Calcif Tissue Int (2008) 82:316–326

DOI 10.1007/s00223-008-9115-8

Ovariectomy Sensitizes Rat Cortical Bone to Whole-Body

Vibration
Alessandro Rubinacci Æ Massimo Marenzana Æ Francesco Cavani Æ
Federica Colasante Æ Isabella Villa Æ Johannes Willnecker Æ Gian Luigi Moro Æ
Luigi Paolo Spreafico Æ Marzia Ferretti Æ Francesca Guidobono Æ
Gastone Marotti

Received: 26 June 2007 / Accepted: 4 February 2008 / Published online: 1 April 2008

Springer Science+Business Media, LLC 2008

Abstract This study was designed to determine the In the OVX-V group, vibration induced a significant
modulatory effect of estrogen on mechanical stimulation in increase compared to the OVX group of the cortical and
bone. Trabecular and cortical bone compartments of medullary areas (P \ 0.01) and of the periosteal
ovariectomized rats exposed to whole-body vibration of (P \ 0.01) and endosteal (P \ 0.05) perimeters at the 3 g
different amplitudes were evaluated by peripheral quanti- vibration. The strain strength index increased in the OVX-V
tative computed tomographic (pQCT) analysis and group significantly (P \ 0.01) at the higher vibration. The
histomorphometry and compared to controls not exposed to results showed that low-amplitude, high-frequency whole-
vibration. Rats underwent whole-body vibration (20 min- body vibration is anabolic to bone in OVX animals. The
utes/day, 5 days/week) on a vibration platform for osteogenic potential is limited to the modeling of the bone
2 months. The control rats were placed on the platform cortex and depends on the amplitude of the vibration.
without vibration for the same time. We divided rats into
six groups: a sham control (SHAM); a sham vibrated Keywords Bone density technology
pQCT

(SHAM-V) at 30 Hz, 0.6 g; a SHAM-V at 30 Hz, 3g; an Mechanical loading
Exercise
Mechanotransduction
ovariectomized control (OVX); an ovariectomized vibrated
(OVX-V) at 30 Hz, 0.6 g; and an OVX-V at 30 Hz, 3g. In
vivo, pQCT analyses of the tibiae were performed at the The mechanical signal that modulates bone metabolism
start of the experiment and after 4 and 8 weeks. After includes high-magnitude strains, at frequencies ranging
8 weeks the tibiae were excised for histomorphometric and 0.5–2 Hz, and strains of low magnitude at high frequencies
for in vitro pQCT analyses. In the SHAM-V group, reaching 30 Hz. Such strains repeatedly impact bone during
vibration had no effect upon the different bone parameters. various activities, such as standing and muscle contraction.
The relevance of these omnipresent, low-level signals was
originally characterized by Rubin and coworkers [1]. They
A. Rubinacci (&)
M. Marenzana
F. Colasante
I. Villa
demonstrated that low-amplitude mechanical signals,
G. L. Moro
L. P. Spreafico applied by use of a ‘‘vibration platform,’’ are associated
Bone Metabolic Unit, Scientific Institute San Raffaele, with modulation of bone mass and morphology by acting
Via Olgettina 60, Milan 20132, Italy
e-mail: a.rubinacci@hsr.it on both phases of bone (re)modeling, i.e., resorption and
formation [1–3]. Such results confirmed the pioneering
F. Cavani
M. Ferretti
G. Marotti observation that chronic vibration tends to increase bone
Department of Anatomy and Histology, University of Modena stiffness and microhardness [4]. Recent studies have
and Reggio Emilia, Modena, Italy
further characterized the impact of low-amplitude, high-
J. Willnecker frequency vibration on bone. It can stimulate new trabec-
Stratec Medizintechnik, Pforzheim, Germany ular bone formation in sheep [5], expression of osteoblastic
genes associated with bone formation and remodeling in
F. Guidobono
Department of Pharmacology, Chemotherapy, and Medical mice [6] and in cultured osteoblasts [7], callus formation in
Toxicology, University of Milan, Milan, Italy a rabbit osteotomy model [8], and new cortical bone

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A. Rubinacci et al.: Lack of Estrogens and Vibration 317

