- Email: [email protected]
Whole-body vibration of mice induces articular cartilage degeneration with minimal changes in subchondral bone
Accepted Manuscript Whole-body vibration of mice induces articular cartilage degeneration with minimal changes in subchondral bone Matthew R. McCann, Cynthia Yeung, Michael A. Pest, Anusha Ratneswaran, Steven I. Pollmann, David W. Holdsworth, Frank Beier, S. Jeffrey Dixon, Dr. Cheryle A. Séguin PII:
S1063-4584(16)30387-9
DOI:
10.1016/j.joca.2016.11.001
Reference:
YJOCA 3885
To appear in:
Osteoarthritis and Cartilage
Received Date: 2 March 2016 Revised Date:
29 July 2016
Accepted Date: 2 November 2016
Please cite this article as: McCann MR, Yeung C, Pest MA, Ratneswaran A, Pollmann SI, Holdsworth DW, Beier F, Dixon SJ, Séguin CA, Whole-body vibration of mice induces articular cartilage degeneration with minimal changes in subchondral bone, Osteoarthritis and Cartilage (2016), doi: 10.1016/j.joca.2016.11.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
1 2
Title: Whole-body vibration of mice induces articular cartilage degeneration with minimal changes in subchondral bone
3 4 5 6 7
Running Title: WBV leads to cartilage degeneration
RI PT
a
SC
Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada. N6A 5C1 b Dentistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada. N6A 5C1 c Imaging Research Laboratories, Robarts Research Institute; Department of Medical Biophysics, and Department of Surgery, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada. N6A 5C1 d Bone and Joint Institute, University of Western Ontario, London, Ontario, Canada. N6A 5C1
M AN U
8 9 10 11 12 13 14 15 16
Authors: Matthew R. McCanna,d, Cynthia Yeunga, d, Michael A. Pesta, d, Anusha Ratneswarana, d, Steven I. Pollmannd, David W. Holdsworthc,d, Frank Beiera,d, S. Jeffrey Dixona,b,d, Cheryle A. Séguina,d
17
30 31 32
TE D
*Corresponding Author: Dr. Cheryle A. Séguin Department of Physiology and Pharmacology Schulich School of Medicine & Dentistry The University of Western Ontario London, Ontario, Canada, N6A 5C1 Email: [email protected]
EP
Keywords: Whole Body Vibration, Knee Joint, Cartilage Degeneration, Subchondral Bone
AC C
18 19 20 21 22 23 24 25 26 27 28 29
33 34 35
1
ACCEPTED MANUSCRIPT
ABSTRACT
37
Objective: Low-amplitude, high-frequency whole-body vibration (WBV) has been
38
adopted for the treatment of musculoskeletal diseases including osteoarthritis; however,
39
there is limited knowledge of the direct effects of vibration on joint tissues. Our recent
40
studies revealed striking damage to the knee joint following exposure of mice to WBV.
41
The current study examined the effects of WBV on specific compartments of the murine
42
tibiofemoral joint over 8 weeks, including microarchitecture of the tibia, to understand
43
the mechanisms associated with WBV-induced joint damage.
44
Design: Ten-week-old male CD-1 mice were exposed to WBV (45 Hz, 0.3 g peak
45
acceleration; 30 min/day, 5 days/week) for 4 weeks, 8 weeks, or 4 weeks WBV followed
46
by 4 weeks recovery. The knee joint was evaluated histologically for tissue damage.
47
Architecture of the subchondral bone plate, subchondral trabecular bone, primary and
48
secondary spongiosa of the tibia was assessed using micro-CT.
49
Results: Meniscal tears and focal articular cartilage damage were induced by WBV; the
50
extent of damage increased between 4 and 8-week exposures to WBV. WBV did not alter
51
the subchondral bone plate, or trabecular bone of the tibial spongiosa; however, a
52
transient increase was detected in the subchondral trabecular bone volume and density.
53
Conclusions: The lack of WBV-induced changes in the underlying subchondral bone
54
suggests that damage to the articular cartilage may be secondary to the meniscal injury
55
we detected. Our findings underscore the need for further studies to assess the safety of
56
WBV in the human population to avoid long-term joint damage.
AC C
EP
TE D
M AN U
SC
RI PT
36
57 58 59
2
ACCEPTED MANUSCRIPT
INTRODUCTION
60
Mechanical loading is critical for maintaining joint tissue homeostasis. Articular
62
cartilage health is paramount to overall joint function and is typically well maintained
63
under normal physiological loading [1]. Moderate physical activity has been shown to
64
sustain cartilage health in human [2] and animal studies [3]. In contrast, insufficient
65
mechanical loading can lead to articular cartilage degeneration; progressive thinning of
66
the articular cartilage is detected in patients with paralysis compared to healthy mobile
67
individuals [4]. Cartilage loss can be detected relatively quickly following changes in
68
mechanical loading; partial load bearing associated with walking with crutches resulted in
69
atrophy of knee joint articular cartilage in just 7 weeks [5]. Conversely, mechanical
70
overloading is perhaps the best-studied initiator of joint degeneration. Articular cartilage
71
is highly susceptible to osteoarthritis (OA) if subjected to acute [6] or chronic [7] impact
72
loading. Altered mechanical loading following injury to the knee joint, modelled in
73
rodents using surgically-induced joint destabilization (e.g. destablization of the medial
74
menicus, anterior cruciate ligament transection), leads to OA-like phenotypes [8].
75
Although the pathological processes mediating the progression of OA remain the focus of
76
ongoing investigation, OA is now generally considered a disease of the entire joint in
77
which articular cartilage loss, meniscus and ligament damage and the periarticular bone
78
changes contribute to the overall disease [9].
SC
M AN U
TE D
EP
AC C
79
RI PT
61
Whole-body vibration (WBV) training was originally implemented clinically as a
80
therapeutic approach to increase bone mass for the treatment of osteoporosis [10], and is
81
capable of increasing muscle strength and power [11] [12]. Underlying these biological
82
effects is the principle that WBV increases bone remodeling [13] and motor unit
3
ACCEPTED MANUSCRIPT
recruitment thresholds in muscle [14]. Contributing to the increasing popularity of WBV
84
training in the fitness industry, studies have reported beneficial effects of WBV on
85
neuromuscular performance in healthy individuals, including flexibility and strength [15].
86
However, recent studies suggest these effects may vary based on age, gender or health
87
status [16].
RI PT
83
Clinical trials have recently evaluated the therapeutic effect of WBV on joint pain
89
and functional performance in patients with chronic knee OA [17]. A number of studies
90
report beneficial effects including reduced pain intensity, increased muscle strength, and
91
reductions in the plasma concentrations of inflammatory markers in OA patients
92
following WBV training [18, 19]. Conversely, some trials reported no significant
93
improvements in pain intensity or other parameters among knee OA patients following
94
WBV [19]. Importantly, in these studies joint health was not directly assessed.
95
Quantification of the direct effects of WBV on knee joint tissues are limited; one study
96
reported the ability of WBV to prevent loss of tibial articular cartilage thickness
97
(determined by MRI) following prolonged immobilization in healthy male subjects [20].
98
A recent meta-analysis evaluating the use of WBV in knee OA management concluded
99
that there were no significant differences in functional performance or patient self-
100
reported pain following WBV, likely related to the diversity of the protocols implemented
101
[19]. Together, these findings suggest the need for for further studies evaluating the
102
safety and efficacy of WBV prior to its therapeutic implementation for the treatment of
103
OA.
AC C
EP
TE D
M AN U
SC
88
104
To address these issues, recent studies from our group evaluated the effect of
105
WBV on joint health using the mouse as a preclinical model. Our findings demonstrated
4
ACCEPTED MANUSCRIPT
that 4 weeks of repeated exposure of healthy young mice to WBV, using protocols
107
reported to be beneficial to bone, induced OA-like knee joint damage marked by
108
degeneration of both the meniscus and articular cartilage as well as activation of MMP-
109
mediated matrix degradation [21]. Building from these findings, the present study was
110
designed to determine: i) if knee joint degeneration was progressive with extended
111
exposure to WBV or was potentiated by cessation of vibration, and ii) if WBV promoted
112
changes in the subchondral bone that would constitute a potential mechanism for the OA-
113
like pathology detected in the knee.
M AN U
SC
RI PT
106
METHODS
114
Whole-Body Vibration. All procedures were approved by the Animal Use Committee at
116
The University of Western Ontario and this study was conducted in accordance the
117
ARRIVE guidelines [22]. Based on protocols of WBV used in our previous studies to
118
model those used clinically [21], 10-week-old male CD-1 mice were exposed to vertical
119
sinusoidal vibrations (frequency of 45 Hz, peak-to-peak amplitude of 74 µm, and 0.3 g
120
peak acceleration) for 30 min/day, 5 days/week for 4 weeks, 8 weeks, or 4 weeks WBV
121
followed by 4 weeks recovery, using a previously described vibration platform [23, 24].
122
Age-matched controls were housed in identical chambers on a sham (non-vibrated)
123
platform to replicate handling and environmental conditions. Following WBV, mice were
124
returned to conventional housing and monitored daily. Twenty-four hours after the final
125
exposure to WBV, mice were euthanized by a lethal dose of sodium pentobarbital. Spinal
126
tissues were isolated to characterize effects of WBV on the intervertebral disc and
127
vertebral bone, detailed in [25].
AC C
EP
TE D
115
128
5
ACCEPTED MANUSCRIPT
Micro-computed Tomography (micro-CT). Right hindlimbs (mid-femur to ankle) were
130
isolated and fixed in 4% paraformaldehyde (PFA) for 24 h and embedded in 1% agarose
131
in PBS in 50 mL conical tubes. Tissues were scanned using a laboratory micro-CT
132
scanner (eXplore Locus, GE Healthcare Biosciences) with an internal calibrating
133
phantom composed of air, water, and synthetic bone-mimicking epoxy (SB3 2.8 x 3.4 x 8
134
mm; Gammex RMI). The scanning protocol consisted of 900 projection images acquired
135
at a 0.4º angle increment, obtained over 165 minutes of gantry rotation. The x-ray tube
136
potential was 80 kVp with a tube current of 450 µA, an exposure time of 4,500 msec, and
137
2 frames per view angle averaged. Images were reconstructed into 3D volumes with 20
138
µm isotropic voxel spacing and linearly rescaled into Hounsfield units using the internal
139
calibration standards, as previously described [26]. Using three-dimensional visualization
140
and analysis software (MicroView, GE Healthcare Biosciences) full volumes were
141
cropped to contain only the tibia and fibula, and reoriented to the same axes. Regions of
142
interest (ROI) were manually outlined to define the subchondral bone plate, subchondral
143
trabecular bone, primary and secondary spongiosa of the tibia, where each compartment
144
was marked within a series of 2D planes and splined together to generate 3D ROIs. Bone
145
morphometry was quantified using the Bone Analysis tool in MicroView, as previously
146
reported [27].
SC
M AN U
TE D
EP
AC C
147
RI PT
129
148
Histological Assessment and Scoring. Following micro-CT, right hind limbs were
149
decalcified for 10 days in 5% EDTA in PBS (pH 7.0), processed, embedded in paraffin
150
wax, and 5 µm coronal sections were obtained. Serial sections were taken every 50 µm to
151
encompass all weight-bearing areas of the femoro-tibial joint. Using established protocols
6
ACCEPTED MANUSCRIPT
[23, 28], sections were stained with 0.1% Safranin O/0.02% fast green to detect
153
proteoglycans and glycosaminoglycans. Sections were imaged on a Leica DM1000
154
microscope with Leica Application Suite (Leica Microsystems). The four quadrants of
155
the knee joint were scored by three independent blinded observers for degeneration using
156
the murine Osteoarthritis Research Society International (OARSI) recommended
157
histopathologic scale [29].
RI PT
152
SC
158
Statistical Analyses. Data are presented from experiments conducted with 5-6 mice per
160
treatment group. Parameters from mice exposed to WBV were compared to those from
161
age-matched non-vibrated sham controls using a parametric, two-tailed, unpaired t-test
162
with a Welch’s correction (4 week time point), or one-way ANOVA with a Tukey post-
163
hoc test (8 week time point). P < 0.05 was considered significant.
