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1.  Sclerostin Antibody Inhibits Skeletal Deterioration Due to Reduced Mechanical Loading 
Sclerostin, a product of the SOST gene produced mainly by osteocytes, is a potent negative regulator of bone formation that appears to be responsive to mechanical loading, with SOST expression increasing following mechanical unloading. We tested the ability of a murine sclerostin antibody (SclAbII) to prevent bone loss in adult mice subjected to hindlimb unloading (HLU) via tail suspension for 21 days. Mice (n = 11–17/group) were assigned to control (CON, normal weight bearing) or HLU and injected with either SclAbII (subcutaneously, 25 mg/kg) or vehicle (VEH) twice weekly. SclAbII completely inhibited the bone deterioration due to disuse, and induced bone formation such that bone properties in HLU-SclAbII were at or above values of CON-VEH mice. For example, hindlimb bone mineral density (BMD) decreased –9.2%±1.0% in HLU-VEH, whereas it increased 4.2%±0.7%, 13.1%±1.0%, and 30.6%±3.0% in CON-VEH, HLU-SclAbII, and CON-SclAbII, respectively (p < 0.0001). Trabecular bone volume, assessed by micro–computed tomography (μCT) imaging of the distal femur, was lower in HLU-VEH versus CON-VEH (p < 0.05), and was 2- to 3-fold higher in SclAbII groups versus VEH (p < 0.001). Midshaft femoral strength, assessed by three-point bending, and distal femoral strength, assessed by micro–finite element analysis (μFEA), were significantly higher in SclAbII versus VEH-groups in both loading conditions. Serum sclerostin was higher in HLU-VEH (134±5 pg/mL) compared to CON-VEH (116±6 pg/mL, p < 0.05). Serum osteocalcin was decreased by hindlimb suspension and increased by SclAbII treatment. Interestingly, the anabolic effects of sclerostin inhibition on some bone outcomes appeared to be enhanced by normal mechanical loading. Altogether, these results confirm the ability of SclAbII to abrogate disuse-induced bone loss and demonstrate that sclerostin antibody treatment increases bone mass by increasing bone formation in both normally loaded and underloaded environments.
PMCID: PMC4076162  PMID: 23109229
2.  Differences in Skeletal Microarchitecture and Strength in African-American and Caucasian Women 
African-American women have a lower risk of fracture than Caucasian women, and this difference is only partially explained by differences in DXA areal bone mineral density (aBMD). Little is known about racial differences in skeletal microarchitecture and the consequences for bone strength. To evaluate potential factors underlying this racial difference in fracture rates, we used high-resolution peripheral quantitative computed tomography (HR-pQCT) to assess cortical and trabecular bone microarchitecture and estimate bone strength using micro-finite element analysis in African-American (n=100) and Caucasian (n=173) women participating in the Study of Women's Health Across the Nation (SWAN). African-American women had larger and denser bones than Caucasians, with greater total area, aBMD, and total volumetric BMD (vBMD) at the radius and tibia metaphysis (p<0.05 for all). African-Americans had greater trabecular vBMD at the radius, but higher cortical vBMD at the tibia. Cortical microarchitecture tended to show the most pronounced racial differences, with higher cortical area, thickness, and volumes in African-Americans at both skeletal sites (p<0.05 for all), and lower cortical porosity in African-Americans at the tibia (p<0.05). African-American women also had greater estimated bone stiffness and failure load at both the radius and tibia. Differences in skeletal microarchitecture and estimated stiffness and failure load persisted even after adjustment for DXA aBMD. The densitometric and microarchitectural predictors of failure load at the radius and tibia were the same in African-American and Caucasian women. In conclusion, differences in bone microarchitecture and density contribute to greater estimated bone strength in African-Americans and probably explain, at least in part, the lower fracture risk of African-American women.
PMCID: PMC3779478  PMID: 23572415
HR-pQCT; bone microarchitecture; microfinite element analysis; African-American; Caucasian
3.  Altered thermogenesis and impaired bone remodeling in Misty mice 
Fat mass may be modulated by the number of brown-like adipocytes in white adipose tissue (WAT) in humans and rodents. Bone remodeling is dependent on systemic energy metabolism and, with age, bone remodeling becomes uncoupled and brown adipose tissue (BAT) function declines. To test the interaction between BAT and bone, we employed Misty (m/m) mice, which were reported be deficient in BAT. We found that Misty mice have accelerated age-related trabecular bone loss and impaired brown fat function (including reduced temperature, lower expression of Pgc1a and less sympathetic innervation compared to wildtype (+/+)). Despite reduced BAT function, Misty mice had normal core body temperature, suggesting heat is produced from other sources. Indeed, upon acute cold exposure (4°C for 6 hr), inguinal WAT from Misty mice compensated for BAT dysfunction by increasing expression of Acadl, Pgc1a, Dio2 and other thermogenic genes. Interestingly, acute cold exposure also decreased Runx2 and increased Rankl expression in Misty bone, but only Runx2 was decreased in wildtype. Browning of WAT is under the control of the sympathetic nervous system (SNS) and, if present at room temperature, could impact bone metabolism. To test whether SNS activity could be responsible for accelerated trabecular bone loss, we treated wildtype and Misty mice with the β-blocker, propranolol. As predicted, propranolol slowed trabecular BV/TV loss in the distal femur of Misty mice without affecting wildtype. Finally, the Misty mutation (a truncation of DOCK7) also has a significant cell-autonomous role. We found DOCK7 expression in whole bone and osteoblasts. Primary osteoblast differentiation from Misty calvaria was impaired, demonstrating a novel role for DOCK7 in bone remodeling. Despite the multifaceted effects of the Misty mutation, we have shown that impaired brown fat function leads to altered SNS activity and bone loss, and for the first time that cold exposure negatively affects bone remodeling.
