1. Whole-body vibration of mice induces progressive degeneration of intervertebral discs associated with increased expression of Il-1β and multiple matrix degrading enzymes 
Matthew R. McCann, Matthew A. Veras, Cynthia Yeung, Gurkeet Lalli, Priya Patel, Kristyn M. Leitch, David W. Holdsworth, S. Jeffrey Dixon, Cheryle A. Séguin 
Osteoarthritis and Cartilage 
Volume 25, Issue 5, Pages (May 2017)
DOI: /j.joca
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

2. Fig. 1
Schematic representation of experimental design. WBV 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. 10-week-old, wild-type male mice (n = 4–6 mice/group) were exposed to 30 min of WBV/day, 5 days/week for 2 weeks, 4 weeks or 8 weeks, or 4 weeks of WBV followed by 4 weeks recovery. 24 h following the last exposure to WBV, mice were sacrificed and assessed compared to age and gender-matched non-vibrated controls.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

3. Fig. 2
Micro-CT analysis of whole-body composition in mice exposed to WBV. A. Representative reconstructed micro-CT images of mice following 8 weeks of WBV, 4 weeks of WBV followed by 4 weeks recovery (WBV/REC), or sham controls. Isotropic surface-rendering of skeletal tissue (indicated in white) is overlaid with a mid-coronal slice where lean tissue is indicated in red and adipose tissue is indicated in yellow. B–G. Values are means ± 95% confidence interval of the indicated parameters. 4 weeks of WBV followed by 4 weeks of recovery resulted in a decrease in % lean tissue mass and corresponding increase in % adipose tissue, compared to controls as well as mice exposed to 8 weeks of WBV. In contrast, total mass, percent skeletal mass, BMC and BMD were not significantly different between treatment groups. n = 6 mice per group.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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4. Fig. 3
Effect of long-term exposure to WBV on the lumbar IVD. Representative histological sections of the lumbar spine stained with Safranin-O/fast green from mice exposed to A. 4 weeks of WBV, B. 8 weeks of WBV or 4 weeks of WBV followed by 4 weeks recovery (WBV/REC) and age-matched non-vibrated SHAM controls. Staining revealed hallmark histological signs of IVD degeneration including the accumulation of mucinous material leading to enhanced glycosaminoglycan staining in the interlamellar matrix of the AF (arrows), loss of a distinct NP-AF boundary, and of focal disruptions in the AF (arrowheads). C. Evaluation of the morphologic grade of tissue degeneration (using a modified Thompson score) demonstrated increased tissue degeneration in the NP at 8 weeks of WBV and 4 weeks of WBV followed by 4 weeks of recovery. The AF of mice subjected to WBV for 8 weeks and 4 weeks of WBV and 4 weeks of recovery also demonstrated increased degeneration scores when compared to age-matched controls. n = 3 IVDs/mouse; 5–6 mice/group.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

5. Fig. 4
Analysis of vertebral bone microarchitecture following WBV. A. Representative high-resolution micro-CT images of L6 vertebral bone from mice exposed to 4 weeks WBV and non-vibrated sham control. The manually-defined ROI (black lines) included the trabecular bone of the weight-bearing region of the vertebral body but excluded the cortical bone and the articulating processes. The volume was determined by extrapolating the shape of the vertebral body. B. Mice subjected to 4 weeks of WBV demonstrate no significant differences in trabecular BMD or BVF compared to sham controls. C. Mice subjected to 8 weeks of WBV or 4 weeks of WBV followed by 4 weeks recovery (WBV/REC) likewise did not show any changes in vertebral trabecular bone morphometry compared to age-matched sham controls. Error bars = mean ± 95% confidence interval. n = 4–6 mice/group.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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6. Fig. 5
The effect of WBV on IVD height. A. Representative high-resolution micro-CT image of the lumbar spine used to quantify the DHI between the L5 and L6 vertebrae, calculated based on measurements of the adjacent L6 vertebrae. DHI was calculated using the following equation: DHI=Discheight+Vertebralboneheightatdepthof25%ofvertebralbonewidthfromanteriorDischeight+Vertebralboneheightatdepthof25%ofvertebralbonewidthfromposterior. B. Exposure of mice to 4 weeks of WBV resulted in a significant decrease in DHI compared to sham controls. C. No significant difference in DHI was detected in mice following 8 weeks of WBV or 4 weeks of WBV followed by 4 weeks recovery (WBV/REC), compared to age-matched non-vibrated sham controls. Error bars = mean ± 95% confidence interval. n = 5–6 mice/group.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