M AN U
159
165
TE D
164
RESULTS
To assess the effects of daily WBV on the knee joint and determine if protocols of
167
WBV used clinically [30] induce long-term joint damage, 10-week-old male CD-1 mice
168
were exposed to sinusoidal WBV (30 min/day, 5 days/week, at 45 Hz, 0.3 g peak
169
acceleration) for 4 weeks, 8 weeks, or 4 weeks of WBV followed by 4 weeks without
170
WBV (recovery) (Figure 1). Consistent with our recent study [21], analysis of Safranin
171
O/fast green stained histological sections demonstrated that 4 weeks of WBV induced
172
knee joint damage in 3 of 6 mice (OARSI score >2), localized to the medial joint
173
compartment in 2 mice and the lateral femoral condyle in the third mouse. In 1 of these
174
mice, articular damage was detected on both the femoral and tibial condyles. The OARSI
AC C
EP
166
7
ACCEPTED MANUSCRIPT
semi-quantitative histologic scoring system assigns a grade between 0-6 to each joint
176
quadrant, where a grade of 2 is characterized by loss of articular cartilage with vertical
177
clefts extending beyond the superficial layer and some loss of surface lamina. WBV-
178
induced damage included evidence of meniscal tears, focal articular cartilage defects with
179
erosion down to the tide mark, loss of glycosaminoglycans in the superficial layer, and
180
osteophyte formation on the medial tibial plateau and femoral condyle (Figure 2A). No
181
joint damage (OARSI >2) was detected in any age-matched sham controls. Despite these
182
findings, no significant differences were detected in the average maximum OARSI scores
183
between mice exposed to 4 weeks of WBV and sham controls.
M AN U
SC
RI PT
175
To determine if WBV-induced joint damage was progressive with continued daily
185
WBV or potentiated by inclusion of a recovery period, mice were exposed to either 8
186
weeks of WBV or 4 weeks of WBV followed by 4 weeks without (recovery). Following
187
8 weeks of WBV, 5 of 6 mice demonstrated articular cartilage damage (OARSI score
188
>2), localized to the medial joint compartment in 2 mice, the lateral joint compartment in
189
1 mouse, and both the medial and lateral joint compartments in 1 mouse. In 3 of these
190
mice, articular damage was detected on both the femoral and tibial condyles. Following 8
191
weeks WBV, joint damage was characterized by severe loss of meniscal tissue, focal
192
articular
193
glycosaminoglycans in the superficial layer of articular cartilage adjacent to focal defects,
194
and osteophyte formation on the medial tibial plateau and femoral condyle (Figure 2B).
195
Focal articular cartilage damage (OARSI score >2) was also detected in 2 of 6 age-
196
matched sham controls, localized to the medial tibial plateau. Exposure of mice to 8
AC C
EP
TE D
184
cartilage
lesions
with
erosion
8
to
the
subchondral
bone,
loss
of
ACCEPTED MANUSCRIPT
197
weeks of WBV induced a significant increase in the average maximum OARSI score in
198
the lateral tibial plateau compared to age-matched sham controls. Articular cartilage damage (OARSI score >2) was detected in only 1 of 5 mice
200
subjected to 4 weeks of WBV followed by 4 weeks of recovery (WBV/REC). Of note
201
however, in this animal the meniscal and articular cartilage damage detected was similar
202
to that of mice exposed to 4 weeks WBV (Figure 2B), suggesting that inclusion of a
203
recovery period failed to mitigate the damage induced by WBV in this animal.
SC
RI PT
199
We next examined if WBV promoted changes to the subchondral bone
205
microarchitecture that would correlate with WBV-induced soft tissue damage. Using
206
high-resolution micro-CT, we first assessed the subchondral bone plate subjacent to the
207
articular cartilage of the tibial plateau since this region showed the most pronounced
208
WBV-induced articular cartilage degeneration (Figure 3A). Subchondral bone plate
209
thickness was measured within a region of interest measuring 500 µm in mediolateral
210
length and 1,150 µm ventrodorsal length, centred on the medial tibial plateau, based on
211
previously reported protocols [31]. No significant differences in bone plate thickness
212
(mm), bone mineral content (BMC, mg), or bone mineral density (BMD, mg/cm3) were
213
detected in the subchondral bone of mice exposed to 4 weeks of WBV (Figure 3B), 8
214
weeks of WBV, or 4 weeks of WBV followed by 4 weeks recovery (Figure 3C).
TE D
EP
AC C
215
M AN U
204
Bone microarchitecture was next assessed in the epiphyseal subchondral
216
trabecular bone in the medial compartment of the tibia. For each specimen, a region of
217
interest was manually outlined in serial planes to capture trabecular bone within this area;
218
the lowest point of the extensor sulcus was extended as a boundary, and the subchondral
9
ACCEPTED MANUSCRIPT
bone plate, growth plate and cortical bone were excluded (Figure 4A). Exposure of mice
220
to 4 weeks of WBV induced a significant increase in the subchondral trabecular bone
221
volume fraction (BVF) and trabecular BMD (mg HA/cm3) compared to non-vibrated
222
sham controls (Figure 4B). In contrast, no significant differences were detected in BVF
223
or BMD (mg HA/cm3) of the subchondral trabecular bone in mice exposed to 8 weeks of
224
WBV or 4 weeks of WBV followed by 4 weeks recovery compared to age-matched sham
225
controls (Figure 4C).
SC
RI PT
219
Previous studies suggest the primary and secondary spongiosa within long bones
227
as sites that demonstrate robust adaptive bone remodelling in response to dynamic
228
mechanical loading [32]. We therefore assessed bone microarchitecture within both the
229
primary and secondary spongiosa of the tibia, and quantified bone properties within each
230
separately to delineate region-specific changes. A region of interest was defined to
231
include the primary spongiosa, manually outlined in serial planes to capture the trabecular
232
bone within 50 µm distal to the growth plate and exclude the cortical bone (Figure 5A).
233
No significant differences were detected in trabecular BVF or trabecular BMD in the
234
primary spongiosa following 4 weeks of WBV (Figure 5B), 8 weeks of WBV or 4 weeks
235
of WBV followed by 4 weeks recovery (Figure 5C). We then moved distally and
236
measured the secondary spongiosa of the tibia, manually outlined in serial planes to
237
capture the trabecular bone within an area extending 1mm from the most distal point of
238
the primary spongiosa, and exclude the cortical bone (Figure 6A). No significant
239
differences were detected in trabecular BVF or trabecular BMD in the secondary
240
spongiosa following 4 weeks of WBV (Figure 6B), 8 weeks of WBV or 4 weeks of
241
WBV followed by 4 weeks recovery (Figure 6C).
AC C
EP
TE D
M AN U
226
10
ACCEPTED MANUSCRIPT
DISCUSSION
242
The reported beneficial effects of WBV on bone mass and muscle strength have
244
led to increased use of WBV devices both clinically and in the sports and fitness industry
245
to treat pain, increase joint mobility, and enhance neuromuscular performance. However,
246
recent conflicting reports have highlighted inconsistencies in the anabolic effects of WBV
247
on bone, in both animal models [21, 33] and human clinical trials [30, 34]. In this report,
248
we investigated the mechanisms associated with our previous reports of WBV-induced
249
damage to the murine femoro-tibial joint (knee) following exposure of mice to protocols
250
of WBV that model those used clinically [21]. Based on histological characterization, our
251
findings suggest progressive degeneration of the meniscus and articular cartilage
252
following exposure to WBV. Between 4 and 8 weeks, progressive degeneration was
253
marked by severe loss of meniscus tissue, an increase in the both the number of mice
254
showing articular cartilage defects and the extent of the articular cartilage damage,
255
resulting in a significant increase in the average maximum OARSI score. Based on
256
detailed micro-CT based examination of the tibia, we show that WBV-induced joint
257
damage is not associated with either thickening of the subchondral bone plate or changes
258
in the trabecular bone of the primary or secondary spongiosa.
SC
M AN U
TE D
EP
AC C
259
RI PT
243
Although loss of articular cartilage is the hallmark pathological feature in OA,
260
disease progression involves biomechanical and biochemical alterations in the
261
composition, structure and functional properties of the articular cartilage and periarticular
262
bone [35]. Within the bone compartment, OA-associated changes include increases in
263
subchondral cortical bone thickness, early bone remodeling in the subchondral trabeculae
264
resulting in decreased trabecular bone mass, formation of osteophytes at the joint margins
11
ACCEPTED MANUSCRIPT
and expansion of the zone of calcified cartilage leading to thinning of the articular
266
cartilage [36, 37]. Studies from our group and others have demonstrated that rodent
267
models of OA likewise demonstrate hallmark bone changes, including thickening of the
268
subchondral bone plate and decreased BMD in the subchondral trabecular bone,
269
particularly in regions adjacent to affected articular cartilage [38-40]. Given the interplay
270
between these tissues, the current study used high-resolution micro-CT to determine if
271
focal defects in articular cartilage induced by WBV were associated with changes to the
272
periarticular bone. Using manually defined regions of interest, we did not detect
273
significant differences in bone microarchitecture in either the subchondral bone plate or
274
primary and secondary spongiosa of the tibia. These findings are consistent with
275
quantification of bone microarchitecture in the vertebral bone of these mice, which
276
likewise demonstrated no change following exposure to WBV [25]. Our findings did
277
however demonstrate a transient increase in subchondral trabecular bone mass after 4
278
weeks of WBV, although no differences were detected between mice exposed to 8 weeks
279
WBV compared to controls. Interestingly, we noted that following 4 weeks of WBV,
280
mice with the highest OARSI scores also showed the highest bone plate thickness, bone
281
mineral content and bone mineral density. This association was not however seen in mice
282
following 8 weeks of WBV and may therefore reflect a transient adaptive response to
283
vibration loading.
SC
M AN U
TE D
EP
AC C
284
RI PT
265
Sclerostin is a marker of osteocyte differentiation that serves as a negative
285
regulator of bone formation by inhibiting the osteo-anabolic Wnt signalling pathway. In
286
humans, loss of function mutations of the human SOST gene (encoding sclerostin) lead to
287
sclerosteosis [41, 42], and deletion of Sost in mice results in a high bone mass phenotype
12
ACCEPTED MANUSCRIPT
[43]. Despite increased subchondral bone, Sost-deficient mice do not demonstrate
289
changes to the articular cartilage in the knee associated with OA [44]. Interestingly, using
290
a femur osteotomy model of fracture healing in aged mice, a recent study demonstrated
291
that WBV increased the expression of Sost in osteocytes, associated with impaired
292
fracture healing [45]. These findings may be in keeping with recent studies by our group
293
[21] and others [46] highlighting inconsistencies in the previously reported anabolic
294
effects of WBV on bone, as well as the confounding effects of age and hormone levels.
295
Furthermore, these findings highlight the potential role of Wnt signaling as a candidate
296
regulator of WBV mechanotransduction that should be investigated in joint tissues such
297
as meniscus and articular cartilage.
M AN U
SC
RI PT
288
Our previous study reported meniscal damage in mice following 2 weeks of
299
WBV, changes that preceded the detection of overt articular cartilage damage [21].
300
Together with the current study, these findings suggest a correlation between meniscus
301
damage and the induction of articular cartilage degeneration following WBV. As such,
302
we speculate that WBV-induced articular cartilage damage may occur following joint
303
destabilization, rather than as a result of changes in the mechanical environment resulting
304
from increased subchondral bone characteristic of OA. Meniscal damage is often
305
associated with pain and joint dysfunction [47], and loss of meniscal function leads to
306
rapid and progressive osteoarthritis [48]. Damage or partial loss of the meniscus
307
influences the displacement and deformation of the meniscus under load [49], resulting in
308
increased focal-point stresses on the articular cartilage surface of the tibial plateau [50]
309
that we propose lead to the formation of the focal cartilage defects we detected in mice
310
exposed to WBV.