PMCID: PMC3743939  PMID: 23553822
bone; brown adipose tissue; DOCK7; Misty; thermogenesis
4.  Vertebral Size, Bone Density, and Strength in Men and Women Matched for Age and Areal Spine BMD 
To explore the possible mechanisms underlying sex-specific differences in skeletal fragility that may be obscured by two-dimensional areal bone mineral density (aBMD) measures, we compared quantitative computed tomography (QCT)-based vertebral bone measures among pairs of men and women from the Framingham Heart Study Multidetector Computed Tomography Study who were matched for age and spine aBMD. Measurements included vertebral body cross-sectional area (CSA, cm2), trabecular volumetric BMD (Tb.vBMD, g/cm3), integral volumetric BMD (Int.vBMD, g/cm3), estimated vertebral compressive loading and strength (Newtons) at L3, the factor-of-risk (load-to-strength ratio), and vertebral fracture prevalence. We identified 981 male-female pairs (1:1 matching) matched on age (± 1 year) and QCT-derived aBMD of L3 (± 1%), with an average age of 51 years (range 34 to 81 years). Matched for aBMD and age, men had 20% larger vertebral CSA, lower Int.vBMD (−8%) and Tb.vBMD (−9%), 10% greater vertebral compressive strength, 24% greater vertebral compressive loading, and 12% greater factor-of-risk than women (p < 0.0001 for all), as well as higher prevalence of vertebral fracture. After adjusting for height and weight, the differences in CSA and volumetric bone mineral density (vBMD) between men and women were attenuated but remained significant, whereas compressive strength was no longer different. In conclusion, vertebral size, morphology, and density differ significantly between men and women matched for age and spine aBMD, suggesting that men and women attain the same aBMD by different mechanisms. These results provide novel information regarding sex-specific differences in mechanisms that underlie vertebral fragility.
PMCID: PMC4149904  PMID: 23955966
5.  The Effects of GATA-1 and NF-E2 Deficiency on Bone Biomechanical, Biochemical, and Mineral Properties 
Journal of cellular physiology  2013;228(7):1594-1600.
Mice deficient in GATA-1 or NF-E2, transcription factors required for normal megakaryocyte (MK) development, have increased numbers of MKs, reduced numbers of platelets, and a striking high bone mass phenotype. Here, we show the bone geometry, microarchitecture, biomechanical, biochemical, and mineral properties from these mutant mice. We found that the outer geometry of the mutant bones was similar to controls, but that both mutants had a striking increase in total bone area (up to a 35% increase) and trabecular bone area (up to a 19% increase). Interestingly, only the NF-E2 deficient mice had a significant increase in cortical bone area (21%) and cortical thickness (27%), which is consistent with the increase in bone mineral density (BMD) seen only in the NF-E2 deficient femurs. Both mutant femurs exhibited significant increases in several biomechanical properties including peak load (up to a 32% increase) and stiffness (up to a 13% increase). Importantly, the data also demonstrate differences between the two mutant mice. GATA-1 deficient femurs break in a ductile manner, whereas NF-E2 deficient femurs are brittle in nature. To better understand these differences, we examined the mineral properties of these bones. Although none of the parameters measured were different between the NF-E2 deficient and control mice, an increase in calcium (21%) and an increase in the mineral/matrix ratio (32%) was observed in GATA-1 deficient mice. These findings appear to contradict biomechanical findings, suggesting the need for further research into the mechanisms by which GATA-1 and NF-E2 deficiency alter the material properties of bone.
PMCID: PMC4128339  PMID: 23359245
6.  Partial Reductions in Mechanical Loading Yield Proportional Changes in Bone Density, Bone Architecture, and Muscle Mass 
Although the musculoskeletal system is known to be sensitive to changes in its mechanical environment, the relationship between functional adaptation and below-normal mechanical stimuli is not well defined. We investigated bone and muscle adaptation to a range of reduced loading using the partial weight suspension (PWS) system, in which a two-point harness is used to offload a tunable amount of body weight while maintaining quadrupedal locomotion. Skeletally mature female C57Bl/6 mice were exposed to partial weight bearing at 20%, 40%, 70%, or 100% of body weight for 21 days. A hindlimb unloaded (HLU) group was included for comparison in addition to age-matched controls in normal housing. Gait kinematics was measured across the full range of weight bearing, and some minor alterations in gait from PWS were identified. With PWS, bone and muscle changes were generally proportional to the degree of unloading. Specifically, total body and hindlimb bone mineral density, calf muscle mass, trabecular bone volume of the distal femur, and cortical area of the femur midshaft were all linearly related to the degree of unloading. Even a load reduction to 70% of normal weight bearing was associated with significant bone deterioration and muscle atrophy. Weight bearing at 20% did not lead to better bone outcomes than HLU despite less muscle atrophy and presumably greater mechanical stimulus, requiring further investigation. These data confirm that the PWS model is highly effective in applying controllable, reduced, long-term loading that produces predictable, discrete adaptive changes in muscle and bone of the hindlimb.