formation in the mouse ulna [9]. The vibratory stimulus led to the conviction that the involvement of sex steroids in
can also prevent bone loss and reductions in bone strength mechanical adaptation, particularly periosteal expansion, is
in ovariectomized rats [10, 11]. Taken together these data more complex than originally believed. Estrogens may
suggest that total-body vibration might differentially affect regulate the rate of bone remodeling, but bone balance at
bone cell activities, thus leading to enhanced bone mass or the surfaces is probably modulated by the prevailing
reduced bone loss associated with estrogen deficiency. mechanical environment [27].
However, it remains to be determined whether estrogens Taken together these data suggest that mechanical
modulate the sensitivity of different bone surfaces to a low- loading can enhance bone (re)modeling at the bone sur-
amplitude, high-frequency strain. The sensitivity of bone to faces and might differentially affect bone cell activities,
the mechanical environment is predicted by the mechano- thus leading to enhanced bone mass or reduced bone loss,
stat theory [12], which introduced the concept that a depending upon estrogen status. This study was therefore
minimum effective strain (MES) must be exceeded in order designed to test the hypotheses that estrogens modulate the
to stimulate an adaptive response to mechanical overload sensitivity of the different bone surfaces to low-amplitude,
[13]. Thinking about how a mechanostat system functions high-frequency strains and that estrogen deficiency might
in bone physiology led to the hypothesis that nonendocrine down- or upregulate the MES set-point depending upon
factors may influence, in one way or another, the which bone surface is considered. To identify the role of
mechanical impact in time and space by modulating MES acceleration in the adaptive response of rat bone to the
set-points in a predictable manner. Thus, the anabolic vibratory stimulus, the loading frequency was kept constant
effects of mechanical load under estrogen replacement while two accelerations were tested. The two accelerations
therapy and the nonresponse of bone to the increased strain applied were within the range 0.3–8 g, which has been
under estrogen deficiency were interpreted as a MES down- shown to be effective and well tolerated in clinical studies
or upregulation, respectively [14]. However, these targeted [28, 29]. For this purpose, the response of the trabecular
shifts of the mechanostat are only able to explain and cortical compartments to total-body vibration was
(re)modeling changes at the bone marrow interface (tra- evaluated in ovariectomized rats by peripheral quantita-
becular and endocortical surfaces) where estrogens and tive computed tomographic (pQCT) analysis and
strains share a common signaling pathway via estrogen histomorphometry.
receptor a (ERa) [15, 16]. It has been shown that stimu-
lation by both strain and estrogen results in activation of
the extracellular signal–regulated kinase (ERK) pathway, Materials and Methods
phosphorylation of ERa, and upregulation of the estrogen-
responsive element [17, 18]. Moreover, mice lacking Strain Gauge Implant and Recording
functional ERa produce less new cortical bone in response
to the same mechanical stimuli as do their ERa+/+ litter- Three 3-month-old female Sprague-Dawley rats, weighing
mates [19]. It follows that at least one strain-related 250–275 g (Charles River Laboratories, Calco, Italy), were
cascade responsible for adaptive control of bone architec- anesthetized and strain gauges (SA06008CL120; Vishay
ture is mediated through ERa, activated by estrogen [20]. Micro-Measurements, Milan, Italy) were implanted
However, the mechanostat model does not predict the according to the technique described by Rabkin et al. [30].
observed changes at the periosteal surface, where estrogens Briefly, a 1.5 cm incision was made over the anterior tibia.
and exercise have opposite effects: exercise enhances A single strain gauge was fixed on the tibial surface by
periosteal bone formation, while estrogen inhibits it [21]. It histoacrylic glue. The lead wire of strain gauge A was
is therefore conceivable that estrogens might down- or passed subcutaneously along the back of the rat in order to
upregulate the MES set-point, depending upon which bone exit at the back of the neck. The implant was waterproofed
surface is considered. In humans as well as in rats, estrogen with polyurethane and RTV silicone rubber according to
deficiency is accompanied by increased bone size at the the supplier’s instructions (Vishay Micro-Measurements).
appendicular skeleton, which might offset the loss in tra- The gauge resistance was checked, and the lead wires
becular bone mass [22–24]. On the other hand, it has also exiting the neck were trimmed to expose a short length of
been reported that treatment with estrogens increased bone wire (1 cm). Strains were measured by a bridge amplifier
size in a young man suffering from aromatase deficiency (P3, ±0.1% accuracy and 1 microstrain resolution; Vishay
[25]. Based on studies of mice carrying a null mutation of Micro-Measurements) equipped with a static strain indi-
ERb, signaling through ERb appears to be the targeted cator and a digital data logger. Strain gauge recording was
upregulator of the MES set-point that affects the mechan- performed for 5 minutes and repeated three times in rats
ical sensitivity of the periosteum, thus balancing ERa placed on the platform vibrating at 0.6 and 3 g peak-to-
signaling at the bone marrow interface [26]. This evidence peak acceleration. The measurements were repeated on the

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same animal 24 hours later and were consistent with the allowed to move freely on the plate, thus adding the
data of the previous day. vibratory stimulus to normal cage activity.

Vibration System and Data Acquisition Vibration Protocol

A National Instruments (Baltimore, MD) PCI-6036E Data Rats were housed in cages under controlled conditions
Acquisition card was used to generate a sine wave to drive (22 ± 2C, 65% humidity, 12/12-hour light/darkness
the vibration and to digitize an analog input signal from the cycle). After receipt, rats were allowed to acclimatize for a
sensor. Software was developed in LabView to control the week before ovariectomy or sham surgery. Ovariectomy
amplitude and frequency of the sine wave output and to was performed under anesthesia obtained with ketamine-
calculate the frequency and amplitude of the digitized input xylazine, 0.075 mL/hg (Pfizer Italia, Milan, Italy), and
signal using a discrete Fourier transform operation. The xylazine, 0.025 mL/hg, i.p. (Rompum; Bayer, Milan,
frequency and amplitude data from the input signal were Italy). A total of 75 rats reached the end of the experiment,
displayed on a chart as a function of time and logged to a text randomly divided into six groups: sham control (SHAM,
file for postprocessing (Science Wares, Falmouth, MA). n = 14); sham vibrated (SHAM-V) at 30 Hz, 0.6 g
An accelerometer (A/120/VT; DJB Instruments, Ver- (n = 11) and at 30 Hz, 3g (n = 10); ovariectomized con-
dun, France), attached to the vibrating plate, was used to trol (OVX, n = 15); ovariectomized vibrated (OVX-V) at
generate the input signal controlling the amplitude. The 30 Hz, 0.6 g (n = 11) and at 30 Hz, 3g (n = 14). The
input signal was amplified by a linear power amplifier (PA number of animals varied from group to group because
100E; Gearing & Watson Electronics, East Sussex, UK) some died as a consequence of the anesthesia required for
and transmitted to an E-M Actuator (PA 100E, Gearing & ovariectomy and/or pQCT analyses. Rats underwent
Watson Electronics) to generate the specific vibration over whole-body vibration on the vibration platform for
the neutral line (Fig. 1a). The actuator and the plate were 20 minutes/day, 5 days/week, for 2 months. The net force
inserted in a clear-walled cylinder on the floor. Vertical acting on the rat tibia (during vibration applied between the
peak-to-peak displacement of the plate occurred without knee joint and ankle joint) was estimated by assuming that
interference by the wall (0.5 mm clearance). Each rat was the body weight is homogeneously distributed on the four

Fig. 1 Schematic
representation of the vibrating
platform control system (A).
PC, personal computer; A/D,
analogic/digital; D/A, digital/
analogic. Typical readings of
the input signal obtained by the
accelerometer at 30 Hz and
0.6 g (B) and at 30 Hz and 3g
(C). The signal remained steady
over the experimental time

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A. Rubinacci et al.: Lack of Estrogens and Vibration 319

legs. Thus, in a 320 g rat, each tibia would bear a load of For the muscle analyses, peel mode 2 with a threshold
80 g, or 0.78 N. Hence, the load increase by the acceler- of 40 mg/cm3 was used to separate fat from muscle and
ation superimposed over gravity yields a peak load of 1.25 contour mode 1 with a threshold of 280 mg/cm3 was used
N at 0.6 g and 3.14 N at 3 g on each tibia. to separate muscle from bone. According to the manufac-
The control rats were placed on the vibration platform turer, the estimated total effective radiation dose of the
without vibration for the same amount of time as the applied single measurement, including the ‘‘scout view’’
vibrated rats. For the longitudinal study, pQCT analyses and two slices, was lower than 10 lSv/exam.
were performed at baseline, before starting the vibration
procedure, and after 4 and after 8 weeks under light Histomorphometry
anesthesia. At the end of the experiment (8 weeks), all rats
were killed by carbon dioxide inhalation and the tibiae Rats were injected i.p. with oxytetracycline (40 mg/kg) on
excised and fixed in buffered formalin for further histo- days 36 and 54 of the experimental period and killed at the
morphometric and pQCT analyses. The experimental end of the eighth week. The left tibia of rats from each
protocol was approved by the local Institutional Animal group (SHAM, SHAM-V at 0.6 and 3g, OVX, OVX-V at at
Care and Use Committee (IACUC license 262, December 0.6 and 3g) was dehydrated in a graded series of ethanol
9, 2004, with subsequent addendum of May 10, 2005). and embedded in polymethylmethacrylate. A single cross
section (150 lm thick) was obtained using a SP 1600
pQCT Measurements diamond saw microtome (Leica, Milan, Italy) cutting sys-
tem from three different regions: metaphyseal (5 mm distal
pQCT measurements were performed using a Stratec to its condyles), mid-diaphyseal, and distal diaphyseal
Research SA+ pQCT scanner (Stratec Medizintechnik, (distal to the fusion of the fibula on the tibia). Metaphyseal
Pforzheim, Germany) with a voxel size of 70 lm and a and mid-diaphyseal sections were microradiographed
scan speed of 3 mm/second. In order to orient the long axes (MicroXray; Italstructure, Como, Italy) and analyzed by
of the bones parallel to the image planes, the anesthetized means of image analysis software (Lucia G; Laboratory
animals (longitudinal study) and the excised bone speci- Imaging, Prague, Czech Republic). The following param-
mens (cross-sectional study) were fixed with a plastic eters were measured: (1) in the metaphyseal region, bone
holder for the pQCT measurements. The correct longitu- volume/tissue volume (BV/TV), trabecular thickness
dinal positioning was determined by means of an initial (Tb.Th), trabecular number (Tb.N), and trabecular separa-
‘‘scout scan.’’ The bones were scanned in the horizontal tion (Tb.S); (2) in the mid-diaphyseal region, total cross-
plane using two consecutive cross-sectional images at sectional area (Tt.Ar), medullary area (Me.Ar), cortical
5 and 25 mm distal to the proximal end of the tibia. The bone area (CtB.Ar), total cross-sectional perimeter
scans were analyzed with pQCT software 6.00B using (Tt.Pm), medullary perimeter (Me.Pm), and cortical width
contour mode 2 and peel mode 2 with a threshold of (Ct.Wi). All sections were also examined under ultraviolet
500 mg/cm3 for the calculation of trabecular and total bone light by means of a Zeiss (Jena, Germany) Axiophot
parameters at the metaphysis and with a threshold of microscope using the previously mentioned image analysis
710 mg/cm3 for cortical bone parameters at the diaphysis. software to evaluate tetracycline labeling at the bone cor-
The different thresholds of 500 and 710 mg/cm3 for the tex. Bone formation rate (BFR) was calculated by dividing
metaphysis and diaphysis, respectively, were established the area of bone between the double labeling for the
to account for partial volume effect. The cortical bone interlabel time at both the endosteal and periosteal sides.
density is lower at the metaphysis than at the diaphysis due Mid-diaphyseal sections were also carefully polished,
to the thinner cortex. The threshold was therefore adjusted etched with a solution of HCl 0.1 N for 60 seconds, and
according to the cortical density to optimize accuracy. gold palladium–coated for backscattered scanning electron
The polar strength strain index (SSI) was calculated by microscopic (SEM) analysis. Nomenclature and abbrevia-
the manufacturer’s software as follows: SSI = Ri = 1,n r2i • tions follow the recommendations of the American Society
aCD/ND • rmax, where r is the distance of a voxel from the for Bone and Mineral Research.
center of gravity, rmax is the maximum distance of a voxel
from the center of gravity, a is the area of a voxel, CD is Statistical Analysis
the cortical density, and ND is the density of normal cor-
tical bone tissue, equal to 1,200 mg/cm3, as measured by Statistical analysis was performed with the statistical
pQCT when no spaces are included. To account for chan- package GraphPad Prism, version 4.00 for Windows
ges in the mineralization of bone, and therefore for changes (GraphPad Software, San Diego, CA, www.graph-
in material properties, the section modulus was normalized pad.com). Data were expressed as mean ± standard error
for this value in the pQCT software. of the mean. The significance of differences between

