. However, it is also likely that high frequencies are able to survive in the denser mineralized matrix of bone. In this case our model for the motion of the cell nucleus is a possible mechanism for bone cells to sense high-frequency loading.

The increased mRNA expression for COX-2 in relation to increasing frequencies of vibration stress suggests a memory response for high-frequency loading. In this study the vibration stress was applied for only 5 min, however, after 2.5 h of postincubation without stress, up-regulation of mRNA expression for COX-2 occurred. Despite a decreased PGE2 release at high frequencies, after 30 min of postincubation, subsequent to the 5 min vibration stress, bone cells maintain the capacity for producing PGE2. This suggests that bone cells compensate for short-term PGE2 production (after 30 min) by increasing mRNA levels for COX-2 for a possible delayed response (or memory effect).

Bone cells do respond to high-frequency vibration stress (i.e., 100 Hz), although fluid shear stresses in vivo might involve lower frequencies (7) . In this study we did not consider in detail the physical differences of the effects of fluid shear stress or vibration stress on bone cell deformation. We performed our studies on 2-dimensional (2-D) cultures, but since the nuclear oscillation was predicted to be in-phase and parallel to the applied translational vibration, nuclear oscillation in 2-D or 3-D cultures are fundamentally the same. However complicated the transfer of forces at the cellular concentration might be in terms of fluid shear stress or vibration stress, our results imply that the joint effect of the frequency and amplitude of loading might play similar roles for different types of stresses on bone cells. Our results suggest that the joint effect of the frequency (at third order) and amplitude (at first order) of loading correlate to the biochemical response of bone cells that contribute to sustained bone metabolism. Furthermore, this response might involve mechanisms that contribute to a specific behavior of bone cells in response to vibration stress enabling recognition of high-frequency loading.


   ACKNOWLEDGMENTS
 
The authors would like to thank R. M. Heethaar for critically reading the manuscript, and C. M. Semeins and J. M. A. de Blieck-Hogervorst for their technical assistance. The Space Research Organization of the Netherlands supported the work of J. J. W. A. van Loon (DESC, MG-057, and SRON grant MG-055) and R. G. Bacabac (SRON grant MG-055) who also received financial assistance from the Netherlands Organization For International Cooperation In Higher Education (Physics Development Project PHL-146).