PMCID: PMC4118556  PMID: 23165526
7.  Irisin Levels Are Lower in Young Amenorrheic Athletes Compared with Eumenorrheic Athletes and Non-Athletes and Are Associated with Bone Density and Strength Estimates 
PLoS ONE  2014;9(6):e100218.
Irisin and FGF21 are novel hormones implicated in the “browning” of white fat, thermogenesis, and energy homeostasis. However, there are no data regarding these hormones in amenorrheic athletes (AA) (a chronic energy deficit state) compared with eumenorrheic athletes (EA) and non-athletes. We hypothesized that irisin and FGF21 would be low in AA, an adaptive response to low energy stores. Furthermore, because (i) brown fat has positive effects on bone, and (ii) irisin and FGF21 may directly impact bone, we hypothesized that bone density, structure and strength would be positively associated with these hormones in athletes and non-athletes. To test our hypotheses, we studied 85 females, 14–21 years [38 AA, 24 EA and 23 non-athletes (NA)]. Fasting serum irisin and FGF21 were measured. Body composition and bone density were assessed using dual energy X-ray absorptiometry, bone microarchitecture using high resolution peripheral quantitative CT, strength estimates using finite element analysis, resting energy expenditure (REE) using indirect calorimetry and time spent exercising/week by history. Subjects did not differ for pubertal stage. Fat mass was lowest in AA. AA had lower irisin and FGF21 than EA and NA, even after controlling for fat and lean mass. Across subjects, irisin was positively associated with REE and bone density Z-scores, volumetric bone mineral density (total and trabecular), stiffness and failure load. FGF21 was negatively associated with hours/week of exercise and cortical porosity, and positively with fat mass and cortical volumetric bone density. Associations of irisin (but not FGF21) with bone parameters persisted after controlling for potential confounders. In conclusion, irisin and FGF21 are low in AA, and irisin (but not FGF21) is independently associated with bone density and strength in athletes.
PMCID: PMC4057451  PMID: 24926783
High-resolution peripheral quantitative computed tomography (HR-pQCT) has recently been introduced as a clinical research tool for in vivo assessment of bone quality. The utility of this technique to address important skeletal health questions requires translation to standardized multi-center data pools. Our goal was to evaluate the feasibility of pooling data in multi-center HR-pQCT imaging trials.
Reproducibility imaging experiments were performed using structure and composition-realistic phantoms constructed from cadaveric radii. Single-center precision was determined by repeat scanning over short (<72hrs), intermediate (3–5mo), and long-term intervals (28mo). Multi-center precision was determined by imaging the phantoms at nine different HR-pQCT centers. Least significant change (LSC) and root mean squared coefficient of variation (RMSCV) for each interval and across centers was calculated for bone density, geometry, microstructure, and biomechanical parameters.
Single-center short-term RMSCVs were <1% for all parameters except Ct.Th (1.1%), Ct.Th.SD (2.6%), Tb.Sp.SD (1.8%), and porosity measures (6–8%). Intermediate-term RMSCVs were generally not statistically different from short-term values. Long-term variability was significantly greater for all density measures (0.7–2.0%; p < 0.05 vs. short-term) and several structure measures: Ct.Th (3.4%; p < 0.01 vs. short-term), Ct.Po (15.4%; p < 0.01 vs. short-term), and Tb.Th (2.2%; p < 0.01 vs. short-term). Multi-center RMSCVs were also significantly higher than short-term values: 2–4% for density and µFE measures (p < 0.0001), 2.6–5.3% for morphometric measures (p < 0.001), while Ct.Po was 16.2% (p < 0.001).
In the absence of subject motion, multi-center precision errors for HR-pQCT parameters were generally less than 5%. Phantom-based multi-center precision was comparable to previously reported in vivo single-center precision errors, although this was approximately 2–5 times worse than ex vivo short-term precision. The data generated from this study will contribute to the future design and validation of standardized procedures that are broadly translatable to multi-center study designs.
PMCID: PMC3577969  PMID: 23074145
HR-pQCT; osteoporosis; precision; bone; microstructure; bone strength; multi-center studies
10.  Variations of CT-Based Trunk Muscle Attenuation by Age, Sex, and Specific Muscle 
Fat accumulation in muscle may contribute to age-related declines in muscle function and is indicated by reduced attenuation of x-rays by muscle tissue in computed tomography scans. Reduced trunk muscle attenuation is associated with poor physical function, low back pain, and increased hyperkyphosis in older adults. However, variations in trunk muscle attenuation with age, sex and between specific muscles have not been investigated.
A cross-sectional examination of trunk muscle attenuation in computed tomography scans was performed in 60 younger (35–50 years) and 60 older (75–87 years) adults randomly selected from participants in the Framingham Heart Study Offspring and Third Generation Multidetector Computed Tomography Study. Computed tomography attenuation of 11 trunk muscles was measured at vertebral levels T8 and L3, and the effects of age, sex, and specific muscle on computed tomography attenuation of trunk muscles were determined.
Muscle attenuation varied by specific muscle (p < .001), was lower in older adults (p < .001), and was generally lower in women than in men (p < .001), although not in all muscles. Age-related differences in muscle attenuation varied with specific muscle (p < .001), with the largest age differences occurring in the paraspinal and abdominal muscles.
