Mechanical loading recovers bone but not muscle lost during unloading.

Space travel and prolonged bed rest are examples of mechanical unloading that induce significant muscle and bone loss. The compromised structure and function of bone and muscle owing to unloading make the reloading period a high risk for injury. To explore interactions between skeletal bone and muscle during reloading, we hypothesized that acute external mechanical loading of bone in combination with re-ambulation facilitates the proportional recovery of bone and muscle lost during hind limb suspension (HLS) unloading. Adult male C57Bl/6J mice were randomly assigned to a HLS or time-matched ground control (GC) group. After 2-weeks of HLS, separate groups of mice were studied at day 14 (no re-ambulation), day 28 (14 days re-ambulation) and day 56 (42 days re-ambulation); throughout the re-ambulation period, one limb received compressive mechanical loading and the contralateral limb served as an internal control. HLS induced loss of trabecular bone volume (BV/TV; -51 ± 2%) and muscle weight (-15 ± 2%) compared to GC at day 14. At day 28, the left tibia (re-ambulation only) of HLS mice had recovered approximately 20% of BV/TV lost during HLS, while the right tibia (re-ambulation and acute external mechanical loading) recovered to GC values of BV/TV (~100% recovery). At day 56, the right tibia continued to recover bone for some outcomes (trabecular BV/TV, trabecular thickness), while the left limb did not. Cortical bone displayed a delayed response to HLS, with a 10% greater decrease in BV/TV at day 28 compared to day 14. In contrast to bone, acute external mechanical loading during the re-ambulation period did not significantly increase muscle mass or protein synthesis in the gastrocnemius, compared to re-ambulation alone. Our results suggest acute external mechanical loading facilitates the recovery of bone during reloading following HLS unloading, but this does not translate to a concomitant recovery of muscle mass.

Protective Effects of Controlled Mechanical Loading of Bone in C57BL6/J Mice Subject to Disuse.

Prolonged reduction in weightbearing causes bone loss. Disuse of bone is associated with recovery from common musculoskeletal injury and trauma, bed rest resulting from various medical conditions, and spaceflight. The hindlimb-suspension rodent model is popular for simulating unloading and disuse. We hypothesized that controlled mechanical loading of the tibia would protect against bone loss occurring from concurrent disuse. Additionally, we hypothesized that areas of high mechanical peak strains (midshaft) would provide more protection than areas of lower strain (distal shaft). Adult C57BL6/J mice were suspended for 3 weeks, with one limb subjected to tibial compression four times per week. µCT imaging was completed at days 0, 11, and 21, in addition to serum analysis. Significant bone loss caused by hindlimb suspension was detected in trabecular bone by day 11 and worsened by day 21 (p < 0.05). Bone loss was also detected in cortical thickness and area fraction by day 21. However, four short bouts per week of compressive loading protected the loaded limb from much of this bone loss. At day 21, we observed a 50% loss in trabecular bone volume/total volume and a 6% loss in midshaft cortical thickness in unloaded limbs, but only 15% and 2% corresponding losses in contralateral loaded limbs (p = 0.001 and p = 0.02). Many bone geometry parameters of the loaded limbs of suspended animals did not significantly differ from non-suspended control limbs. Conversely, this protective effect of loading was not detected in cortical bone at the lower-strained distal shaft. Analysis of bone metabolism markers suggested that the benefits of loading occurred through increased formation instead of decreased resorption. This study uniquely isolates the role of externally applied mechanical loading of the mouse tibia, in the absence of muscle stimulation, in protecting bone from concurrent disuse-related loss, and demonstrates that limited bouts of loading may be highly effective during prolonged disuse. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

LRP receptors in chondrocytes are modulated by simulated microgravity and cyclic hydrostatic pressure.