AC C
EP
TE D
298
13
ACCEPTED MANUSCRIPT
An interesting aspect of this and our previous study is the fact that while severe
312
knee joint damage was induced by WBV (including severe damage to the meniscus, focal
313
articular cartilage defects extending to the subchondral bone, and osteophyte formation
314
on both the femoral and tibial condyles), damage was only detected in a subset of mice at
315
each time point examined. As such, statistical analysis of OARSI scores demonstrated
316
significant WBV-induced damage only following 8 weeks of WBV in a single joint
317
compartment. The absence of a significant increase in OARSI scores following 4 weeks
318
WBV, or in multiple joint compartments at either time point may be associated with the
319
limited sample size used in this study. Of note, the individual cages in which mice are
320
housed during WBV allow for free range of movement. We have observed that during
321
vibration, mice alternate through a variety of postures including rearing on hind limbs,
322
free movement on all 4 limbs, grooming and sleeping. It is tempting to speculate that
323
increased time spent in specific postures may be directly associated with increased joint
324
damage since the magnitude of vibration experienced at skeletal sites likely varies due to
325
muscle contraction, soft tissue damping, or resonance effects [51]. We have recently
326
developed non-invasive techniques to quantify vibration amplitude in the skeleton of live
327
mice using radiographic images of surgically implanted metal marker beads [24]. Future
328
studies will utilize this approach to quantify the vibration amplitude and deformations at
329
specific sites and in various postures. This will allow us to directly relate the biological
330
response of joint tissues to WBV to its biomechanical effects.
AC C
EP
TE D
M AN U
SC
RI PT
311
331
While this and previous studies by our group [21, 25] point to alarming potential
332
for damage induced by whole-body vibration on joint tissues, the preclinical model used
333
is associated with inherent limitations. First, these studies were conducted using a single
14
ACCEPTED MANUSCRIPT
protocol of WBV that does not effectively capture the wide range of parameters currently
335
being used. The variables that control the intensity of vibration are the frequency and
336
amplitude (peak-to-peak displacement), which together determine peak acceleration.
337
Clinical protocols of WBV vary widely in frequency (2.5-90 Hz) and acceleration (0.3-
338
3.6 g), and are implemented for periods ranging from one week to one year. Second, we
339
examined the effects of WBV using young, skeletally mature male CD-1 mice. Previous
340
studies have demonstrated that the ability of WBV to increase bone mineral density and
341
bone formation is dependent on age and genetic background in mouse models [52, 53].
342
As such, physiological factors including age, sex, and genetic background may likewise
343
affect the response of joint tissues to WBV. In fact, a recent study from our group
344
reported a lack of WBV-induced degeneration in either the knee or intervertebral discs of
345
C57Bl/6 mice exposed to the same protocols of WBV [54]. Taken together, these studies
346
underscore the need for further rigorous research to assess the relative contribution of
347
vibration parameters and physiological factors in modulating the effects of WBV on joint
348
health.
TE D
M AN U
SC
RI PT
334
In conclusion, WBV results in progressive degeneration of the meniscus and
350
articular cartilage, damage that may not be repaired following removal of the stimulus.
351
Furthermore, we demonstrate that WBV promotes a transient increase in subchondral
352
trabecular bone; however, no change was detected in morphometry of the subchondral
353
bone plate or primary and secondary spongiosa of the tibia.
AC C
EP
349
354 355
ACKNOWLEDGEMENTS
15
ACCEPTED MANUSCRIPT
356
We thank Dr. Kim Beaucage for her contributions, in particular for her assistance with
357
micro-CT training and protocols.
358
AUTHOR CONTRIBUTIONS
360
All authors were involved in drafting the article or revising it critically for content, and
361
all authors approved the final version. Drs. McCann and Séguin had access to all of the
362
data in the study and take responsibility for the integrity of the data and the accuracy of
363
the data analysis.
364
Study conception and design: M.R.M., M.A.P., A.R., D.W.H., F.B., S.J.D., and C.A.S.
365
Acquisition of data: M.R.M., C.Y., M.A.P., A.R.
366
Analysis and interpretation of data: M.R.M., C.Y., M.A.P., A.R., S.I.P., D.W.H., F. B.,
367
S.J.D., and C.A.S.
SC
M AN U
TE D
368
EP
Role of the funding source: This work was funded by the Natural Sciences and Engineering Research Council of Canada [371546] and Canadian Institutes of Health Research [132377, 312615, 86574, 133575]. Infrastructure was provided in part by the Canada Foundation for Innovation-Leaders Opportunity Fund [25086 to C.A.S]. M.R.M. & M.A.P were supported by CIHR Doctoral Awards. M.R.M., M.A.P. & A.R. were supported in part by the Joint Motion Program - A CIHR Training Program in Musculoskeletal Health Research and Leadership. A.R. was supported by a Doctoral Award from The Arthritis Society. F.B. is the Canada Research Chair in Musculoskeletal Research. C.A.S. is supported by a CIHR New Investigator Award and Early Researcher Award from the Ontario Ministry of Research and Innovation.
AC C
369 370 371 372 373 374 375 376 377 378 379 380 381 382 383
RI PT
359
Competing interest statement: The authors declare that they have no competing interests.
16
ACCEPTED MANUSCRIPT
REFERENCES
384 1.
RI PT
2.
Leong DJ, Hardin JA, Cobelli NJ, Sun HB. Mechanotransduction and cartilage integrity. Ann N Y Acad Sci 2011; 1240: 32-37. Manninen P, Riihimaki H, Heliovaara M, Suomalainen O. Physical exercise and risk of severe knee osteoarthritis requiring arthroplasty. Rheumatology (Oxford) 2001; 40: 432-437. Musumeci G, Castrogiovanni P, Trovato FM, Imbesi R, Giunta S, Szychlinska MA, et al. Physical activity ameliorates cartilage degeneration in a rat model of aging: A study on lubricin expression. Scand J Med Sci Sports 2014. Vanwanseele B, Eckstein F, Knecht H, Stussi E, Spaepen A. Knee cartilage of spinal cord-injured patients displays progressive thinning in the absence of normal joint loading and movement. Arthritis Rheum 2002; 46: 2073-2078. Hinterwimmer S, Krammer M, Krotz M, Glaser C, Baumgart R, Reiser M, et al. Cartilage atrophy in the knees of patients after seven weeks of partial load bearing. Arthritis Rheum 2004; 50: 2516-2520. Borrelli J, Jr., Silva MJ, Zaegel MA, Franz C, Sandell LJ. Single high-energy impact load causes posttraumatic OA in young rabbits via a decrease in cellular metabolism. J Orthop Res 2009; 27: 347-352. Ko FC, Dragomir C, Plumb DA, Goldring SR, Wright TM, Goldring MB, et al. In vivo cyclic compression causes cartilage degeneration and subchondral bone changes in mouse tibiae. Arthritis Rheum 2013; 65: 1569-1578. Fang H, Beier F. Mouse models of osteoarthritis: modelling risk factors and assessing outcomes. Nat Rev Rheumatol 2014; 10: 413-421. Loeser RF, Goldring SR, Scanzello CR, Goldring MB. Osteoarthritis: a disease of the joint as an organ. Arthritis Rheum 2012; 64: 1697-1707. Wysocki A, Butler M, Shamliyan T, Kane RL. Whole-body vibration therapy for osteoporosis: state of the science. Ann Intern Med 2011; 155: 680-686, W206-613. Wilcock IM, Whatman C, Harris N, Keogh JW. Vibration training: could it enhance the strength, power, or speed of athletes? J Strength Cond Res 2009; 23: 593-603. Lam FM, Lau RW, Chung RC, Pang MY. The effect of whole body vibration on balance, mobility and falls in older adults: a systematic review and meta-analysis. Maturitas 2012; 72: 206-213. Totosy de Zepetnek JO, Giangregorio LM, Craven BC. Whole-body vibration as potential intervention for people with low bone mineral density and osteoporosis: a review. J Rehabil Res Dev 2009; 46: 529-542. Pollock RD, Woledge RC, Martin FC, Newham DJ. Effects of whole body vibration on motor unit recruitment and threshold. J Appl Physiol (1985) 2012; 112: 388-395. Karatrantou K, Gerodimos V, Dipla K, Zafeiridis A. Whole-body vibration training improves flexibility, strength profile of knee flexors, and hamstrings-to-quadriceps strength ratio in females. J Sci Med Sport 2013; 16: 477-481. Segal NA, Glass NA, Shakoor N, Wallace R. Vibration platform training in women at risk for symptomatic knee osteoarthritis. PM R 2013; 5: 201-209; quiz 209. Salmon JR, Roper JA, Tillman MD. Does acute whole-body vibration training improve the physical performance of people with knee osteoarthritis? J Strength Cond Res 2012; 26: 2983-2989. Park YG, Kwon BS, Park JW, Cha DY, Nam KY, Sim KB, et al. Therapeutic effect of whole body vibration on chronic knee osteoarthritis. Ann Rehabil Med 2013; 37: 505-515.
3.
4.
8. 9. 10. 11. 12.
13.
14.
TE D
7.
EP
6.
M AN U
SC
5.
AC C
385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431
15.
16. 17.
18.
17
ACCEPTED MANUSCRIPT
23.
24.
25.
26.
27.
28.
29.
RI PT
SC
22.
M AN U
21.
TE D
20.
Li X, Wang XQ, Chen BL, Huang LY, Liu Y. Whole-Body Vibration Exercise for Knee Osteoarthritis: A Systematic Review and Meta-Analysis. Evid Based Complement Alternat Med 2015; 2015: 758147. Liphardt AM, Mundermann A, Koo S, Backer N, Andriacchi TP, Zange J, et al. Vibration training intervention to maintain cartilage thickness and serum concentrations of cartilage oligometric matrix protein (COMP) during immobilization. Osteoarthritis Cartilage 2009; 17: 1598-1603. McCann MR, Patel P, Pest MA, Ratneswaran A, Lalli G, Beaucage KL, et al. Repeated exposure to high-frequency low-amplitude vibration induces degeneration of murine intervertebral discs and knee joints. Arthritis Rheumatol 2015; 67: 21642175. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol 2010; 8: e1000412. McCann MR, Patel P, Beaucage KL, Xiao Y, Bacher C, Siqueira WL, et al. Acute vibration induces transient expression of anabolic genes in the murine intervertebral disc. Arthritis Rheum 2013; 65: 1853-1864. Hu Z, Welch I, Yuan X, Pollmann SI, Nikolov HN, Holdsworth DW. Quantification of mouse in vivo whole-body vibration amplitude from motion-blur using x-ray imaging. Phys Med Biol 2015; 60: 6423-6439. McCann MR. Veras M, Young C, Lalli G, Patel P, Leitch KM, Holdsworth DW, Beier F, Dixon SJ, Sé guin CA. Whole-body vibration of mice induces progressive degeneration of intervertebral discs associated with increased expression of IL-1β and multiple matrix degrading enzymes. Osteoarthritis Cartilage; Manuscript Number: OAC6969R1. Du LY, Umoh J, Nikolov HN, Pollmann SI, Lee TY, Holdsworth DW. A quality assurance phantom for the performance evaluation of volumetric micro-CT systems. Phys Med Biol 2007; 52: 7087-7108. Ulici V, Hoenselaar KD, Agoston H, McErlain DD, Umoh J, Chakrabarti S, et al. The role of Akt1 in terminal stages of endochondral bone formation: angiogenesis and ossification. Bone 2009; 45: 1133-1145. Pest MA, Russell BA, Zhang YW, Jeong JW, Beier F. Disturbed cartilage and joint homeostasis resulting from a loss of mitogen-inducible gene 6 in a mouse model of joint dysfunction. Arthritis Rheumatol 2014; 66: 2816-2827. Glasson SS, Chambers MG, Van Den Berg WB, Little CB. The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the mouse. Osteoarthritis Cartilage 2010; 18 Suppl 3: S17-23. Slatkovska L, Alibhai SM, Beyene J, Hu H, Demaras A, Cheung AM. Effect of 12 months of whole-body vibration therapy on bone density and structure in postmenopausal women: a randomized trial. Ann Intern Med 2011; 155: 668-679, W205. Botter SM, van Osch GJ, Clockaerts S, Waarsing JH, Weinans H, van Leeuwen JP. Osteoarthritis induction leads to early and temporal subchondral plate porosity in the tibial plateau of mice: an in vivo microfocal computed tomography study. Arthritis Rheum 2011; 63: 2690-2699. Sugiyama T, Price JS, Lanyon LE. Functional adaptation to mechanical loading in both cortical and cancellous bone is controlled locally and is confined to the loaded bones. Bone 2010; 46: 314-321. Wenger KH, Freeman JD, Fulzele S, Immel DM, Powell BD, Molitor P, et al. Effect of whole-body vibration on bone properties in aging mice. Bone 2010; 47: 746-755.