Trunk muscle attenuation is lower in older adults than in younger adults in both women and men, but such age-related differences vary widely between muscle groups. The reasons that some muscles exhibit larger age-related differences in fat content than others should be further explored to better understand age-related changes in functional capacity and postural stability.
PMCID: PMC3605905  PMID: 22904095
11.  Mechanical Contributions of the Cortical and Trabecular Compartments Contribute to Differences in Age-Related Changes in Vertebral Body Strength in Men and Women Assessed by QCT-Based Finite Element Analysis 
The biomechanical mechanisms underlying sex-specific differences in age-related vertebral fracture rates are ill defined. To gain insight into this issue, we used finite element analysis of clinical computed tomography (CT) scans of the vertebral bodies of L3 and T10 of young and old men and women to assess age- and sex-related differences in the strength of the whole vertebra, the trabecular compartment, and the peripheral compartment (the outer 2 mm of vertebral bone, including the thin cortical shell). We sought to determine whether structural and geometric changes with age differ in men and women, making women more susceptible to vertebral fractures. As expected, we found that vertebral strength decreased with age 2-fold more in women than in men. The strength of the trabecular compartment declined significantly with age for both sexes, whereas the strength of the peripheral compartment decreased with age in women but was largely maintained in men. The proportion of mechanical strength attributable to the peripheral compartment increased with age in both sexes and at both vertebral levels. Taken together, these results indicate that men and women lose vertebral bone differently with age, particularly in the peripheral (cortical) compartment. This differential bone loss explains, in part, a greater decline in bone strength in women and may contribute to the higher incidence of vertebral fractures among women than men. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179306  PMID: 21542000
12.  The effect of thoracic kyphosis and sagittal plane alignment on vertebral compressive loading 
To better understand the biomechanical mechanisms underlying the association between hyperkyphosis of the thoracic spine and risk of vertebral fracture and other degenerative spinal pathology, we used a previously validated musculoskeletal model of the spine to determine how thoracic kyphosis angle and spinal posture affect vertebral compressive loading. We simulated an age-related increase in thoracic kyphosis (T1-T12 Cobb angle 50° to 75°) during two different activities (relaxed standing and standing with 5 kg weights in the hands) and three different posture conditions: 1) an increase in thoracic kyphosis with no postural adjustment (uncompensated posture), 2) an increase in thoracic kyphosis with a concomitant increase in pelvic tilt that maintains a stable center of mass and horizontal eye gaze (compensated posture), and 3) an increase in thoracic kyphosis with a concomitant increase in lumbar lordosis that also maintains a stable center of mass and horizontal eye gaze (congruent posture). For all posture conditions, compressive loading increased with increasing thoracic kyphosis, with loading increasing more in the thoracolumbar and lumbar regions than in the mid-thoracic region. Loading increased the most for the uncompensated posture, followed by the compensated posture, with the congruent posture almost completely mitigating any increases in loading with increased thoracic kyphosis. These findings indicate that thoracic kyphosis and spinal posture both influence vertebral loading during daily activities, implying that thoracic kyphosis measurements alone are not sufficient to characterize the impact of spinal curvature on vertebral loading.
PMCID: PMC3431452  PMID: 22589006
Kyphosis; Spinal Loading; Posture; Biomechanical Model; Vertebral Fracture
13.  A Biomechanical Model for Estimating Loads on Thoracic and Lumbar Vertebrae 
Biomechanical models are commonly used to estimate loads on the spine. Current models have focused on understanding the etiology of low back pain and have not included thoracic vertebral levels. Using experimental data on the stiffness of the thoracic spine, ribcage, and sternum, we developed a new quasi-static stiffness-based biomechanical model to calculate loads on the thoracic and lumbar spine during bending or lifting tasks.
To assess the sensitivity of the model to our key assumptions, we determined the effect of varying ribcage and sternal stiffness, maximum muscle stress, and objective function on predicted spinal loads. We compared estimates of spinal loading obtained with our model to previously reported in vivo intradiscal pressures and muscle activation patterns.
Inclusion of the ribs and sternum caused an average decrease in vertebral compressive force of 33% for forward flexion and 18% in a lateral moment task. The impact of maximum muscle stress on vertebral force was limited to a narrow range of values. Compressive forces predicted by our model were strongly correlated to in vivo intradiscal pressure measurements in the thoracic (r=0.95) and lumbar (r=1) spine. Predicted trunk muscle activity was also strongly correlated (r=0.95) with previously published EMG data from the lumbar spine.
The consistency and accuracy of the model predictions appear to be sufficient to justify the use of this model for investigating the relationships between applied loads and injury to the thoracic spine during quasi-static loading activities.
PMCID: PMC2949493  PMID: 20655634
Spine; biomechanical model; back injury; muscle activation
14.  Nocturnal oxytocin secretion is lower in amenorrheic athletes than nonathletes and associated with bone microarchitecture and finite element analysis parameters 
Preclinical data indicate that oxytocin, a hormone produced in the hypothalamus and secreted into the peripheral circulation, is anabolic to bone. Oxytocin knockout mice have severe osteoporosis, and administration of oxytocin improves bone microarchitecture in these mice. Data suggest that exercise may modify oxytocin secretion, but this has not been studied in athletes in relation to bone. We therefore investigated oxytocin secretion and its association with bone microarchitecture and strength in young female athletes.
Cross-sectional study of 45 females, 14–21 years (15 amenorrheic athletes (AA), 15 eumenorrheic athletes (EA), and 15 nonathletes (NA)), of comparable bone age and BMI.
We used high-resolution peripheral quantitative CT to assess bone microarchitecture and finite element analysis to estimate bone strength at the weight-bearing distal tibia and non-weight-bearing ultradistal radius. Serum samples were obtained every 60 min, 2300–0700 h, and pooled for an integrated measure of nocturnal oxytocin secretion. Midnight and 0700 h samples were used to assess diurnal variation of oxytocin.
Nocturnal oxytocin levels were lower in AA and EA than in NA. After controlling for estradiol, the difference in nocturnal oxytocin between AA and NA remained significant. Midnight and 0700 h oxytocin levels did not differ between groups. At the tibia and radius, AA had impaired microarchitecture compared with NA. In AA, nocturnal oxytocin correlated strongly with trabecular and cortical microarchitecture, particularly at the non-weight-bearing radius. In regression models that include known predictors of microarchitecture in AA, oxytocin accounted for a substantial portion of the variability in microarchitectural and strength parameters.
Nocturnal oxytocin secretion is low in AA compared with NA and associated with site-dependent microarchitectural parameters. Oxytocin maycontribute to hypoestrogenemic bone loss inAA.
PMCID: PMC3679669  PMID: 23258269
15.  Heritability of Prevalent Vertebral Fracture and Volumetric Bone Mineral Density and Geometry at the Lumbar Spine in Three Generations of the Framingham Study 
Genetic factors likely contribute to the risk for vertebral fractures; however, there are few studies on the genetic contributions to vertebral fracture (VFrx), vertebral volumetric bone mineral density (vBMD) and geometry. Also the heritability (h2) for VFrx and its genetic correlation with phenotypes contributing to VFrx risk have not been established. This study aims to estimate the h2 of vertebral fracture, vBMD and cross-sectional-area (CSA) derived from quantitative computed tomography (QCT) scans, and to estimate the extent to which they share common genetic association in adults of European ancestry from three generations of Framingham Heart Study (FHS) families. Members of the FHS families were assessed for VFrx by lateral radiographs or QCT lateral scout views at 13 vertebral levels (T4-L4) using Genant’s semi-quantitative (SQ) scale (grades 0–3). Vertebral fracture was defined as having at least 25% reduction in height of any vertebra. We also analyzed QCT scans at the L3 level for integral (In.BMD) and trabecular (Tb.BMD) vBMD and cross-sectional area (CSA). Heritability estimates were calculated, and bivariate genetic correlation analysis was performed, adjusting for various covariates. For VFrx, we analyzed 4,099 individuals (148 VFrx cases) including 2,082 women and 2,017 men from 3 generations. Estimates of crude and multivariable-adjusted h2 were 0.43 to 0.69 (P< 1.1×10−2). 3,333 individuals including 1,737 men and 1,596 women from 2 generations had VFrx status and QCT-derived vBMD and CSA information. Estimates of crude and multivariable-adjusted h2 for vBMD and CSA ranged from 0.27 to 0.51. In a bivariate analysis, there was a moderate genetic correlation between VFrx and multivariable-adjusted In.BMD (−0.22) and Tb.BMD (−0.29). Our study suggests vertebral fracture, vertebral vBMD and CSA in adults of European ancestry are heritable, underscoring the importance of further work to identify the specific variants underlying genetic susceptibility to vertebral fracture, bone density and geometry.
PMCID: PMC3375687  PMID: 22222934
vertebral fracture; bone mineral density; heritability; QCT
16.  Microarchitecture Influences Microdamage Accumulation in Human Vertebral Trabecular Bone 
Journal of Bone and Mineral Research  2008;23(10):1613-1618.
It has been suggested that accumulation of microdamage with age contributes to skeletal fragility. However, data on the age-related increase in microdamage and the association between microdamage and trabecular microarchitecture in human vertebral cancellous bone are limited. We quantified microdamage in cancellous bone from human lumbar (L2) vertebral bodies obtained from 23 donors 54–93 yr of age (8 men and 15 women). Damage was measured using histologic techniques of sequential labeling with chelating agents and was related to 3D microarchitecture, as assessed by high-resolution μCT. There were no significant differences between sexes, although women tended to have a higher microcrack density (Cr.Dn) than men. Cr.Dn increased exponentially with age (r = 0.65, p < 0.001) and was correlated with bone volume fraction (BV/TV; r = −0.55; p < 0.01), trabecular number (Tb.N; r = −0.56 p = 0.008), structure model index (SMI; r = 0.59; p = 0.005), and trabecular separation (Tb.Sp; r = 0.59; p < 0.009). All architecture parameters were strongly correlated with each other and with BV/TV. Stepwise regression showed that SMI was the best predictor of microdamage, explaining 35% of the variance in Cr.Dn and 20% of the variance in diffuse damage accumulation. In addition, microcrack length was significantly greater in the highest versus lowest tertiles of SMI. In conclusion, in human vertebral cancellous bone, microdamage increases with age and is associated with low BV/TV and a rod-like trabecular architecture.
