Determination of rat vertebral bone compressive fatigue properties in untreated intact rats and zoledronic-acid-treated, ovariectomized rats

Summary: Compressive fatigue properties of whole vertebrae, which may be clinically relevant for osteoporotic vertebral fractures, were determined in untreated, intact rats and zoledronic-acid-treated, ovariectomized rats. Typical fatigue behavior was found and was similar to that seen in other species. Fatigue properties were comparable between both groups. Introduction: Osteoporosis is often treated with bisphosphonates, which reduce fracture risk. Effects of bisphosphonates on fatigue strength, which may be clinically relevant for vertebral fractures, are unknown. We determined vertebral, compressive fatigue properties in normal and zoledronic acid (ZOL)-treated, OVX rats. Methods: Thirty-five-week old Wistar rats were divided into SHAM-OVX (n = 7) and OVX with ZOL treatment (n = 5; single injection, 20 μg/kg b.w. s.c.). After 16 weeks, vertebral trabecular microarchitecture and cortical thickness were determined using micro-CT. Vertebrae were cyclically compressed in load-control at 2 Hz starting at 0.75% apparent strain. A line parallel to the apparent strain curve was drawn at 0.5% higher offset, after which the intersection was defined as the time to failure and the apparent strain at failure. Data were compared using Student’s t test.Results Morphology and fatigue properties were the same in both groups. Samples failed between 10 min and 15 h. Force–displacement curves displayed typical fatigue behavior. Displacement increased over time due to mostly creep and to decreasing secant stiffness. Conclusions: We established a technique to determine compressive fatigue properties in the rat vertebral body. Our initial results indicate that ZOL-treated OVX rats have similar vertebral fatigue properties as SHAM-OVX controls

Data Mining Activity for Bone Discipline: Calculating a Factor of Risk for Hip Fracture in Long-Duration Astronauts

The factor-of-risk (Phi), defined as the ratio of applied load to bone strength, is a biomechanical approach to hip fracture risk assessment that may be used to identify subjects who are at increased risk for fracture. The purpose of this project was to calculate the factor of risk in long duration astronauts after return from a mission on the International Space Station (ISS), which is typically 6 months in duration. The load applied to the hip was calculated for a sideways fall from standing height based on the individual height and weight of the astronauts. The soft tissue thickness overlying the greater trochanter was measured from the DXA whole body scans and used to estimate attenuation of the impact force provided by soft tissues overlying the hip. Femoral strength was estimated from femoral areal bone mineral density (aBMD) measurements by dual-energy x-ray absorptiometry (DXA), which were performed between 5-32 days of landing. All long-duration NASA astronauts from Expedition 1 to 18 were included in this study, where repeat flyers were treated as separate subjects. Male astronauts (n=20) had a significantly higher factor of risk for hip fracture Phi than females (n=5), with preflight values of 0.83+/-0.11 and 0.36+/-0.07, respectively, but there was no significant difference between preflight and postflight Phi (Figure 1). Femoral aBMD measurements were not found to be significantly different between men and women. Three men and no women exceeded the theoretical fracture threshold of Phi=1 immediately postflight, indicating that they would likely suffer a hip fracture if they were to experience a sideways fall with impact to the greater trochanter. These data suggest that male astronauts may be at greater risk for hip fracture than women following spaceflight, primarily due to relatively less soft tissue thickness and subsequently greater impact force

The associations between QCT-based vertebral bone measurements and prevalent vertebral fractures depend on the spinal locations of both bone measurement and fracture

Summary We examined how spinal location affects the relationships between quantitative computed tomography (QCT)-based bone measurements and prevalent vertebral fractures. Upper spine (T4–T10) fractures appear to be more strongly related to bone measures than lower spine (T11–L4) fractures, while lower spine measurements are at least as strongly related to fractures as upper spine measurements. Introduction Vertebral fracture (VF), a common injury in older adults, is most prevalent in the mid-thoracic (T7–T8) and thoracolumbar (T12–L1) areas of the spine. However, measurements of bone mineral density (BMD) are typically made in the lumbar spine. It is not clear how the associations between bone measurements and VFs are affected by the spinal locations of both bone measurements and VF. Methods A community-based case–control study includes 40 cases with moderate or severe prevalent VF and 80 age- and sex-matched controls. Measures of vertebral BMD, strength (estimated by finite element analysis), and factor of risk (load:strength ratio) were determined based on QCT scans at the L3 and T10 vertebrae. Associations were determined between bone measures and prevalent VF occurring at any location, in the upper spine (T4–T10), or in the lower spine (T11–L4). Results Prevalent VF at any location was significantly associated with bone measures, with odds ratios (ORs) generally higher for measurements made at L3 (ORs = 1.9–3.9) than at T10 (ORs = 1.5–2.4). Upper spine fracture was associated with these measures at both T10 and L3 (ORs = 1.9–8.2), while lower spine fracture was less strongly associated (ORs = 1.0–2.4) and only reached significance for volumetric BMD measures at L3. Conclusions Closer proximity between the locations of bone measures and prevalent VF does not strengthen associations between bone measures and fracture. Furthermore, VF etiology may vary by region, with VFs in the upper spine more strongly related to skeletal fragility.National Institutes of Health (U.S.) (Grants R01AR053986, R01AR/AG041398, T32AG023480, and F31AG041629)National Heart, Lung, and Blood Institute. Framingham Heart Study (NIH/NHLBI Contract N01-HC-25195

Retrospective Study of Serum Sclerostin Measurements in Bed Rest Subjects

Animal models and human studies suggest that osteocytes regulate the skeleton s response to mechanical unloading at the cellular level in part by an increase in sclerostin, an inhibitor of the anabolic Wnt pathway. However, few studies have reported changes in serum sclerostin in humans exposed to reduced mechanical loading. Thus, we determined changes in serum sclerostin and bone turnover markers in healthy adult men who participated in a controlled bed rest study. Seven healthy adult men (31 +/- 3 yrs old) underwent 90-day six-degree head down tilt bed rest at the University of Texas Medical Branch in Galveston's Institute for Translational Sciences - Clinical Research Center (ITS-CRC). Serum sclerostin, PTH, serum markers of bone turnover (bone specific alkaline phosphatase, RANKL/OPG, and osteocalcin), urinary calcium and phosphorus excretion, and 24 hour pooled urinary markers of bone resorption (NTX, DPD, PYD) were evaluated pre-bed rest (BL), bed rest day 28 (BR-28), bed rest day 60 (BR-60), and bed rest day 90 (BR-90). In addition, bone mineral density (BMD) was assessed by dual-energy X-ray absorptiometry (DXA) at BL, BR-60, and post bed rest day 5 (BR+5). Data are reported as mean +/- standard deviation. We used repeated measures ANOVA to compare baseline values to BR-28, BR-60, and BR-90. RESULTS Consistent with prior reports, BMD declined significantly (1-2% per month) at weight-bearing skeletal sites (spine, hip, femur neck, and calcaneus). Serum sclerostin levels were elevated above BL at BR-28 (+29% +/- 20%, p = 0.003), BR-60 (+42% +/- 31%, p < 0.001), and BR-90 (22% +/- 21%, p = 0.07). Serum PTH levels were reduced at BR-28 (-17% +/- 16%, p = 0.02), BR-60 (-24% +/- 14%, p = 0.03), and returned to baseline at BR-90 (-21% +/- 21%, p = 0.14). Serum bone turnover markers did not change, however urinary bone resorption markers and calcium were significantly elevated following bed rest (p < 0.01). CONCLUSION We observed an increase of serum sclerostin associated with decreased serum PTH and elevated bone resorption markers in otherwise healthy men subjected to long-term immobilization

Effect of follower load on motion and stiffness of the human thoracic spine with intact rib cage

Researchers have reported on the importance of the rib cage in maintaining mechanical stability in the thoracic spine and on the validity of a compressive follower preload. However, dynamic mechanical testing using both the rib cage and follower load has never been studied. An in vitro biomechanical study of human cadaveric thoracic specimens with rib cage intact in lateral bending, flexion/extension, and axial rotation under varying compressive follower preloads was performed. The objective was to characterize the motion and stiffness of the thoracic spine with intact rib cage and follower preload. The hypotheses tested for all modes of bending were (i) range of motion, elastic zone, and neutral zone will be reduced with a follower load, and (ii) neutral and elastic zone stiffness will be increased with a follower load. Eight human cadaveric thoracic spine specimen (T1–T12) with intact rib cage were subjected to 5 Nm pure moments in lateral bending, flexion/extension, and axial rotation under follower loads of 0–400 N. Range of motion, elastic and neutral zones, and elastic and neutral zone stiffness values were calculated for functional spinal units and segments within the entire thoracic section. Combined segmental range of motion decreased by an average of 34% with follower load for every mode. Application of a follower load with intact rib cage impacts the motion and stiffness of the human cadaveric thoracic spine. Researchers should consider including both aspects to better represent the physiologic implications of human motion and improve clinically relevant biomechanical thoracic spine testing

Effects of follower load and rib cage on intervertebral disc pressure and sagittal plane curvature in static tests of cadaveric thoracic spines

The clinical relevance of mechanical testing studies of cadaveric human thoracic spines could be enhanced by using follower preload techniques, by including the intact rib cage, and by measuring thoracic intervertebral disc pressures, but studies to date have not incorporated all of these components simultaneously. Thus, this study aimed to implement a follower preload in the thoracic spine with intact rib cage, and examine the effects of follower load, rib cage stiffening and rib cage removal on intervertebral disc pressures and sagittal plane curvatures in unconstrained static conditions. Intervertebral disc pressures increased linearly with follower load magnitude. The effect of the rib cage on disc pressures in static conditions remains unclear because testing order likely confounded the results. Disc pressures compared well with previous reports in vitro, and comparison with in vivo values suggests the use of a follower load of about 400 N to approximate loading in upright standing. Follower load had no effect on sagittal plane spine curvature overall, suggesting successful application of the technique, although increased flexion in the upper spine and reduced flexion in the lower spine suggest that the follower load path was not optimized. Rib cage stiffening and removal both increased overall spine flexion slightly, although with differing effects at specific spinal locations. Overall, the approaches demonstrated here will support the use of follower preloads, intact rib cage, and disc pressure measurements to enhance the clinical relevance of future studies of the thoracic spine

BMP type I receptor inhibition reduces heterotopic ossification

Fibrodysplasia ossificans progressiva (FOP) is a congenital disorder of progressive and widespread postnatal ossification of soft tissues1,2,3,4 and is without known effective treatments. Affected individuals harbor conserved mutations in the ACVR1 gene that are thought to cause constitutive activation of the bone morphogenetic protein (BMP) type I receptor, activin receptor-like kinase-2 (ALK2)5. Here we show that intramuscular expression in the mouse of an inducible transgene encoding constitutively active ALK2 (caALK2), resulting from a glutamine to aspartic acid change at amino acid position 207, leads to ectopic endochondral bone formation, joint fusion and functional impairment, thus phenocopying key aspects of human FOP. A selective inhibitor of BMP type I receptor kinases, LDN-193189 (ref. 6), inhibits activation of the BMP signaling effectors SMAD1, SMAD5 and SMAD8 in tissues expressing caALK2 induced by adenovirus specifying Cre (Ad.Cre). This treatment resulted in a reduction in ectopic ossification and functional impairment. In contrast to localized induction of caALK2 by Ad.Cre (which entails inflammation), global postnatal expression of caALK2 (induced without the use of Ad.Cre and thus without inflammation) does not lead to ectopic ossification. However, if in this context an inflammatory stimulus was provided with a control adenovirus, ectopic bone formation was induced. Like LDN-193189, corticosteroid inhibits ossification in Ad.Cre-injected mutant mice, suggesting caALK2 expression and an inflammatory milieu are both required for the development of ectopic ossification in this model. These results support the role of dysregulated ALK2 kinase activity in the pathogenesis of FOP and suggest that small molecule inhibition of BMP type I receptor activity may be useful in treating FOP and heterotopic ossification syndromes associated with excessive BMP signaling