Mechanical loading is essential for the maintenance of musculoskeletal homeostasis. Cartilage has been demonstrated to be highly mechanoresponsive, but the mechanisms by which chondrocytes respond to mechanical stimuli are not clearly understood. The goal of the study was to determine how LRP4, LRP5, and LRP6 within canonical Wnt-signaling are regulated in simulated microgravity and cyclic hydrostatic pressure, and to investigate the potential role of LRP 4/5/6 in cartilage degeneration. Rat chondrosacroma cell (RCS) pellets were stimulated using either cyclic hydrostatic pressure (1Hz, 7.5 MPa, 4hr/day) or simulated microgravity in a rotating wall vessel (RWV) bioreactor (11RPM, 24hr/day). LRP4/5/6 mRNA expression was assessed by RT-qPCR and LRP5 protein expression was determined by fluorescent immunostaining. To further evaluate our in vitro findings in vivo, mice were subjected to hindlimb suspension for 14 days and the femoral heads stained for LRP5 expression. We found that, in vitro, LRP4/5/6 mRNA expression is modulated in a time-dependent manner by mechanical stimulation. Additionally, LRP5 protein expression is upregulated in response to both simulated microgravity and cyclic hydrostatic pressure. LRP5 is also upregulated in vivo in the articular cartilage of hindlimb suspended mice. This is the first study to examine how LRP4/5/6, critical receptors within musculoskeletal biology, respond to mechanical stimulation. Further elucidation of this mechanism could provide significant clinical benefit for the identification of pharmaceutical targets for the maintenance of cartilage health.

Combination of hindlimb suspension and immobilization by casting exaggerates sarcopenia by stimulating autophagy but does not worsen osteopenia.

Astronauts in space experience a unique environment that causes the concomitant loss of bone and muscle. However, the interaction between these tissues and how osteopenia and sarcopenia affect each other is unclear. We explored this relationship by exaggerating unloading-induced muscle loss using a unilateral casting model in conjunction with hindlimb suspension (HLS). Five-month-old, male C57Bl/6J mice subjected to HLS for 2 weeks displayed a significant decrease in gastrocnemius and quadriceps weight (-9-10%), with a two-fold greater decrease in muscle mass observed in the HLS + casted limb. However, muscle from casted limbs had a higher rate of protein synthesis (+16%), compared to HLS alone, with coordinated increases in S6K1 (+50%) and 4E-BP1 (+110%) phosphorylation. Increased protein content for surrogate markers of autophagy, including LC3-II (+75%), Atg7 (+10%), and Atg5-12 complex (+20%) was only detected in muscle from the casted limb. In proximal tibias, HLS resulted in significant decreases in bone volume fraction (-24% vs -8%), trabecular number (-6% vs +0.3%), trabecular thickness (-10% vs -2%), and trabecular spacing (+8.4% vs +2%) compared to ground controls. There was no further bone loss in casted limbs compared to HLS alone. In tibia midshafts, HLS resulted in decreased total area (-2% vs +1%) and increased bone mineral density (+1% vs -0.3%) compared to ground controls. Cortical bone from casted limbs showed an increase in cortical thickness (+9% vs +2%) and cortical area/total area (+1% vs -0.6%) compared to HLS alone. Our results suggest that casting exacerbates unloading-induced muscle loss via activation of autophagy. Casting did not exacerbate bone loss suggesting that the unloading-induced loss of muscle and bone can be temporally dissociated and the effect of reduced muscle activity plays a relatively minor role compared to reduced load bearing on trabecular bone structure.

Simulated space radiation sensitizes bone but not muscle to the catabolic effects of mechanical unloading.

Deep space travel exposes astronauts to extended periods of space radiation and mechanical unloading, both of which may induce significant muscle and bone loss. Astronauts are exposed to space radiation from solar particle events (SPE) and background radiation referred to as galactic cosmic radiation (GCR). To explore interactions between skeletal muscle and bone under these conditions, we hypothesized that decreased mechanical load, as in the microgravity of space, would lead to increased susceptibility to space radiation-induced bone and muscle loss. We evaluated changes in bone and muscle of mice exposed to hind limb suspension (HLS) unloading alone or in addition to proton and high (H) atomic number (Z) and energy (E) (HZE) (16O) radiation. Adult male C57Bl/6J mice were randomly assigned to six groups: No radiation ± HLS, 50 cGy proton radiation ± HLS, and 50 cGy proton radiation + 10 cGy 16O radiation ± HLS. Radiation alone did not induce bone or muscle loss, whereas HLS alone resulted in both bone and muscle loss. Absolute trabecular and cortical bone volume fraction (BV/TV) was decreased 24% and 6% in HLS-no radiation vs the normally loaded no-radiation group. Trabecular thickness and mineral density also decreased with HLS. For some outcomes, such as BV/TV, trabecular number and tissue mineral density, additional bone loss was observed in the HLS+proton+HZE radiation group compared to HLS alone. In contrast, whereas HLS alone decreased muscle mass (19% gastrocnemius, 35% quadriceps), protein synthesis, and increased proteasome activity, radiation did not exacerbate these catabolic outcomes. Our results suggest that combining simulated space radiation with HLS results in additional bone loss that may not be experienced by muscle.