Received for publication September 13, 2005. Accepted for publication January 4, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Burger, E. H., Klein-Nulend, J. (1999) Mechanotransduction in boneFormula 5ole of the lacuno-canalicular network. FASEB J. 13,S101-S112
  2. Frost, H. M. (2003) Bone’s mechanostat: a 2003 update. Anat Rec. A Discov. Mol. Cell Evol. Biol. 275,1081-1101[CrossRef][Medline]
  3. Huiskes, R., Ruimerman, R., van Lenthe, G. H., Janssen, J. D. (2000) Effects of mechanical forces on maintenance and adaptation of form in trabecular bone. Nature 405,704-706[CrossRef][Medline]
  4. Wolff, J. (1892) Gesetz der transformation der knochen Hirschwald Berlin, Germany.
  5. Turner, C. H., Owan, I., Takano, Y. (1995) Mechanotransduction in bone: role of strain rate. Am. J. Physiol. 269,E438-E442
  6. Tanaka, S. M., Li, J., Duncan, R. L., Yokota, H., Burr, D. B., Turner, C. H. (2003) Effects of broad-frequency vibration on cultured osteoblasts. J Biomech. 36,73-80[CrossRef][Medline]
  7. Bacabac, R. G., Smit, T. H., Mullender, M. G., Dijcks, S. J., Van Loon, J. J. W. A., Klein-Nulend, J. (2004) Nitric oxide production by bone cells is fluid shear stress rate dependent. Biochem. Biophys. Res. Commun. 315,823-829[CrossRef][Medline]
  8. Turner, C. H., Takano, Y., Owan, I., Murrell, G. A. (1996) Nitric oxide inhibitor L-NAME suppresses mechanically induced bone formation in rats. Am. J. Physiol. 270,E634-E639
  9. Pitsillides, A. A., Rawlinson, S. C., Suswillo, R. F., Bourrin, S., Zaman, G., Lanyon, L. E. (1995) Mechanical strain-induced NO production by bone cells: a possible role in adaptive bone (re)modeling?. FASEB J. 9,1614-1622[Abstract]
  10. Chow, J. W. M., Chambers, T. J. (1994) Indomethacin has distinct early and late actions on bone-formation induced by mechanical stimulation. Am. J. Physiol. 267,E287-E292
  11. Jones, D. B., Nolte, H., Scholubbers, J. G., Turner, E., Veltel, D. (1991) Biochemical signal transduction of mechanical strain in osteoblast-like cells. Biomaterial 12,101-110
  12. Forwood, M. R. (1996) Inducible cyclo-oxygenase (COX-2) mediates the induction of bone formation by mechanical loading in vivo. J. Bone Miner. Res. 11,1688-1693[Medline]
  13. Li, J., Burr, D. B., Turner, C. H. (2002) Suppression of prostaglandin synthesis with NS-398 has different effects on endocortical and periosteal bone formation induced by mechanical loading. Calcif. Tissue Internat. 70,320-329[CrossRef][Medline]
  14. Rubin, C. T., Sommerfeldt, D. W., Judex, S., Qin, Y. X. (2001) Inhibition of osteopenia by low magnitude, high-frequency mechanical stimuli. Drug Discov. Today 6,848-858[CrossRef][Medline]
  15. Karcher, H., Lammerding, J., Huang, H. D., Lee, R. T., Kamm, R. D., Kaazempur-Mofrad, M. R. (2003) A three-dimensional viscoelastic model for cell deformation with experimental verification. Biophys. J. 85,3336-3349[Abstract/Free Full Text]
  16. Wang, N., Butler, J. P., Ingber, D. E. (1993) Mechanotransduction across the cell surface and through the cytoskeleton. Science 260,1124-1127[Abstract/Free Full Text]
  17. Kodama, H., Amagai, Y., Sudo, H., Kasai, S., Yamamoto, S. (1981) Establishment of a clonal osteogenic cell line from newborn mouse calvaria. Japanese J. Oral Biol. 23,899-901
  18. Warden, S. J., Turner, C. H. (2004) Mechanotransduction in the cortical bone is most efficient at loading frequencies of 5–10 Hz. Bone 34,261-270[Medline]
  19. Rubin, C., Turner, A. S., Bain, S., Mallinckrodt, C., McLeod, K. (2001) Anabolism. Low mechanical signals strengthen long bones. Nature 412,603-604[Medline]
  20. Midura, R. J., Dillman, C. J., Grabiner, M. D. (2005) Low amplitude, high-frequency strains imposed by electrically stimulated skeletal muscle retards the development of osteopenia in the tibiae of hindlimb suspended rats. Med. Eng. Phys. 27,285-293[CrossRef][Medline]
