Neuromuscular dysfunction, independent of gait dysfunction, modulates trabecular bone homeostasis in mice.

OBJECTIVES: To clarify the effects of neuromuscular dysfunction on hindlimb loading, muscle atrophy, and bone homeostasis. METHODS: We quantified changes to hindlimb loading, muscle atrophy, and bone morphology following either Botulinum toxin A (BTxA) induced muscle paralysis or peripheral nerve injury (PNI) in mice; two in vivo models that we anticipated would differently alter gait and mechanical loading patterns due to their distinct effects on neuromuscular signaling. To confirm the expected behavioral effects of PNI, we assessed mechanical allodynia of the ipsilateral hindlimb using von Frey testing and activity (distance traveled and speed) was monitored in both groups using open field testing. Peak vertical ground reaction forces (GRF) and ankle and knee kinematics during normal locomotion were quantified and used to estimate peak mid-diaphyseal normal strains. Muscle atrophy and trabecular and cortical bone morphology were assessed via high-resolution microCT imaging. RESULTS: BTxA-induced calf paralysis caused severe muscle atrophy and altered gait kinetics and kinematics and reduced gait-induced normal strains. PNI increased mechanical allodynia but did not alter gait, nor did it cause muscle atrophy. We observed that muscle paralysis and PNI both led to severe trabecular bone loss but only BTxA-induced paralysis increased cortical bone resorption. CONCLUSIONS: While mechanical stimuli clearly have essential functions in bone development and adaptation, these data emphasize that neuromuscular signaling, independent of load-induced mechanical strains, may modulate trabecular bone homeostasis in normal and disease states.

Muscle paralysis induces bone marrow inflammation and predisposition to formation of giant osteoclasts.

Transient muscle paralysis engendered by a single injection of botulinum toxin A (BTxA) rapidly induces profound focal bone resorption within the medullary cavity of adjacent bones. While initially conceived as a model of mechanical disuse, osteoclastic resorption in this model is disproportionately severe compared with the modest gait defect that is created. Preliminary studies of bone marrow following muscle paralysis suggested acute upregulation of inflammatory cytokines, including TNF-α and IL-1. We therefore hypothesized that BTxA-induced muscle paralysis would rapidly alter the inflammatory microenvironment and the osteoclastic potential of bone marrow. We tested this hypothesis by defining the time course of inflammatory cell infiltration, osteoinflammatory cytokine expression, and alteration in osteoclastogenic potential in the tibia bone marrow following transient muscle paralysis of the calf muscles. Our findings identified inflammatory cell infiltration within 24 h of muscle paralysis. By 72 h, osteoclast fusion and pro-osteoclastic inflammatory gene expression were upregulated in tibia bone marrow. These alterations coincided with bone marrow becoming permissive to the formation of osteoclasts of greater size and greater nuclei numbers. Taken together, our data are consistent with the thesis that transient calf muscle paralysis induces acute inflammation within the marrow of the adjacent tibia and that these alterations are temporally consistent with a role in mediating muscle paralysis-induced bone resorption.

Biomechanical aspects of the muscle-bone interaction.

There is growing interest in the interaction between skeletal muscle and bone, particularly at the genetic and molecular levels. However, the genetic and molecular linkages between muscle and bone are achieved only within the context of the essential mechanical coupling of the tissues. This biomechanical and physiological linkage is readily evident as muscles attach to bone and induce exposure to varied mechanical stimuli via functional activity. The responsiveness of bone cells to mechanical stimuli, or their absence, is well established. However, questions remain regarding how muscle forces applied to bone serve to modulate bone homeostasis and adaptation. Similarly, the contributions of varied, but unique, stimuli generated by muscle to bone (such as low-magnitude, high-frequency stimuli) remains to be established. The current article focuses upon the mechanical relationship between muscle and bone. In doing so, we explore the stimuli that muscle imparts upon bone, models that enable investigation of this relationship, and recent data generated by these models.

Botulinum Toxin-induced Muscle Paralysis Inhibits Heterotopic Bone Formation.