EP
19.
AC C
432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481
30.
31.
32.
33.
18
ACCEPTED MANUSCRIPT
40.
41.
42.
43.
44.
45.
46.
RI PT
39.
SC
37. 38.
M AN U
36.
TE D
35.
Liphardt AM, Schipilow J, Hanley DA, Boyd SK. Bone quality in osteopenic postmenopausal women is not improved after 12 months of whole-body vibration training. Osteoporos Int 2015; 26: 911-920. Goldring SR. Alterations in periarticular bone and cross talk between subchondral bone and articular cartilage in osteoarthritis. Ther Adv Musculoskelet Dis 2012; 4: 249-258. Patel V, Issever AS, Burghardt A, Laib A, Ries M, Majumdar S. MicroCT evaluation of normal and osteoarthritic bone structure in human knee specimens. J Orthop Res 2003; 21: 6-13. Goldring MB, Goldring SR. Osteoarthritis. J Cell Physiol 2007; 213: 626-634. Holland JC, Brennan O, Kennedy OD, Mahony NJ, Rackard S, O'Brien FJ, et al. Examination of osteoarthritis and subchondral bone alterations within the stifle joint of an ovariectomised ovine model. J Anat 2013; 222: 588-597. Loeser RF, Olex AL, McNulty MA, Carlson CS, Callahan M, Ferguson C, et al. Disease progression and phasic changes in gene expression in a mouse model of osteoarthritis. PLoS One 2013; 8: e54633. McErlain DD, Appleton CT, Litchfield RB, Pitelka V, Henry JL, Bernier SM, et al. Study of subchondral bone adaptations in a rodent surgical model of OA using in vivo micro-computed tomography. Osteoarthritis Cartilage 2008; 16: 458-469. Brunkow ME, Gardner JC, Van Ness J, Paeper BW, Kovacevich BR, Proll S, et al. Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-containing protein. Am J Hum Genet 2001; 68: 577-589. Balemans W, Ebeling M, Patel N, Van Hul E, Olson P, Dioszegi M, et al. Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST). Hum Mol Genet 2001; 10: 537-543. Li X, Ominsky MS, Niu QT, Sun N, Daugherty B, D'Agostin D, et al. Targeted deletion of the sclerostin gene in mice results in increased bone formation and bone strength. J Bone Miner Res 2008; 23: 860-869. Roudier M, Li X, Niu QT, Pacheco E, Pretorius JK, Graham K, et al. Sclerostin is expressed in articular cartilage but loss or inhibition does not affect cartilage remodeling during aging or following mechanical injury. Arthritis Rheum 2013; 65: 721-731. Wehrle E, Liedert A, Heilmann A, Wehner T, Bindl R, Fischer L, et al. The impact of low-magnitude high-frequency vibration on fracture healing is profoundly influenced by the oestrogen status in mice. Dis Model Mech 2015; 8: 93-104. Kiel DP, Hannan MT, Barton BA, Bouxsein ML, Sisson E, Lang T, et al. Low-Magnitude Mechanical Stimulation to Improve Bone Density in Persons of Advanced Age: A Randomized, Placebo-Controlled Trial. J Bone Miner Res 2015; 30: 1319-1328. McNulty AL, Guilak F. Mechanobiology of the meniscus. J Biomech 2015; 48: 14691478. Fairbank TJ. Knee joint changes after meniscectomy. J Bone Joint Surg Br 1948; 30B: 664-670. Yao J, Snibbe J, Maloney M, Lerner AL. Stresses and strains in the medial meniscus of an ACL deficient knee under anterior loading: a finite element analysis with imagebased experimental validation. J Biomech Eng 2006; 128: 135-141. Kurosawa H, Fukubayashi T, Nakajima H. Load-bearing mode of the knee joint: physical behavior of the knee joint with or without menisci. Clin Orthop Relat Res 1980: 283-290.
EP
34.
AC C
482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529
47.
48.
49.
50.
19
ACCEPTED MANUSCRIPT
52.
53.
54.
Zhao L, Dodge T, Nemani A, Yokota H. Resonance in the mouse tibia as a predictor of frequencies and locations of loading-induced bone formation. Biomech Model Mechanobiol 2013. Xie L, Jacobson JM, Choi ES, Busa B, Donahue LR, Miller LM, et al. Low-level mechanical vibrations can influence bone resorption and bone formation in the growing skeleton. Bone 2006; 39: 1059-1066. Xie L, Rubin C, Judex S. Enhancement of the adolescent murine musculoskeletal system using low-level mechanical vibrations. J Appl Physiol (1985) 2008; 104: 1056-1062. Kerr G.J. McCann MR, Branch JK., Ratneswaran A, Pest MA., Beier F, Holdsworth DW, Dixon SJ, Seguin CA. C57BL/6 mice are resistant to whole-body vibration-induced joint degeneration. In Revison at Osteoarthritis Cartilage (Manuscript Number OAC7134).
RI PT
51.
SC
530 531 532 533 534 535 536 537 538 539 540 541 542
AC C
EP
TE D
M AN U
543
20
ACCEPTED MANUSCRIPT
FIGURE LEGENDS
545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572
Figure 1: Schematic representation of experimental design. Whole-body vibration was delivered by an electromagnetic shaker regulated by an open-loop controller set to parameters of 45 Hz, 0.3 g peak acceleration, with a peak-to-peak amplitude of 74 µm. Ten-week-old, wild-type male mice (n=5-6 mice/group) were exposed to 30 mins of WBV/day, 5 days/week for 4 weeks, or 8 weeks, or 4 weeks of WBV followed by 4 weeks recovery (WBV/REC). 24 h following the last exposure to WBV, mice were sacrificed and knee joint tissues were assessed compared to age and gender-matched nonvibrated controls.
SC
M AN U
TE D
EP
574 575 576 577 578 579 580 581 582 583 584 585 586
Figure 2: Histological appearance of mouse medial knee joints after exposure to WBV. A. Representative coronal sections of the medial compartment of the knee joint, stained with Safranin O/fast green from mice exposed to WBV for 4 weeks and nonvibrated sham controls. Images are oriented with the medial meniscus on the left, the femoral condyle on top, and the tibial plateau on the bottom. Microfissures were detected within the medial meniscus in 3 of 6 mice exposed to 4 weeks of WBV (arrows). No meniscal damage was detected in control mice not exposed to WBV. Focal defects in the articular cartilage were detected in 2 of the 5 mice subjected to WBV (arrow heads). B. Histological sections from mice exposed to 8 weeks of WBV or 4 weeks of WBV followed by 4 weeks of recovery and non-vibrated age-matched sham controls. Asterisks indicate developing osteophytes at the medial margin of the joints. No articular cartilage degeneration was detected in the controls. 8 weeks of WBV resulted in severe cartilage erosion and meniscal damage in 4 of 6 mice. 4 weeks of WBV followed by 4 weeks of recovery demonstrated cartilage degeneration but to a lesser degree than that detected following 8 weeks of WBV. Whole joints were evaluated using the OARSI scoring system to quantify the degree of joint degeneration, and maximum OARSI scores are presented corresponding to the medial femoral condyle (MFC), medial tibial plateau (MTP), lateral femoral condyle (LFC) and lateral tibial plateau (LTP). Bars indicate mean ± 95% confidence interval. (n = 5-6 mice per group).
Figure 3: Analysis of tibial bone plate microarchitecture following WBV A. Representative coronal micro-computed tomography (micro-CT) images of the proximal tibia in mice exposed to 4 weeks of WBV and sham controls. Subchondral bone plate thickness was measured within a manually defined 3D region of interest (black lines) measuring 500 µm in mediolateral length and 1,150 µm ventrodorsal length, centred on the medial tibial plateau. B. Morphometric analysis of the subchondral bone plate in mice exposed to 4 weeks of WBV shows no significant difference plate thickness, bone mineral content (BMC), or bone mineral density (BMD, mg/cm3) when compared to nonvibrated sham controls. C. Similarly, 8 weeks of WBV or 4 weeks of WBV followed by 4 weeks of recovery (WBV/REC) resulted in no change in subchondral bone plate thickness, bone mineral content, bone mineral density when compared age-matched nonvibrated sham controls. Bars indicate mean ± 95% confidence interval. (n = 5-6 mice per group).
AC C
573
RI PT
544
21
ACCEPTED MANUSCRIPT
587
RI PT
Figure 4: Analysis of subchondral trabecular bone morphometry in mice exposed to WBV. A. Representative coronal micro-computed tomography (micro-CT) images of the proximal tibia in mice exposed to 4 weeks of WBV and sham controls. Bone morphometry was assessed within a manually defined 3D region of interest (black lines) in the medial subchondral trabecular bone to capture trabecular bone within this area, the lowest point of the extensor sulcus was extended as a boundary, and the subchondral bone plate, growth plate and cortical bone were excluded. B. Mice subjected to 4 weeks of WBV demonstrate a significant increase in bone mineral content (BMC), bone mineral density (BMD, mg/cm3), bone volume fraction (BVF), and trabecular thickness (Th) compared to non-vibrated sham controls. C. Mice, subjected to 8 weeks of WBV or 4 weeks of WBV followed by 4 weeks of recovery (WBV/REC), demonstrate no significant difference in any of the reported parameters compared to age-matched sham controls. Bars indicate mean ± 95% confidence interval. (n = 5-6 mice per group).
SC
588 589 590 591 592 593 594 595 596 597 598 599 600
615 616 617 618 619 620 621 622 623 624 625 626 627
TE D
EP
614
Figure 5: Analysis of bone microarchitecture in the tibial primary spongiosa following WBV. A. Representative coronal micro-computed tomography (micro-CT) images of the proximal tibia in mice exposed to 4 weeks of WBV and sham controls. Bone morphometry was assessed within a manually defined 3D region of interest (white lines) to capture the trabecular bone within 50 µm distal to the growth plate and exclude the cortical bone. B. Mice subjected to 4 weeks of WBV demonstrate no significant change in trabecular bone volume fraction (BVF), bone mineral density (BMD, mg/cm3), or bone mineral content (BMC) when compared to age-matched non-vibrated sham controls. C. Similarly, mice exposed to 8 weeks of WBV or 4 weeks of WBV followed by 4 weeks of recovery (WBV/REC) demonstrated no significant difference in any of the reported parameters compared to age-matched non-vibrated sham controls. Bars indicate mean ± 95% confidence interval. (n = 5-6 mice per group).
Figure 6: Analysis of bone microarchitecture in the tibial secondary spongiosa following WBV. A. Representative coronal micro-computed tomography (micro-CT) images of the proximal tibia in mice exposed to 4 weeks of WBV and sham controls. Bone morphometry was assessed within a manually defined 3D region of interest (white lines) to capture the trabecular bone within an area 50 µm distal from the growth plate extending 970 µm distally and exclude the cortical bone. B. Mice subjected to 4 weeks of WBV demonstrate no significant change in trabecular bone volume fraction (BVF), bone mineral density (BMD, mg/cm3), or bone mineral content (BMC) compared to agematched non-vibrated sham controls. C. Similarly, mice exposed to 8 weeks of WBV or 4 weeks of WBV followed by 4 weeks of recovery (WBV/REC) demonstrated no significant difference in any of the reported parameters compared to age-matched nonvibrated sham controls. Bars indicate mean ± 95% confidence interval. (n = 5-6 mice per group).
