Ind HealthInd HealthINDHEALTHIndustrial Health0019-83661880-8026National Institute of Occupational Safety and Health, Japan258435644466877ih-2014-011710.2486/indhealth.ih-2014-0117Original ArticleEffect of higher frequency components and duration of vibration on bone
tissue alterations in the rat-tail modelPEELUKHANASrikara V.1aGOENKAShilpi1aKIMBrian1KIMJay1BHATTACHARYAAmit2STRINGERKeith F.3BANERJEERupak K.1*Department of Mechanical and Materials Engineering,
University of Cincinnati, USADepartment of Environmental Health, University of Cincinnati,
USADepartment of Pathology, Cincinnati Children’s Hospital
Medical Centre, USA*To whom correspondence should be addressed. E-mail: Rupak.Banerjee@uc.edu
To formulate more accurate guidelines for musculoskeletal disorders (MSD) linked to
Hand-Arm Vibration Syndrome (HAVS), delineation of the response of bone tissue under
different frequencies and duration of vibration needs elucidation. Rat-tails were vibrated
at 125 Hz (9 rats) and 250 Hz (9 rats), at 49 m/s2, for 1D (6 rats), 5D (6
rats) and 20D (6 rats); D=days (4 h/d). Rats in the control group (6 rats for the
vibration groups; 2 each for 1D, 5D, and 20D) were left in their cages, without being
subjected to any vibration. Structural and biochemical damages were quantified using empty
lacunae count and nitrotyrosine signal-intensity, respectively. One-way repeated-measure
mixed-model ANOVA at p<0.05 level of significance was used for
analysis. In the cortical bone, structural damage quantified through empty lacunae count
was significant (p<0.05) at 250 Hz (10.82 ± 0.66) in comparison to the
control group (7.41 ± 0.76). The biochemical damage was significant
(p<0.05) at both the 125 Hz and 250 Hz vibration frequencies. The
structural damage was significant (p<0.05) at 5D for cortical bone
while the trabecular bone showed significant (p<0.05) damage at 20D
time point. Further, the biochemical damage increased with increase in the duration of
vibration with a significant (p<0.05) damage observed at 20D time
point and a near significant change (p=0.08) observed at 5D time point.
Structural and biochemical changes in bone tissue are dependent upon higher vibration
frequencies of 125 Hz, 250 Hz and the duration of vibration (5D, 20D).
Prolonged exposure to hand-transmitted vibrations (HTV) from hand held power tools leads to
Hand-Arm Vibration Syndrome (HAVS) which consists of vascular, sensorineural disorders, and
musculoskeletal disorders (MSD)1). NIOSH
estimates that about half of the 1.2 to 1.5 million US workers who use hand-held powered
tools are at risk of developing HAVS2).
Various bone disorders such as low bone mineral density, bone lesions and carpal bone
abnormalities are caused by vibration3).
For example, it has been reported that in the manufacturing sector, workers using hand-held
powered tools undergo the induction of bone lesions and osteonecrosis of the lunate
(Kienbocks disease)4). In addition, workers
in the mining sector develop osteonecrosis of the carpal scaphoid5). Some studies have reported that the changes observed after
vibration exposure were not different from those found in ageing populations4). Elsewhere, it has been reported that
vibration altered the bone remodeling and affected bone mineralization6). Some studies have reported that low frequency (<40 Hz)
vibrations of high magnitude might be associated with abnormal radiological findings in the
wrist and elbow joints4, 7, 8). Epidemiological
data on the quantitative association between vibration and risks of bone degeneration are
still inconclusive.
Although there have been a few recent studies on the effects of mechanical vibration on
bone9, 10), the range of vibration frequencies reported in these studies are
several orders of magnitude lower than those caused by the pneumatic tools. Pneumatic tool
operators in the workplace are exposed to vibration frequencies in the range of 5.6 Hz to
1,400 Hz. Not all frequencies, magnitudes or durations of vibration cause similar effects.
These effects are extremely subject specific3). It has been reported that the resonance frequency of the human
finger tissue is in the range 100–350 Hz11). In addition, most of the tools, such as grinders and chipping
hammers, used in industrial workplace generate significant vibration components in the
frequency range 100–400 Hz12). However, the current ISO standard 5349-1 and ANSI-standard 2.70
have given greater weight to lower frequencies (<32.5 Hz) and less weight to higher
frequencies (>100 Hz)13). The validity
of this weighting has been questioned by researchers13,14,15,16,17) who suspect a greater risk of vibration-injury in the case of
frequency components higher than the 100 Hz. This ISO guideline, with its higher weightage
to lower frequency range, might lead to an under estimation of the risk of high frequency
components.
The rat-tail bone (caudal vertebra) has been previously used to study changes in bone
tissue morphology and the response of bone under mechanical loading18,19,20,21,22). Moreover, the changes induced in bone tissue can be
better studied using this model since it has been already validated that the rat-tails’
nerves and vasculature surrounding the bone tissue mimic human finger. To the best of our
knowledge, this model has not been studied before for the study of vibration-induced bone
disruption, in relation to HAVS. Currently, there is a gap in the
understanding of how higher-frequency components of vibration (>100 Hz) and the duration
of vibration induce biological alterations in the bone tissue. Hence, there is a
need for elucidation of the cellular basis by which bone responds to
vibrations, which would aid in identification of novel therapeutic approaches for treatment
of musculoskeletal diseases and injuries related to HAVS.
In consideration of the aforementioned factors, in this study we sought to investigate the
changes in bone tissue under the influence of vibration at near resonance frequencies and
also the duration of vibration, using a rat-tail model of vibration injury. We
hypothesized that bone tissue alterations, manifested in the form of
structural damage (cortical and trabecular bone morphology) and biochemical alterations
(nitrotyrosine (NT)-mediated oxidative injury23,
24) are dependent on the resonance
frequencies (125 Hz and 250 Hz) as well as the time duration of vibration exposure. Hence,
in the present study, our aim was two-fold: i) quantification of the effects of
vibration frequency (125 Hz, 250 Hz) on structural and biochemical
(oxidative) alterations in bone tissue; and ii) quantification of the effects of
duration of vibration (1D, 5D, 20D) on structural and biochemical
alterations in bone tissue.
Subjects and Methods
Animal Groups. The animal protocol for this study was approved by the
Institute for Animal Care and Usage Committee (IACUC) at University of Cincinnati (UC). All
procedures were in compliance with the NIH Guide for the Care and Use of Laboratory Animals.
Vibration experiments were performed on non-anesthetized male Sprague-Dawley rats (250 ± 15
gm, Harlan Laboratories, WI). The study consisted of a total of 24 rats (Table 1
The experimental design for the study with the number of animals in each
group
Vibration frequency
1D
5D
20D
*Total rats for frequency analysis
Control
n=2
n=2
n=2
6
125 Hz
n=3
n=3
n=3
9
250 Hz
n=3
n=3
n=3
9
*Rats for day analysis
Control = 2, Vibrated = 6
Control = 2, Vibrated = 6
Control = 2, Vibrated = 6
* number of rats used for the one-way mixed-model data analysis.
). For the control group, 6 rats were utilized (2 each for 1D, 5D, and 20D). 9
rats were assigned to 125 Hz group (3 rats each for 1D, 5D, and 20D) and 9 rats for the
250 Hz group (3 rats each for 1D, 5D, and 20D). The rats were housed in standard cages in a
colony room at 25 ± 1° C with a 12:12 light: dark cycle in the Laboratory for Animal
Medicine and Services (LAMS) at UC and were provided the standard rodent diet and tap
water.
Vibration Protocol. The vibration experiments were done at frequencies of
125 Hz and 250 Hz, for duration of 4 h/day for 1D, 5D and 20D. The rats were placed in
Broome style restrainers resting on a non-vibrating support and the tails were strapped to a
platform. In order to avoid any interference to the experimental vibrations, the platform
material was selected so that it had a natural frequency higher than 1,000 Hz. Three rats
were used at one time for vibration and the tails were symmetrically placed and secured
using duct tape (Fig. 1
Experimental set up for the rat-tail vibration.
). The rats were monitored during the experiments in order to make sure the tail
didn’t bounce on/off the platform. This platform was connected to a mechanical shaker (model
V203; Ling Dynamic Systems, Herts, UK) which generated vertical vibrations (Fig. 1). Control group rats remained in their
respective cages and were not subjected to either the vibration stress or the constraint
(straps) stress on the non- vibrating platform. The required sinusoidal frequency was
produced by a function generator (Model HP 35660A; HP Inc., Palo Alto, CA, USA) connected to
a power amplifier (Model PA 25; Ling Dynamic Systems, Herts, UK). An accelerometer (Model
U352B22; PCB group Inc., Buffalo, NY, USA) was attached to the platform to measure and
maintain the acceleration at a constant value during the experiment. The acceleration used
for both the vibration frequencies was a constant acceleration excitation of 49 m/s2 RMS.
After vibration exposure, rats were returned to their respective cages and housed in the
colony room.
Animal Health Observations. The animals were monitored for health and
behavior during the time of vibration exposure for all the days of vibration. We did not
find any symptoms of chromodacryorrhea (porphyrin secretion) or change in body temperature
or teeth grinding, similar to a previously reported study by Yan et al.,
201025). Also, the animals did not
exhibit any signs of stress or aberrant behavior. Rats in all the groups generally remained
calm in the restrainer during the vibration exposure.
Bone Tissue Processing. After vibration exposure, rats were euthanized
using carbon dioxide as per standard laboratory procedures followed by a secondary physical
method of diaphragm puncture (approved by IACUC at LAMS, UC). The rats were euthanized at
similar intervals (within 3 h) from the last vibration exposure. The rat-tail was first
fixed in 4% paraformaldehyde for 24 h and one bone sample was harvested from the middle part
of the whole tail, from each frequency group. These bone samples were then cleaned of all
adhering soft tissues with the help of a sharp scalpel, careful to avoid any damage to the
underlying bone. The samples were then stored in a neutral 30% sucrose PBS buffer at 4 °C
until they were ready to be prepared for embedding. After dissection, one bone specimen was
decalcified in 10% EDTA solution, according to commonly employed histological methodology.
Decalcification was checked for completion by chemical testing with ammonium oxalate
solution, and it took two weeks for samples to be fully decalcified. Decalcified rat-tail
bone was sectioned longitudinally (on a dental wax substrate) and embedded in paraffin.
Further, material characterization tests were performed to augment the data for biochemical
changes due to frequency effects at the 20D time point only. In order to achieve this, we
harvested a second bone sample from the tails of 20D vibration group rats (Control, 125 Hz,
250 Hz). This bone sample was not decalcified and embedded in acrylic resin
(SamplKwik®, Buehler Ltd., Lake Bluff, IL, USA) and prepared for biochemical
characterization using material tests of Energy Dispersive X-ray analysis (EDX) and
Backscattered Electron microscopy (BSE).
Histological Study for Osteocyte and Empty Lacunae Count. H&E stain
was used to stain 4 µm thick sections from paraffin embedded bone blocks on
glass slides and subsequently assessed under light microscopy for alterations in the bone
structure. After cover-slipping, the slides were assessed at X40 objective magnification for
quantification of i) count of viable osteocytes divided by total count of osteocytes and
empty lacunae26) and ii) empty lacunae in
five randomly selected fields in the diaphysis of cortical bone using NIH ImageJ
program.