BACKGROUND: Short-term muscle atrophy induced by botulinum toxin A (BTxA) has been observed to impair osteogenesis in a rat closed femur fracture model. However, it is unclear whether the underlying mechanism is a direct effect of BTxA on muscle-bone interactions or an indirect effect that is driven by skeletal unloading. Because skeletal trauma in the closed fracture model also leads to disuse atrophy, we sought to mitigate this confounding variable by examining BTxA effects on muscle-bone interactions in two complementary in vivo models in which osteogenesis is induced in the absence of skeletal unloading. The overall aim of this study was to identify a potential strategy to inhibit pathological bone formation and heterotopic ossification (HO). QUESTIONS/PURPOSES: (1) Does muscle paralysis inhibit periosteal osteogenesis induced by a transcortical defect? (2) Does muscle paralysis inhibit heterotopic bone formation stimulated by intramuscular bone morphogenetic protein (BMP) injection? METHODS: Focal osteogenesis was induced in the right hindlimb of mice through surgical initiation of a small transcortical defect in the tibia (fracture callus; n = 7/group) or intramuscular injection of BMP-2 (HO lesion; n = 6/group), both in the presence/absence of adjacent calf paralysis. High-resolution micro-CT images were obtained in all experimental groups 21 days postinduction and total volume (ie, perimeter of periosteal callus or HO lesion) and bone volume (calcified tissue within the total volume) were quantified as primary outcome measures. Finally, these outcome measures were compared to determine the effect of muscle paralysis on inhibition of local osteogenesis in both studies. RESULTS: After a transcortical defect, BTxA-treated mice showed profound inhibition of osteogenesis in the periosteal fracture callus 21 days postsurgery compared with saline-treated mice (total volume: 0.08 ± 0.06 versus 0.42 ± 0.11 mm(3), p < 0.001; bone volume: 0.07 ± 0.05 versus 0.32 ± 0.07 mm(3), p < 0.001). Similarly, BMP-2-induced HO formation was inhibited by adjacent muscle paralysis at the same time point (total volume: 1.42 ± 0.31 versus 3.42 ± 2.11 mm(3), p = 0.034; bone volume: 0.68 ± 0.18 versus 1.36 ± 0.79 mm(3), p = 0.045). CONCLUSIONS: Our data indicate that BTxA-induced neuromuscular inhibition mitigated osteogenesis associated with both a transcortical defect and BMP-2-induced HO. CLINICAL RELEVANCE: Focal neuromuscular inhibition represents a promising new approach that may lead to a new clinical intervention to mitigate trauma-induced HO, a healthcare challenge that is severely debilitating for civilian and war-wounded populations, is costly to both the patient and the healthcare system, and currently lacks effective treatments.

Control of bone resorption in mice by Schnurri-3.

Mice lacking the large zinc finger protein Schnurri-3 (Shn3) display increased bone mass, in part, attributable to augmented osteoblastic bone formation. Here, we show that in addition to regulating bone formation, Shn3 indirectly controls bone resorption by osteoclasts in vivo. Although Shn3 plays no cell-intrinsic role in osteoclasts, Shn3-deficient animals show decreased serum markers of bone turnover. Mesenchymal cells lacking Shn3 are defective in promoting osteoclastogenesis in response to selective stimuli, likely attributable to reduced expression of the key osteoclastogenic factor receptor activator of nuclear factor-κB ligand. The bone phenotype of Shn3-deficient mice becomes more pronounced with age, and mice lacking Shn3 are completely resistant to disuse osteopenia, a process that requires functional osteoclasts. Finally, selective deletion of Shn3 in the mesenchymal lineage recapitulates the high bone mass phenotype of global Shn3 KO mice, including reduced osteoclastic bone catabolism in vivo, indicating that Shn3 expression in mesenchymal cells directly controls osteoblastic bone formation and indirectly regulates osteoclastic bone resorption.

The mechanical consequences of load bearing in the equine third metacarpal across speed and gait: the nonuniform distributions of normal strain, shear strain, and strain energy density.