AC C
602 603 604 605 606 607 608 609 610 611 612 613
M AN U
601
22
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
S1063-4584(16)30387-9
DOI:
10.1016/j.joca.2016.11.001
Reference:
YJOCA 3885
To appear in:
Osteoarthritis and Cartilage
Received Date: 2 March 2016 Revised Date:
29 July 2016
Accepted Date: 2 November 2016
Please cite this article as: McCann MR, Yeung C, Pest MA, Ratneswaran A, Pollmann SI, Holdsworth DW, Beier F, Dixon SJ, Séguin CA, Whole-body vibration of mice induces articular cartilage degeneration with minimal changes in subchondral bone, Osteoarthritis and Cartilage (2016), doi: 10.1016/j.joca.2016.11.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
1 2
Title: Whole-body vibration of mice induces articular cartilage degeneration with minimal changes in subchondral bone
3 4 5 6 7
Running Title: WBV leads to cartilage degeneration
RI PT
a
SC
Department of Physiology and Pharmacology, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada. N6A 5C1 b Dentistry, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada. N6A 5C1 c Imaging Research Laboratories, Robarts Research Institute; Department of Medical Biophysics, and Department of Surgery, Schulich School of Medicine & Dentistry, University of Western Ontario, London, Ontario, Canada. N6A 5C1 d Bone and Joint Institute, University of Western Ontario, London, Ontario, Canada. N6A 5C1
M AN U
8 9 10 11 12 13 14 15 16
Authors: Matthew R. McCanna,d, Cynthia Yeunga, d, Michael A. Pesta, d, Anusha Ratneswarana, d, Steven I. Pollmannd, David W. Holdsworthc,d, Frank Beiera,d, S. Jeffrey Dixona,b,d, Cheryle A. Séguina,d
17
30 31 32
TE D
*Corresponding Author: Dr. Cheryle A. Séguin Department of Physiology and Pharmacology Schulich School of Medicine & Dentistry The University of Western Ontario London, Ontario, Canada, N6A 5C1 Email: [email protected]
EP
Keywords: Whole Body Vibration, Knee Joint, Cartilage Degeneration, Subchondral Bone
AC C
18 19 20 21 22 23 24 25 26 27 28 29
33 34 35
1
ACCEPTED MANUSCRIPT
ABSTRACT
37
Objective: Low-amplitude, high-frequency whole-body vibration (WBV) has been
38
adopted for the treatment of musculoskeletal diseases including osteoarthritis; however,
39
there is limited knowledge of the direct effects of vibration on joint tissues. Our recent
40
studies revealed striking damage to the knee joint following exposure of mice to WBV.
41
The current study examined the effects of WBV on specific compartments of the murine
42
tibiofemoral joint over 8 weeks, including microarchitecture of the tibia, to understand
43
the mechanisms associated with WBV-induced joint damage.
44
Design: Ten-week-old male CD-1 mice were exposed to WBV (45 Hz, 0.3 g peak
45
acceleration; 30 min/day, 5 days/week) for 4 weeks, 8 weeks, or 4 weeks WBV followed
46
by 4 weeks recovery. The knee joint was evaluated histologically for tissue damage.
47
Architecture of the subchondral bone plate, subchondral trabecular bone, primary and
48
secondary spongiosa of the tibia was assessed using micro-CT.
49
Results: Meniscal tears and focal articular cartilage damage were induced by WBV; the
50
extent of damage increased between 4 and 8-week exposures to WBV. WBV did not alter
51
the subchondral bone plate, or trabecular bone of the tibial spongiosa; however, a
52
transient increase was detected in the subchondral trabecular bone volume and density.
53
Conclusions: The lack of WBV-induced changes in the underlying subchondral bone
54
suggests that damage to the articular cartilage may be secondary to the meniscal injury
55
we detected. Our findings underscore the need for further studies to assess the safety of
56
WBV in the human population to avoid long-term joint damage.
AC C
EP
TE D
M AN U
SC
RI PT
36
57 58 59
2
ACCEPTED MANUSCRIPT
INTRODUCTION
60
Mechanical loading is critical for maintaining joint tissue homeostasis. Articular
62
cartilage health is paramount to overall joint function and is typically well maintained
63
under normal physiological loading [1]. Moderate physical activity has been shown to
64
sustain cartilage health in human [2] and animal studies [3]. In contrast, insufficient
65
mechanical loading can lead to articular cartilage degeneration; progressive thinning of
66
the articular cartilage is detected in patients with paralysis compared to healthy mobile
67
individuals [4]. Cartilage loss can be detected relatively quickly following changes in
68
mechanical loading; partial load bearing associated with walking with crutches resulted in
69
atrophy of knee joint articular cartilage in just 7 weeks [5]. Conversely, mechanical
70
overloading is perhaps the best-studied initiator of joint degeneration. Articular cartilage
71
is highly susceptible to osteoarthritis (OA) if subjected to acute [6] or chronic [7] impact
72
loading. Altered mechanical loading following injury to the knee joint, modelled in
73
rodents using surgically-induced joint destabilization (e.g. destablization of the medial
74
menicus, anterior cruciate ligament transection), leads to OA-like phenotypes [8].
75
Although the pathological processes mediating the progression of OA remain the focus of
76
ongoing investigation, OA is now generally considered a disease of the entire joint in
77
which articular cartilage loss, meniscus and ligament damage and the periarticular bone
78
changes contribute to the overall disease [9].
SC
M AN U
TE D
EP
AC C
79
RI PT
61
Whole-body vibration (WBV) training was originally implemented clinically as a
80
therapeutic approach to increase bone mass for the treatment of osteoporosis [10], and is
81
capable of increasing muscle strength and power [11] [12]. Underlying these biological
82
effects is the principle that WBV increases bone remodeling [13] and motor unit
3
ACCEPTED MANUSCRIPT
recruitment thresholds in muscle [14]. Contributing to the increasing popularity of WBV
84
training in the fitness industry, studies have reported beneficial effects of WBV on
85
neuromuscular performance in healthy individuals, including flexibility and strength [15].
86
However, recent studies suggest these effects may vary based on age, gender or health
87
status [16].
RI PT
83
Clinical trials have recently evaluated the therapeutic effect of WBV on joint pain
89
and functional performance in patients with chronic knee OA [17]. A number of studies
90
report beneficial effects including reduced pain intensity, increased muscle strength, and
91
reductions in the plasma concentrations of inflammatory markers in OA patients
92
following WBV training [18, 19]. Conversely, some trials reported no significant
93
improvements in pain intensity or other parameters among knee OA patients following
94
WBV [19]. Importantly, in these studies joint health was not directly assessed.
95
Quantification of the direct effects of WBV on knee joint tissues are limited; one study
96
reported the ability of WBV to prevent loss of tibial articular cartilage thickness
97
(determined by MRI) following prolonged immobilization in healthy male subjects [20].
98
A recent meta-analysis evaluating the use of WBV in knee OA management concluded
99
that there were no significant differences in functional performance or patient self-
100
reported pain following WBV, likely related to the diversity of the protocols implemented
101
[19]. Together, these findings suggest the need for for further studies evaluating the
102
safety and efficacy of WBV prior to its therapeutic implementation for the treatment of
103
OA.
AC C
EP
TE D
M AN U
SC
88
104
To address these issues, recent studies from our group evaluated the effect of
105
WBV on joint health using the mouse as a preclinical model. Our findings demonstrated
4
ACCEPTED MANUSCRIPT
that 4 weeks of repeated exposure of healthy young mice to WBV, using protocols
107
reported to be beneficial to bone, induced OA-like knee joint damage marked by
108
degeneration of both the meniscus and articular cartilage as well as activation of MMP-
109
mediated matrix degradation [21]. Building from these findings, the present study was
110
designed to determine: i) if knee joint degeneration was progressive with extended
111
exposure to WBV or was potentiated by cessation of vibration, and ii) if WBV promoted
112
changes in the subchondral bone that would constitute a potential mechanism for the OA-
113
like pathology detected in the knee.
M AN U
SC
RI PT
106
METHODS
114
Whole-Body Vibration. All procedures were approved by the Animal Use Committee at
116
The University of Western Ontario and this study was conducted in accordance the
117
ARRIVE guidelines [22]. Based on protocols of WBV used in our previous studies to
118
model those used clinically [21], 10-week-old male CD-1 mice were exposed to vertical
119
sinusoidal vibrations (frequency of 45 Hz, peak-to-peak amplitude of 74 µm, and 0.3 g
120
peak acceleration) for 30 min/day, 5 days/week for 4 weeks, 8 weeks, or 4 weeks WBV
121
followed by 4 weeks recovery, using a previously described vibration platform [23, 24].
122
Age-matched controls were housed in identical chambers on a sham (non-vibrated)
123
platform to replicate handling and environmental conditions. Following WBV, mice were
124
returned to conventional housing and monitored daily. Twenty-four hours after the final
125
exposure to WBV, mice were euthanized by a lethal dose of sodium pentobarbital. Spinal
126
tissues were isolated to characterize effects of WBV on the intervertebral disc and
127
vertebral bone, detailed in [25].
AC C
EP
TE D
115
128
5
ACCEPTED MANUSCRIPT
Micro-computed Tomography (micro-CT). Right hindlimbs (mid-femur to ankle) were
130
isolated and fixed in 4% paraformaldehyde (PFA) for 24 h and embedded in 1% agarose
131
in PBS in 50 mL conical tubes. Tissues were scanned using a laboratory micro-CT
132
scanner (eXplore Locus, GE Healthcare Biosciences) with an internal calibrating
133
phantom composed of air, water, and synthetic bone-mimicking epoxy (SB3 2.8 x 3.4 x 8
134
mm; Gammex RMI). The scanning protocol consisted of 900 projection images acquired
135
at a 0.4º angle increment, obtained over 165 minutes of gantry rotation. The x-ray tube
136
potential was 80 kVp with a tube current of 450 µA, an exposure time of 4,500 msec, and
137
2 frames per view angle averaged. Images were reconstructed into 3D volumes with 20
138
µm isotropic voxel spacing and linearly rescaled into Hounsfield units using the internal
139
calibration standards, as previously described [26]. Using three-dimensional visualization
140
and analysis software (MicroView, GE Healthcare Biosciences) full volumes were
141
cropped to contain only the tibia and fibula, and reoriented to the same axes. Regions of
142
interest (ROI) were manually outlined to define the subchondral bone plate, subchondral
143
trabecular bone, primary and secondary spongiosa of the tibia, where each compartment
144
was marked within a series of 2D planes and splined together to generate 3D ROIs. Bone
145
morphometry was quantified using the Bone Analysis tool in MicroView, as previously
146
reported [27].
SC
M AN U
TE D
EP
AC C
147
RI PT
129
148
Histological Assessment and Scoring. Following micro-CT, right hind limbs were
149
decalcified for 10 days in 5% EDTA in PBS (pH 7.0), processed, embedded in paraffin
150
wax, and 5 µm coronal sections were obtained. Serial sections were taken every 50 µm to
151
encompass all weight-bearing areas of the femoro-tibial joint. Using established protocols
6
ACCEPTED MANUSCRIPT
[23, 28], sections were stained with 0.1% Safranin O/0.02% fast green to detect
153
proteoglycans and glycosaminoglycans. Sections were imaged on a Leica DM1000
154
microscope with Leica Application Suite (Leica Microsystems). The four quadrants of
155
the knee joint were scored by three independent blinded observers for degeneration using
156
the murine Osteoarthritis Research Society International (OARSI) recommended
157
histopathologic scale [29].
RI PT
152
SC
158
Statistical Analyses. Data are presented from experiments conducted with 5-6 mice per
160
treatment group. Parameters from mice exposed to WBV were compared to those from
161
age-matched non-vibrated sham controls using a parametric, two-tailed, unpaired t-test
162
with a Welch’s correction (4 week time point), or one-way ANOVA with a Tukey post-
163
hoc test (8 week time point). P < 0.05 was considered significant.