Static Histomorphometry. The H&E stained images were used further for
assessment of changes in trabecular bone microarchitecture by static histomorphometry. The
images were taken at 5× objective magnification and processed in ImageJ. BoneJ plugin27) was used for calculation of the
parameters: trabecular separation (Tb.Sp) and trabecular thickness (Tb.Th). Proximal and
distal trabeculae were evaluated from each section for a total of two sections per rat in
each group.
Immunohistochemistry. The protocol used for immunohistochemial study is
similar to our previous study28). Briefly,
the paraffin-embedded bone tissues were sectioned at 4 µm, affixed to glass
slides and then deparaffinized in xylene and graded series of ethanol, similar to the
H&E stained sections. Sections on the slides were rehydrated, washed and blocked with a
mixture of 2% goat serum and 2% donkey serum. Subsequently, the sections were incubated with
mouse anti-nitrotyrosine primary antibody (Santa Cruz Biotechnology, CA, USA, 1:100, 4 °C)
overnight. Following this, the sections were further incubated in biotinylated goat
anti-mouse secondary antibody (Ventana, CA, USA, 1:100, 30 min, 25 °C) and developed using
peroxidase substrate. Sections were then coverslipped and photographed with a Spot RT Slider
digital camera (Diagnostic Instruments, Sterling Heights, MI, USA) mounted on a Nikon
Eclipse E600 microscope. An intense brown coloration was indicative of a positive signal.
Quantification of NT immuno-staining intensity was done by calculating the GSV (gray scale
value) similar to the method we have reported in earlier study28).
Backscattered Electron Microscopy (BSEM). The resin-embedded bone block
was used for Back Scattered Electron Imaging (BSE)-Scanning Electron Microscopy (SEM) using
a FEI XL30 ESEM model, equipped with a BSE detector. The bone blocks were coated with a thin
layer of Au/Pd to increase the conductivity of the samples and were examined in the electron
microscope under the BSE mode. The working distance was 10 mm, working voltage set to 15 kV
and the spot size was set to 4. The images were acquired for the cortical bone (diaphysis)
for all the groups. A total of 3–5 images were collected in the central part of bone in BSE
mode for each rat in each group. The use of BSE images for mineralization distribution has
already been demonstrated and there are many studies which have assessed the mineralization
distribution curves qualitatively and quantitatively29,30,31). For the assessment of mineralization pattern in the bone, the same
BSE images were opened in ImageJ and were cropped and smoothed. The mineralization
distribution histogram was then plotted in ImageJ (using Analyze > Histogram command) and
the mean grey level values were noted and the mineralization curves were plotted.
Energy Dispersive X-ray Analysis (EDX). Energy Dispersive X-ray Analysis
(EDX) was used along with the BSE to further validate the mineralization changes by
calculating Ca/P ratio. The Ca/P ratio and its spatial distribution can be a useful
predictor of any abnormal alterations occurring in mineralization patterns after vibration
of the bone tissue since Ca/P ratio is highly sensitive to changes in bone mineralization.
Energy-dispersive X-ray spectroscopy (EDX), coupled with Scanning Electron Microscopy (SEM)
in a manner similar to that reported by other researchers32, 33) was used. Images
obtained by SEM (Model: XL-30 ESEM; SN D1398, FEI, Hillsboro, OR, USA) were further used for
EDS (EDAX systems (Mahwah, NJ, USA) analysis to obtain Ca (wt%) and the Ca/P ratio at 4–5
randomly sampled points in the diaphysis of central cortical bone sample, per group.
Statistical Analysis. Based on the suggestions from a biostatistician, in
order to account for the variability between the multiple sections (repeated measures) and
for the variability in data between the rats, a one-way repeated measures mixed-model
analysis of variance (ANOVA) was used. The data was checked for normal distribution prior to
performing the statistical analysis. A compound symmetry covariance structure was assumed
for within the subject (repeated measures) and between the subject (random subject) effects.
Please note that the repeated measures involved obtaining multiple data from a single rat at
a single time point. It didn’t involve a time variation in single rat.
Group mean effects of frequency of vibration on structural damage (osteocyte count) and
biochemical damage (NT signal intensity) were assessed. Similarly, the group mean effects of
duration of vibration on structural damage (osteocyte count) and biochemical damage (NT
signal intensity) were also assessed. In order to assess the interaction effects between the
vibration frequency and days of vibration groups, further statistical analysis was
performed. This analysis was presented in the Appendix
1.
For osteocyte count quantification, we had 36, 34 and 40 data points from the control (6
rats), 125 Hz (9 rats) and 250 Hz groups (9 rats), respectively (Table 1). For NT signal intensity, we had 13 data points for the
control group (6 rats), 23 data points for the 125 Hz group (9 rats) and 18 data points for
the 250 Hz group (9 rats) (Table 1). In all the
analyses, animal was treated as the random effect while frequency was treated as fixed
effect. The number of rats (subjects) is the n-value and each data point is considered an
observation from each rat. Data analysis was performed using SAS 9.1.3 (SAS Institute Inc.,
NC, USA) with p<0.05 used as the probability level to accept statistical
significance. The comparisons were performed using pdiff command. Tukey-kramer and
bonferroni adjustments yielded similar p-values. All values are reported as
mean ± SE.
Power analysis. From Diggle et al., 200234), number of subjects required to compare
means across two groups with repeated measures is given by where, σ=31.67 (obtained from
SAS), this is the common variance in the two groups, the μ1−μ2=174.09–168.1=5.97 (obtained
from SAS), the difference in means of the two groups. The n=1, this is the number of
repeated measures, in this case number of sections from each rat. The lowest number of
sections obtained from a rat were used. The ρ=3.75 (obtained from SAS), this is the
correlation of the repeated measures. The N is the number of rats per group, 2 was used
since that is the least number of rats in the group. Please note that in the above formula,
for each of the parameters minimum values were used to provide a conservative estimate.