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groups and between times in the longitudinal study was (Fig. 2). pQCT analysis showed that ovariectomy also did
assessed by means of a two-way analysis of variance not modify the muscle areas in all groups (data not shown).
(ANOVA) for repeated measures and Bonferroni’s multi- Animal weights increased in all groups during the
ple comparison test. The significance of differences experiment; however, the weight of OVX animals
between groups in the cross-sectional study was assessed increased more than that of SHAM animals, and the weight
by means of a one-way ANOVA and Bonferroni’s multiple gain was not affected by the two vibration regimens
comparison test. SSI dependence on acceleration was (Fig. 3).
assessed by means of a linear regression analysis. Histo-
morphometric data were compared by means of one-way Cross-sectional Evaluation in vitro
ANOVA and the Student-Newmann-Keuls test.
In the SHAM group, vibration at both regimens did not
induce any significant change in all the bone density and
Results size parameters considered, both in the metaphysis
(Table 2) and in the diaphysis (Table 3) of the tibia. In
Vibration System and Strain Evaluation OVX animals, vibration at 3 g induced a significant
increase of the total areas and of the cortical and medullary
The vibration system was tested under both vibration reg- areas (P \ 0.01) as well as the periosteal (P \ 0.01) and
imens (0.6 and 3 g) at each vibration cycle throughout the the endosteal (P \ 0.05) perimeters of the diaphysis
experimental time and found appropriate. Typical readings (Table 3). Tb.BMD was significantly (P \ 0.001) lower in
are shown in Figure 1b, c. In vivo strain data collected the OVX groups than in the SHAM groups, but the dif-
from the anteromedial surface of the tibia showed that the ference was not significantly affected by vibration at both
dynamic strain magnitudes induced at 30 Hz depended regimens (Table 2).
upon the peak-to-peak acceleration, i.e., 0.6 or 3 g. The Polar SSI increased linearly in the OVX-V groups in
mean peak-to-peak strain was 14.07 ± 5.09 le at 0.6 g and proportion to the acceleration applied (F = 11.342,
23.40 ± 7.53 le at 3 g (n = 3). The loading frequency of P \ 0.005), reaching statistical significance (P \ 0.01) at
the platform was transmitted to the strain gauge with no the 3 g vibration regimen (Fig. 4).
alteration as the dominant peak in the frequency domain
coincided with the vibration frequency. However, the peak- Histomorphometry
to-peak strain increased by less than twofold (1.71-fold) in
response to a fivefold increase in the loading amplitude. The static histomorphometric values measured in the tibial
metaphysis are reported in Table 4. Except for Tb.Th,
pQCT Measurements significant differences were observed between the SHAM
and OVX groups, independent of the vibration regimen. No
Longitudinal Evaluation in vivo significant differences were found between the SHAM and
SHAM-V groups or between the OVX and OVX-V groups
The in vivo pQCT measurements of cortical (Ct-BMD) and at both vibration regimens (0.6 and 3 g). The static histo-
trabecular, volumetric bone mineral density (Tb-BMD) at morphometric parameters measured in the mid-diaphyseal
the tibial metaphysis and diaphysis at baseline and at 4 and sections were not statistically different among groups;
8 weeks into the experiment for all groups are shown in nonetheless, a tendency toward an increase of total cross-
Table 1. Ct-BMD of the tibial metaphysis and diaphysis sectional and medullary areas in the OVX compared to the
increased slightly, but significantly, over time in all groups, SHAM group was recognized (Table 4).
whereas Tb-BMD significantly decreased in OVX groups Examination of bone sections under ultraviolet light
and remained steady in the SHAM group. The vibration did showed diffuse labeling at the metaphyseal level that did
not change Ct-BMD and was ineffective at preventing not allow optimal readings of the dynamic parameters. A
trabecular bone loss in OVX rats. No significant differ- measurable tetracycline double labeling was instead pres-
ences in Ct-BMD and Tb-BMD were observed between the ent at the mid-diaphyseal level in most sections, and the
vibrated and nonvibrated groups at all experimental times derived BFR was analyzed as a function of the vibration
and at both vibration regimens applied. To further analyze regimen and estrogen status. No significant differences
the effect of vibration, the trabecular density distribution were found between the vibration regimens in either the
was evaluated. The results showed a shift of the density SHAM or OVX group. However, BFR at the bone surface
distribution in the OVX groups toward lower values, but was significantly (P \ 0.001) greater in the OVX group
the vibration did not significantly change the density dis- (0.00897 ± 0.00399 mm2/day) compared to the SHAM
tribution pattern in the presence or absence of estrogens group (0.00492 ± 0.00291 mm2/day).