M AN U
159
165
TE D
164
RESULTS
To assess the effects of daily WBV on the knee joint and determine if protocols of
167
WBV used clinically [30] induce long-term joint damage, 10-week-old male CD-1 mice
168
were exposed to sinusoidal WBV (30 min/day, 5 days/week, at 45 Hz, 0.3 g peak
169
acceleration) for 4 weeks, 8 weeks, or 4 weeks of WBV followed by 4 weeks without
170
WBV (recovery) (Figure 1). Consistent with our recent study [21], analysis of Safranin
171
O/fast green stained histological sections demonstrated that 4 weeks of WBV induced
172
knee joint damage in 3 of 6 mice (OARSI score >2), localized to the medial joint
173
compartment in 2 mice and the lateral femoral condyle in the third mouse. In 1 of these
174
mice, articular damage was detected on both the femoral and tibial condyles. The OARSI
AC C
EP
166
7
ACCEPTED MANUSCRIPT
semi-quantitative histologic scoring system assigns a grade between 0-6 to each joint
176
quadrant, where a grade of 2 is characterized by loss of articular cartilage with vertical
177
clefts extending beyond the superficial layer and some loss of surface lamina. WBV-
178
induced damage included evidence of meniscal tears, focal articular cartilage defects with
179
erosion down to the tide mark, loss of glycosaminoglycans in the superficial layer, and
180
osteophyte formation on the medial tibial plateau and femoral condyle (Figure 2A). No
181
joint damage (OARSI >2) was detected in any age-matched sham controls. Despite these
182
findings, no significant differences were detected in the average maximum OARSI scores
183
between mice exposed to 4 weeks of WBV and sham controls.
M AN U
SC
RI PT
175
To determine if WBV-induced joint damage was progressive with continued daily
185
WBV or potentiated by inclusion of a recovery period, mice were exposed to either 8
186
weeks of WBV or 4 weeks of WBV followed by 4 weeks without (recovery). Following
187
8 weeks of WBV, 5 of 6 mice demonstrated articular cartilage damage (OARSI score
188
>2), localized to the medial joint compartment in 2 mice, the lateral joint compartment in
189
1 mouse, and both the medial and lateral joint compartments in 1 mouse. In 3 of these
190
mice, articular damage was detected on both the femoral and tibial condyles. Following 8
191
weeks WBV, joint damage was characterized by severe loss of meniscal tissue, focal
192
articular
193
glycosaminoglycans in the superficial layer of articular cartilage adjacent to focal defects,
194
and osteophyte formation on the medial tibial plateau and femoral condyle (Figure 2B).
195
Focal articular cartilage damage (OARSI score >2) was also detected in 2 of 6 age-
196
matched sham controls, localized to the medial tibial plateau. Exposure of mice to 8
AC C
EP
TE D
184
cartilage
lesions
with
erosion
8
to
the
subchondral
bone,
loss
of
ACCEPTED MANUSCRIPT
197
weeks of WBV induced a significant increase in the average maximum OARSI score in
198
the lateral tibial plateau compared to age-matched sham controls. Articular cartilage damage (OARSI score >2) was detected in only 1 of 5 mice
200
subjected to 4 weeks of WBV followed by 4 weeks of recovery (WBV/REC). Of note
201
however, in this animal the meniscal and articular cartilage damage detected was similar
202
to that of mice exposed to 4 weeks WBV (Figure 2B), suggesting that inclusion of a
203
recovery period failed to mitigate the damage induced by WBV in this animal.
SC
RI PT
199
We next examined if WBV promoted changes to the subchondral bone
205
microarchitecture that would correlate with WBV-induced soft tissue damage. Using
206
high-resolution micro-CT, we first assessed the subchondral bone plate subjacent to the
207
articular cartilage of the tibial plateau since this region showed the most pronounced
208
WBV-induced articular cartilage degeneration (Figure 3A). Subchondral bone plate
209
thickness was measured within a region of interest measuring 500 µm in mediolateral
210
length and 1,150 µm ventrodorsal length, centred on the medial tibial plateau, based on
211
previously reported protocols [31]. No significant differences in bone plate thickness
212
(mm), bone mineral content (BMC, mg), or bone mineral density (BMD, mg/cm3) were
213
detected in the subchondral bone of mice exposed to 4 weeks of WBV (Figure 3B), 8
214
weeks of WBV, or 4 weeks of WBV followed by 4 weeks recovery (Figure 3C).
TE D
EP
AC C
215
M AN U
204
Bone microarchitecture was next assessed in the epiphyseal subchondral
216
trabecular bone in the medial compartment of the tibia. For each specimen, a region of
217
interest was manually outlined in serial planes to capture trabecular bone within this area;
218
the lowest point of the extensor sulcus was extended as a boundary, and the subchondral
9
ACCEPTED MANUSCRIPT
bone plate, growth plate and cortical bone were excluded (Figure 4A). Exposure of mice
220
to 4 weeks of WBV induced a significant increase in the subchondral trabecular bone
221
volume fraction (BVF) and trabecular BMD (mg HA/cm3) compared to non-vibrated
222
sham controls (Figure 4B). In contrast, no significant differences were detected in BVF
223
or BMD (mg HA/cm3) of the subchondral trabecular bone in mice exposed to 8 weeks of
224
WBV or 4 weeks of WBV followed by 4 weeks recovery compared to age-matched sham
225
controls (Figure 4C).
SC
RI PT
219
Previous studies suggest the primary and secondary spongiosa within long bones
227
as sites that demonstrate robust adaptive bone remodelling in response to dynamic
228
mechanical loading [32]. We therefore assessed bone microarchitecture within both the
229
primary and secondary spongiosa of the tibia, and quantified bone properties within each
230
separately to delineate region-specific changes. A region of interest was defined to
231
include the primary spongiosa, manually outlined in serial planes to capture the trabecular
232
bone within 50 µm distal to the growth plate and exclude the cortical bone (Figure 5A).
233
No significant differences were detected in trabecular BVF or trabecular BMD in the
234
primary spongiosa following 4 weeks of WBV (Figure 5B), 8 weeks of WBV or 4 weeks
235
of WBV followed by 4 weeks recovery (Figure 5C). We then moved distally and
236
measured the secondary spongiosa of the tibia, manually outlined in serial planes to
237
capture the trabecular bone within an area extending 1mm from the most distal point of
238
the primary spongiosa, and exclude the cortical bone (Figure 6A). No significant
239
differences were detected in trabecular BVF or trabecular BMD in the secondary
240
spongiosa following 4 weeks of WBV (Figure 6B), 8 weeks of WBV or 4 weeks of
241
WBV followed by 4 weeks recovery (Figure 6C).
AC C
EP
TE D
M AN U
226
10
ACCEPTED MANUSCRIPT
DISCUSSION
242
The reported beneficial effects of WBV on bone mass and muscle strength have
244
led to increased use of WBV devices both clinically and in the sports and fitness industry
245
to treat pain, increase joint mobility, and enhance neuromuscular performance. However,
246
recent conflicting reports have highlighted inconsistencies in the anabolic effects of WBV
247
on bone, in both animal models [21, 33] and human clinical trials [30, 34]. In this report,
248
we investigated the mechanisms associated with our previous reports of WBV-induced
249
damage to the murine femoro-tibial joint (knee) following exposure of mice to protocols
250
of WBV that model those used clinically [21]. Based on histological characterization, our
251
findings suggest progressive degeneration of the meniscus and articular cartilage
252
following exposure to WBV. Between 4 and 8 weeks, progressive degeneration was
253
marked by severe loss of meniscus tissue, an increase in the both the number of mice
254
showing articular cartilage defects and the extent of the articular cartilage damage,
255
resulting in a significant increase in the average maximum OARSI score. Based on
256
detailed micro-CT based examination of the tibia, we show that WBV-induced joint
257
damage is not associated with either thickening of the subchondral bone plate or changes
258
in the trabecular bone of the primary or secondary spongiosa.
SC
M AN U
TE D
EP
AC C
259
RI PT
243
Although loss of articular cartilage is the hallmark pathological feature in OA,
260
disease progression involves biomechanical and biochemical alterations in the
261
composition, structure and functional properties of the articular cartilage and periarticular
262
bone [35]. Within the bone compartment, OA-associated changes include increases in
263
subchondral cortical bone thickness, early bone remodeling in the subchondral trabeculae
264
resulting in decreased trabecular bone mass, formation of osteophytes at the joint margins
11
ACCEPTED MANUSCRIPT
and expansion of the zone of calcified cartilage leading to thinning of the articular
266
cartilage [36, 37]. Studies from our group and others have demonstrated that rodent
267
models of OA likewise demonstrate hallmark bone changes, including thickening of the
268
subchondral bone plate and decreased BMD in the subchondral trabecular bone,
269
particularly in regions adjacent to affected articular cartilage [38-40]. Given the interplay
270
between these tissues, the current study used high-resolution micro-CT to determine if
271
focal defects in articular cartilage induced by WBV were associated with changes to the
272
periarticular bone. Using manually defined regions of interest, we did not detect
273
significant differences in bone microarchitecture in either the subchondral bone plate or
274
primary and secondary spongiosa of the tibia. These findings are consistent with
275
quantification of bone microarchitecture in the vertebral bone of these mice, which
276
likewise demonstrated no change following exposure to WBV [25]. Our findings did
277
however demonstrate a transient increase in subchondral trabecular bone mass after 4
278
weeks of WBV, although no differences were detected between mice exposed to 8 weeks
279
WBV compared to controls. Interestingly, we noted that following 4 weeks of WBV,
280
mice with the highest OARSI scores also showed the highest bone plate thickness, bone
281
mineral content and bone mineral density. This association was not however seen in mice
282
following 8 weeks of WBV and may therefore reflect a transient adaptive response to
283
vibration loading.
SC
M AN U
TE D
EP
AC C
284
RI PT
265
Sclerostin is a marker of osteocyte differentiation that serves as a negative
285
regulator of bone formation by inhibiting the osteo-anabolic Wnt signalling pathway. In
286
humans, loss of function mutations of the human SOST gene (encoding sclerostin) lead to
287
sclerosteosis [41, 42], and deletion of Sost in mice results in a high bone mass phenotype
12
ACCEPTED MANUSCRIPT
[43]. Despite increased subchondral bone, Sost-deficient mice do not demonstrate
289
changes to the articular cartilage in the knee associated with OA [44]. Interestingly, using
290
a femur osteotomy model of fracture healing in aged mice, a recent study demonstrated
291
that WBV increased the expression of Sost in osteocytes, associated with impaired
292
fracture healing [45]. These findings may be in keeping with recent studies by our group
293
[21] and others [46] highlighting inconsistencies in the previously reported anabolic
294
effects of WBV on bone, as well as the confounding effects of age and hormone levels.
295
Furthermore, these findings highlight the potential role of Wnt signaling as a candidate
296
regulator of WBV mechanotransduction that should be investigated in joint tissues such
297
as meniscus and articular cartilage.
M AN U
SC
RI PT
288
Our previous study reported meniscal damage in mice following 2 weeks of
299
WBV, changes that preceded the detection of overt articular cartilage damage [21].
300
Together with the current study, these findings suggest a correlation between meniscus
301
damage and the induction of articular cartilage degeneration following WBV. As such,
302
we speculate that WBV-induced articular cartilage damage may occur following joint
303
destabilization, rather than as a result of changes in the mechanical environment resulting
304
from increased subchondral bone characteristic of OA. Meniscal damage is often
305
associated with pain and joint dysfunction [47], and loss of meniscal function leads to
306
rapid and progressive osteoarthritis [48]. Damage or partial loss of the meniscus
307
influences the displacement and deformation of the meniscus under load [49], resulting in
308
increased focal-point stresses on the articular cartilage surface of the tibial plateau [50]
309
that we propose lead to the formation of the focal cartilage defects we detected in mice
310
exposed to WBV.
