Ted S. Gross

Low-Magnitude Mechanical Loading Becomes Osteogenic When Rest Is Inserted Between Each Load Cycle

Strategies to counteract bone loss with exercise have had fairly limited success, particularly those regimens subjecting the skeleton to mild activity such as walking. In contrast, here we show that it is possible to induce substantial bone formation with low‐magnitude loading. In two distinct in vivo models of bone adaptation, we found that insertion of a 10‐s rest interval between each load cycle transformed a locomotion‐like loading regime that minimally influenced osteoblast activity into a potent anabolic stimulus. In the avian ulna model, the minimal mean (+SE) periosteal labeled surface (Ps.LS) observed in the intact contralateral bones (1.6 ± 1.5%) was doubled after 3 consecutive days of low‐magnitude loading (3.8 ± 1.5%; p = 0.03). However, modifying the regimen by inserting 10 s of rest between each load cycle significantly enhanced the periosteal response (21.9 + 4.5%; p = 0.03). In the murine tibia model, 5 consecutive days of 100 low‐magnitude loading cycles did not significantly alter mean periosteal bone formation rate (BFR) compared with contralateral bones (0.011 ± 0.005 μm3/μm2 per day vs. 0.021 ± 0.013 μm3/μm2 per day). In contrast, separating each of 10 of the same loading cycles with 10 s of rest significantly elevated periosteal BFR (0.167 ± 0.049 μm3/μm2 per day; p = 0.01). Endocortical bone formation parameters were not altered by any loading regimen in either model. We conclude that 10 s of rest between each load cycle of a low‐magnitude loading protocol greatly enhances the osteogenic potential of the regimen.

Mechanical loading-related changes in osteocyte sclerostin expression in mice are more closely associated with the subsequent osteogenic response than the peak strains engendered.

SummaryOsteocyte sclerostin is regulated by loading and disuse in mouse tibiae but is more closely related to subsequent local osteogenesis than the peak strains engendered.IntroductionThe purpose of this study was to assess the relationship between loading-related change in osteocyte sclerostin expression, local strain magnitude, and local bone modeling/remodeling.MethodsThe right tibiae of 19-week-old female C57BL/6 mice were subjected to non-invasive, dynamic axial loading and/or to sciatic neurectomy-induced disuse. The sclerostin status of osteocytes was evaluated immunohistochemically, changes in bone mass by micro-computed tomography, new bone formation by histomorphometry, and loading-induced strain by strain gauges and finite element analysis.ResultsIn cortical bone of the tibial shaft, loading engendered strains of similar peak magnitude proximally and distally. Proximally, sclerostin-positive osteocytes decreased and new bone formation increased. Distally, there was neither decrease in sclerostin-positive osteocytes nor increased osteogenesis. In trabecular bone of the proximal secondary spongiosa, loading decreased sclerostin-positive osteocytes and increased bone volume. Neither occurred in the primary spongiosa. Disuse increased sclerostin-positive osteocytes and decreased bone volume at all four sites. Loading reversed this sclerostin upregulation to a level below baseline in the proximal cortex and secondary spongiosa.ConclusionLoading-related sclerostin downregulation in osteocytes of the mouse tibia is associated preferentially with regions where new bone formation is stimulated rather than where high peak strains are engendered. The mechanisms involved remain unclear, but could relate to peak surface strains not accurately reflecting the strain-related osteogenic stimulus or that sclerostin regulation occurs after sufficient signal processing to distinguish between local osteogenic and non-osteogenic responses.