7. Fig. 6
Effect of repeated exposure of WBV on anabolic gene expression in the IVD. SYBR green-based real-time polymerase chain reaction analysis of IVD gene expression. Expression of extracellular matrix genes (Acan, Col1a1, Col2a1, Prg4) and factors associated with their transcriptional regulation (Sox9, Clip) was normalized to expression of the housekeeping gene Hprt and expressed relative to non-vibrated sham controls. A. IVD tissues from mice exposed to 4 weeks of WBV demonstrated a significant increase in the expression of Acan, Col1a1, Col2a1, Prg4, Sox9, Cilp, and Timp1 compared to sham controls. B. There was a significant increase in anabolic genes Acan, Col2a1, Sox9 and Cilp in IVD tissues from mice exposed to 8 weeks WBV compared to non-vibrated sham controls. A significant increase in Prg4 expression was detected in mice exposed to 4 weeks WBV followed by 4 weeks recovery (WBV/REC) compared to age-matched sham controls. Values are presented as the mean ± 95% confidence interval from 5-pooled IVDs per mouse, 4-week timepoint n = 6 and 8-week timepoint n = 5 mice per group, with PCR run in triplicate. 8-week timepoint was analyzed using a one-way ANOVA with multiple comparisons and significance is represented by line over all bars with associated P-value.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
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8. Fig. 7
Effect of repeated exposure of WBV on catabolic gene expression in the IVD. SYBR green-based real-time polymerase chain reaction analysis of IVD gene expression. Genes associated with the induction of IVD degeneration, including matrix degrading enzymes (Mmp13, Mmp3, Adamts4, Adamts5) and the hypertrophic marker Col10a1 were normalized to expression of the housekeeping gene Hprt and expressed relative to non-vibrated sham controls. A. IVD tissues from mice exposed to 4 weeks of WBV demonstrated a significant increase in the expression of Mmp13 and Col10a1 compared to sham controls. B. IVD tissues from mice exposed to 8 weeks of WBV demonstrated a significant increase in the expression of Mmp3, Mmp13 and Adamts5 compared to sham controls; changes not detected in mice exposed to 4 weeks WBV followed by 4 weeks recovery (WBV/REC). Values are presented as the mean ± 95% confidence interval of five pooled IVDs/mouse, 4-week timepoint n = 6 and 8-week timepoint n = 5 mice per group, with PRC run in triplicate. 8-week timepoint was analyzed using a one-way ANOVA with multiple comparisons and were significant across all treatments as indicated by line over all bars with associated P-value.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

9. Fig. 8
Effect of repeated exposure of WBV on pro-inflammatory gene expression in the IVD. SYBR green-based real-time polymerase chain reaction analysis of pro-inflammatory gene expression in IVD tissues from mice exposed to 2, 4 or 8 weeks WBV were normalized to expression of the housekeeping gene Hprt and expressed relative to non-vibrated sham controls. A. Il-1ß expression was upregulated in mice exposed to 2 weeks WBV and 4 weeks WBV, compared to age-matched sham controls. B. No change in Il-6 expression was detected in IVD tissues from mice exposed to 2, 4 or 8 weeks WBV compared to sham controls; however, a significant reduction in Il-6 expression was detected in mice exposed to 4 weeks WBV followed by 4 weeks recovery (WBV/REC) compared to age-matched sham controls. C. No change in Tnfα expression was detected in IVD tissues following WBV at any timepoint examined. Values are presented as the mean ± 95% confidence interval of five pooled IVDs/mouse, 4-week timepoint n = 6 and 8-week timepoint n = 5 mice per group, with PRC run in triplicate. 8-week timepoint was analyzed using a one-way ANOVA with multiple comparisons and were significant across all treatments as indicated by line over all bars with associated P-value.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions

10. Supplemental Fig. 1
Micro-CT analysis of whole-body composition in mice exposed to 4 weeks of WBV. Values are means ± 95% confidence interval of the indicated parameters. Exposure of mice to 4 weeks of WBV did not alter total body mass, or body composition. Following 4 weeks of WBV, a significant decrease was detected in BMD, compared to non-vibrated sham controls n = 6 mice per group.
Osteoarthritis and Cartilage  , DOI: ( /j.joca )
Copyright © 2017 Osteoarthritis Research Society International Terms and Conditions