Whole-body vibration of mice induces articular cartilage degeneration with minimal changes in subchondral bone  M.R. McCann, C. Yeung, M.A. Pest, A. Ratneswaran, S.I. Pollmann, D.W. Holdsworth, F. Beier, S.J. Dixon, C.A. Séguin  Osteoarthritis and Cartilage  Volume 25, Issue 5, Pages 770-778 (May 2017) DOI: 10.1016/j.joca.2016.11.001 Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

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. Ten-week-old, wild-type male mice (n = 5–6 mice/group) were exposed to 30 min 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 non-vibrated controls. Osteoarthritis and Cartilage 2017 25, 770-778DOI: (10.1016/j.joca.2016.11.001) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 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 non-vibrated 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 three of six 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 two of the five 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 four of six 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). Osteoarthritis and Cartilage 2017 25, 770-778DOI: (10.1016/j.joca.2016.11.001) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 3 Analysis of tibial bone plate microarchitecture following WBV A. Representative coronal 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 non-vibrated 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, BMC, BMD when compared age-matched non-vibrated sham controls. Bars indicate mean ± 95% confidence interval. (n = 5–6 mice per group). Osteoarthritis and Cartilage 2017 25, 770-778DOI: (10.1016/j.joca.2016.11.001) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 4 Analysis of subchondral trabecular bone morphometry in mice exposed to WBV. A. Representative coronal 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). Osteoarthritis and Cartilage 2017 25, 770-778DOI: (10.1016/j.joca.2016.11.001) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 5 Analysis of bone microarchitecture in the tibial primary spongiosa following WBV. A. Representative coronal 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). Osteoarthritis and Cartilage 2017 25, 770-778DOI: (10.1016/j.joca.2016.11.001) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions

Fig. 6 Analysis of bone microarchitecture in the tibial secondary spongiosa following WBV. A. Representative coronal 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 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). Osteoarthritis and Cartilage 2017 25, 770-778DOI: (10.1016/j.joca.2016.11.001) Copyright © 2016 Osteoarthritis Research Society International Terms and Conditions