  21. Rubin, C., Xu, G., Judex, S. (2001) The anabolic activity of bone tissue, suppressed by disuse, is normalized by brief exposure to extremely low-magnitude mechanical stimuli. FASEB J. 15,2225-2229[Abstract/Free Full Text]
  22. Kamm, R. D., McVittie, A. K., Bathe, M. (2000) On the role of continuum models in mechanobiology. ASME Internat. Cong.-Mech. Biol. 242,1-9
  23. Guilak, F., Tedrow, J. R., Burgkart, R. (2000) Viscoelastic properties of the cell nucleus. Biochem. Biophys. Res. Commun. 269,781-786[CrossRef][Medline]
  24. Nagano, T. (1999) Practical methods for detection of nitric oxide. Luminescence 14,283-290[CrossRef][Medline]
  25. Klein-Nulend, J., Helfrich, M. H., Sterck, J. G., MacPherson, H., Joldersma, M., Ralston, S. H., Semeins, C. M., Burger, E. H. (1998) Nitric oxide response to shear stress by human bone cell cultures is endothelial nitric oxide synthase dependent. Biochem. Biophys. Res. Commun. 250,108-114[CrossRef][Medline]
  26. Fox, S. W., Chambers, T. J., Chow, J. W. M. (1996) Nitric oxide is an early mediator of the increase in bone formation by mechanical stimulation. Am. J. Physiol. 33,E955-E960
  27. Klein-Nulend, J., Semeins, C. M., Ajubi, N. E., Nijweide, P. J., Burger, E. H. (1995) Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblastsFormula 5orrelation with prostaglandin upregulation. Biochem. Biophys. Res. Commun. 217,640-648[CrossRef][Medline]
  28. Chow, J. W. M., Chambers, T. J. (1994) Indomethacin has distinct early and late actions on bone-formation induced by mechanical stimulation. Am. J. Physiol. 267,E287-E292
  29. Bakker, A. D., Soejima, K., Klein-Nulend, J., Burger, E. H. (2001) The production of nitric oxide and prostaglandin E2 by primary bone cells is shear stress dependent. J. Biomech. 34,671-677[CrossRef][Medline]
  30. Webster, S. S. J. (2001) Integrated bone tissue physiology: anatomy and physiology. Stephen, C. C. eds. Bone Mechanics Handbook ,1-1-1–68 CRC Press Boca Raton.
  31. Mancini, L., Moradi-Bidhendi, N., Brandi, M. L., MacIntyre, I. (1998) Nitric oxide superoxide and peroxynitrite modulate osteoclast activity. Biochem. Biophys. Res. Commun. 243,785-790[CrossRef][Medline]
  32. Buttery, L. D. K., Hukkanen, M. V. J., O’Donnel, A., Polak, J. M., Hughes, F. J. (1995) Nitric oxide dependent and independent induction of prostaglandin synthesis in osteoblasts. Bone 17,560
  33. Hara, S., Yanagi, H., Amagai, H., Endoh, K., Tsuchiya, S., Tomura, S. (2001) Effect of physical activity during teenage years, based on type of sport and duration of exercise, on bone mineral density of young, premenopausal Japanese women. Calcif. Tissue Int. 68,23-30[Medline]
  34. Honda, A., Sogo, N., Nagasawa, S., Shimizu, T., Umemura, Y. (2003) High-impact exercise strengthens bone in osteopenic ovariectomized rats with the same outcome as Sham rats. J. Appl. Physiol. 95,1032-1037[Abstract/Free Full Text]
  35. Taaffe, D. R., Robinson, T. L., Snow, C. M., Marcus, R. (1997) High-impact exercise promotes bone gain in well-trained female athletes. J. Bone Miner. Res. 12,255-260[CrossRef][Medline]
  36. Rubin, C. T., Lanyon, L. E. (1984) Regulation of bone formation by applied dynamic loads. J. Bone Joint Surg. Am. 66,397-402[Abstract/Free Full Text]
  37. Cook, S. D., Salkeld, S. L., Mse, Patron, L. P., Ryaby, J. P., Whitecloud, T. S. (2001) Low-intensity pulsed ultrasound improves spinal fusion. Spine J 1,246-254[CrossRef][Medline]



This article has been cited by other articles:


Home page
 J. Appl. Physiol.Home page
L. Xie, C. Rubin, and S. Judex
Enhancement of the adolescent murine musculoskeletal system using low-level mechanical vibrations
J Appl Physiol, April 1, 2008; 104(4): 1056 - 1062.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Bacabac, R. G.
Right arrow Articles by Klein-Nulend, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Bacabac, R. G.
Right arrow Articles by Klein-Nulend, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS