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.

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 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.

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.

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.

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.

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.

Rest intervals reduce the number of loading bouts required to enhance bone formation.

PURPOSE: As our society becomes increasingly sedentary, compliance with exercise regimens that require numerous high-energy activities each week become less likely. Alternatively, given an osteogenic exercise intervention that required minimal effort, it is reasonable to presume that participation would be enhanced. Insertion of brief rest intervals between each cycle of mechanical loading holds potential to achieve this result because substantial osteoblast function is activated by many fewer loading repetitions within each loading bout. Here, we examined the complementary hypothesis that the number of bouts per week of rest-inserted loading could be reduced from three bouts per week without loss of osteogenic efficacy. METHODS: We conducted a series of 3-wk in vivo experiments that noninvasively exposed the right tibiae of mice to either cyclic (1 Hz) or rest-inserted loading interventions and quantified osteoblast function via dynamic histomorphometry. RESULTS: Although reducing loading bouts from three bouts per week (i.e., nine total bouts) to one bout per week (i.e., three total bouts) effectively mitigated the osteogenic benefit of cyclic loading, the same reduction did not significantly reduce periosteal bone formation parameters induced by rest-inserted loading. The osteogenic response was robust to the timing of the rest-inserted loading bouts (three bouts in the first week vs one bout per week for 3 wk). However, elimination of any single bout of the three one-bout-per-week bouts mitigated the osteogenic response to rest-inserted loading. Finally, periosteal osteoblast function assessed after the 3-wk intervention was not sensitive to the timing or number of rest-inserted loading bouts. CONCLUSIONS: We conclude that rest-inserted loading holds potential to retain the osteogenic benefits of mechanical loading with significantly reduced frequency of bouts of activity while also enabling greater flexibility in the timing of the activity.

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.

Enhancement of Flow-Induced AP-1 Gene Expression by Cyclosporin A Requires NFAT-Independent Signaling in Bone Cells.

Growing evidence suggests that aging compromises the ability of the skeleton to respond to anabolic mechanical stimuli. Recently, we reported that treating senescent mice with Cyclosporin A (CsA) rescued aging-related deficits in loading-induced bone formation. Given that the actions of CsA are often attributed to inhibition of the calcineurin/NFAT axis, we hypothesized that CsA enhances gene expression in bone cells exposed to fluid flow, by inhibiting nuclear NFATc1 accumulation. When exposed to flow, MC3T3-E1 osteoblastic cells exhibited rapid nuclear accumulation of NFATc1 that was abolished by CsA treatment. Under differentiation conditions, intermittent CsA treatment enhanced gene expression of late osteoblastic differentiation markers and activator protein 1 (AP-1) family members. Superimposing flow upon CsA further enhanced expression of the AP-1 members Fra-1 and c-Jun. To delineate the contribution of NFAT in this response, cells were treated with VIVIT, a specific inhibitor of the calcineurin/NFAT interaction. Treatment with VIVIT blocked flow-induced nuclear NFATc1 accumulation but did not recapitulate the CsA-mediated enhancement of flow-induced AP-1 component gene expression. Taken together, our study is the first to demonstrate that CsA enhances mechanically-induced gene expression of AP-1 components in bone cells, and suggests that this response requires calcineurin-dependent mechanisms that are independent of inhibiting NFATc1 nuclear accumulation.

Botulinum toxin induces muscle paralysis and inhibits bone regeneration in zebrafish.