β-Catenin Levels Influence Rapid Mechanical Responses in Osteoblasts

Mechanical loading of bone initiates an anabolic, anticatabolic pattern of response, yet the molecular events involved in mechanical signal transduction are not well understood. Wnt/β-catenin signaling has been recognized in promoting bone anabolism, and application of strain has been shown to induce β-catenin activation. In this work, we have used a preosteoblastic cell line to study the effects of dynamic mechanical strain on β-catenin signaling. We found that mechanical strain caused a rapid, transient accumulation of active β-catenin in the cytoplasm and its translocation to the nucleus. This was followed by up-regulation of the Wnt/β-catenin target genes Wisp1 and Cox2, with peak responses at 4 and 1 h of strain, respectively. The increase of β-catenin was temporally related to the activation of Akt and subsequent inactivation of GSK3β, and caveolin-1 was not required for these molecular events. Application of Dkk-1, which disrupts canonical Wnt/LRP5 signaling, did not block strain-induced nuclear translocation of β-catenin or up-regulation of Wisp1 and Cox2 expression. Conditions that increased basal β-catenin levels, such as lithium chloride treatment or repression of caveolin-1 expression, were shown to enhance the effects of strain. In summary, mechanical strain activates Akt and inactivates GSK3β to allow β-catenin translocation, and Wnt signaling through LRP5 is not required for these strain-mediated responses. Thus, β-catenin serves as both a modulator and effector of mechanical signals in bone cells.

The shock attenuation role of the ankle during landing from a vertical jump.

Three landing surfaces were used to examine a hypothesized increased shock attenuation role of the ankle with increased damping demands. Eleven male recreational basketball players performed three symmetric barefoot countermovement vertical jumps on each surface. Two externally mounted low mass accelerometers (medial calcaneus and distal anterio-medial tibia), a piezoelectric force platform, and high speed cinematography recorded the landing. Accelerometer signal distortion was corrected through the application of a linear spring/damper model of the accelerometer attachment. The model indicated that raw acceleration data were overestimated 68% at the calcaneal attachment and 8% at the tibial attachment. Peak corrected acceleration at metatarsal contact varied little across landing surfaces, and, across surfaces, mean (SD) peak accelerations of 20.8 (9.3) and 14.3 (3.6) gs were recorded at the calcaneus and tibia, respectively. Peak vertical force and ankle joint motion varied little across the surfaces, suggesting that the entrenched kinematics of landing surpassed the introduced range of surface cushioning. Separation of the data by post-metatarsal contact landing style indicated that seven subjects landed with heel contact, with the remaining four attenuating the impact without heel contact. By avoiding the transient associated with the cessation of downward heel motion, the nonheel contact landers effectively reduced exposure to transients by nearly 50%.

Enhanced osteoclastic resorption and responsiveness to mechanical load in gap junction deficient bone.

Emerging evidence suggests that connexin mediated gap junctional intercellular communication contributes to many aspects of bone biology including bone development, maintenance of bone homeostasis and responsiveness of bone cells to diverse extracellular signals. Deletion of connexin 43, the predominant gap junction protein in bone, is embryonic lethal making it challenging to examine the role of connexin 43 in bone in vivo. However, transgenic murine models in which only osteocytes and osteoblasts are deficient in connexin 43, and which are fully viable, have recently been developed. Unfortunately, the bone phenotype of different connexin 43 deficient models has been variable. To address this issue, we used an osteocalcin driven Cre-lox system to create osteoblast and osteocyte specific connexin 43 deficient mice. These mice displayed bone loss as a result of increased bone resorption and osteoclastogenesis. The mechanism underlying this increased osteoclastogenesis included increases in the osteocytic, but not osteoblastic, RANKL/OPG ratio. Previous in vitro studies suggest that connexin 43 deficient bone cells are less responsive to biomechanical signals. Interestingly, and in contrast to in vitro studies, we found that connexin 43 deficient mice displayed an enhanced anabolic response to mechanical load. Our results suggest that transient inhibition of connexin 43 expression and gap junctional intercellular communication may prove a potentially powerful means of enhancing the anabolic response of bone to mechanical loading.

Upregulation of osteopontin by osteocytes deprived of mechanical loading or oxygen.

The pathway(s) by which disuse is transduced into locally mediated osteoclastic resorption remain unknown. We found that both acute disuse (in vivo) and direct hypoxia (in vitro) induced rapid upregulation of OPN expression by osteocytes. Within the context of OPNs role in osteoclast migration and attachment, hypoxia‐induced osteocyte OPN expression may serve to mediate disuse‐induced bone resorption.

RAD001 (Everolimus) inhibits growth of prostate cancer in the bone and the inhibitory effects are increased by combination with docetaxel and zoledronic acid

mTOR activity is increased in advanced prostate cancer (CaP) as a result of a high rate of PTEN mutations. RAD001 (Everolimus) is a new orally available mTOR inhibitor. The objective of our study was to evaluate the effects of RAD001 on the growth of CaP in the bone, both alone and in combination with docetaxel and zoledronic acid.