Substituting the values and solving for Zβ=(−1.46), we get the value of β as 0.072.
Therefore, the power of the test would be (1−β) × 100, which is 92.8%. This is the least
expected power of our comparisons.
Results
The qualitative and quantitative effects of various levels of vibration frequency in bone
tissue were assessed by analyzing the structural damage (H&E stained sections for
osteocyte and empty lacunae count in cortical bone and trabecular bone micro architecture)
and the biochemical damage (NT immunoreactivity obtained by immunohistochemistry). In order
to avoid repetition, first the qualitative effects of the vibration frequency and days of
vibration are presented briefly. This is followed by an explanation of the quantitative
effects of varying vibration frequencies. Subsequently, the quantitative effects of number
of days of vibration is discussed.
Qualitative effects of vibration frequency and days of vibration
H&E staining. The results obtained from H&E staining on control
and vibrated bone sections are shown in Fig.
2
Photomicrographs of the histological examination of cortical bone in the control
and vibrated bone sections using H&E stain. A) Control for 1D; B) 125 Hz for 1D; C) 250 Hz for 1D; D) Control for 5D; E)125 Hz
for 5D; F) 250 Hz for 5D; G) Control for 20D; H) 125 Hz for 20D; I) 250 Hz for 20D.
Osteocytes are represented by black arrows and the white arrows show empty lacunae
in the cortical bone. Magnification is ×40 objective.
. Qualitative observation shows that 250 Hz causes higher number of empty lacunae as
compared to either control or 125 Hz groups. Similarly, when observed across the days of
vibration groups, the number of empty lacunae are higher for 5D group as compared to
control group (Fig. 2).
H&E stained bone sections were also evaluated for qualitative study of trabecular
bone morphology of the control and vibrated bone (Fig.
3
H&E stained images of trabecular bone in the control and vibrated bone
sections. A) Control for 1D; B) 125 Hz for 1D; C) 250 Hz for 1D; D) Control for 5D;
E) 125 Hz for 5D; F) 250 Hz for 5D; G) Control for 20D; H) 125 Hz for 20D; I) 250 Hz
for 20D. T represents trabecular bone and BM represents the bone marrow.
Magnification is ×5 objective.
). The trabecular bone separation (Tb.Sp) appeared higher in the vibrated groups
(Fig. 3C, 3H and 3I) as compared to the
control group (Fig. 3A, 3D and 3G). The
trabecular thickness (Tb.Th) appeared reduced in 1D group (Fig. 3B and 3C) compared to the 5D vibrated groups (Fig. 3D, 3E and 3F). Qualitatively, the Tb.Sp
appears to be higher for the 20D time point (Fig. 3H
and 3I) as compared to either 1D (Fig. 3A, 3B
and 3C) or the 5D group (Fig. 3E and
3F).
Nitrotyrosine staining. Immunohistochemical staining was conducted on
bone sections in order to assess the expression of NT protein, which is a marker of
oxidative injury in the tissue. Figure 4
Representative photomicrographs for NT stained sections for: A) Control for 1D; B)
125 Hz for 1D; C) 250 Hz for 1D; D) Control for 5D; E)125 Hz for 5D; F) 250 Hz for
5D; G) Control for 20D; H) 125 Hz for 20D; I) 250 Hz for 20D. Positive signal in the
osteocytes is represented by black arrows and the black triangles show blood vessels
in the trabecular bone. Magnification is ×40 objective.
presents the qualitative changes observed due to NT immunostaining for all the
groups. For the bone vibrated at 125 Hz and 250 Hz, brown color was also observed in bone
marrow (BM). The cortical bone exhibited very little or no staining; hence, the NT stain
values were not quantified. We observed that most of the positive signal was localized
near the growth plate region of trabecular bone, which was in proximity to the bone marrow
(BM). Qualitative observation shows that vibrated bone groups (125 Hz and 250 Hz)
exhibited increased signal as compared to control bone. Similarly, the NT signal intensity
in bone showed a higher intensity for all the time points of vibration (Fig. 4B, 4E and 4H, Fig. 4C, 4F and 4I).
Quantitative effects of vibration frequency on bone tissue alterations
Quantification of H&E Staining in Cortical Bone. In order to
quantify the extent of structural alteration in the cortical bone, the number of
osteocytes and empty lacunae were counted. The effects of vibration frequency on osteocyte
and empty lacunae counts are summarized in Fig.
5
Bar plots showing the effects of vibration frequency on the parameters quantifying
structural changes in the control and vibrated bone sections. n=30 points from 6
rats for the control group, n=34 points from 9 rats for the 125 Hz group, n=40
points from 9 rats for the 250 Hz group. A) Empty lacunae count. B) Osteocyte count/total.
. The number of empty lacunae in the bone tissue increased with increase in the
vibration frequency (Fig. 5A). The empty lacunae
count for the 250 Hz frequency group (10.82 ± 0.66) was statistically significant
(p<0.05) in comparison to the control group (7.41 ± 0.76, Fig. 5A). For the 125 Hz group (9.09 ± 0.69),
although an increase in the empty lacunae count was observed, the increase was
insignificant (p=0.1) compared to the control group. The increase in
empty lacunae count between 125 Hz and 250 Hz groups showed marginally significant
p-value of 0.07.
Further, the ratio of the number of osteocytes to the total number of osteocytes and
empty lacunae count (denoted as total) is quantified. An opposite trend was observed in
this parameter, with the osteocyte count/total decreasing with an increase in the
vibration frequency (Fig. 5B). The osteocyte
count/total decrease was statistically significant for the 250 Hz frequency group (0.73 ±
0.01) as compared to control group (0.8 ± 0.02, Fig.
5B). The osteocyte count/total remained insignificant (p=0.13)
for the 125 Hz group (0.76 ± 0.02) compared to control. Also, the decrease in count
between 125 Hz and 250 Hz was insignificant (p=0.18). In summary, routine
histology using H&E stain showed that 250 Hz causes highest number of empty lacunae
with a corresponding reduced osteocyte count/total in the cortical bone.
Quantification of the Static Histomorphometry of Trabecular Bone. The
extent of alterations in trabecular bone morphology was quantified using static
histomorphometry. No significance was found for the effects of vibration frequency on
Tb.Sp and Tb.Th considering p<0.05. The value of Tb.Sp for 125 Hz was
0.05 ± 0.005 mm, which remained almost the same (p=0.1) in comparison to
the control group (0.04 ± 0.005 mm). The value of Tb.Sp for 250 Hz was 0.05 ± 0.004 mm,
and showed no significance (p=0.2) compared to the control. Also, the
value of Tb.Th for the 125 Hz group was 0.10 ± 0.007 mm, and for the 250 Hz group was 0.09
± 0.006 mm. Both the groups had insignificant changes compared to control group (0.10 ±
0.007 mm).
Overall, the trabecular bone morphology (represented by trabecular thickness and
separation) was largely unaffected due to the frequency of vibration in comparison to the
control group.
Quantification of the Nitrotyrosine Signal. The nitric oxide-dependent
oxidative injury was quantified based on the NT signal intensity. The bar plots
summarizing the effects of vibration frequency on NT signal intensity in the bone are
shown in Fig. 6
Bar plot showing the effects of vibration frequency on the biological changes
quantified using Nitrotyrosine signal intensity (GSV). n=13 points from 6 rats for
the control group, n=23 points from 9 rats for the 125 Hz group, n=19 points from 9
rats for the 250 Hz group.
. As the vibration frequency increased, the NT signal intensity in the bone
increased. In comparison to the control (157.99 ± 3.47 GSV), the NT Signal Intensity in
trabecular bone was statistically significant for both 125 Hz (168.12 ± 2.74 GSV) and
250 Hz (174.09 ± 2.89 GSV) frequency groups (Fig.
6).
In summary, these results demonstrate that structural disruption in cortical bone was
significant at 250 Hz while the structural damage in the trabecular bone was insignificant
at 125 Hz and 250 Hz. On the contrary, the biochemical changes, assessed by NT signal
intensity, were significant at both 125 Hz and 250 Hz, for the trabecular bone.
Quantitative effects of vibration duration on bone tissue alterations
The effect of days of vibration exposure (1D, 5D, and 10D) on bone alterations were also
assessed using the same methods discussed above. The H&E data and NT signal intensity
data obtained from the control and different time points, 1D, 5D, and 20D (irrespective of
the vibration group) were compared across the number of days of vibration.
Quantification in the Cortical Bone. The effects of days of vibration on
the empty lacunae count (Fig. 7A
Bar plots showing the effects of duration of vibration on parameters quantifying
the structural changes in the bone tissue. n=9 points from 2 rats for the control-1D group, n=11 points from 2 rats for the
control-5D, n=10 points from 2 rats for the control-20D group. n=18 points from 6
rats for the 1D group, n=28 points from 6 rats for the 5D group, n=28 points from 6
rats for the 20D group. A) Empty lacunae count with individual control groups. B)
Osteocyte count/total with individual control groups.
) and Osteocyte count/total (Fig. 7B) are
summarized in Fig. 7. Fig. 7A shows the bar plots comparing the empty lacunae counts in
cortical bone for 1D, 5D and 20D vibration groups with their respective control groups.
Empty lacunae count was statistically significant for 5D vibrated group (11.54 ± 0.64)
compared to its own control group (5.13 ± 1.06). No statistical significance
(p>0.05) was obtained for either the 1D vibrated group (9.3 ± 0.8)
when compared to the 1D control group (8.03 ± 1.12) or the 20D vibrated group (8.91 ±
0.64) compared to the control group at 20D (9.2 ± 1.09). Vibration at 5D time point caused
highest number of empty lacunae.
Figure 7B shows the bar plots comparing the
osteocyte count/total in the cortical bone of the vibrated bone groups with their
respective control groups, at different durations of vibration (1D, 5D and 20D). The
osteocyte count/total was statistically significant for 5D vibrated group (0.72 ± 0.01)
compared to the control group at 5D (0.85 ± 0.02). No statistical significance was
obtained for either 1D vibrated group (0.77 ± 0.02) when compared to the 1D control group
(0.79 ± 0.03) or the 20D (0.76 ± 0.01) compared to the control group at 20D (0.74 ±
0.02).
Overall, the results demonstrate that 5D time point caused highest number of empty
lacunae with a corresponding reduced osteocyte count. Also, the reduction in empty lacunae
count was significant between 5D and 20D time points.
Quantification in the Trabecular Bone. The Tb.Sp was statistically
significant for the 20D group (0.06 ± 0.005 mm) as compared to control group (0.04 ±
0.004 mm). The increase in count between 5D (0.04 ± 0.004 mm) and 20D was also
significant. No significance (p=0.05) was found for the 1D group (0.06 ±
0.005 mm) compared to control. Also, no significance was found (p=0.05)
for the decrease in Tb.Sp between 1D and 5D groups. Similar results were obtained when
comparisons were made between the vibrated groups and their respective control groups.
Overall, these results show that trabecular spacing is dependent on the 20D time point of
vibration and is lesser for vibrated group compared to control group.
The Tb.Th was not statistically significant for the 1D group (0.08 ± 0.008 mm) as
compared to control group (0.10 ± 0.006 mm). Similarly, no significance
(p=0.16) was found for the increase in count between 1D (0.04 ±
0.004 mm) and 5D group (0.10 ± 0.006 mm). Also, no significance was found
(p=0.18) for the decrease in Tb.Th between 1D and 20D group (0.10 ±
0.005 mm). Similar results were obtained when comparisons were made between the vibrated
groups and their respective control groups.
Overall, these results show that the trabecular spacing is dependent on vibration
duration (20D) while trabecular thickness is not dependent on duration of vibration.
Quantification of the biochemical damage.Figure 8
Bar plots showing the effects of duration of vibration on biological changes
quantified using Nitrotyrosine signal intensity (GSV). n=5 points from 2 rats for
the control-1D group, n=5 points from 2 rats for the control-5D, n=3 points from 2
rats for the control-20D group. n 18 points from 6 rats for the 1D group, n=14
points from 6 rats for the 5D group, n=10 points from 6 rats for the 20D group.
shows the bar plot summarizing the effects of duration of vibration on NT signal
intensity in bone. A comparison of NT signal intensity at different time points with their
respective control groups is provided. NT signal intensity in bone was statistically
significant for 20D vibrated group (173.5 ± 3.5) compared to the control group at 20D
(145.0 ± 6.3). The 5D vibrated group (171.4 ± 3.2) showed near significance
p-value of 0.08, when compared to the control group at 5D (160.3 ±
5.4). No statistical significance at p<0.05 was obtained for the 1D
vibrated group (168.4 ± 3.0) when compared to the 1D control group (165.7 ± 5.4).
Overall, these results show that biochemical damage increases with an increase in the
duration of vibration exposure, with a significant effect observed at 20D time point.
Material testing on bone mineralization. In order to augment our
histological methods, we performed material tests to assess the effect to vibration
frequency on mineralization of the bone, at the 20D time point. The chemical composition
of the bone was assessed using the%wt of Ca in the bone and the ratio of Ca/P. These
results are summarized in the Fig. 9
Quantification of mineral content by EDX. (A) Bar plot showing the effect of
vibration frequency on Ca content (wt%); (B) Bar plot showing the effect of
vibration frequency on Ca/P ratio of rat bone.
.
There was a significant decrease in the Ca at 125 Hz (42.99 ± 0.88) in comparison to the
control group, while the 250 Hz (46.09 ± 0.85) remained insignificant compared to the
control (47.79 ± 1.05). The increase in%wt of Ca was also significant between the 125 Hz
and 250 Hz groups (Fig. 9A).
Similarly, the Ca/P ratio was significantly lower for 125 Hz (1.38 ± 0.04) compared to
Control (1.53 ± 0.05, Fig. 9B), while no
significance was found for 250 Hz (1.49 ± 0.04). However, the increase in Ca/P ratio from
125 Hz to 250 Hz was found to be marginally significant (p=0.07).
Further, assessment of the amount of mineralization in the bone tissue was performed.
These results were summarized in Fig. 10
Degree of Mineralization of bone from BSE images. (A) shows the representative
mineralization curve for bone for all the groups; (B) shows bar plot summarizing the
degree of mineralization (denoted by mean grey level) for all the groups.
. The mean grey level is an indicator of mineralization degree and a higher grey
level signals higher mineralization (Fig. 10A).
Quantification of these results show that 250 Hz causes higher degree of heterogeneous
mineralization in bone and the mineralization is not as homogeneous as compared to either
control or 125 Hz. 250 Hz showed significantly higher mineralization (141.18 ± 9.52)
compared to control (111.3 ± 11.4). The increase in mineralization was also significant
between 125 Hz (109.84 ± 11.43) and 250 Hz (Fig.
10B).
In summary, the Ca and Ca/P ratio were significant at 125 Hz while the bone
mineralization was significant at 250 Hz. However, these results were assessed only at the
20D time point. In order to assess the duration effect, further studies are needed.
Discussion
We have studied vibration-induced bone tissue alterations in the form of structural damage,
observed by: i) empty lacunae and osteocyte counts in cortical bone and changes in
trabecular bone structure, and; ii) biochemical damage due to oxidative injury (NT
formation). Results of this study show that the structural damage was significant at
250 Hz for cortical bone while it remained insignificant at both the frequencies
for the trabecular bone. On the other hand, the biochemical damage was significant
at both the 125 Hz and 250 Hz vibration frequencies.
Further, the results of this study show that the structural damage was significant
at 5D time point for the cortical bone while the trabecular bone remained largely
unaffected, with a change in trabecular spacing observed at 20D time point. The biochemical
damage increased with increase in duration of vibration, showing a significant
change at 20D time point and a near significant change at 5D time point.
A change in the NT signal intensity in the individual control groups at 1D, 5D, and 20D
time point was observed. The NT signal intensity values decreased as the duration increased
(Fig. 8B). However, this decrease was not
statistically significant. Further, this decrease in NT signal intensity with an increase in
the duration might be due to the acclimatization of the rats to the new environment and
additionally due to the change in age of the rats. However, such changes need further
investigation.
This study showed that bone alterations are dependent on duration of vibration. Present
literature lacks comparison of NT staining on rat-tail bone tissue at different time
points of vibration. Further, the biochemical response of trabecular bone and its
oxidative damage due to the change in the duration of vibration remained unknown.