Spaceflight and hind limb unloading induce similar changes in electrical impedance characteristics of mouse gastrocnemius muscle

Objective—To assess the potential of electrical impedance myography (EIM) to serve as a marker of muscle fiber atrophy and secondarily as an indicator of bone deterioration by assessing the effects of spaceflight or hind limb unloading. Methods—In the first experiment, 6 mice were flown aboard the space shuttle (STS-135) for 13 days and 8 earthbound mice served as controls. In the second experiment, 14 mice underwent hind limb unloading (HLU) for 13 days; 13 additional mice served as controls. EIM measurements were made on ex vivo gastrocnemius muscle. Quantitative microscopy and areal bone mineral density (aBMD) measurements of the hindlimb were also performed. Results—Reductions in the multifrequency phase-slope parameter were observed for both the space flight and HLU cohorts compared to their respective controls. For ground control and spaceflight groups, the values were 24.7±1.3°/MHz and 14.1±1.6°/MHz, respectively (p=0.0013); for control and HLU groups, the values were 23.9±1.6°/MHz and 19.0±1.0°/MHz, respectively (p=0.014). This parameter also correlated with muscle fiber size (ρ=0.65, p=0.011) for spaceflight and hind limb aBMD (ρ=0.65, p=0.0063) for both groups. Conclusions—These data support the concept that EIM may serve as a useful tool for assessment of muscle disuse secondary to immobilization or microgravity

A two-cohort study on the association between the gut microbiota and bone density, microarchitecture, and strength

The gut microbiome affects the inflammatory environment through effects on T-cells, which influence the production of immune mediators and inflammatory cytokines that stimulate osteoclastogenesis and bone loss in mice. However, there are few large human studies of the gut microbiome and skeletal health. We investigated the association between the human gut microbiome and high resolution peripheral quantitative computed tomography (HR-pQCT) scans of the radius and tibia in two large cohorts; Framingham Heart Study (FHS [n=1227, age range: 32 – 89]), and the Osteoporosis in Men Study (MrOS [n=836, age range: 78 – 98]). Stool samples from study participants underwent amplification and sequencing of the V4 hypervariable region of the 16S rRNA gene. The resulting 16S rRNA sequencing data were processed separately for each cohort, with the DADA2 pipeline incorporated in the16S bioBakery workflow. Resulting amplicon sequence variants were assigned taxonomies using the SILVA reference database. Controlling for multiple covariates, we tested for associations between microbial taxa abundances and HR-pQCT measures using general linear models as implemented in microbiome multivariable association with linear model (MaAslin2). Abundance of 37 microbial genera in FHS, and 4 genera in MrOS, were associated with various skeletal measures (false discovery rate [FDR] ≤ 0.1) including the association of DTU089 with bone measures, which was independently replicated in the two cohorts. A meta-analysis of the taxa-bone associations further revealed (FDR ≤ 0.25) that greater abundances of the genera; Akkermansia and DTU089, were associated with lower radius total vBMD, and tibia cortical vBMD respectively. Conversely, higher abundances of the genera; Lachnospiraceae NK4A136 group, and Faecalibacterium were associated with greater tibia cortical vBMD. We also investigated functional capabilities of microbial taxa by testing for associations between predicted (based on 16S rRNA amplicon sequence data) metabolic pathways abundance and bone phenotypes in each cohort. While there were no concordant functional associations observed in both cohorts, a meta-analysis revealed 8 pathways including the super-pathway of histidine, purine, and pyrimidine biosynthesis, associated with bone measures of the tibia cortical compartment. In conclusion, our findings suggest that there is a link between the gut microbiome and skeletal metabolism