AC C
EP
TE D
298
13
ACCEPTED MANUSCRIPT
An interesting aspect of this and our previous study is the fact that while severe
312
knee joint damage was induced by WBV (including severe damage to the meniscus, focal
313
articular cartilage defects extending to the subchondral bone, and osteophyte formation
314
on both the femoral and tibial condyles), damage was only detected in a subset of mice at
315
each time point examined. As such, statistical analysis of OARSI scores demonstrated
316
significant WBV-induced damage only following 8 weeks of WBV in a single joint
317
compartment. The absence of a significant increase in OARSI scores following 4 weeks
318
WBV, or in multiple joint compartments at either time point may be associated with the
319
limited sample size used in this study. Of note, the individual cages in which mice are
320
housed during WBV allow for free range of movement. We have observed that during
321
vibration, mice alternate through a variety of postures including rearing on hind limbs,
322
free movement on all 4 limbs, grooming and sleeping. It is tempting to speculate that
323
increased time spent in specific postures may be directly associated with increased joint
324
damage since the magnitude of vibration experienced at skeletal sites likely varies due to
325
muscle contraction, soft tissue damping, or resonance effects [51]. We have recently
326
developed non-invasive techniques to quantify vibration amplitude in the skeleton of live
327
mice using radiographic images of surgically implanted metal marker beads [24]. Future
328
studies will utilize this approach to quantify the vibration amplitude and deformations at
329
specific sites and in various postures. This will allow us to directly relate the biological
330
response of joint tissues to WBV to its biomechanical effects.
AC C
EP
TE D
M AN U
SC
RI PT
311
331
While this and previous studies by our group [21, 25] point to alarming potential
332
for damage induced by whole-body vibration on joint tissues, the preclinical model used
333
is associated with inherent limitations. First, these studies were conducted using a single
14
ACCEPTED MANUSCRIPT
protocol of WBV that does not effectively capture the wide range of parameters currently
335
being used. The variables that control the intensity of vibration are the frequency and
336
amplitude (peak-to-peak displacement), which together determine peak acceleration.
337
Clinical protocols of WBV vary widely in frequency (2.5-90 Hz) and acceleration (0.3-
338
3.6 g), and are implemented for periods ranging from one week to one year. Second, we
339
examined the effects of WBV using young, skeletally mature male CD-1 mice. Previous
340
studies have demonstrated that the ability of WBV to increase bone mineral density and
341
bone formation is dependent on age and genetic background in mouse models [52, 53].
342
As such, physiological factors including age, sex, and genetic background may likewise
343
affect the response of joint tissues to WBV. In fact, a recent study from our group
344
reported a lack of WBV-induced degeneration in either the knee or intervertebral discs of
345
C57Bl/6 mice exposed to the same protocols of WBV [54]. Taken together, these studies
346
underscore the need for further rigorous research to assess the relative contribution of
347
vibration parameters and physiological factors in modulating the effects of WBV on joint
348
health.
TE D
M AN U
SC
RI PT
334
In conclusion, WBV results in progressive degeneration of the meniscus and
350
articular cartilage, damage that may not be repaired following removal of the stimulus.
351
Furthermore, we demonstrate that WBV promotes a transient increase in subchondral
352
trabecular bone; however, no change was detected in morphometry of the subchondral
353
bone plate or primary and secondary spongiosa of the tibia.
AC C
EP
349
354 355
ACKNOWLEDGEMENTS
15
ACCEPTED MANUSCRIPT
356
We thank Dr. Kim Beaucage for her contributions, in particular for her assistance with
357
micro-CT training and protocols.
358
AUTHOR CONTRIBUTIONS
360
All authors were involved in drafting the article or revising it critically for content, and
361
all authors approved the final version. Drs. McCann and Séguin had access to all of the
362
data in the study and take responsibility for the integrity of the data and the accuracy of
363
the data analysis.
364
Study conception and design: M.R.M., M.A.P., A.R., D.W.H., F.B., S.J.D., and C.A.S.
365
Acquisition of data: M.R.M., C.Y., M.A.P., A.R.
366
Analysis and interpretation of data: M.R.M., C.Y., M.A.P., A.R., S.I.P., D.W.H., F. B.,
367
S.J.D., and C.A.S.
SC
M AN U
TE D
368
EP
Role of the funding source: This work was funded by the Natural Sciences and Engineering Research Council of Canada [371546] and Canadian Institutes of Health Research [132377, 312615, 86574, 133575]. Infrastructure was provided in part by the Canada Foundation for Innovation-Leaders Opportunity Fund [25086 to C.A.S]. M.R.M. & M.A.P were supported by CIHR Doctoral Awards. M.R.M., M.A.P. & A.R. were supported in part by the Joint Motion Program - A CIHR Training Program in Musculoskeletal Health Research and Leadership. A.R. was supported by a Doctoral Award from The Arthritis Society. F.B. is the Canada Research Chair in Musculoskeletal Research. C.A.S. is supported by a CIHR New Investigator Award and Early Researcher Award from the Ontario Ministry of Research and Innovation.
AC C
369 370 371 372 373 374 375 376 377 378 379 380 381 382 383
RI PT
359
Competing interest statement: The authors declare that they have no competing interests.
16
ACCEPTED MANUSCRIPT
REFERENCES
384 1.
RI PT
2.
Leong DJ, Hardin JA, Cobelli NJ, Sun HB. Mechanotransduction and cartilage integrity. Ann N Y Acad Sci 2011; 1240: 32-37. Manninen P, Riihimaki H, Heliovaara M, Suomalainen O. Physical exercise and risk of severe knee osteoarthritis requiring arthroplasty. Rheumatology (Oxford) 2001; 40: 432-437. Musumeci G, Castrogiovanni P, Trovato FM, Imbesi R, Giunta S, Szychlinska MA, et al. Physical activity ameliorates cartilage degeneration in a rat model of aging: A study on lubricin expression. Scand J Med Sci Sports 2014. Vanwanseele B, Eckstein F, Knecht H, Stussi E, Spaepen A. Knee cartilage of spinal cord-injured patients displays progressive thinning in the absence of normal joint loading and movement. Arthritis Rheum 2002; 46: 2073-2078. Hinterwimmer S, Krammer M, Krotz M, Glaser C, Baumgart R, Reiser M, et al. Cartilage atrophy in the knees of patients after seven weeks of partial load bearing. Arthritis Rheum 2004; 50: 2516-2520. Borrelli J, Jr., Silva MJ, Zaegel MA, Franz C, Sandell LJ. Single high-energy impact load causes posttraumatic OA in young rabbits via a decrease in cellular metabolism. J Orthop Res 2009; 27: 347-352. Ko FC, Dragomir C, Plumb DA, Goldring SR, Wright TM, Goldring MB, et al. In vivo cyclic compression causes cartilage degeneration and subchondral bone changes in mouse tibiae. Arthritis Rheum 2013; 65: 1569-1578. Fang H, Beier F. Mouse models of osteoarthritis: modelling risk factors and assessing outcomes. Nat Rev Rheumatol 2014; 10: 413-421. Loeser RF, Goldring SR, Scanzello CR, Goldring MB. Osteoarthritis: a disease of the joint as an organ. Arthritis Rheum 2012; 64: 1697-1707. Wysocki A, Butler M, Shamliyan T, Kane RL. Whole-body vibration therapy for osteoporosis: state of the science. Ann Intern Med 2011; 155: 680-686, W206-613. Wilcock IM, Whatman C, Harris N, Keogh JW. Vibration training: could it enhance the strength, power, or speed of athletes? J Strength Cond Res 2009; 23: 593-603. Lam FM, Lau RW, Chung RC, Pang MY. The effect of whole body vibration on balance, mobility and falls in older adults: a systematic review and meta-analysis. Maturitas 2012; 72: 206-213. Totosy de Zepetnek JO, Giangregorio LM, Craven BC. Whole-body vibration as potential intervention for people with low bone mineral density and osteoporosis: a review. J Rehabil Res Dev 2009; 46: 529-542. Pollock RD, Woledge RC, Martin FC, Newham DJ. Effects of whole body vibration on motor unit recruitment and threshold. J Appl Physiol (1985) 2012; 112: 388-395. Karatrantou K, Gerodimos V, Dipla K, Zafeiridis A. Whole-body vibration training improves flexibility, strength profile of knee flexors, and hamstrings-to-quadriceps strength ratio in females. J Sci Med Sport 2013; 16: 477-481. Segal NA, Glass NA, Shakoor N, Wallace R. Vibration platform training in women at risk for symptomatic knee osteoarthritis. PM R 2013; 5: 201-209; quiz 209. Salmon JR, Roper JA, Tillman MD. Does acute whole-body vibration training improve the physical performance of people with knee osteoarthritis? J Strength Cond Res 2012; 26: 2983-2989. Park YG, Kwon BS, Park JW, Cha DY, Nam KY, Sim KB, et al. Therapeutic effect of whole body vibration on chronic knee osteoarthritis. Ann Rehabil Med 2013; 37: 505-515.
3.
4.
8. 9. 10. 11. 12.
13.
14.
TE D
7.
EP
6.
M AN U
SC
5.
AC C
385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431
15.
16. 17.
18.
17
ACCEPTED MANUSCRIPT
23.
24.
25.
26.
27.
28.
29.
RI PT
SC
22.
M AN U
21.
TE D
20.
Li X, Wang XQ, Chen BL, Huang LY, Liu Y. Whole-Body Vibration Exercise for Knee Osteoarthritis: A Systematic Review and Meta-Analysis. Evid Based Complement Alternat Med 2015; 2015: 758147. Liphardt AM, Mundermann A, Koo S, Backer N, Andriacchi TP, Zange J, et al. Vibration training intervention to maintain cartilage thickness and serum concentrations of cartilage oligometric matrix protein (COMP) during immobilization. Osteoarthritis Cartilage 2009; 17: 1598-1603. McCann MR, Patel P, Pest MA, Ratneswaran A, Lalli G, Beaucage KL, et al. Repeated exposure to high-frequency low-amplitude vibration induces degeneration of murine intervertebral discs and knee joints. Arthritis Rheumatol 2015; 67: 21642175. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving bioscience research reporting: the ARRIVE guidelines for reporting animal research. PLoS Biol 2010; 8: e1000412. McCann MR, Patel P, Beaucage KL, Xiao Y, Bacher C, Siqueira WL, et al. Acute vibration induces transient expression of anabolic genes in the murine intervertebral disc. Arthritis Rheum 2013; 65: 1853-1864. Hu Z, Welch I, Yuan X, Pollmann SI, Nikolov HN, Holdsworth DW. Quantification of mouse in vivo whole-body vibration amplitude from motion-blur using x-ray imaging. Phys Med Biol 2015; 60: 6423-6439. McCann MR. Veras M, Young C, Lalli G, Patel P, Leitch KM, Holdsworth DW, Beier F, Dixon SJ, Sé guin CA. Whole-body vibration of mice induces progressive degeneration of intervertebral discs associated with increased expression of IL-1β and multiple matrix degrading enzymes. Osteoarthritis Cartilage; Manuscript Number: OAC6969R1. Du LY, Umoh J, Nikolov HN, Pollmann SI, Lee TY, Holdsworth DW. A quality assurance phantom for the performance evaluation of volumetric micro-CT systems. Phys Med Biol 2007; 52: 7087-7108. Ulici V, Hoenselaar KD, Agoston H, McErlain DD, Umoh J, Chakrabarti S, et al. The role of Akt1 in terminal stages of endochondral bone formation: angiogenesis and ossification. Bone 2009; 45: 1133-1145. Pest MA, Russell BA, Zhang YW, Jeong JW, Beier F. Disturbed cartilage and joint homeostasis resulting from a loss of mitogen-inducible gene 6 in a mouse model of joint dysfunction. Arthritis Rheumatol 2014; 66: 2816-2827. Glasson SS, Chambers MG, Van Den Berg WB, Little CB. The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the mouse. Osteoarthritis Cartilage 2010; 18 Suppl 3: S17-23. Slatkovska L, Alibhai SM, Beyene J, Hu H, Demaras A, Cheung AM. Effect of 12 months of whole-body vibration therapy on bone density and structure in postmenopausal women: a randomized trial. Ann Intern Med 2011; 155: 668-679, W205. Botter SM, van Osch GJ, Clockaerts S, Waarsing JH, Weinans H, van Leeuwen JP. Osteoarthritis induction leads to early and temporal subchondral plate porosity in the tibial plateau of mice: an in vivo microfocal computed tomography study. Arthritis Rheum 2011; 63: 2690-2699. Sugiyama T, Price JS, Lanyon LE. Functional adaptation to mechanical loading in both cortical and cancellous bone is controlled locally and is confined to the loaded bones. Bone 2010; 46: 314-321. Wenger KH, Freeman JD, Fulzele S, Immel DM, Powell BD, Molitor P, et al. Effect of whole-body vibration on bone properties in aging mice. Bone 2010; 47: 746-755.