Distributions of normal strain, shear strain, and strain energy density (SED) were determined across the midshaft of the third metacarpal (MCIII, or cannon bone) of 3 adult thoroughbred horses as a function of speed and gait. A complete characterization of the mechanical demands of the bone made through the stride and from mild through the extremes of locomotion was possible by using three 3-element rosette strain gauges bonded at the diaphyseal midshaft of the MCIII and evaluating the strain output with beam theory and finite element analysis. Mean ± sd values of normal strain, shear strain, and SED increased with speed and peaked during a canter (-3560±380 microstrain, 1760±470 microstrain, and 119±23 kPa, respectively). While the location of these peaks was similar across animals and gaits, the resulting strain distributions across the cortex were consistently nonuniform, establishing between a 73-fold (slow trot) to a 330-fold (canter) disparity between the sites of maximum and minimum SED for each gait cycle. Using strain power density as an estimate of strain history across the bone revealed a 154-fold disparity between peak and minimum at the walk but fell to ~32-fold at the canter. The nonuniform, minimally varying, strain environment suggests either that bone homeostasis is mediated by magnitude-independent mechanical signals or that the duration of stimuli necessary to establish and maintain tissue integrity is relatively brief, and thus the vast majority of strain information is disregarded.

Transient muscle paralysis degrades bone via rapid osteoclastogenesis.

A unilateral injection of botulinum toxin A (BTxA) in the calf induces paralysis and profound loss of ipsalateral trabecular bone within days. However, the cellular mechanism underlying acute muscle paralysis-induced bone loss (MPIBL) is poorly understood. We hypothesized that MPIBL arises via rapid and extensive osteoclastogenesis. We performed a series of in vivo experiments to explore this thesis. First, we observed elevated levels of the proosteoclastogenic cytokine receptor activator for nuclear factor-κB ligand (RANKL) within the proximal tibia metaphysis at 7 d after muscle paralysis (+113%, P<0.02). Accordingly, osteoclast numbers were increased 122% compared with the contralateral limb at 5 d after paralysis (P=0.04) and MPIBL was completely blocked by treatment with human recombinant osteoprotegerin (hrOPG). Further, conditional deletion of nuclear factor of activated T-cells c1 (NFATc1), the master regulator of osteoclastogenesis, completely inhibited trabecular bone loss (-2.2±11.9%, P<0.01). All experiments included negative control assessments of contralateral limbs and/or within-animal pre- and postintervention imaging. In summary, transient muscle paralysis induced acute RANKL-mediated osteoclastogenesis resulting in profound local bone resorption. Elucidation of the pathways that initiate osteoclastogenesis after paralysis may identify novel targets to inhibit bone loss and prevent fractures.

A convolutional neural network to characterize mouse hindlimb foot strikes during voluntary wheel running.

Voluntary wheel running (VWR) is widely used to study how exercise impacts a variety of physiologies and pathologies in rodents. The primary activity readout of VWR is aggregated wheel turns over a given time interval (most often, days). Given the typical running frequency of mice (∼4 Hz) and the intermittency of voluntary running, aggregate wheel turn counts, therefore, provide minimal insight into the heterogeneity of voluntary activity. To overcome this limitation, we developed a six-layer convolutional neural network (CNN) to determine the hindlimb foot strike frequency of mice exposed to VWR. Aged female C57BL/6 mice (22 months, n = 6) were first exposed to wireless angled running wheels for 2 h/d, 5 days/wk for 3 weeks with all VWR activities recorded at 30 frames/s. To validate the CNN, we manually classified foot strikes within 4800 1-s videos (800 randomly chosen for each mouse) and converted those values to frequency. Upon iterative optimization of model architecture and training on a subset of classified videos (4400), the CNN model achieved an overall training set accuracy of 94%. Once trained, the CNN was validated on the remaining 400 videos (accuracy: 81%). We then applied transfer learning to the CNN to predict the foot strike frequency of young adult female C57BL6 mice (4 months, n = 6) whose activity and gait differed from old mice during VWR (accuracy: 68%). In summary, we have developed a novel quantitative tool that non-invasively characterizes VWR activity at a much greater resolution than was previously accessible. This enhanced resolution holds potential to overcome a primary barrier to relating intermittent and heterogeneous VWR activity to induced physiological responses.

Beta 2 Adrenergic Receptor Selective Antagonist Enhances Mechanically Stimulated Bone Anabolism in Aged Mice.

The anabolic response of aged bone to skeletal loading is typically poor. Efforts to improve mechanotransduction in aged bone have met with limited success. This study investigated whether the bone response to direct skeletal loading is improved by reducing sympathetic suppression of osteoblastic bone formation via ß2AR. To test this possibility, we treated aged wild-type C57BL/6 mice with a selective ß2AR antagonist, butaxamine (Butax), before each of nine bouts of cantilever bending of the right tibia. Midshaft periosteal bone formation was assessed by dynamic histomorphometry of loaded and contralateral tibias. Butax treatment did not alter osteoblast activity of contralateral tibias. Loading alone induced a modest but significant osteogenic response. However, when loading was combined with Butax pretreatment, the anabolic response was significantly elevated compared with loading preceded by saline injection. Subsequent studies in osteoblastic cultures revealed complex negative interactions between adrenergic and mechanically induced intracellular signaling. Activation of ß2AR by treatment with the ß1, ß2-agonist isoproterenol (ISO) before fluid flow exposure diminished mechanically stimulated ERK1/2 phosphorylation in primary bone cell outgrowth cultures and AKT phosphorylation in MC3T3-E1 pre-osteoblast cultures. Expression of mechanosensitive Fos and Ptgs2 genes was enhanced with ISO treatment and reduced with flow in both MC3T3-E1 and primary cultures. Finally, co-treatment of MC3T3-E1 cells with Butax reversed these ISO effects, confirming a critical role for ß2AR in these responses. In combination, these results demonstrate that selective inhibition of ß2AR is sufficient to enhance the anabolic response of the aged skeleton to loading, potentially via direct effects upon osteoblasts. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

A microCT-based platform to quantify drug targeting.

BACKGROUND: Heterotopic ossification (HO) is a frequent and debilitating complication of traumatic musculoskeletal injuries and orthopedic procedures. Prophylactic dosing of botulinum toxin type A (BTxA) holds potential as a novel treatment option if accurately distributed throughout soft-tissue volumes where protection is clinically desired. We developed a high-resolution, microcomputed tomography (microCT)-based imaging strategy to assess drug distribution and validated this platform by quantifying distribution achieved via a prototype delivery system versus a single-bolus injection. METHODS: We injected an iodine-containing contrast agent (iodixanol 320 mg I/mL) into dissected rabbit musculature followed by microCT imaging and analysis. To contrast the performance of distributed versus bolus injections, a three-dimensional (3D) 64-cm3-printed soft-tissue holder was developed. A centered 2-cm3 volume of interest (VOI) was targeted with a single-bolus injection or an equal volume distributed injection delivered via a 3D-printed prototype. VOI drug coverage was quantified as a percentage of the VOI volume that was < 1.0 mm from the injected fluid. RESULTS: The microCT-based approach enabled high-resolution quantification of injection distribution within soft tissue. The distributed dosing prototype provided significantly greater tissue coverage of the targeted VOI (72 ± 3%, mean ± standard deviation) when compared to an equal volume bolus dose (43 ± 5%, p = 0.031) while also enhancing the precision of injection targeting. CONCLUSIONS: A microCT-based imaging technique precisely quantifies drug distribution within a soft-tissue VOI, providing a path to overcome a barrier for clinical translation of prophylactic inhibition of HO by BTxA. RELEVANCE STATEMENT: This platform will facilitate rapid optimization of injection parameters for clinical devices used to effectively and safely inhibit the formation of heterotopic ossification. KEY POINTS: • MicroCT provides high-resolution quantification of soft-tissue drug distribution. • Distributed dosing is required to maximize soft-tissue drug coverage. • Imaging platform will enable rapid screening of 3D-printed drug distribution prototypes.