PMCID: PMC3276353  PMID: 18518771
microdamage; microcrack; human; vertebral; trabecular bone; microarchitecture; osteoporosis
17.  The Factor-of-Risk Biomechanical Approach Predicts Hip Fracture In Men and Women: The Framingham Study 
Osteoporosis International  2011;23(2):513-520.
Alternative methods of predicting hip fracture are needed since 50% of adults who fracture do not have osteoporosis by BMD measurements. One method, factor-of-risk (φ), computes the ratio of force on the hip in a fall, to femoral strength. We examined the relation between φ and hip fracture in 1,100 subjects from the Framingham Study with measured hip BMD, along with weight, height and age, collected in 1988-89.
We estimated both peak and attenuated force applied to the hip in a sideways fall from standing height, where attenuated force incorporated cushioning effects of trochanteric soft tissue. Femoral strength was estimated from femoral neck BMD, using cadaveric femoral strength data. Sex-specific, age-adjusted survival models were used to calculate hazard ratios (HR) and 95% confidence intervals for the relation between φpeak,φattenuated and their components, with hip fracture.
In 425 men and 675 women (mean age 76 yrs), 136 hip fractures occurred over median follow-up of 11.3 yrs. φ was associated with increased age-adjusted risk for hip fracture. One standard deviation increase in φpeak and φattenuated was associated with HR of 1.88 and 1.78 in men and 1.23 and 1.41 in women, respectively. Examining components of φ, in women, we found fall force and soft tissue thickness were predictive of hip fracture independent of femoral strength, (was estimated from BMD).
Thus, both φpeak and φattenuated predict hip fracture in men and women. These findings suggest additional studies of φ predicting hip fracture using direct measurements of trochanteric soft tissue.
PMCID: PMC3289518  PMID: 21344243
Hip fracture; Factor-of-Risk; bone strength; cohort study; fracture prediction; elderly
18.  Regressions for estimating muscle parameters in the thoracic and lumbar trunk for use in musculoskeletal modeling 
Journal of biomechanics  2011;45(1):66-75.
Musculoskeletal modeling requires information on muscle parameters such as cross-sectional area (CSA) and moment arms. A variety of previous studies have reported muscle parameters in the trunk based on in vivo imaging, but there remain gaps in the available data as well as limitations in the generalizability of such data. Specifically, available trunk muscle CSA data is very limited for older adults, lacking entirely in the thoracic region. In addition, previous studies have made measurements in groups of healthy volunteers or hospital patients who may not be representative of the population in general. Finally, such studies have not reported data for the major muscles connecting the upper limb to the thoracic trunk. In this study, muscle morphology measurements were made for major muscles present in the trunk between vertebral levels T6 and L5 using quantitative computed tomography scans from a community-based sample of 100 men and women aged 36–87. We present regression equations to predict trunk muscle CSA and position relative to the vertebral body in the transverse plane from sex, age, height and weight at vertebral levels T6 to L5. Regressions were also developed for predicting anatomical CSA and muscle moment arms, which were estimated using literature data on muscle line of action. This work thus provides a resource for estimating muscle parameters in the general population for musculoskeletal modeling of the thoraco-lumbar trunk.
PMCID: PMC3242869  PMID: 22018580
Trunk Musculature; Cross-sectional Area; Moment Arm Length; Biomechanical Modeling
19.  Parental Diabetes: The Akita Mouse as a Model of the Effects of Maternal and Paternal Hyperglycemia in Wildtype Offspring 
PLoS ONE  2012;7(11):e50210.
Maternal diabetes and high-fat feeding during pregnancy have been linked to later life outcomes in offspring. To investigate the effects of both maternal and paternal hyperglycemia on offspring phenotypes, we utilized an autosomal dominant mouse model of diabetes (hypoinsulinemic hyperglycemia in Akita mice). We determined metabolic and skeletal phenotypes in wildtype offspring of Akita mothers and fathers.
Both maternal and paternal diabetes resulted in phenotypic changes in wildtype offspring. Phenotypic changes were more pronounced in male offspring than in female offspring. Maternal hyperglycemia resulted in metabolic and skeletal phenotypes in male wildtype offspring. Decreased bodyweight and impaired glucose tolerance were observed as were reduced whole body bone mineral density and reduced trabecular bone mass.
Phenotypic changes in offspring of diabetic fathers differed in effect size from changes in offspring of diabetic mothers. Male wildtype offspring developed a milder metabolic phenotype, but a more severe skeletal phenotype. Female wildtype offspring of diabetic fathers were least affected.
Both maternal and paternal diabetes led to the development of metabolic and skeletal changes in wildtype offspring, with a greater effect of maternal diabetes on metabolic parameters and of paternal diabetes on skeletal development. The observed changes are unlikely to derive from Mendelian inheritance, since the investigated offspring did not inherit the Akita mutation. While fetal programming may explain the phenotypic changes in offspring exposed to maternal diabetes in-utero, the mechanism underlying the effect of paternal diabetes on wildtype offspring is unclear.