EP
19.
AC C
432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481
30.
31.
32.
33.
18
ACCEPTED MANUSCRIPT
40.
41.
42.
43.
44.
45.
46.
RI PT
39.
SC
37. 38.
M AN U
36.
TE D
35.
Liphardt AM, Schipilow J, Hanley DA, Boyd SK. Bone quality in osteopenic postmenopausal women is not improved after 12 months of whole-body vibration training. Osteoporos Int 2015; 26: 911-920. Goldring SR. Alterations in periarticular bone and cross talk between subchondral bone and articular cartilage in osteoarthritis. Ther Adv Musculoskelet Dis 2012; 4: 249-258. Patel V, Issever AS, Burghardt A, Laib A, Ries M, Majumdar S. MicroCT evaluation of normal and osteoarthritic bone structure in human knee specimens. J Orthop Res 2003; 21: 6-13. Goldring MB, Goldring SR. Osteoarthritis. J Cell Physiol 2007; 213: 626-634. Holland JC, Brennan O, Kennedy OD, Mahony NJ, Rackard S, O'Brien FJ, et al. Examination of osteoarthritis and subchondral bone alterations within the stifle joint of an ovariectomised ovine model. J Anat 2013; 222: 588-597. Loeser RF, Olex AL, McNulty MA, Carlson CS, Callahan M, Ferguson C, et al. Disease progression and phasic changes in gene expression in a mouse model of osteoarthritis. PLoS One 2013; 8: e54633. McErlain DD, Appleton CT, Litchfield RB, Pitelka V, Henry JL, Bernier SM, et al. Study of subchondral bone adaptations in a rodent surgical model of OA using in vivo micro-computed tomography. Osteoarthritis Cartilage 2008; 16: 458-469. Brunkow ME, Gardner JC, Van Ness J, Paeper BW, Kovacevich BR, Proll S, et al. Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-containing protein. Am J Hum Genet 2001; 68: 577-589. Balemans W, Ebeling M, Patel N, Van Hul E, Olson P, Dioszegi M, et al. Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST). Hum Mol Genet 2001; 10: 537-543. Li X, Ominsky MS, Niu QT, Sun N, Daugherty B, D'Agostin D, et al. Targeted deletion of the sclerostin gene in mice results in increased bone formation and bone strength. J Bone Miner Res 2008; 23: 860-869. Roudier M, Li X, Niu QT, Pacheco E, Pretorius JK, Graham K, et al. Sclerostin is expressed in articular cartilage but loss or inhibition does not affect cartilage remodeling during aging or following mechanical injury. Arthritis Rheum 2013; 65: 721-731. Wehrle E, Liedert A, Heilmann A, Wehner T, Bindl R, Fischer L, et al. The impact of low-magnitude high-frequency vibration on fracture healing is profoundly influenced by the oestrogen status in mice. Dis Model Mech 2015; 8: 93-104. Kiel DP, Hannan MT, Barton BA, Bouxsein ML, Sisson E, Lang T, et al. Low-Magnitude Mechanical Stimulation to Improve Bone Density in Persons of Advanced Age: A Randomized, Placebo-Controlled Trial. J Bone Miner Res 2015; 30: 1319-1328. McNulty AL, Guilak F. Mechanobiology of the meniscus. J Biomech 2015; 48: 14691478. Fairbank TJ. Knee joint changes after meniscectomy. J Bone Joint Surg Br 1948; 30B: 664-670. Yao J, Snibbe J, Maloney M, Lerner AL. Stresses and strains in the medial meniscus of an ACL deficient knee under anterior loading: a finite element analysis with imagebased experimental validation. J Biomech Eng 2006; 128: 135-141. Kurosawa H, Fukubayashi T, Nakajima H. Load-bearing mode of the knee joint: physical behavior of the knee joint with or without menisci. Clin Orthop Relat Res 1980: 283-290.
EP
34.
AC C
482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529
47.
48.
49.
50.
19
ACCEPTED MANUSCRIPT
52.
53.
54.
Zhao L, Dodge T, Nemani A, Yokota H. Resonance in the mouse tibia as a predictor of frequencies and locations of loading-induced bone formation. Biomech Model Mechanobiol 2013. Xie L, Jacobson JM, Choi ES, Busa B, Donahue LR, Miller LM, et al. Low-level mechanical vibrations can influence bone resorption and bone formation in the growing skeleton. Bone 2006; 39: 1059-1066. Xie L, Rubin C, Judex S. Enhancement of the adolescent murine musculoskeletal system using low-level mechanical vibrations. J Appl Physiol (1985) 2008; 104: 1056-1062. Kerr G.J. McCann MR, Branch JK., Ratneswaran A, Pest MA., Beier F, Holdsworth DW, Dixon SJ, Seguin CA. C57BL/6 mice are resistant to whole-body vibration-induced joint degeneration. In Revison at Osteoarthritis Cartilage (Manuscript Number OAC7134).
RI PT
51.
SC
530 531 532 533 534 535 536 537 538 539 540 541 542
AC C
EP
TE D
M AN U
543
20
ACCEPTED MANUSCRIPT
FIGURE LEGENDS
545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572
Figure 1: Schematic representation of experimental design. Whole-body vibration was delivered by an electromagnetic shaker regulated by an open-loop controller set to parameters of 45 Hz, 0.3 g peak acceleration, with a peak-to-peak amplitude of 74 µm. Ten-week-old, wild-type male mice (n=5-6 mice/group) were exposed to 30 mins of WBV/day, 5 days/week for 4 weeks, or 8 weeks, or 4 weeks of WBV followed by 4 weeks recovery (WBV/REC). 24 h following the last exposure to WBV, mice were sacrificed and knee joint tissues were assessed compared to age and gender-matched nonvibrated controls.
SC
M AN U
TE D
EP
574 575 576 577 578 579 580 581 582 583 584 585 586
Figure 2: Histological appearance of mouse medial knee joints after exposure to WBV. A. Representative coronal sections of the medial compartment of the knee joint, stained with Safranin O/fast green from mice exposed to WBV for 4 weeks and nonvibrated sham controls. Images are oriented with the medial meniscus on the left, the femoral condyle on top, and the tibial plateau on the bottom. Microfissures were detected within the medial meniscus in 3 of 6 mice exposed to 4 weeks of WBV (arrows). No meniscal damage was detected in control mice not exposed to WBV. Focal defects in the articular cartilage were detected in 2 of the 5 mice subjected to WBV (arrow heads). B. Histological sections from mice exposed to 8 weeks of WBV or 4 weeks of WBV followed by 4 weeks of recovery and non-vibrated age-matched sham controls. Asterisks indicate developing osteophytes at the medial margin of the joints. No articular cartilage degeneration was detected in the controls. 8 weeks of WBV resulted in severe cartilage erosion and meniscal damage in 4 of 6 mice. 4 weeks of WBV followed by 4 weeks of recovery demonstrated cartilage degeneration but to a lesser degree than that detected following 8 weeks of WBV. Whole joints were evaluated using the OARSI scoring system to quantify the degree of joint degeneration, and maximum OARSI scores are presented corresponding to the medial femoral condyle (MFC), medial tibial plateau (MTP), lateral femoral condyle (LFC) and lateral tibial plateau (LTP). Bars indicate mean ± 95% confidence interval. (n = 5-6 mice per group).
Figure 3: Analysis of tibial bone plate microarchitecture following WBV A. Representative coronal micro-computed tomography (micro-CT) images of the proximal tibia in mice exposed to 4 weeks of WBV and sham controls. Subchondral bone plate thickness was measured within a manually defined 3D region of interest (black lines) measuring 500 µm in mediolateral length and 1,150 µm ventrodorsal length, centred on the medial tibial plateau. B. Morphometric analysis of the subchondral bone plate in mice exposed to 4 weeks of WBV shows no significant difference plate thickness, bone mineral content (BMC), or bone mineral density (BMD, mg/cm3) when compared to nonvibrated sham controls. C. Similarly, 8 weeks of WBV or 4 weeks of WBV followed by 4 weeks of recovery (WBV/REC) resulted in no change in subchondral bone plate thickness, bone mineral content, bone mineral density when compared age-matched nonvibrated sham controls. Bars indicate mean ± 95% confidence interval. (n = 5-6 mice per group).
AC C
573
RI PT
544
21
ACCEPTED MANUSCRIPT
587
RI PT
Figure 4: Analysis of subchondral trabecular bone morphometry in mice exposed to WBV. A. Representative coronal micro-computed tomography (micro-CT) images of the proximal tibia in mice exposed to 4 weeks of WBV and sham controls. Bone morphometry was assessed within a manually defined 3D region of interest (black lines) in the medial subchondral trabecular bone to capture trabecular bone within this area, the lowest point of the extensor sulcus was extended as a boundary, and the subchondral bone plate, growth plate and cortical bone were excluded. B. Mice subjected to 4 weeks of WBV demonstrate a significant increase in bone mineral content (BMC), bone mineral density (BMD, mg/cm3), bone volume fraction (BVF), and trabecular thickness (Th) compared to non-vibrated sham controls. C. Mice, subjected to 8 weeks of WBV or 4 weeks of WBV followed by 4 weeks of recovery (WBV/REC), demonstrate no significant difference in any of the reported parameters compared to age-matched sham controls. Bars indicate mean ± 95% confidence interval. (n = 5-6 mice per group).
SC
588 589 590 591 592 593 594 595 596 597 598 599 600
615 616 617 618 619 620 621 622 623 624 625 626 627
TE D
EP
614
Figure 5: Analysis of bone microarchitecture in the tibial primary spongiosa following WBV. A. Representative coronal micro-computed tomography (micro-CT) images of the proximal tibia in mice exposed to 4 weeks of WBV and sham controls. Bone morphometry was assessed within a manually defined 3D region of interest (white lines) to capture the trabecular bone within 50 µm distal to the growth plate and exclude the cortical bone. B. Mice subjected to 4 weeks of WBV demonstrate no significant change in trabecular bone volume fraction (BVF), bone mineral density (BMD, mg/cm3), or bone mineral content (BMC) when compared to age-matched non-vibrated sham controls. C. Similarly, mice exposed to 8 weeks of WBV or 4 weeks of WBV followed by 4 weeks of recovery (WBV/REC) demonstrated no significant difference in any of the reported parameters compared to age-matched non-vibrated sham controls. Bars indicate mean ± 95% confidence interval. (n = 5-6 mice per group).
Figure 6: Analysis of bone microarchitecture in the tibial secondary spongiosa following WBV. A. Representative coronal micro-computed tomography (micro-CT) images of the proximal tibia in mice exposed to 4 weeks of WBV and sham controls. Bone morphometry was assessed within a manually defined 3D region of interest (white lines) to capture the trabecular bone within an area 50 µm distal from the growth plate extending 970 µm distally and exclude the cortical bone. B. Mice subjected to 4 weeks of WBV demonstrate no significant change in trabecular bone volume fraction (BVF), bone mineral density (BMD, mg/cm3), or bone mineral content (BMC) compared to agematched non-vibrated sham controls. C. Similarly, mice exposed to 8 weeks of WBV or 4 weeks of WBV followed by 4 weeks of recovery (WBV/REC) demonstrated no significant difference in any of the reported parameters compared to age-matched nonvibrated sham controls. Bars indicate mean ± 95% confidence interval. (n = 5-6 mice per group).
AC C
602 603 604 605 606 607 608 609 610 611 612 613
M AN U
601
22
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT