Contribution of mechanical unloading to trabecular bone loss following non-invasive knee injury in mice

Abstract

Development of osteoarthritis commonly involves degeneration of epiphyseal trabecular bone. In previous studies, we observed 30-44% loss of epiphyseal trabecular bone (BV/TV) from the distal femur within 1 week following non-invasive knee injury in mice. Mechanical unloading (disuse) may contribute to this bone loss; however, it is unclear to what extent the injured limb is unloaded following injury, and whether disuse can fully account for the observed magnitude of bone loss. In this study, we investigated the contribution of mechanical unloading to trabecular bone changes observed following non-invasive knee injury in mice (female C57BL/6N). We investigated changes in gait during treadmill walking, and changes in voluntary activity level using Open Field analysis at 4, 14, 28, and 42 days post-injury. We also quantified epiphyseal trabecular bone using μCT and weighed lower-limb muscles to quantify atrophy following knee injury in both ground control and hindlimb unloaded (HLU) mice. Gait analysis revealed a slightly altered stride pattern in the injured limb, with a decreased stance phase and increased swing phase. However, Open Field analysis revealed no differences in voluntary movement between injured and sham mice at any time point. Both knee injury and HLU resulted in comparable magnitudes of trabecular bone loss; however, HLU resulted in considerably more muscle loss than knee injury, suggesting another mechanism contributing to bone loss following injury. Altogether, these data suggest that mechanical unloading likely contributes to trabecular bone loss following non-invasive knee injury, but the magnitude of this bone loss cannot be fully explained by disuse. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1680-1687, 2016.

Keywords: hindlimb unloading; knee injury; mechanical loading; post-traumatic osteoarthritis; trabecular bone.

© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Figures

Figure 1

Body mass of hindlimb unloaded (HLU) and ground control (GC) mice following non-invasive knee injury or sham injury. There were no significant differences between any of the experimental groups at any time points. Approximately half of the animals subjected to HLU lost weight from day 0 to day 7, but by day 14 their body mass was increased relative to day 0. Data are presented as mean ± standard deviation.

Figure 2

Open Field containment for analysis of voluntary movement (top left). Each mouse was tested for a 24-hour period at 4, 14, 28, and 42 days post-injury. No significant differences were observed between injured and sham mice for any parameters at any time point. Data are presented as mean ± standard deviation.

Figure 3

Gait of injured and sham mice was analyzed during treadmill running using the Treadscan system (left). Average percent of stride spent in stance phase was decreased in injured limbs compared to sham, while percent of stride spent in swing phase was increased (right). Mean data for swing, propel, and brake phases are presented.

Figure 4

(Left) Representative μCT reconstruction frontal sections of mouse distal femurs showing epiphyseal trabecular bone after 14 days of injury or HLU. The volume of interest for analysis is indicated in red. (Right) Trabecular bone volume fraction (top row) and trabecular thickness (bottom row) after 7 or 14 days of injury and/or HLU. Both HLU and knee injury resulted in comparable trabecular bone loss after 7 days, however HLU resulted in further decreases in trabecular bone volume from 7–14 days, while injured GC knees exhibited no further decreases. Data are presented as mean ± standard deviation. Groups that do not share a letter are significantly different from one another (p

Figure 5

Muscle mass of the Tibialis Anterior (top row) and Gastrocnemius and Soleus (bottom row) after 7 or 14 days of injury or HLU. All HLU mice lost comparable muscle mass at 7 and 14 days regardless of injury status. Knee injury alone also resulted in decreased muscle mass, but to a much lesser extent than HLU. Data are presented as mean ± standard deviation. Groups that do not share a letter are significantly different from one another (p

Figure 6

Expression of biomarkers of muscle atrophy MuRF1 (top row) and MAFbx (bottom row) after 7 or 14 days of injury or HLU. HLU resulted in increased levels of MuRF1 and MAFbx relative to GC mice, while knee injury did not result in increased levels of MuRF1 or MAFbx expression at either time point, and in fact often decreased expression in injured mice compared to sham mice. Data are presented as mean ± standard deviation. Groups that do not share a letter are significantly different from one another (p