PMCID: PMC3509145  PMID: 23209676
20.  An essential role for the circadian-regulated gene Nocturnin in osteogenesis: the importance of local timekeeping in skeletal homeostasis 
The role of circadian proteins in regulating whole body metabolism and bone turnover has been studied in detail and has led to the discovery of an elemental system for timekeeping involving the core genes Clock, Bmal1, Per, and Cry. Nocturnin, a peripheral circadian-regulated gene has been shown to play a very important role in regulating adipogenesis by deadenylation of key mRNAs and intra-cytoplasmic transport of PPARγ. The role that it plays in osteogenesis has previously not been studied in detail. In this report we examined in vitro and in vivo osteogenesis in the presence and absence of Nocturnin and show that loss of Nocturnin enhances bone formation and can rescue Rosiglitazone induced bone loss in mice. The circadian rhythm of Nocturnin is likely to be an essential element of marrow stromal cell fate.
PMCID: PMC3285261  PMID: 22082366
Nocturnin; rosiglitazone; PPARγ
21.  β-Arrestin2 Regulates the Differential Response of Cortical and Trabecular Bone to Intermittent PTH in Female Mice 
Cytoplasmic arrestins regulate PTH signaling in vitro. We show that female β-arrestin2-/- mice have decreased bone mass and altered bone architecture. The effects of intermittent PTH administration on bone microarchitecture differed in β-arrestin2-/- and wildtype mice. These data indicate that arrestin-mediated regulation of intracellular signaling contributes to the differential effects of PTH at endosteal and periosteal bone surfaces.
Introduction: The effects of PTH differ at endosteal and periosteal surfaces, suggesting that PTH activity in these compartments may depend on some yet unidentified mechanism(s) of regulation. The action of PTH in bone is mediated primarily by intracellular cAMP, and the cytoplasmic molecule β-arrestin2 plays a central role in this signaling regulation. Thus, we hypothesized that arrestins would modulate the effects of PTH on bone in vivo.
Materials and Methods: We used pDXA, μCT, histomorphometry, and serum markers of bone turnover to assess the skeletal response to intermittent PTH (0, 20, 40, or 80 μg/kg/day) in adult female mice null for β-arrestin2 (β-arr2-/-) and wildtype (WT) littermates (7-11/group).
Results and Conclusions: β-arr2-/- mice had significantly lower total body BMD, trabecular bone volume fraction (BV/TV), and femoral cross-sectional area compared with WT. In WT females, PTH increased total body BMD, trabecular bone parameters, and cortical thickness, with a trend toward decreased midfemoral medullary area. In β-arr2-/- mice, PTH not only improved total body BMD, trabecular bone architecture, and cortical thickness, but also dose-dependently increased femoral cross-sectional area and medullary area. Histomorphometry showed that PTH-stimulated periosteal bone formation was 2-fold higher in β-arr2-/- compared with WT. Osteocalcin levels were significantly lower in β-arr2-/- mice, but increased dose-dependently with PTH in both β-arr2-/- and WT. In contrast, whereas the resorption marker TRACP5B increased dose-dependently in WT, 20-80 μg/kg/day of PTH was equipotent with regard to stimulation of TRACP5B in β-arr2-/-. In summary, β-arrestin2 plays an important role in bone mass acquisition and remodeling. In estrogen-replete female mice, the ability of intermittent PTH to stimulate periosteal bone apposition and endosteal resorption is inhibited by arrestins. We therefore infer that arrestin-mediated regulation of intracellular signaling contributes to the differential effects of PTH on cancellous and cortical bone.
PMCID: PMC1586119  PMID: 15765183
β-arrestin; PTH; knockout; bone architecture; bone remodeling
22.  Regulation of Lean Mass, Bone Mass, and Exercise Tolerance by the Central Melanocortin System 
PLoS ONE  2012;7(7):e42183.
Signaling via the type 4-melanocortin receptor (MC4R) is an important determinant of body weight in mice and humans, where loss of function mutations lead to significant obesity. Humans with mutations in the MC4R experience an increase in lean mass. However, the simultaneous accrual of fat mass in such individuals may contribute to this effect via mechanical loading. We therefore examined the relationship of fat mass and lean mass in mice lacking the type-4 melanocortin receptor (MC4RKO). We demonstrate that MC4RKO mice display increased lean body mass. Further, this is not dependent on changes in adipose mass, as MC4RKO mice possess more lean body mass than diet-induced obese (DIO) wild type mice with equivalent fat mass. To examine potential sources of the increased lean mass in MC4RKO mice, bone mass and strength were examined in MC4RKO mice. Both parameters increase with age in MC4RKO mice, which likely contributes to increases in lean body mass. We functionally characterized the increased lean mass in MC4RKO mice by examining their capacity for treadmill running. MC4R deficiency results in a decrease in exercise performance. No changes in the ratio of oxidative to glycolytic fibers were seen, however MC4RKO mice demonstrate a significantly reduced heart rate, which may underlie their impaired exercise performance. The reduced exercise capacity we report in the MC4RKO mouse has potential clinical ramifications, as efforts to control body weight in humans with melanocortin deficiency may be ineffective due to poor tolerance for physical activity.