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 Table 1 Longitudinal pQCT measurements of volumetric BMD in the tibiae

SHAM SHAM+0.6 g, 30 Hz SHAM+3 g, 30 Hz

Basal 4 Weeks 8 Weeks Basal 4 Weeks 8 Weeks Basal 4 Weeks 8 Weeks

Diaphysis
A. Rubinacci et al.: Lack of Estrogens and Vibration

Ct-BMD 1,306.8 ± 15.1 1,350.6 ± 4.8** 1,369.1 ± 4.8** 1,326.4 ± 5.2 1,352.3 ± 3.0** 1,368.0 ± 3.1** 1,327.2 ± 5.8 1,355.6 ± 4.7* 1,366.5 ± 5.3**
Metaphysis
Tb-BMD 382.9 ± 8.3 378.4 ± 9.7 385.1 ± 10.4 372.9 ± 8.6 378.3 ± 8.2 390.0 ± 8.9 365.2 ± 11.4 366.3 ± 13.3 368.0 ± 13.9
Ct-BMD 1,052.9 ± 9.0 1,116.0 ± 7.1** 1,134.3 ± 11.9** 1,045.9 ± 16.4 1,123.9 ± 10.9** 1,137.8 ± 9.5** 1,077.2 ± 9.9 1,121.7 ± 13.0* 1,154.8 ± 8.8**
OVX OVX+0.6 g, 30 Hz OVX+3 g, 30 Hz
Basal 4 Weeks 8 Weeks Basal 4 Weeks 8 Weeks Basal 4 Weeks 8 Weeks

Diaphysis
Ct-BMD 1,314.4 ± 9.5 1,355.6 ± 24.5* 1,367.4 ± 3.8** 1,325.5 ± 5.8 1,365.1 ± 5.3** 1,370.4 ± 6.5** 1,332.6 ± 5.0 1,354.6 ± 4.9* 1,366.8 ± 12.1*
Metaphysis
Tb-BMD 368.8 ± 8.0 264.0 ± 11.8* 210.7 ± 4.0* 384.6 ± 13.3 280.4 ± 7.9** 229.2 ± 6.1** 360.2 ± 9.6 275.9 ± 11.8** 230.8 ± 13.8**
Ct-BMD 1,040.8 ± 11.9 1,099.9 ± 12.1** 1,133.2 ± 9.8** 1,046.6 ± 15.7 1,089.6 ± 15.8 1,129.7 ± 13.9** 1,070.5 ± 7.5 1,118.6 ± 13.5* 1,126.2 ± 15.9**

Ct-BMD, cortical bone mineral density; Tb-BMD, trabecular bone mineral density
* P \ 0.05, ** P \ 0.01 vs. basal (n = 9–13 rats/group)
321

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322 A. Rubinacci et al.: Lack of Estrogens and Vibration