Hypoxia-inducible factor-1α protein negatively regulates load-induced bone formation.

Mechanical loads induce profound anabolic effects in the skeleton, but the molecular mechanisms that transduce such signals are still poorly understood. In this study, we demonstrate that the hypoxia-inducible factor-1α (Hif-1α) is acutely up-regulated in response to exogenous mechanical stimuli secondary to prostanoid signaling and Akt/mTOR (mammalian target of rapamycin) activation. In this context, Hif-1α associates with ß-catenin to inhibit Wnt target genes associated with bone anabolic activity. Mice lacking Hif-1α in osteoblasts and osteocytes form more bone when subjected to tibia loading as a result of increased osteoblast activity. Taken together, these studies indicate that Hif-1α serves as a negative regulator of skeletal mechanotransduction to suppress load-induced bone formation by altering the sensitivity of osteoblasts and osteocytes to mechanical signals.

Rescuing loading induced bone formation at senescence.

The increasing incidence of osteoporosis worldwide requires anabolic treatments that are safe, effective, and, critically, inexpensive given the prevailing overburdened health care systems. While vigorous skeletal loading is anabolic and holds promise, deficits in mechanotransduction accrued with age markedly diminish the efficacy of readily complied, exercise-based strategies to combat osteoporosis in the elderly. Our approach to explore and counteract these age-related deficits was guided by cellular signaling patterns across hierarchical scales and by the insight that cell responses initiated during transient, rare events hold potential to exert high-fidelity control over temporally and spatially distant tissue adaptation. Here, we present an agent-based model of real-time Ca(2+)/NFAT signaling amongst bone cells that fully described periosteal bone formation induced by a wide variety of loading stimuli in young and aged animals. The model predicted age-related pathway alterations underlying the diminished bone formation at senescence, and hence identified critical deficits that were promising targets for therapy. Based upon model predictions, we implemented an in vivo intervention and show for the first time that supplementing mechanical stimuli with low-dose Cyclosporin A can completely rescue loading induced bone formation in the senescent skeleton. These pre-clinical data provide the rationale to consider this approved pharmaceutical alongside mild physical exercise as an inexpensive, yet potent therapy to augment bone mass in the elderly. Our analyses suggested that real-time cellular signaling strongly influences downstream bone adaptation to mechanical stimuli, and quantification of these otherwise inaccessible, transient events in silico yielded a novel intervention with clinical potential.

A FAK/HDAC5 signaling axis controls osteocyte mechanotransduction.

Osteocytes, cells ensconced within mineralized bone matrix, are the primary skeletal mechanosensors. Osteocytes sense mechanical cues by changes in fluid flow shear stress (FFSS) across their dendritic projections. Loading-induced reductions of osteocytic Sclerostin (encoded by Sost) expression stimulates new bone formation. However, the molecular steps linking mechanotransduction and Sost suppression remain unknown. Here, we report that class IIa histone deacetylases (HDAC4 and HDAC5) are required for loading-induced Sost suppression and bone formation. FFSS signaling drives class IIa HDAC nuclear translocation through a signaling pathway involving direct HDAC5 tyrosine 642 phosphorylation by focal adhesion kinase (FAK), a HDAC5 post-translational modification that controls its subcellular localization. Osteocyte cell adhesion supports FAK tyrosine phosphorylation, and FFSS triggers FAK dephosphorylation. Pharmacologic FAK catalytic inhibition reduces Sost mRNA expression in vitro and in vivo. These studies demonstrate a role for HDAC5 as a transducer of matrix-derived cues to regulate cell type-specific gene expression.

Static Preload Inhibits Loading-Induced Bone Formation.

