Sclerostin antibody treatment causes greater alveolar crest height and bone mass in an ovariectomized rat model of localized periodontitis

IntroductionPeriodontitis and osteoporosis are bone destructive diseases with a high prevalence in the adult population. The concomitant presence of osteoporosis may be a risk factor of progression of periodontal destruction. We studied the effect of sclerostin-neutralizing monoclonal antibody (Scl-Ab) on alveolar bone endpoints in an ovariectomized (OVX) rat model of induced experimental periodontitis.MethodsSixty female, 4-month-old Sprague–Dawley rats underwent sham operation or bilateral OVX and were left untreated for 2 months. Experimental periodontitis (ligature) was established by placing silk sutures subgingival to the right maxillary first and second molar teeth for 4 weeks, and feeding the rats food and high-sugar drinking water during this period. Thereafter, ligatures were removed and 25 mg/kg vehicle or Scl-Ab was administered subcutaneously twice weekly for 6 weeks. Rats were randomized into four groups: (1) Control (Sham + Vehicle), (2) Sham + Ligature + Vehicle, (3) OVX + Ligature + Vehicle, and (4) OVX + Ligature + Scl-Ab. Terminal blood and right maxilla specimens were collected for analyses.ResultsGroup 3 rats showed lower bone volume fraction (BVF) of alveolar bone with higher bone resorption and lower bone formation than Group 2 rats. Group 4 rats had higher alveolar crest height, as assessed by linear distance of cementoenamel junction to the alveolar bone crest and greater alveolar bone mass using Micro CT, than Group 3 rats. Significantly higher values of mineral apposition rate (MAR) and mineralizing surface/bone surface (MS/BS) were also observed in Group 4 rats by analyzing polychrome sequential labeling data. Increased serum osteocalcin and osteoprotegerin, and deceased serum tartrate-resistant acid phosphatase and CTx-1 illustrate the ability of Scl-Ab to increase alveolar bone mass by enhancing bone formation and decreasing bone resorption in an animal model of estrogen deficiency osteopenia plus periodontitis.ConclusionScl-Ab could be a potential bone anabolic agent for improving alveolar crest height and higher alveolar bone mass in conditions where alveolar bone loss in periodontitis is compounded by estrogen deficiency osteopenia.     

Temporal changes in systemic and local expression of bone turnover markers during six months of sclerostin antibody administration to ovariectomized rats

Sclerostin (Scl) is an osteocyte protein that decreases bone formation, and its inhibition by neutralizing antibodies (Scl-Ab) increases bone formation, mass and strength. We investigated the effects of Scl-Ab in mature ovariectomized (OVX) rats with a mechanistic focus on longer-term responses of osteoclasts, osteoblasts and osteocytes. Four-month-old Sprague–Dawley rats had OVX or sham surgery. Two months later, sham controls received sc vehicle while OVX rats received vehicle (OVX-Veh) or Scl-Ab (25 mg/kg) once weekly for 6 or 26 weeks followed by necropsy (n = 12/group). Terminal blood was collected for biochemistry, non-adherent marrow cells were harvested from femurs for ex vivo osteoclast formation assays, and vertebrae and tibiae were collected for dynamic histomorphometry and mRNA analyses. Scl-Ab treatment led to progressively thicker but fewer trabeculae in the vertebra, leading to increased trabecular bone volume and reduced trabecular surfaces. Scl-Ab also increased cortical bone volume in the tibia, via early periosteal expansion and progressive endocortical contraction. Scl-Ab significantly reduced parameters of bone resorption at week 6 relative to OVX-Veh controls, including reduced serum TRACP-5b, reduced capacity of marrow cells to form osteoclasts ex vivo, and > 80% reductions in vertebral trabecular and tibial endocortical eroded surfaces. At week 26, serum TRACP-5b and ex vivo osteoclast formation were no longer reduced in the Scl-Ab group, but eroded surfaces remained > 80% lower than in OVX-Veh controls without evidence for altered skeletal mRNA expression of opg or rankl. Scl-Ab significantly increased parameters of bone formation at week 6 relative to OVX-Veh controls, including increases in serum P1NP and osteocalcin, and increased trabecular, endocortical and periosteal bone formation rates (BFRs). At week 26, surface-referent trabecular BFR remained significantly increased in the Scl-Ab group versus OVX-Veh controls, but after adjusting for a reduced extent of trabecular surfaces, overall (referent-independent) trabecular BFR was no longer significantly elevated. Similarly, serum P1NP and osteocalcin were no longer significantly increased in the Scl-Ab group at week 26. Tibial endocortical and periosteal BFR were increased at week 6 in the Scl-Ab group versus OVX-Veh controls, while at week 26 only endocortical BFR remained increased. The Scl-Ab group exhibited significant increments in skeletal mRNA expression of several osteocyte genes, with sost showing the greatest induction in both the tibia and vertebra. We propose that Scl-Ab administration, and/or the gains in bone volume that result, may have increased osteocytic expression of Scl as a possible means of regulating gains in bone mass.     