Fig. 3 Mean ± standard error of the mean of animal weights in the

different groups at baseline and at 4 and 8 weeks. The two vibration
regimens had no effect on weight. **P \ 0.01 OVX vs. SHAM

potential was dependent upon the amplitude of the vibra-

tory stimuli applied. These results fit the view that
trabecular and cortical compartments display a different
sensitivity to a vibratory stimulus that may be modulated
by the presence of estrogens. In fact, ovariectomy sensi-
tizes periosteal bone apposition to the vibration. At the
vibration amplitude of 3 g, enhanced periosteal apposition
and endosteal resorption in the OVX rats resulted in an
outward shift of bone mass from the neutral axis with a
subsequent improvement of the SSI of the diaphyseal shaft.
Since SSI defines bone resistance, it follows that the
vibratory stimulus is able to work against the trabecular
bone loss due to ovariectomy, enhancing the overall
mechanical competence of the appendicular skeleton. This
result was obtained by cross-sectional pQCT in vitro
measurements, which are less affected by bone edge
detection and positioning problems compared with in vivo
longitudinal evaluation. These data confirmed previous
findings obtained in OVX rats subjected to 25 Hz whole-
body vibration in which vibration did not affect ovariec-
tomy-induced endosteal resorption but preserved cortical
strength by increasing periosteal formation [31]. The geo-
Fig. 2 Distribution of tibial Tb.BMD over the total bone area at
metric changes observed between vibrated and nonvibrated
baseline and after 4 and 8 weeks of daily vibration in the tibiae
(metaphyses) of different groups OVX rats were also apparent by the histomorphometric
analysis of the tibial diaphysis, although the results did not
SEM analysis showed that in all animals the new bone reach statistical significance. Besides quantitative evalua-
laid down beyond labels is regular lamellar bone at both the tion, the histological analysis gave further insights into
endosteal (Fig. 5a) and periosteal (Fig. 5b) sides of the understanding the effect of vibration on bone. SEM anal-
tibia. ysis of the bone sections showed that the new superficial
bone laid down between the two labels was regular
lamellar bone, thus suggesting that the periosteal effect of
Discussion vibration in OVX rats fits with the physiological bone
remodeling pattern. In fact, any nonspecific stimulus upon
The current study has shown that low-amplitude, high- the periosteum is generally associated to the deposition of
frequency whole-body vibration requires the absence of an irregular pattern of lamellae. Furthermore, the histo-
gonadal estrogens to be anabolic to bone at the specific morphometric observation that BFR is significantly
vibration regimens applied. In OVX animals, vibration was activated in OVX rats indicates that OVX expands the bone
effective on modeling of the bone cortex and its osteogenic cellular pool that is the target of the mechanical signal,

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A. Rubinacci et al.: Lack of Estrogens and Vibration 323

Table 2 Cross-sectional evaluation of structural geometric properties at tibial diaphysis evaluated ex vivo after 8 weeks (treatment)
Parameters SHAM SHAM+0.6 g, 30 Hz SHAM+3 g, 30 Hz OVX OVX+0.6 g, 30 Hz OVX+3 g, 30 Hz

Ct-BMD (mg/cm3) 1,380.7 ± 5.8 1,372.6 ± 2.9 1,387.3 ± 2.6 1,380.2 ± 3.6 1,379.2 ± 6.2 1,392.2 ± 4.4
Tt.Ar (mm2) 5.90 ± 0.10 5.67 ± 0.13 5.92 ± 0.15 6.00 ± 0.11 6.28 ± 0.16 6.58 ± 0.09**
2
Md.Cn.Ar (mm ) 0.71 ± 0.03 0.70 ± 0.05 0.67 ± 0.06 0.73 ± 0.04 0.80 ± 0.04 0.88 ± 0.06
2
Ct.Ar (mm ) 4.81 ± 0.10 4.74 ± 0.11 4.83 ± 0.13 4.86 ± 0.09 5.05 ± 0.11 5.27 ± 0.07**
Ct.Th (mm) 0.78 ± 0.01 0.77 ± 0.02 0.79 ± 0.02 0.78 ± 0.01 0.79 ± 0.01 0.80 ± 0.01
Ps.Pm (mm) 8.61 ± 0.07 8.58 ± 0.09 8.62 ± 0.10 8.68 ± 0.08 8.88 ± 0.11 9.09 ± 0.06**
Es.Pm (mm) 3.70 ± 0.05 3.74 ± 0.11 3.68 ± 0.10 3.78 ± 0.07 3.91 ± 0.12 4.06 ± 0.08*
Ct-BMD, cortical bone mineral density; Tt.Ar, total area; Md.Cn.Ar, medullary canal area; Ct.Ar, cortical area; Ct.Th, cortical thickness; Ps.Pm,
periosteal perimeter; Es.Pm, endosteal perimeter
* P \ 0.05, ** P \ 0.01 vs. OVX (n = 9–13 rats/group)

