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.

The means by which muscle function modulates bone homeostasis is poorly understood. To begin to address this issue, we have developed a novel murine model of unilateral transient hindlimb muscle paralysis using botulinum toxin A (Botox). Female C57BL/6 mice (16 weeks) received IM injections of either saline or Botox (n = 10 each) in both the quadriceps and calf muscles of the right hindleg. Gait dysfunction was assessed by multi-observer inventory, muscle alterations were determined by wet mass, and bone alterations were assessed by micro-CT imaging at the distal femur, proximal tibia, and tibia mid-diaphysis. Profound degradation of both muscle and bone was observed within 21 days despite significant restoration of weight bearing function by 14 days. The muscle mass of the injected quadriceps and calf muscles was diminished −47.3% and −59.7%, respectively, vs. saline mice (both P < 0.001). The ratio of bone volume to tissue volume (BV/TV) within the distal femoral epiphysis and proximal tibial metaphysis of Botox injected limbs was reduced −43.2% and −54.3%, respectively, while tibia cortical bone volume was reduced −14.6% (all P < 0.001). Comparison of the contralateral non-injected limbs indicated the presence of moderate systemic effects in the model that were most probably associated with diminished activity following muscle paralysis. Taken as a whole, the micro-CT data implied that trabecular and cortical bone loss was primarily achieved by bone resorption. These data confirm the decisive role of neuromuscular function in mediating bone homeostasis and establish a model with unique potential to explore the mechanisms underlying this relation. Given the rapidly expanding use of neuromuscular inhibitors for indications such as pain reduction, these data also raise the critical need to monitor bone loss in these patients.

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

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/ ␤-catenin signaling has been recognized in promoting bone anabolism, and application of strain has been shown to induce ␤-catenin activation. In this work, we have used a preosteoblastic cell line to study the effects of dynamic mechanical strain on ␤-catenin signaling. We found that mechanical strain caused a rapid, transient accumulation of active ␤-catenin in the cytoplasm and its translocation to the nucleus. This was followed by up-regulation of the Wnt/␤-catenin target genes Wisp1 and Cox2, with peak responses at 4 and 1 h of strain, respectively. The increase of ␤-catenin was temporally related to the activation of Akt and subsequent inactivation of GSK3␤, 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 ␤-catenin or upregulation of Wisp1 and Cox2 expression. Conditions that increased basal ␤-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 GSK3␤ to allow ␤-catenin translocation, and Wnt signaling through LRP5 is not required for these strain-mediated responses. Thus, ␤-catenin serves as both a modulator and effector of mechanical signals in bone cells.Bone tissue undergoes remodeling throughout life to adapt to its mechanical load, resulting in a mass that is optimized for daily mechanical demands. The remodeling process is tightly regulated by a balance between the number of bone-producing osteoblasts and bone-resorbing osteoclasts. Application of mechanical load promotes bone formation, whereas the removal of load leads to bone loss (1, 2). More recently, an important role for Wnt/␤-catenin signaling has been recognized in promoting bone anabolism (3), and interestingly, mice with a loss-of-function mutation in the Wnt co-receptor LRP5 were shown to be resistant to the positive effects of local bone loading (4). Information further linking mechanically induced bone formation with activation of Wnt/␤-catenin processes has emerged with evidence that straining cells causes translocation of ␤-catenin into the cell nucleus (5, 6). Consistent with ␤-catenin activation, osteoblasts respond to mechanical loading with increased expression of Wnt/␤-catenin target genes, including Sfrp1 (secreted frizzled-related protein 1) and cyclin D1 (5).The loss of ␤-catenin has been found to disrupt both skeletal development and postnatal bone acquisition (7,8), establishing the importance of ␤-catenin signaling in osteoblast differentiation and function. ␤-Catenin can be found in at least two different cellular pools, suggesting compartmentalized roles. The pool of ␤-catenin found at the plasma membrane is bound to E-cadherin and ␣-catenin in adherens junctions. The sol...

Physical activity is capable of increasing adult bone mass. The specific osteogenic component of the mechanical stimulus is, however, unknown. Using an exogenous loading model, it was recently reported that circumferential gradients of longitudinal normal strain are strongly associated with the specific sites of periosteal bone formation. Here, we used high-speed running to test this proposed relation in an exercise model of bone adaptation. The strain environment generated during running in a mid-diaphyseal tarsometatarsal section was determined from triplerosette strain gages in six adult roosters (>1 year). A second group of roosters was run at a high speed (1500 loading cycles/day) on a treadmill for 3 weeks. Periosteal surfaces were activated in five out of eight animals. Mechanical parameters as well as periosteal activation (as measured by incorporated fluorescent labels) were quantified site-specifically in 12 30°sectors subdividing a mid-diaphyseal section. The amount of periosteal mineralizing surface per sector correlated strongly (R 2 ‫؍‬ 0.63) with the induced peak circumferential strain gradients. Conversely, peak strain magnitude and peak strain rate were only weakly associated with the sites of periosteal activation. The unique feature of this study is that a specific mechanical stimulus (peak circumferential strain gradients) was successfully correlated with specific sites of periosteal bone activation induced in a noninvasive bone adaptation model. The knowledge of this mechanical parameter may help to design exercise regimens that are able to deposit bone at sites where increased structural strength is most needed. (J Bone Miner

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We examined the hypothesis that peak magnitude strain gradients are spatially correlated with sites of bone formation. Ten adult male turkeys underwent functional isolation of the right radius and a subsequent 4-week exogenous loading regimen. Full field solutions of the engendered strains were obtained for each animal using animal-specific, orthotropic finite element models. Circumferential, radial, and longitudinal gradients of normal strain were calculated from these solutions. Site-specific bone formation within 24 equal angle pie sectors was determined by automated image analysis of microradiographs taken from the mid-diaphysis of the experimental radii. The loading regimen increased mean cortical area (؎SE) by 32.3 ؎ 10.5% ( p ‫؍‬ 0.01). Across animals, some periosteal bone formation was observed in every sector. The amount of periosteal new bone area contained within each sector was not uniform. Circumferential strain gradients (r 2 ‫؍‬ 0.36) were most strongly correlated with the observed periosteal bone formation. SED (a scalar measure of stress/strain magnitude with minimal relation to fluid flow) was poorly correlated with periosteal bone formation (r 2 ‫؍‬ 0.01). The combination of circumferential, radial, and longitudinal strain gradients accounted for over 60% of the periosteal new bone area (r 2 ‫؍‬ 0.63). These data indicate that strain gradients, which are readily determined given a knowledge of the bone's strain environment and geometry, may be used to predict specific locations of new bone formation stimulated by mechanical loading. (J Bone Miner Res 1997;12:982-988)

Transgenic and knockout mice present a unique opportunity to study mechanotransduction pathways in vivo, but the difficulty inherent with applying externally controlled loads to the small mouse skeleton has hampered this approach. We have developed a novel device that enables the noninvasive application of controlled mechanical loads to the murine tibia. Calibration of tissue strains induced by the device indicated that the normal strain environment was repeatable across loading bouts. Two in vivo studies were performed to show the usefulness of the device. Using C57Bl/6J mice, we found that dynamic but not static loading increased cortical bone area. This result is consistent with previous models of bone adaptation, and the lack of adaptation induced by static loading serves as a negative control for the device. In a preliminary study, transgenic mice selectively overexpressing insulin-like growth factor 1 (IGF-1) in osteoblasts underwent a low-magnitude loading regimen. Periosteal bone formation was elevated 5-fold in the IGF-1-overexpressing mice but was not elevated in wild-type littermates, showing the potential for synergism between mechanical loading and selected factors. Based on these data, we anticipate that the murine tibia-loading device will enhance assessment of mechanotransduction pathways in vivo and, as a result, has the potential to facilitate novel gene discovery and optimization of synergies between drug therapies and mechanical loading.