Bimodal distribution of osteocyte lacunar size in the human femoral cortex as revealed by micro-CT

Tomographic reconstructions of sections of human femoral bone were created from x-ray data sets taken using synchrotron radiation of 26.4 keV and with isotropic voxels 1.47 μm on a side. We demonstrate that it is possible to segment the data to isolate both the osteocyte lacunae and the Haversian canals in the bone as well as identifying osteon boundaries. From this information a wealth of data relating to bone structure becomes available. The data were used to map the spatial positions of the osteocyte lacunae, relative to the Haversian canals and of the osteon boundaries. The dimensions and volume of the imaged osteocyte lacunae were measured for close to 10,000 lacunae. When averaged over the 11 osteons measured, osteocyte densities varied from 4 × 10⁴ per mm³ close to the Haversian canals to about 9 × 10⁴ per mm³ at 80% of osteon radius. The nearest-neighbour distances varied from 10 μm to 40 μm with a peak at 23 μm and an approximately normal distribution. The distribution of lacunar long-axis length was also approximately normal with a small positive skew and the peak value was 8 μm with a range from 3 μm to 20 μm. The most significant finding from this study was that the distribution of the measured volumes of osteocyte lacunae had two distinct peaks, one at 200 μm³ and a second at 330 μm³.     

Knee loading protects against osteonecrosis of the femoral head by enhancing vessel remodeling and bone healing

Osteonecrosis of the femoral head is a serious orthopedic problem. Moderate loads with knee loading promote bone formation, but their effects on osteonecrosis have not been investigated. Using a rat model, we examined a hypothesis that knee loading enhances vessel remodeling and bone healing through the modulation of the fate of bone marrow-derived cells. In this study, osteonecrosis was induced by transecting the ligamentum teres followed by a tight ligature around the femoral neck. For knee loading, 5 N loads were laterally applied to the knee at 15 Hz for 5 min/day for 5 weeks. Changes in bone mineral density (BMD) and bone mineral content (BMC) of the femur were measured by pDEXA, and ink infusion was performed to evaluate vessel remodeling. Femoral heads were harvested for histomorphometry, and bone marrow-derived cells were isolated to examine osteoclast development and osteoblast differentiation. The results showed that osteonecrosis significantly induced bone loss, and knee loading stimulated both vessel remodeling and bone healing. The osteonecrosis group exhibited the lowest trabecular BV/TV (p < 0.001) in the femoral head, and lowest femoral BMD and BMC (both p < 0.01). However, knee loading increased trabecular BV/TV (p < 0.05) as well as BMD (p < 0.05) and BMC (p < 0.01). Osteonecrosis decreased the vessel volume (p < 0.001), vessel number (p < 0.001) and VEGF expression (p < 0.01), and knee loading increased them (p < 0.001, p < 0.001 and p < 0.01). Osteonecrosis activated osteoclast development, and knee loading reduced its formation, migration, adhesion and the level of “pit” formation (p < 0.001, p < 0.01, p < 0.001 and p < 0.001). Furthermore, knee loading significantly increased osteoblast differentiation and CFU-F (both p < 0.001). A significantly positive correlation was observed between vessel remodeling and bone healing (both p < 0.01). These results indicate that knee loading could be effective in repair osteonecrosis of the femoral head in a rat model. This effect might be attributed to promoting vessel remodeling, suppressing osteoclast development, and increasing osteoblast and fibroblast differentiation. In summary, the current study suggests that knee loading might potentially be employed as a non-invasive therapy for osteonecrosis of the femoral head.     

Differential temporal effects of sclerostin antibody and parathyroid hormone on cancellous and cortical bone and quantitative differences in effects on the osteoblast lineage in young intact rats