Table 3 Cross-sectional evaluation of structural geometric properties at the metaphysis of tibiae, evaluated ex vivo after 8 weeks
Parameters SHAM SHAM+0.6 g, 30 Hz SHAM+3g, 30 Hz OVX OVX+0.6 g, 30 Hz OVX+3 g, 30 Hz
3
Ct-BMD (mg/cm ) 1,142.4 ± 14.4 1,149.4 ± 9.2 1,147.0 ± 15.3 1,136.1 ± 12.0 1,159.6 ± 12.1 1,149.5 ± 10.4
Tb-BMD (mg/cm3) 396.2 ± 9.3 395.6 ± 9.1 377.9 ± 9.7 251.1 ± 16.08** 250.3 ± 9.7** 252.6 ± 9.8**
Tt.Ar (mm2) 16.54 ± 0.92 15.69 ± 0.50 17.17 ± 0.68 18.29 ± 0.54 17.18 ± 0.87 18.23 ± 0.64
Tb.Ar (mm2) 3.15 ± 0.63 3.36 ± 0.55 4.00 ± 0.70 8.48 ± 0.48** 7.98 ± 0.62** 8.81 ± 0.43**
Ct.Ar (mm2) 8.49 ± 0.67 8.23 ± 0.40 8.57 ± 0.58 7.12 ± 0.16 7.10 ± 0.23 7.00 ± 0.20
Ct.Th (mm) 0.70 ± 0.05 0.69 ± 0.04 0.69 ± 0.04 0.53 ± 0.02** 0.54 ± 0.01* 0.52 ± 0.01**
Ps.Pm (mm) 14.36 ± 0.41 14.19 ± 0.25 14.67 ± 0.29 15.14 ± 0.22 14.78 ± 0.37 15.11 ± 0.27
Es.Pm (mm) 9.98 ± 0.40 9.89 ± 0.32 10.36 ± 0.32 11.79 ± 0.54 11.37 ± 0.39 11.84 ± 0.28
Ct-BMD, cortical bone mineral density; Tb-BMD, trabecular bone mineral density; Tt.Ar, total area; Tb.Ar, trabecular area; Ct.Ar, cortical area;
Ct.Th, cortical thickness; Ps.Pm, periosteal perimeter; Es.Pm, endosteal perimeter
* P \ 0.01, ** P \ 0.001 vs. SHAM (n = 9–13 rats/group)

stimulus, did not result in any new cortical bone formation

[9]; (3) adult female sheep exposed to low-level (0.3 g),
high-frequency (30 Hz) mechanical signals did not show
any change in the bone cortex as assessed by pQCT and
histomorphometry [5].
The demonstration that vibration loses its osteogenic
potential when the estrogen axis is intact is in agreement
with the recent observations that low-dose 17a-ethyny-
lestradiol suppresses the periosteal response to axial
Fig. 4 Mean ± standard error of the mean of SSI obtained by pQCT loading of the ulna of male rats [33] and that ERb knockout
analysis of excised tibiae at the end of the experimental time mice show improved bone formation [26] and increased
(8 weeks) in the different groups. **P \ 0.01 vs. SHAM and OVX periosteal responsiveness [34] after mechanical loading.
Such observations support the view that estrogens act as
suggesting that bone should be in an activated state to negative modulators of the mechanotransduction process at
respond to mechanical stimuli. the periosteal surface via ERb signaling. It is therefore
This study failed to observe any cortical bone response conceivable that estrogens might down- or upregulate the
to the vibratory stimulus in the intact animals, in agreement MES set-point depending upon which signaling pathway is
with the observations of others that (1) low-amplitude (0.1– activated. In a state of estrogen deficiency, as in meno-
0.3 N) vibration at broad frequencies (0–50 Hz) did not pause, the lack of inhibitory ERb signaling might activate a
cause formation of additional cortical bone when applied to compensatory mechanism in order to maintain bone
the female mouse ulna in uniaxial compression [32]; (2) mechanical resistance despite the loss of bone at the
loading the female mouse ulna with a 0.3 N vibration endocortical surface. The outward displacement of the
signal alone, not superimposed upon an osteogenic thinning cortex could be a target for mechanical

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324 A. Rubinacci et al.: Lack of Estrogens and Vibration

Table 4 Static histomorphometric evaluation of the tibiae

SHAM SHAM+0.6 g, 30 Hz SHAM+3 g, 30 Hz OVX OVX+0.6 g, 30 Hz OVX+3 g, 30 Hz

Metaphysis
BV/TV (%) 47.43 ± 7.90 48.38 ± 3.78 46.46 ± 13.34 21.73 ± 6.00** 25.07 ± 6.53* 18.16 ± 5.23*
Tb.Th (lm) 56.69 ± 13.16 60.40 ± 20.11 63.08 ± 15.74 47.07 ± 12.53 60.19 ± 6.26 50.50 ± 14.12
TB.N (/mm2) 6.50 ± 1.47 6.28 ± 1.02 6.01 ± 1.16 3.20 ± 0.94** 3.16 ± 0.59* 2.56 ± 0.96*
Tb.S (lm) 104.46 ± 41.39 102.01 ± 14.04 109.15 ± 45.99 338.45 ± 195.47** 267.25 ± 74.83 411.48 ± 235.02
Diaphysis
Tt.Ar (mm2) 6.00 ± 0.44 5.80 ± 0.54 6.01 ± 0.24 6.12 ± 0.53 6.27 ± 0.64 6.35 ± 0.50
Me.Ar (mm2) 1.44 ± 0.36 1.40 ± 0.25 1.52 ± 0.19 1.50 ± 0.27 1.60 ± 0.46 1.69 ± 0.25
CtB.Ar (mm2) 4.55 ± 0.23 4.40 ± 0.33 4.49 ± 0.30 4.61 ± 0.33 4.66 ± 0.33 4.65 ± 0.31
Tt.Pm (mm) 9.50 ± 0.43 9.17 ± 0.35 9.55 ± 0.34 9.67 ± 0.44 9.74 ± 0.63 9.67 ± 0.37
Me.Pm (mm) 4.46 ± 0.67 4.47 ± 0.32 4.74 ± 0.25 4.73 ± 0.47 4.86 ± 0.76 4.88 ± 0.37
Ct.Wi (lm) 646 ± 48 642 ± 36 622 ± 58 633 ± 31 639 ± 67 635 ± 37
BV/TV, bone volume/total volume; Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.S, trabecular separation; Tt.Ar, total cross-
sectional area; Me.Ar, medullary area; CtB.Ar, cortical bone area; Tt.Pm, total cross-sectional perimeter; Me.Pm, medullary perimeter; Ct.Wi,
cortical width
Mean value ± standard deviation; each value is derived from a single serial cross section taken from the left tibia
* P \ 0.01 vs. basal
** P \ 0.001 vs. SHAM (n = 5–8 rats/group)