Nearly all exogenous loading models of bone adaptation apply dynamic loading superimposed upon a time invariant static preload (SPL) in order to ensure stable, reproducible loading of bone. Given that SPL may alter aspects of bone mechanotransduction (eg, interstitial fluid flow), we hypothesized that SPL inhibits bone formation induced by dynamic loading. As a first test of this hypothesis, we utilized a newly developed device that enables stable dynamic loading of the murine tibia with SPLs ≥ -0.01 N. We subjected the right tibias of BALB/c mice (4-month-old females) to dynamic loading (-3.8 N, 1 Hz, 50 cycles/day, 10 s rest) superimposed upon one of three SPLs: -1.5 N, -0.5 N, or -0.03 N. Mice underwent exogenous loading 3 days/week for 3 weeks. Metaphyseal trabecular bone adaptation (µCT) and midshaft cortical bone formation (dynamic histomorphometry) were assessed following euthanasia (day 22). Ipsilateral tibias of mice loaded with a -1.5-N SPL demonstrated significantly less trabecular bone volume/total volume (BV/TV) than contralateral tibias (-12.9%). In contrast, the same dynamic loading superimposed on a -0.03-N SPL significantly elevated BV/TV versus contralateral tibias (12.3%) and versus the ipsilateral tibias of the other SPL groups (-0.5 N: 46.3%, -1.5 N: 37.2%). At the midshaft, the periosteal bone formation rate (p.BFR) induced when dynamic loading was superimposed on -1.5-N and -0.5-N SPLs was significantly amplified in the -0.03-N SPL group (>200%). These data demonstrate that bone anabolism induced by dynamic loading is markedly inhibited by SPL magnitudes commonly implemented in the literature (ie, -0.5 N, -1.5 N). The inhibitory impact of SPL has not been recognized in bone adaptation models and, as such, SPLs have been neither universally reported nor standardized. Our study therefore identifies a previously unrecognized, potent inhibitor of mechanoresponsiveness that has potentially confounded studies of bone adaptation and translation of insights from our field. © 2018 The Authors. JBMR Plus Published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

32 wk old C3H/HeJ mice actively respond to mechanical loading.

Numerous studies indicate that C3H/HeJ (C3H) mice are mildly responsive to mechanical loading compared to C57BL/6J (C57) mice. Guided by data indicating high baseline periosteal osteoblast activity in 16 wk C3H mice, we speculated that simply allowing the C3H mice to age until basal periosteal bone formation was equivalent to that of 16 wk C57 mice would restore mechanoresponsiveness in C3H mice. We tested this hypothesis by subjecting the right tibiae of 32 wk old C3H mice and 16 wk old C57 mice to low magnitude rest-inserted loading (peak strain: 1235 mu epsilon) and then exposing the right tibiae of 32 wk C3H mice to low (1085 mu epsilon) or moderate (1875 mu epsilon) magnitude cyclic loading. The osteoblastic response to loading on the endocortical and periosteal surfaces was evaluated via dynamic histomorphometry. At 32 wk of age, C3H mice responded to low magnitude rest-inserted loading with significantly elevated periosteal mineralizing surface, mineral apposition rate and bone formation compared to unloaded contralateral bones. Surprisingly, the periosteal bone formation induced by low magnitude rest-inserted loading in C3H mice exceeded that induced in 16 wk C57 mice. At 32 wk of age, C3H mice also demonstrated an elevated response to increased magnitudes of cyclic loading. We conclude that a high level of basal osteoblast function in 16 wk C3H mice appears to overwhelm the ability of the tissue to respond to an otherwise anabolic mechanical loading stimulus. However, when basal surface osteoblast activity is equivalent to that of 16 wk C57 mice, C3H mice demonstrate a clear ability to respond to either rest-inserted or cyclic loading.

Botulinum toxin A-induced muscle paralysis stimulates Hdac4 and differential miRNA expression.