Effect of frequency of vibration. There are primarily three types of bone
cells, namely osteoblasts, osteoclasts and osteocytes, that are expected to respond to
vibratory stimulus. Osteocytes are the differentiated phenotype of osteoblasts and lie
embedded in bone matrix, sensing loading through numerous sensory canalicular networks35). Study of these microscopic osteocytes
under vibration can lead to improved assessment of the macroscopic dynamic characteristics
of bone structure and any injury thereof.
In this study, an increased count of empty lacunae and decreased osteocyte count at 250 Hz
was observed in the cortical bone. A previous study documented a reduced osteocyte density
and higher number of empty lacunae in bones of osteoporotic sheep model36). Since osteocytes are the most abundant cells in bone
matrix and are responsible for providing signals to osteoblasts (bone-forming cells), the
decrease in their count along with increase in empty lacunae indicates that osteocytes are
dying through either necrosis or apoptosis. Necrosis is more common in tissue damage due to
external forces like heat, radiation, hypoxia or crushing of the tissue during handling.
Apoptosis is a programmed phenomenon caused due to a stimulus leading to a specific pathway
of cell death. Higher number of empty lacunae at 250 Hz indicate that this frequency may
result osteocytes to undergo cell death, which might be due to a combination of both
necrosis and apoptosis.
The balance between the osteocytes (bone-forming cells) and the osteoclasts
(bone-resorption cells) is important to determine the bone structure. A previous study37) has shown that low-magnitude vibrations in
the frequency range of 20–90 Hz affect osteocytes, which in turn, respond by inhibiting the
formation of osteoclasts. Another study10)
reported that at frequencies in the range of 5–100 Hz, in a dense bone, osteocyte cells
oppose the presence of osteoclasts. Denser bone is more responsive to higher frequency
(>100 Hz) loading. Hence, when denser bones are subjected to higher frequency vibrations
(>100 Hz), the number of osteoclasts is lowered. These lower numbers of osteoclasts, in
turn, stimulate the activity of osteoblasts. Hence, in denser bones, higher frequency
vibrations (>100 Hz) might not degrade the bone tissue but might maintain it. On the
other hand, less dense bones might suffer from degradation at higher frequency (>100 Hz)
loading since the bone resorption is more pronounced due to the increased presence of
osteoclast activity as opposed to the bone forming activity of osteoblasts. To explore this
phenomenon in detail, further studies are warranted to explore cell death using stains
specific to apoptosis, like cleaved caspase-3 stains or TUNEL assays.
Further, results of the present study on significant NT formation in bone are in agreement
with previous studies. For example, a significant amount of NT staining was noted in femur
metaphyses and chondrocytes in rats, after accumulation of iNOS protein38). Some researchers observed higher oxidative stress in the
subchondral bone due to higher accumulation of NT39). To the best of our knowledge, this is probably the first study to
report NT formation in bone tissue under the influence of higher frequency of vibrations
(125 Hz and 250 Hz), at various durations of vibration.
Effect of vibration on trabecular bone. The structure of bone displays
spatial heterogeneity and is primarily composed of trabecular and cortical bone, which are
structurally different and exhibit different rates of degeneration after a mechanical
stimulus is applied23). In the literature,
there are reports which document that trabecular bone is more sensitive to vibration
loading40). An increased trabecular
thickness and a reduced trabecular spacing have been documented in rat bones when a
mechanical vibration stimulus was applied at a vibration frequency of 50 Hz24). Based on these studies, we assessed
trabecular microarchitecture using two parameters: i) trabecular spacing (Tb.Sp); and ii)
trabecular thickness (Tb.Th). Significant increase in Tb.Sp was observed for vibration at
20D time point; however, Tb.Th did not show significance at 20D. Perhaps, this increase in
spacing might relate edematous changes, wherein excess fluid passes from the blood plasma to
the tissue spaces through damaged endothelium. The discrepancy in the results of Tb.Th might
be due to the higher frequency of vibrations, 125 Hz, and 250 Hz, used in this study as
opposed to the lower frequencies (50 Hz) used in the previous studies32, 33, 38). Further studies are warranted to better evaluate the
response of trabecular bone to different frequencies of vibrations.
Limitations. For this study, normal control group41) has been used as opposed to rats sham-vibrated in
restraints (restraint controls). The lack of restraint in a normal control group introduces
additional bias. The rats in the normal control group have not been exposed to either
constraint or vibration stress. Therefore, the use of restraint controls could be important
to assess any additional variables introduced into the analysis. The authors acknowledge
this as a limitation and consider it to be addressed in future study, when larger sample
sizes are assessed.
The effect of frequency dependence that is near resonance range was investigated to
evaluate vibration-induced bone tissue alterations by exposing rats to vibration of a
constant acceleration of 49 m/s2 RMS. These values were selected since the
vibration frequency responses can be better assessed using rat-tail model42) in workers who use hand held powered tools
are frequently exposed to these vibration parameters (125 Hz, 250 Hz, 49 m/s2).
However, as reported in some studies, high impact vibrations, for example with frequencies
<32.5 Hz might also lead to vibration injury. The effect of these frequencies needs to be
explored in future studies.
The effect of variable vibration amplitude was not considered in the present study. Since
HAVS is a complex disorder with interplay of vibration parameters of frequency, amplitude,
acceleration and duration, future studies are warranted to give better assessment of
vascular damage. In addition, the effect of energy constant condition as opposed to
acceleration constant condition used in this study might somewhat affect the results. This
effect would be explored in future studies. Further, gene expression studies to delineate
the biochemical changes and mineralization studies to assess the material properties of bone
using alkaline phosphatase marker are currently being considered. We also plan to
investigate the role of sclerostin43)
produced by osteocytes under various vibration loading conditions. Lastly, the current study
has a smaller sample size. Hence, the generalizability of the current findings is limited.
The future plan is to augment the current results using a larger sample size.
Conclusions
In summary, structural alterations in the cortical bone occurred significantly at 250 Hz
while biochemical alterations occurred significantly at both 125 Hz and 250 Hz. The
trabecular bone remained unaffected due to change in vibration frequency. These findings
demonstrate that bone damage is dependent on frequencies near resonance: 125 Hz and 250 Hz.
Hence the current ISO guidelines might underestimate the bone damage at frequencies
>100 Hz.
Also, the structural damage was significant at 5D for cortical bone while the trabecular
bone showed significant damage at 20D time point. Further, the biochemical damage increased
with increase in the duration of vibration with a significant damage observed at 20D time
point and a near significant damage observed at 5D time point. Hence, the duration of
vibration exposure might also play a role in elucidating degenerative changes in the bone
tissue.
This work was supported by NIOSH-PRP grant: T42/OH008432-06. We would like to acknowledge
the statistical guidance and advice provided by Dr. Paul Succop, Research Professor,
Biostatistics, Department of Environmental Health, University of Cincinnati. The authors
would like to thank Mr. Soumyarwit Manna for his considerable help with the material
characterization analyses of the bone tissue. The authors are grateful to Dr. Randall
Allemang, Director of the Structural Dynamics Research Laboratory, for granting access to
the vibration equipment.
Appendix 1.Interaction effects on structural and biochemical damage
A summary of the 1-way ANOVA analyses between the groups, Control-1D, 125 Hz-1D,
250 Hz-1D; Control-5D, 125 Hz-5D, 250 Hz-5D; Control-20D, 125 Hz-20D, and 250 Hz-20D, is
presented in Fig. 11
Bar plots showing the effects of vibration frequency and duration on the
structural and biological changes occurring in the interaction groups. A) Empty
lacunae count. n=9 points from 2 rats for the control-1D group, n=11 points from 2
rats for the control-5D, n=10 points from 2 rats for the control-20D group. N=8
points from 3 rats for the 125 Hz-1D group, n=13 points from 3 rats for the
125 Hz-5D group, n=13 points from 3 rats for the 125 Hz-20D group. N=10 points
from 3 rats for the 250 Hz-1D group, n=15 points from 3 rats for the 250 Hz-5D
group, n=15 points from 3 rats for the 250 Hz-20D group. B) Osteocyte count/total.
The sample size is the same as the above. C) Nitrotyrosine signal intensity (GSV).
N=5 points from 2 rats for the control-1D group, n=5 points from 2 rats for the
control-5D, n=3 points from 2 rats for the control-20D group. N=10 points from 3
rats for the 125 Hz-1D group, n=7 points from 3 rats for the 125 Hz-5D group, n=6
points from 3 rats for the 125 Hz-20D group. N=8 points from 3 rats for the
250 Hz-1D group, n=7 points from 3 rats for the 250 Hz-5D group, n=4 points from 3
rats for the 250 Hz-20D group.
. The interaction effects of groups of vibration frequency and days of vibration
on structural parameters, empty lacunae count and Osteocyte count/total is presented in
Fig. 11A, and Fig. 11B, respectively. The interaction effects of groups on
biochemical parameter, NT signal intensity is presented in Fig. 11C.The empty lacunae count was not statistically
significant (p>0.05; Fig.
11A) for 125 Hz-1D group (8.4 ± 1.1) and 250 Hz-1D group (10.1 ± 0.9) compared
to the control group at 1D (8.0 ± 0.9). On the other hand, the 125 Hz-5D group (10.8 ±
0.9) and the 250 Hz-5D group (12.2 ± 0.8) showed significant
(p<0.05) difference in comparison to the 5D control group (5.1 ±
0.9). No statistical significance (p>0.05) was obtained for either
125 Hz-20D group (7.8 ± 0.9) or the 250 Hz-20D group (9.9 ± 0.8) when compared to the
20D control group (9.2 ± 0.9).The osteocyte count/total was not statistically
significant (p>0.05; Fig.
11B) for 125 Hz-1D group (0.78 ± 0.03) and 250 Hz-1D group (0.76 ± 0.02)
compared to the control group at 1D (0.79 ± 0.02). On the other hand, the 125 Hz-5D
group (0.73 ± 0.02) and the 250 Hz-5D group (0.74 ± 0.02) showed significant difference
(p<0.05) in comparison to the 5D control group (0.85 ± 0.02). No
statistical significance (p>0.05) was obtained for either 125 Hz-20D
group (0.78 ± 0.02) or the 250 Hz-20D group (0.74 ± 0.02) when compared to the 20D
control group (0.74 ± 0.02).The NT signal intensity was not statistically significant
(p>0.05; Fig. 11C) for
125 Hz-1D group (167.6 ± 3.6) and 250 Hz-1D group (169.3 ± 3.9) compared to the control
group at 1D (165.7 ± 4.8). On the other hand, the 125 Hz-5D group (171.8 ± 4.0) was
statistical (p<0.05) significance in comparison to the 5D control
group (160.4 ± 3.9) while the 250 Hz-5D group (170.9 ± 4.1) showed borderline
significant (p=0.08) difference in comparison to the 5D control group
(160.4 ± 3.9). Further, statistical significance (p<0.05) was
obtained for both the 125 Hz-20D group (164.4 ± 4.3) and the 250 Hz-20D group (184.8 ±
4.8) when compared to the 20D control group (144.9 ± 5.7).Overall, these results show a
significant change in the structural parameters at both the 125 Hz and 250 Hz at the 5D
timepoint. Similarly, the biochemical damage was significant at 125 Hz vibration at 5D
and borderline significant at 250 Hz-5D. The biochemical damage was significant at the
20D time point for both the 125 Hz and 250 Hz.
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