Intramuscular administration of Botulinum toxin (BTx) has been associated with impaired osteogenesis in diverse conditions of bone formation (eg, development, growth, and healing), yet the mechanisms of neuromuscular-bone crosstalk underlying these deficits have yet to be identified. Motivated by the emerging utility of zebrafish (Danio rerio) as a rapid, genetically tractable, and optically transparent model for human pathologies (as well as the potential to interrogate neuromuscular-mediated bone disorders in a simple model that bridges in vitro and more complex in vivo model systems), in this study, we developed a model of BTx-induced muscle paralysis in adult zebrafish, and we examined its effects on intramembranous ossification during tail fin regeneration. BTx administration induced rapid muscle paralysis in adult zebrafish in a manner that was dose-dependent, transient, and focal, mirroring the paralytic phenotype observed in animal and human studies. During fin regeneration, BTx impaired continued bone ray outgrowth, morphology, and patterning, indicating defects in early osteogenesis. Further, BTx significantly decreased mineralizing activity and crystalline mineral accumulation, suggesting delayed late-stage osteoblast differentiation and/or altered secondary bone apposition. Bone ray transection proximal to the amputation site focally inhibited bone outgrowth in the affected ray, implicating intra- and/or inter-ray nerves in this process. Taken together, these studies demonstrate the potential to interrogate pathological features of BTx-induced osteoanabolic dysfunction in the regenerating zebrafish fin, define the technological toolbox for detecting bone growth and mineralization deficits in this process, and suggest that pathways mediating neuromuscular regulation of osteogenesis may be conserved beyond established mammalian models of bone anabolic disorders.

Distinct cyclosporin a doses are required to enhance bone formation induced by cyclic and rest-inserted loading in the senescent skeleton.

Age-related decline in periosteal adaptation negatively impacts the ability to utilize exercise to enhance bone mass and strength in the elderly. We recently observed that in senescent animals subject to cyclically applied loading, supplementation with Cyclosporin A (CsA) substantially enhanced the periosteal bone formation rates to levels observed in young animals. We therefore speculated that if the CsA supplement could enhance bone response to a variety of types of mechanical stimuli, this approach could readily provide the means to expand the range of mild stimuli that are robustly osteogenic at senescence. Here, we specifically hypothesized that a given CsA supplement would enhance bone formation induced in the senescent skeleton by both cyclic (1-Hz) and rest-inserted loading (wherein a 10-s unloaded rest interval is inserted between each load cycle). To examine this hypothesis, the right tibiae of senescent female C57BL/6 mice (22 Mo) were subjected to cyclic or rest-inserted loading supplemented with CsA at 3.0 mg/kg. As previously, we initially found that while the periosteal bone formation rate (p.BFR) induced by cyclic loading was enhanced when supplemented with 3.0 mg/kg CsA (by 140%), the response to rest-inserted loading was not augmented at this CsA dosage. In follow-up experiments, we observed that while a 30-fold lower CsA dosage (0.1 mg/kg) significantly enhanced p.BFR induced by rest-inserted loading (by 102%), it was ineffective as a supplement with cyclic loading. Additional experiments and statistical analysis confirmed that the dose-response relations were significantly different for cyclic versus rest-inserted loading, only because the two stimuli required distinct CsA dosages for efficacy. While not anticipated a priori, clarifying the complexity underlying the observed interaction between CsA dosage and loading type holds potential for insight into how bone response to a broad range of mechanical stimuli may be substantially enhanced in the senescent skeleton.

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.

Systems-based identification of temporal processing pathways during bone cell mechanotransduction.

Bone has long been established to be a highly mechanosensitive tissue. When subjected to mechanical loading, bone exhibits profoundly different anabolic responses depending on the temporal pattern in which the stimulus is applied. This phenomenon has been termed temporal processing, and involves complex signal amplification mechanisms that are largely unidentified. In this study, our goal was to characterize transcriptomic perturbations arising from the insertion of intermittent rest periods (a temporal variation with profound effects on bone anabolism) in osteoblastic cells subjected to fluid flow, and assess the utility of these perturbations to identify signaling pathways that are differentially activated by this temporal variation. At the level of the genome, we found that the common and differential alterations in gene expression arising from the two flow conditions were distributionally distinct, with the differential alterations characterized by many small changes in a large number of genes. Using bioinformatics analysis, we identified distinct up- and down-regulation transcriptomic signatures associated with the insertion of rest intervals, and found that the up-regulation signature was significantly associated with MAPK signaling. Confirming the involvement of the MAPK pathway, we found that the insertion of rest intervals significantly elevated flow-induced p-ERK1/2 levels by enabling a second spike in activity that was not observed in response to continuous flow. Collectively, these studies are the first to characterize distinct transcriptomic perturbations in bone cells subjected to continuous and intermittent stimulation, and directly demonstrate the utility of systems-based transcriptomic analysis to identify novel acute signaling pathways underlying temporal processing in bone cells.