Sclerostin antibody (Scl-Ab) and parathyroid hormone (PTH) are bone-forming agents that have different modes of action on bone, although a study directly comparing their effects has not been conducted. The present study investigated the comparative quantitative effects of these two bone-forming agents over time on bone at the organ, tissue, and cellular level; specifically, at the level of the osteoblast (Ob) lineage in adolescent male and female rats. Briefly, eight-week old male and female Sprague–Dawley rats were administered either vehicle, Scl-Ab (3 or 50 mg/kg/week subcutaneously), or human PTH (1–34) (75 μg/kg/day subcutaneously) for 4 or 26 weeks. The 50 mg/kg Scl-Ab and the PTH dose were those used in the respective rat lifetime pharmacology studies. Using robust stereological methods, we compared the effects of these agents specifically at the level of the Ob lineage in vertebrae from female rats. Using RUNX2 or nestin immunostaining, location, and morphology, the total number of osteoprogenitor subpopulations, Ob, and lining cells were estimated using the fractionator or proportionator estimators. Density estimates were also calculated referent to total bone surface, total Ob surface, or total marrow volume.Scl-Ab generally effected greater increases in cancellous and cortical bone mass than PTH, correlating with higher bone formation rates (BFR) at 4 weeks in the spine and mid-femur without corresponding increases in bone resorption indices. The increases in vertebral BFR/BS at 4 weeks attenuated with continued treatment to a greater extent with Scl-Ab than with PTH. At 4 weeks, both Scl-Ab and PTH effected equivalent increases in total Ob number (Ob.N). Ob density on the formative surfaces (Ob.N/Ob.S) remained similar across groups while mineral apposition rate (MAR) was significantly higher with Scl-Ab at week 4, reflecting an increase in individual Ob vigor relative to vehicle and PTH. After 26 weeks, Scl-Ab maintained BFR/BS with fewer Ob and lower Ob.N/Ob.S by increasing the Ob footprint (bone surface area occupied by an Ob) and increasing MAR, compared with PTH. The lower Ob.N and Ob.N/Ob.S with Scl-Ab at 26 weeks were associated with decreased osteoprogenitor numbers compared with both vehicle and PTH, an effect not evident at week 4. Osteoprogenitor numbers were generally positively correlated with Ob.N across groups and timepoints, suggesting dynamic coordination between the progenitor and Ob populations. The time-dependent reductions in subpopulations of the Ob lineage with Scl-Ab may be integral to the greater attenuation or self-regulation of bone formation observed at the vertebra, as PTH required more Ob at the formative site with correlative increased numbers of progenitors compared with Scl-Ab indicating potentially greater stimulus for progenitor pool proliferation or differentiation.     

Rapidly growing Brtl/+ mouse model of osteogenesis imperfecta improves bone mass and strength with sclerostin antibody treatment

Osteogenesis imperfecta (OI) is a heritable collagen-related bone dysplasia, characterized by brittle bones with increased fracture risk that presents most severely in children. Anti-resorptive bisphosphonates are frequently used to treat pediatric OI and controlled clinical trials have shown that bisphosphonate therapy improves vertebral outcomes but has little benefit on long bone fracture rate. New treatments which increase bone mass throughout the pediatric OI skeleton would be beneficial. Sclerostin antibody (Scl-Ab) is a potential candidate anabolic therapy for pediatric OI and functions by stimulating osteoblastic bone formation via the canonical Wnt signaling pathway. To explore the effect of Scl-Ab on the rapidly growing OI skeleton, we treated rapidly growing 3 week old Brtl/+ mice, harboring a typical heterozygous OI-causing Gly → Cys substitution on col1a1, for 5 weeks with Scl-Ab. Scl-Ab had anabolic effects in Brtl/+ and led to new cortical bone formation and increased cortical bone mass. This anabolic action resulted in improved mechanical strength to WT Veh levels without altering the underlying brittle nature of the material. While Scl-Ab was anabolic in trabecular bone of the distal femur in both genotypes, the effect was less strong in these rapidly growing Brtl/+ mice compared to WT. In conclusion, Scl-Ab was able to stimulate bone formation in a rapidly growing Brtl/+ murine model of OI, and represents a potential new therapy to improve bone mass and reduce fracture risk in pediatric OI.     

Sclerostin antibody prevented progressive bone loss in combined ovariectomized and concurrent functional disuse