At sufficient dose, intramuscular injection of Botulinum toxin A causes muscle wasting that is physiologically consistent with surgical denervation and other types of neuromuscular dysfunction. The aim of this study was to clarify early molecular and micro-RNA alterations in skeletal muscle following Botulinum toxin A-induced muscle paralysis. Quadriceps were analyzed for changes in expression of micro- and messenger RNA and protein levels after a single injection of 0.4, 2 or 4U Botulinum toxin A (/100g body weight). After injection with 2.0U Botulinum toxin A, quadriceps exhibited significant reduction in muscle weight and increased levels of ubiquitin ligase proteins at 7, 14 and 28 days. Muscle miR-1 and miR-133a/b levels were decreased at these time points, whereas a dose-responsive increase in miR-206 expression at day 14 was observed. Expression of the miR-133a/b target genes RhoA, Tgfb1 and Ctfg, and the miR-1/206 target genes Igf-1 and Hdac4, were upregulated at 28 days after Botulinum toxin A injection. Increased levels of Hdac4 protein were observed after injection, consistent with anticipated expression changes in direct and indirect Hdac4 target genes, such as Myog. Our results suggest Botulinum toxin A-induced denervation of muscle shares molecular characteristics with surgical denervation and other types of neuromuscular dysfunction, and implicates miR-133/Tgf-ß1/Ctfg and miR-1/Hdac4/Myog signaling during the resultant muscle atrophy.

Caveolin-1 knockout mice have increased bone size and stiffness.

UNLABELLED: The skeletal phenotype of the cav-1(-/-) mouse, which lacks caveolae, was examined. muCT 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. INTRODUCTION: The absence of caveolin-1 in cellular membranes causes dysregulated signaling. To understand the role of the caveolar microdomain in bone homeostasis, we examined the skeletal phenotype of 5- and 8-wk-old cav-1(-/-) mice. MATERIALS AND METHODS: High-resolution microCT imaging showed a region-specific effect of cav-1 deficiency on the skeleton. At 5 wk, cav-1(-/-) mice had increased epiphyseal bone volume (+58.4%, p = 0.05); at 8 wk, metaphyseal bone volume was increased by 77.4% (p = 0.008). Cortical bone at the femoral mid-diaphysis showed that the periosteal area of cav-1(-/-) mice significantly exceeded that of cav-1(+/+) mice by 23.9% and 16.3% at 5 and 8 wk, respectively, resulting in increased mechanical properties (I(max): +38.2%, p = 0.003 and I(mi): +23.7%, p = 0.03). RESULTS: Histomorphometry complemented microCT results showing increased bone formation rate (BFR) at trabecular and cortical sites at 5 wk, which supported findings of increased bone at 8 wk in cav-1(-/-) mice. Formal mechanical testing of the femoral diaphysis confirmed increased bone structure: stiffness increased 33% and postyield deflection decreased 33%. Stromal cells from cav-1(-/-) marrow showed a 23% increase in von Kossa-positive nodules; osteoclastogenesis was also modestly increased in cav-1-deficient marrow. Knockdown of cav-1 with siRNA in wildtype stromal cells increased alkaline phosphatase protein and expression of osterix and Runx2, consistent with osteoblast differentiation. CONCLUSIONS: These data suggest that cav-1 helps to maintain a less differentiated state of osteoblast progenitor cells, and the absence of cav-1 causes bone to mature more rapidly. Caveolin-1 may thus be a target for altering skeletal homeostasis.

Rest-inserted loading rapidly amplifies the response of bone to small increases in strain and load cycles.

We hypothesized that a 10-s rest interval (at zero load) inserted between each load cycle would increase the osteogenic effects of mechanical loading near previously identified thresholds for strain magnitude and cycle numbers. We tested our hypothesis by subjecting the right tibiae of female C57BL/6J mice (16 wk, n = 70) to exogenous mechanical loading within a peri-threshold physiological range of strain magnitudes and load cycle numbers using a noninvasive murine tibia loading device. Bone responses to mechanical loading were determined via dynamic histomorphometry. More specifically, we contrasted bone formation induced by cyclic vs. rest-inserted loading (10-s rest at zero load inserted between each load cycle) by first varying peak strains (1,000, 1,250, or 1,600 micro epsilon) at fixed cycle numbers (50 cycles/day, 3 days/wk for 3 wk) and then varying cycle numbers (10, 50, or 250 cycles/day) at a fixed strain magnitude (1,250 micro epsilon). Within the range of strain magnitudes tested, the slope of periosteal bone formation rate (p.BFR/BS) with increasing strain magnitudes was significantly increased by rest-inserted compared with cyclical loading. Within the range of load cycles tested, the slope of p.BFR/BS with increasing load cycles of rest-inserted loading was also significantly increased by rest-inserted compared with cyclical loading. In sum, the data of this study indicate that inserting a 10-s rest interval between each load cycle amplifies bone's response to mechanical loading, even within a peri-threshold range of strain magnitudes and cycle numbers.