PMCID: PMC3407101  PMID: 22848742
23.  Determinants of the Mechanical Behavior of Human Lumbar Vertebrae After Simulated Mild Fracture 
The ability of a vertebra to carry load after an initial deformation and the determinants of this postfracture load-bearing capacity are critical but poorly understood. This study aimed to determine the mechanical behavior of vertebrae after simulated mild fracture and to identify the determinants of this postfracture behavior. Twenty-one human L3 vertebrae were analyzed for bone mineral density (BMD) by dual-energy X-ray absorptiometry (DXA) and for microarchitecture by micro–computed tomography (µCT). Mechanical testing was performed in two phases: initial compression of vertebra to 25% deformity, followed, after 30 minutes of relaxation, by a similar test to failure to determine postfracture behavior. We assessed (1) initial and postfracture mechanical parameters, (2) changes in mechanical parameters, (3) postfracture elastic behavior by recovery of vertebral height after relaxation, and (4) postfracture plastic behavior by residual strength and stiffness. Postfracture failure load and stiffness were 11% ± 19% and 53% ± 18% lower than initial values (p = .021 and p < .0001, respectively), with 29% to 69% of the variation in the postfracture mechanical behavior explained by the initial values. Both initial and postfracture mechanical behaviors were significantly correlated with bone mass and microarchitecture. Vertebral deformation recovery averaged 31% ± 7% and was associated with trabecular and cortical thickness (r = 0.47 and r = 0.64; p = .03 and p = .002, respectively). Residual strength and stiffness were independent of bone mass and initial mechanical behavior but were related to trabecular and cortical microarchitecture (|r| = 0.50 to 0.58; p = .02 to .006). In summary, we found marked variation in the postfracture load-bearing capacity following simulated mild vertebral fractures. Bone microarchitecture, but not bone mass, was associated with postfracture mechanical behavior of vertebrae. © 2011 American Society for Bone and Mineral Research.
PMCID: PMC3179325  PMID: 20928886
24.  Role of Trabecular Microarchitecture and Its Heterogeneity Parameters in the Mechanical Behavior of Ex Vivo Human L3 Vertebrae 
Journal of Bone and Mineral Research  2010;25(11):2324-2331.
Low bone mineral density (BMD) is a strong risk factor for vertebral fracture risk in osteoporosis. However, many fractures occur in people with moderately decreased or normal BMD. Our aim was to assess the contributions of trabecular microarchitecture and its heterogeneity to the mechanical behavior of human lumbar vertebrae. Twenty-one human L3 vertebrae were analyzed for BMD by dual-energy X-ray absorptiometry (DXA) and microarchitecture by high-resolution peripheral quantitative computed tomography (HR-pQCT) and then tested in axial compression. Microarchitecture heterogeneity was assessed using two vertically oriented virtual biopsies—one anterior (Ant) and one posterior (Post)—each divided into three zones (superior, middle, and inferior) and using the whole vertebral trabecular volume for the intraindividual distribution of trabecular separation (Tb.Sp*SD). Heterogeneity parameters were defined as (1) ratios of anterior to posterior microarchitectural parameters and (2) the coefficient of variation of microarchitectural parameters from the superior, middle, and inferior zones. BMD alone explained up to 44% of the variability in vertebral mechanical behavior, bone volume fraction (BV/TV) up to 53%, and trabecular architecture up to 66%. Importantly, bone mass (BMD or BV/TV) in combination with microarchitecture and its heterogeneity improved the prediction of vertebral mechanical behavior, together explaining up to 86% of the variability in vertebral failure load. In conclusion, our data indicate that regional variation of microarchitecture assessment expressed by heterogeneity parameters may enhance prediction of vertebral fracture risk. © 2010 American Society for Bone and Mineral Research.
PMCID: PMC3179283  PMID: 20564249
osteoporosis; vertebra; bone biomechanics; trabecular microarchitecture; heterogeneity
25.  A Proteasome Inhibitor, Bortezomib, Inhibits Breast Cancer Growth and Reduces Osteolysis by Downregulating Metastatic Genes 
The incidence of bone metastasis in advanced breast cancer exceeds 70%. Bortezomib (Bzb), a proteasome inhibitor used for the treatment of multiple myeloma, also promotes bone formation. We tested the hypothesis that proteasome inhibitors can ameliorate breast cancer osteolytic disease.
Experimental Design
To address the potentially beneficial effect of Bzb in reducing tumor growth in the skeleton and counteracting bone osteolysis, human MDA-MB-231 breast cancer (BrCa) cells were injected into the tibia of mice to model bone tumor growth for in vivo assessment of treatment regimens pre- and post-tumor growth.
Controls exhibited tumor growth destroying trabecular and cortical bone and invading muscle. Bzb treatment initiated following inoculation of tumor cells strikingly reduced tumor growth, restricted tumor cells mainly to the marrow cavity, and almost completely inhibited osteolysis in the bone microenvironment over a 3–4 week period demonstrated by 18F-FDG PET, micro-CT scanning, radiography, and histology. Thus, proteasome inhibition is effective in killing tumor cells within bone. Pre-treatment with Bzb for 3 weeks prior to inoculation of tumor cells was also effective in reducing osteolysis. Our in vitro and in vivo studies indicate mechanisms by which Bzb inhibits tumor growth and reduces osteolysis result from inhibited cell proliferation, necrosis and decreased expression of factors that promote BrCa tumor progression in bone.
These findings provide a basis for a novel strategy to treat patients with breast cancer osteolytic lesions, and represent an approach for protecting the entire skeleton from metastatic bone disease.
PMCID: PMC2955762  PMID: 20843837
MDA-MB-231; bone metastasis; prevention of osteolysis; bone anabolic drug; VEGF; MMP9 and Runx2 genes

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