Osteoporosis is characterized by low bone mass and compromised trabecular architecture, and is commonly occurred in post-menopausal women with estrogen deficiency. In addition, prolonged mechanical unloading, i.e., long term bed rest, can exaggerate the bone loss. Sclerostin is a Wnt signaling antagonist and acts as a negative regulator for bone formation. A sclerostin-neutralizing antibody (Scl-Ab) increased bone mineral density in women with postmenopausal osteoporosis and healthy men. The objective of this study was to characterize the condition of bone loss in ovariectomized (OVX) rats with concurrent mechanical unloading and evaluate the effect of sclerostin antibody treatment in mitigating the prospective severe bone loss conditions in this model. Four-month-old OVX- or sham-operated female SD rats were used in this study. They were subjected to functional disuse induced by hind-limb suspension (HLS) or free ambulance after 2 days of arrival. Subcutaneous injections with either vehicle or Scl-Ab at 25 mg/kg were made twice per week for 5 weeks from the time of HLS. μCT analyses demonstrated a significant decrease in distal metaphyseal trabecular architecture integrity with HLS, OVX and HLS + OVX (bone volume fraction decreased by 29%, 71% and 87% respectively). The significant improvements of various trabecular bone parameters (bone volume fraction increased by 111%, 229% and 297% respectively as compared with placebo group) with the administration of Scl-Ab are associated with stronger mechanical property and increased bone formation by histomorphometry. These results together indicate that Scl-Ab prevented the loss of trabecular bone mass and cortical bone strength in OVX rat model with concurrent mechanical unloading. The data suggested that monoclonal sclerostin-neutralizing antibody represents a promising therapeutic approach for severe osteoporosis induced by estrogen deficiency with concurrent mechanical unloading.     

Zoledronate prevents lactation induced bone loss and results in additional post-lactation bone mass in mice

In rodents, lactation is associated with a considerable and very rapid bone loss, which almost completely recovers after weaning. The aim of the present study was to investigate whether the bisphosphonate Zoledronate (Zln) can inhibit lactation induced bone loss, and if Zln interferes with recovery of bone mass after lactation has ceased.Seventy-six 10-weeks-old NMRI mice were divided into the following groups: Baseline, Pregnant, Lactation, Lactation + Zln, Recovery, Recovery + Zln, and Virgin Control (age-matched). The lactation period was 12 days, then the pups were removed, and thereafter recovery took place for 28 days. Zln, 100 μg/kg, was given s.c. on the day of delivery, and again 4 and 8 days later. Mechanical testing, μCT, and dynamic histomorphometry were performed. At L4, lactation resulted in a substantial loss of bone strength (− 55% vs. Pregnant, p < 0.01), BV/TV (− 40% vs. Pregnant, p < 0.01), and trabecular thickness (Tb.Th) (− 29% vs. Pregnant, p < 0.001). Treatment with Zln completely prevented lactation induced loss of bone strength, BV/TV, and Tb.Th at L4. Full recovery of micro-architectural and mechanical properties was found 28 days after weaning in vehicle-treated mice. Interestingly, the recovery group treated with Zln during the lactation period had higher BV/TV (+ 45%, p < 0.01) and Tb.Th (+ 16%, p < 0.05) compared with virgin controls. Similar results were found at the proximal tibia and femur. This indicates that Zln did not interfere with the bone formation taking place after weaning. On this background, we conclude that post-lactation bone formation is not dependent on a preceding lactation induced bone loss.     

Effects of a moderately high-protein diet and interval aerobic training combined with strength-endurance exercise on markers of bone metabolism,microarchitecture and turnover in obese Zucker rats

BackgroundWeight loss is a public health concern in obesity-related diseases such as metabolic syndrome, and the protein level of the diets seem to be crucial for the development and maintenance of bone. The nature of exercise and whether exercise in combination with moderately high-protein dietary interventions could protect against potential bone mass deficits remains unclear.ObjectivesTo investigate the effects of a moderately high-protein diet and interval aerobic training combined with strength-endurance exercise (IASE) protocol on bone status, and to assess potential interaction effects (i.e. diet*IASE).MethodsMale Zucker fatty rats were randomized distributed into 4 groups (n = 8): normoprotein + sedentary; normoprotein + exercise; moderately high-protein + sedentary, and moderately high-protein + exercise. Training groups conducted an IASE program, 5 days/week for 2 months. Markers of bone metabolism were measured in plasma. Parameters of bone mass and 3D outcomes for trabecular and cortical bone microarchitecture were assessed by micro-computed tomography.ResultsFemur length, plasma osteocalcin, sclerostin, osteoprotegerin, receptor activator of nuclear factor kappa-B ligand, insulin, leptin, PTH, uric acid and urinary phosphorus levels were lower in the moderately high-protein compared to the normoprotein groups (all, p < 0.05), whereas plasma alkaline phosphatase, aspartate aminotransferase, alanine transaminase, and urinary uric acid concentrations, and cortical total volume (TV) and bone volume (BV) were higher in the moderately high-protein (all, p < 0.01). Final body weight and alkaline phosphatase levels were lower in the exercise compared to the sedentary (both, p < 0.05), whereas femur length and weight, aminoterminal propeptides of type I procollagen and C-terminal telopeptides of type I collagen concentrations, and cortical TV and BV were higher in the exercise compared to the sedentary groups (all, p < 0.05).ConclusionThe combination of interventions may be effective to enhance trabecular bone microarchitecture and BMD, and has a partial impact on cortical bone in obese rats. Nevertheless, they do not induce any alteration on the bone turnover markers.     