intervention therapy in postmenopausal osteoporosis as 45 Hz there was no significant increase in either cortical or
well as for drug development [35]. trabecular BFR [37]. As pointed out by Judex et al. [37], it
No trabecular effect was observed at the vibration reg- is possible that the adaptive response to low-amplitude,
imens applied, in contrast to the original thought that low- high-frequency mechanical regimens does not follow the
amplitude, high-frequency strain rates are anabolic to rules of strain in the low-frequency domain. It follows that
cancellous, but not to cortical, bone [5]. Besides potential effects upon bone induced by whole-body vibration and the
species-specific differences in the sensitivity to mechanical sensitivity of the bone compartment to a specific vibration
stimuli, it might be possible that different postures on the regimen might be due to the activation of different mech-
vibration platform among the different animal models will anisms of signal transduction, not necessarily working in a
determine different effects of vibration upon the strain rate linear magnitude–dependent manner [38, 39]. How differ-
at the trabecular compartment. Unlike sheep, rats as well as ences in frequency and/or amplitude alter the efficacy of
mice move freely during a cycle of whole-body vibration low-amplitude, high-frequency mechanical stimuli is lar-
and they are, therefore, subjected to a vibratory stimulus gely unknown. How the specific mechanical parameters
that can change according to body posture. Such changes modulate the different bone compartment sensitivities is
can be potentially dampened by the viscoelastic nature of likewise unknown.
the muscle–tendon apparatus [32]. The lack of a linear The increase in Ct-BMD in the SHAM group during the
relationship between the increase in the strain and the experimental time indicates that the animals had not yet
increase in the acceleration is in agreement with this view. reached peak bone mass, in agreement with the observa-
Since the vibratory stimulus was ineffective at altering tions of others [40, 41]. This is an important limitation of
trabecular bone density and preventing postovariectomy the study as it was not possible to discriminate the influ-
trabecular bone loss, it might be that it has not reached the ence of skeletal growth on the whole-body vibration effect.
threshold sensitivity of the trabecular compartment. It is Nevertheless, it should be considered that (1) pQCT does
conceivable that the vibratory stimulus perceived by the not distinguish between material and structural properties
trabecular compartment might require higher frequencies of bone (it follows that as long as the secondary mineral-
to reach the osteogenic threshold and subsequently activate ization process takes place, increasing volumetric density
a transduction pathway of the mechanical stimuli not [material property] does not imply a parallel increment in
necessarily related to matrix strain [36]. It has been bone mass [structural property]) and (2) cross-sectional
reported that both cortical and trabecular BFRs were sig- bone area of rats and humans continues to increase slowly
nificantly increased in vibrated animals compared to throughout life [42]. Therefore, even older rats would
controls when vibration was applied at 90 Hz, while at undergo radial growth and the interpretation of the results

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A. Rubinacci et al.: Lack of Estrogens and Vibration 325

have almost always examined the effect of vibration on

seated subjects, and resonance peaks have typically been
found at frequencies below 10 Hz. Since vibration loading
as a potential treatment for osteoporosis would be applied
to standing subjects at much higher frequencies, it can be
assumed that the risk of low back pain is reduced. More-
over, the exposure of humans to 3 g for 1 minute would be
equivalent to about 30 m/second2, thus within the limits
established by European Union guidelines [44]. Neverthe-
less, it should be taken into account that the actual
accelerations in the human body are much smaller than
those measured on the plate due to the dampening effect of
soft tissue structures, as discussed above. When 8-month
vertical whole-body vibration was applied in a controlled
randomized trial, it was well tolerated up to 8 g [29].
In conclusion, this study strengthens the view that
whole-body vibration has osteogenic potential in a rat
animal model. Since the vibratory stimulus appears to
require the lack of gonadal estrogens to be effective on the
outward displacement of the cortex, it might be effective as
a nonpharmacological adjuvant in the treatment of post-
menopausal osteoporosis by potentially increasing bone
size and reducing the risk of fracture.
The authors thank Erik Karplus (Science Wares, Fal-
mouth, MA), who designed the software for the control and
data acquisition of the vibration system. This study was
supported by the Italian Ministry of Research (COFIN
2004–2006).

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