Caveolin-1 knockout mice have increased bone size and stiffness

The skeletal phenotype of the cav‐1−/− mouse, which lacks caveolae, was examined. μCT and histology showed increased trabecular and cortical bone caused by the gene deletion. Structural changes were accompanied by increased mechanical properties. Cell studies showed that cav‐1 deficiency leads to increased osteoblast differentiation. These results suggest that cav‐1 helps to maintain osteoblast progenitors in a less differentiated state.

Transient muscle paralysis disrupts bone homeostasis by rapid degradation of bone morphology

We have previously shown that transient paralysis of murine hindlimb muscles causes profound degradation of both trabecular and cortical bone in the adjacent skeleton within 3 weeks. Morphologically, the acute loss of bone tissue appeared to arise primarily due to osteoclastic bone resorption. Given that the loss of muscle function in this model is transient, we speculated that the stimulus for osteoclastic activation would be rapid and morphologic evidence of bone resorption would appear before 21 days. We therefore utilized high-resolution in vivo serial micro-CT to assess longitudinal alterations in lower hindlimb muscle volume, proximal tibia trabecular, and tibia mid-diaphysis cortical bone morphology in 16-week-old female C57 mice following transient calf paralysis from a single injection of botulinum toxin A (BtA; 2U/100 g body weight). In an acute study, we evaluated muscle and bone alterations at days 0, 3, 5, and 12 following transient calf paralysis. In a chronic study, following day 0 imaging, we assessed the recovery of these tissues following the maximum observed trabecular degradation (day 12) through day 84 post-paralysis. The time course and degree of recovery of muscle, trabecular, and cortical bone varied substantially. Significant atrophy of lower limb muscle was evident by day 5 of paralysis, maximal at day 28 (-34.1+/-0.9%) and partially recovered by day 84. Trabecular degradation within the proximal tibia metaphysis occurred more rapidly, with significant reduction in BV/TV by day 3, maximal loss at day 12 (-76.8+/-2.9%) with only limited recovery by day 84 (-51.7+/-5.1% vs. day 0). Significant cortical bone volume degradation at the tibia mid-diaphysis was first identified at day 12, was maximal at day 28 (-9.6+/-1.2%), but completely recovered by day 84. The timing, magnitude, and morphology of the observed bone erosion induced by transient muscle paralysis were consistent with a rapid recruitment and prolific activation of osteoclastic resorption. In a broader context, understanding how brief paralysis of a single muscle group can precipitate such rapid and profound bone resorption in an adjacent bone is likely to provide new insight into how normal muscle function modulates bone homeostasis.

Cabozantinib Inhibits Growth of Androgen-Sensitive and Castration-Resistant Prostate Cancer and Affects Bone Remodeling

Cabozantinib is an inhibitor of multiple receptor tyrosine kinases, including MET and VEGFR2. In a phase II clinical trial in advanced prostate cancer (PCa), cabozantinib treatment improved bone scans in 68% of evaluable patients. Our studies aimed to determine the expression of cabozantinib targets during PCa progression and to evaluate its efficacy in hormone-sensitive and castration-resistant PCa in preclinical models while delineating its effects on tumor and bone. Using immunohistochemistry and tissue microarrays containing normal prostate, primary PCa, and soft tissue and bone metastases, our data show that levels of MET, P-MET, and VEGFR2 are increasing during PCa progression. Our data also show that the expression of cabozantinib targets are particularly pronounced in bone metastases. To evaluate cabozantinib efficacy on PCa growth in the bone environment and in soft tissues we used androgen-sensitive LuCaP 23.1 and castration-resistant C4-2B PCa tumors. In vivo, cabozantinib inhibited the growth of PCa in bone as well as growth of subcutaneous tumors. Furthermore, cabozantinib treatment attenuated the bone response to the tumor and resulted in increased normal bone volume. In summary, the expression pattern of cabozantinib targets in primary and castration-resistant metastatic PCa, and its efficacy in two different models of PCa suggest that this agent has a strong potential for the effective treatment of PCa at different stages of the disease.