Site-specific,adult bone benefits attributed to loading during youth: A preliminary longitudinal analysis

We examined site-specific bone development in relation to childhood and adolescent artistic gymnastics exposure, comparing up to 10 years of prospectively acquired longitudinal data in 44 subjects, including 31 non-gymnasts (NON) and 13 gymnasts (GYM) who participated in gymnastics from pre-menarche to ≥ 1.9 years post-menarche. Subjects underwent annual regional and whole-body DXA scans; indices of bone geometry and strength were calculated. Anthropometrics, physical activity, and maturity were assessed annually, coincident with DXA scans. Non-linear mixed effect models centered growth in bone outcomes at menarche and adjusted for menarcheal age, height, and non-bone fat-free mass to evaluate GYM-NON differences. A POST-QUIT variable assessed the withdrawal effect of quitting gymnastics. Curves for bone area, mass (BMC), and strength indices were higher in GYM than NON at both distal radius metaphysis and diaphysis (p < 0.0001). At the femoral neck, greater GYM BMC (p < 0.01), narrower GYM endosteal diameter (p < 0.02), and similar periosteal width (p = 0.09) yielded GYM advantages in narrow neck cortical thickness and buckling ratio (both p < 0.001; lower BR indicates lower fracture risk). Lumbar spine and sub-head BMC were greater in GYM than NON (p < 0.036). Following gymnastics cessation, GYM slopes increased for distal radius diaphysis parameters (p ≤ 0.01) and for narrow neck BR (p = 0.02). At the distal radius metaphysis, GYM BMC and compressive strength slopes decreased, as did slopes for lumbar spine BMC, femoral neck BMC, and narrow neck cortical thickness (p < 0.02). In conclusion, advantages in bone mass, geometry, and strength at multiple skeletal sites were noted across growth and into young adulthood in girls who participated in gymnastics loading to at least 1.9 years post-menarche. Following gymnastics cessation, advantages at cortical bone sites improved or stabilized, while advantages at corticocancellous sites stabilized or diminished. Additional longitudinal observation is necessary to determine whether residual loading benefits enhance lifelong skeletal strength.     

Third metacarpal condylar fatigue fractures in equine athletes occur within previously modelled subchondral bone

Bone modelling and remodelling reduce the risk of fatigue fractures; the former by adapting bone to its loading circumstances, the latter by replacing fatigued bone. Remodelling transiently increases porosity because of the normal delay in onset of the formation phase of the remodelling sequence. Protracted intense loading suppresses remodelling leaving modelling as the only means of maintaining bone strength. We therefore hypothesized that race horses with fatigue fractures of the distal third metacarpal bone (MC3) will have reduced porosity associated with suppressed remodelling while continued adaptive modelling will result in higher volume fraction (BV/TV) at this site. Using high resolution peripheral quantitative computed tomography (HR-pQCT), we measured the distal aspect of the MC3 obtained at postmortem from 13 thoroughbred race horses with condylar fractures of the MC3 (cases), 8 horses without fractures (training controls), 14 horses with a fracture at another site (fractured controls) and 9 horses resting from training (resting controls).Porosity of the subchondral bone of MC3 was lower in cases than resting controls (12 ± 1.4% vs. 18 ± 1.6%, P = 0.017) although areas of focal porosity were observed adjacent to fractures in 6/13 horses. BV/TV of the distal metacarpal epiphysis tended to be higher in horses with condylar fractures (0.79 ± 0.015) than training controls (0.74 ± 0.019, P = 0.070), but also higher in controls with a fracture elsewhere (0.79 ± 0.014) than the training controls (0.74 ± 0.019, P = 0.040). BV/TV was higher in horses over three years of age than those aged two or three years (0.79 ± 0.01 vs. 0.74 ± 0.01, P = 0.016). All metacarpal condylar fractures occurred within focal areas of high BV/TV.We infer that intense training in equine athletes suppresses remodelling of third metacarpal subchondral bone limiting damage repair while modelling increases regional bone volume in an attempt to minimise local stresses but may fail to offset bone fragility.     