Botox induced muscle paralysis rapidly degrades bone.

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.

Mice lacking thrombospondin 2 show an atypical pattern of endocortical and periosteal bone formation in response to mechanical loading.

Thrombospondin 2 (TSP2) is an extracellular matrix (ECM) protein localized to bone. Since mice with a targeted disruption of the TSP2 gene (TSP2-null) have increased bone formation, we hypothesized that mice lacking TSP2 would show an enhanced osteogenic response to mechanical loading. We addressed our hypothesis by subjecting wild-type (WT) and TSP2-null mice to mechanical loading using the non-invasive murine tibia loading device, and statistical comparisons were made between loaded and unloaded bones within genotype, between genotypes, and between the periosteal and endocortical surfaces within genotype. Right tibiae of WT and TSP2-null mice received 5 days of a low-magnitude loading protocol. This low-magnitude loading (inducing approximately 900 and 500 muepsilon at periosteal and endocortical surfaces of WT bones, respectively) affected neither periosteal nor endocortical bone formation rate (BFR/BS) when comparing loaded to intact bones in either WT or TSP2-null mice, nor did it result in any significant differences between WT and TSP2-null. As well, there was no difference between loaded endocortical and periosteal surfaces in WT mice; however, endocortical BFR/BS in TSP2-null loaded tibia was significantly elevated relative to the periosteal BFR/BS-despite peak periosteal strains being significantly greater than endocortical strains in TSP2-null mice (690 versus 460 muepsilon). To confirm this counterintuitive surface-specific response in TSP2-null mice and to induce significant periosteal bone formation, osteogenic potency of the loading protocol was amplified by doubling the number of loading bouts (10 loading days) and loading magnitude (1 Hz, resulting in 1400 and 900 muepsilon peak strain at the periosteal and endocortical surfaces, respectively). Under load, both WT and TSP2-null mice showed significantly increased periosteal mineralizing surface (by nearly three-fold and five-fold, respectively), but mineral apposition rate (MAR) was not statistically changed. The increased MS/BS resulted in a five-fold increase in WT periosteal BFR/BS, but the TSP2-null periosteal BFR/BS was unchanged. Furthermore, this increase in WT loaded periosteal BFR/BS was statistically greater than the WT endocortical BFR/BS. At the endocortical surface of WT mice, loading did not significantly increase bone formation parameters (versus intact). In contrast, at the endocortical surface of TSP2-null mice, loading induced a significant two-fold increase in BFR/BS (versus intact), that was also significantly greater than the endocortical BFR/BS of loaded WT mice. Thus, exogenous loading of TSP2-null mice resulted in highly variable responses that did not reflect the induced strains at the periosteal and endocortical surfaces. While in WT mice, loading resulted in increased periosteal BFR/BS that was greater than the endocortical BFR/BS, in TSP2-null mice loading resulted in endocortical (not periosteal) BFR/BS that was elevated. This reversal in envelope-specific bone formation in TSP2-null mice occurred despite periosteal strains being significantly greater than endocortical (1290 versus 775 muepsilon) and strain distributions being similar to that of WT. These results show that the disruption of a single gene can lead to a reversal in normal pattern of load induced bone formation, and more specifically, that the functional interaction of TSP2 with mechanical loading is highly contextual and specific to the cortical bone envelope examined.