Metaphyseal and diaphyseal bone loss in the tibia following transient muscle paralysis are spatiotemporally distinct resorption events.

When the skeleton is catabolically challenged, there is great variability in the timing and extent of bone resorption observed at cancellous and cortical bone sites. It remains unclear whether this resorptive heterogeneity, which is often evident within a single bone, arises from increased permissiveness of specific sites to bone resorption or localized resorptive events of varied robustness. To explore this question, we used the mouse model of calf paralysis induced bone loss, which results in metaphyseal and diaphyseal bone resorption of different timing and magnitude. Given this phenotypic pattern of resorption, we hypothesized that bone loss in the proximal tibia metaphysis and diaphysis occurs through resorption events that are spatially and temporally distinct. To test this hypothesis, we undertook three complimentary in vivo/µCT imaging studies. Specifically, we defined spatiotemporal variations in endocortical bone resorption during the 3weeks following calf paralysis, applied a novel image registration approach to determine the location where bone resorption initiates within the proximal tibia metaphysis, and explored the role of varied basal osteoclast activity on the magnitude of bone loss initiation in the metaphysis using µCT based bone resorption parameters. A differential response of metaphyseal and diaphyseal bone resorption was observed throughout each study. Acute endocortical bone loss following muscle paralysis occurred almost exclusively within the metaphyseal compartment (96.5% of total endocortical bone loss within 6days). Using our trabecular image registration approach, we further resolved the initiation of metaphyseal bone loss to a focused region of significant basal osteoclast function (0.03mm(3)) adjacent to the growth plate. This correlative observation of paralysis induced bone loss mediated by basal growth plate cell dynamics was supported by the acute metaphyseal osteoclastic response of 5-week vs. 13-month-old mice. Specifically, µCT based bone resorption rates normalized to initial trabecular surface (BRRBS) were 3.7-fold greater in young vs. aged mice (2.27±0.27µm(3)/µm(2)/day vs. 0.60±0.44µm(3)/µm(2)/day). In contrast to the focused bone loss initiation in the metaphysis, diaphyseal bone loss initiated homogeneously throughout the long axis of the tibia predominantly in the second week following paralysis (81.3% of diaphyseal endocortical expansion between days 6 and 13). The timing and homogenous nature are consistent with de novo osteoclastogenesis mediating the diaphyseal resorption. Taken together, our data suggests that tibial metaphyseal and diaphyseal bone loss induced by transient calf paralysis are spatially and temporally discrete events. In a broader context, these findings are an essential first step toward clarifying the timing and origins of multiple resorptive events that would require targeting to fully inhibit bone loss following neuromuscular trauma.

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.

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.

Bone mechanotransduction may require augmentation in order to strengthen the senescent skeleton.

Physical exercise is thought to hold promise as a non-invasive countermeasure against skeletal fragility arising from post-menopausal and age-related osteoporosis. Importantly, mechanical loading and exercise are capable of increasing bone size via periosteal expansion, which by far, is the most effective means of strengthening the structure of a given bone. The focus of this review was to therefore explore whether exercise has the potential to increase periosteal modeling and bone size in the senescent skeleton. A survey of exercise trials in humans suggests that exercise interventions that enhance periosteal modeling in the young skeleton fail to do the same in the elderly skeleton. Underlying this ineffectiveness, in vitro studies indicate that aging lowers basal levels of cell function and degrades bone mechanotransduction at a variety of levels from altered second messenger signaling to gene expression driving proliferation and/or differentiation. Given these age-related alterations, the ultimate efficacy of an exercise intervention may depend upon concurrent supplementation that directly address deficits in signaling and/or cell function. In this context, in vivo animal models of mechanical loading that simulate the muted periosteal adaptation in the elderly hold potential to examine the efficacy of countermeasures. Preliminary in vivo experiments suggest that pharmacologically counteracting age-related deficits in cellular function can restore exercise induced periosteal modeling in the senescent skeleton to levels observed in young animals. If the safety and efficacy of this strategy were to be confirmed for human use, it would enable the utilization of exercise as a viable countermeasure against skeletal fragility at senescence.