Subtle changes in bone mineralization density distribution in most severely affected patients with chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is associated with low aBMD as measured by DXA and altered microstructure as assessed by bone histomorphometry and microcomputed tomography. Knowledge of bone matrix mineralization is lacking in COPD. Using quantitative backscatter electron imaging (qBEI), we assessed cancellous (Cn.) and cortical (Ct.) bone mineralization density distribution (BMDD) in 19 postmenopausal women (62.1 ± 7.3 years of age) with COPD. Eight had sustained fragility fractures, and 13 had received treatment with inhaled glucocorticoids. The BMDD outcomes from the patients were compared with healthy reference data and were correlated with previous clinical and histomorphometric findings.In general, the BMDD outcomes for the patients were not significantly different from the reference data. Neither the subgroups of with or without fragility fractures or of who did or did not receive inhaled glucocorticoid treatment, showed differences in BMDD. However, subgroup comparison according to severity revealed 10% decreased cancellous mineralization heterogeneity (Cn.CaWidth) for the most severely affected compared with less affected patients (p = 0.042) and compared with healthy premenopausal controls (p = 0.021). BMDD parameters were highly correlated with histomorphometric cancellous bone volume (BV/TV) and formation indices: mean degree of mineralization (Cn.CaMean) versus BV/TV (r = 0.58, p = 0.009), and Cn.CaMean and Ct.CaMean versus bone formation rate (BFR/BS) (r = -0.71, p < 0.001). In particular, those with lower BV/TV (< 50th percentile) had significantly lower Cn.CaMean (p = 0.037) and higher Cn.CaLow (p = 0.020) compared with those with higher (> 50th percentile) BV/TV.The normality in most of the BMDD parameters and bone formation rates as well as the significant correlations between them suggests unaffected mineralization processes in COPD. Our findings also indicate no significant negative effect of treatment with inhaled glucocorticoids on the bone mineralization pattern. However, the observed concomitant occurrence of relatively lower bone volumes with lower bone matrix mineralization will both contribute to the reduced aBMD in some patients with COPD.     

Osteoprotegerin is an effective countermeasure for spaceflight-induced bone loss in mice

Bone loss associated with microgravity exposure poses a significant barrier to long-duration spaceflight. Osteoprotegerin-Fc (OPG-Fc) is a receptor activator of nuclear factor kappa-B ligand (RANKL) inhibitor that causes sustained inhibition of bone resorption after a single subcutaneous injection. We tested the ability of OPG-Fc to preserve bone mass during 12 days of spaceflight (SF). 64-day-old female C57BL/6J mice (n = 12/group) were injected subcutaneously with OPG-Fc (20 mg/kg) or an inert vehicle (VEH), 24 h prior to launch. Ground control (GC) mice (VEH or OPG-Fc) were maintained under environmental conditions that mimicked those in the space shuttle middeck. Age-matched baseline (BL) controls were sacrificed at launch. GC/VEH, but not SF/VEH mice, gained tibia BMD and trabecular volume fraction (BV/TV) during the mission (P < 0.05 vs. BL). SF/VEH mice had lower BV/TV vs. GC/VEH mice, while SF/OPG-Fc mice had greater BV/TV than SF/VEH or GC/VEH. SF reduced femur elastic and maximum strength in VEH mice, with OPG-Fc increasing elastic strength in SF mice. Serum TRAP5b was elevated in SF/VEH mice vs. GC/VEH mice. Conversely, SF/OPG-Fc mice had lower TRAP5b levels, suggesting that OPG-Fc preserved bone during spaceflight via inhibition of osteoclast-mediated bone resorption. Decreased bone formation also contributed to the observed osteopenia, based on the reduced femur periosteal bone formation rate and serum osteocalcin level. Overall, these observations suggest that the beneficial effects of OPG-Fc during SF are primarily due to dramatic and sustained suppression of bone resorption. In growing mice, this effect appears to compensate for the SF-related inhibition of bone formation, while preventing any SF-related increase in bone resorption. We have demonstrated that the young mouse is an appropriate new model for SF-induced osteopenia, and that a single pre-flight treatment with OPG-Fc can effectively prevent the deleterious effects of SF on mouse bone.     

Variation in osteocyte lacunar morphology and density in the human femur — a synchrotron radiation micro-CT study

In recent years there has been growing interest in the spatial properties of osteocytes (including density and morphology) and how these potentially relate to adaptation, disease and aging. This interest has, in part, arisen from the availability of increasingly high-resolution 3D imaging modalities such as synchrotron radiation (SR) micro-CT. As resolution increases, field of view generally decreases. Thus, while increasingly detailed spatial information is obtained, it is unclear how representative this information is of the skeleton or even the isolated bone. The purpose of this research was to describe the variation in osteocyte lacunar density, morphology and orientation within the femur from a healthy young male human. Multiple anterior, posterior, medial and lateral blocks (2 mm × 2 mm) were prepared from the proximal femoral shaft and SR micro-CT imaged at the Advanced Photon Source. Average lacunar densities (± standard deviation) from the anterior, posterior, medial and lateral regions were 27,169 ± 1935, 26,3643 ± 1262, 37,521 ± 6416 and 33,972 ± 2513 lacunae per mm³ of bone tissue, respectively. These values were significantly different between the medial and both the anterior and posterior regions (p < 0.05). The density of the combined anterior and posterior regions was also significantly lower (p = 0.001) than the density of the combined medial and lateral regions. Although no difference was found in predominant orientation, shape differences were found; with the combined anterior and posterior regions having more elongated (p = 0.004) and flattened (p = 0.045) lacunae, than those of the medial and lateral regions. This study reveals variation in osteocyte lacunar density and morphology within the cross-section of a single bone and that this variation can be considerable (up to 30% difference in density between regions). The underlying functional significance of the observed variation in lacunar density likely relates to localized variations in loading conditions as the pattern corresponds well with mechanical axes. Lower density and more elongate shapes being associated with the antero-posterior oriented neutral axis. Our findings demonstrate that the functional and pathological interpretations that are increasingly being drawn from high resolution imaging of osteocyte lacunae need to be better situated within the broader context of normal variation, including that which occurs even within a single skeletal element.     

Trabecular microfractures in the femoral head with osteoporosis: Analysis of microcallus formations by synchrotron radiation micro CT

Trabecular bone microfracture pathogenesis and associated healing processes are not well understood. We analyzed the microcalluses that form subsequent to microfractures in patients with osteoporosis (OP) using synchrotron radiation micro CT (SRCT).Subchondral bone columns were extracted from the femoral heads of 11 female patients with a femoral neck fracture. SRCT scanning was performed with 5.9 × 5.9 × 5.9 μm³ voxel size and the microcallus number was measured in a 5-mm cubic subchondral bone region. The trabecular bone microstructure was measured and its relationship to the microcallus number was analyzed. In addition, the degree of mineralization of the microcallus region and that of the rest of the trabecular bone were measured and compared.Microcallus formations were detected in all cases, with a mean microcallus number of 4.9 (range, 2–11). The microcallus number had a significantly negative correlation with bone volume fraction (BV/TV), trabecular thickness (Tb.Th), and degree of mineralization, and had a positive correlation with specific bone surface (BS/BV). The degree of mineralization of the microcallus region was lower than that of the rest of the trabecular bone and had a wider range of values.Microcallus formations were frequently detected in patients with OP, and more prevalent in the bone with thinner trabeculae, suggesting microfractures might occur due to activities of daily living as the OP progresses. The degree of mineralization of microcallus might represent the process of bone healing from immature woven bone to mature trabecular bone.     

The role of bone intrinsic properties measured by infrared spectroscopy in whole lumbar vertebra mechanics: Organic rather than inorganic bone matrix?

Whole bone strength is determined by bone mass, microarchitecture and intrinsic properties of the bone matrix. However, few studies have directly investigated the contribution of bone tissue material properties to whole bone strength in humans. This study assessed the role of bone matrix composition on whole lumbar vertebra mechanics. We obtained 17 fresh frozen human lumbar spines (8 W, 9 M, aged 76 ± 11 years). L3 bone mass was measured by DXA and microarchitecture by μ-CT with a 35 μm-isotropic resolution. Microarchitectural parameters were directly measured: Tb.BV/TV, SMI, Tb.Th, DA, Ct.Th, Ct.Po and radius of anterior cortical curvature. Failure load (N), stiffness (N/mm) and work to failure (N.mm) were extracted from quasi-static uniaxial compressive testing performed on L3 vertebral bodies. FTIRM analysis was performed on 2 μm-thick sections from L2 trabecular cores, with a Perkin-Elmer GXII Auto-image Microscope equipped with a wide band detector. Twenty measurements per sample were performed at 30 1 100 μm of spatial resolution. Each spectrum was collected at 4 cm^− 1 resolution and 50 scans in transmission mode. Mineral and collagen maturity, and mineralization and crystallinity index were measured. There was no association between the bone matrix characteristics and bone mass or microarchitecture. Mineral maturity, mineralization and crystallinity index were not related to whole vertebra mechanics. However, collagen maturity was positively correlated with whole vertebra failure load and stiffness (r = 0.64, p = 0.005 and r = 0.54, p = 0.025, respectively). The collagen maturity (3rd step) in combination with bone mass (i.e. BMC, 1st step) and microarchitecture (i.e. Tb.Th, 2nd step) improved the prediction of whole vertebra mechanical properties in forward stepwise multiple regression models, together explaining 71% of the variability in whole vertebra stiffness (p = 0.001). In conclusion, we demonstrated a substantial contribution of collagen maturity, but not mineralization parameters, to whole bone strength of human lumbar vertebrae that was independent of bone mass and microarchitecture.     

Influence of the weight status on bone mineral content and bone mineral density in a group of Lebanese adolescent girls

AimThe aim of this study was to determine the influence of being overweight on whole-body (WB) bone mineral content (BMC) and bone mineral density (BMD) in a group of Lebanese adolescent girls.MethodsThis study included 32 overweight (BMI > 25 kg/m²) adolescent girls (15.3 ± 2.3 years old) and 24 maturation-matched (15.7 ± 1.7 years old) controls (BMI < 25 kg/m²). Bone mineral area (BMA), BMC, BMD at the WB and body composition (lean mass and fat mass) were assessed by dual-energy X-ray absorptiometry (DXA). Calculation of the ratio BMC/height and bone mineral apparent density (BMAD) were completed for the WB.ResultsExpressed as crude values, BMA, BMC and the ratio BMC/height were higher in overweight adolescent girls compared to controls. After adjusting for body weight, there were no differences in BMC or in the ratio BMC/height between the two groups. However, BMA was lower in overweight girls compared to controls. After adjusting for either lean mass or fat mass, there were no significant differences between the two groups regarding these variables: BMC, BMA, BMD, BMC/height and BMAD.ConclusionThis study suggests that the positive effect of overweight on BMC is due to body weight. In fact, the difference in BMC between the overweight and the control girls disappears after adjusting for body weight. In contrast, overweight girls have lower BMA compared to controls when values are adjusted to body weight.     

Relationships of appendicular LMI and total body LMI to bone mass and physical activity levels in a birth cohort of New Zealand five-year olds

The present study was undertaken to determine whether children with lower bone mass display lower muscle mass for their height than those with high bone mineral content (BMC) and whether appendicular lean mass (bone-free lean mass in arms plus legs) is associated with physical activity and/or BMC in preschool children. 158 children (59% male) from a New Zealand cohort born in 2001–2002 were studied close to their fifth birthday. Body composition was measured by dual energy X-ray absorptiometry (Lunar DPX-L). Lean mass index (LMI) was calculated as lean mass (kg) divided by height in metres squared. Physical activity was assessed objectively by accelerometry (Actical Mini-Mitter). Girls and boys had similar heights, weights and daily accelerometry counts but boys had lower fat mass, and higher lean mass and total body BMC than the girls (P < 0.00l). In both sexes children with greater quantities of total and regional lean mass and higher LMI values had significantly higher bone mass. Appendicular LMI was more strongly associated with BMC than LMI. Accelerometry counts showed no associations with height but were positively associated with lean mass (r = 0.23, P < 0.03), appendicular LMI (r = 0.25 P < 0.01), total body BMC (r = 0.24, P < 0.02) and total body less head BMC (r = 0.27 P < 0.009) in the boys, but not in the girls. Greater time spent in more intense physical activity was also associated with higher appendicular lean mass and TBLH BMC only in the boys. We conclude that children with lower BMC values display not only lower lean mass but also lower total and appendicular lean mass for their height, than those with higher BMC values. The sex differences in associations of accelerometry counts to lean mass and BMC have been noted by others and require further investigation.     

Altered lacunar and vascular porosity in osteogenesis imperfecta mouse bone as revealed by synchrotron tomography contributes to bone fragility

Osteogenesis imperfecta (brittle bone disease) is caused by mutations in the collagen genes and results in skeletal fragility. Changes in bone porosity at the tissue level indicate changes in bone metabolism and alter bone mechanical integrity. We investigated the cortical bone tissue porosity of a mouse model of the disease, oim, in comparison to a wild type (WT-C57BL/6), and examined the influence of canal architecture on bone mechanical performance.High-resolution 3D representations of the posterior tibial and the lateral humeral mid-diaphysis of the bones were acquired for both mouse groups using synchrotron radiation-based computed tomography at a nominal resolution of 700 nm. Volumetric morphometric indices were determined for cortical bone, canal network and osteocyte lacunae. The influence of canal porosity architecture on bone mechanics was investigated using microarchitectural finite element (μFE) models of the cortical bone. Bright-field microscopy of stained sections was used to determine if canals were vascular.Although total cortical porosity was comparable between oim and WT bone, oim bone had more numerous and more branched canals (p < 0.001), and more osteocyte lacunae per unit volume compared to WT (p < 0.001). Lacunae in oim were more spherical in shape compared to the ellipsoidal WT lacunae (p < 0.001). Histology revealed blood vessels in all WT and oim canals. μFE models of cortical bone revealed that small and branched canals, typical of oim bone, increase the risk of bone failure. These results portray a state of compromised bone quality in oim bone at the tissue level, which contributes to its deficient mechanical properties.